Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Regulation of intracellular pH in cultured foetal rat hippocampal pyramidal neurones 1995

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
ubc_1995-0100.pdf
ubc_1995-0100.pdf [ 5.72MB ]
Metadata
JSON: 1.0086732.json
JSON-LD: 1.0086732+ld.json
RDF/XML (Pretty): 1.0086732.xml
RDF/JSON: 1.0086732+rdf.json
Turtle: 1.0086732+rdf-turtle.txt
N-Triples: 1.0086732+rdf-ntriples.txt
Citation
1.0086732.ris

Full Text

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 pH IN C U L T U R E D F O E T A L R A T 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 b y K E I T H A L L E N B A X T E R B . S c . ( C h e m i s t r y ) , T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 9 1 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n 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 ( D e p a r t m e n t o f A n a t o m y ) W e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A F e b r u a r y , 1 9 9 5 © K e i t h A l l e n B a x t e r , 1 9 9 5 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ^ ^ c ^ c w v . ^ The University of British Columbia Vancouver, Canada Date •£^>. JtA^ DE-6 (2/88) 11 A B S T R A C T 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 3 _ , and 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 HCO3", 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 HCO3", 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 Na + . 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 HC0 3 " to the perfusate did not increase the rate of pHj 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 t e m p e r a t u r e , t h i s 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 r e m a i n s a c t i v e , b u t t h e r e g u l a t i o n o f p H j a p p e a r s t o b e s u p p l e m e n t e d b y t h e a c t i v i t y o f a N a + - i n d e p e n d e n t HCO3VCI- e x c h a n g e r . i v TABLE OF CONTENTS P a g e A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i A c k n o w l e d g m e n t s x I N T R O D U C T I O N 1 P h y s i o l o g y , p a t h o p h y s i o l o g y , a n d p H j 3 p H j a n d c e l l e x c i t a b i l i t y 6 p H j a n d i o n i c c o n d u c t a n c e s 9 p H i a n d C a 2 + 1 2 D i s t r i b u t i o n o f p r o t o n s a c r o s s t h e l i m i t i n g m e m b r a n e 1 4 R e g u l a t i o n o f p H j 1 5 I n t r a c e l l u l a r b u f f e r i n g 1 9 O v e r v i e w 2 1 M A T E R I A L S A N D M E T H O D S C e l l p r e p a r a t i o n 2 2 L o a d i n g t h e n e u r o n e s w i t h B C E C F 2 3 E x p e r i m e n t a l s e t u p 2 4 S o l u t i o n s 2 5 C a l c u l a t i o n o f p H j 2 8 A n a l y s i s o f d a t a 3 2 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 f p H , a t r o o m t e m p e r a t u r e 4 2 R e g u l a t i o n o f p H ; a t 3 7 ° C 4 3 N a + - d e p e n d e n t o r - i n d e p e n d e n t a n i o n e x c h a n g e 4 6 M o d u l a t i o n o f p H j b y s h i f t s i n p H 0 a n d t h e a p p l i c a t i o n o f w e a k a c i d s a n d b a s e s 4 6 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 8 6 R e c o v e r y f r o m a n a c i d l o a d a t r o o m t e m p e r a t u r e 8 8 R e c o v e r y f r o m a n a c i d l o a d a t 3 7 ° C 8 9 V P a g e D I S C U S S I O N 1 2 2 R e g u l a t i o n o f p H j a t 3 7 ° C 1 2 2 R e g u l a t i o n o f p H j a t r o o m t e m p e r a t u r e 1 3 0 C o m p a r i s o n o f p H , r e g u l a t i o n a t 3 7 ° C a n d r o o m t e m p e r a t u r e 1 3 3 M o d u l a t i o n o f p H ; b y p H 0 1 3 7 C o n c l u s i o n s 1 4 0 R E F E R E N C E S 1 4 4 v i L I S T O F T A B L E S P a g e 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 3 4 T a b l e 2 C o m p o s i t i o n o f H C 0 3 ' / C 0 2 - 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 a t r o o m t e m p e r a t u r e 3 5 T a b l e 3 C o m p o s i t i o n o f H C 0 3 " / C 0 2 - 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 a t 3 7 ° C .... 3 6 T a b l e 4 C o m p o s i t i o n o f H C 0 3 7 C 0 2 - 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 a t v a r y i n g p H s a t 3 7 ° C 3 7 T a b l e 5 S t e a d y - s t a t e p H j i n H C 0 3 " - f r e e a n d H C O 3 " - c o n t a i n i n g 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 n p H j c a u s e d b y t h e e x p o s u r e t o t h e e x p e r i m e n t a l s o l u t i o n s i n d i c a t e d 4 9 T a b l e 6 p H ; r e c o v e r i e s f r o m a n N H 4 + - i n d u c e d 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 9 5 v i i L I S T O F F I G U R E S P a g e F i g u r e 1 R e l a t i o n s h i p b e t w e e n t h e c o n c e n t r a t i o n o f H C 0 3 " a n d t h e r e s u l t i n g s o l u t i o n p H w h e n e q u i l i b r a t e d w i t h 5 % C 0 2 i n b a l a n c e a i r a t 3 7 ° C 3 9 F i g u r e 2 S a m p l e c a l i b r a t i o n p l o t f o r B C E C F 4 1 F i g u r e 3 D i s t r i b u t i o n o f s t e a d y - s t a t e p H j 5 1 F i g u r e 4 E f f e c t o f 0 [ C l " ] 0 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 0 3 " a t r o o m t e m p e r a t u r e 5 3 F i g u r e 5 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 r o o m t e m p e r a t u r e 5 5 F i g u r e 6 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 r o o m t e m p e r a t u r e 5 7 F i g u r e 7 E f f e c t o f 0 [ N a + ] 0 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 0 3 " a t 3 7 ° C ..; 5 9 F i g u r e 8 E f f e c t o f 0 [ C T ] 0 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 6 1 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 ... 6 3 F i g u r e 1 0 C o m b i n e d e f f e c t o f 0 [ N a + ] 0 a n d E I P A o n s t e a d y - s t a t e p H ; i n t h e a b s e n c e o f H C O 3 - a t 3 7 ° C 6 5 F i g u r e 1 1 E f f e c t o f M G C M A a n d H O E 6 9 4 o n s t e a d y - s t a t e p H , i n t h e a b s e n c e o f H C 0 3 - a t 3 7 ° C 6 7 F i g u r e 1 2 E f f e c t o f 0 [ N a + ] 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 H C O 3 " a t 3 7 ° C 6 9 F i g u r e 1 3 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 F i g u r e 1 4 E f f e c t o f 0 [ C l - ] 0 , a n d t h e c o m b i n e d e f f e c t o f 0 [ C l " ] 0 p l u s 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 3 7 ° C 7 3 F i g u r e 1 5 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 F i g u r e 1 6 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 v i i i P a g e F i g u r e 1 7 E f f e c t o f HCO3VCO2 o n s t e a d y - s t a t e p H j d u r i n g 0 [ N a + ] 0 p e r f u s i o n a t r o o m t e m p e r a t u r e 7 9 F i g u r e 1 8 E f f e c t o f 0 [ C l " ] 0 d u r i n g 0 [ N a + ] 0 p e r f u s i o n 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 0 3 - a t 3 7 ° C 8 1 F i g u r e 1 9 E f f e c t o f c h a n g e s i n p H 0 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 8 3 F i g u r e 2 0 E f f e c t o f p r o p i o n a t e a n d T M 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 r o o m t e m p e r a t u r e 8 5 F i g u r e 2 1 S a m p l e a c i d l o a d w i t h N H 4 C 1 9 4 F i g u r e 2 2 I n i t i a l r a t e o f a c i d l o a d r e c o v e r y a s a f u n c t i o n o f p H j , p r e l o a d p H j , m i n i m u m p H j , a n d n e t p H j d e c r e a s e 9 7 F i g u r e 2 3 p H , r e c o v e r y f r o m a n a c i d l o a d i n t h e a b s e n c e a n d p r e s e n c e o f H C C y a t r o o m t e m p e r a t u r e 9 9 F i g u r e 2 4 E f f e c t o f D I D S o n p H ; r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f H C O 3 " a t r o o m t e m p e r a t u r e 1 0 1 F i g u r e 2 5 E f f e c t o f 0 [ C l " ] 0 o n p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f H C O 3 " a t r o o m t e m p e r a t u r e 1 0 3 F i g u r e 2 6 p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e a b s e n c e a n d p r e s e n c e o f H C 0 3 - a t 3 7 ° C 1 0 5 F i g u r e 2 7 E f f e c t o f 0 [ N a + ] 0 o n p H ; r e c o v e r y f r o m a n a c i d l o a d i n t h e a b s e n c e o f H C 0 3 - a t 3 7 ° C 1 0 7 F i g u r e 2 8 E f f e c t o f E I P A o n p H ; r e c o v e r y f r o m a n a c i d l o a d i n t h e a b s e n c e o f H C 0 3 - a t 3 7 ° C 1 0 9 F i g u r e 2 9 E f f e c t o f D I D S o n p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e a b s e n c e o f H C O 3 - a t 3 7 ° C 1 1 1 F i g u r e 3 0 E f f e c t o f 0 [ N a + ] 0 o n p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f H C 0 3 - a t 3 7 ° C 1 1 3 IX P a g e F i g u r e 3 1 E f f e c t o f E I P A o n p H j 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 o f H C 0 3 - a t 3 7 ° C 1 1 5 F i g u r e 3 2 E f f e c t o f 0 [ C l " ] 0 o n p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f H C 0 3 - a t 3 7 ° C 1 1 7 F i g u r e 3 3 E f f e c t o f D I D S o n p H j r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f H C 0 3 - a t 3 7 ° C 1 1 9 F i g u r e 3 4 E f f e c t o f D I D S o n p H j r e c o v e r y f r o m a n e n h a n c e d a c i d l o a d i n t h e p r e s e n c e o f H C 0 3 - a t 3 7 ° C 1 2 1 F i g u r e 3 5 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 3 " , 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 .... 1 3 9 F i g u r e 3 6 S c h e m a t i c p r e s e n t a t i o n o f p H j r e g u l a t i n g m e c h a n i s m s i n c u l t u r e d f o e t a l 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 a t 3 7 ° C a n d r o o m t e m p e r a t u r e 1 4 3 X ACKNOWLEDGMENTS T h e r e a r e m a n y t o w h i c h I w o u l d l i k e t o e x t e n d m y s i n c e r e s t a p p r e c i a t i o n a n d g r a t i t u d e . F i r s t l y , I a m i n d e b t e d t o D r . J o h n C h u r c h f o r h i s e n d u r i n g g u i d a n c e , e n c o u r a g e m e n t , a n d i n s i g h t . J o h n , y o u h a v e b e e n i n s t r u m e n t a l i n m y g r o w t h a s a s c i e n t i s t a n d a s a n i n d i v i d u a l . F o r t h e i r c o n s i d e r a t i o n a n d t i m e , I t h a n k t h e o t h e r m e m b e r s o f m y s u p e r v i s o r y c o m m i t t e e , 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 P a l a t y . I w o u l d e s p e c i a l l y l i k e t o e x t e n d m y d e e p e s t g r a t i t u d e t o D r . P a l a t y f o r s h a r i n g h i s b r e a d t h o f k n o w l e d g e , e v e n b e y o n d t h e s c o p e o f s c i e n c e . F u r t h e r m o r e , I h u m b l y a c k n o w l e d g e M o n i k a G r u n e r t a n d G a r t h S m i t h f o r t h e i r t e c h n i c a l e x p e r t i s e a n d e x p e r i m e n t a l a s s i s t a n c e . T o t h e F a c u l t y , G r a d u a t e S t u d e n t s , a n d S t a f f i n t h e D e p a r t m e n t o f A n a t o m y , I t h a n k y o u f o r c r e a t i n g a n a t m o s p h e r e t h a t h a s f o s t e r e d a r e l e n t l e s s p u r s u i t o f k n o w l e d g e . I w i l l l o o k f o n d l y u p o n f r i e n d s h i p s a n d m e m o r i e s o f m y t i m e h e r e . I w o u l d p a r t i c u l a r l y l i k e t o e x p r e s s m y h e a r t f e l t a p p r e c i a t i o n t o m y e s t e e m e d f r i e n d a n d c o l l e a g u e , S e a n V i r a n i , f o r h i s p e r s i s t e n t y e t g e n u i n e a b i l i t y t o k e e p m e o n a n e v e n k e e l . F o r e m o s t , a d e b t o f g r a t i t u d e i s o w e d t o m y f a m i l y f o r t h e i r c o n t i n u e d s u p p o r t a n d i n s p i r a t i o n . M u m a n d D a d , I a m e s p e c i a l l y g r a t e f u l t o y O u b o t h f o r y o u r p a t i e n c e a n d u n c o n d i t i o n a l l o v e . F i n a n c i a l s u p p o r t w a s p r o v i d e d b y a n o p e r a t i n g g r a n t t o D r . J o h n C h u r c h f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f C a n a d a . 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 0) shift 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 0 shifts associated with normal neuronal activity, tissue pH 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, 1 9 3 6 ) . R e c e n t e v i d e n c e p o i n t s t o w a r d s p H c h a n g e s a s t h e c a u s e f o r t h e s e C C ^ - i n d u c e d a l t e r a t i o n s i n s e i z u r e a c t i v i t y . A r a m a n d L o d g e ( 1 9 8 7 ) a n d 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 0 , b y e i t h e r d i r e c t t i t r a t i o n w i t h H C 1 o r i n c r e a s i n g t h e p a r t i a l p r e s s u r e o f C 0 2 ( P C O 2 ) , w i l l s u p p r e s s t h e i n d u c t i o n o f s e i z u r e a c t i v i t y i n t h e r a t c o r t e x a n d h i p p o c a m p u s . F O r n a i et al ( 1 9 9 4 ) o b s e r v e d t h a t a c i d i c c o n d i t i o n s , p r o d u c e d b y t h e a p p l i c a t i o n o f l a c t a t e , e x e r t a s i m i l a r a n t i - e p i l e p t i f o r m a c t i o n o n r a t c o r t i c a l n e u r o n e s . I n c o n s t r a s t , a l k a l o s i s i n d u c e s e p i l e p t i f o r m a c t i v i t y ( A r a m a n d L o d g e , 1 9 8 7 ; C h u r c h a n d M c L e n n a n , 1 9 8 9 ; J a r o l i m e k et al, 1 9 8 9 ) . T h e i n d u c t i o n o f m i l d b r a i n a c i d o s i s , p r o d u c e d b y h y p e r c a r b i c v e n t i l a t i o n , h a s a l s o b e e n 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, 1 9 9 3 ) , p o s s i b l y v i a i n h i b i t i o n o f t h e r i s e i n i n t r a c e l l u l a r C a 2 + t h a t n o r m a l l y c h a r a c t e r i z e s t h e o n s e t o f i s c h e m i a ( E b i n e et al, 1 9 9 4 , K r i s t i a n et al, 1 9 9 4 ) . I n a d d i t i o n , h y p o x i a - i n d u c e d s p r e a d i n g d e p r e s s i o n i n h i p p o c a m p a l s l i c e s c a n b e p r e v e n t e d b y e x p o s u r e t o a c i d i c a r t i f i c i a l c e r e b r o s p i n a l f l u i d , w h e r e a s a n a l k a l i n e p H 0 w i l l p r e d i s p o s e n e u r o n e s t o s p r e a d i n g d e p r e s s i o n a f t e r o x y g e n d e p r i v a t i o n ( T o m b a u g h , 1 9 9 4 ) . W h i l s t m i l d e x t r a c e l l u l a r a c i d i t y 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, 1 9 9 3 ) , e x c e s s i v e a c i d o s i s ( p H 0 < 5 . 3 ) h a s b e e n c o r r e l a t e d w i t h t h e d e a t h o f b r a i n t i s s u e f o l l o w i n g c o m p l e t e i s c h e m i a ( K r a i g et al, 1 9 8 7 ) . S i m i l a r l y , N e d e r g a a r d et al ( 1 9 9 1 ) h a v e s h o w n t h a t t h e p r o l o n g e d e x p o s u r e o f n e u r o n e s a n d g l i a t o l a c t i c a c i d o r H C 1 ( p H 0 < 6 . 8 ) p r o d u c e d t o x i c e f f e c t s l e a d i n g t o c e l l d e a t h . 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 t o c h a n g e s i n t i s s u e p H i s r e f l e c t e d i n p H - i n d u c e d a l t e r a t i o n s i n m a n y n o r m a l p h y s i o l o g i c a l e v e n t s . F o r i n s t a n c e , a f a l l i n t h e i n t e r s t i t i a l p H , c a u s e d e i t h e r b y e l e v a t e d H + o r C 0 2 c o n c e n t r a t i o n s , h a s b e e n s h o w n t o h a v e a d e p r e s s a n t e f f e c t o n n e u r o n a l e x c i t a b i l i t y i n t h e h i p p o c a m p a l f o r m a t i o n ( S o m j e n et al, 1 9 8 7 ; B a l e s t r i n o a n d S o m j e n , 1 9 8 8 ; C h u r c h a n d M c L e n n a n , 1 9 8 9 ) . S i m i l a r l y , T a i r a et al ( 1 9 9 3 ) h a v e s h o w n t h a t s y n a p t i c t r a n s m i s s i o n i s s e n s i t i v e t o c h a n g e s i n p H 0 , w h e r e b y a n i n c r e a s e o r d e c r e a s e i n p H 0 w i l l r e d u c e o r p o t e n t i a t e e x c i t a t o r y t r a n s m i s s i o n , 3 r e s p e c t i v e l y . T h e e f f e c t o f t i s s u e p H o n t h e e x c i t a b l e p r o p e r t i e s o f c e l l s m a y i n f a c t r e f l e c t t h e p H - i n d u c e d m o d u l a t i o n o f v a r i o u s i o n i c c o n d u c t a n c e s , s u c h a s C a 2 + , t h r o u g h 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 , i t i s 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 a n d p a t h o l o g i c a l e v e n t s a r e m o d u l a t e d b y f l u c t u a t i o n s i n t h e e x t e r n a l p H . I n m o s t v e r t e b r a t e s t u d i e s , h o w e v e r , t h e i n f l u e n c e o f c h a n g e s i n i n t r a c e l l u l a r p H ( p H j ) o n n e u r o n a l e x c i t a b i l i t y , i n j u r y , o r m o r t a l i t y h a s n o t b e e n t h o r o u g h l y i n v e s t i g a t e d . T h i s i s s u r p r i s i n g s i n c e r e c e n t e v i d e n c e i n d i c a t e s t h a t c h a n g e s i n p H j w i l l a c c o m p a n y c h a n g e s i n p H 0 ( P r e i s s l e r a n d W i l l i a m s , 1 9 8 1 ; A i c k e n , 1 9 8 4 ; T o l k o v s k y a n d R i c h a r d s , 1 9 8 7 ; C h e s l e r , 1 9 9 0 ; O u - y a n g et al, 1 9 9 3 ; K a t s u r a et al, 1 9 9 4 ; S u n a n d V a u g h a n - J o n e s , 1 9 9 4 ) . 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 o n p H 0 i n n e u r o n e s a n d g l i a . T h e f a c t t h a t c h a n g e s i n p H 0 m a y h a v e a s i g n i f i c a n t i m p a c t o n p H ; i m p l i e s a p o s s i b l e r o l e f o r p H j i n s o m e o f t h e a f o r e m e n t i o n e d e v e n t s m o d u l a t e d b y c h a n g e s i n t i s s u e p H . I n d e e d , a s e x p l a i n e d b e l o w , t h e r e a p p e a r s t o b e a n i n t e r d e p e n d e n c e b e t w e e n p H j a n d m a n y 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 o c c u r r e n c e s , w h i c h , t h o u g h e x a m i n e d i n i n v e r t e b r a t e n e u r o n a l a n d v e r t e b r a t e n o n - n e u r o n a l p r e p a r t a t i o n s , h a s n o t b e e n e x t e n s i v e l y s t u d i e d i n m a m m a l i a n c e n t r a l n e u r o n e s . Physiology, pathophysiology, and pHj: M a n y n o r m a l a n d a b n o r m a l c e l l f u n c t i o n s c a n m o d u l a t e , o r a r e m o d u l a t e d b y , t h e i n t r a c e l l u l a r a c i d - b a s e b a l a n c e . F o r i n s t a n c e , p H j c h a n g e s a f f e c t m a n y a s p e c t s o f m u s c l e d y n a m i c s . A n i n t r a c e l l u l a r a l k a l o s i s i n c a r d i a c P u r k i n j e f i b r e s p r o d u c e s a n i n c r e a s e i n t h e 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 i s a s s o c i a t e d w i t h a f a l l 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 a n d V o i p i o ( 1 9 9 0 ) h a v e r e p o r t e d t h a t t h e 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 f i b r e s i s i n c r e a s e d b y a n r e d u c t i o n o f p H j , a n d d e c r e a s e d b y a n e l e v a t i o n o f p H j . C h a n g e s i n p H j a l s o a f f e c t v a s c u l a r t o n e . R a i s i n g p H j i n c r e a s e s t h e t e n s i o n o f r a t v a s c u l a r s m o o t h m u s c l e f i b r e s i n a 4 f a s h i o n t h a t i s i n d e p e n d e n t o f p H 0 ( A u s t i n a n d W r a y , 1 9 9 3 ) . S t u d i e s o n t h e r a t p o r t a l v e i n r e v e a l s i m i l a r r e s u l t s , a n d m a y p r o v i d e a p o s s i b l e e x p l a n a t i o n f o r t h e o b s e r v e d 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 i s a l s o c l o s e l y t i e d t o i n t r a c e l l u l a r a c i d i t y ( B u s a , 1 9 8 6 ) . C e l l s e x p o s e d t o p e r t u r b a t i o n s i n p H , m a y e x p e r i e n c e s h i f t s i n t h e n o r m a l o p e r a t i o n o f i n t r a c e l l u l a r e n z y m e s w h o s e a c t i v i t y k i n e t i c s a r e p H d e p e n d e n t ( B u s a , 1 9 8 6 ) . A c t i v e s i t e s o n e n z y m e s m a y c o n t a i n i o n i z a b l e g r o u p s w h i c h a r e i n v o l v e d i n t h e b i n d i n g o f s u b s t r a t e s a n d c o f a c t o r s ( R o o s a n d B o r o n , 1 9 8 1 ) . F l u c t u a t i o n s i n i n t r a c e l l u l a r p r o t o n l e v e l s w i l l a f f e c t t h e i o n i z a t i o n o f t h e s e g r o u p s , t h u s i n f l u e n c i n g e n z y m e c o n f o r m a t i o n a l s t a t e s a n d t h e a b i l i t y t o f o r m e n z y m e - s u b s t r a t e c o m p l e x e s ( R o o s a n d B o r o n , 1 9 8 1 ) . T h e a r r a n g e m e n t o f c y t o s k e l e t a l p r o t e i n s c a n a l s o b e m o d u l a t e d b y t h e i n t e r n a l p H . T h e p o l y m e r i z a t i o n o f t u b u l i n , f o r e x a m p l e , i n c r e a s e s a s p H ; r i s e s ( B u s a , 1 9 8 6 ) . F u r t h e r m o r e , t h e 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 i s s e n s i t i v e t o i n t r a c e l l u l a r a c i d s h i f t s . 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 t h e a r r a n g e m e n t o f m i c r o f i l a m e n t s , a s o p p o s e d t o a n a l k a l i n i z a t i o n w h i c h p r o m o t e s f i l a m e n t o u s o r g a n i z a t i o n ( B u s a , 1 9 8 6 ) . p H j - d e p e n d e n t v a r i a t i o n s i n t h e s y n t h e s i s o f t h e s e c y t o s k e l e t a l c o m p o n e n t s m a y i n f a c t b e a c o n s e q u e n c e o f p H j - d e p e n d e n t f l u c t u a t i o n s i n t h e a c t i v i t i e s o f e n z y m e s a s s o c i a t e d w i t h t h e s e e l e m e n t s . p H j h a s a l s o b e e n s h o w n t o p l a y a m o d u l a t o r y r o l e i n c e l l u l a r p r o l i f e r a t i o n a n d d e v e l o p m e n t . 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 i n m o u s e t h y m o c y t e s a n d S w i s s 3 T 3 f i b r o b l a s t s s t i m u l a t e d b y t h e a p p l i c a t i o n o f m i t o g e n s , d e m o n s t r a t i n g a p o s s i b l e r e l a t i o n s h i p b e t w e e n p H ; a n d t h e r e g u l a t i o n o f c e l l d i v i s i o n . I t h a s a l s o b e e n o b s e r v e d t h a t a p H j i n c r e a s e a c c o m p a n i e s f e r t i l i z a t i o n o f f r o g , a x o l o t l , a n d s e a u r c h i n o v a ( r e v i e w e d b y R o o s a n d B o r o n , 1 9 8 1 ) . F e r t i l i z a t i o n - i n d u c e d p H j r i s e s h a v e b e e n a t t r i b u t e d t o t h e a c t i v a t i o n o f a N a + / H + e x c h a n g e r , w h i c h a c t s t o e x t r u d e i n t r a c e l l u l a r p r o t o n s ( R o o s a n d B o r o n , 1 9 8 1 ) . S u c h c h a n g e s i n p H j m a y i n t u r n m o d u l a t e 5 t h e c a s c a d e o f b i o s y n t h e t i c p a t h w a y s i n v o l v e d i n e a r l y e m b r y o n i c d e v e l o p m e n t i n c l u d i n g m e t a b o l i s m , s t i m u l u s - r e s p o n s e c o u p l i n g , D N A r e p l i c a t i o n , a n d m i t o s i s ( B u s a a n d N u c c i t e l l i , 1 9 8 4 ) . I n a d d i t i o n t o t h e p r e v i o u s l y m e n t i o n e d a s s o c i a t i o n o f p H 0 w i t h n e u r a l p a t h o l o g i e s , i t 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 i s a m a r k e d d e c l i n e i n p H j , w h i c h i s 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 g l y c o l y s i s ( S i e s j o , 1 9 8 5 ) . A s c e r e b r a l e n e r g y s t a t e s d e t e r i o r a t e d u r i n g a n h y p o x i c i n s u l t , A T P h y d r o l y s i s a l s o c o n t r i b u t e s t o t h e i s c h e m i c - i n d u c e d 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 , w h i c h p r o c e e d s a c c o r d i n g t o t h e f o l l o w i n g r e a c t i o n : A T P + H 2 0 - > A D P + P i + 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 a t 0 . 7 ( W i l k i e , 1 9 7 9 ) . T h e o v e r a l l r e d u c t i o n o f p H , d u r i n g i s c h e m i a m a y t h e n a c t t o p r o t e c t t h e c e l l f r o m e x c e s s i v e d a m a g e . I n d e e d , i n t r a c e l l u l a r a c i d s h i f t s a r e b e l i e v e d t o r e d u c e m e m b r a n e e x c i t a b i l i t y a n d i n h i b i t c e l l u l a r m e t a b o l i s m d u r i n g i s c h e m i a ( T o m b a u g h a n d S a p o l s k y , 1 9 9 3 ) . N e v e r t h e l e s s , a l t h o u g h a n e u r o p r o t e c t i v e f u n c t i o n i s 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 o f i n t r a c e l l u l a r e q u i v a l e n t s h a v e b e e n s h o w n t o i n d u c e b o t h n e u r o n a l a n d g l i a l d e a t h i n c e l l s 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, 1 9 9 1 ) . G l u t a m a t e n e u r o t o x i c i t y m a y a l s o , a t l e a s t i n p a r t , i n v o l v e t h e d e t r i m e n t a l e f f e c t s o f a b n o r m a l l y h i g h i n t r a c e l l u l a r p r o t o n c o n c e n t r a t i o n s i n d u c e d b y g l u t a m a t e r e c e p t o r a c t i v a t i o n ( H a r t l e y a n d D u b i n s k y , 1 9 9 3 ) . I n a d d i t i o n , a m i l d i n t r a c e l l u l a r a c i d o s i s h a s b e e n o b s e r v e d t o a c c o m p a n y n e u r o n a l 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, 1 9 8 5 ) . T h i s a c i d i f i c a t i o n i s t h o u g h t t o o c c u r a s a r e s u l t o f i n c r e a s e d i n t r a c e l l u l a r l a c t i c a c i d p r o d u c t i o n , w h o s e e f f e c t s a r e d e l a y e d o r r e l i e v e d b y N a + / H + e x c h a n g e ( S i e s j o et al, 1 9 8 5 ) . F l u c t u a t i o n s i n p H , h a v e a l s o b e e n n o t e d t o p r o c e e d m a n y o f t h e s e p a t h o l o g i c a l o c c u r e n c e s . F o r e x a m p l e , M a b e et al ( 1 9 8 3 ) h a v e d e m o n s t r a t e d t h a t p H ; r i s e s t o a l k a l i n e l e v e l s i n r a t c o r t i c a l n e u r o n e s i m m e d i a t e l y f o l l o w i n g i s c h e m i c i n s u l t . T h e r e a s o n f o r t h i s p o s t - i s c h e m i c a l k a l o s i s r e m a i n s u n c l e a r , 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 Ca2+-sensitive reduction in pHj follows the depolarization of molluscan 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 GMP into gastropod neurones will also induce cytoplasmic pH 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 3 " (Ganz et al, 1989). Ganz et al (1989) speculated that A V P stimulates both N a + - and HC03"-dependent exchangers, which, depending on the 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 N a + / 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 + ([Ca2 +]j) may serve as an intermediate to growth factor-induced 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), a- amino-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 C a 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 0 does not significantly 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 N e u r o n a l e x c i t a b i l i t y a s s o c i a t e d w i t h L - g l u t a m a t e r e c e p t o r a c t i v a t i o n i s t h e r e f o r e d e p e n d e n t n o t o n l y o n t h e p H o f t h e e x t r a c e l l u l a r e n v i r o n m e n t b u t a l s o o n t h e p H o f t h e i n t r a c e l l u l a r m i l i e u b e c a u s e c h a n g e s i n p H j a r e k n o w n t o m o d u l a t e a w i d e v a r i e t y o f i o n i c c o n d u c t a n c e s ( s e e b e l o w ) . T h e m e c h a n i s m b y w h i c h y - a m i n o b u t y r i c a c i d ( G A B A ) a l t e r s p H j i s u n i q u e a m o n g n e u r o t r a n s m i t t e r s . E x a m i n e d o n c r a y f i s h s k e l e t a l m u s c l e , G A B A i n d u c e s 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 b y a c t i v a t i n g 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 , 1 9 8 7 ) . 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 i s h y d r a t e d i n t o 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 c a r b o n i c a n h y d r a s e . T h e d i s s o c i a t i o n o f c a r b o n i c a c i d i n t o b i c a r b o n a t e l i b e r a t e s i n t r a c e l l u l a r 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 ) . F u r t h e r i n v e s t i g a t i o n o f G A B A - i n d u c e d c h a n g e s i n p H j u s i n g c r a y f i s h s t r e t c h - r e c e p t o r n e u r o n e s p r o d u c e d f i n d i n g s s u p p o r t i n g t h e n o t i o n t h a t 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 G A B A - g a t e d c h a n n e l s ( V o i p i o et al, 1 9 9 1 ) . T h e r e f o r e , t h e r e d u c t i o n o f p H j p r o d u c e d b y G A B A i s u n l i k e t h e a c t i o n s o f o t h e r 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 i t d i r e c t l y i n v o l v e s t h e m o v e m e n t o f a c i d e q u i v a l e n t s a c r o s s t h e p l a s m a m e m b r a n e . pHj and ionic conductances: T h e e v i d e n c e o u t l i n e d a b o v e d e m o n s t r a t e s t h a t n e u r o n a l a c t i v i t y i s a s s o c i a t e d w i t h c h a n g e s i n p H j . I n t u r n , i t i s k n o w n t h a t p H j i s 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 b y m o d u l a t i n g a w i d e v a r i e t y o f i o n i c c o n d u c t a n c e s ( r e v i e w e d b y M o o d y , 1 9 8 4 ) . I n j e c t i n g l o w p H s o l u t i o n s i n t o i s o l a t e d v e n t r i c u l a r m y o c y t e s s h o r t e n s t h e d u r a t i o n a n d a m p l i t u d e o f e v o k e d a c t i o n p o t e n t i a l s , w h e r e a s t h e i n t r a c e l l u l a r a p p l i c a t i o n o f h i g h p H s o l u t i o n s h a s t h e o p p o s i t e e f f e c t ( K u r a c h i , 1 9 8 2 ) . T h e s p e c i f i c c u r r e n t s u n d e r l y i n g p H j - i n d u c e d c h a n g e s i n c e l l e x c i t a b i l i t y h a v e b e e n e x a m i n e d . F o r e x a m p l e , v o l t a g e g a t e d K + c o n d u c t a n c e s i n h u m a n l y m p h o c y t e s a r e e n h a n c e d b y a n e l e v a t i o n i n p H j ( D e u t s c h a n d 1 0 L e e , 1 9 8 9 ) . C o n v e r s e l y , 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 h a s b e e n a s s o c i a t e d w i t h a b l o c k a d e o f i n w a r d r e c t i f y i n g K + c u r r e n t s i n s t a r f i s h o o c y t e s ( M o o d y a n d H a g i w a r a , 1 9 8 2 ) . I n c r a y f i s h s l o w m u s c l e f i b r e s , l o w p H j m e d i a t e s t h e a m p l i f i c a t i o n o f i n w a r d C a 2 + c o n d u c t a n c e s , w h i c h i s b e l i e v e d t o o c c u r a s a r e s u l t 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 t h e o v e r l a p p i n g o u t w a r d K + c u r r e n t s ( M o o d y , 1 9 8 0 ) . T h e d e l a y e d r e c t i f i e r K + c o n d u c t a n c e c a n a l s o b e m o d i f i e d b y a n a c c u m u l a t i o n o f i n t r a c e l l u l a r p r o t o n s i n s q u i d a x o n s ( W a n k e et al, 1 9 7 9 ) . F u r t h e r m o r 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 c o n d u c t a n c e s t h r o u g h C a 2 + - a c t i v a t e d 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 ( 1 9 9 1 ) , i n s t u d y i n g r a t s k e l e t a l m u s c l e , a t t r i b u t e d t o 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 C a 2 + b i n d i n g t o c o n f o r m a t i o n a l s i t e s o n t h e c h a n n e l . K u m e et al ( 1 9 8 9 ) r e p o r t t h a t a f a l l i n p H j c a n a l s o r e d u c e t h e l i k e l i h o o d o f f i n d i n g C a 2 + - a c t i v a t e d K + c h a n n e l s i n a n o p e n s t a t e . I n d e e d , s u p p r e s s i o n o f C a 2 + - a c t i v a t e d K + c o n d u c t a n c e s b y i n t r a c e l l u l a r a c i d o s i s h a v e b e e n o b s e r v e d i n t y p e I c e l l s o f t h e r a t c a r o t i d b o d y ( P e e r s a n d G r e e n , 1 9 9 1 ) , a s w e l l a s C A 1 p y r a m i d a l n e u r o n e s o f t h e r a t h i p p o c a m p u s ( C h u r c h , 1 9 9 2 ) I n a d d i t i o n t o K + c u r r e n t s , o t h e r i o n i c c o n d u c t a n c e s c a n b e e n m o d u l a t e d b y f l u c t u a t i o n s i n p H j . N a + - d e p e n d e n t C a 2 + i n f l u x w a s f i r s t s h o w n t o b e i n h i b i t e d b y a f a l l i n p H j i n s q u i d a x o n s ( B a k e r a n d H o n e r j a g e r , 1 9 7 8 ) . U m b a c h ( 1 9 8 2 ) l a t e r o b s e r v e d d e c r e a s e s i n C a 2 + c h a n n e l p e r m e a b i l i t y i n Paramecium c a u s e d b y 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 ; c o n v e r s e l y , 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 i n c r e a s e d C a 2 + c o n d u c t a n c e s . H i g h v o l t a g e a c t i v a t e d ( H V A ) C a 2 + c u r r e n t s a p p e a r t o b e p a r t i c u l a r l y s e n s i t i v e t o p H j 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 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 w i l l s u p p r e s s w h i l e 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 i l l e n h a n c e H V A C a 2 + c o n d u c t a n c e s i n c a t f i s h r e t i n a l c e l l s . 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 t h a t t h e c o n d u c t i o n k i n e t i c s o f H V A C a 2 + c h a n n e l s a r e i n f l u e n c e d b y p r o t o n s d i r e c t l y 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 s i t e . I n h i b i t i o n o f H V A C a 2 + c u r r e n t s h a s a l s o b e e n 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, 1 9 9 3 ; D i x o n et al, 1 9 9 3 ) . Z e i l h o f e r et al ( 1 9 9 3 ) a t t r i b u t e d t h i s i n h i b i t i o n t o g l u t a m a t e r e c e p t o r - m e d i a t e d 1 1 C a 2 + i n f l u x a n d s u b s e q u e n t C a 2 + - d e p e n d e n t i n a c t i v a t i o n o f C a 2 + c h a n n e l s , w h e r e a s 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 2 + - d e p e n d e n t c h a n g e i n p H , i s r e s p o n s i b l e f o r t h e m o d u l a t i o n o f H V A C a 2 + c u r r e n t s . V a r i a t i o n s i n v o l t a g e - d e p e n d e n t N a + c o n d u c t a n c e s c a u s e d b y c h a n g e s i n p H ; h a v e 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, 1 9 8 0 ; W a n k e et al, 1 9 8 0 ) a n d s q u i d g i a n t a x o n s ( C a r b o n e et al, 1 9 8 1 ) . I n t e r e s t i n g l y , r e s u l t s r e g a r d i n g t h e i n f l u e n c e o f p H j o n N a + c u r r e n t s h a v e b e e n q u i t e v a r i a b l e . I n s q u i d a x o n s , l o w p H ; d e p r e s s e s N a + c o n d u c t a n c e s t h r o u g h t h e e n h a n c e d i n a c t i v a t i o n o f N a + c h a n n e l s , w h e r e a s h i g h p H j r e d u c e s t h i s i n a c t i v a t i o n , t h e r e b y i n c r e a s i n g N a + c o n d u c t a n c e s ( C a r b o n e et al, 1 9 8 1 ) . O p p o s i t e r e s u l t s w e r e a c h i e v e d i n s t u d i e s o f f r o g m u s c l e : l o w e r i n g p H j n e a r l y e l i m i n a t e s c h a n n e l i n a c t i v a t i o n , t h u s e n h a n c i n g N a + c h a n n e l c o n d u c t a n c e s ( N o n n e r et al, 1 9 8 0 ; W a n k e et al, 1 9 8 0 ) . A n a n a l y s i s o f c o n d u c t a n c e k i n e t i c s r e v e a l s t h e p o s s i b i l i t y t h a t t w o ( m e m b r a n e p o t e n t i a l s e n s i t i v e ) p r o t o n b i n d i n g a f f i n i t i e s e x i s t f o r t h e N a + c h a n n e l ( o n e w i t h a p K a o f 4 . 