The role of dihydropydine-sensitive Ca2 + channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body

Neu oscience Vol. 47, No. 2, pp. 463412, 1992 P in ed in G ea B i ain 0306-4522/92 $5.00 + 0.00 Pe gamon P ess plc 0 1992 IBRO THE ROLE OF DIHYDROPYRIDINE-SENSXTIVE Ca*+ CHANNELS IN STIMULUS-EVOKED CATECHOLAMINE RELEASE FROM CH~MORECEPTOR CELLS OF THE CAROTID BODY A. OBEW,* A. ROCHER,* S. FIDONE* and C. CONZALEZQ *Depa men o Physiology, Uni e si y o U ah School o Medicine, 410 Chipe a Way, Resea ch Pa k, Sal Lake Ci y, UT 84108, U.S.A. Depa amen o de Bioquimica y Biologia Molecula y Fisiologia, Facul ad de Medicina, Uni e sidad de Valladolid, C/. Rambn y Cajal s/n, 47005Valladolid, Spain Aha ae -The p esen s udy u ilized an in i o p epa a ion o he abbi ca o id body, wi h issue ca echolamine s o es labeled by incuba ion wi h ‘H- y osine. The goal was o cha ac e ize pha macologi- cally he ol~g~~n&n Ca*+ channels p esen in he ype I (glomus) cells o his a e ial chemo ecep- o o gan, and o elucida e hei ole as pa hways o Ca2+ en y. We ound ha elease o ‘H-dopamine induced by high ex e nal po assium was o e 95% dependen on ex e nal cakium concen a ion and ha his elease was 9&100% inhibi ed by he dihyd opy~~ne an agonis s, nisoldipine and ni endipine, and was po en ia ed by he dihyd opy idine agonis , BayK 8444. The e o e, any s imulus-induced, cakium- dependen elease o 3H-dopamine ha was inhibi ed by nisoldipine and po en ia ed by BayK 8644, was conside ed o be suppo ed by Ca2+ en y in o he cells ia ol age-dependen Ca2+ channels. Signi ican di e ences we e obse ed in he elease o ‘H-dopamine induced by 75 s 25mM K+. On p olonged s imula ion, elease induced by 75 mM K+ was la ge and ansien , whils ha induced by 25 mM K+, al hough mo e mode a e, was sus ained. The elease elici ed by 75 mM K+ was inhibi ed app oxima ely 90% by 1.5 mM Co2+ o 625 nM nisoldipine, while elease by 25 mM K+ was comple ely blocked by 0.6 mM Co*+ o 125 nM nisoldipine. Low PO,-induced elease o 3H-dopamine was 95% dependen on Ca*+, and was inhibi ed by nisoldipine (625 nM) in a manne in e sely p opo ional o he in ensi y o hypoxic s imula ion, i.e. 79% inhibi ion a a PO, o 49 To , and 20% inhibi ion a PO2 o 0 To . BayK 8644 po en ia cd he elease induced by mode a e hypoxic s imuli. Release elici ed by high PCOJlow pH, o by Na+-p opiona e o dini ophenol~n~ining solu ions, was app oxima ely 80% Ca’+-dependen , and he ~hyd~y~din~ ailed o modi y his elease. I is concluded ha ype I mlls possess ol~~de~nd~ Ca ‘+ channels sensi i e o he dihyd opy~di- nes, which in ag eemen wi h p e ious el~ ophysiolo~~l da a should be de ined as L- ype Ca*+ channels. Calcium en y which suppo s he elease o 3H-dopamine elici ed by mode a e hypoxia should occu mainly h ough hese channels while he elease induced by s ong hypoxic s imuli will be Se Ned by Ca2+ en y which occu s in pa ia ol age-dependen Ca2+ channels, and in pa h ough an addi ional pa hway, p obably a Na+/Ca2+ exchange . The insensi i i y o dihyd opy idines o he elease o )H-dopamine induced by high 1DC02/low pH, Na+-p opiona e and dini ophenol may indica e a comple e loss o e icacy o he d ugs o modula e Ca 2+ channels unde hese condi ions o mo e likely, ha o he mechanisms a e ac i a ed, p obably he Na+-Ca’+ exchange . Ca o id body (CB) chemo ecep o s a e hough o be composi e ecep o s in which he ype I (glomus) cells de ec changes in blood PO,, PCO, and pH and espond wi h he elease o neu o ansmi ~ o ac i a e he closely apposed chemosenso y ne e e minals.~** One such neu o ansmi e ha has ecei ed conside able a en ion in ecen yea s and is known o be eleased by he ype I cells is dopamine (DA). This biogenic amine has been shown o be eleased in p opo ion o bo h he in ensi y o s imula ion and he esul an senso y discha ge eco ded om he ca o id sinus ne e $To whom co espondence should be add essed. Abb ~~~~~~ CB, ca o id body; CSN, ca o id sinus ne e; DA, dopamine; DHMA, dihyd ox~~delic acid, DOPAC, dihyd oxyphenyl ace ic acid; NE, no epine- ph ine. (CSN). This ela ionship be ween s imulus in ensi y, CSN senso y discha ge, and DA elease has been shown o be Ca*+-dependen ,@ and is known o occu wi h bo h physiolo~cal (low PO*, low p~)2’,4 .42 and pha macological (cyanide, dini o- phenol, 2-deoxyglucose)3*339 s imuli. In a p e ious s udy,3 i was pos ula ed ha ype I cells migh possess ol age-dependen Ca2+ channels. This sugges ion was p oposed o explain he Ca2+-de- penden elease o DA elici ed by high ex e nal K+.17 I was also shown in a subsequen p elimina y s udy” ha ni endipine, a Ca’+ channel blocke o he dihyd opy idine g oup, ma kedly inhibi ed he high K+ and low PO,-induced elease o DA. Qui e ecen ly, se e al di e en labo a o ies wo king wi h isola ed ype I cells ha e p o ided di ec elec o- physiological e idence o he p esence o ol age- dependen Ca’+ channels in hese cells.