Reciprocal modulation of tyrosine hydroxylasea activity in rat carotid body

572 B ain Resea ch, 172 (1979) 572-576 © Else ie /No h-Holland Biomedical P ess Recip ocal modula ion o y osine hyd oxylasea ac i i y in a ca o id body CONSTANCIO GONZALEZ, YAN KWOK, JAMES W. GIBB and SALVATORE J. FIDONE* Depa men s o Physiology and Biochemical Pha macology and Toxicology, Uni e si y o U ah, Sal Lake Ci y, U ah (U.S.A.) (Accep ed May 5 h, 1979) The ca o id body is an a e ial chemo ecep o o gan esponsi e o blood le els o pO2, pCOe and pH 13. The pa enchymal issue o he ca o id body is composed mainly o wo cell ypes: he glomus o Type I cells, which a e disposed oge he in g oups o glome uli, and he sus en acula o Type II cells, which appea as glial-like elemen s enclosing he glome uli in capsula ashion 3,4. The Type I cells, which ha e abundan dense-co ed esicles and a e known o con ain ca echolaminesl, 2,11,15, ecei e a senso y inne a ion om a e en ibe s o he ca o id sinus ne e 3. Recen s udies ha e also shown he p esence o ecip ocal synapses a hese junc ions be ween a e en ne e e minals and Type I cells TM. In addi ion, hese cells ecei e an e e en inne a ion om bo h p eganglionic and pos ganglionic sympa he ic ibe s which each he ca o id body om he supe io ce ical ganglion 18. The p incipal ca echolamine in he Type I cells o he a ca o id body is dopa- mine (63 ~), he emainde being no epineph ine 11. Al hough he p ecise ole o ca echolamines in he ca o id body emains o be elucida ed, he e is e idence implic- a ing hese subs ances as neu o ansmi e s o modula o s o chemo ecep o ac i i y 5,6,16,~7,2°,24. O pa icula in e es a e he ecen indings in a ca o id body ha hypoxia induces a long- e m inc ease in y osine hyd oxylase (TH) ac i i y, he a e-limi ing enzyme in ca echolamine biosyn hesis 7,9. In hese expe imen s, unanes he- ized animals we e exposed in a chambe o a iable imes o low 02 a mosphe es. When TH ac i i y was assayed 48 h ollowing he hypoxic episode he e was an inc ease in enzyme ac i i y o 50-80 ~ abo e con ol alues (con ol animals we e exposed only o oom ai in he chambe ). This long- e m induc ion o TH could ha e esul ed om ei he di ec ac ion by he hypoxic s imulus upon he Type I cells o he ca o id body, o ins ead i migh ha e a isen e lexy ia he ne ous inne a ion o he o gan. TH ac i i y in o he ca echolamine gic sys ems is known o be subjec o neu al con olS,14,21, 22, and as desc ibed abo e he Type I cells o he ca o id body ecei e a complex ne ous inne a ion (a e en , e e en and ecip ocal synapses). I is hus * To whom co espondence should be add essed a : Depa men o Physiology, Uni e si y o U ah College o Medicine, 410 Chipe a Way, Resea ch Pa k, Sal Lake Ci y, UT 84108, U.S.A. 573 -~ 20 [] Con ol ~- • 10% 02 o .~10. No mal Symp(] h. Dea e Fig. 1. E ec s o hypoxia (10 ~ 02) on TH ac i i y in no mal, sympa hec omized and dea e en ed a ca o id bodies. Unanes he ized animals we e exposed in a chambe o 3 h o ai (clea ba s) o i0~ 02 in N2 (black ba s), and TH ac i i y assayed 48 h la e . Remo al o he supe io ce ical ganglion (Sympa h.) o he ca o id sinus ne e (Dea e .) was pe o med 12-15 days p io o exposu e o he animals in he chambe . Each ba in he igu e ep esen s he mean ± S.E. om 8 a s. impo an o an unde s anding o he ole o ca echolamines in chemo ecep ion o de e mine he con ibu ions o he sympa he ic inne a ion and he ca o id sinus o he obse ed induc ion o TH by hypoxia. In he p esen s udy, we ha e used animals wi h ch onically dene a ed ca o id bodies and we epo he e ha he dual inne a ion (sympa he ic and ca o id sinus ne e) o he ca o id body exe s a ecip ocal con ol o e TH ac i i y in his o gan: sympa hec omy abolishes TH induc ion by hypoxia, while sec ion o he ca o id sinus ne e enhances bo h he esponse o hypoxia and he basal TH le els in he issue. Adul Sp ague-Dawley a s, weighing be ween 180-240 