Molecular and Cellular Endocrinology, 90 (1992) 53-60 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00
53
MOLCEL 02871
Role of angiotensin II receptor subtypes on the regulation of aldosterone in the adrenal glomerulosa zone in the rat
secretion
Greti Aguilera Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA
(Received 26 May 1992; accepted 22 August 1992)
Key words: Aldosterone; Angiotensin II; Angiotensin II receptor subtype; Cyclic AMP; Inositol phosphate; Adrenal glomerulosa
Summary The role of AI1 receptors subtypes, AT, and AT,, in the regulation of aldosterone secretion was studied in adrenal glomerulosa cells and membranes from rats on normal and low sodium intake, using AI1 receptor subtype-specific antagonists. In adrenal glomerulosa cells, more than 90% of the receptors were AT, and there was a good correlation between the potencies of the antagonists to inhibit ligand binding, and AII-stimulated aldosterone production and inositol phosphate formation. The inhibition of basal and ACTH-stimulated CAMP by AI1 was also abolished by the AT,, but not the AT,, antagonist. Sodium restriction for 6 days increased both receptor subtypes in the same proportion, but only the AT, antagonist inhibited AII-stimulated aldosterone production. The data demonstrate that AT, receptor mediates the regulatory actions of AI1 in the adrenal zona glomerulosa.
Introduction Angiotensin II (AID is the major regulator of aldosterone secretion during changes in sodium balance or extracellular volume. AI1 stimulates aldosterone secretion in vivo and in vitro and exerts trophic effects in the zona glomerulosa of the adrenal through binding to plasma membrane receptors (Aguilera and Catt, 1985). The recent development of orally active AI1 receptor antagonists has made possible the identification of two receptor subtypes, type 1 (AT,), recognized by DuP 753, and type 2 (AT,), recognized by PD123177 (Chiu et al., 1989a; Whitebread et al., 1989). Several investigators including Chiu et al. (1990, Garcia-Sainz and Marcia+Silva (1990) and Johnson and Aguilera (1991) have shown that AT, receptors are coupled to calciurn-phospholipid-dependent signalling systems and constitute the majority of AI1 receptors in most target tissues in the adult, including the adrenal glomerulosa and vascular smooth muscle. The subtype-2 AI1 recep-
Correspondence to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, NICHD, NIH, Bldg 10 Rm lON262, Bethesda, MD 20892, USA.
tors, of yet unknown action, are present in the uterus, ovary, adrenal medulla and some brain areas in the adult, and they are widely distributed during late fetal and early postnatal development. Studies by Wong et al. (1990) have shown that injection of the AT, antagonist in rats lowers plasma aldosterone, suggesting that AT, receptors are responsible for aldosterone secretion in the adrenal glomerulosa. However, as reported by Chiu et al. (1989b), a small proportion of AT, receptors is present in the adult adrenal cortex, and selective regulation or interaction between the two receptor subtypes may have a role in modulation of adrenal sensitivity to AII. In addition, as extensively reviewed by Catt et al. (1987), Quinn and Williams (1988) and Spat et al. (1991), the interaction of AI1 with its receptor results in a number of intracellular signals including increases in cytosolic calcium, arachidonic acid metabolites, diacyl glycerol and inositol phosphate formation, activation of protein kinase C and inhibition of adenylate cyclase. The nature of the receptor interaction leading to these different processes and the specific role of the various mechanisms in the steroidogenic effects of AI1 are not clearly understood and may involve different receptor subtypes.
The purpose of these studies was to determine the role of both receptor subtypes on the regulation of adrenal glomerulosa function. The results show that the adrenal effects of AI1 in steroidogenesis, inositol phosphate formation and inhibition of CAMP production, are all mediated solely by AT, receptors. Materials and methods Synthetic [Ile5]AII was purchased from Peninsula Laboratories (Belmont, CA, USA), collagenase type I from Sigma (St. Louis, MO, USA) and other reagents from regular sources. Medium 199 was prepared by the NIH media unit, and monoiodinated lz51[Sar ‘,Ile*]AII by Hazleton Laboratories (Vienna, VA, USA). The AI1 receptor subtype-specific antagonists, DuP 753 (2-nbutyl-4-chloro-5-hydroxymethyl-l-(2’-[1H-tetrazol-5-yl]biphenyl-Cyl)methylimidazole) for AT,, and PD123177 ~l-(4-amino-3-methylphenyl)methyl-3-diphenyl-acetyl4,5,6,7-tetrahydro-III-imidazo(4,5-O)pyridine-6-carboxylic acid) for AT,, were kindly provided by Drs. Andrew Chiu and Ronald D. Smith from DuPont-Merck (Wilmington, DE, USA). Male Sprague-Dawley rats (250-300 g) obtained from Zivic Miller (Zelienople, PA, USA) were maintained in a controlled environment with free access to rat chow and water for at least 4 days. When necessary, groups of 20 rats were placed on a low sodium diet prepared by Ziegler Brothers (Gardners, PA, USA) for 6 days. Rats were killed by decapitation, and the adrenals were immediately removed and cleaned of surrounding fat. Capsules were separated from the inner zones, minced, washed in K-free medium 199 containing 0.1% bovine serum albumin (BSA). Isolated cells were prepared by collagenase digestion and mechanical dispersion as previously described by Douglas et al. (1978). Cell viability was more than 95% as evaluated by trypan blue exclusion. For aldosterone production, cells were resuspended in medium 199 containing 4.5 mM K and 0.2% BSA at a concentration of 100,000 cells per ml. 1 ml aliquots were incubated for 2 h at 37°C with AI1 in the presence and in the absence of the specific receptor subtype antagonists DuP 753 and PD123177, in 12 x 75 mm glass tubes under 95% sir/5% CO,. At the end of the incubation, supernatants were separated by centrifugation for 10 min at 1500 X g and stored at - 20°C for aldosterone RIA using antialdosterone antibodycoated tubes and 1251-aldosterone (Diagnostic Products, CA, USA). For CAMP, cells were resuspended in the same incubation media containing 2 mM isobutylmethylxanthine at a concentration of 200,000 cells/ml. 1 ml aliquots were incubated in the presence of AI1 and the inhibitors for 30 min at 37°C under 95% sir/5% CO,. At the end of the incubation the tubes were boiled for
10 min and supernatants separated by centrifugation and stored at -20°C for CAMP measurement by radioimmunoassay (RIA) as described by Fujita et al. (1979). For measurement of inositol phosphates 40 million cells were washed and resuspended in 2 ml inositol-free medium 199 and added to a 50 ml plastic tube containing 200 PCi [“Hlmyoinositol (Amersham, Arlington Heights, IL, USA). After 3 h incubation, cells were washed 3 times with medium 199 containing 25 nM Hepes and 10 mM LiCl, resuspended in 40 ml of the same medium, and divided into l-ml aliquots in 12 x 75 mm glass tubes. Tubes were placed in a water bath at 37°C and preincubated for 15 min in the presence or absence of AI1 antagonists before addition of AII. After 10 min incubation, 1 ml of methanol/chloroform (2: 1) was added, tubes were vortexed for 30 s, and the aqueous layer separated by centrifugation. Fractions of 0.5 ml were applied to a 3 x 0.6-cm Dowex AGl-X8 column (200-400 mesh; formate form; BioRad Laboratories, Richmond, CA, USA). Fractions containing inositol phosphates were eluted as described by Berridge et al. (1983) and the radioactivity measured in a liquid scintillation counter. AI1 receptors were measured by binding of ‘251[Sar‘,Iles]AII cc isolated adrenal glomerulosa cells or adrenal capsular membranes as previously described by Glossman et al. (1985). Receptor affinity and concentration were calculated by Scatchard analysis of the binding data using the computerized curve fitting program LIGAND designed by Munson and Rodbard (1980). Results AII receptor subtypes in adrenal capsular membranes
