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Ammonia metabolism in the brain Benjamin, Abraham M. 1969-12-31

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AMMONIA M E T A B O L I S M IN T H E BRAIN t  by  A B R A H A M M . BENJAMIN (Hons.), University of Bombay,  1961  B.Sc. (Tech.), University of Bombay,  1963  M.Sc. (Tech.), University of Bombay,  1966  B.Sc.  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F M A S T E R O F SCIENCE in the Department of Bio chemi stry  We accept this thesis as conforming to the required standard,  T H E UNIVERSITY  O F BRITISH  April,  1969  COLUMBIA  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  I further for  shall  the  agree that  his  permission  representatives.  of  this  written  thesis  for extensive  g r a n t e d by  J u n e 30»  gain  Biochemistry  1969.  the  It is understood  for financial  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  Da t e  be  British  available for  permission.  Department of  f u l f i l m e n t of  U n i v e r s i t y of  make i t f r e e l y  s c h o l a r l y p u r p o s e s may  by  in p a r t i a l  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  Study;  this  thesis  Department  copying or  for  or  publication  allowed without  my  ABSTRACT It i s known that the functional activity of the n e r v o u s s y s t e m i s a s s o c i a t e d with ammonia f o r m a t i o n and that the a d m i n i s t r a t i o n of a m m o n i u m salts to e x p e r i m e n t a l a n i m a l s produces convulsions. M e c h a n i s m s , therefore, that c o n t r o l ammonia m e t a b o l i s m i n the b r a i n are of importance for b r a i n c e l l function. The presence of a m m o n i a utilizing m e c h a n i s m s i n the b r a i n maintains the l o w f r e e c e r e b r a l ammonia l e v e l s found i n vivo.  There  is, however, a r a p i d formation of ammonia i n the b r a i n on the death of the a n i m a l and a further l i b e r a t i o n of ammonia takes place when i s o l a t e d b r a i n cortex i s incubated a e r o b i c a l l y i n the absence of glucose.  Studies  of these and other aspects of ammonia m e t a b o l i s m f o r m the subject matter of this thesis. The estimation of ammonia in these studies i s b a s e d on a modification of the diffusion technique of Conway. A m m o n i a and amino a c i d analyses have been c a r r i e d out using the B e c k m a n amino a c i d analyzer. The  r a p i d rate of c e r e b r a l ammonia formation that takes place  when the b r a i n i s r e m o v e d f r o m the a n i m a l i s p a r t i a l l y a r r e s t e d by t r i c h l o r a c e t i c a c i d (TCA), p r e s u m a b l y by the inactivation of c e r e b r a l enzymes. O u r r e s u l t s rule out the p o s s i b i l i t y that glutamine, glutamate,  iii taurine and A T P a r e significant contributors to the i n i t i a l or pre-incubation l e v e l s of ammonia and the evidence f a v o r s the involvement of T C A insoluble components as p r e c u r s o r s of such ammonia. In the presence of glucose the pre-incubation l e v e l s of amino a c i d s of c e r e b r a l cortex s l i c e s of the rat a r e maintained during subsequent aerobic incubation at 3 7 ° C for one hour. In the absence of glucose, however, we have found m a r k e d changes i n the pre-incubation l e v e l s of • >  amino acids of c e r e b r a l cortex s l i c e s under these experimental conditions. A considerable r i s e of ammonia also occurs i n the absence of glucose and this can be l a r g e l y accounted for by a net loss of -NFL^-groups of the amino acid pools of b r a i n s l i c e s .  The significant f a l l i n the c e r e b r a l levels of  glutamate and glutamine under these conditions indicates that for short periods of incubation (one hour), these amino acids m a y s e r v e as m a j o r sources of ammonia formation by r e s p i r i n g b r a i n cortex s l i c e s . O u r findings of a m a r k e d suppression of ammonia f o r m a t i o n by c e r e b r a l cortex s l i c e s incubated for one hour either anaerobically, o r aerobically, i n a g l u c o s e - f r e e m e d i u m in the presence of amytal,  D-  glutamate or a-methylglutamate, i m p l i c a t e the oxidative deamination of c e r e b r a l glutamate a s a m a j o r m e c h a n i s m for a m m o n i a l i b e r a t i o n . D-glutamate also acts by inhibiting the h y d r o l y s i s of glutamine. In the presence of glucose a e r o b i c incubation with 2, 4-dinitrophenol, iodoacetate, malonate, h y d r o x y l a m i n e o r D-glutamate, i n c r e a s e s the rate of ammonia f o r m a t i o n by c e r e b r a l cortex s l i c e s . T h i s i s doubtless due to  diminished activity of c e r e b r a l glutamine synthetase r e q u i r e d for ammonia fixation to occur. We find that the l e v e l of ammonia i n the b r a i n tissue i t s e l f is not m a r k e d l y affected by the presence of absence of glucose.  The  i n c r e a s e d quantity of ammonia f o r m e d by c e r e b r a l cortex s l i c e s i n the absence of glucose is found l a r g e l y in the incubation medium.  This fact  points to the f o r m a t i o n of a m m o n i a i n s p e c i f i c compartment(s), and the retention of ammonia within such compartment(s) up to a c e r t a i n l e v e l . Above this l e v e l there is efflux of ammonia into the incubation medium. Such a conclusion helps to explain the apparent high concentration ratio (tissuermedium) of NH4+ obtained either a e r o b i c a l l y (viz. 42) o r a n a e r o b i c a l l y (viz. 12) at the end of one hour incubation i n the presence of glucose.  T h e r e is some accumulation  of NH^"*" ions i n rat b r a i n cortex  s l i c e s against a concentration gradient. Our finding that ouabain stimulates ammonia f o r m a t i o n i n r e s p i r i n g c e r e b r a l cortex s l i c e s is in a c c o r d with the fact that ouabain inhibits the utilization-of NH^"*" for the biosynthesis of glutamine, p r e s u m a b l y by affecting the transport of NH^"*" to the site of glutamine synthesis.  Ouabain has no effect on the c e r e b r a l glutaminase of the rat.  ACKNOWLEDGMENTS  M y sincere thanks and deep appreciation a r e due to D r . J. H. Quastel,  F.R.S., for h i s guidance and encouragement during the course  of this r e s e a r c h . I also thank my wife, E v a , and M i s s H. E . H a h n for their assistance i n the preparation and typing of t h i s thesis.  T A B L E OF  CONTENTS Page  TITLE PAGE ABSTRACT ACKNOWLEDGMENTS T A B L E OF CONTENTS L I S T O F TABLES.... LIST O F FIGURES ABBREVIATIONS  i ii v vi x xiii xiv  Section 1  INTRODUCTION 1.1 A m m o n i a f o r m a t i o n i n nerve tissue (other than brain) 1.2 A m m o n i a l e v e l s i n the b r a i n i n vivo. 1.3 F a c t o r s affecting ammonia f o r m a t i o n i n the b r a i n A. In vivo studies (i) Conditions producing low b r a i n ammonia levels (ii) Conditions producing high b r a i n ammonia levels B. In vitro studies 1.4 A m m o n i a f o r m a t i o n i n the brain: O r i g i n and utilization m e c h a n i s m s (i) C h e m i c a l changes that occur i n the b r a i n on the application of stimuli to the whole animal (ii) R e c o v e r y p r o c e s s e s that o c c u r i n the b r a i n on the r e m o v a l of the applied stimuli to the whole a n i m a l 1.5 E n z y m e systems i n v o l v e d i n the utilization of ammonia (i) G l u t a m i c dehydrogenase (ii) Glutamine synthetase (iii) Glutamate t r a n s a m i n a s e s 1.6 Scope of the present work  1 1 1 2 2 4 4 6 7  7  9 10 11 12 12 14  Section 2  3  Page M A T E R I A L S AND M E T H O D S 2.1 A n i m a l s 2.2 S a c r i f i c e and b r a i n r e m o v a l 2.3 T i s s u e p r e p a r a t i o n 2.4 M e d i a composition and incubation conditions 2.5 P r o c e d u r e s adopted 2.6 A m m o n i a estimation 2.7 A m i n o a c i d analyzer estimations. Sample p r e p a r a t i o n A c i d i c and neutral amino acids a n a l y s e s A m m o n i a estimation 2.8 D r y weight estimation 2.9 P r o t e i n estimation 2.10 E x p r e s s i o n of r e s u l t s I N I T I A L C O N C E N T R A T I O N O F A M M O N I A IN R A T BRAIN 3.1 T h e total solids, p r o t e i n and ammonia l e v e l s present initially i n two-day old infant brain, and i n adult b r a i n cortex s l i c e s — 3.2 E f f e c t s of i n t r a p e r i t o n e a l injections of glucose, amytal and i p r o n i a z i d on i n i t i a l ammonia contents 3.3 T h e effects of liquid nitrogen and T C A on i n i t i a l ammonia in infant brain.. 3.4 Initial ammonia l e v e l s using T C A to inactivate the c e r e b r a l cortex slice p r i o r to ammonia estimation 3.5 E f f e c t s of various substrates on ammonia estimation with K 7 C O 3 ; (1) L - G l u t a m i n e (2) D- and L - G l u t a m a t e . (3) Adenosine monophosphate (4) T a u r i n e 3.6 F o r m a t i o n of ammonia f r o m exogenous substrates on aerobic incubation with b r a i n tissue. (1) L - G l u t a m i n e (2) D-Glutamate (3) A M P and A T P (4) T a u r i n e 3.7 T h e source of the extra ammonia produced during potassium carbonate estimation i n the absence of T C A 3.8 K i n e t i c s of ammonia formation... 3.9 A m i n o a c i d content of c e r e b r a l cortex s l i c e s  ... ' —  ....  15 15 15 15 16 17 18 20 20 21 23 23 23 23  25  ..  25 25 27  29 30 30 30 32 32 32 33 33 35 35  38 40 44  v m  Section 4  5  Page CONDITIONS A F F E C T I N G A M M O N I A F O R M A T I O N I N • B R A I N DURING.INCUBATION... 4.1 A m m o n i a f o r m a t i o n by infant and adult rat c e r e b r a l cortex s l i c e s ..... 4.2 T i s s u e and incubation m e d i u m ammonia content of adult rat c e r e b r a l cortex s l i c e s on incubation. at 3 7 ° C f o r one hour 4.3 E f f e c t s of amino acids, dipeptides and u r e a on the rate of ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s of adult rat 4.4 T h e effects of glucose, g l y c o l y t i c and c i t r i c a c i d c y c l e i n t e r m e d i a t e s on the rate of ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s . .. 4.5 T i s s u e ammonia content under different experimental conditions 4.6 T h e effects of glucose and pyruvate concentrations on ammonia f o r m a t i o n f r o m r a t b r a i n c e r e b r a l cortex slices 4.7 T h e effects of O.lmM iodoacetate on the s u p p r e s s i o n of ammonia f o r m a t i o n by glucose and pyruvate 4.8 T h e effects of high p o t a s s i u m concentration on the suppression of ammonia formation by different glucose and pyruvate concentrations ........ 4.9 T h e effect of i n h i b i t o r s of oxidative phosphorylation on the rate of ammonia f o r m a t i o n i n b r a i n cortex slices. 4.10 T h e effects of. amytal (ImM) and malonate (5mM) on ammonia f o r m a t i o n by r a t c e r e b r a l cortex s l i c e s i n the presence of low glucose concentrations 4.11 E f f e c t of glutamate d e r i v a t i v e s and h y d r o x y l a m i n e on the rate of ammonia f o r m a t i o n by c e r e b r a l cortex slices 4.12 T h e effects of pre-incubation i n the presence of a m m o n i u m ions on the rate of ammonia f o r m a t i o n i n a Ringer-phosphate m e d i u m 4.13 Studies on the m e c h a n i s m of the inhibitory action of glucose on ammonia f o r m a t i o n 4.14 T h e i r r e v e r s i b l e effects of anaerobic pre-incubation on ammonia f o r m a t i o n T R A N S P O R T O F A M M O N I U M IONS I N T O B R A I N 5.1 T h e effect of exogenous N H 4 + on the accumulation of ammonia i n c e r e b r a l cortex s l i c e s 5.2 C o r r e c t i o n for swelling  47 47  49  51  51 .54  54 57  59  62  62  64  67 69 72 73 74 80  ix Section 6  7  8  Page E F F E C T S O F OUABAIN ON AMMONIA M E T A B O L I S M IN V I T R O . 6.1 E f f e c t s of ouabain (0.0 ImM) on the rate of ammonia formation i n the presence and absence of glucose .". 6.2 E f f e c t s of ouabain on the rate of ammonia f o r m a t i o n i n an incubation m e d i u m containing glutamine i n the presence or absence of glucose 6.3 E f f e c t s of ouabain (0.0ImM) on the rate of ammonia f o r m a t i o n i n the presence of I m M glucose and high p o t a s s i u m i o n concentrations DISCUSSION 7.1 Initial c e r e b r a l ammonia l e v e l s 7.2 E v i d e n c e for the compartmentation of autogenous ammonia formation 7.3 Changes i n the amino acid s p e c t r u m and the f o r m a t i o n of ammonia i n b r a i n cortex s l i c e s (for incubation p e r i o d s of short duration) 7.4 T h e f o r m a t i o n of ammonia i n b r a i n s l i c e s (for incubation periods of long duration) SUMMARY BIBLIOGRAPHY  82  82  84  88 89 89 94  96 109 I l l 118  LIST O F T A B L E S TABLE I. II.  III.  IV.  V.  VI.  VII.  VIII.  IX.  IXa.  X.  XI.  XII. XIII.  Page In vivo l e v e l s of ammonia in the b r a i n  3  I n i t i a l - - t o t a l solids, protein and ammonia contents of 2-day o l d infant and adult b r a i n T h e effects of intraperitoneal injections of glucose, amytal and i p r o n i a z i d on initial ammonia of adult r a t c e r e b r a l cortex s l i c e s  26  ....  26  T h e effects of liquid nitrogen and T C A on i n i t i a l ammonia of infant b r a i n (2-day old) '  28  Initial ammonia l e v e l s in T C A inactivated c e r e b r a l cortex s l i c e s  28  E f f e c t on ammonia f o r m a t i o n by added substrates during the estimation of ammonia i n the p r e s e n c e of K ^ C O ^  31  T h e apparent absence of glutaminase activity at pH 7.4 and r o o m temperature incubation using b r a i n homogenates  34  T h e deamination of glutamine (ImM) by c e r e b r a l cortex s l i c e s and the inhibition of the deamination by D-glutamate  34  T h e deamination of A T P and A M P by c e r e b r a l cortex slices i n a Ringer-Phosphate-Glucose medium  36  Percentage deamination of A T P and A M P : a n a l y s i s of T a b l e IX  36  T h e effects of taurine on ammonia f o r m a t i o n by c e r e b r a l cortex slices incubated f o r one hour at 37°C  37  Initial ammonia values f r o m c e r e b r a l cortex t i s s u e examined under different conditions  39  A m m o n i a f o r m a t i o n with time  41  T h e total, ammonia and amino a c i d content of c e r e b r a l cortex s l i c e s i n i t i a l l y and on incubation i n a R i n g e r Phosphate m e d i u m f o r one hour at 3 7 ° C  45  xi TABLE XIV.  XV.  XVI.  XVII.  XVIII.  XIX.  XX.  XXI.  XXII.  XXIII.  XXIV.  XXV.  Page T o t a l ammonia f o r m a t i o n in infant and adult c e r e b r a l cortex s l i c e s i n one hour at 3 7 ° C i n the presence or absence of glucose (5mM)  48  T i s s u e and media concentrations of ammonia produced under a e r o b i c and anaerobic conditions i n the presence and absence of glucose f r o m adult r a t c e r e b r a l c o r t e x slices  50  T h e effects of amino acids, dipeptides and urea on the f o r m a t i o n of ammonia i n b r a i n cortex s l i c e s  52  T h e effects of g l y c o l y t i c and the c i t r i c a c i d cycle i n t e r mediates on a m m o n i a f o r m a t i o n i n c e r e b r a l c o r t e x slices  53  A m m o n i a content of c e r e b r a l cortex s l i c e s after one hour incubation at 3 7 ° C i n a Ringer-Phosphate m e d i u m  55  T h e effects of O.lmM iodoacetate on the s u p p r e s s i o n of ammonia f o r m a t i o n by glucose and pyruvate i n rat b r a i n cortex s l i c e s  58  T h e effects of high on the s u p p r e s s i o n of ammonia f o r m a t i o n by different glucose concentrations i n r a t c e r e b r a l cortex s l i c e s  60  T h e effects of high K"*" on the pyruvate inhibition of a m m o n i a f o r m a t i o n i n c e r e b r a l cortex s l i c e s  61  T h e effects of inhibitors of oxidative phosphorylation on a m m o n i a f o r m a t i o n i n r a t c e r e b r a l cortex s l i c e s  61  T h e effects of amytal and malonate on ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s of rat i n the presence of low glucose concentrations  63  T h e effects of glutamate, i t s analogs and h y d r o x y l a m i n e on ammonia f o r m a t i o n by c e r e b r a l cortex s l i c e s of rat i n the presence o r absence of glucose  66  T h e effect of a one hour pre-incubation i n the presence of 5mM NH4CI on a m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s of the rat  68  xii TABLE XXVI.  XXVII.  XXVIII.  XXIX.  XXX.  XXXI.  XXXII.  XXXIIa.  XXXIII.  XXXIV.  Page T h e effect of one hour pre-incubation i n the p r e s e n c e of glucose and oxygen on the ammonia f o r m a t i o n by c e r e b r a l cortex s l i c e s i n the p r e s e n c e or absence of glucose  71  T h e effect of a one hour anaerobic pre-incubation at 3 7 ° C on the a e r o b i c f o r m a t i o n of a m m o n i a by c e r e b r a l cortex s l i c e s i n the presence and absence of glucose  71  T h e effect of a m m o n i u m ions added to a Ringer-phosphateglucose m e d i u m on its accumulation i n r a t c e r e b r a l cortex s l i c e s i n an incubation of one hour at 37°C and an atmosphere of p u r e oxygen  76  T h e effect of a m m o n i u m ions added to a R i n g e r -pho sphate m e d i u m on its accumulation a e r o b i c a l l y i n r a t c e r e b r a l cortex slices incubated f o r one hour at 37°C  78  T h e effect of a m m o n i u m ions added to a R i n g e r bicarbonate-glucose m e d i u m on its accumulation a n a e r o b i c a l l y i n r a t c e r e b r a l cortex s l i c e s incubated at 37°C for one hour  79  T h e effects of O.OlmM ouabain on ammonia f o r m a t i o n i n the p r e s e n c e and absence of glucose using c e r e b r a l cortex slices  83  T h e effect of O.OlmM ouabain on the f o r m a t i o n of ammonia f r o m c e r e b r a l cortex s l i c e s i n the p r e s e n c e of glucose and glutamine  85  A n a l y s i s of T a b l e XXXIII: Percentage deamination of added glutamine by b r a i n s l i c e s  86  T h e effects of O.OlmM ouabain on ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s in a Ringer-phosphate m e d i u m containing glutamine (0.5mM) (and no glucose)  87  T h e effects of O.OlmM ouabain on ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s i n the p r e s e n c e of l.OmM glucose and high p o t a s s i u m  87  LIST O F FIGURES FIGURE 1  2  Page T i m e course of the f o r m a t i o n of ammonia b y r a t c e r e b r a l cortex s l i c e s at 37°C i n a Ringer-phosphate m e d i u m in the presence or absence of glucose  43  T h e effects of glucose and pyruvate concentrations on the rate of ammonia f o r m a t i o n by rat b r a i n cortex slices, on incubation i n a Ringer-phosphate m e d i u m for one hour at 37°C  56  ABBREVIATIONS  TCA  Trichloracetic acid  AMP  5'-Phosphate of adenosine  ADP  5'-Diphosphate of adenosine  ATP  5'-Triphosphate of adenosine  Pi  Orthopho sphate  DNP or 2, 4 - D N P  2, 4 - D i n i t r o p h e n o l  NAD+, N A D H  O x i d i z e d and r e d u c e d f o r m s of nicotinamide-adenine dinucleotide.  yABA  Y -amino b u t y r i c a c i d  1.  INTRODUCTION  1.1 A m m o n i a f o r m a t i o n in nerve tissue (other than: brain);. A s e a r l y as 1922, T a s h i r o ( 1 ) demonstrated an evolution of ammonia f r o m nerve f i b r e s . He also showed that while e l e c t r i c a l stimulation i n c r e a s e d ammonia liberation, m e c h a n i c a l i n j u r y d e p r e s s e d it. "Winterstein and H i r s c h b e r g  (2,3) not only c o n f i r m e d these findings, but  a l s o further established the suppressive  effects of anoxia and anaesthesia  on ammonia formation. Observations^ on the effects of stimulation were made by a number of workers. (4.-7).  F o r example, f r o g retina i r r a d i a t e d  with sunlight i n c r e a s e d ammonia f o r m a t i o n when c o m p a r e d to c o n t r o l s left in the dark (5). Isolated nerve on stimulation, e l e c t r i c a l l y o r m e c h a n i cally, by t h e r m a l or o s m o t i c p r e s s u r e changes and by v a r i o u s agents, gave s i m i l a r r e s u l t s ( 6, 7 ). T h e s e observations  chemical  led to the c o n c l u -  sions that ammonia f o r m a t i o n o c c u r s as a r e a c t i o n of nerve f i b r e s to stimulation. The  central, n e r v o u s system, like p e r i p h e r a l nerves, has been  shown to react to stimuli by i n c r e a s e d f o r m a t i o n of ammonia.  Articles  reviewing ammonia m e t a b o l i s m by the c e n t r a l nervous s y s t e m have appeared (8-11 ).  '  1.2 A m m o n i a l e v e l s i n the b r a i n i n vivo  Interest in b r a i n ammonia l e v e l s stems f r o m the fact that a m m o n i a i s a powerful c e r e b r a l i r r i t a n t .  T h e a d m i n i s t r a t i o n of a m m o n i u m salts  2 causes convulsions i n a n i m a l s and it h a s long been thought that ammonia may play a r o l e i n the precipitation of epileptic s e i z u r e s (11-16). >  R i c h t e r and Dawson (16 ) investigating the ammonia l e v e l s i n the b r a i n of young rats k i l l e d by i m m e r s i o n i n l i q u i d a i r c o n f i r m e d the l o w values found by previous w o r k e r s (12,  13, 17, 18).  B y this f r e e z i n g  technique t i s s u e s a r e rapidly fixed and p o s t - m o r t e m changes a r e avoided. U s i n g this technique values of 0.15 - 0.36 m i c r o m o l e s per g. f r e s h weight b r a i n tissue have been obtained i n different species (Table I).  