Proc. Natl. Acad. Sci. USAVol. 75, No. 8, pp. 4057-4061, August 1978Physiological Sciences

Regulation of aldosterone secretion by the renin-angiotensin systemduring sodium restriction in rats

(angiotensin II/adrenal receptors/sodium deficiency/steroidogenesis/converting enzyme inhibitor)

G. AGUILERA AND K. J. CATrEndocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health,Bethesda, Maryland 20014

Communicated by Klaus Hofmann, April 24,1978

ABSTRACT The role of angiotensin II as mediator of thealdosterone response to short periods of sodium restriction wasstudied in rats by administration of a converting enzyme in-hibitor to block formation of the octapeptide throughout theduration of decreased sodium intake. In control animals,short-term sodium restriction caused increased levels of adrenalreceptors for angiotensin II, with enhancement of early and latesteps in aldosterone biosynthesis and elevation of plasma al-dosterone concentration. Each of these changes induced bysodium deficiency was abolished during blockade of angio-tensin II formation by continuous infusion of the convertingenzyme inhibitor, SQ 14,225. The absolute dependence ofadrenal glomerulosa cell responses on angiotensin H formationindicates that the renin-angiotensin system is the primary reg-ulator of aldosterone secretion during physiological fluctuationsin sodium intake.

Although the importance of the renin-angiotensin system inthe control of aldosterone secretion is well established (1-3), theprecise role of angiotensin II in mediating the aldosterone re-

sponse to sodium deficiency has been more difficult to define(4, 5). While it is generally agreed that the presence of therenin-angiotensin system is essential for the normal aldosteroneresponse to sodium deficiency, there has been controversy aboutthe nature and extent of the actions of angiotensin II as a

proximate regulator of zona glomerulosa cells (4, 5). Thus,studies in sheep have suggested that factors other than angio-tensin II are involved in the control of aldosterone secretionduring sodium depletion and repletion (4).An important question remains about the physiological role

of angiotensin II during reduced sodium intake, which is themost frequent stimulus for increased aldosterone secretionunder normal conditions in most animal species. Here also adiscrepancy has been noted, between the effect of angiotensinII infusions on aldosterone secretion in normal animals and thelarger aldosterone responses caused by sodium deficiency (6,7). However, the sensitivity of the adrenal to angiotensin II inseveral species increases during sodium restriction (8-12), andthis could contribute to the relatively greater aldosterone se-cretion that accompanies the increased blood concentrationsof angiotensin II during sodium deficiency.The interaction between angiotensin II and altered adrenal

sensitivity during the aldosterone response to sodium restrictionhas been recently analyzed in rats. Initial studies showed thatprolonged changes in electrolyte intake were accompanied byaltered affinity and concentration of angiotensin II receptorsin the rat adrenal (13). In more recent studies during short-termchanges in sodium intake, salt restriction for as little as 36 hrincreased angiotensin II binding and aldosterone responses in

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4057

the zona glomerulosa (14). Thus, in rats, sodium restriction ledto rapid changes in angiotensin II receptors that accounted forthe change in adrenal sensitivity to the octapeptide. Subse-quently, infusion of angiotensin II into conscious rats for 1.5-6days caused an increase in adrenal receptors for angiotensin IIand in the aldosterone responses of glomerulosa cells to theoctapeptide in vitro (15).

These observations suggested that angiotensin II could serveas the major regulator of aldosterone secretion during physio-ogical fluctuations in dietary electrolyte intake, as well asduring diuretic-induced sodium depletion (16). To test thismechanism, we examined the effects upon adrenal functionof blocking angiotensin II formation with an inhibitor of an-giotensin I converting enzyme in rats during acute sodium re-striction. In these experiments, the inhibitor (SQ 14,225, Squibb)was administered by continuous infusion from osmotic mini-pumps throughout the 4-day period of sodium restriction.