6 , a n d t h e o t h e r w i t h a p K a o f 5 . 6 ) , w h i c h m a y e x p l a i n t h e o b s e r v e d d i f f e r e n c e s i n N a + 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, 1 9 8 0 ) . F u r t h e r m o r e , i n c o n t r a s t t o m o s t o t h e r i o n i c c u r r e n t s , t h e i n h i b i t i o n o f N a + c o n d u c t a n c e s b y p H j i s v o l t a g e d e p e n d e n t ( M o o d y , 1 9 8 4 ) . I o n i c c u r r e n t s a s s o c i a t e d w i t h N a + / C a 2 + e x c h a n g e a r e a l s o s u s c e p t i b l e t o c h a n g e s i n t h e 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 , 1 9 9 3 ) . M e a s u r e d i n g u i n e a - p i g h e a r t C e l l s , 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 s u p p r e s s e s t h e a c t i v i t y o f t h e N a + / C a 2 + e x c h a n g e r , w h e r e a s 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 h a s t h e o p p o s i t e e f f e c t ( D o e r i n g a n d L e d e r e r , 1 9 9 3 ) . M o r e o v e r , t h e r e c o v e r y o f [ C a 2 + ] j i n h i p p o c a m p a l n e u r o n e s a f t e r s t i m u l u s - e v o k e d C a 2 + e n t r y , w h i c h m a y r e q u i r e N a + / C a 2 + e x c h a n g e , i s r e t a r d e d b y 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 ( K o c h a n d B a r i s h , 1 9 9 4 ) . T h e m o d u l a t i o n o f C h c o n d u c t a n c e s b y p H h a s n o t b e e n e x t e n s i v e l y d o c u m e n t e d . B a r n e s a n d B u i ( 1 9 9 1 ) h a v e n o t e d t h a t t h e s e n s i t i v i t y o f C a 2 + - a c t i v a t e d C l " c u r r e n t s i n a m p h i b i a n c o n e p h o t o r e c e p t o r s t o a l t e r a t i o n s i n p H 0 i s p o s s i b l y a c o n s e q u e n c e o f p H - i n d u c e d s h i f t i n C a 2 + c h a n n e l g a t i n g . C h a n g e s i n p H j , o n t h e o t h e r h a n d , h a v e b e e n 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 2 + (Spray et al, 1982; Moody, 1984). 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 0 will 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). pHj and C a 2 + : A complex interdependence appears to exist between pHj and intracellular free C a 2 + . For example, pHj can be modulated by fluctuations in the intracellular concentration of C a 2 + (Ahmed and Connor, 1980). Observed in snail neurones, the intracellular injection of C a 2 + causes an immediate fall in pHj that is proportional to the amount of C a 2 + injected (Meech and Thomas, 1977). Busa and Nuccitelli (1984) have postulated that Ca2+-dependent alterations in pHj may involve the exchange of C a 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 1 3 p r o d u c e a d e c r e a s e i n p H j w i t h o u t a f f e c t i n g t h e m e m b r a n e p o t e n t i a l , w h i c h a v o i d s p o s s i b l e s e c o n d a r y e f f e c t s o n p H j c a u s e d b y c h a n g e s i n i o n d i s t r i b u t i o n a c r o s s t h e m e m b r a n e ( M e e c h a n d T h o m a s , 1 9 7 7 ) . T h e e x p o s u r e o f a v i a n n e u r a l c r e s t c e l l s t o C a 2 + - f r e e 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 i s b e l i e v e d t o o c c u r a s a r e s u l t o f t h e s u b s e q u e n t f a l l i n [ C a 2 + ] j ( D i c k e n s et al, 1 9 9 0 ) . S a n c h e z - A r m a s s et al ( 1 9 9 4 ) h a v e r e c e n t l y r e p o r t e d t h a t a r i s e i n [ C a 2 + ] , c a n i n c r e a s e t h e e f f l u x o f i n t r a c e l l u l a r p r o t o n s v i a t h e s t i m u l a t i o n o f N a + / H + e x c h a n g e ( s e e b e l o w ) . T h i s c o n c l u s i o n i s s u p p o r t e d b y d a t a s h o w i n g t h a t t h e a p p l i c a t i o n o f C a 2 + i o n o p h o r e s o n r a t b r a i n s y n a p t o s o m e s i n d u c e s N a + - 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, 1 9 9 4 ) . p H j h a s i n t u r n b e e n s h o w n t o m o d u l a t e i n t r a c e l l u l a r f r e e C a 2 + ( D i c k e n s et al, 1 9 9 0 ; M a r t i n e z - Z a g u i l a n et al, 1 9 9 1 ) . T h e e x p o s u r e o f b a r n a c l e m u s c l e c e l l s t o C O 2 l e a d s t o a n i n c r e a s e i n [ C a 2 + ] j , w h i c h L e a a n d A s h l e y ( 1 9 7 8 ) a t t r i b u t e t o a C C ^ - i n d u c e d r e d u c t i o n i n p H j . 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 i s t h o u g h t t o d i s p l a c e C a 2 + f r o m i n t r a c e l l u l a r o r g a n e l l e s a n d o t h e r C a 2 + - b i n d i n g p r o t e i n s p r e s e n t i n t h e s a r c o p l a s m ( L e a a n d A s h l e y , 1 9 7 8 ) . 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 C a 2 + i n v a s c u l a r s m o o t h m u s c l e t o t h e r e l e a s e o f C a 2 + f r o m i n t r a c e l l u l a r s o u r c e s . I n t e r e s t i n g l y , p H j h a s a l s o b e e n s h o w n t o m o d u l a t e t h e i n t r a c e l l u l a r l e v e l s o f o t h e r d i v a l e n t c a t i o n s . I n c u l t u r e d c h i c k e n h e a r t c e l l s , f o r e x a m p l e , c h a n g e s i n c y t o s o l i c M g 2 + 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, 1 9 9 2 ) . I n s t u d y i n g t h e r e l a t i o n s h i p b e t w e e n p H , a n d i n t e r n a l f r e e C a 2 + u s i n g f l u o r e s c e n t 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 - d e p e n d e n t c h a n g e s i n [ C a 2 + ] j . I n a g r e e m e n t w i t h p r e v i o u s l y m e n t i o n e d t h e o r i e s , a p H j s h i f t m a y a l t e r C a 2 + f l u x e s a c r o s s v a r i o u s i n t r a c e l l u l a r m e m b r a n e s . H o w e v e r , G a n z et al ( 1 9 9 0 ) c a u t i o n t h a t a r t i f a c t s m a y b e r e s p o n s i b l e f o r p e r c e i v e d c h a n g e s i n c y t o s o l i c C a 2 + l e v e l s b e c a u s e p e r t u r b a t i o n s i n p H j m a y a l t e r : 1 ) t h e a s s o c i a t i o n o f C a 2 + w i t h i t s f l u o r e s c e n t i n d i c a t o r , a n d 2 ) t h e i n t e r a c t i o n o f t h e C a 2 + i n d i c a t o r w i t h i n t e r n a l m e m b r a n e s . U n d e r b o t h o f t h e s e c o n d i t i o n s , a n a p p a r e n t c h a n g e i n [ C a 2 + ] j w o u l d b e 1 4 r e c o r d e d , w h e n , i n f a c t , n o n e a c t u a l l y o c c u r r e d . T h e r e f o r e , i t i s n e c e s s a r y t o t a k e t h e s e f a c t o r s i n t o a c c o u n t w h e n e v a l u a t i n g t h e s i g n i f i c a n c e o f c y t o s o l i c C a 2 + c h a n g e s c a u s e d b y a l t e r i n g 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 i n a v a r i e t y o f c e l l t y p e s h a v e d e t e r m i n e d t h a t p r o t o n s a r e n o t p a s s i v e l y d i s t r i b u t e d a c r o s s t h e p l a s m a m e m b r a n e ( r e v i e w e d b y R o o s a n d B o r o n , 1 9 8 1 ) . T h i s w a s , i n f a c t , f i r s t 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 t h e m i d - 1 9 3 0 ' 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 t o b e 7 . 0 , w h i c h i s m u c h h i g h e r t h a n t h e p r e d i c t e d v a l u e o f 5 . 6 2 b a s e d o n a D 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 s t a t e s t h a t , a t e q u i l i b r i u m , t h e r a t i o s o f a l l d i f f u s a b l e i o n c o n c e n t r a t i o n s o n e i t h e r s i d e o f a p e r m e a b l e m e m b r a n e w i l l b e e q u a l . W i t h a h i g h K + c o n c e n t r a t i o n i n s i d e c e l l s i n c o m p a r i s o n t o t h e o u t s i d e , o n e w o u l d e x p e c t , b a s e d o n t h i s l a w o f m e m b r a n e e q u i l i b r i a , t h a t p r o t o n c o n c e n t r a t i o n s w o u l d b e h i g h e r o n t h e o u t s i d e i n c o m p a r i s o n t o t h e i n s i d e . I f t h i s r u l e h e l d t r u e , t h e n p H j w o u l d r e s t a t 5 . 6 i f p H 0 w a s n e a r n e u t r a l i t y . F u r t h e r c a l c u l a t i o n s o n f r o g m u s c l e b y F e n n a n d M a u r e r ( 1 9 3 5 ) y i e l d e d a v a l u e o f 6 . 9 f o r p H j w h i l e p H 0 r e s t e d a t 7 . 3 4 , b u t e v e n t h i s d i f f e r e n c e w a s n o t l a r g e e n o u g h t o b e e x p l a i n e d s i m p l y b y e q u i l i b r i u m a c r o s s t h e m e m b r a n e . T h i s o b s e r v a t i o n 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 s t u d i e s o n f r o g m u s c l e , c o n c l u d e d t h a t " . . . t h e D o n n a n e q u i l i b r i u m d o e s n o t c o n t r o l , a n d d o e s n o t g r e a t l y i n f l u e n c e , t h e d i s t r i b u t i o n o f h y d r o g e n i o n s a c r o s s t h e f i b r e m e m b r a n e . " C h e s l e r ( 1 9 9 0 ) o u t l i n e s , f r o m a m e m b r a n e p o t e n t i a l p e r s p e c t i v e , t h e u n l i k e l i h o o d o f h a v i n g a p a s s i v e H + d i s t r i b u t i o n a c r o s s t h e p l a s m a m e m b r a n e . I f p r o t o n s p a s s i v e l y m o v e d a c r o s s t h e m e m b r a n e , t h e n t h e 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 a n d e x t r a c e l l u l a r 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 (Em) a s r e p r e s e n t e d b y t h e N e r n s t e q u a t i o n : 1 5 E m =  E H + = — I n J^Tj (Equation 2) w h e r e EH+ i s 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 + , F i s F a r a d a y ' s c o n s t a n t , R i s t h e i d e a l g a s c o n s t a n t , a n d T i s t h e t e m p e r a t u r e . 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 f o r p H 0 a n d 7 . 0 f o r p H j ( s e e C h e s l e r , 1 9 9 0 , f o r a s u m m a r y o f r e s t i n g p H j l e v e l s i n a v a r i e t y o f c e l l s ) , t h e a b o v e e q u a t i o n y i e l d s a n e q u i l i b r i u m p o t e n t i a l f o r t h e H + i o n (EH+) o f - 2 3 . 6 m V a t 2 5 ° C . D u e t o t h e d i f f e r e n c e b e t w e e n EH+ a n d Em, w h i c h n o r m a l l y r e s t s b e t w e e n - 5 0 a n d - 6 0 m V i n e x c i t a b l e c e l l s , a n i n w a r d p r o t o n g r a d i e n t i s e s t a b l i s h e d . T h i s , a n d t h e f a c t t h a t s o m e c e l l u l a r 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 e q u i v a l e n t s ( e . g . g l u c o s e — » 2 l a c t a t e - + 2 H + ) , f o r c e s c e l l s t o c o n t i n u a l l y e x t r u d e a c i d i n o r d e r t o m a i n t a i n p H j n e a r n e u t r a l i t y ( T h o m a s , 1 9 8 4 ) . W h e t h e r a c o n c l u s i o n i s b a s e d o n c a l c u l a t i o n s o f t h e D o n n a n 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 , i t i s c l e a r t h a t p r o t o n s a r e n o t p a s s i v e l y d i s t r i b u t e d a c r o s s t h e l i m i t i n g m e m b r a n e . T h e r e g u l a t i o n o f p H j b y a c i d e x t r u s i o n a n d / o r a c i d b u f f e r i n g i s 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 c e l l t y p e s . Regulation of pHj: A s a n e l e c t r o c h e m i c a l g r a d i e n t f a v o u r s t h e i n f l u x o f p r o t o n s f r o m t h e i n t e r s t i t i a l s p a c e i n t o t h e c y t o p l a s m , c e l l s m u s t c o n t i n u a l l y e x t r u d e a c i d e q u i v a l e n t s i n o r d e r t o m a i n t a i n a c o n s t a n t r e s t i n g p H j . T h e s e e x t r u s i o n m e c h a n i s m s a l s o p a r t i c i p a t e i n t h e r e s t o r a t i o n o f p H j b a c k t o n o r m a l p h y s i o l o g i c a l l e v e l s a f t e r c e l l s h a v e b e e n b u r d e n e d w i t h a n i n d u c e d a c i d l o a d . A c c o r d i n g l y , i n a d d i t i o n t o m o n i t o r i n g s t e a d y - s t a t e c o n d i t i o n s , m a n y s t u d i e s o n p H j r e g u l a t i o n h a v e i n v e s t i g a t e d a c i d e x t r u s i o n m e c h a n i s m s t h r o u g h t h e a n a l y s i 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 i f i c a t i o n . T h e i d e a o f t r a n s - m e m b r a n e f l u x e s o f 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 b y M e s s e t e r a n d S i e s j o ( 1 9 7 1 ) i n t h e i r s t u d y o f r a t b r a i n t i s s u e . T h e s e a u t h o r s n o t e d t h a t t h e 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 i s a t l e a s t 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 - 1 6 i n d u c e d 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 t r a c e l l u l a r p r o t o n l o a d i n g w a s a l s o u s e d b y R o o s ( 1 9 7 5 ) i n s 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 . R o o s r e a s o n e d t h a t i n t r a c e l l u l a r b u f f e r i n g ( d i s c u s s e d b e l o w ) d o e s n o t s u f f i c i e n t l y e x p l a i n w h y p H j i s o n l y m o d e r a t e l y a f f e c t e d b y c o n s i d e r a b l e i n t r a c e l l u l a r p r o t o n l o a d s . P r o t o n e x t r u s i o n m u s t t h e r e f o r e p l a y a s u b s t a n t i a l r o l e i n m a i n t a i n i n g a c o n s t a n t i n t r a c e l l u l a r a c i d - b a s e b a l a n c e ( R o o s , 1 9 7 5 ) . R e c o v e r y f r o m 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 d u c e d b y t h e a d d i t i o n a n d s u b s e q u e n t r e m o v a l o f e x t r a c e l l u l a r N H 4 C 1 , h a s p r o d u c e d a d d i t i o n a l e v i d e n c e s u p p o r t i n g t h e p r e s e n c e o f a c i d e x t r u d i n g m e c h a n i s m s o n v a r i o u s i n v e r t e b r a t e n e u r o n e s , i n c l u d i n g t h e s q u i d g i a n t a x o n ( B o r o n a n d D e W e e r , 1 9 7 6 ) . T h e s t u d y o f i n t r a c e l l u l a r p r o t o n e x t r u s i o n h a s c o n c e n t r a t e d o n t h e i d e n t i f i c a t i o n o f t r a n s p o r t i n g m e c h a n i s m s t h a t e x c h a n g e i n t r a c e l l u l a r o r e x t r a c e l l u l a r i o n s f o r p r o t o n e q u i v a l e n t s . 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 + a n t i p o r t s y s t e m p r e s e n t o n t h e m e m b r a n e s o f r a t i n t e s t i n a l a n d r e n a l c e l l s w h i c h w a s t h o u g h t t o b e i n v o l v e d i n a c i d e x t r u s i o n . T h i s g r o u p o b s e r v e d t h a t t h e a n t i p o r t e r o p e r a t e s i n a n o n - e l e c t r o g e n i c f a s h i o n t o e x c h a n g e i n t r a c e l l u l a r H + i o n s f o r e x t r a c e l l u l a r N a + i o n s ( M u r e r et al, 1 9 7 6 ) . 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 a c t i v i t y o f N a + / H + e x c h a n g e i s s u s c e p t i b l e t o i n h i b i t i o n b y t h e d i u r e t i c d r u g a m i l o r i d e ( l - ( 3 , 5 - d i a m i n o - 6 - c h l o r o p y r a z i n o y l ) g u a n i d i n e ) . T h e a b i l i t y o f t h i s d r u g t o b l o c k N a + / H + e x c h a n g e o n o t h e r c e l l t y p e s , s u c h a s r e n a l o r i n t e s t i n a l e p i t h e l i a l c e l l s , , h a s b e e n e x t e n s i v e l y d o c u m e n t e d ( e . g . S a r d e t e ^ a / , 1 9 8 9 ; T s e et al, 1 9 9 3 ; M r k i c e r a / , 1 9 9 3 ; R o w e e / a / , 1 9 9 4 ) . A s e c o n d a c i d t r a n s p o r t e r h a s b e e n d e s c r i b e d w h i c h i n v o l v e s t h e i n w a r d f l u x o f H C O 3 " 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 ) . T h o m a s s p e c u l a t e d t h a t C l " w a s i n f a c t t h e a n i o n i n q u e s t i o n , a p o s s i b i l i t y t h a t w a s c o n f i r m e d b y R u s s e l l a n d B o r o n ( 1 9 7 6 ) i n t h e i r i n v e s t i g a t i o n o f s q u i d a x o n s . B o t h g r o u p s d e m o n s t r a t e d t h a t t h i s a n i o n e x c h a n g e r , w h i c h o p e r a t e s i n a N a + - i n d e p e n d e n t f a s h i o n , c o u l d b e b l o c k e d b y s t i l b e n e d e r i v a t i v e s s u c h a s S I T S ( 4 - a c e t a m i d o - 4 ' - i s o t h i o c y a n a t o s t i l b e n e - 2 , 2 ' - d i s u l p h o n i c a c i d ) a n d D I D S ( 4 , 4 ' - d i i s o t h i o c y a n a t o s t i l b e n e - 2 , 2 ' - d i s u l p h o n i c a c i d ) . T h e HCO3VCI" 17 e x c h a n g e r a c t s t o r e d u c e i n t r a c e l l u l a r p r o t o n a c c u m u l a t i o n s b y p r e s e n t i n g t h e c y t o s o l 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 3 " i o n s s e q u e s t e r p r o t o n s t o 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 C 0 2 a n d H 2 0 . B o t h C 0 2 a n d H 2 0 f r e e l y d i f f u s e o u t o f t h e c e l l , a n d t h e n 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 H C 0 3 " l e v e l s . T h e n e t r e s u l t o f t h i s m e c h a n i s m i s t h e r e d u c t i o n o f i n t r a c e l l u l a r H + 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 H C 0 3 " a n d s u b s e q u e n t e f f l u x o f C l " , C 0 2 a n d H 2 0 . I n t h e i r e x a m i n a t i o n o f m o u s e s o l e u s m u s c l e f i b r e s , A i c k e n a n d T h o m a s ( 1 9 7 7 ) r e v e a l e d t h e p 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 y p e . T h e s e a u t h o r s c o n c l u d e d t h a t b o t h N a + / H + 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 y t h e t r a n s m e m b r a n e N a + g r a d i e n t , a n d H C 0 3 7 C 1 " e x c h a n g e i n d e p e n d e n t l y m a i n t a i n c o n s t a n t 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 s d 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 s n a i l n e u r o n e s , t h e s e t w o m e c h a n i s m s m a y a l s o o p e r a t e i n c o n j u n c t i o n w i t h o n e a n o t h e r . T h i s p a r t i c u l a r r e g u l a t i n g s y s t e m , c l a s s i f i e d a s N a + - d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e , r e l i e s o n t h e a v a i l a b i l i t y o f e x t r a c e l l u l a r N a + a s a r e q u i s i t e f o r t h e c o u n t e r - t r a n s p o r t o f H C O 3 - 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 H C 0 3 7 C 1 " e x c h a n g e r i s s u s c e p t i b l e t o i n h i b i t i o n w i t h S I T S o r D I D S ( T h o m a s , 1 9 7 7 ) . T h u s , b y t h e e a r l y 1 9 8 0 ' s , t h r e e s e p a r a t e e l e c t r o n e u t r a l m e c h a n i s m s w e r e t a r g e t e d a s s i g n i f i c a n t r e g u l a t o r s o f p H j : N a + / H + e x c h a n g e , N a + - i n d e p e n d e n t H C O 3 7 O " e x c h a n g e , a n d N a + - d e p e n d e n t H C O 3 7 G " e x c h a n g e ( T h o m a s , 1 9 8 4 ) . A l l o f t h e s e t r a n s p o r t e r s h a v e b e e n s h o w n , f o r e x a m p l e , t o b e a c t i v e t o v a r y i n g e x t e n t s i n f r e s h l y i s o l a t e d r a t g l o m e r u l a r m e s a n g i a l c e l l s ( B o y a r s k i et al, 1 9 9 0 a a n d b ) . M o r e o v e r , t h e a c t i v i t i e s o f t h e s e e x c h a n g e r s c a n b e m o d u l a t e d b y t h e a p p l i c a t i o n o f v a r i o u s n e u r o t r a n s m i t t e r s a n d h o r m o n e s , o r f l u c t u a t i o n s i n t h e c o n c e n t r a t i o n o f i n t r a c e l l u l a r i o n s s u c h a s C a 2 + ( s e e a b o v e ) . O t h e r p r o t o n e x t r u d i n g m e c h a n i s m s p r e s e n t o n s o m e c e l l m e m b r a n e s i n c l u d e a N a + / H C 0 3 " t r a n s p o r t e r a n d a p r o t o n p u m p . B o r o n a n d B o u l e p a e p ( 1 9 8 3 ) i d e n t i f i e d a n 1 8 e l e c t r o g e n i c N a + / H C 0 3 _ c o - t r a n s p o r t e r o n t h e b a s o l a t e r a l m e m b r a n e s o f s a l a m a n d e r r e n a l p r o x i m a l t u b u l e s . T h i s m e c h a n i s m , w h i c h i s s e n s i t i v e t o s t i l b e n e d e r i v a t i v e s , i n w a r d l y 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 m e m b r a n e ( B o r o n a n d B o u l e p a e p , 1 9 8 3 ) . A n o n e l e c t r o g e n i c H + p u m p , i n v o l v e d i n g a s t r i c a c i d 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 h o g s t o m a c h . I n r e s p o n s e t o a v a r i e t y o f s e c r e t a g o g u e s , e x t r a c e l l u l a r K + i s e x c h a n g e d f o r i n t r a c e l l u l a r H + w i t h t h e c o n s u m p t i o n o f A T P ( S a c h s , 1 9 8 7 ) . A l s o k n o w n a s t h e H + , K + - A T P a s e , t h i s p r o t o n p u m p i s 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 i s t h e a c i d i f i c a t i o n o f t h e s t o m a c h m i l i e u ( B o r o n , 1 9 8 9 ) . T h o u g h s t u d i e d e x t e n s i v e l y i n i n v e r t e b r a t e s , p H j r e g u l a t i n g m e c h a n i s m s i n v e r t e b r a t e n e u r o n e s h a v e n o t b e e n t h o r o u g h l y i n v e s t i g a t e d , p r i m a r i l y b e c a u s e t h e i r r e l a t i v e l y s m a l l s i z e h a m p e r s t h e u t i l i z a t i o n o f p H - s e n s i t i v e m i c r o e l e c t r o d e s ( C h e s l e r , 1 9 9 0 ) . W i t h t h e a d v e n t o f f l u o r e s c e n t p H d y e s , s t u d i e s o n m a m m a l i a n c e n t r a l n e u r o n e s h a v e r e c e n t l y e m e r g e d . V a r i o u s f l u o r e s c e i n d e r i v a t i v e s , w h o s e s p e c t r a s h i f t a c c o r d i n g t o t h e s u r r o u n d i n g p r o t o n e n v i r o n m e n t , a r e n o w e m p l o y e d a s i n d i c a t o r s o f p H i n b i o l o g i c a l s y s t e m s ( r e v i e w e d b y T s i e n , 1 9 8 9 ) . T h e m o s t p o p u l a r p r o b e o f p H ; i n v e r t e b r a t e n e u r o n e s i s 2 ' , 7 ' - b i s ( c a r b o x y e t h y l ) - 5 ( o r 6 ) - c a r b o x y f l u o r e s c e i n ( B C E C F ) b e c a u s e o f i t s h i g h s e n s i t i v i t y t o s m a l l c h a n g e s i n p H j , e a s e o f i n t r a c e l l u l a r e n t r a p m e n t , a n d p h y s i o l o g i c a l l y r e l e v a n t p K a ( - 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 s y m p a t h e t i c n e u r o n e s , T o l k o v s k y a n d R i c h a r d s ( 1 9 8 7 ) r e p o r t e d t h a t t h e m a i n r e g u l a t o r o f p H j i s a N a + / H + e x c h a n g e r t h a t i s s e n s i t i v e t o i n h i b i t i o n b y a m i l o r i d e . T h o u g h t h e 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 e v i d e n c e f o r t h e p r e s e n c e o f s o m e o t h e r H C 0 3 " - d e p e n d e n t i n t r a c e l l u l a r a c i d r e g u l a t o r , p e r h a p s H C O 3 " s e n s i t i v e N a + / H + e x c h a n g e , w h i c h m a y p l a y a m i n o r r o l e i n p H j r e g u l a t i o n . 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 a l s o s h o w n t h a t t h e N a + / H + a n t i p o r t e r i s t h e p r i m a r y p H j r e g u l a t o r i n r a t b r a i n s y n a p t o s o m e s . G a i l l a r d a n d D u P o n t ( 1 9 9 0 ) h a v e d e m o n s t r a t e d t h a t c u l t u r e d c e r e b e l l a r P u r k i n j e c e l l s 1 9 u t i l i z e N a + / H + e x c h a n g e a n d H C O 3 - / C T e x c h a n g e t o c o n t r o l i n t r a c e l l u l a r a c i d - b a s e l e v e l s . T h i s c o m b i n a t i o n o f a c i d t r a n s p o r t e r s h a s a l s o b e e n i m p l i c a t e d i n t h e m a i n t e n a n c e 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, 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 t h a t p H j i n c u l t u r e d f o e t a l r a t h i p p o c a m p a l n e u r o n e s i s m a i n t a i n e d b y a n a m i l o r i d e i n s e n s i t i v e N a + / H + a n t i p o r t e r , w i t h a m i n o r c o n t r i b u t i o n f r o m a N a + - a n d H C 0 3 " - 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 c h w i e n i n g a n d B o r o n ( 1 9 9 4 ) h a v e a l s o d e m o n s t r a t e d t h a t r e g u l a t i o n o f p H ; i n f r e s h l y i s o l a t e d n e u r o n e s f r o m t h e a d u l t r a t h i p p o c a m p u s i s 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 i n s e n s i t i v e N a + / H + e x c h a n g e r , i n a d d i t i o n t o a D I D S s e n s i t i v e N a + - d e p e n d e n t H C C ^ V C l ' e x c h a n g e r . T h e i n s e n s i t i v i t y o f t h e N a + / H + e x c h a n g e r p r e s e n t o n h i p p o c a m p a l n e u r o n e s t o a m i l o r i d e s u p p o r t s e m e r g i n g e v i d e n c e r e g a r d i n g t h e s t r u c t u r a l d i v e r s i t y o f t h e c a t i o n c o u n t e r - t r a n s p o r t e r ( r e v i e w e d b y C l a r k a n d L i m b i r d , 1 9 9 1 ) . F o u r i s o f o r m s o f t h e N a + / H + e x c h a n g e r h a v e s o f a r b e e n c l o n e d , e a c h r e p r e s e n t i n g u n i q u e m o l e c u l a r c o m p o s i t i o n s , l o c a l i z a t i o n s i n t h e b o d y , a n d 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 i t s a n a l o g u e s ( s e e D i s c u s s i o n ) . T h o u g h r e s u l t s a r e s t i l l q u i t e s p a r s e , i t i s 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 t h e m a m m a l i a n c e n t r a l n e r v o u s s y s t e m i s 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 o f a c i d e x t r u d i n g e x c h a n g e r s . 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 a w 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 a c t s t o 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 t h i s n o t i o n , t h e i n t r a c e l l u l a r m i l i e u , w h i c h i s 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 , i s a b l e t o r e s i s t c h a n g e s i n i t s p H d u e t o t h e b u f f e r i n g c a p a c i t y o f i t s c o n s t i t u e n t s . I n d i s c u s s i n g p H j , i t i s t h e r e f o r e n e c e s s a r y t o i n c l u d e a n e x a m i n a t i o n o f t h e c y t o s o l ' s a b i l i t y t o b u f f e r p r o t o n f l u x e s , b o t h i n t e r m s o f s t e a d y - s t a t e p H j r e g u l a t i o n a n d r e c o v e r y f r o m a c i d o r a l k a l i t r a n s i e n t s . 20 C e l l s a r e i n t e r n a l l y b u f f e r e d b y t h e a c i d - b a s e p a i r s f o r m e d f r o m b i c a r b o n a t e , p r o t e i n s , p h o s p h a t e s , a n d d i p e p t i d e s ( B u r t o n , 1 9 7 8 ) . T o g e t h e r , t h e s e s p e c i e s s e q u e s t e r o r r e l e a s e p r o t o n s t o m i n i m i z e p H j s h i f t s a c c o r d i n g t o t h e e q u a t i o n : M » + H + MHn+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 v a l e n c e o f n+1 ( R o o s a n d B o r o n , 1 9 8 1 ) . T h i s t y p e o f b u f f e r i n g , w h i c h u t i l i z e s 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 , i s k n o w n a s p h y s i o c h e m i c a l b u f f e r i n g ( B o r o n , 1 9 8 9 ) . A n o t h e r m a n n e r b y w h i c h c h a n g e s i n p H ; a r e m i n i m i z e d i s v i a b i o c h e m i c a l r e a c t i o n s . S u c h r e a c t i o n s m a y a c t t o c o n s u m e o r l i b e r a t e H + i o n s i n r e s p o n s e t o i n t r a c e l l u l a r a c i d - b a s e s h i f t s . A c i d p r o d u c t i o n , f o r e x a m p l e , t h a t o c c u r s i n r e s p o n s e t o a l k a l i n e l o a d s m a y i n v o l v e t h e c o n v e r s i o n o f i n t r a c e l l u l a r c a r b o h y d r a t e s i n t o l a c t i c a c i d a c c o r d i n g t o : G l u c o s e — » 2 L a c t a t e " + 2 H + (Equation 4) ( S i e s j o , 1 9 8 5 ) . C o n v e r s e l y , i n r e s p o n s e t o a c i d l o a d s , t h e c o n c e n t r a t i o n s o f l a c t a t e , p y r u v a t e , o r c i t r a t e m a y d e c r e a s e ( S i e s j o a n d M e s s e t e r , 1 9 7 1 ) . A c i d c o n s u m p t i o n , i n t h e c a s e o f l a c t i c a c i d , w o u l d p r o c e e d a s : L a c t a t e " + H + + 3 0 2 - > 3 C 0 2 + 3 H 2 0 (Equation 5) ( S i e s j o , 1 9 8 5 ) . T h e c o n s u m p t i o n o f p r o t o n s b y i n t r a c e l l u l a r a c i d s a n d t h e i r s u b s e q u e n t o x i d a t i o n p r o d u c e s f r e e l y d i f f u s a b l e C 0 2 ( a n d H 2 0 ) , w h i c h c a n r e a d i l y l e a v e t h e c e l l t o r e d u c e t h e e f f e c t s o f a c i d l o a d i n g . 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 i s t h e m o v e m e n t o f a c i d - b a s e e q u i v a l e n t s b e t w e e n t h e c y t o p l a s m a n d t h e i n t e r i o r o f v a r i o u s c y t o s o l i c o r g a n e l l e s ( R o o s a n d B o r o n , 1 9 8 1 ) . 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 , i t i s 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 i n c l u s i o n s , s u c h a s e n d o s o m e s a n d l y s o s o m e s , a r e a b l e t o t r a n s p o r t p r o t o n s a c r o s s t h e i r m e m b r a n e s , p o s s i b l y i n a n e l e c t r o g e n i c f a s h i o n ( B o r o n , 1 9 8 9 ) . T h i s t r a n s p o r t e r h a s b e e n i d e n t i f i e d a s a H + p u m p , d r i v e n b y t h e h y d r o l y s i s o f A T P ( B o r o n , 1 9 8 9 ) . T h e i n n e r m i t o c h o n d r i a l 2 1 m e m b r a n e i s a l s o b e l i e v e d t o b e a n a c t i v e s i t e o f p r o t o n e x c h a n g e ( R o o s a n d B o r o n , 1 9 8 1 ) . R e s p o n d i n g t o 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 s , f o r i n s t a n c e , s u c h b u f f e r i n g w o u l d a c t t o t r a n s p o r t H + i o n s i n t o a c i d i c v e s i c l e s o r l i m i t H + f l u x o u t o f a l k a l i n e o r g a n e l l e s . I t 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 o r g a n e l l a r , d o e s n o t e l i m i n a t e p H , s h i f t s , b u t i n s t e a d a c t s t o m i n i m i z e t h e m . B u f f e r i n g o f f e r s o n l y a s h o r t t e r m a n d p a r t i a l s o l u t i o n t o a c i d - l o a d i n g , b u t w h e n c o m b i n e d w i t h a c i d e x t r u s i o n , c e l l s a r e b e t t e r a b l e t o m a i n t a i n c o n s t a n t p H j l e v e l s . Overview: T i s s u e p H i n t h e c e n t r a l n e r v o u s s y s t e m c a n m o d u l a t e , o r b e m o d u l a t e d b y , m a n y 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 . I t i s 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 t h e s e p r o c e s s e s m a y h a v e s p e c i f i c e f f e c t s o n p H j . C h a n g e s i n p H 0 a n d t h e a p p l i c a t i o n o f n e u r o t r a n s m i t t e r s o r n e u r o m o d u l a t o r s c a n i n f l u e n c e p H j . I n t u r n , f l u c t u a t i o n s i n p H , c a n r e g u l a t e i o n i c c o n d u c t a n c e s i n e x c i t a b l e c e l l s , a l t e r c e l l u l a r m e t a b o l i s m , a n d 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 i s , i n a l l c e l l t y p e s s o f a r s t u d i e d , a c t i v e l y r e g u l a t e d . T h e s e r e g u l a t i n g m e c h a n i s m s h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d i n i n v e r t e b r a t e n e u r o n a l a n d v e r t e b r a t e n o n - n e u r o n a l p r e p a r a t i o n s . A s s t u d i e s o n m a m m a l i a n c e n t r a l n e u r o n e s a r e l i m i t e d , t h i s t h e s i s w i l l e x a m i n e t h e r e g u l a t i o n o f p H j i n p y r a m i d a l n e u r o n e s c u l t u r e d f r o m e m b r y o n i c r a t h i p p o c a m p i , a r e g i o n k n o w n t o b e p a r t i c u l a r l y s e n s i t i v e t o e p i l e p t i f o r m a c t i v i t y a n d i s c h e m i c c o n d i t i o n s . A r a t i o m e t r i c t e c h n i q u e , u t i l i z i n g t h e p H s e n s i t i v e f l u o r o p h o r e B C E C F , w a s e m p l o y e d t o 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 a t r e s t a n d d u r i n g t h e 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 i f i c a t i o n . 2 2 M A T E R I A L S A N D M E T H O D S C e l l p r e p a r a t i o n : N e u r o n a l c e l l c u l t u r e s w e r e p r e p a r e d a c c o r d i n g t o 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 m o d i f i c a t i o n s . H i p p o c a m p a l s e c t i o n s w e r e o b t a i n e d f r o m 1 8 d a y e m b r y o n i c - a g e W i s t a r r a t f o e t u s e s , a n d s t o r e d i n a C a 2 + a n d M g 2 + - f r e e b a l a n c e d s a l t s o l u t i o n ( C M F - B S S ) . T h e C M F - B S S c o n t a i n e d 1 0 % H a n k ' s b a l a n c e s a l t s o l u t i o n , 5 5 . 5 m M g l u c o s e , 1 0 . 0 5 m M 4 - ( - 2 - h y d r o x y e t h y l ) - l - p i p e r a z i n e e t h a n e s u l f o n i c a c i d ( H E P E S ) , 4 . 