Proc. Natl. Acad. Sci. USAVol. 91, pp. 148-152, January 1994Biochemistry

Role of adrenal renin in the regulation of adrenal steroidogenesisby corticotropin

(renin-angiotensin system/adrenal gland/steroids/P450 enzymes/gene expression)

MAIKE SANDER*t, DETLEV GANTENt, AND SYNTHIA H. MELLON*t*Depament of Obstetrics, Gynecology and Reproductive Sciences and the Metabolic Research Unit, University of California, San Francisco, CA 94143; andtGerman Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, and Max Delbrick Center for MolecularMedicine, Berlin-Buch, Germany

Communicated by Seymour Lieberman, September 13, 1993

ABSTRACT The major regulator of mineralocorticoidproduction in the adrenal is angiotensin H produced by theaction ofrenal renin. The discovery that the rodent adrenal alsosynthesizes renin and angiotensinogen suggests there is auto-crine regulation of mineralocorticoid synthesis. The transgenicrat [TGR(mREN2)27] expresses the Ren-2d gene predomi-nantly in the adrenal. Despite suppressed kidney and plasmarenin, these animals develop fulminant hypertension between 5and 15 weeks of age. Corticosteroid concentrations are signif-icantly elevated during hypertension development. We assessedsteroidogenesis in TGR(mREN2)27 rats by analyzing the ex-pression of the mRNAs for three steroidogenic enzymes:P450scc, the rate-limiting step of steroidogenesis; P450cl1l,which converts deoxycorticosterone to corticosterone in thezona fasciculata/reticularis; and P450c11AS, which convertsdeoxycorticosterone to aldosterone in the zona glomerulosa.P450c11AS mRNA, but neither P450c11g3 nor P450scc mRNA,was overexpressed in the adrenal gland of TGR(mREN2)27rats. In situ hybridization with specific probes for P450cl1pand P45Oc11AS mRNA localized the former exclusively to thezona fasciculata and the latter to the zona glomerulosa. InTGR(mREN2)27 rats, the size of the adrenal and number ofP450cllAS-expressing zona glomerulosa cells were about twicethose of a normal Sprague-Dawley rat. Both animals respondto corticotropin similarly; corticotropin had no effect on theexpression of P450scc and P45011, mRNAs, renderedP450c11AS mRNA undetectable, and simultaneously alteredthe morphology of the adrenal cortex, resulting in a lack ofzonaglomerulosa-like cells. Thus, the local renin-angiotensin systemhas a major effect on the basal expression of P450c11ASmRNA, but little effect on the corticotropin-regulated expres-sion of P450scc, P450c11I, and P450c11AS mRNAs.

Adrenal mineralocorticoid production is mainly regulated bythe renin-angiotensin system (RAS). This system is notconfined to the kidney and is also found in various extrarenaltissues (1-3). A self-contained RAS in the adrenal gland mayregulate aldosterone production (4). The hypertensive ratstrain [TGR(mREN2)27] is transgenic for the murine Ren-2dgene, providing an excellent model to study local tissue RAS(5, 6). Introduction of the transgene led to fulminant hyper-tension with blood pressures to 300 torr (1 torr = 133 Pa)despite low plasma and kidney renin concentrations. Thetransgene is highly overexpressed in extrarenal tissues, es-pecially in the zona glomerulosa and fasciculata of theadrenal cortex (5, 7). Plasma steroid concentrations andurinary steroid excretion in TGR(mREN2)27 rats are stimu-lated during the development of hypertension (8). Plasmaaldosterone levels were elevated 9-fold in homozygousTGR(mREN2)27 rats, indicating a possible role of local

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adrenal renin as a stimulus for steroid synthesis in theseanimals (8).We studied the synthesis ofmRNAs for three hormonally

