Potentiation by cyclooxygenase inhibitors of the release of catecholamines from the rabbit carotid body and its reversal by prostaglandin E2

Neu oscience Lel e ,*, 140 (1992) 1 4 I ,~: 1992 Else ie Scien i ic Publishe s I eland L d. All igh s ese ed 0304-3940/92/$ 05.00 NSI, 08648 Po en ia ion by cyclooxygenase inhibi o s o he elease o ca echolamines om he abbi ca o id body and i s e e sal by p os aglandin E2 A. G6mez-Ni io, L. Alma az and C. Gonz ilez Depu amen o de Bioquhnica y Biologia Molecula y l~isiologia, Facul ad de Medicina, Uni e si~kM de ValludolM, l idludolid (Spain) (Recei ed 9 Decembe 1991: Re ised e sion ecei ed 10 Feb ua y 1992: Accep ed 25 Feb ua y 1992) Key wo & Ca o id body: A e ial chemo ecep o ; P os aglandin E.; Ace ylsalicylic acid: lndome hacin Salicyla es, a he high he apeu ic doses used in he ea men o heuma oid a h i is, p oduce an inc ease in en ila ion and augmen he ca o id body eac i i y o hypoxic s imulus, leading o an exagge a ed hype en ila ion du ing hypoxia. These e ec s had been ela ed o he ac ion o salicyla es as uncouple s o oxida i e phospho yla ion. In he p esen s udy, ca ied ou in an in i o p epa a ion o he abbi ca o id body, we show ha ace ylsalicylic acid and indome hacin, wo an i-in lamma o y d ugs ha a e also powe ul inhibi o s o cyclooxygenase, he p os aglandin- syn he izing enzyme, p oduce an inc ease in he [3H]ca echolamine elease e oked by low oxygen s imula ion. The d ugs did no a ec basal no moxic elease, a inding ha sugges s ha a he concen a ion used hese an i-in lamma o y agen s do no ha e uncoupling ac ions, and ha hei e ec s on hypoxic-induced elease o [3H]ca echolamines is media ed by hei speci ic ac ion as cyclooxygenase inhibi o s. In ag eemen wi h his sugges ion we ound ha p os aglandin E~ comple ely p e en ed he e ec s o bo h an i-in lamma o y agen s. In addi ion, ou da a indica e ha endogenously syn he ized p os aglandins a e powe ul modula o s o chemo ecep o cell unc ion. Salicyla es a high doses s imula e en ila ion, he e - ec being media ed by pe iphe al and cen al chemo e- cep o s [6], I has been epo ed also ha salicyla es po- en ia e he ca o id body-media ed hype en ila ion p o- duced by hypoxia in man [10]. The mechanism o ac ion o salicyla es a he ca o id body (CB) le el has been e- la ed o hei well known espi a o y uncoupling e ec [3, 61. On he o he hand, i is well known also ha salicyla es a e po en and i e e sible inhibi o s o cyclooxygenase, an enzyme in ol ed in he syn hesis o p os aglandins [4]. Since i has been epo ed [5] ha p os aglandin E2 in i o inhibi s ongoing chemo ecep o discha ges in he ca o id sinus ne e, we ha e es ed he possibili y ha a leas pa o he CB-media ed e ec s o salicyla e on en- ila ion could be p oduced by a speci ic e ec o he an i- in lamma o y d ugs by inhibi ing p os aglandin syn he- sis. Taking in o conside a ion ha all chemos imulan agen s es ed inc ease in pa allel he ac ion po en ial e- quency in he ca o id sinus ne e and he elease o ca - echolamines (CAs) om chemo ecep o cells [1, 7 9], we Co espondence. C. Gonzalez, Depa amen o de Bioquimica y Biologia Molecula y Fisiologia, Facul ad de Medicina, Uni e sidad de Val- ladolid, 4701)5 Valladolid, Spain. Fax: (34) (83) 42 30 85. ha e measu ed he elease o ca echolamines as an index o he CB chemo ecep o s ac i a ion. The expe imen s we e pe o med in CBs isola ed om adul New Zealand abbi s anes he ized wi h sodium pen oba bi al (30-40 mg/kg, Sigma) adminis e ed ia he la e al ein o he ea . A e acheos omy, he bi u ca- ion o he ca o id a e y was dissec ed, emo ed as a block and placed in a Luci e chambe