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pH Regulat ion in Adul t Rat Carot id BodyGlomus Cel ls

I mpor tance of E xt racel lu la r pH Sodium andPotassiumT . J W I L D IN G , B . C H E N G , a n d A . R o o sF rom the D epa r tm en t o f Ce l l Bio logy and Physio logy , W ash ing ton Un ivers ity Schoo l o fMed icine, S t. Louis , M issouri 63110

B S T R C T T h e c o u r s e o f i n t r a c e l lu l a r p H ( p H i ) w a s f o ll o w e d in s u p e r f u s e d( 3 6 C ) s i n g l e g l o m u s ( t y p e I ) ce l l s o f t h e f r e s h l y d i s s o c i a t e d a d u l t r a t c a r o t i d b o d y .T h e c e ll s h a d b e e n l o a d e d w i t h t h e p H - s e n s i ti v e f lu o r e s c e n t d y e 2 , 7 - ( 2 - c a r b o x y e t h -y l )- 5 ( a n d - 6 ) - c a rb o x y f l u o r e sc e i n . T h e h i g h K + - n i g e r i ci n m e t h o d w a s u s e d f o rc a l i b r a t i o n . T h e p H i o f t h e g l o m u s c e l l a t p H o 7 . 4 0 , w i t h o u t C O ~ , w a s 7 . 2 3 -+ 0 . 0 2( n = 7 0 ) ; i n 5% C O ~ / 2 5 m M H C O ~ , p H i w a s 7 .1 8 _ 0 . 0 8 ( n = 9 ) . T h e p H i w a s v e r ys e n s it i v e t o c h a n g e s i n p H o . W i t h o u t C O 2 , A p H d A p H o w as 0 .8 5 ( p H o 6 . 2 0 - 8 . 0 0 ; 3 2c e ll s) , w h i l e i n C O 2 / H C O ~ t h is r a t i o w a s 0 .8 2 i r r e s p ec t i v e o f w h e t h e r p H o ( 6 . 8 0 -7 . 4 0 ; 1 4 c e l l s) w a s c h a n g e d a t c o n s t a n t P c o 2 o r a t c o n s t a n t [ H C O ~ ] o . T h e g r e a t p H is e n s i t iv i t y o f t h e g l o m u s c e l l t o p H o i s m a t c h e d o n l y b y t h a t o f t h e h u m a n r e d c e l l.A n a c t iv e N a t / H + e x c h a n g e r ( a p p a r e n t K m = 5 8 --- 6 m M ) i s p r e s e n t i n g l o m u sc e ll s : N a t r e m o v a l o r a d d i t i o n o f t h e a m i l o r i d e d e r i v a ti v e 5 - ( N , N - h e x a m e t h y l e n e ) -a m i l o r i d e i n d u c e d p H i t o f a l l b y a s m u c h a s 0. 9. T h e m e m b r a n e o f t h e se c e l ls al s oc o n t a i n s a K / H + e x c h a n g e r . R a i s i n g [ K +] o f r o m 4 . 7 t o 2 5 , 5 0 , o r 1 4 0 m Mr e v e r s i b l y r a i s e d p H i b y 0 . 2 , 0 . 3 , a n d 0 . 6 , r e s p e c t i v e l y . R b h a d n o e f f e c t, b u t i nc o r r e s p o n d i n g c o n c e n t r a t i o n s o f T I + a l k a l i n iz a t i o n w a s m u c h f a s t e r t h a n i n K + .R e d u c i n g [K +] o t o 1 .5 m M l o w e r e d p H i b y 0 . I . T h e s e p H i c h a n g e s w e r e s h o w n n o tt o b e d u e t o c h a n g e s i n m e m b r a n e v o l t ag e , a n d w e r e e v e n m o r e s t r i k in g i n t h ea b s e n c e o f N a + . In t r i n s ic b u f f e r i n g p o w e r ( a m o u n t o f s t r o n g b a s e r e q u i r e d t op r o d u c e , i n t h e n o m i n a l a b s e n c e o f C O 2, a s m a l l p H i r is e ) i n c r e a s e d f r o m 3 t o ~ 21m M a s p H i w a s l o w e r e d , b u t r e m a i n e d n e a r l y u n c h a n g e d b e l o w p h i 6 . 60 . T h e f i t te de x p r e s s i o n a s s u m e d t h e p r e s e n c e o f o n e " e q u i v a l e n t " i n t r a c e l l u l a r b u f f e r ( p K 6 . 4 1,4 1 m M ) . T h e e x c e p t i o n a l p H i s e n s it i vi t y t o p H o s u g g e s t s t h a t t h e p H i o f th e g l o m u sc e l l i s a l i n k i n t h e c h e m o r e c e p t o r ' s r e s p o n s e t o e x t e r n a l a c i d i ty .

Address rep r in t r eques t s to Dr . A . Roos, D epa r tm en t o f Ce l l Bio logy and Physio logy , W ash ing tonUniversity School of M edicine, 660 Sou th E uclid Avenue, St . Louis , MO 63110.

J. GEN. Pm'SlOL. T he Rockefeller University Press 0022-1295/92/1010593/16$2.00Volume 100 October 1992 593-608 593

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594 THE JOURNAL OF GENERAL PHYSIOLOGY VOLUM E 1 1992I N T R O D U C T I O NT h e c a r o t i d b o d y , a s m a l l o r g a n p e r c h e d a t o r n e a r t h e b i f u r c a t i o n o f e a c h o f t h e t w o

c o m m o n c a r o t i d a rt e r i es , m o n i t o r s o x y g e n p r e s s u r e a n d a c i d - b a s e c o n d i t io n s o f t h eb l o o d . D e C a s t r o ( 1 9 2 8 ) w a s t h e f ir s t t o d e m o n s t r a t e t h a t i t s i n n e r v a t i o n b y a b r a n c ho f th e g l o s s o p h a r y n g e a l n e r v e ( c a r o t i d b o d y n e r v e ) is a f f e r e n t in n a t u r e , f r o mw h i c h h e d e d u c e d t h a t t h e c a r o t i d b o d y s e n s e s a l t e r a t i o n s i n t h e b l o o d . S i n c e t h a tt i m e , a w e a l t h o f d a t a h a s b e e n a c c u m u l a t e d o n t h e n e u r a l r e s p o n s e , a n d a l so o n t h ep r o p e r t i e s o f s o m e o f t h e c e ll s o f t h e o r g a n ( f o r r e c e n t w o r k s e e , f o r in s t a nc e ,D u c h e n , C a d d y , K i r b y, P a t t e r s o n , P o n t e , a n d B i s c o e , 19 8 8 ; L 6 p e z - L 6 p e z , G o n z f i le z ,U r e f i a , a n d L 6 p e z - B a r n e o , 1 9 89 ; U r e f ia , L 6 p e z - L 6 p e z , G o n z ~ l e z , a n d L r p e z - B a r -n e o , 1 9 8 9 ; B i s c oe a n d D u c h e n , 1 9 9 0; B u c k l e r , V a u g h a n - J o n e s , P e e r s , a n d N y e ,1 9 9 1 a ; B u c k l e r , V a u g h a n - J o n e s , P e e r s , L a g a d i c - G o s s m a n n , a n d N y e , 1 9 91 b ; P e e r sa n d G r e e n , 1 9 9 1 ; f o r re v i e w s s e e V e r n a , 1 9 7 9; F i d o n e a n d G o n z ~ l e z , 1 9 8 6 ) . Y e t th ep r e c i s e m e c h a n i s m s w h e r e b y t h e c a r o t i d b o d y t r a n s l a te s c h e m i c a l c h a n g e s o f th eb l o o d i n t o n e r v e a c t i vi t y a r e s ti ll f a r f r o m c l e a r .

