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Regulation of intracellular pH in cultured foetal rat hippocampal pyramidal neurones Baxter, Keith Allen 1995

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R E G U L A T I O N O F I N T R A C E L L U L A R p H IN C U L T U R E D F O E T A L R A T HIPPOCAMPAL PYRAMIDAL NEURONES  by K E I T H A L L E N  B A X T E R  B.Sc. (Chemistry), The University of British Columbia,  A THESIS  SUBMITTED  IN PARTIAL  FULFILLMENT  T H E R E Q U I R E M E N T S F O R T H E D E G R E E M A S T E R  1991  O F  O F  O F S C I E N C E in  T H E F A C U L T Y O F G R A D U A T E  S T U D I E S  (Department of Anatomy)  W e accept this thesis as c o n f o r m i n g to the r e q u i r e d standard  T H E U N I V E R S I T Y O F B R I T I S H February,  C O L U M B I A  1995  © Keith Allen Baxter,  1995  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for reference  this or  thesis  thesis by  this  for  his thesis  or  and study. scholarly her  Department  of  ^^c^cwv.^  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  •£^>.  JtA^  I  I further  purposes  gain  the agree agree  may be  representatives.  for financial  permission.  of  shall  It  is  requirements that that  the  for  Library  an  advanced  shall  make  it  permission for extensive  granted  by the  understood  not be allowed  head  that  without  of my  copying  or  my written  11  ABSTRACT  The mechanisms regulating intracellular pH (pHj) were investigated in cultured foetal rat hippocampal pyramidal neurones loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5(or  6)-carboxyfluorescein.  At room temperature  (~20°C), steady-state pHj was 6.85 in the nominal absence of external H C 0 , and _  3  increased to 7.15 in the presence of HCO3". In HCC^-free medium at 37°C, steady-state pHj rested at the substantially higher level of 7.23, whereas in HC03"-containing solutions at 37°C, pHj was reduced to 7.13. Regardless of temperature and in the absence of H C O 3 " , the removal of extracellular N a  +  caused an immediate and sustained  intracellular acidification, suggesting the dominance of a Na -dependent mechanism(s) +  maintaining steady-state pHj.  In HCC^'/CC^-buffered medium at room temperature, a  moderate intracellular acidification was observed during the application of the anion exchanger inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic  acid (DIDS) or after  the removal of H C O 3 " , indicating the contribution of HCO3VCI" exchange to the maintenance of baseline pHj.  Moreover, this anion exchanger could participate in pHj  regulation even in the absence of extracellular N a . At 37°C, however, DIDS did not +  alter steady-state pHj in the presence of HCO3".  Though extremely sensitive to the  removal of extracellular N a at both temperatures, neither steady-state pHj nor the rate of +  pH, restoration from an imposed acid load were influenced by the application of ethylisopropylamiloride, a potent inhibitor of N a / H +  +  exchange.  Following an NH.4 +  induced intracellular acidification, the rate of pHj recovery to baseline levels was faster at 37°C than at room temperature. Furthermore, in contrast to experiments performed at room temperature, the addition of H C 0 " to the perfusate did not increase the rate of pHj 3  recovery at 37°C. The results of this study suggest that at 37°C, the dominant regulator of pHj in hippocampal neurones is a Na -dependent, H C O 3 "-independent acid extrusion +  mechanism (probably an amiloride insensitive variant of the N a / H +  +  exchanger).  At  Ill  r o o m temperature, this N a - d e p e n d e n t acid extrusion m e c h a n i s m remains active, but the +  regulation o f p H j appears to be s u p p l e m e n t e d HCO3VCI- e x c h a n g e r .  b y the activity o f a  Na -independent +  iv  TABLE OF  CONTENTS Page  Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  Acknowledgments  x  INTRODUCTION Physiology, pathophysiology, and pHj pHj a n d cell excitability pHj and ionic conductances pHiandCa Distribution o f protons across the limiting m e m b r a n e Regulation of pHj Intracellular buffering Overview  1 3 6 9 12 14 15 19 21  M A T E R I A L S A N D M E T H O D S Cell preparation L o a d i n g the neurones with B C E C F Experimental setup Solutions Calculation of pHj Analysis of data  22 23 24 25 28 32  2  +  R E S U L T S Steady-state p H ; regulation R e g u l a t i o n o f p H , at r o o m t e m p e r a t u r e R e g u l a t i o n o f p H ; at 3 7 ° C Na -dependent or -independent anion exchange M o d u l a t i o n o f p H j b y shifts in p H a n d the application o f acids and bases pHj recovery from an imposed acid load R e c o v e r y f r o m a n a c i d l o a d at r o o m t e m p e r a t u r e R e c o v e r y f r o m a n a c i d l o a d at 3 7 ° C  42 43 46  +  0  weak 46 86 88 89  V  Page DISCUSSION R e g u l a t i o n o f p H j at 3 7 ° C R e g u l a t i o n o f p H j at r o o m t e m p e r a t u r e C o m p a r i s o n o f p H , r e g u l a t i o n at 3 7 ° C a n d r o o m t e m p e r a t u r e Modulation of p H ; by p H Conclusions  122 122 130 133 137 140  R E F E R E N C E S  144  0  vi  L I S T OF  TABLES  Page Table 1  C o m p o s i t i o n o fH E P E S - b u f f e r e d experimental solutions  Table 2  C o m p o s i t i o n o fH C 0 3 ' / C 0 2 - b u f f e r e d experimental  3 4  solutions at r o o m  temperature  3 5  Table 3  Composition of H C 0 3 " / C 0 2 - b u f f e r e d experimental solutions at37°C  Table 4  C o m p o s i t i o n o fH C 0 7 C 0 - b u f f e r e d experimental solutions a t v a r y i n g pHs at37°C Steady-state p H j i nH C 0 3 " - f r e e a n d HCO3"-containing m e d i a a t r o o m t e m p e r a t u r e a n d a t 3 7 ° C , a n d t h e c h a n g e i np H j c a u s e d b y t h e e x p o s u r e to t h eexperimental solutions indicated  4 9  p H ; recoveries f r o m a nN H  9 5  Table 5  Table 6  3  ....  3 6  2  + 4  - i n d u c e d intracellular acidification  3 7  vii  LIST OF FIGURES  Page Figure 1  Relationship between the concentration o f HC0 " 3  solution p H w h e n equilibratedwith 5 % C 0  2  a n d the resulting  i n b a l a n c e air at 3 7 ° C  3 9  Figure 2  S a m p l e calibrationplot for BCECF  4 1  Figure 3  Distributiono f steady-state p H j  5 1  Figure 4  Effect o f 0 [Cl"] o n steady-state p H j in the presence o f HC0 " room temperature  Figure 5  Figure 6  Figure 7  Figure 8  0  3  at 5 3  E f f e c t o f D I D S o n s t e a d y - s t a t e p H j i n t h e p r e s e n c e o f HCO3" a t r o o m temperature  5 5  S t e a d y - s t a t e p H j i n t h e p r e s e n c e a n d a b s e n c e o f HCO3VCO2 a t r o o m temperature  5 7  Effect of 0[Na ] 3 7 ° C  5 9  +  0  o n steady-state p H j in the absence o f HC0 " ..; 3  at  E f f e c t o f 0 [ C T ] o n s t e a d y - s t a t e p H j i n t h e a b s e n c e o f HCO3" a t 0  37°C  6 1  Figure 9  E f f e c t o f E I P A o n s t e a d y - s t a t e p H j i n t h e a b s e n c e o f HCO3-at 3 7 ° C  Figure 10  C o m b i n e d effect o f 0 [ N a ] a b s e n c e o f HCO3-at 3 7 ° C  F i g u r e 11  +  Effect of MGCMA o f H C 0 - a t 3 7 ° C  0  a n d EIPA o n steady-state p H ; in the 6 5  a n d H O E 694 o n steady-state p H , in the  absence 6 7  3  Figure 12  ... 6 3  Effect of 0[Na ] +  0  o n s t e a d y - s t a t e p H ; i n t h e p r e s e n c e o f HCO3" a t  37°C  6 9  Figure 13  E f f e c t o f E I P A o n s t e a d y - s t a t e p H j i n t h e p r e s e n c e o f H C O 3 " a t 3 7 ° C ... 7 1  Figure 14  Effect o f 0 [Cl-] ,a n d the c o m b i n e d effect o f 0 [Cl"] plus D I D S o n 0  s t e a d y - s t a t e p H j i n t h e p r e s e n c e o f HCO3" a t 3 7 ° C  0  7 3  Figure 15  E f f e c t o f D I D S o n s t e a d y - s t a t e p H j i n t h e p r e s e n c e o f H C O 3 " a t 3 7 ° C ... 7 5  Figure 16  S t e a d y - s t a t e p H j i n t h e p r e s e n c e a n d a b s e n c e o f H C O 3 V C O 2 a t 3 7 ° C ... 7 7  viii  Page Figure 17  F i g u r e 18  E f f e c t o f HCO3VCO2 o n s t e a d y - s t a t e at r o o m temperature Effect of 0 [Cl"] during 0 [Na ] the presence o f H C 0 - a t 3 7 ° C +  0  0  pHj during 0 [Na ] +  0  perfusion 7 9  perfusion o n steady-state  pHj in 8 1  3  Figure 19  Figure 20  Effect of changes in p H HCO3- a t 3 7 ° C  0  o n steady-state  p H j in the presence  of 8 3  Effect o f propionate a n d T M A o n steady-state  p H j in the presence  of  HCO3" a t r o o m t e m p e r a t u r e  8 5  F i g u r e 21  Sample acid load with NH C1  9 4  Figure 22  Initial rate o f acid load recovery as a function o f pHj, p r e l o a d p H j , m i n i m u m p H j , a n d net p H j decrease p H , recovery f r o m a n acid load in the absence a n d presence o f H C C y at r o o m t e m p e r a t u r e  Figure 23  Figure 24  Figure 25  Figure 26  4  Effect o f D I D S o n p H ; recovery f r o m an acid load in the presence HCO3" a t r o o m t e m p e r a t u r e Effect o f 0 [Cl"] o n p H j recovery f r o m an acid load in the o f HCO3" a t r o o m t e m p e r a t u r e 0  9 7 9 9 of 1 0 1  presence 1 0 3  p H j recovery f r o m an acid load in the absence a n d presence HC0 -at37°C  of  Effect of 0 [Na ] ofHC0 -at37°C  absence  1 0 5  3  Figure 27  +  0  o n p H ; recovery f r o m an acid load in the  1 0 7  3  Figure 28  Effect o f E I P A o n p H ; recovery f r o m an acid load in the absence HC0 -at37°C  of  Effect o f D I D S o n p H j recovery f r o m an acid load in the absence HCO3- a t 3 7 ° C  of  1 0 9  3  Figure 29  Figure 30  Effect of 0 [Na ] ofHC0 -at37°C +  3  0  o n p H j recovery f r o m an acid load in the  1 1 1  presence 1 1 3  IX  Page F i g u r e 31  Effect of EIPA on pHj recovery HC0 -at37°C  from  an acid load in the presence o f 115  3  Figure 32  Effect o f 0 [Cl"] o n p H j recovery f r o m an acid load in the presence ofHC0 -at37°C  117  Effect o f D I D S o n p H j recovery f r o m an acid load in the presence o f HC0 -at37°C  119  Effect o f D I D S o n p H j recovery f r o m an enhanced acid load in the presence o f H C 0 - a t 37°C  121  D i a g r a m m a t i c r e p r e s e n t a t i o n o f p H ; r e c o v e r y from a n a c i d l o a d i n t h e p r e s e n c e a n d a b s e n c e o f H C 0 " , a t r o o m t e m p e r a t u r e a n d a t 3 7 ° C ..  139  Schematic presentation of pHj regulating mechanisms in cultured h i p p o c a m p a l p y r a m i d a l n e u r o n e s at 3 7 ° C a n d r o o m t e m p e r a t u r e  143  0  3  Figure 33  3  Figure 34  3  Figure 35  3  Figure 36  foetal  X  ACKNOWLEDGMENTS  T h e r e are m a n y to w h i c h I w o u l d like to extend m y sincerest appreciation gratitude.  Firstly, I a m  encouragement,  indebted to D r . J o h n Church  a n d insight.  for his  enduring  John, you have been instrumental in m y  and  guidance,  g r o w t h as a  scientist a n d as a n individual. For their consideration a n d time, I thank the other m e m b e r s o f m y  supervisory  committee, D r s . K e n B a i m b r i d g e a n d V l a d i m i r Palaty. I w o u l d especially like to  extend  m y deepest gratitude to D r . Palaty for sharing his breadth o f k n o w l e d g e , e v e n b e y o n d scope of science. Furthermore, Ih u m b l y acknowledge Monika  Grunert and Garth  the  Smith  for their technical expertise a n d experimental assistance. To the Faculty, Graduate Students, a n d Staff in the Department o f A n a t o m y , I thank y o u for creating a n atmosphere that has fostered arelentless pursuit o f I will look fondly u p o n friendships a n d memories of m y time here.  knowledge.  Iwould particularly  like to express m y heartfelt appreciation to m y e s t e e m e d friend a n d colleague,  Sean  V i r a n i , for his persistent yet genuine ability to k e e p m e o n a n e v e n keel. F o r e m o s t , ad e b t o f g r a t i t u d e is o w e d to m y f a m i l y f o r their c o n t i n u e d s u p p o r t a n d inspiration.  M u m a n d D a d , I a m especially grateful to y O u b o t h for y o u r patience  and  unconditional love. F i n a n c i a l support w a s p r o v i d e d b y a n operating grant to D r . J o h n C h u r c h f r o m the Medical Research Council of Canada.  1  INTRODUCTION pH fluctuations in the brain have been shown to accompany many physiological and pathophysiological events (reviewed by Chesler, 1990; Chesler and Kaila, 1992). For example, a transient extracellular alkalinization has been demonstrated to follow electrical stimulation of the CA1 region of the rat hippocampus (Voipio and Kaila, 1993). Jarolimek et al (1989) have noted a similar, albeit enhanced, extracellular pH (pH ) shift 0  induced by the application of neurotransmitters to the CA3 region of guinea pig hippocampal slices. Following electrical stimulation of presynaptic pathways in the rat hippocampus, a long-lasting extracellular alkaline shift has also been observed to accompany excitatory synaptic transmission (Krishtal et al, 1987; Gottfried and Chesler, 1994). In addition to the p H shifts associated with normal neuronal activity, tissue pH 0  changes are also associated with various pathologies (reviewed by Siesjo, 1985). For instance, complete brain ischemia has been shown to result in the accumulation of extracellular protons (Harris et al, 1987), caused primarily by the production of lactic acid due to the anaerobic consumption of glycogen and glucose stores (Siesjo et al, 1990). Furthermore, extracellular acidosis has been recorded in the rat parietal cortex during spreading depression (Mutch and Hansen, 1984), whereas biphasic changes in the acid-base balance of the interstitial fluid have been shown to occur during seizure activity in the rat hippocampus (Somjen, 1984; Jarolimek et al, 1989). Not only has it become increasingly apparent that many normal and abnormal events relating to neuronal activity will cause alterations in the tissue pH but, in turn, tissue pH may alter or modulate many of these physiological or pathological occurrences. The clinic implications of changes in tissue pH were described as early as the 1930's. Lennox et al (1936) remarked that voluntary hyperpnea, an action in which one blows off CO2, can cause seizure-like brain patterns in epilepsy-prone patients.  Conversely, the  inhalation of elevated CO2 concentrations has been described as a means of attenuating  2  s e i z u r e a c t i v i t y ( L e n n o x et al,  1936). R e c e n t e v i d e n c e points towards p H c h a n g e s as  cause for these C C ^ - i n d u c e dalterations in seizure activity. Aram  the  and Lodge (1987)  and  V e l i s e k et al ( 1 9 9 4 ) h a v e d e m o n s t r a t e d t h a t t h e l o w e r i n g o f p H , b y e i t h e r d i r e c t t i t r a t i o n 0  with H C 1 or increasing the partial pressure o f C 0  2  (PCO2),  seizure activity in the rat cortex a n d h i p p o c a m p u s .  will suppress the induction o f  F O r n a i et al  (1994) observed  that  acidic conditions, p r o d u c e d b y the application o f lactate, exert a similar anti-epileptiform action o n rat cortical neurones. ( A r a mand L o d g e , 1987;  In constrast, alkalosis induces epileptiform  C h u r c h and M c L e n n a n , 1989;  J a r o l i m e k et al,  activity 1989).  induction o f m i l d brain acidosis, produced b y hypercarbic ventilation, has also s h o w n t o s e r v e a n e u r o p r o t e c t i v e r o l e d u r i n g f o c a l i s c h e m i a ( S i m o n et al, via inhibition o f the rise in intracellular Ca i s c h e m i a ( E b i n e et al, depression  1 9 9 4 , K r i s t i a n et al,  been  1993), possibly  that n o r m a l l y characterizes the onset  2 +  The  of  1994). In addition, hypoxia-induced spreading  in h i p p o c a m p a l slices can be prevented b y  cerebrospinal fluid, whereas an alkaline p H  0  exposure  will predispose  to acidic  neurones  to  artificial spreading  depression after o x y g e n deprivation ( T o m b a u g h , 1994). W h i l s t m i l d extracellular acidity e x e r t s a p r o t e c t i v e e f f e c t d u r i n g n e u r a l p a t h o l o g i e s ( K a k u et al, (pH  <  0  5.3)  1993), excessive  has been correlated with the death o f brain tissue following  i s c h e m i a ( K r a i g et al,  1987).  S i m i l a r l y , N e d e r g a a r d et al  acidosis complete  (1991) have s h o w n that  p r o l o n g e d e x p o s u r e o f neurones a n d glia to lactic acid or H C 1 ( p H  0  the  < 6.8) p r o d u c e d toxic  effects leading to cell death. T h e s u s c e p t i b i l i t y o f t h e s e p a t h o l o g i e s to c h a n g e s i n t i s s u e p H is r e f l e c t e d i n p H induced alterations in m a n y n o r m a l physiological events. interstitial p H , caused either b y elevated H  the  concentrations, has been s h o w n  to  have a depressant effect o n neuronal excitability in the h i p p o c a m p a l formation ( S o m j e n  et  al,  1987;  et  al  Balestrino and Somjen, 1988;  (1993) have  +  or C 0  F o r instance, a fall in  2  Churchand McLennan, 1989).  Similarly, Taira  s h o w n that s y n a p t i c t r a n s m i s s i o n is s e n s i t i v e to c h a n g e s i n p H ,  whereby an increase or decrease in p H  0  0  will reduce or potentiate excitatory  transmission,  3  respectively.  T h e effect o f tissue p H o n the excitable properties o f cells m a y in  reflect the p H - i n d u c e d m o d u l a t i o n o f various ionic conductances, s u c h as C a  2 +  ,  fact  through  v o l t a g e - a n d l i g a n d - g a t e d i o n c h a n n e l s ( O u - Y a n g et al, 1 9 9 4 ) . R e g a r d l e s s o f t h e m e c h a n i s m s i n v o l v e d , it is c l e a r t h a t m a n y p h y s i o l o g i c a l pathological events are m o d u l a t e d b y fluctuations in the external p H . In m o s t studies, however,  the  influence  of changes  in intracellular p H  vertebrate  (pHj) on  excitability, injury, or mortality has not been thoroughly investigated.  and  neuronal  T h i s is s u r p r i s i n g  since recent evidence indicates that changes in p H j will a c c o m p a n y changes in p H (Preissler and Williams, 1981;  A i c k e n , 1984;  1990;  K a t s u r a et al, 1 9 9 4 ;  O u - y a n g et al, 1 9 9 3 ;  T o l k o v s k y and Richards, 1987;  0  Chesler,  Sun and Vaughan-Jones,  1994).  F u r t h e r m o r e , K a t s u r a et al ( 1 9 9 4 ) c o n c l u d e d t h a t t h e r e g u l a t i o n o f p H j i s d e p e n d e n t  on  pH  m a y have asignificant impact  on  pH; implies a possible role for p H j in s o m e o f the aforementioned events m o d u l a t e d  by  0  in neurones a n d glia. T h e fact that c h a n g e s in p H  0  c h a n g e s in tissue p H . Indeed, as explained below, there appears to b e a n  interdependence  between pHj and many  which,  physiological  and pathological  occurrences,  though  examined in invertebrate neuronal and vertebrate non-neuronal prepartations, has  not  been extensively studied in m a m m a l i a n central neurones.  Physiology, pathophysiology, and pHj: Many  n o r m a l a n d a b n o r m a l cell functions can modulate, or are m o d u l a t e d by, the  intracellular acid-base balance.  F o r instance, p H j changes affect m a n y aspects o f  muscle  dynamics. A n intracellular alkalosis in cardiac Purkinje fibres produces an increase in the m u s c l e t w i t c h t e n s i o n , w h e r e a s a n i n t r a c e l l u l a r a c i d o s i s is a s s o c i a t e d w i t h a fall o f m u s c l e f o r c e g e n e r a t i o n ( V a u g h a n - J o n e s et al, 1 9 8 7 ;  B o u n t r a et al, 1 9 8 8 ) .  K a i l a and Voipio  ( 1 9 9 0 ) h a v e r e p o r t e d that the r e s t i n g t e n s i o n i n c r a y f i s h m u s c l e fibres is i n c r e a s e d b y reduction of pHj, and decreased by an elevation of pHj.  Changes in p H j also  an  affect  vascular tone. R a i s i n g p H j increases the tension o f rat vascular s m o o t h m u s c l e fibres in a  4  f a s h i o n that is i n d e p e n d e n t o f p H  0  (Austin and Wray, 1993).  Studies o n the rat portal  v e i n reveal similar results, a n d m a y p r o v i d e a possible explanation for the  observed  d e c r e a s e i n c o n t r a c t i l e a c t i v i t y d u r i n g p a t h o l o g i c a l s i t u a t i o n s s u c h a s h y p o x i a ( T a g g a r t et al, 1 9 9 4 ) . C e l l u l a r e n z y m a t i c a c t i v i t y a n d m e t a b o l i s m is also c l o s e l y t i e d to acidity (Busa, 1986).  intracellular  Cells e x p o s e d to perturbations in p H , m a y experience shifts in the  normal operation of intracellular enzymes  whose  activity kinetics are p H  dependent  ( B u s a , 1986). A c t i v e sites o n e n z y m e s m a y contain ionizable g r o u p s w h i c h are i n v o l v e d in the binding o f substrates  and cofactors (Roos and Boron, 1981).  Fluctuations  intracellular p r o t o n levels will affect the ionization o f these groups, thus  in  influencing  e n z y m e c o n f o r m a t i o n a l states a n d the ability to f o r m enzyme-substrate c o m p l e x e s  (Roos  a n d B o r o n , 1981). T h e arrangement o f cytoskeletal proteins can also be m o d u l a t e d b y the internal p H . 1986).  T h e p o l y m e r i z a t i o n o f tubulin, for example, increases as p H ; rises  (Busa,  F u r t h e r m o r e , the b u n d l i n g a n d c r o s s - l i n k i n g o f m i c r o f i l a m e n t s is sensitive  intracellular a c i d shifts.  to  I n Distyostelium a m o e b a e , a n i n t r a c e l l u l a r a c i d i f i c a t i o n i n h i b i t s  the a r r a n g e m e n t o f microfilaments, as o p p o s e d to a n alkalinization w h i c h  promotes  filamentous organization (Busa, 1986). pHj-dependent variations in the synthesis o f these cytoskeletal c o m p o n e n t s m a y in fact be aconsequence o f p H j - d e p e n d e n t fluctuations the activities o f e n z y m e s associated with these  in  elements.  pHj has also b e e n s h o w n to p l a y a m o d u l a t o r y role in cellular proliferation a n d development.  H e s k e t h et al ( 1 9 8 5 ) , f o r e x a m p l e , h a v e i d e n t i f i e d p H j p e r t u r b a t i o n s  m o u s e thymocytes a n d Swiss 3 T 3 fibroblasts stimulated b y the application o f  in  mitogens,  demonstrating apossible relationship between p H ; a n d the regulation o f cell division.  It  has also been observed that ap H j increase a c c o m p a n i e s fertilization o f frog, axolotl, a n d sea u r c h i n o v a (reviewed b y R o o s a n d B o r o n , 1981). Fertilization-induced p H j rises been  attributed to  the  activation  of a Na /H +  +  exchanger,  which  acts  to  have  extrude  intracellular protons (Roos and B o r o n , 1981). S u c h changes in p H j m a y in turn modulate  5  the cascade o f biosynthetic pathways involved in early embryonic development metabolism,  stimulus-response  Nuccitelli, In  coupling,  D N A  replication, and  mitosis  including (Busa  and  1984). addition  to  the  previously  mentioned  association  of  p H  0  with  neural  p a t h o l o g i e s , it a l s o a p p e a r s t h a t p H j m a y p l a y a n i m p o r t a n t r o l e i n e v e n t s s u c h a s b r a i n i s c h e m i a a n d s e i z u r e s ( S i e s j o , 1 9 8 5 ) . D u r i n g c e r e b r a l i s c h e m i a , t h e r e is a m a r k e d d e c l i n e i n p H j , w h i c h is p r e d o m i n a n t l y c a u s e d b y t h e p r o d u c t i o n o f l a c t i c a c i d d u r i n g a n a e r o b i c glycolysis (Siesjo, 1985). ATP  A s cerebral energy states deteriorate d u r i n g a n h y p o x i c  insult,  hydrolysis also contributes to the i s c h e m i c - i n d u c e d intracellular acidification, w h i c h  p r o c e e d s a c c o r d i n g to the f o l l o w i n g reaction: ATP  +  H  2  0  ->  A D P  +  Pi  +  ntt  (Equation 1)  +  w h e r e n h a s b e e n a p p r o x i m a t e d at 0.7 ( W i l k i e , 1 9 7 9 ) .  T h e overall reduction of p H ,  d u r i n g i s c h e m i a m a y then act to protect the cell f r o m excessive d a m a g e .  Indeed,  intracellular acid shifts are believed to reduce m e m b r a n e excitability a n d inhibit cellular metabolism during ischemia ( T o m b a u g h and Sapolsky, 1993).  Nevertheless, although  n e u r o p r o t e c t i v e f u n c t i o n is a s s o c i a t e d w i t h m i l d a c i d o s i s , e x c e s s i v e a c c u m u l a t i o n s  a of  intracellular equivalents h a v e b e e n s h o w n to i n d u c e b o t h n e u r o n a l a n d glial death in cells c u l t u r e d f r o m r a t f o r e b r a i n s ( N e d e r g a a r d et al,  1991). Glutamate neurotoxicity m a y  also,  at least i n part, i n v o l v e the d e t r i m e n t a l effects o f a b n o r m a l l y h i g h intracellular p r o t o n concentrations induced by glutamate receptor activation (Hartley and Dubinsky, In addition, a m i l d intracellular acidosis has b e e n o b s e r v e d to a c c o m p a n y e p i l e p t i f o r m a c t i v i t y ( S i e s j o et al,  1993).  neuronal  1 9 8 5 ) . T h i s a c i d i f i c a t i o n is t h o u g h t to o c c u r as a result  o f increased intracellular lactic acid production, w h o s e effects are delayed or relieved  by  Na /H  to  +  +  e x c h a n g e ( S i e s j o et  al,  1985).  Fluctuations in p H , have also been noted  proceed m a n y of these pathological occurences. demonstrated  F o r e x a m p l e , M a b e et al  that p H ; rises to alkaline levels in rat cortical n e u r o n e s  f o l l o w i n g i s c h e m i c insult.  (1983)  have  immediately  T h e reason for this post-ischemic alkalosis remains  unclear,  6  but may be a result of the degradation of accumulated intracellular lactic acid, or the resumed production of ATP (Mabe et al, 1983).  pHj and cell excitability: Electrical activity, including the depolarization of cell membranes, can produce intracellular pH shifts in neurones (Chesler, 1990).  Meech and Thomas (1987) have  reported that a Ca -sensitive reduction in pHj follows the depolarization of molluscan 2+  nerve cells. It has also been shown that the application of a depolarizing agent (i.e. high extracellular K ) onto cultured bovine chromaffin cells produces an intracellular +  acidification that can be reduced by lowering extracellular C a  2 +  levels (Rosario et al,  1991). Moreover, trains of action potentials evoked in molluscan nerve cell bodies have been demonstrated to cause a decrease in pHj (Ahmed and Connor, 1980), and the degree of intracellular acidification appears to depend on the frequency of the action potentials (Bountra et al, 1988).  Activity-dependent changes in pHj, in addition to reflecting  changes in intracellular C a  2 +  (see below), may also be caused by the release of acidic  metabolic products (Siesjo, 1985) or the entry of acid equivalents through membrane potential sensitive transport mechanisms (Fitz  et al, 1992).  