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Ammonia and amino acid metabolism and transport in brain in vitro 1972

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A M M O N I A A N D A M I N O A C I D M E T A B O L I S M A N D T R A N S P O R T IN B R A I N IN V I T R O by A b r a h a m M . Benjamin B.Sc . (Hons.), Univer sity of Bombay, 1961 B.Sc . (Tech.), Univer sity of Bombay, 1963 M . S c . (Tech.), University of Bombay, 1966 M . S c , University of B r i t i s h Columbia, 1969 A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemi stry We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A June, 1972 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Biochemistry T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e July 25th, 1972 i i A B S T R A C T Studies have been made of the factors controlling the formation, transport and utilization of ammonia in the brain and its effects on brain metabolism, of the processes promoting amino acid fluxes in brain under a variety of conditions, especially those leading to increased nerve activity, and on the specific locations, and sites of formation, of amino acids in the brain. By using tetrodotoxin (2 tiM) to suppress partly the neuronal efflux of amino acids brought about by the joint action of protoveratrine (5 |iM) and ouabain (0.1 mM), the former drug being used to promote neuronal efflux of amino acids, and the latter being added to diminish re-uptake of amino acids, it has been shown that the major pools of glutamate, aspartate, glycine, serine and probably y-a-minobutyrate, are in the neurons. However, the major pool of glutamine appears to be in the glia. Glutamine formation takes place in the glia and is a process partly controlled by the concentrations of cations (K +, Na"*", C a + + ) within these cells. Fluoroacetate (3 mM) acts mainly in the glia as it suppresses glutamine synthesis, but not the proto- veratrine-stimulated brain respiration. Malonate (2 mM) acts mainly in the neurons since it suppresses the protoveratrine - stimulated respiration but not the synthesis of glutamine. The amino acids, particularly glutamate, y-aminobutyrate, aspartate and glycine, are released from brain cortex slices under conditions associated with brain cell excitation. The release processes are partly or wholly blocked by tetrodotoxin (2 (jM). Tetrodotoxin does not affect the release of glutamine nor does protoveratrine accelerate it. This result is in accord with the con- clusion that the main depot of glutamine lies not in the neurons but in the glia. Protoveratrine brings about an increased rate of formation of glutamine in incubated brain slices, suggesting that glutamate released from the neurons is taken up by the glia and there converted to glutamine. LrGlutamine is more i i i e f f e ctive than L-glutamate as a p r e c u r s o r of y-aminobutyrate i n b r a i n s l i c e s . A s g l u t a m i c a c i d d e c a r b o x y l a s e is l o c a l i z e d i n the neurons, it i s concluded that glutamine r e l e a s e d f r o m the g l i a is taken up by the neurons and there c o n v e r t e d to glutamate and y-aminobutyrate. Changes i n the contents of NH^"*" i n incubated b r a i n s l i c e s a r e a c c o m - panied by quantitatively equivalent changes i n the amino a c i d contents of the ti s s u e . A m y t a l (1 mM) s u p p r e s s e s endogenous glutamate oxidation and enhances the neuronal contents of glutamate and y-aminobutyrate. It d i m i n i s h e s a mmonia l i b e r a t i o n . A m m o n i a is f o r m e d a e r o b i c a l l y by b r a i n c o r t e x s l i c e s i n a g l u c o s e - f r e e m e d i u m l a r g e l y by endogenous glutamate oxidation within the neurons, and a l s o by glutamine h y d r o l y s i s . E x t e r n a l L-glutamate is taken up against a concentration gradient l a r g e l y by the g l i a and i s l e s s effective than endogenous glutamate as a source of ammonia in b r a i n . A m m o n i u m ions a r e not accumulated i n b r a i n s l i c e s against a concent r a t i o n gradient. They a r e p r e s u m a b l y formed, up to a l i m i t i n g concentration, in the neurons indepen- dently of the e x t e r n a l NĤ "*" concentration. A m m o n i u m ions affect both neuronal and g l i a l m e t a b o l i s m and the b r a i n c e l l t r a n s p o r t of Na"*" and in the incubated b r a i n s l i c e s . The d e c r e a s e of i s p a r t l y due to exchange with NĤ "*". The exchange p r o c e s s is most m a r k e d in infant rat b r a i n . The effects of N H ^ + in inhibiting r e s p i r a t i o n , d i m i n i s h i n g A T P concentrations, and changing the ca t i o n i c concentrations at the b r a i n c e l l membrane are more pronounced in the stimulated than i n the unst i m u l a t e d b r a i n t i s s u e . It i s concluded that the effects of on b r a i n m e t a b o l i s m and cation t r a n s p o r t may be explained by its i n h i b i t o r y effect on A T P f o r m a t i o n i n the neurons, by r e m o v a l of a-ketoglutarate and hence by p a r t l y b l o c k i n g the operation of the c i t r i c a c i d c y c l e . T h i s may be one of the r e a s o n s for ammonia t o x i c i t y i n the c e n t r a l nervous system. iv T A B L E O F C O N T E N T S Page T I T L E P A G E i A B S T R A C T i i T A B L E O F C O N T E N T S iv L I S T O F F I G U R E S x i i i L I S T O F T A B L E S xiv A B B R E V I A T I O N S . . . xxi A C K N O W L E D G M E N T S xxi i 1. I N T R O D U C T I O N 1 1.1 Format ion of ammonia by nerve tissue 1 1.2 Fac tors affecting the in vivo levels of ammonia in the b r a i n . . . . i (i) A m m o n i a content of brain in vivo 1 (ii) Fac tors producing low brain ammonia levels in. vivo. 2 (iii) Fac tors producing high brain ammonia levels in vivo 3 1.3 (i) Some chemical changes that occur in the brain on the application of s t imuli to the whole animal 4 (ii) Recovery processes that occur in the brain on the removal of the s t imuli applied to the whole animal 5 (iii) Convulsions and brain energy levels 5 1.4 Fac tors affecting the formation of ammonia in brain tissue in vi t ro . 6 (i) Carbohydrate metabolism and ammonia formation 6 (ii) E lec t ron transport, oxidative phosphorylation and ammonia formation 7 (iii) Effects of exogenous amino acids on ammonia formation 8 1.5 A m m o n i a formation and utilization mechanisms in the b r a i n . . . 9 (i) U r e a 9 (a) U r e a as a source of ammonia 9 (b) Possible formation of urea as a mechanism for ammonia utilization •. 10 (c) Possible importance of urea synthesis in the bra in (85, 95, 100) 11 V Page (ii) (a) A m m o n i a formation f r o m amino acids via aspartate channelled through N A D + or A M P 11 (b) A m m o n i a formation f r o m amino acids via terminal glutamate oxidation 13 (iii) A m m o n i a utilization processes in bra in 15 1.6 Transport of amino acids in brain 16 1.7 (i) Amino acid metabolism in brain 18 (a) Initial cerebral contents of amino acids 20 (b) Pathways of glutamate metabolism 20 (ii) Compartmentation of amino acid metabolism in b r a i n . . . . 24 1.8 Propagation of nerve impulse 26 (i) B r a i n cel l types (168, 169) 26 (ii) Resting potential (164-168) 27 (iii) Act ion potential (164-168) 28 (iv) Sodium-Pump (164-168) 28 (v) Synaptic t ransmiss ion (164-168, 170) 29 (vi) A m i n o acids as putative transmitters (164, 165, 168, 170, 233) 29 (vii) Ca"*"*" ions and excitation 30 (viii) High K+ ion concentration and excitation 30 (ix) Cerebral cortex slices and excitation phenomena 31 1.9 Some properties of drugs and metabolic inhibitors used as tools for this investigation 32 (i) Tetrodotoxin (TTX) 32 (ii) L o c a l anesthetics (166, 176) 35 (iii) Protoveratrine 36 (iv) Barbiturates 37 (v) Cardiac glycosides - Ouabain 39 (vi) Miscellaneous 41 1.10 Objectives of the present work. 42 2. M A T E R I A L S A N D M E T H O D S 43 2.1 A n i m a l s . 43 2.2 Chemicals 43 2.3 Tissue preparation 44 2.4 Media compositions and incubation procedures 44 (i) K r e b s - R i n g e r phosphate medium 44 (ii) K r e b s - R i n g e r bicarbonate medium 45 (iii) M e d i u m II 45 v i P a g e 2.5 A m i n o a c i d a n a l y z e r e s t i m a t i o n s . . 46 (a) S a m p l e p r e p a r a t i o n 46 (i) T i s s u e s a m p l e p r e p a r a t i o n 46 (ii) M e d i u m s a m p l e p r e p a r a t i o n 46 (b) S a m p l e a n a l y s i s 46 (i) A c i d i c and n e u t r a l a m ino a c i d a n a l y s e s 47 (ii) y - A m i n o b u t y r i c a c i d and a m m o n i a 47 ( i i i ) N - A c e t y l a s p a r t a t e 48 (iv) C a l c u l a t i o n s 48 2.6 Water uptake by b r a i n c o r t e x s l i c e s 48 2.7 O x y g en uptake by b r a i n c o r t e x s l i c e s 49 2.8 D r y weight of b r a i n c o r t e x s l i c e s 49 2.9 ^Na~*" i n f l u x i n t o b r a i n c o r t e x s l i c e s 49 2.10 E x p e r i m e n t s w i t h s o d i u m L - [ U - 1 4 C ] g l u t a m a t e 50 2.11 S c i n t i l l a t i o n l i q u i d c o m p o s i t i o n 50 2.12 Na"** and K"*" a s s a y by f l a m e - p h o t o m e t r y 51 2.13 D e t e r m i n a t i o n of A T P c o n c e n t r a t i o n s . 51 2.14 A s s a y of Na"*". K"*~-ATPase of b r a i n homogenates 52 (i) M e d i u m c o m p o s i t i o n 52 (ii) I n c u b a t i o n p r o c e d u r e 52 ( i i i ) E s t i m a t i o n 52 2.15 I s o l a t i o n of s y n a p t o s o m e s f r o m r a t b r a i n c o r t e x 53 2.16 P r o t e i n e s t i m a t i o n . 54 2.17 R e p r o d u c i b i l i t y of r e s u l t s 54 2.18 E x p l a n a t i o n of v a r i o u s t e r m s used i n t h i s t h e s i s 55 (i) I n i t i a l v a l u e s 55 (ii) T o t a l v a l u e s 55 ( i i i ) A d j u s t m e n t 55 (iv) A m m o n i a and a m m o n i u m i o n . . . 56 (v) F l u x 56 (vi) A c t i o n p o t e n t i a l s 56 v i i Page 3 . A M M O N I A F O R M A T I O N IN B R A I N IN V I T R O 57 3.1 A m m o n i a and amino a c i d contents of r a t c e r e b r a l c o r t e x s l i c e s i n i t i a l l y , and after incubation i n the p r e s e n c e or absence of glucos e 57 (i) Changes i n the N H 4 * " and amino a c i d contents of b r a i n s l i c e s incubated i n the p r e s e n c e and absence of glucose.. 57 (ii) Changes i n the i n i t i a l N H 4 + and amino a c i d contents of b r a i n c o r t e x s l i c e s on incubation 60 (iii) P o s s i b l e p r o t e i n breakdown in incubating b r a i n s l i c e s . . . . 61 3.2 A m m o n i a f o r m a t i o n in anoxia 63 3.3 A m m o n i a f o r m a t i o n by infant (2-day-old) r a t b r a i n c o r t e x s l i c e s 66 3.4 Is there a d i r e c t r o l e of aspartate i n the p r o c e s s of ammonia f o r m a t i o n i n the b r a i n ? 68 3.5 E f f e c t s of me t a b o l i c i n h i b i t o r s on the rate of ammonia for m a - tion f r o m endogenous amino a c i d s of r a t c e r e b r a l c o r t e x s l i c e s incubated i n a m e d i u m devoid of glucose. 72 3.6 E f f e c t s of ouabain on the rate of ammonia f o r m a t i o n i n r a t b r a i n cortex s l i c e s incubated i n a g l u c o s e - f r e e medium. 74 3.7 The r o l e of Ca"*"*" i n the f o r m a t i o n of ammonia f r o m L-glutamate by rat b r a i n c o r t e x s l i c e s incubated i n a m e d i u m d -^oid of glucose 76 3.8 A m m o n i a fo r m a t i o n by r a t c e r e b r a l c o r t e x s l i c e s f r o m exo- genous L - g l u t a m i n e i n the absence of glucose 78 3.9 E f f e c t s of tetrodotoxin, l i d o c a i n e and p r o t o v e r a t r i n e on the rate of ammonia f o r m a t i o n by rat b r a i n c o r t e x s l i c e s i n c u - bated i n a g l u c o s e - f r e e m e d i u m 80 3.10 Changes i n the ammonia and amino a c i d contents of i s o l a t e d r a t b r a i n c o r t i c a l synaptosomes on incubation 80 3.11 Summary 83 4 . C O N T R O L M E C H A N I S M S F O R G L U T A M I N E S Y N T H E S I S IN R A T B R A I N C O R T E X IN V I T R O . 85 4.1 E f f e c t s of v a r y i n g s o d i um ion c o n c e n t r a t i o n on the rate of glutamine synthesis i n r a t b r a i n cortex s l i c e s 8 5 4 . 2 E f f e c t s of v a r y i n g p o t a s s i u m ion c o n c e n t r a t i o n on the rate of glutamine synthesis i n r a t b r a i n cortex s l i c e s 86 v i i i P a ge 4 . 3 E f f e c t s of c a l c i u m on the rate of glutamine synthesis by- r a t b r a i n cortex s l i c e s 88 4.4 Glutamine synthesis i n a Na~*"-rich, K**"-free, C a ^ - f r e e , incubation medium. 8 9 4 .5 G l u t a m i n a s e a c t i v i t y and glutamine synthesis 89 4 . 6 E f f e c t s of s o d i u m L-glutamate and N H ^ C l on the i n h i b i t i o n of glutamine synthesis i n r a t b r a i n cortex s l i c e s incubated with glucose either i n M e d i u m II, or in the p r e s e n c e of ouabain. 89 4.7 C o m p a r a t i v e effects of metabolic i n h i b i t o r s (methionine sulfoximine, ouabain, fluoroacetate, malonate, D N P and amytal) on r a t e s of oxygen uptakes, glutamine synthesis and amino a c i d contents of r a t b r a i n cortex s l i c e s incubated i n the p r e s e n c e of glucose. 92 4 .8 E f f e c t s of tetrodotoxin on ouabain i n h i b i t i o n of glutamine synthesis in r a t b r a i n c o r t e x s l i c e s 96 4 . 9 Summary 98 5 . T R A N S P O R T O F L - G L U T A M A T E I N T O B R A I N IN V I T R O 100 5.1 T r a n s p o r t of L - g l u t a m a t e into incubated r a t b r a i n . 100 5.2 The uptake of L - [ U - 1 4 c ] glutamate by r a t c e r e b r a l cortex s l i c e s . 102 5• 3 A p p a r e n t absence of an exchange p r o c e s s between e x t e r n a l L - [ U - I 4 c j glutamate and endogenous glutamate of r a t b r a i n c o r t e x s l i c e s 104 5.4 L o c a t i o n of exogenous L - g l u t a m a t e uptake 10 5 5 .5 E f f e c t s of i n c r e a s i n g e x t e r n a l sodium L-glutamate concen- t r a t i o n s on the t i s s u e and m e d i u m concentrations of amino a c i d s in incubated r a t b r a i n c o r t e x s l i c e s 108 5.6 Summary I l l 6 . T R A N S P O R T O F A M M O N I U M IONS IN B R A I N IN V I T R O 112 6.1 T i s s u e and m e d i u m contents of N"-~4+ of rat b r a i n c o r t e x s l i c e s incubated under v a r y i n g conditions 113 i x P a g e 6.2 T i s s u e / M e d i u m c o n c e n t r a t i o n r a t i o s f o r N H ^ i n the i n c u b a t e d b r a i n s l i c e 115 6.3 E x o g enous N H ^ a c c u m u l a t i o n i n c e r e b r a l c o r t e x s l i c e s of the r a t . 117 6.4 C o m p a r i s o n of the t r a n s p o r t p r o c e s s e s f o r N H ^ and L - g l u t a m a t e into b r a i n c e l l s 121 6.5 R a t e s of N H ^ and g l y c i n e uptakes by b r a i n c o r t e x s l i c e s 123 6.6 E f f e c t s of N H 4 + on g l y c i n e uptake. 123 6.7 E f f e c t s of g l y c i n e on NH4"*" uptake 126 6.8 Uptake (into b r a i n c o r t e x s l i c e s ) of exogenous L - g l u t a m a t e and N H 4 + when p r e s e n t t o g e t h e r i n the i n c u b a t i o n m e d i u m 126 6.9 E f f e c t s of m e t a b o l i c i n h i b i t o r s on the uptake of N H 4 + by i n c u b a t e d r a t b r a i n c o r t e x s l i c e s . 126 6.10 E f f e c t s of i n c r e a s i n g NH4"*" c o n c e n t r a t i o n s on a m ino a c i d con- tent i n , and r e l e a s e f r o m , i n c u b a t e d r a t b r a i n c o r t e x s l i c e s ... 130 6.11 S u m m a r y 133 7. E F F E C T S O F N E U R O T R O P I C D R U G S O N T H E R E L E A S E O F A M I N O A C I D S F R O M T H E B R A I N I N V I T R O 134 7.1 E f f e c t s of p r o t o v e r a t r i n e , ouabain and t e t r o d o t o x i n on a m i n o a c i d content i n , and r e l e a s e f r o m , r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n g l u c o s e - s a l i n e m e d i a 135 7.2 E f f e c t s of p r o t over a t r ine and t e t r o d o t o x i n on a m i n o a c i d content i n , and r e l e a s e f r o m , r a t b r a i n c o r t e x s l i c e s i n c u - b ated i n a g l u c o s e - f r e e m e d i u m 139 7.3 E f f e c t s of p r o t o v e r a t r i n e and t e t r o d o t o x i n on a m i n o a c i d content i n , and r e l e a s e f r o m , r a t b r a i n c o r t e x s l i c e s i n c u - b ated i n c a l c i u m - d e f i c i e n t m e d i a 143 7.4 E f f e c t s of p r o t o v e r a t r i n e and t e t r o d o t o x i n on a m ino a c i d content i n , and r e l e a s e f r o m , r a t b r a i n c o r t e x s l i c e s i n c u - bated i n g l u c o s e - f r e e , c a l c i u m - d e f i c i e n t m e d i a 146 7.5 E f f e c t s of l i d o c a i n e on the r e l e a s e of a m i n o a c i d s f r o m r a t b r a i n s l i c e s i n c u b a t e d i n a v a r i e t y of m e d i a . 149 7.6 E f f e c t s of t e t r o d o t o x i n on the t i s s u e to m e d i u m c o n c e n t r a t i o n r a t i o s of amino a c i d s of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n a v a r i e t y of m e d i a 1 50 X Page 7.7 E f f e c t s of s o d i u m amytal on the r e l e a s e of amino acids f r o m r a t b r a i n cortex s l i c e s 1 52 7.8 E f f e c t s of i n c r e a s e d and of tetrodotoxin on amino a c i d content in, and r e l e a s e from, incubated r a t b r a i n c o r t e x s l i c e s . 1 56 7.9 E f f e c t s of tetrodotoxin on the efflux of amino acids f r o m kidney cortex s l i c e s incubated in the p r e s e n c e of ouabain .... 156 7.10 E f f e c t s of tetrodotoxin on the amino a c i d contents in, and r e l e a s e from, r a t b r a i n cortex s l i c e s incubated i n the p r e - sence of 2,4 dinitrophenol, NH4"*" or 105 m M KC1.. 161 7.11 S u m m ary 164 8. L O C A T I O N S O F A M I N O ACIDS IN B R A I N C O R T E X S L I C E S O F T H E R A T 166 8.1 L o c a t i o n of the glutamate-glutamine system. 166 8.2 E f f e c t s of p r o t o v e r a t r i n e on the s p e c i f i c a c t i v i t i e s of amino aci d s f r o m rat b r a i n c o r t e x s l i c e s incubated i n the p r e s e n c e of [U-14C] glucose 168 8.3 C h a r a c t e r i z a t i o n of amino a c i d compartments in b r a i n . 170 (i) E f f e c t s of the combined p r e s e n c e of p r o t o v e r a t r i n e and ouabain on amino a c i d r e l e a s e 171 (ii) E f f e c t s of tetrodotoxin... 173 (iii) A l t e r a t i o n s i n the contents of i n d i v i d u a l amino acids 174 (a) Glutamate 174 (b) Aspartate... 174 (c) G A B A. 175 (d) G l y c i n e and S e r i n e . 175 (e) Taurine, A l a n i n e and Thre o n i n e 175 (f) G lutamine 175 8.4 E f f e c t s of s o d i u m malonate and s o d i u m f l u o r o a c e t a t e on c e r e b r a l amino a c i d content and r e l e a s e i n the p r e s e n c e of p r o t o v e r a t r i n e 176 8.5 E f f e c t s of L - g l u t a m i n e and sodium L-glutamate on c e r e b r a l amino a c i d content and r e l e a s e 1^2 8.6 Summary *^2 x i P a g e 9. E F F E C T S O F NH 4+ O N B R A I N M E T A B O L I S M A N D T R A N S P O R T I N V I T R O 184 9.1 I n i t i a l contents of Na"*" and K"*" i n the i n f a n t (two-day-old) and a d u l t r a t b r a i n c o r t e x . 184 9.2 E f f e c t s of i n c r e a s i n g N H 4 * c o n c e n t r a t i o n on water and oxygen uptakes, and c a t i o n i c f l u x e s of i n c u b a t e d r a t b r a i n c o r t e x s l i c e s . 186 9.3 A T P a s e s of r a t c e r e b r a l c o r t i c a l homogenates 186 9.4 E f f e c t s of t e t r a m e t h y l a m m o n i u m c h l o r i d e , and the c h l o r i d e s a l t s of Li"*", Rb"*" and Cs"*" on the oxygen and w a t er uptakes, and the Na"*", K"*" f l u x e s i n i n c u b a t e d r a t b r a i n c o r t e x s l i c e s . . . . 188 9.5 E f f e c t s of i n c r e a s i n g c o n c e n t r a t i o n s of NH4"*" on c a t i o n i c f l u x e s i n i n c u b a t e d two-day-old r a t b r a i n c o r t e x s l i c e s 193 9.6 Q u a n t i t a t i v e a s p e c t s of the e f f e c t s of NH4"*" on the N a t K"*" and water contents of i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 195 9.7 E f f e c t s of i n c r e a s i n g NH4"*" c o n c e n t r a t i o n s on the c a t i o n i c contents of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e of 0.1 m M ouabain or i n the absence of g l u c o s e 195 9.8 E f f e c t s of changing m e d i u m i o n i c c o m p o s i t i o n on the Na"*", K"*" contents of i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 1 97 (i) E f f e c t s of Ca++ and K+ 197 (ii) E f f e c t s of i n c r e a s i n g NH4+ 200 ( i i i ) T i s s u e / M e d i u m c o n c e n t r a t i o n r a t i o s f o r Na+ and K+ 200 9.9 E f f e c t s of h i g h m e d i u m K"*" c o n c e n t r a t i o n s on the NH^"*" W . _ _ 1 i n d u c e d changes i n the N a , K contents of i n c u b a t e d r a t b r a i n c o r t e x s l i c e s . 202 9.10 E f f e c t s of i n c r e a s i n g N a - L - g l u t a m a t e c o n c e n t r a t i o n s on water, ^ _j_ 2 a ; — - N a , and K contents of i n c u b a t e d r a t c e r e b r a l c o r t e x s l i c e s . . 205 9.11 E f f e c t s of i n c r e a s i n g c o n c e n t r a t i o n of NH^"*" on A T P contents of r a t c e r e b r a l c o r t e x s l i c e s 207 9.12 E f f e c t s of n e u r o t r o p i c d r u g s on the oxygen and w a t e r uptakes, and Na"*", K"1" f l u x e s of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e of N H 4 C I or of N a - L - g l u t a m a t e . 210 (i) I n c u b a t i o n i n Ca"*""*"-containing m e d i a ( T a b l e 72) 210 (ii) I n c u b a t i o n i n Ca^~~*"-free m e d i a (Table 73) 212 9.13 E f f e c t s of n e u r o t r o p i c d r u g s on the A T P contents of i n c u b a t e d c e r e b r a l c o r t e x s l i c e s 215 x i i P age 9.14 E f f e c t s of s o d i u m fluoroacetate, s o d i u m malonate, tetrodo- toxin and N H 4 C I on the Na+, K"1" and water contents of r a t b r a i n cortex s l i c e s incubated i n the pr e s e n c e of glucose.... 215 9.15 Summary 2 1 8 10. DISCUSSION 220 10.1 A m m o n i a f o r m a t i o n in r a t b r a i n cortex s l i c e s 220 (i) Endogenous glutamate — the p o s s i b l e major so u r c e of a e r o b i c a mmonia f o r m a t i o n 220 (ii) Exogenous L-glutamate — as a p o s s i b l e source of c e r e b r a l ammonia 221 (iii) Glutamine — a sou r c e of ammonia i n b r a i n 222 (iv) M e c h a n i s m of ammonia f o r m a t i o n i n b r a i n i n v i t r o 223 (a) R e a c t i o n s i n the absence of glucose ... 223 (b) R e a c t i o n s i n the p r e s e n c e of glucose 225 (v) Neurons — the p o s s i b l e site of a e r o b i c ammonia f o r m a t i o n 225 10.2 T e t r o d o t o x i n - s e n s i t i v e fluxes of amino acids in the b r a i n i n v i t r o . 228 10.3 L o c a t i o n s of amino a c i d s i n the b r a i n . 232 10.4 N e u r o n - g l i a i n t e r r e l a t i o n s 233 10.5 C o n t r o l of ammonia u t i l i z a t i o n by glutamine synthesis i n b r a i n i n v i t r o . 235 10.6 T r a n s p o r t of N H 4 " * " into b r a i n cortex s l i c e s 237 10.7 T r a n s p o r t of L-glutamate into b r a i n cortex s l i c e s . 238 10.8 E f f e c t s of N H 4 + on b r a i n m e t abolism. 240 11. G E N E R A L R E S U L T S A N D C O N C L U S I O N S 244 R E F E R E N C E S 247 x i i i L I S T O F F I G U R E S F I G U R E Page 1 E f f e c t s of i n c r e a s i n g s o d i um L-glutamate or NH4+ concentrations on t h e i r T i s s u e / M e d i u m co n c e n t r a t i o n r a t i o s of incubated r a t b r a i n cortex s l i c e s 1 2 2 2 E f f e c t s of s o d i u m fluoroacetate and s o d i u m malonate on the p r o t o v e r a t r i n e - s t i m u l a t e d r e s p i r a t i o n of b r a i n cortex s l i c e s 178 x i v L I S T O F T A B L E S T A B L E P a g e 1 Some b r a i n to p l a s m a c o n c e n t r a t i o n r a t i o s of a m i n o a c i d s f o r the cat 19 2 A m m o n i a and amino a c i d changes i n adult r a t b r a i n c o r t e x s l i c e s on i n c u b a t i o n i n the p r e s e n c e or a b s e n c e of g l u c o s e 58 2A Changes i n the glu t a m a t e , g l u t a m i n e , a s p a r t a t e and a m m o n i a contents of b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e or absence of g l u c o s e 59 3 E f f e c t s of a n o x i a on the a m m o n i a and amino a c i d contents of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e o r absence of g l u c o s e 64 3A A m m o n i a and amino a c i d changes of b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e or absence of O 2 65 4 A m m o n i a and amino a c i d changes i n in f a n t r a t b r a i n c o r t e x on i n c u b a t i o n i n the p r e s e n c e o r absence of g l u c o s e .. 67 5 E f f e c t s of s o d i u m m a l o n a t e and amino o x y a c e t a t e on a s p a r t a t e s y n t h e s i s and a m m o n i a f o r m a t i o n i n r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n a g l u c o s e - f r e e m e d i u m 70 6 E f f e c t s of s o d i u m - a m y t a l and 2, 4 - d i n i t r o p h e n o l on the t o t a l a m m o n i a and amino a c i d contents of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n a g l u c o s e - f r e e m e d i u m 73 7 The e f f e c t s of ouabain on the r a t e of a m m o n i a f o r m a t i o n , and the r e l e a s e of a m i n o a c i d s f r o m b r a i n c o r t e x s l i c e s i n c u b a t e d i n a g l u c o s e - f r e e m e d i u m 75 8 E f f e c t s of L - g l u t a m a t e on the f o r m a t i o n of a m m o n i a and a m i n o a c i d s by r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n a m e d i u m d e v o i d of g l u c o s e i n the p r e s e n c e and absence of C a + + 77 9 A m m o n i a f o r m a t i o n f r o m exogenous L - g l u t a m i n e by r a t c e r e b r a l c o r t e x s l i c e s i n c u b a t e d i n a g l u c o s e - f r e e m e d i u m 79 X V T A B L E Page 10 E f f e c t s of n e u r o t r o p i c drugs on the f o r m a t i o n of ammonia, glutamate and glutamine i n r a t b r a i n cortex s l i c e s r e s p i r i n g i n a g l u c o s e - f r e e m e d i u m 81 11 A m m o n i a and amino a c i d changes i n i s o l a t e d r a t b r a i n c o r t e x synaptosomes incubated i n the pr e s e n c e or absence of glucose 82 12 E f f e c t s of the cationic contents of the incubation m e d i u m on the rate of glutamine synthesis i n r a t b r a i n c o r t e x s l i c e s 87 13 E f f e c t s of N H 4 C I and sodium L-glutamate i n r e v e r s i n g the s u p p r e s s e d r a t e s of glutamine synthesis i n r a t b r a i n cortex s l i c e s due to ouabain or changed media co m p o s i t i o n 90 14 E f f e c t s of metabolic i n h i b i t o r s on the rate of glutamine synthesis and amino a c i d contents of incubated r a t b r a i n c o r t e x s l i c e s 93 15 E f f e c t s of ouabain, methionine sulfoximine, K C 1 , 2, 4 - d i n i - trophenol, amytal, and glucose absence, on the r a t e s of oxygen consumption in rat c e r e b r a l c o r t e x s l i c e s 94 16 E f f e c t s of tetrodotoxin on the ouabain s u p p r e s s e d glutamine synthesis of r a t b r a i n c o r t e x s l i c e s 97 17 T r a n s p o r t of sodium L-glutamate into b r a i n c o r t e x s l i c e s of the r a t 101 18 Uptake of sodium L-(U-^^C) glutamate by r a t b r a i n c o r t e x s l i c e s 103 19 E f f e c t s of pr o t o v e r a t r i n e , ouabain and tetrodotoxin on the r a d i o a c t i v i t y of b r a i n c o r t e x s l i c e s p r e l o a d e d with s o d i u m L - ( U - 1 4 C ) glutamate 107 2 0 E f f e c t s of i n c r e a s i n g m e d i u m s o d i u m L-glutamate concen- t r a t i o n s on the amino a c i d contents of incubated rat b r a i n c o r t e x s l i c e s 109 21 E f f e c t s of i n c r e a s i n g m e d i u m s o d i u m L-glutamate concen- tr a t i o n s on the r e l e a s e of amino a c i d s f r o m incubated r a t b r a i n c o r t e x s l i c e s 110 x v i T A B L E ' Page 22 The tissue and m e d i u m concentrations of N H 4 + on incu- bating rat b r a i n cortex s l i c e s under a v a r i e t y of co n d i - tions 114 23 The tissue to m e d i u m concent r a t i o n r a t i o s f or NĤ "*" in r a t b r a i n c o r t e x s l i c e s incubated in a v a r i e t y of media 11° 24 Uptake of N H , + by ra t b r a i n cortex s l i c e s incubated in the j 1 Q presence of glucose 1 1 0 2 5 Uptake of NH^"1" by rat b r a i n c o r t e x s l i c e s incubated i n the absence of glucose 119 26 The uptake of N H 4 * by ra t b r a i n c o r t e x s l i c e s i n anoxia 120 27 Rates of N H 4 * and of gly c i n e uptake by rat b r a i n c o r t e x s l i c e s 124 28 The in h i b i t o r y effects of N H 4 * on the active t r a n s p o r t of glyc i n e into r a t b r a i n c o r t e x s l i c e s 125 29 A b s e n c e of an i n h i b i t o r y effect of g l y c i n e on the uptake of ammonium by r a t b r a i n c o r t e x s l i c e s 127 30 E f f e c t s of the simultaneous p r esence of exogenous s o d i u m L i-glutamate and N H 4 C I on the t r a n s p o r t p r o c e s s e s for ammonium and glutamate into r a t b r a i n c o r t e x s l i c e s 128 31 E f f e c t s of metabolic i n h i b i t o r s on the t r a n s p o r t of Nr^"*" and of g l y c i n e into r a t b r a i n c o r t e x s l i c e s 129 32 E f f e c t s of i n c r e a s i n g m e d i u m N H ^ C l concentrations on the amino a c i d contents of incubated r a t b r a i n c o r t e x s l i c e s 131 33 E f f e c t s of i n c r e a s i n g m e d i u m N H 4 C I concentrations on the r e l e a s e of amino acids f r o m incubated r a t b r a i n cortex s l i c e s 132 34 E f f e c t s of p r o t o v e r a t r i n e , ouabain, l i d o c a i n e and tetrodo- toxi n on the contents of amino acids i n incubated rat b r a i n c o r t e x s l i c e s 136 35 E f f e c t s of protover atr ine, ouabain, l i d o c a i n e and tetrodo- toxin on the r e l e a s e of amino a c i d s f r o m incubated r a t b r a i n c o r t e x s l i c e s 137 x v i i T A B L E Page 36 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin and l i d o c a i n e on contents of amino a c i d s i n r a t b r a i n c o r t e x s l i c e s i n c u - bated i n g l u c o s e - f r e e m edia 140 37 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin and l i d o c a i n e on the r e l e a s e of amino acids f r o m r a t b r a i n c o r t e x s l i c e s i n c u - bated i n g l u c o s e - f r e e media 141 38 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin, lidocaine, and of s o d i u m L-glutamate on the contents of amino a c i d s in r a t b r a i n c o r t e x s l i c e s incubated i n c a l c i u m - d e f i c i e n t media 144 39 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin, lidocaine, and of s o d i u m L-glutamate on the r e l e a s e of amino a c i d s f r o m r a t b r a i n c o r t e x s l i c e s incubated i n c a l c i u m - d e f i c i e n t media 145 40 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin, lidocaine, and of s o d i u m L-glutamate on the contents of amino acids i n r a t b r a i n c o r t e x s l i c e s i n g l u c o s e - f r e e , c a l c i u m - d e f i c i e n t m e dia 147 41 E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin, l i d o c a i n e and of s o d i u m L-glutamate on the r e l e a s e of amino a c i d s f r o m r a t b r a i n c o r t e x s l i c e s incubated i n g l u c o s e - f r e e , c a l c i u m - d e f i c i e n t m e d i a 148 42 E f f e c t s of tetrodotoxin on the t i s s u e to m edium c o n c e n t r a - t i o n r a t i o s of amino acids of r a t b r a i n c o r t e x s l i c e s i n c u - bated under a v a r i e t y of conditions 151 43 E f f e c t s of sodium amytal on the contents of amino acids i n r a t b r a i n c o r t e x s l i c e s incubated i n a v a r i e t y of media 154 44 E f f e c t s of sodium amytal on the r e l e a s e of amino acids f r o m rat b r a i n c o r t e x s l i c e s incubated i n a v a r i e t y of m e d i a 155 45 E f f e c t s of i n c r e a s e d K"*" and of tetrodotoxin on the amino ac i d content i n r a t b r a i n cortex s l i c e s incubated i n v a r i o u s m e dia 1 57 46 E f f e c t s of i n c r e a s e d K~*" and of tetrodotoxin on the r e l e a s e of amino a c i d s f r o m incubated r a t b r a i n c o r t e x s l i c e s 158 xv i i i T A B L E Page 47 E f f e c t s of tetrodotoxin on the contents of amino a c i d s in, and r e l e a s e from, r a t kidney c o r t e x s l i c e s incubated in the p r e s e n c e of ouabain 159 48 T o t a l ammonia and amino a c i d contents of rat kidney c o r t e x s l i c e s i n i t i a l l y p r e s e n c e and on incubation in the p r e s e n c e or absence of glucose 160 49 E f f e c t s of tetrodotoxin on the contents of amino a c i d s i n r a t b r a i n c o r t e x s l i c e s incubated with 2, 4-dinitrophenol, N H 4 C I or KC1 (100 mM) 162 50 E f f e c t s of tetrodotoxin on the r e l e a s e of amino acids f r o m b r a i n c o r t e x s l i c e s incubated with 2, 4-dinitrophenol, N H 4 C I , or KC1 (100 mM) 163 51 S p e c i f i c a c t i v i t i e s of glutamine and glutamate of rat b r a i n c o r t e x s l i c e s d e r i v e d f r o m s o dium (1-^ 4C) acetate and ( U - i 4 C ) glucose 167 52 E f f e c t s of p r o t o v e r a t r i n e (5 (JlM) on the s p e c i f i c a c t i v i t i e s of amino a c i d s of r a t c e r e b r a l c o r t e x s l i c e s incubated i n the pr e s e n c e of (U-^ 4C) gluc o s e 169 53 E f f e c t s of p r o t o v e r a t r i n e , ouabain and tet r o d o t o x i n on amino a c i d content in, and r e l e a s e from, incubated r a t b r a i n c o r t e x s l i c e s 172 54 E f f e c t s of p r o t o v e r a t r i n e on amino a c i d content i n r a t b r a i n c o r t e x s l i c e s incubated i n presence of ouabain 177 55 E f f e c t s of sodium malonate or sodium fluo r o a c e t a t e on amino a c i d content in, and r e l e a s e from, incubated r a t b r a i n s l i c e s in the p r e s e n c e of p r o t o v e r a t r i n e 180 56 E f f e c t s of L-glutamate and L - g l u t a m i n e on a m i n o a c i d content in, and r e l e a s e from, r a t b r a i n cortex s l i c e s 181 57 I n i t i a l N a + and K + contents of 2-day old and adult r a t b r a i n c o r t e x s l i c e s 185 58 E f f e c t s of i n c r e a s i n g N H 4 C I concentrations on oxygen and water uptakes and Na+, contents of incubated r a t b r a i n c o r t e x s l i c e s 187 x i x T A B L E P a g e 2 + 59 E f f e c t s of ca t ions on the M g - A T P a s e a c t i v i t y of h o m o - genates of adult ra t b r a i n c o r t e x 189 60 E f f e c t s of ca t ions on the N a + - K + - M g 2 + - A T P a s e a c t i v i t i e s of h o m o g e n a t e s of adult r a t b r a i n c o r t e x s l i c e s 190 61 E f f e c t s of p r o t o v e r a t r i n e , t e t r o d o t o x i n , o u a b a i n and 2, 4 - d i n i t r o p h e n o l on the A T P a s e a c t i v i t i e s of ra t c e r e b r a l c o r t i c a l h o m o g e n a t e s 191 62 E f f e c t s of N H 4 + , ( C H 3 ) 4 N + , L i + , R b + and C s + on the o x y g e n and water uptakes , and the N a + , K**" contents of i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 192 63 E f f e c t s of i n c r e a s i n g N H 4 C I c o n c e n t r a t i o n s on the o x y g e n and w a t e r uptakes and Na"*", K"** contents of infant (2 day old) r a t b r a i n c o r t e x s l i c e s 194 64 K i n e t i c s of the a l t e r a t i o n s i n the t i s s u e water , Na"*", K"** and NH4"** contents of r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n an N H 4 C I c o n t a i n i n g m e d i u m 196 65 E f f e c t s of i n c r e a s i n g N H 4 C I c o n c e n t r a t i o n s on the o x y g e n a n d water uptakes and the Na+, K + contents of ra t b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e of 0.1 m M o u a b a i n or i n the a b s e n c e of g l u c o s e 198 66 E f f e c t s of m e d i u m c a t i o n contents on the o x y g e n and water uptakes , and Na+, K * l e v e l s in i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 199 67 T i s s u e / M e d i u m c o n c e n t r a t i o n r a t i o s f o r Na**" and i n r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n m e d i a of v a r y i n g s o d i u m , p o t a s s i u m and a m m o n i u m c o n c e n t r a t i o n s 201 68 E f f e c t s of 30 m M K C 1 on the N H 4 C I i n d u c e d c a t i o n i c changes i n i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 203 69 E f f e c t s of 55 m M K C 1 on the N H 4 C I i n d u c e d c a t i o n i c changes i n i n c u b a t e d r a t b r a i n c o r t e x s l i c e s 204 70 E f f e c t s of i n c r e a s i n g s o d i u m L - g l u t a m a t e c o n c e n t r a t i o n s on o x y g e n and water uptakes , and Na+, K"*~ contents of i n c u b a t e d rat b r a i n c o r t e x s l i c e s 206 X X T A B L E Page 71 E f f e c t s of i n c r e a s i n g N H ^ + on A T P concentrations of incubated r a t b r a i n c o r t e x s l i c e s 208 71A A T P concentrations of incubated r a t b r a i n c o r t e x s l i c e s under a v a r i e t y of m e d i a conditions 209 72 E f f e c t s of tetrodotoxin, p r o t o v e r a t r i n e , ouabain, N H 4 C I and sodium-Lr-glutamate on the oxygen and water uptakes, and the Na"*", contents of rat b r a i n cortex s l i c e s incu- bated in a Ca"*""**-containing m e d i u m 211 73 E f f e c t s of tetrodotoxin, p r o t o v e r a t r i n e , N H 4 C I and s o d i u m L-glutamate on the oxygen and water uptakes, and the Na"*", K"*" contents of r a t b r a i n c o r t e x s l i c e s incubated i n a Ca"*"*"- f r e e m e d i u m 213 74 E f f e c t s of NHA"*", p r o t o v e r a t r i n e , Ca*"*"-lack and tetrodotoxin on the A T P contents of incubated r a t b r a i n c o r t e x s l i c e s .... c x o 75 E f f e c t s of sodium fluoroacetate, s o d i u m malonate, t e t r o d o - toxin and N H 4 C I on the oxygen and water uptakes, Na"*" and K"*" contents of incubated r a t b r a i n cortex s l i c e s 217 X X I A B B R E V I A T I O N S A M P adenosine 5' -monopho sphate A D P adenosine 5' -diphosphate A T P adenosine 5' -triphosphate A T P a s e adenosine triphosphatase A c e t y l - C o A acetyl-coenzyme A c p m counts per minute d p m d i s i n t e g r a t i o n s per minute r p m r e v o l u t i o n s per minute DNP, 2, 4-DNP 2, 4-dinitrophenol E G T A ethylene glycol-(diaminoethyl) t e t r a a c e t i c a c i d E D T A ethylene diamine t e t r a a c e t i c a c i d G A B A y -aminobutyric a c i d N AD+ N A D H o x i d i z e d and reduced f o r m s of ni c o t i n a m i d e adenine dinucleotide NADP+, NAD P H ox i d i z e d and reduced f o r m s of ni c o t i n a m i d e adenine dinucleotide phosphate P i orthophosphate QO2 oxygen consumption T C A t r i c h l o r o a c e t i c a c i d T r i s t r i (hydroxymethyl) amino methane T / M tis s u e to m e d i u m concent r a t i o n r a t i o T T X , tetrodotoxin v / v volume/volume w/v weight/volume wt weight A C K N O W L E D G M E N T S To P r o f e s s o r J. H. Quastel, F.R.S., C C , I a m e s p e c i a l l y g r a t e f u l f o r h i s guidance, encouragement, and wise counsel. To P r o f e s s o r s V. J. O'Donnell and G. H. Dixon, my s i n c e r e thanks f o r t h e i r h e l p f u l comments. To my f r i e n d , H elen Halm, my s i s t e r , Juliet, and my wife, E va, my deep gratitude f or t h e i r e x p e r t i s e and m o r a l support, without which I could not have completed this work. To the Department of S o i l Science, the U n i v e r s i t y of B r i t i s h Columbia, my thanks for the use of their A t o m i c A b s o r p t i o n Spectrophotometer. To the M e d i c a l R e s e a r c h C o u n c i l of Canada, my thanks for f i n a n c i a l a s s i s t a n c e through grants made to P r o f e s s o r J . H. Qu a s t e l . T h i s work was c a r r i e d out at the K i n s m e n L a b o r a t o r y of N e u r o l o g i c a l R e s e a r c h , the U n i v e r s i t y of B r i t i s h Columbia. 1 1. I N T R O D U C T I O N 1.1 F o r m a t i o n of ammonia by nerve tissue The f i r s t d e m o n s t r a t i o n of the l i b e r a t i o n of ammonia by nerve f i b r e s was by T a s h i r o i n 1922 (1). He and other w o r k e r s (1-3) showed that e l e c t r i - c a l s t i m u l a t i o n enhanced the rate of ammonia evolution and that m e c h a n i c a l i n j u r y d e p r e s s e d it. W i n t e r s t e i n and H i r s c h b e r g (2, 3) demonstrated the s u p p r e s s i v e effects of anoxia and anesthesia on ammonia f o r m a t i o n by the ne r v o u s system. S i m i l a r o b s e r v a t i o n s were made by a number of other w o r k e r s (4-7). F r o g retina, for example, when i r r a d i a t e d with sunlight exhibited i n c r e a s e d ammonia f o r m a t i o n when compared with that of c o n t r o l s l e f t i n the dark (5). S i m i l a r r e s u l t s were al s o r e p o r t e d when i s o l a t e d n e r v e s were stimulated, not only e l e c t r i c a l l y or mec h a n i c a l l y , but al s o by t h e r m a l or o s m o t i c changes, or by c h e m i c a l agents (6, 7). T h e s e studies l e d r e s e a r c h w o r k e r s i n the f i e l d to conclude that n e r v e f i b r e s l i b e r a t e ammonia as a r e a c t i o n to stimulation. L i k e p e r i p h e r a l nerves, the c e n t r a l n e r v o u s s y s t e m r e a c t s to s t i m u l i by an i n c r e a s e d rate of ammonia fo r m a t i o n . T h e r e a r e d e t a i l e d r e v i e w a r t i c l e s on these and other aspects of ammonia meta- b o l i s m i n the c e n t r a l nervous s y s t e m (8-16). 1.2 F a c t o r s affecting the in vivo l e v e l s of ammonia i n the b r a i n (i) A m m o n i a content of b r a i n in vivo A b n o r m a l l y high l e v e l s of ammonia in the b r a i n are suspected to be in v o l v e d i n the p r e c i p i t a t i o n of e p i l e p t i c s e i z u r e s (17-22) and in hepatic c oma (23-27), as it i s well known that the a d m i n i s t r a t i o n of a m m o n i u m salts to e x p e r i m e n t a l a n i m a l s causes convulsions, c oma and death (22, 23, 28-32). F o r example, R i c h t e r and Dawson (22) showed that i n j e c t i o n of a m m o n i u m c h l o r i d e i n the r a t caused c o n v u l s i o n s when the b r a i n ammonia l e v e l had r i s e n to 9 r n g % (about 5 p.mole/g). The knowledge that ammonia i s a powerful c e r e b r a l i r r i t a n t has st i m u l a t e d i n t e r e s t i n the study of b r a i n 2 ammonia l e v e l s i n vivo (18, 19, 22, 33, 34). D e t e r m i n a t i o n s of the l e v e l s of ammonia in the b r a i n of a n i m a l s i n vivo have to be c a r r i e d out with g r e a t caution as ammonia is a common l a b o r a t o r y contaminant. Moreover,' the p r e s e n c e of l a b i l e substances l i k e glutamine which may give r i s e to ammonia, must be taken into consideration, e s p e c i a l l y when the a l k a l i d i f f u s i o n method fo r the a s s a y of ammonia is employed. It is w e l l known that soon after death t h e r e i s a r a p i d r e l e a s e or " b u r s t " of ammonia i n the b r a i n (8, 36). Thus, i n o r d e r to avoid p o s t - m o r t e m changes, the a n i m a l s a r e often s a c r i f i c e d by im- m e r s i o n i n l i q u i d a i r or l i q u i d n i trogen. U s i n g this f r e e z i n g technique, values of about 0.15 - 0.36 jimole/g f r e s h wt b r a i n have been obtained i n the r a t (22, 35, 36), dog (37, 38), r a b b i t (39), mouse (4), and garden d o rmouse (41). Such low values have led to the suggestion that there may be complete absence of f r e e a mmonia in the b r a i n of the l i v i n g a n i m a l (8). It may be ar g u e d that even r a p i d f r e e z i n g in l i q u i d a i r or n i t r o g e n is not co m p l e t e l y instantaneous (the time of f r e e z i n g v a r i e s d i r e c t l y with the size of the a n i m a l (282) ). A m m o n i a may be produced between the time of i m m e r s i o n and the time of complete f r e e z i n g of the b r a i n . The explosive f o r m a t i o n of ammonia i n the b r a i n after death, p o s s i b l y due to anoxia, becomes evident when heads of decapitated a n i m a l s a r e dropped into l i q u i d a i r . F o r example, there i s about a twofold i n c r e a s e i n ammonia content when the head i s f r o z e n one second after decapitation (22). T h r e e minutes after death, cat b r a i n gave 2 ( j m o l e / g f r e s h wt. Sheep b r a i n gave values of 2.7 and 1.7 |jmole/g f r e s h wt i n grey and white matter r e s p e c t i v e l y when f r e e z i n g took place 5 m i n after death (42). W e i l - M a l h e r b e and G r e e n (43) obtained a value of 5 jimole/g f r e s h wt i n guinea pig b r a i n c o r t e x s l i c e s 20 m i n after death, a value which i s in good agreement with those obtained in other studies (44, 45). In this t h e s i s values of about 1.6 Jimole/g i n i t i a l wet wt are r e p o r t e d for b r a i n c o r t e x of r a t s taken a few minutes after death (Table 2). (ii) F a c t o r s producing low b r a i n ammonia l e v e l s i n vivo The ammonia content of the c e n t r a l n e r v o u s system, analyzed after 3 rapid freezing, is not constant but seems to depend on the state of activity of the brain at the time of the freezing. For example, a reduction in functional activity seems to be associated with a reduced concentration of free ammonia in the brain. Thus, Richter and Dawson (22) showed a significant decrease in brain ammonia levels (to 0.033 nmole/g fresh wt) when rats under pro- longed nembutal narcosis were killed in liquid air. Vladimirova (46) obtained similar effects with urethane or sodium bromide. These reports followed the earlier suggestions and observations on the depression of ammonia formation by nerve fibres under anesthesia (1 -3). Ammonia content of rat brain decreases by about 50 per cent during sleep (47). During hibernation, too, there is a 50 per cent diminution in the brain ammonia levels of garden dormice (41). (iii) Factors producing high brain ammonia levels in vivo Like peripheral nerves (1 - 3), the brain forms ammonia following electrical stimulation (22, 35, 36). This is consistent with the view that any method that enhances cerebral ir r i t a b i l i t y will also enhance the rate of cerebral ammonia formation. Thus, the administration of drugs capable of producing convulsions, like camphor (30), picrotoxin (22, 46), telodrin (49). penta- methylene tetrazole (38, 50), bemigride (38), fluoroacetate (37, 51) or methionine sulfoximine (52) results in increased brain ammonia levels. In some instances the rise in the brain content of ammonia occurs in the precon- vulsive state (22, 37, 52). However, even though ammonium salts are known to cause convulsions when administered to animals (22, 23, 28-32), it is st i l l uncertain whether the increase in brain ammonia levels occuring from endo- genous sources, is the cause or the result of convulsions. Anoxia (22) and high oxygen pressure (51) enhance ammonia levels in brain. Mild stimulation of the central nervous system, like injection of amphetamine (53) or cortico- tropin (54), painful electrical shock to the extremities (35, 36, 47), or certain conditioned reflexes (35, 47), also elevates brain ammonia levels. Though excitement caused by tumbling animals in a revolving drum has a similar 4 e f f e c t (55), R i c h t e r and D a w s o n (22) found no m a r k e d d i f f e r e n c e i n r a t s e x c i t e d by a l l o w i n g t h e m to d r o p f r o m s i d e to si d e i n a g l a s s b e a k e r , w h i l e V r b a (9) found no change i n b r a i n a m m o n i a l e v e l s of r a t s made to undergo p h y s i c a l e x e r c i s e . C e r e b r a l a m m o n i a i n c r e a s e s may not o c c u r i n some c a s e s of p h y s i c a l o r e m o t i o n a l e x c i t e m e n t , owing to an i n c r e a s e d r a t e of g l u t a m i n e f o r m a t i o n i n the b r a i n (9 - 11, 56, 57). I n c r e a s e d b l o o d a m m o n i a l e v e l s a l s o r e s u l t i n enhanced g l u t a m i n e contents i n the b r a i n (22). 1.3 (i) Some c h e m i c a l changes that o c c u r i n the b r a i n on the a p p l i c a t i o n of s t i m u l i to the whole a n i m a l . I n c r e a s e d c e r e b r a l a c t i v i t y p r o d u c e d i n r a t s by p h y s i c a l e x e r t i o n does not r e s u l t i n an enhanced b r a i n c o n c e n t r a t i o n of a m m o n i a (9, 56). However, t h e r e o c c u r s an enhanced c e r e b r a l g l u t a m i n e l e v e l (56) w i t h c o n c o m i t a n t d e c r e a s e s i n the amounts of f r e e g l u t a m a t e (57, 66) and p r o t e i n bound a m i d e n i t r o g e n (67 - 70). S i m i l a r r e s u l t s w e r e o b t a i n e d d u r i n g oxygen i n t o x i c a t i o n (9) and c a r b o n d i s u l p h i d e p o i s o n i n g (70). E v e n d u r i n g e l e c t r i c a l l y i n d u c e d c o n v u l s i o n s , g l u t a m i n e l e v e l s a r e e l e v a t e d (71). A c u t e t e l o d r i n i n t o x i c a t i o n a l s o r e s u l t s i n an i n c r e a s e i n g l u t a m i n e content w i t h a c o n c o m i t a n t d e c r e a s e i n the g l u t a m a t e l e v e l s i n the b r a i n (49). In the e a r l y stages of acute t e l o d r i n i n t o x i c a t i o n the content of f r e e a m m o n i a i n the b r a i n r e m a i n s unchanged, but an i n c r e a s e i s o b s e r v e d l a t e r when s e i z u r e s o c c u r . A c c o r d i n g to Hathway and M a l l i n s o n (49), t e l o d r i n c a u s e s l i b e r a t i o n of a m m o n i a i n the b r a i n and t h i s o c c u r s b e f o r e the onset of c o n v u l s i o n s and throughout t h e i r c o u r s e . a-Ketoglutarate and g l u t a m a t e a r e u t i l i z e d i n an a m m o n i a b i n d i n g m e c h a n i s m w h i c h l a t e r b e c o m e s o v e r w h e l m e d and f r e e a m m o n i a a c c u m u l a t e s i n the c e r e b r a l t i s s u e . It was lon g s u s p e c t e d that the g l u t a m i n y l a m i d e - N of c e r e b r a l p r o t e i n s r e p r e s e n t e d a s o u r c e of endogenous c e r e b r a l a m m o n i a (57, 67, 74, 75). T h i s was l a t e l y found to be t r u e (76). A c c o r d i n g to W h e r r e t t and T o w e r (76), 16 pe r cent of the g l u t a m i n y l r e s i d u e s of c e r e b r a l c o r t i c a l p r o t e i n s are r e a d i l y 5 deaminated i n situ. T h ese r e s u l t s lend support to the view that ammonia i s f o r m e d f r o m p r o t e i n s o u r c e s and is converted to glutamine by condensation with glutamate. B r a i n p r o t e i n m e t a b o l i s m seems c l o s e l y r e l a t e d to that of p e r i p h e r a l nerve, where s t i m u l a t i o n i n c r e a s e s the a c t i v i t y of the n e u t r a l proteinase, d e c r e a s e s p r o t e i n content, and i n c r e a s e s u t i l i z a t i o n of glutamate and l i b e r a t i o n of ammonia (72). (ii) R e c o v e r y p r o c e s s e s that o c cur i n the b r a i n on the r e m o v a l of the s t i m u l i applied to the whole a n i m a l When r a t s p h y s i c a l l y exhausted (by prolonged swimming) are allowed to rest, their c e r e b r a l glutamine l e v e l s d e c r e a s e with concomitant i n c r e a s e s i n their c e r e b r a l p r o t e i n n i t r o g e n (69, 73). A m i d a t i o n of free c a r b o x y l i c groups of c e r e b r a l p r o t e i n s after the a d m i n i s t r a t i o n of N H ^ C l seems to take place (80, 81), but c o n f i r m a t i o n using 1 l a b e l l i n g i s needed (76). W h e r r e t t and Tower (76) were unable to obtain r e a m i d a t i o n of c e r e b r a l p r o t e i n s i n v i t r o by i n c r e a s i n g f r e e pools of ammonia and glutamine of cat c o r t e x s l i c e s . In spite of this, they c o n s i d e r that p r o t e i n d e amidation i s a r e v e r s i b l e p r o c e s s as suggested by Mycek and W a e l s c h (77) as a r e s u l t of the i r studies on t r a n s g l u t a m i n a s e . T r a n s g l u t a m i n a s e is a c a l c i u m - a c t i v a t e d enzyme which c a t a l y z e s (a) the exchange of p r o t e i n amide groups with p r i m a r y amines (like cadaverine, putrescine, histamine, s e r o - tonin, glycinamide, etc.); and (b) the h y d r o l y t i c d e a m i d a t i o n of p r o t e i n bound amide r e s i d u e s (78 - 79). (iii) C o nvulsions and b r a i n energy l e v e l s . No a l t e r a t i o n i n the c e r e b r a l c o r t i c a l A T P l e v e l s of rat s was o b s e r v e d with a m m o n i u m acetate induced convulsions (82), or in hypoxic mice at the onset of s e i z u r e s (83). S i m i l a r l y , no change in the A T P or phosphocreatine 6 l e v e l s were o b s e r v e d i n the b r a i n s of m ice undergoing methionine s u l f o x i m i n e induced s e i z u r e s (52). Such r e s u l t s do not support the postulate of s e v e r a l authors (84, 85) that A T P is a factor i n the induction of convulsions. The apparent absence of c e r e b r a l A T P depletion does not, however, r u l e out the p o s s i b l e depletion of a s m a l l l o c a l i z e d pool of A T P that may be v i t a l f o r n o r m a l b r a i n function. 