The binding inhibition potencies of the receptor subtype-specific antagonists, DuP 753 and PD123177, were determined in adrenal capsular membrane-rich fractions from adult rats. In three experiments, binding of ‘251[Sar’,Ile8]AII to adrenal capsular membranes was inhibited by 100% by the native peptide, AII, and by the peptide antagonist, [Sar’,Ala*]AII, with IC,, values of 1.2 k 0.1 and 0.8 + 0.1 nM, respectively. Concentrations of the AT, receptor antagonist, DuP 753, up to 10 PM inhibited 84 f 3% of the binding with an IC,” of 82 f 6 nM, while the AT, antagonist, PD123177, inhibited only 14 + 1% of the binding with an IC,, of 109 f 7 nM. Higher concentrations of the antagonists caused further binding inhibition, probably due to binding to the heterologous receptor subtype. Scatchard analysis of the binding data, in the presence of increasing concentrations of AII, showed a single class of binding sites with a K, of 1.2 _+0.2 nM and a total receptor concentration of 1623 & 95 fmol/mg (n = 3). In three experiments, measurement
0.8 0.3
t $
0.15
=
a % = -L;
,k2 f?Qc??-\ 500
1000
c
0.3
v,m‘ z”
0.15
!? :
1500
0
0
BOUND All (fmollmg) Fig. 1. Scatchard analysis of AI1 binding to rat adrenal glomerulosa membrane-rich fractions. AT, and AT, were measured in the presence of 10 PM PD123177 or DuP 753, respectively. Data points are the mean of duplicate incubations in one of three similar experiments. TIME
of the receptor concentration in the presence of 10 PM concentrations of the antagonists, PD123177 and DuP 753, revealed values of 1298 f 82 and 230 _t 15 fmol/mg, respectively, indicating that 85 f 4% of the receptors are AT, (Fig. 1). Addition of the sulfhydryl reducing agent dithiothreitol (DTT) (2 mM) caused a marked decrease in the total radioactivity bound. Scatchard analysis of the binding data in the presence and in the absence of the antagonists showed that the effect of DTT was due to a decrease in AT, receptor concentration and an increase in the binding affinity for AT, receptors (Table 1). The effect of guanyl nucleotides on the binding dissociation from AI1 receptor subtypes in adrenal capsular membranes is shown in Fig. 2. Addition of 1 PM unlabeled AI1 at binding equilibrium caused a rapid dissociation of 51.0 & 2.5% of the radioligand binding, with a t,,, of 4.9 rt 0.3 min, followed by a slower decline during the following 2 h. Addition of the non-degradable GTP analog GTP--y-S alone caused a similar decrease of 61.1 &-3.2%, with a t,,, of 4.5 f 0.2 min. Binding dissociation was markedly enhanced by simultaneous addition of the GTP analog and AII, with
IminJ
Fig. 2. Dissociation of ‘%4II from rat adrenal capsule membranerich fractions. AI1 (1 PM), GTP-y-S (10 PM), or their combination were added at binding equilibrium and the reaction terminated at the times indicated. Total receptors (A) were studied in the absence of antagonists, AT, (B) in the presence of 10 PM DuP 753, and AT, (C) in the presence of 10 PM PD123177. Data points are the mean of duplicate incubations in one of four similar experiments. B/T: bound radioactivity/total radioactivity added.
a 91.5 + 1.5% decrease in binding and a t,,, of 3.2 k 0.1 min (Fig. 2A). A similar binding dissociation pattern was observed in the presence of complete blockade of AT, receptors by 10 PM PD123177 (Fig. 2B). However, when AT, receptors were blocked with 10 PM DuP 753, addition of AI1 caused a 34.7 + 2.7% dissociation, with a t,,, of 8.4 + 1.1 min, and GTP-y-S, added alone or in combination with AU, failed to cause any decrease in the binding (Fig. 20. Since in the rat changes in adrenal AI1 receptor content contribute to control of adrenal responsiveness during changes in sodium intake, it was of interest to study the selective regulation of the two receptor subtypes during sodium restriction. As expected, 6 days sodium restriction caused a marked increase in total AI1 receptors in adrenal capsular membranes. Deter-
TABLE 1 EFFECT OF DITHIOTHREITOL LOSA MEMBRANES
(Dl’T) ON THE BINDING OF AI1 TO AI1 RECEPTOR SUBTYPES IN RAT ADRENAL GLOMERU-
Values are the mean and SE from data in three experiments. Angiotension II receptors Total n No D-I-I DTT2mM
1,832+64 1,363 f 58 *
AT, K, 1.6kO.2 1.5+0.2
* p < 0.01 compared with the values without DTT.
n 1,532+41 1,057+64 *
K, 1.6kO.2 1.8kO.3
AT, n
K,
281kll 314+ 18
1.7kO.2 O.S+O.l *
56
mination of AI1 receptor subtypes by Scatchard analysis of the data obtained in the presence of the heterologous antagonist revealed proportional increases in the concentration of both receptor subtypes, and no changes in binding affinity (Table 2). The mean values of three experiments show a small and not significant difference between total and AT, receptors. However, in each experiment AT, receptors accounted for about 85% of the total binding (84.5 + 0.7 and 84.2 f 0.96% for controls and sodium restriction, respectively. Following sodium restriction, there was a significant increase in adrenal capsular AI1 receptors to values 1.74-, 1.85 and 1.78-fold over the control for total, AT, and AT, receptors, respectively. AZZ receptor subtypes in isolated adrenal glomendosa cells
Scatchard analysis of the binding of ‘251[Sar’,Ile8]AII to isolated adrenal glomerulosa cells from rats on normal sodium diet showed a single class of sites with a concentration of 78.0 f 3.2 fmo1/100,000 cells and an affinity of 1.1 + 0.3 nM AII. In contrast to adrenal capsular membranes, in which 15% of the binding corresponds to AT, receptors, isolated adrenal glomerulosa cells contained only about 6.4 + 1.8% AT, receptors. Scatchard analysis of the binding data obtained in the presence of DuP 753 revealed a concentration of AT, receptors of 5.0 f 0.9 fmo1/100,000 cells. In contrast, binding measured in the presence of PD123177 was only slightly lower than the total receptor concentration (72.1 + 2.8 fmo1/100,000 cells). No significant differences in affinity between receptor subtypes were observed (0.9 f 0.2 and 1.8 + 0.8 nM for AT, and AT, receptors, respectively). Similar to the results in adrenal capsular membranes, sodium restriction for 6 days resulted in a marked increase in total AI1 receptors (from 78.0 + 3.2 to 151.4 -t 6.5 fmo1/100,000 cells, p < O.OOl), due to a parallel increase in AT, receptors (from 72.1 f 2.8 to 135.0 + 1.1 fmo1/100,000 cells, p < 0.001) and AT, receptors (from 5.0 k 0.9 to 9.2 + 0.7 fmo1/100,000 cells, p < 0.001) (Fig. 3).
TABLE
100 cells)
125
Fig. 3. Scatchard analysis of total AI1 receptors, AT, and AT, content in collagenase-dispersed adrenal glomerulosa cells of control (closed symbols) and sodium-restricted rats for 6 days (open symbols). Total receptors were measured in the absence of antagonists, AT, in the presence of 10 PM PD123177, and AT, in the presence of 10 PM of DuP 753. Data points are the mean of duplicate incubations in one of three similar experiments.