regulated steroidogenic enzymes in the adrenals ofTGR(m-REN2)27. The synthesis of glucocorticoids and mineralo-corticoids is initiated by conversion of cholesterol to preg-nenolone. This rate-limiting step is mediated by the mito-chondrial cholesterol side-chain cleavage enzyme P450scc,which is regulated by corticotropin (ACTH) and angioten-sin II (All) by separate intracellular pathways (reviewed inrefs. 9 and 10). In addition to this early step, we studied theregulation of the final steps in adrenal steroid biosynthesisleading to formation of corticosterone, 18-hydroxycortico-sterone, and aldosterone, mediated by P450c11. ThecDNAs for the two distinct forms ofP450c11, P450c11f3 (11)and P450c11AS (12), have been cloned. P450c11,8, 11-hydroxylase, is found in the zona fasciculata (13) andconverts 11-deoxycorticosterone (DOC) to corticosteroneand 18-OH DOC (14, 15). P450c11AS, aldosterone syn-thase, is found in the zona glomerulosa (13), has theactivities of P450c11p plus aldosterone synthase activity,and thus is the only enzyme needed to convert DOC toaldosterone (14, 15). P450c11,3 and P450c11AS are inde-pendently regulated in a zone-specific fashion (16, 17).P450c11l,, but not P450c11AS, mRNA abundance wasdiminished by glucocorticoids, whereas low salt and highpotassium diet increased P450c11AS but not P450c11,8mRNA. Since these effects were attenuated by captopril,AII-dependent regulation ofP450c11AS was suggested (17),supporting earlier studies demonstrating stimulation ofaldosterone synthesis by AII (18).We studied the hormonal regulation of adrenal ste-

roidogenesis and the expression and zonal distribution ofthree mitochondrial P450 mRNAs in TGR(mREN2)27. Ourresults indicate that a locally stimulated RAS in the adrenalof TGR(mREN2)27 rats may mediate specific stimulation ofP450c11AS mRNA and may not affect regulation by ACTH.

MATERIALS AND METHODSAnimals. Experiments were performed in male heterozy-

gous transgenic TGR(mREN2)27 rats (5). NormotensiveSprague-Dawley (SD) rats (Zentralinstitut fur Versuchst-ierkunde, Hannover, F.R.G.) were used as controls. Nine-week-old male transgenic (n = 8) and SD rats (n = 8) wereinjected with 0.5 mg of ACTH per kg (Synacthen Depot;Ciba-Geigy) subcutaneously at 10 a.m. for 8 days and also for1 day in SD rats (n = 6). Blood for steroid determinations was

Abbreviations: RAS, renin-angiotensin system; ACTH, corticotropin;DOC, l1-deoxycorticosterone; SD, Sprague-Dawley; All, angioten-sin II.*To whom reprint requests should be addressed at: Department ofObstetrics and Gynecology, Box 0556, University of California, SanFrancisco, CA 94143-0556.

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collected from the aorta under ether anesthesia. Adrenalswere weighed before freezing in liquid nitrogen for RNAisolation or for in situ hybridization. Plasma concentrationsof corticosterone, DOC, 18-OH-corticosterone, and aldoste-rone were determined by RIA after extraction and chroma-tography using specific antibodies as described (8).RNA Probes. The following cDNA fragments were sub-

cloned into pKS (Stratagene) for RNA probe synthesis: A241-bp EcoRI-Pvu II fragment of rP450scc cDNA (19) toprepare a 296-base RNA probe; a 219-bp rat P450cl1pcDNAfragment [bases 213-432 (11)] that distinguishes P450c11f3from P450cllAS (16) to prepare a 280-base P450cll RNAprobe; a 149-bp rat f3actin cDNA fragment (bases 2066-2216of rat cytoplasmic actin cDNA) (20) to prepare a 195-baseRNA probe or a 105-bp rat 3-actin cDNA fragment (bases2066-2171) to prepare a 145-base probe; a 155-bp ratP450c1l13specific cDNA fragment [bases 839-994; (12)] toprepare a 260-base RNA probe; and a 185-bp rat P450c11AS-specific cDNA fragment [bases 809-994 (12)] to prepare a290-base RNA probe. 32p- or 35S-labeled RNA probes weresynthesized by in vitro transcription as described (16).RNA Assays. Total RNA was isolated from tissues and

RNase protection assays were performed as described (21),using 2 ug of adrenal RNA and 5 x 105 cpm of 32P-labeledprobe and 2 x 105 cpm of /actin riboprobe. The RNase-resistant fragments were separated by electrophoresis in 5%or 6% polyacrylamide/7.5 M urea gels followed by autora-diography. In situ hybridization of adrenal tissue was per-formed on 10-,um fixed frozen sections as described (2).