1.4 F a c t o r s affecting the f o r m a t i o n of a mmonia in b r a i n t i s s u e i n v i t r o The r e s u l t s of our e a r l i e r work (62) on the f a c t o r s c o n c e r n e d with the r a t e of ammonia l i b e r a t i o n by r a t b r a i n c o r t e x s l i c e s incubated i n oxygen at 3 7 ° C for one hour i n K r e b s - R i n g e r phosphate medium, were explained by changes in amino acid metabolism, p a r t i c u l a r l y those of glutamate and g l u t a - mine (see s e c t i o n 1.5 (ii) (b) ). A s u m mary of our o b s e r v a t i o n s is given below. (i) Carbohydrate m e t a b o l i s m and ammonia f o r m a t i o n A m m o n i a f o r m a t i o n i s s u p p r e s s e d by v a r i o u s exogenous su b s t r a t e s that a r e capable of supporting c e r e b r a l r e s p i r a t i o n . Thus, with glucose, lactate (58 - 62) or pyruvate (62), where the rate of the o p e r a t i o n of the c i t r i c a c i d c y c l e is high, there is a m a x i m a l s u p p r e s s i o n of ammonia l i b e r a t i o n . The m i n i m u m (threshold) c o n c e n t r a t i o n f o r this s u p p r e s s i o n i s about I m M f o r glucose and about 2.5 m M for pyruvate (62). T h i s may be c o r r e l a t e d with the fact that g l y c o l y s i s of one molecule of glucose y i e l d s two m o l e c u l e s of p y r u - vate. In the p r e s e n c e of glucose but not of pyruvate, the addition of iodo- acetate (0.1 mM), by i t s s u p p r e s s i v e effect on t r i o s e phosphate dehydrogenase, impedes the operation of the c i t r i c a c i d c y c l e and thereby the energy dependent glutamine synthesis. It enhances the output of ammonia by more than 50 per cent (62). In the absence of glucose, iodoacetate only s l i g h t l y i n h i b i t s endo- genous ammonia f o r m a t i o n (62, 64); this may be due to a s m a l l d i m i n u t i o n 7 i n endogenous oxygen consumption (64). A c e t a t e i s only o x i d i z e d feebly by b r a i n c o r t e x s l i c e s (226) and hence has l i t t l e or no d i m i n i s h i n g effect on ammonia formation. S i m i l a r l y , c i t r a t e or a-ketoglutarate (62), acetoacetate or succinate (our unpublished o b s e r v a - tions), do not suppress the f o r m a t i o n of ammonia. However, oxaloacetate causes high r e s p i r a t o r y r a t e s (227) and i s capable of p a r t i a l l y s u p p r e s s i n g a mmonia l i b e r a t i o n . T h i s i s p r e s u m a b l y due to its r e l a t i v e l y r a p i d influx into the b r a i n c e l l and to its p a r t i c i p a t i o n i n the c i t r i c a c i d c y c l e . In the p r e s e n c e of c e r t a i n i n h i b i t o r s , e.g., 2, 4-dinitrophenol (62, 64), f l u o r o a c e t a t e (63), methionine s u l f o x i m i n e (65) or iodoacetate (64), the sup- p r e s s i v e effects of glucose i s l e s s marked. Malonate (5 mM), an i n h i b i t o r of the c i t r i c a c i d c y c l e at the s u c c i n i c dehydrogenase stage (228), enhances a mmonia l i b e r a t i o n only i n the p r e s e n c e of t h r e s h o l d concentrations (1 mM) of g l u c o s e . T h i s i s also found to be true with 100 m M KC1 (62). K i n i and Q u a s t e l (213, 229) demonstrated a m a r k e d stim u l a t i o n in the o p e r a t i o n of the c i t r i c a c i d c y c l e i n K"*"-stimulated c e l l s so that i n the p r e s e n c e of 1 m M (but not 5 mM) glucose, the i n t e r m e d i a t e s i n the breakdown of glucose a r e o x i d i z e d too fast to allow for the n o r m a l rate of (energy requiring) ammonia fi x a t i o n . (ii) E l e c t r o n transport, oxidative p h o s p h o r y l a t i o n and ammonia f o r m a t i o n . In the absence of glucose, adult c e r e b r a l cortex s l i c e s y i e l d g r e a t e r amounts of ammonia than those of the infant b r a i n (62), p r e s u m a b l y because oxygen uptake by infant b r a i n i s lower. M i l s t e i n et al., (230) showed that the oxygen consumption per mg m i t o c h o n d r i a l p r o t e i n i n c r e a s e s with the age of the rat. E l e c t r o n t r a n s p o r t i n h i b i t o r s , e.g., azide, a r s e n i t e or cyanide, sup- p r e s s autogenous ammonia f o r m a t i o n (43, 63, 64). A m y t a l (1 mM) in h i b i t s e l e c t r o n t r a n s p o r t between N A D H and the c y t ochrome s y s t e m (219 - 221) and 8 concom i t a n t l y s u p p r e s s e s ammonia f o r m a t i o n i n a g l u c o s e - f r e e m e d i u m (62). The e l e c t r o n t r a n s p o r t i n h i b i t o r s p r e s u m a b l y act by s u p p r e s s i n g the regenera- t i o n of NAD + r e q u i r e d for glutamate oxidation. A d i r e c t consequence of the i n h i b i t i o n of the r e s p i r a t o r y chain i s a d i m i n i s h e d rate of A T P synthesis. B oth 2, 4-dinitrophenol (0.1 mM) (62, 63), or s o d i u m s a l i c y l a t e (5 mM) (62), s u p p r e s s the endogenous f o r m a t i o n of ammonia, but enhance the l i b e r a t i o n of ammonia i n the p r e s e n c e of glucose, p r e s u m a b l y by in h i b i t i n g the A T P - r e q u i r i n g g l u t a m i n e synthesis. The rate of ammonia pr o d u c t i o n i s s u p p r e s s e d under anoxia (43, 62, 244), doubtless by an i n h i b i t i o n of the oxidation of endogenous glutamate (62). (iii) E f f e c t s of exogenous amino ac i d s on ammonia f o r m a t i o n . The addition of L-glutamate to b r a i n c o r t e x s l i c e s r e s p i r i n g i n a n o r m a l K r e b s - R i n g e r phosphate m e d i u m r e s u l t s i n a d i m i n i s h e d r a t e of ammonia f o r m a t i o n (62, 103), accompanied by an enhanced rate of glutamine s y n t h e s i s (58, 103). F i v e m M D-glutamate (62, 138, 231), or 5 m M a - m e t h y l glutamate i n h i b i t s ammonia l i b e r a t i o n i n a g l u c o s e - f r e e medium, p r e s u m a b l y by i n h i b i t i n g endogenous glutamate oxidation. In the p r e s e n c e of glucose, D-glutamate enhances the rate of ammonia l i b e r a t i o n (62) by in h i b i t i n g glutamine synthetase (103). The rate of autogenous ammonia f o r m a t i o n i s unaffected by taurine, y -aminobutyrate (GABA), L - or D - a s p a r t a t e g l y c i n e or g l y c y l - L - a s p a r t a t e . However, with L - a l a n i n e or L - a r g i n y l - L - g l u t a m a t e there seems to be a s m a l l i n h i b i t i o n (62). L-Glutamine is deamidated by b r a i n s l i c e s . Net dea m i d a t i o n o c c u r s to a g r e a t e r extent i n the absence of glucose than in its presence, as g l u t a - mine r e s y n t h e s i s is promoted by the pr e s e n c e of glucose (62). 9 1.5 A m m o n i a f o r m a t i o n and u t i l i z a t i o n m e c h a n i s m s in the b r a i n (i) U r e a (a) U r e a as a s o u r c e of ammonia. Urea i s present i n mammalian b r a i n at a c o n c e n t r a t i o n of about 5.0 m M (41, 88, 100). Can it s e r v e as a s o u r c e of ammonia in the b r a i n ? Godin et al., (41) have shown that in hi b e r n a t i n g garden d o r m i c e , both the ammonia and u r e a l e v e l s f a l l to about 50 per cent of the c o n t r o l (awake) a n i m a l s . A c c o r d i n g to these workers, the d e c r e a s e d b r a i n a mmonia l e v e l i n h i b e r n a t i n g a n i m a l s i s due to r e d u c e d f u n c t i o n a l a c t i v i t y . D u r i n g hibernation, the i n t e r n a l body temperature, the blood flow, the oxygen consumption and the u t i l i z a t i o n of d i f f e r e n t m e t a b o l i c s u b s t r a t e s a r e a l l reduced. It i s co n c e i v a b l e that b r a i n u r e a l e v e l f a l l s due to a f a l l i n the blood l e v e l of urea, as a r e s u l t of a dr o p i n a mmonia formation, a r i s i n g p a r t l y through d i m i n i s h e d b a c t e r i a l a c t i o n i n the i n t e s t i n e and partly due to l o w e r e d n i t r o g e n m e t a b o l i s m i n the h i b e r n a t i n g a n i m a l s . Thus, lov/ered c e r e b r a l u r e a i s due to l o w e r e d blood u r e a l e v e l s (see s e c t i o n 1.5 (i) (b)); it i s not acc o m p a n i e d by an i n c r e a s e d a m m o n i a l e v e l i n the b r a i n . M o r e o v e r , u r e a s e e m s not to be the sou r c e of a m m o n i a i n b r a i n d u r i n g t e l o d r i n - i n d u c e d s e i z u r e s , since u r e a l e v e l s r e m a i n unchanged under these conditions (49). F u r t h e r m o r e , i n v i t r o studies showed that the addition of u r e a to r e s p i r i n g b r a i n cortex s l i c e s "of the r a t incubated in oxygen for one hour at 3 7 ° C in a n o r m a l R i n g e r phosphate m e d i u m (glucose free) had l i t t l e or no eff e c t on the rate of ammonia f o r m a t i o n (62). A d d i t i o n a l l y , u r e a s e seems not to be pr e s e n t in m a m m a l i a n t i s s u e s so f a r examined (86). It seems unlikely, therefore, that u r e a is a sou r c e of ammonia in b r a i n . 10 (b) P o s s i b l e f o r m a t i o n of u r e a as a m e c h a n i s m for a mmonia u t i l i z a t i o n . U r e a i s not synthesized i n b r a i n f r o m NH4* and (100). Some of the i n t e r m e d i a t e s of the K r e b s - H e n s e l e i t u r e a c y c l e (87) are p r e s e n t in b r a i n . Contents of these i n t e r m e d i a t e s in cat b r a i n c a l c u l a t e d in t e r m s of jjmole/g i n i t i a l wet wt, f r o m the data of T a l l a n , M o o r e and Stein (given as mg/100 g) (88), a r e as follows: ornithine, 0.046; c i t r u l l i n e , 0.23; arginine, 0.2; and urea, 4.2. C a r b a m y l phosphate has not yet been detected i n b r a i n . S e v e r a l of the enzymes that p a r t i c i p a t e in the synthesis of u r e a c y c l e meta- b o l i t e s (namely, a r g i n i n o succinate synthetase, a r g i n i n o s u c c i n a s e and arginase) are a l s o present i n crude p r e p a r a t i o n s of b r a i n (89» 90, 100). It s e ems that part of the u r e a c y c l e and its mediating enzymes o c c u r i n b r a i n . M e t a b o l i c studies in vivo show that L.-proline i s c o n v e r t e d to g l u t a m i c acid, a r g i n i n e and ornithine (91); and L - a r g i n i n e (92), or c i t r u l l i n e (93) to urea. However, the enzyme that c a t a l y z e s the f o r m a t i o n of c i t r u l l i n e f r o m c a r b a m y l phosphate and ornithine (viz., ornithine t r a n s c a r b a m y l a s e ) does not operate i n the c e n t r a l nervous s y s t e m (93, 100). C a r b a m y l phosphate synthetase, the enzyme that f i x e s ammonia to CO-> i n the p r e s e n c e of A T P , i s l i k e w i s e absent in the b r a i n (100). It is doubtful therefore, whether the K r e b s - H e n s e l e i t cycle per se o c c u r s i n b r a i n at any rate to any s i g n i f i c a n t extent. The r e l a t i v e l y high c o n c e n t r a t i o n of u r e a i n b r a i n is probably due to p a s s i v e d i f f u s i o n f r o m the blood. A b r a i n to p l a s m a co n c e n t r a t i o n r a t i o of • about one (0.8 as c a l c u l a t e d f r o m the data of T a l l a n et a l . (88) see T a b l e 1), has been found (see also r e f e r e n c e 281). It may be pointed out that i n the l i v e r the c o n c e n t r a t i o n of u r e a is not much di f f e r e n t f r o m that of b r a i n (5.16 iamole/g for l i v e r v e r s u s 4.5 (amole/g for b r a i n (100) ). A c c o r d i n g to B l a s s (see r e f e r e n c e 49). b r a i n s y n t h e s i z e s u r e a at a m a x i m u m rate of 1 (imole/g of t i s s u e / h r , whereas the rate for glutamine synthesis i s 10-20 nmole/g of t i s s u e / h r (95). The above considerations lead to the conclusion that removal of ammonia in the brain does not occur by urea formation. The observation that hepatectomy in the dog leads to cessation of urea production points to the l iver as possibly the only major source of urea in the mammalian organisms (96, 97). (c) Possible importance of urea synthesis in the brain (86, 95, 100). The metabolism of ci trull ine and argininosuccinate in the bra in via urea formation seems to be important for brain development. Relatively large amounts of citrulline and argininosuccinate are present in the brain, cerebrospinal fluid, and plasma, and are excreted in the urine in ' c i t r u l l i n u r i a ' and'argininosuccinic ac iduria ' , respectively. These diseases of the central nervous system are due to the relevant enzymes being absent in brain . The conditions are accompanied by mental retardation. (ii) (a) A m m o n i a formation f r o m amino acids via aspartate channelled through N A D + or A M P . A c c o r d i n g to Buniatian and co-workers (14), ammonia is formed in bra in and other organs f r o m amino acids . They have proposed a mechanism by means of which amino acids through aspartate supply amino groups to desamino- N A D ( D - N A D + ) . N A D + thus fo rmed on deamination yields ammonia and desamino-NAD"*". The sequence of reactions proposed is as follows: Glutamate > Aspartate D -NAD*t^ -r N A D succinate ^ x N A D 1 " + fumarate v N H o + D - N A D " 1 " * I 12 T h i s p r o c e s s is a v a r i a t i o n of the scheme suggested by another R u s s i a n w orker. A c c o r d i n g to K o m e t i a n i (81, 98), the r e a m i n a t i o n of I M P by a s p a r - tate and the deamination of A M P thu.- formed, is a m e c h a n i s m by which ammonia i s produced f r o m amino a c i d s . The r e s u l t s leading to this hypo- t h e s i s were obtained using m i t o c h o n d r i a l p r e p a r a t i o n s (14). The amount of ammonia f o r m e d f r o m N A D * and aspartate by mito- c h o n d r i a of r a t b r a i n (14) seems to be i n s u f f i c i e n t to account for the f o r m a t i o n of ammonia by b r a i n t i s s u e in v i t r o . T h i s i s evident f r o m the following calcu- l a t i o n s (see r e f e r e n c e (14) f o r values): 5.28 jf0 . 3 |ig ammonia/mg p r o t e i n / 2 hr at 37°C, was obtained on the addition of NAD"*" (1.4 p.moles) and aspartate (26 |amoles) to mit o c h o n d r i a . The co n t r o l value was 3.07 + 0.44. With aspartate or N A D * present alone, the values r e c o r d e d were 3.45 + 0.3, or 3.63 _+ 0.3, r e s p e c t i v e l y . T a k i n g standard deviations into consideration, it s eems that a l i t t l e o ver 1 (ig ammonia/mg p r o t e i n i s f o r m e d i n two hours by b r a i n m i t o c h o n d r i a f r o m N A D * and aspartate. T h i s amounts to 0.03 l-lmole a mmonia f o r m e d per mg m i t o c h o n d r i a l p r o t e i n / h r . E v e n if the p r o t e i n content of b r a i n was wholly m i t o c h o n d r i a l , then only 3 (jmole ammonia/100 mg p r o t e i n and hence per g wet wt b r a i n would be f o r m e d under these conditions. T h i s value i s too s m a l l to account for the l i b e r a t i o n of ammonia by b r a i n under a v a r i e t y of incubation conditions that w i l l be r e c o r d e d in this work. Moreover, the addition of L - a s p a r t a t e (5 mM) to r e s p i r i n g b r a i n c o r t e x s l i c e s was without any effect on the rate of ammonia l i b e r a t i o n (62). Again, r a i s i n g the endogenous t i s s u e l e v e l of aspartate by incubating c e r e b r a l c o r t e x s l i c e s in a glucose f r e e m e d i u m containing 105 m M KC1, or l o w e r i n g it by the use of metabolic i n h i b i t o r s such as malonate or amino . oxyacetic acid, does not affect the rate of ammonia forma t i o n . These experiments w i l l be d e s c r i b e d in g r e a t e r d e t a i l i n Chapter 3. With guinea pig b r a i n s l i c e s , T a k a g a k i (99) obtained no d e c r e a s e i n 13 endogenous l e v e l s of a d e n y l i c compounds, and concluded that the c o n v e r s i o n of adenylic a c i d to i n o s i n i c a c i d by adenylic deaminase i s quantitatively a r e l a t i v e l y minor p r o c e s s . It may be mentioned that f r e e a d e n y l i c compounds a r e p r e s e n t at r e l a t i v e l y low l e v e l s i n the b r a i n and t h e r e f o r e cannot be con- s i d e r e d as major s o u r c e s for c e r e b r a l ammonia f o r m a t i o n (43). (ii) (b) A m m o n i a f o r m a t i o n f r o m amino acids v i a t e r m i n a l glutamate oxidation. The o r i g i n and m e c h a n i s m of ammonia f o r m a t i o n is b r a i n s l i c e s r e s p i r i n g in a g l u c o s e - f r e e incubation m e dium is s t i l l a subject of much con- t r o v e r s y . A c c o r d i n g to the e a r l y work of Weil-Malherbe and c o - w o r k e r s (43, 75) none of the six deaminating b r a i n enzymes present in b r a i n (e.g., g l u t a m i c dehydrogenase, glutaminase, amine oxidase, a d e n y l i c deaminase, adenosine deaminase or hexosamine deaminase), can account for ammonia f o r m a t i o n . T h e s e w o r k e r s suggested that a mmonia f o r m a t i o n o c c u r s l a r g e l y by a r e a c t i o n c l o s e l y l i n k e d to p r o t e o l y s i s , since guinea pig b r a i n s l i c e s incubated for 5 h r s gave s i g n i f i c a n t i n c r e a s e s in n o n - p r o t e i n n i t r o g e n . In t h e i r studies, they a s s u m e d a single o r i g i n f r o m which f r e e ammonia is l a r g e l y d e r i v e d . On the other hand, a c c o r d i n g to V r b a et a L (67, 101), a mmonia f o r m a t i o n i s too complex a p r o c e s s to be explained on the b a s i s of s i m p l e p r o t e o l y s i s . T hese w o r k e r s showed that not more than 25 per cent of the a mmonia f o r m e d by guinea pig b r a i n s l i c e s in incubations of 4 h r s and 6 h r s d u r a t i o n can be accounted for as coming f r o m p r o t e i n amide nitrogen, and c o n s i d e r the source of the l a r g e r p a r t of ammonia as s t i l l being unknown. In t h e i r studies they also o b s e r v e d some i n c r e a s e s in n o n - p r o t e i n n i t r o g e n and l i p i d nitrogen, and some d e c r e a s e s of n i t r o g e n i n the n u c l e i c a c i d and p r o t e i n f r a c t i o n s . That some ammonia may p o s s i b l y be d e r i v e d f r o m p r o t e i n amide 14 n i t r o g e n i s supported by the d e m o n s t r a t i o n that 16 per cent of the g l u t a m i n y l r e s i d u e s of c e r e b r a l c o r t i c a l p r o t e i n s a r e r e a d i l y deamidated (76). However, even this does not account f o r the major p o r t i o n of ammonia f o r m e d in v i t r o by b r a i n c o r t e x s l i c e s (67, 76, 101). In the studies of T a k a g a k i et a l . (64), the incubation of guinea pig b r a i n s l i c e s was r e s t r i c t e d to one hour because it was fe l t that ammonia f o r m e d at the end of 4 h r s might be the r e s u l t of a s e r i e s of highly c o m p l i c a t e d r e a c t i o n s , much of which in v o l v e d p r o t e o l y s i s . Glutamate d e c r e a s e s in the b r a i n concomitant with an i n c r e a s e in ammonia f o r m a t i o n when incubations a r e c a r r i e d out in a g l u c o s e - f r e e medium; a c c o r d i n g to these workers, the d e c r e a s e i n glutamate c o n c e n t r a t i o n accounts for 50 per cent of the endo- genous oxygen uptake. However, it appears that their e s t i m a t i o n s of glutamate and ammonia were confined to the c e r e b r a l tissue, the contents of these sub- stances in the incubation m e d i u m not being c o n s i d e r e d . M o r e o v e r , the p o s s i b l e c o n v e r s i o n of glutamate to other amino acids such as aspartate, Y-aminobutyrate, and alanine, were not taken into account. In e a r l i e r work (62, 65) we have shown that the total p r o d u c t i o n of a mmonia (i.e., i n both t i s s u e and incubation medium) by b r a i n s l i c e s incubated f o r s h o r t p e r i o d s of time i n the absence of glucose, i s l a r g e l y due to amino a c i d metabolism, i n which endogenous glutamate oxidation is the t e r m i n a l p r o c e s s . These studies showed how the i n d i v i d u a l l e v e l s of ammonia and amino a c i d s v a r i e d on incubating r a t b r a i n cortex s l i c e s in oxygen for one hour at 3 7 ° C i n the p r e s e n c e or absence of glucose. They i n d i c a t e d that the d i m i n u t i o n in the glutamate and glutamine l e v e l s in the b r a i n c o r t e x s l i c e s accounts l a r g e l y f or the l i b e r a t i o n of ammonia. Changes in the o b s e r v e d r a t e s of ammonia formation, on incubating rat b r a i n c o r t i c a l s l i c e s f o r one hour under a v a r i e t y of conditions, could be explained on the b a s i s of g l u t a - mate oxidation and glutamine synthesis. T h i s has a l r e a d y been mentioned in s e c t i o n 1.3. Subsequent to our work (62, 65), a r e c e n t r e p o r t by W e i l - M a l h e r b e and G o r d o n (10 2) supports our view that changes in the l e v e l s of 15 amino acids ( p a r t i c u l a r l y glutamate) i n guinea pig b r a i n c o r t e x s l i c e s , can account for the l i b e r a t i o n of ammonia. However, the contents of amino ac i d s found i n i t i a l l y in their s l i c e s are about 30 - 50 per cent of the va l u e s r e p o r t e d i n the l i t e r a t u r e (102, 233). A l s o , th e i r r a t e of ammonia f o r m a t i o n is 40 - 50 per cent of the values they r e p o r t e d i n 1955 (43). The o r i g i n , m e c h a n i s m and f a c t o r s affecting ammonia f o r m a t i o n i n incubated b r a i n c o r t e x s l i c e s , w i l l be dealt with in Chapter 3, i n which an attempt w i l l be made to c h a r a c t e r i z e the compartments in the b r a i n t i s s u e m a i n l y r e s p o n s i b l e for ammonia pr o d u c t i o n (Chapter 10). (iii) A m m o n i a u t i l i z a t i o n p r o c e s s e s i n b r a i n . T h e r e appear to be at l e a s t three m e c h a n i s m s of ammonia u t i l i z a t i o n i n the b r a i n . (a) U r e a f o r m a t i o n . A s mentioned e a r l i e r , c o n v e r s i o n of ammonia to u r e a i n b r a i n o c c u r s either not at a l l or only at a v e r y s m a l l r a t e . (b) A m i d a t i o n of p r o t e i n s . A c c o r d i n g to a number of workers, deamidation of g l u t a m i n y l r e s i d u e s i n p r o t e i n (9-12, 57, 67, 74, 75) c a t a l y z e d by the c a l c i u m r e q u i r i n g t r a n s g l u t a m i n a s e (78, 79) o c c u r s i n b r a i n (see se c t i o n 1.4 (ii)). Though there i s g e n e r a l consensus among w o r k e r s i n the f i e l d that the r e a m i d a t i o n p r o c e s s ( u t i l i z i n g either N H ^ + ions or glutamine, should take place, the actual d e m o n s t r a t i o n of its o c c u r r e n c e i s s t i l l a matter of doubt (76, 80, 81). (c) Glutamine synthesis. The f o r m a t i o n of glutamine seems to be the m a j o r p r o c e s s of ammonia u t i l i z a t i o n i n b r a i n . I n c r e a s e d glutamine synthesis o c c u r s in vivo d u r i n g i n c r e a s e d functional a c t i v i t y (9-11, 56, 57) brought about by p h y s i c a l e x e r c i s e , con- v u l s i v e agents, or i n f u s i o n of ammonium s a l t s . A m m o n i a 16 l i b e r a t i o n by b r a i n t i s s u e is inhibited under conditions leading to high rates of glutamine synthesis; e.g., in the p r e s e n c e of g l u c o s e (58-62) or glutamate (103). One of the objectives of our work i s to understand in g r e a t e r d e t a i l the c o n t r o l m e c h a n i s m for glutamine syn t h e s i s and its actual l o c a t i o n i n the b r a i n c e l l s . 1.6 T r a n s p o r t of amino a c i d s i n b r a i n . T r a n s p o r t of amino a c i d s i n b r a i n has usually been in v e s t i g a t e d by the use of t i s s u e s l i c e s as they are able to accumulate amino acids against a con- c e n t r a t i o n gradient. In the intact animal, however, the b r a i n is only able to concentrate amino acids to a s m a l l extent or not at a l l . The d i f f e r e n c e between_in vivo and in v i t r o e x p e r i m e n t s l e d to the concept of a blood b r a i n b a r r i e r . R e c e n t l y i t has been found that, though there is but l i t t l e i n c r e a s e i n the amounts of amino a c i d s in b r a i n on i n c r e a s i n g blood amino a c i d concentra- tions, there i s a r a p i d movement of amino a c i d s into and out of the b r a i n (15, 104-107) as shown by the use of l a b e l l e d amino a c i d s . The supply of amino a c i d s to the b r a i n f r o m the blood is of obvious i m p o r t a n c e f or the n o r m a l fu n c t i o n a l a c t i v i t y of the b r a i n . In addition to the i r i n c o r p o r a t i o n into protein, they are needed for the synthesis of biogenic amines whose uptake i s much s m a l l e r than the c o r r e s p o n d i n g amino a c i d g i v i n g r i s e to t h e m (104). P a s s i v e d i f f u s i o n leads only to a co n c e n t r a t i o n within the c e l l no higher than that outside, except under c i r c u m s t a n c e s where binding of the substance to a c e l l constituent takes place in the c e l l or i n a c e l l compartment (104). A c t i v e transport, on the other hand, r e q u i r e s energy, being l e s s in the absence of oxygen and glucose, and a l s o in the p r e s e n c e of m e t a b o l i c i n h i b i t o r s l i k e cyanide and 2, 4 d i n i t r ophenol. It u t i l i z e s A T P and i n v o l v e s movement against a c o n c e n t r a t i o n gradient. It i s subject to s a t u r a t i o n k i n e t i c s , the rate of uptake r e a c h i n g a l i m i t with i n c r e a s e i n the concent r a t i o n of amino a c i d available, owing to sat u r a t i o n of c a r r i e r s i t e s . Competition also o c c u r s 17 between amino ac i d s d u r i n g t r a n s p o r t into b r a i n s l i c e s . A m i n o ac i d s may- compete with e ach other for a common c a r r i e r but may b r i n g about the sup- p r e s s i o n of each other's t r a n s p o r t i n d i r e c t l y by d i m i n i s h i n g the A T P l e v e l s (104, 106, 113). U n l i k e competitive inhibition, non-competitive i n h i b i t i o n of t r a n s p o r t p r o c e s s e s does not involve a s p e c i f i c c a r r i e r . It can be produced, f o r example, by i n t e r f e r e n c e with metabolic p r o c e s s e s within the c e l l upon which energy f o r t r a n s p o r t i s dependent, or by damage to the c e l l membrane, and i s u n r e l a t e d to the c o n c e n t r a t i o n of substrate amino a c i d (106). The uptake of amino ac i d s i s a m e a s u r e m e n t of the net t r a n s p o r t and has been b a s e d e s s e n t i a l l y on the amount by which the c o n c e n t r a t i o n of a p a r t i c u l a r f r e e amino a c i d of the t i s s u e has i n c r e a s e d over a defined p e r i o d (usually one hour). ' Both influx and efflux p r o c e s s e s occur when the steady state is reached. E i t h e r of these p r o c e s s e s may be produced by p a s s i v e d i f f u s i o n or by active t r a n s p o r t or p r o c e s s e s such as f a c i l i t a t e d d i f f u s i o n (107). P a s s i v e d i f f u s i o n may also o c c u r at the same time as active t r a n s p o r t (108). T h e r e may be a factor in the influx and efflux s y s t e m that i n v o l v e s counter t r a n s p o r t (106, 109) or exchange d i f f u s i o n (110, 111) i n which movement of substrate in one d i r e c t i o n causes movement at the same time i n the opposite d i r e c t i o n . T h i s latter p r o c e s s may be of s p e c i a l i n t e r e s t in b r a i n as it may be r e l a t e d to the blood b r a i n b a r r i e r phenomenon in which net t r a n s f e r i s impeded i n vivo inspite of r a p i d interchange between blood and b r a i n (106). A c t i v e t r a n s p o r t of amino ac i d s in b r a i n i s s o d i u m dependent (113, 104, 105). H i gh p o t a s s i u m inh i b i t s amino a c i d uptake (114) by d i m i n i s h i n g A T P l e v e l s (115). A b s e n c e of c a l c i u m r e t a r d s the rate of amino a c i d uptake, p r e - sumably by l o w e r i n g A T P l e v e l s . Inhibition also o c c u r s with high c a l c i u m salts (10 mM), p r e s u m a b l y due to the i n h i b i t i o n of N a t K"*"-stimulated A T P a s e . Ouabain inhibits both the active t r a n s p o r t of amino ac i d s and the Na"*", K*- s t i m u l a t e d A T P a s e . (section 1.9 (v)). Ouabain p r e s u m a b l y acts by its af f i n i t y f o r the Na"*" t r a n s p o r t ' c a r r i e r (at the K ^ - s i t e ) and thus bl o c k s t r a n s p o r t coupled with this c a r r i e r (section 1.9 (v)). Ouabain has l i t t l e or no effects on 18 c e r e b r a l A T P l e v e l s (11 5). Ouabain (112, 280), anoxia, N a + l a c k and r e s p i r a t o r y chain i n h i b i t o r s , enhance the rate of amino a c i d efflux f r o m b r a i n (104, 105, 205). M o r e i n f o r m a t i o n on t r a n s p o r t p r o c e s s e s f or i n d i v i d u a l amino acids and other compounds of b i o c h e m i c a l i n t e r e s t i n b r a i n and i n other t i s s u e s of a v a r i e t y of sp e c i e s may be obtained f r o m the r e v i e w s by Qu a s t e l (104, 105) and Naeme (106). Table 1 giv e s b r a i n / p l a s m a c o n c e n t r a t i o n r a t i o s of amino acids calcu- l a t e d f r o m the data (for the cat) of T a l l a n , M o o r e and Stein (88, see al s o 95, 116). In our work, our i n t e r e s t i n t r a n s p o r t p r o c e s s e s has been c h i e f l y con- f i n e d to the t r a n s p o r t of amino ac i d s ( p a r t i c u l a r l y that of glutamate), and that of ammonia in r a t b r a i n c o r t e x s l i c e s . 1.7 (i) A m i n o a c i d m e t a b o l i s m in b r a i n . G l u t a m i c a c i d o c c upies a c e n t r a l p o s i t i o n i n b r a i n metabolism, i n view of its high concentration, its involvement i n many b i o c h e m i c a l r e a c t i o n s , i t s r e l a t i v e l y high rate of oxidation, and its p o s s i b l e r o l e as a t r a n s m i t t e r . Glutamate, together with the c l o s e l y r e l a t e d amino acids, viz., glutamine, aspartate, G A B A and al s o alanine, contribute up to 70 per cent of the total amino a c i d n i t r o g e n of adult b r a i n . In this r e s p e c t the f r e e amino a c i d pool of b r a i n is unlike that of other a n i m a l t i s s u e s . While s p e c i a l attention w i l l be g i v e n to the above m e n t i o n e d group of amino acids, the amino acids glycine, serine, threonine,taurine and N - a c e t y l aspartate, that together make up much of the r e m a i n i n g amino a c i d n i t r o g e n of the f r e e amino a c i d pool, w i l l a l s o be dealt with i n studies r e p o r t e d i n this t h e s i s . 19 T A B L E 1. Some b r a i n to p l a s m a c o n c e n t r a t i o n r a t i o s of amino acids f o r the cat. T h e s e c a l c u l a t i o n s were made f r o m the data of T a l l a n , M o o r e and Stein (88) g i v e n as mg%. C o n v e r s i o n of ( i m o l e / g to )j,mole/ml t i s s u e water was done by a s s u m i n g the t i s s u e water content to be 8 0 % that of the i n i t i a l f r e s h weight b r a i n . B r a i n P l a s m a m M B r a i n / P l a s m a Hmole /g (Jmole / ml Glutamate 8.74 10.92 0.123 89.0 Glutamine > 3.42 >4.27 >0.427 10.0 A s p a r t a t e 2.23 2.79 0.0076 368.0 A s p a r a g i n e 0.20 0.25 0.068 3.7 G A B A 2.27 2.84 < 0.02 > 142.0 A l a n i n e 0.95 1.19 0.845 1.4 G l y c i n e 1.35 1.69 0.307 5.5 S e r i n e 0.73 0.91 0.20 4.6 T h r e o n i n e 0.22 0.27 0.118 2.3 V a l i n e 0.18 0.22 0.21 1.1 L e u c i n e 0.14 0.17 0.123 1.4 Isoleucine 0.09 0.11 0.092 1.2 Methionine 0.11 0.14 0.034 4.1 A r g i n i n e 0.08 0.10 0.08 1.2 O r n i t h i n e 0.045 0.06 0.015 4.0 C i t r u l l i n e < 0.023 <0.03 ^0.00 57 <C 5.3 U r e a 4.17 5.21 6.67 0.8 20 (a) I n i t i a l c e r e b r a l contents of amino a c i d s The i n i t i a l l e v e l s of amino acids, d e t e r m i n e d in these studies and given in T a b l e s 2 and 4 for adult and infant (2 day old) r a t brain, a r e i n c l o s e agreement with those r e p o r t e d i n the l i t e r a t u r e (125-131). Infant r a t b r a i n has high concentrations of taurine and phosphoethanolamine (see r e f e r e n c e 130, 131), but have low l e v e l s of glutamate and its m e t a b o l i c a l l y d e r i v e d amino a c i d s . The a c t i v i t i e s of the enzymes i n v o l v e d in the f o r m a - t i o n and i n t e r c o n v e r s i o n of these amino a c i d s a r e also low (15, 163). A d u l t v a l u e s of amino acids and of t h e i r r e l a t e d enzymes ar e r e a c h e d as the b r a i n m a tures and at about the time of the complete laying down of m y e l i n (129, 130). A n i m a l s such as the guinea pig, i n which the infant stage shows many mature c h a r a c t e r i s t i c s , show only s m a l l v a r i a t i o n s in amino a c i d l e v e l s with age (129). (b) Pathways of glutamate m e t a b o l i s m A d e t a i l e d account of the s y n t h e s i s and c a t a b o l i s m of amino acids i s beyond the scope of this t h e s i s . Only the main aspects pertinent to the pre- sent study w i l l be d i s c u s s e d . F o r more d e t a i l e d i n f o r m a t i o n there a r e many us e f u l r e v i e w s (8, 11, 12, 15, 86, 95, 106, 117-120). Glutamate i s c l o s e l y r e l a t e d to the c i t r i c a c i d c y c l e due to i t s f o r m a - t i o n by t r a n s a m i n a t i o n of a-ketoglutarate. Glutamate t r a n s a m i n a s e s ; Glutamate can r e v e r s i b l y t r a n s a m i n a t e with the keto a c i d s , oxaloacetate and pyruvate to y i e l d aspartate and alanine r e s p e c t i v e l y . These reactions, shown below, ar e quantitatively the dominant t r a n s a m i n a t i o n s o c c u r r i n g in b r a i n t i s s u e (122, 123). They are c a t a l y z e d by s p e c i f i c t r a n s a m i n a s e s r e q u i r i n g p y r i d o x a l phosphate as co-enzyme (124). glutamate + oxaloacetate ^ ^ a -ketoglutarate + aspartate (1) glutamate + pyruvate . _ a-ketoglutarate + alanine (2) 21 When the rate of f o r m a t i o n of f r e e ammonia i s high, r e d u c t i v e a n i m a - t i o n of a-ketoglutarate, mediated by glutamate dehydrogenase, o c c u r s and a r a p i d synthesis of glutamate takes place, followed by the synthesis of g l u t a - mine. Glutamate, however, t r a n s a m i n a t e s with pyruvate and oxaloacetate f o r m i n g alanine and aspartate, and r e g e n e r a t i n g o:-ketoglutarate which again undergoes r e d u c t i v e a m i n a t i o n i n p r e s e n c e of excess ammonia. Thus, a flow of ammonia into amino acids (glutamate, glutamine, alanine and aspartate) takes place. When the amount of ammonia f o r m e d i s low, a -ketoglutarate i s m a i n l y c o n v e r t e d to glutamate by t r a n s a m i n a t i o n with alanine or aspartate f o r m i n g pyruvate or oxaloacetate which enter the c i t r i c a c i d c y c l e . Glutamate can also t r a n s a m i n a t e d i r e c t l y with a-ketoglutarate, each compound being converted to the other. T h i s allows the r a p i d exchange of l a b e l l e d c a r b o n between glutamate and m e m b e r s of the c i t r i c a c i d c y c l e (121). The G A B A shunt. Glutamate i s also f o r m e d f r o m a-ketoglutarate and G A B A by t r a n s a m i n a t i o n (by the enzyme G A B A t r a n s a m i n a s e ) . S u c c i n i c •semialdehyde f o r m e d i n the r e a c t i o n enters the c i t r i c a c i d c y c l e on conver- s i o n to succinate by the NAD"*" r e q u i r i n g enzyme s u c c i n i c semialdehyde dehydrogenase. These r e a c t i o n s including the d e c a r b o x y l a t i o n of glutamate by glutamate d e c a r b o x y l a s e constitute the G A B A shunt (116, 119). It r e p r e s e n t s an alternate pathway of a-ketoglutarate oxidation i n the meta- b o l i s m of carbohydrate i n the c e n t r a l nervous s y s t e m of mammals or the p e r i p h e r a l n e r v e s of anthropods. A c c o r d i n g to Bala z s _ e t a l . (116), 8 per cent of the a-ketoglutarate of the c i t r i c a c i d c y c l e may be d i v e r t e d to such a route. Haber (279) had a l r e a d y concluded that l e s s than 10 per cent of car b o h y d r a t e oxidation i n b r a i n took place v i a the G A B A shunt. The r e a c t i o n s constituting the G A B A shunt are depicted below (Reactions 3, 4, 5). 22 Glutamate > G A B A + C 0 2 (3) G A B A + a-ketoglutarate^=z±r Glutamate + S u c c i n i c semialdehyde (4) H z O S u c c i n i c semialdehyde + NAD +^- "'Succinate + N A D H + H + (5) The sum of the r e a c t i o n s : a-ketoglutarate + N A D + + H 2 0 = Succinate + N A D H + H + C O 2 Glutamate dehydrogenase. Glutamate i s f o r m e d r e v e r s i b l y f r o m a - k e t o g l u t a r a t e and ammonium ion by the enzyme glutamate dehydrogenase a c c o r d i n g to the following equations (8, 95) (Reactions 6 and 7 ) . Glutamate + NAD +^=zrz=^: a - i m i n o g l u t a r a t e + N A D H + H + (6) a-iminoglutarate + ^ 0 ^ . a -ketoglutarate +NH3 (7) non-enzymic In the presence of ammonium sal t s the e q u i l i b r i u m of r e a c t i o n (6) is much in favour of glutamate f o r m a t i o n only 1.4 per cent being o x i d i z e d at p H 7 .4 (8). In the absence of ammonium salts, a - i m i n o g l u t a r a t e spontaneously h y d r o l y z e s to a-ketoglutarate and ammonia. Thus the supply or r e m o v a l of ammonia may regulate the rate of oxidation of glutamate (8, 133) . The p r e s e n c e of a-ketoglutarate inh i b i t s c e r e b r a l ammonia f o r m a t i o n (42). T h e s e c o n c l u s i o n s were r e a c h e d f r o m r e a c t i o n s i n homogenous solutions d e r i v e d f r o m b r a i n (8, 4 2 , 132) . In b r a i n s l i c e s i n the presence of glucose, the re d u c t i v e amination of ot-ketoglutarate i s favo u r e d (102) . However, in the absence of glucose, as endogenous substrates of incubated b r a i n c o r t i c a l s l i c e s a r e depleted, the NAD + /NADH r a t i o i n c r e a s e s while the A T P / A D P r a t i o d e c r e a s e s . Increase of A D P r e s u l t s i n i n c r e a s e o f N A D + (V N A D H + A D P + P i — — — > N A D + + A T P ) . These changes favour the oxidation of 23 glutamate by glutamate dehydrogenase and ammonia and a-ketoglutarate a r e f o r m e d i n the r e a c t i o n . G l utamine synthetase. Glutamate combines with the ammonium ion, through the a c t i v i t y of the enzyme glutamine synthetase (58, 103) in the pre- sence of A T P (134, 136) a c c o r d i n g to the following r e a c t i o n (Reaction 8). ++ + + Glutamate + N H 4 + + A T P — ^ G l u t a m i n e + A D P + P i (8) Ox i d i z a b l e substrates l i k e glucose lactate and pyruvate, s u p p r e s s ammonia f o r m a t i o n in b r a i n both by compe t i t i o n with endogenous o x i d i z a b l e nitrogenous m a t e r i a l and by the f o r m a t i o n of glutamine (58-62). The p r e - sence of glucose b r i n g s about a disappearance of ammonia with incubated b r a i n s l i c e s (58). Inhibition of A T P synthesis (e.g., by 2,4 di n i t r o p h e n o l (62, 64) or fluoroacetate (63) ) and thereby d i m i n i s h e d glutamine synthesis causes i n c r e a s e d ammonia output. Glutamine synthetase is l o c a l i z e d i n the m i c r o s o m a l f r a c t i o n of b r a i n c e l l s (15). F o r the b r a i n enzyme the K m f o r L-glutamate is 2.5 mM; f o r A T P , 2.3 mM; and for N H 4 + , 0.18 m M (95). The low value for N H 4 + is si g n i f i c a n t in r e l a t i o n to the r o l e of glutamine synthetase in the c e r e b r a l u t i l i z a t i o n of ammonia. Glutaminase. Glutamate i s f o r m e d f r o m glutamine with the r e l e a s e of ammonia by the m i t o c h o n d r i a l enzyme glutaminase (15). The p r o c e s s is as follows (Reaction 9). H z O Gluta m i n e >- Glutamate + N H 4 + (9) Glutami n a s e is strongly inhibited by D- or L-glutamate (103). Phos- phate and other polyvalent anions such as arsenate and sulphate activate the enzyme (137, 138). In the absence of phosphate, glutaminase a c t i v i t y of guinea pig b r a i n homgenates is strongly enhanced by the pre s e n c e of t r i - 24 c a r b o x y l i c acids like c i t r a t e , l e s s strongly by that of d i c a r b o x y l i c acids, and slig h t l y , if at a l l , by that of m o n o c a r b o x y l i c acids l i k e lactate or propionate (138) . The enzyme f r o m b r a i n and kidney has a l l o s t e r i c p r o p e r t i e s (15, 138, 139) . (ii) C ompartmentation of amino a c i d m e t a b o l i s m i n b r a i n The concept of m e t a b o l i c compartmentation i s the outcome of o b s e r - vations (116, 1 4 1 - 1 4 8 ) that cannot be explained by the s i m p l e p r e c u r s o r - product r e l a t i o n as d e s c r i b e d by Z i l v e r s m i t e_t al. (140) . It i m p l i e s the pre- sence of two or m o re d i s t i n c t pools of a given metabolite in a given t i s s u e not n e c e s s a r i l y in e q u i l i b r i u m with each other, each maintaining quantitatively and/or q u a l i t a t i v e l y its own patterns of m e t a b o l i s m . M e t a b o l i c compartmen- tation i n b r a i n was f i r s t d e s c r i b e d for the glutamate-glutamine system. B e r l and C l a r k e (141) have r e c e n t l y r e v i e w e d the r e l e v a n t l i t e r a t u r e . E a r l y work on this subject was c a r r i e d out in vivo by W a e l s c h and c o - w o r k e r s ( 1 4 1 - 1 4 3 ) . They studied the effects of intravenously, i n t r a - c e r e b r a l l y and i n t r a c i s t e r n a l l y a d m i n i s t e r e d ^ C - g l u t a m a t e for short t i m e i n t e r v a l s ( 2 - 5 min, in o r d e r to avoid e q u i l i b r a t i o n of isotope through a l l m e t a b o l i c pools) and found that the s p e c i f i c a c t i v i t y of glutamine i s o l a t e d f r o m b r a i n t i s s u e was g r e a t e r than that of glutamate. T h i s o b s e r v a t i o n was a l s o found to be true f o r i n t r a v e n o u s l y administered 1 5 j \ f _ a m r n o n i u m acetate 14 or C l a b e l l e d bicarbonate. On the assumption that glutamate i s the only p r e c u r s o r of glutamine, these o b s e r v a t i o n s indicated that glutamine must be d e r i v e d f r o m a compartment of glutamate not i n e q u i l i b r i u m with the r e s t of the glutamate present i n the b r a i n . In other words, glutamate exists i n at l e a s t two d i s t i n c t pools, one (the s m a l l e r ) pool being p a r t i c u l a r l y d i s p o s e d to the f o r m a t i o n of glutamine. M e a s u r e m e n t s of the r a t i o of the s p e c i f i c r a d i o a c t i v i t y of glutamine to that of glutamate showed that l a b e l l e d ketogenic p r e c u r s o r s such as acetate, butyrate, propionate and acetoacetate give r i s e to a r a t i o g r e a t e r than one. 25 With l a b e l l e d g l y c o g e n i c substances such as glucose, pyruvate, lactate or g l y c e r o l , the s p e c i f i c a c t i v i t y of glutamate i s g r e a t e r than that of glutamine. T h e s e effects are accounted for by assuming the p r e s e n c e of at l e a s t two pools in the b r a i n c e l l s , in which the c i t r i c a c i d c y c l e operates, which d i f f e r in their a c c e s s i b i l i t i e s to substrates that show metabolic compartmentation (144). Compartmentation of glutamate m e t a b o l i s m has also been de m o n s t r a t e d i n v i t r o using b r a i n c o r t e x s l i c e s p r e p a r e d and incubated under c e r t a i n con- d i t i o n s (145). The amino acids ly s i n e , leucine, GABA, p r o l i n e and phenylalanine and a l s o ethanol exhibit m e t a b o l i c compartmentation i n the f o r m a t i o n of glutamate and glutamine (146, 148, 283, 284). The assumption of the p r e s e n c e of two pools of glutamate in b r a i n n e c e s s i t a t e s the p r e s e n c e of two pools of a-ketoglutarate f r o m which they a r e d e r i v e d . A s the p h y s i c a l l o c a t i o n of these pools has not been c h a r a c t e r - ized, B e r l and C l a r k e (141) have c a l l e d the p r o c e s s (involving the c i t r i c a c i d cycle) that generates a l a r g e glutamate pool but only a s m a l l glutamine pool, "the energy c y c l e " . The p r o c e s s (also i n v o l v i n g the c i t r i c a c i d cycle) which is p r e d o m i n a n t l y active i n the synthesis of glutamine, they have c a l l e d "the synthetic c y c l e " . "The energy c y c l e " i s p r e s u m a b l y r e s p o n s i b l e for a l a r g e r pool of A T P . A s e a r c h for m e t a b o l i c i n h i b i t o r s that s e l e c t i v e l y affect one of these pools is beginning to prove s u c c e s s f u l . L a h i r i and Q u a s t e l (63) showed that while f l u o r o a c e t a t e has l i t t l e effect on oxygen consumption, it s t r o n g l y inh i b i t s the synthesis of glutamine and concluded that at low (convulsive) c o n c e n t r a - tions it affects c e r e b r a l a mmonia metabolism. A c c o r d i n g to C l a r k e et a l . (150), the phenomenon can be explained if f l u o r oacetate inhibits o p erations of "the synthetic c y c l e " with r e l a t i v e l y no effect on those of "the energy c y c l e " . Work on the action of malonate and n e u r o t r o p i c agents l i k e p r o t o v e r a t r i n e , on the operations of the two pools, w i l l be presented in this t h e s i s (Chapter 8). 26 M e t a b o l i c compartmentation has not yet been s a t i s f a c t o r i l y defined i n s t r u c t u r a l or c e l l u l a r t e r m s . M i t o c h o n d r i a , heterogenous with r e s p e c t to thei r enzymes (232), may offer a p o s s i b l e explanation to this problem. But mi t o c h o n d r i a may be d e r i v e d f r o m d i f f e r e n t c e l l types (e.g., neuron and glia) or d i f f e r e n t portions of the same c e l l (e.g., nerve c e l l body and nerve ending). Some evidence i n favour of neurons and g l i a as r e s p o n s i b l e f o r metabolic c ompartmentation has been pre s e n t e d by Rose (159) using his neuronal and g l i a l p r e p a r a t i o n s . However, at the p r e s e n t time f r a c t i o n a t i o n p r o c e d u r e s f o r the s e p a r a t i o n of c e l l types such as neurons and g l i a a r e somewhat d r a s t i c and cannot be expected to give pure p r e p a r a t i o n s . M o r e o v e r , it is u n c e r t a i n how f a r the procedu r e affects the p r o p e r t i e s of the f r a c t i o n s . M e t a b o l i c compartmentation of the glu'.amate-glutamine s y s t e m i s not ma n i f e s t in i m m a t u r e r a t b r a i n (149), or i n octopus b r a i n that contains l i t t l e g l i a (160). C ompartmentation of amino a c i d s following neuronal degeneration has been r e p o r t e d (161). B r i e f l y stated, such i n d i r e c t r e s u l t s i n d i c a t e that the " l a r g e " glutamate compartment may c o n s i s t of ne u r o n a l p r o c e s s e s (116, 149, 154-156), while the " s m a l l " glutamate pool a s s o c i a t e d with high glutamine content i s thought to c o n s i s t of g l i a l t i s s u e (161, 162). In studies r e p o r t e d in this thesis m o r e d e f i n i t i v e attempts w i l l be made to c h a r a c t e r i z e the compartments of amino acids i n b r a i n c o r t e x s l i c e s of the rat using p h a r m a c o l o g i c a l agents, r a t h e r than fractionations, f o r l o c a l i z a t i o n of the compartments. 1.8 Pr o p a g a t i o n of nerve impulse N e u r o p h y s i o l o g i c a l aspects of excitation have been e x t e n s i v e l y r e v i e w e d (164-168, 176, 262). Only m a t e r i a l r e l e v a n t to this work w i l l be pr e s e n t e d here. (i) B r a i n c e l l types (168, 169) The c e l l s of the c e n t r a l n e r v o u s s y s t e m may be di v i d e d into two c l a s s e s on the b a s i s of whether they a r e excitable (neurons) or not (glia). E a c h 27 n e u r on i s made up of (1) dendrites which are c o v e r e d by synaptic p r o c e s s e s ; (2) the c e l l body r e s p o n s i b l e for the p r o p e r functioning of the neuron; and (3) an axon whose t e r m i n a l b r a n c h e s f o r m synaptic connections with other neurons. G l i a c o n s i s t s of three types, namely, as t r o c y t e s , o l i g o d e n d r o g l i a and m i c r o g l i a , and are capable of p r o l i f e r a t i o n . They act as i n s u l a t o r s because of their high r e s i s t a n c e membrane and a r e e s s e n t i a l for the l a y i n g down and maintenance of the m y e l i n sheath. It i s thought that they offer s t r u c t u r a l support, supply n u t r i t i o n f r o m the blood, and ma i n t a i n the c h e m i c a l environment of the neuron (30 5). T h e r e are about ten t i m e s m o re g l i a l c e l l s than neurons i n the b r a i n (171). The volume occupied by the g l i a l c e l l s , however, i s about the same as that occupied by the neurons (95). T h e r e is agreement that the neurons a r e r e s p o n s i b l e for much of the oxygen consumption of b r a i n (175, 264, 268- 271). In the immature b r a i n n e u r o n a l p r o c e s s e s are not developed. The growth of such p r o c e s s e s c o i n c i d e s with myelination. (ii) R e s t i n g potential (164-168) When a membrane separating two solutions is p e r m e a b l e to one io n i c s p e c i e s a potential difference, E, i s set up a c r o s s the membrane whose magni- tude is given by the fol l o w i n g N e r n s t equation (at 38°C and f o r a monovalent ion): , , _ , [penetrating ion out s i d e l ,. E = 61.5 log Jf1 6 -r1- m i l l i v o l t s 10[penetrating ion i n s i d e j Under r e s t i n g conditions the i n t e r i o r of nerve and m u s c l e f i b r e s is 69-75 mV negative with r e s p e c t to the outside. T h i s i s caused by the s e l e c t i v e permea- b i l i t y of the c e l l membrane to the flow of K~*\ Under these conditions the o membrane pore si z e of the neuron is in t e r m e d i a t e (3A) between the hydrated d i a m e t e r of K+ (2.2 A) and that for N a + (3.4 A) and N a + is p r a c t i c a l l y i m - p e r m e a b l e (164). The nature of the charge on the membrane is another fa c t o r c o n t r o l l i n g p e r m e a b i l i t y of ions (164). 