9 5 m M H E P E S N a + - s a l t , a n d 2 m M N a H C 0 3 . T h e h i p p o c a m p i w e r e s u b s e q u e n t l y t r a n s f e r r e d i n t o 6 m L o f a n e n z y m a t i c s o l u t i o n c o n t a i n i n g a l i q u o t s o f t r y p s i n a n d D N A s e d i s s o l v e d i n C M F - B S S , f o r 2 0 m i n u t e s a t 3 7 ° C . T h e e n z y m e - b a s e d d i s s o c i a t i o n w a s f o l l o w e d b y a t r i t u r a t i o n p r o c e d u r e i n w h i c h t i s s u e w a s s u s p e n d e d i n a t e s t t u b e c o n t a i n i n g D N A s e , C M F - B S S , a n d 1 0 % f o e t a l b o v i n e s e r u m ( F B S ) , a n d w a s m e c h a n i c a l l y s i p h o n e d 2 0 t i m e s t h r o u g h f i r e p o l i s h e d p a s t e u r p i p e t t e s o f d e c r e a s i n g t i p d i a m e t e r . T h e t r i t u r a t e d m i x t u r e 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 1 m 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 a t 1 5 0 g f o r 4 t o 5 m i n u t e s . T h e s u p e r n a t a n t w a s r e m o v e d a n d t h e r e s u l t i n g c e l l p e l l e t w a s r e - s u s p e n d e d i n 6 m L o f D u l b e c c o ' s M o d i f i e d E a g l e ' s M e d i u m ( D - M E M ) c o n t a i n i n g 1 0 % F B S ( D - M E M / F B S ) . A l l D - M E M s o l u t i o n s w e r e b u f f e r e d b y 2 2 m M N a H C 0 3 , 1 0 m M H E P E S , a n d 5 % C 0 2 i n a i r . A 5 0 u L s a m p l e o f t h e c e l l s u s p e n s i o n w a s r e m o v e d f r o m t h e D - M E M s u s p e n s i o n a n d m i x e d i n t o 4 1 0 u L C M F - B S S a n d 4 0 u , L t r y p a n b l u e . T h e l i v i n g p y r a m i d a l c e l l s w e r e t h e n c o u n t e d o n a h e m o c y t o m e t e r ( N e u b a u e r ) c h a m b e r . I n o r d e r t o c o m p u t e t h e p e r c e n t d i l u t i o n t o b e a p p l i e d t o t h e t o t a l c e l l s u s p e n s i o n , t h i s s a m p l i n g c o u n t w a s m u l t i p l i e d b y a f a c t o r o f 0 . 0 1 . T h i s d i l u t i o n c o n s t a n t a c c o u n t e d f o r t h e n u m b e r o f c h a m b e r s o n t h e h e m o c y t o m e t e r , t h e s i z e o f t h e c o v e r s l i p s , a n d t h e f i n a l d e n s i t y ( 1 0 5 c e l l s / c m 2 ) a t w h i c h t h e c o v e r s l i p s w e r e t o b e p l a t e d . T h e c e l l - c o n t a i n i n g m e d i u m w a s s u b s e q u e n t l y d i l u t e d w i t h D - M E M / F B S b y t h e f a c t o r d e t e r m i n e d i n t h e s a m p l e c o u n t 2 3 c a l c u l a t i o n . C o v e r s l i p s , p r e v i o u s l y c o a t e d w i t h p o l y - D - l y s i n e a n d l a m i n i n f o r c e l l a d h e s i o n a n d g r o w t h , w e r e t h e n p l a t e d w i t h 0 . 2 m L o f t h e d i l u t e d c u l t u r e m e d i u m . A f t e r 1 h o u r , t h e c o v e r s l i p s w e r e t r a n s f e r r e d f a c e d o w n i n t o 6 - w e l l c u l t u r e p l a t e s c o n t a i n i n g 2 m L o f D - M E M / F B S i n e a c h w e l l , a n d s t o r e d i n a 5 % C 0 2 i n a i r e n v i r o n m e n t a t 3 5 ° C . T h e c e l l s w e r e c u l t u r e d i n a f a c e - d o w n p o s i t i o n d u e t o t h e h i g h r a t e o f m o r t a l i t y a s s o c i a t e d w i t h f a c e - u p g r o w t h ( s e e B r e w e r a n d C o t m a n , 1 9 8 9 ) . A f t e r 3 t o 4 h o u r s o f i n c u b a t i o n , h a l f o f t h e c u l t u r e m e d i u m w a s r e p l a c e d b y 1 m L o f s e r u m - f r e e D - M E M c o n t a i n i n g 5 m g / L t r a n s f e r r i n , 6 . 2 p g / L p r o g e s t e r o n e , 8 . 8 m g / L p u t r e s c i n e , 5 . 1 9 p g / L s e l e n i u m , a n d 5 m g / L i n s u l i n . T h i s D - M E M / F B S r e p l a c e m e n t p r o c e d u r e w a s r e p e a t e d s e v e n d a y s a f t e r t h e i n i t i a l c u l t u r e d a t e . T h e p r e s e n c e o f n o n - n e u r o n a l c e l l s w a s c h e c k e d f o l l o w i n g 2 t o 3 d a y s o f s t o r a g e , a n d t h e c u l t u r e s w e r e t r e a t e d w i t h 5 - f l u o r o d e o x y u r i d i n e t o a r r e s t g l i a l m u l t i p l i c a t i o n . E x p e r i m e n t s w e r e p e r f o r m e d a t b o t h r o o m t e m p e r a t u r e a n d 3 7 ° C u s i n g 6 t o 1 4 d a y o l d c u l t u r e s . Loading the neurones with B C E C F : B C E C F a c e t o x y m e t h y l e s t e r ( B C E C F - A M ) , t h e c e l l - p e r m e a n t f o r m o f t h e f l u o r e s c e n t 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 f r o m M o l e c u l a r P r o b e s I n c . ( E u g e n e , O r e g o n ) . T h e f l u o r e s c e n t p r o b e w a s p r e p a r e d i n a d v a n c e a s a 1 . 0 m M s t o c k i n a n h y d r o u s D M S O , s e p a r a t e d i n t o 6 0 p L a l i q u o t s , a n d s t o r e d a t - 6 0 ° C . L o a d i n g m e d i u m , m a d e u p o n t h e d a y o f t h e e x p e r i m e n t , c o n t a i n e d t h e s a m e 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 p l a c e o f N a C l . 5 p . L o f t h e 1 . 0 m M B C E C F - A M s t o c k w a s t h a w e d a n d d i l u t e d t o 2 u M i n 2 . 5 m L o f t h e l o a d i n g m e d i u m c o n t a i n e d i n a s i n g l e w e l l o f a 6 - w e l l t i s s u e c u l t u r e p l a t e . A n 1 8 m m c o v e r s l i p , p l a t e d w i t h t h e h i p p o c a m p a l n e u r o n e s , w a s p l a c e d f a c e - u p i n t h e d y e - c o n t a i n i n g m e d i u m f o r 3 0 m i n u t e s a t r o o m t e m p e r a t u r e . T h e c o v e r s l i p w a s t h e n m o u n t e d i n a t e m p e r a t u r e - c o n t r o l l e d p e r f u s i o n c h a m b e r s o a s t o f o r m t h e b a s e o f t h e 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 a r 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. E x p e r i m e n t a l s e t u p : 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 pH 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 BCECF 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 2 5 e x c i t a t i o n r a d i a t i o n w a s r e f l e c t e d b y a l o n g b a n d - p a s s d i c h r o i c m i r r o r ( F T - 4 9 5 ) a n d w a s f o c u s e d t h r o u g h a x 4 0 N e o f l u a r o b j e c t i v e ( n u m e r i c a l a p e r t u r e 0 . 7 5 ) o n t o t h e c e l l s i n t h e r e c o r d i n g c h a m b e r . T h e e m i t t e d f l u o r e s c e n t l i g h t p a s s e d b a c k t h r o u g h t h e d i c h r o i c b e a m s p l i t t e r b e f o r e b e i n g f i l t e r e d b y a 5 1 0 n m l o n g - p a s s f i l t e r , t h e w a v e l e n g t h a t w h i c h t h e e m i s s i o n w a s m o n i t o r e d . F l u o r e s c e n c e e m i s s i o n s w e r e m e a s u r e d b y a n i n t e n s i f i e d c h a r g e - c o u p l e d d e v i c e c a m e r a m o u n t e d o n t o t h e 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 s e t b y m a x i m i z i n g t h e i m a g e i n t e n s i t y w h i l e m i n i m i z i n g t h e p o s s i b i l i t y o f c a m e r a s a t u r a t i o n , a n d w a s h e l d c o n s t a n t t h r o u g h o u t a n e x p e r i m e n t . I m a g e s w e r e d i g i t i z e d t o 8 b i t r e s o l u t i o n w i t h a 5 1 2 x 4 8 0 p i x e l f r a m e s i z e . D u r i n g a c q u i s i t i o n , a s i n g l e i m a g e w a s c a p t u r e d f o r e a c h o f t h e t w o e x c i t a t i o n w a v e l e n g t h s . A v i d e o t e r m i n a l s e q u e n t i a l l y d i s p l a y e d e a c h p s e u d o c o l o u r e d i m a g e , w h i c h w e r e u s e d n o t o n l y t o v i s u a l l y m o n i t o r t h e p r o g r e s s o f t h e s t u d y , b u t t o a l s o s e l e c t a r e a s f o r a n a l y s i s . T h e s e s e l e c t e d r e g i o n s o f i n t e r e s t ( R O I ' s ) , 1 0 p i x e l s b y 1 0 p i x e l s i n s i z e , w e r e s e t a t t h e s t a r t o f t h e 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 9 9 ) n e u r o n a l s o m a t a h a v i n g a n a p p r o x i m a t e p y r a m i d a l s h a p e : o n e d e f i n e d l o n g p r o c e s s , a n d t w o o r m o r e s h o r t e r p r o c e s s e s . T o a i d i n t h e r o u g h f o c u s i n g o f t h e n e u r o n e s a n d t h e s e l e c t i o n o f t h e R O I ' s , t h e c e l l p o p u l a t i o n w a s v i s u a l i z e d u n d e r p h a s e i l l u m i n a t i o n u s i n g a 1 2 V , 1 0 0 W h a l o g e n l a m p . T h r o u g h o u t t h e c o u r s e o f a n e x p e r i m e n t , t h e c o m p u t e r c a l c u l a t e d a n d g r a p h i c a l l y d i s p l a y e d t h e e m i s s i o n i n t e n s i t i e s f o r b o t h e x c i t a t i o n w a v e l e n g t h s , a n d t h e r a t i o o f t h e e m i t t e d f l u o r e s c e n c e f o r a l l , o r a c h o s e n s i n g l e , r e g i o n ( s ) o f i n t e r e s t . I n a l l c a s e s , t h e r e c o r d e d v a l u e s r e f l e c t e d t h e m e a n e m i t t e d i n t e n s i t y w i t h i n e a c h R O I , c o m p u t e d i n r e a l t i m e . Solutions: T h e s o l u t i o n s u t i l i z e d d u r i n g t h e c o u r s e o f t h e s e e x p e r i m e n t s a r e l i s t e d i n T a b l e s 1 t o 4 . A C o r n i n g 2 4 0 p H m e t e r , c a l i b r a t e d d a i l y , w a s e m p l o y e d t o m e a s u r e a l l 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 1 0 m M H E P E S a n d t h e n 2 6 t i t r a t e d t o t h e a p p r o p r i a t e p H w i t h 1 0 M N a O H , e x c e p t w h e n n o t e d . T h e N a + - f r e e s a l i n e {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 + s a l t s f o u n d i n t h e s t a n d a r d 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 u s e o f 1 0 M H C 1 t o l o w e r t h e p H t o t h e 7 . 4 r a n g e . C l " w a s r e m o v e d t h r o u g h t h e u s e o f s o d i u m , p o t a s s i u m , a n d h e m i - c a l c i u m g l u c o n a t e i n p l a c e o f N a C l , K C 1 , a n d C a C l 2 , r e s p e c t i v e l y (solution 3). T h e a d d i t i o n N H 4 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 w a s a c h i e v e d t h r o u g h e q u i m o l a r r e p l a c e m e n t o f N a C l . C a l i b r a t i o n s o l u t i o n s w e r e p r e p a r e d u s i n g t h e i o n o p h o r e n i g e r i c i n , a c a t i o n - h y d r o g e n e x c h a n g e r t h a t i s h i g h l y s e l e c t i v e f o r K + ( C h a i l l e t a n d B o r o n , 1 9 8 5 ) . N i g e r i c i n w a s p r e p a r e d a s a 1 0 m M s t o c k s o l u t i o n i n e t h a n o l , d i v i d e d i n t o 1 0 0 p L v o l u m e s , a n d t h e n s t o r e d a t - 6 0 ° C . W h e n n e e d e d , a 1 0 m M a l i q u o t w a s d i l u t e d t o 1 0 p M i n a s o l u t i o n c o n t a i n i n g a c o n c e n t r a t i o n o f 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). N i g e r i c i n - c o n t a i n i n g s o l u t i o n s w e r e t i t r a t e d t o a p p r o p r i a t e p H s w i t h 1 0 M K O H , w i t h t h e e x c e p t i o n o f t h e p H 5 . 5 s o l u t i o n e m p l o y e d d u r i n g f u l l c a l i b r a t i o n s ( s e e b e l o w ) t h a t r e q u i r e d 1 M H C 1 . R e g a r d l e s s o f t h e t e m p e r a t u r e a t 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 p e r f o r m e d , a l l H E P E S 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 a t r o o m t e m p e r a t u r e ( 1 8 ° C t o 2 2 ° C ) . I n o r d e r t o a c c o u n t f o r t h e p H f l u c t u a t i o n a s s o c i a t e d w i t h r a i s i n g t h e s o l u t i o n t e m p e r a t u r e , t h e p H a t r o o m t e m p e r a t u r e ( p H R T ) w a s a d j u s t e d t o r e f l e c t t h e 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 p H c h a n g e , s u c h t h a t a t 3 7 ° C t h e 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 d i f f e r e n t p H s f o r H C 0 3 " / C 0 2 - f r e e , H E P E S - b u f f e r e d s o l u t i o n s a t 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 r e l a t e d b y t h e e q u a t i o n : p H 3 7 = 0 . 1 8 + 0 . 9 6 x p H R X (Equation 6) T h i s e q u a t i o n w a s d e t e r m i n e d d u r i n g p r e l i m i n a r y e x p e r i m e n t s ( n = 8 ) i n w h i c h t h e p H s o f H E P E S b u f f e r e d s o l u t i o n s , p r e p a r e d a t 2 2 ° C , w e r e c o m p a r e d t o t h e r e s u l t i n g p H w h e n h e a t e d t o 3 7 ° C . T h e c o m p o s i t i o n o f s o l u t i o n s b u f f e r e d b y a c o m b i n a t i o n o f H C 0 3 " a n d C 0 2 a t r o o m t e m p e r a t u r e a r e s u m m a r i z e d i n T a b l e 2 . A l l H C 0 3 " - c o n t a i n i n g s o l u t i o n s , a t r o o m 2 7 t e m p e r a t u r e a n d 3 7 ° C , w e r e e q u i l i b r a t e d w i t h 5 % C 0 2 i n b a l a n c e a i r . F o r e x p e r i m e n t s a t r o o m t e m p e r a t u r e , t h e s t a n d a r d H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m c o n t a i n e d 2 6 . 0 m M N a H C 0 3 (solution 6), r e s u l t i n g i n a p H o f 7 . 3 2 ± 0 . 0 1 ( m e a n ± s t a n d a r d e r r o r o f t h e m e a n , n = 1 9 ) . T h e p r e p a r a t i o n o f N a + - f r e e s a l i n e w a s a c c o m p l i s h e d b y r e p l a c i n g N a C l a n d N a H C 0 3 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 ) . 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) a n d N H 4 C 1 (solution 11) w e r e a d d 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 N a C l ; w h e n e q u i l i b r a t e d w i t h 5 % CO2, t h e s e m i x t u r e s r e s u l t e d i n p H s o f 7 . 3 0 ( n = l ) , 7 . 3 1 ( n = l ) , a n d 7 . 3 2 ( n = 2 ) , r e s p e c t i v e l y . A t 3 7 ° C , t h e c o n c e n t r a t i o n o f b i c a r b o n a t e i n t h e s t a n d a r d m e d i u m w a s r e d u c e d t o 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 . 3 6 ± 0 . 0 1 ( m e a n ± S . E . M . , n = 1 9 ) . N a + - f r e e (solution 13; p H 0 7 . 3 8 ± 0 . 0 1 , n = 4 ) , C l ' - f r e e (solution 14; p H 0 7 . 3 8 ± 0 . 0 1 , n = 7 ) , a n d N H 4 C l - c o n t a i n i n g (solution 16; p H 0 7 . 3 5 ± 0 . 0 1 , n = 7 ) s o l u t i o n s w e r e p r e p a r e d i n a n s i m i l a r f a s h i o n t o t h e i r r o o m t e m p e r a t u r e c o u n t e r p a r t s . A m i x t u r e l a c k i n g b o t h N a + a n d C l - w a s f o r m e d u s i n g f r e e c h o l i n e b a s e , c h o l i n e b i c a r b o n a t e , f r e e g l u c o n i c a c i d , p o t a s s i u m a n d h e m i - c a l c i u m g l u c o n a t e , a n d n o r m a l c o n c e n t r a t i o n s o f M g S 0 4 a n d D- g l u c o s e (solution 15; p H 0 7 . 3 8 , 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 a d j u s t t h e c o n c e n t r a t i o n o f N a H C 0 3 v i a i s o o s m o t i c s u b s t i t u t i o n w i t h N a C l ( T a b l e 4 ) . P r e l i m i n a r y e x p e r i m e n t s e s t a b l i s h e d t h a t , a t 3 7 ° C , t h e s o l u t i o n p H w a s r e l a t e d t o i t s b i c a r b o n a t e c o n c e n t r a t i o n ( i n m M ) b y t h e e q u a t i o n : p H = 6 . 0 3 + 1 . 0 3 x l o g [ H C 0 3 - ] (Equation 7) T h i s f o r m u l a w a s d e r i v e d f r o m a s e r i e s o f p H v e r s u s c o n c e n t r a t i o n o f H C 0 3 _ d a t a p o i n t s 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, 18, 20, 21, a n d 22 ( T a b l e 4 ) . T h e N H 4 C l - c o n t a i n i n g s o l u t i o n a t 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 b y 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 4 C 1 (solution 19). T h e p H s o f a l l s o l u t i o n s w e r e r e - m e a s u r e d a t t h e 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 e x p e r i m e n t . 2 8 E t h y l i s o p r o p y l a m i l o r i d e ( E I P A ) w a s p r e p a r e d a s a 5 0 m M s t o c k s o l u t i o n i n d i m e t h y l s u l p h o x i d e ( D M S O ) p r i o r t o a 1 i n 1 0 0 0 d i l u t i o n i n t h e p e r f u s i o n s o l u t i o n . 4 , 4 ' - d i i s o t h i o c y a n a t o s t i l b e n e - 2 , 2 ' - d i s u l p h o n i c a c i d ( D I D S ) w a s d i s s o l v e d i n D M S O a t a c o n c e n t r a t i o n o f 1 0 0 m M , a n d u s e d a t a f i n a l c o n c e n t r a t i o n o f 2 0 0 u M . A l l s t o c k s o l u t i o n s w e r e p r e p a r e d o n t h e d a y o f t h e e x p e r i m e n t , a n d t h e f i n a l c o n c e n t r a t i o n o f D M S O i n t h e p e r f u s i o n s o l u t i o n n e v e r e x c e e d e d 0 . 5 % . C o n t r o l e x p e r i m e n t s d e m o n s t r a t e d t h a t , a t t h i s c o n c e n t r a t i o n , D M S O h a d n o e f f e c t o n p H ; ( d a t a n o t s h o w n ) . 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 ( S t . L o u i s , M i s s o u r i ) , w i t h t h e e x c e p t i o n o f 3 - m e t h y l s u l f o n y l - 4 - p i p e r i d i n o b e n z o y l g u a n i d i n e h y d r o c h l o r i d e ( H O E 6 9 4 ) , a n d 5 - ( A f - m e t h y l - / V - g u a n i d i n o c a r b o n y l m e t h y l ) a m i l o r i d e ( M G C M A ) . H O E 6 9 4 w a s o b t a i n e d f r o m H o e c h s t A . G . ( F r a n k f u r t , G e r m a n y ) , w h i l e M G C M A w a s a g e n e r o u s g i f t f r o m D r . V . P a l a t y ( 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 B r i t i s h C o l u m b i a ) ; b o t h c h e m i c a l s w e r e p r e p a r e d a s 1 0 0 m M s t o c k s o l u t i o n s i n D M S O , a n d u t i l i z e d a t a f i n a l c o n c e n t r a t i o n o f 1 0 0 u M . Calculation of pHj: E x p e r i m e n t a l r e s u l t s w e r e s t o r e d i n c o m p u t e r - g e n e r a t e d d a t a f i l e s c o n t a i n i n g p i x e l i n t e n s i t i e s f o r e a c h r e g i o n o f i n t e r e s t . D u r i n g a c q u i s i t i o n p e r i o d s t h e f o l l o w i n g i n f o r m a t i o n w a s s t o r e d : t h e i n t e n s i t y o f t h e f l u o r e s c e n t s i g n a l a f t e r e x c i t a t i o n a t 4 8 8 n m , t h e 4 6 0 n m - i n d u c e d f l u o r e s c e n t s i g n a l , a n d a r a t i o (I488/I460) ° f m e fluorescence i n t e n s i t i e s . U t i l i z i n g a s t a n d - a l o n e D O S b a s e d g r a p h i n g p r o g r a m ( A T T O G R A F , A t t o I n s t r u m e n t s I n c , v e r s i o n 5 . 4 1 ) , r e g i o n s o f i n t e r e s t c o r r e s p o n d i n g t o n e u r o n e s t h a t r e m a i n e d v i a b l e t h r o u g h o u t a n e x p e r i m e n t w e r e s e l e c t e d f o r a n a l y s i s . V i a b i l i t y w a s j u d g e d b y t h e c a p a c i t y o f t h e n e u r o n e s t o r e t a i n t h e fluorescent i n d i c a t o r ( a s j u d g e d b y r a w i n t e n s i t y v a l u e s ) 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 t h e e x p e r i m e n t ( s e e S c h w i e n i n g a n d B o r o n , 1 9 9 2 ; S c h w i e n i n g a n d B o r o n , 1 9 9 4 ) . 2 9 T h e d e t e r m i n a t i o n o f p H j w a s i n i t i a t e d b y t h e s u b t r a c t i o n o f b a c k g r o u n d f l u o r e s c e n c e i n t e n s i t i e s f r o m t h e r a w i n t e n s i t y v a l u e s i n e a c h s e l e c t e d R O I . B a c k g r o u n d l e v e l s w e r e d e t e r m i n e d b y m e a s u r i n g t h e f l u o r e s c e n c e s i g n a l i n a r e g i o n d e v o i d o f c e l l u l a r p r o c e s s e s a t e a c h e x c i t a t i o n w a v e l e n g t h . T r a n s f o r m a t i o n o f t h e b a c k g r o u n d - c o r r e c t e d r a t i o s i n t o p H j v a l u e s u t i l i z e d c o n v e r s i o n e q u a t i o n s d e r i v e d f r o m in situ c a l i b r a t i o n e x p e r i m e n t s ( F i g u r e 2 ) . I n s u c h e x p e r i m e n t s , t h e n e u r o n e s w e r e e x p o s e d t o v a r i e t y o f H E P E S - b u f f e r e d s o l u t i o n s a t 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 ( F i g u r e 2 A ) . A l l c a l i b r a t i o n s o l u t i o n s c o n t a i n e d 1 0 u M o f t h e i o n o p h o r e n i g e r i c i n , w h i c h w a s a d d e d t o a s o l u t i o n c o n t a i n i n g h i g h c o n c e n t r a t i o n s o f K + (solution 5, T a b l e 1 ) . E a c h s o l u t i o n w a s t i t r a t e d t o a d i f f e r e n t p H i n t h e 5 . 5 t o 8 . 5 r a n g e u s i n g 1 0 M K O H o r 1 M H C 1 . N i g e r i c i n i s a c h a r g e d e l e c t r o n c a r r i e r t h a t a c t s t o b a l a n c e p H j a n d p H 0 i f t h e i n t r a c e l l u l a r a n d e x t r a c e l l u l a r K + a c t i v i t i e s a r e e q u a l ( C h a i l l e t a n d B o r o n , 1 9 8 5 ) . T h u s , i n t h e p r e s e n c e o f h i g h e x t r a c e l l u l a r K + c o n c e n t r a t i o n s , p H j w a s c o n t r o l l e d m e r e l y b y t h e p H o f t h e s u p e r f u s i n g m e d i u m . T h e r e s u l t i n g i n t e n s i t y r a t i o s p r o d u c e d b y e x p o s i n g t h e n e u r o n e s t o v a r i o u s p H s o l u t i o n s c o n t a i n i n g 1 0 p M n i g e r i c i n w e r e u s e d t o c o n s t r u c t a c a l i b r a t i o n c u r v e . F o l l o w i n g s u b t r a c t i o n o f b a c k g r o u n d f l u o r e s c e n c e v a l u e s f r o m t h e 4 8 8 a n d 4 6 0 n m - i n d u c e d f l u o r e s c e n c e s i g n a l s , t h e r a t i o s ( 1 4 8 8 ^ 4 6 0 ) w e r e n o r m a l i z e d s u c h t h a t t h e e n s u i n g c u r v e p a s s e d t h r o u g h u n i t y a t p H 7 . 0 ( F i g u r e 2 B . ) . A f u l l c a l i b r a t i o n e x p e r i m e n t r e s u l t e d i n t h e d e t e r m i n a t i o n o f p a r a m e t e r s f i t t i n g a s t a n d a r d c u r v e w h i c h c o u l d t h e n b e u s e d t o t r a n s f o r m o t h e r 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 . T h e d e r i v a t i o n o f t h e e q u a t i o n f i t t i n g t h i s s t a n d a r d c u r v e s t e m s f r o m t h e H e n d e r s o n - H a s s e l b a l c h e x p r e s s i o n f o r t h e d i s s o c i a t i o n o f a w e a k a c i d : [A1 p H = p K a + l o g f-—^ (Equation 8) 3 0 w h e r e [ A - ] i s 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 ] i s t h e n e u t r a l f o r m o f t h e a c i d , a n d K a i s t h e a c i d d i s s o c i a t i o n c o n s t a n t . T a k i n g i n t o c o n s i d e r a t i o n t h e t o t a l a c i d c o n c e n t r a t i o n , d e n o t e d b y [ T o t a l ] , a s e q u a l i n g t h e s u m o f t h e i o n i z e d [ A - ] a n d n o n - i o n i z e d [ H A ] f o r m s o f t h e a c i d , t h e a b o v e e q u a t i o n b e c o m e s : p H = p K + l o g L ) , (Equation 9) [ T o t a l ] - [ 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 f l u o r e s c e n c e i n t e n s i t i e s a t 4 8 8 n m a n d 4 6 0 n m . T h u s t h e t o t a l a c i d c o n c e n t r a t i o n i s p r o p o r t i o n a l t o t h e m a x i m a l o b t a i n a b l e r a t i o ( " b " ) . S u b s t i t u t i n g t h e s e v a r i a b l e s i n t o E q u a t i o n 9 y i e l d s : R 1Q(PH-PKa) p H = p K a + l o g ^ - ^ - , o r R = b - 1 + 1 0 ( p H - P K a ) (Equation 10) I f R i s c o n s t r a i n e d t o p a s s t h r o u g h u n i t y a t p H 7 . 0 , t h e n t h e v a l u e o f R a t p H 7 . 0 m u s t b e 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 n , c a n b e e x p r e s s e d a s : j Q C p H - p K J 1Q(7-PKa) R n = b - b v . + 1 (Equation 11) l + 1 0 ( p p a ) 1 + 1 0 ( p a ) T h e f i t t e d v a l u e s f o r b a n d p K a v a r i e d w i t h t h e s e t u p o f t h e m i c r o s c o p e . F o r t h i s r e a s o n , a n y c h a n g e s t o t h e e x p e r i m e n t a l e q u i p m e n t ( f o r e x a m p l e , t h e r e p l a c e m e n t o f t h e m e r c u r y a r c b u r n e r ) w a s a c c o m p a n i e d b y t h e e x e c u t i o n o f a f u l l c a l i b r a t i o n e x p e r i m e n t , a n d r e v i s e d c a l i b r a t i o n p a r a m e t e r s w e r e d e t e r m i n e d . . E q u a t i o n 1 1 w a s s i m p l i f i e d b y d e t e r m i n i n g t h e t h e o r e t i c a l m a x i m u m a n d m i n i m u m o b t a i n a b l e v a l u e s f o r t h e n o r m a l i z e d r a t i o . T h e s e v a l u e s , s y m b o l i z e d b y R n ( m a x ) a n d R n ( m i n ) , c a n b e r e p r e s e n t e d a s : 1 0 ( 7 - p K J R , . = l + b - b TZ—TTT~ (Equation 12) ±vn(max) u j + 1 Q ( 7 - p K a ) * 1 / a n d 3 1 1Q(7-PKa) R „ ( m i n ) = 1 - b ' 1 + 1 0 ( 7 - P K a ) (Equation 13) U s i n g t h e d e t e r m i n e d v a l u e s f o r b a n d p K a , t h e m a x i m u m a n d m i n i m u m n o r m a l i z e d r a t i o s w e r e c a l c u l a t e d . I n o r d e r t o 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 , i t i s n e c e s s a r y t o e x p r e s s t h e r e g r e s s i o n e q u a t i o n a s a f u n c t i o n o f p H . M a n i p u l a t i o n o f E q u a t i o n 1 1 y i e l d s : R „ J Q ( P H - P K J _ 1 _ U 10( 7-PKa> ' (Equation 14) l i b b 1 Q ( 7 " p K a ) 1 R S u b s t i t u t i n g R n ( m a x ) a n d R n ( m j n ) i n t o E q u a t i o n 1 4 g i v e s r i s e t o : IO"*--*. ' = ( R „ - R n ( m i n ) ) / ( R n ( m a x ) - R n ) (Equation 15) I s o l a t i n g t h e p H t e r m u t i l i z i n g a l o g a r i t h m i c m a n i p u l a t i o n p r o d u c e s t h e f o l l o w i n g e q u a t i o n : p H = l o g [ ( R n - R n ( m i n ) ) / ( R n ( m a x ) - R n ) ] + p K a (Equation 16) E q u a t i o n 1 6 w a s t h e n u t i l i z e d i n t h e c o n v e r s i o n o f a l l 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 , 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 n ( m j n ) , R n ( m a x ) > a n Q l p K a . T h e s e f a c t o r s w e r e c a l c u l a t e d f o r e a c h f u l l c a l i b r a t i o n e x p e r i m e n t . F o r t h e s e v e n f u l l c a l i b r a t i o n e x p e r i m e n t s u t i l i z e d i n a n a l y z i n g a l l e x p e r i m e n t s , t h e m e a n v a l u e s o f p K a , R n ( m j n ) , a n d R n ( m a x ) w e r e 6 . 9 8 ± 0 . 0 2 , 0 . 4 9 ± 0 . 0 2 , a n d 1 . 4 9 ± 0 . 0 2 , r e s p e c t i v e l y . F u r t h e r m o r e , t h e v a l u e s o f t h e s e c a l c u l a t e d p a r a m e t e r s d i d n o t a p p e a r t o b e d e p e n d e n t o n c h a n g e s i n t h e e x p e r i m e n t a l t e m p e r a t u r e , w h i c h t y p i c a l l y v a r i e d b e t w e e n 2 0 ° C a n d 3 0 ° C . F o r e x a m p l e , i n a c a l i b r a t i o n p e r f o r m e d a t 3 0 ° C , t h e d e t e r m i n e d p K a v a l u e w a s 6 . 9 8 , w h e r e a s i n a s e p a r a t e s t u d y a t 2 1 ° C , p K a w a s f o u n d t o b e 6 . 9 7 . M o s t e x p e r i m e n t s w e r e c o n c l u d e d b y e x p o s i n g t h e n e u r o n e s t o a s i n g l e p H 7 . 0 n i g e r i c i n - c o n t a i n i n g s o l u t i o n ( s e e F i g u r e s 4 , 7 , a n d 1 2 ) . T h e r e s u l t i n g ( b a c k g r o u n d - c o r r e c t e d ) r a t i o a t p H j 7 . 0 w a s u s e d a s t h e n o r m a l i z a t i o n f a c t o r f o r t h a t p a r t i c u l a r 3 2 e x p e r i m e n t . 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 i s t h a t i t 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 . A f t e r d i v i d i n g a l l e x p e r i m e n t a l l y - d e r i v e d ( b a c k g r o u n d - c o r r e c t e d ) i n t e n s i t y r a t i o s b y t h e d e t e r m i n e d n o r m a l i z a t i o n v a l u e , e a c h R n w a s c o n v e r t e d t o p H j u t i l i z i n g E q u a t i o n 1 6 a n d t h e a p p r o p r i a t e f i t t e d c a l i b r a t i o n p a r a m e t e r s . A n a l y s i s o f d a t a : E a c h e x p e r i m e n t t y p i c a l l y r e q u i r e d t h e a n a l y s i s o f 1 0 o r m o r e r e g i o n s o f i n t e r e s t , 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 m t 0 P ^ i e m p l o y i n g e i t h e r a D O S - b a s e d t r a n s f o r m a t i o n p r o g r a m ( c o u r t e s y o f D r . K . A b d e l - H a m i d , D e p a r t m e n t o f P h y s i o l o g y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) o r p e r s o n a l l y d e s i g n e d 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 l e v e l s a r e r e p o r t e d f o r 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 b o t h r o o m t e m p e r a t u r e a n d 3 7 ° C . A t s t e a d y - s t a t e , a n y p e r t u r b a t i o n s i n p H j w e r e m e a s u r e d r e l a t i v e t o t h e r e s t i n g p H j b e f o r e t h e c h a n g e . I n e x p e r i m e n t s d e s i g n e d t o a n a l y z e t h e r e s t o r a t i o n o f p H j b a c k t o s t e a d y - s t a t e l e v e l s a f t e r a n i m p o s e d a c i d l o a d , 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 w a s f i t t e d t o a s i n g l e e x p o n e n t i a l f u n c t i o n h a v i n g a f o r m a t : p H j = a + b ( l - 1 0 ( _ c t ) ) (Equation 17) w h e r e a , b , a n d c a r e t h e e x p o n e n t i a l p a r a m e t e r s . T h e d i f f e r e n t i a t e d f o r m o f E q u a t i o n 1 7 r e p r e s e n t s t h e c h a n g e i n p H j a s a f u n c t i o n o f t i m e , a n d w a s u s e d t o a n a l y z e t h e r e c o v e r y 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 - = -bcl 0 ( _ c t ) (Equation 18) di R e c o v e r y r a t e s 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 a f t e r t h e p e a k a c i d i f i c a t i o n , a n d a t 5 0 % a n d 8 0 % r e c o v e r y r e l a t i v e t o t h e s t e a d y - s t a t e p H j b e f o r e t h e i n d u c e d a c i d l o a d . 3 3 S t a t i s t i c a l c o m p a r i s o n s w e r e c a r r i e d o u t u s i n g S t u d e n t ' s t t e s t w i t h a 9 5 % c o n f i d e n c e l i m i t . I f a p r e c o n c e i v e d d i r e c t i o n a l i t y e x i s t e d i n m a k i n g a c o m p a r i s o n , a o n e - t a i l e d t e s t w a s u s e d , o t h e r w i s e t h e t w o - t a i l e d v e r s i o n w a s u t i l i z e d . I n a l l c a s e s , u n p a i r e d t v a l u e s w e r e c a l c u l a t e d , w i t h s u p p l e m e n t a l p a i r e d d a t a a d d e d w h e n a p p r o p r i a t e . A n y i n d i c a t e d e r r o r s a r e e x p r e s s e d a s t h e s t a n d a r d e r r o r o f t h e m e a n ( S . E . M . ) , w i t h t h e a c c o m p a n y i n g n v a l u e r e f e r r i n g t o t h e n u m b e r o f c e l l p o p u l a t i o n s ( i . e . n u m b e r o f c o v e r s l i p s ) a n a l y z e d . P e r i o d i c a l l y , v a r i a t i o n s i n t h e e m i s s i o n i n t e n s i t i e s a r o s e w h i c h w e r e c a u s e d b y 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, 1 9 8 8 a ) . I n o r d e r t o s m o o t h t h e r e s u l t i n g g r a p h i c a l r e p r e s e n t a t i o n o f t h e p H j v e r s u s t i m e r e c o r d , a m o v i n g a v e r a g e ( 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, 1 9 8 8 a ) . 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 ( a l l c o n c e n t r a t i o n s i n m M ) : S o l u t i o n 1 2 3 . 4 5 S t a n d a r d N a + f r e e C h f r e e NH4CI H i g h K + N a C l 1 3 6 . 5 - - 1 1 6 . 5 - K C 1 3 . 0 3 . 0 - 3 . 0 - C a C l 2 2 . 0 2 . 0 - 2 . 0 2 . 0 N a H 2 P 0 4 1 . 5 - 1 . 5 1 . 5 1 . 5 M g S 0 4 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 N a G l u - - 1 3 6 . 5 - 1 0 . 0 K G l u - - 3 . 0 - 1 3 0 . 5 > / 2 C a G l u - - 4 . 0 - - D - g l u c o s e 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 N M D G + - 1 3 6 . 5 - - - NH4CI - - - 2 0 . 0 - H E P E S 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 T i t r a t e d 1 0 M 1 0 M 1 0 M 1 0 M 1 0 M w i t h : N a O H H C 1 N a O H N a O H K O H A b b r e v i a t i o n s : N a G l u , s o d i u m g l u c o n a t e ; K G l u , p o t a s s i u m g l u c o n a t e ; ' / i C a h e m i - c a l c i u m g l u c o n a t e ; N M D G + , A ^ - m e t h y l - D - g l u c a m i n e . 3 5 T a b l e 2 : C o m p o s i t i o n o f H C 0 3 7 C 0 2 - 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 a t r o o m t e m p e r a t u r e ( a l l c o n c e n t r a t i o n s i n m M ) : S o l u t i o n 6 7 8 9 10 11 S t a n d a r d N a + f r e e C l - f r e e P R O P T M A N H 4 C I N a C l 1 2 0 . 5 - - 1 0 0 . 5 1 1 0 . 5 1 0 0 . 5 N a H C 0 3 2 6 . 0 - 2 6 . 0 2 6 . 0 2 6 . 0 2 6 . 0 K C 1 3 . 0 3 . 0 - 3 . 0 3 . 0 3 . 0 C a C l 2 2 . 0 2 . 0 - 2 . 0 2 . 0 2 . 0 N a H 2 P 0 4 1 . 5 - 1 . 5 1 . 5 1 . 5 1 . 5 M g S 0 4 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 D - g l u c o s e 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 N H 4 C I - - - - - 2 0 . 0 N a G l u - - 1 2 0 . 5 - - - K G l u - - 3 . 0 - - - V i C a G l u - - 4 . 0 - - - C h o l i n e H C 0 3 - 2 6 . 0 - - - - C h o l i n e C l - 1 2 0 . 5 - - - - P R O P - - - 2 0 . 0 - - T M A - - - - 1 0 . 0 - f i n a l p H 7 . 3 2 ± 0 . 0 1 7 . 3 5 7 . 3 3 ± 0 . 0 1 7 . 3 0 7 . 3 1 7 . 3 2 ( n = 1 4 ) ( n = l ) ( n = 2 ) ( n = l ) ( n = l ) ( n = 2 ) A l l H C 0 3 " - c o n t a i n i n g s o l u t i o n s w e r e e q u i l i b r i a t e d w i t h 5 % C 0 2 i n b a l a n c e a i r . p H s a r e r e p o r t e d a s t h e m e a n ± S . E . M . A b b r e v i a t i o n s : N a G l u , s o d i u m g l u c o n a t e ; K G l u , p o t a s s i u m g l u c o n a t e ; M - C a G l u , h e m i - c a l c i u m g l u c o n a t e ; P R O P , p r o p i o n a t e ; T M A , t r i m e t h y l a m i n e . 3 6 T a b l e 3 : C o m p o s i t i o n o f H C 0 3 7 C 0 2 - 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 a t 3 7 ° C ( a l l c o n c e n t r a t i o n s i n m M ) : S o l u t i o n 12 13 14 7 5 16 S t a n d a r d N a + f r e e C h f r e e N a + a n d C h f r e e N H 4 C 1 N a C l 1 2 6 . 5 - - - 1 0 6 . 5 N a H C 0 3 2 0 . 0 - 2 0 . 0 - 2 0 . 0 K C 1 3 . 0 3 . 0 - - 3 . 0 C a C l 2 2 . 0 2 . 0 - - 2 . 0 N a H 2 P 0 4 1 . 5 - 1 . 5 - 1 . 5 M g S 0 4 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 D - g l u c o s e 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 N H 4 C 1 - - - - 2 0 . 0 N a G l u - - 1 2 6 . 5 - - K G l u - - 3 . 0 3 . 0 - ! / 2 C a G l u - - 4 . 0 4 . 0 - G l u c o n i c a c i d - - - 1 2 6 . 5 - C h o l i n e H C 0 3 - 2 0 . 0 - 2 0 . 0 - C h o l i n e C l - 1 2 6 . 5 - - - C h o l i n e b a s e - - - 1 2 6 . 5 - f i n a l p H 7 . 3 6 ± 0 . 0 1 7 . 3 8 ± 0 . 0 1 7 . 3 8 ± 0 . 0 1 7 . 3 8 7 . 3 5 ± 0 . 0 1 ( n = 1 9 ) ( n = 4 ) (n=7) ( n = l ) ( n = 7 ) A l l H C 0 3 ' - c o n t a i n i n g s o l u t i o n s w e r e e q u i l i b r i a t e d w i t h 5 % C 0 2 i n b a l a n c e d a i r . R e p o r t e d p H s a r e g i v e n a s t h e m e a n ± S . E . M . A b b r e v i a t i o n s : N a G l u , s o d i u m g l u c o n a t e ; K G l u , p o t a s s i u m g l u c o n a t e ; V i C a G l u , h e m i - c a l c i u m g l u c o n a t e . 