‘6~“~32~33~” 463 NSC 4112-l The p esen s udy was unde aken o cha ac e ize pha macologically he ype(s) o ol age-dependen Ca’+ channels p esen in he chemo ecep o ype I cells o he CB, and o de ine he ole o hese Ca” channeIs in he esponse o di e en o ms o chemo ecep o s imula ion. Since he in ol emen o ol age-dependen Ca*+ channels in a cellula esponse couples a ol age dependency o ha esponse,” he p esen s udy should e eal which chemo ecep o s imuli p oduce signi ican changes in he memb ane po en ial o ype I cells. Calcium en y in o hese cells was assessed by measu ing he Ca’ ’ _ dependen elease o DA om he issue. We chose his indi ec me hod, ins ead o he measu emen o 45Ca2 luxes, because i has been ound ha , o a a ie y o issues, he e exis s a e y close ela ionship be ween Ca2+ in lux and he in ensi y o he issue’s Ca*+ -dependen esponse.6.‘3.29.3* Fu he~o e, he small size o he CB and he cellula he e ogenei y o he issue ( ype I and II cells, endo helial cells, neu ons, e c.) make he measu emen o 4 Ca’ ’ luxes bo h echnically di icul and o ques ionable signih- cance. The elease o DA, on he o he hand, is a pa ame e speci ic o he ype I ceils.‘” EXPERIMENTAL PROCEDURES The p esen s udy u ilized an in ci o CB p epa a ion am adul (1.5-2.5 kg) New Zealand abbi s. The animals we e anes he ized wi h Na+-pen oba bi one (30-40 mg/kg, i. .), acheo omized, and bo h ca o id bi u ca ions we e exposed and emo ed o u he dissec ion in a luci e chambe illed wi h ice-cold 100% O,-equilib a ed Ty ode’s solu ion (in mmol/l: NaCI, 140; KCl, 5; CaCI,, 2; MgCl,, 1.1; HEPES, IO mM; glucose, 5.5 mM; pH adius ed wi h I N NaOH o 7.42). Ca o id bodies we e ileaned o su ounding issue and incuba ed o 2 h wi h 3 H- y osine (20 l M, 30 Ci/mmol), as p e iously desc ibed.lg Unde hese condi ions, he o gans syn hesized app oxima ely 20-22 pmol o 3H-ca echoi- amine/mg issue (i.e. abou IOpmol, o mo e han 5 x IO5 d.p.m./CB). Following incuba ion, he CBS we e ans e ed o new ials con aining 4 ml o y osine- ee media and incuba ed o an addi ional 2 h o wash ou he apidly eleasable pool o labeled ca echolamine;’ du ing his pe iod he medium (4 ml) was eplaced by p ewa med esh solu ions e e y 30 min and he samples we e disca ded. The ea e , he collec ion o incuba ion media o analysis was g ouped in s imula ion cycles; one s imula ion cycle consis ed o a pe iod o incuba ion wi h he s imulus ( es ) solu ion, p eceded by a simila pe iod and ollowed by se e al pos s imulus pe iods o incuba ion wi h s anda d solu ion o de e mine basal esponses and allow he p ep- a a ion o eco e , espec i ely. The du a ion o each o hese pe iods in any gi en expe imen is p o ided in he Resul s sec ion. The numbe o s imula ion cycles pe expe imen a ied om one o ou depending on he na u e o he es incuba ion media. The s imulus-e oked elease declined in successi e p esen a ion o he s imulus in pa allel o he basal elease and he e o e he a ios o e oked o basal elease emained cons an h oughou he expe - imen s.*’ The incuba ion media we e con inuously bubbled wi h he app op ia e gas mix u e, and he POZ was moni- o ed wi h an 0, elec ode in selec ed expe imen s. Unde hese expe imen al condi ions, he h eshold o DA elease by low O2 was app oxima ely 75 To O (i.e. no mal saline equilib a ed wi h IO% 0,/90% N,). All co lec ed samples we e added o a ca ie mix u e con aining 0.3 M ace ic acid, I mM asco bic acid, and looI I F=l -l OHMA B 55mM 5 %O, Fig. 1. P o ile o ‘H-ca echols (‘H-CA) eleased om he ca o id body ollowing incuba ion wi h ‘H- y osine. ‘H-Ca echols p esen in he alumina elua es we e aken as 100%. No e ha he ela i e p opo ions o ‘H-DA and ‘H-DOPAC a e e e sed unde basal (B) s s imulus condi ions (55 mM K+ and 5% 0,/95% N;equilib a ed media; IO mm), owing o sa u a ion o deg ada ion mechan- isms du ing s imula ion. None heless, DA + DOPAC s ill ep esen o e 85% o 3H-ca echols unde all condi ions. 100 GM unlabeled DA. Radiolabeled cd echols in he incu- ba ion media we e analysed by adso p ion o alumina a pH 8.6, and elu ion wi h I N HCI.” Since he CB ecei es a ich sympa he ic inne a ion,” he ‘Hsa echols eleased may ha e wo o igins, he ype I cells and he sympa he ic ne e endings. 