28 The K concent r a t i o n i n s i d e the c e l l i s g r e a t e r than that i n the body f l u i d s outside, and the co n v e r s e is true for Na*. Thus, f o r cat motoneurons the c o n c e n t r a t i o n of K* in the neurons i s 27 times higher than that outside, while the concentration for Na"*" is 10 times higher outside than inside (165). (iii) A c t i o n potential (164-168) A c t i o n potentials are generated i n an a l l or none p r o c e s s when the membrane potential f a l l s to the t h r e s h o l d l e v e l . Thus, when the membrane is d e p o l a r i z e d by an outward flow of current, its Na"*" p e r m e a b i l i t y i m m e d i a t e l y r i s e s and there is a net inward movement of Na* ions down the s o d i u m con- c e n t r a t i o n gradient. If the i n i t i a l d e p o l a r i z a t i o n i s l a r g e enough (i.e., the t h r e s h o l d potential for f i r i n g is reached), N a * enters f a s t e r than K* can leave and this causes the potential to dr o p s t i l l f u r t h e r i n c r e a s i n g the N a * p e r m e a b i l i t y even more, i n the following manner: I n c r e a s e i n N a * p e r m e a b i l i t y D e p o l a r i z a t i o n of membrane The entry of s o d i u m i s halted when the membrane potential r e a c h e s a l e v e l c l o s e to (Ejsj a), the e q u i l i b r i u m potential for sodium, when the net i n w a r d d r i v i n g f o r c e acting on s o d i u m ions becomes z e r o . It i s s t i l l u n c e r - t a i n whether N a and K+ t r a v e r s e the same channels (166). However, the Na"*" and K* channels can be d i f f e r e n t i a l l y blocked; e.g., by tetrodotoxin (190, 193, 194) and t e t r a e t h y l a m m o n i u m ions (167, 172) r e s p e c t i v e l y . (iv) Sodium-Pump (164-168) A f t e r the conduction of the nerve impulse, the sodium-pump r e - e s t a b l i s h e s the r e s t i n g potential by ejecting N a* f r o m and r e t u r n i n g K* to E n t r y of N a * 29 the n e r v e t i s s u e . A T P i s u t i l i z e d as the s o u r c e of energy. The sodium- pump i s b e l i e v e d to be operated by the Na +, K + - s t i m u l a t e d M g + + - d e p e n d e n t A T P a s e present i n c e l l membranes. These and other aspects of the sodium- pump w i l l be dealt with i n a l a t e r s e c t i o n (section 1.9 (v)). (v) Synaptic t r a n s m i s s i o n (164-168, 170) I n t e r c e l l u l a r c o m m u n i c a t i o n at the synaptic or n e r v e ending is achieved, i t i s p r e s e n t l y understood, through the action of c h e m i c a l t r a n s - m i t t e r s . F o r a substance to be c o n s i d e r e d a t r a n s m i t t e r , the following c r i t e r i a should be s a t i s f i e d . It must be synthesized and stored i n the p r e - synaptic n e r v e t e r m i n a l (not n e c e s s a r i l y in v e s i c l e s ) . It should be r e l e a s e d i n adequate quantities on the s t i m u l a t i o n of the p r e s y n a p t i c n e r v e s . It must be capable of i n t e r a c t i n g with r e c e p t o r s on the post-synaptic n e u r on or effector c e l l and br i n g about t r a n s i e n t a l t e r a t i o n s in the p e r m e a b i l i t y of the post synaptic membrane towards ions in the immediate environment. Its ac t i o n on post synaptic s t r u c t u r e s when applied d i r e c t l y should be i d e n t i c a l with the n o r m a l t r a n s m i t t e r action. In some cases at least, an ina c t i v a t i n g enzyme should be pr e s e n t in the synaptic cl e f t . B l o c k i n g and com p e t i t i v e agents should affect its n o r m a l action, and its action on d i r e c t a p p l i c a t i o n i n a s i m i l a r manner. A c e t y l c h o l i n e , and the biogenic amines, noradrenaline, dopamine and serotonin, are str o n g l y i m p l i c a t e d i n c e n t r a l t r a n s m i s s i o n . (vi) A m i n o ac i d s as putative t r a n s m i t t e r s (164, 165, 168, 170, 233) Glutamate, and r e l a t e d substances such as aspartate, excite many c e n t r a l neurons when i o n t o p h o r e t i c a l l y applied. Since glutamate i s in v o l v e d i n m e t a b o l i s m of c o r t i c a l neurons, enzymes f o r its synthesis are obv i o u s l y p r e s e n t i n the b r a i n , and although there is no evidence for the p r e s e n c e of a d e s t r u c t i v e enzyme, analogous to choline e s t e r a s e i n the synaptic cleft, a r a p i d uptake of excess glutamate into neighbouring n e r v e c e l l s may serv e to ensure the n e c e s s a r y b r e v i t y of its t r a n s m i t t e r action. L i k e L-glutamate 30 and L - a s p a r t a t e , D L - h o m o c y s t e a t e and N-methyl aspartate reduce the t h r e s h o l d for f i r i n g and a r e t h e r e f o r e c o n s i d e r e d to be e x c i t a t o r y amino acids (164, 170). G A B A and gl y c i n e a r e i n h i b i t o r y amino acids, r a i s i n g the t h r e s h o l d for f i r i n g , by h y p e r p o l a r i z a t i o n caused by an i n c r e a s e in the post synaptic intake of K* and/or c h l o r i d e (170). C u r t i s and Johnston (170) have r e v i e w e d the evidence for the p a r t i c i p a t i o n of amino acids as t r a n s m i t t e r s in the v e r t e b r a t e and i n v e r t e b r a t e n e r v o u s system. The m e c h a n i s m of action of amino acids as t r a n s m i t t e r s is not known. It is b e l i e v e d that they cause changes i n the io n i c p e r m e a b i l i t y of the post synaptic membrane by r e a c t i o n s with s p e c i f i c r e c e p t o r s i t e s . (vii) C a * * ions and ex c i t a t i o n T h e r e is a twenty-fold i n c r e a s e i n c a l c i u m p e r m e a b i l i t y accompanying the propagation of i m p u l s e s along giant n e r v e f i b r e s . The inflow of Ca"*"*", however, is too s m a l l to convey a p p r e c i a b l e c u r r e n t or to modify the c h a r a c t e r of the ac t i o n potential (172). N e r v e f i b r e s f i r e spontaneously when e x t e r n a l Ca"*""*" i s reduced. Con- v e r s e l y , a r i s e in e x t e r n a l Ca"*"*" tends to s t a b i l i z e the nerve membrane and to r a i s e the t h r e s h o l d for e x c i t a t i o n (166-168). C a * * may achieve this by bl o c k i n g t r a n s m e m b r a n e channels to N a * by binding with the membrane phos p h o l i p i d l a y e r ; d i s l o c a t i o n of Ca"*"*" f r o m this bond might then p e r m i t f r e e passage of N a * (174). ++ Ca is known to be r e q u i r e d for t r a n s m i t t e r r e l e a s e (173). It causes an i n c r e a s e d r e l e a s e of ac e t y l c h o l i n e at the n e u r o m u s c u l a r junction. Mn** and Mg"*"*" reduce Ca"*"*" entry and inhibit t r a n s m i t t e r r e l e a s e (172, 173). (viii) High K ion co n c e n t r a t i o n and e x c i t a t i o n H i g h e x t e r n a l K"*" causes d e p o l a r i z a t i o n (177). However, if the high K"*" i s applied simultaneously over the whole su r f a c e of a fib r e , e x c i t a b i l i t y i s u s u a l l y d e p r e s s e d or abolished. A maintained d e p o l a r i z a t i o n , p roduced by high K+, r a i s e s the t h r e s h o l d f o r e x c i t a t i o n and g r e a t l y affects the p e r m e a - b i l i t y p r o p e r t i e s of the nerve t i s s u e to s o d i u m and p o t a s s i u m ions (168). S o m e t i mes the ap p l i c a t i o n of K* to excitable c e l l s make them generate pu l s e s . S t i m u l a t o r y effects of this kind a r i s e if the applied solutions do not penetrate u n i f o r m l y into the t i s s u e and one p a r t of the nerve is d e p o l a r i z e d m u ch more than another (168). In addition, to i t s d e p o l a r i z i n g effect, K* appears to have a potentiating a c t i o n on t r a n s m i t t e r r e l e a s e (156, 178). (ix) C e r e b r a l c o r t e x s l i c e s and ex c i t a t i o n phenomena The use of c e r e b r a l c o r t e x s l i c e s to study e x c i t a t i o n phenomena i s r a p i d l y gaining support. L i and M c l l w a i n (179) used m i c r o e l e c t r o d e s to demonstrate the maintenance of r e s t i n g membrane potentials of -55 to -60 mV i n c e r e b r a l c o r t e x s l i c e s of the guinea pig, values approaching those o b s e r v e d i n vivo. G i b s o n and M c l l w a i n (180) have been able to c o r r e l a t e potential d i f f e r e n c e s a c r o s s c o r t i c a l c e l l membranes, as c a l c u l a t e d f r o m the N e r n s t equation f r o m e l e c t r o l y t e a n a l y s i s , with potential d i f f e r e n c e s m e a s u r e d d i r e c t l y by m i c r o e l e c t r o d e s i n s e r t e d into the incubated s l i c e s . M o r e o v e r , c o r t i c a l c e l l s a r e d e p o l a r i z e d i n v i t r o by an i n c r e a s e d e x t e r n a l K* or the ap p l i c a t i o n of e l e c t r i c a l pulses, without l o s i n g the a b i l i t y to r e p o l a r i z e on the r e m o v a l of the applied s t i m u l i (180). Y amamoto and M c l l w a i n (181) obtained spike potentials i n the i s o l a t e d p i r i f o r m c o r t e x of guinea pig brain, which a r e said to be akin to action potentials (224, 264). The tetrodotoxin s e n s i t i v i t y of such evoked potentials i n c e r e b r a l s l i c e s (265) lends support to this view. S i m i l a r l y , changes in the tet r o d o t o x i n - s e n s i t i v e i o n i c balance of rat b r a i n c o r t e x s l i c e s (presumably at the ne u r o n a l membrane) on incubation under c e r t a i n conditions, led Okamoto and Q u a s t e l (182) to suggest that action potentials may be generated in incubated b r a i n s l i c e s . 32 S l i c e s of b r a i n a c t i v e l y accumulate s e r o t o n i n (183), n o r e p i n e p h r i n e (184), a c e t y l c h o l i n e (185) and amino acids (104-106, 113, 114, 231, 252, 295) against c o n c e n t r a t i o n g r a d i e n t s . P r e s u m a b l y , this may be one method for the r e m o v a l of r e l e a s e d t r a n s m i t t e r substance f r o m their site of action. Many studies have demonstrated that the stimulation of b r a i n e l e c t r i c a l l y or c h e m i c a l l y (e.g., with p r o t o v e r a t r i n e ) d e p o l a r i z e s the n e r v e membrane and m a r k e d l y enhances the r e l e a s e of l a b e l f r o m the b r a i n s l i c e p r e - l o a d e d with l a b e l t r a n s m i t t e r substrate (e.g., 186, 187). Our e a r l i e r studies showed that the ac t i v a t i o n of the s o d i u m c u r r e n t s y s t e m i n b r a i n s l i c e s under c e r t a i n conditions of incubation (182), concomitantly affects the r e l e a s e of endogenous amino acids f r o m the t i s s u e (188). T h e s e and later studies a r e d e s c r i b e d i n d e t a i l i n th i s t h e s i s . The above c o n s i d e r a t i o n s (and others that w i l l be apparent in due course) support the v a l i d i t y of employing the i s o l a t e d b r a i n s l i c e f o r the study of nerve function and m e t a b o l i s m . E l l i o t , i n his r e v i e w (234) on "the use of b r a i n s l i c e s " , has d e s c r i b e d the usefulness of the t i s s u e s l i c e technique i n the d i s c o v e r y and e l u c i d a t i o n of metabolic p r o c e s s e s . 1.9 Some p r o p e r t i e s of drugs and metabolic i n h i b i t o r s used as tools for this i n v e s t i g a t i o n Only a b r i e f account of the p r o p e r t i e s of some of the c h e m i c a l sub- stances used as tools i n these studies w i l l be pres e n t e d here. (i) T e t r o d o t o x i n ( T T X ) Tetrodotoxin, a potent n o n - p r o t e i n neurotoxin found i n Japanese puffer f i s h and C a l i f o r n i a n newts, s u p p r e s s e s at conce n t r a t i o n s as low as 0.3 (imolar the generation of action potentials i n a v a r i e t y of t i s s u e s i n c l u d i n g f r o g m y e l i n a t e d n e r v e f i b r e s and lob s t e r and squid giant axons, and hence the a s s o c i a t e d influx of N a + ions (190, 193, 194). Its m e c h a n i s m of ac t i o n 33 r e s e m b l e s that of l o c a l anesthetics but it has a potency more than a 100,000 t i m e s that of cocaine (189). It acts on the outer, r a t h e r than the i n t e r n a l , s u r f a c e of the membrane s e l e c t i v e l y b l o c k i n g the inward movements of N a * ions accompanying the generation of action potentials. It seems to have no effect on K* ion movements (195, 196). E a r l i e r studies of T T X concerned with i t s chemistry, p h a r m a c o l o g y (in i n vivo studies) and its e l e c t r o p h y s i o l o g i c a l effects using i s o l a t e d n e r v e and m u s c l e have been r e v i e w e d by a number of w o r k e r s (189-192). Only r e c e n t l y has T T X been used i n the study of m e t a b o l i s m and c a t i o n i c t r a n s p o r t p r o c e s s e s i n i s o l a t e d b r a i n t i s s u e by Q u a s t e l and his co-workers, working m a i n l y with r a t b r a i n (182, 188, 192, 197, 198, 205) and by M c l l w a i n ' s group using c h i e f l y the b r a i n of the guinea pig (199, 201-204). While T T X has no effect on the rate of r e s p i r a t i o n of r a t b r a i n c o r t e x s l i c e s incubated i n a p h y s i o l o g i c a l glucose saline medium, it c o m p l e t e l y b l o c k s the s t i m u l a t e d r e s p i r a t i o n due to the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s (197, 199) or the p r e s e n c e of p r o t o v e r a t r i n e (182), or when c a l c i u m ions are omitted f r o m the incubation m e d i u m (197). However, it has no effect on the p o t a s s i u m stimu l a t e d r e s p i r a t i o n (197). A p p l i c a t i o n of e l e c t r i c a l i m p u l s e s (182, 202) or the p r e s e n c e of p r o t o - v e r a t r i n e (182) b r i n g s about a T T X - s e n s i t i v e influx of Na"*"* in b r a i n c o r t e x s l i c e s . T he K* content of incubated e l e c t r i c a l l y s t i m u l a t e d s l i c e s is g r e a t e r i n the p r e s e n c e of T T X than i n its absence (199, 200). T T X has a s i m i l a r effect with p r o t o v e r a t r i n e stimulated s l i c e s (182). Chan and Quastel (198) showed that the inh i b i t i o n of acetate oxidation by r a t c e r e b r a l cortex s l i c e s is completely s u p p r e s s e d by T T X . They con- cluded that T T X exerts its a c t i o n on acetate m e t a b o l i s m i n d i r e c t l y by its effects on N a * movement. It is w e l l known that Li-glutamate excites nervous t i s s u e (section 1.8 34 (vii) ). A n influx of Na ions into incubated c e r e b r a l c o r t e x s l i c e s takes p l a c e when 5 m M sodium L-glutamate is added to the incubation m e d i u m (95, 182, 201, 263). T h i s i s only p a r t l y inh i b i t e d by T T X (182) during a shor t i n i t i a l p e r i o d of incubation (202). A c c o r d i n g to R a m s a y and M c l l w a i n (203), low concentrations of T T X are capable of inhibiting Ca influx both i n the p r e s e n c e or absence of L-glutamate. It also causes a detectable d i m i n u t i o n in Ca efflux (203). Chelating agents, e.g., 5 m M E D T A b r i n g about i n c r e a s e s i n the influx of N a * into, and efflux of K* from, incubated guinea pig c e r e b r a l cortex s l i c e s and these changes a r e p a r t i a l l y p revented by T T X (204). R e c e n t e x p e r i m e n t s of Okamoto and Qua s t e l (182) have shown that under a v a r i e t y of incubation conditions, the i n c r e a s e of Na+ influx and of water uptake that take place i n r a t b r a i n c o r t e x s l i c e s are s u p p r e s s e d either wholly or p a r t i a l l y by s m a l l concentrations of T T X (3 |JM). T h e i r e x p e r i m e n t s l e d to the c o n c l u s i o n that action potentials, or a c t i v a t i o n of the N a * c u r r e n t a r e generated in b r a i n c o r t e x s l i c e s i n v i t r o . T h i s o c c u r s for example, i n the absence of glucose, or i n the p r e s e n c e of 0.1 m M ouabain or of 10 (-1 M p r o t o v e r a t r i n e or on the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s or a c c o r d i n g to Shankar and Q u a s t e l (192, 20 5) at the onset of anoxia. Under these conditions T T X a f f e c t s both the ca t i o n i c fluxes and water uptake. However, T T X has no effect on the enhanced N a * influx and water uptake i n the p r e s e n c e of 30 (JM 2, 4-dinitrophenol or of 100 m M KC1 (182). Recently, Shankar and Q u a s t e l (192, 20 5) have shown that T T X (2 |jM) enhances the rate of an a e r o b i c g l y c o l y s i s i n r a t c e r e b r a l c o r t e x s l i c e s when added b e f o r e the onset of anoxia. They also showed that the effects of T T X on a n a e r o b i c g l y c o l y s i s a r e s p e c i f i c for mature c e r e b r a l t i s s u e and r e q u i r e the i n t e g r i t y of the b r a i n c e l l f o r its action. The s t i m u l a t o r y effect of T T X on a n a e r o b i c g l y c o l y s i s is i n d i r e c t . It s u p p r e s s e s N a * influx into, and K* efflux from, c e r e b r a l c o r t e x s l i c e s that occur at the onset of anoxia. The enhanced K*/Na* concent r a t i o n r a t i o activates the rate l i m i t i n g pyruvate 35 k i n a s e thereby stimulating the rate of anae r o b i c g l y c o l y s i s . The effect of T T X on anaerobic g l y c o l y s i s is reduced or abo l i s h e d i n the p r e s e n c e of pr o t o v e r a t r i n e , high K +, NĤ "** or L-glutamate (192). In studies r e p o r t e d here, T T X w i l l be used to inhibit N a + - m o v e m e n t into b r a i n c o r t e x s l i c e s when these a r e incubated in such a manner as to b r i n g about the activ a t i o n of the sodium c u r r e n t at the c e l l membrane or the gener a t i o n of action potentials. (ii) L o c a l anesthetics (166, 176) L o c a l anesthetics r e v e r s i b l y block the nerve impulse by r e d u c i n g the membrane p e r m e a b i l i t y to s o d i u m ions. I n c r e a s e d e x t r a c e l l u l a r s o d i u m d e c r e a s e s anesthetic action. M o s t l o c a l anesthetics w i l l d e p o l a r i z e the membrane at concentrations higher than are needed for blocking action, and w i l l a l s o s l i g h t l y reduce the p o t a s s i u m p e r m e a b i l i t y of the membrane. L i d o c a i n e i s said to be a hundred t i m e s more effective on Na"*" channels than on K* channels (176). L o c a l anesthetics behave li k e C a + + , s t a b i l i z i n g the nerve membrane, r a i s i n g the t h r e s h o l d f o r excitation, a b o l i s h i n g spontaneous activity, and bl o c k i n g conduction without d e p o l a r i z a t i o n . F e i n s t e i n (206) states that l o c a l a nesthetics may act p r i m a r i l y by inh i b i t i n g r e l e a s e of Ca"*"** f r o m sites to which it is bound to the membrane. They thereby prevent secondary changes i n the Na"*" and K* p e r m e a b i l i t i e s , and consequently suppress the gene r a t i o n and propagation of the nerve impulse. The suggestion has been made that both l o c a l anesthetics and Ca*"** act c o m p e t i t i v e l y on the s y s t e m c a r r y i n g N a* through the membrane. F o r example, i n c r e a s e d e x t r a c e l l u l a r Ca"*""*" d e c r e a s e s p r o c a i n e effects, whereas p r o c a i n e completely r e v e r s e s the d e p o l a r i z a t i o n c aused by r e m o v a l of e x t e r n a l Ca"*"*" (167, 176). L o c a l anesthetics a r e said to compete with a c e t y l c h o l i n e for r e c e p t o r s i t e s on the post-synaptic membrane (176). 36 The r e s u l t s d e s c r i b e d above have been obtained c h i e f l y in studies employing i s o l a t e d nerve fibres-. However, studies have a l s o been c a r r i e d out with b r a i n cortex s l i c e s with s i m i l a r r e s u l t s . Thus, Chan and Q u a s t e l (198) showed that cocaine (0.2 mM), l i d o c a i n e (0.5 and 1 mM), and p r o c a i n e (1 and 3 mM) block the i n c r e a s e d influx of N a * due to e l e c t r i c a l s t i m u l a t i o n without affecting either the content of Na"*" found in unstimulated b r a i n tissue, or the a c t i v i t y of membrane Na*, K * - A T P a s e . These l o c a l a nesthetics were al s o found to a b o l i s h the d e p r e s s e d oxidation of ( 1 - ^ C ) acetate to ̂^CO^, and the enhanced oxygen uptake brought about by the a p p l i c a t i o n of e l e c t r i c a l p u lses. Such effects were not obtained with the unstimulated s l i c e s at the concentrations studied. T h i s l e d to the c o n c l u s i o n that l o c a l anesthetics at low concentrations act on s o d i u m channels that become a v a i l a b l e following the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s . They inhibit e l e c t r i c a l l y s t i m u l a t e d r e s p i r a t i o n p r e s u m a b l y by b l o c k i n g the influx of Na"*" ions and thereby the s t i m u l a t e d a c t i v i t y of the N a * s e n s i t i v e membrane A T P a s e . T h i s leads to s u p p r e s s i o n of the rate of f o r m a t i o n of A D P that p a r t l y c o n t r o l s the r a t e of m i t o c h o n d r i a l r e s p i r a t i o n . Thus, l i k e T T X , l o c a l anesthetics exert t h e i r effects on m e t a b o l i s m i n d i r e c t l y , by t h e i r action on c a t i o n i c movements i n s t i m u l a t e d nervous t i s s u e . In studies r e p o r t e d be re, l i d o c a i n e (0.5 mM) w i l l be used as a r e p r e - sentative of the l o c a l anesthetic group of drugs, for s u p p r e s s i n g the a c t i v a t i o n of the N a * c u r r e n t s y s t e m generated i n b r a i n s l i c e s incubated under s p e c i f i c conditions. (iii) P r o t o v e r a t r i n e It i s well known that v e r a t r i n e alkaloids, of which p r o t o v e r a t r i n e i s a member, generate action potentials in nervous t i s s u e (167, 207). Shanes has shown that in r e s t i n g nerve, v e r a t r i n e a l k a l o i d s cause a net r e l e a s e of K* ions and an uptake of an equivalent amount of Na"*" ions (208). However, the mode of action of v e r a t r i n e drugs is not yet c l e a r . T h e r e is some evidence 37 suggesting that the b a s i c effect of v e r a t r i n e compounds i s a d i s p l a c e m e n t of Ca*"*" ions f r o m the c e l l s u r f a c e (209). thus affecting the st a b i l i t y and con s e - quently the p e r m e a b i l i t y of the excitable c e l l membrane to the flow of ions. W o l l e n b e r g e r (211) showed that p r o t o v e r a t r i n e at low concentrations (about 2 |iM) stimulates r e s p i r a t i o n and a e r o b i c g l y c o l y s i s , and in h i b i t s a n a e r o b i c g l y c o l y s i s i n guinea pig b r a i n c o r t e x s l i c e s incubated i n a p h y s i o - l o g i c a l glucose s a l i n e medium. Under these conditions it also b r i n g s about a T T X - s e n s i t i v e influx of N a * ions into, and efflux of K* ions from, r a t c e r e b r a l c o r t e x s l i c e s (182, 210). Such cationic fluxes a r e accompanied by an enhanced oxygen consumption (182). The enhanced uptake of oxygen due to p r o t o v e r a t r i n e i s inhibited by T T X (182), cocaine (213) or malonate (226). P r o t o v e r a t r i n e may al s o stimulate the i n c r e a s e d r e s p i r a t i o n due to e l e c t r i c a l s t i m u l a t i o n (212). It i n c r e a s e s y i e l d s of l a b e l l e d glutamate, glutamine, G A B A and aspartate f r o m l a b e l l e d glucose i n incubated b r a i n s l i c e s , and these effects are r e v e r s e d by cocaine (213). A s p r o t o v e r a t r i n e has effects on incubated b r a i n s l i c e s c l o s e l y r e s e m b l i n g those of e l e c t r i c a l i m p u l s e s (211, 182), and as it is known to generate a c t i o n potentials in nerve t i s s u e (167, 207), it w i l l be used exten- s i v e l y i n studies r e p o r t e d in this t h e s i s . (iv) B a r b i t u r a t e s T h e r e are at pr e s e n t two schools of thought concerning the mode of acti o n of b a r b i t u r a t e s on nerve function. A c c o r d i n g to one group of workers, b a r b i t u r a t e s e x e r t th e i r effects by " d i m i n i s h i n g i o n i c movements of e x c i t a t i o n " (214), or "on membrane p a r a m e t e r s " (215), or by "cutting down inw a r d s o d ium l e a k a g e " (216). However, another group of w o r k e r s have obtained r e s u l t s not c l e a r l y e x p l i c a b l e by such statements. With the b a r b i - turates amytal (0.25 and 0.5 mM) and pentothal (0.1 and 0.2 mM) at concentra- tions that do not affect the content of ̂ 2j^ a+ o r ^ n e r e s p i r a t i o n of unstimulated 38 r a t b r a i n c o r t e x s l i c e s incubated for one hour, Chan and Q u a s t e l (198) found no a b o l i t i o n of the enhanced influx of 22j\f a+ following e l e c t r i c a l s t i m u l a t i o n . H i l l m a n et a l . (217) s i m i l a r l y found that phenobarbital was without effect on the N a * influx obtained by the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s to guinea pig c e r e b r a l cortex s l i c e s . Thus, the effects of b a r b i t u r a t e s under the given e x p e r i m e n t a l conditions were d i f f e r e n t f r o m those of l o c a l anesthetics or of T T X which completely block the i n c r e a s e d influx of 22]\ja+ that o c c u r s on the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s . L i k e l o c a l anesthetics or T T X , b a r b i t u r a t e s at s m a l l concentrations n e v e r t h e l e s s suppress e l e c t r i c a l l y s t i m u l a t e d b r a i n r e s p i r a t i o n . None of these drugs at these concentrations affects the a c t i v i t y of membrane bound Na +, K + - A T P a s e (198). Unlike the l o c a l anesthetics or T T X , b a r b i t u r a t e s do not a l l e v i a t e the d e p r e s s e d oxidation of ( 1 - ^ C ) acetate to I^QO brought about by the influx of N a * and the efflux of K + due to the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s (198). However, in co n t r a s t to the l o c a l anesthetics and T T X , the b a r b i t u r a t e s inhibit the r a t e of ( l - ^ C ) acetate oxidation to ^^CO^ in unstimulated r a t b r a i n s l i c e s . Chan and Q u a s t e l (198) have concluded that b a r b i t u r a t e s (at the concen- t r a t i o n s quoted and under the incubation conditions studied) do not d i r e c t l y affect the movement of Na+ and K* a c r o s s the b r a i n c e l l membrane; rather, they act by s u p p r e s s i n g c e l l e n e r g e t i c s . Such s u p p r e s s i n g a c t i o n also explains the i n h i b i t o r y effects of b a r b i t u r a t e s on the s t i m u l a t i o n of r a t b r a i n c o r t e x r e s p i r a t i o n brought about by the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s or by the p r e s e n c e of high K~*~ concentrations (218), or with p r o t o v e r a t r i n e , or in a m e d i u m devoid of C a + * (210). It i s w e l l known following the work of M i c h a e l i s and Quastel (219), and E r n s t e r and c o - w o r k e r s (220, 221), that the barbiturate, amytal, as w e l l as c e r t a i n other hypnotics at low concentrations, s u p p r e s s the oxidation of N A D H and hence the generation of A T P in the c e l l . In view of the g r e a t amount of work which has been c a r r i e d out with amytal, both in n e u r o c h e m i c a l and n e u r o p h y s i o l o g i c a l studies, it has been used in the studies r e p o r t e d in this t h e s i s as r e p r e s e n t a t i v e of b a r b i t u r a t e s 39 i n work on the t r a n s p o r t and m e t a b o l i s m of amino acids and ammonia in b r a i n s l i c e s incubated under conditions leading to the a c t i v a t i o n of the s o d i u m c u r r e n t or to the g e n e r a t i o n of action potentials. (v) C a r d i a c g l y c o s i d e s - Ouabain The p r o c e s s whereby N a * is extruded f r o m the c e l l and accumu- late d against a concentration gradient is u s u a l l y r e f e r r e d to as the "Sodium- Pump". T h i s c a r r i e r mediated p r o c e s s maintains the p o l a r i t y of non- excitable membranes, and r e s t o r e s that of excitable ones which have under- gone d e p o l a r i z a t i o n , d u r i n g the gene r a t i o n of n e r v e i m p u l s e s . R e p o l a r i z a t i o n i s a c hieved at the expense of m e t a b o l i c energy d e r i v e d f r o m a e r o b i c meta- b o l i s m . Thus, i n h i b i t i o n of A T P f o r m a t i o n by anoxia, cyanide, DNP, or i n the absence of glucose, causes a r e d i s t r i b u t i o n of cations e l i m i n a t i n g t h e i r c o n c e n t r a t i o n g r a d i e n t s (104, 10 5). A c t i v e uptake of K* i s N a * dependent. M o r e o v e r , the effects of i n c r e a s e d K* on the m e t a b o l i s m of intact t i s s u e (e.g. i n c r e a s e d r e s p i r a t i o n ) r e q u i r e s the pr e s e n c e of N a * (104, Z18). Thus, the k i n e t i c s of b r a i n m e t a b o l i s m a r e g r e a t l y influenced by the op e r a t i o n .of the sodium-pump which c o n t r o l s the c e l l u l a r l e v e l s of N a * and K*. It i s known, for example, that i n c r e a s e d N a * s u p p r e s s e s acetate oxidation by its i n h i b i t o r y effect on acetate c o n v e r s i o n to a c e t y l CoA (198), and that i n c r e a s e d K * enhances pyruvate kinase a c t i v i t y (192, 262, 299, 300) and the oxidation of acetate (198). Skou (222, 223) r e p o r t e d that a membrane bound enzyme ( A T P a s e ) that h y d r o l y z e s A T P to A D P and P i , r e q u i r e s N a * and K * f o r a c t i v a t i o n and is dependent on the p r e s e n c e of M g + + . T h i s N a * K * - a c t i v a t e d Mg** dependent A T P a s e and the active t r a n s p o r t of N a * and K* a c r o s s the membrane, have many fea t u r e s i n common, namely, t h e i r l o c a t i o n at the c e l l membrane, their a c t i v a t i o n by the simultaneous p r e s e n c e of both N a * and K* their r e q u i r e m e n t fo r energy, and inh i b i t i o n by ouabain (104, 105, 223). 40 Ouabain i s held to act (see r e f e r e n c e s 115, 262) by inhibiting the Re- activate d dephosphorylation (Reaction 11) of an int e r m e d i a t e f o r m e d f r o m A T P and the enzyme A T P a s e (En) i n the p r e s e n c e of N a + (Reaction 10). E n + A T P + Na +- > ( E n ~ P ) Na + A D P (10) in ( E n — P ) Na- K + o u t > E n + P i + N a + + K+ (11) out i n M u c h of the c e l l u l a r energy is s a i d to involve the o p e r a t i o n of the sodium-pump (see r e f e r e n c e s 104, 224). A D P r e l e a s e d by the action of the Na*, R e a c t i v a t e d A T P a s e c o n t r o l s energy production by enhancing r e s p i r a - tion. T h i s it does through the r e a c t i o n : N A D H + A D P + P i > N A D + + A T P . However, in the p r e s e n c e of ouabain, when both cation t r a n s p o r t and the A T P a s e a c t i v i t y a s s o c i a t e d with i t are inhibited, there i s not n e c e s s a r i l y a concomitant d i m i n u t i o n in the r e s p i r a t i o n of incubated c e r e b r a l c o r t e x s l i c e s (112, 182, 224). Inhibition may be o b s e r v e d in incubations of lengthy durations (as o b s e r v e d i n our studies). In an ex p e r i m e n t of one hour there i s only a s m a l l drop in the A T P l e v e l s of r a t b r a i n c o r t e x s l i c e s i n c u - bated in p r e s e n c e of 0.1 m M ouabain (115). P r e s u m a b l y , when h i g h energy l e v e l s f a l l below a c e r t a i n l e v e l r e s p i r a t o r y i n h i b i t i o n by ouabain takes place (225). T h i s view is supported by the observations that ouabain ++ s u p p r e s s e s the s t i m u l a t e d r e s p i r a t i o n due to the absence of C a (224) or the p r e s e n c e of high (112) or on the a p p l i c a t i o n of e l e c t r i c a l i m p u l s e s (200) and in our studies in the p r e s e n c e of 5 (J.M p r o t o v e r a t r i n e ( b y 4 8 % ) . Such conditions a r e known to b r i n g about a f a l l i n the c e l l u l a r l e v e l s of high energy compounds. Tower (224) has shown that, in a medium devoid of Ca"*~+, the c a l c i u m content of cat cortex s l i c e s incubated for one hour f a l l s to 30 per cent of the c o n t r o l l e v e l s whether or not ouabain is present. In the p r e s e n c e of Ca , however, ouabain s i g n i f i c a n t l y enhances the t i s s u e l e v e l of Ca"*"*" by 36 per cent — which p r i m a r i l y o c c u r s in the m i t o c h o n d r i a l compartment. T h i s could 41 be due either to an i n c r e a s e d membrane p e r m e a b i l i t y to Ca or to an inhibi- t i o n of the active t r a n s p o r t m e c h a n i s m for the e x t r u s i o n of c a l c i u m . It is a well known fact that ouabain inhibits the active t r a n s p o r t of many compounds of b i o l o g i c a l i n t e r e s t (104, 10 5). It has been mentioned a l r e a d y that ouabain inh i b i t s the a c c u m u l a t i o n of amino ac i d s against c o n c e n t r a t i o n g r a d i e n t s (section 1.6). T h i s is so because active t r a n s p o r t of m a t e r i a l s a c r o s s membranes i s a p r o c e s s dependent on the o p e r a t i o n of the sodium-pump (104). One r e s u l t of the i n h i b i t i o n of active t r a n s p o r t by ouabain or by N a * lack, i s leakage of amino acids and other substances f r o m the b r a i n (104, 112, 280). The s o d i u m influx and the water uptake that o c c u r in b r a i n c o r t e x s l i c e s of the r a t incubated in the p r e s e n c e of 0.1 m M ouabain is p a r t l y sup- p r e s s e d by T T X (182). A concomitant r e t e n t i o n of K* may o c c u r with T T X under these conditions. In the work p r e s e n t e d in this t hesis ouabain is m a i n l y used (a) to throw li g h t on the c o n t r o l m e c h a n i s m for glutamine synthesis i ^ b r a i n cortex s l i c e s (Chapter 4); (b) to study t r a n s p o r t p r o c e s s e s of ammonia and amino acids i n i s o l a t e d b r a i n (Chapters 5 and 6); and (c) to block the re-uptake p r o c e s s of amino acids that a r e r e l e a s e d f r o m b r a i n s l i c e s incubated under conditions leading to the g e n e r a t i o n of action potentials or the a c t i v a t i o n of s o d i u m c u r r e n t at the b r a i n c e l l membrane (Chapters 7 and 8). (vi) M i s c e l l a n e o u s A d d i t i o n a l metabolic i n h i b i t o r s have also been used i n the studies r e p o r t e d in this t h e s i s . 42 (a) Malonate, w e l l known to inhibit the operation of the c i t r i c a c i d c y c l e by competition at the s u c c i n i c dehydrogenase stage (228). (b) F l u o r oacetate, which f o r m s f l u o r o c i t r ate and inhibits the o p e r a - t i o n of the c i t r i c a c i d c y c l e by i n h i b i t i o n of aconitase (63, 150). (c) Methionine sulfoximine, which inhibits the synthesis of glutamine by competitive i n h i b i t i o n of glutamine synthetase (15, 65, 146). (d) A m i n o oxyacetate, which inh i b i t s enzyme s y s t e m s r e q u i r i n g p y r i d o x a l d e r i v a t i v e s as co-enzymes. It i s used in these studies m a i n l y for s u p p r e s s i n g the c o n v e r s i o n of glutamate to aspartate by t r a n s a m i n a t i o n (147, 279. 304). (e) 2, 4-Dinitrophenol, w e l l known to uncouple oxidation f r o m phos- phorylation, and which leads to low A T P l e v e l s (115). (f) Ethane d i o x y b i s (ethylamine) t e t r a acetate ( E G T A ) , which s p e c i - f i c a l l y chelates c a l c i u m ions and is used for complete r e m o v a l of C a * * f r o m the incubation m e d i u m (306, 307). 1.10 O b j e c t i v e s of the present work The a i m of the p r e s e n t i n v e s t i g a t i o n has been to throw f u r t h e r li g h t on the p r o c e s s e s c o n t r o l l i n g ammonia f o r m a t i o n and a c c u m u l a t i o n in b r a i n and its effects on b r a i n metabolism, on the p r o c e s s e s c o n c e r n e d with amino a c i d fluxes in b r a i n under v a r i o u s conditions a s s o c i a t e d with i n c r e a s e d n e r v e activity, and on the s p e c i f i c l o c a t i o n s of amino acids in the b r a i n . 43 2. M A T E R I A L S A N D M E T H O D S 2.1 A n i m a l s A d u l t r a t s (usually male) of the W i s t a r strain, weighing 150-200 g, were used and were obtained f r o m the V i v a r i u m , Department of Zoology, or f r o m the A n i m a l Unit, F a c u l t y of Medicine, U n i v e r s i t y of B r i t i s h Columbia. In some experim e n t s 2-day old infant r a t s of the same s t r a i n were used. A l l a n i m a l s had f r e e a c c e s s to food and water. Infant r a t s were s e p a r a t e d f r o m the i r mothers before the star t of the experiment. 2.2 C h e m i c a l s A l l common l a b o r a t o r y c h e m i c a l s were of "reagent g r a d e " and were used without f u r t h e r p u r i f i c a t i o n . S o d i u m ( U - ^ C ) glutamate was obtained f r o m V o l k R a d i o c h e m i c a l Co., I l l i n o i s , U.S.A., and ^ N a C l was obtained f r o m the R a d i o c h e m i c a l Centre, A m e r sham, England. Glucose-6-phosphate dehydrogenase (E. C. 1.1.1.49) ( f r o m yeast), and hexokinase (E. C.2.7.1.1.) ( f r o m yeast) were obtained f r o m Calbiochem, L o s Angeles, C a l i f o r n i a . T e t r o d o t o x i n and sodium f l u o r oacetate were obtained f r o m Calbiochem; l i d o c a i n e (base) f r o m A s t r a P h a r m a c e u t i c a l s ; amytal f r o m E l i L i l l y Co., M o n t r e a l ; p r o t o v e r a t r i n e f r o m K&cK L a b o r a t o r i e s , Plainview, N.Y.; ouabain f r o m N u t r i t i o n a l B i o c h e m i c a l s Coporation, Cleveland, Ohio; amino o x y a c e t i c a c i d f r o m E a s t m a n Kodak Co., Rochester, N.Y.; d i s o d i u m malonate f r o m Matheson C o l e m a n and B e l l , Norwood, N.J.; E G T A f r o m K o c h and Light, Colnbrook, B u c k i n g h a m s h i r e , U.K.; D L - m e t h i o n i n e - D L - s u l f o x i m i n e f r o m C a l i f o r n i a C o p o r a t i o n for B i o c h e m i c a l R e s e a r c h , L o s Angeles; tr i e t h a n o l a m i n e and t r i s A T P f r o m S i g m a C h e m i c a l Co., St. Lo u i s , Mo., ; and N A D P + f r o m C a l b i o c h em. 44 2.3 T i s s u e p r e p a r a t i o n Rats were k i l l e d by decapitation. The b r a i n s were r e m o v e d and c e r e b r a l c o r t e x s l i c e s were p r e p a r e d using a Stadie-Riggs ti s s u e s l i c e r . In some experiments, rat kidney c o r t e x s l i c e s were p r e p a r e d in a s i m i l a r manner. One d o r s a l and one l a t e r a l b r a i n s l i c e ( f i r s t s l i c e s ) weighing a total 80-100 mg i n i t i a l wet weight and not m o re than 0.4 mm thick, were used for incubation. The s l i c e s were q u i c k l y weighed on a t o r s i o n balance (to give the i n i t i a l wet weight) and suspended i n c h i l l e d m a n o m e t r i c v e s s e l s containing the appropriate incubation media. Infant (2-day old rat) b r a i n c o r t e x s l i c e s were p r e p a r e d by cutting by hand, only s l i c e s of the t e m p o r a l - p a r i e t a l p o r t i o n of each h e m i s p h e r e being used. A s the infant b r a i n i s s m a l l , it was found expedient to use s l i c e s up t o . l mm thick. A c c o r d i n g to Itoh and Q u a s t e l (23 5), the r a t e s of oxygen con- sumption or 1 4 C 0 2 f o r m a t i o n f r o m l 4 C - l a b e l l e d substrates by infant r a t b r a i n c o r t e x s l i c e s are not s i g n i f i c a n t l y lower with t h i c k n e s s of 0.9-1 m m than those with t h i c k n e s s e s of 0.4 - 0.5 mm, which is p r o b a b l y l a r g e l y due to the fact that the rate of r e s p i r a t i o n in infant r a t b r a i n s l i c e s i s s u f f i c i e n t l y low to allow adequate oxygenation of the b r a i n c e l l s even with s l i c e s 1 mm thick (237). 2.4 M e d i a compositions and incubation p r o c e d u r e s (i) K r e b s - R i n g e r phosphate m e d i u m T h i s had the following c o m p o s i t i o n unless otherwise stated: 128 m M - N a C l ; 5 m M - KC1; 2.8 m M - C a C l 2 ; 1.3 m M - M g S 0 4 ; and 10 m M - N a 2 H P 0 4 adjusted to pH 7.4 with HC1. G l u c o s e when added was 10 mM. In a K - f r e e m e d i u m KC1 was omitted. In a Ca - f r e e m e d i u m C a C l 2 was omitted. To ensure complete absence of f r e e c a l c i u m ions in the m e d i u m during incubation, 3 mM E G T A was added when stated. V a r i o u s sub- stances were added to the incubation m e d i u m in a f i n a l volume of 3 ml. Cups 45 containing r o l l s of f i l t e r paper moistened with 0.2 ml 2 0 % K O H were p l a c e d i n the cen t r e w e l l s to absorb evolved CC" 2. The incubation was c a r r i e d out in a conventional W a r b u r g m a n o m e t r i c apparatus i n an atmosphere of 0 2 at 3 7 ° C for a p e r i o d of us u a l l y one hour. The f l a s k s were oxygenated for 5 minutes and t h e r m a l l y e q u i l i b r a t e d for 7 minutes p r i o r to the commencement of the incubation. Readings of the r a t e s of oxygen consumption were r o u t i n e l y taken to ensure that the b r a i n s l i c e s under i n v e s t i g a t i o n exhibited n o r m a l r e s p i r a t o r y a c t i v i t i e s . (ii) K r e b s - R i n g e r bicarbonate m e d i u m T h i s had the following composition: 128 m M - Na C l ; 5 m M - KC1; 2.8 m M - C a C l 2 ; 1.3 m M - M g S 0 4 ; 1.4 m M - KH-jPO^; and 28 m M - NaHCG^; i n a f i n a l volume of 3 m l . G a s s i n g was c a r r i e d out for 5 minutes either w i t h 0 2 / C 0 2 (95/5%) or N 2 / C 0 2 (95/5%) mixtures, depending on whether the incubation was c a r r i e d out a e r o b i c a l l y or a n a e r o b i c a l l y r e s p e c t i v e l y . A f t e r 7 minutes t h e r m a l e q u i l i b r a t i o n the incuba- ti o n was c a r r i e d out f o r one hour at 37°C. It should be stated that no s i g n i f i - cant d i f f e r e n c e s were found i n the amino a c i d patterns, or the cat i o n i c fluxes, of rat b r a i n c o r t e x s l i c e s incubated a e r o b i c a l l y i n a K r e b s - R i n g e r m e d i u m whether this was buffered with phosphate ions or with b i c a r b o n a t e ions. (iii) M e d i u m II T h i s m e d i u m was a Na+-rich, C a + + and K+-free m e d i u m having the foll o w i n g composition: 178 m M - Na C l ; 1.3 m M - M g S 0 4 ; 10 m M - N a 2 H P 0 4 adjusted to p H 7.4 with HC1. 46 2. 5 A m i n o a c i d a n a l y z e r e s t i m a t i o n s (a) Sample p r e p a r a t i o n (i) T i s s u e sample p r e p a r a t i o n A t the end of the p e r i o d of incubation, the Warburg m a n o m e t r i c v e s s e l s were p l a c e d i n ice, the t i s s u e q u i c k l y removed, and adhering m e d i u m d r a i n e d off by blotting with f i l t e r paper. The s l i c e s were q u i c k l y weighed (to give the f i n a l wet weight) and then homogenized in 3 m l of 5% T C A . The debr i s , separated f r o m the supernatant by c e n t r i f u g a t i o n and decantation, was washed with 5 % TCA, re c e n t r i f u g e d , and the washings added to the super- natant. The supernatant was then e x t r a c t e d three t i m e s with equal v o l u m e s of d i e t h y l ether to remove T C A . T r a c e s of ether l e f t behind were evaporated by c a r e f u l l y blowing i n ni t r o g e n gas (bubbled through NH^SO^ to ensure com- plete absence of p o s s i b l e contaminating ammonia) and the samples were made up to a known volume (4-6 ml). (ii) M e d i u m sample p r e p a r a t i o n A t the end of the incubation 2.0 or 2.5 m l of the m e d i u m were mixed with 2 or 2.5 m l of 1 0 % T C A and centrifuged. The supernatant was e x t r a c t e d with d i e t h y l ether, the ether r e m o v e d as d e s c r i b e d e a r l i e r , and samples were made up to known volumes (4-6 ml). (b) Sample a n a l y s i s A m i n o acids present i n aliquots (1-2 ml) of the samples were separated on a p p r o p r i a t e columns of a B e c k m a n 120B, A m i n o A c i d A n a l y z e r , operated as d e s c r i b e d i n the B e c k m a n manual. The a c c e l e r a t e d method was used to separate the amino acids which were detected by the n i n h y d r i n r e a c t i o n . 47 (i) A c i d i c and n e u t r a l amino a c i d a n a l y s e s The amino acids, taurine, a s p a r t i c acid, threonine, glutamine and serine, g l u t a m i c acid, glycine, and alanine, were separated on a 50 x 0.9 c m column of sulfonated p o l y s t y r e n e — 8 % d i v i n y l benzene copolymer ion exchange r e s i n (Type 50A - p a r t i c l e size 25-31 \i) at 5 0 ° C by elution with 0.067 M s o d i u m buffer pH 3.28. Under these conditions glutamine, asparagine and serine co- elute. The f r e e asparagine pool in b r a i n is v e r y s m a l l (0.1 - 0.2 pmole/g i n i t i a l wet wt) when c o m p a r e d with those of glutamine and serine (22, 88, 239), and was not estimated. V a l u e s for s e r i n e were obtained in a number of e x p e r i m e n t s by the following procedure. The sample containing glutamine and s e r i n e was heated with 1 0 % T C A at 7 5 ° C for 75 minutes (22). T h i s b r i n g s about h y d r o l y s i s of the glutamine while the s e r i n e i s unaffected. T C A is r e m o v e d with diethy l ether and the sample p r e p a r e d as d e s c r i b e d e a r l i e r . Glutamine values a r e obtained by estimating either (1) the d i f f e r e n c e between the a r e a s of the peaks, at the g l u t a m i n e - s e r i n e p o s i t i o n of the amino a c i d p r o f i l e s , obtained before and after h y d r o l y s i s , or (2) the i n c r e a s e in the a r e a of the ammonia peak. It should be mentioned that glutamine h y d r o l y z e d by this method y i e l d s ammonia and p y r r o l i d o n e c a r b o x y l i c a c i d . A s m a l l p r o p o r t i o n of this c y c l i c compound i s f u r t h e r h y d r o l y z e d to g l u t a m i c a c i d (238). Thus, we find that about 22 per cent of the glutamine h y d r o l y z e d appears as glutamic a c i d under these c o n d i - tions. T h i s is in a c c o r d with the r e s u l t s of Dobkin and M a r t i n (236). (ii) y - A m i n o b u t y r i c a c i d and ammonia A n aliquot (usually 1 ml) of the sample was eluted with 0.127 M s o d i u m c i t r a t e buffer pH 4.26 at 5 0 ° C f r o m a 5.5 x 0 . 9 c m column of sulfonated p o l y s t y r e n e - 8 % d i v i n y l benzene copolymer (Beckman r e s i n , Type 15A - p a r t i c l e s i z e 19-25 u). The advantage of this method for a mmonium assa y is the s e p a r a t i o n of l a b i l e ammonia producing components, l i k e glutamine, f r o m f r e e ammonia in the sample. T h e r e i s also tot a l r e c o v e r y of ammonia f r o m the column on elution with buffer r e s u l t i n g in g r e a t e r a c c u r a c y i n the 48 ammonia e s t i m a t i o n . (iii) N - A c e t y l a s p a r t a t e T h i s amino a c i d d e r i v a t i v e is m e a s u r e d as aspartate after h y d r o l y s i s with 0.5 volume of 5.5 NHC1 at 100°C for 30 minutes, followed by r e m o v a l of HC1. Under these conditions, glutamine i s r e c o v e r e d as glutamate and ammonia. The amino acids, taurine,aspartate, glutamate,glycine and alanine, were unaffected by this p r o c e d u r e . HC1 i s r e m o v e d by evaporating the sample to dr y n e s s . The d r i e d sample i s d i s s o l v e d in 0.067 M s o d i u m c i t r a t e buffer pH 2.2, and ana l y z e d with the A m i n o A c i d A n a l y z e r . (iv) C a l c u l a t i o n s The a r e a s of the amino a c i d peaks were m e a s u r e d by the height-width method. H e r e the height of the peak was m u l t i p l i e d by the width which is mea- s u r e d at half height. The width of the peak was m e a s u r e d by counting the num- ber of dots p r i n t e d above the half height of the peak. The constant (i.e., a r e a of the peak) per (jmole for each amino a c i d was obtained f r o m an amino a c i d p r o f i l e of a standard amino a c i d m i x t u r e . F r o m these values the c o n c e n t r a - t i o n of each amino a c i d was computed. The constants and peak po s i t i o n s were i n v a r i a b l y d e t e r m i n e d for each new batch of buffer or n i n h y d r i n reagent p r e p a r e d . A f t e r the e s t i m a t i o n of amino acids i n a sample was completed, the 50 c m column (and sometimes the short column) was r e g e n e r a t e d with 0.2 N N a O H and e q u i l i b r a t e d with the a p p r o p r i a t e buffer b e f o r e r e u s e . 2.6 Water uptake by b r a i n c o r t e x s l i c e s I mmediately after incubation the s l i c e s were r e m o v e d f r o m the m e d i a and d r a i n e d . They were sp r e a d without folding on an i c e - c o l d c l e a n g l a s s s u r f a c e and excess of f l u i d around the s l i c e s and on the g l a s s was abs o r b e d 49 with s t r i p s of f i l t e r paper. The d i f f e r e n c e between the f i n a l wet weight and the i n i t i a l wet weight gives a m e a s u r e of the i n c r e a s e of water uptake. The total water content of the t i s s u e is equal to the sum of the water taken up by the t i s s u e during the p e r i o d of incubation and the amount of water o r i g i n a l l y p r e s e n t (section Z.8). 2.7 Oxygen uptake by b r a i n c o r t e x s l i c e s Oxygen uptakes ( Q Q 2 w e r e c a l c u l a t e d as given in " M a n o m e t r i c T e c h n i q u e s " (240) in t e r m s of n l/mg d r y wt tissue, and c o n v e r t e d to ^imole/g i n i t i a l wet wt on m u l t i p l i c a t i o n by a f a c t o r which for the adult r a t was 200/22.4 and for the infant r a t 120/22.4. 2.8 D r y weight of b r a i n cortex s l i c e s Weighed cortex s l i c e s were d r i e d at 1 1 0 ° C to constant weights. F o r the adult rat, d r y wt = 2 0 % wet wt; f o r 2-day old infant rat, d r y wt = 1 2 % wet wt. with a standard deviation ̂  + 5%. 22 + 2.9 N a influx into b r a i n cortex s l i c e s . In e xperiments on ^2]sj a+ fluxes, 0.5 \ld of 2 2^ a+ w a s p l a c e d i n the m a i n compartment of the W a r b u r g m a n o m e t r i c v e s s e l with 3 m l of the incuba- ti o n medium. Sodium ion c o n c e n t r a t i o n in the m e d i u m was u s u a l l y 148 mequiv/1 unless otherwise stated. A f t e r the incubation, the b r a i n s l i c e s were q u i c k l y removed, l i g h t l y blotted with s t r i p s of f i l t e r paper to r e m o v e adhering fluid, weighed to estimate swelling, and homogenized in 3 m l of 5 % T C A . A f t e r standing in the c o l d for two hours with o c c a s i o n a l mixing, the homogenate was centrifuged. 22 + F o r the e s t i m a t i o n of r a d i o a c t i v i t y due to Na in the m e d i u m at the 50 end of the incubation period, 0.4 m l m e d i u m was mixed with 1.6 m l 5% T C A and centrifuged. 0.5 m l aliquots of the T C A e x t r a c t s of tissue or m e d i u m was m i x e d with 10 m l s c i n t i l l a t i o n l i q u i d and a s s a y e d for 22j^ a+ using the a p p r o p r i a t e settings on a N u c l e a r Chicago M o d e l M a r k I l i q u i d s c i n t i l l a t i o n counter. The value of counts per minute (cpm) was converted to that of d i s i n t e g r a t i o n s per minute (dpm) by m u l t i p l i c a t i o n with the factor 100/percentage e f f i c i e n c y of counting. P e r c e n t a g e e f f i c i e n c y was obtained by the channel r a t i o method and 22 + was g e n e r a l l y about 30 per cent for Na . 7 7 + The c o n c e n t r a t i o n of Na i n the t i s s u e s l i c e was c a l c u l a t e d f r o m the r a d i o a c t i v i t y found in unit weight of the t i s s u e s l i c e (dpm/g) d i v i d e d by the 7 7 4" s p e c i f i c r a d i o a c t i v i t y of ^ N a i n the incubation m e d i u m (dpm/H equiv sodium in 1 m l incubation medium). 2.10 E x p e r i m e n t s with s o d i um L - [ U - 1 4 C ] glutamate In e x p e r i m e n t s with l a b e l l e d glutamate, L . - f U - ^ C ] glutamate of speci- f i c a c t i v i t y 0.067 nCi/^imole (or 122,300 cpm/i_imole) and c o n c e n t r a t i o n of 5 m M was p l a c e d in the m a i n compartment of Warburg m a n o m e t r i c v e s s e l s containing 3 m l incubation medium. A t the end of the incubation period, the b r a i n s l i c e s were r e m o v e d and counting was c a r r i e d out on the T C A e x t r a c t s of ti s s u e and m e d i u m as mentioned e a r l i e r i n section 2.9. Counting e f f i c i e n c y 14 f o r C was found to be 83 per cent. 2.11 S c i n t i l l a t i o n l i q u i d c o m p o s i t i o n T h i s c o n s i s t e d of a m i x t u r e of equal volumes of toluene, dioxane and 9 5 % (v/v) ethanol containing 2, 5 diphenyloxazole (5g/l), 1, 4 - b i s - ( 4 - m e t h y l - 5-phenyloxazol-2-yl)-benzene (0.05 g/1) and napthalene (80 g/1). 51 2.12 Na and K assay by f lame-photometry The t i s s u e contents of u n l a b e l l e d N a * and K* were d e t e r m i n e d as f o l l o w s . B r a i n cortex s l i c e s , either i n i t i a l l y or at the end of the incubation period, were homogenized in 3 m l 5% T C A after r e m o v i n g adhering f l u i d by s t r i p s of f i l t e r paper and weighing to d e t e r m i n e the extent of water uptake. A f t e r standing in the c o l d for two h o u r s the homogenates were c e n t r i f u g e d and the supernatants were di l u t e d with d i s t i l l e d water to give f i n a l c ation c o n c e n t r a t i o n s of (0.05 - 0.1 p. equiv/ml). The atomic a b s o r p t i o n s (% absorbance) were m e a s u r e d at 294.3 and 383.3 n m f o r N a * and K* r e s p e c - t i v e l y , with a P e r k i n - E l m e r model 303 atomic a b s o r p t i o n spectrophotometer. The cation concentrations were computed f r o m standard plots of known con- c e n t r a t i o n s of N a * and K* v e r s u s Absorbance, c a r r i e d out s i m u l t a n e o u s l y with each set of determinations. G e n e r a l l y good agreement was found between N a * contents of a sample obtained using the flame photometer and 22 that obtained by m e a s u r i n g N a (also 182). 