0013-7227/91/1295-2305S03.00/0 Endocrinology Copyright© 1991 by The Endocrine Society
Vol. 129, No. 5 Printed in U.S.A.
Atrial Natriuretic Peptide Inhibition of Calcium Ionophore A23187-Stimulated Aldosterone Secretion in Rat Adrenal Glomerulosa Cells* DAVID P. LOTSHAWf, ROBERTO FRANCO-SAENZ, AND PATRICK J. MULROW Department of Medicine, Medical College of Ohio, Toledo, Ohio 43699
ABSTRACT. The effect of atrial natriuretic peptide (ANP) on calcium ionophore A23187-stimulated aldosterone secretion was investigated using collagenase-dispersed rat adrenal glomerulosa cell suspensions. A23187 treatment induced a dose-dependent stimulation of aldosterone secretion, exhibiting an EC6o of approximately 75 nM. In agreement with the presumed action of A23187 as a Ca2+ ionophore, stimulation was dependent on the extracellular Ca2+ concentration, being completely inhibited in nominally Ca2+-free medium. In such Ca2+-free medium, stimulation of aldosterone secretion by bath applied 25-hydroxycholesterol was not inhibited, indicating that cells and biosynthetic pathway enzymes were not inhibited by low extracellular Ca2+ levels. A23187-induced aldosterone secretion was also inhibited by more than 90% when cells were simultaneously treated with ANP. Maximal ANP inhibition of A23187-stimulated aldoste-
rone secretion was not overcome by concentrations of A23187 up to 10 /*M or by increasing the extracellular Ca2+ concentration from 1.25 to 5 mM in the presence of A23187 and ANP. Addition of A23187 to ACTH-, angiotensin II-, or K+-stimulated glomerulosa cells did not overcome ANP-induced inhibition of aldosterone secretion stimulated by these secretagogues. In contrast to ANP inhibition of Ca2+-dependent A23187 stimulation of aldosterone secretion, ANP inhibition of dBcAMP-stimulated aldosterone secretion was readily overcome by increasing the dBcAMP concentration. These results indicated that ANP selectively and noncompetetively inhibited an intracellular step necessary for Ca2+-dependent stimulation of the early pathway of aldosterone biosynthesis in rat adrenal glomerulosa cells. (Endocrinology 129: 2305-2310, 1991)
A
TRIAL natriuretic peptide (ANP) is a hormone secreted primarily from the atria that exerts effects on multiple target organs, the net effect of which is a reduction of blood volume and arterial blood pressure (1). The zona glomerulosa layer of the adrenal cortex is one such target tissue, and here ANP acts as a potent inhibitor of hormone-stimulated aldosterone secretion (2, 3). Although several studies have investigated ANP-induced inhibition of aldosterone secretion, the mechanism(s) of this inhibition has not been elucidated. The primary effect of ANP on aldosterone secretion is thought to be mediated through receptor-induced inhibition of hormonal stimulation of the early pathway of aldosterone biosynthesis (4-7). Hormone stimulation of the early pathway involves the regulation of a complex Received May 6,1991. Address all correspondence and requests for reprints to: Dr. Patrick J. Mulrow, Department of Medicine, Medical College of Ohio, CS 10008, Toledo, Ohio 43699. * An abstract of these data was published previously in the Program of the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990, p. 399. This work was supported in part by a grant from the American Heart Association, Ohio Affiliate, Inc. (Columbus, OH). t Present address: T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506-0225.
and poorly understood system of cholesterol mobilization and delivery to the inner mitochondrial membrane for the initial side-chain cleavage reaction converting cholesterol to pregnenolone (8, 9). Therefore, most studies have focused on possible ANP effects on hormone-coupled second messenger systems that stimulate aldosterone biosynthesis. These studies have suggested that ANP-induced inhibition occurs subsequent to stimulatory hormone binding to its receptor and initiation of second messenger cascades. For example, ANP was shown to be a potent inhibitor of angiotensin-II-stimulated aldosterone secretion, but did not affect angiotensin-II receptor binding (4, 6), angiotensin-II-induced phosphatidylinositol hydrolysis (4, 10, 11), or angiotensin-II-induced elevation of intracellular Ca2+ (10,12,13). The effect of ANP on hormone-stimulated aldosterone secretion has been reported to correlate with the sensitivity of stimulation to inhibition by Ca2+ channel blockers (14). Several studies have examined the effect of ANP on hormone-stimulated Ca2+ fluxes across the glomerulosa cell plasma membrane, but contradictory results have been obtained. Most studies employing 45Ca2+ flux have reported no effect of ANP on angiotensin-IIor K+-induced Ca2+ influx or efflux (10,13,15); however, one study reported that ANP inhibited ACTH-, angio-
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INHIBITION OF ALDOSTERONE SECRETION
tensin-II-, and K+-stimulated Ca2+ influx (14). In contrast to most results of radiolabeled Ca2+ flux, electrophysiological experiments indicated that ANP inhibited a voltage-dependent T-type Ca2+ current in the glomerulosa cells (16). In the present study we have attempted to further define the mechanism of ANP-induced inhibition of aldosterone secretion by using the Ca2+ ionophore A23187 to elevate intracellular Ca2+, bypassing plasma membrane control of Ca2+ flux.
Materials and Methods All protocols employing animals were approved by the institutional animal care committee at the Medical College of Ohio. Isolated adrenal zona glomerulosa cell suspensions were prepared using a collagenase dispersion method similar to that previously described (15). Briefly, female Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing between 175-200 g, were killed, and adrenal glands were removed. The adrenal glands were dissected free of adherent fat, and capsules containing the zona glomerulosa layer were removed, minced into small pieces, and rinsed several times with fresh medium. The capsule fragments were digested in a modified Hanks' salts-medium 199 (M199; Sigma, St. Louis, MO) containing 1.5 mg/ml collagenase (Cooper Biochemicals, Malvern, PA) and 25 g/ml DNase (Sigma) for 1 h at 37 C in a shaking water bath. Hanks' saltsM199 was modified to contain 3.5 mM K+, 1.25 mM Ca2+, 0.2% BSA, 50 U/ml penicillin, 50 Mg/ml streptomycin, and 5 mM HEPES buffer, pH 7.4; the medium and atmosphere over the medium were equilibrated with 100% O2. Digested capsules were triturated, and the cell suspension was filtered through a 10-nm nylon mesh screen to obtain isolated cells. Isolated cells were washed four times with fresh medium by repeating a centrifugation-resuspension cycle, then equilibrated at 37 C in a shaking water bath at a cell density of approximately 200,000 cells/ml for between 1-2 h before experimentation. The cell preparation consisted primarily of zona glomerulosa cells contaminated by approximately 10% fasciculata cells; less than 10% of the total cells were stained by trypan blue. After the equilibration period, cells were pelleted by centrifugation and resuspended in fresh M199. The cell suspension was aliquoted to a cell density of approximately 50,000 cells/ ml in a final volume of 1 ml containing drugs and hormones or appropriate vehicle. Cells were incubated over a variable duration of up to 1.5 h at 37 C in a shaking water bath. Incubations were terminated by rapidly lowering the temperature of the cell suspensions in an ice bath; cells were pelleted by centrifugation, and the supernatants were stored at -20 C until assayed for aldosterone content. Aldosterone in the medium was quantified without prior extraction, using a commercial aldosterone RIA (Diagnostic Products, Los Angeles, CA). The inter- and intrassay coefficients of variation at an aldosterone concentration of 100 pg/ ml were 6.9% and 5.4%, respectively. A23187, 25-hydroxycholesterol, (Bu)2cAMP (dBcAMP), angiotensin-II, and ACTH, were obtained from Sigma. Rat ANP was obtained from Peninsula (Belmont, CA). A23187 was dis-
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solved in dimethylsulfoxide (DMSO), and the final DMSO concentration did not exceed 0.1%; 25-hydroxycholesterol was dissolved in ethanol, and the final ethanol concentration did not exceed 0.1%.
