PROSTAGLANDINS
Prcetaglandins
and Rain
Release:
I. SthbtionofReninReleaseFmnRa&itRenal CorticalslicesbyPG12 A. R. hhortm, K. Misono, J. Hollifield,J. C. Fro&h, T. Inagami and J. A. Oates DeparbmtsofPhamacolo9y,BiochemistxyandMedicine VanderbiltUniversitySchcclofMedicine Nashville,Tennessee 37232
Prcstaglandinshavebeenshcwntibeinvolvedinthe ~snofreninsecretianinavarietycdsi~ati~. Both arachidcmicacid~prostaghdinem@mmxidehavebeenshcm toreleasereninfrancorticalslicesandtobe convertedto PG12bycorticalmiC~. InthepresentstudiesEGI was famdtocauseatimdependentincrease inrenirlreleas2 fraTl rabbitrenalcarticalslices, a systmisulated frananyindirect effectsthatresultfrunthe adninistrationofproetaglandins invivo. Thestirmlaticmwaslinearupto 3Oxninutes andeff&~%arangeof concentraticmsfmn10-7 Mto 10-5 M. At similar concentrations6-keto-prostagl.andinF was not active on these slices. Thus, it is proposed +&a&' PGI exerts adirecteffecton the release of renin franaxtical. GelL andmaybe themsdiatorof arachidonateorprcstaglandin er&peroxidestimlatedreninsecreticm.
The authorswish to thankGlendaRye for technical assistancewith the rmin assay. Prcetacyclin,6-keto-F anl PC8 were kindly suppliedbyDr.J.PikeofTheUpjohnC& This wori4was supportedby NIH grants HL-14192 and @KL5431. Dr. oates is the Joe and Morris Wrthan Professorof InvestigativeM&him.
DECEMBER
1977 VOL. 14 NO. 6
1095
PROSTAGLANDINS
?zntroduction
Evidence for the participationof the renal prostaglandin systeminregulation of renin releasewas provided& Larssonet al -? (1) whodemnstrated thatarachidcmic acid infused innon-hypotensive doses near the renal arteriesof the rabbit caused an increasein plasma renin activity (PRA). Intheseexperimntsinhibitionof prostaglandinsynthesisby indamathacinpreventedthe rise inrenin asscciatedwitharachidona~ infusion. W,theinhibitionby indcmathacin,of elevatedPRA obsemed infumsemideinduceddiuresis in man (2) in rabbits (3) and in patientswith &utter's syndrans (4) suggeststhe iqortauceofprostaglaudins in the control of renin secretion. The directeffectofprostaglaudins on rehinreleasehas also beend~~tratedininvitro~imentsusingrahbitrendlcorti-cal slices (5). In these studies arachidonicacid stimlated renin releasewhichcouldbeblockedby indmthacin. Aprostaglaudin 'crosamasaud amjorproductof arachidonicacid in several tissues (6-9). Based on these findings, we investigated the fateof arachidonicacid inrabbitrenal cortex aud found that prostacyclin(PG12)is formed in substantialmuhts (10). Thus, we have studied the effect of PG12 on renin release both invivo in thedog (11) aud ihvitrousiugslices franthe rabbiTGiZcortex. Intheseex$GGiZiiEs releaseofrenin fran renal cortical sliceswas investigatedtodetermine thedirecteffect of PG12 on reuin release free franother influenceswhichare operative-invivo. Materials P-rostacyclin as the scdiumsaltwas suppliedby The DpjohucO. Stable solutions (1 ng/ml) ware preparedby dissolvingthe salt in 9:l ethanol-O.05M tris buffer pH 9.0 aud stored at -700 C. Dilutions were made into ice cold 0.5 M tris buffer pH 9.0 and used inmediately. PGE2 and 6-keto-F were dissolvedin 1:9 ethanol0.05 M tris buffer pH 7.4. Ara&idonic acid franNuCheck Prep dilutedtoa was neutralizedwith a one m1a.r excess of Na CO final concentrationof15 rq/mlwith O.lMso&u~%'phosphate buffer pH 7.4, ccmplexedwith fatty acid free albumin (3 moles of arachidonate/mle of albumin)and used inmediately (all steps were perconvertedtothe fonmadunderN2 atrrpsphere).Indamethacinwas sodium salt prior to use by neutralizationwith a one molar excess and diluting to a final concentrationof 1.78 q/ml with of Na CO 0.1 M2so&um phosphatebuffer pH 7.4. Isoproterenol(1 - isoprotermolbitartrate) was obtained franSigm aud L- prOpranOlOl fran Ayerst ResearchLaboratories.
