Intracellular Ca2 + deposits and catecholamine secretion by chemoreceptor cells of the rabbit carotid body

42 INTRACELLULAR Ca2 +DEPOSITS AND CATECHOLAMINE SECRETION BY CHEMORECEPTOR CELLS OF THE RABBIT CAROTID BODY Ana Obeso, Asuncion Roche , Jose Ramon Lopez-Lopez, and Cons ancio Gonzalez Depa amen o de Bioquimica y Biologia Molecula yFisiologia Facul ad de Medicina, Uni e sidad de Valladolid 47005 Valladolid, Spain 1. INTRODUCTION The pi o al ole o in acellula ee [Ca2 +] luc ua ions in he con ol o cellula unc ions such as con ac ion and sec e ion, including he elease o neu o ansmi e s, was ecognized many decades ago (see Rubin, 1982). Mo e ecen ly, he lis o cellula unc ions igge ed o modula ed by he le els o Ca2+ jhas g own eno mously. Addi ional unc ions egula ed by [Ca2+l include neu onal exci abili y, synap ic plas ici y, gene ex- p ession, cellula me abolism, cell di ision and di e en ia ion, and p og ammed cell dead (Mille , 1991; Clapham, 1995). Pa alleling he g ow h in his lis o Ca2+ -con olled unc- ions, amul iplici y o cellula mechanisms aimed a main aining es ing ee [Ca2+l in he ange o 100 nM o mos cells has been desc ibed, allowing inc eases in Ca2+ jle els ha a e speci ic in hei magni ude, ime cou se and spa ial dis ibu ion, acco ding o he cell unc ion ac i a ed (Toescu, 1995). Since Ca2+canno be me abolized, cells egula e hei cy oplasmic le els o ee Ca2+ h ough nume ous binding p o eins and in lux and e lux mechanisms (Fig 1). Ca2+in lux o cell cy oplasm om he ex acellula milieu occu s ia ol age o ecep o ope a ed channels o ia ye ill-de ined capaci a i e pa hways; he Na+/Ca2+exchange can also p oduce in some ci cums ances ne in lux o Ca2+(Mille , 1991; Clapham, 1995). Ca2+e - lux o he ex acellula space occu s agains elec ochemical g adien s, and he eby he pumping ou o Ca2+is di ec ly (Ca2+pump) o indi ec ly (Na+/Ca2 +) coupled o he hy- d olysis o ATP. In addi ion o he ex acellula space, he e a e in acellula s o es, ep esen ed p i- ma ily by he smoo h endoplasmic e iculum, capable o accumula ing Ca 2+a e y high concen a ions (mM ange) ia speci ic Ca2+ ATPases (Fig 1). The smoo h endoplasmic F on ie s in A e ial Chemo ecep ion, edi ed by Zapa a e al. Plenum P ess, New Yo k, 1996 279 280 A. Obeso e al. Na Figu e 1. Model o he egula ion o cellula calcium homeos asis. See ex o an explana ion. e iculum o many cells may possess wo speci ic mechanisms capable o mobilizing he accumula ed Ca2+in esponse o adequa e signals, he inosi ol 1,4,5- iphospha e ecep- o s (IP3R) ha on IP] binding a e ensambled o p oduce aCa2+channel, and he yano- dine ecep o -channel complex (RyR) equally pe mean o Ca2+(Pozzan e ai, 1994; Simpson e aI, 1995). Many ex acellula signals, ho mones and neu o ansmi e s, pos- sess ecep o s in he plasma cell memb ane ha ac i a e phospholipase Cleading o he genesis o IP] in he cell in e io and o he elease o Ca2+ om he endoplasmic e icu- lum ia he IP]R (Clapham, 1995); he RyR a e ac i a ed by local inc eases in ee Ca2+; p oduced by Ca2+en e ing ia plasma cell memb ane (Lopez-Lopez e ai, 1995), and ep- esen he subs a e o he long ime known p ocess o Ca2+ -induced Ca2 + elease (Simp- son e aI, 1995). The e o e, he endoplasmic e iculum may unc ion as asink o as a sou ce o ee cy oplasmic Ca2+in di e en unc ional s a es o he cells, and addi ion- ally he sink o sou ce unc ion o he endoplasmic e iculum migh a y om cell o cell, depending on he deg ee o exp ession o he epe oi e o he molecules in ol ed in he endoplasmic e iculum handling o