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Cerebral metabolism in anoxia and the effects of some neurotropic drugs Shankar, Raj 1971

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CEREBRAL METABOLISM IN ANOXIA AND THE EFFECTS OF SOME NEUROTROPIC DRUGS  by  RAJ B.Sc,  SHANKAR  U n i v e r s i t y o f Gorakhpur, 1964  M.Sc., U n i v e r s i t y o f Lucknow, 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of Biochemistry  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1971  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y s h a l l I  f u r t h e r agree  in p a r t i a l  fulfilment of  the requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia,  make i t  freely available  that permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s of  representatives.  this  thesis  It  for financial  i s understood that gain s h a l l  written permission.  Department of The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  Date  %tvA  <^WW  Columbia  V^l  |  \  copying o r p u b l i c a t i o n  not be allowed without my  ABSTRACT The  effects  neurotropic transport slices more and  drugs  fully  been  mode  of  tetrodotoxin anaerobic of  studied  in  of  action  general various  glycolysis  of  of  an  on  incubated  effort  conditions.  also  studied  The  under  processes  has the  brain  in  these  in  c e r e b r a l metabolism.  out  on  the  glucose  in  contents  cation these  the  cerebral  under  a  to  rates  of  presence  TTX  anaerobic  of  Ca  .  such  glycolysis  fold, + +  of  the  Such  that  obtained  bral  cortex  as  on  of  effect the  slices.  )  of  rates  yM,  of  the  anaerobic a  the  were  amino  of  variety  were changes  acids  the  drug, the  in  and  glycolysis at  rate  slices the  of  the  glycolysis  low of two  to  absence  far greater  metabolism  anaerobic  carried  conditions.  cortex  is  changes  also  anaerobic  greater  TTX  study  and  enhances  being  drugs.  concomitant  that  cerebral  aerobic The  anoxia,  contents  incubation  showed 2  understand  under  Experiments  effects an  , K  r e l a t e d to  the  cortex  +  conditions  v a r i e t y of of  on  during  been  slices  (Na  transport  Measurement  concentrations  three  were  to  other  and  cerebral  + of  and  certain neurotropic  approach drugs  (TTX)  glycolysis,  incubated  cerebral metabolic  The action  on  processes,  have  the  of  than cereof  kidney or  medulla  o f acetone  affected of  mature  cerebral  the brain  rate  when  high  present  conditions, «n4  loss  decline  bral  cortex  slices  lead  on a n a e r o b i c  the  brain  of and  medium.  an i n f l u x o f N a  anoxic  i n t h e K /Na i s observed.  ratio These that  by  the i n i t i a l  t o be m a i n t a i n e d K /Na  the anaerobic  cereex-  g l y c o l y s i s a r e due t o i t s a c t i o n  the onset  +  a  the effects of  about  cellular  tissue.  and other  cell  o f TTX,  into,Or  of the  i n brain  brought  +  these  conditions,  t h e changes +  NH^  +  cerebral  r e s u l t i n g i n the  K  or  Under  membrane  tends  on  or  cell  presence  the  o f anoxia,  L-glutamate  to the conclusion  TTX  10  the incubated  o f TTX under  slower  integrity  i t i s added  the onset  i s either  much  specific  o r no e f f e c t i n i n c r e a s i n g  after  from,  +  the effects  action.  g l y c o l y s i s when  there  of K  that  slices  are not  and r e q u i r e s  i n the incubation  the presence  periments  tissue,  protoveratrine,  +  brain  glycolysis are  for i t s  or later, K ,  from  indicating  has l i t t l e  of anaerobic  minutes,  are  cells  o l d r a t brain  extracts  by t h e drug,  TTX  In  powder  2-day  TTX on t h e a n a e r o b i c  for of  slices,  +  due t o o n l y  ratio.  prevention  permeabilities  high  to Na  o f anoxia. rate  g l y c o l y s i s are thought  +  In the  of glycolysis  a slow  The e f f e c t s  at  decline i n  of K  +  t o be  and N a  +  mediated  iv. largely which  by c h a n g e s i n t h e p y r u v a t e  i s e n h a n c e d by K TTX  appears  activity,  a n d d i m i n i s h e d by N a . +  to affect  metabolism o f b r a i n affects  +  kinase  t h e a e r o b i c and a n a e r o b i c  i n vitro  i n t h e same way a s i t  the generation action  potentials  i . e . by  + diminishing results are  the i n f l u x  lead  generated  fests  i t s effect  greater former the by Na  o f TTX on t h e N a  are the s i t e  TTX a r e p r o b a b l y found  from at  the incubated  brain.  K /Na +  a t the  +  of anoxia.  mani-  glycolysis.  because the  Consequently,  ratio  brought  those  about  o f the K / +  as a whole.  to i t s effects  on t h e N a  the e f f l u x  cerebral  cells  o f TTX.  much g r e a t e r t h a n  dependent o f the a c t i v i t y normally  tissue  c o n t e n t s may be  +  in glial  of action  TTX a l s o p r e v e n t s  the onset  and K  +  i n the tissue  In a d d i t i o n fluxes,  cerebral  These  potentials  by a h i g h e r r a t e o f a n a e r o b i c  i n the neurons than  ratio  of K .  T h e s e a r e b l o c k e d b y TTX w h i c h  changes i n the n e u r o n a l  +  and e f f l u x  i n the incubated  of anoxia.  effect  o f Na  t o the conclusion that action  onset  The  +  and K  +  o f amino a c i d s  cortex s l i c e s  This effect  +  that  occurs  o f TTX i s i n -  of the transport processes  o p e r a t i n g on t h e amino a c i d  uptake i n t o the  V.  L o c a l a n e s t h e t i c s , o u a b a i n , a m y t a l and r e s e r p i n e a l s o increase the r a t e of anaerobic b r a l cortex s l i c e s .  g l y c o l y s i s of cere-  L o c a l a n e s t h e t i c s a c t i n a manner  s i m i l a r t o TTX, a l t h o u g h much h i g h e r c o n c e n t r a t i o n s a r e required.  The e f f e c t s o f o u a b a i n i n a C a - f r e e + +  a r e much g r e a t e r than i n a C a - c o n t a i n i n g + +  medium  medium. I t  i s suggested t h a t the i n c r e a s e i n the r a t e o f anaerobic g l y c o l y s i s due t o o u a b a i n i s p o s s i b l y m e d i a t e d by an i n c r e a s e i n c e l l ATP c o n c e n t r a t i o n under a n o x i a , as a r e s u l t of i n h i b i t i o n of Na  +  , K -ATPase. +  o f a m y t a l and r e s e r p i n e on t h e a n a e r o b i c  The a c t i o n s glycolysis of  c e r e b r a l c o r t e x s l i c e s a r e p o s s i b l y m e d i a t e d by membrane c a t i o n changes, b u t f u r t h e r work i s n e c e s s a r y to support  this conclusion.  TABLE OF CONTENTS  v i Page  T I T L E PAGE  i  ABSTRACT  i i  TABLE OF CONTENTS L I S T OF FIGURES  v i x i i  L I S T OF TABLES  xvi  L I S T OF SCHEMES  xx  ABBREVIATIONS..  xxi  ACKNOWLEDGEMENTS.  xxii  Chapter 1  2  INTRODUCTION 1.1 E f f e c t o f A n o x i a on B r a i n M e t a b o l i s m 1.2 Glucose Metabolism i n B r a i n 1.3 Effects of Local Anesthetics 1.4 E f f e c t s o f T e t r o d o t o x i n on t h e N e r v o u s S y s t e m 1.5 E f f e c t s o f o t h e r N e u r o t r o p i c Drugs o n t h e Nervous System 1.6 Biochemistry o f the Developing Brain 1.7 T r a n s p o r t o f Amino A c i d s a n d S u g a r s a c r o s s t h e B r a i n C e l l Membrane 1.8 Objectives  1 1 6 19 26  MATERIALS AND METHODS 2.1 Experimental Animals 2.2 Chemicals . 2.3 Preparation of Brain Slices 2.4 Incubation Procedure 2.5 I n c u b a t i o n Medium 2.6 P r e p a r a t i o n o f Kidney Medulla 2.7 P r e p a r a t i o n o f Caudate Nucleus 2.8 P r e p a r a t i o n o f A c e t o n e Powder E x t r a c t s 2.9 P r e p a r a t i o n o f Synaptosomes 2.10 P r e p a r a t i o n o f M i c r o s o m e s a n d A s s a y o f Na , K -ATPase 2.11 M e a s u r e m e n t o f G l y c o l y s i s 2.12 M e a s u r e m e n t o f Amino A c i d E f f l u x a n d U p t a k e . . 2.13 M e a s u r e m e n t o f NAD+ a n d NADI-1 2.14 M e a s u r e m e n t o f CAMP P r o d u c t i o n 2.15 M e a s u r e m e n t o f ATP L e v e l s  49 49 49 50 51 52 54 54 54 55  +  +  31 43 45 48  55 57 59 60 62 63  vii 2.16 2.17 2.18 2.19 3  22 Measurement o f Na Influx Measurement o f N a and K M i s c e l l a n e o u s Methods R e p r o d u c i b i l i t y of the Results +  63 64 64 66  +  RATE LIMITING FACTORS IN ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES AND ACETONE POWDER EXTRACTS 67 3.1 E f f e c t s o f C a l c i u m on the A n a e r o b i c G l y c o l y s i s of C e r e b r a l C o r t e x S l i c e s 67 3.2 E f f e c t s o f E x t e r n a l l y Added NAD+ and ATP on the A n a e r o b i c G l y c o l y s i s o f C e r e b r a l C o r t e x Slices.. 72 3.3 Movement o f NAD A c r o s s the C e l l Membrane.. 74 3.4 E f f e c t s o f NAD on A e r o b i c G l y c o l y s i s 77 3.5 E f f e c t s o f C i t r a t e and AMP on t h e A n a e r o b i c G l y c o l y s i s o f Cerebral Cortex S l i c e s 78 3.6 E f f e c t s o f C y c l i c AMP and D i b u t y r y l c y c l i c AMP on t h e A n a e r o b i c G l y c o l y s i s c f t h e Cerebral Cortex S l i c e s 81 3.7 Effects of varying Cation Concentrations on the A n a e r o b i c G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 83 3.8 E f f e c t s o f L-Glutamate and o f Ammonium Ions on the A n a e r o b i c G l y c o l y s i s o f C e r e b r a l Cortex S l i c e s 85 3.9 E f f e c t o f NAD and ATP, i n a Sodium-Free Medium, on the A n a e r o b i c G l y c o l y s i s o f Cerebral Cortex S l i c e s 87 3.10 Rate L i m i t i n g F a c t o r s o f G l y c o l y s i s i n t h e Acetone Powder E x t r a c t s 89 3.11 Summary o f Chapter 3 91 +  +  +  4  EFFECTS OF TETRODOTOXIN ON CEREBRAL METABOLISM AND TRANSPORT IN ANOXIA 94 4.1 E f f e c t s o f T e t r o d o t o x i n on t h e A n a e r o b i c G l y c o l y s i s o f G u i n e a P i g and R a t C e r e b r a l 94 Cortex S l i c e s 4.2 E f f e c t s o f C a l c i u m on T e t r o d o t o x i n S t i m u l a t e d Anaerobic G l y c o l y s i s 95 4.3 E f f e c t o f T e t r o d o t o x i n on MAD Movements A c r o s s t h e C e l l Membrane 98 4.4 E f f e c t s o f T e t r o d o t o x i n on t h e Amino A c i d E f f l u x from t h e C e r e b r a l C o r t e x S l i c e s . . . 100 4.5 E f f e c t o f T e t r o d o t o x i n on the Uptake o f Amino A c i d s under A n a e r o b i c C o n d i t i o n s . . . 102 4.6 E f f e c t o f T e t r o d o t o x i n a t D i f f e r e n t Glucose C o n c e n t r a t i o n s on t h e A n a e r o b i c G l y c o l y s i s of C e r e b r a l C o r t e x S l i c e s 105 4.7 G l u c o s e T r a n s p o r t i n C e r e b r a l C o r t e x S l i c e s 105 4.8 E f f e c t s o f T e t r o d o t o x i n , i n the Presence o f Some Amino A c i d s , on the A n a e r o b i c G l y c o l y s i s of Cerebral Cortex S l i c e s 107 4.9 E f f e c t s o f C i t r a t e , AMP and NH^ on t h e Tetrodotoxin Stimulation of Glycolysis of R a t C e r e b r a l C o r t e x S l i c e s 109 +  +  viii 4.10 E f f e c t s o f P h o s p h o l i p a s e s on t h e T e t r o d o t o x i n Stimulated G l y c o l y s i s of the r a t Cerebral Cortex S l i c e s 4.11 E f f e c t s o f T e t r o d o t o x i n on A n a e r o b i c G l y c o l y s i s o f K i d n e y M e d u l l a S l i c e s and Acetone Powder E x t r a c t s 4.12 E f f e c t s o f T e t r o d o t o x i n on t h e A n a e r o b i c G l y c o l y s i s of Developing B r a i n Cortex Slices 4.13 E f f e c t o f T e t r o d o t o x i n i n P r e s e n c e o f G l u t a m a t e , A s p a r t a t e and NH + on t h e Anaerobic G l y c o l y s i s of Developing Cortex S l i c e s 4.14 E f f e c t s o f T e t r o d o t o x i n on t h e A e r o b i c G l y c o l y s i s of A d u l t Rat C e r e b r a l Cortex Slices 4.15 Summary o f Chapter 4  112 114 117  4  5  FURTHER STUDIES ON THE MECHANISM OF ACTION OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES 5.1 E f f e c t s o f P r e - i n c u b a t i o n i n oxygen on t h e Tetrodotoxin Stimulated Glycolysis of Cerebral Cortex S l i c e s 5.2 E f f e c t s o f T e t r o d o t o x i n a f t e r V a r i o u s P e r i o d s o f A n a e r o b i o s i s on t h e A n a e r o b i c G l y c o l y s i s of Cerebral Cortex S l i c e s 5.3 E f f e c t s o f T e t r o d o t o x i n i n t h e P r e s e n c e o f P y r u v a t e on t h e A n a e r o b i c G l y c o l y s i s o f Cerebral Cortex S l i c e s 5.4 E f f e c t s o f G l u c o s e A d d i t i o n under V a r i o u s C o n d i t i o n s on t h e S t i m u l a t i o n o f A n a e r o b i c G l y c o l y s i s by T e t r o d o t o x i n 5.5 E f f e c t o f A e r o b i c I n c u b a t i o n w i t h D i n i t r o p h e n o l on t h e T e t r o d o t o x i n S t i m u l a t i o n o f A n a e r o b i c G l y c o l y s i s , and on t h e ATP l e v e l o f the Rat C e r e b r a l Cortex S l i c e s . . 5.6 E f f e c t s o f T e t r o d o t o x i n and Ouabain on t h e ATP C o n t e n t o f Guinea P i g C e r e b r a l C o r t e x Slices 5.7 E f f e c t s o f R a i s i n g ATP L e v e l by A e r o b i c I n c u b a t i o n w i t h Adenosine on t h e T e t r o d o t o x i n S t i m u l a t e d G l y c o l y s i s o f the Rat Cerebral Cortex S l i c e s 5.8 E f f e c t s o f T e t r o d o t o x i n on cAMP P r o d u c t i o n i n Cerebral Cortex S l i c e s 5.9 E f f e c t o f P r o t o v e r a t r i n e on t h e T e t r o d o toxin Stimulated Glycolysis  117 120 124  127 127 128 130 134  136 139  139 142 143  ix 22 5.10 E f f e c t s o f T e t r o d o t o x i n on t h e Na Transport i n t h e Rat C e r e b r a l C o r t e x S l i c e s 5.11 E f f e c t s o f T e t r o d o t o x i n , a t V a r i o u s C a t i o n C o n c e n t r a t i o n s o f t h e Medium, on t h e Anaerob i c G l y c o l y s i s of Cerebral Cortex S l i c e s 5.12 E f f e c t s o f T e t r o d o t o x i n on t h e Na+ and K L e v e l s o f Incubated C e r e b r a l C o r t e x S l i c e s under A n o x i a 5.13 E f f e c t s o f T e t r o d o t o x i n on t h e A n a e r o b i c G l y c o l y s i s and Na^2 T r a n s p o r t i n t h e Caudate N u c l e u s o f Rat 5.14 E f f e c t s o f T e t r o d o t o x i n on t h e A n a e r o b i c G l y c o l y s i s o f Synaptosomal P r e p a r a t i o n s o f Rat B r a i n 5.15 E f f e c t s o f P r e - i n c u b a t i o n i n Oxygen on t h e N a and K C o n t e n t s o f C e r e b r a l C o r t e x S l i c e s under A n o x i a i n t h e P r e s e n c e o f Tetrodotoxin 5.16 E f f e c t s o f T e t r o d o t o x i n on t h e N a and K l e v e l s o f I n f a n t Rat and G u i n e a P i g C e r e b r a l Cortex S l i c e s 5.17 E f f e c t s o f T e t r o d o t o x i n on t h e N a and K Levels o f Kidney Medulla S l i c e s 5.18 E f f e c t s o f T e t r o d o t o x i n i n t h e P r e s e n c e o f C h e l a t i n g Agents on t h e A n a e r o b i c G l y c o l y s i s of C e r e b r a l Cortex S l i c e s 5.19 E f f e c t s o f A e r o b i c P r e - i n c u b a t i o n w i t h E t h a n o l on t h e T e t r o d o t o x i n S t i m u l a t i o n o f A n a e r o b i c G l y c o l y s i s of Cerebral Cortex S l i c e s 5.20 E f f e c t s o f T e t r o d o t o x i n on t h e Na+ and K Content o f the 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 o f G l u t a m a t e , A s p a r t a t e , Homoc y s t e i a t e and NH.+ 5.21 E f f e c t s o f T e t r o d o t o x i n on t h e P y r u v a t e and Phosphoenol-pyruvate Contents o f C e r e b r a l C o r t e x s l i c e s under A n o x i a 5.22 Summary o f C h a p t e r 5 +  +  145 150 155 157 157  +  +  +  +  +  159 161 16 5 165 167  +  6  EFFECTS OF OUABAIN AND LOCAL ANESTHETICS ON THE CEREBRAL METABOLISM AND TRANSPORT UNDER ANOXIA 6.1 E f f e c t s o f o u a b a i n on t h e A n a e r o b i c G l y c o l y s i s of Cerebral Cortex S l i c e s 6.2 E f f e c t s o f Ouabain on t h e A n a e r o b i c G l y c o l y s i s o f I n f a n t Rat B r a i n C o r t e x S l i c e s 6.3 E f f e c t s o f Ouabain and T e t r o d o t o x i n on t h e A n a e r o b i c G l y c o l y s i s o f Rat C e r e b r a l C o r t e x S l i c e s a t D i f f e r e n t Concentrations of Na i n t h e I n c u b a t i o n Medium 6.4 E f f e c t s o f Ouabain on A n a e r o b i c G l y c o l y s i s o f Acetone Powder o f B r a i n  170 170 174 177 178 180  +  183 183  6.5  E f f e c t s o f Ouabain i n t h e Presence o f LG l u t a m a t e , C i t r a t e , AMP and N H on t h e Anaerobic G l y c o l y s i s o f Cerebral Cortex Slices E f f e c t s o f O u a b a i n on t h e A e r o b i c G l y c o l y s i s of Cerebral Cortex S l i c e s E f f e c t s o f O u a b a i n on t h e ATP C o n t e n t s o f Cerebral Cortex S l i c e s E f f e c t s o f A d d i t i o n o f Ouabain a f t e r V a r i o u s P e r i o d s o f A n a e r o b i o s i s on t h e A n a e r o b i c G l y c o l y s i s o f Rat C e r e b r a l Cortex S l i c e s . . E f f e c t s o f A d d i t i o n o f Ouabain and T e t r o d o t o x i n t o g e t h e r on t h e A n a e r o b i c G l y c o l y s i s of C e r e b r a l Cortex S l i c e s E f f e c t s o f Ouabain on t h e N a and K Contents o f Rat C e r e b r a l Cortex S l i c e s Under A n o x i a E f f e c t s o f Ouabain, C a a n d NAD o n t h e Microsomal Na , K -ATPase E f f e c t s o f O u a b a i n on Amino A c i d E f f l u x e s from t h e C e r e b r a l C o r t e x S l i c e s under Anoxia E f f e c t s o f P r o c a i n e a n d L i d o c a i n e on t h e Anaerobic G l y c o l y s i s o f Rat C e r e b r a l Cortex S l i c e s E f f e c t s o f L i d o c a i n e on t h e A n a e r o b i c G l y c o l y s i s o f I n f a n t Rat and Guinea P i g Cerebral Cortex S l i c e s E f f e c t s o f L i d o c a i n e o n t h e N a and K Contents o f C e r e b r a l Cortex S l i c e s Summary o f C h a p t e r 6 +  4  6.6 6.7 6.8  6.9  6.10  6.11  +  + +  +  6.12  6.13 6.14 6.15 6.16  +  +  +  +  +  E F F E C T S OF VARIOUS NEUROTROPIC DRUGS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX S L I C E S 7.1 A c t i o n o f P y r r o l e and P y r i d i n e on t h e Anaerobic G l y c o l y s i s o f Cerebral Cortex Slices 7.2 E f f e c t s o f P y r r o l e on t h e Na+ a n d K Contents o f Guinea P i g C e r e b r a l Cortex S l i c e s under Anoxia 7.3 E f f e c t s o f A m y t a l and P e n t o t h a l on t h e Anaerobic G l y c o l y s i s of Cerebral Cortex Slices 7.4 E f f e c t s o f C h l o r p r o m a z i n e on t h e A n a e r o b i c G l y c o l y s i s o f Cerebral Cortex S l i c e s 7.5 E f f e c t s o f Amphetamine a n d N i a l a m i d e on t h e Anaerobic G l y c o l y s i s o f Rat C e r e b r a l Cortex S l i c e s +  xi 7.6 7.7 8  E f f e c t o f R e s e r p i n e on the A n a e r o b i c G l y c o l y s i s of C e r e b r a l Cortex S l i c e s Summary o f Chapter 7  DISCUSSION...... 8.1 E f f e c t o f C a l c i u m Ions on C e r e b r a l Anaerobic G l y c o l y s i s 8.2 E f f e c t s o f Exogenous N u c l e o t i d e s on the Anaerobic G l y c o l y s i s 8.3 E f f e c t s o f C a t i o n s on the A n a e r o b i c G l y colysis 8.4 A n a e r o b i c G l y c o l y s i s o f Acetone Powder Extracts of B r a i n 8.5 E f f e c t s o f T e t r o d o t o x i n on A n a e r o b i c G l y c o l y s i s of B r a i n 8.6 E f f e c t s o f Ouabain on C e r e b r a l M e t a b o l i s m and T r a n s p o r t 8.7 E f f e c t s o f L o c a l A n e s t h e t i c s on the Anaerobic G l y c o l y s i s 8.8 E f f e c t s o f Other N e u r o t r o p i c Drugs on the Anaerobic G l y c o l y s i s 8.9 General Conclusions BIBLIOGRAPHY  219 222 224 224 226 228 229 230 246 250 251 253 258  xii L I S T OF FIGURES FIGURE 1  2  3  4  5  6  Page E f f e c t o f c a l c i u m on t h e a n a e r o b i c g l y c o l y s i s o f guinea p i g c e r e b r a l cortex s l i c e s  68  Effect of different concentrations of c a l c i u m on t h e a n a e r o b i c g l y c o l y s i s o f cerebral cortex s l i c e s  69  E f f e c t o f e x o g e n o u s NAD o n t h e N A D a n d NADH c o n c e n t r a t i o n s o f r a t c e r e b r a l c o r tex s l i c e s under anoxia  75  E f f e c t o f e x o g e n o u s N A D o n t h e NAD a n d NADH c o n c e n t r a t i o n s o f a e r o b i c a l l y i n cubated c e r e b r a l c o r t e x s l i c e s from r a t . .  76  E f f e c t o f NAD i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f 2 , 4 - d i n i t r o p h e n o l on the a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l cortex s l i c e s  79  E f f e c t s o f c i t r a t e a n d AMP o n t h e a n aerobic glycolysis of r a t cerebral cortex slices  80  +  +  +  +  +  7  E f f e c t s o f c y c l i c AMP a n d d i b u t y r y l c y c l i c AMP on t h e a n a e r o b i c g l y c o l y s i s o f g u i n e a pig cerebral cortex s l i c e s 82  8  Effect of varying concentrations of K , Li a n d N a on t h e a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s +  +  9  10  11  +  E f f e c t s o f glutamate and N H on t h e anaerobic g l y c o l y s i s of r a t cerebral cortex s l i c e s  84  +  4  A n a e r o b i c g l y c o l y s i s by a c e t o n e extracts of r a t brain  8  6  powder  E f f e c t of varying concentrations of rate l i m i t i n g f a c t o r s on t h e a n a e r o b i c glyc o l y s i s o f b r a i n a c e t o n e powder e x t r a c t s  90  92  xii i  12  E f f e c t s o f t e t r o d o t o x i n on the a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s . .  96  13  E f f e c t s of d i f f e r e n t concentrations of t e t r o d o t o x i n on the a n a e r o b i c g l y c o l y s i s of c e r e b r a l cortex s l i c e s  97  E f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s of c a l c i u m on the a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i n the p r e s e n c e of t e t r o d o t o x i n  99  14  15  E f f e c t of v a r y i n g glucose concentrations on the a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s i n the p r e s e n c e and absence o f t e t r o d o t o x i n  106  16  E f f e c t s of t e t r o d o t o x i n on the a n a e r o b i c g l y c o l y s i s of r a t kidney medulla s l i c e s . . .  115  17  E f f e c t s o f t e t r o d o t o x i n and o u a b a i n on the a n a e r o b i c g l y c o l y s i s o f acetone powder e x t r a c t s from r a t b r a i n  116  E f f e c t s of d i f f e r e n t concentrations of t e t r o d o t o x i n on the a n a e r o b i c g l y c o l y s i s o f newly b o r n g u i n e a p i g c e r e b r a l c o r t e x slices  119  E f f e c t s o f t e t r o d o t o x i n and o u a b a i n on the aerobic g l y c o l y s i s of r a t c e r e b r a l c o r t e x slices  121  E f f e c t o f a e r o b i c p r e - i n c u b a t i o n on the t e t r o d o t o x i n s t i m u l a t i o n of a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l c o r t e x s l i c e s . .  129  E f f e c t of a d d i t i o n of t e t r o d o t o x i n a f t e r v a r y i n g time p e r i o d s of a n o x i a on the anaerobic g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s  131  E f f e c t s o f t e t r o d o t o x i n , i n the p r e s e n c e o f p r o t o v e r a t r i n e , on the a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s . .  146  18  19  20  21  22  xiv 23  24  25  26  27  28  29  30  31  32  33  34  E f f e c t s of a d d i t i o n of high concentrations o f c a t i o n s on t h e t e t r o d o t o x i n s t i m u l a t e d anaerobic g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s  154  E f f e c t s o f t e t r o d o t o x i n on t h e s o d i u m and potassium concentrations of guinea p i g c e r e b r a l c o r t e x s l i c e s under anoxia  156  E f f e c t s o f t e t r o d o t o x i n , c a l c i u m and o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s o f r a t synaptosomes  160  E f f e c t s o f t e t r o d o t o x i n and c a l c i u m s o d i u m and p o t a s s i u m c o n c e n t r a t i o n s cerebral cortex slices  162  on t h e of r a t  E f f e c t s o f t e t r o d o t o x i n on t h e s o d i u m and p o t a s s i u m c o n c e n t r a t i o n s o f newly born guinea p i g c e r e b r a l cortex s l i c e s  163  E f f e c t s o f t e t r o d o t o x i n on t h e s o d i u m and p o t a s s i u m c o n c e n t r a t i o n s o f two day o l d rat cerebral cortex s l i c e s  164  E f f e c t s o f t e t r o d o t o x i n on t h e s o d i u m and potassium concentrations of r a t kidney medulla s l i c e s  166  E f f e c t s of d i f f e r e n t concentrations o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s cerebral cortex s l i c e s  179  of of  E f f e c t of varying calcium concentration i n t h e p r e s e n c e o f o u a b a i n on t h e a n a e r o b i c g l y c o l y s i s of c e r e b r a l cortex s l i c e s  181  E f f e c t s o f o u a b a i n and t e t r o d o t o x i n on t h e anaerobic g l y c o l y s i s of r a t cerebral cortex s l i c e s at varying concentrations of sodium  184  E f f e c t of a d d i t i o n of ouabain a f t e r v a r y i n g t i m e p e r i o d s o f a n o x i a on t h e a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s . . .  189  E f f e c t s o f o u a b a i n and t e t r o d o t o x i n t o g e t h e r on t h e a n a e r o b i c g l y c o l y s i s o f r a t cerebral cortex s l i c e s  190  XV  35  E f f e c t s o f o u a b a i n and t e t r o d o t o x i n and o u a b a i n t o g e t h e r on t h e sodium and potassium concentrations of r a t c e r e b r a l cortex s l i c e s  192  36  E f f e c t o f L i d o c a i n e on t h e a n a e r o b i c g l y c o l y s i s of cerebral cortex s l i c e s  199  37  E f f e c t s o f p y r r o l e and p y r i d i n e on t h e anaerobic g l y c o l y s i s of cerebral cortex slices  206  E f f e c t o f p y r r o l e on t h e sodium and potassium concentrations of guinea p i g c e r e b r a l c o r t e x s l i c e s under a n o x i a  210  39  E f f e c t o f a m y t a l on t h e a n a e r o b i c g l y c o l y s i s of cerebral cortex s l i c e s  212  40  E f f e c t o f c h l o r p r o m a z i n e on t h e a n a e r o b i c g l y c o l y s i s of r a t cerebral cortex s l i c e s .  217  41  E f f e c t s o f amphetamine and n i a l a m i d e on the a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l cortex s l i c e s  218  E f f e c t s o f r e s e r p i n e on t h e a n a e r o b i c g l y c o l y s i s of cerebral cortex s l i c e s  220  38  42  xvi L I S T OF TABLES TABLE 1  2  3  4  5  6  Page E f f e c t o f c a l c i u m on t h e a n a e r o b i c g l y c o l y s i s o f i n f a n t r a t and g u i n e a pig cerebral cortex s l i c e s  71  E f f e c t o f e x o g e n o u s n u c l e o t i d e s on t h e anaerobic g l y c o l y s i s o f cerebral cortex s l i c e s  73  E f f e c t s o f c a l c i u m a n d N A D on t h e a n aerobic g l y c o l y s i s o f guinea p i g cereb r a l c o r t e x s l i c e s i n a sodium f r e e medium  88  +  E f f e c t s o f t e t r o d o t o x i n o n t h e amino a c i d content o f r a t cerebral cortex s l i c e s under anoxia  103  E f f e c t s o f t e t r o d o t o x i n on t h e u p t a k e o f amino a c i d s u n d e r a n a e r o b i c conditions  104  E f f e c t s o f t e t r o d o t o x i n on t h e g l u c o s e transport i n r a t cerebral cortex s l i c e s under anoxia  10 8  7  E f f e c t s o f t e t r o d o t o x i n , i n the presence o f some amino a c i d s , o n t h e a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s . . . . 110  8  E f f e c t s o f c i t r a t e , AMP and N H i n the p r e s e n c e o f t e t r o d o t o x i n on t h e a n a e r o bic g l y c o l y s i s of r a t cerebral cortex slices I l l  9  E f f e c t s o f t e t r o d o t o x i n i n the presence o f p h o s p h o l i p a s e s on t h e a n a e r o b i c g l y c o l y s i s o f r a t c e r e b r a l c o r t e x s l i c e s 113  10  11  +  4  E f f e c t o f t e t r o d o t o x i n on t h e a n a e r o b i c glycolysis of infant r a t cerebral cortex s l i c e s  118  E f f e c t s o f tetrodotoxin i n the presence o f some amino a c i d s o r N H on t h e a n aerobic g l y c o l y s i s of cerebral cortex s l i c e s from i n f a n t animals  122  +  4  xvii  11A  E f f e c t s o f t e t r o d o t o x i n on t h e a e r o b i c g l y c o l y s i s of r a t cerebral cortex s l i c e s in the presence of cyanide  123  E f f e c t s o f t e t r o d o t o x i n i n the presence o f p y r u v a t e on t h e a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s  133  E f f e c t s o f a d d i t i o n o f g l u c o s e under v a r i o u s c o n d i t i o n s on t h e t e t r o d o t o x i n s t i m u l a t i o n of anaerobic g l y c o l y s i s of rat cerebral cortex s l i c e s  135  E f f e c t s of incubation with 2,4-dinitrop h e n o l on t h e t e t r o d o t o x i n s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s and t h e ATP c o n tents of r a t cerebral cortex s l i c e s  138  E f f e c t s o f t e t r o d o t o x i n a n d o u a b a i n on t h e ATP c o n t e n t o f g u i n e a p i g c e r e b r a l c o r t e x s l i c e s under anoxia  140  E f f e c t o f aerobic pre-incubation i n ade n o s i n e on t h e t e t r o d o t o x i n s t i m u l a t i o n of anaerobic g l y c o l y s i s o f r a t c e r e b r a l cortex s l i c e s  141  17  E f f e c t s o f t e t r o d o t o x i n on t h e cAMP f o r mation i n the c e r e b r a l cortex s l i c e s  144  18  Effect influx  12  13  14  15  16  22  19  20  o f some n e u r o t r o p i c d r u g s o n Na i n r a t cerebral cortex s l i c e s  148  E f f e c t s o f t e t r o d o t o x i n on t h e a n a e r o b i c g l y c o l y s i s a n d Na22 t r a n s p o r t i n r a t cerebral cortex s l i c e s i n a chloridef r e e medium  149  E f f e c t o f presence o f d i f f e r e n t concentration of K on t h e t e t r o d o t o x i n s t i m u l a t i o n o f anaerobic g l y c o l y s i s of r a t cerebral cortex s l i c e s  1^1  +  21  E f f e c t s of tetrodotoxin at varying cation c o n c e n t r a t i o n o f t h e medium on t h e a n aerobic g l y c o l y s i s of r a t cerebral cortex slices -*-52  xviii  22  23  24  25  26  27  28  E f f e c t s o f t e t r o d o t o x i n on t h e a n a e r o b i c g l y c o l y s i s and Na22 t r a n s p o r t i n c a u d a t e nucleus of r a t  158  E f f e c t s o f EDTA and EGTA on t h e t e t r o d o toxin stimulation of anaerobic g l y c o l y s i s of c e r e b r a l cortex s l i c e s  16 8  E f f e c t o f e t h a n o l on t h e t e t r o d o t o x i n stimulation of anaerobic g l y c o l y s i s of rat cerebral cortex s l i c e s  169  E f f e c t s o f t e t r o d o t o x i n on t h e s o d i u m a n d potassium concentrations o f r a t cerebral c o r t e x s l i c e s i n t h e p r e s e n c e o f some amino a c i d s and NH^*  171  E f f e c t s o f t e t r o d o t o x i n on t h e p y r u v a t e and phosphoenol pyruvate content o f r a t c e r e b r a l cortex s l i c e s  173  E f f e c t o f o u a b a i n on t h e a n a e r o b i c g l y c o l y sis of developing r a t cerebral cortex s l i c e s .  182  E f f e c t s of ouabain i n the presence of g l u t a mate, c i t r a t e , N H a n d AMP o n t h e a n a e r o b i c glycolysis of r a t cerebral cortex s l i c e s  186  +  4  ++ 29  E f f e c t s o f o u a b a i n , Ca a n d NAD microsomal N a , K -ATPase +  30  31  32 33  + on t h e 19  +  E f f e c t s o f o u a b a i n and o u a b a i n + t e t r o d o t o x i n t o g e t h e r on t h e amino a c i d e f f l u x f r o m r a t c e r e b r a l c o r t e x s l i c e s under a n o x i a  196  E f f e c t s o f p r o c a i n e and l i d o c a i n e on t h e anaerobic g l y c o l y s i s of r a t cerebral cortex slices E f f e c t s o f l i d o c a i n e on t h e s o d i u m and potassium contents of cerebral cortex s l i c e s .  4  ±  2  J  O  °1  E f f e c t o f p y r r o l e on t h e a n a e r o b i c g l y c o l y s i s of guinea p i g 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 o f some amino a c i d s ^8 2  34  E f f e c t s o f some n e u r o t r o p i c d r u g s on t h e sodium and p o t a s s i u m c o n t e n t s o f r a t c e r e b r a l cortex slices ^14  xix 35  36  E f f e c t s o f some n e u r o t r o p i c drugs on t h e sodium and p o t a s s i u m c o n t e n t s o f newly born g u i n e a p i g c e r e b r a l c o r t e x s l i c e s . . . .  216  E f f e c t s o f some amines on t h e a n a e r o b i c g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s . .  221  XX  Page  LIST OF SCHEMES 1  The G l y c o l y t i c Pathway  2  P r e p a r a t i o n o f Synaptosomes by S u c r o s e Gradient Centrifugation  9 56  xx i ABBREVIATIONS  AMP  Adenosine  monophosphate  ADP  Adenosine  diphosphate  ATP  Adenosine  triphosphate  cAMP, c y c l i c  AMP  3',5'-cyclic  adenosine  monophosphate Dibutyryl  cyclic  6  AMP N  2* , 0  -Dibutyryl  s' -5' - c y c l i c  DNP EDTA EGTA  adenosinephosphate  2,4-Dinitrophenol Ethylenediaminetetraacetic  acid  Ethanedioxybis(ethylamine) a-KG NAD  +  NADH  tetraacetate ct-Ketoglutarate Nicotinamide  adenine  Nicotinamide  adenine d i -  nucleotide,  dinucleotide  reduced  form  xxii ACKNOWLE DGEMENTS I am most g r a t e f u l t o my s u p e r v i s o r , P r o f e s s o r J.H.  Q u a s t e l , F.R.S., f o r h i s c a r e f u l g u i d a n c e and  encouragement t h r o u g h o u t t h e c o u r s e o f t h i s work. I would l i k e t o e x p r e s s  my s i n c e r e thanks t o  P r o f e s s o r s V . J . O'Donnell and S.C. Sung f o r c r i t i c a l reading of the manuscript. I am i n d e b t e d t o my f r i e n d , Dr. C.B.R. S a s t r y for  h i s u s e f u l advice during the preparation of the  manuscript. Thanks a r e due t o Dr. D.S. Grewaal and Mr. A.M. Benjamin f o r r e a d i n g p a r t s o f t h e t h e s i s .  Mr. Benjamin  a l s o h e l p e d me i n t h e amino a c i d a n a l y s e s . Thanks a r e extended t o t h e Department o f S o i l Science, t h e U n i v e r s i t y o f B r i t i s h Columbia, f o r perm i t t i n g me t o use t h e i r Atomic A b s o r p t i o n  Spectro-  photometer . I wish a l s o t o express  my thanks t o M i s s Joanne  A l l a n f o r help i n t y p i n g the manuscript. I w i s h t o thank t h e M e d i c a l Research C o u n c i l o f Canada f o r f i n a n c i a l a s s i s t a n c e through g r a n t s made t o Professor Quastel.  Thanks a r e a l s o due t o t h e U n i -  v e r s i t y o f B r i t i s h Columbia f o r f i n a n c i a l a s s i s t a n c e i n t h e form o f a U.B.C. Graduate F e l l o w s h i p  (1968-1971).  CHAPTER  1  INTRODUCTION  1.1 E F F E C T S  OF A N O X I A  The is  t o gain  operating not  be  major  information  developed  t o study  the animal  ural  changes.  few of  minutes  oxygen  i s made  incubation  oxygen  to  the brain  be  maintained  tissue  by  o f the animal  can  techniques have  been  o f the  brain  and  struct-  factor  influencing  during  the  during  for i n vitro  to the tissue  desired  of metabolic  i s one major  (slices)  available  o f Hypoxia  deprivation  during  have  time  the  studies,  first  preparation even  when  subsequent  Metabolism  carried  i n vivo.  o u t on t h e e f f e c t s When  cerebral  i n vivo  oxygen  function  utilization  of  s o f a r a s i s known  at present,  four  during  ATP, g l y c o g e n  b r i e f periods  Anoxia - oxygen l a c k ; hypoxia - oxygen d e f i c i e n c y ; l o c a l anemia due t o m e c h a n i c a l o b s t r u c t i o n (mainly narrowing) t o the blood supply.  s t i l l  energy  and g l u c o s e .  of hypoxia  supply  may  through  phosphocreatine,  substances  been  or cut off,  f o r a short have,  on B r a i n  on t h e b r a i n  i s reduced  that  components-  *  mechanisms  Removal  i s s p e c i a l l y important  investigations  of  these  o f oxygen*  the death  Effects  Many  in  in_ v i t r o .  metabolism  procedure.  (i)  reserves  experiments  result i n a variety  This  the cerebral  biochemical  A l l the information  i n vivo  the brain  may  after  on t h e b r a i n  and a number o f t e c h n i q u e s  Lack  changes.  brain.  by d o i n g  available  from  METABOLISM  concerning  i n the l i v i n g  obtained  BRAIN  aim of i n v e s t i g a t i o n s  presently  these  ON  have  major Changes been  ischemia arterial  -  studied  reported  2  -  as  measures  of  metabolic  As  early  in  1944,  that  as  i n the  phosphocreatine  dog,  r a t e s under  Gurdjian,  cerebral  breakdown  occurs  Stone  lactic  when  these and  acid  the  conditions.  Webster"*"  level  oxygen  rises  and  saturation  of  2 the  cerebral  workers the  venous  showed  blood  that  cerebral  phosphocreatine  Jilek,  Fischer,  biochemical  hypoxia  and  anoxia  newborn  r a t i s more  younger glycogen In  (4-12  and  a  older rats  lactic  acid  conclusion hypoxic  are that  this  ability.  the  metabolic  and  observed  conditions. acid  Thorn,  similar For  while  Samson^  extended  tides.  They  shift  during  ATP  of  these  of  20th  and  the  decreased  studies further  in  to  to of  In of  hypoxia.  the  adjust  do  anoxia under  to  anaerobic not 4  and  both  (Pi)  ischemia, these  and  anoxia  triphosphate n u c l e o t i d e s to  lactic  decreased  include other  the  possess  studied  Lolley  confirmed of  general  exhaustion  phosphate  to  This  life.  Mueldener  gradually.  to  the  of  acid  animals  under  brain  increase i n  intensity  creatine  that  l e d them  and  the the  day  i s able  taking place  of  these until  anoxia.  lactic  adult  brain  to  i n o r g a n i c phosphate  content  content  in  tissue  of  complete  Pfleiderer  the  changes  example,  i n c r e a s e d , the  rapidly  tissues  Scholl,  processes  adult  Their results  the  studied  showed  the  rise  ,  unchanged  reaction  the  study  decomposed. 3  i s almost  nervous  later  remain  and  about  increasing  the  a  Trojan  i n glycogen  less.  immature  whereas  at  three-fold  c o n d i t i o n s by  glycolysis,  than  there  decrease  much  and  development  rapidly  days)  levels  structural  resistant  two-to the  and  In  completely  Krulich  during  decreases  rats  ATP  i s almost  functional,  resistance  i s reduced.  observation that  and nucleo-  there  is  monophos-  a  phates  and  pattern than  of  that  anoxia  a  very  changes of  when  ATP. they  Studies and  Schulz  whole  6  and  are  i n GTP NAD  and  to  out  Stewart, the  changes  measure  phosphocreatine.  content  remained  some by  Passonneau rabbit  the  was  more  The  complex  unaffected  until  late  extent.  Lowry,  i n the  of  of  UTP  levels  decrease  with  ischemia  reserves  breakdown  carried  , and  brain  during  rapid  3  Passonreau, and  sciatic high  Lowry nerve  energy  metabolic  7  Hasselberger , with  showed  that  compounds  rates.  the  and  energy  Gatefield,  Lowry,  g Schulz  and  supply  to  Passonneau  , used  several different  histologically  defined  changes  high  in  the  was  depletion of  all  regions  and  metabolic  rates  that  were  they  cortex  and  energy  there  assessed about  but  Cortex  and  of  anoxia  ed  a  on  findings. chemical confirmed  changes  same  in cerebral  Anoxia  this  behaviour the  and  to  and  ATP  the  three  investigate  ischemia  there  from  nearly  lactate.  i n metabolites  depressed  on  of  been  cortex,  The indicated  cerebellar  i n the  Metabolism  carried  concerned  morphology  the  in vitro  Using  During  of  blood  brain,  cerebellum  from  been  cerebral  metabolizing  mouse  accumulation  more  blocking  Amnion's of  the  horn.  Cerebral  Slices  have  whereby  mouse  of  phosphocreatine  much  of  of  of  compounds.  an  s t u d i e s have  some  method  the  Effects  Many  was  method  regions  layers  glycogen,  medulla  (ii)  slices  the  be  method  and  observations  of  of  Cohen ^ 1  the  made  cortex a  cerebral  cortex  with  effects  chemistry.  correlated  cerebral  with  exclusively  morphology  might  out  number  develop-  cortex  slices  biochemical  detailed  slices of  Cohen  cerebral with  the 9  study  i n anoxia  workers  of  the  and  t h a t ATP  level  declines results  slowly during of  Albaum,  concentration As  bolic  requirements  a  i s the  reservoir, at  observed  that  however,  scattered  effects  ation  of  The  of  energy  be  f o r ATP  relatively  substance  d i m i n u t i o n , the  anoxia,  to  hour such  phosphate  be  the  f o r energy  a  into  result that  of  to  the  of  to  Cohen"^  also damage; s t i l l  alterations  amount  of  RNA  does  Other  meta-  i n the i n c o r p o r and  nucleic  d i m i n i s h e d ATP  hypoxia  serves  neurons  anoxia.  lipids  meta-  remain  neuronal  decrease  than  meet  morphological  period as  that  the  slowly  intact  Despite  two  to  phosphocreatine  minutes.  a  more  i n widespread  of  g e n e r a l c o n c l u s i o n was  mechanisms  of  while  should  results  radioactive  considered  claimed  phosphocreatine.  islands  during  bolic  were  40  i n contrast  decreases  source  brain  of  was  Chinn"*""*" who  tendency  expense  Nissl  decrease  i n the  anoxia  after  indicating not  the  and  immediate  the  elevated  persist  Noell  -  This  phosphocreatine  ATP.  as  ATP  of  anoxia.  4  damages  acids,  synthesis.  cerebral  production.  12 Swanson  studied  the  effects  the  electrically  stimulated guinea  and  he  that  suggested  systems  i n the  hypoxia  cerebral  of  oxygen  pig cerebral  damages  the  cortex slices.  deprivation cortex  energy  He  on  slices  utilizing  attempted  to  explain  13 the  results  persists tissue  of  after  loses  Mcllwain  that  decreased  cessation  of  electrical  i t s ability  to  respond  anaerobic  glycolysis  stimulation  aerobically,  and  both  the in  12 respiration speculated structure cation  and that,  occurs  glycolysis, with so  to  hypoxia,  that  gradients across  the their  electrical alterations  cells  can  membranes  no or  stimulation. i n the longer that  Swanson  membrane maintain  the  cell  membranes  become i n e x c i t a b l e changes t h a t the  period  deficit  stimulation  impulse.  processes  transport.  Lack  influx  and  electrical  K  studied  technique  t h a t d u r i n g the  such  efflux.  stimulation  He  the h e l p  of energy +  structural  i n d u c e d by h y p o x i a w i t h and  concluded  pathways, i s i n s u f f i c i e n t  utilization  +  of a nerve  of hypoxia, p r o d u c t i o n o f h i g h energy  non-oxidative  Na  i n t e r f e r e n c e w i t h the t r a n s i e n t  o c c u r upon p a s s a g e  functional  electrical  by  5 -  phosphates,  to m a i n t a i n the  as t h o s e i n v o l v e d  i n active  i n the b r a i n  results  This effect  accompanies  of  cell  through energy  cation  i n increased  i s made more a p p a r e n t i f  the hypoxia.  However,  12 experiments  carried  out  with reoxygenated  short period  of hypoxia  preservation  o f t h e mechanisms o f b o t h  and  energy  hypoxia  production.  (60 m i n u t e s )  (15 m i n u t e s ) ,  Experiments  suggest  slices,  suggest active  that  after  only  t h e r e i s some  cation  transport  with prolonged periods of  t h a t an  irreversible  p h o s p h o r y l a t i n g mechanisms t a k e s p l a c e .  Jennings,  damage t o  the  Kaltenbach  14 and  Sommers  ischemic  have  found  that mitochondria, isolated  c a n i n e myocardium,  oxidative  and  show a 60  phosphorylating capacity  from  percent decrease i n after  15 m i n u t e s  of  ischemia. Other cortex  changes have a l s o been o b s e r v e d  i n the  cerebral  a f t e r p e r i o d s o f a n a e r o b i o s i s . Thus M c l l w a i n , 15 Thomas and B e l l f o u n d t h a t t h e l e v e l o f cozymase i n t h e g u i n e a pig cerebral cortex s l i c e s decreases during anaerobiosis while it  slices  remains  constant during aerobic period  after  an i n i t i a l  drop.  16 Thomas incubated uptake  concluded  that creatine  phosphate  cerebral  cortex s l i c e s  d u r i n g a n a e r o b i o s i s and  of K , +  glutamate,  a s c o r b a t e and  i s lost  creatine  from  the that  i s diminished.  -  Quastel brain  -  13 7 h i s coworkers have shown t h a t a c c u m u l a t i o n  and  cell,  ascorbic  6  a g a i n s t a c o n c e n t r a t i o n g r a d i e n t , of  acid  and  amino a c i d s i s c o m p l e t e l y  \ i n the  thiamine,  suppressed  under  anaerobic conditions. 1.2  GLUCOSE METABOLISM IN As  noted  earlier,  endogenous g l y c o g e n process,  the  anaerobic  first  study.  lactate  glycolysis,  I t i s proposed,  i s the  obtained  glucose  through  from  by  formation  o p e r a t i o n of the  oxygen, through process  produces  and  during anoxia.  the e f f e c t s  of  of  the  t h e r e f o r e , to d i s c u s s i n and  This  thereon  topic  the  i t s regulation.  the  citric  CoA  acid  formation  out  and  of l a c t i c the  conditions i s lactic  affecting  carbohydrate  acid.  latter  product  acid.  metabolism  be  sequence  The  in brain  substance  absence  of  Whereas t h e produces tissues  of g l y c o l y s i s ,  the end  anaerobic  i n the  Most animal  the p r o c e s s  can  o x i d a t i o n of t h i s  cycle,or,  consumed.  In b r a i n ,  ATP  w e l l known g l y c o l y t i c  3 8 m o l e s o f ATP,  of carrying  varying extents.  s y n t h e s i s o f ATP.  of acetyl  p e r mole o f g l u c o s e  capable  glucose  of the major consequences of g l u c o s e metabolism, i n tissue,  either  from  c o n d i t i o n s i s the major  t h e mammalian  ATP  and  p l a c e , mechanisms o f g l y c o l y s i s One  by  formation  increases several fold  various environmental present  BRAIN  2 moles  though  and  of  are  of g l y c o l y s i s  process  former  to under  conditions  have r e c e n t l y  been  17-20 reviewed  .  In t h i s  s e c t i o n we  will.be  the n o n - o x i d a t i v e metabolism of glucose Brain ration  i s c h a r a c t e r i z e d by  of g l y c o l y t i c  more t h a n  90  percent  enzymes and of the  total  in  mostly  of h i g h  been e s t i m a t e d  glucose  with  brain.  the presence i t has  concerned  consumed i n  concentthat brain  proceeds  through  the  7 -  glycolytic  pathway  21  .  The  reverse  formation 17  of  glucose  from  The  pyruvate  i s negligible  e n z y m i c makeup o f b r a i n  i n brain  (see B a l a z s  for glycolytic  ).  enzymes 17  differs  quantitatively  compared t h e those  relevant data  present  kinase  from  in liver.  activity  that of other on  tissues.  t h e enzyme a c t i v i t i e s  Thus t h e  available  i s about twenty times  data  higher  Balazs  has  i n brain  with  show t h a t h e x o -  in brain  than  in  22 liver  .  Activities  phosphoglycerate brain  than  glycolysis that  the  brain  kinase  in liver. and  of a l l other kinases and  pyruvate  These enzymic  kinase)  compared  from  t o t h a t i n the  glucose  from  pentose  phosphate c y c l e  are  steps are  have been d i s c u s s e d l a t e r .  synthesis of glucose  as  (phosphofructokinase,  liver  control points i n  I t has  pyruvate  also higher i n  been  mentioned  i s negligible  or kidney.  in  Formation  of  r e q u i r e s r e v e r s a l of c e r t a i n r e a c t i o n s of 23 24 t h e g l y c o l y t i c pathway ' . The a c t i v i t i e s o f enzymes 17 c a t a l y z i n g t h e s e r e a c t i o n s seem t o be v e r y low i n b r a i n so are t h o s e o f enzymes n e e d e d f o r t h e s y n t h e s i s o f g l y c o g e n , and o f 17  t o the  pyruvate  A number o f i n v e s t i g a t i o n s  c o n c l u s i o n t h a t pentose  minor r o l e It glycolytic  i n the  central  s h o u l d be process  i n the  p o i n t e d out  rations  by  nervous  brain,  system  (CNS)  the  localization  Passonneau  organ  greatly  rations.  An  glycolytic  and  therefore, of  changes i n the b r a i n  must w a i t  from the  of  various  , true l o c a l  are q u i t e d i f f e r e n t  understanding,  of  i s most  of i n t e r m e d i a t e s i n v a r i o u s p a r t s of the  edly vary  a  o f a d u l t mammals.  t h a t f o r an u n d e r s t a n d i n g  a heterogeneous 25  L o w r y and  have l e d  p h o s p h a t e pathway p l a y s o n l y  pointed out  enzymes i n v o l v e d i n s u c h As  .  important.  concent-  brain  undoubt-  average  concent-  finer  details  of  histochemical studies  - 8 of  individual cells (i)  part  Properties of  the  (a)  Hexokinase;  for  its activity  including  and  of  of  cells.  Some E n z y m e s  involved  in  Glycolysis  Brain  Brain and  glucose,  hexokinase  (HK)  requires  i t phosphorylates  fructose  and  ATP  several  mannose  as  and  sugar  well  as  Mg  + +  substrates  D-glucosamine  26 and  2-deoxyglucose  .  It  is  .  G-6-P  inhibited  by  glucose  6-phosphate  acts  non-competitive  27-29 (G-6-P) and HK  is  and  ADP  thought  activity  partially  to  by  play  a  regulatory  competing  r e l i e v e d by  as  f o r ATP  phosphate  a  role.  site.  G-6-P  G-6-P  ions.  Km  for  and  inhibitor  ADP  inhibit  inhibition  may  be  glucose  is  in  the  Km  have  18 range been and  0.04-0.1 mM  of  reported  difference plasmic (b)  were  HK  However,  sheep  34  Bachelard  cytoplasmic  as  from  .  into  between  brain  able two  the  enzymes and  separate  activities  with  higher  human b r a i n both  f r a c t i o n s but of  .  Thompson  mitochondrial  could the  33  not  HK  and  detect  any  major  mitochondrial  and  cyto-  enzymes.  Phosphofructokinase; well  as  studied. group  to  32  from This  of  ATP  a  variety  enzyme  to  Phosphofructokinase of  t i s s u e s , has  catalyzes  fructose  the  been  transfer  6-phosphate  (PFK)  (F-6-P)  from  brain,  extensively  of  the  terminal  i n the  presence  of  ++ Mg  .  other  PFK  acts  transfer  account  for  brain.  Whilst  specifically reaction.  i t s rate ATP  on  It  shows  limiting  and  F-6-P  citrate  and  kinetic  e f f e c t s on are  does  catalyze  properties  the  strong  not  which  any may  glycolysis in  inhibitors  of  PFK,  + the as  enzyme well  Lowry  as  and  is  stimulated  cyclic  AMP  Passonneau  35  by  (see have  F - 6 - P , ADP, Lowry  and  proposed  AMP,  NH^,  Passonneau a  model  for  phosphate  35  and  brain  *  Balazs PFK  ions  17  that  ).  -  Scheme  1  : THE  9  GLYCOLYTIC  PATHWAY  Activators  Inhibitors GLUCOSE + GLUC0SE-6-PH0SPHATE  i  1  FRUCTOSE-6-PHOSPHATE cAMP,  AMP,  ADP,  P i  + .  ATP,  Citrate  FRUCTOSE-l-6-DIPHOSPHATE GLYCERALDEHYDE-3PHOSPHATE NAD^" ^  ^  ^  DIHYDROXYACETONE PHOSPHATE Iodoacetate  * * 1,3-DIPHOSPHOGLYCERIC  ACID  J f 3-PH0SPH0GLYCERIC 2-3-Diphosphoglycerate  J  ACID  ^  2-PHOSPHOGLYCERIC  ACID  J  F7  PHOSPHOENOL P Y R U V I C K ,  Fructose-1-6-  +  diphosphate NAD  +  I t PYRUVIC  ACID Ca  + +  ,  Na  ACID  + LACTIC  PPi, Ca  Oxamate ACID  +  +  +  may  account  f o r a number o f k i n e t i c  They suggest  that  activators.  binding  properties in  Their  lead  may  of glycolysis  o f phosphoenol  pyruvate  as compared  +  with  to greater a f f i n i t y  inhibitors  , NH^  +  that  of other  (see l a t e r  kinase  i n this  ( P K ) , w h i c h i s one o f t h e  (PEP) t o p y r u v a t e .  