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.     

Adrenocorticotropin increases interleukin-6 release from rat adrenal zona glomerulosa cells.

Interleukin-6 (IL-6) is produced by adrenal zona glomerulosa cells; its release is stimulated by several secretagogues, including IL-1 alpha, IL-1 beta, and angiotensin II. The present study reports that ACTH (0.1-100 nM) increased the release of IL-6 from primary cultures of rat adrenal cells in a concentration-dependent manner. This increase was accompanied by an increase in cAMP content in cell extracts and in the incubation medium. The dynamics of IL-6 release from the adrenal cells also were investigated using a perifusion system; approximately 50 min were required for the effects of IL-1 alpha, IL-1 beta, and ACTH on IL-6 release to become apparent. Following withdrawal of the secretagogues, IL-6 release returned to basal levels within 90-120 min. In some experiments, the adrenal zona glomerulosa was separated from the zona fasciculata/reticularis to determine the origin of secretagogue-stimulated IL-6 release. PGE2 and forskolin increased IL-6 release from both cell types, but maximal release from zona glomerulosa cells was more than 10-fold greater than that from zona fasciculata/reticularis cells. ACTH (0.1-100 nM) increased intracellular cAMP levels in cells from both cell types in a concentration-dependent manner, but increased IL-6 release only from zona glomerulosa cells. Dexamethasone, an inhibitor of IL-6 production in several tissues, had no effect on either basal or stimulated IL-6 production in the adrenal. Because IL-1 beta is produced primarily by tissues of the immune system, whereas ACTH is a classical endocrine hormone, we investigated the effect of interaction of these proteins on IL-6 release from the adrenal. Together, IL-1 beta and ACTH stimulation of IL-6 release was greater than the sum of the effects of each substance separately; however, IL-1 beta did not potentiate the effect of ACTH on cAMP levels. Similarly, IL-1 beta potentiated IL-6 release stimulated by forskolin and (Bu)2cAMP. Thus, the adrenal may be an important convergence point between the immune and endocrine systems, and because IL-6 release is regulated by IL-1 alpha, IL-1 beta, ACTH, and angiotensin II, and this cytokine stimulates corticosterone release, IL-6 may play an important paracrine role in integrating the signals derived from these systems.     

Effects of prolonged cyclosporine-A treatment on the morphology and function of rat adrenal cortex

The effects of prolonged (30 day) treatment with daily therapeutical doses of cyclosporine A (CSA) (20 mg/kg) on the function and morphology of adrenal cortex were studied in adult male rats. CSA-treated animals developed a notable hypertension, along with a striking rise in PRA, which was not coupled with significant changes in the plasma concentrations of aldosterone and corticosterone (hyperreninemic hypoaldosteronism). Morphometry showed that zona glomerulosa (ZG) and zona fasciculata, and their parenchymal cells were atrophic. Isolated capsular (ZG) and inner (zona fasciculata/reticularis) cells displayed reduced basal and stimulated secretory responses. However, while the response of ZG cells to angiotensin II was almost completely suppressed (96%), basal steroid secretion of isolated cells, as well as the aldosterone and corticosterone response of ZG cells to potassium and ACTH, and corticosterone production of inner cells in response to ACTH were decreased by only about 30-40%. The hypothesis is advanced that CSA exerts a dual effect on rat adrenal cortex: 1) a general inhibitory effect on the growth and steroidogenic capacity of adrenocortical cells, which manifests itself only after very prolonged treatment and may be caused by an impairment of protein synthesis; and 2) an acute effect involving the specific blockade of the angiotensin-II-induced stimulation of the secretory activity of ZG cells.     

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.     