3 7 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 2 - 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 a t v a r y i n g p H s a t 3 7 ° C ( a l l c o n c e n t r a t i o n s i n m M ) : S o l u t i o n 17 18 . 19 20 21 22 p H 6 . 5 p H 6 . 8 p H 6 . 8 p H 7 . 0 p H 7 . 8 p H 8 . 0 s t a n d a r d s t a n d a r d N H 4 C I s t a n d a r d s t a n d a r d s t a n d a r d N a C l 1 4 3 . 5 1 4 0 . 7 1 2 0 . 7 1 3 7 . 5 1 0 1 . 5 6 1 . 5 N a H C C - 3 3 . 0 5 . 8 5 . 8 9 . 0 4 5 . 0 8 5 . 0 K C 1 3 . 0 3 . 0 3 . 0 3 . 0 3 . 0 3 . 0 C a C l 2 2 . 0 2 . 0 2 . 0 2 . 0 2 . 0 2 . 0 N a H 2 P 0 4 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 M g S 0 4 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 1 . 5 D - g l u c o s e 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 1 0 . 0 N H 4 C I - - 2 0 . 0 - - - f i n a l p H 6 . 5 6 6 . 7 9 ± 0 . 0 1 6 . 8 0 7 . 0 0 ± 0 . 0 1 7 . 7 5 8 . 0 2 ( n = 2 ) ( n = 3 ) ( n = l ) ( n = 3 ) ( n = 2 ) ( n = 2 ) A l l H C 0 3 - - c o n t a i n i n g s o l u t i o n s w e r e e q u i l i b r i a t e d w i t h 5 % C 0 2 i n b a l a n c e d a i r . p H ' s a r e i n d i c a t e d a s t h e m e a n ± S . E . M . 3 8 Figure 1. Relationship between the concentration of HCO3- and the resulting solution pH when equilibrated with 5% CO2 in balance air at 37°C. F o l l o w i n g e q u i l i b r a t i o n w i t h 5 % C 0 2 , t h e p H s o f s o l u t i o n s w e r e m e a s u r e d a t 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 H C O 3 - ( s e e T a b l e 4 f o r s o l u t i o n r e c i p e s ) . D a t a w a s d e r i v e d f r o m a s i n g l e e x p e r i m e n t . T h e c u r v e w a s f o r m e d f r o m a l o g a r i t h m i c g r o w t h l e a s t s q u a r e s r e g r e s s i o n f i t t o t h e d a t a p o i n t s h a v i n g t h e e q u a t i o n : p H = 6 . 0 3 + 1 . 0 3 x l o g [ H C O 3 - ] 3 9 8.50 T 8.25 - 0 10 20 30 40 50 60 70 80 90 100 [HCO3-] (mM) 4 0 F i g u r e 2. S a m p l e c a l i b r a t i o n p l o t f o r 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 a t p H 0 ( a n d t h e r e f o r e p H j ) 5 . 5 5 , 6 . 0 2 , 6 . 5 0 , 7 . 0 0 , 7 . 5 1 , 7 . 9 6 , a n d 8 . 4 1 . 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 i s 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 t r a c e , w h i c h 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 2 9 c e l l s 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 . T h e r e s u l t i n g 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 ) w e r e n o r m a l i z e d t o 1 . 0 0 a t p H , 7 . 0 0 . B . P l o t o f p H j a g a i n s t t h e r e s u l t i n g n o r m a l i z e d r a t i o ( R n ) . S t a n d a r d e r r o r b a r s a r e i n d i c a t e d ( n = 3 c o v e r s l i p s ) . T h e c u r v e i s a r e s u l t o f a n o n - l i n e a r l e a s t s q u a r e s r e g r e s s i o n f i t t o E q u a t i o n 1 6 . F o r t h i s p a r t i c u l a r c a l i b r a t i o n , t h e v a l u e s o f R m a x , R m j n , a n d p K a w e r e 1 . 5 4 2 , 0 . 4 9 1 , a n d 7 . 0 2 7 , r e s p e c t i v e l y . 41 4 2 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 f p H j a t r o o m t e m p e r a t u r e : I n 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 p H 0 7 . 3 2 (solution 1, T a b l e 1 ) , s t e a d y - s t a t e p H j r e s t e d a t 6 . 8 5 ± 0 . 0 4 ( n = 2 5 ) 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 . A t t h e s a m e p H 0 b u t i n t h e p r e s e n c e o f H C 0 3 " , (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 s u b s t a n t i a l l y h i g h e r l e v e l o f 7 . 1 5 ± 0 . 0 3 ( n = 2 2 ; T a b l e 5 ; F i g u r e 3 B ) . T h i s s u g g e s t s 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 - s t a t e p H ; a t r o o m t e m p e r a t u r e . T h e e q u i m o l a r r e p l a c e m e n t o f c o n s t i t u e n t i o n s i n t h e p e r f u s i o n m e d i u m , o r a p p l i c a t i o n o f p h a r m a c o l o g i c a l a g e n t s , p r o v i d e d i n s i g h t i n t o t h i s H C 0 3 " - d e p e n d e n t m e c h a n i s m ( s e e T a b l e 5 ) . I n t h e p r e s e n c e o f H 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 " ( [ C l ~ ] 0 ) (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 4 p H u n i t s ( n = 2 ; F i g u r e 4 ) . A s d e p i c t e d i n F i g u r e 5 , t h e a p p l i c a t i o n o f 2 0 0 u M D I D S , a n i n h i b i t o r o f H C 0 3 7 C 1 " e x c h a n g e , r e d u c e d p H j b y 0 . 0 8 ± 0 . 0 4 p H u n i t s ( n = 3 ) . F i g u r e 5 a l s o d e m o n s t r a t e s t h a t p H j i m m e d i a t e l y r e t u r n e d t o n o r m a l r e s t i n g l e v e l s w h e n D I D S w a s w a s h e d f r o m t h e e x t r a c e l l u l a r m e d i u m . A c c o r d i n g t o t h e s e r e s u l t s , i t w o u l d a p p e a r t h a t 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 p l a y a r o l e r e g u l a t i n g s t e a d y - s t a t e p H j a t r o o m t e m p e r a t u r e . T o f u r t h e r i n v e s t i g a t e t h e r o l e o f H C 0 3 " i n m a i n t a i n i n g s t e a d y - s t a t e p H j a t r o o m t e m p e r a t u r e , t h e n e x t s e r i e s o f e x p e r i m e n t s e x p l o r e d t h e m o d u l a t i o n o f p H j d u r i n g 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, T a b l e 2 ) p e r f u s i o n m e d i a a t a c o n s t a n t p H 0 . A s s h o w n i n F i g u r e 6 A , s u c h a m a n o e u v r e w a s m a r k e d b y a n i n i t i a l a c i d i f i c a t i o n d u e t o t h e i n f l u x o f C 0 2 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 t o c a r b o n i c a c i d . T h i s b r i e f f a l l i n p H ; w a s f o l l o w e d b y a s u s t a i n e d a l k a l i n i z a t i o n , p r e s u m a b l y d u e t o t h e a c t i v a t i o n o f H C 0 3 " - d e p e n d e n t a c i d e x t r u s i o n 43 m e c h a n i s m s . T h i s r e s u l t i s r e f l e c t e d i n t h e m o r e a l k a l i n e r e s t i n g p H j o b s e r v e d i n e x p e r i m e n t s p e r f o r m e d i n t h e p r e s e n c e o f H C O 3 - a s c o m p a r e d w i t h e x p e r i m e n t s 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 a n d B ) . A s s h o w n i n F 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 v a l u e d u r i n g p e r f u s i o n w i t h H C C ^ ' - c o n t a i n i n g m e d i u m w a s i n h i b i t e d b y 2 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 H E P E S - b u f f e r e d p e r f u s i o n c o n d i t i o n s . T h e s e r e s u l t s s u g g e s t t h e c o n t r i b u t i o n o f s o m e f o r m o f H C 0 3 " / C 1 " e x c h a n g e t o t h e 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 a t r o o m t e m p e r a t u r e . A d e t a i l e d a n a y l s i s 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 3 7 ° C ( s e e b e l o w ) . 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 e x t r a c e l l u l a r 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 ( s e e F i g u r e 1 7 ) , w h i c h i n d i c a t e s t h a t 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 e x t r u d e r c o n t r i b u t e s t o t h e p r e s e r v a t i o n o f a s t a b l e r e s t i n g p H j . Regulation of steady state pHj at 3 7 ° 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 ; p H 0 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 t e m p e r a t u r e , c h a n g e s t o t h e i o n i c c o m p o s i t i o n o f t h e p e r f u s i n g m e d i u m h a d a m o d e r a t e i n f l u e n c e o n s t e a d y - s t a t e p H j . 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. A s s h o w n i n F i g u r e 7 , 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 + ( [ N a + ] 0 ) f r o m t h e 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 . 5 3 ± 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 [ N a + ] 0 c a u s e d a r e t u r n o f p H j t o s t e a d y - s t a t e l e v e l s . T h i s r e s u l t s u g g e s t s t h e p r e s e n c e 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 h i c h a r e o p e r a t i o n a l 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 . H o w e v e r , t h e r e m o v a l o f [ C l " ] 0 (solution 3, T a b l e 3 ) d i d n o t s i g n i f i c a n t l y c h a n g e t h e s t e a d y s t a t e p H j ( n = 3 ; F i g u r e 8 ) , s u g g e s t i n g t h e 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 3 7 ° C . 4 4 F i g u r e 9 s h o w s t h a t t h e a p p l i c a t i o n o f 5 0 u M E I P A , a p 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 N a + / H + e x c h a n g e i n a w i d e v a r i e t y o f c e l l t y p e s ( C l a r k a n d L i m b i r d , 1 9 9 1 ) , d i d n o t a l t e r t h e r e s t i n g p H j ( n = 3 ) . S i m i l a r l y , t h e a p p l i c a t i o n o f E I P A a f t e r 5 m i n u t e s o f [ N a + ] 0 - f r e e p e r f u s i o n d i d n o t i n f l u e n c e t h e a c i d i f i c a t i o n c a u s e d b y [ N a + ] 0 r e m o v a l ( n = 3 ; F i g u r e 1 0 ) . F i g u r e 1 0 a l s o d e m o n s t r a t e s t h a t p H j r e b o u n d e d b a c k t o i t s s t e a d y - s t a t e v a l u e a f t e r [ N a + ] 0 w a s r e t u r n e d t o t h e p e r f u s i o n s o l u t i o n , d e s p i t e t h e c o n t i n u e d p r e s e n c e o f E I P A . 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, 1 9 9 1 ) , a n d H O E 6 9 4 , a n o v e l i n h i b i t o r o f N a + / H + e x c h a n g e ( S c h m i d et al, 1 9 9 2 ; W o l l et al, 1 9 9 3 ) , w e r e b o t h a p p l i e d a t 1 0 0 p M b u t w e r e a l s o f o u n d t o h a v e n o e f f e c t o n s t e a d y - s t a t e p H j a t 3 7 ° C ( n = 3 f o r e a c h c o m p o u n d ; F i g u r e 1 1 ) . I t 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 i s n o t s e n s i t i v e t o 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 o f N a + / H + e x c h a n g e . 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 a t p H 0 7 . 3 6 (solution 12, T a b l e 3 ) , t h e s t e a d y - s t a t e p H j w a s 7 . 1 3 ± 0 . 0 1 ( n = 4 4 ; F i g u r e 3 D ) , a v a l u e l o w e r t h a n i n 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 t h e s a m e t e m p e r a t u r e . R e m o v i n g [ N a + ] 0 f r o m t h e p e r f u s i n g s o l u t i o n u n d e r H C 0 3 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, T a b l e 3 ) c a u s e d a 0 . 6 5 ± 0 . 0 4 p H u n i t f a l l i n p H j ( n = 8 ; T a b l e 5 ) . A s s h o w n i n F i g u r e 1 2 , p H ; f e l l r a p i d l y o n e x p o s u r e t o [ N a + ] 0 - f r e e m e d i u m , r e a c h e d a m i n i m u m i n l e s s t h a n 1 0 m i n u t e s , a n d i m m e d i a t e l y r e t u r n e d t o s t e a d y - s t a t e l e v e l s u p o n t h e r e - i n t r o d u c t i o n o f [ N a + ] 0 . T h i s r e s u l t i n d i c a t e s t h e d o m i n a n c e o f N a + - d e p e n d e n t a c i d e x t r u d e r s r e g u l a t i n g s t e a d y - s t a t e p H j a t 3 7 ° C , p o s s i b l y a N a + / H + e x c h a n g e r . H o w e v e r , a p p l i c a t i o n o f 5 0 p M E I P A o v e r a 1 0 m i n u t e p e r i o d d i d n o t a l t e r r e s t i n g p H j ( n = 3 ; F i g u r e 1 3 ) , w h i c h p r e v e n t s a m o r e p r e c i s e d e s c r i p t i o n o f t h i s 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 i t s d e p e n d e n c e o n N a + a n d c a p a c i t y t o o p e r a t e i n t h e p r e s e n c e o r a b s e n c e o f H C 0 3 " . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h p r e v i o u s o b s e r v a t i o n s s h o w i n g t h e i n a b i l i t y o f 5 0 p M E I P A t o i n f l u e n c e s t e a d y - s t a t e 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 1 0 ) . 4 5 A s s t e a d y s t a t e p H j a t r o o m t e m p e r a t u r e a p p e a r e d t o b e d e p e n d e n t o n H C 0 3 7 C 1 " e x c h a n g e , 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 t 3 7 ° C t o t 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 f b l o c k e r s o f t h e c a t i o n e x c h a n g e r . S h o w n i n F i g u r e 1 4 A , r e p l a c i n g [ C l " ] 0 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 3 " {solution 14, T a b l e 3 ) c a u s e d a g r a d u a l p H j i n c r e a s e o f 0 . 1 9 ± 0 . 0 1 p H u n i t s ( n = 5 ) . T h i s 0 [ C l ' ] 0 - i n d u c e d r i s e i n p H j a t 3 7 ° C w a s s i m i l a r t o , t h o u g h s l i g h t l y s m a l l e r t h a n , t h e i n c r e a s e i n p H j i n d u c e d b y t h e s a m e m a n o e u v r e 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 4 ) . U p o n s u b s t i t u t i o n o f [ C l " ] 0 b a c k i n t o t h e p e r f u s i n g b u f f e r , p H j r e t u r n e d t o i t s s t e a d y - s t a t e l e v e l . T h e i n t r o d u c t i o n o f 2 0 0 u . M D I D S , a p p l i e d i n c o m b i n a t i o n w i t h 0 [ C l " ] 0 p e r f u s i o n , a b o l i s h e d t h e 0 [ C l " ] 0 - i n d u c e d p H j r i s e ( n = 3 ; F i g u r e 1 4 A ) . F u r t h e r m o r e , t h e a p p l i c a t i o n o f D I D S 5 m i n u t e s a f t e r t h e r e m o v a l o f [ C l " ] 0 p r e v e n t e d t h e s u s t a i n e d a l k a l i n i z a t i o n a s s o c i a t e d w i t h t h e a b s e n c e o f [ C l " ] 0 a n d r e s u l t e d i n a d e c l i n e o f p H j b a c k t o w a r d s s t e a d y s t a t e l e v e l s d e s p i t e c o n t i n u e d p e r f u s i o n w i t h C l " - f r e e m e d i u m ( n = 4 ; F i g u r e 1 4 B ) . W h e n [ C l " ] 0 w a s r e - i n t r o d u c e d t o t h e p e r f u s i o n s o l u t i o n i n t h e c o n t i n u e d p r e s e n c e o f 2 0 0 u M D I D S , p H j c o n t i n u e d t o f a l l , o v e r s h o o t i n g s t e a d y - s t a t e p H j l e v e l s t o r e s t - 0 . 0 5 p H u n i t s b e l o w n o r m a l l e v e l s . O n c e t h e D I D S w a s r e m o v e d , p H j s l o w l y r e t u r n e d t o b a s e l i n e l e v e l s . 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 t s 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 ( T a b l e 5 , a n d F i g u r e 1 5 ) . 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 H C O 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 i n w h i c h s t e a d y - s t a t e p H j w a s s i g n i f i c a n t l y r e d u c e d b y 2 0 0 u M D I D S ( n = 3 ; 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" e x c h a n g e r m a y b e p r e s e n t , s t e a d y - s t a t e p H ; i s p r i m a r i l y g o v e r n e d b y t h e a c t i v i t y o f t h e N a + / H + e x c h a n g e r . 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 e e 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 w e r e i n v e s t i g a t e d a t 3 7 ° C . I n t e r e s t i n g l y , t h e n e t a l k a l i n i z a t i o n t h a t o c c u r r e d o n t h e t r a n s i t i o n 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 4 6 3 7 ° C ( n = 1 3 ; F i g u r e 1 6 ) . I n f a c t , t h e s t e a d y - s t a t e p H ; i n H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m a t t h i s t e m p e r a t u r e w a s s i g n i f i c a n t l y l o w e r t h a n t h e o b s e r v e d l e v e l u n d e r H C 0 3 " - f r e e H E P E S - b u f f e r e d c o n d i t i o n s ( T a b l e 5 ) . I n c o n t r a s t t o t h o s e 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 ( s e e F i g u r e 6 B ) , t h e a p p l i c a t i o n 2 0 0 p M D I D S a t 3 7 ° C d i d n o t a f f e c t t h e p H ; r e s p o n s e t o t h e i n t r o d u c t i o n o f 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 ( F i g u r e 1 6 ) , a g a i n i n d i c a t i n g t h e r e l a t i v e u n i m p o r t a n c e o f H C 0 3 7 C 1 ~ 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 o f s t e a d y - s t a t e p H ; a t t h i s t e m p e r a t u r e . N a + - d e p e n d e n t o r - i n d e p e n d e n t a n i o n e x c h a n g e : B y r e s p o n d i n g t o c h a n g e s i n t h e e x t r a c e l l u l a r c o n c e n t r a t i o n s o f b o t h H C 0 3 " a n d C l " , e s p e c i a l l y a t r o o m t e m p e r a t u r e , t h e n e u r o n e s e m p l o y e d i n t h e s e e x p e r i m e n t s i n d i c a t e d t h e i r a b i l i t y t o r e g u l a t e p H ; t h r o u g h a n i o n e x c h a n g e . T o d e t e r m i n e w h e t h e r t h e s u s p e c t e d H C 0 3 7 C 1 " e x c h a n g e r p r e s e n t o n t h e s e n e u r o n e s w a s d e p e n d e n t o n e x t r a c e l l u l a r N a + , a n e x p e r i m e n t w a s p e r f o r m e d i n t h e a b s e n c e o f H C 0 3 " a t r o o m t e m p e r a t u r e i n w h i c h [ N a + ] 0 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 ) . A s i l l u s t r a t e d i n F i g u r e 1 7 , t h i s c a u s e d p H j t o f a l l , b u t t h e s u b s e q u e n t i n t r o d u c t i o n o f H C 0 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 o f [ N a + ] 0 ( n = 3 ) . S i n c e p H j r e c o v e r e d i n t h e p r e s e n c e o f H C 0 3 " a n d i n t h e a b s e n c e [ N a + ] 0 , t h i s r e s u l t s u g g e s t s t h a t N a + - i n d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e w a s b e i n g u t i l i z e d b y t h e n e u r o n e s t o r e g u l a t e p H j b a c k t o r e s t i n g l e v e l s . U s i n g a n a l t e r n a t i v e a p p r o a c h , e x t r a c e l l u l a r N a + w a s a g a i n e l i m i n a t e d b u t n o w f r o m H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m a t 3 7 ° C (solution 13, T a b l e 3 ) . A f t e r l e t t i n g p H j f a l l t o a p l a t e a u , p e r f u s a t e d e v o i d o f [ C l " ] 0 a n d [ N a + ] 0 (solution 15, T a b l e 1 5 ) w a s i n t r o d u c e d , w h i c h c a u s e d a 0 . 1 4 ± 0 . 0 3 i n c r e a s e i n p H j ( n = 3 ; F i g u r e 1 8 ) . T h i s 0 [ C l " ] 0 - i n d u c e d 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 i n t h e a b s e n c e o f e x t e r n a l N a + w a s s i m i l a r t o , t h o u g h s m a l l e r t h a n , t h e 0 [ C l " ] 0 - i n d u c e d a l k a l i n i z a t i o n o b s e r v e d i n t h e c o r r e s p o n d i n g e x p e r i m e n t p e r f o r m e d i n t h e p r e s e n c e o f [ N a + ] 0 ( s e e F i g u r e 1 4 A ) . I n t h e a b s e n c e o f [ N a + ] 0 , t h e r e t u r n o f [ C l " ] 0 p r o d u c e d a b r i e f a c i d i f i c a t i o n 4 7 f o l l o w e d b y a g r a d u a l p H j r e c o v e r y t o w a r d s s t e a d y - s t a t e l e v e l s . T h i s s l o w r e c o v e r y w a s p r o b a b l y t h e r e s u l t o f t h e a c t i v a t i o n o f N a + - i n 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 . 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 t h e s e n e u r o n e s a r e a b l e t o r e g u l a t e p H j u t i l i z i n g a N a + - i n d e p e n d e n t f o r m o f t h e H C 0 3 7 C 1 " e x c h a n g e r . M o d u l a t i o n o f p H j b y s h i f t s i n p H 0 a n d t h e a p p l i c a t i o n o f w e a k a c i d s a n d b a s e s : T h e s t e a d y - s t a t e p H j o f t h e n e u r o n e s p e r f u s e d w i t h m e d i a c o n t a i n i n g H C 0 3 ~ e q u i l i b r a t e d w i t h 5 % CO2 i n b a l a n c e a i r a t 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 t h e p H o f t h e e x t r a c e l l u l a r e n v i r o n m e n t . I n c r e a s i n g p H 0 f r o m 7 . 3 5 t o 7 . 7 5 a n d t h e n 8 . 0 2 c a u s e d p H j t o r e a c h l e v e l s o f 7 . 4 1 ± 0 . 0 1 a n d 7 . 5 4 ± 0 . 0 1 , r e s p e c t i v e l y ( n = 3 ; F i g u r e 1 9 A ) . R e d u c i n g p H 0 b e l o w 7 . 3 5 r e s u l t e d i n a d e c r e a s e i n p H ; b e l o w n o r m a l r e s t i n g l e v e l s : w h e n p H 0 w a s l o w e r e d t o 7 . 0 2 a n d 6 . 5 6 , p H ; s t a b i l i z e d a t 6 . 9 0 ± 0 . 0 7 a n d 6 . 5 3 ± 0 . 0 1 , r e s p e c t i v e l y ( n = 3 ) . A s s h o w n i n F i g u r e 1 9 B , a 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 r e l a t i o n s h i p b e t w e e n p H 0 a n d p H j y i e l d e d t h e f o l l o w i n g r e l a t i o n s h i p : p H j = 1 . 9 9 0 + 0 . 6 9 9 x p H o (Equation 19) T h e m o d u l a t i o n o f p H 0 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 H C 0 3 " h a d a s i m i l a r e f f e c t o n p H j ( d a t a n o t s h o w n ) . U n d e r t h e s e c o n d i t i o n s , t h e r e l a t i o n s h i p r e p r e s e n t i n g t h e d e p e n d e n c e o f p H j o n p H 0 w a s : p H , = 1 . 2 4 0 + 0 . 8 0 7 x p H o (Equation 20) T h e s e r e s u l t s s u g g e s t t h a t p H j i s s t e e p l y d e p e n d e n t o n p H 0 a n d t h a t p H j i s n o t r e g u l a t e d b a c k t o n o r m a l s t e a d y - s t a t e v a l u e s u n t i l p H 0 i s n o r m a l i z e d . T h e e x t r a c e l l u l a r 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 a t c o n s t a n t p H 0 h a s b e e n s h o w n t o a l t e r p H j ( S h a r p a n d T h o m a s , 1 9 8 1 ; R o o s a n d B o r o n , 1 9 8 1 ) . A c c o r d i n g l y , 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 t o h i p p o c a m p a l 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 i n H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m i n o r d e r t o i n v e s t i g a t e t h e i r a b i l i t y t o m o d u l a t e p H j a t a c o n s t a n t p H 0 . 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 9, T a b l e 2 ) , t h e u n d i s s o c i a t e d f o r m o f t h e a c i d r e a d i l y c r o s s e d t h e c e l l m e m b r a n e , 4 8 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 t o i t s n e g a t i v e c h a r g e , r e m a i n e d r e l a t i v e l y m e m b r a n e i m p e r m e a n t . 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 t o i t s p K a t o r e l e a s e p r o t o n s , t h u s a c i d i f y i n g t h e c e l l ' s i n t e r i o r ( F i g u r e 2 0 A ) . S i n c e t h e a m o u n t o f a c i d e n t e r i n g t h e c e l l s w a s m i n i m a l w h e n c o m p a r e d t o t h e t o t a l e x t r a c e l l u l a r p r o p i o n a t e c o n c e n t r a t i o n , p H j w a s l o w e r e d w h i l e p H 0 w a s m a i n t a i n e d a t 7 . 3 2 . T h e i n i t i a l p H j d e c r e a s e w a s 0 . 2 1 ± 0 . 0 5 p H u n i t s ( n = 3 ) , a f t e r w h i c h p H j g r a d u a l l y i n c r e a s e d t o w a r d s n o r m a l s t e a d y - s t a t e l e v e l s d u e e i t h e r t o t h e s l o w p e r m e a t i o n o f 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 t h r o u g h t h e m e m b r a n e o r t h e a c t i v a t i o n o f a c i d e x t r u s i o n m e c h a n i s m s . T h e l a t t e r i s t h e 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 t h e 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 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 f o l l o w e d b y g r a d u a l r e t u r n t o n o r m a l s t e a d y - s t a t e p H j l e v e l s . F i g u r e 2 0 B i l l u s t r a t e s t h e o p p o s i t e c h a n g e i n p H ; a t 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 t h e 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 e x t r a c e l l u l a r T M A c a u s e d a i m m e d i a t e 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 o f 0 . 3 8 ± 0 . 0 4 p H u n i t s w h i c h g r a d u a l l y r e t u r n e d t o w a r d s b a s e l i n e ( n = 3 ) . O n c e r e m o v e d , t h e i n t r a c e l l u l a r m i l i e u w a s b r i e f l y a c i d i f i e d f o l l o w e d b y a p H j r e c o v e r y t o r e s t i n g b a s e l i n e l e v e l s . 4 9 T a b l e 5 : S t e a d y - s t a t e p H ; i n H C 0 3 " - 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 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 n p H j c a u s e d b y e x p o s u r e t o t h e e x p e r i m e n t a l s o l u t i o n s i n d i c a t e d . T e m p p H j A p H j n H E P E S b u f f e r : (solution 1) r o o m 6 . 8 5 ± 0 . 0 4 2 5 H C 0 3 - / C 0 2 b u f f e r : (solution 6) r o o m 7 . 1 5 ± 0 . 0 3 2 2 0 [ C l " ] 0 (solution 8) r o o m 0 . 2 8 ± 0 . 0 4 2 2 0 0 p M D I D S ( i n solution 6) r o o m - 0 . 0 8 ± 0 . 0 4 3 H E P E S b u f f e r : (solution 1) 3 7 ° C 7 . 2 3 ± 0 . 0 3 2 9 0 [ N a + ] 0 (solution 2) 3 7 ° C - 0 . 5 3 ± 0 . 0 5 5 0 [ C T ] 0 (solution 3) 3 7 ° C 0 . 0 0 ± 0 . 0 2 3 5 0 p M E I P A ( i n solution 1) 3 7 ° C 0 . 0 1 ± 0 . 0 1 3 H C 0 3 - / C 0 2 b u f f e r : (solution 12) 3 7 ° C 7 . 1 3 ± 0 . 0 1 4 4 0 [ N a + ] 0 (solution 13) 3 7 ° C - 0 . 6 5 ± 0 . 0 4 8 0 [ C T ] 0 (solution 14) 3 7 ° C 0 . 1 9 ± 0 . 0 1 5 5 0 p M E I P A ( i n solution 12) 3 7 ° C 0 . 0 1 ± 0 . 0 2 3 2 0 0 p M D I D S ( i n solution 12) 3 7 ° C - 0 . 0 1 ± 0 . 0 1 3 0 [ C l " ] 0 + 2 0 0 p M D I D S (solution 14) 3 7 ° C 0 . 0 1 ± 0 . 0 1 2 S o l u t i o n r e c i p e s a r e r e f e r e d t o i n p a r e n t h e s e s ( s e e T a b l e s 1 t o 3 ) . T h e e x p e r i m e n t a l t e m p e r a t u r e ( T e m p ) w a s e i t h e r r o o m t e m p e r a t u r e ( 1 8 - 2 2 ° C ) o r 3 7 ° C . p H j i s t h e s t e a d y - s t a t e p H , u n d e r t h e l i s t e d 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 i s t h e c h a n g e i n p H j ( i n p H u n i t s ) c a u s e d b y e x p o s u r e t o t h e i n d i c a t e d e x p e r i m e n t a l s o l u t i o n s . V a l u e s a r e r e p o r t e d a s 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 . 5 0 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 HC0 3 " (solution 12, pH 7.36). The mean steady-state pHj under each 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. 5 1 ^ — i ' i I I I I r-1 1 , " i 1 I I I I I 1 =1 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 5 2 Figure 4. Effect of 0 [Cl"] 0 on steady-state pHj in the presence of H C O 3 " at room temperature. T h e r e m o v a l o f [ C l " ] 0 (solution 8) a t a c o n s t a n t p H 0 ( 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 b a r a b o v e t h e t r a c e r e s u l t e d i n a n - 0 . 3 p H u n i t i n c r e a s e i n r e s t i n g p H j ( n = 2 ) . p H j r e t u r n e d t o 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 ~ ] 0 . S h o w n a l s o i s a o n e p o i n t c a l i b r a t i o n w i t h 1 0 u M n i g e r i c i n a t p H 7 . 0 0 . T h e t r a c e i s 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 0 c e l l s 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 . 53 0 10 20 30 40 50 60 70 80 90 Time (minutes) 54 Figure 5. Effect of DIDS on steady-state pHj in the presence of H C O 3 - at room temperature. T h e a d d i t i o n 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 t r a c e c a u s e d a n ~ 0 . 1 p H u n i t 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 ( n = 3 ) , w h i l e p H 0 w a s m a i n t a i n e d a t 7 . 3 2 . p H j w a s r e s t o r e d t o 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 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 3 0 c e l l s 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 . 5 5 10 20 30 Time (minutes) 40 50 60 5 6 Figure 6. Steady-state pHj in the presence and absence of HC0 3 "/C02 at room temperature. 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 0 7 . 3 2 ) t o H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m (solution 6, p H 0 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 t r a n s i t i o n b a c k i n t o 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 H C 0 3 " / C 0 2 - b u f f e r e d m e d i u m w a s a b o l i s h e d b y t h e p r e s e n c e 2 0 0 p M D I D S ( n = 5 ) . R a t h e r , t h e r e w a s a n a c i d i f i c a t i o n f o l l o w e d b y a s l o w r e c o v e r y . E a c h t r a c e , r e c o r d e d f r o m s e p a r a t e c o v e r s l i p s , i s d a t a o b t a i n e d f r o m 1 0 c e l l s s i m u l t a n e o u s l y . T i m e ( m i n u t e s ) 58 Figure 7. Effect of 0 [Na+]0 on steady-state pHj in the absence of H C 0 3 " at 37°C. The replacement of extracellular N a + with N M D G + {solution 2) at p H 0 7.35 caused an ~0.5 pH 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 pH 7.00. The trace is a mean of data obtained from 6 cells recorded on a single coverslip. 5 9 Time (minutes) 6 0 F i g u r e 8 . E f f e c t o f 0 [ C l ] 0 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 0 3 " a t 3 7 ° C . 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 0 7 . 3 3 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 t r a c e d i d n o t c h a n g e r e s t i n g p H j l e v e l s ( n = 3 ) . T h e 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 2 6 c e l l s 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 . 6 1 Time (minutes) 6 2 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 0 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 a l t e r r e s t i n g p H j l e v e l s ( n = 3 ) . T h e t r a c e i s 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 6 c e l l s 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 .  64 Figure 10. Combined effect of 0 [ N a + ] 0 and E I P A on steady-state pHj in the absence 0fHCO 3 - at 37°C. The application of 50 pM EIPA 5 minutes after the removal of extracellular Na + (solution 2) did not reverse the fall in pHj caused by 0 [Na+]0, nor the return to resting pHj levels after the re-introduction of [Na+]0 (n-3). Throughout the experiment, pH 0 was maintained at 7.33. The trace is a mean of data obtained from 23 cells recorded on a single coverslip. 65 T i m e ( m i n u t e s ) 6 6 F i g u r e 11. E f f e c t o f M G C M A a n d H O E 6 9 4 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 0 3 - a t 3 7 ° C . A . T h e a p p l i c a t i o n o f 1 0 0 u M M G C M A a t p H 0 7 . 3 6 d i d n o t s i g n i f i c a n t l y c h a n g e s t e a d y - s t a t e p H j ( n = 3 ) . B . S i m i l a r l y , t h e a p p l i c a t i o n o f 1 0 0 u M H O E 6 9 4 ( p H 0 7 . 3 8 ) h a d n o e f f e c t 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 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 8 c e l l s , w h e r e a s t r a c e B 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 1 3 c e l l s , e a c h 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 . Time (minutes) 68 Figure 12. Effect of 0 [Na +] 0 on steady-state pHj in the presence of HCO3- at 37°C. The removal of extracellular N a + (solution 13, p H 0 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 Na + . Also shown is a one point calibration with 10 pM nigericin at pH 7.00. The trace is a mean of data obtained from 35 cells recorded on a single coverslip. 6 9 10 20 30 40 50 60 70 80 T i m e ( m i n u t e s ) 70 Figure 13. Effect of E I P A on steady-state pHj in the presence of H C O 3 - at 3 7 ° C . The addition of 50 uM EIPA to solution 12 at p H 0 7.32 did not significantly alter resting pHj levels (n=3). This result was also observed in the absence of H C 0 3 " as shown in Figure 9. The trace is a mean of data simultaneously obtained from 30 cells recorded on a single coverslip. 71 Time (minutes) 72 Figure 14. Effect of 0 [ C l ] 0 , and the combined effect of 0 [ C T ] 0 plus DIDS on steady-state pHj in the presence of H C O 3 - at 3 7 ° C . A . The removal of extracellular Cl" (solution 14) at p H 0 7.36 produced an -0.20 pH unit intracellular alkalinization (n=5). This 0 [Cl"]0-induced pHj increase was completely inhibited by 200 uM DIDS (n=3). This trace is a mean of data obtained from 16 cells recorded on a single coverslip. B. At p H 0 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. 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 0 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. 75 T i m e ( m i n u t e s ) 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 0 7.35) to H C O 3 V C O 2 - buffered medium (solution 12, p H 0 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 HC03"-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. 7 7 Time (minutes) 78 Figure 17. Effect of H C 0 3 - / C 0 2 on steady-state pHj during 0 [Na + ] 0 perfusion at room temperature. [Na + ] 0 was removed from the HCC^'-free buffered media (solution 2) at p H 0 7.35, resulting in an immediate fall in pHj. The addition of H C 0 3 " during the period of continued 0 [Na + ] 0 (solution 7) perfusion caused pH, to gradually recover (n=3). pHj immediately returned to normal levels when [Na + ] 0 was re-introduced to the perfusion medium. The trace is a mean of data obtained from 34 cells recorded on a single coverslip. 7 9 80 Figure 18. Effect of 0 [Cl"]0 during 0 [Na +] 0 perfusion on steady-state pHj in the presence of H C 0 3 " at 37°C. The removal of external N a + from the HC03"/C02-buffered medium at 37°C (solution 13, p H 0 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 +] 0 (solution 15) caused an -0.15 pH unit increase in pHj (n=3). The re-introduction of [Cl"]0 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. 8 1 Time (minutes) 82 Figure 19. Effect of changes in p H 0 on steady-state pHj in the presence of H C O 3 " at 37°C. A . I n c r e a s i n g p H 0 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 0 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 p H 0 . 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 i s : pHj = 1.99 + 0.699xpHo (R 2 = 0.978, n=3 c o v e r s l i p s )  8 4 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 0 ( 7 . 3 0 ) c a u s e d 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 o f - 0 . 2 0 p H u n i t s ( n = 3 ) f o l l o w e d b y a g r a d u a l r e c o v e r y t o b a s e l i n e l e v e l s . T h e r e m o v a l o f p r o p i o n a t e f r o m t h e e x t r a c e l l u l a r 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 0 ( 7 . 3 1 ) c a u s e d 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 l k a l i n i z a t i o n o f - 0 . 