Inhibition of AZZ binding and aldosterone production by receptor subtype antagonists
The role of the AI1 receptor subtypes on steroidogenesis was studied by examining the relationship between binding inhibition potency of the AI1 receptor subtype antagonists and their ability to inhibit aldosterone production in isolated adrenal glomerulosa cells (Fig. 4). Addition of the peptide antagonist [Sar’,Ala’]AII, 100 nM, resulted in complete inhibition of the binding and AU-stimulated aldosterone production. Addition of increasing concentrations of the AT, antagonist DuP 753 caused a maximum 90.5 _t 2.4% decrease in binding (IC,, 82.0 f 3.2 nM) (Fig. 4A). Despite the partial binding inhibition, DuP 753 completely inhibited AII-stimulated aldosterone production, with an ED,, of 86.2 + 3.1 nM, similar to the binding inhibition potency. The AT, antagonist, PD123177, caused only minor binding inhibition with a maximum of 7.4 + 1.3% with 1 and 10 PM, and an
2
CHANGES TION Values
J 50 75 BOUND All (fmol/105
IN AI1 RECEPTOR
are the mean
Diet
SUBTYPES
and SE of data obtained Angiotension
IN ADRENAL by Scatchard
ZONA analysis
* p < 0.001 compared
in three
MEMBRANES
FOLLOWING
SODIUM
RESTRIC-
experiments.
II receptors
Total
Normal sodium Low sodium
GLOMERULOSA
AT,
AT,
n
Kd
n
Kd
n
Kd
1,671+ 140 4,582 + 390 *
1.3kO.3 1.3kO.4
1,418* 117 4,055 f 504 *
1.2 * 0.3 1.3 f 0.3
262k 11 729+ 7.3 *
0.9 f 0.4 1.0+0.4
with the values of normal
sodium
diet.
ED,, of about 100 nM. Higher concentrations, 0.1 mM, caused a further inhibition, probably due to interaction with AT, receptors. Incubation of the cells with the AT, antagonist, up to concentrations of 100 PM, had no effect on AII-stimulated aldosterone production (Fig. 4B). The effect of AI1 receptor subtype-specific antagonists on the stimulation of aldosterone production by increasing concentrations of AI1 is shown in Fig. 5. Incubation of the cells with AI1 resulted in the characteristic dose-dependent increase in aldosterone production. In six of ten experiments, the effect showed the typical biphasic pattern often seen with AII, with a maximum stimulation between 0.3 and 10 nM AI1 followed by a decrease of about 30% with higher concentrations of AII. In the additional four experiments aldosterone secretion reached maximum with 1 nM AI1 and remained in a plateau with supramaximal AI1 concentrations. In all experiments, simultaneous incubation with 10 PM DuP 753 completely inhibited aldosterone production by submaximal and maximal stimulatory concentrations of AIL Amounts of AI1 higher than 10 nM progressively reversed the inhibition, indicating that DuP 753 acts as a competitive antagonist (Fig. 5A and B). In contrast, addition of 10 PM of the AT, antagonist had little effect on AIIstimulated aldosterone production. In six of ten experi-
/All
1nM
PDl23177
ANTAGONIST
CONCENTRATION
IM)
Fig. 4. Binding competition (A) and aldosterone inhibition (B) curves by the peptide antagonist [Sar’,Ala8jAII, and the non-peptide receptor subtype-specific antagonists in collagenase-dispersed adrenal glomerulosa cells in rats on normal sodium diet. Data points are the mean of duplicate incubations in one of four similar experiments.
AlI CONCENTRATION
(M)
Fig. 5. Effect of AI1 receptor subtype-specific antagonist on the aldosterone response to increasing concentrations of AI1 in collagenase-dispersed rat adrenal glomerulosa cells. Panel A is representative of seven experiments in which the AT, antagonist had no effect on AILstimulated aldosterone production, and panel B is representative of three experiments in which AI1 showed biphasic stimulation and PD123177 inhibited the maximum response.
ments, addition of PD123177 had no significant effect on the stimulatory effect of AI1 up to 1 nM (Fig. 5A). However, in four of the experiments in which AI1 showed a biphasic response, in the presence of PD123177 aldosterone production reached a plateau with 1 nM AII, without attaining the maximum stimulation levels observed in the control cells (Fig. 5B). In three experiments, the ED,, for the stimulation of aldosterone production by AI1 was unchanged by PD123177, and in one experiment PD123177 caused a slight decrease in the ED,, for AII. Similar to the results in rats on normal sodium diet, in two experiments in isolated adrenal glomerulosa cells from sodium-restricted rats for 6 days, AII-stimulated aldosterone secretion was completely inhibited by DuP 753, and not significantly affected by PD123177 (not shown). Effect of AII receptor subtype antagonists on inositol phosphate and CAMP The effects of AI1 and AI1 antagonists on inositol phosphate formation are shown in Fig. 6. Treatment of the cells with 100 nM AI1 resulted in a time-dependent increase in inositol phosphate production. In three experiments, inositol trisphosphate (IP3) increased by 2.5 f 0.3-fold at 30 s and progressively decreased to near-basal values by 10 min. Inositol bisphosphate (IP,)
58
m’b
0 CONTROL APD 123177 . DUP 753
2-
x E B
It has been previously shown that AI1 inhibits basal and ACTH-stimulated CAMP production. To determine which AI1 receptor subtype is involved in this action of AII, the effect of the specific antagonists on CAMP accumulation was analyzed in adrenal glomerulosa cells treated with AI1 in the absence and in the presence of ACTH. Consistent with previous reports, AI1 caused a significant decrease in basal and ACTHstimulated CAMP accumulation (Table 3). This effect was abolished by the peptide AI1 antagonist, [Sar’, AlaX]AII, and the AT, antagonist, DuP 753, but it was completely unaffected by the AT, antagonist, PD123177, indicating that the inhibitory effect of AI1 on CAMP is also mediated by AT, receptors.
l-
&-
r z
? 0
// /I
x z
i
Discussion
k Of
,
II
30
60N TIME
300
600
(set)
Fig. 6. Effect of 10 PM concentrations of the AI1 receptor subtype antagonists, DuP 753 (0) and PD123177 (A 1, on AII-stimulated inositol monophosphate (II’,), inositol bisphosphate (IP,) and inosito1 trisphosphate (IP,) accumulation in collagenase-dispersed adrenal glomerulosa cells from rats on normal sodium diet. Data points are the mean of duplicate incubations in one of three similar experiments.
increased by 2.0 + 0.2-fold at 1 min and remained in a plateau up to 10 min, and inositol monophosphate (IP,) showed a continuous increase reaching levels of 9.3 f 1.7-fold the basal at 10 min. Similar to the effects in steroidogenesis, addition of 10 PM DuP 753 completely inhibited the stimulation of IP,, IP, and IP,. The AT, antagonist, PD123177, had no effect on inosito1 phosphate formation at any time of incubation with AII.
TABLE
These in vitro studies provide further evidence that the effects of AI1 on cellular signalling systems and steroidogenesis in the adrenal glomerulosa cell are mediated by AT, receptors. As shown by the present data and studies by Chiu et al. (19891, Chang et al. (19891, Balla et al. (1991) and Wiest et al. (1991) both AI1 receptor subtypes are present in the adrenal glomerulosa. However, only the AT, antagonist inhibited the stimulatory effect of AI1 on inositol phosphate formation and aldosterone production, with a potency in agreement with its binding inhibition activity. The binding properties of both receptor subtypes in the adrenal glomerulosa were consistent with observations by Dudley et al. (1990), Speth et al. (19901, Tsusumi et al. (1991) and Puce11 et al. (1991) in other tissues in relation to their sensitivity to sulfhydryl reducing agents and guanyl nucleotides. With respect to the effect of disulfhydryl reducing agents, it has been shown by Whitebread et al. (1989), Chiu et al. (1989) and Speth et al. (1990) that binding to AT, receptors is increased by DTT, while binding to AT, receptors is inhibited. A novel finding in these experiments was that DTT increases the binding to AT, receptors by increasing the affinity but not receptor number.
3
EFFECT OF AI1 ANTAGONISTS GLOMERULOSA CELLS
ON THE INHIBITORY
Values are the mean and SE of the data in four pmol/105 cells for ACTH-stimulated accumulation. CAMP accumulation
EFFECT
experiments.
