Transforming growth factor-beta 1 inhibits aldosterone and stimulates adrenal renin in cultured bovine zona glomerulosa cells.

Transforming growth factors-beta (TFG beta s) are multifunctional peptides that affect proliferation, differentiation, and many other functions in a variety of cell types. In this study we examined the effect of TGF beta 1 on aldosterone and adrenal renin production using cultured bovine adrenal zona glomerulosa cells. Collagenase-dispersed zona glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h. The cells during this 24-h period were exposed to TGF beta 1, ACTH, and (Bu)2cAMP (dbcAMP). It was observed that TGF beta 1 at 1 nM 1) inhibited basal aldosterone secretion from 680.0 +/- 40.0 to 270.0 +/- 10.0 pg/10(6) cells.h, 2) inhibited ACTH- and dbcAMP-stimulated aldosterone production, 3) increased levels of active renin in the cells from 17.8 +/- 2.5 to 70.7 +/- 4.4 pg angiotensin-I/10(6) cells.h and prorenin from 270.0 +/- 5.0 to 970.0 +/- 90 pg angiotensin-I/10(6) cells.h, 4) stimulated prorenin in the medium synergistically in combination with ACTH and dbcAMP, and 5) had no significant effect on basal cAMP production, but significantly inhibited the ACTH-stimulated production of cAMP. These observations show that TGF beta 1 is a potent inhibitor of basal and ACTH- and cAMP-stimulated aldosterone production and inhibits ACTH-stimulated cAMP production. Contrary to its effect on aldosterone, TGF beta 1 stimulates the synthesis and release of adrenal renin and prorenin. TGF beta 1 may act as an autocrine or paracrine regulator of aldosterone production.     

Origin of aldosterone in trypsin-stimulated rat adrenal zona glomerulosa incubations.

The time-course for the in-vitro secretion of aldosterone and 18-hydroxycorticosterone (18-OH-B) by rat adrenal whole capsular tissue (largely zona glomerulosa) was studied under control and stimulated conditions. The stimulatory effect of trypsin was relatively delayed, and the steroids were significantly enhanced only after 1 h, in contrast to the actions of ACTH, which produced effects after 15 or 30 min. Tissue-sequestered 18-hydroxydeoxycorticosterone (t-18-OH-DOC), which is not affected by ACTH, was significantly depleted by trypsin, but secreted 18-OH-DOC was not consistently affected by either stimulant. In contrast to the apparent mobilization of t-18-OH-DOC, the conversion of exogenously added [3H]18-OH-DOC to [3H]18-OH-B was inhibited by trypsin, and aldosterone was unaffected. When trilostane was added to inhibit de-novo steroidogenesis, under conditions in which the steroid secretory response to ACTH is completely inhibited, aldosterone and 18-OH-B secretion was still stimulated by trypsin although yields were lower. Compared with controls, trilostane reduced t-18-OH-DOC concentrations, and trypsin caused a further depletion. In other studies, glomerulosa plasma membrane enriched preparations were homogenized and centrifuged, and the supernatants were dialysed and added to incubations of dispersed zona glomerulosa cells in the presence or absence of stimulators of aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

c-Fos mediates angiotensin II-induced aldosterone production and protein synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AngII), potassium ion, and ACTH are the main factors controlling aldosterone biosynthesis in adrenal glomerulosa cells. AP-1 response elements for the immediate early gene products, c-Fos and c-Jun, have been identified, among others, in the promoter of the steroidogenic acute regulatory (StAR) protein gene, whose expression is acutely regulated by activators of aldosterone production. In bovine glomerulosa cells, AngII treatment led to a rapid and transient increase in c-fos mRNA expression, c-Fos protein expression, and c-Fos phosphorylation. Inhibition of the ERK1/2 MAPK pathway abolished the effect of AngII on c-fos mRNA, protein, and phosphorylation. EMSA and chromatin immunoprecipitation experiments demonstrated that c-Fos binds with c-Jun to the proximal StAR promoter and that AngII treatment increases the amount of c-Fos bound to the promoter. Overexpression of a dominant-negative form of c-Fos with adenoviral vectors inhibited StAR mRNA and StAR protein expression as well as aldosterone biosynthesis in response to AngII. The dominant-negative c-Fos also prevented the increase in protein synthesis induced by AngII in glomerulosa cells, as assessed by [(3)H]leucine incorporation. These results indicate that AngII rapidly induces c-Fos expression and posttranslational modifications. Furthermore, a heterodimeric c-Fos/c-Jun complex binds to the proximal StAR promoter in glomerulosa cells, thus activating StAR gene expression and acute aldosterone biosynthesis. Finally, c-Fos also contributes to other functional responses to the hormone, such as protein synthesis.     

Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells.

Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.     

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.     

Mechanism of cholinergic stimulation of aldosterone secretion in bovine adrenal glomerulosa cells

The effect of cholinergic stimulation on aldosterone secretion was examined in bovine adrenal glomerulosa cells. Both acetylcholine and carbachol stimulated aldosterone secretion in a dose-dependent manner. Acetylcholine-induced secretion was inhibited by atropine but not by hexamethonium, suggesting that cholinergic agonists act on muscarinic receptors. The mechanisms of cholinergic agonist action were compared with those of angiotensin II. Like angiotensin II, carbachol generated calcium signal in glomerulosa cells. When [3H]inositol-labeled cells were stimulated by carbachol, there was an immediate increase in [3H]inositol trisphosphate, followed by a relatively slow increase in [3H]inositol bisphosphate. Carbachol increased the cytosolic concentration of calcium transiently but not intracellular cAMP. Carbachol caused a rapid 3-fold increase in 45Ca fractional efflux ratio in 45Ca-prelabeled cells both in the presence and absence of extracellular calcium. Carbachol also increased calcium influx; however, carbachol-induced influx was smaller than that of angiotensin II. In a perifusion system, the time course of carbachol-induced aldosterone secretion was biphasic. However, when calcium influx was increased to a value similar to that in angiotensin II-treated cells by combination of carbachol and BAY K-8644, this combination induced a monophasic and sustained secretory pattern. These results indicate that muscarinic cholinergic agonists stimulate aldosterone secretion via the calcium messenger system, and the biphasic secretory response to cholinergic agonist is due to a smaller increase in calcium influx.     

Effects of cholesterol and lipoproteins on aldosterone secretion by bovine zona glomerulosa cells

Freshly isolated bovine adrenocortical cells were pretreated with various concentrations of cholesterol and of high- (HDL) and low-density lipoprotein (LDL) fractions of known cholesterol content and then incubated in medium alone with and without angiotensin II. Preincubation with cholesterol (323 mumol/l) caused basal aldosterone synthesis to increase from 0.89 +/- 0.08 to 2.77 +/- 0.22 pmol/10(6) cells per hour (+/- S.E.M.) but did not significantly affect angiotensin-stimulated synthesis. Human HDL containing cholesterol at a final concentration of 129-647 mumol/l increased both basal and angiotensin-stimulated aldosterone synthesis. In HDL-treated cells, both the threshold response and responses to increasing concentrations of angiotensin were raised. Human LDL had no effect on basal or stimulated aldosterone synthesis nor did LDL alter the effects of HDL when cells were incubated with HDL and LDL in combination. Qualitatively similar results were obtained with bovine lipoproteins. These studies show that, in short-term incubations of fresh tissue, the supply of cholesterol may be a limiting factor in aldosterone synthesis and that HDL rather than LDL is the preferred source. These observations are discussed in relation first to the mechanisms by which cholesterol/HDL might augment steroid responses and secondly to other studies with cultured cells which have demonstrated a role for LDL.     

