Changes in expression of angiotensin receptor subtypes in the rat aorta during development

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CHANGES IN EXPRESSION OF ANGIOTENSIN RECEPTOR SUBTYPES IN THE RAT AORTA DURING DEVELOPMENT Mohan Viswanathan, Keisuke Tsutsumi, Fernando M.A. Correa, and Juan M. Saavedra Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bldg. 10; Rm. 2D-45, 9000 Rockville Pike, Bethesda, MD 20892 Received

July

29,

1991

Quantitativeautoradiography was usedto characterizeangiotensin AT, andAT, receptors,in the rat aorta at three developmentalages;embryonicday 18 (El8), and postnatalweeks2 and 8. The expressionof angiotensinreceptorswas higher in the aorta of El8 and 2-week-oldrat. A majorproportionof the angiotensinreceptorsexpressedin the aorta at these two ageswas AT, (84 and 81% respectively). Conversely,in the aortaof 8-week-oldrats, AT, wasthe predominant angiotensinreceptorsubtype(71%). In 8-week-oldrats,the AT, subtypewasalsopresent(28%). In pre-andpostnatalrats,[‘251]Sar’-angiotensin II bindingto AT, receptorswassensitiveto GTPySwhereasbindingto AT, receptorswasnot. AT, receptors mayserve an importantroleduringstagesof rapidgrowthof the aorta,andalsohave a significantfunction in the adultvasculature. 0 1991 Academic Press,Inc.

Vasoconstriction,mediatedthroughspecificangiotensin(AT) receptorsin the vascularsmooth muscle,is oneof thewell recognizedphysiological functionsof angiotensin II (ANG)(1). AT receptorshave recentlybeenclassifiedinto two distinctsubtypes,AT, and AT,, basedon their affinitiesfor peptidicand nonpeptidicligandsandalsoby theirdifferentialsensitivities to dithiothreitol(2). VascularAT receptorsare currentlybelievedto be exclusivelyAT,. Rataorticsmoothmusclecellsmaintainedin cultureexpressonly AT, receptors(3,4), andthe AT, antagonist,DuP753, inhibitsANG-inducedcontractionin the rabbitaorta (5,6). We have usedquantitativeautoradiography to studyAT receptorsin the aortaof fetal (El 8) young (2-week-old)and adult(8-week-old)rats. We reportthat bothsubtypesof AT receptorsare presentin the rat aorta,andthatduringdevelopment,thereisa dramaticchangein theexpressionof AT, andAT, receptors in this organ.

MATERIALSAND METHODS Animals Sprague-Dawleyrats from Zivic-Miller(AllistonPark, PA) were usedin the study. Pregnant females,1O-day-oldmalepupsalongwith lactatingfemales,and 7-week-oldmalesweremaintainedin a 12:12h light-darkcycle. Foodandwaterwereprovidedad libitum.Ratfetuseswereremovedfrom 18day pregnantfemalesafter decapitatingthem. Fetuseswere decapitatedandfrozen in isopentaneat -30 “C. Piecesof descending(thoracic)aorta (1 cm long) were taken from 2 and 8-week-oldmaleskilled by decapitation,and werealsofrozen in isopentane.The tissueswere kept frozenat -70 ‘C.