2.13 D e t e r m i n a t i o n of A T P c o n c e n t r a t i o n s A T P was d e t e r m i n e d a c c o r d i n g to the method of G r e e n g a r d (241). The t i s s u e s l i c e s at the t e r m i n a t i o n of the experiment were ex t r a c t e d with 2 m l 6% (w/v) p e r c h l o r i c a c i d and c e n t r i f u g e d . One m l of the supernatant was n e u t r a l i z e d with K 2 C O 3 and made up to 4 ml. 0.3 m l aliquots were taken f o r each a s s a y i n a f i n a l volume of 2 ml containing 0.215 mmole glucose; 9.4 n mole N A D P + ; 3 (jmole M g C l 2 ; 1.2 p mole E D T A (pH 7.4); 24 pmole tr i e t h a n o l - amine buffer (pH 8.0); 0.5 units hexokinase; and 1 unit glucose-6-phosphate dehydrogenase. Incubation was c a r r i e d out at r o o m temperature for 30 minutes. N A D P H was m e a s u r e d with an A minco-Bowman s p e c t r o f l u o r i m e t e r with e x c i t a t i o n wavelength 365 n m and f l u o r e s c e n c e wavelength 460 n m. One unit of hexokinase was that amount of enzyme ca t a l y z i n g the c o n v e r s i o n of 1 p mole of glucose per minute at pH 8.5 at 25°C. One unit of glucose-6-phosphate 52 dehydrogenase was that amount of enzyme ca t a l y z i n g the r e d u c t i o n of 1 n m o le of N A D P + per minute at p H 7.4 at 25°C. 2.14 A s s a y of Na"*", K ^ - A T P a s e of b r a i n homogenates (i) M e d i u m comp o s i t i o n The m e d i u m used for the a s s a y of N a * K + - A T P a s e had the following composition; 0.1 m M - d i s o d i u m E D T A ; 1 m M - M g C ^ ; 58 m M - N a C l ; 15 m M - KC1; 3 m M - t r i s A T P ; 95 m M - t r i s - C l (pH 7.6); whenever the c o n c e n t r a t i o n of Na"*" or K"*" was changed, the t o n i c i t y of the m e d i u m was maintained by a l t e r i n g an equivalent amount of t r i s - C l in the medium. (ii) Incubation p r o c e d u r e Incubation was c a r r i e d out i n centrifuge tubes, each containing 3 m l of the medium, with an amount of b r a i n c o r t i c a l homogenate i n c o l d d i s t i l l e d water equivalent to 1 mg of the o r i g i n a l wet t i s s u e - c o r t e x s l i c e . The tubes were shaken in a water bath shaker at 3 7 ° C for one hour. (iii) E s t i m a t i o n Na*, K * - A T P a s e a c t i v i t y was m e a s u r e d in t e r m s of the amount of phosphate produced by the h y d r o l y s i s of A T P by the enzyme p r e p a r a t i o n . The h y d r o l y s i s was brought about by two components: (1) a M g * * - a c t i v a t e d A T P a s e ; (2) a N a * K + - s t i mulated A T P a s e r e q u i r i n g Mg"*"*" for a c t i v i t y . The incubation was t e r m i n a t e d by the addition of 0.2 m l 1 0 0 % (w/v) TCA, and the m i x t u r e was centrifuged. To 0.5 m l of the supernatant was added 2.0 m l d i s t i l l e d water and 2.5 ml of a colour reagent p r e p a r e d by d i s s o l v i n g 4.0 g FeSC>4 * n 100 m l of 1 % a mmonium molybdate in 1.15 N H 2 S O 4 a c c o r d i n g to the method of Bonting et a l . (242). The r e s u l t i n g blue colour was r e a d within 2 h o u r s at 700 n m on a B e c k m a n M o d e l D U spectrophotometer. Substrate and enzyme blanks were in c l u d e d in each determination, together with phos- 53 phate standards to convert o p t i c a l density to (Jmole P i (phosphate) r e l e a s e d by one mg i n i t i a l wet wt t i s s u e / h o u r at 37°C. The Na +, K + - A T P a s e a c t i v i t y was e s t i m a t e d as the d i f f e r e n c e between the a c t i v i t y of b r a i n homogenate prep a r a - • i 4-4- tion incubated in the p r e s e n c e of Na"*", K and Mg and that incubated i n the absence of N a + and K + . In the following text, only mean values f o r P i l i b e r a t i o n a r e given, since the deviations f r o m the mean were never g r e a t e r than + 5 per cent. 2.15 Isolation of synaptosomes f r o m r a t b r a i n c o r t e x Synaptosomes (or pinched-off n e r v e t e r m i n a l s ) were i s o l a t e d e s s e n - t i a l l y by the method of G r a y and "Whittaker (243). B r a i n c o r t e x of the r a t was homogenized i n 0.32 M s u c r o s e ( 1 0 % w/v), in a teflon h o mogenizer r e v o l v i n g at 1725 r p m (5 up-down strokes), and c e n t r i f u g e d at 1,000 g for 10 minutes. The crude n u c l e a r p e l l e t separated f r o m the supernatant-Sp was washed twice by r e s u s p e n s i o n i n 0.32 M s u c r o s e and r e c e n t r i f u g e d as b e f o r e . The p e l l e t - ~Pi obtained in the f i n a l washing containing l a r g e m y e l i n fragments, n u c l e i and c e l l d e b r i s , was d i s c a r d e d . The washings were m i x e d with super- natant-S i and c e n t r i f u g e d at 17,000 g f o r one hour. The supernatant-S2 containing m i c r o s o m e s and soluble c e l l constituents, was d i s c a r d e d . The crude m i t o c h o n d r i a l p e l l e t - P 2 (containing s m a l l m y e l i n and membrane frag - ments, m i t o c h o n d r i a and synaptosomes) was suspended in 0.32 M s u c r o s e and c e n t r i f u g e d at 100, 000 g for 45 minutes into a density gradient c o n s i s t i n g of equal volumes of 0.8 M and 1.2 M s u c r o s e . The following f r a c t i o n s were obtained. A. A f r a c t i o n floating between 0.32 and 0.8 M s u c r o s e ( s m a l l m y e l i n fragments). T h i s was d i s c a r d e d . B. A f r a c t i o n f l o a t i n g between 0.8 and 1.2 M s u c r o s e ( S Y N A P T O S O M E S ) . T h i s f r a c t i o n was c a r e f u l l y r e m o v e d with a pasteur pipette d i l u t e d and c e n t r i f u g e d in i s o t o n i c s u c r o s e (0.32 M). The p e l l e t obtained 54 was suspended i n i s o t o n i c K r e b s -Ringer phosphate m e d i u m and used. C. A pellet, below 1.2 M sucrose, containing m i t o c h o n d r i a was d i s c a r d e d . The incubation p r o c e d u r e f o r synaptosom.es was the same as that d e s c r i b e d for b r a i n c o r t e x s l i c e s (section 2.4 (i) ). However, the W a r b u r g m a n o m e t r i c v e s s e l s were shaken v e r y slowly to maintain the i n t e g r i t y of the synaptosome. A t the end of the incubation period, T C A was added to give a f i n a l c o n c e n t r a t i o n of 5 per cent, and samples were p r e p a r e d for amino a c i d a n a l y s i s as d e s c r i b e d i n section 2.5. P r i o r to incubation, p r o t e i n was measured, in an aliquot of the synaptosomal suspension used for incubation, as f o l l o w s . 2.16 P r o t e i n e s t i m a t i o n The method of L o w r y et a l . (249) using the F o l i n - C i o c a l t e u reagent (2 50) was employed f o r e s t i m a t i n g protein, the blue c o l o r f o r m e d being r e a d at 750 n m on a B e c k m a n M o d e l D U Spectrophotometer. Bovine albumin was used as a standard. 2.17 R e p r o d u c i b i l i t y of r e s u l t s E a c h e x p e r i m e n t was c a r r i e d out at l e a s t four t i m e s and the r e s u l t s found to be r e p r o d u c i b l e . C o n t r o l s were always ru n simultaneously. In our work r e s u l t s are e x p r e s s e d as means of the values obtained and the standard deviations f r o m the mean are given. 55 2.18 E x p l a n a t i o n of v a r i o u s t e r m s used i n this t h e s i s (i) I n i t i a l values " I n i t i a l v a l u e s " are the values (of NĤ "*", Na"**, K"*~ or amino acids) obtained p r i o r to incubation. (ii) T o t a l values The t e r m " t o t a l " has been used as follows: (a) T o t a l values (i.e., those of t i s s u e + medium) are the sums of the tis s u e and m e d i u m concentrations of a p a r t i c u l a r substance (e.g., a mmonia or amino acid) e x p r e s s e d as pmole/g i n i t i a l wet wt. (b) Sometimes it has been used to e x p r e s s the s u m of a number of constituents i n a given t i s s u e or medium; e.g., total t i s s u e N H -N + amino acid-N, or total t i s s u e Na+ + K+ contents. These 4 e x p r e s s i o n s of total values are made c l e a r i n the text. (iii) A d j u s t m e n t "Adjustment" i s a t e r m used to signify a change made to maintain the i s o t o n i c i t y of the incubation medium. F o r example, (a) when substances i n the f o r m of the i r s o d i u m salts (e.g., s o d i u m L-glutamate) are added to the incubation medium, the m e d i u m N a * ion c o n c e n t r a t i o n i s maintained at 148 p equiv per m l by app r o p r i a t e r e d u c t i o n s in'the m e dium N a C l ; (b) when i n c r e a s i n g concentrations of KC1 are added to the medium, the medium i s "ad j u s t e d " by an equivalent r e d u c t i o n in the N a C l content, thus maintaining the total m e d ium (Na"1" + K + ) ion concentration at 153 pequiv per ml. 56 (iv) A m m o n i a and ammonium ion Since the pKa for the r e a c t i o n N H 4 + ^ = ^ N H 3 + H + i s 9.3 at p h y s i o l o g i c a l pH (7.4) at which our experiments are c a r r i e d out, a l a r g e p r o p o r t i o n (97%) of the total ( N H 3 + NĤ "*") i s p r e s e n t i n the f o r m of the a m m o n i um ion (278). When the t e r m s "ammonia" and "ammonium i o n s " are used in the following text, i t is understood that the f o r m e r t e r m r e p r e s e n t s a m m o n i u m ions. (v) F l u x The t e r m s " i n f l u x " and "efflux", i n this thesis, are used to de s c r i b e , r e s p e c t i v e l y , the "net" i n w a r d or outward movement of a substance f r o m the ti s s u e i n a given i n t e r v a l of time. (vi) A c t i o n potentials The "generation of act i o n p o t e n t i a l s " i s used interchangeably with the 'activation of the N a ^ - c u r r e n t system". It i s p r e s u m e d to o c c u r i n b r a i n c o r t e x s l i c e s under c e r t a i n conditions of incubation and is i d e n t i f i e d by sensi- t i v i t y to T T X (section 1.8 (viii) ). 57 3. A M M O N I A F O R M A T I O N IN B R A I N IN V I T R O 3.1 A m m o n i a and amino a c i d contents of ra t c e r e b r a l cortex s l i c e s i n i t i a l l y , and after incubation i n the presence or absence of glucose V a l u e s of the contents of amino acids i n r a t b r a i n c o r t e x s l i c e s , f r e s h l y p r e p a r e d and im m e d i a t e l y p r i o r to incubation, a r e given in Table 2. The s e values a r e i n agreement with those a l r e a d y r e c o r d e d i n the l i t e r a t u r e (125-131). T o t a l (tissue + medium) values for ammonia and amino a c i d s found on incubating b r a i n s l i c e s in K r e b s - R i n g e r phosphate m e d i u m in O^ at 3 7 ° C for v a r i o u s p e r i o d s of time, with or without the addition of 10 m M glucose, are als o r e c o r d e d in T a b l e 2. The following c o n c l u s i o n s may be made. (i) Changes i n the N H 4 * and amino a c i d contents of b r a i n s l i c e s incubated i n the p r e s e n c e and absence of gl u c o s e . The f o r m a t i o n of (16.81 - 6.33) = 10.48 pmole ammonia/g t i s s u e i s su p p r e s s e d i n the p r e s e n c e of glucose i n a one hour incubation p e r i o d . T h i s s u p p r e s s i o n by glucose i s r e f l e c t e d (1) i n an enhanced glutamate content, viz., (10.11 - 3.55) = 6.56 pmole/g, and (2) in an enhanced glutamine content, viz., (6.35 - 1.79) = 4.56 pmole/g ( c o r r e s p o n d i n g to 9-12 patom amino acid-N/g) which together gives an i n c r e a s e of 15.68 p a t o m amino acid-N/g. On sub- t r a c t i n g f r o m this value, the value due to a d i m i n i s h e d aspartate l e v e l , viz., (9.13 - 4.14) = 4.99, a value of 10.69 p a t o m amino acid-N/g is obtained. T h i s completely accounts f o r the s u p p r e s s i o n of ammonia production by glucose. T h e r e are r e l a t i v e l y s m a l l changes in the l e v e l s of other amino acids under these conditions. T h e r e f o r e , it seems that the f a l l i n the l e v e l of N H ^ + due to glucose i s about equal to the r i s e i n glutamate plus the r i s e in glutamine minus the f a l l i n aspartate l e v e l s . A s i m i l a r c o n c l u s i o n may be drawn on c o n s i d e r i n g the ammonia, glutamate, glutamine and aspartate l e v e l s of b r a i n c o r t e x s l i c e s incubated for four hours. These c a l c u l a t i o n s are given in T a b l e 2A. T A B L E 2. Ammonia and amino acid changes in adult rat brain cortex slices on incubation in the presence or absence of glucose. Rat brain cortex slices were incubated in Krebs-Ringer phosphate medium with or without glucose (lOmM) in at 3 7 ° C for varying periods of t ime. Initial values ( i .e . values prior to incubation) of ammonia and amino acids and the total (tissue + medium) values on incubation are expressed as umole/g initial wet wt. INITIAL I N C U B A T I O N P E R I O D 15 min. 30 min. One hour Four hours No Glucose No Glucose No Glucose Glucose No Glucose Glucose Glutamate 11.83 1 . 0 9 8.10 + 0.06 5.58 + 0. 18 3.55 + 0.17 10.11 + 0.36 2.57 + 0.24 6.44 + 0. 51 Glutamine 4.40 + 0.15 3.47 + 0.10 2.07 + 0.15 1.79 + 0.20 6.35 + 0.18 2.10 + 0.20 11.40 + 0. 50 G A B A 2.01 + 0.10 2.56 + 0.26 2.95 + 0.07 2.32 + 0.32 2.81 + 0.08 1.40 + 0.15 1.75 + 0. 18 Aspartate 3.36 + 0.34 8.14 + 0.10 8.70 + 0.05 9.13 + 0.47 4.14 + 0.46 8.47 + 0.54 2.49 0. 14 Alanine 0.61 + 0.08 0.86 + 0.04 0 . 9 0 + 0.05 0.73 + 0.05 1 . 2 9 + 0.01 1.67 + 0. 10 1.04 + 0. 10 Glycine 0.79 + 0.06 1.00 + 0.03 1. 13 + 0.02 1.47 + 0.03 1.53 + 0.11 2.15 + 0.19 1.68 + 0. 15 Sc rine 1.25 + 0.05 1.55 + 0 . 0 9 1.88 + 0.14 2.23 + 0.03 1.82 + 0.08 3.32 + 0 . 1 9 2.27 + 0. 16 Threonine 0.52 + 0.11 0.74 + 0.01 0.89 + 0.03 0.95 + 0.05 0.67 + 0.04 1.51 + 0.15 1. 10 + 0.20 Taurine 5.81 + 0.71 5.62 + 0.28 6.02 + 0.13 5.57 + 0.39 5.44 + 0.24 6.13 + 0.50 5.72 + 0 . 2 9 Lysine 0.2 0.25 0.25 0.35 + 0.11 0.25 + 0.03 1.50 + 0.30 0.80 + 0. 10 Ammonia 1.58 + 0.14 8.32 + 0.28 11.90 + 0.40 16.81 + 0.07 6.33 + 0.45 24.20 + 1.26 6. 82 + 0.45 Total : N H 4 + - N + amino acid - N 36.76 44.08 44.28 46.69 47.09 57.12 52.91 T A B L E 2A. Changes in the glutamate, glutamine, aspartate and ammonia contents of bra in cortex slices incubated in the presence or absence of glucose. Total (tissue+medium) values of ammonia and amino acids (pmole /g initial wet wt.) of bra in cortex slices used for these calculations were taken f r o m T A B L E 2. One Hour Four Hour No Glucose Glucose A - N H 2 No Glucose Glucose A - N H z Glutamate 3.55 10. 11 -6 . 56 2.57 6.44 -3 . 87 Glutamine 1. 79 6.35 -9 . 12 2. 10 11.40 -18. 60 Aspartate 9.13 4. 14 +4.99 8.47 2.49 +5.98 A mmonia 16.81 6.33 +10.48 24.20 6. 82 + 17.23 Tota l - N H 2 33.07 33.28 -0. 21 39.44 38.55 +0.89 60 F r o m the above c o n s i d e r a t i o n s , it i s evident that much of the ammonia turn o v e r i n incubated b r a i n s l i c e s i s due to changes in b r a i n amino ac i d s . (ii) Changes in the i n i t i a l N H ^ + and amino a c i d contents of b r a i n c o r t e x s l i c e s on incubation. In the absence of glucose, the r i s e i n ammonia f r o m b r a i n s l i c e s incu- bated f o r one hour is accompanied by s i g n i f i c a n t f a l l s i n the glutamate and glutamine l e v e l s f r o m t h e i r i n i t i a l values. T h e r e i s an accompanying r i s e in the l e v e l of aspartate. Thus, i n the absence of glucose there is a l i b e r a t i o n of (16.81 - 1.58) = 15.23 pmole ammonia/g t i s s u e / h o u r . Concomitantly, g l u t a - mate l e v e l s f a l l by (11.83 - 3.55) = 8.28 pmole/g, and glutamine f a l l s by (4.40 - 1.79) = 2.61 |jmole/g. The d i m i n u t i o n i n the l e v e l s of these amino a c i d s could account for about 90 per cent (or 13.5 patoms-N/g) of the tot a l a mmonia l i b e r a t e d . However, it i s probable that some of the aspartate formed, i.e., (9.13 - 3.36) = 5.77 pmole/g i s d e r i v e d f r o m glutamate by t r a n s a m i n a t i o n . It i s t h e r e f o r e evident that at l e a s t (13.50 - 5.77) = 7.73 pmole/g, i.e., 50 per cent, of the tot a l ammonia li b e r a t e d , is accounted for by breakdown of g l u t a - mate and glutamine in the b r a i n t i s s u e (assuming no p r o t e i n breakdown takes place). In the absence of glucose, ammonia is continuously being l i b e r a t e d , though it i s evident that the r a t e of its f o r m a t i o n d i m i n i s h e s with the time of incubation. F o r example, l e s s than 8 |imole ammonia/g i n i t i a l wet wt t i s s u e i s l i b e r a t e d i n the three hours incubation subsequent to the f i r s t hour incubation, c o m p a r e d to 15|jmole/g in the f i r s t hour. T h e r e seems to be a d i r e c t r e l a t i o n - ship between the rate of ammonia f o r m a t i o n and the r a t e of oxygen consumption- The rat e of the l i b e r a t i o n of ammonia by rat b r a i n c o r t e x s l i c e s incubated i n a g l u c o s e - f r e e m e d i u m p a r a l l e l s the rate of i t s oxygen consumption. While the rate of oxygen consumption by the s l i c e s is a l m o s t constant in the p r e s e n c e of 10 m M glucose, even at the end of a four hour incubation period, the rate f a l l s with time in the absence of glucose (Table 15). The drop in the rate of oxygen 61 uptake in a glucose-free medium probably coincides with the fall in the levels of endogenous substrates, including such substrates as glutamate, capable of liberating ammonia on oxidation. In the presence of glucose, there is only a small diminution in the init ial level of glutamate of brain slices incubated for one hour, but the content of glutamine is increased. In a four hour incubation period, the rate of endo- genous glutamate oxidation in brain tissue is diminished in the presence of glucose and ammonia is utilized in the synthesis of glutamine. Glucose maintains high levels of glutamate and glutamine in incubated slices by generating a -ketoglutar ate (315) which forms glutamate, either by reductive amination with NH4*, or by transamination with amino acids such as aspartate. Moreover , glucose oxidation generates N A D H , which favors reductive amina- tion of CY -ketoglutar ate over the reverse reaction of glutamate oxidation. Fur thermore , glucose generates A T P required for glutamine synthesis. These facts account for the inhibitory effect of glucose on ammonia formation in brain slices during incubation. (iii) Possible protein breakdown in incubating brain s l ices . In order to obtain information concerning the kinetics of the r ise in aspartate level accompanying the fall in glutamate level, rat brain cortex slices were incubated aerobically at 3 7 ° C in K r e b s - R i n g e r phosphate glucose- free medium for 15 minutes and 30 minutes. Results of the total (tissue + medium) ammonia and amino acid changes are given in Table 2. F r o m this table it may be calculated that 83 per cent (or 4.78 (imole/g) of the total r i se in the aspartate level occurring in one hour, takes place in the f i rs t 15 minutes, and 92 per cent (or 5.24 |jmole/g) at the end of 30 minutes. Only 43 per cent (or 3.73 (imole/g) of the total diminution in the level of glutamate, that is obtained in one hour, occurs in the f i rs t 15 minutes and 73 per cent (or 6.25 (-imole/g) at the end of 30 minutes incubation. These and other results in Table 2 indicate that, if the bulk of the aspartate is derived f r o m endogenous glutamate, by transamination with oxaloacetate, then under these conditions 62 glutamate f r o m some p r e c u r s o r s o u r c e (possibly proteins) d i r e c t l y and/or following glutamine h y d r o l y s i s , enters i t s i n i t i a l f r e e pool, undergoes oxida- ti o n and l i b e r a t e s ammonia in the p r o c e s s . P o s s i b l y , some aspartate may appear as a r e s u l t of p r o t e i n breakdown. That p r o t e i n breakdown may indeed o c c u r i n incubating b r a i n s l i c e s i s i m p l i c a t e d by the continuous r i s e i n the l e v e l s of c e r t a i n amino acids such as glycine, serine, threonine or l y s i n e with i n c r e a s i n g t i m e s of incubation (0 to 4h). It i s to be noted, however, that the l e v e l of glutamate (and glutamine) continues to f a l l with time of incubation i n d i c a t i n g its (glutamate) continuous oxidation. The value of the total amino acid-N, i n c l u d i n g the value f o r ammonia, is enhanced f r o m the i n i t i a l l e v e l by about 10 patoms/g i n i t i a l wet wt on incu- bation for one hour, either i n the presence or absence of glucose. T h i s p o s s i b l y r e f l e c t s c e r e b r a l p r o t e i n breakdown on incubation. It i s to be noted that the l e v e l of taurine (an amino a c i d d e r i v a t i v e not a constituent of pr o t e i n s ) r e m a i n s , within e x p e r i m e n t a l e r r o r , unchanged during incubation of b r a i n s l i c e s i n the presence or absence of glucose, even i n four hour incubations. I n c r e a s e s i n the amino a c i d content of incubated b r a i n t i s s u e are in a c c o r d with the o b s e r v a t i o n s made by W e i l - M a l h e r b e and Go r d o n (102), and Jones and M c l l w a i n (267). It should be mentioned that the i n i t i a l l e v e l of N - a c e t y l aspartate (5.72 + 0.24 pmole/g) in r a t b r a i n cortex s l i c e s i s unaffected by incubation i n the p r e s e n c e or absence of glucose, which i s i n agreement with many r e s u l t s r e p o r t e d in the l i t e r a t u r e (247, 248, 291). Th e r e f o r e , the r i s e in the aspartate content of b r a i n s l i c e s incubated i n a g l u c o s e - f r e e m e d i u m is not due to a f a l l i n the N - a c e t y l aspartate content of the t i s s u e . However, this view is opposed to that of Buniatian et al., (273) who obtained a d i m i n u t i o n i n N - a c e t y l aspartate of b r a i n ti s s u e incubated i n the absence of glucose. These studies lend support to the view that the bulk of the ammonia f o r m e d i n b r a i n cortex s l i c e s incubated i n g l u c o s e - f r e e m edia o c c u r s f r o m 63 glutamine and by t e r m i n a l glutamate oxidation. 3.2 A m m o n i a f o r m a t i o n in anoxia. The tota l (tissue + medium) ammonia and amino a c i d contents of rat b r a i n c o r t e x s l i c e s incubated at 3 7 ° C for one hour i n K r e b s - R i n g e r b i c a r b o n - ate m e d i u m with or without the addition of 10 m M glucose i n an atmosphere of N2/CO2 mixture ( 9 5 % / 5 % ) , are g i v e n i n Table 3. F r o m these r e s u l t s it i s seen that the rate of ammonia f o r m a t i o n in a g l u c o s e - f r e e m e d i u m i s s u p p r e s s e d i n the absence of oxygen (compared with values obtained on incubation i n the pr e s e n c e of oxygen - T a b l e 2). T h i s is i n a c c o r d with e a r l i e r findings (43, 244). Such r e s u l t s support our view (62) that ammonia f o r m a t i o n i n b r a i n depends g r e a t l y on glutamate oxidation. The value of total amino acid-N, including the value f o r ammonia, i s enhanced by anaerobic incubation and is a l m o s t equal i n one hour to that obtained at the end of one hour a e r o b i c incubations ( T a b l e s 3 and 2). T h i s enhancement i s l i k e l y to be due to p r o t e i n breakdown because there a r e s m a l l i n c r e a s e s i n the amounts of most amino acids apart f r o m glutamine (Table 3A). The net f a l l i n the l e v e l of glutamine obtained i n anoxia, may p r e s u m a b l y be due to the fact that the rate of its h y d r o l y s i s by glutaminase exceeds the rate of its f o r m a t i o n by the A T P r e q u i r i n g glutamine synthetase. T h i s p r e s u m a b l y accounts f o r a p o r t i o n of the ammonia l i b e r a t e d a n a e r o b i c a l l y . A balance sheet r e p r e s e n t i n g the changes i n the total (tissue + medium) ammonia and amino a c i d contents of b r a i n s l i c e s incubated (with or without glucose) i n the p r e s e n c e or absence of oxygen, is given in Table 3A. F r o m th i s T a b l e it is evident that much of the ammonia turnover in b r a i n is due to changes in the b r a i n amino a c i d s . M o r e o v e r , it i s evident that, i n the absence of glucose, the enhanced glutamate content of b r a i n s l i c e s incubated a n a e r o b i - c a l l y over the ti s s u e value of glutamate obtained a e r o b i c a l l y , n e a r l y accounts f o r the d i m i n i s h e d rate of ammonia f o r m a t i o n under anoxia. F u r t h e r m o r e , the accompanying i n c r e a s e i n G A B A content, and perhaps part of the i n c r e a s e 6 4 T A B L E 3. E f f e c t s of anoxia on the ammonia and amino a c i d contents of r a t b r a i n c o r t e x s l i c e s incubated i n the p r e s e n c e or absence of g l u c o s e . Rat b r a i n c o r t e x s l i c e s were incubated i n K r e b s - R i n g e r b i c a r b o n a t e m e d i u m at 3 7 ° C for one hour in an atmosphere of N 2/CO;? (95% : 5 % ) . T o t a l (tissue + medium) values of ammonia and amino a c i d s a r e e x p r e s s e d as jj.mole/g i n i t i a l wet wt. A d d i t i o n s to the incubation m e d i u m No G l u c o s e G l u c o s e Glutamate 12.40 + 0.22 13. 4 3 + 0.27 G l u t a m i n e 2. 88 + 0. 18 3. 08 + 0.10 G A B A 3. 06 + 0 . 1 9 3 . 63 + 0.34 A s p a r t a t e 4. 34 + 0.03 4. 30 + 0. 15 A l a n i n e 1. 3 6 + 0. 18 1. 88 + 0.30 G l y c i n e 1. 66 + 0. 13 1. 73 + 0. 15 S e r i n e 2. 76 + 0. 16 2. 89 0.07 T h r e o n i n e 0. 76 + 0.05 0. 77 + 0.04 T a u r i n e 5. 8 9 + 0.11 5 . 86 + 0 . 0 9 A m m o n i a 7. 10 + 0.31 6. 59 + 0.80 T o t a l : N H 4 + - N + amino a c i d -N 4 5 . 0 9 47. 34 T A B L E 3A. Ammonia and amino acid changes of brain cortex slices incubated in the presence or absence of G ^ . Total (tissue + medium) values of ammonia and amino acids (umole/g initial wet wt.)used for the calculations were taken from T A B L E 2 (for O ) and T A B L E 3 (for N ? ) . No Glucose Glucose o 2 N 2 A - N H 2 o 2 N 2 A- N H 2 Glutamate 3.55 12.40 -8. 85 10. 11 13.43 -3.32 Glutamine 1.79 2.88 -2. 18 6.35 3.08 +6.54 G A B A 2.32 3.06 -0. 74 2.81 3. 63 -0. 82 Aspartate 9.13 4.34 +4.79 4. 14 4.30 -0. 16 Alanine 0.73 1.36 -0.63 1 . 2 9 1.88 -0.59 Glycine 1.47 1.66 - 0 . 1 9 1.53 1.73 -0.20 Serine 2.23 2.76 -0.53 1.82 2.89 -1.07 Threonine 0.95 0.76 +0,19 0. 67 0. 77 -0. 10 Ammonia 16.81 7.10 +9.17 6.33 6.59 +0.26 Total -NH2 40.77 39.20 +1.57 41.40 41.38 +0.02 66 i n alanine, must occur by glutamate m etabolism. These data f u r t h e r substantiate the dependence of a e r o b i c a mmonia f o r m a t i o n l a r g e l y on endogenous glutamate oxidation. 3.3 A m m o n i a f o r m a t i o n by infant (2-day-old) r a t b r a i n c o r t e x s l i c e s In e a r l i e r studies, we have shown (62) that the rate of ammonia forma- t i o n by 2-day-old r a t b r a i n c o r t e x s l i c e s , incubated for one hour i n a glucose- f r e e K r e b s - R i n g e r phosphate medium, i s lower than that of the adult. In view of the p o s s i b i l i t y that f o r m a t i o n of ammonia by adult rat b r a i n c o r t e x i s depen- dent on t e r m i n a l endogenous glutamate oxidation, a study was made of the changes in the total (tissue + medium) ammonia and amino a c i d l e v e l s of infant r a t b r a i n incubated i n the pr e s e n c e or absence of glucose. R e s u l t s r e p o r t e d i n T a b l e 4 show the following f a c t s . 1. V a l u e s of the contents of amino acids i n infant c o r t e x , f r e s h l y pre- p a r e d and p r i o r to incubation a r e in agreement with those r e p o r t e d i n the l i t e r a t u r e (11, 130, 131, 311). The total i n i t i a l amino a c i d - N including the value for ammonia, are r e m a r k a b l y c l o s e to that found for the adult (Table 2). However, i n agreement with the r e s u l t s of other w o r k e r s (129-131), the lower l e v e l s of g l u t a - mate (3 5 % of the adult), aspartate ( 6 0 % of the adult), and glutamine ( 6 5 % of the adult) are compensated for by the high l e v e l s of taurine ( 3 0 0 % of the adult). The amino acids, c l o s e l y r e l a t e d to the c i t r i c a c i d cycle, a r e c o n s i d e r a b l y lower i n the infant b r a i n . 2. In the absence of glucose, the rate of ammonia f o r m a t i o n i n infant r a t b r a i n i s 40 per cent that of the adult. T h e r e i s a r i s e in the aspartate l e v e l accompanying the f a l l s i n glutamate and glutamine l e v e l s . 3. In the p r e s e n c e of glucose, the i n i t i a l content of glutamate i s l i t t l e changed in infant cortex incubated for one hour. Unlike the adult 67 T A B L E 4. A m m o n i a and amino a c i d changes i n infant r a t b r a i n c o r t e x on inc u b a t i o n i n the p r e s e n c e or absence of g l u c o s e . Infant (2-year old) r a t b r a i n c o r t e x s l i c e s were incubated i n K r e b s - R i n g e r m e d i u m with or without glucose i n 0 2 at 3 7 ° C for one hour. I n i t i a l values ( i . e . v a l u e s p r i o r to incubation) of ammonia and amino acids and the t o t a l (tissue + medium) values are e x p r e s s e d as p.mole/g i n i t i a l wet wt. I N I T I A L O N I N C U B A T I O N No G l u c o s e G l u c o s e Glutamate 4. 15 + 0.05 1. 91 + 0.12 4.00 + 0.23 G l u t a m i n e 2. 91 + 0.05 0. 34 + 0.07 0.85 0. 15 G A B A 1. 76 + 0.12 1. 65 + 0.05 2.16 + 0.30 A s p a r t a t e 2. 02 + 0.10 5. 62 + 0.06 2.52 + 0 .02 A l a n i n e 1.. 26 + 0. 02 0. 93 + 0.30 1.25 + 0. 20 G l y c i n e 1. 01 + 0.03 1. 63 + 0.19 1.71 + 0.03 S e r i n e 1. 30 + 0.04 2. 26 + 0.20 2.37 + 0.40 T h r e o n i n e 0. 40 + 0. 15 1. 08 + 0. 18 0.95 + 0. 12 T a u r i n e 17. 60 + 0.30 19. 03 + 1.20 18 .91 + 2.34 L y s ine 0. 31 + 0.03. 0. 59 + 0. 02 0.42 + 0. 11 A m m o n i a 0. 76 + 0.02 7. 04 + 0.75 5.34 + 0.64 T o t a l : NH4+-N + amino a c i d -N 36. 49 42.42 41. 33 68 b r a i n , however, infant b r a i n glutamine l e v e l f a l l s . A m m o n i a ut i l i z a t i o n , by glutamine synthesis, seems to occur only f e e b l y in the incubated b r a i n t i s s u e of infant rat. T h i s p r e s u m a b l y is not only due to the low a c t i v i t i e s of the r e l e v a n t enzymes i n v o l v e d i n glutamine synthesis (15, 163), but may also be due to the r e l a t i v e l y low rate of the operation of the c i t r i c a c i d cycle, known to occur in the infant r a t b r a i n (235), and the r e s u l t a n t low rate of f o r m a t i o n of a-ketoglutarate and, therefore, of glutamate. 4. It was also found that the oxygen consumption i n the infant b r a i n (per unit wet wt t i s s u e is c o n s i d e r a b l y lower than in the adult (see also r e f e r e n c e 235). F o r example, i n the p r e s e n c e of 10 m M glucose, the value for the infant b r a i n i s 46 + 2 [imole/g i n i t i a l wet wt/hour, and that f o r the adult i s 102 _+ 4. In the absence of glucose, the values for the infant and adult b r a i n c o r t e x s l i c e s are 42 +_ 3, and 68 +_ 3, r e s p e c t i v e l y . 5. C o m p a r e d to the i n i t i a l , the total amino a c i d - N i n c l u d i n g the value fo r ammonia, is enhanced (by about 5-6 patoms/g i n i t i a l wet wt) on incubation for one hour either in the p r e s e n c e or absence of glucose. T h i s is p o s s i b l y due to g radual p r o t e i n breakdown (but at a rate lower than that in the adult - T a b l e s 4 and 2). T h e r e ar e enhanced l e v e l s of some amino acids, e.g., glycine, serine, threonine or lysine, i n infant r a t b r a i n incubated in the p r e s e n c e or absence of glucose, which may also r e f l e c t some p r o t e i n break- down during incubation. 3.4 Is there a d i r e c t r o l e of aspartate in the p r o c e s s of ammonia f o r m a t i o n in the b r a i n ? A s mentioned e a r l i e r (section 1.5 (ii) a), some w o r k e r s (14, 46, 98) studying the o r i g i n and m e c h a n i s m of ammonia formation, have i n t h e i r model a s s i g n e d a c e n t r a l r o l e to aspartate. A c c o r d i n g to this model, a l l ammonia 69 l i b e r a t e d emanates f r o m aspartate through a c y c l i c set of r e a c t i o n s i n v o l v i n g either d e samino-NAD or i n o s i n i c a c i d . The cont r i b u t i o n of other amino acids to ammonia production o c c u r s through aspartate subsequent to t r a n s a m i n a t i o n with oxaloacetate. In other words, other amino acids play the r o l e of a " r e s e r v o i r " , maintaining aspartate l e v e l s . In view of the fact that incubation of b r a i n cortex s l i c e s of the rat, i n a m e d i u m devoid of glucose r e s u l t s i n enhanced aspartate l e v e l s accompanying ammonia formation, experiments were c a r r i e d out to observe whether aspartate actually plays a d i r e c t r o l e i n the f o r m a t i o n of ammonia in the incubated b r a i n t i s s u e . When adult rat b r a i n c o r t e x s l i c e s are incubated i n 0 2 f o r one hour i n K r e b s - R i n g e r phosphate g l u c o s e - f r e e m e d i u m in the pr e s e n c e of 100 m M KC1, the tota l (tissue + medium) l e v e l of aspartate is enhanced by 40 per cent (Table 5). T h i s i n c r e a s e i n aspartate i s not accompanied by a p a r a l l e l d e c r e a s e in the glutamate l e v e l , nor i s it a ccompanied by an enhanced rate of ammonia f o r m a t i o n . In fact, high i n v a r i a b l y causes a s m a l l but s i g n i f i c a n t f a l l i n the rate of ammonia l i b e r a t i o n . T h i s f a l l a ccompanies a f a l l in the rate of r e s p i r a t i o n under these conditions, which may, i n turn, be due to the l o s s of some endogenous glutamate to the incubation m e d i u m (i.e., a l o s s of 2.75 + 0.03 jjmole/g c o m pared with 0.90 _+0.07 for the contr o l ) . T o t a l (tissue + medium) glutamine l e v e l s f a l l under these conditions, while G A B A l e v e l s (not shown) are unaffected. T h e r e i s a s m a l l but s i g n i f i c a n t r i s e in the l e v e l of alanine (i.e., a r i s e of 0.32 _+ 0 .02 |jmole/g over the value of 1.0 1 _+ 0.0 5 for the contro l ) . I n c r e a s i n g the time of incubation under these conditions to four h o u r s does not furth e r enhance the total (tissue + medium) aspartate l e v e l , nor does it affect the l i b e r a t i o n of ammonia. T h e s e r e s u l t s show that an i n c r e a s e in the endogenous aspartate l e v e l does not n e c e s s a r i l y accompany, or r e s u l t in, s t i m u l a t i o n of the l i b e r a t i o n of ammonia in b r a i n t i s s u e . It may be mentioned that there was no f a l l of N - a c e t y l a s p a r t a t e to compensate f or the r i s e of aspartate under these conditions. The i n h i b i t i o n of aspartate formation, f r o m endogenous sources, i n T A B L E 5. Effects of sodium malonate and amino oxyacetate on aspartate synthesis and ammonia formation in rat brain cortex slices incubated in a glucose-free m e d i u m . Rat bra in cortex slices were incubated aerobically in (glucose-free) K r e b s - R i n g e r phosphate medium at 3 7 ° C for one hour in the presence or absence of KC1 (lOOmM) with or without the addition of sodium malonate (5mM) or sodium amino oxyacetate (5mM). QO2 values are expressed as H mole oxygen consumed per g initial wet wt. Total (tissue + medium) ammonia and amino acids are expressed as p ,mole/g init ial wet wt. Additions to the incubation medium Q0 2 Aspartate Glutamate Glutamine Ammonia N i l Malonate Amino oxyacetate 71.2 + 4.8 60.3 + 3.9 63.6 + 3.0 8.58 + 0.54 7.31 + 0.11 5.30 + 0.47 3.20 + 0.26 3.44 + 0.27 3.58 + 0.07 2.49 + 0.34 2.58 + 0.38 3.58 + 0.07 17.23 + 0.11 16.80 + 0.38 17.71 + 0.11 With KC1 KC1 + malonate KC1 + amino- oxyacetate 54.5 + 6.2 45.6 + 1.0 52.6 + 1.0 12.20 + 0.18 8.60 +. 0.64 5.06 + 0.28 3.85 + 0.05 4.87 + 0.04 4.98 + 0.11 1.22 + 0.12 1.33 + 0.22 3.69 + 0.27 16.04 + 0.16 15.25 + 0.07 16.96 + 1.01 71 b r a i n s l i c e s is another way of studying the effects of aspartate on the rate of ammonia l i b e r a t i o n . A s p a r t a t e f o r m a t i o n i n r a t b r a i n c o r t e x s l i c e s incubated i n K r e b s - R i n g e r phosphate (glucose-free) m e d i u m may be s u p p r e s s e d by meta- b o l i c i n h i b i t o r s such as malonate or amino-oxyacetate (Table 5). 1. Malonate (5 mM), by inhibiting the rate of operation of the c i t r i c a c i d cycle, l i m i t s the supply of oxaloacetate r e q u i r e d for aspartate f o r m a t i o n by t r a n s a m i n a t i o n . T h i s i s s p e c i a l l y evident i n the p r e s e n c e of 100 m M KC1 when the rate of aspartate f o r m a t i o n i s c o n s i d e r a b l y s u p p r e s s e d . However, there is no concomitant s u p p r e s s i o n of the rate of ammonia f o r m a t i o n under these conditions. 2. Amino-oxyacetate (5 mM) inhibits aspartate f o r m a t i o n both i n the p r e s e n c e or absence of 100 m M KC1 (in a g l u c o s e - f r e e medium) p r e s u m a b l y by d i r e c t l y i n h i b i t i n g the p y r i d o x a l phosphate r e q u i r i n g t r a n s a m i n a s e r e a c t i o n (147, 279, 304). The i n h i b i t i o n in the rate of aspartate f o r m a t i o n is not accompanied by any i n h i b i t i o n of the rate of ammonia fo r m a t i o n . It seems, therefore, that the evidence does not support the view that aspartate i s a major source of ammonia in r a t b r a i n c o r t e x s l i c e s under our e x p e r i m e n t a l conditions. In view of the lack of supporting evidence f o r the r o l e of aspartate as an immediate ammonia p r e c u r s o r i n b r a i n s l i c e s , i n our experi- ments, and in view of the positive evidence f a v o r i n g glutamate (+glutamine) as the immediate p r e c u r s o r , i t seems u n n e c e s s a r y to c o n s i d e r aspartate as playing a major r o l e in ammonia f o r m a t i o n . N e v e r t h e l e s s , these r e s u l t s do not constitute evidence that a s p a r - tate plays no r o l e i n the p r o c e s s of c e r e b r a l ammonia forma t i o n . 72 3.5 E f f e c t s of metabolic i n h i b i t o r s on the rate of ammonia f o r m a t i o n f r o m endogenous amino acids of rat c e r e b r a l c o r t e x s l i c e s incubated in a m e d i u m devoid of glucose. C e r e b r a l cortex s l i c e s of the rat were incubated i n 0 2 in K r e b s - R i n g e r phosphate m e d i u m at 3 7 ° C for one hour with and without the addition of 2, 4-dinitr ophenol (DNP) (0.1 mM) or s o d i um amytal (1 mM). R e s u l t s are given in T a b l e 6. In the p r esence of D N P there i s a s m a l l d i m i n u t i o n (3 |imole/g/hour) in the rate of ammonia formation, which i s c h i e f l y r e f l e c t e d i n accompanying enhanced l e v e l s of glutamate (by 1.83 p.mole/g) and G A B A (by 1.46 |imole/g). D N P seems not to i m p a i r the breakdown of protein, since the total amino a c i d n i t r o g e n (including the value for ammonia) i s not affected when c o m p a r e d with the value of the c o n t r o l . It was also found that the rate of oxygen consumption, i s d i m i n i s h e d f r o m the c o n t r o l value of 70 + 7 |imole/g to 50 + Z [imole/g i n i t i a l wet wt t i s s u e i n the p r e s e n c e of 0.1 m M DNP. With 1 m M amytal, the rate of ammonia f o r m a t i o n is s u p p r e s s e d by 5.36 |imole/g/hour. T h i s i s accompanied by enhanced l e v e l s of glutamate (by 3.31 )jmole/g) and G A B A (by 1.83 (imole/g). Under these conditions, however, there seems to be a s m a l l d i m i n u t i o n in the breakdown of p r o t e i n since the total amino a c i d - n i t r o g e n (including the value for ammonia) i s s l i g h t l y lower than that f o r the c o n t r o l . It was also found that amytal (1 mM) l o w e r e d the rate of oxygen consumption f r o m the c o n t r o l value of 68 jf 5 (jmole/g to the value of 41 + 3 (imole/g i n i t i a l wet wt t i s s u e . T h ese data on the r a t e s of NĤ "*" formation, which are in agreement with our e a r l i e r r e s u l t s on the i n h i b i t o r y effects of D N P or amytal by b r a i n t i s s u e incubated in a g l u c o s e - f r e e m e d i u m (62), are not wholly explained by i n c r e a s e d glutamate l e v e l s . A n elevated G A B A content also takes place due, obviously, to glutamate d e c a r b o x y l a t i o n taking place at a g r e a t e r rate when the endogenous glutamate c o n c e n t r a t i o n i s i n c r e a s e d . 73 T A B L E 6. E f f e c t s of s o d i u m - a m y t a l and 2, 4 - d i n i t r ophenol on the total a mmonia and amino a c i d contents of r a t b r a i n c o r t e x s l i c e s incubated i n a glucose f r e e medium. Rat c e r e b r a l c o r t e x s l i c e s were incubated i n K r e b s - R i n g e r phosphate m e d i u m i n G"2 at 3 7 ° C for one hour i n the p r e s e n c e or absence of a m y t a l (ImM) or 2, 4 - d i n i t r o p h e n o l (0. 1 mM), T o t a l (tissue + medium) values a r e e x p r e s s e d as jj, m o l e / g i n i t i a l wet wt. A d d i t i o n to the incubation m e d i u m N i l A m y t a l 2,4 din i t r o p h e n o l G l u t a m a t e 3. 62 + 0.26 6.93 + 0.25 5. 45 + 0. 26 G l u t a m i n e 2. 22 + 0. 14 1.97 + 0. 12 1. 80 + 0. 10 G A B A 1. 92 + 0. 02 3. 75 + 0. 17 3. 38 + 0. 08 A s p a r t a t e 9. 07 1. 00 7.45 + 0. 08 9. 08 + 1. 08 A l a n i n e 0. 84 + 0. 03 0.93 + 0. 09 1. 38 +_ 0. 05 G l y c i n e 1. 40 + 0.03 1.27 + 0. 08 1. 58 + 0. 03 S e r i n e 2. 21 + 0.05 2. 09 + 0. 10 2. 34 + 0. 18 T h r e o n i n e 0. 86 + 0. 05 0.78 + 0. 02 0. 87 + 0. 04 T a u r i n e 6. 16 + 1. 04 6.10 + 0.52 6. 20 + 0. 82 A m m o n i a 17. 16 + 0.16 11. 80 + 0.20 14. 17 + 1. 16 T o t a l : N H 4 + - N + amino a c i d -N 47. 68 44.94 47. 85 74 3.6 E f f e c t s of ouabain on the rate of ammonia f o r m a t i o n i n r a t b r a i n cortex s l i c e s incubated i n a g l u c o s e - f r e e m e dium If endogenous glutamate oxidation is a major factor in the c e r e b r a l p r o d u c t i o n of ammonia, i t s depletion f r o m c e r e b r a l c o r t e x s l i c e s (by in h i b i t i o n of its re-uptake mechanism) should b r i n g about a reduced rate of ammonia f o r m a t i o n and a concomitant i n h i b i t i o n i n the rate of r e s p i r a t i o n in a g l u c o s e - f r e e medium. T h i s i n fact o c c u r s when rat b r a i n c o r t e x s l i c e s are incubated i n O-, for one hour at 3 7 ° C i n K r e b s - R i n g e r phosphate m e d i u m in p r e s e n c e of ouabain (0.1 and 1.0 mM). R e s u l t s given in Table 7 show the following f a c t s . 1. The content of amino a c i d - N (including the value f o r ammonia) i s the same in the presence or absence of ouabain, i n d i c a t i n g that ouabain does not i n t e r f e r e with the p r o t e i n breakdown p r o c e s s . T h i s is f u r t h e r supported by the fact that the total (tissue + medium) amounts of amino a c i d s such as glycine, serine or threonine, are unaffected by ouabain, though their r a t e s of r e l e a s e into the i n c u - bation m e dium a r e c o n s i d e r a b l y enhanced. 2. The rate of ammonia f o r m a t i o n is inhibite d by 4.74 and 6.74 pmole/g i n i t i a l wet wt ti s s u e / h o u r with 0.1 and 1.0 m M ouabain r e s p e c t i v e l y . With 0.01 m M ouabain the value is 1.7 5 pmole/g (not shown). 3. The inh i b i t i o n of the content of ammonia f o r m a t i o n by ouabain can be accounted for by the gain of glutamate and the amino a c i d s d e r i v e d f r o m it, viz., aspartate and G A B A . T h e r e i s c o n s i d e r a b l e l o s s of these amino acids f r o m the tiss u e and most of the gain takes place i n the medium. 4. It was also found that the l o s s of t i s s u e glutamate and its metabolites under these conditions is accompanied by a drop in the rate of oxygen consumption f r o m c o n t r o l values of 75 + 4 pmole/g i n i t i a l wet wt tissue to values of 49 + 2 and 4 2 + 1 pmole/g with 0.1 mM and 1.0 m M ouabain r e s p e c t i v e l y . T A B L E 7. The effects of ouabain on the rate of ammonia formation, and the release of amino acids from brain cortex slices incubated in a glucose-free medium. Rat brain cortex slices were incubated in a Krebs-Ringer phosphate medium devoid of glucose in O2 at 3 7 ° C for one hour in the presence or absonce of ouabain. Tissue values are expressed as jjmole/g initial wet wt. and medium values as U m o l e / g initial wet wt. /3 m l . Addition to the incubation medium NIL OUABAIN (0. ImM) OUABAIN (l.OmM) TISSUE M E D I U M T O T A L TISSUE M E D I U M T O T A L TISS UE MEDIUM T O T A L Glutamate 1. 78 + 0. 33 0.78 + 0.05 2.56 0.90 + 0.09 3.60 + 0. 12 4.50 0. 65 + 0.03 4.42 + 0.06 5. 07 Glutamine 0. 17 + 0.04 2.08 + 0.11 2.25 0.50 0.07 1.75 + 0. 05 2.25 0.41 + 0.01 1.74 + 0.21 2. 15 G A B A 1.22 + 0. 14 0.10 + 0.08 1.32 0.43 + 0.02 2.20 + 0. 08 2.63 0.41 + 0. 01 2.27 + 0.05 2. 68 Aspartate 7.27 + 0.30 1.91 + 0.58 9.18 3.96 + 0.38 7. 10 + 0. 15 11.06 2.81 + 0. 06 7.21 0.21 10. 02 Alanine 0. 18 + 0.02 0.55 + 0.05 0. 73 0.09 + 0.06 1 .09 + 0. 01 1.18 0. 10 + 0.02 1.08 + 0.03 1. 18 Glycine 0.77 + 0. 14 0.67 + 0.04 1.44 0.30 + 0.06 1.34 + 0.01 1.64 0.28 + 0.02 1.33 0.02 1. 61 Serine 0.86 + 0.16 1.18 + 0.06 2.04 0.33 + 0.06 1.97 + 0.01 2.30 0.27 + 0.01 1.95 + 0.03 2. 22 Threonine 0.23 + 0.03 0.53 + 0.06 0.76 0.14 + 0.05 0.66 + 0.03 0.80 0. 12 + 0.03 0.74 _+ 0.03 0. 86 Taurine 2.99 + 0.26 2.83 + 0.21 5.82 1 .94 + 0.16 4.21 + 0.11 6.15 1.55 + 0.04 4.54 + 0.04 6.09 Ammonia 2.17 + 0.14 15.71 + 1.35 17.88 2.02 + 0.24 11.12 + 0.02 13.14 1.85 + 0.15 9 .29 + 0.5J 11. 14 Total : N H 4 + - N + amino acid-N 17.81 28.42 46.23 . 11. 11 36. 79 47.90 8.86 36.31 45. 17 76 5. A t l e a s t 4.3 P-mole ammonia/g tissue, must be d e r i v e d f r o m glutamine under these conditions (assuming no glutamine directly- d e r i v e d f r o m proteins enters the f r e e amino a c i d pool on incubation) as the d i f f e r e n c e between the i n i t i a l and incubation values i s 4.40 - 2.25 = 2.15 (Tables 2 and 7). 6. In spite of the ouabain induced amino a c i d r e l e a s e f r o m the tissue, m a r k e d changes i n glutamate and aspartate contents (tissue + medium) take place. Thus, c o n s i d e r a b l e c o n v e r s i o n of glutamate to aspartate and G A B A o c c u r s i n p r e s e n c e of ouabain. 3.7 The r o l e of Ca"*"* i n the f o r m a t i o n of ammonia f r o m L-glutamate by rat b r a i n cortex s l i c e s incubated in a medium devoid of glucose. The evidence so f a r supports the r o l e of endogenous glutamate oxidation as a major factor i n the a e r o b i c generation of ammonia by b r a i n t i s s u e . E x p e r i m e n t s were then c a r r i e d out to observe whether added L-glutamate enhances the rate of ammonia f o r m a t i o n . In c o n f i r m a t i o n of our e a r l i e r studies (62) and those of others (58, 103), the addition of L-glutamate (2.5 mM) to r e s p i r i n g b r a i n c o r t e x s l i c e s in a n o r m a l K r e b s - R i n g e r phosphate m e d i u m (containing Ca"*"*) i s found not to enhance the amount of f r e e ammonia f o r m e d in one hour. In fact, a di m i n u t i o n takes place (Table 8). R e s u l t s given i n Table 8 show that L-glutamate addition r e s u l t s i n accompanying i n c r e a s e s i n the rate of glutamine synthesis. T h i s i s i n a c c o r d with e a r l i e r findings (58, 103). We now find that, if C a * * is omitted f r o m the incubation medium, the ra t e of ammonia f o r m a t i o n f r o m L - g l u t a m a t e is substantially enhanced above that i n a C a * * containing m e dium and that of glutamine is d i m i n i s h e d (Table 8). The addition of L-glutamate to r e s p i r i n g b r a i n c o r t e x s l i c e s in the presence of C a * * enhances the c o n t r o l rate of oxygen consumption f r o m 70 + T A B L E 8. Effects of L-glutamate on the formation of ammonia and amino acids by rat brain cortex slices incubated in a medium devoid of glucose in the presence and absence of C a + + . Rat brain cortex slices were incubated aerobically in Krebs-Ringer phosphate medium for one hour at 3 7 ° C in the presence or absence of 2. 8mM C a C l 2 with or without the addition of 2. 5mM sodium L-glutamate. Total (tissue + medium) values are expressed as umole /g initial wet wt. Tissue values only of glutamate* are given. Additions to the incubation medium Nil Sodium L-glutamate 1 2.5 mM) Ca++ - f r e e Ca"*"+- containing Ca++ - free Ca++- containing Glutamate *1.62 + 0.12 *2.03 + 0.16 *4.48 + 0.02 *6.36 + 0.08 Glutamine 1.46 + 0.13 2.22 + 0.14 3.22 + 0.50 8.24 + 0.38 Aspartate 10.01 + 0.24 9.07 + 1.00 20.31 + 0.45 20.00 + 0.30 G A B A 1.87 + 0.09 1.92 + 0.02 2.22 +_ 0.32 2.17 + 0.20 Alanine 0.75 + 0.11 0.84 + 0.03 0.90 + 0.03 1.03 + 0.13 Glycine 1.23 + 0.05 1.40 + 0.03 1.38 + 0.02 1.53 + 0.12 Serine 1.75 + 0.09 2.21 + 0.05 1.96 + 0.03 2.22 + 0.10 Threonine 0.68 + 0.08 0.86 + 0.05 0.77 + 0.02 0.94 + 0.03 Taurine 6.28 + 0.20 6.61 + 1.04 6.64 + 0.30 6.20 + 0.43 Ammonia 17.15 + 0.43 17.16 + 0.26 19.24 + 0.62 12.68 + 0.28 78 2 pmole/g i n i t i a l wet wt/hour to a value of 97 +_ 3, and in the absence of C a + + f r o m the c o n t r o l r a t e of 68 + 2 to a value of 88 + 2. That L-glutamate can support b r a i n r e s p i r a t i o n is a well known fact. These r e s u l t s point to the profound effects of Ca"1""*" on glutamine syn- t h e s i s i n b r a i n (see Chapter 4). The i n c r e a s e d l i b e r a t i o n of ammonia in the absence of C a + + is due to a d i m i n i s h e d a b i l i t y of the ti s s u e to synthesize gluta- mine under these conditions. In the pr e s e n c e of external L-glutamate, Ca**"* has l i t t l e or no effects on the total (tissue + medium) l e v e l s of a l l amino acids d e t e r m i n e d except (as mentioned above) that of glutamine. In the pr e s e n c e of L-glutamate, Ca"*"*" enhances (1) the tissue content of glutamate (Table 8); (2) the rate of r e s p i r a - tion; and (3) the tissue A T P content (Table 71 A). A probable i n c r e a s e i n glutamine synthetase a c t i v i t y takes place, r e s u l t i n g i n an enhanced glutamine and a d i m i n i s h e d ammonia content. 3.8 A m m o n i a f o r m a t i o n by rat c e r e b r a l cortex s l i c e s f r o m exogenous L - g l u t a m i n e in the absence of glucose. R e s u l t s s u m m a r i z e d i n Table 9 show that, i n the absence of glucose, the rate of ammonia f o r m a t i o n f r o m 5 m M L- g l u t a m i n e by incubated r a t b r a i n c o r t e x s l i c e s , i s about 17-18 jamole/g i n i t i a l wet wt ti s s u e i n one hour, in the p r e s e n c e or absence of Ca*'"'", or in the pr e s e n c e of 0.1 m M ouabain, or i n the N a ^ - r i c h K**" and C a ^ - f r e e incubation m e d i u m - M e d i u m II. The fact that a constant rate of ammonia f o r m a t i o n o c c u r s f r o m e x t e r n a l l y - a d d e d L - g l u t a m i n e i m p l i e s that the ac t i v i t y of glutaminase is unaltered under such v a r y i n g media conditions. It i s evident, f r o m the r e s u l t s presented so far, that both endogenous and e x t e r n a l L - g l u t a m i n e can support high rates of ammonia f o r m a t i o n i n b r a i n t i s s u e incubated i n glucose free-media. 79 TABLE 9. Ammonia formation from exogenous L-glutamine by rat cerebral cortex slices incubated in glucose-free media. Cerebral cortex slices of the rat were incubated aerobically in a variety of media in the presence or absence of 5mM L-glutamine for one hour at 37°C. Total (tissue + medium) values of ammonia are expressed as [imole/g initial wet wt. Medium Medium L-Glutamine Total Ammonia Ammonia formed from L-glutamine Krebs-Ringer phosphate + 17.16 + 0.26 34.70 + 4.90 17. 54 Krebs-Ringer phosphate + 0. ImM ouabain + 13.14 + 0.22 30.20 + 4.00 18.65 Ca-free Krebs-Ringer phosphate + 17.15 + 0.43 35.80 + 5.60 17.06 Medium II + 14.90 + 0.80 32.00 + 2.20 17.10 80 3.9 E f f e c t s of tetrodotoxin, l i d o c a i n e and p r o t o v e r a t r i n e on the rate of ammonia f o r m a t i o n by r a t b r a i n cortex s l i c e s incubated in a glucose- f r e e medium. Rat b r a i n cortex s l i c e s were incubated in 0 2 for one hour at 3 7 ° C i n K r e b s - R i n g e r phosphate g l u c o s e - f r e e medium. The effects of n e u r o t r o p i c d rugs on the rate of ammonia f o r m a t i o n under these conditions were studied. R e s u l t s are r e c o r d e d in T a b l e 10. The addition of p r o t o v e r a t r i n e (5 )JlM) d i m i n i s h e s the rate of ammonia formation, some of which appears in an enhanced glutamine l e v e l . These effects of p r o t o v e r a t r i n e are completely abolished by the p r e s e n c e of tetrodo- toxin (2 LiM) or of l i d o c a i n e (0.5 mM), and o c c u r in the presence, but not in the absence, of c a l c i u m . The i m p l i c a t i o n of these findings w i l l be dealt with l a t e r . It i s to be noted that T T X or l i d o c a i n e t h e m s e l v e s have l i t t l e or no effects on the rates of glutamine and NĤ "*" f o r m a t i o n under these conditions. 