ANG II-stimulated Aldosterone Secretion Research Articles

The Endocrine‐Disrupting Chemical Di‐(2‐ethylhexyl) phthalate (DEHP) Increases Corticosteroid Secretion in Human Adrenocortical Cells

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

MON-035 Aldosterone Secretion in a Human Adrenal Cell Line Is Enhanced by the Endocrine-Disrupting Chemical Di-(2-Ethylhexyl) Phthalate (DEHP)

Di-(2-ethylhexyl) phthalate (DEHP) is an endocrine-disrupting chemical that is widely used as a plasticizer in the production of polyvinyl chloride containing plastics. DEHP is a component of many common household items (toys, packaging, furniture, etc.) and medical devices, resulting in significant and chronic human exposure. Human exposure to DEHP has been linked to androgen disruption and developmental toxicity, including cryptorchidism, decreased anogenital distance, and decreased testosterone levels. Other adrenal corticosteroids are also susceptible to the endocrine-disrupting effects of DEHP. One study has demonstrated that fetal exposure to DEHP results in altered adrenal aldosterone production in adult rats. Due to these potential effects of DEHP on adrenal cortex steroidogenesis, the focus of our studies is to characterize the effect of DEHP on aldosterone secretion in a human adrenocortical cell line. HAC15 cells were exposed to vehicle or various concentrations of DEHP (10 nM-30 μM). After 72 h, cell viability, aldosterone secretion, and gene expression of enzymes and cofactors involved in aldosterone synthesis was examined. No effects on cell viability were observed with the concentrations studied. Basal aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 75.6±3.5; 10 μM DEHP, 7735.2±151.3; n=8). Ang II-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 4278.9±110.2; 10 μM DEHP, 18651.7±572.2; n=8). ACTH-stimulated aldosterone secretion was significantly increased from 100 nM-30 μM DEHP, with maximal secretion at 10 μM (in pg/mL: vehicle, 124.0±3.7; 10 μM DEHP, 23455.8±272.7; n=8). Gene expression of most enzymes and cofactors involved in aldosterone synthesis were increased by 10 μM DEHP. Aldosterone synthase (CYP11B2) had the greatest induction at 175.2±2.0 fold. These data support the evidence that DEHP disrupts the regulation of aldosterone secretion and provides further evidence that DEHP is an endocrine disruptor.

Angiotensin II-activated protein kinase D mediates acute aldosterone secretion

Dysregulation of the renin-angiotensin II (AngII)-aldosterone system can contribute to cardiovascular disease, such that an understanding of this system is critical. Diacylglycerol-sensitive serine/threonine protein kinase D (PKD) is activated by AngII in several systems, including the human adrenocortical carcinoma cell line NCI H295R, where this enzyme enhances chronic (24 h) AngII-evoked aldosterone secretion. However, the role of PKD in acute AngII-elicited aldosterone secretion has not been previously examined. In primary cultures of bovine adrenal glomerulosa cells, which secrete detectable quantities of aldosterone in response to secretagogues within minutes, PKD was activated in response to AngII, but not an elevated potassium concentration or adrenocorticotrophic hormone. This activation was time- and dose-dependent and occurred through the AT1, but not the AT2, receptor. Adenovirus-mediated overexpression of constitutively active PKD resulted in enhanced AngII-induced aldosterone secretion; whereas overexpression of a dominant-negative PKD construct decreased AngII-stimulated aldosterone secretion. Thus, we demonstrate for the first time that PKD mediates acute AngII-induced aldosterone secretion.

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.

RGS2 Is Regulated by Angiotensin II and Functions as a Negative Feedback of Aldosterone Production in H295R Human Adrenocortical Cells