g, we e housed 3 pe cage, a 26 °C wi h ad libi um ood and wa e , and a no mal da k-ligh hy hm. In some animals, he supe io ce ical ganglion o he ca o id sinus ne e was emo ed 12-15 days p io o sac i icing he animals o assay o TH ac i i y. Unanes he ized a s we e exposed o 3 h ei he o ai (con ol animals) o o 10 ~ 02 in N2 (hypoxic animals) in a luci e chambe con inuously lushed wi h he gi en gas mix u e (2-4 a s pe exposu e; chambe olume, 14 li e s; lushing a e, 4 li e s/ ain; gases ce i ied zk 0.5 ~ 02 o a ed pu i y, IAP, Po land). Fo y-eigh hou s ollowing exposu e, he animals we e anes he ized wi h sodium pen oba bi al (i.p.), he achea cannula ed, and he a ea o he ca o id bi u ca ion exposed wi h he aid o a dissec ing mic oscope. Ca o id bodies we e quickly emo ed om he animals and immedia ely cleaned o su ounding connec i e issue in a dissec ing chambe illed wi h ice-cold physiological saline. The specimens we e weighed on a Cahn Elec obalance (sensi i i y, ~ 50 ng) equipped wi h a humidi ied chambe o p e en d ying o he issues. All samples we e placed in glass homogenize s con aining 0.2~ T i on X-100 and kep on ice un il assayed o TH ac i i y, which was pe o med acco ding o he me hod desc ibed by Naga su e al. 19. The p inciple o his me hod elies on he eco e y and measu emen 574 o i ia ed wa e p oduced du ing he hyd oxyla ion o L-[3,5-ZH] y osine. The assay medium con ained he ollowing, exp essed as inal concen a ions in a ol. o 100 #l: 1 mM e ous ammonium sul a e; 2 mM 2-amino-6,7-dime hyl-4 hyd oxy-5,6,7,8- e ahyd op e ine dihyd ochlo ide (DMPH4; Ald ich); 100 mM 2-me cap oe hanol (Calbiochem); 200 mM sodium ace a e bu e , pH 6.0; 0.1 mM L- y osine; 0.4 #Ci L- [3,5-3H] y osine (Ame sham); and 50 /~l o issue homogena e (o 80 #l ini ial homogena e ol.). The assay eac ion was allowed o p oceed o 15 ain in a me abolic shake (250 pm; New B unswick) a 37 °C, and was s opped by addi ion o 0.1 ml o 10 ~ TCA. The acidi ied mix u e was passed h ough a 7 × 30 mm pu i ied Dowex 50- H + column (Bio-Rad Labo a o ies); 1.8 ml o dis illed wa e was used o wash he column, and he combined e luen s we e collec ed in scin illa ion coun ing ials. Fi een ml o scin illa ion luid (23 g PPO, 2 li e s oluene and 1 li e T i on X-100) we e added o each ial p io o coun ing in a Packa d liquid scin illa ion spec o- me e . Blanks we e p epa ed wi h 50 #l o dis illed wa e in place o he issue homogena e and p ocessed in iden ical manne . TH ac i i y is exp essed as nmol/h/mg issue, and he alues gi en a e means 4- S.E. As epo ed p e iously, TH ac i i y in no mally inne a ed ca o id bodies was inc eased when he enzyme ac i i y was assayed 48 h ollowing a 1-3 h hypoxic episode7, 9. These da a a e shown in Fig. 1 (no mal) and we e ob ained om a s exposed o 3 h o 10 700 02 in N2 (black ba ) o o ai (clea ba ). The inc ease in TH ac i i y abo e con ol alues in a s exposed o hypoxia was 43 ~ (P < 0.01). Basal TH le els in ch onically sympa hec omized ca o id bodies we e unchanged om con ol alues (4.97 4- 0.49 s 5.25 4- 0.46 nmol/h/mg issue, espec i ely, P > 0.05). Howe e , he inc ease in TH ac i i y seen in no mal animals 48 h ollowing exposu e o l0 ~ 02 was absen in ca o id bodies dep i ed o hei sympa he ic inne a ion (7.57 4- 0.65 s 4.88 4- 0.56 nmol/h/mg issue, espec i ely, Fig. 1). In con as o he esul s ob ained wi h sympa hec omized animals, a ca o id bodies ch onically dea e en ed by emo al o he ca o id sinus ne e exhibi ed an inc ease in bo h basal TH ac i i y and he pe cen ise in TH ollowing hypoxia (Fig. 1, Dea e .). Basal TH ac i i y mo e han doubled in he dea e en ed ca o id bodies (10.64 4- 1.04 nmol/h/mg dea e en ed ca o id body, P < 0.01) compa ed wi h he no mal con ols. Fo y-eigh hou s ollowing exposu e o hypoxia, a s wi h dea e - en ed ca o id bodies exhibi ed a 71 ~ inc ease in TH ac i i y abo e he al eady ele a ed