and Rb  ++  role  section).  enzymes, c a t a l y z e s  of t h i s  these  t o p l a y an i m p o r t a n t  to other tissues,  properties  s t i m u l a t e d by K  and p o s s i b l y  also act synergistically:  of the g l y c o l y t i c  the i n t e r e s t i n g is  may  Kinase; Pyruvate  most i m p o r t a n t  seven,  f o r combination  o f t h e enzyme a r e t h o u g h t  the r e g u l a t i o n  the b r a i n ,  at least  o f t h e enzyme.  a r r a n g e m e n t on t h e enzyme a r e s u c h  the a c t i v a t o r s  (c) P y r u v a t e  ion  separate s i t e s  o f one i n h i b i t o r  inhibitors;  properties  t h e enzyme p o s s e s s  as many a s t w e l v e , or  10 -  the convers-  Its activity in  i s very high.  enzyme i s t h a t (see A x e l r o d  36  One o f  i t s activity ) and i s  + 37-39 inhibited.by (d) L a c t i c  Na  Dehydrogenase: L a c t i c  dehydrogenase  (LDH) i s p r e s e n t  17 in  the b r a i n  at a very high a c t i v i t y  pyruvate accumulation in  vivo  : there i s very  i n the i s o l a t e d  little  incubated brain  cortex or  e v e n d u r i n g c o n v u l s i o n s , w h i c h shows t h e g r e a t  capacity  40 of t h i s in  enzyme  .  several different  mammalian b r a i n , while  type V  inhibits  type  (muscle  (H a n d M t y p e )  is  LDH  H type  from b r a i n  molecular I  (heart  t y p e , M)  i s a t e t r a m e r and i s p r e s e n t 17  forms  (isozymes)  t y p e , H)  In a d u l t  i s the dominant  and h y b r i d s o f d i f f e r e n t  are present to a lesser a t lower  .  extent.  Excess 17  c o n c e n t r a t i o n s than M type  a p r o g r e s s i v e change i n t h e LDH i s o z y m e  .  form subunits pyruvate There  pattern during  maturation which c o r r e l a t e s w i t h the r e s i s t a n c e . 41 anoxia  brain  o f the animal t o  (ii) The almost by  Localization glycolytic  entirely  high  speed  of  11  Glycolytic  enzymes,  present  in  -  the  centrifugation  with  Enzymes  the  exception  supernatant  of  in  the of  HK,  fractions  b r a i n homogenates  Brain are  obtained  (see  Quastel  19 ). of  Several  groups  glycolytic  of  enzymes  workers  in  the  have  s t u d i e d the  sub-cellular  distribution  preparations  of  brain  42-45 Cerebral bound  to  HK  occurs  partly  mitochondria"^'^.  in  the  However,  cytoplasm  sufficient  and  partly  cytoplasmic  34 HK  i s available  Particulate  HK  to  account  activity  f o r normal  rates  i s exclusively  of  glycolysis  mitochondrial  and  43-44 from  30  to  over  75  Earlier activity  of  percent  workers  of  have  the  total  shown  r a t b r a i n homogenates  activity  that  some  varies  46 '  glycolytic  i s associated with  the  mito-  43 chondrial fraction . Such i n i t i a l o b s e r v a t i o n s are r e l a t e d to the presence of nerve ending p a r t i c l e s , which contains glycolytic enzymes, have  in this  shown  obtain  a  fraction  that, with  fraction  17  .  Abood,  hypertonic  rich  Brunngraber  sucrose,  i n mitochondria  and  Taylor  i t is possible  which  i s almost  49  to  devoid  of  45 glycolytic presence  activity.  of  glycolysis  conventionally of  this  from  fraction Studies  enzymes cerebral  and  igations  show  with have  that of  the  other been areas  cerebellar  parts  (iii)  in  and  Abood  cell  done of  contents  the  brain.  due  fraction,  largely  the  prepared  to  contamination  components. the  brain  cortex,  of  be  on  the  Controls  established that  mitochondrial  r a t b r a i n , may  in different  different  Tanaka  and of  distribution  and  in  Ammons'  the  Carbohydrate  glycolytic  various  horn.  glycolytic  of  These  enzymes  Metabolism  layers  in  invest-  vary  the  of  in  Brain  The  rate  of glucose u t i l i z a t i o n  i n cerebral  tissue i n  50 vitro  i s much l e s s t h a n  anoxia,  the rate  of glucose  maximum p o s s i b l e evident  that  individual processes use  cerebral (1)  the various  i n cerebral cortex  tissues  regulation  through ionic (a)  i n vivo  .  utilization  Even i n v i t r o ,  rates  From t h i s i t i s  must be s u b j e c t  mechanisms.  t o c o n t r o l by  Most o f t h e r e g u l a t o r y  g l y c o l y s i s have b e e n i n v e s t i g a t e d  slices  i n vitro.  G l y c o l y t i c rates  of the concentrations  of metabolic  c o n t r o l o f enzyme a c t i v i t i e s  can account  l a c t a t e Kg  intermediates  and (2) r e g u l a t i o n  o f oxygen consumption  f o r the oxidation  of the  min  Since,  lactate  production  actual  rate  ena  of i n h i b i t i o n  the  Pasteur e f f e c t . The  i s only  10-20 p e r c e n t  rate  respiratory  1 mmole Kg  1 mmole  that -1 min  of t h i s value.  aerobic .  p r o c e s s o f r e s p i r a t i o n a l o n e i s n o t , however, I t i s possible  of lactate production rate  while  at a constant value.  there  i s found  t o be a d e c r e a s e  while  t h e c o n t e n t s o f ADP, AMP  the concentrations  However,  T h e phenom-  o f g l y c o l y s i s by r e s p i r a t i o n i s u s u a l l y  f o r the Pasteur e f f e c t .  aerobic  0.4 mmole  under a n o x i a , a p p r o x i m a t e l y  w o u l d be l e s s t h a n  the  i n brain  of approximately  l a c t a t e Kg "*"min ^ i s f o r m e d , i t w o u l d be e x p e c t e d -1  rate  in.the  contents.  slices  in  with the  a r e c o n t r o l l e d by two d i f f e r e n t m e c h a n i s m s :  P a s t e u r E f f e c t : The r a t e  ible  under  i s much l e s s t h a n t h e  by t h e enzymes i n v o l v e d .  regulatory  of brain  that  called  respons-  t o increase the  still  maintaining  Under t h e s e  the  conditions  i n t h e ATP c o n t e n t o f t h e t i s s u e and P i i n c r e a s e .  are considered  o f g l y c o l y s i s by s t i m u l a t i o n  to bring  -These  a b o u t an  changes increased  o f some o f t h e g l y c o l y t i c  enzymes  (see  inhibition  later).  of  PFK  concentrations  Racker  by  of  ATP  ADP  has,  together  and  Pi  may  in  fact,  with  the  account  concluded  rate  for  that  limiting  the  Pasteur  effect. 6  (b)  Regulation  have  studied  substrates vivo.  of  AMP  and  PFK.  the  of  ischemia  cofactors  of  the  of  aerobic  the  step.  that  controlling  in  guinea  changes  ing  "non-equilibrium" caused  concluded  3-phosphate control  in  that  and  known  HK  for  by  that  HK,  PFK,  cerebral  PFK  a  time  large  increase  51 Newsholme  form  under  largely  in  the  anoxia  from  the  supporting  studied cortex  with of  the  the  and  the  led  accel-  increase  ATP  in  inhibition  evidence  They  for  changes  of  of  incubation.  glyceraldehyde  subject  regulatory  catalyz-  i n rates  conditions  They  of  correlated  f o r enzymes  possibly are  control  slices.  substrates  slices. a  in brain  to  metabolic  further  system.  suggested I t has  been  that  inhibition is relieved of adenylate kinase, a cause  cortex  together  long  '  (G-3-PDH)  dehydrogenase the  PK  known  intermediates  found  overcoming  the  PFK. 5152  of  alteration  of  coworkers ' of  pathway  attributed to  cerebral  reactions  his  amount  levels  provided  and  concentrations  the  those  of  Newsholme pig  the  glycolysis  in  HK  and  resulted  was  capable  of  and  changes  also  on  state with  This  are  roles  Rolleston  They  the  Lowry  glycolytic  steady  conditions  that  PFK  Pi  the  K i n e t i c evidence  glycolysis  Enzymes:  effects  conclusion  eration  of  and  under  the  ADP,  the  Glycolytic  Comparison  found to  of  25  in  ATP i n h i b i t s PFK and t h a t t h i s 35 53-56 b y AMP ' . Because of the presence s m a l l c h a n g e i n t h e ATP concentration may the  concentration  of  AMP.  Rolleston  and  52 '  concentration  pointed of  ATP  out  will  that tend  any to  condition  increase  the  decreasing activity  the of  PFK  -  while of  the changes  ATP.  The r e s u l t a n t  decrease of  HK.  PFK  i n AMP  concentration  increased  the concentration Thus,  according  are linked The  will  activity  i n a  controlling role  o f PFK  the  i s itself  workers,  regulatory  amplify  o f PFK w i l l  o f F-6-P which  to these  together  -  14  effect  tend  an  to  inhibitor  the a c t i v i t y  o f HK  and  system.  has been  established  i n a  35  wide a  variety  wide  PFK  by  acid  of tissues  .  I t has been  variety  o f compounds a f f e c t  citrate  may  cycle  anoxia,  on  result  the rate  or during  i n increased The  rate  concentration formation of  PFK  activity,  added  .  citric  formed  be e x p e c t e d  during to  activity  of phosphate  phosphate  may  Inhibition of of the  T h e NH^  that  35  o f g l y c o l y s i s may  i n cerebral  affect  of glycolysis"^.  electrical  mentioned  activity.  i n a modifying  .  result  PFK  already  also  be  affected  i n t h e medium"*"^ '  extracts  i s dependent  and i s p r o p o r t i o n a l  by t h e  ' ^® . upon  Lactate  the presence  t o the phosphate  concent-  50 ration  up  G-3-PDH  t o 1 5 - 2 0 mM  sensitive HK  by  maximal about  known  to inhibition  '  enzymatic  required  or  has been  G-6-P  phosphate the  Further,  and i t i s an a c t i v a t o r It  of  .  a  .  f o r a long  b y ADP  G-6-P,  activity  because rate  o f PFK  27  29  '  28  i s non-competitive '  possible,  by  as w e l l time  i s a as  that  itself,  as c a l c u l a t e d  5-6% o f the maximal  rate  3 (  28  3  HK i s  .  Inhibition  relieved to  from  from  i t  i s  1-3% o f t h e  the t o t a l  possible  by  suppress  to the extent  of g l y c o l y s i s i s only  for  HK ^' ^.  i s unlikely  cell  substrate  brain  a n d b y G-6-P a n d c a n be  i n the brain  the rate  phosphate  HK  activity,  t h e a m o u n t o f HK  18  found in  i n the cytoplasm  vivo,  produce  about  .  G-6-P,  50-70%  at the concentrations  inhibition  o f HK  under  present  optimal  28 conditions of  30 31a. '  inhibition  the  total  to account  '  .  o f HK b y t h e s e  f o r the observed  substances  cerebral  clear  that  i s not s u f f i c i e n t  glycolytic  rates.  The  o f t h e enzymes a n d i n h i b i t o r s i n t h e b r a i n  j u s t as important  the  From t h e e x p e r i m e n t a l d a t a on t h e d e g r e e s  o f HK by G-6-P a n d ADP, i t i s q u i t e  inhibition  localization be  15 -  as t h e i r  quantities  cell  f o r an e x p l a n a t i o n  may of  regulation of cerebral glycolysis. It  is  greatly  Na :  K  +  +  has already  been p o i n t e d  out that  the a c t i v i t y  a f f e c t e d by t h e p r e s e n c e o f c a t i o n s , n o t a b l y  being  stimulatory  and N a  +  being  inhibitory.  Ca  K  o f PK +  and i s also  + +  known t o i n h i b i t t h e a c t i v i t y o f PK, b o t h i n t h e b r a i n a n d o t h e r mammalian t i s s u e s ^ / 5 9 ^ B y g r a v e ^ observed that the i n h i b i t i o n 8  of  g l y c o l y s i s by a d d e d C a  is  l a r g e l y accounted  competition  with Mg  i n extracts  + +  of Ehrlich ascites  f o r by t h e i n h i b i t i o n  + +  and K . +  Considering  o f PK a c t i v i t y  cells due t o  the e f f e c t s of cations 59  on  PK a c t i v i t y  i n cerebral  t i s s u e s , Takagaki  concluded  that  + in  the presence o f K  tissue, of K the is  +  Ca be  i n this  a c t i v e and a s m a l l  o f Ca  similar to that changes i n C a  found + +  (1.3mM)  inhibits  Howsver,  PK i n v i t r o ,  i n t h e i n t a c t t i s s u e and, t h e r e f o r e ,  Takagaki  i n the a e r o b i c a l l y incubated  responsible  found i n t h e cerebral  change i n t h e c o n c e n t r a t i o n  , which  concentration 59  t h e enzyme a c t i v i t y . + +  normally  r a n g e , may n o t c h a n g e t h e enzyme a c t i v i t y . ++ 61  concentration  small in  PK i s f u l l y  concentration  may b r i n g  about l a r g e  has f u r t h e r c o n c l u d e d cerebral  changes that  t i s s u e may, i n p a r t ,  f o r t h e p r e s e n c e o f PK i n a h i g h l y  i n h i b i t e d state,  in  addition to the c e l l u l a r organization of the s l i c e s , G l y c o l y s i s i n Cerebral Cortex S l i c e s : (c) E f f e c t s o f C a t i o n C o n t e n t s a n d o f E l e c t r i c a l S t i m u l a t i o n  on  Quastel glycolysis cations  19  16 -  has reviewed  the e f f e c t s  i n b r a i n and has p o i n t e d  t r a n s p o r t changes a t t h e c e l l  ion  concentration  glycolytic  i n the c e l l ,  process  It  ionic  o f two f o l d  membrane  and d i r e c t  causing effects  action -  changed on t h e  itself.  h a s b e e n known f o r a l o n g  metabolism o f i s o l a t e d the  i o n s on  out that the e f f e c t s of  must b e i n t e r p r e t e d i n t h e l i g h t  ion  of various  time t h a t  brain tissue i n vitro  environment.  carbohydrate  i s d e p e n d e n t on  A s e a r l y as i n 1935, A s h f o r d  62  and Dixon  + found  t h a t t h e p r e s e n c e o f lOOmM K  oxygen consumption cortex  slices,  and o f a e r o b i c  respiring  increase  glycolysis  i n a Ringer  both  the rate of  of rabbit brain  medium a t 3 7 ° .  Dickens  63 and  Greville  respiration  further studied and a e r o b i c  the e f f e c t  glycolysis  o f n e u t r a l s a l t s on  of r a t brain cortex  slices.  63 They  confirmed  addition the  found  +  that omitting  rate of respiration.  respiration K  the observations  explain  of C a  as t h e o n l y  i s increased  i s suppressed.  clearly  +  the e f f e c t  and Dixon  f r o m t h e medium  The e f f e c t  With N a  glycolysis  glycolysis  + +  c o u l d b e o v e r c o m e by u s i n g  (up t o lOOmM).  aerobic  Ca  of Ashford  concentrations of  c a t i o n , the rate o f  but that of  Dickens  increases  i n lowering the  + +  high  and i n  anaerobic  and G r e v i l l e  of neutral salts  were u n a b l e t o  on a e r o b i c  glycolysis. 64 With c e l l - f r e e b r a i n p r e p a r a t i o n s , Racker and Krimsky o b s e r v e d t h a t N a i s a s t r o n g i n h i b i t o r o f r e s p i r a t i o n and 37 65 g l y c o l y s i s (see U t t e r ) • T a k a g a k i and T s u k a d a studied the +  effects  o f lOOmM K  a Na -free +  there  +  medium.  i s no i n c r e a s e  formation  or glucose  on c e r e b r a l c o r t e x They o b s e r v e d  slices  metabolizing i n  that i n response  i n t h e r a t e o f oxygen u p t a k e , utilization,  although  t o lOOmM lactic  i n the usual  K , +  acid  medium  s u c h an i n c r e a s e When N a  in K  i s omitted  +  +  increased  lactic  utilization  increase  an  i n the glucose  formation,  while  Na  i s replaced  +  Li  medium^.  +  glycolysis  the metabolism  a c i d formation  significantly;  considerably.  glycolysis  In contrast  glucose of K  and t h e l a c t i c  causes  +  acid  i s n o t changed.  i n brain slices  by c h o l i n e o r K  and  the omission  utilization  the r e s p i r a t i o n  Anaerobic  -  f r o m t h e medium, t h e o x y g e n u p t a k e i s n o t  changed b u t t h e a e r o b i c  increase  iv  i s increased i f  but i s s l i g h t l y  +  to aerobic  depressed i n a  glycolysis,  i s d e p r e s s e d by t h e a d d i t i o n o f h i g h  K  anaerobic to a  +  Ringer  ,.62 medium The  effect  of K ,  under a e r o b i c  +  metabolism o f c e r e b r a l cortex fact  that K  partly  +  stimulates  phosphate  K  *4~  the i n t e r a c t i o n  19  .  an i n c r e a s e The e f f e c t  of a diminished  bolizing high  may be p a r t l y  6  glucose  level  or pyruvate  concentration  , the content  cell  K  +  found  Brain  be t h e  slices  i n a normal balanced  of phosphocreatine.  69  o f ADP and  also partly  o f ATP.  of phosphocreatine  Magee and R o s s i t e r  may  and  o f membrane  i n the concentration  of high  due t o t h e  o f PEP and A D P ^  due t o i t s s t i m u l a t i o n o f t h e a c t i v i t y  ATPase, c a u s i n g  result  slices  c o n d i t i o n s , on t h e  media  metamaintain  In the presence of high  i s diminished  that high K  +  inhibits  68  .  Findlay,  the m c o r p o r a -  32 tion  of P i  cortex  i n the various  phosphate  fraction  of the b r a i n  slices. 70 In  of  anaerobic  stimulated Mg  1937, Q u a s t e l  ++  glycolysis  and Wheatley  observed  of brain cortex  by t h e a d d i t i o n o f C a  , t o a medium i n w h i c h Na  +  + +  ,  and K  slices  that the r a t e  i s markedly  and t o a l e s s e r e x t e n t  +  are the only  by  cations.  Adams and on  the  Quastel  anaerobic  71  glycolysis  r . T. o f tumour s l i c e s . and  Wheatley  the  r a t e of b r a i n  Ca is  from  after due  and  m  , 71 They  study  of guinea  confirmed  f u r t h e r observed glycolysis  o v e r c o m e by  i s o b t a i n e d by  to pyruvate  t h a t the  t o 7.0.  brain  treatment.  and  effect  on  of  suspended,  The  slices  T h i s was  d u r i n g the  are  ++  Ca  Quastel  absence  slices  of  cortex  inhibitory  e s t a b l i s h e d i n the  s u b j e c t i n g the  accumulation  effects  the o b s e r v a t i o n s of  d e c r e a s i n g t h e pH  a period of anerobic  of the  pig cerebral  t h e medium, i n w h i c h t h e b r a i n  largely  effect  made a d e t a i l e d  same  to  oxygenation  thought  the p e r i o d o f  to  be  oxygenation  71 - pyruvate glycolysis. the  being  a w e l l known a c c e l e r a t o r o f c e r e b r a l 19 Quastel suggested t h a t i t i s q u i t e p o s s i b l e t h a t  diminished r a t e of c e r e b r a l  from  the  brain  a b s e n c e o f Ca  cells  with  ++  i s due  corresponding  anaerobic t o an  accelerate anaerobic  medium"^"*".  Their efficiency  glycolysis  can  be,  i n f l o w o f Na  +  resulting into  suppression of g l y c o l y t i c  A number o f o r g a n i c b a s e s aniline  glycolysis  such  brain  as p y r r o l e ,  glycolysis  in replacing  Ca  + +  i n general, correlated with  the rate.  pyridine  in a  Ca  + +  -free  for brain their  and  anaerobic  dissociation  4 - 4 - 7 1  constants Studying slices,  Takagaki  aerobic glycolysis 59  observed  t h a t Ca  many g l y c o l y t i c  enzymes i n c l u d i n g  n a m e l y HK,  G-3-PDH and  that  Ca  ascites  + +  PFK, has  inhibitory  tumour c e l l  glycolysis  of i n t a c t  Application  PK.  effect  extracts^ tumour  i n the ++  cerebral  inhibits  the  four c o n t r o l l i n g I t has  on  the  while  cortex activities enzymes,  a l r e a d y been glycolysis  they  have no  of  mentioned Ehrlich  effect  on  c e l l s ^ ' ^ .  of e l e c t r i c a l  impulses  to the  of  nerve  or  the  cerebral  tissue  leads  across  the c e l l  to  f o l d increase  2-3  to well  membrane.  ation  activity  i n the Na  o f ATP.  increase of  There  i n ADP.  the tissue  sis  o f Na  known movements o f  Even  + greater  19 -  a few minutes content.  +  , K  ATPase  i n the c e l l  respiration  On  This  leads  results i n  and hence g r e a t e r  As a r e s u l t , d u r i n g  i s increased.  excitation  +  i s a fall  slices,  cations  the other  l e v e l o f ATP  electrical  as w e l l  hand  utiliz-  stimulation  as a e r o b i c  anaerobic  and  glycoly-  glycolysis i s  50  suppressed  by e l e c t r i c a l  1.3 E F F E C T S OF L O C A L Much o f t h i s tetrodotoxin anesthetic the  ANESTHETICS thesis  (TTX) on b r a i n 12 8  effects  effects  .  metabolism.  In this  As t h e p r i m a r y  the  blockade  the  transmission  of nerve  section  impulse  Electrochemical  TTX h a s p o t e n t  t h e mode o f a c t i o n  conduction,  and  system w i l l  a short  impulse w i l l  be  i s  account of  follow.  Effects  of  section.  o f the Nerve aspects  of  local  of the l o c a l anesthetic  i n a separate  Transmission  the effects  on t h e n e r v o u s  action  o f the nerve  be d i s c u s s e d (i)  i s concerned with  of l o c a l anesthetics  discussed.  TTX w i l l  stimulation.  Impulse:  of excitation  has been  extensive-  72-74 ly  reviewed  .  is  a steady  difference  part  of the c e l l  to  the transitory  of  a nerve  difference the  as w e l l  of potential  (the resting  impulse  changes  as i n other  between  potential).  potential).  For the c a t motoneurons,  of Na  +  i s i n  during The  t h e n e u r o n a l membrane  i n the concentration  cells,  the inner This  i n this potential  (the action  s e t up a c r o s s  difference  membrane.  In excitable  there  and  outer  contrast  propagation  potential i s the result of  and K  +  across the  the concentration  of  K  +  inside the  the neurons  20 -  i s 27 t i m e s h i g h e r t h a n t h a t o u t s i d e w h i l e  concentrations of Na  +  i s 10 t i m e s h i g h e r o u t s i d e t h a n  75 that  inside The  to  K  the  +  than  nerve  resting  membrane  i s at least  50 t i m e s more  permeable  to Na .  D u r i n g t h e g e n e r a t i o n o f an a c t i o n p o t e n t i a l , + + becomes more p e r m e a b l e t o Na . As a r e s u l t Na +  flushes  inside  the c e l l  outside  the c e l l .  and a l m o s t  Hodgkin  76  a t t h e same m a g n i t u d e K  has c a l c u l a t e d  movements a s s o c i a t e d w i t h t h e e l e c t r i c a l  t h a t Na  activity  and K  +  moves  +  o f t h e unmyel-12  inated The of  nerve  changes  fibers  a r e i n t h e range  o f 3.5-4.5 x 10  2 M p e r cm .  i n t h e membrane p e r m e a b i l i t y d u r i n g t h e g e n e r a t i o n  an a c t i o n p o t e n t i a l  as r e s u l t i n g from 77 a change i n t h e p o r e s i z e o f c e r t a i n c h a n n e l s . U s i n g TTX a s a t o o l , i t h a s b e e n c a l c u l a t e d t h a t t h e r e a r e a b o u t 13, o r + 2 78 7 9 f e w e r , Na c h a n n e l s p e r u o f t h e membrane s u r f a c e ' . It is still  Na  +  uncertain  w h e t h e r Na  + and K c h a n n e l s (ii) It  have b e e n v i s u a l i z e d  +  and K  +  c a n be d i f f e r e n t i a l l y  Calcium ions  blocked  75  and E x c i t a t i o n :  h a s b e e n known f o r a l o n g t i m e  that nerves  s p o n t a n e o u s l y when t h e c o n c e n t r a t i o n o f C a has  75 t h e same c h a n n e l s .  traverse  l e d to the suggestion that  + +  the increase  fire  i s reduced i n the Na  +  and t h i s perm-  ++ eability or  carriers  cross in  o c c u r s because  d e p o l a r i z a t i o n removes Ca from s i t e s 80 81 + i n t h e membrane ' . One c a n v i s u a l i z e t h a t Na  t h e membrane  the presence  through  special  channels which  o f Ca  On t h e o t h e r h a n d , i f a n e r v e containing threshold  are blocked  h i g h e r than normal i s raised  Ca  + +  i s bathed  in a  c o n c e n t r a t i o n , the  a n d t h e n e r v e may e v e n  solution firing  75 become i n e x c i t a b l e .  It  has been  proposed  transmembrane heads from  that  channels  of the external this  bond  would  Ca  +  21 may  +  to Na by  be thought  loose  +  phospholipids then  permit  o f as  binding  layer;  blocking  t o the polar  dislocation  rotational  of  Ca  +  +  movement o f t h e  + 82 83 polar  head  and f r e e  (iii) As  passage  The Sodium  has been  o f an i m p u l s e  efflux  +  nerve ment the  from  '  Pump;  discussed  generation of K  o f Na  earlier  results  the nerve  i n this  i n the i n f l u x  cell.  After  of Na  the and  +  the conduction  of the  impulse, t h e r e s t i n g p o t e n t i a l i s r e - e s t a b l i s h e d b y move+ + o f Na t o o u t s i d e the nerve c e l l , and o f K to inside of  cell.  which  This  utilizes  i s achieved ATP  by t h e so c a l l e d  to eject Na  and r e t u r n  +  the sodium  pump  i s operated  b y Na  "Sodium  K .  Pump"  I t i s believed  +  + that  chapter,  + ,  K  -activated  .  This  ATPase  84 which  i s present  later  i n detail. (iv)  Site  There  i n the c e l l  a n d Mode  i s very  membrane  of Action  little  doubt  of Local  that  site  will  be  discussed  Anesthetics: of action  of  local  85 anesthetics nerve  i s a t the nerve  membrane  action  i s bathed  membrane  with  p o t e n t i a l becomes  .  a local  smaller  Thus  when  anesthetic  and smaller  and  an  isolated  solution, the eventually  75 disappears. Further,  I t reappears  the external  sheat/and  Schwann  The  anesthetics  local  only  when  supporting  cell,  the drug  structure  i s not required  may  i n fact  block  i s washed o u t  of the fiber, i t s  f o r the blocking the impulse  effect.  conduction  86 quicker  i n unstripped  fibers,  the myelinated  only  a t the nodes  nerves fibers  of Ranvier;  .  In contrast  permit this  to  non-myelinated  access  to local  anesthetics  exposure  i s s t i l l  sufficient  -  22 -  87 88 to block  impulse It  block to  conduction  i sgenerally  the conduction  the generation  transient  rise  '  believed  that  by i n t e r f e r i n g  the l o c a l  with  anesthetics  theprocess  o f an a c t i o n p o t e n t i a l , n a m e l y  i n thepermeability  the large •  o f t h e membranes 89  w h i c h a r i s e s on d e p o l a r i z a t i o n o f t h e membrane  fundamental  to Na , +  .  I t has been  well  established that thee f f e c t i s the result o f reduction i n 90 96 tshte dc ur pt ho et e nc ta ir ar ly i n g' c a.p a cL io tc ya l oafn e h estyisctse m a t l soo N rae d u e i ntgh ean i n ca rc etaisoen i n + + K c o n d u c t a n c e b u t t h e e f f e c t on K c o n d u c t a n c e i s much l e s s +  + 90 91 97 98 t h a t o n t h e Na c o n d u c t a n c e ' ' '  than  anesthetic  concentration  conductance  99  .  Increasing  h a s an i n c r e a s i n g e f f e c t  on t h e K  +  + on t h e K  T h u s , 0.5mM l i d o c a i n e h a s no e f f e c t  c o n d u c t a n c e b u t 3.5mM l i d o c a i n e l o w e r s i t t o 75% o f i t s o r i g i n a l value;  l i d o c a i n e i s 100 t i m e s more e f f e c t i v e  than on K  effects of local  conductance  tion  such  membrane  anesthetics  i nexcitable cells a s human  of the l o c a l  suggested  +  on t h e N a  +  that  r e d blood  anesthetic  local  differ cells  (RBC)  1 0 0  (both  +  .  High  concentra-  and i t has been  b a s e makes t h e r e d c e l l  In a d d i t i o n t o nerve  + block  and K  from those i n i n e x c i t a b l e  causes hemolysis  anesthetic  more p e r v i o u s .  anesthetics muscle  channels  channels.  +  The  cells  on N a  t h e movements o f Na  fibers,  local  + and K  i n t h e r e s t i n g s t a t e and d u r i n g  i n t h e membrane o f generation  of the  89 action  p o t e n t i a l ) , f r o g s k i n and o t h e r  tissues  A number o f n a t u r a l l y o c c u r r i n g acetylcholine, key  ATP, C a  + +  ,  substances  such as  a n d t h i a m i n e have b e e n t h o u g h t  r o l e s i n the conduction  o f t h e nerve  impulse  to play  and t h e r e  have  b e e n a number o f r e p o r t s  23 -  on i n t e r a c t i o n o f l o c a l  anesthetics 89  w x t h t h e above a g e n t s .  However, as R i t c h i e and G r e e n g a r d  have p o i n t e d  o u t , any t h e o r y  anesthetics,  b a s e d on an a n t a g o n i s m between  and  a naturally occurring  diversity  stabilizing  time In  capable  local  anesthetics  i s weakened by t h e v e r y of antagonizing  respects  local  anesthetics  t h e e x c i t a b l e membranes.  constants  f o r the rise  and f a l l  behave  ient quantity conductance  both  + +  carrying  i s present,  completely  72,73,91,103^ Ca  I  ^  t  a  s  the l o c a l  with  + +  They i n c r e a s e t h e  of Na  conductance ^ .  +  1  anesthetic w i l l  2  i f sufficblock the  that  the l o c a l  anesthetic  a c t on t h e same s y s t e m w h i c h i s r e s p o n s i b l e f o r  the Na  through the nerve membrane ^.  +  In f r o g  1  n e r v e s , d e p o l a r i z a t i o n i n the absence o f  and  c a n be p r e v e n t e d by l o c a l 92 Goldman have shown t h a t ,  and  local  + +  Ca  no change i n t h e r e s t i n g p o t e n t i a l  keen p r o p o s e d  desheathed myelinated Ca  local  like  addition to increasing the e x c i t a t i o n threshold,  and  local  activity.  I n many in  substance,  o f the s t r u c t u r e s  anesthetic  on t h e mode o f a c t i o n o f  anesthetics  compete  anesthetics ^'  Blaustein ++ i n l o b s t e r g i a n t a x o n s , Ca 1  1 (  ^.  f o r t h e same s i t e  on t h e  107 membrane. local Ca  + +  On t h e o t h e r  anesthetics  may  from t h e s i t e s  t h i s way,  local +  hand F e i s t e i n  a c t p r i m a r i l y by i n h i b i t i n g  that  release of  t o w h i c h i t i s bound i n t h e membrane.  anesthetics  may p r e v e n t  the secondary  In  changes  +  in  t h e Na  of  t h e wave o f e x c i t a t i o n a l o n g  and K  There sociated  has proposed  permeability,  is still  molecular  and thus p r e v e n t the c e l l  a controversy  propagation  membrane.  as t o whether the u n d i s -  form o f the drug o r i t s c a t i o n i c form i s  responsible that  f o r the nerve b l o c k i n g  i n a l l studies  local  anesthetics  s o l u t i o n where  cation  found  De J o n g  s h e a t h was  concluded  left  t o be most e f f e c t i v e  intact,  i n alkaline  t h e b a s e p r e d o m i n a t e s , w h e r e a s i n most s h e a t h was  removed, a n e s t h e t i c s  i n neutral or s l i g h t l y  studies  were  more  a c i d s o l u t i o n where t h e  predominates. (v)  Biochemical  Several local  i n which the nerve  were  i n which the nerve effective  action.  showed  on t h e m e t a b o l i s m o f i s o l a t e d  as on t h a t o f h o m o g e n a t e s . that  Anesthetics:  s t u d i e s h a v e been c a r r i e d o u t on t h e e f f e c t s o f  anesthetics  as w e l l  E f f e c t s of Local  cocaine  depressed  nervous  tissue 108 Niwa  As e a r l y as i n 1919,  carbon dioxide  production  i n the  109 sciatxc  nerve  of the f r o g .  procaine  inhibited  rabbit.  These  anesthetics that  Sherif  studies  l e d Watt" ""^ t o s t u d y 1  anesthetics cocaine  oxidation  and g l u c o s e  of succinate  t h e s e d r u g s , n u p e r c a i n e was the i n h i b i t i o n  such  o f r e s p i r a t i o n was  of these  this  the for  and showed  this  glycolysis.  and s t a t e d t h a t  that  He  local  observed  butacaine, the  In order  g l y c o l y s i s of r a t  t e t r a c a i n e and nupercaine unable to give  the i n c r e a s e  to  studied  t h e y h a d no i n h i b i t o r y  Watt was  Of  n o t due t o t h e  enzymes, W a t t ^ " ^  d r u g s on t h e a n a e r o b i c  system; i n c o n t r a s t ,  anaerobic  inhibited  t h e most e f f e c t i v e .  the  in  the e f f e c t s of  by r a t b r a i n h o m o g e n a t e s .  o f some o f t h e g l y c o l y t i c  homogenates  and  nerves of the  as n u p e r c a i n e ,  and p r o c a i n e  inhibition  brain  cocaine  on t h e r e s p i r a t i o n o f b r a i n h o m o g e n a t e s .  t e t r a c a i n e , metycaine,  effect  that  r e s p i r a t i o n i n the s c i a t i c  a number o f l o c a l  show t h a t  showed  effect  stimulated  any  explanation  i n the anaerobic  glycolysis  might  be due t o t h e m a i n t e n a n c e  glycolysis the  b y some  inhibition  inhibition oxidase  He  high  rate  concluded  anesthetics  was  o f cytochrome  of  that due t o  c-cytochrome  stage.  procaine,  and Quastel ''" 1  lidocaine,  active  respiration  of r a t brain  glucose.  They  the resting  Results of  local  at the oxidation  cologically  on  mechanism.  o f r e s p i r a t i o n by  possibly  Geddes ics  unexplained  of the original  observed  1  tetracaine  concentrations,  had l i t t l e  cortex  workers  the l o c a l  and d i b u c a i n e , inhibit  slices  the K  brain  further  cortex  indicated  these  drugs  as i n h i b i t o r s o f K  parallel  their  anesthetic  a t pharmastimulated  +  of  concentrations  respiration. that  stimulation  +  anesthet-  i n the presence  o r no e f f e c t a t t h e s e  or unstimulated  of these  that  the  potencies  of brain  cortex  activities. 112  Fink, consumption depressed compared  cell  by v o l a t i l e ,  that  system  brain  anesthetics  cycle. the  The f a c t  effects ulated  nerve.  and b a r b i t u r a t e that  o f Geddes  cocaine) of active local  may  find inhibit  acetate  of  cells  oxygen  was  anesthetics.  and Q u a s t e l  block  that  as t h a t  1  1  They and  1  certain  of the  local  the condensing  into  anesthetics  o f the r e s t i n g nerve by t h e i r  the rate  o f t h e r e s p i r a t i o n o f t h e mouse  and Walsh  that  are exerted  that  a n e s t h e t i c s i s as great 113  Ryman  the entry  found  o f mouse h e t e r o p l o i d  the s e n s i t i v i t y  (e.g.,  metabolism  local  results with  to local  slices.  blocking  and Simpson  of a suspension  their  concluded  Kenny  the c i t r i c  have  makes  little  i t likely  o f i o n movement  enzyme,  acid action that  i n the  on  their stim-  -  1.4  EFFECTS TTX  known  to  similar  OF  is man  to  TETRODOTOXIN  one  of  the  and  has  a  local  26  ON  most  -  THE  NERVOUS  toxic  mechanism  anesthetics  non-protein  of  but  SYSTEM  action  has  substances  which  potency  of  seems more  very  than  114 100,0  00  times  Japanese  puffer  chemistry TTX such  as  that  fish  depression  . - As now  muscles. is  generation  the  of  property (i)  of  of of  These  as  abolished  tissues cells, by  action  of  TTX. of  Nerve  e f f e c t of  TTX  the  TTX.  potential  on  the  actions  as  in  of and in  is  nerve  block  detail  concerned, on  fibers the  of  considered  Neuromuscular  hypo-  i t acts  of  vivo  respiratory  vitro  that  the  fact, is  to  in  discussed  generation  In  .  reflexes  been  show  such  Its  suppression  TTX  to  the  newt.  leading  spinal  have  evidence  the  Action  Isolated  these  as  aspects  the  excitable In  such  in  reviewed"*""^  respiration  responses  far  been  s k e l e t a l muscle,  sufficient  electrically  main  Californian  pharmacological  variety  tensive effects. 115  potential  found  a  evoked  the  the  is  has  stimulus  is  in  TTX  have  paralysis  there  and  .  pharmacology  the  Kao  cocaine  and  failure,  by  of  and  action the  to  be  Junction  the  and  on  Preparations: 117  Furukawa, TTX  on  the  rations  to  and  neuromuscular  preparation. region  Sasooka  They  high  enough  junction  showed  acetylcholine to  Hosoya  is  studied on  the  frog  the  nerve  that  response  of  the  end  not  affected  by  TTX  at  block  the  action  effects  potential.  of  sartorius plate  concentThis  study  -27  was  followed  by  the  work  of  -  Narahasi,  Deguchi,  Urakawa  and  118 Ohkubo on  in  frog  which  muscle  they  fiber  analysed  membrane.  microelectrodes,  they  ration,  applied  make  producing resting The is  the  action  concluded  stabilized  that  cathodal  resistance  by  that  mode  With  the  the  the  i n a c t i v a t i o n of  at  of  of  TTX  intracellular  10  Mconcent-  ineffective in  resting potential little  presence the  action  aid  TTX,  undergoes in  of  current  p o t e n t i a l whereas  membrane  authors  observed  the  Na  or  of  no  change.  TTX  the  carrying  +  and  membrane  mechanism.  119 However, voltage the  i t remained clamp  above  TTX,  at  experiments  hypothesis.  a  very  production ing  low  and  with  These  Moore  lobster  studies  concentration,  through  mechanism  carrying  for Narahashi,  and  giant  mechanism.  that  blocks  These  i t has  findings  axons  confirmed the  i t s selective inhibition showed  Scott  no  to  the  of  the  been  do  verify  fact  action  e f f e c t on  have  to  that  potential Na  carry-  +  the  K  further  +  extended  122-125 by  a  number  of  workers  120 Kao has  now  nerve  been  Kao  and  a l . with  high  Fuhrman  shown  blocking  Moore, et  and  be  identical  action  on  the  Fuhrman  121  tarichotoxin of  significantly  blocking  and There  shown  to  concentration  toxin,  had  thus  Ca  to  and, + +  ,  some be  with  in  the  studies  of  that  that  with of  against  the  the  TTX  acts  effect  rather being  than to  the  block  internal surface the  inward  of  movement  the of  axon Na  +  119  a  the Na  +  toxin. on  124 outer  strong  Narahashi  observed  reversibility  doubt  a  (which  Takata,  concomitantly  protection  little  exerted  nerves.  addition,  applied the  tarichotoxin  TTX)  sciatic  extended  improves  gives  seems  frog  that  the  127 '  , i t s  accompanying  -  the  generation  of action  28  -  potential. 126  Pullman, effects  Lavender  o f TTX  solution  i n dog.  directly  significant Using  and Aho  studied  Tetrodotoxin  into  one  differential  renal  artery  increase  c r a y f i s h abdominal  was  i n K  nerve  the d i r e c t infused  i n dilute  and caused and M g  +  fibers,  renal  highly  excretions.  + +  Ogura  and  Mori  128 have  shown  that  ive  i n the sheathed  was  more  the c a t i o n i c  form  o f TTX  form,  and t h a t  due  Effects  Metabolism Until  concerned logical  o f TTX  either  effects  most  with on  one, they  to guanidyl  nervous  on  Cerebral  suggested i s the  active  more  effects  simultaneously,  r e s u l t s o f two  opened  the p o s s i b i l i t y  of using  phenomenon  with  or with  the i s o l a t e d nerves  TTX  Cation  Content  Tissues:  of the experiments  i n vivo  and t r a n s p o r t  solution i t  tissue  the E x c i t a b i l i t y ,  almost  metabolism  group,  effect-  form.  of Isolated  recently  i s more  and i n n e u t r a l  TTX p e n e t r a t e s  i n i t suncharged  (ii) and  preparation,  s o l u t i o n TTX  e f f e c t i v e i n the desheathed  that  rapidly  i n alkaline  TTX  electrophysio-  and muscle. studies  as a  were  tool  I n 1967,  appeared  which  t o study  the  i n the i s o l a t e d  cerebral  tissues. Chan  and Quastel"*" ^ 3  respiratory place  on  TTX  the K also  +  that  of the r a t brain  upon the a p p l i c a t i o n  effect that  increase  showed  stimulated  inhibits  3 yM  cortex  of e l e c t r i c a l respiration.  increase  TTX  inhibits slices  impulses They  i n the rate  the  that  takes  but has  further  no  observed  of respiration  that  -  29  -  ++ occurs over,  when Ca their  oxidation influx  Na  of Na ,  ible  slices  Chan and Q u a s t e l  during  electrical  metabolic guinea  similarly  responses  greatly  respiration was  induced  blocks this  acetate  to the concentthe  inhibits  that  TTX  by e l e c t r i c a l slices.  He  influx  i s respons-  found  b y 0.4uM T T X  aerobic  due  at small  TTX  brain.  the  stimulation i n the  also  showed  to electrical  that  large  stimulation are  b u t i t h a s no e f f e c t on t h e tissue; K  +  t o be i n s e n s i t i v e t o TTX.  were observed w i t h  of  on i s o l a t e d  of the unstimulated  found  that  More-  e f f e c t s o f TTX  i n t h e r e s p i r a t i o n due diminished  impulses,  by TTX  concluded  medium.  the i n h i b i t i o n  s t i m u l a t i o n and t h a t  p i g cerebral cortex  increase  ation  blocked  f o r the potent metabolic 129 Mcllwam  that  by e l e c t r i c a l  i s completely  +  +  from the i n c u b a t i o n  r e s u l t s demonstrated  i n brain  rations. of  i s omitted  glycolysis.  stimulated  respir-  Similar effects  The K  content  +  of the  e l e c t r i c a l l y stimulated s l i c e s i s greater i n the presence of 129 129 TTX . B e c a u s e TTX i s a g u a n i d i n e d e r i v a t i v e , M c l l w a i n studied  a number  of guanidine  d e r i v a t i v e s and showed  some o f t h e m a r e a s e f f e c t i v e a s T T X ,  b u t a t much  that  only  higher  concentrations. 131 Swanson ally it  stimulated  +  guinea  t h e e f f e c t s o f TTX  p i g cerebral cortex  affects the cationic shifts  electrical K  studied  stimulation.  and c r e a t i n e  electrical  Cerebral  phosphate,  stimulation.  which  and g a i n  slices  normally  cortex  on t h e  t o see i f  take  slices  place  lose  non-inulin Na  T h e s e e f f e c t s were shown  electric-  +  with  non-inulin during  t o be  prevented  -  by  1 yM TTX.  H i s experiments  t h a t TTX d i r e c t l y of both N a  and K ,  +  It  interferes which  +  i s well  30 -  strongly  support the conclusion  w i t h t h e p a s s i v e d o w n h i l l movement  o c c u r upon e l e c t r i c a l  known t h a t  glutamate  causes  stimulation. excitation of  50 the nervous of  5mM  .  L-glutamate  cerebral  induced  Electrical  causes  cortex slices  confirmed  short  tissue  132  t h a t TTX a l m o s t  electrically,  initial  Extrusion  period,  an i n f l u x .  or the addition  into  the incubated  and R o d r i g u e z  completely prevents increase  and t h a t the N a  i tpartly influx  +  inhibits,  i n d u c e d by  electrical  +  These  of Na  +  M c l l w a i n , Harvey  of Na , following  e d by TTX.  stimulation  s t u d i e s were f u r t h e r  134  i n Na , +  during a  glutamate.  stimulation, extended  by  i s unaffectPull,  133 Mcllwain  and Ramsay  amate a c t i n g observed  and  i n a way s i m i l a r  that C a  the c a t i o n caused  , who c o n s i d e r e d t h e p o s s i b i l i t y . .  + +  to chelating  act synergistically  movements on t h e a d d i t i o n  an i n c r e a s e  agents.  of glut133  They  w i t h TTX i n r e s t r i c t i n g  o f glutamates. 5mM EDTA + + and a d e c r e a s e i n K ,  i n i n t r a c e l l u l a r Na  t h e s e c h a n g e s were p a r t i a l l y  b l o c k e d by TTX.  134 Ramsay and M c l l w a i n Ca  + +  studied  the e f f e c t s  movement i n i n c u b a t e d g u i n e a p i g c e r e b r a l  induced o f TTX  by g l u t a m a t e .  They  found  o f TTX on  cortex  slices  t h a t v e r y low c o n c e n t r a t i o n s 45  (66-330nM) a r e c a p a b l e o f i n h i b i t i n g  Ca i n f l u x ,  both  i n t h e p r e s e n c e and absence o f L - g l u t a m a t e . TTX a l s o c a u s e d a detectable diminution of C a efflux. 135 + Okamoto and Q u a s t e l r e p o r t e d t h a t Na i n f l u x and + +  water 0.1 mM  uptake  i n r a t cerebral  ouabain,  cortex slices  o r 10 yM p r o t o v e r a t r i n e ,  i n the presence of  or e l e c t r i c a l  stimu-  -  lation  absence of  glucose  s u p p r e s s e d by  3 yM  On  effect  Na  30  uM  or  the  on  the  TTX. influx  +  dinitrophenol  or  the  and  of  -  31  is partially other  h a n d , TTX  water uptake  100  mM  or  wholly has  i n the  no  presence  of  K . +  136 I t o k a w a and (as w e l l as  showed t h a t p e r f u s i o n  w i t h o u a b a i n and  promoted r e l e a s e c o r d s and  Cooper  the  been e a r l i e r 1.5  of  LSD-25) a t  radioactive  low  thiamine  TTX  concentrations,  from the  sciatic  n e r v e s o f b u l l - f r o g s and 35 . i n j e c t e d with S -thiamine.  E F F E C T S OF  with  spinal  r a t s which  OTHER NEUROTROPIC DRUGS ON  THE  had  NERVOUS  SYSTEM (i)  Ouabain.  (a)  I n h i b i t i o n o f Na,K-ATPase .by  It Na  has  been p r e v i o u s l y  i s extruded  +  cell  from the  i s described  as  the  cell  stated and  K  Ouabain;  that  I t has  + that  through the  membrane a g a i n s t  energy dependent  the  137  p e r i p h e r a l nerve of both Na by  the  and  +  K  cardiac  Since crab,  glycoside  ence i n s u p p o r t of  the  processes  s u c h as  their  the  well  , which takes  this  gradient,  is  in  which  place  the requires  enzyme i s i n h i b i t e d i s overwhelming this  evid-  enzyme i n  the  140-145 .  This  A T P a s e and  the  transport  + and  K  have a number o f  localization,  i n the  cell,  features their  + energy  been  ATPase i s p r e s e n t  ouabain, there  '  + m e c h a n i s m f o r Na  K  c o n t r o l l i n g r o l e of  137 transport  an  inside  138139 ' showed t h a t  f o r a c t i v a t i o n and  +  and  a concentration Skou  which  +  t r a n s f e r o f Na  .  p r o c e s s by  i s accumulated  +  "Sodium Pump".  established  the  utilization,  a c t i v a t i o n by  Na  i n common  properties  + and  K  , and  ouabain  of  -  inhibition  137  As  early  glycosides of  cation  32 -  a s i n 195 3,  Schatzman  a t low c o n c e n t r a t i o n s transport.  This  was  146  showed  are s p e c i f i c  later  that  cardiac  inhibitors  confirmed  by a  number  147-149 of  workers  lated  .  form  seems  o f the Na , K -ATPase, +  intermediate Na  Ouabain  during  +  the operation  activate the formation  +  iate  while  K  stimulate  +  t o a c t on which  the'phosphory-  appear  of this  t o a c t as an  enzyme ^ 1  o f the phosphorylated  i t s breakdown.  intermed-  The a c t i o n  of  K  +  i  is  prevented  between 3 Na  +  by ouabain.  the transport  i s transported  inside  the c e l l  is  very  Na  high  1  6  of Na  Thus  +  relation  i n erythrocytes  and 2 K  +  i s transported  o f ATP h y d r o l y z e d ^ 1  162^  +  .  Yoshida,  activity Nukada  0.0ImM o u a b a i n c a u s e s a n a l m o s t + + of K a n d e x t r u s i o n o f Na from cortex  h  and K .  molecule  , K -ATPase 3  +  i s a stoichiometric  to the outside  f o reach  + The  There  i n the cerebral  and F u j i s a w a  complete  block  the guinea  1 6  ^  cortex  showed  that  o f uptake  pig cerebral  slices. In  recent  concerning  years  the binding  many  of ouabain  + glycosides Laughter  1 6  t o t h e Na ^  studied  heart  muscle  state  o f t h e enzyme  glycoside  enzyme  binding.  studies  have  been  and o t h e r  carried out  cardiac  + , K  -ATPase.  the binding  of t r i t i a t e d  and concluded i s probably Their  data  Schwartz,  that  the  of primary support  Matsui  and  digoxin  to the  conformational significance i n  the concept  o f an  -  "allosteric of  type"  phosphorylated  which  33  -  o f enzyme and t h e y e n z y m e may  the conformational  state  that  the  formation  be one o f a number o f ways i n  nature  o f t h e e n z y m e may  be  altered.  166 Charnock  and P o t t e r that  using  enzyme  cortex  concluded  o u a b a i n may  lation  and d e p h o s p h o r y l a t i o n  nature  and amount o f t h e c a t i o n s  from t h e guinea p i g  inhibit  both  phosphory-  o f t h e enzyme d e p e n d i n g present.  Yoda  and  on t h e Hokin  16 7 observed beef brain that the  of  enzyme  the sugar  Other  the binding  linkage  to the  results  indicated  Their with  studies  '  the 3 p o s i t i o n of  s t a t e s , and t h a t  +  i n Relation  t h e enzyme  whether  the  binding  depends on t h e  and other f a c t o r s . E f f e c t s o f Ouabain and Cations K -ATPase  irreversible  indicate that  i s reversible or irreversible  temperature (b)  glycosides  an i m p o r t a n t r o l e i n t h e 156 16 8  exist i n several ouabain  of cardiac  i s irreversible.  i n glycoside  steroid plays  binding. can  that  to Brain  on  Na , +  Metabolism  and  Transport: As for  Na  +  and K  K -ATPase +  has  energy  +  derived  transport,  i s affected  i n the regulation  metabolism  and as t h e a c t i v i t y  by t h e c a t i o n  l e d to the suggestion  involved  from glucose  that  o f Na  +  concentrations,  N a , K - A T P a s e may +  i s utilized  +  , this  be  of respiration i n the cerebral  -  34  -  169 cortex  slices.  whittam  + the  activity  the  incubated  energy  of  and  found  a  parallelism  between  +  Na  , K  -ATPase  rat brain  production  Blond  by  and  a  homogenates  respiration  part  of  the  suggesting  i s geared  to  respiration  that  the  of  rate  i t s rate  of  of  170 utilization. of  ouabain  slices the  on  but  rates amino  the  and  Quastel  respiration  i t partially  amount  ouabain.  Gonda  of  of  observed  the  depending  a  find  only  unstimulated  suppressed  suppression  They  could  K  on  pronounced  little  brain  cortex  stimulated  +  the  effect  respiration,  concentration  effect  of  ouabain  of  on  the  o f t r a n s f o r m a t i o n o f g l u c o s e i n t o amino a c i d s , and a c i d and c r e a t i n e t r a n s p o r t i n t h e c e r e b r a l c o r t e x  on slices.  171 Using frog brain, e f f e c t s o f o u a b a i n on  the to  the  is  a  K  concentrations.  small  increase by  +  dePiras glucose  but  in  low  showed t h a t according  concentrations  K  consistent stimulation of  respiration  ouabain.  At  and Z a d u n a i s k y metabolism d i f f e r  With  due  guinea  to  pig  high  K  oxygen  is  +  cerebral cortex  observed  in  begin  to  fall.  after  short  effects to  a  and  respiration  be  of  but  in  K  after  incubation  There  the  ouabain  there  but  the  inhibited  slices,  with by  also  a  of  the  content  +  and  reversed was  causes  40-60  phosphate  partially  f r e s h medium. decrease  that  Creatine  periods  could  ,  Swanson  . .  Mcllwain  increase  +  uptake  completely  172 and  of  an  minutes ATP  initial respiration  levels  glycosides  also and  transferring  great  these  the  increase  ouabain  fall  in  treated  tissue the  Na  +  slices  16 4 (cf. rical  Yoshida  et  a l .  s t i m u l a t i o n and  ineffective  in  that  presence  in  the  the  ).  These  the  recovery  presence of  effects  of  ouabain  are  process  ouabain  1 3 1  creatine  .  augmented is  by  elect-  completely  Swanson phosphate  1 3 1  noted  levels  continue  to drop a f t e r e l e c t r i c a l Swanson  and  the  35  rise  cerebral  173  observed  i n Na  cortex  content of  +  slices  m a r k e d i n a Ca  that  i n the  stimulation  the  the  has  increased  incubated  loss of  guinea  - f r e e medium.  difficult loss  that  access  when C a  of  This  was  ouabain to  i s present  + +  K  +  pig  p r e s e n c e o f o u a b a i n i s most explained  + suggestion  ceased.  the  i n the  Na,  by  the  + K-ATPase i s more  medium  1 3 1  '  1 7 3  '  1 7  ^.  The  of K ,  i n the p r e s e n c e of o u a b a i n , i s a l s o slow i n a Na 175 + f r e e medium ( C h o l i n e C h l o r i d e was s u b s t i t u t e d f o r Na ). 175 T h e s e r e s u l t s l e d Swanson and S t a h l to conclude t h a t i n the +  +  presence of Na + Na,  the site  there  +  i s a Na -induced  + K-ATPase w h i c h a l l o w s  of  s t r u c t u r a l change  +  accessibility  of  ouabain  in  to i t s  inhibition. 176 Tower  showed t h a t  mitochondrial of  Ca  the  than  + +  f r a c t i o n from such s l i c e s , the  increased  or  controls.  uptake of  of p r e p a r a t i o n eability  ouabain t r e a t e d  and  Stahl  Ca  + +  m i g h t be  and  does n o t due  to  an  inhibition  mechanism f o r e x t r u s i o n 137  of  Ca  Quastel cation that  transport  the  various direct was  stimuli, e f f e c t on  pointed  ation CoA  fluxes  by  and  out  , while and at  K  +  Swanson  1 7 7  of  the  the  an  artifact  membrane p e r m active  transport  the  c h a r a c t e r i s t i c s of  cell  membrane, as  brain,  e f f e c t b r a i n m e t a b o l i s m l a r g e l y by  the  membrane bound A T P a s e . influx  may  of Na  a c t i o n on  a f f e c t the  +  may  activity  of  their  Nevertheless,  suppress  acetate  concluded  a r e s u l t of  may  that  that  .  