Regulator of G protein signaling (RGS) proteins interact with Galpha-subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. Angiotensin (Ang) II interacts with its G protein-coupled receptor in zona glomerulosa adrenal cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. We studied Ang II-mediated regulation of RGS2, the role of RGS2 in steroidogenesis, and the intracellular signal events involved in H295R human adrenal cells. We report that both H295R cells and human adrenal gland express RGS2 mRNA. In H295R cells, Ang II caused a rapid and transient increase in RGS2 mRNA levels quantified by real-time RT-PCR. Ang II effects were mimicked by calcium ionophore A23187 and blocked by calcium channel blocker nifedipine. Ang II effects also were blocked by calmodulin antagonists (W-7 and calmidazolium) and calcium/calmodulin-dependent kinase antagonist KN-93. RGS2 overexpression by retroviral infection in H295R cells caused a decrease in Ang II-stimulated aldosterone secretion but did not modify cortisol secretion. In reporter assays, RGS2 decreased Ang II-mediated aldosterone synthase up-regulation. These results suggest that Ang II up-regulates RGS2 mRNA by the calcium/calmodulin-dependent kinase pathway in H295R cells. RGS2 overexpression specifically decreases aldosterone secretion through a decrease in Ang II-mediated aldosterone synthase-induced expression. In conclusion, RGS2 expression is induced by Ang II to terminate the intracellular signaling cascade generated by Ang II. RGS2 alterations in expression levels or functionality could be implicated in deregulations of Ang II signaling and abnormal aldosterone secretion by the adrenal gland.

Estradiol attenuates angiotensin-induced aldosterone secretion in ovariectomized rats.

Estrogen replacement therapy significantly reduces the risk of cardiovascular disease in postmenopausal women. Previous studies indicate that estradiol (E2) decreases angiotensin II (AT) receptor density in the adrenal and pituitary in NaCl-loaded rats. We used an in vivo model that eliminates the potentially confounding influence of ACTH to determine whether the E2-induced decrease in adrenal AT receptor expression affects aldosterone responses to angiotensin II (Ang II). Female rats were ovariectomized, treated with oil (OVX) or E2 (OVX+E2; 10 microg, s.c.) for 14 days, and fed a NaCl-deficient diet for the last 7 days to maximize adrenal AT receptor expression and responsiveness. On days 12-14 rats were treated with dexamethasone (DEX; 25 microg, i.p., every 12 h) to suppress plasma ACTH. On day 14 aldosterone secretion was measured after a 30-min infusion of Ang II (330 ng/min). Ang II infusion increased the peak plasma aldosterone levels to a lesser degree in the OVX+E2 than in the OVX rats (OVX, 1870 +/- 290 pg/ml; OVX+E2, 1010 +/- 86 pg/ml; P < 0.05). Ang II-induced ACTH and aldosterone secretion was also studied in rats that were not treated with DEX. In the absence of DEX, the peak plasma aldosterone response was also significantly decreased (OVX, 5360 +/- 1200 pg/ml; OVX+E2, 2960 +/- 570 pg/ml; P < 0.05). However, E2 also reduced the plasma ACTH response to Ang II (P < 0.05; OVX, 220 +/- 29 pg/ml; OVX+E2, 160 +/- 20 pg/ml), suggesting that reduced pituitary ACTH responsiveness to Ang II contributes to the effect of E2 on Ang II-induced aldosterone secretion. Adrenal AT1 binding studies confirmed that E2 significantly reduces adrenal AT1 receptor expression in both the presence and absence of DEX in NaCl-deprived rats. These results indicate that E2-induced decreases in pituitary and adrenal AT1 receptor expression are associated with attenuated pituitary ACTH and adrenal aldosterone responses to Ang II and suggest that estrogen replacement therapy may modulate Ang II-stimulated aldosterone secretion as part of its well known cardioprotective actions.

Direct modulation of basal and angiotensin II-stimulated aldosterone secretion by hydrogen ions.

