Effects of low pH on synthesis and release of catecholamines in the cat carotid body in vitro

178 B ain Resea ch, 309 (1984) 178-181 Else ie BRE 20344 E ec s o low pH on syn hesis and elease o ca echolamines in he ca ca o id body in i o R. RIGUAL 1, E. GONZALEZ 1, S. FIDONE 2 and C. GONZALEZ 1 l Depa amen o de Fisiolog a y Bioquimica, Facul ad de Medicina, Valladolid (Spain) and 2Depa men o o Physiology, School o Medicine, Uni e si y o U ah, Sal Lake Ci y, UT (U. S. A.) (Accep ed May 1s , 1984) Key wo ds: ca o id body -- dopamine -- no ad enaline -- syn hesis -- elease -- acidosis The a es o dopamine and no ad enaline syn hesis in he ca ca o id body (c.b.) a e 5.9 _+ 0.58 pmol/c.b./2 h and 0.3 + 0.02 pmol/c.b./2 h, espec i ely. The syn hesis is doubled when he o gans a e incuba ed a pH 7. Simila ly, low pH induces a elease o do- pamine om he c.b. which is p opo ional o inc eased ac i i y in he ca o id sinus ne e. The ca o id body (c.b.) is a chemo ecep o o gan ac i a ed by en i onmen al low pO2, low pH and high pCO2. I possesses clus e s o cells o wo di e - en ypes, ype I (glomus) and ype II (sus en acula ) cells. Senso y ne e ibe s pene a e in o he clus e s and hei endings o m synap ic-like con ac s wi h ype I cells. Type I cells possess dense co e esicles in hei cy oplasm which con ain ca echolamines (CA). The possible ole o CA as neu o egula o s be ween ype I cells and senso y endings is a ma e o dispu e because he mos abundan CA in he c.b., dopamine (DA), beha es as an exci a o y o inhibi o y agen depending on he dose employed 21 o he animal spe- cies used 16. Fu he , a dec ease in CA con en (an in- dex o CA elease) a e hypoxic s imula ion has no always been obse ed (see e . 6 o a e iew). In a e y ecen a icle by Fi zge ald e a1.10, he e ec o se e e hype capnic acidosis has been s udied in he ca c.b. and no dec ease in CA con en was obse ed; simila indings ha e been epo ed o he a c.b. is. In ecen yea s 7-9 we ha e used an in i o p epa- a ion5 o measu e CA elease and ha e epo ed ha hypoxia eleases DA om abbi and ca c.b.s pa allel o he inc eased elec ical ac i i y in he ca- o id sinus ne e (c.s.n.). In he p esen epo we show, wi h he same p epa a ion, ha low pH en- hances he a e o bo h DA and NE syn hesis and ha his s imulus also elici s an inc ease in DA elease concomi an wi h he inc eased ac i i y o he c.s.n. Ca s we e anes he ized wi h sodium pen oba bi- one (30-40 mg/kg i.p.) and a i icially espi ed while he ca o id a ea was exposed. To s udy he syn- hesis o CA, he ca o id bi u ca ion was emo ed and placed in a luci e chambe illed wi h ice-cold 100% 02 equilib a ed Ty ode 2 (in mmol: NaC1, 112; KCI, 4.7; CaCI2, 2.2; MgC12, 1.1; sodium glu ama e, 42; Hepes, 5; glucose 5.6, adjus ed o pH = 7.4 wi h 1 N NaOH). The c.b.s, we e dissec ed ou , ho - oughly cleaned o su ounding connec i e issue and incuba ed a 37 °C o 2 h in small ials placed in a me abolic shake . The incuba ion medium (0.5 ml) was 100% O2-equilib a ed Ty ode con aining 10 -4 M 6-MPH4 (Sigma) and 40/~M 3,5 [3H] y osine (spec. ac . 2 Ci/mmol, Ame sham). The inal pH o he me- dia was 7.4 (con ol) and 7.0 (expe imen al). A he end o incuba ion, he o gans we e homogenized and he supe na an s p ocessed o TLC as desc ibed by Fleming and Cla k 12, excep ha he pla es we e s ained wi h po assium e icyanide o minimize quenching. In some expe imen s he incuba ion me- dia we e passed h ough alumina and all he ca echol compounds elu ed wi h 1 N HCI; he elua es we e d ied in a acuum concen a o (Sa an ) and esus- pended in 40 ~1 o he sol en used o TLC and ch o- Co espondence: C. Gonzalez, Depa amen o de Fisiologia y Bioquimica, Facul ad de Medicina, Valladolid, Spain. 0006-8993/84/$03.00 ~) 1984 Else ie Science Publishe s B.V. 179 TABLE I E ec s o low pH on [3H]CA-syn hesis in he c.b. The da a (pmol/c.b.) a e means + S.E. o 8 pai s o c.b. Di e - ence was es ed by he pai ed - es . pH = 7.4 pH = 7 P [3H]Ty osine 7.2 ___ 0.95 6.5 + 0.60 n.s. [3H]NE 0.3 + 0.02 0.7 + 0.08 0.005 pH]DA 5.9 + 0.58 12.5 + 2.50 0.02 ma og aphed. To s udy he elease o DA and he senso y ne e discha ge, he cleaned c.b.s and hei own ca o id si- nus ne es (c.s.n.) we e incuba ed as be o e (pH = 7.4) wi h [3H] y osine o high speci ic ac i i y (20 Ci/mmol). A he end o incuba ion hey we e moun ed in a supe usion chambe as desc ibed else- whe eS, 9. The ac ion po en ials om he c.s.n, we e led h ough an AC-coupled p eampli ie o an oscillo- scope. The ampli ied signals we e also led h ough a window disc imina o o a digi al coun e p in e which ead he o al numbe o ne e impulses e- co