3H-DA is syn he ized and s o ed exclusi ely in ype I cells while ‘H-no epineph ine (NE) is syn he ized and s o ed, bo h in ype I cells and in he sympa he ic endings;” he e o e o assess he o igin o he 3H-ca echols eleased i was necessa y o iden i y i s chemical na u e. To do so, he alumina elua es we e acuum-concen a ed o d yness, e- susnended in 40 u1 o mobile obaseZ con ening 5 UR o u&beled DA, N’E, dihyd oxyphenyl ace ic acid <kPAC) and dihyd oxymandelic acid (DHMA), and sepa a ed using hin-laye ch oma og aphy acco ding o he me hod o Fleming and Cla k.22 The labeled compounds we e elu ed and coun ed as p e iously desc ibed.s5 The p o iles o eleased ‘H-ca echols unde basal condi ions and in esponse o high K+ and low PO, s imula ion a e shown in Fig. I. Obse-me ha ega dless o he expec ed a i- a ions18,2i.” m he ‘H-DA o “H-DOPAC a ios be ween basal and s imulus samples, ‘H-DA + 3H-DOPAC ( he main ca aboli e o ‘H-DA in he CB) 4 ep esen o e 85% o he o al 3H-ca echols eleased, indica ing ha ype I cehs a e hei main sou ce. Because he same analy ical p o ile was ound in pilo expe imen s o all he s imuli es ed in his s udy, in mos o he eplica e expe imen s he alumina elua es we e di ec ly coun ed and “H-ca echol elease was aken as a measu e o ‘H-DA elease om ype I cells. Resul s a e exp essed as c.p.m. o 3H-DA p esen in each collec ed sample, as he a io o s imulus-e oke elease o basal elease (e oked/basal); o as pe cen o issue con en . Ma e ials. 3,S-3H-Ty osine, 4@-@ Ci/mmol was pu - chased om New England Nuclea and aluminum oxide (alumna) om Se a (Heidel~ g). Nisol~pine and ni- endipine we e a gene ous gi om Miles Labo a o ies, and BayK 8644 was p o ided by P o . A. Ga cia (U.A.M. Spain). RESULTS The Ca2+ dependency o 3H-DA elease obse ed in his s udy is summa ized in Table I. The esul s a e simila o hose p e iously epo ed o he ca CB.3,38-40 Ahbough no shown in he able, mos o Ca o id body Ca 2+ channels and ca echolamine elease Table 1. Ca2+ dependency o 3H-dopamine elease induced by di e en s imuli Pe cen age educ ion S imulus No mal Ca2+ 0 M Ca2+ in 0 M Ca2+ High K+ (40mM) 105 + 8.2 4.80 + 0.38* 95.60 Hypoxia (2% 0,) 28.6 3.3 0.69 + 0.05* 97.50 High PCO,/pH 6.6 1.60*0.12 0.31 0.09* 81.00 Na+-P opiona e (15 mM) 1.02*0.11 0.21 0.06* 80.00 Dini opy idine (2.5 x 10d4 M) 47.65 + 3.82 9.00 + 1.3; 79.00 Cyanide (1 Oe4 M) 4.00 0.66 0.93 0.13* 76.75 Ve a idine (5 x lo-’ M) 16.9 + 1.4 1.6 + 0.09; 90.50 Ouabain ( 10e5 M) 11.2& 1.5 0.80 + 0.21* 93.00 Da a a e exp essed as he a ios o e oked o basal elease. Excep o e a idine and ouabain, he p o ocol in hese expe imen s was as ollows: a s imula ion cycle wi h Caz+-con aining media was applied i s , hen wo s imula ion cycles wi h nominally ee Ca2+ media, ollowed by a s imula ion cycle wi h Ca2+con aining media; he incuba ion wi h he es solu ions was 10 min in all s imula ion cycles. In he expe imen s wi h e a idine, pai s o CBS we e incuba ed in Ca2+-con ain- ing and in nominally ee Ca2+ media 30 min p io o and du ing he applica ion o e a idine (20min); media we e changed and collec ed o analysis e e y 10 min. The expe imen s wi h ouabain we e iden ical excep o he du a ion o he exposu e o he es solu ion (30min). In all he cases he da a a e means S.E. o he mean o six indi idual alues. Di e ences o he mean e oked elease in Ca2+-con aining s Ca 2+- ee media a e s a is ically signi ican (*P < 0.001; pai ed S uden ’s - es ). 465 he s imuli we e es ed a a ying in ensi ies, and he Ca*+ dependency o elease was p esen a all s imu- lus in ensi ies. Time cou se o high K+-induced elease o ‘H- dopamine The ime cou se o ‘H-DA elease induced by 75 and by 25 mM K+ a e compa ed in Fig. 2A; in bo h cases he media we e changed and collec ed o g30. 2 320. -0 % I L 10. $ ,I 0. A. n lcon ol q S imulus -3min , , + , 75mM K’ 25mM K’ B. a’““ 75mM K’ “1 25mM K’ 32 e = 50 s I I l n - ._ a E oJ L-s O_~ 3 9 15 3 9 15 min Fig. 2. Time cou se o K+-induced elease o ‘H-DA. (A) Two single s imulus cycles o 75 (le ) and 25 ( igh ) mM K+. (B) Mean ac ional elease (* S.E.) o ‘H-DA in esponse o 75 and 25 mM K+. To al ‘H-DA eleased du ing he 15&n incuba ion wi h high K+ was aken as lOO%, and he ac ion collec ed in each 3-mi pe iod is exp essed as a pe cen o his o al. Da a a e om six expe imen s. analysis e e y 3 min o a o al o 15 min. As shown in Fig. 2B, mos o he ‘H-DA elease e oked by 75 mM K+ was collec ed in he i s 3-min ac ion, while wi h 25 mM K+, elease emained nea ly con- s an o he du a ion o he s imulus. These esul s would sugges ha Ca*+ en y