illed wi h ice-cold saline. The CBs we e iden i ied and cleaned o su ound- ing connec i e issues unde a dissec ing scope. The ea - e he ca echolamine deposi s o he CBs we e labeled by incuba ing he o gans wi h he labeled na u al p ecu so [3H] y osine as p e iously desc ibed [11]. A e his load- ing incuba ion, he CBs we e incuba ed in p ecu so - ee solu ion o 2 h (solu ion enewal e e y 30 min), and inally he collec ion o he incuba ing media o analysis in hei [3H]CA con en was s a ed (see Fig. 1 o p o o- col o sample collec ion). The isola ion and quan i ica- ion o [3H]CA in he collec ed media and in he CB a he end o he elease expe imen s was pe o med as p e i- ously desc ibed [11]. The incuba ing solu ion in hese ex- pe imen s was a COjHCO3-bu e ed saline wi h he ol- lowing composi ion (in mM): NaCI 116, KC1 5, CaCI, 2, MgC12 1.1, NaHCO3 24, HEPES 10, glucose 5.5. The solu ions we e equilib a ed wi h a 5% CO2-con aining gas mix u e (see esul s) and adjus ed, i needed, a a pH A 4000 T [] 20~ 0 2 • 5% 02 o 3000 ooo 3 1000 o Con ol 6 o x~ o g l 3: B 4000 - ASA 100/~M 2000 1000 0 2.5 2.0 1.5 0.5 0.0 Expe imen al Con ol Expe imen el Fig. l. E ec s o ace ylsalicylic acid on low oxygen-induced elease o [3H]CA in he abbi CB. Single expe imen . A shows he elease o [3H]CA om a CB (con ol) incuba ed wi h a 20% 02/5% CO2-equili- b a ed solu ion (emp y ba s), and subjec ed o wo pe iods o 10 min o hypoxic s imula ion (incuba ion in a solu ion equilib a ed wi h 5% 02/ 5% CO> illed ba s). B: ano he CB (expe imen al) was simila ly incu- ba ed bu he 10 min pe iod p io o and du ing he second hypoxic s imula ion he incuba ing solu ions con ained 100/IM o ace ylsali- cylic acid (ASA). C shows he e oked elease (cpm abo e he ho izon al lines h oughou he ba s), exp essed as pe cen o [3H]CA issue con- en , o bo h p esen a ions o he s imulus ($1 and $2) and o con ol and expe imen al CBs. D shows he a ios o he e oked elease in he second s imulus o ha in he i s s imulus ($2/S, a ios) o con ol and expe imen al CBs. o 7.40. S a is ical signi icance o he di e ences ob- se ed was assessed by using a - es o unpai ed da a. Fig. 1 shows he elease o [3H]CA om a pai o CBs subjec ed o wo consecu i e hypoxic s imula ions (incu- ba ion o 10 min in a 5% 02/5% CO2 es N2-equili- b a ed solu ion; illed ba s). In he expe imen al CB (Fig. 1B), he 10 min p io o and du ing he second p esen a- ion o he s imulus he solu ion con ained 100 ~ M o ace ylsalicylic acid. I is e iden ha ace ylsalicylic acid po en ia es low oxygen-induced elease o [3H]CA. The e ec o he d ug a his concen a ion on he basal no moxic elease (20% 02/5% CO2-equilib a ed solu- ions) was somewha a iable. In some expe imen s we obse ed a mode a e inc ease in he basal elease while in o he s (like he one in he igu e) he e was no any signi - ican e ec . No e ec on he basal elease was obse ed wi h lowe concen a ions o ace ylsalicylic acid (20/IM), Fig. 1C shows he s imulus-induced elease (abo e ho i- zon al lines in A and B) o con ol and expe imen al CBs and o bo h p esen a ions o he s imuli, S~ and S> espec i ely, exp essed as pe cen o endogenous [3H]CA con en . In Fig. 1D a e ep esen ed he SJS~ a ios o he [3H]CA-induced elease o bo h CBs. This o m o p e- 4 - 3 (/3 O4 U3 2 Con ol ASA (100/zM) Indo (2/zM) Fig. 2. E ec s o ace ylsalicylic acid and indome hacin on low oxygen- induced elease o [3H]CA. The igu e shows means _+ S,E.M. o he $2/S~ a ios ound in con ol, ace ylsalicylic acid (ASA; 100 #M) and indome hacin (Indo; 2 /IM)-incuba ed CBs. P o ocols and hypoxic s imulus in ensi y as in Fig. 1. *P < 0.005. sen ing he da a elimina es indi