1096
DECEMBER 1977 VOL. 14 NO. 6
PROSTAGLANDINS Kidney slices
NewZealandmalerakbitsw@hing2to3kgwrefedastandarddietbeforekilLingbycervicaldislocati~.Thekidneys wrequicklyremmed, deca~ulat&,washedinKrebbs-Ringerbicxrbonate(KRB)bufferpJ-I 7.4 amtajninglg/literof glucose and slicedusingaStaddie-Riggsmicrotcm.c.orticalslices (approxinmtely 0.5 mnthick) m preparedfmneach flat surface ofthe~~beingcautiousnottoincludeanyoftherendl medulla. Foreache.xperimntslicesfranonetotwoanimalswere mixed,washedwithbufferandrandcmlydistributed into flasks containing 5ml of KRBbuffer. JncubationJ?mcedure Incubations wzre &me at 25'C underan atmsphereof 95% 0-5%(X inashakingincubatmr. AfteralOminutepreincubation p&d, hbufferwas rapidlyaspiratedardreplaazdwith fresh buffer before startingthetestperid. Afteranexperimntthe tissuefraneach flaskwas blotteddry and weighed(200-400 IKJof tissue/flask). Aliqmts for the reninassay (250~1) were mmved, centrifuged and storedat -700untilassayed. Appropriate correctianswremde for changes inrenincmcentrationresulting franremvingaliquotsinthetimcowseexperhrents. PeninAssay Afteranappropriatedilutimof the aliquotfranthe slice incubaticnmedium, 20 1~1mreused forangiotensin I (A 1 generation. The A generatimassaymediumwas canposedof 240 ~1 of 0.12M made - 8 nW EDNA bufferpH 5.0 containing 0.25n mles of Al equivalents of purifiedhcg reninsubstrate(MilesLaboratories,Inc.). In addition 5 ~1 of 0.27M diisop~lfluorophos&ate (inisopropanol) was acWedto inhibitangiotensinase activity. The sampleswere irumbatedfor 60 minutesat 37O C afterwhich theywF?retransferedtoanicebathanddilutedwithO.lMtris acetatehffer pH 7.4. Aliquots according to Haber-et al (12)using NewEnglandNuclea~~Corporation. boundAlusingagamnacounter, thequantityofA generatedwas derivedfrcxnastandadcurveandusedtocalcula&e reninactivity inthe sliceimubationnrzdim. Valuesare reportedasngA1 produced/min& slice. Inorder tovalidatethe useof the renalslice systemfor investigatingreninrelease,~_~~theres~of thy slicesto isopro~l (4 x 10 M) and propranolol(1.9x 10 M) (13)as a standardstimulusagainstwhichtocmparethe effect
DECEMBER
1977 VOL. 14 NO. 6
1097
PROSTAGLANDINS
of PGI2. Figurelshows the time course of stianrlationofrenin abovecontrolandtheeffectiveblockade releasebyisoproterenol by the antagonist, propranolol. The sttiation of reninreleaseby .B- agonistsis well -ted and has previously been demonstrated in slicesystems (13,141.As seen,the responsetoisoproterenolisapproximately linearup to 60 minutesof incubation at whichth the release of reninis apphtely 200 - 400%of the controlvalues. There is alsoa non-specific releaseof reninfrcrn controlslicesthat continued throughout the experirfent. In each of the following experinentsisoproterenol (4 x 10W6M)was includedas a positive control. The responsiveness of thisrenalslicesystemto prostaglandin mediatedreleaseof reninwas denonstrated by the arachidonic acid stimulated releaseof reninto approximately 250% (Fig.2) of amtrolvaluesduring6Ominutesincubation, a findingconsistentwith thatof Weberet al (5). The dependence of this effectupon a cyclooxygenasederivedn&aholite of arachidonatewas confirsedby its inhibition by indanethacin.Thus in the followingexparinwts, ws investigated the roleof FG12 in the reninsystem. ReSliLt.9 In these expertits FG12 (5x lo-6M)stimulated reninrelease fromcorticalslicesto 176 + 30% of control(t SD, n=9) after 30 minutesof incubation.A timedependentstimulation of renin releaseby PC1 (5 x 10D6M)is shownin Fig. 3. Althoughit has been reprted &atPG12is destroyedin 2OminutesatpH 7.5 and 220 C (71,the effectwas linearup to 30 minutesof incubation and was as effective as 4 x 10m6Misoproterenol. In theseexperixnents the reqonse to PGI was 206 _+19% at 5 x lO"6Mand 173 f 8% at 5 x 10S7M. similar¢ration of PGE (2.~0-7~, 2.&r10-6,2.~~ 10B5M)producedcnly a minimalchangein &in releasewhichwas not significantly differentfrcan control(90+ ll%, 115 f 21%, 107 + 20%, respectively).