ded du ing a collec ion pe iod. The supe usa es we e collec ed in ials con aining a ca ie solu ion (0.3 M ace ic acid, 1 mM asco bic acid) and p ocess- ed wi h alumina o adso b he labeled ca echol com- pounds ([3H]DA and [3H]DOPAC) which we e elu ed wi h 1 N HC120 and measu ed by liquid scin il- la ion coun ing. In some expe imen s he alumina elua es we e d ied in a acuum concen a o , esus- pended and u he analyzed by TLC o e i y he iden i y o he adioac i e compounds. Lowe ing he pH o he incuba ion media om 7.4 o 7, p oduced a signi ican inc ease in [3H]NE and [3H]DA con en wi hou any signi ican change in ee [3H] y osine con en (Table I). The only [3H]ca- echol measu able in he incuba ion media was [3H]DOPAC, in amoun s ep esen ing abou 25% o he [3H]DA p esen in he c.b.s o each g oup. The elease o [3H]DA (measu ed as [3H]DA + [3H]DOPAC) was dependen on he pH o he supe - usion media. Fig. 1 shows he ime-cou se o low pH-induced elease o DA om ype I cells and ac i - i y in he c.s.n, in a single expe imen . The p opo - ionali y be ween bo h pa ame e s is e iden in Fig. 2 which summa izes he esul s o 6-8 expe imen s. 100 $ E u ) d E. (J 50- pH66 pH 7 500~ 250- pH 6.6 pH 7 Fig. 1. Bo h c.b.s om one ca p e iously loaded wi h [3H]DA we e placed in he supe usion chambe and he elec ical ac i i y o he c.s.n, o one o hem was con inuously moni o ed. Supe usa es we e collec ed e e y 5 o 10 min. The op o he igu e shows he change in c.s.n, ac i i y induced by 10 min o supe usion a low pH. The lowe pa shows he change in [3H]DA elease (measu ed as [3H]DA + [3H]DOPAC) induced by he same expe imen al maneu e . 180 A -o 200. O _o "I i] I iiiii! 1 ! ~.n J .20o ~g qd O z U loo D Fig. 2. Means + S.E. o he c.s.n, ac i i y and elease o [3H]DA a 3 di e en pHs ob ained in 6-8 expe imen s. Re- sul s a e exp essed as % o ha ob ained a pH 7.4 (100%). Ou esul s clea ly show ha he a e o syn hesis and elease o DA by he c.b. inc eases when he ex- acellula pH is educed; he e is a pa allelism be- ween he pH-induced elease o DA om ype I cells and he pH induced ac i i y in he c.s.n, which sug- ges s a possible linkage be ween bo h e en s. The s imula o y e ec o low pH on CA syn hesis in he c.b. con o ms o indings in o he s uc- u esl,3as, TM. In ag eemen wi h he esul s ob ained in synap osomes TM, ou da a sugges ha he in- c eased hyd ogen ion concen a ion modi ies he ki- ne ic pa ame e s o y osine hyd oxylase in he c.b. In addi ion, ou di ec measu emen o he a e o CA syn hesis in he c.b. explains he indings o Han- baue ]3, who epo ed an inc ease in CA con en in he a c.b. a e exposu e o he animals o a hype - capnic a mosphe e. The elease o DA by he c.b. is e y sensi i e o ex acellula pH when compa ed o o he s uc u es; o example, acco ding o Can u e al. 4, he ou pu o CA om he dog ad enal medulla inc eases only when pH d ops o 6.85-6.95 and acco ding o o he au ho s T, pH mus d op below 6.8 o de ec an in- c ease in ad enal medulla y sec e ion. In s ia al slices a pH o 6.5 did no p oduce any signi ican in- c ease in DA elease ]9. I was epo ed in a ecen publica ion ]0 ha hy- poxia eleases DA in he ca c.b. in i o whe eas hy- pe capnic acidosis does no . I was concluded ha he p ocess o chemosenso y ac i a ion induced by hese s imuli mus be di e en . Howe e , i should be no ed ha e en in ense hype capnic acidosis p oduc- es only a modes inc ease in c.s.n, ac i i y when com- pa ed wi h hypoxia 11. The e o e, i he p opo ional- i y be ween DA elease and c.s.n, ac i i y seen wi h hypoxia7,8, 9 holds o acidosis, i should be di icul o de ec changes in DA con en in he c.b. induced by acidosis. As shown in his pape he e is also a pa allelism be ween acidosis-induced elease o DA and ac i i y in he c.s.n., sugges ing ha acidosis, like hypoxia, ac s ia ype I cells. Howe e , when he da a ob- ained wi h acidosis a e compa ed wi h hose o hy- poxia 9 i appea s ha o a gi en le el o ac i i y in he c.s.n, he elease o DA is highe wi h hypoxia; his seems o imply ei he ha acidosis also di ec ly ac i a es he senso y ne e endings, as seen in o he s uc u es 14, o ha acidosis makes he c.s.n, endings mo e sensi i e o he neu o egula o s eleased om ype I cells. As a whole, he da a show ha ype I cells de ec he na u al s imulus o acidosis and ha DA seems o play an impo an ole in he gene a ion o chemo ecep o impulses. We hank P o esso s C. Eyzagui e, B. He e os and R. Fi zge ald o hei c i icisms du ing he p ep- a a ion o he manusc ip . The wo k was suppo ed by a g an om he C.A.I.C.T. o Spain and by a USPHS G an NS-12636. 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