reviewing  brain  the  contents  stated  a p p e a r t o be  r e s p i r a t o r y c o n t r o l i n the  its inhibiting that  + +  and  have h i g h e r  increased  p o s s i b l y to  slices,  acetate  conversion PK.  to  i t  oxidacetyl  Rolleston of  0.1  mM  ouabain,  production These ment 0.1 of  by  ouabain mM  inhibition both, Na , +  i n a manner  i n the  and a e r o b i c l a c t i c  . 0 0 1 mM  acid  i s increased.  ouabain.  to conclude  pig cerebral  that  Measure-  the  o f t h e known  of  addition  cortex slices  G-3-PDH o r p h o s p h o g l y c e r a t e  independent  presence  intermediates i n the presence  workers  to guinea  that,  cortex slices  with  of glycolytic  of either  causes  kinase, or  action  o f ouabain  on  K -ATPase. +  (ii) (a)  Protoveratrine Site  Veratrum purposes  aration  a n d Mode  of  and r e l a t e d  f o r many y e a r s .  alkaloids,  is  p i g cerebral  l e d these  ouabain  observed  utilization  are not observed  of the l e v e l  0.1  glucose  guinea  effects  mM  and Newsholme  the f i r s t  was  excitable  plants  They  one  have  contain  t o be  protoveratrine.  alteration  Action been  used  a number  for medicinal  of hypertensive  obtained i n crystalline  The major  i n the permeabilities  effect  of  prep-  protoveratrine  o f membranes  o f a number  of  cells. 178  Frank fibers,  t h e membrane  potential delays  observed  that  i n the veratrine-treated  becomes permeable  and the enhanced  repolarization.  inward  Using  to Na  movement  +  muscle  following  of Na  c r u s t a c e a n axons,  +  an  action  considerably  Wright  and  17 9 Tomita  concluded  chemical They  or metabolic  suggested  mechanism Ca  that  o r Na  that  o r may  veratrine  reaction  veratrine  itself  by may  compete  action  i s the result  the alkaloid inhibit  for sites  i n the  the Na  +  of  membrane.  extrusion  i n t h e membrane  .  Shanes  73  suggested  that  an i n t e r a c t i o n  takes  place  with  -  between  37  -  t h e a l k a l o i d s a n d membrane  channels,  affecting  i n turn  lipids  t h e membrane  which  affect  the  permeability.  ionic  However,  180 Kupchan site"  and F l a c k e  have  or "receptor"  has been  l o i d s and that there , _ . ,186, and Q u a s t e l ). (b)  pointed  a r e no  Biochemical Veratrum  out that  recognized specific  no  specific  "binding  f o r the veratrum  antagonists  known  Effects of Protoveratrine  and  alka-  (cf.  Kini  Other  Alkaloids 181  Wollenberger (protoveratrine of  respiration  pig  cerebral  the  normal  lasting  These  the effects  Omission  of K  The by  from  which  inhibition  washed  cerebral  concentrations .  while  significant  hours  stimulation  i n the incubated  increase  aerobic  inhibiting  the  guinea  glycolysis  to  anaerobic  a r e c h a r a c t e r i s t i c o f t h e b r a i n and + 62 of high K r e p o r t e d by A s h f o r d and Dixon  t h e medium g i v e s  i s n o t enhanced  of anaerobic  the presence  They  alkaloids  effects  resemble  glycolysis  level  veratrum  produce  f o r several  slices.  anaerobic  +  that  and v e r a t r i d i n e )  cortex  glycolysis.  observed  which  slices  to strong  f u r t h e r by  glycolysis  of nicotinamide cortex  rise  182  .  may  protoveratrine be p a r t i a l l y  The n e t uptake  i s inhibited  stimulate  aerobic  respiration  181  prevented  of K  +  by  by p r o t o v e r a t r i n e a t and a e r o b i c  glycoly-  182  sis 183 Wollenberger on  electrically  that  in the  stimulated  simultaneous  veratrine  studied  exposure  and t o b r i e f  the rate  increases  caused  cerebral  cortex  of cerebral  condenser  of respiration  the effects  which  by t h e drug  pulses  of protoveratrine slices  cortex bring  i s greater  and  slices about  than  noted to  an  protoincrease  t h e sum o f  and by t h e p u l s e s  individually.  18 4 Quastel of  rat brain  is  diminished  addition of  of  38  -  185 '  has  noted  that protoveratrine  respiration i s highly by  malonate.  5 yM  rat brain  Kini  protoveratrine  cortex  i n the  s e n s i t i v e to n a r c o t i c s  and  i n the  Quastel  found  presence of  glucose-U-C  which d i f f e r s  absence of p r o t o v e r a t r i n e .  and  that  to a e r o b i c a l l y incubated 14  t o a r a d i o a c t i v e amino a c i d p a t t e r n obtained  stimulation  the  slices  gives from  rise  that  These e f f e c t s  are  18 0  antagonized  by  (iii)  cocaine  ( c f . K u p c h a n and  Amphetamines  While  discussing  and  the  Flacke  ).  Nialamide  mechanism o f  release  of  norepine-  187  phrine  (NE),  disquieting  Brodie, to  Cho,  realize  that  physiological-biochemical drug  that  acts  on  the  S t e p h a n o and at present  level  nervous  the  Gessa we  stated:  cannot d e s c r i b e  mechansim o f  system".  "It is  I f one  at  action of  knows  any  the  mechanism of  a c t i o n of  e v e n a s i n g l e d r u g t h e n i t may  provide  framework on  which  study  could  based.  the  Amphetamine  study because of plicity  of  i t s simplicity  sites  release  of  strongly is  also  serotonin  an  inhibitor  between  e f f e c t s on  and  the  i n recent  be  thorough multi-  years  catecholamines, from t h e i r  not  appear to a f f e c t the  ( 5 - h y d r o x y t r y p t a m i n e , 5-HT) u p t a k e o f NE  by  MAO  the  i t  has  the was  stimulating first  .  neuronal  uptake  or  Amphetamine  n e u r o n a l membrane  o f monoamine o x i d a s e  enzyme i n i n a c t i v a t i n g t h e  Parallelism  drugs  for a  in structure  e f f e c t s , and  release  t h e y do  a f f e c t s the  important  their  but  other  a  studied.  Amphetamines storage  a c t i o n of  i s a s u i t a b l e candidate  i t s biological  been e x t e n s i v e l y  of  a  (MAO)  which i s  and  an  a c t i o n o f monoamines i n e f f e c t of  shown by  CNS-,  amphetamines and 188 18 9 Mann and Q u a s t e l '  Other  e f f e c t s of  these  basic  in  amphetamines  effects  and  have  are  presumably  recently  been  linked  up  discussed  with  in  detail  ,190 monograph  a  D-Amphetamine 1-amphetamine.  A  i s at  number  least  of  20  times  derivatives  more  of  active  these  than  compounds  have  191 been  prepared  with  hydroxylation activity. cerebral recently  of  different  phenyl  Unlike  biological  group  or  the  activity  side  chain  .  Usually  reduces  the  amphetamine,  p-chloroamphetamine causes a 192 serotonin depletion in rats . Amphetamine has b e e n s h o w n t o i n c r e a s e t h e l e v e l s o f 5-HT i n mouse  , • 193 brain Nialamide with  this  drug  i s  a MAO  causes  inhibitor  increased  and  treatment  accumulation  of  of  the  amines  in  animals the  , . 194 brain (iv)  Barbiturates 195  In certain  1932,  Quastel  barbiturates  the  r e s p i r a t i o n of  was  later  the  drug  anesthetic  by  as  other  demonstrated anesthetic  tissue preparations.  other  were  doses.  Wheatley  well  brain  confirmed required  as  and  workers.  relatively  Barbiturates  The  large  This  suppress  observation  concentrations  compared  however,  drugs  that  at  with  anesthetic  of  the doses,  suppress K as w e l l as e l e c t r i c a l l y s t i m u l a t e d r e s p i r a t i o n ' . I t i s now w e l l k n o w n , f o l l o w i n g the work o f M i ± a e i s and QuasteL +  1  9  1  9  6  7  281  282-283 and  amytal of the  NADH  Ernster  and  as  as  well  and  hence  processes  These  co-workers certain other the  which  e f f e c t s have  , that hypnotics  generation  require been  ATP  of are  discussed  by  ATP  in  the  barbiturate  suppress the  affected Quastel  by 196  the  cell.  '  these 197  oxidation Thus a l l drugs.  Barbiturates concentrations do  they  that  inhibit  do  not  -  a f f e c t c e r e b r a l Na ,  K -ATPase  +  suppress  the  40  stimulated  increased  Na  brain  influx  +  due  +  at  r e s p i r a t i o n , nor to  electrical  198 stimulation  n o t show a l l e v i a t i o n o f t h e 14 14 d e p r e s s i o n o f o x i d a t i o n o f (1-C ) acetate to C 0^ b r o u g h t 198 a b o u t by t h e a p p l i c a t i o n o f e l e c t r i c a l p u l s e s . They d i f f e r in  this  .  T h e y a l s o do  respect  to  local  anesthetics  and  TTX.  199  Webb and respiration  of b r a i n  a c c o m p a n i e d by glycolysis. the  Elliott  suspensions  a considerable  The  normal r a t e  of  anaerobic  i s consistent with  to decrease  that  the  rate  unaffected  the  (1 mM by  i n the  glycolysis. vary  case of  The  from drug (v)  to  thiazine  i n the  50  of  of  barbiturates rate  of  a m y t a l and than  aerobic exceeding rate  by  the  drug.  information  on  the  relief  of  respiratory chain inhibitors 199  Elliott  by  further  brain  low  observed  suspensions  drug  but  is inhibited  t h o s e w h i c h c a u s e maximum at which  is  concentrations  pentobarbital),  concentrations  is  percent  aerobic  these e f f e c t s occur  drug.  and  of  CPZ  since  (CPZ) i n the  1950"s was  t h e n CPZ  as w e l l  d e r i v a t i v e s have b e e n e x t e n s i v e l y  treatment  by  r a t e , equal to or  present  accelerated  Chlorpromazine  importance  inhibition  g l y c o l y s i s , o c c u r s when t h e  Webb and  greater  Introduction peutic  increase  anaerobic g l y c o l y s i s of  slightly  concentrations  the  slices  presence of  i n ATP.)  of  or  and  i s i n h i b i t e d about  of P a s t e u r e f f e c t i n the due  that  maximum g l y c o l y t i c  oxygen consumption (This  found  a number o f m e n t a l  Lindan, Quastel  and  of as  used  great other  therapheno-  i n the  clinical  disorders.  Sved^^  have shown t h a t  CPZ  at  low  41  concentration uptake.  The by  binding  power  CPZ.  CPZ  with  i t s uncoupling  tions  that  the  electrically  reduced  in  depresses  do  stimulation  +  stimulated  differs  tissue of  not  K  from  the  of  cerebral  respiration is  the  proteins  oxidative  affect  -  narcotics  but  in  resembles  phosphorylation  r e s p i r a t i o n of  oxygen  also i t s  the  at  high  narcotics  concentra-  unstimulated  194 brain the  cortex  slices  .  It  has  been  shown  that  CPZ  uncouples  oxidative phosphorylation i n the m i t o c h o n d r i a ^ ^ 204^ CPZ i s an a n t a g o n i s t o f f l a v i n a d e n i n e d i n u c l e o t i d e  205 (FAD) CPZ  .  206  .  is  claimed  Dawkins,  electron of  FAD  CPZ,  Judah  transport together  to  and  between  with  antagonize  20V  Rees  the  have  NADH a n d  EEG  shown  that  cytochrome  i t s suppression  effect  effects  C.  on  of  CPZ  inhibits  This  effect  membrane  20 8—210 permeability  •^4.oxidative  , may  account  for  i t s ability  to  uncouple  v, u 1 4 . - 194,211 phosphorylation 212  CPZ  inhibits  the  release  of  acetylcholine  in  cat  brain  194 and  stabilizes  the  acetylcholine  containing  synaptic  vesicles  213 Maickel  has  Considerable levels of  NE  of  shown  that  information  NE,  dopamine  in heart  and  CPZ  inhibits  exists  and  5-HT  adrenals  and  acetylcholinesterase.  on  the  in  brain.  also  influence CPZ  cause  of  CPZ  on  diminishes  complete  uptake  release  194 particle  bound  adrenalin  in  adrenal  gland  the  preparations  of  196 '  214 Magee  and  Rossiter  have  shown  that  low  concentrations  32 of  CPZ  various  bring  about  changes  phospholipids  of  in  the  guinea  incorporation  pig  cerebral  of  cortex  P  into slices.  215 Mule on and  studied  the  effect  of  phospholipd-facilitated Ca concluded  that  an  CPZ +  +  alteration  and  others  transport in  the  CNS  acting  drugs  which  is  inhibited,  binding  of  ions  to  -  phospholipids the  within  pharmacological  the  42  neuronal  action  of  -  membrane  CNS  acting  22 the  rates  of  penetration  of  may  be  drugs.  involved CPZ  in  decreases  42  Na  and  K  into  cerebral  tissues  216 , a drug.  fact  that  Buzard ory  effect  on  especially  +  , K  +  ATPase  CPZ  rather +  , K  workers  218  CPZ  enzyme  .  Akera  light  to  with  ouabain  of  a marked  inhibitor  observed  a  be  the  l i g h t was  this  inhibit-  homogenates,  a  sulphydryl  the  more  of  effective  free-radical f o r the  later  free-radical  microsomal  inhibition  even  responsible In  of  that  semiquinone  CPZ  interacting  action  rat brain  enhanced  (UV)  may  of  219  Brody  that  has  i s an  that  the  CPZ  of  incubation.  in vitro.  i n contrast  used  of  activity  (-SH)  groups to  inhibition  study  inhibits  a  of  220  ,  these  enzyme on  which  i s bound  plant  Rauwolfia  serpentina  a  drug.  the  different  site.  Reserpine  Preparations been  to  itself  1 mM  CPZ  and  concluded  CPZ  mechanism  glycolysis  Ultraviolet  -ATPase  (vi)  that  period  solution  than  by  the  concentrations  concluded  activity  on  shown  later  authors  +  bear  anaerobic  activity.  these  Na  high  of  and  of  has  the  -ATPase  exposure  217  i n the  At Na  might  i n India  from  the  for centuries  as  calming  have  Reserpine 221  has  been  and  the  studied  shown effects since The  the from  CNS  t o be  reserpine  principal  reserpine  on  alkaloid  CNS  has  of  been  this  plant  extensively  then.  most  found  the  of  the  important  i n vivo  amine cause  storage  biochemical effect  i s the sites  depletion  of  release  of  in brain. these  NE,  of  5-HT  reserpine and  Prolonged  amines  from  the  in  dopamine  treatment brain.  by  Presumably  these  effects  43 -  a r e due t o c h a n g e s i n t h e p e r m e a b i l i t i e s 222  of  certain  neuronal  membranes.  Giachetti  and Shore  have  shown t h a t r e s e r p i n e g r e a t l y  enhances  adrenergic neuronal  t o t h e o u t w a r d movement  membrane  the permeability of o f amines.  Reserpine  i n t e r f e r e s w i t h t h e u p t a k e o f t h e a m i n e s by t h e amine 223 storage granules . Whether r e s e r p i n e a c t s a t t h e n e u r o n a l 224 membrane o r s t o r a g e g r a n u l e membrane i s c o n t r o v e r s i a l  + Vascular  be , by  tissue  Na  + and K  contents  h a v e b e e n shown t o 225 changed a f t e r treatment o f the animal w i t h r e s e r p i n e The a c t i o n s o f r e s e r p i n e have b e e n r e v i e w e d i n d e t a i l ' . . 194,223,226,227 a number o f w o r k e r s .  1.6 BIOCHEMISTRY Profound  OF THE DEVELOPING  b i o c h e m i c a l changes take  o f mammals d u r i n g d e v e l o p m e n t . with  BRAIN place i n the b r a i n  H e r e we w i l l  be c o n c e r n e d  t h e p r o b l e m s w h i c h a r e more p e r t i n e n t t o t h e  reported brain  i n this  thesis.  Various  aspects  have b e e n d i s c u s s e d by M c l l w a i n The  time  50  only  experiments  of the developing a n d Himwich  a t w h i c h t h e maximum b r a i n  growth  22 8  occurs  229 differs is  from  species to species  maximum a t 5-15 d a y s a f t e r  .  birth,  In c o n t r a s t t o the r a t , i n guinea occurs  before b i r t h  with  a peak a t 10  rate  days.  p i g s t h e maximum g r o w t h while  i s maximum a t a b o u t t h e t i m e o f  the major events  system are the formation ation.  with  t h e growth  a peak a t 17 d a y s b e f o r e b i r t h ,  i n man t h e g r o w t h r a t e o f b r a i n , . ,.,229 birth Functionally,  In r a t s ,  i n the d e v e l o p i n g  o f s y n a p s e s and t h e p r o c e s s  The m y e l i n a t i o n p e r i o d c o i n c i d e s w i t h  nervous  of myelin-  the period of  greatest first to  n e t increment  occurs  44 -  i n the b r a i n weight  c l o s e t o the nerve  cell  nerve  terminal  fibers  lacking  Myelination spreads  However, e v e n i n a d u l t  are often not t o t a l l y  portions usually  .  body a n d s l o w l y  the terminal p o r t i o n of the nerve.  life,  230  myelin  myelinated,  their  (see D a v i s o n and  231 Peters the  f o rdetails).  time  a t which  animal. at  synaptic  while  i n the r a t a sharp  functions occurs  is  m  (except guinea  from  animal t o  i n c r e a s e i n t h e number o f  d u r i n g t h e 3 r d a n d 4 t h week o f  changes, there occurs 50 244  t h e e n z y m i c make up o f t h e b r a i n  o f major importance  brain  of myelination,  p i g c o n s i d e r a b l e synapses a r e p r e s e n t  232 development Concomitant with these  change  the process  synapses a r e formed d i f f e r s  In the guinea  birth,  As w i t h  i n the i n f a n t  pig).  '  .  r a t h e r than  The c i t r i c  acid  cycle  a marked  Glycolysis  i n the adult enzymes  increase  markedly d u r i n g development and o x i d a t i v e metabolism  of  greatly  brain  increases during maturation.  monophosphate pathway i s o f l i t t l e brain are  although  channeled  through  this  a decrease  pathway i n t h e i n f a n t  changes i n t h e e l e c t r i c a l increases while Na decrease  extremely neonatal  +  with  i n the q u a n t i t i e s  changes o c c u r activity  diminishes. development.  utilized  brain.  of electrolytes 50  i n water content d u r i n g  However, s e v e r a l i m p o r t a n t  space  i n the a d u l t  considerable portions o f the glucose  There i s a decrease together with  importance  The h e x o s e  development  that are related  during maturation.  Chloride content Activity  o f Na  t h e maximum a t a b o u t  +  content  and c h l o r i d e , K -ATPase i s  low i n t h e newborn r a t a n d i n c r e a s e s r a p i d l y development with  K  to the  50 d a y s  during after  birth  234  partly noted  .  The  increase  for a large during  i n the  portion  brain  in vitro  -  activity the  maturation.  enzyme s y s t e m i s e q u a l l y efficient  of  45  The  of  this  increased  In  the  enzyme may  energy  increase  important.  stimulator  of  account  metabolism  i n the  addition, K  mitochondrial is  an  oxygen consumption  in  +  the 235  mature r a t b r a i n This  but  phenomenon may  is inefficient be  directly  i n a new  r e l a t e d to  born r a t  the  brain  changes i n ATPase  .. 233 activity A l t h o u g h Na infant  rat brain,  cellular 1.7  K  +  and  -K  ATPase a c t i v i t y  i t may  low  TRANSPORT OF  Na  be  sufficient  i s very  low  to maintain  in a  the  high  +236  AMINO ACIDS AND  SUGARS ACROSS THE  BRAIN  CELL  MEMBRANE Although influences the  the  influence  across  in  1958,  transport of Na  animal  year period  i t has  cell  on  +  1958  small  Na .  This  was  other  compounds s u c h as  glucose  237  .  solutes  the  and  intestine  of  first  1963  2 37  .  time  the  i n the  Riklis  Quastel  and by  s y s t e m s may The  be  driven  transport  by  reactions  of  downhill  amino a c i d s  of  include  dependent a c t i v e  a parallel  ,  3-0-methyl  + port  2 38  presence  a number o f w o r k e r s t o  +  five  isolated  the  and  +  solutes  identified  absorption  t h a t Na  Na  membrane,  a v a r i e t y of  amino a c i d s , u r a c i l  i s evidence  that  cell  d e p e n d s m a r k e d l y on  s o o n e x t e n d e d by  There  across  transport  showed t h a t g l u c o s e  guinea p i g +  of  membranes was  between  first  b e e n known f o r a l o n g  Na  and  trans-  239-241 flux  sugars  at 137  the  brain  cell  membrane has  been r e v i e w e d  r e c e n t l y by  Quastel  242 Although  there  i s some e x c h a n g e d i f f u s i o n ,  the  ammo  acids  '  are  usually  Several acids  accumulated  drugs  i n vivo  a r e known as w e l l  acids penetrate ponding  a concentration  to affect  the t r a n s p o r t  as i n c e r e b r a l c o r t e x  the b r a i n  cells  gradient. o f amino  slices.  more e a s i l y  L-amino  than the c o r r e s -  D-isomers. The  is  against  u p t a k e o f amino a c i d s  e n e r g y d e p e n d e n t and t h e i r  by c e r e b r a l c o r t e x  accumulation  under  slices  aerobic 137  conditions  i s blocked  by 2 , 4 - d i n i t r o p h e n o l  or iodoacetate  '  243  244 ' . U p t a k e o f g l y c i n e by c e r e b r a l c o r t e x s l i c e s u n d e r a wide v a r i e t y o f e x p e r i m e n t a l conditions i s proportional to 245 the of  level  o f ATP  .  There  i s a reduction  i n cerebral  uptake  one amino a c i d i n t h e p r e s e n c e o f a n o t h e r w h i c h may be  largely  due t o t h e m u t u a l c o m p e t i t i o n f o r a common c a r r i e r ( o r 137 s i t e ) a t t h e b r a i n c e l l membrane The a c t i v e t r a n s p o r t o f amino a c i d s i n b r a i n i s a l s o + 245 246 247 + Na dependent ' ' . High concentrations o f K diminish the  c e r e b r a l u p t a k e o f amino a c i d s  by l o w e r i n g  t h e ATP  level  137 A l s o , t h e r e i s l e s s u p t a k e o f amino a c i d s i n t h e a b s e n c e ++ ++ o f Ca o r i n t h e p r e s e n c e o f 10 mM Ca ; t h e l a t t e r may be  + due  to inhibition  o f Na  + ,K - A T P a s e .  Ouabain i n h i b i t s the  u p t a k e o f amino a c i d s , w h i c h i s c o n s i s t e n t w i t h  the r o l e o f  137 this  enzyme i n amino a c i d t r a n s p o r t p r o c e s s e s  presence  of ouabain,  increased  1 7  ^.  respiratory the  efflux  Anaerobiosis,  efflux  lack of Na , +  In the  i n t o medium i s  the presence  chain  i n h i b i t o r or chlorpromazine also 137 o f amino a c i d s  Although across  t h e amino a c i d s  .  i t i s w e l l e s t a b l i s h e d that glucose  the i n t e s t i n a l  wall  i s Na  +  and e n e r g y  of a  increases  transport  dependent,  there  -  is  no  evidence  that Na  47  -  mediate or  +  is essential for  glucose  137 transport  xnto the  Very of  little  carbohydrate  properties glucose  of  of  of  information  transport  transport  processes  i n the  i n suboptimal  of  i s a v a i l a b l e on and  i n the  doubtful  pentoses, evidence  brain  bral  glucose  .  cortex  slices.  emerged t o  Studies  process that of has  rather  u p t a k e and  been s u g g e s t e d  transport observed the  brain may  the  t o be  Consideration regulating  be  case  the  of  be  of  concentration  the  is a  space of  the  order  5  factors likely  a possible  50  of of  mM  rate K  +  glucose  or  control point of  i n the  increased  rate  glycolysis.  inhibitor transport  to operate  in  condition  g i v i n g lower r a t e  was  not  glucose  Rolleston  is rapid  increasing  medium f r o m 3 t o This  2-  indicate  2051249 ' ' .  cyanide),  14  mM.  glucose metabolism  3 mM  C  facilitated  for glucose of  a  cere-  uptake of  carbohydrate metabolism i n d i c a t e that  t h a t when t h e  presence  the  a number o f  a competitive  a p p a r e n t Km of  of  view t h a t  than d i f f u s i o n is  non-raffinose  as  and  However, i n t h e  Other experiments  regarded  rapid  from  transport  than d i f f u s i o n .  d e o x y g l u c o s e may  glucose  the  on  renders data  support  Kinetic properties  deoxyglucose i n d i c a t e that  the kinetic  under normal c o n d i t i o n s  concentrations  analogs i n t o the  specificity  l e s s on  f a c i l i t a t e d or c a r r i e r mediated process r a t h e r , ,137,248 involved 248 Bachelard has s t u d i e d t h e u p t a k e o f labelled  the  owing t o the  significance.  has  even 248  brain  have b e e n d i f f i c u l t  glucose  glucose  such s t u d i e s  cell  such p r o c e s s e s  metabolism of use  brain  10  (in  the  mM  observed  51  causes  under 51  of metabolism.  Rolleston  concluded  that  the rate  limiting  of entry  factor  of glucose  i n glucose  into  the tissue  may  become  a  metabolism. OBJECTIVES  The the  effect  anaerobic  aim of the present o f TTX and o t h e r  glycolysis,  cerebral  cortex  cerebral  metabolic  action The  general  various see  whether  i n an e f f o r t  processes  during  has been  on a n a e r o b i c  their  drugs  on  under  activities  study  of incubated more  fully  a n d t h e mode o f these  to investigate  glycolysis  to  cerebral  t o understand anoxia  neurotropic drugs  approach  drugs  neurotropic  has been  and on t r a n s p o r t p r o c e s s e s  slices,  of certain  investigation  conditions.  the action  of brain  may b e a f f e c t e d  slices  of  and t o  by a v a r i e t y  of  22 conditions. were  The c a t i o n  s t u d i e d under  drugs  and these  brain  metabolism.  the  various  then  following of  acids  t o t h e accompanying  two c h a p t e r s  slices.  i n the cerebral (Chapter  Chapters  out  with  TTX  i n affecting  last  other  chapter  experiments conclusions.  drugs  (Chapter reported  also  carried  3) d e a l w i t h cortex  4 a n d 5) d e a l  i nthe  o u t on  they  8) p r e s e n t s  resemble  with  thesis  The  the effects glycolysis carried  or differ  of the brain a general  the rates  slices.  and on a n a e r o b i c  metabolism  i n this  of various  changes  6 and 7 i n c l u d e experiments  t o s e e how  anoxic  were  (Chapter  TTX on t h e t r a n s p o r t p r o c e s s e s  brain  transport  and glucose.  experiments  glycolysis  o f Na  conditions i n the presence  related  t r a n s p o r t o f amino  anaerobic  and r a t e s  Some e x p e r i m e n t s  Preliminary of  contents,  slices.  from The  discussion of the  and o f the r e s u l t s  and  of  48a  STRUCTURE  OF  Mol.  For  details  see  Mosher  TETRODOTOXIN  Wt.  322  of chemistry et a l  1  1  4  ,  Kao  and 1  1  5  pharmacology and  Evans  1 1 6  .  CHAPTER MATERIALS 2.1 EXPERIMENTAL The guinea  AND METHODS  ANIMALS  animals  used  i n the experiments  were r a t s a n d  pigs.  Adult Wistar r a t s , obtained or  2  either  from  weighing  Columbia.  200-250  g , were  t h e N a t i o n a l L a b o r a t o r y C o . , Edmonton  t h e Department o f Zoology  British  about  V i v a r i u m , The U n i v e r s i t y o f  I n f a n t r a t s were s u p p l i e d b y t h e  Vivarium. A d u l t male g u i n e a p i g s , w e i g h i n g newly born obtained  from  University All until  guinea p i g s , the Animal  of British  the adult  used,  Unit,  300-350 g , a n d  short hair  Faculty  strain,  were  o f M e d i c i n e , The  Columbia.  animals  had f r e e  whereas t h e i n f a n t  the mother s h o r t l y 2.2  of English  about  access  animals  before s t a r t  t o f o o d and water  were s e p a r a t e d  from  o f the experiment.  CHEMICALS All  common l a b o r a t o r y  and were u s e d w i t h o u t  c h e m i c a l s were o f " r e a g e n t  any f u r t h e r  purification.  NAD , NADH, NADP , ATP, ADP, AMP, A d e n o s i n e , +  +  phosphoenol  pyruvate,  lithium  lactate,  cAMP,  epinephrine, norepin-  ephrine,  h i s t a m i n e , t y r a m i n e , LDH ( b e e f h e a r t ) , LDH  muscle),  g l u t a m i c dehydrogenase  kinase  ( r a b b i t muscle)  obtained  (beef l i v e r ) ,  (rabbit  pyruvate  a n d p h o s p h o l i p a s e s A and C were  from Calbiochem,  the product  grade"  Los Angeles.  o f Sigma C h e m i c a l  T r i e t h a n o l a m i n e was  Co., S t . L o u i s .  (HK) a n d g l u c o s e 6 - p h o s p h a t e d e h y d r o g e n a s e  Hexokinase  (G-6-PDH) were  purchased e i t h e r Dibutyryl acetyl  50  -  from Calbiochem  cAMP was  o r f r o m Sigma C h e m i c a l  o b t a i n e d from Schwarz B i o R e s e a r c h  c h o l i n e was  a p r o d u c t o f M a t h e s o n Co.  Co.  and  I n c . , Norwood,  Ohio. The C  14  r a d i o c h e m i c a l s : sodium-22  sulfate  and  glucose-2-C  14  (as c h l o r i d e ) ,  adenine-8-  were t h e p r o d u c t s o f  Radio-  14 c h e m i c a l C e n t r e , Amersham, E n g l a n d . 14 and  glycme-2-C  Co.,  Chicago.  -glutamic acid  were o b t a i n e d f r o m V o l k  T e t r o d o t o x i n was and  U-C  purchased  Radiochemxcal  from Calbiochem.  Ouabain  r e s e r p i n e were o b t a i n e d f r o m N u t r i t i o n a l B i o c h e m i c a l s  Co.; New  protoveratrine York;  f r o m K and  K Laboratories, Plainview,  c h l o r p r o m a z i n e from P o u l e n c , , M o n t r e a l ; d-  1-amphetamines  from Smith, K l i n e  and  French  and  Laboratories,  IAC, M o n t r e a l ; p e n t o t h a l from A b b o t t L a b o r a t o r i e s , M o n t r e a l ; nialamide •  ;  £  obtained British 2.3  from P f i z e r  I n c . , B r o o k l y n , N.  l i Lilly.-,  P r o c a i n e and  Y.  and  lidocaine  f r o m D e p a r t m e n t o f P h a r m a c o l o g y , The  BRAIN  quickly.  The  b r a i n s were d i s s e c t e d  i n c u b a t i o n medium slicing.  The  were a b o u t  discarded. each  ( w i t h o u t any  0.4  O n l y one  mm  slicer  thick  top d o r s a l  h e m i s p h e r e were u s e d .  The  and  additions  cerebral cortex  the S t a d i e - R i g g s t i s s u e  slices  were University  of  SLICES  R a t s were s t u n n e d by b l o w a t t h e b a c k and  with  from  Columbia.  PREPARATION OF  until  amytal  and and  kept i n the except glucose)  slices and  selected  were p r e p a r e d  a moist razor.  thinner one  decapitated  or t h i c k e r  lateral slices  slice were  The ones from  kept  on  an  ice-cold  medium slice used  each  i n each Guinea  until  hemisphere  pig cerebral  as  parietal  lobes  for rats,  i n the  dorsal  weighing  prepared  except  first  from  them.  When  weighing  about  40-55  of the high  cerebral  placed One  cortex  were  because  -  cortex  rate  slices  that  mg,  was  then  the slices  were  guinea  pigs  were  used,  one  mg  used  i n each  was  of glycolysis  and  of each  Cerebral  vessel  slices.  method.  about  only  i n the guinea p i g  different  removed,  slices  c u t o u t and  slightly  born  lateral  prepared i n the  for lateral  (1-3 d a y o l d ) s l i c e s  weighing  60-75  were  r a t brain  portion  and one  about  Infant  were  incubation  vessel.  same way  a  dish  i n the Warburg v e s s e l . from  slice  petri  51  one  slice  After was  hemisphere. 70-90  cortex  guinea pigs  mg,  were  slices  were  slices  used  from  prepared  prepared  decapitation,  c u t from  Two  were  brains  temporal-parietal from  f o r each  older  by  infant  each  brain,  vessel. rats  i n t h e same way  and  as  newly  f o r adult  brain. 2.4  INCUBATION The the  the were  slices  nearest  ation made  mg  medium  were and  weighed then  quickly  suspended  on  a torsion  i n 3 ml  i n the Warburg v e s s e l .  side  arm  during  carried  out using  a t 37°C  percent C0  the incubation  2  i n an  (N :C0 ) 2  2  atmosphere or  95  either  i n from  Incubations  Warburg manometric  o f 95  percent 0 : 2  percent  N :  5 percent  to  incub-  were  or tipped  period.  a conventional  balance  precooled  Additions  a t the beginning of the experiment  apparatus 5  PROCEDURE  2  C0  2  (C^iCC^)  as i n d i c a t e d  52 -  f o r each  experiment.  I n some  experi-  m e n t s , h o w e v e r , t h e g a s p h a s e was c h a n g e d d u r i n g t h e incubation. 2.5 INCUBATION  MEDIUM  In the experiments cortex  slices  described i n this  were i n c u b a t e d i n v a r i o u s m e d i a  upon t h e n a t u r e o f t h e e x p e r i m e n t . s o l u t i o n s were made up a t 5 t i m e s and  stored  thesis,  a t 4°C.  The f i n a l  the cerebral  depending  The r e q u i r e d  salt  isotonic concentrations  composition of the v a r i o u s  media a r e g i v e n below. (a) K r e b - R i n g e r Krebs-Ringer  Medium b i c a r b o n a t e medium was u s e d  experiments.  I t c o n t a i n e d 119 mM  CaCl ,  M g S 0 , 1.2 mM  2  1.2 mM  Krebs-Ringer CaCl (pH  and M g S 0  2  7.4) was  4  phosphate  that  occur before  5 mM  and 25 mM  4  K C l , 3.6  mM  NaHC0 . 3  medium c o n t a i n e d K C l , N a C l , Na-phosphate  added i n p l a c e o f NaHCO-j.  buffer  Phosphate  buffer  i n c u b a t i o n medium a t t h e e n d t o  the p r e c i p i t a t i o n may  2  a s a b o v e , b u t 10 mM  was a d d e d t o t h e f i n a l avoid  KR" P0  4  NaCl,  f o r anaerobic  of C a  + +  and M g  + +  (as p h o s p h a t e s ) ,  dilution.  ++ (b) Ca  -free  Medium  I n most o f t h e a n a e r o b i c e x p e r i m e n t s medium u s e d Quastel . 7 1  and  29 mM  the C a  + +  -free  was s i m i l a r t o t h a t d e s c r i b e d by Adams a n d I t c o n t a i n e d 149 mM N a , 5 mM K , 125 mM C l ~ +  —  HCO^-  as, under these  Ca  ++  and Mg  conditions,  ++  +  were o m i t t e d f r o m  anaerobic g l y c o l y t i c  showed g r e a t e s t s e n s i t i v i t i e s  t h e medium rates  to v a r i o u s types o f neuro-  -  tropic  53 -  d r u g s and gave t h e most c o n s i s t e n t  results.  Omission  o f p h o s p h a t e h a d no a p p a r e n t e f f e c t i n o u r e x p e r i m e n t s . (c) K - f r e e  Medium  +  K -free  medium was p r e p a r e d b y o m i t t i n g  +  Ca  + +  -free  2 9 mM (d)  +  154 mM N a , 125 mM +  C l ~ and  C l - f r e e Medium - f r e e medium was p r e p a r e d by u s i n g  and K  cations SO^ (e)  I t contained  HCO~.  Cl Na  medium.  K C l from the  instead  +  of chloride s a l t s .  was t h e same a s t h a t  was u s e d  of C a  + +  sulfate salts of  I t s composition f o r  -free  medium b u t t h e  a t h a l f the concentration  of C l .  L i " " - c o n t a i n i n g Medium l  The  composition  o f L i - c o n t a i n i n g medium was s i m i l a r +  ++ to  that  o f Ca  was r e p l a c e d specified  - f r e e medium e x c e p t by L i C l .  that  some, o r a l l , N a C l  The c o n c e n t r a t i o n s  i n the experiments described  of L i  +  are  below.  (f) H i g h - K  +  Medium  High-K  +  medium was p r e p a r e d by r e p l a c i n g  some, o r a l l ,  N a C l by K C l , o r by r e p l a c i n g N a C l a n d N a H C 0 b y K C l and ++ + KHCO^, i n t h e C a - f r e e medium. I n some c a s e s , where Na 3  was a l s o  t o be r e d u c e d  increasing of (g)  salt  K  +  i n concentration  a t t h e same t i m e ) ,  solution)  was a d d e d t o m a i n t a i n  Na -free  Medium  Na -free  medium c o n t a i n e d  +  +  10 mM  sucrose  tris-HCl buffer  26 0 mM  (pH 7 . 4 ) .  (without (twice  further the m o l a r i t y  tonicity.  sucrose,  6 mM  K C l and  -  2.6  PREPARATION Kidney the  medulla  f o r the  blow  then  on  and  head  each  used  and  quickly the  discarded. mm  was  as  experiments.  the  removed  0.5-1  MEDULLA  preparation  Subsequently  2.7  KIDNEY  glycolysis  used by  OF  54 -  Two  thick  and  the  the  kidney  tissue;  was  cortex of  weighing  tissue  following  viscera  each  halves  control  The  of  and  a  was the  rats  into  cut  were  two  120-140  mg,  was  were  equal the  medulla,  of  stunned  kidneys  o f f from  kidney  about  procedure  opened;  cut  i n some  halves. medulla  each  were  used  in  vessel.  PREPARATION The brains along  OF  rats  CAUDATE  were  quickly fissura  NUCLEUS  stunned  removed,  by  a  each  blow  brain  longitudinalis  at was  cerebri  the  back  and  the  cut  into  two  halves  and  the  caudate  250 nucleus the  was  located  caudate  nucleus  quickly 2.8  and  PREPARATION Acetone  placed OF  and was  then  i n the  ACETONE  powder  dissected cut  into  Warburg  POWDER  extracts  out.  Each  half  three pieces,  of weighed  vessel.  EXTRACTS  of  the  rat cerebral  cortex  was  251 252 prepared  by  the  brain  cortices  After  filtering  the  resulting  4°C  until 100  method  of  Harpur  and  were  removed  and  and  further  washing  powder  was  stored  Quastel  ground  '  .  in ice-cold  with  ice-cold  i n a vacuum  Rat  acetone. acetone,  dessicator  at  10  ml  used.  mg  of  a  mM  cysteine  of  the  solution, and  acetone  usually 156  mM  powder  was  extracted  containing  100  NaCl.  extract  The  mM  with  nicotinamide, was  33  centrifuged  -  and on  1 ml o f s u p e r n a t a n t was t a k e n i n e a c h v e s s e l . dilution  t o 3 m l , gave a f i n a l  nicotinamide, additions 2.9  55 -  11 mM  cysteine  concentration  a n d 52 mM N a C l .  o f 33 mM  Other  were made a s i n d i c a t e d i n t h e r e s u l t s .  PREPARATION OF SYNAPTOSOMES 253 The the  method d e v e l o p e d  by W h i t t a k e r  254 '  a s shown i n  Scheme 2 was u s e d f o r t h e p r e p a r a t i o n  After  separation  gradient,  o f synaptosomes.  o f s u b c e l l u l a r p a r t i c l e s on a  the various of a pasteur  help  the  f r a c t i o n having density the residue  used f o r experiments  density  l a y e r s were c a r e f u l l y r e m o v e d  the  sucrose,  2.10  This,  pipette.  with  After centrifugation of  b e t w e e n 0.8 a n d 1.2 M o f  ( s y n a p t o s o m e s ) were r e s u s p e n d e d a n d and p r o t e i n  determination.  PREPARATION OF MICROSOMES AND ASSAY OF N a , K - A T P a s e M i c r o s o m e s f r o m g u i n e a p i g b r a i n were p r e p a r e d by t h e 255 +  method o f H o k i n a n d Y o d a after decapitation in  an a l l g l a s s  + 2 mM E D T A ) .  .  Brains  were r e m o v e d  homogenizer  i n Sucrose-EDTA  The homogenate was f i r s t  a t 700 g and t h e s e d i m e n t d i s c a r d e d .  was  centrifuged  resuspended The 15  min.  again  a t 105000 g i n r o t o r  The r e s i d u e  residue  model  The r e s i d u e  was  homogenization.  was c e n t r i f u g e d  was d i s c a r d e d  f o r5  The s u p e r n a t a n t  30 i n a S p i n c o  i n 0.25 M s u c r o s e w i t h hand  suspended r e s i d u e  (0.25 M s u c r o s e  centrifuged  u l t r a c e n t r i f u g e f o r 10 m i n .  centrifuged  quickly  a n d c e r e b r a l h e m i s p h e r e s were h o m o g e n i z e d  min  L preparative  +  a t 8500 g f o r  and t h e s u p e r n a t a n t was  f o r 20 m i n a t 105000 g .  The r e s u l t i n g  ( m i c r o s o m a l f r a c t i o n ) was s u s p e n d e d i n s u c r o s e -  - 56 -  Scheme 2 F r a c t i o n a t i o n o f Synaptosomes Gradient  by S u c r o s e  Centrifugation  4 g w h o l e b r a i n (3 r a t s ) + 0.32 M s u c r o s e (36 m l ) , h o m o g e n i z e , 1725 r e v o l u t i o n s p e r min i n a t e f l o n h o m o g e n i z e r . 5 up a n d down s t r o k e s . C e n t r i f u g e a t 1000 g f o r 10 m i n .  P r e c i p i t a t e (Ppt) +0.32 M s u c r o s e (32 ml) s u s p e n d and c e n t r i f u g e a t 1000 g f o r 10 m i n  4.  S u p e r n a t a n t (Sup)  4, Sup.  Ppt.  Rotor  35 m l x 2 # 30 S p i n c o , 15000 g 45 m i n  4 Ppt. + 0.32 M s u c r o s e 2 ml x 2, h a n d h o m o g e n i z e (8 ml) 4 6 ml L a y e r 1 m l o v e r 0.8 M s u c r o s e , 2 m l + 1.2 M s u c r o s e , 2 ml a n d c e n t r i f u g e f o r 45 m i n a t 100,000 g i n r o t o r # SW39L (without brakes) A  B  Fraction  Fraction  C  4 Sup.  4 2 ml  Fraction  E q u a l Volume o f 0.3 2 M s u c r o s e , t h e n c e n t r i f u g e a t 100,000 g f o r 30 m i n 4 Ppt, SYNAPTOSOMES.  4 Supt. S u s p e n d i n 0.32 M s u c r o s e a n d u s e .  -  EDTA c o r r e s p o n d i n g used  either The  t o 0.5  f o r assay  tubes  57  -  ml/g  of o r i g i n a l  immediately  f o r assay  or  o f ATPase c o n t a i n e d  imidazole-HCl buffer  Na ATP, 2  20  min  80 mM at  N a C l and  37°C.  60  (pH mM  A l l assays  and  other  The  r e a c t i o n was  10%  trichloroacetic  7.1),  KCl.  centrifuged  and  terminated acid  by  2 mM  (TCA),  p h o s p h a t e was  the  ml  yg  was  of  2  2  tubes  measured  was  in duplicates  i n the  addition  the  mM  Incubation period out  -20°C.  protein),  MgCl ,  indicated the  0.5  (25  were c a r r i e d  a d d i t o n s were made as  and  stored frozen at  medium c o n t a i n i n g m i c r o s o m a l s u s p e n s i o n 80 mM  brain  results.  o f 0.5  were  i n the  ml  of  cooled, supernatant  256 by  t h e method o f S t a n t o n  time  data,  actual from 2.11  the  zero time  these  values  and  From o p t i c a l  density  l i b e r a t e d were t h e n  and  calculated  GLYCOLYSIS  Manometric  estimated  by m e a s u r i n g c a r b o n  dioxide  glycolysis  (C0 )  phere of N : C 0 . 2  phase, each molecule  easily  of C0  2  2  i n t h e medium and  of l a c t i c i n the gas  measured w i t h  acid  assays,  i n the  produced  phase.  C0  atmos-  2  gaseous  will  s i n c e one  can  measure  release  so e v o l v e d  t h e h e l p o f t h e manometer.  m a n o m e t r i c method i s much more c o n v e n i e n t lactate  i n an  from  I t i s w e l l known t h a t when t h e r e i s  2  e q u i l i b r i u m between C 0  molecule  was  evolution  2  a b i c a r b o n a t e medium i n t h e W a r b u r g a p p a r a t u s  be  vs  standards.  Lactate production during anaerobic  one  (OD)  i n t e r c e p t s were d e t e r m i n e d  amounts o f p h o s p h a t e  MEASUREMENT OF (a)  .  than  lactate  the  can  The actual  production  -  continuously Unless  over  was  carried  were  taken  the to  ymoles  after  readings  of lactate t o 22.4  established  that  obtained (b)  by d i v i d i n g litres  by manometric  readings  f o rone hour.  Results  (by c a l c u l a t i o n  b y 22.4  a t NTP).  cortex slices  and e q u i l i b r a t i o n  constants)  product  After  were  (1 m o l e  I t has been  converted o f a gas well  during anaerobic  i s lactate  and enzymatic  from  glyco-  and that  values  closely  agree,  assays  Enzymatic For  medium, this of  f o r the f i r s t  2  Manometric  produced  and f l a s k  t h e major  of cerebral  2  closed  10 m i n .  of lactate  2  N :C0  with  20 m i n p e r i o d  this  corresponds  lysis  gassed  o f 80 m i n  were  i n the incubation bath.  o u t f o ranother  manometric  the experiments  t h e s t o p p e r s were  as y l C0  obtained  were  shaking,  periods  incubation period.  stated,  The v e s s e l s  10 m i n , w h i l e gassing  the entire  otherwise  duration.  58 -  t h e measurement and a e r o b i c a l l y ,  method,  NAD  of lactate  lactate  the enzymatic  i s oxidized  a n d LDH a n d t h e p y r u v a t e  hydrazine-glycine  buffer.  production i n a method  to pyruvate so formed  NADH p r o d u c e d  Na -free +  was u s e d .  In  i n the presence  i s trapped by  c a n be  measured  257 spectrophotometrically For (v/v)  K CC>2 2  in  t h e sample  perchloric u s  -"- 9 n  a total  sample, 1  assay,  1.3  M glycine  or  acid  phenol volume  fluorimetrically.  was d e p r o t e i n i z e d w i t h  and the supernatant  r e d as i n d i c a t o r . o f 2.94  neutralized  The r e a c t i o n  ml, contained  1 ml o f  ml o f h y d r a z i n e - g l y c i n e b u f f e r pH  9.5),  0.11  ml o f NAD  +  6 percent  (40  (0.4  with  mixture,  diluted M hydrazine,  mg/ml) a n d . 0 3 m l  -  of  LDH  (1250 I.U./ml).  temperature increase  The m i x t u r e  i n cuvettes  with  calculated  ally  ml  of hydrazine-glycine buffer  The amount  NADH p r o d u c e d  after  fluorimetrically off.  fluorescent  was m e a s u r e d  (as above),  t h e a d d i t i o n o f enzyme  until  free  with  Amino A c i d  the help  The  25 y l N A D  2  +  a n d 50 y l o f tubes.  was m e a s u r e d  The amount  of lactate  of the standards. UPTAKE  were i n c u b a t e d  f o r 30 m i n i n a  Ca  + +  -  At the end o f the incubation, the s l i c e s  h o m o g e n a t e was l e f t supernatant  precipitate  contained  Efflux  cerebral slices  medium.  In  w a v e l e n g t h w a s 3 6 5 nm a n d  w a v e l e n g t h w a s 4 6 0 nm.  were homogenized w i t h  the  fluorimetric-  the increase i n fluorescence  The e x c i t a t i o n  calculated  The  o f NADH.  i n B a u s c h a n d Lomb c o l o r i m e t e r  MEASUREMENT OF A M I N O A C I D E F F L U X AND (a)  model  of lactate  coefficient  (20 I . U . p e r a s s a y ) ,  samples o r standards  was  a Beckman  i n 2.1 m l , t h e r e a c t i o n m i x t u r e  (2 m g v / m l ) , 2 0 y l L D H  2.12  time.  lactate  and t h e  an Aminco-Bowman s p e c t r o p h o t o f l u o r o m e t e r .  cases,  levelled  path  a t room  cuvette, without the  from the e x t i n c t i o n  some e x p e r i m e n t s  using  these  1 cm l i g h t  A control  s a m p l e , was i n c l u d e d e v e r y  In  was i n c u b a t e d  i n OD w a s f o l l o w e d a t 3 4 0 n m u s i n g  DU s p e c t r o p h o t o m e t e r .  was  59 -  2.5 m l o f 5%  i n cold  two s u p e r n a t a n t s ether  TCA.  f o r 30 m i n a n d t h e n  was t r a n s f e r r e d t o a n o t h e r  washed w i t h  3 ml o f d i e t h y l  (w/v) c o l d  1 ml of cold  were mixed  centrifuged;  tube, the  TCA a n d c e n t r i f u g e d .  and e x t r a c t e d t h r i c e  t o remove TCA.  The  Ether  with  was removed  -  from  the water  after  a  removed.  acids  sulfonated  quantitatively i n  the supernatant after  analyses.  TCA a n d t h e s u p e r n a t a n t f r a c t i o n as  suitable  aspartic  resin  acid,  and alanine)  d i l u t i o nf o r  Conditions  using  - 8% d i v i n y l - b e n z e n e  (type  50 A - p a r t i c l e s i z e  the buffer,  before  acid, on  a 50 c m  copolymer 2 5 - 3 1 y) a t  u s e d w a s 0.2 N s o d i u m c i t r a t e  with  Amino A c i d  glutamic  were s e p a r a t e d  analyzer,  The column was r e g e n e r a t e d w i t h  equilibrated (b)  glycine  polystyrene  exchange  3.3.  was u s e d  with  120 B a m i n o a c i d  50°C; t h e e l u a n t pH  from  (taurine,  + serine,  Beckman model  ion  deproteinized  extract,  analyses.  amino  glutamine a  tube,  and the e x t r a c t  The  f o ramino a c i d  medium was t r a n s f e r r e d  TCA was r e m o v e d  amino a c i d  a i r a n d t h e TCA f r e e  d i l u t i o n , was u s e d  incubation  centrifuge  above  phase by blowing  suitable  The  60 -  buffer,  0.2 N N a O H , a n d  each use.  Influx o f incubation  are given with  t h e r e s u l t s and  22 the 2.13  samples were p r e p a r e d  MEASUREMENT OF N A D The  +  AND  method o f Lowry  and co-workers  extraction  and assay  (a)  Extraction  o f NADH  a  ing  o f NAD  the end of incubation,  dilution of at least 0.5 mM  cysteine.  at  60°C w i t h  one hour  to  t h e NaOH s o l u t i o n  -influx  studies.  NADH  the  At  as f o r Na  +  was u s e d f o r  a n d NADH.  the tissue  1:100 i n i c e - c o l d  was h o m o g e n i z e d a t 0 . 0 2 N NaOH  The homogenate was h e a t e d of homogenization. slightly  before  use.  contain-  f o r 10 m i n  Cysteine  was added  Usually  10-25 y l  -  of the e x t r a c t for  each  after  in  o f t h e t i s s u e was u s e d  +  1:50 d i l u t i o n ,  0.1 M N a S 0 2  6 0°C. is  o f NAD  the end o f i n c u b a t i o n ,  at least  and  1:1000 d i l u t i o n  assay.  (b) E x t r a c t i o n At  61 -  This  t h e t i s s u e was h o m o g e n i z e d ,  i n a m i x t u r e o f 0.01 M H S 0 2  w i t h subsequent  4  h e a t i n g f o r 45 m i n a t  r e d u c e s NADase a c t i v i t y  ordinarily  not disturbing  4  to a point  a t which i t  a l t h o u g h some a c t i v i t y may  26 3 persist  .  dilution (c) A s s a y To tris  Usually  15 y l o f t h e e x t r a c t  was u s e d  f o r each  o f NAD  a n d NADH  +  after  assay.  a 200 yl o f t h e c y c l i n g  mixture, containing  HC1 pH 8.4, 100 mM L i - l a c t a t e ,  0.05 M NH^Ac, 400 yg/ml b o v i n e  tube,  liver  0-30 y l o f t i s s u e  L-glutamic  min.  a n d Lomb  After  t o a b o i l i n g water  cycling, bath f o r 2  T h e t u b e s were t h e n c o o l e d i n i c e a n d 200 y l o f  phosphate:LDH s o l u t i o n  (0.65 M Na H P 0 , 0.15 M K H P 0 2  1.5 y g i / o f r a b b i t m u s c l e t o each  tube.  Subsequently  HC1 was a d d e d .  After  4  temperature  4  and  t h e t u b e s were i n c u b a t e d f o r c o o l e d i n i c e and then  a few m i n a t room  2 m l o f 6 N NaOH was added w i t h i m m e d i a t e t u b e s were t h e n h e a t e d  2  LDH) a n d 200 y g o f NADH was a d d e d  15 m i n a t room t e m p e r a t u r e , 5N  dehydro-  e x t r a c t was a d d e d ;  c y c l i n g was c o n t i n u e d f o r 1 h r a t 37°C. t h e t u b e s were t r a n s f e r r e d  0.2 M  0.3 mM ADP, 5 mM a-KG,  g e n a s e a n d 45 yg/ml b e e f h e a r t LDH i n a B a u s c h colorimeter  1:1000  50 y l o f  temperature, mixing.  a t 60°C f o r 10 m i n , b r o u g h t  The a t room  a n d t h e f l u o r e s c e n c e was r e a d i n an A m i n c o -  -  62 -  Bowman s p e c t r o p h o t o f l u o r o m e t e r . NaOH, t h e t u b e s since  were kept  i n dark  t i l l  t h e f l u o r e s c e n t form o f NAD  destruction  by  linear  formation  w i t h NAD  included  the addition of readings  were  taken,  i s sensitive to  +  light.  As p y r u v a t e  2.14  After  during  cycling  i snot strictly  o r NADH c o n c e n t r a t i o n s , s t a n d a r d s  +  with each  were  s e tof determinations.  MEASUREMENT OF CAMP P R O D U C T I O N 264 The method to  measure  cortex  of Shimizu,  the production  Daly  and Creveling  was u s e d  o f cAMP i n t h e i n c u b a t e d  cerebral  slices.  Cerebral  cortex slices  Warburg v e s s e l ,  were  incubated  f o r 40 m i n i n a  u n d e r a n a t m o s p h e r e o f O^CO,, i n a  Krebs14  Ringer  bicarbonate  sulfate slices  a n d 2 0 mM were washed  medium c o n t a i n i n g 2 yC glucose. i n a cold  After  of  the incubation, slices  5 percent  supernatant of  cold  (w/v) i c e - c o l d  total  50  radioactivity  was  extracted twice with ether was removed by b l o w i n g  a PEI cellulose developed  plate  tube  y lof this  determining  for a  w e r e h o m o g e n i z e d i n 1.5 m l  was removed t o a n o t h e r  cAMP w a s a d d e d .  another  the results.  TCA a n d c e n t r i f u g e d .  for  ether  and were i n c u b a t e d  p e r i o d , as d e s c r i b e d w i t h  After  incubation, the  medium, t r a n s f e r r e d t o  Warburg v e s s e l c o n t a i n i n g drugs, desired  aerobic  o f adenine-8-C  The  a n d 150 y g (100 y l ) solution  while  was  the rest  taken of i t  t o remove TCA, e x c e s s  of  a i r a n d 100 y l was s p o t t e d o n  (Brinkman). The chromatograms were 302 i n 0.25 M L i C l , t h e s p o t s were v i s u a l i z e d by  - 63 UV and the r a d i o a c t i v i t y of cAMP spots determined,  after  s c r a p p i n g , i n 10 ml o f s c i n t i l l a t i o n medium, by a N u c l e a r Chicago Mark  i liquid  s c i n t i l l a t i o n counter.  l a n t medium c o n t a i n e d , i n 3 l i t r e s ,  The s c i n t i l -  e q u a l volumes o f toluene,  dioxan and 95% (v/v) e t h a n o l , 15 g 2 , 5 - d i p h e n y l o x a z o l e , 150 mg 1,4-bis-(4-methyl-5-phenoxaxol-2-yl) g  benzene and 240  naphthalene.  2.15 MEASUREMENT OF ATP LEVELS At the end o f the i n c u b a t i o n , the s l i c e s homogenized i n 6% HCIO^. for of  30 min.  were q u i c k l y  The homogenate was l e f t over i c e  The supernatant  obtained a f t e r  centrifugation  the homogenate was n e u t r a l i z e d w i t h a p r e c a l c u l a t e d  volume o f 5 M I^CO^, the KClO^ p r e c i p i t a t e was c e n t r i f u g e d off  and ATP was assayed i n t h i s supernatant e i t h e r by the 265  s p e c t r o p h o t o m e t r i c method o f Lamprecht and T r a u t s c h o l d 266 or by t h e f l u o r i m e t r i c method o f Greengard these methods u t i l i z e the dehydrogenase system presence NADP  +  .  