An examination of possible mechanisms of angiotensin II-stimulated steroidogenesis.

Dog and rat adrenal glomerulosa cells and subcellular fractions have been utilized to evaluate the mechanism of angiotensin II- and angiotensin III-induced aldosterone production. The effects of angiotensin, ACTH, and potassium have been compared on cyclic AMP and cyclic GMP in isolated glomerulosa cells and adenylate cyclase activity in subcellular fractions. The effect of angiotensin II has also been assessed on Na+-K+-activated ATPase of plasma membrane enriched fractions of dog and rat adrenals. We have demonstrated no effect of angiotensin II or angiotensin III on either adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-dependent ATPase activity over a wide range of concentrations. Potassium ion in concentrations that stimulate significant aldosterone production was also without effect. The negative effects of angiotensin and potassium were contrasted against a positive correlation between an ACTH-induced effect on aldosterone production, adenylate cyclase, and cyclic AMP accumulation. These studies have served to demonstrate that neither adenylate cyclase, cyclic AMP, cyclic GMP, or Na+-K+-activated ATPase seem to be directly involved in the mechanism of action of angiotensins on aldosterone production in the rat and dog adrenal glomerulosa.     

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells.

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 x 10(-9) or 17.5 x 10(-9) M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10(-11) or 10(-8) M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10(-4) or 10(-3) M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10(-6) M) of forskolin were decreased 50-70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10(-5) M) of forskolin. The secretory responses to Ang II (10(-8) M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.     

The pharmacologic control of adrenal steroidogenesis

The control of cortisol secretion by ACTH and of aldosterone secretion by angiotensin is exerted upon separate cell populations in the adrenal cortex. Cells of the zona faciculata and the zona glomerulosa, while sharing common steroidogenic pathways, are affected differently by hormones and drugs. Fasciculata cells demonstrate increased cAMP formation and cortisol output primarily in response to ACTH. ACTH receptors, when occupied by hormone, transmit an activating signal to membrane-bound adenylate cyclase by a mechanism that may require the translocation of Ca²⁺. Although the precise way in which increased intracellular cAMP leads to increased steroidogenesis is unknown, protein phosphorylation and new protein synthesis are probably involved. Glomerulosa cells also respond to ACTH, but are uniquely responsive to physiological concentrations of angiotensin II and K⁺. The responsiveness of these cells to angiotensin may be governed by alterations in receptor number. Whether occupied angiotensin receptors activate steroidogenesis via cAMP is uncertain, but alterations in Ca²⁺ distribution within the cell may again be involved. Dopamine probably exerts a tonic inhibitory effect on glomerulosa cell function. Competitive inhibitory analogs for both ACTH and angiotensin II are available, but thus far all inhibitors have retained weak agonist properties. Because the regulatory processes for both cortisol and aldosterone are complex, a wide variety of drugs can affect rates of steroidogenesis $\text{in}$$\text{vivo}$.     

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells.

An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of glucagon and glucagon-like peptide-1 on glucocorticoid secretion of dispersed rat adrenocortical cells.

The effects of glucagon and glucagon-like peptide-1 (GLP-1) on the secretory activity of rat adrenocortical cells have been investigated in vitro. Neither hormones affected basal or agonist-stimulated aldosterone secretion of dispersed rat zona glomerulosa cells or basal corticosterone production of zona fasciculata-reticularis (inner) cells. In contrast, glucagon and GLP-1 partially (40%) inhibited ACTH (10(-9) M)-enhanced corticosterone secretion of inner cells, maximal effective concentration being 10(-7) M. The effect of 10(-7) M glucagon or GPL-1 was suppressed by 10(-6) M Des-His1-[Glu9]-glucagon amide (glucagon-A) and exendin-4(3-39) (GPL-1-A), which are selective antagonists of glucagon and GLP-1 receptors, respectively. Glucagon and GLP-1 (10(-7) M) decreased by about 45-50% cyclic-AMP production by dispersed inner adrenocortical cells in response to ACTH (10(-9) M), but not to the adenylate cyclase activator forskolin (10(-5) M). Again this effect was blocked by 10(-6) M glucagon-A or GLP-1-A. The exposure of dispersed inner cells to 10(-7) M glucagon plus GLP-1 completely suppressed corticosterone response to ACTH (10(-9) M). However, they only partially inhibited (by about 65-70%) both corticosterone response to forskolin (10(-5) M) or dibutyryl-cyclic-AMP (10(-5) M) and ACTH (10(-9) M)-enhanced cyclic-AMP production. Quantitative HPLC showed that 10(-7) M glucagon or GLP-1 did not affect ACTH-stimulated pregnenolone production, evoked a slight rise in progesterone and 11-deoxycorticosterone release, and markedly reduced (by about 55%) corticosterone secretion of dispersed inner adrenocortical cells. In light of these findings the following conclusion are drawn: (i) glucagon and GLP-1, via the activation of specific receptors, inhibit glucocorticoid response of rat adrenal cortex to ACTH; and (ii) the mechanism underlying the effect of glucagon and GLP-1 is probably two-fold, and involves both the inhibition of the ACTH-induced activation of adenylate cyclase and the impairment of the late steps of glucocorticoid synthesis.     