These  values a r e c o n s i d e r a b l y lower than those obtained when the severed head of the decapitated animal i s dropped into l i q u i d a i r .  F o r example, there  i s about a twofold i n c r e a s e in ammonia content when the head i s f r o z e n one second after decapitation (16 ). Cat b r a i n gave 2.0 m i c r o m o l e s per g. f r e s h weight three minutes after death  while sheep b r a i n gave values of  2.7 and 1.7 m i c r o m o l e s per g. f r e s h weight i n grey and white matter r e s p e c t i v e l y when freezing was done within five minutes after death (19 ). W e i l - M a l h e r b e and G r e e n ( 20 ) obtained 5.0 m i c r o m o l e s per g. f r e s h weight in slices of b r a i n cortex fixed twenty minutes after death, which i s in good agreement with values obtained by other w o r k e r s ( 21, 22) and with r e s u l t s reported in this thesis. 1.3 F a c t o r s affecting ammonia formation in the b r a i n . A.  In vivo studies The ammonia content of the central nervous system, analyzed after r a p i d fixation i n l i q u i d a i r or liquid nitrogen, i s not constant  3 T A B L E I.  In vivo l e v e l s of ammonia in the b r a i n  Species Rat  Amount of a m m o n i a iamoles/g f r e s h wt.  Reference  0.16  16  0.36  23  0.26  24  0.20  25  Rabbit  0.18  26  Mice  0.32  27  Garden Dormice  0.3 5  28  Dog  4  but depends on the state of activity of the brain at the time of fixation. (i)  Conditions producing low brain ammonia levels. When Tashiro ( 1 ) observed a greater formation of ammonia  during electrical stimulation of nerve fibres, he speculated that anaesthesia would depress it.  This was shown to be true by  Winterstein and Hirschberg ( 2 ), and later workers confirmed this observation (3, example,  16, 29).  Richter and Dawson ( 16 ), for  showed a significant decrease in brain ammonia levels  (0.033 micromoles per g. wet weight) when rats under prolonged nembutal narcosis (thirty minutes or longer) were killed in liquid air. Vladimirova obtained similar effects with urethane and sodium bromide (30  ).  Ammonia concentration of rat brain decreases by 50 per cent during sleep (31  ). During hibernation, too, there is a 50 per  cent lowering of ammonia levels in the brain ( 28 ). Thus, a reduction in functional activity is associated with a reduced concentration of free ammonia in the brain. (ii)  Conditions producing high brain ammonia levels. In confirmation of earlier reports ( 1 - 3 ) with nerve, a  number of workers have established the induction of ammonia formation in the brain during electrical stimulation ( 16, 23 ).  5 T h i s i s consistent with the view that any method that i n c r e a s e s c e r e b r a l i r r i t a b i l i t y w i l l produce greater amounts of ammonia i n the b r a i n . Thus, a number of drugs capable of producing convulsions, telodrin ( 3 3 (35  e.g., camphor ( 30 ), p i c r o t o x i n ( 16, 32 ), ), pentamethylene tetrazole ( 34  ) and b e m i g r i d e  ) i when injected into a n i m a l s r e s u l t e d i n i n c r e a s e d b r a i n  ammonia l e v e l s . In some instances the r i s e o c c u r r e d in the preconvulsive state ( 16, 35). T h i s effect was m a r k e d in the b r a i n of dogs after i n j e c t i o n s of fluoracetate, where the c e r e b r a l ammonia l e v e l r o s e and reached a m a x i m u m at the time of the convulsions (24 ). T h i s i n c r e a s e by fluoracetate on ammonia formation has also been demonstrated i n v i t r o on incubation of b r a i n cortex s l i c e s i n the p r e s e n c e of glucose ( 3 6  ). However,  it i s not known definitely whether an i n c r e a s e i n b r a i n ammonia i s a r e s u l t or the cause of convulsions.  Injections of a m m o n i u m  c h l o r i d e i n the r a t caused convulsions when the b r a i n ammonia l e v e l reached 9.0mg per cent (about 5.0 m i c r o m o l e s per g. f r e s h weight) pressure  ( 16 )• Both anoxia ( 16 ) and high oxygen  ( 3 5 ) r e s u l t i n i n c r e a s e s in ammonia l e v e l s i n the  brain.  M i l d e r stimulation of the c e n t r a l nervous system, like injection of amphetamine (37  ) or c o r t i c o t r o p i n ( 38 ), painful  e l e c t r i c shock to the e x t r e m i t i e s ( 23, 31 ) o r c e r t a i n conditioned r e f l e x e s ( 23, 30 )» a l s o elevated ammonia l e v e l s . Though emotional excitement caused by tumbling i n a r e v o l v i n g d r u m  had a s i m i l a r effect ( 39 )» R i c h t e r and Dawson found no m a r k e d difference i n rats excited by allowing t h e m to d r o p f r o m side to side i n a glass beaker ( 16 ), while V r b a ( 9 ) found no change i n r a t s made to undergo p h y s i c a l e x e r c i s e .  Cerebral  ammonia  i n c r e a s e s may not occur i n some cases of p h y s i c a l of emotional excitement owing to i n c r e a s e d f o r m a t i o n of glutamine (9-11, 40, 41); a r i s e i n c e r e b r a l glutamine usually a c c o m p a n i e s a r i s e i n blood ammonia. B.  In v i t r o studies W o r k on 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 s l i c e s and the f a c t o r s  affecting its.formation has been c a r r i e d out to throw light on the following p r o b l e m s : (i)  T h e o r i g i n of ammonia in the brain;  (ii) T h e m e c h a n i s m of its formation; (iii) T h e mode of utilization of the ammonia. How far these p r o b l e m s have, been solved w i l l be d i s c u s s e d later i n this thesis. A m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s i n v i t r o has two components ( 2 0 ) as follows: (1)  Spontaneous ammonia f o r m a t i o n following decapitation.  (2)  A m m o n i a f o r m a t i o n that depends on (i)  s t r u c t u r a l integrity of the tissue (e.g., b r a i n homogenates  produce ammonia l e s s efficiently than b r a i n s l i c e s though they  7 give higher initial values ( 2 0  )  (ii) M e t a b o l i c activity of the tissue (e.g., ammonia f o r m a t i o n i n the b r a i n i s inhibited (a) by the presence of glucose and lactate (42-45 ); (b) by the presence of substances such as cyanide or arsenite that inhibit electron transport, dinitrophenol that inhibits oxidative phosphorylation and (c) by a n a e r o b i o s i s  2:4  (20, 46,47 );  ( 20, 46 )•  In the p r e s e n c e of c e r t a i n inhibitors, e.g., ( 46  or  )• fluoracetate ( 36  ) and iodoacetate  ( 47  2:4-dinitrophenol ), the  suppressive  effects of glucose on ammonia f o r m a t i o n i s l e s s marked.  1. 4  A m m o n i a f o r m a t i o n in the brain:  (i)  O r i g i n and utilization m e c h a n i s m s .  C h e m i c a l changes that o c c u r 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 . I n c r e a s e d c e r e b r a l activity produced in rats by p h y s i c a l exertion  does not r e s u l t in free ammonia ( 9 , 40  ). However, it was  shown  that under such conditions free glutamine l e v e l s are i n c r e a s e d in the b r a i n ( 40 (41, 48  ) with concomitant d e c r e a s e s in free glutamate  ) and protein bound amide nitrogen ( 49,  r e s u l t s are obtained during oxygen intoxication ( 9 disulphide poisoning (52  52 ). S i m i l a r ) or carbon  ). E v e n during e l e c t r i c a l l y induced  8 convulsions, glutamine l e v e l s are elevated ( 5 3  ). Acute  t e l o d r i n intoxication, too, r e s u l t s in'an i n c r e a s e i n glutamine content with a concomitant decrease in glutamate l e v e l s i n the b r a i n ( 33 ). In the e a r l y stages of acute t e l o d r i n intoxication the content of free ammonia 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 l a t e r in the s e i z u r e pattern. A c c o r d i n g to Hathway and M a l l i n s o n ( 33  ), t e l o d r i n causes  l i b e r a t i o n of ammonia i n the b r a i n and this o c c u r s before the onset of convulsions and throughout their course, a-ketoglutarate and glutamate are u t i l i z e d i n an ammonia-binding m e c h a n i s m which later b e c o m e s overwhelmed and free a m m o n i a  accumulates  i n the c e r e b r a l tissue. These r e s u l t s would lend support to the v i e w that ammonia i s f o r m e d f r o m protein s o u r c e s and i s converted to glutamine by fixation with glutamate. Such findings are also substantiated by i n v i t r o studies with b r a i n homogenate s ( 9  )  o  r  c e r e b r a l cortex s l i c e s ( 5 4  ) in  incubations of long duration (3-4 hours). Besides, under such conditions W e i l - M a l h e r b e  et al., on c o n s i d e r i n g the six de-  aminating enzymes known to occur in the brain, d i s m i s s e d them as major f a c t o r s in the p r o c e s s of ammonia f o r m a t i o n . enzymes they c o n s i d e r e d were: glutaminase,  The  glutamic  dehydrogenase, adenylic and adenosine deaminases, amine oxidase ( 20  ) and hexosamine deaminase ( 5 5  a c c o r d i n g to W e i l - M a l h e r b e  and G r e e n ( 20  ). Thus,  )• s m a l l m o l e c u l e s  l i k e amino acids f r o m the f r e e pool may as ammonia p r e c u r s o r s .  not be involved significantly  F r o m these considerations it would appear  that ammonia formation o c c u r s by splitting part of the protein amide bonds, perhaps f r o m glutamine moieties of the b r a i n proteins, as b r a i n protein i s r e l a t i v e l y r i c h in glutamine ( 1 6  ).  However, V r b a  r e p o r t e d that only 25 per cent of the ammonia f o r m e d f r o m guinea pig c e r e b r a l cortex s l i c e s i n the absence of substrate can be accounted for by the splitting of p r o t e i n amide bonds in a four hour incubation (49,  54  ). W e i l - M a l h e r b e and G r e e n ( 20 ) suggested that ammonia  f o r m a t i o n i s l i n k e d with p r o t e o l y s i s because both ammonia and nonprotein nitrogen were i n c r e a s e d under s i m i l a r conditions. V r b a ( 56 too, obtained evidence  showing that the T C A  insoluble f r a c t i o n of  b r a i n s l i c e s during substrateless incubations l o s t some amide groups with concomitant significant i n c r e a s e s i n non-protein and l i p i d nitrogen. However, V r b a does not consider ammonia f o r m a t i o n simply as a consequence of o r d i n a r y proteolytic  processes.  (ii) 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 applied stimuli to the whole a n i m a l . When rats p h y s i c a l l y exhausted (by prolonged  swimming) a r e  allowed to rest, their c e r e b r a l glutamine l e v e l s d e c r e a s e with a concomitant i n c r e a s e in their c e r e b r a l protein nitrogen ( 51,  56  It should be mentioned here that glutamine has been i m p l i c a t e d i n the synthesis of polypeptides and proteins ( 9, 57 - 60).  Thus,  ).  ),  changes that occur i n the f r e e glutamine l e v e l s i n the b r a i n on the application of stimuli to the whole animal, or the r e m o v a l of the applied stimuli, appear to be d i r e c t l y associated with b r a i n protein metabolism.  1.5  E n z y m e systems involved i n the utilization of ammonia. A m m o n i a in excess of the n o r m a l i s toxic to the c e n t r a l n e r v o u s  s y s t e m ( 11-16, 61, 62).  The m e c h a n i s m s by which e x c e s s i v e ammonia  i s reduced i n amount centre around glutamic a c i d metabolism. T h i s i s understandable when i t i s r e a l i z e d that glutamic a c i d i s one of the  few  amino acids that occur i n appreciable concentrations i n the b r a i n and accounts for close to 50 per cent of a-amino nitrogen of protein f r e e b r a i n tissue f i l t r a t e s (8, 19,  63,  64).  A l s o , it i s known that glutamic  a c i d d e c r e a s e s during strychnine convulsions (65), c o m a ( 66  in hypoglycemic  ), and in epileptogenic f o c i of cat cortex produced i n vivo by  f r e e z i n g (67).  M o r e o v e r , glutamate injections inhibit  c h l o r i d e convulsions ( 68  ammonium  ), while the infusion of a m m o n i u m salts leads  to m a r k e d i n c r e a s e s i n protein amide groups (69) concentrations of rat, cat and dog b r a i n ( 70 - 72  and i n the glutamine ). T h e s e and  other  f a c t s mentioned e a r l i e r i m p l i c a t e glutamine as an end product of c e r e b r a l ammonia utilization. The m a i n enzymes i m p l i c a t e d in ammonia utilization are as follows:  11 (i)  glutamic  dehydrogenase  (ii) glutamine synthetase (iii) glutamate t r a n s a m i n a s e s (i)  Glutamic dehydrogenase: T h i s enzyme s p e c i f i c a l l y catalyzes the oxidative deamination  of glutamic a c i d (the only amino a c i d oxidized to any appreciable extent i n the b r a i n (42, 73 ), a c c o r d i n g to the following equations (8): glutamic a c i d  + NAD  •*»* a - i m i n o g l u t a r i c a c i d + N A D H  +  a - i m i n o g l u t a r i c a c i d + H.O — — — Z  +H ....(1) +  a-ketoglutaric a c i d + N H „ 3  :—  enzymic In the presence of a m m o n i u m salts the e q u i l i b r i u m of reaction (1) i s much in favor of glutamic acid, only 1.4 p e r cent being oxidized at p H 7.4.  In the absence of a m m o n i u m salts, i m i n o g l u t a r i c a c i d  hydrolyzes  spontaneously to a-ketoglutarate  and ammonia.  Thus, the  supply o r r e m o v a l of a m m o n i a can condition the oxidation of glutamic acid, a-ketoglutaric a c i d inhibits ammonia formation,  which i s due  to the fact that the enzyme f a v o r s the reductive amination of a-ketoglutarate  (42).  G l u t a m i c a c i d thus appears to be p a r t i c u l a r l y  adapted for the synthetic r e a c t i o n which o c c u r s spontaneously a s soon as sufficient ammonia i s available (74).  (2)  (ii) Glutamine synthetase: T h i s enzyme (42, 73 ) catalyzes the endergonic f o r m a t i o n (75, 76 ) of glutamine a c c o r d i n g to the following equation:  glutamic a c i d + NH^ + A T P  M  g  (  M  n  )  > glutamine + A D P + P i  (3)  O x i d i z a b l e substrates such as glucose suppress ammonia f o r m a t i o n (42 -45)  by e x e r t i n g  a 'sparing action' on endogenous  oxidizable nitrogenous m a t e r i a l s , by checking autolytic breakdown of adenine nucleotides (8)  and by f o r m i n g glutamine (77,  78).  B r a i n s l i c e s can b r i n g about a disappearance of added ammonia i n the p r e s e n c e of glucose ( 4 2 ). However, any method that inhibits A T P synthesis (e.g., 2, 4-DNP (20, 46), fluoracetate ( 36 ), and thereby lowered glutamine formation) concomitantly i n c r e a s e s ammonia output.  Glutamine p r e s u m a b l y i s an end product of the  ammonia binding m e c h a n i s m .  It m a y s e r v e as a storage or transport  f o r m of ammonia and may be h y d r o l y z e d at the sites of ammonia utilization ( 79 ). In this connection it is perhaps pertinent to note that glutamine passes much m o r e f r e e l y through the blood b r a i n b a r r i e r than glutamate ( 80 ).  (iii) Glutamate transaminases: T r a n s a m i n a t i o n r e a c t i o n s ( 8 ) utilizing glutamate as the a m i n o group donor, and the keto-acids, pyruvate and oxaloacetate as acceptor m o l e c u l e s (to y i e l d alanine and a s p a r t i c a c i d respectively)  serve as a u x i l i a r y m e c h a n i s m s a s s i s t i n g ammonia u t i l i z a t i o n i n the b r a i n . T h e r e a c t i o n s shown below are the only t r a n s a m i n a t i o n s that o c c u r to a considerable extent i n b r a i n tissue (81, 82), and a r e catalyzed by specific t r a n s a m i n a s e s which r e q u i r e pyridoxine derivative s as coenzymes (83). glutamic a c i d + p y r u v a t e — - — ^ a - k e t o g l u t a r i c a c i d + alanine glutamic a c i d + oxaloacetate  s  .(4)  a - k e t o g l u t a f i c a c i d + aspartate.....(5)  When the f o r m a t i o n of f r e e a m m o n i a i s high, the reductive amination of a-ketoglutarate mediated by glutamic dehydrogenase o c c u r s and a r a p i d synthesis of glutamic a c i d r e s u l t s . If the rate of ammonia f o r m a t i o n exceeds the glutamine synthesizing capacity,, then glutamic a c i d t r a n s a m i n a t e s with p y r u v i c or o x a l o a c e t i c acids and the a-ketoglutarate r e g e n e r a t e d can again accept ammonia so that a steady flow of a m m o n i a into the t r a n s a m i n a t i o n s y s t e m i s ensured. In the absence of ammonia, a-ketoglutarate accepts an amino group f r o m alanine or aspartate and the keto a c i d (pyruvate o r oxaloacetate) r e l e a s e d r e - e n t e r s the c i t r i c a c i d c y c l e (8). Thus, these amino a c i d s (alanine and aspartate) act as t e m p o r a r y s t o r e houses for excess ammonia  ensuring gradual d i s p o s a l during p e r i o d s  of restitution. E a r l y r e p o r t s c l a i m e d that o r a l a d m i n i s t r a t i o n of glutamic a c i d d e c r e a s e d petit m a l s e i z u r e s , i n c r e a s e d mental a l e r t n e s s and  a r o u s e d patients f r o m i n s u l i n coma ( 84, 85).  However, no  convincing evidence of a relationship between these c l i n i c a l effects and glutamic a c i d m e t a b o l i s m i n the c e n t r a l nervous s y s t e m has yet been put f o r w a r d ( 85, 86 ).  1.6 Scope of the present work. The work presented here i s a p r e l i m i n a r y  study of c e r e b r a l  ammonia m e t a b o l i s m and f a c t o r s affecting i t s f o r m a t i o n i n vitro, i n attempts to throw further light on the unsettled o r i g i n and obscure m e c h a n i s m of c e r e b r a l ammonia formation.  15  2. M A T E R I A L S AND  2.1  METHODS  Animal s Adult rats of the W i s t a r  strain and  2-day old infant r a t s were  used i n these studies. M a l e rats were usually employed. J o s a n et al., ( 22)  observed no m a r k e d difference between the a m m o n i a  contents  and rates of formation of adult m a l e or female rat b r a i n s .  2.2  S a c r i f i c e and b r a i n r e m o v a l Adult rats were k i l l e d by decapitation. The whole b r a i n  was  r a p i d l y e x c i s e d f r o m the severed head and placed i n c h i l l e d R i n g e r solution. With infant rats the head was b r a i n was  cut off with s c i s s o r s before the  removed. In some cases, p a r t i c u l a r l y during the  determination  of i n i t i a l ammonia l e v e l s i n infant b r a i n , the a n i m a l s were i m m e r s e d i n liquid nitrogen. T h i s procedure f r o z e the a n i m a l s i n a few seconds and produced a r a p i d fixation of metabolites i n the b r a i n (16  ). B e f o r e the  s a c r i f i c e of adult rats, i n some instances i n t r a p e r i t o n e a l injections were made i n attempts to prevent the i n i t i a l b u r s t s of ammonia, details of which w i l l be mentioned below.  2.3  T i s s u e preparation The b r a i n was  c e r e b e l l u m was  r e m o v e d f r o m the chilled R i n g e r m e d i u m and the  d i s s e c t e d f r e e f r o m the h e m i s p h e r e s .  C e r e b r a l cortex  s l i c e s p r e p a r e d by a Stadie-Riggs microtome, were placed on p e t r i  16 dishes c h i l l e d externally with ice.  C a r e was taken that the s l i c e s were  not more than 0.3mm thick, but not so thin that they tended to disintegrate when shaken i n the W a r b u r g m a n o m e t r i c apparatus. C o r t e x s l i c e s f r o m each h e m i s p h e r e of adult r a t b r a i n (two i n number) were weighed quickly on a t o r s i o n balance (average weight 80lOOmg wet weight) and suspended into chilled m a n o m e t r i c v e s s e l s containing the incubation media.  2.4  M e d i a composition and incubation conditions ( 87 ) (i)  Ringer-phosphate medium: The b a s a l m e d i u m had the following composition:  R i n g e r solution: N a C l , and M g S 0  4  142.6mM; KC1, 5.7mM; C a C l ^ 3.06mM;  . 7 H O , 1.43mM. z  Phosphate buffer: N a H P 0 2  4 >  l O m M made to pH 7.4 with N H C 1 .  Incubation i n a Ringer-phosphate m e d i u m was c a r r i e d out i n a conventional W a r b u r g m a n o m e t r i c apparatus at 37°C for one hour unless otherwise stated. T h e gaseous phase was pure oxygen. The centre well contained a s m a l l g l a s s tube, c l o s e d at one end, containing 0.2ml of 20 per cent K O H and a r o l l of filter paper. Oxygen was passed through the apparatus f o r five minutes after which the m a n o m e t e r s were placed i n the W a r b u r g apparatus and t h e r m a l l y equilibrated for seven minutes.  17 (ii) Ringer-bicarbonate medium: The b a s a l m e d i u m had the following composition: R i n g e r solution: NaCl, MgSO„.7HO, 4 2 &  141.8mM; KC1, 5.65mM; C a C l ^  1.4ZmM; K H , P O „ , 2 4  B i c a r b o n a t e buffer: NaHCO^,  3.02mM;  1.42mM.  28mM.  Incubation i n a R i n g e r - b i c a r b o n a t e m e d i u m was c a r r i e d out at 3 7 ° C for one hour. G a s s i n g with a  /CO^ (95/5%) mixture was o  c a r r i e d out f o r five minutes before t h e r m a l equilibration at 37 C for seven  minutes.  The incubation media were five t i m e s the strength of the final solutions and were diluted after additions, or modifications, as mentioned i n the text. T h e final volume of a l l incubation media was always 3.0ml.  