Both o f  hexokinase-glucose-6-phosphate  coupled t o NADP  +  r e d u c t i o n i n the  o f g l u c o s e and o t h e r c o f a c t o r s .  The amount o f  reduced i s p r o p o r t i o n a l t o the amount o f ATP p r e s e n t .  2.16 MEASUREMENT OF N a 22 0.5 yC  Na  2 2  INFLUX  was t i p p e d i n from the s i d e arm o f t h e  Warburg v e s s e l a f t e r a s h o r t p e r i o d o f p r e i n c u b a t i o n , as i n d i c a t e d i n the experiments  d e s c r i b e d below.  d e s i r e d p e r i o d o f i n c u b a t i o n , the s l i c e s  After a  were removed and  q u i c k l y washed t w i c e , i n a n o n - r a d i o a c t i v e i n c u b a t i o n medium, and  homogenized i n 2.5 ml o f 5% (w/v) c o l d TCA.  - 64 -  After and  one hour  i n the cold,  t h e homogenate was  centrifuged  0.5 m l o f t h e s u p e r n a t a n t w a s p l a c e d i n 10 m l o f  illation Chicago  medium f o rc o u n t i n g r a d i o a c t i v i t y . Mark  I liquid  scintillation  A  scint-  Nuclear  c o u n t e r was  used.  22 The as Na  results  o f Na  influx  experiments  are expressed  , yequivalent/gjA i n i t i a l  wet weight,  and were c a l -  22 c u l a t e d by m u l t i p l y i n g t h e t i s s u e ^ cpm/g t i s s u e ^ , ^ + c p m / y l o f medium 2gy N a  c  o  n  t  e  Na n  volume f 4incubation  4 -  Q  n e  2  fluid  (yequiv/yl)  2 . 1 7 M E A S U R E M E N T S OF N a The  tissue  determined Samples below:  +  AND  K  +  contents of unlabelled  w i t h an atomic  absorption  f o rthese  assays  were p r e p a r e d  the tissue  slices  were removed from  p o i n t e d f o r c e p s and t h e a d h e r i n g  with  filter  paper  3 m l o f 5% c o l d precipitate suitably tissue/ml  i n cold  centrifuged  The a t o m i c  +  a n d 3 8 3 . 3 nm f o r N a  +  as d e s c r i b e d  +  and K  +  was removed  were homogenized i n f o r an hour.  The  f o r K  +  a n d 0.2 mg o f  a b s o r p t i o n was m e a s u r e d a t  and K , +  were  Warburg v e s s e l s  fluid  respectively,  m o d e l 303 a t o m i c - a b s o r p t i o n  contents of Na  were  +  o f fand t h e supernatant  (2 mg o f t i s s u e / m l  forNa ).  2.18 MISCELLANEOUS  with a  spectrophotometer.  calculated  from  standards  s e t of determinations. METHODS  Determination of Protein Protein  was d e t e r m i n e d 48  Farr  the slices  TCA and l e f t  diluted  run w i t h each  (a)  strips;  was t h e n  Perkin-Elmer The  and K  +  spectrophotometer.  with  294.3  Na  and R a n d a l l  by t h e method o f Lowry, Rosebrough,  65  -  (b) D e t e r m i n a t i o n o f W a t e r At  t h e end  Uptake  of the i n c u b a t i o n , s l i c e s  f r o m t h e medium and  excess  removed w i t h  paper  The  slices  difference weight (c) Use  filter  not  of f l u i d strips  were t h e n w e i g h e d on between the i n i t i a l  was  of  were  a torsion weight  removed  completely  touching the  p r o v i d e d t h e measure o f w a t e r  slices.  balance;  and  the  the  final  uptake.  Cyanide  Whenever c y a n i d e was ized  -  w i t h HCl  t o pH  t o l e t pH  used,  i t s solution  was  neutral-  7.0-7.5 b e f o r e u s e , c a r e b e i n g  go b e l o w  taken  neutrality. 258  (d) M e a s u r e m e n t o f A c i d The  slices  5 percent for  (w/v)  30 min  material. with  Labile  Phosphate  were h o m o g e n i z e d q u i c k l y TCA  and  f o r complete Adenine  t h e homogenate was  left  extraction  soluble  50 mg  Norit A  treatments w i t h p y r i d i n e , HCl a t 2 0°C  ice-cold  of acid  n u c l e o t i d e s were a b s o r b e d  approximately  10 min  in  (purified  and  by  in ice  treatment  by s u c c e s s i v e  distilled  water) f o r  a c c o r d i n g t o t h e method o f C r a n e  and  259 Lipmann The  labile  the supernatant  phosphate was  assayed  washed c h a r c o a l w i t h bath  and  subsequent  of the n u c l e o t i d e f r a c t i o n  4 ml  after  of N HCl  cooling  and  treatment  of  in a boiling  centrifuging,  by  in  the water the  260  method o f  Bartlett  (e) M e a s u r e m e n t o f P h o s p h o e n o l PEP  and  pyruvate  (PEP)  p y r u v a t e were d e t e r m i n e d  by  and  Pyruvate  fluorimetric  - 66 -  adaptation 2.19  of  the  REPRODUCIBILITY Each were  OF  values  different Control  days  and  in typical  the  are  given.  Czok  and  Eckert  out  several  RESULTS carried  There  were  variations  i n experiments  with  different  were,  g i v e n below  obtained  of  was  found  experiments  results  mean  THE  experiment  reproducible.  absolute  The  method  are  averages  experiments.  carried  batches  therefore,  The  in  of  always of  times  and  the out  on  animals. carried  several  deviations  out.  values from  CHAPTER  RATE  LIMITING CORTEX  SLICES  rate  limiting  The glucose  metabolism,  glutamate,  acetone  were  powder  3.1 E F F E C T S CORTEX The  rate  ACETONE  factors  such  operating  i n anaerobic  A T P , AMP,  +  and cations,  extracts  G L Y C O L Y S I S OF C E R E B R A L  POWDER E X T R A C T S  as NAD ,  carried  out both  CAMP,  will  now b e  with  brain  cerebral  citrate,  considered. slices  and  of brain.  O F C A L C I U M ON T H E A N A E R O B I C  G L Y C O L Y S I S OF CEREBRAL  SLICES  effect of Ca  discussed  IN ANAEROBIC AND  aspartate  Experiments  the  FACTORS  3  +  i n Chapter  on c e r e b r a l  +  1.  o f anaerobic  Results  g l y c o l y s i s has been  given  i n Figure  g l y c o l y s i s decreases  1 show  that  progressively  ++ with  time.  medium obic is  Furthermore,  has a marked  the addition  stimulatory  g l y c o l y s i s o f guinea  i n agreement  i  with  Ca  t o a Ca -free'  e f f e c t on t h e r a t e  p i gcerebral  earlier  o f 4 mM  ++  findings  cortex  o f anaer-  slices.  of Quastel  This  and co-  70,71  workers The  effects  ++ of rat  Ca  greater  of different  concentrations  ++ t o a Ca  o r guinea  Figure  of the addition  2.  -free  medium,  p i gcerebral  I t i s clear  stimulatory  guinea  p i gcerebral  guinea  p i gbrain  effect  on t h e r a t e  from  on a n a e r o b i c  cortex these  slices  are given i n  results  that  e f f e c t on t h e a n a e r o b i c cortex  slices  the presence  than  o f even  of anaerobic  glycolysis of  Ca  +  has a  glycolysis of  on t h a t 1 mM  +  Ca  +  of rat. +  has a  g l y c o l y s i s whereas  With marked  i n r a t i t  - 68 -  FIGURE  1  EFFECT OF CALCIUM ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX SLICES 150,  ,  Time,in  minutes  A l l v e s s e l s c o n t a i n e d 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o time and l a c t a t e p r o d u c t i o n was measured m ^ n o m e t r i c a l l y as giv<piji i n t h e m a t e r i a l s and methods.( O ) Ca - f r e e medium; ( @ ) 4 mM Ca added t o a Ca - f r e e medium.  FIGURE  EFFECT  69 -  2  OF DIFFERENT CONCENTRATION OF CALCIUM GLYCOLYSIS OF CEREBRAL  CORTEX  ON THE  ANAEROBIC  SLICES  150 •H  -P O  0  I  I  I  t  0  1  2  3  Calcium  added,in  I  4  mM  I n c u b a t i o n s w e r e c a r r i e d o u t i n a C a + ± f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o t i m e and l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and m e t h o d s . ( B ) Rat; ( ® ) G u i n e a p i g .  -  is  less  effective.  anaerobic  70  -  The a c c e l e r a t i n g  glycolysis of brain  action  slices  o f Ca  on t h e  i s i n contrast  to  that  71 on Ca  other +  slices.  reduces  guinea  Ca  +  on  newly  born  Table  1.  of that  of Ca  glycolysis  pig  shown  This  to a Ca  +  + +  effect of  glycolysis of  -free  medium  glycolysis i n incubated r a t  of different  concentrations  g l y c o l y s i s o f immature  cerebral  i n Table  o f 1-3  .  slices.  the anaerobic  Results  +  of the addition  guinea  slices  on t h e a e r o b i c  of aerobic  p i g brain  results  +  as tumour  Addition  the rate  The of  such  i s the reverse  +  brain  or  tissues,  cortex  1 show  that  day o l d r a t b r a i n  slices  r a t , and  a r e shown i n  the rate  of  anaerobic  i s practically  insensitive  to  the addition of d i f f e r e n t concentrations u p t o 4 mM o f ++ ++ Ca . Some s t i m u l a t i o n b y C a o c c u r s i n 14-day o l d r a t brain. the  With  newly  presence  of Ca  accelerating  born + +  a t 2 mM  approaching  those  slices.  that  the brain  like  a mature  is  very  with  and  +  +  has a very  brain.  guinea known  i n metabolism would This  will  be  further  glycolysis.  300%, w i t h guinea with  values p i g cerebral our  p i g i s more that  i n i t sneurophysiological and i t would  slices,  striking  i s consistent  I t i s well  r a t brain,  brain.  than  f o r the adult  born  cortex  of anaerobic  i s more  finding  o f a newly  differences  adult  Ca  obtained  This  immature  the adult  major  however,  p i g cerebral  e f f e c t on t h e r a t e  Stimulation  cortex  ,  guinea  or  infant  between  discussed  less  r a t brain  character  be e x p e c t e d exist  knowledge  compared that infant  i n Chapter  8.  71 TABLE  E F F E C T OF OF  I N F A N T RAT  C A L C I U M ON AND  THE  1  ANAEROBIC  GLYCOLYSIS  GUINEA P I G CEREBRAL CORTEX  SLICES  Lactate produced, ymoles p e r g i n i t i a l wet wt (20-80  min)  Additions  2-day o l d rat  24.6  Control  7-day o l d rat  14-day o l d rat  Newly born guxnea p i g  ± 1.8  2 2 . 8 ± 1.4  24.5 ± 1.4  32.2 ± 4 . 5 92.5  Ca  + +  ,  ImM  2 4 . 1 + 1.4  24.5 ± 3.5  30.5 ± 5.3  Ca  + +  ,  2mM  32.2 + 4 . 5  25.0  ± 2.7  42.0 ± 5.8  Ca  + +  ,  4mM  28.6  ± 1.7  29.2 ± 5 . 3  ±12.5  137.5 ± 6.7  3 3 . 5 ± 3 . 5 117.5 ± 4 . 1  I n c u b a t i o n s were c a r r i e d o u t i n a Ca f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods.  -  3.2 E F F E C T S OF E X T E R N A L L Y  72 -  ADDED NAD+ AND A T P ON THE  G L Y C O L Y S I S OF C E R E B R A L Various are  needed  CORTEX S L I C E S  i . e . NAD+, N A D H , A T P a n d A D P ,  f o r the operation  glycolytic rate  coenzymes,  pathway.  of anaerobic  of various  The e s t a b l i s h e d  steps  fact  glycolysis of incubated  time  may b e d u e t o t h e l o s s  coenzymes w i t h  time  of incubation  of cations  The ATP l e v e l and  may b e c o m e  lation  13  aerobic  the  both  anoxia  o f NAD  +  +  marked  o f NAD  + NADH under  facts, the effect  The e x p e r i m e n t s were  2 demonstrate  + +  stimulating  ,  medium, on cortex  carried out  as i n t h e absence o f C a that  together  i n h i b i t o r y e f f e c t on t h e a n a e r o b i c p i gcerebral  .  e f f e c t on t h e p i g cerebral  o f 2.5 mM A T P h a s  however,  + +  the presence o f  r a t and guinea  the addition  ATP + C a  r a t and guinea  +  glycolysis of cerebral  as w e l l  anoxia  Moreover,  constant  of these  effects.  f o rt h e phosphory-  6-phosphate.  but stays  g l y c o l y s i s o f both while  limiting  a n d ATP t o t h e i n c u b a t i o n  has a marked  slices,  factor  the level  I n view  shown i n T a b l e  no e f f e c t .  both  and fructose  was s t u d i e d .  mM N A D  cortex  limiting  of anaerobic  anaerobic  or  rapidly during  i n the presence  Results 0.5  falls  conditions.  rate  slices  i n the brain  during  addition  or to the loss or rate  a rate  slices  o f such  exert  h a s shown t h a t  diminishes  that the  also  o f glucose  Mcllwam  of  which  ofthe  brain  decreases with  gain  ANAEROBIC  cortex  have  little  a  glycolysis of  slices.  73 TABLE  EFFECT  2  OF EXOGENOUS N U C L E O T I D E S  ANAEROBIC  G L Y C O L Y S I S OF CEREBRAL  ON T H E CORTEX  SLICES  ymoles  Lactate produced p e r g i n i t i a l wet wt  (30-90 m i n )  Additions Rat  Guinea p i g  Control  26.4  45.5  ATP,  27.0  56 . 0  ATP, 2.5mM + C a , ImM  12.1  36.8  NAD ,  62.7  60.0  47. 8  57.0  2.5mM  + +  +  0.5mM  N A D , 0.5mM + C a , ImM +  + +  I n c u b a t i o n s were c a r r i e d o u t i n a C a - f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s w e r e made a t 30 m i n a n d l a c t a t e p r o d u c t i o n w a s m e a s u r e d e n z y m a t i cally. R e s u l t s a r e m e a n o f 2-4 d e t e r m i n a t i o n s with v a l u e s w i t h i n ± 7%.  -  3.3  MOVEMENT  OF NAD  Results  exert  I t also  incubation  i n Chapter  effect appears  that  t h a t NAD  nucleotides  membrane b a r r i e r ,  slices  w e l l as aerobic  experiments  as  effects  o f added  w e l l a s NADH c o n t e n t  incubated 3.  under  the  content later  other  hand,  steadily in  first  i t remains  anoxia.  NAD  a  level  relatively  The  NADH  high  level  incubation.  +  +  slices  incubated i n  i s a marked decrease  relatively decreases  i n the  present  +  On t h e  slightly but o f 0.5 mM  NAD  +  concentration  i n the s l i c e ) , remains a t  a n d shows o n l y  however r i s e s  during  constant.  In the presence  t h e i n c u b a t i o n medium, t h e NAD  (measured a s t o t a l  slices,  20 min o f anaerobiosis;  t h e NADH c o n t e n t  during  NAD  c o n d i t i o n s , a r e shown i n F i g u r e  that, i n brain  during  period  3 a n d 4. +  +  +  these  0.5 mM N A D , o n t h e t o t a l  absence o f added NAD , t h e r e NAD  of  anaerobic  o f t h e r a tc e r e b r a l c o r t e x  anaerobic  I t c a n be s e e n  outto  the r a t  under  Results  a r e shown i n F i g u r e s  As i t  t o cross the  were c a r r i e d  place  conditions.  glyco-  tothe  o f NAD+ i n t o  can take  +  may i n f a c t  the brain c e l l .  experiments  cerebral  The  , added  a r e unable  the transport  cortex  slices,  NAD  on t h e r a t e o f a n a e r o b i c  observe whether  as  3.2 s h o w t h a t  m e d i u m , may p e n e t r a t e  believed  cell  MEMBRANE  i n the cerebral cortex  a limiting  lysis.  is  ACROSS THE C E L L  reported  concentration,  74 -  during  a slow  the later  I t c a n n o t be a s c e r t a i n e d  decline. period of  from these  results  - 75 -  FIGURE 3 EFFECT OF EXOGENOUS NAD ON THE NAD +  +  AND NADH CONCENTRATIONS  OF  RAT CEREBRAL CORTEX SLICES UNDER ANOXIA  200  160  •H 4J  •i-i  120 _  C •H  &>  U Q) Ch Cn  3-  <  Q  M O Q  2  10  20 30 40 50 60 I n c u b a t i o n t i m e , i n minutes  I n c u b a t i o n s were c a r r i e d ou£ under N2~eo_ i n a Ca - f r e e medium c o n t a i n i n g 20mM g l u c o s e . N A D ,when p r e s e n t i n t h e i n c u b a t i o n medium,was added a t zero t i m e . ( » ) N A D c o n e , 0 . 5 mM NAD was added t o the medium;( n ) N A D cone.,no exogenous N A D was added to the medium;( * ) N A D H cone, 0.5 mM N A D was added t o the medium; ( O ) N A D H c o n e , n o exogenous N A D was added t o the medium. +  FIGURE EFFECT  OF EXOGENOUS N A D  +  OF A E R O B I C A L L Y I N C U B A T E D  4  ON T H E N A D CEREBRAL  +  AND NADH  CORTEX  SLICES  CONCENTRATIONS FROM RAT  240  O  Incubation  time,in  minutes  present  I n c u b a t i o n s were c a r r i e d ou£ under 02:CO„ i n a Ca - f r e e medium c o n t a i n i n g 20mM g l u c o s e . N A D ,when i n the incubation m e d i u m , w a s a d d e d a t z e r o t i m e . ( © ) NA^ c o n e , n o e x o g e n o u s NAD w a s a d d e d t o t h e m e d i u m ; ( O ) NAD c o n e , 0.5 mM NAD was a d d e d t o t h e m e d i u m ; ( m )NADH c o n e , 0.5 mM NAD w a s a d d e d t o t h e m e d i u m ; ( O ) NADH c o n e , n o e x o g e n o u s NAD w a s a d d e d t o t h e m e d i u m . +  +  -  w h e t h e r some NAD cortex  slices  as  i s e x t e r n a l l y bound t o t h e c e r e b r a l  o r whether i t i s w h o l l y  (an ATP b i n d i n g The  an i n c r e a s e d  During NAD  +  the  aerobic  (Figure first  o f NAD  +  glycolysis  under a n a e r o b i c  conditions  4 ) , t h e NAD  content  i s slightly  that  conditions.  of the tissue f a l l s i n I t then  increases  i n NADH.  In the  +  NADH l e v e l  ).  as w e l l  show  o f 0.5 mM e x o g e n o u s NAD , t h e c e r e b r a l than those  268  i n t h e absence o f added  concomitant decrease  i s higher  +  the c e l l  from b r a i n  i n t h e NADH l e v e l  20 m i n o f i n c u b a t i o n .  slowly with presence  i s a rise  rate of anaerobic  must e n t e r  +  intracellular  p r o t e i n h a s been i s o l a t e d  f a c t s that there  some NAD  77 -  level  of the controls, while the  lower d u r i n g  the i n i t i a l  period. 26 9  It  h a s b e e n shown by Weidemann, Hems a n d K r e b s  t h a t e x t e r n a l l y a d d e d AMP a n d ATP a f f e c t s of r a t kidney. penetrate cell  author,  the content  It +  Permeability  are able t o of brain  a s s u b s t a n t i a l amounts o f ATP b i n d t o 26 8 .  Moreover, washing the  o f e n d o g e n o u s ATP a n d g i v e  i n a diminution lower  values.  E F F E C T S OF N A D  NAD  nucleotides  t o remove t h e bound ATP may r e s u l t  observed 3.4  t h e membrane b a r r i e r .  b r a i n membrane p r o t e i n s  slices of  these  membrane t o ATP h a s n o t b e e n i n v e s t i g a t e d by t h e  present the  Presumably  the metabolism  +  ON AEROBIC GLYCOLYSIS  h a s b e e n shown i n C h a p t e r  stimulates  3. 2 t h a t  the rate of anaerobic  exogenous  glycolysis of  -  cerebral NAD  cortex  slices.  We a l s o s t u d i e d t h e e f f e c t o f  on t h e r a t e o f a e r o b i c  +  brain  slices.  glycolysis operation in  78 -  glycolysis  I t i s known t h a t  i s reduced  the presence  the rate o f aerobic  under a e r o b i c  of the Pasteur  effect  given  DNP-stimulated under these on 3.5  1.2).  of respiratory inhibitors  glycolysis  i n Figure  c o n d i t i o n s due t o  (Chapter  agents such as 2 , 4 - d i n i t r o p h e n o l aerobic  i n incubated  5 show t h e e f f e c t s glycolysis.  c o n d i t i o n s , NAD  or uncoupling  (DNP), t h e r a t e o f  i s considerably  aerobic  However,  increased.  Results  o f e x t e r n a l NAD I t c a n be s e e n  h a s no s t i m u l a t i n g  +  +  on that,  effect  the rate of glycolysis.  E F F E C T S OF CITRATE AND AMP ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX S L I C E S Citrate  glycolytic markedly  i s known t o e x e r t  process  inhibits  inhibitory  effects  and i t has been r e p o r t e d the a c t i v i t y  of  on t h e  that i t  phosphofructokinase  35 xn  xsolated  systems  and  15 mM c i t r a t e  rat  cerebral cortex  inhibition  The the of  1.2).  slices  glycolysis  importance  The  a c t i v i t y of  activity.  o f AMP on t h e a n a e r o b i c slices,  i n connection  6.  by c i t r a t e i s  t h e known s u p p r e s s i v e  rat cerebral cortex  o f 5 mM  glycolysis of  i s shown i n F i g u r e  on p h o s p h o f r u c t o k i n a s e effect  The e f f e c t  on t h e r a t e o f a n a e r o b i c  o f anaerobic  consistent with citrate  (Chapter  with  glycolysis of  a s shown i n F i g u r e  6, i s  i t s stimulation of the  -  79 -  FIGURE 5 EFFECT OF NAD IN THE PRESENCE OF VARYING CONCENTRATION OF 2,4-DINITROPHENOL ON THE AEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES +  150  0.025 Concentration  0.05 0.075 of 2,4-dinitrophenol,in  0.10 mM  I n c u b a t i o n s were c a r r i e d out i n a Krebs-Ringer b i c a r b o n a t e medium c o n t a i n i n g 20 mM glucose,under 0„:CO„.Additions were made a t 30 min and l a c t a t e produced was determined e n z y m a t i c a l l y a t the end o f 90 min i n c u b a t i o n p e r i o d . ( ® ) N A D was not added to the medium;(n)0.5 mM NAD was added t o the i n c u b a t i o n medium.  - 80 -  FIGURE 6 EFFECTS OF CITRATE AND RAT  AMP ON THE ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX SLICES  4J 4->  20  30  40  50  60  70  80  Time, i n minutes ++ I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t z e r o time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , a s g i v e n i n t h e m a t e r i a l s and methods.(O ) c o n t r o l ; ( @ ) 5 m M c i t r a t e ; ( A ) 1 5 mM c i t r a t e and ( B ) 2 mM AMP.  -  activity an  of phosphofructokinase  inhibition  cortex  81 -  of the anaerobic  slices.  I t i s evident  t h a t mask i t s s t i m u l a t i o n 3.6 E F F E C T OF C Y C L I C AMP ANAEROBIC  AND  36  .  The d a t a  g l y c o l y s i s o f the c e r e b r a l t h a t AMP  has o t h e r  AMP  effects  of phosphofructokinase. DIBUTYRYL C Y C L I C AMP  GLYCOLYSIS OF THE CEREBRAL CORTEX  Cyclic  indicate  (cAMP) i s known  ON  THE  SLICES  t o a f f e c t a number o f  enzyme s y s t e m s i n mammalian t i s s u e s , b a c t e r i a  2 7  ^ as w e l l  271 as  those i n plants  .  There i s evidence  of  a number o f hormones m i g h t be m e d i a t e d  that  the a c t i o n  through  cAMp' , 72  273 cAMP i s an a c t i v a t o r o f p h o s p h o r y l a s e diminishes  and i t a l s o  t h e i n h i b i t o r y e f f e c t o f ATP on p h o s p h o f r u c t o -  kinase , causing  a stimulation  of phosphofructokinase  35 activity ian  .  tissues  Brain  has t h e h i g h e s t 274  to synthesise  t o enhance t h e a c t i v i t y studied of are  .  cAMP i s known  of phosphofructokinase,  the guinea p i g c e r e b r a l cortex shown i n F i g u r e  cortex  Since  among mammal-  we  t h e e f f e c t s o f cAMP on t h e a n a e r o b i c g l y c o l y s i s  enhances t h e r a t e slices.  whose a c t i o n has  cAMP  ability  no  7.  slices  and t h e r e s u l t s  I t c a n be s e e n t h a t  o f anaerobic  1 mM  cAMP  g l y c o l y s i s of the cerebral  Under s i m i l a r c o n d i t i o n s ,  dibutyryl  cAMP,  i s s i m i l a r t o cAMP i n a number o f s y s t e m s ,  effect. 275  D i t t m a n n and Herrmann presence and  0.5-1.0 mM  cAMP,  have shown t h a t  the rates  r e s p i r a t i o n of r a b b i t cerebral  creased.  of aerobic cortex  i n the glycolysis  slices  are i n -  -  EFFECTS  OF  CYCLIC  GLYCOLYSIS  "H  60  i  OF  82  r  FIGURE  7  AMP  DIBUTYRYL  AND  GUINEA  PIG  CYCLIC  CEREBRAL  CORTEX  AMP  THE  ANAEROBIC  SLICES  ;  ;  ON  ,  B  o 00  Time  ,i n  minutes  I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y , a s given i n the materials and m e t h o d s . ( O ) c o n t r o l ; ( • )ImM c y c l i c A M P ; ( ® ) 1 mM dibutyryl cyclic AMP.  -  83 -  3.7 E F F E C T S OF VARYING CATION CONCENTRATIONS GLYCOLYSIS OF CEREBRAL CORTEX It  has been mentioned  metabolism i n v i t r o concentration  ON THE  SLICES  i n Chapter  1 that cerebral  i s g r e a t l y i n f l u e n c e d by t h e  of cations  i n t h e i n c u b a t i o n medium. 36  required  f o r the a c t i v i t y  activity  o f pyruvate kinase  .increase  in K  concentration effect  +  increases  greatly  up t o 50 mM  59  on t h e a n a e r o b i c  glycolysis  is  +  The  with  but a high  h a s b e e n shown t o h a v e an  +  K  301 ' .  of pyruvate kinase  concentration  of K  ANAEROBIC  inhibitory  of cerebral  cortex  62 slices ration  .  However, i n t h e l a t t e r  of Na  when t h e K  +  +  study,  the concent-  i n t h e i n c u b a t i o n medium was n o t c h a n g e d  content  was i n c r e a s e d .  Sodium i o n s a r e 5 3a  known t o i n h i b i t  the a c t i v i t y  of hexokinase  pyruvate k i n a s e  ' ^ ' ^ ' ^1^  Hence t h e e f f e c t s o f  varying  3 7  3  3  concentrations  anaerobic  glycolysis  3  of Na  and K  +  and  on t h e r a t e s o f  +  of cerebral cortex  slices  + studied.  The e f f e c t  studied.  The r e s u l t s  Figure  evidence  of K  was  also  a r e shown i n  a t t h e same t i m e d e c r e a s i n g t h e +  rate of c e r e b r a l anaerobic of K , +  marked i n h i b i t o r y  slices.  L i  L i  i n c r e a s i n g the concent-  o f N a , h a s a marked  concentrations has  experiments  indicates that  , while  concentration the  o f these  with  8.  The ration  o f r e p l a c i n g Na  were  +  +  acts  stimulating effect  glycolysis.  some i n h i b i t i o n effect  At very  was o b s e r v e d .  on t h e g l y c o l y s i s  i n a manner s i m i l a r  to Na . +  on  high Na  +  of brain These  data  -  FIGURE  84  -  8  EFFECT OF VARYING CONCENTRATIONS OF K , L i +  ANAEROBIC GLYCOLYSIS OF  +  AND Na ON THE +  RAT CEREBRAL CORTEX SLICES  150  (0  "3  ui 0  i  30 K ,Li  +  +  i  i  i  60 90 120 o r Na C o n e , i n mM  i  150  I n c u b a t i o n s were c a r r i e d o u t i n t h e p r e s e n c e o f 20 mM g l u c o s e . The medium c o n t a i n e d e i t h e r Na ,K +29 mM Na L i + 2 9 mM Na . When r e q u i r e d , t h e medium was made i s o t o n i c w i t h s u c r o s e . L a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y ( o )Na cone.; ( ®)K cone.+ 29 mM Na ; ( • ) L i cone. +29 mM Na . +  +  f  -  show t h a t w h i l e  85 -  considering effects  cation  on c e r e b r a l a n a e r o b i c  ration  of other cations present  medium must a l s o  be t a k e n  3.8 E F F E C T S OF L-GLUTAMATE ANAEROBIC  glycolysis,  into  AND  of a  particular the concent-  i n the i n c u b a t i o n account.  OF AMMONIUM IONS ON  GLYCOLYSIS OF CEREBRAL CORTEX  THE  SLICES. 277  During observed ation  h i s s t u d i e s on g l u t a m i n e  t h a t L-glutamate  of lactic  investigated  acid.  on  addition  of glutamate  glycolysis.  of pyruvate.  who  are active i n  This effect  amino a c i d  n e r v o u s system, and i t s p o s s i b l e e f f e c t s  was  further 27 8  i s reversed  Because o f t h e p o s s i b l e r o l e  a s an e x c i t a t o r y  movements, i t s e f f e c t  form-  by W e i l - M a l h e r b e  D- a n d L - g l u t a m a t e  anaerobic  Krebs  the anaerobic  T h i s phenomenon was  i n greater detail  showed t h a t b o t h inhibiting  inhibits  formation,  i n the c e n t r a l on c a t i o n  on t h e r a t e o f a n a e r o b i c  glycolysis  reinvestigated. R e s u l t s , w h i c h a r e shown i n F i g u r e 9, d e m o n s t r a t e  that of  glutamate  cerebral  inhibits  the rate of anaerobic  cortex s l i c e s  i n a Krebs-Ringer  glycolysis  bicarbonate  ++ medium as w e l l a s i n a Ca effect  o f glutamate  in  + +  a Ca  -containing  - f r e e medium.  However, t h e  i n a C a - f r e e medium i s l e s s (Krebs-Ringer  D - g l u t a m a t e was a l s o e f f e c t i v e  bicarbonate)  than  that  medium.  i n d i m i n i s h i n g the r a t e o f  anaerobic  g l y c o l y s i s but to a lesser extent. 278 + Weil-Malherbe n o t e d t h a t NH^ i n h i b i t s the anaerobic g l y c o l y s i s o f guinea p i g c e r e b r a l cortex  slices.  FIGURE  EFFECTS  OF  GLUTAMATE OF  RAT  AND  9  NH*  CEREBRAL  ON  THE  CORTEX  ANAEROBIC  GLYCOLYSIS  SLICES  I n c u b a t i o n s w e r e c a r r i e d o u t i n a m e d i u m c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o p p t r i c a l l y a s g i v e n i n t h e m a t e r i a l s and methods.( O ) C a -free medium,control;(©) Krebs-Ringer bicarbonate medium,control; ( A ) K r e ^ - R i n g e r b i c a r b o n a t e m e d i u m , w i t h 5 mM L - g l u t a m a t e ; ( A ) C a -free m e d i u m , w i t h 5 mM L - g l u t a m a t e ; ( B ) K r e ^ s - R i n g e r b i c a r b o n a t e m e d i u m . w i t h 5 mM D - g l u t a m a t e ; ( a ) C a - f r e e m e d i u m , w i t h 5mM NH..  -  86  -  -  However,  87  h i s r e s u l t s were  e x p e r i m e n t s .1-3 mM some e x p e r i m e n t s , glycolysis  was  -  not consistent;  NH^" " h a d l i t t l e  o r no e f f e c t w h i l e , i n  1  considerable  observed  inhibition  a t 1 mM  Under or  the given  anaerobic  experimental  no e f f e c t on t h e r a t e  will  be shown  marked the  on c e r e b r a l  later,  latter  NH^" " t o a  Ca  1  + +  -free  g l y c o l y s i s i s s h o w n i n Figure conditions,  i n Chapter  i s stimulated  anaerobic  +  of anaerobic  e f f e c t on t h e r a t e  of  NH^ .  T h e e f f e c t s o f a d d i t i o n o f 5mM medium  i n some  i t has  little  glycolysis.  4, t h a t  of anaerobic  NH*  has  I t  a  g l y c o l y s i s when  by c e r t a i n d r u g s ,  such  as  tetrodotoxin. 3.9  E F F E C T OF N A D ANAEROBIC  +  AND  ATP,  G L Y C O L Y S I S OF C E R E B R A L  The e f f e c t s o f N A D lysis are  of cerebral  shown  glycolysis  3.  slices  i n a Na -free + +  .  as  compared  t o a medium  of  Na  +  and K .  Thus  dependence  3.7), r e p l a c i n g N a stimulatory  containing  the anaerobic  +  As has been by K  +  anaerobic  the rate  i s itself  on t h e p r e s e n c e  medium.  of  of  greatly  normal  reduced  concentration  g l y c o l y s i s shows of cations  shown  a  i n the  earlier  i n the incubation  e f f e c t on t h e r a t e  these  e i t h e r i n the presence  Further,  i n control slices,  incubation  that  glyco-  medium  +  medium,  +  glycolysis,  marked  i n a Na -free  have no e f f e c t on t h e r a t e  THE  SLICES  on t h e a n a e r o b i c  I t c a n be s e e n  i n the absence o f C a  +  CORTEX  a n d ATP  +  cortex  i n Table  nucleotides  or  I N A S O D I U M - F R E E M E D I U M , ON  (Chapter  medium h a s a  of anaerobic g l y c o l y s i s .  9  88 TABLE  3  EFFECTS OF CALCIUM AND NAD ANAEROBIC CORTEX  Addition  +  ON THE  GLYCOLYSIS OF GUINEA P I G CEREBRAL S L I C E S IN A SODIUM FREE MEDIUM  Incubation period  Lactate produced, y m o l e s p e r g i n i t i a l w e t wt  Control  0-30 min  18.1  Control  0-90 min  20.8  Ca  + +  ,  ImM  0-90 min  23.0  Ca  + +  ,  4mM  0-90 min  20 .2  0.5mM  0-90 m i n  24.5  0-90 min  23.5  NAD , +  NAD , 0.5mM + C a , ImM +  + +  I n c u b a t i o n s were c a r r i e d o u t i n a medium c o n t a i n i n g 260mM s u c r o s e , 6mM K C l , 20mM g l u c o s e a n d lOmM T r i s - H C l b u f f e r (pH 7 . 4 ) . L a c t a t e p r o d u c t i o n was m e a s u r e d enzymatically. A d d i t i o n s w e r e made a t 30 m i n . R e s u l t s a r e a v e r a g e s o f 2-4 d e t e r m i n a t i o n s w i t h v a l u e s w i t h i n ± 7%.  -  3.10  RATE L I M I T I N G  89  F A C T O R S OF  -  GLYCOLYSIS  IN  THE  ACETONE  POWDER E X T R A C T S To  maintain  optimal  powder e x t r a c t s , or to  add  various  rates  ground  of  glycolysis  by  acetone  cerebral tissues, i t is  coenzymes and  co-factors  + Thxs  i s necessary  b e c a u s e NAD  lost  q u i c k l y from  the  to  the  necessary  suspensions.  + , ATP,  K  tissue during  and  phosphate  preparation  of  are  the  brain  acetone powder. A l t h o u g h ATPase i s v e r y low i n the 279 28 0 acetone powders , t h e r e i s a h i g h NADase a c t i v i t y in the  brain  For  this  t i s s u e which reason,  p e r s i s t s even  i t i s necessary  to  i n the add  acetone  powders.  nicotinamide,  which  28 0 is  a  strong  inhibitor  Results glycolysis ATP,  Mg  the  rate  given  by  of  K .  Under  +  anaerobic  imately  constant  extract  taken.  glycolysis  i n Figure  by  10  and  the  glycolysis  Moreover, the  acetone  the  This  will  be  further discussed  e f f e c t s of  of Na  and  ATP, +  and ATP  or  such are  NAD  +  the  limiting  rate  of  of  without  approx-  the  rate  of  of  higher  brain  slices.  the  different  8. of  as  When t h e  is  K , +  Na , +  concentration  change of  glycolysis  +  quantity  componenets 11.  NAD ,  the  the  concentration  i s increased, the  of  of  conditions,  extracts i s  i n Chapter  shown i n F i g u r e  concentration,  by  quantity  changing rate  with  p o w d e r e x t r a c t s i s much  a corresponding  +  course  experimental  magnitude  of  NAD  extracts.  time  i t i s proportional to  that  cofactors  show t h e  given  than  The  , i n the  acetone powder e x t r a c t s f o r t i f i e d  and  + +  o f NADase  the  increased.  FIGURE ANAEROBIC  GLYCOLYSIS  BY  90  -  10  ACETONE  POWDER E X T R A C T S  OF  RAT  BRAIN  12.5  Time,  i n minutes  I n c ; u b a t i o n | w e r e c a r r i e d o u t i n a m e d i u m c o n t a i n i n g 52 mM N a ,28 mMK ,4 mM Mg ,33 mM c y s t e i n e , 3 3 mM nicotinamide, 0.5 mM NAD a n d 0.8 mM A T P . L a c t a t e p r o d u c t i o n w a s m e a s u r e d manometrically. A l l t h e v e s s e l s c o n t a i n e d 20 mM glucose. (O)20 mg a c e t o n e p o w d e r e x t r a c t p e r v e s s e l ; ( © ) 4 0 mg a c e t o n e powder e x t r a c t p e r v e s s e l . +  -  Increase  of Na  concentration of Na  K  i s low.  +  has an i n h i b i t o r y e f f e c t and i n c r e a s i n g  +  has a s l i g h t  increased  91 -  i s more  +  These  stimulating  marked  when  experiments  may e x e r t  limiting  effects  anaerobic  g l y c o l y s i s by t h e a c e t o n e  given  i n this  compounds  process  o f g l y c o l y s i s on t h e r a t e s  the cerebral  Ca  +  +  powder  extracts.  describe  limiting  the action o f  effects  of anaerobic  ,  i ti s found  adult  brain.  anaerobic  in  presence  It  i s thought (4)  + +  added  -free  of Ca  that  Movement  studied.  +  +  on t h e  glycolysis  o f acetone  to Ca  NAD  +  +  slices. o f anaerobic  t h e marked  stimulates  +  the rate  medium.  I t appears  that  NAD  +  cross  cerebral  o r no  The a d d i t i o n  of anaerobic  the brain  of  of  o f ATP has l i t t l e  the rate  across  response  to that  i s d u e t o ATP c h e l a t i o n  o f NAD  marked  glycolysis of  i s similar  +  additon  inhibits  o f Quastel and  has a  +  cortex  whereas  incubation  this  +  of anaerobic  r a tb r a i n ,  glycolysis while  i n a Ca  Ca  work  e f f e c t on t h e rate  p i gbrain  Externally  that  p i gcerebral  of infant guinea  (3)  of the earlier  has l i t t l e  infant  effect  on t h e speed o f  and o f e x t r a c t s  e f f e c t on t h e r a t e  glycolysis  been  7 1  r a tand guinea (2)  the  rate  slices  In confirmation  stimulatory  of  have  cortex  coworkers'^'  adult  different  of brain.  (1) his  that  chapter  various  powder  that  of  OF CHAPTER 3  Results  of  +  The e f f e c t  the concentration  demonstrate  cofactors  SUMMARY  rate  effect.  K  cell  of  o f ATP  glycolysis. Ca  + +  .  membrane h a s  the brain  cell  -  FIGURE  92 -  11  E F F E C T OF VARYING CONCENTRATIONS THE  OF RATE LIMITING FACTORS ON  ANAEROBIC GLYCOLYSIS OF BRAIN ACETONE POWDER EXTRACTS 1500 ATP,0.8 mM;NAD ,0.5 mM;Na ,52 mM. +  "g 1250  +  ATP, 0.8 mM;NAD ,0.5mM;Na ,106 +  in  +  mM.  i LD  ATP,0.8,mM;NAD ,0.2 mM, Na ,52 mM.  U  1o  1000  ATP.0.4 mM;Na ,52 mM;NAD ,0.5 mM +  +  04  c o •p o (0  cn 750 u  w O rH  O  500  n O •O O U  Ch <D  +J  250  nJ -P O  10  30  20  40  50  60  K c o n c e n t r a t i o n , i n mM I n c u b a t i o n s were c a r r i e d o u t a s c j i v e n w i t h F i g u r e 10 e x c e p t t h a t t h e c o n c e n t r a t i o n s o f K , Na, NAD and ATP were v a r i e d . +  +  -  93 -  membrane u n d e r b o t h a n a e r o b i c (5) N A D has  +  h a s no e f f e c t  been s t i m u l a t e d (6)  Citrate  anaerobic  by  on t h e a e r o b i c  glycolysis  at  t h e t i m e when K  action  The e f f e c t s  glycolysis  of b r a i n . those  +  +  i s raised, K  of Na  brought  The e f f e c t s  Whilst  inhibitor cortex  +  +  the rate of  when t h e  h a s a marked  stimulatory  glycolysis.  and K  +  are also apparent  during  a b o u t by t h e a c e t o n e powder e x t r a c t s of these  ions  glycolysis  i t i s confirmed  NH^"" a t e q u i v a l e n t 1  a r e n o t as l a r g e as by b r a i n t i s s u e  slices.  t h a t L - g l u t a m a t e i s an  o f the rate of anaerobic  slices,  (100 mM) o f  i n t h e i n c u b a t i o n medium i s d e c r e a s e d  shown i n a n a e r o b i c  (9)  Cyclic  I t i s shown t h a t  on t h e r a t e o f a n a e r o b i c  (8) the  concentration  glycolysis.  of Na  slices.  medium, i n h i b i t s  +  concentration  that  effect.  K , when a d d e d t o a R i n g e r anaerobic  glycolysis  o f the rate of  of cerebral cortex  I t i s known t h a t a h i g h  cerebral  conditions.  2,4-dinitrophenol.  a n d AMP a r e i n h i b i t o r s  AMP h a s a s t i m u l a t o r y (7)  and a e r o b i c  glycolysis  of the cerebral  concentrations  has  no  effect. (10)  I t i s evident  limiting  effects  slices.  In p a r t i c u l a r ,  effects  that a variety of factors exert  on a n a e r o b i c  glycolysis  of cerebral  rate  cortex  a t t e n t i o n i s drawn t o t h e marked  + + ++ o f Na , K and Ca  CHAPTER EFFECTS  OF  TETRODOTOXIN AND  It puffer the  has been fish  neurotoxin,  generation  able  been  i n r e s p i r a t i o n brought  electric  impulses  of  anaerobic  cerebral  of  kidney been  specific of  cortex  these  property  slices  cell  OF  GUINEA  Results  was  of C a  +  powder  whether  membrane  as w e l l  In view  the  will  P I G AND of a  yM T T X o n t h e r a t e  tissue  of the  RAT  typical  ON  on t h e glycolysis  addition,  of brain  have  and i f the i n t e g r i t y  required  for action. light  on  below.  THE ANAEROBIC G L Y C O L Y S I S  CEREBRAL CORTEX experiment,  of anaerobic  In  o u t , t o throw  be d e s c r i b e d  TETRODOTOXIN  rate  as o f r a t  act similarly  extracts  of  t h e e f f e c t o f TTX i s a  i s also  carried  the  from the  +  increases  +  3).  TTX b l o c k s  investigated.  of excitable  of experiments  4.1 E F F E C T S  +  pig,  and TTX m i g h t  +  and acetone  problems,  OF  +  to ascertain  the brain  Results  Ca  of excit-  by a p p l i c a t i o n  as by o m i s s i o n  (Chapter  suppresses  t h e e f f e c t o f TTX on t h e a n a e r o b i c  medulla  used  Ca  TTX, t h e  i n a variety  about  Moreover,  slices  that  tissue,  cerebral  ^.  1 that  shown t h a t  g l y c o l y s i s of guinea  cortex  possibility brain  as w e l l 1 3  METABOLISM  ANOXIA  potentials  I t has also  medium  IN  i n Chapter  increase  incubation  CEREBRAL  i n low c o n c e n t r a t i o n s ,  of action  tissues.  ON  TRANSPORT  mentioned  4  SLICES  showing  the e f f e c t o f 2  glycolysis of r a t cerebral  -  cortex  slices  bicarbonate that the Ca  ++  due  TTX  increases  to the fact  + +  -free  that  m  as i n a  i n Figure  (Figure  glycolysis  containing  i n a Krebs-Ringer  Ca  The with  seen  i n both  i s greater  ++  .  This  same  medium  effects  guinea  i n the  i s partly  of g l y c o l y s i s  bicarbonate  12).  are observed  Krebs-Ringer I t c a n be  increase  the c o n t r o l values  medium  12.  of anaerobic  that  rates  effects  of anaerobic  cortex rate  of three  slices  a r e shown  of anaerobic  (without than i n  o f TTX  on  pig cerebral  glycolysis  i n the absence  cortex  rates  obtained  about  t h e same.  EFFECTS  slices  OF  ANAEROBIC The  i n both  cor-  than  f o r t h e two  CALCIUM  animals,  with  although  the rate  i n guinea  slices;  that  increased  conof  p i g cer-  t h e maximum  i n the presence  TETRODOTOXIN  on  o f TTX, a r e  STIMULATED  GLYCOLYSIS  effects  of addition  calcium,  to a Ca  the  of anaerobic  rate  ON  I t i s evident  i s greater  i n r a t cortex  o f TTX  p i g and r a t c e r e -  13.  increases  the animals, o f TTX  concentrations  of guinea  i n Figure  glycolysis  o f TTX  ebral  different  glycolysis  centration  4.2  as w e l l  slices.  bral the  -  the percentage  glycolysis  The the  the rate  than  are greater Ca  -free,  are given  However,  - f r e e medium  anaerobic tex  medium,  media.  TTX) the  i n a Ca  95  + +  -free  medium  of d i f f e r e n t  concentrations  i n the presence  glycolysis  of cerebral  o f 2yM  TTX,  of on  EFFECTS  OF  FIGURE T E T R O D O T O X I N ON CEREBRAL  80  96  -  12 THE ANAEROBIC  CORTEX  G L Y C O L Y S I S OF  RAT  SLICES  1  .  Time,in  minutes  I n c u b a t i o n s w e r e c a r r i e d o u t i n t h e p r e s e n c e o f 20 mM g l u c o s e . T T X ( 2 uM),when p r e s e n t , w a s a d d e d a t z e r o t i m e a n d l a c t a t e prod u c t i o n was m e a s u r e d m a n o m e ^ r i c a l l y as given i n t h e m a t e r i a l s and m e t h o d s . ( O ) c o n t r o l , C a -free medium;(A)control, K r e b s - R i n g e r b i c a r b o n a t e m e d i u m ; ( ® ) 2 uM T T X p r e s e n t , C a -free m e d i u m ; ( S ) 2 uM T T X p r e s e n t , K r e b s - R i n g e r b i c a r b o n a t e m e d i u m .  - 97 FIGURE EFFECTS  13  OF D I F F E R E N T CONCENTRATIONS  ANAEROBIC  OF T E T R O D O T O X I N  G L Y C O L Y S I S OF C E R E B R A L C O R T E X  TTX  concentration , i n  ++  ON  THE  SLICES  uM  I n c u b a t i o n s were c a r r i e d o u t i n a C a " ' - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . T T X w a s p r e s e n t f r o m t h e s t a r t o f t h e e x p e r i m e n t a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as given i n the m a t e r i a l s and m e t h o d s . ( O ) G u i n e a p i g c e r e b r a l c o r t e x slices; ( 9 ) r a t cerebral cortex slices.  -  cortex  slices  98 -  a r e shown i n F i g u r e  14.  With  guinea  pigs,  ++ the  presence  further values  increase  with  with  acid  the  labile  gives  cell.  were  higher  i n these  results  will  EFFECT CELL It  some  than  increase remain  i s observed; the (as o b t a i n e d  conditions, measured  t h e amount  ( n o t shown),  o f TTX  i n the presence  the corresponding  controls.  tissues  measured  were  OF TETRODOTOXIN  has been  t h e NAD  +  also  i n Chapter ON N A D  shown i n C h a p t e r  +  ATP  of  concent-  and the  5.6.  MOVEMENTS  the  decline from  required  ACROSS  THE  the tissue increase  e f f l u x o f NAD o f NAD  +  +  under  slices  time  anoxia.  decreases  there  As NAD  o f g l y c o l y s i s might into  t h e medium.  the rate  during  level  cortex  the rate o f  i s decrease  +  f o r g l y c o l y s i s , i t seemed  of the rate  TTX might  3 that  A t t h e same  concentration  cofactors  decline  alone.  +  obtained  be d i s c u s s e d  the  the  +  status  of incubation.  that  Ca  MEMBRANE  time  +  4 mM  with  i n d i c a t i o n o f the energy  with  NAD  glycolysis  t h e same  was a l s o  glycolysis of cerebral  of  causes  decreased.  anaerobic  in  with  experimental  The values  rations  4.3  obtained  phosphate  conditions,  of anaerobic  or are slightly  t h e same  this  these  r a t s , no such  TTX alone)  since  that  of glycolysis either  Under of  , under  i n the rate  approaching  However, rates  o f Ca  observed  t o us  that  be due t o l o s s  I t seemed  o f g l y c o l y s i s by  the incubation.  i s one o f  possible  blocking  Although the  b y u s was o n l y  20% d u r i n g  the  FIGURE EFFECTS  OF  ANAEROBIC  99 -  14  D I F F E R E N T CONCENTRATIONS  OF  G L Y C O L Y S I S OF CEREBRAL CORTEX OF T E T R O D O T O X I N  Ca  cone.,in  CALCIUM SLICES  ON  THE  IN THE  PRESENCE  mM  ++  I n c u b a t i o n s w e r e c a r r i e d o u t jj-iji a C a - f r e e medium c o n t a i n i n g 2 0 mM g l u c o s e . 2 uM T T X a n d C a ,when p r e s e n t , w e r e a d d e d a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d manometrically as g i v e n i n t h e m a t e r i a l s a n d methods.  - 100 -  20-60 min i n c u b a t i o n p e r i o d , t h i s l o s s may s t i l l be s i g n i f i c a n t , i f i t occurs o n l y from the e x t r a m i t o c h o n d r i a l compartment  (cytoplasm) where g l y c o l y s i s i s t a k i n g p l a c e .  However, i n the presence of 2 yM TTX, c o n t r a r y t o the above assumption, no i n c r e a s e i n the NAD c e r e b r a l c o r t e x s l i c e s was observed. NAD  +  +  l e v e l o f the  We a l s o assayed the  l e v e l i n the medium a t the end o f the i n c u b a t i o n  p e r i o d , by the c y c l i n g method o f Lowry c o u l d be d e t e c t e d  2 61 ~ 2 6 3 , but no NAD  i n the i n c u b a t i o n medium  H~  (results not  shown). The  presence o f NAD  +  i n the i n c u b a t i o n medium was 284  a l s o t e s t e d by the method o f Q u a s t e l  and Wheatley  attempts t o show i t s presence were n o t s u c c e s s f u l . these experiments l e d t o the f i n d i n g t h a t some  but However,  lactic  dehydrogenase appears i n the medium i n which the c e r e b r a l c o r t e x s l i c e s are i n c u b a t e d .  Stern, Eggleston,  Hems and  2 85 Krebs  have shown t h a t g l u t a m i c - a s p a r t i c transaminase  d i f f u s e s from i n c u b a t e d medium.  guinea p i g c o r t e x s l i c e s i n t o the  I t thus appears t h a t c e r t a i n enzymes may leak  i n t o t h e medium from i n c u b a t e d  b r a i n s l i c e s but i t i s  u n c e r t a i n whether t h i s i s simply  due t o the breakdown o f  damaged c e l l s . 4.4  EFFECTS OF TETRODOTOXIN  ON THE AMINO ACID  EFFLUX FROM THE CEREBRAL CORTEX SLICES As has been shown i n Chapter 4.3, the p o s s i b i l i t y the e f f e c t o f TTX on the a n a e r o b i c  that  g l y c o l y s i s might be due t o  -  blocking  of  conditions ration  of  NAD  anoxia,  ATP,  active  Hence,  intermediates incubation  of  to  -  been  owing  to  ruled  the  transport  the  For  thus  example,  increase  the  the  compounds  that  of  anaerobic  If  TTX  of  in  some o f  into  rate  under  concent-  diminish  out  like  However, cell  exist  leak  reduce  rate  in  mechanisms  possibilities  and  out.  fall  g l y c o l y s i s might  medium  glycolysis. known  e f f l u x has  of  activity.  101  the  the  anaerobic  pyruvate  are  glycolysis  of  71 cerebral such  cortex  compounds  indirectly,  slices from  an  .  the  slices,  accelerating  blocks  then  the  i t may  e f f e c t on  the  efflux  of  exert,  rate  of  anaerobic  glycolysis. Energy activity rated leak  during  against into  acids  brain  cell  TTX,  in  are  as  and  the  a  present in  gradient  with  that  in this  The  the  TTX  acids.  of  may  the  slices show  tend  transport  movements are  and  of . the  the  be  glutamic  large  that  at  more acid  amounts.  this  to  affected  influence  e f f e c t may  in  concent-  brain  movement  acid  in  acids  active  also  This  section  in  cation  these  aspartic  brain  diminish  r e s u l t amino  because  amino case  processes  medium.  associated  reported  The  conclusion  correct. The  as  is  e f f l u x of  these  results is  incubation  membrane  pronounced  and  concentration  i t is possible  cerebral  as  transport  anoxia  a  the  amino  by  dependent  in  the  amino  acid  incubation  concentrations medium,  in  the  in  the  tissue,  presence  and  as  well  absence  of  - 102 -  2 yM TTX a r e g i v e n i n T a b l e 4.  I t can be seen t h a t the  c o n c e n t r a t i o n s of a number o f amino a c i d s a r e h i g h e r i n the i n c u b a t e d s l i c e s i n the presence o f TTX than i n i t s absence.  The most marked e f f e c t o c c u r s w i t h g l u t a m i c  and a s p a r t i c a c i d s .  Thus, f o r these a c i d s , the t i s s u e /  medium r a t i o s , i n the presence of g l u c o s e  are 1, w h i l s t  i n the a d d i t i o n a l presence o f TTX the r a t i o s a r e 2.5 and 2.0 r e s p e c t i v e l y .  The same e f f e c t of TTX was observed  when g l u c o s e i s absent from t h e i n c u b a t i o n medium although to a l e s s e r e x t e n t .  T h i s w i l l be d i s c u s s e d f u r t h e r i n  Chapter 8. 4.5 EFFECT OF TETRODOTOXIN UNDER ANAEROBIC  ON THE UPTAKE OF AMINO ACIDS  CONDITIONS  As TTX d i m i n i s h e s the e f f l u x o f amino a c i d s  from  i n c u b a t e d c e r e b r a l c o r t e x s l i c e s under a n a e r o b i c c o n d i t i o n s (previous s e c t i o n ) , i t was d e c i d e d t o study t h e r a t e s o f uptake o f amino a c i d s  (glutamic a c i d and g l y c i n e ) under the  14 same c o n d i t i o n s .  C  - l a b e l l e d amino a c i d s were used f o r  these experiments, r e s u l t s o f which are shown i n T a b l e 5. When no c a r r i e r  ( u n l a b e l l e d ) amino a c i d i s p r e s e n t i n the  i n c u b a t i o n medium, the r a d i o a c t i v i t y taken up by t h e s l i c e s i s more than doubled by t h e presence of TTX. A s i m i l a r e f f e c t i s o b t a i n e d i n the presence o f c a r r i e r amino a c i d s .  Glutamic a c i d i s m e t a b o l i z e d i n the b r a i n  t i s s u e but the i n c r e a s e d uptake o f r a d i o a c t i v e g l u t a m i c a c i d i n these experiments  can n o t r e s u l t wholly from the metabolism.  10 3 TABLE EFFECTS OF TETRODOTOXIN  ON AMINO ACID  CONTENT OF RAT CEREBRAL CORTEX SLICES UNDER ANOXIA  Additions Amino Acids  +Glucose -TTX  +Glucose +TTX  -Glucose -TTX  -Glucose +TTX  TAURINE  Tissue Medium  2.25 + .01 3.21 ± .01  2.3 ± .05 3.76 ± .34  2.34 ± .03 3.26 ± .34  2.39 ± .03 2.97 ± .20  ASPARTIC ACID  Tissue Medium  0.96 ± .05 1.24 + .2  1.65 ± .02 0.82 + .17  1.11 ± .04 1.47 ± .20  1.55 ± .20 1.14  GLUTAMINE + SERINE  Tissue Medium  1.31 ± .07 3.07 + .22  1.60 ± .10 2.52 ± .10  1.01 3.27 ±  1.16 ± 2.8 ±  GLUTAMIC ACID  Tissue Medium  6.67 ± .5 6.10 ± .34  9.1 ± .20 3.54 ± .44  5.63 ± .06 7.25 ± .65  7.68 ± .80 4.60 ±1.2  GLYCINE  Tissue Medium  0.41 + .05 0.62 ± .04  0.57 ± .02 .46 ± .04  0.41 ± .07 0.72 ± .18  0.42 ± .02 0.56 ± .02  ALANINE  Tissue Medium  0.36 ± .05 0.64 ± .02  0.56 ± .02 0.61 ± .01  0.36 ± .23 0.83 ± .17  0.33 ± .11 0.54 ± .10  .07  Incubations were c a r r i e d out f o r 30 min i n a C a ~ f r e e medium under N :C022uM TTX or 20mM g l u c o s e , when p r e s e n t , were added a t zero time. A t the end of i n c u b a t i o n , t i s s u e as w e l l as medium was a n a l y z e d f o r amino a c i d s , as g i v e n i n the m a t e r i a l s and methods. R e s u l t s a r e expressed as ymoles o f amino a c i d p e r g i n i t i a l wet wt o f the s l i c e s . +  2  1 5  104  TABLE  5  EFFECTS OF TETRODOTOXIN ON THE UPTAKE OF AMINO ACIDS UNDER ANAEROBIC CONDITIONS BY RAT CEREBRAL CORTEX SLICES A.  Uptake o f U - C  14  L-Glutamate 15 min I n c u b a t i o n  Additions r  NO TTX  WITH 2yM TTX  B.  Uptake  Amino acid uptake  No carrier  cpm p e r wet wt  3 7 7 00 ' 0  umoles p e r g wet wt  cpm p e r r wet wt  0  lh  0  Q  1  >  Q  0  4  178,750 ' 4.18  9  815,000 '  ymoles p e r g wet wt  o f 2-C  >  w i t h 5mM L-Glutamate  241,700 ' 5.56  2  30 min I n c u b a t i o n No carrier  w i t h 5mM L-Glutamate  466,000 '  221,500 5.16  0.0024  1080,000  293,000 ' 6.85  ,0055  Glycine  15 min I n c u b a t i o n  Additions  NO TTX  WITH 2yM TTX  Amino acid uptake cpm p e r g wet wt ymoles p e r g wet wt cpm p e r g wet wt ymoles p e r g wet wt  No carrier  w i t h 2mM Glycine  186,500  133,750  0.022 377,500 0.044  1.85 207,000 2. 86  10 4A TABLE  5  (Continued)  I n c u b a t i o n s were c a r r i e d out f o r 15 min i n a Ca f r e e medium c o n t a i n i n g 20mM g l u c o s e under N2:C02« L a b e l l e d amino a c i d s , w i t h or without c a r r i e r were t i p p e d i n from the s i d e arm o f the Warburg v e s s e l . TTX, when p r e s e n t , was added a t zero time. Total cpm per ml o f the medium were 214000 f o r C glutamate and 145000 f o r C - g l y c i n e . Amino a c i d uptake was c a l c u l a t e d by d i v i d i n g cpm/g by s p e c i f i c a c t i v i t i e s and were not c o r r e c t e d f o r s w e l l i n g o r i n t r a c e l l u l a r space. Each v a l u e r e p r e s e n t averages of d u p l i c a t e d e t e r m i n a t i o n s w i t h i n ± 7%. lh  1 4  - 105 -  because s i m i l a r r e s u l t s a r e o b t a i n e d w i t h g l y c i n e which metabolizes  a t a much slower r a t e than an e q u i v a l e n t  q u a n t i t y o f glutamate. acid  (e.g., c o n v e r s i o n  under anaerobic  Moreover, the metabolism o f g l u t a m i c t o glutamine) i s g r e a t l y reduced  c o n d i t i o n s , and there i s normally  no uptake o f amino a c i d s a g a i n s t a c o n c e n t r a t i o n  l i t t l e or gradient  under such c o n d i t i o n s . 