MATERIALS AND METHODSAll experiments were performed in 200- to 250-g maleSprague-Dawley rats (Charles River, Inc., MA) maintained onnormal sodium diet for at least 4 days after arrival in the labo-ratory. Three groups of 15-20 animals were then placed for 4additional days on normal sodium intake (0.31-0.37% Na+),low sodium diet (0.06-0.1% Na+), and low sodium diet withsimultaneous infusion of the converting enzyme inhibitor, SQ14,225. The inhibitor was administered by continuous infusionfrom intraperitoneal osmotic minipumps (Alza) at a rate of 1.4Mg/min. In the first of three such experiments, animals onnormal and low sodium intake were implanted with emptysilastic tubing as controls for the minipump-infused group. Inthe latter two experiments, no implants were present in thecontrol animals The effectiveness of the inhibitor in producingblockade of converting enzyme activity was confirmed bymeasurement of the pressor responses to angiotensin I and an-giotensin II in normal and SQ 14,225-infused rats. Blood pres-sures were measured via an intracarotid catheter and transducerin pentobarbital-anesthetized, pentolinium/atropine-treated,nephrectomized rats (17). The infusion of SQ 14,225 completelyblocked the pressor response to angiotensin I, whereas the re-sponse to angiotensin II was not affected (Table 1). No effectsof the converting enzyme inhibitor at concentrations up to 10-4M were observed in the binding assay used to measure angio-tensin II receptors or in the action of angiotensin II on aldo-sterone production in isolated adrenal cells (18). Thus, thepossibility of interactions between the inhibitor and adrenalreceptors during infusion studies could be excluded.

Plasma aldosterone concentrations were measured by ra-dioimmunoassay after extraction and LH-20 chromatography(19). Plasma renin activity and blood angiotensin II concen-

4058 Physiological Sciences: Aguilera and Catt

Table 1. Effect of converting enzyme inhibition by SQ 14,225 onpressor responses to angiotensin I and II

SQ 14,225-Controls infused

Basal 93.3 ± 7.6 95.0 + 4.9Angiotensin II 133.5 ± 11.6 139.0 + 5.1Angiotensin I 133.3 i 11.6 95.0 + 4.9

Values are the mean + SEM of blood pressures (mm Hg) measuredin groups of three animals after intravenous injection of 50 ng of eachpeptide.

trations were measured as described (20, 21). Adrenal capsulartissue was homogenized in 20 mM Tris-HCl, pH 7.4/250 mMsucrose/l mM EDTA/1% bovine serum albumin. The mito-chondrial fraction used for aldosterone biosynthetic studies wasobtained by centrifugation at 6000 g. The supernate was sub-sequently centrifuged at 30,000 X g in order to obtain themembrane-rich fraction used in the angiotensin-binding studies.The conversion of endogenous mitochondrial cholesterol topregnenolone was measured by incubating aliquots of the mi-tochondrial suspension at 370 for 15 min in 1 ml of 3 mMTris-HCI, pH 7.4/20 mM KCI/1 mM EDTA/10 mM sodiummalate/10 mM sodium isocitrate/1% bovine serum albumin.Metabolism of pregnenolone during incubation was blockedby the addition of 10,uM cyanoketone (Winthrop 19,578), andpregnenolone formation was measured by direct radioimmu-noassay with a highly specific antibody (22). The conversionof corticosterone to aldosterone was measured by incubatingaliquots of the mitochondrial suspension with 20 ,ug of unlabeledcorticosterone for 30 min at 370 in the medium describedabove. The aldosterone formed was measured by radioimmu-noassay after extraction with 10 vol of methylene chloride andfractionation by LH-20 Sephadex column chromatography.The content of endogenous cholesterol in the mitochondria wasmeasured by a colorimetric technique with o-ph1dialdehyde(23). Binding of 125I-labeled angiotensin II to the 6000-30,000X g membrane-rich pellet was determined as described forparticulate adrenal fractions (24). Association constants andreceptor concentration were calculated from equilibriumbinding data derived at 200 with monoiodoangio-tensin II by computer analysis as described (25).