3.10 Changes in the ammonia and amino a c i d contents of i s o l a t e d r a t b r a i n c o r t i c a l synaptosomes on incubation. A m i n o a c i d contents of synaptosomes i s o l a t e d f r o m the b r a i n c o r t e x of the rat, f r e s h l y p r e p a r e d and i m m e d i a t e l y p r i o r to incubation, are given in Table 11. A l s o g i v e n in this Table a r e the contents of ammonia and amino acids found on incubation i n G"2 at 3 7 ° C for one hour in K r e b s - R i n g e r phos- phate m e d i u m with or without the addition of 10 m M glucose. The following fa c t s are evident f r o m the r e s u l t s . 1. A t the end of the incubation period, either in the p r e s e n c e or absence of glucose, the total amino acid-N, i n c l u d i n g the value f o r ammonia, is i n c r e a s e d by about 6 |aatom/100 mg synaptosomal prot e i n . T h i s is p r o b a b l y due to p r o t e i n breakdown. P r o t e o l y s i s is also r e f l e c t e d in the i n c r e a s e d l e v e l s of c e r t a i n amino ac i d s such as glycine, serine, threonine or l y s i n e . 81 TABLE 10. Effects of neurotropic drugs on the formation of ammonia, glutamate and glutamine in rat brain cortex slices respiring in a glucose-free medium. Rat brain cortex slices were incubated aerobically in Krebs-Ringer phosphate medium at 37°C for one hour in the presence or absence of neurotropic drugs. Total (tissue + medium) values are expressed as U m o l e/g initial wet wt. Additions to the incubation medium Ammonia Glutamate Glutamine Nil 17. 10 + 0.58 2.93 + 0.21 2. 25 + 0.21 + TTX (2aM) 16. 80 + 0.39 3.61 + 0.23 1. 81 + 0. 02 + lidocaine (0.5mM) 19.41 + 0. 11 2. S4 + 0.17 1. 01 + 0.05 + protoveratrine (5|jM) 11. 79 + 0.28 3.44 + 0.20 3. 78 + 0. 08 + protoveratrine (5uM) 13. 01 + 0.25 3.10 -\_ 0.04 2. 04 + 0. 07 + TTX (2UM) + protoveratrine (5 JJM) 18. 51 + 0. 19 2.94 + 0. 02 1. 83 + 0. 14 + lidocaine (0.5mM) 82 T A B L E 11. A m m o n i a and amino a c i d changes i n i s o l a t e d r a t b r a i n c o r t e x synaptosomes incubated i n the p r e s e n c e or absence of g l u c o s e . Synaptosomes f r o m r a t b r a i n cortex, i s o l a t e d by the method of G r a y and Whittaker (243), were incubated i n 0 2 at 37 ° C f or one hour i n K r e b s - R i n g e r phosphate m e d i u m with or without the addi t i o n of glucose (lOmM). I n i t i a l v a l ues ( i . e . p r i o r to incubation) and the total (cells + medium) values of a m m o n i a and amino a c i d s obtained on incubation a r e e x p r e s s e d as p.mole/ 100 mg s y n a p t o s o m a l p r o t e i n . I N I T I A L O N I N C U B A T I O N No glucose G l u c o s e ' Glutamate 5. 54 + 0. 21 0. 92 + 0. 24 3. 77 + 0. 17 G l u t a m i n e 1. 30 + 0. 16 0. 72 + 0. 13 0.70 + 0. 14 G A B A 1.26 + 0. 13 0. 75 + 0. 13 1.27 + 0.07 A s p a r t a t e 2.34 + 0. 18 3. 51 + 0.37 2. 83 + 0.22 A l a n i n e 0. 21 + 0.07 0. 79 + 0.04 0.91 + 0. 05 G l y c i n e 0. 27 + 0. 03 0. 70 + 0. 08 0.69 + 0.02 Se r i n e 0.39 + 0. 08 0. 98 + 0. 08 0.97 + 0.07 T h r e o n i n e 0. 20 + 0. 07 0. 64 + 0. 10 0.62 + 0. 02 T a u r i n e 2.41 + 0. 08 2. 44 + 0. 20 2.43 + 0. 11 L y s ine - 0. 40 + 0. 10 0.45 + 0. 01 A m m o n i a 2.31 + 0.37 11. 21 + 0. 16 8. 01 + 0. 19 T o t a l : N H 4 + - N + amino a c i d -N 17.52 23. 78 23 .35 83 2. In the absence of glucose, the r i s e in the l e v e l of ammonia i s accompanied by s i g n i f i c a n t f a l l s i n the glutamate, glutamine and G A B A l e v e l s , p art of which may account for the r i s e in the content of aspartate. 3. In the p r e s e n c e of glucose, the total a-amino + ammonia nit r o g e n after one hour incubation i s the same as that i n the absence of glucose. However, there are r i s e s in the l e v e l s of glutamate and G A B A which a r e balanced by f a l l s i n those of aspartate and NH^*. 4. Synaptosomes are unable to f o r m glutamine f r o m l i b e r a t e d a mmonia i n the pr e s e n c e of glucose. In this r e s p e c t synapto- somes do not behave i n a manner s i m i l a r to b r a i n c o r t e x s l i c e s incubated under i d e n t i c a l conditions. It is known that there i s l i t t l e or no glutamine synthetase a c t i v i t y in i s o l a t e d n e r v e endings (245, 246). 5. It was also found that the r a t e s of oxygen uptakes by synaptosomes incubated i n the p r e s e n c e and absence of glucose are 64 + 2 and 24 _+ 1 pmoles/lOO mg synaptosomal p r o t e i n r e s p e c t i v e l y . 3.11 Summary 1. When the oxygen consumption of r a t b r a i n c o r t e x s l i c e s r e s p i r i n g i n g l u c o s e - f r e e media i s s u p p r e s s e d (as, e.g., in the p r e s e n c e of 0.1 m M DNP, or 1 m M amytal, or 0.1 m M or 1.0 m M ouabain, or in the absence of oxygen), there i s an accompanying d i m i n u t i o n i n the l o s s of endogenous glutamate and a d i m i n i s h e d rate of ammonia fo r m a t i o n . Infant rat brain, which r e s p i r e s at a l e s s e r rate than adult per g f r e s h wt, also shows s m a l l e r l o s s e s of glutamate and lower r a t e s of ammonia f o r m a t i o n . 84 Although, endogenous glutamate seems to be the major source of a e r o b i c ammonia f o r m a t i o n i n b r a i n s l i c e s incubated i n a n o r m a l K r e b s - R i n g e r phosphate g l u c o s e - f r e e medium, exogenous Li-glutamate d i m i n i s h e s the rate of ammonia f o r m a t i o n and i n c r e a s e s that of glutamine synthesis. When Ca"*""*" i s omitted f r o m the incubation medium, the rate of ammonia f o r m a t i o n in the pr e s e n c e of L-glutamate is sub s t a n t i a l l y enhanced above that in a Ca"*"*" containing medium, and that of glutamine i s d i m i n i s h e d . B oth endogenous and exogenous L - g l u t a m i n e can serve as s o u r c e s of ammonia i n b r a i n s l i c e s r e s p i r i n g i n g l u c o s e - f r e e media. The aspartate content of b r a i n s l i c e s , incubated i n a g l u c o s e - f r e e medium, i s enhanced by over 40 per cent i n the p r e s e n c e of 105 m M KC1. It i s d i m i n i s h e d in the presence of 5 m M malonate or 5 m M amino oxyacetic a c i d with or without high p o t a s s i u m ion c o n c e n t r a - tions. These conditions have l i t t l e or no effects on the rate of ammonia l i b e r a t i o n . These r e s u l t s do not support the view that aspartate is a d i r e c t p r e c u r s o r of t i s s u e ammonia. P r o t o v e r a t r i n e (5 |-tM), b r i n g s about a d i m i n i s h e d f o r m a t i o n of ammonia together with an enhanced synthesis of glutamine i n b r a i n s l i c e s incubated i n a g l u c o s e - f r e e m e d i u m containing Ca"*"*". T h i s p r o c e s s i s T T X (2 iaM) and l i d o c a i n e (0.5 mM) se n s i t i v e . B r a i n c o r t e x synaptosomes f o r m ammonia a e r o b i c a l l y , l a r g e l y f r o m their pools of glutamate and glutamine. Unlike b r a i n cortex s l i c e s , synaptosomes do not s e e m to synthesize glutamine in the pr e s e n c e of glucose. P r o t e o l y s i s s eems to o c c u r in incubated rat b r a i n c o r t e x s l i c e s under a l l our incubation p r o c e d u r e s . 85 4. CONTROL, M E C H A N I S M S F O R G L U T A M I N E S Y N T H E S I S IN R A T B R A I N C O R T E X IN V I T R O Gonda and Qu a s t e l (112) have shown that ouabain at concentrations (e.g., 0.01 mM), not i n h i b i t o r y to r e s p i r a t i o n i n h i b i t s the synthesis of l a b e l l e d glutamine f r o m l a b e l l e d g l ucose. The in h i b i t i o n of glutamine synthesis (Table 12, Condition II) i s acc o m p a n i e d by an enhanced l i b e r a t i o n of ammonia (62) into the incubation m e d i u m (Table 22). However, it was also shown (112) that ouabain does not inhibit glutamine synthetase i s o l a t e d f r o m b r a i n t i s s u e . NH^* p a r t i a l l y r e v e r s e d the i n h i b i t o r y effect of ouabain on glutamine synthesis i n b r a i n cortex s l i c e s . T h i s l e d to the suggestion that ouabain s u p p r e s s e s the t r a n s p o r t of NH^* to the site of glutamine synthesis (112). However, it l a t e r became c l e a r that ammonium ions are not accumulated against a con c e n t r a t i o n gradient (see Chapter 6). In the lig h t of this new information, the m e c h a n i s m of ouabain action on the synthesis of glutamine in intact t i s s u e s t i l l r e m a i n e d to be found. F u r t h e r e x p e r i m e n t a l work was t h e r e f o r e c a r r i e d out and the r e s u l t s are gi v e n below. 4.1 E f f e c t s of v a r y i n g sodium ion con c e n t r a t i o n on the rate of glutamine synthesis i n ra t b r a i n c o r t e x s l i c e s One of the consequences of the i n h i b i t i o n of the s o d i u m pump by ouabain i s an enhanced s o d i u m ion concentration. However, tis s u e Na"*" con c e n t r a t i o n may al s o be changed by a l t e r i n g the sodium concentration of the incubation medium. ( T i s s u e / M e d i u m c o n c e n t r a t i o n r a t i o f or N a * was found to be 0.79, within wide ranges of m e d i u m N a * and K* concentrations. See Table 67). Thus, we were able to r a i s e the N a * content of b r a i n c o r t i c a l s l i c e s (without r e s o r t i n g to the use of ouabain) by incubating t hem i n K r e b s - R i n g e r phosphate gluc o s e m e d i u m containing a high co n c e n t r a t i o n of N a C l . Rat b r a i n cortex s l i c e s were incubated in O^ at 3 7 ° C for one hour in glucose containing media v a r y i n g in N a C l concentrations. The r e s u l t s obtained 86 (Table 12, Condition III) show that while the r e s p i r a t o r y r a t e s and water uptakes are l i t t l e affected by changes in the salt concentrations, the synthesis of glutamine i s inhibited at high concentrations of N a C l in the incubation medium. T h e r e is also an accompanying dim i n u t i o n in the T i s s u e / M e d i u m con c e n t r a t i o n r a t i o of glutamine under these conditions. 4.2 E f f e c t s of v a r y i n g p o t a s s i u m ion c o n c e n t r a t i o n on the rate of glutamine synthesis i n r a t b r a i n c o r t e x s l i c e s . A n o ther consequence of the i n h i b i t i o n of the s o d i um pump by ouabain i s a d i m i n i s h e d tissue p o t a s s i u m i o n concentration. It i s p o s s i b l e to r a i s e the l e v e l of K"*" i n c o r t i c a l s l i c e s by incubating t hem in a m e d i u m containing h i g h K*. However, Gonda and Q u a s t e l (112) were unable to r e v e r s e the i n h i b i t o r y effect of ouabain on the synthesis of glutamine, with lOOmM KC1. In fact, high K* enhanced the i n h i b i t o r y effect of ouabain on glutamine sy n t h e s i s . T h i s is undoubtedly due to a f a l l i n the t i s s u e A T P l e v e l known to occur with 100 m M KC1 (115), and although A T P l e v e l s i n s l i c e s a r e not known to d i m i n i s h m a r k e d l y with ouabain (112, 115), the additional p r e s e n c e of 100 m M KC1 may g r e a t l y reduce the A T P con c e n t r a t i o n owing to the depleted phosphocreatine r e s e r v e (251). It should also be noted that 100 m M KC1 enhances the uptakes of Na"* and water by the t i s s u e (182). Incubating r a t b r a i n cortex s l i c e s f o r one hour i n at 3 7 ° C i n K r e b s - R i n g e r phosphate glucose medium, devoid (initially) of KC1, r e s u l t s i n a f a l l of t i s s u e K"** (see Table 66) and A T P l e v e l s (115), with l i t t l e accompanying effects on oxygen and water uptakes. T h e r e i s a di m i n u t i o n i n the r a t e of glutamine synthesis due to absence of (Table 12, Condition IV). H igher concentrations of m e d i u m KC1 than 5 mM, do not affect the amount of g l u t a - mine synthesized i n one hour (Table 12), although the T i s s u e / M e d i u m concen- t r a t i o n r a t i o of glutamine is much i n c r e a s e d . The effect of K~*" on glutamine re t e n t i o n in the tissue i s i n a c c o r d with the recent r e s u l t s of M a c h i y a m a et a l . (156). T A B L E 12. Effects of the cationic contents of the Incubation medium on the rate of glutamine synthesis in rat brain cortex slices. Rat brain cortex slices were incubated in 0 2 at 37°C for one hour in incubation media of varying composition with glucose present. The tissue and total glutamine levels (expressed as umole/g initial wet wt.) and that for the medium (expressed as urnole/g initial wet wt./3 ml . ) are given. Oxygen uptakes are expressed as umole/g initial wet wt. and water uptakes as ul/100 mg initial wet wt. Condition MEDIUM COMPOSITION (mM) Oxygen Uptake Water Uptake Glutamine Content Total Content of glutamine % Control NaCl KC1 C a C l 2 E G T A Ouabain TISSUE MEDIUM T O T A L I (Control) 128 5 2.8 - - 101 + 6 14.0 + 1.5 3.45 + 0.30 2.92 + 0.28 6.37 100 II 128 5 128 5 2.8 2.8 - 0.01 0.1 98 + 3 96 + 2 19.2 + 1.0 38.0 + 1.1 0.31 + 0.05 0.71 + 0.01 1.86 + 0.12 1.64 + 0.07 2.17 2.35 34 37 i n 78 5 178 5 2.8 2.8 - 90 + 3 9 5 + 2 22.2 + 0.4 15.9 + 1.1 4.38 + 0.15 1.96 + 0.21 2.97 + 0.15 3.01 + 0.20 7.35 4.97 115 77 IV 128 128 30 128 55 128 105 2.8 2. 8 2.8 2.8 - - 110 + 1 122 + 2 126 + 2 144 + 2 18.3 + 0.5 17.5 + 1.4 31.8 + 3.0 38.1 + 0.5 1.58 + 0.05 4.06 + 0.40 4.93 + 0.05 5.38 + 0.22 2.70 + 0.20 2.40 + 0.30 1.29 + 0.07 1.43 + 0.25 4.28 6.46 6.22 6.81 67 101 98 107 v 128 5 128 5 ~ 3 128 + 5 134 + 5 18.0 + 2.0 19.3 + 0.5 1.96 + 0.20 1.05 + 0.04 2.41 + 0.14 1.96 + 0.07 4.3 7 3.01 68 47 VI 178 •- 3 - 105 + 3 26.9 + 1.1 0.56 + 0.01 1.42 + 0.07 1.98 31 00 -si 88 4.3 E f f e c t s of c a l c i u m on the r a t e of g l u t a m i n e s y n t h e s i s by r a t b r a i n c o r t e x s l i c e s G l u t a m i n e synthetase, a M g r e q u i r i n g enzyme, i s c o m p e t i t i v e l y i n h i b i t e d by Ca**(135). However, r e s u l t s g i v e n i n T a b l e 12 ( C o n d i t i o n V), show that the i n c u b a t i o n of r a t b r a i n c o r t e x s l i c e s i n a K r e b s - R i n g e r phos- phate g l u c o s e m e d i u m c o n t a i n i n g Ca"*"* b r i n g s about a h i g h e r net g l u t a m i n e s y n t h e s i s than i n a m e d i u m d e v o i d of the c a t i o n . The d i m i n i s h e d s y n t h e s i s due to the absence of C a * * i s even m o r e m a r k e d by the p r e s e n c e of E G T A i n the i n c u b a t i o n medium. In the absence of C a * * the t i s s u e g a i n s N a * and l o s e s K * (see T a b l e 66). A s a consequence of su c h i o n i c changes, the s o d i u m pump i s s t i m u l a t e d . T h e r e i s a f a l l i n the A T P l e v e l (see T a b l e 74). A d i m i n i s h e d A T P content of the t i s s u e , due to the absence of c a l c i u m ions, may p o s s i b l y be the r a t e l i m i t i n g f a c t o r i n the s y n t h e s i s of g l u t a m i n e . On t h i s view, the e f f e c t of c a l c i u m i n m a i n t a i n i n g h i g h A T P l e v e l s i n the t i s s u e may be r e s p o n s i b l e i n d i r e c t l y f o r ++ the a p p a r e n t l y anomalous b e h a v i o r of Ca R e s u l t s r e p o r t e d e a r l i e r ( C hapter 3) a r e f u r t h e r e v i d e n c e f o r the i m - p o r t a n t r o l e of C a * * i n the s y n t h e s i s of g l u t a m i n e i n the i n t a c t b r a i n t i s s u e . T h e s e r e s u l t s obtained w i t h r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n a g l u c o s e f r e e medium, d e m o n s t r a t e the r e q u i r e m e n t of C a * * f o r the s y n t h e s i s of g l u t a m i n e f r o m e x t e r n a l l y added g l u t a m a t e ( s e c t i o n 3.7), and f o r the s t i m u l a t o r y e f f e c t of 5 |_lM p r o t o v e r a t r i n e on g l u t a m i n e s y n t h e s i s w i t h an a c c o m p a n y i n g s u p p r e s s i o n of a m m o n i a f o r m a t i o n ( s e c t i o n 3 . 9 ) . A l l these r e s u l t s a r e u n d e r s t a n d a b l e on +4- the b a s i s of C a being n e c e s s a r y to m a i n t a i n a s u f f i c i e n t l y h i g h A T P c o n - c e n t r a t i o n i n the b r a i n t i s s u e to b r i n g about an o p t i m a l r a t e of g l u t a m i n e s y n t h e s i s . 89 4.4 Glutamine synthesis in a Na" l"-rich, K -free, Ca f - f r e e , incubation medium. F r o m the above considerations, it would be expected that the inhibition in the rate of glutamine synthesis in brain slices would be most evident in a Na"*-rich, K~* and C a ^ - f r e e K r e b s - R i n g e r phosphate glucose medium (Medium II) for absence of K + (115) or C a + + (Table 74) leads to a marked f a l l in the A T P concentration. This is, in fact, what occurs as shown in Table 12 (Condition VI). Under these conditions, the water uptake is enhanced over the control value (Condition I), but the oxygen consumption is little affected . 4.5 Glutaminase activity and glutamine synthesis We have seen that, when bra in cortex slices are incubated in K r e b s - Ringer phosphate glucose medium in the absence of Ca*'*, or in the presence of ouabain, or in glucose containing M e d i u m II, there is an inhibition of the rate of glutamine synthesis as shown by the diminished total (tissue + medium) levels of glutamine at the end of the incubation period. Evidence was p r e - sented earl ier (Chapter 3) showing that the incubation of brain tissue in such media does not promote glutamine breakdown by enhancement of glutaminase activity (section 3.8). The fact that the rate of ammonia formation f r o m 5 m M L-glutamine, added to glucose free media containing ouabain or devoid of C a + + is constant (17-18 Llmole/g initial wet wt/hr), demonstrates that brain glutaminase activity is unaffected by these conditions. 4.6 Effects of sodium L-glutamate and N H ^ C l on the inhibition of glutamine synthesis in rat brain cortex slices incubated with glucose either in M e d i u m II, or in the presence of ouabain Results given in Table 13 show that in the presence of 5 m M L-glutamate, or of 5 m M N H ^ C l , the rate of glutamine synthesis by rat brain cortex slices T A B L E 13. Effects of N H ^ C l and sodium L-glutamate in reversing the suppressed rates of glutamine synthesis in rat brain cortex slices due to ouabain or changed media composition. Rat brain cortex slices were incubated in O2 at 3 7 ° C for one hour in the presence of glucose under media conditions given below. N H 4 C I or L-glutamatc when present were 5 m M and Ouabain O . l m M , Tissue glutamate levels, the total and tissue glutamine levels are expressed as umole/g initial wet wt. and those for the medium as |iinole/g initial wet wt./3 m l . Oxygen uptakes (QO?) are given as nmolc/g initial wet wt. and water uptakes as (jl/100 mg. initial wet wt. Medium II was a N a + r ich, K + and Ca free medium (see Section 2.4 (iii)). Water Tissue* Glutamine Content Total Content MEDIA COMPOSITION of glutamine u p t a K e oiutamate TISSUE M E D I U M Total % Control Krcbs-Rinner phosphate-glucose (Control) 101 + 6 14.0 + 1.5 8.44 + 0.21 3.45 + 0.30 2.92 + 0.28 6.37 100 + ouabain 98 + 3 38.0 + 1.1 4. 95 _+ 0.23 0. 71 + 0.01 1 . 64 + 0. 07 2.35 37 + L-glutamate 117 + 10 34. 7 + 0.7 23.50 _+ 0.17 5 .67 + 0. 17 4.86 + 0.19 10.34 163 + L-glutamatc + ouabain 98 + 1 37.9 + 1.5 12.48 + 1.12 1.20 + 0. 06 4. 76 + 0.22 5 .96 94 + N H 4 C I 111 + 1 14. 6 + 0.1 5.98 + 0.16 4. 78 + 0.19 5.10 + 0.21 9.88 155 + N H 4 C I + ouabain 88 + 2 49.5 + 0.3 3. 71 + 0.11 1.68 _+ 0. 03 4.92 + 0.20 6.68 104 + L-glutamate + N H 4 C I 104 + 3 35.9 + 0.7 14.52 + 0.18 5.16 + 0. 15 7.37 + 0.84 12.53 197 + L-glutamatc + N H 4 C I + 84 + 3 41.2 + 1.2 4. 82 + 0. 14 0.73 + 0. 04 5.01 + 0.43 5.74 90 ouabain Medium II - glucose 105 + 3 26.9 + 1.1 9.09 + 0.68 0.56 + 0.01 1.42 + 0.07 1.98 31 + L-glutamate 106 + 2 37.5 + 0.3 18.77 + 0.63 2.82 + 0.38 4.62 + 0.14 7.44 116 + N H 4 C I 108 1 30.5 + 1.5 7.85 + 0.13 2.79 + 0.41 4.17 + 0 .14 6.96 109 + N H 4 C I + L-glutamate 109 + 1 43.5 + 1.5 13.70 + 0 .40 2.51 + 0.33 5.05 + 0.40 7.56 119 91 incubated i n 0 2 for one hour at 3 7 ° C i n a n o r m a l K r e b s - R i n g e r phosphate gluco s e medium, i s enhanced. T h i s i s also found to be true when the incu- bation is c a r r i e d out i n glucose containing M e d i u m II. Both L - g l u t a m a t e and NH4"* cause some ele v a t i o n of the rate of r e s p i r a t i o n of the b r a i n s l i c e . Glutamate and/or NĤ ~** only p a r t i a l l y r e v e r s e s the i n h i b i t i o n caused by ouabain or by incubation i n M e d i u m II. However, the r e v e r s a l seems to be g r e a t e r in M e d i u m II, than i n the m e d i u m containing ouabain. F o r example, when both L-glutamate and NH^"* are present, the percentage i n c r e a s e s i n glutamine synthesis (calculated on the b a s i s of L-glutamate + NH^* f r e e controls) are (90 - 37) = 53 i n the ouabain containing incubation m e d i u m and (119 - 31) = 88 i n M e d i u m II. F r o m these and other r e s u l t s r e p o r t e d in T a b l e 13 it seems that the rate of glutamine synthesis i s dependent on the oua b a i n - s e n s i t i v e t r a n s p o r t of N H ^ " ^ and/or glutamate to the site of glutamine synthesis. Since the K m for N H 4 is e x t r e m e l y low, 0.18 m M (95) and a l s o since NH^ are not a c t i v e l y accumulated i n b r a i n s l i c e s (Chapter 6), it i s re a s o n a b l e to conclude that the t r a n s p o r t of glutamate, which i s known to be impeded by ouabain (Table 18), may be a r a t e - l i m i t i n g f a c t o r . Another factor l i m i t i n g glutamine synthesis i s the c e l l u l a r l e v e l of A T P , not only r e q u i r e d for active t r a n s p o r t (of glutamate) but also for the synthesis of glutamine. Since ouabain affects the t i s s u e A T P l e v e l s only s l i g h t l y (115), and since the addition of L - g l u t a m a t e does not com- pl e t e l y r e v e r s e the i n h i b i t i o n of glutamine synthesis by ouabain, it follows that i n h i b i t i o n of glutamate t r a n s p o r t cannot be the whole explanation for the ouabain effect. It seems l i k e l y that there is an ouabain induced f a l l of A T P l e v e l in the s p e c i f i c compartment of glutamine synthesis as a r e s u l t of the changed t i s s u e Na /K con c e n t r a t i o n r a t i o . Studies were c a r r i e d out with a v a r i e t y of metabolic i n h i b i t o r s to throw f u r t h e r light on this phenomenon. 92 4.7 Co m p a r a t i v e effects of met a b o l i c i n h i b i t o r s (methionine sulfoximine, ouabain, fluoroacetate, malonate, D N P and amytal) on r a t e s of oxygen uptakes, glutamine synthesis and amino a c i d contents of r a t b r a i n cortex s l i c e s incubated i n the presence of glucose The r e s u l t s of a study of the effects of metabolic i n h i b i t o r s on the total (tissue + medium) ra t e s of glutamine synthesis and oxygen uptakes of rat b r a i n c o r t e x s l i c e s incubated i n K r e b s - R i n g e r phosphate glucose m e d i u m are given i n T a b l e s 14 and 15, r e s p e c t i v e l y . A l s o given i n Table 14 are the total (tissue + medium) values f o r ammonia and amino acids obtained on incubation of the b r a i n t i s s u e i n the p r e s e n c e of the i n h i b i t o r s . The r e s u l t s are s u m m a r i z e d below. 1. Methionine sulfoximine (5 mM), a known inhibitor of glutamine synthetase, i s without any effects on oxygen consumption, but inhib i t s the f o r m a t i o n of glutamine and b r i n g s about an i n c r e a s e d l i b e r a t i o n of ammonia. Glutamate and other amino a c i d contents a r e l i t t l e a ffected. 2. Ouabain (0.01 mM), has no effect on oxygen consumption i n one hour, but d e p r e s s e s it by 25 per cent in four hours. It s u p p r e s s e s the rate of glutamine synthesis and concomitantly enhances ammonia output i n one and four hours. G A B A l e v e l s are elevated i n one hour, but not s i g n i f i c a n t l y i n 4 hour incubation, while alanine l e v e l s are enhanced i n both the one and four hour incubation p e r i o d s . Glutamate and other amino a c i d l e v e l s do not change s i g n i f i c a n t l y under these conditions. 3. F l u o r oacetate (3 mM), does not affect r e s p i r a t i o n but inhi b i t s the f o r m a t i o n of glutamine i n one hour and elevates the rate of ammonia f o r m a t i o n . The G A B A content is elevated but the aspartate l e v e l f a l l s to a s m a l l extent. Glutamate, and other amino a c i d contents s e e m not to be affected with f l u o r o a c e t a t e . T A B L E 14. Effects of metabolic inhibitors on the rate of glutamine synthesis and amino acid contents of incubated rat brain cortex sl ices . Rat brain cortex slices were incubated in Krebs-Ringer phosphate glucose medium in at 3 7 ° C , in the presence or absence of ouabain (0. ImM), DL-mcthioninc DL-sulfoximine (5mM), sodium malonate (2mM), sodium fluoroacetate (3mM), sodium amytal (ImM), and D N P ( O . l m M ) . Total (tissue + medium) values are expressed as Limole/g initial wet wt. Addition to the Incubation Medium Glutamate Glutamine G A B A Aspartate Alanine Glycine Ammonia One hour incubation N i l 10.11 + 0.30 6.35 + 0. 18 2.81 + 0. 12 3.84 + 0.21 1. 29 + 0. 02 1.53 + 0.11 5.12 0.35 + methionine ) sulfoximine ) 10.91 + 0.18 2.50 + 0.20 2.81 + 0. 10 4.03 + 0.17 1. 41 + 0. 07 1.41 + 0.07 11.20 + 0.20 + ouabain 11.29 + 0.14 2.35 + 0.08 4.32 + 0 .29 3.88 + 0.27 1. 92 ± 0. 08 1.61 + 0.11 10.92 + 0.29 + fluoroacetate 11.46 + 0.32 3. 64 + 0. 12 3.63 0. 15 3.23 + 0.23 1. 35 0. 02 1.47 0.03 10.61 + 0.21 + nvilonate 8.75 + 0.26 6.00 + 0.28 3.98 + 0. 12 1.20 + 0.03 1. 17 + 0. 10 1.23 + 0.06 6.67 + 0.44 + amytal 10.62 + 0.42 2.94 _+ 0.20 6.03 + 1.11 2.50 + 0.04 1. 53 + 0.09 1.15 + 0. 19 8.00 + 0. 14 + DNP 9.69 + 0.40 3.48 + 0. 15 4.50 + 0.38 2.59 + 0.25 2. 47 0. 12 1.53 + 0.06 11.43 + 1.01 Four hour incubation Nil 6.82 + 0.51 11.40 + 0.50 1.85 + 0. 18 2.49 + 0.14 1. 04 + 0.01 1.68 + 0.10 6.66 0.25 + methionine ) sulfoximine ) 7.95 + 0.80 1.26 + 0.40 1 .95 + 0.08 3.76 + 0.08 3. 40 + 0. 11 1.62 + 0.13 20.32 + 1.02 + ouabain 6.52 + 0.33 1.96 + 0.24 2.25 + 0.23 2.64 + 0.05 2. 76 + 0. 19 1.74 + 0.14 18.20 + 0.30 + amytal 10.82 + 0.42 2.41 + 0.21 7.75 + 0.32 2.38 + 0.30 2. 96 0 .09 2.16 + 0.11 9 .92 + 0.58 +DNP 8.42 + 0.14 3.42 + 0.12 4.15 + 0.56 2 .53 + 0.05 3. 52 + 0.02 2. 13 + 0.08 16.20 + 0.50 T A B L E 15. E f f e c t s of ouabain , methionine sulfoximine, KC1, 2,4 dinitrophenol, amytal, and glucose absence, on the rates of oxygen consumption i n r a t c e r e b r a l c o r t e x s l i c e s . C e r e b r a l c o r t e x s l i c e s of the rat were incubated i n K r e b s - R i n g e r phosphate glucose m e d i u m at 37°C f o r v a r i o u s periods of time . C o n t r o l values f o r oxygen consumption (QO^) a r e e x p r e s s e d as p m o l e / g i n i t i a l wet wt. Other values of Q O £ are given as percentages of the c o n t r o l , with standard deviations f r o m the mean not exceeding + 5%. T i m e C o n t r o l Q o 2 - % C o n t r o l (min) Q O z Ouabain (0. ImM) Methionine Sulfoximine (5mM) KCl(lOOmM) 2, 4 - D N P (O.lmM) A m y t a l (ImM) Gl u c o s e f r e e m e d i u m 15 24 + 2 108 98 123 96 72 89 30 46 + 3 106 101 124 93 55 83 45 71 + 1 102 102 123 90 48 73 60 9 6 + 2 103 102 128 85 43 66 90 141 + 5 - 103 127 79 - 55 120 192 + 7 88 103 127 71 26 47 180 283 + 12 88 104 121 55 20 38 240 376 + 14 75 101 - 41 16 31 95 Malonate (2 mM), which st r o n g l y in h i b i t s r e s p i r a t i o n , has l i t t l e or no effect on the rate of glutamine synthesis or ammonia fo r m a t i o n . It strongly inhibits aspartate f o r m a t i o n and enhances that of G A B A . T h e r e are diminutions in glutamate and g l y c i n e l e v e l s . F u r t h e r experiments c a r r i e d out with malonate and fluo r o a c e t a t e w i l l be d e s c r i b e d l a t e r (Chapter 8). D N P at a con c e n t r a t i o n of 0.1 mM, which i n h i b i t s oxygen uptake, in h i b i t s glutamine synthesis and at the same time enhances ammonia, G A B A and alanine l e v e l s in one and four hour i n c u b a - ti o n p e r i o d s . Glutamate is l i t t l e a f fected but aspartate l e v e l s f a l l d u ring the one hour incubation. D N P (0.01 mM) sti m u l a t e s r e s p i r a t i o n with no effects on glutamine and ammonia l e v e l s . A m y t a l (1 mM), b r i n g s about a strong i n h i b i t i o n of oxygen con- sumption, accompanied by a s u p p r e s s e d rate of glutamine synthesis, which i s only p a r t i a l l y accountable for by i n c r e a s e d a m m o n i a production. T h e r e i s a substantial i n c r e a s e i n the G A B A l e v e l . The content of glutamate is, unaffected i n one hour but a higher l e v e l i s obtained in four hours. The i n h i b i t i o n of the rate of glutamine synthesis is doubtless p a r t l y due to the in h i b i t i o n of the N A D * - l i n k e d glutamate oxidation, through s u p p r e s s i o n of N A D H oxidation, and t h e r e f o r e of ammonia r e l e a s e f r o m glutamate, and p a r t l y due to the s u p p r e s s e d ge n e r a t i o n of A T P . A n elevated endogenous glutamate concen t r a t i o n r e s u l t s in an enhanced G A B A content a c c o r d i n g to the following scheme. C 0 0 , ^ ±, 2 ^ 1 Glutamate + s u c c i n i c s f i m i a l H s h y d s ^ ^ G A B A + cv-keto glutarate NAD + N H A + N A D + H -^s u c c i n at e-<- 96 Since both a-ketoglutarate and s u c c i n i c semiaLdehyde r e q u i r e N A D * for oxidation, the operation of the c i t r i c a c i d c y c l e and its G A B A by-pass are both s u p p r e s s e d i n the p r e s e n c e of amytal, due to a g r e a t l y reduced NAD+/NADH con c e n t r a t i o n r a t i o . Inter conver sion of glutamate and G A B A may r e v e r s i b l y occur, but it i s the i r r e v e r s i b l e d e c a r b o x y l a t i o n of glutamate that t r i p s the balance in f a v o r of G A B A accumulation. 4.8 E f f e c t s of tetrodotoxin on ouabain in h i b i t i o n of glutamine synthesis in r a t b r a i n c o r t e x s l i c e s . Chan and Quastel (198) have demonstrated that the i n h i b i t i o n of acetate oxidation in incubated r a t b r a i n cortex s l i c e s due to sodium influx induced by e l e c t r i c a l stimulation, i s r e v e r s e d by T T X . Okamoto and Q u a s t e l (182) have shown that the influx of N a * that o c c u r s on incubating b r a i n s l i c e s i n the p r e s e n c e of ouabain (0.1 mM) i s p a r t i a l l y s u p p r e s s e d by T T X . Such eff e c t s of T T X may be p r e s u m e d to be due to its action on the s o d i u m c u r r e n t s y s t e m at the b r a i n c e l l membrane and t h e r e f o r e confined to the neurons. Since we have shown that the ti s s u e N a * and K* a r e f a c t o r s con- t r o l l i n g the synthesis of glutamine, and also since ouabain is known to affect the c a t i o n i c concentrations of b r a i n t i s s u e (see Table 65), the question a r i s e s to whether T T X w i l l r e v e r s e (at l e a s t p a r t i a l l y ) the in h i b i t i o n of glutamine synthesis due to ouabain. R e s u l t s obtained with 0 .0 1 m M ouabain are given i n T a b l e 16. They show that T T X (2 |iM) has l i t t l e or no effect on the ouabain s u p p r e s s e d synthesis of glutamine in r a t b r a i n cortex s l i c e s incubated i n 0 2 at 3 7 ° C for one hour i n K r e b s - R i n g e r phosphate glucose medium. (This was also found to be true when 0.1 m M ouabain was used.) The t i s s u e l e v e l s of glutamate are not d i m i n i s h e d by 0.01 m M ouabain, so that glutamate and NH^* concen t r a t i o n s are not l i k e l y to be l i m i t i n g f a c t o r s i n the synthesis of glutamine. The above r e s u l t s lead to the suggestion that glutamine synthesis o c c u r s T A B L E 16. E f f e c t s of tetrodotoxin on the ouabain s u p p r e s s e d glutamine synthesis of rat b r a i n c o r t e x s l i c e s . Rat b r a i n c o r t e x s l i c e s were incubated i n a K r e b s - R i n g e r phosphate glucose m e d i u m containing (0. OlmM) ouabain with or without the addition of tetrodotoxin (2[iM) at 37°C f o r one hour. T i s s u e values a r e e x p r e s s e d as p m o l e s / g i n i t i a l wet wt. and m e d i u m values as p m o l e s / g i n i t i a l wet wt. / 3 m l . medium. Ouabain Ouabain + T e t r o d o t o x i n T i s s u e M edium T o t a l T i s s u e M e d i u m T o t a l Glutamate 9. 93 + 0.53 0. 95 + 0.07 10. 88 9.41 + 0.03 0. 72 + 0.22 10. 13 Glutamine 0. 23 + 0. 04 2. 11 + 0.27 2. 34 0.43 + 0.06 2.36 + 0. 08 2. 79 A s p a r t a t e 3. 76 + 0.40 0. 50 + 0.10 4. 26 3.63 + 0.33 0.45 + 0. 05 4.08 G A B A 2. 47 + 0.47 0. 19 + 0.01 2. 66 2. 83 + 0.59 0. 10 + 0. 06 2.93 G l y c i n e 0. 85 + 0. 03 0. 62 + 0.03 1. 47 0.95 + 0.10 0.49 + 0.08 1.44 A l a n i n e 0. 70 + 0.05 1. 14 + 0 .09 1. 84 0.78 + 0.05 1.10 + 0o 05 1. 88 A m m o n i a 1. 35 + 0. 08 8. 03 + 0.83 9. 38 1.40 + 0.02 8.19 + 0. 13 9.59 98 i n those b r a i n c e l l s that a r e unaffected by T T X , viz., the glia , but i s by no means proof. T h i s suggestion w i l l be c o n s i d e r e d again when c o n s i d e r i n g the effects of T T X on ouabain induced r e l e a s e of amino acids other than glutamine f r o m b r a i n s l i c e s (Chapter 7). 4.9 Summary 1. The synthesis of glutamine by r a t b r a i n cortex s l i c e s incubated i n the p r e s e n c e of glucose i s suppressed under a v a r i e t y of condi- tions that enhance the tiss u e Na*/K* co n c e n t r a t i o n ratio, e.g., the p r e s e n c e i n the incubation m e d i u m of ouabain (0.1 or 1.0 mM), or metabolic i n h i b i t o r s such as D N P (0.1 mM), amytal (1 mM), or fluoroacetate (1 mM), or when C a " ^ i s omitted. Under these conditions there i s an enhanced rate of ammonia f o r m a t i o n . T h i s i s unlikely to be due to an enhanced glutaminase a c t i v i t y as the rate of ammonia l i b e r a t i o n f r o m added L - g l u t a m i n e i s unaffected by these i n h i b i t o r s . 2. A l t h o u g h Ca"** i s known to inhibit c o m p e t i t i v e l y the Mg** r e q u i r i n g glutamine synthetase i s o l a t e d f r o m b r a i n tissue, it enhances the rate of glutamine synthesis i n b r a i n c o r t e x s l i c e s incubated i n the p r e s e n c e of glucose or L-glutamate. 3. R e s u l t s suggest that ouabain inhibits the synthesis of glutamine by s u p p r e s s i n g the t r a n s p o r t of glutamate to the site of glutamine synthesis and by inducing a l o c a l i z e d drop i n the energy l e v e l of the compartment concerned with much of the synthesis of glutamine in the b r a i n . 4. The inhibi t i o n of glutamine synthesis i n the pr e s e n c e of D N P (0.1 mM), methionine sulfoximine (5 mM), flu o r o a c e t a t e (3 mM), or ouabain (0.1 mM) r e s u l t s i n an enhanced ammonia formation, under conditions where there i s li t t l e or no change in the glutamate 99 content of the t i s s u e . T h i s suggests that glutamine synthesis o c c u r s i n a compartment separate f r o m that containing the mai n bulk of the endogenous glutamate of the b r a i n t i s s u e . 5. A l t h o u g h te t r o d o t o x i n (2 pM) is known to d i m i n i s h the enhanced t i s s u e Na+/K"'* con c e n t r a t i o n r a t i o due to ouabain (0.01 or 0.1 mM), it has l i t t l e or no effect on the ouabain induced s u p p r e s s i o n of glutamine synthesis. T h i s suggests that glutamine synthesis o c c u r s mainly i n a T T X - i n s e n s i t i v e compartment of the b r a i n . 100 5. T R A N S P O R T O F L - G L U T A M A T E I N T O B R A I N IN V I T R O It i s a w e l l known fact that c e r e b r a l cortex s l i c e s r e s p i r i n g in physio- l o g i c a l glucose saline m e d i u m accumulate L-glutamate against a c o n c e n t r a - t i o n gradient (106, 231, 252, 295). We have shown e a r l i e r that the oxidation of endogenous glutamate accounts to a l a r g e extent f o r the l i b e r a t i o n of ammonia and the r e s p i r a t i o n of b r a i n s l i c e s incubated i n the absence of gluco s e (Chapter 3). However, even i n the absence of C a * * when the u t i l i z a t i o n of ammonia (through the synthesis of glutamine) i s s e r i o u s l y impeded, rat b r a i n s l i c e s give only r e l a t i v e l y s m a l l i n c r e a s e s i n the rate of ammonia f o r m a t i o n when L-glutamate i s added to a g l u c o s e - f r e e m e d i u m (see T a b l e 8). T h i s could be due to the slow t r a n s p o r t (or penetration) to the site of ammonia f o r m a t i o n where p r e s u m a b l y the major pool of endogenous glutamate i s present. We have c a r r i e d out ex p e r i m e n t s i n an attempt to throw light on the actual site in the b r a i n of the major pool of exogenous L- g l u t a m a t e taken up by the t i s s u e . 5.1 T r a n s p o r t of L-glutamate into incubated r a t b r a i n R e s u l t s of the effects of v a r y i n g the m e d i u m L-glutamate c o n c e n t r a t i o n s on the acc u m u l a t i o n of this amino a c i d into r a t b r a i n cortex s l i c e s r e s p i r i n g i n O z at 3 7 ° C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m a r e gi v e n i n Table 17. The t i s s u e values of glutamate obtained, in t e r m s of (imole/g i n i t i a l wet wt, a r e c o r r e c t e d for swelling so as to give values of concentrations i n the t i s s u e water. F o r i n i t i a l m e d i u m glutamate concentra- tions of 2.5, 5, 10, 20 and 30 mM, values f or the t i s s u e to m e d i u m concentra- ti o n r a t i o s for glutamate are 9.0, 5.7, 2.9. 1.9 and 1.6 r e s p e c t i v e l y . It i s , therefore, evident that exogenous glutamate i s accumulated against a concen- t r a t i o n gradient i n r a t b r a i n c o r t e x s l i c e s . It may be noted f r o m values i n T a b l e 17 that the active component of glutamate t r a n s p o r t has r e a c h e d its satu r a t i o n l e v e l at an e x t e r n a l glutamate concentration between 2.5 and 5.0 mM. Increments of glutamate in the m e d i u m above this c o n c e n t r a t i o n T A B L E 17. T r a n s p o r t of sodium-L-glutamate into b r a i n c o r t e x s l i c e s of the r a t . Rat b r a i n c o r t e x s l i c e s were incubated i n 0 2 at 37°C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m containing i n c r e a s i n g concentrations of sodium-L-glutamate. The m e d i u m Na* c o n c e n t r a t i o n was adjusted to 148 (aequiv/ml by appropriate reductions of m e d i u m N a C l . Values of tiss u e glutamate obtained, as p m o l c / g i n i t i a l wet wt. (Tg), were converted to \smole/ml t i s s u e water (T) by talcing t i s s u e s w e l l i n g into c o n s i d e r a t i o n . (Tg-E) values were obtained by sub t r a c t i n g the endogenous t i s s u e glutamate (E=8.44 pmole/g) f r o m the glutamate contents of s l i c e s incubated with e x t e r n a l glutamate; these values were then converted to nmole/ml t i s s u e water (T*) taking s w e l l i n g into c o n s i d e r a t i o n . M e d i u m L - glutamate (mM) p m o l e / g i n i t i a l wet wt. Water Uptake. H 1/100 mg i n i t i a l wet wt. Tis s u e glutamate |imole/ m l M e d i u m glutamate (mM) after incubation M T i s s u e glutamate M e d i u m glutamate T i s s u e glutamate T g Net glutamate uptake (Tg - E) T T* T / M T*/M N i l 8.44 + 0.5S 14.8 + 0.6 8.9 0.018 493.00 2.5 16. 60 + 1. 00 8.16 33.8 + 0.4 14.6 7.2 1.62 + 0. 07 9 .01 4.44 5.0 23.50 + 0. 70 15.06 34.0 + 0.7 20.5 13.2 3.59 + 0. 03 5.71 3.66 10.0 27. 60 + 2.00 19.16 35.2 + 1.9 24.0 16.6 8.25 + 0. 10 2.90 2.01 20.0 38.00 + 2.80 29.56 31.8 + 2.9 34.0 26.4 18.00 1.89 1.47 30. 0 49.20 + 1.40 40.76 1 31.2 + 0.8 44.3 36.7 27.50 1.61 1.33 102 only r e s u l t in p a s s i v e inflow of glutamate into the t i s s u e . F o r example, an i n c r e a s e i n the m e d i u m glutamate conce n t r a t i o n f r o m 5 to 30 m M (i.e., by 25 mM), enhances the ti s s u e concentration by about the same value. V a l u e s a r e also given i n T a b l e 17, for the net i n c r e a s e s of t i s s u e glutamate. T h e s e are obtained by subtracting the t i s s u e c o n c e n t r a t i o n s of glutamate on incubation i n the absence of e x t e r n a l glutamate, f r o m those obtained on incubation with e x t e r n a l l y added glutamate. The t i s s u e to m e d i u m concentration r a t i o s f o r glutamate, using net uptake values, are given in F i g u r e 1 i n which a c o m p a r i s o n is made of the uptake of glutamate with that of N H 4 + . 5.2 The uptake of L - [ U - ^ C ] glutamate by r a t c e r e b r a l c o r t e x s l i c e s R at c e r e b r a l c o r t e x s l i c e s were incubated at 37° C i n under a v a r i e t y of conditions i n the p r e s e n c e or absence of 5 mM, L - f U - ^ c ] glutamate (of s p e c i f i c a c t i v i t y 1.223 x 10^ cpm/|_imole). A t the end of the incubation period, the glutamate contents of the t i s s u e were m e a s u r e d by the f o l l o w i n g two methods: 1. L i q u i d s c i n t i l l a t i o n counting. T h i s method gives a m e a s u r e of the r a d i o a c t i v e glutamate taken up by the t i s s u e . 2. A m i n o A c i d A n a l y z e r e s t i m a t i o n s . T h i s method gives a m e a s u r e of the tota l glutamate content of the t i s s u e by the n i n h y d r i n r e a c t i o n . The s p e c i f i c a c t i v i t y of the m e d i u m glutamate was l i t t l e affected at the end of the incubation p e r i o d by the incubation procedure, though there were f a l l s i n the m e d i u m concentrations of glutamate. R e s u l t s given i n T a b l e 18, show that the t i s s u e to m e d i u m con c e n t r a t i o n r a t i o s f o r l a b e l l e d glutamate are c o n s i d e r a b l y lower than those for total glutamate content of the t i s s u e . However, it i s evident that, if only net glutamate uptakes are T A B L E 18. Uptake of sodium L - ( U - C) glutamate by rat brain cortex slices. Rat brain cortex slices were incubated in C>2 at 3 7 ° C in glucose containing media under conditions given below. Medium II was a N a + - r i c h K + and C a + + - f r c e medium. Ouabain when added was (0. ImM) and sodium L - ( U - * 4 C ) glutamate was 5mM of specific activity 1.223 x 10^ cpm/ umole. Medium N a + concentrations was adjusted to 148 uequiv/ml. Labelled and Total tissue glutamate determined respectively by liquid scintillation counting and the Amino Acid Analyzer, as umole/g initial wet wt., were converted to umolc /ml tissue water (T), taking tissue swelling into consideration. Net glutamate uptake in umole/g initial wet wt. was obtained by subtracting the tissue value of glutamate after incubation in the absence of external L-glutamatc, from that value obtained at the end of the incubation period in the presence of external L-glutamate; these values were then converted to u m o l e / m l tissue water (T*) taking tissue swelling into consideration. Water Tissue Glutamate Medium T / M Conditions o f incubation Uptake Labelled Total glutamate Ul / lOOmg u m o l e / g U m o l e / U m o l e / g U m o l e / Net glutamate (mM) Labelled Total J )',t initial m l . m l . Uptake after M~ wet wt. u m o l e / g umole / incubation T T m l . T * M A . Krebs - Ringer phosphate glucose (i) 30 min. incubation Control - - - 9.39 + 0.23 - - + L - ( U - 1 4 C ) glutamate 23.6 + 0.5 12.50 + 0.42 12.10. 20.60 + 0.02 19 .90 11.21 10.82 3 . 9 2 + 0.08 3. 1 5.1 2.8 (ii) 60 min . incubation Control - - - 8.44 + 0.33 - + L - ( U - 1 4 C ) glutamate 34.0 + 0.7. 15.40 + 0.30 13.42 23.50 ,+ 0.70 20.48 15.06 13. 14 3.59 + 0.03 3.7 5.7 3.7 + ouabain + ouabain + L - ( U - 1 4 C ) glutamate - - - 4.95 + 0.26 - - 37.9 + 1.9 7.73 + 0.95 6.50 12.48 + 1.12 10.60 7.53 6.42 3.65 + 0 . 0 9 1.8 2.9 1.8 B. Medium 11 - glucose 60 min . incubation Control - - - 9 . 0 9 + 0.68 - - + L - ( U - 1 4 C ) glutamate 37.8 + 0.3 11.20 + 0.20 9.50 18.77 + 0.63 15.98 9.68 8.24 3.22 + 0.08 3.0 5.0 2.6 104 u s e d to c a l c u l a t e the t i s s u e to m e d i u m concent r a t i o n r a t i o s f o r glutamate, the t i s s u e to m e d i u m r a t i o s for both l a b e l l e d and total glutamate are identi- c a l . T h e s e r e s u l t s were obtained in glucose containing m e d i a du r i n g incu- bation p e r i o d s of 30 or 60 minutes, either i n the p r e s e n c e of ouabain (0.1 mM), or when the b r a i n s l i c e s are incubated i n a N a + - r i c h , K + and C a + + - f r e e m e d i u m (Medium II). The r e s u l t s suggest that glutamate taken up f r o m the m e d i u m accumu- la t e s within the t i s s u e i n a compartment d i s t i n c t f r o m another which contains the bulk of the endogenous glutamate. R e s u l t s given l a t e r w i l l indicate that the major pool of endogenous glutamate l i e s i n the neurons (Chapter 8), while that of exogenous glutamate l i e s i n the g l i a . 5.3 A p p a r e n t absence of an exchange p r o c e s s between e x t e r n a l L - [U-1^C] glutamate and endogenous glutamate of rat b r a i n c o r t e x s l i c e s . E x p e r i m e n t s were c a r r i e d out to observe the extent of exchange taking place between the bulk of the glutamate i n i t i a l l y p r e s e n t and that taken up f r o m the incubation medium. B r a i n c o r t e x s l i c e s of the rat were incubated at 3 7 ° C for 30 minutes in K r e b s - R i n g e r phosphate glucose m e d i u m with or without the addition of 5 mM, L - f U - l ^ c J glutamate (of s p e c i f i c a c t i v i t y 1.Z23 x 10 ̂  cpm/pmole). A t the end of the incubation p e r i o d the t i s s u e contained 1.53 x 10^ cpm/g i n i t i a l wet wt. T h e r e was also p r esent 20.6 pmole glutamate/g i n i t i a l wet wt t i s s u e . T h i s c o r r e s p o n d s to a s p e c i f i c a c t i v i t y 0.75 x 10^ cpm/pmole, as s u m i n g that the en t i r e r a d i o a c t i v i t y is located i n the glutamate. The amount of glutamate absorbed by the t i s s u e f r o m the incubation m e d i u m must then be (1.53 x 10^/1.223 x 10^) = 12.5 pmole/g, as the s p e c i f i c a c t i v i t y of the incuba- t i o n m e d i u m i s 1.223 x 10^ cpm/nmole. T h e r e f o r e , 20.6 — 12.5 = 8.1 (imole glutamate/g do not exchange with m e d i u m glutamate because, had there been f r e e exchange between l a b e l l e d glutamate taken up by the t i s s u e and the pool 105 of endogenous glutamate in the tissue, the s p e c i f i c a c t i v i t y of glutamate within the t i s s u e would equal that outside i t . Now, in the absence of e x t e r n a l glutamate, we find that the total glutamate 10.16 l i m o l e / g i n i t i a l wet wt t i s s u e i s d i s t r i b u t e d between t i s s u e (9.39 + 0.23 Limole/g) and m e d i u m (0.77 + 0.08 |jmole/g/3 ml). T h e r e f o r e , it appears that (8.1 x 100)/9.39, or about 87 per cent, of the endogenous t i s s u e glutamate does not exchange with l a b e l l e d glutamate added to the incubation medium. A c t u a l l y , the percentage i s higher because the total t i s s u e r a d i o a c t i v i t y was used to estimate the l a b e l l e d g l u t a - mate in the tissue, and the r e l a t i v e l y s m a l l amounts of r a d i o a c t i v i t y c o n t r i - buted by glutamate metabolites were not taken into account. The s p e c i f i c a c t i v i t y of the incubation m e d i u m was but l i t t l e changed by the incubation with c o r t e x s l i c e s for 30 minutes. 5.4 L o c a t i o n of exogenous L-glutamate uptake. R a t b r a i n cortex s l i c e s were loaded with r a d i o a c t i v e L-glutamate by p r e - i n c u b a t i o n i n 0 2 at 3 7 ° C for 30 minutes in K r e b s - R i n g e r phosphate glu c o s e m e d i u m containing 5 m M L - f U - ^ ^ c J glutamate of s p e c i f i c a c t i v i t y 1.223 x 10^ cpm/(imole. A t the end of the incubation period, the s l i c e s were qui c k l y removed, l i g h t l y blotted to soak up adhering fluid, weighed on a t o r s i o n balance to measure water uptake, and incubated i n at 3 7 ° C f o r one hour i n one of the following incubation media: A. K r e b s - R i n g e r phosphate glucose medium. B. M e d i u m A, containing p r o t o v e r a t r i n e (5 L M ) and ouabain (0.1 mM). C. M e d i u m B, containing T T X (2 |iM). The p r e s e n c e of p r o t o v e r a t r i n e ensures the a c t i v a t i o n of the s o d i u m c u r r e n t i n b r a i n s l i c e s (182) with r e s u l t a n t effluxes of amino acids (310), while that of ouabain b l o c k s the re-uptake of the r e l e a s e d amino acids against a concen- t r a t i o n g r a d i e n t (104, 112). The p r e s e n c e of T T X by its s e l e c t i v e action on 106 the neurons s u p p r e s s e s the neuronal efflux of amino acids f r o m b r a i n c o r t e x s l i c e s brought about by the joint action of p r o t o v e r a t r i n e and ouabain. E x a m i n a t i o n of the r e s u l t s i n Table 19 show the following f a c t s . 1. The apparent s p e c i f i c a c t i v i t y of glutamate i n the incubation m e d i u m during the one hour incubation following the loading of the t i s s u e with l a b e l l e d glutamate, is much higher than that of the glutamate in the o r i g i n a l incubation medium. T h i s is due to the fact that, during the one hour incubation period, the l a b e l l e d glutamate taken up o r i g i n a l l y by the t i s s u e has been conv e r t e d to other l a b e l l e d amino acids (e.g., aspartate, alanine, glutamine, (Table 20), which a l s o appear i n the incubation m e d i u m (Table 21). The apparently high s p e c i f i c a c t i v i t y i s i l l u s o r y , as only a s m a l l p r o p o r t i o n of the r a d i o a c t i v i t y i n the m e d i u m is due to glutamate. 2. The pr e s e n c e of p r o t o v e r a t r i n e and ouabain b r i n g s about r e l e a s e of a r e l a t i v e l y l a r g e quantity of r a d i o a c t i v i t y f r o m the tissue, together with a c o n s i d e r a b l e r e l e a s e of glutamate. The apparent s p e c i f i c a c t i v i t y of glutamate in the incubation m e d i u m i s much lower than that found i n the absence of the drugs. P r e s u m a b l y this i s due to the r e l e a s e by the drugs of glutamate (both l a b e l l e d and unlabelled) f r o m its l o c a t i o n i n the t i s s u e . 3. A d d i t i o n of T T X s u p p r e s s e s r e l e a s e of glutamate, i n p r e s e n c e of p r o t o v e r a t r i n e and ouabain, f r o m the tissue, without affecting the r e l e a s e of r a d i o a c t i v i t y . T h i s would indicate that T T X is blo c k i n g the r e l e a s e of unlabelled glutamate. T h i s p r e s u m a b l y l i e s i n the neurons as T T X i s b e l i e v e d to act only upon the neurons. T h i s evidence indicates that a l a r g e r pool of endogenous glutamate l i e s i n the neurons. It w i l l be shown l a t e r that this pool i s actually the major pool of glutamate (Chapter 8). T A B L E 19. Effects of protoveratrine ouabain and tetrodotoxin on the radioactivity of brain cortex slices preloaded with sodium L - ( U - ^ C ) glutamate. Rat brain cortex slices were preincubatcd aerobically in Krebs-Ringer phosphate glucose medium with 5mM sodium L - f U - ^ C ) glutamate of specific activity 1.223 x 10"' cpm/umole at 37°C for 30 min. The slices were then incubated aerobically in a Krebs-Ringer phosphate glucose medium at 37°C for one hour, in the presence or absence of protoveratrine (5uM) ouabain (O.lmM) and T T X (2uM). The contents of radioactive glutamate were estimated with the Liquid Scintillation Counter and the amounts of glutamate were estimated with' the Amino Acid Analyzer. Incubation Conditions TISSUE (glutamate) M E D I U M (glutamate) Total radioactivity cprn/g ( x l 0 ° ) Amount U mole/g cpm per U mole (xlO6) Total radioactivity cpm/g/3ml (xl0b) Amount U m o l e / g/3ml cpm per U m o l e (xlO6) Pre-incubation 1.53 + 0,05 20.6 + 0.2 0.075 Incubation: Additions to Krebs-Ringer phosphate glucose medium A . Nil B. protoveratrine + ouabain C . protoveratrine + ouabain + T T X 0.57 + 0.03 0.19 + 0.01 0.38 + 0.01 9 .0 + 0.6 4.1 + 0.4 6.9 + 0.1 0.063 0.046 0.055 0.509 + 0.05 0.902 + 0.01 0.898 + 0.02 1.53 + 0.03 10.50 + 0.34 5.25 + 0.01 0.332 0.086 0.171 108 4. T T X bri n g s about a retention of r a d i o a c t i v i t y i n the b r a i n c o r t e x s l i c e s , incubated in p r e s e n c e of p r o t o v e r a t r i n e and ouabain, equal to (0.38 - 0 .19) (100) /1.53 = 12.4 per cent of the r a d i o a c t i v i t y o r i g i n a l l y taken up (Table 19). T h i s amounts to 1.55 pmole glutamate/g i n i t i a l wet wt tissue, i f it i s assumed that the r a d i o - a c t i v i t y r e t a i n e d by T T X i s wholly due to glutamate. The amount w i l l be s m a l l e r as the value must include r a d i o a c t i v i t y due to glutamate metabolites. T h i s r e s u l t i n d i c a t e s that T T X has r e l a t i v e l y l i t t l e affect i n s u p p r e s s i n g the r e l e a s e of l a b e l l e d glutamate f r o m the t i s s u e and, therefore, that much of the l a b e l l e d glutamate l i e s not i n the neurons, but in the non-excitable c e l l s , i.e., the g l i a . 