Range
OF AI1 ON CAMP PRODUCTION of CAMP values
was 1.1-2.1
IN ISOLATED
pmol/lO’
RAT ADRENAL
cells for basal,
(% inhibition)
AI1 10 nM
AI1 10 nM + [Sar,AlajAII
AI1 10 nM + DuP 753
AI1 10 nM + PD123177
(10 nM)
20.3 + 5.1 38.9 f 9.2
1.5k2.4 * 2.42 1.8 *
2.5 k 2.0 * 1.7k2.5 *
23.5 k 7.9 40.6+9.4
* p < 0.0001 compared
with AI1 in the absence
Basal ACTH
of antagonists.
and 4.7-10.8
59
It should be noted that the concentration of AT, receptors in isolated glomerulosa cells was markedly lower than that in adrenal capsular membranes. It is unlikely that the lower AT, receptor content is the consequence of preferential damage of this receptor subtype during enzymatic digestion, because AT, receptors are preserved following collagenase dispersion of the cells in other tissues containing AT, receptors, such as fetal skin or adrenal capsule from neonatal rats. The lack of effect of the AT, antagonist could be attributed to the low receptor content in the adrenal cell preparation. However, the AT, antagonist is equally ineffective in inhibiting steroidogenesis in adrenal glomerulosa cells with more abundant AT, receptors, as shown by Feuillan (1991) in 7-day-old rats, or by the present experiments in sodium-restricted rats. Therefore it is likely that the function of AT, receptors, if any, is not directly related to the acute steroidogenic activity of AII. Similar dependency of aldosterone secretion on AT, receptors has been shown in vivo by Wong et al. (1990) and in vitro by Chang et al. (1989) and Balla et al. (1991). In conjunction with activation of steroidogenesis, interaction of AI1 with its receptor results in a number of intracellular events including calcium and phospholipid turnover, activation of protein kinase C and inhibition of adenylate cyclase (Catt et al., 1987; Spat et al., 1991). Consistent with reports by Garcia-Sainz and Macias-Silva (1990) in hepatocytes, and Johnson and Aguilera (1991) in fetal skin fibroblasts, in the present study AH-stimulated inositol phosphates in adrenal glomerulosa cells were also inhibited by the AT, antagonist. This is in agreement with the view that phospholipid breakdown has a mediatory role in the steroidogenie effect of AII. On the other hand, the present experiments, as well as reports by Balla et al. (19911, show that the inhibitory effect of AI1 on adenylate cyclase, which is unrelated to stimulation of steroidogenesis, was also mediated by AT, receptors. One of the objectives of these studies was to determine whether differential regulation of the AI1 receptor subtypes influences sensitivity of the adrenal glomerulosa to AII. This hypothesis is not supported by the present experiments in rats receiving altered sodium diet, in which differential changes in receptor subtypes were not correlated with changes in adrenal sensitivity to AII. Consistent with the increases in AT, mRNA reported by Iwai and Inagami (19921, binding to AT, receptors to adrenal capsular membranes and cells from sodium-restricted rats was increased. However, there was a proportional increase in AT, receptors which are not involved in the acute steroidogenic effects of AI1 and moreover, blockade of AT, receptors did not shift the AI1 dose-response for aldosterone production. Similarly, Feuillan (1991) has shown identical aldosterone response curves to increasing
concentrations of AII, in the presence and in the absence of AT, antagonist, in adrenal cells from neonatal rats which contain about equal number of AT, and AT, receptors. Although AT, and AT, receptors are present in the zona glomerulosa, there is no information about the cellular localization of the subtypes. It is possible that AT, receptors are present in the cells responsible for steroidogenesis, while AT, receptors may by associated with other cell types, such as fibroblasts or germinal cells of the adrenal capsule. It is conceivable that AT, receptors have a role in cell growth and differentiation in the adrenal glomerulosa zone. Consistent with this possibility is the fact that the number of AT, receptors in the adrenal capsule is higher in conditions associated with adrenal growth such as sodium restriction and during fetal and neonatal development. Studies by Dzau et al. (1991) in vascular smooth muscle cells have shown that AI1 has stimulatory and inhibitory effects in cell proliferation depending upon the conditions under which the cells are exposed to the peptide. A number of studies by Taubman et al. (1989), Bobick et al. (1990), Dzau et al. (19911, and others, show that AI1 stimulates the expression of platelet-derived growth factor (PDGF) and growth-related proto-oncogenes, and to potentiate the mitogenic effect of epidermal growth factor in cultured kidney tubular cells (Norman et al., 1987). The demonstration by Feuillan et al. (19911, Millan et al. (1991) and Grady et al. (1991) that AT, receptors are transiently expressed during development suggests that the effects of AI1 in growth are mediated by AT, receptors. However, at least in cultured vascular smooth muscle cells, Chiu et al. (1990) have shown that the hypertrophic responses to AI1 are mediated by AT, and not by AT, receptors. In summary, these studies show that the actions of AI1 on intracellular signalling systems and steroidogenesis in the adrenal glomerulosa cell are mediated by type 1 angiotensin II receptors. Further studies are needed to determine the precise cellular localization and the physiological role of AT, receptors in adrenal glomerulosa function. Acknowledgements The author would like to thank Drs. Andrew Chiu and Ronald D. Smith from DuPont, Wilmington, DE, USA, for their generous supply of the non-peptide AI1 antagonists used in this study. References Aguilera, G. and Catt, K.J. (1985) in The Adrenal Gland and Hypertension (Mantero, F., Biglieri, E.G., Funder, J.W. and Scoggins, B.A., eds.), pp. 33-53, Raven Press, New York.
60
Balla, T., Baukal, A.J., Eng, S. and Catt, K.J. (1991) Mol. Pharmacol. 40, 401-406. Berridge, M., Dawson, R., Downwes, C. and Irvine, R. (1983) Biochem. J. 212, 473-482. Bobick, A., Grinpukel, S., Grooms, A. and Jackman, G. (1990) Biochem. Biophys. Res. Commun. 166, 580-588. Catt, K.J., Carson, M.C., Hausdorff, W.P., Leach-Harper, C.M., Baukal, A.J., Guillemette, G., Bala, T. and Aguilera, G. (1987) J. Steroid Biochem. 77, 915-927. Chang, R.S.L. and Lotti, V.J. (1989) Mol. Pharmacol. 29, 347-351. Chiu, A.T., McCall, D.E., Nguyen, T.T., Carini, D.J., Duncia, J.V., Herblin, W.F., Uyeda, R.T., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M. (1989a) Eur. J. Pharmacol. 170, 117-118. Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L.J., Wong, P.C., Wexler, R.R., Johnson, A.L. and Timmermans, P.B.M.W.M. (1989b) Biochem. Biophys. Res. Commun. 165, 196-202. Chiu, A.T., Roscoe, W.A. and Timmermans, P.P.M.W.M. (1990) Receptor 1, 133-140. Chiu, A.T., Carini, D.J., Duncia, J.V., Leung, K.H., McCall, D.E., Price, W.A., Wong, P.C., Smith, R.D., Wexler, R.R. and Timmermans, P.B.W.M.W. (1991) Biochem. Biophys. Res. Commun. 177, 209-217. Douglas, J., Aguilera, G., Kondo, T. and Catt, K.J. (1978) Endocrinology 102, 685-696. Dudley, D.T., Panek, R.L., Major, T.C., Lu, G.H., Bruns, R.F., Klinkefus, B.A., Hodges, J.C. and Weishaar, R.E. (1990) Mol. Pharmacol. 38, 370-377. Dzau, V.J., Gibbons, G.H. and Pratt, R.E. (1991) Hypertension 18, II,lOO-11,105. Feuillan, P. (1991) 74th Annual Meeting Endocrine Society, Washington, DC, abstract 676. FeuiIIan, P., Millan, M.A. and Aguilera, G. (1991) Fed. Proc. 5, 2915. Fujita, K., Aguilera, G. and Catt, K.J. (1979) J. Biol. Chem. 25, 8567-8574. Garcia-Sainz, J.A. and Macias-Silva, M. (1990) Biochem. Biophys. Res. Commun. 172, 780-785.