RESULTSDuring sodium restriction, marked and rapid changes occurredin plasma renin activity and in blood angiotensin II and plasmaaldosterone concentrations. In addition, the responses to sodiumdeficiency were significantly altered when animals were treatedsimultaneously with the converting enzyme inhibitor. Plasmarenin activity rose from 4.0 t 0.6 to 12.5 + 0.9 ng/ml per hrafter 4 days of sodium restriction, and was further increasedto 19.5 ± 1.0 ng/ml per hr in-sodium-deficient animals infusedwith SQ 14,225. Significant increases in blood angiotensin ii,from the basal value of 9.1 t 1.4 pg/ml, were observed at 36hr (to 36.8 t 6.5 pg/ml) and after 4 days of sodium restriction(to 36.0 t 6.7 pg/ml). There was a concomitant rise in plasmaaldosterone, from the basal value of 12.5 ng/100 ml to 63ng/100 ml on the fourth day of sodium restriction, and thisincrease was completely abolished by infusion of the convertingenzyme inhibitor (Fig. 1). Thus, the aldosterone response in vivoto sodium restriction was completely dependent on formationof angiotensin II and was prevented when formation of theoctapeptide was blocked by the converting enzyme inhibi-tor.

F

6

o31

60 -I

I.

0 40h

II

ro 20~CL

Control

I

Low Lowsodium sodiurn

t SQ 1 4,225

FIG. 1. Inhibition of the plasma aldosterone response to sodiumrestriction during blockade of angiotensin I converting enzyme. Ratswere placed on low sodium diet (0Q06-0.1%) for 4 days, and one groupreceived continuous intraperitoneal infusions of converting enzymeinhibitor (SQ 14,225) from Alzet osmotic minipumps. The values ineach group correspond to the mean + SEM of plasma aldosteronevalues in 19 rats.

To analyze the activities of sites in the aldosterone biosyn-thetic pathway that are known to be influenced by angiotensinII and sodium deficiency, we performed studies on early andlate steps in steroid synthesis. As noted above, the activity of theearly portion of the biosynthetic pathway in glomerulosa cellswas analyzed during sodium deficiency by measuring preg-nenolone formation by isolated mitochondria in the presenceof cyanoketone (Winthrop 19,578) to prevent further steroidmetabolism.

In sodium-restricted rats, the formation of pregnenolonefrom endogenous cholesterol was significantly increased (P <0.02) from the control value of 1.6 : 0.1 to 2.1 : 0.1 Mug/mg ofmitochondrial protein per 15 min. This elevation in pregnen-olone synthesis was completely abolished in animals infusedwith SQ 14,225 during sodium restriction, when the rate ofpregnenolone formation was 1.5 : 0.1 Mg/mg per 15 min (Fig.2). To determine whether the increased rate of pregnenolonesynthesis during sodium restriction was accompanied by anincrease in available cholesterol substrate, we measured themitochondrial content of cholesterol in the same adrenals. Thepattern of changes observed in the total cholesterol content ofmitochondria during sodium deficiency was similar to that ofpregnenolone, with an increase from 43.3 h 2.5 Mug/mg ofprotein in the controls to 60.2 ± 4.9 in the sodium-deficient ratsthat was reduced by infusion of SQ 14,225 to a value of 39.9 ±Mg/mg of protein (Fig. 3).The late portion of the aldosterone biosynthetic pathway was

examined by assay of the conversion of unlabeled corticosteroneto aldosterone in the glomerulosa mitochondrial fraction, andshowed an elevation during sodium restriction, with completeblockade of this response in animals treated with the convertingenzyme inhibitor (Fig. 4). The percent of corticosterone con-verted to aldosterone per mg of mitochondrial protein was 2.9± 0.2 in the controls, 5.8 ± 0.3 in the sodium-restricted rats, and3.2 + 0.2 when the rats were simultaneously infused with SQ14,225.