basal le el ( o 18.21 4- 1.67 nmol/h/mg/ issue, P < 0.01). Thus, hypoxia p oduced a g ea e inc ease in TH ac i i y in dea e en ed ca o id bodies han in no mally inne a ed o gans (71 ~ s 43 ~, espec i ely, P < 0.01). These esul s demons a e ha he ca o id sinus ne e and he sympa he ic inne a ion o he ca o id body exe a ecip ocal con ol o e long- e m changes in TH ac i i y which esul om al e a ions in a e ial oxygen ensions. Thus, loss o TH induc ion by hypoxia ollows ch onic sympa hec omy o he ca o id body, and enhan- cemen o he TH esponse occu s wi h dea e en a ion o he o gan. Ou esul s di e om hose epo ed by Hanbaue e alP, who ound no change in basal TH ac i i y in a ca o id body one week a e ansec ion o he ca o id sinus ne e. Also, hey obse ed ha he induc ion o TH ac i i y ollowing hypoxia was abolished ollowing 575 dea e en a ion o he ca o id body, a he han a e sympa hec omy, as we epo he e. Howe e , in hei expe imen s a s we e subjec ed o one hou o se e e hypoxia (5 ~ Oz), which may p oduce a di e en p o ile o au onomic adjus men han exposu e o 3 h o mode a e hypoxia (10 ~ 02). Fu he mo e, Hanbaue and cowo - ke s employed a di e en me hod o TH assay han we used, and epo ed much lowe enzyme ac i i ies han we ound. The appa en disc epancies be ween ou da a and hose o Hanbaue e alp ha e been discussed elsewhe e 7. Loss o TH induc ion by hypoxia ollowing sympa hec omy o he ca o id body sugges s ha he sympa he ic ne ous sys em may play a key ole in e lexly media ing TH induc ion in his o gan. This in e p e a ion i s consis en wi h ou p e ious inding ha TH ac i i y in a supe io ce ical ganglion, like ha in he ca o id body, is also inc eased ollowing hypoxia 7. Fu he mo e, ou p esen da a sugges ha basal TH le els in he ca o id body appea o be onically egula ed, a leas in pa , by he ca o id sinus ne e. This la e phenomenon migh occu by means o he ecip ocal synap ic o ganiza ion which exis s be ween a e en ne e e minals and Type I cells 18. Howe e , a ecen s udy by Vazquez-Nin e al.Z3 shows ha ch onic dea e en a ion o he ca ca o id body esul s in sp ou ing and p oli e a ion o sympa he ic ne e e minals on Type I cells. I such we e also he case in a ca o id body, he inc eased TH ac i i y ollowing dea e en a ion migh eside in he newly- o med ne e e - minals. Howe e , sympa hec omy alone does no signi ican ly educe ca o id body TH (Fig. 1), and he ac i i y o he enzyme doubles ollowing dea e en a ion o he o gan. Consequen ly, i is imp obable ha newly o med sympa he ic e minals could con ain a o al TH ac i i y equi alen o ha p esen in he no mal o gan. Thus, i is easonable o conside ha he ca o id sinus ne e i sel is esponsible o he obse ed onic supp ession o TH ac i i y in he ca o id body. Inc ease in ca o id body TH ollowing dea e en a ion ag ees wi h he ecen inding by Hanbaue and Hells om 1° ha ca o id body no epineph ine con en was inc eased by 70 ~ a 5-7 days ollowing sec ion o he ca o id sinus ne e. Fu he mo e, hese in es iga o s also showed in ano he epo ha eco e y o no mal ca o id body dopamine s o es a e hyp0xia- induced deple ion o his ca echolamine occu ed mo e apidly in ch onically dea e - en ed ca o id bodies han in no mally inne a ed o gans 1~. In his ega d, i is in e es ing o no e ha ch onically dea e en ed ca ca o id bodies show an inc ease in he concen a ion o dense-co ed esicles in Type I cells, as well as p oli e a ion o eac i e Type I cells con aining la ge dense g anules~L This wo k was suppo ed by Public Heal h Se ice G an s NS 12636, NS 07938 and DA 00869. 1 Chiocchio, S. R., Bisca di, A. M. and T amezzani, J. H., Ca echolamines in he ca o id body o he ca , Na u e (Lond.), 212 (1966) 834-835. 2 Dea naley, D. P., Fillenz, M. and Woods, R. I., The iden i ica ion o dopamine in he abbi 's ca o id body, P oc. i y. Soc. B, 170 (1968) 195-203. 