inac i a es apidly du ing exposu e o 75 mM K+ bu ha en y is sus ained du ing exposu e o 25 mM K +. The unsus ained elease o 3 H-DA du ing exposu e o 75 mM K+ is, in ac , due o inac i a ion o Ca*+, as con i med by Fig. 3. The i s panel o Fig. 3 illus a es a ypical p o ile o declining elease in 75 mM K + . In a second CB (middle panel) emo al o Ca*+ om he incuba ion medium no only abol- ished he immedia e elease o 3H-DA, bu also p e en ed i s subsequen elease ollowing e-in o- duc ion o Ca*+ in o he medium. This sugges s ha p io depola iza ion o he cells in a Ca*+- ee I 0 W2l" d u3min lh+__li oca++ .lonon 75mM 71mM 71mM Fig. 3. E ec s o 0 M Ca2+ and ionomycin (30pM) on ‘H-DA elease. induced by 75 mM K+. The expe imen shows ha nei he deple ion o eleasable ‘H-DA s o es, no adap a ion o exocy o ic ac i i y a e esponsible o accom- moda ion o elease induced by 75 mM K+. 466 . Con ol D 2 5mM K+ -3mm .OCae . Nis Fig. 4. E ec s o OM Caz4 and nisoldipine (125 nM) on IH-DA elease induced b 25 mM K+. The exne imen demons a es he in ol e&W o dih~d opy~dine~sensi i e Ca*+ channels in he sus ained elease o ‘H-DA induced by 25 mM K+. medium p e en ed subsequen en y o CaZ appa - en ly by inac i a ing he pa hways o Ca*+ en y. Da a ob ained using a hi d CB { igh panel), ap- pea ed o suppo his no ion: in oduc ion o iono- mycin, a Ca2+ ionopho e, du ing he declining phase o elease eac i a ed he elease p ocess. This p ecluded he possibili y ha exhaus ion o he eleasable ‘H-DA pool, o loss o sensi i i y o Ca’ + by he exocy o ic machine y, was esponsible o he unsus ained elease no mally obse ed du ing exposu e o 75 mM K+ . Ve y di e en esul s we e seen wi h 25 mM K+. The le panel o Fig. 4 shows he no mal ime cou se o )H-DA elease du ing exposu e o 25 mM K+; in he middle panel, emo al o Ca2+ du ing he ini ial exposu e o 25 mM K+ abolished he inc eased e- lease, bu did no p e en eac i a ion o inc eased elease upon ~in odu~ ion o Ca*+. Nisoldipine educed elease when in oduced la e in he incu- ba ion pe iod (Fig. 4, igh panel), indica ing ha sec e ion depends on Ca2+ en y in o ype I cells ia a dihyd opy idine-sensi i e pa hway (see also below}. These indings also indica e ha , unlike he case o 75 M I(+, p edepola iza ion wi h 25mM K+ does no inac i a e he pa hway o Ca*+ en y. 10, i 75mM I<+ K+ Pha macology o high K’ -induced kus . I)/ ‘H- dopamine The e ec s o di e en concen a ions o Ca? ! in he incuba ion media on he basal (5 mM K + ) and high K+ (25 and 75 mM)~ oked elease o 3 H-DA a e shown in Fig. 5. In hese expe imen s, 25 mM and 75 mM K + we e applied o 10 min, and he media we e sampled o analysis e e y 10 min. In he le o he igu e, he plo o 3H-DA elease s Ca’ concen- a ion shows ha basal elease did no change app eciably a Low Ca’+, bu ended o dec ease as Ca + was inc eased. E oked elease in 25 mM K * was abolished (o e 95%) in 0 mM Ca”, was maxi- mum a 0.5 mM bu hen declined a highe Ca”. The elease e oked by 75 mM K’ was likewise abolished in 0 mM Ca +, and inc eased d ama ically wi h inc easing Ca2+. To he igh in Fig. 5 a plo o he a io o e oked eiease o basal elease as a unc ion o Ca*+ illus a es he educ ion o he e oked esponse o 25 mM K+ a 10 mM Ca2+. and he p og essi e inc ease in he e oked elease wi h 75 mM K+ wi h inc easing Ca’ ’ These changes in CB 3 H-DA sec e ion wi h inc easing Ca2 i a e simila o hose obse ed wi h o he issues.” They p e- sumably e lec he inc ease in he h eshold o Ca: ’ channel ac i a ion wi h inc easing Cal’ ha was e iden wi h small depola iza ions (25 mM K+), bu which disappea ed upon s onge depola iza ions (75 mM K+).27,4’ The inse in he igu e shows he p o ocol o sample collec ion o analysis. Cd2+ and Co’+ , well-known Ca2+ channel block- e s, p oduced a dose-dependen inhibi ion o he ‘H-DA elease induced by 25 and 75 mM K+. as shown in Fig. 6. Bo h esponses o ele a ed K ’ we e comple ely blocked by 100 pm Cd’+. Va ia ion in he e ec o di e en Cd2+ concen a ions on basal elease p ecluded an accu a e calcula ion o an c,, o elease e oked by 2.5 mK K A. bu did no in e e e signi ican ly in calcula ing a alue o he much la ge elease e oked by 75 mM K + . Fo his we ound an O mM Ca+’ 0 5 10 mM Ca+* Fig. 5. E ec o Ca2+ concen a ion on 3H-DA elease unde es ing condi ions and in esponse o high K+. Le : &ease exp essed as pe cen o issue con en . Righ : a io o e oked o basal elease. Inse : sequence o medium enewal and collec ion o analysis. Each da a poin ep esen s he mean + S.E. o a leas ou samples. Ca o id body Ca + channels and ca echolamine