FG12was effectiveover the rangeof 10q7to 10B5rrolar(Fig. 4). The responseto thesedosesreflectsthe net effectresulting franboth nonspecific breakdownof FG12 in the incubation aedium, nWebol.ismbythe slicesanddistribution to the celltypeonwhich that the aqueousbreakdcwn it exertsitsaction. The possibility (6-keto-prostaglandin Fla),mightbe prcductofFGI 6-keto-FGFl, responsible fos'thereninreleaseobserved PGI was excludedby l-T at 3.4 x 10' M ar& 6.8 x lo-6Mdid desonstrating that 6-keto-PGFlcl notsignificantlychange reninreleasefromcontrollevels(102f 3% and 94 f 7% of CXmtrolrespectively).
1098
DECEMBER
1977 VOL. 14 NO. 6
PROSTAGLANDINS
REVIN SECRETION FROM RABBIT RENAL CORTICAL SLICES IN RESPONSE TO ISOPROTERENOL
*Or 70-
TIME (min)
Figure
1
Slices (200-300~&flask) were incubatedwith 4 x 10% isoproterenol (0) , 19 x 10'6M propranolol(A) or isop~l plus Each pmpranolol (0). Controlflasks (A) amtaimdonlybuffer. pointrepresentsman+sEMof five incubations.
DECEMBER
1977 VOL. 14 NO. 6
I099
PROSTAGLANDINS
THE EFFECT OF ARACHIDONIC INDOMETHACIN ON RENIN
ACID AND REtEASE
Isoproterenol
Arochidonote Arochidonote Indomethacin Indomethocin
Figure
2
incubated with the albumin amplex of sodium arachi(2.5 x 10’3M) , with isop~terenol (4 x lO'%II , with indomthacin (5 x 10’5M) or with inda~thacin (5 x 10-k) plus aradidonate (2.5 x lo-3MI . T.rdaWthacinwas~lOminutes pltiortoadditionofarabidcma~. DataiSpreserbdaSIEan+ SEM-(Ih3) of the percent increaseinreleaseabovethecontrol value after 60 minutes of inckaticx~. slices
were
donate
1100
DECEMBER 1977VOL.14N0.6
PROSTAGLANDINS
RENIN
RELEASE
IN RESPONSE
TO PG12
PO12
CONTROL
I
I
I
I
I
20’
IO'
1
I
I
30’
TIME OF INCUBATION (min)
Figure 3 responseto XI2 (5 x lo'% as a fumtion of RZIliJlld~in b-3) ofthepercent time. Eadlpointrepresents theman+sEM increaseinreninreleaseabovetheCoEtrolvdlueat~time.
DECEMBER 1977 VOL. 14 NO. 6
1101
PROSTAGLANDINS
DOSE
I
10-a
RESPONSE
1
IO-7
I
IO-6
TO PGIz
I
IO-5
I
J
IO-4
PGIz (Ml
Figure 4 The dose responsecurvewas obtainedby incubatingFGI2at the concentratims indicatedfor 30 minutes. Dataispresentedas msan+range (n=3)ofpxcentincreaseabovecontmlafter 30 minu&ofincubatim.