idual a ia ions in he absolu e amoun s o he e oked elease and allows a be - e dissec ion o he e ec s o he d ug. In his pa icula expe imen ace ylsalicylic acid aised he SJS, a io o 350% o ha seen in he con ol CB, The es o he expe imen s o be p esen ed we e pe o med ollowing his p o ocol. Fig. 2 shows mean $2/S, a ios ob ained in 8 con ol CBs, 4 CBs ea ed wi h 100 ~ M ace ylsalicylic acid and 8 CBs ea ed wi h 2/IM indome hacin, ano he an i-in lamma o y agen and po en inhibi o o cycloox- ygenase. I is e iden ha bo h agen s inc eased ma k- edly he elease o [3H]CA induced by hypoxic s imula- ion (P< 0.005 in bo h cases). A his concen a ion in- dome hacin did no modi y he basal no moxic elease o [3H]CA. A he concen a ions used, nei he ace ylsalicylic acid no indome hacin ha e e ec as uncouple s o oxida i e phospho yla ion [3], bu hey inhibi ma kedly cyclo- oxygenase, he p os aglandin-syn hesizing enzyme [4]. The e o e we es ed o he e e sibili y o he e ec s p o- duced by bo h agen s by he addi ion o p os aglandin E 2. We chose his pa icula p os aglandin because i is known ha i inhibi s he s imulus-induced elease o CA in di e en ca echolamine gic s uc u es and in di e en species including he abbi [12]. Table I shows ha 300 nM o p os aglandin Ez applied simul aneously wi h in- dome hacin comple ely e e sed he e ec o he la e agen . The same e e sibili y was obse ed o he ace yl- salicylic acid e ec (no shown). P os aglandin E z, a hese concen a ions, did no modi y he basal elease o [3H]CA. TABLE I REVERSIBILITY OF INDOMETHACIN EFFECTS BY PROSTAGLANDIN E, P o ocols o he expe imen s as in Fig. 1. P os aglandin E e (PGE~) was added o he incuba ion solu ion o he expe imen al CBs a he same ime as indome hacin. Da a a e means _+ S.E.M. o 5, 6 and 4 alues o con ol, indome hacin and indome hacin plus PE z, espec i ely. Expe imen al S~ S, SjS~ condi ion Con ol 5.(10 + 0.45 4.17 + 0.46 0.84 _+ 0.06 lndome hacin (2 uM) 4.80 + 0.52 10.42 +_ 1.71 * 2.25 _+ 0.44* lndome hacin + PGE 2 (300 nM) 5.28 + 0.36 1.34 + 0.39* 0.24 _+ 0.06* *P < 0.01 in all he cases. The p esen esul s show ha ace ylsalicylic acid and indome hacin po en ia ed low pO2-induced elease o [3H]CA wi hou a ec ing he basal elease. This e ec is comple ely e e sed by simul aneous applica ion o p os aglandin E2, sugges ing ha he ac ions o hese an i-in lamma o y d ugs a e no he esul o hei un- coupling p ope ies, bu p oduced ia inhibi ion o cy- clooxygenase wi h he subsequen dec ease in en- dogenous p os aglandin p oduc ion. Suppo ing his in- e p e a ion, we ha e ound ha a e age p os aglandin E2 eleased o he incuba ing medium om 7 pai s o CBs was 134.5 + 46.9 pmol/mg p o . in basal no moxic condi ions (20% O~/5% CO2), and 198.7 _+ 62.0 in hy- poxic condi ions (7% 02/5% CO2) (P<0.02). In wo addi- ional CBs incuba ed in he p esence o indome hacin, p os aglandin E, elease was hal ed in no moxia and he hypoxia-induced elease was abolished. These da a show, o he i s ime, ha p os aglandins a e modula o s o chemo ecep o cell unc ion and sug- ges ha , a leas in pa , he inhibi o y ac ions o p os a- glandin E, on ca o id sinus ne e ac i i y obse ed by Belmon e and McQueen [5] we e media ed ia chemo e- cep o cells and no solely media ed by changes in he CB blood low. I should be men ioned, howe e , ha in he same a icle hese au ho s epo ed ha , con a y o he in i o si ua ion, in he ascula ly isola ed CB p epa a- ion when he spon aneous ac i i y in he ca o id sinus ne e was low, p os aglandin E~ did no ha e any e ec . This la es inding is in ag eemen wi h he obse a ion on he lack o " e ec o p os aglandin E, on he basal elease in