4 0 p H u n i t s ( n = 3 ) f o l l o w e d b y a s l o w r e c o v e r y t o b a s e l i n e l e v e l s . T h e r e m o v a l o f T M A c a u s e d 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 f o l l o w e d b y a r 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 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 1 2 c e l l s , w h e r e a s t r a c e B 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 2 0 c e l l s . Time (minutes) 8 6 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 T h e i n v e s t i g a t i o n o f p H j r e g u l a t o r y m e c h a n i s m s w a s e x p a n d e d b y i n d u c i n g 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 , w h i l e m a i n t a i n i n g a c o n s t a n t p H 0 , a n d s t u d y i n g t h e s u b s e q u e n t r e c o v e r y . T h e e x a m i n a t i o n o f a c i d l o a d r e c o v e r i e s p r o v i d e s a d d i t i o n a l i n f o r m a t i o n o n m e c h a n i s m s r e g u l a t i n g p H j , s i n c e e x c e s s i v e i n t r a c e l l u l a r p r o t o n s w i l l a c t i v a t e a c i d e x t r u s i o n m e c h a n i s m s ( R o o s a n d B o r o n , 1 9 8 1 ) . A c i d t r a n s i e n t s w e r e p r o d u c e d u s i n g t h e N H 4 + - p r e p u l s e t e c h n i q u e ( B o r o n a n d D e W e e r , 1 9 7 6 ) , i n w h i c h t h e n e u r o n e s w e r e e x p o s e d t o N H 4 + t h r o u g h t h e a d d i t i o n o f 2 0 m M N H 4 C 1 t o t h e p e r f u s i o n s o l u t i o n (solution 4, T a b l e 1 ; solution 11, T a b l e 2 ; solution 16, T a b l e 3 ) . A s s h o w n i n F i g u r e 2 1 , t h i s e x p o s u r e c a u s e s 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 l k a l i n i z a t i o n d u e t o t h e p a s s i v e i n f l u x o f N H 3 , t h e d i s s o c i a t e d f o r m o f N H 4 + , a n d i t s i m p e n d i n g h y d r a t i o n t o f o r m N H 4 + a n d O H ' . T h e m e m b r a n e e x h i b i t s a s l i g h t p e r m e a b i l i t y t o N H 4 + , a n d t h u s t h e i n i t i a l p H , r i s e i s s l o w l y d a m p e n e d b y t h e i n f l u x o f e x t r a c e l l u l a r N H 4 + d r i v e n b y a n i n w a r d l y - d i r e c t e d e l e c t r o c h e m i c a l g r a d i e n t ( B o r o n a n d D e W e e r , 1 9 7 6 ) . T h e r e m o v a l o f t h e e x t e r n a l N H 4 C 1 , 3 m i n u t e s a f t e r i t s o r i g i n a l a p p l i c a t i o n , r e s u l t s i n a n e x o d u s o f t h e h i g h l y p e r m e a b l e N H 3 , l e a v i n g b e h i n d s i g n i f i c a n t c o n c e n t r a t i o n s o f N H 4 + , t r a p p e d i n t h e c e l l s b y t h e o p p o s i n g e l e c t r i c a l g r a d i e n t g e n e r a t e d b y t h e m e m b r a n e p o t e n t i a l . T h e d i s s o c i a t i o n o f i n t r a c e l l u l a r N H 4 + i n t o N H 3 l i b e r a t e s p r o t o n s , t h u s f o r c i n g p H j t o f a l l b e l o w i t s i n i t i a l r e s t i n g v a l u e . T h e r e c o v e r y f r o m t h i s i m p o s e d a c i d i f i c a t i o n b a c k t o s t e a d y - s t a t e p H ; l e v e l s i s 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 t h e m e c h a n i s m s i n v o l v e d i n p H j r e g u l a t i o n , a s f i r s t 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 S i e s j o ( 1 9 7 1 ) . D a t a g e n e r a t e d f r o m t h e 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 . T h e i n i t i a l r a t e o f p H ; r e c o v e r y t o r e s t i n g l e v e l s w a s q u a n t i f i e d 1 0 s e c o n d s a f t e r t h e p e a k a c i d i f i c a t i o n . T h e i n s t a n t a n e o u s r e c o v e r y r a t e s w e r e a l s o d e t e r m i n e d a t t 5 0 a n d t 8 0 , w h i c h a r e d e f i n e d a s t h e t i m e a t w h i c h p H j r e c o v e r e d t o 5 0 % a n d 8 0 % o f i t s p r e - a c i d l o a d l e v e l , r e s p e c t i v e l y . p H j a t t 5 Q w a s c a l c u l a t e d b y t a k i n g t h e r e s t i n g p H j b e f o r e t h e a p p l i c a t i o n o f 87 N H 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 8 0 was determined by subtracting 20% of the NH 4 +-induced net pHj decrease from the 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 pH units (n=7) in the absence and presence of HC0 3 ~, respectively. These values markedly 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 4 + exposure in solutions buffered with HCO37CO2. 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. This phenomenon is graphically depicted in Figure 22A. 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. 8 8 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 3 " {solution 1, T a b l e 1 ) r e c o v e r e d f r o m a n N H 4 + - i n d u c e d a c i d i f i c a t i o n a t a n m a x i m u m i n i t i a l r a t e o f 1 . 3 1 x I O " 3 p H u n i t s / s e c o n d ( n = 6 ; 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 HCO3" (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 s i g n i f i c a n t i n c r e a s e i n t h e a c i d e x t r u s i o n r a t e 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 t h e r e s t o r a t i o n . U n d e r t h e s e c o n d i t i o n s , t h e i n i t i a l r a t e o f 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 i f i c a t i o n w a s 2 . 5 5 x I O - 3 p H u n i t s / s e c o n d ( n = 7 ; 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 e a r l i e r r e g a r d i n g t h e r e g u l a t i o n o f s t e a d y - s t a t e p H j 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 ) , F i g u r e 2 3 a l s o i l l u s t r a t e s t h a t t h e r e s t o r a t i o n o f p H j f r o m a n a c i d l o a d i n 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 l e v e l f o u n d w h e n t h e n e u r o n e s w e r e b e i n g p e r f u s e d w i t h H E P E S b u f f e r e d m e d i u m . I n i t i a l 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 i n t h e p r e s e n c e o f H C 0 3 " w e r e 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 ; r e c o v e r y f o l l o w i n g a n a c i d l o a d . F i g u r e 2 4 i l l u 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 2 0 0 p M D I D S d u r i n g r e c o v e r y c a u s e d a s i g n i f i c a n t r e d u c t i o n i n t h e r a t e o f a c i d e f f l u x ( n = 3 ) , a s 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 o f s t e a d y - s t a t e p H j a t r o o m t e m p e r a t u r e . T h e i n i t i a l r a t e f e l l f r o m 2 . 5 5 x 1 0 ~ 3 p H u n i t s / s e c o n d t o 0 . 9 5 x 1 0 " 3 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 o f 2 0 0 p M D I D S w a s a l s o n o t a b l y r e d u c e d f r o m a c o n t r o l l e v e l o f 1 . 3 8 x 1 0 - 3 p H 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 1 0 - 3 p H u n i t s / s e c o n d ( T a b l e 6 , r o w d). A q u a l i t a t i v e l y s i m i l a r e f f e c t w a s o b s e r v e d i n t h e t 8 0 r a t e ( T a b l e 6 , r o w d). A s D I D S i s a n i n h i b i t o r o f H C 0 3 7 C 1 " e x c h a n g e ( T h o m a s , 1 9 7 6 a ) , t h e n t h e d e p l e t i o n o f [ C l " ] j d u r i n g p H j r e c o v e r y s h o u l d s i m i l a r l y a l t e r t h e r a t e o f a c i d e x t r u s i o n . F i g u r e 2 5 A d e p i c t s t h e a b i l i t y o f t h e n e u r o n e s t o r e c o v e r f r o m a n a c i d l o a d w h i l e b e i n g p e r f u s e d w i t h a [ C l " ] 0 - f r e e s o l u t i o n (solution 8, T a b l e 2 ) . U n d e r t h e s e 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 i n f a c t i n c r e a s e d t o 3 . 1 4 x 1 0 " 3 p H u n i t s / s e c o n d ( 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 8 9 0 [ C l - ] 0 - i n d u c e d a c c e l e r a t i o n o f t h e H C 0 3 7 C 1 - e x c h a n g e r . T h i s r e s u l t a l s o s u g g e s t s t h a t t h e d u r a t i o n o f 0 [ C l " ] 0 p e r f u s i o n w a s n o t l o n g e n o u g h t o s u f f i c i e n t l y d e p l e t e i n t r a c e l l u l a r C l " s t o r e s , w h i c h w o u l d h a v e l i k e l y p r o d u c e d a s i m i l a r d e c r e a s e i n t h e p H j r e c o v e r y r a t e 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 s u p p o r t e d b y t h e e x p e r i m e n t s h o w n i n F i g u r e 2 5 B i n w h i c h t h e r a p i d r e c o v e r y s e e n o n r e m o v i n g e x t r a c e l l u l a r C l " w a s h i n d e r e d w h e n t h e n e u r o n e s w e r e s i m u l t a n e o u s l y e x p o s e d t o 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 t h e i n i t i a l r e c o v e r y r a t e t o 1 . 1 8 x 1 0 " 3 p H u n i t s / s e c o n d ( 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 1 0 " 3 p H 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 i n d u c e d 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 s . T h e i n a b i l i t y o f 2 0 0 u M D I D S t o c o m p l e t e l y b l o c k p H j r e s t o r a t i o n ( s e e F i g u r e 2 4 ) i n d i c a t e s t h a t o t h e r a c i d t r a n s p o r t e r s , s u c h a s 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 e x t r u s i o n m e c h a n i s m , 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 a t r o o m t e m p e r a t u r e . T h e p a r t i c i p a t i o n o f t h i s l a t t e r m e c h a n i s m i n t h e r e c o v e r y o f p H j a f t e r a n i m p o s e d a c i d i f i c a t i o n i s d e s c r i b e d i n d e t a i l a t 3 7 ° C ( s e e b e l o w ) . 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 HCO3" (solution 1, T a b l e 1 ) , t h e n e u r o n e s r e c o v e r e d f r o m t h e a c i d i f y i n g N H 4 + p r e p u l s e a t a n i n i t i a l r a t e o f 5 . 5 6 x 1 0 " 3 p H u n i t s / s e c o n d ( n = 1 2 ; T a b l e 6 , r o w / ; F i g u r e 2 6 ) . T h i s i s h i g h e r t h a n t h e i n i t i a l r a t e o f 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 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 6 , r o w a ) . C o r r e s p o n d i n g v a l u e s f o r t h e t 5 g a n d t g 0 r a t e s w e r e a l s o h i g h e r a t 3 7 ° C t h a n a t r o o m t e m p e r a t u r e ( T a b l e 6 ) . p H j r e s t o r a t i o n w a s a b o l i s h e d w h e n [ N a + ] 0 w a s r e m o v e d f r o m t h e 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 ( n = 2 ; F i g u r e 2 7 ) , a s 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 N a + - 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 - s t a t e p H j ( s e e a b o v e ) . H o w e v e r , s t u d i e d o n 6 c e l l p o p u l a t i o n s , t h e a p p l i c a t i o n o f 5 0 u . M E I P A d i d n o t i n f l u e n c e t h e a b i l i t y o f t h e n e u r o n e s t o r e c o v e r f r o m 9 0 t h e i n d u c e d 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 ( F i g u r e 2 8 ; T a b l e 6 , r o w h). T w o o t h e r c o m p o u n d s w e r e t e s t e d f o r t h e i r a b i l i t y t o i n h i b i t 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 v i a b l o c k a d e o f t h e p u t a t i v e N a + / H + e x c h a n g e r p r e s e n t o n t h e s e n e u r o n e s . T h e a p p l i c a t i o n o f 1 0 0 p M M G C M A ( n = 3 ) o r 1 0 0 p M H O E 6 9 4 ( n = 3 ) f a i l e d t o a f f e c t t h e r a t e o f s t e a d y - s t a t e p H j r e s t o r a t i o n f r o m a n N H 4 + - i n d u c e d a c i d i f i c a t i o n ( d a t a n o t s h o w n ) . S i m i l a r l y , a s i l l u s t r a t e d i n F i g u r e 2 9 , 2 0 0 p M D I D S d i d n o t i n f l u e n c e t h e r a t e 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 t h i s c o n t r o l e x p e r i m e n t w a s 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 D I D S - s e n s i t i v e a n i o n e x c h a n g e r , i t 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 s p u r i o u s e f f e c t s o n p H j r e c o v e r y . T h o u g h t h e i n s t a n t a n e o u s i n i t i a l r a t e , t 5 0 r a t e , a n d t g n r a t e f o r t h e r e c o v e r y f r o m a n a c i d l o a d i n t h e p r e s e n c e o f D I D S s e e m e d t o d e c l i n e i n c o m p a r i s o n t o 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 u n d e r 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 ) . T h e s i m i l a r i t y b e t w e e n a c i d l o a d 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 i s b e t t e r d e p i c t e d b y t h e a c t u a l 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). T h e f a c t 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 p r e m i s e t h a t s t i l b e n e d e r i v a t i v e s o n l y i n h i b i t H C 0 3 " - d e p e n d e n t a c i d t r a n s p o r t e r s ( T h o m a s , 1 9 7 6 a ; R u s s e l l a n d B o r o n , 1 9 7 6 ) . 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 3 7 ° C , h i p p o c a m p a l n e u r o n e s r e c o v e r 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 u t i l i z i n g a N a + - d e p e n d e n t , E I P A - i n s e n s i t i v e a c i d e x t r u s i o n m e c h a n i s m . I n t e r e s t i n g l y , 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 p e r f u s i n g 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 n o t a l t e r t h e r a t e o f a c i d l o a d r e c o v e r y a t 3 7 ° C . T h e i n i t i a l r a t e o f p H ; r e s t o r a t i o n i n t h e p r e s e n c e o f HCO3" a t 3 7 ° C w a s 4 . 7 7 x 1 0 " 3 p H u n i t s / s e c o n d ( n = 1 6 ; T a b l e 6 , r o w j). I n a d d i t i o n , t h e a b i l i t y o f t h e n e u r o n e s t o r e c o v e r 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 h i l e b e i n g 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 n e a r l y h a l t e d b y 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 + ( n = 3 ; 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, HC03--independent mechanism governing pHj regulation in these cells at 37°C. However, in the absence of [Na+]0 (solution 13, Table 3) a small amount of pHj recovery occurred (see Figure 30), presumably caused by pHj regulatory mechanisms not requiring extracellular Na + . 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 H C O 3 7 O " exchange towards the regulation of pHj at 37°C, as already suggested by results from the steady- state pHj experiments. In the absence of [Cl"]0, pHj did however recover to a slightly 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"3 pH units/second with corresponding reductions in the t 5 0 and t 8 0 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 H C O 3 7 C I " exchange to pH; recovery from an NH4+-induced acid load at 37°C. 9 2 T o d e t e r m i n e w h e t h e r t h e s e n s i t i v i t y t o D I D S w a s p e r h a p s d e p e n d e n t o n t h e l e v e l o f 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 d u c e d b y t h e N H 4 + p r e p u l s e , t h e p r e l o a d p H j w a s r e d u c e d 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 + - p r e p u l s e a t p H 0 6 . 8 (solution 19, T a b l e 4 ) , p H j t y p i c a l l y f e l l t o a l e v e l o f 6 . 4 ( F i g u r e 3 4 ) , a v a l u e t h a t w a s a p p r o x i m a t e l y 0 . 2 5 p H u n i t s l o w e r t h a n w a s t y p i c a l l y a c h i e v e d w h e n p H 0 r e s t e d a t 7 . 3 6 . T h e i n i t i a l , t 5 0 , a n d t 8 0 r a t e s 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 d n o t i n h i b i t t h e r a t e o f p H j r e s t o r a t i o n w h e n p H 0 w a s m a i n t a i n e d a t 6 . 8 ( n = 2 ; 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 i n a m a n n e r t h a t i s i n d e p e n d e n t o f t h e a c t i v i t y o f H C 0 3 7 C 1 " e x c h a n g e . I n s u m m a r y , t h e e x p e r i m e n t s o u t l i n e d a b o v e h a v e d e m o n s t r a t e d t h a t t h e a c t i v i t i e s o f t h e a c i d e x t r u s i o n m e c h a n i s m s p r e s e n t o n c u l t u r e d f o e t a l 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 a r e d e p e n d e n t o n t e m p e r a t u r e . A t r o o m t e m p e r a t u r e , b o t h 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 e x t r u s i o n m e c h a n i s m ( p o s s i b l y a N a + / H + e x c h a n g e r ) a n d a H C O 3 V C I " e x c h a n g e r h a v e b e e n s h o w n t o b e i n v o l v e d i n r e s t o r i n g p H j b a c k t o r e s t i n g l e v e l s f o l l o w i n g a n i m p o s e d a c i d l o a d . P r e s u m a b l y , t h i s a n i o n e x c h a n g e r i s t h e s a m e N a + - i n d e p e n d e n t H C O 3 V C I " c o u n t e r - t r a n s p o r t e r r e g u l a t i n g s t e a d y - s t a t e p H j a t r o o m t e m p e r a t u r e . H o w e v e r , a t 3 7 ° C , t h e d o m i n a n t m e c h a n i s m t h a t a c t s t o r e t u r n p H , t o b a s e l i n e v a l u e s a f t e r a n a p p l i e d a c i d i f i c a t i o n a p p e a r s t o b e a N a + - d e p e n d e n t , HCO3"- i n d e p e n d e n t a c i d e x t r u d e r . T h e s e r e s u l t s a r e i n a g r e e m e n t w i t h t h o s e o b t a i n e d 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 w h i c h N a + - i n d e p e n d e n t H C O 3 7 C I " e x c h a n g e w a s s i m i l a r l y o b s e r v e d t o b e a p p r e c i a b l y a c t i v e o n l y a t r o o m t e m p e r a t u r e . 9 3 F i g u r e 21. S a m p l e a c i d l o a d w i t h N H 4 C 1 . 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 e t 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 e i 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 4 + a n d O H - . T h i s r i s e i n p H ; q u i c k l y p l a t e a u s d u e t o t h e g r a d u a l i n f l u x e x t r a c e l l u l a r N H 4 + . 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 H 4 C 1 r e s u l t s i n a n i m m e d i a t e f a l l i n p H j t o a c i d i c l e v e l s , c a u s e d b y t h e f a s t e f f l u x o f N H 3 ; t h e r e m a i n i n g N H 4 + d i s s o c i a t e s t o r e l e a s e H + i o n s . A f t e r p e a k a c i d i f i c a t i o n i s r e a c h e d , p H j r e c o v e r s t o p r e - a c i d l o a d l e v e l s . T h i s s a m p l e t r a c e i s m e a n o f d a t a o b t a i n e d f r o m 6 c e l l s 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 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 HCO3" (solutions 1 a n d 4, p H 0 7 . 3 6 ) . 9 4 0 5 10 15 20 25 30 T i m e ( m i n u t e s ) 9 5 co 13 ° s oo O -*-' O CU CO CO 13 ° s u~i O +-> O CU CO M 13 1 S T 8 o o 42 O O 1 CO X •a fi H H EG fi £ OH P 2 «? o o X £ EG c S ft 8 .2 © . . CO x .-S OH -H» H H 0) CD o CD 13 CO EG & CU CO cd eu 2 EC a o leg r- m m CN CN CN VO m vo m m T f in m CN OS VO »—i CN T f os in CN r-~ © 00 m T f CN © vo VO <—i in •—i T f •—i m >—1 CN CN m OS vo 1—1 41 41 41 -H -H -H -H -H 4H 4H 4H 41 41 41 T f 00 in VD T f ,—< vo © in T f OS in oo (N oo oo in OS *—1 in vo m m in CN in OO m *—< T f T f CN CN T f i—i vo in in OS oo © CN in OS CN in CN r~- CN T f 00 •—i in rn -H +1 +1 +1 +1 -H +\ -H -H -H 4H 4H 41 4H 4H vo cn o\ , — i r-- os m in in 00 T f © r- t> oo 00 00 O 00 OS r- VD in m VD CN m , — i ' 1 * — < . — I CN in in oo m T f in vo m T f vo vo 00 T f os o o o o m •—i m CN CN © in © © © © © © © © o © © © © © © -H -H -H 4H +1 i -H -HH -HH • -H 41 4H 4H 4H 41 in © in m o o CN m (N vo CN vo © oo ,—< m VO OS m m OS •—< © CN CN in © in rn © © © O © 1 CN ^ © CN rn vo os CN m in vo OS © CN 00 CN CN © 00 1-H © *—> o CN vo CN CN m 00 m rn os © © © o © © © © © © © © © © -H -H -H 4H -H -H i -H -H -H i 41 4H 41 41 4H 41 m oo m m o m os vo vo m r—i m 00 os rn 00 vo »- T f in (N r- vo oo OS in in © ^ © © rn rn CN CN CN CN CN © T f vd m r- T f T f 00 T f in m 00 VO in os OS 00 CN m <—l o m © T f in in vo >—1 os vo © © © © © ^ H © © © © © © © -H -H 4H -H -H -H i -H 4H -HH i 41 41 41 41 41 41 i—< in T f in 00 vo T f m CN vo © CN CN oo in os i — i in vq os 00 T f os © i—i vo ^ CN rn © r-H in <n rn T f T f T f T f t> os rf CN r- T f m © m m T f oo m m m OS vo O o o o o © © © © © © © © © © © © © © © © © © o © © © © © © ©* © -H -H -H -H -H -H -H -H -H -H -H 4H 4H 4H 4H 4H 4H oo o m CN m vo , -H VD T f _ m © m T f © m vo vo T f T f T f VO vo VO VD in vo in T f T f T f in T f © © © © © © © O © © © © © © © © o in m o © o © © © © © -H -H -H 4H CN 00 r—1 in m CN © © © © a a a a a U O o O u O O O u u O O o Q o Q o Q o o o Q 0 o r~~ o r-- o r- O r-- o r- o r~- o r-- o O r- o r- o SH *H SH l-l m m m m m m m m m m m m GO w PH W 00 o 4 Q + O O CN u © W PH 00 < Q +c« S I 1—' o © © m CN O o W oo Q i—i Q O CD w oo 00 S Z7 oo oo ^ vd '—1 o 1—11—i i—i ; T | pr] U o m © Q Q &, &i X S K O R, * cu cu ^ ^ w co OO § 4H o a oo o «2 & to O CO fi CU o o C o o cu cu s o t H ^ f i ^ o co +̂ CU co > >H g ^ 3 ^ , , cd cd "O CO DH 00 ^ H 3 co 2 U S O > O OO Q Q fi <+H CU (U a O cu m co fi ts cd o _ OH o> CU a ^ O CU VH ca fi + CU w ft B cu Q H-H o co <U T3 © s ° >, B ' o r*H <+H 13 T 3 CU o ,1> .S cu fi +-> E ° cu fi "S cu SI ^ td x~ OH CO i s _CU co cu 8 § a I S ? OH . a a - a OH fi cd cu fi fi SH CU OH CU > o o cu SH X OH O co O cu cd CU co „ ^ Q "±3 H H 00 oo Q H H Q cu S fi "1 < CO - n 3 2 o co S SH SH © Cd <N td 13 -° CU CU CO 1 F a HJ « 8.1 CU £3 2 fi CH* O CU «U -H* ^ O j H CU O .fi SH CU T3 ™ h b »i .— o cu H U o cd ft d H *3 "Si "cd « ^ 3 »H C3 Pi OH A . . 13 -H< O0 ^O Q -a cu ™4 *H 13 cu s ^ O + H <u cd » - H f i a co ^ CU CO cu co O O CU SH CU > X X OH OH 96 Figure 22. Initial rate of acid load recovery as a function of pHj , preload pHj, minimum pHj , and net pHj 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. 9 7 0.006 0.005 T3 fl 8 0.004 0.003 > o o (-1 o u 0.002 0.001 4- 0.000 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 p H } 0.006 0.005 % 0.004 •a ffi 0.003 & > 0.002 o <D s-i O u 0.001 0.000 B. • O • • • o o o • • # o o ° • • • o o o ••• o 93 • i i i o 1 1 6.2 0.006 6.4 6.6 6.8 7.0 7.2 7.4 0.000 0.3 0.4 0.5 0.6 N e t p H j d e c r e a s e 9 8 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 HCO37CO2 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 0 7 . 3 3 ) , p H j r e c o v e r e d f r o m a n N H 4 + - i n d u c e d a c i d l o a d a t a n i n i t i a l r a t e o f 1 . 3 1 x 1 0 ' 3 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 " 3 p H u n i t s / s e c o n d ( n = 7 ) . T h e t r a c e i s 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 a s i n g l e c o v e r s l i p . 9 9 T i m e ( m i n u t e s ) 1 0 0 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 0 7 . 3 2 , t h e a p p l i c a t i o n o f 2 0 0 p M D I D S , a p 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 H C O 3 - / C T e x c h a n g e , s l o w e d t h e p H j r e c o v e r y f r o m a n i n d u c e d 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 w h e n a p p l i e d d u r i n g 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 t r a c e ( n = 3 ) . I n t h e p r e s e n c e o f D I D S , t h e i n i t i a l r a t e o f p H j r e c o v e r y w a s 0 . 9 5 x 1 0 " 3 p H u n i t s / s e c o n d . T h e 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 2 2 c e l l s 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 . 1 0 1 0 5 10 15 20 25 30 35 40 45 T i m e ( m i n u t e s ) 1 0 2 Figure 25. Effect of 0 [Cl"]0 on pHj recovery from an acid load in the presence of H C O 3 " at room temperature. A . A t p H 0 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 t h e r a t e p H j r e c o v e r y f r o m a n a c i d l o a d ( n = 3 ) . U n d e r [ C l " ] 0 - f r e e 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 i n c r e a s e d t o 3 . 1 4 x 1 0 " 3 p H u n i t s / s e c o n d f r o m 2 . 5 5 x 1 0 ' 3 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 ~ ] 0 . 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 e x p o s u r e t o 0 [ C l " ] 0 w a s b l o c k e d b y t h e s i m u l t a n e o u s a p p l i c a t i o n o f 2 0 0 p M D I D S . I n t h e a b s e n c e o f [ C l " ] 0 a n d t h e p r e s e n c e o f D I D S , t h e i n i t i a l r a t e o f p H j r e c o v e r y w a s 1 . 1 8 x 1 0 " 3 p H u n i t s / s e c o n d ( n = 2 ) . T r a c e A i s m e a n o f d a t a o b t a i n e d f r o m 2 6 c e l l s , w h e r e a s t r a c e B 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 3 4 c e l l s , e a c h r e c o r d e d o n a s e p a r a t e c o v e r s l i p . 103 T i m e ( m i n u t e s ) 1 0 4 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 0 7 . 3 6 ) , p H , r e c o v e r e d f r o m a n N H 4 + - i n d u c e d i n t r a c e l l u l a r a c i d l o a d a t a n i n i t i a l r a t e o f 5 . 5 6 x 1 0 " 3 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 H C O 3 V C O 2 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 i n i t i a l r a t e o f 4 . 7 7 x 1 0 " 3 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 s a m e a n o f d a t a o b t a i n e d f r o m 7 c e l l s 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 2 3 , w h i c h i s a s i m i l a r e x p e r i m e n t 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 . 105 T i m e ( m i n u t e s ) 1 0 6 Figure 27. Effect of 0 [Na +] 0 on pHj recovery from an acid load in the absence of H C 0 3 - at 37°C. 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 0 7 . 3 2 ) c o m p l e t e l y h a l t e d t h e a b i l i t y o f p H j t o r e c o v e r f r o m a n i n d u c e d a c i d l o a d ( n = 2 ) . O n c e [ N a + ] 0 w a s r e - i n t r o d u c e d t o 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 i t s r e c o v e r y t o 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 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 1 0 c e l l s 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 . 1 0 7 T i m e ( m i n u t e s ) 1 0 8 F i g u r e 2 8 . E f f e c t o f E I P A o n p H j r e c o v e r y f r o m a n a c i d l o a d 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 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 3 7 ° C ( p H 0 7 . 3 5 ) d i d n o t a f f e c t t h e r a t e o f p H j r e c o v e r y d u r i n g t h e r e s t o r a t i o n o f p H j t o n o r m a l l e v e l s a f t e r a n i n d u c e d 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 ( n = 6 ) . T h e 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 3 0 c e l l s 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 . 109 Time (minutes) 1 1 0 F i g u r e 2 9 . E f f e c t o f D I D S o n p H j r e c o v e r y f r o m a n a c i d l o a d 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 2 0 0 p M D I D S d u r i n g t h e r e s t o r a t i o n o f p H j d i d n o t s i g n i f i c a n t l y a f f e c t t h e r a t e 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 t h e a b s e n c e o f H C 0 3 " a t 3 7 ° C ( p H 0 7 . 3 7 ) . T h e 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 2 3 c e l l s r e c o r d e d f r o m 1 o f t h e 3 c e l l p o p u l a t i o n s s t u d i e d . I l l T i m e ( m i n u t e s ) 1 1 2 Figure 30. Effect of 0 [ N a + ] 0 on pHj recovery from an acid load in the presence of H C 0 3 - at 3 7 ° C . At p H 0 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 +] 0, possibly due to the activity of Na+-independent recovery mechanisms. The re-addition of [Na +] 0 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. 1 1 3 T i m e ( m i n u t e s ) 1 1 4 Figure 31. Effect of EIPA on pHj recovery from an acid load in the presence of H C O 3 - at 37°C. I n 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 0 7 . 3 4 ) , t h e a d d i t i o n o f 5 0 p M E I P A d u r i n g t h e p e r i o d o f p H j r e s t o r a t i o n d i d n o t a l t e r t h e r a t e o f 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 i f i c a t i o n ( n = 3 ) . T h e 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 2 5 c e l l s 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 . 1 1 5 116 Figure 32. Effect of 0 [Cl~]0 on pH; recovery from an acid load in the presence of H C 0 3 - at 37°C. 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 i n c r e a s e t h e r a t e o f 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 i f i c a t i o n ( n = 7 ) . T h o u g h p H 0 w a s h e l d c o n s t a n t a t 7 . 3 7 , p H j d i d r e c o v e r t o a s 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 " ] 0 , b u t r e t u r n e d t o n o r m a l s t e a d y - s t a t e l e v e l s w h e n [ C l " ] 0 w a s r e - i n t r o d u c e d t o t h e p e r f u s i o n m e d i u m . T h e 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 2 8 c e l l s 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 2 5 , w h i c h s h o w s t h e e f f e c t o f e x p o s u r e t o 0 [ C l " ] 0 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 . 117 6.6 -| 1 1 1 1 1 1 0 10 20 30 40 50 60 Time (minutes) 118 Figure 33. Effect of DIDS on pHj 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 (pH 0 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 10~3 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). 1 1 9 1 2 0 Figure 34. Effect of DIDS on pH, recovery from an enhanced acid load in the presence of H C O 3 - at 37°C. T h e p H o f t h e p e r f u s 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 r e d u c e t h e r e s t i n g p H j ( s e e F i g u r e 1 9 ) . T h i s m a n o e u v r e a l s o l o w e r e d t h e m i n i m u m p H j r e a c h e d d u r i n g t h e i n d u c e d 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 . U n d e r d r u g - f r e e c o n d i t i o n s a t p H 0 6 . 8 ( f i r s t a c i d l o a d ) , t h e i n i t i a l r a t e o f p H , r e c o v e r y w a s 7 . 1 2 x 1 0 " 3 p H u n i t s / s e c o n d ( n = 3 ) . T h e p r e s e n c e o f 2 0 0 p M D I D S d u r i n g p H j r e s t o r a t i o n f a i l e d t o s l o w t h e r a t e o f r e c o v e r y ( n = 2 ) . T h e 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 8 c e l l s 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 . 121 Time (minutes) 1 2 2 D I S C U S S I O N p H j r e g u l a t i o n , s t u d i e d i n v a r i o u s c e l l t y p e s , h a s b e e n t h e s o u r c e o f c o n s i d e r a b l e i n v e s t i g a t i o n . T h e p r e d o m i n a n t m e c h a n i s m s r e g u l a t i n g p H j i n t h e l i m i t e d n u m b e r o f v e r t e b r a t e n e u r o n e t y p e s s t u d i e d t o d a t e a p p e a r t o b e a N a + / H + e x c h a n g e r , a N a + - i n d e p e n d e n t H C 0 3 7 C 1 ~ e x c h a n g e r , a n d a N a + - d e p e n d e n t H C 0 3 7 O ~ e x c h a n g e r ( s e e , f o r e x a m p l e , O u - y a n g et al, 1 9 9 3 ; R a l e y - S u s m a n et al, 1 9 9 1 ; S c h w i e n i n g a n d B o r o n , 1 9 9 4 ) . 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, 1 9 9 4 ) , t h o u g h t h e s e a p p e a r t o p l a y a m i n o r r o l e . V e r y f e w s t u d i e s , h o w e v e r , h a v e c o m p a r a t i v e l y e x a m i n e d t h e r e g u l a t i o n o f p H ; a t r o o m t e m p e r a t u r e a n d 3 7 ° C . T h e r e s u l t s p r e s e n t e d i n t h i s t h e s i s u n c o v e r s o m e s t r i k i n g d i f f e r e n c e s i n p H , r e g u l a t i o n c a u s e d b y t e m p e r a t u r e . A c c o r d i n g l y , t h i s d i s c u s s i o n w i l l n o t o n l y e x a m i n e t h e m e c h a n i s m s r e g u l a t i n g t h e i n t r a c e l l u l a r p r o t o n e n v i r o n m e n t i n c u l t u r e d r a t h i p p o c a m p a l n e u r o n e s a t 3 7 ° C a n d a t r o o m t e m p e r a t u r e , b u t 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 t h e 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 a t t h e s e t w o t e m p e r a t u r e s . R e g u l a t i o n o f p H ; a t 3 7 ° C : S t e a d y - s t a t e p H j r e s u l t i n g f r o m t h e p e r f u s i o n o f t h e n e u r o n e s a t 3 7 ° C w i t h 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 ( p H 0 7 . 3 4 ) w a s 7 . 2 3 . R e s t i n g p H j i n t h e n o m i n a l a b s e n c e o f H C 0 3 " a t 3 7 ° C ( p H 0 7 . 3 5 t o 7 . 4 ) h a s b e e n r e p o r t e d t o r e s i d e a t 7 . 0 3 i n c u l t u r e d r a t s y m p a t h e t i c n e u r o n e s ( T o l k o v s k y a n d R i c h a r d s , 1 9 8 7 ) , 7 . 0 0 i n c u l t u r e d r a t 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, 1 9 9 1 ) , 7 . 0 0 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, 1 9 9 3 ) , a n d 6 . 7 4 i n f r e s h l y i s o l a t e d C A 1 p y r a m i d a l n e u r o n e s f r o m r a t h i p p o c a m p i ( S c h w i e n i n g a n d B o r o n , 1 9 9 4 ) . H o w e v e r , G a i l l a r d a n d D u p o n t ( 1 9 9 0 ) h a v e s h o w n t h a t s t e a d y - s t a t e p H j i s 7 . 3 7 i n c u l t u r e d r a t c e r e b e l l a r P u r k i n j e c e l l s p e r f u s e d w i t h H E P E S - b u f f e r e d s o l u t i o n s ( p H 0 7 . 