I n e a r l y i n vi v o s t u d i e s f r o m t h i s la b o r a t o r y ( H o r n b e i n a n d R o o s , 1 9 6 3 ) i n w h i c h w er e c o r d e d t h e i n t e g r a t e d e l e c tr i c al a c t iv it y o f t he c a t c a r o t id b o d y n e r v e , w e d e m o n -s t r a t e d t h e p r e e m i n e n c e o f a n e l e v a t e d a r te r i a l H i o n c o n c e n t ra t i o n , r a t h e r t h a n o fP c o 2 , a s a c h e m o r e c e p t o r s t i m u l u s . W e r a i s e d t h e p o s s i b i l i t y t h a t t h i s r e s p o n s e t oa c i di ty m i g h t b e m e d i a t e d v i a c h a n g e s o f H i o n c o n c e n t r a t i o n w i t h in t h e c h e m o s e n s -i n g c e l ls o f th e c a r o t i d b o d y . A t t h e t i m e , l i t tl e i n f o r m a t i o n w a s a v a i l a b le a b o u t t h ep h y s i o l o gy o f i ts v a ri o u s c e ll t y p es , a n d t h e r e w a s n o s t a n d a r d m e t h o d f o r m e a s u r i n gi n t r a c e ll u l a r p H ( p H i) . T h e r e i s n o w r e a s o n a b l e ( t h o u g h n o t u n a n i m o u s a n d ,a d m i t t e d l y , l a r g e l y c i r c u m s t a n t i a l ) e v i d e n c e t h a t t h e s o - c a l l e d g l o m u s c e l ls ( a ls ok n o w n a s t y p e I ce l ls ) , w h i c h a r e b y f a r t h e m o s t n u m e r o u s ( V e r n a , 1 9 7 9) , s e r v e at r a n s d u c e r f u n c ti o n , t h e n e r v e e n d i n g s i n t h e i r i m m e d i a t e v i c in i ty b e i n g t h ep o s t s y n a p t i c s t r u c t u r e s ( s e e , f o r i n s t a n c e , V e r n a , 1 9 7 9; D u c h e n e t a l ., 1 9 8 8 ). A ls o ,o p t i c a l m e t h o d s m a k e i t p o s s i b l e t o f o l lo w p H i i n r e l a t iv e l y s m a l l c e l ls s u c h a s g l o m u sc e l ls ( ~ 1 0 I~ m d i a m e t e r ) . I n t h i s w o r k w e h a v e s t u d i e d t h e r e l a t i o n s h i p b e t w e e ne x t r a c e l l u l a r p H ( p H o ) a n d p H i i n i s o la t e d a d u l t r a t g l o m u s c e l ls in b o t h t h e p r e s e n c ea n d ( n o m i n a l ) a b s e n c e o f C O 2 . W e a l s o e x a m i n e d t h e e f f e c t o n p H i o f c h a n g e s i ne x t r a c e l l u l a r [ N a + ] a n d [ K + ].

A p r e l i m i n a r y r e p o r t o n t h i s w o r k h a s b e e n p u b l i s h e d ( W i l d in g , C h e n g , a n d R o o s ,1991) .

M E T H O D SAdult Sprague-Dawley ra ts (~ 30 0 g ; Har la n Sprague-Dawley , Inc. , Ind ianapo l is , IN) wereanesthet ized wi th sod ium pen toba rb i ta l (1 mg/kg) . Th e two caro t id bod ies were d issected f reeand pu t in 2 m l o f enzyme-con tain ing sal t solu tion (pH ~ 7 .4) o f the fo l lowing composi t ion ( inmillimoles pe r liter): 140 Na +, 4.7 K +, 2 Mg 2+, 3 N-methy l-D-g lucamm onium+ (NM DG+), 146.7CI- , 10 HEPES, an d 5 g lucose (NMDG was subst i tu ted fo r C a 2, which was omit ted to p reve n tclum ping of the cells). Th e solution c onta ined 4 m g each of class II collagenase (WorthingtonBiochemical Corp . , F reeho ld , N ) and type XIV pro tea se (S igma Chem ical Co . , S t. Lou is , MO).Th e cel ls, kep t a t 37C, were t r i tu rated twice and then cen tr i fuged fo r 10 min at 800 g . The

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WILDING ET AL. IntraceUularpH o f Glomus ells 595pellet was resu spen ded in 2 ml of the same salt solution contai ning 4 mg each of class IIcollagenase and hyaluro nidase (Sigma Chemical Co.), 10 mg of al bumi n (Sigma Chemica l Co.),and 1.4 mg of DNAse I (Sigma Chemical Co.). Tri tur atio n was repe ated and the s uspens ion wasagain centrifuged. The pellet was resuspended, washed with enzyme-free salt solution, andcentrifuged twice. This procedure is a modification of that of Biscoe, Duchen, Eisner, O'Neill,and Valdeolmillos (1989). An aliquot of the cell susp ension was placed o n a coverslip, coatedwith the adhesive polyphenolic protein Cell Tak (Collaborative Research, Inc., Bedford, MA),that fo rmed the b ottom of a shallow chamber (surface area ~ 75 mm 2, dept h ~ 1 mm) fastenedto the stage of a Nikon Diaphot inverted microscope. After a 30-min period required for thecells to settle, most of the cham ber's cont ents was replaced by a salt solution that now cont ained2 mM Ca 2+ and 10 ~M o f the acetoxymethyl ester o f 2',7'-bis(2-carboxy ethyl)-5- (and -6)carboxyfluorescein (BCECF; Molecular Probes, Inc., Eugene, OR) (Rink, Tsien, and Pozzan,1982). The ester readily enters the cell where esterases remove the acetoxymethyl groups,trapping an ion whose fluorescence is pH sensitive. The dye was stored at -20C as a 10-ramsolution in water-free DMSO. After 30 rain of dye-lo ading, superfus ion by gravity at a rate of 4ml /m in was started with dye-free soluti on from one o f an assembly of six 140-ml syringes whichwere kept in a thermostatically controlled water bath. Chamber temperature, continuouslymoni tore d with a min iatu re probe, was 36---36.5C. Some studies were done at ro om t empera-ture ( ~ 22C); these will be separa tely described. CO2 -impermeab le Saran tubi ng connect ed thesyringes through a manifold to the chamber. The CO2-free experiments were usually startedwith norm al superfusate (pH 7.40) of the following compos ition (millimoles per liter): 140Na +, 4.7 K +, 2 Mg2+, 2 Ca2+, 146.7 CI-, 10 HEPES, and 5 glucose (modified Tyrode's). Theeffect on pHi of raisi ng or lowering pHo, of pulsi ng with NH4CI, or of changin g Na +, CI-, or K+concentrat ions was then studied. At pHo < 6.8 0 the buff er was 10 mM PIPES; at pHo 8.00 thebuffer was 10 mM glycylglycine. Whe n [Na+]o was reduced , NMDG was used as subs titute .When [C1-]o was reduced or omitted, glucur onate was the substi tute (except that the Ca 2+ andMg~ salts were gluconates; to com pensa te for bin ding, the Ca~+ concentr atio n was increased to5 raM). In most exper imen ts in which [K+]owas raised, [Cl-]o was kept constant; in a few, [Cl-]owas reciprocally reduc ed to ma inta in [K+]o x [Cl-]o constant. Th e thalliu m (TI +) Tyro de wasmade entirely from nitrate salts. K+ and TI + were su bstituted for Na +.

The intrinsic (i.e., nonb icar bona te) buffe ring power, 13 (Roos and Boron, 1981), wasmeas ured i n 32 cells (14 rats) between pHi 5.90 and 7.70. These pHi' s were achieved by varyingpHo (6.20-8.20). At a stable pHi value, buffering power was evaluated with small pulses ofNH4CI (0.8-5 mM, the higher concentrations at the more acidic pH's), which were applied for1-2 min. After the pulses pHi always returned to the original value.

In ex perim ents with CO2 the syringes' contents were continuo usly gassed with 1, 5, or 10%CO2 in 02. Preliminary experiments showed negligible loss of COs between syringe andchamber. The effect of hypoxia was examine d by switching to a superfusate gassed with 2% 02,the rema inder being either N2 or a N2 CO 2 mixture. The effect of 0.5 mM SITS (4-acetamido-4'-iso thiocya natosti lbene 2,2'-dis ulfonic acid; Pfalz and Bauer Inc., Waterbury, CT) on pHirecovery from CO2 was studied by preexposing the cells to the drug for 1 h. This should resultin irreversible binding to the membrane. The subsequent solutions were SITS-free since thedrug's fluorescence interferes with pHi measurements. The effect on pHi of 5-(N,N-hexameth-ylene)- amiloride (HMA; Research Biochemicals Inc., Natick, MA), a powerful inhib itor ofNa/ H + exchange (Simchowitz and Cragoe, 1986), was also examined. Here, the pr oblem ofthe drug's fluorescence cannot be solved so easily, since its reversible binding propertiesrequire it to remain present in the superfusate. We could not use concentrations greater than15 I~M without no ticeab le optical interf erence . Since, in 140 mM Na +, the dr ug at this low

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WILD ING ET AL. IntracellularpH of Glomus Cells6 o F, , o r j j w r / . - - -1 . 0 0 I - . . . . . . . . . . . . . . . . . . . . . . . .~ QS O

I I I I J I5.5 60 6 .5 7 .0 7 .5 8 .0PHisc r ibed by : E49o/E440 = (10PHi-pK + A /B ( l + 10PHi'pK), wh e r e7 .14 _ . 0 .022 , A = 0 .227 - 0 .0053 , an dB = 0 .589 - - 0 .0070 .

