UBC Theses and Dissertations

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UBC Theses and Dissertations

Ammonia and amino acid metabolism and transport in brain in vitro Benjamin, Abraham Moses 1972

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A M M O N I A AND AMINO ACID 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 . S c . (Hons.),  U n i v e r sity of Bombay,  1961  B . S c . (Tech.),  U n i v e r sity of Bombay,  1963  M.Sc.  U n i v e r s i t y of Bombay,  1966  M.Sc,  (Tech.),  U n i v e r s i t y of B r i t i s h C o l u m b i a ,  1969  A thesis submitted in p a r t i a l fulfilment of the r e q u i r e m e n t s for the degree of Doctor of Philosophy in the D e p a r t m e n t of B i o c h e m i stry  We accept this thesis as conforming to the r e q u i r e d  standard  T H E UNIVERSITY  O F BRITISH C O L U M B I A  June,  1972  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  this  thesis  in  at  University  the  make  that  it  purposes  written  for  may  be  It  financial  for  of  July 25th, 1972  of  Columbia,  British  by  gain  Columbia  for  the  understood  Biochemistry  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  extensive  granted  is  fulfilment  available  permission.  Department  Date  freely  permission  representatives. thesis  partial  shall  Head o f  be  requirements  reference copying  that  not  the  of  I agree and this  or  allowed  without  that  study. thesis  my D e p a r t m e n t  copying  for  or  publication my  ii  ABSTRACT  Studies have been made of the f a c t o r s controlling the  formation,  t r a n s p o r t and utilization of a m m o n i a i n the b r a i n and its effects on b r a i n metabolism, of the p r o c e s s e s  promoting amino a c i d fluxes in b r a i n under  a v a r i e t y of conditions, e s p e c i a l l y those leading to i n c r e a s e d nerve activity, and on the s p e c i f i c locations, and sites of formation,  of amino acids in the  brain. By using tetrodotoxin (2 tiM) to s u p p r e s s partly the neuronal efflux of amino acids brought about by the joint action of p r o t o v e r a t r i n e (5 |iM) ouabain (0.1 mM),  and  the f o r m e r drug being used to promote n e u r o n a l efflux of  amino acids, and the latter being added to d i m i n i s h re-uptake of amino acids, it has been shown that the major pools of glutamate, aspartate, glycine, s e r i n e and probably y-a-minobutyrate, are in the neurons. However, the major pool of glutamine appears to be i n the g l i a .  Glutamine  formation  takes place i n the g l i a and i s a p r o c e s s p a r t l y c o n t r o l l e d by the of cations (K , +  Na"*", C a  + +  ) within these c e l l s .  concentrations  F l u o r o a c e t a t e (3 mM)  acts  m a i n l y i n the g l i a as it s u p p r e s s e s glutamine synthesis, but not the protoveratrine-stimulated brain respiration.  Malonate (2 mM)  acts m a i n l y i n  the neurons since it s u p p r e s s e s 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 but not the synthesis of glutamine. The  amino acids, p a r t i c u l a r l y glutamate, y-aminobutyrate,  aspartate  and glycine, are r e l e a s e d f r o m b r a i n cortex s l i c e s under conditions a s s o c i a t e d with b r a i n c e l l excitation. by tetrodotoxin (2 (jM).  The  release processes  are partly or wholly  blocked  Tetrodotoxin does not affect the r e l e a s e of glutamine  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 it.  T h i s r e s u l t is i n a c c o r d with the con-  c l u s i o n that the main depot of glutamine l i e s not in the neurons but in the glia. P r o t o v e r a t r i n e brings about an i n c r e a s e d rate of f o r m a t i o n of glutamine in incubated b r a i n slices,  suggesting that glutamate r e l e a s e d f r o m the neurons is  taken up by the g l i a and there converted to glutamine.  LrGlutamine is more  iii  effective than L - g l u t a m a t e as a p r e c u r s o r As  glutamic  of y-aminobutyrate  a c i d d e c a r b o x y l a s e i s l o c a l i z e d i n the n e u r o n s ,  in brain  slices.  it i s c o n c l u d e d  t h a t g l u t a m i n e r e l e a s e d f r o m the g l i a i s t a k e n up by the n e u r o n s a n d t h e r e converted  y-aminobutyrate.  to g l u t a m a t e a n d  C h a n g e s i n the c o n t e n t s  of NH^"*" i n i n c u b a t e d  brain slices are accom-  p a n i e d b y q u a n t i t a t i v e l y e q u i v a l e n t c h a n g e s i n the a m i n o a c i d c o n t e n t s A m y t a l (1 m M )  tissue.  s u p p r e s s e s endogenous glutamate oxidation and  e n h a n c e s the n e u r o n a l contents ammonia liberation. a glucose-free neurons,  of the  of g l u t a m a t e a n d y-aminobutyrate.  Ammonia  It d i m i n i s h e s  i s 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  m e d i u m l a r g e l y by endogenous g l u t a m a t e o x i d a t i o n within the  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 - g l u t a m a t e i s t a k e n up  a g a i n s t a c o n c e n t r a t i o n g r a d i e n t l a r g e l y b y the g l i a and i s l e s s e f f e c t i v e than e n d o g e n o u s g l u t a m a t e a s a s o u r c e of a m m o n i a i n b r a i n .  A m m o n i u m ions a r e  not a c c u m u l a t e d i n b r a i n s l i c e s against a c o n c e n t r a t i o n gradient. presumably  formed,  up to a l i m i t i n g c o n c e n t r a t i o n ,  They are  i n the n e u r o n s i n d e p e n -  d e n t l y of t h e e x t e r n a l NH^"*" c o n c e n t r a t i o n .  A m m o n i u m ions affect both n e u r o n a l 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"*" a n d of  i n the i n c u b a t e d b r a i n s l i c e s .  i s p a r t l y due to e x c h a n g e w i t h NH^"*".  m a r k e d i n infant rat b r a i n . diminishing A T P  the b r a i n c e l l m e m b r a n e unstimulated  The exchange p r o c e s s  T h e e f f e c t s of N H ^  concentrations,  The decrease  +  is most  i n inhibiting r e s p i r a t i o n ,  a n d c h a n g i n g the c a t i o n i c c o n c e n t r a t i o n s at  a r e m o r e p r o n o u n c e d i n the s t i m u l a t e d t h a n i n t h e  brain tissue.  It i s c o n c l u d e d that t h e e f f e c t s of  on b r a i n  m e t a b o l i s m a n d c a t i o n t r a n s p o r t m a y be e x p l a i n e d b y i t s i n h i b i t o r y e f f e c t o n A T P formation  i n t h e n e u r o n s , b y r e m o v a l of a - k e t o g l u t a r a t e  p a r t l y b l o c k i n g t h e o p e r a t i o n of t h e c i t r i c a c i d c y c l e . r e a s o n s f o r a m m o n i a t o x i c i t y i n the c e n t r a l n e r v o u s  and hence by  T h i s m a y be one of t h e system.  iv TABLE  OF  CONTENTS Page  TITLE PAGE  i  ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST O F FIGURES  xiii  LIST O F T A B L E S  xiv  ABBREVIATIONS...  xxi  ACKNOWLEDGMENTS  1.  xxii  INTRODUCTION  1  1.1  F o r m a t i o n of a m m o n i a by nerve tissue  1  1.2  F a c t o r s affecting the in vivo l e v e l s of a m m o n i a i n the b r a i n . . . .  i  (i) (ii) (iii)  A m m o n i a content of b r a i n in vivo F a c t o r s producing low b r a i n a m m o n i a l e v e l s in. v i v o . F a c t o r s producing high b r a i n a m m o n i a l e v e l s in vivo  1 2 3  (i)  Some c h e m i c a l changes that occur in the b r a i n on the application of s t i m u l i to the whole a n i m a l R e c o v e r y p r o c e s s e s that occur 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 Convulsions and b r a i n energy l e v e l s  1.3  (ii) (iii) 1.4  F a c t o r s affecting the f o r m a t i o n of a m m o n i a in b r a i n t i s s u e in v i t r o . (i) (ii)  5 5 6  Carbohydrate m e t a b o l i s m and a m m o n i a f o r m a t i o n E l e c t r o n transport, oxidative phosphorylation and ammonia formation E f f e c t s of exogenous amino acids on a m m o n i a formation  7  A m m o n i a f o r m a t i o n and utilization m e c h a n i s m s in the b r a i n . . .  9  (i)  9 9  (iii)  1.5  4  Urea (a) U r e a as a source of a m m o n i a (b) P o s s i b l e f o r m a t i o n of urea as a m e c h a n i s m for a m m o n i a utilization •. (c) P o s s i b l e importance of urea synthesis in the b r a i n (85, 95, 100)  6  8  10 11  V  Page (ii)  (iii)  (a)  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 aspartate channelled through N A D + or A M P (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 A m m o n i a utilization processes in b r a i n  13 15  1.6  T r a n s p o r t of amino acids i n b r a i n  16  1.7  (i)  18 20 20 24  (ii) 1.8  Amino acid metabolism in brain (a) Initial c e r e b r a l contents of amino acids (b) Pathways of glutamate m e t a b o l i s m Compartmentation of amino acid m e t a b o l i s m i n b r a i n . . . .  Propagation of nerve i m p u l s e  26  (i) (ii) (iii) (iv) (v) (vi)  26 27 28 28 29  B r a i n c e l l types (168, 169) Resting potential (164-168) A c t i o n potential (164-168) S o d i u m - P u m p (164-168) Synaptic t r a n s m i s s i o n (164-168, 170) A m i n o acids as putative t r a n s m i t t e r s (164, 165, 168, 170, 233) (vii) Ca"*"*" ions and excitation (viii) H i g h K + ion concentration and excitation (ix) C e r e b r a l cortex s l i c e s and excitation phenomena  1.9  1.10 2.  11  29 30 30 31  Some p r o p e r t i e s of drugs and metabolic inhibitors used as tools for this investigation  32  (i) (ii) (iii) (iv) (v) (vi)  32 35 36 37 39 41  Tetrodotoxin ( T T X ) L o c a l anesthetics (166, 176) Protoveratrine Barbiturates C a r d i a c g l y c o s i d e s - Ouabain Miscellaneous  Objectives of the present work.  42  MATERIALS AND METHODS  43  2.1  Animals.  43  2.2  Chemicals  43  2.3  Tissue preparation  44  2.4  M e d i a compositions and incubation p r o c e d u r e s  44  (i) (ii) (iii)  44 45 45  K r e b s - R i n g e r phosphate m e d i u m K r e b s - R i n g e r bicarbonate m e d i u m M e d i u m II  vi Page 2.5  Amino acid analyzer estimations..  46  (a)  46 46 46 46 47 47 48 48  (b)  Sample preparation (i) T i s s u e s a m p l e p r e p a r a t i o n (ii) M e d i u m s a m p l e p r e p a r a t i o n Sample analysis (i) A c i d i c a n d n e u t r a l a m i n o a c i d a n a l y s e s (ii) y - A m i n o b u t y r i c a c i d a n d a m m o n i a (iii) N - A c e t y l a s p a r t a t e (iv) C a l c u l a t i o n s  2.6  W a t e r 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 e n uptake by b r a i n c o r t e x s l i c e s  49  2.8  Dry  w e i g h t 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  Experiments with sodium L - [ U -  50  2.11  Scintillation liquid composition  50  2.12  Na"** a n d 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  Determination  51  2.14  A s s a y of Na"*". K"*~-ATPase of b r a i n h o m o g e n a t e s  52  (i) (ii)  M e d i u m composition Incubation procedure  52 52  (iii)  Estimation  52  of A T P  1 4  C ] glutamate  concentrations.  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  Protein estimation.  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 u s e d i n t h i s t h e s i s  55  (i)  Initial values  55  (ii) (iii) (iv) (v) (vi)  Total values Adjustment A m m o n i a and a m m o n i u m i o n . . . Flux Action potentials  55 55 56 56 56  vii Page 3.  AMMONIA  3.1  FORMATION  57  IN B R A I N I N V I T R O  Ammonia  a n d a m i n o a c i d c o n t e n t s of r a t c e r e b r a l c o r t e x  initially,  a n d a f t e r i n c u b a t i o n i n the p r e s e n c e  slices  or absence of 57  glucose (i)  C h a n g e s i n the N H 4 * " and a m i n o a c i d contents  of b r a i n  s l i c e s i n c u b a t e d i n t h e p r e s e n c e a n d a b s e n c e of g l u c o s e . . (ii) (iii)  C h a n g e s i n the i n i t i a l N H 4 +  b r a i n cortex s l i c e s on incubation  60  P o s s i b l e protein breakdown i n incubating b r a i n slices....  61  3.2  Ammonia  formation in anoxia  3.3  Ammonia  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  63  66  slices 3.4  I s t h e r e a d i r e c t r o l e of a s p a r t a t e i n t h e p r o c e s s  of a m m o n i a 68  f o r m a t i o n i n the b r a i n ? 3.5  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 o n the r a t e of a m m o n i a f o r m a t i o n f r o m e n d o g e n o u s a m i n o a c i d s of r a t c e r e b r a l c o r t e x 72  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 . 3.6  E f f e c t s of o u a b a i n o n the r a t e of a m m o n i a f o r m a t i o n i n r a t brain cortex slices incubated i n a glucose-free medium.  3.7  3.8  3.9  3.10  3.11  4.  m e d i u m d -^oid of g l u c o s e  76  A m m o n i a formation by r a t 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 i n the a b s e n c e of g l u c o s e  78  E f f e c t s of t e t r o d o t o x i n , l i d o c a i n e a n d p r o t o v e r a t r i n e o n t h e r a t e of a m m o n i a f o r m 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 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  C h a n g e s i n the a m m o n i a a n d a m i n o a c i d c o n t e n t s  of i s o l a t e d  rat b r a i n c o r t i c a l s y n a p t o s o m e s on incubation  80  Summary  83  CONTROL MECHANISMS FOR  GLUTAMINE  S Y N T H E S I S IN  RAT 85  IN V I T R O .  E f f e c t s of v a r y i n g s o d i u m i o n c o n c e n t r a t i o n o n the r a t e of 85  glutamine synthesis i n rat b r a i n cortex slices 4.2  74  T h e r o l e of Ca"*"*" i n the f o r m a t i o n o f a m m o n i a f r o m L - g l u t a m a t e 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  BRAIN CORTEX 4.1  57  a n d a m i n o a c i d c o n t e n t s of  E f f e c t s of v a r y i n g p o t a s s i u m i o n c o n c e n t r a t i o n of g l u t a m i n e s y n t h e s i s i n r a t b r a i n c o r t e x s l i c e s  on the r a t e 86  viii Page 4.3  4.4  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 byrat brain cortex slices G l u t a m i n e s y n t h e s i s i n a Na~*"-rich,  K**"-free,  Ca^-free, 8 9  incubation medium. 4.5  G l u t a m i n a s e a c t i v i t y and g l u t a m i n e s y n t h e s i s  4.6  E f f e c t s of s o d i u m L - g l u t a m a t e a n d N H ^ C l on the i n h i b i t i o n of g l u t a m i n e s y n t h e s i 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 b a t e d w i t h g l u c o s e e i t h e r i n M e d i u m II, o r i n the p r e s e n c e of  C o m p a r a t i v e 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 ( m e t h i o n i n e sulfoximine, ouabain, fluoroacetate, malonate, D N P and a m y t a l ) on r a t e s of o x y g e n u p t a k e s , g l u t a m i n e s y n t h e s i s a n d a m i n o a c i d c o n t e n t 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 92  i n the p r e s e n c e of g l u c o s e . 4.8  4.9  5.  E f f e c t s of t e t r o d o t o x i n on o u a b a i n i n h i b i t i o n of g l u t a m i n e synthesis in rat b r a i n cortex slices  96  Summary  98  TRANSPORT OF  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  Transport  5.2  T h e u p t a k e of L - [ U - 1 4 c ] g l u t a m a t e by r a t c e r e b r a l c o r t e x slices.  102  A p p a r e n t a b s e n c e of a n e x c h a n g e p r o c e s s b e t w e e n e x t e r n a l L - [ U - I 4 c j g l u t a m a t e a n d e n d o g e n o u s g l u t a m a t e of r a t b r a i n cortex slices  104  5• 3  of L - g l u t a m a t e i n t o i n c u b a t e d r a t b r a i n .  5.4  L o c a t i o n of e x o g e n o u s L - g l u t a m a t e u p t a k e  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 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 the t i s s u e and m e d i u m c o n c e n t r a t i o n s of a m i n o acids in incubated rat b r a i n cortex slices  5.6  6.  89  89  ouabain. 4.7  88  Summary  TRANSPORT OF 6.1  100  10 5  108 I l l  A M M O N I U M IONS I N B R A I N IN V I T R O  T i s s u e a n d m e d i u m c o n t e n t s of N"-~4 of r a t b r a i n c o r t e x slices incubated under varying conditions  112  +  113  ix Page 6.2 6.3 6.4  T i s s u e / M e d i u m concentration incubated brain slice Exogenous N H ^ the r a t .  ratios for N H ^  i n the 115  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 117  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 ^ L - g l u t a m a t e into b r a i n c e l l s and  and 121  6.5  R a t e s of N H ^  6.6  E f f e c t s of N H 4  6.7  E f f e c t s of g l y c i n e on NH4"*" u p t a k e  126  6.8  U p t a k e (into b r a i n c o r t e x s l i c e s ) of e x o g e n o u s L - g l u t a m a t e a n d N H 4 w h e n 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  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 o n the u p t a k e of N H 4 + incubated rat b r a i n cortex slices.  126  +  g l y c i n e u p t a k e s by b r a i n c o r t e x s l i c e s  on g l y c i n e uptake.  123  +  6.9 6.10 6.11  7.  7.2  7.3  7.4  7.5 7.6  by  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 i n o a c i d c o n t e n t 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  Summary  133  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 M I N O ACIDS F R O M THE B R A I N IN V I T R O 7.1  123  RELEASE  OF 134  E f f e c t s of p r o t o v e r a t r i n e , o u a b a i n a n d t e t r o d o t o x i n on a m i n o a c i d content 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 incubated in glucose-saline media  135  E f f e c t s of p r o t o v e r a t r i n e a n d t e t r o d o t o x i n on a m i n o a c i d c o n t e n t 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 in a glucose-free m e d i u m  139  E f f e c t s of p r o t o v e r a t r i n e a n d t e t r o d o t o x i n on a m i n o a c i d c o n t e n t 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 calcium-deficient media  143  E f f e c t s of p r o t o v e r a t r i n e a n d t e t r o d o t o x i n on a m i n o a c i d c o n t e n t 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 in glucose-free, c a l c i u m - d e f i c i e n t media  146  E f f e c t s of l i d o c a i n e on t h e 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  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 a m i n o 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 a m y t a l o n t h e r e l e a s e of a m i n o a c i d s rat brain cortex  7.8  from  slices  1 52  E f f e c t s of i n c r e a s e d a n d of t e t r o d o t o x i n on a m i n o a c i d content in, and r e l e a s e f r o m , incubated r a t b r a i n c o r t e x slices.  7.9  1 56  E f f e c t s of t e t r o d o t o x i n o n t h e e f f l u x of a m i n o a c i d s kidney cortex  7.10  s l i c e s incubated  r a t brain cortex  s e n c e of 2,4 d i n i t r o p h e n o l ,  8.  s l i c e s incubated  NH4"*" o r 105 m M  i n the p r e -  KC1..  161  Summary  164  L O C A T I O N S O F AMINO ACIDS IN BRAIN C O R T E X SLICES THE RAT  OF  8.1  L o c a t i o n of t h e g l u t a m a t e - g l u t a m i n e s y s t e m .  8.2  E f f e c t s of p r o t o v e r a t r i n e o n t h e s p e c i f i c a c t i v i t i e s of a m i n o acids f r o m rat brain cortex  8.3  8.5  8.6  slices incubated  166 166  i n the p r e s e n c e  of [ U - 1 4 C ] g l u c o s e  168  C h a r a c t e r i z a t i o n of a m i n o a c i d c o m p a r t m e n t s i n b r a i n .  170  (i)  8.4  156  E f f e c t s of t e t r o d o t o x i n o n t h e a m i n o a c i d c o n t e n t s i n , a n d release from,  7.11  from  i n the p r e s e n c e of o u a b a i n ....  (ii)  E f f e c t s of t h e c o m b i n e d p r e s e n c e of p r o t o v e r a t r i n e a n d o u a b a i n on a m i n o a c i d r e l e a s e E f f e c t s of t e t r o d o t o x i n . . .  171 173  (iii)  A l t e r a t i o n s i n t h e c o n t e n t s of i n d i v i d u a l a m i n o a c i d s  174  (a)  Glutamate  174  (b)  Aspartate...  174  (c)  G A B A.  175  (d) (e) (f)  G l y c i n e and S e r i n e . T a u r i n e , A l a n i n e and T h r e o n i n e Glutamine  175 175 175  E f f e c t s of s o d i u m m a l o n a t e a n d s o d i u m f l u o r o a c e t a t e o n c e r e b r a l a m i n o a c i d c o n t e n t a n d r e l e a s e i n t h e p r e s e n c e of protoveratrine  176  E f f e c t s of L - g l u t a m i n e a n d s o d i u m L - g l u t a m a t e on c e r e b r a l a m i n o a c i d content and r e l e a s e  1^2  Summary  *^2  xi Page 9.  E F F E C T S OF IN VITRO 9.1  NH + ON  B R A I N M E T A B O L I S M AND  4  TRANSPORT 184  I n i t i a l c o n t e n t s of Na"*" a n d adult rat b r a i n cortex.  K"*" i n the i n f a n t (two-day-old)  and 184  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 w a t e r a n d o x y g e n u p t a k e s , a n d 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 slices.  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 h o m o g e n a t e s  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 , a n d t h e c h l o r i d e s a l t s of Li"*", Rb"*" a n d Cs"*" on the o x y g e n a n d w a t e r u p t a k e s , a n d 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  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  Q u a n t i t a t i v e a s p e c t s of t h e e f f e c t s of NH4"*" o n the N a t a n d w a t e r c o n t e n t 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  195  9.2  9.5 9.6 9.7  9.8  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 c o n t e n t 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 0.1 m M o u a b a i n o r i n t h e a b s e n c e of g l u c o s e E f f e c t s of c h a n g i n g m e d i u m i o n i c c o m p o s i t i o n o n t h e Na"*", K"*" c o n t e n t 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 (i) (ii) (iii)  9.9  E f f e c t s of Ca++ a n d K + E f f e c t s of i n c r e a s i n g NH4+ 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+ W  .  _  Na  9.12  1 97 197 200 200  1  _  , K  c o n t e n t s of i n c u b a t e d r a t 202  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 o n ^  9.11  and K+  195  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^"*" i n d u c e d c h a n g e s i n the N a brain cortex s l i c e s .  9.10  K"*"  _j_  , and K  2  a  ;  —  water, -  c o n t e n t s 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 . .  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^"*" o n A T P of r a t c e r e b r a l c o r t e x s l i c e s  205  contents 207  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 o x y g e n a n d w a t e r u p t a k e s , a n d Na"*", K"" 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 t h e p r e s e n c e of N H 4 C I o r of N a - L - g l u t a m a t e .  210  (i) (ii)  210 212  1  9.13  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) I n c u b a t i o n i n Ca^~~*"-free m e d i a ( T a b l e 73)  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 t h e A T P cerebral cortex slices  c o n t e n t s of i n c u b a t e d 215  xii Page 9.14  E f f e c t s of s o d i u m f l u o r o a c e t a t e , toxin and N H 4 C I  o n t h e Na+,  s o d i u m malonate,  1  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 9.15  10.  tetrodo-  K"" a n d w a t e r c o n t e n t s of r a t of g l u c o s e . . . .  215  Summary  2  DISCUSSION 10.1  formation in rat b r a i n cortex slices  220  E n d o g e n o u s g l u t a m a t e — t h e p o s s i b l e m a j o r s o u r c e of aerobic ammonia formation  220  (ii)  E x o g e n o u s L - g l u t a m a t e — as a p o s s i b l e s o u r c e of  (iii)  Glutamine — a source  (iv)  M e c h a n i s m of a m m o n i a f o r m a t i o n i n b r a i n i n v i t r o  cerebral ammonia  (v)  221 of a m m o n i a i n b r a i n  (a)  R e a c t i o n s i n the a b s e n c e of g l u c o s e  (b)  R e a c t i o n s i n the p r e s e n c e  of g l u c o s e  N e u r o n s — t h e p o s s i b l e s i t e of a e r o b i c  11.  222 223 ...  223 225  ammonia  formation 10.2  8  220  Ammonia (i)  1  225  T e t r o d o t o x i n - s e n s i t i v e f l u x e s of a m i n o a c i d s i n t h e b r a i n in vitro.  228  10.3  L o c a t i o n s of a m i n o a c i d s i n the b r a i n .  232  10.4  Neuron-glia interrelations  233  10.5  C o n t r o l of a m m o n i a u t i l i z a t i o n b y g l u t a m i n e  synthesis in  brain i n vitro.  235  10.6  T r a n s p o r t of N H 4 " * " i n t o b r a i n c o r t e x s l i c e s  237  10.7  T r a n s p o r t of L - g l u t a m a t e i n t o b r a i n c o r t e x s l i c e s .  238  10.8  E f f e c t s of N H  240  GENERAL  REFERENCES  + 4  on b r a i n m e t a b o l i s m .  R E S U L T S AND  CONCLUSIONS  244  247  x i i i  LIST  OF  FIGURES  FIGURE  1  Page  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 o r concentrations  on t h e i r T i s s u e / M e d i u m  r a t i o s of i n c u b a t e d 2  NH4+  concentration 122  r a t b r a i n cortex slices  E f f e c t s of s o d i u m f l u o r o a c e t a t e a n d s o d i u m  malonate  o n 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  slices  178  xiv  LIST OF  TABLES  TABLE 1  2  2A  3  3A  4  5  6  7  8  Page S o m e b r a i n t o 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 acids for the cat  19  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  C h a n g e s i n the g l u t a m a t e , g l u t a m i n e , a s p a r t a t e a n d 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 incubated i n the p r e s e n c e o r a b s e n c e of g l u c o s e  59  E f f e c t s of a n o x i a on the a m m o n i a a n d a m i n o a c i d c o n t e n t 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 t h e p r e s e n c e o r a b s e n c e of g l u c o s e  64  A m m o n i a a n d a m i n o a c i d c h a n g e s 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 t h e p r e s e n c e o r a b s e n c e of O 2  65  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 o n i n c u b a t i o n i n t h e p r e s e n c e o r a b s e n c e of g l u c o s e ..  67  E f f e c t s of s o d i u m m a l o n a t e a n d a m i n o o x y a c e t a t e o n aspartate synthesis 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 cortex slices incubated i n a glucose-free m e d i u m  70  E f f e c t s of s o d i u m - a m y t a l a n d 2, 4 - d i n i t r o p h e n o l o n t h e t o t a l a m m o n i a a n d a m i n o a c i d c o n t e n t s of r a t b r a i n c o r t e x slices incubated i n a glucose-free m e d i u m  73  T h e e f f e c t s of o u a b a i n o n t h e r a t e of a m m o n i a f o r m a t i o n , a n d 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 incubated i n a glucose-free m e d i u m  75  E f f e c t s of L - g l u t a m a t e on t h e f o r m a t i o n of a m m o n i a a n d amino acids by r a t b r a i n cortex slices incubated 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 t h e p r e s e n c e a n d a b s e n c e of C a  77  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 cortex slices incubated i n a glucose-free medium  79  + +  9  X V  TABLE 10  Page 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 f o r m a t i o n g l u t a m a t e and g l u t a m i n e i n r a t b r a i n c o r t e x respiring in a glucose-free  11  Ammonia  medium  13  14  81  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  cortex synaptosomes incubated  12  of a m m o n i a , slices  i n the p r e s e n c e o r a b s e n c e  of g l u c o s e  82  E f f e c t s of the c a t i o n i c c o n t e n t s of the i n c u b a t i o n m e d i u m on t h e r a t e of g l u t a m i n e s y n t h e s i s i n r a t b r a i n c o r t e x slices  87  E f f e c t s of N H 4 C I a n d s o d i u m L - g l u t a m a t e 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 g l u t a m i n e s y n t h e s i s i n r a t b r a i n c o r t e x s l i c e s due t o o u a b a i n o r c h a n g e d m e d i a c o m p o s i t i o n  90  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 o n the r a t e of g l u t a m i n e  s y n t h e s i s and a m i n o a c i d contents  of i n c u b a t e d  rat brain  cortex slices 15  16  93  E f f e c t s of o u a b a i n , m e t h i o n i n e s u l f o x i m i n e , K C 1 , 2, 4 - d i n i t r o p h e n o l , a m y t a l , a n d g l u c o s e a b s e n c e , o n t h e r a t e s of oxygen consumption in rat c e r e b r a l cortex slices E f f e c t s of t e t r o d o t o x i n on the o u a b a i n s u p p r e s s e d g l u t a m i n e s y n t h e s i s of r a t b r a i n c o r t e x s l i c e s  17  Transport  of s o d i u m L - g l u t a m a t e i n t o b r a i n c o r t e x  97 slices 101  of the r a t 18  19  U p t a k e of s o d i u m L - ( U - ^ ^ C ) g l u t a m a t e by r a t b r a i n slices  cortex  21  103  E f f e c t s of p r o t o v e r a t r i n e , o u a b a i n a n d t e t r o d o t o x i n o n 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 w i t h s o d i u m L - ( U - C ) glutamate  107  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 - g l u t a m a t e c o n c e n t r a t i o n s on the a m i n o a c i d c o n t e n t s of i n c u b a t e d r a t b r a i n cortex slices  109  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 - g l u t a m a t e c o n c e n t r a t i o n s on the r e l e a s e of a m i n o a c i d s f r o m i n c u b a t e d r a t brain cortex slices  110  1 4  20  94  xvi TABLE 22  '  Page  T h e t i s s u e a n d m e d i u m c o n c e n t r a t i o n s of N H  + 4  on incu-  b a t i n g r a t b r a i n c o r t e x s l i c e s u n d e r a v a r i e t y of c o n d i tions 23  114  T h e 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 f o r NH^"*" 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 v a r i e t y of m e d i a  24  U p t a k e of N H ,  +  11°  by 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 g l u c o s e 25  j1 Q 1  1  0  U p t a k e of NH^" " b y 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 1  a b s e n c e of g l u c o s e  119  26  T h e uptake of N H 4 * b y r a t b r a i n c o r t e x s l i c e s i n a n o x i a  120  27  R a t e s of N H 4 * a n d o f g l y c i n e u p t a k e b y r a t b r a i n c o r t e x slices  124  T h e i n h i b i t o r y e f f e c t s of N H 4 * o n the a c t i v e t r a n s p o r t 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  125  28  29  A b s e n c e of a n i n h i b i t o r y e f f e c t o f g l y c i n e o n the u p t a k e of a m m o n i u m by r a t b r a i n c o r t e x s l i c e s  30  E f f e c t s of t h e s i m u l t a n e o u s Li-glutamate  p r e s e n c e of e x o g e n o u s  127 sodium  a n d N H 4 C I o n 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 a n d g l u t a m a t e into r a t b r a i n c o r t e x s l i c e s 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 o n the t r a n s p o r t of Nr^"*" a n d of g l y c i n e i n t o r a t b r a i n c o r t e x s l i c e s  32  34  35  129  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 c o n c e n t r a t i o n s o n t h e a m i n o a c i d c o n t e n t 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  33  128  131  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 c o n c e n t r a t i o n s on t h e r e l e a s e of a m i n o a c i d s 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 slices  132  E f f e c t s of p r o t o v e r a t r i n e , o u a b a i n , l i d o c a i n e a n d t e t r o d o t o x i n on the c o n t e n t s o f a m i n o a c i d s i n i n c u b a t e d r a t b r a i n cortex slices  136  E f f e c t s of p r o t o v e r a t r ine, ouabain,  lidocaine and tetrodo-  t o x i n on the r e l e a s e of a m i n o a c i d s f r o m i n c u b a t e d r a t brain cortex slices  137  xvii  TABLE 36  Page 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 and l i d o c a i n e on  c o n t e n t s of a m i n o 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 -  37  38  39  40  bated i n glucose-free m e d i a  140  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 and 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 c o r t e x s l i c e s i n c u bated in glucose-free media  141  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 , l i d o c a i n e , a n d of s o d i u m L - g l u t a m a t e on the c o n t e n t s of a m i n o a c i d s i n r a t b r a i n cortex slices incubated in calcium-deficient media  144  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 , l i d o c a i n e , a n d of s o d i u m L - g l u t a m a t e o n 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 cortex slices incubated i n calcium-deficient media  145  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 ,  l i d o c a i n e , a n d of  s o d i u m L - g l u t a m a t e o n t h e c o n t e n t s of a m i n o a c i d s i n r a t brain cortex slices i n glucose-free,  41  42  calcium-deficient  media  147  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 , l i d o c a i n e a n d of s o d i u m L - g l u t a m a t e o n 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 cortex slices incubated i n glucose-free, calciumdeficient media  148  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  concentra-  t i o n r a t i o s of a m i n o 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 -  43  44  45  46  b a t e d u n d e r a v a r i e t y of c o n d i t i o n s  151  E f f e c t s of s o d i u m a m y t a l on the c o n t e n t s of a m i n o 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 b a t e d i n a v a r i e t y of m e d i a  154  E f f e c t s of s o d i u m a m y t a l 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 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 media  155  E f f e c t s of i n c r e a s e d K"*" a n d of t e t r o d o t o x i n on the a m i n o acid 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 media  1 57  E f f e c t s of i n c r e a s e d K~*" a n d of t e t r o d o t o x i n on the r e l e a s e of a m i n o a c i d s f r o m i n c u b a t e d  rat brain cortex slices  158  xv i i i  TABLE 47  Page E f f e c t s of t e t r o d o t o x i n on the c o n t e n t s and  release from,  of a m i n o a c i d s i n ,  r a t kidney cortex slices incubated  i n the  p r e s e n c e of o u a b a i n 48  159  T o t a l a m m o n i a and a m i n o a c i d contents  of r a t k i d n e y  cortex  s l i c e s i n i t i a l l y p r e s e n c e a n d on i n c u b a t i o n i n the p r e s e n c e o r a b s e n c e of g l u c o s e 49  160  E f f e c t s of t e t r o d o t o x i n on t h e c o n t e n t s b r a i n cortex slices incubated or KC1  50  NH4CI  (100 m M )  162  E f f e c t s of t e t r o d o t o x i n on 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 w i t h 2, 4 - d i n i t r o p h e n o l , N H 4 C I , or KC1  51  of a m i n o a c i d s i n r a t  w i t h 2, 4 - d i n i t r o p h e n o l ,  (100 mM)  163  S p e c i f i c a c t i v i t i e s of g l u t a m i n e a n d g l u t a m a t e 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 - ^ C ) acetate and ( U - C ) glucose 4  i 4  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) o n the s p e c i f i c a c t i v i t i e s of a m i n o 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 i n c u b a t e d  i n the  p r e s e n c e of ( U - ^ C ) g l u c o s e  169  4  53  E f f e c t s of p r o t o v e r a t r i n e , o u a b a i n a n d t e t r o d o t o x i n on a m i n o a c i d content in, and r e l e a s e f r o m , i n c u b a t e d r a t brain cortex slices  54  172  E f f e c t s of p r o t o v e r a t r i n e on a m i n o a c i d c o n t e n t cortex slices incubated  55  57  58  in rat brain  i n p r e s e n c e of o u a b a i n  177  E f f e c t s of s o d i u m m a l o n a t e o r s o d i u m f l u o r o a c e t a t e on a m i n o a c i d c o n t e n t in, a n d r e l e a s e f r o m ,  56  167  incubated r a t  b r a i n 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  180  E f f e c t s of L - g l u t a m a t e and L - g l u t a m i n e o n a m i n o a c i d c o n t e n t 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  181  Initial N a and K contents brain cortex slices  185  +  +  of 2-day o l d a n d a d u l t r a t  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 o x y g e n a n d w a t e r u p t a k e s and Na+, c o n t e n t s of i n c u b a t e d r a t b r a i n cortex slices  187  xix  TABLE  Page 2+  59  E f f e c t s of c a t i o n s o n the M g  - A T P a s e a c t i v i t y of h o m o -  g e n a t e s of a d u l t r a t b r a i n c o r t e x 60  189  E f f e c t s of c a t i o n s o n the N a - K + - M g +  -ATPase  2 +  activities  of h o m o g e n a t e s of a d u l t r a t b r a i n c o r t e x s l i c e s 61  E f f e c t s of p r o t o v e r a t r i n e ,  tetrodotoxin,  190  o u a b a i n and  2, 4 - d i n i t r o p h e n o l o n the A T P a s e a c t i v i t i e s of r a t cortical homogenates 62  E f f e c t s of N H  4  +  ,  (CH ) N , 3  4  +  o x y g e n and w a t e r u p t a k e s ,  Li , Rb +  +  and C s  +  cerebral 191  o n the  a n d the N a , K**" c o n t e n t s +  of  incubated rat brain cortex slices 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 and w a t e r  64  65  192  u p t a k e s a n d Na"*",  on the o x y g e n  K"** c o n t e n t s of i n f a n t (2 d a y old)  rat brain cortex slices  194  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 w a t e r , Na"*", K"** a n d NH4"** c o n t e n t 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 an N H 4 C I c o n t a i n i n g m e d i u m  196  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 o n the o x y g e n a n d w a t e r u p t a k e s a n d the Na+, K c o n t e n t 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 0.1 m M o u a b a i n o r i n the a b s e n c e of g l u c o s e  198  E f f e c t s of m e d i u m c a t i o n c o n t e n t s on the o x y g e n a n d w a t e r uptakes, a n d 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 slices  199  +  66  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**" a n d in 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 sodium,  68  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  E f f e c t s of 30 m M K C 1 o n the N H 4 C I i n d u c e d c a t i o n i c changes in incubated rat b r a i n cortex slices  69  203  E f f e c t s of 55 m M K C 1 o n the N H 4 C I i n d u c e d c a t i o n i c changes in incubated rat b r a i n cortex slices  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 on o x y g e n and water uptakes,  a n d Na+,  incubated rat b r a i n c o r t e x s l i c e s  204  concentrations  K"*~ c o n t e n t s  of 206  XX  TABLE 71  Page E f f e c t s of i n c r e a s i n g N H ^ incubated  71A  ATP  +  on A T P  concentrations  of  rat brain cortex slices  concentrations  of i n c u b a t e d  208 rat brain cortex  slices  u n d e r a v a r i e t y of m e d i a c o n d i t i o n s 72  E f f e c t s of t e t r o d o t o x i n ,  protoveratrine,  209 ouabain,  NH4CI  a n d s o d i u m - L r - g l u t a m a t e on the o x y g e n a n d w a t e r u p t a k e s , a n d the Na"*",  contents  b a t e d i n a Ca"*""**-containing 73  E f f e c t s of t e t r o d o t o x i n ,  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 medium  protoveratrine,  211 N H 4 C I and  sodium  L - g l u t a m a t e o n the o x y g e n a n d w a t e r u p t a k e s , and t h e Na"*", K"*" c o n t e n t 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 Ca"*"*"-  free medium 74  75  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 a n d t e t r o d o t o x i n o n the A T P c o n t e n t 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 .... E f f e c t s of s o d i u m f l u o r o a c e t a t e , s o d i u m m a l o n a t e , t e t r o d o t o x i n and N H 4 C I on the o x y g e n and w a t e r u p t a k e s , Na"*" a n d K"*" c o n t e n t 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  213  c  x  o  217  X X I  ABBREVIATIONS  AMP  a d e n o s i n e 5' - m o n o p h o s p h a t e  ADP  a d e n o s i n e 5' - d i p h o s p h a t e  ATP  a d e n o s i n e 5' - t r i p h o s p h a t e  ATPase  adenosine triphosphatase  Acetyl-CoA  acetyl-coenzyme  cpm  counts p e r minute  dpm  disintegrations p e r minute  rpm  revolutions p e r minute  DNP,  2, 4 - D N P  A  2, 4 - d i n i t r o p h e n o l  EGTA  ethylene glycol-(diaminoethyl) tetra acetic acid  EDTA  ethylene diamine tetra acetic acid  GABA  y -aminobutyric acid  NAD+  NADH  NADP+,  NAD P H  o x i d i z e d a n d r e d u c e d f o r m s of n i c o t i n a m i d e adenine dinucleotide o x i d i z e d a n d r e d u c e d f o r m s of n i c o t i n a m i d e a d e n i n e dinucleotide  phosphate  Pi  orthophosphate  QO2  oxygen  TCA  trichloroacetic  Tris  t r i (hydroxymethyl) amino methane  T/M  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  TTX  ,  consumption  tetrodotoxin  v/v  volume/volume  w/v  weight/volume  wt  weight  acid  ACKNOWLEDGMENTS  T o P r o f e s s o r J . H. Q u a s t e l , for h i s guidance, encouragement,  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 and wise  counsel.  