Glossman, H., Baukal, A.J., Aguilera, G. and Catt. K.J. (I9851 Methods Enzymol. 109, 110-126. Grady, E.F., Sechi, L.A., Griffin, C.A., Schambelan, M. and Kalinyak, J.E. (1991) J. Clin. Invest. 88, 921-933. Iwai, N. and Inagami, T. (1992) Biochem. Biophys. Res. Commun. 182, 1094-1099. Johnson, M.C. and Aguilera, G. (1991) Endocrinology 129, 12661274. Millan, M.A., Carvallo, P., Izumi, S.-I., Catt, K.J. and Aguilera, G. (1989) Science 244, 1340-1342. Millan, M.A., Jacobowitz, D.M., Aguilera, G. and Catt, K.J. (1991) Proc. Natl. Acad. Sci. USA 88, 11440-11444. Munson, P.J. and Rodbard, D. (1980) Ann. Biochem. 107, 220-239. Norman, J., Badie-Dezfoly, B., Nord, E.P., Kurtz, I., Schlosser, A., Chaudhari, A. and Fine, L.G. (1987) Am. J. Physiol. 253, F299F309. Pucell, A.G., Hodges, J.C., Sen, I., Bumpus, M.F. and Hussain, A. (1991) Endocrinology 128, 1947-1959. Quinn, S.J. and Williams, G.H. (1988) Annu. Rev. Physiol. 50, 409-431. Spat, A., Enyedi, P., Hajnoczky, G. and Hunyady, L. (1991) Exp. Physiol. 76, 859-885. Speth, R.C. and Kim, K.H. (1990) Biochem. Biophys. Res. Commun. 169, 997-1006. Taubman, M.B., Berk, B.C., Izumo, S., Tsuda, T., Alexander, R.W. and Nadal-Ginard, R. (1989) J. Biol. Chem. 264, 526-530. Tsusumi, K., Stromberg, C., Viswannathan, M. and Saavedra, J.M. (1991) Endocrinology 129, 1075-1082. Whitebread, S., Mele, M., Kamber, B. and De Gasparo, M. (1989) Biochem. Biophys. Res. Commun. 163, 284-291. Wiest. S.A., Rampersaud, A., Zimmerman, K. and Steinberg, M.I. (1991) J. Cardiovasc. Pharmacol. 17, 177-184. Wong, P.C., Price, W.A., Chiu, A.T., Carini, D.J., Duncia, J.V., Johnson, A.L., Wexler, R.R. and Timmermans, P.B.M.W.M. (1990) Hypertension 15, 823-834.
53
MOLCEL 02871
Role of angiotensin II receptor subtypes on the regulation of aldosterone in the adrenal glomerulosa zone in the rat
secretion
Greti Aguilera Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA
(Received 26 May 1992; accepted 22 August 1992)
Key words: Aldosterone; Angiotensin II; Angiotensin II receptor subtype; Cyclic AMP; Inositol phosphate; Adrenal glomerulosa
Summary The role of AI1 receptors subtypes, AT, and AT,, in the regulation of aldosterone secretion was studied in adrenal glomerulosa cells and membranes from rats on normal and low sodium intake, using AI1 receptor subtype-specific antagonists. In adrenal glomerulosa cells, more than 90% of the receptors were AT, and there was a good correlation between the potencies of the antagonists to inhibit ligand binding, and AII-stimulated aldosterone production and inositol phosphate formation. The inhibition of basal and ACTH-stimulated CAMP by AI1 was also abolished by the AT,, but not the AT,, antagonist. Sodium restriction for 6 days increased both receptor subtypes in the same proportion, but only the AT, antagonist inhibited AII-stimulated aldosterone production. The data demonstrate that AT, receptor mediates the regulatory actions of AI1 in the adrenal zona glomerulosa.
Introduction Angiotensin II (AID is the major regulator of aldosterone secretion during changes in sodium balance or extracellular volume. AI1 stimulates aldosterone secretion in vivo and in vitro and exerts trophic effects in the zona glomerulosa of the adrenal through binding to plasma membrane receptors (Aguilera and Catt, 1985). The recent development of orally active AI1 receptor antagonists has made possible the identification of two receptor subtypes, type 1 (AT,), recognized by DuP 753, and type 2 (AT,), recognized by PD123177 (Chiu et al., 1989a; Whitebread et al., 1989). Several investigators including Chiu et al. (1990, Garcia-Sainz and Marcia+Silva (1990) and Johnson and Aguilera (1991) have shown that AT, receptors are coupled to calciurn-phospholipid-dependent signalling systems and constitute the majority of AI1 receptors in most target tissues in the adult, including the adrenal glomerulosa and vascular smooth muscle. The subtype-2 AI1 recep-
Correspondence to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, NICHD, NIH, Bldg 10 Rm lON262, Bethesda, MD 20892, USA.
tors, of yet unknown action, are present in the uterus, ovary, adrenal medulla and some brain areas in the adult, and they are widely distributed during late fetal and early postnatal development. Studies by Wong et al. (1990) have shown that injection of the AT, antagonist in rats lowers plasma aldosterone, suggesting that AT, receptors are responsible for aldosterone secretion in the adrenal glomerulosa. However, as reported by Chiu et al. (1989b), a small proportion of AT, receptors is present in the adult adrenal cortex, and selective regulation or interaction between the two receptor subtypes may have a role in modulation of adrenal sensitivity to AII. In addition, as extensively reviewed by Catt et al. (1987), Quinn and Williams (1988) and Spat et al. (1991), the interaction of AI1 with its receptor results in a number of intracellular signals including increases in cytosolic calcium, arachidonic acid metabolites, diacyl glycerol and inositol phosphate formation, activation of protein kinase C and inhibition of adenylate cyclase. The nature of the receptor interaction leading to these different processes and the specific role of the various mechanisms in the steroidogenic effects of AI1 are not clearly understood and may involve different receptor subtypes.
