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(Hypertension. 1998;31:463.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Different Signaling Pathways Mediate Stimulated Secretions Of Endogenous Ouabain and Aldosterone From Bovine Adrenocortical Cells

Jui R. Shah; James Laredo; Bruce P. Hamilton; John M. Hamlyn

From the Departments of Physiology (J.R.S., J.M.H) and Medicine (B.P.H.), University of Maryland at Baltimore; the Department of Surgery (J.L.), Beth Israel Hospital, Harvard University, Boston; and the Veterans Administration Medical Center (B.P.H.), Baltimore.

Correspondence to John M. Hamlyn, PhD, Department of Physiology, School of Medicine, University of Maryland at Baltimore, 655 West Baltimore Street, Baltimore, MD 21201. E-mail jhamlyn{at}umabnet.ab.umd.edu

	Abstract

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Angiotensin II stimulates secretion of corticosteroids and an ouabain-like compound from adrenocortical cells. The angiotensin AT₁ and AT₂ receptor subtypes have been linked with stimulated secretion of aldosterone and endogenous ouabain, respectively, but the second messenger mechanisms involved in the latter secretion are not known. Accordingly, we investigated the effects of several pharmacological agents that affect signaling pathways on the basal and stimulated secretions of aldosterone and endogenous ouabain from primary cell cultures of bovine adrenocortical cells. The AT₂ receptor antagonist, PD 123319, blocked the effects of angiotensin II on secretion of endogenous ouabain but not aldosterone. Treatment of the cells with either dibutyryl cAMP, a membrane permeant analog, or the phorbol ester tetradecanoyl phorbol acetate stimulated aldosterone secretion but had no effect on the secretion of endogenous ouabain. On the other hand, the membrane permeant analog, 8BcGMP, maximally activated secretion of endogenous ouabain whereas incubation of cells with sodium orthovanadate blocked angiotensin II stimulated secretion of endogenous ouabain. Neither 8BcGMP nor sodium orthovanadate affected the basal or stimulated components of aldosterone secretion. These results show that the secretions of aldosterone and endogenous ouabain from bovine adrenocortical cells are mediated by different intracellular signaling mechanisms and provide evidence that the adrenal secretions of these steroids are regulated differently.

Key Words: angiotensin II receptors • sodium pump • phosphotyrosine phosphatase • signal transduction • steroids • cardiac glycosides

Abbreviations: 8BcGMP = 8-bromoguanosine 3':5'-cyclic monophosphate • ACTH = adrenocorticotropic hormone • AII = angiotensin II • AT₁ = angiotensin II type 1 • AT₂ = angiotensin II type 2 • BAC = bovine adrenocortical cells • BAG = bovine adrenal glomerulosa • DAG = diacylglycerol • dbcAMP = N⁶-2'-o-dibutyryladenosine 3':5'-cyclic monophosphate • DTFA = ditrifluoroacetate • EO = endogenous "ouabain" • IP₃ = 1,4,5-inositol trisphosphate • MAP = mitogen-activated protein • PD123319 = (s)-1-{[4-(dimethylamino)-3-methylphenyl]methyl}-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid • PBS = phosphate buffered saline • PKC = protein kinase C • PLC = phospholipase C • PTPase = phosphotyrosine phosphatase • RIA = radioimmunoassay • TPA = tetradecanoyl phorbol acetate • ZG = zona glomerulosa

	Introduction

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An endogenous ouabain-like steroid (EO) that is a specific inhibitor of the sodium-potassium pump has been identified in the circulation and tissues of humans and other mammals.^1–3 Various studies have implicated the adrenal gland as a source of EO.^4–6 In addition, mixed bovine adrenocortical cells (BAC) and adrenal glomerulosa (BAG) cells in primary cell culture secrete EO and its secretion is stimulated by both adrenocorticotropic hormone (ACTH) and angiotensin II (AII).^7,8 The primary source of both EO and aldosterone in the bovine adrenal gland appears to be the zona glomerulosa (ZG).⁸ Recently, we suggested that the stimulation of EO and aldosterone secretions from adrenocortical cells by AII is mediated by different AII receptors with the former secretion being linked specifically with activation of AT₂ receptors.⁹ The intracellular mechanisms involved in EO secretion are not known, and the objective of this study was to define some of the key elements distal to the AT₂ receptor that mediate basal and stimulated EO secretion. The results show that the signaling events that underlie the secretions of aldosterone and EO are mediated by distinct signal pathways.