T o P r o f e s s o r s V . J . O ' D o n n e l l a n d G . H. D i x o n ,  my  s i n c e r e thanks f o r  their helpful comments.  To my f r i e n d ,  H e l e n Halm, m y  sister,  Juliet,  a n d m y wife, E v a , m y  deep gratitude f o r t h e i r e x p e r t i s e and m o r a l support, without w h i c h I c o u l d not have c o m p l e t e d this work.  T o the D e p a r t m e n t  of S o i l S c i e n c e , t h e U n i v e r s i t y of B r i t i s h  Columbia,  m y t h a n k s f o r t h e u s e of t h e i r A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t e r .  T o t h e M e d i c a l R e s e a r c h C o u n c i l of C a n a d a ,  my thanks for f i n a n c i a l  a s s i s t a n c e t h r o u g h g r a n t s m a d e to P r o f e s s o r J . H. Q u a s t e l .  T h i s w o r k w a s 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 Research,  t h e U n i v e r s i t y of B r i t i s h  Columbia.  1  1.  1.1  INTRODUCTION  F o r m a t i o n of a m m o n i a b y n e r v e t i s s u e  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 a m m o n i a b y n e r v e f i b r e s  w a s b y T a s h i r o i n 1922 (1). H e a n d o t h e r w o r k e r s (1-3) s h o w e d t h a t e l e c t r i cal  stimulation  enhanced  i n j u r y d e p r e s s e d it. suppressive nervous  t h e r a t e of a m m o n i a e v o l u t i o n a n d that m e c h a n i c a l  Winterstein  and H i r s c h b e r g  e f f e c t s of a n o x i a a n d a n e s t h e s i a  system.  w o r k e r s (4-7).  Similar Frog  exhibited increased  observations  on a m m o n i a f o r m a t i o n  by the  w e r e m a d e b y a n u m b e r of o t h e r  retina, f o r example,  ammonia formation  (2, 3) d e m o n s t r a t e d t h e  when  i r r a d i a t e d with sunlight  w h e n c o m p a r e d w i t h t h a t of c o n t r o l s  l e f t i n t h e d a r k (5). S i m i l a r r e s u l t s w e r e a l 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,  but also by  or  osmotic  research  not only e l e c t r i c a l l y or mechanically,  changes,  o r by c h e m i c a l  agents  (6, 7).  These  thermal  studies l e d  w o r k e r s i n t h e f i e l d to c o n c l u d e that n e r v e f i b r e s l i b e r a t e a m m o n i a  as a r e a c t i o n to s t i m u l a t i o n .  Like peripheral 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 t o s t i m u l i b y a n i n c r e a s e d r a t e of a m m o n i a f o r m a t i o n . are  detailed review  articles  on these  and other  aspects  There  of a m m o n i a  meta-  b o l i s m i n the c e n t r a l n e r v o u s s y s t e m (8-16).  1.2  Factors  (i)  a f f e c t i n g t h e i n v i v o l e v e l s of a m m o n i a i n the b r a i n  Ammonia  c o n t e n t of b r a i n i n v i v o  A b n o r m a l l y h i g h l e v e l s of a m m o n i a i n the b r a i n a r e s u s p e c t e d  to b e  i n 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) a n d i n h e p a t i c (23-27),  as i t i s w e l l known  that the a d m i n i s t r a t i o n o f a m m o n i u m  experimental animals causes convulsions, For  example,  chloride  c o m a and death  coma  s a l t s to  (22, 23, 28-32).  R i c h t e r a n d D a w s o n (22) s h o w e d that i n j e c t i o n of a m m o n i u m  i n the r a t caused  convulsions  r i s e n t o 9 r n g % (about 5 p.mole/g).  w h e n the b r a i n  ammonia  T h e k n o w l e d g e that  level had  ammonia  is a  p o w e r f u l c e r e b r a l i r r i t a n t h a s s t i m u l a t e d i n t e r e s t i n the s t u d y of b r a i n  2  ammonia levels  i n vivo  (18, 19, 22, 33, 34).  a m m o n i a i n the b r a i n of a n i m a l s  in vivo  D e t e r m i n a t i o n s of the l e v e l s of  h a v e t o b e c a r r i e d out w i t h g r e a t  caution as a m m o n i a i s a c o m m o n l a b o r a t o r y contaminant. p r e s e n c e of l a b i l e s u b s t a n c e s l i k e g l u t a m i n e w h i c h m a y m u s t be t a k e n i n t o c o n s i d e r a t i o n ,  M o r e o v e r , ' the  g i v e r i s e to a m m o n i a ,  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 m e t h o d  f o r the a s s a y of a m m o n i a i s e m p l o y e d .  It i s w e l l k n o w n that s o o n a f t e r  t h e r e i s a r a p i d r e l e a s e o r " b u r s t " of a m m o n i a i n t h e b r a i n (8, 36). o r d e r to a v o i d p o s t - m o r t e m changes,  death  Thus, i n  the a n i m a l s a r e often s a c r i f i c e d by i m -  m e r s i o n i n liquid air or liquid nitrogen.  U s i n g this f r e e z i n g technique, values  of about 0.15 - 0.36 j i m o l e / g f r e s h wt b r a i n h a v e b e e n o b t a i n e d i n t h e r a t (22, 35, 36),  d o g (37, 38),  r a b b i t (39),  m o u s e (4), a n d g a r d e n d o r m o u s e (41). S u c h  l o w v a l u e s h a v e l e d to the s u g g e s t i o n t h a t t h e r e m a y be c o m p l e t e f r e e a m m o n i a i n t h e b r a i n o f the l i v i n g a n i m a l (8).  a b s e n c e of  It m a y b e a r g u e d that  e v e n r a p i d f r e e z i n g i n l i q u i d a i r o r n i t r o g e n i s not c o m p l e t e l y i n s t a n t a n e o u s (the t i m e of f r e e z i n g v a r i e s d i r e c t l y w i t h the s i z e of the a n i m a l (282) ). Ammonia complete  m a y be p r o d u c e d b e t w e e n t h e t i m e of i m m e r s i o n f r e e z i n g of t h e b r a i n .  b r a i n a f t e r death,  a n d t h e t i m e of  T h e e x p l o s i v e f o r m a t i o n of a m m o n i a i n t h e  p o s s i b l y d u e t o a n o x i a , b e c o m e s e v i d e n t w h e n h e a d s of  decapitated animals are dropped  into l i q u i d a i r .  F o r example,  there i s  a b o u t a t w o f o l d i n c r e a s e i n a m m o n i a c o n t e n t w h e n t h e h e a d i s f r o z e n one s e c o n d a f t e r d e c a p i t a t i o n (22). 2 ( j m o l e / g f r e s h wt.  a f t e r death,  cat b r a i n gave  S h e e p b r a i n g a v e v a l u e s of 2.7 a n d 1.7 | j m o l e / g  wt i n g r e y a n d w h i t e m a t t e r a f t e r d e a t h (42).  T h r e e minutes  respectively  Weil-Malherbe  fresh  when f r e e z i n g took p l a c e 5 m i n  a n d G r e e n (43) o b t a i n e d a v a l u e of 5 j i m o l e / g  f r e s h wt i n g u i n e a p i g b r a i n c o r t e x s l i c e s  20 m i n a f t e r death,  a value which  i s i n g o o d a g r e e m e n t w i t h t h o s e o b t a i n e d i n o t h e r s t u d i e s (44, 45).  In t h i s  t h e s i s v a l u e s o f about 1.6 J i m o l e / g i n i t i a l w e t wt a r e r e p o r t e d f o r b r a i n c o r t e x of r a t s t a k e n a f e w m i n u t e s  (ii)  The  a f t e r d e a t h ( T a b l e 2).  F a c t o r s producing low brain ammonia levels i n vivo  a m m o n i a c o n t e n t of t h e c e n t r a l n e r v o u s  system, analyzed after  3  r a p i d freezing, is not constant but seems to depend on the state of activity of the b r a i n at the time of the f r e e z i n g .  F o r example, a reduction in functional  activity seems to be associated with a reduced concentration in the b r a i n .  of free a m m o n i a  Thus, R i c h t e r and Dawson (22) showed a significant d e c r e a s e  in b r a i n ammonia l e v e l s  (to 0.033 nmole/g f r e s h wt) when rats under p r o -  longed nembutal n a r c o s i s were k i l l e d in liquid a i r . s i m i l a r effects with urethane or s o d i u m bromide. e a r l i e r suggestions and observations by n e r v e f i b r e s under anesthesia  Vladimirova  These r e p o r t s followed the  on the d e p r e s s i o n  (1 - 3 ) .  (46) obtained  of a m m o n i a f o r m a t i o n  A m m o n i a content of rat b r a i n  d e c r e a s e s by about 50 per cent during sleep (47).  D u r i n g hibernation, too,  there is a 50 per cent diminution in the b r a i n ammonia l e v e l s of garden d o r m i c e (41). (iii)  F a c t o r s producing high b r a i n a m m o n i a l e v e l s in vivo  L i k e p e r i p h e r a l n e r v e s (1 - 3), the b r a i n f o r m s a m m o n i a following e l e c t r i c a l stimulation (22,  35, 36).  T h i s is consistent with the view that any  method that enhances c e r e b r a l i r r i t a b i l i t y w i l l also enhance the rate of c e r e b r a l a m m o n i a formation. convulsions,  Thus, the a d m i n i s t r a t i o n of drugs capable of producing  like camphor (30), p i c r o t o x i n (22, 46), t e l o d r i n (49). penta-  methylene t e t r a z o l e (38, 50), b e m i g r i d e (38), fluoroacetate (37, methionine sulfoximine  51) or  (52) r e s u l t s i n i n c r e a s e d b r a i n a m m o n i a l e v e l s . In  some instances the r i s e i n the b r a i n content of ammonia o c c u r s in the preconv u l s i v e state (22, 37, 52). However, even though a m m o n i u m salts a r e known to cause convulsions  when a d m i n i s t e r e d to a n i m a l s (22,  23, 28-32), it is s t i l l  u n c e r t a i n whether the i n c r e a s e in b r a i n ammonia l e v e l s o c c u r i n g f r o m endogenous sources, is the cause or the r e s u l t of convulsions. oxygen p r e s s u r e (51) enhance a m m o n i a l e v e l s i n brain.  A n o x i a (22) and high M i l d stimulation of  the c e n t r a l nervous system, like injection of amphetamine (53) or c o r t i c o t r o p i n (54), painful e l e c t r i c a l shock to the e x t r e m i t i e s (35,  36, 47), or c e r t a i n  conditioned r e f l e x e s (35, 47), also elevates b r a i n ammonia l e v e l s .  Though  excitement caused by tumbling a n i m a l s in a revolving d r u m has a s i m i l a r  4  e f f e c t (55), R i c h t e r a n d D a w s o n (22) f o u n d n o 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 s i d e i n a g l a s s b e a k e r ,  while  V r b a (9) f o u n d n o c h a n g e 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 m a d e to u n d e r g o physical exercise.  Cerebral ammonia increases  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 , glutamine formation  may not occur i n some  o w i n g to a n i n c r e a s e d  i n t h e b r a i n (9 - 11, 56, 57).  r a t e of  Increased blood ammonia  l e v e l s a l s o r e s u l t i n e n h a n c e d g l u t a m i n e c o n t e n t s i n t h e b r a i n (22). 1.3  (i) S o m e c h e m i c a l c h a n g e s t h a t o c c u r i n t h e b r a i n o n t h e a p p l i c a t i o n of s t i m u l i to the w h o l e 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  o f a m m o n i a (9, 56).  However,  t h e r e o c c u r s a n e n h a n c e d 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 t h e a m o u n t s of f r e e g l u t a m a t e (57, 66) a n d p r o t e i n b o u n d 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 obtained during oxygen i n t o x i c a t i o n  (9) a n d 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 convulsions,  amide  (70).  Even during electrically induced  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).  Acute telodrin intoxication  also r e s u l t s i n an i n c r e a s e i n glutamine content with a concomitant d e c r e a s e i n t h e 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 observed later when s e i z u r e s occur.  A c c o r d i n g to H a t h w a y  a n d 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 t h e b r a i n a n d t h i s o c c u r s b e f o r e t h e o n s e t of c o n v u l s i o n s a-Ketoglutarate  and throughout t h e i r c o u r s e .  and glutamate a r e u t i l i z e d i n an a m m o n i a binding  mechanism  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 free a m m o n i a a c c u m u l a t e s i n the cerebral tissue.  It w a s l o n g s u s p e c t e d t h a t t h e 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  proteins  r e p r e s e n t e d a s o u r c e o f e n d o g e n o u s c e r e b r a l a m m o n i a (57, 67, 74, 7 5 ) . T h i s w a s l a t e l y f o u n d t o be t r u e (76). per  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  c e n t of t h e g l u t a m i n y l r e s i d u e s o f 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  5  deaminated  i n situ.  T h e s e r e s u l t s l e n d s u p p o r t to the v i e w that a m m o n i a  is formed  from  p r o t e i n s o u r c e s and is c o n v e r t e d to g l u t a m i n e by c o n d e n s a t i o n with glutamate. B r a i n protein metabolism  s e e m s c l o s e l y r e l a t e d t o that of p e r i p h e r a l n e r v e ,  w h e r e s t i m u l a t i o n i n c r e a s e s t h e a c t i v i t y of the n e u t r a l p r o t e i n a s e , 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 o f g l u t a m a t e a n d l i b e r a t i o n o f a m m o n i a (72).  (ii)  Recovery  p r o c e s s e s that o c c u r i n the b r a i n o n 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 rats physically exhausted to r e s t ,  (by p r o l o n g e d s w i m m i n g ) a r e a l l o w e d  their c e r e b r a l glutamine levels decrease with concomitant i n c r e a s e s  i n t h e i r 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 f r e e c a r b o x y l i c g r o u p s a d m i n i s t r a t i o n of N H ^ C l 1  of c e r e b r a l p r o t e i n s a f t e r t h e  s e e m s to t a k e p l a c e (80, 81), b u t c o n f i r m a t i o n u s i n g  l a b e l l i n g i s n e e d e d (76).  W h e r r e t t a n d T o w e r (76) w e r e u n a b l e t o o b t a i n  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 b y i n c r e a s i n g f r e e p o o l s of a m m o n i a a n d g l u t a m i n e of c a t c o r t e x s l i c e s .  In s p i t e of t h i s ,  they c o n s i d e r that p r o t e i n  d e a m i d a t i o n i s a r e v e r s i b l e p r o c e s s a s s u g g e s t e d b y M y c e k a n d W a e l s c h (77) a s a r e s u l t of t h e i r s t u d i e s o n t r a n s g l u t a m i n a s e . calcium-activated enzyme which catalyzes  Transglutaminase isa  (a) the e x c h a n g e of p r o t e i n a m i d e  g r o u p s with p r i m a r y a m i n e s (like c a d a v e r i n e , p u t r e s c i n e , h i s t a m i n e , tonin,  glycinamide,  sero-  etc.); a n d (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 b o u n d  a m i d e r e s i d u e s (78 - 79).  (iii) C o n v u l s i o n s a n d b r a i n e n e r g y  levels.  N o 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 r a t s w a s  observed  w i t h a m m o n i u m a c e t a t e i n d u c e d c o n v u l s i o n s (82), o r i n h y p o x i c m i c e at t h e o n s e t o f s e i z u r e s (83).  Similarly,  no c h a n g e i n the A T P o r p h o s p h o c r e a t i n e  6  levels were observed  i n the b r a i n s of m i c e u n d e r g o i n g m e t h i o n i n e  induced  s e i z u r e s (52).  authors  (84,  sulfoximine  S u c h r e s u l t s do not s u p p o r t the p o s t u l a t e of s e v e r a l  85) that A T P  i s a f a c t o r i n the i n d u c t i o n of c o n v u l s i o n s .  a p p a r e n t a b s e n c e of c e r e b r a l A T P  d e p l e t i o n d o e s not,  p o s s i b l e d e p l e t i o n of a s m a l l l o c a l i z e d p o o l of A T P  however,  that m a y  The  r u l e out the  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 a f f e c t i n g the f o r m a t i o n of a m m o n i a i n 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 o u r e a r l i e r w o r k (62) on the f a c t o r s c o n c e r n e d w i t h the  r a t e of a m m o n i a 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 i n c u b a t e d i n o x y g e n at 3 7 ° C f o r one h o u r i n K r e b s - R i n g e r  phosphate medium,  changes in amino acid metabolism,  p a r t i c u l a r l y t h o s e of g l u t a m a t e and g l u t a -  m i n e ( s e e s e c t i o n 1.5 (ii) (b) ).  A  were  explained  s u m m a r y of o u r o b s e r v a t i o n s  by  is given  below.  (i)  C a r b o h y d r a t e m e t a b o l i s m and a m m o n i a  A m m o n i a formation is suppressed  by v a r i o u s e x o g e n o u s s u b s t r a t e s that  a r e c a p a b l e of s u p p o r t i n g c e r e b r a l r e s p i r a t i o n . (58 - 62) o r p y r u v a t e c y c l e i s high,  formation  Thus,  with glucose,  lactate  (62), w h e r e the r a t e of the o p e r a t i o n of the c i t r i c a c i d  t h e r e i s a m a x i m a l s u p p r e s s i o n of a m m o n i a l i b e r a t i o n .  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 g l u c o s e a n d a b o u t 2.5 m M  for pyruvate  (62).  f a c t t h a t g l y c o l y s i s of one  m o l e c u l e of g l u c o s e  T h i s may  The  ImMfor  be c o r r e l a t e d w i t h the  y i e l d s two  molecules  of p y r u -  vate. In the p r e s e n c e of g l u c o s e but not of p y r u v a t e , a c e t a t e (0.1 mM),  by i t s s u p p r e s s i v e  the a d d i t i o n of i o d o -  e f f e c t on t r i o s e p h o s p h a t e d e h y d r o g e n a s e ,  i m p e d e s 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 and t h e r e b y the e n e r g y d e p e n d e n t glutamine c e n t (62).  synthesis.  It e n h a n c e s the output of a m m o n i a by m o r e t h a n  In the a b s e n c e of g l u c o s e ,  g e n o u s a m m o n i a f o r m a t i o n (62,  64);  50  per  iodoacetate only slightly inhibits endothis may  be due  to a s m a l l  diminution  7  i n e n d o g e n o u s o x y g e n c o n s u m p t i o n (64).  A c e t a t e i s o n l y o x i d i z e d f e e b l y by b r a i n c o r t e x s l i c e s (226) and h a s l i t t l e o r no d i m i n i s h i n g e f f e c t on a m m o n i a f o r m a t i o n . or  a-ketoglutarate  tions),  Similarly,  (62), a c e t o a c e t a t e o r s u c c i n a t e (our u n p u b l i s h e d  do not s u p p r e s s the f o r m a t i o n of a m m o n i a .  However,  T h i s i s p r e s u m a b l y due  of c e r t a i n i n h i b i t o r s ,  f l u o r o a c e t a t e (63), m e t h i o n i n e  e.g.,  suppressing  cycle.  2, 4 - d i n i t r o p h e n o l (62,  M a l o n a t e (5 mM),  a n i n h i b i t o r of  t h e 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 d e h y d r o g e n a s e s t a g e (228), a m m o n i a l i b e r a t i o n o n l y i n the p r e s e n c e  demonstrated a marked  mM  KC1  (62).  Kini  of  and  s t i m u l a t i o n i n the o p e r a t i o n of t h e  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 t h a t i n the p r e s e n c e n o t 5 mM)  enhances  of t h r e s h o l d c o n c e n t r a t i o n s (1 mM)  T h i s i s a l s o f o u n d to be t r u e w i t h 100  Q u a s t e l (213, 229)  64),  s u l f o x i m i n e (65) o r i o d o a c e t a t e (64), the s u p -  p r e s s i v e e f f e c t s of g l u c o s e i s l e s s m a r k e d .  glucose.  observa-  to i t s r e l a t i v e l y r a p i d i n f l u x  i n t o the b r a i n c e l l a n d to i t s p a r t i c i p a t i o n i n t h e c i t r i c a c i d  In the p r e s e n c e  citrate  oxaloacetate  c a u s e s h i g h r e s p i r a t o r y r a t e s (227) a n d i s c a p a b l e of p a r t i a l l y ammonia liberation.  hence  of 1 m M  (but  g l u c o s e , the i n t e r m e d i a t e s i n t h e b r e a k d o w n of g l u c o s e a r e o x i d i z e d  too f a s t to a l l o w f o r the n o r m a l r a t e of ( e n e r g y r e q u i r i n g ) a m m o n i a f i x a t i o n .  (ii)  E l e c t r o n t r a n s p o r t , o x i d a t i v e p h o s p h o r y l a t i o n and  ammonia  formation.  In the a b s e n c e of g l u c o s e ,  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  a m o u n t s of a m m o n i a t h a n t h o s e of the i n f a n t b r a i n (62), p r e s u m a b l y b e c a u s e o x y g e n u p t a k e by i n f a n t b r a i n i s l o w e r . oxygen consumption per mg  M i l s t e i n et a l . , (230)  s h o w e d t h a t the  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 w i t h the age  of  the r a t .  E l e c t r o n transport inhibitors,  e.g.,  p r e s s a u t o g e n o u s a m m o n i a f o r m a t i o n (43, e l e c t r o n t r a n s p o r t between  azide, a r s e n i t e or cyanide, 63,  64).  A m y t a l (1 mM)  N A D H and the c y t o c h r o m e s y s t e m (219  sup-  inhibits - 221)  and  8  concomitantly The  suppresses ammonia formation  i n a glucose-free  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  t i o n of N A D  A  +  (62).  the r e g e n e r a -  required for glutamate oxidation.  d i r e c t c o n s e q u e n c e of t h e i n h i b i t i o n of t h e r e s p i r a t o r y c h a i n i s a  diminished  r a t e of A T P  synthesis.  o r s o d i u m s a l i c y l a t e (5 m M ) ammonia,  (62),  B o t h 2, 4 - d i n i t r o p h e n o l  (0.1 m M )  b u t e n h a n c e the l i b e r a t i o n of a m m o n i a i n the p r e s e n c e of g l u c o s e ,  The  r a t e of a m m o n i a p r o d u c t i o n  doubtless  synthesis.  i s s u p p r e s s e d u n d e r a n o x i a (43,  The  formation.  a d d i t i o n of L - g l u t a m a t e t o 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  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 ammonia formation  in a  r a t e of  (62, 103), a c c o m p a n i e d by a n e n h a n c e d r a t e of g l u t a m i n e  (58, 103).  Five mM  D - g l u t a m a t e (62, 138, 231), o r 5  a-methyl glutamate inhibits ammonia liberation i n a glucose-free p r e s u m a b l y by inhibiting endogenous glutamate oxidation. of g l u c o s e ,  62,  b y a n i n h i b i t i o n of t h e o x i d a t i o n of e n d o g e n o u s g l u t a m a t e (62).  (iii) E f f e c t s of e x o g e n o u s a m i n o a c i d s o n a m m o n i a  synthesis  (62, 63),  s u p p r e s s the e n d o g e n o u s f o r m a t i o n o f  p r e s u m a b l y b y i n 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  244),  medium  mM medium,  In the p r e s e n c e  D - g l u t a m a t e e n h a n c e s the r a t e of a m m o n i a l i b e r a t i o n (62) b y  i n h i b i t i n g g l u t a m i n e s y n t h e t a s e (103).  The  r a t e of a u t o g e n o u s a m m o n i a f o r m a t i o n  y-aminobutyrate (GABA), However,  with L-alanine  is unaffected by taurine,  L - or D-aspartate glycine or g l y c y l - L - a s p a r t a t e . or L - a r g i n y l - L - g l u t a m a t e  there  s e e m s to be a s m a l l  i n h i b i t i o n (62).  L - G l u t a m i n e i s d e a m i d a t e d by b r a i n s l i c e s . to a g r e a t e r  Net d e a m i d a t i o n o c c u r s  e x t e n t i n the a b s e n c e of g l u c o s e t h a n i n i t s p r e s e n c e ,  m i n e r e s y n t h e s i s i s p r o m o t e d b y t h e p r e s e n c e of g l u c o s e (62).  as g l u t a -  9  1.5  Ammonia  (i)  Urea  (a) Urea 88,  f o r m a t i o n a n d u t i l i z a t i o n m e c h a n i s m s i n the b r a i n  U r e a a s a s o u r c e of a m m o n i a .  i s p r e s e n t i n mammalian b r a i n a t a c o n c e n t r a t i o n of a b o u t  100).  C a n i t s e r v e a s a s o u r c e o f a m m o n i a i n the b r a i n ?  (41) h a v e s h o w n that i n h i b e r n a t i n g g a r d e n d o r m i c e ,  5.0 m M Godin  these workers,  D u r i n g h i b e r n a t i o n , the i n t e r n a l b o d y  t h e b l o o d f l o w , t h e o x y g e n c o n s u m p t i o n a n d the u t i l i z a t i o n of  different metabolic substrates are a l l reduced.  It i s c o n c e i v a b l e t h a t 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 t h e b l o o d l e v e l of u r e a , in a m m o n i a formation,  as a r e s u l t of a d r o p  arising partly through diminished bacterial action i n  the i n t e s t i n e a n d p a r t l y d u e 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 animals. (see  According  the d e c r e a s e d b r a i n a m m o n i a 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 . temperature,  et a l . ,  both the a m m o n i a and  u r e a l e v e l s f a l l to a b o u t 50 p e r c e n t of t h e c o n t r o l (awake) a n i m a l s . to  (41,  Thus,  i n the h i b e r n a t i n g  l o v / e r e d c e r e b r a l u r e a i s d u e to l o w e r e d b l o o d u r e a l e v e l s  s e c t i o n 1.5 (i) (b)); i t i s n o t a c c o m p a n i e d by a n i n c r e a s e d a m m o n i a  level  i n the b r a i n .  Moreover,  u r e a s e e m s n o t to be the s o u r c e o f a m m o n i a i n b r a i n d u r i n g  telodrin-induced seizures,  since urea levels remain  unchanged under these  c o n d i t i o n s (49).  Furthermore,  i n v i t r o s t u d i e s s h o w e d t h a t t h e a d d i t i o n of u r e a 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 "of the r a t i n c u b a t e d i n o x y g e n f o r one h o u r 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 f r e e ) h a d little o r no  e f f e c t o n t h e r a t e of a m m o n i a f o r m a t i o n (62).  Additionally,  urease  s e e m s n o t to be p r e s e n t i n m a m m a l i a n  tissues  s o f a r e x a m i n e d (86).  It s e e m s u n l i k e l y , brain.  t h e r e f o r e , that u r e a i s a s o u r c e of a m m o n i a i n  10  (b)  Possible formation  of u r e a as a m e c h a n i s m f o r  ammonia  utilization. U r e a i s not s y n t h e s i z e d the i n t e r m e d i a t e s  in brain from  of the K r e b s - H e n s e l e i t  C o n t e n t s of t h e s e i n t e r m e d i a t e s i n i t i a l wet (88), and  wt,  NH4* a n d  u r e a , 4.2.  u r e a c y c l e (87) a r e p r e s e n t  i n c a t b r a i n c a l c u l a t e d i n t e r m s of  o r n i t h i n e , 0.046;  Carbamyl  citrulline,  phosphate has  0.23;  b o l i t e s (namely,  arginino succinate  arginase) are also present  synthetase,  jjmole/g  of u r e a c y c l e m e t a -  of b r a i n (89»  and  90,  100).  o r n i t h i n e (91); and  L-arginine  (92),  o r n i t h i n e (viz.,  of c i t r u l l i n e  100).  the e n z y m e that f i x e s a m m o n i a to CO->  Krebs-Henseleit  cycle per  from  ornithine t r a n s c a r b a m y l a s e ) does  n o t o p e r a t e i n the c e n t r a l n e r v o u s s y s t e m (93,  i s l i k e w i s e a b s e n t i n the b r a i n (100).  acid,  o r c i t r u l l i n e (93) to u r e a .  H o w e v e r , the e n z y m e t h a t c a t a l y z e s the f o r m a t i o n p h o s p h a t e and  It  its m e d i a t i n g e n z y m e s o c c u r i n b r a i n .  s t u d i e s i n v i v o s h o w that L . - p r o l i n e i s c o n v e r t e d to g l u t a m i c  a r g i n i n e and  0.2;  in brain.  arginino succinase  in crude preparations  s e e m s that p a r t of the u r e a c y c l e and  synthetase,  in brain.  arginine,  not y e t b e e n d e t e c t e d  S e v e r a l of the e n z y m e s that p a r t i c i p a t e i n the s y n t h e s i s  carbamyl  S o m e of  f r o m the d a t a of T a l l a n , M o o r e and S t e i n ( g i v e n as m g / 1 0 0 g)  a r e as f o l l o w s :  Metabolic  (100).  Carbamyl  phosphate  i n the p r e s e n c e of  It i s d o u b t f u l t h e r e f o r e ,  se o c c u r s i n b r a i n at any  ATP,  whether  r a t e to any  the  significant  extent.  The passive  d i f f u s i o n f r o m the b l o o d .  a b o u t one has  relatively high concentration  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  (0.8 as c a l c u l a t e d f r o m the d a t a of T a l l a n et a l . (88) s e e  b e e n f o u n d (see a l s o r e f e r e n c e  l i v e r the c o n c e n t r a t i o n  (see r e f e r e n c e of t i s s u e / h r ,  49).  281).  It m a y  According  u r e a at a m a x i m u m r a t e of 1  w h e r e a s the r a t e f o r g l u t a m i n e s y n t h e s i s i s 10-20  (95).  T a b l e 1),  be p o i n t e d out that i n the  ( a m o l e / g f o r b r a i n (100) ).  brain synthesizes  to  r a t i o of •  of u r e a i s not m u c h d i f f e r e n t f r o m t h a t of b r a i n  i a m o l e / g f o r l i v e r v e r s u s 4.5  tissue/hr  A  of u r e a i n b r a i n i s p r o b a b l y due  to  (5.16 Blass  (imole/g  n m o l e / g of  The above considerations lead to the c o n c l u s i o n that r e m o v a l of a m m o n i a in the b r a i n does not o c c u r by u r e a f o r m a t i o n .  The o b s e r v a t i o n  that hepatectomy in the dog leads to cessation of u r e a production points to the l i v e r as p o s s i b l y the only major source of u r e a in the m a m m a l i a n o r g a n i s m s (96,  97). (c)  P o s s i b l e importance of u r e a synthesis i n the b r a i n (86,  95,  100).  The m e t a b o l i s m of c i t r u l l i n e and argininosuccinate in the b r a i n v i a u r e a f o r m a t i o n seems to be important for b r a i n development.  Relatively  l a r g e amounts of c i t r u l l i n e and argininosuccinate are present in the b r a i n , c e r e b r o s p i n a l fluid,  and p l a s m a , and are e x c r e t e d i n the urine i n  ' citrullinuria' and'argininosuccinic aciduria', respectively.  These diseases  of the c e n t r a l nervous s y s t e m are due to the relevant e n z y m e s being absent in b r a i n . (ii)  The conditions are a c c o m p a n i e d by mental r e t a r d a t i o n . (a)  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 aspartate channelled through N A D  +  or A M P .  A c c o r d i n g to B u n i a t i a n and c o - w o r k e r s (14), b r a i n and other organs f r o m amino a c i d s .  a m m o n i a is f o r m e d i n  They have p r o p o s e d a m e c h a n i s m  by means of which amino acids through aspartate supply amino groups to desamino- NAD  (D-NAD ). +  a m m o n i a and desamino-NAD"*".  NAD  +  thus fo r m e d on deamination yields  The sequence of reactions p r o p o s e d i s as  follows: Glutamate  > Aspartate D -NAD*t^ -r  NAD  succinate  ^  x  NAD " 1  +  fumarate  v N H o* +  D - N AI D " " 1  12  This process worker.  i s a v a r i a t i o n of the s c h e m e s u g g e s t e d b y a n o t h e r  According  t o K o m e t i a n i (81, 98),  t a t e a n d the d e a m i n a t i o n of A M P  the r e a m i n a t i o n  thu.- f o r m e d ,  ammonia is produced f r o m amino acids.  of I M P b y a s p a r -  is a m e c h a n i s m by which  T h e r e s u l t s l e a d i n g to t h i s h y p o -  thesis were obtained using m i t o c h o n d r i a l preparations  The  a m o u n t of a m m o n i a f o r m e d f r o m N A D *  (14).  and aspartate by mito-  c h o n d r i a o f r a t b r a i n (14) s e e m s t o b e i n s u f f i c i e n t to a c c o u n t f o r the of a m m o n i a b y b r a i n t i s s u e i n v i t r o . l a t i o n s (see r e f e r e n c e 2 h r at 3 7 ° C ,  formation  T h i s i s e v i d e n t f r o m the f o l l o w i n g c a l c u -  (14) f o r v a l u e s ) :  5.28 j f 0 . 3 |ig a m m o n i a / m g p r o t e i n /  w a s o b t a i n e d o n t h e a d d i t i o n of NAD"*" (1.4 p.moles) a n d a s p a r t a t e  (26 |amoles) to m i t o c h o n d r i a .  T h e c o n t r o l v a l u e w a s 3.07 + 0.44.  a s p a r t a t e o r N A D * p r e s e n t alone, 3.63  Russian  the values r e c o r d e d  were  With  3.45 + 0.3, o r  _+ 0.3, r e s p e c t i v e l y .  Taking  s t a n d a r d d e v i a t i o n s into c o n s i d e r a t i o n , i t s e e m s t h a t a l i t t l e  o v e r 1 (ig a m m o n i a / m g p r o t e i n i s f o r m e d i n t w o h o u r s b y b r a i n from NAD*  and aspartate.  T h i s a m o u n t s to 0.03 l-lmole a m m o n i a  per mg mitochondrial protein/hr. wholly  mitochondrial,  mitochondria  then only  formed  E v e n i f the p r o t e i n c o n t e n t o f b r a i n w a s  3 (jmole a m m o n i a / 1 0 0 m g p r o t e i n and hence  p e r g w e t wt b r a i n w o u l d be f o r m e d u n d e r t h e s e c o n d i t i o n s .  This value is  too s m a l l to a c c o u n t f o r t h e l i b e r a t i o n o f a m m o n i a b y b r a i n u n d e r a v a r i e t y of i n c u b a t i o n c o n d i t i o n s that w i l l b e r e c o r d e d a d d i t i o n of L - a s p a r t a t e  in this work.  Moreover, the  (5 m M ) t o 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 w a s w i t h o u t a n y  e f f e c t o n the r a t e o f a m m o n i a 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 a s p a r t a t e b y i n c u b a t i n g c e r e b r a l c o r t e x s l i c e s i n a g l u c o s e m e d i u m c o n t a i n i n g 105 m M  KC1,  or lowering  free  i t b y the u s e of m e t a b o l i c  i n h i b i t o r s s u c h a s m a l o n a t e o r a m i n o . o x y a c e t i c a c i d , d o e s n o t a f f e c t the r a t e of a m m o n i a f o r m a t i o n .  T h e s e e x p e r i m e n t s w i l l be d e s c r i b e d i n g r e a t e r  d e t a i l i n C h a p t e r 3.  With guinea pig b r a i n s l i c e s ,  T a k a g a k i (99) o b t a i n e d n o d e c r e a s e i n  13  e n d o g e n o u s l e v e l s of a d e n y l i c c o m p o u n d s ,  and  c o n c l u d e d that the  conversion  o f a d e n y l i c a c i d to i n o s i n i c a c i d by a d e n y l i c d e a m i n a s e i s q u a n t i t a t i v e l y a relatively minor process.  It m a y  be m e n t i o n e d that f r e e a d e n y l i c  a r e p r e s e n t at r e l a t i v e l y l o w l e v e l s i n the b r a i n and  t h e r e f o r e c a n n o t be  s i d e r e d as m a j o r s o u r c e s f o r c e r e b r a l a m m o n i a f o r m a t i o n  (ii)  (b)  Ammonia formation  compounds con-  (43).  f r o m amino acids via terminal  glutamate oxidation.  The  o r i g i n and  m e c h a n i s m of a m m o n i a f o r m a t i o n  is b r a i n slices  r e s p i r i n g i n a g l u c o s e - f r e e i n c u b a t i o n m e d i u m i s s t i l l a s u b j e c t of m u c h c o n troversy.  According  to the e a r l y w o r k of W e i l - M a l h e r b e a n d  c o - w o r k e r s (43,  75)  n o n e of the s i x d e a m i n a t i n g b r a i n e n z y m e s p r e s e n t i n b r a i n (e.g.,  glutamic  dehydrogenase, glutaminase, amine oxidase,  adenosine  adenylic deaminase,  d e a m i n a s e or h e x o s a m i n e d e a m i n a s e ) , can a c c o u n t f o r a m m o n i a T h e s e w o r k e r s s u g g e s t e d that a m m o n i a f o r m a t i o n  occurs  formation.  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 , s i n c e g u i n e a p i g b r a i n s l i c e s i n c u b a t e d gave significant increases in non-protein  nitrogen.  for 5 hrs  In t h e i r s t u d i e s ,  they  a s s u m e d a single origin f r o m which free ammonia is l a r g e l y derived.  On formation  the o t h e r hand,  a c c o r d i n g to V r b a et a L  i s too c o m p l e x a p r o c e s s  proteolysis.  (67,  101),  ammonia  to be e x p l a i n e d on the b a s i s of  T h e s e w o r k e r s s h o w e d that not m o r e t h a n 25 p e r  simple  c e n t of the  a m m o n i a f o r m e d by g u i n e a p i g b r a i n s l i c e s i n i n c u b a t i o n s of 4 h r s and d u r a t i o n c a n be a c c o u n t e d f o r as c o m i n g f r o m p r o t e i n a m i d e n i t r o g e n , c o n s i d e r the s o u r c e of the l a r g e r p a r t of a m m o n i a as s t i l l b e i n g t h e i r studies they a l s o o b s e r v e d s o m e i n c r e a s e s i n n o n - p r o t e i n lipid nitrogen,  and  6 hrs and  unknown. nitrogen  s o m e 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  protein fractions.  T h a t s o m e a m m o n i a 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 a m i d e  In  and  14  nitrogen is supported  b y the d e m o n s t r a t i o n that 16 p e r c e n t of the  glutaminyl  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 d e a m i d a t e d (76).  However,  e v e n t h i s d o e s n o t a c c o u n t f o r the m a j o r p o r t i o n of a m m o n i a f o r m e d i n v i t r o b r a i n c o r t e x s l i c e s (67,  76,  by  101).  In the s t u d i e s of T a k a g a k i et a l . (64), the i n c u b a t i o n of g u i n e a p i g b r a i n s l i c e s was  r e s t r i c t e d to one h o u r b e c a u s e i t was  f e l t that a m m o n i a  f o r m e d at the end of 4 h r s m i g h t be the r e s u l t of a s e r i e s of h i g h l y reactions,  m u c h of w h i c h i n v o l v e d p r o t e o l y s i s .  G l u t a m a t e d e c r e a s e s i n the  b r a i n c o n c o m i t a n t w i t h an i n c r e a s e i n a m m o n i a f o r m a t i o n a r e c a r r i e d out i n a g l u c o s e - f r e e m e d i u m ;  complicated  when incubations  a c c o r d i n g to t h e s e w o r k e r s ,  d e c r e a s e i n g l u t a m a t e c o n c e n t r a t i o n a c c o u n t s f o r 50 p e r c e n t of the genous oxygen uptake.  However,  stances  the c o n t e n t s  i n the i n c u b a t i o n m e d i u m not b e i n g c o n s i d e r e d .  possible conversion Y-aminobutyrate,  65) we  aspartate,  i n b o t h t i s s u e a n d i n c u b a t i o n m e d i u m ) by b r a i n s l i c e s  acid metabolism,  sub-  the  h a v e s h o w n that the t o t a l p r o d u c t i o n  f o r s h o r t p e r i o d s of t i m e i n the a b s e n c e of g l u c o s e ,  process.  of t h e s e  a l a n i n e , w e r e not t a k e n i n t o a c c o u n t .  In e a r l i e r w o r k (62, a m m o n i a (i.e.,  of g l u t a m a t e  Moreover,  of g l u t a m a t e to o t h e r a m i n o a c i d s s u c h as  and  endo-  i t a p p e a r s that t h e i r e s t i m a t i o n s  a n d a m m o n i a w e r e c o n f i n e d to t h e c e r e b r a l t i s s u e ,  the  i s l a r g e l y due  of  incubated  to a m i n o  i n w h i c h e n d o g e n o u s g l u t a m a t e o x i d a t i o n i s the t e r m i n a l  T h e s e s t u d i e s s h o w e d how  the i n d i v i d u a l l e v e l s of a m m o n i a  and  a m i n o a c i d s v a r i e d on i n c u b a t i n g r a t b r a i n c o r t e x s l i c e s i n o x y g e n f o r  one  h o u r at 3 7 ° C 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 . d i m i n u t i o n i n the g l u t a m a t e and  g l u t a m i n e l e v e l s i n the b r a i n c o r t e x  a c c o u n t s l a r g e l y f o r the l i b e r a t i o n of a m m o n i a . r a t e s of a m m o n i a f o r m a t i o n ,  T h e y i n d i c a t e d t h a t the  C h a n g e s i n the  slices  observed  on i n c u b a t i n g r a t b r a i n c o r t i c a l s l i c e s f o r  one  h o u r u n d e r a v a r i e t y of c o n d i t i o n s , c o u l d be e x p l a i n e d on the b a s i s of g l u t a m a t e o x i d a t i o n and g l u t a m i n e s y n t h e s i s . i n s e c t i o n 1.3. M a l h e r b e and  S u b s e q u e n t to o u r G o r d o n (10 2) s u p p o r t s  T h i s has  w o r k (62,  65),  already been mentioned a r e c e n t r e p o r t by  Weil-  o u r v i e w that c h a n g e s i n the l e v e l s of  15  amino acids (particularly glutamate) i n guinea pig b r a i n c o r t e x slices, a c c o u n t f o r t h e l i b e r a t i o n of a m m o n i a .  