4 ) . T h e r e f o r e , t h e v a l u e o f r e s t i n g p H j d e t e r m i n e d i n 1 2 3 t h e 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 3 ~ - f r e e c o n d i t i o n s i s w e l l w i t h i n t h e r e p o r t e d r a n g e f o r v e r t e b r a t e n e u r o n e s . T h e 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 w e r e a b l e t o s u s t a i n a s t a b l e p H j d u r i n g e x p o s u r e t o H E P E S 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 , t h u s i n d i c a t i n g a s i g n i f i c a n t c o n t r i b u t i o n 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 t o w a r d s t h e m a i n t e n a n c e o f n o r m a l p H j l e v e l s a t 3 7 ° C . U n d e r H C C ^ - f r e e c o n d i t i o n s , 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 + p r o d u c e d a m a r k e d a n d s u s t a i n e d a c i d i f i c a t i o n , w h i c h w a s r e v e r s e d b y t h e r e - a d d i t i o n o f [ N a + ] 0 t o t h e p e r f u s i o n m e d i u m . T h e s e o b s e r v a t i o n s i n d i c a t e t h e p r e s e n c e o f a 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 o p e r a t i n g i n d e p e n d e n t l y o f H C 0 3 " . A p r o b a b l e c a n d i d a t e f o r t h i s m e c h a n i s m i s t h e N a + / H + e x c h a n g e r , w h i c h . r e m o v e s i n t r a c e l l u l a r p r o t o n s i n e x c h a n g e f o r 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 [ N a + ] 0 t h i s a n t i p o r t e r i s n o t a b l e t o f u n c t i o n , c a u s i n g a n a c c u m u l a t i o n o f i n t r a c e l l u l a r a c i d e q u i v a l e n t s . A s m a l l p o r t i o n o f t h e s e e q u i v a l e n t s m a y o r i g i n a t e o u t s i d e o f t h e m e m b r a n e a n d l e a k t h r o u g h t o t h e c y t o p l a s m d o w n a n e l e c t r o c h e m i c a l g r a d i e n t , b u t m o s t a r e l i k e l y g e n e r a t e d f r o m n o r m a l c e l l u l a r m e t a b o l i s m . T h e a c i d i f i c a t i o n r e s u l t i n g f r o m t h e r e m o v a l o f [ N a + ] 0 c o u l d a l s o b e e x p l a i n e d b y a r e v e r s a l o f t h e N a + / H + a n t i p o r t e r , a l t h o u g h i t w a s n o t p o s s i b l e t o t e s t t h i s i n v i e w o f t h e l a c k o f a p h a r m a c o l o g i c a l i n h i b i t o r . T o c o n f i r m t h e p r e s e n c e o f t h e N a + / H + e x c h a n g e r o n t h e s e n e u r o n e s , a u s e f u l t e s t w o u l d h a v e b e e n t o i n h i b i t i t s a c t i v i t y u t i l i z i n g k n o w n p h a r m a c o l o g i c a l b l o c k e r s . U n f o r t u n a t e l y , t h e a p p l i c a t i o n o f v a r i o u s N a + / H + e x c h a n g e i n h i b i t o r s , i n c l u d i n g 2 p o t e n t a m i l o r i d e a n a l o g u e s a n d o n e n o v e l i n h i b i t o r , f a i l e d t o a l t e r 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 3 7 ° C . E I P A , a p p l i e d a t 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 r e s t i n g p H ; , n o r d i d i t i n h i b i t t h e r e c o v e r y o f p H ; a f t e r a 0 [ N a + ] 0 - i n d u c e d a c i d i f i c a t i o n . S i m i l a r l y , 1 0 0 p M M G C M A , w h i c h h a s b e e n s h o w n t o i n h i b i t t h e N a + / H + a n t i p o r t e r i n 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, 1 9 9 1 ) , a n d 1 0 0 p M H O E 6 9 4 , a n o v e l i n h i b i t o r 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, 1 9 9 2 ) , w e r e b o t h i n c a p a b l e o f p r o d u c i n g a c h a n g e i n b a s e l i n e p H j . T o e x a m i n e w h e t h e r t h e 1 2 4 p r e s e n c e o f H C 0 3 " w a s r e q u i r e d f o r t h e m a n i p u l a t i o n o f p H j b y E I P A , t h e c a t i o n e x c h a n g e i n h i b i t o r w a s a p p l i e d t o n e u r o n e s p e r f u s e d w i t h H C C ^ V C C ^ - b u f f e r e d m e d i u m . A g a i n , t h e a p p l i c a t i o n o f 5 0 p M E I P A d i d n o t i n d u c e a c h a n g e i n p H j . T h e i n s e n s i t i v i t y o f 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 i n 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 t o a m i l o r i d e a n d i t s 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 a d d i t i o n a l s t u d i e s . 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 ) h a v e o b s e r v e d t h a t a m i l o r i d e , o r a d d i t i o n a l a n a l o g u e s t e s t e d , w e r e u n a b l e t o m o d u l a t e t h e a c t i v i t y o f t h e s u s p e c t e d N a + / H + e x c h a n g e r p r e s e n t o n t h e c e l l s u s e d i n t h e i r e x p e r i m e n t s . I n c o n t r a s t , i t h a s b e e n d e m o n s t r a t 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 c o m p l e t e l y s e n s i t i v e t o a m i l o r i d e i n c u l t u r e d r a t P u r k i n j e c e l l s ( G a i l l a r d a n d D u p o n t , 1 9 9 0 ) a n d s y m p a t h e t i c n e u r o n e s ( T o l k o v s k y a n d R i c h a r d s , 1 9 8 7 ) , a n d p a r t i a l l y s e n s i t i v e t o a m i l o r i d e 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, 1 9 9 3 ) . O t h e r c a t i o n e x c h a n g e i n h i b i t o r s , s u c h a s 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 , 1 9 8 1 ) , w e r e n o t e x a m i n e d d u e t o i n t e r f e r e n c e w i t h t h e f l u o r e s c e n c e s i g n a l e m i t t e d b y t h e B C E C F - l o a d e d n e u r o n e s . A l t h o u g h i n i t i a l l y r e p o r t e d t o i n h i b i t N a + / H + e x c h a n g e i n c u l t u r e d h i p p o c a m p a l p y r a m i d a l c e l l l o a d e d w i t h B C E C F ( R a l e y - S u s m a n et al, 1 9 9 1 ) , t h i s f i n d i n g h a s b e e n q u e s t i o n e d b y o t h e r s ( e . g . O u - y a n g et al, 1 9 9 3 ; S c h w i e n i n g a n d B o r o n , 1 9 9 4 ) d u e t o t h i s t e c h n i c a l l i m i t a t i o n . A n a n a l y s i s o f t h e k i n e t i c p r o p e r t i e s o f c a t i o n c o u n t e r - t r a n s p o r t o n v a r i o u s c e l l t y p e s h a s r e v e a l e d t h a t L i + i s a s u b s t r a t e f o r t h e o p e r a t i o n o f t h e N a + / H + a n t i p o r t e r ( A r o n s o n , 1 9 8 5 ) . T h e s u b s t i t u t i o n o f [ N a + ] 0 w i t h [ L i + ] 0 h a s t h e r e f o r e b e e n u t i l i z e d a s a n a l t e r n a t i v e m e a n s o f s u g g e s t i n g t h e p r e s e n c e o f N a + / H + e x c h a n g e o n c e l l s i n s e n s i t i v e t o a m i l o r i d e ( R a l e y - S u s m a n et al, 1 9 9 1 ) . T h e l a t t e r a u t h o r s , d e m o n s t r a t e d t h a t t h e r e p l a c e m e n t o f [ N a + ] 0 w i t h [ L i + ] 0 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 a b i l i t y o f c u l t u r e d h i p p o c a m p a l n e u r o n e s t o r e g u l a t e p H j , w h i c h i n d i c a t e 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 w a s l i k e l y t h e N a + / H + e x c h a n g e r , d e s p i t e i t s i n s e n s i t i v i t y t o a m i l o r i d e . I n d e e d , p r e l i m i n a r y r e s u l t s o n t h e 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 i n d i c a t e t h a t t h e r e p l a c e m e n t o f [ N a + ] 0 w i t h [ L i + ] 0 d o e s n o t j e o p a r d i z e t h e m a i n t e n a n c e o f a s t a b l e 1 2 5 r e s t i n g p H j i n H C 0 3 - - f r e e m e d i u m a t 3 7 ° C . A c c o r d i n g l y , t h e 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 e x t r u s i o n m e c h a n i s m r e g u l a t i n g p H j o n t h e s e n e u r o n e s m a y b e a n a m i l o r i d e - i n s e n s i t i v e v a r i a n t o f t h e N a + / H + e x c h a n g e r , s i m i l a r t o t h a t 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 i s o f o r m s o f t h e N a + / H + e x c h a n g e r ( N H E ) h a v e r e c e n t l y b e e n d i s t i n g u i s h e d ( M r k i c et al, 1 9 9 3 ) b a s e d o n t h e i r a m i l o r i d e s e n s i t i v i t y a n d t i s s u e l o c a l i z a t i o n . T h e N H E - 1 f o r m , p r e s e n t o n t h e b a s o l a t e r a l s u r f a c e s o f i n t e s t i n a l a n d k i d n e y e p i t h e l i a , i s 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, 1 9 8 9 ) . R e l a t i v e a m i l o r i d e s e n s i t i v i t y h a s a l s o b e e n o b s e r v e d w i t h t h e N H E - 2 i s o f o r m , w h i c h h a s b e e n l o c a l i z e d i n t h e i n t e s t i n e , k i d n e y , a n d a d r e n a l g l a n d ( T s e et al, 1 9 9 1 ) . 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 a n d E I P A ( T s e et al, 1 9 9 3 ) , i s 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 i t 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, 1 9 9 2 ) . F i n a l l y , a n N H E - 4 i s o f o r m h a s b e e n d e t e c t e d b y O r l o w s k i et al ( 1 9 9 2 ) i n m a n y m a m m a l i a n t i s s u e s , i n c l u d i n g t h e b r a i n , t h o u g h i t s s e n s i t i v i t y t o a m i l o r i d e a n d i t s a n a l o g u e s h a s n o t b e e n d o c u m e n t e d . I t i s 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 , i f a n y , m a y b e p r e s e n t o n t h e c u l t u r e d h i p p o c a m p a l n e u r o n e s e m p l o y e d i n t h e p r e s e n t e x p e r i m e n t s , t h o u g h t h e i r r e s i s t a n c e t o E I P A , M G C M A , a n d H O E 6 9 4 i s a p p a r e n t . S t e a d y - s t a t e p H ; r e s u l t i n g f r o m t h e p e r f u s i o n o f t h e s e n e u r o n e s w i t h H C 0 3 7 C 0 2 - b u f f e r e d m e d i u m a t 3 7 ° C ( p H 0 7 . 3 6 ) w a s 7 . 1 3 . T h i s v a l u e c o m p a r e s w e l l w i t h t h a t r e p o r t e d i n p r e v i o u s s t u d i e s o n m a m m a l i a n c e n t r a l n e u r o n e s m a i n t a i n e d u n d e r s i m i l a r c o n d i t i o n s . A t 3 7 ° C a n d i n t h e p r e s e n c e o f H C 0 3 " , i t 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 a t 7 . 1 6 i n c u l t u r e d r a t c e r e b e l l a r g r a n u l e c e l l s ( P o c o c k a n d R i c h a r d s , 1 9 8 9 ) , 7 . 1 8 i n m i x e d n e u r o n a l c u l t u r e s f r o m v a r i o u s r a t b r a i n r e g i o n s ( R i c h a r d s a n d P o c o c k , 1 9 8 9 ) , 7 . 0 6 i n c u l t u r e d r a t c e r e b e l l a r P u r k i n j e c e l l s ( G a i l l a r d a n d D u p o n t , 1 9 9 0 ) , 7 . 1 7 i n c u l t u r e d r a t 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, 1 9 9 1 ) , 7 . 0 9 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, 1 9 9 3 ) , a n d 7 . 0 3 i n a c u t e l y d i s s o c i a t e d a d u l t r a t h i p p o c a m p a l C A 1 p y r a m i d a l n e u r o n e s ( S c h w i e n i n g a n d B o r o n , 1 9 9 4 ) . I n t h e n e u r o n e s e m p l o y e d i n t h i s 1 2 6 s t u d y , b a s e l i n e p H j w a s 0 . 1 0 p 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 H C O 3 " t h a n i n t h e a b s e n c e o f H C O 3 " a t 3 7 ° C . I n d e e d , 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 t o 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 a t 3 7 ° C d i d n o t i n d u c e t h e n e t a l k a l i n i z a t i o n o b s e r v e d i n t h e s a m e e x p e r i m e n t 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 ( s e e b e l o w ) . T h o u g h a n u m b e r o f s t u d i e s o n v e r t e b r a t e n e u r o n e s h a v e d e m o n s t r a t e d t h a t r e s t i n g p H j i s h i g h e r i n t h e p r e s e n c e t h a n i n t h e a b s e n c e o f H C 0 3 " a t 3 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 d B 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 . G a i l l a r d a n d D u p o n t ( 1 9 9 0 ) , f o r e x a m p l e , r e p o r t e d t h a t s t e a d y - s t a t e p H , i n r a t P u r k i n j e c e l l s b u f f e r e d b y H C O 3 V C O 2 r e s i d e s 0 . 2 1 p H u n i t s b e l o w t h e r e s t i n g p H ; i n 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 . S i m i l a r l y , R i c h a r d s a n d P o c o c k ( 1 9 8 9 ) , i n t h e i r e x a m i n a t i o n o f r a t b r a i n n e u r o n e s , 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 a l k a l i n i z a t i o n . A c c o r d i n g l y , t h e l e v e l o f s t e a d y - s t a t e p H j i n t h e n e u r o n e s u s e d i n t h i s 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 e p 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 e n o t u n l i k e t h o s e r e p o r t e d i n o t h e r s t u d i e s o n v e r t e b r a t e c e n t r a l n e u r o n e s . 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 p r e s e n c e o f H C O 3 " i n i t i a t e d a r a p i d a n d s u s t a i n e d 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 , a r e s u l t s i m i l a r t o t h a t o b t a i n e d i n t h e a b s e n c e o f H C O 3 " ( s e e a b o v e ) . T h e r e - a d d i t i o n o f [ N a + ] 0 r e l i e v e d t h i s a c i d - l o a d , a n d a n i m m e d i a t e r e c o v e r y t o s t e a d y - s t a t e p H j l e v e l s w a s o b s e r v e d . T h e s e o b s e r v a t i o n s i n d i c a t e t h a t , e v e n i n t h e p r e s e n c e o f HCO3", t h e 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 a t 3 7 ° C i s l a r g e l y d e p e n d e n t o n [ N a + ] 0 . I n n e u r o n e s b u f f e r e d b y H C 0 3 7 C 0 2 - c o n t a i n i n g s o l u t i o n s , 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 " c a u s e d a 0 . 1 9 p H u n i t a n d a 0 . 1 4 p H u n i t 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 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 [ N a + ] 0 , r e s p e c t i v e l y . B y r e m o v i n g [ C l " ] 0 , t h e g r a d i e n t f o r C l " a c r o s s t h e m e m b r a n e w o u l d b e i n c r e a s e d a n d m a y r e s u l t 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 H C O 3 7 O " 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 ) . T h i s w o u l d c a u s e a n i n f l u x o f e x t r a c e l l u l a r HCO3" w h i c h w o u l d p r o d u c e t h e o b s e r v e d 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 . I n t h e p r e s e n c e o f [ N a + ] 0 , t h i s 0 [ C l " ] 0 - i n d u c e d a l k a l i n i z a t i o n w a s a b o l i s h e d b y 2 0 0 u M D I D S , w h i c h a d d s w e i g h t t o t h e p o s s i b i l i t y t h a t a H C 0 3 " / C 1 " 1 2 7 e x c h a n g e r i s p r e s e n t o n t h e s e n e u r o n e s . F u r t h e r m o r e , t h i s a n i o n e x c h a n g e r m a y p r e d o m i n a n t l y f u n c t i o n i n a N a + - i n d e p e n d e n t m a n n e r s i n c e t h e a m p l i t u d e o f t h e 0 [ C T ] 0 - i n d u c e d p H j r i s e h a d a s i m i l a r m a g n i t u d e w h e t h e r [ N a + ] 0 w a s p r e s e n t o r n o t . I t i s p o s s i b l e t h a t , a t 3 7 ° C , N a + - i n d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e m a y o p e r a t e t o m a i n t a i n s t e a d y - s t a t e p H j a t a l e v e l s l i g h t l y b e l o w t h a t w h i c h i s o b s e r v e d i n t h e a b s e n c e o f HCO3". H o w e v e r , t h e a p p l i c a t i o n o f 2 0 0 p M D I D S d i d n o t a f f e c t b a s e l i n e p H j i n 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 t o t h e t r a n s i t i o n f r o m H C C ^ ' - f r e e t o 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 i n a b i l i t y o f D I D S t o a f f e c t p H j i n t h e s e s i t u a t i o n s d o e s n o t s u p p o r t t h e p r e s e n c e o f a n 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 " ] 0 , i t i s 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 p r e s e r v i n g t h e s t e a d y - s t a t e p H j i n t h e s e c e l l s . I n f a c t , r e c e n t e v i d e n c e s u g g e s t s t h a t a D I D S - s e n s i t i v e C 1 7 H + c o - t r a n s p o r t e r i s i n v o l v e d i n p H j r e g u l a t i o n i n r a t b r a i n s y n a p t o s o m e s ( M a r t i n e z - Z a g u i l a n et al, 1 9 9 4 ) . H o w e v e r , t h e l i k e l i h o o d o f t h i s c o - t r a n s p o r t e r b e i n g p r e s e n t o n h i p p o c a m p a l n e u r o n e s i s r e m o t e , s i n c e t h e e x p o s u r e t o 0 [ C l " ] 0 i n t h e a b s e n c e o f H C 0 3 " a t 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 s t e a d y - s t a t e 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 " ] 0 - 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 t h e s i m u l t a n e o u s a p p l i c a t i o n o f D I D S , w h i c h s u g g e s t s t h a t a n i o n e x c h a n g e i s i n f a c t 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 l i t t l e t o t h e 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 a t 3 7 ° C . A c l e a r e r i n d i c a t i o n o f t h e m a n n e r i n w h i c h c u l t u r e d h i p p o c a m p a l n e u r o n e s r e g u l a t e p H j a t 3 7 ° C w a s a c h i e v e d t h r o u g h t h e a n a l y s i s o f a c i d l o a d r e c o v e r y e x p e r i m e n t s . T h e a d d i t i o n a n d s u b s e q u e n t r e m o v a l o f 2 0 m M N H 4 C 1 f r o m t h e e x t r a c e l l u l a r p e r f u s i o n m e d i u m p r o v i d e d a c o n v e n i e n t m e a n s o f l o w e r i n g p H , . T h e r a t e o f p H j r e s t o r a t i o n t o w a r d s i t s r e s t i n g l e v e l w a s t h e r e f o r e e x a m i n e d a s a m e a n s o f e x p a n d i n g t h e c h a r a c t e r i z a t i o n o f p H j r e g u l a t i n g m e c h a n i s m s a n d , i n a d d i t i o n , w a s e m p l o y e d t o p r o v i d e i n f o r m a t i o n o n a c t i v i t y r a t e s . A c i d e x t r u s i o n r a t e s a r e o f t e n r e p o r t e d 1 2 8 a s t h e r a t e o f c h a n g e o f t h e i n t r a c e l l u l a r p r o t o n c o n c e n t r a t i o n a s a f u n c t i o n o f t i m e . T h e s e n e t H + f l u x e s (JH+) a r e u s u a l l y c a l c u l a t e d a s t h e p r o d u c t t h e t o t a l i n t r a c e l l u l a r b u f f e r i n g c a p a c i t y (PX) 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 a c i d i f i c a t i o n ( s e e B o y a r s k i et al, 1 9 8 8 a ) . F o r r e a s o n s e x p l a i n e d b e l o w , a b s o l u t e b u f f e r i n g c a p a c i t i e s w e r e n o t d e t e r m i n e d i n t h e p r e s e n t e x p e r i m e n t s . A c c o r d i n g l y , t h i s d i s c u s s i o n o f a c i d l o a d r e c o v e r y r a t e s w i l l b e l i m i t e d t o t h e r a t e o f p H j c h a n g e m e a s u r e d i n p H u n i t s p e r s e c o n d . I n 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 , t h e r e m o v a l o f [ N a + ] 0 d u r i n g a c i d 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 r e s t o r a t i o n . T h e i n a b i l i t y o f p H j t o r e c o v e r i n 0 [ N a + ] 0 s u g g e s t s t h a t p H , i s 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 , H C O 3 " i n 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 . T h e m o s t l i k e l y c a n d i d a t e f o r s u c h a m e c h a n i s m , a s d i s c u s s e d a b o v e , i s t h e N a + / H + e x c h a n g e r . E I P A , w h e t h e r i n t h e a b s e n c e o r p r e s e n c e o f e x t e r n a l H C O 3 " , 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 i s c o n s i s t e n t w i t h p r e v i o u s r e s u l t s d e m o n s t r a t i n g t h e 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 p H j i n t h e s e n e u r o n e s t o a m i l o r i d e a n a l o g u e s . T h e r a t e 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 w a s n o t s i g n i f i c a n t l y d i f f e r e n t 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 , i n t h e a b s e n c e 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 , w h i c h l i k e l y r e f l e c t s t h e f a c t t h a t s t e a d y - s t a t e p H j i s h 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 a t 3 7 ° C . T h e s i m i l a r i t y b e t w e e n a c i d l o a d r e c o v e r y r a t e 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 H C O 3 " 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 r e c o v e r f r o m a c i d i c c h a n g e s i n p H , a t 3 7 ° C , p o s s i b l y d u e t o a n i n c r e a s e i n t h e n e u r o n a l b u f f e r i n g c a p a c i t y ( s e e b e l o w ) . 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 H C O 3 7 C O 2 , t h e r e m o v a l o f [ N a + ] 0 d i d n o t c o m p l e t e l y i n h i b i t t h e a b i l i t y o f t h e s e n e u r o n e s t o r e c o v e r f r o m a n N H 4 + - i n d u c e d a c i d i f i c a t i o n . T h o u g h s m a l l , t h e o b s e r v e d r e c o v e r y o f p H j i n t h e a b s e n c e o f [ N a + ] 0 i n d i c a t e s 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 r e g u l a t o r o p e r a t i n g a t 3 7 ° C . S i n c e t h i s N a + - i n d e p e n d e n t r e c o v e r y w a s n o t p r e s e n t i n s i m i l a r e x p e r i m e n t s c o n d u c t e d i n t h e 1 2 9 a b s e n c e o f e x t e r n a l H C 0 3 " , 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 H C O 3 " t o f u n c t i o n e f f e c t i v e l y . 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 H C O 3 7 Q " 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 b e p r e s e n t t h o u g h r e l a t i v e l y i n a c t i v e a t 3 7 ° C , o r H C C ^ ' - d e p e n d 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 . I n f a c t , i t 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 l e v e l s a t 3 7 ° C . I n 5 o f 9 n e u r o n a l p o p u l a t i o n s s t u d i e d , D I D S s i g n i f i c a n t l y s l o w e d r e c o v e r y r a t e s f r o m a n i m p o s e d a c i d l o a d , w h i c h r e v e a l s a d e p e n d e n c e o f p H j r e s t o r a t i o n 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 t h i s t r a n s p o r t e r h a s p r e v i o u s l y b e e n s h o w n t o r e m a i n r e l a t i v e l y q u i e s c e n t 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 a t 3 7 ° C , t h e s e 5 e x p e r i m e n t s u n c o v e r a p o s s i b l e r e l a t i o n s h i p b e t w e e n t h e a c t i v i t y o f t h e a n i o n e x c h a n g e r a n d t h e l e v e l o f p H j . S u c h a r e l a t i o n s h i p b e t w e e n p H , a n d t h e r a t e o f a c i d e x t r u s i o n h a s 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 r a t b r a i n . H o w e v e r , t h e r e m a i n i n g 4 n e u r o n a l p o p u l a t i o n s e x p o s e d t o D I D S d u r i n g r e c o v e r y f r o m a n a c i d l o a d d i d n o t e x h i b i t a r e d u c t i o n i n p H , r e c o v e r y r a t e s . F u r t h e r m o r e , t h e r e m o v a l o f [ C l ' ] 0 d u r i n g r e c o v e r y d i d n o t a l t e r t h e r a t e o f p H j r e s t o r a t i o n a t 3 7 ° C . T a k e n a s a w h o l e , 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 e a c t i v i t y o f 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 p r e s e n t o n t h e s e n e u r o n e s m a y b e o v e r s h a d o w e d b y t h e o p e r a t i o n o f t h e N a + / H + t r a n s p o r t e r o r t h e i n c r e a s e d e f f i c i e n c y o f i n t r a c e l l u l a r b u f f e r i n g s y s t e m s ( s e e b e l o w ) . C e l l s w i t h a n i n c r e a s e d a b i l i t y t o r e s i s t p H ; p e r t u r b a t i o n s t h r o u g h m o r e e f f i c i e n t o r g a n e l l a r , m e t a b o l i c , o r p h y s i o c h e m i c a l b u f f e r i n g w o u l d p r e s u m a b l y b e l e s s r e l i a n t o n a c i d e x t r u d i n g e x c h a n g e r s f o r i m m e d i a t e 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 i f i c a t i o n . T h i s h y p o t h e s i s i s a p o s s i b l e e x p l a n a t i o n f o r t h e a p p a r e n t i n a c t i v i t y o f t h e N a + - i n d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e r a t 3 7 ° C . N e v e r t h e l e s s , b y i m p o s i n g a l a r g e i n t r a c e l l u l a r a c i d l o a d , t h e p r o b a b i l i t y o f a c t i v a t i n g t h e o t h e r w i s e d o r m a n t a n i o n e x c h a n g e r t o a i d i n p H , r e g u l a t i o n a p p e a r s t o i n c r e a s e . 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 a c t i v a t e d b y l o w e r i n g p H j t o e x t r e m e l e v e l s , i m p o s e d a c i d i f i c a t i o n s w e r e e m p l o y e d a t p H 0 6 . 8 i n s t e a d o f t h e n o r m a l 7 . 3 5 . T h e l o w e r i n g o f p H 0 i n d e e d c a u s e d a r e d u c t i o n i n t h e 1 3 0 m i n i m u m p H j r e a c h e d d u r i n g t h e N H 4 + - i n d u c e d a c i d l o a d . A s a r e s u l t , t h e r a t e o f p H j r e c o v e r y i n c r e a s e d , b u t t h e a p p l i c a t i o n o f D I D S d u r i n g t h i s p r o c e d u r e h a d n o a p p r e c i a b l e e f f e c t . T h o u g h t h i s s u g g e s t s a p o s s i b l e c o n n e c t i o n b e t w e e n t h e r a t e o f p H j r e c o v e r y a n d p H 0 , t h e o b s e r v e d 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 o u l d n o t b e a t t r i b u t e d t o t h e a c t i v a t i o n o f N a + - i n d e p e n d e n t HCO3VCI" e x c h a n g e . I n s u m m a r y , t h e r e g u l a t i o n o f p H j a t 3 7 ° C ( p H 0 7 . 3 - 7 . 4 ) i n c u l t u r e d h i p p o c a m p a l n e u r o n e s i s p r i m a r i l y g o v e r n 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 , H C 0 3 " - i n 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 . T h e m o s t l i k e l y c a n d i d a t e f o r t h i s m e c h a n i s m i s a n a m i l o r i d e - i n s e n s i t i v e N a + / H + e x c h a n g e r , a s s u g g e s t 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 ) . R e g u l a t i o n o f p H j a t r o o m t e m p e r a t u r e : A t r o o m t e m p e r a t u r e , a n d i n n e u r o n e s s u p e r f u s e d w i t h H C C > 3 " - f r e e H E P E S - b u f f e r e d m e d i u m ( p H 0 7 . 3 2 ) , s t e a d y - s t a t e p H j w a s 6 . 8 5 , w h i c h i s c o n s i d e r a b l y l o w e r t h a n t h e v a l u e o f p H j 7 . 2 3 o b s e r v e d a t 3 7 ° C u n d e r s i m i l a r b u f f e r i n g c o n d i t i o n s . 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 [ N a + ] 0 f r o m t h e p e r f u s a t e r e s u l t e d i n a f a l l i n p H j s i m i l a r t o t h a t o b s e r v e d a t 3 7 ° C ( s e e F i g u r e 1 7 ) s u g g e s t i n g t h a t t h e N a + - d e p e n d e n t , H C C ^ ' - i n 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 d e s c r i b e d a t 3 7 ° C c o n t i n u e s t o o p e r a t e a t r o o m t e m p e r a t u r e . H o w e v e r , i n c o n t r a s t t o o b s e r v a t i o n s a t 3 7 ° C , t h e a d d i t i o n o f H C O 3 " t o t h e p e r f u s i o n s o l u t i o n c a u s e d p H j t o r i s e t o t h e s u b s t a n t i a l l y h i g h e r l e v e l o f 7 . 1 5 . M o r e o v e r , 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 f r o m H C 0 3 " - f r e e t o 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 a t c o n s t a n t p H 0 w a s b l o c k e d b y t h e a p p l i c a t i o n o f t h e a n i o n e x c h a n g e i n h i b i t o r D I D S . 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 H C O 3 7 C O 2 a t r o o m t e m p e r a t u r e , 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 H C O 3 7 O " e x c h a n g e . I n e x p e r i m e n t s c a r r i e d o u t i n t h e a b s e n c e o f [ N a + ] 0 , t h e t r a n s i t i o n i n t o f r o m H C C ^ ' - f r e e t o H C 0 3 " - c o n t a i n i n g m e d i u m s i m i l a r l y p r o d u c e d a n i n c r e a s e i n p H j , w h i c h f u r t h e r 1 3 1 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 a t r o o m t e m p e r a t u r e i s N a + - i n d e p e n d e n t . 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 , t h e 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 u n i t s . T h i s a c i d i f i c a t i o n w a s l i k e l y a r e s u l t o f t h e i n h i b i t i o n o f t h e N a + - i n d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e w h i c h t h e r e f o r e m u s t p a r t i c i p a t e i n t h e r e g u l a t i o n o f p H j a t t h i s t e m p e r a t u r e . F u r t h e r m o r e , t h e r e m o v a l o f [ C l " ] 0 c a u s e d a n a p p r o x i m a t e 0 . 3 p H u n i t 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 , p r o b a b l y d u e t o a 0 [ C l " ] 0 - 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 d e s c r i b e d p r e v i o u s l y b y G a i l l a r d a n d D u p o n t ( 1 9 9 0 ) . T h e s e r e s u l t s s u g g e s t t h a t t h e 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 a c t i v i t y o f t h i s a n i o n 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 t h u s a c t s a s a c e l l a l k a l i n i z i n g m e c h a n i s m , i s 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 r e s t i n g p H , o b s e r v e d i n t h e p r e s e n c e a s o p p o s e d t o t h e a b s e n c e o f H C 0 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 a t r o o m t e m p e r a t u r e w e r e , i n g e n e r a l , s l o w e r t h a n t h o s e f o u n d a t 3 7 ° C ( s e e F i g u r e 3 5 ) . A t t h i s r e d u c e d t e m p e r a t u r e a n d i n c o n t r a s t t o r e s u l t s o b t a i n e d a t 3 7 ° C , t h e r a t e o f p H , 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 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 , t h e a d d i t i o n o f H C 0 3 " t o t h e p e r f u s i o n m e d i u m a t 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 s u b s t a n t i a l e n h a n c e m e n t o f t h e r e c o v e r y r a t e , w h i c h p o s s i b l y r e f l e c t s t h e a c t i v a t i o n o f 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" e x c h a n g e ( e i t h e r N a + - d e p e n d e n t o r N a + - i n d e p e n d e n t ) h a s a l s o b e e n d e m o n s t r a t e d i n l a m p r e y r e t i c u l o s p i n a l 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 ( C h e s l e r , 1 9 8 6 ) . T h e p r e s e n c e o f H C O 3 7 C O 2 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 ( s e e R o o s a n d B o r o n , 1 9 8 1 ) , w h i c h w o u l d i n t u r n c o n t r i b u t e t o r e d u c e t h e m a g n i t u d e a n d d u r a t i o n o f i m p o s e d a c i d t r a n s i e n t s . I n d e e d , a c o m p a r i s o n o f t h e n e t d e c r e a s e s i n p H j c a u s e d b y t h e N H 4 + p r e p u l s e a t 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 ) i n d i c a t e s t h a t a 1 3 2 g r e a t e r a c i d i f i c a t i o n w a s o b t a i n e d u n d e r H C 0 3 " - f r e e c o n d i t i o n s t h a n u n d e r c o n d i t i o n s 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 . H o w e v e r , t h e e f f e c t s o f b u f f e r i n g c a p a c i t y o n H C 0 3 ' - i n d u c e d i n c r e a s e s i n p H , r e c o v e r y r a t e s a t r o o m t e m p e r a t u r e w e r e l i k e l y o v e r s h a d o w e d b y t h e c o n t r i b u t i o n o f a n i o n e x c h a n g e . I n f a c t , s e v e r a l o t h e r o b s e r v a t i o n s i n d i c a t e t h a t t h e N a + - i n d e p e n d e n t H C O 3 7 O " e x c h a n g e r i s a c t i v e l y i n v o l v e d i n t h e r e c o v e r y o f p H , f r o m a n i m p o s e d a c i d l o a d a t t h i s 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", 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 t h a t 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 r e s u l t s u g g e s t s t h a t N a + - i n d e p e n d e n t H C O 3 7 Q " 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"- c o n t a i n i n g s t e a d y - s t a t e p e r f u s i o n a t r o o m t e m p e r a t u r e F u r t h e r m o 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 , t h e r e m o v a l o f [ C l " ] 0 e n h a n c e d t h e r a t e o f p H j r e c o v e r y . A s n o t e d a b o v e , r e m o v a l o f e x t e r n a l C l " l i k e l y i n c r e a s e s t h e a c t i v i t y o f H C 0 3 7 C 1 " e x c h a n g e a n d t h u s t h e 0 [ C l " ] 0 - i n d u c e d i n c r e a s e i n t h e p H j r e c o v e r y r a t e m a y b e d u e t o a n a c c e l e r a t i o n o f t h e a n i o n e x c h a n g e r . T h i s p o s s i b i l i t y i s c o n f i r m e d b y t h e o b s e r v e d i n h i b i t o r y i n f l u e n c e o f D I D S o n t h e r a t e o f p H j r e c o v e r y a t r o o m t e m p e r a t u r e d u r i n g H C O 3 7 C O 2 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 " ] 0 . H o w e v e r , t h e f a c t t h a t D I D S d i d n o t c 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 i n d i c a t e s t h a t o t h e r r e g u l a t o r s o f p H j , s u c h a s t h e N a + - d e p e n d e n t , H C O 3 " - i n 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 , m a y a l s o b e o p e r a t i o n a l a t r o o m t e m p e r a t u r e . O v e r a l l , t h e r e s u l t s o f t h i s s t u d y s u g g e s t t h a t , a t r o o m t e m p e r a t u r e , a c o m b i n a t i o n o f 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 e x t r u s i o n m e c h a n i s m a n d a 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 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 n e u r o n e s a t s t e a d y - s t a t e a s w e l l a s d u r i n g p H j r e c o v e r y f r o m a n i n d u c e d 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 . 1 3 3 Comparison of pHj regulation at 37°C and room temperature: B a s e d e i t h e r o n s t e a d y - s t a t e p H j o b s e r v a t i o n s o r o n d a t a f r o m a c i d - l o a d r e c o v e r i e s , t h e 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 a t 3 7 ° C o r a t r o o m t e m p e r a t u r e w e r e v e r y s i m i l a r . B o t h a t 3 7 ° C a n d a t 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 t o b e p r e d o m i n a n t 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 , H C 0 3 " - i n 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 o b a b l y a n a m i l o r i d e - i n s e n s i t i v e v a r i a n t o f t h e N a + / H + e x c h a n g e r . T h e d o m i n a n c e o f N a + / H + 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 a v a r i e t y o f v e r t e b r a t e c e n t r a l n e u r o n e s a t 3 7 ° C ( N a c h s h e n a n d D r a p e a u , 1 9 8 8 ; 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 ) . 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 p r i m a r y a c i d e x t r u s i o n m e c h a n i s m i n C A 1 p y r a m i d a l n e u r o n e s a c u t e l y d i s s o c i a t e d f r o m 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 . T h e f i n d i n g s o f 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 c u l t u r e d f o e t a l h i p p o c a m p a l n e u r o n e s i s n o t d e p e n d e n t o n [ N a + ] 0 , 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 o f t h i s a n i o n e x c h a n g e r a p p e a r s t o b e m i n i m a l 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 a t 3 7 ° C . A s t h e 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 w e r e c u l t u r e d f r o m h i p p o c a m p i o b t a i n e d f r o m f o e t a l r a t s , t h e d i f f e r e n c e b e t w e e n t h e p r e s e n t r e s u l t s a n d t h o s e r e p o r t e d b y S c h w i e n i n g a n d B o r o n ( 1 9 9 4 ) m a y r e f l e c t d e v e l o p m e n t a l c h a n g e s . 