Statistical Analysis. Differences in hormone concentrationsand adrenal weights in untreated rats were determined by ttest for independent variables. Treatment effects and differ-ences in effects between groups were assessed by two-wayanalysis of variance, followed by t test for paired variables.Statistics were done on IBM computer using the CRUNCHstatistic software program.

RESULTSPlasma Steroids and Adrenal Weight. In TGR(mREN2)27

compared with SD rats, plasma aldosterone concentrationswere elevated 14-fold, 18-OH corticosterone was elevated6-fold, and corticosterone was elevated 2-fold (Table 1).Differences in plasma DOC concentrations between the twogroups were not significant. Adrenal weight/body weightindex was about 50% higher in TGR(mREN2)27 rats com-pared to SD rats. After 8 days of ACTH injection, plasmaDOC and corticosterone concentrations increased similarlyin transgenic and SD rats. In SD rats, DOC was significantlyelevated after 1 day of ACTH treatment, whereas plasmacorticosterone concentration was similar to untreated ani-mals. One day after one injection of ACTH, plasma aldoste-rone was significantly higher than in untreated controls. SDrats had the typical biphasic pattern of plasma aldosteroneconcentration following ACTH stimulation. One day afterone injection ofACTH, plasma aldosterone was significantly

higher than in untreated controls. The initial rise in plasmaaldosterone was followed by a drop below control levels after8 days of ACTH. Plasma aldosterone also decreased after 8days of ACTH in the transgenic rats. In contrast to aldoste-rone, 18-OH corticosterone did not decrease after 8 days ofACTH treatment in SD rats. Plasma 18-OH corticosteronewas elevated 3-fold after 1 day and elevated 6-fold after 8 daysof ACTH injection in SD rats and decreased after 8 days ofACTH in transgenic rats, although the difference was notsignificant. Adrenal weight increased in both strains after 8days of ACTH treatment. Adrenal weight in SD rats in-creased within 1 day of ACTH injection.mRNA Expression. To evaluate whether the elevations in

plasma corticosterone, 18-OH corticosterone, and aldoste-rone concentrations in TGR(mREN2)27 rats are secondary toincreased abundance of the mRNAs for the enzymes medi-ating their synthesis, we compared the expression ofP450scc, P450cl11,, and P450cllAS mRNAs in 10-week-oldmale transgenic rats (heterozygous and homozygous) withthat in SD rats by RNase protection assays (Fig. 1). Despitethe elevated plasma corticosterone levels in heterozygoustransgenic rats, there was no difference in expression ofP450scc and P450cl1p mRNAs among the three groups. Incontrast, the abundance of P450cllAS mRNA was increasedin heterozygous and homozygous transgenic rats. The aver-age amount of P450cllAS mRNA was twice as great intransgenic rats compared to SD rats. In heterozygous animalsthe abundance of P450cllAS mRNA significantly differedamong individuals. In all cases, the amount of P450cllASmRNA paralleled the plasma aldosterone concentration, andthe greatest accumulation of P450cllAS mRNA was ob-served in rats with extremely high plasma aldosterone con-centrations (up to 613.2 ng/100 ml). Thus expression ofP450cllAS mRNA, but not ofP450scc and P450c11, mRNA,is specifically stimulated in TGR(mREN2)27 rats.To determine whether the observed changes in plasma

steroid concentrations after ACTH treatment can be attrib-uted to stimulation or suppression of specific mRNA syn-thesis, we measured the expression of P450scc, P450c11,B,and P450cllAS mRNAs (Fig. 2). Despite a 15-fold increasein plasma DOC concentrations in SD rats and a 9-foldincrease in transgenic rats after 8 days of ACTH treatment,the abundance of P450scc and P450c11,8 mRNAs remainedunchanged in both strains. In contrast, the 8 days of ACTHtreatment rendered P450cllAS mRNA undetectable in SDrats, but there was still very low but detectable expression ofP450cllAS mRNA in the transgenic rats after the sameregimen. To determine ifACTH causes a biphasic pattern ofP450cllAS mRNA expression like plasma aldosterone con-centrations, we examined the expression of this mRNA 24 hrafter one injection of ACTH in SD rats. Despite elevatedplasma aldosterone levels, the amount of P450cllAS mRNAwas markedly reduced after one ACTH injection.