elease 461 ICd”lpM ICd’lmM Fig. 6. E ec s o Cd*+ (le ) and Co2+ ( igh ) on ‘H-DA elease unde es ing condi ions (5 mM K+) and du ing high K+ (25 and 75 mM). Expe imen al p o ocol as in Fig. 5. Cd2+ o Co*+ was in oduced 10 min be o e and du ing he incuba ion wi h high K+. Release is exp essed as pe cen o issue con en . Da a poin s a e mean + SE. o ou o six expe imen s. GQ o lOpM, which is lowe han he 56 PM obse ed o PC12 cells by F eedman e d2’ We ound ha Co2+ a any concen a ion did no app eciably modi y he basal elease, bu a 0.6 mM comple ely abolished he elease e oked by 25 mM K+, and a 1.5 mM inhibi ed 93% o he elease induced by 75 mM K+. The q,, o he Co2+ e ec on he 75 mM Kc-induced elease was 0.3 mM, wi h maximum inhibi ion a 1.5 mM. Bo h alues a e compa able o hose epo ed o PC12 cell~.~~ Nei he nisoldipine (Fig. 7A) no ni endipine (Fig. 7B) modi ied he basal elease 6 ‘H-DA. Howe e , bo h exhibi ed a dose-dependen inhibi o y e ec on he elease induced by 25 and 75 mM K+. A lob9 M, nisoldipine inhibi ed 50% and 10% o he elease induced by 25 and 75 mM K+, espec i ely. As shown in he inse , his di e ence was main ained h ough- ou he ange o nisoldipine concen a ions (Fig. 7A). 0 lo-’ lo-’ lo-’ Nisoldipine (Mb Ni endipine, on he o he hand, equi ed highe concen a ions o inhibi elease, and exhibi ed less ma ked di e ences in i s e ec s on 25 and 75 mM K+-induced elease (inse Fig. 7B). These da a demons a e ha he ol age-dependen Ca2+ chan- nels in he CB ype I cells a e dihyd opy idine sensi i e. Figu e 8 illus a es u he p oo o he dihyd opy idine sensi i i y o he Ca2+ channels in ype I cells; BayK 8644, a Ca2+ channel agonis belonging o he dihyd opy idine amily,49 ma kedly po en ia ed he elease o ‘H-DA by 30 mM K+. E ec s o dihyd opy idines on low PO,-induced elease o ‘H-dopamine The pa icipa ion o Ca2+ channels in he esponse o low PO, was assessed using s imuli o mode a e in ensi y (10 min incuba ion wi h 7% O2 equilib a ed media; PO2 = 49 To ). The esul s o a single expe - imen wi h wo s imula ion cycles a e shown in Fig. 9A (le ). In oduc ion o nisoldipine (625 nM) in o he media 10 min p io o p esen a ion o he second s imulus inhibi ed he esponse. The inhibi ion o he e oked elease om ou such expe imen s had a mean o 79 7% (Fig. 9A, igh ). In Fig. 9B, BayK 8644 was used in a simila p o ocol and p oduced a i e- old inc ease in he elease o 3 H-DA in esponse o 7% 02. These da a implica e dihyd opy idine- sensi i e Ca2 + channels in he media ion o Ca2+ en y du ing low PO,-induced elease o ‘H-DA. The e ec s o 625 nM nisoldipine on he low PO induced elease o ‘H-DA a e summa ized in Fig. 10. The inhibi o y e ec o his dihyd opy idine dec eased as he in ensi y o he low PO, s imulus was inc eased. E en a high s imulus in ensi ies (e.g. 2% 02/98% N2 media) o e 95% o ‘H-DA elease was Ca2+-dependen (see Table l), sugges ing ha as he in ensi y o hypoxic s imula ion inc eases, he ole 4 -75mM K+ -25” ” .--_ 5 ” - 2 Ni endipine hl) Fig. 7. E ec s o nisoldipine (le ) and ni endipine ( igh ) on he elease o 3H-DA unde es ing condi ions (5 mM K+) and du ing high K+ (25 and 75 mM) s imula ion. Expe imen al p o ocol as in p e ious igu e, nisoldipine and ni endipine we e p esen in he incuba ion media 10 min p io and du ing high K+ s imula ion. Release is exp essed as pe cen o issue con en . Inse s: dose- esponse inhibi ion cu es. All da a poin s a e means + SE. o ou o six alues. C 3 30mM K+ c-3 BAY-K Fig. 8. E a o he dihyd opy idine Ca* + channel agonis BayK 8644 (1~ M) on 30 mM K+ induced elease o 3 H-DA. No e ha BayK 8644 did no a ec basal elease. o Ca2+ channels in media ing Ca2+ en y becomes less impo an . To es he possibili y ha he loss o e icacy o nisoldipine wi h inc easing s eng h o he hypoxic s imuli is ela ed o he p opo ionally inc easing acidi ica ion ha is p oduced du ing low PO, s imu- la ion,15.& we u ilized a s ong hypoxic s imulus o sho e du a ion (2% 0,; 3 min). The a ionale o hese expe imen s was ha acidic s imuli e oke Ca’+- dependen elease ha was insensi i e o dihyd opy- idines (see Table 1 and below) and ha in acellula acidi ica ion p oduced unde low 0, inc eased d a- ma ically be ween 3 and 10 min (see Re . 15). In hese expe imen s (Fig. 1 l), pai s o CBS we e subjec ed o wo cycles o low PO* s imula ion; one CB se ed as n7%0, 2 _-Iom,n ; P x : 40 E- 2’ 0 ,,h, d u 20 d z . 