1102
DECEMBER
1977 VOL. 14 NO. 6
PROSTAGLANDINS
Discussion In these studies,PGI2was shmnto increase the releaseof renin franrabbitreMlccarticalslices,asystemisolatedfram indirectinfluencessuchasintrarendLherodyMmiceffectsat themaculadensa,rmalsynpa~eticnervms amtrolandnultiple lxmxxalagents. Arachidonicacid,whi&is active instirmlatingrminsecr_etion fran the renal cortex (5), is comerted bytheprostaglandin cyclaxygemse to a metabolite,PGG2 (16),that is also effective in releasingrenin (5). %!hmerecentlyfcundthatarxhidonate furthermetabolizedtoFG1 andPGGare PGEandpGF byrenal corticd micrcsamesfran the r&it (lo,?:0_$ data an&hat of others (5) suggests thatPGE2is notactivewhilePGF hasbeen shmn to inhibit renin release (5,151. The effect of2?hrcmboxaneA2 or B2is notkrmn in this systmvhowever, insignificant anrxlntsof~~B2aref~franPGG2orarachidcolate byrenalcorticalmicros~s (17). Thus, the formatianof FGI2 intherenal cortex andthe stimlationofreninreleasebythx prostaglandinsuggestthatPGI2is themtabolite of arachidcmic acidwhichmdiates the secret~onof rmin.
References 1. Larsson,C., Weber, P., ad Angga?zd, E., Eur. J. Pharm. 2, 391-394 (1974). 2. F&lich, J. G., Hollifield,J. W. Do-is, J. C., -lick, B. L., seyberth, H., Midelakis, A. M. and Oates, J. A., Circ. F&es.2, 447-452 (1976). 3. Oliw, E., Kover, G., Larsson,C., and Anggard, E., Eur. J. Pharm. 2, 95-100 (1976). 4. Gill, J. R., Frglich, J. C., Bawden, R. E., Taylor, A. A., Keiser, H. R., Seyberth, H. W., Oates, J. A., amI Bartter, F. C., Am. J. Med. 61, 43-51 (1976). 5. Weber, P. C., Larsson, C., Anggard,E., Hanberg,M., Corey, E. J., Nicolaori,K. C., and Samuelsson,B., Cir. Res. 2, 868-874 (1976). 6. Mmcada, S., Gryglewski,R., Bunding, S., ard Vane, J. R., Nature 263, 663-665 (1976).
DECEMBER
1977 VOL. 14 NO. 6
1103
PROSTAGLANDINS
7.
Johnson, R. A., Mxton, D. R., Kinner, J. H., Corman, R. R., MXui.se, J. C., Sun, F. F., attaker, N., Bunting, S., Salntm;J., ticada; S., a&Vane, J. R., Prostagiandins~, 915-928 (1976).
and 8. Isakson,P. C., Amixam, R., Denny, S. Acad. Sci. ~,~'l~i~los Needleman,P., Proc.Natl. (1977). 9. Pace-Asciak,C. R., and Rangaraj,G., Biochim.BiC&S. -486, 579-582 (1977).
Acta
M., Oates, J. A ., and Frijli&, J. C. 10. Horton, A. R., Sanigel, Proetaglandins13, 1021 (1977). J. A., Br&, R. A., Nies, A. S., Gerkins,J. F., 11. Gerber, Hollifield,J., and Oates, J. A., Submitted. 12. Har+=r, E., Kaerner,T., Page, L. B., Klimm, B., and pwnode, A., J. Clin. Endocrinol.Metab. 29, 1349-1355 (1969). 13. Misono, K., Horton, R., Inagami,T., and Hollifield,J. w. Ehdocrinol.Sot. 58th Annual Meetings (abstr.)229, (1976). 14. weinberger, M. H., PDi, U., an3 Henry, D. P., Circ. l&s. 2, 318-324 (1975). 15. t&d, M. E., at-dMichelakis,A. M., J%anmcologist I_&,198 (1974). 16. m,M. and Samwls%n,B., USA, 70, 899-903 (1973).
Proc. Natl. Acad.
Sci.