Interestingly,  in vivo  stimulation of cortical astrocytes produces an cytoplasmic alkalinization (Chesler and Kraig, 1987). This observation suggests that the intra-neuronal acidification caused by electrical stimulation may occur as a result of proton transfer between neurones and their supporting structures. It has recently become apparent that the application of neuromodulators and neurotransmitters, including hormones and excitatory amino acids, can alter pH; (reviewed by Chesler, 1990). Barber et al (1989), for instance, have shown that pHj in cultured canine enteric endocrine cells can be altered by the application of epinephrine and somatostatin in a manner independent of their established effects on cAMP production.  Epinephrine, acting on the P2-adrenergic receptor, activates a N a / H +  +  7  exchanger which leads to an intracellular alkalinization, whereas somatostatin inhibits this exchanger producing a cytosolic acidification (Barber et al, 1989). The activation of other cell surface receptors has also been shown to modify N a / H exchange independent +  +  of the concomitant changes in cAMP. Stimulation of prostaglandin E l and parathyroid hormone receptors on a variety of non-neuronal preparations results in a pHj rise by enhancing N a / H +  exchange (Ganz et al, 1990).  +  In contrast, the activation of D 2  dopaminergic receptors act to reduce pHj via an inhibition of this exchanger (Ganz et al, 1990).  Though all of the above neuromodulators or neurotransmitters alter pH; in a  manner independent of any associated fluctuations in intracellular cyclic nucleotide levels, Conner and Hockberger (1984) have shown that the injection of cyclic A M P or cyclic G M P into gastropod neurones will also induce cytoplasmic p H changes.  Two  other cell surface receptors that, when activated, alkalinize the interior of NG108-15 cells by accelerating N a / H exchange are muscarinic cholinergic and 5-opiate receptors (Isom +  +  et al, 1987). Moreover, Ludt et al (1991) have indicated that protein kinase C, which is linked to muscarinic receptor activation, is involved in pHj modulation of primate renal cells through the regulation of a HCO3VCI" exchanger. Other hormones and growth factors have been shown to produce fluctuations in pHj.  Arginine vasopressin (AVP) raises steady-state pHj in renal mesangial cells in the  absence of extracellular HCO3", whereas A V P reduces pH; in the presence of extracellular H C 0 " (Ganz et al, 1989). 3  Ganz et al (1989) speculated that A V P  stimulates both N a - and HC0 "-dependent exchangers, which, depending on the +  3  composition of the interstitial fluid, will act to increase or decrease pHj. Indeed, the exposure of rat mesangial cells to A V P , epidermal growth factor, or serotonin has recently been shown to cause increases in the activities of various acid extusion mechanisms, including the N a / H , Na -independent H C O 3 7 O " , and Na -dependent +  HCO3VCI"  +  +  +  exchangers (Ganz and Boron, 1994). The application of a combination of  mitogens (platelet-derived growth factor, vasopressin, and insulin) has been reported to  8  cause an increase in pHj by stimulating the N a / H exchanger present on mouse 3T3 cells +  +  (Schuldiner and Rozengurt, 1982). Other mitogenic activators, such as epidermal growth factor and serum growth factor, have been demonstrated to alter N a / H +  +  exchange in  human diploid fibroblasts (Moolenaar et al, 1982). The tumour promoter, okadaic acid, which inhibits protein phosphatase activity, is also believed to stimulate fibroblast Na /H +  +  exchange which leads to an intracellular alkalinization (Sardet et al, 1991).  Interestingly, studies on mouse thymocytes and Swiss 3T3 fibroblasts have indicated that intracellular C a  2 +  ([Ca ]j) may serve as an intermediate to growth factor-induced 2+  changes in pHj (Hesketh et al, 1985). Extracellular changes in pH have been shown to modulate ionic conductances through channels activated by excitatory amino acids. Traynelis and Cull-Candy (1991), for example, have reported that conductances through /V-methyl-D-aspartate (NMDA), aamino-3-hydroxy-5-methyl-4-isoxazole propionate  (AMPA),  and  kainate  receptor  channels can be inhibited by accumulations of extracellular protons. Furthermore, the blockade of the N M D A receptor by interstitial protons occurs well within the physiological pH range (Vyklicky et al, 1990; Giffard et al, 1990; Tang et al, 1990; Gottfried and Chesler, 1994).  The application of excitatory amino acids has more  recently been linked to alterations in pHj. N M D A , quisqualate, and kainate have been shown  to  produce  concentration-dependent  intracellular  acidifications  in  frog  motoneurones (Endres et al, 1986). Irwin et al (1994) have proposed that an influx of Ca  2 +  may be required for the internal acidosis of foetal rat hippocampal neurones induced  by the activation of N M D A receptors. Moreover, increasing p H does not significantly 0  alter the degree of intracellular acidification caused by N M D A , suggesting that the agonist-induced fall in pHj is not a consequence of transmembrane proton fluxes (Irwin et al, 1994). It appears that the activation of metabotropic receptors may also contribute to the intracellular acidosis resulting from the application of glutamate possibly by modulating the activity of a Na /HC03" cotransporter (Amos and Richards, 1994). +  9  Neuronal  excitability  associated  with  L-glutamate  receptor  activation  i s therefore  dependent not only o n the p H o f the extracellular environment but also o n the p H o f the intracellular m i l i e u because changes in p H j are k n o w n to m o d u l a t e a w i d e variety o f ionic c o n d u c t a n c e s (see The  below).  mechanism  by which y-aminobutyric acid (GABA)  a m o n g neurotransmitters.  alters p H j i s u n i q u e  E x a m i n e d o n crayfish skeletal muscle,  GABA  induces  intracellular acidification b y activating a H C C ^ 'c o n d u c t a n c e ( K a i l a a n d V o i p i o ,  an  1987).  T h e a p p l i c a t i o n o f G A B A , a c c o r d i n g t o K a i l a et al ( 1 9 9 0 ) , l e a d s t o a n i n f l u x o f CO2, w h i c h is h y d r a t e d into c a r b o n i c a c i d t h r o u g h a c a t a l y z e d r e a c t i o n i n v o l v i n g anhydrase.  T h e dissociation  o f carbonic acid into bicarbonate liberates  carbonic  intracellular  p r o t o n s , a n d i n c o n c e r t w i t h a n i n c r e a s e i n m e m b r a n e p e r m e a b i l i t y t o HCO3", a d e c l i n e i n p H j i s p r o d u c e d ( K a i l a et al, 1 9 9 0 ) .  Further investigation of G A B A - i n d u c e d changes  in  pHj using crayfish stretch-receptor neurones p r o d u c e d findings supporting the notion that t h e i n t r a c e l l u l a r a c i d i f i c a t i o n i s m e d i a t e d b y a n e t e f f l u x o f HCO3" t h r o u g h c h a n n e l s ( V o i p i o et al, 1 9 9 1 ) .  GABA-gated  Therefore, the reduction o f p H j p r o d u c e d b y G A B A  unlike the actions o f other h o r m o n e s  a n d t r a n s m i t t e r s i n t h a t it d i r e c t l y i n v o l v e s  m o v e m e n t o f acid equivalents across the p l a s m a  is the  membrane.  pHj and ionic conductances: The  evidence  outlined above demonstrates  that n e u r o n a l a c t i v i t y is  associated  w i t h c h a n g e s i n p H j . I n t u r n , it is k n o w n t h a t p H j is a b l e t o i n f l u e n c e c e l l e x c i t a b i l i t y  by  modulating a wide variety of ionic conductances (reviewed by M o o d y , 1984).  Injecting  l o w p H solutions into isolated ventricular myocytes shortens the duration a n d  amplitude  o f e v o k e d action potentials, whereas the intracellular application o f h i g h p H solutions the  opposite  changes  effect (Kurachi, 1982).  in cell excitability  have  been  T h e specific examined.  currents underlying For example,  voltage  has  pHj-induced gated  conductances in h u m a n lymphocytes are enhanced b y an elevation in p H j (Deutsch  K  +  and  10  Lee, 1989). Conversely, an intracellular acidification has been associated with a of inward rectifying K crayfish  slow  conductances,  currents in starfish oocytes ( M o o d y a n d H a g i w a r a , 1982).  +  muscle  blockade  fibres,  low  pHj mediates  the  amplification  In  of inward  Ca  w h i c h is b e l i e v e d to o c c u r as a result o f p r o t o n - i n d u c e d i n h i b i t i o n o f  overlapping outward K  +  currents ( M o o d y , 1980).  T h e delayed rectifier K  +  2 +  the  conductance  can also be modified by an accumulation of intracellular protons in squid axons  (Wanke  et al,  through  Ca  2 +  1979).  Furthermore, an intracellular acidification inhibits conductances  -activated K  +  c h a n n e l s i n p a n c r e a t i c B - c e l l s ( C o o k et al,  1 9 8 4 ) w h i c h L a u r i d o et  al  (1991), in studying rat skeletal muscle, attributed to a p r o t o n - i n d u c e d w e a k e n i n g o f b i n d i n g to c o n f o r m a t i o n a l sites o n the channel. pHj can also reduce the likelihood o f finding Ca Indeed,  suppression  of Ca  2 +  -activated K  +  K u m e et al 2 +  -activated K  conductances  Ca  (1989) report that a fall in channels i n a n o p e n state.  +  by intracellular acidosis  have  b e e n o b s e r v e d in type I cells o f the rat carotid b o d y (Peers a n d G r e e n , 1991), as well C A 1 p y r a m i d a l neurones o f the rat h i p p o c a m p u s ( C h u r c h , In addition to K  fluctuations in pHj. Na -dependent Ca +  2 +  in Ca  acidification;  2 +  channel  conversely,  permeability  can been modulated  1978).  U m b a c h ( 1 9 8 2 ) later  i n Paramecium c a u s e d  by  an intracellular alkalinization increased Ca  High voltage activated ( H V A ) Ca  2 +  by  i n f l u x w a s first s h o w n to b e i n h i b i t e d b y a fall  in pHj in squid axons (Baker and Honerjager, decreases  an  observed  intracellular conductances.  2 +  currents a p p e a r to b e particularly sensitive to  pHj  s h i f t s ( K a i b a r a a n d K a m e y a m a , 1 9 8 8 ) . T a k a h a s h i et al ( 1 9 9 3 ) h a v e d e m o n s t r a t e d t h a t intracellular acidification will suppress while an intracellular alkalinization will H V A C a the  2  +  c o n d u c t a n c e s i n catfish retinal cells.  conduction  kinetics  of H V A Ca  o c c u p y i n g a c h a n n e l r e g u l a t o r y site.  2 +  channels  are influenced  Inhibition of H V A Ca  2 +  by  Z e i l h o f e r et al  protons  ( 1 9 9 3 ) attributed this inhibition to glutamate  that  directly  currents has also 1993;  an  enhance  P r o d ' h o m et al ( 1 9 8 7 ) h a v e r e p o r t e d  a s s o c i a t e d w i t h t h e a c t i v a t i o n o f g l u t a m a t e r e c e p t o r s ( Z e i l h o f e r et al, 1993).  as  1992)  currents, other ionic conductances  +  2 +  D i x o n et  been al,  receptor-mediated  11  Ca  influx and subsequent  2 +  Ca  -dependent inactivation of Ca  2 +  D i x o n et al ( 1 9 9 3 ) s u g g e s t e d t h a t a g l u t a m a t e - i n d u c e d C a responsible for the modulation o f H V A C a  whereas  - d e p e n d e n t c h a n g e i n p H , is  currents.  2 +  Variations in voltage-dependent N a  2 +  channels,  2 +  conductances caused by changes in p H ; have  +  b e e n s t u d i e d i n f r o g s k e l e t a l m u s c l e ( N o n n e r et al,  1980;  W a n k e et al,  1980) and squid  g i a n t a x o n s ( C a r b o n e et al,  1981). Interestingly, results regarding the influence o f p H j o n  Na  quite  +  currents have  conductances  been  variable.  through the enhanced  In squid axons,  inactivation of N a  reduces this inactivation, thereby increasing N a  +  W a n k e et al,  1980).  +  p H ; depresses  channels, whereas  +  conductances  Opposite results were achieved in studies o f frog muscle: channel inactivation, thus enhancing N a  low  a  ( N o n n e r et  +  a  contrast to m o s t other ionic currents, the inhibition of Na  Na /Ca +  2 +  heart  eliminates al,  1980;  +  Cells, a n  exchanger,  +  neurones after stimulus-evoked C a  2 +  recovery  of [Ca  activity  the  opposite  2 +  ]j in  entry, w h i c h m a y require Na /Ca +  2 +  voltage are  also  Measured in  the  an intracellular alkalinization has M o r e o v e r , the  (one  observed  exchange  2 +  intracellular acidification suppresses  (Doering and Lederer, 1993).  channel  c o n d u c t a n c e s b y p H j is  Ionic currents associated with Na /Ca  whereas  two  1980). Furthermore, in  susceptible to changes in the c y t o p l a s m i c p H ( D o e r i n g a n d L e d e r e r , 1993). guinea-pig  1981).  o f 5.6), w h i c h m a y e x p l a i n the  c o n d u c t a n c e s e n s i t i v i t i e s t o p H , ( W a n k e et al,  dependent ( M o o d y , 1984).  +  +  pHj  A n analysis o f conductance kinetics reveals the possibility that  o f 4.6, a n d the other w i t h a p K  differences in N a  al,  lowering p H j nearly  ( m e m b r a n e potential sensitive) p r o t o n b i n d i n g affinities exist for the N a with a p K  high  ( C a r b o n e et  channel conductances  N a  of  the effect  hippocampal exchange,  is  retarded b y an intracellular acidification ( K o c h and Barish, 1994). The modulation of C h conductances by p H has not been extensively B a r n e s a n d B u i (1991) have noted that the sensitivity o f Ca a m p h i b i a n c o n e photoreceptors to alterations in p H i n d u c e d shift in C a  2 +  channel gating.  0  2 +  documented.  -activated C l " currents  is p o s s i b l y a c o n s e q u e n c e  in  of p H -  C h a n g e s in p H j , o n the other hand, have  been  12  observed to significantly affect basolateral Cl" conductances in colonic epithelial cells (Chang et al, 1991). These results, along with the others outlined above, indicate a common link between cell excitability and pHj in many preparations. Not only is the intracellular proton environment shifted by the electrical behavior of the cell, but pHj has the ability to modulate many ionic conductances that underlie basic membrane excitability. Finally, gap junctional conductances are also susceptible to changes in pHj (Spray and Bennett, 1985). In fact, it is suggested that intracellular protons may modulate these conductances more effectively than other intracellular ions, including C a 1982;  Moody, 1984).  2 +  (Spray et al,  A decrease in Lucifer yellow dye-coupling in guinea pig  hippocampal slices has been associated with a fall in pHj (MacVicar and Jahnsen, 1985). Conversely, Church and Baimbridge (1991) have shown that there is an increased incidence of dye-coupling caused by the exposure of rat hippocampal pyramidal neurones to high pH extracellular medium, presumably related to the fact that raising p H will 0  result in an increase in pHj (see Discussion). pHj transients in amphibian embryos have been similarly correlated with coupling changes in a manner that is independent of the extracellular proton milieu (Spray and Bennett, 1985; Busa, 1986).  p H j and C a  2 +  :  A complex interdependence appears to exist between pHj and intracellular free Ca . 2 +  For example, pHj can be modulated by fluctuations in the intracellular  concentration of C a  2 +  (Ahmed and Connor, 1980).  intracellular injection of C a amount of C a  2 +  2 +  Observed in snail neurones, the  causes an immediate fall in pHj that is proportional to the  injected (Meech and Thomas, 1977). Busa and Nuccitelli (1984) have  postulated that Ca -dependent alterations in pHj may involve the exchange of C a 2+  2 +  for  protons by various intracellular organelles, such as the mitochondria or smooth endoplasmic reticulum. Furthermore, slow C a  2 +  iontophoresis has been observed to  13  produce a decrease in p H j without affecting the m e m b r a n e potential, w h i c h possible  avoids  secondary effects o n p H j caused b y changes in ion distribution across  m e m b r a n e ( M e e c h a n d T h o m a s , 1977). T h e exposure o f avian neural crest cells to  the Ca  2 +  free m e d i a i n d u c e s a c y t o p l a s m i c a c i d i f i c a t i o n , w h i c h is b e l i e v e d to o c c u r as a result the subsequent fall in [Ca  2 +  ] j ( D i c k e n s et al,  recently reported that a rise in [ C a the stimulation o f Na /H +  1990).  s h o w i n g that the application o f C a  T h i s c o n c l u s i o n is s u p p o r t e d b y  leads to a n increase in [ C a reduction in pHj.  2 +  1994).  1991).  2 +  ( D i c k e n s et  T h e exposure o f barnacle m u s c l e cells to  CO2  2  +  from  2 +  - b i n d i n g proteins present in the sarcoplasm  muscle  to the release o f C a  2 +  f r o m intracellular  (Lea  sources.  p H j has also b e e n s h o w n to m o d u l a t e the intracellular levels o f  divalent cations.  al,  S i s k i n d et al ( 1 9 8 9 ) a l s o c r e d i t t h e l o w p H j - i n d u c e d r i s e i n i n t e r n a l  in vascular smooth  Interestingly,  +  ]j, w h i c h L e a a n d A s h l e y (1978) attribute to a C C ^ - i n d u c e d  intracellular organelles and other C a  Ca  2  A n e l e v a t i o n i n c y t o s o l i c p r o t o n s is t h o u g h t to d i s p l a c e C a  and Ashley, 1978).  data +  p H j has in t u r n b e e n s h o w n to m o d u l a t e intracellular free C a M a r t i n e z - Z a g u i l a n et al,  via  i o n o p h o r e s o n rat b r a i n s y n a p t o s o m e s induces N a -  2 +  d e p e n d e n t i n c r e a s e s i n p H j ( S a n c h e z - A r m a s s et al,  1990;  of  have  ] , can increase the efflux o f intracellular protons  2 +  e x c h a n g e (see below).  +  S a n c h e z - A r m a s s et al ( 1 9 9 4 )  -  other  In cultured c h i c k e n heart cells, for e x a m p l e , c h a n g e s in cytosolic  h a v e b e e n i n v e r s e l y c o r r e l a t e d w i t h i n d u c e d s h i f t s i n p H j ( F r e u d e n r i c h et al, In studying the relationship b e t w e e n p H , a n d internal free Ca  2 +  Mg  2 +  1992).  using  fluorescent  i n d i c a t o r s i n a v a r i e t y o f c e l l t y p e s , G a n z et al ( 1 9 9 0 ) r a i s e d t h r e e p o s s i b i l i t i e s f o r p H j dependent changes in [Ca shift m a y alter C a  2  +  2 +  ]j.  In agreement with previously mentioned theories, a p H j  fluxes across various intracellular membranes.  H o w e v e r , G a n z et  (1990) caution that artifacts m a y be responsible for perceived c h a n g e s in cytosolic levels b e c a u s e perturbations i n p H j m a y alter: fluorescent membranes.  indicator, and  2)  the  1)  the association o f Ca  interaction o f the  Ca  2 +  2 +  indicator with  U n d e r both of these conditions, an apparent change in [Ca  2 +  with  al Ca  2 +  its  internal  ]j would  be  14  r e c o r d e d , w h e n , in fact, n o n e actually o c c u r r e d .  T h e r e f o r e , it is n e c e s s a r y t o t a k e  factors into account w h e n evaluating the significance o f cytosolic Ca  changes  2 +  these caused  b y altering p H j .  Distribution of protons across the limiting membrane:  M e a s u r e m e n t s o f p H j in avariety o f cell types have determined that protons  are  not passively distributed across the p l a s m a m e m b r a n e (reviewed b y R o o s a n d B o r o n , 1981).  T h i s w a s , i n fact, first o b s e r v e d i n m u s c l e b y F e n n a n d C o b b i n the  mid-1930's.  W i t h a n e x t r a c e l l u l a r p H o f 7.0, F e n n a n d C o b b ( 1 9 3 4 ) m e a s u r e d p H j to b e 7.0, w h i c h is m u c h higher than the predicted value o f 5.62 based o n aD o n n a n e q u i l i b r i u m w i t h K T h e D o n n a n r u l e states that, at e q u i l i b r i u m , the ratios o f all d i f f u s a b l e i o n o n either side o f apermeable m e m b r a n e will be equal. inside cells in c o m p a r i s o n to the outside, membrane  With ahigh K  +  concentration of  w o u l d be higher o n the outside  c o m p a r i s o n to the inside. If this rule h e l d true, t h e n p H j w o u l d rest at 5.6 i f p H neutrality.  .  concentrations  one w o u l d expect, based o n this l a w  equilibria, that proton concentrations  +  0  was  in near  Further calculations on frog muscle by F e n n and M a u r e r (1935) yielded a  value o f 6.9 for p H j w h i l e p H  0  rested at 7.34, b u t e v e n this d i f f e r e n c e w a s n o t  e n o u g h to be explained s i m p l y b y equilibrium across the m e m b r a n e .  This  large  observation  w a s c o n c u r r e d w i t h b y H i l l ( 1 9 5 5 ) w h o , i n studies o n f r o g m u s c l e , c o n c l u d e d that "...the D o n n a n equilibrium does not control, a n d does not greatly influence, the distribution o f h y d r o g e n ions across the fibre m e m b r a n e . " C h e s l e r ( 1 9 9 0 ) outlines, f r o m am e m b r a n e potential perspective, the u n l i k e l i h o o d of having apassive H moved  across  +  distribution across the p l a s m a m e m b r a n e .  If protons  the m e m b r a n e , then the distribution o f intracellular a n d  passively  extracellular  p r o t o n s w o u l d b e g o v e r n e d b y t h e r e s t i n g m e m b r a n e p o t e n t i a l (E ) a s r e p r e s e n t e d b y m  Nernst equation:  the  15  E  m  where E  H+  =  E  H  +  =  —  I n J^Tj  (Equation 2)  is t h e e q u i l i b r i u m p o t e n t i a l f o r H  c o n s t a n t , a n d T is the t e m p e r a t u r e .  +  , F is F a r a d a y ' s c o n s t a n t , R is the i d e a l  G i v e n a p p r o x i m a t e v a l u e s o f 7.4 for p H  gas  a n d 7.0  for  pHj (see Chesler, 1990, for a s u m m a r y o f resting p H j levels i n a variety o f cells),  the  above equation yields an equilibrium potential for the H D u e to the difference b e t w e e n E  H+  mV  a n d E,  +  i o n (E ) H+  0  o f -23.6 m V at 2 5 ° C .  w h i c h n o r m a l l y rests b e t w e e n -50 a n d  m  i n e x c i t a b l e cells, a n i n w a r d p r o t o n g r a d i e n t is e s t a b l i s h e d .  s o m e cellular m e t a b o l i c p r o c e s s e s p r o d u c e a c i d equivalents (e.g.  This, a n d the fact glucose — » 2 lactate  2 H ) , forces cells to continually extrude acid in order to m a i n t a i n p H j near +  (Thomas,  1984).  Whether  a conclusion  is b a s e d  on  calculations  o f the  e q u i l i b r i u m f u n c t i o n o r t h e N e r n s t e q u a t i o n , it is c l e a r t h a t p r o t o n s a r e n o t distributed across the limiting m e m b r a n e .  -60 that +  -  neutrality Donnan passively  T h e regulation of p H j by acid extrusion and/or  a c i d b u f f e r i n g is t h e r e f o r e a c o m m o n p r o p e r t y o f m o s t cell t y p e s .  Regulation of  pHj:  A s a n electrochemical gradient favours the influx o f protons f r o m the space into the cytoplasm, cells m u s t continually extrude acid equivalents maintain a constant resting pHj.  These extrusion mechanisms  interstitial in order  also participate in  to the  restoration o f p H j b a c k to n o r m a l physiological levels after cells h a v e b e e n b u r d e n e d w i t h an induced acid load.  A c c o r d i n g l y , in addition to m o n i t o r i n g steady-state  conditions,  m a n y studies o n p H j regulation have investigated acid extrusion m e c h a n i s m s through the analysis of p H j recovery f r o m an imposed acidification. T h e idea of trans-membrane fluxes of H  +  o r HCO3" w a s i n i t i a l l y s u g g e s t e d  by  M e s s e t e r a n d Siesjo (1971) in their study o f rat brain tissue. T h e s e authors noted that the e x t r u s i o n o f a c i d e q u i v a l e n t s is at least p a r t i a l l y r e s p o n s i b l e f o r t h e r e c o v e r y f r o m a C 0  2  -  16  induced intracellular acidification. (1975)  Intracellular proton loading was also used b y  i ns t u d y i n g p H j r e g u l a t i o n i n r a t d i a p h r a g m m u s c l e .  Roos  Roos  reasoned  that  intracellular buffering (discussed below) does not sufficiently explain w h y p H j i s only moderately affected b y considerable intracellular proton loads.  Proton extrusion  therefore play a substantial role in maintaining a constant intracellular acid-base (Roos, 1975).  must  balance  R e c o v e r y f r o m an intracellular acidification, induced b y the addition a n d  subsequent removal of extracellular NH C1, has produced additional evidence supporting 4  the presence o f acid extruding m e c h a n i s m s o n various invertebrate neurones, the squid giant a x o n ( B o r o n a n d D e W e e r ,  including  1976).  T h e study o f intracellular proton extrusion has concentrated o n the  identification  o f transporting m e c h a n i s m s that e x c h a n g e intracellular or extracellular ions for p r o t o n equivalents.  M u r e r et al ( 1 9 7 6 ) f i r s t i d e n t i f i e d a N a / H +  +  antiport system present o n  the  m e m b r a n e s o f rat intestinal a n d renal cells w h i c h w a s thought to be i n v o l v e d in acid extrusion. to  T h i s g r o u p observed that the antiporter operates in a non-electrogenic  exchange  intracellular H  +  ions for extracellular N a  F u r t h e r m o r e , J o h n s o n et al ( 1 9 7 6 ) f o u n d t h a t t h e susceptible  t o inhibition  b y t h ediuretic  +  ions  ( M u r e r et al,  activity o f N a / H +  drug  amiloride  chloropyrazinoyl)guanidine). T h e ability o f this d r u g to b l o c k N a / H +  T s e et al, 1 9 9 3 ;  M r k i c e r a / , 1993;  1976).  exchange i s  +  (l-(3,5-diamino-6+  exchange on  cell t y p e s , s u c h as r e n a l o r intestinal epithelial cells,, h a s b e e n e x t e n s i v e l y (e.g. S a r d e t e ^ a / , 1989;  fashion  Rowee/a/,  other  documented  1994).  A second acid transporter has been described w h i c h involves the inward flux HCO3" i n e x c h a n g e f o r a n i n t r a c e l l u l a r a n i o n ( T h o m a s , 1 9 7 6 a ) .  of  T h o m a s speculated  that  Cl" w a s in fact the anion in question, a possibility that w a s c o n f i r m e d b y Russell  and  B o r o n (1976) in their investigation o f squid axons.  B o t h groups demonstrated that this  anion exchanger, which operates in a Na -independent fashion, could be blocked +  stilbene derivatives s u c h as S I T S  by  (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic  acid) and D I D S (4,4'-diisothiocyanatostilbene-2,2'-disulphonic  acid).  The  HCO3VCI"  17  exchanger acts t o reduce intracellular proton accumulations b y presenting t h e cytosol w i t h HCO3-, w h i c h e n t e r s t h e c e l l i n e x c h a n g e f o r C l " . H C 0 " i o n s s e q u e s t e r p r o t o n s t o 3  f o r m H2CO3 w h i c h t h e n , u t i l i z i n g t h e e n z y m e c a r b o n i c a n h y d r a s e , d i s s o c i a t e s i n t o and H 0 .  Both C 0  2  and H  2  2  0  T h e net result o fthis m e c h a n i s m i s the  3  C l " ,C 0  2  2  freely diffuse o u to f t h e cell, a n d then c o m b i n e t o  r e g e n e r a t e e x t r a c e l l u l a r HC0 " l e v e l s . of intracellular H  C 0  reduction  i o n c o n c e n t r a t i o n s v i a t h e i n f l u x o f HC0 " a n d s u b s e q u e n t e f f l u x o f  +  3  and H 0 . 