5.5 E f f e c t s of i n c r e a s i n g e x t e r n a l s o d i um L-glutamate concentrations on the t i s s u e and m e d i u m concentrations of amino ac i d s i n incubated r a t b r a i n cortex s l i c e s . In addition to the work on the uptake of e x t e r n a l L - g l u t a m a t e by incubate r a t b r a i n s l i c e s , studies were also made of the effects of i n c r e a s i n g m e d i u m concentrations of L - g l u t a m a t e (up to 30 mM) on the concentrations of amino aci d s i n the t i s s u e (Table 20), and in the m e d i u m (Table 21). The m e d i u m N a C l was adjusted to give a m e d i u m s o d i u m co n c e n t r a t i o n of 148 |iequiv/ml. The r e s u l t s given i n T a b l e s 20 and 21 show that the t i s s u e and m e d i u m and, hence, the total l e v e l s of aspartate, GABA, alanine, and glutamine, a r e enhanced by i n c r e a s i n g e x t e r n a l L - g l u t a m a t e concentrations. Other amino acids, e.g., glycine (shown), taurine, serine, or threonine (not shown), a r e l i t t l e affected under these conditions. Neither a r e the concentrations of ammonia i n t i s s u e or m e d i u m affected. M o s t of the change in the concentra- ti o n of glutamine, alanine or aspartate, i s brought about by the lowest m e d i u m concen t r a t i o n s (i.e., 2.5 mM) of L-glutamate investigated. I n c r e a s i n g T A B L E 20. E f f e c t s of i n c r e a s i n g m e d i u m sodium L-glutamate concentrations on the amino a c i d contents of incubated rat b r a i n cortex s l i c e s . B r a i n c o r t e x s l i c e s of the r a t were incubated a e r o b i c a l l y i n K r e b s - R i n g e r phosphate glucose m e d i u m containing i n c r e a s i n g m e d i u m sodium L-glutamate concentrations at 37°C f o r one hour. T h e m e d i u m s o d i u m was adjusted to 148 p e q u i v / m l by appropriate reductions of m e d i u m N a C l . Amounts of amino acids i n the t i s s u e a r e e x p r e s s e d as |jmole/g i n i t i a l wet wt. M e d i u m L-glutamate (mM) Glutamate Glutamine G A B A As p a r t a t e A l a n i n e G l y cine N i l 8. 44 + 0. 58 3. 07 + 0. 26 2. 44 + 0.04 2. 72 + 0.14 1. 09 + 0.11 1. 06 + 0.01 2.5 16. 60 + 1. 00 5. 27 + 0. 35 3. 51 + 0.04 4. 28 + 0.86 1. 92 + 0.25 1. 02 + 0.02 5.0 23. 50 + o. 70 5. 67 + 0. 17 3. 64 + 0.07 5. 03 + 0.10 1. 90 + 0. 04 0. 98 + 0.05 10.0 27. 60 + 2. 00 5. 06 + 0. 80 4. 62 + 0.02 3. 91 + 0.23 1. 87 + 0.02 1. 05 + 0.01 20.0 38. 00 + 2. 80 5. 44 + 0. 38 4. 70 + 0.10 4. 30 + 0.32 1. 61 + 0.08 0. 96 + 0.04 30.0 49. 20 + 1. 40 4. 97 + 0. 58 6. 60 + 0.20 4. 39 + 0.71 1. 55 + 0. 11 0. 93 + 0.20 T A B L E 21. E f f e c t s of i n c r e a s i n g m e d i u m sodium L-glutamate concentrations on the r e l e a s e of amino acids f r o m incubated r a t b r a i n cortex s l i c e s . The e x p e r i m e n t a l conditions were as d e s c r i b e d i n T a b l e 20. Amounts of amino acids i n the m e d i u m are e x p r e s s e d as p m o l e / g i n i t i a l wet wt. t i s s u e / 3 m l . medium. M e d i u m L - glutamate (mM) Glutamate Glutamine G A B A A s p a r t a t e A l a n i n e G l y c i n e N i l 0.63 + 0.10 3. 34 + 0.01 0.03 + 0.01 0. 54 + 0.01 0. 60 + 0.05 0.34 + 0. 04 2.5 1.62 + 0.07 m M 4. 66 + 0.16 0.03 + 0.01 3. 88 + 0. 73 1. 39 + 0.03 0.38 + 0. 04 5.0 3.59 + 0.03 m M 4. 87 + 0 .19 0.15 + 0.03 4. 12 + 0. 02 1. 75 + 0.16 0.44 + 0. 04 10.0 8.25 + 0.10 m M 5. 42 + 0.12 0.22 + 0.01 5. 64 + 0.30 1. 64 + 0.23 0.37 + 0. 07 20.0 - 5. 86 + 0.06 0.45 + 0.02 7. 36 + 0.40 2. 24 + 0.31 0.53 + 0. 02 30.0 7. 10 + 0.45 5. 01 + 0.08 2. 46 + 0.17 0.46 + 0. 02 I l l m e d i u m glutamate concentrations, enhances the G A B A l e v e l s in the t i s s u e and effects a s m a l l r e l e a s e of G A B A into the incubation medium. With 2.5 mM, ex t e r n a l L-glutamate, there i s about a twofold i n c r e a s e i n the tissue, or medium, content of alanine; the t i s s u e and m e d i u m contents of aspartate a r e i n c r e a s e d by 50 per cent and 700 per cent, r e s p e c t i v e l y . 5.6 S u m m a r y 1. T h e r e i s an apparent absence of a f r e e exchange p r o c e s s between endogenous glutamate and l a b e l l e d L -glutamate taken up by b r a i n c o r t e x s l i c e s against a concentration gradient. L e s s than 13 per cent of the endogenous glutamate appears to have f r e e a c c e s s to l a b e l l e d L-glutamate taken up by the t i s s u e . 2. A d d i t i o n of T T X s u p p r e s s e s the neuronal r e l e a s e of glutamate brought about by the joint action of p r o t o v e r a t r i n e and ouabain f r o m b r a i n s l i c e s p r e - l o a d e d with l a b e l l e d L -glutamate without affec t i n g the r e l e a s e of r a d i o a c t i v i t y . T h i s suggests that the endogenous pool of glutamate l i e s i n T T X - s e n s i t i v e compart- m e n t s ) , i.e., the neurons, while L-glutamate taken up by the ti s s u e i s l a r g e l y p r e s e n t i n a T T X - i n s e n s i t i v e compartment(s), i.e., the g l i a . Under these conditions T T X b r i n g s about a m a x i m a l re t e n t i o n of 12.4 per cent of the r a d i o a c t i v i t y o r i g i n a l l y taken up by the t i s s u e amounting to a value for tis s u e glutamate no l a r g e r than 1.55 (imole/g i n i t i a l wet wt. 3. R e s u l t s on the effects of i n c r e a s i n g e x t e r n a l L - g l u t a m a t e con- centrations on the amino a c i d content in, and r e l e a s e from, incubated b r a i n c o r t e x s l i c e s , are given. 112 6. T R A N S P O R T O F A M M O N I U M IONS IN B R A I N IN V I T R O A c c o r d i n g to Ja c o b s et a l . (253, 254), the rate of t r a n s p o r t of ammonium ions into e r y t h r o c y t e s cannot be m e a s u r e d by present methods as ammonia p a s s e s the membrane so r a p i d l y . However, P o s t and J o l l y (255) used an i n d i r e c t method in their studies and showed that the addition of N H 4 to N a + - f i l l e d r e d c e l l s br ings about a high rate of Na"*" t r a n s p o r t (outward). Saturating the t r a n s p o r t s y s t e m with K"* (by incubating with e x t e r n a l K"*) produces only a s m a l l i n c r e a s e in Na"* t r a n s p o r t by NH4"*" ions. They concluded that NĤ "** substitutes d i r e c t l y for i n the s o d i u m pump of ery t h r o c y t e s , but r e q u i r e a con c e n t r a t i o n 3-7 tim e s that of to produce a com p a r a b l e effect. Tower and his colleagues (256, 257) studied the t r a n s p o r t of NH^* in cat b r a i n c o r t e x s l i c e s . T h e y incubated the t i s s u e at 3 7 ° C f o r one hour i n the p r e s e n c e of 10 m M N H ^ C l and, since they obtained a higher i n t e r n a l (i.e., tissue) than e x t e r n a l (i.e., medium) NH^"* concentration at the end of the incubation period, they concluded that there was an " a c t i v e " or energy- + + dependent uptake of f r e e NH^ . In a t y p i c a l e x p e r i m e n t c i t e d (256), the N H 4 content ( c o r r e c t e d for swelling) was 11.35 (imole/g while the m e d i u m concen- t r a t i o n at the end of the incubation was 8.52 (imole/ml. T h i s gave an apparent "NH^** s p a c e " in the t i s s u e of 133 per cent, which they s a i d was c l e a r l y an i m p o s s i b l e value unless i n t e r p r e t e d to mean an " a c t i v e " c o n c e n t r a t i o n of N H 4 + i n the i n t r a c e l l u l a r spaces of the t i s s u e . The work with rat b r a i n c o r t e x s l i c e s r e p o r t e d in this chapter i s i n agreement with the values of NĤ "*" obtained by Tower and coworkers, but our i n t e r p r e t a t i o n d i f f e r s c o n s i d e r a b l y f r o m t h e i r s . We conclude that NH 4"* ions are not taken up against a concentration in b r a i n c o r t e x s l i c e s , but endogenous NH."* is f o r m e d within s p e c i f i c compartments in the b r a i n t i s s u e . independently of the concentration of NĤ "*" i n the incubation medium. The su m of the t i s s u e endogenous NĤ "*" and that which has entered by p a s s i v e i i d i f fusion, gives the appearance of " a c t i v e " N H ^ t r a n s p o r t . Recently- p u b l i s h e d data of Wherett and Tower (76) support our i n t e r p r e t a t i o n . The r e s u l t s of experiments d i r e c t e d to throw light on N H ^ + t r a n s p o r t into b r a i n a r e d e s c r i b e d below. 6.1 T i s s u e and medium contents of NH."*" of ra t b r a i n c o r t e x s l i c e s 4 incubated under v a r y i n g conditions. Some of the studies on the d i s t r i b u t i o n between tissue and m e d i u m of the a mmonia formed, when b r a i n c o r t e x s l i c e s of the ra t are incubated i n K r e b s - R i n g e r phosphate m e d i u m under v a r y i n g incubation conditions, are gi v e n i n Table 22. It i s evident f r o m the r e s u l t s i n this Table, that the ammonia contents of the t i s s u e r e m a i n a p p r o x i m a t e l y constant under many di f f e r e n t incubation conditions. The r e s u l t s show that the v a r i a t i o n i n the r a t e s of ammonia f o r m a t i o n by r a t c e r e b r a l c o r t e x s l i c e s incubated i n media of d i f f e r i n g contents, are l a r g e l y confined to the NH^* contents of the incuba- t i o n medium. F o r example, the incubation m e d i u m concent r a t i o n of NĤ "*" in the absence of glucose is more than three t i m e s that in its p r e s e n c e . M o r e - over, the in h i b i t i o n of ammonia u t i l i z a t i o n i n the pr e s e n c e of glucose ( l a r g e l y due to the in h i b i t i o n of the synthesis of glutamine) by ouabain, methionine sulfoximine, fluoroacetate, and 2, 4-dinitrophenol, manifests i t s e l f in i n c r e a s e d m e d i a NH^* contents, while the t i s s u e concentrations r e m a i n more or l e s s the same. These facts are understandable if a mmonia is f o r m e d i n s p e c i f i c compartmentf s) of the b r a i n c e l l s and above a l i m i t i n g c o n c e n t r a t i o n i n such compartments, NĤ "*" i s r e l e a s e d into the c y t o p l a s m and thence into the incubation medium. T h i s i n t e r p r e t a t i o n would explain why a major p r o - p o r t i o n of the ammonia f o r m e d by c e r e b r a l c o r t e x s l i c e s d uring r e s p i r a t i o n under v a r i o u s conditions i s found i n the medium while the t i s s u e r e t a i n s a constant ammonia l e v e l . T A B L E 22. The t i s s u e and m e dium concentrations of N H ^ + on incubating r a t b r a i n c o r t e x s l i c e s under a v a r i e t y of conditions. Rat b r a i n c o r t e x s l i c e s were incubated at 37°C i n K r e b s - R i n g e r phosphate m e d i u m in the p r e s e n c e or absence of glucose o r metabolic i n h i b i t o r s f o r p e r i o d s of t i m e as given below. T i s s u e NH^_+ contents are e x p r e s s e d as p m o l e / g i n i t i a l wet wt. t i s s u e and m e d i u m NH4* contents as l i m o l e / g i n i t i a l wet wt. t i s s u e / 3 m l . A d d i t i o n s to the incubation m e dium T i s s u e N H A + M e d i u m N H 4 + T o t a l N i l ; one hour incubation 2. 66 + 0.27 14. 60 + 0.32 17. 26 + ouabain (O.OlmM) 2. 65 + 0. 15 13.85 + 0.25 16. 50 + m e t h i o n i n e - s u l f o x i m i n e (5mM) 2. 95 + 0.11 16. 76 + 0. 05 1 9 . 7 1 C a ^ - f r e e ; two hour incubation 3. 14 + 0. 13 19.35 + 0.18 22.49 G l u c o s e (lOmM); one hour incubation 1. 89 + 0.16 4. 13 + 0.28 6. 02 + ouabain (O.OlmM) 2. 15 + 0. 05 7.55 + 0.36 9. 70 (0. ImM) 2. 12 + 0. 15 8.80 + 0.44 1 0 . 9 2 + m e t h i o n i n e - s u l f o x i m i n e (5mM) 2. 06 + 0.11 9.36 + 0. 07 11.42 + s o d i u m flu o r c a c e t a t e (ImM) 2. 22 + 0.23 8.62 + 0. 58 10. 84 + ouabain (O.lmM) + fluoroacetate (ImM) 2. 38 + 0.26 8.89 + 0.65 11.27 + 2,4 D N P (0. ImM) 1. 96 + 0.22 9 . 9 0 + 0.55 11. 86 + s o d i u m malonate (2mM) 2. 30 + 0.34 5.40 + 0.20 7. 70 G l u c o s e (lOmM); four h o u r incubation 1. 73 + 0.05 4.93 + 0. 10 6.66 + m e t h i o n i n e - s u l f o x i m i n e (5mM) 2. 9 0 + 0.42 17.42 + 0.50 20. 32 1 i 5 6.2 T i s s u e / M e d i u m concentration r a t i o s for N H 4 in the incubated b r a i n s l i c e . F u r t h e r evidence i n support of the concept of compartmentation of ammonia f o r m a t i o n in b r a i n c e l l s i s obtained f r o m a study of the T i s s u e / M e d i u m concentration r a t i o s for ammonia at the end of one hour incuba- tion p e r i o d s . The T i s s u e / M e d i u m concen t r a t i o n r a t i o s are l a r g e r than one, under a l l incubation conditions tested. R e s u l t s of a few t y p i c a l examples a r e g i v e n i n Table 23. These r e s u l t s are c a l c u l a t e d f r o m values r e p o r t e d i n Table 22, taking swelling of the tiss u e into c o n s i d e r a t i o n . F r o m these r e s u l t s i t can be seen that even in the pr e s e n c e of 10 |iM ouabain (a potent i n h i b i t o r of active t r a n s p o r t p r o c e s s e s ) in a glucose containing medium, the r a t i o is as high as 9. If the T i s s u e / M e d i u m co n c e n t r a t i o n r a t i o s were due to a c c u m u l a t i o n of NH^* against a concent r a t i o n gradient, a l l evidence (104) in d i c a t e s that the p r e s e n c e of ouabain would reduce this to near unity. A n a e r o b i c incubations (see T a b l e 26) or incubation for lengthy durations (e.g., 2 hours), have l i t t l e effects on the tissue ammonia content, and the T i s s u e / M e d i u m concen t r a t i o n r a t i o s for NĤ "*" are c o n s i d e r a b l y l a r g e r than one on incubation of the s l i c e s with 0.1 m M D N P ( T / M = 5.2), or with 2 m M s o d i u m malonate ( T / M = 12.3), or in the absence of glucose ( T / M = 4.0). Such conditions are known to reduce energy dependent t r a n s p o r t against a con c e n t r a t i o n gradient. The fact that high values are s t i l l r e a c h e d is evidence against active t r a n s p o r t . The r e l a t i v e l y high T i s s u e / M e d i u m co n c e n t r a t i o n r a t i o s of NH^"* f o r b r a i n c o r t e x s l i c e s incubated with glucose ( T / M = 14.5) is held to be due to the fact that while the tissue NH^* concentration i s l i t t l e affected, the total rate of N H 4 " * * f o r m a t i o n is much diminished, so that r e l a t i v e l y l i t t l e N H 4 " * i s r e l e a s e d into the incubation medium. T h i s r e s u l t s in a high T i s s u e / M e d i u m r a t i o for ammonia. T A B L E 23. T h e ti s s u e to medium concentration r a t i o s f or NH4 i n r a t b r a i n cortex s l i c e s incubated i n a v a r i e t y of media. T i s s u e values of ammonia are obtained f r o m T a b l e 22 and e x p r e s s e d as ^ m o l e / m l tis s u e water,taking into c o n s i d e r a t i o n the swe l l i n g of the tissue at the end of the incubation p e r i o d . M e d i u m values of ammonia, f r o m T a b l e 22, a r e e x p r e s s e d as p m o l e / m l taking the average i n i t i a l wet wt. of the t i s s u e i n v e s t i g a t e d to be 100 mg. The medium NH4* concentration ( p m o l e / m l = mM) is the m e d i u m value (Table 22) div i d e d by 3 x 1 0 = 30. Add i t i o n s to K r e b s - R i n g e r phosphate m edium T i s s u e NH 4+ |i mole / g i n i t i a l wet wt. T i s s u e N H 4 + \1 m o l e / m l T M e d i u m N H 4 + m M M _T_ M N i l ; one hour incubation 2.66 2. 13 0.49 4.4 + ouabain (O.OlmM) 2.65 2. 10 0.46 4.6 + me t h i o n i n e - s u l f o x i m i n e (5mM) 2.95 2.36 0.56 4.2 C a ^ - f r e e ; two hour incubation 3.14 2. 12 0.64 3.3 Gl u c o s e (lOmM); one hour incubation 1.89 2. 02 0. 14 14. 5 + ouabain (0. OlmM) 2.15 1 . 9 6 0.25 7. 8 (O.lmM) 2.12 1. 80 0 . 2 9 6.2 + met h i o n i n e - s u l f o x i m i n e (5mM) 2.06 2. 15 0.31 6.9 + s o d i u m fluoroacetate (ImM) 2.22 2.30 0 . 2 9 7 . 9 + ouabain (0. ImM) + fluoroacetate (ImM) 2.38 2. 06 0.30 6 . 9 + 2, 4-DNP (0. ImM) 1 . 9 6 1. 70 0.33 5.2 + s o d i u m malonate (2mM) 2.30 2.23 0. 18 12.3 i 17 6.3 Exogenous N H ^ + a c c u m u l a t i o n in c e r e b r a l c o r t e x s l i c e s of the r a t Having demonstrated the existence of a concentration of NĤ "*" i n c e r e b r a l c o r t e x s l i c e s incubated i n v a r i o u s media, e x p e r i m e n t s were c a r r i e d out to examine the accumulation of NH^* within r a t b r a i n c o r t e x incubated f o r one hour at 3 7 ° C i n the p r e s e n c e of v a r y i n g concentrations of N H ^ C l added to the incubation medium. These studies were c a r r i e d out under the following incubation conditions, the water uptakes by the t i s s u e being m e a s u r e d at the end of the incubation. 1. A e r o b i c a l l y in the p r e s e n c e of 10 m M glucose ( T a b l e 24). 2. A e r o b i c a l l y in the absence of glucose (Table 25). 3. A n a e r o b i c a l l y in the p r e s e n c e of 10 m M gluco s e (Table 26). It i s seen f r o m r e s u l t s g i v e n in T a b l e 24, that when the N H ^ + content of the t i s s u e found in the absence of added N H ^ C l is subtracted f r o m the t i s s u e NĤ "*" contents obtained at the end of the one hour incubation with e x t e r n a l l y added N H 4 + , the T i s s u e / M e d i u m concent r a t i o n r a t i o s a re app r o x i m a t e l y unity. The value of unity is also obtained when the incubation i s c a r r i e d out a e r o b i c a l l y i n the absence of glucose (Table 25), or even a n a e r o b i c a l l y i n the p r e s e n c e of glucose (Table 26). T h i s evidence i n d i c a t e s that there i s only p a s s i v e d i f f u s i o n of N H , + f r o m the incubation m e dium into the s l i c e . The 4 + i n i t i a l c oncentrations of NH^ i n the incubation m e d i u m were used i n the calcu- l a t i o n s of the T i s s u e / M e d i u m co n c e n t r a t i o n r a t i o s . T h i s i s p e r m i s s i b l e since the u t i l i z a t i o n of NĤ "*~ is s u f f i c i e n t l y low not to affect within e x p e r i m e n t a l e r r o r , the m e d i u m NH^ concentration. T A B L E 24. Uptake of N H 4 * by rat b r a i n cortex s l i c e s incubated i n the p r e s e n c e of glucose. Rat b r a i n c o r t e x s l i c e s were incubated in at 37°C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m containing i n c r e a s i n g concentrations of N H 4 C I . Values of t i s s u e N H 4 + obtained as p m o l e / g i n i t i a l wet wt. (Tg) were conver t e d to }jmole/ml t i s s u e water (T) by taking t i s s u e s w e l l i n g into c o n s i d e r a t i o n . (Tg-E) values a r e obtained by subtracting the endogenous tissue N H 4 content (E = 2.5 Umole/g) f r o m the N H 4 contents of s l i c e s incubated with external N H 4 + ; these values were then converted to p.mole/ml t i s s u e water (T*) taking swelling into c o n s i d e r a t i o n . M e d i u m N H 4 C I (mM) pmole/g i n i t i a l wet wt. Water Uptake p l / 1 0 0 mg i n i t i a l wet wt. T i s s u e N H 4 + \l m o l e / m l ti s s u e water M e d i u m N H 4 C I (mM) afte r i n cubation M T i s s u e N H 4 + M e d i u m N H 4 + T i s s u e N H 4+ T g Net N H 4 + uptake (Tg - E) T / M T*/M T T* N i l 2.50 _+ 0 . 2 0 11.6 + 1.3 2.75 0.13 + 0 . 0 1 2 1 . 2 0 2. 0 4.14 + 0 . 19 1. 64 14.6 + 0.3 4.48 1.74 1.81 + 0 . 1 0 2.48 0.96 5.0 6.68 + 0.40 4. 18 14.6 + 0.2 7.06 4.42 4.40 + 0 . 1 0 1.60 1.00 10. 0 12.42 + 0 . 1 2 9 . 9 2 26.1 + 1.1 11.70 9.32 9.23 + 0 . 2 1 1.27 1 .01 2 0 . 0 26.30 + 0.50 23. 80 40.0 + 4.0 21.90 19.80 19.40 + 0.40 1. 13 1 . 0 2 30. 0 40.40 + 0 . 2 0 37 . 9 0 47.8 + 0.6 3.1.70 29.70 2 9 . 2 0 + 1.00 1 . 0 9 1 . 0 2 co T A B L E 25. Uptake of N H 4 by ra t b r a i n c o r t e x s l i c e s incubated i n the absence of glu c o s e . Rat b r a i n c o r t e x s l i c e s were incubated i n 0 2 at 37°C for one hour in g l u c o s e - f r e e K r e b s - R i n g e r phosphate m e d i u m containing i n c r e a s i n g concentrations of N H ^ C l . C a l c u l a t i o n s were done as d e s c r i b e d i n T a b l e 24. The m e d i u m N H 4 " * concentration was e s s e n t i a l l y unchanged at the end of the incubation p e r i o d . E = 3.66 ( i m o l e / g . M e d i u m N H 4 C 1 M(mM) (i mole / g i n i t i a l wet wt. Water Uptake |i 1/100 mg i n i t i a l wet wt. T i s s u e N H 4 + p, m o l e / m l tiss u e water T i s s u e NH 4+ M e d i u m N H 4 + T i s s u e N H 4+ T g Net • N H 4 + uptake (Tg - E) T / M T*/M T T* N i l 3.66 + 0.18 45.2 + 1 . 2 . 2 . 9 2 2 6.19 + 0.15 2.53 44.2 + 0.8 4.98 2. 03 2.4.9 1.01 5 10.10 + 0.22 6.44 48.2 + 1.2 7.88 5. 02 1.58 1. 00 10 16.00 + 1.20 12. 34 43.0 + 1.3 13. 00 10. 05 1. 30 1.01 20 31.40 + 1.40 27. 74 53.7 + 2.0 23.50 20. 76 1.18 1.04 30 40.10 + 2.70 36.44 42.0 + 1.4 33. 60 29.98 1. 12 1.00 T A B L E 26. The uptake of N H 4 by rat bra in cortex slices in anoxia. Rat b r a i n cortex slices were incubated at 3 7 ° C for one hour in K r e b s - R i n g e r bicarbonate glucose medium containing increasing concentrations of NH4CI in an atmosphere of N^/CO^ (95%/5%). Water uptake was 50 + 3 p l /100 mg initial wet wt. under these conditions. The medium concentration of N H 4 + was essentially unchanged at the end of the incubation p e r i o d . T * = (T - 2. 02) | jmole /ml . tissue water. Medium N H 4 C I M(mM) Tissue N H 4 + T * |imole / m l . T i s sue N H 4 + / M e d i u m N H 4 + p mole Ig initial wet wt. | imole/ml T T / M T * / M N i l 2.63 + 0.26 2.02 - 12.24 - 2 5.28 + 0.59 4.06 2. 04 2.03 1.02 5 9.20 + 0.76 7.06 5.04 1.41 1. 01 10 15.42 + 0.40 11.82 9.84 1.18 0.98 121 6.4 C o m p a r i s o n of the t r a n s p o r t p r o c e s s e s for NH^* and L-glutamate into b r a i n c e l l s . E x p e r i m e n t s were c a r r i e d out to compare the uptake of NH^"* ions with that of an amino acid known to be a ccumulated against a c o n c e n t r a t i o n gradient. Glutamate was chosen for the following r easons: 1. It i s p r e s e n t i n high concentrations i n b r a i n cortex s l i c e s and its l e v e l does not s u b s t a n t i a l l y change on incubation for one hour i n a glucose containing m e d i u m (Table 2). 2. We have de m o n s t r a t e d that exogenous l a b e l l e d L -glutamate only slowly exchanges with the bulk of the glutamate (endogenous) pre s e n t in the t i s s u e (section 5.3). T h i s i s i n a c c o r d with the r e s u l t s of e x p e r i m e n t s c a r r i e d out in this l a b o r a t o r y (258) showing that t i s s u e glutamate l a b e l l e d with r a d i o a c t i v e g l u c o s e does not exchange f r e e l y with c o l d glutamate in a subsequent incubation. 3. We have also d e m o n s t r a t e d that the uptake of l a b e l l e d glutamate by c e r e b r a l c o r t e x s l i c e s is l e s s than the total t i s s u e glutamate m e a s u r e d by the n i n h y d r i n r e a c t i o n using the A m i n o A c i d A n a l y z e r . The d i f f e r e n c e between these two values is about equal to the t i s s u e glutamate content found at the end of the incubation p e r i o d with no glutamate added to the incubation m e d i u m (section 5.2). The r e s u l t s g i v e n in F i g u r e 1 show that, in c o n t r a s t to NĤ ."*", there i s evidence of a c c u m u l a t i o n of glutamate against a concen- t r a t i o n gradient, even when the endogenous content of glutamate i s subtracted f r o m the f i n a l tissue values after incubation. Rat b r a i n cortex s l i c e s were incubated in O 2 at 37°C for one hour in Krebs-Ringer phosphate glucose medium in the presence of increasing concentrations of sodium-L-glutamate or N H 4 C L Values of T i s s u e / Medium concentration ratios (T/M) for L-glutamate and NHv* are taken f r o m Tables 17 and 24 respectively. Medium concentrations (mM) of glutamate or are the concentrations at the beginning of the incu- bation. • Values for L-glutamate; total tissue glutamate contents used to derive T/M concentration ratios for glutamate. O Values for L-glutamate; net glutamate uptake values used to derive T/M concentration ratios lor glutamate. A Values for NH_j+; total tissue NH^ + contents used to derive T/M concentration ratios for N H 4 ' . A Values for NH.jT; net N H ^ uptake values used to derive T/M concentration ratios for N H ^ . 1 6.5 Rates of N H ^ and glycine uptakes by brain cortex s l ices . The kinetics of the passive (?) accumulation of NH^"* were studied and compared with the kinetics of the active uptake of glycine in rat brain cortex slices incubated in 0 2 at 3 7 ° C in K r e b s - R i n g e r phosphate glucose medium. Glycine (5 mM) was used instead of glutamate because in brain it undergoes relatively little metabolic change under the given experimental conditions (114, 128, our observation). F r o m the results given in Table 27, it is seen that, while the amount of glycine taken up by bra in cortex slices against a concentration gradient increases with time, that of N H ^ * reaches its maximum before the commence ment of the incubation. This is further evidence for a rapid movement of N H ^ * into cerebral cortex s l ices . It is to be noted that the concentration of glycine in the tissue (like that of NH^"*") very rapidly attains that in the incuba- tion medium, before the incubation commenced. This is obviously due to a rapid passive component of (glycine) uptake. 6.6 Effects of NH4"*" on glycine uptake. It is a well known fact that the active transport of one substrate into bra in tissue may be competitively inhibited by another substrate, especially when they share the same carr ier system (113, 114). Using labelled glycine (2 mM), Nakazawa and Quastel (114) showed that N H 4 * inhibits the active transport of glycine into cerebral cortex s l ices . We have confirmed this finding and have likewise shown the inhibition occurs also with cationic (KC1, 30 mM) stimulated slices (Table 28). This effect of N H ^ * has been presumed to be due to a fall in the A T P levels, and indeed a smal l drop in the cellular A T P levels does occur with 10 m M N H ^ C l (see Table 71), even though the synthesis of glutamine is enhanced in the presence of N H 4 * ions (section 6.10). T A B L E 27. Rates of NH^ and of glycine uptake by ra t b r a i n c o r t e x s l i c e s . Incubations w e r e ' c a r r i e d out i n G"2 at 37°C f or var i o u s p e r i o d s of t i m e i n K r e b s - R i n g e r phosphate glucose m e d i u m i n the p r e s e n c e either of l O m M N H 4 C I or of 5mM g l y c i n e . Q 0 2 values are e x p r e s s e d as ( i m o l e s / g i n i t i a l wet wt. t i s s u e . T i s s u e contents of ammonia or of glycine a r e e x p r e s s e d as |jmole/g i n i t i a l wet wt. and | i m o le /ml. t i s s u e water. P e r i o d of Incubation T i s s u e N H 4 + content of rat b r a i n cortex s l i c e s at the end of the incubation p e r i o d i n l O m M N H 4 C I ( Q 0 2 = 114 + 4) T i s s u e glycine content of rat b r a i n c o r t e x s l i c e s at the end of the incubation p e r i o d i n 5 m M glycine (QO? = 113 ± 2) Umole/g |i m o l e / m l (imole/g ( i m o l e / m l A f t e r (5 min) oxygenation 8.80 + 0.10 10.04 2.02 + 0.22 2.30 A f t e r (7 min) e q u i l i b r a t i o n 11.05 + 0.57 12.30 6.41 + 0.77 7.00 (i . e . at z e r o time) 15 m i n 11. 73 + 0 . 0 1 12. 76 13.60 + 1.40 14.41 30 m i n 11.15 + 0.15 11.84 23.55 + 2.35 24.30 45 m i n 12. 05 + 0.55 11.99 24.80 + 1.20 25.00 60 m i n 11.64 + 0.32 11. 05 30.00 + 0.22 27.83 T A B L E 28. The i n h i b i t o r y effects of N H 4 + on the active t r a n s p o r t of glycine into r a t b r a i n cortex s l i c e s . Incubation of ra t b r a i n c o r t e x s l i c e s was c a r r i e d out i n 0 2 at 37° C f o r one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m containing 5mM glycine with or without the addition of lOmM, N H 4 C I or of 25mM KC1. Oxygen uptakes ( Q 0 2 ) a r e e x p r e s s e d as (jmole/g i n i t i a l wet wt. t i s s u e . T i s s u e and m e d i u m contents of gl y c i n e d e t e r m i n e d at the end of the incubation are given below. Ad d i t i o n s to K r e b s - R i n g e r Phosphate Glucose M e d i u m Q 0 2 T i s s u e G l y c i n e Content M e d i u m G l y c i n e after incubation M(mM) T_ M Umole/g i n i t i a l wet wt. p.mole/ml. T G l y c i n e 113 + 9 30.0 + 0.2 27.8 4.72 + 0.13 5.9 G l y c i n e + N H 4 C 1 108 + 8 20.0 + 1.2 17.2 5.33 + 0.15 3.2 G l y c i n e + KC1 146 + 2 28.7 + 2.8 24.4 4.67 + 0.05 5.2 G l y c i n e + KC1 + NH 4C1 140 + 4 17.8 + 1.6 14.7 4.81 + 0.27 3.1 126 6.7 E f f e c t s of glycine on NH^ uptake. The effects of glycine (5 mM) on N H 4 + t r a n s p o r t (NH 4C1, 10 mM) in r a t b r a i n c o r t e x s l i c e s i n the p r e s e n c e or absence of 30 m M KC1 are given i n T a b l e 29. It i s evident that g l y c i n e does not affect the uptake of NĤ ."* into the t i s s u e . P o s s i b l y this i s because the t r a n s p o r t of N H 4 + i s a p a s s i v e p r o c e s s . 6.8 Uptake (into b r a i n c o r t e x s l i c e s ) of exogenous L-glutamate and NH^ 4" when p r e s e n t together in the incubation medium. T h i s study c a r r i e d out with L-glutamate, is s i m i l a r to the work d e s c r i b e d above for gl y c i n e (sections 6.6 and 6.7). R e s u l t s obtained are shown i n T a b l e 30. Thus, while N H ^ C l (5 mM) strongly in h i b i t s the uptake of L -glutamate (5 mM) f r o m the incubation medium, L-glutamate has no effect on the uptake of N H 4 + . The NH^"1" i n h i b i t i o n is attributed to the f a l l i n c e l l A T P . I n c r e a s e d t i s s u e u t i l i z a t i o n of glutamate to f o r m glutamine i s i n s u f f i c i e n t to account for the f a l l in t i s s u e glutamate conce n t r a t i o n (Table 13). 6.9 E f f e c t s of metabolic i n h i b i t o r s on the uptake of NH4,"* by incubated r a t b r a i n c o r t e x s l i c e s . E x p e r i m e n t s were c a r r i e d out to observe effects of metabolic i n h i b i t o r s on NH^* uptake into the b r a i n . In Table 31, the effects of v a r i o u s metabolic i n h i b i t o r s on gl y c i n e and NH^"* t r a n s p o r t into r a t b r a i n cortex s l i c e s a re shown. G l y c i n e was used to test the potency of some of the i n h i b i t o r s on the p r o c e s s of active t r a n s p o r t . The addition of f l u o r o a c e t a t e (1 mM), or malonate (2 mM), or D N P (0.1 mM), or ouabain (0.1 mM), has l i t t l e or no effect on the NH^"* con- ce n t r a t i o n s of b r a i n cortex s l i c e s incubated in 0 2 at 3 7 ° C for one hour in K r e b s - R i n g e r phosphate glucose m e d i u m containing 10 m M N H ^ C l . On the other hand, t h e r e i s a m a r k e d i n h i b i t i o n of glycine uptake in the p r e s e n c e of D N P (0.1 mM) 127 T A B L E 2 9 . A b s e n c e of an i n h i b i t o r y effect of gly c i n e on the uptake of a mmonium by r a t b r a i n c o r t e x s l i c e s . Incubation of r a t b r a i n c o r t e x s l i c e s was c a r r i e d out i n 0 2 at 3 7 ° C for one hour i n K r e b s - R i n g e r phosphate m e d i u m containing l O m M N H 4 C I . G l y c i n e and KC1 when added were 5 m M and 2 5 m M r e s p e c t i v e l y . T i s s u e contents of a m m o n i u m (umole/g) at the end of the in c u b a t i o n a r e g i v e n below. 1 A d d i t i o n s to K r e b s - R i n g e r phosphate g l u c o s e m e d i u m T i s s u e A m m o n i u m Limole/g i n i t i a l wet wt. j i m o l e / m l N H 4 C 1 12.42 + 0,12 11.70 N H 4 C I + G l y c i n e 13.35 + 0 . 1 0 11.60 N H 4 C 1 + K C 1 + 14 . 0 0 + 0.40 11.64 G l y c i n e 1 2 8 T A B L E 3 0. E f f e c t s of the simultaneous p r e s e n c e of exogenous s o d i u m L - g l u t a m a t e and N H ^ C l on the t r a n s p o r t p r o c e s s e s f o r a m m o n i u m and glutamate into rat b r a i n c o r t e x s l i c e s . Incubation of r a t b r a i n c o r t e x s l i c e s was done i n 0 2 for one hour at 37 C i n K r e b s - R i n g e r phosphate glucose m e d i u m with or without the addi t i o n of N H 4 C 1 (5mM) o r s o d i u m L - g l u t a m a t e (5mM). T i s s u e contents of a m m o n i u m or glutamate at the end of the inc u b a t i o n p e r i o d a r e e x p r e s s e d as ^ m o l e / g i n i t i a l wet wt. t i s s u e . A d d i t i o n s to K r e b s - R i n g e r phosphate g l u c o s e m e d i u m T i s s u e Contents O f A m m o n i u m Glutamate N H 4 C I L - G l u t a m a t e L - G l u t a m a t e + N H 4 C I 6.68 + 0,40 6.65 + 0.07 23,70 + 0.70 14.52 + 0,18 T A B L E 31. E f f e c t s of m e t a b o l i c i n h i b i t o r s on the t r a n s p o r t of NH4+ and of g l y c i n e into r a t b r a i n c o r t e x s l i c e s . Rat b r a i n c o r t e x s l i c e s were incubated i n at 37°C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m with c i t h e r l O m M N H ^ C l or 5mM glycine. Metabolic i n h i b i t o r s were added as indicated. T i s s u e contents of a mmonia or g l y c i n e are given i n urnole/g i n i t i a l wet wt. t i s s u e and u m o l e / m l . t i s s u e water. A d d i t i o n s to K r e b s - R i n g e r phosphate gluc o s e m e d i u m T i s s u e N H 4 + contents of rat b r a i n cortex s l i c e s at the end of one hour incubation in l O m M NH .4CI T i s s u e glycine contents of rat b r a i n c o r t e x s l i c e s at the end of one hour incubation in 5mM glycine Umole/g u m o l e / m l Umole/g U m o l e/ml No i n h i b i t o r 12.42 + 0.12 11. 70 2 9 . 80 + 0.20 27. 00 + s o d i u m fluoroacetate (ImM) 12.45 + 0.05 11.80 - - + s o d i um malonate (2mM) 12.76 + 0.20 11.18 - - + 2, 4 - D N P (0. ImM) 13.72 + 0.44 11.45 10.28 + 0.14 8. 72 + ouabain (0. ImM) 14.51 + 0.31 11.66 7.39 + 0.14 6. 33 G l u c o s e absent 16.00 + 1.20 13.00 10.68 + 0. 10 8. 68 tNJ 130 or g l u c o s e - l a c k , and a n e a r l y complete in h i b i t i o n of the active t r a n s p o r t com- ponent of g l y c i n e uptake with 0.1 m M ouabain. The above r e s u l t s support the c o n c l u s i o n that N H 4 " * uptake into b r a i n i s by p a s s i v e d i f f u s i o n . 6.10 E f f e c t s of i n c r e a s i n g NH„"'~ concentrations on amino a c i d content in, a 4 and r e l e a s e from, incubated r a t b r a i n c o r t e x s l i c e s . E x p e r i m e n t s were also c a r r i e d out to observe the effects of i n c r e a s i n g m e d i u m N H ^ C l concentrations (tissue, m e d i u m and total) of r a t b r a i n c o r t e x s l i c e s incubated i n 0 2 at 3 7 ° C f o r one hour i n K r e b s - R i n g e r phosphate glucose medium. It i s evident f r o m r e s u l t s given in T a b l e s 32 and 33 that Nrl .4^" ions have profound effects on amino a c i d m e t a b o l i s m . A s expected, there i s a r i s e i n glutamine of both the t i s s u e and medium. The rate of glutamine synthesis, however, seems to have r e a c h e d i t s m a x i m u m value with the lowest l e v e l of N H ^ C l co n c e n t r a t i o n (2 mM) used. T h e r e a r e substantial f a l l s i n the t i s s u e l e v e l s of glutamate and aspartate, the f a l l s i n c r e a s i n g with i n c r e a s i n g concen- t r a t i o n s of e x t e r n a l N H 4 " * . T o t a l (tissue + medium) G A B A l e v e l s are enhanced with i n c r e a s i n g e x t e r n a l N H 4 * * c o n c e n t r a t i o n s . The r e l e a s e of amino a c i d s to the incubation m e d i u m i s l i t t l e affected by e x t e r n a l N H ^ C l concentrations of 10 m M or lower. A t concentrations of 20 or 30 m M N H ^ C l, s i g n i f i c a n t i n c r e a s e s i n the m e d i u m c o n c e n t r a t i o n s of a l l amino acids m e a s u r e d occur. The r e l e a s e of amino acids to the incubation m e d i u m co i n c i d e s with r e l a t i v e l y l a r g e f a l l s i n r e s p i r a t i o n (Table 58), and A T P l e v e l s (Table 71) of the t i s s u e . It would appear that the effects of r e l a t i v e l y high l e v e l s of N H 4 * in the incubations m e d i u m are l a r g e l y due to the f a l l of c e l l A T P accompanying glutamine synthesis. T h e r e appears, however, to be a s p e c i f i c effect on the f o r m a t i o n and r e l e a s e of G A B A . T A B L E 32. Effects of increasing medium N H 4 C I concentrations on the amino acid contents of incubated rat brain cortex slices. Cerebral cortex slices of the rat were incubated in at 37°C for one hour in Krebs-Ringer phosphate glucose medium containing increasing medium N H 4 C I concentrations. Tissue contents of amino acids are expressed as Umole/g initial wet wt. tissue. Initial Medium NH 4C1 (mM) Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine Nil 8.48 ± 0.24 3.26 + 0 . 0 9 1. 80 + 0 . 0 9 2.93 + 0.01 0 . 9 0 + 0. 06 0.90 + 0.08 4.49 + 0.15 2.0 6.37 + 0.25 4.82 + 0. 12 1. 86 + 0.03 1.98 + 0.24 0. 89 + 0. 06 0.89 + 0. 10 4. 04 + 0.04 5.0 5 . 9 8 + 0.16 4. 78 + 0 . 19 2. 13 + 0.14 1.86 + 0.10 0.89 + 0. 04 0.79 + 0. 17 4. 18 + 0. 11 10. 0 5.67 + 0.15 5.32 + 0.23 2. 30 + 0.14 1.66 + 0.06 0. 81 + 0. 03 0.95 + 0.06 3.21 + 0.06 20. 0 4.34 + 0.18 4.07 + 0. 17 2. 60 + 0. 15 1.18 + 0.04 0.52 + 0. 11 0.41 + 0. 10 2.31 + 0.48 30.0 2.71 ± 0.07 3.98 + 0. 11 3. 10 + 0 . 12 0.89 + 0.03 0.42 + 0. 05 0.42 + 0.02 1.75 + 0.20 T A B L E 33. Effects of increasing medium NH^Cl concentrations on the release of amino acids from incubated rat brain cortex slices. The experimental conditions were as described in Table 32. Medium contents of amino acids are expressed as Umole/g initial wet wt. tissue/3 ml. medium. Initial Medium N H 4 C I (mM) Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine Nil 0.54 + 0 . 0 9 3. 13 + 0. 14 0.04 + 0.02 0.32 + 0.02 0.44 + 0.20 0.50 + 0.03 1. 96 0.02 2.0 0.44 + 0.05 5.23 + 0.16 0.04 + 0.03 0.35 + 0.01 0. 38 + 0. 06 0.65 + 0.05 2. 16 + 0.08 5.0 0.53 + 0. 07 5. 10 + 0.21 0.08 + 0.04 0.34 + 0.02 0. 38 0. 06 0. 76 + 0. 11 2. 06 0.02 10.0 0.63 + 0.14 4.41 + 0. 13 0.09 + 0.03 0.37 _+ 0.04 0.57 + 0. 15 0.99 + 0. 17 3. 01 + 0. 1 1 20.0 1.39 + 0. 17 5.50 + 0 . 0 9 0.22 + 0.08 0.58 + 0.06 0.73 + 0. 04 1.11 + 0. 04 3. 44 + 0. 32 30.0 2.72 + 0.10 6.51 + 0.22 0.34 + 0.10 1.04 0.04 0.89 + 0.06 1.15 + 0.21 4. 58 + 0.28 133 6.11 S ummary 1. The NH * concentr a t i o n of incubated rat b r a i n cortex s l i c e s 4 i s l i t t l e affected i n the pr e s e n c e or absence of glucose, or in the p r e s e n c e of meta b o l i c i n h i b i t o r s (such as sodium f l u o r o - acetate, s o d i u m malonate, ouabain, D N P or methionine sulfoximine), or under anoxia. 2. The enhanced r a t e s of NĤ "*" f o r m a t i o n by incubated b r a i n t i s s u e i n the absence of glucose, or in the p r e s e n c e of meta- b o l i c i n h i b i t o r s , a re r e f l e c t e d l a r g e l y i n the enhanced NH^* concentrations of the incubation medium. 3. The NĤ "*" con c e n t r a t i o n of b r a i n c o r t e x s l i c e s incubated with e x t e r n a l N H 4 * i s l i t t l e affected i n the absence of glucose or of oxygen, i n the pr e s e n c e of ouabain or of amino acids such as g l y c i n e or s o d i u m L-glutamate, or in the p r e s e n c e of meta- b o l i c i n h i b i t o r s such as s o d i u m fluoroacetate, s o d i um malonate or DNP. The T i s s u e / M e d i u m concentration r a t i o s f or NĤ "*" become unity when the ammonium con c e n t r a t i o n of the t i s s u e found i n the absence of added N H 4 + i s subtracted f r o m the tis s u e N H 4 + concentrations obtained with e x t e r n a l l y added N H 4 * . 4 . It is concluded that NH^* ions are not taken up against a con- c e n t r a t i o n g r a d i e n t i n b r a i n cortex s l i c e s , but endogenous NH^* i s f o r m e d within s p e c i f i c compartments in the b r a i n t i s s u e independent of the co n c e n t r a t i o n of NH^* i n the e x t e r n a l medium. 5. N H ^ + ions inhibit the active t r a n s p o r t of gly c i n e or L-glutamate p r e s u m a b l y by l o w e r i n g the A T P con c e n t r a t i o n of the t i s s u e . 6. R e s u l t s on the effects of i n c r e a s i n g e x t e r n a l NH^* concen t r a t i o n s on the amino a c i d content in, and r e l e a s e from, incubated b r a i n c o r t e x s l i c e s , a r e given. 134 7. E F F E C T S O F N E U R O T R O P I C D R U G S O N T H E R E L E A S E O F A M I N O A C I D S F R O M T H E B R A I N IN V I T R O The generation of action potentials, or an act i v a t i o n of the Na*- c u r r e n t at the b r a i n c e l l membrane, i s p r e s u m e d to occur in incubated b r a i n c o r t e x s l i c e s under c e r t a i n conditions. It m a n i f e s t s i t s e l f i n an enhanced t i s s u e l e v e l of Na+ and a net efflux of t i s s u e K**\ The c r i t e r i o n used to e s t a b l i s h its o c c u r r e n c e i s its a b o l i t i o n by n e u r o t r o p i c drugs that b l o c k the N a * - c u r r e n t system. A n important tool for this purpose i s tetrodotoxin ( T T X ) . T T X appears not to affect non-excitable c e l l s and only affects c e l l s in the state of excit a t i o n . T T X i s not known to have any d i r e c t effects on metabolic events in the b r a i n . It does not i t s e l f affect the ionic, and as we w i l l show, amino a c i d contents of b r a i n s l i c e s incubated i n a n o r m a l K r e b s - R i n g e r glucose m e d i u m (Introduction, Section 1.9 (i) )• The T T X - s e n s i t i v e N a ^ - c u r r e n t s y s t e m i s activated i n b r a i n c o r t e x s l i c e s i n the p r e s e n c e of p r o t o v e r a t r i n e , or ouabain, or i n the absence of glucos e or of Ca**, or on the a p p l i c a t i o n of e l e c t r i c a l p u l s e s (182), or at the onset of anoxia (192, 205). It should be noted that, while the ac t i v a t i o n of the T T X - s e n s i t i v e N a + - c u r r e n t s y s t e m r e s u l t s in a l t e r e d t i s s u e i o n i c concentrations, not a l l a l t e r a t i o n s in the t i s s u e l e v e l s of cations (as, for example, due to DNP, or high K + or L-glutamate (182) ) are the outcome of an activated Na"*"-current s y s t e m that i s T T X - s e n s i t i v e . It w i l l now be demonstrated that some incubation conditions and a number of ne u r o t r o p i c drugs have profound effects on the r e l e a s e of amino a c i d s f r o m incubated b r a i n t i s s u e . The r e s u l t s of these experiments and thei r p o s s i b l e i m p l i c a t i o n s i n our understanding of the fluxes of c e r e b r a l amino acids are d e s c r i b e d below. 135 7.1 E f f e c t s of p r o t o v e r a t r i n e , ouabain and tetrodotoxin on amino a c i d content in, and r e l e a s e from, rat b r a i n c o r t e x s l i c e s incubated i n glucose - saline media . R e s u l t s r e c o r d e d i n T a b l e s 34 and 35 show the values of the amino aci d s p r e s e n t in the s l i c e s (Table 34) and i n the incubation m e d i u m (Table 35) after incubation of the s l i c e s in a p h y s i o l o g i c a l s a l i n e - g l u c o s e m e d i u m i n for one hour. It w i l l be obs e r v e d that the incubation brought about r e l a t i v e l y s m a l l changes i n the total (i.e., t i s s u e and medium) quantities of amino acids. T h e r e was a r i s e i n glutamine content f r o m 4.40 (imole/g to 6.37 (imole/g, and a f a l l of glutamate content f r o m 11.83 (imole/g to 9.97 (imole/g. T h e r e were s m a l l r i s e s i n the contents of aspartate, y-aminobutyrate, g l y c i n e and alanine. In view of the fact that glutamate i s o x i d i z e d under these conditions (112), g i v i n g r i s e to substantial quantities of aspartate, it i s evident that a steady state i s achi e v e d i n the b r a i n t i s s u e during a e r o b i c incubation in the glucose m e d i u m whereby glutamate l o s s by oxidation is compensated by its gain by other p r o - c e s s e s ; e.g., t r a n s a m i n a t i o n of cv-ketoglutarate by other amino ac i d s i n the t i s s u e . The steady state is p r e s u m a b l y such that efflux of amino acids f r o m the t i s s u e to the incubation m e d i u m i s b alanced by active uptake p r o c e s s e s f r o m m e d i u m to t i s s u e . A d d i t i o n of p r o t o v e r a t r i n e (5 |iM), known to affect the k i n e t i c s of glucose m e t a b o l i s m i n incubated b r a i n s l i c e s (211, 212, 213), and to generate action potentials in nervous t i s s u e (167, 207) brought about s i g n i f i c a n t f a l l s i n the t i s s u e contents of glutamate and aspartate (Table 34) with no c o r r e s p o n d i n g i n c r e a s e s i n the medium (Table 35), and ve r y s m a l l changes (if any) in the quantities of the other amino acids investigated. The changes due to proto- v e r a t r i n e were ab o l i s h e d by the pr e s e n c e of tetrodotoxin (2 )iM) which, itse l f , c a u s e d no changes in the amino a c i d content of tiss u e and m e d i u m (Tab l e s 34 and 3 5). The changes, brought about by pr o t o v e r a t r i n e , in the t i s s u e contents T A B L E 34. Effects of protoveratrine, ouabain, lidocaine and tetrodotoxin on the contents of amino acids in incubated rat brain cortex slices. Amounts of amino acids (expressed as umolc /g initial wet wt. tissue) present in rat brain cortex slices incubated in 0 2 for one hour at 37°C in Krebs-Ringer phosphate medium containing 10 m M glucose in the presence or absence of protoveratrine (5 uM), ouabain (O.lmM), lidocaine (0. 5mM) and tetrodotoxin (2 UM). Additions to the incubation medium Glutamate Glutamine G A B A Aspartate Glycine Alanine Nil 9.20 + 0.14 3.45 + 0.30 2.56 + 0.20 3.45 + 0.23 1. 03 + 0.15 0.75 0.05 Tetrodotoxin 8.82 + 0.01 2.88 + 0.11 2.58 + 0.16 3.64 + 0.20 0.88 + 0.03 0.82 + 0.02 Protoveratrine 6.00 + 0.83 4.10 + 0.39 2.18 + 0.27 2.15 + 0.12 1.01 + 0.06 0.85 + 0.06 Protoveratrine 8.83 + 0.01 3.46 + 0.03 2.60 + 0.16 3.51 + 0.15 1.10 + 0. 10 0.75 + 0.02 + Tetrodotoxin Ouabain 4.95 0.26 0.71 + 0.05 1.39 + 0.06 1.75 + 0.32 0.59 + 0. 03 0.42 + 0.02 Ouabain 6.94 + 0.36 0.53 + 0.04 1.71 + 0.06 3.32 + 0.02 0. 82 + 0.03 0.45 + 0.03 + Tetrodotoxin Initial values prio L 1 - 11. 83 + 1 .09 4.40 + 0.15 2.01 + 0.10 3.36 + 0.34 0. 79 + 0. 06 0.61 + 0.08 to incubation Lidocaine 8.86 + 0.27 3.23 + 0.26 2.25 + 0.14 3.12 + 0.28 0.98 + 0. 11 0.73 0.13 Protoveratrine 9.21 + 0.27 4.11 + 0.04 2.40 + 0.02 3.08 + 0.21 1.08 + 0.03 0.75 + 0.02 + Lidocaine T A B L E 35. Effects of protoveratrine, ouabain, lidocaine and tetrodotoxin on the release of amino acids from incubated rat brain cortex slices. Amounts of amino acids (umole/g initial wet wt. tissue) in the incubation medium (3 ml . ) after incubation of rat brain cortex slices in lor one hour at 37°C in 3 ml Krebs-Ringer phosphate medium containing 10 m M glucose in the presence or absence of protoveratrine (5uM), ouabain (O.lmM), lidocaine (0.5mM) and tetrodotoxin (2 U M ) . Additions to the incubation medium Glutamate Glutamine G A B A Aspartate Glycine Alanine Nil 0.77 + 0.13 2.92 + 0.28 0.03 + 0.02 0.43 + 0.05 0.36 + 0 . 06 0.44 + 0. 04 Tetrodotoxin 0.87 + 0.02 2.62 + 0.13 0.04 + 0.03 0.38 + 0.06 0.27 + 0. 02 0.38 + 0.02 Protoveratrine 0.76 + 0.08 2.83 + 0.66 0.10 + 0.04 0.42 + 0.01 0.36 + 0. 08 0.47 + 0.09 Protoveratrine 0. 60 + 0.06 3. 65 + 0.03 0.05 + 0.01 0.40 + 0.04 0. 19 + 0. 05 0.37 + 0.09 + Tetrodotoxin Ouabain 6.34 + 0.40 2.41 + 0.12 2.93 + 0.35 2.13 + 0.05 1. 12 + 0. 08 1.50 + 0.06 Ouabain 2.18 + 0.36 1.65 + 0.08 2.42 + 0.26 0.95 + 0.26 0.84 + 0. 16 1.15 + 0.03 + Tetrodotoxin Lidocaine 0.84 + 0.13 2.44 + 0.12 0.04 + 0.02 0.50 + 0.11 0.49 + 0. 10 0.35 + 0. 11 Pr oto vc r atr ine 0.62 + 0.02 2.77 + 0.08 0.10 + 0.05 0.36 + 0.04 0.29 + 0.02 0.37 + 0.10 + Lidocaine 138 of glutamate and aspartate could not be due to d i m i n i s h e d r a t e s of c o n v e r s i o n of glucose to glutamate and aspartate as it has been shown (213) that the y i e l d s of l a b e l l e d glutamate and aspartate f r o m l a b e l l e d glucose by incubated r a t b r a i n c o r t e x s l i c e s are enhanced by the p r e s e n c e of p r o t o v e r a t r i n e (5 uM). P o s s i b l y the d i m i n i s h e d t i s s u e contents of glutamate and aspartate were due to their r e m o v a l by oxidation d u r i n g the p r o c e s s of enhanced r e s p i r a t i o n that takes place i n b r a i n t i s s u e i n the p r e s e n c e of p r o t o v e r a t r i n e (211-213). The fact that no m a r k e d changes i n the concentrations of amino ac i d s in the i n c u - bation m e d i u m o c c u r r e d in the p r e s e n c e of p r o t o v e r a t r i n e may only r e f l e c t the fact that the r e l e a s e p r o c e s s e s were balanced by the uptake p r o c e s s e s . E x p e r i m e n t s were, therefore, c a r r i e d out with ouabain p a r t l y because th i s substance i s thought to activate the Na~*"-curr ent s y s t e m at the membrane i n r a t b r a i n s l i c e s incubated in a p h y s i o l o g i c a l saline glucose m e d i u m (182) and p a r t l y because it b l o c k s the active t r a n s p o r t of amino ac i d s into b r a i n s l i c e s (112, 114). Results, given in T a b l e s 34 and 35, show that incubation i n the p r e s e n c e of ouabain brought about extensive changes i n the amount of amino ac i d s i n t i s s u e and medium. T h e r e were l a r g e f a l l s i n the t i s s u e contents of a l l the amino a c i d s investigated and c o r r e s p o n d i n g r i s e s in the amino a c i d concentrations in the incubation medium. The total (tissue and medium) contents of glutamate (11.29 umole/g) or of aspartate (3.88 umole/g) in p r e s e n c e of ouabain were not m a r k e d l y changed f r o m the values (9.97 umole /g and 3.88 umole/g r e s p e c t i v e l y ) obtained in the absence of ouabain. However, the total (tissue and medium) content of glutamine was d i m i n i s h e d ( f r o m 6.37 Umole/g to 3.12 umole/g) and that of G A B A was i n c r e a s e d f r o m 2.59 Umole/g to 4.32 umole/g. A d d i t i o n of T T X (2 |iM) brought about a substantial r e d u c t i o n of the stimulating effect of ouabain on the r e l e a s e of glutamate and aspartate, but had only minor effects on the r e l e a s e of the other amino acids (Table 3 5). 139 These r e s u l t s lead to the i n f e r e n c e that action potentials generated by ouabain and s u p p r e s s e d by T T X (182) bring about the r e l e a s e of glutamate and aspartate f r o m r a t b r a i n c o r t e x s l i c e s incubated in a p h y s i o l o g i c a l saline glucose medium. T h i s becomes evident under the given incubation conditions because the uptake p r o c e s s e s a r e blocked by ouabain. 