17. Nhorton, A. R., Smigel,M., Frijlich, J. C., and Cates, J.A., tobesubmitted. Received
1104
10/l/77 - Approved
11/15/77
DECEMBER
1977 VOL. 14 NO. 6
Prcetaglandins
and Rain
Release:
I. SthbtionofReninReleaseFmnRa&itRenal CorticalslicesbyPG12 A. R. hhortm, K. Misono, J. Hollifield,J. C. Fro&h, T. Inagami and J. A. Oates DeparbmtsofPhamacolo9y,BiochemistxyandMedicine VanderbiltUniversitySchcclofMedicine Nashville,Tennessee 37232
Prcstaglandinshavebeenshcwntibeinvolvedinthe ~snofreninsecretianinavarietycdsi~ati~. Both arachidcmicacid~prostaghdinem@mmxidehavebeenshcm toreleasereninfrancorticalslicesandtobe convertedto PG12bycorticalmiC~. InthepresentstudiesEGI was famdtocauseatimdependentincrease inrenirlreleas2 fraTl rabbitrenalcarticalslices, a systmisulated frananyindirect effectsthatresultfrunthe adninistrationofproetaglandins invivo. Thestirmlaticmwaslinearupto 3Oxninutes andeff&~%arangeof concentraticmsfmn10-7 Mto 10-5 M. At similar concentrations6-keto-prostagl.andinF was not active on these slices. Thus, it is proposed +&a&' PGI exerts adirecteffecton the release of renin franaxtical. GelL andmaybe themsdiatorof arachidonateorprcstaglandin er&peroxidestimlatedreninsecreticm.
The authorswish to thankGlendaRye for technical assistancewith the rmin assay. Prcetacyclin,6-keto-F anl PC8 were kindly suppliedbyDr.J.PikeofTheUpjohnC& This wori4was supportedby NIH grants HL-14192 and @KL5431. Dr. oates is the Joe and Morris Wrthan Professorof InvestigativeM&him.
DECEMBER
1977 VOL. 14 NO. 6
1095
PROSTAGLANDINS
?zntroduction
Evidence for the participationof the renal prostaglandin systeminregulation of renin releasewas provided& Larssonet al -? (1) whodemnstrated thatarachidcmic acid infused innon-hypotensive doses near the renal arteriesof the rabbit caused an increasein plasma renin activity (PRA). Intheseexperimntsinhibitionof prostaglandinsynthesisby indamathacinpreventedthe rise inrenin asscciatedwitharachidona~ infusion. W,theinhibitionby indcmathacin,of elevatedPRA obsemed infumsemideinduceddiuresis in man (2) in rabbits (3) and in patientswith &utter's syndrans (4) suggeststhe iqortauceofprostaglaudins in the control of renin secretion. The directeffectofprostaglaudins on rehinreleasehas also beend~~tratedininvitro~imentsusingrahbitrendlcorti-cal slices (5). In these studies arachidonicacid stimlated renin releasewhichcouldbeblockedby indmthacin. Aprostaglaudin 'crosamasaud amjorproductof arachidonicacid in several tissues (6-9). Based on these findings, we investigated the fateof arachidonicacid inrabbitrenal cortex aud found that prostacyclin(PG12)is formed in substantialmuhts (10). Thus, we have studied the effect of PG12 on renin release both invivo in thedog (11) aud ihvitrousiugslices franthe rabbiTGiZcortex. Intheseex$GGiZiiEs releaseofrenin fran renal cortical sliceswas investigatedtodetermine thedirecteffect of PG12 on reuin release free franother influenceswhichare operative-invivo. Materials P-rostacyclin as the scdiumsaltwas suppliedby The DpjohucO. Stable solutions (1 ng/ml) ware preparedby dissolvingthe salt in 9:l ethanol-O.05M tris buffer pH 9.0 aud stored at -700 C. Dilutions were made into ice cold 0.5 M tris buffer pH 9.0 and used inmediately. PGE2 and 6-keto-F were dissolvedin 1:9 ethanol0.05 M tris buffer pH 7.4. Ara&idonic acid franNuCheck Prep dilutedtoa was neutralizedwith a one m1a.r excess of Na CO final concentrationof15 rq/mlwith O.lMso&u~%'phosphate buffer pH 7.4, ccmplexedwith fatty acid free albumin (3 moles of arachidonate/mle of albumin)and used inmediately (all steps were perconvertedtothe fonmadunderN2 atrrpsphere).Indamethacinwas sodium salt prior to use by neutralizationwith a one molar excess and diluting to a final concentrationof 1.78 q/ml with of Na CO 0.1 M2so&um phosphatebuffer pH 7.4. Isoproterenol(1 - isoprotermolbitartrate) was obtained franSigm aud L- prOpranOlOl fran Ayerst ResearchLaboratories.