3 De Cas o, F., Su la s uc u e e l'inne a ion du sinus ca o idien de l'homme e des mammi e es. Nou eaux ai s su l'inne a ion e la onc ion du glomus ca o icum. E udes ana omiques e physiologiques, T ab. Lab. ln es , biol. Uni . Mad ., 25 (1928) 331-380. 576 4 De Kock, L. L., In aglome ula issues o he ca o id body, Ae a ana . (Basel), 21 (1954) 101-116. 5 Fillenz, M., The unc ion o he Type I cell o he ca o id body. In M. J. Pu es (Ed.), The Pe iph- e al A e ial Chemo ecep o s, Camb idge Uni e si y P ess, 1975, pp. 133-142. 6 Gonzalez, C. and Fidone, S., Inc eased elease o [ZH]dopamine du ing low 02 s imula ion o abbi ca o id body in i o, Neu osci. Le ., 6 (1977) 95-99. 7 Gonzalez, C., Kwok, Y., Gibb, J. and Fidone, S. J., E ec s o hypoxia on y osine hyd oxylase ac i i y in a ca o id body, J. Neu ochem., in p ess. 8 Guido i, A., Zi ko ic, B., P ei e , R. and Cos a, E., In ol emen o 3',5'-cyclic adenosine monophospha e in he inc ease o y osine hyd oxylase ac i i y elici ed by cold exposu e, Naunyn-Schmiedebe g's A ch. exp. Pa h. Pha m., 278 (1973) 195-206. 9 Hanbaue , I., Lo enbe g, W. and Cos a, E., Induc ion o y osine 3-mono-oxygenase in ca o id body o a s exposed o hypoxic condi ions, Neu opha macology, 16 (1977) 277-282. 10 Hanbaue , I. and Hells om, S., The egula ion o dopamine and no ad enaline in he ca ca o id body and i s modi ica ion by dene a ion and by hypoxia, J. Physiol. (Lond.), 282 (1978) 21-34. 11 Hells om, S. and Koslow, S. H., Biogenic amines in ca o id body o adul and in an a s -- a gas ch oma og aphic-mass spec ome ic assay, Ac a physiol, scand., 93 (1975) 540-547. 12 Hells om, S., Hanbaue , 1. and Cos a, E., Selec i e dec ease o dopamine con en in a ca o id body du ing exposu e o hypoxic condi ions, B ain Resea ch, 118 (1976) 352-355. 13 Heymans, C., Bouchae , J. J. and Dau ebande, L., Sinus ca o idien e e lexes espi a oi es. II. In luences espi a oi es e lexes de l'acidose, de l'alcalose, de l'anhyd ide ca bonique, de l'ion hyd ogene e de l'anoxemie. Sinus ca o idiens e echanges espi a oi es dans les poumons e au dela des poumons, A ch. in . Pha macodyn., 39 (1930) 400-448. 14 K e nansky, R., Gewi z, G. P., Weise, V. K. and Kopin, 1. J., Ca echolamine syn hesizing enzymes in he a ad enal gland du ing exposu e o cold, Ame . J. Physiol., 220 (1971) 928-931. 15 Le e , J. D. and Boyd, J. D., Osmiophile g anules in he glomus cells o he abbi ca o id body, Na u e (Lond.), 179 (1957) 1082-1083. 16 Llados, F. and Zapa a, P., E ec s o dopamine analogues and an agonis s on ca o id body chemosenso s in si u, J. Physiol. (Lond.), 274 (1978) 487-499. 17 Llados, F. and Zapa a, P., E ec s o ad enocep o s imula ing and blocking agen s on ca o id body chemosenso y inhibi ion, J. Physiol. (Lond.), 274 (1978) 501-509. 18 McDonald, D. and Mi chell, R. A., The inne a ion o glomus cells, ganglion cells and blood essels in he a ca o id body: a quan i a i e ul as uc u al analysis, J. Neu ocy ol., 4 (1975) 177-230. 19 Naga su, T., Le i , M. and Uden iend, S., A apid and simple adicassay o y osine hyd o- xylase ac i i y, Analy . Biochem., 9 (1964) 122-126. 20 Sampson, S. R. and Vid uk, E. H., Hype pola izing e ec s o dopamine on chemo ecep o ne e endings om ca and abbi ca o id bodies in i o, J. Physiol. (Land.), 268 (1977) 211-221. 21 Thoenen H., Muelle , R. A. and Axel od, J., Inc eased y osine hyd oxylase ac i i y a e d ug- induced al e a ion o sympa he ic ansmission, Na u e (Land.), 221 (1969) 1264. 22 Thoenen, H., Muelle , R. A. and Axel od, J., T ans-synap ic induc ion o ad enal y osine hyd o- xylase, J. Pha macol. exp. The ., 169 (1969) 249-254. 23 Vazquez-Nin, G. H., Cos e o, I., Eche e ia, O. M., Aguila , R. and Ba oso-Moguel, R., Inne - a ion o he ca o id body. An expe imen al quan i a i e s udy, Ac a ana . (Basel), 102 (1978) 12-28. 24 Zapa a, P., E ec s o dopamine on ca o id chemo- and ba o ecep o s in i o, J. Physiol. (Lond.), 244 (1975) 235-251.