Specific receptor-mediated inhibition by synthetic atrial natriuretic factor of hormone-stimulated steroidogenesis in cultured bovine adrenal cells

The effect of synthetic atrial natriuretic factor (ANF) on adrenal steroidogenesis has been studied in primary culture of bovine adrenal cells. ANF-(8-33) produced a potent 40-70% inhibition of angiotensin II-, ACTH-, PGE1-, and forskolin-stimulated secretion of aldosterone production from zona glomerulosa cells with an ED50 of 120 pM. An equipotent inhibitory effect of the natriuretic factor on cortisol production was also observed in cultured zona fasciculata cells. Nicotine-stimulated secretion of catecholamines from medullary cells was only slightly inhibited by the factor at doses above 10 nM. [125I]iodo-ANF-(8-33) binding to glomerulosa membranes displayed an apparent affinity of 100-150 pM for specific receptor sites and was not inhibited by angiotensin II or ACTH. Conversely, the natriuretic factor had no affinity for angiotensin II receptor sites. The results demonstrate that part of the natriuretic effect of this new factor might be due to inhibition of adrenal steroidogenesis by action through a distinct receptor.     

Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II.

The production of aldosterone by isolated canine zona glomerulosa cells was measured after the incubation of cell suspensions with angiotensin II and ACTH, and during changes in extracellular potassium concentration. Adrenal cell suspensions were prepared by collagenase digestion and physical dispersion of the capsular layer of the dog adrenal cortex, and aldosterone production was determined by direct radioimmunoassay of cell incubation media. The isolated dog adrenal cells were highly responsive to angiotensin II, with aldosterone production significantly stimulated by concentrations of the octapeptide as low as 10(-11)M. Thus, the steroidogenic response of zona glomerulosa cells was consistently observed at peptide concentrations within the physiological range of angiotensin II in dog plasma, i.e., 2.0-5.0 X 5.0 X 10(-11)M. The maximum aldosterone response of 3-8 times the basal level of steroid production was induced by 3 X 10(-10)M angiotensin II, and a decrease in aldosterone production occurred at peptide concentrations above 10(-9)M. The aldosterone response of isolated adrenal cells to ACTH was consistently less sensitive than their response to angiotensin II, by a factor of 10-20 fold. Aldosterone production was significantly increased by 10(-10)M ACTH, and reached a maximum at 10(-8)M ACTH. By contrast with angiotensin II, ACTH usually evoked a higher maximal level of aldosterone production, and did not produce a decline in steroidogenesis at peptide concentrations above the level which caused maximum stimulation of aldosterone formation. Changes in the potassium concentration of cell incubation media were also accompanied by marked effects upon aldosterone synthesis which was abolished in the absence of potassium and became detectable in the presence of 0.5 mM K+. After remaining constant between 2.5 and 4.0 mM K+, aldosterone production rose sharply above 4.5 mM K+ and reached a maximum at 8 mM K+. These observations provide direct evidence that aldosterone production by zona glomerulosa cells is influenced by changes in angiotensin II levels within the normal plasma range.     

The 21-hydroxylase activity in the glomerulosa and fasciculata of the adrenal cortex in congenital adrenal hyperplasia

In the two clinical syndromes of congenital adrenal hyperplasia due to a 21-hydroxylation defect of adrenal steroidogenesis, the simple virilizing and the salt-wasting forms, the 21-hydroxylase activity was studied considering the zona fasciculata and the zona glomerulosa of the adrenal cortex as two separate glands under different regulation. To test this hypothesis, we stimulated adrenal steroidogenesis by ACTH infusion or dietary sodium restriction in eight patients with congenital adrenal hyperplasia (four patients with the simple virilizing form and four with the salt-wasting form of congenital adrenal hyperplasia) and in six normal children. Both the 17-hydroxy and 17-deoxy pathways of adrenocortical steroid biosynthesis were examined by measuring serum concentrations of 17-hydroxyprogesterone, cortisol, progesterone, deoxycorticosterone, corticosterone, and aldosterone and the excretion of free deoxycorticosterone, 18-hydroxydeoxycorticosterone, corticosterone, 18-hydroxycorticosterone, cortisol, and aldosterone. We considered the steroids 18-hydroxycorticosterone and aldosterone to be primarily of zona glomerulosa origin. These studies indicated that the zona fasciculata of both the salt-wasting and the simple virilizing forms is defective in 21-hydroxylation of 17-hydroxy and 17-deoxy steroids. The zona glomerulosa demonstrated deficient 21-hydroxylation only in the salt-wasting form, whereas in the simple virilizing form, the glomerulosa was spared this defect.     