Ca2+(Simpson e ai, 1995). A cy oplasmic le els o ee Ca2+highe han 500 nM (Clapham, 1995), i.e., du ing cell ac i a ion, mi ochon- d ia may accumula e Ca2 +, he eby unc ioning as e ec i e sinks o educe cy oplasmic ee Ca2+ concen a ion. Finally, many cy osolic cons i uen s including p o eins, and small o ganic molecules can bind Ca2+ions con ibu ing o modula e local le els o cy o- plasmic ee Ca2+(Clapham, 1995; Toescu, 1995). The p o eins wi h he capaci y o bind Ca2+may in um acqui e ca aly ic ac i i ies when hei binding si es a e occupied by he egula ing ion. In he ca o id body (CB) chemo ecep o cells, ou knowledge on he ole o Ca2+in cell signalling is a he es ic ed (see Gonzalez e ai, 1994). I was only in 1968 ha Eyzagui e and Zapa a showed he Ca2+dependency o ca o id sinus ne e (CSN) dis- cha ges elici ed by hypoxia, acidi y o low in e up ion. In 1975, again Eyzagui e's g oup (Eyzagui e e ai, 1975) epo ed ha ace ylcholine was incapable o gene a ing ac- In acellula Ca l +Deposi s and Ca echolamine Sec e ion by Chemo ecep o Cells 281 ion po en ials in he CSN in Caz+- ee solu ions. Some yea s la e , a ew ul as uc u al s udies showed he appea ance o omega exocy o ic p o iles and/o coa ed pi s and es- icles in chemo ecep o cells o a CBs incuba ed in Caz+-con aining (bu no in Caz+- ee) ich K solu ions o in he p esence o he Caz+ionopho e A23187, concluding ha exocy- osis was Caz+e-dependen (G onblad e ai, 1980). Consis en wi h ha , i was la e shown ha he elease o dopamine (DA) elici ed by hypoxia and high K was Ca Z -dependen in >95% (Fidone e ai, 1982; Alma az e ai, 1986; Obeso e ai, 1992). Mo e ecen ly, h ee di e en labo a o ies (Gonzalez e ai, 1993; Lopez-Ba neo e ai, 1993; Buckle & Vaughan-Jones, 1994) ha e used luo escen dyes o measu e [Caz+l in isola ed chemo e- cep o cells, and ha e consis en ly ound ha mo e han 95% o he [Caz+l ise p oduced by hypoxia is due o Caz+en e ing om he ex acellula space. In he case o acidic s imula ion he Ca Z dependence o he elease o DA was app oxima ely 80% (Obeso e ai, 1992) and he Ca2+; signal was educed by asimila pe cen age in Caz+- ee media (Buckle &Vaughan-Jones, 1993). A a iance wi h hose indings, Biscoe and Duchen (1990) epo ed ha up o 40% o he hypoxic Caz + j ise was due o Ca2+ en e ing he cy o- plasm om in acellula s o es, and Biscoe e al (1989) could no de ec a ise in ee Ca z + j du ing acidic s imula ion. In addi ion, Lahi i e al (1995) ha e ecen ly communica ed ha he anoxic CSN discha ge was be e p ese ed in Caz+- ee media con aining hapsiga gin han in i s absence, concluding ha Caz + om in acellula deposi s seems o con ibu e o he anoxic chemo ecep ion. 2. METHODS Using an in i o p epa a ion o he abbi CB whose ca echolamine deposi s ha e been labeled by p io incuba ion o he o gans wi h hei na u al p ecu so 3H- y osine (Fi- done &Gonzalez, 1982), we ha e s udied he signi icance o in acellula Caz+s o es as possible sou ces o Caz+ o he sec e o y esponse, and as possible bu e s (sinks) o he Ca z+en e ing he cell cy oplasm du ing s imula ion. The ools used in he expe imen s in- clude: ATP and b adykinin as possible gene a o s o IP3,and he eby as possible ac i a- o s o he IP3 R; yanodine a submic omola concen a ion and ca eine as ac i a o s o he RyR; hapsiga gin as ablocke o he e iculum ATPase; u henium ed as ablocke o he mi ochond ial Ca2+ unipo e ; and, ionomycin as a Ca z+ionopho e ha allows apid equilib a ion o Ca z+ h oughou cellula memb anes. The expe imen s ha e been pe - o med in Caz+-con aining and in Ca2+- ee