Results A23187-stimulated aldosterone secretion Treatment of glomerulosa cell suspensions with the calcium ionophore A23187 induced a dose-dependent stimulation of aldosterone secretion (Fig. 1). A23187 stimulation exhibited an EC5o of approximately 75 nM for incubations of 1-h duration, and maximal secretory responses occurred at A23187 concentrations of approximately 0.1 nM and above. This experiment was repeated four times using different batches of A23187, and similar results were obtained each time, indicating a steep doseresponse relationship between 50-100 nM A23187. The stimulatory effect of A23187 on aldosterone secretion has been reported previously to be biphasic. When examined in continous flow perifusion systems, A23187 evoked a large increase in the secretory rate that declined to a much lower, yet stable, rate after 15-20 min of stimulation (17, 18). In the static cell suspensions employed in the present study, A23187-induced aldosterone secretion also exhibited a biphasic response (Fig. 2). A23187 treatment induced an initial rapid increase in the rate of aldosterone secretion that diminished to an apparently stable, but much reduced, rate after approximately 15 min of stimulation. Under similar conditions, stimulation with elevated extracellular K+ (10 mM) induced a much larger increase in the rate of aldosterone secretion that remained constant for up to 1 h of treatment (data not shown). This experiment was repeated three times, and similar results were obtained each time. The absolute magnitude of aldosterone secretion induced by the secretagogues was somewhat variable throughout the course of these experiments, as can be seen when comparing Figs. 1 and 2. The reasons for this 200
100
a o
id A23187,
moles/liter
FIG. 1. Concentration dependence of A23187-induced aldosterone secretion in isolated suspensions of rat adrenal glomerulosa cells. Cells were incubated for 1 h in the presence of the indicated concentration of A23187; all samples contained 0.05% DMSO. Symbols indicate the mean ± SEM of triplicate determinations at each concentration from a single experiment. Lines were fit to the data by eye.
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INHIBITION OF ALDOSTERONE SECRETION
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stimulated aldosterone secretion approximately 5-fold greater than 15 ^g/g/ml 25-hydroxycholesterol treatment. Pregnenolone-induced stimulation was also not affected by the nominally Ca2+-free medium (data not shown). Effect ofANP on A23187 stimulation |
o
20
40 Time, min.
FlG. 2. Time course of aldosterone secretion from glomerulosa cell suspensions in the absence (D) and presence of either 0.1 jtM A23187 (0) or 1.0 nM A23187 (O). Separate cell aliquots were incubated for 0, 15, 30, 45, and 60 min. Each symbol represents the mean ± SEM of triplicate determination from a single experiment. Lines were fit to the data by eye.
variability were uncertain. Nevertheless, the patterns of secretion and relative order of maximal secretagogue potency remained constant; A23187 < angiotensin II < K+ < ACTH. Effect of low extracellular calcium
Decreasing extracellular Ca2+ has been shown to inhibit stimulation of aldosterone secretion induced by any of several different treatments (18-20). In agreement with previous studies (19, 21), omission of Ca2+ from the incubation medium completely inhibited A23187-induced aldosterone secretion (Table 1). This result indicated that A23187 stimulated aldosterone secretion by translocating Ca2+ across the plasma membrane, down its concentration gradient. The nominal Ca2+ concentration of the Ca2+-free medium was 4 /xU, based on atomic absorption spectroscopy. Inclusion of 0.5 mM EGTA in the nominally Ca2+-free medium did not further suppress aldosterone secretion in either the presence or absence of 0.5 ixM A23187. The activity of the biosynthetic pathway enzymes did not appear to be inhibited by low extracellular Ca2+, as omission of extracellular Ca2+ in the presence or absence of EGTA did not inhibit conversion of 25-hydroxycholesterol to aldosterone (Table 1). Additionally, the effect of the nominally Ca2+-free medium was tested on aldosterone secretion in response to treatment with 1 /ig/ml pregnenolone. Treatment with 1 pregnenolone in the presence of 1.25 mM Ca2+
Simultaneous treatment of the glomerulosa cell suspensions with ANP and A23187 substantially inhibited A23187-induced aldosterone secretion (Fig. 3). Low concentrations of ANP (10 and 100 pM) inhibited the response to A23187, but inhibition was overcome by higher concentrations of A23187. In the presence of 1 nM ANP, a maximally effective concentration for inhibition of hormone-stimulated aldosterone secretion, inhibition was not overcome by A23187 tested at concentrations of up to 10 /xM. This result suggested that maximally effective ANP doses noncompetitively inhibited the aldosterone response to A23187. The effect of ANP on A23187stimulated aldosterone secretion was tested in four separate experiments. The effect of increasing the extracellular Ca2+ concentration on A23187 stimulation of aldosterone secretion and ANP inhibition of stimulation was tested (Table 2). Raising the extracellular Ca2+ concentration from 1.25 to 5 mM increased the secretory response to A23187. This elevation of the extracellular Ca2+ concentration did not affect ANP-induced inhibition of aldosterone secretion stimulated by 0.1 or 1.0 nM A23187. This paradigm was tested in four separate experiments. Effect of ANP on dBcAMP stimulation We also tested the effect of ANP on cAMP-mediated stimulation of aldosterone secretion by treating cells with the membrane-permeant analog dBcAMP. Bath-applied dBcAMP induced a dose-dependent stimulation of aldosterone secretion, inducing maximal secretion at a bath concentration of approximately 1 mM and exhibiting an EC50 of approximately 0.1 mM (Fig. 4). Simultaneous treatment with 10 nM ANP partially inhibited the stimulation of aldosterone secretion by low concentrations of dBcAMP (Fig. 4). At dBcAMP concentrations of 0.5 mM or higher, 10 nM ANP did not inhibit the
TABLE 1. Effect of extracellular calcium on aldosterone secretion Aldosterone secretion (ng/h-10"6 cells)
Treatment Control 0.5 MM A23187 15 jig/ml 25-OH cholesterol
1.25 mM Ca++
0Ca ++
0 Ca++ + 0.5 mM EGTA
1.98 ± 0.62 43.13 ± 1.19 26.50 ± 1.95
2.12 ± 0.23 1.67 ± 0.03 26.20 ± 2.60
1.67 ± 0.03 2.02 ± 0.65 26.78 ± 3.80
Data indicates the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations in media containing the indicated calcium concentration. 0 Ca++ indicates a nominally calcium-free solution in which the calcium concentration was determined to be 4.0 j*M using atomic absorption spectroscopy.
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INHIBITION OF ALDOSTERONE SECRETION
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2
0
10
10 A23187,
10 moles/liter
1 0
FIG. 3. The effect of ANP on the stimulation of aldosterone secretion by increasing concentrations of A23187 in isolated glomerulosa cell suspensions. Cell suspensions were incubated for 1 h in the presence of either A23187 alone (•) or A23187 plus ANP at 10 pM (0), 100 pM (O), or 1 nM (A). Symbols indicate the mean ± SEM of triplicate determinations from a single experiment. Lines were fit to the data by eye. TABLE 2. Effect of extracellular calcium on ANP inhibition of A23187 stimulation of aldosterone secretion Aldosterone secretion (ng/h-HT6 cells)
Treatment 0.1 nM A23187 0.1 fiM A23187 + 1 nM ANP 1.0 /zM A23187 1.0 MM A23187 + 1 nM ANP
1.25 mM Ca
5.0 mM Ca
88.0 ± 9.8 4.0 ± 0.4 84.3 ± 4.6 5.2 ± 0.6
122.8 ± 6.1 6.7 ± 0.6 118.9 ± 4.2 5.0 ± 0.3
Data indicate the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations from a single experiment.