The purpose of these studies was to determine the role of both receptor subtypes on the regulation of adrenal glomerulosa function. The results show that the adrenal effects of AI1 in steroidogenesis, inositol phosphate formation and inhibition of CAMP production, are all mediated solely by AT, receptors. Materials and methods Synthetic [Ile5]AII was purchased from Peninsula Laboratories (Belmont, CA, USA), collagenase type I from Sigma (St. Louis, MO, USA) and other reagents from regular sources. Medium 199 was prepared by the NIH media unit, and monoiodinated lz51[Sar ‘,Ile*]AII by Hazleton Laboratories (Vienna, VA, USA). The AI1 receptor subtype-specific antagonists, DuP 753 (2-nbutyl-4-chloro-5-hydroxymethyl-l-(2’-[1H-tetrazol-5-yl]biphenyl-Cyl)methylimidazole) for AT,, and PD123177 ~l-(4-amino-3-methylphenyl)methyl-3-diphenyl-acetyl4,5,6,7-tetrahydro-III-imidazo(4,5-O)pyridine-6-carboxylic acid) for AT,, were kindly provided by Drs. Andrew Chiu and Ronald D. Smith from DuPont-Merck (Wilmington, DE, USA). Male Sprague-Dawley rats (250-300 g) obtained from Zivic Miller (Zelienople, PA, USA) were maintained in a controlled environment with free access to rat chow and water for at least 4 days. When necessary, groups of 20 rats were placed on a low sodium diet prepared by Ziegler Brothers (Gardners, PA, USA) for 6 days. Rats were killed by decapitation, and the adrenals were immediately removed and cleaned of surrounding fat. Capsules were separated from the inner zones, minced, washed in K-free medium 199 containing 0.1% bovine serum albumin (BSA). Isolated cells were prepared by collagenase digestion and mechanical dispersion as previously described by Douglas et al. (1978). Cell viability was more than 95% as evaluated by trypan blue exclusion. For aldosterone production, cells were resuspended in medium 199 containing 4.5 mM K and 0.2% BSA at a concentration of 100,000 cells per ml. 1 ml aliquots were incubated for 2 h at 37°C with AI1 in the presence and in the absence of the specific receptor subtype antagonists DuP 753 and PD123177, in 12 x 75 mm glass tubes under 95% sir/5% CO,. At the end of the incubation, supernatants were separated by centrifugation for 10 min at 1500 X g and stored at - 20°C for aldosterone RIA using antialdosterone antibodycoated tubes and 1251-aldosterone (Diagnostic Products, CA, USA). For CAMP, cells were resuspended in the same incubation media containing 2 mM isobutylmethylxanthine at a concentration of 200,000 cells/ml. 1 ml aliquots were incubated in the presence of AI1 and the inhibitors for 30 min at 37°C under 95% sir/5% CO,. At the end of the incubation the tubes were boiled for
10 min and supernatants separated by centrifugation and stored at -20°C for CAMP measurement by radioimmunoassay (RIA) as described by Fujita et al. (1979). For measurement of inositol phosphates 40 million cells were washed and resuspended in 2 ml inositol-free medium 199 and added to a 50 ml plastic tube containing 200 PCi [“Hlmyoinositol (Amersham, Arlington Heights, IL, USA). After 3 h incubation, cells were washed 3 times with medium 199 containing 25 nM Hepes and 10 mM LiCl, resuspended in 40 ml of the same medium, and divided into l-ml aliquots in 12 x 75 mm glass tubes. Tubes were placed in a water bath at 37°C and preincubated for 15 min in the presence or absence of AI1 antagonists before addition of AII. After 10 min incubation, 1 ml of methanol/chloroform (2: 1) was added, tubes were vortexed for 30 s, and the aqueous layer separated by centrifugation. Fractions of 0.5 ml were applied to a 3 x 0.6-cm Dowex AGl-X8 column (200-400 mesh; formate form; BioRad Laboratories, Richmond, CA, USA). Fractions containing inositol phosphates were eluted as described by Berridge et al. (1983) and the radioactivity measured in a liquid scintillation counter. AI1 receptors were measured by binding of ‘251[Sar‘,Iles]AII cc isolated adrenal glomerulosa cells or adrenal capsular membranes as previously described by Glossman et al. (1985). Receptor affinity and concentration were calculated by Scatchard analysis of the binding data using the computerized curve fitting program LIGAND designed by Munson and Rodbard (1980). Results AII receptor subtypes in adrenal capsular membranes
The binding inhibition potencies of the receptor subtype-specific antagonists, DuP 753 and PD123177, were determined in adrenal capsular membrane-rich fractions from adult rats. In three experiments, binding of ‘251[Sar’,Ile8]AII to adrenal capsular membranes was inhibited by 100% by the native peptide, AII, and by the peptide antagonist, [Sar’,Ala*]AII, with IC,, values of 1.2 k 0.1 and 0.8 + 0.1 nM, respectively. Concentrations of the AT, receptor antagonist, DuP 753, up to 10 PM inhibited 84 f 3% of the binding with an IC,” of 82 f 6 nM, while the AT, antagonist, PD123177, inhibited only 14 + 1% of the binding with an IC,, of 109 f 7 nM. Higher concentrations of the antagonists caused further binding inhibition, probably due to binding to the heterologous receptor subtype. Scatchard analysis of the binding data, in the presence of increasing concentrations of AII, showed a single class of binding sites with a K, of 1.2 _+0.2 nM and a total receptor concentration of 1623 & 95 fmol/mg (n = 3). In three experiments, measurement
0.8 0.3
t $
0.15
=
a % = -L;
,k2 f?Qc??-\ 500
1000
c
0.3
v,m‘ z”
0.15
!? :
1500
0
0
BOUND All (fmollmg) Fig. 1. Scatchard analysis of AI1 binding to rat adrenal glomerulosa membrane-rich fractions. AT, and AT, were measured in the presence of 10 PM PD123177 or DuP 753, respectively. Data points are the mean of duplicate incubations in one of three similar experiments. TIME
of the receptor concentration in the presence of 10 PM concentrations of the antagonists, PD123177 and DuP 753, revealed values of 1298 f 82 and 230 _t 15 fmol/mg, respectively, indicating that 85 f 4% of the receptors are AT, (Fig. 1). Addition of the sulfhydryl reducing agent dithiothreitol (DTT) (2 mM) caused a marked decrease in the total radioactivity bound. Scatchard analysis of the binding data in the presence and in the absence of the antagonists showed that the effect of DTT was due to a decrease in AT, receptor concentration and an increase in the binding affinity for AT, receptors (Table 1). The effect of guanyl nucleotides on the binding dissociation from AI1 receptor subtypes in adrenal capsular membranes is shown in Fig. 2. Addition of 1 PM unlabeled AI1 at binding equilibrium caused a rapid dissociation of 51.0 & 2.5% of the radioligand binding, with a t,,, of 4.9 rt 0.3 min, followed by a slower decline during the following 2 h. Addition of the non-degradable GTP analog GTP--y-S alone caused a similar decrease of 61.1 &-3.2%, with a t,,, of 4.5 f 0.2 min. Binding dissociation was markedly enhanced by simultaneous addition of the GTP analog and AII, with
IminJ
Fig. 2. Dissociation of ‘%4II from rat adrenal capsule membranerich fractions. AI1 (1 PM), GTP-y-S (10 PM), or their combination were added at binding equilibrium and the reaction terminated at the times indicated. Total receptors (A) were studied in the absence of antagonists, AT, (B) in the presence of 10 PM DuP 753, and AT, (C) in the presence of 10 PM PD123177. Data points are the mean of duplicate incubations in one of four similar experiments. B/T: bound radioactivity/total radioactivity added.
a 91.5 + 1.5% decrease in binding and a t,,, of 3.2 k 0.1 min (Fig. 2A). A similar binding dissociation pattern was observed in the presence of complete blockade of AT, receptors by 10 PM PD123177 (Fig. 2B). However, when AT, receptors were blocked with 10 PM DuP 753, addition of AI1 caused a 34.7 + 2.7% dissociation, with a t,,, of 8.4 + 1.1 min, and GTP-y-S, added alone or in combination with AU, failed to cause any decrease in the binding (Fig. 20. Since in the rat changes in adrenal AI1 receptor content contribute to control of adrenal responsiveness during changes in sodium intake, it was of interest to study the selective regulation of the two receptor subtypes during sodium restriction. As expected, 6 days sodium restriction caused a marked increase in total AI1 receptors in adrenal capsular membranes. Deter-
TABLE 1 EFFECT OF DITHIOTHREITOL LOSA MEMBRANES
(Dl’T) ON THE BINDING OF AI1 TO AI1 RECEPTOR SUBTYPES IN RAT ADRENAL GLOMERU-
Values are the mean and SE from data in three experiments. Angiotension II receptors Total n No D-I-I DTT2mM
1,832+64 1,363 f 58 *
AT, K, 1.6kO.2 1.5+0.2
* p < 0.01 compared with the values without DTT.