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0006-291X3/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BindinaAssays Crosssections(16 pm) of the fetusat the level of the thorax containingascendingand descending aorta,andcrosssectionsof theaortafrom2 andB-week-old rats(referredto asyoungandadult, respectively)werecut in a cryostatat -17‘C, thawmountedon gelatin-coated glassslidesanddriedovernight in a desiccatorat 4 ‘C. The bindingconditionsusedhavebeendescribed(7,8). TheAT agonist,[‘251]Sar’-ANG (Peninsula, Belmont,CA; iodinatedby NewEnglandNuclear,Wilmington,DE;specificactivity, 2200Ci/mmol)wasused asthe ligand. Tissuesectionswerepreincubated for 15 minin 10 mMphosphatebuffer (pH7.4) containing 120 mM NaCI, 5 mM NqEDTA, 0.005%bacitracin(SigmaChemicalCo., St. Louis,MO), and 0.2% proteinase-free bovineserumalbumin(Sigma),followedby incubationfor 120minin freshbuffercontaining the appropriateconcentrationof the ligand. After incubation,the sectionswerewashed4 times,for 1 min each, in fresh ice-cold50 mM Tris-HCIbuffer (pH7.6), followedby 30 set in distilledwaterat 0 ‘C. To characterizeANGreceptorsubtypes(9),consecutivesectionswereincubatedwith[‘251]Sar’-ANG (5xlV” M) and increasingconcentrationsof ANG, AT, antagonistDuP 753 (2-n-butyl-4-chloro-5hydroxymethyl-1-[2’(1 H-tetrazol-5-yl)biphenyl-4-yl-methyl]imidazole, from DuPont,Wilmington,DE) or AT, competitor PD 123177 [l-(4-amino-3-methyl-phenyl)methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-l Himidazo[4,5-Clpyridine-6-carboxylic acid-2HCI,from ParkeDavis,Ann Arbor, Ml). The possibleregulationof AT receptorsubtypesby guaninenucleotidebindingproteinswasstudied by incubatingconsecutivesectionsin the presenceof increasingconcentrationsof a nonhydrolysable analogueof GTP,guanosine-5’-O-(3-thiotriphosphate) (GTPyS).Theseincubations weredonewhileexposing the sectionsto either DuP753 or PD 123177(lo” M) in orderto differentiatethe effect of GTPyS on the specificreceptorsubtype.Preincubationand incubationbuffersin theseexperimentsconsistedof 50 mM phosphatebuffer (pH7.4)containing120mMNaCI,10mMMgCI,,0.005%bacitracin,and0.2%proteinasefree bovineserumalbumin. QuantitativeAutoradioaraohvSectionsweredriedafterwashingandexposedto Hyperfilm-[3H] (Amersham Corporation,Arlington Heights,IL), along with 16 pm sectionsof [‘251]-labeled Micro-scalestandards (Amersham).[‘251]Sar’-ANG bindingwasquantifiedasdescribedusingcomputerized microdensitometry (10). IC,, valueswerecalculatedwith the GraphPadlnplotprogram(GraphPad,San Diego,CA). RESULTS AutoradiographyrevealedAT bindingin the aortaof the fetal, young,andadultrats. ANG inhibited [‘251]Sar’-ANG bindingin a monophasic mannerin the aortaat allthreeages,with completedisplacement at 1O6M (I&; fetus,1.2 X 10.’ M; young,3.6 X 10.’M; adult,3.2 X 10.’ M). NeitherDuP753 nor PD 123177 wasableto completelyinhibitANGbindingindicatingthe presenceof morethanonesubtypeof the receptor in thistissue(Fig. 1). A majorproportionof the bindingin the aortaof fetus andyoung ratswas displaced 8-Week-old

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F~JJ. Competition of angiotensin II, DuP 753, and PD 123177 for specific [‘251]Sar’-ANG binding in the rat aorta. Competition curves were obtained from consecutive sections of aorta from El 8 fetuses, and 2 and 8-week-old rats, incubated in the presence of 5 X 10” M [‘251]Sar’-ANG and increasing concentrations of angiotensin II (circles) or AT receptor competitors, DuP 753 (squares) and PD 123177 (triangles). Results are expressed as the mean + S.E.M of groups of five rats, measured individually. 1362

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Fiq. Angiotensin receptor subtypes in the rat aorta during development. A: apparent amount of AT receptors during development. AT, specific [‘251]Sar’-ANG binding; AT, and AT,, specific binding not displaced by 1W5M PD 123177 or DuP 753, respectively. B: proportions of AT receptor subtypes in the aorta during development. Values are percentage of specific [‘?]Sar’-ANG binding.

by PD 123177,while that in the adult aorta was displacedby DuP 753. The apparentnumberof AT receptorsin the fetalaortawas6 timeshigherthanthat in youngratsand20 timeshigherthanthat in adult rats(Fig.2A). Theproportionsof thetwo subtypesof AT receptors,whenquantified,revealedthat morethan 60%of the receptorsexpressedin the aortaof the fetusandyoungratsandabout30%inthe adultwereAT, (Fig.28). That the restof the AT bindingwasto AT, receptorswasconfirmedby the completedisplacement of bindingwhenboth DuP753 and PD 123177were usedtogether(Fig.3). While AT, receptorsshoweda uniformdistributionover the entiresonicwall of the fetal (Fig. 4), youngand adultrats,AT, receptorsseemedto be restrictedto the medialayer(Fig.5). Thiswasespecially conspicuous in the aortaof adultrats(Fig.5). The identificationof the celltype(s)expressingthesereceptor subtypesin the aortic wall was not possiblewith quantitativeautoradiographybecauseof the inherent limitationin the resolutionof the technique.

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Fiq. Competition of PD 123177 or DuP 753 for specific [“‘I]Sar’-ANG binding in rat aorta. In the aorta from El8 fetus and 2-week-old rats, AT, receptors were blocked by DuP 753 (10.’ M), whereas in 8week-old rats AT, receptors were blocked by PD 123177 (IO’ M). Results are expressed as the mean f S.E.M. of groups of five rats, measured individually. 1363

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F~J& Autoradiography of AT receptors in the fetal rat aorta. A: transverse section stained with hematoxylin and eosin. B-E: enlarged view (magnification X2) of area marked in A. B: stained with hematoxylin and eosin. C: consecutive section showing total ANG binding after incubation with 5 X 10” M [‘251]Sar’-ANG. D: consecutive section incubated as in C, in the presence of 1U5 M DuP 753. E: consecutive section incubated as in C, in the presence of IO5 M PD 123177. Arrow points to descending aorta, and arrow head to ascending aorta.