Proc. Nati. Acad. Sci. USA 75 (1978)

Proc. Nati. Acad. Sci. USA 75 (1978) 4059

C.-.2 E

E.

04.-o0

SE:a..

0-Control Low Low

sodium sodium+SQ 14,225

FIG. 2. Pregnenolone formation by the adrenal glomerulosamitochondrial fraction obtained from control and sodium-restrictedrats with and without infusion ofSQ 14,225. Each bar represents themean + SEM of results obtained in three experiments.

The extent to which these changes in the steroid biosyntheticpathway and aldosterone production were related to alteredcell receptors for angiotensin II was examined by equilibriumbinding studies with 125I-labeled angiotensin II in a particulatefraction of adrenal capsules from groups of normal, sodium-deficient, and SQ 14,225-treated rats. In a typical experiment(Fig. 5), the previously observed rise in angiotensin II receptorsites was evident after 4 days of sodium restriction and wascompletely abolished by infusion-of the converting enzymeinhibitor throughout the period of sodium deficiency. Themean (-SEM) receptor content in three experiments was sig-nificantly increased (P < 0.02) from 1097 4 83 to 1.723 + 166fmol/mg of protein., while in the SQ 14,225-treated rats thevalue was similar to the controls (1108 I 51). No changes in the

0

0.

C0

00

Control Low Lowsodium sodium

+SQ 14,225

FIG. 4. Conversion of corticosterone to aldosterone by theglomerulosa mitochondrial fraction from control and sodium-re-stricted rats, with and without infusion ofSQ 14,225. Each bar rep-resents the mean + SEM of results obtained in three experiments.

angiotensin II receptor affinity were observed between thegroups. The K. values were 1.30 + 0.10, 1.26 + 0.04, and 1.36+ 0.10 X 109 M-1 in the controls, sodium-restricted, and so-dium-restricted and SQ 14,225-infused animals, respective-ly.

DISCUSSIONThese studies were performed to clarify the role of angiotensinII in the acute aldosterone response to sodium restriction, asopposed to the more severe stimulus imposed by sodium de-pletion. Although there is no dispute about the essential role ofthe renin-angiotensin system in sustaining the aldosterone re-sponse to established sodium deficiency (5), there is debatewhether angiotensin acts in long-term sodium depletion as apermissive factor or a primary regulator (26). The acute effects

80

T60[-

C

4..C._m

oL04uO 40[-

20 _-

T

Control Low Lowsodium sodium

+SQ 14,225

FIG. 3. Cholesterol content of mitochondrial- fractions fromadrenal glomerulosa of control and sodium-restricted rats, with andwithout infusion ofSQ 14,225. Each bar represents the mean ± SEMof results obtained in three experiments.

0 2500 5000Total angiotensin 11, pM

FIG. 5. Binding of 12I-labeled angiotensin II to adrenal glom-erulosa particles from control (0) and sodium-restricted rats with (0)and without (0) infusion ofSQ 14,225. Similar results were obtainedin each of three experiments.

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4060 Physiological Sciences: Aguilera and Catt

of angiotensin II infusion on aldosterone secretion are transientin certain species (12, 27), and infusions of the peptide do notquantitatively reproduce the aldosterone response to equivalentperiods of sodium deficiency (2, 6, 9, 28). However, it is wellknown that the action of angiotensin II upon aldosterone se-cretion is enhanced by sodium deficiency in several species(8-12), and this could account for the apparent discrepancybetween the response to angiotensin II infusion and that to so-dium restriction or depletion.We have recently observed that the actions of angiotensin