Inhibition by vanadyl of adenylate cyclase activation reactions in rat adrenal membranes

In rat adrenal dispersed cells, both vanadyl and vanadate inhibited ACTH-stimulated steroidogenesis and the formation of cAMP, whereas these compounds did not inhibit the cAMP-dependent steroidogenesis. Then, the membrane fraction was prepared and activated by various secretagogues including ACTH, Gpp(NH)p, GTP gamma S, and forskolin. The cyclase activity was inhibited by vanadyl but not by vanadate in the presence of these stimulators. Based on these results, we conclude that cationic vanadyl acts against a metal-requiring step in the adenylate cyclase system containing G-protein and the catalytic subunit. In addition, we believe that vanadyl is a useful tool to investigate adenylate cyclase systems.     

ACTH-induced refractoriness in cultured adrenal cell line (Y1).

Treatment of cultured mouse adrenal cells Y₁ with ACTH induced cell refractoriness to further hormonal stimulation. When ACTH was added to the cells every 2 hours the first addition increased the levels of 20αOH-progesterone and cAMP secreted into the medium. Upon the second and third additions of ACTH the levels of 20αOH-progesterone and cAMP secreted were greatly diminished and upon the fourth addition of ACTH were absent. Prolonged incubation (14 hours) with different concentrations of ACTH (5 × 10^?11 M to 10^?6 M) induced a dose-related steroidogenic refractoriness to further ACTH stimulation, 10^?8 M ACTH inducing complete refractoriness. The number of ACTH binding sites of cell particles prepared from desensitized cells was similar to that of the control but ACTH failed to stimulate the adenylate cyclase of desensitized cells, whereas the enzyme responded fully to NaF and Gpp(NH)p. The cAMP phosphodiesterase activity was similar in both desensitized and control cells. In addition the steroidogenic response to dibutyryl cAMP of desensitized cells was abolished. Thus, ACTH-induced adrenal cell desensitization seems to be related to at least two phenomena : a defect in the “coupling” between the hormone-receptor sites and the adenylate cyclase and an alteration of certain steps beyond cAMP formation.     

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

Regulation of aldosterone production from zona glomerulosa cells by ANG II and cAMP: evidence for PKA-independent activation of CaMK by cAMP

Aldosterone production in zona glomerulosa (ZG) cells of adrenal glands is regulated by various extracellular stimuli (K(+), ANG II, ACTH) that all converge on two major intracellular signaling pathways: an increase in cAMP production and calcium (Ca(2+)) mobilization. However, molecular events downstream of the increase in intracellular cAMP and Ca(2+) content are controversial and far from being completely resolved. Here, we found that Ca(2+)/calmodulin-dependent protein kinases (CaMKs) play a predominant role in the regulation of aldosterone production stimulated by ANG II, ACTH, and cAMP. The specific CaMK inhibitor KN93 strongly reduced ANG II-, ACTH-, and cAMP-stimulated aldosterone production. In in vitro kinase assays and intact cells, we could show that cAMP-induced activation of CaMK, using the adenylate cyclase activator forskolin or the cAMP-analog Sp-5,6-DCI-cBIMPS (cBIMPS), was not mediated by PKA. Activation of the recently identified cAMP target protein Epac (exchange protein directly activated by cAMP) by 8-pCPT-2'-O-Me-cAMP had no effect on CaMK activity and aldosterone production. Furthermore, we provide evidence that cAMP effects in ZG cells do not involve Ca(2+) or MAPK signaling. Our results suggest that ZG cells, in addition to PKA and Epac/Rap proteins, contain other as yet unidentified cAMP mediator(s) involved in regulating CaMK activity and aldosterone secretion.     