4.6 EFFECT OF TETRODOTOXIN AT DIFFERENT GLUCOSE CONCENTRATIONS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES When the r a t e o f g l y c o l y s i s i s h i g h , the r a t e o f glucose  e n t r y i n the c e l l may become r a t e l i m i t i n g .  can be t r u e under anaerobic amount o f g l u c o s e  c o n d i t i o n s , as a c o n s i d e r a b l e  i s almost e x c l u s i v e l y m e t a b o l i z e d  through the g l y c o l y t i c pathway. anaerobic  This  The e f f e c t o f TTX on the  g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s was,  therefore, investigated at d i f f e r e n t concentrations of glucose.  The r e s u l t s o f these experiments a r e shown i n  F i g u r e 15. glucose  I t can be seen t h a t , when t h e c o n c e n t r a t i o n o f  i s i n c r e a s e d , the r a t e o f anaerobic  glycolysis i n  the presence o f TTX p r o g r e s s i v e l y i n c r e a s e s u n t i l a maximum i s o b t a i n e d  a t about 50 mM.  g l y c o l y s i s , i n the c o n t r o l s l i c e s  The r a t e s o f anaerobic  ( i . e . without  TTX) do not  show any s i g n i f i c a n t i n c r e a s e w i t h v a r y i n g glucose  concent-  r a t i o n s above about 5 mM. 4.7 GLUCOSE TRANSPORT IN CEREBRAL CORTEX SLICES 14 The  e f f e c t s o f 2 yM TTX on the glucose-2-C  transport  -  FIGURE  106  -  15  EFFECT OF VARYING GLUCOSE CONCENTRATION ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES IN THE PRESENCE AND ABSENCE  •P  OF  TETRODOTOXIN  100 -  0  20 Glucose  40  60  80  100  c o n c e n t r a t i o n , i n mM  I n c u b a t i o n s were c a r r i e d o u t i n a C a - f r e e medium.TTX( 2 uM) and glucose,when p r e s e n t , w e r e added a t z e r o time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , a s g i v e n i n t h e m a t e r i a l s and methods.(©)TTX a b s e n t ; ( O ) 2 uM TTX p r e s e n t . + +  -  107 -  in  t h e r a tc e r e b r a l c o r t e x  slices  is  evident  of radioactivity  greater ever, sis,  in  which  i n the presence the presence  increased  metabolites  the  fact  that  colysis,  o f TTX b r i n g s  slices  rate  glucose  of glucose  the  enzymes  I t  How-  glycolyradio-  therefore,  of  of glycolydemonstrate  obtained  concentrations  i n the  i s n o t due t o  entry  into  the slices.  that with  high  rates  gradient  results  with  of gly-  increased  i n a greater  and t h e r e f o r e  that  iodoacetate,  experiments  of glycolysis  of glucose  TTX.  no i n c r e a s e d  These  glucose  concentrations glycolytic  with  i s due t o t h e presence  due t o t h e f a c t  the increased  about  6.  present i s  blocked  i n t h e absence  i n the tissue.  facilitation  which  i n Table  I t may b e c o n c l u d e d ,  the greater  i s probably  incubated  of iodecetate  o f TTX a t h i g h  unspecific  been  radioactivity,  t h e TTX t r e a t e d  presence  have  i n the slices.  tic  It  t h e amount  i n slices  activity the  that  are given  external  saturation of  i n an o p t i m a l  rate of  glycolysis. 4.8  EFFECTS ACIDS,  OF T E T R O D O T O X I N , ON T H E A N A E R O B I C  IN THE PRESENCE GLYCOLYSIS  OF SOME AMINO  OF C E R E B R A L  CORTEX  SLICES It the  i swell  brain,  known  glutamate  that,  o f a l l t h e amino  and i t s immediate  acids  metabolites  present i n are of  28 6 outstanding amate slices is  on t h e anaerobic has been  known  nerve  importance  glycolysis  discussed  t o cause  286 cell  to the brain  while  earlier  excitation  The e f f e c t  of the cerebral i n Chapter  and thus  i t h a s no s u c h  .  3.  of glutcortex  L-glutamate  depolarization ofthe  effect  on t h e g l i a l  cells.  108 TABLE  6  EFFECTS OF TETRODOTOXIN ON THE GLUCOSE TRANSPORT IN RAT CEREBRAL CORTEX SLICES UNDER ANOXIA 5mM Additions cpm per g. wet wt  »  glucose Glucose uptake ymoles/g  20mM g l u c o s e cpm p e r g wet wt  Glucose uptake ymoles/g  None  183,000  3.2  174,000  12.1  2yM TTX  201,000  3.5  198,000  13.7  Iodoacetate, 0: 2mM  183,000  3.2  185,000  12.8  Iodoacetate, 0. 2mM + TTX, 2yM  173,000  3.0  166 ,000  11.9  I n c u b a t i o n s were c a r r i e d out a n a e r o b i c a l l y i n a Ca ~t_ f r e e medium f o r 15 min. TTX, i o d o a c e t a t e or c o l d g l u c o s e , when p r e s e n t , were added a t zero time. 0.5yC o f g l u c o s e - 2 - C was added and i n c u b a t i o n was c a r r i e d out f o r another 5 min. T o t a l cpm i n the i n c u b a t i o n medium was 289000/ml. Glucose uptake was c a l c u l a t e d by d i v i d i n g cpm/g by s p e c i f i c a c t i v i t i e s and were not c o r r e c t e d f o r s w e l l i n g o r i n t r a c e l l u l a r space. Each value r e p r e s e n t averages of d u p l i c a t e d e t e r m i n a t i o n s w i t h i n + 5%. +  1h  -  Mcllwain the  and  his  cationic  slices  induced  partly  inhibits  It  was  thought  of  glutamate  of  cerebral  are 5  shown  mM  by  i n Table  therefore, to acids  on  7.  i s evident  It  of  inhibits TTX  in a  on  Ca  medium.  action  of  cerebral  caused  +  i n the  both  bicarbonate  i n Na  + +  the  by  of  that  rate of as  while  5  AND  NH*^  the  TTX.  also  TTX  effects glycolysis The  results  presence  anaerobic as  on  glutamate.  glycolysis,  well  mM  TTX  that  5 mM  i n the  anaerobic  -free  showed  anaerobic  presence  the  of  cortex  examine  D-Glutamate  TTX,  effects  authors  slices  effect  diminished,  These  amino  which  the  incubated  increase  other  cortex  accelerating  the  desirable,  L-glutamate,  Ringer  of  studied  glutamate.  the  and  accelerating is  coworkers  changes  -  109  in a  of  the  glycolysis Krebs-  inhibits  L-aspartate  the  has  no  effect. 4.9  EFFECTS  OF  CITRATE,  STIMULATION As have of  pointed  therefore, anaerobic The  RAT  i n Chapter  3,  citrate,  conditions,  on  are  given  5 mM  NH*  had  the  slices.  ascertain  stimulated  i n the  a  of  presence  i n Table have  rate of  effects  8.  marked  slight  of  AMP  were TTX  and  these be  whereas  the  SLICES  NH^  glycolysis  carried  on  inhibitory  effect.  CORTEX  anaerobic  I t can  glycolysis,  TETRODOTOXIN  CEREBRAL  Experiments  the  anaerobic only  THE  OF  glycolysis  and  ON  GLYCOLYSIS  effects  cortex to  results  citrate TTX  out  inhibitory  cerebral  OF  AMP  out,  rate  of  compounds. seen  that  effects AMP,  on  under  15  mM  the these  110 TABLE  EFFECTS OF  OF  7  TETRODOTOXIN,  SOME AMINO A C I D S , ON  GLYCOLYSIS  OF  CEREBRAL FROM  IN THE THE  PRESENCE  ANAEROBIC  CORTEX  SLICES  RAT  L a c t a t e produced ymoles p e r g i n i t i a l wet wt  (20-80 min)  Additions  C a i f r e e medium +  Krebs-Ringer b i c a r b o n a t e medium  None  31.7 ± 4.3  38.0 ± 4.4  5mM L-Glutamate  20.9 + 2.3  28.6 ± 3.7  5mM  D-Glutamate  5mM  L-Aspartate  34.4 ± 3.8 38.4 ± 2.0  2yM T T X  72.8 ±17.0  65.0  5mM L-Glutamate + 2yM T T X  22.4 ± 2.5  38.4 ± 4.4  5mM D-Glutamate + 2uM T T X 5mM L - A s p a r t a t e + 2yM T T X  43.5 ± 2.4 76.5 ±15.0  A l l v e s s e l s c o n t a i n e d 20mM g l u c o s e . Additions were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods.  I l l  TABLE  8  EFFECTS OF CITRATE, AMP AND  NH\  +  IN THE PRESENCE OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  Additions  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min) No TTX  None 15mM 2mM  4.9  63.0  +  6.7  17. 8 + 3.5  16.9  +  2.0  25.0 citrate AMP  5mM NH4CI  +  w i t h 2yM TTX  15.5  +  5.0  50.5  +  3.1  29.9  +  5.8  37.0  +  4.3  I n c u b a t i o n s were c a r r i e d out i n a Ca i f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured manometric a l l y , as g i v e n i n the m a t e r i a l s and methods.  -  4.10  112  -  EFFECTS OF PHOSPHOLIPASES ON THE TETRODOTOXIN STIMULATED GLYCOLYSIS OF THE RAT CEREBRAL CORTEX SLICES The  s i t e o f a c t i o n o f TTX appears 124  o u t e r s u r f a c e o f the c e l l membrane  t o be l o c a t e d a t the  127 ' .  Phospholipids  are known t o be major and important membrane c o n s t i t u e n t s . 287 Cuthbert  has shown the importance  i n some a s p e c t s o f drug a c t i o n .  o f membrane 288  Heilbronn  e f f e c t o f p h o s p h o l i p a s e s on the uptake  s t u d i e d the  o f a t r o p i n e and  a c e t y l c h o l i n e by mouse b r a i n c o r t e x s l i c e s . demonstrate  lipids  His results  t h a t the p h o s p h o l i p a s e s decrease the uptake o f  a t r o p i n e and p a r t i c u l a r l y t h a t o f a c e t y l c h o l i n e by the slices.  The e f f e c t o f the enzyme was time-dependent, and,  up t o a c e r t a i n l i m i t ,  concentration-dependent.  In view o f t h e f a c t t h a t s i t e o f a c t i o n o f TTX might be a t the c e l l membrane o f c e r e b r a l c o r t e x c e l l s ,  and t h a t  TTX might be a c t i n g by i n t e r a c t i o n w i t h the membrane c o n s t i t u e n t s , the e f f e c t o f TTX on the a n a e r o b i c  glycolysis  of the p h o s p h o l i p a s e t r e a t e d c e r e b r a l c o r t e x s l i c e s was studied.  R e s u l t s o f these experiments  a r e shown i n T a b l e 9.  I t can be seen t h a t , w i t h p r o g r e s s i v e l y h i g h e r c o n c e n t r a t i o n s of p h o s p h o l i p a s e A, the percentage g l y c o l y s i s by TTX i s decreased.  s t i m u l a t i o n of a n a e r o b i c  TTX i s , however,  still  e f f e c t i v e d u r i n g the e a r l y p e r i o d (20-50 minutes) o f the experiment.  T h i s i s t r u e , even a t p h o s p h o l i p a s e A concent-  r a t i o n o f 40 u n i t s / v e s s e l  (3 m l ) , p r e s e n t from zero time up  113 TABLE  EFFECTS OF  OF  TETRODOTOXIN  PHOSPHOLIPASES OF  RAT  TTX  IN THE  PRESENCE  THE ANAEROBIC  CEREBRAL  None 2yM  ON  Amount o f Phospholipase, I.U. p e r v e s s e l (3 m l )  Additions  9  CORTEX  GLYCOLYSIS  SLICES  ymoles  Lactate produced p e r g i n i t i a l wet  20-50 m i n  wt  50-80 m i n  22.5  ± 4.8  10.5  ± 1.4  44.9  ±13  30.8 ±10  Phospholipase  A  1  12.1  ± 3.1  5.8 ± 2.6  Phospholipase + 2yM T T X  A  1  51.8  ± 1.4  Phospholipase  C  1  20.9  ± 0.4  Phospholipase + 2yM T T X  C  1  33.5  ± 1.3  Phospholipase  A  5  13.6  ± 0.5  Phospholipase + 2yM T T X  A  5  37.5  ± 2.7  28.0  ± 2.7  Phospholipase  A  20  25.0  ± 1.7  12.9  ± 0.5  Phospholipase + 2yM T T X  A  20  45.1  ± 4.0  12.3  ± 1.6  Phospholipase  A  40  22.3  ± 1.3  Phospholipase + 2yM T T X  A  40  50.5  ± 1.4  14.3  Phospholipase  A  60  20.3  ± 0.7  11.2 ± 0.5  Phospholipase + 2uM T T X  A  60  22.1  ±11. 8  17.9  33.9  7.4 ± 1.5 11.8  +  ± 2.9  7.8 ± 1.1  8.5 ± 1.3  Incubations were c a r r i e d o u t i n a C a f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods. +  ± 2.3  ± 1.8  ± 1.0  - 114 -  to  the end o f experiment.  Phospholipase C, a t the concent-  r a t i o n t e s t e d , i s more p o t e n t than p h o s p h o l i p a s e A i n a f f e c t i n g the TTX s t i m u l a t e d g l y c o l y s i s o f c e r e b r a l c o r t e x slices. act  These r e s u l t s l e a d to the c o n c l u s i o n t h a t TTX may  by combining  w i t h p h o s p h o l i p i d c o n s t i t u e n t s o f the c e l l  membrane which a r e s l o w l y a t t a c k e d by p h o s p h o l i p a s e s . However, i t i s p o s s i b l e t h a t the p r o d u c t s o f p h o s p h o l i p a s e a c t i v i t y may i n h i b i t the a c t i o n o f TTX.  T h i s i s y e t t o be  resolved. 4.11  EFFECTS OF TETRODOTOXIN ON ANAEROBIC GLYCOLYSIS OF KIDNEY MEDULLA SLICES AND ACETONE POWDER EXTRACTS Experiments  were c a r r i e d o u t t o see i f the accelerating  e f f e c t o f TTX on a n a e r o b i c g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s i s s p e c i f i c f o r the b r a i n t i s s u e . kidney medulla  s l i c e s have h i g h g l y c o l y t i c r a t e s and hence  i t was s e l e c t e d f o r examination.  The e f f e c t s o f TTX on the  a n a e r o b i c g l y c o l y s i s o f kidney medulla F i g u r e 16.  I t i s known t h a t  s l i c e s are given i n  From t h i s d a t a i t can be concluded t h a t TTX has  a b s o l u t e l y no e f f e c t on the a n a e r o b i c g l y c o l y s i s o f kidney medulla.  T h i s o b s e r v a t i o n demonstrates t h a t the e f f e c t o f  TTX on c e r e b r a l c o r t e x s l i c e s i s s p e c i f i c , and t h a t the anaerobic g l y c o l y s i s of a l l tissues i s not s e n s i t i v e to i t . The e f f e c t o f TTX and ouabain on the a n a e r o b i c g l y c o l y s i s o f acetone powder e x t r a c t s i s shown i n F i g u r e 17. r e s u l t s prove  The  t h a t TTX has no e f f e c t on the p r o c e s s o f c e l l  f r e e a n a e r o b i c g l y c o l y s i s and t h a t a membrane phenomenon i s  -  FIGURE  115 -  16  EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT KIDNEY MEDULLA SLICES  I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e ( • ) w i t h 2 uM TTX.  13.(O)control;  -  FIGURE E F F E C T S OF T E T R O D O T O X I N OF A C E T O N E  M  116 -  17 AND  O U A B A I N ON THE A N A E R O B I C  POWDER E X T R A C T S FROM RAT  GLYCOLYSIS  BRAIN  1000  20  30  40 Time,  I n c u b a t i o n c o n d i t i o n s were ( O ) w i t h 2 jaM T T X ; ( « ) w i t h  50  60  70  81)  i n minutes same a s i n F i g u r e 10 pM o u a b a i n .  10.(±)control;  - 117 -  involved are  i n the mechanism  clearly  4.12  necessary  EFFECTS  OF TETRODOTOXIN  Effects  Table slices  from  rats  slices  from  with  behaviour 14-day  cells  o f TTX. GLYCOLYSIS  SLICES  of different that  Intact  THE ANAEROBIC  CORTEX  I t i s evident  the g l y c o l y t i c  However,  ON  o f TTX on t h e a n a e r o b i c  slices 10.  o f TTX.  f o r the action  OF D E V E L O P I N G BRAIN  cortex  of action  glycolysis ages  of cerebral  are given i n  2 o r 7-day o l d r a t b r a i n  i s unaffected  o l d animals  by TTX.  show a n  appreciable  response. The  effects  anaerobic cortex  of different  glycolysis  slices  glycolysis  concentrations  o f newly  born  a r e a-lso- s h o w n i n F i g u r e  o f i n f a n t guinea  s e n s i t i v e t o TTX and even  effective  a s 10  yM T T X .  the presence  tested.  guinea  o f TTX i s c o n s t a n t  Experiments  discussed  later,  pigs  t o drugs  tested.  results  to appreciate  4.13  pigs  EFFECT  i s much  AND  mature  NH*  CORTEX  ON  during  other  slices  than  of  are  which  period will  be  of the infant the adult  i n considering  known  fact  that  compared  with  newly  I N P R E S E N C E OF  glycolysis  the time  drugs,  THE ANAEROBIC  anaerobic  uM T T X i s a s  the rate  greater  the well  The  cortex  the s e n s i t i v i t y  OF T E T R O D O T O X I N  DEVELOPING have  some  I t i s important  are very  ASPARTATE  We  with  show t h a t  tissues  guinea  Moreover,  0.2  pig cerebral  18.  p i g cerebral  extremely  in  guinea  o f TTX on t h e  brain  these newly  born  born  rats.  GLUTAMATE,  G L Y C O L Y S I S OF  SLICES  shown e a r l i e r  i n this  chapter  (4.8  and  4.9)  118 TABLE 10 EFFECT OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF INFANT RAT CEREBRAL CORTEX SLICES L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80  Additions  2-day o l d rat  min)  7-day o l d rat  14-day o l d rat  25  +  1.5  21.2  +  0.45  25.4 + 3.1  0. 2yM T T X  28.6  +  1.8  26.0  +  2.2  42.5 + 1.3  2yM T T X  29.9  +  5.9  27.7 + 1.3  48.3 + 9.9  None  29. 8 + 0.9  30.0  + +  35.8 + 3.5  31.3 + • 1.3  45.3 + 4.4  + +  31.7 + 0.9  34.4 + 0.4  54.5 + 6.2  + +  29.5 + 3.5  29.0  55.8 + 6.7  lOyM T T X 2yM T T X + ImM C a 2yM T T X + 2mM C a 2yM T T X + 4mM  Ca  +  0.5  51.4  +  I n c u b a t i o n s were c a r r i e d out i n a Ca t f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods.  6.6  -  119 -  150  Time,in minutes FIGURE  18  EFFECTS OF DIFFERENT CONCENTRATIONS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF NEWLY BORN GUINEA PIG CEREBRAL CORTEX SLICES ++ I n c u b a t i o n s were c a r r i e d out i n a Ca - f r e e medium c o n t a i n i n g 2 0 mM glucose.TTX,when present,was added a t zero time and l a c t a t e p r o d u c t i o n was measured manometrically,as g i v e n i n the m a t e r i a l s and methods.( • ) c o n t r o l ; ( • )0.2 uM TTX; (• ) 2 uM T T X ; ( A ) 1 0 pM TTX.  -  that  t h e TTX  shows  sensitivity  desirable, allied old to  therefore,  more  light  o f these  clear that  the  rate  rat  brain  4.14  on TTX  these  ADULT The  OF  RAT  compounds have  CEREBRAL  cerebral  cortex  slices  cortex  ON  CORTEX  slices  when  2-week  that  slices  o f TTX. 11.  I t  effects  on  2-week o l d of the adult. GLYCOLYSIS  OF  SLICES  a r e shown  of aerobic  The  THE A E R O B I C  o f TTX on t h e a e r o b i c  on t h e r a t e  cerebral  inhibitory  than  and  cortex  i n Table  glycolysis.  s e n s i t i v e t o NH*  of  of action  a r e shown  TETRODOTOXIN  effects  pig cerebral  slices  I t was  of these  glycolysis  on t h e mechanism  o f TTX a c c e l e r a t e d  EFFECTS  effect  guinea  cortex  a s t o NH*.  the effects  stimulated  experiments  i s more  of cerebral  as w e l l  t o study  r a t and newly-born  throw  -  glycolysis  to glutamate  substances  Results is  stimulated  120  glycolysis  i n Figure  glycolysis  the l a t t e r  19.  of r a t  TTX has no  of the r a t  are incubated  i n a  ++ Krebs-Ringer  bicarbonate  medium.  medium,  TTX has an i n h i b i t o r y  lysis.  I n a Ca  the is  rate  effect  - f r e e medium,  of lactate production,  greater  than  that  Moreover,  under  on  i n a Ca  -free  the aerobic  glyco-  aerobic  conditions  i n the absence  i n a Krebs-Ringer  o f the drug,  medium.  The  i n h i b i t o r y e f f e c t o f TTX on t h e r a t e o f a e r o b i c glycolysis ++ ++ i n a Ca - f r e e medium i s s i m i l a r t o t h a t o f Ca and analogous  to that  slices  incubated  Quastel  ).  Results on  on t h e r e s p i r a t i o n under  given  the aerobic  similar  i n Table  glycolysis  11A  of r a t brain  conditions  show  (Chan  the e f f e c t s  i n the presence  cortex  and  o f TTX  of cyanide.  In  -  121  -  FIGURE 19 EFFECTS OF TETRODOTOXIN AND OUABAIN ON THE AEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  4J  •H +J •H  U  CD CU  W CD iH O  1  'O  o o u  cu 0)  +J rO 4J O fO  20 40 60 Time,in minutes  80  A l l v e s s e l s c o n t a i n e d 20 mM glucose.A:Krebs-Ringer bicarbonate medium.B:Ca - f r e e medium.Additions were made a t zero time and l a c t a t e p r o d u c t i o n was measured e n z y m a t i c a l l y . ( v ) c o n t r o l ; (• )2 pM TTX; ( • ) 10 pM ouabain. +  122 TABLE 11 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF SOME AMINO ACIDS OR NHi*"" ON THE 1  ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES FROM INFANT ANIMALS  Additions  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min) 2-week o l d rat  newly born guinea p i g  None  25.5 + 3.1  34.4  5mM L-glutamate  19.0  +  3.1  22.3 + 0.9  5mM D-glutamate  19.2  +  1.8  32.2  +  2.3  2yM TTX  48.3 + 9.9  129.5  +  6.7  5mM L - A s p a r t a t e  17.4  +  1.3  25.9  +  3.1  5mM NH4CI  13.9  +  2.2  +  2.7  -  2yM TTX + 5mM L-glutamate  25.5 + 0.5  51.5  + 5mM D-glutamate  29. 5 + 4.0  98.1 + 3.1  + 5mM L - A s p a r t a t e  28.6  +  4.0  + 5mM NH4CI  17.0  +  3.1  Incubation  113.7  +  +  -  c o n d i t i o n s were same as i n T a b l e 10.  1.8 3.5  123 TABLE  EFFECTS  OF  GLYCOLYSIS  Medium  Krebs-Ringer bicarbonate Ca -free + +  TETRODOTOXIN  OF  THE  11A  RAT  ON  CEREBRAL  PRESENCE  OF  THE  CORTEX  AEROBIC SLICES  IN  CYANIDE  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min) No T T X  w i t h 2yM T T X  112.9 + 6.7  117.2 + 9 . 6  63.8 ± 8.9  103.1 ± 2.5  A l l v e s s e l s c o n t a i n e d 20mM g l u c o s e . A d d i t i o n s were made a t z e r o time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n t h e m a t e r i a l s and methods, (gaseous phase was O 2 : C 0 2 i n t h e s e experiments)  -  a  Ca  -free  electron  medium, u n d e r  transport  effective However,  chain  i n further  due  to optimal  rates  OF CHAPTER  Tetrodotoxin  anaerobic cortex  conditions,  i s blocked  i n a Krebs-Ringer  effective i n further  1.  aerobic  enhancing  not  SUMMARY  124 -  by c y a n i d e ,  the rate  bicarbonate  enhancing  of glycolysis. medium, TTXi s  pig 2.  (TTX) m a r k e d l y  These  2  cortex  The rate  effects  accelerates  guinea  p i g cerebral  slightly  inhibitory effect.  slices,  suppression 4.  markedly  from under 5.  by t h e presence  cerebral  suppressed  uptake  the guinea  of Ca  +  acid  cortex  +  i n  +  In rats the has a  glycolysis of cerebral  o f TTX, i s n o t due t o under  anoxic  conditions.  or of aspartic  slices,  b y 2 yM T T X .  of radioactive  the incubation  with  no e f f e c t or i t  o f t h e e f f l u x o f NAD  incubated  increased  slices.  of anaerobic  The e f f l u x o f g l u t a m i c  the is  has e i t h e r  The a c c e l e r a t i o n  cortex  on Ca  by t h e a d d i t i o n  cortex  of Ca  3.  ++  g l y c o l y s i s i n presence o f  enhanced  addition  +  t o those  slices.  p i g cerebral +  the rate o f  o f TTX a r e s i m i l a r  and coworkers  o f anaerobic  yM T T X i s f u r t h e r  the  itself.  4  by Q u a s t e l  cerebral  possibly  of glycolysis i n the controls  71 reported  TTXi s  the glycolysis,  g l y c o l y s i s o f r a t and guinea  slices.  when t h e  due t o TTX a l s o  glutamic  acid  medium, by t h e c e r e b r a l  acid  anaerobiosis, causes and g l y c i n e ,  cortex  slices  anoxia. In the presence  of increasing  from  concentrations  of  -  glucose, the  the  stimulation  presence  rate  of  ration due  of  anaerobic may  the  shown 6.  by The  to  TTX  unspecific cortex  reversed  by  citrate  even  in  the  no  medulla  glucose  above  the  when  concent-  effect is of  not  glucose  presence  the  of  entry  TTX  as  glucose.  of  or  L-glutamate,  NH*. accelerating  e f f e c t on slices  by  addition  stimulation  8.  the  to  glycolysis is partially,  presence  decreased.  kidney  of  or  The  labelled  the  considerably has  high,  facilitation  slices  with  g l y c o l y s i s , due  indicating that  limiting.  stimulation  percentage  TTX  enhanced,  is effective in  glycolysis the  an  experiments  D-glutamate, TTX  rate  cerebral  completely  7.  is  -  anaerobic  glycolysis is  become  however,  into  TTX,  of  125  or  of  during  the of  the  rate  of  anaerobic  phospholipases, the  later  anaerobic  acetone  although  period  is  glycolysis  powder  of  extracts  of  brain. 9. of  TTX 2-  or  markedly of  has  7-day at  maximum  glycolysis many m a t u r e TTX  little  about brain by  of  10.  The  the  rat 14th  no  brain  but  day.  This  growth  slices  e f f e c t on  of  and  adult  rate  of  brain,  aerobic  by  anaerobic  period  glycolysis  coincides  myelination.  newly  g l y c o l y s i s of  the  i t s effectiveness  born  characteristics, i s very  stimulated  that  old  or  infant  The  guinea  brain  L-glutamate,  g l y c o l y s i s of  is  with  to  which  shows  TTX.  The  inhibited  D-glutamate  the  that  anaerobic  pigs,  sensitive  increases  cerebral  like  or  cortex  -  slices  i n a Krebs-Ringer  126 -  b i c a r b o n a t e medium  i s unaffected  ++ by  TTX, w h i l s t  depressed The cyanide However, TTX  i n a Ca  -free  medium  i ti s slightly  by TTX. rate  of aerobic glycolysis  i n a Ca  + +  -free  medium  i n a Krebs-Ringer  has no  effect.  i n the presence  of  i s i n c r e a s e d by TTX.  medium  under  t h e same  conditions,  CHAPTER FURTHER  S T U D I E S ON  TETRODOTOXIN OF It  ON  MECHANISM  CORTEX  shown t h a t  greatly  enhances  cortex  menon  i s c h a r a c t e r i s t i c o f mature  sults  o f experiments,  mode o f a c t i o n  slices  SLICES  the rate  of cerebral  OF  GLYCOLYSIS  the presence  lysis  the  OF A C T I O N  THE ANAEROBIC  CEREBRAL  has been  concentrations,  THE  5  o f TTX,  of anaerobic  and, moreover,  that  cerebral  tissue.  c a r r i e d out t o throw  further  o f TTX on t h e b r a i n  a t low glyco-  this  pheno-  The r e light  tissue, will  now  on be  described. 5.1  EFFECTS  OF  PRE-INCUBATION  TETRODOTOXIN CEREBRAL As that in  a brief  a marked  glycolysis. tumours from  CORTEX early  anaerobic  studies  1928, R o s e n t h a l  of incubation  increase This  GLYCOLYSIS  OF  and L a s n i t z k i  i n presence  i n the subsequent rate  phenomenon was normal t i s s u e s , i t was  of general  further  2  8  showed  9  o f oxygen  of  results  anaerobic  studied  i n  liver,  i n c l u d i n g b r a i n , 290-294  a  concluded  g l y c o l y s i s by previous  phenomenon  THE  SLICES  as  period  and other  these  STIMULATED  I N O X Y G E N ON  that  the stimulation  incubation  importance and occurs  i n oxygen  n  (  j  of  i s a  i n a l lnormal  adult  ? 91 tissues.  x  definitely  However,  i t has not been  t h e changes  associated  possible  with  to  the aerobic  define pre-incuba-  -  128  t i o n of the t i s s u e s . 2 9 2 In  view o f the above, i t was  considered d e s i r a b l e to  see i f the a n a e r o b i c g l y c o l y s i s , which has been s t i m u l a t e d by p r e v i o u s oxygenation,  can be f u r t h e r enhanced by TTX.  r e s u l t s o f these experiments  The  are shown i n F i g u r e 20.  I t can  be seen t h a t when the s l i c e s are p r e - i n c u b a t e d a e r o b i c a l l y , i n the absence of TTX,  the drug i s s t i l l  the r a t e o f a n a e r o b i c g l y c o l y s i s . pre-incubation  enhancing  Longer p e r i o d s o f a e r o b i c  (40 minutes) lower the t o t a l amount o f the  l a c t a t e produced in  effective in  d u r i n g the subsequent a n a e r o b i c p e r i o d , b o t h  the c o n t r o l as w e l l as i n the T T X - t r e a t e d s l i c e s .  However,  the s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s i n the presence w^ 5.2  s t i l l very  of  TTX  apparent.  EFFECTS OF TETRODOTOXIN AFTER VARIOUS PERIODS OF ANAEROBIOSIS ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES The r e s u l t s o f experiment  s t r a t e t h a t TTX  shown i n F i g u r e 20 demon-  i s e f f e c t i v e a f t e r the p r e l i m i n a r y p r e - i n c u b a -  t i o n o f the s l i c e s i n oxygen.  The r a t e of a n a e r o b i c  glycolysis  a t t a i n e d w i t h the a e r o b i c a l l y i n c u b a t e d s l i c e s i s g r e a t e r than t h a t found w i t h the c o r r e s p o n d i n g s l i c e s which were not exposed to  oxygen.  Hence i t was  thought  d e s i r a b l e t o c a r r y out e x p e r i -  ments, i n which the s l i c e s have been p r e - i n c u b a t e d f o r v a r y i n g p e r i o d s i n n i t r o g e n , t o see i f TTX  is still  c i n g the r a t e o f a n a e r o b i c g l y c o l y s i s .  e f f e c t i v e i n enhan-  R e s u l t s of a t y p i c a l  - 129 FIGURE 20 EFFECT OF AEROBIC PREINCUBATION ON THE TETRODOTOXIN  STIMULATION  OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES 125 .  20  .  30  40 50 60 Time,in minutes  70  80  C e r e b r a l c o r t e x s l i c e s were p r e i n c u b a t e d i n a Krebs-Ringer b i c a r b o n a t e medium c o n t a i n i n g 20 mM g l u c o s e f o r 20 min under 02:00-.They were then t r a n s f e r r e d t o another s e t o f v e s s e l s c o n t a i n i n g , i n a d d i t i o n , 2 pM TTX.Lactate p r o d u c t i o n was measured m a n o m e t r i c a l l y under subsequent a n a e r o b i c p e r i o d , as g i v e n i n the m a t e r i a l s and m e t h o d s . ( O ) c o n t r o l ; ( • ) 2 pM TTX.  -  experiment, periods ment  shows t h a t  sively TTX,  of the cerebral  as t h e p e r i o d  slices  t o respond  ineffective.  cortex  are  important  very  anaerobic  5.3  brief  changes  drops  fact  I N THE  G L Y C O L Y S I S OF  that  that  occur  h a d no  b y TTX,  10 m i n " p e r i o d ,  i t was  that  changes  few m i n u t e s  effect  effect  t h e 5 min cortex  i n the  of  anoxia,  o f TTX on t h e r a t e  PRESENCE THE  OF  PYRUVATE  CEREBRAL  a r e no quickly.  of the cerebral  effect  cortex  of anoxia  mechanisms dependent operative.  number  i n stimulating the cortex  raises the question  i n the cerebral  longer A  i s not effective  o f anoxia  The major  the oxidative  TTX  glycolysis  periods  conditions.  oxygen  progres-  of cerebral  the f i r s t  f o r the enhancing  TETRODOTOXIN  o f anaerobic  after  a  anaerobic  the addition of  2 min o f anoxia  demonstrates  during  the  experi-  SLICES  The  that  slices,  THE ANAEROBIC  CORTEX  rate  experiment  This  decreases  before  the ability  varying  glycolysis.  E F F E C T S OF ON  slices,  glycolysis  t o TTX and a f t e r  This  cerebral  of  cortex  although  reduced  21.  o f TTX i n e n h a n c i n g  the stimulation of anaerobic considerably  i n "after  i n Figure  of anaerobiosis, Thus,  period  "tipped  a r e shown  the ability  i s increased.  -  TTX h a s b e e n  of anaerobiosis,  glycolysis,  on  i n which  130  o f ATP  slices  slices  as t o t h e under  on a l i v i n g  tissue i s  on t h e p r e s e n c e  In brain, dependent  the c e l l processes  anaerobic  of  ATP  level  are  impaired  - 131 FIGURE  21  EFFECT OF ADDITION OF TETRODOTOXIN AFTER VARYING TIME PERIODS OF ANOXIA ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  80  Time,in minutes ++ I n c u b a t i o n s were c a r r i e d out i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . ( B ) N o a d d i t i o n ; ( O ) 2 pM TTX,from zero time; ( • ) 2 pM TTX added a t 2min; ( A )2 pM TTX added a t 5 min; ( • )2 pM TTX added a t 10 min .The gas phase was anaerobic throughout the experiment , and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y as g i v e n i n the m a t e r i a l s and methods.  and  this  results,  contents, cation  W!e TTX  incubated  and  anoxia,  was,  of  may  substances,  the  One  of  pyruvate  of  anaerobic  over,  to  the  quently  of  amino  efflux  slices  of  higher  the  that  rates  were  venting  efflux  the  of  of  cation maintain  might  the  be  of  the  i n the  out  pyruvate  from  under  and  anaerobic  c a t i o n s , as  of  such  greatly  as  rate  the  enhances  cortex  and,  glycolysis.  from  on  concentration i n  pyruvate,  i f TTX  a  Tetrodo-  addition the  slices.  pre-incubation results  see  of  the  glycolysis.  their  tissue^l  to  acids  efflux  cerebral  aerobic  presence  stimulating effect  raising  anaerobic  carried  of  t o have  preventing  the  occurs  rate  i n c u b a t i o n medium  pyruvate  experiments  that  metabolites  thereby  glycolysis  accumulation  pump t o  the  compound  i t i s thought  sodium  that  various  by  regulating  such  the  cerebral  4.4  reduce  glycolysis,  cell.  of  i n the  i n Chapter  t h e r e f o r e , thought  anaerobic  i n changes  inability  cortex  Efflux  -  solute concentrations.  shown  cerebral  of  the  suppresses  conditions.  toxin  of  have  markedly  result  f o r example,  because  gradients  132  hence,  Morein subse-  Therefore,  could  incubated  in  rate  act by  cerebral  precortex  slices. Results the  anaerobic  sence  of  increased  i n Table  glycolysis  pyruvate; by  given  TTX,  the the  of  rate  12  cerebral of  show t h e cortex  anaerobic  stimulatory effect  effects slices  Ca  + +  TTX  i n the  glycolysis in a  of  i s  -free  on  pre-  further medium  133 TABLE 12 EFFECTS OF TETRODOTOXIN IN THE PRESENCE OF PYRUVATE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  L a c t a t e produced ymoles per g i n i t i a l wet wt (20-80 min) Additions  Krebs-Ringer b i c a r b o n a t e medium  Ca + f r e e medium +  None  38.0 ± 4.4  31.7 ±  2yM TTX  65  76  ImM  4.3  Pyruvate  113.4 ± 6.3  75  ± 14.9  ImM Pyruvate + 2yM TTX  124.1 ± 5.4  117.9  ± 14.3  lOmM Pyruvate lOmM Pyruvate + 2yM TTX  70.1 ± 12.7 128.1 ±  A l l v e s s e l c o n t a i n e d 20mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods.  6.5  - 134  b e i n g almost a d d i t i v e .  -  These r e s u l t s show t h a t t h e e f f e c t o f  TTX on t h e a n a e r o b i c g l y c o l y s i s can not be due t o i n h i b i t i o n o f t h e e f f l u x o f p y r u v a t e from t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s , s i n c e i n t h a t c a s e , an a d d i t i v e e f f e c t on a n a e r o b i c g l y c o l y s i s w o u l d not have been 5.4  observed.  EFFECTS OF GLUCOSE ADDITION UNDER VARIOUS CONDITIONS ON THE  STIMULATION OF ANAEROBIC GLYCOLYSIS BY  TETRODOTOXIN Experiments  were c a r r i e d out t o o b t a i n more i n f o r m a t i o n  c o n c e r n i n g t h e c o n d i t i o n s t h a t might a f f e c t t h e TTX  stimulation  of anaerobic g l y c o l y s i s i n c e r e b r a l cortex s l i c e s .  I t i s well  known t h a t v e r y l i t t l e endogenous energy r e s e r v e s a r e p r e s e n t i n t h e b r a i n t i s s u e and t h a t exogenous g l u c o s e i s t h e major s o u r c e o f energy  (see Chapter 1 ) .  I t i s possible to deplete  s l i c e s o f ATP by i n c u b a t i n g them i n t h e absence o f g l u c o s e o r i n the presence  of uncoupling agents.  out t o see i f TTX enhancing  Experiments  were c a r r i e d  i s e f f e c t i v e , under such c o n d i t i o n s , i n  t h e a n a e r o b i c g l y c o l y s i s o f such  slices.  R e s u l t s i n T a b l e 13 show t h e e f f e c t s o f a d d i t i o n o f g l u c o s e a f t e r p e r i o d s o f a n a e r o b i o s i s and a e r o b i o s i s on t h e s t i m u l a t i o n of g l y c o l y s i s .  TTX  I t can be seen from t h e s e e x p e r i -  ments t h a t g l u c o s e s h o u l d be p r e s e n t b e f o r e t h e s t a r t o f a n a e r o b i o s i s t o o b t a i n an e f f e c t o f TTX  (Table 13A).  Once t h e  s l i c e s have been exposed t o a n o x i a , t h e g l y c o l y s i s o f t h e c e r e -  135 TABLE 13 EFFECTS OF ADDITION OF GLUCOSE UNDER VARIOUS CONDITIONS ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min)  A. 2yM TTX from s t a r t  B.  Time o f addition of Glucose  ImM Pyruvate p r e s e n t from s t a r t  Absent  0 min  60.3 ± 4.5  Present  0 min  129.5 ± 5.0  No Pyruvate 29.0 ±  4.9  83.5 ± 10.7  Absent  15 min  18. 8  17.6 ±  1.8  Present  15 min  22.3  16.5 ±  4.0  2yM  Time o f addition of Glucose  TTX from s t a r t  ImM Pyruvate from s t a r t  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (30-90 min)  Absent  0 min  Present  68.8 ± 4.5  Present  0 min  Present  190.2 ± 0.9  Absent  10 min  Present  54.9 ± 2.7  Present  10 min  Present  180.8 ± 4.4  135A  TABLE  13  (Continued)  2yM TTX from s t a r t  Time of addition of Glucose  ImM Pyruvate from s t a r t  L a c t a t e produced ymoles per g i n i t i a wet wt (40-100 min)  Absent  0 min  Present  79.0 + 2 . 7  Present  0 min  Present  174.6 ± 3 . 6  Absent  25 min  Present  85.7 ± 1 . 3  Present  25 min  Present  153.1  + 1.0  When p r e s e n t , f i n a l c o n c e n t r a t i o n of g l u c o s e was 20mM. I n c u b a t i o n s were c a r r i e d out i n a Ca "t- f r e e medium. (A) I n c u b a t i o n was a n a e r o b i c . (B) F i r s t 10 min p e r i o d was a e r o b i c f o l l o w e d by a n a e r o b i o s i s . (C) F i r s t 15 min p e r i o d was a n a e r o b i c f o l l o w e d by 10 min a e r o b i c p e r i o d and subsequent a n a e r o b i o s i s . L a c t a t e p r o d u c t i o n was measured manometrioally. +  - 136 -  b r a l c o r t e x s l i c e s i s not a f f e c t e d by TTX, i r r e s p e c t i v e o f whether TTX i s p r e s e n t  from t h e v e r y b e g i n n i n g  the medium l a t e r on.  However, when the p r e l i m i n a r y i n c u b a t i o n ,  without  glucose  and then g l u c o s e  i s a e r o b i c , and i s i n t h e presence o f pyruvate, i s added t o t h e i n c u b a t i o n medium, a s t i m u l a t i o n  o f g l y c o l y s i s by TTX takes p l a c e response o f a n a e r o b i c  (Table 13B).  The l o s s o f  g l y c o l y s i s t o TTX, a f t e r a p e r i o d o f  a n a e r o b i o s i s i n t h e absence o f g l u c o s e , due  o r i s added t o  t o a permanent damage t o t h e t i s s u e .  does n o t appear t o be Thus, a f t e r a p e r i o d  o f a n a e r o b i o s i s , i f t h e s l i c e s a r e exposed t o oxygen b r i e f l y , then t h e a b i l i t y o f t h e s l i c e s t o have i n c r e a s e d r a t e o f g l y colysis,  i n t h e presence o f TTX, d u r i n g subsequent  periods i s regained  anaerobic  (Table 13C). The s i g n i f i c a n c e o f these  experiments w i l l be d i s c u s s e d i n Chapter 8. 5.5  EFFECT OF AEROBIC INCUBATION WITH DINITROPHENOL ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS, AND ON THE ATP LEVEL OF THE RAT CEREBRAL CORTEX SLICES From t h e experiments j u s t r e p o r t e d i t appears t h a t  the ATP c o n c e n t r a t i o n i n t h e s l i c e s might be important  f o r the  a c t i v i t y o f TTX, s i n c e t h e i r a b i l i t y t o respond t o TTX ( a f t e r a b r i e f period o f anaerobiosis) bation.  i s r e g a i n e d by a e r o b i c i n c u -  Hence experiments were c a r r i e d out t o d e f i n e more  c l e a r l y t h e exact r o l e o f ATP i n t h e s t i m u l a t i o n o f anaerobic g l y c o l y s i s o f c e r e b r a l c o r t e x s l i c e s by TTX.  I n these  experi-  - 137 -  merits, ATP l e v e l s were measured under a v a r i e t y o f c o n d i t i o n s and t h e s e were t h e n r e l a t e d t o t h e r a t e s o f a n a e r o b i c g l y c o l y s i s . T a b l e 14A shows t h a t t h e ATP c o n t e n t o f a e r o b i c a l l y incubated c e r e b r a l c o r t e x s l i c e s decreases i n t h e presence o f 2,4-dinitrophenal  (DNP).  When t h e s l i c e s a r e i n c u b a t e d anaero-  b i c a l l y f o r 15 min f o l l o w e d b y 10 min a e r o b i c i n c u b a t i o n , t h e subsequent ATP l e v e l i s r e l a t i v e l y h i g h ( T a b l e 14B). a n a e r o b i c p e r i o d r e d u c e s t h e ATP l e v e l s i g n i f i c a n t l y  A further (Table 14C).  However, t h e ATP l e v e l i n t h e s l i c e s exposed t o TTX, i s h i g h e r b o t h i n t h e p r e s e n c e o r absence o f DNP t h a n i n t h o s e n o t so exposed.  From t h e d a t a on subsequent  rates o f anaerobic  g l y c o l y s i s under t h e s e as w e l l a s under s i i g h t l y d i f f e r e n t conditions  (see T a b l e 14C), i t c a n be seen t h a t t h e r a t e o f  anaerobic g l y c o l y s i s o f t h e DNP-treated s l i c e s i s s l i g h t l y t h a n t h o s e w h i c h were n o t exposed t o i t .  lower  The r a t e s o f g l y c o l y -  s i s a r e h i g h e r when p y r u v a t e i s p r e s e n t from t h e b e g i n n i n g o f the experiment b e f o r e t h e a d d i t i o n o f g l u c o s e .  When g l u c o s e  i s added a f t e r t h e a e r o b i c i n c u b a t i o n p e r i o d , t h e r a t e s o f g l y c o l y s i s a r e lower than t h a t o f those s l i c e s , incubated i n a medium, t o w h i c h g l u c o s e h a d been added b e f o r e t h e a e r o b i c period.  The s i g n i f i c a n c e o f t h e s e r e s u l t s w i l l be d i s c u s s e d  i n C h a p t e r 8.  138 TABLE 14 EFFECTS OF INCUBATION WITH (DNP) ON THE TETRODOTOXIN  2,4-DINITROPHENOL STIMULATION OF  ANAEROBIC GLYCOLYSIS AND THE ATP CONTENTS OF RAT CEREBRAL CORTEX SLICES  A.  E f f e c t o f DNP on the ATP c o n c e n t r a t i o n o f c e r e b r a l c o r t e x s l i c e s i n c u b a t e d a e r o b i c a l l y f o r one hour i n a Krebs-Ringer phosphate medium. DNP was added a t zero time and the medium c o n t a i n e d 20mM g l u c o s e .  Additions  B.  ATP . . . , . , . . ymoles p e r g m i t r a l wet wt  None  1.57  O.lmM DNP  0.66  E f f e c t s o f DNP on the ATP c o n c e n t r a t i o n i n the presence o f TTX. Incubations were c a r r i e d o u t f o r 25 minutes i n a C a free medium c o n t a i n i n g 20mM g l u c o s e . F i r s t 15 min p e r i o d was a n a e r o b i c f o l l o w e d by 10 min a e r o b i c p e r i o d . Additions were made a t zero time. + +  Additions  ATP ymoles p e r g i n i t i a l wet wt  None  1.48  O.lmM DNP  1.16  2  1.42  yM TTX  O.lmM DNP + 2yM TTX  1  7  138A TABLE 14 (Continued) C.  E f f e c t s o f DNP and TTX on the ATP c o n t e n t and a n a e r o b i c g l y c o l y s i s of cerebral cortex s l i c e s . I n c u b a t i o n s were c a r r i e d out i n a C a - f r e e medium. First 15 min p e r i o d was a n a e r o b i c f o l l o w e d by 10 min a e r o b i c p e r i o d and subsequent a n a e r o b i o s i s . L a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y from 40-10 0 min. TTX and DNP were p r e s e n t from zero time. L a c t a t e produced i s expressed as ymoles o f l a c t a t e p e r g i n i t i a l wet wt o f t h e s l i c e s . +  Addition  ImM Pyruvate from  No Pyruvate ATP content at 35 min ymoles p e r g initial wet wt  start  Lactate produced Glucose added a t 25 min  Lactate produced Glucose at zero time  Lactate produced Glucose added a t 15 min  114.7 ± 3.1  79.5 ± 8.0  86.6 ± 9.8  49.6 ± 7.1  None  0.45  37.9 ± 1.78  DNP, O.lmM  0. 43  26.8 ± 1.3  TTX, 2yM  0.64  146  ± 1.3  178.6 ± 7.0  159.8 ± 6.7  TTX, 2uM + DNP, O.lmM  0.59  117  ± 6.3  136.2 ± 2.2  101  ±16.9  -  5.6  139  EFFECTS OF TETRODOTOXIN AND  -  OUABAIN ON THE  ATP  CONTENT OF GUINEA PIG CEREBRAL CORTEX SLICES As has been mentioned i n Chapter  4, the c o n c e n t r a t i o n s  o f a c i d l a b i l e phosphates i n the i n c u b a t e d c e r e b r a l c o r t e x s l i c e s are r a i s e d i n the presence  o f both TTX and  ouabain.  Under these c o n d i t i o n s the r a t e s of g l y c o l y s i s are a l s o h i g h e r . T a b l e 1 5 shows the e f f e c t o f ouabain and TTX on the ATP o f the i n c u b a t e d c e r e b r a l c o r t e x s l i c e s . the presence  t e r s 6 and 5.7  I t can be seen t h a t i n  of b o t h these drugs the l e v e l of ATP  i s increased.  i n the  slices  These r e s u l t s w i l l be f u r t h e r d i s c u s s e d i n Chap-  8.  EFFECTS OF RAISING ATP  LEVEL BY AEROBIC INCUBATION  WITH ADENOSINE ON THE  TETRODOTOXIN STIMULATED  GLYCOLYSIS OF THE  CEREBRAL CORTEX SLICES  RAT  Abadom and S c h o l e f i e l d and o t h e r s an i n c r e a s e i n the ATP  2 4 5 , 2 9 5  have r e p o r t e d  (7 min phosphate) content when the  c e r e b r a l c o r t e x s l i c e s are i n c u b a t e d a e r o b i c a l l y i n t h e o f adenosine. if  contents  Experiments  presence  were c a r r i e d out, t h e r e f o r e , t o see  s l i c e s t r e a t e d i n such a manner show more r e s p o n s i v e n e s s t o  the e f f e c t s o f TTX on the a n a e r o b i c g l y c o l y s i s . experiments  In t h e s e  (see T a b l e 1 6 ) , the s l i c e s were p r e - i n c u b a t e d  a e r o b i c a l l y f o r 40 min  i n a Krebs-Ringer  c o n t a i n i n g 1 RM adenosine  bicarbonate  and 20niM g l u c o s e ;  s l i c e s were t r a n s f e r r e d t o another  solution  subsequently  the  s e t of v e s s e l s c o n t a i n i n g TTX.  140  TABLE  EFFECTS ON  OF T E T R O D O T O X I N  THE ATP  CEREBRAL  Addition  15  CONTENT  CORTEX  AND  OF G U I N E A P I G  SLICES  , ymoles  UNDER  TTX,  lOyM  2yM  o u a b a i n , lOyM + ImM C a T T X , 2yM + ImM C a  +  +  +  +  ANOXIA  .. .. . i n i t i a l w e t wt.  per g  None ouabain,  OUABAIN  n  0.46  ±  0.06  0.71  +  0.05  0.6 8 ±  0.02  K  _ °'  n  °-  n  5  0  n en °6  0  1  + n  Q 0  9  n  1  I n c u b a t i o n s were c a r r i e d o u t f o r 90 min under N : C 0 2 i n a C a + - f r e e m e d i u m c o n t a i n i n g 20mM g l u c o s e . ATP was m e a s u r e d e n z y m a t i c a l l y , a s d e s c r i b e d i n the m a t e r i a l s and methods. +  2  141 TABLE 16 EFFECT OF AEROBIC PREINCUBATION IN ADENOSINE ON THE TETRODOTOXIN STIMULATION OF ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min)  Addition  s l i c e s not incubated w i t h adenosine 80.4 ± 6.7  None 2yM TTX, p r e s e n t from zero time  132  TTX added a f t e r 15 min anaerobiosis, 2yM f i n a l concentration  84. 8  ± 1.5  s l i c e s incubated w i t h adenosine 102.1  ± 4.5  136.1  ± 4.0  92.2  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d f o r 40 min under 0 2 : C 0 i n a Krebs-Ringer b i c a r b o n a t e medium c o n t a i n i n g 20mM g l u c o s e . They were then t r a n s f e r r e d t o a C a i f r e e medium c o n t a i n i n g 20mM g l u c o s e , w i t h or without TTX. A n a e r o b i c g l y c o l y s i s was then measured m a n o m e t r i c a l l y d u r i n g subsequent 20-80 min a n a e r o b i c p e r i o d , as d e s c r i b e d i n the m a t e r i a l s and methods. 2  +  -  The  anaerobic  142 -  g l y c o l y s i s was t h e n measured.  These r e s u l t s  with  the adenosine t r e a t e d s l i c e s sr>w t h a t t h e r e i s no i n c r e a s e i n response t o TTX, although that o f the non-treated min  the g l y c o l y t i c rates are higher  slices.  When TTX i s added, a f t e r 15  a n a e r o b i o s i s t o t h e adenosine t r e a t e d s l i c e s , t h e r e i s no  a c c e l e r a t i n g e f f e c t on t h e r a t e o f a n a e r o b i c 5.8  than  glycolysis.  EFFECTS OF TETRODOTOXIN ON cAMP PRODUCTION IN CEREBRAL CORTEX SLICES C y c l i c AMP has been shown t o i n c r e a s e t h e r a t e o f  a e r o b i c as w e l l as anaerobic F u r t h e r , Mansour and Stone  glycolysis i n brain  (Chapter 3 ) .  have shown t h a t some drugs such  as LSD-25 i n c r e a s e t h e p r o d u c t i o n o f cAMP i n l i v e r f l u k e s , and t h i s i n turn increases the rate o f l a c t a t e production, by a c t i v a t i o n o f p h o s p h o f r u c t o k i n a s e  activity.  Mcllwain  c o w o r k e r s ^ ^ e s t a b l i s h e d t h a t when t h e c e r e b r a l c o r t e x 0  are e l e c t r i c a l l y stimulated, considerably increased.  formation  presumably and h i s  slices  o f cAMP i n t h e s l i c e s i s  As has been mentioned i n Chapter 1,  e l e c t r i c a l s t i m u l a t i o n a l s o increases the rate of aerobic glycolysis.  Hence experiments were c a r r i e d out t o see i f t h e  s t i m u l a t i o n o f anaerobic  g l y c o l y s i s of cerebral cortex  by TTX i s due t o i n c r e a s e d f o r m a t i o n o f cAMP. the f o r m a t i o n  slices  I f TTX i n c r e a s e s  o f cAMP then i t may f a c i l i t a t e t h e phosphofruc-  tokinase step, e s p e c i a l l y at the beginning  o f t h e experimsnt,  by d e c r e a s i n g  phosphofructokinase  t h e degree o f i n h i b i t i o n o f  -  by ATP;  -  143  at a l a t e r stage, when ATP  not have any  effect.  The  l e v e l decreases,  i t should  i n c r e a s e d r a t e of g l y c o l y s i s  at  the  s t a r t o f the experiment w i l l tend t o keep the r a t e of d e c l i n e o f ATP of  content  slow, and  t h i s may  r e s u l t i n an i n c r e a s e d r a t e  glycolysis. O C^. A  We  used the method o f Shimizu et.aJL.  the p r o d u c t i o n o f cAMP.  t o measure  i n t h i s method, as d e s c r i b e d i n Chapter  2, the c e r e b r a l c o r t e x s l i c e s are f i r s t p r e - i n c u b a t e d  i n oxygen  i n the presence o f C  14 -adenine t o get a p o o l o f C - A T P .  The  14  e f f e c t of drugs on cAMP can be e a s i l y measured r a t e s of c o n v e r s i o n  by o b s e r v i n g  o f C - A T P t o C -cAMP under v a r i o u s 14  the  condi-  14  tions. R e s u l t s i n Table t i o n of adenine-8-C i n i t s ATP et.al. ^ 2  cAMP, was  4  14  17A  show t h a t the r a t e s o f i n c o r p o r a -  i n the c e r e b r a l c o r t e x s l i c e s as w e l l as  pool increases with time. that histamine confirmed.  i n the presence o f TTX  The  o b s e r v a t i o n o f Shimizu  g r e a t l y i n c r e a s e s the p r o d u c t i o n  However, any  increased production  c o u l d not be observed  shows t h a t the e f f e c t o f TTX  on the anaerobic  c e r e b r a l c o r t e x s l i c e s i s not due  o f cAMP  (Table 17B). glycolysis  of  This o f the  t o an i n c r e a s e i n the concen-  t r a t i o n o f cAMP i n the b r a i n c e l l s . 5.9  EFFECT OF PROTOVERATRINE ON THE  TETRODOTOXIN  STIMULATED GLYCOLYSIS of Na  +  e i nother c r e a s ehand, s the TTX i n f il s ux i n t oI t e i x cs iknown t a b l e tc he altl sp .r o t'o v e r a tOnr i nthe 7 2  1 3 5  144 TABLE 17 EFFECTS OF TETRODOTOXIN ON THE cAMP FORMATION  A.  IN THE CEREBRAL CORTEX SLICES  Incorporation of adenine-8-C sulfate into r a t cerebral c o r t e x s l i c e s and i t s ATP p o o l . 14  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n Krebs-Ringer b i c a r b o n a t e c o n t a i n i n g 20mM g l u c o s e and 2yc.of a d e n i n e - 8 - C s u l f a t e (51.5 mc/mM). A t the end o f the i n c u b a t i o n s l i c e s were homogenized i n 5% TCA. A f t e r d e p r o t e i n i z a t i o n , TCA was removed w i t h e t h e r and a p o r t i o n was counted f o r t o t a l r a d i o a c t i v i t y w h i l e another p o r t i o n was s p o t t e d w i t h c a r r i e r ATP on a PEI c e l l u l o s e p l a t e and developed i n IM L i C l ; r a d i o a c t i v i t y i n ATP spots were d e t e r m i n e d ^ a f t e r s c r a p p i n g , i n a Mark I l i q u i d s c i n t i l l a t i o n counter. 1h  Adenine-8-C incorporated, x l O c.p.m. p e r g i n i t i a l wet wt 114  Incubation time i n min  4  Total  ATP  5  171  5.0  10  288  16.9  20  471  42.7  40  1043  144A TABLE  17  (Continued)  B.  Conversion slices.  of C 1 1  ATP  t o cAMP  i n guinea  p i g cerebral  cortex  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d as i n A. A f t e r 40 rain, t h e p u l s e l a b e l l e d s l i c e s were t r a n s f e r r e d t o a Ca t_ f r e e m e d i u m c o n t a i n i n g ImM c a f f e i n e a n d 20mM g l u c o s e , w i t h a n d w i t h o u t 2yM T T X . I n c u b a t i o n s were c a r r i e d o u t f o r a n o t h e r 10 m i n i n N : C 0 and t o t a l r a d i o a c t i v i t y i n the s l i c e s a s w e l l a s t h a t i n cAMP w a s d e t e r m i n e d a s g i v e n i n the m a t e r i a l s and methods. +  2  2  c.p.m. p e r g  initial  wet wt  Additions Total,  xlO  5  cAMP,  xlO  ImM  caffeine  103  ± 7  159  ± 26  ImM +  caffeine 2yM T T X  102  ± 8  95  ± 45  3  -  known t o b l o c k cells  the generation  -  145  o f t h e a c t i o n p o t e n t i a l s i n such  (Chapter 1 ) . As noted by Wollenberger,  protovera-  t r i n e i n h i b i t s the r a t e of anaerobic g l y c o l y s i s of c e r e b r a l cortex s l i c e s .  