can  H o w e v e r , the contents of a m i n o  a c i d s f o u n d i n i t i a l l y i n t h e i r s l i c e s a r e a b o u t 30 - 50 p e r c e n t of the v a 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 , t h e i r r a t e of a m m o n i a  formation  i s 40 - 50 p e r cent of the v a l u e s t h e y r e p o r t e d i n 1955 (43).  The  origin,  m e c h a n i s m and factors affecting a m m o n i a f o r m a t i o n i n  incubated brain cortex slices, attempt  w i l l be d e a l t w i t h i n C h a p t e r  3, i n w h i c h a n  w i l l be m a d e t o c h a r a c t e r i z e the c o m p a r t m e n t s i n the b r a i n t i s s u e  mainly responsible for ammonia production (Chapter 10).  (iii) A m m o n i a  utilization processes i n brain .  T h e r e a p p e a r t o b e a t l e a s t t h r e e m e c h a n i s m s of a m m o n i a  utilization  i n the b r a i n . (a)  Urea formation.  A s mentioned earlier,  c o n v e r s i o n of a m m o n i a  to u r e a i n b r a i n o c c u r s e i t h e r n o t a t a l l o r o n l y a t a v e r y  small  rate. (b)  Amidation  of p r o t e i n s .  deamidation  A c c o r d i n g to a n u m b e r of w o r k e r s ,  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,  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 i n b r a i n ( s e e s e c t i o n 1.4 ( i i ) ) .  57, 67, 74, 75) (78, 79) o c c u r s  Though there is general consensus  a m o n g w o r k e r s i n t h e f i e l d t h a t the r e a m i d a t i o n p r o c e s s either N H ^  +  (utilizing  i o n s o r g l u t a m i n e , s h o u l d take p l a c e , the a c t u a l  demonstration  of i t s o c c u r r e n c e i s s t i l l a m a t t e r  of d o u b t (76, 80,  81). (c)  Glutamine synthesis.  T h e f o r m a t i o n of g l u t a m i n e  s e e m s to be  the m a j o r p r o c e s s o f a m m o n i a u t i l i z a t i o n i n b r a i n . glutamine activity  synthesis occurs  (9-11,  v u l s i v e agents,  Increased  in vivo during i n c r e a s e d functional  56, 57) b r o u g h t  about by p h y s i c a l e x e r c i s e ,  o r i n f u s i o n of a m m o n i u m s a l t s .  Ammonia  con-  16  l i b e r a t i o n b y b r a i n t i s s u e i s i n h i b i t e d u n d e r c o n d i t i o n s l e a d i n g to h i g h r a t e s of g l u t a m i n e s y n t h e s i s ; e.g., i n the p r e s e n c e of g l u c o s e (58-62) o r g l u t a m a t e (103).  O n e of the o b j e c t i v e s of o u r w o r k i s to  u n d e r s t a n d i n 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 f o r g l u t a m i n e s y n t h e s i s a n d i t s a c t u a l l o c a t i o n i n the b r a i n c e l l s .  1.6  use  Transport  of a m i n o a c i d s i n b r a i n .  Transport  of a m i n o a c i d s i n b r a i n h a s u s u a l l y b e e n i n v e s t i g a t e d b y t h e  of t i s s u e s l i c e s as t h e y a r e a b l e to a c c u m u l a t e a m i n o a c i d s a g a i n s t a con-  centration gradient. concentrate  In the i n t a c t a n i m a l , h o w e v e r ,  the b r a i n i s o n l y a b l e to  a m i n o a c i d s to a s m a l l e x t e n t o r n o t at a l l .  The difference  b e t w e e n _ i n v i v o a n d i n v i t r o e x p e r i m e n t s l e d to t h e c o n c e p t of a b l o o d barrier.  Recently  i t h a s b e e n f o u n d that, t h o u g h t h e r e i s b u t l i t t l e i n c r e a s e i n  the a m o u n t s of a m i n o a c i d s i n b r a i n o n i n c r e a s i n g b l o o d a m i n o a c i d tions, (15,  brain  concentra-  t h e r e i s a r a p i d m o v e m e n t of a m i n o a c i d s i n t o a n d out of t h e b r a i n  104-107) as s h o w n b y the u s e of l a b e l l e d a m i n o a c i d s .  T h e s u p p l y of a m i n o a c i d s to t h e b r a i n f r o m the b l o o d i s of o b v i o u s i m p o r t a n c e f o r the n o r m a l f u n c t i o n a l a c t i v i t y of the b r a i n .  In a d d i t i o n to t h e i r  i n c o r p o r a t i o n i n t o p r o t e i n , t h e y a r e n e e d e d f o r t h e s y n t h e s i s of b i o g e n i c a m i n e s w h o s e u p t a k e i s m u c h s m a l l e r t h a n the c o r r e s p o n d i n g g i v i n g r i s e to t h e m  amino acid  (104).  P a s s i v e d i f f u s i o n l e a d s o n l y to a c o n c e n t r a t i o n w i t h i n the c e l l no than that outside,  e x c e p t u n d e r c i r c u m s t a n c e s w h e r e b i n d i n g of the s u b s t a n c e  to a c e l l c o n s t i t u e n t t a k e s p l a c e i n the c e l l o r i n a c e l l c o m p a r t m e n t A c t i v e transport,  on t h e o t h e r hand, r e q u i r e s e n e r g y ,  a b s e n c e of o x y g e n a n d g l u c o s e ,  against a concentration gradient.  available,  being  It u t i l i z e s A T P  (104).  l e s s i n the  a n d a l s o i n the p r e s e n c e of m e t a b o l i c  l i k e c y a n i d e a n d 2, 4 d i n i t r o p h e n o l .  of u p t a k e r e a c h i n g  higher  and i n v o l v e s  inhibitors  movement  It i s s u b j e c t t o s a t u r a t i o n k i n e t i c s ,  the r a t e  a l i m i t w i t h i n c r e a s e i n the c o n c e n t r a t i o n of a m i n o a c i d  o w i n g to s a t u r a t i o n of c a r r i e r  sites.  Competition also  occurs  17  between a m i n o a c 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 acids  c o m p e t e w i t h e a c h o t h e r f o r a c o m m o n c a r r i e r but m a y  b r i n g a b o u t the  p r e s s i o n of e a c h o t h e r ' 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 (104,  106,  113).  Unlike competitive  transport processes for  example,  d o e s not i n v o l v e a s p e c i f i c c a r r i e r .  by i n t e r f e r e n c e w i t h m e t a b o l i c  processes  It c a n be  has  levels  produced,  w i t h i n the c e l l u p o n  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 s u b s t r a t e a m i n o a c i d  The  sup-  i n h i b i t i o n , n o n - c o m p e t i t i v e i n h i b i t i o n of  w h i c h e n e r g y f o r t r a n s p o r t i s d e p e n d e n t , o r by d a m a g e to the c e l l and  may-  membrane,  (106).  u p t a k e of a m i n o a c 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  b e e n b a s e d e s s e n t i a l l y on the a m o u n t b y w h i c h 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 a m i n o a c i d of the t i s s u e h a s ( u s u a l l y one h o u r ) . ' B o t h i n f l u x and state is r e a c h e d .  efflux p r o c e s s e s  E i t h e r of t h e s e p r o c e s s e s  d i f f u s i o n o r by a c t i v e t r a n s p o r t or p r o c e s s e s Passive  diffusion may  T h e r e may  also occur  109)  s u b s t r a t e i n one direction.  may  occur  w h e n the  be p r o d u c e d b y  steady  passive  s u c h as f a c i l i t a t e d d i f f u s i o n  111)  (108).  i n w h i c h m o v e m e n t of  d i r e c t i o n c a u s e s m o v e m e n t at the s a m e t i m e i n the may  (107).  e f f l u x s y s t e m that i n v o l v e s c o u n t e r  o r e x c h a n g e d i f f u s i o n (110,  T h i s latter p r o c e s s  period  at the s a m e t i m e as a c t i v e t r a n s p o r t  be a f a c t o r i n the i n f l u x a n d  t r a n s p o r t (106,  increased over a defined  opposite  be of s p e c i a l i n t e r e s t i n b r a i n as i t  may  be r e l a t e d to the b l o o d b r a i n b a r r i e r p h e n o m e n o n i n w h i c h net t r a n s f e r i s i m p e d e d i n v i v o i n s p i t e of r a p i d i n t e r c h a n g e  b e t w e e n b l o o d and b r a i n  (106).  A c t i v e t r a n s p o r t of a m i n o a c i d s i n b r a i n i s s o d i u m d e p e n d e n t (113, 105).  H i g h p o t a s s i u m i n h i b i t s a m i n o a c i d u p t a k e (114) by d i m i n i s h i n g  l e v e l s (115).  ATP  A b s e n c e of c a l c i u m r e t a r d s the r a t e of a m i n o a c i d uptake,  s u m a b l y by l o w e r i n g s a l t s (10 mM),  ATP  levels.  p r e s u m a b l y due  Inhibition also occurs  to the i n h i b i t i o n of N a t  with high  K"*"-stimulated  104,  pre-  calcium ATPase.  O u a b a i n i n h i b i t s b o t h the a c t i v e t r a n s p o r t of a m i n o a c i d s and the Na"*", K*s t i m u l a t e d A T P a s e . ( s e c t i o n 1.9 for  the Na"*"  coupled  (v)).  O u a b a i n p r e s u m a b l y a c t s by i t s a f f i n i t y  t r a n s p o r t ' c a r r i e r (at the K ^ - s i t e ) and  with this c a r r i e r  ( s e c t i o n 1.9  (v)).  thus b l o c k s  O u a b a i n has  transport  l i t t l e o r no e f f e c t s o n  18  c e r e b r a l A T P l e v e l s (11 5).  O u a b a i n (112, 280), a n o x i a ,  r e s p i r a t o r y chain inhibitors, (104,  105,  e n h a n c e the r a t e of a m i n o a c i d e f f l u x f r o m b r a i n  on t r a n s p o r t p r o c e s s e s f o r i n d i v i d u a l amino  o t h e r c o m p o u n d s of b i o c h e m i c a l  a v a r i e t y of s p e c i e s and N a e m e  +  205).  M o r e information and  N a l a c k and  acids  i n t e r e s t i n b r a i n a n d i n o t h e r t i s s u e s of  m a y b e o b t a i n e d f r o m the r e v i e w s b y Q u a s t e l  (104, 105)  (106).  Table 1 gives brain/plasma concentration  r a t i o s of a m i n o a c i d s c a l c u -  l a t e d f r o m the d a t a (for t h e cat) of T a l l a n , M o o r e  a n d S t e i n (88, s e e a l s o 95,  116).  In o u r work,  o u r 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 h a s b e e n c h i e f l y con-  f i n e d to t h e t r a n s p o r t o f a m i n o a c i d s ( p a r t i c u l a r l y t h a t of g l u t a m a t e ) , of a m m o n i a i n r a t b r a i n c o r t e x  1.7  (i) A m i n o  and that  slices.  acid metabolism in brain.  Glutamic acid occupies a central position in brain metabolism, of i t s h i g h c o n c e n t r a t i o n ,  its involvement i n many biochemical  r e l a t i v e l y h i g h r a t e of o x i d a t i o n , Glutamate, aspartate,  together  reactions, i t s  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 .  w i t h the c l o s e l y r e l a t e d a m i n o a c i d s ,  G A B A and also alanine,  viz., glutamine,  c o n t r i b u t e up to 70 p e r c e n t of t h e t o t a l  a m i n o a c i d n i t r o g e n of a d u l t b r a i n . of b r a i n i s u n l i k e that of o t h e r  In t h i s r e s p e c t the f r e e a m i n o a c i d p o o l  animal tissues.  While s p e c i a l attention will  b e g i v e n to the a b o v e m e n t i o n e d g r o u p o f a m i n o a c i d s , t h e a m i n o glycine,  serine,  threonine,taurine  i n view  and N - a c e t y l aspartate,  acids  that t o g e t h e r  make  u p m u c h of the r e m a i n i n g a m i n o a c i d n i t r o g e n of the f r e e a m i n o a c i d p o o l , a l s o be d e a l t w i t h i n s t u d i e s r e p o r t e d  i n this thesis.  will  19  TABLE  1.  Some b r a i n to p l a s m a concentration ratios of amino acids f o r the c a t .  T h e s e c a l c u l a t i o n s w e r e m a d e f r o m the data of T a l l a n , M o o r e a n d S t e i n (88) g i v e n a s m g % .  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  d o n e b y a s s u m i n g t h e t i s s u e w a t e r c o n t e n t t o b e 8 0 % t h a t of t h e i n i t i a l f r e s h weight b r a i n .  Plasma  Brain Hmole Glutamate Glutamine  /g  8.74 > 3.42  (Jmole  /ml  mM  Brain/ Plasma  10.92  0.123  89.0  >4.27  >0.427  10.0 368.0  Aspartate  2.23  2.79  0.0076  Asparagine  0.20  0.25  0.068  GABA  2.27  2.84  Alanine  0.95  1.19  0.845  1.4  Glycine  1.35  1.69  0.307  5.5  Serine  0.73  0.91  0.20  4.6  Threonine  0.22  0.27  0.118  2.3  Valine  0.18  0.22  0.21  1.1  Leucine  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  Arginine  0.08  0.10  0.08  1.2  Ornithine  0.045  0.06  0.015  4.0  < 0.023  <0.03  Citrulline Urea  4.17  5.21  < 0.02  ^0.00 57 6.67  3.7 > 142.0  <C  5.3 0.8  20  (a) The  Initial c e r e b r a l contents  of a m i n o a c i d s  i n i t i a l l e v e l s of a m i n o a c i d s , d e t e r m i n e d i n t h e s e s t u d i e s  g i v e n i n T a b l e s 2 a n d 4 f o r a d u l t a n d i n f a n t (2 d a y  old) r a t b r a i n , a r e i n  c l o s e a g r e e m e n t w i t h t h o s e 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 h a s h i g h c o n c e n t r a t i o n s of t a u r i n e and p h o s p h o e t h a n o l a m i n e r e f e r e n c e 130,  131),  but h a v e l o w l e v e l s of g l u t a m a t e and  derived amino acids. t i o n and  The  (see  its m e t a b o l i c a l l y  a c t i v i t i e s of the e n z y m e s i n v o l v e d i n the  i n t e r c o n v e r s i o n of t h e s e a m i n o a c i d s a r e a l s o l o w  v a l u e s of a m i n o a c i d s and  and  (15,  forma-  163).  of t h e i r r e l a t e d e n z y m e s a r e r e a c h e d  Adult  as the b r a i n  m a t u r e s a n d at a b o u t t h e t i m e of t h e c o m p l e t e l a y i n g d o w n of m y e l i n (129,  130).  A n i m a l s s u c h as the g u i n e a p i g , i n w h i c h the i n f a n t s t a g e s h o w s m a n y  mature  characteristics,  age  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  (129). (b) A  P a t h w a y s of g l u t a m a t e  metabolism  d e t a i l e d a c c o u n t of the s y n t h e s i s and  b e y o n d the s c o p e of t h i s t h e s i s . s e n t s t u d y w i l l be d i s c u s s e d . useful reviews  (8, 11,  12,  O n l y the m a i n a s p e c t s p e r t i n e n t to the  pre-  more detailed information there are  many  For  15,  c a t a b o l i s m of a m i n o a c i d s i s  86,  95,  106,  117-120).  G l u t a m a t e i s c l o s e l y r e l a t e d t o the c i t r i c a c i d c y c l e due t i o n by t r a n s a m i n a t i o n  to i t s f o r m a -  of a - k e t o g l u t a r a t e .  Glutamate transaminases;  Glutamate can r e v e r s i b l y  transaminate  w i t h the k e t o a c i d s , o x a l o a c e t a t e and p y r u v a t e  to y i e l d a s p a r t a t e and  respectively.  a r e q u a n t i t a t i v e l y the d o m i n a n t  T h e s e r e a c t i o n s , s h o w n below,  transaminations  o c c u r r i n g i n b r a i n t i s s u e (122,  123).  alanine  T h e y a r e 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 p h o s p h a t e as c o - e n z y m e  (124).  glutamate + oxaloacetate ^  ^ a -ketoglutarate + aspartate  (1)  glutamate + pyruvate  _  (2)  .  a-ketoglutarate  + alanine  21  W h e n the r a t e of f o r m a t i o n of f r e e a m m o n i a i s h i g h , t i o n of a-ketoglutarate,  reductive anima-  mediated by glutamate dehydrogenase,  occurs  and a  r a p i d s y n t h e s i s of g l u t a m a t e t a k e s p l a c e , f o l l o w e d b y t h e s y n t h e s i s of g l u t a mine. forming  Glutamate,  however, t r a n s a m i n a t e s  alanine and aspartate,  undergoes reductive amination  is  mainly  forming  and oxaloacetate  and r e g e n e r a t i n g o:-ketoglutarate w h i c h a g a i n i n p r e s e n c e of e x c e s s  of a m m o n i a i n t o a m i n o a c i d s ( g l u t a m a t e , takes place.  with pyruvate  glutamine,  ammonia.  Thus,  a flow  alanine and aspartate)  W h e n the a m o u n t of a m m o n i a f o r m e d i s low, a - k e t o g l u t a r a t e  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  pyruvate  with alanine or aspartate  o r oxaloacetate w h i c h enter the c i t r i c acid  Glutamate can also transaminate c o m p o u n d b e i n g c o n v e r t e d to t h e o t h e r .  cycle.  d i r e c t l y with a-ketoglutarate,  each  T h i s a l l o w s the r a p i d e x c h a n g e of  l a b e l l e d c a r b o n b e t w e e n g l u t a m a t e a n d 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  GABA  shunt.  Glutamate is also formed 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 t h e e n z y m e G A B A t r a n s a m i n a s e ) .  • s e m i a l d e h y d e f o r m e d i n the r e a c t i o n e n t e r s  the c i t r i c a c i d c y c l e on conver-  s i o n t o s u c c i n a t e by the NAD"*" r e q u i r i n g e n z y m e s u c c i n i c dehydrogenase.  Succinic  semialdehyde  T h e s e r e a c t i o n s i n c l u d i n g the d e c a r b o x y l a t i o n of g l u t a m a t e  by g l u t a m a t e d e c a r b o x y l a s e  c o n s t i t u t e t h e G A B A s h u n t (116,  119). It  r e p r e s e n t s a n a l t e r n a t e p a t h w a y of a - k e t o g l u t a r a t e o x i d a t i o n i n the  meta-  b o l i s m of c a r b o h y d r a t e  o r the  i n the c e n t r a l n e r v o u s s y s t e m o f m a m m a l s  p e r i p h e r a l n e r v e s of a n t h r o p o d s .  A c c o r d i n g to B a l a z s _ e t a l . (116),  8 p e r cent  of the a - k e t o g l u t a r a t e of the c i t r i c a c i d c y c l e m a y b e d i v e r t e d t o s u c h a r o u t e . H a b e r (279) h a d a l r e a d y c o n c l u d e d  that l e s s t h a n 10 p e r c e n t of c a r b o h y d r a t e  o x i d a t i o n i n b r a i n t o o k p l a c e v i a t h e G A B A shunt. the G A B A s h u n t a r e d e p i c t e d b e l o w ( R e a c t i o n s  The reactions constituting  3, 4, 5).  22  Glutamate  GABA  >GABA  +  C 0  + a-ketoglutarate^=z±r  (3)  2  (4)  Glutamate + Succinic semialdehyde H O z  Succinic semialdehyde + NAD ^-  "'Succinate + N A D H  +  The  sum  + H  (5)  +  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 isformed reversibly  from  a - k e t o g l u t a r a t e a n d a m m o n i u m i o n b y the e n z y m e g l u t a m a t e d e h y d r o g e n a s e a c c o r d i n g to the f o l l o w i n g e q u a t i o n s  (8, 95) ( R e a c t i o n s 6 a n d 7 ) .  Glutamate + NAD ^=zrz=^: a-iminoglutarate + N A D H + H +  + ^ 0 ^ .  a-iminoglutarate  (6)  +  a - k e t o g l u t a r a t e +NH3  (7)  non-enzymic In t h e p r e s e n c e  of a m m o n i u m s a l t s t h e e q u i l i b r i u m of r e a c t i o n (6) i s  m u c h i n f a v o u r of g l u t a m a t e f o r m a t i o n o n l y 1.4 p e r c e n t b e i n g o x i d i z e d at p H 7 . 4 (8).  I n the a b s e n c e o f a m m o n i u m s a l t s , a - i m i n o g l u t a r a t e  h y d r o l y z e s to a - k e t o g l u t a r a t e a n d a m m o n i a .  T h u s t h e s u p p l y o r r e m o v a l of  a m m o n i a m a y r e g u l a t e the r a t e of o x i d a t i o n of g l u t a m a t e (8, presence  of a-ketoglutarate  conclusions were reached f r o m b r a i n (8, 4 2 , 1 3 2 ) .  133).  The  inhibits c e r e b r a l a m m o n i a f o r m a t i o n (42).  These  f r o m reactions i nhomogenous solutions derived In b r a i n s l i c e s i n t h e p r e s e n c e  r e d u c t i v e a m i n a t i o n of ot-ketoglutarate a b s e n c e of g l u c o s e ,  spontaneously  i s f a v o u r e d (102).  of g l u c o s e , t h e H o w e v e r , i n the  as e n d o g e n o u s s u b s t r a t e s o f i n c u b a t e d b r a i n c o r t i c a l  s l i c e s a r e depleted, the N A D + / N A D H r a t i o i n c r e a s e s w h i l e the A T P / A D P ratio decreases. ADP  I n c r e a s e 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  + Pi———>NAD  +  + ATP).  +  (V N A D H  T h e s e c h a n g e s f a v o u r t h e o x i d a t i o n 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 .  Glutamine synthetase.  G l u t a m a t e c o m b i n e s w i t h the a m m o n i u m i o n ,  t h r o u g h the a c t i v i t y of the e n z y m e g l u t a m i n e  s y n t h e t a s e (58, 103) i n t h e p r e -  s e n c e o f A T P (134, 136) a c c o r d i n g to t h e f o l l o w i n g r e a c t i o n ( R e a c t i o n 8).  ++ Glutamate + N H  + 4  + ATP  +  +  —^Glutamine  + ADP + Pi  (8)  O x i d i z a b l e s u b s t r a t e s like glucose lactate and pyruvate,  suppress  a m m o n i a f o r m a t i o n i n b r a i n both by c o m p e t i t i o n with endogenous o x i d i z a b l e n i t r o g e n o u s m a t e r i a l a n d b y the f o r m a t i o n of g l u t a m i n e s e n c e of g l u c o s e b r i n g s about a d i s a p p e a r a n c e b r a i n s l i c e s (58). (62,  (58-62).  The pre-  of a m m o n i a w i t h i n c u b a t e d  I n h i b i t i o n of A T P s y n t h e s i s (e.g., b y 2,4 d i n i t r o p h e n o l  64) o r f l u o r o a c e t a t e (63) ) a n d t h e r e b y d i m i n i s h e d g l u t a m i n e  causes  increased ammonia  synthesis  output.  G l u t a m i n e s y n t h e t a s e i s 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). ATP,  F o r t h e b r a i n e n z y m e the K m  2.3 mM;  and for N H  + 4  ,  0.18 m M  for L-glutamate  (95).  s i g n i f i c a n t i n r e l a t i o n to the r o l e of g l u t a m i n e  i s 2.5 mM; f o r  The low value f o r N H  + 4  is  s y n t h e t a s e i n the c e r e b r a l  u t i l i z a t i o n of a m m o n i a .  Glutaminase.  Glutamate isformed f r o m  glutamine  of a m m o n i a b y t h e m i t o c h o n d r i a l e n z y m e g l u t a m i n a s e  (15).  w i t h the r e l e a s e The process is  a s f o l l o w s ( R e a c t i o n 9). H O z  Glutamine  >- G l u t a m a t e + N H  Glutaminase  (9)  + 4  i s s t r o n g l y inhibited by D- or L - g l u t a m a t e  (103).  Phos-  phate and other p o l y v a l e n t anions such as a r s e n a t e and sulphate activate the e n z y m e (137, 138). In t h e a b s e n c e of p h o s p h a t e ,  glutaminase  a c t i v i t y of  g u i n e a p i g b r a i n h o m g e n a t e s i s s t r o n g l y e n h a n c e d by the p r e s e n c e  of t r i -  24  c a r b o x y l i c acids like citrate, slightly, (138).  i f at a l l ,  l e s s s t r o n g l y by that of d i c a r b o x y l i c a c i d s ,  by t h a t of m o n o c a r b o x y l i c  a c i d s l i k e l a c t a t e or  and  propionate  a l l o s t e r i c p r o p e r t i e s (15,  138,  The  e n z y m e f r o m b r a i n a n d k i d n e y has  (ii)  C o m p a r t m e n t a t i o n of a m i n o a c i d m e t a b o l i s m i n b r a i n  The  c o n c e p t of m e t a b o l i c c o m p a r t m e n t a t i o n i s the o u t c o m e of o b s e r -  139).  v a t i o n s (116,  1 4 1 - 1 4 8 ) t h a t c a n n o t be e x p l a i n e d b y the s i m p l e  p r o d u c t r e l a t i o n as d e s c r i b e d b y Z i l v e r s m i t e_t a l . ( 1 4 0 ) . s e n c e of two  a n d / o r q u a l i t a t i v e l y i t s own t a t i o n i n b r a i n was C l a r k e (141)  Metabolic  f i r s t d e s c r i b e d f o r the g l u t a m a t e - g l u t a m i n e  have r e c e n t l y reviewed  co-workers (141-143).  compartmensystem.  Berl  the r e l e v a n t l i t e r a t u r e .  c a r r i e d out i n v i v o b y W a e l s c h  T h e y s t u d i e d the e f f e c t s of i n t r a v e n o u s l y ,  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 min,  each maintaining quantitatively  p a t t e r n s of m e t a b o l i s m .  E a r l y w o r k on t h i s s u b j e c t w a s  intervals (2-5  It i m p l i e s the p r e -  o r m o r e d i s t i n c t p o o l s of a g i v e n m e t a b o l i t e i n a g i v e n t i s s u e  not n e c e s s a r i l y i n e q u i l i b r i u m with e a c h other,  and  precursor-  and  intra-  ^ C - g l u t a m a t e for short time  i n o r d e r to a v o i d e q u i l i b r a t i o n of i s o t o p e t h r o u g h a l l  m e t a b o l i c p o o l s ) and f o u n d t h a t the s p e c i f i c a c t i v i t y of g l u t a m i n e f r o m b r a i n t i s s u e was  g r e a t e r t h a n t h a t of g l u t a m a t e .  isolated  This observation  a l s o f o u n d to be t r u e f o r i n t r a v e n o u s l y a d m i n i s t e r e d 1 5 j \ f _  a  m  r  n  o  n  i  u  m  was  acetate  14 or  C labelled bicarbonate.  p r e c u r s o r of g l u t a m i n e ,  On  the a s s u m p t i o n that g l u t a m a t e i s the o n l y  t h e s e o b s e r v a t i o n s i n d i c a t e d that g l u t a m i n e  must  be  d e r i v e d f r o m a c o m p a r t m e n t of g l u t a m a t e not i n e q u i l i b r i u m w i t h t h e r e s t of the g l u t a m a t e p r e s e n t i n the b r a i n . l e a s t two  d i s t i n c t p o o l s , one  to the f o r m a t i o n of  In o t h e r w o r d s ,  g l u t a m a t e e x i s t s i n at  (the s m a l l e r ) p o o l b e i n g p a r t i c u l a r l y  disposed  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 g l u t a m i n e  to  t h a t of g l u t a m a t e s h o w e d t h a t l a b e l l e d k e t o g e n i c p r e c u r s o r s s u c h as a c e t a t e , butyrate,  p r o p i o n a t e a n d a c e t o a c e t a t e g i v e r i s e to a r a t i o g r e a t e r t h a n  one.  25  With labelled glycogenic substances glycerol,  s u c h as g l u c o s e ,  the s p e c i f i c a c t i v i t y of g l u t a m a t e  T h e s e effects are accounted p o o l s i n the b r a i n c e l l s ,  pyruvate,  lactate or  i s g r e a t e r t h a n t h a t of g l u t a m i n e .  f o r by a s s u m i n g the p r e s e n c e  of at l e a s t  i n w h i c h the c i t r i c a c i d c y c l e o p e r a t e s ,  i n t h e i r a c c e s s i b i l i t i e s to s u b s t r a t e s t h a t s h o w m e t a b o l i c  two  which differ  compartmentation  (144).  C o m p a r t m e n t a t i o n of g l u t a m a t e  m e t a b o l i s m has  also been d e 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 i n c u b a t e d under c e r t a i n d i t i o n s (145).  The  amino acids lysine,  leucine,  con-  G A B A , p r o l i n e and p h e n y l a l a n i n e  a n d a l s o e t h a n o l e x h i b i t m e t a b o l i c c o m p a r t m e n t a t i o n i n t h e f o r m a t i o n of glutamate  and g l u t a m i n e  The  assumption  (146,  ized,  As  B e r l and  283,  of the p r e s e n c e  n e c e s s i t a t e s the p r e s e n c e are derived.  148,  of two  synthetic cycle".  The  p o o l but only a s m a l l g l u t a m i n e  a c t i v e i n the s y n t h e s i s of g l u t a m i n e , "The  energy  they have c a l l e d  pool,  "the  c y c l e " is presumably responsible for a  ATP.  s e a r c h f o r m e t a b o l i c i n h i b i t o r s t h a t s e l e c t i v e l y a f f e c t one successful.  t h e s y n t h e s i s of g l u t a m i n e  of t h e s e  L a h i r i a n d Q u a s t e l (63) s h o w e d t h a t  w h i l e f l u o r o a c e t a t e h a s l i t t l e e f f e c t on o x y g e n c o n s u m p t i o n ,  it s t r o n g l y i n h i b i t s  a n d c o n c l u d e d that at l o w ( c o n v u l s i v e ) c o n c e n t r a -  t i o n s it a f f e c t s c e r e b r a l a m m o n i a m e t a b o l i s m .  of  acid  p r o c e s s ( a l s o i n v o l v i n g the c i t r i c a c i d c y c l e ) w h i c h  p o o l s i s b e g i n n i n g to p r o v e  (150),  in brain  C l a r k e (141) h a v e c a l l e d the p r o c e s s ( i n v o l v i n g the c i t r i c  is predominantly  A  p o o l s of g l u t a m a t e  the p h y s i c a l l o c a t i o n of t h e s e p o o l s h a s not b e e n c h a r a c t e r -  cycle".  l a r g e r p o o l of  of two  p o o l s of a - k e t o g l u t a r a t e f r o m w h i c h t h e y  c y c l e ) that g e n e r a t e s a l a r g e g l u t a m a t e "the e n e r g y  284).  A c c o r d i n g to C l a r k e et a l .  the p h e n o m e n o n c a n be e x p l a i n e d i f f l u o r o a c e t a t e i n h i b i t s o p e r a t i o n s  "the s y n t h e t i c c y c l e " w i t h r e l a t i v e l y no e f f e c t o n t h o s e of "the e n e r g y  cycle".  W o r k o n the a c t i o n of m a l o n a t e a n d n e u r o t r o p i c a g e n t s l i k e p r o t o v e r a t r i n e , on the o p e r a t i o n s of the two  pools,  w i l l be p r e s e n t e d i n t h i s t h e s i s ( C h a p t e r 8).  26  M e t a b o l i c c o m p a r t m e n t a t i o n h a s n o t yet b e e n s a t i s f a c t o r i l y d e f i n e d i n structural or cellular terms. t h e i r e n z y m e s (232),  Mitochondria,  h e t e r o g e n o u s w i t h r e s p e c t to  m a y o f f e r a p o s s i b l e e x p l a n a t i o n to t h i s p r o b l e m .  m i t o c h o n d r i a m a y be d e r i v e d f r o m  But  d i f f e r e n t c e l l t y p e s (e.g., n e u r o n a n d g l i a )  o r d i f f e r e n t p o r t i o n s of the s a m e c e l l (e.g., n e r v e  c e l l body and nerve  ending).  S o m e e v i d e n c e i n f a v o u r of n e u r o n s a n d g l i a a s r e s p o n s i b l e f o r m e t a b o l i c c o m p a r t m e n t a t i o n h a s b e e n p r e s e n t e d b y R o s e (159) u s i n g h i s n e u r o n a l and g l i a l p r e p a r a t i o n s . H o w e v e r ,  a t the p r e s e n t t i m e f r a c t i o n a t i o n  procedures  f o r t h e s e p a r a t i o n of c e l l t y p e s s u c h a s n e u r o n s a n d g l i a a r e s o m e w h a t d r a s t i c a n d c a n n o t b e e x p e c t e d to g i v e p u r e p r e p a r a t i o n s . M o r e o v e r , h o w f a r the p r o c e d u r e  it is u n c e r t a i n  a f f e c t s t h e p r o p e r t i e s of the f r a c t i o n s .  c o m p a r t m e n t a t i o n of the g l u ' . a m a t e - g l u t a m i n e i m m a t u r e r a t b r a i n (149),  Metabolic  s y s t e m i s not m a n i f e s t i n  o r i n o c t o p u s b r a i n t h a t c o n t a i n s l i t t l e g l i a (160).  C o m p a r t m e n t a t i o n of a m i n o a c i d s f o l l o w i n g n e u r o n a l d e g e n e r a t i o n h a s b e e n r e p o r t e d (161). glutamate  B r i e f l y s t a t e d , s u c h i n d i r e c t r e s u l t s i n d i c a t e t h a t the  c o m p a r t m e n t m a y c o n s i s t of n e u r o n a l p r o c e s s e s  w h i l e the " s m a l l " glutamate  pool a s s o c i a t e d with high glutamine  t h o u g h t to c o n s i s t of g l i a l t i s s u e (161, m o r e definitive attempts  (116,  149, 154-156), content i s  162). In s t u d i e s r e p o r t e d i n t h i s t h e s i s  w i l l b e m a d e to c h a r a c t e r i z e t h e c o m p a r t m e n t s of  a m i n o a c i d s i n b r a i n c o r t e x s l i c e s of the r a t u s i n g p h a r m a c o l o g i c a l r a t h e r than fractionations,  1.8  "large"  agents,  f o r l o c a l i z a t i o n of t h e c o m p a r t m e n t s .  P r o p a g a t i o n of n e r v e  impulse  N e u r o p h y s i o l o g i c a l a s p e c t s of e x c i t a t i o n h a v e b e e n e x t e n s i v e l y reviewed  (164-168,  presented  176, 262). O n l y  m a t e r i a l r e l e v a n t to t h i s w o r k w i l l b e  here.  (i)  B r a i n c e l l t y p e s (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 m a y be d i v i d e d into two c l a s s e s  on the b a s i s of w h e t h e r t h e y a r e e x c i t a b l e ( n e u r o n s ) o r n o t ( g l i a ) .  Each  27  n e u r o n i s m a d e u p of (1) d e n d r i t e s w h i c h a r e c o v e r e d b y s y n a p t i c p r o c e s s e s ; (2) t h e c e l l b o d y r e s p o n s i b l e f o r t h e p r o p e r f u n c t i o n i n g of t h e n e u r o n ; a n d (3) a n a x o n w h o s e t e r m i n a l b r a n c h e s neurons.  form  synaptic connections with other  G l i a c o n s i s t s of t h r e e t y p e s , n a m e l y ,  and m i c r o g l i a ,  a n d a r e c a p a b l e of p r o l i f e r a t i o n .  b e c a u s e of t h e i r h i g h r e s i s t a n c e m e m b r a n e  environment  It i s t h o u g h t that t h e y o f f e r  supply n u t r i t i o n f r o m the blood,  and m a i n t a i n the c h e m i c a l  of t h e n e u r o n (30 5).  There (171).  They act as insulators  and a r e e s s e n t i a l f o r the l a y i n g  d o w n a n d m a i n t e n a n c e of the m y e l i n s h e a t h . s t r u c t u r a l support,  astrocytes,oligodendroglia  a r e a b o u t t e n t i m e s m o r e g l i a l c e l l s t h a n n e u r o n s i n the b r a i n  T h e v o l u m e o c c u p i e d by the g l i a l c e l l s ,  a s that o c c u p i e d b y the n e u r o n s (95).  There  however,  i s about the s a m e  i s a g r e e m e n t that the n e u r o n s  a r e r e s p o n s i b l e f o r m u c h of the o x y g e n c o n s u m p t i o n of b r a i n (175, 271).  264, 268-  In t h e i m m a t u r e b r a i n n e u r o n a l p r o c e s s e s a r e n o t d e v e l o p e d .  The  g r o w t h of s u c h p r o c e s s e s c o i n c i d e s with m y e l i n a t i o n .  (ii)  R e s t i n g p o t e n t i a l (164-168)  When a m e m b r a n e  s e p a r a t i n g two s o l u t i o n s i s p e r m e a b l e to one i o n i c  species a potential difference,  E, i s s e t up a c r o s s t h e m e m b r a n e w h o s e m a g n i -  t u d e i s g i v e n b y t h e f o l l o w i n g N e r n s t e q u a t i o n (at 3 8 ° C a n d f o r a m o n o v a l e n t i o n ) :  E  , , _ , [penetrating ion outsidel ,. = 61.5 l o g Jf -r - m i l l i v o l t s 10[penetrating i o n i n s i d e j 1  6  1  U n d e r r e s t i n g c o n d i t i o n s the i n t e r i o r of n e r v e a n d m u s c l e f i b r e s i s 6 9 - 7 5 n e g a t i v e w i t h r e s p e c t to t h e o u t s i d e .  mV  T h i s i s c a u s e d b y the s e l e c t i v e p e r m e a -  b i l i t y o f the c e l l m e m b r a n e to t h e f l o w of K~*\  U n d e r t h e s e c o n d i t i o n s the o  m e m b r a n e p o r e s i z e o f the n e u r o n i s i n t e r m e d i a t e (3A) b e t w e e n the h y d r a t e d diameter  of K + (2.2 A) a n d that f o r N a  p e r m e a b l e (164).  +  (3.4 A ) a n d N a  T h e n a t u r e of t h e c h a r g e  +  is practically i m -  on the m e m b r a n e i s a n o t h e r  f a 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 i o n s (164).  28  The  K  c o n c e n t r a t i o n i n s i d e t h e c e l l i s g r e a t e r t h a n that i n the b o d y  f l u i d s outside,  a n d the c o n v e r s e i s t r u e f o r N a * .  Thus,  f o r cat motoneurons  the c o n c e n t r a t i o n of K * i n the n e u r o n s i s 27 t i m e s h i g h e r t h a n that o u t s i d e , w h i l e t h e c o n c e n t r a t i o n f o r Na"*" i s 10 t i m e s h i g h e r  o u t s i d e t h a n i n s i d e (165).  (iii) A c t i o n p o t e n t i a l (164-168)  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 i n an a l l o r none p r o c e s s m e m b r a n e p o t e n t i a l f a l l s t o the t h r e s h o l d l e v e l . i s d e p o l a r i z e d b y a n o u t w a r d f l o w of c u r r e n t ,  Thus,  when the  w h e n the m e m b r a n e  i t s Na"*" p e r m e a b i l i t y  immediately  r i s e s a n d t h e r e i s a n e t i n w a r d m o v e m e n t of N a * i o n s d o w n t h e s o d i u m c o n centration gradient.  If t h e 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 e n o u g h (i.e., t h e  t h r e s h o l d potential for f i r i n g i s reached),  N a * enters f a s t e r than K * c a n  l e a v e a n d t h i s c a u s e s the p o t e n t i a l to d r o p s t i l l f u r t h e r i n c r e a s i n g t h e N a * p e r m e a b i l i t y e v e n m o r e , i n the f o l l o w i n g m a n n e r :  Increase  in Na*  permeability Depolarization of  The  membrane  E n t r y of N a *  e n t r y of s o d i u m i s h a l t e d when the m e m b r a n e p o t e n t i a l r e a c h e s  a l e v e l c l o s e t o (Ejsj ), a  the e q u i l i b r i u m p o t e n t i a l f o r s o d i u m ,  w h e n the n e t  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 b e c o m e s zero.  It i s s t i l l u n c e r -  tain whether N a  H o w e v e r , the  Na"*" (190,  and K*  and K +  traverse  the s a m e c h a n n e l s (166).  c h a n n e l s c a n be d i f f e r e n t i a l l y b l o c k e d ;  193, 194) a n d t e t r a e t h y l a m m o n i u m i o n s (167,  (iv)  e.g., b y t e t r o d o t o x i n 172) r e s p e c t i v e l y .  S o d i u m - P u m p (164-168)  A f t e r the c o n d u c t i o n  of t h e n e r v e i m p u l s e ,  the s o d i u m - p u m p r e -  e s t a b l i s h e s the r e s t i n g p o t e n t i a l b y e j e c t i n g N a * f r o m and r e t u r n i n g K * to  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 a s the s o u r c e of e n e r g y .  p u m p i s b e l i e v e d to be o p e r a t e d b y the N a , K - s t i m u l a t e d +  A T P a s e present  in cell membranes.  Mg  +  T h e s e and other  The sodium+ +  -dependent  a s p e c t s of t h e s o d i u m -  p u m p w i l l b e d e a l t w i t h i n a l a t e r s e c t i o n ( s e c t i o n 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 s y n a p t i c o r n e r v e ending i s achieved, mitters.  it is presently understood,  t h r o u g h the a c t i o n of c h e m i c a l t r a n s -  F o r a s u b s t a n c e to be c o n s i d e r e d  c r i t e r i a should be satisfied.  a transmitter,  It m u s t be s y n t h e s i z e d  the f o l l o w i n g  a n d s t o r e d i n the p r e -  s y n a p t i c 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 i n v e s i c l e s ) .  It s h o u l d be r e l e a s e d  i n a d e q u a t e q u a n t i t i e s o n t h e s t i m u l a t i o n of the p r e s y n a p t i c be  c a p a b l e of i n t e r a c t i n g w i t h r e c e p t o r s  nerves.  o n the p o s t - s y n a p t i c  It m u s t  neuron or  e f f e c t o r c e l l a n d b r i n g a b o u t t r a n s i e n t a l t e r a t i o n s i n the p e r m e a b i l i t y  of t h e  p o s t s y n a p t i c m e m b r a n e t o w a r d s i o n s i n the i m m e d i a t e e n v i r o n m e n t . I t s a c t i o n on post synaptic  structures when applied d i r e c t l y should be identical  w i t h the n o r m a l t r a n s m i t t e r a c t i o n . e n z y m e should be p r e s e n t agents should  I n s o m e c a s e s at l e a s t , a n i n a c t i v a t i n g  i n the s y n a p t i c c l e f t .  Blocking  competitive  affect its n o r m a l action, and its action on d i r e c t application i n  a s i m i l a r manner.  Acetylcholine,  d o p a m i n e and serotonin,  a n d the b i o g e n i c a m i n e s ,  are strongly implicated i n central  (vi) A m i n o a c i d s a s p u t a t i v e t r a n s m i t t e r s  Glutamate,  noradrenaline, transmission.  (164, 165, 168, 170, 233)  and r e l a t e d substances such as aspartate,  c e n t r a l n e u r o n s when i o n t o p h o r e t i c a l l y applied. i n m e t a b o l i s m of c o r t i c a l n e u r o n s , present  and  i n the b r a i n ,  d e s t r u c t i v e enzyme,  excite  many  Since glutamate i s involved  enzymes for its synthesis  are obviously  a n d a l t h o u g h t h e r e i s n o e v i d e n c e f o r the p r e s e n c e of a analogous to c h o l i n e e s t e r a s e  i n the s y n a p t i c c l e f t ,  r a p i d uptake of e x c e s s g l u t a m a t e into n e i g h b o u r i n g n e r v e c e l l s m a y e n s u r e t h e n e c e s s a r y b r e v i t y of i t s t r a n s m i t t e r a c t i o n .  Like  a  s e r v e to  L-glutamate  30  and  L-aspartate,  D L - h o m o c y s t e a t e a n d N - m e t h y l a s p a r t a t e r e d u c e 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 a c i d s (164, 170). G A B A threshold for firing,  to b e e x c i t a t o r y a m i n o  and g l y c i n e a r e i n h i b i t o r y a m i n o acids, r a i s i n g the  b y h y p e r p o l a r i z a t i o n c a u s e d by a n i n c r e a s e i n the p o s t  s y n a p t i c i n t a k e of K * a n d / o r c h l o r i d e (170).  C u r t i s a n d J o h n s t o n (170)  have  r e v i e w e d t h e e v i d e n c e f o r the p a r t i c i p a t i o n of a m i n o a c i d s 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 s y s t e m .  T h e m e c h a n i s m of  a c t i o n of a m i n o a c i d s a s t r a n s m i t t e r s i s n o t k n o w n .  It i s b e l i e v e d that t h e y  c a u s e c h a n g e s i n the i o n i c p e r m e a b i l i t y of t h e p o s t s y n a p t i c m e m b r a n e b y r e a c t i o n s with specific r e c e p t o r  (vii)  C a * * ions and excitation  T h e r e i s a twenty-fold the p r o p a g a t i o n however,  sites.  