Glucose, when present, was 5.0mM unless otherwise  stated. A l l solutions were made to pH 7.4 with N a O H b e f o r e use. 2.5 P r o c e d u r e s  adopted  E s t i m a t i o n s were c a r r i e d out to determine the i n i t i a l ammonia content and the r a t e s of ammonia f o r m a t i o n during one hour incubation under v a r i o u s conditions.  Homogenization was c a r r i e d out i n i c e - c o l d  R i n g e r m e d i u m f o r about one minute in a P o t t e r - E l v e h j e m homogenizer. The  ammonia  content of the tissue,  as the i n i t i a l ammonia  content.  at the time of gassing,  was taken  T h i s did not v a r y beyond the  18 experimental e r r o r between 10-30 estimation used  minutes under the conditions of  here.  Initial ammonia values.  The  t i s s u e s were placed in  W a r b u r g v e s s e l s containing 3.0ml Ringer-phosphate flasks  were  stoppered and K^CO^  from  medium.  the side a r m  The  added  immediately. Ammonia  content of the tissue.  after incubation and  the side a r m  was  Ammonia  from  removed Ringer  W a r b u r g v e s s e l containing 3.0ml water  medium, i m m e d i a t e l y  stoppered and K  CO,  from  added. content of the incubation media.  estimated after the s l i c e s were removed. and K^CO^  s l i c e s were  quickly washed by dipping i n a c h i l l e d  m e d i u m and p l a c e d i n another or Ringer-phosphate  The  the side a r m  was  T o t a l ammonia values. f l a s k s after incubation was  The  This  was  f l a s k s were  added into the m a i n  stoppered  compartment.  T h e s e were the values found i n the  completed  without removing the tissue.  T h e s e were also the sums of the ammonia  contents of tissue  and  m e d i u m as obtained s e p a r a t e l y .  2.6  A m m o n i a estimation A  modified method of B r a g a n c a et_ al_.,  p r i n c i p l e being the same as the m i c r o (88).  The  (21)  was  used,  diffusion method of Conway  W a r b u r g m a n o m e t r i c v e s s e l was  used,  ammonia  the  •  19  l i b e r a t e d by K For  CO  being absorbed by a c i d i n the centre well.  i n i t i a l (tissue,  media or total) ammonia  estimations,  the centre well of a W a r b u r g v e s s e l contained 0 . 2 m l N H 2 S O 4 and a contained 0 . 3 m l  r o l l of f i l t e r paper.  The  side a r m  (saturated) solution.  The  v e s s e l s were tightly c l o s e d with  -j  CO  y  2  solution f r o m the side a r m  was  quickly  3  added into the main compartment and  mixed.  0 . 1 m l of lOOg per cent  K„CO„.  2  rubber  -  stoppers and the K  In some cases  K^CO^  The  TCA  pH  was  r o s e to  10.5.  added p r i o r to  -  3  Under the above conditions ammonia present i n the test solutions diffused in the centre well where it was on the f i l t e r paper.  The  temperature overnight.  absorbed by the a c i d  v e s s e l s were allowed to stand at r o o m T h i s was  found  ammonia diffusion and absorption. duplicated with accuracy,  to be  Ammonia  experimental  sufficient for complete estimations could be  deviation being _+ 5 per  cent.  A f t e r the overnight incubation i n the p r e s e n c e of K ^ C O ^ r o l l of f i l t e r paper was placed i n graduated  taken out of the centre well of the f l a s k  tubes.  The  a number of t i m e s using P a s t e u r to the graduated  and  centre well was pipettes.  tubes containing the filter  C o l o r was  the  developed  by adding  The  and  washed c a r e f u l l y washings were added  paper.  1.0ml N e s s l e r ' s reagent  2 . 0 m l of 2N N a O H in a total volume of 1 0 . 0 m l .  (87)  A f t e r standing  20 for twenty minutes,  the c o l o r intensity was  a B a u s c h and  c o l o r i m e t e r or a B e c k m a n spectrophotometer  Lomb  the ammonia content s i m i l a r l y and  computed f r o m  obtained  For  no  a  ammonia values treated  Warburg  v e s s e l containing  tissue preparation was  conditions as i n the experimental Whenever a new two  standard  substance (in the absence of tissue) was vessel.  used f o r the f i r s t time with "the test  set aside and  T h i s was  treated i d e n t i -  done i n o r d e r  whether the test substance contained  For  example,  (see a l s o ref. 21), deaminated.  2.7  methionine  All  10-12  per  cent  sulfoximine i s also p a r t i a l l y  estimations  preparation. samples were homogenized i n the presence of 3 per  in a Potter-Elvehjem  homogenizer f o r one  f e r r e d to graduated centrifuge tubes and minutes.  hydrolyzed  during  C o r r e c t i o n s were always made for such effects.  A m i n o a c i d analyzer Sample  TCA  and  glutamine was  to a s c e r t a i n  t r a c e s of ammonia,  (ii) the l a b i l i t y of the test substance to a l k a l i used estimation.  same  vessels.  test substance was  c a l l y to the experimental  3.0ml  treated under the  W a r b u r g v e s s e l s containing a l l solutions together  (i)  and  c o n c u r r e n t l y with a l l experiments.  the reagent blank  d i s t i l l e d water and  determined at 425my. on  The  d e b r i s was  separated  minute, then t r a n s -  centrifuged for fifteen  f r o m the  cent  supernatant by  ' decantation,  21  washed with d i s t i l l e d water,  washings added to the a known volume and  supernatant.  supernatant  and  were evaporated by  The  made up to  some l i p i d m a t e r i a l .  Traces  of  c a r e f u l l y blowing i n f i l t e r e d a i r  and the volume readjusted to i t s o r i g i n a l l e v e l . put on appropriate  was  the  extracted t h r i c e with equal v o l u m e s of ether.  T h i s r e m o v e d much of the T C A ether left behind  The  r e c e n t r i f u g e d and  columns of a B e c k m a n 120B  A l i q u o t s were then Amino acid analyzer.  estimations a r e b a s e d on the method of Spackman et al., (89) j  using the p r i n c i p l e of ion exchange chromatography developed M o o r e and  Stein (90).  preparations,  The  details of the reagents  the operation of the B e c k m a n  120B,  and  and the  by  column calculation  of r e s u l t s a r e as given i n the B e c k m a n Instruction Manual AIM-2.' The  e s s e n t i a l conditions used f o r the a c i d i c and  and  ammonia determinations, A c i d i c and The  neutral amino acids,  a r e as follows:  neutral amino acids  analyses.  amino acids, v i z . taurine, a s p a r t i c acid, glutamic "acid,  glutamine, glycine and alanine, that were estimated i n the studies r e p o r t e d fin this thesis, were separated on a column (50 x 0.9cm& sulphonated  poly-  styrene  - 8%  divinyl benzene copolymer ion exchange r e s i n (Type  particle  size,  25-31p.) at 50°C by  buffer at pH  3.28.  mixing manifold. very  The The  elution with a 0.2N  eluate then  sodium citrate  m i x e d with n i n h y d r i n i n a  m i x t u r e then flowed  50A-  through a long length of  s m a l l b o r e tubing i n the boiling water of the r e a c t i o n bath for  fifteen minutes during which time the n i n h y d r i n r e a c t i o n takes place. In the presence  of amino acids, ammonia and  blue diketohydrindylidene - diketohydrindamine color being f o r m e d with imino constant environmental  certain amines i s f o r m e d (a y e l l o w  a c i d s like proline).  By  maintaining  conditions the color intensity can be  proportional to the quantity of amino acid present.  made  Using a  colori-  meter containing three photometer units each consisting of a light source,  a lens, an i n t e r f e r e n c e f i l t e r ,  photovoltaic cell,  a slit,  the cuvette and  an e l e c t r i c a l c u r r e n t i s generated  the  p r o p o r t i o n a l to  the density of color in the effluent-ninhydrin mixture.  The  e l e c t r i c a l c u r r e n t i s then used to d r i v e a multipoint r e c o r d e r which plots the r e s u l t s of the a n a l y s i s as absorbance v e r s u s time. diketohydrindylidene-diketohydrindamine through  As  f r o m each amino a c i d  the  passes  the c o l o r i m e t e r , light to the photovoltaic c e l l i s reduced,  resulting in a reduction of e l e c t r i c a l output and a movement on the r e c o r d e r pen.  T h r e e multipoint c u r v e s are plotted  simultaneously  consisting of a s e r i e s of peaks, each peak corresponding  to a  specific  amino acid.  The  height-width method was  used to integrate these peaks.  H e r e the height of the peak i s multiplied by the width which i s m e a s u r e d at half height.  The  width of a peak i s m e a s u r e d in t e r m s  of time by counting the number of dots printed above the half height of the peak.  The  constant per m i c r o m o l e (i.e. the a r e a of the peak per  m i c r o m o l e ) for each amino acid i s obtained f r o m an amino profile of a standard amino a c i d mixture.  acid  F r o m these values the  23 concentration of each amino acid is computed. The constants were invariably obtained for each new batch of buffer or ninhydrin reagent prepared. After the estimation of the acidic and neutral amino acids in a sample was completed, the 50cm column was regenerated with 0.2 N NaOH and equilibrated with a 0.2 N sodium citrate buffer at pH 3.28,  before  re-use. Ammonia estimation. Ammonia was estimated by elution at room temperature with a 0.35N sodium citrate buffer at pH 5.28 from a 15cm column (resin Type 15A,  particle size 19-25 microns).  2.8 D r y weight estimation Weighed cerebral cortex slices were dried on a steam bath (at about 100°C) or an electric oven at 110° C to constant weights.  2.9  Protein estimation  (91)  The Lowry method (92) (93)  was employed for protein estimation,  at 750m)J.  2.10  using the Folin-Ciocalteu reagent the blue color being read  Albumin was used as standard for expressing the results.  Expression of Results The results obtained are expressed as micromoles ammonia  per gram fresh weight tissue under the given conditions, otherwise  stated.  unless  Medium ammonia concentration is either  expressed  as m M  ammonia obtained on incubation with lOOmg tissue,  o r as  m i c r o m o l e s of ammonia that would be obtained on incubation with g r a m f r e s h weight tissue. The  mean and standard deviation values were calculated.  M o s t r e s u l t s a r e averages  of not l e s s than three  experiments.  Standard deviation  where  x  =  experimental value  x  =  mean of experimental values obtained under s i m i l a r conditions  n  =  number of experiments  3.  INITIAL C O N C E N T R A T I O N  3.1 T h e total solids,  T a b l e II.  IN R A T  BRAIN  protein and ammonia l e v e l s present  in two-day o l d infant brain, The  O F AMMONIA  and i n adult brain,  initially  cortex slices.  r e s u l t s of these determinations a r e s u m m a r i z e d i n  It i s seen that the i n i t i a l ammonia  mental conditions used  content under the e x p e r i -  i s about 5.0 m i c r o m o l e s p e r g r a m wet  Ammonia  was estimated by the modified method of B r a g a n c a  and T C A  was not used here.  weight. et al.(21)  S i m i l a r values were obtained by W e i l -  M a l h e r b e and other w o r k e r s with adult r a t s ( 20 -22 ). However, experiments extremely  designed to give v a l u e s close to i n vivo l e v e l s gave  low quantities of ammonia  i n the b r a i n of r a t and other  species (0.2-0.3 m i c r o m o l e s p e r g r a m wet weight: Experiments  that follow a r e t h e r e f o r e attempts  see Introduction).  to control and under-  stand the high values obtained under these experimental conditions. It should be mentioned that on a d r y weight b a s i s (dry weight infant b r a i n equals 13.15 per cent wet weight; d r y weight adult b r a i n equals 20.4 percent wet weight),  the adult initial ammonia  i s lower  (35 percent) than that of infant b r a i n .  3.2 E f f e c t s of intraperitoneal injections of glucose, i p r o n i a z i d on initial ammonia (i)  Glucose:  amytal and  contents.  (dose : lOOmg/lOOg body weight) was used  it i s known to inhibit the i n v i t r o f o r m a t i o n of ammonia  because (42 -45 ).  T A B L E II.  Initial --total solids, protein and a m m o n i a contents of 2-day old infant and adult brain. %  Total Solids  Infant  13.15 + 0.68  Adult  20.40 + 0.68  T A B L E III.  Amytal Iproniazid  A m m o n i a content  mg/g wet wt mg/g d r y wt |jmoles/g wet wt Hmoles/g d r y wt 69.55 +2.90 529.0 + 21.0 114.90 + 1.30 564.0 +  6.4  5.06 + 0.49  38.50 +  3.72  5.10 + 0.29  25.00 +  1.42  T h e effects of intraperitoneal injections of glucose, amytal and i p r o n i a z i d on i n i t i a l ammonia of adult rat c e r e b r a l cortex s l i c e s .  T e s t Substance Gluco se  P r o t e i n content  D o s e per lOOg body wt (mg)  A m m o n i a Content Control A n i m a l s Injected with Not injected  physiological  saline  100.0 12.5 1.0  5.35 + 0.17  5.68 + 0.18  A n i m a l s injected with test substance 5.80  + 0.35  6.15  + 0.77  4.91 + 0.46  27 The  a n i m a l s were s a c r i f i c e d 30  (ii) A m y t a l ( 94)  was  used because it has  anaesthesia r e s u l t s i n low The  dose given was  minutes after injections. been established  i n i t i a l b r a i n ammonia l e v e l s ( 2,  12.5mg/100g body weight and  the  that  16  ).  animals  were s a c r i f i c e d about 30 minutes later when they were anae sthetised.. (iii) I p r o n i a z i d (dose : lmg/lOOg body wt) an  was  t r i e d because it i s  effective inhibitor of ammonia l i b e r a t i o n f r o m c e r e b r a l  (95,  96  )•  The  a n i m a l s were  amines  s a c r i f i c e d 30 minutes after  injection. The  r e s u l t s s u m m a r i z e d in T a b l e ITJ show that these  injections had experimental  3.3  The  no  effect on the i n i t i a l ammonia l e v e l s under  our  conditions.  effects of l i q u i d nitrogen and  TCA  on i n i t i a l ammonia in  infant b r a i n . When i n t r a p e r i t o n e a l injections of known c e r e b r a l i n h i b i t o r s failed to reduce the i n i t i a l ammonia level, achieve this a i m nitrogen and, was  were t r i e d .  p o t a s s i u m carbonate, another  other means to  Infant rats were dropped i n l i q u i d  when completely frozen,  d i s s e c t e d while it was  ammonia  were removed.  still f r o z e n and  The  ammonia estimations,  were c a r r i e d out on weighed portions.  set of experiments the  brain with  In  effects of homogenizing infant b r a i n  28  T A B L E IV.  T h e effects of liquid nitrogen and T C A on i n i t i a l ammonia of infant b r a i n (2-day old).  Condition  Ammonia  Content  C e r e b r a l tissue (control)  5.19 + 1.17  C e r e b r a l tissue f r o m f r o z e n a n i m a l  5.39 +  C e r e b r a l tissue homogenized i n T C A (3%)  2.35  + 0.12  C e r e b r a l tissue f r o m f r o z e n a n i m a l homogenized i n T C A (3%)  2.07  + 0.17  T A B L E V.  Initial ammonia l e v e l s i n T C A inactivated c e r e b r a l cortex slice s.  T o t a l ammonia estimated in T i s s u e slice TCA  0.51  (control)  inactivated tissue slice  Ammonia  Content  6.40  + 0.48  4.30  + 0.15  T o t a l NH^ values include ammonia contents of the incubation (3 mins.) media.  29 tissue i n T C A  (3%) f r o m f r o z e n or unfrozen  a n i m a l s on ammonia  content were studied. F r e e z i n g i n liquid N  followed by T C A  2  extraction of the f r o z e n  b r a i n i s a method that has been employed by other  workers  investigating i n vivo ammonia l e v e l s i n b r a i n ( 16, 23-28). our  method of estimation,  p r e l i m i n a r y treatment with l i q u i d nitrogen  was without any effect on the ammonia used,  Using  content,  but when T C A  there was a m a r k e d reduction of the i n i t i a l ammonia  amounting to about 50 percent.  was  level  These results are summarized i n  Table TV),  3.4 Initial ammonia l e v e l s using T C A to inactivate the c e r e b r a l cortex .slice p r i o r to ammonia  estimation.  Weighed brain, cortex  s l i c e s were inactivated by dipping i n a  Ringer-phosphate m e d i u m containing 3 percent  TCA,  were dipped i n a Ringer-phosphate m e d i u m devoid minutes standing the s l i c e s were removed, ammonia  estimation,  ammonia  i n the m e d i a was  are given i n Table values  of ammonia,  corked  V.  while the controls  of T C A .  placed i n f l a s k s ready f o r  and p o t a s s i u m carbonate added. also estimated.  A f t e r three  T o t a l ammonia  The values  It i s seen that T C A inactivated s l i c e s gave  68 percent  that of the control.  30 3.5 E f f e c t s of v a r i o u s substrates on ammonia e s t i m a t i o n with K „ C O „ . — 3 2  U s i n g c e r e b r a l c o r t e x slices,  the effects of the following  substrates on the initial ammonia l e v e l s were then t r i e d .  This  was  done to observe whether such substances affected ammonia f o r m a t i o n during the procedure of p o t a s s i u m carbonate e s t i m a t i o n of ammonia. R e s u l t s a r e given i n T a b l e VI. (1)  L -Glutamine. The  s u p p r e s s i o n of i n i t i a l ammonia by T C A was thought at  f i r s t to be due to suppression of glutaminase activity. the  Though  pH o p t i m u m for this enzyme i n b r a i n i s 8.8 ( 8, 73 ),  it  seemed possible that under the conditions of ammonia e s t i m a t i o n used here  (pH 10.5) some glutaminase activity i s retained and the  glutamine present i n the tissue i s h y d r o l y z e d to give ammonia. In  vivo the glutamine l e v e l in r a t b r a i n i s about  per g r a m "wet weight.(  5.5 m i c r o m o l e s  (16, also see T a b l e XIII).  However,  both with slices and homogenates no a p p r e c i a b l e i n c r e a s e i n ammonia (2)  o c c u r r e d on addition of l.OmM glutamine.  D- and L-Glutamate If glutaminase was indeed responsible f o r the high i n i t i a l  ammonia inspite of the high pH, may  then inhibitors of this  be expected to lower ammonia  that b r a i n glutaminase,  levels.  enzyme  It i s known ( 73)  active at pH 9.0, i s strongly inhibited by  T A B L E VI.  E f f e c t on a m m o n i a f o r m a t i o n by added substrates during the estimation of a m m o n i a in the p r e s e n c e of K _ C O . A m m o n i a f o r m a t i o n during estimation Controls  T e s t substances  Condition of c e r e b r a l tissue  Tissue  L-Glutamine (lmM)  Slice  5.94  +0.26  3.22 + 0.20  7.20  + 0.27  3.80  + 0.38  Homogenate  5.30  +0.30  3.30  +0.00  5.27  + 0.42  3.43  + 0.71  -  T i s s u e + test substance 3%  -  TCA  3%  TCA  D-Glutamate  (5mM)  Slice  6.15 + 0.52  2.60  + 0.38  6.50 +0.45  2.29 + 0.83  L-Glutamate  (5mM)  Slice  6.15 +0.52  2.60  +0.38  7.15  + 0.05  2.24 + 0.50  D- + L - G l u t a m a t e (5mM each)  Slice  6.15+0.52  2.60  +0.38  7.78  + 1.18  2.25 + 1.25  AMP  Slice  6.00 +0.04  3.13  +0.27  7.53  +0.75  3.23 + 1.14  Slice  5.78  2.31  + 0.18  5.36  + 0.22  3.06  (2mM)  Taurine  (5mM)  +0.07  + 0.08  both i s o m e r s of glutamate. separately,  or together,  were then investigated.  The effects of D- and L-glutamate  i n the p r e s e n c e o r absence of T C A , E x p e r i m e n t s showed that ammonia  l e v e l s do not d i m i n i s h under these conditions (Table VI). Moreover,  glutamic  dehydrogenase seems to be inactive under  these conditions. (3)  Adenosine monophosphate The  p o s s i b i l i t y that nucleotide deaminases ( 97, 98)  active under these conditions had to be investigated.  are  The  nucleotide pool i s r e l a t i v e l y s m a l l i n the b r a i n (20, 99). Added A M P formation (4)  (2.0mM) gives little or no i n c r e a s e i n a m m o n i a  under the given experimental  conditions.  Taurine A  possible deamination of taurine under these conditions  seems  not to occur. It should ammonia  3.6  also be mentioned here that glucose  formation  does not affect  under these conditions.  F o r m a t i o n of ammonia f r o m exogenous substrates on a e r o b i c incubation with b r a i n tissue. In addition to a study of the effects of substrates on the  i n i t i a l c e r e b r a l ammonia l e v e l estimated  by K  CO  , studies were  c a r r i e d out to demonstrate their possible effects on ammonia  formation in vitro.  These  studies are d e s c r i b e d below and the r e s u l t s  appear i n T a b l e s VII - X. (1)  L-Glutamine E x p e r i m e n t s were c a r r i e d out to observe the effects of the  addition of glutamine i n the p r e s e n c e of whole b r a i n homogenates under p h y s i o l o g i c a l pH  at r o o m temperature.  were n i l and four hours,  after which K  CO 2  side a r m s f o r the ammonia estimation. TCA  Incubation t i m e s was  added f r o m the  3 One  set of f l a s k s had  (3%) present p r i o r to the addition of homogenates.  Glutamine  was  present at a concentration of l.OmM.  r e s u l t s are given i n T a b l e VII.  The  In the p r e s e n c e of T C A  the  f o r m a t i o n of additional ammonia both i n the p r e s e n c e and of exogenous glutamine i s prevented. i n ammonia,  Though there are i n c r e a s e s  estimated after 4 hours incubation at r o o m  ture, both in the p r e s e n c e or absence  absence  of glutamine,  tempera-  only an  insignificant amount can be attributed to that coming f r o m added glutamine.  