300r
S
200
100
0.2
0.4
0.6
0.8
1.0
Dibutyryl cAMP, mM
FlG. 4. The concentration dependence of dBcAMP-induced aldosterone secretion from isolated glomerulosa cell suspensions in the absence (D) and presence (O) of 10 nM ANP. Cells were incubated for 1 h; symbols indicate the mean ± SEM of triplicate determinations from a single experiment. Lines were fit to the data by eye.
stimulation of aldosterone secretion. This result was observed in two separate experiments. ANP inhibition of hormone plus A23187 stimulation Hormonal stimulation of glomerulosa cells may result in the activation of multiple second messenger systems involved in stimulation of aldosterone secretion. We, therefore, examined whether the reported ANP inhibition of Ca2+ influx (14, 16) was a primary component of ANP-mediated inhibition of the ACTH-, angiotensin
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II-, or K+-stimulated aldosterone secretion. This was accomplished by treating glomerulosa cells with hormone alone and in combination with A23187, ANP, or A23187 plus ANP (Table 3). Using this paradigm, stimulation of aldosterone secretion by 10 pM ACTH, 1 nM angiotensinII, or 10 mM K+ was partially inhibited by simultaneous treatment with 1 nM A2 3187. However, 10 nM ANP caused a much greater inhibition of hormone-stimulated aldosterone secretion than that observed with A23187. Addition of 1 iiM A23187 plus 10 nM ANP resulted in further inhibition of secretion relative to that caused by ANP alone. This paradigm was performed in two separate experiments, in which similar results were obtained. Discussion The results of the present study, in agreement with previous studies (19, 21), demonstrated that the Ca2+ ionophore A23187 caused a dose-dependent Ca2+-dependent stimulation of aldosterone secretion from glomerulosa cells. These results suggested that elevation of the intracellular free Ca2+ concentration is sufficient to initiate a cascade of intracellular reactions, resulting in the stimulation of aldosterone secretion. The dependence of A23187-induced stimulation on extracellular Ca2+ indicated that stimulation was mediated by A23187 translocation of extracellular Ca2+ down its concentration gradient, across the plasma membrane, to increase the intracellular free Ca2+ concentration. A23187 would also have been expected to release sequestered Ca2+ from intracellular stores. Apparently, the absence of sufficient extracellular Ca2+ precluded the latter effect from stimulating aldosterone secretion, possibly due to a rapid depletion of cellular Ca2+ pools. ANP inhibition of A23187-stimulated aldosterone secretion suggested that a primary mechanism by which ANP inhibits hormone-stimulated aldosterone secretion is through blockade of an intracellular step in a Ca2+activated cascade of events, leading to stimulation of the early pathway of aldosterone secretion. ANP appeared to noncompetitively inhibit this intracellular Ca2+-activated process. This was indicated by the inability of increasing the A23187 concentration alone or in combination with increased extracellular Ca2+ to overcome maximal ANP inhibition of aldosterone secretion. Increasing A23187 and extracellular Ca2+ would have been expected to induce a greater increase in intracellular free Ca2+; the increased extracellular Ca2+ concentration would also have been expected to reduce depletion of sequestered intracellular Ca2+ pools that may be important for processes such as protein synthesis (22) or for stimulation of steroidogenesis (23). The hypothesis that ANP acted through inhibition of an intracellular Ca2+-activated pathway is dependent
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TABLE 3. Effect of A23187 on ANP inhibition of hormone-stimulated aldosterone secretion Aldosterone secretion (ng/h-HT6 cells) Stimulus
10 pM ACTH 1 nM angiotensin II 10 mM K+
Control
1.0 MM A23187
10 nM ANP
1.0 MM A23187 + 10nM ANP
24.0 ± 0.2 14.4 ± 1.8 24.8 ± 1.8
14.5 ± 0.2 11.5 ± 1.1 14.8 ± 1.2
6.9 ± 0.5 2.7 ± 0.5 2.2 ± 0.5
4.5 ± 0.1 1.7 ±0.1 1.7 ± 0.2
Data indicate the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations from a single experiment. Control values indicate aldosterone secretion in the presence of stimulus alone, and subsequent columns indicate aldosterone secretion in the presence of stimulus plus the indicated additions.
upon our interpretation of the mechanism of A23187 stimulation, i.e. by increasing the intracellular free Ca2+ concentration. The involvement of intracellular Ca2+ as a mediator of hormone-induced aldosterone secretion is well established (24), although the events occurring subsequent to increased intracellular Ca2+ are poorly understood. The primary evidence indicating that A23187induced aldosterone secretion is mediated by increased intracellular Ca2+ is the observation that decreasing the extracellular Ca2+ concentration inhibited A23187 stimulation. In support of this observation, A23187-stimulated aldosterone secretion has also been reported to be inhibited by calmodulin antagonists (25). Reduction of extracellular Ca2+ has been shown previously to inhibit stimulation of aldosterone secretion by all of the commonly tested secretagogues (26) through effects on either or both of the early and late pathways (20). In the present study reduction of extracellular Ca2+ did not significantly inhibit basal aldosterone secretion or the ability of the cell suspensions to convert 25-hydroxycholesterol to aldosterone. These observations indicated that cells remained viable, and enzymes of the biosynthetic pathway were not adversely affected by low extracellular Ca2+. ANP inhibition of an intracellular Ca2+-activated step is consistent with the results of several previous studies, which collectively indicated ANP must inhibit angiotensin-II-induced aldosterone secretion subsequent to angiotensin-II-stimulated Ca2+ influx (4, 6, 10-13). Angiotensin-II stimulation has been shown to induce a sustained increase in Ca2+ infux, and this sustained Ca2+ influx is thought to be primarily responsible for angiotensin-II-induced aldosterone secretion (18, 27-29). ANP-induced inhibition of ACTH-stimulated aldosterone secretion may also be consistent with ANP inhibition of an intracellar Ca2+-dependent process. The inhibitory effect of ANP on ACTH-stimulated aldosterone secretion can be overcome by increasing the concentration of ACTH (30). It may be interpreted that higher doses of ACTH are required to overcome ANP inhibition of ACTH-stimulated cAMP synthesis (31). However, ACTH also has been shown to stimulate Ca2+ influx (14, 32, 33), and this effect appears to be independent of
ACTH-stimulated cAMP synthesis (34). The cAMP second messenger system is reported to act synergistically with the Ca2+ second messenger system in stimulating aldosterone (17;, 20, 35). Thus, if ANP acted through inhibition of an intracellular Ca2+-dependent reaction as well as partially inhibiting cAMP synthesis, then the observed ANP-induced shift to the right of the ACTH dose-response carve for aldosterone secretion might be expected. It remains to be determined whether ANP inhibition of dBcAMP-stimulated aldosterone secretion, observed in the present study and in previous reports (4, 20), represented a Ca2+-dependent component of dBcAMP-induced stimulation of aldosterone. ANP has been reported to inhibit T-type Ca2+ channels in glomerulosa cells (16). If ANP acted only by inhibiting T-type Ca2+ channels, it would not have been expected to inhibit A23187-induced aldosterone secretion, as observed in the present study. Furthermore, A23187 might have been expected to reduce the inhibitory effects of ANP on hormone- or K+-stimulated aldosterone secretion, and this result was not observed in the present study. Previously, A23187 was not found to overcome ANP inhibition of aldosterone secretion induced by either phorbol ester (10) or phorbol ester and angiotensin-II (11). In addition to the stimulatory effect of A23187 on aldosterone secretion, A23187 was shown to inhibit stimulation of aldosterone secretion by other hormones in the present as well as previous studies (21). This inhibitory effect of A23187 may have accounted for the biphasic nature of A23187-induced aldosterone secretion observed in the static cell suspension method employed in the present study as well as in perifusion systems (17, 18). The mechanism of A23187-induced inhibition of aldosterone secretion is unknown; however, A23187 treatment has been found to inhibit protein synthesis in glomerulosa cells (36) as well as to generally suppress phospholipid turnover (37). Both processes have been implicated in pathways leading to stimulation of steroidogenesis (8, 9, 23). On the other hand, A23187 has been shown to act synergistically with phorbol esters (18) and forskolin (17), changing a transient A23187-induced al-
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dosterone secretory response to a sustained response. These results might suggest that forskolin and phorbol esters produce effects that oppose the inhibitory effects of A23187, as opposed to potentiating stimulatory effects. It is also of interest that this synergism has only been observed in bovine glomerulosa cells and not in those from the rat.(38). In summary, the ability of ANP to inhibit A23187induced aldosterone secretion provides compelling evidence for ANP-mediated inhibition of an intracellular process necessary for the Ca2+ second messenger system to stimulate aldosterone biosynthesis in the adrenal glomerulosa cells. Therefore, the effects of ANP on glomerulosa cells may provide a useful tool for defining intracellular reactions important for the stimulation of aldosterone biosynthesis.