Endothelium-derived steroidogenic factor enhances angiotensin II-stimulated aldosterone release by bovine zona glomerulosa cells

Endothelium-derived steroidogenic factor (EDSF) is an endothelial peptide that stimulates aldosterone release from bovine adrenal zona glomerulosa (ZG) cells. The regulation of aldosterone release by combinations of EDSF and angiotensin II (AII) or EDSF and ACTH was investigated. Endothelial cells (ECs) and EC-conditioned media (ECCM) increased aldosterone release from ZG cells, an activity attributed to EDSF. AII (10(-12) to 10(-8) M) and ACTH (10(-12) to 10(-9) M) also stimulated the release of aldosterone from ZG cells. The stimulation by AII, but not ACTH, was greatly enhanced when ZG cells were coincubated with ECs. AII was metabolized by ECs to peptides identified by mass spectrometry as angiotensin (1-7) and angiotensin IV. There was very little metabolism of AII by ZG cells. Neither of these two AII metabolites altered aldosterone release from ZG cells, so they could not account for the enhanced response with ECs. AII-induced aldosterone release from ZG cells was enhanced by ECCM but not cell-free conditioned medium. This enhanced response was not due to increased EDSF release from ECs by AII. The synergistic effect of EDSF and AII was apparent when AII was added during or after the generation of ECCM and not observed when the AII component of the enhancement was blocked by the AII antagonist, losartan. These studies indicate that EDSF enhances the steroidogenic effect of AII. In the adrenal gland, ECs are in close anatomical relationship with ZG cells and may sensitize ZG cells to the steroidogenic action of AII by releasing EDSF.     

A potential role for phospholipase-D in the angiotensin-II-induced stimulation of aldosterone secretion from bovine adrenal glomerulosa cells.

The mechanism by which angiotensin-II (Ang II) stimulates aldosterone secretion from adrenal glomerulosa cells involves a phospholipase-C-mediated increase in phosphoinositide turnover and diacylglycerol (DAG) production. Because agonist-induced activation of phospholipase-D (PLD) also contributes to elevations in DAG in other cell types, the ability of Ang II to stimulate PLD activity in cultured bovine adrenal glomerulosa cells was examined. Ang II elicited significant increases in the levels of phosphatidic acid and, in the presence of ethanol, of phosphatidylethanol, a more specific marker for PLD activation. The potential role of this increased PLD activity in the regulation of aldosterone secretion was examined by investigating the ability of exogenous PLD to alter secretory rates. PLD alone dose-dependently increased aldosterone secretion from 5.9 +/- 0.5 to 135 +/- 48 pg/min.mg protein. In the presence of the calcium channel agonist Bay K 8644, which by itself had only a modest effect on aldosterone production, the stimulatory action of PLD was enhanced, yielding a secretory rate (442 +/- 119 pg/min.mg protein) that was approximately 60% of that elicited by 10 nM Ang II (763 +/- 182 pg/min.mg protein). Exogenous PLD also induced a significant increase in DAG levels (from 0.76 +/- 0.03 to 1.10 +/- 0.1 nmol/mg protein), which was not altered by the addition of Bay K 8644. However, PLD did not stimulate inositol phosphate production. These data indicate that 1) Ang II activates PLD; 2) exogenous PLD can elevate aldosterone secretory rates and DAG levels without eliciting phosphoinositide hydrolysis; and 3) the stimulatory action of exogenous PLD on aldosterone secretion is enhanced in the presence of Bay K 8644. Thus, PLD-induced DAG production may play an important role in the Ang II-mediated stimulation of aldosterone secretion from the adrenal zona glomerulosa.     

Signal transduction mechanisms involved in carbachol-induced aldosterone secretion from bovine adrenal glomerulosa cells.