In Situ Hybridization. We determined directly how the twodistinct P450cll mRNAs are distributed by in situ hybrid-ization of adrenal sections from TGR(mREN2)27 and SD rats

Table 1. Plasma steroids and adrenal weight after ACTH treatment of TGR(mREN2)27 and SD rats

TGR(mREN2)27 SD

Steroid and adrenal weight Control ACTH (8 days) Control ACTH (1 day) ACTH (8 days)DOC (ng/100 ml) 1352 ± 244 12,281 ± 1914*** 832.4 ± 124.4 4162 ± 1145* 12,238 ± 673.5***Corticosterone (,ug/100 ml) 41.0 ± 3.4tt 56.6 ± 3.3** 23.0 ± 2.8 21.2 ± 3.2 57.2 ± 2.0***18-OH-B (ng/100 ml) 930.0 ± 217.0ttt 523.7 ± 73.2 150.4 ± 25.1 402.9 ± 50.4** 850.3 ± 50.3***Aldosterone (ng/100 ml) 189.0 ± 75.Ottt 34.0 ± 9.4** 13.5 ± 3.8 44.3 ± 4.5** 11.2 ± 1.6Adrenal weight/Bw (mg/100 g) 14.7 ± 1.Ott 47.9 ± 6.5 * 9.6 ± 0.4 13.7 ± 0.9** 56.7 ± 4.1***

18-OH-B, 18-hydroxycorticosterone; Bw, body weight. Values represent mean ± SEM of n = 6 for SD rats treated with ACTH for 1 dayand n = 8 rats for all other groups.*, P < 0.05; **, P < 0.005; ***, P < 0.0005 for comparisons between control and ACTH treatment within each strain of rats.tt, P < 0.005; ttt, P < 0.0005 for comparisons between TGR and SD control rats.

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FIG. 1. RNase protection assay of adrenal RNA from SD andTGR(mREN2)27 rats. RNA (2 Mug) from the adrenals of each animalwas combined with 5 x 105 cpm of 32P-labeled rat P450scc (a), ratP450cll (b), rat P450cllAS (c), and 2 x 105 cpm of rat ,B-actin probe.The lane marked tRNA contains 50 ,ug of tRNA that was treatedidentical to adrenal samples. Markers (in nucleotides) are 32P-labeledSau3A-digested pUC19 DNAs. Probe 1 represents P450scc in a,P450c11,8 in b, and P450cllAS in c. Probe 2 represents rat ,B-actin.SD, SD rats; TGR het, heterozygous TGR(mREN2)27; TGR hom,homozygous TGR(mREN2)27. Nine rats were analyzed for eachgroup and two representative animals from each group are shown.

using specific mRNA probes for P450c11ll and P450cllAS(Fig. 3). As noted in Table 1, the overall size of the adrenalsfrom TGR(mREN2)27 rats was greater than in SD rats. Thezonal distribution of P450scc, P450c11ll, and P450cllASmRNAs was similar in transgenic and SD rats. P450sccmRNA was found throughout the adrenal cortex (Fig. 3 A andB), P450cll, mRNA was only expressed in the zona fascic-ulata (Fig. 3 C and D), and P450cllAS mRNA was foundexclusively in the zona glomerulosa (Fig. 3 E and F). The sizeof the zona glomerulosa from TGR(mREN2)27 rats wasnearly twice that in SD rats (Fig. 3 G and H) and wasaccompanied by an increase in the amount of P450cllAS

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mRNA found there (Fig. 3 E and F). Although the whole zonaglomerulosa increased in size, only the outer half of the zonecontained P450cllAS mRNA (Fig. 3F).

After ACTH treatment, the zonal distribution of thesemRNAs changed dramatically (Fig. 3 I-P). There was noapparent cellular delineation in the cortex, and it appearedthat the cortex was composed of one cell type. We detectedno P450cllAS mRNA nor zona glomerulosa-like cells in theadrenal cortex of ACTH-treated transgenic (Fig. 3 N and P)and SD rats (Fig. 3 M and 0). The signals obtained in thesesections were equivalent to those obtained using a sense