597F IG U R E 1 . I n t r a c e U u l a r c a l i -b r a t i o n of BCECF: r e l a t ionsh ipb e t we e n p Hi a n d t h e r a t i o o femit ted l ight in tensi t ies a t exci-t a t ion wave leng ths 490 and 440n m . T h e e m i t t e d l i g h t wa s m e a -su red a t 520-560 n m ; t h e r a -t i o s we r e n o r m a l i z e d a t p H7 . 10 . 8 8 m e a s u r e m e n t s f r o m 1 9g lomus ce l l s . The cu rve i s de -A and B a re cons tan t s , pK =

R E S U L T SExperiments in the Nominal Absence of C02

Relation between pHi and pHo. I n s o l u ti o n s b u f f e r e d w i t h 1 0 m M H E P E S t o p H 7 . 4 0 ,t h e p H i o f t h e g l o m u s c e l l s w a s 7 . 2 3 --- 0 . 0 2 ( m e a n +-- S E M ; n = 7 0 ) . W h e n p H o w a sc h a n g e d f r o m 7 . 4 0 t o v a l u e s r a n g i n g f r o m 6 . 2 0 t o 8 . 0 0 ( 3 2 c el ls ), p h i c h a n g e d i n t h es a m e d i r e c t i o n a n d a t t a i n e d v a l u e s b e t w e e n 6 . 0 8 a n d 8 . 1 8 . T h e n e w s t e a d y s t a t e w a sr e a c h e d i n 5 - 1 5 m i n a n d w a s re v e r si b l e ( F ig . 2 ). A t p H o v a l u e s l es s t h a n 6 . 9 0 p H i a n dp H o w e r e n e a r l y i d e n t ic a l ; a t h i g h e r v a l u e s p H i w a s s li g ht ly l o w e r t h a n p H o . T h es e ns it iv i ty o f p H i t o c h a n g e s i n p H o w a s s tr i k in g : t h e r a t i o A p H i / A p H o a m o u n t e d t o0 . 8 5 o v e r t h e e n t i re p H r a n g e s t u d ie d . T h e r e l a t i o n s h i p is g i v e n b y p H i = 0 . 9 9 +0 . 8 5 ( p H o ) , w i t h a c o r r e l a t i o n c o e f f i c i e n t o f 0 . 9 9 5 ( F i g. 3 ) .

Effect of Na + removal and of HMA. U p o n r e m o v a l o f N a + ( r e p l a c em e n t w i thN M D G ) , p H i fe l l i n ~ 1 5 m i n f r o m t h e c o n t r o l v a l u e ( 7 . 2 7 - 0 . 0 7 ; n = 1 1 ) t o a n e ws t e a d y v a l u e o f 6 . 3 5 --+ 0 . 1 0 . W h e n N a w a s r e t u r n e d , p H i r e c o v e r e d t o c l o s e t o it so r i g i n a l v a l ue . R e d u c i n g [ N a + ] o t o 5 0 o r 2 0 m M r e v e rs i b ly l o w e r e d p H i t o 7 . 0 6 -0 . 0 3 ( n = 9 ) o r 6 . 8 6 -+ 0 . 0 5 ( n = 1 0 ), r e s p e c ti v e l y . I n n i n e c e l l s b a t h e d i n 5 0 m MN a , th e e f f e c t o f 1 5 I ~M H M A , a n a m i l o r i d e d e r iv a t iv e , w a s c o m p a r e d w i t h t h a t o fN a + r e m o v a l . T h e f al l in p H i i n d u c e d b y H M A w a s a l w a ys s l o w e r a n d o f l es sm a g n i t u d e ( F i g. 4) . I t i s n o t c l e a r t o w h a t e x t e n t t h i s d i f f e r e n c e i s d u e t o i n c o m p l e t e

Exp. ?'957 . 4 0 I 5 r ai n I

P H i 7 . 0 0 ~I mM6 . 6 C ~ 4 c J ,z4o p H 6 . 8 0 7 . 4 0 1 1] o l i b r o t i o n 6.80, pHi fa l ls by 0 .54 to as t eady va lue and then r e tu rns . Th i s i s fo l lowed by an NH 4CI pu l se ( fo r me asu r ing b u f fe r ing

power ) . The expe r imen t ends wi th a ca l ib ra t ion in 140 mM K + and 10 IxM n ige r i c in , p H 7 .10 .No te the pHi peak tha t p recedes the s t ab le f ina l ca l ib ra t ion va lue .

F IGURE 2 . Respo nse o f pHi toex te rna l ac id i f i ca t ion and toNH4Cl . The p lo t s in th i s f igu reand in Figs . 4 , 6 , 7 , and 9 werede r ived f rom the op t i ca l mea -su remen t s a s desc r ibed unde rM e t h o d s . W h e n , i n t h e n o m i n a la b s e n c e o f C O2 , p Ho is t r a n -s i en t ly lowered f rom 7 .40 to

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598 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y V O L U M E 1 19925

ZB0 PH= .985 +0,848 PHo) A y7 .4 0 ~ , . , ~ T

pH Z00 ~6 .6 0 4 ~ ~6.20

I I I I I I I I I I6 . 2 0 6 6 0 7 . 0 0 7 4 - 0 7 . 80pHo

FGURE3 . Re la t ionsh ip be tween pHia n d p H o i n t h e n o m i n a l a b s e n c e o fCOz. M eans -+ SEM; 32 cel ls . Correla-t ion coef f ic ien t = 0 .995 . Each exper i -ment was star ted at pHo 7.40.

i n h i b i ti o n b y th e d r u g h i g h e r c o n c e n t r a t i o n s c o u l d n o t b e u s e d s i n c e th e y i n te r f e r ew i t h th e o p t i c a l m e a s u r e m e n t s ; s e e M e t h o d s ) o r t o tr a n s i e n t r e v e r sa l o f t h e N a + / H e x c h a n g e r u p o n e x t e r n a l N a + r e m o v a l . T h e o b s e r v e d c e ll a ci d i fi c a ti o n m i g h t b e d u et o t h e u n m a s k i n g e i t h e r o f m e t a b o l i c H i o n p r o d u c t i o n , a m e c h a n i s m t h a t e x c h a n g e se x t e r n a l H + f o r i n t e r n a l K + s e e D i s c u s s io n ) , o r a c o m b i n a t i o n o f t h e s e .

T h e e f f ec t o f v a r i o u s N a + c o n c e n t r a t i o n s o n t h e r a t e o f p H i r e c o v e r y r e la t iv e t o t h er a t e o f r e c o v e r y at 1 4 0 m M N a + w a s d e t e r m i n e d a f t e r t h e c el ls h a d b e e n a c i di f ie d b yN a + r e m o v a l . A ll m e a s u r e m e n t s w e r e m a d e a t n e ar l y t h e s a m e p H i 6 . 4 3 - 6 . 5 4 ) .I n t e r v a l s a t d i f f e r e n t [ N a + ] o w e r e s e p a r a t e d b y N a + - f r e e p e r i o d s . 2 5 ce l ls f r o m 1 3 r a t sy i e l d e d 64 m e a s u r e m e n t s . W h e n e x p r e s s e d b y s i m p l e M i c h a e l i s - M e n t e n k i n e ti c s, t h ea p p a r e n t K m o f t h e [ N a + ] o - d e p e n d e n t r e c o v e r y w a s 5 8 -+ 6 m M F i g. 5 ). T h e r a t e o fr e m o v a l o f H + e q u i v a l e n t s f r o m t h e c e l ls m i l l i m o l e s p e r l i t e r c e ll w a t e r p e r m i n u t e ) ,o b t a i n e d a s t h e p r o d u c t o f t h e n o n n o r m a l i z e d p H i r e c o v e r y r a t e a n d i n t r in s i cb u f f e r i n g p o w e r 1 9 - 2 1 r a M ; se e b e l ow ) , a m o u n t e d t o 7 .1 + 1 .2 n = 2 6 ) a t 1 4 0 m MN a + , 5 .3 +-- 1 . 0 n = 1 1 ) a t 4 0 m M N a , a n d 1 .0 + - 0 .1 7 n = 7 ) a t 1 0 m M N a + .Ef fec t o f changing [K ]o . W h e n t h e c a l i b r at i o n s o l u t i o n c o n t a i n i n g 1 4 0 m M K +a n d n i g e r i c i n p H 7 . 10 ) w a s a p p l i e d a t th e c o n c l u s i o n o f e a c h e x p e r i m e n t , t h ee v e n t u a l l y s t a b l e p H i r e a d i n g w a s o f t e n p r e c e d e d b y a t r a n s i e n t r i s e i n p H i s e e F i g s.2 a n d 6 ). T o a n a l y z e th i s p h e n o m e n o n , w e e x a m i n e d t h e e ff e c t o n p H i w h e n , a t p H o7 . 4 0 , [ K + ]o w a s r a is e d t o 1 4 0 m M w i t h o u t n i g e r i c i n . T h i s r e s u l t e d i n a re v e r s i b l e r is eo f p H i f r o m 7 . 3 8 0 . 0 8 t o 7 . 9 8 0 . 1 6 n = 6 ) i n ~ 5 m i n . T h e r e s u l t s w e r e t h e s a m ea t e i t h e r c o n s t a n t [ K + ] o x [ C l - ] o p r o d u c t o r c o n s t a n t [ C l - ] o . S m a l l e r e l e v a t i o n s o f[ K + ]o 5 0 a n d 2 5 r a M ) r e v e r s ib l y r a i s e d p H i b y s m a l l e r a m o u n t s : 0 . 3 4 0 . 0 4 n = 7 )a n d 0 . 2 1 + 0 . 0 3 n = 1 1 ), r e s p e c t i v e l y F i g . 6 ) . T h e K + - i n d u c e d a l k a l i n i z a t io n a l s oo c c u r r e d i n c e ll s w h o s e p H i h a d f i rs t b e e n r a i s e d t o 7 . 8 0 ) o r l o w e r e d t o 6 . 8 0 ) b yc h a n g i n g p H o .