I n view o f t h e i r o p p o s i t e  e f f e c t s on t h e  anaerobic g l y c o l y s i s of c e r e b r a l cortex s l i c e s , t o study t h e i r e f f e c t s t o g e t h e r . are shown i n F i g u r e 22.  Results  I t i s evident  these drugs a r e a n t o g o n i s t i c .  i t was  decided  o f such an experiment  that the e f f e c t s of  Thus 5 uM TTX p r o t o v e r a t r i n e  i n h i b i t s t h e enhanced g l y c o l y s i s caused by 2 yiM TTX t o a considerable extent.  When t h e c o n c e n t r a t i o n  was f u r t h e r i n c r e a s e d , p l e t e l y abolished 5.10  of protoveratrine  up t o 20 uM, the e f f e c t o f TTX i s com-  ( r e s u l t s not shown).  EFFECTS OF TETRODOTOXIN ON THE Na-22 TRANSPORT IN THE  RAT CEREBRAL CORTEX SLICES I t has been w e l l e s t a b l i s h e d t h a t t h e movement o f  Na  +  during the generation  o f an a c t i o n p o t e n t i a l i s b l o c k e d  by TTX (Chapter 1 ) . F u r t h e r , reverse sis  both glutamate and p r o t o v e r a t r i n e  t h e s t i m u l a t i n g e f f e c t o f TTX on the a n a e r o b i c g l y c o l y -  (Chapters 4.8 and 5.9).  increase the i n f l u x of N a over, N a  +  glycolysis  +  These two agents a r e knownto i n the c e r e b r a l cortex s l i c e s .  More-  has i n h i b i t o r y e f f e c t on t h e r a t e o f a n a e r o b i c (Chapters 1.2 and 3.7).  t h a t TTX might a c t by p r e v e n t i n g  Hence, i t appeared t o us  t h e i n f l u x o f Na , i n the +  - 146 FIGURE  22  EFFECTS OF TETRODOTOXIN,IN THE PRESENCE OF  PROTOVERATRINE,ON  THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  60  '  ,  Time, i n minutes I n c u b a t i o n s were c a r r i e d out i n a C a - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m t r i c a l l y as g i v e n i n the m a t e r i a l s and m e t h o d s . ( O ) c o n t r o l ; ( A ) 5 uM p r o t o v e r a t r i n e ; ( • )2 uM TTX; ( B ) 5 uM p r o t o v e r a t r i n e and 2 uM TTX. + +  -  cerebral  cortex  therefore,  slices,  t o study  absence  o f TTX.  in  Table  18.  These  on  pp t h e Na""  influx  If of  +  The r e s u l t s  medium b y s u l f a t e glycolysis  the influx  results under  then  have  of TTX  these  less  glycolysis.  Moreover  control  slices  medium.  This  due  t o suppression 2  There  i s very  under  these  (not  are given  shown).  i n greater  i n Table  the rate  little  rate  of Na  +  water  sulfate  uptake  conditions, indicating  +  19.  anaerobic  due t o 2  9  8  4  The  anaero-  ) ;  I t i s seen of  that  anaerobic  glycolysis to that  glycolysis Furthermore,  medium  TTX  results  i n the  i n theC l ~  out the p o s s i b i l i t y  anaerobic  effect  suppression  of  S0  the rate  of the Na -influx.  transport i n a  summarized  of the incubation  of anaerobic  rules  and  little  to the  l a r g e as compared  thus  decided,  conditions.  conditions.  i n increasing  o f TTX on c e r e b r a l  TTX on N a ^  experimental  these  are  TTX has v e r y  the influx  under  experiment  effect  experiments  C l " i s replaced by  i s not very  the  of  when  effective  that  I t was  i n the presence  the chloride  result that  effect  experiments  i s s t i l l  show  2 2  o f TTX i s due o n l y  ( i t i s known  should  of Na  the given  should  i s suppressed  of anoxia.  of these  replacing  biosis  -  a t the onset  the effect  Na -influx,  147  that  i s solely the  effect  i s negligible.  by the cerebral the suppression  cortex of Na  slices, +  influx  148 TABLE 18 EFFECT OF SOME NEUROTROPIC DRUGS ON Na  INFLUX IN RAT CEREBRAL CORTEX  2 2  SLICES  yequivalents Na , corresponding to N a s l i c e s , p e r g i n i t i a l wet wt +  Additions  10 min  None 0.2yM TTX 2yM TTX lOyM TTX  15 min  170 + 13  177 ±  4  162 ± 10  151 ±  8  151 ±  149 ±  5  7  2 2  in  60 min 208 ± 12  143  O.lmM L i d o c a i n e  189 ±  2  0.25mM Amytal  186 ±  2  lOyM ouabain  179 ±  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a Ca f r e e medium c o n t a i n i n g 20mM glucose under N : C 0 . A d d i t i o n s were made a t zero time. A f t e r 15 min, 0.5 yc o f N a was added from the s i d e arm and N a i n f l u x was determined a f t e r v a r i o u s i n t e r v a l s , (10 min, 15 min o r 60 min), as g i v e n i n the m a t e r i a l s and methods. The above v a l u e s have n o t been c o r r e c t e d for swelling. 2  2 2  2  2 2  149 TABLE  EFFECTS  OF  TETRODOTOXIN  GLYCOLYSIS CEREBRAL  AND  Na  CORTEX  2 2  ON  THE  TRANSPORT  SLICES  FREE  Additions  19  IN A  ANAEROBIC IN  RAT  CHLORIDE  MEDIUM  Lactate produced ymoles p e r g i n i t i a l wet wt (20-80 min)  yequivalent Na corresponding to Na , p e r g i n i t i a l wet wt +  2 2  None  14.7  ±1.0  61.5±1.5  2yM T T X  38.0 ± 1.8  60.0 ± 1.7  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C l ~ f r e e m e d i u m c o n t a i n i n g 20mM g l u c o s e . Lactate p r o d u c t i o n was d e t e r m i n e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. For Na influx e x p e r i m e n t s , c o n d i t i o n s w e r e same a s T a b l e 18 except that incubation time i n N a w a s 15 m i n . 2 2  2 2  - 150  5.11  -  EFFECTS OF TETRODOTOXIN, AT VARIOUS CATION CONCENTRATIONS OF THE  MEDIUM, ON  THE  ANAEROBIC GLYCOLYSIS  OF CEREBRAL CORTEX SLICES Ashford 100 mM  K, +  and D i x o n  6 2  showed t h a t , i n the presence o f  both r e s p i r a t i o n and a e r o b i c g l y c o l y s i s o f the  b r a l c o r t e x s l i c e s are i n c r e a s e d . aerobic g l y c o l y s i s , anaerobic Chapter 1.2).  We  cere-  However, i n c o n t r a s t t o  g l y c o l y s i s i s depressed  have seen i n Chapter 3 t h a t when K  t r a t i o n i s increased with a corresponding  +  (see concen-  decrease i n N a  con-  +  c e n t r a t i o n , t h e r e i s a c o n s i d e r a b l e i n c r e a s e i n the r a t e o f anaerobic  glycolysis.  Experiments were c a r r i e d out,  therefore,  t o observe whether under t h e s e v a r y i n g c a t i o n c o n c e n t r a t i o n s , TTX  is still  e f f e c t i v e i n s t i m u l a t i n g the r a t e o f  g l y c o l y s i s o f the c e r e b r a l c o r t e x  anaerobic  slices.  T a b l e 20 shows the e f f e c t o f TTX  on the  anaerobic  g l y c o l y s i s of r a t c e r e b r a l cortex s l i c e s i n a C a - f r e e + +  t o which a d d i t i o n l K a  +  has been added.  r e s u l t s t h a t , when the K decreasing  +  I t i s evident  concentration i s increased  the c o r r e s p o n d i n g  Na  +  c o n c e n t r a t i o n , TTX  e f f e c t i v e i n i n c r e a s i n g the r a t e o f anaerobic However, when N a  c o n c e n t r a t i o n i s decreased  +  the i n c r e a s e o f K  +  i s i n c r e a s e d by TTX has  medium,  from  these  without i s not  glycolysis. i n proportion to  c o n c e n t r a t i o n , then the r a t e o f g l y c o l y s i s (Table 21).  Under these c o n d i t i o n s ,  p r o g r e s s i v e l y l e s s e f f e c t as N a  +  i s decreased  until  a  TTX  151 TABLE 20 EFFECT OF PRESENCE OF DIFFERENT CONCENTRATION OF K  +  ON THE TETRODOTOXIN STIMULATION OF  ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  K , added t o Ca free medium, i n mM +  + +  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min) No TTX  2yM TTX  0  26.3 ± 0.4  77.7 ± 1.8  33.3  19.7 ± 1.3  21.0 ± 1.3  66.6  20.1 ± 3.6  24.5 ± 2.4  21.0 ± 0.9  23.2 ± 2.7  100  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a Ca f r e e medium c o n t a i n i n g 20mM g l u c o s e . Additions were made a t zero time and l a c t a t e p r o d u c t i o n was measured manometrically as g i v e n i n the m a t e r i a l s and methods.  152 TABLE  EFFECTS CATION  OF  TETRODOTOXIN  CONCENTRATION  THE  ANAEROBIC CEREBRAL  Na  K  +  2yM TTX  +  21  OF  AT  VARYING  T H E MEDIUM  G L Y C O L Y S I S OF CORTEX  ON  RAT  SLICES  Lactate produced y m o l e s p e r g i n i t i a l w e t wt (20-80 min)  149  5  0  21  149  5  +  72  89  60  0  59  89  60  +  70  29  60  0  127  29  60  +  144  29  125  0  107  29  125  . +  130  0  154  0  115  0  154  + •  114  H i g h p o t a s s i u m medium was p r e p a r e d b y r e p l a c i n g some o r a l l N a by K a s d e s c r i b e d i n C h a p t e r 2.5 f . M e d i u m c o n t a i n e d 20mM g l u c o s e , T T X w a s a d d e d a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i cally. V a l u e s a r e a v e r a g e s o f two d e t e r m i n a t i o n s w i t h i n ± 7%. +  +  -  153  -  maximum r a t e o f g l y c o l y s i s i s o b t a i n e d when most o f t h e Na"" was 1  r e p l a c e d b y K ( T a b l e 2 1 ) . When t h e r a t e o f g l y c o l y s i s i s v e r y +  high  (i.e., Na  such as C a  + +  +  i s completely  r e p l a c e d by K ) , t h e n o t h e r  agents  +  showed no s t i m u l a t o r y e f f e c t .  As t h e r a t e o f a n a e r o b i c g l y c o l y s i s i n t h e p r e s e n c e of h i g h c o n c e n t r a t i o n s  of K  +  ( i n p r e s e n c e o f 149 M N a ) i s n o t 111  +  i n f l u e n c e d by TTX, i t was d e c i d e d t o s t u d y t h e i r e f f e c t s a t a l a t e r s t a g e i . e . , when t h e e f f e c t o f TTX has a l r e a d y been established. F i g u r e 23. Li  +  The r e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n I t i s e v i d e n t t h a t , when s o l u t i o n s o f K , N a o r +  +  a r e t i p p e d i n f r o m t h e s i d e arm o f t h e Warburg v e s s e l  giving a f i n a l concentration  o f 100 M m  ( i n addition to that  a l r e a d y p r e s e n t ) , t h e r a t e o f g l y c o l y s i s , w h i c h i s h i g h due t o t h e p r e s e n c e o f TTX, i s r e d u c e d s i g n i f i c a n t l y . tested, K depressing  +  Of t h e c a t i o n s  i s t h e most e f f e c t i v e , f o l l o w e d by L i  +  and N a , i n +  t h e T T X - s t i m u l a t e d g l y c o l y s i s . Under same c o n d i t i o n s  t h e r e i s an i n c r e a s e d i n f l u x o f N a  2 2  (preliminary r e s u l t s ) .  The e f f e c t o f c a t i o n s on a n a e r o b i c g l y c o l y s i s does n o t appear t o be due t o changes i n t h e t o n i c i t y s i n c e a d d i t i o n s o f s u c r o s e , a t 66, 133, o r 200 mM f i n a l c o n c e n t r a t i o n s ,  have l i t t l e o r no  e f f e c t on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f b r a i n s l i c e s liminary  experiments). The l a c k o f e f f e c t o f TTX i n t h e p r e s e n c e o f h i g h  and  (pre-  149 mM N a  +  may be due t o i n c r e a s e d i n f l u x o f N a .  w i l l be f u r t h e r d i s c u s s e d  +  i n C h a p t e r 8.  This  K  +  - 154 FIGURE  23  EFFECTS OF ADDITION OF HIGH CONCENTRATIONS OF CATIONS ON THE TETRODOTOXIN STIMULATED ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  Time , i n minutes I n c u b a t i o n s were c a r r i e d out i n a C a - f r e e medium c o n t a i n i n g 20 mM glucose.Arrow p o i n t s o u t time a t which 1 M s o l u t i o n o f KCl,NaCl o r L i C l was t i p p e d i n so as t o g i v e a f i n a l c o n c e n t r a t i o n o f 100 mM.Lactate p r o d u c t i o n was measured m a n o m e t r i c a l l y , a s g i v e n i n the m a t e r i a l s and methods.(—•) c o n t r o l , ( )2 uM TTX from zero time,no c a t i o n was added; ( ) 2 uM TTX from zero- time, Na was t i p p e d i n ; ( )2 uM TTX from z<pro t i m e , L i was t i p p e d i n ; ( )2 p.M TTX from zero time,K was t i p p e d i n . + +  - 155 -  5.12  EFFECTS OF TETRODOTOXIN ON THE N a  +  and K+ LEVELS OF  INCUBATED CEREBRAL CORTEX SLICES UNDER ANOXIA I t i s w e l l known t h a t t h e e f f e c t s o f e l e c t r i c a l  stimu-  l a t i o n on c a t i o n movements i n i n c u b a t e d b r a i n s l i c e s a r e b l o c k e d by TTX, as shown by i t s m e t a b o l i c e f f e c t s ^ ^ ' l -> 2  on t h e i n f l u x o f N a as noted while Na  + 1 3 2  (see Chapter  and on t h e e f f l u x o f K  1), K  +  a n G  + 1 2 9  stimulate pyruvate  i n h i b i t s both h e x o k i n a s e  +  3<  : i t  '  .  kinase  and p y r u v a t e  5 3 a  1 3 1  s  effects Also,  6 7 , 3  ®^  kinase.37-39.  I n view o f these marked e f f e c t s o f t h e s e c a t i o n s on t h e metabolism of cerebral t i s s u e ,  i t seemed q u i t e p o s s i b l e t h a t t h e s e might  p l a y an important r o l e , under t h e c o n d i t i o n s o f anoxia, when a c t i o n p o t e n t i a l s might be g e n e r a t e d . the K / N a +  +  TTX c o u l d g r e a t l y  influence  r a t i o o f t h e c e r e b r a l c o r t e x s l i c e s by b l o c k i n g t h e  movements o f t h e s e c a t i o n s .  An i n c r e a s e d K / N a +  c e l l w i l l r e s u l t i n a greater r a t e o f anaerobic However, as a l r e a d y mentioned  2 2  i n f l u x under a n o x i a i n t h e  I n view o f t h i s ,  t o measure both t h e K  +  and N a  c o r t e x s l i c e s under a n o x i a .  glycolysis.  (see 5.9), we have been unable t o  d e t e c t l a r g e changes i n t h e N a presence o f TTX.  r a t i o i n the  +  +  i t was c o n s i d e r e d n e c e s s a r y  contents of incubated c e r e b r a l  The r e s u l t s o f these  experiments  are d e s c r i b e d below. The  e f f e c t s o f 2 uM TTX on t h e N a  the g u i n e a p i g c e r e b r a l c o r t e x s l i c e s , c o n d i t i o n s , a r e shown i n F i g u r e 24.  +  and K  +  level i n  i n c u b a t e d under anoxic  I t w i l l be seen t h a t , i n  - 156 -  FIGURE  24  EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF GUINEA PIG CEREBRAL CORTEX SLICES UNDER ANOXIA  %  3  0  0  -  0  10  20  30  40  50  60  70  80  Time,in minutes I n c u b a t i o n s were c a r r i e d out i n a C a - f r e e medium c o n t a i n i n g 2Q_ mM glucose.TTX,when present,was added a t zero time.Na and K contents were determined as g i v e n i n the m a t e r i a l s and methods. ( "j-Na, c o n t r o l ; ( O )Na ,with 2 pM TTX; ( • )K , c o n t r o l ; ( • ) K,with 2 uM TTX. + +  -  157  -  t h e p r e s e n c e o f TTX, t h e amount o f K  +  retained i n the s l i c e s  i s twice the concentration i n the c o n t r o l s . t h e r e i s a r e d u c t i o n i n t h e amount o f N a .  S i m i l a r r e s u l t s on  +  the K  +  A t t h e same t i m e ,  content are obtained w i t h r a t c e r e b r a l cortex  These r e s u l t s make i t r e a s o n a b l e of TTX on t h e a n a e r o b i c  t o conclude t h a t the e f f e c t  g l y c o l y s i s of the c e r e b r a l cortex  s l i c e s may be due t o an i n c r e a s e i n t h e K / N a +  5.13  slices.  +  ratio.  EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS AND N a  2 2  TRANSPORT I N THE CAUDATE NUCLEUS OF RAT  As TTX i s b e l i e v e d t o a c t o n l y on e x c i t a b l e c e l l s , i t i s l i k e l y t h a t t h e e f f e c t o f TTX w i l l be c o n f i n e d t o t h e neurons.  Caudate n u c l e u s  i s a p a r t o f b r a i n which has r e l a -  t i v e l y few g l i a l c e l l s .  E x p e r i m e n t s were c a r r i e d o u t ,  t h e r e f o r e , on t h e e f f e c t s o f TTX on t h e r a t e o f a n a e r o b i c g l y c o l y s i s and on N a  2 2  transport i n the  caudate  nucleus.  R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n T a b l e 22. anaerobic  g l y c o l y s i s o f caudate nucleus  The  i s increased by the  p r e s e n c e o f TTX b u t t h e magnitude o f s t i m u l a t i o n i s n o t g r e a t e r than t h a t observed w i t h t h e c e r e b r a l c o r t e x s l i c e s . TTX h a s no e f f e c t on t h e N a  2 2  influx.  Moreover,  These r e s u l t s w i l l be  f u r t h e r d i s c u s s e d i n C h a p t e r 8. 5.14  EFFECTS OF TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF SYNAPTOSOMAL PREPARATIONS OF RAT BRAIN As mentioned e a r l i e r ,  of anaerobic  2 pM TTX a c c e l e r a t e s t h e r a t e  g l y c o l y s i s o f a d u l t r a t s and g u i n e a p i g s b u t n o t  158  TABLE 22 EFFECTS OF TETRODOTOXIN  ON THE ANAEROBIC  GLYCOLYSIS AND Na .TRANSPORT IN CAUDATE 22  NUCLEUS OF RAT  Additions  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min)  y e q u i v a l e n t Na, corresponding t o N a , per g i n i t i a l wet wt 2 2  None  26.8 ± 1.0  101+2  2yM TTX  62.5  104 ± 2  + 5.8  Incubations were c a r r i e d out i n a medium c o n t a i n i n g 20mM g l u c o s e . TTX was added a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods. F o r N a experiments, c o n d i t i o n s were same as i n Table 18 except t h a t i n c u b a t i o n time i n N a was 15 min. 2 2  2 2  -  o f 2-day o l d r a t b r a i n .  -  159  The s e n s i t i v i t y of t h e r a t b r a i n  slices  t o TTX i n c r e a s e s c o n s i d e r a b l y a t about 14th day a f t e r b i r t h , w h i c h c o i n c i d e s w i t h t h e t i m e o f maximum b r a i n growth and myelination.  I t has a l s o been mentioned  (Chapter 1.6) O c: O  that the  O C A  nerve endings are not m y e l i n a t e d . W h i t t a k e r ^ h  a  s  developed  t e c h n i q u e s by w h i c h i t i s p o s s i b l e t o s e p a r a t e t h e n e r v e e n d i n g s from o t h e r s u b c e l l u l a r p a r t i c l e s .  As t h e n e r v e e n d i n g s and  receptors are developed i n the b r a i n during maturation, e x p e r i ments were c a r r i e d o u t t o see whether t h e a n a e r o b i c g l y c o l y s i s o f t h e n e r v e e n d i n g p a r t i c l e s i s a f f e c t e d by TTX and o t h e r d r u g s . R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e 25.  These  e x p e r i m e n t s showed t h a t TTX and o u a b a i n have l i t t l e o r no  effect  on t h e r a t e o f a n a e r o b i c g l y c o l y s i s o f synaptosomes, w h i l e 4 ~-M Ca  + +  5.15  has an i n h i b i t o r y  action.  EFFECTS OF PRE-INCUBATION I N OXYGEN ON THE K  +  AND  Na  +  CONTENTS OF CEREBRAL CORTEX SLICES UNDER ANOXIA IN THE PRESENCE OF TETRODOTOXIN B r i e f aerobic pre-incubation of brain s l i c e s  increases  t h e subsequent r a t e o f a n a e r o b i c g l y c o l y s i s , b o t h i n t h e p r e s e n c e and absence o f TTX, oxygenated s l i c e s  as compared t o t h a t o b t a i n e d w i t h t h e non-  ( S e c t i o n 5.1).  E x p e r i m e n t s were c a r r i e d o u t ,  t h e r e f o r e , t o e v a l u a t e t h e e f f e c t s o f s h o r t (10 min) p r e - i n c u b a t i o n on t h e N a  +  and K  s l i c e s i n t h e p r e s e n c e o f TTX.  +  aerobic  content of the c e r e b r a l c o r t e x I t i s e v i d e n t from t h e r e s u l t s  FIGURE  25  EFFECTS OF TETRODOTOXIN,CALCIUM AND OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF RAT  SYNAPTOSOMES  I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured manometrically as g i v e n i n the m a t e r i a l s and methods.( • ) c o n t r o l ; ( A ) 1 0 uM ouabain; ( O ) 2 uM TTX ; ( B ) 4 mM Ca"." 4  4  - 160 FIGURE  30  25  40 50 60 T i m e , i n minutes  70  80  - 161 -  shown i n F i g u r e 26 t h a t t h e r e t e n t i o n o f K  i n t h e oxygenated  s l i c e s d u r i n g t h e subsequent p e r i o d o f a n o x i a i s much g r e a t e r . I n t h e p r e s e n c e o f 2 uM o f TTX, t h e r e i s v e r y l i t t l e K  +  and l e s s g a i n o f N a  +  during the anaerobic  There i s an i n i t i a l d r o p i n t h e K nary aerobic p e r i o d .  increased retention of K  +  incubation.  content during the p r e l i m i -  +  The e f f e c t o f C a  a l s o shown i n t h e same f i g u r e .  loss of  on t h e K  + +  +  level i s  I t i s c l e a r t h a t t h e r e i s some  by t h e s l i c e s i n t h e presence o f C a , + +  b u t i t was not as e f f e c t i v e as TTX. Because t h e r e s u l t s w i t h c a t i o n c o n t e n t s  a r e more  c l e a r c u t i n t h e oxygenated s l i c e s , i n a l l subsequent e x p e r i m e n t s r e p o r t e d below t h e technique study the r e t e n t i o n o f K  +  of p r i o r oxygenation  and u p t a k e o f N a  +  was u s e d t o  by incubated b r a i n  slices. 5.16  EFFECTS OF TETRODOTOXIN ON THE N a  AND K  +  +  LEVELS OF  INFANT RAT AND GUINEA PIG CEREBRAL CORTEX SLICES R e s u l t s o f e x p e r i m e n t s on t h e e f f e c t o f TTX on t h e anaerobic  g l y c o l y s i s o f d e v e l o p i n g b r a i n c o r t e x s l i c e s have  been g i v e n i n C h a p t e r 4.12. to  I t was o f i n t e r e s t , t h e r e f o r e ,  o b s e r v e w h e t h e r TTX a f f e c t s N a  +  and K  +  contents  of the  i n f a n t r a t , as w e l l as i n f a n t g u i n e a p i g , c e r e b r a l c o r t e x  slices.  The r e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e s 27 and 28. It  i s e v i d e n t t h a t TTX has l i t t l e o r no e f f e c t on t h e c a t i o n  content  o f i n f a n t (2-day o l d ) r a t b r a i n s l i c e s b u t t h e i n f a n t  - 162 FIGURE  26  E F F E C T S OF T E T R O D O T O X I N AND C A L C I U M ON T H E SODIUM P O T A S S I U M C O N C E N T R A T I O N S O F RAT' C E R E B R A L CORTEX S L I C E S  AND  300  250 .  4J  tj 200 iH (0  •H -P •H C  •  H  150  U  <u Ch  cn +» g 100  H  >  •H  d  (D  3_  50 O  0  5 (  AEROBIC  10 )  5  10  30  ( A N A E R O B I C Time,  i n minutes  ++  I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . I n i t i a l 10 m i n p e r i o d w a s a e r o b i c ( 0 _ : C O _ ) f o l l o w e d by a n a e r o b i c p e r i o d ( N 2 ^ - A d d i t i o n s w e r e made a t zero time.Vertical bars represent standard d e v i a t i o n s . ( • ) N a , : C 0  2  c o n t r o l ; (• ]Na, .with  (A ) 4mM C a  ,K .  2 uM  +  TTX; ( O ) K , c o n t r o l ; ( • ) K , w i t h +  +  2 uM r  TTX.  -  163 -  175  150  -  4-» +j 1 2 5 CD  f0  •H +J •H 100  n CD  to  +J  c  75  -  CD  >  •H 3  cr  r  CD  3.  f  50  O  2  25  5 10 (A N A E R 0 B ) Time,in minutes FIGURE 27 : EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF NEWLY BORN GUINEA PIG CEREBRAL CORTEX S L I C E S . I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 2 6 . ( A ) N a ,control; ( A ) Na , w i t h 2 uM TTX; . ( © ) , c o n t r o l ; ( O ) K , w i t h 2 uM TTX. 0  5 (AEROBIC  10  )  +  +  K  +  - 164 FIGURE 28 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF TWO DAY OLD RAT CEREBRAL CORTEX SLICES  0 (  5 10 AEROBIC )(  5 10 A N A E R O Time,in minutes  B  I  C  30 )  I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 26 . ( • )Na*. c o n t r o l ; ( • ) N a ,with 2 uM TTX; ( A ) K , c o n t r o l ; K ,with 2 pM TTX ( A )•  -  165  -  g u i n e a p i g shows a marked i n c r e a s e i n t h e K decrease i n Na 5.17  +  content,  +  i n the presence of  EFFECTS OF TETRODOTOXIN ON THE  Na  +  c o n t e n t , and  a  TTX.  and K  LEVELS OF  +  KIDNEY MEDULLA SLICES . K i d n e y m e d u l l a was 4.11). the K  As has and N a  +  +  u s e d as a c o n t r o l t i s s u e  (Chapter  been shown i n F i g u r e 29, TTX has no e f f e c t contents  of the kidney medulla s l i c e s .  on  This  r e s u l t i s c o n s i s t e n t w i t h the c o n c l u s i o n t h a t the e f f e c t of  TTX  i s s p e c i f i c t o the nervous t i s s u e . 5.18  EFFECTS OF TETRODOTOXIN I N THE AGENTS ON THE  PRESENCE OF CHELATING  ANAEROBIC GLYCOLYSIS OF CEREBRAL  CORTEX SLICES Chan and Q u a s t e l - ^ O have shown t h a t t h e i n c r e a s e i n r e s p i r a t i o n b r o u g h t about by t h e absence o f C a by TTX. Ca  + +  We  + +  is  have a l s o seen, i n C h a p t e r 4, t h a t TTX  i n i n c r e a s i n g the anaerobic  prevented acts  g l y c o l y s i s and s u p p r e s s i n g  enhanced a e r o b i c g l y c o l y s i s c a u s e d by t h e absence of I t has been known f o r a l o n g t i m e , t h a t Ca b i o l o g i c a l membranes.  + +  may  + +  the  .  the  a c t by  and t h u s change i t s p e r -  To t e s t t h i s p o s s i b i l i t y , i t was  t o s t u d y t h e e f f e c t s o f TTX  Ca  stabilizes  I t i s p o s s i b l e t h a t TTX  i n t e r - a c t i n g w i t h membrane-bound C a meability.  like  on a n a e r o b i c  considered desirable  g l y c o l y s i s i n the  p r e s e n c e o f c h e l a t i n g a g e n t s EDTA and EGTA (EDTA c h e l a t e s b o t h Ca  + +  and M g  + +  w h i l e EGTA i s more s p e c i f i c i n b i n d i n g  Ca ). + +  - 166 FIGURE  29  EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT KIDNEY MEDULLA SLICES  150  01  I 1  l  0  5  (  AEROBIC)  I 5  0  (  J 10  A N A E R O Time, i n minutes  I  313  B  I  C  )  I n c u b a t i o n c o n d i t i o n s were same as i n Figure. 26. ( • ) N a , c o n t r o l ; ( • ) Na , with 2 uM TTX; ( • ) K , c o n t r o l ; ( 0 ) K ,with 2 pM TTX. +  -  Results  o f experiments,  ineffective thought about of  i n the presence  that  this  TTX o r t h a t  5.19  there  TTX i s  OF  occurs  of these  presence regain which  large influx  of C a  excessive  to  ethanol. to  to  not been  TTX.  exposed  of K , +  show  rule  out the p o s s i b i l i t y  with  t h e mode this.  any  effect  i n which  as t h a t  have  which  GLYCOLYSIS  This  inhibition  then  the slices  experiments  that  o f TTX.  with  +  have  directly  slices  was  stated  K -ATPase by +  been  show  However,  F u r t h e r work  the  i n the do n o t  +  glycolysis  a r e shown  may  K  action  should  the case. ethanol  lost  of Na ,  which  of ethanol  shown t h a t ,  have  obtained  to ethanol.  i s indeed  of action  ON  QQ  o f TTX on a n a e r o b i c  of typical  this  slices  partial  they  settle  that  masks  depolarization  and L e B l a n c ^ ^  i n the presence  Results  which  brought  +  SLICES  extent  I f the effects  oxygen,  +  I t i s  of Na  P R E - I N C U B A T I O N WITH ETHANOL  cerebral  through  the retention  under  Kalant  i t t o t h e same  be mediated  +  TTX i s  agents.  S T I M U L A T I O N OF A N A E R O B I C  CORTEX  of ethanol,  have  show t h a t  chelating  O  Israel,  23,  ineffective.  TETRODOTOXIN CEREBRAL  i n Table  chelation  E F F E C T S OF A E R O B I C THE  -  i s due t o t h e v e r y  by t h e complete  condition  given  167  a r e due incubated,  less  i n Table this  response 24;  does n o t  interfere  i s necessary  to  168 TABLE  EFFECTS  OF  TETRODOTOXIN GLYCOLYSIS  OF  ymoles  EDTA  23  AND  EGTA  STIMULATION CEREBRAL  OF  ON  THE  ANAEROBIC  CORTEX  SLICES  Lactate produced p e r g i n i t i a l w e t wt  (20-80 m i n )  Additions  None  Rat  Guinea P i g  24.4 ± 2.8  30.7 ± 7.6  2yM  TTX  81.1  ± 7.6  ImM  EDTA  11.3  ImM  EGTA  24.9  ImM +  EDTA 2yM T T X  17.8 + 4.3  14.9  ± 4.6  ImM +  EGTA 2yM T T X  27.3  37.5  ± 8.0  141.8 ±14.5 11.8 ± 0.8  ± 3.5  ± 1.9  17.7 ± 2.9  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C a f r e e m e d i u m c o n t a i n i n g 20mM g l u c o s e . Additions w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was measured manometrically as given i n t h e m a t e r i a l s and methods. +  +  169 TABLE  EFFECT  OF  ETHANOL  STIMULATION RAT  Additions  None 200mM 2yM  Ethanol TTX  2uM T T X + 200mM E t h a n o l  24  ON  THE  OF A N A E R O B I C  CEREBRAL CORTEX  TETRODOTOXIN G L Y C O L Y S I S OF SLICES  Lactate produced umoles p e r g i n i t i a l wet wt  23.7  ± 0.5  36.2  ± 4.5  107.2  ± 2.0  82.5  ± 3.1  (30-90 m i n )  I n c u b a t i o n s were c a r r i e d o u t i n a Ca - f r e e m e d i u m c o n t a i n i n g 20mM g l u c o s e . F i r s t 10 m i n i n c u b a t i o n w a s a e r o b i c (02:C02) f o l l o w e d b y anaerobiosis. A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods.  -  5.20  170  -  EFFECTS OF TETRODOTOXIN ON THE N a  +  AND K  +  CONTENT OF  THE CEREBRAL CORTEX SLICES IN THE PRESENCE OF GLUTAMATE, ASPARTATE, HOMOCYSTEIATE AND  NHA  +  We have shown i n Chapter 4 . 8 t h a t i n t h e presence o f glutamate and  NH4  +  ,  TTX i s much l e s s e f f e c t i v e i n i n c r e a s i n g  t h e a n a e r o b i c g l y c o l y s i s o f the c e r e b r a l c o r t e x s l i c e s .  Under  t h e same c o n d i t i o n s a s p a r t a t e was found t o have no e f f e c t . Hence experiments were c a r r i e d out t o see i f t h e a a t i o n concent r a t i o n of the s l i c e s , glutamate, N H 4  i n t h e presence o f TTX, i s a f f e c t e d by  and o t h e r amino a c i d s .  experiments a r e g i v e n i n T a b l e 2 5 L- or D-glutamate  The r e s u l t s o f t h e s e  I t w i l l be seen t h a t when  0  i s present, the r e t e n t i o n o f K  the s l i c e s than i n those i n c u b a t e d i n t h e acids.  +  i s less i n  absence o f t h e amino  I n t h e presence o f NH^"", TTX had a s i g n i f i c a n t b u t 1  s m a l l e r e f f e c t on t h e K  +  levels.  Under t h e same c o n d i t i o n s ,  when L - a s p a r t a t e or DL-homocysteate tention of K  +  i s present, increased r e -  i n t h e presence o f TTX i s e v i d e n t .  These  results  demonstrate t h a t t h e e f f e c t s o f glutamate and NH^* on t h e TTX s t i m u l a t i o n o f a n a e r o b i c g l y c o l y s i s may be mediated through changes 5 21 0  i n the c e r e b r a l concentrations of N a  +  and K . +  EFFECTS OF TETRODOTOXIN ON THE PYRUVATE AND PHOSPHOENOL-PYRUVATE  CONTENTS OF CEREBRAL  CORTEX SLICES UNDER ANOXIA We have seen, i n s e c t i o n s 5.12 and 5.15, t h a t i n t h e  171 TABLE 25 EFFECTS OF TETRODOTOXIN ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF RAT CEREBRAL CORTEX SLICES IN THE PRESENCE OF SOME AMINO ACIDS AND NHi*"" 1  Aerobic Addition  5mM LGlutamate  5mM LGlutamate + 2yM TTX  Cation  5mM DGlutamate + 2yM TTX  5mM LAspartate  5mM LAspartate + 2yM TTX  5mM DLHomocisteiate  5mM DLHomocysteiate + 2yM TTX  5 min  10 min  30 min  160  170  195  220  42  25  21  16  130  155  175  220  48  42  37  23  147  175  200  232  46  29  20  15  150  155  165  215  49  43  36  23  145  180  190  220  40  27  21  15  140  135  170  210  K+  48  42  38  30  Na  153  200  200  223  48  27  20  14  150  160  175  235  47  46  38  45  Na K  1  +  Na + K  +  Na"  1  5mM DGlutamate  10 min  Anaerobic  K"  1  Na + K  +  Na" K Na +  1  K Na + K  +  171A TABLE  25  (Continued)  Aerobic Addition  10 m i n  Na 5mM  rc 5mM  M  +  NH^Cl  MN I IH 4m CI  2yM  TTX  Anaerobic  Cation  K  N  a  K  +  +  1  10 m i n  30  min  125  145  170  210  38  24  16  15  +  +  5 min  2  4  0  Q  1  2  3  5  5  1  4  3  Q  0  1  1  8  5  8  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e 26. E a c h v a l u e r e p r e s e n t a v e r a g e s o f two e x p e r i m e n t s w i t h i n ± 7%. F o r c o n t r o l s , s e e F i g u r e 26. Results a r e e x p r e s s e d as y e q u i v a l e n t s p e r g i n i t i a l wet wt.  -  presence  172  -  o f 2 uM TTX, t h e r e i s an i n c r e a s e i n t h e K / N a +  +  r a t i o o f t h e i n c u b a t e d c e r e b r a l c o r t e x s l i c e s a f t e r t h e onset of anoxia.  I f t h e e f f e c t o f TTX on t h e a n a e r o b i c  glycolysis  i s due t o t h e f a c i l i t a t i o n o f pyruvate k i n a s e s t e p because o f an i n c r e a s e i n t h e K / N a +  +  ratio.., then i n t h e presence  t h e r e s h o u l d be a decrease i n t h e phosphoenol-pyruvate c o n t e n t and an i n c r e a s e i n t h e pyruvate Experiments above h y p o t h e s i s .  o f TTX (PEP)  content.  were c a r r i e d out t h e r e f o r e t o t e s t t h e  F o r these experiments, c o n d i t i o n s  maximum e f f e c t on t h e K / N a +  +  under which  r a t i o i s o b t a i n e d , were s e l e c t e d .  As shown i n Table 26, i n t h e presence  o f TTX t h e c o n c e n t r a t i o n  o f pyruvate i n t h e c e r e b r a l c o r t e x s l i c e s i s indeed i n c r e a s e d . Phosphoenol-pyruvate extremely  c o n t e n t s i n t h e i n c u b a t e d s l i c e s were  low and a c c u r a t e d e t e r m i n a t i o n was t h e r e f o r e not  possible. The  i n c r e a s e i n pyruvate c o n t e n t s i n t h e presence o f  TTX thus supports t h e view t h a t t h e e f f e c t s o f TTX on t h e a n a e r o b i c g l y c o l y s i s i s due t o t h e f a c i l i t a t i o n o f t h e pyruvate kinase step;  presumably i t i s not due t o b l o c k o f pyruvate  e f f l u x from t h e s l i c e s d u r i n g i n c u b a t i o n , as TTX i s e f f e c t i v e i n i n c r e a s i n g t h e r a t e o f a n a e r o b i c g l y c o l y s i s even i n t h e presence  o f pyruvate  ( S e c t i o n 5.3).  173  TABLE  EFFECTS AND  OF  26  TETRODOTOXIN  ON  PHOSPHOENOL PYRUVATE CEREBRAL  mymoles  CORTEX  per  THE  PYRUVATE  CONTENT  OF  RAT  SLICES  g  initial  wet  wt  Additions  Zero  2  time  Pyruvate  PEP  107  6 2 + 5  ± 6  None  52  ± 8  LOW  yM  79  ± 6  LOW  TTX  Incubations were c a r r i e d o u t i n a C a free m e d i u m c o n t a i n i n g 20mM g l u c o s e f o r 10 m i n a e r o b i c a l l y f o l l o w e d b y 20 m i n a n a e r o b i o s i s . P y r u v a t e a n d PEP were m e a s u r e d a t t h e b e g i n n i n g and e n d o f t h e i n c u b a t i o n p e r i o d , as g i v e n i n the m a t e r i a l s and methods. +  +  t  -  SUMMARY  OF  CHAPTER Prior  increases  t h e subsequent  the absence  that  found  2.  oxygenation  min,  rate  and presence  of anaerobic  of the cerebral after  cerebral  cortex  retention directly The  ATP c o n t e n t s  the  presence  4. cerebral with  cortex  time.  aerobic slices  levels,  anoxia  slices,  glycolysis  glycolysis  i s blocked  glycolysis  on a n a e r o b i c  of the  through  the  glycolysis  by  (See Chapter  cortex  are higher i n  than  slices  i n t h e absence  from  o f TTX.  o f adenine  the pulse  8).  ATP  into the increases  labelled .  ATP  under  conditions.  anaerobic  effect  anoxia.  i s n o t i n c r e a s e d b y T T X , ."  Protoveratrine reverses  inhibitory  10  i s unlikely.  o f cAMP,  5.  the  i f i t i s added  incubation, uptaie  anaerobic  than  the anaerobic  and i t s i n c o r p o r a t i o n i n t o  The f o r m a t i o n  the incubated  slices  of  of the cerebral  o f TTX under  During  i n increasing  Its effect  ATP  both  greater  do n o t seem t o b e m e d i a t e d  of pyruvate. influencing  glycolysis,  o f TTX on t h e anaerobic  slices  slices  slices.  cortex  the onset  The e f f e c t s  cortex  o f TTX, t o a v a l u e  i n t h e non-oxygenated  or later,  3.  of  of the cerebral  TTX i s n o t e f f e c t i v e  glycolysis  -  5  1.  in  174  the effects  of the cerebral  cortex  o f TTX on t h e  slices.  o f p r o t o v e r a t r i n e on c e r e b r a l by TTX.  Moreover, anaerobic  - 175 -  6  TTX has l i t t l e e f f e c t on t h e N a ^  0  c e r e b r a l c o r t e x s l i c e s under 7.  z  i n f l u x into the  anoxia.  TTX h a s no a c c e l e r a t i n g e f f e c t on a n a e r o b i c  l y s i s when h i g h K  i s present  +  t a i n i n g normal N a  i n t h e i n c u b a t i o n medium c o n -  concentration.  +  t h e same t i m e t h a t K  +  glyco-  I f the Na  i s reduced a t  +  i s increased, the r a t e of anaerobic  g l y c o l y s i s i n c r e a s e s and TTX h a s p r o g r e s s i v e l y l e s s e f f e c t with increasing K + 8.  concentration.  +  +  +  K , L i o r Na  (100 mJM f i n a l c o n c e n t r a t i o n )  added t o t h e i n c u b a t i o n medium, i n w h i c h t h e a n a e r o b i c  when glyco-  l y s i s o f c e r e b r a l c o r t e x s l i c e s h a s been a c c e l e r a t e d by TTX, has  an i n h i b i t o r y e f f e c t on t h e TTX s t i m u l a t e d g l y c o l y s i s .  9.  K  of the a n a e r o b i c a l l y incubated  cerebral  c o r t e x s l i c e s i s i n c r e a s e d i n t h e p r e s e n c e o f TTX.  There i s  +  content  decrease i n t h e N a 10.  content  a t t h e same t i m e .  TTX i n c r e a s e s t h e a n a e r o b i c 22  n u c l e u s b u t Na 11.  +  i n f l u x i s not s u p p r e s s e d .  TTX and o u a b a i n have no e f f e c t on t h e a n a e r o b i c  g l y c o l y s i s o f synaptosomes b u t C a 12.  g l y c o l y s i s o f caudate  + +  has an i n h i b i t o r y a c t i o n .  The e f f e c t s o f TTX on t h e K  +  and N a  +  content  i s much  more a p p a r e n t i f , a f t e r t h e a d d i t i o n o f TTX, t h e s l i c e s a r e incubated  f o r a s h o r t p e r i o d i n oxygen p r e l i m i n a r y t o t h e  onset o f anoxia.  -  13.  176 -  TTX h a s l i t t l e o r no e f f e c t on t h e N a  c o n t e n t s o f 2-day o l d r a t  +  and K  +  c e r e b r a l cortex s l i c e s but i n f a n t  g u i n e a p i g s a r e s i m i l a r t o a d u l t a n i m a l s i n r e t a i n i n g more K and t a k i n g up l e s s N a 14.  +  i n t h e presence  +  o f TTX.  TTX has no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s o f  c e r e b r a l c o r t e x s l i c e s i n t h e presence  o f c h e l a t i n g agents,  EDTA and EGTA. 15.  A e r o b i c p r e - i n c u b a t i o n o f t h e s l i c e s i n t h e presence  of ethanol decreases s i s i n t h e presence 16.  t h e subsequent r a t e o f a n a e r o b i c  glycoly-  o f TTX.  Ammonium i o n s , L - g l u t a m a t e and D-glutamate r e d u c e  t h e e f f e c t o f TTX on t h e r e t e n t i o n o f K s l i c e s under a n o x i a  +  by t h e c e r e b r a l c o r t e x  (see T a b l e 2 5 ) , w h i l e L - a s p a r t a t e and  D L - h o m o c y s t e i a t e have no e f f e c t . 17.  I n t h e presence  o f TTX, t h e r e i s an i n c r e a s e i n t h e  p y r u v a t e c o n t e n t o f t h e c e r e b r a l c o r t e x s l i c e s under a n o x i a . T h i s s u p p o r t s t h e h y p o t h e s i s t h a t t h e e f f e c t s o f TTX on a n a e r o b i c g l y c o l y s i s might be due i n d i r e c t l y t o t h e f a c i l i t a t i o n o f t h e pyruvate kinase step.  CHAPTER EFFECTS  OF OUABAIN  AND  METABOLISM It  was  L O C A L A N E S T H E T I C S ON  AND  TRANSPORT  shown i n C h a p t e r s  increases  the rate  Na  of the incubated  ratio  concluded  that  responsible anaerobic the  effects  resemble  those  anesthetics  action  (Chaper  on a n a e r o b i c  1^  which the  effects  cell  tissue.  that  the e f f e c t s  of  o f membrane  showed lactic  that  acid  slices.  o f Na  As noted,  bound  ATPase. .  lOOyM o u a b a i n  inhibitor  + ,  they  ouabain  effects  increases  of glyceraldehyde  -ATPase, across  has marked  incubated a r e due t o i n h i b -  Rolleston  concluded  K  ion gradients  and Newsholme  the formation  i n the a e r o b i c a l l y incubated  Furthermore,  local  investigated.  of aerobically  of these  some o f  processes  f o r maintaining  Most  +  on c e r e b r a l  inhibitor  membrane.  K /  o f TTX on t h e  g l y c o l y s i s were  on t h e metabolism  cerebral  an  strong  i s responsible  brain  ition 52  i s a  as  I t was  + Ouabain  greatly  ratio i s  +  of the fact  anesthetics  o f TTX  TTX  tissue. +  f o r the stimulating  of local  CEREBRAL  ANOXIA  i n the K /Na  In view  THE  g l y c o l y s i s as w e l l  cerebral  increase  glycolysis.  UNDER  4 and 5 t h a t  of anaerobic  this  6  cerebral  that  ouabain  3-phosphate  of  cortex may  be  dehydrogenase.  181 Wollenberger the  anaerobic  manner  that  some  digitalis  glycolysis of cerebral  similar to that  ouabain light  found  of present  cortex  of protoveratrine.  on t h e a n a e r o b i c  g l y c o l y s i s was  knowledge  alkaloids slices  inhibit i n a  The e f f e c t o f  investigated  a s t o i t s mode o f a c t i o n .  i n the The  - 178 -  results  of experiments  anesthetics 6.1  will  EFFECTS  OF  CEREBRAL  be  greatly  CORTEX  ON  carried  enhances  tions  o f ouabain  i s shown  r a t cerebral  cortex  a  high  and  out with  A dose  10  i n a Ca rate  response  increase  stimulatory  results  Ringer  using  medium  decided,  slices,  medium.  30.  the rate  These  even  guinea  with  Ca  are contrary other  to those  digitalis  alkaloids  that,  concentp i g brain ouabain,  additional  i n c u b a t i o n medium  results  show  effect  than  stands  i n contrast to the effect  that  medium,  that  taking place  i n a Krebs-Ringer  of these  o f ouabain,  + +  less  i t was  i n the presence  -con-  Krebs-  Ca  + +  -free Krebs-  stimulatory  medium.  This  anaerobic  medium,  (Chapter  a  balanced  -free  bicarbonate  results,  of a  o f TTX on t h e  s t i m u l a t i o n i s observed  view  h a s much  + +  I t was  using  i n place  i n a Ca  Ca  slices.  i n a normal  ouabain  by  and a  where  con-  4.1).  decided  t o study  of different  +  glyco-  1 yM  reported  +  concentra-  ouabain  i n i t s c o n c e n t r a t i o n had no  bicarbonate  siderable  in a  of anaerobic  increasing  i s obtained  that  I t i s evident  However, w i t h  Ringer  effects  fordifferent  f o r incubation of the brain  bicarbonate  glycolysis  showed  glycolysis  t h e r e f o r e , t o c a r r y out experiments  medium.  In  ouabain  effect.  Wollenberger taining  -free  uM  curve  i n Figure  of glycolysis  further  These  + +  local  G L Y C O L Y S I S OF  of anaerobic  increases progressively with  rations  and  Chapter.  THE ANAEROBIC  the rate  medium.  lysis  i n this  ouabain  SLICES  -free  with  out with  considered  OUABAIN  Experiments it  carried  the  concentra-  - 179 FIGURE  30  EFFECTS OF DIFFERENT CONCENTRATIONS OF OUABAIN ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES  150 C •H  e o  CO  I  o  CN  125 -  •P  rd •H +1 •H  100 -  c  U  0) Ou CO  T3 CD O 3 tJ O  U Ou  CD •p ra -p  o  rd  20 40 60 Ouabain c o n c e n t r a t i o n , i n  uM  80  100  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a medium c o n t a i n i n g 2 0 mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured manome^rically as given i n t h e m a t e r i a l s and m e t h o d s . ( • ) G u i n e a pig,Ca -free medium;(•)rat,Ca -free m e d i u m ; ( O ) r a t , K r e b s - R i n g e r b i c a r b o n a t e medium. +  -  tions  of Ca  slices.  + +  ,  -  on t h e a n a e r o b i c  In these  experiments,  Figure  31,different  to  + +  a Ca  180  -free  glycolysis results  of which  concentrations of C a  medium  from  the start  of cerebral  +  a r e shown i n  ( 1 - 4 mM)  +  cortex  were  of the experiment  added  (see  ++ Figure  2 for results  strate  that  glycolytic when  both  when rate  cerebral 6.2  OF OUABAIN  BRAIN  The  primary  CORTEX  In infant  brain  are  given  has  little  old  r a t brain.  old  r a t brain  ouabain. very lysis  alone.  together  of anaerobic  of ouabain  they  the  Thus, have  glycolysis  G L Y C O L Y S I S OF  an  of the  INFANT  i s known  t o be on t h e N a ,  +  during  These  t h e 2-3 w e e k  period  on t h e a n a e r o b i c  results  the anaerobic  after  glycolysis  and the  indicate  on t h e a n a e r o b i c  However,  i s very  +  therefore, investigated  27.  +  the Na ,K -ATPase a c t i v i t y  of ouabain  o r no e f f e c t  that  glycolysis  glycolysis  The e f f e c t to that  o f ouabain f o r adult  r a t brain  i n the presence brain,  ouabain of  of  2-day  2-week  ++ i n the presence  o f Ca  of Ca  +  +  i . e . the anaerobic  i n the presence  of ouabain  of  results  i s considerably stimulated i n the presence  of infant  diminished  demon-  to ouabain,  ouabain  THE ANAEROBIC  rats,  was,  i n Table  similar  results  SLICES  action  The e f f e c t s  developing  are present  ON  but increases rapidly  birth.  i n addition  as with  on t h e r a t e  These  slices.  RAT  +  low  action  EFFECTS  alone).  i s present  +  substances  cortex  K -ATPase.  +  Ca  i s not as high  these  antagonistic  Ca  with  of i s glyco-  i s also  FIGURE EFFECT OUABAIN  181 31  OF VA-RYING C A L C I U M C O N C E N T R A T I O N ON  THE ANAEROBIC  IN THE PRESENCE  G L Y C O L Y S I S OF C E R E B R A L C O R T E X  OF SLICES  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a Ca - f r e e medium c o n t a i n i n g 10 uM o u a b a i n , 2 0 mM g l u c o s e a n d d i f f e r e n t c o n c e n t r a t i o n s o f Ca . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as given i n t h e m a t e r i a l s and methods.  182 TABLE 27 EFFECT OF  OF  OUABAIN  DEVELOPING  ON RAT  THE  ANAEROBIC  CEREBRAL  GLYCOLYSIS  CORTEX  SLICES  L a c t a t e produced ymoles per g i n i t i a l wet wt (20-80 min) Addition  2 day o l d  7 day o l d  14 day o l d  None  25 .0 + 1.5  21. 2 + 0.45  25 .4 + 3.1  lyM ouabain  38 .4 + 4.4  30. 8 + 1.7  31 .7 + 1.9  lOyM ouabain  41 .1 + 1.8  38. 8 + 5.8  64 .7 + 2.3  lOOyM ouabain  32 .6 + 1.4  46. 1 + 8.5  87 .0 + 8.0  lOyM ouabain + ImM C a  36 .1 + 1.9  29. 4 + 1.3  39 .7 + 3.6  lOyM ouabain + 2mM C a  36 .0 + 3.1  35. 3 + 6.2  46 .2 + 7.1  lOyM ouabain + 4mM C a  40 .1 + 2.3  35. 7 + 3.0  46 .0 + 2.7  + +  + +  + +  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C a f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y as g i v e n i n the m a t e r i a l s and methods.  -  6.3  EFFECTS  OF  OUABAIN  GLYCOLYSIS  OF  RAT  CONCENTRATIONS  Sodium pyruvate  ions  lysis  were  of Na  made  tions  of Na  of cerebral  influx  +  glycolysis. those  evident  that  The r e s u l t s out with  of Na , +  are higher  concentrations  6.4  EFFECTS  of  OF  than  OUABAIN  those  ON  of experiments  with  ANAEROBIC  concentra-  as w e l l  32.  as o f  I ti s  glycolysis normal  G L Y C O L Y S I S OF  i s also  carried  Na  +  ACETONE  o u t on t h e e f f e c t s o f  glycolysis of brain 17.  These  acetone  results  show  not effective i n increasing  glycolysis of brain  the e f f e c t of ouabain cell.  an  o r o f TTX, a t low  of anaerobic  obtained  glyco-  BRAIN  TTX, o u a b a i n  the  of ouabain,  i n the  anaerobic  TTX a r e shown i n F i g u r e  a r e shown i n F i g u r e  Hence  at different  investigations  the rates  of  investigations  o f these  extract  anaerobic  slices,  induces  of cerebral  on t h e a n a e r o b i c  of  of anaerobic  on r a t e s  ouabain  for  MEDIUM  3.7, an i n c r e a s e  As ouabain  of ouabain  AT D I F F E R E N T  the a c t i v i t y  the rate  cortex  ANAEROBIC  (See C h a p t e r 8 ) .  POWDER OF Results  THE  INCUBATION  slices.  i n the presence  concentrations attained  mM)  ON  SLICES  to influence  cerebral  (30-150  carried  IN THE  decreases  +  of the effects  of Na  +  CORTEX  shown i n C h a p t e r  cortex  into  +  OF N a  As  TETRODOTOXIN  CEREBRAL  a r e known  kinase.  concentration  AND  183 -  acetone  powder  seems t o r e q u i r e  powder that,  as  the  rate  extracts.  the i n t e g r i t y  -  FIGURE  184  -  32  EFFECTS OF OUABAIN AND TETRODOTOXIN ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES AT VARYING CONCENTRATIONS OF SODIUM  125  ^  I  I  0  0  I  I  30 60 90 Na c o n c e n t r a t i o n , i n mM +  1  120  1—  150  I n c u b a t i o n medium c o n t a i n e d 5 K ,20'mM glucose and v a r y i n g c o n c e n t r a t i o n s o f Na .29 mM Na was p r e s e n t as HCO^.When Na c o n c e n t r a t i o n was below 149 mM,sucrose was used t o r e p l a c e Na . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measure manometrically as g i v e n i n the m a t e r i a l s and methods . ( • ) c o n t r o l ; ( n ) 2 uM TTX and ( O ) 1 0 pM ouabain.  -  6.5  EFFECTS  OF  OUABAIN  C I T R A T E , AMP CEREBRAL  We the  rate  have  the  ouabain  the  inhibition  glutamate  may  or  inhibitory TTX, which  NH  K  +  6.6  4  +  efflux  +  CORTEX The  whether  i n the were,  i n the presence  i n increasing  and t h i s  of  the  experiments  (Table  effect  on  i s e x p l a i n e d by  The i n h i b i t o r y  conditions.  while  effect  has very  little  AMP  of  thus  THE  under  have  action  these  obtained  i n the  t h e Na  (See C h a p t e r  AEROBIC  little  to that  o f ouabain  on t h e g l y c o l y s i s  ON  +  stimulating  In the presence  OUABAIN  and NH ^  i s i n contrast  i s already high,and  OF  that  Experiments  strong inhibitory  glycolysis  This  h a s no e f f e c t  EFFECTS  has a  4 a n d 5,  i s suppressed  i s effective  effect.  ^.  THE  t o t h e l o w e r i n g o f ATP c o n c e n t r a t i o n  our experimental  and  i n Chapters  glycolysis  inhibitory.  be due  o f NH  G L Y C O L Y S I S OF  or of glutamate.  citrate  under  presence  L-GLUTAMATE,  o f p h o s p h o f r u c t o k i n a s e by c i t r a t e  i s less  with  OF  ANAEROBIC  Results of these  stimulated  glutamate  no  THE  earlier,  ouabain  that  PRESENCE  out t o observe  glycolysis.  indicate  -  SLICES  citrate  carried  2 8)  ON  stimulated  substances  anaerobic  IN THE  NH*  noted  o f NH*,  therefore, these  CORTEX  o f TTX  presence  AND  185  +  influx  conditions 4.9).  G L Y C O L Y S I S OF  CEREBRAL  SLICES  effects  o f 10  yM  ouabain  on  the aerobic glycolysis  ++ rat as  cerebral  cortex slices  i n a Krebs-Ringer  (Figure  19).  i n c u b a t e d i n a Ca  b i c a r b o n a t e medium  I t i s evident that  a t 10  was  -free  as  well  investigated  yM c o n c e n t r a t i o n ,  of  186 TABLE  E F F E C T S OF O U A B A I N  28  I N THE  G L U T A M A T E , C I T R A T E , NHi+ ANAEROBIC  +  PRESENCE  AND  G L Y C O L Y S I S OF RAT CORTEX  AMP  ON  OF THE  CEREBRAL  SLICES  L a c t a t e produced ymoles per g i n i t i a l wet wt (20-80 Additions  Ca  + +  f r e e medium  Krebs-Ringer b i c a r b o n a t e medium  31.7  ±  4.3  38.0  ±  4.4  ouabain  95.9  +  3.6  58.5  ±  6.3  lOuM ouabain  99.1  ±  3.1  20.9  ± 2.3  28.6  ±  3.7  63.8  ±  41.7  ±  4.5  34.4  ±  3.8  56.7  ±  2.7  None 10yM +  5mM  5mM  NHi+  +  L-Glutamate  lOuM ouabain + 5mM 5mM 10yM  3.0  L-Glutamate  D-Glutamate ouabain  + 5mM  D-Glutamate 102.6  ±  3.0 1.8  lOOyM ouabain  82.6  ±  lOOyM ouabain + 5mM L-Glutamate 5mM c i t r a t e 15mM c i t r a t e  13.8  ± 2.3  12.5  ± 0.9  lOyM ouabain + 5mM c i t r a t e  52.2  ±  3.1  lOyM ouabain +15mM c i t r a t e  22.3  ±  4.0  15.5  ±  5.0  83.9  ±16.5  AMP  2mM  lOyM ouabain + AMP 2mM  min)  I n c u b a t i o n c o n d i t i o n s were same as i n T a b l e 27.  -  ouabain  has l i t t l e  glycolysis. lysis the  + +  -free  on t h e r a t e  of  medium, however,  suppressed  e f f e c t s of ouabain  6.7  -  o r no e f f e c t  In a Ca  i s slightly  187  b y TTX w h i c h  aerobic  aerobic  glyco-  i s i n contrast to  (See C h a p t e r 8 ) .  E F F E C T S OF O U A B A I N  ON  THE A T P C O N T E N T S OF C E R E B R A L  CORTEX  SLICES M e a s u r e m e n t o f ATP conditions (Table  rate 6-  t h e ATP  anoxia, limiting  phosphate.  the  i s i n fact  15) i n t h e p r e s e n c e  As under  there  contents  speed  content  show t h a t u n d e r  an i n c r e a s e of  of the t i s s u e i s very  f o r the phosphorylation thus  of anaerobic  exert  glycolysis.  i s consumed by Na  of  ouabain  on t h e a n a e r o b i c  of  the increased  utilization. out  that  As a  This  will  6.8  E F F E C T S OF A D D I T I O N OF O U A B A I N OF A N A E R O B I O S I S ON CORTEX  has been  and  fructose  effect  on  considerable  may  be t h e r e s u l t  o f ATP due t o i t s d e c r e a s e d  of anaerobic  be d i s c u s s e d  become  , K -ATPase, t h e e f f e c t  i n t h e ATP c o n t e n t glycolysis  reduced  +  rate  It  limiting  However, t h e p o s s i b l i l i t y  increase  CEREBRAL  of glucose  glycolysis  concentration  much  o f A T P may  a rate  + a m o u n t o f ATP  concentration  ouabain.  