solu ions. The e ec s o he d ugs ha e been es ed on hei abili y o igge asec e o y esponse in basal no moxic condi ions, o es o a ole o in acellula Caz+s o es as sou ces o Caz+ o he sec e o y esponse, and on hei abili y o modi y he in ensi y and ime cou se o he sec e o y esponse elici ed by hypoxia and high K . In his way, we ha e es ed o he capaci y o he in acellula s o es o ac as sou ces o Ca2+, o as sinks o he Caz+en e ing om he ex acellula space. I should be no ed ha mos o he expe imen s ha e been pe o med sampling he incuba ing solu ions o hei analyses in 3H-ca echolamine eH-CA) con en e e y 2min, he eby p o iding ahigh sensi i i y o he o e all p ocedu es capable o de ec ing <6 mol o labelled CA «200 cpm), Which a e co ec ion o he speci ic ac i i y o 3 H-CA in he cells, ep esen s app oxima ely 120 mol o o al CA elease, o an amoun equi alen o he basal elease/2 min in no moxic condi ions. Du ing s imula ion (hypoxia, high K+ e ) he elease inc eases ma kedly, and consequen ly he sensi i i y o he adioiso opic me hod o de ec he e ec o ad ug on he s imulus induced elease dec eases. A. Obeso e al. Figu e 2. Time cou se o he disappea ance o high ex e - nal K+ induced elease o 3H-ca echolamines upon emo al o calcium om he ex acellula space. Emp y ba s: e- lease esponse in Ca2+-con aining solu ions a he begin- ning and he end o he expe imen s. Slashed ba s: elease esponse a e incuba ion in Ca2+- ee solu ions o he in- dica ed pe iods. Release esponse (o dina e) is exp essed as imes basal elease in no mal K+ solu ions. 3. RESULTS AND DISCUSSION Figu e 2shows he ime cou se o ex acellula Ca 2 +washing ou . In designing hese expe imen s, he ad an age ha he elease induced by high ex e nal K+ is almos o ally dependen on Ca2 +en e ing om he ex acellula space was aken. The e o e, he mini- mum ime o incuba ion in Ca 2 + ee medium equi ed o abolish high K induced elease would be equi alen o he ime equi ed o wash ou Ca2+ions om he incuba ing ial and he CB ex acellula space. As shown in Figu e 2, one min o incuba ion in 0Ca2 + e- duced ma kedly he elease esponse, a e 4min in 0Ca2+ he elease was p ac ically abolished, and on ein oduc ion o Ca 2+ he elease esponse eco e ed comple ely. In a simila expe imen using hypoxic s imula ion (2% 02-equilib a ed solu ions, 2min), he ime cou se o inhibi ion was compa able; in ac , a e 4min in 0Ca2 + he elease induced by hypoxia was comple ely abolished. These expe imen s con i m p e iously epo ed indings wi h longe imes o incuba ion in Ca 2+ ee solu ions (e.g. Obeso e ai, 1992), and sugges ha in acellula Ca2+s o es do no playa signi ican ole in media ing he se- c e o y esponse o hypoxia, unless chemo ecep o cells ha e an unusually as -emp ying in acellula Ca 2 +deposi s. On he o he hand, he p esen indings a e consis en wi h ob- se a ions o Fidone (see Fidone e ai, 1977) and To ance (1977) showing ha he hal - ime o bica bona e and suc ose wash ou , espec i ely, was close o hal aminu e. Nex we pe o med ase ies o expe imen s aimed a mobilizing in acellula Ca 2 + s o es. In a i s g oup o expe imen s we es ed o he abili y o ATP o mobilize Ca 2 + s o es. In a ecen s udy, Spe gel &Lahi i (1993) showed ha ATP and analogs, bu no adenosine, we e able o inc ease CSN discha ges in adose-dependen manne , concluding ha he CB exp esses su ace P2 ype ATP ecep o s. Since P2 ecep o s a e coupled o 13 ype phospholipase C, i should be expec ed ha hei ac i a ion will lead o he o ma ion o IP3and ac i a ion o IP 3R. A TP a 100 and 1500 IlM did no a ec he