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 d e m o n s t r a t e d i n e i t h e r a c u t e l y d i s s o c i a t e d o r c u l t u r e d f o e t a l r a t h i p p o c a m p a l n e u r o n e s a t 3 7 ° C , a c t i v e l y r e g u l a t e s p H j i n a c u t e l y d i s s o c i a t e d a d u l t n e u r o n e s . H o w e v e r , t h i s o b s e r v a t i o n m a y r e f l e c t t h e f a c t t h a t H C 0 3 " / C 1 " e x c h a n g e i s s i m p l y n o t a c t i v e i n f o e t a l n e u r o n e s a t 3 7 ° C , r a t h e r t h a n b e i n g c o m p l e t e l y a b s e n t . 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 t h a t b o t h a d u l t a n d f o e t a l n e u r o n e s e x p r e s s m R N A f o r t h i s a n i o n e x c h a n g e r . T h e r e s u l t s o f t h e p r e s e n t i n v e s t i g a t i o n s u g g e s t t h a t , r a t h e r t h a n b e i n g a b s e n t f r o m c u l t u r e d f o e t a l r a t 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 , t h e N a + - i n d e p e n d e n t HCO3VCI" 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 a t a r e d u c e d t e m p e r a t u r e . T h e r e w e r e , i n a d d i t i o n , o t h e r d i f f e r e n c e s i n s t e a d y - s t a t e p H j c a u s e d b y c h a n g i n g t h e t e m p e r a t u r e o f 134 t h e p e r f u s i o n m e d i u m f r o m 3 7 ° 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 w e r e 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 i n c o m p a r i s o n t o 3 7 ° C . I n a d d i t i o n , r e s t i n g p H j a t 3 7 ° 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 h i g h e r i n t h e p r e s e n c e 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 e n h a n c e d w h e n t h e t e m p e r a t u r e o f t h e p e r f u s a t e w a s i n c r e a s e d t o 3 7 ° C . 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 i n t h e r e g u l a t o r s o f p H j m a y r e f l e c t d i f f e r e n t i a l a c t i v i t i e s o f t h e a c i d e x t r u s i o n m e c h a n i s m s p r e s e n t , o r p e r t u r b a t i o n s i n t h e i n t r a c e l l u l a r b u f f e r i n g c a p a c i t y . B u f f e r i n g c a p a c i t y i s t h e a b i l i t y o f c e l l s t o r e s i s t c h a n g e s i n p H j i m p o s e d b y t h e 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 t o t a l 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 T ) i s d e f i n e d 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 c a u s e d 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 H C O 3 7 C O 2 i n a s y s t e m ( p b ) . p ^ , , a t c o n s t a n t C 0 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 H C O 3 V C O 2 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 . 3 x [ H C C » 3 ~ ] ( s e e R o o s a n d B o r o n , 1981). 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 . s u l p h a t e s , p h o s p h a t e s , a n d p r o t e i n s ) , b i o c h e m i c a l b u f f e r i n g , a n d o r g a n e l l a r b u f f e r i n g ( s e e I n t r o d u c t i o n ) . T o c a l c u l a t e t h e t o t a l 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 , a k n o w n c o n c e n t r a t i o n o f w e a k a c i d o r b a s e i s a p p l i e d t o t h e c e l l s a n d , u s i n g t h e d i s s o c i a t i o n c o n s t a n t , t h e 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 s c a l c u l a t e d ; t h e e n s u i n g c h a n g e i n p H j i s t h e n u s e d t o d e t e r m i n e t h e b u f f e r i n g c a p a c i t y . E x p e r i m e n t s i n w h i c h T M A o r p r o p i o n a t e w e r e a p p l i e d t o t h e 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 p r o v i d e d a p 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 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 . 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 T M A 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 p r o p i o n a t e i n d u c e d 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 . A c c o r d i n g t o t h e a b o v e r a t i o n a l e , t h e i m p o s e d c h a n g e s i n p H j s h o u l d h a v e p r o v i d e d s u f f i c i e n t i n f o r m a t i o n t o c a l c u l a t e b u f f e r i n g p o w e r . H o w e v e r , e r r o r s a r e i n t r o d u c e d i n t o t h e c o m p u t a t i o n o f b u f f e r i n g v a l u e s b y t h e o p e r a t i o n o f p H j 1 3 5 r e g u l a t i n g m e c h a n i s m s w h i c h a r e a c t i v a t e d a s s o o n a s a n i n t r a c e l l u l a r a c i d o r a l k a l i l o a d i s i m p o s e d o n t h e c e l l s . T h i s p r o b l e m c a n o n l y b e a l l e v i a t e d b y t h e a p p l i c a t i o n o f p h a r m a c o l o g i c a l i n h i b i t o r s w h i c h a c t t o b l o c k t h e s e r e g u l a t o r s ( s e e R o o s a n d B o r o n , 1 9 8 1 ; V a u g h a n - J o n e s a n d W u , 1 9 9 0 ) . U n f o r t u n a t e l y , t h e p h a r m a c o l o g i c a l b l o c k a d e o f 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 t h e i n s e n s i t i v i t y o f t h e n e u r o n e s t o i n h i b i t o r s o f c a t i o n e x c h a n g e , w h i c h a p p e a r s t o b e t h e d o m i n a n t m e c h a n i s m o f p H j r e g u l a t i o n . D e s p i t e t h e s e d i f f i c u l t i e s , a n a p p r o x i m a t i o n o f i n t r a c e l l u l a r b u f f e r i n g c a p a c i t y i n t h e s e n e u r o n e s w a s o b t a i n e d b y a n a l y z i n g d a t a r e s u l t i n g f r o m t h e a p p l i c a t i o n o f N H 4 + . W h e n e x p o s e d t o e x t r a c e l l u l a r N H 4 C 1 , p H j i n c r e a s e s d u e t o t h e i n f l u x o f N H 3 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 t o f o r m N H 4 + a n d O H " . K n o w i n g t h e c o n c e n t r a t i o n o f t h e a p p l i e d N H 4 C I , 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 PT. T h e l i m i t a t i o n o f t h i s t e c h n i q u e , i n a d d i t i o n t o t h e p r e v i o u s l y d i s c u s s e d i n a b i l i t y t o p r e v e n t e r r o r s i n t r o d u c e d b y t h e a c t i v a t i o n o f p H j r e g u l a t i n g m e c h a n i s m s , i s t h a t i s d o e s n o t 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 4 " 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 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 N H 4 C I , 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 t o a n e l e c t r o c h e m i c a l d r i v i n g f o r c e . M o r e o v e r , t h e r e i s e v i d e n c e t h a t a N a + / K + p u m p , i f p r e s e n t , c a n c a r r y N H 4 + i n t o c e l l s i n p l a c e o f K + ( s e e B o r o n , 1 9 8 9 ) . S i n c e t h e e x p e r i m e n t s p e r f o r m e d c o u l d n o t e x c l u d e t h e e f f e c t s o f t h e s e p h e n o m e n a , t h e r e s u l t s c o u l d n o t b e u s e d t o f o r m a l l y d e t e r m i n e b u f f e r i n g p o w e r . H o w e v e r , b y m e a s u r i n g t h e 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 N H 4 + u n d e r t h e v a r i o u s e x p e r i m e n t a l c o n d i t i o n s , a n i n d i c a t i o n o f t h e n e u r o n a l a b i l i t y t o b u f f e r i m p o s e d p H j s h i f t s w a s o b t a i n e d ( s e e c o l u m n 3 , T a b l e 6 ) . T h e m a g n i t u d e o f t h e p H j i n c r e a s e r e s u l t i n g f r o m a 3 m i n u t e e x p o s u r e t o N H 4 + w a s g r e a t e s t i n n e u r o n e s l a c k i n g H C 0 3 " a t r o o m t e m p e r a t u r e . T h i s i n c r e a s e w a s a t t e n u a t e d b y t h e p r e s e n c e o f H C 0 3 " d u r i n g t h e a p p l i c a t i o n o f N H 4 + . T h e r e f o r e , a t r o o m t e m p e r a t u r e , t h e s e n e u r o n e s a r e b e t t e r a b l e t o r e s i s t p H ; c h a n g e s w h e n H C 0 3 " a n d C 0 2 1 3 6 a r e p r e s e n t i n t h e e x t r a c e l l u l a r m e d i u m . C 0 2 - i n d u c e d e n h a n c e m e n t o f i n t e r n a l b u f f e r i n g 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 v a r i e t y 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 , 1 9 7 6 b ) , a n d t h u s i n c r e a s e d r e s i s t a n c e t o a l k a l i n e p H j s h i f t s p r o b a b l y r e f l e c t s t h e c o n t r i b u t i o n o f H C 0 3 7 C 0 2 - 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 b ) . I n c r e a s i n g t h e t e m p e r a t u r e o f t h e p e r f u s i o n m e d i u m t o 3 7 ° C a l s o h a d a p r o f o u n d e f f e c t o n t h e 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 . 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 4 + a t 3 7 ° C p r o d u c e d a p H ; r i s e l e s s 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 t h e p e r f u s i o n s o l u t i o n a t 3 7 ° C , t h e p H j r i s e w a s f u r t h e r r e d u c e d t o a p p r o x i m a t e l y 0 . 1 o f a p H u n i t . T h e i n t e r n a l b u f f e r i n g c a p a c i t y a t 3 7 ° C i s t h e r e f o r e a l s o e n h a n c e d b y t h e p r e s e n c e o f C 0 2 a n d HCO3-. F u r t h e r m o r e , t h e r e s u l t s s u g g e s t t h a t , w h e t h e r i n t h e p r e s e n c e o r a b s e n c e o f 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 a t 3 7 ° C . T e m p e r a t u r e c o n s i d e r a t i o n s r e g a r d i n g i n t r a c e l l u l a r b u f f e r i n g h a v e n o t b e e n w e l l d o c u m e n t e d ( s e e R o o s a n d B o r o n , 1 9 8 1 ) . H o w e v e r , B u r t o n ( 1 9 7 8 ) i n d i c a t e s t h a t t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t s o f a c i d - b a s e p a i r s w h i c h c o n t r i b u t e t o p h y s i o c h e m i c a l b u f f e r i n g m a y b e s i g n i f i c a n t l y a l t e r e d b y f a c t o r s s u c h a s t e m p e r a t u r e . I n a d d i t i o n t o p h y s i o c h e m i c a l b u f f e r i n g , t h e r e m a y a l s o b e a n i n c r e a s e i n b i o c h e m i c a l a n d o r g a n e l l a r b u f f e r i n g a s s o c i a t e d w i t h a n e l e v a t i o n i n t e m p e r a t u r e . A c c o r d i n g l y , t h e e f f e c t s o f t e m p e r a t u r e o n t h e r e g u l a t i o n o f p H j m a y r e f l e c t , a t l e a s t i n p a r t , t h e t e m p e r a t u r e d e p e n d e n c e o f i n t r a c e l l u l a r b u f f e r i n g m e c h a n i s m s , a l t h o u g h o t h e r m e c h a n i s m s a r e l i k e l y t o p a r t i c i p a t e i n t h e o b s e r v e d t e m p e r a t u r e e f f e c t s , i n c l u d i n g t e m p e r a t u r e - d e p e n d e n t c h a n g e s i n a c i d e x t r u s i o n r a t e s a n d p a s s i v e f l u x e s o f a c i d e q u i v a l e n t s ( s e e R o o s a n d B o r o n , 1 9 8 1 ) . I n s u m m a r y , 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 , t h e r e s u l t i n g s t e a d y - s t a t e p H j d u r i n g p e r f u s i o n w i t h H C 0 3 " / C 0 2 - b u f f e r e d m e d i u m ( p H 0 7 . 3 5 ) i s a p p r o x i m a t e l y 7 . 1 5 . T h i s v a l u e m a y b e t h e o p t i m a l l e v e l a s s u m e d b y p H j u n d e r c o n d i t i o n s t h a t a l l o w f o r t h e e x p r e s s i o n o f a l l p H j r e g u l a t i n g m e c h a n i s m s . W h e n HCO3" i s r e m o v e d a t e i t h e r t e m p e r a t u r e , p H , d e v i a t e s f r o m t h i s o p t i m a l v a l u e . A t r o o m t e m p e r a t u r e , p H j f a l l s 1 3 7 s u b s t a n t i a l l y w h e n s w i t c h e d t o H E P E S - b u f f e r e d p e r f u s i o n s o l u t i o n s , w h e r e a s a t 3 7 ° C , p H j i n c r e a s e s , a l b e i t o n l y s l i g h t l y , w h e n HCO3" i s n o t p r e s e n t . T h e a b i l i t y o f t h e 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 H C O 3 " a t 3 7 ° C m a y r e f l e c t a t e m p e r a t u r e - d e p e n d e n t a c c e n t u a t i o n o f t h e i n t e r n a l b u f f e r i n g p o w e r . A t r o o m t e m p e r a t u r e , t h i s 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 i s d i m i n i s h e d , a n d t h u s t h e n e u r o n e s m a y r e s o r t t o o t h e r m e a n s , s u c h a s i n c r e a s e d a c t i v i t y o f N a + - i n d e p e n d e n t H C 0 3 7 C T e x c h a n g e , t o r e g u l a t e s t e a d y - s t a t e p H j t o t h e o p t i m a l l e v e l . I n t r a c e l l u l a r b u f f e r i n g t h e r e f o r e c o n t r i b u t e s t o 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 i s d e p e n d e n t o n t h e e x p e r i m e n t a l t e m p e r a t u r e , a n d t h u s m a y e x p l a i n t h e 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 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 a n d a t 3 7 ° C . M o d u l a t i o n o f p H j b y p H 0 : I n t h e p r e s e n c e o f H C 0 3 " , a s h i f t i n p H 0 c a u s e d a q u a l i t a t i v e l y s i m i l a r s h i f t i n p H j , a t b o t h r o o m t e m p e r a t u r e a n d a t 3 7 ° C . T h i s o b s e r v a t i o n d e m o n s t r a t e s t h e d e p e n d e n c e o f p H j o n p H 0 , w h i c h h a s b e e n d o c u m e n t e d i n o t h e r v e r t e b r a t e n e u r o n e s ( T o l k o v s k y a n d R i c h a r d s , 1 9 8 7 ; N a c h s h e n a n d D r a p e a u , 1 9 8 8 ; O u - y a n g et al, 1 9 9 3 ) a n d i n d i c a t e s t h a t n e u r o n a l p H j c a n n o t b e r e g u l a t e d b a c k t o n o r m a l l e v e l s u n t i l p H 0 i s n o r m a l i z e d . I n t u r n , t h i s s u g g e s t s t h e p o s s i b i l i t y t h a t t h e m e c h a n i s m s r e s p o n s i b l e f o r t h e m a i n t e n a n c e o f p H j m a y b e s u b j e c t t o m o d u l a t i o n b y t h e e x t r a c e l l u l a r p r o t o n e n v i r o n m e n t . I n d e e d , a s d i s c u s s e d a b o v e , a d e c r e a s e i n p H 0 c a u s e s a n i n c r e a s e i n t h e r a t e o f r e c o v e r y f r o m a n i n d u c e d 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 . M o r e o v e r , t h e d e p e n d e n c e o f p H j o n p H 0 s u g g e s t s t h a t c h a n g e s i n n e u r o n a l e x c i t a b i l i t y w h i c h t o d a t e h a v e b e e n a t t r i b u t e d t o c h a n g e s i n p H 0 ( s e e I n t r o d u c t i o n ) s h o u l d b e c a r e f u l a n a l y z e d s i n c e t h e y m a y i n f a c t r e f l e c t t h e a c c o m p a n y i n g a l t e r a t i o n i n p H j , r a t h e r t h a n j u s t b e i n g d u e t o c h a n g e s i n p H 0 p e r s e . 1 3 8 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. T h e f i g u r e s h o w s p H j r e c o v e r y f r o m a n N H 4 + - i n d u c e d a c i d i f i c a t i o n 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 ( • ) , 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 n t h e p r e s e n c e o f H C O 3 - 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 3 " a t 3 7 ° C ( A ) . p H 0 r a n g e d f r o m 7 . 3 3 t o 7 . 3 6 u n d e r t h e f o u r c o n d i t i o n s . T h e s o l i d l i n e s r e p r e s e n t a l e a s t 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 s e p a r a t e e x p e r i m e n t s u n d e r t h e c o n d i t i o n s s p e c i f i e d . L i n e s b e g i n a t t h e m i n i m u m p H , r e a c h e d d u r i n g t h e a c i d i f i c a t i o n , a n d r e p r e s e n t t h e t r e n d f o u n d i n a l l e x p e r i m e n t s c o n d u c t e d u n d e r s i m i l a r c o n d i t i o n s . 1 3 9 T i m e ( m i n u t e s ) 1 4 0 C o n c l u s i o n s : T h e m e c h a n i s m s o p e r a t i n g t o r e g u l a t e p H j w e r e e x a m i n e d a t s t e a d y - s t a t e o r d u r i n g 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 i f i c a t i o n i n c u l t u r e d f o e t a l r a t 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 . T e m p e r a t u r e w a s f o u n d t o e x e r t a p r o f o u n d e f f e c t o n t h e r e l a t i v e a c t i v i t i e s o f t h e s e m e c h a n i s m s ( s e e F i g u r e 3 6 ) . A t 3 7 ° C , t h e p r i m a r y r e g u l a t o r o f p H ; a p p e a r s t o b e a N a + - d e p e n d e n t , H C C ^ ' - i n 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 o b a b l y a n a m i l o r i d e i n s e n s i t i v e v a r i a n t o f t h e N a + / H + e x c h a n g e r ) . A t r o o m t e m p e r a t u r e , t h e r e s u l t s o f t h i s s t u d y s u g g e s t t h a t p H j i s r e g u l a t e d b y a N a + - i n d e p e n d e n t HCO37CI" e x c h a n g e r , w h i c h p r o b a b l y a c t s t o s u p p l e m e n t t h e a c t i v i t y o f t h e s a m 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 o b s e r v e d a t 3 7 ° C . T h e d a t a d o n o t s u p p o r t n o r e x c l u d e t h e e x i s t e n c e o f a N a + - d e p e n d e n t H C 0 3 7 C 1 " e x c h a n g e r o n t h e s e n e u r o n e s . H o w e v e r , i f p r e s e n t , t h i s N a + - d e p e n d e n t a n i o n t r a n s p o r t e r w o u l d p r e s u m a b l y p l a y a m i n o r r o l e i n t h e 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 a c i d e x t r u s i o n m e c h a n i s m g o v e r n i n g p H j , e s p e c i a l l y a t 3 7 ° C . A s n o t e d i n t h e I n t r o d u c t i o n , p H j i s a n i m p o r t a n t m o d u l a t o r o f m a n y 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 e v e n t s . T h e i n v e s t i g a t i o n o f t h e m e c h a n i s m s t h a t a r e d i r e c t l y r e s p o n s i b l e f o r r e g u l a t i n g p H j m a y t h e r e f o r e h e l p t o p r o v i d e i n f o r m a t i o n r e g a r d i n g t h e r o l e o f p H j i n s u c h e v e n t s . F u t u r e e x p e r i m e n t a l d i r e c t i o n s i n c l u d e a n i n v e s t i g a t i o n o f t h e p o s s i b l e m o d u l a t i o n o f p H j r e g u l a t o r y m e c h a n i s m s b y n e u r o t r a n s m i t t e r c a n d i d a t e s , a n d w a y s i n w h i c h t h e e f f e c t s o f a p p l i e d n e u r o m o d u l a t o r s a r e i n t u r n r e g u l a t e d b y p H j . D e m o n s t r a t e d o n n o n - n e u r o n a l c e l l t y p e s , t h e a c t i v a t i o n o f v a r i o u s c e l l s u r f a c e r e c e p t o r s , i n c l u d i n g P - a d r e n e r g i c , o ^ - a d r e n e r g i c , s o m a t o s t a t i n , D 2 - d o p a m i n e r g i c , a n d m u s c a r i n i c c h o l i n e r g i c r e c e p t o r s , d i r e c t l y r e g u l a t e s t h e a c t i v i t y o f 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, 1 9 9 0 ) . A c c o r d i n g l y , i t 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 , s o m a t o s t a t i n , d o p a m i n e , a n d s e r o t o n i n , o n t h e p H j r e g u l a t i n g m e c h a n i s m s p r e s e n t o n h i p p o c a m p a l n e u r o n e s . S u c h a n i n v e s t i g a t i o n w o u l d i n c l u d e a n e x a m i n a t i o n o f t h e 1 4 1 e f f e c t s o f i n t r a c e l l u l a r s e c o n d m e s s e n g e r s o n p H , , s i n c e c h a n g e s i n c y t o p l a s m i c l e v e l s o f c A M P , C a 2 + , a n d c e r t a i n p r o t e i n k i n a s e s h a v e b e e n s h o w n t o m o d u l a t e p H j i n a v a r i e t y o f p e r i p h e r a l c e l l t y p e s ( G r i n s t e i n a n d R o t h s t e i n , 1 9 8 6 ) . I n a r e c e n t s t u d y o f r a t b r a i n s y n a p t o s o m e s , S a n c h e z - A r m a s s et al ( 1 9 9 4 ) h a v e i n d i c a t e d t h a t C a 2 + , a c t i n g i n t r a c e l l u l a r l y , m a y p l a y a n i m p o r t a n t r o l e i n t h e m o d u l a t i o n o f N a + / H + e x c h a n g e . p H , w a s n o t a l t e r e d b y e n h a n c i n g t h e c y t o s o l i c l e v e l s o f p r o t e i n k i n a s e C o r k i n a s e A ( S a n c h e z - A r m a s s et al, 1 9 9 4 ) . I n a d d i t i o n , i t i s 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 a p h y s i o l o g i c a l l y - r e l e v a n t i n t e r d e p e n d e n c e e x i s t s b e t w e e n p H j a n d C a 2 + . F o r e x a m p l e , 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 2 + c u r r e n t s i n c a t f i s h h o r i z o n t a l c e l l s a r e s u p p r e s s e d d u r i n g g l u t a m a t e a p p l i c a t i o n a s a r e s u l t o f a n a s s o c i a t e d i n c r e a s e i n i n t r a c e l l u l a r p r o t o n l e v e l s . 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 t h a t a n N M D A - i n d u c e d i n t r a c e l l u l a r a c i d o s i s o b s e r v e d i n r a t h i p p o c a m p a l n e u r o n e s i s d e p e n d e n t o n C a 2 + i n f l u x r e s u l t i n g f r o m t h e N M D A a p p l i c a t i o n . T h e r e f o r e , f u r t h e r 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 i t s a b i l i t y t o m o d u l a t e s o m e o f t h e i n t r a c e l l u l a r p r o c e s s e s w h i c h t h u s f a r h a v e b e e n a t t r i b u t e d t o c h a n g e s i n c y t o s o l i c C a 2 + a n d o t h e r i n t r a c e l l u l a r s e c o n d m e s s e n g e r s . 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, HC03"-independent acid extrusion mechanism observed at 37°C. 1 4 3 3 7 ° C Room temperature OUT I IN 1 Na + 1 HCO, OUT 1 IN 1 N a + - ^ S*-1 H+ 1 HC0 3 - OTc- i 1 4 4 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 . ( 1 9 8 0 ) . I n t r a c e l l u l a r p H c h a n g e s i n d u c e d b y c a l c i u m 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, 4 0 3 - 4 2 6 . A i c k e n , C . C . ( 1 9 8 4 ) . D i r e c t m e a s u r e m e n t o f i n t r a c e l l u l a r p H a n d b u f f e r i n g p o w e r i n s m o o t h m u s c l e c e l l s o f g u i n e a - p i g v a s d e f e r e n s . Journal of Physiology 349, 5 7 1 - 5 8 5 . A i c k e n , C . C , a n d T h o m a s , R . C . ( 1 9 7 7 ) . A n i n v e s t i g a t i o n o f t h e i o n i c m e c h a n i s m o f i n t r a c e l l u l a r p H r e g u l a t i o n i n m o u s e s o l e u s m u s c l e f i b r e s . Journal of Phyiology 273, 2 9 5 - 3 1 6 . A m o r o s o , S . , D i R e n z o , G . , T a g l i a l a t e l a , M . , C a n z o n i e r o , L . M . T . , C r a g o e , E . J . , a n d A n n u n z i a t o , L . ( 1 9 9 1 ) . C y t o p l a s m i c a l k a l i n i z a t i o n i n d u c e d b y i n s u l i n t h r o u g h a n a c t i v a t i o n o f N a + - H + a n t i p o r t e r i n h i b i t s t y r o s i n e h y d r o x y l a s e a c t i v i t y i n s t r i a t a l s y n a p t o s o m e s . Biochemical Pharmacology 41, 1 2 7 9 - 1 2 8 2 . A m o s , B . J . , a n d R i c h a r d s , C . D . ( 1 9 9 4 ) . E f f e c t o f g l u t a m a t e o n i n t r a c e l l u l a r p H o f r a t n e u r o n e s m a i n t a i n e d i n c u l t u r e . Journal of Physiology 479.P, 4 3 P . A r a m , J . A . , a n d L o d g e , D . ( 1 9 8 7 ) . E p i l e p t i f o r m a c t i v i t y i n d u c e d b y a l k a l o s i s i n r a t n e o c o r t i c a l s l i c e s : b l o c k b y a n t a g o n i s t s o f T V - m e t h y l - D - a s p a r a t e . Neuroscience Letters 83, 3 4 5 - 3 5 0 . A r o n s o n , P . S . ( 1 9 8 5 ) . K i n e t i c p r o p e r t i e s o f t h e p l a s m a m e m b r a n e N a + - H + e x c h a n g e r . Annual Review of Physiology 47, 5 4 5 - 5 6 0 . A r o n s o n , P . S . , a n d B o u n d s , S . E . ( 1 9 8 0 ) . H a r m a l i n e i n h i b i t i o n o f N a - d e p e n d e n t t r a n s p o r t i n r e n a l m i c r o v i l l u s m e m b r a n e v e s i c l e s . American Journal of Physiology 238, F 2 1 0 - F 2 1 7 . A u s t i n , C , a n d W r a y , S . ( 1 9 9 3 ) . E x t r a c e l l u l a r p H s i g n a l s a f f e c t v a s c u l a r t o n e b y r a p i d t r a n s d u c t i o n i n t o i n t r a c e l l u l a r p H c h a n g e s . Journal of Physiology 466, 1 - 8 . B a k e r , P . F . , a n d H o n e r j a g e r , P . ( 1 9 7 8 ) . I n f l u e n c e o f c a r b o n d i o x i d e o n l e v e l o f i o n i s e d c a l c i u m i n s q u i d a x o n s . Nature 273, 1 6 0 - 1 6 1 . B a l e s t r i n o , M . , a n d S o m j e n , G . G . ( 1 9 8 8 ) . C o n c e n t r a t i o n o f c a r b o n d i o x i d e , i n t e r s t i t i a l p H a n d s y n a p t i c t r a n s m i s s i o n i n h i p p o c a m p a l f o r m a t i o n o f t h e r a t . Journal of Physiology 396, 2 4 7 - 2 6 6 . B a n k e r , G . A . , a n d C o w a n , W . M . ( 1 9 7 7 ) . R a t h i p p o c a m p a l n e u r o n s i n d i s p e r s e d c e l l c u l t u r e . Brain Research 126, 3 9 7 - 4 2 5 . 1 4 5 B a r b e r , D . L . , M c G u i r e , M . E . , a n d G a n z , M . B . ( 1 9 8 9 ) . P - a d r e n e r g i c a n d s o m a t o s t a t i n r e c e p t o r s r e g u l a t e N a - H e x c h a n g e i n d e p e n d e n t o f c A M P . Journal of Biological Chemistry 264, 2 1 0 3 8 - 2 1 0 4 2 . B a r n e s , S . , a n d B u i , Q . ( 1 9 9 1 ) . M o d u l a t i o n o f c a l c i u m - a c t i v a t e d c h l o r i d e c u r r e n t v i a p H - i n d u c e d c h a n g e s o f c a l c i u m c h a n n e l p r o p e r t i e s i n c o n e p h o t o r e c e p t o r s . Journal of Neuroscience 11, 4 0 1 5 - 4 0 2 3 . B o r o n , W . F . ( 1 9 8 9 ) . C e l l u l a r b u f f e r i n g a n d i n t r a c e l l u l a r p H . I n : The Regulation of Acid-Base Balance, p p . 3 3 - 5 6 . E d s . D . W . S e l d i n , a n d G . G i e b i s c h . R a v e n P r e s s . B o r o n , W . F . , a n d B o u l p a e p , E . L . ( 1 9 8 3 ) . I n t r a c e l l u l a r p H r e g u l a t i o n i n t h e r e n a l p r o x i m a l t u b u l e o f t h e s a l a m a n d e r . Journal of General Physiology 81, 5 3 - 9 4 . B o r o n , W . F . , a n d D e W e e r , P . ( 1 9 7 6 ) . I n t r a c e l l u l a r p H t r a n s i e n t s i n s q u i d g i a n t a x o n s c a u s e d b y C 0 2 , N H 3 , a n d m e t a b o l i c i n h i b i t o r s . Journal of General Physiology 67, 9 1 - 1 1 2 . B o u n t r a , C , K a i l a , K . , a n d V a u g h a n - J o n e s , R . D . ( 1 9 8 8 ) . E f f e c t o f r e p e t i t i v e a c t i v i t y u p o n i n t r a c e l l u l a r p H , s o d i u m a n d c o n t r a c t i o n i n s h e e p c a r d i a c P u r k i n j e f i b r e s . Journal of Physiology 398, 3 4 1 - 3 6 0 . B o y a r s k i , G . , G a n z , M . B . , S t e r z e l , R . B . , a n d B o r o n , W . F . ( 1 9 8 8 a ) . p H r e g u l a t i o n i n s i n g l e g l o m e r u l a r m e s a n g i a l c e l l s . I . A c i d e x t r u s i o n i n a b s e n c e a n d p r e s e n c e o f HCO3". American Journal of Physiology 255, C 8 4 4 - C 8 5 6 . B o y a r s k i , G . , G a n z , M . B . , S t e r z e l , R . B . , a n d B o r o n , W . F . ( 1 9 8 8 b ) . p H r e g u l a t i o n i n s i n g l e g l o m e r u l a r m e s a n g i a l c e l l s . I I . N a + - d e p e n d e n t a n d - i n d e p e n d e n t 0 " - H C 0 3 ~ e x c h a n g e r s . American Journal of Physiology 255, C 8 5 7 - C 8 6 9 . B r e w e r , G . J . , a n d C o t m a n , C . W . ( 1 9 8 9 ) . S u r v i v a l a n d g r o w t h o f h i p p o c a m p a l n e u r o n s i n d e f i n e d m e d i u m a t l o w d e n s i t y : a d v a n t a g e s o f a s a n d w i c h c u l t u r e t e c h n i q u e o r l o w o x y g e n . Brain Research 494, 6 5 - 7 4 . B r i g h t , G . R . , F i s h e r , G . W . , R o g o w s k a , J . , a n d T a y l o r , D . L . ( 1 9 8 7 ) . F l u o r e s c e n c e r a t i o i m a g i n g m i c r o s c o p y : t e m p o r a l a n d s p a t i a l m e a s u r e m e n t s o f c y t o p l a s m i c p H . Journal of Cell Biology 104, 1 0 1 9 - 1 0 3 3 . B u r t o n , R . F . ( 1 9 7 8 ) . I n t r a c e l l u l a r b u f f e r i n g . Respiration Physiology 33, 5 1 - 5 8 . B u s a , W . B . ( 1 9 8 6 ) . M e c h a n i s m s a n d c o n s e q u e n c e s o f p H - m e d i a t e d c e l l r e g u l a t i o n . Annual Review of Physiology 48, 3 8 9 - 4 0 2 . B u s a , W . B . , a n d N u c c i t e l l i , R . ( 1 9 8 4 ) . M e t a b o l i c r e g u l a t i o n v i a i n t r a c e l l u l a r p H . American Journal of Physiology 246, R 4 0 9 - R 4 3 8 . 1 4 6 C a r b o n e , E . , T e s t a , P . L . , a n d W a n k e , E . ( 1 9 8 1 ) . I n t r a c e l l u l a r p H a n d i o n i c c h a n n e l s i n t h e Loligo vulgaris g i a n t a x o n . Biophysical Journal 35, 3 9 3 - 4 1 3 . C h a i l l e t , J . R . , a n d B o r o n , W . F . ( 1 9 8 5 ) . I n t r a c e l l u l a r c a l i b r a t i o n o f a p H - s e n s i t i v e d y e i n i s o l a t e d , p e r f u s e d s a l a m a n d e r p r o x i m a l t u b u l e s . Journal of General Physiology 86, 7 6 5 - 7 9 4 . C h a n g , D . , K u s h m a n , N . L . , a n d D a w s o n , D . C . ( 1 9 9 1 ) . I n t r a c e l l u l a r p H r e g u l a t e s b a s o l a t e r a l K + a n d C l " c o n d u c t a n c e s i n c o l o n i c e p i t h e l i a l c e l l s b y m o d u l a t i n g C a 2 + a c t i v a t i o n . Journal oj General Physiology 98, 1 8 3 - 1 9 6 . C h e s l e r , M . ( 1 9 8 6 ) . 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 i n r e t i c u l o s p i n a l n e u r o n e s o f t h e l a m p r e y , Petromyzon marinus. Journal of Physiology 381, 2 4 1 - 2 6 1 . C h e s l e r , M . ( 1 9 9 0 ) . T h e r e g u l a t i o n a n d m o d u l a t i o n o f p H i n t h e n e r v o u s s y s t e m . Progress in Neurobiology 34, 4 0 1 - 4 2 7 . C h e s l e r , M . , a n d K a i l a , K . ( 1 9 9 2 ) . M o d u l a t i o n o f p H b y n e u r o n a l a c t i v i t y . Trends in Neuroscience 15, 3 9 6 - 4 0 2 . C h e s l e r , M . , a n d K r a i g , R . P . ( 1 9 8 7 ) . I n t r a c e l l u l a r p H o f a s t r o c y t e s i n c r e a s e s r a p i d l y w i t h c o r t i c a l s t i m u l a t i o n . American Journal of Physiology 22, R 6 6 6 - R 6 7 0 . C h u r c h , J . ( 1 9 9 2 ) . A c h a n g e f r o m H C 0 3 - - C 0 2 - t o H E P E S - b u f f e r e d m e d i u m m o d i f i e s m e m b r a n e p r o p e r t i e s o f r a t C A 1 p y r a m i d a l n e u r o n e s in vitro. Journal of Physiology 4 5 5 , 5 1 - 7 1 . C h u r c h , J . , a n d B a i m b r i d g e , K . G . ( 1 9 9 1 ) . E x p o s u r e t o h i g h - p H m e d i u m i n c r e a s e s t h e i n c i d e n c e a n d e x t e n t o f d y e c o u p l i n g b e t w e e n r a t h i p p o c a m p a l C A 1 p y r a m i d a l n e u r o n s in vitro. Journal of Neuroscience 11, 3 2 8 9 - 3 2 9 5 . C h u r c h , J . , a n d M c L e n n a n , H . ( 1 9 8 9 ) . E l e c t r o p h y s i o l o g i c a l p r o p e r t i e s o f r a t C A 1 p y r a m i d a l n e u r o n e s in vitro m o d i f i e d b y c h a n g e s i n e x t r a c e l l u l a r b i c a r b o n a t e . Journal of Physiology 415, 8 5 - 1 0 8 . C l a r k , J . D . , a n d L i m b i r d , L . L . ( 1 9 9 1 ) . ' N a + - H + e x c h a n g e r s u b t y p e s : a p r e d i c t i v e r e v i e w . American Journal of Physiology 261, C 9 4 5 - C 9 5 3 . C o n n e r , J . A . , a n d H o c k b e r g e r , P . P . ( 1 9 8 4 ) . I n t r a c e l l u l a r p H c h a n g e s i n d u c e d b y i n j e c t i o n s o f c y c l i c n u c l e o t i d e s i n t o g a s t r o p o d n e u r o n e s . Journal of Physiology 354, 1 6 3 - 1 7 2 . C o o k , D . L . , I k e u c h i , M . , a n d F u j i m o t o , W . Y . ( 1 9 8 4 ) . L o w e r i n g o f p H j i n h i b i t s C a 2 + - a c t i v a t e d 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 . N a t u r e 311, 2 6 9 - 2 7 1 . D e u t s c h , C , a n d L e e , S . C . ( 1 9 8 9 ) . M o d u l a t i o n o f K + c u r r e n t s i n h u m a n l y m p h o c y t e s b y p H . Journal of Physiology 413, 3 9 9 - 4 1 3 . 1 4 7 D i c k e n s , C . J . , G i l l e s p i e , J . I . , a n d G r e e n w e l l , J . R . ( 1 9 9 0 ) . M e a s u r e m e n t o f i n t r a c e l l u l a r c a l c i u m a n d p H i n a v i a n n e u r o n a l c r e s t c e l l s . Journal of Physiology 428, 5 3 1 - 5 4 4 . D i x o n , D . B . , T a k a h a s h i , K . - L , a n d C o p e n h a g e n , D . R . ( 1 9 9 3 ) . L - g l u t a m a t e s u p p r e s s e s H V A c a l c i u m c u r r e n t i n c a t f i s h h o r i z o n t a l c e l l s b y r a i s i n g i n t r a c e l l u l a r p r o t o n c o n c e n t r a t i o n . Neuron 11, 261-211. D o e r i n g , A . E . , a n d L e d e r e r , W . J . ( 1 9 9 3 ) . T h e m e c h a n i s m b y w h i c h c y t o p l a s m i c p r o t o n s i n h i b i t t h e s o d i u m - c a l c i u m e x c h a n g e r i n g u i n e a - p i g h e a r t c e l l s . Journal of Physiology 466, 4 8 1 - 4 9 9 . E b i n e , Y . , F u j i w a r a , N . , a n d S h i m o j i , K . ( 1 9 9 4 ) . M i l d a c i d o s i s i n h i b i t s t h e r i s e i n i n t r a c e l l u l a r C a 2 + c o n c e n t r a t i o n i n r e p o n s e t o o x y g e n - g l u c o s e d e p r i v a t i o n i n r a t h i p p o c a m p a l s l i c e s . Neuroscience Letters 168, 1 5 5 - 1 5 8 . E n d r e s , W . , B a l l a n y i , K . , S e r v e , G . , a n d G r a f e , P . ( 1 9 8 6 ) . E x c i t a t o r y a m i n o a c i d s a n d i n t r a c e l l u l a r p H i n m o t o n e u r o n s o f t h e i s o l a t e d f r o g s p i n a l c o r d . Neuroscience Letters 72, 5 4 - 5 8 . F e n n , W . O . , a n d C o b b , D . M . ( 1 9 3 4 ) . T h e p o t a s s i u m e q u i l i b r i u m i n m u s c l e . Journal of General Physiology 17, 6 2 9 - 6 5 6 . F e n n , W . O . , a n d M a u r e r , F . W . ( 1 9 3 5 ) . T h e p H o f m u s c l e . Protoplasma 28, 3 3 7 - 3 4 5 . F i t z , J . G . , L i d o f s k y , S . D . , X i e , M . - H . , a n d S c h a r s c h m i d t , B . F . ( 1 9 9 2 ) . T r a n s m e m b r a n e e l e c t r i c a l p o t e n t i a l d i f f e r e n c e r e g u l a t e s N a + / H C 0 3 " c o t r a n s p o r t a n d i n t r a c e l l u l a r p H i n h e p a t o c y t e s . Proceedings of the National Academy of Science, U.S.A. 8 9 , 4 1 9 7 - 4 2 0 1 . F o r n a i , F . , D y b d a l , D . J . , P r o c t o r , M . R . , a n d G a l e , K . ( 1 9 9 4 ) . F o c a l i n t r a c e r e b r a l e l e v a t i o n o f L - l a c t a t e i s a n t i c o n v u l s a n t . European Journal of Pharmacology 254, R l - R 2 . F r e u d e n r i c h , C . C , M u r p h y , E . , L e v y , L . A . , L o n d o n , R . E . , a n d L i e b e r m a n , M . ( 1 9 9 2 ) . I n t r a c e l l u l a r p H m o d u l a t e s c y t o s o l i c f r e e m a g n e s i u m i n c u l t u r e d c h i c k e n h e a r t c e l l s . American Journal of Physiology 262, C 1 0 2 4 - C 1 0 3 0 . G a i l l a r d , S . , a n d D u P o n t , J . - L . ( 1 9 9 0 ) . I o n i c c o n t r o l o f i n t r a c e l l u l a r p H i n r a t c e r e b e l l a r P u r k i n j e c e l l s m a i n t a i n e d i n c u l t u r e . Journal of Physiology 425, 7 1 - 8 3 . G a n z , M . B . , a n d B o r o n , W . F . ( 1 9 9 4 ) . L o n g - t e r m e f f e c t s o f g r o w t h f a c t o r s o n p H a n d a c i d - b a s e t r a n s p o r t i n r a t g l o m e r u l a r m e s a n g i a l c e l l s . American Journal of Physiology 266, F 5 7 6 - F 5 8 5 . G a n z , M . B . , P a c h t e r , J . A . , a n d B a r b e r , D . L . ( 1 9 9 0 ) . M u l t i p l e r e c e p t o r s c o u p l e d t o a d e n y l a t e c y c l a s e r e g u l a t e N a - H - e x c h a n g e i n d e p e n d e n t o f c A M P . Journal of Biological Chemistry 265, 8 9 8 9 - 8 9 9 2 . 