PH

5 0 N o I O N I 5 0 N o 1 5 0 N a 1 5 p M I . I I M A7 2 0 r p ~ 7 4 0

6 e t k / . .6 6 0 L ~ 5 m i n j Exp 609

FIGURE 4. Th e pH i of a cel l in50 m M Na fa l ls r ap id ly u ponNa + remova l , and then re tu rnswhen Na + i s r eadmi t ted . Upo napp ly ing 15 IxM HMA the pHiagain falls, but the fall is slowerand le ss p ronounced .

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WILDING ET AL

1 00

~ 0 5 0~._~n~ 0 0 0

lntracellular pH of G lomus Cells

Kmi i i3 0 6 0 - - o L N o j 1 2 o 4 0

599

FIGURE 5. Effect of Na + con-centration on the pHi recoveryrate relative to the recoveryrate in 140 mM Na +. 25 cellswere initially acidified by expo-sure to Na+-free medium. Allmeasurements are at pH~ 6.43-6.54. Measurements on eachcell included one in 140 mMNa +. The apparent Km for Na +(Michaelis-Menten kinetics) is58 + 6 raM.

The question arose whether, by reducing external potassium below its normalvalue, pHi could be driven in the opposite direction. Indeed, lowering [K+]o from 4.7to 1.5 mM reversibly re duc ed pHi by 0.11 _+ 0.03 (n = 5). In the absence o f Na +(which lowers phi as described above) all these [K+]o effects were more pronounced:50 mM K + increas ed pHi by 0.66 +- 0.05 (n = 3), while 1.5 mM K + reduc ed it by0.40 -+ 0.06 (n = 6). (The studies in 1.5 mM K+/ 0 Na were d on e at pHo 7.70[pHi ~6.9], rather than at 7.40, in order to obviate intracellular acidity beyondcalibration range [see Fig. 1]).

These responses o f pHi to changes in external K + were not the result o f changes inmembra ne voltage that almost certainly must have occurred. Depolarization in eithergramicid in (1 I~M; Sigma Chemical Co., St. Louis , MO) or Ba 2+ (2 mM) in C1--freemedium had no effect on pHi (for the depolarizing effect of gramicidin, see Silmanand Karlin [1968] an d Podleski and Cha nge ux [1969]; for the effect of Ba 2+ inC1--free solutions, see Astion, Obaid, and Orkand [1989]). Moreover, in cellsexpo sed to 2 mM Ba2+/0 CI - for 30 min or more , t he rise of pHi with 50 mM K + wassimilar to that in nor mal c hloride without Ba 2+, and the fall in pHi up on redu cing[K+]o to 1.5 mM was also unaffected .Effect o f NH4Cl When we pul sed cells (pHo 7.40) for ~ 5 min with 20 m MNH4CI (substituted for NaC1) we observed the expected steep rise of pHi, alwaysfollowed by pla teau acidificat ion (i.e., a slow fall). Up on NH4C1 removal , the pHiunde rsh oot below the initial value was followed by near- compl ete recovery. Quali-

tatively similar transients were obta ined in 140, 50, and 20 mM Na+; of course, theinitial values before the pulse were substantially lower at the re duce d N a + conce ntra-tions (7.06 at 50 mM and 6.86 at 20 mM). When the solution after the pulse wasNa+-free, there was no recovery. In the prese nce of 50 mM Na +, 15 I~M HMA, adde d

Z 8 O r t 5rain

I 5 K I olibrot ion

FIGURE 6. The pHi transiently risesby 0.44 when [K+]o is transiently in-creased from 4.7 to 50 mM at con-stant [Cl-]o (pHo 7.40, nominal ab-sence of CO2). The end of the recordshows the calibration.

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WILDINGET AL. IntraceUular H of Glomus Cells 601T h e p H i - p H o r e l a ti o n s h i p i n th e p H o r a n g e 6 . 2 0 - 8 . 0 0 c o u l d b e e x p r e s s e d b y p H i =2 . 3 2 + 0 . 6 4 5 ( p H o ) ( n = 1 3) . T h u s , p H i a t r o o m t e m p e r a t u r e i s n o t q u i t e a s s en s i ti v et o p H o a s a t b o d y t e m p e r a t u r e . U p o n r e m o v a l o f N a + , t h e f a ll in p H i a v e r a g e d 0 . 5 8( n = 4 ) , s o m e w h a t le s s t h a n a t b o d y t e m p e r a t u r e . T h e s e n si ti v it y o f in t r in s i c b u f f e r i n gp o w e r t o p H i b e t w e e n 6 . 1 0 a n d 7 . 4 0 ( p H o 6 .2 0 -- -8 .0 0 ; n = 1 3 ) w a s n e a r l y t h e s a m e a tr o o m t e m p e r a t u r e a s a t 3 6 C . A g a in , N a + r e m o v a l w a s w i t h o u t e f fe c t .

Experiments with C02-containing SolutionsRelat ion between pH i an d pHo in the presence of C02. W i t h c el ls in C O 2 / H C O ~ - b u f f e r e dm e d i a ( 3 6 C ), w e e x a m i n e d t h e r e l a t i o n s h i p b e t w e e n s t e a d y - s t a t e p H o a n d p H i , j u s ta s w e h a d w i t h c el ls i n C O 2 - f re e s o l u t io n s . T h e p H o w a s c h a n g e d e i t h e r a t c o n s t a n tP c o 2 o r a t c o n s t a n t [ H C O ~ ] . ( a) I n 5 % C O 2 / 9 5 % 0 2 , a r e d u c t i o n o f [H C O ~ ] o f r o m~ 2 5 t o 5 m M ( p H o 7 .4 0 a n d 6 . 8 0 ) p r o m p t l y l o w e r e d p H i f r o m 7 . 1 8 - 0 . 0 8 to 6 .6 9 - -0 . 0 6 ( n = 9 ) . T h i s i s i l l u s t r a t e d i n F i g . 9 A . (b ) A t 5 m M [ H C O ~ ] o , a n i n c r e a s e i n P c o 2

A 5 % c o Z.2SmM I IC0~ 5 r aM H C 0 3 p i l e6 .8 0

pi loT.40

I 6 ra in IE xp . 5 4 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B 5 m M HCO~5 COz P H 6 7 J~ < '/ o C 0 2 , oH o 7. 41

Exp. 5 6 5

FIGURE 9. (A) Th e pH i falls by 0.4when, a t constan t Pco~ (5%), pHo islowered by 0.6 when HCO~ concen-t r a t ion i s r educed f rom ~ 25 t o 5 m M .B) The pHi rises by 0.66 when, atcons t an t H C O ~ concen t r a t i on (5mM), pHo is ra ised by 0.7 when Pco~is reduced f rom 5 to 1%.

f r o m 1 t o 5 % ( p H o 7 . 4 2 a n d 6 . 7 1) p r o m p t l y l o w e r e d p H i f r o m 7 . 2 6 --- 0 . 0 4 t o 6 . 6 7 -0 . 0 3 ( n = 5 ). F ig . 9 B s h o w s a n e x p e r i m e n t i n w h i c h t h e s e q u e n c e o f t h e s e c o n d i t i o n sw a s r e v e r s e d . B o t h a t c o n s t a n t P c o 2 a n d a t c o n s t a n t H C O ~ t h e r e s p o n s i v e n e s s o f p H it o p H o ( A p H i / A p H o ) w a s 0 .8 2 . T h i s r a t i o i s n e a r l y t h e s a m e a s t h a t o b t a i n e d ( o v e r aw i d e r p H r a n g e ) i n t h e n o m i n a l a b s e n c e o f C O 2 ( 0. 8 5; F i g . 1 0) .W h e n , a t u n c h a n g e d p H o 7 . 4 0, b o t h b i c a r b o n a t e c o n c e n t r a t i o n a n d P c o2 w e r ei n c r e as e d 1 0 -f ol d, f ro m 1 % C O 2 / 5 m M H C O ~ t o 1 0 % C O 2 / 5 0 m M H C O ~ , t h e p H i( 7 .1 7 _ 0 . 0 5 ; n = 8 ) t r a n s i e n t l y f e l l b y a n a v e r a g e o f 0 . 2 5 , b u t t h e n r e t u r n e d t o t h eo r i g i n a l v a lu e . C o m p l e t e r e c o v e r y w a s a l s o o b s e r v e d w h e n P c o 2 w a s i n c r e a s e df iv e fo ld , f r o m 1 % C O 2 / 5 m M H C O ~ t o 5% C O ~ / 2 5 m M H C O ~ .Effect of changing f rom C02- free to C02-contain ing media. C h a n g i n g t h e b u ff e r f r o m1 0 m M H E P E S ( n o m i n a l a b s e n c e o f C O 2 ) t o 5 % CO2/25 m M H C O ~ a t u n c h a n g e d

