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.     

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)     

Studies on the intracellular mechanism of action of alpha-melanocyte-stimulating hormone on rat adrenal zona glomerulosa.

The intracellular mechanisms of action of alpha-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of alpha-MSH there was significant stimulation of aldosterone secretion at 10(-10) mol/l, although cyclic AMP was not increased until high concentrations of alpha-MSH were used (10(-6) mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10(-11) mol/l and levels of cyclic AMP were elevated at 10(-9) mol ACTH/l. When rat adrenal whole capsules were incubated with alpha-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not. When [3H]inositol-loaded glomerulosa cells were stimulated with alpha-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of alpha-MSH which stimulated secretion of aldosterone. Significantly increased levels of [3H]IP3 were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [3H]IP3 production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of alpha-MSH.     

Effects of some hydroxylated sterols on the steroid production in isolated rat adrenal glomerulosa and fasciculata/reticularis cells in the presence of potassium, angiotensin II or ACTH

22S-OH-cholesterol and 25-OH-cholesterol are good aldosterone precursors in isolated rat adrenal glomerulosa cells. In the two concentrations tested (25 and 50 microM) 25-OH-cholesterol stimulated the aldosterone production in a (not-linear) dose-dependent way. An increase in the 22S-OH-cholesterol concentration from 25 microM to 50 microM led to a decrease in the aldosterone production. The exogenous substrate deoxycorticosterone, entering the steroidogenic pathway after the cholesterol side-chain cleavage, is a much better substrate than the sterols mentioned. These results suggest that the cholesterol side-chain cleavage is the rate-limiting step in the aldosterone production from both sterols. We found no effect of the sterols on the potassium-induced aldosterone synthesis. This might be explained by the existence of separate pools of steroid intermediates within the adrenal cell. In vitro a difference in steroid production rates exists between glomerulosa and fasciculata/reticularis cells. This may arise from differences in availability of endogenous steroid precursors like cholesterol. However similar differences can be observed if exogenous substrates like 25-OH-cholesterol or 22S-OH-cholesterol are used. These results therefore suggest that enzymatic activities in the steroidogenic pathway are more important than the cholesterol concentration in regulating steroid production in isolated rat adrenal glomerulosa and fasciculata/reticularis cells.     

Lipoxygenase products as common intermediates in cyclic AMP-dependent and -independent adrenal steroidogenesis in rats

Aldosterone secretion from adrenal glomerulosa cells can be stimulated by angiotensin II (AII), extracellular potassium and ACTH. Mitochondria from these cells respond to intracellular factors generated by AII (cyclic AMP (cAMP)-independent steroidogenesis) and ACTH (cAMP-dependent steroidogenesis), suggesting that the two-signal-transduction mechanisms are linked by a common intermediate. We have evaluated this hypothesis by stimulating mitochondria from the unstimulated zona glomerulosa with a subcellular post-mitochondrial fraction (PMF) obtained from the zona glomerulosa after stimulation with AII or from the fasciculata gland after stimulation with ACTH; the subcellular fractions were also tested on mitochondria from fasciculata cells. PMFs obtained after incubation of adrenal zona glomerulosa with or without AII (0.1 microM) or ACTH (0.1 nM) were able to increase net progesterone synthesis 4.5-fold in mitochondria isolated from unstimulated rat zona glomerulosa. AII-pretreated PMFs from the zona glomerulosa also stimulated steroidogenesis by mitochondria from zona fasciculata cells. Separate experiments showed that inhibitors of arachidonic acid release and metabolism (bromophenacyl bromide, nordihydroguaiaretic acid, caffeic acid or esculetin) blocked corticosterone production in fasciculata cells stimulated with ACTH, suggesting that arachidonic acid could be the common intermediate in the actions of AII and ACTH on steroid synthesis. Evidence to support this concept was obtained from experiments in which the formation of an activated PMF by treatment of zona fasciculata with ACTH was blocked by the presence of the same inhibitors. Moreover, the inhibitory effects of these substances on PMF activation by ACTH were overcome by exogenous arachidonic acid and, in addition, arachidonic acid release was stimulated by ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.     