0 id BAVK - -7%O, _BAYK Fig. 9. Dihyd opy idine and low P&-induced elease o ‘H-DA. (A) Le : e ec o nisoldipine (625 nM) on elease induced by 7% O,-equilib a ed media (10 min; black ba s). (A) Righ : e oked o basal elease a ios om ou expe - imen s (x + S.E.). (B) Le : e ec o 1 pM BayK 8644 was es ed. (B) Righ : mean e oked o basal elease a ios. Di e ences o he mean e oked elease in con ol s es condi ions we e s a is ically signi ican , *P < 0.01; pai ed S uden ’s - es . 100. 0 7x02 WO, 2%4 N, Fig. 10. Dec easing po ency o nisoldipine (625 nM) in inhibi ing he elease o ‘H-DA induced by inc easingly s ong hypoxic s imuli. Expe imen al p o ocol as in Fig. 9. Du a ion o low PO, incuba ions was 10 min. Means + SE. o he mean o six o mo e samples. a con ol while he second was used o assess he e ec o nisoldipine du ing he second s imulus cycle. The a ios o e oked elease in he i s and second cycles om ou such expe imen s a e shown in he igh hand panel o Fig. 1 I. I is e iden om a compa ison wi h he esul s o Fig. 10, ha sho en- ing he du a ion o he s imulus inc eased he e icacy o nisoldipine inhibi ion o he low PO>-induced elease (29% s 73%). E sec s o dihyd opy idines on he elease q ‘H-dopa - mine induced by d@e en s imuli In addi ion o high K+ (a depola izing agen use ul in e ealing he pa icipa ion o ol age-dependen Ca2+ channels in depola iza ion-media ed ansmi e elease) and low PO2 ( he p incipal na u al s imulus o he ca o id chemo ecep o s), we also examined he e ec s o he dihyd opy idines on he sec e o y esponse e oked by o he na u al and pha macologi- cal s imuli o he ca o id chemo ecep o s (Table 2). S imuli ha e oke only a mode a e elease o ‘H-DA (20% C02/pH 6.6 and Na -p opiona e) we e em- ployed o es he e ec s o he Cal+ channel agonis , Fig. 11. E ec o nisoldipine on 3 H-DA elease induced by se e e hypoxia. Du a ion o s imulus (2% Oz i i N,), 3 min (black ba s). Le : compa ison o con ol ( op) and nisoldip- ine (625 nM)- ea ed (bo om) ca o id bodies. Righ : a ios o e oked elease (second/ i s s imulus p esen a ion). Means + S.E. o such ou pai ed expe imen s. Ca o id body Ca *+ channels and ca echolamine elease 469 Table 2. Dihyd opy idine sensi i i y o 3Hdopamine elease e oked by di e en s imuli Pe cen age S imulus Con ol Dih d ou idine e ec 20% CO,/pH 6.6 NaP (15mM) Dini opy idine (2.5 x 10e4 M) Cyanide (2.5 x IO-” M) 1.6kO.17 1.5 0.21 - l.OkO.16 1.1 +0.17 48.0 7.0 47.4 8.5$ 17.8 2.1 11.4 l.o**$ -36% Vk a idine (5 x 10m5 M) 20 min 22.8 + 4.0 16.4 + 1.9.1 -28% Ve a idine (5 x 10e5 M) 3 min 9.5 * 1.6 5.0 + 0.9**i -47% - BayK 8644, 1 PM; Snisoldipine, 625 nM. Expe imen al p o ocols as in Fig. 9; du a ion o exposu e o es solu ions 10 min in all he cases excep when indica ed o he wise. Da a a e means SE. o he means o ou o mo e indi idual alues. Di e ences o he mean e oked elease in con ol s es condi ions we e s a is ically signi ican o cyanide and e a idine (*P < 0.05; **P < 0.02, pai ed - es ). BayK 8644 (1 p M); s onge pha macological s imuli we e used o es he e ec s o he an agonis nisoldip- ine (625 nM). The p o ocol o hese expe imen s was iden ical o ha desc ibed abo e o he expe imen s o Fig. 9. Ou indings om hese expe imen s, summa ized in Table 2, a e as ollows: (i) i appea s ha high PCO,/low pH, Na -p opiona e, and he p o onopho e dini ophenol, do no igge he ac i- a ion o ol age-dependen Ca2+ channels because elease is una ec ed by he dihyd opy idines. I is no ewo hy ha in o he p epa a ions hese h ee s imuli p oduce in acellula acidi ica ion and ail o app eciably modi y he memb ane po en ial.2SsM This sugges s ha he coupling be ween he inc ease in in acellula H+ and he Ca2+ dependen elease o DA om ype I cells ollow di e en pa hways han hose used by low PO,; (ii) cyanide is a powe ul s imulan classically used wi h ca o id chemo ecep- a s,’ bu i s mechanism o ac ion, al hough p obably ela ed o his o oxic hypoxia, is no unde s ood.9~‘0~39 Wha e e i s p ecise mechanism o ac ion, i is likely ha i in ol es depola iza ion o he ype I cells, because cyanide-induced elease o 3H-DA is pa - ially sensi i e o blocke s o ol age-dependen Ca2+ channels; (iii) i has ecen ly been shown ha abbi ype I cells a e exci able cells ha possess e odo- oxin-sensi i e Na channels.