basal no moxic elease o DA ei he in Ca 2+con aining o in Ca2+ ee solu ions sugges ing ha he ATP ecep o s esponsible o he ac i a ion o CSN discha ges a e loca ed in as uc u e di e - en om chemo ecep o cells. B adykinin, apo en mobilize o Ca2 + om in acellula In acellula Ca 2+Deposi s and Ca echolamine Sec e ion by Chemo ecep o Cells 283 deposi s and po en sec e agogue in ch oma in cells (Augus ine &Nehe , 1992), was also wi hou e ec on he basal elease o 3H-CA om he CB, sugges ing ha chemo ecep o cells do no possess b adykinin ecep o s o ha he endoplasmic e iculum Ca2+s o es a e eally small. The same was ue o he es o he agen s es ed including ionomycin in Ca2+- ee solu ions. In conclusion, he pa icipa ion o endoplasmic e iculum and mi ochond ia as sou ces o Ca2+capable o al e ing he sec e o y esponse o chemo ecep o cells appea o be o sec- onda y impo ance, implying ha chemo ecep o cells mus ely almos exclusi ely on plasma memb ane mechanisms o main ain hei Ca2 +homeos asis. Al hough he p ecise iden- i y o he Ca2 +s o es si es is no ully clea ,i appea s ha he smoo h endoplasmic e iculum (SER) may be he p incipal place whe e he in acellula Ca2+ATPase is loca ed and he eby he SER could be he main place whe e Ca2+is s o ed (Pozzan e aI, 1994). I his is he case in chemo ecep o cells ou indings would co ela e wi h he appa en ly ea SER p o iles p e- sen in he cells ha appea o be loca ed mainly in he p ocesses o chemo ecep o cells (Ve na, 1975; McDonald, 1981). I should be men ioned ha pape s consis en wi h ou con- clusion ha e been p esen ed in his Symposium. Thus, He e al (1996) communica ed ha en- do helin Iinc eases up o i e imes he no moxic basal le els o IP 3 ,and he e o e i should be expec ed ha IP 3 R o he endoplasmic e iculum ep esen s he a ge o he IP3 o med. Al- hough he cellula elemen in which he inc ease o IP3had occu ed was no iden i ied, i was shown ha endo helin Idid no a ec he basal elease o CA o he Ca2 +jle els in chemo ecep o cells, bu s ill endo helin Iwas e ec i e in po en ia ing he elease o DA and he Ca2+j ise p oduced by hypoxia. Those indings imply ha chemo ecep o cells possess en- do helin ecep o s, and he e o e i could be expec ed ha IP 3has inc eased in chemo ecep o cells. The e o e, he lack o e ec on he basal elease o CA and Ca2+jwould imply ha he cellula deposi s o Ca 2+sensi i e o IP3a e eally small. The e ec s obse ed du ing s imula- ion would be p oduced ia ol age-dependen Ca2+channels, as i is he case in o he sys- ems. These pieces o in o ma ion u ge o explo e he dynamics o in acellula Ca2+s o es in amo e di ec manne and o co ela e he da a wi h o he unc ions o he chemo ecep o cells, besides he elease o neu o ansmi e s. 4. ACKNOWLEDGMENTS Suppo ed by Spanish DGICYT G an PB92/0267. 5. REFERENCES Alma az L, Gonzalez & abe so A(1986) E ec s o high po assium on he elease o eH]dopamine om he ca ca- o id body in i o. JPhysiol, London 379: 293-307 Augus ine GJ &Nehe E(1992) Calcium equi emen s o sec e ion in bo ine ch oma in cells. JPhysiol, London 450:247-271 Biscoe TJ &Duchen MR (1990) Responses o ype Icells dissocia ed om he abbi ca o id body o hypoxia. J Physiol, Lond 428: 39-59 Biscoe TJ, Duchen MR, Eisne DA, O'Neill SC &Valdeolmillos M(1989) Measu emen s o in acellula Ca2+ in dissocia ed ype Icells o he abbi ca o id body. JPhysiol, London 416: 421-434 Buckle KJ &Vaughan-Jones RD (1993) E ec s o acidic s imuli on in acellula calcium in isola ed ype Icells o he neona al a ca o id body. 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