7.2 E f f e c t s of p r o t o v e r a t r i n e and tetrodotoxin on amino a c i d content in, and r e l e a s e from, r a t b r a i n c o r t e x s l i c e s incubated i n a g l u c o s e - f r e e medium. In view of the fact that a c t i o n potentials appear to be generated, or the Na~*~-current s y s t e m activated, i n K r e b s - R i n g e r phosphate m e d i u m devoid of glucose (182), e x p e r i m e n t s were c a r r i e d out to observe the effects of l a c k of glucos e on the amino a c i d contents of b r a i n t i s s u e and on their r e l e a s e f r o m the t i s s u e i n an incubation p e r i o d of one hour. R e s u l t s given i n T a b l e s 36 and 37 show that the tissue contents of a l l amino acids investigated, save aspartate, were m a r k e d l y redu c e d f r o m the n o r m a l (Tables 34 and 35 ) and that the con- tents of a l l amino acids i n the incubation medium, save glutamine, were i n c r e a s e d , p a r t i c u l a r l y with GABA, aspartate and g l y c i n e . The f a l l i n the t i s s u e c o n c e n t r a t i o n of glutamate was to be expected, as this amino a c i d f o r m s a major f u e l of the b r a i n i n the absence of o r g a n i c substrates f r o m the incuba- tion m e d i u m (see. Chapter 3). M o r e o v e r , as glutamate oxidation i s a c c o m - panied by m a r k e d production of aspartate (Table 2), the r i s e i n the tiss u e con- c e n t r a t i o n of aspartate was a l s o to be expected. It was evident that, with e v e r y amino a c i d investigated, the rat i o of t i s s u e c o n c e n t r a t i o n to the m e d i u m con c e n t r a t i o n is g r e a t l y d i m i n i s h e d f r o m the n o r m a l by incubation of the t i s s u e i n the g l u c o s e - f r e e medium. T h i s r e s u l t i s perhaps p a r t l y due to the fact that there i s depletion of t i s s u e A T P in the absence of glucose with consequent s u p p r e s s i o n of active uptake of the amino a c i d s . The p o s s i b i l i t y that the generation of action potentials (on an ac t i v a t i o n of the N a ^ - c u r r e n t system) plays a s i g n i f i c a n t r o l e in the r e l e a s e of amino ac i d s T A B L E 36. Effects of protoveratrine, tetrodotoxin and lidocaine on contents of amino acids in rat brain cortex slices incubated in glucose-free media. Amounts of amino acids (umole/g initial wet wt. tissue) present in rat brain cortex slices incubated in O 2 f ° r o n c hour at 37°C in Krebs-Ringer phosphate medium in the absence of glucose with or without the addition of protoveratrine (5uM), lidocaine (0.5mM) and tetrodotoxin (2|iM). Additions to the glucose-free incubation medium Glutam ate Glutamine G A B A Aspartate Glycine Alanine Nil 2.03 + 0.33 0.51 + 0.20 1.38 + 0.22 7.79 + 0.27 0.62 + 0. 12 0.17 + 0.04 Tetrodotoxin 3.14 + 0.34 0.48 + 0.20 2.04 + 0.05 8.60 + 0.04 0.91 + 0. 11 0.20 + 0.03 Protoveratrine 1.12 + 0.10 1.17 + 0. 10 0.65 + 0.14 5.68 + 0.42 0.36 + 0. 10 0.07 + 0.02 Protoveratrine 2.57 + 0.01 0.34 + 0.03 1.20 + 0.02 8.43 + 0.01 0. 82 + 0.02 0.08 + 0.01 + Tetrodotoxin Lidocaine 2.36 + 0.11 0.38 + 0.10 1.45 + 0.12 8.41 + 0.21 0. 86 + 0.04 0.21 + 0. 11 Protoveratrine 1.88 + 0.30 0.45 + 0.04 0 . 9 0 + 0.18 8.14 + 0.74 0. 70 + 0.06 0.12 + 0.03 + Lidocaine T A B L E 3 7 . Effects of protoveratrine,tetrodotoxin and lidocaine on the release of amino acids from rat brain cortex slices incubated in glucose-free media. Amounts of amino acids (umole/g initial wet wt. tissue) present in the incubation medium (3 ml . ) after incubation of rat brain cortex slices in O 2 for one hour at 3 7 ° C in the absence of glucose with or without the addition of protoveratrine (5uM), tetrodotoxin (2 uM) and lidocaine (0.5mM). Additions to the glucose-free incubation medium Glutamate Glutamine G A B A Aspartate Glycine Alanine Nil 0.96 + 0.12 1.74 + 0.10 0.41 + 0.08 1.98 + 0.13 0. 70 + 0. 10 0.52 + 0.10 Tetrodotoxin 0.47 + 0.11 1.33 + 0.22 0.35 + 0.04 0.78 + 0.06 0.52 + 0. 12 0.36 + 0.09 Protoveratrine 2.32 + 0.30 2. 61 + 0.18 0.93 + 0.07 4.31 + 0.32 1.02 + 0. 10 0.72 + 0.03 Protoveratrine 0.53 + 0.03 1.79 + 0.04 0.42 + 0.02 1.21 + 0.07 0.47 + 0. 01 0.33 + 0.02 + Tetrodotoxin Lidocaine 0.48 + 0.06 0.81 + 0.05 0.32 + 0.05 0.89 + 0.10 0. 61 + 0. 07 0.53 + 0.05 Protoveratrine 1.06 + 0.32 1.28 + 0.10 0.63 + 0.02 1.86 + 0.52 0.65 + 0. 06 0.54 + 0.06 + Lidocaine 142 f r o m the b r a i n tissue, incubated i n a g l u c o s e - f r e e medium, was shown by the effects of the addition of T T X (2 uM) to the incubation medium. T T X brought about significant retention in the ti s s u e of glutamate, GABA, aspar- tate and gly c i n e (Table 36), and reduced concentrations of amino acids, p a r t i c u l a r l y glutamate and aspartate, in the incubation m e d i u m (Table 37). The amino a c i d c o n c e n t r a t i o n r a t i o (tissue to medium) was s i g n i f i c a n t l y i n c r e a s e d , with glutamate, GABA, aspartate and glycine, when T T X was pr e s e n t in the incubation medium. A d d i t i o n of p r o t o v e r a t r i n e (5 uM) to the g l u c o s e - f r e e incubation m e d i u m brought about an even m ore pronounced l o w e r i n g of the t i s s u e con- c e n t r a t i o n of glutamate, GABA, and gly c i n e (Table 36). The t i s s u e aspartate c o n c e n t r a t i o n was also diminished, but the ti s s u e concentration of glutamine was enhanced. These changes i n t i s s u e concentrations of amino acids, brought about by pr o t o v e r a t r i n e , were accompanied by notable i n c r e a s e s i n the concentration of amino acids, p a r t i c u l a r l y glutamate, G A B A and aspartate i n the incubation m e dium (Table 37). That these effects of p r o t o v e r a t r i n e were l a r g e l y due to the ge n e r a t i o n of action potentials was shown by the fact that addition of T T X either abolished, or g r e a t l y reduced, the ac t i o n of proto- v e r a t r i n e i n promoting amino a c i d r e l e a s e f r o m the t i s s u e into the incubation m e d i u m (Tables 36 and 37). The amino a c i d c o n c e n t r a t i o n r a t i o (tissue to medium) found i n the p r e s e n c e of p r o t o v e r a t r i n e , was enhanced by the addition of T T X f r o m 14 to 143 with glutamate, f r o m 21 to 86 with GABA, f r o m 39 to 209 with aspartate, f r o m 10 to 52 with glycine; it f e l l , however, f r o m 13 to 5 with glutamine (Table 42). A noteworthy effect of the pr e s e n c e of pr o t o - v e r a t r i n e was the c o n s i d e r a b l e r i s e i n the total content of glutamine. A value of 2.25 umole/g (0.51 + 1.74) found i n the absence of p r o t o v e r a t r i n e r o s e to 3.78 umole/g (1.17 + 2.61) in the p r e s e n c e of the drug (Tables 36 and 37; see als o T a b l e 10). Such a m a r k e d r i s e did not oc c u r when T T X was present; nor was it so c l e a r l y evident when glucose was pr e s e n t i n the incubation m e d i u m (Tables 34 and 35). 143 7.3 E f f e c t s of p r o t o v e r a t r i n e and tetrodotoxin on amino a c i d content in, and r e l e a s e from, rat b r a i n c o r t e x s l i c e s incubated i n c a l c i u m - d e f i c i e n t media. A b s e n c e of C a ^ * f r o m the gl u c o s e - c o n t a i n i n g incubation m e d i u m brought about r e l a t i v e l y s m a l l changes f r o m the n o r m a l i n the amino a c i d contents of the t i s s u e s l i c e s after a e r o b i c incubation f o r one hour ( T a b l e s 34 and 38), though there seemed to be a s i g n i f i c a n t f a l l in the t i s s u e content of glutamine. A s e r i e s of e x p e r i m e n t s was c a r r i e d out with E G T A added to the c a l c i u m - d e f i c i e n t incubation m e d i u m in or d e r to secure as complete a r e m o v a l as p o s s i b l e of c a l c i u m ions f r o m the medium. The r e s u l t s showed no s i g n i f i c a n t changes of amino a c i d contents in either the t i s s u e or the incubation m e d i u m f r o m those found in the absence of E G T A . A d d i t i o n of p r o t o v e r a t r i n e (5 M.M) to the c a l c i u m - d e f i c i e n t m e d i u m brought about changes i n the t i s s u e contents of amino acids s i m i l a r to those found i n the n o r m a l incubation m e d i u m (Tables 34 and 38), but it caused sub- st a n t i a l i n c r e a s e s i n the efflux of glutamate, GABA, and aspartate ( T a b l e s 3 5 and 39). These effects of p r o t o v e r a t r i n e were a b o l i s h e d by the addition of T T X (Table 39). The amino a c i d co n c e n t r a t i o n r a t i o s (tissue: medium) found i n the p r e s e n c e of p r o t o v e r a t r i n e , i n the c a l c i u m - d e f i c i e n t media, rose, on addition of T T X with glutamate f r o m 80 to 468, with G A B A f r o m 86 to 950, with aspartate f r o m 102 to 226, with g l y c i n e f r o m 32 to 76 (Table 42). No s i g n i f i c a n t changes due to T T X o c c u r r e d with glutamine or alanine. The effects of p r o t o v e r a t r i n e i n promoting T T X - s e n s i t i v e amino a c i d effluxes i n incubated r a t b r a i n cortex s l i c e s were c o n s i d e r a b l y g r e a t e r i n the c a l c i u m - d e f i c i e n t m e d i u m than i n the n o r m a l incubation medium, the amino a c i d s most m a r k e d l y affected being glutamate, GABA, aspartate and g l y c i n e . The total quantity of glutamine found in the t i s s u e and medium, at the end of the incubation p e r i o d of one hour, was g r e a t e r i n the p r e s e n c e of p r o t o - T A B L E 38. Effects of protoveratrine, tetrodotoxin, lidocaine and of sodium L-glutamate on the contents of amino acids in rat brain cortex slices incubated in calcium-deficient media. Amounts of amino acids (umole/g initial wet wt, tissue) in rat brain cortex slices incubated in Krebs-Ringer phosphate medium containing 10 m M glucose in 0 2 for one hour at 37°C but with C a ^ + omitted. When present protoveratrine was 5 u M ; tetrodotoxin, 2 u M ; lidocaine, 0 .5mM; and sodium L-glutamate, 2 . 5 m M ; Additions tc the Ca^ + deficient incubation medium Glutamate Glutamine G A B A Aspartate Glyc ine Alanine Nil 9.75 + 0.34 1.96 + 0.02 2.30 + 0.05 2.95 + 0.19 0.88 + 0. 17 0.73 + 0.23 Tetrodotoxin 10.87 + 0.31 2.00 + 0.02 2.44 + 0.10 3.21 + 0.28 0. 76 + 0.12 0.68 + 0.27 Protoveratrine 7.07 + 0.68 2.53 + 0.27 1.55 + 0.06 2.43 + 0.35 0.65 + 0. 11 0.55 + 0.10 Protoveratrine 9.37 + 0.09 1.50 + 0.20 2.56 + 0.48 3.70 + 0.48 0.91 + 0. 02 0.85 + 0.02 + Tetrodotoxin Protoveratrine 10.25 + 0.44 1.83 + 0.20 2.70 + 0.12 3.47 + 0.06 0.81 + 0.03 0.S2 + 0.12 + Lidocaine L-Glutamate 20.17 + 0.09 3.81 + 0.20 3.21 + 0.34 5.69 + 0.07 0.81 + 0.03 0.98 + 0.04 L-Glutamate 22.61 + 1.39 3.82 + 0.21 3.85 + 0.02 6.30 + 0.30 0.99 + 0.08 1.06 + 0.12 + Tetrodotoxin T A B L E 39. Effects of protoveratrine, tetrodotoxin, lidocaine and of sodium L-glutamate on the release of amino acids from rat brain cortex slices incubated in calcium-deficient media. Amounts of amino acids (p.mole/g initial wet wt. tissue) present in the calcium deficient incubation medium (3 ml . ) after incubation of rat brain cortex slices in O2 for one hour at 37°C. When present protoveratrine was 5 u M ; tetrodotoxin, 2 uM; lidocaine, 0 .5mM; and sodium L-glutamate 2 .5mM; Additions to the C a 2 + deficient incubation Glutamate Glutamine G A B A Aspartate Gly cine Alanine medium Nil 1.17 + 0.11 2.41 + 0. 14 0.05 + 0.03 0.45 + 0.05 0.44 + 0.10 0.70 + 0.21 Tetrodotoxin 0.72 + 0.04 2.46 + 0.19 0.03 + 0.02 0.31 + 0.01 0.34 + 0.03 0.80 + 0.06 Protoveratrine 2.63 + 0.30 3.09 + 0.34 0.55 + 0.05 0.71 + 0.04 0. 60 + 0. 10 0.76 + 0.14 Protoveratrine + Tetrodotoxin 0.60 + 0.04 2.21 + 0.04 0.08 + 0.04 0.49 + 0.13 0.36 + 0. 10 0.83 + 0.21 Protoveratrine + Lidocaine 0.83 + 0.06 2.46 + 0.03 0.05 + 0.02 0.51 0.01 0.32 0.01 0.68 + 0.02 L-Glutamatc 1.73 + 0.06 m M 3.39 + 0.18 0.26 + 0.04 2.73 + 0.03 0.38 + 0.07 1.34 + 0.50 L -Glutamate + Tetrodotoxin 1.73 + 0.05 m M 3.69 + 0.11 0.16 + 0.02 2.73 + 0.05 0.26 + 0.02 0.50 + 0.22 146 v e r a t r i n e than i n its absence (Tables 38 and 39). The values of 4.37 umole/g was i n c r e a s e d i n the p r e s e n c e of p r o t o v e r a t r i n e to 5.62 umole/g. It was the only amino acid, among those investigated, that showed a r i s e i n total value on incubation i n the p r e s e n c e of p r o t o v e r a t r i n e . The r i s e was abolished by the addition of T T X . E x p e r i m e n t s were c a r r i e d out to observe whether the addition of s o d i u m L-glutamate (2.5 mM) to the c a l c i u m - d e f i c i e n t m e d i u m would r e s u l t i n i n c r e a s e d effluxes of amino ac i d s f r o m the incubated b r a i n s l i c e s . R e s u l t s g i v e n i n T a b l e s 38 and 39 show that there were s i g n i f i c a n t l y i n c r e a s e d amounts of glutamine, GABA, and aspartate, both i n the t i s s u e and the medium, after incubation f o r one hour, but that these amounts were not m a r k e d l y affected by the p r e s e n c e of T T X . 7.4 E f f e c t s of p r o t o v e r a t r i n e and tetrodotoxin on amino a c i d content in, and r e l e a s e from, rat b r a i n c o r t e x s l i c e s incubated i n g l u c o s e - f r e e , c a l c i u m - d e f i c i e n t , media. 2 + A b s e n c e of both glucose and Ca f r o m the incubation m e d i u m brought about a m a r k e d l y high efflux of amino acids, p a r t i c u l a r l y glutamate, G A B A and aspartate f r o m b r a i n c o r t e x s l i c e s incubated a e r o b i c a l l y f o r one hour (Table 41), the efflux being g r e a t e r than that found i n an incubation m e d i u m 2 + dev o i d only of glucose (Table 37) or of Ca (Table 39). The r a t e s of r e l e a s e of the amino ac i d s were g r e a t l y d i m i n i s h e d by the p r e s e n c e of T T X (Table 41). A m i n o a c i d c o n c e n t r a t i o n r a t i o s (tissue: medium) rose, on addition of T T X (Tab l e s 40 and 41) with glutamate f r o m 23 to 117, with G A B A f r o m 23 to 168, with aspartate f r o m 49 to 210, and with g l y c i n e f r o m 12 to 34 (Table 42). A d d i t i o n of p r o t o v e r a t r i n e to the g l u c o s e - f r e e , C a ^ * - d e f i c i e n t medium, i n c r e a s e d the efflux of a l l amino ac i d s except glutamine and alanine (Table 41), and the efflux was d i m i n i s h e d by T T X . A m i n o a c i d co n c e n t r a t i o n r a t i o s (tissue:medium), found with p r o t o v e r a t r i n e present, r o s e on addition of T T X (Table 42) with glutamate f r o m 17 to 56, with G A B A f r o m 6 to 114, with T A B L E 40. Effects of protoveratrine, tetrodotoxin, lidocaine and of sodium L-glutamate on the contents of amino acids in rat brain cortex slices in glucose-free, calcium-deficient media. Amounts of amino acids (Umole/g initial wet wt. tissue) in rat brain cortex slices incubated in Krebs-Ringer phosphate media in O 2 for one hour at 3 7 ° C with Ca^"*" omitted and in absence of glucose. When present protoveratrine was 5 u M ; tetrodotoxin, 2 uM; lidocaine, 0 .5mM; and sodium L-glutamate, 2 . 5 m M ; Additions to the incubation medium Glutamate Glutamine G A B A Aspartate Glycine Alanine Nil 1.62 + 0.12 0.22 + 0.06 0.80 + 0.17 6.39 + 0.42 0.33 + 0. 02 0.07 + 0.05 Tetrodotoxin 3.01 + 0.38 0.17 + 0.03 1.73 + 0.29 8.04 + 0.06 0. 62 + 0. 13 0.21 + 0.04 Lidocaine 2.56 + 0.12 0.10 + 0.02 1.84 + 0.07 8.02 + 0.12 0.79 + 0.05 0.11 + 0.01 Protoveratrine 1.48 + 0.07 0.37 + 0.02 0.29 + 0.03 6.63 + 0.37 0.27 + 0. 02 0.02 + 0.01 Protoveratrine 2.39 + 0.08 0.27 + 0.07 1.45 + 0.01 7.67 + 0.37 0.50 + 0.02 0.05 + 0.01 + Tetrodotoxin L-Glutamate 5.14 + 0.02 0.75 + 0.09 1.37 + 0.30 9.65 + 0.20 0.51 + 0.07 0.12 + 0.03 L-Glutamate 9.33 + 0.60 0.75 + 0.07 2.52 + 0.23 12.05 + 0.20 0.82 + 0.02 0.22 + 0.03 + Tetrodotoxin T A B L E 41. Effects of protoveratrine, tetrodotoxin, lidocaine and of sodium L-glutamate on the release of amino acids from rat brain cortex slices incubated in glucose-free, calcium deficient media. Amounts of amino acids (umole/g initial wet wt. tissue) present in the incubation medium (3 ml . ) after incubation of rat brain cortex slices in C>2 for one hour at 3 7 ° C . When present protoveratrine was 5 u M ; tetrodotoxin, 2UM;lidocaine, 0.5mM;and sodium L-glutamate, 2 .5mM; Additions to the incubation medium Glutamate Glutamine G A B A Aspartate Glycine Alanine Nil 2.08 + 0.19 1.24 + 0.19 1.07 + 0.08 3.92 + 0.18 0.80 + 0.03 0.60 + 0.06 Tetrodotoxin 0.77 + 0.12 0.75 + 0.14 0.31 + 0.05 1.15 + 0.10 0.54 _+ 0. 04 0.45 + 0.07 Lidocaine 0.97 + 0.13 0.90 + 0.04 0.41 + 0.08 1.61 + 0.10 0.58 + 0. 05 0.55 + 0.09 Protoveratrine 2.68 + 0.42 0.97 + 0.01 1.39 + 0.01 4.81 + 0.06 0.93 + 0. 05 0.47 + 0.04 Protoveratrine 1.29 + 0.19 0.80 + 0.10 0.38 + 0.06 2.02 + 0.16 0.57 + 0.03 0.51 + 0.01 + Tetrodotoxin L-Glutamate 2.15 + 0.02 3.01 + 0.18 1.25 + 0.13 12.07 + 0.22 0. 74 + 0. 02 0.49 + 0.01 m M L-Glutamate 1.96 + 0.07 2.41 + 0.26 0.70 + 0.17 6.02 + 0.51 0.37 + 0.08 0.84 + 0.29 +Tetrodotoxin m M 149 aspartate f r o m 41 to 114, and with g l y c i n e f r o m 9 to 26. No si g n i f i c a n t changes o c c u r r e d with glutamine or alanine. A d d i t i o n of sodium L-glutamate to the gl u c o s e - f r e e , c a l c i u m - d e f i c i e n t , medium, l e d to i n c r e a s e s in the t i s s u e contents of amino acids, p a r t i c u l a r l y glutamate, glutamine, G A B A and aspartate after an incubation p e r i o d of one hour, and also i n the contents of these amino acids, p a r t i c u l a r l y aspartate, i n the incubation m e d i u m (Tab l e s 40 and 41). The p r e s e n c e of T T X l e d to some re t e n t i o n of the amino acids (apart f r o m glutamine) i n the t i s s u e and to d i m i n i s h e d values of the contents of these amino acids i n the incubation medium. Q u a l i t a t i v e l y , s i m i l a r r e s u l t s were obtained when Ca"*"*" was p r e s e n t in the g l u c o s e - f r e e medium. 7.5 E f f e c t s of l i d o c a i n e on the r e l e a s e of amino acids f r o m r a t b r a i n s l i c e s incubated i n a v a r i e t y of media. L i d o c a i n e and other l o c a l anesthetics are known to have s u p p r e s s i v e e f f e c t s s i m i l a r to those of T T X on the i n c r e a s e d r e s p i r a t i o n and the a l t e r e d i o n i c balance that o c c u r s i n b r a i n s l i c e s when the N a * - c u r r e n t s y s t e m is activated. It was, therefore, not s u r p r i s i n g to find that the T T X - s e n s i t i v e r e l e a s e of amino acids f r o m r a t b r a i n c o r t e x s l i c e s incubated in 0 2 at 3 7 ° C for one hour i n c e r t a i n media, was al s o l i d o c a i n e - s e n s i t i v e . The r e l e v a n t r e s u l t s a r e given i n T a b l e s 34 - 41 and are b r i e f l y s u m m a r i z e d as follows: 1. Incubation i n a n o r m a l K r e b s - R i n g e r phosphate glucose medium. L i k e TTX, li d o c a i n e (0.5 mM) does not affect amino a c i d contents in, or r e l e a s e from, r a t b r a i n cortex s l i c e s . The dimi n u t i o n of t i s s u e contents of glutamate and aspartate brought about by the addition of p r o t o v e r a t r i n e (5 uM) i s r e v e r s e d by lidocaine ( T a b l e s 34 and 3 5). 150 2. Incubation i n a g l u c o s e - f r e e K r e b s - R i n g e r phosphate medium. L i k e TTX, l i d o c a i n e enhances the t i s s u e contents of amino a c i d s and in h i b i t s their r e l e a s e in g l u c o s e - f r e e m e d i u m i n the p r e s e n c e or absence of p r o t o v e r a t r i n e (Tables 36 and 37). The i n c r e a s e of the total content of glutamine due to p r o t o v e r a t r i n e does not o c c u r i n the p r e s e n c e of l i d o c a i n e . 3. Incubation in Ca"*"*-def i c i e n t K r e b s - R i n g e r phosphate glu c o s e medium. The changes i n the t i s s u e contents of amino acids and the i n c r e a s e d r e l e a s e of glutamate, G A B A and aspartate f r o m b r a i n c o r t e x s l i c e s brought about by the addition of p r o t o v e r a t r i n e a r e abolished i n the p r e s e n c e of l i d o c a i n e ( T a b l e s 38 and 39). 4. Incubation i n a C a ^ ^ - d e f i c i e n t g l u c o s e - f r e e K r e b s - R i n g e r phosphate medium. The r e l e a s e of amino acids, p a r t i c u a r l y glutamate, G A B A and aspartate, is g r e a t l y d i m i n i s h e d by l i d o c a i n e (Table 41), and this is accompanied by i n c r e a s e d t i s s u e contents of these amino acids (Table 40). 7.6 E f f e c t s of tetrodotoxin on the t i s s u e to m e dium concentration r a t i o s of amino acids of r a t b r a i n cortex s l i c e s incubated in a v a r i e t y of media. It i s seen f r o m the r e s u l t s given in T a b l e 42, that incubation of r a t b r a i n c o r t e x s l i c e s f o r one hour i n a v a r i e t y of m e d i a that b r i n g about a c t i v a - t i o n of the Na~*~-curr ent or the g e n e r a t i o n of action potentials (182) gives T i s s u e / M e d i u m concentration r a t i o s for glutamate, GABA, aspartate and g l y c i n e that a r e g r e a t l y enhanced by the p r e s e n c e of T T X (2 uM). L i d o c a i n e behaves in a s i m i l a r manner to T T X . However, it i s noteworthy that the v a l u e s for glutamine a r e unaffected by the p r e s e n c e of these drugs. These drugs, therefore, do not affect the r e l e a s e of glutamine under v a r i o u s incuba- t i o n conditions. M o r eover, the T i s s u e / M e d i u m concen t r a t i o n r a t i o f or glutamine i s not d i m i n i s h e d by the p r e s e n c e of p r o t o v e r a t r i n e (5 uM). T h i s T A B L E 42. Effects of tetrodotoxin on the tissue to medium concentration ratios of amino acids of rat brain cortex slices incubated under a variety of conditions. Brain cortex slices of the rat were incubated in 0 2 at 37°C for one hour in a variety of Krebs-Ringer phosphate medium that activates the sodium current system. Ratios of the concentrations of amino acids in the tissue (umole/g initial wet wt.) to those in the incubation medium (umole/ml) are given below. They were calculated from the values of amino acid concentrations in the tissue given in Tables 34, 36, 38 and 40 and from the amounts of amino acids released, from the quantity of tissue investigated (lOOmg) into the total volume (3ml) of the incubation medium (Tables 35, 37, 39 and 41). Incubation Conditions T T X (2UM) T I S S U E / M E D I U M Glutamine Glutamate G A B A Aspartate Glycine Glucose medium - 36 354 2560 246 86 + 33 306 2580 289 99 Glucose medium + - 43 236 66 150 84 protoveratrine (5QM) + 40 440 156 278 174 Glucose, C a + + - f r e e medium - 25 250 1353 197 60 + 25 453 2440 311 68 Glucose, Ca ++-free medium _ 25 80 86 102 32 + protoveratrine (5uM) + 25 468 950 226 76 Glucose-free medium + - 13 14 21 39 10 protoveratrine (5|iM) + 5 143 86 209 52 Glucose-free, C a + + - f r e e - 12 23 23 49 12 medium + 7 117 168 210 34 Glucose-free, C a + + - f r e e medium - 10 17 6 41 9 + protoveratrine (5 uM) + 10 56 114 114 26 Glucose medium + - 9 23 14 25 16 ouabain (0. ImM) + 10 95 21 104 29 Glucose medium + ouabain (O.lmM) - 9 9 6 12 6 + protoveratrine (5uM) + 8 88 20 91 25 152 means that p r o t o v e r a t r i n e does not a c c e l e r a t e the r e l e a s e of glutamine f r o m b r a i n s l i c e s . It i s i n f e r r e d that the mai n depot of glutamine l i e s i n T T X and p r o t o v e r a t r i n e i n s e n s i t i v e b r a i n c e l l s , viz., g l i a . It i s to be noted that T T X has no enhancing effect on the T i s s u e / M e d i u m con c e n t r a t i o n r a t i o s of rat b r a i n c o r t e x s l i c e s , incubated i n a n o r m a l p h y s i o - l o g i c a l g l u c o s e saline medium. 7.7 E f f e c t s of s o d i u m amytal on the r e l e a s e of amino acids f r o m r a t b r a i n c o r t e x s l i c e s . A s mentioned i n the Introduction (Section 1.9 (iv)), the anesthetic effect of b a r b i t u r a t e s i s b e l i e v e d either to be brought about d i r e c t l y by a blo c k i n the N a ^ - c u r r e n t s y s t e m or to be the r e s u l t of the i r effects on c e l l e n e r g e t i c s . T h i s l a t t e r view was fu r t h e r supported when Chan and Q u a s t e l (198) f a i l e d to o b s e r v e a s u p p r e s s i v e effect of amytal or pentothal, on the 22]^ a influx into e l e c t r i c a l l y s t i m u l a t e d b r a i n s l i c e s though the i n c r e a s e d rate of r e s p i r a t i o n under these conditions was inhibited. Shankaran and Q u a s t e l (210) s i m i l a r l y found that the ^ N a influx into s l i c e s of the ra t b r a i n c o r t e x due to pr o t o v e r a t r i n e , or to g l u c o s e - l a c k i s u n a l t e r e d by these b a r b i t u r a t e s . These workers, however, found that the influx of ̂ 2]\j a+ into b r a i n t i s s u e due to (10 uM) p r o t o v e r a t r i n e (added to a glucose saline medium) i s p a r t i a l l y s u p p r e s s e d by amytal if C a * * i s c ompletely omitted f r o m the incubation m e d i u m (i.e., C a ^ - f r e e + (3 mM) E G T A ) . They obtained f u r t h e r evidence supporting the concept that b a r b i - turates act by s u p p r e s s i n g the gene r a t i o n of A T P in the mi t o c h o n d r i a . T h ey thereby inhibit the m i t o c h o n d r i a l A T P - d e p e n d e n t uptake of f r e e Ca f r o m the c y t o p l a s m or r e l e a s e C a * ^ a l r e a d y bound to m i t o c h o n d r i a (308). Increase of c y t o p l a s m i c C a con c e n t r a t i o n may then affect the k i n e t i c s of Na and K movements through the b r a i n c e l l membrane. A s i t has been shown e a r l i e r i n this chapter, that the r e l e a s e of amino 153 a c i d s f r o m incubated b r a i n t i s s u e accompanying the a c t i v a t i o n of the s o d i u m c u r r e n t s y s t e m at the b r a i n c e l l membrane, is both T T X and l i d o c a i n e sensi- tive, we have c a r r i e d out experi m e n t s to investigate the effects of amytal on the efflux of amino ac i d s . The con c e n t r a t i o n of 0.25 m M amytal was chosen as this i s the anesthetic c o n c e n t r a t i o n of amytal for the r a t (309). Rat b r a i n cortex s l i c e s were incubated in O., at 3 7 ° C for one hour, with or without the addition of 0.2 5 m M amytal, i n the following media: A. G l u c o s e - f r e e K r e b s - R i n g e r phosphate m e d i u m (with or without C a present). In this condition ^ N a influx into b r a i n s l i c e s i s unaffected by am y t a l (210). ++ B. Ca -free (3 m M E G T A containing) K r e b s - R i n g e r phosphate m e d i u m containing p r o t o v e r a t r i n e (10 LiM). In this condition 2 2 0.25 m M am y t a l p a r t i a l l y s u p p r e s s e s the Na influx into b r a i n s l i c e s (210). T a b l e s 43 and 44 show the amino a c i d contents of t i s s u e and m e d i u m at the end of the incubation p e r i o d . It i s evident f r o m these r e s u l t s that only under condition B i s the r e some s u p p r e s s i o n of the glutamate, G A B A and aspartate r e l e a s e . T o t a l (tissue + medium) glutamine l e v e l s are l o w e r e d i n the p r e s e n c e of amytal, p r e s u m a b l y due to a low e r e d A T P content, a c c o m - panying a d i m i n i s h e d rate of r e s p i r a t i o n (210). A m y t a l has no effect on the efflux of amino acids i n the C a ^ ^ - f r e e E G T A containing glucose saline m e d i u m (re s u l t s not shown). It was thus evident that amytal, at its anesthetic concentration, d e p r e s s e s the efflux of amino acids f r o m b r a i n s l i c e s under the condition that it d e p r e s s e s the influx of sodium ions; i.e., when an a c t i v a t i o n of the s o d i u m c u r r e n t s y s t e m at the b r a i n c e l l membrane has o c c u r r e d by the p r e s e n c e of p r o t o v e r a t r i n e i n a C a ^ - f r e e incubation medium. T A B L E 43. Effects of sodium amytal on the contents of amino acids in rat brain cortex slices incubated in a variety of media. Rat brain cortex slices were incubated in at 37°C for one hour in a variety of media in the presence or absence of 0.25mM sodium amytal. Tissue contents of amino acids are expressed as Umole/g initial wet wt. Incubation Media Medium Amytal Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine Glucose-free Krebs-Ringer phosphate + 2.03 + 0.33 2.01 + 0.06 0.51 + 0.20 0.23 + 0.02 1.38 + 0.22 1.45 + 0 .09 7.79 + 0.27 7.37 + 0.05 0.62 + 0.12 0.64 + 0.06 0. 17 + 0. 04 0.22 + 0.04 2.97 + 0.24 3.00 + 0.16 Glucose-frce C a + + - f r e e Krebs- Ringer phosphate + 1.62 + 0.12 1.80 + 0.05 0.22 + 0.06 0.24 + 0.02 0.80 + 0.17 0.92 + 0.08 6.39 + 0.42 6.08 + 0.48 0.33 + 0.02 0.45 + 0.05 0.07 + 0.05 0 .09 + 0.05 2.39 + 0.16 2.47 + 0.11 C a + + - f r e c Krebs- Ringer phosphate- glucose + 3mM E G T A + protovera- trine (10UM) + 5.90 + 0.14 7.67 + 0.33 2.15 + 0.17 1.77 + 0.13 1.43 + 0.18 2.37 + 0 .29 3.12 + 0.10 3.00 + 0. 17 0.53 + 0.01 0.67 + 0.03 0.61 + 0.02 0.70 + 0.04 3.05 + 0.48 3.19 + 0.21 T A B L E 44. Effects of sodium amytal on the release of amino acids from rat brain cortex slices incubated in a variety of media. Amount of amino acids (Umole/g initial wet wt./3 ml.) present in incubation media after incubation of rat brain cortex slices in O for one hour at 37°C in the presence or absence of 0.25mM sodium amytal. Incubation Media Medium Amytal Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine Glucose-free Krebs-Ringer phosphate + 0.96 + 0. 12 1.06 + 0 .09 1.74 + 0.10 1.39 + 0.02 0.41 + 0.08 0.46 + 0.04 1.98 + 0. 13 2.20 + 0.15 0.70 + 0.10 0.94 + 0.10 0.52 + 0.10 0.86 + 0.05 2.71 + 0.15 2.84 + 0.12 Glucosc-frce C a ^ - f r c e Krebs- Ringer phosphate + 2.08 + 0 .19 2.27 + 0. 16 1.24 + 0.19 1 .09 + 0.04 1.07 + 0.08 1.14 + 0.06 3.92 + 0.18 3.88 + 0.06 0.80 + 0.06 0 . 9 0 + 0.14 0.60 + 0.08 0.71 + 0.05 3.28 + 0.30 3.19 + 0.20 Ca ++-frec Krcbs- Ringcr phosphate - glucose + 3mM EG'i A + protovera- trine (10 uM) + 3.34 + 0.17 2.22 + 0.23 2.34 + 0.09 1.71 + 0.08 0.82 + 0.13 0.35 + 0 .09 1.16 + 0.02 0.83 + 0.08 0.61 + 0.08 0.54 + 0.06 0.88 + 0.05 0.90 + 0.04 3.62 + 0.62 3.10 + 0.32 Ui Ui 156 7.8 E f f e c t s of i n c r e a s e d K + and of tetrodotoxin on amino a c i d content in, and r e l e a s e from, incubated rat b r a i n cortex s l i c e s . Incubation of r a t b r a i n c o r t e x s l i c e s in K r e b s - R i n g e r phosphate m e d i u m containing 10 m M glucose, i n which K"*" was i n c r e a s e d to 50 mequiv/1, brought about a s i g n i f i c a n t i n c r e a s e i n the t i s s u e content of G A B A and an i n c r e a s e d efflux of G A B A into the incubation m e d i u m (Tables 45 and 46). T h e r e was some re t e n t i o n of glutamine i n the t i s s u e s l i c e s . L e s s definite changes o c c u r r e d with the other amino a c i d s examined. The i n c r e a s e d efflux of G A B A due to i n c r e a s e d was r e d u c e d to some extent by T T X (Table 45). In a c a l c i u m - d e f i c i e n t medium, i n c r e a s e d brought about no s i g n i f i - cant changes i n the glutamate content of t i s s u e or m e d i u m in c o n t r a s t to the effects of p r o t o v e r a t r i n e (Tables 38, 39, 45, 46). It would appear, therefore, that the changes i n amino a c i d fluxes i n r a t b r a i n c o r t e x s l i c e s incubated with i n c r e a s e d K* may d i f f e r m a r k e d l y f r o m those brought about by the p r e s e n c e of p r o t o v e r a t r i n e . When glucose was absent f r o m the incubation medium, i n c r e a s e d K^" p r o m o t e d an i n c r e a s e d efflux of glutamate, GABA, aspartate, g l y c i n e and alanine. The efflux of glutamine seemed not to be changed (Tables 37 and 46). A c c o m p a n y i n g changes took place i n the t i s s u e contents of amino ac i d s ( T a b l e s 36 and 45) and the total (tissue + medium) content of aspartate was c o n s i d e r a b l y i n c r e a s e d . The addition of T T X caused no s i g n i f i c a n t changes i n the contents of amino ac i d s both in t i s s u e and medium, except for a s m a l l r e d u c t i o n of the efflux of G A B A . 7.9 E f f e c t s of tetrodotoxin on the efflux of amino acids f r o m kidney cort e x s l i c e s incubated in the p r e s e n c e of ouabain. R e s u l t s given i n Table 47 show that the efflux of amino acids f r o m kidney c o r t e x s l i c e s of the r a t incubated in 0 2 at 3 7 ° C for one hour in K r e b s - R i n g e r phosphate gluc o s e medium, containing 0.1 m M ouabain, was not T A B L E 45. Effects of i n c r e a s e d K + and of tetrodotoxin on the amino a c i d content i n rat b r a i n cortex s l i c e s incubated i n various m e d i a . Amounts of amino acids ( u m o l e / g ini t ia l wet wt. tissue) present i n rat b r a i n cortex s l i c e s incubated in O2 for one hour at 37°C in K r e b s - R i n g e r phosphate m e d i u m with or without 10 m M g l u c o s e . When present , tetrodotoxin was 2uM. Addit ions to the incubation m e d i u m Glutamate Glutamine G A B A Aspar ta te G l y c i n e A l a n i n e 45 m M KC1 45 m M KC1 + T e t r o d o t o x i n 8.38 + 0.49 8.31 + 0.13 4.93 + 0.05 5.94 + 0.18 3.98 + 0.04 3.80 + 0.40 2.31 + 0.13 2.12 + 0.16 1.03 + 0.05 1.00 + 0.04 1.13 + 0.06 1.13 + 0.02 C a 2 + absent 45 m M KC1 45 m M KC1 + T e t r o d o t o x i n 9.45 + 0.19 9.37 + 0.17 1.45 + 0.09 1.06 + 0.10 2..93 + 0.34 2.74 + 0.03 2.33 + 0.08 2.42 + 0.23 0.67 + 0.03 0.71 + 0.05 0.57 + 0.04 0.48 + 0.04 G l u c o s e absent 45 m M KC1 45 m M KC1 + T e t r o d o t o x i n 1.29 + 0.27 1.27 + 0.16 0.30 + 0.09 0.34 + 0.08 0.72 + 0.36 0.76 + 0.28 7.08 + 0.32 7.78 + 0.06 0.48 + 0.07 0.49 + 0.07 0.18 + 0.04 0.17 + 0.04 T A B L E 46. Effects of increase K + and of tetrodotoxin on the release of amino acids from incubated rat brain cortex slices. Amounts of amino acids (umole/g initial wet wt. tissue) in the incubation medium after incubation of rat brain cortex slices in 0 2 for one hour at 3 7 ° C in 3 ml Krebs-Ringer phosphate medium with or without 10 m M glucose. When present,tetrodotoxin was 2 u M . Additions to the incubation medium Glutamate Glutamine G A B A Aspartate Glyc ine Alanine 45 m M KC1 45 m M KC1 + Tetrodotoxin 0.88 + 0.08 0.60 + 0.02 1 . 2 9 + 0.07 0.88 + 0.05 0.63 + 0.02 0.32 + 0.01 0.43 + 0.01 0.41 + 0.02 0.67 + 0.08 0.47 + 0.01 0.77 + 0.03 0.67 + 0.02 C a 2 + absent 45 m M KC1 45 m M KC1 + Tetrodotoxin 1 .20 + 0.13 1.24 + 0 . 0 9 3.77 + 0.07 3.82 + 0.18 0.34 + 0.04 0.21 + 0.01 0.57 + 0.06 0.59 + 0.02 0.54 + 0.11 0.59 + 0.06 1 .04 + 0.01 1.05 + 0.02 Glucose absent 45 m M KC1 45 m M KC1 + Tetrodotoxin 2.63 + 0.21 2.52 + 0.04 1.45 + 0 . 0 9 1.34 + 0.04 0.74 + 0.05 0.53 + 0.12 6.07 + 0.93 5.33 + 0.50 1.16 + 0.06 1.10 + 0.07 1.00 + 0.02 0 . 9 1 + 0.05 T A B L E 47. E f f e c t s of tetrodotoxin on the contents of amino acids in, and r e l e a s e from, r a t kidney c o r t e x s l i c e s incubated in the p r e s e n c e of ouabain. Rat kidney c o r t e x s l i c e s were incubated at 37°C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m containing ouabain (O.lmM) in the p r e s e n c e or absence of T T X (2uM). T i s s u e values a r e e x p r e s s e d as u m o l e / g i n i t i a l wet wt. and m e d i u m values as Umole/g i n i t i a l wet wt./3ml. Ouabain Ouabain + T e t r o d o t o x i n T i s s u e M e d ium T i s s u e M e d i u m Glutamate 1.37 + 0.04 2 . 0 9 + 0. 06 1.39 + 0.16 2. 22 + 0.07 A s p a r t a t e 1.05 + 0.07 0.44 + 0.03 1.08 + 0.01 0. 45 + 0. 01 A l a n i n e 0.38 + 0.03 2.31 + 0.25 0.42 + 0.16 2. 15 + 0. 04 G l y c i n e 0 . 6 0 + 0.05 2.28 + 0.12 0.54 + 0.01 2. 14 + 0. 08 T h r e o n i n e 0.21 + 0.01 1.56 + 0.15 0.20 + 0.04 1. 46 + 0. 02 T a u r ine 2.65 + 0 . 2 9 3 . 9 2 + 0.32 2.55 + 0.28 4. 15 + 0.20 Glutamine + Serine ) 0 . 9 0 + 0.01 6.95 + 0.75 0.82 + 0.14 6. 79 + 0. 15 + A s p a r a g i n e ) A m m o n i a 1.97 + 0.05 13.06 + 0. 06 2.15 + 0.16 13 . 74 0.50 160 T A B L E 48. T o t a l ammonia and amino a c i d contents of rat kidney c o r t e x s l i c e s i n i t i a l l y p r e s e n t and on incubation i n the p r e s e n c e o r absence of gl u c o s e . Rat k i d n e y c o r t e x s l i c e s were incubated at 3 7 ° C f o r one hour i n K r e b s - R i n g e r phosphate m e d i u m i n the p r e s e n c e or absence of gl u c o s e . I n i t i a l values ( i . e . values p r i o r to incubation) of ammonia and amino ac i d e s and the to t a l (tissue + medium) values a r e e x p r e s s e d as ( i m o l e s / g i n i t i a l wet wt. • I N I T I A L ON I N C U B A T I O N No G l u c o s e G l u c o s e Glutamate 5. 85 + 0.09 3.48 + 0. 03 3.91 + 0.04 A s p a r t a t e 1. 76 + 0 . 0 9 1. 00 + 0.03 1.24 + 0.05 A l a n i n e 0. 79 + 0.04 2 . 2 9 + 0.03 2. 22 + 0.17 G l y c i n e 2.00 + 0. 12 3.65 + 0. 68 2. 63 + 0.51 T h r e o n i n e 0.75 + 0. 04 2. 18 + 0.05 2. 10 + 0.10 T aur ine 6 . 9 2 + 0.28 6.53 + 0. 25 6. 68 + 0.27 Gl u t a m i n e + S e r i n e ) + A s p a r a g i n e ) 2. 35 + 0.01 8. 83 ± 0.21 3. 72 + 0 . 1 9 L y s i n e 0. 87 + 0.03 2.38 + 0. 12 2. 57 + 0.06 A m m o n i a 3.90 + 0. 06 17. 39 -f 0. 04 16.31 + 0.09 161 a f f e c t e d by the p r e s e n c e of T T X (2 |jM). T h i s i s i n d i r e c t c o n t r a s t to the e f f e c t s of T T X on the e f f l u x of a m i n o a c i d s f r o m r a t b r a i n c o r t e x s l i c e s i n c u b a t e d under s i m i l a r m e d i u m c o n d i t i o n s (see T a b l e s 34 and 35). T h i s s u p p o r t s the c o n c l u s i o n that T T X - s e n s i t i v e e f f l u x e s of amino a c i d s a r e con- f i n e d to e x c i t a b l e t i s s u e s . It may be noted that G A B A was not d e t e c t e d i n k i d n e y c o r t e x s l i c e s . M o r e o v e r , the oxygen uptake (92 + 9 ( i m o l e / g i n i t i a l wet wt), the t o t a l ( t i s s u e + medium) a m i n o a c i d contents, and the r a t e of a m m o n i a f o r m a t i o n , w e r e l i t t l e a f f e c t e d by the p r e s e n c e of g l u c o s e (Table 48). K i d n e y c o r t e x s l i c e s do not t a k e up water on i n c u b a t i o n even i n the p r e s e n c e of 0.1 m M ouabain, o r i n the absence of g l u c o s e . However, l a r g e i n c r e a s e s i n the c o n c e n t r a t i o n of g l y c i n e , t h r e o n i n e and l y s i n e , o c c u r on i n c u b a t i o n , p o s s i b l y due to p r o t e i n b r e a k d o w n ( T a b l e 48). 7.10 E f f e c t s of t e t r o d o t o x i n on the amino a c i d contents in, and r e l e a s e f r o m , r a t b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e of 2, 4 d i n i t r o - phenol, N H 4 + or 10 5 m M KC1. Okamoto and Q u a s t e l (182) have shown that the i n c r e a s e s i n water uptake, 22j\j a i n f l u x , and the r a t e of r e s p i r a t i o n of b r a i n c o r t e x s l i c e s , found i n the p r e s e n c e of D N P (0.03 mM) or KC1 (105 mM), a r e u n a f f e c t e d by T T X (3 (iM). The i n c r e a s e s i n uptakes of water and Na"*", and the l o s s of K"^ f r o m b r a i n c o r t e x s l i c e s i n c u b a t e d i n the p r e s e n c e of N H ^ C l (10 mM), a r e a l s o u n a f f e c t e d by T T X (3 (iM) (Chapter 9). Under these c o n d i t i o n s , t h e r e f o r e , a c t i o n p o t e n t i a l s or a c t i v a t i o n of the s o d i u m c u r r e n t s e e m not to be g e n e r a t e d . If the v i e w i s c o r r e c t that a m ino a c i d s a r e r e l e a s e d f r o m n e u r o n s o n l y when a c t i o n p o t e n t i a l s a r e g e n e r a t e d , o r when T T X - s e n s i t i v e a c t i v a t i o n s of the s o d i u m c u r r e n t s y s t e m o c c u r , then the r e l e a s e of a m i n o a c i d s b r o ught about by the a d d i t i o n of DNP, KC1 (high) or N H ^ C l to the i n c u b a t i o n medium, s h o u l d not be a f f e c t e d by T T X . E x p e r i m e n t s show that this' i s the case. T A B L E 49. Effects of tetrodotoxin on the contents of amino acids in rat brain cortex slices incubated with 2, 4 dinitrophenol, NH 4C1 or KC1 (lOOmM). Amounts of amino acids (Mmolc/g initial wet wt. tissue) present in rat brain cortex slices incubated in 0-> at 37°C for one hour in Krcbs-Ringcr phosphate-glucose medium. Tetrodotoxin when present in the medium was 2 uM. Add it ion to the incubation mod ium T T X Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine DNP (0. ImM) + 4.87 + 0.27 4.81 + 0.14 0.89 + 0.10 0.84 + 0.12 3.67 + 0.09 4. 10 + 0.25 1.14 + 0.11 1.14 + 0.02 0.65 + 0.09 0.57 + 0.03 0.83 + 0.06 0. 75 + 0. 04 1.92 + 0.18 2.07 + 0.20 NH 4C1 (30mM) + 3.09 + 0.31 3.80 + 0.20 3.27 + 0.26 2.99 + 0.10 4.31 + 0.26 4.65 + 0.02 1.26 + 0.08 1.35 + 0.03 0.68 + 0.08 0.76 + 0.02 0.73 + 0.07 0.71 + 0.02 1.88 + 0.15 1.92 + 0.01 KC1 (lOOmM) + 7.77 + 0.19 7.72 + 0.18 5.38 + 0.22 4.49 + 0.29 3.40 + 0.06 3.66 + 0.30 1.95 + 0.03 1.62 + 0.06 0.80 + 0.01 0.78 + 0.03 1. 57 + 0. 01 1.37 + 0.04 3.31 +• 0.05 3.48 + 0.38 T A B L E 50. Effects of tetrodotoxin on the release of amino acids from brain cortex slices incubated with 2,4 dinitrophenol, NH 4C1 or KC1 (lOOmM). Amounts of amino acids (umole/g initial wet wt./3 ml.) in the incubation medium after incubation of rat brain cortex slices in G*2 for one hour at 37°C in Krebs-Ringer phosphate glucose medium. Tetrodotoxin when present in the medium was 2uM. Addit ion to the incubation medium TTX Glutamate Glutamine GABA Aspartate Glycine Alanine Taurine DNP (0. ImM) + 3.44 + 0.22 3.14 + 0.06 2.47 + 0.32 2.37 + 0.19 1.40 + 0.08 1.07 + 0.03 1.06 +0.12 0.90 + 0.03 0.84 + 0.03 0.75 + 0.06 1.59 + .0.09 1.47 + 0.05 3.33 + 0.17 3.15 + 0.05 NH 4C1 (30mM) + 2.22 + 0.16 2.22 + 0.12 6.58 + 0.42 5.37 + 0.16 0.34 + 0.04 0.31 + 0.06 1.02 + 0.09 0.92 + 0.02 1.00 + 0.07 0.90 + 0.05 1.46 + 0.07 1.40 + 0.02 4.03 + 0.05 4.06 + 0. 14 KC1 (lOOmM) + 1.05 + 0.06 0.96 + 0.02 1.43 + 0.25 1.14 + 0.10 0.78 + 0.03 0.85 + 0.03 0.47 + 0.04 0.36 + 0.02 0.60 + 0.01 0.56 + 0.01 1.16 + 0.06 1.08 + 0.01 3.89 + 0.19 4.06 _+ 0.23 164 In o r d e r to ensure adequate efflux of amino acids f r o m r a t b r a i n c o r t e x s l i c e s incubated i n O., at 37 ° C for one hour i n K r e b s - R i n g e r phosphate glucose medium, r e l a t i v e l y high concentrations of D N P (0.1 mM), or N H ^ C l (30 mM), or KC1 (10 5 mM), were used. R e s u l t s are r e c o r d e d i n T a b l e s 49 and 50. F r o m these r e s u l t s it i s seen that T T X (2 uM) has no effect on the r e l e a s e of amino acids, f r o m the tissue, due to 30 m M N H ^ C l (Table 50). In the p r e s e n c e of 0.1 m M DNP, the net efflux of amino acids, with the p o s s i b l e exception of GABA, is al s o unaffected by T T X . The efflux of amino a c i d s i s f u r t h e r enhanced by 10 5 m M KC1, when c o m p a r e d with that brought about by 50 m M KC1 (Tables 46 and 50). A n i n c r e a s e in the efflux of GABA, and a retention i n the t i s s u e of glutamine, i s al s o evident with 10 5 m M KC1. However, the r e l e a s e of amino acids f r o m the t i s s u e by 10 5 m M KC1 i s unaffected by T T X . It was also found that the enhanced r e l e a s e of amino a c i d s f r o m incubated b r a i n c o r t e x s l i c e s i n a g l u c o s e - f r e e m e d i u m in the p r e s e n c e of this c o n c e n t r a t i o n of KC1, i s also not affected by T T X . 7.11 S u m m a r y 1. A m i n o acids, p a r t i c u l a r l y glutamate, y-aminobutyr ate, aspartate, and glycine, are r e l e a s e d f r o m r a t b r a i n c o r t e x s l i c e s under s p e c i f i c incubation conditions; e.g., i n the pr e s e n c e of p r o t o - v e r a t r i n e ( e s p e c i a l l y i n a Ca -d e f i c i e n t medium), or of ouabain, or i n the absence of glucose. The p r o c e s s e s of r e l e a s e a r e p a r t i a l l y or wholly s u p p r e s s e d by tetrodotoxin. It is i n f e r r e d that, during the generation of ac t i o n potentials or the ac t i v a t i o n of the s o d i u m c u r r e n t s y s t e m at the c e l l membrane in b r a i n s l i c e s , there i s a r e l e a s e of glutamate, GABA, aspartate, and g l y c i n e f r o m the neurons. 2. T e t r o d o t o x i n does not affect the r e l e a s e of glutamine under v a r i o u s incubation conditions, nor does p r o t o v e r a t r i n e a c c e l e r a t e i t . It i s 165 i n f e r r e d that the main depot of glutamine l i e s not in the neurons, but i n the g l i a . 3. P r o t o v e r a t r i n e b r i n g s about an i n c r e a s e d rate of f o r m a t i o n of glutamine i n incubated b r a i n s l i c e s . 4. I n c r e a s e d K"*" (50 mequiv/ml) i n the incubation m e d i u m leads to a r e l e a s e of GABA, the p r o c e s s being p a r t l y s u p p r e s s e d by T T X . 5. Incubation of b r a i n s l i c e s in medium devoid of glucose leads to an i n c r e a s e p r o d u c t i o n of aspartate but to d i m i n i s h e d t i s s u e contents of glutamate, glutamine and g l y c i n e . 6. The effects of l i d o c a i n e on the r e l e a s e of amino ac i d s f r o m s l i c e s a r e q u a l i t a t i v e l y s i m i l a r to those of tetrodotoxin. 7. T e t r o d o t o x i n has l i t t l e or no effect on the ouabain induced r e l e a s e of amino ac i d s f r o m kidney cort e x s l i c e s (unlike b r a i n c o r t e x s l i c e s). 8. T e t r o d o t o x i n does not affect the r e l e a s e of amino acids f r o m s l i c e s incubated i n the pr e s e n c e of N H 4 C I (30 mM), D N P (0.1 mM), or K C 1 (10 5 mM). 9. A m y t a l feebly s u p p r e s s e s the r e l e a s e of glutamate, G A B A and aspartate f r o m b r a i n c o r t e x s l i c e s incubated with p r o t o v e r a t r i n e in a p h y s i o l o g i c a l glucose saline m e d i u m devoid of Ca"*"*. 10. It is suggested that drugs which suppress the T T X - s e n s i t i v e a c t i v a t i o n of the s o d i u m c u r r e n t at the c e l l membrane con- comitantly suppress the outward movement of amino acids f r o m the b r a i n t i s s u e . 166 8. L O C A T I O N S O F A M INO A C I D S IN B R A I N C O R T E X S L I C E S O F T H E R A T 8.1 L o c a t i o n of the glutamate-glutamine system. Compartmentation of the glutamate-glutamine system, i n brain, f i r s t became evident f r o m in vivo studies with c e r t a i n l a b e l l e d p r e c u r s o r s , when it was found that the s p e c i f i c a c t i v i t y of i s o l a t e d glutamine was g r e a t e r than that of glutamate. L a t e r , this phenomenon was o b s e r v e d i n i s o l a t e d b r a i n c o r t e x s l i c e s . The phenomenon i s man i f e s t e d when l a b e l l e d acetate, but not l a b e l l e d glucose, i s p r e s e n t as p r e c u r s o r s (see Chapter 1.7 (ii) ). U s i n g the r a d i o a c t i v e amino a c i d data of Gonda and Q u a s t e l (2 59) obtained with l a b e l l e d acetate, and that of L a h i r i and Q u a s t e l (63) obtained with l a b e l l e d glucose, and using also our values concerning the contents of amino a c i d s i n b r a i n cortex s l i c e s , incubated under i d e n t i c a l conditions, we have made c a l c u l a t i o n s of the s p e c i f i c a c t i v i t i e s of glutamate and glutamine d e r i v e d f r o m l a b e l l e d acetate or l a b e l l e d g l u cose. In these experiments, b r a i n c o r t e x s l i c e s of the r a t were incubated in O-y at 3 7 ° C for one hour in K r e b s - R i n g e r phosphate m e d i u m containing 5 m M glucose in the p r e s e n c e of either [1 - ^ C ] acetate ( s p e c i f i c activity, 2 x 10^ cpm/umole), or [U -*^C] glucose ( s p e c i f i c activity, 4 x 10^ cpm/umole). L a b e l l e d amino acids were separated by two d i m e n s i o n a l paper chromatography and th e i r r a d i o a c t i v i t i e s were e s t i m a t e d (63, 259). The contents of the amino aci d s were m e a s u r e d by the A m i n o A c i d A n a l y z e r . R e s u l t s of the c a l c u l a t i o n s g i v e n i n Table 51 show that the s p e c i f i c a c t i v i t y of glutamine d e r i v e d f r o m l a b e l l e d acetate i s g r e a t e r than that of glutamate (280 c o m p a r e d to 58 m u g atom C/umole). The r e v e r s e i s true when l a b e l l e d glucose i s used as substrate, i.e., the s p e c i f i c a c t i v i t y of glutamine is lower than that of glutamate (674 c o m p a r e d to 934 m u g atom C/umole). In other words, the r a t i o of the s p e c i f i c a c t i v i t i e s of glutamine to glutamate i s g r e a t e r than one (viz., 4.8) when l a b e l l e d acetate i s employed, but lower than one (viz., 0.72) when l a b e l l e d T A B L E 51, S p e c i f i c a c t i v i t i e s of glutamine and glutamate of r a t b r a i n c o r t e x s l i c e s d e r i v e d f r o m s o d i u m (1-* 4C) acetate and ( U - ^ C ) glucose. Rat b r a i n c o r t e x s l i c e s were incubated at 37°C for one hour in K r e b s - R i n g e r phosphate glucose (5mM) medium, in the p r e s e n c e of either I m M sodium ( 1 - ^ C ) acetate (specific a c t i v i t y , 2 x 1 0 ^ cpm/umole) o r (U-* 4C) glucose ( s p e c i f i c a c t i v i t y , 4 x 10^ cpm/umole). R a d i o a c t i v i t y data f o r acetate were obtained f r o m the values given by Gonda and Quastel (259) and for glucose f r o m the values given by L a h i r i and Quastel (63). Contents of amino acids were estimated by the Amino A c i d A n a l y z e r . R a d i o a c t i v i t y m U g atom of C/g Content u m o l e / g , S p e c i f i c A c t i v i t y mug atom of C / u m o l e R e l a t i v e S p e c i f i c A c t i v i t y Glutamine Glutamate ( 1 - 1 4 C ) Acetate Glutamine Glutamate 970 + 80 530 + 70 3.45 + 0.30 9.20 + 0. 14 280 58 4.80 ( U - 1 4 C ) Gl u c o s e Glutamine Glutamate 2320 + 110 8580 + 200 3.45 + 0.30 9.20 + 0.14 674 934 0.72 168 glucose is used. These r e s u l t s are in a c c o r d with those a l r e a d y r e p o r t e d (see s e c t i o n 1.7 (ii) ) and may be taken to i m p l y the p r e s e n c e of two pools of c i t r i c a c i d c y c l e i n t e r m e d i a t e s both a c c e s s i b l e to glucose but only one f r e e l y a c c e s s i b l e to acetate. The lat t e r pool of c i t r i c a c i d c y c l e i n t e r m e d i a t e s has evidently a r e l a t i v e l y h i g h rate of glutamine synthesis and p r e s u m a b l y main- tains only a r e l a t i v e l y s m a l l pool of the total t i s s u e glutamate. 8.2 E f f e c t s of p r o t o v e r a t r i n e on the s p e c i f i c a c t i v i t i e s of amino acids f r o m r a t b r a i n c o r t e x s l i c e s incubated i n the p r e s e n c e of r u - 1 4 c i gluco se. C a l c u l a t i o n s were also made, as d e s c r i b e d above, to obtain s p e c i f i c a c t i v i t i e s of amino acids of rat c e r e b r a l c o r t e x s l i c e s incubated in the p r e s e n c e or absence of p r o t o v e r a t r i n e , with the radioactive amino a c i d data of K i n i and Q u a s t e l (213), and with our v a l u e s of amino a c i d contents of c o r t e x s l i c e s incubated under i d e n t i c a l conditions. S l i c e s of c e r e b r a l c o r t e x were incubated at 37 C for one hour i n K r e b s - R i n g e r phosphate m e d i u m containing glucose (5 m M and s p e c i f i c a c t i v i t y 2 x 10"* cpm/|jmole) i n the p r e s e n c e or absence of p r o t o v e r a t r i n e (5 |iM). A n a l y s e s for amino a c i d r a d i o a c t i v i t i e s and contents were c a r r i e d out as mentioned i n s e c t i o n 8.1. R e s u l t s given in Table 52 show that accompanying an i n c r e a s e d r e s p i r a t i o n in the p r e s e n c e of p r o t o v e r a t r i n e (211-213, see Chapter 9). t h e r e i s an i n c r e a s e d u t i l i z a t i o n of glucose r e f l e c t e d p a r t l y i n enhanced s p e c i f i c a c t i v i t i e s of glutamate (117%), G A B A (124%), and aspartate (105%). With glutamine, however, the i n c r e a s e i n the s p e c i f i c a c t i v i t y i s only s m a l l i n c o m p a r i s o n ( 2 4 % ) . These r e s u l t s may indicate that the major p o r t i o n of the amino acids, glutamate, G A B A and aspartate, but not glutamine, d e r i v e d f r o m l a b e l l e d glucose, is n e u r o n a l as p r o t o v e r a t r i n e i s a s s u m e d to act only on e x c i t a b l e c e l l s . However, this e x p e r i m e n t g i v e s no clue as to the s i z e of the n e u r o n a l pool of these amino a c i d s . It i s to be noted that p r o t o v e r a t r i n e does not in h i b i t glutamine synthesis; in fact, it enhances i t . T h i s o c c u r s T A B L E 52. E f f e c t s of p r o t o v e r a t r i n e (5|a M) on the s p e c i f i c a c t i v i t i e s of amino acids of r a t c e r e b r a l c o r t e x s l i c e s incubated i n the p r e s e n c e of (U- C) glucose. S l i c e s of rat b r a i n cortex were incubated for one hour at 3 7°C i n K r e b s - R i n g e r phosphate-glucose (5mM) medium. The s p e c i f i c a c t i v i t y of glucose was 2 x 1 0 ^ cpm/(imole. R a d i o a c t i v i t y data were c a l c u l a t e d f r o m values given by K i n i and Quastel (213) and are e x p r e s s e d as cpm/ml t i s s u e water. Contents of amino acids are e x p r e s s e d as p, m o l e / m l t i s s u e water and s p e c i f i c a c t i v i t i e s as cpm/|imole. Glutamate Glutamine G A B A A s p a r t a t e C o n t r o l - R a d i o a c t i v i t y 70.0 x 10 3 16.2 x 1 0 3 12.1 x 1 0 3 16.9 x 10 3 Amino A c i d Content 11.5 4.25 3.2 4.31 S p e c i f i c A c t i v i t y 6.0 x 10 3 3.8 x 1 0 3 3.8 x 1 0 3 3.9 x 10 3 P r o t o v e r a t r i n e R a d i o a c t i v i t y 98.6 x 10 3 24.4 x 10 3 22.9 x 10 3 21.6 x 10 3 A m i n o A c i d Content 7.5 5.12 2.72 2.69 S p e c i f i c A c t i v i t y 13.1 x 10 3 4.7 x 1 0 3 8.5 x 1 0 3 8.0 x 10 3 % Change of S p e c i f i c A c t i v i t y f r o m C o n t r o l +117 +24 +124 +105 170 i n spite of a d i m i n i s h e d t i s s u e A T P content in the p r e s e n c e of p r o t o v e r a t r i n e (see T a b l e 74). The f a l l i n A T P due to p r o t o v e r a t r i n e p r e s u m a b l y o c c u r s in the neurons as the p r o t o v e r a t r i n e induced Na"*" influx r e s u l t s in an i n c r e a s e i n the a c t i v i t y of the n e uronal N a + pump, with an accompanying i n c r e a s e in the rate of A D P c o n t r o l l e d m i t o c h o n d r i a l r e s p i r a t i o n . The u n d i m i n i s h e d rate of the A T P dependent glutamine synthesis i n the p r e s e n c e of p r o t o v e r a t r i n e may i m p l y that the neurons are not the major compartment of glutamine sy n t h e s i s . 