Disturbances in acid-base balance in vivo are associated with changes in plasma aldosterone concentration, and in vitro changes in extracellular pH (pH(o)) influence the secretion of aldosterone by adrenocortical tissue or glomerulosa cells. There is considerable disparity, however, as to the direction of the effect. Furthermore, the mechanisms by which pH(o) independently affects aldosterone secretion or interacts with other secretagogues are not defined. Thus, bovine glomerulosa cells maintained in primary monolayer culture were used to examine the direct effects of pH(o) on cytosolic free calcium concentration ([Ca(2+)](i))( )and aldosterone secretion under basal and angiotensin II (AngII)-stimulated conditions. pH(o) was varied from 7.0 to 7.8 (corresponding inversely to changes in extracellular H(+) concentration from 16 nM to 100 nM). Whereas an elevation of pH(o) from 7.4 to 7.8 had no consistent effect, reductions of pH(o) from 7.4 to 7.2 or 7.0 caused proportionate increases in aldosterone secretion that were accompanied by increases in transmembrane Ca(2+) fluxes and [Ca(2+)](i). These effects were abolished by removal of extracellular Ca(2+). A decrease in pH(o) from 7.4 to 7.0 also enhanced AngII-stimulated aldosterone secretion. This effect was more pronounced at low concentrations of AngII and was manifested as an increase in the magnitude of the secretory response with no effect on potency. In contrast to its effect on AngII-stimulated aldosterone secretion, a reduction of pH(o) from 7.4 to 7.0 inhibited the Ca(2+) signal elicited by low concentrations (</=1x10(-10) M) of AngII, but did not affect the increase in [Ca(2+)](i) caused by a maximal concentration (1x10(-8) M) of AngII. These data suggest that pH(o) (i.e. H(+)) has multiple effects on aldosterone secretion. It independently increases aldosterone secretion through a mechanism involving Ca(2+) influx and an increase in [Ca(2+)](i). Also, it modulates the action of AngII by both decreasing the magnitude of the AngII-stimulated Ca(2+) signal and increasing the sensitivity of a more distal site to intracellular Ca(2+). The latter action appears to be a more important determinant in the effects of pH(o) on AngII-stimulated aldosterone secretion.

Angiotensin II stimulates both aldosterone secretion and DNA synthesis via type 1 but not type 2 receptors in bovine adrenocortical cells.

Angiotensin II (Ang II) type 2 receptor (AT2) has been shown to counteract the type 1 receptor (AT1)-mediated biological actions of Ang II in the cardiovascular system. The biological significance of AT2 receptor in the adrenals however remains unknown. In the present study, we investigated the roles of AT1 and AT2 receptor subtypes in the regulation of aldosterone secretion and DNA synthesis in bovine adrenocortical zona glomerulosa cells in vitro. Ang II (1 mumol/l)-stimulated aldosterone secretion was completely suppressed by AT1 antagonist CV-11974 but not affected by AT2 receptor antagonist PD-123319. Effects on DNA synthesis were investigated by determining the incorporation of BrdU into the nuclei of the cultured zona glomerulosa cells. Ang II (1 mumol/l)-stimulated DNA synthesis of the cells was also completely suppressed by CV-11974 but not by PD-123319. These results suggest that AT1 receptor but not AT2 receptor is the predominant receptor subtype which mediates the Ang II-stimulated aldosterone secretion and cell growth in bovine adrenocortical cells.

-Melanocyte-stimulating hormone-induced inhibition of angiotensin II receptor-mediated events in the rat adrenal zona glomerulosa

It is well established that ACTH and angiotensin II (Ang II) stimulate aldosterone secretion from rat adrenal zona glomerulosa cells in vitro and mediate their steroidogenic effects via the cyclic AMP (cAMP) pathway and phosphoinositide turnover respectively. alpha-MSH also stimulates aldosterone secretion from zona glomerulosa cells in vitro, and recent studies from our laboratory have shown that its steroidogenic effects are mediated by increases in inositol 1,4,5-trisphosphate (IP3) production. alpha-MSH also stimulates adenylyl cyclase activity, but only at concentrations that are supramaximal for stimulation of steroidogenesis. The observation that alpha-MSH-stimulated IP3 accumulation declines as the activity of adenylyl cyclase increases prompted further studies on the interactions of cAMP and phosphoinositide production. The effects of alpha-MSH and ACTH on Ang II-stimulated steroidogenesis and IP3 accumulation were studied. On addition of increasing concentrations of ACTH, both the aldosterone and IP3 responses to Ang II were significantly inhibited; however, only high concentrations of alpha-MSH achieved this effect. These results suggest that cAMP or a cAMP-dependent event is able to inhibit phospholipase C activity. This hypothesis was tested by measuring IP3 production in Ang II-stimulated zona glomerulosa cells exposed to two different concentrations of alpha-MSH: 1 nmol/l, which stimulates the generation of IP3, and 1 mumol/l, which activates adenylyl cyclase. It was found that this high concentration of alpha-MSH significantly inhibited Ang II-stimulated aldosterone secretion and IP3 levels. In addition, alpha-MSH reduced 125I-labelled Ang II binding to rat adrenal zona glomerulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of ET-1 potentiation of angiotensin II stimulation of aldosterone production