increase in calcium permeability  of i m p u l s e s along giant n e r v e f i b r e s .  accompanying  T h e i n f l o w of Ca"*"*",  i s t o o s m a l l t o c o n v e y a p p r e c i a b l e c u r r e n t o r to m o d i f y t h e c h a r a c t e r  of the a c t i o n p o t e n t i a l (172).  N e r v e f i b r e s f i r e s p o n t a n e o u s l y w h e n e x t e r n a l Ca"*""*" i s r e d u c e d . versely,  Con-  a r i s e i n e x t e r n a l Ca"*"*" t e n d s to s t a b i l i z e t h e n e r v e m e m b r a n e a n d  to r a i s e the t h r e s h o l d f o r e x c i t a t i o n (166-168).  C a * * may achieve  this by  b l o c k i n g t r a n s m e m b r a n e c h a n n e l s to N a * by b i n d i n g with the m e m b r a n e phospholipid layer;  d i s l o c a t i o n of Ca"*"*" f r o m t h i s b o n d m i g h t t h e n p e r m i t  f r e e p a s s a g e of N a * (174).  ++ Ca  i s k n o w n t o b e r e q u i r e d f o r t r a n s m i t t e r r e l e a s e (173). It  c a u s e s a n i n c r e a s e d r e l e a s e of a c 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 j u n c t i o n . M n * * a n d Mg"*"*" r e d u c e Ca"*"*" e n t r y a n d i n h i b i t t r a n s m i t t e r r e l e a s e (172, (viii)  High K  173).  i o n c o 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"*" c a u s e s d e p o l a r i z a t i o n (177). K"*" i s a p p l i e d s i m u l t a n e o u s l y  However,  o v e r the w h o l e s u r f a c e of a f i b r e ,  i f the h i g h excitability  is usually depressed or abolished. h i g h K+,  produced by  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 a n d g r e a t l y a f f e c t s the p e r m e a -  bility properties  of the n e r v e t i s s u e to s o d i u m a n d p o t a s s i u m i o n s  S o m e t i m e s the a p p l i c a t i o n o f K * pulses.  A maintained depolarization,  Stimulatory  (168).  to e x c i t a b l e c e l l s m a k e t h e m g e n e r a t e  e f f e c t s of t h i s k i n d a r i s e i f t h e a p p l i e d s o l u t i o n s do n o t  p e n e t r a t e u n i f o r m l y i n t o the t i s s u e a n d one p a r t of t h e n e r v e i s d e p o l a r i z e d m u c h m o r e t h a n a n o t h e r (168).  In a d d i t i o n , to i t s d e p o l a r i z i n g a c t i o n on t r a n s m i t t e r  (ix) C e r e b r a l  The  release  cortex  support.  a p p e a r s to h a v e a p o t e n t i a t i n g  (156, 178).  s l i c e s and excitation phenomena  u s e of c e r e b r a l c o r t e x  r a p i d l y gaining  effect, K *  s l i c e s to study e x c i t a t i o n p h e n o m e n a i s  L i a n d M c l l w a i n (179) u s e d m i c r o e l e c t r o d e s to  d e m o n s t r a t e t h e m a i n t e n a n c e of r e s t i n g m e m b r a n e  p o t e n t i a l s of -55 t o -60  in cerebral cortex  s l i c e s of the g u i n e a p i g ,  observed i n vivo.  G i b s o n a n d M c l l w a i n (180) h a v e b e e n a b l e t o c o r r e l a t e  values a p p r o a c h i n g those  potential differences a c r o s s c o r t i c a l cell membranes, the N e r n s t e q u a t i o n f r o m e l e c t r o l y t e a n a l y s i s ,  as calculated  K*  cortical cells a r e depolarized  i n v i t r o by a n i n c r e a s e d  o r t h e 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 ,  r e p o l a r i z e on the r e m o v a l of the a p p l i e d  from  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 i n t o the i n c u b a t e d Moreover,  mV  slices. external  w i t h o u t l o s i n g the a b i l i t y t o  s t i m u l i (180).  Yamamoto  and  M c l l w a i n (181) o b t a i n e d s p i k e p o t e n t i a l s 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, The (265)  tetrodotoxin  w h i c h a r e s a i d to be a k i n to a c t i o n p o t e n t i a l s (224, 264). s e n s i t i v i t y of s u c h e v o k e d p o t e n t i a l s i n c e r e b r a l  lends support to t h i s view.  S i m i l a r l y , c h a n g e s i n the t e t r o d o t o x i n -  s e n s i t i v e i o n i c b a l a n c e of r a t b r a i n c o r t e x m e m b r a n e ) on incubation (182)  slices  s l i c e s ( p r e s u m a b l y at the n e u r o n a l  under c e r t a i n conditions,  led O k a m o t o and Q u a s t e l  to s u g g e s t that a c t i o n p o t e n t i a l s m a y be g e n e r a t e d i n i n c u b a t e d b r a i n  slices.  32  S l i c e s of b r a i n a c t i v e l y a c c u m u l a t e s e r o t o n i n (183), (184),  a c e t y l c h o l i n e (185) a n d a m i n o a c i d s (104-106,  against concentration gradients.  Presumably,  norepinephrine  113, 114, 231, 252, 295)  t h i s m a y be one m e t h o d f o r 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 s u b s t a n c e f r o m t h e i r s i t e of a c t i o n .  Many  s t u d i e s h a v e d e m o n s t r a t e d that the s t i m u l a t i o n of b r a i n e l e c t r i c a l l y o r chemically  (e.g.,  w i t h 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 m e m b r a n e a n d  m a r k e d l y e n h a n c e s the r e l e a s e of l a b e l f r o m t h e b r a i n s l i c e p r e - l o a d e d l a b e l t r a n s m i t t e r s u b s t r a t e (e.g.,  186, 187).  t h a t 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 c o n d i t i o n s o f i n c u b a t i o n (182),  showed  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  concomitantly  a m i n o a c i d s f r o m the t i s s u e (188).  O u r e a r l i e r studies  with  a f f e c t s the r e l e a s e of e n d o g e n o u s  T h e s e and later studies a r e d e s c r i b e d i n  detail i nthis thesis.  The  a b o v e c o n s i d e r a t i o n s (and o t h e r s t h a t w i l l be a p p a r e n t i n d u e  course) support  the v a l i d i t y of e m p l o y i n g t h e i s o l a t e d b r a i n s l i c e f o r t h e  s t u d y of n e r v e f u n c t i o n a n d m e t a b o l i s m . use  of b r a i n s l i c e s " ,  Elliot,  has d e s c r i b e d the usefulness  i n t h e d i s c o v e r y a n d e l u c i d a t i o n of m e t a b o l i c  1.9  i n h i s r e v i e w (234)  o n "the  of the t i s s u e s l i c e t e c h n i q u e  processes.  S o m e p r o p e r t i e s of d r u g s a n d m e t a b o l i c  inhibitors used as tools  for this i n v e s t i g a t i o n  O n l y a b r i e f a c c o u n t of t h e p r o p e r t i e s of s o m e of t h e c h e m i c a l stances  u s e d a s t o o l s i n t h e s e s t u d i e s w i l l be p r e s e n t e d  (i)  here.  Tetrodotoxin (TTX)  Tetrodotoxin,  a potent n o n - p r o t e i n  f i s h a n d C a l i f o r n i a n newts, the g e n e r a t i o n myelinated  sub-  neurotoxin  found i n J a p a n e s e puffer  s u p p r e s s e s at c o n c e n t r a t i o n s  a s l o w a s 0.3 ( i m o l a r  o f a c t i o n p o t e n t i a l s 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  n e r v e f i b r e s and l o b s t e r and squid giant axons,  a s s o c i a t e d i n f l u x of N a  +  i o n s (190, 193, 194).  and hence the  I t s m e c h a n i s m of a c t i o n  33  r e s e m b l e s that of l o c a l a n e s t h e t i c s b u t i t h a s a p o t e n c y m o r e t h a n a 100,000 t i m e s t h a t of c o c a i n e  (189).  s u r f a c e of the m e m b r a n e  It a c t s o n t h e outer,  s e l e c t i v e l y b l o c k i n g t h e i n w a r d m o v e m e n t s of N a *  i o n s a c c o m p a n y i n g the g e n e r a t i o n e f f e c t on K *  r a t h e r t h a n the i n t e r n a l ,  of a c t i o n p o t e n t i a l s .  i o n m o v e m e n t s (195, 196).  E a r l i e r s t u d i e s of T T X  c o n c e r n e d with i t s c h e m i s t r y ,  (in i n v i v o s t u d i e s ) a n d i t s e l e c t r o p h y s i o l o g i c a l e f f e c t s and  It s e e m s to h a v e no  pharmacology  using isolated nerve  m u s c l e h a v e b e e n r e v i e w e d by a n u m b e r of w o r k e r s (189-192).  Only  r e c e n t l y h a s T T X b e e n u s e d i n the s t u d y of m e t a b o l i s m a n d c a t i o n i c t r a n s p o r t processes  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 a n d h i s c o - w o r k e r s ,  working  m a i n l y w i t h r a t b r a i n (182, 188, 192, 197, 198, 205) a n d b y M c l l w a i n ' s g r o u p u s i n g c h i e f l y the b r a i n of the g u i n e a p i g (199, 201-204).  While  h a s n o e f f e c t o n the r a t e 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 in a physiological glucose  saline medium,  it c o m p l e t e l y  TTX  incubated  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 d u e to t h e a p p l i c a t i o n o f e l e c t r i c a l i m p u l s e s (197, 199) o r 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), i n c u b a t i o n m e d i u m (197).  o r when c a l c i u m ions a r e o m i t t e d  f r o m the  H o w e v e r , i t h a s no e f f e c t o n the p o t a s s i u m  s t i m u 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) o r 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 slices.  The K*  c o n t e n t of i n c u b a t e d  i n f l u x of Na"*"* i n b r a i n  electrically stimulated slices is greater  i n t h e p r e s e n c e of T T X t h a n i n i t s a b s e n c e (199, 200). effect with p r o t o v e r a t r i n e stimulated  Chan and Quastel  cortex  slices  TTX  has a s i m i l a r  (182).  (198) s h o w e d that the i n h i b i t i o n of a c e t a t 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 s c o m p l e t e l y  s u p p r e s s e d by T T X .  oxidation They con-  c l u d e d that T T X e x e r t s i t s a c t i o n o n a c e t a t e m e t a b o l i s m i n d i r e c t l y b y i t s effects on N a *  movement.  It i s w e l l k n o w n that L i - g l u t a m a t e e x c i t e s n e r v o u s t i s s u e ( s e c t i o n 1.8  34  (vii) ).  A n i n f l u x of N a  p l a c e when 5 m M (95,  i o n s into incubated c e r e b r a l c o r t e x s l i c e s  s o d i u m L - g l u t a m a t e i s a d d e d to the i n c u b a t i o n  182, 201, 263).  T h i s i s only p a r t l y i n h i b i t e d by T T X  s h o r t i n i t i a l p e r i o d of i n c u b a t i o n (202). (203),  According  l o w c o n c e n t r a t i o n s of T T X a r e c a p a b l e  i n the p r e s e n c e o r a b s e n c e of L - g l u t a m a t e . diminution in Ca  e f f l u x (203).  to R a m s a y a n d  of i n h i b i t i n g C a  TTX  Mcllwain  influx both  It a l s o c a u s e s a d e t e c t a b l e  a b o u t i n c r e a s e s i n the i n f l u x of N a * into, a n d e f f l u x of K *  by  medium  (182) d u r i n g a  C h e l a t i n g a g e n t s , e.g., 5 m M  g u i n e a p i g c e r e b r a l cortex s l i c e s and these  takes  EDTA  from,  bring  incubated  changes a r e p a r t i a l l y  prevented  (204).  R e c e n t e x p e r i m e n t s of O k a m o t o and Q u a s t e l  (182) h a v e s h o w n t h a t  u n d e r a v a r i e t y of i n c u b a t i o n c o n d i t i o n s , the i n c r e a s e of N a +  i n f l u x a n d of  w a t e r u p t a k e t h a t take p l a c e i n r a t b r a i n c o r t e x s l i c e s a r e s u p p r e s s e d e i t h e r wholly  o r p a r t i a l l y b y s m a l l c o n c e n t r a t i o n s of T T X  (3 |JM). T h e i r  experiments  l e d t o t h e c o n c l u s i o n that a c t i o n p o t e n t i a l s , o r a c t i v a t i o n of t h e N a * are generated in brain cortex slices in vitro. the a b s e n c e of g l u c o s e ,  This occurs  o r i n t h e p r e s e n c e o f 0.1 m M  current  f o r example, i n  o u a b a i n o r of 10 (-1 M  p r o t o v e r a t r i n e o r on t h e 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 o r a c c o r d i n g to S h a n k a r a n d Q u a s t e l (192, 20 5) at the o n s e t of a n o x i a .  Under these  TTX  H o w e v e r , T T X h a s no  a f f e c t s b o t h the c a t i o n i c f l u x e s a n d w a t e r u p t a k e .  conditions  e f f e c t o n t h e e n h a n c e d N a * i n f l u x a n d w a t e r u p t a k e i n t h e p r e s e n c e of 30 (JM 2, 4 - d i n i t r o p h e n o l o r of 100 m M  Recently,  KC1  S h a n k a r and Q u a s t e l  e n h a n c e s t h e r a t e of a n a e r o b i c  (192, 20 5) h a v e s h o w n that T T X (2 |jM)  glycolysis i n rat c e r e b r a l cortex slices  a d d e d b e f o r e t h e o n s e t of a n o x i a . on a n a e r o b i c  (182).  when  T h e y a l s o s h o w e d that the e f f e c t s of T T X  glycolysis a r e specific for mature c e r e b r a l tissue and require  the i n t e g r i t y of the b r a i n c e l l f o r i t s a c t i o n . on a n a e r o b i c  glycolysis is indirect.  efflux from,  c e r e b r a l c o r t e x s l i c e s that o c c u r  T h e s t i m u l a t o r y e f f e c t of T T X  It s u p p r e s s e s N a * i n f l u x into,  and K *  at the o n s e t of a n o x i a .  e n h a n c e d K * / N a * c o n c e n t r a t i o n r a t i o a c t i v a t e s the r a t e l i m i t i n g  The  pyruvate  35  k i n a s e t h e r e b y s t i m u l a t i n g t h e r a t e of a n a e r o b i c TTX  on a n a e r o b i c  glycolysis.  T h e e f f e c t of  g l y c o l y s i s i s r e d u c e d o r a b o l i s h e d i n the p r e s e n c e o f  protoveratrine, high K ,  NH^"** o r L - g l u t a m a t e  +  In s t u d i e s r e p o r t e d h e r e ,  (192).  T T X w i l l be u s e d to inhibit N a - m o v e m e n t +  into b r a i n cortex s l i c e s when these a r e incubated  i n s u c h a m a n n e r a s to  b r i n g a b o u t t h 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 t h e c e l l m e m b r a n e o r t h e generation  (ii)  of a c t i o n p o t e n t i a l s .  L o c a l a n e s t h e t i c s (166, 176)  L o c a l a n e s t h e t i c s r e v e r s i b l y b l o c k the n e r v e i m p u l s e by r e d u c i n g the m e m b r a n e p e r m e a b i l i t y to s o d i u m ions. d e c r e a s e s anesthetic action. m e m b r a n e at c o n c e n t r a t i o n s  Increased  extracellular sodium  M o s t l o c a l a n e s t h e t i c s w i l l d e p o l a r i z e the higher than a r e needed for blocking action, and  w i l l a l s o s l i g h t l y r e d u c e the p o t a s s i u m p e r m e a b i l i t y of the m e m b r a n e . L i d o c a i n e i s s a i d t o be a h u n d r e d t i m e s m o r e e f f e c t i v e o n Na"*" c h a n n e l s t h a n on K *  channels  (176).  L o c a l anesthetics behave like C a  + +  ,  s t a b i l i z i n g the n e r v e  membrane,  r a i s i n g the t h r e s h o l d f o r e x c i t a t i o n , a b o l i s h i n g s p o n t a n e o u s a c t i v i t y , a n d blocking 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)  s t a t e s that l o c a l  a n e s t h e t i c s m a y a c t p r i m a r i l y b y i n h i b i t i n g r e l e a s e of Ca"*"** f r o m s i t e s t o w h i c h i t i s b o u n d to the m e m b r a n e .  They thereby prevent secondary  changes  i n the Na"*" a n d K * p e r m e a b i l i t i e s , a n d c o n s e q u e n t l y s u p p r e s s t h e g e n e r a t i o n and  propagation  o f the n e r v e i m p u l s e .  The suggestion  has been made that  b o t h l o c a l a n e s t h e t i c s a n d Ca*"** a c t 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 * t h r o u g h the m e m b r a n e . procaine  effects,  F o r example,  whereas procaine  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  completely  c a u s e d b y r e m o v a l of e x t e r n a l Ca"*"*" (167,  reverses  the d e p o l a r i z a t i o n  176).  L o c a l a n e s t h e t i c s a r e s a i d to c o m p e t e w i t h a c e t y l c h o l i n e f o r r e c e p t o r s i t e s o n the p o s t - s y n a p t i c  membrane  (176).  36  The  r e s u l t s d e s c r i b e d above have been obtained  e m p l o y i n g i s o l a t e d n e r v e fibres-.  However,  studies have a l s o been c a r r i e d  out w i t h b r a i n c o r t e x s l i c e s w i t h s i m i l a r r e s u l t s . (198)  s h o w e d that c o c a i n e  (1 and  3 mM)  (0.2 mM),  b l o c k the i n c r e a s e d i n f l u x of N a *  o r the a c t i v i t y o f m e m b r a n e Na*,  Thus,  l i d o c a i n e (0.5 a n d  w i t h o u t a f f e c t i n g e i t h e r the c o n t e n t  chiefly in studies  due  C h a n and  1 mM),  and  procaine  to e l e c t r i c a l s t i m u l a t i o n  of Na"*" f o u n d i n u n s t i m u l a t e d  K*-ATPase.  Quastel  b r a i n tissue,  These local anesthetics  were  a l s o f o u n d to a b o l i s h the d e p r e s s e d o x i d a t i o n of ( 1 - ^ C ) a c e t a t e to ^^CO^, a n d the e n h a n c e d o x y g e n u p t a k e b r o u g h t a b o u t b y the a p p l i c a t i o n of e l e c t r i c a l pulses.  S u c h e f f e c t s w e r e not o b t a i n e d  concentrations low  studied.  concentrations  w i t h the u n s t i m u l a t e d  T h i s l e d to the c o n c l u s i o n t h a t l o c a l a n e s t h e t i c s at  a c t on s o d i u m c h a n n e l s that b e c o m e a v a i l a b l e f o l l o w i n g  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  r e s p i r a t i o n p r e s u m a b l y b y b l o c k i n g the i n f l u x of Na"*" s t i m u l a t e d a c t i v i t y of the N a * suppression  r e s p i r a t i o n . Thus,  e f f e c t s on m e t a b o l i s m i n d i r e c t l y ,  of A D P  like TTX,  stimulated  ions and t h e r e b y  sensitive membrane A T P a s e .  of the r a t e of f o r m a t i o n  mitochondrial  s l i c e s at the  the  T h i s l e a d s to  that p a r t l y c o n t r o l s the r a t e of local anesthetics exert their  by t h e i r a c t i o n on c a t i o n i c m o v e m e n t s i n  stimulated nervous tissue.  In s t u d i e s r e p o r t e d be r e , l i d o c a i n e (0.5 mM)  w i l l be u s e d as a  s e n t a t i v e of t h e l o c a l a n e s t h e t i c g r o u p of d r u g s , f o r s u p p r e s s i n g of the N a *  current s y s t e m generated i n b r a i n slices incubated  repre-  the a c t i v a t i o n  under  specific  conditions.  (iii) P r o t o v e r a t r i n e  It i s w e l l k n o w n t h a t v e r a t r i n e a l k a l o i d s , of w h i c h p r o t o v e r a t r i n e i s a member,  g e n e r a t e a c t i o n p o t e n t i a l s i n n e r v o u s t i s s u e (167,  s h o w n that i n r e s t i n g n e r v e , i o n s and  207).  Shanes  v e r a t r i n e a l k a l o i d s c a u s e a n e t r e l e a s e of  an u p t a k e of an e q u i v a l e n t a m o u n t of Na"*" i o n s (208).  m o d e of a c t i o n of v e r a t r i n e d r u g s i s not y e t c l e a r .  However,  has  K* the  T h e r e is some evidence  37  s u g g e s t i n g that t h e b a s i c e f f e c t of v e r a t r i n e c o m p o u n d s i s a d i s p l a c e m e n t of Ca*"*" i o n s f r o m the c e l l s u r f a c e (209). t h u s a f f e c t i n g t h e s t a b i l i t y a n d c o n s e q u e n t l y the p e r m e a b i l i t y of t h e e x c i t a b l e c e l l m e m b r a n e to the f l o w of i o n s .  Wollenberger  (211) s h o w e d that p r o t o v e r a t r i n e at l o w c o n c e n t r a t i o n s  (about 2 |iM) s t i m u l a t e s r e s p i r a t i o n a n d a e r o b i c g l y c o l y s i s ,  and inhibits  anaerobic g l y c o l y s i s i n guinea pig b r a i n cortex s l i c e s incubated i n a physiological glucose saline medium.  U n d e r these c o n d i t i o n s it a l s o b r i n g s about  a T T X - s e n s i t i v e i n f l u x of N a * i o n s into, c e r e b r a l c o r t e x s l i c e s (182, 210).  a n d e f f l u x of K * i o n s f r o m , r a t  Such cationic fluxes a r e a c c o m p a n i e d by  an e n h a n c e d o x y g e n c o n s u m p t i o n (182).  T h e e n h a n c e d u p t a k e of o x y g e n due  to p r o t o v e r a t r i n e i s i n h i b i t e d b y T T X (182),  c o c a i n e (213) o r m a l o n a t e  (226).  P r o t o v e r a t r i n e m a y a l s o s t i m u l a t e the i n c r e a s e d r e s p i r a t i o n d u e t o e l e c t r i c a l s t i m u l a t i o n (212). GABA  It i n c r e a s e s y i e l d s o f l a b e l l e d g l u t a m a t e ,  glutamine,  and aspartate f r o m labelled glucose i n incubated b r a i n slices, and  these effects a r e r e v e r s e d by cocaine  As  (213).  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  r e s e m b l i n g t h o s e of e l e c t r i c a l i m p u l s e s  (211,  closely  182), a n d a s i t i s k n o w n to  g e n e r a t e a c t i o n p o t e n t i a l s i n n e r v e t i s s u e (167,  207), i t w i l l be u s e d  exten-  s i v e l y i n studies r e p o r t e d i n 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 a r e at p r e s e n t two s c h o o l s of t h o u g h t c o n c e r n i n g t h e m o d e of a c t i o n of b a r b i t u r a t e s o n n e r v e f u n c t i o n .  A c c o r d i n g to one g r o u p of w o r k e r s ,  b a r b i t u r a t e s e x e r t t h e i r effects by " d i m i n i s h i n g i o n i c m o v e m e n t s of e x c i t a t i o n " (214), o r " o n m e m b r a n e p a r a m e t e r s " inward  s o d i u m l e a k a g e " (216).  However,  (215),  or by "cutting down  a n o t h e r g r o u p of w o r k e r s h a v e  o b t a i n e d r e s u l t s n o t c l e a r l y e x p l i c a b l e by s u c h s t a t e m e n t s . turates amytal  (0.25 a n d 0.5 m M )  With the b a r b i -  a n d p e n t o t h a l (0.1 a n d 0.2 m M )  t i o n s that d o n o t a f f e c t the c o n t e n t of ^ 2 j ^ + a  o  r  ^  n e  at c o n c e n t r a -  r e s p i r a t i o n of u n s t i m u l a t e d  38  rat b r a i n cortex slices incubated  f o r one  hour,  C h a n and Q u a s t e l  (198)  found  no a b o l i t i o n of the e n h a n c e d i n f l u x of 22j\f + f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n . a  Hillman  et a l . (217)  the N a *  i n f l u x o b t a i n e d 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 g u i n e a  pig  s i m i l a r l y f o u n d that p h e n o b a r b i t a l  c e r e b r a l cortex slices.  experimental TTX  b l o c k the i n c r e a s e d i n f l u x of 22]\j + that o c c u r s  respiration.  L i k e l o c a l anesthetics or T T X ,  nevertheless  K -ATPase  +  +  However,  the e f f l u x of K  due  +  b r o u g h t a b o u t by the i n f l u x  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  i n c o n t r a s t to the l o c a l a n e s t h e t i c s a n d  t h e r a t e of ( l - ^ C ) a c e t a t e o x i d a t i o n to ^^CO^ slices.  C h a n and Q u a s t e l  t r a t i o n s q u o t e d and  TTX,  the b a r b i t u r a t e s i n h i b i t  in unstimulated  rat brain  u n d e r the i n c u b a t i o n c o n d i t i o n s s t u d i e d ) do not  suppressing  (198).  (198) h a v e c o n c l u d e d that b a r b i t u r a t e s (at the c o n c e n -  a f f e c t the m o v e m e n t of N a + t h e y a c t by  a f f e c t s the a c t i v i t y  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 o x i d a t i o n of ( 1 - ^ C ) a c e t a t e to I^QO and  brain  (198).  U n l i k e the l o c a l a n e s t h e t i c s o r T T X ,  of N a *  the  barbiturates  suppress electrically stimulated  N o n e of t h e s e d r u g s at t h e s e c o n c e n t r a t i o n s  of m e m b r a n e b o u n d N a ,  given  on  a  small concentrations  on  d i f f e r e n t f r o m t h o s e of l o c a l a n e s t h e t i c s o r of  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 . at  without effect  T h u s , the e f f e c t s of b a r b i t u r a t e s u n d e r the  conditions were  which completely  was  and  K*  directly  a c r o s s the b r a i n c e l l m e m b r a n e ;  cell energetics.  Such suppressing  rather,  action also  e x p l a i n s the i n h i b i t o r y e f f e c t s 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 b r o u g h t a b o u t 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 the p r e s e n c e of h i g h K~*~  concentrations  m e d i u m d e v o i d of C a * (210). +  M i c h a e l i s and barbiturate,  Quastel amytal,  (219),  (218),  or w i t h p r o t o v e r a t r i n e ,  and E r n s t e r and  c o - w o r k e r s (220,  as w e l l as c e r t a i n o t h e r h y p n o t i c s  In v i e w of the g r e a t a m o u n t of w o r k w h i c h h a s  at l o w of A T P  221),  that the  concentrations, i n the  b e e n c a r r i e d out  b o t h i n 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 s t u d i e s ,  u s e d i n the s t u d i e s r e p o r t e d  or i n a  It i s w e l l k n o w n f o l l o w i n g the w o r k of  s u p p r e s s the o x i d a t i o n of N A D H and h e n c e the g e n e r a t i o n  amytal,  by  it h a s  cell.  with been  i n t h i s 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 w o r k on the t r a n s p o r t a n d m e t a b o l i s m of a m i n o a c i d s a n d a m m o n i a i n b r a i n slices incubated  u n d e r c o n d i t i o n s l e a d i n g to the a c t i v a t i o n of t h e  s o d i u m c u r r e n t o r to t h e g e n e r a t i o n  of action potentials.  (v)  Cardiac  glycosides - Ouabain  The  process  w h e r e b y N a * i s e x t r u d e d f r o m the c e l l a n d  accumu-  l a t e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t i s u s u a l l y r e f e r r e d to a s t h e " S o d i u m Pump".  This c a r r i e r mediated process  excitable membranes,  m a i n t a i n s the p o l a r i t y of n o n -  a n d r e s t o r e s that of e x c i t a b l e o n e s w h i c h h a v e u n d e r -  gone d e p o l a r i z a t i o n , d u r i n g the g e n e r a t i o n is achieved bolism.  a t the e x p e n s e of m e t a b o l i c  of n e r v e i m p u l s e s .  energy derived f r o m aerobic  T h u s , i n h i b i t i o n of A T P f o r m a t i o n b y a n o x i a ,  the a b s e n c e of g l u c o s e , concentration Moreover,  Repolarization  cyanide,  meta-  DNP,  or in  c a u s e s a r e d i s t r i b u t i o n of c a t i o n s e l i m i n a t i n g t h e i r  g r a d i e n t s (104,  10 5).  A c t i v e u p t a k e of K * i s N a * d e p e n d e n t .  t h e e f f e c t s of i n c r e a s e d K * o n t h e m e t a b o l i s m of i n t a c t 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 t h e p r e s e n c e of N a * (104,  Z18). T h u s ,  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 i n f l u e n c e d b y t h e o p e r a t i o n .of the s o d i u m - p u m p w h i c h c o n t r o l s t h e c e l l u l a r l e v e l s of N a * a n d K*. known, f o r e x a m p l e ,  that i n c r e a s e d N a * s u p p r e s s e s a c e t a t e o x i d a t i o n b y i t s  i n h i b i t o r y effect on acetate c o n v e r s i o n K * enhances pyruvate kinase acetate  t o a c e t y l C o A (198),  a c t i v i t y (192,  a n d that i n c r e a s e d  262, 299, 300) a n d t h e o x i d a t i o n of  (198).  Skou  (222, 223) r e p o r t e d t h a t a m e m b r a n e b o u n d e n z y m e  that h y d r o l y z e s  A T P to A D P a n d P i , r e q u i r e s N a * a n d K * f o r  d e p e n d e n t o n the p r e s e n c e of M g  + +  .  many features i n common,  namely,  a c t i v a t i o n and i s  the m e m b r a n e ,  t h e i r l o c a t i o n a t the c e l l m e m b r a n e ,  a c t i v a t i o n b y the s i m u l t a n e o u s p r e s e n c e of b o t h N a * a n d K * energy,  (ATPase)  T h i s N a * K * - a c t i v a t e d M g * * dependent  A T P a s e a n d the a c t i v e t r a n s p o r t o f N a * a n d K * a c r o s s  for  It i s  a n d i n h i b i t i o n b y o u a b a i n (104,  105,  223).  their  have their  requirement  40  O u a b a i n i s h e l d to a c t (see r e f e r e n c e s 115, 262) b y i n h i b i t i n g t h e R e activated dephosphorylation ATP  En  ( R e a c t i o n 11) of a n i n t e r m e d i a t e f o r m e d  a n d the e n z y m e A T P a s e (En) i n the p r e s e n c e of N a  + A T P  +  ( E n — P ) Na-  Na -  >  +  in  K  +  o  u  t  >En  ( E n ~ P ) Na  +  Pi + Na  (Reaction  +  from 10).  + ADP  (10)  + K+ in  +  out  (11)  M u c h of the c e l l u l a r e n e r g y i s s a i d t o i n v o l v e the o p e r a t i o n of the s o d i u m - p u m p (see r e f e r e n c e s 104, 224). A D P r e l e a s e d b y the a c t i o n of the Na*, tion.  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 e n e r g y production by enhancing T h i s i t d o e s t h r o u g h the r e a c t i o n :  ATP.  However,  NADH  i n the p r e s e n c e of ouabain,  + ADP  + Pi  respira-  > NAD  +  +  when both c a t i o n t r a n s p o r t  a n d 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 w i t h i t a r e i n h i b i t e d ,  there i s not  n e c e s s a r i l y a c o n c o m i t a n t d i m i n u t i o n i n the r e s p i r a t i o n 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 (112,  182, 224). I n h i b i t i o n m a y be o b s e r v e d  l e n g t h y d u r a t i o n s (as o b s e r v e d  i n i n c u b a t i o n s of  i n o u r s t u d i e s ) . In a n e x p e r i m e n t of one h o u r  t h e r e i s o n l y a s m a l l d r o p i n 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 b a t e d i n p r e s e n c e of 0.1 m M  o u a b a i n (115).  Presumably,  when h i g h energy  l e v e l s f a l l b e l o w 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 o u a b a i n takes p l a c e (225).  This view is supported  b y the o b s e r v a t i o n s that o u a b a i n  ++ suppresses  the s t i m u l a t e d r e s p i r a t i o n d u e to the a b s e n c e of C a  p r e s e n c e of h i g h  (224) o r t h e  (112) o r 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)  a n d i n o u r s t u d i e s i n 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  c o n d i t i o n s a r e k n o w n to b r i n g a b o u t a f a l l i n the c e l l u l a r l e v e l s of h i g h  energy  compounds. T o w e r (224) h a s s h o w n that, i n a m e d i u m d e v o i d of Ca"*~ , t h e c a l c i u m +  c o n t e n t of c a t c o r t e x s l i c e s i n c u b a t e d f o r o n e h o u r f a l l s to 30 p e r c e n t of the c o n t r o l l e v e l s whether or not ouabain is present. however,  In the p r e s e n c e of C a  ,  o u a b a i n s i g n i f i c a n t l y e n h a n c e s the t i s s u e l e v e l of Ca"*"*" b y 36 p e r  cent — w h i c h p r i m a r i l y o c c u r s i n the m i t o c h o n d r i a l c o m p a r t m e n t .  This could  41  be due  e i t h e r to a n i n c r e a s e d  m e m b r a n e p e r m e a b i l i t y to C a  or  t i o n of the a c t i v e t r a n s p o r t m e c h a n i s m f o r the e x t r u s i o n of  to an  inhibi-  calcium.  It i s a w e l l k n o w n f a c t that o u a b a i n i n h i b i t s the a c t i v e t r a n s p o r t of m a n y c o m p o u n d s of b i o l o g i c a l i n t e r e s t (104,  10 5).  It h a s b e e n m e n t i o n e d  that o u a b a i n i n h i b i t s the a c c u m u l a t i o n of a m i n o a c i d s a g a i n s t gradients  ( s e c t i o n 1.6).  concentration  T h i s i s so b e c a u s e a c t i v e t r a n s p o r t of  materials  across  m e m b r a n e s i s a p r o c e s s d e p e n d e n t on the o p e r a t i o n of the  (104).  One  lack,  already  sodium-pump  r e s u l t of the i n h i b i t i o n of a c t i v e t r a n s p o r t by o u a b a i n o r by  i s l e a k a g e of a m i n o a c i d s a n d  other  Na*  s u b s t a n c e s f r o m the b r a i n (104,  112,  280).  The  s o d i u m i n f l u x and the w a t e r u p t a k e that o c c u r i n b r a i n  s l i c e s of the r a t i n c u b a t e d pressed  by T T X  under these  (182). A  i n the p r e s e n c e of 0.1  mM  c o n c o m i t a n t r e t e n t i o n of K *  cortex  ouabain is p a r t l y supmay  occur with  TTX  conditions.  In the w o r k p r e s e n t e d i n t h i s t h e s i s o u a b a i n i s m a i n l y u s e d (a)  to t h r o w l i g h t on the c o n t r o l m e c h a n i s m f o r g l u t a m i n e  synthesis  i ^ b r a i n c o r t e x s l i c e s ( C h a p t e r 4); (b)  to study t r a n s p o r t p r o c e s s e s of a m m o n i a a n d i s o l a t e d b r a i n ( C h a p t e r s 5 and  (c)  6);  amino acids in  and  to b l o c k the r e - u p t a k e p r o c e s s of a m i n o a c i d s that a r e f r o m b r a i n slices incubated generation  u n d e r c o n d i t i o n s l e a d i n g to the  of a c t i o n p o t e n t i a l s o r the a c t i v a t i o n o f s o d i u m  c u r r e n t at the b r a i n c e l l m e m b r a n e ( C h a p t e r s 7 and  (vi)  8).  Miscellaneous  A d d i t i o n a l m e t a b o l i c i n h i b i t o r s h a v e a l s o b e e n u s e d i n the reported  released  in this thesis.  studies  42  (a)  (b)  (c)  Malonate,  w e l l k n o w n to i n h i b i t the o p e r a t i o n of the c i t r i c  c y c l e by c o m p e t i t i o n at the s u c c i n i c d e h y d r o g e n a s e s t a g e  (228).  F l u o r o a c e t a t e , w h i c h f o r m s f l u o r o c i t r ate a n d i n h i b i t s the  opera-  t i o n of the c i t r i c a c i d c y c l e b y i n h i b i t i o n of a c o n i t a s e (63,  150).  Methionine  s u l f o x i m i n e , w h i c h i n h i b i t s the s y n t h e s i s of  b y c o m p e t i t i v e i n h i b i t i o n of g l u t a m i n e (d)  acid  A m i n o oxyacetate,  synthetase  (15,  which inhibits enzyme systems  p y r i d o x a l d e r i v a t i v e s as c o - e n z y m e s .  glutamine  65,  146).  requiring  It i s u s e d i n t h e s e s t u d i e s  m a i n l y f o r s u p p r e s s i n g the c o n v e r s i o n of g l u t a m a t e to a s p a r t a t e b y t r a n s a m i n a t i o n (147, (e)  2, 4 - D i n i t r o p h e n o l , phorylation,  (f)  279.  304).  w e l l k n o w n to u n c o u p l e o x i d a t i o n f r o m p h o s -  and w h i c h l e a d s to l o w A T P  levels  (115).  Ethane d i o x y b i s (ethylamine) t e t r a acetate ( E G T A ) ,  which  speci-  f i c a l l y c h e l a t e s c a l c i u m i o n s and i s u s e d f o r c o m p l e t e r e m o v a l of C a * * f r o m the i n c u b a t i o n m e d i u m (306,  1.10  O b j e c t i v e s of the p r e s e n t  The  aim  on the p r o c e s s e s  307).  work  of the p r e s e n t i n v e s t i g a t i o n h a s b e e n to t h r o w f u r t h e r l i g h t c o n t r o l l i n g a m m o n i a f o r m a t i o n and a c c u m u l a t i o n  a n d i t s e f f e c t s on b r a i n m e t a b o l i s m ,  on the p r o c e s s e s  concerned  in b r a i n with a m i n o  a c i d f l u x e s in b r a i n under v a r i o u s c o n d i t i o n s a s s o c i a t e d with i n c r e a s e d activity,  a n d on the s p e c i f i c l o c a t i o n s of a m i n o a c i d s i n the b r a i n .  nerve  43  2.  2.1  M A T E R I A L S AND  METHODS  Animals  A d u l t r a t s ( u s u a l l y m a l e ) of the W i s t a r s t r a i n ,  w e i g h i n g 150-200 g,  w e r e u s e d a n d w e r e o b t a i n e d f r o m the V i v a r i u m , D e p a r t m e n t of Z o o l o g y , f r o m the A n i m a l Unit, In  F a c u l t y of M e d i c i n e , U n i v e r s i t y of B r i t i s h  or  Columbia.  s o m e e x p e r i m e n t s 2 - d a y o l d i n f a n t r a t s of the s a m e s t r a i n w e r e u s e d . A l l  a n i m a l s h a d f r e e a c c e s s to f o o d and w a t e r .  Infant r a t s w e r e s e p a r a t e d f r o m  t h e i r m o t h e r s b e f o r e t h e s t a r t of the e x p e r i m e n t .  2.2  Chemicals  A l l c o m m o n l a b o r a t o r y c h e m i c a l s w e r e of " r e a g e n t g r a d e " a n d  were  used without further p u r i f i c a t i o n .  S o d i u m ( U - ^ C ) g l u t a m a t e was Illinois,  U.S.A., and ^ N a C l  A m e r sham,  was  obtained f r o m Volk R a d i o c h e m i c a l  o b t a i n e d f r o m the R a d i o c h e m i c a l C e n t r e ,  England.  Glucose-6-phosphate  d e h y d r o g e n a s e ( E . C. 1.1.1.49) ( f r o m y e a s t ) ,  h e x o k i n a s e (E. C.2.7.1.1.) ( f r o m y e a s t ) w e r e o b t a i n e d f r o m Angeles,  Co.,  Calbiochem,  and Los  California.  T e t r o d o t o x i n and s o d i u m fluor oacetate w e r e obtained f r o m 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 ; Montreal;  Calbiochem;  amytal f r o m E l i L i l l y  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,  Co.,  N.Y.;  o u a b a i n 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 C o p o r a t i o n , C l e v e l a n d , Ohio; a m i n o o x y a c e t i c a c i d f r o m E a s t m a n K o d a k Co., f r o m M a t h e s o n C o l e m a n and B e l l , Light, from  Colnbrook,  R o c h e s t e r , N.Y.;  N o r w o o d , N.J.;  Buckinghamshire,  U.K.;  E G T A f r o m K o c h and  DL-methionine-DL-sulfoximine  California Coporation for B i o c h e m i c a l Research,  triethanolamine and N A D P  +  from  and t r i s A T P C a l b i o c h em.  disodium malonate  Los Angeles;  f r o m S i g m a C h e m i c a l Co.,  St. L o u i s ,  Mo., ;  44  2.3  Tissue preparation  R a t s w e r e k i l l e d by d e c a p i t a t i o n . T h e c e r e b r a l cortex slices were prepared some experiments, manner. total  One  using a Stadie-Riggs  The  (to g i v e the i n i t i a l wet  in a  In  similar  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 ) w e i g h i n g a  i n i t i a l wet  used for incubation.  and  tissue s l i c e r .  rat kidney cortex slices were prepared  d o r s a l and  80-100 mg  brains were removed  w e i g h t a n d n o t m o r e t h a n 0.4  mm  thick,  s l i c e s w e r e q u i c k l y w e i g h e d on a t o r s i o n  weight) and  were balance  suspended in chilled m a n o m e t r i c v e s s e l s  c o n t a i n i n g the a p p r o p r i a t e i n c u b a t i o n m e d i a .  I n f a n t ( 2 - d a y o l d rat) b r a i n c o r t e x s l i c e s w e r e p r e p a r e d  b y c u t t i n g by  hand, o n l y 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 e a c h h e m i s p h e r e used. to.l  As  mm  the i n f a n t b r a i n i s s m a l l , i t was  thick.  According  found  e x p e d i e n t to use  to Itoh and Q u a s t e l (23 5),  being  slices  up  the r a t e s of o x y g e n con-  s u m p t i o n or 1 C 0 2 f o r m a t i o n f r o m l C - l a b e l l e d s u b s t r a t e s by infant r a t 4  4  b r a i n c o r t e x s l i c e s a r e n o t s i g n i f i c a n t l y l o w e r w i t h t h i c k n e s s of 0.9-1 t h a n t h o s e w i t h t h i c k n e s s e s of 0.4  - 0.5  mm,  mm  w h i c h i s p r o b a b l y l a r g e l y due  to  the f a c t t h a t the r a t e of r e s p i r a t i o n i n i n f a n t 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 l o w to a l l o w a d e q u a t e o x y g e n a t i o n thick  2.4  of the b r a i n c e l l s e v e n w i t h s l i c e s 1  mm  (237).  Media compositions  (i)  Krebs-Ringer  and  incubation  procedures  phosphate m e d i u m  T h i s h a d the f o l l o w i n g c o m p o s i t i o n  unless otherwise  128  2.8  mM  7.4  w i t h HC1.  and  10 m M  was  10 mM.  C a C l 2 was  mM  - NaCl;  - Na HP0 2  In a K omitted.  4  5 mM  - KC1;  a d j u s t e d to p H  - f r e e m e d i u m KC1 To  was  - CaCl ; 2  omitted.  stated:  1.3  Glucose  mM  - MgS0 ; 4  when added  In a C a  -free medium  e n s u r e c o m p l e t e a b s e n c e of f r e e c a l c i u m i o n s i n the  m e d i u m during incubation,  3 mM  E G T A was  added when stated.  Various  s t a n c e s w e r e a d d e d to the i n c u b a t i o n m e d i u m i n a f i n a l v o l u m e of 3 m l .  subCups  45  c o n t a i n i n g r o l l s of f i l t e r p a p e r m o i s t e n e d i n t h e c e n t r e w e l l s to a b s o r b  The apparatus The  w i t h 0.2 m l 2 0 % K O H  were placed  e v o l v e d CC" . 2  i n c u b a t i o n w a s c a r r i e d out i n a c o n v e n t i o n a l W a r b u r g i n a n a t m o s p h e r e of 0  2  manometric  a t 3 7 ° C f o r a p e r i o d of u s u a l l y o n e h o u r .  