‘7*32~33~53 We u he showed ha he ‘H-DA elease induced by e a- idine, a Na channel ac i a o , is Na and Ca2+ dependen , and e odo oxin sensi i e.45 I was he e- o e o in e es o explo e he e ec s o nisoldipine on he e a idine-induced elease. Table 2 shows ha his elease is pa ially sensi i e o he dihy opy idine, and ha he sensi i i y inc eases as e a idine exposu e dec eases. DISCUSSION The esul s show ha high K+-induced elease o ‘H-DA om ype I cells is o e 95% dependen on Ca2+, is inhibi ed in a dose-dependen manne by Cd2+ and Co’+ and is modula ed by dihyd opy idi- nes. We also ound ha low PO,-induced elease is likewise Ca2+ dependen , modula ed by dihyd opy- idines, and ha his modula ion is dependen on he in ensi y o hypoxic s imula ion. The elease o 3H- DA induced by acidi ying s imuli also exhibi ed a ma ked Ca2+ dependence, bu was insensi i e o modula ion by dihyd opy idines. In a p e ious publica ion om ou labo a o y, be o e i was known ha ype I cells we e elec ically exci able, Alma az e ale3 pos ula ed he exis ence o ol age-dependen Ca*+ channels in hese cells o explain he elease o ‘H-DA induced by high K+. Qui e ecen ly, he p esence o Ca2+ channels in ype I cells has been di ec ly con i med by se e al g oups in elec ophysiological s udies o eshly dissocia ed cells.L6~17*32~33*53 Analysis o he Ca2+ ail cu en s, eco ded a ins an aneous epola iza ions, e ealed a la ge componen wi h a apid decay and a small componen which deac i a ed slowly, equi alen o abou 95% and 5% o he o al Ca2+ cu en ampli- ude, espec i ely. 53 Howe e , because o a slow ime cou se o inac i a ion, insensi i i y o he closing kine ics o long condi ioning pulses and he labili y o Ca2+ cu en ollowing in acellula dialysis du ing whole-cell pa ch-clamp eco dings, wo Ca2+ cu en s could no be esol ed. I was he e o e concluded ha mos , i no all, o he Ca2+ cu en was media ed by apidly deac i a ing (L- ype) Ca2 + channels.53 Ou p esen da a a e consis en wi h hese elec ophysio- logical indings in showing ha Ca2+ en y in o ype I cells ac i a ed by high K+ a e dihyd opy idine sensi i e, a cha ac e is ic o L-channels;35*52 his con- clusion also ag ees wi h he epo ed po en ia ion o Ca2+ cu en s eco ded in dissocia ed chemo ecep o cells by BayK 8641.26 Howe e , Akaike e uZ.‘*~ ha e desc ibed Ca2+ channels ha beha e kine ically as T- ype channels bu none heless a e sensi i e o dihy- d opy idines. In o de o explain di e ences in he ime cou se o he sec e o y esponse induced by 25 mM and 75 mM K+, Alma az e al.’ pos ula ed he exis ence o wo ypes o Ca2+ channels in ype I cells. Howe e , he a ailable da a mo e likely a o he exis ence o a single ype o ol age-dependen Ca2+ channel in ype I cell~,‘~ despi e he inding in he p esen s udy o di e en sensi i i ies o nisoldipine and Co2+ in he elease esponse induced by 25 mM and 75 mM K +, and he wo componen s ound in he analysis o he 470 A. OHESO / (I/. Ca’ ‘- ail cu en s 53 bo h o which could be iewed as compa ible wi h ;he exis ence o wo ypes o Ca’? channels. Wi h he knowledge ha ype I cells gene - a e ac ion po en ials, ‘7.3’,” i is possible now o explain di e ences in he ime cou se o elease in esponse o 25 and 75 mM K+ wi h a single se o Cal+ channels o he L- ype. The la ge, ansien elease in esponse o 75 mM K i would esul om he maximal, o nea maximal, ac i a ion o Ca’+ channels ha wi hin a ew seconds comple ely inac i- a e.53 The magni ude o he sus ained depola iza ion wi h 75 mM K+ ensu es con inued inac i a ion, wi h he consequence ha he sec e o y esponse subsides. A he same ime, he high le el o depola iza ion should a o he en y o Ca* + ia he Na +/Ca’ + exchange ,” and he eby accoun o bo h he small esponse o 75 mM K+ la e in he s imulus pe iod, as well as he small di e ence be ween he Ca2+ depen- dency and he sensi i i y o channel blocke s. The mode a e, sus ained elease in esponse o 25 mM K’ would esul om he depola iza ion o ype I cells o he ac i a ion h eshold o Na+ and Can’ channels, and he consequen gene a ion o ac ion po en ials.” Repola iza ion o he ac ion po en ial esul ing om he ac i a ion o ol age-ga ed K+ channels, bo h Ca2+ dependen and independen ”,” will limi he en y o Ca 2+ o he du a ion o he ac ion po en ials. A he same ime epola iza ion will allow he emo al o inac i a ion om Ca2+ channels, he eby pe mi ing hei ec ui men by successi e ac ion po en ials. The slow kine ics o Ca” channel ac i- a ion and he in e spike in e als will limi he en y o Ca*+ and he e o e he elease esponse. Dihyd opy idine inhibi ion and po en ia ion o low PO,-induced elease o 3 H-DA is e idence o he pa icipa ion o ol age-ga ed Ca2+- channels in he physiological esponse o he ca o id chemo eocep- o s. A he same ime he da a indica e he exis ence o addi ional pa hways o Ca2 + en y du ing hypoxic s imula ion: low PO,-induced elease o “H-DA is o e 95% Ca 2+ dependen a all in ensi ies o he s imulus and he blockade o he elease by dihy- d opy idines anges om 79% wi h mild hypoxic s imulus o abou 20% unde anoxia. The ecen inding ha chemo ecep o cells exhibi a K + cu en ha is e e sibly inhibi ed by low oxygen p ess- u e’6.3?.33 sugges s a possible mechanism o he depola iza ion equi ed o ac i a e he Ca2+ channels. The addi ional pa hways o Ca2+ en y ac i a ed du ing hypoxic s imula ion could be ela ed o he concomi an acidi ica ion p oduced unde low O2 ha is pa allel o he in ensi y and du a ion o he hypoxia.