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FIG. 3. In situ hybridization of rat adrenal sections from SD and TGR(mREN2)27 rats. Ten-micron sections were hybridized with 35S-labeledcRNA probes for rat P450scc (A, B, I, and J), rat P450cl1p (C, D, K, and L), and rat P450c11AS (E, F, M, and N). Dark-field photomicrographs(A-F and I-N) show positive signals as white dots on a black background. Sections from untreated SD rats (A, C, E, and G) and untreatedTGR(mREN2)27 rats (B, D, F, and H) were probed for P450scc (A and B), P450c11(3 (C and D), and P450c11AS (E and F); sections from animalstreated with ACTH for 8 days [SD, I, K, M, and 0; TGR(mREN2)27, J, L, N, and P] were probed for P450scc (I and J), P450c11p (K and L),and P450c11AS (M and N). Sections were also stained with hematoxylin/eosin and photographed by light microscopy (G, H, 0, and P) to showtissue architecture. The region of the zona glomerulosa is indicated by "g" and zona fasciculata/reticularis is indicated by "f/r" in G, H, 0,and P; the adrenal capsule and the region below it are indicated by "c" in O and P, and the adrenal medulla is indicated by "m" inP. AU dark-fieldphotomicrographs were taken for the same length of time, and all photomicrographs are at the same magnification. (Bar in A = 200 pm.)

RNA probe (data not shown). P450cl1,8 mRNA was ex-pressed throughout the entire adrenal cortex to the capsule(Fig. 3K, SD rat; Fig. 3L, transgenic rat). There may havebeen a slight decrease in the abundance of P450c11, mRNAin the transgenic rat (Fig. 3 D vs. L) compared to the SD rat(Fig. 3 C vs. K). P450scc mRNA was also expressed through-out the cortex (Fig. 3 I and J) with a slight decrease inabundance in SD (Fig. 3 A vs. I) and TGR(mREN2)27 rats(Fig. 3 B vs. J). Similar results were obtained in SD rats afteronly 24 hr of ACTH treatment. These data indicate thatACTH changes the pattern ofP450cll gene expression oftheouter zone of the adrenal cortex from a glomerulosa-like tofasciculata-like pattern, and these changes are also associ-ated with changed morphology. In addition to these changesin adrenal cortex morphology, ACTH caused a change in thecenter ofthe adrenal gland in TGR(mREN2)27 rats only (Fig.3P). Immunocytochemistry using an anti-tyrosine hydroxy-lase antibody showed a strong signal, demonstrating that thisregion was adrenal medulla (data not shown).

DISCUSSIONThe adrenal RAS causes adrenal hypertrophy and increasesthe relative abundance of the zona glomerulosa, resulting inelevated plasma steroid concentrations but not resulting instimulation of P450scc and P450cll,8 mRNAs. The adrenalsfrom hypertensive transgenic rats and normotensive SD ratswere morphologically similar, but the overall size of theadrenals from the transgenic rat was about 50% greater thanfrom SD rats (Table 1). This increase in adrenal size mayaccount for the increased corticosterone production. The

relative abundances ofthe mRNAs for P450scc and P450cl1,Bdid not change in the transgenic adrenals, and the immuno-cytochemical staining for these proteins was equivalent (percell) in the adrenals of SD and transgenic rats (data notshown). Thus the increased plasma corticosterone in thetransgenic rats is probably due to more adrenal tissue ratherthan to a qualitative change in that tissue. In contrast, therelative abundance of P450cllAS mRNA did increase in thetransgenic rat adrenals, suggesting that the increased plasmaaldosterone in these animals was due to a combination ofincreased adrenal cell number and increased aldosterone-synthetic capacity per cell. These effects may be mediated byAII. AII stimulates P450cllAS mRNA expression, withoutaffecting P450cl1j3 mRNA expression (16, 17). P450cll-specific immunohistochemical studies confirm these data,showing increased amounts of the P450cllAS protein in ratsgiven a low salt diet (13, 22). AII stimulates adrenal cellgrowth as well as augmenting aldosterone synthesis (23). Ourdata demonstrating increased accumulation of P450cllASmRNA, increased adrenal mass, and increased size of theglomerulosa in transgenic rats provide additional evidencefor a locally activated adrenal RAS in TGR(mREN2)27 rats.Two phenomena remained unchanged in SD and transgenic

rats, suggesting that the adrenal RAS does not affect regu-lation of adrenal steroid synthesis by ACTH. Corticosteronelevels increased after short- and long-term ACTH treatment,and ACTH had a biphasic effect on plasma aldosteroneconcentration, initially increasing after 1 day of ACTH anddecreasing after 8 days of treatment ("aldosterone-escapephenomenon") (24). There was no biphasic effect onP450cllAS mRNA abundance in eitherSD or transgenic rats,

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since P450cllAS mRNA expression was suppressed afteronly injection of ACTH. This finding supports previousresults (25), describing a decrease in the abundance ofcapsular P450cll mRNA after ACTH treatment. However,that study employed a manual separation ofglomerulosa fromthe rest of the cortex and did not distinguish betweenP450c11I3 and P450cllAS mRNA.