2  In their examination o fm o u s e soleus m u s c l e fibres, A i c k e n a n d T h o m a s  (1977)  r e v e a l e d t h ep o s s i b l e p r e s e n c e o f m u l t i p l e p H j r e g u l a t i n g s y s t e m s i n a s i n g l e c e l l T h e s e authors c o n c l u d e d that both Na /H +  transmembrane N a  +  type.  e x c h a n g e , w h i c h i s p r i m a r i l y d r i v e n b yt h e  +  gradient, a n d HC0 7C1" exchange independently maintain  constant  3  r e s t i n g l e v e l s o f p H j ( A i c k e n a n d T h o m a s , 1 9 7 7 ) . A sd e m o n s t r a t e d b y T h o m a s ( 1 9 7 7 ) o n snail neurones, these t w o m e c h a n i s m s m a y also operate i nconjunction w i t h one This particular regulating system,  classified  relies o n t h eavailability o f extracellular N a  +  HC0 7C1"  as Na -dependent +  3  +  separate  regulators o fpHj: N a / H +  +  HC0 7C1"  +  exchanger is susceptible t o inhibition with S I T S o rD I D S (Thomas, 1977). 1980's, three  electroneutral  exchange,  a s a requisite for t h ecounter-transport o f  HCO3- a n d C l " . S i m i l a r t o t h e N a - i n d e p e n d e n t s u b t y p e , t h e N a - d e p e n d e n t  early  another.  mechanisms  were  targeted  3  Thus, b y the a s significant  e x c h a n g e , N a - i n d e p e n d e n t HCO37O" e x c h a n g e , a n d N a -  d e p e n d e n t HCO37G" e x c h a n g e  +  +  (Thomas, 1984).  A l l o fthese transporters have  been  shown, for example, t o b e active t o varying extents i n freshly isolated r a t glomerular mesangial exchangers  cells  ( B o y a r s k i et al,  c a nb e modulated  1990a  a n db ) .  M o r e o v e r , t h e activities  b y t h e application o f various  o f these  neurotransmitters a n d  hormones, o r fluctuations i n t h econcentration o f intracellular ions such a s Ca  2 +  (see  above). Other proton extruding mechanisms present o n s o m e cell m e m b r a n e s include a N a / H C 0 3 " transporter and a proton p u m p . +  B o r o n and Boulepaep (1983) identified a n  18  electrogenic Na /HC03 +  proximal tubules.  co-transporter o n the basolateral m e m b r a n e s o f salamander renal  _  T h i s m e c h a n i s m , w h i c h is s e n s i t i v e to s t i l b e n e d e r i v a t i v e s ,  d i r e c t s e x t r a c e l l u l a r N a , HCO3", a n d a n e t n e g a t i v e c h a r g e a c r o s s t h e p l a s m a +  (Boron and Boulepaep, 1983).  A nonelectrogenic  H  inwardly membrane  p u m p , involved in gastric  acid  s e c r e t i o n , w a s f i r s t d e s c r i b e d b y S a c h s et al ( 1 9 7 6 ) , s t e m m i n g f r o m s t u d i e s o n t h e  hog  stomach.  for  +  In r e s p o n s e to a variety o f secretagogues, extracellular K  intracellular H ATPase,  +  +  is e x c h a n g e d  with the c o n s u m p t i o n o f A T P (Sachs, 1987). A l s o k n o w n as the  H ,K +  +  t h i s p r o t o n p u m p is e x c l u s i v e l y f o u n d o n g a s t r i c p a r i e t a l c e l l s w h o s e p r i n c i p a l  f u n c t i o n is t h e a c i d i f i c a t i o n o f the s t o m a c h m i l i e u ( B o r o n , 1 9 8 9 ) . Though vertebrate  studied  neurones  extensively  have  i ni n v e r t e b r a t e s ,  p H j regulating  not been thoroughly investigated,  mechanisms i n  primarily because  their  relatively small size hampers the utilization o f pH-sensitive microelectrodes  (Chesler,  1990).  neurones  W i t h the advent o f fluorescent p H dyes, studies o n m a m m a l i a n central  have recently emerged.  V a r i o u s fluorescein derivatives, w h o s e spectra shift a c c o r d i n g to  the s u r r o u n d i n g p r o t o n environment, are n o w e m p l o y e d as indicators o f p H in biological systems (reviewed  by Tsien, 1989).  n e u r o n e s i s2',7'-bis(carboxyethyl)-5(or high  sensitivity  t o small  physiologically relevant p K  changes a  T h e most popular probe of p H ; in 6)-carboxyfluorescein (BCECF) i np H j , ease  vertebrate  because of  o fi n t r a c e l l u l a r e n t r a p m e n t ,  its and  ( - 6 . 9 8 ) ( R i n k et al, 1 9 8 2 ) . U t i l i z i n g B C E C F i n c u l t u r e d r a t  sympathetic neurones, T o l k o v s k y a n d Richards (1987) reported that the m a i n regulator o f pHj i sa Na /H +  e x c h a n g e r that i ssensitive to inhibition b y amiloride.  +  Though  the  e x c h a n g e o f HCO3" f o r C l " d o e s n o t a p p e a r t o b e a c t i v e i n t h e s e c e l l s , t h e a u t h o r s p r o v i d e evidence for the presence o f s o m e other H C 0 " - d e p e n d e n t intracellular acid regulator, 3  perhaps  HCO3" s e n s i t i v e N a / H  regulation.  play a m i n o r role in  pHj  N a c h s h e n a n d D r a p e a u ( 1 9 8 8 ) a n d S a n c h e z - A r m a s s et al ( 1 9 9 4 ) h a v e  also  +  s h o w n that the Na /H +  +  +  exchange, which may  a n t i p o r t e r is the p r i m a r y p H j r e g u l a t o r i n rat b r a i n s y n a p t o s o m e s .  Gaillard a n d D u P o n t (1990) have demonstrated that cultured cerebellar Purkinje cells  19  utilize Na /H +  +  e x c h a n g e a n d HCO3-/CT e x c h a n g e to c o n t r o l i n t r a c e l l u l a r  levels. T h i s combination o f acid transporters has also been implicated in the o f p H j i n c u l t u r e d r a t c o r t i c a l n e u r o n e s ( O u - y a n g et al,  acid-base  maintenance  1 9 9 3 ) . R a l e y - S u s m a n et al ( 1 9 9 1 )  h a v e o b s e r v e d that p H j i n c u l t u r e d foetal rat h i p p o c a m p a l n e u r o n e s is m a i n t a i n e d b y amiloride insensitive Na /H +  +  antiporter, with a m i n o r contribution f r o m a N a -  3  Schwiening and Boron (1994) have  that regulation o f p H ; in freshly isolated neurones +  addition to aD I D S sensitive N a - d e p e n d e n t HCC^VCl'  exchanger.  +  +  +  also  f r o m the adult  h i p p o c a m p u s is p o s s i b l y g o v e r n e d b y a n a m i l o r i d e insensitive N a / H  the Na /H  and  +  HC0 "-dependent acid extrusion mechanism. demonstrated  an  rat  exchanger,  in  T h e insensitivity  of  +  e x c h a n g e r present o n h i p p o c a m p a l neurones to a m i l o r i d e supports  emerging  evidence regarding the structural diversity o f the cation counter-transporter (reviewed Clark and Limbird, 1991).  F o u r isoforms o f the Na /H +  +  exchanger have so far  by been  cloned, each representing unique molecular compositions, localizations in the body, s e n s i t i v i t i e s t o a m i l o r i d e a n d its a n a l o g u e s (see D i s c u s s i o n ) .  T h o u g h results are still quite  s p a r s e , it is b e c o m i n g i n c r e a s i n g l y e v i d e n t t h a t t h e r e g u l a t i o n o f n e u r o n a l p H j i n mammalian extruding  central nervous  and  s y s t e m is m a i n t a i n e d b y v a r i o u s c o m b i n a t i o n s  of  the acid  exchangers.  Intracellular buffering: A n y c h e m i c a l s y s t e m t h a t c o n t a i n s aw e l l p r o p o r t i o n e d m i x t u r e o f a c i d s a n d b a s e s acts to b u f f e r a n y d i s p l a c e m e n t o f p H . B y this n o t i o n , the i n t r a c e l l u l a r m i l i e u , w h i c h is r i c h i n m a n y p r o t o n a c c e p t o r s a n d d o n a t o r s , is a b l e to resist c h a n g e s i n its p H d u e to b u f f e r i n g c a p a c i t y o f its c o n s t i t u e n t s .  the  I n d i s c u s s i n g p H j , it is t h e r e f o r e n e c e s s a r y  to  include a n e x a m i n a t i o n o f the cytosol's ability to buffer p r o t o n fluxes, b o t h in terms  of  steady-state p H j regulation a n d recovery f r o m acid or alkali transients.  20  Cells are internally buffered b y the acid-base  pairs formed f r o m  bicarbonate,  proteins, phosphates, and dipeptides (Burton, 1978). Together, these species sequester or release protons to m i n i m i z e p H j shifts a c c o r d i n g to the M» + H +  MH  equation: >  n+1  (Equations)  w h e r e M i s a w e a k b a s e h a v i n g a v a l e n c e o f n, a n d M H i s i t s c o n j u g a t e a c i d h a v i n g a valence  o f n+1  (Roos  and Boron, 1981).  This type  o f buffering, w h i c h utilizes a  b a l a n c e d d i s t r i b u t i o n o f i n t r a c e l l u l a r w e a k a c i d s a n d b a s e s , is k n o w n as buffering (Boron,  1989).  A n o t h e r m a n n e r b y w h i c h changes in p H ; are m i n i m i z e d reactions.  physiochemical  S u c h reactions  may  intracellular acid-base shifts.  act to c o n s u m e  or liberate  H  is v i a  biochemical  ions in response  to  Acid production, for example, that occurs in response  to  +  alkaline loads m a y involve the conversion o f intracellular carbohydrates into lactic  acid  a c c o r d i n g to: Glucose (Siesjo, 1985).  —»  2Lactate"  + 2 H  (Equation 4)  +  C o n v e r s e l y , in r e s p o n s e to acid loads, the concentrations  of  lactate,  pyruvate, or citrate m a y decrease (Siesjo a n d Messeter, 1971). A c i d c o n s u m p t i o n , in the case o f lactic acid, w o u l d proceed Lactate" (Siesjo, 1985).  + H +  + 30  2  as: ->  3 C 0  2  (Equation 5)  + 3 H 0 2  T h e consumption o f protons b y intracellular acids a n d their  oxidation produces freely diffusable C 0  2  subsequent  (and H 0 ) , w h i c h c a n readily leave the cell to 2  reduce the effects o f acid loading. A f i n a l f o r m o f i n t r a c e l l u l a r b u f f e r i n g is t h e m o v e m e n t  of acid-base  equivalents  b e t w e e n the cytoplasm a n d the interior o f various cytosolic organelles ( R o o s a n d B o r o n , 1981).  K n o w n a s o r g a n e l l a r b u f f e r i n g , it is t h o u g h t t h a t m a n y i n t r a c e l l u l a r  s u c h as e n d o s o m e s a n d l y s o s o m e s , are able to transport protons across their  inclusions, membranes,  possibly in an electrogenic fashion (Boron, 1989). This transporter has been identified a H  +  p u m p , driven b y the hydrolysis o f A T P (Boron, 1989).  T h e inner  as  mitochondrial  21  m e m b r a n e is a l s o b e l i e v e d to b e a n a c t i v e site o f p r o t o n e x c h a n g e ( R o o s a n d 1981).  Boron,  R e s p o n d i n g to intracellular acidifications, for instance, s u c h buffering w o u l d  to transport H  +  ions into acidic vesicles or limit H  +  act  flux out o f alkaline organelles.  It s h o u l d b e e m p h a s i z e d t h a t b u f f e r i n g , w h e t h e r p h y s i o c h e m i c a l , b i o c h e m i c a l , o r organellar, d o e s not eliminate p H , shifts, b u t instead acts to m i n i m i z e t h e m .  Buffering  offers o n l y a short t e r m a n d partial solution to acid-loading, but w h e n c o m b i n e d w i t h a c i d extrusion, cells are better able to m a i n t a i n constant p H j levels.  Overview: Tissue p H in the central nervous system can modulate, or be m o d u l a t e d by,  many  p h y s i o l o g i c a l a n d p a t h o l o g i c a l p r o c e s s e s . It is b e c o m i n g i n c r e a s i n g l y a p p a r e n t t h a t m a n y o f these processes m a y have specific effects o n p H j . C h a n g e s in p H  0  a n d the application  of neurotransmitters or neuromodulators can influence pHj. In turn, fluctuations in p H , can  regulate  ionic  conductances  in excitable  cells,  alter cellular m e t a b o l i s m ,  and  m o d u l a t e s e i z u r e a c t i v i t y a n d n e u r o d e g e n e r a t i v e p r o c e s s e s . p H j is, i n a l l c e l l t y p e s s o f a r studied,  actively  investigated studies  on  regulated.  These  regulating  mechanisms  in invertebrate neuronal and vertebrate mammalian  central neurones  are  have  been  extensively  non-neuronal preparations.  limited, this thesis  will examine  regulation o f p H j in p y r a m i d a l neurones cultured f r o m e m b r y o n i c rat h i p p o c a m p i , r e g i o n k n o w n to be particularly sensitive to epileptiform activity a n d i s c h e m i c A ratiometric technique, utilizing the p H sensitive fluorophore BCECF,  from an induced acidification.  the a  conditions.  was employed  d e t e r m i n e p H j r e g u l a t i n g m e c h a n i s m s o p e r a t i n g w h i l e at rest a n d d u r i n g the  A s  to  recovery  22  MATERIALS AND METHODS  Cell preparation: N e u r o n a l cell cultures w e r e p r e p a r e d a c c o r d i n g to B a n k e r a n d C o w e n ( 1 9 7 7 ) w i t h s o m e m i n o r modifications. H i p p o c a m p a l sections w e r e obtained f r o m 18 d a y e m b r y o n i c age W i s t a r rat foetuses, a n d stored in a Ca BSS).  The CMF-BSS  10.05  m M  HEPES  2 +  and Mg  2  +  - f r e e b a l a n c e d salt solution ( C M F -  c o n t a i n e d 1 0 % H a n k ' s b a l a n c e salt solution, 55.5 m M  4-(-2-hydroxyethyl)-l-piperazineethanesulfonic  Na -salt, and 2 m M NaHC0 . +  3  acid  glucose,  ( H E P E S ) , 4.95  The hippocampi were subsequently  transferred  into 6 m L o f an enzymatic solution containing aliquots o f trypsin a n d D N A s e in CMF-BSS,  for 2 0 m i n u t e s at 3 7 ° C .  dissolved  T h e enzyme-based dissociation was followed by a  trituration p r o c e d u r e i n w h i c h tissue w a s s u s p e n d e d in a test tube c o n t a i n i n g CMF-BSS,  m M  D N A s e ,  a n d 1 0 % foetal bovine s e r u m ( F B S ) , a n d was mechanically siphoned 20  t h r o u g h fire polished pasteur pipettes o f decreasing tip diameter.  times  T h e triturated mixture  w a s t h e n d i l u t e d w i t h a n a d d i t i o n a l 1m L o f 1 0 % F B S , a n d c o l d c e n t r i f u g e d at 150 g for 4 to 5 minutes.  T h e supernatant was  removed  a n d the resulting cell pellet w a s  suspended in 6 m L of Dulbecco's Modified Eagle's M e d i u m (D-MEM) F B S ( D - M E M / F B S ) . All D - M E M HEPES, and 5% C 0  2  re-  containing  solutions were buffered b y 22 m M N a H C 0 , 3  10%  10  m M  in air.  A 50 u L sample o f the cell suspension was r e m o v e d f r o m the D-MEM and m i x e d into 410 u L CMF-BSS  and 40 u,L trypan blue.  were then counted on a hemocytometer (Neubauer) chamber. percent dilution to be applied to the total cell suspension, multiplied b y a factor o f 0.01.  suspension  T h e living p y r a m i d a l cells In order to c o m p u t e  the  this sampling count  was  This dilution constant accounted for the n u m b e r  of  chambers o n the hemocytometer, the size o f the coverslips, a n d the final density  (10  c e l l s / c m ) at w h i c h the c o v e r s l i p s w e r e to b e plated.  was  2  subsequently  diluted with D-MEM/FBS  T h e cell-containing m e d i u m  b y the factor determined in the sample  5  count  23  calculation.  Coverslips, previously coated  with poly-D-lysine  a n d laminin for cell  adhesion a n d growth, were then plated with 0.2 m L o f the diluted culture m e d i u m .  After  1 hour, the coverslips were transferred face d o w n into 6-well culture plates containing 2 mL The  o f D-MEM/FBS cells were  in each well, a n d stored in a5 % C 0  cultured in a face-down  2  i n air environment at 35°C.  position d u e to the h i g h rate o f mortality  associated with face-up growth (see B r e w e r a n d C o t m a n , 1989).  After 3to 4 hours o f  incubation, half o f the culture m e d i u m w a s replaced b y 1m L o f serum-free containing 5 m g / L transferrin, 6.2 p g / L progesterone, selenium, a n d 5m g / Linsulin.  This D-MEM/FBS  s e v e n days after the initial culture date.  D-MEM  8.8 m g / L putrescine, 5.19 p g / L  replacement procedure was  repeated  T h e presence o f non-neuronal cells w a s checked  following 2to 3days o f storage, a n d the cultures were treated with 5-fluorodeoxyuridine to arrest glial multiplication. E x p e r i m e n t s w e r e p e r f o r m e d at b o t h r o o m temperature a n d 37°C u s i n g 6to 1 4 d a y o l d cultures.  L o a d i n g the neurones with B C E C F :  BCECF fluorescent  acetoxymethyl  ester  (BCECF-AM),  the cell-permeant  form  o f  h y d r o g e n i o n i n d i c a t o r B C E C F ( R i n k et al, 1 9 8 2 ) , w a s o b t a i n e d  the from  Molecular Probes Inc. (Eugene, Oregon). T h e fluorescent probe w a s prepared in advance as a 1.0 m M stock i n a n h y d r o u s D M S O , -60°C.  separated into 6 0 p L aliquots , a n d stored at  Loading medium, made u p o n the day o f the experiment, contained the  same  e l e m e n t s a s solution 1 ( T a b l e 1 ) w i t h t h e i s o o s m o t i c a d d i t i o n o f 3 . 0 m M N a H C 0 3 i n place o f NaCl.  5p . Lo f t h e 1.0 m M B C E C F - A M  stock w a s thawed a n d diluted to 2 u M  in 2.5 m L o f the loading m e d i u m contained in asingle well o f a6-well tissue  culture  plate. A n 18 m m coverslip, plated with the h i p p o c a m p a l neurones, w a s placed face-up i n the dye-containing m e d i u m for 3 0 minutes at r o o m temperature.  T h e coverslip w a s then  mounted in atemperature-controlled perfusion chamber so as to f o r m the base o f the c h a m b e r . T h e n e u r o n e s w e r e p e r f u s e d a t ar a t e o f 2 . 4 m L / m i n u t e f o r 1 5 m i n u t e s w i t h t h e  24  initial experimental buffer at the appropriate temperature prior to the start of an experiment. The polyethylene perfusion line was contained within an aluminum block that was heated when necessary to raise the perfusate temperature to 31°C.  During  perfusion with HCC^VCC^-buffered solutions, the atmosphere in the recording chamber consisted of 5% CO2  in balance air.  Experimental setup: pH values were measured utilizing the dual-excitation fluorescence ratio method, employing an Attofluor Digital Fluorescence System (Atto Instruments Inc.) operating in conjunction with a Zeiss Axiovert 10 microscope (Carl Zeiss Canada Ltd.). B C E C F was used as a dual-excitation indicator, with the ratio of the emitted fluorescence intensities from excitations at 488 nm and 460 nm providing the pH determination. Exciting the dye at 488 nm, the emitted fluorescence, measured at 510 nm, was p H sensitive. The dye was subsequently excited at 460 nm, a wavelength in close proximity to the indicator's isoexcitation point, and thus at this wavelength the emitted fluorescence was nearly completely insensitive to pH. The ratiometric method has been shown to substantially reduce signal errors caused by variations in optical path length, dye concentration, dye leakage, and photobleaching (Bright et al, 1989). The limits and potential artifacts of fluorescence ratio imaging microscopy have been discussed by Bright et al (1987) and Silver era/(1992). The source of the excitation photons was a 100 W mercury arc burner whose light path was interrupted by a computer actuated high speed shutter. The shutter served to restrict the illumination of the B C E C F to periods of data acquisition (usually once every 10 to 60 seconds) in order to minimize any photo-induced damage to the dye or cells. Such degradation was also reduced by placing variable neutral density filters in the light path. 488 and 460 nm short band-pass filters were mounted on a computer-controlled filter changer which, during excitation, sequentially interrupted the light path.  The  25  excitation radiation was reflected b y a long band-pass dichroic mirror (FT-495) a n d  was  focused through a x40 N e o f l u a r objective (numerical aperture 0.75) onto the cells in the recording chamber.  T h e emitted fluorescent light passed b a c k through the dichroic b e a m  splitter b e f o r e b e i n g filtered b y a 5 1 0 n m l o n g - p a s s filter, the w a v e l e n g t h at w h i c h emission was  the  monitored.  Fluorescence emissions were measured b y an intensified charge-coupled  device  c a m e r a m o u n t e d o n t o the m i c r o s c o p e . T h e c a m e r a g a i n w a s set b y m a x i m i z i n g the intensity while m i n i m i z i n g the possibility o f c a m e r a saturation, a n d w a s held throughout an experiment. pixel f r a m e size.  image constant x  480  During acquisition, a single image was captured for each o f the  two  excitation wavelengths.  I m a g e s w e r e digitized to 8 bit resolution w i t h a 512  A video terminal sequentially displayed each  pseudocoloured  i m a g e , w h i c h w e r e u s e d not o n l y to visually m o n i t o r the progress o f the study, but to also select areas for analysis. T h e s e selected regions o f interest (ROI's), 10 pixels b y 10 i n size, w e r e set at the start o f the e x p e r i m e n t o v e r m u l t i p l e ( m a x i m u m 99)  pixels  neuronal  somata having an approximate pyramidal shape: one defined long process, and two  or  m o r e shorter processes. T o aid in the r o u g h focusing o f the neurones a n d the selection o f the ROI's, the cell population w a s visualized u n d e r phase illumination using a 12 V ,  100  W  T h r o u g h o u t the course o f an experiment, the computer calculated  and  graphically displayed the emission intensities for both excitation wavelengths, a n d  the  halogen lamp.  ratio o f the emitted fluorescence for all, or a c h o s e n single, region(s) o f interest. cases, the  recorded values reflected  the  mean  emitted  intensity  within  each  In all R O I ,  c o m p u t e d in real time.  Solutions:  T h e solutions utilized during the course o f these experiments are listed in Tables 1 to 4.  A  Corning  2 4 0 p H meter, calibrated daily, w a s e m p l o y e d to m e a s u r e all p H s .  T h o s e s o l u t i o n s l a c k i n g b i c a r b o n a t e ( T a b l e 1) w e r e b u f f e r e d b y 10 m M H E P E S  and then  26  titrated to the appropriate p H w i t h 10 M NaOH,  except w h e n noted.  {solution 2) w a s p r e p a r e d b y e q u i m o l a r s u b s t i t u t i o n o f a l l N a  The Na -free +  salts f o u n d in the  +  saline standard  m e d i u m (solution 1) w i t h J V - m e t h y l - D - g l u c a m i n e ( N M D G ) , w h i c h t h e n r e q u i r e d t h e  use  +  o f 10 M  H C 1 to l o w e r the p H to the 7.4 range.  sodium, potassium,  and hemi-calcium gluconate in place of NaCl,  r e s p e c t i v e l y (solution 3). was  achieved  selective for K  +  K C 1 , and  of  CaCl , 2  T h e a d d i t i o n N H C 1 (solution 4) t o t h e H E P E S b u f f e r e d s a l i n e 4  through equimolar replacement  prepared using the  C l " was r e m o v e d through the use  ionophore  of NaCl.  Calibration solutions  nigericin, a cation-hydrogen  (Chaillet and Boron, 1985).  solution in ethanol, divided into 100  exchanger  that is  were highly  N i g e r i c i n w a s p r e p a r e d as a 10 m M  p L volumes,  a n d t h e n stored at - 6 0 ° C .  stock W h e n  needed, a 10 m M aliquot w a s diluted to 10 p M i n a solution containing a concentration K  +  n e a r i n t r a c e l l u l a r l e v e l s (solution 5).  appropriate p H s with 10 M  N i g e r i c i n - c o n t a i n i n g solutions w e r e titrated to  K O H , w i t h the e x c e p t i o n o f the p H 5.5 solution  d u r i n g full calibrations (see b e l o w ) that r e q u i r e d 1 M  employed  HC1.  R e g a r d l e s s o f the t e m p e r a t u r e at w h i c h a g i v e n e x p e r i m e n t w a s b e i n g all HEPES  of  performed,  b u f f e r e d m e d i a w e r e p r e p a r e d at r o o m t e m p e r a t u r e ( 1 8 ° C to 2 2 ° C ) .  In order  to account for the p H fluctuation associated with raising the solution temperature, the  p H  at r o o m t e m p e r a t u r e ( p H  p H  R  T  ) w a s adjusted to reflect the e n s u i n g t e m p e r a t u r e - i n d u c e d  c h a n g e , s u c h that at 3 7 ° C the d e s i r e d p H w o u l d b e r e a c h e d ( p H  3 7  ). T h e different p H s for  H C 0 3 " / C 0 2 - f r e e , H E P E S - b u f f e r e d solutions at r o o m t e m p e r a t u r e a n d 3 7 ° C w e r e b y the  related  equation: pH  3 7  = 0.18 + 0 . 9 6 x p H  R  (Equation 6)  X  This equation was determined during preliminary experiments (n=8) in w h i c h the p H s HEPES  b u f f e r e d solutions, p r e p a r e d at 2 2 ° C , w e r e c o m p a r e d to the resulting p H  heated to  of  when  37°C.  The composition of solutions buffered by a combination of HC0 " 3  r o o m t e m p e r a t u r e are s u m m a r i z e d i n T a b l e 2.  and C 0  A l l H C 0 " - c o n t a i n i n g solutions, at 3  2  at  room  27  temperature and 37°C, were equilibrated with 5 % C 0 room  temperature,  NaHC03  the  i n b a l a n c e air. F o r e x p e r i m e n t s  2  standard H C 0 7 C 0 2 - b u f f e r e d m e d i u m  contained  3  (solution 6),  resulting in a p H o f 7.32 ± 0.01  26.0  at m M  (mean ± standard error of  the  mean, n=19).  T h e preparation o f Na -free saline was accomplished b y replacing  NaCl  and NaHC0  w i t h c h o l i n e c h l o r i d e a n d c h o l i n e b i c a r b o n a t e , r e s p e c t i v e l y (solution 7 ) .  +  3  S o l u t i o n s l a c k i n g C l w e r e p r o d u c e d b y s u b s t i t u t i n g g l u c o n a t e i n p l a c e o f C h (solution 8). -  P r o p i o n a t e (solution 9),  t r i m e t h y l a m i n e (solution 10)  added b y equimolar substitution of NaCl;  when  a n d N H C 1 (solution 11)  were  4  CO2,  equilibrated with 5 %  m i x t u r e s resulted i n p H s o f 7.30 (n=l), 7.31 (n=l), a n d 7.32 (n=2),  these  respectively.  A t 3 7 ° C , the concentration o f bicarbonate in the standard m e d i u m w a s r e d u c e d to 2 0 . 0 m M (solution 12,  T a b l e 3), y i e l d i n g a p H o f 7.36 ± 0.01  N a - f r e e (solution 13;  p H  +  0  (mean ± S.E.M., n=19).  7 . 3 8 ± 0 . 0 1 , n = 4 ) , C l ' - f r e e (solution 14;  n = 7 ) , a n d N H C l - c o n t a i n i n g (solution 16; 4  p H  0  p H  0  7.38 ±  0.01,  7.35 ± 0 . 0 1 , n=7) solutions w e r e prepared  in a n similar fashion to their r o o m temperature counterparts. A m i x t u r e l a c k i n g b o t h N a and C l  -  w a s f o r m e d using free choline base, choline bicarbonate, free gluconic  potassium and hemi-calcium gluconate, and normal concentrations of M g S 0 g l u c o s e (solution 15;  p H  0  7.38,  4  +  acid,  and  D-  n=l).  I n o r d e r t o v a r y t h e p H o f a s o l u t i o n b u f f e r e d b y HCO3VCO2, i t w a s n e c e s s a r y t o adjust the concentration o f NaHC0 Preliminary experiments  via isoosmotic substitution with NaCl  (Table  4).  established that, at 3 7 ° C , the s o l u t i o n p H w a s r e l a t e d to  its  3  bicarbonate concentration (in m M ) b y the equation: p H = 6.03 + 1.03 x l o g [ H C 0 - ]  (Equation 7)  3  This f o r m u l a was derived f r o m a series o f p H versus concentration o f HC0 t h a t a r e s h o w n i n F i g u r e 1 , a n d w a s e m p l o y e d t o c r e a t e solutions 17, ( T a b l e 4).  18,  data points  _ 3  20,  21,  and  T h e N H C l - c o n t a i n i n g s o l u t i o n at p H 6.8 w a s i s o o s m o t i c a l l y b a l a n c e d 4  s u b s t i t u t i o n o f N a C l w i t h 2 0 m M N H C 1 (solution 19). 4  m e a s u r e d at the a p p r o p r i a t e t e m p e r a t u r e f o l l o w i n g e a c h  22 by  T h e p H s o f all solutions w e r e reexperiment.  28  Ethylisopropylamiloride (EIPA) was dimethylsulphoxide (DMSO)  p r e p a r e d as a 50 m M  stock solution  prior to a 1 in 1000 dilution in the p e r f u s i o n solution.  