the tissue concentration  I t may  i n i t s  anaerobic  c a n n o t be  may  be due t o a  i n the presence  further i n Chapter  greater  of ouabain.  8.  AFTER VARIOUS  THE A N A E R O B I C  ruled  PERIODS  G L Y C O L Y S I S OF  RAT  SLICES  shown e a r l i e r  that  a d d i t i o n o f TTX,  10 m i n o r  -  more on  after  the onset  the rate  attributed during  therefore at  of anoxia,  of anaerobic t o the loss  the f i r s t  of K  intervals  its  action  with  TTX.  33)  has p r o g r e s s i v e l y l e s s  in  anoxia on  of anoxia.  are important  the anaerobic  6.9  EFFECTS TOGETHER  results  slices  I n v e s t i g a t i o n s were of  i n order  ouabain,  t o compare  show t h a t  ouabain  on t h e r a t e  of  shows t h a t during  changes  after  occurring  the early  f o r the enhancing  (Figure  anaerobic  t o t h e i n c u b a t i o n medium  slices  action  was  cortex  of the addition  effect  This  cortex  effect  the brain  of anoxia,  These  i f i t i s added  the cerebral  This  few min o f a n o x i a .  time  onset  h a s no s t i m u l a t i n g  from  +  made o n t h e e f f e c t s  glycolysis  -  glycolysis.  various  the  188  effect  period of of  ouabain  glycolysis.  OF A D D I T I O N O F O U A B A I N ON  THE ANAEROBIC  AND  TETRODOTOXIN  G L Y C O L Y S I S OF  CEREBRAL  CORTEX  SLICES Since rate  both  ouabain  of anaerobic  combined results increase  effects (Figure  obtained  6.10  EFFECTS CEREBRAL  It  glycolysis of these  34)  the rate  that  i n a Ca  drugs  show t h a t ,  of glycolysis  when  either  OF OUABAIN CORTEX  has been  and TTX were  drug  ON  SLICES  shown t h a t  found + +  were  -free  these  to a value i s present  +  UNDER  medium, t h e  studied.  together,  THE N a a n d  to increase the  K  +  These drugs  higher  than  alone.  CONTENTS  OF  RAT  ANOXIA  i n the presence  o f TTX, t h e r e i s  -  FIGURE  18 9  -  33  EFFECT OF ADDITION OF OUABAIN AFTER VARYING TIME PERIODS OF ANOXIA ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES  125  20  30  40 Time,in  50 60 minutes  70  80  I n c u b a t i o n s were c a r r i e d out i n a Ca - f r e e medium c o n t a i n i n g 20 mM glucose.Ouabain was added a f t e r v a r y i n g p e r i o d s o f a n a e r o b i o s i s so as t o g i v e a f i n a l c o n c e n t r a t i o n of 10 uM.Lactate p r o d u c t i o n was measured manometrically as g i v e n i n the m a t e r i a l s and methods.Ouabain added a t ( • ) 0 t i m e , ( _ ) 5 min o r ( 9 ) 10 min a f t e r onset o f anoxia. (O)control.  - 190 EFFECTS  FIGURE 34 OF O U A B A I N AND T E T R O D O T O X I N  ANAEROBIC  GLYCOLYSIS  OF  T O G E T H E R ON  RAT' C E R E B R A L C O R T E X  THE  SLICES  150  •P  125 -  St  •P rH t0 •H -P 100 •H C •H tn QJ  Cu CO QJ  -a o 3 o M  Ou 0)  •p fO •p o (0  30  40  50 Time,  60  70  80  i n minutes  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e 22.(•)control; ( O ) 2 uM T T X * A ) 10 uM o u a b a i n ; ( B ) 2 pM T T X a n d 10 uM o u a b a i n .  -  an  increase i n K  cerebral  cortex  but a decrease slices  to  diminish either  it  has i n f a c t ,  under  by ouabain.  i n the presence  presence  slight when  of K  of  ouabain  increase anoxia,  i n K  of Na  +  since Na , +  K +  were, t h e r e f o r e ,  35 i n d i c a t e  under  +  there  t h a t , i nt h e  i s no i n c r e a s e d  i s either  no e f f e c t ,  content.  On t h e o t h e r  and a s l i g h t  the influx  + o f Na ;  or the influx  effects  anoxia,  a n d TTX a r e p r e s e n t +  i s n o t known  ouabain.  under  increase i n the Na  Ouabain  of the  of cerebral cortex slices  and t h e r e  +  +  content  Studies  shown i n F i g u r e  o f ouabain  retention  of K  the opposite  made o f t h e c a t i o n c o n t e n t s  The r e s u l t s  i n Na  anoxia.  the efflux  just  ATPase i s b l o c k e d  anoxia  -  191  drop  together, i n t h e Na  there +  or only  a  hand,i s an  content.  Under  i s very  large,  i n cerebral tissue  22  and  any e f f e c t It  of  on Na  i s evident  ouabain  -influx  from  on a n a e r o b i c  these  In  a c t i o n on t h e Na this  respect,its  n o t be s e e n  experiments  glycolysis  + its  could  (Table 18).  that the effect  i s not mediated  through  + and K  contents  of the brain  mode o f a c t i o n d i f f e r s  cells.  from t h a t o f  TTX. 6 . 1 1 E F F E C T S OF O U A B A I N , Na ,  Ga  + +  AND N A D  +  ON THE  MICROSOMAL  K -ATPase  +  +  Results  reported  i n S e c t i o n 6.1 s h o w t h a t , i n t h e  ++ presence  o f Ca  increasing cortex  , ouabain  i s not very  the rate of anaerobic  slices.  Furthermore,  effective i n  glycolysis  ouabain  and C a  of cerebral + +  seem t o  -  192  -  I  EFFECTS THE  OF  SODIUM  OUABAIN AND  FIGURE 35 AND T E T R O D O T O X I N  POTASSIUM CONCENTRATIONS CORTEX  0  AND  5  10  ( AEROBIC  )(  5  OUABAIN OF  TOGETHER  RAT  CEREBRAL  I  C  ON  SLICES  10  A N A E R O B Time , i n minutes  30 )  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e 26. A d d i t i o n s we^e made a t z e r o t i m e . ( B ) N a w i t h 10 pM o u a b a i n ; ( • ) N a , w i t h 10 pip o u a b a i n a n d 2 uM T T X ; ( C ) K , w i t h 10 uM ouabain;(O)K , w i t h 10 uM o u a b a i n a n d 2 pM T T X . C o n t r o l s a r e same a s i n F i g u r e 2 6 . f  -  antagonize major  each  effect  ATPase  pointed  a n d i t may  lysis.  I t was  decreasing  Under  (Section  the rate  suggested  that  of utilization  K -  +  anaerobic  limiting  that the  of Na ,  +  conditions,  6 . 7 ) , t h e ATP  become r a t e  further  established  i s the i n h i b i t i o n  1.5).  out  -  I t i s well  of ouabain  (see Chapter  already falls  other.  193  as  concentration  f o r anaerobic  glyco-  ouabain  a c t by  might  o f ATP b y N a ,  K -  +  +  ATPase. Our Ca  ++  the  results  would  be e x p l a i n e d  inhibitory  K -ATPase +  induced  on t h e a n t a g o n i s m  effect  ouabain  i f , i n the presence  o f ouabain  i s diminished.  changes  between  While  i n the Na  +  and K  o f Ca  o n t h e membrane studying  ,  bound  Nat  the ouabain  content,  +  ++  and  as w e l l  as on 131  the  respiration  of the cerebral  cortex  slices,  Swanson  '  173 came presence not  to a similar o f Ca  accessible In  study  view  ++  conclusion  , a l l t h e Na  o f these  the effects  activity  that,  under  o f Na  onistic  (Table  +  and stated -ATPase  considerations,  of Ca  +  +  and ouabain  that  i n the  o f t h e membrane i s  i t was when  decided  present  to  together  on  + , K  -ATPase.  our experimental  preparations,  , K  to ouabain.  + the  +  the effects 29).  This  The r e s u l t s  conditions  of ouabain was  true  i n brain  and C a  even  obtained  +  when  +  indicate  microsomal  are not antagmicrosomal  prep-  19 4 TABLE 29 EFFECTS OF OUABAIN, C a  + +  AND NAD ON +  THE MICROSOMAL N a , K -ATPase +  +  A c t i v i t y o f N a , K -ATPase . . , ymoles P i p e r mg p r o t e i n p e r hour +  Additions Experiment 1 None  76.8  ImM C a  + +  31.9  2mM C a  + +  30.8  4mM C a  + +  27.5  lOyM ouabain lOyM ouabain + ImM C a + +  lOyM ouabain + 2mM C a + +  lOyM ouabain + 4mT4 C a  49.6 O 2  D  '  8  2  6  ocT *  ot  6  -  7  q  + +  Experiment 2 None  57.6  lOOyM ouabain  30.1  0.2mM NAD  +  60.4  0.4mM NAD  +  72.5  0.6mM NAD  +  72.5  0.8mM NAD  +  63.2  Microsomal p r e p a r a t i o n s were prepared and ATPase a c t i v i t y assayed as g i v e n i n the m a t e r i a l s and methods. A d d i t i o n s were made b e f o r e t h e a d d i t i o n of the enzyme t o the i n c u b a t i o n m i x t u r e . Each v a l u e r e p r e s e n t averages o f d u p l i c a t e d e t e r m i n a t i o n ,  -  arations Ca  + +  are  preincubated  in  the  presence  of  either  ouabain  or  . Other  inhibit  6.12  r e s u l t s given  the  discussed  Na ,  TTX cortex  OF  blocks  therefore, efflux these  OUABAIN  of  the  under  amino  the  acids  presence are  released  incubation presence  are  of  medium.  of  AMINO  acids  also  will  from  incubated  not  be  THE  cerebral  brain  the  Results  i s evident of  tissue amino  reduced  were,  affects  amounts  e f f l u x of  considerably  the  slices. It  considerable  the  from  30.  ouabain,  is  does  +  Experiments  incubated  i n Table  the  aspect  4.4).  i f ouabain  the  shown  NAD  ANOXIA  amino  see  However,  ouabain  This  (Chapter  from  that  A C I D E F F L U X E S KRQM  UNDER  anoxia to  show  8.  e f f l u x of  acids  experiments  ON  SLICES  c a r r i e d out  29  activity.  i n Chapter  CORTEX  slices  Table  +  further  EFFECTS  in  K -ATPase  +  CEREBRAL  in  -  195  by  that  amino into  acids  the  of  the in  the  presence  of  TTX. The acid  above  findings  e f f l u x by  anaerobic brain  TTX  cannot  glycolysis.  cell  membrane  potentials  which  indicate  by  be  TTX  that  due  to  the  an  presumably  abolishing  results  in  PROCAINE  AND  increase acts  the  increased  inhibition in  of  the  directly  rate  on  of  the  generation  of  release  tissue  of  amino  the  action amino  acids. 6.13  EFFECTS LYSIS It  OF  has  respects,  OF  RAT  been  the  CEREBRAL pointed  mode  of  LIDOCAINE CORTEX  out  action  THE  ANAEROBIC  GLYCO-  1.3)  in  SLICES  earlier of  ON  local  (Chapter  anesthetics  that are  many  similar  196 TABLE  EFFECTS  OF O U A B A I N  TOGETHER RAT  CEREBRAL  Additions  Amino Acids  ON  AND  30  OUABAIN  T H E AMINO CORTEX  ACID  SLICES  +  TETRODOTOXIN  EFFLUX UNDER  FROM ANOXIA  -Glucose +ouabain  +Glucose +ouabain  -Glucose +ouabain +TTX  +Glucose +ouabain +TTX  TAURINE  Tissue Medium  2. 0 0 ± 0 • 0 1 3.55+0.11  2.47±0.25 3.30+0.08  1 . 5 8 ± 0 .36 3.6810.34  2, 1 1 1 0 . 1 1 3, 0 0 ± 0 . 0 3  ASPARTIC ACID  Tissue Medium  0.94+0.08 1.88±0.36  0.98±0.19 1.6510.07  0.8610.22 2.18±0.27  1.5310.13 0.97±0.15  GLUTAMINE +• S E R I N E  Tissue Medium  0.7010.06 3.3710.13  1.0310.11 2.8010.08  0.42±0.07 3.54±0.37  1.4910.28 2.7410.18  GLUTAMIC ACID  Tissue Medium  3.8210.40 8.2 ± 0 . 4 0  5.4111.05 6.9510.10  3.1010.46 8.0510.60  8.2410.61 3.5910.15  GLYCINE  Tissue Medium  0.2310.04 0.9310.21  0.3710.05 0.8510.05  1.0810-08  0.4510.03 0.4710.04  ALANINE  Tissue Medium  0. 1 7 1 0 1, 2 8 1 0  0.2510.05 0 . 8810.03  1.0510-16  0.4810.04 0 .5610.02  02 21  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n T a b l e 4. For controls without a n y d r u g , s e e T a b l e 4. Conc e n t r a t i o n o f o u a b a i n w a s l O y M a n d o f T T X w a s 2yM.  -  to  that  of  potential and  K .  cortex  The  obic  glycolysis a  required  to  TTX. of  slices  The local  of  higher  may  be  after have  no  of  effect  OF  RAT  GUINEA  local  ure  36).  markedly born found  of  brain  increases  with  pig TTX,  case  between are  Under  PIG  of  ON  added these  in  slices  Na  see  is  of  the  i f  that  of  anaer-  increased. is  obtained  stimulatory  with  effects  TTX.  TTX  and  local  the  incubation  conditions,  of  TTX.  of  to  +  presence  rate  anesthetic  the  anaerobic  be  anesthetics  these  glycolysis  ANAEROBIC  were  CORTEX  carried  lidocaine will  seen of  the  obtained that  2-day rate  cerebral this  THE  CEREBRAL  slices  glycolysis  guinea  the  latter  rate  that,  local  of  to  that  l i d o c a i n e , the  of  action  glycolysis  to  s t i m u l a t i o n as  in  experiments  It will  anaerobic  as  out  anaerobic  diminishes  +  LIDOCAINE  anesthetic  glycolysis  +  movements  carried  show  of  medium drugs  of  the  slices.  EFFECTS AND  same Ca  the  the  similar  31  or  generation  cerebral cortex  anoxia. on  cortex  Further the  the  of  manner  similarities  when  min  cerebral 6.14  of  anesthetics,  15  rate  procaine  the  presence  seen  the  concentration  obtain  Additional  affect  i n Table  rat  the  therefore,  in a  given  -  block  they  were,  anesthetics  However,  so  affect  results  local  drugs  i n doing  anesthetics  cerebral  of  and  These  Experiments  +  local  TTX.  197  must  out  to  increase from  old  of  rat  slices.  related  to  INFANT  observe the  whether  anaerobic  i n f a n t animals  brain  anaerobic  OF  SLICES  l i d o c a i n e has  cortex be  GLYCOLYSIS  no  effect  slices  but  glycolysis As the  with  the  maturity  of  (Figon  the  i t newly  results of  the  198 TABLE 31 EFFECTS OF PROCAINE AND LIDOCAINE ON THE ANAEROBIC GLYCOLYSIS OF RAT CEREBRAL CORTEX SLICES L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 min) Additions  Ca  + +  f r e e medium  Krebs-Ringer b i c a r b o n a t e medium 42.4 ± 6.7  24.6  ± 1.4  O.OlmM P r o c a i n e  40.6  ± 6.8  O.OlmM L i d o c a i n e  45.6  ± 2.0  0.1 mM P r o c a i n e  66.0  ± 1.0  58.0  ± 8.9  0.1 mM L i d o c a i n e  93.7 ±17.0  70.5  ± 3.1  None  A l l v e s s e l s c o n t a i n e d 20mM g l u c o s e . Additions were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y , as g i v e n i n the m a t e r i a l s and methods.  x  - 199 FIGURE  36  EFFECT OF LIDOCAINE ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES  150 Newly born guinea p i g  0.25 0.5 0.75 L i d o c a i n e c o n c e n t r a t i o n , i n mM  1.0  ++ C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a Ca - f r e e medium c o n t a i n i n g 20 mil glucose . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured manometrically as g i v e n i n the m a t e r i a l s and methods.  -  brain 6.15  200  -  tissue. EFFECTS  OF  CEREBRAL  LIDOCAINE  CORTEX  ON  THE  Na  +  AND  K  CONTENTS  +  SLICES  Since  the a c t i o n  of local  glycolysis  i s similar  to that  anesthetics o f TTX  and  on  anaerobic  since  i n the  + of  the  latter  thought  there  desirable +  on  i s an  increase  to observe  i n the K  the e f f e c t s  and  K  contents  slices.  A  was  i n these  relatively  of  high  incubated  slices rat  that  concentration  experiments  cerebral  anesthetics on  glycolysis in  there  cortex block  ratio.  of  as  SUMMARY  the  of  This OF  i s s t i l l  cerebral  local  t o be will  CHAPTER  Ouabain, rate  i t was  anesthetics  at  The  of  results  expected  as  brain  with the  adult local  potentials the  increase  that  anaerobic l i d o c a i n e but  i n tissue of the  K /Na +  +  high  in infant r a t brain  in glycolysis  i s not  i n the brains  further discussed  of  ^-depend-  mature  i n Chapter  8.  6  low  concentrations  of anaerobic cortex  step  It i s  ratio  unspecific effect  the case  be  /Na  a f f e c t e d by  anesthetic,or  of lidocaine  +  However,  an  cortex  32).  of action  anoxia.  limiting  appears  a r e t o be  o l d r a t i s not  there  of  i n K  lidocaine.  the generation  2-day  the rate  animals.  1.  of  P o s s i b l y , t h i s i s an  concentration  ent,  local  ( 0 . 5 mM)  (See T a b l e  increase  slices  the onset  i t s presence  slices  i s an  i n the presence  develop  ratio,  cerebral  + evident  + /Na  of  presence  +  t h e Na  used  OF  slices  glycolysis in a  Ca  + +  (1-100  of both  -free  yM)  greatly  r a t and  medium.  increases  guinea p i g  201 TABLE  EFFECTS AND  OF  POTASSIUM  32  L I D O C A I N E ON CONTENTS  OF  THE  SODIUM  CEREBRAL  CORTEX  SLICES  Age of Rats  2-day o  l  d  Aerobic  Anaerobic  Cation  10 m i n Na K  +  Na Adult K  +  +  +  5 min  10 m i n  30 m i n  100  111  120  142  56  57  49  43  127  130  132  155  54  48  40  35  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e 26. E a c h v a l u e r e p r e s e n t a v e r a g e s o f two e x p e r i m e n t s w i t h i n ± 7%. F o r c o n t r o l s s e e F i g u r e 26 a n d 28. R e s u l t s a r e e x p r e s s e d as y e q u i v a l e n t s p e r g i n i t i a l wet wt. 0.5mM l i d o c a i n e w a s p r e s e n t f r o m t h e s t a r t of the experiment.  -  2.  Ouabain  has very  obic  glycolysis  Krebs-Ringer added on  i n their  3.  medium.  -free  effects  about 4.  increasing  Thus,  Ca  Na  +  Citrate  cortex  glycolysis  +  i s  +  of  ouabain  slices i s are antagonist-  anaerobic  glycolysis.  on t h e a n a e r o b i c  slices  i n a the  increases  Ca  + +  -free  anaerobic  considerably at  and TTX s i g n i f i c a n t l y even  when  most  increase  of the Na  +  of s t i m u l a t i o n decreases  the rate of i s replaced with  on t h e a n a e r o b i c  glycolysis  of  extracts.  ( 1 5 mM)  o f ATP.  inhibits  h a s some AMP  ( 1 0 yM)  the ouabain  inhibitory  a n d NH*  of anaerobic  Ouabain  effect  i n a  concentration.  glutamate  stimulation  Ca  anaer-  week.  has no e f f e c t  powder  when  and ouabain  o r no e f f e c t  r a t brain  The degree  Ouabain  lowering  the enhancing of cerebral  on t h e  incubated  Moreover,  o l d r a t brain  glycolysis,  sucrose.  while  slices  on t h e c e r e b r a l  of infant  ouabain  anaerobic  6.  medium.  medium,  has l i t t l e  the second  acetone  stimulating effect  I t s e f f e c t i v e n e s s i n enhancing  Both  5.  -  cortex  glycolysis  o f 2-day  glycolysis  7.  + +  reduced.  Ouabain  glycolysis  by  of cerebral  the anaerobic  considerably ic  little  bicarbonate  to a Ca  202  have  glycolysis  has l i t t l e  of incubated  brain  stimulated  effect, little by  possibly  glycolys due t o  o r no e f f e c t  on t h e  ouabain.  o r no e f f e c t  on t h e a e r o b i c  slices.  ++ 8.  In the presence  anoxia,  the c e l l  o f ouabari  content  o f ATP  i n a Ca  -free  i s increased.  medium,  under  -  9.  I f ouabain  increasing effect  i s added  periods  203  t o t h e i n c u b a t i o n medium  of anoxia,  on t h e r a t e  -  after  i t has p r o g r e s s i v e l y  of anaerobic  glycolysis  i n a  Ca  lesser + +  -free  medium.  ++ 10.  When  medium,  ouabain  the rate  obtained  when  and TTX a r e b o t h of anaerobic  either  drug  present  glycolysis  i s present  Ouabain  cerebral TTX,  cortex  however,  tissue TTX  h a s no  slices  reduces  i n presence  acts  effect  the K  +  of  o r Na  Ouabain  and Ca  both  on t h e i n h i b i t i o n  13.  Ouabain  the  cerebral  blocks  i n a Ca  cerebral greater  that  cortex  -free  + +  action  increased  efflux  by t h e  medium.  Thus  +  t h e Na  , K  -ATPase i n  are present  of Na ,  +  +  , K  K -ATPase +  +  together, i s  the e f f l u x  o f amino  anoxia.  o f amino  acids  i n the presence amino  i n the presence  o f t h e Na -pump^as +  no  observed.  -ATPase.  under  acids  However,  acid o f TTX  a result  from  TTX  of  content  s t i l l  ouabain, of the  i s n o t due t o o f an  increased  glycolysis.  i s presumably  potentials  gain  +  slices  slices  of anaerobic  t h e Na  both  the the increased  operation  TTX  When  not affect  cortex  the e f f l u x  indicating  rate  not inhibit  does  anoxia.  and t h e N a  inhibit  antagonism  does  under  +  preparations.  NAD  of the  ouabain.  microsomal  +  content  medium  ++ 12.  that  +  -free  loss  o f ouabain  independently  + +  than  alone.  on t h e K  in a Ca  -free  i s higher  + 11.  i n a Ca  acting  induced o f amino  by b l o c k i n g t h e g e n e r a t i o n  by a n o x i a , acids.  that  result  i n an  of  14.  Procaine  rate  of  mature  anaerobic brain.  cellular  ratio  anesthetics brain  and  during  -  204  -  lidocaine  act  like  g l y c o l y s i s of  In  the  of  K /Na +  resemble anoxia.  presence +  TTX  TTX  cerebral of  0.5  i s increased. in  mM  in increasing  the  cortex  from  slices  lidocaine, Thus  their metabolic  the  the  local  effects  on  CHAPTER  EFFECTS  OF VARIOUS  NEUROTROPIC  GLYCOLYSIS  It of  was  bases,  rate  pointed  such  OF  DRUGS ON  CEREBRAL CORTEX  out i n Chapter  as p y r r o l e  of anaerobic  7  1 that  and p y r i d i n e ,  g l y c o l y s i s of guinea  THE  ANAEROBIC  SLICES  Ca  +  and a number  +  greatly  enhance t h e  p i g cerebral  cortex  71 slices  (Adams a n d Q u a s t e l  Quastel were  on t h e e f f e c t s  confirmed  tissues such  3).  as TTX, l o c a l 4-6).  and  anesthetics  these  their  +  bases  action  drugs  as  well  of  these  7.1  experiments  The  effects  glycolysis in  Figure  amines  were  AND  reported  studies  that  TTX  g l y c o l y s i s by  cells.  In view o f  the e f f e c t s  changes  of  various  i n the cation  In addition, reserpine  also  are described  CORTEX  effects of  and  studied.  amphetamine The  results  below.  P Y R I D I N E ON  THE ANAEROBIC G L Y C O L Y S I S  SLICES  o f some  of guinea 37.  cell.  drugs  g l y c o l y s i s , t o see whether  through  as b a r b i t u r a t e s ,  as b i o g e n i c  were  these  t o study  anaerobic  i n the brain  CEREBRAL  from  cerebral  of various  and ouabain  i n the brain  decided  on c e r e b r a l  such  effects  and  glycolysis,  to infant  a c t on a n a e r o b i c  ratio  +  A C T I O N OF P Y R R O L E OF  extended  too i s mediated  concentrations other  may  o f Adams  on a n a e r o b i c  +  I t became c l e a r  the K /Na  findings  In addition,  r e s u l t s , i t was  organic  +  The  anesthetics  (Chapters  affecting  of Ca  and f u r t h e r  (Chapter  local  ).  organic  bases  on t h e a n a e r o b i c  p i g and r a t c e r e b r a l  I t i s seen  that  the rate  slices  a r e shown  of anaerobic  glycolysis  FIGURE EFFECTS  OF  P Y R R O L E AND OF  206 -  37 PYRIDINE  CEREBRAL  CORTEX  ON  THE  ANAEROBIC  GLYCOLYSIS  SLICES  125 Pyridine,Guinea p i g  Pyrrole,Guinea p i g  Pyridine,Rat  JL  0  10 Concentration  20 of pyrrole  and p y r i d i n e , i n  30 mM  I n c u b a t i o n s were c a r r i e d o u t i n a C a - f r e e medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s w e r e made a t z e r o t i m e a n d l a c t a t e p r o d u c t i o n was m e a s u r e d m a n o m e t r i c a l l y as g i v e n i n t h e m a t e r i a l s and methods. + +  -  of  guinea  presence  pig cerebral of pyrrole  207  cortex  -  slices  and p y r i d i n e ,  i s enhanced  confirming  i n the  the  findings  71 of  Adams  has  and Q u a s t e l  pig. are  slices  less  a t equivalent  D-glutamate  I t i s evident  glycolysis response  effect  on  stimulation  i t has no  CEREBRAL In  several sis  OF  or L-aspartate, results  that  ON  slices  SLICES  to  i n Table of  of  o r no  stimulatory  D-glutamate  i s partial-  medium  i n  preventing  Moreover, i f after  15 m i n  anaero-  effect. +  UNDER  ANOXIA  AND  K  +  i t has been of pyrrole  o f TTX.  As  g l y c o l y s i s i s presumably +  mM  i n the presence  has no e f f e c t  section,  and K  of 5  stimulation  has l i t t l e  THE N a  the action  +  cortes  slices  o f TTX i n showing  Thus,  glycolysis.  stimulatory  to that  on t h e N a  guinea  cortex  i s shown  to that  acids.  pyrrole  PYRROLE  respects,  of  i n the presence  to the incubation  the previous  anaerobic effect  pyrrole,  L-aspartate  CORTEX  i s similar  p i g cerebral  o f g l y c o l y s i s by p y r r o l e .  biosis,  EFFECTS  r a t cerebral  i s similar  the anaerobic  i s added  PIG  guinea  these  30 mM  effective while  pyridine  glycolysis of r a t cerebral  that  t o d i f f e r e n t amino  pyrrole  7.2  from  by p y r r o l e  L-glutamate,  that  concentrations.  e f f e c t o f 30 mM  L-glutamate,  5 mM  than  evident  t o i t se f f e c t on t h a t  appears  responsive  The  33.  i n contrast  In g e n e r a l , i t  drugs,  the  I t i s also  no e f f e c t on t h e a n a e r o b i c  cortex  ly  .  contents  CONTENTS  OF  shown t h a t ,  on a n a e r o b i c  the action mediated  GUINEA  i n glycoly-  o f TTX on  through i t s  of cerebral  cortex  slices,  208 TABLE 33 EFFECT OF PYRROLE ON THE ANAEROBIC GLYCOLYSIS OF GUINEA PIG CEREBRAL CORTEX SLICES IN THE PRESENCE OF SOME AMINO ACIDS  . Additions  L a c t a t e produced ymoles p e r g i n i t i a l wet wt (20-80 mi  None  48.4 ± 12.5  30mM P y r r o l e 5mM L - G l u t a m a t e  94.3 ± 16.7 25.5 ±  4.0  47.4 ±  5.7  51.7 ±  5.0  5mM L - A s p a r t a t e +30mM P y r r o l e  pc q + "  o o  5mM D-Glutamate  31.5 ±  6.9  5mM D - G l u t a m a t e +30mM P y r r o l e  66.5 ±  1.5  43.3 ±  1.0  30mM P y r r o l e + 5mM L - G l u t a m a t e 5mM  L-Aspartate  30mM P y r r o l e , tipped i n after 15 min anoxia  C e r e b r a l c o r t e x s l i c e s were i n c u b a t e d i n a C a f r e e medium c o n t a i n i n g 20mM g l u c o s e . A d d i t i o n s were made a t zero time (except one case, as shown) and l a c t a t e p r o d u c t i o n was measured manometrically as g i v e n i n t h e m a t e r i a l s and methods.  - 209 -  i t was d e s i r a b l e t o study the e f f e c t s o f p y r r o l e on the c a t i o n i c concentrations. given  i n Figure  38.  30 mM p y r r o l e , there but  Results  o f these experiments a r e  I t can be seen t h a t , i n the presence o f i s an i n c r e a s e i n the r e t e n t i o n o f K  a decrease i n the uptake o f Na .  +  These r e s u l t s i n d i c a t e  t h a t the a c t i o n o f p y r r o l e on a n a e r o b i c g l y c o l y s i s may a l s o be mediated through changes i n the c a t i o n 7.3  concentration.  EFFECTS OF AMYTAL AND PENTOTHAL ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES Barbiturates  have long been shown t o d i m i n i s h  the brain  195 tissue respiration barbiturates  .  As noted, i n Chapter L5 both t h e  (amytal and p e n t o t h a l )  as w e l l as c e r t a i n hypnot-  ics,  suppress the o x i d a t i o n o f NADH, and hence the g e n e r a t i o n 281—283 o f ATP i n the c e l l . Using suspensions o f c e r e b r a l 199 t i s s u e , Webb and E l l i o t t rate of aerobic  showed t h a t , amytal enhances the  g l y c o l y s i s and suppresses the r e s p i r a t i o n .  T h i s i s i n agreement w i t h p r e s e n t amytal on o x i d a t i v e p r o c e s s e s . o n l y observe v e r y  little  g l y c o l y s i s of the c e l l The  However, these workers  suspensions.  mechanism o f b a r b i t u r a t e a c t i o n on t h e nerve c e l l i s They a r e known t o suppress e l e c t -  a c t i v i t y o f e x c i t e d nerve c e l l s but so f a r no s i g n i f i c a n t  e f f e c t o f the b a r b i t u r a t e s , a t a n e s t h e t i c Na  +  could  e f f e c t o f amytal on the a n a e r o b i c  s t i l l n o t f u l l y understood. rical  knowledge on the e f f e c t s o f  movement i n the c e r e b r a l c o r t e x  c o n c e n t r a t i o n , on  s l i c e s have been observed  198 As i t was found t h a t TTX, and l o c a l  anesthetics,  FIGURE  38  EFFECT OF PYRROLE ON THE SODIUM AND POTASSIUM CONCENTRATIONS OF GUINEA PIG CEREBRAL CORTEX SLICES UNDER ANOXIA  300 r  1  Time,in minutes I n c u b a t i o n c o n d i t i o n s were same as i n Ficjure 2 4 . ( » ) N a c o n t r o l ; (O)Na. ,with 30 mM p y r r o l e ; ( • )K ,with 30 mM pyrrole;( B ) K ,control.  -  greatly  increase  cerebral effects  cortex  the rate slices,  of barbiturate  of  cerebral  of  sodium  tissue.  amytal  as w e l l  Figure  39.  I t i s seen  cerebral but  anesthetics  a s on i n f a n t that  has  r a tb r a i n  greatly slices  from  have  mature  small  used  pigs  adult  r a t and  are given i n  the anaerobic  from  infant doses  c h a r a c t e r i s t i c s ) show v e r y  exceeding  glycolysis of  r a tand i n f a n t  effect but infant  presence  of traces  may b e e n o u g h  conditions  can increase  oxidation since  o f NADH.  preliminary  mM  azide,  effect  guinea p i g  r a tb r a i n .  In  ( 0 . 2 5 mM)  amytal  guinea  pigs  (that  considerable  sensitivity  slices.  Moreover,  anoxia,  rules  this  experiments blocks  have  Amytal rate  amytal  that  oxidation  o f NADH  experiments  these  i s unlikely the presence o f h a s no  glycolysis of cerebral i s added  15 m i n a f t e r  amytal  might  o r any u n i d e n t i f i e d showed  under  chain,  i t h a s no e f f e c t o n t h e a n a e r o b i c that  suppress  by b l o c k i n g t h e  possibility shown  mixture  and thus  the respiratory  of anaerobic when  formed  the glycolytic  out the p o s s i b i l i t y  Other  NADH  i n the ^rCC^  t h e ATP l e v e l .  However,  which  on t h e r a t e  o f oxygen  to oxidize  by i n c r e a s i n g  the  concentrations  a t concentrations  a t anesthetic  glycolysis  of  glycolysis  the drug. The  0.5  t o study the  glycolysis of adult guinea  enhances  slices,  a relatively  desirable  on t h e a n a e r o b i c  amytal,  i t h a s no e f f e c t on s l i c e s  adult  to  cortex  glycolysis of r a t  i t was t h o u g h t  on t h e a n a e r o b i c  rat,  anesthetic,  of anaerobic  The e f f e c t s o f d i f f e r e n t  infant  the  211 -  that  amytal  cortex  the onset  glycolysis. a c t by  This  blocking  metabolite. i s less  effective i n  -  FIGURE  212  -  39  EFFECT OF AMYTAL ON THE ANAEROBIC GLYCOLYSIS OF CEREBRAL CORTEX SLICES  175 Newly b o r n g u i n e a p i g  Two day o l d r a t  J.  JL  JL  0.25 0.5 0.75 A m y t a l c o n c e n t r a t i o n , i n mM I n c u b a t i o n c o n d i t i o n s were same as i n F i g u r e 37  1.0  - 213  a Krebs-Ringer  -  b i c a r b o n a t e medium when compared w i t h i t s  a c t i o n i n a C a - f r e e medium.  E f f e c t s of p e n t o t h a l on  a n a e r o b i c g l y c o l y s i s resembles  t h a t of amytal ( p r e l i m i n a r y  + +  experiments). From the above i t appears  t h a t the e f f e c t s of  on a n a e r o b i c g l y c o l y s i s might be mediated through in c a t i o n i c concentrations.  F u r t h e r experiments  out t o examine t h i s p o s s i b i l i t y .  The  results  showed t h a t a t a n e s t h e t i c doses, amytal no e f f e c t on the K  contents.  +  r e d u c t i o n i n the N a  +  amytal changes  were c a r r i e d  (Table  34)  (or p e n t o t h a l )  has  However, there i s some  c o n t e n t s of the s l i c e s under our  experimental c o n d i t i o n s . With i n f a n t guinea p i g s , a s l i g h t i n c r e a s e i n the contents i n the presence I t appears (0.25 mM)  o f amytal was  observed  amytal  e f f e c t on a n a e r o b i c g l y c o l y s i s of  r a t b r a i n - e s p e c i a l l y when compared w i t h the l a r g e e f f e c t s due  +  (Table 35).  that while anesthetic concentrations of have but l i t t l e  K  relatively  t o the l o c a l a n e s t h e t i c s o r TTX  - increasing  c o n c e n t r a t i o n s o f the b a r b i t u r a t e s do b r i n g about i o n i c changes t h a t r e s u l t i n i n c r e a s e d r a t e s o f a n a e r o b i c These d o u b t l e s s are r e l a t e d t o the m o d i f i c a t i o n by  glycolysis. barbitur-  ates but more work i s r e q u i r e d t o e s t a b l i s h the nature of these changes. 7.4  EFFECTS OF CHLORPROMAZINE ON  THE  ANAEROBIC GLYCOLYSIS OF  CEREBRAL CORTEX SLICES Chlorpromazine  i s known to suppress  the anaerobic g l y c o 217'  l y s i s i n the c e l l  f r e e e x t r a c t s from b r a i n  .  The e f f e c t s of  214 TABLE 34 EFFECTS OF SOME NEUROTROPIC DRUGS ON THE SODIUM AND POTASSIUM CONTENTS OF RAT CEREBRAL CORTEX SLICES  Aerobic Addition  Cation  0.25mM Amytal  O.lmM Pentothal  Na + K+ Na K  +  +  0.25mM Na Chlorpromazine K  1  +  i ^ 12uM  Reserpine  Na+ K  +  10 min  Anaerobic 5 min  10 min  30 min  125  140  145  175  44  31  26  20  120  140  147  170  42  30  24  17  132  125  145  175  45  31  25  16  115  140  135  147  46  40  32  34  I n c u b a t i o n c o n d i t i o n s a r e same as i n F i g u r e 26. Each v a l u e r e p r e s e n t averages o f two experiments w i t h i n ± 7%. F o r c o n t r o l s see F i g u r e 26. R e s u l t s are expressed as u e q u i v a l e n t s p e r g i n i t i a l wet wt.  -  different  concentrations of  glycolysis Figure any  215  of  40.  adult  chlorpromazine  rat cerebral  It i s evident  significant  -  that  stimulating  cortex  on  the  slices  chlorpromazine  effect  on  anaerobic  are  shown  does  the  rate  Na  and  not  of  in  have  anaerobic  glycolysis. Effects  of  adult  r a t and  given  i n Tables  chlorpromazine  infant 34  and  promazine,  at  Na  contents  and  +  7.5  K  +  EFFECTS  the  OF  system  their  by  storage  of  The  biogenic The amide was  on  AND  the  the  CORTEX  drugs.  has  no  of  the  the  sites.  As  i n Chapter  system  i s an  central  uptake  r e l e a s e s the  Nialamide  application  the  SLICES  and  of  on  ANAEROBIC  THE  granules  result  effect  ON  It inhibits  (MAO).  are  slices.  storage  oxidase  slices  of  cortex  potent  noted  contents  +  anesthetics, chlor-  cerebral  most  K  cortex  NIALAMIDE  CEREBRAL of  +  of  1.5,  i s an  these  i n c r e a s e i n the  of  nernor-  amines i t also  inhibitor  drugs  amount  to  of  the  free  amines. effects the  relatively of  net  nervous  studied  lysis  RAT  the  monoamine  central  Unlike  incubated  i s one  inhibits MAO.  of  stimulating  epinephrine from  35.  concentration tested,  OF  Amphetamine  the  pig cerebral  AMPHETAMINE  GLYCOLYSIS  vous  guinea  on  of  both  anaerobic  cerebral  or  no  and  1-amphetamine  glycolysis  ( F i g u r e 41) . small  d-  As  cerebral  i s evident^ these  effects  cortex  of  on  slices.  the  rate  of  as  well  cortex drugs  as  nial-  slices have  anaerobic  glyco-  216 TABLE 35  E F F E C T S OF SOME NEUROTROPIC DRUGS ON THE  SODIUM AND POTASSIUM CONTENTS OF  NEWLY BORN GUINEA PIG CEREBRAL CORTEX SLICES  Aerobic Addition  10 m i n  0.25mM Amytal  Anaerobic  Cation  Na  K  0.25mM Na Chlorpromazme  i  5 min  10 m i n  30 m i n  125  140  145  175  47  42  38  36  127  145  167  175  44  33  29  21  I n c u b a t i o n c o n d i t i o n s a r e same a s i n F i g u r e 27. E a c h v a l u e r e p r e s e n t a v e r a g e s o f two e x p e r i m e n t s w i t h i n ± 7%. F o r c o n t r o l s , s e e F i g u r e 27. R e s u l t s a r e e x p r e s s e d as y e q u i v a l e n t s p e r g i n i t i a l w e t wt.  - 217 -  FIGURE EFFECT  OF OF  CHLORPROMAZINE RAT  40 ON  THE ANAEROBIC G L Y C O L Y S I S  CEREBRAL CORTEX  SLICES  100  0  0.25  0.5  Chlorpromazine Incubation  conditions  were  concentration  same  as i n F i g u r e  0.75 f  i n mM 37.  1.0  -  FIGURE EFFECTS  OF A M P H E T A M I N E GLYCOLYSIS  OF  AND RAT  218  -  41 NIALAMIDE CEREBRAL  ON  THE  ANAEROBIC  CORTEX  SLICES  I n c u b a t i o n c o n d i t i o n s w e r e same a s i n F i g u r e amine;(A)d-amphetamine;(O)nialamide.  37.(9)1-Amphet-  -  7.6  EFFECT  OF  CEREBRAL  CORTEX  Reserpine sites  so  that  monoamine  may  +  fluxes The  lysis  anaerobic  Since  Na  anoxia that in was  in  the  a  and  of  degradation i t has  treatment  of  storage  amines  reserpine  by  no  with  from  reser-  cerebral  a f f e c t s Na  and  +  membrane.  on  (Figure  the  rate  4 2).  rat  of  anaerobic  I t was  markedly  drugs  cationic  K  were the  found  enhance  and  the  infant  glyco-  that  rate  guinea  a f f e c t anaerobic  fluxes,  the  +  reserpine, anaerobic action of  of  the  low  of  pig  cere-  glycolysis  e f f e c t s of  cerebral  These  presence  uM  +  ratio  of of  possible by  out  contents  investigated.  K /Na  anaerobic  release  their  reserpine  by on  +  indirectly  the  cell  adult  to  However,  whether  reserpine  number  the  thought  carried  OF  slices.  + the  GLYCOLYSIS  from  susceptible  reserpine  g l y c o l y s i s of  cortex  modifying  of  amines  depletion  brain  investigated  concentrations  bral  the  of  Prolonged  cause  e f f e c t s of  ANAEROBIC  1.5).  MAO.  i s uncertain  across  were  more  (Chapter  e f f e c t on  It  THE  release  are  therefore  tissue. K  they  ON  SLICES  causes  oxidase  inhibitory pine  RESERPINE  -  219  as  amine to  the  that  the  glycolysis of  The  have  It  rat may  no  exert  36).  therefore  therefore, the  unlike the  mediated  concluded  It  effect  on  experiments be  increase  tissue.  were,  that  under  showed  an  this  e f f e c t s on  These to  is  amines  indicate  large  34)  cerebral  biogenic  is unlikely is  there  Investigations  results  (Table  reserpine  amines.  reserpine  slices  (Table  reserpine,  e f f e c t s of  amines  results  incubated  release.  glycolysis. these  12  cortex  that  rate show  of that  through  the  reserpine  -  220  FIGURE EFFECTS  OF  RESERPINE  ON  CORTEX  THE  -  42 ANAEROBIC  SLICES  GLYCOLYSIS  OF  CEREBRAL  221 TABLE 36 EFFECTS OF SOME AMINES ON THE GLYCOLYSIS OF RAT  ANAEROBIC  CEREBRAL CORTEX SLICES  L a c t a t e produced ymoles per g i n i t i a l wet wt None  25.6  0.0ImM E p i n e p h r i n e  ±  2.3  (20-80  21.0  0.1  mM  Epinephrine  29.0  +  2.4  1  mM  Tyramine  30.6  ±  1.0  0.1  mM  Norepinephrine  2 8.6  ±  4.5  1  mM  Norepinephrine  30.8  ±  5.0  1  mM  Histamine  22.8  ±  2.4  50  yM Paraxon  26.8  ±  1.3  50  yM Paraxon + ImM A c e t y l c h o l i n e  29.0  ±  3.6  Incubations were c a r r i e d out i n a Ca free medium c o n t a i n i n g 20 mM g l u c o s e . A d d i t i o n s were made a t zero time and l a c t a t e p r o d u c t i o n was measured m a n o m e t r i c a l l y as g i v e n i n the m a t e r i a l s and methods.  mm)  -  may  exert  effects  on  cation  well-known  effects  on  SUMMARY  CHAPTER  7  1.  OF  Observations  the  rate  slices 2.  in a  In  pig  of  the  -free  presence cortex  of  mM  4.  In  a  cerebral  by  Ca  rations, Infant  guinea  pyrrole,  lose  i t s  less that  K  pig  cerebral  cortex  confirmed. the  incubated  and  +  the  gain  action  through  guinea  less  of  Na  +  pyrrole  increase  in  the  on K / +  cell.  inhibited  -free  cortex of  are  medium  infant  by  resembles  5  has  mM  TTX  glutamate  cerebral slightly  slices  i t has  well no  stimulated but  of  TTX  cortex of on  or  the  as  not  at  by  on  of guinea  the  pig  anaerobic  anesthetic  local  slices  rate  infant  effect  Its effects,  those  effect  concent-  anesthetics.  were  found  to  be  very  amytal. the  under  K  +  content  anoxia,  of  the  while  the  incubated Na  +  content  decreased.  The  effect  of  cerebral  cortex  slices  respiratory  while  presence  cortex  increases  a d u l t r a t as  cerebral  no  amytal  rats.  than  pig the  of  slices  less  to  Amytal  rat  + +  guinea  sensitive 5.  being  glycolysis  glycolysis  a  mM  i s mediated  brain  on  aspartate.  anaerobic  6.  the  as  that pyrrole increases  been  Pyrrole stimulated glycolysis  glycolysis  is  30  of  have  I t i s concluded  Na  5  of  slices  glycolysis  3.  Quastel,  medium,  anoxia.  ratio  and  Ca  + +  well  storage.  glycolysis  anaerobic +  amine  Adams  -  t r a n s p o r t , as  anaerobic  cerebral  under  of  222  amytal  on  seems  inhibitor.  the to  anaerobic be  glycolysis  independent  of  of  i t s action  as  7.  Chlorpromazine  has  + glycolysis cortex 8. or  slices  Dno  or  and  Na  little  and  under  on  no  effect  on  the  anaerobic  + K  content  the  of  the  incubated  cerebral  anoxia.  L-amphetamine,  effect  or  as  anaerobic  well  as  nialamide,  glycolysis  of  has  little  cerebral  cortex  slices. 9.  Reserpine,  glycolysis the  low  cerebral  cortex  slices.  incubated  cerebral  cortex  slices,  is  increased  Epinephrine, have  glycolysis  cerebral  of  reserpine i s not  from  storage  under  sites.  little  or  cortex  mediated  The  K /Na  i n the  +  the +  anaerobic  ratio  presence  of  of 12  yM  anoxia.  norepinephrine,  acetylcholine  of  concentrations, increases  of  reserpine, 10.  at  no  histamine,tyramine  stimulatory action  slices. through  Presumably the  and  on the  release of  anaerobic action  amines  CHAPTER  8  DISCUSSION  8.1.  EFFECT  OF  Results  IONS  described  pounds,  e.g.  actions  on  the  as  as  of  well  CALCIUM  cations,  in  ON  CEREBRAL  Chapter  nucleotides  anaerobic acetone  3  show  etc.,  extracts  that  exert  g l y c o l y s i s of  powder  ANAEROBIC a  variety  rate  cerebral from  GLYCOLYSIS of  com  limiting  cortex  brain.  slices  Thus  the  ++ presence rate  of  of  Ca  an  anaerobic  the  incubation  g l y c o l y s i s of  medium  cerebral  greatly cortex  enhances  slices  the  (Fig-  ++ ures  1  and  slices  +  The  differs  extracts, Ca  2).  e f f e c t of  markedly  where  from  i t inhibits  on  that  on  the  guinea  pig  cerebral  cerebral  glycolysis ^. 6  slices  On  the  tumour  other  hand,  i n t a c t tumour c e l l s ^ ' anaerobic g l y c o l y s i s 6  +  cortex  cortex  Ehrlich ascites  has no e f f e c t on t h e g l y c o l y s i s o f . The e f f e c t o f C a on t h e r a t e o f  +  +  of  Ca  has  been  studied  in  7  some  71 detail  by  factory workers, a  Adams  however, to  dissociation  a  with  common It  Ca  site  they  phenomenon  found  that  a  + +  ,  and  number  gave  additive  +  of  no  also  at  of  correlated  I t was  +  could  this  constants.  not  offer  that  found  satis-  time.  organic  these  a  These  bases  act  effects with that  stimulatory  a  mixture  effect  in  their of  suggesting  action.  is possible,  effects  are  ity  brought  about  some  but  for  Ca  the  is  Quastel  explanation  s i m i l a r way  bases  &  as  pointed  associated  increased  by  the  with  of  by  Adams  changes  ions.  retention  out  In K  +  the by  &  Quastel,  i n membrane presence  the  of  incubated  that  permeabilCa  + +  ,  there  cerebral  cor  -  tex Ca  slices  +  (Figure  26)  and t h i s  has a s t i m u l a t o r y  +  cerebral A  cortex view  pattern  225 -  may  be one o f t h e r e a s o n s  e f f e c t on t h e a n a e r o b i c  why  glycolysis of  slices.  that  Ca  of the c e l l  +  acts  +  by becoming  membrane  i n such  part  a way  of the structural  that  active  trans-  71 port to  of glucose  be  true  energy  as t h e r e  assisted)  as  the rate  by  increasing Under  is  much  the  into  transport  of cerebral  anaerobic  a strong  therefore  a n d may  +  into  i s active  brain  from  t h e ATP  (i.e.  i n vitro,  of microsomal  5 mM  t o 50  content  and  cell  factor f o r  6-phosphate.  Na ,K -ATPase, +  mM.  of the  limiting  and f r u c t o s e  the anaerobic  i s unlikely  glycolysis i s not affected  become a r a t e  of glucose  stimulate  there  concentration  conditions,  inhibitor  that  of glucose  anaerobic  the glucose  diminished  i s facilitated  i s no e v i d e n c e  phosphorylation  is  the neuron  +  Ca  +  +  a n d i t may  g l y c o l y s i s by d e c r e a s i n g  the  +  l o s s o f A T P b y N a ,K - A T P a s e a c t i v i t y . However, i t i s u n c e r t a i n as t o w h e t h e r e x t e r n a l C a i n h i b i t s t h e enzyme i n b r a i n slices. +  It ation with  • i s now  medium  i i « w e l l known  results  concomitant  (Na ) +  retention tration, the  The (Table is  of K , +  +  +  from  influx  a n d may,  i s prevented  and p o s s i b l e  increase  along  i n the c e l l  f o r the increased  the  i n the nerve  In the presence  3  rate  cell  of C a with  ATP  incub-  +  +  this  the  concen-  of g l y c o l y s i s of  slices.  effects of C a  n o t known  of N a  _ _ ++ o f Ca  depolarization ^.  cortex  1) p r o v i d e  ,, , . . that omission  i n an i n f l u x  be r e s p o n s i b l e  cerebral  +  some  t o respond  +  +  on t h e g l y c o l y s i s o f d e v e l o p i n g  interesting features. t o the presence  Infant  of high  K  +  brain  r a t brain  or  electrical  -  stimulation aerobic to Ca  + +  235  .  Results  .  week),  The r e s p o n s i v e n e s s  Infant  sensitive  brain  herein  increases  and m y e l i n a t i o n  show t h a t  during  thean-  t o the presence  o f Ca  like  t o be  t h e 2nd extremely  i n the incubation  a s i s w e l l known t h a t  g u i n e a p i g i s more m a t u r e t h a n and behaves  the period of  (ie. during  g u i n e a p i g b r a i n was f o u n d  i s not unexpected,  infant  reported  g l y c o l y s i s o f 2-day o l d r a t b r a i n does n o t r e s p o n d  maximum b r a i n g r o w t h  this  226 -  an adult  brain  that  medium;  the brain of  of infantr a t  (Chapter 1 ) .  ++ Although glycolytic case with the  Ca  lysis.  the brain  + +  ,  would  inhibits  extracts, this  as i n t h a t  medium w i l l  and t h i s  t h a t Ca  free  slices  However, t h i s  extracts  8.2.  i s evidence  enzymes i n c e l l  incubation  lular  both  there  by  + +  Ca  from  + +  result i n decline  i n the intracel-  r e s u l t i n greater  rate  i s not true.  Ca  of the  i s n o tthe  event removing  of glyco-  The g l y c o l y s i s o f c e l l - f r e e  and synaptosomes behave s i m i l a r l y  are inhibited  some  i n t h e sense  that  .  E F F E C T S OF EXOGENOUS N U C L E O T I D E S  ON THE A N A E R O B I C  GLYCO-  LYSIS It cerebral  i s known t h a t  the rate  cortex  decreases  slices  out i n Chapter  of anaerobic  progressively with  has  been p o i n t e d  the  l o s s o f co-enzymes o r due t o a change  3, t h i s  position that  of the cerebral cortex slices. + t i s s u e NAD + NADH l e v e l d e c r e a s e s  view  of this  fact,  glycolysis of  experiments were  time.  As  d e c r e a s e may b e d u e t o i n t h e c a t i o n i c comMcllwain during  has found 15 anoxia . In  c a r r i e d o u t t o examine  -  whether  t h e NAD  becomes  rate  given  incubation of  concentration  limiting  i n Table  the cerebral no e f f e c t  the  site  gated  (Figures  NAD  NAD  t o the outer conditions, within  of  anaerobic  membrane,  sibly  with  glycolysis. NADase  o f NADase was u s e d  i n the intact  cell,  it  i s i n homogenates  be  due t o t h e l o c a l i z a t i o n  tures  such  aerobic  glycolysis  of  NAD  i n the presence  an  optimal  this  +  +  i n presence  i n spite  powder  binding  of  under t h e  i n the rate  of the fact cell  and no  experiments.  Pos-  as r a p i d l y as  extracts.  This  might  i n subcellular struc-  h a s no e f f e c t  on t h e r a t e o f  o f DNP.  lack  o f DNP may  of glycolysis  consider-  This  of  be due t o t h e f a c t  i s obtained  effect that  i n the presence of  substance. The  bral  rate  NAD  investi-  possibility  increase  these  o f NADase  reaching  i n the concentration  i s n o t degraded  +  or acetone  as lysosomes.  The  i n the brain  during  NAD  that  out because,  occurs  i s present  conditions,  has been  +  found  concomitant  This  tothe  glycolysis  before  due t o mere  i s ruled  +  t h e same  the cell.  i s an i n c r e a s e  the cell  considerable  inhibitor  o f NAD  a r e an a r t i f a c t  there  o f NAD  i ti s hydrolyzed  can penetrate  +  results  NADH  ATP, under  slices  Results  of anaerobic  3 a n d 4) a n d i t h a s b e e n  these  of  the rate  Transport  cortex  glycolysis.  the addition  slices.  of glycolysis.  that  that  increases  but possibly  exogenous  same  f o ranaerobic  cortex  able  +  of the cerebral  2 indicate that  medium  has  227 -  effect  cortex  o f NAD  slices  +  on t h e a n a e r o b i c  glycolysis  i s n o t due t o i n h i b i t i o n  of cere-  o f ATPase  because  -  NAD  has no e f f e c t  +  results  point  ditions  the c e l l  limiting In  is  on t h e a c t i v i t y  to the p o s s i b i l i t y NAD  on t h e r a t e  by ATP, w h i c h  natively, sence  8.3.  of Ca  ,  + +  2.5 mM  of anaerobic  ,  EFFECTS  slices  i s well  A T P may  OF  rate  known  incubation Addition  ON  dependence  tex  slices.  However, r e s u l t s ,  of  K  of  Na  +  into  increased ulatory  the brain  cell  action  medium  +  i s also  This  i n the incubation  med-  with  Ca  that  i n the  + +  .  Alterpre-  the t i s s u e unhydrolyzed  and  activity. GLYCOLYSIS  of cerebral  f o r the presence  135  When  +  cortex  of cations i n  t o a Ringer  glycolysis  and K  8, +  +  the Na  +  decreased  indicate  glycolysis  i s enhanced.  i n considerable  may  then  outweigh  when  influx  effect  concentration  a t the time  that  concentrations  and t h e i n h i b i t o r y of Na  cor-  i s increased  an i n c r e a s e d  result  med-  of cerebral  shown i n F i g u r e  that  medium  cell  of K  of anaerobic  concentration of K .  2).  i s reduced  +  the fact  i n the incubation  +  ation  of Na  the rate by  +  (Table  likely)  of anaerobic  the concentration  i s explained  h a s an i n h i b i t o r y  concentration  the rate  This  rate-  (Table 3 ) .  of a high  time,  be  con-  ATP  glycolysis  inhibits  t h e same  anaerobic  i n fact  THE ANAEROBIC  ium  when  +  (but less  of anaerobic  medium  These  may  t o combine  penetrate  CATIONS  shows s t r o n g  under  glycolysis  the phosphofructokinase  The  the  + +  enzyme.  glycolysis.  i t i s possible  of C a  inhibit  at  that  p o s s i b l y due t o c h e l a t i o n o f C a  ium  of this  concentration  +  f o r anaerobic  the presence  effect  228 -  K  of the  the  i n the +  stimincub-  i s increased,  -22q-  less  Na  enters  the  concentration  of  intracellular  Na  and  this  brain  K .  cell  There  +  compared  +  presumably  in  the  results with  results  in  little  the a  presence  Decreasing  the  extracellular  Na ,  increasing  the  K  without  +  in  greater  the  K /Na +  of  ratio of  of  ratio  +  anaerobic rate  rate  anaerobic  has  glycolysis  anaerobic  pyruvate  centration  results  It  59  Over in  .  of  a  Na  +  L-glutamate  causes  responsible  for  a  mM  increased  the  doubling  the  the  inhibits  slices.'  Na -influx +  action  It  on  the  this the  +  +  activity K  con-  +  pyruvate  i s now  and  K /Na  the  a c t i v a t i o n by  L-glutamate  of  of  The  in  in  +  Thus  rate  changes  inhibits  i t s inhibitory  the  high  increase  large  on  K  results  32).  of  two-fold  cortex  itself  slices.  range,  the  anaerobic  presence  through  0-50  that  cerebral  effect  i n the  in  concentration  (Figure  cerebral cortex  mediated  i s confirmed  glycolysis  controlling  of  kinase.  activity  glycolysis  high  cellular  of  concentration,  +  glycolysis  i s presumably  kinase  a  in  rate  glycolysis.  a  increase  increase  greater  of  K  +301  anaerobic  well  is  known  that  doubtless  anaerobic  glyco-  lysis . 8.4.  ANAEROBIC From  the  GLYCOLYSIS  rates  powder  extracts,  a  higher  much  the This the  rate  be  due  glycolytic  of  be  the  glycolysis  observed  glycolysis  amount to  A C E T O N E POWDER E X T R A C T S  anaerobic  i t can  corresponding may  of  OF  of  fact  intermediates  the that and  that  in  with  i s obtained brain  slices  i n the  the such  as  may  extracts,  (Figures  be  BRAIN  acetone  compared  cell-free  coenzymes  OF  10  to &  extracts readily  11).  -  230 ~  a v a i l a b l e t o the enzymes as compared t o the b r a i n where the s p e c i f i c mentalization  slices,  l o c a l i z a t i o n o f the enzymes and compart-  of the m e t a b o l i t e s must be p l a y i n g an import-  ant r o l e i n d e t e r m i n i n g t h e o v e r a l l r a t e o f g l y c o l y s i s . When t h e r a t e of g l y c o l y s i s i s h i g h , der  extracts, K  +  as i n the acetone pow-  does n o t have v e r y l a r g e e f f e c t s on the  rate of anaerobic g l y c o l y s i s . 