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.     

Zonal distribution and regulation of adrenal renin in a transgenic model of hypertension in the rat.

The hypertensive transgenic rat [TGR (mRen-2)27] is a genetic model of hypertension in which transfection of the Ren-2 mouse renin gene into rats results in severe hypertension. These transgenic rats express a high level of renin in the adrenal gland, and the hypertension is ameliorated by treatment with angiotensin-converting enzyme inhibitors. In this study we investigated the distribution of adrenal renin in the TGR rat and examined the regulation of adrenal renin in a monolayer culture of adrenal cells. High concentrations of active renin and prorenin were found in the adrenal capsular (glomerulosa) and decapsular (fasciculata-medullary) portions of the TGR adrenal. This is in contrast with the Sprague-Dawley (S-D) rat, in which adrenal renin is found mostly in the active form and located primarily in the glomerulosa cells. The zonal distribution of aldosterone was also different in the TGR, with substantial amounts of aldosterone in the zona fasciculata as well as in the glomerulosa, while in the S-D rat, aldosterone is limited to the zona glomerulosa. In the primary monolayer culture of glomerulosa cells, TGR cells had significantly higher levels of active renin and prorenin and showed an increased response to ACTH and high potassium in the medium. Renin activity in the medium was predominantly in the form of prorenin and significantly higher than that in the S-D rat. Cultured fasciculata cells from TGR also produce renin that is stimulated by ACTH, but not by a high potassium concentration. Renin activity in the adrenal homogenate, medium, and plasma from TGR rats was completely inhibited by the renin inhibitor (CP 71362; 1 microM), but only slightly inhibited (12.3 +/- 3%) by a monoclonal antibody that inhibits renin activity from S-D rat tissues by 79.2 +/- 2.5%, suggesting that renin in the plasma and adrenal glands from TGR appears to derive primarily from mouse renin. In conclusion, the TGR (mRen-2)27 rats have higher than normal levels of adrenal renin, and the cultured cells show an exaggerated renin response to ACTH and potassium. The distribution of the renin enzyme in the adrenal zones of the TGR is similar to the distribution of mouse adrenal renin.     

Molecular mechanism of cytochrome P-450-dependent aldosterone biosynthesis in the adrenal cortex.

In the adrenal cortex, the potent mineralocorticoid, aldosterone, is produced in the zoba glomerulosa but not in the zona fasciculata/reticularis. In rodents and humans, two distinct species of P-450(C18) (aldosterone synthase) and P-450(11beta) (11beta-hydroxylase) are expressed in the adrenal cortex. The selective expression of cytochrome P-450 species in different zones contributes to zone specificity of aldosterone synthesis. In the cow and pig, only one molecular species of P-450(11beta) having both 11beta-hydroxylase and aldosterone synthase activity is expressed throughout the adrenal cortex. P-450(11beta) in the zona fasciculata/reticularis catalyzes the formation of corticosterone but not that of aldosterone from 11-deoxycorticosterone; the same enzyme in the zona glomerulosa produces aldosterone from the same substrate, indicating that a local factor in mitochondria is likely to be involved in the selective suppression of the aldosterone synthetic activity of P-450(11beta) in the zona fasciculata/reticularis. The zone specificity of aldosterone synthesis catalyzed by P-450(11beta) in the bovine adrenal cortex appears to be due to differences in interactions between P-450(11beta) and P-450(SCC) in mitochondria in different cortical zones. Thus, two modes exist for aldosterone biosynthesis in mammals: rodent-human and bovine-porcine modes.     

Effects of prolonged infusion of basic fibroblast growth factor and IGF-I on adrenocortical differentiation in the autotransplanted adrenal: an immunohistochemical study.