References 1. Brenner BM, Ballerman BJ, Gunning ME, Zeidal ML 1990 Diverse biological actions of atrial natriuretic peptide. Physiol Rev 70:665699 2. Atarashi K, Mulrow PJ, Franco-Saenz R, Snajdar R, Rapp J 1984 Inhibition of aldosterone production by an atrial extract. Science 224:992-994 3. De Lean A, Racz K, Gutkowska J, Nguyen T-T, Cantin M, Genest J 1984 Specific receptor-mediated inhibition by synthetic atrial natriuretic factor of hormone-stimulated steroidogenesis in cultured bovine adrenal cells. Endocrinology 115:1636-1638 4. Goodfriend TL, Elliott ME, Atlas SA 1984 Actions of synthetic atrial natriuretic factor on bovine adrenal glomerulosa. Life Sci 35:1675-1682 5. Campbell WB, Currie MG, Needleman P 1985 Inhibition of aldosterone biosynthesis by atriopeptins in rat adrenal cells. Circ Res 57:113-118 6. Racz K, Kuchel 0, Cantin M, De Lean A 1985 Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex. FEBS Lett 192:19-22 7. Schiebinger RJ, Kern DC, Brown RD 1988 Effect of atrial natriuretic peptide on ACTH, dibutyryl cAMP, angiotensin II, and potassium-stimulated aldosterone secretion by rat adrenal glomerulosa cells. Life Sci 42:919-926 8. Simpson ER, Waterman MR 1983 Regulation by ACTH of steroid hormone biosynthesis in adrenal cortex. Can J Biochem Cell Biol 61:692-707 9. Jefcoate CR, McNamara BC, DiBartolomeis MJ 1986 Control of steroid synthesis in adrenal fasciculata cells. Endocr Res 12:315— 350 10. Isales CM, Bollag WB, Kiernan LC, Barret PQ 1989 Effect of ANP on sustained aldosterone secretion stimulated by angiotensin II. Am J Physiol 256:C89-C95 11. Ganguly A, Chiou S, West LA, Davis JS 1989 Atrial natriuretic factor inhibits angiotensin-induced aldosterone secretion: not through cGMP or interference with phospholipase C. Biochem Biophys Res Commun 159:148-154 12. Capponi AM, Lew PD, Jarnot L, Valloton MB 1986 Effect of atrial natriuretic peptide on the stimulation by angiotensin II of various target cells. J Hypertension [Suppl] 4:S61-S65 13. Apfeldorf WJ, Isales CM, Barrett PQ 1988 Atrial natriuretic peptide inhibits the stimulation of aldosterone secretion but not the transient increase in intracellular free calcium concentration induced by angiotensin II addition. Endocrinology 122:1460-1465 14. Chartier L, Schiffrin EL 1987 Role of calcium in effects of atrial natriuretic peptide on aldosterone production in adrenal glomerulosa cells. Am J Physiol 252:E485-E491 15. Takagi M, Takagi M, Franco-Saenz R, Shier D, Mulrow PJ 1988 Effects of atrial natriuretic factor on calcium fluxes in adrenal
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glomerulosa cells. Hypertension 11:433-439 16. McCarthy RT, Isales CM, Bollag WB, Rasmussen H, Barrett PQ 1990 Atrial natriuretic peptide differentially modulates T- and Ltype calcium channels. Am J Physiol 258:F473-F478 17. Kojima I, Kojima K, Rasmussen H 1985 Intracellular calcium and adenosine 3',5'-cyclic monophosphate as mediators of potassiuminduced aldsoterone secretion. Biochem J 228:69-76 18. Kojima I, Kojima K, Rasmussen H 1985 Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells. J Biol Chem 260:9177-9184 19. Foster R, Lobo MV, Rasmussen H, Marusic ET 1981 Calcium: its role in the mechanism of action of angiotensin II and potassium in aldosterone production. Endocrinology 109:2196-2201 20. Schiebinger RJ, Braley LM, Menachery A, Williams GH 1986 Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat. J Endocrinol 110:315-325 21. Fakunding JL, Catt KJ 1982 Calcium-dependent regulation of aldosterone production in isolated adrenal glomerulosa cells: effects of the ionophore A23187. Endocrinology 110:2006-2010 22. Brostrom CO, Brostrom MA 1990 Calcium-dependent regulation of protein synthesis in intact mammalian cells. Annu Rev Physiol 52:577-590 23. Pederson RC 1985 Polypeptide activators of cholesterol side chain cleavage. Endocr Res 10:533-561 24. Quinn SJ, Williams GH 1988 Regulation of aldosterone secretion. Annu Rev Physiol 50:409-426 25. Wilson JX, Aguilera G, Catt KJ 1984 Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells. Endocrinology 115:1357-1363 26. Fakunding JL, Chow R, Catt KJ 1979 The role of calcium in the stimulation of aldosterone production by adrenocorticotropin, angiotensin II, and potassium in isolated glomerulosa cells. Endocrinology 105:327-333 27. Capponi AM, Lew PD, Vallotton MB 1987 Qualitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine glomerulosa cells. Biochem J 247:335-340 28. Pratt JH, Rothrock JK, Dominguez JH 1989 Evidence that angiotensin II and potassium collaborate to increase cytosolic calcium and stimulate the secretion of aldosterone. Endocrinology 125:2463-2469 29. Quinn SJ, Enyedi P, Tillotson DL, Williams GH 1990 Cytosolic calcium and aldosterone response patterns of rat adrenal glomerulosa cells stimulated by vasopressin: comparison with angiotensin II. Endocrinology 127:541-548 30. Atarashi K, Mulrow PJ, Franco-Saenz R 1985 Effect of atrial peptides on aldosterone production. J Clin Invest 76:1807-1811 31. Anand-Srivastava MB, Genest J, Cantin M 1985 Inhibitory effect of atrial natriuretic factor on adenylate cyclase activity in adrenal cortical membranes. FEBS Lett 181:199-202 32. Kojima I, Kojima K, Rasmussen H 1985 Characteristics of angiotensin II-, K+-, and ACTH-induced calcium influx in adrenal glomerulosa cells. J Biol Chem 260:9171-9176 33. Payet MD, Benabderrazik M, Gallo-Payet N 1987 Excitationsecretion coupling: ionic currents in glomerulosa cells: effects of adrenocorticotropin and K+ channel blockers. Endocrinology 121:875-882 34. Kojima I, Ogata E 1986 Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell. J Biol Chem 261:9832-9838 35. Kojima I, Kojima K, Rasmussen H 1985 Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion. J Biol Chem 260:4248-4256 36. Farese RV, Sabir MA, Larson RE 1981 A23187 inhibits adrenal protein synthesis and the effects of adrenocorticotropin (ACTH) on steroidogenssis and phospholipid metabolism in rat adrenal cells in vitro: further evidence implicating phospholipids in the steroidogenic action of ACTH. Endocrinology 108:1243-1250 37. Farese RV, Larson RE, Sabir MA, Gomez-Sanchez CE 1983 Effect of angiotensin II, K+, adrenocorticotropin, serotonin, adenosine 3',5'-monophosphate, guanosine 3',5'-monophosphate, A23187, and EGTA on aldosterone synthesis and phospholipid metabolism in the rat adrenal zona glomerulosa. Endocrinology 113:1377-1386 38. Spat A 1988 Stimulus secretion coupling in angiotensin-stimulated adrenal glomerulosa cells. J Steroid Biochem 29:443-453
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Vol. 129, No. 5 Printed in U.S.A.