	Methods

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Adrenocortical Cell Culture
Bovine adrenal glands were obtained from a local slaughterhouse and transported to the laboratory on ice in phosphate buffered saline (PBS). Processing of the glands to produce either mixed (20% zona glomerulosa and 80% zona fasciculata) adrenocortical cells or an enriched preparation containing >98% zona glomerulosa (ZG) cells were performed by methods described elsewhere.^8,9 Mixed cells were used when comparison of the secretions with those stimulated by ACTH were desired; otherwise, ZG cells were used as indicated in the figure legends. In this protocol, cells were grown (37°C in 5% CO₂) in DMEM supplemented with horse and fetal bovine serum and antibiotics and plated at approximately 4 million cells per well. Cell culture media was replaced at 24 hour intervals. Typically, secretion experiments were performed between 48 to 72 hours following plating. All experiments were performed using 3 or 4 separate cell cultures as indicated in the figure legends. All experiments were conducted with simultaneous measurements of aldosterone and EO secretion with assay measurements in duplicate.

Steroid Secretion Experiments
For secretion studies, the culture media was removed, and cells were washed twice with buffer (containing in mmol/L; NaCl 154, KCl 5, CaCl₂ 1, MgCl₂ 5.5, Glucose 5.6, HEPES 1.8-NaOH; pH 7.4). Secretion buffer, either alone or containing various pharmacological agents, was added to the cells and incubated at 37°C in 5% CO₂ for 2 or 4 hours. In experiments using PD123319, cells were pretreated for 1 hour with the antagonist. Following incubation, the secretion media was collected and assayed for aldosterone and endogenous ouabain. The cells were rinsed twice with buffer and dissolved with 2 mL of 1% sodium dodecyl sulfate for assay of total cell protein (BCA method, Pierce).

Steroid Radioimmunoassays
Aldosterone was measured without extraction using a commercially available RIA kit (Diagnostic Products Corp.). This RIA has no cross-reactivity to ouabain at concentrations 1000 fold in excess of those present in typical samples. The intra- and interassay coefficients of variation were 5.5 and 8.9% respectively.

Endogenous ouabain was measured using samples extracted with disposable Bond Elut C-18 columns (Varian). The extracted samples were assayed by RIA utilizing a polyclonal rabbit antiserum (R8) raised against ouabain conjugates by methods described elsewhere.⁴ The assay shows full reactivity with ouabain and human EO but exhibits no significant cross-reactivity (<0.01%) with cortisol, aldosterone, ACTH, angiotensin II, or any of the pharmacological agents used in this study. We have shown previously that >95% of the immunoreactivity measured in extracts prepared under these conditions is due to EO and its aglycone.⁹ Typical intra- and interassay coefficients of variation in the present experiments were 6.2 and 9.6% respectively.

Materials
PD123319 in the DTFA form was a gift of Dr. David Taylor of Warner-Lambert and was repurified by HPLC to remove DTFA before use. Collagenase Type I and DNAse Type I were obtained from Worthington Biochemicals. Percoll was obtained from Pharmacia. Fetal bovine serum, and DMEM were obtained from GIBCO-BRL. All other chemicals used were obtained from Sigma Chemical Co.

Statistical Analyses
Statistical evaluations of significance were performed using analysis of variance and Fisher’s specific test. Results were expressed as means±SEM, and P<.05 was used to indicate significance unless otherwise noted.

	Results

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In cultured bovine adrenal glomerulosa cells (BAG) cells, angiotensin II (10 nmol/L) stimulated the secretions of aldosterone and EO significantly over basal levels. As expected, PD123319 (2 µmol/L), a selective antagonist at the AT₂ receptor, prevented the AII mediated increase in EO secretion, while having no effect on the stimulated aldosterone secretion (Fig 1), showing that AT₂ receptors mediate the AII stimulation of EO secretion from ZG cells.

View larger version (17K): [in this window] [in a new window]   Figure 1. Angiotensin II stimulates EO secretion via AT2 receptors. Bovine adrenal zona glomerulosa cells were incubated in serum free media (basal) or containing angiotensin II (AII, 10 nmol/L), PD123319, (PD, 2 µmol/L), or both. Amounts of EO and aldosterone secreted are mean±SEM; **P<.05, *P<.01 vs basal, n=3 experiments.