However, b r a i n s l i c e s incubated at 37°C f o r one  hour  s i m i l a r m e d i u m conditions,  under  produce  ammonia  from  added glutamine. (2)  D-Glutamate. R e s u l t s i n T a b l e VIII show that c e r e b r a l c o r t e x s l i c e s  incubated at 37°C for one hour i n a Ringer-phosphate-glucose  34  T A B L E VII. T h e apparent absence of glutaminase activity_at pH 7.4 and r o o m temperature incubation using b r a i n homogenates. Additions to a R i n g e r -Pho sphate Medium  Ammonia formation [ncubation time, after which K^CO^ was added 0  4 hour s  —  5.30 + 0.30  13.45 + 0.25  T C A (3%)  3.30 + 0.00  3.12 + 0.40  (ImM)  5.27 + 0.42  14.78 + 0.83  Glutamine (ImM) + T C A (3%)  3.43 + 0.71  3.21 + 1.41  Glutamine  T A B L E VIII. T h e deamiflation of glutamine (ImM) by c e r e b r a l cortex s l i c e s and the inhibition of the deamination by D-Glutamate. Additions to a R i n g e r - P h o s p h a t e Glucose Medium  Total N H  3  formation  6.30 + 0.15  Nil D-Glutamate  (5mM)  8.71 + 0.72  L-Glutamine  (ImM)  15.55+ 0.95  L - G l u t a m i n e (ImM) + D-Glutamate  Incubation one hour at 37 C.  (5mM)  11.70 + 0.70  35 m e d i u m produce ammonia f r o m added glutamine. inhibition of this glutaminase (70%) (3)  A  significant  activity by addition of D-glutamate  takes place.  AMP  and A T P  AMP  has no substantial effect on i n i t i a l ammonia  Using  c e r e b r a l cortex s l i c e s i n a  (Table VI).  Ringer-phosphate-glucose  m e d i u m and incubation at 3 7 ° C f o r one hour, both A M P b r i n g about i n c r e a s e s i n ammonia f o r m a t i o n AMP may  (Table IX).  i s deaminated to a greater extent than A T P imply h y d r o l y s i s p r i o r to deamination,  and A T P That  (Table IXa)  since adenylic  deaminase only attacks the monophosphate of adenosine ( 1 0 0 ). W e i l - M a l h e r b e and G r e e n ( 20 ) using d i a l y z e d or washed b r a i n suspension, ATP, (4)  have r e p o r t e d  ADP  from  and adenosine.  Taurine. Taurine  containing (10  significant ammonia f o r m a t i o n  1  i s found i n high concentrations i n brain, r a t b r a i n  5.0 m i c r o m o l e s f r e e taurine p e r g r a m f r e s h weight  , also see Table XIII).  T a u r i n e i s converted to  isethionie a c i d i n r a t b r a i n ( 1 0 2 ) and heart m u s c l e  ( 1 0 3 ).  T h i s involves a replacement of an amino by a h y d r o x y l group. The  m e c h a n i s m of the c o n v e r s i o n h a s not yet been elucidated;  however, transamination  i s indicated.  T h e p o s s i b i l i t y that  T A B L E IX.  T h e deamination of A T P and A M P G l u c o s e medium. Nucleotide Concentration (mM)  Nucleotide Concentration (mM) 1.0 2.0 4.0  Total ammonia formation f r o m ATP 8.95 16.30 20.90 31.65  Nil 1.0 2.0 4.0  T A B L E IXa.  by c e r e b r a l cortex s l i c e s i n a R i n g e r - P h o s p h a t e -  + 0.22 + 1.90 + 0.40 + 3.75  P e r c e n t a g e deamination of A T P and A M P : Theoretical yield for complete deamination \X m o l e s 3.0 6.0 12.0  AMP 7.23 21.80 26.80 42.30  + 0.50 + 1.20 + 3.80 + 0.50  a n a l y s i s of T a b l e IX.  Amount of a m m o n i a produced f r o m nucleotide & percentage deamination Percentage Percentage ATP AMP Deamination Deamination 0.611 0.920 1.870  20.4 15.3 15.6  0.985 1.200 2.330  32.80 20.00 19.40  ON  37  T A B L E X. T h e effects of taurine on a m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s incubated for one hour at 37°C. Additions to a R i n g e r - P h o sphate Medium  T o t a l ammonia  Nil Taurine  (5mM)  Glucose G l u c o s e + taurine  (5mM)  7  formation  17.10  +  1.50  17.35  +  0.95  7.60  +  0.35  7.68  +  0.88  38  taurine can y i e l d free ammonia i n vitro i n the p r e s e n c e of c e r e b r a l cortex slices was then studied.  However, f r o m T a b l e  it can be seen that taurine i s without any effect on ammonia f o r m a t i o n when added to the incubation m e d i u m i n the p r e s e n c e or absence  of glucose.  3.7 T h e source of the extra ammonia produced carbonate e s t i m a t i o n i n the absence Experiments ammonia  Set a:  without incubation.  following p r e p a r a t i o n s were made: Homogenates of c e r e b r a l c o r t e x s l i c e s i n a R i n g e r phosphate  Set b:  of T C A .  were c a r r i e d out to explore the source of the  found i n the brain, The  during p o t a s s i u m  medium.  Supernatant of Set a, p r e p a r e d by centrifuging under c h i l l e d conditions f o r 15 minutes.  The supernatant  contained the soluble c e l l components including  soluble  proteins. Set c:  Supernatant of Set b after precipitation with T C A (final concentration 3%) followed by centrifuging off the precipitate.  Set d:  Homogenates of c e r e b r a l cortex s l i c e s i n a R i n g e r phosphate  m e d i u m containing 3 %  TCA.  X  T A B L E XI.  Initial a m m o n i a values f r o m c e r e b r a l c o r t e x tissue examined under different conditions.  Initial ammonia v a l u e s estimate d by  Condition:  K C O ^ method  Amino acid analyzer  Homogenate  5.35 + 0.36  -  b.  Supernatant of a (includes soluble components)  4.47  -  c.  Supernatant of b after precipitation of c e r t a i n soluble components with T C A  2.54 + 0.16  d.  Homogenate p r e p a r e d i n T C A  3.43 + 0.39  e.  Supernatant of d.  2.50 + 0.14  Set  (Tissue i n Ringer-Phosphate  a.  medium)  2  + 0.38  2.76  + 0.12  3.08  + 0.07  40  Set e:  Supernatant  of Set d.  R e s u l t s s u m m a r i z e d i n Table X I TCA The  show that the r e m o v a l of  insoluble components r e s u l t s in a lower addition of T C A  not totally (70%)  initial ammonia level.  to b r a i n homogenates (Set d) substantially but  suppresses  ammonia formation.  these experiments  that T C A  may  breakdown of T C A  insoluble components.  It seems  be i m m o b i l i z i n g an However,  from  enzymatic further work i s  r e q u i r e d to throw m o r e light on the p r o c e s s .  3.8  K i n e t i c s of a m m o n i a f o r m a t i o n Using  c e r e b r a l c o r t e x slices,  ammonia with time of incubation, was m e d i u m at 3 7 ° C i n the presence  the a e r o b i c f o r m a t i o n of c a r r i e d out i n a  Ringer-phosphate  or absence of glucose.  V a l u e s given  i n T a b l e XII compare well with those of other w o r k e r s ( 20, The flasks,  effects of the addition of T C A  on the completion  (3%) to  25).  experimental  of incubation, were also investigated.  From  T a b l e XII.it i s seen that T C A ,  when added to a  Ringer-phosphate  m e d i u m devoid of glucose, has  little or no effect on the total amount  of ammonia present in the incubation flask. However, i n the presence the end  of one  of glucose, the addition of T C A  hour incubation give s a m a r k e d l y lower  m i c r o m o l e s per g r a m wet  value  at  (4.23  weight) than control f l a s k s (7.0 3 m i c r o m o l e s  41  T A B L E XII.  A m m o n i a f o r m a t i o n with time. Total ammonia f o r m a t i o n  T i m e of incubation (hour s)  Glucose TCA  0.25  9.26 + 1.17  0.50  14.10 +0.82  *1.00  TCA _  _  -  -  -  17.90 +0.05  2.63 + 0.35  6.65 + 0.40  2.75 +0.01  1.00  17.00 + 0.19  16.50 + 1.25  7.03 + 0.17  4.23 +0.07  2.00  21.68 +0.68  22.50 + 0.07  6.83 + 0.30  7.30 +0.97  3.00  22.93 + 0.17  23.20 + 0.23  7.59 + 0.21  8.05 + 0.36  4.00  24.57 + 0.36  25.57 + 1.02  7.02 + 0.75  7.80 + 0.47  T C A (final concentration 3 % ) , when added, was at the end of the incubation period, except f o r * when it was added b e f o r e incubation commenced.  per g r a m wet weight).  This d i f f e r e n c e , v i z . (7.03 - 4.23) = 2.8  m i c r o m o l e s p e r g r a m wet weight, i s s m a l l e r than the ammonia difference obtained when T C A is added to the flask before the one hour incubation commences, v i z . (6.65 - 2.75) = 3.9 m i c r o m o l e s per g r a m wet weight, and completely disappears i n 2, 3 and 4 hour incubations.  This implies that during incubation i n the presence of  glucose, ammonia f o r m a t i o n continues up to a point, being only prevented by the addition of T C A .  In other words, it appears that  there are at least two m e c h a n i s m s ( o r sources) by which ammonia formation takes place.  One, that is dependent on time of incubation,  occurs not only i n the presence of glucose but even to some extent i n the presence of K2CO3 and is inhibited by T C A .  The other, i s sup-  p r e s s e d b y glucose and also on the addition of K2CO3 or T C A . • Since the final addition of T C A neither affects the g l u c o s e sensitive ammonia formation,  nor the ammonia formation.in a m e d i u m  devoid of glucose, its use has been discontinued i n the experiments: that follow.  A g r a p h i c a l representation of ammonia f o r m a t i o n with time of incubation i n the p r e s e n c e or absence of glucose, with or without t e r m i n a l addition of T C A , appears i n F i g u r e 1.  28.0 r-  24.0  -  20.0 00  cn © i — <  O  8  16.0  -  ^a. s o  .1-1  £  12.0 -  r H  O  c o  s  8.0  O  4.0 -  0.0  FIGURE  1. T i m e c o u r s e of the f o r m a t i o n of a m m o n i a b y r a t c e r e b r a cortex  ;  s l i c e s at 3 7 ° C i n a R i n g e r - p h o s p h a t e  m e d i u m in  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 .  O  A m m o n i a f o r m a t i o n i n the a b s e n c e of g l u c o s e  o  S a m e a s (©) e x c e p t that T C A ( 3 % ) w a s a d d e d at the end o the i n c u b a t i o n  A  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 g l u c o s e  A  S a m e as (A) e x c e p t that T C A (3%) w a s a d d e d at the e n d o  (5mM)  the i n c u b a t i o n E x t r a p o l a t i o n to the i n v i v o (0 .3 n m o l e s / g  wet w e i g h t ) .  a m m o n i a c o n t e n t of b r a i n  44  3. 9 A m i n o acid content of c e r e b r a l cortex s l i c e s .  Initial values of amino acid content of c e r e b r a l cortex s l i c e s were determined.  P a r a l l e l studies on the total (tissue plus medium)  amino acid content at the end of one hour incubation at 37° C i n the presence or absence of glucose, were also c a r r i e d out. are given i n Table XIII.  The results  They show that the i n i t i a l values are not  substantially changed on incubation of s l i c e s with glucose.  In the  absence of glucose however, the values of taurine, glutamate  and  glutamine levels f a l l , while the aspartate and alanine levels r i s e . Glutamate is known to be converted to aspartate either v i a the c i t r i c a c i d cycle (104) or by a pathway independent  of it (105, 106).  Oxalo-  acetate and pyruvate are converted to aspartate and alanine r e s p e c t i v e l y by t r a n s a m i n a t i o n reactions with glutamate (8, 80-82).  Taurine is  converted to isethionic a c i d on losing an amino group, p r e s u m a b l y by a t r a n s a m i n a t i o n r e a c t i o n (101-103).  Under the following e x p e r i m e n t a l conditions of a m m o n i a and amino acid a n a l y s i s , v i z . (1)  initial,  (2)  one hour incubation at 37° C,  T A B L E XIII.  The total, ammonia and amino acid content of cerebral cortex slices initially and on incubation in a Ringer-Phosphate medium for one hour at 37°C. yimoles per gram wet weight Incubation: one hour at 37°C  Compound e stimated  Initial  + Glucose  *Change  No glucose  *Change  16.90 + 2.40  + 13.82  Ammonia  3.08 + 0.07  5.33 + 0.67  + 2.25  Taurine  5.05+0.69  5.00 + 0.41  - 0.05  2.93 + 0.09  - 2.12  Aspartate  2.97 +0.48  3.00 + 0.12  + 0.03  7.33 + 0.28  + 4.36  5.42+0.52  4.86 + 0.21  - 1.12  2.00 + 0.30  - 6.84  11.35+0.86  10.02 +0.84  - 1.33  2.87 + 0.13  - 8.48  Glycine  1.30 +0.13  1.50 + 0.17  + 0.20  1.05 + 0.16  - 0.25  Alanine  0.60 +0.12  0.62 + 0.21  + 0.02  1.10 +0.11  + 0.50  35.19 + 3.42  35.19 + 2.85  0.00  36.18 + 3.77  - 0.99  Glutamine  x  Glutamate  Total -NH^ present in the free pool *  Change in amide and a-amino nitrogen over initial values.  x Glutamine values taken twice for obtaining total -NH^, present in the free pool. A l l estimations carried out with the amino acid analyzer.  46  (3)  one hour incubation at 3 7 C i n the presence of glucose,  i t i s seen that the amount of -NH  groups present i n the amide and alpha  amino groups of the m a i n amino a c i d s i n brain, when added to the ammonia content, give values which are r e m a r k a b l y  equal. In other  words, the l o s s of amino groups i s about equal to the a m m o n i a formation. Thus, it i s justifiable to conclude that, under these conditions, r i s e of ammonia i s due to l o s s of amino a c i d nitrogen.  Conclusion It may  be concluded f r o m these r e s u l t s that there a r e at l e a s t  two m e c h a n i s m s by which ammonia forrration o c c u r s i n b r a i n tissue. that o c c u r s immediately  after death of the animal, p r e s u m a b l y f r o m  insoluble m a t e r i a l . Its f o r m a t i o n i s inhibited by T C A .  The  One TCA-  other o c c u r s  during aerobic incubation of b r a i n c o r t e x s l i c e s i n a g l u c o s e - f r e e medium. E v i d e n c e obtained shows that a m m o n i a f o r m a t i o n by c e r e b r a l cortex 7  s l i c e s incubated at 3 7 ° C for one hour can l a r g e l y be accounted f o r by the l o s s of amino a c i d nitrogen of c e r e b r a l amino a c i d s .  47 4. CONDITIONS A F F E C T I N G A M M O N I A  FORMATION  IN B R A I N D U R I N G I N C U B A T I O N  4.1 A m m o n i a f o r m a t i o n by infant and adult r a t c e r e b r a l cortex s l i c e s . E s t i m a t i o n s were made of the rate of a m m o n i a f o r m a t i o n by two day old infant and adult rat c e r e b r a l cortex s l i c e s i n the presence or o absence of glucose, on incubation a e r o b i c a l l y at 37 C for one hour. Studies were also made of the rate of ammonia f o r m a t i o n by adult rat c e r e b r a l cortex s l i c e s under anaerobic conditions. R e s u l t s of these experiments  on the b a s i s of wet weight and d r y weight a r e given in  T a b l e XIV.  "With r e f e r e n c e to estimates given i n t e r m s of wet weight  tissue,  i t should be noted that the water content of the b r a i n changes  during growth; the concentration of solids n e a r l y doubles II).  O n a d r y weight b a s i s the p r o t e i n  (107,  Table  contents i n infant b r a i n and  c e r e b r a l cortex s l i c e s of adult rats a r e found to be v e r y  similar  (Table II). /  It can be seen f r o m  Table X I V that, on a wet weight basis,  a e r o b i c incubation with glucose gives s i m i l a r values f o r the rate of ammonia  f o r m a t i o n i n either infant or adult cortex slices,  the absence of glucose infant b r a i n gives lower  whereas i n  values than the adult.  On a d r y weight basis, however, the r e v e r s e i s true.  The  rate of ammonia f o r m e d i n the absence of glucose i s about the same within experimental  error,  while,  with glucose,  adult b r a i n cortex  48  T A B L E XIV.  T o t a l ammonia f o r m a t i o n i n infant and adult c e r e b r a l cortex slices i n one hour at 3 7 ° C i n file p r e s e n c e or absence of glucose (5mM).  Conditions I  T o t a l ammonia production p.mole s/g ^imoles/g wet wt. d r y wt.  Ammonia formation on d r y weight b a s i s ( F i n a l - Initial)  Aerobic A . Infant (i) (ii) G l u c o s e  80.5 + 12.5  42.0  7.20 + 0.70  54.7+  5.3  16.2  17.80 + 1.00  87.4+  4.9  62.4  8.00 +0.50  39.2+  2.4  14.2  8.55 + 0.74  41.9 + 3.62  16.9  8.52 +0.27  41.7 + 1.32  16.7  10.55 + 1.65  B. A d u l t (i) " (ii) G l u c o s e  [I A n a e r o b i c A. Adult (i) (ii) G l u c o s e  Initial values obtained f r o m T a b l e II, i.e. Infant Adult  = 38.5 + 3.72 = 25.00 + 1.42  49 s l i c e s give lower values.  O n subtracting initial  values of ammonia f r o m those (1)  after one hour incubation, i t i s seen:  T h e rate of ammonia f o r m a t i o n aerobically,  in one hour by infant b r a i n  i n the absence of glucose,  adult c e r e b r a l cortex d r y weight b a s i s .  (non-incubation)  i s 65 percent that of  s l i c e s (under s i m i l a r conditions) on a  (On a wet weight b a s i s the percentage i s  about 45.) (2)  About  16 m i c r o m o l e s ammonia p e r g r a m d r y weight a r e  produced i n one hour when (i)  infant or adult c e r e b r a l cortex s l i c e s a r e incubated a e r o b i c a l l y i n the presence of glucose; or,  (ii) adult c e r e b r a l cortex  s l i c e s a r e incubated a n a e r o b i c a l l y  in the presence or absence of glucose.  4.2 T i s s u e and incubation m e d i u m ammonia content c e r e b r a l cortex  of adult r a t  o slices on incubation at 37 C f o r one hour.  V a l u e s of tissue and m e d i u m content  of ammonia on incubation,  a e r o b i c a l l y or anaerobically, i n the presence or absence of glucose, are given i n T a b l e X V .  V a l u e s of total ammonia produced a e r o b i c a l l y  in the p r e s e n c e of glucose, of glucose,  o r a n a e r o b i c a l l y i n the presence or absence  a r e about the same.  While the tissue contents  f o r m e d a e r o b i c a l l y , i n the absence or presence of glucose,  of ammonia a r e not  50  T A B L E XV.  T i s s u e and media concentrations of ammonia produced under a e r o b i c and anaerobic conditions i n the presence and absence of glucose f r o m adult r a t c e r e b r a l cortex slices.  Incubation conditions I  Total  formed--(imoles /g^ wet weight s Tissue  Media  (*)  Aerobic 14.76 + 1.12 (0.492)  18.Z3 + 1.25  3.47 +0.13  7.95 + 1.28  3.52 + 0.35  4.43+0.93  (0.148)  (i) -  8.55 j f 0 . 7 4  2.47 + 0.26  6.08 +0.48  (0.210)  (ii) Glucose  8.52 + 0.27  2.97 + 0.26  5.55+0.21  (0.185)  (i) (ii) Glucose  II  Ammonia  Anaerobic  (*) M e d i a ammonia i n mM,produced by lOOmg f r e s h weight t i s s u e .  51  different, the concentration of ammonia in the incubation medium in the absence of glucose is threefold that in its presence.  This results  in the total ammonia formation in the absence of glucose being at least twice the total ammonia formation in its presence. Anaerobically, there i s a greater leakage of ammonia into the incubation medium, thereby leaving the tissue with a lower  ammonia  content.  4.3  Effects of amino acids, dipeptides and urea on the rate of ammonia formation i n cerebral cortex slices of adult rat. The addition of urea or glycine only slightly increases the  aerobic formation of ammonia, when added to a Ringer-phosphate medium, and that of taurine, y ABA, L-aspartate or glycyl-L-aspartate is without effect.  There is only a slight diminution in the rate of  ammonia formation in the presence of added L-alanine, D-aspartate or Li-arginyl-L-glutamate.  The presence of L-glutamate has a larger  inhibitory effect on the rate of ammonia formation under similar conditions, and D-glutamate i s even more effective.  The results are  given in Table XVI.  4.4 The effects of glucose, glycolytic and citric acid cycle intermediates on the rate of ammonia formation in cerebral cortex slices. It may be seen from Table XVII that either lactate or pyruvate added to a Ringer-phosphate medium at concentrations of 5mM  or  52  T A B L E XVI.  T h e effects of amino acids, dipeptides and u r e a on the f o r m a t i o n of ammonia i n b r a i n cortex s l i c e s .  A d d i t i o n to a R i n g e r -Pho sphate Medium (5.0mM)  T o t a l ammonia f o r m a t i o n  17.37  +  0.34  Glucose  8.18  +  0.51  Glycine  18.36  +  0.16  L-alanine  15.50  +  0.30  Taurine  17.35  +  0.95  v -amino butyrate  17.18  +  0.32  L-glutamate  14.64  +  0.20  D-glutamate  11.44  +  1.00  L-aspartate  17.10  +  0.90  D-aspartate  16.62  +  0.63  Glycyl-L-aspartate  17.60  +  0.65  L - a r g i n y l -L -glutamate  15.50  +  0.97  Urea  19.38  +  0.22  Nil  T A B L E XVII. T h e effects of g l y c o l y t i c and the c i t r i c a c i d cycle i n t e r m e d i a t e s on a m m o n i a f o r m a t i o n i n c e r e b r a l cortex s l i c e s . Total Substrate(s) added to Ringer - Phosphate Medium  ammonia  formation  Controls Nil  Glucose  Test Substrate  Pyruvate  (5mM)  17.57 +0.73  8.31 + 0.03  7.30 +0.27  D L -lactate  (5mM)  17.37^ + 0.55  8.22 + 0.28  8.30 +0.80  D L -lactate  (lOmM)  17.37 +0.55  8.22 + 0.28  8.32 +0.28  Acetate  (5mM)  17.57 +0.73  8.31 + 0.03  16.70 +0.63  P y r u v a t e (5mM) + Acetate  (5mM)  17.57 +0.73  8.31 + 0.03  7.31 + 0.24  Citrate  (5mM)  18.09+0.61  6.30 + 0.15  15.00 +0.90  a-ketoglutarate  (5mM)  18.09+0.61  6.30 + 0.15  16.25+0.35  Oxaloacetate  (5mM)  17.05 +0.85  6.43 + 0.02  11.03 + 1.67  54 greater,  suppresses the rate of ammonia f o r m a t i o n to about the same  extent as that due to 5mM oxaloacetate,  glucose  (see a l s o F i g u r e  the inhibition of a m m o n i a f o r m a t i o n  Citrate only slightly  2). With  5mM  i s not as marked.  suppresses the rate of ammonia f o r m a t i o n and  acetate or a-ketoglutarate has little or no effect.  4.5 T i s s u e ammonia  content under different experimental  conditions.  