diisothiocyanatostilbene-2,2'-disulphonic  acid (DIDS)  was  dissolved  c o n c e n t r a t i o n o f 100 m M , a n d u s e d at a final c o n c e n t r a t i o n o f  200  4,4'-  in DMSO  a t a  u M .  stock  A l l  solutions w e r e prepared o n the day o f the experiment, a n d the final concentration DMSO  i nthe  perfusion  solution  never  exceeded  d e m o n s t r a t e d that, at this c o n c e n t r a t i o n , D M S O  0.5%.  in  Control  of  experiments  h a d no effect o n p H ; (data not  shown).  C o m p o u n d s w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o m p a n y (St. L o u i s , M i s s o u r i ) , w i t h the exception o f 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride ( H O E 694),  and 5-(Af-methyl-/V-guanidinocarbonylmethyl) amiloride (MGCMA).  was obtained from Hoechst A . G . (Frankfurt, Germany), while MGCMA  H O E  694  was a generous  gift f r o m D r . V . Palaty ( D e p a r t m e n t o f A n a t o m y , U n i v e r s i t y o f British C o l u m b i a ) ; c h e m i c a l s w e r e p r e p a r e d as 100 m M stock solutions in D M S O ,  both  a n d utilized at a final  concentration of 100 u M .  Calculation of pHj: E x p e r i m e n t a l results w e r e stored in computer-generated data files containing pixel intensities  for each  region  o f interest.  During acquisition  periods  the  following  i n f o r m a t i o n w a s stored: the intensity o f the fluorescent signal after excitation at 4 8 8 the  460  nm-induced fluorescent  s i g n a l , a n d a r a t i o (I488/I460) ° f  m  fluorescence  e  intensities.  Utilizing a stand-alone D O S based graphing program (ATTOGRAF,  Instruments  Inc, version  5.41), regions  o f interest c o r r e s p o n d i n g t on e u r o n e s  remained viable throughout an experiment were selected for analysis. j u d g e d b y the capacity o f the neurones to retain the  fluorescent  Viability  i n d i c a t o r (as j u d g e d  r a w intensity values) t h r o u g h o u t the entire course o f the e x p e r i m e n t (see S c h w i e n i n g Boron, 1992;  nm,  Schwiening and Boron, 1994).  Atto that was by and  29  The  determination  of pHj was  initiated b y  the  of  background  fluorescence intensities f r o m the r a w intensity values in each selected R O I .  Background  levels w e r e determined b y measuring the fluorescence cellular p r o c e s s e s at e a c h excitation w a v e l e n g t h .  subtraction  signal in a region devoid  Transformation o f the  corrected ratios into p H j values utilized conversion equations calibration e x p e r i m e n t s ( F i g u r e 2).  background-  d e r i v e d f r o m in  +  (solution 5,  +  Each  K O H or 1  N i g e r i c i n is a c h a r g e d e l e c t r o n c a r r i e r that acts to b a l a n c e p H j a n d p H  intracellular a n d extracellular K  was  T a b l e 1).  s o l u t i o n w a s titrated to a different p H i n the 5.5 to 8.5 r a n g e u s i n g 10 M  to  (Figure  A l l calibration solutions contained 10 u M o f the ionophore nigericin, w h i c h  a d d e d to a solution containing h i g h concentrations o f K  HC1.  situ  In such experiments, the neurones were exposed  v a r i e t y o f H E P E S - b u f f e r e d solutions at r o o m t e m p e r a t u r e h a v i n g d i f f e r i n g p H s 2A).  of  M  if the  0  activities are equal (Chaillet a n d B o r o n , 1985). T h u s , in  the presence o f high extracellular K  concentrations, p H j was controlled merely by  the  T h e resulting intensity ratios p r o d u c e d b y exposing the neurones to various  p H  +  p H o f the superfusing m e d i u m .  solutions  containing  10  p M  nigericin were  used  Following subtraction of background fluorescence i n d u c e d fluorescence signals, the ratios (1488^460)  to  construct  a calibration  values f r o m the 488  curve.  and 460  n m -  n o r m a l i z e d such that the  ensuing  c u r v e p a s s e d t h r o u g h u n i t y at p H 7.0 ( F i g u r e 2 B . ) . A full c a l i b r a t i o n e x p e r i m e n t  resulted  w e r e  in the determination o f parameters fitting a standard curve w h i c h c o u l d then be u s e d transform other n o r m a l i z e d ratios into p H , values.  to  T h e derivation o f the equation fitting  this standard curve stems f r o m the H e n d e r s o n - H a s s e l b a l c h expression for the  dissociation  of a weak acid:  [1 A  p H  = p K  a  + l o g f-—^  (Equation  8)  30  w h e r e [ A ] is t h e c o n c e n t r a t i o n o f t h e i o n i z e d f o r m o f t h e a c i d , [ H A ] is t h e n e u t r a l f o r m -  of the acid, a n d K  a  is the a c i d d i s s o c i a t i o n constant.  T a k i n g into consideration the total  acid concentration, denoted b y [Total], as equaling the s u m o f the ionized [A ] a n d n o n -  ionized [ H A ] forms o f the acid, the above equation becomes:  pH =p K +l o g  )  L  ,  (Equation 9)  [Total]-[A J  F o r B C E C F , t h e c o n c e n t r a t i o n o f t h e i o n i z e d f o r m i s p r o p o r t i o n a l t o t h e r a t i o ("R") o f t h e fluorescence  intensities at 4 8 8 n m a n d 4 6 0 n m .  T h u s the total acid concentration  p r o p o r t i o n a l to the m a x i m a l obtainable ratio ("b").  Substituting these variables  is into  Equation 9 yields: R  pH=p K  a  1Q(P -P a) H  + l o g ^ - ^ -  , o r  R  =  b  -  1  +  1 0  K  (pH- K P  (Equation 10)  a )  I f R is c o n s t r a i n e d to p a s s t h r o u g h u n i t y at p H 7.0, t h e n the v a l u e o f R at p H 7.0 m u s t  be  s u b t r a c t e d f r o m t h e E q u a t i o n 1 0 , f o l l o w e d b y t h e a d d i t i o n o f 1. T h i s n o r m a l i z e d R t e r m , n o w d e n o t e d R ,c a n b e e x p r e s s e d as: n  jQCpH-pKJ  R  1Q( -P a) 7  =b  n  K  -b l +1 0  ( p  p  T h e fitted values for ba n d p K  a  .+1  v  1 +10  a)  (  p  (Equation 11)  a)  varied with the setup o f the microscope.  F o r this reason,  a n y c h a n g e s to the experimental e q u i p m e n t (for e x a m p l e , the r e p l a c e m e n t o f the m e r c u r y arc burner) was revised  a c c o m p a n i e d b y the execution  calibration parameters  were  o f a full calibration experiment,  determined.  . Equation  1 1w a s  and  simplified  by  determining the theoretical m a x i m u m a n d m i n i m u m obtainable values for the normalized ratio. T h e s e values, s y m b o l i z e d b y R ( n  R,  . = l + b - b  n(max)  ±v  and  u  max  ) a n d R ( i ), c a n b e r e p r e s e n t e d a s : n  m  n  10(7-pKJ  (Equation 12)  TZ—TTT~  j  +  1  Q(7-pK ) a  *  1  /  31  1Q( -P a) 7  R  „  ( m i  n)  =  1  -  '  b  1  +  K  (Equation 13)  (7- K )  1 0  P  a  U s i n g the determined values for b a n d p K , the m a x i m u m a n d m i n i m u m  normalized  a  ratios w e r e calculated. I n o r d e r to c r e a t e a n e q u a t i o n w h i c h c o n v e r t s n o r m a l i z e d r a t i o s i n t o p H v a l u e s , it is n e c e s s a r y to e x p r e s s the r e g r e s s i o n e q u a t i o n as a f u n c t i o n o f p H .  Manipulation of  E q u a t i o n 11 yields:  JQ( H- KJ P  P  n  b  the  K  1Q(7  "  (Equation 14)  1 R  pKa)  ) a n d R ( j ) into E q u a t i o n 14 g i v e s rise to:  m a x  n  IO"*--*.' = ( R „ - R  Isolating  7  _  lib Substituting R (  10( -P a> '  1_U  R„  m  n  (  n  m  i  n  )  ) / ( R  n  (  m  a  x  - R  )  n  (Equation 15)  )  p H term utilizing a logarithmic manipulation produces  the  following  equation: pH =log[(R  n  -R  n  (  m  i  n  )  )/(R  n  (  m  a  x  )  - R ) ] +p K n  (Equation 16)  a  E q u a t i o n 16 w a s then utilized in the conversion o f all n o r m a l i z e d ratios into p H u s i n g t h e p r e d e t e r m i n e d p a r a m e t e r s f o r R ( j ) , R ( x)> n  m  calculated for each full calibration experiment.  n  n  p K .  a n Q l  ma  These factors  a  F o r the seven full calibration  6.98 ± 0.02, 0.49 ± 0.02, a n d 1.49 ± 0.02, respectively.  n  m  n  n  which typically varied between 20°C  calibration p e r f o r m e d at 3 0 ° C , the d e t e r m i n e d p K s t u d y at 2 1 ° C ,p K  a  w a s f o u n d to b e  a  m a x  )  and  30°C.  experimental  For example,  corrected) ratio at p H j 7.0  was  in a  value w a s 6.98, w h e r e a s in a separate  6.97.  M o s t experiments w e r e c o n c l u d e d b y exposing the neurones to a single p H n i g e r i c i n - c o n t a i n i n g solution (see  were  Furthermore, the values o f these  calculated parameters d i d not appear to be d e p e n d e n t o n c h a n g e s in the temperature,  were  experiments  utilized in analyzing all experiments, the m e a n values o f p K , R ( j ), a n d R ( a  values,  F i g u r e s 4, 7, a n d 12).  T h e resulting  7.0  (background-  u s e d as the n o r m a l i z a t i o n factor for that  particular  32  experiment.  A s o u t l i n e d b y B o y a r s k i et al ( 1 9 8 8 ) , t h e a d v a n t a g e o f t h i s n o r m a l i z a t i o n  s t e p is t h a t it p r o v i d e s a o n e - p o i n t c a l i b r a t i o n f o r e a c h c e l l p o p u l a t i o n s t u d i e d . dividing  all  experimentally-derived  (background-corrected)  determined normalization value, each R  n  intensity  ratios  After by  the  w a s c o n v e r t e d to p H j utilizing E q u a t i o n 16  and  the appropriate fitted calibration parameters.  Analysis of data: E a c h e x p e r i m e n t typically required the analysis o f 10 or m o r e regions o f interest, a n d t h u s i t b e c a m e n e c e s s a r y t o a u t o m a t e t h e c o n v e r s i o n o f I488/I46O either  a  DOS-based  transformation  Department of Physiology,  program  (courtesy  of  Dr.  P ^ i  mt0  K .  employing Abdel-Hamid,  University of British Columbia) or personally  designed  V i s u a l B a s i c m a c r o s r u n n i n g i n M i c r o s o f t E x c e l 5.0. A b s o l u t e p H j levels are r e p o r t e d for n e u r o n e s u n d e r s t e a d y - s t a t e c o n d i t i o n s i n t h e p r e s e n c e a n d a b s e n c e o f HCO3-, a t room temperature and 37°C.  A t steady-state, any perturbations in p H j w e r e  relative to the resting p H j before the  both  measured  change.  In experiments designed to analyze the restoration o f p H j b a c k to  steady-state  levels after a n i m p o s e d acid load, the recovery portion o f the e x p e r i m e n t w a s fitted to  a  single exponential function having a format: pHj = a + b ( l - 1 0  ( _ c t )  )  w h e r e a, b, a n d c are the e x p o n e n t i a l p a r a m e t e r s .  (Equation 17) T h e differentiated f o r m o f E q u a t i o n 17  represents the c h a n g e in p H j as a function o f time, a n d w a s u s e d to analyze the  recovery  r a t e (dpH^/df) a t a n y p o i n t d u r i n g t h e r e s t o r a t i o n t o s t e a d y - s t a t e p H j l e v e l s : ^S-  di  = -bcl  0  ( _ c t )  (Equation 18)  R e c o v e r y rates w e r e d e t e r m i n e d i m m e d i a t e l y after the p e a k a c i d i f i c a t i o n , a n d at 5 0 % 8 0 % r e c o v e r y relative to the steady-state p H j before the i n d u c e d acid load.  and  33  Statistical  comparisons  were  carried out  using  Student's  t test w i t h  a  95%  confidence limit. If a preconceived directionality existed in m a k i n g a comparison, a  one-  tailed test w a s used, otherwise the two-tailed version w a s utilized. In all cases, u n p a i r e d t values were  calculated, with supplemental  indicated errors are expressed accompanying  as the standard error o f the m e a n  referring to the n u m b e r  appropriate.  A n y  (S.E.M.), with  o f cell populations  (i.e.  the  number  of  Periodically, variations in the emission intensities arose w h i c h were caused  by  coverslips)  n value  paired data added w h e n  analyzed.  b r i e f f l u c t u a t i o n s i n t h e i n c i d e n t r a d i a t i o n ( s e e B o y a r s k i et al,  1988a). In order to  the resulting graphical representation o f the p H j versus time record, a m o v i n g ( p e r i o d = 3 ) w a s a p p l i e d t o a l l p l o t s ( B o y a r s k i et al,  1988a).  smooth average  T a b l e 1: C o m p o s i t i o n o f H E P E S - b u f f e r e d e x p e r i m e n t a l s o l u t i o n s (allconcentrations in m M ) :  Solution  1 N a C l KC1 CaCl N a H P 0 M g S 0 N a G l u K G l u >/ Ca G l u D-glucose N M D G + 2  2  4  4  2  NH4CI  H E P E S Titrated with:  2  Standard 136.5 3.0 2.0 1.5 1.5 -  Na  -  3 free  +  free  -  -  3.0 2.0 1.5 -  -  -  10.0 10.0 1 0 M N a O H  Ch  10.0 136.5 10.0 1 0 M HC1  . 4  1.5 1.5 136.5 3.0 4.0 10.0 10.0 10 M N a O H  NH4CI  116.5 3.0 2.0 1.5 1.5  10.0  20.0 10.0 1 0 M N a O H  5 High K + -  2.0 1.5 1.5 10.0 130.5  10.0 10.0 1 0 M K O H  Abbreviations: N a G l u , sodium gluconate; K G l u , potassium gluconate; hemi-calcium gluconate; NMDG , A^-methyl-D-glucamine. +  '/iCa  35  T a b l e 2: C o m p o s i t i o n o f H C 0 7 C 0 - b u f f e r e d e x p e r i m e n t a l s o l u t i o n s at r o o m (all concentrations i n m M ) : 3  2  temperature  Solution 6 Standard 120.5 26.0 3.0 2.0 1.5 1.5 10.0 -  N a C l N a H C 0 K C 1 CaCl N a H P 0 M g S 0 D-glucose 3  2  2  4  4  NH4CI  N K V C C P T  a G l u G l u iCa G l u holine H C 0 holine C l R O P M A final p H  3  7 Na  8 free  +  1.5 1.5 10.0  9 P R O P 100.5 26.0 3.0 2.0 1.5 1.5 10.0  -  -  Cl- free  -  -  -  26.0  3.0 2.0 1.5 10.0 -  -  -  -  -  -  -  -  7.32 ± 0 . 0 1 (n=14)  26.0 120.5 7.35 (n=l)  120.5 3.0 4.0 -  7.33 ± 0 . 0 1 (n=2)  10 T M A 110.5 26.0 3.0 2.0 1.5 1.5 10.0 -  -  20.0 7.30 (n=l)  11 NH4CI  100.5 26.0 3.0 2.0 1.5 1.5 10.0 20.0  -  -  10.0 7.31 (n=l)  -  -  7.32 (n=2)  A l l H C 0 " - c o n t a i n i n g solutions w e r e equilibriated w i t h 5 % C 0 in balance air. p H s are reported as the m e a n ± S.E.M. Abbreviations: N a G l u , sodium gluconate; K Glu, potassium gluconate; M-Ca G l u , hemi-calcium gluconate; PROP, propionate; T M A , trimethylamine. 3  2  36  T a b l e 3: C o m p o s i t i o n o f H C 0 7 C 0 2 - b u f f e r e de x p e r i m e n t a l solutions at 3 7 ° C (allconcentrations in m M ) : 3  Solution  12 Standard N a C l N a H C 0 K C 1 CaCl N a H P 0 M g S 0 D-glucose N H C 1 N a G l u K G l u !/ Ca G l u Gluconic acid Choline H C 0 Choline C l Choline base final p H 3  2  2  4  4  4  2  3  126.5 20.0 3.0 2.0 1.5 1.5 10.0 7.36 ± 0 . 0 1 (n=19)  13 Na  +  14 free  3.0 2.0 1.5 10.0 20.0 126.5 7.38 ± 0 . 0 1 (n=4)  Ch  free  20.0 1.5 1.5 10.0 126.5 3.0 4.0 7.38 ± 0 . 0 1 (n=7)  16  75 Na Ch  and free  +  -  N H C 1 4  106.5 20.0 3.0 2.0 1.5 1.5 10.0 20.0 7.35 ± 0 . 0 1 (n=7)  1.5 10.0 3.0 4.0 126.5 20.0 126.5 7.38 (n=l)  All H C 0 ' - c o n t a i n i n g solutions were equilibriated with 5 % C 0 in b a l a n c e d air. R e p o r t e d p H s are g i v e n as the m e a n ± S.E.M. Abbreviations: N a G l u , s o d i u m gluconate; K G l u , potassium gluconate; ViCa G l u , hemi-calcium gluconate. 3  2  37  T a b l e 4: C o m p o s i t i o n o f H C 0 3 / C 0 - b u f f e r e d e x p e r i m e n t a l solutions at v a r y i n g p H s at (all concentrations in m M ) : _  2  37°C  Solution  N a C l NaHCC-3 K C 1 CaCl N a H P 0 M g S 0 D-glucose 2  2  4  4  NH4CI  final p H  17  18  p H 6.5 standard 143.5 3.0 3.0 2.0 1.5 1.5 10.0  p H 6.8 standard 140.7 5.8 3.0 2.0 1.5 1.5 10.0  -  -  6.56 (n=2)  6.79 ± 0 . 0 1 (n=3)  . 19 pH  6.8  NH4CI  120.7 5.8 3.0 2.0 1.5 1.5 10.0 20.0 6.80 (n=l)  20  21  22  p H 7 . 0 standard 137.5 9.0 3.0 2.0 1.5 1.5 10.0  p H 7.8 standard 101.5 45.0 3.0 2.0 1.5 1.5 10.0  p H 8 . 0 standard 61.5 85.0 3.0 2.0 1.5 1.5 10.0  -  -  -  7.00 ± 0 . 0 1 (n=3)  7.75 (n=2)  8.02 (n=2)  All H C 0 - c o n t a i n i n g solutions were equilibriated with 5 % C 0 are indicated as the m e a n ± S.E.M. -  3  2  i n b a l a n c e d air.  pH's  38  Figure 1. Relationship between the concentration of HCO3- and the resulting solution p H when equilibrated with 5 % CO2 in balance air at 37°C. F o l l o w i n g equilibration with 5 % C 0 , the p H s o f solutions were measured at 3 7 ° C c o n t a i n i n g 3 . 0 m M , 5 . 8 m M , 9 . 0 m M , 2 0 . 0 m M , 4 5 . 0 m M , a n d 8 5 . 0 m M HCO3- ( s e e T a b l e 4f o r solution recipes). D a t a w a s derived f r o m asingle experiment. T h e curve w a s f o r m e d f r o m a logarithmic g r o w t h least squares regression fit to t h e data points h a v i n g the equation: pH = 6 . 0 3 + 1 . 0 3 x l o g [ H C O - ] 2  3  39  8.50  T  8.25 -  0  10  20  30  40  50  60  [HCO3-] (mM)  70  80  90  100  40  F i g u r e 2.  Sample calibration plot for B C E C F .  A . C e l l s w e r e e x p o s e d t o H E P E S - b u f f e r e d s o l u t i o n s (solution 5, T a b l e 1 ) c o n t a i n i n g 1 0 p M n i g e r i c i n at p H ( a n d therefore p H j ) 5.55, 6.02, 6.50, 7.00, 7.51, 7.96, a n d 8.41. T h e d u r a t i o n o f e a c h e x p o s u r e is i n d i c a t e d b y t h e b a r s a b o v e t h e trace, w h i c h is a m e a n o f data obtained f r o m 29 cells recorded o n a single coverslip. T h e resulting b a c k g r o u n d s u b t r a c t e d r a t i o s OUgg/T^o) n o r m a l i z e d to 1.00 at p H , 7.00. B . P l o t o f p H j against the resulting n o r m a l i z e d ratio (R ). Standard error bars are indicated (n=3 coverslips). T h e c u r v e is a result o f a n o n - l i n e a r least s q u a r e s r e g r e s s i o n fit to E q u a t i o n 16. F o r this particular calibration, the values o f R , R j , a n d p K were 1.542, 0.491, a n d 7.027, respectively. 0  w e r e  n  m a x  m  n  a  41  42  R E S U L T S  S T E A D Y - S T A T E  p H ;  R E G U L A T I O N  R e g u l a t i o n o fp H j a t r o o m  temperature:  In HC03 -free H E P E S buffered m e d i u m atp H _  0  7 . 3 2 (solution 1, T a b l e 1 ) , s t e a d y -  state p H j rested at 6 . 8 5± 0 . 0 4(n=25) a s s h o w n i n T a b l e 5 a n d F i g u r e 3 A . pH  A tthe same  b u t i n t h e p r e s e n c e o f H C 0 " , (solution 6, T a b l e 2 ) t h e b a s e l i n e p H j r e s i d e d a t t h e  0  3  substantially higher level o f 7.15 ± 0 . 0 3 (n=22;  Table 5; Figure 3B).  This suggests a  s u b s t a n t i a l c o n t r i b u t i o n o f HCO3 " - d e p e n d e n t m e c h a n i s m s t o t h e m a i n t e n a n c e o f s t e a d y state p H ; a t r o o m temperature.  T h ee q u i m o l a r r e p l a c e m e n t o f constituent i o n s i n t h e  perfusion m e d i u m , o r application o f pharmacological agents, p r o v i d e d insight into HC0 "-dependent mechanism (seeTable 5). I n t h epresence o fHC0 ", 3  3  this  theremoval o f  e x t r a c e l l u l a r C l " ( [ C l ~ ] ) (solution 8, T a b l e 2 ) r e s u l t e d i n a r e v e r s i b l e p H j i n c r e a s e o f 0 . 2 8 0  ± 0 . 0 4p Hunits (n=2;  Figure 4). A s depicted i n Figure 5, t h eapplication o f 2 0 0u M  D I D S , a ninhibitor o fHC0 7C1" 3  exchange, r e d u c e d p H j b y 0 . 0 8± 0 . 0 4p Hunits  Figure 5 also demonstrates that p H j immediately returned t o n o r m a l resting levels D I D S w a swashed f r o m t h eextracellular m e d i u m .  (n=3). when  A c c o r d i n g t o these results, it w o u l d  appear that C l " a n d H C 0 3 " - d e p e n d e n t m e c h a n i s m s m a y play a role regulating  steady-state  pHj atr o o m temperature. To further investigate t h erole o fHC0 " 3  i n maintaining steady-state p H j at r o o m  temperature, t h e next series o f experiments explored t h e modulation o f p H j during t h e t r a n s i t i o n f r o m H C 0 3 " - f r e e (solution 1, T a b l e 1 ) i n t o H C O 3 " - c o n t a i n i n g  (solution 6,  Table 2 ) perfusion media at a constant p H . A s shown i nFigure 6 A , such a  manoeuvre  was  subsequent  0  m a r k e d b y a n initial acidification d u e t o t h e influx o f C 0  hydration to carbonic acid.  2  a n dits  T h i s brief fall i n p H ; w a s followed  b y a  sustained  alkalinization, presumably d u e to t h e activation o f HC03"-dependent acid  extrusion  43  mechanisms. experiments  T h i s result i sr e f l e c t e d i n t h em o r e performed  i n t h epresence  alkaline resting p H j observed i n  o f HCO3- a s c o m p a r e d  with  experiments  c o n d u c t e d i n t h e a b s e n c e o f HCO3" a t r o o m t e m p e r a t u r e ( s e e T a b l e 5; F i g u r e s 3 A B).  A ss h o w n i nF i g u r e 6 B , t h e t e n d e n c y o f p H j t o s h i f t t o w a r d s a m o r e a l k a l i n e  and value  d u r i n g perfusion w i t h H C C ^ ' - c o n t a i n i n gm e d i u m w a s inhibited b y2 0 0 u . M D I D S . O n r e t u r n t o HCO3"- a n d D I D S - f r e e m e d i u m , t h e r e w a s a t r a n s i e n t i n c r e a s e i n p H j d u e t o t h e e f f l u x o f CO2, a f t e r w h i c h p H j f e l l t o t h e n o r m a l r e s t i n g l e v e l s o b s e r v e d u n d e r buffered perfusion conditions. HC0 "/C1" 3  exchange  HEPES-  T h e s e results s u g g e s t the c o n t r i b u t i o n o fs o m e f o r m o f  t o the maintenance  o fsteady-state  p H j a tr o o m temperature.  A  detailed anaylsis o f N a - d e p e n d e n t a c i d e x t r u s i o n m e c h a n i s m s w a s c a r r i e d o u t a t 37°C +  (see below).  H o w e v e r , a t r o o m t e m p e r a t u r e a n d i n t h e a b s e n c e o f HCO3", t h e r e m o v a l o f  extracellular N a  +  {solution 2, T a b l e 1 ) r e s u l t e d i n a n i m m e d i a t e i n t r a c e l l u l a r a c i d i f i c a t i o n  (see F i g u r e 17), w h i c h indicates that a N a - d e p e n d e n t , H C 0 3 " - i n d e p e n d e n t a c i d extruder +  contributes t othe p r e s e r v a t i o n o f a stable resting p H j .  Regulation of steady state pHj at  37°C:  I n n o m i n a l l y H C 0 3 " - f r e e H E P E S b u f f e r e d m e d i u m a t 3 7 ° C (solution 1, T a b l e 1 ; pH  7 . 3 4 ) , s t e a d y - s t a t e p H j w a s m a i n t a i n e d a t 7 . 2 3 ± 0 . 0 3 ( n = 2 9 ; s e e F i g u r e 3C). A t t h i s  0  temperature, changes t othe ionic composition o f the perfusing m e d i u m h a d a moderate influence o nsteady-state  pHj.  T h e s e r e s u l t s a r e s u m m a r i z e d T a b l e 5.  F i g u r e 7 , the r e m o v a l o fextracellular N a  +  ([Na ] ) +  0  A sshown i n  f r o m the H E P E S - b u f f e r e d  m e d i u m  (solution 2, T a b l e 2 ) c a u s e d a 0.53 ± 0 . 0 5 p H u n i t f a l l i n p H j ( n = 5 ) ; t h e r e - i n t r o d u c t i o n o f [Na ] +  0  caused a return o f p H j t osteady-state levels.  Na -dependent +  conditions.  acid extrusion mechanisms  H o w e v e r , the r e m o v a l o f[Cl"]  c h a n g e the steady state p H j (n=3;  0  T h i s result suggests the presence o f  w h i c h are operational under  steady-state  (solution 3, T a b l e 3 ) d i d n o t  significantly  F i g u r e 8), s u g g e s t i n g the a b s e n c e o f C F - d e p e n d e n t p H j  r e g u l a t i n g m e c h a n i s m s o p e r a t i n g u n d e r H C 0 3 " - f r e e c o n d i t i o n s a t 37°C.  44  Figure 9 s h o w s that the application o f 50 u M EIPA, a p h a r m a c o l o g i c a l inhibitor of Na /H +  +  exchange in a wide variety o f cell types (Clark a n d Limbird, 1991), did not  alter the resting p H j (n=3).  Similarly, the application o f EIPA  after 5 minutes o f [ N a ] +  0  free perfusion d i d not influence the acidification caused b y [ N a ]  removal (n=3;  +  10).  0  Figure  F i g u r e 1 0 a l s o d e m o n s t r a t e s that p H j r e b o u n d e d b a c k to its s t e a d y - s t a t e v a l u e  [Na ]  w a s returned to the perfusion solution, despite the c o n t i n u e d presence o f  +  0  M G C M A , a n o t h e r a m i l o r i d e a n a l o g u e ( A m o r o s o et al, inhibitor of N a  / H e x c h a n g e ( S c h m i d et al,  +  +  1992;  EIPA.  1991), and H O E 694, a  W o l l et al,  after  novel  1993), were both applied  at 1 0 0 p M b u t w e r e also f o u n d to h a v e n o effect o n steady-state p H j at 3 7 ° C (n=3 each compound;  F i g u r e 11).  for  It t h e r e f o r e a p p e a r s t h a t t h e N a - d e p e n d e n t a c i d e x t r u s i o n +  m e c h a n i s m p r e s e n t o n t h e s e n e u r o n e s is n o t sensitive to i n h i b i t i o n b y k n o w n b l o c k e r s Na /H +  of  exchange.  +  I n H C 0 3 " - c o n t a i n i n g p e r f u s i o n m e d i u m at p H steady-state p H j w a s 7.13 ± 0.01 (n=44;  3  exposure  to [Na ] -free +  0  Removing [Na ] +  7 C 0 2 b u f f e r i n g c o n d i t i o n s (solution 13,  0.04 p H unit fall in p H j (n=8;  T a b l e 5).  T a b l e 3),  0  f r o m the perfusing  T a b l e 3) c a u s e d a 0.65  A s s h o w n in F i g u r e 12, p H  ;  fell rapidly  m e d i u m , reached a m i n i m u m in less than 10 minutes,  i m m e d i a t e l y returned to steady-state  levels u p o n the re-introduction o f [Na+] . +  +  +  exchanger.  p r e c i s e d e s c r i p t i o n o f this a c i d e x t r u s i o n m e c h a n i s m , o t h e r t h a n its d e p e n d e n c e o n 3  T h e s e results are  w i t h p r e v i o u s observations s h o w i n g the inability o f 50 p M E I P A to influence p H , i n t h e a b s e n c e o f HCO3" a t 3 7 ° C ( s e e F i g u r e s 9 a n d  10).  and  over a  F i g u r e 13), w h i c h prevents a  a n d capacity to operate in the presence or absence o f HC0 ".  on  steady-state  However, application of 50 p M EIPA  10 m i n u t e p e r i o d d i d not alter resting p H j (n=3;  ±  This  0  result indicates the d o m i n a n c e o f N a - d e p e n d e n t acid extruders regulating p H j at 3 7 ° C , p o s s i b l y a N a / H  the  Figure 3D), a value lower than in HC03"-free,  H E P E S - b u f f e r e d m e d i u m at the s a m e t e m p e r a t u r e . solution under H C 0  7 . 3 6 (solution 12,  0  more N a  +  consistent steady-state  45  A s steady state p H j a t r o o m temperature a p p e a r e d t o b e d e p e n d e n t o n exchange,  f u r t h e r s t u d i e s e x p l o r e d t h e s e n s i t i v i t y o f p H j a t3 7 ° C  HC0 7C1" 3  t ot h e r e m o v a l o f  c o n s t i t u e n t i o n s a n d t h e a p p l i c a t i o n o fb l o c k e r s o ft h e c a t i o n e x c h a n g e r .  S h o w n i n Figure  1 4 A , r e p l a c i n g [ C l " ] w i t h g l u c o n a t e i n t h e p r e s e n c e o f H C 0 " {solution 14, T a b l e 3 ) 0  3  c a u s e d a g r a d u a l p H j increase o f0.19 ± 0.01 p H units (n=5). T h i s 0 [ C l ' ] - i n d u c e d rise i n 0  p H j a t 3 7 ° C w a ss i m i l a r t o ,t h o u g h slightly s m a l l e r than, t h eincrease i n p H j i n d u c e d b y the s a m e m a n o e u v r e a tr o o m t e m p e r a t u r e (see F i g u r e 4 ) . U p o n substitution o f[ C l " ] into t h e perfusing buffer, p H j returned t o its steady-state level.  0  back  T h ei n t r o d u c t i o n o f  200 u . M D I D S , applied i ncombination with 0 [Cl"] perfusion, abolished t h e0 [Cl"] 0  i n d u c e d p H j rise (n=3;  Figure 14A).  0  Furthermore, t h eapplication o fD I D S 5 minutes  after t h e r e m o v a l o f [Cl"] prevented t h e sustained alkalinization associated w i t h t h e 0  a b s e n c e o f [ C l " ] a n d resulted i na d e c l i n e o fp H j b a c k t o w a r d s s t e a d y state levels despite 0  continued perfusion with Cl"-free m e d i u m (n=4;  Figure 14B).  