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.     

Vasoactive intestinal peptide stimulates adrenal aldosterone and corticosterone secretion

Vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers have been demonstrated in the rat adrenal cortex in close association with zona glomerulosa cells, suggesting neural regulation of adrenocortical cell function (5). The present studies were undertaken to study the possible role of VIP in the regulation of steroid hormone secretion from the outer zones of the normal rat adrenal cortex. Capsule-glomerulosa preparations, consisting of the capsule, zona glomerulosa, and a small but variable portion of the zona fasciculata, were perifused in vitro. To assess the secretory responsiveness of the capsule-glomerulosa preparation, aldosterone and corticosterone release were measured after stimulation with ACTH and angiotensin II. ACTH (10(-12)-10(-8) M) stimulated dose-dependent increases in aldosterone secretion (1.9- to 36.9-fold increases over basal values) and corticosterone secretion (1.4- to 14.0-fold increases over basal values). Angiotensin II (10(-8)-10(-5) M) stimulated dose-dependent increases in aldosterone secretion (1.6- to 8.8-fold increases over basal values). VIP (10(-6)-10(-4) M) stimulated dose-dependent increases in both aldosterone (1.7- to 41.0-fold) and corticosterone secretion (1.8- to 5.3-fold). However, glucagon and (N-Ac-Tyr1-D-Phe2)GRF-(1-29)NH2, peptides structurally related to VIP, stimulated neither aldosterone nor corticosterone secretion, indicating that VIP effects are likely to be specific for this peptide. It is noteworthy that in this preparation, the stimulation of corticosteroid secretion by VIP at 10(-5) and 10(-4) M was comparable to those by 10(-6) M angiotensin II and 10(-9) M ACTH, respectively. These results support the hypothesis that the VIP innervation of the adrenal cortex may contribute directly to the regulation of adrenal steroidogenesis.     

Distribution of extracellular signal-regulated protein kinases 1 and 2 in the rat adrenal and their activation by angiotensin II

The adrenal gland of the rat is continuously regenerated through proliferation of a stem cell population in the outer part of the gland. To clarify the location of proliferative events within the adrenal gland, and the factors that stimulate them, rat adrenal capsule preparations, consisting of capsule, zona glomerulosa (ZG) and the outer zona fasciculata (ZF) were maintained in vitro under different conditions of stimulation, for varying periods. Sites of proliferation were identified by 5-bromo-2'-deoxy-uridine (BrdU) staining, and the distribution of classical MAP kinase (MAPK) family members, extracellular signal-regulated kinase (ERK) 1 and 2, immunoreactivity was determined using immunocytochemistry. BrdU staining was limited to the outer glomerulosa and the capsule, where it was enhanced by angiotensin II, but not by a high potassium ion concentration nor by ACTH. In contrast, ERK1/2 immunoreactivity was distributed throughout the ZG and in the medulla, with none detectable in the ZF and reticularis. Furthermore, angiotensin II, potassium ions and ACTH were all shown to induce ERK1 and ERK2 phosphorylation in the ZG. Treatment of adrenal capsule tissue with the specific MAPK kinase inhibitor PD98059 revealed inhibition of ERK1/2 phosphorylation, but no effect on angiotensin II-induced aldosterone secretion. Although the distribution and activation of the MAPK pathway suggest a link with proliferation, the findings clearly designated only the outer part of the glomerulosa and capsule as a potential stem cell population. Further functions should be sought for the apparently silent major part of the glomerulosa.     

In vitro responses of focal hyperplastic tissue of the human adrenal zona fasciculata to ACTH.