Incubationof sectionswithincreasingconcentrations of GTPySdecreasedDuP-sensitive AT binding (AT, receptors)from the fetal, young and adult aorta (IC,,; 3.8, 1.7, and 1.OX 10.’ M respectively). PD 123177-sensitive AT binding(AT, receptors)was not affectedby the guaninenucleotide(Fig. 6). DISCUSSION Characterizationby autoradiography and displacement with selectivecompetitorsrevealedfor the first timethat the aortacontainsbothAT, andAT, receptors,andthat in fetal andyoungrats,mostof the AT receptorsbelongto the AT, type. We alsoreportthat AT, receptorsin the aortaat all agesare insensitive to guaninenucleotides,a findingconsistentwith our observations in someof the otherperipheraltissuesof the rat fetus (11). Aortic AT, receptors, like AT, receptorsin other

tissues(11,12) and AT receptorsin

vascularsmoothmusclecellsin culture(13),aresensitiveto guaninenucleotides, whichindicatesa possible associationto G-proteins(14), andthat thisassociationmayalreadybe presentduringthe embryoniclife. 1364

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Fiq. Autoradiography of AT receptors in young and adult rat aorta. A-E: consecutive sections of aorta from a 2-week-old rat. F-J: consecutive sections from an 8-week-old rat. A and F: sections stained with hematoxylin and eosin. 13and G: Sections showing binding after incubation with 5 x 10 ” M [‘25f]Sar’ANG (total binding). C and H: sectrons incubated as in B and G, in the presence of 1O-5M DuP 753. D and I: sections incubated as In B and G, in the presence of 10 5 M PD 123177. E and J: sectrons incubated as in B and G, in the presence of 5 x 106 M ANG (nonspecific binding). Thin arrow pornts to tunica adventitia, and arrowhead to tunica media. 1365

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F~J& Effectsof GTPy?? on ANGbindingin rataorta. Consecutive sections of El8 fetus and aorta from 2 and B-week-old rats were incubated with 5 X 10” M [‘251]Sar’-ANG in the presence of 1O-5M DuP 753, to block AT, receptors (open circles) or 1O5 PD 123177, to block AT, receptors (closed circles), and increasing concentrations of GTPyS. Results are expressed as the mean f S.E.M. of groups of five rats, measured individually.

The expressionof AT receptorsin the rat aortaismanytimeshigherduringfetal lifethanafter birth, andthe AT receptornumberisseveraltimeshigherin immature,younganimalswhencomparedto adultrats. Our findingsare consistentwith previousdata of a markedactivationof the renin-angiotensin systemin the late gestationfetus (1516) indicatinga rolefor ANG duringgestation.In culturedvascularsmoothmuscle cells,ANG induceshypertrophyand/orhyperplasia(17) and stimulatesthe synthesisof type V collagen, associatedwith cell growthand tissuestructuralremodeling(18). ANG hasbeen suggestedto mediate vascularhypertrophythrougha non-pressor relatedmechanism (19) and to promotevasculargrowth(20). Most of the AT receptorsin the aorta of the fetus and youngrat belongto the AT, type. The functional significanceof AT, receptors,and specifically,the significanceof the presenceof large numbersof AT, receptorsin a rapidlygrowingstructure,hasnotyet beenclarified.However,theirmarkedexpressionin fetal aorta, and in other fetal tissues(11) indicatesthe possibilityof a specializedroleof AT, receptorsduring organdevelopment.

AT, receptorsare responsible for the vascularcontractionproducedby ANG, sinceANG-induced aortic contractionscan be completelyinhibitedby the AT, antagonistDuP 753 and not affectedby AT, competitorPD 123177(5,6),andin hypertensiveratsadministration of DuP753reducesbloodpressure(21). In adult rats,the proliferativeresponseto vascularinjury issuppressed by angiotensinconvertingenzyme inhibition(22),andANGstimulatescollagensynthesis(18). Thesedata indicateparticipationof ANG in the mechansims of vasculartissuerepair. The hypertrophyof vascularsmoothmuscleproducedby ANG is antagonizedby DuP 753 (23) suggestingthe role of AT, receptorsin this phenomenon.Our results demonstrate, for the firsttime,that in additionof AT, receptors,AT, receptorsarealsoexpressedin the aorta of the adult rat. Whetherthe role of ANG in vasculargrowthin adultlife issolelydependenton stimulation of AT, receptors,or dependson the interplayof AT, andAT, receptorstimulation,remainsto be studied. 1366

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