1096
DECEMBER 1977 VOL. 14 NO. 6
PROSTAGLANDINS Kidney slices
NewZealandmalerakbitsw@hing2to3kgwrefedastandarddietbeforekilLingbycervicaldislocati~.Thekidneys wrequicklyremmed, deca~ulat&,washedinKrebbs-Ringerbicxrbonate(KRB)bufferpJ-I 7.4 amtajninglg/literof glucose and slicedusingaStaddie-Riggsmicrotcm.c.orticalslices (approxinmtely 0.5 mnthick) m preparedfmneach flat surface ofthe~~beingcautiousnottoincludeanyoftherendl medulla. Foreache.xperimntslicesfranonetotwoanimalswere mixed,washedwithbufferandrandcmlydistributed into flasks containing 5ml of KRBbuffer. JncubationJ?mcedure Incubations wzre &me at 25'C underan atmsphereof 95% 0-5%(X inashakingincubatmr. AfteralOminutepreincubation p&d, hbufferwas rapidlyaspiratedardreplaazdwith fresh buffer before startingthetestperid. Afteranexperimntthe tissuefraneach flaskwas blotteddry and weighed(200-400 IKJof tissue/flask). Aliqmts for the reninassay (250~1) were mmved, centrifuged and storedat -700untilassayed. Appropriate correctianswremde for changes inrenincmcentrationresulting franremvingaliquotsinthetimcowseexperhrents. PeninAssay Afteranappropriatedilutimof the aliquotfranthe slice incubaticnmedium, 20 1~1mreused forangiotensin I (A 1 generation. The A generatimassaymediumwas canposedof 240 ~1 of 0.12M made - 8 nW EDNA bufferpH 5.0 containing 0.25n mles of Al equivalents of purifiedhcg reninsubstrate(MilesLaboratories,Inc.). In addition 5 ~1 of 0.27M diisop~lfluorophos&ate (inisopropanol) was acWedto inhibitangiotensinase activity. The sampleswere irumbatedfor 60 minutesat 37O C afterwhich theywF?retransferedtoanicebathanddilutedwithO.lMtris acetatehffer pH 7.4. Aliquots according to Haber-et al (12)using NewEnglandNuclea~~Corporation. boundAlusingagamnacounter, thequantityofA generatedwas derivedfrcxnastandadcurveandusedtocalcula&e reninactivity inthe sliceimubationnrzdim. Valuesare reportedasngA1 produced/min& slice. Inorder tovalidatethe useof the renalslice systemfor investigatingreninrelease,~_~~theres~of thy slicesto isopro~l (4 x 10 M) and propranolol(1.9x 10 M) (13)as a standardstimulusagainstwhichtocmparethe effect
DECEMBER
1977 VOL. 14 NO. 6
1097
PROSTAGLANDINS
of PGI2. Figurelshows the time course of stianrlationofrenin abovecontrolandtheeffectiveblockade releasebyisoproterenol by the antagonist, propranolol. The sttiation of reninreleaseby .B- agonistsis well -ted and has previously been demonstrated in slicesystems (13,141.As seen,the responsetoisoproterenolisapproximately linearup to 60 minutesof incubation at whichth the release of reninis apphtely 200 - 400%of the controlvalues. There is alsoa non-specific releaseof reninfrcrn controlslicesthat continued throughout the experirfent. In each of the following experinentsisoproterenol (4 x 10W6M)was includedas a positive control. The responsiveness of thisrenalslicesystemto prostaglandin mediatedreleaseof reninwas denonstrated by the arachidonic acid stimulated releaseof reninto approximately 250% (Fig.2) of amtrolvaluesduring6Ominutesincubation, a findingconsistentwith thatof Weberet al (5). The dependence of this effectupon a cyclooxygenasederivedn&aholite of arachidonatewas confirsedby its inhibition by indanethacin.Thus in the followingexparinwts, ws investigated the roleof FG12 in the reninsystem. ReSliLt.9 In these expertits FG12 (5x lo-6M)stimulated reninrelease fromcorticalslicesto 176 + 30% of control(t SD, n=9) after 30 minutesof incubation.A timedependentstimulation of renin releaseby PC1 (5 x 10D6M)is shownin Fig. 3. Althoughit has been reprted &atPG12is destroyedin 2OminutesatpH 7.5 and 220 C (71,the effectwas linearup to 30 minutesof incubation and was as effective as 4 x 10m6Misoproterenol. In theseexperixnents the reqonse to PGI was 206 _+19% at 5 x lO"6Mand 173 f 8% at 5 x 10S7M. similar¢ration of PGE (2.~0-7~, 2.&r10-6,2.~~ 10B5M)producedcnly a minimalchangein &in releasewhichwas not significantly differentfrcan control(90+ ll%, 115 f 21%, 107 + 20%, respectively).