8.5.  EFFECTS OF TETRODOTOXIN ON ANAEROBIC GLYCOLYSIS OF BRAIN TTX  cortex  e x e r t s marked e f f e c t s on t h e metabolism o f b r a i n  s l i c e s i n c u b a t e d a e r o b i c a l l y under a v a r i e t y o f con130  ditions.  I t was shown by Chan and Q u a s t e l  , and by M c l l -  129 warn  , i n d e p e n d e n t l y and a t about t h e same time, i n 1967,  t h a t t h e drug suppresses the i n c r e a s e d a p p l i c a t i o n of e l e c t r i c a l impulses. small concentrations, f l u x of Na  +  r e s p i r a t i o n induced by  Moreover, the drug a t  suppresses t h e e l e c t r i c a l l y  induced i n -  as shown d i r e c t l y by measurements of the i n f l u x  of l a b e l l e d N a  +  and i n d i r e c t l y by the s t i m u l a t o r y  the drug on t h e c e r e b r a l o x i d a t i o n i t e d by the i n f l u x o f N a  +  a c t i o n of  of a c e t a t e which i s i n h i b -  due t o e l e c t r i c a l  stimulation. 135  R e c e n t l y , i t has been shown by Okamoto and Q u a s t e l tetrodotoxin i n f l u x of Na bation  i n h i b i t s both the i n c r e a s e d +  that  water uptake and  t h a t occurs under a v a r i e t y of a e r o b i c  conditions,  incu-  f o r example i n the presence o f p r o t o v e r a -  t r i n e or of ouabain o r i n t h e absence o f g l u c o s e . cluded  t h a t under such c o n d i t i o n s  erated  i n the i n c u b a t e d b r a i n s l i c e s , or t h a t t h e r e  I t was con-  a c t i o n p o t e n t i a l s a r e geni s some  -  activation for  of a specific  t h e marked Results  effects shown  concentrations obic in  +  +  -  o f TTX m a r k e d l y  be r e g a r d e d  the cation  of  the  these  carrying  that  as i n a C a  system  the presence the rate cortex  + +  interest.  of  -free  resulting  anaerboth  medium.  The a n o x i c  there  o f low  slices,  to the generation  conditions  account  i n vitro.  cerebral  e f f e c t o f TTX on a n a e r o b i c  rat  and g u i n e a  the  values  TTX  both  pig cerebral  obtained  together  i n the guinea  14).  This  tatively  condition  of  i s  This  action  stimulation  i n depolarization  t o the animal  Thus,  of a mixture  t h e same  p i g the rate  i s possibly  glycolysis differs  slices.  i n the presence  or slightly  i s further  due t o t h e f a c t  different effects  according  cortex  are either  while  on c e r e b r a l  that  with r a t of C a  +  +  anaerobic  +  and  +  reduced  increased Ca  i n  (Figure  exerts  quali-  glycolysis  species.  Influx: Because  system  during  TTX  potentials  the onset influx  i s a specific  an a c t i o n  on a n a e r o b i c  action  the  as w e l l  to  cell.  The  at  show  system,  stimulates  as one l e a d i n g  as under  of  act  12  i s of considerable  potentials,  1  current  of tetrodotoxin  i n Figure  containing  phenomenon  Na" "  sodium  g l y c o l y s i s of the incubated  a Ca  can  23i -  i n h i b i t o r of the Na -carrying  potential,  +  i t was  g l y c o l y s i s by a b o l i s h i n g and hence  of anoxia.  of labelled Na  during  anoxia  that  the generation  the concomitant  However, l a r g e r +  thought  movement  effects  i t may of  of Na  o f TTX on  were n o t observed  +  -  (Table the to  18).  Thus,  percentage assign  by  Cl  Media  rates as  is  role  influx  i s not large  enough  i n enhancing  the pyruvate  kinase  +  concentration.  i n the absence  o f TTX  I t i s well  i s replaced influx  under  by  of Na  a variety  the  slices  brain  of Na .  medium  i s not greater  centration  of C l  from  results  responsible  A  possibilities mentioned  that  as  of these  medium  cortex  i n this  cortex  factors  of the rate  may  ,  there  cortex t o be  of the reduced  i n which  i n the c e l l  i n the rate slices  Na  normal to  +  are solely  i n the presence  a number  o f TTX.  As  other  i t has  of factors  of anaerobic  Changes  glyco-  out to test  o f TTX.  con-  conclude  of anaerobic  were c a r r i e d  f o r the rate  that  glycolysis of  It i s difficult  chapter,  slices.  i s found  well  of glycolysis i n a C l -free  i n a medium  changes  as  SO^  cerebral  i n spite  t o t h e mode o f a c t i o n  limiting  cerebral  TTX  increased  known  impermeable  of anaerobic  the rate  than  not r e s u l t i n  the incubated  the rate  of experiments  earlier  rate  into  +  f o r the increase  number  19).  the r e l a t i v e l y  i s present.  of the cerebral  does  (Table  Moreover,  +  SO^  i n a C l -free  influx  these  by  of conditions.  i n increasing  vation  studied,  glycolysis - either  effective  each  was  of anaerobic  slices  the  o f Na  of C l  reduced  become  t  Substitution  Cl  lysis  of Na  i t sdecreased  i n the presence  when  -  the influx  suppression  i t the major  activity -free  when  232  may  glycolysis of  i n the concentration  r e s u l t i n an i n h i b i t i o n  o f g l y c o l y s i s , and hence  been  these  or  of  acti-  were  investi-  - 23 3 -  gated  i n an e f f o r t t o throw more l i g h t on the mechanism o f  enhancement of a n a e r o b i c g l y c o l y s i s by the presence o f TTX. These experiments w i l l now be d i s c u s s e d . Role of N u c l e o t i d e s .  As has been mentioned, t h e r e i s a  marked d e c l i n e i n t h e c o n c e n t r a t i o n o f NAD c e r e b r a l c o r t e x s l i c e s under anoxia l i m i t i n g e f f e c t on t h e anaerobic f o r the d e c l i n e i n t h e NAD  +  + NADH of the  +  and i t may have a r a t e -  glycolysis.  The e x a c t  reason  + NADH l e v e l i s n o t known b u t  p o s s i b l y i t may be due t o a v a r i e t y o f causes such as ( i ) + breakdown of NAD under anoxia, t i s s u e during anaerobic The  rapid  + ( i i ) l o s s o f NAD from t h e  incubation.  second p o s s i b i l i t y has been r u l e d out by us as t h e r e  i s no evidence  i f t h e r e i s a leakage  the i n c u b a t i o n medium.  o f NAD  +  (or NADH) i n t o  I f i t does l e a k out i n t h e medium  under the g i v e n experimental  c o n d i t i o n s then i t i s degraded so  q u i c k l y t h a t i t can n o t be d e t e c t e d i n our experiments. Furthermore, t h e r e i s no i n c r e a s e i n t h e NAD  l e v e l of the  c e r e b r a l c o r t e x s l i c e s i n t h e presence o f TTX (Chapter 4.3). Hence, t h e p o s s i b i l i t y t h a t t h e e f f e c t of TTX on anaerobic g l y c o l y s i s i s due t o an i n c r e a s e i n the c e l l NAD t o reduced  +  l e v e l due  e f f l u x i s c o n s i d e r e d most u n l i k e l y .  As e a r l y as 1928 i t was demonstrated t h a t a s h o r t p r e i n c u b a t i o n i n oxygen markedly i n c r e a s e s the subsequent r a t e 289 of anaerobic  glycolysis  .  T h i s has been shown f o r a number  of a d u l t t i s s u e s , i n c l u d i n g b r a i n , b u t the reason  for this i s  290-294 not c l e a r of anaerobic  .  I t has been s t a t e d t h a t the i n c r e a s e d r a t e  g l y c o l y s i s of the c e r e b r a l c o r t e x s l i c e s a f t e r an,  -  aerobic  preincubation  234  might  -  be  the  result  of  accumulation  of  71 pyruvate ATP  during  concentration,  anaerobic One the  of  ATP  content  lysis  acid  was  cerebral  Experiments extent  to  cerebral w e r e made  increase  in  the  to  low  a  very a  rate  the  and in  limiting  anoxia  out ATP  is  role  on  due  as  effect  and  to  a  the  many  de-  pointed on  of  phos-  conditions,  and,  limiting  loss  concentration,  anaerobic  level  the  the  out  glyco-  that  there  the  ATP  content  the  presence  ATP  content  TTX  out,  on  of  of  the these  was  ATP  TTX  existed the  under that  to  in  the  of  the  anaerobic TTX  may  slices.  ascertain  i s responsible glycolysis  experiments,  the  15)  incubated  anaerobic  induced of  increase  (Table  of  therefore,  level  In  there  i s an  the for  the  of  conditions  either a  decrease  cerebral  cortex  the  ATP  content  of  TTX  in  the  incubation  medium  (Tables  i s added  to  the  incubation  medium  after  presence  of  effect  mitochondrial  indirectly  possibility  cell of  of  the  slices. that  in  us  carried the  rate  the  anaerobiosis.  by  hence  action  such  are  Under  slices  were  cortex  an  anoxia  However,  discussed.  carry  content.  phosphate  which  stimulating  of  observed  increasing  been  i n decreased  subsequent  and  not  a  consequences  exert  cortex  conditions,  and  falls  i t may  labile  by  ATP  .  exert  tissue to  effects  during It  the  period  can  has  resulting  i n the  earlier,  which  major  of  other  crease  act  the  capacity  the  aerobic  glycolysis,  phorylation of  the  or  slices 13  14). When T T X  10  or  -  15  minutes  the  of anoxia,  anaerobic  changes, such  taking place  sponsible  ive if  of glucose  i s added  the  absence  TTX  i s s t i l l  anaerobic if  from  after  TTX.  ditions  results  which  show  should  creases  t h e ATP  TTX  time  either  be g r e a t l y  be t h e ATP  i s not  effectHowever,  aerobic, i n  result  then  rate of i s obtained  i s f o l l o w e d by of  TTX  a  glucose.  but glucose  then  then  of pyruvate,  i s added  i s not  effect-  glycolysis.  the presence  of  o r an a e r o b i c  t o show  glucose preincu-  any r e s p o n s e  i n f l u e n c e d under  these  to con-  concentration of the cerebral carried  out, i n the presence  the oxidative phosphorylation contents  and  the subsequent  of anoxia,  that  anaerobic  o f TTX,  by t h e a d d i t i o n zero  arer e -  +  glycolysis.  (15 m i n )  f o r the s l i c e s  Experiments  uncouples  from  some  o u t f o r 15 m i n i n  similar  of the experiment  t h a t would  slices.  and then  A  on  anoxia,  of K ,  i s made  the rate of anaerobic  i s necessary One  13).  period  a period  the beginning  bation  of anaerobic  (Table  i s present  i n increasing These  loss  i s carried  i n increasing  the preliminary anoxic  ive  and/or  but i n the presence  effective  glycolysis  immediately  few m i n o f  but i n the presence  of glucose  shows t h a t  o f t e t r o d o t o x i n on t h e  the rate  10 m i n a e r o b i c p e r i o d  tex  content  10 m i n i n c u b a t i o n p e r i o d  When p y r u v a t e  This  t o t h e i n c u b a t i o n medium,  i n increasing the f i r s t  stimulating effect  the f i r s t  I f anaerobiosis  absence  glucose  i t h a s no  during  i n ATP  -  (Figure 21).  f o r the effects  glycolysis. the  then  glycolysis  as decrease  235  (Table  1 4 ) , show  of  and hence  t h a t when  cor-  the  DNP,  deaero-  -  bic  23  preincubation i s carried  then the  the percent  -  out i n the presence  stimulation  presence  6  of anaerobic  o f DNP,  glycolysis i n  o f TTX i s n o t a f f e c t e d .  experiments  lead  to the conclusion that  responsible  f o rthe effect  These  the c e l l  o f TTX on t h e speed  ATP i s n o t  of anaerobic  glycolysis. Experiments  carried  slices  show  that  crease  i n the rate  although  due t o a r i s e  These  slices  when  i ti s added higher  slices,  higher  rate  ference brain Role  production  slices  after  there  i s an i n -  i n controls,  pos-  o f TTX i s u n a f f e c t e d . similar  i n showing  t o t h e non-  the effects  o f TTX  15 m i n o f a n o x i a .  content  o f ATP i n t h e i n c u b a t e d  of glycolysis  cerebral  o f T T X , may b e t h e r e s u l t  itself  t h e energy  rather  than  utilization  that  of a  o f an  processes  cor-  inter-  ofthe  Pyruvate  anaerobic  lysis,  i s known  glycolysis  affecting  important  and  slices,  cells. of  hence  such  i n a manner  i n the presence  Pyruvate  by  respond  o f TTX w i t h  adenosine-preincubated  i n ATP, t h e e f f e c t  preincubated  The tex  also  with  of lactate  sibly  adenosine  out with  to increase considerably the rate of  and i thas been  t h e NAD /NADH +  ratio.  f o rthe regulation  t h e NAD /NADH  TTX a r e p r e s e n t  colysis  i s additive.  +  together, This  +  their  i t might a c t ratio i s  of anaerobic  the efflux  ratio.  shows  that  A s NAD /NADH  of the rate  T T X may a c t b y p r e v e n t i n g affecting  stated  o f pyruvate and  However, when effect  that  both  glyco-  pyruvate  on anaerobic  gly-  these  must  agents  -  be  affecting  slices out  by d i f f e r e n t  Role  The TTX  that  of pyruvate  of Glucose  when  creased.  The r a t e  the  effect slices  when  a maximum  b y TTX  (Table  high,  the concentration This  required  the  brain  Role  when  to saturate  cell  or c e l l  i s de-  until  50 mM  about  results  becomes  obtained.  entry  demonstrate  into that,  glycolysis i s a rate that  limiting  with  concentration  the glucose  i s  of glucose  due t o t h e f a c t external  glucose  of glycolysis  of anaerobic  of glucose  high  by  i n the presence  These  the rate  glycolysis  glycolysis,  rate  6).  i s doubtless  of glycolysis,  are  slices.  concentration  i s n o t due t o f a c i l i t a t i o n  conditions  rates  the incubated  glucose  increases  under  factor.  rule  o f TTX i s due t o t h e  from  of anaerobic  TTX, p r o g r e s s i v e l y  This  cortex  experiments  stimulation of anaerobic  the external  concentration,  These  the effect efflux  of cerebral  Concentration  percentage  falls  glycolysis  mechanisms.  the p o s s i b i l i t y  suppression  of  the anaerobic  237 -  of  utilization  high glucose  system i n  compartments.  o f cAMP As  cerebral  shown i n C h a p t e r cortex  creased;  instead  demonstrate glycolysis  that  i n the presence  a decrease the effect  when  i t has been action  formation  These  o f TTX on t h e r a t e formation  shown t h a t  i nthe  o f TTX i s n o t i n -  i s observed.  i s n o t due t o i n c r e a s e d  Recently, ditions,  slices  5 . 8 , t h e cAMP  under  of  results anaerobic  o f cAMP. a variety of  p o t e n t i a l s are generated  con-  and d e p o l a r i z -  -  ation  occurs,  trine, of  such  increase  ect  effects  slices  or e l e c t r i c a l  i n the cerebral cortex  the  Effects  caused  by b a t r a c h o t o x i n  n o t been  o f TTX  of high  K  ,  protovera-  stimulation, the  slices  o f T T X o n t h e cAMP  have  ^  as i n t h e p r e s e n c e  batrachotoxin  cAMP  238  i s increased i s blocked  contents  production (297 ,300);  by TTX.  of cerebral  Dir-  cortex  reported.  i n the Presence  of L-glutamate,  NH^  and  +  Proto-  veratrine A of  number  of experiments  TTX on t h e a n a e r o b i c  the  glycolysis  movements o f c a t i o n s  transport  and/or  veratrine, influx obic out  of Na  +  other  yM p r o t o v e r a t r i n e , rate  of anaerobic  sively  +  decreases  (Figure  2 yM  with  to direct  also  inhibits  slices.  that  22).  Thus,  protostimulate  the rate  glycolysis,  of  drugs  stimulatory  i n the  a r e antago-  effect  o f TTX  increasing concentrations  anaer-  carried  i n the presence  and t h e e f f e c t  on  on t h e  Experiments  these  Thus,  effects  effects  of metabolites.  TTX has l e s s  glycolysis  the  be due t o e f f e c t s  on t h e a n a e r o b i c  o f p r o t o v e r a t r i n e , show  t o each  might  that  a c t i o n p o t e n t i a l s and t o  of K ,  o f TTX  indirectly,  than  of cerebral cortex  on t h e e f f e c t s  nistic  level  t o generate  and e f f l u x  glycolysis  presence  rather  cellular  known  show,  of  of 5 on t h e  progresprotovera-  trine. Similarly,  L-glutamate  + of bic  Na  i s known  to stimulate  the entry  286 into  brain  glycolysis  glutamate  cells  *  The e f f e c t  of the cerebral cortex  i s also present  o f TTX  slices  i n the incubation  on t h e a n a e r o -  i s abolished i f medium  from the  start has  of the experiment.  no e f f e c t  increase These  experiments  ebral  results  cortex  slices  (Figures  out that  of Na  +  the presence  glycolysis  K  while  likely  that  cortex  slices  +  Because  Na  +  content  +  7  cer-  of the These o f TTX,  affected;  thus  of the cerebral  i s an i n h i b i t o r  the K  on  i n the presence  3  5  o f TTX on t h e  investigated.  of  cortex  anaerobic  ' ^ , i ti s therefore  the anaerobic  by i n c r e a s i n g  3  +  ratio  +  slices ^ .  anoxia.  and K  +  i s an a c t i v a t o r  TTX s t i m u l a t e s  cortex  are definitely  +  i s increased.  of  and K  that  o f TTX t h e K / N a  1  due t o i t se f f e c t  were d i r e c t l y  of cations  NH^" ", T T X  NH^"*" i s k n o w n t o  the effect  a t the onset  2 4 , 2 6 , 2 7 ) show  level  o f 5mM  the cerebral  e f f e c t s o f TTX on t h e N a  cellular  slices  from  +  o f TTX on C o n t e n t s  cerebral  Na  point  glycolysis.  g l y c o l y s i s i s presumably  The  in  of K  c a t i o n i c movements  Effects  the  on a n a e r o b i c  the efflux  anaerobic  In the presence  glycolysis of cerebral  content  and decreasing  the  content.  +  As TTX  has been  are best  fore  the onset  cortex show  slices  that during  is  probably  of  K  i s measured  before  some  time  i t may  are preincubated  under  little  preliminary  the metabolic  When t h e K such loss  anoxic  due t o t h e f a c t s  TTX t a k e s  membrane  i s very  the subsequent  during  earlier,  i fthe slices  o f anoxia.  there  TTX  that  seen  mentioned  level  +  period  (a) t h a t  +  the results  i n the presence of  (Figures  there  26,27).  This  i s decreased  loss  aerobic  incubating  t o bind  to the sites  affect  i n oxygen be-  of the cerebral  conditions, of K  effects of  period,  a n d (b)  at the brain  t h e c a t i o n movements.  cell  Thus, i f  -  the  initial K  period  loss  of  and  very  effective  +  centration  of  m a t e o r NH^ ,  slices  of  K  and  evidence  rate  anaerobic  by  a  the  the  measured  that  less  these  anoxic,  by  +  in affecting  further  ed  Na  cations,  +  under  of  pf  showed  +  tention  i s wholly  gain  there  is  considerable  before  is  sufficient  colysis.  Can  observed umed  i n the  gluta-  less  in  the  i n the  presence  of  increase  i n the  gain  25).  (Table  since  TTX  follows tissue the  are  of  of  K  The  cerebral  this  the  of  site on  in  of  the  +  i n our  measured of  may  the  cortex  rethe  results  are  when  TTX  is  the  affect-  in  the  Na  slices  /Na  ratio  TTX,  under  rate  glycolysis  K  is  electrically the  Na  and  greater  the  whole  cations  of  TTX  may  the  of  on  then  in  If  gly-  ass-  neurons  and  e x c i t a b l e neurons, K  found  i f they  they  i n the occur  actual  i n the  be  anoxia,  i t is  the  tissue,  the  the  concentrations  +  and  the  of  anaerobic  Na  and  considerably  effects  of  and  +  experiments, in  K  +  quantitatively?  in  these vary  large  con-  by  +  involved  presence  increased  results  changes  As  Na  These  are  presence  p r o p o r t i o n a t e l y much  were  and  +  cells. of  only  composition Na  the  the  neurons.  age  i n the  changes  major  acts  that  trations  the  the  increase  explain  explain  that  the  slices  to  be  agents.  Qualitatively, cortex  could The  +  cerebral  TTX  c a t i o n movements.  c a t i o n movements  glycolysis  v a r i e t y of  there  slices  is  decrease  conditions  that  -  240  K  whole  only  in  concen-  give  the  aver-  concentration  neurons  anaerobic  readily  i t  and  glial  glycolysis  explained  on  basis. Results  of  experiments  on  the  caudate  nucleus  (Table  22)  -  indicate  that  the  presence  the  same The  i t sa n a e r o b i c  observed  conclusion  that  glycolysis  i s mediated  is  supported  there  due  i f there  to increased Results  fects K  +  of  a  with  higher  until  centration tration  centration colysis  that  inhibition  (Na  of glycolysis by N a  of Na  conditions  the incubation  +  medium.  be  kinase  step  theef-  K  i n the Na  +  content  Under  since  i t s  When  without  increase  i n the K  sucrose),  Under  then  having  The r a t e  these  i s then  decreases  i s obtained.  stimulated  cat-  and t h e degree o f  i s low.  32).  and  glycolysis  concentration  +  +  of both  medium  by  i s markedly  o f TTX  would  of anaerobic  i s not observed  +  the controls  (Figure  ratio  +  26).  o f TTX p r o g r e s s i v e l y  i s replaced than  which  out, to explain  when  decrease  i s decreased +  +  the concentration  Thus,  i n the incubation +  i n the K /Na  (Table  of g l y c o l y s i s i s obtained  rate  slices.  on t h e a n a e r o b i c  content  f o r the regulation  i n the presence  i s about  i n the presence  cell  in  glycolysis at different Na  that  a corresponding  cortex  of the pyruvate  carried  slices.  i s higher  centrations these  cortex  of Na  increase  i n the brain  indicate  an optimal  conditions,  +  o f TTX  i n the pyruvate  on t h e a n a e r o b i c  rate  stimulation  in  K  are important  raised  through  increased  of stimulation  the cerebral  the e f f e c t  of experiments  o f TTX  cerebral  with  i s facilitation  concentrations  ions  glycolysis i s also  by t h e f a c t  i s an i n c r e a s e  expected  -  o f TTX, b u t t h e m a g n i t u d e  as t h a t  further  241  +  conof  ( 1 4 9 mM)  of glycolysis  by t h e p r e s e n c e conditions,  con-  the concen-  the rate  normal  these  glycon-  under o f TTX  presumably,  - 242 -  prevention  of the e f f l u x of c e l l u l a r K  ant p a r t .  We have seen  t r a t i o n of K  +  (Table 2 0)  p l a y s a very  import-  t h a t when the concen-  i n the i n c u b a t i o n medium i s h i g h , i n a d d i t i o n  t o 149 mM N a , then TTX i s n o t e f f e c t i v e i n i n c r e a s i n g t h e +  r a t e o f anaerobic  g l y c o l y s i s o f the c e r e b r a l c o r t e x  I t i s known t h a t high K the c e r e b r a l t i s s u e  135  +  causes i n c r e a s e d  slices.  i n f l u x of N a  + and when 149 mM Na i s p r e s e n t  +  into  i n the  i n c u b a t i o n medium, there i s no s t i m u l a t i o n o f g l y c o l y s i s because o f i n c r e a s e d N a of N a  +  +  influx.  T h i s i n c r e a s e i n the i n f l u x  i s presumably not i n h i b i t e d by TTX and thus,  respect,  i t differs  from the N a  +  i n this  i n f l u x caused by p r o t o v e r a -  t r i n e , e l e c t r i c a l s t i m u l a t i o n or a n a e r o b i o s i s . Results  obtained  w i t h kidney medulla s l i c e s on the e f -  f e c t s of TTX on anaerobic •ehow and  g l y c o l y s i s and N a  +  and K  +  contents  t h a t TTX had no e f f e c t on e i t h e r o f them (Figures 16 29). T h i s shows t h a t the e f f e c t of TTX on anaerobic  gly-  c o l y s i s i s n o t an u n s p e c i f i c phenomenon and perhaps i s conf i n e d only t o c e r e b r a l t i s s u e . der  The r e s u l t s w i t h acetone pow-  e x t r a c t s of b r a i n demonstrate t h a t the i n t e g r i t y of b r a i n  c e l l membrane i s r e q u i r e d f o r TTX t o show any e f f e c t on the r a t e o f anaerobic  g l y c o l y s i s and TTX does not a c t by a f f e c t -  i n g any o f the enzymes of the g l y c o l y t i c pathway. E f f e c t s on Amino A c i d T r a n s p o r t TTX  Under Anaerobic  Conditions  markedly suppresses the e f f l u x of amino a c i d s from  the incubated  cerebral cortex  slices.  pronounced i n the case o f g l u t a m i c are p r e s e n t  in relatively  T h i s e f f e c t i s very  and a s p a r t i c a c i d s which  l a r g e amounts (Table 4 ) . Abadom  - 24 3 -  and  Scholefie  have shown t h a t when the ATP content  cerebral cortex s l i c e s i s higher,  of the  t h e r e i s g r e a t e r uptake of  g l y c i n e from the i n c u b a t i o n medium.  One can argue t h a t i n the  presence of TTX, g r e a t e r amounts of ATP are p r e s e n t s l i c e s due t o a h i g h e r r a t e of anaerobic  i n the  g l y c o l y s i s and as a  r e s u l t the t r a n s p o r t system works more e f f i c i e n t l y , i n g r e a t e r uptake o f amino a c i d s .  resulting  However, such a p o s s i b i l i t y  i s r u l e d out by the f a c t t h a t there i s an i n c r e a s e d  reten-  t i o n of amino a c i d s even i n the presence of a m i x t u r e of ouabain and TTX  (Table 30).  Ouabain i s known t o b l o c k the  a c t i v e uptake of amino a c i d s and i t s a c t i o n i s not by TTX.  Thus TTX must be a c t i n g independently  s e n s i t i v e amino a c i d t r a n s p o r t system. contents  antagonized  of the ouabain  The i n c r e a s e d t i s s u e  of amino a c i d s i n the presence of TTX i s even apparent,  i n the absence of g l u c o s e ,  although  to a l e s s e r extent.  Experiments w i t h l a b e l l e d amino a c i d s  (Table 5) showed  t h a t t h e r e i s a s i g n i f i c a n t i n c r e a s e i n the uptake by the ana e r o b i c a l l y incubated  s l i c e s i n the presence of TTX.  a b l y t h i s i s due t o two reasons:  Presum-  ( i ) i n c r e a s e d uptake of amino  a c i d s by the s l i c e s because of the g r e a t e r r a t e of  anaerobic  g l y c o l y s i s and hence the h i g h e r ATP l e v e l and ( i i ) because of i n h i b i t o r y e f f e c t of TTX on the e f f l u x of amino a c i d s from the  slices.  E f f e c t s of TTX i n the Presence of C h e l a t i n g Agents and Phospholipases .  TTX has l i t t l e  or no e f f e c t on the r a t e of  anaerobic  g l y c o l y s i s i n the presence of EDTA and EGTA (Table 23).  In  the Na  presence into  +  This it  influx  appears  lating Na  and  +  K  o f Na that  +  substances  efflux  i s only  must  This  through  on  influx  cerebral  inhibited  by  TTX  through  their  133  the cerebral  i t s e f f e c t s on  .  of  Thus che-  actions  further  on  supports  anaerobic  the K  the our  glyco-  and N a  +  of  tissue.  i n the presence  conclusion  o f TTX  i s a rapid  from-the  +  o f TTX,  be m e d i a t e d  the action  i s mediated  of K  there  partly  the action  movements.  +  that  lysis  a rapid  agents,  and  view  of these  con-  +  tents . Results pholipases cubation, obic the  t h e e f f e c t s o f TTX  (Table  9)  show  phospholipase  that  A  has  g l y c o l y s i s of cerebral later  part  glycolysis more  i s considerably  binds  indicate that  which  finding  further  stituents Effects  i s slowly  o f TTX  brain  shows  glycolysis  on  Developing  of  TTX  2-day  acts  has  on  c a r r i e d o u t on  little  o r no  old r a t brain.  contents  2-day  of  20  or These and  constituents, This  o f membrane  con-  metabolism.  Brain  and  of  anaerobic  per vessel.  the cerebral  o l d r a t i s s e n s i t i v e t o TTX.  +  anaerduring  the phospholipases.  2-week K  the  of i n -  a t t h e membrane  out the importance o f TTX  on  i n the presence  TTX  phos-  period  However,  has p h o s p h o l i p i d by  of  (50-80 m i n ) , t h e  of phospholipase  of experiments that  effect  slices.  reduced  which  i n the action  Results  cortex  attacked  points  the early  no m a r k e d  possibly  to the receptors  i n the presence  during  of the incubation  International Units  results  and  on  the developing  effect  However,  on  the  old r a t cerebral  anaerobic  the brain  Investigations cortex  rat  from  on t h e  slices  Na  show  - 245 -  that the  there  presence  brain The  receptors  tive  a r e formed  i n f a n t guinea  aerobic  glycolysis  there  brain, sence  pig brain  a s 10  This  too, the increase  Effects  i s mediated  o f TTX on A e r o b i c  The  rates  creased  when  of aerobic  Ca  presumably  +  +  decrease  i n ATP  o f ATP  medium  (containing Ca  this who  Thus,  i s analogous showed  emission TTX.  values  medium.  that  of C a  +  through  + +  )  of the  on i t s m e t a b o l i s m .  period  old r a t  of  life.  are extremely  with  sensi-  i n i n c r e a s i n g an-  +  i n the presence  of  and l e s s  of  uptake  the i n f a n t guinea p i g  glycolysis  increase  glycolysis  i n the  i n the K /Na +  from Na  +  of Na , +  of brain  slices  the incubation influx  preratio.  +  K -ATPase. +  of the c e l l  This  In a  2 yM T T X h a s n o e f f e c t - f r e e medium  approaching i n this  regard,  obtained TTX  acts  inhibitory Krebs-Ringer  on t h e a e r o b i c  i n a like  by Chan  i n respiration,  the incubation  results i n a  i t is suppressed  those  medium,  This  corresponding  and t h e  i s diminished.  are i n -  medium.  and t h e  t o the r e s u l t s obtained  from  maturity  Glycolysis  the increase +  slices  i n anaerobic  on g l y c o l y s i s  b u t i n a Ca  resulting  the later  shows t h a t  ratio i n  +  i n t h e 2-day  retention of K  concentration  effect  Ringer  any e f f e c t  Furthermore,  due t o g r e a t e r  i n the activity  the  yM.  i s omitted  increase  glycolysis,  that  i  during  i s an i n c r e a s e d  o f TTX  +  yM T T X i s a s e f f e c t i v e  by t h e s l i c e s .  +  i n the K /Na  r e s u l t s show  f o r TTX t o h a v e  t o T T X a n d 0.2  TTX,  These  increase  f o r TTX a r e n o t d e v e l o p e d  and they The  is  o f TTX.  i s required  brain  Na  i s no s i g n i f i c a n t  KrebsCa  and  brought  by TTX,  +  +  and  Quastel^ about  by  i s suppressed  by  -  8.6  EFFECTS As  the  through  ever, not  the  be an  into  +  the  ative  to  enters  the  and  view  the  of  of  of  Na  ouabain  on  Na  on  , K  of  this  in  K  conditions  K  that  comes  these  has 35),  is  limited.  These  lation  of  anaerobic  glycolysis  a  f r e e medium  the  cationic + Na  the  speed  and  ing  very for  phate. + Na  K  of  Under a  a  since  great  K . +  therefore,  under  not  rate  to  to  of  Na  the  favours  the  of  Na ,  K -  +  the  +  stimu-  slices  in  i t s effects  these  +  under  on  cortex  due  oper-  due  effect  which  cerebral  of  Further,  operation  be  of  amount  indicate that  cannot  condi-  i s not  slices. no  can-  ouabain  conditions  or  How-  influx  efflux  -ATPase  result the  of  medi-  on  conditions,  +  the  to  and  , K  little  results  ouabain  contents,  an  brain  conditions,  by  and  a  considerable  ATPase  Ca  to  and  the  on  is  aerobic  aerobic  of  (Figure  fluxes  under  ouabain  contents under  out  TTX  glycolysis  under  leads  a  of  concentrations.  +  and,  Na  TRANSPORT  slices  anaerobic  slices the  cortex K  AND  effects  cation  drug  as  concentration +  the  the  cortex  extent  and  +  -ATPase  conditions,  same  and  cerebral  i n the  i n ATP  anaerobic Na  earlier,  cerebral  anaerobic  fall  discussed  presence  Under  a  METABOLISM  i t s effects  inhibitor the  CEREBRAL  changes  to  -  ON  glycolysis  effect  due  tions, Na  OUABAIN  been  anaerobic  ated  is  has  OF  246  contents anaerobic  anaerobic low  the  level  do  a  a  limiting  role  on  glycolysis.  conditions, (Table  15)  phosphorylation  Since  play  considerable  of  the  and  ATP  concentration  i t may  glucose  amount  of  become  rate  and  fructose  ATP  i s consumed  falls limit-  6-phosby  + , K  -ATPase  i n the  brain  cell,  the  inhibition  of  this  en-  -  zyme  i n the presence  concentration This  may  sugar  ouabain  An  increase  i n t h e ATP  observed  this  noted  with  of  glycolysis,  diminished higher  Ca  +  fall  effective  values  glycolysis +  ltion  o f Na  with  + +  .  of cerebral  , K -ATPase,  that  ouabain  medium,  +  of  itself,  an i n c r e a s e d  K -ATPase,  rate  leads t o  +  of C a  medium,  results i n  +  show  +  that  i t i s n o t as  of ouabain  cortex slices  then  these  i n the presence  the data  slight  of Na ,  as i t i s i n  on t h e  anaer-  i s due t o t h e  i s added  This  to guinea  ,  ouabain  does  conclusion  inhib-  When C a  of C a  followed after i s omitted  i n respiration  + +  ,  t o the n o ti n -  i s i n agreewho  pig cerebral  i n the presence  i n respiration  a decrease  + +  o f Swanson and h i s c o - w o r k e r s  i n a medium  depression.  observations lead  of Ca  +  rise  be  +  ment w i t h  initial  of glycolysis  ouabain,  I f the action  K -ATPase completely.  incubating  rate  the anaerobic glycolysis  hibit  when  but i t cannot  i n the presence  that  +  i n the presence  c o n c e n t r a t i o n , and t h i s  conclusion Na ,  of anaerobic  i s due t o t h e i n h i b i t i o n  i n the incubation  i n increasing of C a  available for  f o r ATP.  of ouabain  i s present  absence  obic  i n t h e ATP  effects +  Possibly  due t o i n h i b i t i o n  observed  The when  TTX.  content  ( T a b l e 15)  increase  cell  i n i t s utilization.  i n greater rate  +  as  i n a greater  a g r e a t e r amount o f ATP  K -ATPase o r due t o a h i g h e r  +  the  make  whether  result  due t o a d e c r e a s e  i s i n fact  ascertained Na ,  will  phosphorylations resulting  glycolysis. of  -  7  of ouabain  p f ATP  i n turn  24  observed  cortex  there  slices  i s an  45 o r 60 m i n b y  from  the  i n the presence  a  incubation of ouabain  was  24 8 -  -  '  172 observed  .  These  results  were  e x p l a i n e d as  + the Ca  +  incomplete by  +  enzyme  inhibition  ouabain, to  which  respond  phosphates  and  to  o f Na  allowed  cation  thereby  resulting  from  + , K,-ATPase  i n the  the uninhibited  shifts  by  stimulating  presence  portion  consuming  respiration  high '  of  in  the  effects  presence  show  that,  show  any  of  creasing  later  Ca  from When  fall  ++  on  the  i t has  '  dose  centrations  ouabain  rat  cerebral  the  sensitivities  to  the  stimulating  ouabain  pig cerebral  curve on  +  Na , +  Na  but  ,K  the  ouabain  slices,  -ATPase  incubation i t has  that  -ATPase results  do  the  not acces-  is different  as  medium  in-  progressively This  when  may  ouabain  effects  of  be is  less  due  to  added  different  anaerobic glycolysis  to  after  effect.  f o r the  the  slices  of  and  +  tissue  , K  then  cortex slices  cortex  +  +  29),  anaerobic glycolysis.  response  of  t o Na  c o n c e n t r a t i o n s so  more  (Table  of  +  microsomal p r e p a r a t i o n s .  i s added  of  inhibition  in brain  anaerobiosis,  rate  no  the  studied  Perhaps  ouabain  on  preparations, Ca  i n the  periods of  A  were  +  and  those  i n t h e ATP  guinea  +  antagonism.  compared  a  Ca  ouabain  i n microsomal  sibilities  effects  of  of  this  energy  + The  of  are  ouabain.  K -ATPase +  to  more  show  sensitive  This  that  the  than  i s probably  ouabain  con-  differs  the because  with  the  163 species  have ase  of  the  in fact differ  animal  shown  with  Results brain  cortex-  that  the of  used.  Bonting,  ouabain  species of experiments  slices  show  that  Cravaggio  inhibition the  animal,  carried the  out  response  and  curves and with of  Hawkins  of  tissue  +  Na  , K  + -ATP-  used.  developing r a t infant  rat  anaer-  -  obic  glycolysis  249 -  t o ouabain  increases  with  + this  i s related  energy that  metabolism  the effect  stimulation of  brain  of ouabain  , K  Other  by t h e p r e s e n c e  that  o f NH^  i s i n accord with  the conclusion  play  a minor  ouabain  a  partial  ate  inhibition  and t h i s  lysis,  the Na  the rate  lated  ouabain..  when  either  Under  these  observed  affects ently In  Like  medium,  increased  of these  conditions, (Figure  drugs  then when  ATP  medium.  +  movements  glycolysis.  However,  TTX  of  L-glutam-  stimulated  glyco-  by c i t r a t e ,  These  the rate  i n K /Na +  results  of both  utilization  ouabain  of anaerobic with  the  +  ratio  o f Na  and K  (Na , +  may  +  K /Na  +  s t i l l  that  TTX  independ-  K -ATPase). +  and TTX, a c o m b i n a t i o n  and i n c r e a s e d  rate  (Figure 34).  demonstrate  pump  stimu-  are present to-  i s present alone  an i n c r e a s e  35).  medium i s  i s markedly  compared  o f t h e o p e r a t i o n o f t h e sodium  creased  and N a  +  and TTX b o t h  t h e p a s s i v e d o w n h i l l movements  the presence  K  i s inhibited  glycolysis  ouabain  i n the incubation  found  t h e TTX  due  o f p h o s p h o f r u c t o k i n a s e a c t i v i t y ( T a b l e 28).  When  i s further  extracts  i s not at  i n the presence  glycolysis  of anaerobic  glycolysis  be  from  concentration of the incubation  +  reduced,  gether  observed  stimulated  to inhibition  When  by  was  a  observed.  the former  stimulated  i s y e t t o be r e s o l v e d .  ouabain  probably  with  powder  differs  that  show  since  i n the incubation  +  This  role  experiments  phenomenon  i s not  glycolysis  by t h e f a c t  and g e n e r a l  of acetone  of ouabain,  stimulated  glycolysis  affected  -ATPase  i s a membrane  of anaerobic glycolysis  ouabain  stimulated  i n Na  possibly  +  d u r i n g development.  i n the presence  The  all  to the rise  age and  ratio  must  of debe  -  responsible  for  the  very  250  _  high  rates  of  anaerobic  glycolysis  obtained. Furthermore, place or  on  the  absence  These  the  onset  of  efflux  of  of  ouabain,  No aerobic  from  the  significant  out  across  action effect  g l y c o l y s i s and  of of  this  the  brain  ouabain 10  uM  is in  which  in  blocked  that  the  acids,  either  is partially point  substances  independent  amino  anaerobiosis,  results further  movement  of  the  by  on  presence  TTX  action cell  takes  of  (Table  30).  TTX  the  on  membrane  Na ,  K -ATPase.  +  ouabain  +  was  agreement  is  observed  with  the  on  findings  52 of  Rolleston  8.7  EFFECTS The  nervous local tex  bic  OF  modes tissue  Newsholme LOCAL ANESTHETICS  of  seem  like  shows TTX,  onset  of  anoxia,  ratio  of  presence  the of  observed  cerebral  are  0.5  mM  was  observed  slices  the  As  with  observed slices  presumably  rates  of  this  anaerobic  infant rat  increase cerebral  the  with cor-  drugs. of  anaero-  31).  When  medium  after  increasing TTX,  i s also  on  cerebral  rate  (Table  in  TTX  these  the  incubation  GLYCOLYSIS  obtained  between  ineffective  cortex  and  g l y c o l y s i s of  increase  cortex to  ANAEROBIC  Results  similarities  l i d o c a i n e , an with  THE  anesthetics  anaerobic  added  l i d o c a i n e and increased  local  anesthetics  glycolysis.  of  also  are  ON  identical.  cerebral  they  presence  of  be  the  local  anesthetics  the  on  g l y c o l y s i s of  anaerobic  to  further  local  of  action  anesthetics  slices  Thus,  and  the  the  rate  K /Na +  increased  the  +  in  the  i s responsible  for  glycolysis. in  the  cortex  K /Na +  In +  slices  the  ratio (Table  - 251 -  32);  however, no s t i m u l a t i o n of a n a e r o b i c g l y c o l y s i s was ob-  served w i t h i n f a n t r a t c e r e b r a l cortex Perhaps the i n f a n t  (2-day old)  slices  (Figure 36).  r a t cerebral cortex g l y c o l y s i s  i s n o t as s e n s i t i v e as t h a t of the a d u l t t o changes i n the K and  Na  this  +  concentrations.  Further  +  work i s necessary t o c l a r i f y  point.  8.8.  EFFECTS OF OTHER NEUROTROPIC DRUGS ON THE ANAEROBIC GLY-  LYSIS Results  of experiments c a r r i e d out w i t h p y r r o l e show t h a t  p o s s i b l y i t s a c t i o n on the anaerobic g l y c o l y s i s of guinea p i g c e r e b r a l cortex Na  +  s l i c e s i s mediated through an i n c r e a s e  r a t i o resulting i n stimulation  cerebral cortex  of pyruvate k i n a s e .  inK / +  The  s l i c e s from r a t s were found, however, t o be  more r e s i s t a n t t o the o r g a n i c  bases t e s t e d than those of guinea  p i g s l i c e s and the reason f o r t h i s d i f f e r e n c e between these two animals i s not c l e a r . Preliminary  r e s u l t s o b t a i n e d w i t h amytal and r e s e r p i n e  i n t h e i r e f f e c t s on the a n a e r o b i c g l y c o l y s i s o f c e r e b r a l s l i c e s , a r e of some i n t e r e s t .  Thus i t was d i s c o v e r e d  cortex  t h a t the  a n a e r o b i c g l y c o l y s i s o f a d u l t r a t b r a i n s l i c e s , and e s p e c i a l l y those from the i n f a n t guinea p i g b r a i n s l i c e s i s enhanced by amytal.  At anesthetic  doses  (0.25  mM), however, the anaerobic  g l y c o l y s i s of r a t b r a i n s l i c e s i s n o t much a f f e c t e d by amytal (Figure 3 9 ) . The  a c c e l e r a t i v e e f f e c t s of amytal on anaerobic  glyco-  l y s i s of b r a i n does not seem t o be an a r t i f a c t due t o i t s w e l l known a c t i o n i n s u p p r e s s i n g NADH o x i d a t i o n .  Thus, as has been  -  pointed the  out earlier,  N :C02  mixture  2  Pasteur under  effect,  these  glycolysis;  could  conditions,  (See  Chapter 7 ) .  found  their  anionic  infant mM  and K  +  guinea  forms  dissolve  of  adult  At this  with  guinea adult  of cerebral  was  i n the K /Na does  the oxidation  the rate since  of  azide,  to inhibit  o f NADH  anaerobic under the  slices  n o t appear  lipids  was  pig brain  and  i s more  permeabilities^  study  of  0.25  g l y c o l y s i s of by t h e drug. amytal  concentration  e f f e c t on t h e c a t i o n i c  Further  with  sensitive to  and a h i g h e r  i n  thereby  i n the presence  affected  been  axons  observed  the anaerobic  i s n o t much  have  barbiturates  affect ionic  of barbiturates  contents  i s necessary  to  on t h e a n a e r o b i c  set-  glyco-  slices.  also  +  They  i n lobster  that  concentration  cortex  the trans-  ganglia.  suggested  r a tbrain  cortex  +  +  slices.  g l y c o l y s i s of brain  increase  i n  o f g l y c o l y s i s due t o  i n t h e membrane  a greater  of action  Reserpine  as an i m p u r i t y  conductances  and thus  +  slices  have  r a t brain  +  concentration,  cortex  could  t h e mode  +  p i g cerebral  the infant  amytal  serpine  stated  I t has been  i n the K  of  obic  the Na  increase  compared  lysis  been  An  Perhaps  tle  increase  i s unlikely  of C a  cerebral  when  this  the binding  amytal.  rat  rates  by p r e v e n t i n g  would  have  excitation.  affect  lower  o f impulses, i n t h e s y m p a t h e t i c  t o reduce  during  of oxygen  has no e f f e c t on t h e a n a e r o b i c g l y c o l y s i s .  Barbiturates mission  give  and amytal  however,  -  the presence  conditions,  same  252  found slices,  ratio  was  to increase  the rate  and i n i t s p r e s e n c e , observed.  of  anaer-  t o o , an  The e f f e c t o f r e -  t o b e d u e t o i t s known  e f f e c t on t h e  -  release have The  o f amines  very  little  effects  pected  ronal mode  and thereby  membrane. of action cortex  nialamide  have  anaerobic From  cluded of  that  some d r u g s results these  to  8.9  this  GENERAL  t h e neu-  amphetamine  stimulatory  specific with  effects  cortex  i n this  thesis,  drugs  and  on t h e r a t e s  slices. i t c a n be  can a f f e c t  cortex  anaesthetics, that  the  With  experimental  i n the presence  i n the c e l l  pyrrole,  amytal  affect  con-  the rates  slices.  increase  t o o may  membrane,  K /Na +  and  +  reserpine  the ionic  b u t f u r t h e r work i s  of  fluxes necessary  matter. .  CONCLUSIONS abolishes  the  generation  potentials i n a v a r i e t y of excitable tissues.  concentrations,  anaerobic  across  of  on t h e b a s i s  drugs  of action  glycolysis  TTX, a t low c o n c e n t r a t i o n s ,  action  similar  cell  the generation  of the cerebral  results  a r e unex-  on t h e anaerobic  as TTX and l o c a l  the brain  contents  the  discussed  i s a  glycolysis.  to clarify  of cerebral  glycolysis  these  +  C h l o r .promazine,  glycolysis  there  and K  alone  i s i n progress  no s i g n i f i c a n t  Preliminary  1. of  slices.  c a n be e x p l a i n e d  settle  +  as amines  of anaerobic  to block  work  of reserpine  shown t h a t  across  on t h e N a  a number o f n e u r o t r o p i c  drugs  ratio.  on t h e r a t e s  Further  such  sites  t h e movements o f c a t i o n s  the results  anaerobic  have  effect  storage  i t i s n o t known  cerebral  of  their  of reserpine  since  potential  from  25 3 -  glycolysis  fold.  Such  an e f f e c t  tained  on t h e a e r o b i c  such  a s 2 yM,  of cerebral o f TTX  cortex  i s much  metabolism  i t enhances slices  greater  of cerebral  the rate of  two t o  than  At  that  cortex  threeob-  slices.  -  2.  The  old  rat  the  e f f e c t of  anaerobic  brain  Furthermore, extracts integrity  are  TTX  specific  is  the  brain  of  the  not  affected for  anaerobic  i s not  cell  -  g l y c o l y s i s of  slices  since  from  254  is  kidney by  medulla  TTX.  mature  .This  cerebral  g l y c o l y s i s of  affected  required  by  for  TTX,  or  2-day  shows  tissue.  acetone  powder  i t follows  i t s action  that  on  that  the  cerebral  metabolism. 3. obic ium at  TTX  containing the  same  centage  high  4.  that  lead  into,  and  the  onset  the  steady  K  to  that to  is  +  in  lack high  added  a of  K  in  the the  If the  rate  of  the  rate  Na  is  +  of  e f f e c t of  in  cortex  TTX  due  to  slices  a  the in  medreduced  anaerobic  smaller  be  anaer-  incubation  progressively  may  +  of  K  cerebral  from  +  and  i t is  glycolysis. and  occur  after  conclusion  state,  +  has  cerebral  anoxic  anoxia  Na  increased,  TTX  and  is  the  efflux  the  is,present  +  concentration.  The  +  e f f e c t on  per-  medium  increased  presence  of  concentration.  +  the  of  K  +  and  occur K  anaerobic  due  Na  When T T X  ments  fluxes  Na  when  normal  affects  in  normal  time  external  longer  high  acceleration.  Na-influx  are  accelerating  increases  containing  ges  no  g l y c o l y s i s when  glycolysis  of  has  K  +  at  i t s action  that the  i f TTX not  at  The  there  is  incubated is  anoxia,  metabolism.  added  effects  onset the  of  an  of  TTX  diminishing  of  anoxia. cell  no experi-  i n f l u x of  Na  tissue  +  at  establishment  increasing  by  brain  i t  These  cerebral  after  effective in  contents the  15.min  are the  These  membrane,  due  the to  of  rate chan-  cationic effects  of  possibly  TTX  involving ability  phospholipids,  to cations.  high  rate  slow  decline  and  K  ated by  Th s,  of g l y c o l y s i s tends i n the cellular  by changes  5.  ance  i s also  of o r i g i n a l  direct  by Na  In the presence  concentration  effects  high  o f TTX  i n t h e perme-  o f TTX,  t o be m a i n t a i n e d + + K /Na ratio.  kinase  the  due  initial  to only  The e f f e c t s  g l y c o l y s i s are considered  i n pyruvate  and d i m i n i s h e d  i n changes  i n the presence  u  on t h e a n a e r o b i c  +  K  resulting  activity  o f Na  t o be  which  a +  medi-  i s enhanced  . o f TTX, u n d e r  increased rate  anoxia,  b u t t h i s may  ATP  be due t o m a i n t e n -  of glycolysis rather  on t h e energy  the c e l l  utilizing  than  due t o  processes  of the  cell. 6. brain  TTX a f f e c t s  i n vitro  tials.  This  incubated blocked obic  i n t h e same way  shows  that  cerebral  action  tissue  and a n a e r o b i c  as i t e f f e c t s potentials  at the onset  by TTX and m a n i f e s t s  itself  metabolism  of  the action  poten-  are generated  i n the  of anoxia.  i n the higher  This  i s  rate  of  anaer-  glycolysis. 7.  that  The above  conclusion  i n the presence  tamate, bated  which  brain,  thermore, EGTA, TTX  the aerobic  lead  of agents  to larger  TTX does  also  such  results  lysis  of cerebral 8.  cortex  +  i n greatly  by  or  increased  such  L-glu-  incu-  metabolism.  agents  the rate  the fact  i n isolated  the anoxic  of chelating  i s i n e f f e c t i v e i n enhancing  supported  as p r o t o v e r a t r i n e  i n f l u x of Na  not affect  i n the presence  which  i s further  Fur-  a s EDTA a n d  i n f l u x of Na , +  of anaerobic  glyco-  slices.  The e f f e c t o f TTX  on t h e N a  +  and K  +  contents  may  be  -  •greater  i n the neurons  than  are  regarded  as t h e s i t e  the  neuronal  K /Na +  -  i n glial  of action  ratio  +  256  brought  cells,  o f TTX. about  + greater sue  than  t h e changes  because Thus,  the  changes i n  b y T T X may  be  much  +  i n t h e K /Na  ratio  found  i n the t i s -  as whole.  + 9. TTX  In addition  also  prevents  cerebral TTX  cortex  to i t s effects  the efflux  slices  i s independent  port  colysis cell  on t h e Na  o f amino  a t the onset  acids  and K  from  of anoxia.  of the operation  The a c c e l e r a t i n g  i s presumably  contents, 11.  fluxes,  .the i n c u b a t e d This  o f t h e amino  effects  by  mediated  a mechanism  i n anoxia  o f some  slices  be e x p l a i n e d  may 12.  The r a t e  i n a Ca  10  pM  of  anaerobic  in  a Ca  + +  effect  acid  of  trans-  ouabain.  13.  glycolysis  + +  that  +  and N a  gly+  o f TTX. glucose  concentrations.  as p y r r o l e ,  which  The also  p i g cerebral  cortex  lines.  glycolysis  i s increased  -containing  of cerebral  i n the presence  medium,  i s not observed  cortex of  the stimulation  t o t h e same  extent  as  medium.  The e f f e c t  of the Na , +  brain  on s i m i l a r  medium  i n the K  of guinea  of anaerobic  In a Ca  -free  such  on t h e a n a e r o b i c  +  TTX on c e r e b r a l  higher  glycolysis  -free  +  resembling  organic, bases,  anaerobic  + +  of C a  by changes  b u t a t much  accelerate  slices  effect  Local anesthetics act like  breakdown  tion  +  system. 10.  the  former  cell.  of ouabain  K -ATPase, +  Under  which  anoxic  i s presumably consumes  much  conditions there  due t o  inhibi-  o f t h e ATP i n i s a f a l l i n  -  ATP  concentration  phosphorylation K -ATPase in  of  results  +  sults  and  greater  257  i t may  glucose in  an  -  become and  F-6-P.  enhanced  availability  rate  of  limiting  The  for  inhibition  ATP  level.  ATP  for  This  sugar  in  the of  Na ,  turn  re-  +  phosphorylat  tion  reactions.  play  a  less  Under  important  these part  conditions,  in  the  cation  regulation  of  changes  may  anaerobic  gly-  colysis. 14. anaerobic  Amytal  indicate  changes  in  fect.  amytal  of  work or  15. little  the  It. i s  Further  or  glycolysis  reserpine  g l y c o l y s i s of  results  centration  and  of  that  cation amytal  less is  cerebral  their  needed  e f f e c t i v e than  necessary  to  of  may  than  or  establish  a  stimulatory brain  amphetamines  con-  significant efanesthetics.  mode o f  effects  slices.  on  the  and rate  of  through  anesthetic  local the  rate  Preliminary  mediated  produce  TTX  the  slices. be  Larger to  increase  action  reserpine.  Chlorpromazine, no  may  cortex  action  contents. are  also  nialamide of  have  anaerobic  of .  - 258 -  BIBLIOGRAPHY 1.  E . S. G u r d j i a n , W. E . Stone and J . E. Webster, A r c h . N e u r o l . 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