R e s u l t s given i n Table XVIII show that the ammonia  contents o  of c e r e b r a l cortex  slices,  incubated a e r o b i c a l l y f o r one hour at 37 C  i n a Ringer-phosphate m e d i u m containing different substrates, same within experimental  e r r o r . ^ T h i s i m p l i e s that the v a r i a t i o n i n  the rate of ammonia formation,  obtained a e r o b i c a l l y under a v a r i e t y  of media conditions, i s seen only i n the ammonia incubation  contents  incubations,  slightly lower tissue ammonia  it should be noted (Table XV), give contents,  due to the g r e a t e r leakage of  into the incubation m e d i u m under these  4.6 T h e effects of glucose  conditions.  and pyruvate concentrations on a m m o n i a  f o r m a t i o n f r o m r a t b r a i n c e r e b r a l cortex  slices.  E x p e r i m e n t s were c a r r i e d out to find the lowest concentration of glucose ammonia  of the  medium.  Anaerobic  ammonia  a r e the  formation  (threshold)  and pyruvate that would be inhibitory to  i n an incubation of one hour at 37°C.  It i s seen  55  T A B L E XVIII.  A m m o n i a content of c e r e b r a l cortex s l i c e s after one hour incubation at 37° C i n a Ringer-Phosphate medium.  Additions to a Ring e r -Pho sphate Medium (5mM)  Ammonia in tissue  Nil  3.87 + 0.13  Glucose  3.74+ 0.30  2-deoxy glucose  4.00 + 0.10  Pyruvate  3.72 + 0.60  Citrate  3.37 + 0.17  a-ketoglutarate  3.90 + 0.36  Anaerobic (Ringer H C O ^  3.16 + 0.16  medium)  F I G U R E 2.  T h e effects of glucose and pyruvate concentrations on the rate of a m m o n i a f o r m a t i o n by rat b r a i n cortex s l i c e s on incubation i n a Ringer-phosphate m e d i u m for one hour at 3 7 ° C .  57 f r o m F i g u r e 2 that l.OmM glucose i s about the m i n i m u m concentration r e q u i r e d f o r m a x i m a l suppression of ammonia formation,  while f o r  pyruvate  concentrations  the concentration i s about 2.5mM.  Above these  the rate of ammonia f o r m a t i o n i s not f u r t h e r depressed.  These  threshold l e v e l s (viz. l.OmM f o r glucose and 2.5mM f o r pyruvate) a r e utilized i n further e x p e r i m e n t s c a r r i e d out to obtain a further insight into the m e c h a n i s m s of ammonia  formation.  4.7 T h e effects of O.lmM iodoacetate on the s u p p r e s s i o n of ammonia f o r m a t i o n by glucose and pyruvate. The  i n c r e a s e i n the rate of ammonia f o r m a t i o n by c e r e b r a l  cortex s l i c e s i n an incubation m e d i u m containing glucose and iodoacetate ( 47 ), p r e s u m a b l y o c c u r s by the inhibition of glucose metabolism.  T h e inhibition of phosphoglyceraldehyde  by iodoacetate d e c r e a s e s  the supply of pyruvate  and thereby^ the oxidation of pyruvate  through  dehydrogenase  formed f r o m  the c i t r i c a c i d  glucose cycle/  whereby i n t e r m e d i a t e s (e.g. a-ketoglutarate) a r e f o r m e d resulting i n ammonia  fixation.  With this i n mind, the effect of iodoacetate on the  rate of a m m o n i a formation i n the presence  of pyruvate  was then t r i e d .  It i s seen f r o m the r e s u l t s i n T a b l e X I X that, while iodoacetate i n c r e a s e s the rate of a m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s i n an incubation m e d i u m containing 5mM effect when 5mM  pyruvate  glucose,  i s the substrate.  it h a s little or no  58  T A B L E XDC.  T h e effects of O.lmM iodoacetate on the s u p p r e s s i o n of ammonia f o r m a t i o n by glucose and pyruvate i n rat b r a i n cortex s l i c e s .  Additions to a Ringer - Phosphate Medium  Total ammonia f o r m a t i o n 17.60 + 0.40  Nil Iodoacetate  (O.lmM)  Glucose  (5mM)  G l u c o s e (5mM) + Iodoa cetate (O.lmM) Pyruvate  (5mM)  P y r u v a t e (5mM) + Iodoacetate (O.lmM)  16.77 + 1.33 9.00 + 0.17 13.48 + 0.23 9.92 + 0.35 10.18 + 0.15  59 4.8 T h e effects of high potassium i o n concentration on the suppression of ammonia f o r m a t i o n by different glucose and pyruvate concentrations. The r e s u l t s f o r glucose and pyruvate a r e given i n T a b l e s X X and XXI respectively. In the presence of 5mM  glucose, the addition of lOOmM KC1 to a  Ringer-phosphate m e d i u m has no effect on the rate of ammonia tion.  T h i s i s consistent with the observed  effects of high  potassium  concentrations on ammonia f o r m a t i o n i n the p r e s e n c e of glucose guinea p i g b r a i n slices i n a four hour incubation ( 9, 54, 108). i s a l s o found to be true i n the presence of 5mM potassium  pyruvate  using This  and high  i n experiments r e p o r t e d h e r e (Table X X I ) .  In the presence of the t h r e s h o l d l e v e l of glucose potassium  forma-  (ImM),  a high  i o n concentration strikingly elevates the rate of ammonia  f o r m a t i o n i n one hour.  T h e r e s u l t i s consistent with the c o n c l u s i o n  that i n the presence of a high concentration of KC1 there i s a m a r k e d i n c r e a s e i n the operation of the c i t r i c a c i d c y c l e ( 77,  78 ) so that the  intermediates f r o m glucose a r e oxidized too fast to allow f o r the n o r m a l "rate of ammonia fixation. A s expected on this view, the threshold l e v e l of pyruvate  (2.5mM) gives only v e r y  ammonia f o r m a t i o n over  s m a l l i n c r e a s e s i n the rate of  c o n t r o l values when incubation i s c a r r i e d out  i n the presence of a high potassium  ion concentration.  60  T A B L E XX.  T h e effects of high K on the suppression of a m m o n i a f o r m a t i o n b y different glucose concentrations i n rat c e r e b r a l cortex s l i c e s . +  A d d i t i o n s to a Ringer -Pho sphate Medium  Total ammonia f o r m a t i o n 16.88 + 0.12  Nil KC1  (lOOmM)  16.46 + 0.36  Glucose  (0.5mM)  11.75 + 0.35  (lOOmM)  15.11 + 0.71  (l.OmM)  7.71 + 0.53  (lOOmM)  13.55 + 0.45  (5.0mM)  8.68 + 0.00  (lOOmM)  8.24 + 0.55  Glucose  (0.5mM) + KC1  Glucose Glucose  (l.OmM) + KC1  Glucose Glucose  (5.0mM) + KC1  61 T A B L E XXI.  T h e effects of high K on the pyruvate inhibition of ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s . +  Additions to a Ringer - Phosphate Medium  Total ammonia f o r m a t i o n 17.50 + 0.23  Nil Pyruvate  (2.5mM)  10.15+ 0.30  P y r u v a t e (2.5mM) + KC1  (lOOmM)  11.91 + 0.47  Pyruvate  (lO.OmM)  9.61 + 0.68  P y r u v a t e (10.0 mM)  T A B L E XXII.  9.12 + 0.73  + KC1 (lOOmM)  T h e effects of inhibitors of oxidative phosphorylation on ammonia f o r m a t i o n i n r a t c e r e b r a l cortex s l i c e s . Total Ammonia Formation  Inhibitor Used  A d d i t i o n s to a R i n ger-Phosphate M e d i u m Glucose Glucose (5mM) Nil Inhibitor (5mM) and Inhibitor  D N P (O.lmM) 18.10 +0.72  13.94 + 1.00  9.18 +0.47  13.98 +0.86  Salicylate (5mM)  14.80 + 0.10. 8.80 +0.20  11.70 +0.00  17.75 + 0.05  In the absence of glucose high potassium  i s only  slightly  inhibitory to the rate of ammonia f o r m a t i o n i n a one hour incubation. However, f o r a longer incubation p e r i o d (4 hours), r e p o r t e d a considerable lowering of ammonia potassium  i o n concentrations,  V r b a (108)  formation by high  while at the same time there" was a  diminution i n the rate of oxygen uptake.  4.9 T h e effect of inhibitors of oxidative phosphorylation of ammonia f o r m a t i o n i n b r a i n cortex  on the rate  slices.  R e s u l t s i n T a b l e X X I I c o n f i r m the fact that 2,4-dinitrophenol (DNP) inhibits the rate of ammonia f o r m a t i o n i n the absence of glucose  (20, 46).  In the p r e s e n c e of glucose,  rate of a m m o n i a formation, and thereby  DNP  elevates the  p r e s u m a b l y by inhibiting A T P  the c o n v e r s i o n of glutamate to glutamine.  are obtained here with N a - s a l i c y l a t e (5mM),  synthesis  Similar results  known to uncouple  oxidative phosphorylation (109. However, with salicylate the i n c r e a s e i n the rate of ammonia f o r m a t i o n i n the p r e s e n c e of glucose i s not so markec  4.10  T h e effects of amytal (ImM), and malonate (5mM)  on ammonia  f o r m a t i o n by r a t c e r e b r a l cortex s l i c e s i n the presence o.f low glucose  concentrations.  In vivo, the ammonia l e v e l i n the b r a i n i s d e p r e s s e d nembutal n a r c o s i s (16).  by  However, intraperitoneal injections of  63  T A B L E XXm.  T h e effects of a m y t a l and malonate on a m m o n i a f o r m a t i o n i n c e r e b r a l cortex s l i c e s of r a t i n the p r e s e n c e of l o w glucose concentrations.  Additions to a Ringer -Pho sphate Medium  Total ammonia formation 17.90 + 0.20  Nil Glucose  (0.25mM)  16.68 + 0.08  Amytal  (ImM)  11.50 + 0.35  G l u c o s e (0.25mM) + A m y t a l (ImM)  11.10 + 0.00  Glucose  (ImM)  8.41 +  G l u c o s e (ImM) + A m y t a l  (ImM)  8.90 + 0.32  G l u c o s e (ImM) + Malonate (5mM)  11.68 + 0.33  1.01  64 of amytal (12.5mg/100g body wt) do not lower i n i t i a l ammonia our e x p e r i m e n t a l conditions (Table III). investigated.  presumably  Its effect i n v i t r o was then  It i s seen f r o m T a b l e X X U J that I m M  inhibits the rate of ammonia  under  amytal  f o r m a t i o n i n the absence  strongly  of glucose,  by blocking the oxidation of endogenous substances that  l i b e r a t e ammonia.  Amytal  (ImM) does not affect the r e d u c e d rate of  ammonia f o r m a t i o n obtained i n the p r e s e n c e of I m M  glucose.  This  may be understood i f glucose oxidation still p r o c e e d s  sufficiently  r a p i d l y to produce i n t e r m e d i a t e s r e s u l t i n g i n ammonia  fixation.  Malonate, a c i d cycle,  as may be expected, by i t s s u p p r e s s i o n of the c i t r i c  elevates the rate of ammonia f o r m a t i o n i n the presence of  l.OmM glucose.  Malonate  i n the absence  of glucose has been shown  to be without effect on both the rate of ammonia f o r m a t i o n and the rate of oxygen uptake ( 47 the  ), indicating that i n the absence  o f glucose  rate of operation of the c i t r i c a c i d cycle i s greatly reduced.  4.11 E f f e c t of glutamate d e r i v a t i v e s and of h y d r o x y l a m i n e on the rate of ammonia The  f o r m a t i o n by c e r e b r a l cortex  r e s u l t s of these studies c a r r i e d out i n a Ringer-phosphate  m e d i u m i n the presence or absence (a)  slices.  In the absence  of glucose a r e given in T a b l e X X I V .  of glucose.  It i s seen f r o m T a b l e X X I V that, while L-glutamate, Li-glutamate -y -methyle ster or L - g l u t a m a t e - y - ethyl ester added at  5mM  l e v e l s inhibit the rate of ammonia formation, the inhibition i s  m o r e pronounced with D L - a - m e t h y l g l u t a m a t e  or D-glutamate. In  the p r e s e n c e of h y d r o x y l a m i n e (5mM), however, the rate of ammonia f o r m a t i o n i s elevated under the e x p e r i m e n t a l conditions used.  This  i s c o n t r a r y to the r e s u l t s r e p o r t e d by W e i l - M a l h e r b e and G r e e n (20 ) where a 42 percent inhibition of ammonia f o r m a t i o n was shown to o c c u r i n a four hour incubation with guinea pig b r a i n s l i c e s . the concentration of hydroxylamine they used was I m M  However,  i n the one  experiment they c a r r i e d out. (b)  In the p r e s e n c e of glucose. A m o n g the glutamate analogs tried,  D-glutamate  i s the only  one that elevates the rate of ammonia f o r m a t i o n i n a R i n g e r phosphate  glucose medium.  T h i s is understandable as D-glutamate  i s known to inhibit glutamine synthetase (73,110). of 5mM  hydroxylamine,  In the p r e s e n c e  a considerable i n c r e a s e i n the rate of  ammonia f o r m a t i o n occurs, which c o m p a r e s well with the rate of ammonia f o r m a t i o n i n a substrate free incubation medium.  Under  these conditions there i s o b s e r v e d a concomitant drop i n the rate of oxygen uptake by c e r e b r a l cortex s l i c e s to that value seen i n the absence  of glucose.  A f a l l i n the rate of oxidation of glucose (111)  brought about by hydroxylamine, of ammonia formation.  would  r e s u l t i n an i n c r e a s e d rate  It i s also possible that h y d r o x y l a m i n e  competes with ammonia i n the glutamine synthetase r e a c t i o n ( 112 ).  T A B L E XXIV.  T h e effects of glutamate, its analogs and h y d r o x y l a m i n e on a m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s of r a t i n the p r e s e n c e or absence of glucose.  Total ammonia f o r m a t i o n A d d i t i o n s to a L Ringer-Phosphate E x p e r i m e n t s using the test substance  Medium  +(5mM) T e s t Substance  Controls Glucose  Glucose  L-glutamate  17.89+0.41  8.48 + 0.32  15.16 +0.64  7.41 + 0.13  D-glutamate  18.09 +0.61  6.30 + 0.15  12.23 + 1.37  8.71 +0.82  D L - a l p h a - m e t h y l glutamate  17.05 + 0.85  6.43 + 0.02  12.18 + 1.32  4.17 + 1.02  L-glutamate-gamma-ethyl ester  17.95 + 0.20  7.45 + 0.10  15.16 +0.39  5.22 +0,49  L-glutamate-gamma-methyl ester  17.20 +0.02  7.57 + 0.23  14.87 + 1.10  6.32 +0.19  Hydroxylamine  17.15 + 0.05  7.59 + 0.23  19.20 + 1.12  17.93 +0.91  67 4.12  The  effects of pre-incubation in the p r e s e n c e of a m m o n i u m  on the rate of ammonia f o r m a t i o n It was  i n a Ringer-phosphate  ions  medium.  thought that a one hour pre-incubation of r a t c e r e b r a l o  cortex s l i c e s at 37 C i n a Ringer-phosphate  m e d i u m containing  NH4CI (5mM), might r e s u l t i n a r e v e r s a l of the p r o c e s s of a m m o n i a f o r m a t i o n by ammonia producing endogenous substrates. idea stems f r o m the knowledge that exogenous NH^+  This  i n the incubation  m e d i u m increases glutamine and protein amide groups of b r a i n tissue ( 113,  114 ). The  details of the experiment were as follows:  R a t b r a i n cortex  s l i c e s were pre-incubated  for one hour at 3 7 ° C  i n the following media: Medium A l  :  Ringer-phosphate  Medium B l  :  R i n g e r -phosphate -NH^CI (5mM)  Medium CI  :  Same as B l .  A f t e r the one hour  medium.  incubation was  completed the s l i c e s were  f r o m the W a r b u r g m a n o m e t r i c vessels, and  s u c c e s s i v e l y i n three beakers  medium.  The  medium.  washed by dipping  removed  momentarily  containing 25ml Ringer-phosphate  t i s s u e s f r o m media A l , B l and C I were placed  r e s p e c t i v e l y i n 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 f r e s h media A2,  B2  and  C2,  hour at 37°C.  as d e s c r i b e d below, The  and allowed  to incubate  for one  time lapse between the end of the f i r s t and the  68  T A B L E XXV.  T h e effect of a one hour pre-incubation i n the presence of 5mM N H 4 C I on ammonia f o r m a t i o n by c e r e b r a l cortex s l i c e s of the rat.  Conditions  Ammonia  formation  P r e - i n c u b a t i o n control medium. Medium A l -  (Ringer-phosphate)  14.40 + 0.80  Total ammonia formation. M e d i u m A 2 - T i s s u e incubated i n R i n g e r phosphate, t r a n s f e r r e d to a Ringer-phosphate medium.  8.52+ 0.29  M e d i u m B 2 - T i s s u e incubated i n R i n g e r phosphate-NH4CI, transferred to a Ringer-phosphate medium.  15.11  M e d i u m C 2 - T i s s u e incubated i n R i n g e r phosphate-NH^Cl, t r a n s f e r r e d to a R i n g e r - p h o s p h a t e - T C A medium.  10.21 + 0.09  Subsequent incubation —  1 hr. at 37° C.  + 0.11  69 beginning of the second  incubation was 15 minutes.  Medium A2  : Ringer-phosphate  medium.  Medium B2  : Same as A 2 .  M e d i u m C2  : Same as A 2 + 3 % t r i c h l o r a c e t i c acid.  A m m o n i a estimations were c a r r i e d out on m e d i u m A l . ammonia was determined i n A2, B2 and C2. i n Table X X V .  F o r NH4  +  Total  The results a r e given  r e v e r s a l to occur a value of 24. 6 ( i . e.  14. 4 + 10.2) u m o l e s / g wet wt. ( T a b l e X X V ) should be obtained i n the second incubation; however, the value obtained is only 15.11 m i c r o moles p e r g r a m wet weight.  Hence, the results do not indicate the  inhibition by added NH4+ of the breakdown of a m m o n i a - f o r m i n g endogenous substances.  4.13  Studies on the m e c h a n i s m of the inhibitory action of glucose on ammonia  formation.  Since the f o r m a t i o n of ammonia by c e r e b r a l cortex s l i c e s i s inhibited i n the p r e s e n c e  of glucose ( 42 -44  ), i t was thought that  the inhibition of ammonia f o r m a t i o n by a one hour pre-incubation of o c e r e b r a l cortex s l i c e s at 37 C i n an incubation m e d i u m containing glucose might be r e v e r s e d on placing in a glucose c a r r i e d out.  free incubation medium.  s l i c e s treated i n such a manner T h e following experiment was  70  Rat c e r e b r a l cortex slices w e r e pre-incubated i n a R i n g e r o phosphate-glucose (5mM)  m e d i u m f o r one hour at 37 C.  was then separated f r o m the pre-incubation medium,  T h e tissue  washed by  dipping m o m e n t a r i l y and s u c c e s s i v e l y into two b e a k e r s containing 25ml Ringer-phosphate medium,  placed into W a r b u r g  manometric  v e s s e l s containing f r e s h Ringer-phosphate m e d i u m with o r without glucose,  and incubated for one hour at 37°C.  T h e time lapse between  the end of the f i r s t and the beginning of the second incubations was 20 or 15 minutes,  depending r e s p e c t i v e l y upon whether  minute gassing was done using pure oxygen.  or not a 5  J o s a n et a l . ( 22)  o b s e r v e d no difference in the ammonia producing capacity of c e r e b r a l tissue when either a i r or oxygen were used as the gas phase. P r e - i n c u b a t i o n media  a m m o n i a and (total) ammonia  after the completion of the second incubation, F o r a complete  s  formed  a r e given i n T a b l e X X V I .  " s p a r i n g a c t i o n " of glucose on a m m o n i a - f o r m i n g  substrates to occur, about 18 m i c r o m o l e s ammonia p e r g r a m wet weight should be produced i n the second incubation. moles NH^  per g r a m wet weight i s produced.  However, about 9-11 m i c r o T h e r e s u l t s indicate  that inspite of the inhibition of ammonia f o r m a t i o n during pre-incubation i n a glucose medium,  the rate of f o r m a t i o n of ammonia under these  conditions c o r r e s p o n d s to the rate of ammonia f o r m a t i o n i n the second hour of a two hour incubation i n a glucose free incubation  medium.  71 T A B L E XXVI.  The effect of one hour pre-incubation in the presence of glucose and oxygen on the ammonia formation by cerebral cortex slices in the presence or absence of glucose. Ammonia formation Oxygen Air  Conditions  Pre-incubation medium estimated after one hour incubation. 4.60 + 1.05  . 4.12+0.5  Ringer-phosphate medium  9.58 + 0.29  10.90 + 1.83  Ringer-phosphate-gluco se medium  4.58 +0.82  Ringer-phosphate-glucose  medium  Total ammonia formation in the second hour. (incubation using either air or oxygen as the gaseous phase)  5.40  +0.41  T A B L E XXVn. The effect of a one hour anaerobic pre-incubation at 3 7 ° C on the aerobic formation of ammonia by cerebral cortex slices in the presence and absence of glucose.  Ammonia formation  Conditions Pre-incubation medium estimated after one hour. (anaerobic incubation) Ringer-HCO^-glucose (5mM) medium  6.78 +  0.75  5.65+  0.67  5.83 +  0.43  Total ammonia estimated after the second hour incubation. Ringer-phosphate medium Ringer-phosphate-glucose  medium  72 4.14  The i r r e v e r s i b l e - effects of anaerobic  pre-incubation on a m m o n i a  formation. A  one-hour pre-incubation of c e r e b r a l cortex s l i c e s i n a  Ringer-bicarbonate  m e d i u m was c a r r i e d out. T h e s l i c e s were then  separated f r o m the medium, bicarbonate  washed f r e e f r o m adhering  Ringer-  (by dipping m o m e n t a r i l y and s u c c e s s i v e l y into two b e a k e r s  containing 25ml of a R i n g e r  solution),  resuspended i n f r e s h R i n g e r -  phosphate m e d i u m contained i n W a r b u r g v e s s e l s and incubated after a 5 minute oxygenation  f o r one hour at 37°C.  the end of the f i r s t and the beginning 20 minutes.  The time l a p s e between  of the second incubation was  T h e pre-incubation m e d i u m ammonia,  found after the completion  and ammonia  of the second incubation, were  R e s u l t s a r e given i n Table X X V I I .  estimated.  It can be seen f r o m these  results  that a one hour anaerobic p r e - i n c u b a t i o n at 37° C completely and i r r e v e r s i b l y inhibits the a m m o n i a - f o r m i n g m e c h a n i s m operating on incubation.  Conclusion It m a y be concluded f r o m the results i n this s e c t i o n that the rate of f o r m a t i o n of ammonia depends upon the p r o p e r functioning of e l e c t r o n transport and oxidative phosphorylation p r o c e s s e s , and is inhibited when a m m o n i a u t i l i z i n g p r o c e s s e s a r e operative.  While the tissue concentration  of ammonia is maintained at a constant l e v e l , the variations i n the rate of ammonia f o r m a t i o n a r e seen for the most part i n the incubation medium.  73 5.  T R A N S P O R T O F A M M O N I U M IONS I N T O B R A I N Studies on a m m o n i u m ion uptake were f i r s t c a r r i e d out with  erythrocytes, (115-118  where  swelling and h e m o l y s i s commonly  occur  ). The t r a n s p o r t of a m m o n i u m ions i n e r y t h r o c y t e s  have been a s s u m e d to depend upon the p e r m e a b i l i t y of the r e d c e l l membrane to anions (chloride, bicarbonate)  (115, 116).  