It is thought that ACTH stimulates adrenal steroidogenesis via activation of adenylyl cyclase and subsequent elevation of cellular cAMP levels. To investigate whether adenylyl cyclase mediates the AII stimulation of EO secretion, we treated BAC cells with dibutyryl cAMP (dbcAMP), a membrane permeant analog of cAMP. In preliminary work, dose response studies showed that the minimal concentration of dbcAMP required to stimulate aldosterone secretion above basal was 0.2 mmol/L, while maximal effects were found using 3 mmol/L. At the latter concentration, dbcAMP stimulated aldosterone secretion to levels comparable to those seen with ACTH or AII stimulation (Fig 2). However, dbcAMP had no effect on EO secretion at any concentration tested.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of dibutyryl cAMP (dbcAMP) on the basal secretions of EO and aldosterone secretion from mixed bovine adrenocortical cells. Cells were incubated for 2 hours in secretion medium alone, or containing AII (10 nmol/L), ACTH (10 nmol/L), or dbcAMP (3 mmol/L). Data are means±SEM; *P<.01 versus basal, n=4 experiments.

Angiotensin II stimulates aldosterone secretion by activating phospholipase C (PLC), by generation of 1,4,5-inositol trisphosphate (IP₃) and diacylglycerol (DAG), and by activation of PKC and Ca²⁺ channels. Activation of PKC is itself sufficient to stimulate aldosterone secretion. To determine whether PKC is involved in mediating AII stimulated EO secretion, we used the phorbol ester tetradecanoyl phorbol acetate (TPA, 100nmol/L) and followed its effect on the basal secretions of aldosterone and EO. In agreement with other reports, TPA was about half as effective as ACTH or AII in stimulating aldosterone secretion but, under these conditions, had no effect on the basal secretion of EO (Fig 3).

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of tetradecanoyl phorbol acetate (TPA) on the basal secretions of EO and aldosterone from mixed bovine adrenocortical cells. Cells were incubated for 2 hours in secretion medium alone, or containing AII (10 nmol/L), ACTH (10 nmol/L), or TPA (100 nmol/L). Data are means±SEM; *P<.01 versus basal, n=4 experiments.

Guanylyl cyclase is present in adrenocortical cells, but its role as part of the second messenger pathway in corticosteroid secretion is controversial. To determine whether this system affects EO secretion, we treated cells with the membrane permeant analog 8-bromo cGMP (8BcGMP, 0.2 mmol/L). The analog significantly stimulated basal EO secretion, but had no effect on aldosterone secretion in BAG cells (Fig 4). Moreover, the effects of AII and 8BcGMP were not additive when cells were treated with both agents. Furthermore, when the effects of AII on EO secretion were eliminated by blocking the AT₂ receptor with PD123319, 8BcGMP raised EO secretion to levels comparable to those seen with AII alone.

View larger version (20K): [in this window] [in a new window]   Figure 4. Stimulation of EO secretion by 8-bromo-cGMP. ZG cells were incubated for 4 hours in secretion medium alone (basal), or containing AII (10 nmol/L), PD (2 µmol/L), 8-bromocGMP (8BcGMP, 0.2 mmol/L), or combinations thereof. EO and aldosterone secreted are means±SEM; *P<.01 and **P<.05 versus basal, n=4 experiments.

Activation of the AT₂ receptor in PC12W cells stimulates protein phosphotyrosine phosphatases (PTPases). Accordingly, we treated cells with the PTPase inhibitor sodium vanadate (Na₃VO₄, 100 µmol/L). Remarkably, Na₃VO₄ had no effect on the basal secretion of aldosterone or EO, whereas this phosphatase inhibitor blocked the effects of AII on EO but not aldosterone secretion (Fig 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. Sodium orthovanadate inhibits stimulated secretion of EO from bovine adrenal ZG cells. Cells were incubated for 4 hours in secretion medium alone, or containing AII (10 nmol/L), Na3VO4 (100 µmol/L), or both. Data are mean±SEM; *P<.01 and **P<.05 versus basal, n=4 experiments.