FG12was effectiveover the rangeof 10q7to 10B5rrolar(Fig. 4). The responseto thesedosesreflectsthe net effectresulting franboth nonspecific breakdownof FG12 in the incubation aedium, nWebol.ismbythe slicesanddistribution to the celltypeonwhich that the aqueousbreakdcwn it exertsitsaction. The possibility (6-keto-prostaglandin Fla),mightbe prcductofFGI 6-keto-FGFl, responsible fos'thereninreleaseobserved PGI was excludedby l-T at 3.4 x 10' M ar& 6.8 x lo-6Mdid desonstrating that 6-keto-PGFlcl notsignificantlychange reninreleasefromcontrollevels(102f 3% and 94 f 7% of CXmtrolrespectively).
1098
DECEMBER
1977 VOL. 14 NO. 6
PROSTAGLANDINS
REVIN SECRETION FROM RABBIT RENAL CORTICAL SLICES IN RESPONSE TO ISOPROTERENOL
*Or 70-
TIME (min)
Figure
1
Slices (200-300~&flask) were incubatedwith 4 x 10% isoproterenol (0) , 19 x 10'6M propranolol(A) or isop~l plus Each pmpranolol (0). Controlflasks (A) amtaimdonlybuffer. pointrepresentsman+sEMof five incubations.
DECEMBER
1977 VOL. 14 NO. 6
I099
PROSTAGLANDINS
THE EFFECT OF ARACHIDONIC INDOMETHACIN ON RENIN
ACID AND REtEASE
Isoproterenol
Arochidonote Arochidonote Indomethacin Indomethocin
Figure
2
incubated with the albumin amplex of sodium arachi(2.5 x 10’3M) , with isop~terenol (4 x lO'%II , with indomthacin (5 x 10’5M) or with inda~thacin (5 x 10-k) plus aradidonate (2.5 x lo-3MI . T.rdaWthacinwas~lOminutes pltiortoadditionofarabidcma~. DataiSpreserbdaSIEan+ SEM-(Ih3) of the percent increaseinreleaseabovethecontrol value after 60 minutes of inckaticx~. slices
were
donate
1100
DECEMBER 1977VOL.14N0.6
PROSTAGLANDINS
RENIN
RELEASE
IN RESPONSE
TO PG12
PO12
CONTROL
I
I
I
I
I
20’
IO'
1
I
I
30’
TIME OF INCUBATION (min)
Figure 3 responseto XI2 (5 x lo'% as a fumtion of RZIliJlld~in b-3) ofthepercent time. Eadlpointrepresents theman+sEM increaseinreninreleaseabovetheCoEtrolvdlueat~time.
DECEMBER 1977 VOL. 14 NO. 6
1101
PROSTAGLANDINS
DOSE
I
10-a
RESPONSE
1
IO-7
I
IO-6
TO PGIz
I
IO-5
I
J
IO-4
PGIz (Ml
Figure 4 The dose responsecurvewas obtainedby incubatingFGI2at the concentratims indicatedfor 30 minutes. Dataispresentedas msan+range (n=3)ofpxcentincreaseabovecontmlafter 30 minu&ofincubatim.