II upon the adrenal glomerulosa cell include changes in themembrane receptors for angiotensin, as well as enhancementof the biosynthetic responses to the polypeptide in vivo and invitro (15). After administration of angiotensin II for periods of36 hr to 6 days, significant rises in receptor sites and blood al-dosterone levels were observed, with an increase in the in vitroresponses of adrenal cells isolated from the zona glomerulosaof the peptide-treated animals (15). Such findings are consistentwith earlier observations on the ability of renin or angiotensinII treatment to enhance subsequent aldosterone response to theoctapeptide in vvo (28, 29) and could provide an explanationfor the increase in adrenal sensitivity that occurs during ele-vation of blood angiotensin II levels in sodium-deficient ani-mals. In the rat adrenal, sodium restriction also led to significantchanges in glomerulosa receptor sites, with an early rise inbinding affinity (at 36 hr) and a subsequent rise in receptorcontent at the fourth day of sodium restriction. These changeswere accompanied by corresponding increases in the sensitivityand magnitude of the aldosterone responses to angiotensin II,both in vvo and in isolated cells studied in vitro (14).

These findings emphasize the ability of angiotensin II tomodulate its receptor sites and steroidogenesis in the zonaglomerulosa, as well as to evoke the acute aldosterone responsesthat accompany short-term elevations of blood angiotensin II.They also raise the possibility that angiotensin II could be re-sponsible for mediating all of the changes seen during sodiumdeficiency, i.e., the enhanced sensitivity of the adrenal as wellas the direct regulation of aldosterone biosynthesis and secre-tion. It has been noted above that prolonged infusions of an-giotensin II in rats and human beings did not quantitativelyreproduce the aldosterone response to an equivalent period ofsodium restriction. This discrepancy is not large (the differencein response being about 2-fold) and might result from the useof continuous infusion of angiotensin II instead of the normalepisodic and circadian secretory pattern that is characteristicof the renin-angiotensin system in normal and sodium-deficientstates (30, 31). As in other hormone-regulated cells, mainte-nance of a high sustained level of angiotensin II may be a lesseffective stimulus of cell responses than intermittent elevationsof the peptide. A significant proportion of the circulating an-giotensin II in certain species may be the des-Aspl-heptapeptideof angiotensin II, in addition to the [Aspl]octapeptide. Thissituation may in fact apply in the rat, a species in which theheptapeptide has been reported to comprise up to 60% of thecirculating angiotensin activity (32). Whichever form of an-giotensin II is predominant in the individual species, it is likelythat angiotensin I is either the major biologically active peptideor its precursor, and that the effects of blocking convertingenzyme apply equally well to both of the active angiotensinpeptides.The present observations have extended the analysis of an-

giotensin's role in sodium deficiency by blocking formation ofthe peptide in sodium-deprived rats. Infusion of the convertingenzyme inhibitor can lead to increased plasma bradykininlevels, as well as the anticipated decrease in angiotensin II

formation (33). However, no effects of bradykinin upon an-giotensin-stimulated aldosterone production have been detectedin isolated adrenal glomerulosa cells (unpublished observations)and there is no reason to believe that actions of bradykinin areinvolved in the present study. The results have clearly shownthat all aspects of the adrepal response to sodium restrictioncould be inhibited during blockade of angiotensin II production.The mitochondrial accumulation of cholesterol and its con-version to pregnenolone, as well as the conversion of cortico-sterone to aldosterone, were unchanged during sodium re-striction in animals given the converting enzyme inhibitor,whereas each of these steps was increased in the untreated so-dium-deficient control rats. Also, the induction of angiotensinII receptors during sodium restriction was completely abolishedby administration of the inhibitor. The absence of these char-acteristic adrenal responses during inhibition of convertingenzyme activity provides an unequivocal demonstration of thecentral role of the renin-angiotensin system in mediating theadrenal response to sodium restriction.