flasks were oxygenated for 5 minutes and t h e r m a l l y equilibrated for 7  m i n u t e s p r i o r to t h e c o m m e n c e m e n t of t h e i n c u b a t i o n .  R e a d i n g s o f the r a t e s  of o x y g e n c o n s u m p t i o n w e r e r o u t i n e l y t a k e n to e n s u r e t h a t the b r a i n s l i c e s u n d e r investigation exhibited n o r m a l r e s p i r a t o r y  (ii)  Krebs-Ringer  bicarbonate  activities.  medium  T h i s h a d the f o l l o w i n g c o m p o s i t i o n :  128 1.4 m M  mM  - NaCl;  - KH-jPO^;  5 mM  - KC1;  a n d 28 m M  2.8 m M  - CaCl ;  - MgS0 ; 4  - N a H C G ^ ; i n a f i n a l v o l u m e of 3 m l .  w a s c a r r i e d out f o r 5 m i n u t e s e i t h e r w i t h 0 / C 0 2  mixtures,  1.3 m M  2  2  (95/5%) or N / C 0 2  2  Gassing (95/5%)  d e p e n d i n g o n w h e t h e r t h e i n c u b a t i o n w a s c a r r i e d out a e r o b i c a l l y o r  anaerobically respectively.  A f t e r 7 m i n u t e s t h e r m a l e q u i l i b r a t i o n the i n c u b a -  t i o n w a s c a r r i e d out f o r one h o u r at 3 7 ° C .  It s h o u l d be s t a t e d t h a t no s i g n i f i -  c a n t d i f f e r e n c e s w e r e f o u n d i n t h e a m i n o a c i d p a t t e r n s , o r the c a t i o n i c 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 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  medium  whether this was b u f f e r e d with phosphate ions or with b i c a r b o n a t e i o n s .  (iii)  M e d i u m II  T h i s m e d i u m was a N a + - r i c h , following 178  Ca  +  +  and K+-free m e d i u m having the  composition: mM  - NaCl;  p H 7.4 w i t h H C 1 .  1.3 m M  - MgS0 ; 4  10 m M  - Na HP0 2  4  a d j u s t e d to  46  2. 5  A m i n o acid analyzer  (a)  Sample  (i)  estimations  preparation  Tissue sample  preparation  A t the e n d of the p e r i o d of i n c u b a t i o n , the W a r b u r g v e s s e l s w e r e p l a c e d i n i c e , the t i s s u e q u i c k l y r e m o v e d , d r a i n e d off by blotting with f i l t e r p a p e r .  manometric  and adhering  The slices were quickly weighed  (to g i v e the f i n a l wet w e i g h t ) a n d t h e n h o m o g e n i z e d i n 3 m l of 5 % debris, washed natant.  s e p a r a t e d f r o m the supernatant with 5 %  TCA,  Traces  super-  with equal  of e t h e r l e f t b e h i n d w e r e  was  volumes evaporated  i n n i t r o g e n g a s ( b u b b l e d t h r o u g h N H ^ S O ^ to e n s u r e c o m -  p l e t e a b s e n c e of p o s s i b l e c o n t a m i n a t i n g up to a k n o w n v o l u m e  (ii)  The  by c e n t r i f u g a t i o n and decantation,  was t h e n e x t r a c t e d t h r e e t i m e s  of d i e t h y l e t h e r to r e m o v e T C A . by c a r e f u l l y blowing  TCA.  r e c e n t r i f u g e d , a n d t h e w a s h i n g s a d d e d to the  The supernatant  medium  ammonia) and the s a m p l e s w e r e made  (4-6 m l ) .  M e d i u m sample preparation  A t the e n d of t h e i n c u b a t i o n 2.0 o r 2.5 m l of the m e d i u m w e r e w i t h 2 o r 2.5 m l of 1 0 % with d i e t h y l ether,  TCA  and c e n t r i f u g e d .  the e t h e r r e m o v e d  The supernatant  as d e s c r i b e d e a r l i e r ,  was  mixed extracted  and s a m p l e s w e r e  m a d e up to k n o w n v o l u m e s (4-6 m l ) .  (b)  Sample analysis  A m i n o a c i d s p r e s e n t i n a l i q u o t s (1-2 m l ) of the s a m p l e s w e r e o n a p p r o p r i a t e c o l u m n s of a B e c k m a n 120B, a s d e s c r i b e d i n the B e c k m a n m a n u a l .  Amino A c i d Analyzer,  The accelerated method  was  separated operated u s e d to  s e p a r a t e the a m i n o a c i d s w h i c h w e r e d e t e c t e d b y the n i n h y d r i n r e a c t i o n .  47  (i)  The  A c i d i c and n e u t r a l a m i n o a c i d  analyses  amino acids, taurine, a s p a r t i c acid, threonine, glutamine  and  s e r i n e , g l u t a m i c a c i d , g l y c i n e , a n d a l a n i n e , w e r e s e p a r a t e d on a 50 x 0.9 c o l u m n of s u l f o n a t e d p o l y s t y r e n e — 8 % r e s i n (Type buffer pH elute.  50A  3.28.  The  cm  divinyl benzene c o p o l y m e r ion exchange  - p a r t i c l e s i z e 25-31 \i) at 5 0 ° C by e l u t i o n w i t h 0.067 M U n d e r these c o n d i t i o n s glutamine, a s p a r a g i n e and  free asparagine  sodium  serine co-  p o o l i n b r a i n i s v e r y s m a l l (0.1 - 0.2 p m o l e / g i n i t i a l  wet  wt) w h e n c o m p a r e d w i t h t h o s e of g l u t a m i n e  was  not e s t i m a t e d .  and  s e r i n e (22,  88,  239),  V a l u e s f o r s e r i n e w e r e o b t a i n e d i n a n u m b e r of  by the f o l l o w i n g p r o c e d u r e .  The  at 7 5 ° C  sample containing glutamine  heated with 1 0 %  TCA  f o r 75 m i n u t e s (22).  of the g l u t a m i n e  while the s e r i n e i s unaffected.  and  and  experiments  serine  was  T h i s b r i n g s about h y d r o l y s i s  TCA  e t h e r a n d the s a m p l e p r e p a r e d a s d e s c r i b e d e a r l i e r .  is r e m o v e d with diethyl Glutamine values  are  o b t a i n e d b y e s t i m a t i n g e i t h e r (1) the d i f f e r e n c e b e t w e e n the a r e a s of the p e a k s , 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 a m i n o a c i d p r o f i l e s , and after h y d r o l y s i s ,  o r (2) the i n c r e a s e i n t h e a r e a of the a m m o n i a p e a k .  s h o u l d be m e n t i o n e d t h a t g l u t a m i n e 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 .  h y d r o l y z e d by t h i s m e t h o d y i e l d s A  s m a l l p r o p o r t i o n of t h i s c y c l i c  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). c e n t of the g l u t a m i n e tions.  hydrolyzed appears  T h u s , we  It  ammonia compound  f i n d t h a t a b o u t 22  per  as g l u t a m i c a c i d under these c o n d i -  T h i s i s i n a c c o r d w i t h the r e s u l t s of D o b k i n a n d M a r t i n (236).  (ii)  An  y -Aminobutyric  a c i d and  ammonia  a l i q u o t ( u s u a l l y 1 ml) of the s a m p l e was  citrate buffer pH polystyrene  - 8%  p a r t i c l e s i z e 19-25  4.26  at 5 0 ° C  from  divinyl benzene u).  The  a  5.5  x  copolymer  0.9  e l u t e d w i t h 0.127 cm  column  (Beckman  the  column  on  elution  Type  a d v a n t a g e of t h i s m e t h o d f o r a m m o n i u m  f r o m f r e e a m m o n i a in the s a m p l e .  M  sodium  of s u l f o n a t e d  resin,  i s the s e p a r a t i o n of l a b i l e a m m o n i a p r o d u c i n g c o m p o n e n t s ,  from  obtained before  like  15A  -  assay  glutamine,  T h e r e i s a l s o t o t a l r e c o v e r y of a m m o n i a  w i t h b u f f e r r e s u l t i n g i n g r e a t e r a c c u r a c y i n the  48  ammonia estimation.  (iii) N - A c e t y l a s p a r t a t e  T h i s amino acid derivative i s m e a s u r e d as aspartate after h y d r o l y s i s w i t h 0.5 v o l u m e of 5.5 N H C 1 at 1 0 0 ° C f o r 30 m i n u t e s , HC1.  Under these conditions, glutamine  ammonia. alanine,  The amino acids,  i s r e c o v e r e d as glutamate and  taurine,aspartate, glutamate,glycine and  were unaffected by this procedure.  the s a m p l e to d r y n e s s .  f o l l o w e d b y r e m o v a l of  H C 1 i s r e m o v e d by e v a p o r a t i n g  T h e d r i e d s a m p l e i s d i s s o l v e d i n 0.067 M  sodium  c i t r a t e b u f f e r p H 2.2, a n d a n a l y z e d w i t h 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 method.  a r e a s of the a m i n o a c i d p e a k s w e r e m e a s u r e d b y t h e h e i g h t - w i d t h  H e r e t h e h e i g h t of the p e a k w a s m u l t i p l i e d b y the w i d t h w h i c h i s mea-  s u r e d at half height.  T h e w i d t h of the p e a k w a s m e a s u r e d by c o u n t i n g t h e num-  b e r o f d o t s p r i n t e d a b o v e t h e h a l f h e i g h t of t h e peak.  T h e c o n s t a n t (i.e., a r e a  of the p e a k ) p e r ( j m o l e f o r e a c h a m i n o a c i d w a s o b t a i n e d f r o m a n a m i n o a c i d p r o f i l e of a s t a n d a r d a m i n o a c i d m i x t u r e . t i o n of e a c h a m i n o a c i d was computed. invariably determined  F r o m these v a l u e s the c o n c e n t r a -  The constants and peak positions were  f o r e a c h n e w b a t c h of b u f f e r o r n i n h y d r i n r e a g e n t  prepared.  A f t e r the e s t i m a t i o n of a m i n o a c i d s 50 c m c o l u m n ( a n d s o m e t i m e s  i n a sample was completed,  the  t h e s h o r t c o l u m n ) w a s r e g e n e r a t e d w i t h 0.2 N  N a O H a n d e q u i l i b r a t e d w i t h the a p p r o p r i a t e b u f f e r b e f o r e r e u s e .  2.6  W a t e r uptake b y b r a i n c o r t e x s l i c e s  I m m e d i a t e l y a f t e r i n c u b a t i o n the s l i c e s w e r e 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 .  T h e y w e r e s p r e a d without f o l d i n g on a n 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 a r o u n d t h e s l i c e s a n d o n the g l a s s w a s  absorbed  49  w i t h s t r i p s of f i l t e r p a p e r .  The  d i f f e r e n c e b e t w e e n the f i n a l wet w e i g h t and  i n i t i a l wet w e i g h t g i v e s a m e a s u r e of the i n c r e a s e of w a t e r  the  uptake.  T h e t o t a l w a t e r c o n t e n t of the t i s s u e i s e q u a l to the s u m  of the w a t e r  t a k e n up b y the t i s s u e d u r i n g the p e r i o d of i n c u b a t i o n a n d the a m o u n t of w a t e r o r i g i n a l l y p r e s e n t ( s e c t i o n Z.8).  2.7  O x y g e n uptake by b r a i n c o r t e x s l i c e s  Oxygen uptakes  (Q Q  w  e  r  e  2  c a l c u l a t e d as g i v e n i n " M a n o m e t r i c  (240) i n t e r m s of n l / m g d r y wt t i s s u e ,  Techniques"  and c o n v e r t e d to ^ i m o l e / 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 b y a f a c t o r w h i c h f o r the a d u l t r a t was  200/22.4  a n d f o r the i n f a n t r a t 120/22.4.  2.8  Dry  w e i g h t of b r a i n c o r t e x s l i c e s  W e i g h e d c o r t e x s l i c e s w e r e d r i e d at 1 1 0 ° C the a d u l t r a t , d r y wt = 2 0 %  wet  wt;  w e t wt. w i t h a s t a n d a r d d e v i a t i o n ^ +  2.9  22  Na  +  to c o n s t a n t w e i g h t s .  f o r 2-day o l d i n f a n t r a t , d r y wt = 5%.  on ^2]sj + f l u x e s , a  0.5 \ld  of 2 2 ^ + a  w  a  s  p l a c e d i n the  m a i n c o m p a r t m e n t 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 w i t h 3 m l  mequiv/1  S o d i u m i o n c o n c e n t r a t i o n i n the m e d i u m was  unless otherwise stated.  were quickly removed, adhering fluid, 5%  TCA.  12%  influx into b r a i n c o r t e x s l i c e s .  In e x p e r i m e n t s  tion medium.  For  of the i n c u b a -  usually  148  A f t e r the i n c u b a t i o n , the b r a i n s l i c e s  l i g h t l y b l o t t e d w i t h s t r i p s of f i l t e r p a p e r to r e m o v e  w e i g h e d to e s t i m a t e s w e l l i n g , and h o m o g e n i z e d i n 3 m l of  A f t e r s t a n d i n g i n the c o l d f o r two h o u r s w i t h o c c a s i o n a l m i x i n g ,  the h o m o g e n a t e was  centrifuged.  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  22  Na  +  i n the m e d i u m at the  50  e n d of the i n c u b a t i o n p e r i o d , 0.4  m l m e d i u m was  m i x e d w i t h 1.6  ml  5%  TCA  and c e n t r i f u g e d .  0.5  m l a l i q u o t s of the T C A  w i t h 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 f o r 22j^ + a  s e t t i n g s on a N u c l e a r  m i n u t e (dpm)  was  u s i n g the a p p r o p r i a t e  was  The  c o n v e r t e d to t h a t of d i s i n t e g r a t i o n s  per  b y m u l t i p l i c a t i o n w i t h the f a c t o r 1 0 0 / p e r c e n t a g e e f f i c i e n c y of  Percentage  e f f i c i e n c y was  g e n e r a l l y about 30 p e r c e n t f o r  The  mixed  Chicago M o d e l M a r k I liquid scintillation counter.  v a l u e of c o u n t s p e r m i n u t e (cpm)  counting.  e x t r a c t s of t i s s u e o r m e d i u m w a s  c o n c e n t r a t i o n of  7 7  Na  +  o b t a i n e d by the c h a n n e l r a t i o m e t h o d 22  Na  and  + .  i n the t i s s u e s l i c e was  calculated f r o m  the  r a d i o a c t i v i t y f o u n d i n u n i t w e i g h t of the t i s s u e s l i c e (dpm/g) d i v i d e d b y the  4"  7 7  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 i n c u b a t i o n m e d i u m (dpm/H e q u i v  sodium  in 1 ml incubation medium). 2.10  Experiments  with s o d i u m L - [ U -  In e x p e r i m e n t s  was  C]  glutamate  with l a b e l l e d glutamate,  f i c a c t i v i t y 0.067 n C i / ^ i m o l e 5 mM  1 4  L . - f U - ^ C ] glutamate  of s p e c i -  (or 122,300 cpm/i_imole) and c o n c e n t r a t i o n of  p l a c e d i n the m a i n c o m p a r t m e n t of W a r b u r g 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 i n c u b a t i o n p e r i o d , the  b r a i n s l i c e s w e r e r e m o v e d a n d c o u n t i n g was  c a r r i e d out on the T C A  of t i s s u e and m e d i u m a s m e n t i o n e d e a r l i e r i n s e c t i o n 2.9.  Counting  extracts efficiency  14 for  2.11  C was  f o u n d to be 83 p e r  Scintillation liquid  cent.  composition  T h i s c o n s i s t e d of a m i x t u r e of e q u a l v o l u m e s of t o l u e n e , d i o x a n e 95%  (v/v) e t h a n o l c o n t a i n i n g 2, 5 d i p h e n y l o x a z o l e (5g/l),  5-phenyloxazol-2-yl)-benzene  (0.05 g/1) and n a p t h a l e n e  and  1, 4 - b i s - ( 4 - m e t h y l (80 g/1).  51  2.12  Na  and K  The  t i s s u e c o n t e n t s of u n l a b e l l e d N a *  follows. period,  a s s a y by  flame-photometry  B r a i n cortex slices,  and K *  were determined  as  e i t h e r i n i t i a l l y o r at the end of the i n c u b a t i o n  were homogenized in 3 ml  s t r i p s of f i l t e r p a p e r a n d w e i g h i n g A f t e r s t a n d i n g i n the c o l d f o r two  5%  TCA  after r e m o v i n g a d h e r i n g f l u i d  to d e t e r m i n e  the e x t e n t of w a t e r  by  uptake.  h o u r s the h o m o g e n a t e s w e r e c e n t r i f u g e d  a n d the s u p e r n a t a n t s w e r e d i l u t e d w i t h d i s t i l l e d w a t e r to g i v e f i n a l c a t i o n c o n c e n t r a t i o n s of (0.05  - 0.1 p. e q u i v / m l ) .  a b s o r b a n c e ) w e r e m e a s u r e d at 294.3 a n d tively, The  with a P e r k i n - E l m e r  m o d e l 303  atomic  between Na*  for Na*  22  Na  D e t e r m i n a t i o n of A T P  ATP  respec-  spectrophotometer.  s t a n d a r d p l o t s of k n o w n  con-  c a r r i e d out s i m u l t a n e o u s l y  G e n e r a l l y g o o d a g r e e m e n t was  was  determined  (also  found  concentrations  a c c o r d i n g to the m e t h o d of G r e e n g a r d (241).  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 .  neutralized with K 2 C O 3  3 (jmole M g C l ;  +  2  a m i n e b u f f e r (pH 8.0); 0.5 dehydrogenase. N A D P H was  One  and m a d e up to 4 m l .  f o r e a c h a s s a y i n a f i n a l v o l u m e of 2 m l nmole N A D P ;  m l of the s u p e r n a t a n t 0.3  ml  aliquots were  c o n t a i n i n g 0.215  1.2 p m o l e E D T A  units hexokinase;  I n c u b a t i o n was  were extracted with 2  The ml  was taken  mmole glucose;  9.4  ( p H 7.4); 24 p m o l e t r i e t h a n o l -  and 1 u n i t  glucose-6-phosphate  c a r r i e d out at r o o m t e m p e r a t u r e  f o r 30  minutes.  m e a s u r e d with an A m i n c o - B o w m a n 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 w a v e l e n g t h 365 n m a n d f l u o r e s c e n c e w a v e l e n g t h 460 of h e x o k i n a s e  and  182).  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 e x p e r i m e n t (w/v)  and K *  (%  c o n t e n t s of a s a m p l e o b t a i n e d u s i n g the f l a m e p h o t o m e t e r  that o b t a i n e d by m e a s u r i n g  6%  absorptions  absorption  and K* v e r s u s A b s o r b a n c e ,  w i t h e a c h s e t of d e t e r m i n a t i o n s .  atomic  383.3 n m  cation concentrations were computed f r o m  c e n t r a t i o n s of N a *  2.13  The  was  n m.  One  unit  that a m o u n t of e n z y m e c a t a l y z i n g the c o n v e r s i o n of 1 p m o l e  of g l u c o s e p e r m i n u t e at p H  8.5  at 2 5 ° C .  One  u n i t of  glucose-6-phosphate  52  d e h y d r o g e n a s e was of N A D P  2.14  that a m o u n t of e n z y m e c a t a l y z i n g the r e d u c t i o n of 1 n m o l e  p e r m i n u t e at p H  +  Assay  7.4  at 2 5 ° C .  of Na"*", K ^ - A T P a s e of b r a i n  homogenates  (i)  Medium  The  m e d i u m u s e d f o r the a s s a y of N a *  composition; 15 m M  0.1  - KC1;  composition  mM  - disodium EDTA;  3 mM  - tris ATP;  95 m M  the c o n c e n t r a t i o n of Na"*" o r K"*" was  K - A T P a s e h a d the f o l l o w i n g +  1 mM  - MgC^;  58 m M  - tris  - C l (pH 7.6);  - NaCl;  whenever  c h a n g e d , the t o n i c i t y of the m e d i u m  was  m a i n t a i n e d by a l t e r i n g an e q u i v a l e n t a m o u n t of t r i s - C l i n the m e d i u m .  (ii)  Incubation  I n c u b a t i o n was the m e d i u m ,  procedure  c a r r i e d out i n c e n t r i f u g e tubes,  e a c h c o n t a i n i n g 3 m l of  w i t h a n a m o u n t of b r a i n c o r t i c a l h o m o g e n a t e i n c o l d  w a t e r e q u i v a l e n t to 1 m g  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 .  w e r e s h a k e n i n a w a t e r b a t h s h a k e r at 3 7 ° C f o r one  distilled The  tubes  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 w a s phosphate produced h y d r o l y s i s was  i n c u b a t i o n was m i x t u r e was distilled FeSC>4 *  by the h y d r o l y s i s of A T P  b r o u g h t a b o u t b y two  A T P a s e ; (2) a N a *  100  b y the e n z y m e p r e p a r a t i o n .  components:  centrifuged.  ml  of 1 %  ml  T o 0.5  ml  100%  (w/v)  m l of the s u p e r n a t a n t was  TCA,  a d d e d 2.0  and  a m m o n i u m m o l y b d a t e i n 1.15 The  N  H2SO4  g  a c c o r d i n g to  r e s u l t i n g b l u e c o l o u r was  on a B e c k m a n M o d e l D U s p e c t r o p h o t o m e t e r .  the  ml  of a c o l o u r r e a g e n t p r e p a r e d by d i s s o l v i n g 4.0  the m e t h o d of B o n t i n g et a l . (242). 2 h o u r s at 700 n m  The  +  t e r m i n a t e d by the a d d i t i o n of 0.2  The  (1) a M g * * - a c t i v a t e d  K - s t i m u l a t e d A T P a s e r e q u i r i n g Mg"*"*" f o r a c t i v i t y .  w a t e r a n d 2.5 n  m e a s u r e d i n t e r m s of the a m o u n t of  read within Substrate  and e n z y m e b l a n k s w e r e i n c l u d e d in each determination, together with phos-  53  p h a t e s t a n d a r d s to c o n v e r t o p t i c a l d e n s i t y to (Jmole P i ( p h o s p h a t e ) 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 3 7 ° C .  The  Na ,  K - A T P a s e activity  +  +  e s t i m a t e d as the d i f f e r e n c e b e t w e e n the a c t i v i t y of b r a i n h o m o g e n a t e •  t i o n i n c u b a t e d i n the p r e s e n c e a b s e n c e of N a  +  and K .  and M g  a n d that i n c u b a t e d i n the  In the f o l l o w i n g text, o n l y m e a n v a l u e s f o r P i  +  l i b e r a t i o n a r e given,  prepara-  4-4-  i  of Na"*", K  s i n c e the d e v i a t i o n s f r o m the m e a n w e r e n e v e r  than +  5 per  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  greater  cent.  S y n a p t o s o m e s (or p i n c h e d - o f f n e r v e t e r m i n a l s ) w e r e i s o l a t e d t i a l l y by the m e t h o d of G r a y a n d "Whittaker h o m o g e n i z e d i n 0.32 at  1725  The  was  rpm  M  (243).  s u c r o s e ( 1 0 % w/v),  (5 u p - d o w n s t r o k e s ) ,  B r a i n c o r t e x of the r a t  in a teflon homogenizer  a n d c e n t r i f u g e d at 1,000  g f o r 10  c r u d e n u c l e a r p e l l e t s e p a r a t e d f r o m the s u p e r n a t a n t - S p  t w i c e b y r e s u s p e n s i o n i n 0.32  M  was  natant-S i and  discarded.  The  containing m i c r o s o m e s and  minutes. washed  The  s o l u b l e c e l l c o n s t i t u e n t s , was  The  fragments,  washings were m i x e d with  c e n t r i f u g e d at 17,000 g f o r one h o u r .  was  revolving  s u c r o s e and r e c e n t r i f u g e d a s b e f o r e .  p e l l e t - ~Pi o b t a i n e d i n the f i n a l w a s h i n g c o n t a i n i n g l a r g e m y e l i n n u c l e i a n d c e l l d e b r i s , was  essen-  super-  supernatant-S2 discarded.  The  c r u d e m i t o c h o n d r i a l p e l l e t - P 2 ( c o n t a i n i n g s m a l l m y e l i n and m e m b r a n e f r a g ments,  m i t o c h o n d r i a and  s y n a p t o s o m e s ) was  s u s p e n d e d i n 0.32  M  sucrose  a n d c e n t r i f u g e d at 100, 000  g f o r 45 m i n u t e s into a d e n s i t y g r a d i e n t c o n s i s t i n g  of e q u a l v o l u m e s of 0.8 M  and  1.2 M  sucrose.  The  following fractions were  obtained.  A.  A  f r a c t i o n f l o a t i n g b e t w e e n 0.32  fragments). B.  A  T h i s was  M  sucrose (small myelin  discarded.  f r a c t i o n f l o a t i n g b e t w e e n 0.8  T h i s f r a c t i o n was  a n d 0.8  and  1.2 M  sucrose  (SYNAPTOSOMES).  c a r e f u l l y r e m o v e d with a pasteur pipette diluted  a n d c e n t r i f u g e d i n i s o t o n i c s u c r o s e (0.32 M ) .  The  pellet obtained  54  was  suspended in isotonic K r e b s -Ringer  phosphate m e d i u m  and  used. C.  A  pellet,  b e l o w 1.2  M  sucrose,  containing mitochondria  was  discarded. The  i n c u b a t i o n p r o c e d u r e f o r s y n a p t o s o m . e s was  d e s c r i b e d f o r b r a i n c o r t e x s l i c e s ( s e c t i o n 2.4  the s a m e a s t h a t  ( i ) ). H o w e v e r ,  the  Warburg  m a n o m e t r i c v e s s e l s w e r e s h a k e n v e r y s l o w l y to m a i n t a i n t h e i n t e g r i t y of the synaptosome.  A t the end  of the i n c u b a t i o n p e r i o d ,  f i n a l c o n c e n t r a t i o n of 5 p e r cent,  and  a n a l y s i s a s d e s c r i b e d i n s e c t i o n 2.5. measured,  i n an a l i q u o t of the  TCA  was  a d d e d to g i v e a  samples were prepared  for amino acid  P r i o r to i n c u b a t i o n , p r o t e i n  synaptosomal suspension  was  used for incubation,  as f o l l o w s .  2.16  Protein estimation  The  m e t h o d of L o w r y et a l . (249) u s i n g the F o l i n - C i o c a l t e u r e a g e n t  (2 50) was  e m p l o y e d f o r e s t i m a t i n g p r o t e i n , the b l u e c o l o r f o r m e d b e i n g  at 750  on a B e c k m a n M o d e l D U  nm  u s e d as a  2.17  Spectrophotometer.  Bovine albumin  read was  standard.  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 f o u n d to be r e p r o d u c i b l e . work results are expressed  c a r r i e d out at l e a s t f o u r t i m e s and the  Controls were always run  simultaneously.  as m e a n s of the v a l u e s o b t a i n e d a n d the  d e v i a t i o n s f r o m the m e a n a r e  given.  results In o u r standard  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 u s e d i n t h i s t h e s i s  (i)  Initial values  " I n i t i a l v a l u e s " a r e t h e v a l u e s (of NH^"*", Na"**, K"*~ o r a m i n o a c i d s ) o b t a i n e d p r i o r to i n c u b a t i o n .  (ii)  Total values  The  t e r m " t o t a l " has been used as follows:  (a)  T o t a l v a l u e s (i.e., t h o s e of t i s s u e + m e d i u m ) a r e the s u m s of the t i s s u e a n d m e d i u m c o n c e n t r a t i o n s of a p a r t i c u l a r a m m o n i a o r a m i n o acid) e x p r e s s e d  (b)  substance  (e.g.,  a s p m o l e / g i n i t i a l wet wt.  S o m e t i m e s i t h a s b e e n u s e d to e x p r e s s  the s u m o f a n u m b e r of  c o n s t i t u e n t s i n a g i v e n t i s s u e o r m e d i u m ; e.g., t o t a l t i s s u e NH  -N + a m i n o a c i d - N , o r t o t a l t i s s u e N a + + K + c o n t e n t s . 4 e x p r e s s i o n s of t o t a l v a l u e s a r e m a d e c l e a r i n the t e x t .  These  (iii) A d j u s t m e n t  " A d j u s t m e n t " i s a t e r m u s e d to s i g n i f y a c h a n g e m a d e to m a i n t a i n the i s o t o n i c i t y of the i n c u b a t i o n m e d i u m . the f o r m of t h e i r s o d i u m s a l t s (e.g., incubation medium, p equiv per m l  F o r example,  (a) w h e n s u b s t a n c e s i n  s o d i u m L - g l u t a m a t e ) a r e a d d e d to t h e  the m e d i u m N a * i o n c o n c e n t r a t i o n i s m a i n t a i n e d  by a p p r o p r i a t e r e d u c t i o n s  in'the m e d i u m N a C l ;  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 K C 1 a r e a d d e d to the m e d i u m , " a d j u s t e d " by an e q u i v a l e n t r e d u c t i o n i n the N a C l content, the t o t a l m e d i u m (Na" " + K ) 1  +  at 148  (b) w h e n  the m e d i u m i s thus  i o n c o n c e n t r a t i o n at 153 p e q u i v  maintaining  per ml.  56  (iv)  A m m o n i a and a m m o n i u m i o n  S i n c e the p K a f o r t h e r e a c t i o n NH  + 4  ^ = ^ N H  3  + H  +  i s 9.3 at p h y s i o l o g i c a l p H (7.4) a t w h i c h o u r e x p e r i m e n t s a r e c a r r i e d out, a l a r g e p r o p o r t i o n ( 9 7 % ) of the t o t a l ( N H 3 + NH^"*") i s p r e s e n t i n t h e f o r m of the a m m o n i u m i o n (278).  W h e n the t e r m s  " a m m o n i a " and " a m m o n i u m i o n s " a r e  u s e d i n the f o l l o w i n g text, i t i s u n d e r s t o o d that the f o r m e r t e r m ammonium  (v)  represents  ions.  Flux  The t e r m s respectively,  " i n f l u x " and "efflux",  i n this thesis,  a r e u s e d to d e s c r i b e ,  the " n e t " i n w a r d o r o u t w a r d m o v e m e n t of a s u b s t a n c e f r o m the  t i s s u e i n a g i v e n i n t e r v a l of t i m e .  (vi)  A c t i o n potentials  The  " g e n e r a t i o n o f a c t i o n p o t e n t i a l s " i s u s e d i n t e r c h a n g e a b l y w i t h 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 " . c o r t e x s l i c e s under  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 e r t a i n c o n d i t i o n s of i n c u b a t i o n and i s i d e n t i f i e d by s e n s i -  t i v i t y to T T X ( s e c t i o n 1.8 (viii) ).  57  3.  3.1  AMMONIA  F O R M A T I O N IN B R A I N  IN V I T R O  A m m o n i a a n d a m i n o a c i d c o n t e n t s of r a t c e r e b r a l c o r t e x initially,  a n d a f t e r i n c u b a t i o n i n the p r e s e n c e  slices  o r a b s e n c e of g l u c o s e  V a l u e s of the c o n t e n t s o f a m i n o 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 , f r e s h l y p r e p a r e d and immediately  p r i o r to i n c u b a t i o n , a r e g i v e n i n T a b l e 2.  T h e s e v a l u e s a r e i n a g r e e m e n t w i t h t h o s e 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) v a l u e s f o r a m m o n i a a n d a m i n o a c i d s found on incubating b r a i n slices in K r e b s - R i n g e r v a r i o u s p e r i o d s of t i m e ,  with o r without  a l s o r e c o r d e d i n T a b l e 2.  (i)  p h o s p h a t e m e d i u m i n O^ a t 3 7 ° C f o r t h e a d d i t i o n of 10 m M  glucose, a r e  T h e f o l l o w i n g c o n c l u s i o n s m a y be m a d e .  C h a n g e s i n the N H * a n d a m i n o a c i d contents of b r a i n s l i c e s 4  i n c u b a t e d i n the p r e s e n c e  a n d a b s e n c e of g l u c o s e .  T h e f o r m a t i o n o f (16.81 - 6.33) = 10.48 p m o l e a m m o n i a / g t i s s u e i s suppressed  i n the p r e s e n c e  of g l u c o s e i n a o n e h o u r i n c u b a t i o n p e r i o d .  suppression by glucose is reflected (10.11 - 3.55) = 6.56 p m o l e / g , (6.35  - 1.79)  (1) i n a n e n h a n c e d g l u t a m a t e  a n d (2) i n a n e n h a n c e d g l u t a m i n e  (9.13  content, v i z . , content, v i z . ,  = 4.56 p m o l e / g ( c o r r e s p o n d i n g to 9-12 p a t o m a m i n o a c i d - N / g )  w h i c h t o g e t h e r g i v e s a n i n c r e a s e of 15.68 tracting  This  patom amino acid-N/g.  On sub-  f r o m t h i s v a l u e , the v a l u e d u e to a d i m i n i s h e d a s p a r t a t e l e v e l , v i z . ,  - 4.14)  = 4.99,  a v a l u e of 10.69  patom amino acid-N/g  is obtained.  This  c o m p l e t e l y a c c o u n t s f o r the s u p p r e s s i o n of a m m o n i a p r o d u c t i o n b y g l u c o s e . T h e r e a r e r e l a t i v e l y s m a l l c h a n g e s i n the l e v e l s of o t h e r a m i n o a c i d s u n d e r these conditions. to  Therefore,  i t s e e m s t h a t the f a l l i n the l e v e l of N H ^  g l u c o s e i s a b o u t e q u a l t o the r i s e i n g l u t a m a t e  m i n u s the f a l l i n a s p a r t a t e l e v e l s . c o n s i d e r i n g the a m m o n i a ,  A  glutamate,  cortex slices incubated for four hours.  +  due  p l u s the r i s e i n g l u t a m i n e  s i m i l a r c o n c l u s i o n m a y be d r a w n on glutamine  a n d a s p a r t a t e l e v e l s of b r a i n  T h e s e c a l c u l a t i o n s a r e g i v e n i n T a b l e 2A.  T A B L E 2.  A m m o n i a 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 K r e b s - R i n g e r phosphate medium with or without glucose (lOmM) in at 3 7 ° C for varying periods of t i m e . 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  INCUBATION 15 m i n . No Glucose  30 m i n . No Glucose  PERIOD  One hour No Glucose Glucose  Four hours No Glucose Glucose  Glutamate  11.83  1.09  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  GABA  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  0.90  + 0.05  0.73  + 0.05  1.29  + 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  + 0.09  1.88  + 0.14  2.23  + 0.03  1.82  + 0.08  3.32  + 0.19  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  + 0.29  0.35  + 0.11  0.25  + 0.03  1.50  + 0.30  0.80  + 0. 10  16.81  + 0.07  6.33  + 0.45  24.20  + 1.26  6. 82  + 0.45  0.25  0.2  Lysine Ammonia  1.58  Total: N H - N + amino acid - N 4  +  + 0.14  36.76  8.32  + 0.28  44.08  0.25 11.90  + 0.40  44.28  46.69  47.09  57.12  52.91  T A B L E 2A.  Changes i n the glutamate, glutamine, aspartate and a m m o n i a contents of b r a i n cortex slices incubated i n the presence or absence of glucose.  T o t a l (tissue+medium) values of ammonia and amino acids ( p m o l e / g initial wet wt.) of b r a i n cortex s l i c e s used f o r these calculations were taken f r o m T A B L E 2.  F o u r Hour  One Hour No Glucose  Glucose 10. 11  No G l u c o s e  Glucose  A-NHz  - 6 . 56  2.57  6.44  - 3 . 87  A-NH  2  Glutamate  3.55  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  16.81  6.33  +10.48  24.20  6. 82  + 17.23  33.07  33.28  - 0 . 21  39.44  38.55  +0.89  A mmonia  Total - N H  2  60  F r o m the a b o v e c o n s i d e r a t i o n s , turnover  i n i n c u b a t e d b r a i n s l i c e s i s due  (ii)  C h a n g e s i n the i n i t i a l N H ^  i t i s e v i d e n t that m u c h of the  ammonia  to c h a n g e s i n b r a i n a m i n o a c i d s .  a n d a m i n o a c i d c o n t e n t s of b r a i n  +  c o r t e x s l i c e s on incubation.  In the a b s e n c e of g l u c o s e , b a t e d f o r one  the r i s e i n a m m o n i a f r o m b r a i n s l i c e s i n c u -  h o u r i s a c c o m p a n i e d by  s i g n i f i c a n t f a l l s i n the g l u t a m a t e  glutamine levels f r o m their initial values. t h e l e v e l of a s p a r t a t e . (16.81 - 1.58)  = 15.23  m a t e l e v e l s f a l l by 1.79)  = 2.61  pmole ammonia/g tissue/hour.  The  = 8.28  (9.13  - 3.36)  However, = 5.77  pmole/g,  it is probable  gluta-  and g l u t a m i n e f a l l s b y (4.40  -  amino acids could  p a t o m s - N / g ) of the t o t a l a m m o n i a  t h a t s o m e of the a s p a r t a t e f o r m e d , i.e.,  p m o l e / g i s d e r i v e d f r o m g l u t a m a t e by t r a n s a m i n a t i o n .  i s t h e r e f o r e e v i d e n t t h a t at l e a s t (13.50 - 5.77) cent,  Concomitantly,  d i m i n u t i o n i n the l e v e l s of t h e s e  a c c o u n t f o r a b o u t 90 p e r c e n t (or 13.5 liberated.  T h e r e i s an a c c o m p a n y i n g r i s e in  T h u s , i n the a b s e n c e of g l u c o s e t h e r e i s a l i b e r a t i o n of  (11.83 - 3.55)  |jmole/g.  and  of the t o t a l a m m o n i a l i b e r a t e d ,  = 7.73  pmole/g,  i.e., 50  It  per  i s a c c o u n t e d f o r by b r e a k d o w n of g l u t a -  m a t e and g l u t a m i n e i n the b r a i n t i s s u e ( a s s u m i n g no p r o t e i n b r e a k d o w n  takes  place).  In the a b s e n c e of g l u c o s e ,  a m m o n i a is continuously being  t h o u g h i t i s e v i d e n t that the r a t e of i t s f o r m a t i o n d i m i n i s h e s incubation.  For  example,  liberated,  w i t h the t i m e of  l e s s t h a n 8 | i m o l e a m m o n i a / 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 t h r e e h o u r s i n c u b a t i o n s u b s e q u e n t to the f i r s t h o u r i n c u b a t i o n , c o m p a r e d to 1 5 | j m o l e / g i n the f i r s t h o u r .  T h e r e s e e m s to be a d i r e c t r e l a t i o n -  s h i p b e t w e e n the r a t e of a m m o n i a f o r m a t i o n a n d the r a t e of o x y g e n c o n s u m p t i o n The  r a t e of the l i b e r a t i o n of a m m o n i a 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  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 r a t e of i t s o x y g e n c o n s u m p t i o n .  W h i l e the  r a t e of o x y g e n c o n s u m p t i o n by the s l i c e s i s a l m o s t c o n s t a n t i n the p r e s e n c e of 10 m M  glucose,  e v e n at the end  of a f o u r h o u r i n c u b a t i o n p e r i o d ,  w i t h t i m e i n the a b s e n c e of g l u c o s e  ( T a b l e 15).  The  the r a t e  falls  d r o p i n the r a t e of o x y g e n  61  uptake in a g l u c o s e - f r e e m e d i u m probably coincides with the f a l l in the l e v e l s of endogenous substrates,  including such substrates as glutamate,  capable of  l i b e r a t i n g a m m o n i a on oxidation. In the p r e s e n c e of glucose,  there is only a s m a l l diminution in the  i n i t i a l l e v e l of glutamate of b r a i n s l i c e s incubated for one hour, of glutamine is i n c r e a s e d .  but the content  In a four hour incubation period, the rate of endo-  genous glutamate oxidation in b r a i n tissue is d i m i n i s h e d in the p r e s e n c e of glucose and a m m o n i a is u t i l i z e d in the synthesis of glutamine. maintains high l e v e l s of glutamate generating a -ketoglutar ate (315)  Glucose  and glutamine in incubated s l i c e s by  which f o r m s glutamate,  either by reductive  amination with NH4*, or by t r a n s a m i n a t i o n with amino acids such as aspartate. Moreover,  glucose oxidation generates N A D H ,  which f a v o r s reductive amina-  tion of CY -ketoglutar ate over the r e v e r s e r e a c t i o n of glutamate Furthermore,  oxidation.  glucose generates A T P r e q u i r e d for glutamine synthesis.  These  facts account for the inhibitory effect of glucose on a m m o n i a f o r m a t i o n in b r a i n s l i c e s during incubation. (iii) P o s s i b l e protein breakdown in incubating b r a i n s l i c e s . In o r d e r to obtain i n f o r m a t i o n concerning the kinetics of the r i s e i n aspartate l e v e l accompanying the f a l l in glutamate l e v e l , rat b r a i n cortex s l i c e s were incubated a e r o b i c a l l y at 3 7 ° C in K r e b s - R i n g e r phosphate f r e e m e d i u m for 15 minutes and 30 minutes.  glucose-  R e s u l t s of the total (tissue +  medium) a m m o n i a 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 s e in the aspartate l e v e l o c c u r r i n g in one hour,  takes place in the f i r s 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, obtained in one hour,  that is  o c c u r s in the f i r s t 15 minutes and 73 per cent (or  (-imole/g) at the end of 30 minutes incubation.  6.25  These and other r e s u l t s in  Table 2 indicate that, if the bulk of the aspartate is d e r i v e d 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 following glutamine  source  (possibly proteins) d i r e c t l y  and/or  h y d r o l y s i s , e n t e r s i t s i n i t i a l f r e e pool, u n d e r g o e s oxida-  t i o n and l i b e r a t e s a m m o n i a i n the p r o c e s s . a p p e a r a s a r e s u l t of p r o t e i n b r e a k d o w n .  Possibly, some aspartate  That protein breakdown may  o c c u r i n i n c u b a t i n g 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  may indeed  r i s e i n the  l e v e l s of c e r t a i n a m i n o a c i d s s u c h a s g l y c i n e , s e r i n e , t h r e o n i n e o r l y s i n e w i t h i n c r e a s i n g t i m e s o f i n c u b a t i o n (0 t o 4h).  It i s t o b e noted,  however,  that  the l e v e l of g l u t a m a t e (and g l u t a m i n e ) c o n t i n u e s to f a l l w i t h t i m e of i n c u b a t i o n i n d i c a t i n g i t s (glutamate) continuous  The  oxidation.  v a l u e of the t o t a l a m i n o a c i d - N ,  i n c l u d i n g the v a l u e f o r a m m o n i a ,  i s e n h a n c e d f r o m the i n i t i a l l e v e l b y a b o u t 10 p a t o m s / g i n i t i a l w e t wt o n i n c u b a t i o n f o r one hour,  e i t h e r i n the p r e s e n c e o r a b s e n c e of g l u c o s e .  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 b r e a k d o w n on incubation.  This  It i s to be n o t e d  t h a t t h e l e v e l of t a u r i n e (an a m i n o a c i d d e r i v a t i v e not a c o n s t i t u e n t of p r o t e i n s ) remains,  within experimental  e r r o r , u n c h a n g e d d u r i n g i n c u b a t i o n of b r a i n  s l i c e s i n t h e p r e s e n c e o r a b s e n c e of g l u c o s e ,  even i n four hour incubations.  I n c r e a s e s i n the a m i n o a c i d c o n t e n t of i n c u b a t e d b r a i n t i s s u e a r e i n a c c o r d the o b s e r v a t i o n s Mcllwain  m a d e by W e i l - M a l h e r b e a n d G o r d o n (102),  and J o n e s and  (267).  It s h o u l d be m e n t i o n e d that the i n i t i a l l e v e l of N - a c e t y l (5.72  with  aspartate  + 0.24 p m o l e / g ) i n r a t b r a i n c o r t e x s l i c e s i s u n a f f e c t e d by i n c u b a t i o n  i n the p r e s e n c e o r a b s e n c e of g l u c o s e , w h i c h i s i n a g r e e m e n t w i t h m a n y r e s u l t s r e p o r t e d i n the l i t e r a t u r e (247, 248, 291).  T h e r e f o r e , the r i s e i n the  a s p a r t a t e c o n t e n t of b r a i n 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 i s n o t due  t o a f a l l i n t h e N - a c e t y l a s p a r t a t e c o n t e n t o f the t i s s u e .  However, this  v i e w i s o p p o s e d t o that of B u n i a t i a n e t a l . , (273) who o b t a i n e d a d i m i n u t i o n i n N - a c e t y l a s p a r t a t e of b r a i n t i s s u e i n c u b a t e d i n the a b s e n c e of g l u c o s e .  T h e s e s t u d i e s l e n d s u p p o r t to the v i e w that the b u l k of the a m m o n i a f o r m e d i n brain cortex slices incubated i n glucose-free media occurs  from  63  glutamine  3.2  a n d by t e r m i n a l g l u t a m a t e o x i d a t i o n .  Ammonia  The  formation in anoxia.  t o t a l ( t i s s u e + m e d i u m ) a m m o n i a a n d a m i n o a c i d c o n t e n t 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 at 3 7 ° C  f o r one h o u r i n K r e b s - R i n g e r  ate m e d i u m w i t h o r w i t h o u t the a d d i t i o n of 10 m M  N2/CO2  mixture  (95%/5%),  a r e g i v e n i n T a b l e 3.  