Trophic and steroidogenic effects of water deprivation on the adrenal gland of the adult female rat

The effects of a 3-day water deprivation were studied in adult female rats in order to know what are the different zones of the adrenal gland and the hormonal factors involved in the growth and the activity of the adrenal gland. Water deprivation significantly increased plasma renin activity (PRA), plasma Angiotensin II (AII), vasopressin (AVP), epinephrine, aldosterone and corticosterone concentrations but did not modify the plasma adrenocorticotropin hormone (ACTH) level. Water deprivation significantly increased the absolute weight of the adrenal capsule containing the zona glomerulosa without modification of the density of cells per area unit suggesting that the growth of the adrenal capsule was due to a cell hyperplasia of the zona glomerulosa. Water deprivation significantly increased the density of AII type 1 (AT₁) receptors in the adrenal capsule but did not modify the density of AII type 2 (AT₂) receptors in the adrenal capsule and core containing the zona fasciculata, the zona reticularis and the medulla. The treatment of dehydrated female rats with captopril, which inhibits the angiotensin converting enzyme (ACE) in order to block the production of AII, significantly decreased the absolute weight of the adrenal capsule, plasma aldosterone and the density of AT₁ receptors in the adrenal capsule. The concentration of corticosterone in the plasma, the density of AT₂ receptors and the density of cells per unit area in the zona glomerulosa of the adrenal capsule were not affected by captopril-treatment. In conclusion, these results suggest that AII seems to be the main factor involved in the stimulation of the growth and the secretion of aldosterone by the adrenal capsule containing the zona glomerulosa during water deprivation. The low level of plasma ACTH is not involved in the growth of the adrenal gland but is probably responsible for the secretion of corticosterone by the zona fasciculata.     

Angiotensin stimulates adenylate cyclase activity via prostaglandins in the adrenal glomerulosa membrane

The effects of angiotensin II (A II) on adenylate cyclase activities in membranes of the zona glomerulosa (the capsular fraction) and the zona fasciculata (the decapsulated fraction) from rat adrenocortical glands were investigated. A time- and GTP-dependent stimulation by A II of adenylate cyclase activity was observed in the capsular fraction but not in the decapsulated fraction. The activation of adenylate cyclase by ACTH and A II was additive. Stimulation by A II was inhibited by an angiotensin antagonist, DD-3487 (DD). Moreover, the addition of a prostaglandin antagonist, a mixture of polyesters of polyphloretin phosphate (PPP) and an inhibitor of prostaglandin synthesis, indomethacin, was effective in inhibiting A II-induced stimulation of the capsular adenylate cyclase activity, suggesting that the activation of A II receptors located on the capsular membrane leads to the release of prostaglandins, which in turn stimulates the adenylate cyclase.     

Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands.

nurr77 and nurr-1 are growth factor-inducible members of the steroid/thyroid hormone receptor gene superfamily. In order to gain insight into the potential roles of nur77 in the living organism, we used pharmacologic treatments to examine the expression of nur77 in the mouse adrenal gland. We found that nur77 and nurr-1 are induced in the adrenal gland upon treatment with pentylene tetrazole (Ptz; Metrazole). This induction is separable into distinct endocrine and neurogenic mechanisms. In situ hybridization analysis demonstrates that nur77 expression upon Ptz treatment in the adrenal cortex is localized primarily to the inner cortical region, the zona fasciculata-reticularis, with minimal induction in the zona glomerulosa. This induction is inhibitable by pretreatment with dexamethasone, indicating involvement of the hypothalamic-pituitary-adrenal axis in the activation of adrenal cortical expression. When mice were injected with adrenocorticotrophic hormone (ACTH), nur77 expression in the adrenal gland spanned all cortical layers including the zona glomerulosa, but medullary expression was not induced. Ptz also induces expression of both nur77 and nurr-1 in the adrenal medulla. Medullary induction is likely to have a neurogenic origin, as nur77 expression was not inhibitable by dexamethasone pretreatment and induction was seen after treatment with the cholinergic neurotransmitter nicotine. nur77 is also inducible by ACTH, forskolin, and the second messenger analog dibutyryl cyclic AMP in the ACTH-responsive adrenal cortical cell line Y-1. Significantly, Nur77 isolated from ACTH-stimulated Y-1 cells bound to its response element whereas Nur77 present in unstimulated cells did not. Moreover, Nur77 in ACTH-treated Y-1 cells was hypophosphorylated at serine 354 compared with that in untreated cells. These results, taken together with the previous observation that dephosphorylation of serine 354 affects DNA binding affinity in vitro, show for the first time that phosphorylation of Nur77 at serine 354 is under hormonal regulation, modulating its DNA binding affinity. Thus, ACTH regulates Nur77 in two ways: activation of its gene and posttranslational modification. A promoter analysis of nur77 induction in Y-1 cells indicates that the regulatory elements mediating ACTH induction differ from those required for induction in the adrenal medullary tumor cell line PC12 and in 3T3 fibroblasts.     