Adrenocortical regeneration after adrenal autotransplantation provides a model for the study of local autocrine/paracrine mechanisms involved in the growth and differentiation of the adrenal cortex. To study the possible involvement of some growth factors, namely basic fibroblast growth factor (bFGF, FGF-2) and insulin-like growth factor I (IGF-I), in cell differentiation, immunohistochemical and ultrastructural studies were carried out on adrenal autotransplants in adult male rats. To distinguish between fasciculata and glomerulosa-like cells with accuracy, tissue sections were immunostained with IZAb, which recognizes the inner zone antigen (IZAg) present in fasciculata and reticularis cells but absent from the glomerulosa, and by electron microscopy. IGF-I-treated animals exhibited a clear glomerulosa-like zone that was devoid of IZAb immunostaining. In this outer subcapsular area, ultrastructural examination showed cells containing mitochondria with irregular cristae resembling those of the fetal or immature glomerulosa cells. In contrast, no significant morphological differences were observed in bFGF-treated animals when compared with those from saline-treated controls, in both of which, IZAb immunostaining occurred in almost all adrenocortical cells, with no clear zonation or glomerulosa, as seen in the intact animal. Plasma aldosterone and corticosterone concentrations were lower in autotransplanted control animals than in intact controls, although plasma renin activities were similar. IGF-I treatment significantly increased aldosterone concentrations, whereas corticosterone and plasma renin activity were reduced. bFGF infusion further reduced plasma aldosterone, although plasma renin activity and corticosterone were unaffected. These results suggest that the two growth factors have different effects on zonal differentiation and function in the autotransplanted gland. In particular, bFGF, by reducing glomerulosa function, appears partly to replicate the actions of ACTH in normal animals. In contrast, IGF-I enhances the glomerulosa secreting phenotype and diminishes that of the fasciculata/reticularis, possibly replicating the actions of angiotensin II or a low sodium diet.     

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.     

Differential production of adrenal steroids by purified cells of the human adrenal cortex is relative rather than absolute

OBJECTIVES: The adrenal cortex produces aldosterone, cortisol and androgens in response to ACTH and angiotensin II. To define the differential response of morphologically distinct cells of the adrenal cortex, we examined the phenotypical and functional characteristics of human adrenocortical cells. RESULTS: Tumour growth factor-beta receptor-1 (TGFbeta-R1) and CYP-11 were found to be expressed predominantly in the zona fasciculata, whereas human leukocyte antigen (HLA-DR) and CYP-17 were localised to the zona reticularis. The angiotensin II receptor, AT-1, was found to be predominantly expressed in the zona glomerulosa. Adrenocortical cells, separated by density, yielded two distinct fractions which displayed differential growth patterns. Lipid-rich cells of fraction I expressed TGFbeta-R1 and produced significantly more cortisol relative to androstenedione than unseparated or fraction II cells, whereas lipid-poor cells of fraction II expressed HLA-DR and produced more androstenedione relative to cortisol in the presence of ACTH. Aldosterone production by fraction II was significantly greater than fraction I or unseparated cells. TGFbeta-R1-positive fasciculata-type cells separated into fraction I and HLA-DR-positive cells consistent with reticularis cells separated into fraction II. Aldosterone-producing cells indicative of glomerulosa cells separated into fraction II. CONCLUSIONS: Our findings are consistent with the concept that all adrenocortical cells are capable of producing a range of steroids, but the relative production of cortisol, androgen and aldosterone differs.     

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.     

Transforming growth factor-beta 1 inhibits aldosterone and stimulates adrenal renin in cultured bovine zona glomerulosa cells.