In fact,  O r s k o v (115 ) showed that the rate of h e m o l y s i s of m a m m a l i a n e r t h r o c y t e s i n solutions of a m m o n i u m fold by the addition of bicarbonate. ammonium  chloride i s i n c r e a s e d  Thus,  many-  the active t r a n s p o r t of  (NH .+) itself cannot be m e a s u r e d because N H 4 4  ions pass  through the membrane r a p i d l y a s shown by J a c o b s and c o - w o r k e r s ( 116-118 )• However, P o s t and J o l l y ( 119 ).  an i n d i r e c t method has been employed by  They showed that the addition of a m m o n i u m  ions to N a - f i l l e d red cells induces m a x i m a l active s o d i u m transport i n the absence of external potassium.  Saturating the t r a n s p o r t s y s t e m  with p o t a s s i u m ions (by incubating with external potassium) produces only a s m a l l i n c r e a s e i n s o d i u m transport by a m m o n i u m ions.  However,  a m m o n i u m ions do not facilitate p o t a s s i u m i o n transport i n low s o d i u m cells and, presumably, cannot substitute for sodium. their r e s u l t s , N H 4  +  A c c o r d i n g to  ions appear to substitute d i r e c t l y for K+ (in  erythrocytes) and r e q u i r e a concentration 3 - 7 times greater than p o t a s s i u m to produce a comparable effect.  74  In brain, though the ammonia i n the n o r m a l The  state, during  content i n vivo i s v e r y low  convulsions  a d m i n i s t r a t i o n of a m m o n i u m  salts,  l e v e l s ( 120 ) b r i n g s about experimental to a r i s e i n b r a i n ammonia. across  it i s markedly  increased.  without affecting the A T P convulsions  i n a n i m a l s due  T h i s i m p l i e s the passage of ammonia  the blood b r a i n b a r r i e r ( 121 ). W o r k on the influx of N H „ + ions i n cat b r a i n cortex 4  has~been c a r r i e d out by T o w e r and colleagues studies demonstrate a competition  slices  (114 ) and their  of a m m o n i u m ions with  potassium  ions f o r t r a n s p o r t into the c e l l . W o r k reported here i s a p r e l i m i n a r y of a m m o n i u m ions by r a t b r a i n cortex  5.1 T h e effect of exogenous NH^+ i n c e r e b r a l cortex These 1)  study on the uptake  slices.  on the accumulation of ammonia  slices.  studies were c a r r i e d out under the following conditions:  a e r o b i c a l l y i n the presence of glucose  (Table X X V I I I )  where  the percentage swelling of the b r a i n slice i s taken as 16.8 (122 2)  );  a e r o b i c a l l y i n the absence of glucose the percentage  3)  (Table X X I X )  swelling i s taken as 44 ( 123 );  a n a e r o b i c a l l y i n the presence of glucose the percentage  where  (Table X X X ) where  swelling i s taken a s 50 ( 122 - 124  ).  It i s seen f r o m the r e s u l t s r e c o r d e d and X X X that, i f the tissue ammonia NH,+ to the medium, 4  i n Tables XXVUI,  XXIX  content i n the absence of added  i s not deducted f r o m the tissue  ammonia  contents obtained at the end of the one-hour incubation with external NH^t  there appears a m a r k e d uptake of ammonia against a concen-  tration gradient. 30mM  F o r example,  i n T a b l e XXVIII,  concentrations of N H ^ C l added to a  2, 5, 10, 20 and  Ringer-phosphate-glucose  m e d i u m give t i s s u e : m e d i u m concentration r a t i o s of 3.74, 2.33,  1.87,  1.55 and 1.31 r e s p e c t i v e l y (where the tissue water content i s taken a s 80%  (Table U), a l l values being c o r r e c t e d f o r swelling (section 5.2)).  It should be mentioned that the concentrations of ammonia  i s taken,  after incubation i s completed, a s the added NH.+ concentration. ' 4  These  values do not significantly change e s p e c i a l l y at the high NH^+ concentration used.  Since the tissue ammonia  contents under a v a r i e t y of o  media conditions at the end of one-hour incubation at 37 C a r e about the  same (Table XVIII),  the tissue value with no added NH^+ i n the  incubation m e d i u m i s subtracted f r o m the tissue ammonia  values •+  found when incubation i s c a r r i e d out i n the p r e s e n c e of external NH.. 4 For  example,  i n Table XXVIII,  (mM) when deductions 35.09, f o r external N H  the tissue ammonia  concentrations  a r e made, a r e 3.36, 7.54, 14.59,  26.89 and  + concentrations of 2, 5, 10, 20 and 30mM 4 r e s p e c t i v e l y . The deduction of the control NH^+ value (i.e., the tissue NH.+ concentration found with no added NH,+ i n the incubation 4 4  T A B L E XXVIII. T h e effect of a m m o n i u m ions added to a Ringer-phosphate-glucose m e d i u m on i t s accumulation i n r a t c e r e b r a l cortex s l i c e s i n an incubation of one hour at 3 7 ° C and an atmosphere of pure oxygen.  NH4CI added to the incubation medium  Weight of cerebral cortex slice s  mM  mg  Tissue ammonia at the end of the incubation |i mole s  T i s s u e ammonia after c o r r e c t i o n for 16.8% swelling calculated a s  Tissue medium  T i s s u e (x) ammonia content after control a m m o n i a value deducted  T i s s u e (x) medium  mM  mM  0 (control)  67 + 12  0.259 + 0.028  4.11  2  72 + 13  0.520 + 0.071  7.47  3.74  3.36  1.68  5  71 + 12  0.802 + 0.026  11.65  2.33  7.54  1.51  10  75 +  1.352 + 0.026  18.70  1.87  14.59  1.45  20  89  2.670  31.00  1.55  26.89  1.35  30  79  2.948  39.20  1.31  35.09  1.17  8  41.6  T h e c o n t r o l m e d i u m contained 0.099 + 0.025mM a m m o n i a at the end of the one hour incubation. With l O m M NH4+ the amount of swelling did not change and i s 16.9% (122).  -4 ON  77 medium)  seems to be a l o g i c a l procedure a s there i s no evidence  the bulk of the autogenous ammonia i s cytoplasmic  that  and can therefore  be used i n a s s u m i n g a concentration gradient f o r externally applied ammonia.  In fact,  the evidence  already  secured  (Table XVITJ) indicates  "N  that the autogenous tissue ammonia i s r e m a r k a b l y constant under a variety of conditions, a fact most easily understood i f the autogenous ammonia  i s f o r m e d i n one (or a l i m i t e d number) compartment of the  c e l l and that above a l i m i t i n g concentration a m m o n i a l e a k s into the cytoplasm  and thence into the incubation medium.  t i s s u e : m e d i u m concentration ratio of 12 i s obtained  Furthermore, i n an  a  anaerobic  incubation of c e r e b r a l cortex s l i c e s i n the absence of added  NH.+  4 (Table X X X ) .  T h i s i s a very unlikely value as there i s no case of  so high a concentration ratio being obtained i n b r a i n c e l l s under anaerobic  conditions.  T h i s i m p l i e s that a major portion of the a m m o n i a  i s not locked up i n the cytoplasm.  O n the other hand, i f a m m o n i a  i s f o r m e d i n specific compartments, f r o m which a m m o n i a above a constant  concentration,  leaks  the r e s u l t i s understandable.  T A B L E XXIX.  N H C 1 added to the incubation medium 4  mM  T h e effect of a m m o n i u m ions added to a Ringer-phosphate m e d i u m on i t s accumulation a e r o b i c a l l y i n r a t c e r e b r a l cortex s l i c e s incubated f o r one hour at 37°C.  Weight of cerebral cortex slices mg  T i s s u e ammonia at the end of the incubation p. m o l e s  T i s s u e ammonia after c o r r e c t i o n for 4 4 % swelling calculated as  Tissue medium  T i s s u e (x) ammonia content after control ammonia value deducted  T i s s u e (x) medium  mM  mM  --  61+3  0.236 + 0.008  3.03  10.10  2  68 + 11  0.490 + 0.046  5.80  2.90  2.77  1.36  5  62+5  0.716 + 0.025  9.32  1.86  6.29  1.26  10  62+5  1.100 + 0.026  14.30  1.43  11.27  1.13  0 (control)  T h e control m e d i u m contained 0.30 + 0.02mM ammonia at the end of the one hour incubation.  oo  T A B L E XXX.  NH4CI added to the incubation medium mM  T h e effect of a m m o n i u m ions added to a R i n g e r - b i c a r b o n a t e - g l u c o s e m e d i u m on i t s accumulation a n a e r o c i a l l y i n r a t c e r e b r a l cortex s l i c e s incubated at 3 7 ° C f o r one hour.  Weight of cerebral cortex slice s mg  T i s s u e ammonia at the end of the incubation p, m o l e s  T i s s u e ammonia after c o r r e c t i o n for 5 0 % swelling calculated as  Tissue . medium  T i s s u e (x) ammonia content after control ammonia deducted  T i s s u e (x) medium  mM  mM  <  0 (control)  9 0 + 6.0  0.237 + 0.026  2.02  12.24  2  67 + 3.0  0.427 + 0.059  4.89  2.45  2.87  1.44  5  70 + 5.0  0.7 58 + 0.076  8.33  1.66  6.31  1.26  10  78 + 8.0  1.340 + 0.040  12,90  1.29  10.88  1.09  T h e control m e d i u m  contained 0.165 + 0.004mM a m m o n i a at the end of the one hour incubation.  The  work of T o w e r and h i s colleagues ( 114 ) shows that  an a e r o b i c incubation of cat c e r e b r a l cortex  s l i c e s with l O m M  NH.C1  4 i n the m e d i u m gives a tissue: m e d i u m ratio of 1.67 (tissue  values  being c o r r e c t e d f o r swelling and a s s u m i n g 8 0 % water content of the b r a i n tissue). has they The  T h i s uptake of NH^+ against a concentration  gradient  indicated to them an active t r a n s p o r t of a m m o n i u m ions, which say, competes with p o t a s s i u m ions for the t r a n s p o r t  carrier.  t i s s u e : m e d i u m ratio obtained i n our studies with rat b r a i n  cortex s l i c e s incubated  i n a m e d i u m containing  l O m M N H ^ C l i s 1.45  (Table X X V f f i ) , which i s g r e a t e r than the ratio 1.13 obtained a e r o b i c a l l y i n the absence of glucose 1.09  (Table XXIX),  obtained a n a e r o b i c a l l y (Table X X X ) , both with  NH^Cl.  or the ratio lOmM  external  The gradient obtained a e r o b i c a l l y i n the absence of glucose,  or anaerobically, may be attributed to passive diffusion of a m m o n i u m ions into the b r a i n slice.  Thus,  though some uptake of a m m o n i u m  ions against a concentration gradient i s indicated here,  an energy-  dependent t r a n s p o r t of a m m o n i u m ions into b r a i n c e l l s cannot be confirmed. 5.2 C o r r e c t i o n for swelling. Taking,  f o r example,  the c o r r e c t i o n for tissue N H ^ as follows.  the control experiment of Table  XXX,  concentrations due to swelling i s made  81  Assuming to  be  80%  contain  (Table  increases hour  content is  II), t h e n  content  90mg  of adult c e r e b r a l  wet weight  cortex  slice  cerebral cortex  slices  72|il o f w a t e r .  Under  one  the water  anaerobic  conditions a  the tissue water incubation,  50%  t i s s u e s w e l l i n g ( 1 2 2 - 124 )  b y 45p.l t o a v a l u e  at which time  o f 117p,l  the cerebral tissue  i s 0.237^imoles and the i n c u b a t i o n  medium  at the end of ammonia  ammonia  content  0.l65mM.  Thus,  ammonia  concentration  i n the c e r e b r a l tissue  0.237 corrected  Hence, equals  in  f o r swelling equals  the tissue:medium  M °l s/rnl, m  concentration  e  ratio  which equals 2.02  equals  ,. ,  12.24.  2.02mM.  which ,  Conclusion  Results  given  of autogenous a m m o n i a  i n this  s e c t i o n point to a  formation  i n cerebral  compartmentation  cortex  slices.  82  6.  E F F E C T S O F OUABAIN ON AMMONIA M E T A B O L I S M  IN V I T R O .  The work of Gonda and Q u a s t e l (125) using c e r e b r a l c o r t e x s l i c e s of the rat, showed that ouabain at a concentration^not inhibitory -5 to respiration (10  M), inhibits the f o r m a t i o n of l a b e l l e d glutamine f r o m  l a b e l l e d glucose. However, when a glutamine synthetase s y s t e m isolated f r o m r a t b r a i n was used, no inhibition of glutamine synthesis by ouabain was found. It was concluded f r o m these studies that ouabain may inhibit the transport of NH^"*" into the mitochondria, a major site of glutamine synthesis. 6.1  E f f e c t s of ouabain (0.0 ImM) on the rate of ammonia f o r m a t i o n i n the p r e s e n c e and absence of glucose. It i s seen f r o m T a b l e X X X I that ouabain, at a concentration  (0.0 ImM) not inhibitory to the r e s p i r a t i o n of c e r e b r a l c o r t e x s l i c e s (125), i n c r e a s e s the rate of ammonia f o r m a t i o n at the end of one hour at 3 7 ° C i n a Ringer-phosphate-gluco se medium. In the absence of glucose, ouabain only slightly inhibits ammonia formation, when c o m p a r e d to the c o r r e s p o n d i n g controls.  83  T A B L E XXXI.  The effects of O.OlmM ouabain on ammonia f o r m a t i o n i n the presence and absence of glucose using c e r e b r a l cortex s l i c e s .  A d d i t i o n s to a R i n g e r phosphate m e d i u m  T o t a l ammonia f o r m a t i o n  Nil  17.78 +  0.22  Ouabain  16.94 +  0.27  Glucose G l u c o s e + ouabain  8.57  +  0.17  10.28 +  0.06  84 .2 E f f e c t s of ouabain (0.0 ImM)  on the rate of ammonia f o r m a t i o n  i n an incubation m e d i u m containing glutamine i n the presence or absence  of glucose.  i . . . In the presence of glucose. To  support the contention that ouabain,  by blocking the  r e synthesis of glutamine i n c e r e b r a l c o r t e x s l i c e s elevates the ammonia formation, the following e x p e r i m e n t s were c a r r i e d out. One- and four-hour incubations were c a r r i e d out and the effects of ouabain were studied using a Ringer-phosphate-glucose m e d i u m containing L-glutamine. a r e analyzed i n Table XXXIIa. that,  T h e r e s u l t s given i n T a b l e X X X I I F r o m these r e s u l t s i t i s seen  ouabain i n the p r e s e n c e of glutamine,  the rate of a m m o n i a f o r m a t i o n . incubation,  substantially i n c r e a s e s  However, i n a four-hour  glutamine i s still not quantitatively  In the absence  deaminated.  of glucose.  It i s seen f r o m T a b l e XXXIII that ouabain does not contribute to the i n c r e a s e of ammonia f r o m glutamine i n the absence of glucose.  This result  glutaminase. However,  shows that ouabain does not activate  If it did, i t would work i n the absence  more ammonia i s f o r m e d  one hour i n the absence  of glucose.  f r o m 0.5mM glutamine i n  of glucose than i n i t s presence (about  85  C T A B L E XXXII.  -  T h e effect of O.OlmM ouabain on the f o r m a t i o n of ammonia f r o m c e r e b r a l cortex s l i c e s i n the p r e s e n c e of glucose and glutamine.  Additions to a Ringer-phosphateglucose m e d i u m  T o t a l ammonia f o r m a t i o n 1 hour  4 hours  Nil  7.88 + 1.03  10.95+ 1.10  Ouabain  9.45 + 1.85  21.50+ 2.09  Glutamine (0.5mM)  9.92  + 0.96  --  Glutamine (0.5mM) + ouabain  12.48 + 0.90  --  Glutamine  14.94 + 1.44  18.24+ 1.00  18.55  29.34 + 3.69  (l.OmM)  Glutamine (l.OmM) + ouabain  + 0.75  T A B L E XXXIIa.  A n a l y s i s of T a b l e XXXIII: P e r c e n t a g e deamination of added glutamine by b r a i n slice s.  Ringer-pho s pha te glucose m e d i u m  T h e o r e t i c a l ammonia f r o m added glutamine micromole s  A m m o n i a obtained f r o m added glutamine micromole s  Percentage Deamination  One hour incubation Glutamine (0.5mM)  1.5  0.117  7.8  Glutamine (0.5mM) + ouabain  1.5  0.230  Glutamine (1.0 mM)  3.0  0.279  Glutamine (l.OmM) + ouabain  3.0  0.462  16.1  3.0  0.452  15.1  3.0  1.031  34.4  15.3 9.3  F o u r hour incubation Glutamine  (l.OmM)  Glutamine (l.OmM) + ouabain  87  T A B L E XXXIII.  T h e effects of 0.0 I m M ouabain on ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s i n a Ringer-phosphate m e d i u m containing glutamine (0.5mM) (and no glucose).  Additions to a R i n g e r phosphate m e d i u m  T o t a l ammonia f o r m a t i o n  Nil  19.23  +  0.25  Ouabain  18.60  +  0.10  Glutamine  23.36  +  0.21  Glutamine + ouabain  22.89 +  0.29  T A B L E XXXIV.  The effects of 0.0 I m M ouabain on ammonia f o r m a t i o n i n c e r e b r a l cortex s l i c e s i n the p r e s e n c e of l.OmM glucose and high potassium.  A d d i t i o n s to a R i n g e r phosphate m e d i u m Glucose  T o t a l ammonia f o r m a t i o n  8.56  +  0.68  Glucose + ouabain  13.53  +  0.85  G l u c o s e + potassium?  13.47  +  0.47  G l u c o s e + p o t a s s i u m + ouabain  13.32 +  0.22  * as KC1 (lOOmM)  88  4^moles/g wet 6.3  weight c o m p a r e d to 1.6y.moles/g wet  E f f e c t s of ouabain i n the presence  (O.OlmM) on the rate of ammonia  of ImM  glucose and high p o t a s s i u m  weight). formation ion  concentrations. F r o m the r e s u l t s i n T a b l e X X X I V it i s seen that both or high p o t a s s i u m  ouabain  r e v e r s e s p a r t i a l l y the ammonia f o r m a t i o n by  ImM  glucose to about the same extent, but together their effects are not additive.  T h i s can be explained on the b a s i s that high  greatly a c c e l e r a t e s ImM  glucose u t i l i z a t i o n so that, i n effect,  s y s t e m b e c o m e s a no-glucose However,  potassium  while high p o t a s s i u m  one--i.e., ouabain i n the p r e s e n c e  w i l l have no of 5mM  the effect.  glucose does  not r e v e r s e even p a r t i a l l y the s u p p r e s s i o n by glucose of ammonia formation,  ouabain  in the presence  ammonia f o r m a t i o n by  of 5mM  1.6iimoles/g wet  c o m p a r e d to a value of about 5|imoles/g otherwise  glucose i n c r e a s e s  weight.(Table X X X I ) when wet  weight obtained under  s i m i l a r conditions (Table X X X I V ) .  Conclusion F r o m the r e s u l t s in this section i t may  be  concluded that  ouabain inhibits the synthesis (but not the breakdown) of glutamine i n c e r e b r a l cortex slices, NH.+  p r e s u m a b l y by inhibiting the t r a n s p o r t of  into the site(s) of glutamine  synthesis.  8 7. DISCUSSION  7.1 Initial c e r e b r a l ammonia l e v e l s . A m m o n i a l e v e l i n the b r a i n p r i o r to incubation (called here, i n i t i a l level) i s around 5.0(J.moles/g wet weight tissue (Table II). Such a l e v e l i s high when compared to the ammonia content of b r a i n obtained by r a p i d l y f r e e z i n g the a n i m a l in liquid a i r o r nitrogen,  followed  i n v a r i a b l y by extracting the f r o z e n b r a i n with T C A before the e s t i m a tion of ammonia by the a l k a l i diffusion method of Conway ( 88 ). T h e c e r e b r a l ammonia l e v e l s of a number of species (Table I) obtained by such treatment (0.2 - 0.3p.moles/g wet weight) a r e a s s u m e d to be i n vivo levels, and i t i s possible, as has been suggested ( 8 )J that i n r e a l i t y f r e e ammonia i s absent in c e r e b r a l tissue. However, "Weil-Malherbe and other investigators ( 20, wet weight under conditions  22)  obtained values around 5.0|jmoles/g  s i m i l a r to those used i n this study. Attempt  have therefore been made (section 3) to account for the ammonia concentration of the b r a i n and to devise methods affecting rates of c e r e b r a l ammonia formation. Intraperitoneal injections of glucose, amytal or i p r o n i a z i d failed to lower i n i t i a l ammonia l e v e l s (Table III). F r e e z i n g  infant rats  i n l i q u i d nitrogen also failed to lower the i n i t i a l l e v e l s . However, these values a r e significantly reduced (about 50 percent) on treating the tissue with 3 percent T C A p r i o r to the a l k a l i estimation of a m m o n i a either  90 with o r without fixation with liquid nitrogen (Table IV). S i m i l a r low values are obtained using the B e c k m a n amino aeid analyzer (Table XI) where K  CO Cd  i s not needed. In this method the sample p r e p a r a t i o n J  c a l l s for ether extraction to r e m o v e the T C A  used for deproteinization.  T h u s the r e s u l t s for the i n i t i a l a m m o n i a content of the b r a i n indicate that, i n the absence of T C A  treatment, an i n c r e a s e d rate of ammonia  f o r m a t i o n takes place. T h a t T C A  itself i f not an i n t e r f e r i n g factor i n  the estimation i s shown by the r e s u l t s obtained by (1)  r e m o v i n g the c e r e b r a l cortex s l i c e s f r o m an incubation  m e d i u m containing T C A  (after tissue inactivation) whereby the  N H + i s not affected (Table V); 4  (2)  addition of 0.1ml, 100 g. %  .  TCA  to 0.3ml saturated K  i n the side tube of a W a r b u r g v e s s e l (a value 6.10+ - -  CO  0.25nmoles/g wet  weight i s obtained as compared with the control 5.88+0.63 where no T C A  was  used).  The p o s s i b i l i t y of some enzyme activity (liberating ammonia) o c c u r r i n g at pH  10.5 (which i s the pH at which the b r a i n i s incubated i n  the presence of K  CO C*  considered. 9.0 ( 73  ), but destroyed on T C A  treatment,  was  «J  F o r example, b r a i n glutaminase having a pH optimum at  ) can still r e t a i n some activity at pH  10.5.  However, v e r y  s m a l l i n c r e a s e s a r e obtained on addition of L - g l u t a m i n e to the tissue i m m e d i a t e l y p r i o r to the estimation with K  CO,. Cd  A l s o , the i n i t i a l  J  l e v e l s are not d e p r e s s e d either by D-glutamate or L-glutamate  known  inhibitors of glutaminase.  