Aldosterone and corticosterone stimulation by ACTH in isolated rat adrenal glomerulosa cells: interaction with vasopressin

An interaction between ACTH and vasopressin on steroidogenesis was observed in isolated rat adrenal zona glomerulosa cell preparations. 1. The presence of 10(-11) M vasopressin further increased by 52% the output of aldosterone produced by 10(-12) M ACTH on those cells. 2. At a pharmacological concentration of ACTH (10(-7) M), the aldosterone output was increased 5 fold while the addition of 10(-12) M or 10(-8) M vasopressin decreased it by 17% and 48% respectively. 3. Vasopressin also produced a dose-dependent inhibition of the stimulatory effect of ACTH on the output of corticosterone. 4. We have thus shown for the first time, that vasopressin acts directly on adrenal zona glomerulosa cell preparations to modify the aldosterone output by modulating the action of ACTH. It is postulated that, in addition to other known aldosterone regulating factors, ACTH and vasopressin might synergistically act to regulate the secretion of aldosterone in vivo.     

Proopiomelanocortin-derived peptides, phosphoinositides, cAMP, and aldosterone secretion

Since the intracellular messengers of various proopiomelanocortin-derived peptides remain ambiguous at best, we have investigated the possible involvement of phosphoinositide metabolism in aldosterone secretion evoked by alpha-MSH, beta-LPH, as well as ACTH in rat and calf adrenal glomerulosa cells. We have also examined the cAMP responses in the adrenal glomerulosa cells to alpha-MSH comparing it with those of ACTH. Our results showed that neither alpha-MSH, beta-LPH, nor ACTH increased inositol triphosphate (IP3) or other inositol phosphates in adrenal glomerulosa cells while increasing aldosterone secretion from the same cells. Angiotensin II, known to cause hydrolysis of the phosphoinositides, increased IP3 in these adrenal cells in a dose-dependent manner. Both ACTH and alpha-MSH raised the cAMP levels in the calf adrenal glomerulosa cells, although the magnitude of the increase of cAMP in response to ACTH was greater. These findings suggest that IP3 as a mediator of alpha-MSH- and beta-LPH-induced aldosterone secretion is not likely and other mediator(s) may be involved.     

Leukotrienes as common intermediates in the cyclic AMP dependent and independent pathways in adrenal steroidogenesis

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and adrenocorticotropin (ACTH). Since the mitochondria can recognize factors generated by AII (cyclic-AMP-independent) and ACTH (cyclic AMP dependent), it is reasonable to postulate the existence of a common intermediate in spite of a different signal transduction mechanism. We have evaluated this hypothesis by stimulation of mitochondria from glomerulosa gland with fractions isolated from glomerulosa gland stimulated with AII or from fasciculata gland stimulated with ACTH; the same fractions were tested using mitochondria from fasciculata cells. Postmitochondrial fractions (PMTS) obtained after incubation of adrenal zona glomerulosa with or without AII (10(-7) M) or ACTH (10(-10) M), were able to increase net progesterone synthesis 5-fold in mitochondria isolated from non-stimulated rat zona glomerulosa. In addition, AII in zona glomerulosa produced in vitro steroidogenic fractions that were able to stimulate mitochondria from zona fasciculata cells. Inhibitors of arachidonic acid release and metabolism blocked corticosterone production in fasciculata cells stimulated with ACTH. This concept is supported by the experiment in which bromophenacylbromide and nordihydroguaiaretic acid also blocked the formation of an activated PMTS. In fact, non-activated PMTS, in the presence of exogenous arachidonic acid AA, behaved as an activated PMTS from ACTH stimulated cells. We suggest that the mechanisms of action of ACTH and AII involve an increase in the release of AA and an activation of the enzyme system which converts AA in leukotriene products.     