The temporal cAMP, cortisol and aldosterone responses to ACTH of focal hyperplasia of the zona fasciculata and of normal human adrenocortical tissue were investigated. ACTH significantly increased cAMP levels (1 min) and cortisol output (2 min) in normal adrenal tissue but not in hyperplastic tissue. However, following ACTH treatment cortisol and aldosterone production were depressed in the abnormal adrenal tissue below the untreated or the ACTH stimulated normal adrenal tissue. In addition, basal cortisol and aldosterone production of the hyperplastic adrenal tissue was elevated above that of the normal adrenal tissue. These findings suggest that the cAMP second messenger concept may be only one of several mechanisms in the modulation of human adrenocortical function.     

A role for phospholipase D in angiotensin II-induced protein kinase D activation in adrenal glomerulosa cell models

The mineralocorticoid aldosterone plays an important role in regulating blood pressure, with excess causing hypertension and exacerbating cardiovascular disease. Previous studies have indicated a role for both phospholipase D (PLD) and protein kinase D (PKD) in angiotensin II (AngII)-regulated aldosterone production in adrenal glomerulosa cells. Therefore, the relationship between AngII-activated PLD and PKD was determined in two glomerulosa cell models, primary bovine zona glomerulosa (ZG) and HAC15 human adrenocortical carcinoma cells, using two inhibitors, 1-butanol and the reported PLD inhibitor, fluoro-2-indolyl des-chlorohalopemide (FIPI). FIPI was first confirmed to decrease PLD activation in response to AngII in the two glomerulosa cell models. Subsequently, it was shown that both 1-butanol and FIPI inhibited AngII-elicited PKD activation and aldosterone production. These results indicate that PKD is downstream of PLD and suggest that PKD is one of the mechanisms through which PLD promotes aldosterone production in response to AngII in adrenal glomerulosa cells.     

Studies on cyclic nucleotides in the adrenal gland. VIII. Effects of angiotensin on adenosine 3',5'-monophosphate and steroidogenesis in the adrenal cortex.

Effects of angiotensin II on corticoid biogenesis and cAMP levels in the zona fasciculata-reticularis (the decapsulated fraction) and the zona glomerulosa (the capsular fraction) from the rat adrenal gland have been studied. Angiotensin II exclusively stimulated steroidogenesis in the zona glomerulosa without stimulation of the cAMP system, suggesting that steroidogenic action of this polypeptide does not involve the adenylate cyclase system. Angiotensin II was also found to stimulate cAMP-phosphodiesterase activity in the zona glomerulosa. An elevation of calcium concentration in the incubation medium has been observed to be effective in stimulating the production of aldosterone and cAMP by the capsular fraction. Angiotensin II caused a significant enhancement of the steroidogenic response of the capsular fraction to increasing calcium concentration regardless of the response of the cAMP system to calcium. This steroidogenic effect of angiotensin II was completely abolished by calcium antagonists added to the incubation medium without any inhibitory effect on the calcium-induced accumulation of tissue cAMP. These results suggest that angiotensin II acts on the adrenal II acts on the adrenal glomerulosa cell to increase intracellular calcium, which in turn directly stimulates steroidogenesis concomitant with the increased activity of phosphodiesterase.     

Dissecting human adrenal androgen production.

The human adrenal cortex produces aldosterone, cortisol and the so-called adrenal androgens, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). Within the adult adrenal, the zona glomerulosa produces aldosterone, the zona fasciculata cortisol and the zona reticularis both DHEA and DHEAS. The processes regulating aldosterone and cortisol synthesis are well defined; however, the mechanisms regulating the production of DHEA(S) remain elusive. The emphasis of this review is based on increasing evidence that cytochrome b(5), DHEA sulfotransferase and 3 beta-hydroxysteroid dehydrogenase play crucial roles in regulating production of DHEA(S). Insight into the mechanisms that regulate the synthesis of these key components of DHEA(S) synthesis should provide important clues to the regulation of adrenal androgen biosynthesis.     