In cultured bovine adrenal glomerulosa cells, diacylglycerol content remains elevated for up to 75 min following the removal of angiotensin II. This maintained increase could provide a mechanism by which angiotensin II pretreatment may prime cells to secrete aldosterone in response to the calcium channel agonist Bay K 8644. In the present study we find that carbachol failed both to produce this persistent diacylglycerol elevation and to exert a priming effect. In addition, because carbachol was also a less potent activator of phospholipase D than angiotensin II, our results implicate phospholipase D in the maintained increase in diacylglycerol content observed following stimulation with and removal of angiotensin II. Carbachol also elicited changes in the radiolabeled levels of both myristate- and arachidonate-containing diacylglycerol. However, the rapid decline in diacylglycerol content following carbachol removal resembled the rapid fall in arachidonate-diacylglycerol; we therefore proposed that the diacylglycerol species generated with carbachol stimulation contains predominantly arachidonic acid. In summary, our results suggest that prolonged elevations in diacylglycerol content following removal of hormones such as angiotensin II, as well as the identity of the diacylglycerol species itself, may be important in the regulation of cellular responses.     

The conversion of corticosterone to aldosterone is the site of the oxygen sensitivity of the bovine adrenal zona glomerulosa.

The dissociation of renin and aldosterone observed during hypoxia in vivo has been attributed to a direct inhibition of low oxygen on adrenal zona glomerulosa function. We have demonstrated that the adrenal zona glomerulosa production of aldosterone in vitro is directly proportional to a wide range of oxygen concentrations in the physiological range but that cortisol production from coincubated fasciculata cells is not oxygen sensitive. The present study examined the hypothesis that the sensitivity to O2 is limited to the aldosteronogenic late pathway. In order to localize the site of oxygen sensitivity, we measured endogenous pregnenolone production (early pathway) and the conversion of exogenous corticosterone to aldosterone (ALDO) (i.e. 18-hydroxylase activity) in adrenal cells treated with cyanoketone (3-beta-hydroxy-steroid dehydrogenase inhibitor). Acutely dispersed bovine adrenal glomerulosa cells (four experiments in pentuplicate) were incubated under low (5%) vs. normal (21%) O2 in the presence of cyanoketone (CK; 1 microM) and/or the following: corticosterone (500 ng/ml), angiotensin II (ANG II; 10 nM), or dibutyryl cAMP (1 mM). Conversion of exogenous corticosterone to ALDO in the presence of CK was inhibited by 41 +/- 1% under low O2. This was similar to the inhibitory effect of low O2 on ANG II-stimulated aldosterone production from endogenous precursors in the absence of CK (52 +/- 11% inhibition). Basal, ANG II-, and cAMP-stimulated endogenous pregnenolone production was not significantly reduced by low O2. In another experiment, glomerulosa cells were incubated under 5, 13, or 50% vs. 21% O2 in the presence of CK (1 microM) and different concentrations of corticosterone (10-1000 ng/ml). ALDO production was significantly inhibited by low O2 when corticosterone was greater than or equal to 500 ng/ml and ALDO was significantly augmented by high O2 when added corticosterone was 1000 ng/ml. We conclude that the conversion of corticosterone to ALDO (i.e. 18-hydroxylase) appears to be the primary site of oxygen sensitivity since 1) pregnenolone production was unaffected and 2) the magnitude of the inhibition of the conversion of corticosterone to ALDO by low O2 in the presence of CK was similar to the inhibition of ALDO production from endogenous precursors in the absence of CK. These studies demonstrate that oxygen sensitivity of the steroidogenic pathway is a unique, constitutive property of 18-hydroxylase, the enzyme which catalyzes the conversion of corticosterone to ALDO. We propose that the sensitivity of 18-hydroxylase to oxygen accounts for the dissociation of renin and aldosterone during hypoxia in vivo.     

Kinetics of cytosolic calcium and aldosterone responses in rat adrenal glomerulosa cells