n 1,532+41 1,057+64 *
K, 1.6kO.2 1.8kO.3
AT, n
K,
281kll 314+ 18
1.7kO.2 O.S+O.l *
56
mination of AI1 receptor subtypes by Scatchard analysis of the data obtained in the presence of the heterologous antagonist revealed proportional increases in the concentration of both receptor subtypes, and no changes in binding affinity (Table 2). The mean values of three experiments show a small and not significant difference between total and AT, receptors. However, in each experiment AT, receptors accounted for about 85% of the total binding (84.5 + 0.7 and 84.2 f 0.96% for controls and sodium restriction, respectively. Following sodium restriction, there was a significant increase in adrenal capsular AI1 receptors to values 1.74-, 1.85 and 1.78-fold over the control for total, AT, and AT, receptors, respectively. AZZ receptor subtypes in isolated adrenal glomendosa cells
Scatchard analysis of the binding of ‘251[Sar’,Ile8]AII to isolated adrenal glomerulosa cells from rats on normal sodium diet showed a single class of sites with a concentration of 78.0 f 3.2 fmo1/100,000 cells and an affinity of 1.1 + 0.3 nM AII. In contrast to adrenal capsular membranes, in which 15% of the binding corresponds to AT, receptors, isolated adrenal glomerulosa cells contained only about 6.4 + 1.8% AT, receptors. Scatchard analysis of the binding data obtained in the presence of DuP 753 revealed a concentration of AT, receptors of 5.0 f 0.9 fmo1/100,000 cells. In contrast, binding measured in the presence of PD123177 was only slightly lower than the total receptor concentration (72.1 + 2.8 fmo1/100,000 cells). No significant differences in affinity between receptor subtypes were observed (0.9 f 0.2 and 1.8 + 0.8 nM for AT, and AT, receptors, respectively). Similar to the results in adrenal capsular membranes, sodium restriction for 6 days resulted in a marked increase in total AI1 receptors (from 78.0 + 3.2 to 151.4 -t 6.5 fmo1/100,000 cells, p < O.OOl), due to a parallel increase in AT, receptors (from 72.1 f 2.8 to 135.0 + 1.1 fmo1/100,000 cells, p < 0.001) and AT, receptors (from 5.0 k 0.9 to 9.2 + 0.7 fmo1/100,000 cells, p < 0.001) (Fig. 3).
TABLE
100 cells)
125
Fig. 3. Scatchard analysis of total AI1 receptors, AT, and AT, content in collagenase-dispersed adrenal glomerulosa cells of control (closed symbols) and sodium-restricted rats for 6 days (open symbols). Total receptors were measured in the absence of antagonists, AT, in the presence of 10 PM PD123177, and AT, in the presence of 10 PM of DuP 753. Data points are the mean of duplicate incubations in one of three similar experiments.
Inhibition of AZZ binding and aldosterone production by receptor subtype antagonists
The role of the AI1 receptor subtypes on steroidogenesis was studied by examining the relationship between binding inhibition potency of the AI1 receptor subtype antagonists and their ability to inhibit aldosterone production in isolated adrenal glomerulosa cells (Fig. 4). Addition of the peptide antagonist [Sar’,Ala’]AII, 100 nM, resulted in complete inhibition of the binding and AU-stimulated aldosterone production. Addition of increasing concentrations of the AT, antagonist DuP 753 caused a maximum 90.5 _t 2.4% decrease in binding (IC,, 82.0 f 3.2 nM) (Fig. 4A). Despite the partial binding inhibition, DuP 753 completely inhibited AII-stimulated aldosterone production, with an ED,, of 86.2 + 3.1 nM, similar to the binding inhibition potency. The AT, antagonist, PD123177, caused only minor binding inhibition with a maximum of 7.4 + 1.3% with 1 and 10 PM, and an
2
CHANGES TION Values
J 50 75 BOUND All (fmol/105
IN AI1 RECEPTOR
are the mean
Diet
SUBTYPES
and SE of data obtained Angiotension
IN ADRENAL by Scatchard
ZONA analysis
* p < 0.001 compared
in three
MEMBRANES
FOLLOWING
SODIUM
RESTRIC-
experiments.
II receptors
Total
Normal sodium Low sodium
GLOMERULOSA
AT,
AT,
n
Kd
n
Kd
n
Kd
1,671+ 140 4,582 + 390 *
1.3kO.3 1.3kO.4
1,418* 117 4,055 f 504 *
1.2 * 0.3 1.3 f 0.3
262k 11 729+ 7.3 *
0.9 f 0.4 1.0+0.4
with the values of normal
sodium
diet.
ED,, of about 100 nM. Higher concentrations, 0.1 mM, caused a further inhibition, probably due to interaction with AT, receptors. Incubation of the cells with the AT, antagonist, up to concentrations of 100 PM, had no effect on AII-stimulated aldosterone production (Fig. 4B). The effect of AI1 receptor subtype-specific antagonists on the stimulation of aldosterone production by increasing concentrations of AI1 is shown in Fig. 5. Incubation of the cells with AI1 resulted in the characteristic dose-dependent increase in aldosterone production. In six of ten experiments, the effect showed the typical biphasic pattern often seen with AII, with a maximum stimulation between 0.3 and 10 nM AI1 followed by a decrease of about 30% with higher concentrations of AII. In the additional four experiments aldosterone secretion reached maximum with 1 nM AI1 and remained in a plateau with supramaximal AI1 concentrations. In all experiments, simultaneous incubation with 10 PM DuP 753 completely inhibited aldosterone production by submaximal and maximal stimulatory concentrations of AIL Amounts of AI1 higher than 10 nM progressively reversed the inhibition, indicating that DuP 753 acts as a competitive antagonist (Fig. 5A and B). In contrast, addition of 10 PM of the AT, antagonist had little effect on AIIstimulated aldosterone production. In six of ten experi-
/All
1nM
PDl23177
ANTAGONIST
CONCENTRATION
IM)
Fig. 4. Binding competition (A) and aldosterone inhibition (B) curves by the peptide antagonist [Sar’,Ala8jAII, and the non-peptide receptor subtype-specific antagonists in collagenase-dispersed adrenal glomerulosa cells in rats on normal sodium diet. Data points are the mean of duplicate incubations in one of four similar experiments.
AlI CONCENTRATION
(M)
Fig. 5. Effect of AI1 receptor subtype-specific antagonist on the aldosterone response to increasing concentrations of AI1 in collagenase-dispersed rat adrenal glomerulosa cells. Panel A is representative of seven experiments in which the AT, antagonist had no effect on AILstimulated aldosterone production, and panel B is representative of three experiments in which AI1 showed biphasic stimulation and PD123177 inhibited the maximum response.
ments, addition of PD123177 had no significant effect on the stimulatory effect of AI1 up to 1 nM (Fig. 5A). However, in four of the experiments in which AI1 showed a biphasic response, in the presence of PD123177 aldosterone production reached a plateau with 1 nM AII, without attaining the maximum stimulation levels observed in the control cells (Fig. 5B). In three experiments, the ED,, for the stimulation of aldosterone production by AI1 was unchanged by PD123177, and in one experiment PD123177 caused a slight decrease in the ED,, for AII. Similar to the results in rats on normal sodium diet, in two experiments in isolated adrenal glomerulosa cells from sodium-restricted rats for 6 days, AII-stimulated aldosterone secretion was completely inhibited by DuP 753, and not significantly affected by PD123177 (not shown). Effect of AII receptor subtype antagonists on inositol phosphate and CAMP The effects of AI1 and AI1 antagonists on inositol phosphate formation are shown in Fig. 6. Treatment of the cells with 100 nM AI1 resulted in a time-dependent increase in inositol phosphate production. In three experiments, inositol trisphosphate (IP3) increased by 2.5 f 0.3-fold at 30 s and progressively decreased to near-basal values by 10 min. Inositol bisphosphate (IP,)
58
m’b
0 CONTROL APD 123177 . DUP 753
2-
x E B
It has been previously shown that AI1 inhibits basal and ACTH-stimulated CAMP production. To determine which AI1 receptor subtype is involved in this action of AII, the effect of the specific antagonists on CAMP accumulation was analyzed in adrenal glomerulosa cells treated with AI1 in the absence and in the presence of ACTH. Consistent with previous reports, AI1 caused a significant decrease in basal and ACTHstimulated CAMP accumulation (Table 3). This effect was abolished by the peptide AI1 antagonist, [Sar’, AlaX]AII, and the AT, antagonist, DuP 753, but it was completely unaffected by the AT, antagonist, PD123177, indicating that the inhibitory effect of AI1 on CAMP is also mediated by AT, receptors.
l-
&-
r z
? 0
// /I
x z
i
Discussion
k Of
,
II
30
60N TIME
300
600
(set)
Fig. 6. Effect of 10 PM concentrations of the AI1 receptor subtype antagonists, DuP 753 (0) and PD123177 (A 1, on AII-stimulated inositol monophosphate (II’,), inositol bisphosphate (IP,) and inosito1 trisphosphate (IP,) accumulation in collagenase-dispersed adrenal glomerulosa cells from rats on normal sodium diet. Data points are the mean of duplicate incubations in one of three similar experiments.
increased by 2.0 + 0.2-fold at 1 min and remained in a plateau up to 10 min, and inositol monophosphate (IP,) showed a continuous increase reaching levels of 9.3 f 1.7-fold the basal at 10 min. Similar to the effects in steroidogenesis, addition of 10 PM DuP 753 completely inhibited the stimulation of IP,, IP, and IP,. The AT, antagonist, PD123177, had no effect on inosito1 phosphate formation at any time of incubation with AII.