W h e n [Cl"] w a s re0  introduced to t h eperfusion solution i n t h econtinued presence o f 2 0 0 u MD I D S , p H j c o n t i n u e d t o fall, overshooting steady-state p H j levels t o rest -0.05  p H units  below  O n c e t h eD I D S w a sremoved, p H j slowly returned to baseline  levels.  A p p l i e d a l o n e , 2 0 0 u M D I D S d i d n o ts i g n i f i c a n t l y a l t e r s t e a d y - s t a t e p H j a t 3 7 ° C  (Table  n o r m a l levels.  5, a n d F i g u r e 15).  T h i s r e s u l t d i f f e r s f r o m t h a t o b s e r v e d i n HCO3 " - c o n t a i n i n g m e d i u m a t  r o o m t e m p e r a t u r e i nw h i c h steady-state p H j w a ssignificantly r e d u c e d b y 2 0 0 u M  D I D S  F i g u r e 5 ) . T h e s e r e s u l t s s u g g e s t t h a t a t 3 7 ° C , t h o u g h a D I D S - s e n s i t i v e HCO3VCI"  (n=3;  exchanger m a y b e present, steady-state p H ; is primarily governed b y t h eactivity o f the Na /H +  +  exchanger. I n a m a n n e r s i m i l a r t o e x p e r i m e n t s p e r f o r m e d a t r o o m t e m p e r a t u r e , t h ee f f e c t s o n  p H j c a u s e d b y t h e t r a n s i t i o n f r o m H C 0 3 " - f r e e (solution 1, T a b l e 1 ) t o H C O 3 " - c o n t a i n i n g (solution 12, T a b l e 3 ) p e r f u s i o n m e d i a a t c o n s t a n t p H  0  were  investigated  a t 37°C.  Interestingly, t h e n e t alkalinization that occurred o nthe transition f r o m a H C 0 3 " - f r e e t o a H C 0 3 " - c o n t a i n i n g m e d i u m a t r o o m t e m p e r a t u r e ( s e e F i g u r e 6 A )w a s n o t o b s e r v e d a t  46  3 7 ° C ( n = 1 3 ; F i g u r e 16). this temperature was HEPES-buffered  In fact, the steady-state p H ; i n H C 0 7 C 0 2 - b u f f e r e d m e d i u m at 3  significantly lower than the observed  c o n d i t i o n s ( T a b l e 5).  level under  HC0 "-free 3  In contrast to those experiments p e r f o r m e d  r o o m t e m p e r a t u r e (see F i g u r e 6 B ) , the a p p l i c a t i o n 2 0 0 p M D I D S at 3 7 ° C d i d n o t  at  affect  the p H ; response to the introduction o f H C 0 " - c o n t a i n i n g perfusion m e d i u m (Figure  16),  again  towards  the  B y r e s p o n d i n g to c h a n g e s in the extracellular concentrations o f b o t h H C 0 "  and  3  indicating  the  relative  unimportance  of  HC0 7C1~  exchange  3  m a i n t e n a n c e o f steady-state p H ; at this t e m p e r a t u r e .  Na -dependent or -independent anion exchange: +  3  Cl", especially  at r o o m t e m p e r a t u r e ,  the  neurones  employed  in these  experiments  indicated their ability to regulate p H ; t h r o u g h a n i o n e x c h a n g e . T o determine w h e t h e r suspected HC0 7C1"  exchanger present on these neurones was dependent o n extracellular  3  Na ,  a n experiment w a s p e r f o r m e d in the absence o f HC0 "  +  3  which [Na ]  the  at r o o m t e m p e r a t u r e  in  w a s r e m o v e d i n i t i a l l y f r o m t h e p e r f u s i o n s o l u t i o n (solution 2, T a b l e 1 ) .  +  0  illustrated in F i g u r e 17, this c a u s e d p H j to fall, but the s u b s e q u e n t introduction o f  A s  HC0 " 3  (solution 7 , T a b l e 2 ) r e s u l t e d i n a s l o w i n c r e a s e i n p H j d e s p i t e t h e c o n t i n u e d a b s e n c e [Na ] +  0  (n=3).  Since p H j recovered in the presence o f HC0 "  this result suggests that N a - i n d e p e n d e n t HC0 7C1" +  3  neurones  to  regulate  extracellular N a  +  0  in p H j (n=3;  p H j b a c k to  0  exchange was being utilized by Using  an  alternative  the  approach, at  T a b l e 3). A f t e r letting p H j fall to a plateau, perfusate d e v o i d o f [ C l " ]  F i g u r e 18). +  levels.  +  3  (solution 15,  of external N a  resting  [Na ] ,  was again eliminated but n o w from H C 0 7 C 0 2 - b u f f e r e d m e d i u m  +  3 7 ° C (solution 13, and [Na ]  a n d in the absence  3  of  T a b l e 15) w a s introduced, w h i c h c a u s e d a 0.14 ± 0.03  increase  This 0 [Cl"] -induced intracellular alkalinization in the absence 0  w a s similar to, t h o u g h smaller than, the 0 [ C l " ] - i n d u c e d alkalinization 0  observed in the corresponding experiment performed in the presence o f [ N a ] +  0  (see  Figure 14A). In the absence o f [Na ] , the return o f [Cl"] p r o d u c e d a brief acidification +  0  0  0  47  f o l l o w e d b y a gradual p H j recovery towards steady-state levels.  This slow recovery  probably the result o f the activation o f Na -independent acid extrusion  was  mechanisms.  +  Overall, these results suggest that these neurones are able to regulate p H j utilizing a N a +  independent f o r m o f the HC0 7C1"  exchanger.  3  M o d u l a t i o n o f p H j b y shifts in p H The  steady-state  0  a n d the application of w e a k acids a n d  p H j o f the neurones perfused with m e d i a containing  e q u i l i b r a t e d w i t h 5 % CO2  Increasing p H  0  Figure 19A).  b e l o w 7.35 resulted in a decrease in p H ; b e l o w n o r m a l resting levels:  pH  0  Reducing  when p H  l o w e r e d to 7.02 a n d 6.56, p H ; stabilized at 6.90 ± 0.07 a n d 6.53 ± 0.01, (n=3).  3  f r o m 7.35 to 7.75 a n d then 8.02 c a u s e d p H j to  r e a c h levels o f 7.41 ± 0.01 a n d 7.54 ± 0.01, respectively (n=3; 0  HC0 ~  i n b a l a n c e air at 3 7 ° C w a s s t r o n g l y i n f l u e n c e d b y the p H o f the  extracellular environment.  pH  bases:  was  0  respectively  A s s h o w n in Figure 19B, a linear regression analysis o f the relationship  between  a n d p H j yielded the following relationship: pHj = 1.990 + 0 . 6 9 9 x p H  The modulation of pH  0  (Equation 19)  o  at r o o m t e m p e r a t u r e i n the p r e s e n c e o f H C 0 " 3  o n p H j (data not shown). dependence of pHj on p H  0  h a d a similar effect  U n d e r these conditions, the relationship representing  the  was:  pH, = 1.240 + 0 . 8 0 7 x p H  (Equation 20)  o  T h e s e results s u g g e s t that p H j is steeply d e p e n d e n t o n p H b a c k to n o r m a l steady-state values until p H  0  0  a n d that p H j is n o t  regulated  is n o r m a l i z e d .  T h e extracellular a p p l i c a t i o n o f w e a k acids o r b a s e s at constant p H s h o w n to alter p H j ( S h a r p a n d T h o m a s , 1981;  Roos and Boron, 1981).  0  has  Accordingly,  p r o p i o n a t e a n d t r i m e t h y l a m i n e ( T M A ) w e r e a p p l i e d to h i p p o c a m p a l n e u r o n e s at temperature in H C 0 7 C 0 2 - b u f f e r e d m e d i u m in order to investigate 3  been  room  their ability  to  m o d u l a t e p H j a t a c o n s t a n t p H . I n e x p o s i n g t h e n e u r o n e s t o 2 0 m M p r o p i o n a t e (solution 0  9,  T a b l e 2), the undissociated f o r m o f the a c i d readily crossed the cell  membrane,  48  w h e r e a s t h e d i s s o c i a t e d f o r m o f t h e a c i d , d u e to its n e g a t i v e c h a r g e , r e m a i n e d membrane impermeant. pK  a  O n c e a c r o s s t h e m e m b r a n e t h e a c i d d i s s o c i a t e d a c c o r d i n g to its  to release protons, thus acidifying the cell's interior ( F i g u r e 2 0 A ) .  of acid entering  the  cells was  minimal when  propionate concentration, p H j was lowered while p H pHj  decrease was  0.21  relatively  ±  0.05  c o m p a r e d to 0  the  Since the total  extracellular  w a s m a i n t a i n e d at 7.32.  T h e initial  p H units (n=3), after w h i c h p H j gradually  t o w a r d s n o r m a l steady-state levels d u e either to the s l o w p e r m e a t i o n o f the f o r m o f the acid through the m e m b r a n e or the activation o f acid extrusion  amount  increased dissociated  mechanisms.  T h e latter is the m o s t l i k e l y e x p l a n a t i o n b e c a u s e the r e m o v a l o f p r o p i o n a t e c a u s e d a b r i e f intracellular alkalinization followed b y gradual return to n o r m a l steady-state p H j levels. F i g u r e 2 0 B illustrates the o p p o s i t e c h a n g e i n p H ; at r o o m t e m p e r a t u r e r e s u l t i n g f r o m the a p p l i c a t i o n o f 1 0 m M T M A , a w e a k o r g a n i c b a s e {solution 10,  T a b l e 2). T h e a p p l i c a t i o n  o f extracellular T M A caused a immediate intracellular alkalinization o f 0.38 ± 0.04 units w h i c h gradually returned towards baseline (n=3).  p H  O n c e r e m o v e d , the intracellular  m i l i e u w a s briefly acidified f o l l o w e d b y a p H j r e c o v e r y to resting baseline  levels.  49  T a b l e 5: S t e a d y - s t a t e p H ; i n H C 0 " - f r e e a n d H C 0 3 " - c o n t a i n i n g m e d i a at r o o m t e m p e r a t u r e a n d at 3 7 ° C , a n d the c h a n g e i n p H j c a u s e d b y e x p o s u r e to the experimental solutions indicated. 3  T e m p  ApHj  pHj  room  6.85 ±  H C 0 - / C 0 b u f f e r : (solution 6) 0 [ C l " ] (solution 8) 2 0 0 p M D I D S ( i n solution 6)  room room room  7.15  H E P E S b u f f e r : (solution 1) 0 [Na ] (solution 2) 0 [ C T ] (solution 3) 5 0 p M E I P A ( i n solution 1)  3 3 3 3  7 7 7 7  ° ° ° °  C C C C  7.23 ±  H C 0 - / C 0 b u f f e r : (solution 12) 0 [Na ] (solution 13) 0 [ C T ] (solution 14) 5 0 p M E I P A ( i n solution 12) 2 0 0 p M D I D S ( i n solution 12) 0 [ C l " ] + 2 0 0 p M D I D S (solution 14)  3 3 3 3 3 3  7 7 7 7 7 7  ° ° ° ° ° °  C C C C C C  7.13 ± 0 . 0 1  H E P E S buffer: 3  (solution 1)  2  0  +  0  0  3  2  +  0  0  0  n  0.04  25  ±0.03 0.04 0.04  22 2 3  -0.53 ± 0.05 0.00 ± 0.02 0.01 ± 0 . 0 1  29 5 3 3  -0.65 0.19 0.01 -0.01 0.01  44 8 5 3 3 2  0.28 ± -0.08 ± 0.03  S o l u t i o n recipes are refered to i n parentheses (see T a b l e s 1 to temperature ( T e m p ) w a s either r o o m temperature (18 - 2 2 ° C ) or 3 state p H , u n d e r the listed b u f f e r i n g c o n d i t i o n s , a n d A p H j is the units) c a u s e d b y exposure to the indicated experimental solutions. t h e m e a n o f n c o v e r s l i p s (i.e. c e l l p o p u l a t i o n s ) s t u d i e d , ± S.E.M.  ± 0.04 ± 0 . 0 1 ± 0.02 ± 0 . 0 1 ± 0 . 0 1  3). T h e e x p e r i m e n t a l 7 ° C . p H j is the s t e a d y change in p H j (in p H V a l u e s are reported as  50  Figure 3. Distribution of steady-state pHj. A.  Distribution of steady-state pHj at room temperature in the absence of HCO3"  (solution 1, pH 7.32). B. Distribution of steady-state pHj at room temperature in the presence of HCO3" (solution 6, pH 7.32). C . Distribution of steady-state pHj at 37°C in the absence of HCO3" (solution 1, pH 7.34). D. Distribution of steady-state pH; at 37°C in the presence of H C 0 " (solution 12, pH 7.36). The mean steady-state pHj under each 3  of the four conditions is indicated (± S.E.M.), where n equals the total number of coverslips (i.e. cell populations) studied. The solid lines represent the least squares Gaussian fit to the data.  51  ^ —  i '  i  I  I  I  I  r-  1  1  ,  6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4  "  i  6.8  1  6.9  I  I  I  I  I  7.0  7.1  7.2  7.3  7.4  1  7.5  = 1  7.6  52  Figure 4. Effect of 0 [Cl"] on steady-state pHj in the presence of H C O 3 " at room temperature. 0  T h e r e m o v a l o f [ C l " ] (solution 8) a t a c o n s t a n t p H ( 7 . 3 1 ) f o r t h e p e r i o d i n d i c a t e d b y t h e bar above the trace resulted in an -0.3 p H unit increase in resting p H j (n=2). p H j returned to n o r m a l l e v e l s w i t h t h e r e - i n t r o d u c t i o n o f [ C l ~ ] . S h o w n a l s o is ao n e p o i n t c a l i b r a t i o n w i t h 10 u M n i g e r i c i n at p H 7.00. T h e trace is am e a n o f d a t a s i m u l t a n e o u s l y o b t a i n e d f r o m 2 0 c e l l s r e c o r d e d o n as i n g l e c o v e r s l i p . 0  0  0  53  0  10  20  30  40  50  Time (minutes)  60  70  80  90  54  Figure 5. Effect of DIDS on steady-state pHj in the presence of H C O 3 - at room temperature.  The addition trace caused at 7.32. p H j data obtained  o f 2 0 0 u M D I D S t o solution 6 f o r t h e p e r i o d i n d i c a t e d b y t h e b a r a b o v e t h e a n ~0.1 p H unit intracellular acidification (n=3), while p H was maintained w a s r e s t o r e d to n o r m a l l e v e l s o n r e m o v a l o f D I D S . T h e t r a c e is am e a n o f f r o m 3 0 c e l l s r e c o r d e d o n as i n g l e c o v e r s l i p . 0  55  10  20  30  Time (minutes)  40  50  60  56  Figure 6. Steady-state pHj in the presence and absence of H C 0 " / C 0 2 at room temperature. 3  A . T h e t r a n s i t i o n f r o m H E P E S - b u f f e r e d m e d i u m {solution 1, p H 7 . 3 2 ) t o H C 0 7 C 0 2 b u f f e r e d m e d i u m (solution 6, p H 7 . 3 2 ) a t r o o m t e m p e r a t u r e c a u s e d a b r i e f a c i d i f i c a t i o n , p r e s u m a b l y c a u s e d b y CO2 i n f l u x , f o l l o w e d b y a n e t a l k a l i n i z a t i o n o f ~ 0 . 3 p H u n i t s (n=7). T h e transition b a c k into H E P E S - b u f f e r e d m e d i u m w a s m a r k e d b y a m o m e n t a r y a l k a l i n i z a t i o n d u e t o CO2 e f f l u x , f o l l o w e d b y a f a l l i n p H j t o t h e n o r m a l r e s t i n g l e v e l s f o u n d i n t h e a b s e n c e o f HCO3". B. T h e n e t a l k a l i n i z a t i o n c a u s e d b y t h e t r a n s i t i o n i n t o HC03"/C02-buffered m e d i u m w a s abolished b y the presence 2 0 0 p M D I D S (n=5). Rather, there w a s a n acidification followed b y a s l o w recovery. E a c h trace, recorded f r o m separate coverslips, is data obtained f r o m 1 0 cells simultaneously. 0  0  3  Time  (minutes)  58  Figure  7.  Effect of 0 [Na+] on steady-state p H j in the absence of H C 0 " at 37°C. 0  3  The replacement of extracellular N a with N M D G {solution 2) at p H 7.35 caused an ~0.5 p H unit fall in pHj (n=5). The re-introduction of N a produced a rapid return to steady-state pHj levels. Shown also is a one point calibration with 10 u M nigericin at p H 7.00. The trace is a mean of data obtained from 6 cells recorded on a single coverslip. +  +  0  +  59  Time (minutes)  60  F i g u r e 8. E f f e c t o f 0 [ C l ]  0  at 37°C.  on steady-state p H j in the absence of HC0 " 3  T h e r e p l a c e m e n t o f e x t r a c e l l u l a r C l " w i t h g l u c o n a t e {solution 3) a t p H 7 . 3 3 f o r t h e period indicated b y the bar above the trace did not change resting p H j levels (n=3). T h e t r a c e is a m e a n o f d a t a o b t a i n e d f r o m 2 6 cells r e c o r d e d o n a s i n g l e c o v e r s l i p . C o m p a r e w i t h F i g u r e 4. 0  61  Time (minutes)  62  F i g u r e 9. E f f e c t o f E I P A o n s t e a d y - s t a t e p H j i n t h e a b s e n c e o f H C O 3 - a t 3 7 ° C . T h e a p p l i c a t i o n o f 5 0 p M E I P A t o solution 1 a t p H 7 . 3 5 f o r t h e p e r i o d i n d i c a t e d b y t h e s o l i d b a r d i d n o t s i g n i f i c a n t l y alter r e s t i n g p H j levels ( n = 3 ) . T h e t r a c e is a m e a n o f d a t a simultaneously obtained f r o m 6 cells recorded o n a single coverslip. 0  64  Figure 10. C o m b i n e d effect of 0 [ N a ] +  0  and E I P A on steady-state p H j in the absence  0fHCO - at 37°C. 3  The application of 50 pM EIPA 5 minutes after the removal of extracellular N a (solution 2) did not reverse the fall in pHj caused by 0 [Na ] , nor the return to resting pHj levels after the re-introduction of [Na ] (n-3). Throughout the experiment, p H was maintained at 7.33. The trace is a mean of data obtained from 23 cells recorded on a single coverslip. +  +  0  +  0  0  65  T i m e (minutes)  66  F i g u r e 11. Effect of M G C M A H C 0 - at 3 7 ° C .  a n d H O E 694 o n steady-state  p H j in the absence  of  3  A. T h e application of 100 u M MGCMA at p H 7.36 d i d not significantly c h a n g e steadystate p H j (n=3). B . Similarly, the application o f 100 u M H O E 694 ( p H 7.38) h a d n o effect o n n o r m a l r e s t i n g p H j l e v e l s ( n = 3 ) . T r a c e A is a m e a n o f d a t a o b t a i n e d f r o m 8 cells, w h e r e a s t r a c e B is a m e a n o f d a t a o b t a i n e d f r o m 13 cells, e a c h r e c o r d e d o n s e p a r a t e coverslips. 0  0  Time (minutes)  68  Figure 12. Effect of 0 [Na ] on steady-state pHj in the presence of HCO3- at 37°C. +  0  The removal of extracellular N a (solution 13, p H 7.35) produced an -0.60 pH unit fall in pHj (n=8). pHj rebounded to normal resting levels with the re-introduction of extracellular N a . Also shown is a one point calibration with 10 p M nigericin at pH 7.00. The trace is a mean of data obtained from 35 cells recorded on a single coverslip. +  0  +  69  10  20  30  40  Time  (minutes)  50  60  70  80  70  Figure 13. Effect of E I P A on steady-state p H j in the presence of H C O 3 - at 3 7 ° C .  The addition of 50 u M EIPA to solution 12 at p H 7.32 did not significantly alter resting pHj levels (n=3). This result was also observed in the absence of H C 0 " as shown in Figure 9. The trace is a mean of data simultaneously obtained from 30 cells recorded on a single coverslip. 0  3  71  Time (minutes)  72  F i g u r e 14.  Effect of 0 [ C l ] , a n d the combined effect of 0 [ C T ] plus D I D S on 0  0  steady-state p H j in the presence of H C O 3 - at 3 7 ° C .  A . The removal of extracellular Cl" (solution 14) at p H 7.36 produced an -0.20 pH unit intracellular alkalinization (n=5). This 0 [Cl"] -induced pHj increase was completely inhibited by 200 u M DIDS (n=3). This trace is a mean of data obtained from 16 cells recorded on a single coverslip. B. At p H 7.36, the addition of 200 u.M DIDS 5 minutes after the removal of extracellular Cl" returned pHj to normal resting levels, with a small overshoot to acidic values (n=4). This trace is a mean of data obtained from 37 cells recorded on a different coverslip to A . 0  0  0  Time (minutes)  74  Figure 15. Effect of DIDS on steady-state pHj in the presence of H C O 3 - at 37°C. The application of 200 p M DIDS for the period indicated by the bar above the trace in the presence of HCO3" at 37°C (pH 7.33) did not significantly alter steady-state pHj (n=3). This result differs from that observed at room temperature (see Figure 5), in which DIDS reduced pHj by -0.10 pH units. This trace is a mean of data obtained from 44 cells recorded on the same coverslip. 0  75  Time (minutes)  76  Figure 16. Steady state pHj in the presence and absence of H C O 3 V C O 2 at 37°C. The transition from HEPES-buffered medium (solution 1, p H 7.35) to H C O 3 V C O 2 buffered medium (solution 12, p H 7.35) at 37°C caused an intracellular acidification such that the resulting pHj in the presence of H C O 3 " remained -0.1 pH units lower than resting pHj in the absence of H C O 3 " (n=13). With the re-introduction of HEPES-buffered medium, pHj briefly increased followed by a decline to the normal resting levels found in the absence of H C O 3 " . The presence of 200 u M DIDS did not influence the manner in which pHj responded to the transition from HCC^'-free to HC0 "-containing perfusion media at 37°C (n=3). The trace is a mean of data obtained from 16 cells recorded on the same coverslip. Compare with Figures 6A and B. 0  0  3  77  Time (minutes)  78  Figure 17. Effect of H C 0 - / C 0 on steady-state pHj during 0 [Na ] perfusion at room temperature. +  3  2  0  [ N a ] was removed from the HCC^'-free buffered media (solution 2) at p H 7.35, resulting in an immediate fall in pHj. The addition of H C 0 " during the period of continued 0 [ N a ] (solution 7) perfusion caused pH, to gradually recover (n=3). pHj immediately returned to normal levels when [ N a ] was re-introduced to the perfusion medium. The trace is a mean of data obtained from 34 cells recorded on a single coverslip. +  0  0  3  +  0  +  0  79  80  Figure 18. Effect of 0 [Cl"] during 0 [Na ] perfusion on steady-state pHj in the +  0  0  presence of H C 0 " at 3 7 ° C . 3  The removal of external N a from the HC03"/C0 -buffered medium at 37°C (solution 13, p H 7.37) caused a similar fall in pHj to that shown in Figure 12. The additional removal of extracellular Cl" during a sustained period of perfusion with 0 [Na ] (solution 15) caused an -0.15 pH unit increase in pHj (n=3). The re-introduction of [Cl"] caused pHj to fall back to a level observed prior to its removal, followed by a gradual increase in pHj probably due to the activity of Na -independent acid extrusion mechanisms (see Figure 17). pHj quickly recovered to normal levels when N a was again added to the perfusion medium. The trace is a mean of data obtained from 8 cells recorded on a single coverslip. +  2  0  +  0  0  +  +  81  Time (minutes)  82  Figure 19. Effect of changes in p H on steady-state pHj in the presence of H C O 3 " at 37°C. 0  A . I n c r e a s i n g p H f r o m a n o r m a l l e v e l o f 7.35 (solution 12) t o 7.75 (solution 21) a n d 8.02 (solution 22) c a u s e d a s i m i l a r t h o u g h s m a l l e r i n c r e a s e i n pHj. D e c r e a s i n g p H t o 7.02 (solution 20) a n d 6.56 (solution 17) c a u s e d pHj t o f a l l t o a c i d i c l e v e l s . T h i s t r a c e i s a m e a n o f d a t a o b t a i n e d f r o m 35 c e l l s o n a s i n g l e c o v e r s l i p . B. L i n e a r r e g r e s s i o n a n a l y s i s o f t h e d e p e n d e n c e o f pHj o n pH . T h e e q u a t i o n d e s c r i b i n g t h i s r e l a t i o n s h i p is: pHj = 1.99 + 0.699xpH (R = 0.978, n=3 coverslips) 0  0  0  2  o  84  Figure 20. Effect of propionate and T M A on steady-state pHj in the presence of H C O 3 " at room temperature.  A . T h e a p p l i c a t i o n o f 2 0 m M p r o p i o n a t e (solution 9) a t a c o n s t a n t p H ( 7 . 3 0 ) c a u s e d a n immediate intracellular acidification o f -0.20 p H units (n=3) followed b y a gradual r e c o v e r y to baseline levels. T h e r e m o v a l o f propionate f r o m the extracellular m e d i u m c a u s e d p H , t o r a p i d l y i n c r e a s e a f t e r w h i c h i t r e c o v e r e d t o n o r m a l r e s t i n g l e v e l s . B. T h e a p p l i c a t i o n o f 1 0 m M T M A (solution 10) a t a c o n s t a n t p H ( 7 . 3 1 ) c a u s e d a n i m m e d i a t e intracellular alkalinization o f -0.40 p H units (n=3) followed b y a s l o w recovery to baseline levels. T h e r e m o v a l o f T M A caused an immediate intracellular acidification f o l l o w e d b y ar e t u r n t o s t e a d y - s t a t e p H j l e v e l s . R e c o r d e d o n s e p a r a t e c o v e r s l i p s , t r a c e A is am e a n o f d a t a o b t a i n e d f r o m 1 2 c e l l s , w h e r e a s t r a c e B is am e a n o f d a t a o b t a i n e d f r o m 2 0 cells. 0  0  Time (minutes)  86  p H , R E C O V E R Y F R O M A N I M P O S E D A C I D  L O A D  The investigation of p H j regulatory mechanisms was expanded by inducing intracellular acidification, while maintaining a constant p H , a n d studying the  subsequent  0  recovery.  an  T h e examination of acid load recoveries provides additional information  mechanisms  regulating p H j , since excessive intracellular protons  on  will activate  acid  extrusion m e c h a n i s m s (Roos a n d B o r o n , 1981). Acid transients w e r e p r o d u c e d using NH  + 4  -prepulse technique  e x p o s e d to N H  + 4  (Boron and D e  Weer,  through the addition o f 20  (solution 4, T a b l e 1 ; solution 11,  1976), in w h i c h the neurones  m M NH C1 4  T a b l e 2 ; solution 16,  to the p e r f u s i o n  the were  solution  T a b l e 3). A s s h o w n i n F i g u r e 21,  this e x p o s u r e causes a n i m m e d i a t e intracellular alkalinization d u e to the passive influx o f N H , the dissociated f o r m o f N H  +  3  4  ,  a n d its i m p e n d i n g h y d r a t i o n to f o r m N H  + 4  and O H ' .  The m e m b r a n e exhibits a slight permeability to NH ,  a n d thus the initial p H , rise  slowly d a m p e n e d b y the influx o f extracellular N H  driven by an  +  4  + 4  electrochemical gradient (Boron and D e Weer, 1976). NH C1, 4  3 minutes  permeable N H by  the  3  opposing  inwardly-directed  T h e r e m o v a l o f the  external  after its o r i g i n a l a p p l i c a t i o n , results i n a n e x o d u s o f t h e  , leaving behind significant concentrations o f NH electrical  gradient  dissociation of intracellular NH b e l o w its i n i t i a l r e s t i n g v a l u e .  + 4  generated  into N H  3  by  the  + 4  ,  highly  trapped in the  membrane  is  cells  potential.  The  liberates protons, thus forcing p H j to  fall  T h e recovery f r o m this i m p o s e d acidification b a c k  to  s t e a d y - s t a t e p H ; l e v e l s is a n e s t a b l i s h e d m e a n s o f a n a l y z i n g the m e c h a n i s m s i n v o l v e d i n p H j regulation, as first d e m o n s t r a t e d b y M e s s e t e r a n d Siesjo (1971). D a t a g e n e r a t e d f r o m the a c i d l o a d e x p e r i m e n t s a r e s u m m a r i z e d i n T a b l e 6.  The  initial rate o f p H ; r e c o v e r y to resting levels w a s quantified 10 s e c o n d s after the  peak  acidification. T h e instantaneous r e c o v e r y rates w e r e also d e t e r m i n e d at t  5 0  and t , 8 0  which  a r e d e f i n e d as t h e t i m e at w h i c h p H j r e c o v e r e d to 5 0 % a n d 8 0 % o f its p r e - a c i d l o a d l e v e l , respectively.  p H j at t Q w a s calculated b y t a k i n g the resting p H j b e f o r e the a p p l i c a t i o n o f 5  87  NH  + 4  (the "preload pH,"), and then subtracting 50% of the resulting net change in pH;  (the "net pHj decrease").  The net pHj decrease was calculated by subtracting the  minimum pH, reached during the acidification from the preload pH{. Similarly, pHj at t  80  was determined by subtracting 20% of the NH -induced net pHj decrease from the +  4  preload pHj. As an indicator of intracellular buffering power, the increase in pHj (the "pH, increase") caused by the 3 minute exposure to N H  + 4  was also measured by taking  the difference between the preload pHj and the pHj immediately prior to the removal of extracellular N H  + 4  (see Table 6). At room temperature, the mean pHj increases after the  3 minute exposure to N H  + 4  were 0.52 ± 0.05 pH units (n=6) and 0.38 ± 0.01 p H units  (n=7) in the absence and presence of H C 0 ~ , respectively. These values markedly 3  decreased when studies were performed at 37°C. In cells exposed to a solution buffered with HEPES, the 3 minute application of N H  + 4  elicited a 0.27 ± 0.03 pH unit rise (n=12),  whereas pHj increased by only 0.11 ±0.01 pH units (n=16) during the N H solutions buffered with HCO37CO2.  + 4  exposure in  These results suggest that both an increase in  temperature and the presence of HCO3" lead to an enhanced intracellular buffering capacity (see Roos and Boron, 1981). In any given experiment, the rate of pHj recovery was greatest at the acid peak and then declined, in a linear fashion, as pHj recovered to normal levels. phenomenon is graphically depicted in Figure 22A.  