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602 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y V O L U M E 1 992p H o 7 . 4 0 tr a n s i e n t l y r e d u c e d p H i f r o m 7 . 2 9 + 0 . 0 4 t o 7 .0 5 + 0 . 0 4 ( n = 1 2 ). W i t h i n5 - 1 5 m i n p H i u s u a l l y p a r t i a ll y r e c o v e r e d t o a n e w s t e a d y s t at e ( 7 . 1 7 -+ 0 . 0 5 ) , w h i c hw a s s li g h t ly b u t s i g n i fi c a n t l y l o w e r t h a n t h e o r i g i n a l v a l u e i n H E P E S ( P < 0 . 0 0 5 ) . As i m il a r p H i c o u r s e w a s o b s e r v e d u p o n s w i t c h in g f r o m H E P E S t o 1 0 C O ~ / 5 0 m MH C O ~ : a t r a n s i e n t f a l l f r o m 7 . 2 5 -+ 0 . 0 5 t o 6 . 8 7 + 0 . 0 6 ( n = 1 1 ) w a s f o ll o w e d b yr e c o v e r y t o 7 . 1 1 0 . 0 9 , 0 . 1 4 l e s s t h a n t h e i n i t i a l v a l u e . T h u s , t h e s t e a d y - s t a t e v a l u e si n 1 , 5 , a n d 1 0 C O 2 a r e n e a r l y t h e s a m e a n d s o m e w h a t lo w e r t h a n u n d e r n o m i n a l l yC O 2 - f r e e c o n d i t i o n s a t t h e s a m e p H o ( 7 . 4 0 ) .

S i m il ar e x p e r i m e n t s ( 1 0 m M H E P E S t o 5 C O 2 / 2 5 m M H C O ~ ) w e re d o n e a ft e rt h e ce ll s h a d b e e n e x p o s e d f o r 1 h t o H E P E S - b u f f e r e d m e d i u m c o n t a i n i n g 0 . 5 m MS I T S ; t h e s u b s e q u e n t s o l u t i o n s w e r e S I T S - f r e e . T h e i n i t i al p H i w a s 7 . 2 5 - + 0 . 0 6( n = 1 4 ), n e a r l y t h e s a m e a s w i t h o u t S I T S . T h e a v e r a g e C O s - i n d u c e d i n i t ia l fa ll ( 0 . 2 7 )w a s a ls o s i m i la r t o t h e f al l w i t h o u t S I T S . T h e s u b s e q u e n t p H i r e c o v e r y o f 0 . 0 6 w a s h a l fo f th a t s e e n w i t h o u t S I T S , b u t t h e d i f f e r e n c e w a s n o t q u i t e s i g n i f i c a n t(0 .05 < P < 0 .1 ) .

PHi

ZSO 4 0ZOO6.606 2 0 ~ , ,, ,x

I62O

1 1 X~ X

I I I I I I I I6.60 7.00 7.40 7.80pHo

FIGURE 10 . Com par i son o f pHi -pHore la t ionsh ips in g lomus ce l l s and redcells . The circles pertain to glomuscells in CO2/HCOs: (3, pHo wasc h a n g e d a t c o n s t a n t H C O ~ - c o n c e n -t ra t ion (5 mM ); O , pHo was changeda t cons tan t Pc02 (5 ) . X represen t sd a t a o b t a i n e d f r o m o x y g e n a t e d h u -ma n re d ce l ls ; pHo was chang ed e i -t h e r b y c h a n g i n g P c o 2 o r w i th N a O H

or HC I ( f rom Fund er an d W ie th , 1966). Th e s ix po in t s have been a rb i tr a r i ly cho .sen us ing theseau tho rs ' r eg ress ion , [H+]o /[H+]i = 3 .094 -0 .335 pHo (cor re la t ion coef f icien t = 0 .995 -+ 0 .015 ;pHo range 6 .32-8 .64) . T he dashed l ine (same as in F ig . 3 ) r ep resen t s the pHi -pHo re la t ionsh ipin g lomus ce l ls in the n omin a l absence o f CO2.

Ef fec t o f Cl - removal . U n d e r C l - - f r e e c o n d i t i o n s ( p H o 7 . 4 0 ) t h e c o u r s e o f p H iu p o n c h a n g i n g f r o m H E P E S t o 5 C O 2 / H C O ~ - b u f f e r e d s u p e r f u sa t e w a s n e a rl y t h es a m e a s i n t h e p r e s e n c e o f C I - : p H i fe l l f r o m 7 . 3 5 t o 7 . 1 3 a n d t h e n p a r t i a ll yr e c o v e r e d t o 7 . 21 ( n = 3 ). I n a g r e e m e n t w i t h r e c e n tl y p u b l i s h e d w o r k b y B u c k l e r e t al .( 1 9 9 1 a ), C I - r e m o v a l i n t h e m i d s t o f t h e e x p o s u r e t o C O s r a i s e d p H i f r o m 7 . 0 5 -+0 . 0 4 t o 7 . 5 2 -+ 0 . 1 2 ( n = 8 ) , a v a l u e h i g h e r t h a n t h e p r e c e d i n g p H i i n C I - f r e eH E P E S - b u f f e r e d m e d i u m ( 7 .2 3 - 0 . 07 ) . T h i s e f fe c t w a s r e v e rs i b le a n d a b o l i s h e d b yp r e t r e a t m e n t w i t h 0 . 5 m M S I T S .

High [K ]o . I n t w o c e ll s, r a i s i n g K + c o n c e n t r a t i o n f r o m 4 . 7 t o 2 5 m M i n t h ep r e s e n c e o f 5 C O 2 / H C O 3 ( p H o 7 .4 0 ) r e s u l t e d i n a p H i ri s e f r o m 7 . 0 6 to 7. 4 1 ,s i m i l a r t o t h e r i se s e e n i n t h e a b s e n c e o f C O 2 .

Studies at room temperature. C h a n g i n g t h e b u f f er f r o m H E P E S t o 5 C O s /H C O ~ ( p H o 7 . 4 0 ) r e s u l t e d i n a p H i f a l l f r o m 7 . 2 9 + - 0 . 0 5 t o 7 . 0 0 -+ 0 . 0 3 ( n = 1 0 ), n o td i f f e r e n t f r o m o u r f i n d i n g s a t 3 6 C . H o w e v e r , i n s i x o f t h e s e c e l l s t h e r e w a s n or e c o v e r y . A t r o o m t e m p e r a t u r e , a s a t 3 6 C , r e m o v a l o f C 1 - d u r i n g C O s e x p o s u r e l e dt o a ri se i n p H i t h a t c o u l d b e b l o c k e d b y 1 5 0 ~ M D I D S ( 4 , 4 ' - d ii s o t h i o c y a n a t o s ti l b e n e

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WILDING ET AL. IntraceUular H of Glomus ells 603d i s u l f o n i c a c i d ). A t th i s c o n c e n t r a t i o n , t h e d r u g ' s f l u o r e s c e n c e d o e s n o t i n t e r f e r e w i tht h e p H i m e a s u r e m e n t .

Effect of HypoxiaW h e n c e ll s, o r i g i n a ll y i n 1 0 C O s , 5 0 m M H C O ~ ( p H o 7 .4 0 ) , w e r e e x p o s e d t o ah y p o x i c g a s m i x t u r e (2 0 2 , 1 0 C O 2 , r e m a i n d e r N 2 ), n o c h a n g e in p H i w a so b s e r v e d . In t h e n o m i n a l a b se n c e o f C O 2 , h y p o x i a (2 0 2 , 9 8 N 2 a l s o h a d n oe f f e c t e i t h e r a t p H o 7 . 4 0 o r 6 . 8 0 .

D I S C U S S I O NT h e a v e r a g e p H i o f f r e s h ly d i s so c i a t e d a d u l t r a t g l o m u s c e l ls in H E P E S b u f f e r ( p H o7 . 4 0 , 3 6 C ) w a s 7 . 2 3 . T h i s i s i n r e a s o n a b l e a g r e e m e n t w i t h t h e p H i o f n e o n a t a l r a tc e l ls ( 7 . 3 0 ) a f t e r o v e r n i g h t c u l t u r e i n H E P E S ( B u c k l e r e t al ., 1 9 9 1 a ) . I n 5 C O 2 /H C O ~ ( p H o 7 . 4 0 ) t h e p H i w a s 7 .1 7 . T h i s c l o s el y r e s e m b l e s t h e p H i o f m o s t o t h e rm a m m a l i a n c e l l s , s u c h a s m o u s e a n d r a t s k e l e t a l m u s c l e f i b e r s , s h e e p P u r k i n j e c e l l s ,g u i n e a p i g v a s d e f e r e n s s m o o t h m u s c l e ( se e A i c k in , 1 0 8 4 f o r r e f e r e n c e s ) , h u m a nn e u t r o p h i l s ( S i m c h o w i t z a n d R o o s , 1 0 8 5 ) , a n d h u m a n r e d c e l ls ( F u n d e r a n d W i e t h ,1066) . Ra t r en a l m esan g i a l ce l l s have a s l i gh t l y h i g he r pH i ( 7 .26 ; Boya r sky e t a l. ,1988) .