bicarbon-  g l u c o s e i n a n a t m o s p h e r e of F r o m these r e s u l t s it i s  s e e n t h a t t h e r a t e of a m m o n i a 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 i s s u p p r e s s e d i n the a b s e n c e o f o x y g e n presence  ( c o m p a r e d w i t h v a l u e s o b t a i n e d o n i n c u b a t i o n i n the  of o x y g e n - T a b l e 2).  T h i s i s i n a c c o r d w i t h e a r l i e r f i n d i n g s (43, 244).  S u c h r e s u l t s s u p p o r t o u r v i e w (62) t h a t a m m o n i a f o r m a t i o n i n b r a i n d e p e n d s g r e a t l y on g l u t a m a t e o x i d a t i o n .  The  v a l u e of t o t a l a m i n o a c i d - N ,  i n c l u d i n g the value f o r a m m o n i a , i s  e n h a n c e d b y a n a e r o b i c i n c u b a t i o n a n d i s a l m o s t e q u a l i n one h o u r t o t h a t o b t a i n e d a t the e n d o f one h o u r a e r o b i c i n c u b a t i o n s ( T a b l e s 3 a n d 2).  This  e n h a n c e m e n t i s l i k e l y to b e due to p r o t e i n b r e a k d o w n b e c a u s e t h e r e a r e s m a l l i n c r e a s e s i n the a m o u n t s of m o s t a m i n o a c i d s a p a r t f r o m g l u t a m i n e The  n e t f a l l i n the l e v e l of g l u t a m i n e  obtained i n anoxia,  (Table 3A).  m a y p r e s u m a b l y be due  to t h e f a c t t h a t t h e r a t e of i t s h y d r o l y s i s b y g l u t a m i n a s e  e x c e e d s the r a t e of i t s  f o r m a t i o n b y the A T P r e q u i r i n g g l u t a m i n e  This presumably  synthetase.  a c c o u n t s f o r a p o r t i o n of the a m m o n i a l i b e r a t e d a n a e r o b i c a l l y .  A  balance  s h e e t r e p r e s e n t i n g t h e c h a n g e s i n the t o t a l ( t i s s u e + m e d i u m )  a m m o n i a a n d a m i n o a c i d c o n t e n t s of b r a i n s l i c e s i n c u b a t e d (with o r w i t h o u t g l u c o s e ) i n the p r e s e n c e  o r a b s e n c e of o x y g e n ,  i s g i v e n i n T a b l e 3A.  From  t h i s T a b l e i t i s e v i d e n t t h a t m u c h o f the a m m o n i a t u r n o v e r i n b r a i n i s d u e to c h a n g e s i n the b r a i n a m i n o a c i d s .  Moreover,  of g l u c o s e ,  c o n t e n t of b r a i n s l i c e s i n c u b a t e d a n a e r o b i -  the e n h a n c e d g l u t a m a t e  c a l l y o v e r t h e t i s s u e v a l u e of g l u t a m a t e  i t i s e v i d e n t that, i n the a b s e n c e  obtained aerobically,  f o r the d i m i n i s h e d r a t e of a m m o n i a f o r m a t i o n u n d e r a n o x i a . the a c c o m p a n y i n g i n c r e a s e i n G A B A content,  nearly  accounts  Furthermore,  a n d p e r h a p s p a r t of the i n c r e a s e  64  TABLE  3.  E f f e c t s of a n o x i a o n t h e a m m o n i a a n d a m i n o 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 t h e p r e s e n c e  or  absence of glucose. Rat b r a i n cortex slices were incubated i n K r e b s - R i n g e r  bicarbonate  m e d i u m at 3 7 ° C f o r o n e h o u r i n a n a t m o s p h e r e o f N 2 / C O ; ? ( 9 5 % : 5 % ) . Total as  ( t i s s u e + m e d i u m ) v a l u e s of a m m o n i a a n d a m i n o a c i d s a r e  expressed  jj.mole/g i n i t i a l w e t wt.  A d d i t i o n s to the i n c u b a t i o n m e d i u m No  Glucose  12.40  Glutamate  +  0.22  Glucose  13. 4 3  +  0.27  Glutamine  2. 88  +  0. 18  3. 08  +  0.10  GABA  3. 06  +  0.19  3 . 63  +  0.34  Aspartate  4. 34  +  0.03  4. 30  +  0. 15  Alanine  1. 3 6  +  0. 18  1. 88  +  0.30  Glycine  1. 66  +  0. 13  1. 73  +  0. 15  Serine  2. 76  +  0. 16  2. 89  Threonine  0. 76  +  0.05  0. 77  +  0.04  Taurine  5. 8 9  +  0.11  5 . 86  +  0.09  Ammonia  7. 10  +  0.31  6. 5 9  +  0.80  Total:  NH  + 4  + amino acid  -N -N  4 5 . 09  0.07  4 7 . 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 f r o m T A B L E 2 (for O ) and T A B L E 3 (for N ) . ?  No  o  2  Glucose N  2  Glucose A-NH  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  GABA  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  1.29  1.88  -0.59  Glycine  1.47  1.66  -0.19  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  in alanine,  must occur  by g l u t a m a t e m e t a b o l i s m .  T h e s e d a t a f u r t h e r s u b s t a n t i a t e t h e d e p e n d e n c e of a e r o b i c  ammonia  f o r m a t i o n l a r g e l y on e n d o g e n o u s g l u t a m a t e o x i d a t i o n .  3.3  Ammonia  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  slices  In e a r l i e r s t u d i e s , we h a v e s h o w n (62) that the r a t e of a m m o n i a f o r m a tion by 2-day-old r a t b r a i n cortex slices, f r e e K r e b s - R i n g e r phosphate medium, of the p o s s i b i l i t y that f o r m a t i o n  i n c u b a t e d f o r one h o u r i n a g l u c o s e -  i s l o w e r t h a n that of t h e a d u l t .  In v i e w  of a m m o n i a b y a d u l t r a t b r a i n c o r t e x i s depen-  d e n t o n t e r m i n a l e n d o g e n o u s g l u t a m a t e o x i d a t i o n , a s t u d y w a s m a d e o f the c h a n g e s i n t h e t o t a l ( t i s s u e + m e d i u m ) a m m o n i a a n d a m i n o a c i d l e v e l s of i n f a n t r a t b r a i n i n c u b a t e d i n t h e p r e s e n c e o r a b s e n c e of g l u c o s e .  Results reported in  T a b l e 4 show the f o l l o w i n g f a c t s .  1.  V a l u e s of the c o n t e n t s  of a m i n o a c i d s i n i n f a n t c o r t e x , f r e s h l y p r e -  p a r e d a n d p r i o r to i n c u b a t i o n a r e i n a g r e e m e n t w i t h t h o s e i n t h e l i t e r a t u r e (11, 130, 131, 311).  The total initial amino  a c i d - N i n c l u d i n g the v a l u e f o r a m m o n i a , t h a t f o u n d f o r t h e a d u l t ( T a b l e 2).  reported  a r e r e m a r k a b l y c l o s e to  However, i n agreement with  the r e s u l t s of o t h e r w o r k e r s (129-131),  the l o w e r l e v e l s of g l u t a -  m a t e (3 5 % of the adult), a s p a r t a t e ( 6 0 % of the adult), a n d g l u t a m i n e (65%  of the adult) a r e c o m p e n s a t e d f o r b y the h i g h l e v e l s of t a u r i n e  (300%  of t h e a d u l t ) .  T h e a m i n o a c i d s , c l o s e l y r e l a t e d to t h e c i t r i c  a c i d c y c l e , a r e c o n s i d e r a b l y l o w e r i n the infant b r a i n . 2.  I n t h e a b s e n c e of g l u c o s e , rat  t h e r a t e of a m m o n i a f o r m a t i o n i n i n f a n t  b r a i n i s 40 p e r c e n t that of the a d u l t .  T h e r e i s a r i s e i n the  a s p a r t a t e l e v e l a c c o m p a n y i n g the f a l l s i n g l u t a m a t e a n d g l u t a m i n e levels. 3.  In the p r e s e n c e of g l u c o s e ,  the i n i t i a l c o n t e n t of g l u t a m a t e i s l i t t l e  c h a n g e d i n i n f a n t c o r t e x i n c u b a t e d f o r one h o u r .  U n l i k e the adult  67  TABLE  4.  A m m o n i a and a m i n o a c i d changes i n infant 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 a b s e n c e 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 w e r e i n c u b a t e d i n K r e b s - R i n g e r m e d i u m with o r without g l u c o s e i n 0  2  at 3 7 ° C f o r o n e h o u r .  Initial values  ( i . e . v a l u e s p r i o r to incubation) of a m m o n i a and a m i n o a c i d s and the total (tissue + m e d i u m ) values a r e e x p r e s s e d  a s p.mole/g i n i t i a l wet wt.  INITIAL  ON No  Glucose  INCUBATION Glucose  + 0.05  1. 91  + 0.12  4.00  + 0.23  2. 91  + 0.05  0. 34  +  0.07  0.85  0. 15  GABA  1. 76  + 0.12  1. 65  + 0.05  2.16  + 0.30  Aspartate  2. 02  + 0.10  5. 62  + 0.06  2.52  + 0 .02  Alanine  1..26  + 0. 02  0. 93  + 0.30  1.25  + 0. 20  Glycine  1. 01  + 0.03  1. 63  + 0.19  1.71  + 0.03  Serine  1. 30  + 0.04  2. 26  + 0.20  2.37  + 0.40  Threonine  0. 4 0  + 0. 15  1. 08  + 0. 18  0.95  + 0. 12  17. 60  + 0.30  19. 03  + 1.20  Glutamate  4. 15  Glutamine  Taurine  18.91  + 2.34  L y s ine  0. 31  + 0.03.  0. 59  + 0. 02  0.42  + 0. 11  Ammonia  0. 76  + 0.02  7. 04  + 0.75  5.34  + 0.64  Total: NH4+-N + amino acid  -N  36. 49  42.42  4 1 . 33  68  brain,  however,  utilization,  infant b r a i n glutamine l e v e l f a l l s .  by g l u t a m i n e s y n t h e s i s ,  s e e m s to o c c u r o n l y  i n the i n c u b a t e d b r a i n t i s s u e of i n f a n t r a t . n o t o n l y due  to the l o w  This  feebly  presumably  a c t i v i t i e s of the r e l e v a n t e n z y m e s  in glutamine synthesis r e l a t i v e l y low  Ammonia  (15,  163),  but m a y  r a t e of the o p e r a t i o n  a l s o be due  of the c i t r i c  involved  to the  acid cycle,  to o c c u r i n the i n f a n t r a t b r a i n (235), and the r e s u l t a n t l o w f o r m a t i o n of a - k e t o g l u t a r a t e 4.  It was  and,  therefore,  wt t i s s u e i s c o n s i d e r a b l y  (see a l s o r e f e r e n c e  5.  235).  For  example,  wt/hour,  and  glucose,  the v a l u e s f o r the i n f a n t and and  that f o r the a d u l t i s 102  68  +_ 3,  ammonia,  i n the p r e s e n c e of 10  _+ 4.  + 2 [imole/g  o r a b s e n c e of g l u c o s e ,  e.g.,  glycine,  2).  There  are  serine, presence  also r e f l e c t some protein break-  incubation.  i n the p r o c e s s of a m m o n i a  brain?  mentioned earlier  the o r i g i n and  wt)  to g r a d u a l p r o t e i n b r e a k d o w n (but  w h i c h may  Is t h e r e a d i r e c t r o l e of a s p a r t a t e  studying  are  value  p a t o m s / g i n i t i a l wet  t h r e o n i n e o r l y s i n e , i n i n f a n t r a t b r a i n i n c u b a t e d i n the  As  wet  h o u r e i t h e r i n the p r e s e n c e or a b s e n c e of  e n h a n c e d l e v e l s of s o m e a m i n o a c i d s ,  f o r m a t i o n i n the  slices  i n c l u d i n g the  at a r a t e l o w e r t h a n that i n the a d u l t - T a b l e s 4 a n d  3.4  initial  adult b r a i n c o r t e x  (by about 5-6  T h i s i s p o s s i b l y due  down during  mM  In the a b s e n c e of  the t o t a l a m i n o a c i d - N  is enhanced  on i n c u b a t i o n f o r one glucose.  adult  respectively.  C o m p a r e d to the i n i t i a l , for  r a t e of  of g l u t a m a t e .  l o w e r t h a n i n the  g l u c o s e , the v a l u e f o r the i n f a n t b r a i n i s 46  +_ 3,  known  a l s o f o u n d t h a t the o x y g e n c o n s u m p t i o n i n the i n f a n t b r a i n  (per u n i t wet  42  is  ( s e c t i o n 1.5  (ii) a),  s o m e w o r k e r s (14,  m e c h a n i s m of a m m o n i a f o r m a t i o n ,  a s s i g n e d a c e n t r a l r o l e to a s p a r t a t e .  46,  98)  have i n t h e i r m o d e l  A c c o r d i n g to t h i s m o d e l ,  all ammonia  69  l i b e r a t e d e m a n a t e s f r o m a s p a r t a t e t h r o u g h a c y c l i c s e t of r e a c t i o n s i n v o l v i n g either d e s a m i n o - N A D or inosinic acid. to  T h e c o n t r i b u t i o n of o t h e r a m i n o  ammonia production occurs through aspartate  with oxaloacetate. "reservoir",  In o t h e r w o r d s ,  maintaining  acids  s u b s e q u e n t to t r a n s a m i n a t i o n  o t h e r a m i n o a c i d s p l a y t h e r o l e of a  aspartate levels.  In v i e w of t h e f a c t that i n c u b a t i o n  of b r a i n c o r t e x s l i c e s o f the r a t , i n a m e d i u m d e v o i d of g l u c o s e enhanced aspartate levels accompanying ammonia formation,  results in  experiments  w e r e c a r r i e d out to o b s e r v e w h e t h e r a s p a r t a t e a c t u a l l y p l a y s a d i r e c t r o l e i n the f o r m a t i o n  of a m m o n i a i n the i n c u b a t e d b r a i n t i s s u e .  When adult rat b r a i n cortex s l i c e s a r e incubated i n 0  2  f o r one h o u r i n  K r e b s - R i n g e r p h o s p h a t e 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 of 100 m M  KC1,  the t o t a l ( t i s s u e + m e d i u m ) l e v e l of a s p a r t a t e i s e n h a n c e d b y 40 p e r c e n t ( T a b l e 5).  T h i s i n c r e a s e i n a s p a r t a t e i s not a c c o m p a n i e d by a p a r a l l e l 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 , formation.  n o r i s i t a c c o m p a n i e d b y a n e n h a n c e d r a t e of a m m o n i a  In f a c t , h i g h  i n v a r i a b l y c a u s e s 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 r a t e of a m m o n i a l i b e r a t i o n . r e s p i r a t i o n under these  T h i s f a l l a c c o m p a n i e s a f a l l i n the r a t e of  c o n d i t i o n s , w h i c h may,  i n t u r n , b e due t o the l o s s of  s o m e e n d o g e n o u s g l u t a m a t e to the i n c u b a t i o n m e d i u m (i.e., 0.03 j j m o l e / g c o m p a r e d w i t h 0.90 _+0.07 f o r the c o n t r o l ) . medium) glutamine levels fall under these shown) a r e u n a f f e c t e d . a l a n i n e (i.e., control).  a l o s s of 2.75 + T o t a l (tissue +  c o n d i t i o n s , w h i l e G A B A l e v e l s (not  T h e r e i s a s m a l l b u t s i g n i f i c a n t r i s e i n t h e l e v e l of  a r i s e of 0.32 _+ 0 .02 | j m o l e / g o v e r the v a l u e of 1.0 1 _+ 0.0 5 f o r t h e  I n c r e a s i n g the t i m e of i n c u b a t i o n u n d e r t h e s e c o n d i t i o n s to f o u r  h o u r s d o e s not f u r t h e r enhance the total (tissue + m e d i u m ) a s p a r t a t e l e v e l , n o r d o e s it a f f e c t t h e l i b e r a t i o n o f a m m o n i a .  T h e s e r e s u l t s s h o w that a n i n c r e a s e  i n the e n d o g e n o u s a s p a r t a t e l e v e l d o e s n o t n e c e s s a r i l y a c c o m p a n y , in,  s t i m u l a t i o n of t h e l i b e r a t i o n of a m m o n i a i n b r a i n t i s s u e .  or r e s u l t  It m a y be  m e n t i o n e d that t h e r e w a s no f a l l of N - a c e t y l a s p a r t a t e to c o m p e n s a t e f o r the r i s e of a s p a r t a t e u n d e r t h e s e  The  conditions.  i n h i b i t i o n of a s p a r t a t e f o r m a t i o n ,  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 a m m o n i a f o r m a t i o n in rat b r a i n cortex s l i c e s incubated in a g l u c o s e - f r e e m e d i u m .  Rat b r a i n cortex slices were incubated a e r o b i c a l l y i n (glucose-free) 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 in the p r e s e n c e or absence of KC1 (lOOmM) with or without the addition of s o d i u m malonate (5mM) or sodium amino oxyacetate (5mM). QO2 values are e x p r e s s e d as H mole oxygen consumed per g initial wet wt. T o t a l (tissue + medium) a m m o n i a and amino acids 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.  A d d i t i o n s to the incubation m e d i u m  Q0  Aspartate  2  Glutamate  Glutamine  Ammonia  Nil  71.2  +  4.8  8.58  +  0.54  3.20  +  0.26  2.49  +  0.34  17.23  +  0.11  Malonate  60.3  +  3.9  7.31  +  0.11  3.44  +  0.27  2.58  +  0.38  16.80  +  0.38  A m i n o oxyacetate  63.6  +  3.0  5.30  +  0.47  3.58  +  0.07  3.58  +  0.07  17.71  +  0.11  With K C 1  54.5  +  6.2  12.20  +  0.18  3.85  +  0.05  1.22  +  0.12  16.04  +  0.16  KC1 + malonate  45.6  +  1.0  8.60  +.  0.64  4.87  +  0.04  1.33  +  0.22  15.25  +  0.07  KC1 + a m i n o oxyacetate  52.6  +  1.0  5.06  +  0.28  4.98  +  0.11  3.69  +  0.27  16.96  +  1.01  71  b r a i n s l i c e s i s a n o t h e r way of s t u d y i n g t h e e f f e c t s of a s p a r t a t e on t h e r a t e of ammonia liberation.  Aspartate  formation i n rat brain cortex slices  in K r e b s - R i n g e r phosphate (glucose-free) m e d i u m may  incubated  be s u p p r e s s e d by m e t a -  b o l i c i n h i b i t o r s s u c h a s m a l o n a t e o r a m i n o - o x y a c e t a t e ( T a b l e 5).  1.  M a l o n a t e (5 mM),  b y i n h i b i t i n g the r a t e of 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 , l i m i t s t h e s u p p l y of o x a l o a c e t a t e r e q u i r e d f o r a s p a r t a t e f o r m a t i o n by t r a n s a m i n a t i o n . p r e s e n c e o f 100 m M  K C 1 w h e n the r a t e of a s p a r t a t e f o r m a t i o n i s  considerably suppressed. suppression 2.  T h i s i s s p e c i a l l y e v i d e n t i n the  However,  t h e r e i s no c o n c o m i t a n t  of t h e r a t e o f a m m o n i a f o r m a t i o n  A m i n o - o x y a c e t a t e (5 m M )  under these  conditions.  i n h i b i t s a s p a r t a t e f o r m a t i o n b o t h i n the  p r e s e n c e o r a b s e n c e of 100 m M  K C 1 (in a g l u c o s e - f r e e  medium)  p r e s u m a b l y b y 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 p h o s p h a t e 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). of a s p a r t a t e f o r m a t i o n r a t e of a m m o n i a  T h e i n h i b i t i o n i n the r a t e  i s n o t a c c o m p a n i e d b y a n y i n h i b i t i o n of t h e  formation.  It s e e m s , t h e r e f o r e , t h a t the e v i d e n c e that a s p a r t a t e i s a m a j o r s o u r c e s l i c e s under our e x p e r i m e n t a l  d o e s n o t s u p p o r t the v i e w  of a m m o n i a i n r a t b r a i n c o r t e x  conditions.  In v i e w of the l a c k of s u p p o r t i n g e v i d e n c e as a n i m m e d i a t e a m m o n i a p r e c u r s o r ments,  f o r the r o l e of a s p a r t a t e  i nbrain slices,  a n d i n v i e w of the p o s i t i v e e v i d e n c e  ( + g l u t a m i n e ) as the i m m e d i a t e p r e c u r s o r ,  i n our experi-  favoring glutamate it seems unnecessary  to c o n s i d e r a s p a r t a t e a s p l a y i n g a m a j o r r o l e i n a m m o n i a Nevertheless,  t h e s e r e s u l t s do  t a t e p l a y s no r o l e i n t h e p r o c e s s  not constitute evidence of c e r e b r a l a m m o n i a  formation.  that  aspar-  formation.  72  3.5  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 r a t e of a m m o n i a f o r m a t i o n  from  e n d o g e n o u s a m i n o 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 i n c u b a t e d i n a m e d i u m devoid  of g l u c o s e .  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 w e r e i n c u b a t e d i n 0  2  in  Krebs-Ringer  p h o s p h a t e m e d i u m at 3 7 ° C f o r one  h o u r w i t h a n d w i t h o u t the a d d i t i o n of  2, 4 - d i n i t r o p h e n o l ( D N P )  o r s o d i u m a m y t a l (1 mM).  given in Table  (0.1 mM)  are  6.  In the p r e s e n c e of D N P  t h e r e i s a s m a l l d i m i n u t i o n (3 | i m o l e / g / h o u r ) i n  the r a t e of a m m o n i a f o r m a t i o n ,  which is chiefly reflected in accompanying  e n h a n c e d l e v e l s of g l u t a m a t e (by 1.83 DNP  Results  p.mole/g) a n d G A B A  (by 1.46  |imole/g).  s e e m s n o t to i m p a i r the b r e a k d o w n of p r o t e i n , s i n c e the t o t a l a m i n o  acid  n i t r o g e n ( i n c l u d i n g the v a l u e f o r a m m o n i a ) i s not a f f e c t e d w h e n c o m p a r e d  with  the v a l u e of the c o n t r o l .  It was  a l s o f o u n d that the r a t e of o x y g e n c o n s u m p t i o n ,  i s d i m i n i s h e d f r o m the c o n t r o l v a l u e of 70 + 7 | i m o l e / g wet  wt t i s s u e i n the p r e s e n c e of 0.1  With 1 mM  amytal,  5.36  |imole/g/hour.  3.31  )jmole/g) and G A B A  mM  to 50 + Z [ i m o l e / g  initial  DNP.  the r a t e of 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  T h i s i s a c c o m p a n i e d b y e n h a n c e d l e v e l s of g l u t a m a t e (by (by 1.83  (imole/g).  Under these conditions, however,  t h e r e s e e m s to be a s m a l l d i m i n u t i o n i n the b r e a k d o w n of p r o t e i n s i n c e the t o t a l a m i n o a c i d - n i t r o g e n ( i n c l u d i n g the v a l u e f o r a m m o n i a ) i s s l i g h t l y l o w e r t h a n t h a t f o r the c o n t r o l .  It was  a l s o f o u n d that a m y t a l (1 mM)  r a t e of o x y g e n c o n s u m p t i o n f r o m the c o n t r o l v a l u e of 68 jf v a l u e of 41  + 3 ( i m o l e / g i n i t i a l wet  wt  l o w e r e d the  5 ( j m o l e / g to t h e  tissue.  T h e s e d a t a on the r a t e s of NH^"*" f o r m a t i o n ,  which are in agreement  w i t h o u r 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 e f f e c t s of D N P  o r a m y t a l by b r a i n  t i s s u e 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 (62), a r e not w h o l l y e x p l a i n e d  by  i n c r e a s e d glutamate levels.  due,  obviously,  An  e l e v a t e d G A B A content a l s o takes place  to g l u t a m a t e d e c a r b o x y l a t i o n  t a k i n g p l a c e at a g r e a t e r r a t e w h e n  the e n d o g e n o u s g l u t a m a t e 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  TABLE  6.  E f f e c t s o f s o d i u m - a m y t a l a n d 2, 4 - d i n i t r o p h e n o l o n t h e t o t a l a m m o n i a a n d a m i n o a c i d c o n t e n t s of r a t b r a i n c o r t e x  slices  incubated i n a glucose free medium. Rat c e r e b r a l cortex s l i c e s w e r e 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 f o r o n e h o u r i n t h e p r e s e n c e o r a b s e n c e o f a m y t a l ( I m M ) o r 2, 4 - d i n i t r o p h e n o l (0. 1 mM), T o t a l ( t i s s u e + m e d i u m ) v a l u e s 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 i n c u b a t i o n m e d i u m Amytal  Nil  2,4 d i n i t r o p h e n o l  Glutamate  3. 62  + 0.26  6.93  + 0.25  5. 45  + 0. 26  Glutamine  2. 22  + 0. 14  1.97  + 0. 12  1. 80  + 0. 10  GABA  1. 92  + 0. 02  3. 75  + 0. 17  3. 38  + 0. 08  Aspartate  9. 07  1. 00  7.45  + 0. 08  9. 08  + 1. 08  Alanine  0. 84  + 0. 03  0.93  + 0. 09  1. 38  +_ 0. 05  Glycine  1. 40  + 0.03  1.27  + 0. 08  1. 58  + 0. 03  Serine  2. 21  + 0.05  2. 09  + 0. 10  2. 34  + 0. 18  Threonine  0. 86  + 0. 05  0.78  + 0. 02  0. 87  + 0. 04  Taurine  6. 16  + 1. 04  6.10  + 0.52  6. 20  + 0. 82  17. 16  + 0.16  11. 80  + 0.20  14. 17  + 1. 16  Ammonia  Total: NH4 -N + a m i n o a c i d -N +  47. 68  44.94  47. 85  74 3.6  E f f e c t s o f o u a b a i n on the r a t e of a m m o n i a f o r m a t i o n cortex slices incubated i n a glucose-free  in rat brain  medium  If e n d o g e n o u s g l u t a m a t e o x i d a t i o n i s a m a j o r f a c t o r i n the c e r e b r a l p r o d u c t i o n of a m m o n i a ,  i t s d e p l e t i o n 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 i n h i b i t i o n  of i t s r e - u p t a k e m e c h a n i s m ) s h o u l d b r i n g about a r e d u c e d r a t e of a m m o n i a f o r m a t i o n a n d a c o n c o m i t a n t i n h i b i t i o n i n the r a t e of r e s p i r a t i o n i n a g l u c o s e free medium.  This i n fact occurs  when r a t b r a i n c o r t e x s l i c e s a r e incubated  i n O-, f o r one h o u r a t 3 7 ° C i n K r e b s - R i n g e r p h o s p h a t e m e d i u m i n p r e s e n c e o f o u a b a i n (0.1 a n d 1.0 m M ) .  1.  R e s u l t s g i v e n i n T a b l e 7 show the f o l l o w i n g f a c t s .  T h e content of a m i n o a c i d - N  ( i n c l u d i n g the value f o r a m m o n i a ) i s  the s a m e i n the p r e s e n c e o r a b s e n c e of o u a b a i n ,  i n d i c a t i n g that  o u a b a i n d o e s n o t i n t e r f e r e w i t h the p r o t e i n b r e a k d o w n T h i s i s further supported  b y the f a c t t h a t the t o t a l ( t i s s u e + m e d i u m )  a m o u n t s o f a m i n o a c i d s s u c h as g l y c i n e , unaffected by ouabain,  process.  serine or threonine, a r e  though t h e i r r a t e s of r e l e a s e into the i n c u -  bation m e d i u m a r e c o n s i d e r a b l y enhanced. 2.  T h e r a t e of a m m o n i a f o r m a t i o n  i s i n h i b i t e d b y 4.74 a n d 6.74 p m o l e / g  i n i t i a l wet wt t i s s u e / h o u r w i t h 0.1 a n d 1.0 m M W i t h 0.01 m M 3.  ouabain r e s p e c t i v e l y .  o u a b a i n the v a l u e i s 1.7 5 p m o l e / g (not shown).  T h e i n h i b i t i o n of t h e c o n t e n t of a m m o n i a f o r m a t i o n b y o u a b a i n c a n be a c c o u n t e d f o r b y t h e g a i n of g l u t a m a t e a n d the a m i n o a c i d s d e r i v e d f r o m it, v i z . , a s p a r t a t e a n d G A B A .  There i s considerable  l o s s of t h e s e a m i n o a c i d s f r o m the t i s s u e a n d m o s t of the g a i n  takes  p l a c e i n the m e d i u m . 4.  It w a s a l s o f o u n d that the l o s s of t i s s u e g l u t a m a t e a n d i t s m e t a b o l i t e s u n d e r t h e s e c o n d i t i o n s i s a c c o m p a n i e d b y a d r o p i n the r a t e of o x y g e n c o n s u m p t i o n f r o m c o n t r o l v a l u e s of 75 + 4 p m o l e / g wet  wt t i s s u e to v a l u e s of 49 + 2 a n d 4 2 + 1  and  1.0 m M  ouabain r e s p e c t i v e l y .  initial  p m o l e / g w i t h 0.1  mM  T A B L E 7.  The effects of ouabain on the rate of ammonia formation, and the release of amino acids f r o m brain cortex slices incubated in a glucose-free medium.  Rat b r a i n cortex slices were incubated in a K r e b s - R i n g e r 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 w t . / 3 m l .  Addition to the incubation NIL TISSUE  MEDIUM  medium  O U A B A I N (0. ImM) TOTAL  TISSUE  MEDIUM  O U A B A I N (l.OmM) TOTAL  TISS UE  MEDIUM  TOTAL  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  GABA  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  46.23  .  Total: N H 4 - N + amino a c i d - N +  17.81  28.42  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 a m m o n i a / g t i s s u e ,  m u s t be d e r i v e d  from  g l u t a m i n e u n d e r t h e s e c o n d i t i o n s ( a s s u m i n g no g l u t a m i n e d i r e c t l y d e r i v e d f r o m p r o t e i n s e n t e r s the f r e e a m i n o a c i d p o o l o n i n c u b a t i o n ) a s the d i f f e r e n c e b e t w e e n the i n i t i a l a n d i n c u b a t i o n v a l u e s i s 4.40 2.25 = 2.15 ( T a b l e s 2 a n d 7). 6.  I n s p i t e of the o u a b a i n i n d u c e d a m i n o a c i d r e l e a s e f r o m the t i s s u e , m a r k e d changes  i n glutamate and aspartate contents (tissue +  m e d i u m ) take p l a c e . to a s p a r t a t e a n d G A B A  3.7  Thus,  c o n s i d e r a b l e c o n v e r s i o n of g l u t a m a t e  o c c u r s i n p r e s e n c e of o u a b a i n .  T h e r o l e of Ca"*"* i n the f o r m a t i o n o f a m m o n i a f r o m L - g l u t a m a t e b y 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 .  The as a major  e v i d e n c e s o f a r s u p p o r t s the r o l e of e n d o g e n o u s g l u t a m a t e  oxidation  f a c t o r i n the a e r o b i c g e n e r a t i o n of a m m o n i a by b r a i n t i s s u e .  E x p e r i m e n t s w e r e t h e n c a r r i e d out t o o b s e r v e w h e t h e r a d d e d e n h a n c e s the r a t e of a m m o n i a f o r m a t i o n .  L-glutamate  I n c o n f i r m a t i o n of o u r e a r l i e r  s t u d i e s (62) a n d t h o s e o f o t h e r s (58, 103), the a d d i t i o n of L - g l u t a m a t e 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 i n a n o r m a l K r e b s - R i n g e r p h o s p h a t e  (2.5  medium  ( c o n t a i n i n g Ca"*"*) i s f o u n d n o t t o e n h a n c e t h e a m o u n t of f r e e a m m o n i a i n one h o u r .  In fact,  mM)  formed  a d i m i n u t i o n takes p l a c e ( T a b l e 8).  R e s u l t s g i v e n i n T a b l e 8 s h o w that L - g l u t a m a t e a d d i t i o n r e s u l t s i n accompanying  i n c r e a s e s i n the r a t e of g l u t a m i n e s y n t h e s i s .  This isin accord  w i t h e a r l i e r f i n d i n g s (58, 103).  We  n o w f i n d that,  if Ca**  i s o m i t t e d f r o m the i n c u b a t i o n m e d i u m , the  r a t e of a m m o n i a f o r m a t i o n f r o m L - g l u t a m a t e i s s u b s t a n t i a l l y e n h a n c e d that i n a C a * *  above  c o n t a i n i n g m e d i u m a n d that of g l u t a m i n e i s d i m i n i s h e d ( T a b l e 8).  T h e a d d i t i o n of L - g l u t a m a t e 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 i n the p r e s e n c e of C a * *  enhances  the c o n t r o l r a t e of o x y g e n c o n s u m p t i o n  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 K r e b s - R i n g e r phosphate medium for one hour at 3 7 ° C in the presence or absence of 2. 8mM C a C l with or without the addition of 2. 5 m M sodium L-glutamate. Total (tissue + medium) values are expressed as u m o l e / g initial wet wt. Tissue values only of glutamate* are given. 2  Additions to the incubation medium Ca++ - f r e e  Nil Ca"*" - containing +  Sodium L-glutamate 12.5 mM) Ca++ - free Ca++- containing  Glutamate  *1.62  + 0.12  *2.03  + 0.16  Glutamine  1.46  + 0.13  2.22  + 0.14  3.22  + 0.50  8.24  + 0.38  10.01  + 0.24  9.07  + 1.00  20.31  + 0.45  20.00  + 0.30  1.92 + 0.02  2.22  +_ 0.32  2.17  + 0.20  Aspartate  *4.48 + 0.02  *6.36 + 0.08  GABA  1.87 + 0.09  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  17.15  + 0.43  17.16  + 0.26  19.24  + 0.62  12.68  + 0.28  Ammonia  78  2 p m o l e / g i n i t i a l wet w t / h o u r to a v a l u e of 97 +_ 3, a n d i n the a b s e n c e f r o m the c o n t r o l r a t e of 68 + 2 to a v a l u e of 88 + 2.  That  of C a  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 p o i n t to t h e p r o f o u n d e f f e c t s of Ca"""*" on g l u t a m i n e  t h e s i s i n b r a i n ( s e e C h a p t e r 4). absence  of C a  syn-  1  +  +  T h e i n c r e a s e d l i b e r a t i o n of a m m o n i a i n the  i s due to a d i m i n i s h e d a b i l i t y of the t i s s u e to s y n t h e s i z e g l u t a -  mine under these conditions.  In the p r e s e n c e of e x t e r n a l L - g l u t a m a t e ,  Ca**"* h a s l i t t l e o r no e f f e c t s  o n the t o t a l ( t i s s u e + m e d i u m ) l e v e l s of a l l a m i n o a c i d s d e t e r m i n e d e x c e p t ( a s mentioned enhances tion;  a b o v e ) that of g l u t a m i n e .  In the p r e s e n c e of L - g l u t a m a t e ,  Ca"*"*"  (1) the t i s s u e c o n t e n t of g l u t a m a t e ( T a b l e 8); (2) the r a t e of r e s p i r a -  a n d (3) t h e t i s s u e A T P c o n t e n t ( T a b l e 71 A ) .  glutamine synthetase activity takes place,  A probable increase i n  resulting i n an enhanced glutamine  and a d i m i n i s h e d a m m o n i a content.  3.8  Ammonia  formation by rat c e r e b r a l cortex slices f r o m  L - g l u t a m i n e i n the a b s e n c e  exogenous  of g l u c o s e .  R e s u l t s s u m m a r i z e d i n T a b l e 9 s h o w that, i n the a b s e n c e the  r a t e of a m m o n i a f o r m a t i o n f r o m 5 m M  brain  cortex slices,  i s a b o u t 17-18 j a m o l e / g  i n the p r e s e n c e o r a b s e n c e or  L-glutamine  of g l u c o s e ,  by incubated r a t  i n i t i a l wet wt t i s s u e i n o n e h o u r ,  of Ca*'"'", o r i n t h e p r e s e n c e of 0.1 m M  ouabain,  i n the N a ^ - r i c h K**" a n d C a ^ - f r e e i n c u b a t i o n m e d i u m - M e d i u m II.  The  f a c t that a c o n s t a n t r a t e of a m m o n i a 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 a c t i v i t y of g l u t a m i n a s e i s u n a l t e r e d u n d e r  such  varying media conditions.  It i s e v i d e n t , f r o m the r e s u l t s p r e s e n t e d so f a r , that b o t h e n d o g e n o u s and e x t e r n a l L - g l u t a m i n e  c a n s u p p o r t h i g h r a t e s of a m m o n i a f o r m a t i o n i n  brain tissue incubated i n glucose free-media.  79  T A B L E 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  17.16 + 0.26 Krebs-Ringer phosphate  +  Krebs-Ringer phosphate + 0. ImM ouabain  34.70 + 4.90  17. 54  13.14 + 0.22 18.65 +  30.20 + 4.00 17.15 + 0.43  Ca-free Krebs-Ringer phosphate  +  35.80 + 5.60  17.06  14.90 + 0.80 Medium II +  32.00 + 2.20  17.10  80  3.9  E f f e c t s of t e t r o d o t o x i n , ammonia formation free  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 r a t e of  by r a t b r a i n c o r t e x  K r e b s - R i n g e r phosphate g l u c o s e - f r e e  are recorded  The  in Table  2  The  f o r one  under these conditions were  of c a l c i u m .  The  a b o l i s h e d by the p r e s e n c e of t e t r o d o -  and  occur  i n the p r e s e n c e ,  but not i n  or l i d o c a i n e t h e m s e l v e s have little or  e f f e c t s o n the r a t e s of g l u t a m i n e a n d NH^"*" f o r m a t i o n  C h a n g e s i n the a m m o n i a and c o r t i c a l s y n a p t o s o m e s on  a m i n o a c i d contents  of i s o l a t e d r a t b r a i n  incubation.  i m m e d i a t e l y p r i o r to i n c u b a t i o n ,  A l s o given in this Table  a c i d s f o u n d on i n c u b a t i o n i n G"  2  no  under these conditions.  of s y n a p t o s o m e s i s o l a t e d f r o m the b r a i n  of the r a t , f r e s h l y p r e p a r e d and 11.  These  i m p l i c a t i o n of t h e s e f i n d i n g s w i l l be d e a l t w i t h  It i s to be n o t e d that T T X  A m i n o a c i d contents  studied.  the r a t e of a m m o n i a  s o m e of w h i c h a p p e a r s i n an e n h a n c e d g l u t a m i n e l e v e l .  the a b s e n c e ,  in Table  h o u r at 3 7 ° C i n  e f f e c t s of n e u r o t r o p i c  a d d i t i o n 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  t o x i n (2 LiM) o r of l i d o c a i n e (0.5 mM),  3.10  glucose-  10.  e f f e c t s of p r o t o v e r a t r i n e a r e c o m p l e t e l y  later.  in 0  medium.  d r u g s on the r a t e of a m m o n i a f o r m a t i o n  formation,  in a  medium.  Rat b r a i n cortex s l i c e s were incubated  Results  slices incubated  a r e the c o n t e n t s  at 3 7 ° C f o r one  are  of a m m o n i a and  hour in K r e b s - R i n g e r  p h a t e m e d i u m w i t h o r w i t h o u t the a d d i t i o n of 10 m M  cortex  glucose.  The  given amino phos-  following  f a c t s a r e e v i d e n t f r o m the r e s u l t s .  1.  A t the end  of the i n c u b a t i o n p e r i o d ,  a b s e n c e of g l u c o s e , for ammonia,  the  e i t h e r i n the p r e s e n c e  total amino acid-N,  i n c l u d i n g the  i s i n c r e a s e d by a b o u t 6 |aatom/100 mg  p r o t e i n . T h i s i s p r o b a b l y due  to p r o t e i n b r e a k d o w n .  or value  synaptosomal Proteolysis  i s a l s o r e f l e c t e d i n the i n c r e a s e d l e v e l s of c e r t a i n a m i n o a c i d s s u c h as g l y c i n e ,  serine, threonine  or lysine.  81  T A B L E 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  Glutamate  Glutamine  17. 10 + 0.58  2.93 + 0.21  2.25 + 0.21  + T T X (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) + T T X (2 M)  13. 01 + 0.25  3.10 -\_ 0.04  2.04 + 0. 07  + protoveratrine (5 JJM) + lidocaine (0.5mM)  18. 51 + 0. 19  2.94 + 0. 02  1.83 + 0. 14  Nil  Ammonia  U  82  TABLE  11.  Ammonia  and amino acid changes i n isolated rat b r a i n c o r t e x  s y n a p t o s o m e s i n c u b a t e d i n the p r e s e n c e  o r a b s e n c e of g l u c o s e .  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 , i s o l a t e d b y the m e t h o d of G r a y a n d Whittaker  (243), w e r e i n c u b a t e d i n 0  2  at 3 7 ° C f o r o n e h o u r i n K r e b s - R i n g e r  p h o s p h a t e m e d i u m w i t h o r without the a d d i t i o n of g l u c o s e (lOmM).  Initial  v a l u e s ( i . e . p r i o r to incubation) and the total (cells + m e d i u m ) values of a m m o n i a and amino acids obtained on incubation a r e e x p r e s s e d 100 m g  a s p.mole/  synaptosomal protein.  INITIAL  ON No  INCUBATION  glucose  Glucose  '  Glutamate  5. 54  + 0. 21  0. 92  + 0. 24  3. 77  + 0. 17  Glutamine  1. 30  + 0. 16  0. 72  + 0. 13  0.70  + 0. 14  GABA  1.26  + 0. 13  0. 75  + 0. 13  1.27  + 0.07  Aspartate  2.34  + 0. 18  3. 51  + 0.37  2. 83 + 0.22  Alanine  0. 21  + 0.07  0. 79  + 0.04  0.91  + 0. 05  Glycine  0. 27  + 0. 03  0. 70  + 0. 08  0.69  + 0.02  Serine  0.39  + 0. 08  0. 98  + 0. 08  0.97  + 0.07  Threonine  0. 20  + 0. 07  0. 64  + 0. 10  0.62  + 0. 02  Taurine  2.41  + 0. 08  2.44 + 0. 20  2.43  + 0. 11  -  0. 40  + 0. 10  0.45  + 0. 01  11. 21  + 0. 16  8. 01  + 0. 19  L y s ine 2.31  Ammonia  Total: NH4 -N +  + amino acid  -N  + 0.37  17.52  23. 78  23 .35  83  2.  In the a b s e n c e accompanied  of g l u c o s e , the r i s e i n the l e v e l of a m m o n i a i s  b y s i g n i f i c a n t f a l l s i n the g l u t a m a t e ,  and G A B A levels,  glutamine  p a r t of w h i c h m a y a c c o u n t f o r the r i s e i n t h e  c o n t e n t of a s p a r t a t e . 3.  In the p r e s e n c e of g l u c o s e , the t o t a l a-amino  + ammonia  nitrogen  a f t e r one h o u r i n c u b a t i o n i s the s a m e a s that i n t h e a b s e n c e of glucose.  H o w e v e r , t h e r e a r e r i s e s i n the l e v e l s of g l u t a m a t e a n d  G A B A w h i c h a r e b a l a n c e d b y f a l l s i n t h o s e of a s p a r t a t e a n d 4.  S y n a p t o s o m e s a r e unable to f o r m g l u t a m i n e f r o m a m m o n i a i n t h e p r e s e n c e of g l u c o s e .  NH^*.  liberated  In t h i s r e s p e c t  synapto-  s o m e s do n o t b e h a v e i n a m a n n e r s i m i l a r t o b r a i n c o r t e x s l i c e s incubated under identical conditions.  It i s k n o w n that t h e r e i s  l i t t l e o r no g l u t a m i n e s y n t h e t a s e a c t i v i t y i n i s o l a t e d n e r v e  endings  (245, 246). 5.  It w a s a l s o f o u n d that the r a t e s of o x y g e n u p t a k e s b y s y n a p t o s o m e s incubated i n the p r e s e n c e and absence 24 _+ 1 p m o l e s / l O O m g  3.11  of g l u c o s e a r e 64 + 2 a n d  synaptosomal protein respectively.  Summary  1.  W h e n the o x y g e n c o n s u m p 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  respiring  i n g l u c o s e - f r e e m e d i a i s s u p p r e s s e d (as, e.g., i n the p r e s e n c e o f 0.1 m M  DNP,  or 1 m M  i n the a b s e n c e the  amytal,  of oxygen),  o r 0.1 m M  o r 1.0 m M  ouabain, or  t h e r e i s an a c c o m p a n y i n g d i m i n u t i o n i n  l o s s of e n d o g e n o u s g l u t a m a t e a n d a d i m i n i s h e d r a t e of a m m o n i a  formation.  Infant r a t b r a i n ,  w h i c h r e s p i r e s at a l e s s e r r a t e t h a n  a d u l t p e r g f r e s h wt, a l s o s h o w s s m a l l e r l o s s e s of g l u t a m a t e a n d l o w e r r a t e s of a m m o n i a f o r m a t i o n .  84  A l t h o u g h , e n d o g e n o u s g l u t a m a t e s e e m s to be the m a j o r s o u r c e of aerobic ammonia formation i n b r a i n slices incubated i n a n o r m a l Krebs-Ringer Li-glutamate  phosphate g l u c o s e - f r e e medium,  exogenous  d i m i n i s h e s t h e r a t e of a m m o n i a f o r m a t i o n a n d  i n c r e a s e s that of g l u t a m i n e  synthesis.  W h e n Ca"*""*" i s o m i t t e d f r o m the i n c u b a t i o n m e d i u m , a m m o n i a f o r m a t i o n i n the p r e s e n c e  of L - g l u t a m a t e i s s u b s t a n t i a l l y  e n h a n c e d a b o v e that i n a Ca"*"*" c o n t a i n i n g m e d i u m , glutamine  the r a t e of  a n d that of  i s diminished.  B o t h endogenous and exogenous L - g l u t a m i n e  c a n s e r v e as s o u r c e s  of a m m o n i a 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 m e d i a .  The  a s p a r t a t e c o n t e n t of b r a i n s l i c e s ,  medium, KC1.  