This  These data, though obtained from a  single experiment, represent the trend found in all neurones under all buffering conditions. The rate of pHj recovery was not related to. the preload pHj nor the minimum pHj reached during acidification in 14 randomly selected experiments (Figure 23B). Likewise, as illustrated in Figure 23 C, the net decrease in pHj caused by the N H  + 4  prepulse was not a factor in evaluating the rate of pHj restoration. In accordance with these observations, is was not necessary to replicate exact acid load conditions throughout these acid load recovery studies.  88  Recovery from an acid load at room temperature: N e u r o n e s p e r f u s e d a t r o o m t e m p e r a t u r e i n t h e a b s e n c e o f H C 0 " {solution 1, 3  T a b l e 1) r e c o v e r e d f r o m a n N H  + 4  1.31 x I O " p H u n i t s / s e c o n d (n=6; 3  HCO3"  - i n d u c e d acidification at a n m a x i m u m initial rate  of  T a b l e 6 , r o w a). F i g u r e 2 3 s h o w s t h a t t h e a d d i t i o n o f  (solution 6, T a b l e 2 ) d u r i n g t h e r e c o v e r y p o r t i o n o f t h e e x p e r i m e n t c a u s e d a  significant restoration.  increase  in the  acid extrusion  rate throughout  the  entire  course  of  U n d e r these conditions, the initial rate o f p H j recovery f r o m a n  acidification was 2.55 x IO  - 3  p H units/second (n=7;  the  induced  T a b l e 6 , r o w b). A s e x p e c t e d f r o m  d a t a p r e s e n t e d earlier r e g a r d i n g the r e g u l a t i o n o f steady-state p H j at r o o m  temperature  (see F i g u r e 6 A ) , F i g u r e 23 also illustrates that the restoration o f p H j f r o m a n a c i d l o a d in t h e p r e s e n c e o f HCO3" c o n t i n u e d t o a h i g h e r r e s t i n g p H j t h a n t h e p r e v a i l i n g s t e a d y - s t a t e level f o u n d w h e n the neurones were being perfused with HEPES  buffered medium.  Initial e x p e r i m e n t s p e r f o r m e d at r o o m t e m p e r a t u r e i n the p r e s e n c e o f H C 0 "  were  3  c o n d u c t e d t o a s s e s s t h e c o n t r i b u t i o n o f t h e HCO37CI" e x c h a n g e r t o t h e r a t e o f p H ; recovery following an acid load.  F i g u r e 24 illustrates that the application o f  2 0 0 p M  D I D S d u r i n g recovery caused a significant reduction in the rate o f acid efflux (n=3),  as  e x p e c t e d g i v e n t h e c o n t r i b u t i o n o f HCO37G" e x c h a n g e t o w a r d s t h e m a i n t e n a n c e  of  steady-state p H j at r o o m temperature.  T h e initial rate fell f r o m 2.55  x 10~  p H  3  u n i t s / s e c o n d t o 0 . 9 5 x 1 0 " p H u n i t s / s e c o n d ( T a b l e 6 , r o w d). T h e t5Q r a t e i n t h e p r e s e n c e 3  of 200  p M D I D S was  also n o t a b l y r e d u c e d f r o m a control level o f 1.38  u n i t s / s e c o n d ( T a b l e 6 , r o w b) t o 0 . 6 3  x 10  - 3  qualitatively similar effect w a s observed in the t inhibitor of HC0 7C1" 3  x 10  p H  - 3  p H u n i t s / s e c o n d ( T a b l e 6 , r o w d). 8 0  A  r a t e ( T a b l e 6 , r o w d). A s D I D S i s a n  e x c h a n g e ( T h o m a s , 1976a), then the depletion o f [Cl"]j d u r i n g p H j  r e c o v e r y s h o u l d similarly alter the rate o f acid extrusion.  Figure 2 5 A depicts the ability  of the neurones to recover f r o m a n acid load w h i l e b e i n g perfused w i t h a [Cl"] -free 0  s o l u t i o n (solution 8, T a b l e 2 ) . increased to 3.14 x 10"  3  U n d e r these conditions, the initial rate o f recovery in fact  p H units/second (n=3;  T a b l e 6, r o w c), w h i c h m a y i n d i c a t e a  89  0 [Cl-] -induced acceleration o f the HC0 7C10  3  exchanger.  T h i s result also suggests that  the d u r a t i o n o f 0 [Cl"] perfusion w a s not l o n g e n o u g h to sufficiently deplete intracellular 0  Cl" stores, w h i c h w o u l d h a v e likely p r o d u c e d asimilar decrease in the p H j recovery rate a s t h a t c a u s e d b y t h e a p p l i c a t i o n o f D I D S ( s e e T a b l e 6 , r o w d). T h e f o r m e r p o s s i b i l i t y i s supported b y the experiment s h o w n in Figure 2 5 B in w h i c h the rapid recovery seen r e m o v i n g extracellular C l "was hindered w h e n the neurones were simultaneously  on  exposed  to 2 0 0 u M D I D S (n=2). T h i s m a n o e u v r e r e d u c e d the initial r e c o v e r y rate to 1.18 x 10" pH  units/second  ( T a b l e 6 , r o w e) c o m p a r e d t o a n i n i t i a l r a t e o f 3 . 1 4  x 10"  3  p H  3  u n i t s / s e c o n d i n t h e a b s e n c e o f D I D S ( T a b l e 6 , r o w c). O v e r a l l , t h e s e r e s u l t s s u g g e s t t h a t HCO37CI" e x c h a n g e i s u t i l i z e d b y t h e s e n e u r o n e s a t r o o m t e m p e r a t u r e t o r e c o v e r f r o m induced intracellular acidifications.  T h e inability o f 2 0 0 u M D I D S to c o m p l e t e l y  block  pHj restoration (see F i g u r e 24) indicates that other a c i d transporters, s u c h as a N a +  dependent, H C 0 3 " - i n d e p e n d e n t acid extrusion m e c h a n i s m , contribute to the regulation o f p H j at r o o m t e m p e r a t u r e .  T h e participation o f this latter m e c h a n i s m i n the r e c o v e r y  p H j after a n i m p o s e d a c i d i f i c a t i o n is d e s c r i b e d i n d e t a i l at 3 7 ° C (see  of  below).  Recovery from an acid load at 37°C:  A t 3 7 ° C a n d i n t h e a b s e n c e o f H C O 3 " (solution 1, T a b l e 1 ) , t h e recovered f r o m the acidifying NH units/second (n=12;  + 4  T a b l e 6, r o w / ;  p r e p u l s e at a n initial rate o f 5.56 F i g u r e 26).  neurones x 10"  p H  3  T h i s is h i g h e r t h a n the initial rate  of  r e c o v e r y u n d e r i d e n t i c a l b u f f e r i n g c o n d i t i o n s at r o o m t e m p e r a t u r e (see T a b l e 6, r o w  a).  Corresponding values for the t g a n d tg 5  0  rates w e r e also h i g h e r at 3 7 ° C  t e m p e r a t u r e ( T a b l e 6). p H j restoration w a s a b o l i s h e d w h e n [ N a ] +  0  t h a n at  was r e m o v e d f r o m the  p e r f u s a t e (solution 2, T a b l e 1 ) d u r i n g t h e r e c o v e r y p o r t i o n o f t h e e x p e r i m e n t F i g u r e 27), as expected  given the contribution o f N a - d e p e n d e n t m e c h a n i s m s to  m a i n t e n a n c e o f steady-state p H j (see above).  +  room  (n=2; the  H o w e v e r , studied o n 6cell populations, the  application o f 50 u . M EIPA d i d not influence the ability o f the neurones to recover f r o m  90  the  induced  intracellular acidification (Figure 28;  T a b l e 6 ,r o w  h).  T w o  other  c o m p o u n d s w e r e tested for their ability to inhibit p H j r e c o v e r y f r o m a n i n d u c e d a c i d l o a d via blockade  o f the  putative  application of 100 p M M G C M A  Na /H +  exchanger  +  present  on  these neurones.  The  (n=3) or 100 p M H O E 694 (n=3) failed to affect the rate  of steady-state p H j restoration f r o m an N H 4 - i n d u c e d acidification (data not  shown).  +  Similarly, as illustrated in Figure 29, 200 p M D I D S d i d not influence the rate o f p H j r e c o v e r y i n n e u r o n e s i n t h e a b s e n c e o f HCO3" ( n = 3 ) .  A s this control experiment  c o n d u c t e d i n t h e a b s e n c e o f HCO3", w h i c h i s a c o n s t i t u e n t i o n o f t h e  DIDS-sensitive  a n i o n e x c h a n g e r , it d e m o n s t r a t e s t h a t t h e a p p l i c a t i o n o f D I D S d o e s n o t c a u s e effects o n p H j recovery. T h o u g h the instantaneous initial rate, t  5 0  was  spurious  rate, a n d tgn rate for the  recovery f r o m a n acid load in the presence o f D I D S s e e m e d to decline in c o m p a r i s o n to c o n t r o l v a l u e s ( T a b l e 6 , r o w i), s u c h d i f f e r e n c e s w e r e n o t s t a t i s t i c a l l y s i g n i f i c a n t p a i r e d a n d u n p a i r e d / - t e s t c a l c u l a t i o n s (P > 0 . 0 5 ) .  under  T h e similarity between acid  r e c o v e r i e s i n t h e p r e s e n c e o r a b s e n c e o f 2 0 0 p M D I D S is better d e p i c t e d b y t h e t i m e r e q u i r e d f o r 5 0 % a n d 8 0 % p H , r e s t o r a t i o n ( T a b l e 6 , r o w i v e r s u s r o w j).  load actual  The  fact  t h a t D I D S h a d n o e f f e c t o n p H ; r e c o v e r y i n t h e a b s e n c e o f HCO3" s u p p o r t s t h e  premise  that stilbene derivatives only inhibit H C 0 " - d e p e n d e n t acid transporters ( T h o m a s ,  1976a;  3  Russell and Boron, 1976). 37°C,  O v e r a l l , t h e s e r e s u l t s s u g g e s t t h a t i n t h e a b s e n c e o f HCO3" a t  hippocampal neurones  recover f r o m an induced acidification utilizing a N a +  dependent, EIPA-insensitive acid extrusion mechanism. Interestingly,  a s s h o w n i n F i g u r e 2 6 , t h e a d d i t i o n o f HCO3" t o t h e  perfusing  m e d i u m (solution 12, T a b l e 3 ) t h r o u g h o u t t h e e n t i r e c o u r s e o f p H j r e s t o r a t i o n d i d  not  alter the rate o f a c i d l o a d r e c o v e r y at 3 7 ° C .  the  p r e s e n c e o f HCO3" a t 3 7 ° C w a s 4 . 7 7  x  T h e initial rate o f p H ; restoration i n  10" p H units/second ( n = 1 6 ; 3  T a b l e 6 , r o w j).  addition, the ability o f the neurones to recover f r o m a n i m p o s e d acidification w h i l e p e r f u s e d w i t h a HCO3-/CO2 b u f f e r e d s o l u t i o n w a s extracellular N a  +  (n=3;  In  being  nearly halted b y the r e m o v a l  of  F i g u r e 3 0 ) . T h e s e r e s u l t s r e f l e c t t h e l a c k o f i n f l u e n c e o f a HCO3"  91  / C l exchanger and the possible dominance of a Na -dependent, HC0 -independent -  +  -  3  mechanism governing pHj regulation in these cells at 37°C. However, in the absence of [Na ] (solution 13, Table 3) a small amount of pHj recovery occurred (see Figure 30), +  0  presumably caused by pHj regulatory mechanisms not requiring extracellular N a . +  Illustrated in Figure 31, the rate of pHj recovery was unaffected by the presence of 50 U.M EIPA (n=3; Table 6, row /), a result similar to that obtained at room temperature. The inability of EIPA to influence pH; has been previously demonstrated under steady-state conditions (see Figures 9 and 13). In contrast to a similar study performed at room temperature (see Table 6, row c; Figure 25), acid load recovery at 37°C was not affected by the removal of Cl" from the extracellular perfusate (solution 14, Table 3). Illustrated in Figure 32 and Table 6 (row m),  this result may reflect a diminished cellular dependence on  HCO37O"  exchange  towards the regulation of pHj at 37°C, as already suggested by results from the steadystate pHj experiments. In the absence of [Cl"] , pHj did however recover to a slightly 0  higher level than was present before the induced acidification, a result that is reflected by previous observations regarding 0 [Cl"]-induced increases in resting pHj at this temperature (see Figure 14A). Though the activity of the H C O 3 7 O " exchanger may not be pronounced at 37°C, the application of 200 uM DIDS inhibited recovery from an imposed acid load on 5 out of 9 coverslips examined (Figure 33A). When affected by DIDS, the initial rate of pHj recovery was reduced to 1.02 x 10" pH units/second with 3  corresponding reductions in the t  50  and t  80  rates (Table 6, row o). Illustrated in Figure  33B, acid load recovery in the remaining 4 cell populations (one of which was a sister culture of a population that did respond to the drug) was not influenced by the presence of 200 (J.M DIDS (Table 6, row n). The point and duration of DIDS application during the pHj restoration did not appear to influence the ability of this anion exchange blocker to inhibit acid load recoveries. These results suggest a variable contribution of exchange to pH; recovery from an NH -induced acid load at 37°C. +  4  HCO37CI"  92  To determine whether the sensitivity to D I D S w a s perhaps dependent o n t h e level of intracellular acidification induced b y the NH  p r e p u l s e , t h e p r e l o a d p H jw a s r e d u c e d  + 4  b y l o w e r i n g t h e p H o f t h e H C C ^ ' - c o n t a i n i n g p e r f u s i o n m e d i u m t o 6 . 8 a t 3 7 ° C {solution 18, T a b l e 4 ) . A f t e r e x p o s u r e t o t h e N H  + 4  -prepulse at p H  0  6 . 8 (solution 19, T a b l e 4 ) , p H j  typically fell t o a level o f 6.4 (Figure 3 4 ) , a value that w a s a p p r o x i m a t e l y 0 . 2 5 p H units lower than w a s typically achieved when p H  0  rested at 7.36. T h einitial, t  5 0  , a n dt  8  rates  0  o f r e c o v e r y w e r e a c c e l e r a t e d u n d e r t h e s e c o n d i t i o n s ( n = 3 ; T a b l e 6 , r o w p), b u t 2 0 0 p M D I D S d i dn o t inhibit t h e rate o f p H j restoration w h e n p H  w a s maintained at 6.8 (n=2;  0  T a b l e 6 , r o w q). T h i s r e s u l t i n d i c a t e s t h a t t h e r a t e o f p H j r e c o v e r y i s s e n s i t i v e t o p H  0  in a  m a n n e r that is independent of the activity o f HC0 7C1" exchange. 3  In s u m m a r y , t h e experiments outlined above have demonstrated that t h e activities of the acid extrusion mechanisms  present o n cultured foetal h i p p o c a m p a l  neurones are dependent o n temperature.  A t r o o m temperature, both a Na -dependent, +  HC0 "-independent acid extrusion mechanism 3  HCO3VCI" e x c h a n g e r  +  temperature.  (possibly  a Na /H +  +  exchanger)  a n d a  have been s h o w n to b e involved i n restoring p H jback to resting  levels following a n imposed acid load. Na -independent  pyramidal  HCO3VCI"  Presumably, this anion exchanger is t h e s a m e  counter-transporter regulating  steady-state p H j at  r o o m  H o w e v e r , at 37°C, t h e d o m i n a n t m e c h a n i s m that acts to return p H , to  baseline values after a n applied acidification appears to b e a N a - d e p e n d e n t , +  independent acid extruder.  HCO3"-  T h e s e results a r e i n agreement w i t h those obtained  under  s t e a d y - s t a t e c o n d i t i o n s , i n w h i c h N a - i n d e p e n d e n t HCO37CI" e x c h a n g e w a s s i m i l a r l y +  observed to b e appreciably active only at r o o m temperature.  93  F i g u r e 21.  Sample acid load with  NH C1. 4  T h e a d d i t i o n o f 20 m M NH4CI t o t h e p e r f u s i o n m e d i u m f o r t h e p e r i o d i n d i c a t e d b y t h e b a r a b o v e t h et r a c e p r o d u c e s a n i n i t i a l a l k a l i n i z a t i o n d u e t o t h ei n f l u x o f m e m b r a n e p e r m e a b l e NH3, a n d i t s s u b s e q u e n t h y d r a t i o n i n t o N H a n d O H . T h i s rise i n p H ; q u i c k l y plateaus d u et ot h e g r a d u a l influx extracellular N H . The removal o f extracellular NH C1 results in a n i m m e d i a t e fall in p H j t oacidic levels, caused b y the fast efflux o f N H ; t h eremaining N H dissociates t o release H ions. After peak acidification i sreached, p H j recovers t opre-acid load levels. T h i s sample trace i s m e a n o f data obtained f r o m 6 cells recorded o na single coverslip a tr o o m temperature i n the a b s e n c e o f H C O 3 " (solutions 1 a n d 4, p H 7 . 3 6 ) . +  -  4  +  4  4  +  3  +  4  0  94  0  5  10  15  Time  (minutes)  20  25  30  95 r- m m CN OS »—i  co 13  <—i 41  °oo sO  T f  (N OO  -*-' O CU  CN  T f  VO os in T f  m  T f  cu cu  in m CN  r-~  CN  -H in  CN  © >—  1  vo  m T f CN © vo CN CN m OS vo 4H 4H 4H 41 41 in T f OS in vo m m in CN  00 -H  ©  CN  T f  i—i  co OO  VO  CO  °u~isO CU CO  41  oo  T  8  o o 42 OO  CO  1  X  •a fi  HH  £2  EG fi  «?OH oo  P  .2 ©  .  CO  x .-S  0) OH  CO  CD o CD EG 13  &  CU CO  cd eu HH  vo  -H  m  +1 +1 +1 +1 ,—i cn o\ r-t> oo 00 00 ,—i  in o  in oo m o o o  ©  -H in  m  '  1  T f  *  —  <  2 EC  ©  T f  ©  ©  ©  -H -H 4H in m o  ©  ©  VO OS m © © O  vo  os CN m in © *—> o CN  1-H  ©  -H  ©  £ EG c S ft 8  -H»  oo  m  X  .  in OS  CN r~- CN  00  +\ -H m in  os O  m ©  o © -H -H 4H -H  ©  ©  oo m m o rn 00 vo »-  00  in  vo m  ^  in  CU co  00  i  m  -  ©  -H  -H -H -H -H o m CN m T f  m  ©  ©  ©  T f  T f  ©  ©  ©  o  ©  m  © ©  © o -H -H -H -H  vo ,-H VD T f VO vo VO VD ©  ©  O  ©  -H  -H  00  ©  ©  o o o SH *H Q Q Q l-l o  o  GO  W  O  O  CN ©  ©  ©  H - H i 41 41 41 41 41 41 CN vo © CN CN oo T f os © i—i vo 00 T f T f T f t> os  T f  m © ©  oo m  T f  © ©  © ©  m  © ©  © ©  m © ©  m © m in vo in T f ©  ©  ©  ©  T f  ©  T f  T f  ©  ©  m  vo  ©  o  in  o  o  W PH  fi ts  cd o  _  o  o  o  o  I  —' o  ©  1  ©  m CN  o  .  SI ^  td  SH  Q  oW  O  CD  w  Z7 oo oo  S  oo  ^  00 vd  '— o —1—i i—i ;T| pr] 1  o  m  1  1  ©  Q  S  Q  K  &, &i  O  R,  OH  OH  QQ HH HH  Q  OH  fi  00 cd cu  -  n  CU  CO  *  co  S  SH SH  ©  Cd <N  CO 3  td  13 . .-° 13  2 CoU1 « 8.1 O0 a  CU  i—i  X  cu fi "S cu  § a oocu I S ?fifi X a CU CcoU oo„ a -cocu a "±3  F  Q  U  fi + > E °  ^  oo  O  o  O CU OH CO VH ca s _CC UU co icu >  O O  O O o  CU  a ^ x~  <  O  T3  .S cu  cu S fi "1  Q  S  OH  o> CU  cd  00  c«  co  OH  T f  rr-- r- r~- r-m m m m m m rm m  o  O  <+H  SH  © © ©* ©  -H -H 4H 4H 4H 4H 4H 4H  _  r*H  8  OS vo  r~~ m r-- m rm m  0  m  ©  Q  a 13 ,1>  1  ©  o  (U  O cu  VO in os OS 00 CN >— vo os vo  in  u O O O u u  +  u  vd  U O o O  +  w  T f  ©  < Q  os  ©  00  4  00  OO  U  CU  r—1  CN  a a a a  in in  ©  ^ H  ©  vo m r—im vo oo OS CN CN CN ©  00  00  co DH <U  S O T3 © > O Q fi s ° <+H >, B '  4H  -H  m  ©  CO  2  © ©  ©  CN  PH  ©  m o  © ©  o  ©  ©  r-  T f  in in  T f  o o o  m vq os <n rn  ©  i 41 4H 41 41 4H 41  CN  ©  ©  m  o  o o  3 co  CN CN © 00 rn os  00  -H  rn CN  i -H 4H  -H  ©  vo  in m in  vo  (N  os  in (N  T f  ©  r H  ©  -H -H  T f  ©  in  00  ©  -H  ©  CN m  ^H  CN r-  in o  ©  ©  OS © CN CN  ©  CN rn ©  ©  vo vo  ©  -H 41 4H 4H 4H 41  ^  T f  o m  rf  ©  1  ©  Q  H-H  vo vo 00 T f os •—im CN CN © in ©  B  cu  T f  ©  CU w  o o ft  CN  •  ^  fi +  fi CU  r- VD in m VD CN  o H -H  >H  , , cd & cd "O O  CO  ©  T f  vo CN vo © oo ,—< CN in © in rn © CN rn  ©  -H 4H -H -H in T f in 00 os i — i in  o SH Q  leg  «2 to  -H 4H 4H 41 4H 4H  -H in  o  ^ f i  3 ^  CN in OS CN •—i in rn  © CN •—< CN  00  -H i—<  oo vo  OS  ©  -H -H H CN m  ©  ©  ©  i  T f  T f  ©  O  ©  +1 o  rn  r-  ^  ©  ©  ©  <—l  m  ©  m m  a  a o  OS  .—I  ©  tH  o a > oo o g  C  13  M  S  in  r-  +-> O  1  vo  in -H  s  o § 4H ^co +^  1—1  CO  13  cu cu ^  ^ w  in •—i•—i m 41 41 -H -H -H ,—< in VD T f 00 *—1 oo oo in OS *—< T f T f m  vo m  CN CN VO m  HJ  £3  2 fi  Q  -H<  ^O  -a  cu  ™4 *H CH* O CU 13 cu «U -H* ^ O j H CU O + H O .fi SH <u cd CU T3 ™ » - H f i h b »i ^ Cco U  — . o cu HU o cdH ft d *3  "Si  "cd «»H C^3 3  Pi  OH  A  s^  a CO  cu  co O O CU CU  >  SH  OH  OH  X X  96  F i g u r e 22.  Initial rate of acid load recovery as a function of p H j , preload p H j ,  m i n i m u m p H j , and net p H j decrease.  A. Based on a single experiment at 37°C in the absence of H C O 3 - , though indicative of all recoveries, the initial instantaneous rate of recovery was maximal at the minimum pHj reached during the acid load. The rate of recovery decreased to 0 in a linear fashion as pHj returned to the preload level. B. The initial rates of recovery from 14 randomly chosen experiments (9 in the presence and 5 in the absence of H C O 3 " at 37°C) as a function of the preload pHj (O), and the minimum pHj reached during the acid load (•). C. The initial rates of recovery of the same 14 experiments shown in B as a function of the net pHj decrease, which is the difference between the preload pHj and the minimum pHj reached during the acid load.  97  0.006  0.005 T3 fl  8  0.004  0.003  0.002 > o o 0.001  (-1  o u  4-  0.000  6.4  6.5  6.6  6.7  pH  B. 0.005  % 0.004  7.0  7.1  }  O  •  •• • • • • •• ••• •  o  ffi 0.003  &  > 0.002 o <D s-i 0.001  0.000 6.2  i  i  i  6.4  6.6  6.8  o  o o  #  •a  u  6.9  0.006  0.006  O  6.8  o  °  o o o o 93 o 1 7.0  1 7.2  0.000 7.4  0.3  0.4  0.5  Net pHj decrease  0.6  98  Figure 23. pHj recovery from an acid load in the absence and presence of H C O 3 " at room temperature.  I n t h e a b s e n c e o f H C O 3 7 C O 2 b u f f e r a t r o o m t e m p e r a t u r e (solution 1 a t p H 7 . 3 3 ) , p H j r e c o v e r e d f r o m a n N H - i n d u c e d a c i d l o a d at a n initial rate o f 1.31 x 10' p H u n i t s / s e c o n d ( n = 6 ) . R e c o v e r y f r o m a n a c i d l o a d w a s f a s t e r i n t h e p r e s e n c e o f HCO3" (solution 12) U n d e r t h e l a t t e r c o n d i t i o n s , t h e i n i t i a l r a t e o f r e c o v e r y w a s 2 . 5 5 x 1 f j " p H u n i t s / s e c o n d ( n = 7 ) . T h e t r a c e is a m e a n o f d a t a s i m u l t a n e o u s l y o b t a i n e d f r o m 2 6 c e l l s r e c o r d e d o n as i n g l e c o v e r s l i p . 0  +  3  4  3  99  Time (minutes)  100  Figure 24. Effect of DIDS on pHj recovery from an acid load in the presence of H C O 3 - at room temperature.  A t p H 7.32, the a p p l i c a t i o n o f 2 0 0 p M D I D S , ap h a r m a c o l o g i c a l i n h i b i t o r o f HCO3-/CT exchange, slowed the p H j recovery f r o m an induced intracellular acidification w h e n applied during the period indicated b y the bar above the trace (n=3). In the presence o f D I D S , t h e i n i t i a l rate o f p H j r e c o v e r y w a s 0 . 9 5 x 10" p H u n i t s / s e c o n d . T h e t r a c e is a m e a n o f d a t a o b t a i n e d f r o m 2 2 cells r e c o r d e d o n as i n g l e c o v e r s l i p . 0  3  101  0  5  10  15  20  25  Time (minutes)  30  35  40  45  102  Figure 25. Effect of 0 [Cl"] on pHj recovery from an acid load in the presence of H C O 3 " at room temperature. 0  A . A t p H 7 . 3 3 , t h e r e m o v a l o f e x t r a c e l l u l a r C l " a t r o o m t e m p e r a t u r e (solution 8) e n h a n c e d the rate p H j recovery f r o m a n acid load (n=3). U n d e r [Cl"] -free conditions, the initial rate o f recovery increased to 3.14 x 10" p H units/second f r o m 2.55 x 10' p H u n i t s / s e c o n d i n t h e p r e s e n c e o f [ C l ~ ] . B. T h e i n c r e a s e i n t h e r e c o v e r y r a t e c a u s e d b y exposure to 0 [ C l " ] w a s b l o c k e d b y the simultaneous application o f 200 p M D I D S . In the a b s e n c e o f [ C l " ] a n d the p r e s e n c e o f D I D S , the initial rate o f p H j r e c o v e r y w a s 1.18 x 10" p H u n i t s / s e c o n d ( n = 2 ) . T r a c e A is m e a n o f d a t a o b t a i n e d f r o m 2 6 cells, w h e r e a s t r a c e B is am e a n o f d a t a o b t a i n e d f r o m 3 4 c e l l s , e a c h r e c o r d e d o n as e p a r a t e c o v e r s l i p . 0  0  3  0  0  0  3  3  103  Time  (minutes)  104  Figure 26. pHj recovery from an acid load in the absence and presence of H C O 3 " at 37°C.  I n t h e a b s e n c e o f HCO3" a t 3 7 ° C (solution 1 a t p H 7 . 3 6 ) , p H , r e c o v e r e d f r o m a n N H i n d u c e d intracellular a c i d l o a d at a n initial rate o f 5.56 x 10" p H u n i t s / s e c o n d ( n = 1 2 ) . T h e p r e s e n c e o f HCO3" (solution 12) d i d n o t s i g n i f i c a n t l y a l t e r t h e r e s t o r a t i o n r a t e a t t h i s t e m p e r a t u r e ; c e l l s b u f f e r e d b y HCO3VCO2 r e c o v e r e d f r o m a n i m p o s e d a c i d i f i c a t i o n w i t h a n initial rate o f 4 . 7 7 x 10" p H u n i t s / s e c o n d ( n = 1 6 ) . T h e t r a c e i sa m e a n o f d a t a o b t a i n e d f r o m 7cells r e c o r d e d o n as i n g l e c o v e r s l i p . C o m p a r e w i t h F i g u r e 2 3 , w h i c h is a similar e x p e r i m e n t p e r f o r m e d at r o o m temperature. +  0  4  3  3  105  Time (minutes)  106  Figure 27. Effect of 0 [Na ] on pHj recovery from an acid load in the absence of H C 0 - at 3 7 ° C . +  0  3  T h e r e m o v a l o f e x t r a c e l l u l a r N a i n t h e a b s e n c e o f HCO3" (solution 2 a t p H 7.32) c o m p l e t e l y halted the ability o f p H j to recover f r o m a n i n d u c e d acid load (n=2). Once [ N a ] w a s r e - i n t r o d u c e d to t h e p e r f u s i o n m e d i u m , p H , r e s u m e d its r e c o v e r y to n o r m a l r e s t i n g l e v e l s . T h e t r a c e is a m e a n o f d a t a o b t a i n e d f r o m 10 c e l l s r e c o r d e d o n a s i n g l e coverslip. +  0  +  0  107  Time  (minutes)  108  F i g u r e 28. Effect of E I P A at 3 7 ° C .  on p H j recovery f r o m an acid load in the absence  of  HCO3-  The 37°C norm data  a d d i t i o n o f 5 0 u M E I P A t o t h e H E P E S b u f f e r e d p e r f u s i o n m e d i u m {solution 1) a t ( p H 7.35) d i d not affect the rate o f p H j recovery d u r i n g the restoration o f p H j to a l l e v e l s after a n i n d u c e d intracellular a c i d i f i c a t i o n ( n = 6 ) . T h e trace is a m e a n o f obtained f r o m 30 cells recorded o n a single coverslip. 0  109  Time (minutes)  110  Figure 29. Effect of D I D S on p H j recovery f r o m a n acid load in the absence HCO3- a t 3 7 ° C .  of  T h e application o f 200 p M D I D S during the restoration o f p H j d i d not significantly affect the rate o f p H j r e c o v e r y i n n e u r o n e s p e r f u s e d i n the a b s e n c e o f H C 0 " at 3 7 ° C ( p H 7.37). T h e trace is a m e a n o f d a t a o b t a i n e d f r o m 2 3 cells r e c o r d e d f r o m 1 o f the 3 cell populations studied. 3  0  Ill  Time  (minutes)  112  F i g u r e 30.  Effect of 0 [ N a ] on p H j recovery from an acid load in the presence of +  0  H C 0 - at 3 7 ° C . 3  At p H 7.36, the removal of extracellular N a in the presence of HCO3" at 37°C (solution 13) at the point of peak acidification caused a near complete inhibition of pHj recovery (n=3). However, some pHj recovery did occur despite the absence of [Na ] , possibly due to the activity of Na -independent recovery mechanisms. The re-addition of [Na ] resulted in a rapid restoration of pHj to steady-state levels. The trace is a mean of data obtained from 37 cells recorded on a single coverslip. +  0  +  0  +  +  0  113  Time (minutes)  114  Figure 31. Effect of EIPA on pHj recovery from an acid load in the presence of H C O 3 - at 3 7 ° C .  In of fr re  n e u r o n e s e x p o s e d t o s o l u t i o n s c o n t a i n i n g HCO37CO2 a t 3 7 ° C ( p H 7 . 3 4 ) , t h e a d d i t i o n 50 p M E I P A d u r i n g the period o f p H j restoration d i d not alter the rate o f p H j recovery o m a n i n d u c e d a c i d i f i c a t i o n ( n = 3 ) . T h e trace is am e a n o f d a t a o b t a i n e d f r o m 2 5 cells c o r d e d o n asingle coverslip. 0  115  116  Figure 32. Effect of 0 [Cl~] on pH; recovery from an acid load in the presence of H C 0 - at 3 7 ° C . 0  3  T h e r e m o v a l o f e x t r a c e l l u l a r C l " i n t h e p r e s e n c e o f HCO3" a t 3 7 ° C (solution 14) d i d n o t increase the rate o f recovery f r o m an i n d u c e d acidification (n=7). T h o u g h p H w a s held c o n s t a n t at 7.37, p H j d i d r e c o v e r to as l i g h t l y h i g h e r r e s t i n g v a l u e i n t h e a b s e n c e o f [ C l " ] , but returned to n o r m a l steady-state levels w h e n [Cl"] w a s re-introduced to the p e r f u s i o n medium. T h e t r a c e is a m e a n o f d a t a o b t a i n e d f r o m 2 8 cells r e c o r d e d o n a s i n g l e coverslip. C o m p a r e w i t h F i g u r e 25, w h i c h s h o w s the effect o f exposure to 0 [Cl"] o n p H j r e c o v e r y i n t h e p r e s e n c e o f HCO3" a t r o o m t e m p e r a t u r e . 