To evaluate the relationship between cytosolic calcium (Cai) and aldosterone production, rat adrenal zona glomerulosa (ZG) cells were studied during long-term stimulation by different secretagogues. Cai was measured in single ZG cells using microspectrofluorimetry, and aldosterone was determined in cell populations using a superfusion system. For external potassium (K+), Cai increases are sustained, with only a slight decrement over time, a feature shared by aldosterone production. The relationship between aldosterone output and Cai is nonlinear, with a Cai value for half-maximal stimulation of approximately 500 nM. Furthermore, the sustained changes in Cai with external K+ indicate that ZG cells can use an amplitude-based Cai signal to stimulate aldosterone production. Cai changes stimulated by angiotensin-II (Ang-II) show a complex dose-response pattern, with high concentrations (greater than or equal to 1 nM) of Ang-II eliciting a peak-plateau signal and lower doses (0.1 nM to 10 pM) producing repeated Cai oscillations. The peak amplitude of the Cai response in individual cells is not dose dependent, with the ZG cell experiencing peak levels repeatedly at the lowest Ang-II concentrations. However, the Cai transients are more frequent with increasing Ang-II concentrations between 0.1 nM and 10 pM. When integrated over time, the mean Cai signal also shows only modest dose-dependency during the sustained phase of Ang-II stimulation. Unlike the integrated Cai signal, aldosterone production increases steeply between 10 pM and 0.1 nM Ang-II, indicating that the Cai signal is likely to be frequency-based. Conversely, the steroid response to high Ang-II closely mirrors the kinetics of the more sustained Cai signals, including the diminished Cai and aldosterone levels during sustained stimulation with the highest Ang-II doses. Arginine vasopressin stimulated Cai and aldosterone responses, which closely resemble those elicited by 0.1 nM Ang-II, except that both Cai and aldosterone return to basal values within 20 min of continuous presentation of arginine vasopressin. Each ZG secretagogue produces a distinct pattern of Cai and aldosterone response. In addition, Cai response patterns can be divided into two general classes: a sustained Cai response, which appears to modulate cell activation by the amplitude of the Cai signal, and an oscillating Cai response, which uses the frequency of the Cai transients to control the magnitude of stimulation.     

Effect of angiotensin II on aldosterone and its precursor steroid production in adrenal zona glomerulosa cells from heparin-treated rats

To assess the nature of the heparin-induced aldosterone deficiency, we investigated the stimulatory effect of angiotensin II (AII) on aldosterone and its precursor steroids in adrenal zona glomerulosa cells from heparin-treated rats compared with those in the cells from vehicle-treated rats. Heparin-treated rats had low plasma aldosterone levels, high plasma renin activity and plasma AII levels, and normal plasma corticosterone level 6 weeks after the treatment (1500 IU/kg, twice daily). Basal aldosterone production, when corrected to a uniform number of cells per group, was similar in the cells from heparin- and vehicle-treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to AII; an increase by 4 orders of magnitude in the threshold dose for AII and a decrease in the maximum AII-stimulated level. The maximum AII-stimulated levels, but not the basal levels, of pregnenolone, corticosterone and 18-OHB production were low in the cells from heparin-treated rats. ACTH caused a similar stimulatory effect on aldosterone production in the cells from heparin- and vehicle-treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to potassium; an increase by one order of magnitude in the threshold dose for potassium and a decrease in the maximum potassium-stimulated level, presumably because of the glomerulosa hyporesponsiveness to AII. These results suggest that our heparin-treated rats have selective impairment of adrenal zona glomerulosa cells, involving the specific receptors and the aldosterone biosynthesis, to AII.     

Effect of osmolarity on aldosterone production by rat adrenal glomerulosa cells

The effect of osmotic changes on aldosterone production, [Ca2+]i and voltage-gated Ca2+ currents, was studied in cultured rat glomerulosa cells. Alteration of osmolarity by sucrose addition in the 250-330 mosM range did not influence aldosterone production per se, but it substantially affected K+-stimulated aldosterone production. Hyposmosis markedly increased the hormone response evoked by raising [K+] from 3.6 to 5 mM, whereas hyperosmosis had a mild decreasing effect. Cytoplasmic [Ca2+]i, measured in single glomerulosa cells, did not show detectable change in response to either hyposmotic or hyperosmotic exposure, but the [Ca2+]i signal evoked by elevation of [K+] to 5 mM was augmented in hyposmotic solution. The osmosensitivity of the transient (T)-type and long-lasting (L)-type voltage-gated Ca2+ currents was studied using the nystatin-perforated voltage-clamp technique. Lowering osmolarity to 250 mosM significantly increased the amplitude of the T-type current, and it had a transient augmenting effect on L-type current amplitude. Hyperosmotic solution (330 mosM) reduced L-type current amplitude but did not evoke significant change in T-type current. These results indicate that the responsiveness of rat glomerulosa cells to physiological elevation of [K+] is remarkably influenced by changes in osmolarity by means of modulating the function of voltage-gated Ca2+ channels.     