1102
DECEMBER
1977 VOL. 14 NO. 6
PROSTAGLANDINS
Discussion In these studies,PGI2was shmnto increase the releaseof renin franrabbitreMlccarticalslices,asystemisolatedfram indirectinfluencessuchasintrarendLherodyMmiceffectsat themaculadensa,rmalsynpa~eticnervms amtrolandnultiple lxmxxalagents. Arachidonicacid,whi&is active instirmlatingrminsecr_etion fran the renal cortex (5), is comerted bytheprostaglandin cyclaxygemse to a metabolite,PGG2 (16),that is also effective in releasingrenin (5). %!hmerecentlyfcundthatarxhidonate furthermetabolizedtoFG1 andPGGare PGEandpGF byrenal corticd micrcsamesfran the r&it (lo,?:0_$ data an&hat of others (5) suggests thatPGE2is notactivewhilePGF hasbeen shmn to inhibit renin release (5,151. The effect of2?hrcmboxaneA2 or B2is notkrmn in this systmvhowever, insignificant anrxlntsof~~B2aref~franPGG2orarachidcolate byrenalcorticalmicros~s (17). Thus, the formatianof FGI2 intherenal cortex andthe stimlationofreninreleasebythx prostaglandinsuggestthatPGI2is themtabolite of arachidcmic acidwhichmdiates the secret~onof rmin.
References 1. Larsson,C., Weber, P., ad Angga?zd, E., Eur. J. Pharm. 2, 391-394 (1974). 2. F&lich, J. G., Hollifield,J. W. Do-is, J. C., -lick, B. L., seyberth, H., Midelakis, A. M. and Oates, J. A., Circ. F&es.2, 447-452 (1976). 3. Oliw, E., Kover, G., Larsson,C., and Anggard, E., Eur. J. Pharm. 2, 95-100 (1976). 4. Gill, J. R., Frglich, J. C., Bawden, R. E., Taylor, A. A., Keiser, H. R., Seyberth, H. W., Oates, J. A., amI Bartter, F. C., Am. J. Med. 61, 43-51 (1976). 5. Weber, P. C., Larsson, C., Anggard,E., Hanberg,M., Corey, E. J., Nicolaori,K. C., and Samuelsson,B., Cir. Res. 2, 868-874 (1976). 6. Mmcada, S., Gryglewski,R., Bunding, S., ard Vane, J. R., Nature 263, 663-665 (1976).
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7.
Johnson, R. A., Mxton, D. R., Kinner, J. H., Corman, R. R., MXui.se, J. C., Sun, F. F., attaker, N., Bunting, S., Salntm;J., ticada; S., a&Vane, J. R., Prostagiandins~, 915-928 (1976).
and 8. Isakson,P. C., Amixam, R., Denny, S. Acad. Sci. ~,~'l~i~los Needleman,P., Proc.Natl. (1977). 9. Pace-Asciak,C. R., and Rangaraj,G., Biochim.BiC&S. -486, 579-582 (1977).
Acta
M., Oates, J. A ., and Frijli&, J. C. 10. Horton, A. R., Sanigel, Proetaglandins13, 1021 (1977). J. A., Br&, R. A., Nies, A. S., Gerkins,J. F., 11. Gerber, Hollifield,J., and Oates, J. A., Submitted. 12. Har+=r, E., Kaerner,T., Page, L. B., Klimm, B., and pwnode, A., J. Clin. Endocrinol.Metab. 29, 1349-1355 (1969). 13. Misono, K., Horton, R., Inagami,T., and Hollifield,J. w. Ehdocrinol.Sot. 58th Annual Meetings (abstr.)229, (1976). 14. weinberger, M. H., PDi, U., an3 Henry, D. P., Circ. l&s. 2, 318-324 (1975). 15. t&d, M. E., at-dMichelakis,A. M., J%anmcologist I_&,198 (1974). 16. m,M. and Samwls%n,B., USA, 70, 899-903 (1973).
Proc. Natl. Acad.
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17. Nhorton, A. R., Smigel,M., Frijlich, J. C., and Cates, J.A., tobesubmitted. Received
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