Atrial Natriuretic Peptide Inhibition of Calcium Ionophore A23187-Stimulated Aldosterone Secretion in Rat Adrenal Glomerulosa Cells* DAVID P. LOTSHAWf, ROBERTO FRANCO-SAENZ, AND PATRICK J. MULROW Department of Medicine, Medical College of Ohio, Toledo, Ohio 43699
ABSTRACT. The effect of atrial natriuretic peptide (ANP) on calcium ionophore A23187-stimulated aldosterone secretion was investigated using collagenase-dispersed rat adrenal glomerulosa cell suspensions. A23187 treatment induced a dose-dependent stimulation of aldosterone secretion, exhibiting an EC6o of approximately 75 nM. In agreement with the presumed action of A23187 as a Ca2+ ionophore, stimulation was dependent on the extracellular Ca2+ concentration, being completely inhibited in nominally Ca2+-free medium. In such Ca2+-free medium, stimulation of aldosterone secretion by bath applied 25-hydroxycholesterol was not inhibited, indicating that cells and biosynthetic pathway enzymes were not inhibited by low extracellular Ca2+ levels. A23187-induced aldosterone secretion was also inhibited by more than 90% when cells were simultaneously treated with ANP. Maximal ANP inhibition of A23187-stimulated aldoste-
rone secretion was not overcome by concentrations of A23187 up to 10 /*M or by increasing the extracellular Ca2+ concentration from 1.25 to 5 mM in the presence of A23187 and ANP. Addition of A23187 to ACTH-, angiotensin II-, or K+-stimulated glomerulosa cells did not overcome ANP-induced inhibition of aldosterone secretion stimulated by these secretagogues. In contrast to ANP inhibition of Ca2+-dependent A23187 stimulation of aldosterone secretion, ANP inhibition of dBcAMP-stimulated aldosterone secretion was readily overcome by increasing the dBcAMP concentration. These results indicated that ANP selectively and noncompetetively inhibited an intracellular step necessary for Ca2+-dependent stimulation of the early pathway of aldosterone biosynthesis in rat adrenal glomerulosa cells. (Endocrinology 129: 2305-2310, 1991)
A
TRIAL natriuretic peptide (ANP) is a hormone secreted primarily from the atria that exerts effects on multiple target organs, the net effect of which is a reduction of blood volume and arterial blood pressure (1). The zona glomerulosa layer of the adrenal cortex is one such target tissue, and here ANP acts as a potent inhibitor of hormone-stimulated aldosterone secretion (2, 3). Although several studies have investigated ANP-induced inhibition of aldosterone secretion, the mechanism(s) of this inhibition has not been elucidated. The primary effect of ANP on aldosterone secretion is thought to be mediated through receptor-induced inhibition of hormonal stimulation of the early pathway of aldosterone biosynthesis (4-7). Hormone stimulation of the early pathway involves the regulation of a complex Received May 6,1991. Address all correspondence and requests for reprints to: Dr. Patrick J. Mulrow, Department of Medicine, Medical College of Ohio, CS 10008, Toledo, Ohio 43699. * An abstract of these data was published previously in the Program of the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990, p. 399. This work was supported in part by a grant from the American Heart Association, Ohio Affiliate, Inc. (Columbus, OH). t Present address: T. H. Morgan School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506-0225.
and poorly understood system of cholesterol mobilization and delivery to the inner mitochondrial membrane for the initial side-chain cleavage reaction converting cholesterol to pregnenolone (8, 9). Therefore, most studies have focused on possible ANP effects on hormone-coupled second messenger systems that stimulate aldosterone biosynthesis. These studies have suggested that ANP-induced inhibition occurs subsequent to stimulatory hormone binding to its receptor and initiation of second messenger cascades. For example, ANP was shown to be a potent inhibitor of angiotensin-II-stimulated aldosterone secretion, but did not affect angiotensin-II receptor binding (4, 6), angiotensin-II-induced phosphatidylinositol hydrolysis (4, 10, 11), or angiotensin-II-induced elevation of intracellular Ca2+ (10,12,13). The effect of ANP on hormone-stimulated aldosterone secretion has been reported to correlate with the sensitivity of stimulation to inhibition by Ca2+ channel blockers (14). Several studies have examined the effect of ANP on hormone-stimulated Ca2+ fluxes across the glomerulosa cell plasma membrane, but contradictory results have been obtained. Most studies employing 45Ca2+ flux have reported no effect of ANP on angiotensin-IIor K+-induced Ca2+ influx or efflux (10,13,15); however, one study reported that ANP inhibited ACTH-, angio-
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INHIBITION OF ALDOSTERONE SECRETION
tensin-II-, and K+-stimulated Ca2+ influx (14). In contrast to most results of radiolabeled Ca2+ flux, electrophysiological experiments indicated that ANP inhibited a voltage-dependent T-type Ca2+ current in the glomerulosa cells (16). In the present study we have attempted to further define the mechanism of ANP-induced inhibition of aldosterone secretion by using the Ca2+ ionophore A23187 to elevate intracellular Ca2+, bypassing plasma membrane control of Ca2+ flux.