‘4~15~5’ The da a p esen ed in Figs 10 and I 1 suppo his con en ion; as he in ensi y and du a ion o he hypoxic s imuli inc ease he sensi i i y o dihyd opy idines dec eases and ends owa ds he insensi i i y o he elease esponse obse ed wi h pu e acidic s imuli (see Table 2). I would appea hen ha unde in i o condi ions he pa icipa ion o dihyd opy idine-sensi i e ol age-ga ed Ca’ ’ chan- nels du ing s ong hypoxia is limi ed o he ini ial momen s o he s imulus and he ea e he pa hways o Ca” en y ac i a ed by acidic s imuli a e hose ha suppo he Ca*’ -dependen elease o ‘H-DA. In appa en con adic ion wi h his in e p e a ion is he epo ed inhibi ion by low pH o he $-sensi i e K + cu en in dissocia ed chemo ecep o cells;” mo e ecen ly, howe e , i has been also shown ha Na’ and Ca2+ cu en s in hese cells a e simila ly inhibi ed by low pH32 (see also Re . 36). I mus be poin ed ou also ha in ui o, physiological hypoxia IS accompanied by sys emic alkalosis. Si ua ions com- pa able o he in i o condi ions a e seen i i :ico a ex emely low POZ, and in ce ain lung pa hologies and ci cula o y shock. Rega ding he na u e o he pa hways used o in lux o Ca’+ in o chemo ecep o cells du ing acidic s imula ion, ou da a a e compa ible wi h wo possi- bili ies: (i) Ca’+ en e s he cells ia he ol age-ga ed Ca*+ channels, which became insensi i e o dihy- d opy idines unde he speci ic condi ions se by he s imuli, and (ii) Ca 2+ in lux in o chemo ecep o cells occu s h ough a dihyd opy idine-insensi i e pa h- way. Al hough i is no possible o dis inguish be- ween hese al e na i es wi hou di ec eco dings o Ca2+ cu en s unde he same condi ions, he e a e o he da a ha a o he second al e na i e. Fo example, on heo e ical g ounds i should no be expec ed ha dini ophenol p oduces any signi ican depola iza ion a he plasma memb ane le el. In ac i has been shown ha dini ophenol p oduces an in ense acidi ica ion in lymphocy es wi hou any app eciable change in hei memb ane po en ial.” I has also been shown ha he o he wo acidi ying s imuli used in he p esen s udy do no p oduce any signi ican change in he memb ane po en ial o snail neu ons.” The e o e, i appea s inconcei able ha hese s imuli ac i a e ol age-ga ed Ca’ ’ channels. In addi ion, a ecen publica ion om ou g oup sugges ed ha he Ca’+-dependen elease o ‘H-DA elici ed by acidi ying s imuli would be media ed by in lux o Ca’ + ia he Na + Ca’ + exchange c3” i was sugges ed also ha he accumula ion o Na + accom- panying p o on ex usion du ing an acidic load, would be esponsible o he e e sal o he Na’ / Ca2+ exchange .39 Con i ma ion o hese sugges ions has been ob ained.” The elease o DA induced by acidic s imuli was ound o be Na+-dependen and inhibi ed by blocke s o he Na + -dependen p o on- ex uding mechanisms indica ing ha en y o Na+ is he d i ing o ce o Ca ‘+ en y du ing acidic s imu- la ion. This in o ma ion, coupled o he comple e insensi i i y o he acidic s imuli elease esponse o BayK 8644 and o nisoldipine, suppo s he con- clusion ha ansduc ion o he acidic s imuli does no in ol e depola iza ion o ype I cells no pa ici- pa ion o ol age-ga ed Ca’ ’ channels. The ime-dependence o nisoldipine on e a idine- induced 3H-DA elease, i.e. he inhibi ion p oduced by he Ca2+ -channel block dec eased wi h he p o- Ca o id body Ca *+ channels and ca echolamine elease 471 longed exposu e o e a idine (see Table 2), may be explained as ollows. I is concei able ha , a e an ini ial bu s o Ca’+ in h x ia Ca*+ channels, he sus ained depola iza ion p oduced by he alkaloid pe manen ly inac i a es he ol age-dependen Ca2+ channels in he chemo ecep o cell~.~~ Wi h ime, he depola iza ion i sel , and he in acellula accumu- la ion o Na+ p oduced by e a idine,4s12 will igge Na+/Ca2+ exchange ha will p o ide he in lux o Ca2+ o suppo he e a idine-induced elease.” A Na+/Ca2+ an ipo e may also be an impo an mechanism in he sec e o y esponse induced by cyanide.” Acknowledgemen s-This wo k was suppo ed by g an s PB89/0358, FISS 89/0340 and Jun a de Cas illa y Leon 1101/89 (Spain), and by USPHS G an s NS12636 and NS07938. REFERENCES 1. 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