	Discussion

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Recent work has shown that adrenocortical AT₁ and AT₂ receptors mediate the stimulatory effects of AII on aldosterone and EO secretion, respectively.⁹ The present results demonstrate that the secretions of EO and aldosterone are activated by different intracellular second messenger analogs and are differentially sensitive to modulators of protein kinase C and phosphotyrosine phosphatase. The results are consistent with the view that the post-receptor signaling mechanisms that govern stimulated EO and aldosterone secretion from bovine zona glomerulosa cells are different.

In the current study, concomitant measurements of EO and aldosterone secretion were made in all cell cultures in each of the experiments. Secretion of aldosterone verifies that EO secretion arose from cells whose functional integrity was established by a classical indicator of overall adrenocortical function. In agreement with previous reports from this laboratory, AII consistently elevated the secretions of both EO and aldosterone from cultured cells.^7–9 Pretreatment of the cells with PD123319, a specific AT₂ receptor antagonist in doses known to block the AT₂ and not the AT₁ receptor, antagonized the effects of AII on EO but not aldosterone secretion consistent with a functional role for the AT₂ receptor in steroid secretion.⁹ Basal EO secretion was not affected by PD123319 alone or in the presence of AII. Moreover, in other work, losartan did not affect AII stimulated EO secretion under conditions where the stimulated secretions of aldosterone and cortisol were suppressed completely.⁹ Others have suggested that AT₁ and AT₂ receptors may exhibit crosstalk based on their opposite effects on MAP kinases in cultured neonatal neurons.¹⁰ The extent to which these receptors and their signaling cascades interact in adrenocortical cells is unclear, but are not apparent with our conditions and end points.

Angiotensin II receptors exist in at least two different subtypes, type 1 (AT₁) and type 2 (AT₂), based on their ligand selectivity.¹¹ Many of the known physiological effects of AII, including the rise in both aldosterone secretion and blood pressure, are mediated by the AT₁ receptor. Both the AT₁ and AT₂ receptor subtypes are present on adrenal glomerulosa and medulla cells¹² and have been linked with distinct signal transduction mechanisms in neurons and astrocytes.¹³ In PC12W cells, AT₂ receptors affect PTPase activity and apoptosis.^14–16 Sodium vanadate (Na₃VO₄) is a transition analog of phosphate and a potent inhibitor of PTPases.¹⁷ In our experiments, the stimulatory effects of AII on EO secretion were blocked completely by orthovanadate (Fig 5). Under these conditions, orthovanadate did not affect basal or stimulated aldosterone secretion. Vanadate is also a fairly potent inhibitor of the isolated Na, K-ATPase. However, in healthy cells, free [Mg²⁺] is fairly low, and vanadate is converted to the vanadyl (VO²⁺) cation so that the sodium pump does not become inhibited under these conditions.¹⁸ Accordingly, the effects of vanadate on EO secretion that we describe are not attributable to inhibition of the sodium pump, but are more compatible with the view that a critical event following activation of AT₂ receptors involves stimulation of PTPase activity.

In the adrenal cortex, AII stimulates aldosterone secretion via events involving activation of phospholipase C,^19,20 elevation of IP₃ and DAG,^21,22 and release of calcium from an intracellular pool.¹⁹ The initial rise in [Ca²⁺]_i is believed to initiate an early steroidogenic response mediated by Ca²⁺-calmodulin dependent protein kinases whereas the sustained stimulation of aldosterone secretion by AII requires maintained influx of Ca²⁺ via T-type voltage dependent channels and heightened activity of DAG sensitive protein kinase C.^22–27 Although protein kinase C activators (such as tetradecanoyl phorbol acetate) stimulate aldosterone and cortisol secretion, their maximal stimulatory effects are typically 30 to 50% of that observed with AII.^28–30 Consistent with the aforementioned studies, direct activation of PKC by the phorbol ester TPA in our experiments raised aldosterone secretion to levels about 50% of those seen with AII stimulation. Under these conditions, no effect of TPA was observed on EO secretion. These results suggest that, in contrast to aldosterone secretion, PKC either is not a component of the EO signaling pathway or that it is not a sufficient stimulus to its secretion.