Materials and Methods All protocols employing animals were approved by the institutional animal care committee at the Medical College of Ohio. Isolated adrenal zona glomerulosa cell suspensions were prepared using a collagenase dispersion method similar to that previously described (15). Briefly, female Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing between 175-200 g, were killed, and adrenal glands were removed. The adrenal glands were dissected free of adherent fat, and capsules containing the zona glomerulosa layer were removed, minced into small pieces, and rinsed several times with fresh medium. The capsule fragments were digested in a modified Hanks' salts-medium 199 (M199; Sigma, St. Louis, MO) containing 1.5 mg/ml collagenase (Cooper Biochemicals, Malvern, PA) and 25 g/ml DNase (Sigma) for 1 h at 37 C in a shaking water bath. Hanks' saltsM199 was modified to contain 3.5 mM K+, 1.25 mM Ca2+, 0.2% BSA, 50 U/ml penicillin, 50 Mg/ml streptomycin, and 5 mM HEPES buffer, pH 7.4; the medium and atmosphere over the medium were equilibrated with 100% O2. Digested capsules were triturated, and the cell suspension was filtered through a 10-nm nylon mesh screen to obtain isolated cells. Isolated cells were washed four times with fresh medium by repeating a centrifugation-resuspension cycle, then equilibrated at 37 C in a shaking water bath at a cell density of approximately 200,000 cells/ml for between 1-2 h before experimentation. The cell preparation consisted primarily of zona glomerulosa cells contaminated by approximately 10% fasciculata cells; less than 10% of the total cells were stained by trypan blue. After the equilibration period, cells were pelleted by centrifugation and resuspended in fresh M199. The cell suspension was aliquoted to a cell density of approximately 50,000 cells/ ml in a final volume of 1 ml containing drugs and hormones or appropriate vehicle. Cells were incubated over a variable duration of up to 1.5 h at 37 C in a shaking water bath. Incubations were terminated by rapidly lowering the temperature of the cell suspensions in an ice bath; cells were pelleted by centrifugation, and the supernatants were stored at -20 C until assayed for aldosterone content. Aldosterone in the medium was quantified without prior extraction, using a commercial aldosterone RIA (Diagnostic Products, Los Angeles, CA). The inter- and intrassay coefficients of variation at an aldosterone concentration of 100 pg/ ml were 6.9% and 5.4%, respectively. A23187, 25-hydroxycholesterol, (Bu)2cAMP (dBcAMP), angiotensin-II, and ACTH, were obtained from Sigma. Rat ANP was obtained from Peninsula (Belmont, CA). A23187 was dis-
Endo • 1991 Vol 129 • No 5
solved in dimethylsulfoxide (DMSO), and the final DMSO concentration did not exceed 0.1%; 25-hydroxycholesterol was dissolved in ethanol, and the final ethanol concentration did not exceed 0.1%.
Results A23187-stimulated aldosterone secretion Treatment of glomerulosa cell suspensions with the calcium ionophore A23187 induced a dose-dependent stimulation of aldosterone secretion (Fig. 1). A23187 stimulation exhibited an EC5o of approximately 75 nM for incubations of 1-h duration, and maximal secretory responses occurred at A23187 concentrations of approximately 0.1 nM and above. This experiment was repeated four times using different batches of A23187, and similar results were obtained each time, indicating a steep doseresponse relationship between 50-100 nM A23187. The stimulatory effect of A23187 on aldosterone secretion has been reported previously to be biphasic. When examined in continous flow perifusion systems, A23187 evoked a large increase in the secretory rate that declined to a much lower, yet stable, rate after 15-20 min of stimulation (17, 18). In the static cell suspensions employed in the present study, A23187-induced aldosterone secretion also exhibited a biphasic response (Fig. 2). A23187 treatment induced an initial rapid increase in the rate of aldosterone secretion that diminished to an apparently stable, but much reduced, rate after approximately 15 min of stimulation. Under similar conditions, stimulation with elevated extracellular K+ (10 mM) induced a much larger increase in the rate of aldosterone secretion that remained constant for up to 1 h of treatment (data not shown). This experiment was repeated three times, and similar results were obtained each time. The absolute magnitude of aldosterone secretion induced by the secretagogues was somewhat variable throughout the course of these experiments, as can be seen when comparing Figs. 1 and 2. The reasons for this 200
100
a o
id A23187,
moles/liter
FIG. 1. Concentration dependence of A23187-induced aldosterone secretion in isolated suspensions of rat adrenal glomerulosa cells. Cells were incubated for 1 h in the presence of the indicated concentration of A23187; all samples contained 0.05% DMSO. Symbols indicate the mean ± SEM of triplicate determinations at each concentration from a single experiment. Lines were fit to the data by eye.
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INHIBITION OF ALDOSTERONE SECRETION
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stimulated aldosterone secretion approximately 5-fold greater than 15 ^g/g/ml 25-hydroxycholesterol treatment. Pregnenolone-induced stimulation was also not affected by the nominally Ca2+-free medium (data not shown). Effect ofANP on A23187 stimulation |
o
20
40 Time, min.
FlG. 2. Time course of aldosterone secretion from glomerulosa cell suspensions in the absence (D) and presence of either 0.1 jtM A23187 (0) or 1.0 nM A23187 (O). Separate cell aliquots were incubated for 0, 15, 30, 45, and 60 min. Each symbol represents the mean ± SEM of triplicate determination from a single experiment. Lines were fit to the data by eye.
variability were uncertain. Nevertheless, the patterns of secretion and relative order of maximal secretagogue potency remained constant; A23187 < angiotensin II < K+ < ACTH. Effect of low extracellular calcium
Decreasing extracellular Ca2+ has been shown to inhibit stimulation of aldosterone secretion induced by any of several different treatments (18-20). In agreement with previous studies (19, 21), omission of Ca2+ from the incubation medium completely inhibited A23187-induced aldosterone secretion (Table 1). This result indicated that A23187 stimulated aldosterone secretion by translocating Ca2+ across the plasma membrane, down its concentration gradient. The nominal Ca2+ concentration of the Ca2+-free medium was 4 /xU, based on atomic absorption spectroscopy. Inclusion of 0.5 mM EGTA in the nominally Ca2+-free medium did not further suppress aldosterone secretion in either the presence or absence of 0.5 ixM A23187. The activity of the biosynthetic pathway enzymes did not appear to be inhibited by low extracellular Ca2+, as omission of extracellular Ca2+ in the presence or absence of EGTA did not inhibit conversion of 25-hydroxycholesterol to aldosterone (Table 1). Additionally, the effect of the nominally Ca2+-free medium was tested on aldosterone secretion in response to treatment with 1 /ig/ml pregnenolone. Treatment with 1 pregnenolone in the presence of 1.25 mM Ca2+
Simultaneous treatment of the glomerulosa cell suspensions with ANP and A23187 substantially inhibited A23187-induced aldosterone secretion (Fig. 3). Low concentrations of ANP (10 and 100 pM) inhibited the response to A23187, but inhibition was overcome by higher concentrations of A23187. In the presence of 1 nM ANP, a maximally effective concentration for inhibition of hormone-stimulated aldosterone secretion, inhibition was not overcome by A23187 tested at concentrations of up to 10 /xM. This result suggested that maximally effective ANP doses noncompetitively inhibited the aldosterone response to A23187. The effect of ANP on A23187stimulated aldosterone secretion was tested in four separate experiments. The effect of increasing the extracellular Ca2+ concentration on A23187 stimulation of aldosterone secretion and ANP inhibition of stimulation was tested (Table 2). Raising the extracellular Ca2+ concentration from 1.25 to 5 mM increased the secretory response to A23187. This elevation of the extracellular Ca2+ concentration did not affect ANP-induced inhibition of aldosterone secretion stimulated by 0.1 or 1.0 nM A23187. This paradigm was tested in four separate experiments. Effect of ANP on dBcAMP stimulation We also tested the effect of ANP on cAMP-mediated stimulation of aldosterone secretion by treating cells with the membrane-permeant analog dBcAMP. Bath-applied dBcAMP induced a dose-dependent stimulation of aldosterone secretion, inducing maximal secretion at a bath concentration of approximately 1 mM and exhibiting an EC50 of approximately 0.1 mM (Fig. 4). Simultaneous treatment with 10 nM ANP partially inhibited the stimulation of aldosterone secretion by low concentrations of dBcAMP (Fig. 4). At dBcAMP concentrations of 0.5 mM or higher, 10 nM ANP did not inhibit the
TABLE 1. Effect of extracellular calcium on aldosterone secretion Aldosterone secretion (ng/h-10"6 cells)
Treatment Control 0.5 MM A23187 15 jig/ml 25-OH cholesterol
1.25 mM Ca++
0Ca ++
0 Ca++ + 0.5 mM EGTA
1.98 ± 0.62 43.13 ± 1.19 26.50 ± 1.95
2.12 ± 0.23 1.67 ± 0.03 26.20 ± 2.60
1.67 ± 0.03 2.02 ± 0.65 26.78 ± 3.80
Data indicates the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations in media containing the indicated calcium concentration. 0 Ca++ indicates a nominally calcium-free solution in which the calcium concentration was determined to be 4.0 j*M using atomic absorption spectroscopy.