Pyridine nucleotide redox state parallels production of aldosterone in potassium-stimulated adrenal glomerulosa cells.

Extracellular potassium ions (K+) raise the intracellular concentration of free Ca2+ ([Ca2+]i) by gating voltage-dependent Ca2+ channels and stimulate aldosterone production in adrenal glomerulosa cells. The pathway leading from calcium influx to increased steroid synthesis has not been completely elucidated. In the present study we demonstrate that the reduction of pyridine nucleotides known to be required for steroid hydroxylation is enhanced by K+ (4.1-8.4 mM) in single rat glomerulosa cells. The action of K+ was strictly dependent on the presence of extracellular Ca2+. Amytal, a blocker of site I of the mitochondrial respiratory chain, abolished the K+ effect, indicating a mitochondrial origin for the recorded changes. Supraphysiological K+ concentration (18 mM) resulted in a further increase in [Ca2+]i, while steroidogenesis was decreased as measured in cell suspensions. However, a possible explanation for this dichotomy is provided by the finding that the level of reduced pyridine nucleotides also decreased at supraphysiological K+ concentration.     

Actions of angiotensin II antagonists upon aldosterone production by isolated adrenal glomerulosa cells.

The biological activities of angiotensin II antagonists upon basal and angiotensin II-stimulated aldosterone production were evaluated in an isolated canine glomerulosa cell preparation. The most potent competitive antagonist of angiotensin II-stimulated aldosterone production was the [Sar1, Ile8]derivative of angiotensin II. However, this peptide was also a partial agonist at concentrations required to inhibit the steroidogenic effect of angiotensin II on dog adrenal cells, and never reduced aldosterone production to basal levels. On a molar basis, the [Sar1, Ala8] and [Sar1, Gly8]derivatives of angiotensin II were relatively less potent as competitive inhibitors of angiotensin II-stimulated aldosterone production. However, the [Ala8] and [Gly8]-analogues did not exhibit significant agonist activity and were therefore more effective antagonists of angiontensin II-stimulated aldosterone production. These results suggest that increased length of the aliphatic side chain at the C-terminus of angiotensin II antagonists is accompanied by enhanced affinity for the receptor site, but also by increased agonist activity upon aldosterone synthesis. The actions of angiotensin II and [Des-Asp1]angiotensin II upon aldosterone production were inhibited identically and completely by [Sar1, Ala8]angiotensin II, and identically, though incompletely, by lower concentrations of [Sar1, Ile8]angiotensin II. The heptapeptide antagonist [Des-Asp1, Ile8]angiotensin II was much less potent than [Sar1, Ile8]angiotensin II as an inhibitor of the actions of both the heptapeptide and octapeptide agonists. The antagonist activity of six angiotensin II analogues at the adrenal level, determined by the concentration required for 50% inhibition of maximum aldosterone secretion, correlated well with their antagonist activity measured upon isolated smooth muscle. These observations demonstrate that the octapeptide antagonists are more effective than the heptapeptide antagonists upon angiotensin II-stimulated aldosterone production, and that angiotensin II receptors in smooth muscle and adrenal cortex exhibit generally similar responses to angiotensin II antagonists. Also, these results do not support the proposal that the [Des-Asp1]heptapeptide is an important intermediate in the action of angiotensin II upon adolesterone production in the adrenal glomerulosa cells. The production of aldosterone by dispersed zona glomerulosa cells in vitro provides a highly sensitive and biologically appropriate response for evaluation of the agonist and antagonist properties of angiotensin II analogues upon the adrenal gland.     