Calcium-dependent Desensitization of Adenylate Cyclase in Rat Cerebral Cortical Slices

Incubation of slices of rat cerebral cortical grey matter in Krebs-Ringer bicarbonate-glucose buffer induced a rapid decline in the responsiveness of the adenylate cyclase in subsequently prepared membrane preparations to stimulation by various activators of the enzyme. The loss of responsiveness was time- and temperature-dependent, showed an absolute dependence on extracellular calcium ions, and was mimicked by the presence of serine proteases in the incubation medium. The resultant adenylate cyclase preparation was partially responsive to activation by fluoride and guanylylimidodiphosphate but had become virtually unresponsive to activation by ganglioside, trypsin, or beta-adrenergic agonists. The loss of responsiveness of adenylate cyclase was not altered if slices were incubated with depolarizing agents, putative neurotransmitters, receptor blockers, serine protease inhibitors, or adenosine deaminase. The nature of the calcium-dependent mechanism involved in the loss responsiveness of membranal adenylate cyclase is unknown. A suggested mechanism for the loss of sensitivity is the action of a membrane-bound, calcium-dependent protease.     

Vasopressin: a potent growth factor in adrenal glomerulosa cells in culture

Previous studies have shown that vasopressin stimulates the mitotic activity in adrenal zona glomerulosa cells in intact as well as in hypophysectomized rats. (Payet and Isler, Cell and Tissue Res. 172, 1976; Payet and Lehoux, J. steroid Biochem. 12, 1980). We now report that this effect is direct and specific, since vasopressin stimulates the mitotic activity of rat adrenal zona glomerulosa cells in primary cultures. These cells were prepared by dissociation with collagenase in the culture medium MEM-d-Valine. Isolated cells were placed in 3.5 diameter petri dishes in MEM-d-valine medium containing 15% fetal calf serum and antibiotics for two days and 5% fetal calf serum for subsequent cultures. The medium was changed at 24 hr intervals. The hormones were added 3 days after the culture was started. The mitogenic effect of vasopressin was found to be dependent both on time and hormone concentrations. Vasopressin (10(-11) M) stimulated thymidine incorporation 4.8 +/- 0.6-fold after 2 days of treatment and 5.3 +/- 1.6-fold after 8 days. When ACTH (10(-11) M) was added together with vasopressin (10(-11) M) the mitogenic effect was enhanced at 6.5 +/- 1.9-fold after 2 days and 12.9 +/- 6.9-fold after 8 days of treatment. The aldosterone and corticosterone outputs were also stimulated by the combined presence of vasopressin and ACTH in the incubation medium; a maximal effect was observed between 6 and 8 days of treatment. Vasopressin (10(-11) M) + ACTH (10(-11) M) stimulated the aldosterone output 7-fold and that of corticosterone by 18-fold. When added alone, vasopressin, as well as ACTH alone had only a small effect on the aldosterone output. However, ACTH alone stimulated the corticosterone output 10-fold. In conclusion, vasopressin is an important and specific growth factor of the adrenal zona glomerulosa cells. In addition, together with ACTH vasopressin stimulates the aldosterone and corticosterone output both in vivo and in vitro in primary cell cultures.     

Interaction of ACTH, beta-endorphin and alpha-melanocyte stimulating hormone in relation to the corticosteroid production of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells.

The combined effects of ACTH, beta-endorphin (beta-EP) and alpha-MSH were studied on the corticosteroidogenesis of isolated rat adrenocortical zona fasciculata and zona glomerulosa cells. beta-EP potentiated the effects of ACTH and alpha-MSH on the zona fasciculata corticosterone production but inhibited those on the zona glomerulosa aldosterone production. beta-EP did not affect the combined action of 4 x 10(-11) M ACTH and 5 x 10(-9) M alpha-MSH on the zona fasciculata or the zona glomerulosa cells, but it inhibited the stimulatory action of the combination of 1.6 x 10(-10) M ACTH and 10(-9) M alpha-MSH on the zona glomerulosa aldosterone production. An interaction of ACTH, beta-EP and alpha-MSH in relation to the zona fasciculata and zona glomerulosa corticosteroid production was found.