Disabled-2 is expressed in adrenal zona glomerulosa and is involved in aldosterone secretion

The differentiation of the adrenal cortex into functionally specific zones is probably due to differential temporal gene expression during fetal growth, development, and adulthood. In our search for adrenal zona glomerulosa-specific genes, we found that Disabled-2 (Dab2) is expressed in the zona glomerulosa of the rat adrenal gland using a combination of laser capture microdissection, mRNA amplification, cDNA microarray hybridization, and real-time RT-PCR. Dab2 is an alternative spliced mitogen-regulated phosphoprotein with features of an adaptor protein and functions in signal transduction, endocytosis, and tissue morphogenesis during embryonic development. We performed further studies to analyze adrenal Dab2 localization, regulation, and role in aldosterone secretion. We found that Dab2 is expressed in the zona glomerulosa and zona intermedia of the rat adrenal cortex. Low-salt diet treatment increased Dab2-long isoform expression at the mRNA and protein level in the rat adrenal gland, whereas high-salt diet treatment did not cause any significant modification. Angiotensin II infusion caused a transient increase in both Dab2 isoform mRNAs in the rat adrenal gland. Dab2 overexpression in H295R human adrenocortical cells caused an increase in aldosterone synthase expression and up-regulated aldosterone secretion under angiotensin II-stimulated conditions. In conclusion, Dab2 is an adrenal gland zona glomerulosa- and intermedia-expressed gene that is regulated by aldosterone secretagogues such as low-salt diet or angiotensin II and is involved in aldosterone synthase expression and aldosterone secretion. Dab2 may therefore be a modulator of aldosterone secretion and be involved in mineralocorticoid secretion abnormalities.     

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.     

Effects of prolactin administration on the zona glomerulosa of the rat adrenal cortex: stereology and plasma hormone concentrations

The effects of short and prolonged treatments with prolactin on the morphology and hormone secretion of the rat adrenal zona glomerulosa were investigated by coupled morphometric and radioimmunological techniques. Short-term prolactin administration did not alter plasma aldosterone concentration or zona glomerulosa morphology. Conversely, chronic prolactin treatment caused both a notable hypertrophy of zona glomerulosa cells and a significant rise in the blood level of aldosterone. The possibility is discussed that prolactin may be involved in the control of the growth and steroidogenic capacity of the rat adrenal zona glomerulosa.     

Expression and localization of human dopamine D2 and D4 receptor mRNA in the adrenal gland, aldosterone-producing adenoma, and pheochromocytoma

Aldosterone secretion is evidently regulated by a dopaminergic inhibitory mechanism. Pharmacological characterization and autoradiographic studies revealed D2-like receptors in the adrenal cortex, especially in the zona glomerulosa. However, the subtype of the dopamine receptors involving this regulation has not been elucidated. To investigate which subtype of receptors expresses in the adrenal cortex, we examined the messages of D2-like receptors, D2, D3, and D4, by RT-PCR and in situ hybridization of adrenal glands and adrenal neoplasm. Both D2 and D4 receptors were expressed in normal adrenal glands, pheochromocytoma, and aldosterone-producing adenoma. However, the D2 receptors were not universally expressed, in contrast with the D4 receptors that were detected in all cases of aldosterone-producing adenoma and adrenal remnant. No D3 receptor message was detected by RT-PCR in any adrenal sample. Both D2 and D4 receptors were expressed in significant amounts in the adrenal medulla and pheochromocytoma. In the adrenal cortex, the expression of the D2 receptors was in the zona glomerulosa and zona reticularis, with no different signal intensities between the two zones. D4 receptors were mainly localized in the zona glomerulosa and, to a lesser extent, in the zona reticularis. Both receptors were expressed at low levels in the zona fasciculata. In aldosterone-producing adenoma, the expression of D2 and D4 was especially found in nonzona fasciculata-like cells. To elucidate which dopamine receptor regulates aldosterone secretion, the effects of specific D2 and D4 antagonists, raclopride and clozapine, respectively, were examined in cultured NCI-H295 cells. Dopamine further increased angiotensin II-induced aldosterone secretion by 20%. In the presence of 1 microM dopamine and angiotensin II, 10(-5)-10(-7) M clozapine decreased aldosterone levels by 40-55%. The decrease in aldosterone secretion by clozapine was completely reversed when raclopride was added simultaneously. These data suggest that dopamine exerts dual effects on aldosterone secretion in NCI-H295 cells. Activation of D4 receptors can increase aldosterone secretion, whereas an inhibitory effect is mediated via D2 receptors. In summary, we demonstrated the existence of both D2 and D4 receptors in the human adrenal gland and adrenal neoplasm. Both receptors play significant roles in the modulation of aldosterone secretion, but in opposite directions.     

Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium

The stimulation of aldosterone secretion from adrenal glomerulosa cells by angiotensin II (AII), potassium, and ACTH is highly dependent on the extracellular calcium concentration. To evaluate the role of voltage-dependent calcium channels in aldosterone production, we analyzed the actions and binding of calcium channel antagonists in collagenase-dispersed adrenal glomerulosa cells and membrane-rich particles. In rat glomerulosa cells, nifedipine caused dose-dependent inhibition of the aldosterone responses to AII and potassium, with half-maximum inhibitory concentration (IC50) of 100 nM, but had no effect on ACTH or 8-bromo-cAMP stimulated steroidogenesis in adrenal glomerulosa and fasciculata cells. Binding studies with [3H]nitrendipine in adrenal glomerulosa cells revealed a high affinity site with dissociation constant (Kd) of 0.4 +/- 0.1 nM, similar to that described in other tissues but about 100-fold lower than the IC50 for blockade of aldosterone production. However, Scatchard analysis of binding data from three of seven experiments in isolated adrenal glomerulosa cells revealed a low affinity site with Kd of 130 nM, in agreement with the IC50 for the effect of nifedipine on aldosterone production. In rat adrenal particles, nitrendipine-binding sites were located in the adrenal capsule and medulla and were undetectable in the zona fasciculata. Furthermore, there was a close correlation (r = 0.92) between the concentrations of nitrendipine-binding sites and AII receptors in the different zones of the adrenal in rat, dog, and cow, suggesting a functional relationship between AII receptors and calcium channels. These studies have shown a major and selective role of voltage-dependent calcium channels in the control of aldosterone secretion by the major physiological regulators, AII and potassium.     

Long-term trophic action of alpha-melanocyte-stimulating hormone on the zona glomerulosa of the rat adrenal cortex

The effects of alpha-melanocyte-stimulating hormone (alpha-MSH) on the rat adrenal cortex were investigated by coupled morphometric and radioimmunological techniques. Short-term alpha-MSH administration provoked a significant increase in the aldosterone plasma level along with a notable lipid droplet depletion in zona glomerulosa cells. Long-term alpha-MSH treatment induced a notable hypertrophy of zona glomerulosa cells and a further rise in the blood concentration of aldosterone. alpha-MSH did not affect zona fasciculata morphology and corticosterone plasma level. The possibility is discussed that alpha-MSH may be specifically involved in the control of the growth and steroidogenic capacity of rat adrenal zona glomerulosa.     

Biosynthetic ad morphological evidence for inhibition of aldosterone production following administration of ACTH to sheep

The effect of ACTH administration for 1-5 days on the morphology and steroidogenic capability of sheep adrenal tissue has been examined. During this period of treatment there was a gradual decline in the in vitro conversion of 3H-labelled precursors to products of solely zona glomerulosa origin (aldosterone and 18-hydroxycorticosterone) while conversion to products of zona fasciculata origin (17-hydroxyprogesterone, 11-deoxycortisol and cortisol) was stimulated throughout. Conversion to DOC, 18-hydroxydeoxycorticosterone and corticosterone (steroids produced by both the zona glomerulosa and the zona fasciculata) declined after initial stimulation. Within 2--3 days of the commencement of treatment, the zona glomerulosa showed a progressive decrease in cell number associated with disruption of cords and cell separation. Ultrastructurally, it was found that typical zona glomerulosa cells had almost disappeared. The majority of residual cells in this area had a structure intermediate between zona glomerulosa and zona fasciculata cells. The similarity in time-course of the alterations in both the morphological and biosynthetic characteristics suggests that the decline in aldosterone output caused by ACTH administration to sheep results from the loss of adrenal zona glomerulosa cells, predominantly due to selective cellular degeneration.     

Serotonin increases interleukin-6 release and decreases tumor necrosis factor release from rat adrenal zona glomerulosa cells in vitro