incubated i n a glucose-free  i s e n h a n c e d b y o v e r 40 p e r c e n t i n the p r e s e n c e  It i s d i m i n i s h e d i n the p r e s e n c e  of 5 m M  malonate or 5  a m i n o o x y a c e t i c a c i d w i t h or without h i g h p o t a s s i u m tions.  of 105 mM  ion concentra-  T h e s e c o n d i t i o n s h a v e l i t t l e o r no e f f e c t s o n the r a t e of  ammonia liberation.  T h e s e r e s u l t s do n o t s u p p o r t the v i e w t h a t  a s p a r t a t e i s 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 a b o u t a d i m i n i s h e d f o r m a t i o n of ammonia  t o g e t h e r w i t h a n e n h a n c e d s y n t h e s i s of g l u t a m i n e i n  b r a i n 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 c o n t a i n i n g Ca"*"*". T h i s p r o c e s s i s T T X (2 iaM) a n d l i d o c a i n e (0.5 mM)  sensitive.  B r a i n cortex synaptosomes f o r m ammonia aerobically, f r o m t h e i r p o o l s of g l u t a m a t e a n d g l u t a m i n e . slices,  largely  Unlike brain cortex  s y n a p t o s o m e s do not s e e m to s y n t h e s i z e g l u t a m i n e  presence  i n the  of g l u c o s e .  P r o t e o l y s i s s e e m s to o c c u r 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 under a l l our incubation  procedures.  mM  slices  85  4.  CONTROL, MECHANISMS RAT  Gonda and Quastel (e.g., 0.01 mM),  BRAIN  FOR GLUTAMINE CORTEX  SYNTHESIS IN  IN V I T R O  (112) h a v e s h o w n that o u a b a i n a t c o n c e n t r a t i o n s  n o t 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 s y n t h e s i s of l a b e l l e d  glutamine f r o m labelled glucose.  T h e i n h i b i t i o n of g l u t a m i n e  synthesis  ( T a b l e 12, C o n d i t i o n II) i s a c c o m p a n i e d b y a n e n h a n c e d l i b e r a t i o n of a m m o n i a (62) i n t o the i n c u b a t i o n m e d i u m  ( T a b l e 22).  H o w e v e r , i t w a s a l s o s h o w n (112)  that o u a b a i n d o e s n o t inhibit g l u t a m i n e synthetase NH^*  isolated f r o m brain tissue.  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 g l u t a m i n e  in brain cortex slices. t r a n s p o r t of N H ^ *  synthesis  T h i s l e d t o t h e s u g g e s t i o n that o u a b a i n s u p p r e s s e s t h e  to t h e s i t e o f g l u t a m i n e s y n t h e s i s (112).  However, it later  b e c a m e c l e a r that a m m o n i u m i o n s a r e n o t a c c u m u l a t e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t ( s e e C h a p t e r 6).  In t h e l i g h t of t h i s n e w i n f o r m a t i o n ,  the m e c h a n i s m  of o u a b a i n a c t i o n on the s y n t h e s i s o f g l u t a m i n e i n i n t a c t t i s s u e s t i l l r e m a i n e d to be f o u n d .  Further  experimental  work was therefore c a r r i e d  out a n d the  r e s u l t s a r e given below.  4.1  E f f e c t s of v a r y i n g  s o d i u m i o n c o n c e n t r a t i o n o n the r a t e of g l u t a m i n e  synthesis i nrat b r a i n cortex  slices  One of the c o n s e q u e n c e s of t h e i n h i b i t i o n o f t h e s o d i u m p u m p b y o u a b a i n i s an e n h a n c e d s o d i u m i o n c o n c e n t r a t i o n . H o w e v e r , t i s s u e Na"*" c o n c e n t r a t i o n m a y a l s o b e c h a n g e d b y a l t e r i n g t h e s o d i u m c o n c e n t r a t i o n of t h e i n c u b a t i o n 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 o r N a * w a s f o u n d to b e 0.79,  w i t h i n w i d e r a n g e s of m e d i u m N a * a n d K * Thus,  concentrations.  S e e T a b l e 67).  we w e r e a b l e to r a i s e the N a * c o n t e n t of b r a i n c o r t i c a l s l i c e s  r e s o r t i n g to t h e u s e of o u a b a i n ) by i n c u b a t i n g t h e m i n K r e b s - R i n g e r glucose  phosphate  m e d i u m c o n t a i n i n g a h i g h c o 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 glucose  (without  i n O^ at 3 7 ° C f o r one h o u r i n  containing media varying i n N a C l concentrations.  The results  obtained  86  ( T a b l e 12, C o n d i t i o n III) s h o w that w h i l e t h e r e s p i r a t o r y r a t e s a n d w a t e r u p t a k e s a r e l i t t l e a f f e c t e d by c h a n g e s i n t h e s a l t c o n c e n t r a t i o n s , of g l u t a m i n e i s i n h i b i t e d medium.  a t h i g h c o n c e n t r a t i o n s of N a C l i n t h e i n c u b a t i o n  T h e r e i s a l s o a n a c c o m p a n y i n g d i m i n u t i o n i n the  c o n c e n t r a t i o n r a t i o of g l u t a m i n e u n d e r t h e s e  4.2  the s y n t h e s i s  Tissue/Medium  conditions.  E f f e c t s of v a r y i n g p o t a s s i u m i o n c o n c e n t r a t i o n glutamine synthesis in rat b r a i n cortex  on the r a t e of  slices.  A n o t h e r c o n s e q u e n c e of the i n h i b i t i o n of the s o d i u m p u m p b y o u a b a i n is a d i m i n i s h e d tissue p o t a s s i u m ion concentration.  It i s p o s s i b l e to r a i s e  t h e l e v e l of K"*" i n c o r t i c a l s l i c e s b y i n c u b a t i n g t h e m i n a m e d i u m c o n t a i n i n g h i g h K*.  However,  Gonda and Quastel  (112) w e r e u n a b l e t o r e v e r s e t h e  i n h i b i t o r y e f f e c t of o u a b a i n on t h e s y n t h e s i s of g l u t a m i n e , In fact,  h i g h K * e n h a n c e d the i n h i b i t o r y e f f e c t of o u a b a i n o n g l u t a m i n e  synthesis. to o c c u r  T h i s i s u n d o u b t e d l y due to a f a l l i n the t i s s u e  w i t h 100 m M  K C 1 (115),  of 100 m M  A T P  l e v e l known  and although A T P l e v e l s i n s l i c e s a r e not  k n o w n t o d i m i n i s h m a r k e d l y w i t h o u a b a i n (112,  115), t h e a d d i t i o n a l p r e s e n c e  K C 1 m a y g r e a t l y r e d u c e the A T P c o n c e n t r a t i o n o w i n g to t h e  depleted phosphocreatine r e s e r v e KC1  with l O O m M KC1.  (251).  It s h o u l d a l s o be n o t e d t h a t 100  mM  e n h a n c e s the u p t a k e s of Na"* a n d w a t e r b y the t i s s u e (182).  I n c u b a t i n g r a t b r a i n c o r t e x s l i c e s f o r one h o u r i n Ringer  phosphate glucose  medium,  at 3 7 ° C i n K r e b s -  d e v o i d ( i n i t i a l l y ) 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 T a b l e 66) a n d A T P l e v e l s (115), effects on oxygen and water uptakes.  with little  accompanying  T h e r e i s a d i m i n u t i o n i n the r a t e of  g l u t a m i n e s y n t h e s i s due t o a b s e n c e of  ( T a b l e 12, C o n d i t i o n I V ) .  Higher  c o n c e n t r a t i o n s of m e d i u m K C 1 t h a n 5 mM,  do n o t a f f e c t the a m o u n t of g l u t a -  m i n e s y n t h e s i z e d i n one h o u r ( T a b l e 12),  a l t h o u g h the 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 of g l u t a m i n e i s m u c h i n c r e a s e d . r e t e n t i o n i n the t i s s u e i s i n a c c o r d et a l . (156).  T h e e f f e c t of K~*" o n g l u t a m i n e  w i t h the r e c e n t r e s u l t s of M a c h i y a m a  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 at 3 7 ° C for one hour in incubation media of varying composition with glucose present. The tissue and total glutamine levels (expressed as u m o l e / g initial wet wt.) and that for the medium (expressed as urnole/g initial wet wt./3 m l . ) are given. Oxygen uptakes are expressed as u m o l e / g initial wet wt. and water uptakes as ul/100 mg initial wet wt. 2  M E D I U M COMPOSITION  Condition  (mM)  EGTA  Ouabain  Oxygen Uptake  2.8  -  -  101 + 6  5 5  2.8 2.8  -  78 178  5 5  2.8 2.8  128 128 128 128  30 55 105  v  128 128  5 5  VI  178  NaCl  KC1  CaCl  128  5  II  128 128  in  IV  I (Control)  2.8 2. 8 2.8 2.8  2  0.01 0.1  -  -  ~  3 •-  3  -  98  Glutamine Content  Water Uptake  + 3  96 + 2 90 + 3 95+2  TISSUE  MEDIUM  TOTAL  Total Content of glutamine % Control  14.0  +  1.5  3.45  +  0.30  2.92  +  0.28  6.37  100  19.2 38.0  + +  1.0 1.1  0.31 0.71  + +  0.01  0.05  1.86 1.64  + +  0.12 0.07  2.17 2.35  34 37  22.2 15.9  + +  0.4 1.1  4.38  1.96  + +  0.15 0.21  2.97 3.01  + 0.15 + 0.20  7.35 4.97  115 77  + + + +  0.20 0.30 0.07 0.25  4.28 6.46 6.22 6.81  67 101 98 107  110 122 126 144  + + + +  1 2 2 2  18.3 17.5 31.8 38.1  + + + +  0.5 1.4 3.0 0.5  1.58 4.06 4.93 5.38  + + + +  0.05 0.40 0.05 0.22  2.70 2.40  128 134  + 5 + 5  18.0 19.3  + +  2.0 0.5  1.96 + 1.05  +  0.20 0.04  2.41  1.96  + +  0.14 0.07  4.3 7 3.01  68 47  105  + 3  26.9  +  1.1  0.56  +  0.01  1.42  +  0.07  1.98  31  1.29 1.43  00 -si  88  4.3  E f f e c t s of c a l c i u m on t h e r a t e of g l u t a m i n e s y n t h e s i s b y r a t b r a i n cortex  slices  G l u t a m i n e synthetase, i n h i b i t e d by Ca**(135).  a Mgrequiring  enzyme, i s competitively  H o w e v e r , 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),  s h o w t h a t t h e 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 p h o s phate glucose  m e d i u m c o n t a i n i n g Ca"*"* b r i n g s a b o u t a h i g h e r n e t g l u t a m i n e  s y n t h e s i s t h a n i n a m e d i u m d e v o i d of t h e c a t i o n .  The d i m i n i s h e d  synthesis  due t o t h e a b s e n c e of C a * * i s e v e n m o r e m a r k e d b y t h e 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 m e d i u m . I n t h e a b s e n c e of C a * * t h e t i s s u e g a i n s N a * a n d l o s e s K * As  (see Table  a c o n s e q u e n c e of s u c h i o n i c c h a n g e s , t h e s o d i u m p u m p i s s t i m u l a t e d .  i s a f a l l i n t h e A T P l e v e l ( s e e T a b l e 74).  66).  There  A d i m i n i s h e d A T P c o n t e n t of t h e  t i s s u e , due t o t h e a b s e n c e of c a l c i u m i o n s , m a y p o s s i b l y be t h e r a t e l i m i t i n g f a c t o r i n t h e s y n t h e s i s of g l u t a m i n e . maintaining high  ATP  O n t h i s v i e w , t h e e f f e c t of c a l c i u m i n  l e v e l s i n the t i s s u e m a y 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 a n o m a l o u s b e h a v i o r  of C a  R e s u l t s r e p o r t e d e a r l i e r ( C h a p t e r 3) a r e f u r t h e r e v i d e n c e f o r t h e i m p o r t a n t r o l e of C a * * i n t h e s y n t h e s i s of g l u t a m i n e i n t h e i n t a c t b r a i n t i s s u e . These r e s u l t s obtained with rat b r a i n cortex s l i c e s incubated i n a glucose  free  m e d i u m , d e m o n s t r a t e t h e r e q u i r e m e n t of C a * * f o r t h e 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 a d d e d 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 effect  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 a n a c c o m p a n y i n g 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).  suppression  A l l these r e s u l t s a r e understandable on  +4-  the b a s i s of C a  b e i n g n e c e s s a r y to m a i n t a i n  a sufficiently high A T P  c e n t r a t i o n i n t h e b r a i n t i s s u e t o b r i n g a b o u t an o p t i m a l r a t e of g l u t a m i n e synthesis.  con-  89  4.4  Glutamine synthesis in a Na" "-rich,  K -free,  l  Ca - f r e e , f  incubation  medium. F r o m the above considerations,  it would be expected that the inhibition  in the rate of glutamine synthesis i n b r a i n s l i c e s 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 m e d i u m  ( M e d i u m II) for absence of K  +  f a l l i n the A T P concentration. T a b l e 12 (Condition VI).  (115)  or C a  + +  (Table 74) leads to a m a r k e d  T h i s is, in fact,  what o c c u r s as shown i n  Under these conditions, the water uptake is enhanced  over the c o n t r o l value (Condition I), but the oxygen consumption is little affected .  4.5  G l u t a m i n a s e activity and glutamine synthesis We have seen that, when b r a i n cortex s l i c e s are incubated i n K r e b s -  R i n g e r phosphate glucose m e d i u m i n the absence of Ca*'*,  or in the p r e s e n c e  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 d i m i n i s h e d total (tissue + medium) l e v e l s of glutamine at the end of the incubation p e r i o d .  E v i d e n c e was p r e -  sented e a r l i e r (Chapter 3) showing that the incubation of b r a i n t i s s u e i n such m e d i a does not promote glutamine breakdown by enhancement of glutaminase activity (section 3.8). L-glutamine, Ca  + +  The fact that the rate of a m m o n i a f o r m a t i o n f r o m 5 m M  added to glucose f r e e m e d i a containing ouabain or devoid of  is constant (17-18 Llmole/g i n i t i a l wet wt/hr),  demonstrates that b r a i n  glutaminase activity is unaffected by these conditions.  4.6  E f f e c t s of s o d i u m L - g l u t a m a t e and N H ^ C l on the inhibition of glutamine synthesis in rat 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 presence of ouabain  R e s u l t s given i n Table 13 show that i n the presence of 5 m M L - g l u t a m a t e , or of 5 m M N H ^ C l ,  the rate of glutamine synthesis by rat b r a i n cortex s l i c e s  T A B L E 13.  Rat brain N H 4 C I or levels are (QO?) are Ca free  Effects of N H ^ C l and sodium L-glutamate in reversing the suppressed rates of glutamine synthesis in rat b r a i n cortex slices due to ouabain or changed media composition.  cortex slices were incubated in O2 at 3 7 ° C for one hour in the presence of glucose under media conditions given below. L-glutamatc when present were 5 m M and Ouabain O . l m M , T i s s u e glutamate l e v e l s , the total and tissue glutamine expressed as u m o l e / g initial wet wt. and those for the medium as |iinole/g initial wet w t . / 3 m l . Oxygen uptakes given as n m o l c / g initial wet wt. and water uptakes as (jl/100 m g . initial wet wt. Medium II was a N a r i c h , K and medium (see Section 2.4 (iii)).  MEDIA  +  COMPOSITION  K r c b s - R i n n e r phosphate-glucose (Control) + ouabain + L-glutamate + L-glutamatc + ouabain + NH4CI  + N H 4 C I + ouabain + L-glutamate + N H 4 C I + L-glutamatc + N H 4 C I + ouabain Medium II -  glucose  + L-glutamate + NH4CI  + N H 4 C I + L-glutamate  Water  Tissue*  uptaKe  oiutamate  TISSUE  MEDIUM  3.45  + 0.30  2.92  + 0.28  0. 71 5.67 1.20 4. 78 1.68 5.16  + 0.01  1 . 64 + 0. 07 4.86 + 0.19 4. 76 + 0.22 5.10 + 0.21 4.92 + 0.20 7.37 + 0.84  + 6  14.0  + 1.5  98 117 98 111 88 104  +  3 + 10 + 1 + 1 + 2 + 3  38.0 34. 7 37.9 14. 6 49.5 35.9  + 1.1 + 0.7  4. 82  + 0. 14  0.73  + 0. 04  5.01  +  1.5 0.1 + 0.3 + 0.7 + +  + 0.21  4. 95 _+ 23.50 _+ 12.48 + 5.98 + 3. 71 + 14.52 +  0.23 0.17 1.12 0.16 0.11 0.18  T o t a l Content  Glutamine Content  101  8.44  +  + + + _+ +  0. 17 0. 06 0.19 0. 03 0. 15  Total  of glutamine % Control  6.37  100  2.35 10.34 5.96 9.88 6.68 12.53  163 94 155 104 197  0.43  5.74  90  37  84  +  3  41.2  + 1.2  105  +  3  26.9  + 1.1  9.09  + 0.68  0.56  + 0.01  1.42  + 0.07  1.98  31  106 108 109  + 2  37.5 30.5 43.5  + 0.3 + 1.5 + 1.5  18.77 7.85 13.70  + 0.63 + 0.13 + 0.40  2.82 2.79 2.51  + 0.38 + 0.41 + 0.33  4.62 4.17 5.05  +  0.14  7.44 6.96 7.56  116 109 119  1 + 1  + 0.14 + 0.40  91  incubated glucose  in 0  2  f o r one h o u r at 3 7 ° C i n a n o r m a l K r e b s - R i n g e r  medium,  i s enhanced.  b a t i o n i s c a r r i e d out i n g l u c o s e and  NH4"*  phosphate  T h i s i s a l s o f o u n d to b e t r u e w h e n t h e i n c u c o n t a i n i n g M e d i u m II.  Both L-glutamate  c a u s e s o m e e l e v a t i o n of t h e r a t e of r e s p i r a t i o n of t h e b r a i n s l i c e .  G l u t a m a t e a n d / o r NH^~** o n l y p a r t i a l l y r e v e r s e s b y o u a b a i n o r b y i n c u b a t i o n i n M e d i u m II. be g r e a t e r  the i n h i b i t i o n c a u s e d  H o w e v e r , t h e r e v e r s a l s e e m s to  i n M e d i u m II, t h a n i n t h e m e d i u m c o n t a i n i n g o u a b a i n .  w h e n b o t h L - g l u t a m a t e a n d NH^"*  a r e present,  F o r example,  the p e r c e n t a g e i n c r e a s e s i n  g l u t a m i n e s y n t h e s i s ( c a l c u l a t e d o n the b a s i s o f L - g l u t a m a t e + N H ^ *  free  c o n t r o l s ) a r e (90 - 37) = 53 i n t h e o u a b a i n c o n t a i n i n g i n c u b a t i o n m e d i u m a n d (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 Table  13 i t s e e m s that t h e  r a t e of g l u t a m i n e s y n t h e s i s i s d e p e n d e n t o n the o u a b a i n - s e n s i t i v e of N H ^ " ^ for N H 4  and/or  g l u t a m a t e to t h e s i t e of g l u t a m i n e s y n t h e s i s .  i s e x t r e m e l y low, 0.18 m M  S i n c e the  (95) a n d a l s o s i n c e N H ^  18),  may  i s the c e l l u l a r l e v e l of A T P ,  Another factor limiting glutamine  (Table  synthesis  not o n l y r e q u i r e d f o r a c t i v e t r a n s p o r t (of g l u t a m a t e )  but a l s o f o r t h e s y n t h e s i s of g l u t a m i n e . l e v e l s o n l y s l i g h t l y (115),  to c o n c l u d e  w h i c h i s k n o w n to b e i m p e d e d b y o u a b a i n  be a r a t e - l i m i t i n g f a c t o r .  Km  a r e not  a c t i v e l y a c c u m u l a t e d i n b r a i n s l i c e s ( C h a p t e r 6), i t i s r e a s o n a b l e that t h e t r a n s p o r t of g l u t a m a t e ,  transport  Since ouabain a f f e c t s the t i s s u e A T P  a n d s i n c e t h e a d d i t i o n of L - g l u t a m a t e d o e s n o t c o m -  p l e t e l y r e v e r s e t h e i n h i b i t i o n of g l u t a m i n e s y n t h e s i s b y o u a b a i n ,  it follows  that i n h i b i t i o n of g l u t a m a t e t r a n s p o r t c a n n o t be the w h o l e e x p l a n a t i o n f o r t h e ouabain effect.  It s e e m s l i k e l y that t h e r e i s a n o u a b a i n i n d u c e d  f a l l of A T P  l e v e l i n the s p e c i f i c c o m p a r t m e n t of g l u t a m i n e s y n t h e s i s a s a r e s u l t of t h e changed tissue Na  /K  concentration  ratio.  S t u d i e s w e r e c a r r i e d out w i t h a v a r i e t y of m e t a b o l i c t h r o w f u r t h e r light on this p h e n o m e n o n .  i n h i b i t o r s to  92  4.7  C o m p a r a t i v e 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 ( m e t h i o n i n e ouabain,  fluoroacetate, malonate, D N P  uptakes,  glutamine  a n d a m y t a l ) o n r a t e s of o x y g e n  s y n t h e s i s a n d a m i n o a c i d c o n t e n t 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  The  sulfoximine,  of g l u c o s e  r e s u l t s of a s t u d y of the 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 t h e t o t a l  ( t i s s u e + m e d i u m ) r a t e s of g l u t a m i n e  synthesis and oxygen uptakes  cortex slices incubated i n K r e b s - R i n g e r i n T a b l e s 14 a n d 15, r e s p e c t i v e l y .  of r a t b r a i n  phosphate glucose m e d i u m a r e g i v e n  A l s o g i v e n i n T a b l e 14 a r e t h e t o t a l  ( t i s s u e + m e d i u m ) v a l u e s f o r a m m o n i a a n d a m i n o a c i d s obtained on i n c u b a t i o n o f the b r a i n t i s s u e i n t h e p r e s e n c e  of the i n h i b i t o r s .  The results are  s u m m a r i z e d below.  1.  Methionine synthetase,  s u l f o x i m i n e (5 mM),  a k n o w n i n h i b i t o r of g l u t a m i n e  i s w i t h o u t a n y e f f e c t s o n o x y g e n c o n s u m p t i o n , but  i n h i b i t s the f o r m a t i o n of g l u t a m i n e l i b e r a t i o n of a m m o n i a .  a n d b r i n g s a b o u t an i n c r e a s e d  G l u t a m a t e and other amino acid  contents  are little affected. 2.  O u a b a i n (0.01 mM),  h a s no e f f e c t o n o x y g e n c o n s u m p t i o n i n one  hour, but d e p r e s s e s  i t b y 25 p e r c e n t i n f o u r h o u r s .  the r a t e of g l u t a m i n e  synthesis and concomitantly  a m m o n i a output i n one a n d f o u r h o u r s .  It s u p p r e s s e s  enhances  G A B A levels are  e l e v a t e d i n one h o u r , but n o t s i g n i f i c a n t l y i n 4 h o u r i n c u b a t i o n , w h i l e a l a n i n e l e v e l s a r e e n h a n c e d i n b o t h t h e one a n d f o u r h o u r incubation periods.  G l u t a m a t e a n d o t h e r a m i n o 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 c o n d i t i o n s . 3.  F l u o r o a c e t a t e (3 mM),  does not affect r e s p i r a t i o n but inhibits  t h e f o r m a t i o n of g l u t a m i n e ammonia formation.  i n one h o u r a n d e l e v a t e s t h e r a t e of  The G A B A  c o n t e n t i s e l e v a t e d but t h e  a s p a r t a t e l e v e l f a l l s to a s m a l l e x t e n t .  Glutamate,  and other  a m i n o a c i d c o n t e n t s s e e m not to be a f f e c t e d w i t h 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 s l i c e s .  Rat brain cortex slices were incubated in K r e b s - R i n g e r phosphate glucose medium in at 3 7 ° C , in the presence or absence of ouabain (0. ImM), D L - m c t h i o n i n c D L - s u l f o x i m i n e (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  GABA  Aspartate  Alanine  Glycine  Ammonia  One hour incubation Nil + methionine ) sulfoximine ) + ouabain + fluoroacetate + nvilonate + amytal + DNP  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  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  11.29 11.46 8.75 10.62 9.69  + + + + +  2.35 3. 64 6.00 2.94 3.48  + 0.08 + 0. 12 + 0.28 _+ 0.20 + 0. 15  4.32 3.63 3.98 6.03 4.50  + 0.29 0. 15 + 0. 12 + 1.11 + 0.38  3.88 3.23 1.20 2.50 2.59  + 0.27 + 0.23 + 0.03 + 0.04 + 0.25  1. 1. 1. 1. 2.  ±  0. 08 0. 02 + 0. 10 + 0.09 0. 12  1.61 1.47 1.23 1.15 1.53  + 0.11 0.03 + 0.06 + 0. 19 + 0.06  10.92 10.61 6.67 8.00 11.43  + 0.29 + 0.21 + 0.44  0.25  0.14 0.32 0.26 0.42 0.40  92 35 17 53 47  + 0. 14 + 1.01  Four hour incubation Nil + methionine sulfoximine + ouabain + amytal +DNP  ) )  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  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  6.52 10.82 8.42  + 0.33 + 0.42 + 0.14  1.96 2.41 3.42  + 0.24 + 0.21 + 0.12  2.25 7.75 4.15  + 0.23 + 0.32 + 0.56  2.64 2.38 2.53  + 0.05 + 0.30 + 0.05  2. 76 2. 96 3. 52  + 0. 19 0.09 + 0.02  1.74 2.16 2. 13  + 0.14  18.20 9.92 16.20  + 0.30 + 0.58 + 0.50  + 0.11 + 0.08  TABLE  15.  E f f e c t s o f o u a b a i n , m e t h i o n i n e s u l f o x i m i n e , K C 1 , 2,4 d i n i t r o p h e n o l , and glucose absence,  on the r a t e s of oxygen c o n s u m p t i o n  amytal,  in rat cerebral cortex  slices. 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 w e r e i n c u b a t e d i n K r e b s - R i n g e r p h o s p h a t e 3 7 ° C f o r v a r i o u s p e r i o d s of t i m e . as p m o l e / g i n i t i a l wet wt.  C o n t r o l values f o r oxygen consumption  g l u c o s e m e d i u m at  (QO^) a r e e x p r e s s e d  O t h e r v a l u e s of Q O £ a r e g i v e n a s p e r c e n t a g e s of t h e c o n t r o l ,  with  s t a n d a r d d e v i a t i o n s f r o m the m e a n not e x c e e d i n g + 5 % .  Qo Time  2  -  %  Control  Control  (min)  QO  z  Ouabain  Methionine  (0.  Sulfoximine  ImM)  2, 4 KCl(lOOmM)  DNP  (O.lmM)  Amytal  Glucose  (ImM)  free  (5mM)  medium 123  96  72  89  101  124  93  55  83  102  102  123  90  48  73  103  102  128  85  43  66  15  24  +  2  108  30  46  +  3  106  45  71  + 1  60  9 6 + 2  98  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  M a l o n a t e (2 mM),  which strongly inhibits respiration,  has little  o r n o e f f e c t o n t h e r a t e of g l u t a m i n e s y n t h e s i s o r a m m o n i a formation.  It s t r o n g l y i n h i b i t s a s p a r t a t e f o r m a t i o n  that of G A B A . levels.  There are diminutions  Further  and enhances  i n g l u t a m a t e and g l y c i n e  e x p e r i m e n t s c a r r i e d out w i t h m a l o n a t e a n d  f l u o 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 ( C h a p t e r 8). DNP  at a c o n c e n t r a t i o n of 0.1 mM,  w h i c h i n h i b i t s o x y g e n uptake,  i n h i b i t s g l u t a m i n e s y n t h e s i s a n d a t the s a m e t i m e ammonia,  enhances  G A B A a n d a l a n i n e l e v e l s i n one a n d f o u r h o u r  tion periods.  G l u t a m a t e i s little affected but aspartate  f a l l d u r i n g the one hour i n c u b a t i o n .  DNP  (0.01 mM)  sumption,  levels.  b r i n g s a b o u t a s t r o n g i n h i b i t i o n of o x y g e n c o n -  a c c o m p a n i e d b y a s u p p r e s s e d r a t e of g l u t a m i n e  w h i c h i s only p a r t i a l l y accountable production. The  levels  stimulates  r e s p i r a t i o n w i t h no e f f e c t s on g l u t a m i n e a n d a m m o n i a  A m y t a l (1 mM),  incuba-  for by i n c r e a s e d  synthesis,  ammonia  T h e r e i s a s u b s t a n t i a l i n c r e a s e i n the G A B A  level.  c o n t e n t of g l u t a m a t e is, u n a f f e c t e d i n one h o u r b u t a h i g h e r  l e v e l i s obtained i n four hours.  T h e i n h i b i t i o n of the r a t e of  g l u t a m i n e s y n t h e s i s i s d o u b t l e s s p a r t l y due to the i n h i b i t i o n of t h e N A D * - l i n k e d glutamate oxidation, through s u p p r e s s i o n oxidation,  of N A D H  a n d t h e r e f o r e of a m m o n i a r e l e a s e f r o m g l u t a m a t e ,  p a r t l y due to t h e s u p p r e s s e d g e n e r a t i o n of A T P .  An  elevated  e n d o g e n o u s g l u t a m a t e c o n c e n t r a t i o n r e s u l t s i n an e n h a n c e d  GABA  c o n t e n t a c c o r d i n g to t h e f o l l o w i n g s c h e m e .  ,  ^  C0  ±,  ^  1  Glutamate + succinic s f i m i a l H s h y d s ^ NAD N H A  +  +  NAD  0  ^ GABA +  -^s u c c i n at e-<-  and  2  + cv-keto glutarate H  96  S i n c e b o t h a-ketoglutarate N A D * f o r oxidation, its G A B A  by-pass  and succinic semiaLdehyde r e q u i r e  t h e 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 a n d  a r e b o t h s u p p r e s s e d i n t h e p r e s e n c e of  amytal,  due to a g r e a t l y r e d u c e d N A D + / N A D H c o n c e n t r a t i o n  ratio.  I n t e r c o n v e r s i o n of g l u t a m a t e a n d G A B A  may  reversibly  o c c u r , b u t i t 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 g l u t a m a t e t h a t t r i p s the b a l a n c e i n f a v o r of G A B A  4.8  accumulation.  E f f e c t s of t e t r o d o t o x i n o n o u a b a i n i n h i b i t i o n o f g l u t a m i n e  synthesis  in r a t b r a i n cortex s l i c e s .  C h a n a n d Q u a s t e l (198) h a v e d e m o n s t r a t e d  that the i n h i b i t i o n o f  a c e t a t e o x i d a t i o n 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 d u e to s o d i u m i n d u c e d by e l e c t r i c a l s t i m u l a t i o n ,  i s r e v e r s e d by T T X .  influx  O k a m o t o and  (182) h a v e s h o w n that the i n f l u x of N a * that o c c u r s on i n c u b a t i n g b r a i n i n t h e p r e s e n c e o f o u a b a i n (0.1 m M )  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 .  Quastel slices Such  e f f e c t s of T T X m a y be p r e s u m e d t o be d u e t o i t s a c t i o n o n the s o d i u m c u r r e n t s y s t e m at t h e b r a i n c e l l m e m b r a n e a n d t h e r e f o r e c o n f i n e d to the neurons.  S i n c e we h a v e s h o w n t h a t t h e t i s s u e N a * a n d K *  a r e f a c t o r s con-  t r o l l i n g t h e s y n t h e s i s of g l u t a m i n e , a n d a l s o s i n c e o u a b a i n i s k n o w n to a f f e c t the c a t i o n i c c o n c e n t r a t i o n s of b r a i n t i s s u e ( s e e T a b l e 65), t h e q u e s t i o n a r i s e s to w h e t h e r 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 i n h i b i t i o n of g l u t a m i n e s y n t h e s i s d u e to o u a b a i n . i n T a b l e 16.  R e s u l t s o b t a i n e d w i t h 0 .0 1 m M  ouabain are given  T h e y s h o w that T T X (2 |iM) h a s l i t t l e o r no e f f e c t o n the o u a b a i n  s u p p r e s s e d s y n t h e s i s of g l u t a m i n e 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 0 at 3 7 ° C f o r o n e h o u r i n K r e b s - R i n g e r p h o s p h a t e a l s o f o u n d t o be t r u e w h e n 0.1 m M  glucose medium.  o u a b a i n w a s used.)  g l u t a m a t e a r e n o t d i m i n i s h e d b y 0.01 m M  ouabain,  2  (This was  T h e t i s s u e l e v e l s of  so that g l u t a m a t e a n d N H ^ *  c o n c e n t r a t i o n s a r e n o t 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 s y n t h e s i s of glutamine.  The  a b o v e r e s u l t s l e a d to t h e s u g g e s t i o n that g l u t a m i n e s y n t h e s i s o c c u r s  TABLE  16.  E f f e c t s o f t e t r o d o t o x i n o n t h e o u a b a i n s u p p r e s s e d g l u t a m i n e s y n t h e s i s of r a t b r a i n cortex  slices.  Rat b r a i n c o r t e x s l i c e s w e r e 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. O l m M ) o u a b a i n w i t h o r w i t h o u t t h e a d d i t i o n o f t e t r o d o t o x i n (2[iM) a t 3 7 ° C f o r o n e h o u r .  Tissue  v a l u e s a r e e x p r e s s e d a s p m o l e s / g i n i t i a l wet wt. a n d m e d i u m v a l u e s a s p m o l e s / g i n i t i a l wet wt. / 3 m l . medium.  Ouabain Tissue  Medium  Ouabain + Total  Tissue  Tetrodotoxin Medium  Total  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  Aspartate  3. 76  + 0.40  0. 50  + 0.10  4. 26  3.63  + 0.33  0.45  + 0. 05  4.08  GABA  2. 47  + 0.47  0. 19  + 0.01  2. 66  2. 83  + 0.59  0. 10  + 0. 06  2.93  Glycine  0. 85  + 0. 03  0. 62  + 0.03  1. 47  0.95  + 0.10  0.49  + 0.08  1.44  Alanine  0. 70  + 0.05  1. 14  + 0.09  1. 84  0.78  + 0.05  1.10  + 0o 05  1. 88  Ammonia  1. 35  + 0. 08  8. 03  + 0.83  9. 38  1.40  + 0.02  8.19  + 0. 13  9.59  98  i n t h o s e b r a i n c e l l s that a r e u n a f f e c t e d b y T T X , v i z . , t h e g l i a , means proof.  T h i s s u g g e s t i o n w i l l be c o n s i d e r e d a g a i n w h e n  the e f f e c t s of T T X o n o u a b a i n i n d u c e d r e l e a s e  b u t i s b y no considering  of a m i n o a c i d s o t h e r t h a n  g l u t a m i n e f r o m b r a i n s l i c e s ( C h a p t e r 7).  4.9  Summary  1.  T h e s y n t h e s i s of g l u t a m i n e b y 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 t h e p r e s e n c e of g l u c o s e i s s u p p r e s s e d u n d e r a v a r i e t y of c o n d i t i o n s that e n h a n c e t h e t i s s u e N a * / K *  c o n c e n t r a t i o n r a t i o , e.g.,  the p r e s e n c e i n the i n c u b a t i o n m e d i u m o f o u a b a i n (0.1 o r 1.0 o r m e t a b o l i c i n h i b i t o r s s u c h as D N P o r f l u o r o a c e t a t e (1 mM),  (0.1 mM),  mM),  a m y t a l (1 mM),  or when C a " ^ i s omitted.  Under these  c o n d i t i o n s t h e r e i s a n e n h a n c e d r a t e of a m m o n i a f o r m a t i o n .  This  i s u n l i k e l y to be d u e to a n e n h a n c e d g l u t a m i n a s e a c t i v i t y a s t h e r a t e of a m m o n i a l i b e r a t i o n f r o m a d d e d L - g l u t a m i n e i s u n a f f e c t e d by these i n h i b i t o r s .  2.  A l t h o u g h Ca"** i s k n o w n to i n h i b i t c o m p e t i t i v e l y the M g * * r e q u i r i n g glutamine synthetase isolated f r o m b r a i n tissue, i t e n h a n c e s t h e r a t e of g l u t a m i n e s y n t h e s i s i n 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 t h e p r e s e n c e of g l u c o s e o r L - g l u t a m a t e .  3.  R e s u l t s s u g g e s t that o u a b a i n i n h i b i t s the s y n t h e s i s of g l u t a m i n e by s u p p r e s s i n g t h e t r a n s p o r t of g l u t a m a t e to t h e s i t e of g l u t a m i n e s y n t h e s i s a n d by i n d u c i n g a l o c a l i z e d d r o p i n t h e e n e r g y l e v e l of the c o m p a r t m e n t c o n c e r n e d w i t h m u c h of the s y n t h e s i s of g l u t a m i n e i n the b r a i n .  4.  T h e i n h i b i t i o n of g l u t a m i n e s y n t h e s i s i n the p r e s e n c e of D N P (0.1 mM),  m e t h i o n i n e s u l f o x i m i n e (5 mM),  o r o u a b a i n (0.1 m M )  f l u o r o a c e t a t e (3 mM),  r e s u l t s i n an enhanced a m m o n i a f o r m a t i o n ,  u n d e r c o n d i t i o n s w h e r e t h e r e i s l i t t l e o r no c h a n g e i n the g l u t a m a t e  99  content  of t h e t i s s u e .  T h i s s u g g e s t s that g l u t a m i n e  occurs  ina compartment  separate  synthesis  f r o m that c o n t a i n i n g t h e m a i n  b u l k of the e n d o g e n o u s g l u t a m a t e of t h e b r a i n t i s s u e .  5.  A l t h o u g h t e t r o d o t o x i n (2 p M ) i s k n o w n to d i m i n i s h t h e e n h a n c e d t i s s u e Na /K"'* c o n c e n t r a t i o n r a t i o due to o u a b a i n (0.01 o r 0.1 +  i t h a s l i t t l e o r no e f f e c t o n t h e o u a b a i n i n d u c e d glutamine synthesis. occurs  s u p p r e s s i o n of  T h i s s u g g e s t s that g l u t a m i n e  synthesis  m a i n l y i n a T T X - i n s e n s i t i v e c o m p a r t m e n t of the b r a i n .  mM),  100  5.  TRANSPORT OF  L-GLUTAMATE  INTO BRAIN  IN V I T R O  It i s a w e l l k n o w n f a c t that c e r e b r a l c o r t e x s l i c e s r e s p i r i n g i n p h y s i o logical glucose  saline m e d i u m accumulate L-glutamate against a  t i o n g r a d i e n t (106, 231, 252, 295).  We  concentra-  have shown e a r l i e r that the o x i d a t i o n  of e n d o g e n o u s g l u t a m a t e a c c o u n t s to a l a r g e e x t e n t f o r the l i b e r a t i o n of a m m o n i a a n d t h e r e s p i r a t i o n of b r a i n s l i c e s i n c u b a t e d i n t h e a b s e n c e of glucose  ( C h a p t e r 3).  H o w e v e r , e v e n i n the a b s e n c e of C a * *  u t i l i z a t i o n of a m m o n i a impeded,  w h e n the  (through the s y n t h e s i s of g l u t a m i n e ) i s s e r i o u s l y  r a t b r a i n s l i c e s g i v e o n l y 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 t h e r a t e of  ammonia formation T a b l e 8).  w h e n L - g l u t a m a t e i s a d d e d to a g l u c o s e - f r e e m e d i u m ( s e e  T h i s c o u l d be due t o t h e s l o w t r a n s p o r t ( o r p e n e t r a t i o n ) to the s i t e  of a m m o n i a f o r m a t i o n glutamate i s present.  where presumably We  the m a j o r  p o o l of e n d o g e n o u s  h a v e c a r r i e d out e x p e r i m e n t s i n a n a t t e m p t to  t h r o w l i g h t o n t h e a c t u a l s i t e i n the b r a i n of the m a j o r  p o o l of e x o g e n o u s  L - g l u t a m a t e t a k e n up b y t h e t i s s u e .  5.1  T r a n s p o r t of L - g l u t a m a t e i n t o i n c u b a t e d r a t b r a i n  R e s u l t s of t h e e f f e c t s of v a r y i n g the m e d i u m L - g l u t a m a t e  concentrations  o n the a c c u m u l a t i o n of t h i s a m i n o a c i d into r a t b r a i n c o r t e x s l i c e s in O  z  a t 3 7 ° C f o r one h o u r i n K r e b s - R i n g e r p h o s p h a t e g l u c o s e  given i n Table (imole/g  17.  T h e t i s s u e v a l u e s of g l u t a m a t e obtained,  respiring  medium are in t e r m s of  i n i t i a l wet wt, a r e c o r r e c t e d f o r s w e l l i n g so a s to g i v e v a l u e s of  c o n c e n t r a t i o n s i n the t i s s u e w a t e r . t i o n s of 2.5, 5, 10, 20 a n d 30 mM,  F o r initial m e d i u m glutamate v a l u e s f o r t h e t i s s u e to m e d i u m  concentraconcentra-  t i o n r a t i o s f o r g l u t a m a t e a r e 9.0, 5.7, 2.9. 1.9 a n d 1.6 r e s p e c t i v e l y . therefore,  e v i d e n t that e x o g e n o u s g l u t a m a t e i s a c c u m u l a t e d a g a i n s t a c o n c e n -  tration gradient i n rat brain cortex slices. Table  It i s ,  It m a y be n o t e d f r o m v a l u e s i n  17 that the a c t i v e c o m p o n e n t of g l u t a m a t e t r a n s p o r t h a s r e a c h e d i t s  s a t u r a t i o n l e v e l at a n e x t e r 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 b e t w e e n 2.5 a n d 5.0 m M .  Increments  of g l u t a m a t e i n t h e m e d i u m a b o v e t h i s  concentration  T A B L E 17.  T r a n s p o r t of s o d i u m - L - g l u t a m a t e  Rat b r a i n cortex slices were incubated i n 0  2  into b r a i n c o r t e x s l i c e s of the r a t .  at 3 7 ° C f o r o n e h o u r i n K r e b s - R i n g e r p h o s p h a t e g l u c o s e  m e d i u m c o n t a i n i n g 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 s o d i u m - L - g l u t a m a t e .  The medium Na* concentration  w a s a d j u s t e d to 148 ( a e q u i v / m l b y a p p r o p r i a t e r e d u c t i o n s o f m e d i u m N a C l . o b t a i n e d , as p m o l c / g  i n i t i a l wet wt. (Tg), w e r e c o n v e r t e d t o \smole/ml  V a l u e s of t i s s u e  tissue water  glutamate  (T) b y 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 . ( T g - E ) v a l u e s w e r e o b t a i n e d b y s u b t r a c t i n g t h e e n d o g e n o u s t i s s u e g l u t a m a t e (E=8.44 p m o l e / g ) f r o m t h e g l u t a m a t e c o n t e n t s o f s l i c e s i n c u b a t e d w i t h e x t e r n a l g l u t a m a t e ; t h e s e v a l u e s w e r e t h e n c o n v e r t e d to n m o l e / m l t i s s u e w a t e r  pmole/ g initial Medium Lglutamate  wet wt. Tissue  Net glutamate uptake (Tg - E )  glutamate (mM)  W a t e r Uptake.  Tg  Nil  8.44  2.5 5.0 10.0 20.0 30. 0  16. 60  + 0.5S + 1. 00  23.50  +  0. 70  15.06  27. 60  +  2.00  19.16  38.00  + 2.80  29.56  49.20 + 1.40  40.76  1  Tissue glutamate  Medium  Tissue  glutamate  |imole/ m l  glutamate  Medium  glutamate  H 1/100 m g  (mM) after  i n i t i a l wet  incubation  wt. T  14.8 + 0.6  8.16  (T*) t a k i n g s w e l l i n g into c o n s i d e r a t i o n .  33.8 + 0.4 34.0 + 0.7 35.2 + 1.9 31.8 + 2.9 31.2 + 0.8  8.9  T*  T/M  M 0.018  14.6  7.2  20.5 24.0  13.2 16.6  34.0 44.3  26.4 36.7  1.62 3.59 8.25  + 0. 07 + 0. 03 + 0. 10  18.00 27.50  493.00 9 .01 5.71  2.90 1.89 1.61  T*/M  4.44 3.66 2.01 1.47 1.33  102  o n l y r e s u l t i n p a s s i v e i n f l o w of g l u t a m a t e i n t o the t i s s u e .  F o r example,  i n c r e a s e i n the m e d i u m g l u t a m a t e c o n c e n t r a t i o n f r o m 5 to 30 m M 25 mM),  (i.e.,  an by  e n h a n c e s the t i s s u e c o n c e n t r a t i o n by a b o u t the s a m e v a l u e .  V a l u e s a r e a l s o g i v e n i n T a b l e 17, f o r the net i n c r e a s e s of t i s s u e glutamate.  T h e s e a r e o b t a i n e d by s u b t r a c t i n g the t i s s u e c o n c e n t r a t i o n s of  g l u t a m a t e o n i n c u b a t i o n i n the a b s e n c e of e x t e r n a l g l u t a m a t e , f r o m t h o s e o b t a i n e d on i n c u b a t i o n w i t h e x t e r n a l l y a d d e d g l u t a m a t e .  T h e 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 f o r glutamate, u s i n g net uptake values,  are  g i v e n i n F i g u r e 1 i n w h i c h a c o m p a r i s o n i s m a d e of the uptake of g l u t a m a t e w i t h that of N H  5.2  4  +  .  T h e u p t a k e of L - [ U - ^ C ] g l u t a m a t e by r a t c e r e b r a l c o r t e x  R a t c e r e b r a l c o r t e x s l i c e s w e r e i n c u b a t e d at 3 7 ° C i n  x 10^  under  a  L-fU-^c]  v a r i e t y of c o n d i t i o n s i n the p r e s e n c e o r a b s e n c e of 5 mM, g l u t a m a t e (of s p e c i f i c a c t i v i t y 1.223  slices  cpm/|_imole).  A t the e n d of t h e  i n c u b a t i o n p e r i o d , the g l u t a m a t e c o n t e n t s of the t i s s u e w e r e m e a s u r e d b y the f o l l o w i n g two  1.  methods:  L i q u i d scintillation counting.  This method gives a measure  of the r a d i o a c t i v e g l u t a m a t e t a k e n up by the t i s s u e . 2.  Amino Acid Analyzer  estimations.  This method gives a  m e a s u r e of the t o t a l g l u t a m a t e c o n t e n t of the t i s s u e by the ninhydrin reaction.  