This observation is of particular significance in the physio-logical control of aldosterone secretion, a process which hasoften been studied in animals (usually dogs or sheep) subjectedto quite severe sodium depletion or long-term sodium restric-tion. It is clear that a distinction should be made between so-dium depletion, induced by diuretic treatment or salivary loss,and sodium restriction imposed by reduced sodium intake. Theformer case is accompanied by sodium loss and extracellularfluid volume changes, and the stimulus to aldosterone secretionis more intense and probably also more complex. During sodi-um restriction, the degree of sodium loss and fluid volumechange is much less, particularly during the first few days ofsodium deprivation. Even after periods of sodium restrictionfor up to 6 weeks in rats, no changes in plasma sodium andpotassium concentration are detectable (13).

For these reasons, we studied the short-term response tolow-sodium intake in rats as a model for the physiologicalregulation of aldosterone secretion during fluctuations in di-etary salt content. Recent studies in sodium-depleted dogsshowed that the aldosterone response to acute, diuretic-inducedsodium loss could be abolished by treatment with an angiotensinII antagonist (the Sarl, Ala8 derivative). Also, plasma aldosteronelevels in chronically sodium-depleted dogs were markedly re-duced by administration of the nonapeptide converting enzymeinhibitor (SQ 20,881), though not to completely normal levels(16). These, and numerous earlier studies (3-5, 34-36), haveemphasized the importance of renin and angiotensin II in thecontrol of aldosterone secretion during sodium deficiency.Despite this, a controversy had remained about the precise roleof angiotensin II, regarded by some as a primary regulator ofthe aldosterone response (5) and by others as a permissive factorin aldosterone secretion (26). These discrepant views have arisenin part from the complexity of aldosterone regulation in long-term sodium deficiency and in animals subjected to variousforms of sodium depletion, sometimes with accompanyingchanges in other regulating factors such as blood volume andplasma potassium concentration. Also, different animal modelshave been used, and results derived under anesthesia havefrequently differed from those obtained in conscious animals(13, 37, 38).

Until recently, the rat was not regarded as a satisfactory an-imal model for studies on the control of aldosterone secretion,largely due to initial misconceptions about the responsivenessof the rat adrenal to angiotensin II. However, several earlierreports had indicated that angiotensin II would indeed stimulatealdosterone secretion by the rat adrenal (9,39), and more recent

Proc. Natl. Acad. Sci. USA 75 (1978)

Proc. Nati. Acad. Sci. USA 75(1978) 4061

studies have shown that the zona glomerulosa cells of the ratadrenal are highly sensitive to angiotensin H in vivo and in vitro(14, 18, 36, 38, 40). In the present study performed on consciousrats, it has been possible to analyze the role of angiotensin IIduring simple dietary restriction of sodium intake, to providethe most physiologically appropriate conditions for examinationof this control mechanism. Our results have indicated that an-giotensin II is the mediator of the physiological response in al-dosterone secretion during the onset of sodium deficiency.The actions of angiotensin II upon the zona glomerulosa are

complex, and include a trophic effect as well as an acute stim-ulating action on aldosterone secretion. The trophic actions ofangiotensin II include effects on angiotensin II receptors andenzymes in the steroid biosynthetic sequence, as well as stim-ulation of glomerulosa cell differentiation and multiplication.Both of these actions, by leading to changes in acute steroido-genic capacity and long-term glomerulosa cell mass, contributeto the enhanced sensitivity of the adrenal seen during sodiumdeficiency (3, 14) and angiotensin II infusion (10, 15). Themultiple actions of angiotensin II on glomerulosa cell structureand function clearly increase the complexity of analyzing al-dosterone regulation in various animal models. However,during short periods of sodium deficiency, as demonstrated bythe present studies, there remains little reason to doubt thatangiotensin II functions as the major physiological regulatorof aldosterone secretion.

We are grateful to Dr. Z. P. Horovitz and the Squibb Institute forMedical Research for providing the converting enzyme inhibitor (SQ14,225: D-2-methyl-3-mercaptopropanoyl-L-proline) that made itpossible to perform these studies. The cyanoketone and pregnenoloneantiserum used in assays of pregnenolone formation were generouslyprovided by the Sterling-Winthrop Research Institute and by Dr. C.Strott.

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