0  0  0  0  117  6.6 -| 0  1  10  1 20  1 30  Time (minutes)  1 40  1  50  1 60  118  Figure 33.  Effect of DIDS on p H j recovery from an acid load in the presence of  H C O 3 - at 37°C.  The application of DIDS had a variable effect on the rate of recovery from an induced acidification in the presence of HCO3" at 37°C ( p H 7.37). A . The rate of recovery was inhibited by 200 p M DIDS in 5 out of 9 coverslips studied. pHj restoration was significantly slowed as soon as DIDS was added to the extracellular solution. The initial pHj recovery rate on those cell populations affected by DIDS was 1.02 x 10~ pH units/second. The removal of DIDS resulted in an increase in the recovery rate until steady-state pHj levels were attained. This trace is a mean of data obtained from 49 cells recorded on a single coverslip. B. On the remaining 4 coverslips, the application of 200 p M DIDS failed to slow the restoration towards resting pHj levels. This trace is a mean of data obtained from 28 cells recorded on a separate coverslip to the one used in trace A . Compare with the same experiment performed at room temperature (Figure 24). 0  3  119  120  Figure 34.  Effect of DIDS on p H , recovery from an enhanced acid load in the  presence of H C O 3 - at 37°C.  T h e p H o f the perfus reduce the resting p H reached during the in 6.8 (first a c i d load), (n=3). T h e presence recovery (n=2). T h e coverslip.  i o n m e d i u m w a s l o w e r e d t o 6 . 8 0 (solutions 18 and 19) i n o r d e r t o j (see F i g u r e 19). T h i s m a n o e u v r e also l o w e r e d the m i n i m u m p H j d u c e d intracellular acidification. U n d e r d r u g - f r e e c o n d i t i o n s at p H the initial rate o f p H , recovery w a s 7.12 x 10" p H units/second o f 200 p M D I D S during p H j restoration failed to s l o w the rate o f t r a c e is a m e a n o f d a t a o b t a i n e d f r o m 8 c e l l s r e c o r d e d o n a s i n g l e 0  3  121  Time (minutes)  122  D I S C U S S I O N  pHj regulation, studied in various cell types, has b e e n the source o f investigation.  considerable  T h e predominant m e c h a n i s m s regulating p H j in the limited n u m b e r  vertebrate n e u r o n e types studied to date appear to be a Na /H +  independent HC0 7C1~  exchanger, and a Na -dependent HC0 7O~  e x a m p l e , O u - y a n g et al,  1993;  3  +  +  3  R a l e y - S u s m a n et al,  1991;  N a +  e x c h a n g e r (see,  for  1994), though these  V e r y few studies, however, have comparatively  the r e g u l a t i o n o f p H ; at r o o m t e m p e r a t u r e a n d 3 7 ° C .  a  Schwiening and Boron, 1994).  O t h e r m e c h a n i s m s h a v e b e e n d e s c r i b e d ( e . g . M a r t i n e z - Z a g u i l a n et al, appear to play a m i n o r role.  exchanger,  of  examined  T h e results presented in this thesis  uncover s o m e striking differences in p H , regulation caused by temperature.  Accordingly,  this discussion will not only e x a m i n e the m e c h a n i s m s regulating the intracellular proton e n v i r o n m e n t i n c u l t u r e d rat h i p p o c a m p a l n e u r o n e s at 3 7 ° C a n d at r o o m t e m p e r a t u r e ,  but  w i l l a l s o a d d r e s s p o s s i b l e r e a s o n s u n d e r l y i n g the d i f f e r e n c e s i n p H j r e g u l a t i o n at t h e s e two  temperatures.  R e g u l a t i o n o f p H ; a t 37°C: Steady-state p H j resulting f r o m the perfusion o f the neurones HC0 "-free HEPES  buffered m e d i u m ( p H  absence of HC0 "  at 3 7 ° C  3  3  ( p H  0  0  7.34) w a s 7.23.  at 3 7 ° C  with  Resting p H j in the n o m i n a l  7.35 to 7.4) h a s b e e n r e p o r t e d to reside at 7.03  in  cultured rat sympathetic neurones ( T o l k o v s k y a n d R i c h a r d s , 1987), 7.00 in cultured rat h i p p o c a m p a l n e u r o n e s ( R a l e y - S u s m a n et al, ( O u - y a n g et al,  1991), 7.00 in cultured rat cortical  neurones  1993), a n d 6.74 in freshly isolated C A 1 p y r a m i d a l neurones f r o m rat  hippocampi (Schwiening and Boron, 1994).  However, Gaillardand Dupont (1990)  have  s h o w n that s t e a d y - s t a t e p H j is 7 . 3 7 i n c u l t u r e d rat c e r e b e l l a r P u r k i n j e cells p e r f u s e d  with  HEPES-buffered  solutions ( p H  0  7.4).  Therefore, the value o f resting p H j determined  in  123  the p r e s e n t e x p e r i m e n t s u n d e r H C 0 ~ - f r e e c o n d i t i o n s is w e l l w i t h i n the r e p o r t e d r a n g e f o r 3  vertebrate  neurones.  The  neurones e m p l o y e d in this study w e r e able to sustain a stable p H j d u r i n g  exposure to HEPES  b u f f e r e d (i.e. H C C ^ V C C ^ - f r e e )  m e d i u m , thus indicating a significant  contribution o f H C C ^ ' - i n d e p e n d e n t m e c h a n i s m s towards the maintenance o f n o r m a l p H j levels at 3 7 ° C . U n d e r H C C ^ - f r e e  conditions, the r e m o v a l o f extracellular N a  produced  +  a m a r k e d a n d sustained acidification, w h i c h was reversed b y the re-addition o f [ N a ]  to  +  the perfusion m e d i u m . T h e s e observations indicate the presence o f a N a - d e p e n d e n t  acid  +  extrusion mechanism operating independently of HC0 ".  A probable candidate for this  3  m e c h a n i s m is the N a / H +  +  0  exchanger, which.removes intracellular protons in exchange for  extracellular N a . In the absence o f [ N a ] +  this a n t i p o r t e r is n o t a b l e to f u n c t i o n , c a u s i n g  +  0  an accumulation of intracellular acid equivalents.  A small portion of these  equivalents  m a y originate outside o f the m e m b r a n e a n d leak t h r o u g h to the c y t o p l a s m d o w n  an  electrochemical gradient, but most are likely generated f r o m n o r m a l cellular metabolism. The  acidification resulting f r o m the r e m o v a l o f [ N a ] +  reversal o f the Na /H +  +  0  could also be explained by  a  a n t i p o r t e r , a l t h o u g h it w a s n o t p o s s i b l e to test t h i s i n v i e w o f t h e  lack o f a pharmacological inhibitor. To c o n f i r m the presence o f the Na /H +  would  have  b e e n to  exchanger o n these neurones, a useful test  +  i n h i b i t its a c t i v i t y u t i l i z i n g k n o w n  Unfortunately, the application o f various Na /H +  +  pharmacological  blockers.  exchange inhibitors, including 2 potent  a m i l o r i d e a n a l o g u e s a n d one n o v e l inhibitor, failed to alter steady-state p H j in the a b s e n c e o f HCO3" a t 3 7 ° C . pH;,  E I P A , a p p l i e d at a c o n c e n t r a t i o n o f 5 0 p M , d i d n o t i n f l u e n c e  n o r d i d it i n h i b i t t h e r e c o v e r y o f p H ; after a 0  Similarly, 100 p M MGCMA,  [Na ] -induced  r a t s t r i a t a l s y n a p t o s o m e s ( A m o r o s o et al,  acidification.  +  0  w h i c h has b e e n s h o w n to inhibit the N a / H +  +  antiporter in  1991), and 100 p M H O E 694, a novel inhibitor  o f N a / H e x c h a n g e f i r s t s t u d i e d o n b r a i n c a p i l l a r y e n d o t h e l i a l c e l l s ( S c h m i d et al, +  resting  +  were both incapable of producing a change in baseline pHj.  1992),  T o examine whether  the  124  presence of HC0 "  was  3  required for the manipulation o f p H j b y E I P A ,  the  e x c h a n g e inhibitor w a s applied to neurones perfused w i t h H C C ^ V C C ^ - b u f f e r e d Again, the application o f 50 p M EIPA did not induce a change in p H j . T h e of the N a - d e p e n d e n t  acid extrusion mechanism  medium. insensitivity  in hippocampal  pyramidal  n e u r o n e s to a m i l o r i d e a n d its a n a l o g u e s h a s r e c e n t l y b e e n r e p o r t e d i n t w o  additional  +  studies.  R a l e y - S u s m a n et al  present  cation  (1991) and Schwiening and Boron (1994) have  observed  that amiloride, or additional a n a l o g u e s tested, w e r e u n a b l e to m o d u l a t e the activity o f the suspected Na /H +  +  exchanger present o n the cells used in their experiments.  I n c o n t r a s t , it  h a s b e e n d e m o n s t r a t e d that the r e g u l a t i o n o f p H j is c o m p l e t e l y s e n s i t i v e to a m i l o r i d e i n cultured rat P u r k i n j e cells (Gaillard a n d D u p o n t ,  1990)  and  sympathetic  neurones  ( T o l k o v s k y a n d R i c h a r d s , 1987), a n d partially sensitive to a m i l o r i d e in cultured cortical neurones  ( O u - y a n g et  al,  1993).  rat  Other cation exchange inhibitors, such  as  h a r m i l i n e ( A r o n s o n a n d B o u n d s , 1981), w e r e not e x a m i n e d d u e to interference w i t h  the  fluorescence signal emitted b y the BCECF-loaded to inhibit N a / H +  +  1993;  BCECF  1991), this f i n d i n g has b e e n q u e s t i o n e d b y others (e.g. O u - y a n g  et  S c h w i e n i n g a n d B o r o n , 1994) d u e to this technical limitation.  An  analysis o f the kinetic properties o f cation counter-transport o n various  types has revealed that L i  is a substrate f o r the o p e r a t i o n o f the N a / H  +  +  (Aronson, 1985). T h e substitution o f [Na ] +  replacement  (Raley-Susman of [Na ] +  hippocampal neurones  et  al,  1991).  with [Li ] +  0  0  antiporter  with [Li ] has therefore b e e n utilized as a n 0  +  amiloride  +  The  +  exchange o n cells insensitive  latter authors, d e m o n s t r a t e d  d i d not significantly alter the ability o f  that  +  extrusion m e c h a n i s m present was likely the Na /H +  +  e x c h a n g e r , d e s p i t e its i n s e n s i t i v i t y  Indeed, preliminary results o n the neurones e m p l o y e d in this study +  with [Li ] +  0  0  to the  cultured  to regulate p H j , w h i c h indicates that the N a - d e p e n d e n t  that the replacement o f [ N a ]  cell  +  0  alternative m e a n s o f suggesting the presence o f Na /H  amiloride.  A l t h o u g h initially reported  exchange in cultured h i p p o c a m p a l pyramidal cell loaded with  ( R a l e y - S u s m a n et al, al,  neurones.  acid to  indicate  does not jeopardize the maintenance o f a stable  125  resting p H j i n H C 0 - - f r e e m e d i u m at 3 7 ° C . 3  Accordingly, the Na -dependent,  independent acid extrusion mechanism regulating p H j on these neurones amiloride-insensitive variant o f the Na /H +  +  HC0 "-  +  3  m a y  be  an  exchanger, similar to that p r o p o s e d b y R a l e y -  S u s m a n et al ( 1 9 9 1 ) , a n d S c h w i e n i n g a n d B o r o n ( 1 9 9 4 ) . F o u r isoforms o f the N a / H +  ( M r k i c et al,  exchanger ( N H E ) have recently been  +  distinguished  1993) based o n their amiloride sensitivity a n d tissue localization. T h e N H E -  1 f o r m , p r e s e n t o n the b a s o l a t e r a l s u r f a c e s o f intestinal a n d k i d n e y epithelia, is s e n s i t i v e t o a m i l o r i d e ( S a r d e t et al, with the NHE-2  1989).  Relative amiloride sensitivity has also b e e n  observed  isoform, w h i c h has b e e n localized in the intestine, kidney, a n d adrenal  g l a n d ( T s e et al,  1991).  A n N H E - 3 s u b t y p e , w h i c h i s hyper-resistant t o a m i l o r i d e  E I P A ( T s e et al,  1 9 9 3 ) , is p r e d o m i n a n t l y e x p r e s s e d i n t h e k i d n e y a n d i n t e s t i n e , t h o u g h it  h a s b e e n d e t e c t e d i n m i n u t e c o n c e n t r a t i o n s i n t h e h e a r t a n d b r a i n ( O r l o w s k i et al, Finally, an N H E - 4 isoform has mammalian  been detected by  tissues, including the brain, though  analogues has not been documented.  O r l o w s k i et  its s e n s i t i v i t y  al  (1992)  1992). in  7.36)  w a s 7.13.  present  a n d H O E 6 9 4 is a p p a r e n t .  Steady-state p H ; resulting f r o m the perfusion o f these neurones with H C 0 0  3  7 C 0 2 -  This value compares well with  reported in previous studies o n m a m m a l i a n central neurones maintained under conditions.  its  It is n o t k n o w n w h i c h o f t h e s e f o u r N H E i s o f o r m s ,  experiments, t h o u g h their resistance to E I P A , M G C M A ,  ( p H  many  to amiloride a n d  if any, m a y be present o n the cultured h i p p o c a m p a l neurones e m p l o y e d in the  b u f f e r e d m e d i u m at 3 7 ° C  and  A t 37°C a n d in the presence o f HC0 ", 3  that  similar  it h a s b e e n r e p o r t e d t h a t p H j r e s t s at  7.16 in cultured rat cerebellar granule cells ( P o c o c k a n d R i c h a r d s , 1989), 7.18 in m i x e d n e u r o n a l cultures f r o m various rat brain regions (Richards a n d P o c o c k , 1989), 7.06  in  cultured rat cerebellar Purkinje cells (Gaillard a n d D u p o n t , 1990), 7.17 in cultured rat h i p p o c a m p a l n e u r o n e s ( R a l e y - S u s m a n et al, ( O u - y a n g et  al,  1993), a n d 7.03  1991), 7.09 in cultured rat cortical  in acutely dissociated  pyramidal neurones (Schwiening and Boron, 1994).  neurones  adult rat h i p p o c a m p a l  C A 1  In the neurones e m p l o y e d in this  126  s t u d y , b a s e l i n e p H j w a s 0 . 1 0p H u n i t s l o w e r i n t h e p r e s e n c e o f HCO3" t h a n i n t h e a b s e n c e o f HCO3" a t 3 7 ° C .  Indeed,  perfusion m e d i u m at 37°C  t h e transition  from  H C 0 " - f r e e t o HC0 "-containing 3  3  d i dn o t induce t h e n e t alkalinization observed i n t h e s a m e  experiment performed at r o o m temperature (seebelow).  T h o u g h a n u m b e r o f studies o n  vertebrate neurones have demonstrated that resting p H j is higher i n t h e presence than i n the absence o f H C 0  3  " a t3 7 ° C ( R a l e y - S u s m a n  et al, 1 9 9 1 ; O u - y a n g et al, 1 9 9 3 ;  S c h w i e n i n g a n dB o r o n , 1 9 9 4 ) , o t h e r s h a v e i n d i c a t e d t h e o p p o s i t e . (1990), f o r example, HCO3VCO2  reported that steady-state p H , i n r a t Purkinje cells buffered b y  resides  0.21 p H units  buffered) medium. brain neurones, alkalinization.  Gaillard a n d Dupont  below  t h e resting  p H  ;  i n HC0 --free  (HEPES-  3  S i m i l arl y , R i c h a r d s a n dP o c o c k (1989), i n their e x a m i n a t i o n o fr a t  i n d i c a t e t h a t t h e removal o f e x t e r n a l H C O 3 " c a u s e s a n i n t r a c e l l u l a r Accordingly, t h e level o f steady-state p H j i n t h e neurones  used i n this  i n v e s t i g a t i o n o b s e r v e d i n t h ep r e s e n c e o f H C 0 3 " - c o n t a i n i n g e x t e r n a l m e d i a a r en o t u n l i k e those reported i nother studies o nvertebrate central T h e removal o f extracellular N a  +  neurones.  i n t h e p r e s e n c e o f HCO3" i n i t i a t e d a r a p i d a n d  sustained intracellular acidification, a result similar t o that obtained i n t h e absence o f HCO3" ( s e e a b o v e ) .  T h e re-addition o f [Na ] +  0  relieved this acid-load, a n d a n i m m e d i a t e  recovery to steady-state p H j levels w a sobserved. in the presence dependent  o f HCO3", t h e m a i n t e n a n c e  o n [Na ] . +  0  I n neurones  removal o f extracellular C l " caused  T h e s e observations indicate that,  o f steady-state p H j a t3 7 ° C  i s largely  buffered b y H C 0 7 C 0 - c o n t a i n i n g solutions, t h e 3  2  a 0 . 1 9 p H unit a n d a 0 . 1 4 p H unit  a l k a l i n i z a t i o n i n t h e p r e s e n c e a n da b s e n c e o f [ N a ] , r e s p e c t i v e l y . 0  the membrane  intracellular  B y removing [Cl"] ,  +  the gradient f o r C l " across  even  0  would b e increased  a n d m a y result i n a  d i r e c t i o n a l r e v e r s a l o f t h e HCO37O" e x c h a n g e r ( s e e G a i l l a r d a n d D u p o n t , 1 9 9 0 ) . would  cause  a n influx  o f extracellular  HCO3" w h i c h  would  produce  the  This  observed  intracellular alkalinization. I nt h epresence o f [ N a ] , this 0 [Cl"] -induced alkalinization +  0  0  w a s abolished b y 2 0 0 u MD I D S , w h i c h adds w e i g h t t o t h e possibility that a HC0 "/C1" 3  127  exchanger  i spresent  on  these  neurones.  F u r t h e r m o r e , this  anion  exchanger  m a y  predominantly function in aNa -independent m a n n e r since the amplitude o f the 0  [CT] -  +  i n d u c e d p H j rise h a d asimilar m a g n i t u d e whether [ N a ] +  0  0  was present or not.  It is p o s s i b l e that, at 3 7 ° C , N a - i n d e p e n d e n t H C 0 7 C 1 " e x c h a n g e m a y o p e r a t e +  3  m a i n t a i n s t e a d y - s t a t e p H j at a l e v e l slightly b e l o w that w h i c h is o b s e r v e d i n the o f HCO3".  to  absence  H o w e v e r , the application o f 200 p M D I D S d i d not affect baseline p H j in  n e u r o n e s p e r f u s e d i n t h e p r e s e n c e o f HCO3", n o r d i d i t i n f l u e n c e t h e m a n n e r i n w h i c h p H j r e s p o n d e d to the transition f r o m H C C ^ ' - f r e e to H C C ^ ' - c o n t a i n i n g p e r f u s i o n m e d i a .  T h e  inability o f D I D S to affect p H j in these situations does not support the presence o f  an  active N a - i n d e p e n d e n t H C O 3 7 C T e x c h a n g e r a t 3 7 ° C . A s r e s t i n g p H j c a n b e a l t e r e d b y +  f l u c t u a t i o n s i n [ C l " ] , it is p o s s i b l e t h a t s o m e o t h e r C h - d e p e n d e n t m e c h a n i s m a s s i s t s i n 0  p r e s e r v i n g the steady-state p H j i n these cells. DIDS-sensitive synaptosomes  C17H  +  co-transporter  In fact, recent e v i d e n c e suggests that a  i s involved  ( M a r t i n e z - Z a g u i l a n et al, 1 9 9 4 ) .  0  3  pHj  regulation  in  rat  brain  H o w e v e r , the likelihood o f this  transporter being present on hippocampal neurones 0 [Cl"] in the absence o f HC0 "  in  i sr e m o t e , s i n c e t h e e x p o s u r e  coto  at 3 7 ° C d i d n o t c a u s e a n y c h a n g e i n steady-state p H j .  M o r e o v e r , i n t h e p r e s e n c e o f HCO3", a n y 0 [ C l " ] - i n d u c e d a l k a l i n i z a t i o n w a s b l o c k e d b y 0  the simultaneous  application o f D I D S , w h i c h suggests that anion exchange  is i n  p r e s e n t o n t h e s e n e u r o n e s , t h o u g h l i k e l y c o n t r i b u t e s little to the m a i n t e n a n c e o f state p H j at A regulate  experiments.  steady-  37°C.  clearer indication o f the m a n n e r in w h i c h cultured h i p p o c a m p a l pHj  fact  a t37°C T h e  was  achieved  addition and  through  subsequent  the  analysis  of  removal of 20  acid  m M  load  NH C1 4  neurones recovery from  the  extracellular perfusion m e d i u m p r o v i d e d a convenient m e a n s o f lowering p H , . T h e rate of p H j restoration towards  its r e s t i n g l e v e l w a s  therefore  examined  expanding the characterization o f p H j regulating m e c h a n i s m s  as a m e a n s  and, in addition,  of was  e m p l o y e d to p r o v i d e i n f o r m a t i o n o n activity rates. Acid extrusion rates are often r e p o r t e d  128  as the rate o f c h a n g e o f the intracellular p r o t o n concentration as a function o f time. net H  f l u x e s (JH+)  +  a r e  These  usually calculated as the p r o d u c t the total intracellular buffering  c a p a c i t y (P ) a n d t h e r a t e o f p H j c h a n g e d u r i n g r e c o v e r y f r o m a n i m p o s e d X  ( s e e B o y a r s k i et al, 1 9 8 8 a ) .  acidification  F o r reasons explained below, absolute buffering  w e r e not determined in the present experiments.  capacities  A c c o r d i n g l y , this discussion o f acid  l o a d r e c o v e r y rates will b e limited to the rate o f p H j c h a n g e m e a s u r e d in p H units  per  second. In HC03"-free HEPES  b u f f e r e d m e d i u m at 3 7 ° C , the r e m o v a l o f [ N a ] +  0  during  acid l o a d r e c o v e r y c o m p l e t e l y b l o c k e d p H j restoration. T h e inability o f p H j to recover in 0 [Na ] +  s u g g e s t s that p H , is h i g h l y r e g u l a t e d b y t h e a c t i v i t y o f a N a - d e p e n d e n t , +  0  HCO3"  independent acid extrusion mechanism. T h e most likely candidate for such a mechanism, as d i s c u s s e d a b o v e , is the N a / H +  exchanger.  +  EIPA, whether in the absence or presence  o f e x t e r n a l HCO3", d i d n o t i n h i b i t t h e r e c o v e r y f r o m a n i m p o s e d i n t r a c e l l u l a r a c i d o s i s , w h i c h is c o n s i s t e n t w i t h p r e v i o u s results d e m o n s t r a t i n g the i n s e n s i t i v i t y o f s t e a d y - s t a t e pHj in these neurones to amiloride  analogues.  T h e rate o f p H j recovery f r o m a n i m p o s e d acid load w a s not significantly different i n t h e p r e s e n c e o r a b s e n c e o f HCO3" a t 3 7 ° C ( s e e F i g u r e 3 5 ) .  H o w e v e r , in the absence o f  HCO3", p H j d i d r e c o v e r f r o m t h e i n d u c e d a c i d i f i c a t i o n t o a m o r e a l k a l i n e p H ,  which  likely reflects the fact that steady-state p H j i sh i g h e r u n d e r H C 0 3 " - f r e e c o n d i t i o n s  at  37°C.  of  T h e similarity b e t w e e n acid load recovery rates in the presence a n d absence  HCO3" s u g g e s t s t h a t t h e s e n e u r o n e s a r e n o t d e p e n d e n t o n t h e a n i o n e x c h a n g e r t o  recover  f r o m a c i d i c c h a n g e s i n p H , at 3 7 ° C , p o s s i b l y d u e to a n i n c r e a s e i n the n e u r o n a l b u f f e r i n g capacity (see below). [Na ]  d i d not completely inhibit the ability o f these neurones to recover f r o m a n  +  0  induced acidification. [Na ]  H o w e v e r , i n s o l u t i o n s b u f f e r e d b y HCO37CO2, t h e r e m o v a l  T h o u g h small, the observed recovery o f p H j in the absence  indicates a n a d d i t i o n a l N a - i n d e p e n d e n t p H j regulator o p e r a t i n g at 3 7 ° C .  +  +  0  NH  of + 4  -  of  Since  this N a - i n d e p e n d e n t recovery was not present in similar experiments conducted in the +  129  a b s e n c e o f e x t e r n a l HC0 ", t h i s m e c h a n i s m l i k e l y r e q u i r e s HCO3" t o f u n c t i o n  effectively.  3  P o s s i b i l i t i e s i n c l u d e N a - i n d e p e n d e n t HCO37Q" e x c h a n g e , w h i c h h a s b e e n s h o w n t o +  be  p r e s e n t t h o u g h relatively inactive at 3 7 ° C , o r H C C ^ ' - d e p e n d e n t intracellular b u f f e r i n g . I n fact, it a p p e a r s t h a t b o t h o f t h e s e f a c t o r s m a y b e i n v o l v e d i n p H j r e c o v e r y f r o m a c i d i c levels at 37°C.  In 5 o f 9 neuronal populations studied, D I D S significantly  slowed  recovery rates f r o m a n i m p o s e d acid load, w h i c h reveals a dependence o f p H j restoration o n t h e N a - i n d e p e n d e n t HCO37CI" e x c h a n g e r .  A s this transporter has previously  +  s h o w n to r e m a i n relatively quiescent u n d e r steady-state c o n d i t i o n s at 37°C, experiments uncover a possible relationship between the activity o f the anion  been  these 5 exchanger  and the level o f p H j . S u c h a relationship b e t w e e n p H , a n d the rate o f acid extrusion  has  b e e n d e m o n s t r a t e d b y O u - y a n g et al ( 1 9 9 3 ) o n n e u r o n e s i s o l a t e d f r o m t h e c o r t e x o f t h e rat brain. recovery  H o w e v e r , the r e m a i n i n g 4 neuronal populations e x p o s e d to D I D S from  an  Furthermore, the  acid  did not  removal of [Cl']  restoration at 37°C. the  load  0  exhibit  a reduction  during recovery  did not  during  in p H , recovery alter the  rates.  rate o f  T a k e n as a w h o l e , these results s u g g e s t that, at 37°C, the activity  Na -independent +  HCO37CI" e x c h a n g e r  o v e r s h a d o w e d b y the operation o f the Na /H +  intracellular b u f f e r i n g s y s t e m s (see b e l o w ) .  present  o nthese  neurones  of  m a y b e  transporter or the increased efficiency  +  pHj  of  Cells with a n increased ability to resist p H ;  perturbations through m o r e efficient organellar, metabolic, or physiochemical  buffering  w o u l d p r e s u m a b l y be less reliant o n acid extruding exchangers for i m m e d i a t e  recovery  f r o m a n i n d u c e d a c i d i f i c a t i o n . T h i s h y p o t h e s i s is a p o s s i b l e e x p l a n a t i o n f o r the  apparent  inactivity o f the Na -independent +  HC0 7C1" 3  exchanger  at 3 7 ° C .  Nevertheless,  i m p o s i n g a large intracellular acid load, the probability o f activating the  by  otherwise  d o r m a n t a n i o n e x c h a n g e r to aid in p H , regulation appears to increase. I n a n a t t e m p t t o t e s t w h e t h e r t h e N a - i n d e p e n d e n t HCO37CI" e x c h a n g e r c o u l d b e +  activated b y l o w e r i n g p H j to e x t r e m e levels, i m p o s e d acidifications w e r e e m p l o y e d pH  0  6.8 instead o f the n o r m a l 7.35.  The lowering of p H  0  at  indeed caused areduction in the  130  m i n i m u m p H jreached during the N H  + 4  - i n d u c e d acid load.  A s a result, t h e rate o f p H j  r e c o v e r y increased, b u tt h e application o f D I D S d u r i n g this p r o c e d u r e h a dn o appreciable effect.  T h o u g h this suggests a possible connection b e t w e e n t h e rate o f p H j r e c o v e r y a n d  pH , t h e o b s e r v e d increase i n t h e recovery rate c o u l d n o t b e attributed to t h e activation o f 0  N a - i n d e p e n d e n t HCO3VCI"  exchange.  +  In summary, the regulation of pHj at 37°C ( p H  7.3 -7.4) i n cultured hippocampal  0  neurones is primarily governed b y t h e activity o f a Na -dependent, +  acid extrusion mechanism. insensitive  Na /H +  +  HC03"-independent  T h em o s t likely candidate f o rthis m e c h a n i s m is a n a m i l o r i d e -  exchanger,  a s suggested  b yR a l e y - S u s m a n  et  al  (1991)  a n d  S c h w i e n i n g a n dB o r o n (1994).  Regulation o fp H j at r o o m  temperature:  At r o o m temperature, buffered m e d i u m ( p H  0  a n d in neurones  superfused  w i t h HCC>3"-free  HEPES-  7.32), steady-state p H j w a s 6.85,w h i c h is considerably lower  the value o f p H j 7.23 observed at 37°C under similar buffering conditions. o f HCO3", t h e r e m o v a l o f [ N a ] +  0  than  Inthe absence  f r o m t h e perfusate resulted i n a fall i n p H j similar t o that  o b s e r v e d at 37°C (see F i g u r e 17) suggesting that t h e N a - d e p e n d e n t , +  HCC^'-independent  acid extrusion m e c h a n i s m described at 37°C continues to operate at r o o m However, i n contrast to observations  temperature.  a t 3 7 ° C , t h e a d d i t i o n o f HCO3" t o t h e p e r f u s i o n  solution caused p H jto rise to t h e substantially higher level o f 7 . 