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 g l u t a m a t e was  l i t t l e a f f e c t e d at  the e n d of the i n c u b a t i o n p e r i o d by the i n c u b a t i o n p r o c e d u r e , w e r e f a l l 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 g l u t a m a t e . Table  18,  though  there  Results given in  s h o w t h a t 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 f o r l a b e l l e d  g l u t a m a t e a r e c o n s i d e r a b l y l o w e r t h a n t h o s e f o r t o t a l g l u t a m a t e c o n t e n t of the t i s s u e .  However,  i t i s e v i d e n t that, i f o n l y n e t g l u t a m a t e u p t a k e s a r e  T A B L E 18.  Uptake of sodium L - ( U -  C) glutamate by rat brain cortex s l i c e s .  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 m e d i u m . Ouabain when added was (0. ImM) and sodium L - ( U - * C ) glutamate was 5 m M of specific activity 1.223 x 10^ cpm/ umole. Medium N a concentrations was adjusted to 148 u e q u i v / m l . Labelled and T o t a l tissue glutamate determined respectively by liquid scintillation counting and the Amino A c i d Analyzer, as u m o l e / g initial wet wt., were converted to u m o l c / m l 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, f r o m 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. +  +  + +  4  +  Water Uptake Ul/lOOmg initial wet wt.  Conditions o f incubation  A.  K r e b s - Ringer phosphate  1 4  (ii) 60 m i n . incubation Control + L - ( U - C ) glutamate + ouabain + ouabain + L - ( U - C ) glutamate 1 4  B.  Total  umole/g  Umole/  Umole/g  Umole/  ml.  ml.  T  T  Net glutamate Uptake u m o l e / g umole / ml.T*  Medium glutamate (mM) after incubation M  T/M Labelled Total  J )',t M~  glucose  (i) 30 m i n . incubation Control + L - ( U - C ) glutamate  1 4  Tissue Glutamate Labelled  -  -  23.6  +  0.5  12.50  +  34.0  + 0.7.  15.40  37.9  +  1.9  7.73  37.8  +  0.3  11.20  -  -  -  0.42  12.10.  +  0.30  13.42  +  0.95  6.50  0.20  9.50  -  -  9.39 20.60  + 0.23 + 0.02  -  -  19.90  11.21  -  10.82  3.92  +  0.08  3. 1  5.1  2.8  8.44 + 23.50 ,+ 4.95 +  0.33 0.70 0.26  20.48  15.06  13. 14  3.59  +  0.03  3.7  5.7  3.7  12.48  1.12  10.60  7.53  6.42  3.65  + 0.09  1.8  2.9  1.8  + 0.68 + 0.63  15.98  8.24  3.22  +  3.0  5.0  2.6  +  -  -  Medium 11 - glucose 60 m i n . incubation Control + L - ( U - C ) glutamate 1 4  -  -  +  -  9.09  18.77  -  -  9.68  0.08  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 c o n c e n t r a t i o n r a t i o s f o r g l u t a m a t e , the  t i s s u e to m e d i u m r a t i o s f o r b o t h l a b e l l e d and t o t a l g l u t a m a t e a r e i d e n t i -  cal.  T h e s e r e s u l t s w e r e obtained i n g l u c o s e containing m e d i a d u r i n g incu-  b a t i o n p e r i o d s of 30 or 60 m i n u t e s , or  e i t h e r i n the p r e s e n c e of o u a b a i n (0.1  w h e n the b r a i n s l i c e s a r e i n c u b a t e d i n a N a - r i c h , +  K  +  and  Ca  + +  mM),  -free  m e d i u m ( M e d i u m II).  T h e r e s u l t s s u g g e s t that g l u t a m a t e t a k e n up f r o m the m e d i u m a c c u m u l a t e s within the t i s s u e i n a c o m p a r t m e n t d i s t i n c t f r o m another w h i c h c o n t a i n s the  b u l k of the e n d o g e n o u s g l u t a m a t e .  Results given later  the  m a j o r p o o l of e n d o g e n o u s g l u t a m a t e l i e s i n the n e u r o n s  will indicate  that  ( C h a p t e r 8), w h i l e  t h a t of e x o g e n o u s g l u t a m a t e l i e s i n the g l i a .  5.3  A p p a r e n t a b s e n c e of a n e x c h a n g e p r o c e s s b e t w e e n e x t e r n a l L - [ U - 1 ^ C ] g l u t a m a t e a n d e n d o g e n o u s g l u t a m a t e of r a t b r a i n c o r t e x  slices.  E x p e r i m e n t s w e r e c a r r i e d out to o b s e r v e the e x t e n t of e x c h a n g e t a k i n g p l a c e b e t w e e n the b u l k of the g l u t a m a t e i n i t i a l l y p r e s e n t a n d that t a k e n up the  from  incubation medium.  B r a i n c o r t e x s l i c e s of the r a t w e r e i n c u b a t e d at 3 7 ° C f o r 30  minutes  i n K r e b s - R i n g e r p h o s p h a t e g l u c o s e m e d i u m w i t h o r w i t h o u t the a d d i t i o n of 5 mM, At  L - f U - l ^ c J g l u t a m a t e (of s p e c i f i c a c t i v i t y 1.Z23  x 10 ^  cpm/pmole).  the e n d of the i n c u b a t i o n p e r i o d the t i s s u e c o n t a i n e d 1.53 x 10^  i n i t i a l wet wt. tissue. assuming  T h e r e was  cpm/g  a l s o p r e s e n t 20.6 p m o l e g l u t a m a t e / g i n i t i a l wet  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^  wt  cpm/pmole,  that the e n t i r e r a d i o a c t i v i t y i s l o c a t e d i n the g l u t a m a t e .  The  amount  of g l u t a m a t e a b s o r b e d by the t i s s u e f r o m the i n c u b a t i o n m e d i u m m u s t t h e n be (1.53 x 10^/1.223 x 10^) = 12.5 p m o l e / g , t i o n m e d i u m i s 1.223 glutamate/g  x 10^ c p m / n m o l e .  as the s p e c i f i c a c t i v i t y of the i n c u b a T h e r e f o r e , 20.6 — 12.5 = 8.1 ( i m o l e  do not e x c h a n g e w i t h m e d i u m g l u t a m a t e b e c a u s e ,  had t h e r e b e e n  f r e e e x c h a n g e b e t w e e n l a b e l l e d g l u t a m a t e t a k e n up by the t i s s u e a n d the p o o l  105  of e n d o g e n o u s g l u t a m a t e i n the t i s s u e , the  t i s s u e would  glutamate,  we  e q u a l that o u t s i d e i t .  the s p e c i f i c a c t i v i t y of g l u t a m a t e w i t h i n Now,  i n the a b s e n c e of e x t e r n a l  f i n d that the t o t a l g l u t a m a t e 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 b e t w e e n t i s s u e (9.39 + 0.23 L i m o l e / g ) a n d m e d i u m (0.77 + 0.08 |jmole/g/3 m l ) .  T h e r e f o r e , i t a p p e a r s that (8.1 x 100)/9.39,  or about  87 p e r cent, of t h e e n d o g e n o u s t i s s u e g l u t a m a t e d o e s n o t e x c h a n g e w i t h l a b e l l e d g l u t a m a t e a d d e d t o the i n c u b a t i o n m e d i u m . b e c a u s e the t o t a l t i s s u e r a d i o a c t i v i t y was m a t e i n the t i s s u e ,  Actually,  the p e r c e n t a g e i s h i g h e r  u s e d to e s t i m a t e the l a b e l l e d g l u t a -  a n d t h e r e l a t i v e l y s m a l l a m o u n t s of r a d i o a c t i v i t y  b u t e d b y g l u t a m a t e m e t a b o l i t e s w e r e not t a k e n into a c c o u n t . a c t i v i t y of the i n c u b a t i o n m e d i u m was c o r t e x s l i c e s f o r 30  5.4  The  contri-  specific  but l i t t l e c h a n g e d by the i n c u b a t i o n w i t h  minutes.  L o c a t i o n of e x o g e n o u s L - g l u t a m a t e  uptake.  Rat b r a i n cortex s l i c e s were loaded with radioactive L-glutamate pre-incubation  in 0  2  at 3 7 ° C  f o r 30 m i n u t e s i n K r e b s - R i n g e r  glucose m e d i u m containing 5 mM 1.223  x 10^  cpm/(imole.  were quickly removed,  by  phosphate  L - f U - ^ ^ c J g l u t a m a t e of s p e c i f i c a c t i v i t y  A t the e n d of the i n c u b a t i o n p e r i o d , l i g h t l y b l o t t e d to s o a k up a d h e r i n g f l u i d ,  a t o r s i o n b a l a n c e to m e a s u r e w a t e r uptake, one h o u r i n one of the f o l l o w i n g i n c u b a t i o n  and i n c u b a t e d i n  the s l i c e s weighed  on  at 3 7 ° C f o r  media:  A.  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 .  B.  M e d i u m A,  c o n t a i n i n g p r o t o v e r a t r i n e (5 L M )  C.  M e d i u m B,  containing T T X  a n d o u a b a i n (0.1  mM).  (2 | i M ) .  T h e 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 s the a c t i v a t i o n of the s o d i u m  current  i n b r a i n s l i c e s (182) w i t h r e s u l t a n t e f f l u x e s of a m i n o a c i d s (310), w h i l e that of o u a b a i n b l o c k s the r e - u p t a k e of the r e l e a s e d a m i n o a c i d s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t (104,  112).  T h e p r e s e n c e of T T X  by i t s s e l e c t i v e a c t i o n on  106  the n e u r o n s  s u p p r e s s e s t h e n e u r o n a l e f f l u x of a m i n o a c i d s f r o m b r a i n  cortex  s l i c e s b r o u g h t a b o u t b y the j o i n t a c t i o n of p r o t o v e r a t r i n e a n d o u a b a i n .  E x a m i n a t i o n of the r e s u l t s i n T a b l e 19 s h o w the f o l l o w i n g f a c t s .  1.  T h e a p p a r e n t s p e c i f i c a c t i v i t y of g l u t a m a t e i n t h e i n c u b a t i o n m e d i u m d u r i n g t h e one h o u r i n c u b a t i o n f o l l o w i n g t h e l o a d i n g o f the t i s s u e w i t h l a b e l l e d g l u t a m a t e ,  i s m u c h h i g h e r t h a n t h a t of  the g l u t a m a t e i n the o r i g i n a l i n c u b a t i o n m e d i u m .  T h i s i s d u e to  the f a c t that, d u r i n g t h e one h o u r i n c u b a t i o n p e r i o d ,  the l a b e l l e d  g l u t a m a t e t a k e n up o r i g i n a l l y b y t h e t i s s u e h a s b e e n c o n v e r t e d t o o t h e r l a b e l l e d a m i n o a c i d s (e.g.,  aspartate,  alanine,  glutamine,  ( T a b l e 20), w h i c h a l s o a p p e a r i n t h e i n c u b a t i o n m e d i u m ( T a b l e 21). The  apparently high specific activity i s illusory,  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 t h e m e d i u m i s d u e to g l u t a m a t e .  2.  T h e p r e s e n c e of p r o t o v e r a t r i n e a n d o u a b a i n b r i n g s a b o u t  release  of a r e l a t i v e l y l a r g e q u a n t i t y of r a d i o a c t i v i t y f r o m the t i s s u e , together  with a considerable  r e l e a s e of g l u t a m a t e .  The apparent  s p e c i f i c a c t i v i t y of g l u t a m a t e i n the i n c u b a t i o n m e d i u m i s m u c h l o w e r t h a n t h a t f o u n d i n t h e a b s e n c e of t h e d r u g s . t h i s i s d u e to t h e r e l e a s e b y t h e d r u g s  Presumably  of g l u t a m a t e (both l a b e l l e d  a n d u n l a b e l l e d ) f r o m i t s 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 g l u t a m a t e ,  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 a n d o u a b a i n , f r o m the t i s s u e , w i t h o u t a f f e c 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 w o u l d i n d i c a t e that T T X i s  b l o c k i n g t h e r e l e a s e of u n l a b e l l e d g l u t a m a t e .  This  presumably  l i e s i n the n e u r o n s a s T T X i s b e l i e v e d t o a c t o n l y u p o n the n e u r o n s . T h i s e v i d e n c e i n d i c a t e s that a l a r g e r p o o l of e n d o g e n o u s g l u t a m a t e l i e s i n the n e u r o n s .  It w i l l be s h o w n l a t e r that t h i s p o o l i s  a c t u a l l y the m a j o r p o o l of g l u t a m a t e ( C h a p t e r 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 m i n . 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  Pre-incubation  TISSUE (glutamate) Total radioactivity Amount cprn/g U mole/g (xl0°)  cpm per U mole (xlO ) 6  1.53  + 0,05  20.6  + 0.2  0.075  0.57  + 0.03  9.0  + 0.6  0.063  0.19  + 0.01  4.1  + 0.4  0.046  6.9  + 0.1  0.055  M E D I U M (glutamate) Total radioactivity Amount cpm per cpm/g/3ml Umole U m o l e / g/3ml (xl0 ) (xlO ) b  6  Incubation: Additions to K r e b s - R i n g e r phosphate glucose medium A. Nil B. protoveratrine + ouabain C . protoveratrine + ouabain + TTX  0.38 + 0.01  0.509 + 0.05 0.902 + 0.01 0.898 + 0.02  1.53 10.50 5.25  + 0.03  0.332  + 0.34  0.086  + 0.01  0.171  108  4.  T T X b r i n g s a b o u t a r e t e n t i o n of r a d i o a c t i v i t y i n t h e b r a i n c o r t e x slices,  i n c u b a t e d 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 a n d o u a b a i n ,  e q u a l to (0.38 - 0 .19) (100) /1.53 = 12.4 p e r c e n t of t h e r a d i o a c t i v i t y o r i g i n a l l y t a k e n up ( T a b l e 19). glutamate/g initial  wet wt t i s s u e ,  T h i s a m o u n t s to 1.55 p m o l e i f i t i s a s s u m e d t h a t 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 w h o l l y due to g l u t a m a t e .  The amount  w i l l be s m a l l e r a s t h e v a l u e m u s t i n c l u d e r a d i o a c t i v i t y d u e t o 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 a f f e c t 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 g l u t a m a t e f r o m the t i s s u e and, t h e r e f o r e , t h a t m u c h of the l a b e l l e d g l u t a m a t e l i e s n o t i n t h e n e u r o n s , b u t i n the n o n - e x c i t a b l e i.e.,  5.5  cells,  the g l i a .  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 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 o n the t i s s u e a n d m e d i u m c o n c e n t r a t i o n s of a m i n o a c i d s i n i n c u b a t e d r a t b r a i n cortex  slices.  In a d d i t i o n to t h e w o r k o n t h e u p t a k e of e x t e r n a l L - g l u t a m a t e b y i n c u b a t e rat  brain slices,  s t u d i e s w e r e a l s o m a d e of t h e e f f e c t s o f i n c r e a s i n g m e d i u m  c o n c e n t r a t i o n s of L - g l u t a m a t e (up to 30 m M )  o n t h e c o n c e n t r a t i o n s of a m i n o  a c i d s i n the t i s s u e ( T a b l e 20), a n d i n the m e d i u m ( T a b l e 21).  The  medium  N a C l w a s a d j u s t e d to g i v e a m e d i u m s o d i u m c o n c e n t r a t i o n of 148 | i e q u i v / m l .  The and,  hence,  r e s u l t s g i v e n i n T a b l e s 20 a n d 21 s h o w t h a t the t i s s u e a n d m e d i u m t h e t o t a l l e v e l s of a s p a r t a t e ,  GABA,  alanine, and glutamine,  enhanced by i n c r e a s i n g external L-glutamate concentrations. acids,  e.g., g l y c i n e (shown), t a u r i n e , s e r i n e , o r t h r e o n i n e  little affected under these c o n d i t i o n s . ammonia i ntissue or m e d i u m affected. t i o n of glutamine,  alanine or a s p a r t a t e ,  c o n c e n t r a t i o n s (i.e.,  2.5 m M )  are  Other amino  (not shown), a r e  N e i t h e r a r e the c o n c e n t r a t i o n s of M o s t of the c h a n g e i n t h e c o n c e n t r a i s b r o u g h t a b o u t by t h e l o w e s t  of L - g l u t a m a t e i n v e s t i g a t e d .  Increasing  medium  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 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 o n t h e a m i n o a c i d of i n c u b a t e d r a t b r a i n c o r t e x  contents  slices.  B r a i n c o r t e x s l i c e s of the r a t w e r e 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 p h o s p h a t e g l u c o s e m e d i u m c o n t a i n i n g i n c r e a s i n g m e d i u m 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 at 3 7 ° C f o r o n e h o u r . to 148 p e q u i v / m l b y a p p r o p r i a t e r e d u c t i o n s o f m e d i u m N a C l .  T h e m e d i u m s o d i u m was  adjusted  A m o u n t s of a m i n o a c i d s i n the t i s s u e a r e  e x p r e s s e d a s | j m o l e / g i n i t i a l wet wt.  Medium Glutamine  GABA  Aspartate  8. 44 + 0. 58  3. 07 + 0. 26  2. 44 + 0.04  2. 72 + 0.14  1. 09  + 0.11  1. 06  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  L-glutamate  Glutamate  Alanine  Gly cine  (mM) Nil  + 0.01  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 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 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 incubated rat b r a i n cortex s l i c e s .  The  experimental  expressed  c o n d i t i o n s w e r e a s d e s c r i b e d i n T a b l e 20.  a s p m o l e / g i n i t i a l wet wt. t i s s u e / 3 m l .  Amounts of amino acids i n the m e d i u m are  medium.  Medium L - glutamate  Glutamate  Glutamine  GABA  Aspartate  Alanine  Glycine  (mM) Nil  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  -  5. 86  + 0.06  0.45  + 0.02  7. 36  + 0.40  2. 24  + 0.31  0.53  + 0. 02  7. 10  + 0.45  5. 01  + 0.08  2. 46  + 0.17  0.46  + 0. 02  20.0 30.0  Ill  m e d i u m glutamate concentrations,  e n h a n c e s the G A B A  a n d e f f e c t s a s m a l l r e l e a s e of G A B A mM,  external L-glutamate,  or medium,  into the i n c u b a t i o n m e d i u m .  W i t h 2.5  t h e r e i s a b o u t a t w o f o l d i n c r e a s e i n the t i s s u e ,  c o n t e n t of a l a n i n e ;  the t i s s u e a n d m e d i u m c o n t e n t s of a s p a r t a t e  a r e i n c r e a s e d by 50 p e r c e n t a n d 700 p e r cent,  5.6  l e v e l s i n the t i s s u e  respectively.  Summary  1.  T h e r e i s a n a p p a r e n t a b s e n c e of a f r e e e x c h a n g e p r o c e s s b e t w e e n endogenous g l u t a m a t e and l a b e l l e d L - g l u t a m a t e t a k e n up by b r a i n cortex slices against a concentration gradient.  L e s s t h a n 13 p e r  c e n t of the e n d o g e n o u s g l u t a m a t e a p p e a r s to h a v e f r e e a c c e s s to l a b e l l e d L - g l u t a m a t e t a k e n up b y t h e 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 n e u r o n a l r e l e a s e of g l u t a m a t e b r o u g h t a b o u t by the j o i n t a c t i o n of p r o t o v e r a t r i n e a n d o u a b a i n 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 - g l u t a m a t e without a f f e c 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 s u g g e s t s t h a t the  e n d o g e n o u s p o o l of g l u t a m a t e l i e s i n T T X - s e n s i t i v e ments),  i.e., the n e u r o n s ,  w h i l e L - g l u t a m a t e t a k e n up b y the  tissue i slargely present i n a T T X - i n s e n s i t i v e i.e.,  the g l i a .  compart-  compartment(s),  U n d e r these conditions T T X b r i n g s about a  m a x i m a l r e t e n t i o n o f 12.4 p e r c e n t 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 t a k e n up b y t h e t i s s u e a m o u n t i n g t o a v a l u e f o r t i s s u e  glutamate  no l a r g e r t h a n 1.55 ( i m o l e / g i n i t i a l wet wt. 3.  R e s u l t s on t h e e f f e c t s o f 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 c e n t r a t i o n s o n t h e a m i n o a c i d c o n t e n t in, a n d r e l e a s e incubated brain cortex slices,  are given.  con-  from,  112  6.  TRANSPORT OF AMMONIUM  According  to J a c o b s et a l . (253,  IONS I N B R A I N  IN V I T R O  254), the r a t e of t r a n s p o r t of  a m m o n i u m i o n s into e r y t h r o c y t e s c a n n o t be m e a s u r e d b y p r e s e n t as a m m o n i a  p a s s e s the m e m b r a n e so r a p i d l y .  However,  methods  P o s t and J o l l y  (255) u s e d an i n d i r e c t m e t h o d i n t h e i r s t u d i e s a n d s h o w e d that the a d d i t i o n of N H  to N a - f i l l e d r e d c e l l s  br ings  +  4  (outward).  a b o u t a h i g h r a t e of Na"*"  transport  S a t u r a t i n g the t r a n s p o r t s y s t e m w i t h K"* (by i n c u b a t i n g  with  e x t e r n a l K"*) p r o d u c e s o n l y a s m a l l i n c r e a s e i n Na"* t r a n s p o r t b y NH4"*" i o n s . They concluded erythrocytes, comparable  that NH^"** s u b s t i t u t e s d i r e c t l y f o r  i n the s o d i u m p u m p of  b u t r e q u i r e a c o n c e n t r a t i o n 3-7 t i m e s t h a t of  to p r o d u c e a  effect.  T o w e r a n d h i s c o l l e a g u e s (256, cat b r a i n cortex slices. the p r e s e n c e o f 10 m M  257) s t u d i e d the t r a n s p o r t of N H ^ * i n  T h e y i n c u b a t e d the t i s s u e at 3 7 ° C f o r one h o u r i n NH^Cl  and, s i n c e t h e y o b t a i n e d a h i g h e r  internal  (i.e., t i s s u e ) t h a n e x t e r n a l (i.e., m e d i u m ) NH^"* c o n c e n t r a t i o n at the e n d o f the i n c u b a t i o n p e r i o d , t h e y c o n c l u d e d d e p e n d e n t u p t a k e of f r e e N H ^  +  .  that t h e r e was an " a c t i v e " or e n e r g y -  I n a t y p i c a l e x p e r i m e n t c i t e d (256),  c o n t e n t ( c o r r e c t e d f o r s w e l l i n g ) w a s 11.35  (imole/g  impossible NH  + 4  T h i s gave an apparent  w h i c h they s a i d was c l e a r l y an  v a l u e u n l e s s i n t e r p r e t e d to m e a n a n " a c t i v e " c o n c e n t r a t i o n of  i n the i n t r a c e l l u l a r The  + 4  w h i l e the m e d i u m c o n c e n -  t r a t i o n a t the e n d of the i n c u b a t i o n w a s 8.52 ( i m o l e / m l . "NH^** s p a c e " i n the t i s s u e of 133 p e r cent,  the N H  s p a c e s of t h e t i s s u e .  w o r k with r a t 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 i n this chapter  is in  a g r e e m e n t w i t h the v a l u e s of NH^"*" o b t a i n e d b y T o w e r a n d c o w o r k e r s , b u t our  interpretation differs considerably f r o m theirs.  We  c o n c l u d e t h a t NH "* 4  i o n s a r e n o t t a k e n up a g a i n s t a c o n c e n t r a t i o n i n b r a i n c o r t e x s l i c e s , b u t e n d o g e n o u s NH."* i s f o r m e d w i t h i n s p e c i f i c c o m p a r t m e n t s i n the b r a i n t i s s u e . independently sum  of the c o n c e n t r a t i o n of NH^"*" i n the i n c u b a t i o n m e d i u m .  of the t i s s u e e n d o g e n o u s NH^"*" a n d that w h i c h h a s e n t e r e d  The  by p a s s i v e  ii  d i f f u s i o n , g i v e s the a p p e a r a n c e o f " a c t i v e " N H ^ p u b l i s h e d d a t a of W h e r e t t a n d T o w e r (76) s u p p o r t  transport.  Recently-  our i n t e r p r e t a t i o n .  r e s u l t s of e x p e r i m e n t s d i r e c t e d to t h r o w l i g h t o n N H ^  +  The  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 m e d i u m contents incubated  of NH."*" 4 of r a t b r a i n c o r t e x  slices  under v a r y i n g conditions.  S o m e o f the s t u d i e s on t h e d i s t r i b u t i o n b e t w e e n t i s s u e a n d m e d i u m o f the a m m o n i a f o r m e d ,  w h e n b r a i n c o r t e x s l i c e s of the r a t a r e i n c u b a t e d 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, a r e given i n Table  22.  a m m o n i a contents  It i s e v i d e n t f r o m the r e s u l t s i n t h i s T a b l e , of t h e 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  different incubation conditions.  that t h e  constant under many  T h e r e s u l t s s h o w t h a t the v a r i a t i o n i n t h e  r a t e s of a m m o n i a f o r m a t i o n b y 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 m e d i a of d i f f e r i n g c o n t e n t s , tion medium.  a r e l a r g e l y c o n f i n e d to t h e N H ^ *  contents  of the i n c u b a -  F o r e x a m p l e , t h e i n c u b a t i o n m e d i u m c o n c e n t r a t i o n of NH^"*" i n  the a b s e n c e of g l u c o s e  i s m o r e than t h r e e t i m e s that i n its p r e s e n c e .  o v e r , the i n h i b i t i o n of a m m o n i a u t i l i z a t i o n i n t h e p r e s e n c e of g l u c o s e due  to t h e i n h i b i t i o n of the s y n t h e s i s of g l u t a m i n e ) b y o u a b a i n ,  sulfoximine,  f l u o r o a c e t a t e , a n d 2, 4 - d i n i t r o p h e n o l ,  i n c r e a s e d m e d i a NH^* o r l e s s the same.  contents,  manifests  (largely  methionine itself in  while the t i s s u e c o n c e n t r a t i o n s r e m a i n  more  These facts a r e understandable if a m m o n i a is f o r m e d i n  s p e c i f i c c o m p a r t m e n t f s) of t h e b r a i n c e l l s a n d a b o v e a l i m i t i n g in such compartments,  NH^"*" i s r e l e a s e d i n t o the c y t o p l a s m  the i n c u b a t i o n m e d i u m .  under various conditions level.  concentration  and thence into  T h i s i n t e r p r e t a t i o n w o u l d e x p l a i n why a m a j o r  p o r t i o n of the a m m o n i a f o r m e d b y c e r e b r a l c o r t e x s l i c e s  constant a m m o n i a  More-  during  pro-  respiration  i s f o u n d i n the m e d i u m w h i l e t h e t i s s u e r e t a i n s a  T A B L E 22.  The  t i s s u e and m e d i u m c o n c e n t r a t i o n s of N H ^  on i n c u b a t i n g r a t b r a i n c o r t e x s l i c e s  +  u n d e r a v a r i e t y of c o n d i t i o n s . R a t b r a i n c o r t e x s l i c e s w e r e i n c u b a t e d at 3 7 ° C i n K r e b s - R i n g e r p h o s p h a t e m e d i u m i n the p r e s e n c e o r absence of glucose o r metabolic are expressed tissue/3  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 g i v e n b e l o w .  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  Tissue  hour incubation  + ouabain  C a ^ - f r e e ; two  (5mM)  hour incubation  G l u c o s e ( l O m M ) ; one h o u r i n c u b a t i o n + ouabain  (O.OlmM) (0.  ImM)  + sodium fluorcacetate  (5mM) (ImM)  + ouabain (O.lmM) + fluoroacetate DNP  (0.  (2mM)  (lOmM); four h o u r incubation  + methionine-sulfoximine  (5mM)  A  Medium  2. 95 3. 14  + 0. 13  19.35  1. 89 2. 15 2. 06  + + + +  2. 22  +  2. 38  + 0.26 + 0.22 + 0.34  1. 96 2. 30  ImM)  + s o d i u m malonate  (ImM)  NH +  14. 60  2. 12  + methionine-sulfoximine  Glucose  contents  NH4* c o n t e n t s a s l i m o l e / g i n i t i a l wet  + 0.27 + 0. 15 + 0.11  2. 66 2. 65  (O.OlmM)  + methionine-sulfoximine  + 2,4  +  ml.  A d d i t i o n s to the i n c u b a t i o n m e d i u m N i l ; one  T i s s u e NH^_  1. 73 2.  9 0  +  + 0.32 + 0.25 16. 76 + 0. 05 13.85  0.16  4. 13  0. 05  7.55  0. 15  8.80  0.11  9.36  0.23  8.62  + 0.05 + 0.42  NH4  8.89 9 . 9 0  5.40 4.93 17.42  + 0.18 + + + + + + + +  Total 17. 26 16. 50 19.71  22.49  0.28  6. 02  0.36  9. 70  0.44  10.92  0. 07  11.42  0. 58  10. 84  0.65  11.27  0.55  11. 86  0.20  7. 70  + 0. 10 + 0.50  6.66 20. 32  wt.  1i5  T i s s u e / M e d i u m concentration ratios forN H 4  6.2  brain  i n the i n c u b a t e d  slice.  F u r t h e r evidence ammonia formation  i n support  of the c o n c e p t of c o m p a r t m e n t a t i o n of  i n b r a i n c e l l s i s o b t a i n e d f r o m a study of t h e 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 a m m o n i a a t t h e e n d of one h o u r tion periods.  T h e 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 a r e l a r g e r t h a n one,  under a l l incubation conditions tested. are given in Table in Table  incuba-  23.  R e s u l t s of a f e w t y p i c a l  examples  These results are calculated f r o m values  22, t a k i n g s w e l l i n g of the t i s s u e i n t o c o n s i d e r a t i o n .  From  r e s u l t s i t c a n be s e e n that e v e n i n t h e p r e s e n c e of 10 | i M o u a b a i n i n h i b i t o r of a c t i v e t r a n s p o r t p r o c e s s e s ) r a t i o i s a s h i g h as 9. to  accumulation  in a glucose  reported these  (a p o t e n t  c o n t a i n i n g m e d i u m , the  If the 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 w e r e d u e  of N H ^ *  against a concentration gradient,  a l l e v i d e n c e (104)  i n d i c a t e s that the p r e s e n c e of o u a b a i n w o u l d r e d u c e t h i s to n e a r u n i t y . Anaerobic (e.g.,  incubations (see T a b l e  2 hours),  26) o r i n c u b a t i o n f o r l e n g t h y  durations  h a v e l i t t l e e f f e c t s o n t h e t i s s u e a m m o n i a content,  and the  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 NH^"*" a r e c o n s i d e r a b l y l a r g e r t h a n one  o n i n c u b a t i o n of the s l i c e s w i t h 0.1 m M  DNP  ( T / M = 5.2),  s o d i u m m a l o n a t e ( T / M = 12.3), o r i n the a b s e n c e of g l u c o s e  o r with  2  mM  ( T / M = 4.0).  S u c h c o n d i t i o n s a r e k n o w n to r e d u c e e n e r g y d e p e n d e n t t r a n s p o r t a g a i n s t a concentration gradient. evidence  T h e f a c t that h i g h v a l u e s a r e s t i l l r e a c h e d i s  against active transport.  The  r e l a t i v e l y h i g h 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 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 the f a c t that w h i l e the t i s s u e N H ^ *  ( T / M = 14.5) i s h e l d to be due to  c o n c e n t r a t i o n i s little affected, the total  r a t e of N H 4 " * * f o r m a t i o n i s m u c h d i m i n i s h e d , i s r e l e a s e d i n t o the i n c u b a t i o n m e d i u m . ratio for ammonia.  so that r e l a t i v e l y l i t t l e  T h i s results in a high  NH4"*  Tissue/Medium  T A B L E 2 3 . T h e 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 f o r NH4 v a r i e t y of m e d i a .  i n rat b r a i n cortex slices incubated  T i s s u e v a l u e s o f a m m o n i a a r e o b t a i n e d f r o m T a b l e 22 a n d e x p r e s s e d  as ^ m o l e / m l tissue water,taking  c o n s i d e r a t i o n t h e s w e l l i n g of the t i s s u e at t h e e n d of t h e i n c u b a t i o n p e r i o d . f r o m T a b l e 22, a r e e x p r e s s e d be  100 m g .  3x10  The medium  in a  into  M e d i u m v a l u e s of a m m o n i a ,  a s p m o l e / m l t a k i n g the a v e r a g e i n i t i a l w e t wt. o f the t i s s u e i n v e s t i g a t e d t o  NH4*  concentration (pmole/ml = mM)  i s t h e m e d i u m v a l u e ( T a b l e 22) d i v i d e d b y  = 30.  A d d i t i o n s to K r e b s - R i n g e r phosphate  N i l ; one h o u r  medium  incubation  Tissue  NH + 4  |i m o l e / g i n i t i a l wet wt.  Tissue  NH  + 4  Medium N H  \1 m o l e / m l  mM  T  M  + 4  _T_ M  2.66  2. 13 2. 10  0.49 0.46  4.4  2.65 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  G l u c o s e ( l O m M ) ; one h o u r i n c u b a t i o n  1.89 2.15  2. 02  0. 14  14. 5  1.96  0.25  7. 8  2.12  1. 80  0.29  6.2  2.06  2. 15  0.31  6.9  + s o d i u m fluoroacetate (ImM)  2.22  2.30  0.29  + o u a b a i n (0. I m M ) + f l u o r o a c e t a t e ( I m M )  2.38  2. 06  0.30  7.9 6.9  + ouabain  (O.OlmM)  + methionine-sulfoximine  (5mM)  + o u a b a i n (0. O l m M ) (O.lmM) + methionine-sulfoximine  (5mM)  + 2, 4 - D N P (0. I m M )  1.96  1. 70  0.33  + sodium malonate  2.30  2.23  0. 18  (2mM)  4.6  5.2 12.3  i 17  6.3  Exogenous N H ^  accumulation  +  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  H a v i n g d e m o n s t r a t e d the e x i s t e n c e of a c o n c e n t r a t i o n of NH^"*" i n c e r e b r a l c o r t e x slices incubated i n v a r i o u s media, out to e x a m i n e the a c c u m u l a t i o n one  h o u r at 3 7 ° C  experiments were  carried  of N H ^ * within r a t b r a i n c o r t e x incubated f o r  i n the p r e s e n c e of v a r y i n g c o n c e n t r a t i o n s of N H ^ C l a d d e d t o  the i n c u b a t i o n m e d i u m .  T h e s e s t u d i e s w e r e c a r r i e d out u n d e r the f o l l o w i n g i n c u b a t i o n c o n d i t i o n s , the w a t e r u p t a k e s b y the t i s s u e b e i n g m e a s u r e d at the e n d of the i n c u b a t i o n .  1.  A e r o b i c a l l y i n t h e p r e s e n c e of 10 m M  glucose (Table  2.  A e r o b i c a l l y i n the a b s e n c e of g l u c o s e  (Table  3.  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 10 m M  24).  25).  glucose  ( T a b l e 26).  It i s s e e n f r o m r e s u l t s g i v e n i n T a b l e 24, that w h e n the N H ^  +  content  of the t i s s u e f o u n d i n the a b s e n c e of a d d e d N H ^ C l i s s u b t r a c t e d f r o m the t i s s u e NH^"*" c o n t e n t s o b t a i n e d a t the e n d of the one h o u r i n c u b a t i o n w i t h e x t e r n a l l y added N H  + 4  The  ,  the 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 a r e a p p r o x i m a t e l y  v a l u e of u n i t y i s a l s o o b t a i n e d w h e n the i n c u b a t i o n i s c a r r i e d out  a e r o b i c a l l y i n the a b s e n c e o f g l u c o s e  ( T a b l e 25),  the p r e s e n c e of g l u c o s e  This evidence  ( T a b l e 26).  p a s s i v e d i f f u s i o n of N H ,  +  or even anaerobicallyi n i n d i c a t e s that t h e r e i s only  f r o m the i n c u b a t i o n m e d i u m i n t o the s l i c e .  i n i t i a l c o n c e n t r a t i o n s of N H ^  i n t h e i n c u b a t i o n m e d i u m w e r e u s e d i n the c a l c u -  l a t i o n s o f the 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 .  This is permissible  the u t i l i z a t i o n of NH^"*~ i s s u f f i c i e n t l y l o w n o t to a f f e c t w i t h i n the m e d i u m  NH^  The  +  4  error,  unity.  concentration.  experimental  since  T A B L E 24.  U p t a k e of N H 4 * b y 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  Rat b r a i n c o r t e x s l i c e s were incubated containing increasing concentrations were converted obtained  in  at 3 7 ° C f o r o n e h o u r i n K r e b s - R i n g e r p h o s p h a t e g l u c o s e  of N H 4 C I .  medium  V a l u e s of t i s s u e N H 4 + o b t a i n e d a s p m o l e / g i n i t i a l w e t wt.  to } j m o l e / m l t i s s u e w a t e r (T) b y t a k i n g 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 .  b y s u b t r a c t i n g the e n d o g e n o u s t i s s u e N H 4 c o n t e n t  incubated  i n the p r e s e n c e o f g l u c o s e .  (Tg-E) values a r e  ( E = 2.5 U m o l e / g ) f r o m the N H 4 c o n t e n t s  with external N H 4 + ; these values were then converted  (Tg)  of slices  t o p . m o l e / m l t i s s u e w a t e r (T*) t a k i n g s w e l l i n g  into c o n s i d e r a t i o n .  pmole/g initial N H 4 C I  4  wet wt.  Medium Tissue  NH + 4  Water Uptake Net NH  pl/100 mg +  4  (mM) Nil 2. 0 5.0  2.50  _+ 0 . 2 0  4.14 6.68  + 0.19 + 0.40  10. 0 20. 0  12.42 26.30  30. 0  40.40  Medium  N H 4 C I  (mM) after  Tissue Medium  NH  + 4  NH4+  incubation  i n i t i a l wet  uptake Tg  Tissue N H + \l m o l e / m l tissue water  wt.  (Tg - E )  T  T*  T*/M  0.13  + 0.01  21.20  1.81 4.40  + 0.10 + 0.10  2.48 1.60  0.96 1.00  9.32  9.23  + 0.21  1.27  19.80  19.40  + 0.40  1. 13  29.20  + 1.00  1.09  1.01 1.02 1.02  1. 64  11.6 14.6  + 1.3 + 0.3  4. 18  14.6  + 0.2  + 0.12  9.92  26.1  + 1.1  11.70  + 0.50  23. 80  40.0  + 4.0  21.90  + 0.20  37.90  47.8  + 0.6  3.1.70  29.70  2.75 4.48 7.06  T/M  M  1.74 4.42  co  25. Uptake of N H 4  TABLE  b y r a t b r a i n c o r t e x s l i c e s incubated i n the a b s e n c e 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 0  2  at 3 7 ° C f o r o n e h o u r i n g l u c o s e - f r e e K r e b s - R i n g e r p h o s p h a t e  m e d i u m c o n t a i n i n g 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 N H ^ C l .  C a l c u l a t i o n s w e r e done a s d e s c r i b e d i n T a b l e 24.  T h e m e d i u m N H 4 " * c o n c e n t r a t i o n w a s e s s e n t i a l l y u n c h a n g e d at t h e e n d o f t h e i n c u b a t i o n p e r i o d . E = 3.66 ( i m o l e / g .  Tissue N H  (i m o l e / g i n i t i a l NH C1 4  Tissue  NH + 4  Net • NH uptake (Tg - E ) +  4  M(mM)  Tg  Nil 2  3.66  + 0.18  6.19  + 0.15  5  10.10  + 0.22  10  16.00  + 1.20  20  31.40  + 1.40  30  40.10  + 2.70  |i 1/100  mg  +  4  p, m o l e / m l  Water Uptake  wet wt.  Medium  Tissue  NH + 4  Medium N H 4 +  tissue water  i n i t i a l wet wt. T  T*  T/M  T*/M  45.2  + 1.2  2.53  44.2  + 0.8  4.98  2.4.9  1.01  6.44  48.2  + 1.2  43.0  + 1.3  5. 02 10. 05  1.58 1. 30  1. 00  12. 34  7.88 13. 00  27. 74 36.44  53.7  + 2.0  23.50  20. 76  1.18  1.04  42.0  + 1.4  33. 60  29.98  1. 12  1.00  .2.92 2. 03  1.01  T A B L E 26.  T h e uptake of N H  4  by rat b r a i n cortex slices i n a n o x i a .  Rat b r a i n c o r t e x s l i c e s were incubated at 3 7 ° C for one hour in K r e b s - R i n g e r bicarbonate glucose m e d i u m containing i n c r e a s i n g concentrations of NH4CI i n an atmosphere of N^/CO^ (95%/5%). Water uptake was 50 + 3 p l / 1 0 0 m g initial wet wt. under these conditions. The m e d i u m concentration of N H 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 . T * = (T - 2. 02) | j m o l e / m l . t i s s u e w a t e r . 4  Tissue NH +  Medium  4  T*  NH4CI  M(mM)  p mole Ig initial wet wt.  |imole/ml T  |imole / m l .  -  T i s sue N H  4  +  +  /Medium  NH  T/M  T*/M  12.24  -  Nil  2.63  +  0.26  2.02  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  4  +  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  f o r 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 w e r e c a r r i e d out to c o m p a r e the u p t a k e of NH^"* w i t h that of a n a m i n o a c i d k n o w n to be a c c u m u l a t e d a g a i n s t a gradient.  1.  G l u t a m a t e was  ions  concentration  c h o s e n f o r the f o l l o w i n g r e a s o n s :  It i s p r e s e n t i n h i g h c o n c e n t r a t i o n s  in brain cortex  slices  and  i t s l e v e l d o e s not s u b s t a n t i a l l y c h a n g e o n i n c u b a t i o n f o r one in a glucose 2.  We  hour  c o n t a i n i n g m e d i u m ( T a b l e 2).  h a v e d e m o n s t r a t e d that e x o g e n o u s l a b e l l e d L - g l u t a m a t e  only  s l o w l y e x c h a n g e s w i t h the b u l k of the g l u t a m a t e ( e n d o g e n o u s ) present  i n the t i s s u e ( s e c t i o n 5.3).  This is in accord  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 i n t h i s l a b o r a t o r y  with  the  (258)  s h o w i n g that t i s s u e g l u t a m a t e l a b e l l e d w i t h r a d i o a c t i v e  glucose  d o e s not e x c h a n g e f r e e l y w i t h c o l d g l u t a m a t e i n a s u b s e q u e n t incubation. 3.  We  h a v e a l s o d e m o n s t r a t e d that the u p t a k e of l a b e l l e d g l u t a m a t e  b y c e r e b r a l c o r t e x s l i c e s i s l e s s t h a n the t o t a l t i s s u e g l u t a m a t e 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 u s i n g the A m i n o A c i d The  d i f f e r e n c e b e t w e e n t h e s e two  values  t i s s u e g l u t a m a t e c o n t e n t f o u n d at the end  Analyzer.  i s a b o u t e q u a l to the of the i n c u b a t i o n  period  w i t h no g l u t a m a t e a d d e d to the i n c u b a t i o n m e d i u m ( s e c t i o n 5.2). The  results given in F i g u r e  1 s h o w that,  i n c o n t r a s t to NH^."*",  t h e r e i s e v i d e n c e of a c c u m u l a t i o n of g l u t a m a t e a g a i n s t a c o n c e n tration gradient,  e v e n w h e n the e n d o g e n o u s c o n t e n t of g l u t a m a t e  i s s u b t r a c t e d f r o m the f i n a l t i s s u e v a l u e s a f t e r i n c u b a t i o n .  R a t b r a i n c o r t e x s l i c e s w e r e i n c u b a t e d i n O 2 at 3 7 ° C 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 m e d i u m i n the p r e s e n c e 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 s o d i u m - L - g l u t a m a t e o r N H 4 C L V a l u e s of 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 (T/M) f o r L-glutamate and NHv* a r e t a k e n f r o m T a b l e s 17 and 24 r e s p e c t i v e l y . M e d i u m c o n c e n t r a t i o n s (mM) of g l u t a m a t e or a r e the c o n c e n t r a t i o n s at the b e g i n n i n g of the i n c u bation. •  V a l u e s f o r L - g l u t a m a t e ; total t i s s u e g l u t a m a t e contents u s e d to d e r i v e T / M c o n c e n t r a t i o n r a t i o s f o r g l u t a m a t e . O V a l u e s f o r L - g l u t a m a t e ; net glutamate uptake v a l u e s u s e d to d e r i v e T / M c o n c e n t r a t i o n r a t i o s l o r g l u t a m a t e . A V a l u e s for NH_j ; total t i s s u e N H ^ contents u s e d to d e r i v e T / M concentration ratios for N H 4 ' . A V a l u e s f o r NH.j ; net N H ^ uptake v a l u e s u s e d to d e r i v e T / M concentration ratios for N H ^ . +  T  +  1  6.5  Rates of N H ^  and glycine uptakes by b r a i n cortex s l i c e s .  The kinetics of the passive (?)  accumulation of NH^"* were studied  and c o m p a r e d with the kinetics of the active uptake of gl