Sensitivity o f pH i to pHoA p r i n c i p a l f e a t u r e o f o u r w o r k i s t h e s t r ik i n g r e s p o n s i v e n e s s o f th e p H i o f t h e g l o m u sc e ll t o c h a n g e s i n e x t e r n a l p H i n b o t h t h e a b s e n c e a n d p r e s e n c e o f C O2 . F o ri n s t a n c e , r e d u c i n g p H o i n C O 2 - f r e e s o l u ti o n f r o m 7 . 4 0 t o 6 . 2 0 c a u s e d p H i t o fa l l f r o m7 . 2 3 t o 6 . 2 3 ; r a i s i n g p H o t o 8 . 0 0 i n c r e a s e d p H i t o 7 . 8 4 . T h e r a t i o A p H i / A p H oa m o u n t e d t o 0 . 8 5 a n d w a s c o n s t a n t o v e r t h e e n t i r e p H o r a n g e ( 6 . 2 0 - 8 . 0 0 ; F i g . 3 ).N e a r l y t h e s a m e h i g h r a t i o ( 0 . 82 ) w as f o u n d i n m e d i a b u f f e r e d w it h C O ~ / H C O ~ .H e r e , p H i a l t e r a ti o n s a c h i e v e d e i t h e r b y c h a n g e s i n [ H C O ~ ] o a t c o n s t a n t P c o z o r b yc h a n g e s i n PCO2 a t c o n s t a n t [ H C O ~ ] o h a d t h e s a m e e f f e c t o n p h i (F ig. 10) . S imi laro b s e r v a ti o n s in C O 2 / H C O ~ - c o n t a i n i n g m e d i a h a v e b e e n m a d e b y B u c k l e r e t a l.( 1991b) .

T h i s s e n s it iv i ty o f p H i t o c h a n g e s i n p H o i s q u i te u n u s u a l , a s i s sh o w n b y ac o m p a r i s o n w i th o t h e r c e ll s. F o r e x a m p l e , A p H i / A p H o i n g u i n e a p i g s m o o t h m u s c lei s 0 .38 ( A i ck in , 1984) ; i n m ou se so l eus musc l e , 0 .39 ( A i ck i n and T ho m as , 1977) ; i ns h e e p P u r k i n j e f ib e r s , 0 . 4 0 ( E ll is a n d T h o m a s , 1 0 7 6 ) ; a n d i n h u m a n n e u t r o p h i l s , 0 . 3 0( S i m c h o w i tz a n d R o o s , 1 9 8 5 ) ( a ll m e a s u r e m e n t s i n C O 2 / H C O ~ - ). T h e h u m a n r e d c e l ls t a n d s o u t a s a n e x c e p t i o n . I n t h is c e ll , th e A p H i / A p H o r a t i o , a s w e l l a s t h e a c t u a l p H iv a l u e s a t t h e r e s p e c t i v e p H o ' s ( F u n d e r a n d W i e t h , 1 9 6 6 ) , c l o s el y m a t c h t h o s e o f t h eg l o m u s c e l l . F i g . l 0 ( s e e l e g e n d f o r d e t a i l s ) s h o w s t h e c o m p a r i s o n . Y e t t h e m e c h a -n i s m s o f H i o n d i s t r i b u t i o n i n t h e t w o c e l l t y p e s a r e p r o b a b l y q u i t e d i f f e r e n t . I n t h er e d c e ll , H , C 1 , a n d H C O a i o n s a r e a t e l e c t r o c h e m i c a l e q u i li b r i u m . T h e i r d i s t r i b u t i o n s( a n d m e m b r a n e v o l ta g e , V m) a r e d e t e r m i n e d b y t h e n e t c h a r g e c o n c e n t r a t i o n s o f t h ei m p e r m e a n t i o n s o n b o t h s i d es o f t h e m e m b r a n e ( D o n n a n e q u i l ib r iu m ) . I n g l o m u sc e ll s, o n t h e o t h e r h a n d , t h e H i o n s a r e a l m o s t c e r t a in l y n o t a t e q u i l ib r i u m . T h i s i si n d ir e c tl y d e m o n s t r a t e d b y o u r o b s e r v a t io n t h a t e i t h e r N a + r e m o v a l o r a p p l i c a t i o n o f

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604 THE JOURNA L OF GENERAL PHYSIOLOGY VOLUM E 1 992H M A , a n i n h i b i t o r o f N a + / H + e x c h a n g e , s h a r p l y r e d u c e d p H i ( F ig . 4) , i n di c a t i n gt h a t i n n o r m a l , N a + - c o n t a i n i n g m e d i a a v i g o r o u s H + - r e m o v i n g m e c h a n i s m i s i nf o r ce . W e h a v e n o t m e a s u r e d m e m b r a n e v o l t a g e ; Vm m e a s u r e m e n t s b y o t h e r s ( f o rr e f e r e n c e s s e e P e e r s a n d G r e e n , 1 9 91 ) , o b t a i n e d b y i m p a l i n g t h e s e s m a l l c e ll s, m u s tb e l o o k e d u p o n w i t h s o m e s k e p t ic i s m b e c a u s e o f p os s i b l e ce l l d a m a g e . L . A . F i e b e ra n d E . W . M c C l e s k e y h a v e m e a s u r e d t h e Vm o f d is s o c i a t e d a d u l t r a t g l o m u s c e l l sw i t h o u t d i s r u p t i n g t h e c e l l m e m b r a n e ( u n p u b l i s h e d o b s e r v a t i o n s ). T h e y u s e d o n - c e l lp a t c h p i p e t t e s t o m e a s u r e c u r r e n t s ( I ) t h r o u g h s i n gl e C a 2+ c h a n n e l s a t d i f f e r e n tp i p e t t e v o l t a g e s ( V ) . F r o m t h e I V r e l a t i o n s h i p , t h e c e l l s ' r e s t i n g v o l t a g e c o u l d b ed e r i v e d b y r e p e a t i n g t h e p r o c e d u r e i n 1 4 0 m M K + t h a t w a s a s s u m e d t o d e p o l a r i z et h e c e l l 's r e s t i n g V m tO z e r o ( t h e a s s u m p t i o n w a s v a l i d a t e d b y t h e u n c h a n g e d I Vr e l a t i o n s h i p o f t h e e x c i s e d p a t c h i n 1 4 0 m M K + ) . A t p H o 7 . 3 0 , r e s t i n g v o l t a g e i n s ixc el ls r a n g e d b e t w e e n - 1 1 a n d - 2 3 m V ; i n t h e r e m a i n i n g f o ur , b e t w e e n - 4 3 a n d- 6 2 m V . I f t h e v o l t a g e h e t e r o g e n e i t y i s d u e t o t h e d a m a g e d c o n d i t i o n o f s o m e o f t h ec e l ls , t h e m o r e n e g a t i v e v a l u e s s h o u l d m o s t a c c u r a t e l y r e f l e c t i n v iv o c o n d i t i o n s ; i f t h eh e t e r o g e n e i t y w e r e a l s o t o e x i s t i n v iv o , t h e H i o n s i n t h e l o w - v o l t a g e c e l ls w o u l d b e i nn e a r - e q u i l i b r i u m . S u c h e q u i l i b r i u m w o u l d b e h a r d t o e x p l a i n i n v i ew o f t h e p r e s e n c eo f a n a c t iv e N a t / H + e x c h a n g e r a s w e j u s t d i s cu s se d . T h e s e d i re c t m e a s u r e m e n t si n d i c a t e t h a t i n s o m e o f t h e g l o m u s c e l ls , i f n o t i n a ll , t h e H i o n s a r e n o t i ne q u i l i b r i u m .