1 5 . M o r e o v e r , t h e n e t alkalinization caused b y the transition f r o m H C 0 " - f r e e to H C 0 " - c o n t a i n i n g  perfusion  m e d i u m at constant p H  inhibitor  3  DIDS.  0  3  w a s blocked b y the application o f the anion exchange  T h u s , w h e n t r a n s f e r r i n g i n t o m e d i a b u f f e r e d b y HCO37CO2 a t r o o m  temperature,  t h e l i k e l y c a u s e o f t h e a l k a l i n i z i n g t e n d e n c y w a s t h e a c t i v a t i o n o f HCO37O" In experiments carried o u t i n t h e absence o f [Na ] , t h e transition into f r o m +  0  to H C 0 3 " - c o n t a i n i n g m e d i u m  similarly produced  a n increase  exchange. HCC^'-free  i n p H j ,w h i c h  further  131  s u g g e s t s t h a t t h e a c t i v i t y o f t h e HCO3VCI" e x c h a n g e r  at r o o m t e m p e r a t u r e i s N a +  independent. U n d e r s t e a d y - s t a t e c o n d i t i o n s i n t h e p r e s e n c e o f HCO3" a t r o o m t e m p e r a t u r e ,  the  a p p l i c a t i o n o f 2 0 0 p M D I D S c a u s e d a r e d u c t i o n i n p H j o f a p p r o x i m a t e l y 0.1 p H  units.  T h i s acidification w a s likely a result o f the inhibition o f the N a - i n d e p e n d e n t  3  HC0 7C1"  +  e x c h a n g e w h i c h therefore m u s t participate i n the regulation o f p H j at this  temperature.  F u r t h e r m o r e , the r e m o v a l o f [ C l " ] c a u s e d a n a p p r o x i m a t e 0.3  intracellular  0  p H unit  a l k a l i n i z a t i o n , p r o b a b l y d u e t o a 0 [ C l " ] - i n d u c e d e n h a n c e m e n t o f HCO37CI" e x c h a n g e a s 0  described previously by Gaillard and Dupont (1990).  T h e s e results suggest that  the  m a i n t e n a n c e o f s t e a d y - s t a t e p H j i n t h e p r e s e n c e o f HCO3" a t r o o m t e m p e r a t u r e i s a t l e a s t p a r t i a l l y r e g u l a t e d b y N a - i n d e p e n d e n t HCO37CI" e x c h a n g e .  T h e activity o f this anion  +  c o u n t e r - t r a n s p o r t e r , w h i c h e x c h a n g e s i n t r a c e l l u l a r C l " f o r e x t r a c e l l u l a r HCO3" a n d a c t s as a cell a l k a l i n i z i n g m e c h a n i s m , is p r e s u m a b l y r e s p o n s i b l e f o r t h e h i g h e r p H , o b s e r v e d in the presence as o p p o s e d to the a b s e n c e o f  thus  resting  HC0 ". 3  R a t e s o f p H j r e c o v e r y f r o m a n i m p o s e d a c i d l o a d at r o o m t e m p e r a t u r e w e r e , general, s l o w e r t h a n t h o s e f o u n d at 3 7 ° C (see F i g u r e 35).  A t this reduced  in  temperature  a n d i n contrast to results o b t a i n e d at 3 7 ° C , the rate o f p H , r e c o v e r y f r o m a n  induced  a c i d i f i c a t i o n a l s o a p p e a r e d t o d e p e n d o n t h e p r e s e n c e o f HCO3". A s s e e n i n F i g u r e 3 5 , the addition o f HC0 " 3  t o the p e r f u s i o n m e d i u m at r o o m t e m p e r a t u r e r e s u l t e d i n a  substantial e n h a n c e m e n t o f the r e c o v e r y rate, w h i c h possibly reflects the activation N a - i n d e p e n d e n t HCO37CI" e x c h a n g e .  T h e d e p e n d e n c e o f p H j r e c o v e r y o n HCO3VCI"  +  exchange (either N a - d e p e n d e n t +  of  or Na -independent) +  has also been demonstrated  l a m p r e y reticulospinal n e u r o n e s at r o o m t e m p e r a t u r e (Chesler, 1986).  The presence  in of  HCO37CO2 c o u l d a l s o a c t t o a u g m e n t t h e a p p a r e n t i n t r a c e l l u l a r b u f f e r i n g p o w e r  (see  R o o s a n d B o r o n , 1981), w h i c h w o u l d in turn contribute to r e d u c e the m a g n i t u d e  and  duration o f i m p o s e d acid transients. caused b y the NH  + 4  Indeed, a c o m p a r i s o n o f the net decreases in p H j  p r e p u l s e at r o o m t e m p e r a t u r e ( c o l u m n 4, T a b l e 6) indicates that a  132  greater acidification w a s obtained under H C 0 3 " - f r e e conditions than under w h e r e HCO3" w a s p r e s e n t i n t h e p e r f u s a t e . HC03'-induced  increases  H o w e v e r , t h eeffects o fbuffering capacity o n  i n p H ,recovery  rates  a tr o o m  overshadowed b y thecontribution o fanion exchange. indicate that t h e N a - i n d e p e n d e n t +  conditions  temperature  were  I n fact, several other  HCO37O" e x c h a n g e r  i sactively  recovery o f p H , f r o m a n i m p o s e d acid load at this temperature.  likely  observations  involved i n the  I n the presence o f  HCO3", p H j w a s r e s t o r e d t o a h i g h e r r e s t i n g l e v e l a f t e r t h e i m p o s e d a c i d i f i c a t i o n t h a n  s e e n i n t h e a b s e n c e o f HCO3" ( s e e F i g u r e 2 3 ) .  T h i s result suggests that  that  Na -independent +  HCO37Q" e x c h a n g e , w h i l e p a r t i c i p a t i n g i n t h e r e s t o r a t i o n o f p H j f o l l o w i n g a n i n d u c e d a c i d l o a d , c o n t i n u e s t o r e g u l a t e p H , t o t h e h i g h e r r e s t i n g l e v e l o b s e r v e d u n d e r HCO3"containing steady-state perfusion at r o o m temperature HCO3" a t r o o m t e m p e r a t u r e , t h e r e m o v a l o f [ C l " ]  0  Furthermore, i n thepresence o f  e n h a n c e d t h erate o fp H j recovery.  noted above, r e m o v a l o fexternal C l " likely increases t h eactivity o fHC0 7C1" 3  and thus t h e 0 [Cl"] -induced increase  exchange  i n t h e p H jrecovery rate m a y b e d u e t o a n  0  acceleration o f the anion exchanger.  A s  T h i s p o s s i b i l i t y i sc o n f i r m e d b y t h e o b s e r v e d  inhibitory influence o f D I D S o n t h e rate o f p H j recovery at r o o m temperature  during  HCO37CO2 p e r f u s i o n i n t h e p r e s e n c e o r a b s e n c e o f [ C l " ] . H o w e v e r , t h e f a c t t h a t D I D S 0  d i d n o tc o m p l e t e l y b l o c k a c i d l o a d r e c o v e r y indicates that other regulators o fp H j , s u c h a s the  Na -dependent, +  HCO3"-independent  acid  extrusion  mechanism,  m a y also b e  operational atr o o m temperature. O v e r a l l , t h eresults o fthis study s u g g e s t that, a t r o o m temperature, a c o m b i n a t i o n of  a Na -dependent, +  HC0 "-independent 3  acid  extrusion  mechanism  a n d aN a  +  -  i n d e p e n d e n t HCO37CI" e x c h a n g e r c o n t r i b u t e t o t h e r e g u l a t i o n o f p H j i n h i p p o c a m p a l neurones at steady-state as well as during p H j recovery f r o m a n induced acidification.  intracellular  133  Comparison of pHj regulation at 37°C and room temperature:  Based  either  on  steady-state  pHj  observations  o ro n  data  from  acid-load  r e c o v e r i e s , the c o n c l u s i o n s r e g a r d i n g p H j r e g u l a t i o n at 3 7 ° C o r at r o o m t e m p e r a t u r e very similar.  B o t h at 3 7 ° C a n d at r o o m t e m p e r a t u r e , p H j a p p e a r s to b e  regulated  the  by  activity  of  a Na -dependent, +  HC03"-independent  m e c h a n i s m , probably an amiloride-insensitive variant o f the Na /H +  dominance of Na /H +  +  predominantly acid  extrusion  exchanger.  +  T h e  e x c h a n g e o v e r p H j r e g u l a t i o n h a s b e e n d e m o n s t r a t e d i n av a r i e t y o f  vertebrate central n e u r o n e s at 3 7 ° C( N a c h s h e n a n d D r a p e a u , 1988; 1991;  were  R a l e y - S u s m a n et al,  O u - y a n g et al, 1 9 9 3 ) . H o w e v e r , S c h w i e n i n g a n d B o r o n ( 1 9 9 4 ) c o n c l u d e d t h a t t h e  primary acid extrusion mechanism in C A 1 pyramidal neurones acutely dissociated a d u l t r a t h i p p o c a m p i a t 3 7 ° C i s a N a - d e p e n d e n t HCO3VCI" e x c h a n g e r . +  from  T h e findings  t h e p r e s e n t s t u d y , h o w e v e r , i n d i c a t e t h a t t h e HCO37CI" e x c h a n g e r p r e s e n t o n  cultured  foetal h i p p o c a m p a l n e u r o n e s is n o t d e p e n d e n t o n [ N a ] , a n d f u r t h e r m o r e , t h e a c t i v i t y +  0  this a n i o n e x c h a n g e r a p p e a r s to b e m i n i m a l u n d e r steady-state c o n d i t i o n s at 3 7 ° C .  A s  neurones e m p l o y e d in this study w e r e cultured f r o m h i p p o c a m p i obtained f r o m  foetal  dissociated  or  cultured foetal  reflect the fact that HC0 "/C1" 3  demonstrated  rat h i p p o c a m p a l neurones  regulates p H j in acutely dissociated adult neurones.  in  a t37°C,  either actively  H o w e v e r , this observation  e x c h a n g e is s i m p l y n o t a c t i v e i n foetal n e u r o n e s at  rather than being completely absent.  and  I n f a c t , R a l e y - S u s m a n et al ( 1 9 9 3 )  h a v e r e p o r t e d t h a t HCO37CI" e x c h a n g e , w h i c h c o u l d n o t b e acutely  m a y 37°C,  I n d e e d , R a l e y - S u s m a n et al ( 1 9 9 3 ) r e p o r t e d  both adult a n d foetal neurones express mRNA  of the  rats, the difference b e t w e e n the present results a n d those reported b y S c h w i e n i n g B o r o n (1994) m a y reflect developmental changes.  of  for this anion  that  exchanger.  T h e results o f the present investigation suggest that, rather t h a n b e i n g absent f r o m cultured foetal rat h i p p o c a m p a l p y r a m i d a l neurones, the N a - i n d e p e n d e n t +  e x c h a n g e a c t i v e l y r e g u l a t e s n e u r o n a l p H j , b u t o n l y at ar e d u c e d t e m p e r a t u r e .  HCO3VCI" There were,  in addition, other differences in steady-state p H j caused b y changing the temperature  of  134  t h e p e r f u s i o n m e d i u m f r o m 37°C t o r o o m t e m p e r a t u r e .  I n t h e a b s e n c e o f HCO3", p H j  r e s t e d a p p r o x i m a t e l y 0.4 p H u n i t s l o w e r w h e n e x p e r i m e n t s  were performed a t r o o m  t e m p e r a t u r e i n c o m p a r i s o n t o 37°C. I n a d d i t i o n , r e s t i n g p H j a t 37°C w a s h i g h e r i n t h e a b s e n c e o f HCO3", w h e r e a s a t r o o m t e m p e r a t u r e p H ; w a s  higher in the presence o f  HCO3". p H j r e c o v e r y r a t e s f r o m i n d u c e d a c i d i f i c a t i o n s w e r e a l s o s u b s t a n t i a l l y  w h e n the temperature o f the perfusate was  i n c r e a s e d t o 37°C.  enhanced  Temperature-induced  c h a n g e s in the regulators o f p H j m a y reflect differential activities o f the acid  extrusion  m e c h a n i s m s present, or perturbations in the intracellular buffering capacity. B u f f e r i n g c a p a c i t y i st h e a b i l i t y o f c e l l s to resist c h a n g e s i n p H j i m p o s e d b y  the  a p p l i c a t i o n o f w e a k a c i d s o r b a s e s . T h e total i n t r a c e l l u l a r b u f f e r i n g p o w e r ( p ) is d e f i n e d T  a s t h e a m o u n t o f a c i d o r b a s e r e q u i r e d t o c h a n g e p H j b y o n e u n i t ( C h e s t e r , 1990). Pj i s a c o m b i n a t i o n o f t h e i n t r i n s i c b u f f e r i n g c a p a c i t y (Pj), a n d t h e b u f f e r i n g p o w e r s p e c i f i c a l l y b y t h e p r e s e n c e o f HCO37CO2 i na s y s t e m ( p ) . b  caused  p^,, a t c o n s t a n t  C0  2  t e n s i o n s , v a r i e s d i r e c t l y w i t h t h e c o n c e n t r a t i o n o f HCO3", s u c h t h a t t h e c o n t r i b u t i o n m a d e b y t h e p r e s e n c e o f HCO3VCO2 t o t h e b u f f e r i n g e q u a t i o n i s 2.3x[HCC»3~] ( s e e R o o s Boron,  P, i n c l u d e s n o n - b i c a r b o n a t e p h y s i o c h e m i c a l b u f f e r i n g ( e . g .  1981).  phosphates, Introduction).  and  proteins),  biochemical  buffering,  and  organellar  sulphates,  buffering  T o c a l c u l a t e the total i n t r a c e l l u l a r b u f f e r i n g p o w e r , ak n o w n  (see  concentration  o f w e a k acid or base i s applied t othe cells and, using the dissociation constant, a m o u n t o f b a s e o r a c i d e n t e r i n g t h e c e l l i sc a l c u l a t e d ;  and  the  the ensuing change in p H j i s then  u s e d to d e t e r m i n e the buffering capacity. E x p e r i m e n t s in w h i c h T M A or propionate  were  a p p l i e d to t h e n e u r o n e s e m p l o y e d i n this s t u d y p r o v i d e d ap o t e n t i a l m e a n s o f c a l c u l a t i n g intracellular buffering power. T M A  I n t h e p r e s e n c e o f HCO3" a t r o o m t e m p e r a t u r e , 10 m M  i n d u c e d a n i n t r a c e l l u l a r a l k a l i n i z a t i o n , w h e r e a s 20 m M  propionate induced a n  intracellular acidification. A c c o r d i n g to the a b o v e rationale, the i m p o s e d c h a n g e s in p H j should have provided sufficient information t ocalculate buffering power.  However,  errors are introduced into the computation o f buffering values b y the operation o f p H j  135  regulating m e c h a n i s m s w h i c h are activated as s o o n as a n intracellular acid or alkali load is i m p o s e d o n t h e cells.  T h i s p r o b l e m can only be alleviated b y the application  p h a r m a c o l o g i c a l inhibitors w h i c h act to b l o c k these regulators (see R o o s a n d 1981;  Vaughan-Jones and W u , 1990).  of  Boron,  Unfortunately, the pharmacological blockade  of  all a c i d e x t r u d i n g m e c h a n i s m s w a s n o t p o s s i b l e i n t h e p r e s e n t e x p e r i m e n t s d u e t o  the  insensitivity o f the neurones to inhibitors o f cation e x c h a n g e , w h i c h appears to b e  the  dominant m e c h a n i s m of p H j regulation. Despite these difficulties, a n a p p r o x i m a t i o n o f intracellular buffering capacity these neurones was obtained b y analyzing data resulting f r o m the application o f W h e n e x p o s e d to extracellular N H C 1 ,  p H j increases d u e to the influx o f N H  4  s u b s e q u e n t h y d r a t i o n to f o r m N H  + 4  in  NH and  3  and O H " . K n o w i n g the concentration o f the  its  applied  NH4CI, t h e m a g n i t u d e o f t h i s i n d u c e d a l k a l i n i z a t i o n c o u l d b e u s e d t o e s t i m a t e P .  The  T  limitation o f this technique, in addition to the previously discussed inability to errors introduced b y the activation o f p H j regulating mechanisms,  .  + 4  prevent  i sthat i sdoes  not  a c c o u n t f o r t h e a p p e a r a n c e o f i n t r a c e l l u l a r NH4 " f r o m s o u r c e s o t h e r t h a n i n f l u x i n g N H  .  4  3  D u r i n g e x p o s u r e t o e x t r a c e l l u l a r NH4CI, N H 4 w i l l p a s s i v e l y d i f f u s e i n t o t h e c e l l d u e  to  an electrochemical driving force.  if  +  present,  can carry NH  + 4  M o r e o v e r , t h e r e i se v i d e n c e that a N a / K +  into cells in place  of K  +  (see  pump,  +  Boron, 1989).  Since  experiments p e r f o r m e d could not exclude the effects o f these p h e n o m e n a , the c o u l d not be used to formally determine buffering power.  results  However, by measuring  a l k a l i n e c h a n g e s i n p H j r e s u l t i n g f r o m a 3 m i n u t e a p p l i c a t i o n o f NH4 u n d e r t h e  the  the  various  +  e x p e r i m e n t a l conditions, a n indication o f the n e u r o n a l ability to buffer i m p o s e d p H j shifts w a s o b t a i n e d (see c o l u m n 3, T a b l e 6). The m a g n i t u d e o f the p H j increase resulting f r o m a 3 m i n u t e e x p o s u r e to was  greatest in neurones  lacking HC0 " 3  attenuated b y the presence o f HC0 " 3  a tr o o m temperature.  This increase  NH  + 4  was  d u r i n g t h e a p p l i c a t i o n o f NH4 . T h e r e f o r e , a t r o o m +  temperature, these neurones are better able to resist p H ; c h a n g e s w h e n H C 0 " 3  and  C 0  2  136  are present in the extracellular m e d i u m .  C 0 - i n d u c e d enhancement of internal buffering 2  p o w e r h a s b e e n d e m o n s t r a t e d i n a variety o f n e u r o n a l p r e p a r a t i o n s (e.g. T h o m a s ,  1976b),  and thus increased resistance to alkaline p H j shifts p r o b a b l y reflects the contribution o f H C 0 7 C 0 - d e p e n d e n t b u f f e r i n g p r o c e s s e s (i.e. p ) . 3  2  Increasing the temperature o f  b  the  p e r f u s i o n m e d i u m to 37°C also h a d a p r o f o u n d effect o n the apparent buffering capacity. I n t h e a b s e n c e o f HCO3", a 3 m i n u t e a p p l i c a t i o n o f N H  at 3 7 ° C p r o d u c e d a p H ; rise less  + 4  t h a n t h a t o b s e r v e d a t r o o m t e m p e r a t u r e i n t h e p r e s e n c e o f HCO3". W i t h HCO3" p r e s e n t i n the p e r f u s i o n s o l u t i o n at 3 7 ° C , the p H j rise w a s f u r t h e r r e d u c e d to a p p r o x i m a t e l y 0.1 o f a pH  unit.  T h e internal b u f f e r i n g c a p a c i t y at 3 7 ° C  presence of C 0  2  a n d HCO3-.  is t h e r e f o r e a l s o e n h a n c e d b y  the  F u r t h e r m o r e , the results s u g g e s t that, w h e t h e r i n  the  p r e s e n c e o r a b s e n c e o f HCO3", t h e r e l a t i v e b u f f e r i n g p o w e r o f t h e s e n e u r o n e s i s g r e a t e s t at 3 7 ° C .  Temperature considerations regarding intracellular buffering have not been well  d o c u m e n t e d (see R o o s a n d B o r o n , 1981).  H o w e v e r , B u r t o n (1978) indicates that  the  apparent dissociation constants o f acid-base pairs w h i c h contribute to  physiochemical  buffering m a y be significantly altered b y factors s u c h as temperature.  In addition  physiochemical buffering, there m a y also be an increase in biochemical a n d buffering associated with an elevation temperature  in temperature.  o n the regulation o f p H j m a y  organellar  Accordingly, the effects  reflect, at least i n part, the  to  of  temperature  dependence o f intracellular buffering mechanisms, although other m e c h a n i s m s are likely to participate in the  observed  temperature  effects, including  temperature-dependent  changes in acid extrusion rates a n d passive fluxes o f acid equivalents Boron,  (see R o o s  and  I n s u m m a r y , at r o o m t e m p e r a t u r e a n d at 3 7 ° C , the resulting steady-state  pHj  1981).  during perfusion with HC03"/C0 -buffered medium ( p H 2  0  7.35) is a p p r o x i m a t e l y  7.15.  This value m a y be the optimal level a s s u m e d b y p H j under conditions that allow for the expression  o f all p H j regulating mechanisms.  W h e n HCO3" i s r e m o v e d  temperature, p H , deviates f r o m this optimal value.  at  A t r o o m temperature, p H j  either falls  137  substantially w h e n s w i t c h e d to H E P E S - b u f f e r e d pHj increases,  albeit only slightly, w h e n  p e r f u s i o n solutions, w h e r e a s at  HCO3"  is n o t present.  37°C,  T h e ability of  n e u r o n e s e m p l o y e d i n t h i s s t u d y t o m a i n t a i n p H j d e s p i t e t h e a b s e n c e o f HCO3" a t m a y reflect a temperature-dependent  the 37°C  accentuation o f the internal buffering power.  A t  r o o m t e m p e r a t u r e , this a p p a r e n t b u f f e r i n g c a p a c i t y is d i m i n i s h e d , a n d t h u s the  neurones  m a y resort to other m e a n s , s u c h as increased activity o f N a - i n d e p e n d e n t  HC0 7CT  +  exchange, to regulate  steady-state p H j to the o p t i m a l level.  3  Intracellular  buffering  t h e r e f o r e c o n t r i b u t e s to t h e m a i n t e n a n c e o f r e s t i n g p H j i n a m a n n e r t h a t is d e p e n d e n t the experimental temperature, a n d thus m a y explain the differences  in p H j  on  regulation  o b s e r v e d at r o o m t e m p e r a t u r e a n d at 3 7 ° C .  Modulation of p H j by p H  0  :  In the presence o f HC0 ", 3  a shift in p H  at b o t h r o o m t e m p e r a t u r e a n d at 3 7 ° C .  0  caused a qualitatively similar shift in p H j ,  This observation demonstrates the dependence  of  pHj o n p H , w h i c h has been documented in other vertebrate neurones (Tolkovsky  and  Richards, 1987;  that  0  N a c h s h e n and Drapeau, 1988;  O u - y a n g et al, 1 9 9 3 ) a n d i n d i c a t e s  neuronal p H j cannot be regulated b a c k to n o r m a l levels until p H  0  is n o r m a l i z e d . I n t u r n ,  this suggests the possibility that the m e c h a n i s m s responsible for the maintenance o f p H j m a y  b e subject to m o d u l a t i o n b y the extracellular p r o t o n e n v i r o n m e n t .  discussed above, a decrease in p H induced intracellular acidification.  0  Indeed,  as  causes a n increase in the rate o f recovery f r o m  an  M o r e o v e r , the dependence o f p H j o n p H  0  suggests  that c h a n g e s in n e u r o n a l excitability w h i c h to date h a v e b e e n attributed to c h a n g e s i n p H (see  Introduction)  should  be  careful analyzed  since they  m a y  in fact  a c c o m p a n y i n g alteration in p H j , rather than just being due to c h a n g e s in p H  0  reflect p e r se.  0  the  138  Figure 35. Diagrammatic representation of pHj recovery from an acid load in the presence and absence of HCO3", at room temperature and at 37°C.  The figure s h o w s p H j recovery f r o m an N H - i n d u c e d acidification in the absence o f HCO3" a t r o o m t e m p e r a t u r e ( • ) , i n t h e p r e s e n c e o f HCO3" a t r o o m t e m p e r a t u r e ( • ) , in t h e p r e s e n c e o f HCO3- a t 3 7 ° C ( • ) , a n d i n t h e a b s e n c e o f H C 0 " a t 3 7 ° C (A). p H r a n g e d f r o m 7.33 to 7.36 u n d e r the four conditions. T h e solid lines represent a least s q u a r e s e x p o n e n t i a l b e s t fit t o d a t a p o i n t s i n d i c a t e d , w h i c h w e r e o b t a i n e d f r o m f o u r separate e x p e r i m e n t s u n d e r the c o n d i t i o n s specified. L i n e s b e g i n at the m i n i m u m p H , reached during the acidification, a n d represent the trend f o u n d in all experiments conducted under similar conditions. +  4  3  0  139  Time (minutes)  140  Conclusions: The during  mechanisms  recovery  from  pyramidal neurones.  operating to regulate p H j were examined a n induced  acidification i n cultured foetal  at steady-state o r rat hippocampal  Temperature w a s found to exert a p r o f o u n d effect o n the relative  activities o f these m e c h a n i s m s (see Figure 36). A t 37°C, t h e p r i m a r y regulator o f p H ; appears to b e a Na -dependent, HCC^'-independentacid extrusion mechanism  (probably  +  an amiloride insensitive variant o f the Na /H +  +  exchanger).  A t r o o m temperature, the HCO37CI"  results o f this study suggest that p H j is regulated b y a N a - i n d e p e n d e n t +  exchanger, w h i c h probably acts to supplement t h e activity o f t h e s a m e acid extrusion mechanism observed at 37°C.  Na -dependent +  T h edata d o not support n o r exclude the  existence o f a N a - d e p e n d e n t HC0 7C1" exchanger o n these neurones. +  3  However,  if  present, this N a - d e p e n d e n t anion transporter w o u l d p r e s u m a b l y play a m i n o r role i n the +  r e g u l a t i o n o f p H j d u e t o t h e d e m o n s t r a t e d d o m i n a n c e o f a HCO3 " - i n d e p e n d e n t  acid  extrusion m e c h a n i s m governing p H j , especially at 37°C. As  noted  i n the Introduction,  physiological a n d pathological events. directly responsible  p H j is a n important  o f  for regulating p H j m a y therefore  the  possible  o f  T h e investigation o f the mechanisms  regarding the role o f p H j i n such events. investigation  modulator  help  to provide  many  that a r e  information  Future experimental directions include a n  modulation  o f  p H j regulatory  neurotransmitter candidates, a n d w a y s i n w h i c h the effects o f applied  mechanisms  b y  neuromodulators  are i n turn regulated b y p H j . Demonstrated o n non-neuronal cell types, the activation o f various cell surface receptors, i n c l u d i n g P-adrenergic, o^-adrenergic, somatostatin, D dopaminergic, a n d muscarinic cholinergic receptors, directly regulates the activity N a  +  / H e x c h a n g e ( I s o m etal, 1 9 8 9 ; B a r b e r et al, +  1 9 8 9 ; G a n z et al,  1990).  2  o f  Accordingly,  it w i l l b e o f i n t e r e s t t o e x a m i n e t h e e f f e c t s o f n e u r o t r a n s m i t t e r s , i n c l u d i n g n o r e p i n e p h r i n e , somatostatin,  dopamine, a n d serotonin, o n the p H j regulating mechanisms  hippocampal neurones.  present  o n  S u c h a n investigation w o u l d include a n examination o f the  141  effects o f intracellular second messengers o n p H , , since changes in cytoplasmic levels cAMP,  Ca  2 +  of  , a n d certain protein kinases h a v e b e e n s h o w n to m o d u l a t e p H j in a variety o f  peripheral cell types (Grinstein a n d Rothstein, 1986).  In a recent study o f rat brain  synaptosomes,  indicated  Sanchez-Armass  et  al  (1994)  have  that  intracellularly, m a y play a n important role in the m o d u l a t i o n o f Na /H +  was  not altered b y enhancing the cytosolic  ( S a n c h e z - A r m a s s et al,  1994).  levels  C a  increase in intracellular p r o t o n levels.  p H ,  exists between  pHj and Ca 2  2 +  +  .  For  observed  influx resulting f r o m the N M D A  associated  in rat h i p p o c a m p a l neurones application.  Therefore,  the intracellular processes w h i c h thus far h a v e b e e n attributed to c h a n g e s in 2 +  and other intracellular second  messengers.  a  currents in catfish  that is  further  s t u d i e s s h o u l d i n c l u d e t h e i n v e s t i g a t i o n o f p H j i n t e r m s o f its a b i l i t y to m o d u l a t e s o m e  Ca  A  example,  M o r e o v e r , I r w i n et al ( 1 9 9 4 ) h a v e s u g g e s t e d  intracellular acidosis 2 +  acting  or kinase  horizontal cells are suppressed d u r i n g glutamate application as a result o f a n  on Ca  ,  exchange.  +  of protein kinase C  D i x o n et al ( 1 9 9 3 ) h a v e d e m o n s t r a t e d t h a t h i g h v o l t a g e a c t i v a t e d C a  dependent  +  I n a d d i t i o n , it is b e c o m i n g i n c r e a s i n g l y a p p a r e n t t h a t  physiologically-relevant interdependence  an NMDA-induced  2  of  cytosolic  142  Figure 36. Schematic presentation of pHj regulating mechanisms in cultured foetal hippocampal pyramidal neurones at 37°C and room temperature.  At 37°C, the dominant regulator in pHj in these hippocampal neurones is a N a dependent, HCC>3"-independent acid extrusion mechanism, which is probably a N a / H exhanger. Though an anion exchanger was found to be present, its activity at 37°C appears to be minimal. At room temperature, pHj is regulated by a Na -independent HCO3VCI" exchanger acting to supplement the activity of the same Na -dependent, HC0 "-independent acid extrusion mechanism observed at 37°C. +  +  +  +  3  +  143  37 ° C  Room temperature  OUT I IN  1Na  OUT  1 IN  1 Na -^ +  +  S*-1 H+ 1 HCO,  1 HC0 3  i  OTc-  144  R E F E R E N C E S  A h m e d , Z . ,a n d C o n n o r , J . A . (1980). Intracellular p H changes induced b y calcium i n f l u x d u r i n g e l e c t r i c a l a c t i v i t y i n m o l l u s c a n n e u r o n s . Journal of General Physiology 75, 403-426. 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