While A M P  and taurine do not-contribute any  ammonia to initial values, glucose does not prevent its f o r m a t i o n (initially) (Table VI). P a r a l l e l studies using these substrates were a l s o c a r r i e d out. Homogenates left standing at r o o m temperature f o r four h o u r s produce 8|jmoles/g wet weight, with an i n c r e a s e of only l j j m o l e / g wet weight, of ammonia f r o m added L-glutamine (ImM) (Table VII). However, a g r e a t e r rate of ammonia f o r m a t i o n o c c u r s i n the presence of L - g l u t a m i n e using c e r e b r a l cortex s l i c e s after one hour incubation at 37°C. T h i s rate i s strongly inhibited (70%) by D-glutamate (Table VIII). Rat b r a i n cortex s l i c e s a l s o f o r m considerable amounts of ammonia f r o m A T P and A M P when they a r e added to the incubation m e d i u m (Table IX). T h e s e r e s u l t s a r e consistent with the o c c u r r e n c e of adenylic deaminase i n brain, but it should be noted that T a k a g a k i (126) failed to observe deamination of AMP  i n the presence of guinea pig b r a i n cortex s l i c e s .  T a u r i n e i s known  to occur i n high concentrations i n b r a i n (101, T a b l e XIII). It i s converted to isethionic a c i d (102), but i t has no effects on the rate of ammonia f o r m a t i o n i n the p r e s e n c e or absence of glucose (Table X ) . TCA  extraction of f r o z e n b r a i n r e s u l t s i n a s u p p r e s s i o n of the  r a p i d initial rate of ammonia formation, before incubation at 3 7 ° C i s allowed to take place. P r e s u m a b l y this o c c u r s by an inactivation of enzyme system(s) that d i r e c t l y , or indirectly, contribute(s) to the  92  ammonia f o r m e d post-mortem. A c c o r d i n g to R i c h t e r arid Dawson ( 16)» the ammonia b u r s t after death i s neither d e r i v e d f r o m glutamine nor f r o m nucleotides, and from'-evidence r e p o r t e d i n T a b l e VI, i t i s seen that neither glutamine nor A M P  contribute ammonia to i n i t i a l a m m o n i a  l e v e l s . T h e i n c r e a s e in the i n i t i a l ammonia during the K  GO 2  estimation 3  .is seen to a r i s e f r o m T C A - i n s o l u b l e c e l l components, for r e m o v a l of c e l l d e b r i s and T C A - i n s o l u b l e m a t e r i a l gives lower i n i t i a l ammonia values (Table XI). In this connection i t should be noted that the existence of a v e r y unstable neutral proteinase i n b r a i n (which could not be extracted) active for only a short time after deathjhas been reported by A n s e l l and R i c h t e r ( 127 )- B a s e d on e x p e r i m e n t s c a r r i e d out i n vivo, V r b a efa_l.( 48, 50, 51 )  conclude that the source of  ammonia i s an a m i d i c functional group of c e r e b r a l proteins. A kinetic study of the p r o c e s s of ammonia f o r m a t i o n i n the presence or absence of glucose and-of the effects of T C A addition on the completion of the incubation was then c a r r i e d out (Table XII, F i g u r e 1). T C A has little or no effect when i t i s added at the end of incubations when the bathing m e d i u m was devoid of glucose.  However,  with glucose present i n the m e d i u m a one hour incubation gives lower ammonia values with T C A than i n the absence of T C A ; while the ammonia values without T C A after two, three or four hours, a r e about the same as the ammonia values with T C A . T h i s i m p l i e s that ammonia f o r m a t i o n during incubation in the p r e s e n c e of glucose continues up to a  93 point, and during its f o r m a t i o n is only prevented by the addition of T C A . F u r t h e r experiments show that ammonia f o r m e d i n one hour 'on., subtracting the  i n i t i a l values , is about l6umoles/g d r y weight  under the following conditions ( T a b l e XIV). (1)  A e r o b i c incubation of either infant or adult c e r e b r a l cortex s l i c e s i n the p r e s e n c e of glucose.  (2)  Anaerobic  incubation of c e r e b r a l cortex s l i c e s i n the p r e s e n c e  or absence of glucose. F r o m the w o r k of W e i l - M a l h e r b e and D r y s d a l e ( 5 5 ) it i s evident that the anaerobic f o r m a t i o n of ammonia occurs up to a point ( 2 hours), after which no further ammonia f o r m a t i o n o c c u r s .  Thus, the ammonia f o r m e d  during aerobic incubation i n the presence of glucose, m a y s e e m to be r e l a t e d to, or originate f r o m , the same source as the ammonia f o r m e d during anaerobic incubation.  It m a y be concluded f r o m these results that there a r e at least two distinct m e c h a n i s m s (or p r o c e s s e s ) by which ammonia f o r m a t i o n occurs.  One that occurs i m m e d i a t e l y after death of the animal. Its  f o r m a t i o n is inhibited by T C A , but o c c u r s to some extent i n the p r e s e n c e of  K 2 C O 3 .  It p o s s i b l y a r i s e s f r o m amide groups of c e r e b r a l proteins.  However, further work is r e q u i r e d for complete c h a r a c t e r i z a t i o n . The other occurs during aerobic incubation i n a m e d i u m devoid of added substrate.  Its formation is inhibited during anaerobic incubations, o r  during aerobic incubations i n the presence of glucose.  94 7.2  E v i d e n c e f o r the compartmentation of autogenous ammonia  formation.  The ammonia contents of c e r e b r a l cortex s l i c e s at the end of one hour a e r o b i c incubation at 3 7 ° C i n a v a r i e t y of incubation m e d i a a r e r e m a r k a b l y constant (Table XVIII). ammonia formation, medium.  T h e d i f f e r e n c e s i n the r a t e s of  therefore, appear to be manifested i n the incubation  F o r example, the incubation m e d i u m concentration of a m m o n i a  i n the absence of glucose i s three t i m e s that i n i t s presence (Table X V ) . T h e s e facts a r e most easily understood i f the autogenous ammonia i s f o r m e d i n one (or a l i m i t e d number) compartment of the c e l l and above a l i m i t i n g concentration leaks into the cytoplasm and thence into the incubation medium.  T h i s would explain why a major p r o p o r t i o n ( 7 5 - 8 0 % ) of the  ammonia f o r m e d by c e r e b r a l cortex s l i c e s during endogenous r e s p i r a t i o n i s found in the m e d i u m while the tissue maintains a constant a m m o n i a level. Concomitant with ammonia formation i n the absence of glucose there i s a continuous efflux of N H „ + when the c e l l ammonia r e a c h e s an  4 o p t i m u m l e v e l . T h i s optimum l i m i t of c e l l ammonia i s also reached i n the presence of glucose and since the rate of ammonia f o r m a t i o n i s d i m i n i s h e d under these conditions, the efflux of NH^+ i s g r e a t l y reduced. T h u s the constant tissue ammonia value r e p r e s e n t s the l i m i t beyond which the efflux of NH^+ it i s f o r m e d .  o c c u r s f r o m specific c e l l u l a r compartment(s) i n which  It should be noted that, though the f o r m a t i o n of ammonia  i s suppressed anaerobically, there i s a greater ammonia leakage f r o m  the c e r e b r a l cortex slice into the incubation m e d i u m c o m p a r e d to an a e r o b i c incubation i n the p r e s e n c e of glucose the tissue ammonia  (Table X V ) .  Consequently,  content obtained at the end of an anaerobic  tion i s consistently found to be slightly lower than i t s a e r o b i c  incubacounter-  part. F u r t h e r evidence  in support  f o r the compartmentation of  ammonia f o r m a t i o n i n b r a i n c e l l s i s obtained f r o m  tissue:medium  concentration r a t i o s of ammonia at the end of an anaerobic  incubation.  A t the end of one hour incubation at 3 7 ° C a tissue:medium r a t i o of about 1 2 i s obtained cytoplasm  (Table X X X ) .  T h i s r u l e s out the p r e s e n c e i n the  of a m a j o r portion of the autogenous tissue a m m o n i a  there i s no case of so high a concentration ratio being b r a i n c e l l s under anaerobic  conditions.  obtained i n  O n the other hand,  r e s u l t s can be explained i f a m m o n i a i s f o r m e d i n specific f r o m which ammonia leaks above a constant  since  concentration.  these compartments) Thus, it is  seen that the results point favourably to the compartmentation of autogenous a m m o n i a  formation.  96 7.3  Changes in the amino acid spectrum and the formation of ammonia in brain cortex slices (for incubation periods of short duration). The  origin of ammonia. The  origin of ammonia in respiring brain slices, during their  incubation in a glucose free incubation medium, is a subject of much controversy. According to Weil-Malherbe and co-workers ( 20 ), none of the six deaminating brain enzymes are likely to be involved. Glutamic dehydrogenase, they maintain,  is strongly in favour of  the reductive amination of a-ketoglutarate, while glutaminase can at the most account for only a small proportion of the ammonia formed.  Since hexosamine, catecholamines and adenosine  nucleotides in brain occur only in small amounts, the enzymes that mediate their deamination have been ruled out as contributors to ammonia formation.  These workers suggest that ammonia  formation occurs by a reaction closely linked to proteolysis, since guinea pig brain slices incubated for five hours give significant increases in non-protein nitrogen.  In their studies they assume a  single origin from which free ammonia is derived. On  the other hand, according to Vrba et al.(49)  ammonia  formation is too complex a process to be explained on the basis of simple proteolysis. These workers showed that not more than 25 percent of the ammonia formed by guinea pig brain slices in  97  four h o u r s can be accounted f o r as coming f r o m p r o t e i n amide nitrogen, as  and  consider the source  still being unknown.  of the l a r g e r p a r t of a m m o n i a  T h e y did not,  however,  c o n s i d e r the  possible f o r m a t i o n of some of the ammonia f r o m the entire amino a c i d pool present in b r a i n s l i c e s . In the guinea was  studies of T a k a g a k i e t _ a L ( 47  ) the incubations of  pig b r a i n s l i c e s were r e s t r i c t e d to one hour because i t  felt that ammonia f o r m e d at the end of four h o u r s might be  r e s u l t of a s e r i e s of highly complicated being due  to p r o t e o l y s i s .  decarboxylase)  reactions, the g r e a t e r part  Glutamate (estimated by  decreases  a  squash  i n the b r a i n concomitant with an  increase  in ammonia f o r m a t i o n when incubations a r e c a r r i e d out i n a glucose f r e e medium; and,  a c c o r d i n g to these workers,  i n glutamate concentrations, endogenous oxygen uptake. estimations of glutamate and tissue,  accounts However,  i t seems that their  ammonia were made i n the c e r e b r a l  Glutamate (-8.48(_imoles),  aspartate (+4.36timole s), and  considered.  studies of the concentration of amino a c i d s show the  following changes i n amino acids i n one glucose.  decrease  for 50 percent of the  while the incubation m e d i u m contents were not  However,  the  hour i n the absence of  glutamine  (-3.42iim.oles),  alanine (+0. 50p.moles), taurine (- 2.12p.moles)  glycine (-0.25|imoles), a l l e x p r e s s e d  per g r a m wet  weight.  The  net change i n amino-nitrogen (refer to T a b l e XIII) j u s t i f i e s our conclusion that f o r short incubation periods ammonia f o r m a t i o n can be attributed to a l o s s of amino a c i d nitrogen. Thus, though incubations i n a substrateless m e d i u m r e s u l t i n i n c r e a s e s i n the aspartate and  the alanine levels,  the l e v e l s of taurine, glutamine and  glutamate m a r k e d l y drop.  T h a t only an insignificant amount of  y A B A a p p e a r s on the decarboxylation of glutamate i n the absence of glucose  was demonstrated b y H a b e r (111) who obtained  only  s m a l l i n c r e a s e s of labelled y-A-BA f r o m l a b e l l e d L-glutamate i n r a t c e r e b r a l cortex s l i c e s .  Thus, i t i s seen that the changes i n  amino a c i d nitrogen account substantially f o r the i n c r e a s e of a m m o n i a i n short incubation p e r i o d s . In the light of these  results,  the f o r m a t i o n  of a m m o n i a i n  r e s p i r i n g b r a i n s l i c e s f o r short incubation p e r i o d s one  (e.g.,  up to  hour) w i l l now be d i s c u s s e d .  Carbohydrate m e t a b o l i s m and ammonia E v e n i n the absence of glucose s l i c e s and CO_  i s evolved  formation.  oxygen i s absorbed by b r a i n  (128) with a concomitant l i b e r a t i o n of  C*  ammonia and  (Table XVII).  Endogenous r e s p i r a t i o n f a l l s with time  this i s accompanied by a f a l l i n the rate of a m m o n i a  tion (Table XII).  Glucose i s known to maintain  tion and at the same time s u p p r e s s e s ammonia  forma-  cerebral respiraformation  99 (Table XVTJ).  E x p e r i m e n t s with b r a i n suspensions  ( 129 ) and  s l i c e s ( 130 ) show that under these conditions glucose used instead of endogenous substrates. -evidence  i s being  Though there i s some  that both p r o t e i n and l i p i d may be u t i l i z e d to support  endogenous b r a i n r e s p i r a t i o n (1.3.1 ), r e s u l t s (for short incubation periods) r e p o r t e d i n this t h e s i s (Table XIII) and by other  workers  (47"  a s sub-  ) i m p l i c a t e f r e e ami-no acids, p a r t i c u l a r l y glutamate,  s t r a t e s f o r endogenous oxidations. Let  u s consider the changes taking place i n the b r a i n i n the  absence of glucose. and pyruvate  Glutamate transaminates  with  oxaloacetate  to give i n c r e a s e s i n aspartate and alanine (Table XIII)  and the a-ketoglutarate f o r m e d i n these r e a c t i o n s enters the c i t r i c a c i d cycle enabling oxygen consumption to o c c u r f r o m a-Ketoglutarate  i s a l s o f o r m e d under these  operation of glutamic  conditions by the  dehydrogenase and ammonia i s l i b e r a t e d i n  the r e a c t i o n (Reaction a). glutamine  by glutaminase  Ammonia  i s also l i b e r a t e d f r o m  (Reaction b) and glutamate f o r m e d i n  the r e a c t i o n can undergo further degradation to give and  ammonia  aminations  a-ketoglutarate.  (Reaction a).  a-ketoglutarate  A s p a r t a t e and alanine through t r a n s -  with a-ketoglutarate (Reaction c), followed by the  action of glutamic dehydrogenase, (Reaction a).  can give r i s e to a m m o n i a  100 a.  glutamate  b.  glutamine  c.  aspartate,  ot-ketoglutarate + •>(alanine)  glutamate +  NH  NH  q-ketoglutarate  glutamate  «i-ketoacids  Since ammonia f o r m e d i n these reactions cannot be due  to a l a c k of both c i t r i c a c i d cycle intermediates and  energy compounds,  i t accumulates and  utilized high  diffuses into the incubation  m e d i u m (Table X V ) . Now  l e t us consider reactions in the presence  Glucose,  as a source f o r c i t r i c a c i d cyle  r i c h energy source,  can  of glucose.  i n t e r m e d i a t e s and  a  either d i r e c t l y prevent the breakdown of  glutamate or, i f m e t a b o l i s m i s viewed as a dynamic r e m o v e ammonia f o r m e d by the glutamine  process,  synthetase r e a c t i o n .  T h i s i s borne out by the fact that the addition of glucose to the incubation m e d i u m prevents ammonia liberation, while maintaining c e r e b r a l free amino a c i d incubation l e v e l s (Table Lactate has  simultaneously  concentrations to p r e -  XIII).  also been shown to have a s i m i l a r effect to  glucose i n preventing the f a l l of f r e e glutamate l e v e l s (47 ). Thus,  with glucose,  pyruvate  or lactate where the rate of  operation of the c i t r i c a c i d cycle i s high, and with a continued high rate of f o r m a t i o n of a-ketoglutarate and thereby  glutamate  101  by t r a n s a m i n a t i o n and of glutamine by fixation of a m m o n i a i n the presence of optimal A T P , the s u p p r e s s i o n of ammonia l i b e r a t i o n i s the greatest (Table XVII).  T h e m i n i m u m l e v e l s (threshold) f o r  this suppression to occur i s around I m M  and 2.5mM f o r glucose  and pyruvate r e s p e c t i v e l y (Figure 2), which i s consistent with the fact that g l y c o l y s i s of one molecule of glucose y i e l d s two of pyruvate. Iodoacetate only slightly inhibits ammonia f o r m a t i o n (Table XIX), but this may be due to a slight diminution i n oxygen uptake ( 47 ). In the p r e s e n c e of glucose iodoacetate however affects both g l y c o l y s i s and oxygen uptake ( 132, 133  ) by i t s attack on t r i o s e phosphate  dehydrogenase. Thus,the c i t r i c a c i d c y c l e i s impeded by diminution of pyruvate f o r m a t i o n and hence fixation of ammonia does not occur. The r e s u l t s show that i n the presence of glucose, but not pyruvate, iodoacetate i n c r e a s e s ammonia f o r m a t i o n by m o r e than 5 0 % . Acetate i s only oxidized feebly by s l i c e s ( 134, 135 ) and hence has little or no effect on ammonia fixation. S i m i l a r l y , citrate and a - k e t o g l u t a r a t e have little effect i n r e p l a c i n g glucose owing to p e r m e a b i l i t y b a r r i e r ( 136 ) and like acetate do not suppress ammonia formation. However, oxaloacetate affords high r e s p i r a t o r y r a t e s ( 137 ) and, consequently,  inhibits quite strongly ammonia formation.  T h i s i s due to r e l a t i v e l y r a p i d influx into the b r a i n c e l l and p a r t i c i pation i n the c i t r i c a c i d c y c l e .  102 Malonate, an inhibitor of the c i t r i c a c i d c y c l e ( 138 ) at the succinic dehydrogenase stage, i n c r e a s e s ammonia f o r m a t i o n with ImM  glucose (Table XXIII), but was shown to be without any effect  i n the absence of glucose ( 20, 47 ), p r e s u m a b l y because i t does not affect endogenous r e s p i r a t i o n ( 47 ). In the p r e s e n c e of glucose malonate elevates the rate of ammonia f o r m a t i o n by inhibiting the c i t r i c a c i d cycle and, consequently,  suppressing the u t i l i z a t i o n of  ammonia. L a h i r i and Quastel (36)~have r e p o r t e d a s i m i l a r effect on ammonia f o r m a t i o n when the c i t r i c a c i d cycle i s inhibited by f l u o r acetate i n the p r e s e n c e of glucose.  E l e c t r o n transfer, oxidative phophorylation and a m m o n i a f o r m a t i o n . In the absence of glucose the oxidation of the ammonia-forming p r e c u r s o r s i n adult c e r e b r a l cortex s l i c e s r e s u l t s i n a g r e a t e r y i e l d of ammonia than infant c e r e b r a l s l i c e s (62 vs. 42p.moles/g d r y weight) (Table XIV), p r e s u m a b l y because oxygen uptake by infant b r a i n i s lower. M i l stein et al. ( 139) showed that the oxygen consumption p e r mg  m i t o c h o n d r i a l protein i n c r e a s e s with the age of the r a t .  A number of investigator s have r e p o r t e d a s u p p r e s s i o n of the fate of ammonia f o r m a t i o n by 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 (20, 47). Thus, azide, a r senite, cyanide, etc., inhibit ammonia f o r m a t i o n in a substrateless  incubation medium.  Amytal, a b a r b i t u r a t e that  inhibits e l e c t r o n t r a n s f e r between N A D H and the cytochrome system,  103  strongly suppresses the rate of ammonia f o r m a t i o n i n the absence of glucose, and whatever little ammonia i s f o r m e d under these conditions i s suppressed i n the presence of glucose (Table XXIII). The mode of action of amytal i n suppressing a m m o n i a f o r m a t i o n may be due to l a c k of NAD+, which h a s to be present f o r activity of glutamic dehydrogenase. A m m o n i a f o r m a t i o n has been shown to be dependent on the s t r u c t u r a l integrity of the b r a i n tissue, since the rate of a m m o n i a f o r m a t i o n i n c e l l d i s p e r s i o n s i n much d e p r e s s e d (20 ). F u r t h e r evidence on the dependence of ammonia f o r m a t i o n on the proper functioning of the e l e c t r o n chain i s obtained f r o m p r e incubation experiments.  Though there i s little change i n the amino  a c i d s p e c t r u m of b r a i n cortex s l i c e s incubated for one hour i n the presence of glucose, the rate of ammonia f o r m a t i o n by slices, pre-incubated in a glucose m e d i u m f o r one hour and t r a n s f e r r e d to a glucose free medium, i s lower than if no pre-incubation was c a r r i e d out (Table XXVI).  That this may be due to an i m p a i r m e n t of  oxidative p r o c e s s e s leading to ammonia f o r m a t i o n i s substantiated by the observation that an anaerobic pre-incubation of c e r e b r a l cortex s l i c e s r e s u l t s i n a complete and i r r e v e r s i b l e damage to the ammonia f o r m i n g s y s t e m (Table XXVII).  In this connection it should  be noted that a sixty minute exposure of rabbit b r a i n cortex s l i c e s to anoxia r e s u l t s i n an i r r e v e r s i b l e damage to the mitochondria and phosphorylating mechanisms, and a diminution i n A T P and creatine phosphate l e v e l s (140). A d i r e c t consequence of the inhibition of the r e s p i r a t o r y chain i s a d i m i n i s h e d rate of A T P synthesis. Both D N P and salicylate, (109) compounds that reduce c e l l u l a r A T P levels, suppress the endogenous f o r m a t i o n of ammonia. In the p r e s e n c e of glucose the suppression of a m m o n i a f o r m a t i o n is not complete i n the p r e s e n c e of either of these i n h i b i t o r s of A T P formation.  