Endothelin-1 (ET-1) exerts the following two types of aldosterone-stimulating actions on glomerulosa cells: ET-1-mediated direct stimulation of aldosterone secretion (per se effect) and potentiation of the aldosterone secretion to angiotensin II (ANG II; potentiation effect). The role of Ca2+ and protein kinase C (PKC) systems in these two effects was investigated. Incubations of calf cultured adrenal zona glomerulosa cells in low-Ca2+ media or in the presence of the Ca2+ channel antagonist verapamil reduced the aldosterone secretion to ET-1. When cells were preincubated with ET-1 in a low-Ca2+ media or in the presence of the Ca2+ channel antagonist verapamil, washed, and incubated in media with normal Ca2+, ANG II showed potentiation of ANG II-stimulated aldosterone secretion. The PKC inhibitors H-7 and staurosporine did not decrease ET-1-stimulated aldosterone secretion, but they inhibited the potentiation effect of ET-1 on ANG II-mediated aldosterone secretion. Adrenocorticotropic hormone desensitization or prolonged phorbol ester stimulation of PKC resulting in desensitization also resulted in the abolition of the ET-1-mediated ANG II potentiation of aldosterone secretion. The PKC inhibitors did not affect ANG II-stimulated aldosterone secretion. We conclude that ET-1 exerts a direct stimulation of aldosterone secretion through a mechanism dependent on Ca2+ and potentiates ANG II-mediated aldosterone stimulation through a mechanism involving PKC.

The Effect of Oxygen on Aldosterone Release from Bovine Adrenocortical Cellsin Vitro:PO2versus Steroidogenesis*

Hypoxia decreases plasma aldosterone in vivo without a decrease in PRA, angiotensin II (ANG II), ACTH, or cortisol. The present study evaluated whether this could be due to a direct, specific inhibitory effect on the zona glomerulosa related to the magnitude of the decrease in oxygen (O2). Bovine adrenocortical cells were dispersed with collagenase and studied in vitro within 48 h. Cells were stimulated for 2 h with ANG II (0.1-1000 nM) or (Bu)2cAMP (0.3-3 mM) under oxygen levels ranging from 0 to 100% O2 (PO2 from 66 +/- 4 to 561 +/- 46 torr) vs. a reference gas mixture (21% O2 PO2 approximately 140 torr). Exposure to 123 +/- 8, 110 +/- 12, 100 +/- 16, and 66 +/- 4 torr led to 27%, 30%, 40% and 70% inhibition, respectively, of 3 nM ANG II-stimulated aldosterone secretion as compared to 140 +/- 16 torr (reference). Exposure to hyperoxia (288 +/- 36 to 561 +/- 46 torr) led to a small (10%) increase in ANG II-stimulated aldosterone secretion which was not statistically significant. The P50 (half-maximal PO2) for aldosteronogenesis was approximately 95 torr. The results for other doses of ANG II and for cAMP were similar. The inhibitory effect of low O2 was reversed by returning the cells to reference conditions (140 +/- 16 torr). Cortisol secretion was not significantly affected by changes in oxygen tension. We conclude that small changes in O2 within the physiological range directly and specifically inhibit aldosteronogenesis in a dose-dependent manner with a P50 of approximately 95 torr. Inhibition of cAMP-stimulated aldosterone secretion suggests a postreceptor site of action. This direct, reversible, and specific effect on the zona glomerulosa of the adrenal cortex may account for the dissociation of renin and aldosterone during hypoxia in vivo.

Dissociation of osmotic and ionic modulation of aldosterone secretion.