Previously, we showed that ACTH stimulates EO secretion from bovine adrenocortical cells.^7–9 Adrenocortical ACTH receptors are coupled, in part, to adenylyl cyclase. A rise in cellular cAMP, induced either by cell permeant analogs or by ACTH, stimulates PKA activity and activates aldosterone secretion maximally.^22,31 In our experiments, treatment of BAC cells with a maximally effective concentration of the cell permeant analog, dbcAMP, raised aldosterone secretion to levels similar to those observed with AII, whereas EO secretion remained unaffected. Therefore, the present results suggested that neither cAMP nor PKA appear to be critical components of the EO signaling pathway.

In view of our earlier results with ACTH, the absence of an effect of dbcAMP on EO secretion was surprising, and we investigated alternative intracellular mediators. ACTH may elevate cellular cGMP and cAMP, and the former may be more important in stimulated corticosterone secretion from rat adrenocortical cells.^32,33 Moreover, activation of AT₂ receptors in neuroblastoma cells activates soluble guanylyl cyclase and increases cellular cGMP (34). In our preparations, the membrane permeant analog 8BcGMP raised basal EO secretion to levels comparable to those observed with AII. Further, when the two stimulants were coapplied, no further enhancement of EO secretion was observed consistent with the notion that cGMP may be a component of the AT₂ signaling pathway. In addition, the stimulatory effects of 8BcGMP on EO secretion persisted in the presence of PD123319 (Fig 4) suggesting that the effects of the analog occur at a point distal to the AT₂ receptor. In contrast, 8BcGMP had no effect on basal or AII stimulated aldosterone secretion. Collectively, these observations indicate that cellular cGMP may specifically and maximally activate EO secretion, and, although not determined directly by this work, they raise the possibility that AT₂ receptors may be linked with guanylyl cyclase. Further work will be necessary to define the relationship between adrenocortical AT₂ receptors and guanylyl cyclase activity in ZG cells.

The present results show that ACTH stimulates EO secretion and suggest that the cellular mediator may not be cAMP. These results inferred the existence of either two different ACTH receptor subtypes or the coupling of a common receptor to two intracellular messengers. Two classes of ACTH receptors with high and low affinity have been reported in adrenocortical cells,³⁵ and it has been suggested that high concentrations of ACTH activate adenylyl cyclase and raise cAMP levels, whereas physiologically relevant amounts of this peptide stimulate a high affinity receptor not linked to cAMP or aldosterone secretion.³⁶ The data from our experiments with the cell permeant analogs are compatible with the hypothesis that ACTH activates aldosterone and EO secretion via different signaling mechanisms and suggest that the regulation of EO and aldosterone secretion is distinct. In accord with this conclusion, Goto et al have described a hypertensive patient with ACTH excess secondary to ectopic corticotropin syndrome in which EO and cortisol were elevated with normal aldosterone.³⁷ In addition, we have found elevated circulating levels of aldosterone and EO in approximately 50% of hypertensive patients with surgically confirmed primary hyperaldosteronism, whereas the remaining patients exhibited only increased circulating levels of aldosterone.³⁸ Furthermore, in a preliminary communication, we have described a hypertensive patient with an adrenocortical tumor in whom the circulating levels of EO were elevated, whereas the classical adrenocortical steroids were normal.³⁹ The aforementioned clinical data suggest not only that tumors of this zone may exhibit at least three functionally distinct subtypes but raise the possibility that alterations in steroid specific signal transduction pathways may underlie the pathophysiology.

Zona glomerulosa cells express both AT₁ and AT₂ receptors and appear to be the site from which aldosterone and EO originate.⁸ The observations that the secretion of either steroid can be activated from ZG cells independently and that the cells in this layer express aldosterone synthase suggest that EO and part of the aldosterone are secreted by the same cells.^9,40 The rise in intracellular Ca²⁺ in bovine ZG cells following ACTH or AII stimulation is confined to discrete subcellular domains but the physiological significance of this phenomenon remains unknown.⁴¹ Because calcium influx is a trigger for EO (J.L. and J.M.H., unpublished data, 1996) and aldosterone secretion,^23,28 the aforementioned observations raise the possibility that the synthesis and secretion of EO and aldosterone arise from spatially distinct regions in the same cell.

In summary, previous work with AII receptor antagonists and agonists revealed distinct effects of AT₁ and AT₂ receptors on steroid secretion in bovine adrenocortical cells. The present results demonstrate that many of the post-receptor events involved in the stimulated secretions of EO and aldosterone by bovine adrenocortical cells are mediated by specific signaling mechanisms consistent with the view that their secretions are regulated differently.

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