These  uncouplers  d i m i n i s h the y i e l d of l a b e l l e d glutamine f r o m l a b e l l e d glucose i n the p r e s e n c e of rat b r a i n cortex slices, a p r o c e s s well known to depend on A T P (42, 73, 77, 78 ). However, salicylate e x e r t s a slighter effect than D N P  (141) which may be r e f l e c t e d i n i t s lower  capacity to accumulate ammonia (Table XXII). The effect of a high concentration of potassium ions on ammonia formation may be explained on the b a s i s of its effect on the c e l l u l a r energy and the r e s p i r a t i o n of r a t b r a i n cortex s l i c e s . In a glucose f r e e medium,oxygen uptake i n an incubation p e r i o d of one hour, and therefore the rate of ammonia formation, a r e only slightly i m p a i r e d i n the p r e s e n c e of high K+ (lOOmM). However, i n a four hour incubation V r b a and F o l b e r g e r ( 108) r e p o r t e d a m a r k e d inhibition of the rate of ammonia f o r m a t i o n at the same time  105 as a substantial diminution of oxygen uptake. K i n i and Quastel (77, 78) demonstrated a m a r k e d i n c r e a s e i n the operation of the c i t r i c a c i d cycle i n K - s t i m u l a t e d b r a i n c e l l s , so that i n the p r e s e n c e of I m M  (but  +  not 5mM)  glucose the intermediates a r e oxidized too fast to allow for;  the n o r m a l rate of ammonia fixation (Table XX). H i g h K  +  ion concentra  tion also speeds up the rate of f o r m a t i o n of lactate f r o m glucose.  This  probably explains why the K*-effect on ammonia f o r m a t i o n i n the presence of threshold l e v e l s of pyruvate i s not so m a r k e d (Table XXI).  A m i n o a c i d effects on ammonia formation. In v i e w of the fact that there i s a profound change i n the amino a c i d s p e c t r u m of c e r e b r a l cortex s l i c e s after a e r o b i c incubation i n a g l u c o s e - f r e e incubation medium, it may be thought that addition of amino a c i d s to the incubation m e d i u m might affect the f o r m a t i o n of ammonia by c e r e b r a l cortex s l i c e s .  Thus, addition of glutamate  might be expected to i n c r e a s e the rate of ammonia formation. However, the oxidation of L-glutamate by b r a i n s l i c e s r e s u l t s i n a d e c r e a s e i n the rate of ammonia f o r m a t i o n (Table X X I V ) , with a concomitant i n c r e a s e i n amide f o r m a t i o n (42, 73). The r e a c t i o n has been formulated as follows: 2 glutamic a c i d + j O  — — ^  glutamine + a-ketoglutaric a c i d + H  O  It indicates that the p r o c e s s of deamination of glutamate a l s o involves the p r o c e s s of glutamine synthesis. Thus, addition of L-glutamate  106  does not n e c e s s a r i l y involve ammonia l i b e r a t i o n if energy i s available to enable fixation of ammonia to take place with f o r m a t i o n o f glutamine. M o r e o v e r , it i s a well known fact that both i s o m e r s of glutamate are inhibitors of the glutaminase r e a c t i o n ( 73 ).  The  presence therefore of glutamate both i n the incubation m e d i u m and i n the c e r e b r a l tissue bathed i n such a medium, may  r e t a r d the  further deamidation of glutamine. T o w e r has pointed out that glutamate may  be concentrated i n  the m i t o c h o n d r i a ( 142 ). It seems probable in view of r e s u l t s r e p o r t e d i n this thesis that the f o r m a t i o n of ammonia i s confined to the oxidative m e c h a n i s m s present and its rate may  depend on the  structural integrity of the mitochondria. F o r m a t i o n of ammonia f r o m glutamine and glutamate by mitochondria of r a t l i v e r and kidney has recently been r e p o r t e d ( 143 ). However, evidence on the compartmentation  of glutamic a c i d m e t a b o l i s m i n b r a i n s l i c e s has  r e c e n t l y appeared ( 144 ). That i s to say, glutamate exists i n b r a i n i n at least two distinct pools. One of these pools of glutamate i s p a r t i c u l a r l y concerned with the f o r m a t i o n of glutamine, and in such a pool the breakdown of glutamine f o r m e d f r o m external L-glutamate would be inhibited by the presence of excess L-glutamate. The inhibition of glutamine breakdown by glutamate analogs and derivative s (viz. L - g l u t a m a t e - y - m e t h y l  ester,  L-glutamate-y-  ethyl ester, and DL-a-methyl glutamate) r e s u l t s i n a further  107  s u p p r e s s i o n of a m m o n i a f o r m a t i o n i n the p r e s e n c e of glucose (Table X X I V ) .  With, a - m e t h y l glutamate, B r a g a n c a et al,( 1 4 5 ) T  showed^ that i n addition to the inhibition of glutamine breakdown, this compound i s capable of f o r m i n g a - m e t h y l glutamine i n a r e a c t i o n analogous to the glutamine synthetase reaction. In the absence of glucose the glutamate analogs a l l inhibit the rate of a m m o n i a formation, the inhibition being greatest with D - g l u t a m a t e and a - m e t h y l glutamate. Though W e i l - M a l h e r b e and G r e e n ( 20 ) have reported that D -glutamate i s without any effect on 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 medium, our r e s u l t s on the m a r k e d s u p p r e s s i o n of a m m o n i a f o r m a t i o n by D-glutamate i s i n a g r e e m e n t with those of T a k a g a k i et aj* ( 146 ), who  also obtained a concomitant  inhibition of oxygen uptake with this i n h i b i t o r .  It i s p o s s i b l e that  D - g l u t a m a t e and a - m e t h y l glutamate (and to a l e s s e r extent the other glutamate derivatives) in addition to inhibiting the glutaminase r e a c t i o n also inhibit g l u t a m i c dehydrogenase. T h e r e i s no m a r k e d change i n the rate of a m m o n i a f o r m a t i o n on the addition of glycine, L-aspartate, taurine or y A B A (Table XVI). However, glutamine added to the incubation m e d i u m i s deamiriated by b r a i n s l i c e s and there i s m o r e ammonia f o r m e d by the operation of this r e a c t i o n i n the absence of glucose than in its presence.  This  suggests that re synthesis of glutamine o c c u r s i n the p r e s e n c e of glucose since c i t r i c a c i d c y c l e i n t e r m e d i a t e s and the n e c e s s a r y  108 energy supply a r e available. T h e inhibition of the f o r m a t i o n of l a b e l l e d glutamine f r o m l a b e l l e d glucose by ouabain, h a s been shown to o c c u r i n c e r e b r a l c o r t e x s l i c e s (125). The addition of glutamine to a Ringer-phosphate-glucose m e d i u m i n the p r e s e n c e of ouabain gives a m a r k e d i n c r e a s e i n the rate of ammonia f o r m a tion over the control (i.e., i n the absence of ouabain) (Table XXXII). It i s concluded that ouabain inhibits the r e s y n t h e s i s of glutamine i n the p r e s e n c e of glucose i n b r a i n cortex s l i c e s . It may  be noted  that ouabain does not activate glutaminase, for i f it did, it would also act on glutamine i n the absence of glucose (Table XXX111). Since ouabain has been shown to be without any effect on glutamine synthetase isolated f r o m b r a i n tissue (125), it i s c o n s i d e r e d that ouabain affects glutamine biosynthesis by changing the fluxes of a m m o n i u m ions at the m e m b r a n e s of either the m i t o c h o n d r i a or the m i c r o s o m e s , in which compartments glutamine synthetase i s held to be active (141, 147).  109  7.4 T h e formation of ammonia i n b r a i n s l i c e s , (for incubation p e r i o d s of long duration. M o s t of the work r e p o r t e d i n this thesis h a s involved studies of ammonia f o r m a t i o n during incubation p e r i o d s of one hour; during longer periods of incubation i n a g l u c o s e - f r e e incubation m e d i u m l a r g e r quantities of ammonia a r e l i b e r a t e d (9, 20). A s c e r e b r a l proteins contain a l a r g e p r o p o r t i o n of c e r e b r a l c e l l u l a r nitrogen, they may serve as a source of ammonia after the i n t r a c e l l u l a r f r e e a m i n o a c i d pools ( p a r t i c u l a r l y those of glutamate and glutamine) have diminished i n quantity. In a four hour incubation 25%  of the total ammonia f o r m e d can be accounted f o r on the b a s i s  of p r o t e i n amide nitrogen. (49 )• P r o t e i n m e t a b o l i s m i n the b r a i n i s i n a dynamic state ( 148).  In the p r e s e n c e of glucose, p r o t e i n  ( 114 ) and amino acids l e v e l s (Table XIII) r e m a i n constant since both a r e synthesized as fast as they a r e utilized.  In the absence of  glucose, however, there i s a net c a t a b o l i s m of protein ( 149 )N o n - p r o t e i n nitrogen has been shown to i n c r e a s e i n an incubation of four hours i n a glucose-free m e d i u m ( 20, 49).  A m i n o acids,  p a r t i c u l a r l y glutamate and glutamine, that f o r m during p r o t e i n catabolism,  can serve as sources of ammonia in the way they serve  during incubation p e r i o d s of short duration. B r a i n p r o t e i n metab o l i s m i s c l o s e l y r e l a t e d to that of p e r i p h e r a l nerve, where it h a s  .  110  been shown that stimulation i s a s s o c i a t e d with an i n c r e a s e d activity of the neutral proteinase,  a d e c r e a s e d p r o t e i n content,  increased  utilization of glutamate and l i b e r a t i o n of ammonia ( 1 50 ). T h e r e seems to be a r e l a t i o n between the rate of p r o t e i n b r e a k down and that of ammonia formation.  Conditions that inhibit protein  c a t a b o l i s m are apparently identical with the conditions that inhibit the subsequent f o r m a t i o n of ammonia. A neutral proteinase of whole r a t b r a i n i s m o r e active i n an atmosphere of oxygen than i n an atmosphere of nitrogen; and the r e q u i r e m e n t of energy f o r p r o t e i n c a t a b o l i s m indicates a p r o c e s s different f r o m simple h y d r o l y s i s ( 148).  Conditions that inhibit i n c o r p o r a t i o n of amino  a c i d s into proteins, or d i m i n i s h c e l l energetics, o r the addition of cyanide, azide, DNP, also inhibit protein breakdown ( 148).  such as  as well as amino a c i d analogs, W e i l - M a l h e r b e and D r y s d a l e  (55 ) showed that after an i n i t i a l l a g (about two hours), or the p r e s e n c e of D N P  anaerobiosis  i n an aerobic incubation,  anaerobiosis,  completely inhibits  the subsequent formation of ammonia. M o r e o v e r , though protein synthesis o c c u r s in the m i c r o s o m e s , protein c a t a b o l i s m i n b r a i n tissue has been shown to occur i n the mitochondria ( 148 ). With an i r r e v e r s i b l e damage to the mitochondria, ammonia f o r m a t i o n  does  not take place, p o s s i b l y due to the inhibition of the protein b r e a k down system.  Ill  8. 1.  SUMMARY  T h e i n i t i a l (pre-incubation) ammonia content of adult r a t c e r e b r a l cortex s l i c e s i s about 5y.moles/g wet weight tissue. value was obtained by a diffusion technique i n which K ^ C O ^  This  was  used to l i b e r a t e the ammonia f r o m the tissue. 2.  Intraperitoneal injection of glucose, amytal o r i p r o n i a z i d into adult rats, or the r a p i d f r e e z i n g of infant r a t s i n l i q u i d nitrogen, i s without effect on the i n i t i a l a m m o n i a content of c e r e b r a l tissue. However, i f the tissue i s treated with t r i c h l o r a c e t i c a c i d ( T C A , 3 % ) , the i n i t i a l ammonia content i s d i m i n i s h e d by 5 0 % giving i n i t i a l ammonia values, by the a l k a l i assay,  comparable with those obtained  by use of the B e c k m a n amino a c i d a n a l y z e r in which ammonia i s m e a s u r e d by the ninhydrin reaction. 3.  T h e estimation of the initial ammonia content of c e r e b r a l c o r t e x s l i c e s i s not significantly affected by the addition of L-glutamine, D - o r L-glutamate,  4.  AMP,  taurine or glucose.  A 5 0 % diminution of the i n i t i a l ammonia content o c c u r s when the T C A - i n s o l u b l e components a r e r e m o v e d f r o m the tissue p r i o r to ammonia estimation.  5.  It i s suggested that T C A i m m o b i l i z e s an enzyme system(s) of c e r e b r a l tissue partly r e s p o n s i b l e for the high i n i t i a l a m m o n i a value s.  112  6.  T h e a d d i t i o n of T C A p e r i o d i s without  at the end of the e x p e r i m e n t a l  significant effect on the a s s a y  incubation  of cerebral ammonia  f o r m a t i o n when b r a i n slices a r e incubated either i nthe presence o r absence of glucose.  7.  T h e p r e - i n c u b a t i o n concentrations of the amino  acids of r a t  c e r e b r a l cortex slices a r e not significantly changed on incubation of the t i s s u e f o r one h o u r a t 37°C i n the p r e s e n c e o f g l u c o s e .  8.  I n t h e a b s e n c e of g l u c o s e m a r k e d l e v e l s of a m i n o incubation. glutamine weight),  changes i n the p r e - i n c u b a t i o n  acids of c e r e b r a l c o r t e x s l i c e s o c c u r  These  during  subsequent  a r e a s f o l l o w s : t a u r i n e (-2. 12 u m o l e s / g w e t w e i g h t ) ,  (-3. 4 2 u m o l e s / g w e t w e i g h t ) ,  g l u t a m a t e (-8. 4 8 u m o l e s / g w e t  g l y c i n e (-0.25 u m o l e s / g w e t w e i g h t ) , a s p a r t a t e (+4. 36 u m o l e s /  g w e t w e i g h t ) a n d a l a n i n e (+0. 5 u m o l e s / g w e t w e i g h t ) , t h e r e b e i n g change  o f (-12. 83 u m o l e s  NH2  - N/g wet weight).  A  considerable rise i n  a m m o n i a f o r m a t i o n ( + 1 3 . 82 u m o l e s / g w e t w e i g h t ) a l s o o c c u r s these conditions. amino-nitrogen  9.  a  under  T h i s c a n b e l a r g e l y a c c o u n t e d f o rb y the n e t l o s s of  g r o u p s of the a m i n o  acid pools i n the b r a i n s l i c e s .  C e r t a i n substrates that support c e r e b r a l r e s p i r a t i o n inhibit the rate of c e r e b r a l a m m o n i a formation. at 5 m M  levels suppress  Glucose,  pyruvate  the rate of a m m o n i a f o r m a t i o n by r a t  c e r e b r a l c o r t e x s l i c e s to about the s a m e extent. acetate  lactate or  i s l e s s inhibitory and acetate,  5mM  oxalo-  citrate or a-ketoglutarate  .has l i t t l e o r n o e f f e c t o n 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  owing  113  probably to p e r m e a b i l i t y b a r r i e r s . 10.  -  T h e t h r e s h o l d l e v e l f o r the m a x i m u m suppression of the rate of ammonia f o r m a t i o n by r a t  c e r e b r a l cortex s l i c e s i s l.OmM for  glucose and 2.5mM for pyruvate.  T h i s i s consistent with the fact  that g l y c o l y s i s of one molecule of glucose y i e l d s two of pyruvate. 11.  T h e rate of ammonia formation by c e r e b r a l cortex s l i c e s of the r a t i s elevated by O.lmM iodoacetate i n the presence of glucose but not i n the presence of pyruvate.  T h e s e facts indicate that c e r e b r a l  oxidation of pyruvate may be responsible for the s u p p r e s s i o n of the rate of ammonia formation.  In the absence of added substrate,  iodoacetate i s only slightly inhibitory to the rate of ammonia f o r m a tion which may be due to a slightly diminished oxygen uptake. 12.  In the absence of glucose the y i e l d of ammonia, in one hour, f r o m adult rat c e r e b r a l cortex s l i c e s (on a d r y weight basis) i s greater (by about 50%) than that f r o m infant rats.  T h i s i s consistent  with the known fact that the rate of c e r e b r a l oxidation of glucose i n the adult r a t i s greater than that i n the infant rat. 13.  T h e addition of lOOmM KC1 to a Ringer-phosphate m e d i u m i n c r e a s e s the rate 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 cortex s l i c e s in the p r e s e n c e of I m M glucose (threshold level), but not i n the p r e s e n c e of 5mM  glucose.  T h i s i s probably because high K+ ion  114  concentration i n c r e a s e s the rate of glucose breakdown i n b r a i n s l i c e s , so that with initially I m M b e c o m e s deprived of glucose.  glucose, the b r a i n tissue soon  H i g h K+ i o n concentration also speeds  the rate of formation of lactate f r o m glucose.  T h i s probably  explains  why K+ ions affect ammonia f o r m a t i o n i n the p r e s e n c e of I m M glucose but not i n the presence of 2.5mM pyruvate.  In the absence  of added substrate high K^" i o n concentration i s only slightly inhibitory to the rate of ammonia formation i n one hour. 14.  T h e inhibition of the c i t r i c a c i d c y c l e by malonate (5mM) i n the p r e s e n c e of I m M  glucose, i n c r e a s e s the rate of ammonia  formation  by c e r e b r a l cortex s l i c e s by suppressing the f o r m a t i o n of. c i t r i c a c i d c y c l e intermediates, 15.  r e q u i r e d for ammonia fixation to occur.  A m y t a l (ImM) d i m i n i s h e s the rate of a m m o n i a f o r m a t i o n by c e r e b r a l cortex s l i c e s in the absence of glucose.  This presumably  o c c u r s because amytal suppresses the oxidation of glutamate. 16.  In the presence of glucose, uncouplers (e.g. DNP,  O.lmM; or salicylate, 5mM)  of oxidative phosphorylation  i n c r e a s e s the rate of ammonia  f o r m a t i o n by b r a i n cortex s l i c e s by inhibiting the synthesis of glutamine (an A T P dependent p r o c e s s ) . In the absence of glucose there i s a diminution i n the rate of ammonia f o r m a t i o n by these inhibitor s.  115  17.  Anaerobic-sis s u p p r e s s e s the rate of ammonia formation, probably by suppressing the oxidation of glutamate.  It i r r e v e r s i b l y damages  the ammonia f o r m i n g s y s t e m present i n c e r e b r a l cortex s l i c e s . 18.  With the exception of glutamine, none of the n a t u r a l l y o c c u r r i n g amino a c i d s (e.g. alanine, glycine, yA~BA, taurine, L-aspartate) of c e r e b r a l tissue when added to incubated c e r e b r a l cortex s l i c e s significantly affects the rate of a m m o n i a formation.  T h e glutamate  d e r i v a t i v e s ( D L - a - m e t h y l glutamate,  L-glutamate-y-methylester,  and L-glutamate-y-ethylester) at 5mM  l e v e l s inhibit the rate of  ammonia f o r m a t i o n both i n the presence of glucose (by suppressing glutamine h y d r o l y s i s ) a n d i n the absence of glucose (by suppressing glutamate oxidation). H y d r o x y l a m i n e (5mM) i n c r e a s e s the rate of ammonia f o r m a t i o n both i n the presence or absence of glucose, p r e s u m a b l y by competing with ammonia for glutamine 19.  synthetase.  D-glutamate has the following effects on the ammonia m e t a b o l i s m of c e r e b r a l cortex s l i c e s : a) it suppresses the rate of ammonia f o r m a t i o n i n the absence of glucose, doubtless by inhibiting glutamic dehydrogenase; b) i t elevates the rate of ammonia f o r m a t i o n i n the presence of glucose by inhibiting glutamine  synthetase;  c) it  suppresses the rate of ammonia f o r m a t i o n f r o m L-glutamine in the presence of glucose by inhibiting 20.  AMP  glutaminase.  or A T P i n the p r e s e n c e of b r a i n cortex s l i c e s b r i n g s about  an i n c r e a s e i n the rate of ammonia formation.  116  21.  R e s u l t s obtained point to a possible compartmentation of the site of ammonia f o r m a t i o n and the retention (within the compartment(s)) of ammonia up to a l i m i t i n g level, above which the efflux of ammonia into the incubation m e d i u m takes place. Such a c o n c l u s i o n helps to explain the fact that the b r a i n tissue ammonia i s not m a r k e d l y affected by the presence o r absence of glucose, a s t±ie i n c r e a s e d quantity of ammonia f o r m e d i n the absence of glucose i s found l a r g e l y i n the incubation medium. It also explains the apparent high concentration ratio (tissue:medium) of NH^+ at the end of one hour incubation i n the p r e s e n c e of glucose,  under  a e r o b i c or anaerobic conditions where the concentration ratio (tissue:medium) i s 42 o r 12 r e s p e c t i v e l y . 22.  E v i d e n c e obtained f r o m the examination of NH.+ influx f r o m 4 the incubation medium, shows some accumulation of NH..+ ions i n 4 r a t b r a i n cortex slices against a concentration gradient.  23.  Ouabain, at a concentration of O.OlmM which has no effect on the rate of r e s p i r a t i o n of rat b r a i n cortex slices, a) i n c r e a s e s the rate of ammonia formation i n the presence of glucose but has little effect i n the absence of glucose, b) i n c r e a s e s the rate of a m m o n i a f o r m a t i o n f r o m added glutamine i n the p r e s e n c e of glucose but i t does not affect the rate of ammonia f o r m a t i o n f r o m added glutamine i n the absence of glucose.  T h e s e r e s u l t s a r e consistent with the  117  known fact that O.OlmM ouabain inhibits the rate of bio syn the sis of glutamine by rat c e r e b r a l cortex s l i c e s r e s p i r i n g i n a R i n g e r phosphate m e d i u m containing glucose. 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