Molecular evidence of organic ion transporters in the rat adrenal cortex with adrenocorticotropin-regulated zonal expression

Experimental evidence suggested that secretion of steroid hormones from adrenocortical cells involves carrier-mediated transport: Cortisol release from, and uptake of p-[3H]aminohippurate into, bovine adrenocortical cells showed properties of the renal p-[3H]aminohippurate/anion exchanger OAT1. Other poly-specific transporters such as organic anion-transporting polypeptides (oatps) and organic cation transporters (OCTs) could also be involved in steroid hormone release. A homology-cloning procedure was established to detect these transporters in rat adrenal gland cDNA. PCR revealed the presence of OAT1, oatp1, oatp2, and oatp3. In situ hybridization localized OAT1 in the outer zona fasciculata, oatp3 in the zona glomerulosa, and oatp1 and oatp2 in the inner zona fasciculata and outer zona reticularis. An OCT2-specific probe produced signals in the zona glomerulosa and outer zona fasciculata. Pretreatment of rats with ACTH increased the expression of OAT1 mRNA that spread to all zones, and hypophysectomy strongly decreased it. A less pronounced regulation was detected for OCT2 and oatp3. Specific antibodies confirmed the localization of OAT1 in the outer zona fasciculata, supporting a possible role of OAT1 in cortisol release. The zonated distribution of transporters furthermore suggest that oatp1-3 and OCT2 may be important for the endocrine function of rat adrenocortical cells.     

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.     

Actions of desacetyl-alpha-melanocyte-stimulating hormone on human adrenocortical cells

The responses of human adrenocortical cells to stimulation by ACTH(1-24), desacetyl-alpha-MSH, alpha-MSH and angiotensin II amide have been compared. Both desacetyl-alpha-MSH, thought to be the major form of the peptide in the human pituitary and in circulating plasma, and alpha-MSH caused a significant stimulation of aldosterone, corticosterone and cortisol secretion. Significant stimulation of the production of these steroids was obtained with desacetyl-alpha-MSH at a concentration of 1 nmol/l, while the response to alpha-MSH was considerably less sensitive, with a minimum effective concentration of 0.1 mumol/l. These values compared with minimum effective concentrations of 1 pmol/l for ACTH and 0.1 mumol/l for angiotensin II amide. Although cell types were not separated, it is possible to conclude that none of the peptides showed any specificity for the zona glomerulosa, and in each case the same minimum effective concentration of peptide was required for both aldosterone and cortisol secretion. Yields of steroid obtained under conditions of maximal stimulation by ACTH(1-24), alpha-MSH and desacetyl-alpha-MSH were at least three to five times the basal output of aldosterone, four to eight times that for corticosterone and more than seven to sixteen times that for cortisol. Angiotensin II amide was a relatively poor stimulant with maximal stimulation only 1.5 x basal. In these experiments the minimum effective concentration for desacetyl-alpha-MSH (1 nmol/l) was close to the circulating concentration of desacetyl-alpha-MSH (0.3 nmol/l) in man, and it is thus possible that this peptide may have a physiological role in the control of adrenocortical function.     

Stimulation of steroidogenesis by forskolin in rat adrenal zona glomerulosa cell preparations.

The actions of forskolin have been investigated to determine to what extent its effects on steroidogenesis in rat adrenal preparations are dependent on activation of adenylate cyclase. In zona glomerulosa preparations, stimulation of both aldosterone and corticosterone production was obtained at concentrations of forskolin between 1 and 10 mumol/l. The effects of 10 mumol forskolin/l were additive with those of low doses (1 pmol/l) of corticotrophin (ACTH), but not with those of high doses (1 nmol/l) of ACTH. In contrast, in zona fasciculata/reticularis cells, doses of forskolin up to 10 mumol/l produced no significant stimulation of corticosterone production either alone or in the presence of ACTH (1 pmol/l and 1 nmol/l). The response to 1 nmol ACTH/l was attenuated in the presence of forskolin (10 mumol/l) in both zona glomerulosa and zona fasciculata/reticularis cell preparations. Cyclic AMP production increased progressively with dose up to 100 mumol forskolin/l in zona glomerulosa cells, whereas corticosterone production was maximal between 10 and 30 mumol forskolin/l and decreased at 100 mumol forskolin/l. In zona fasciculata/reticularis cells, cyclic AMP production was also increased by forskolin (1 and 10 mumol/l). The stimulation of zona glomerulosa steroidogenesis by forskolin (1-10 mumol/l) and ACTH (1-100 pmol/l) were both reduced by the adenylate cyclase inhibitor, N6-phenylisopropyladenosine (100 mumol/l). The calcium channel inhibitor, nifedipine, only reduced the steroidogenic response to forskolin (3 mumol/l) at doses of 300 mumol/l whereas the response to 8.4 mmol K+/l was inhibited at 10 mumol nifedipine/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged infusions of Ile5-angiotensin-II in sodium replete and deplete man: effects on aldosterone, ACTH, cortisol, blood pressure, and electrolyte balance.