Effects of angiotensin II, adrenocorticotropin, and potassium on aldosterone production in adrenal zona glomerulosa cells from streptozotocin-induced diabetic rats

Hyporeninemic hypoaldosteronism has been shown to occur in streptozotocin-induced chronic diabetic rats with normokalemia. To test the nature of the aldosterone deficiency, we investigated the responses of aldosterone production to angiotensin II (AII), ACTH, and potassium in adrenal zona glomerulosa cells from diabetic rats at 6 weeks after an injection of streptozotocin compared with those in the cells from control rats. In diabetic rats, plasma glucose was high and plasma immunoreactive insulin was low. Diabetic rats also had low levels of PRA and plasma AII, low levels of plasma aldosterone, and normal levels of plasma corticosterone and plasma potassium. The zona glomerulosa width was narrower in diabetic rats than in control rats. Basal aldosterone production, when corrected to an uniform number of cells per group, was similar in the cells from control and diabetic rats. Cells from diabetic rats showed a less sensitive and lower response of aldosterone production to AII, increases in the threshold and the ED50, and a decrease in the maximal AII-stimulated aldosterone level. ACTH, however, caused a similar effect on aldosterone production in the cells from control and diabetic rats. Cells from diabetic rats exhibited a less sensitive response of aldosterone production to potassium and a tendency to be low in the maximal potassium-stimulated aldosterone level, presumably attributable to the impairment of adrenal zona glomerulosa cells to AII. We conclude that the hypoaldosteronism observed in our diabetic rats may be secondary to the deficiency of AII.     

Dopaminergic binding and inhibitory effect in the bovine adrenal zona glomerulosa

Dopaminergic mechanisms may be involved in the regulation of aldosterone secretion in humans and in the rat. Whether these effects are indirect or are exerted directly at the adrenal level has not yet been resolved. We now report the identification of dopaminergic binding sites in the bovine adrenal zone glomerulosa using [3H]spiperone, a butyrophenone with high affinity for D2 dopamine receptors. Specific [3H]spiperone binding (defined as binding displaceable by 10 microns (+)-butaclamol) reached equilibrium within 20 minutes at 22 degrees C, was reversible, and was heat labile (60 degrees C). Binding was of high affinity and saturable with a Kd of 1.8 +/- 0.2 nM and maximal specific binding of 38 +/- 8 fmol/mg (means +/- SEM; n = 18). [3H]Spiperone binding was unaffected by coincubation with angiotensin II, adrenocorticotropic hormone, or KCl. Binding characteristics, including a dissociation constant at the nanomolar range, greater potency of the D2-agonist LY 171555 relative to the D1-agonist SKF 38393 in inhibiting [3H]spiperone binding, and lack of stimulation of cyclic adenosine 3',5'-monophosphate by dopamine (10(-4) M), were consistent with a predominantly D2-receptor. In vitro studies with collagenase-dispersed adrenal zona glomerulosa cells showed that dopamine (10(-4) M) attenuated angiotensin II-stimulated aldosterone secretion. These observations are consistent with a direct inhibitory effect of dopamine on aldosterone secretion in the adrenal zona glomerulosa.     

Effects of alpha-human atrial natriuretic polypeptide, sodium nitroprusside and dibutyryl cyclic GMP on aldosterone production in bovine zona glomerulosa cells

When bovine adrenal zona glomerulosa cells were incubated with alpha-human atrial natriuretic polypeptide (alpha-hANP), the basal aldosterone production in the cells was hardly affected, although the angiotensin II- or K+-stimulated production was completely inhibited. alpha-hANP was found to cause the generation of cyclic GMP in the cells. When the cells were incubated with sodium nitroprusside, the drug inhibited the angiotensin II- or K+-stimulated aldosterone production, and also generated cyclic GMP in the cells. In contrast, dibutyryl cyclic GMP was found to be a stimulator of the aldosterone response rather than an inhibitor. The results obtained in this study cast doubt on the role of cyclic GMP as an intracellular second messenger for the action of ANP on aldosterone secretion.