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INHIBITION OF ALDOSTERONE SECRETION
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2
0
10
10 A23187,
10 moles/liter
1 0
FIG. 3. The effect of ANP on the stimulation of aldosterone secretion by increasing concentrations of A23187 in isolated glomerulosa cell suspensions. Cell suspensions were incubated for 1 h in the presence of either A23187 alone (•) or A23187 plus ANP at 10 pM (0), 100 pM (O), or 1 nM (A). Symbols indicate the mean ± SEM of triplicate determinations from a single experiment. Lines were fit to the data by eye. TABLE 2. Effect of extracellular calcium on ANP inhibition of A23187 stimulation of aldosterone secretion Aldosterone secretion (ng/h-HT6 cells)
Treatment 0.1 nM A23187 0.1 fiM A23187 + 1 nM ANP 1.0 /zM A23187 1.0 MM A23187 + 1 nM ANP
1.25 mM Ca
5.0 mM Ca
88.0 ± 9.8 4.0 ± 0.4 84.3 ± 4.6 5.2 ± 0.6
122.8 ± 6.1 6.7 ± 0.6 118.9 ± 4.2 5.0 ± 0.3
Data indicate the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations from a single experiment.
300r
S
200
100
0.2
0.4
0.6
0.8
1.0
Dibutyryl cAMP, mM
FlG. 4. The concentration dependence of dBcAMP-induced aldosterone secretion from isolated glomerulosa cell suspensions in the absence (D) and presence (O) of 10 nM ANP. Cells were incubated for 1 h; symbols indicate the mean ± SEM of triplicate determinations from a single experiment. Lines were fit to the data by eye.
stimulation of aldosterone secretion. This result was observed in two separate experiments. ANP inhibition of hormone plus A23187 stimulation Hormonal stimulation of glomerulosa cells may result in the activation of multiple second messenger systems involved in stimulation of aldosterone secretion. We, therefore, examined whether the reported ANP inhibition of Ca2+ influx (14, 16) was a primary component of ANP-mediated inhibition of the ACTH-, angiotensin
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II-, or K+-stimulated aldosterone secretion. This was accomplished by treating glomerulosa cells with hormone alone and in combination with A23187, ANP, or A23187 plus ANP (Table 3). Using this paradigm, stimulation of aldosterone secretion by 10 pM ACTH, 1 nM angiotensinII, or 10 mM K+ was partially inhibited by simultaneous treatment with 1 nM A2 3187. However, 10 nM ANP caused a much greater inhibition of hormone-stimulated aldosterone secretion than that observed with A23187. Addition of 1 iiM A23187 plus 10 nM ANP resulted in further inhibition of secretion relative to that caused by ANP alone. This paradigm was performed in two separate experiments, in which similar results were obtained. Discussion The results of the present study, in agreement with previous studies (19, 21), demonstrated that the Ca2+ ionophore A23187 caused a dose-dependent Ca2+-dependent stimulation of aldosterone secretion from glomerulosa cells. These results suggested that elevation of the intracellular free Ca2+ concentration is sufficient to initiate a cascade of intracellular reactions, resulting in the stimulation of aldosterone secretion. The dependence of A23187-induced stimulation on extracellular Ca2+ indicated that stimulation was mediated by A23187 translocation of extracellular Ca2+ down its concentration gradient, across the plasma membrane, to increase the intracellular free Ca2+ concentration. A23187 would also have been expected to release sequestered Ca2+ from intracellular stores. Apparently, the absence of sufficient extracellular Ca2+ precluded the latter effect from stimulating aldosterone secretion, possibly due to a rapid depletion of cellular Ca2+ pools. ANP inhibition of A23187-stimulated aldosterone secretion suggested that a primary mechanism by which ANP inhibits hormone-stimulated aldosterone secretion is through blockade of an intracellular step in a Ca2+activated cascade of events, leading to stimulation of the early pathway of aldosterone secretion. ANP appeared to noncompetitively inhibit this intracellular Ca2+-activated process. This was indicated by the inability of increasing the A23187 concentration alone or in combination with increased extracellular Ca2+ to overcome maximal ANP inhibition of aldosterone secretion. Increasing A23187 and extracellular Ca2+ would have been expected to induce a greater increase in intracellular free Ca2+; the increased extracellular Ca2+ concentration would also have been expected to reduce depletion of sequestered intracellular Ca2+ pools that may be important for processes such as protein synthesis (22) or for stimulation of steroidogenesis (23). The hypothesis that ANP acted through inhibition of an intracellular Ca2+-activated pathway is dependent
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TABLE 3. Effect of A23187 on ANP inhibition of hormone-stimulated aldosterone secretion Aldosterone secretion (ng/h-HT6 cells) Stimulus
10 pM ACTH 1 nM angiotensin II 10 mM K+
Control
1.0 MM A23187
10 nM ANP
1.0 MM A23187 + 10nM ANP
24.0 ± 0.2 14.4 ± 1.8 24.8 ± 1.8
14.5 ± 0.2 11.5 ± 1.1 14.8 ± 1.2
6.9 ± 0.5 2.7 ± 0.5 2.2 ± 0.5
4.5 ± 0.1 1.7 ±0.1 1.7 ± 0.2
Data indicate the mean ± SEM of triplicate determinations of aldosterone secretion during 1-h incubations from a single experiment. Control values indicate aldosterone secretion in the presence of stimulus alone, and subsequent columns indicate aldosterone secretion in the presence of stimulus plus the indicated additions.