It appears that the effects ofACTH in vitro and in vivo aredifferent. Despite markedly elevated plasma corticosteronelevels, ACTH did not stimulate P450scc or P450c11f3 mRNAexpression (25, 26), shown in other systems in vitro. Incontrast, ACTH stimulation of cultured adrenal cells in-creases P450scc and P450cll mRNA accumulation in vitro(27, 28). Other differences are seen in the half-life of theproteins; ACTH prolongs the half-life of P450c11 protein invitro (29), whereas we found by immunocytochemistry thatACTH did not affect the amount of P450scc or P450c11/3 invivo (data not shown). ACTH may increase steroidogenesisby other mechanisms-e.g., increasing the availability offreecholesterol (30), facilitating transport of cholesterol intomitochondria (31), and promoting the binding of cholesterolto P450scc (32). All of these mechanisms plus hypertrophy ofthe adrenal gland due to ACTH stimulation may contribute toincreased steroid synthesis.The adrenal RAS increases P450cllAS mRNA expression

and leads to highly increased plasma 18-OH corticosteroneand aldosterone levels. In vitro studies suggested that 18-hydroxylation of corticosterone to 18-OH corticosterone is aunique activity of P450cllAS (14, 15, 33). Our results areinconsistent with this concept, since 8 days of ACTH treat-ment increased plasma 18-OH corticosterone concentrationsand decreased plasma aldosterone concentrations andP450cllAS mRNA abundance. These data therefore suggestthat P450c11p in the zona fasciculata possibly contributes to18-hydroxylation of corticosterone to yield 18-OH cortico-sterone or that there exists an additional P450cll generesponsible for this activity. Earlier studies demonstratingconversion of [14C]corticosterone to 18-OH-[14C] corticoste-rone by homogenates or mitochondria from bovine adrenalfasciculata/reticularis support this notion (34).

In situ hybridization localized P450cllAS mRNA to thezona glomerulosa and P450cllp mRNA to the zona fascic-ulata, consistent with immunocytochemical data (13). SD andtransgenic rats showed cell-specific expression of the twoP450cll mRNAs, but the size of the zona glomerulosa andthe abundance of P450cllAS mRNA in that zone are greaterin the transgenic rat. The inner region of these glomerulosacells adjacent to fasciculata cells does not express eitherP450cllAS or P450c11,3 mRNA. The conversion from fas-ciculata- to glomerulosa-type cells may therefore be at leasta two-step process involving a change in morphology fol-lowed by a change (increase or decrease) in the expression ofat least two steroidogenic mRNAs. ACTH markedly andrapidly changed the zonal distribution ofthe P450c11 mRNAsand the morphology of the adrenal in the transgenic and SDrats. The outer zone (glomerulosa) was undetectable by lightmicroscopy and by in situ hybridization for P450cllASmRNA as early as 24 hr after one ACTH injection. Simul-taneously, P450cll,8 mRNA was detected throughout theentire adrenal cortex. These changes in the enzyme patternafter ACTH are in keeping with the morphological change ofglomerulosa cells to fasciculata-like cells (35, 36) and dem-onstrate further that the local RAS appears to have no effecton the ACTH-induced morphological changes.These experiments thus demonstrate an effect of adrenal

renin on the growth and function of the rat adrenal. It is stillunclear if the effects of adrenal renin are direct or if adrenalAll or some other factor mediates these events.

We are grateful to Prof. P. Vecsei for providing the facilities forsteroid measurements and thank P. Ruf for his skillful technicalassistance. This work was supported by grants from the NationalInstitutes of Health (HD 27970) and from the American HeartAssociation, California Affiliate (Grant-in-Aid, 91-115) and NationalCenter (91015940), to S.H.M.

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