T h e H C O ~ i o n i s n o t r e q u i r e d f o r t h e p H i r e s p o n s i v e n e s s t o p H o , s i n c e t h ep H i - p H o r e l a t i o n s h i p w i th a n d w i t h o u t C O 2 / H C O ~ w a s n e ar l y t h e s a m e . T h a t l e a ve sa t l e a s t t h r e e m e c h a n i s m s t o a c c o u n t f o r t h e s e n s i t iv i t y o f p H i t o c h a n g e s i n p H o : ( a )E l e c t r o d i f f u s i o n o f H i o n s . F o r e x a m p l e , r e d u c i n g p H o f r o m 7 . 4 ( p H i 7 .2 ) t o 6 . 8e s t a b l i s h e s ( o r i n c r e a s e s ) a n i n w a r d e l e c t r o c h e m i c a l H i o n g r a d i e n t . A s s u m i n g t h a tVm = - 6 0 m V a n d c e l l d i a m e t e r = 1 0 I~ m , a v e r y l a r g e H + p e r m e a b i l i t y , 3 1 0 - 2c m ' s - l , w o u l d , h o w e v e r , b e r e q u i r e d t o a c c o u n t f o r t h e o b s e r v e d i n it i a l r a t e o f f a l l i np H i ( ~ 0 . 2 6 p e r m i n u t e ) . M o r e o v e r , d e p o l a r i z a t i o n s h o u l d r e d u c e t h e r a t e o f i n t e r n a la c i d i f ic a t i o n . Y e t e x p o s u r e t o a d e p o l a r i z i n g s o l u t i o n ( 0 C I - , 2 m M B a 2+ ; A s t i o n e t a l. ,1 9 89 ) h a d n o e f f e c t o n t h is r a t e . T h e r e f o r e , a l t h o u g h e l e c t r o di f f u s io n c a n n o t b e r u l e do u t , o t h e r m e c h a n i s m s m u s t b e o p e r a t i v e a s w e ll . (b ) C h a n g e s i n a c ti v it y o f t h eN a + / H + e x c h a n g e r . I t s r a t e m i g h t b e r e d u c e d i n r e s p o n s e t o e x t e r n a l a c i d if i c at i on( r e v e rs a l w o u l d s e e m u n l ik e l y a t n o r m a l [ N a+ ] o. H o w e v e r , t h e e x c h a n g e r w a s f o u n dt o b e s t i l l f u n c t i o n i n g e v e n a f t e r p H o h a d b e e n r e d u c e d t o 6 . 8 0 ( p H i 6 . 6 0 ) : w h e n[ N a +] o w a s t h e n r e m o v e d , p H i f e ll f u r t h e r b y ~ 0 . 6 . T h i s c o r r e s p o n d s t o a b o u t t h es a m e i n c r e a s e i n i n t r a c e l l u l a r H i o n s a s w h e n N a + w a s r e m o v e d a t p H o 7 . 4 0, t a k i n gb u f f e r i n g p o w e r i n t o a c c o u n t . T h e r e f o r e , a r a t e r e d u c t i o n o f th e e x c h a n g e r u n d e rt h e s e c o n d i t i o ns s e e m s u n l ik e l y. M i g h t t h e i n c r e a s e i n p H i u p o n r a i s i ng p H o h a v eb e e n d u e t o s t i m u l a t e d N a + / H + e x c h a n g e ? W h e n p H o w as r a is e d f r o m 7 . 40 t o 8 . 0 0i n t h e a b s e n c e o f N a + , p H i i n c r e a s e d f r o m 6 . 5 0 __ 0 . 1 2 t o 6 . 9 0 -+ 0 . 1 0 ( n = 7 ). T h i si n d i c a t e s a r a ti o , A p H i / A p H o , o f 0 . 6 7 , s i g n if i c a n tl y l e s s t h a n t h a t i n t h e p r e s e n c e o fN a + ( 0 .8 5 ; F i g . 3 ). T h u s , a c o n t r i b u t i o n o f N a + / H + e x c h a n g e t o t h e o b s e r v e d r i s e i np H i c a n n o t b e d i s m i s s ed . (c ) K + / H + e x c h a n g e . A s will b e s h o w n b e l ow , t h e m e m b r a n eo f t he g l o m u s c e l l p r o b a b l y c o n t a in s a n e l e c t r o n e u t r a l K + / H + e x c h a n g e r . R a i s i n ge x t r a c e l l u l a r [ H + ] m i g h t i n c r e a s e e x c h a n g e r - m e d i a t e d H + in f l u x ( a n d K + e f f iu x ) . I ts h o u l d b e a d d e d t h a t t h e l ow i n t ra c e l l u la r b u f f e r i n g p o w e r o f t h e g l o m u s c e l l ( se e F i g.

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WILDING ET AL. IntraceUularpH of Glomus Cells 6058 ) w o u l d e n h a n c e p H i c h a n g e s d u e t o t r a n s m e m b r a n e H + m o v e m e n t s r e g a rd l e s s o fm e c h a n i s m .

K H E x c ha n g eI n t h e c a l i b r a ti o n p r o c e d u r e a t t h e e n d o f e a c h e x p e r i m e n t , t h e c e ll s w e r e e x p o s e d t oa N a + - f r e e s o l u t i o n c o n t a i n i n g 1 4 0 m M K + a n d 1 0 ~ M n i g e r i c i n a t p H o 7 . 1 0 . I nn e a r l y e v e r y c a s e t h e e v e n t u a l l y s t a b l e p H i w h i c h , it i s a s s u m e d , c l o s e l y a p p r o a c h e sp H o , w a s p r e c e d e d b y a s t e e p a n d t r a n s i e n t r i s e ; t h i s is i l l u s t r a t e d i n F i gs . 2 a n d 6 . W ek n o w t h a t r e m o v a l o f N a + b y i ts e l f p r o d u c e s a f a U i n p H i ( F i g . 4 ) . W e s u s p e c t e d t h a tt h e t r a n s i e n t r i s e w a s c a u s e d b y t h e e l e v a t e d [ K + ]o . I n d e e d , s i m p l e s u b s t i t u t i o n o f 2 0,5 0 , o r 1 4 0 m M N a + b y e q u a l a m o u n t s o f K p r o d u c e d a r e v e r s i b l e ri s e in p H i ( s e eF i g. 6 ) w h i c h , i n g e n e r a l , w a s g r e a t e r t h e h i g h e r [ K+ ]o . L o w e r i n g [ K + ]o f r o m 4 . 7 t o1 .5 m M p r o d u c e d a r e v e r s i b l e fa l l i n p H i . W e h a v e s h o w n ( s e e R e s u l t s) t h a t t h e s ee f f e ct s a r e n o t d u e t o c h a n g e s i n m e m b r a n e v o l t a g e . T h e y s u g g e s t t h e p r e s e n c e o f am e m b r a n e - b o u n d K + / H e x c h a n ge r . R e c en t ly , B o n a n n o ( 19 9 1) h a s p r o v i d e d e v i-d e n c e f o r th e p r e s e n c e o f s u c h a n e x c h a n g e r i n r a b b i t c o r n e a l e p i t h e l i u m . T h ee x c h a n g e r i s p r o b a b l y e l e c t r o n e u t r a l : w e f o u n d t h a t t h e r e s p o n s i v e n e s s o f [ H ] i t oc h a n g e s i n [K + ]o w a s n o t a f f e c te d b y d e p o l a r i z a t i o n . U n d e r n o r m a l s t e a d y - s ta t ec o n d i t i o n s ( [K + ]i 1 4 0 m M , [ K + ]o 4 . 7 m M , p H i 7 . 2, p H o 7 . 4 ) t h is m e c h a n i s m w o u l dp r o d u c e a n e t e f f l u x o f K + in e x c h a n g e f o r n e t i n f l u x o f H + a n d t h u s w o u l d a c i d i fyt h e c e ll . C e l l ul a r a l k a l i n iz a t i o n d u e t o t h e N a + / H + e x c h a n g e r w o u l d b a l a n c e t h isp r o c e s s . W h e n [ K+ ]o is r a i s e d , u n i d i r e c t i o n a l K + i n fl u x , a n d t h u s H + e f f l u x w h i c h i sc o u p l e d t o it , s h o u l d i n c r e a se . T h e K / H + e x c h a n g e r s e e m s t o b e s e p a r a t e f r o m t h eN a + / H + e x c h a n g e r : i n t h e a b s e n c e o f N a + t h e r i s e i n p H i i n d u c e d b y 5 0 m M K + w a su n a f f e c t e d b y H M A ( 1 5 I xM ) .

I t i s i m p o r t a n t t o p o i n t o u t t h a t i n th e s e N a + - f r e e e x p e r i m e n t s t h e s t e a d y - s ta t er a t i o s o f t h e H a n d K i o n s a c ro s s th e m e m b r a n e a r e n o t t h e s a m e , a s w o u l d b ee x p e c t e d i f t h e K + / H + e x c h a n g e r w e r e t h e s o l e r e g u l a t o r o f p H i . T h e r a t i o[ H + ] i / [ H + ] o w a s a l w a y s s i g n if i c a n t ly l e s s t h a n [ K + ] i/ [ K + ]o . T h u s , N a + r e m o v a l i n o n es p e c i f ic e x p e r i m e n t l o w e r e d p H i t o 6 . 4 0 ( p H o 7 . 4 0) , y i e l d i n g a n H + r a t i o o f 1 0,w h e r e a s t h e K + r a t i o w a s 1 4 0 / 4 . 7 ~ 3 0 . E v e n g r o s s d e v i a t i o n s f r o m t h e a s s u m e d[ K ] i c a n n o t e x p l a i n t h i s d i s c r e p a n c y . O n e c o u l d r a i s e t h e o b j e c t i o n t h a t a t t h e s e l o wp H i v a l u e s t h e d y e i s a n i m p r e c i s e i n d ic a t o r . H o w e v e r , w h e n N a + w a s r e m o v e d a t a ne l e v a t e d p H o o f 7 . 7 0 ( a v e r a g e p H i 6 . 8 3 , n = 4 ) , t h e d i s c r e p a n c y i n t h e r a t i o sr e m a i n e d . A d d i t i o n a l i n s e r t i o n o f K + / H + e x c h a n g e r s b y a d d i n g n i g e r ic i n f u r t h e rr e d u c e d p H i , a n d t h e H + a n d K + r a ti o s w e r e n o w 2 0 a n d 3 0 , r e s pe c t iv e l y ; c l o s e r t h a nw i t h o u t t h e d r u g b u t s t i l l n o t i d e n t i c a l . I t i s a p p a r e n t t h a t e v e n i n t h e a b s e n c e o fN a + / H + e x c h a n g e a l k a li n i z in g f a c t o r s m u s t h a v e b e e n p r e s e n t . O n e s u c h f a c t o rm i g h t b e o u t w a r d e l e c t r o d i f fu s i o n o f H i o ns f r o m t h e a c i d if i e d c yt o so l . I n t h e a b s e n c eo f m e m b r a n e v o l t a g e m e a s u r e m e n t s , t h e c o n t r i b u t i o n o f t h is f a c to r c a n n o t b ee v a l u a t e d .