Angiotensin II (Ile5) was infused for 72 h into 4 sodium replete (3 ng/kg/min) and 8 sodium deplete (3 or 6 ng/kg/min) healthy young men after appropriate control periods, and the effects on aldosterone secretion, plasma cortisol, ACTH, renin activity, plasma and urinary electrolytes, and blood pressure were assessed. Sustained contrived elevation of plasma angiotensin II levels in sodium replete subjects to the range of moderate sodium depletion led to a sustained increase in plasma and urinary aldosterone levels, which further and significantly increased between the 1st and 2nd days of angiotensin II infusion, when gross sodium retention during infusion was prevented. This additional increase may be explained as the expression of a "trophic" effect of angiotension II on the zona glomerulosa. In the sodium deplete state, the absolute increment of aldosterone secretion for a given elevation of angiotensin II levels diring infusion was larger than in sodium replete subjects. This confirms the conclusions from previous short-term angiotensin II infusion experiments that sodium deficiency sensitizes the zona glomerulosa against angiotensin II. The "trophic" effect of angiotensin II on the adrenal gland seems to be one mechanism by which the sensitization is brought about, but insufficient for its full explanation. Since plasma ACTH and cortisol, plasma sodium and potassium concentrations, and potassium blance did not change significantly across sodium depletion or angiotensin II infusion, the mechanism of sensitization awaits its full elucidation. The effect of angiotensin II on blood pressure was blunted by soidum depletion. The opposite shifts in sensitivity against angiotensin II of the zona glomerulosa and of resistance blood vessels with changes in the sodium state seem to be an effective and important means in the regulation of body sodium.     

Dependence of aldosterone stimulation in adrenal glomerulosa cells on calcium uptake: effects of lanthanum nd verapamil

The calcium dependence of steroidogenic responses to angiotensin II, potassium, and ACTH was analyzed in isolated adrenal glomerulosa cells incubated with inhibitors of calcium uptake. Both lanthanum and verapamil reduced the stimulation of aldosterone production by each regulator in a dose-dependent manner, with only a moderate decrease in basal steroid production. The stimulation of aldosterone and cAMP production by ACTH was blocked by both antagonists, and the degree of inhibition was dependent on the concentration of ACTH employed. Increasing concentrations of verapamil caused an increase in th ACTH concentration required for half-maximal stimulation as well as a reduction in the maximum production of aldosterone. Aldosterone production by glomerulosa cells in response to angiotensin II or potassium was also decreased by lanthanum and verapamil, with no change in the concentration of angiotensin II required for half-maximal stimulation of steroidogenesis. Stimulation of pregnenolone synthesis by angiotensin II was also inhibited by verapamil, indicating that calcium is required for the action of angiotensin II at an early step in the steroidogenic pathway. The inhibitory action of verapamil upon angiotensin-stimulated aldosterone production was overcome by increasing concentrations of calcium. Neither of the calcium antagonists affected the binding of angiotensin II to glomerulosa cells, placing the calcium requirement for the action of angiotensin II at a postreceptor locus. These results provide further evidence that angiotensin II and potassium increase aldosterone production in adrenal glomerulosa cells through a calcium-dependent mechanism, and indicate that calcium uptake is an essential requirement for the stimulation of aldosterone production by these two effectors.