Although other investigators have suggested that reductions in either Na or chloride concentration stimulate aldosterone secretion, we previously found that small reductions in NaCl (3-7 mM) that enhanced angiotensin II-(ANG II) and [K]- but not adrenocorticotropic hormone (ACTH)-stimulated aldosterone secretion are due to a change in osmolality. In the present study, aldosterone secretion by an isolated perfused canine adrenal gland was stimulated by low doses of ANG II or ACTH or by small increases in perfusate [K], and during this stimulation, replacing 25 mM NaCl with an isosmotic amount of mannitol enhanced aldosterone secretion induced by each of the above secretagogues. Choline chloride significantly enhanced ANG II-stimulated aldosterone secretion when used in place of 25 mM NaCl, but sodium methylsulfate did not. Large isosmotic reductions in [NaCl] failed to alter ACTH-stimulated cortisol secretion or the conversion of either exogenous corticosterone or 11-deoxycorticosterone to aldosterone. Thus, reductions in Na, but not in chloride concentration, specifically enhance the ability of the adrenal glomerulosa to secrete aldosterone in response to ANG II, K, and ACTH by an action on some site in the steroidogenic cascade that is sensitive to ANG II, potassium, and ACTH.

Effect of cyclosporin on blood pressure and renin-aldosterone axis in rats.

Although hypertension is a frequent complication of cyclosporin A (CSA) therapy in clinical practice, little experimental information is available on the nature and the mechanism of this form of hypertension. We studied the effect of currently recommended therapeutic dosages of CSA, i.e., 5 (CSA5) and 20 (CSA20) mg.kg-1.day-1, on blood pressure and the renin-aldosterone system (RAS) in spontaneously hypertensive rats (SHR). Influence of in vivo CSA treatment on in vitro angiotensin II (ANG II)-stimulated aldosterone secretion by isolated adrenal glomerulosa cells (AGC) was also measured. CSA treatment in SHR resulted in a consistent increase in systolic blood pressure. This increase in blood pressure occurred in the absence of significant changes in creatinine clearance in CSA5 rats, whereas in CSA20 rats a significant reduction in creatinine clearance was observed. Sodium balance and serum calcium and magnesium concentrations were not different between the control group and either of the two CSA-treated groups of rats. Plasma renin concentration (PRC) and inactive renin (IR) were markedly elevated, but plasma renin substrate remained unchanged with CSA administration. Despite the presence of hyperreninemia, plasma aldosterone was not elevated, suggesting that CSA may induce relative adrenal resistance to ANG II. This possibility was tested using AGC isolated from CSA-treated rats. ANG II-stimulated aldosterone secretion in AGC was diminished by low dose and aborted by high dose CSA-treatment. Thus CSA administration in SHR induces a predictable increase in blood pressure in association with "hyperreninemic hypoaldosteronism."

Cyclosporin A-induced hyperreninemic hypoaldosteronism. A model of adrenal resistance to angiotensin II.

We studied the effects of cyclosporin A on the renin-aldosterone axis in Sprague-Dawley rats. Two weeks of intragastric administration of cyclosporin A (5 mg/kg/day or or 20 mg/kg/day) resulted in large increases in plasma renin concentration (23 +/- 5, 70 +/- 12, and 79 +/- 11 ng/ml/hr in control rats and rats receiving 5 mg and 20 mg of cyclosporin A, respectively), with no parallel increments in plasma aldosterone. In vitro angiotensin II (ANG II)-stimulated aldosterone secretion by zona glomerulosa cells obtained from cyclosporin A-treated rats was also reduced (4.8 +/- 0.5, 1.5 +/- 0.2, and 0.2 +/- 0.2 ng/10(5) cells in control rats and rats receiving 5 mg and 20 mg of cyclosporin A, respectively). In contrast, in vitro aldosterone response to graded increments of potassium (3.7-10.7 mmol/L) or adrenocorticotropic hormone (ACTH) (10(-11)-10(-8) M) was preserved in cyclosporin A-treated rats. When added in vitro to zona glomerulosa cells from untreated rats, cyclosporin A also attenuated ANG II-stimulated aldosterone secretion, but did not affect potassium or ACTH-mediated aldosterone production. Thus, cyclosporin A-induced hyperreninemic hypoaldosteronism in the rat depends on opposing renal and adrenal effects, with a direct or feedback stimulation of renin secretion and a specific blockade of ANG II-mediated aldosterone production.