8.3 C h a r a c t e r i z a t i o n of amino a c i d compartments i n b r a i n . The r e s u l t s obtained above are i n a c c o r d with those of i n v e s t i g a t i o n s that have shown the existence of at l e a s t two separate pools in b r a i n t i s s u e that d i f f e r i n t h e i r contents of amino acids and in their amino a c i d flux r a t e s . S e g r e g a t i o n of glutamate and GABA, and other amino acids into " s m a l l " or " l a r g e " compartments o c c u r s as d emonstrated by the r e s u l t s of a v a r i e t y of studies of amino a c i d m e t a b o l i s m i n both in v i t r o and in vivo (116, 141-158). The " s m a l l " compartment seems to be a s s o c i a t e d with a r e l a t i v e l y s m a l l content of glutamate, but p o s s e s s e s the major amount of glutamine. The " l a r g e " compartment appears to contain the major amount of glutamate. However, the p h y s i c a l boundaries of these compartments have not been e s t a - b l i s h e d . I n d i r e c t evidence has suggested that the " l a r g e " compartment may c o n s i s t of n e uronal s t r u c t u r e s (116, 149. 154-156), while the " s m a l l " pool a s s o c i a t e d with high glutamine content may c o n s i s t of g l i a l t i s s u e (161, 162). In Chapter 7 we have shown that, under s p e c i f i c incubation conditions where action potentials or a c t i v a t i o n of the s o d i um c u r r e n t s y s t e m at the c e l l m embrane are thought to be generated in r a t b r a i n c o r t e x s l i c e s (182), there i s an i n c r e a s e d efflux of amino acids f r o m the tissue into the medium. T h i s r e l e a s e is bl ocked p a r t i a l l y or wholly by the p r e s e n c e of T T X . A s it is known that g l i a a r e not e l e c t r i c a l l y excitable c e l l s and do not generate action potentials (260, 261), it i s i n f e r r e d that the effects of T T X , 171 which a b o l i s h e s the acti v a t i o n of the sodium, c u r r e n t s y s t e m at the n e u r o n a l membrane (190, 193. 194), are confined to the neurons. We have c a r r i e d out e x p e r i m e n t s to determine the p r e c i s e incubation conditions that a r e most effective i n r e l e a s i n g amino acids f r o m incubated r a t b r a i n c o r t e x s l i c e s and we have estimated how much of this r e l e a s e i s ab o l i s h e d by the p r e s e n c e of T T X . The amino a c i d that is r e t a i n e d i n the b r a i n t i s s u e by the addition of T T X is c o n s i d e r e d to be located i n the neurons. Its amount r e p r e s e n t s a m i n i m u m value of the amino a c i d content of the neurons in the incubated b r a i n cortex s l i c e s . E x p e r i m e n t s c a r r i e d out on these l i n e s may give, therefore, m e a s urements of m i n i m u m values of the contents of a v a r i e t y of amino ac i d s i n the neurons. The r e s u l t s of these e x p e r i m e n t s are d e s c r i b e d below. (i) E f f e c t s of the combined p r e s e n c e of p r o t o v e r a t r i n e and ouabain on amino a c i d r e l e a s e . It was found, after a s e r i e s of exper i m e n t s u t i l i z i n g v a r i o u s incuba- t i o n conditions, that the o p t i m a l condition f o r the r e l e a s e of amino acids f r o m r a t b r a i n c o r t e x s l i c e s , incubated under a e r o b i c conditions at 3 7 ° C for one hour, was to use an incubation m e d i u m c o n s i s t i n g of K r e b s - R i n g e r phosphate m e d i u m containing 10 m M glucose together with p r o t o v e r a t r i n e (5 uM) and ouabain (0.1 mM). The p r e s e n c e of p r o t o v e r a t r i n e e n s u r e d the g e n e r a t i o n of act i o n potentials in the b r a i n s l i c e s with r e s u l t a n t efflux of amino ac i d s (see Chapter 7), while that of ouabain blocked the re-uptake of the r e l e a s e d amino aci d s against a c o n c e n t r a t i o n g r a d i e n t (104, 112). R e s u l t s are shown in T a b l e 53, which r e c o r d s values of the contents of amino ac i d s i n the t i s s u e and i n the incubation m e d i u m at the end of the incubation p e r i o d . It w i l l be o b s e r v e d that the p r e s e n c e of p r o t o v e r a t r i n e and ouabain c a u s e d a l o s s f r o m the b r a i n t i s s u e of 26.10 - 7.74 - 18.36 umole/g of the total amino acids investigated, i.e., a l o s s of 70 per cent. A t the same time, the T A B L E 5 3 . Effects of protoveratrine, ouabain and tetrodotoxin on amino acid content in, and release from, incubated rat brain cortex slices. Rat brain cortex slices were incubated in 3 ml Krebs-Ringer phosphate medium containing lOmM glucose in at 37°C for one hour in the presence or absence of mixtures of protoveratrine (5uM), ouabain (O.lmM) and tetrodotoxin (2 uM) as shown below. Tissue values of amino acid are expressed as Limole/g initial wet wt. and medium values as (imole/g wet wt. /3 ml. Amino Acid No Addition Tetrodotoxin Protoveratrine + Ouabain Protoveratrine + Ouabain + Tetrodotoxin Tissue Medium Total Tissue Medium Total Tissue Medium Total Tis sue Medium Total Glutamate 9.20 + 0.14 0. 77 + 0.14 9.97 8.82 + 0.01 0.87 + 0.02 9.69 2.52 + 0.11 8,33 + 0,48 10.85 7.14 + 0.12 2.42 + 0. 16 9.56 Glutamine 3.45 + 0.30 2.92 + 0.28 6.37 2.88+0.11 2.62 + 0.13 5.50 0.74 + 0.04 2.56+0.41 3. 30 0.53 + 0.03 2.07 + 0. 25 2.60 GABA 2.56 + 0.20 0.03 + 0.02 2.59 2.58+0.16 0.04 + 0.03 2.62 0.63 + 0.02 3.23 + 0.15 3.86 1.47 + 0.13 2.18 + 0.25 3.65 Aspartate 3.45 + 0.23 0.43 + 0.05 3.88 3.64 + 0.20 0.38 + 0.06 4.02 1.20 + 0.07 2.92 + 0.18 4. 12 3.28 + 0.19 1.09 + 0. 02 4.37 Glycine 1.03 + 0. 15 0.36 + 0.06 1.39 0.88 + 0.03 0.27 + 0.02 1.15 0.25 + 0.01 1.22+0.04 1.47 0.66 + 0.03 0.81 + 0. 05 1.47 Alanine 0.75 + 0.05 0.44 + 0.04 1.19 0.82 + 0.02 0.38 + 0.02 1.20 0.26 + 0. 01 1.59 + 0.14 1.85 0.46 + 0.05 1.28 + 0. 13 1.74 Taurine 4.10 + 0.35 1.89+0.08 5.99 3.92 + 0.01 2.05 + 0.01 5.97 1.76 + 0. 13 4.49 + 0.71 6.25 2.07 + 0.50 3.89 + 0. 35 5.96 So rine 1.12 + 0. 13 0.82 + 0. 05 1.94 0.96 + 0.12 0.67 + 0.09 1.63 0.28 + 0. 01 1.76+0.15 2. 04 0.72 + 0.02 1.33 + 0. 06 2.05 Threonine 0.44 + 0.06 0.28 + 0.05 0.72 0.48 + 0.03 0.26 + 0. 02 0.74 0.10+ 0.01 0.66 + 0.01 0.76 0.21 + 0.01 0.60 + 0.04 0.81 T O T A L 26.10 7.94 34.04 24.98 7.54 32.52 7.74 26.76 34.50 16.54 15. 67 32.21 tNJ 173 incubation m e d i u m (3 ml) gained amino acids to the extent of 26.76 - 7.94 = 18.82 umole/g, a value a p p r o x i m a t e l y equal to that l o s t by the t i s s u e . The t i s s u e l o s s e s i n c u r r e d by the i n d i v i d u a l amino acids were as follows: glutamate (73%), glutamine (78%), G A B A (75%), aspartate (65%), g l y c i n e (74%), alanine (65%), taurine (57%), s e r i n e (75%), threonine ( 7 7 % ) . Thus, a l l the t i s s u e amino acids s u f f e r e d substantial l o s s e s , taurine being r a t h e r l e s s affected than the others. The total (tissue + medium) quantity of amino acids was not s i g n i f i - cantly affected by the combined action of p r o t o v e r a t r i n e and ouabain. However, the tot a l (tissue + medium) value f o r glutamine f e l l by 48 per cent, whereas that f o r G A B A i n c r e a s e d by 49 per cent, that f o r alanine i n c r e a s e d by 55 p e r cent, and that f o r glutamate i n c r e a s e d only s l i g h t l y (9%) (Table 53). (ii) E f f e c t s of tetrodotoxin T e t r o d o t o x i n (2 uM) e x e r c i s e d l i t t l e or no effects, within the range of e x p e r i m e n t a l e r r o r , on the contents of amino acids both i n the t i s s u e and i n the incubation m e d i u m under the n o r m a l incubation conditions. It brought about, however, large changes in the p r e s e n c e of p r o t o v e r a t r i n e and ouabain. R e s u l t s given in Tabl e 53 show that, on the addition of T T X , the total quantity of amino acids i n tissue, n a m e l y 24.98 umole/g, was r e d u c e d by the combined presence of p r o t o v e r a t r i n e and ouabain to 16.54 umole/g, i.e., a l o s s of 8.44 umole/g equivalent to 34 per cent of the t i s s u e content. The gain i n amino acids i n the m e d i u m was 15.67 - 7.74 = 8.13 umole/g, a p p r o x i m a t e l y equal to the l o s s f r o m the t i s s u e . T h e r e f o r e , T T X r e d u c e d a l o s s of 70 per cent of amino acids f r o m the tissue, brought about by p r o t o v e r a t r i n e and ouabain, to 34 per cent. The i m p l i c a t i o n i s that at l e a s t 36 per cent of the t i s s u e content of amino ac i d s i s lo c a t e d i n the neurons. T h i s value i s a m i n i m u m value, as it i s unlikely that the T T X causes a complete block of the n e u r o n a l efflux of amino acids, or that the block i s equally e f f e c t i v e with a l l amino ac i d s or with a l l conditions that a r e p r e s u m e d to generate action potentials. 174 (iii) A l t e r a t i o n s i n the contents of i n d i v i d u a l amino a c i d s . (a) Glutamate C o n s i d e r i n g some of the amino acids i n d i v i d u a l l y , it i s seen f r o m T a b l e 53 that, with glutamate, the tissue content was r e d u c e d by p r o t o - v e r a t r i n e and ouabain i n p r e s e n c e of T T X f r o m 8.8Z umole/g to 7.14 umole/g, i.e., a l o s s of 1.68 umole/g or 1 9 per cent of the t i s s u e content of glutamate. Since the l o s s amounted to 73 per cent i n the absence of T T X , i t follows that the difference, i.e., 73-19 = 54 per cent, r e p r e s e n t s the percentage of the t i s s u e glutamate l o c a t e d in the neurons. T h i s constitutes good evidence that at l e a s t half of the glutamate in the b r a i n c o r t e x s l i c e s i s p r e s e n t in the neurons. C o n s i d e r a t i o n s of the glutamate contents i n the incubation m e d i u m (Table 53) give r i s e to the same c o n c l u s i o n . Increase of the c o n c e n t r a t i o n of glutamate i n the medium, due to the p r e s e n c e of p r o t o v e r a t r i n e and ouabain, amounted to 8.33 -0.77 = 7.56 umole/g, while with T T X added, the i n c r e a s e amounted to 2.42 -0.87 = 1.55 umole/g. The difference, 6.01 umole/g, r e p r e s e n t e d the amount r e t a i n e d by the neurons and which, therefore, con- stituted (6.01 x 100)/(9.20)= 65 per cent of the glutamate p r e s e n t i n the t i s s u e at the end of the incubation p e r i o d . (b) A s p a r t a t e The ti s s u e content of aspartate was r e d u c e d by p r o t o v e r a t r i n e and ouabain, i n p r e s e n c e of T T X , f r o m 3.64 umole/g to 3.28 umole/g, i.e., a l o s s of 0.36 umole/g or of 10 per cent of the t i s s u e content of aspartate (Table 53). A s the l o s s amounted to 65 per cent in the absence of T T X , it appears that the difference, i.e., 65 - 10 = 55 per cent, r e p r e s e n t s the percentage of the t i s s u e aspartate located in the neurons. I n c r e a s e of the c o n c e n t r a t i o n of a s p a r t a t e in the m e d i u m i n the p r e s e n c e of p r o t o v e r a t r i n e and ouabain amounted to 2.49 umole/g (Table 53), while, with T T X added, the i n c r e a s e amounted to 0.71 umole/g. The difference, 1.78 umole/g, r e p r e s e n t s the amount r e t a i n e d by the neurons and t h e r e f o r e constitutes (1.78 x 100)/(3.45) = 51 per cent of 175 the amount of aspartate n o r m a l l y present i n the t i s s u e under the given e x p e r i - mental condition. It seems, therefore, that as with glutamate, the neurons are the site of the major pool of aspartate. (c) G A B A Ca l c u l a t i o n s s i m i l a r to those given above, f r o m the values r e c o r d e d i n T a b l e 53, indicate that at l e a s t 32 per cent of the t i s s u e content of G A B A is l o c a t e d i n the neurons. The actual amount must be c o n s i d e r a b l y g r e a t e r than this because r e s u l t s , r e c o r d e d i n the p r e c e d i n g Chapter 7, of the effects of T T X on G A B A efflux brought about by p r o t o v e r a t r i n e under c e r t a i n incuba- t i o n conditions, ind i c a t e d a m o r e complete block of the r e l e a s e of G A B A . (d) G l y c i n e and S e r i n e C a l c u l a t i o n s made i n the manner d e s c r i b e d above f r o m the r e s u l t s i n T a b l e 53 show that at l e a s t 50 per cent of the tissue content of either g l y c i n e or s e r i n e l i e s i n the neurons. P r e s u m a b l y , therefore, the neurons a r e loca- tions of the major pools of g l y c i n e and of s e r i n e . (e) Taurine, A l a n i n e and T h r e o n i n e S i m i l a r c a l c u l a t i o n s show that at l e a s t 21 per cent of the t i s s u e con- tent of alanine, 10 per cent of the t i s s u e content of taurine, and 6 per cent of the the t i s s u e content of threonine l i e in the neurons. It is not p o s s i b l e to decide, f r o m these values, whether the neurons are the l o c a t i o n s of the major pools of these amino a c i d s . (f) Glutamine T u r n i n g to a c o n s i d e r a t i o n of glutamine, it i s seen (Table 53) that the percentage f a l l i n t i s s u e glutamine in p r e s e n c e of p r o t o v e r a t r i n e and ouabain 176 was (3.45 - 0 .74) (100) / (3.4 5) = 78 per cent, and the percentage f a l l , with T T X added, was (2.88 - 0.53) (100) / (2.88) = 81 per cent. Thus, the p r e s e n c e of T T X had no d i m i n i s h i n g effect on the p r o p o r t i o n of glutamine r e l e a s e d f r o m the b r a i n t i s s u e in p r e s e n c e of p r o t o v e r a t r i n e and ouabain. It appears, therefore, that glutamine is not r e t a i n e d in the b r a i n t i s s u e by T T X under conditions where the r e l e a s e of glutamate and other amino acids is g r e a t l y affected. R e s u l t s , r e c o r d e d i n T a b l e 54, show, moreover, that the p r e s e n c e of p r o t o v e r a t r i n e (5 jiM) has no effect on the t i s s u e content of glutamine under conditions where it causes s i g n i f i c a n t f a l l s i n the t i s s u e contents of glutamate, GABA, aspartate, g l y c i n e and alanine. 8.4 E f f e c t s of s o d i um malonate and sodium fl u o r o a c e t a t e on c e r e b r a l amino a c i d content and r e l e a s e i n the p r e s e n c e of p r o t o v e r a t r i n e . It has been suggested (150) that the s u p p r e s s i o n of c e r e b r a l glutamine synthesis by low concentrations of f l u o r o a c e t a t e (63) i s due to the l o c a l i z a t i o n of its effect i n a s p e c i a l compartment where glutamine synthesis takes place. The f l u o r o a c e t a t e i s c o n s i d e r e d to block the o p e r a t i o n of the c i t r i c a c i d c y c l e i n this compartment, thereby r e d u c i n g the amount of A T P a v a i l a b l e for gluta- mine synthesis. E x p e r i m e n t s were, therefore, c a r r i e d out to observe the effects of two c i t r i c a c i d c y c l e i n h i b i t o r s , s o d i u m malonate and s o d i um fluoroacetate, on both the r e s p i r a t i o n and on the contents of amino ac i d s of b r a i n s l i c e s i n - cubated in a m e d i u m containing p r o t o v e r a t r i n e . It is w e l l known that proto- v e r a t r i n e b r i n g s about stim u l a t i o n of b r a i n r e s p i r a t i o n . The s t i m u l a t e d r e s p i r a t i o n i s abolished by T T X (182) and is considered, therefore, to be a s s o c i a t e d with the neurons. R e s u l t s given in F i g u r e 2 show that the p r o t o v e r a t r i n e stimulated r e s p i r a t i o n was blocked by malonate (2 mM) but not by fluoroacetate (3 mM). 177 T A B L E 54. E f f e c t s of p r o t o v e r a t r i n e on amino a c i d content i n r a t b r a i n c o r t e x s l i c e s incubated i n p r e s e n c e of ouabain. Rat b r a i n c o r t e x s l i c e s were incubated i n 3 m l K r e b s - R i n g e r phosphate m e d i a containing l O m M glucose i n 0 2 at 3 7 ° C f o r one hour in the p r e s e n c e of p r o t o v e r a t r i n e (5 uM) or of ouabain (O.lmM) as shown below. Values of a m i n o a c i d s are given as Umole/g i n i t i a l wet wt. Ouabain Ouabain and P r o t o v e r a t r i n e G lutamate 4. 95 + 0.26 2. 52 + 0.11 G l u t a m i n e 0. 71 + 0.04 0. 74 + 0.04 G A B A 1. 39 + 0.06 0. 63 + 0.02 A s p a r t a t e 1. 75 + 0.22 1. 20 + 0.07 G l y c i n e 0. 59 + 0.03 0.25 + 0.01 A l a n i n e 0. 42 + 0. 02 0.26 + 0.01 178 o o —< CO a 3 cn fi O O rvj O 14 12 10 0 15 30 45 60 T i m e (minutes) F I G U R E 2. E f f e c t s of s o d i u m fluoroacetate and s o d i u m malonate on the p r o t o v e r a t r i n e - s t i m u l a t e d r e s p i r a t i o n of b r a i n cortex s l i c e s . R e s p i r a t i o n of rat b r a i n cortex incubated in O-? at 3 7 ° C in K r e b s - R i n g e r phosphate glucose for v a r i o u s per iods of t ime in the p r e s e n c e or absence of protoveratr ine (5^.M), s o d i u m fluoroacetate (3mM) or s o d i u m malonate (2mM). O No additions O With malonate A With fluoroacetate • P r o t o v e r a t r i n e added • P r o t o v e r a t r i n e + malonate added A P r o t o v e r a t r i n e + fluoroacetate added 179 T h i s r e s u l t is consistent with the c o n c l u s i o n that fluoroacetate, i n c o n t r a s t to malonate at the concentrations i n v e s t i g a t e d does not suppress the opera- t i o n of the c i t r i c a c i d c y c l e in the neurons. R e s u l t s given in T a b l e 55 indicate that malonate (2 mM) and fluoro- acetate (3 mM) brought about d i f f e r e n t effects on the amounts of amino ac i d s in, and on t h e i r r e l e a s e from, b r a i n s l i c e s incubated in the p r e s e n c e of p r o t o v e r a t r i n e . The major effect of fluoroacetate (3 mM) was to b r i n g about a d i m i n u t i o n in the total (tissue + medium) amount of glutamine, amounting to 2.4 5 (imole/g, accompanied by an i n c r e a s e i n that of glutamate amounting of 2.45 (imole/g. T h e r e was an i n c r e a s e i n the amount of G A B A amounting to 1.71 (imole/g, but a s l i g h t l y d i m i n i s h e d value of aspartate. T h e r e were m i n o r changes in the other amino a c i d s . T h e s e r e s u l t s with flu o r o a c e t a t e r e s e m b l e those obtained i n e a r l i e r studies of the amount of l a b e l l e d amino a c i d s f o r m e d f r o m l a b e l l e d glucose (63). F l u o r o a c e t a t e caused a s i g n i f i c a n t r i s e i n tissue, but not medium, glutamate and a s i g n i f i c a n t f a l l i n tissue, but not medium, glutamine. The r i s e in G A B A was m o s t l y confined to the t i s s u e . The major effect of malonate was to d i m i n i s h the total (tissue + medium) amount of aspartate by 1.59 jamole/g, and to i n c r e a s e that of G A B A by 1.92 (imole/g. T h e r e were no s t a t i s t i c a l l y s i g n i f i c a n t effects on the total amounts of glutamine or other amino acids (Table 55). Malonate caused s i g n i f i c a n t r i s e s in the medium contents of glutamate and glutamine with accompanying f a l l s i n the t i s s u e contents of these amino a c i d s . It brought about a l a r g e f a l l i n the t i s s u e value of aspartate with a r e l a t i v e l y s m a l l change in the m e d i u m content of aspartate. T h e r e o c c u r r e d a l a r g e r i s e i n t i s s u e G A B A with a small, but significant, change in the m e d i u m content of G A B A . The t i s s u e content of alanine was a l s o s i g n i f i c a n t l y d i m i n i s h e d by malonate. Changes that took place i n the other amino acids investigated were not statis- t i c a l l y s i g n i f i c a n t . T A B L E 55. Effects of sodium malonate or sodium flouroacetate on amino acid content in, and release from, incubated rat brain slices in the presence of protoveratrine. Rat brain slices were incubated in 3 ml Krebs-Ringer phosphate medium containing 10 mM glucose in O 2 at 37°C for one hour in the presence of protoveratrine (5UM) with or without sodium malonate (2mM) or sodium fluoroacetate (3mM). Tissue values of amino acids arc expressed as Umole/g initial wet wt. and medium values as Umole/g wet wt./3ml. Amino Acid Protoveratrine Protoveratrine + Malonate Protoveratrine + Fluoroacetate Tissue Medium Total Tis sue Medium Total Tis sue Medium Total Glutamate 7.04 + 0.06 0.78 + 0.06 7.82 6.07 ± 0.37 1.22 + 0.01 7 .29 9. 31 + 0.69 0.96 + 0. 10 10.27 Glutamine 3.56 + 0.07 2. 73 + 0.25 6 .29 2. 77 + 0.54 3.88 + 0.09 6.65 0.91 + 0.17 2.93 ± 0.59 3.84 GABA 2 .92 + 0. 37 0.06 + 0.03 2.98 4.65 0.01 0.25 + 0. 03 4 .90 4. 58 + 0.47 0. 11 + 0.02 4.69 Aspartate 1.98 + 0.10 0.51 + 0.01 2.49 0.53 + 0.15 0.37 + 0.05 0 .90 1. 39 + 0.04 0.53 + 0.08 1 .92 Glycine 0.89 + 0.07 0.30 + 0.03 1.19 0.79 + 0.07 0.53 + 0.07 1.32 0.98 _+ 0.07 0.48 + 0.03 1.46 Alanine 1.07 + 0.01 0.59 + 0.06 1.66 0.66 _+ 0.04 0.79 + 0.01 1.45 1. 04 + 0.01 0.86 + 0.07 1 .90 Taurine 3.61 + 0.28 2.40 + 0.10 6.01 2.98 + 0.32 3.17 + 0.05 6.15 3.52 + 0. 08 3.10 + 0.12 6.62 Serine 0.97 + 0.08 0.70 + 0.01 1.67 0.86 + 0.07 0.99 + 0. 13 1.85 1.07 + 0.08 0.95 + 0.05 2.02 Threonine 0.51 + 0.02 0.25 + 0.02 0.76 0.37 0.06 0.38 + 0. 02 0.75 0.35 + 0.03 0.37 + 0.03 0.72 T O T A L 22.55 8.32 30.87 19.68 11. 58 31.26 23.15 10. 29 33.44 Co o T A B L E 56. Effects of L-glutamate and L-glutamine on amino acid content in, and release from, rat brain cortex slices. Rat brain cortex slices were incubated in 3 ml Krebs-Ringer phosphate medium containing lOmM glucose, but in absence of C a + + , at 37°C for one hour with or without L-glutamate (2.5mM) or L-glutamine (2.5mM). Tissue values of amino acids are expressed as (imole/g initial wet wt., and medium values as umole/g initial wet wt./3ml. Amino Control L-Glutamate L-Glutamine Acid Tis sue Medium Total Tissue Medium Total Tissue Medium Total Glutamate 9.75 + 0.34 1.17 + 0. 11 10.92 20.17 + 0.09 (1.73 + 0. 06 mM) 10.10 + 0.08 2.27 + 0.28 12. 37 Glutamine 1.96 + 0.02 2.41 + 0.14 4.37 3.81 + 0.20 3.39 + 0.18 7.20 12.22 + 0.09 (1.62 + 0.06 mM) GABA 2.30 + 0.05 0.05 + 0.03 2.35 3.21 + 0.34 0.26 +_ 0.04 3.47 4.04 + 0.06 0.03 0.01 4. 07 Aspartate 2.95 + 0.19 0.45 + 0.05 3.40 5.69 + 0.07 2.73 + 0.03 8.42 2.96 + 0.23 0.63 0.01 3.59 Ammonia 1.47 + 0.09 5.69 + 0.63 7.16 1.48 + 0.45 4.88 + 0.36 6.36 1.51 + 0. 12 9.93 + 0.49 11.44 182 8.5 E f f e c t s of L-glutamine and sodium L-glutamate on c e r e b r a l amino a c i d content and r e l e a s e . E x p e r i m e n t s were c a r r i e d out to observe the effects of the addition of L - g l u t a m i n e (2.5 mM) to the incubation m e d i u m on the amino a c i d contents of rat b r a i n c o r t e x s l i c e s . K r e b s - R i n g e r phosphate solution containing 10 m M glucose, but devoid of Ca^^, was used as the incubation medium, as with this m e d i u m the addition of L-glutamate was found to promote the efflux of G A B A and aspartate. R e s u l t s are shown in Table 56, which also g i v e s comparable r e s u l t s obtained with s o d i u m L-glutamate (2.5 mM). They indicate that the addition of L - g l u t a m i n e gave r i s e to an i n c r e a s e d value of the ti s s u e content of G A B A but no i n c r e a s e d efflux of GABA, to an i n c r e a s e d quantity of ammonia and to a s m a l l r i s e i n the m e d i u m c o n c e n t r a t i o n of glutamate. T h e r e was no i n c r e a s e d f o r m a t i o n of aspartate. A d d i t i o n of glutamate (2.5 mM), however, ca u s e d a l e s s e r i n c r e a s e of t i s s u e G A B A but an i n c r e a s e d efflux of GABA, i n c r e a s e d f o r m a t i o n and efflux of both aspartate and glutamine, and a some- what d i m i n i s h e d f o r m a t i o n of ammonia. Thus, the addition of glutamine and of L-glutamate to the incubation m e d i u m brought about s i g n i f i c a n t l y d i f f e r e n t e f f e c t s both i n amino a c i d content and amino a c i d effluxes i n r a t b r a i n c o r t e x s l i c e s. 8.6 S u m m a r y 1. By using tetrodotoxin to suppr e s s the ne u r o n a l efflux of amino a c i d s f r o m r a t b r a i n c o r t e x s l i c e s brought about by the joint a ction of p r o t o v e r a t r i n e and ouabain, the la t t e r being added to d i m i n i s h re-uptake of amino acids, it is shown that the major pools of glutamate, aspartate, glycine, s e r i n e and p r o b a b l y Y -aminobutyrate, a r e in the neurons. 2. The major pool of glutamine l i e s not i n the neurons but in the g l i a . 183 3. The citric acid cycle inhibitors, fluoroacetate and malonate, exert different effects on amino acid contents in, and on amino acid releases from, brain slices incubated in presence of protoveratrine. Fluoroacetate (3 mM) diminishes the content of glutamine, increases that of glutamate and y-aminobutyrate, and does not affect respirat ion. Malonate (Z mM) diminishes aspartate and y-aminobutyrate content, suppresses respiration, and does not affect glutamine content. 4. Glutamine is a more effective precursor of y-aminobutyrate than glutamate. 184 9. E F F E C T S O F NH^ ON B R A I N M E T A B O L I S M A N D T R A N S P O R T IN V I T R O It i s a w e l l known fact that ammonium ions e x e r t profound effects on b r a i n c e l l m e t a b o l i s m and function. The maintenance of extremely" low con- centrations of f r e e N H ^ by an ef f i c i e n t u t i l i z a t i o n m e c h a n i s m in the n o r m a l a n i m a l e n s u r e s the pr o p e r o p e r a t i o n of such p r o c e s s e s . Injection of ammo- n i u m salts may l e a d to convulsions. T h i s has been attributed to an imbalance of ammonia m e t a b o l i s m i n the c e n t r a l nervous system, but no s a t i s f a c t o r y explanation i s yet a v a i l a b l e . D i s e a s e s like hepatic coma and ep i l e p s y are known to be accompanied by elevated blood and b r a i n l e v e l s of ammonia. We have al r e a d y c o n s i d e r e d some aspects of the formation, t r a n s p o r t and u t i l i z a t i o n of NH^* in the b r a i n . In this chapter, the r e s u l t s of studies of the effects of N H 4 * " on m e t a b o l i s m and t r a n s p o r t w i l l be d e s c r i b e d . T h e i r p o s s i b l e b e a r i n g on ammonia t o x i c i t y w i l l be c o n s i d e r e d . 9.1 I n i t i a l contents of N a * and K"*~ in the infant (two-day-old) and adult rat b r a i n cortex. R e s u l t s i n Table 57 show the Na"*" and K* contents of two day old r a t b r a i n c o r t e x s l i c e s f r e s h l y p r e p a r e d and found p r i o r to i n c u b a t i o n . Values also given for the b r a i n c o r t e x s l i c e s of adult r a t are in a c c o r d with data in the l i t e r a t u r e (263, 264). The infant r a t b r a i n cortex content of Na"** ex- p r e s s e d in t e r m s of u e q u i v / m l t i s s u e water is about the same as that of the adult but the K"*" con c e n t r a t i o n i s lower. The sum of the Na"*" a n c j K* con- centrations f o r the infant b r a i n i s about the same as that used i n the incuba- tion m e d i u m (viz., 153 uequiv/ml), but that of the adult is 20 per cent higher. 185 T A B L E 57. I n i t i a l Na and K contents of 2-day o l d and adult r a t b r a i n c o r t e x s l i c e s . N a + and K + contents of infant and adult r a t b r a i n c o r t e x s l i c e s f r e s h l y p r e p a r e d , and found i m m e d i a t e l y p r i o r to incubation a r e given below and are e x p r e s s e d as Umole/g i n i t i a l wet wt. and as u m o l e / m l . t i s s u e water. Water content of 2-day o l d c o r t e x of r a t b r a i n is 88%, wh i l s t that of adult r a t b r a i n c o r t e x s l i c e s is 80% of the i n i t i a l f r e s h wt. Rat B r a i n C o r t e x N a + Content Content N a + + K + Liequiv/g U e q u i v / m l liequiv/g U e q u i v / m l U e q u i v / m l 2-day A d u l t 60. 8 + 0.8 . 69.1 53. 6 + 3.0 67.0 77. 2 £ 1.9 87. 6 91.4 + 1.6 114.0 156. 7. 181. 0 186 9.2 E f f e c t s of i n c r e a s i n g NH^* concentration on water and oxygen uptakes, and cationic fluxes of incubated rat b r a i n c o r t e x s l i c e s . When r a t b r a i n cortex s l i c e s are incubated in 0 2 at 3 7 ° C for one hour in a n o r m a l K r e b s - R i n g e r phosphate glucose medium, they take up water and Na"* from, and lose K"* to, the surrounding m e dium (Table 58, compare with i n i t i a l values i n Table 57). I n c r e a s i n g the c o n c e n t r a t i o n of N H 4 C I i n the m e d i u m f u r t h e r enhances the t i s s u e content of Na~* and concomitantly d i m i n i s h e s that of K*. The water uptake by the b r a i n s l i c e s i n c r e a s e with m e d i u m N H 4 C I concentrations above 5 mM. Since we have shown that N H 4 " * i s not concentrated against a concentration gradient (Chapter 6), the l o s s of t i s s u e K* due to m e d i u m N H 4 + , cannot be explained s i m p l y by N H 4 ~ * - K ^ exchange. F o r example, with 10 m M N H 4 C I in the incubation medium, there i s a t i s s u e l o s s of about 32 uequiv K"*/rnl t i s s u e water/hour for a gain of 10 uequiv N H 4 * / m l / h o u r . M o r e o v e r , simple N H 4"*-K"* exchange would not account for the a l t e r a t i o n s in N a * and water contents of the b r a i n s l i c e s . + + + + A s NH^ can r e p l a c e K i n the a c t i v a t i o n of the Na , K - A T P a s e (223, s e c t i o n 9.3), and as N H 4 * i n h i b i t s the active t r a n s p o r t of amino a c i d s such as g l y c i n e or glutamate (Chapter 5), it seems p o s s i b l e that NH^"* ions may act by uncoupling A T P a s e a c t i v i t y f r o m t r a n s p o r t p r o c e s s e s . E v i d e n c e to be presented below, however, shows that this p o s s i b i l i t y is u n l i k e l y . It i s to be noted that i n c r e a s e of NH^"* i n the incubation m e d i u m to high l e v e l s (e.g., 20 or 30 mM) leads to s i g n i f i c a n t f a l l s i n b r a i n r e s p i r a t i o n . 9.3 A T P a s e s of rat c e r e b r a l c o r t i c a l homogenates In view of the fact that N H 4 " * ions e x e r t profound effects on the c a t i o n i c contents of incubated r a t c e r e b r a l cortex s l i c e s (Table 58), studies were c a r r i e d out on the effects of v a r i o u s cations including N H 4 " * on the A T P a s e a c t i v i t i e s of adult r a t b r a i n c o r t i c a l homogenates. T A B L E 58. E f f e c t s of i n c r e a s i n g NH4CI concentrations on oxygen and water uptakes and Na"*, K + contents of incubated r a t b r a i n cortex s l i c e s . Rat b r a i n c o r t e x s l i c e s were incubated i n 0-> at 37°C for one hour in K r e b s - R i n g e r phosphate glucose m e d i u m 4- .1., with i n c r e a s i n g concentrations of N H ^ C l . T i s s u e concentrations of Na or K"1" at the end of the incubation a r e g i v e n as aequi v / g i n i t i a l wet wt. ti s s u e and as ue'quiv/ml t i s s u e water. Oxygen uptake (QO2) i s e x p r e s s e d as ( i m o l e / g i n i t i a l wet wt. t i s s u e and water uptake as (jl/100mg i n i t i a l wet wt. M e d i u m NH4CI (mM) Q 0 2 Water Uptake T i s s u e Contents (Uequiv/(?) (Uequiv/ml) Na+ K+ T o t a l Na+ K + T o t a l N i l 101 + 6 14.0 + 1.5 109.3 + 1.3 58.5 + 3.8 167.8 116.3 62.2 178.5 1 104 + 3 14.6 + 2.6 116. 0 + 6.0 44.8 + • 7.4 160.8 122.6 47.4 170. 0 2 111 + 3 14.8 + 1.7 118. 1 + 4.5 42.7 + 5.1 160.8 124.6 45. 0 169.6 5 107 + 3 18. 6 + 2.5 128.4 + 0.4 35.5 + 2.2 163 . 9 130.2 36. 0 166.2 10 107 + 6 24.4 + 1.6 135. 0 + 1.7 31.5 + 2.8 166.5 129.3 30. 2 159.5 20 90 + 1 42.4 + 0.4 177.2 + 1.8 19.0 + 0.4 196.2 144.8 15.5 160.3 30 78 + 1 50.8 + 3.3 192.1 + 3.3 16.3 + 0.1 208.4 146 . 9 12. 5 159.4 00 188 1. M g ^ - A T P a s e . None of the c h l o r i d e salts of the cations t r i e d + + + + + + (viz., Na , K , NH^ , L i , Rb , or Cs ) has any s i g n i f i c a n t effect on the M g + + - A T P a s e a c t i v i t y (Table 59). 2. Na +, K4", ( M g + + ) - A T P a s e . R e s u l t s i n T a b l e 60 show the dependence of the a c t i v i t y of this A T P a s e on Mg N a * and K*. A t 15 m M (chloride) salt concentrations, NH^*, L i * Rb , or Cs can substitute for K in the p r e s e n c e of Na though L i ^ has a r e l a t i v e l y s m a l l effect. None of these cations can substitute for N a * in the p r e s e n c e of K*. T h e s e r e s u l t s a r e i n a c c o r d with those of Skou (222, 223). C a * * and ouabain inhibit the Na*, K ^ - A T P a s e but have no effect on the Mg**-ATPase (222, 223). P r o t o v e r a t r i n e , DNP, and T T X have no effect on the Na"*", K * - A T P a s e while only p r o t o v e r a - t r i n e may have a s m a l l i n h i b i t o r y effect on the M g + + - A T P a s e (Table 61). The af f i n i t y constants ( K m ) for N a * K + - A T P a s e obtained i n our studies is 0.7 uequiv K * / m l and 3.7 u e q u i v / m l when NH^"1" i s substituted f o r K*. 9.4 E f f e c t s of t e t r a m e t h y l ammonium chlo r i d e , and the c h l o r i d e salts of L i , Rb and Cs on the oxygen and water uptakes, and the Na1", K"*" fluxes in incubated r a t b r a i n cortex s l i c e s . S ince L i C l , R b C l and C s C l , l i k e N H^Cl, can r e p l a c e K C 1 in activating the Na-K**~-Mg + +ATPase, it was of i n t e r e s t to compare the effects of these c h l o r i d e salts (Li"*", Rb*, C s + ) on the cationic fluxes at the b r a i n c e l l mem- brane, with those brought about by the p r e s e n c e of N H 4 C I . Rat b r a i n cortex s l i c e s were incubated in O-, at 3 7 ° C for one hour in K r e b s - R i n g e r phosphate glucose m e d i u m in the p r e s e n c e of (CH^J^NCl, L i C l , R b C l or C s C l . F r o m the r e s u l t s given in Table 62, it i s seen that the t i s s u e K* content is d i m i n i s h e d i n the p r e s e n c e of these salts. However, 189 T A B L E 59. E f f e c t s of cations on the M g i + - A T P a s e a c t i v i t y of homogenates of adult r a t b r a i n c o r t e x . Incubations of r a t b r a i n c o r t i c a l homogenates were c a r r i e d out f o r one hour at 3 7°C i n the fo l l o w i n g a s s a y s y s tems E D T A , 0. ImM; T r i s - C l buffer (pH 7.6), 95mM; T r i s - A T P , 3mM; (See Methods, S e c t i o n 2. 14). When p r e s e n t MgCl-, was ImM; C a C l ? , 2.8mM; N a C l , 58mM; L i C l , R b C l , C s C l , N H C l , KC1, 15mM. M g 2 * A T P a s e a c t i v i t i e s ( i . e . a c t i v i t i e s i n the absence of combined Na+ and K + ) given below, a r e means of 4 det e r m i n a t i o n s , with standard deviations not g r e a t e r than+_ 5%; they a r e e x p r e s s e d as u mole P i r e l e a s e d f r o m A T P p e r hour p e r mg wet wt. b r a i n c o r t e x . A d d i t i o n s to the a s s a y s y s t e m U mole P i / h / m g wet wt. N i l 0.04 Mg2+ 0. 76 M g 2 + + Ca2 + 0.69 M g 2 + + N a + 0.86 Mg2+ + K+ 0.73 Mg2+ + N H 4 + 0.71 Mg2+ + L i + 0. 77 M g 2 + + R b + 0. 75 M g 2 + + Cs + 0.75 190 T A B L E 60. E f f e c t s of cations on the N a + - K + - M g ' i + A T P a s e a c t i v i t i e s of homogenates of adult r a t b r a i n c o r t e x s l i c e s . Incubations of r a t b r a i n c o r t i c a l homogenates were c a r r i e d out f o r one hour at 3 7 ° C i n the f o l l o w i n g a s s a y system; E D T A , O.lmM; T r i s - C l , b uffer (pH 7.6), 95mM; T r i s - A T P , 3mM; (See Methods, s e c t i o n 2. 14). When p r e s e n t M g C l £ was ImM; Ca C l 2 , 2.8mM; N a C l , 58mM; L i C l , R b C l , C s C l , NH 4C1, KC1, 15mM; Values given below are means of 4 de t e r m i n a t i o n s with standard deviations not g r e a t e r than 5% and a r e e x p r e s s e d as umole P i r e l e a s e d f r o m A T P p e r hour p e r mg wet wt. b r a i n c o r t e x . A d d i t i o n s to the a s s a y s y s t e m u m o l e P i / h / m g wet wt. Na+ + K + Mg2 + + N a + + K+ M g 2 + + Na+ + K + + C a 2 + 0.04 2.36 0.76 M g 2 + + N a + + N H 4 + M g 2 + + N H 4 + + K+ M g 2 + + N a + + K+ + N H 4 + M g 2 + + N a + + K + + N H 4 + + C a 2 + 2.45 0. 78 2.51 0.76 M g 2 + + N a + + L i + M g 2 + + L i + + K + M g 2 + + Li ( 5 8 equiv/1) + K + M g 2 + + Na+ + K + + L i + 1.34 0.76 0. 76 2.45 M g 2 + + N a + + R b + M g 2 + + R b + + K + M g 2 + + N a + + K+ + R b + 2.10 0.81 2.36 M g 2 + + N a + + Cs + M g 2 + + C s + + K + M g 2 + + Na+ + K + + Cs + 2. 00 0.80 2.41 191 T A B L E 61. E f f e c t s of p r o t o v e r a t r i n e , tetrodotoxin, ouabain and 2, 4 d i n i t r o p h e n o l on the A T P a s e a c t i v i t i e s of rat c e r e b r a l c o r t i c a l homogenates. Incubations of ra t c o r t i c a l homogenates were c a r r i e d out at 3 7 ° C f o r one hour i n the f o l l o w i n g a s s a y s y s t e m f o r the e s t i m a t i o n of M g 2 + A T P a s e a c t i v i t y : E D T A , O.lmM; T r i s - C l buffer (pH 7.6), 95mM; T r i s A T P , 3mM; and M g C l 2 , ImM. F o r the d e t e r m i n a t i o n of the N a + , K + - A T P a s e + M g 2 + A T P a s e a c t i v i t y N a C l (58mM) and KC1 (15mM) were a l s o p r e s e n t . N a + , K + - A T P a s e is obtained by the d i f f e r e n c e of the two e s t i m a t i o n s . R e s u l t s a r e means of at l e a s t 4 d e t e r m i n a t i o n s with s t a n d a r d deviations not g r e a t e r than + 5%. Values a r e gi v e n as umole P i r e l e a s e d f r o m A T P pe r hour p e r m g wet wt. b r a i n c o r t e x . A d d i t i o n s to the a s s a y s y s t e m M g 2 + - A T P a s e N a + , K + - A T P a s e N i l 0. 76 1. 67 P r o t o v e r a t r i n e (5|jM) 0.56 1. 88 T e t r o d o t o x i n (3uM) 0. 73 1. 71 Ouabain (0.0 ImM) 0. 64 0.94 (0. ImM) 0. 62 0. 70 D N P (O.OlmM) 0. 73 1. 70 (0. ImM) 0. 76 1. 67 C a C l 2 (2.8mM) 0. 72 0. 14 C a C l 2 (2.8mM) 0.52 0.02 + P r o t o v e r a t r i n e (5 uM) T A B L E 62. E f f e c t s of N H 4 + , ( C H 3 ) 4 N + , L i + , R b + and C s * on the oxygen and water uptakes, and the Na"*", K + contents of incubated r a t b r a i n cortex s l i c e s . Rat b r a i n c o r t e x s l i c e s were incubated i n at 37 ° C for one hour in K r e b s - R i n g e r phosphate glucose m e d i u m containing the c h l o r i d e salts of NH 4*, ( C H o ^ N * , L i * , Rb and C s * at concentrations g i v e n below. Oxygen uptake (QO2) is e x p r e s s e d as umole/g i n i t i a l wet wt. ti s s u e and water uptake as Ul/lOOmg i n i t i a l wet wt. Add i t i o n s to the incubation m e d i u m Q 0 2 Water Uptake T i s s u e (uec] Contents [uiv/g) Na + K+ N i l (Control) 103 + 2 14.5 + 1. 6 110. 6 + 1. 4 59. 2 + 4.3 N H 4 C 1 5 107 + 3 18.6 + 2. 5 128.4 + 0. 4 35. 5 + 2.2 10 108 + 6 24.4 + 1. 2 135. 0 + 1. 7 31. 5 + 2.8 20 90 + 1 42.4 + 0. 4 177.2 + 1. 8 19 . 0 + 0.4 ( C H 3 ) 4 N C 1 10 105 + 2 12.6 + 2. 5 110.9 + 2. 1 49. 6 + 3. 1 20 104 + 2 10.6 + 2. 2 110. 7 + 4. 3 40. 6 + 4.4 L i C l 5 107 + 9 13.6 + 2. 9 109.8 + 4. 0 53. 4 + 4.4 10 111 + 4 14.5 + 0. 9 112. 0 + 3. 0 48. 7 + 3. 1 R b C l 5 105 + 6 13.2 + 1. 8 114. 0 + 4. 7 32. 0 0. 1 10 105 + 2 13.5 + 1. 6 113. 6 + 0. 4 27. 9 + 0.2 C s C l 5 105 + 2 9.9 + 1. 0 108. 1 + 2. 7 38. 0 + 1.8 10 131 + 5 12.5 + 1. 4 110. 8 + 0. 4 32. 3 + 1.2 NO CM 193 the d i m i n u t i o n i n tissue K* l e v e l s due to L i C l or (CH-jJ^NCl may be s i m p l y due to an exchange of L i * or ( C ^ J ^ N " * for K"*, as there i s no evidence that L i * or (CH^^N"* i s a c t i v e l y accumulated i n the t i s s u e . The f a l l i n the t i s s u e K"* l e v e l s due to Rb + or Cs"* r e s e m b l e s that due to NH^"*, but although Rb + (unlike NH^"*) is a c t i v e l y t r a n s p o r t e d into b r a i n c e l l s (224), it i s not known whether this i s the case with Cs"* ions. However, neither Rb*, Cs"*, L i " * or (CH^^N"* (at 5 or 10 mM), unlike NH4"*, b r i n g s about i n c r e a s e in the water uptake or the Na"* content of the incubated b r a i n t i s s u e . It i s t h e r e f o r e evident that the effects of N H ^ C l a r e d i f f e r e n t f r o m those of the a l k a l i metal salts ( L i + , Rb"*, or Cs"*) and of ( C H 3 ) 4 N C 1 . None of these salts affect the rate of r e s p i r a t i o n , except C s C l at 10 mM, which causes a stim u l a t i o n . 9.5 E f f e c t s of i n c r e a s i n g concentrations of NH^"* on cationic fluxes i n incubated two-day-old rat b r a i n c o r t e x s l i c e s . Infant (two-day-old) r a t b r a i n c o r t e x s l i c e s were incubated i n 0 2 at 3 7 ° C for one hour i n K r e b s - R i n g e r phosphate glucose m e d i u m containing i n c r e a s i n g concentrations of N H ^ C l . F r o m the r e s u l t s g i v e n i n T a b l e 63, it is seen that the l o s s of K"* f r o m the t i s s u e i s equivalent to the concen- t r a t i o n of N H 4 C I in the medium. T h i s may simply be explained as due to an exchange of NH4"* f o r K + in the infant b r a i n . T h e r e i s no evidence of accu m u l a t i o n of N H 4 against a concentration g r a d i e n t in infant b r a i n . Water is not taken up by incubated infant rat b r a i n c o r t e x s l i c e s i n the p r e s e n c e or absence of N H 4 * in the incubation medium. The content of water i n two day old r a t b r a i n i s , however, high (about 88%) so that i n c r e a s e d water uptake would not be expected. The r e s p i r a t o r y rate of infant b r a i n c o r t e x s l i c e s i s unaffected by NH^* in the incubation medium. The b r a i n Na"* content i n incubated infant r a t c o r t e x i s enhanced to some extent with m e d i u m N H ^ C l concentrations of 20 and 30 mM. Com- T A B L E 63. E f f e c t s of i n c r e a s i n g N H 4 C I concentrations on the oxygen and water uptakes and Na+, K + contents of infant (2-day old) r a t b r a i n cortex s l i c e s . Incubation of two-day old rat b r a i n c o r t e x s l i c e s were c a r r i e d out i n K r e b s - R i n g e r phosphate glucose m e d i u m in O2 at 3 7 ° C f o r one hour with i n c r e a s i n g m e d ium N H 4 C I c o n c e n t r a t i o n s . T i s s u e contents of N a + or K + at the end of the incubation are e x p r e s s e d as u e q u i v / g i n i t i a l wet wt. ti s s u e , and as Uequiv/ml t i s s u e water. Oxygen uptake (QO2) is given i n t e r m s of u m o l e / g i n i t i a l wet wt. t i s s u e . Water uptake was found to be (0. 0 _+ 2.0) u l / 1 0 0 mg i n i t i a l wet wt. and water content 88%. M e d i u m N H 4 C 1 (mM) Q O 2 T i s s u e Contents (Uequiv/g) (Uequiv/m 1) Na + K+ T o t a l Na+ K+ T o t a l N i l 72 + 4 82.3 + 0.5 55.7 + 0.5 138.0 93.5 63.3 156. 8 2.0 73 + 4 82. 0 + 1 . 6 53 . 9 + 1. 1 135 . 9 93.2 61.3 154.5 5.0 68 + 5 84. 7 + 3.5 50.3 + 0.7 135.0 96.3 57.2 153. 5 10.0 79 + 5 84. 0 + 2.0 48 . 9 + 1.1 132 . 9 95.5 55 . 6 151.5 20.0 82 + 6 91.1 + 1.3 36.3 + 2 . 9 127.4 103.5 41.3 144.8 30.0 81 + 1 98.5 + 2 . 6 22.7 + 0 . 6 121.2 111 . 9 28.1 137. 7 195 p a r e d with the value for incubated adult b r a i n t i s s u e (Table 58), the i n c r e a s e i s r e l a t i v e l y s m a l l . F o r example, with 20 m M N H 4 C I in the incubation medium, the i n c r e a s e s in the Na"*" contents of incubated infant and adult r a t b r a i n c o r t e x s l i c e s i s 9 and 68 uequiv/g/hour, r e s p e c t i v e l y . 9.6 Quantitative aspects of the effects of NH4"* on the Na"*, K"* and water contents of incubated r a t b r a i n c o r t e x s l i c e s . If N H 4 * ions e x e r t t h e i r effects on N a * and K"* contents of incubated adult brain, by uncoupling the Na"*, K"*-ATPase a c t i v i t y f r o m active t r a n s p o r t p r o c e s s e s , it would be expected that they would act without time lag. E x p e r i - ments were c a r r i e d out to obse r v e whether N H 4 " * ions act with or without a t i m e l a g . R a t b r a i n cortex s l i c e s were incubated in 0 2 at 3 7 ° C in K r e b s - R i n g e r phosphate glucose m e d i u m with or without 10 m M N H 4 C I for v a r i o u s p e r i o d s of time. A t the end of the incubation periods, the Na +, K"*, NH^"* and water contents of the t i s s u e were analyzed. R e s u l t s given i n Table 64 show that the i n c r e a s e i n water and Na"* contents of the tissue, due to NH^"*, only o c c u r s after about 30 minutes incubation. While the ti s s u e NĤ "*" content r e a c h e s its m a x i m u m l e v e l within a few minutes of incubation, t i s s u e K"* content f a l l s s i g n i f i c a n t l y (i.e., beyond the value expected by K"*-NH4"* exchange) only after 15 minutes incubation and then continues to f a l l . Such r e s u l t s + + a r e only to be expected if the effect of N H 4 , on K r e l e a s e f r o m the brain, i s due to a ti m e - c o n s u m i n g metabolic p r o c e s s and are not cons i s t e n t with an immediate action of N H 4 " * at the c e l l membrane leading to an "uncoupling" of A T P a s e a c t i v i t y f r o m cationic t r a n s p o r t . 9.7 E f f e c t s of i n c r e a s i n g N H 4 " * concentrations on the c a t i o n i c contents of rat b r a i n cortex s l i c e s incubated in the p r e s e n c e of 0.1 m M ouabain or in the absence of glucose. The fact that N H 4 + e x e r t s its effects by metabolic changes, is shown TABLE 64. Kinetics of the alterations in the tissue water, Na , K and NH^ contents of rat brain cortex slices incubated in an NH^Cl containing medium. Rat brain cortex slices were incubated in O2 at 37°C for one hour in Krebs-Ringer phosphate glucose medium with or without lOmM NrLCl. Water uptake is expressed as ul/100 mg. initial wet wt. Time Water Uptake Tissue Contents U e q u i v/g initial wet wt. Uequiv/ml tissue water Na T K + N H 4 + Na + K + NH4+ After Oxygenation (5 min) Nil 5.3 + 0.1 101.5 + 1.9 62.4 + 7.2 119.0 73.2 + NH4C1 7.6 + 0.2 95.6 + 7.0 60.0 + 4.7 8.8 + 0.1 109.1 68.5 10.0 After Equilibration (7 min) (i. e. zero time) Nil 6.2 + 1.8 105.5 0.5 54.3 + 4.5 122.4 63. 0 + N H 4 C I 9.7 + 0.7 104.6 + 8.4 45.3 + 1.5 11.1 + 0.6 116.6 50. 5 12.3 15 min. incubation Nil 8.2 + 1.4 100.6 + 0.6 58.0 + 2.5 114. 1 65.8 + N H 4 C I 11.9 0.2 99.8 + 7.4 43.0 + 2. 8 11.7 + 0. 1 109.0 46. 8 12.8 30 min. incubation Nil 11.9 + 0.8 108.5 + 4.5 58.5 + 1.5 118.1 63. 7 + NH4C1 14.2 + 0.4 110.5 + 4.5 40.3 0.9 11.2 + 0.2 117.3 42.8 11.8 45 min. incubation Nil 12.3 + 1.5 104.9 4.7 60.3 + 3.9 113.7 65.3 + N H 4 C I 20.5 + 0.3 122.0 + 1.0 36.6 + 2.2 12. 1 + 0.6 121.4 36.4 12.0 60 min. incubation Nil 14.9 + 0..7 110.0 + 0.8 57.4 + 1.1 115.9 60.5 + N H 4 C I 25.3 + 1.5 133.0 + 1.6 30.5 + 3.3 11.6 + 0.3 126.3 29.0 11.1 197 also by the r e s u l t s of experi m e n t s c a r r i e d out in the presence of 0.1 m M ouabain (Table 65). L i k e ouabain, NH^"* i s known to inhibit active t r a n s p o r t p r o c e s s e s ( T a b l e s 28, 30 and 31), but, unlike ouabain, it s t i m u l a t e s Na*, K * - A T P a s e (Ta b l e s 61 and 6 y j . F r o m the r e s u l t s given i n T a b l e 65, it i s evident that the drop i n t i s s u e K"* l e v e l s due to 0.1 m M ouabain i s f u r t h e r potentiated by NH^"*. Thus, with 2 m M N H 4 C I in the medium, the drop i n K"*, namely, (19.8 - 14.1) - 5.7 u e q u i v / m l tis s u e water i s g r e a t e r than that allowed for by the 1:1 NH^"* for K* exchange. The effect i s also evident with 5 m M NH^Cl, but the ti s s u e has under this c i r c u m s t a n c e (i.e., with 5 m M m e d i u m KC1) a l m o s t r e a c h e d i t s m i n i m u m concent r a t i o n of K* so that f u r t h e r i n c r e a s e of the m e d i u m NH.* con c e n t r a t i o n cannot f u r t h e r d i m i n i s h the K"* 4 content of the tis s u e . The tiss u e Na"* concentrations (uequiv/ml t i s s u e water) with or without .dded N H^Cl, does not change as it has r e a c h e d i n the p r e s e n c e of 0.1 m M ouabain, the N a * concentration of the incubation medium. S i m i l a r r e s u l t s were obtained on incubation of b r a i n c o r t e x s l i c e s i n C>2 f o r one hour at 3 7 ° C i n g l u c o s e - f r e e K r e b s - R i n g e r phosphate m e d i u m containing i n c r e a s i n g concentrations of N H ^ C l (Table 65). Thus, a f a l l of (19.9 - 13.5) = 6.4 uequiv K"* / m l tiss u e water o c c u r s with a concomitant gain, by the tissue, of only 2 uequiv NH^^/ml ti s s u e water. The Na"1" l e v e l in the ti s s u e has a l r e a d y attained the m e d i u m concentration of N a * so that f u r t h e r i n c r e a s e of t i s s u e Na"* cannot o c c u r . 9.8 E f f e c t s of changing m e d i u m io n i c c o m p o s i t i o n on the Na"*, K* contents of incubated r a t b r a i n c o r t e x s l i c e s . (i) E f f e c t s of Ca"*"* and K"*. R e s u l t s given i n Table 66 show that r a t b r a i n cortex s l i c e s incubated i n f o r one hour at 3 7 ° C i n a p h y s i o l o g i c a l glucose saline m e d i u m in the absence of K"* or Ca"*"*, gain N a * and lose K* to the incubation medium. In the absence f r o m the m e d i u m of both C a + + and K"*, the tiss u e K* content is T A B L E 65. E f f e c t s of i n c r e a s i n g N H 4 C I concentrations on the oxygen and water uptakes and the N a + , K + contents of r a t b r a i n cortex s l i c e s incubated i n the p r e s e n c e of 0 . I m M ouabain or in the absence of glucose. Contents of Na"*" and K + i n r a t b r a i n cortex s l i c e s incubated i n for one hour at 3 7 ° C i n the p r e s e n c e of i n c r e a s i n g N H 4 C I concentrations i n K r e b s - R i n g e r phosphate medium: A) with glucose and 0 . I m M ouabain p r e s e n t ; B) without glucose. A d d i t i o n s to the incubation m e d i u m Q O z U mole/g i n i t i a l wet wt. Water Uptake u l / 1 0 0 m g i n i t i a l wet wt. T i s s u e Contents U mole/ g i n i t i a l wet wt. umole / m l ti.ssue water Na + K+ N a + K+ A) G l u c o s e + Ouabain 94 + 5 38.7 + 1.7 172.6 + 2.1 23.5 + 0. 9 145.4 19.8 + N H 4 C I , 2mM 100 + 2 42.8 + 0. 1 179.7 + 0.5 17.3 + 1. 0 146.3 14. 1 5 m M 94 + 8 40.4 + 3.5 178.4 + 1.4 13.1 + 0 . 1 148.2 10.9 l O m M 85 + 3 43.3 + 1. 1 183. 0 + 6. 8 12.5 + 0 . 5 148.4 10.1 2 0 mM 69 + 5 43.2 + 3.6 181.8 + 6.4 11.8 + 0 . 2 147.6 9 . 5 B) N i l 69 + 3 43.4 + 0.4 1 9 0 . 9 + 2.5 24.6 + 0 . 1 154.7 1 9 . 9 + NI-I4C1, 2mM 64 + 1 46.6 + 0. 1 198.6 + 8.6 17.1 + 1. 0 1 5 6 . 9 13.5 5mM 58 + 1 48.2 + 1.2 198.2 + 4.7 17.5 + 0 . 4 154.6 13.7 l O m M 53 + 2 43.6 + 1.3 189.7 + 0.6 16.4 + 0 . 1 153.5 13.3 20mM 50 + 2 53. 7 + 0.5 204.4 + 4.2 16.1 + 0 . 1 1 5 2 . 9 12.0 T A B L E 66. Effects of medium cation contents on the oxygen and water uptakes, and Na , K levels in incubated rat brain cortex slices. Incubations of rat brain cortex slices were carried out in O 2 at 37°C for one hour under media conditions given below. When adjustments in the incubation media were made, the concentration of NaCl was diminished by an amount equal to the concentration of KC1 added. Tissue N a + and K contents at the end of the incubation are expressed as umole/g initial wet wt., oxygen consumption ( Q O 2 ) as umole/g initial wet wt. and water uptake as ul/100 mg initial wet wt. Changes made to a Krebs-Ringer phosphate trlucose medium Medium N a + U equiv/ml Q ° 2 Water Uptake Tissue Content (Umolc /g) N a + K+ Control 148 101 + 6 14.0 + 1.5 109.3 + 1.3 58.5 + 3.8 K + - f r c e 148 94 + 3 22.6 + 2.6 146.0 + 1.0 35.5 + 6.5 C a + + - free 148 128 + 5 18.0 + 2.0 136.6 + 3.3 37.4 + 4.9 K + + - f r c e , C a + + - f r e e 148 138 + 2 19.2 _+ 1.0 144.8 + 5.4 26.6 + 6.2 KC1 (+25mM) Adjusted 118 147 J. 2 22.7 + 2.7 94.4 + 3.0 80.5 r 2.3 123 141 + 3 22.3 + 0.4 100.7 + 1.5 . 73.4 + 3.3 Not adjusted 148 127 + 1 15.1 + 2.9 110.7 3.9 92.6 + 2.8 KC1 (+50mM) Adjusted 98 131 + 1 40.4 + 0.4 91.2 + 0.3 107.3 + 1.3 Not adjusted 148 133 + 2 35.8 + 1.4 135.5 + 1.2 120.0 + 4.0 KC1 (+100mM) Not adjusted 148 137 + 3 38.0 + 2.0 138.8 + 2.4 - 200 further diminished while the tissue N a * concentration approaches that of the incubation medium. It is to be noted that media, initially free of K