W e h a v e n o i n f o r m a t i o n a b o u t t h e k i n e t ic p r o p e r t i e s o f th e K + / H + e x c h a n g e ro t h e r t h a n t h a t t h e i n it ia l r a t e o f a lk a l i ni z a t io n c o n t i n u e d t o i n c r e a s e o v e r t h e e n t i r er a n g e o f [K + ]o t h r o u g h w h i c h i t c o u l d b e s t u d i e d ( 7 - 1 4 0 m M ) .

W e c o m p a r e d t h e p H i e f f e c t o f ra i s i n g [ K+ ]o w i t h t h e e f f e c t o f R b + o r T I + i n t h ec o n t i n u e d p r e s e n c e o f 4 . 7 m M K + . I n e a c h c e l l K + w a s f i rs t t r a n s i e n t l y i n c r e a s e d , a n d

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606 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y V O L U M E 1 992t h e n t h e s a m e a m o u n t o f R b + o r T l + w a s a d d e d . 5 0 m M R b + h a d n o e f f e c t o n p H i .O n t h e o t h e r h a n d , t h e i n i ti a l r a t e s o f a l k a l i n i z a t i o n d u e t o 5 - 4 0 m M T 1 + w e r e t h r e et o s i x t i m e s t h o s e p r o d u c e d b y K + . A p p a r e n t l y t h e e x c h a n g e r ' s a f f in i t y f o r T1 g r e a t l ye x c e e d s t h a t f o r K + . T h e s i m i l a r it y i n s e q u e n c e o f e f f e c ti v e n e s s o f t h e t h r e e i o n s t ot h a t o b s e r v e d b y H i l l e ( 1 9 7 3 ) i n K + c h a n n e l p e r m e a b i l i t y o f m y e l i n a t e d f r o g n e r v ef i b e r m i g h t p o i n t t o s o m e c o m m o n f e a t u r e o f m o l e c u l a r s t r u c t u r e o f c a r r i e r a n dc h a n n e l .

Effect of Na on RecoveryW h e n , a t p H o 7 .4 0 , t h e p H i w a s l o w e r e d e i t h e r b y N a + r e m o v a l o r b y a n N H 4 C Ip r e p u l s e , t h e r a t e o f t he s u b s e q u e n t r e c o v e r y d u e t o N a + / H + e x c h a n g e w a sd e p e n d e n t o n [ N a ] o. W i t h o u t N a + t h e r e w a s n o r e c o v e r y , a s w a s al s o f o u n d b yB u c k l e r e t a l. ( 1 9 91 a ) . T h i s N a + d e p e n d e n c e a g r e e s w i t h w h a t h a s b e e n f o u n d i nm a n y o t h e r c e l l t y p e s (s e e , f o r i n s t a n c e , T h o m a s , 1 9 77 ) . T h e r e l a t i o n s h i p b e t w e e n[ N a +] o a n d r a t e o f p H i r e c o v e r y c o u ld b e e x p r e s s e d b y s i m p l e M i c h a e l i s- M e n t e nk i n e ti c s , w i t h a n a p p a r e n t K m o f 5 8 m M ( F ig . 5 ). T h i s r e p r e s e n t s a r a t h e r l o w a ff i n it yo f t h e e x c h a n g e r : K m i n r a t m e s a n g i a l c e l ls is 2 7 m M ( B o y a r s k y e t a l ., 1 9 8 8) a n d K mi n f r o g m u s c l e ( e x p o s e d t o 5 0 m M K ) i s 12 m M ( P u t n a m , R o o s , a n d W i l d in g , 1 9 8 6 ).

C1- RemovalT h e a b s e n c e o f c h lo r i d e h a d n o e f f e c t o n r e s t i n g p H i , p H i s e n s it iv i ty t o p H o c h a n g e s( f r o m 7 . 4 0 t o 6 . 8 0 o r 7 . 8 0) , o r p H i r e s p o n s e t o a p u l s e o f C O 2 . I f , h o w e v e r , c h l o r i d ew a s r e m o v e d i n t h e m i d s t o f C O 2 e x p o s u r e , t h e r e w a s a d is t i n c t r is e i n p H i ; t h is w a sp r e v i o u s l y o b s e r v e d b y B u c k l e r e t a l . (1 9 9 1 a ) . T h e y f o u n d t h i s r i s e t o b e N a +i n d e p e n d e n t a n d a s c r i b e d it t o e x c h a n g e o f e x t e r n a l H C O ~ f o r i n t e r n a l C 1- . W ec o n f i r m e d t h a t d i s u l f o n i c s t i l b e n e s ( D I D S a n d S I T S ) i n h i b i t t h e r i s e .

Buffering PowerB e t w e e n p H i 7 . 7 0 a n d 6 . 6 0 t h e i n t r in s i c b u f f e r i n g p o w e r , [ 3, o f g l o m u s c e l ls f el l w i t hi n c r e a s i n g p H i ; b u t b e l o w 6 . 6 0, [3 d i d n o t a p p e a r t o c h a n g e . A c u r v i l i n e a r r e g r e s s i o nr e p r e s e n t i n g t h e p r e s e n c e o f o n e e q u i v a l e n t i n t ra c e l l u la r b u f f e r f it s o u r d a t a b e t t e rt h a n a l i n e a r r e g r e s s i o n . O t h e r o b s e r v e r s w o r k i n g e i t h e r w i th g l o m u s c e l ls o r w it ho t h e r c e l l t y p e s h a v e f i t t e d t h e i r b u f f e r i n g d a t a t o a s t r a i g h t l i n e w i t h n e g a t i v e s l o p e( g l o m u s c e l l s : B u c k l e r e t a l . , 1 9 9 1 a ; b a r n a c l e m u s c l e f i b e r s : B o r o n , M c C o r m i c k , a n dR o o s , 1 9 7 9; r a t r e n a l m e s a n g i a l c e ll s: B o y a r s k y e t a l ., 1 9 8 8 ; f r o g m u s c l e : A m o r e n a ,W i l d i n g , M a n c h e s t e r , a n d R o o s , 1 9 9 0 ; s h e e p P u r k i n j e f ib e r s : V a u g h a n - J o n e s a n d W u ,1 9 90 ). W e f o u n d t h a t N a + r e m o v a l d i d n o t s i gn i fi c a nt ly a f f e c t th e p H i - b u f f e r i n gp o w e r r e l a t i o ns h i p , n o r d i d l o w e r i n g t e m p e r a t u r e t o 2 2 C.

HypoxiaF i na l ly , w e f o u n d t h a t h y p o x i a ( 2 % 0 2 ) h a d n o o b s e r v a b l e e f f e c t o n p h i i n e i th e r t h ep r e s e n c e o r a b s e n c e o f C O 2. T h i s a g r e e s w i t h t h e l a c k o f e f fe c t o f 2 m M c y a n i d e( B i s c o e e t a l. , 1 9 8 9 ) , a n d s e e m s t o i n d i c a t e t h a t i n t h e c h a i n o f e v e n t s l i n k i n ge x t r a c e l l u l a r h y p o x i a t o n e r v e d i s c h a r g e , p H i d o e s n o t p l a y a r o l e .

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W1LDING ET aL. ln tracellular p H of Glomus Cells 6 0 7Some years ago, Hornbein a nd Roos 1963) present ed evidence that the excitatoryeffect of blood extraceUular) acidity on carotid body nerve activity is produced

through changes in pHo rather than through changes in Pcoz or bicarbonateconcentration per se. The exceptional sensitivity of the pHi of the isolated glomuscell to pHo, which is nearly the same in the presence and absence of COs, makes therole of pHi as a link between extracellular acidity and chemoreceptor activity anattractive possibility.Original version received 2 Au gust 1991 and accepted version received 22 Ju ne 1992

R E F E R E N C E SA i c k in , 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

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( c a r b o x y- S N A R F - 1 ) f o r i n t r a c e l l u l a r p H m e a s u r e m e n t i n s m a l l , i s o la t e d c e ll s. Pflizgers Archly4 1 7 : 2 3 4 - 2 3 9 .

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