di e en p epa a ions, including ou s, in which his p os aglandin inhibi s he s imulus-induced elease [2, 121. Rega ding he mechanism o ac ion o salicyla es on he p oduc ion o hype en ila ion, i will appea ha he apid hype en ila ion obse ed immedia ely (wi hin 1 min) a e a high dose o he d ug [6] could in ac be due o an uncoupling e ec , bu he long las ing hype eac- i i y o hypoxia [10] can be adequa ely explained on he basis o ou indings. Thus, an inhibi ion o p os a- glandin syn hesis will, on he a i al o a hypoxic s imu- lus, lead o an exagge a ed elease o ca echolamines (and p obably o o he neu o ansmi e s) and a concu - en exagge a ed ac i i y in he ca o id sinus ne e and en ila o y esponse. The mechanism by which p os aglandin E 2 p oduces i s inhibi o y e ec emains o be es ablished. Due o he ac ha i only a ec s he s imulus-induced elease, and ha he e ec can be a leas pa ially abolished by in- c easing he ex acellula concen a ion o Ca > [12], i is emp ing o sugges ha p os aglandins in e e e wi h he s imulus-ac i a ed in lux o Ca > in o chemo ecep o cells. We wan o hank M ~' de los Llanos B a o o ech- nical assis ance. Wo k suppo ed by G an s DGICYT 89/0358 and Jun a de Cas illa y Leon 1101/89. 1 Fidone, S., Gonz;ilez, C. and Yoshizaki, K., E ec s o low oxygen on he elease o dopamine om he abbi ca o id body in i o, J. Physiol., 333 (1982) 93 110. 2 Hedq is , P. and Pe sson, N.-A., P os aglandin ac ion on ad ene - gic and choline gic esponses in he abbi and guinea pig in es ine. In O. Almg en, A. Ca lsson and J. Engel {Eds.), Chemical Tools in Ca echolamine Resea ch, Vol. II, No h-Holland. Ams e dam, 1975, pp. 211-218. 3 lnsel, P.A.. Analgesic an ipy e ics and an i-in lamma o y agen s: d ugs employed in he ea men o heuma oid a h i is and gou . In A. Goodman Gilman, T.W. Rail, A.S. Nies and P. Taylo (Eds.). The Pha macological Basis o The apeu ics, Pe gamon, New Yo k. NY, 1990. pp. 638 681. 4 Kulmacz, R.J. and Lands, W.E.M., Cyclo-oxygenase: measu e- men , pu i ica ion and p ope ies. In C. Benede o, R.G. Mc- Donald-Gibson, S. Nigam and T.F. Sla e (Eds.), P os aglandins and Rela ed Subs ances, IRL P ess, Ox o d, 1987, pp. 209 227. 5 McQueen, D.S. and Belmon e, C., The e ec s o p os aglandins E 2, A, and F2~ on ca o id ba o ecep o s and chemo ecep o s, Qua . J. Exp. Physiol., 59 (1974) 63 71. 6 McQueen, D.S., Ri chie, I.M. and Bi ell, G.J., A e ial chemo e- cep o s in ol emen in salicyla e-induced hype en ila ion in a s, B . J. Pha macol., 98 i1989} 413 424. 70beso, A., Alma az, L, and Gonzalez, C., E ec s o 2-deoxy-D- glucose on in i o ca ca o id body, B ain Res., 371 (1986) 25-36. 80beso, A., Alma az, L. and Gonzalez, C., E ec s o cyanide and uncouple s on chemo ecep o ac i i y and ATP con en o he ca ca o id body, B ain Res., 481 (1989) 250-257. 9 Rigual, R., L6pez-L6pez, J.R. and Gonz ilez, C., Release o dopa- mine and chemo ecep o discha ge induced by ow pH and high pCO: s imula ion o he ca ca o id body, J. Physiol., 433 (1991) 5 9 531. 10 Riley, D.J., Legawiec, B.A., San iago, T.V. and Edelman, N.H., Ven ila o y esponses o hype capnia and hypoxia du ing con inu- ous aspi in inges ion, J. Appl. Physiol., 43 ( 977) 971 976. 11 Roche , A., Obeso, A., He e os, B. and Gonz~ilez, C., Ac iw~ ion o he elease o dopamine in he ca o id body by e a idinc. Ex.i- dence o he p esence o ol age-dependen Na ~ channels in ype 1 cells, Neu osci. Le ., 94 (1988) 274 -278. 12 S j~i ne, L., Role o p os aglandins and cyclic adenosine mono- phospha e in elease. In D.M. Pa on (Ed.), The Release o Ca echol- amines ' om Ad ene gic Neu ons, Pe gamon. Oxl'o d, pp. I I 1 142.