TABLE
These in vitro studies provide further evidence that the effects of AI1 on cellular signalling systems and steroidogenesis in the adrenal glomerulosa cell are mediated by AT, receptors. As shown by the present data and studies by Chiu et al. (19891, Chang et al. (19891, Balla et al. (1991) and Wiest et al. (1991) both AI1 receptor subtypes are present in the adrenal glomerulosa. However, only the AT, antagonist inhibited the stimulatory effect of AI1 on inositol phosphate formation and aldosterone production, with a potency in agreement with its binding inhibition activity. The binding properties of both receptor subtypes in the adrenal glomerulosa were consistent with observations by Dudley et al. (1990), Speth et al. (19901, Tsusumi et al. (1991) and Puce11 et al. (1991) in other tissues in relation to their sensitivity to sulfhydryl reducing agents and guanyl nucleotides. With respect to the effect of disulfhydryl reducing agents, it has been shown by Whitebread et al. (1989), Chiu et al. (1989) and Speth et al. (1990) that binding to AT, receptors is increased by DTT, while binding to AT, receptors is inhibited. A novel finding in these experiments was that DTT increases the binding to AT, receptors by increasing the affinity but not receptor number.
3
EFFECT OF AI1 ANTAGONISTS GLOMERULOSA CELLS
ON THE INHIBITORY
Values are the mean and SE of the data in four pmol/105 cells for ACTH-stimulated accumulation. CAMP accumulation
EFFECT
experiments.
Range
OF AI1 ON CAMP PRODUCTION of CAMP values
was 1.1-2.1
IN ISOLATED
pmol/lO’
RAT ADRENAL
cells for basal,
(% inhibition)
AI1 10 nM
AI1 10 nM + [Sar,AlajAII
AI1 10 nM + DuP 753
AI1 10 nM + PD123177
(10 nM)
20.3 + 5.1 38.9 f 9.2
1.5k2.4 * 2.42 1.8 *
2.5 k 2.0 * 1.7k2.5 *
23.5 k 7.9 40.6+9.4
* p < 0.0001 compared
with AI1 in the absence
Basal ACTH
of antagonists.
and 4.7-10.8
59
It should be noted that the concentration of AT, receptors in isolated glomerulosa cells was markedly lower than that in adrenal capsular membranes. It is unlikely that the lower AT, receptor content is the consequence of preferential damage of this receptor subtype during enzymatic digestion, because AT, receptors are preserved following collagenase dispersion of the cells in other tissues containing AT, receptors, such as fetal skin or adrenal capsule from neonatal rats. The lack of effect of the AT, antagonist could be attributed to the low receptor content in the adrenal cell preparation. However, the AT, antagonist is equally ineffective in inhibiting steroidogenesis in adrenal glomerulosa cells with more abundant AT, receptors, as shown by Feuillan (1991) in 7-day-old rats, or by the present experiments in sodium-restricted rats. Therefore it is likely that the function of AT, receptors, if any, is not directly related to the acute steroidogenic activity of AII. Similar dependency of aldosterone secretion on AT, receptors has been shown in vivo by Wong et al. (1990) and in vitro by Chang et al. (1989) and Balla et al. (1991). In conjunction with activation of steroidogenesis, interaction of AI1 with its receptor results in a number of intracellular events including calcium and phospholipid turnover, activation of protein kinase C and inhibition of adenylate cyclase (Catt et al., 1987; Spat et al., 1991). Consistent with reports by Garcia-Sainz and Macias-Silva (1990) in hepatocytes, and Johnson and Aguilera (1991) in fetal skin fibroblasts, in the present study AH-stimulated inositol phosphates in adrenal glomerulosa cells were also inhibited by the AT, antagonist. This is in agreement with the view that phospholipid breakdown has a mediatory role in the steroidogenie effect of AII. On the other hand, the present experiments, as well as reports by Balla et al. (19911, show that the inhibitory effect of AI1 on adenylate cyclase, which is unrelated to stimulation of steroidogenesis, was also mediated by AT, receptors. One of the objectives of these studies was to determine whether differential regulation of the AI1 receptor subtypes influences sensitivity of the adrenal glomerulosa to AII. This hypothesis is not supported by the present experiments in rats receiving altered sodium diet, in which differential changes in receptor subtypes were not correlated with changes in adrenal sensitivity to AII. Consistent with the increases in AT, mRNA reported by Iwai and Inagami (19921, binding to AT, receptors to adrenal capsular membranes and cells from sodium-restricted rats was increased. However, there was a proportional increase in AT, receptors which are not involved in the acute steroidogenic effects of AI1 and moreover, blockade of AT, receptors did not shift the AI1 dose-response for aldosterone production. Similarly, Feuillan (1991) has shown identical aldosterone response curves to increasing
concentrations of AII, in the presence and in the absence of AT, antagonist, in adrenal cells from neonatal rats which contain about equal number of AT, and AT, receptors. Although AT, and AT, receptors are present in the zona glomerulosa, there is no information about the cellular localization of the subtypes. It is possible that AT, receptors are present in the cells responsible for steroidogenesis, while AT, receptors may by associated with other cell types, such as fibroblasts or germinal cells of the adrenal capsule. It is conceivable that AT, receptors have a role in cell growth and differentiation in the adrenal glomerulosa zone. Consistent with this possibility is the fact that the number of AT, receptors in the adrenal capsule is higher in conditions associated with adrenal growth such as sodium restriction and during fetal and neonatal development. Studies by Dzau et al. (1991) in vascular smooth muscle cells have shown that AI1 has stimulatory and inhibitory effects in cell proliferation depending upon the conditions under which the cells are exposed to the peptide. A number of studies by Taubman et al. (1989), Bobick et al. (1990), Dzau et al. (19911, and others, show that AI1 stimulates the expression of platelet-derived growth factor (PDGF) and growth-related proto-oncogenes, and to potentiate the mitogenic effect of epidermal growth factor in cultured kidney tubular cells (Norman et al., 1987). The demonstration by Feuillan et al. (19911, Millan et al. (1991) and Grady et al. (1991) that AT, receptors are transiently expressed during development suggests that the effects of AI1 in growth are mediated by AT, receptors. However, at least in cultured vascular smooth muscle cells, Chiu et al. (1990) have shown that the hypertrophic responses to AI1 are mediated by AT, and not by AT, receptors. In summary, these studies show that the actions of AI1 on intracellular signalling systems and steroidogenesis in the adrenal glomerulosa cell are mediated by type 1 angiotensin II receptors. Further studies are needed to determine the precise cellular localization and the physiological role of AT, receptors in adrenal glomerulosa function. Acknowledgements The author would like to thank Drs. Andrew Chiu and Ronald D. Smith from DuPont, Wilmington, DE, USA, for their generous supply of the non-peptide AI1 antagonists used in this study. References Aguilera, G. and Catt, K.J. (1985) in The Adrenal Gland and Hypertension (Mantero, F., Biglieri, E.G., Funder, J.W. and Scoggins, B.A., eds.), pp. 33-53, Raven Press, New York.
60
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