Interleukin-6 (IL-6) and tumor necrosis factor (TNF) are secreted by rat adrenal zona glomerulosa cells. Serotonin increases the release of aldosterone, corti-costerone, and cortisol from the adrenal cortex. Therefore, the effects of serotonin on IL-6 and TNF release from rat adrenal zona glomerulosa cells were investigated. Cultures of rat adrenal zona glomerulosa cells were enzymatically prepared and cultured for 4–6 d. The cells were then exposed to serum-free RPMl-1640 medium containing vehicle (RPMl medium alone), serotonin, and/or endotoxin, interleukin-1β, or adrenocorticotrophic hormone (ACTH). Following a 5-h incubation, medium was removed from the cells, and IL-6 and TNF content of this medium determined with bioassays. Serotonin (1–1000 nM) increased basal IL-6 release from zona glomerulosa cells, but inhibited basal TNF release from these cells. Endotoxin and interleukin-1β (IL-1β) increased IL-6 and TNF release from zona glomerulosa cells. Serotonin potentiated IL-6 release stimulated by endotoxin and IL-1β, but inhibited TNF release stimulated by these agents. Serotonin potentiated ACTH-stimulated IL-6 release. Serotonin had no effect on IL-6 release from rat anterior pituitary cells. Because IL-6, TNF, and serotonin modify the release of aldosterone and glucocorticoids from adrenal cells, the stimulatory effects of serotonin on aldosterone and glucocorticoid release may be mediated in part by the effects of serotonin on IL-6 and TNF release from adrenal cells.     

Adrenal renin: A possible local hormonal regulator of aldosterone production

Summary The complete renin-angiotensin system is present in the adrenal cortex; prorenin, renin, angiotensinogen, angiotensin I and II, and converting enzyme. Most of the renin found is probably synthesized there since the renin concentration increases after nephrectomy, and the mRNA for renin is present. The renin-angiotensin system has the highest activity in the zona glomerulosa cells, the site of aldosterone formation. A low-sodium dist or a high-potassium diet, or nephrectomy markedly inereases the adrenal renin concentration in the zona glomerulosa cells without any effect on the fasciculata-medullary cells. There is a close correlation between adrenal renin and aldosterone production. The adrenal renin angiotensin system may be a local regulator of aldosterone production.     

Production of aldosterone and its precursor steroids by the adrenal zona glomerulosa in dextran sulfate-treated rats

Heparin and heparinoids are known to produce selective aldosterone deficiency in man and experimental animals. To assess the nature of the hypoaldosteronism caused by heparin and heparinoids, we investigated the production of aldosterone and its precursor steroids in response to angiotensin II (AII), ACTH or potassium in adrenal zona glomerulosa cells from dextran sulfate-treated rats compared with that in the cells from vehicle-treated rats. Dextran sulfate-treated rats had a decrease in plasma aldosterone and a reduction in the width of the zona glomerulosa 4 weeks after the treatment (40 mg/day, intramuscularly). In these rats, PRA and plasma AII tended to be high, and plasma corticosterone was normal. Basal aldosterone production, when corrected to a uniform number of cells per group, was similar in cells from dextran sulfate- and vehicle-treated rats. The cells from dextran sulfate-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 maximal AII-stimulated level. The maximal AII-stimulated levels, but not the basal levels, of pregnenolone, corticosterone and 18-hydroxycorticosterone production were low in the cells from dextran sulfate-treated rats. ACTH produced a similar stimulatory effect on aldosterone production in the cells from dextran sulfate- and vehicle-treated rats. The cells from dextran sulfate-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 long-term treatment with dextran sulfate in rats produces selective impairment of adrenal zona glomerulosa cells, involving the specific receptors and the aldosterone biosynthesis, to AII in addition to a reduction in the glomerulosa width.     

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.     

Expression of prostaglandin E receptor EP4 subtype in rat adrenal zona glomerulosa: involvement in aldosterone release

We examined the role of prostaglandin (PG) E receptors in the secretion of aldosterone. PGE2 is known to exert its various biological functions by binding to PGE receptors. There are four subtypes of PGE receptors, EP1, EP2, EP3, and EP4. Among the PGE receptors EP2 and EP4 subtypes are coupled to Gs protein and stimulate adenylyl cyclase. In this study, PGE2 caused a dose-dependent increase in aldosterone production from the rat adrenal zona glomerulosa cells in vitro accompanied with an increase in intracellular cAMP concentration. A specific agonist for EP2, butaprost, did not increase the cAMP production or the aldosterone release, suggesting the possibility that EP4 mediates the secretion of aldosterone by PGE2. Northern blot hybridization analysis disclosed that EP4 gene was expressed in the rat adrenal gland but that EP2 gene was not. In situ hybridization revealed that EP4 mRNA is present abundantly in the zona glomerulosa of rat adrenal gland. These findings suggest that the PGE2-EP4 system is involved in the regulation of aldosterone secretion from the rat adrenal gland.     