1 4 8 G a n z , M . B . , R a s m u s s e n , J . , B o l l a g , W . B . , a n d R a s m u s s e n , H . ( 1 9 9 0 ) . E f f e c t o f b u f f e r s y s t e m s a n d p H j o n t h e m e a s u r e m e n t o f [ C a 2 + ] j w i t h f u r a - 2 . FASEB Journal 4, 1 6 3 8 - 1 6 4 4 . G i f f a r d , R . G . , M o n y e r , H . , C h r i s t i n e , C . W . , a n d C h o i , D . W . ( 1 9 9 0 ) . A c i d o s i s r e d u c e s N M D A r e c e p t o r a c t i v a t i o n , g l u t a m a t e n e u r o t o x i c i t y , a n d o x y g e n - g l u c o s e d e p r i v a t i o n n e u r o n a l i n j u r y i n c o r t i c a l c u l t u r e s . Brain Research 506, 3 3 9 - 3 4 2 . G o t t f r i e d , J . A . , a n d C h e s l e r , M . ( 1 9 9 4 ) . E n d o g e n o u s H + m o d u l a t i o n o f N M D A r e c e p t o r - m e d i a t e d E P S C s r e v e a l e d b y c a r b o n i c a n h y d r a s e i n h i b i t i o n i n r a t h i p p o c a m p u s . Journal of Physiology 478.3, 3 7 3 - 3 7 8 . H a r r i s , R . J . , R i c h a r d s , P . G . , S y m o n , L . , H a b i b , A . - H . A . , a n d R o s e n s t e i n , J . ( 1 9 8 7 ) . . p H , K + , a n d PO2 o f t h e e x t r a c e l l u l a r s p a c e d u r i n g i s c h a e m i a o f p r i m a t e c e r e b r a l c o r t e x . Journal of Cereberal Blood Flow and Metabolism 7, 5 9 9 - 6 0 4 . H a r t l e y , Z . , a n d D u b i n s k y , J . M . ( 1 9 9 3 ) . C h a n g e s i n i n t r a c e l l u l a r p H a s s o c i a t e d w i t h g l u t a m a t e e x c i t o t o x i c i t y . Journal of Neuroscience 13, 4 6 9 0 - 4 6 9 9 . H e s k e t h , T . R . , M o o r e , J . P . , M o r r i s , J . D . H . , T a y l o r , M . V . , R o g e r s , J . , S m i t h , G . A . , a n d M e t c a l f e , J . C . ( 1 9 8 5 ) . A c o m m o n s e q u e n c e o f c a l c i u m a n d p H s i g n a l s i n t h e m i t o g e n i c s t i m u l a t i o n o f e u k a r y o t i c c e l l s . Nature 313, 4 8 1 - 4 8 4 . H i l l , A . V . ( 1 9 5 5 ) . T h e i n f l u e n c e o f t h e e x t e r n a l m e d i u m o n t h e i n t e r n a l p H o f m u s c l e . Proceedings of the Royal Society London B Series 144, 1 - 2 2 . I r w i n , R . P . , L i n , S . - Z . , L o n g , R . T . , a n d P a u l , S . M . ( 1 9 9 4 ) . 7 V - m e t h y l - D - a s p a r t a t e i n d u c e s a r a p i d , r e v e r s i b l e , a n d c a l c i u m - d e p e n d e n t i n t r a c e l l u l a r a c i d o s i s i n c u l t u r e d f e t a l r a t h i p p o c a m p a l n e u r o n s . Journal of Neuroscience 14, 1 3 5 2 - 1 3 5 7 . I s o m , L . L . , C r a g o e , E . J . , a n d L i m b i r d , L . E . ( 1 9 8 7 ) . M u l t i p l e r e c e p t o r s l i n k e d t o i n h i b i t i o n o f a d e n y l a t e c y c l a s e a c c e l e r a t e N a + / H + e x c h a n g e i n n e u r o b l a s t o m a x g l i o m a c e l l s v i a a m e c h a n i s m o t h e r t h a n d e c r e a s e d c A M P a c c u m u l a t i o n . Journal of Biological Chemistry 262, 1 7 5 0 4 - 1 7 5 0 9 . J a r o l i m e k , W . , M i s g e l d , U . , a n d L u x , H . D . ( 1 9 8 9 ) . A c t i v i t y d e p e n d e n t a l k a l i n e a n d a c i d t r a n s i e n t s i n g u i n e a p i g h i p p o c a m p a l s l i c e s . Brain Research 505, 2 2 5 - 2 3 2 . J o h n s o n , J . D . , E p e l , D . , a n d P a u l , M . ( 1 9 7 6 ) . I n t r a c e l l u l a r p H a n d a c t i v a t i o n o f s e a u r c h i n e g g s a f t e r f e r t i l i z a t i o n . Nature 262, 6 6 1 - 6 6 4 . K a i b a r a , M . , a n d K a m e y a m a , M . ( 1 9 8 8 ) . I n h i b i t i o n o f t h e c a l c i u m c h a n n e l b y i n t r a c e l l u l a r p r o t o n s i n s i n g l e v e n t r i c u l a r m y o c y t e s o f t h e g u i n e a - p i g . Journal of Physiology 403, 6 2 1 - 6 4 0 . 1 4 9 K a i l a , K . , S a a r i k o s k i , J . , a n d V o i p i o , J . ( 1 9 9 0 ) . M e c h a n i s m o f a c t i o n o f G A B A o n i n t r a c e l l u l a r p H a n d o n s u r f a c e p H i n c r a y f i s h m u s c l e f i b r e s . Journal of Physiology 427, 2 4 1 - 2 6 0 . K a i l a , K . , a n d V o i p o i , J . ( 1 9 8 7 ) . P o s t s y n a p t i c f a l l i n i n t r a c e l l u l a r p H i n d u c e d b y G A B A - a c t i v a t e d b i c a r b o n a t e c o n d u c t a n c e s . Nature 330, 1 6 3 - 1 6 5 . K a i l a , K . , a n d V o i p i o , J . ( 1 9 9 0 ) . D e p e n d e n c e o f i n t r a c e l l u l a r f r e e c a l c i u m a n d t e n s i o n o n m e m b r a n e p o t e n t i a l a n d i n t r a c e l l u l a r p H i n s i n g l e c r a y f i s h m u s c l e f i b r e s . PFliigers Archiv 416, 5 0 1 - 5 1 1 . K a k u , D . A . , G i f f a r d , R . G . , a n d C h o i , D . W . ( 1 9 9 3 ) . N e u r o p r o t e c t i v e e f f e c t s o f g l u t a m a t e a n t a g o n i s t s a n d e x t r a c e l l u l a r a c i d i t y . Science 260, 1 5 1 6 - 1 5 1 8 . K a t s u r a , K . , K r i s t i a n , T . , N a i r , R . , a n d S i e s j o , B . K . ( 1 9 9 4 ) . R e g u l a t i o n o f i n t r a - a n d e x t r a c e l l u l a r p H i n t h e r a t b r a i n i n a c u t e h y p e r c a p n i a : a r e - a p p r a i s a l . Brain Research 651, 4 7 - 5 6 . K o c h , R . A . , a n d B a r i s h , M . E . ( 1 9 9 4 ) . P e r t u r b a t i o n o f i n t r a c e l l u l a r c a l c i u m a n d h y d r o g e n i o n r e g u l a t i o n i n c u l t u r e d m o u s e h i p p o c a m p a l n e u r o n s b y r e d u c t i o n o f t h e s o d i u m i o n c o n c e n t r a t i o n g r a d i e n t . Journal of Neuroscience 14, 2 5 8 5 - 2 5 9 3 . K r a i g , R . P . , P e t i t o , C . K . , P l u m , F . , a n d P u l s i n e l l i , W . A . ( 1 9 8 7 ) . H y d r o g e n i o n s k i l l b r a i n a t c o n c e n t r a t i o n s r e a c h e d i n i s c h e m i a . Journal of Cereberal Blood Flow and Metabolism 7, 3 7 9 - 3 8 6 . K r i s h t a l , O . A . , O s i p c h u k , Y . V . , S h e l e s t , T . N . , a n d S m i r n o f f , S . V . ( 1 9 8 7 ) . R a p i d e x t r a c e l l u l a r p H t r a n s i e n t s r e l a t e d t o s y n a p t i c t r a n s m i s s i o n i n r a t h i p p o c a m p a l s l i c e s . Brain Research 436, 3 5 2 - 3 5 6 . K r i s t i a n , T . , K a t s u r a , K . , G i d o , G . , S i e s j o , B . K . ( 1 9 9 4 ) . T h e i n f l u e n c e o f p H o n c e l l u l a r c a l c i u m i n f l u x d u r i n g i s c h e m i a . Brain Research 641, 2 9 5 - 3 0 2 . K u m e , H . , T a k a g i , K . , S a t a k e , T . , T o k u n o , H . , a n d T o m i t a , T . ( 1 9 9 0 ) . E f f e c t s o f i n t r a c e l l u l a r p H o n c a l c i u m - a c t i v a t e d p o t a s s i u m c h a n n e l s i n r a b b i t t r a c h e a l s m o o t h m u s c l e . Journal of Physiology 424, 4 4 5 - 4 5 7 . K u r a c h i , Y . ( 1 9 8 2 ) . T h e e f f e c t s o f i n t r a c e l l u l a r p r o t o n s o n t h e e l e c t r i c a l a c t i v i t y o f s i n g l e v e n t r i c u l a r c e l l s . Pflugers Archiv 394, 2 6 4 - 2 7 0 . L a u r i d o , C , C a n d i a , S . , W o l f f , D . , a n d L a t o r r e , R . ( 1 9 9 1 ) . P r o t o n m o d u l a t i o n o f a C a 2 + - a c t i v a t e d K + c h a n n e l f r o m r a t s k e l e t a l m u s c l e i n c o r p o r a t e d i n t o p l a n a r b i l a y e r s . Journal of General Physiology 98, 1 0 2 5 - 1 0 4 3 . L e a , T . J . , a n d A s h l e y , C . C . ( 1 9 7 8 ) . I n c r e a s e i n f r e e C a 2 + i n m u s c l e a f t e r e x p o s u r e t o C 0 2 . Nature 275, 2 3 6 - 2 3 8 . 1 5 0 L e n n o x , W . G . , G i b b s , F . A . , a n d G i b b s , E . L . ( 1 9 3 6 ) . E f f e c t o n t h e e l e c t r o - e n c e p h a l o g r a m o f d r u g s a n d c o n d i t i o n s w h i c h i n f l u e n c e s e i z u r e s . Archives of Neurology and Psychiatry 36, 1 2 3 6 - 1 2 5 0 . L u d t , J . , T 0 n n e s s e n , T . I . , S a n d v i g , K . , O l s n e s , S . ( 1 9 9 1 ) . E v i d e n c e f o r i n v o l v e m e n t o f p r o t e i n k i n a s e C i n 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 b y C l ' / H C C ^ " a n t i p o r t . Journal of Membrane Biology 119, 1 7 9 - 1 8 6 . M a b e , H . , B l o m q v i s t , P . , a n d S i e s j o , B . K . ( 1 9 8 3 ) . I n t r a c e l l u l a r p H i n t h e b r a i n f o l l o w i n g t r a n s i e n t i s c h e m i a . Journal of Cereberal Blood Flow and Metabolism 3, 1 0 9 - 1 1 4 . M a c V i c a r , B . A . , a n d J a h n s e n , H . ( 1 9 8 5 ) . U n c o u p l i n g o f C A 3 p y r a m i d a l n e u r o n s b y p r o p i o n a t e . Brain Research 330, 1 4 1 - 1 4 5 . M a r t i n e z - Z a g u i l a n , R . , G i l l i e s , R . J . , a n d S a n c h e z - A r m a s s , S . ( 1 9 9 4 ) . R e g u l a t i o n o f p H i n r a t b r a i n s y n a p t o s o m e s . I I . R o l e o f C 1 - . Journal of Neurophysiology 71, 2 2 4 9 - 2 2 5 7 . M a r t i n e z - Z a g u i l a n , R . , M a r t i n e z , G . M . , L a t t a n z i o , F . , a n d G i l l i e s , R . J . ( 1 9 9 1 ) . S i m u l t a n e o u s m e a s u r e m e n t o f i n t r a c e l l u l a r p H a n d C a 2 + u s i n g t h e f l u o r e s c e n c e o f S N A R F - 1 a n d f u r a - 2 . American Journal of Physiology 260, C 2 9 7 - C 3 0 7 . M e e c h , R . W . , a n d T h o m a s , R . C . ( 1 9 7 7 ) . T h e e f f e c t o f c a l c i u m i n j e c t i o n o n t h e i n t r a c e l l u l a r s o d i u m a n d p H o f s n a i l n e u r o n e s . Journal of Physiology 265, 8 6 7 - 8 7 9 . M e e c h , R . W . , a n d T h o m a s , R . C . ( 1 9 8 7 ) . V o l t a g e - d e p e n d e n t i n t r a c e l l u l a r p H i n Helix aspersa n e u r o n e s . Journal of Physiology 390, 4 3 3 - 4 5 2 . M e s s e t e r , K . , a n d S i e s j o , B . K . ( 1 9 7 1 ) . T h e i n t r a c e l l u l a r p H i n t h e b r a i n i n a c u t e a n d s u s t a i n e d h y p e r c a p n i a . Acta Physiologica Scandinavica 83, 2 1 0 - 2 1 9 . M o o d y , W . ( 1 9 8 0 ) . A p p e a r a n c e o f c a l c i u m a c t i o n p o t e n t i a l s i n c r a y f i s h s l o w m u s c l e f i b r e s u n d e r c o n d i t i o n s o f l o w i n t r a c e l l u l a r p H . Journal of Physiology 302, 3 3 5 - 3 4 6 . M o o d y , W . ( 1 9 8 4 ) . E f f e c t s o f i n t r a c e l l u l a r H + o n t h e e l e c t r i c a l p r o p e r t i e s o f e x c i t a b l e c e l l s . Annual Review of Neuroscience 7, 2 5 7 - 2 7 8 . M o o d y , W . J . , a n d H a g i w a r a , S . ( 1 9 8 2 ) . B l o c k o f i n w a r d r e c t i f i c a t i o n b y i n t r a c e l l u l a r H + i n i m m a t u r e o o c y t e s o f t h e s t a r f i s h Mediaster aequalis. Journal of General Physiology 79, 1 1 5 - 1 3 0 . M o o l e n a a r , W . H . , d e L a a t , S . W . , M u m m e r y , C . L . , a n d v a n d e r S a a g , P . T . ( 1 9 8 2 ) . N a + / H + e x c h a n g e i n t h e a c t i o n o f g r o w t h f a c t o r s . I n : Ions, Cell Proliferation, and Cancer, p p . 1 5 1 - 1 6 2 . E d s . A . L . B o y t o n , W . L . M c K e e h a n , a n d J . F . W h i t f i e l d . A c a d e m i c P r e s s . 1 5 1 M r k i c , B . , T s e , C . - M . , F o r g o , J . , H e l m l e - K o l b , C , D o n o w i t z , M . , a n d M u r e r , H . ( 1 9 9 3 ) . I d e n t i f i c a t i o n o f P T H - r e s p o n s i v e N a / H - e x c h a n g e r i s o f o r m s i n a r a b b i t p r o x i m a l t u b u l e c e l l l i n e ( R K P C - 2 ) . Pflugers Archiv 424, 3 7 7 - 3 8 4 . M u r e r , F L , H o p f e r , U . , a n d K i n n e , R . ( 1 9 7 6 ) . S o d i u m / p r o t o n a n t i p o r t i n b r u s h - b o r d e r - m e m b r a n e v e s i c l e s i s o l a t e d f r o m r a t s m a l l i n t e s t i n e a n d k i d n e y . Biochemical Journal 154, 5 9 7 - 6 0 4 . M u t c h , W . A . C , a n d H a n s e n , A . J . ( 1 9 8 4 ) . E x t r a c e l l u l a r p H c h a n g e s d u r i n g s p r e a d i n g d e p r e s s i o n a n d c e r e b r a l i s c h e m i a : m e c h a n i s m s o f b r a i n p H r e g u l a t i o n . Journal of Cereberal Blood Flow and Metabolism 4, 1 7 - 2 7 . N a c h s h e n , D . A . , a n d D r a p e a u , P . ( 1 9 8 8 ) . T h e r e g u l a t i o n o f c y t o s o l i c p H i n i s o l a t e d p r e s y n a p t i c n e r v e t e r m i n a l s f r o m r a t b r a i n . Journal of General Physiology 91, 2 8 9 - 3 0 3 . N e d e r g a a r d , M . , G o l d m a n , S . A . , D e s a i , S . , a n d P u l s i n e l l i , W . A . ( 1 9 9 1 ) . A c i d - i n d u c e d d e a t h i n n e u r o n s a n d g l i a . Journal of Neuroscience 11, 2 4 8 9 - 2 4 9 7 . N o n n e r , W . , S p a l d i n g , B . C , a n d H i l l e , B . ( 1 9 8 0 ) . L o w i n t r a c e l l u l a r p H a n d c h e m i c a l a g e n t s s l o w i n a c t i v a t i o n g a t i n g i n s o d i u m c h a n n e l s o f m u s c l e . Nature 284, 3 6 0 - 3 6 3 . O r l o w s k i , J . , K a n d a s a m y , R . A . , a n d S h u l l , G . E . ( 1 9 9 2 ) . M o l e c u l a r c l o n i n g o f p u t a t i v e m e m b e r s o f t h e N a / H e x c h a n g e r g e n e f a m i l y . Journal of Biological Chemistry 267, 9 3 3 1 - 9 3 3 9 . O u - y a n g , Y . , K r i s t i a n , T . , M e l l e r g a r d , P . , a n d S i e s j o , B . K . ( 1 9 9 4 ) . T h e i n f l u e n c e o f p H o n g l u t a m a t e - a n d d e p o l a r i z a t i o n - i n d u c e d i n c r e a s e s o f i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n i n c o r t i c a l n e u r o n s i n p r i m a r y c u l t u r e . Brain Research 646, 6 5 - 7 2 . O u - y a n g , Y . , M e l l e r g a r d , P . , a n d S i e s j o , B . K . ( 1 9 9 3 ) . 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 i n s i n g l e r a t c o r t i c a l n e u r o n s i n v i t r o : a m i c r o s p e c t r o f l u o r o m e t r i c s t u d y . Journal of Cereberal Blood Flow and Metabolism 13, 8 2 7 - 8 4 0 . P e e r s , C , a n d G r e e n , F . K . ( 1 9 9 1 ) . I n h i b i t i o n o f C a 2 + - a c t i v a t e d K + c u r r e n t s b y i n t r a c e l l u l a r a c i d o s i s i n i s o l a t e d t y p e I c e l l s o f t h e n e o n a t a l r a t c a r o t i d b o d y . Journal of Physiology 437, 5 8 9 - 6 0 2 . P o c o c k , G . , a n d R i c h a r d s , C . D . ( 1 9 8 9 ) . H y d r o g e n i o n r e g u l a t i o n i n r a t c e r e b e l l a r g r a n u l e c e l l s m a i n t a i n e d i n t i s s u e c u l t u r e . Journal of Physiology 412, 6SP. P r e i s s l e r , M . , a n d W i l l i a m s , J . A . ( 1 9 8 1 ) . P a n c r e a t i c a c i n a r c e l l f u n c t i o n : m e a s u r e m e n t o f i n t r a c e l l u l a r i o n s a n d p H a n d t h e i r r e l a t i o n t o s e c r e t i o n . Journal of Physiology 321, 4 3 7 - 4 4 8 . 1 5 2 P r o d ' h o m , B . , P i e t r o b o n , D . , a n d H e s s , P . ( 1 9 8 7 ) . D i r e c t m e a s u r e m e n t o f p r o t o n t r a n s f e r r a t e s t o a g r o u p c o n t r o l l i n g t h e d i h y d r o p y r i d i n e - s e n s i t i v e C a 2 + c h a n n e l . Nature 329, 2 4 3 - 2 4 6 . R a l e y - S u s m a n , K . M . , C r a g o e , E . J . , S a p o l s k y , R . M . , a n d K o p i t o , R . R . ( 1 9 9 1 ) . 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 i n c u l t u r e d h i p p o c a m p a l n e u r o n e s b y a n a m i l o r i d e - i n s e n s i t i v e N a + / H + e x c h a n g e r . Journal of Biological Chemistry 266, 2 7 3 9 - 2 7 4 5 . R a l e y - S u s m a n , K . M . , S a p o l s k y , R . M . , a n d K o p i t o , R . B . ( 1 9 9 3 ) . C T / H C O 3 - e x c h a n g e f u n c t i o n d i f f e r s i n a d u l t a n d f e t a l r a t h i p p o c a m p a l n e u r o n s . Brain Research 614, 3 0 8 - 3 1 4 . R i c h a r d s , C . D . , a n d P o c o c k , G . ( 1 9 8 9 ) . H y d r o g e n i o n r e g u l a t i o n i n c u l t u r e d m a m m a l i a n C N S n e u r o n e s . Acta Physiologica Scandinavica Supplementum 136, 3 6 . R i n k , T . J . , T s i e n , R . Y . , a n d P o z z a n , T . ( 1 9 8 2 ) . C y t o p l a s m i c p H a n d f r e e M g 2 + i n l y m p h o c y t e s . Journal of Cell Biology 95, 1 8 9 - 1 9 6 . R o o s , A . ( 1 9 7 5 ) . I n t r a c e l l u l a r p H a n d d i s t r i b u t i o n o f w e a k a c i d s a c r o s s c e l l m e m b r a n e s . A s t u d y o f D- a n d L - l a c t a t e a n d o f D M O i n r a t d i a p h r a g m . Journal of Physiology 249, 1 - 2 5 . R o o s , A . , a n d B o r o n , W . F . ( 1 9 8 1 ) . I n t r a c e l l u l a r p H . Physiological Reviews 61, 2 9 6 - 4 3 4 . R o s a r i o , L . M . , S t u t z i n , A . , C r a g o e , E . J . , a n d P o l l a r d , H . B . ( 1 9 9 1 ) . M o d u l a t i o n o f i n t r a c e l l u l a r p H b y s e c r e t a g o g u e s a n d t h e N a + / H + a n t i p o r t e r i n c u l t u r e d b o v i n e c h r o m a f f i n c e l l s . Neuroscience 41, 2 6 9 - 2 7 6 . R o w e , W . A . , L e s h o , M . , J . , a n d M o n t r o s e , M . H . ( 1 9 9 4 ) . P o l a r i z e d N a + / H + e x c h a n g e f u n c t i o n i s p l i a b l e i n r e s p o n s e t o t r a n s e p i t h e l i a l g r a d i e n t s o f p r o p i o n a t e . Proceedings of the National Academy of Science, U.S.A. 9 1 , 6 1 6 6 - 6 1 7 0 . R u s s e l l , J . M . , a n d B o r o n , W . F . ( 1 9 7 6 ) . R o l e o f c h l o r i d e t r a n s p o r t i n 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 . Nature 264, 7 3 - 7 4 . S a c h s , G . ( 1 9 8 7 ) . T h e g a s t r i c p r o t o n p u m p : t h e H + , K + , A T P a s e . I n : Physiology of the Gastrointestinal Tract, Second Edition, p p . 8 6 5 - 8 8 1 . E d . L . R . J o h n s o n . R a v e n P r e s s . S a c h s , G . , C h a n g . H . H . , R a b o n , E . , S c h a c k m a n , R . , L e w i n , M . , a n d S a c c o m a n i , G . ( 1 9 7 6 ) . A n o n e l e c t r o g e n i c H + p u m p i n p l a s m a m e m b r a n e s o f h o g s t o m a c h . Journal of Biological Chemistry 251, 7 6 9 0 - 7 6 9 8 . S a n c h e z - A r m a s s , S . , M a r t i n e z - Z a g u i l a n , R . , a n d M a r t i n e z , G . M . ( 1 9 9 4 ) . R e g u l a t i o n o f p H i n r a t b r a i n s y n a p t o s o m e s . I . R o l e o f s o d i u m , b i c a r b o n a t e , a n d p o t a s s i u m . Journal of Neurophysiology 71, 2 2 3 6 - 2 2 4 8 . 1 5 3 S a r d e t , C , F a f o u r n o u x , P . , a n d P o u y s s e g u r , J . ( 1 9 9 1 ) . a - t h r o m b i n , e p i d e r m a l g r o w t h f a c t o r , a n d o k a d a i c a c i d a c t i v a t e t h e N a + / H + e x c h a n g e r , N H E - 1 , b y p h o s p h o r y l a t i n g a s e t o f c o m m o n s i t e s . Journal of Biological Chemistry 266, 1 9 1 6 6 - 1 9 1 7 1 . S a r d e t , C , F r a n c h i , A . , a n d P o u y s s e g u r , J . ( 1 9 8 9 ) . M o l e c u l a r c l o n i n g , p r i m a r y s t r u c t u r e , a n d e x p r e s s i o n o f t h e h u m a n g r o w t h f a c t o r - a c t i v a t a b l e N a + / H + a n t i p o r t e r . Cell 56, 2 7 1 - 2 8 0 . S c h m i d , A . , S c h o l z , W . , L a n g , H . - J . , a n d P o p p , R . ( 1 9 9 2 ) . N a + / H + e x c h a n g e i n p o r c i n e c e r e b r a l 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 i s i n h i b i t e d b y a b e n z o y l g u a n i d i n e d e r i v a t i v e . Biochemical and Biophysical Research Communications 184, 1 1 2 - 1 1 7 . S c h u l d i n e r , S . , a n d R o z e n g u r t , E . ( 1 9 8 2 ) . N a + / H + a n t i p o r t i n s w i s s 3 T 3 c e l l s : m i t o g e n i c s t i m u l a t i o n l e a d s t o c y t o p l a s m i c a l k a l i n i z a t i o n . Proceedings of the National Academy of Science, U.S.A. 79, 7 7 7 8 - 7 7 8 2 . S c h w i e n i n g , C . J . , a n d B o r o n , W . F . ( 1 9 9 2 ) . F r a c t i o n a l r a t e o f d y e s i g n a l d e c r e a s e a s a n a i d i n a s s e s s i n g m e m b r a n e p e r m e a b i l i t y . Journal of Physiology 452, 1 8 7 P . S c h w i e n i n g , C . J . , a n d B o r o n , W . F . ( 1 9 9 4 ) . 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 i n p y r a m i c a l n e u r o n e s f r o m t h e r a t h i p p o c a m p u s b y N a + - d e p e n d e n t C l ' - H C C ^ " e x c h a n g e . Journal of Physiology 475.1, 5 9 - 6 7 . S h a r p , A . P . , a n d T h o m a s , R . C . ( 1 9 8 1 ) . T h e e f f e c t s o f c h l o r i d e s u b s t i t u t i o n o n i n t r a c e l l u l a r p H i n c r a b m u s c l e . Journal of Physiology 312, 7 1 - 8 0 . S i e s j o , B . K . ( 1 9 8 5 ) . A c i d - b a s e h o m e o s t a s i s i n t h e b r a i n : p h y s i o l o g y , c h e m i s t r y , a n d n e u r o c h e m i c a l p a t h o l o g y . I n : Progress in Brain Research, V o l . 6 3 , p p . 1 2 1 - 1 5 4 . E d s . K . K o g u r e , K . - A . H o s s m a n , B . K . S i e s j o , a n d F . A . W e l s h . E l s e v i e r . S i e s j o , B . K . , E k h o l m , A . , K a t s u r a , K , a n d T h e a n d e r , S . ( 1 9 9 0 ) . A c i d - b a s e c h a n g e s d u r i n g c o m p l e t e b r a i n i s c h e m i a . Stroke 21 ( S u p p l I I I ) , 1 9 4 - 1 9 9 . S i e s j o , B . K . , a n d M e s s e t e r , K . ( 1 9 7 1 ) . F a c t o r s d e t e r m i n i n g i n t r a c e l l u l a r p H . I n : Ion Homeostasis of the brain. Alfred Benzon Symposium III, p p . 2 4 4 - 2 6 9 . E d s . B . K . S i e s j o , a n d S . C . S o r e n s e n . M u n k s g a a r d , C o p e n h a g e n . S i e s j o , B . K . , v o n H a n w e h r , R . , N e r g e l i u s , G . , N e v a n d e r , G . , a n d I n g v a r , M . ( 1 9 8 5 ) . E x t r a - a n d I n t r a c e l l u l a r p H i n t h e b r a i n d u r i n g s e i z u r e s a n d i n t h e r e c o v e r y p e r i o d f o l l o w i n g t h e a r r e s t o f s e i z u r e a c t i v i t y . Journal of Cereberal Blood Flow and Metabolism 5, 4 7 - 5 7 . S i l v e r , R . A . , W h i t a k e r , M . , a n d B o l s o v e r , S . R . ( 1 9 9 2 ) . I n t r a c e l l u l a r i o n i m a g i n g u s i n g f l u o r e s c e n t d y e s : a r t e f a c t s a n d l i m i t s t o r e s o l u t i o n . Pflugers Archiv 420, 5 9 5 - 6 0 2 . 1 5 4 S i m o n , R . P . , N i i r o , M . , a n d G w i n n , R . ( 1 9 9 3 ) . B r a i n a c i d o s i s i n d u c e d b y h y p e r c a r b i c v e n t i l a t i o n a t t e n u a t e s f o c a l i s c h e m i c i n j u r y . Journal of Pharmacology and Experimental Therapeutics 267, 1 4 2 8 - 1 4 3 1 . S i s k i n d , M . S . , M c C o y , C . E . , C h o b a n i a n , A . , a n d S c h w a r t z , J . H . ( 1 9 8 9 ) . 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 c a l c i u m b y c e l l p H i n v a s c u l a r s m o o t h m u s c l e c e l l s . American Journal of Physiology 256, C 2 3 4 - C 2 4 0 . S o m j e n , G . G . ( 1 9 8 4 ) . A c i d i f i c a t i o n o f i n t e r s t i t i a l f l u i d i n h i p p o c a m p a l f o r m a t i o n c a u s e d b y s e i z u r e s a n d b y s p r e a d i n g d e p r e s s i o n . Brain Research 311, 1 8 6 - 1 8 8 . S o m j e n , G . G . , A l l e n , B . W . , B a l e s t r i n o , M . , a n d A i t k e n , P . G . ( 1 9 8 7 ) . P a t h o p h y s i o l o g y o f p H a n d C a 2 + i n b l o o d s t r e a m a n d b r a i n . Canadian Journal of Physiology 65, 1 0 7 8 - 1 0 8 5 . S p r a y , D . C , a n d B e n n e t t , M . V . L . ( 1 9 8 5 ) . P h y s i o l o g y a n d p h a r m a c o l o g y o f g a p j u n c t i o n s . Annual Review of Physiology 47, 2 8 1 - 3 0 3 . S p r a y , D . C , S t e r n , J . H . , H a r r i s , A . L . , a n d B e n n e t t , M . V . L . ( 1 9 8 2 ) . G a p j u n c t i o n a l c o n d u c t a n c e s : c o m p a r i s o n o f s e n s i t i v i t i e s t o H a n d C a i o n s . Proceedings of the National Academy of Science, U.S.A. 7 9 , 4 4 1 - 4 4 5 . S u n , B . , a n d V a u g h a n - J o n e s , R . D . ( 1 9 9 4 ) . E f f e c t o f e x t r a c e l l u l a r p H o n i n t r a c e l l u l a r p H i n i s o l a t e d g u i n e a - p i g v e n t r i c u l a r m y o c y t e s . Journal of Physiology 475.P, 8 3 P . T a g g a r t , M . , A u s t i n , C , a n d W r a y , S . ( 1 9 9 4 ) . A c o m p a r i s o n o f t h e e f f e c t s o f i n t r a c e l l u l a r a n d e x t r a c e l l u l a r p H o n c o n t r a c t i o n i n i s o l a t e d r a t p o r t a l v e i n . Journal of Physiology 475, 2 8 5 - 2 9 2 . T a i r a , T . , S m i r n o v , S . , V o i p i o , J . , a n d K a i l a , K . ( 1 9 9 3 ) . I n t r i n s i c p r o t o n m o d u l a t i o n o f e x c i t a t o r y t r a n s i m i s s i o n i n r a t h i p p o c a m p a l s l i c e s . NeuroReport 4, 9 3 - 9 6 . T a k a h a s h i , K . - I . , D i x o n , D . B . , a n d C o p e n h a g e n , D . R . ( 1 9 9 3 ) . M o d u l a t i o n o f a s u s t a i n e d c a l c i u m c u r r e n t b y i n t r a c e l l u l a r p h i n h o r i z o n t a l c e l l s o f f i s h r e t i n a . Journal of General Physiology 101, 6 9 5 - 7 1 4 . T a n g , C . - M . , D i c h t e r , M . , a n d M o r a d , M . ( 1 9 9 0 ) . M o d u l a t i o n o f 7 V - m e t h y l - D - a s p a r t a t e c h a n n e l b y e x t r a c e l l u l a r H + . Proceedings of the National Academy of Science, U.S.A. 87, 6 4 4 5 - 6 4 4 9 . T h o m a s , R . C . ( 1 9 7 6 a ) . I o n i c m e c h a n i s m s o f t h e H + p u m p i n s n a i l n e u r o n e . Nature 262, 5 4 - 5 5 . T h o m a s , R . C . ( 1 9 7 6 b ) . T h e e f f e c t o f C 0 2 o n t h e i n t r a c e l l u l a r p H a n d b u f f e r i n g p o w e r o f s n a i l n e u r o n e s . Journal of Physiology 255, 7 1 5 - 7 3 5 . 1 5 5 T h o m a s , R . C . ( 1 9 7 7 ) . T h e r o l e o f b i c a r b o n a t e , c h l o r i d e a n d s o d i u m i o n s i n t h e 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 i n s n a i l n e u r o n e s . Journal of Physiology 273, 3 1 7 - 3 3 8 . T h o m a s , R . C . ( 1 9 8 4 ) . E x p e r i m e n t a l d i s p l a c e m e n t o f i n t r a c e l l u l a r p H a n d t h e m e c h a n i s m o f i t s s u b s e q u e n t r e c o v e r y . Journal of Physiology 354, 3P-22P. T o l k o v s k y , A . M . , a n d R i c h a r d s , C . D . ( 1 9 8 7 ) . N a + / H + e x c h a n g e i s t h e m a j o r m e c h a n i s m o f p H r e g u l a t i o n i n c u l t u r e d s y m p a t h e t i c n e u r o n s : m e a s u r e m e n t s i n s i n g l e c e l l b o d i e s a n d n e u r i t e s u s i n g a f l u o r e s c e n t p H i n d i c a t o r . Neuroscience 22, 1 0 9 3 - 1 1 0 2 . T o m b a u g h , G . C . ( 1 9 9 4 ) . M i l d a c i d o s i s d e l a y s h y p o x i c s p r e a d i n g d e p r e s s i o n a n d i m p r o v e s n e u r o n a l r e c o v e r y i n h i p p o c a m p a l s l i c e s . Journal of Neuroscience 14(9), 5 6 3 5 - 5 6 4 3 . T o m b a u g h , G . C . , a n d S a p o l s k y , R . M . ( 1 9 9 3 ) . E v o l v i n g c o n c e p t s a b o u t t h e r o l e o f a c i d o s i s i n i s c h e m i c n e u r o p a t h o l o g y . Journal ofNeurochemistry 61, 7 9 3 - 8 0 3 . T r a y n e l i s , S . F . , a n d C u l l - C a n d y , S . G . ( 1 9 9 1 ) . P h a r m a c o l o g i c a l p r o p e r t i e s a n d H + s e n s i t i v i t y o f e x c i t a t o r y a m i n o a c i d r e c e p t o r c h a n n e l s i n r a t c e r e b e l l a r g r a n u l e n e u r o n e s . Journal of Physiology 433, 7 2 7 - 7 6 3 . T s e , C . - M . , L e v i n e , S . A . , Y u n , C . H . C , B r a n t , S . R . , P o u y s s e g u r , J . , M o n t r o s e , M . H . , a n d D o n o w i t z , M . ( 1 9 9 3 ) . F u n c t i o n a l c h a r a c t e r i s t i c s o f a c l o n e d e p i t h e l i a l N a + / H + e x c h a n g e r ( N H E 3 ) : r e s i s t a n c e t o a m i l o r i d e a n d i n h i b i t i o n b y p r o t e i n k i n a s e C . Proceedings of the National Academy of Science, U.S.A. 90, 9 1 1 0 - 9 1 1 4 . T s e , C . M . , W a t s o n , A . J . M . , M a , A . I . , P o u y s s e g u r , J . , a n d D o n o w i t z , M . ( 1 9 9 1 ) . C l o n i n g a n d f u n c t i o n a l e x p r e s s i o n o f a s e c o n d n o v e l r a b b i t i l e a l v i l l u s e p i t h e l i a l c e l l N a + / H + e x c h a n g e r ( N H E - 2 ) . Gastroenterology 100, A 2 5 8 . T s i e n , R . Y . ( 1 9 8 9 ) . F l u o r e s c e n t i n d i c a t o r s o f i o n c o n c e n t r a t i o n . I n : Methods in Cell Biology, Volume 30, p p . 1 2 7 - 1 5 6 . E d s . Y . W a n g , a n d D . L . T a y l o r . A c a d e m i c P r e s s . U m b a c h , J . A . ( 1 9 8 2 ) . C h a n g e s i n i n t r a c e l l u l a r p H a f f e c t c a l c i u m c u r r e n t s i n Paramecium caudatum. Proceedings of the Royal Society of London B 216, 2 0 9 - 2 2 4 . V a u g h a n - J o n e s , R . D . , E i s n e r , D . A . , a n d L e d e r e r , W . J . ( 1 9 8 7 ) . E f f e c t s o f c h a n g e s o f i n t r a c e l l u l a r p H o n c o n t r a c t i o n i n s h e e p c a r d i a c P u r k i n j e f i b e r s . Journal of General Physiology 89, 1 0 1 5 - 1 0 3 2 . V a u g h a n - J o n e s , R . D . , a n d W u , M . - L . ( 1 9 9 0 ) . p H d e p e n d e n c e o f i n t r i n s i c H + b u f f e r i n g p o w e r i n t h e s h e e p c a r d i a c P u r k i n j e f i b r e . Journal of Physiology 425, 4 2 9 - 4 4 8 V e l i s e k , L . , D r e i e r , J . P . , S t a n t o n , P . K . , H e i n e m a n n , U . , a n d M o s h e , S . L . ( 1 9 9 4 ) . L o w e r i n g o f e x t r a c e l l u l a r p H s u p p r e s s e s l o w - M g 2 + - i n d u c e s s e i z u r e s i n c o m b i n e d e n t o r h i n a l c o r t e x - h i p p o c a m p a l s l i c e s . Experimental Brain Research 101, 4 4 - 5 2 . 1 5 6 V o i p i o , J . , a n d K a i l a , K . ( 1 9 9 3 ) . I n t e s t i t i a l PCO2 a n d p H i n r a t h i p p o c a m p a l s l i c e s m e a s u r e d b y m e a n s o f a n o v e l f a s t C 0 2 / H + - s e n s i t i v e m i c r o e l e c t r o d e b a s e d o n a P V C - g e l l e d m e m b r a n e . PFliigers Archiv 4 2 3 , 1 9 3 - 2 0 1 . V o i p i o , K . , P a s t e r n a c k , M . , R y d q v i s t , B . , a n d K a i l a , K . ( 1 9 9 1 ) . E f f e c t o f y - a m i n o b u t y r i c a c i d o n i n t r a c e l l u l a r p H i n t h e c r a y f i s h s t r e t c h - r e c e p t o r n e u r o n e . Journal of Experimental Biology 1 5 6 , 3 4 9 - 3 6 1 . V y k l i c k y , L . , V l a c h o v a , V . , a n d K r u s e k , J . ( 1 9 9 0 ) . T h e e f f e c t o f e x t e r n a l p H c h a n g e s o n r e p o n s e s t o e x c i t a t o r y a m i n o a c i d s i n m o u s e h i p p o c a m p a l n e u r o n e s . Journal of Physiology AM, 4 9 7 - 5 1 7 . W a n k e , E . , C a r b o n e , E . , a n d T e s t a , P . L . ( 1 9 7 9 ) . K + c o n d u c t a n c e m o d i f i e d b y a t i t r a t a b l e g r o u p a c c e s s i b l e t o p r o t o n s f r o m t h e i n t r a c e l l u l a r s i d e o f t h e s q u i d a x o n m e m b r a n e . Biophysical Journal 2 6 , 3 1 9 - 3 2 4 . W a n k e , E . , C a r b o n e , E . , a n d T e s t a , P . L . ( 1 9 8 0 ) . T h e s o d i u m c h a n n e l a n d i n t r a c e l l u l a r H + b l o c k a g e i n s q u i d a x o n s . Nature 2 8 7 , 6 2 - 6 3 . W i l k i e , D . R . ( 1 9 7 9 ) . G e n e r a t i o n o f p r o t o n s b y m e t a b o l i c p r o c e s s e s o t h e r t h a n g l y c o l y s i s i n m u s c l e c e l l s : a c r i t i c a l v i e w . Journal of Molecular and Cellular Cardiology 1 1 , 3 2 5 - 3 3 0 . W 6 1 1 , E . , R i t t e r , M . , O f f n e r , F . , L a n g , H . - J . , S c h o l k e n s , B . , H a u s s i n g e r , D . , a n d L a n g , F . ( 1 9 9 3 ) . E f f e c t s o f H O E 6 9 4 - a n o v e l i n h i b i t o r o f N a + / H + e x c h a n g e - o n N I H 3 T 3 f i b r o b l a s t s e x p r e s s i n g t h e RAS o n c o g e n e . European Journal of Pharmacology - Molecular Pharmacology Section 2 4 6 , 2 6 9 - 2 7 3 . Z e i l h o f e r , H . U . , M i i l l e r , T . H . , a n d S w a n d u l l a , D . ( 1 9 9 3 ) . I n h i b i t i o n o f h i g h v o l t a g e - a c t i v a t e d c a l c i u m c u r r e n t s b y L - g l u t a m a t e r e c e p t o r - m e d i a t e d c a l c i u m i n f l u x . Neuron 1 0 , 8 7 9 - 8 8 7 .

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
France 5 0
Russia 3 0
China 3 14
United States 2 0
Finland 1 0
Japan 1 0
City Views Downloads
Unknown 8 0
Beijing 3 0
Ashburn 2 0
Helsinki 1 0
Tokyo 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items