upon our interpretation of the mechanism of A23187 stimulation, i.e. by increasing the intracellular free Ca2+ concentration. The involvement of intracellular Ca2+ as a mediator of hormone-induced aldosterone secretion is well established (24), although the events occurring subsequent to increased intracellular Ca2+ are poorly understood. The primary evidence indicating that A23187induced aldosterone secretion is mediated by increased intracellular Ca2+ is the observation that decreasing the extracellular Ca2+ concentration inhibited A23187 stimulation. In support of this observation, A23187-stimulated aldosterone secretion has also been reported to be inhibited by calmodulin antagonists (25). Reduction of extracellular Ca2+ has been shown previously to inhibit stimulation of aldosterone secretion by all of the commonly tested secretagogues (26) through effects on either or both of the early and late pathways (20). In the present study reduction of extracellular Ca2+ did not significantly inhibit basal aldosterone secretion or the ability of the cell suspensions to convert 25-hydroxycholesterol to aldosterone. These observations indicated that cells remained viable, and enzymes of the biosynthetic pathway were not adversely affected by low extracellular Ca2+. ANP inhibition of an intracellular Ca2+-activated step is consistent with the results of several previous studies, which collectively indicated ANP must inhibit angiotensin-II-induced aldosterone secretion subsequent to angiotensin-II-stimulated Ca2+ influx (4, 6, 10-13). Angiotensin-II stimulation has been shown to induce a sustained increase in Ca2+ infux, and this sustained Ca2+ influx is thought to be primarily responsible for angiotensin-II-induced aldosterone secretion (18, 27-29). ANP-induced inhibition of ACTH-stimulated aldosterone secretion may also be consistent with ANP inhibition of an intracellar Ca2+-dependent process. The inhibitory effect of ANP on ACTH-stimulated aldosterone secretion can be overcome by increasing the concentration of ACTH (30). It may be interpreted that higher doses of ACTH are required to overcome ANP inhibition of ACTH-stimulated cAMP synthesis (31). However, ACTH also has been shown to stimulate Ca2+ influx (14, 32, 33), and this effect appears to be independent of
ACTH-stimulated cAMP synthesis (34). The cAMP second messenger system is reported to act synergistically with the Ca2+ second messenger system in stimulating aldosterone (17;, 20, 35). Thus, if ANP acted through inhibition of an intracellular Ca2+-dependent reaction as well as partially inhibiting cAMP synthesis, then the observed ANP-induced shift to the right of the ACTH dose-response carve for aldosterone secretion might be expected. It remains to be determined whether ANP inhibition of dBcAMP-stimulated aldosterone secretion, observed in the present study and in previous reports (4, 20), represented a Ca2+-dependent component of dBcAMP-induced stimulation of aldosterone. ANP has been reported to inhibit T-type Ca2+ channels in glomerulosa cells (16). If ANP acted only by inhibiting T-type Ca2+ channels, it would not have been expected to inhibit A23187-induced aldosterone secretion, as observed in the present study. Furthermore, A23187 might have been expected to reduce the inhibitory effects of ANP on hormone- or K+-stimulated aldosterone secretion, and this result was not observed in the present study. Previously, A23187 was not found to overcome ANP inhibition of aldosterone secretion induced by either phorbol ester (10) or phorbol ester and angiotensin-II (11). In addition to the stimulatory effect of A23187 on aldosterone secretion, A23187 was shown to inhibit stimulation of aldosterone secretion by other hormones in the present as well as previous studies (21). This inhibitory effect of A23187 may have accounted for the biphasic nature of A23187-induced aldosterone secretion observed in the static cell suspension method employed in the present study as well as in perifusion systems (17, 18). The mechanism of A23187-induced inhibition of aldosterone secretion is unknown; however, A23187 treatment has been found to inhibit protein synthesis in glomerulosa cells (36) as well as to generally suppress phospholipid turnover (37). Both processes have been implicated in pathways leading to stimulation of steroidogenesis (8, 9, 23). On the other hand, A23187 has been shown to act synergistically with phorbol esters (18) and forskolin (17), changing a transient A23187-induced al-
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INHIBITION OF ALDOSTERONE SECRETION
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dosterone secretory response to a sustained response. These results might suggest that forskolin and phorbol esters produce effects that oppose the inhibitory effects of A23187, as opposed to potentiating stimulatory effects. It is also of interest that this synergism has only been observed in bovine glomerulosa cells and not in those from the rat.(38). In summary, the ability of ANP to inhibit A23187induced aldosterone secretion provides compelling evidence for ANP-mediated inhibition of an intracellular process necessary for the Ca2+ second messenger system to stimulate aldosterone biosynthesis in the adrenal glomerulosa cells. Therefore, the effects of ANP on glomerulosa cells may provide a useful tool for defining intracellular reactions important for the stimulation of aldosterone biosynthesis.
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glomerulosa cells. Hypertension 11:433-439 16. McCarthy RT, Isales CM, Bollag WB, Rasmussen H, Barrett PQ 1990 Atrial natriuretic peptide differentially modulates T- and Ltype calcium channels. Am J Physiol 258:F473-F478 17. Kojima I, Kojima K, Rasmussen H 1985 Intracellular calcium and adenosine 3',5'-cyclic monophosphate as mediators of potassiuminduced aldsoterone secretion. Biochem J 228:69-76 18. Kojima I, Kojima K, Rasmussen H 1985 Role of calcium fluxes in the sustained phase of angiotensin II-mediated aldosterone secretion from adrenal glomerulosa cells. J Biol Chem 260:9177-9184 19. Foster R, Lobo MV, Rasmussen H, Marusic ET 1981 Calcium: its role in the mechanism of action of angiotensin II and potassium in aldosterone production. Endocrinology 109:2196-2201 20. Schiebinger RJ, Braley LM, Menachery A, Williams GH 1986 Unique calcium dependencies of the activating mechanism of the early and late aldosterone biosynthetic pathways in the rat. J Endocrinol 110:315-325 21. Fakunding JL, Catt KJ 1982 Calcium-dependent regulation of aldosterone production in isolated adrenal glomerulosa cells: effects of the ionophore A23187. Endocrinology 110:2006-2010 22. Brostrom CO, Brostrom MA 1990 Calcium-dependent regulation of protein synthesis in intact mammalian cells. Annu Rev Physiol 52:577-590 23. Pederson RC 1985 Polypeptide activators of cholesterol side chain cleavage. Endocr Res 10:533-561 24. Quinn SJ, Williams GH 1988 Regulation of aldosterone secretion. Annu Rev Physiol 50:409-426 25. Wilson JX, Aguilera G, Catt KJ 1984 Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells. Endocrinology 115:1357-1363 26. Fakunding JL, Chow R, Catt KJ 1979 The role of calcium in the stimulation of aldosterone production by adrenocorticotropin, angiotensin II, and potassium in isolated glomerulosa cells. Endocrinology 105:327-333 27. Capponi AM, Lew PD, Vallotton MB 1987 Qualitative analysis of the cytosolic-free-Ca2+-dependency of aldosterone production in bovine glomerulosa cells. Biochem J 247:335-340 28. Pratt JH, Rothrock JK, Dominguez JH 1989 Evidence that angiotensin II and potassium collaborate to increase cytosolic calcium and stimulate the secretion of aldosterone. Endocrinology 125:2463-2469 29. Quinn SJ, Enyedi P, Tillotson DL, Williams GH 1990 Cytosolic calcium and aldosterone response patterns of rat adrenal glomerulosa cells stimulated by vasopressin: comparison with angiotensin II. Endocrinology 127:541-548 30. Atarashi K, Mulrow PJ, Franco-Saenz R 1985 Effect of atrial peptides on aldosterone production. J Clin Invest 76:1807-1811 31. Anand-Srivastava MB, Genest J, Cantin M 1985 Inhibitory effect of atrial natriuretic factor on adenylate cyclase activity in adrenal cortical membranes. FEBS Lett 181:199-202 32. Kojima I, Kojima K, Rasmussen H 1985 Characteristics of angiotensin II-, K+-, and ACTH-induced calcium influx in adrenal glomerulosa cells. J Biol Chem 260:9171-9176 33. Payet MD, Benabderrazik M, Gallo-Payet N 1987 Excitationsecretion coupling: ionic currents in glomerulosa cells: effects of adrenocorticotropin and K+ channel blockers. Endocrinology 121:875-882 34. Kojima I, Ogata E 1986 Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell. J Biol Chem 261:9832-9838 35. Kojima I, Kojima K, Rasmussen H 1985 Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion. J Biol Chem 260:4248-4256 36. Farese RV, Sabir MA, Larson RE 1981 A23187 inhibits adrenal protein synthesis and the effects of adrenocorticotropin (ACTH) on steroidogenssis and phospholipid metabolism in rat adrenal cells in vitro: further evidence implicating phospholipids in the steroidogenic action of ACTH. Endocrinology 108:1243-1250 37. Farese RV, Larson RE, Sabir MA, Gomez-Sanchez CE 1983 Effect of angiotensin II, K+, adrenocorticotropin, serotonin, adenosine 3',5'-monophosphate, guanosine 3',5'-monophosphate, A23187, and EGTA on aldosterone synthesis and phospholipid metabolism in the rat adrenal zona glomerulosa. Endocrinology 113:1377-1386 38. Spat A 1988 Stimulus secretion coupling in angiotensin-stimulated adrenal glomerulosa cells. J Steroid Biochem 29:443-453
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