Long-term effects of ACTH combined with angiotensin II on steroidogenesis and adrenal zona glomerulosa morphology in the rat

To test the hypothesis that the trophic action of angiotensin II on the adrenal zona glomerulosa may allow a sustained stimulation of aldosterone by ACTH by preventing the morphological changes of the zona glomerulosa cells into zona fasciculata-like elements we investigated the effects in rats of a 6-day treatment with ACTH (100 micrograms/kg/day) alone or combined with angiotensin II (300 ng/kg/day) on corticosterone and aldosterone production and adrenal morphology. The responsiveness of both steroids to an acute ACTH dose was also studied on the last day of long-term treatment. Morphologic data showed that prolonged ACTH treatment stimulated the growth of zona glomerulosa cells, though it transformed the tubulo-lamellar cristae of mitochondria into a homogeneous population of vesicles. Angiotensin II furthered the trophic effects of ACTH but prevented the mitochondrial transformation. Despite its ability to conserve the well differentiated aspect of the zona glomerulosa cells, the administration of angiotensin II was unable to prevent the fall in the secretion of aldosterone caused by chronic ACTH treatment and its subsequent unresponsiveness to ACTH stimulation.     

Specific effects of agonists of the calcium messenger system on secretion of 'late-pathway' steroid products by intact tissue and dispersed cells of the rat adrenal zona glomerulosa

Recent data have implicated the phosphatidylinositol/calcium second-messenger system in the control of aldosterone secretion by the adrenal zona glomerulosa. However, in the rat adrenal there are few reports of a direct effect of protein kinase C activation on steroid secretion, while the effects of calcium mobilization may be variable. Since the rat adrenal zona glomerulosa is sensitive to the mode of tissue preparation, these mechanisms were reinvestigated in intact (non-dispersed) capsular tissue and collagenase-dispersed zona glomerulosa cells. Steroidogenesis in the intact zona glomerulosa was markedly affected by agonists of the calcium messenger system. Most notably, aldosterone and 18-hydroxycorticosterone (18-OH-B) secretion were stimulated by A23187 (100 nmol to 10 mumols/l) and BAY K 8644 (500 nmol/l). Phorbol 12-myristate 13-acetate (TPA; 1 pmol to 1 mumol/l) stimulated aldosterone secretion at all doses and caused a dose-dependent increase in 18-OH-B and 18-hydroxydeoxycorticosterone (18-OH-DOC) secretion. Corticosterone secretion was slightly increased in the presence of A23187 but not by TPA or BAY K 8644. Production of 18-OH-DOC was unaffected by A23187 and BAY K 8644. The calcium channel antagonist verapamil (10 mumols/l) inhibited ACTH-stimulated aldosterone secretion by the intact zona glomerulosa but had no effect on corticosterone secretion. Verapamil (10 mumols/l) also inhibited the increase in aldosterone secretion by collagenase-dispersed zona glomerulosa cells stimulated by ACTH (100 fmol to 100 nmol/l), angiotensin II (100 pmol to 10 nmol/l) and potassium (5.9 and 8.4 mmol/l); stimulated corticosterone secretion was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholecystokinin stimulates aldosterone secretion from dispersed rat zona glomerulosa cells, acting through cholecystokinin receptors 1 and 2 coupled with the adenylate cyclase-dependent cascade

Cholecystokinin is a regulatory peptide, that acts through two subtypes of receptors, 1 and 2. RT-PCR demonstrated the expression of both cholecystokinin receptors 1 and 2 genes in the zona glomerulosa, but not the zona fasciculata-reticularis, of rat adrenals. Autoradiography demonstrated the presence of abundant [(125)I]cholecystokinin-binding sites in the zona glomerulosa, but not the zona fasciculata-reticularis, which were displaced by both cholecystokinin receptor 1- and 2-selective antagonists (cholecystokinin 1-A and 2-A). Cholecystokinin increased basal aldosterone secretion from dispersed zona glomerulosa cells without affecting corticosterone secretion from zona fasciculata-reticularis cells. The aldosterone response to cholecystokinin was blunted by cholecystokinin 1-A and 2-A, which when added together abolished it. ACTH-stimulated aldosterone production was not affected by cholecystokinin; in contrast, cholecystokinin potentiated aldosterone response to both angiotensin II and K(+). Cholecystokinin enhanced cAMP, but not IP(3), release by dispersed zona glomerulosa cells. The aldosterone response to cholecystokinin was abolished by the adenylate cyclase inhibitor SQ-22536 and the PKA inhibitor H-89, but not by either the PLC inhibitor U-73122 or the PKC inhibitor calphostin C. In conclusion, our study provides evidence that cholecystokinin, acting through cholecystokinin receptors 1 and 2 coupled with the adenylate cyclase/PKA cascade, exerts a sizeable secretagogue action on rat zona glomerulosa cells.     

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.