Transforming growth factors-beta (TFG beta s) are multifunctional peptides that affect proliferation, differentiation, and many other functions in a variety of cell types. In this study we examined the effect of TGF beta 1 on aldosterone and adrenal renin production using cultured bovine adrenal zona glomerulosa cells. Collagenase-dispersed zona glomerulosa cells were incubated in PFMR-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h. The cells during this 24-h period were exposed to TGF beta 1, ACTH, and (Bu)2cAMP (dbcAMP). It was observed that TGF beta 1 at 1 nM 1) inhibited basal aldosterone secretion from 680.0 +/- 40.0 to 270.0 +/- 10.0 pg/10(6) cells.h, 2) inhibited ACTH- and dbcAMP-stimulated aldosterone production, 3) increased levels of active renin in the cells from 17.8 +/- 2.5 to 70.7 +/- 4.4 pg angiotensin-I/10(6) cells.h and prorenin from 270.0 +/- 5.0 to 970.0 +/- 90 pg angiotensin-I/10(6) cells.h, 4) stimulated prorenin in the medium synergistically in combination with ACTH and dbcAMP, and 5) had no significant effect on basal cAMP production, but significantly inhibited the ACTH-stimulated production of cAMP. These observations show that TGF beta 1 is a potent inhibitor of basal and ACTH- and cAMP-stimulated aldosterone production and inhibits ACTH-stimulated cAMP production. Contrary to its effect on aldosterone, TGF beta 1 stimulates the synthesis and release of adrenal renin and prorenin. TGF beta 1 may act as an autocrine or paracrine regulator of aldosterone production.     

Responses of rat fetal adrenals to synthetic adrenocorticotrophic hormone and alpha-melanocyte-stimulating hormone in-vivo and in-vitro studies

The response of the adrenals from rat fetuses at 16, 18 and 20 days of gestation to 1-24 ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in vitro. The response to 1-24 ACTH increased as gestation progressed. By the end of fetal life, corticosterone release induced by ACTH from whole adrenals was greater than that observed with adrenal tissue from non-pregnant adult female rats. High doses of alpha-MSH also stimulated adrenal activity but the response to ACTH was always higher than that to alpha-MSH. The effect of 1-24 ACTH and alpha-MSH on fetal adrenal growth was also compared in vivo. The adrenal atrophy induced by fetal hypophysectomy on day 17 of gestation could be prevented by i.m. administration of 10 microgram 1-24 ACTH or alpha-MSH. However, the adrenal growth was greater in ACTH-treated fetuses than in alpha-MSH treated ones. Later in gestation, between days 19 and 20, 1-24 ACTH but not alpha-MSH was able to prevent atrophy induced by fetal hypophysectomy. These findings are discussed in relation to the literature on levels of ACTH and alpha-MSH in the plasma and pituitary glands of the rat throughout the last third of gestation. High levels of ACTH in the fetal circulation contrast sharply with very weak or undetectable concentrations of alpha-MSH. Since the present data suggest that both trophic and steroidogenic activities of ACTH were greater than those of alpha-MSH, it may be concluded that ACTH but not alpha-MSH plays a major physiological role during gestation in the regulation of both fetal adrenal growth and function in the rat.     

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.     

Effects of bromocriptine on pituitary and adrenal cortex in pre-adrenarchal rabbits

The effect of bromocriptine on the morphological picture and steroid content of the adrenal gland, and on certain pro-opiomelanocortin (C) peptides in the pituitary gland was evaluated in preadrenarchal rabbits. Eighteen immature male rabbits (5 weeks of age), were treated for 10 days with saline (n = 10,2 ml sc) or bromocriptine mesylate (n = 8, 3 mg/kg sc) two times/day. After the last administration all animals received dexamethasone (0.25 mg im) and the next morning, 60 min after ACTH injection (0.25 mg im), plasma was drawn and they were sacrificed. Adrenals and pituitaries were immediately removed. For each animal, one adrenal gland was fixed, dehydrated and embedded in paraffin for histology; the other one was stored in saline for determination of androstenedione (A), dehydroepiandrosterone (DHA), 17-OH progesterone (17 P), and cortisol. Steroids were analyzed by RIA after previous extraction and celite-ethyleneglycol chromatography, or directly (cortisol). The immunoreactivities (ir) related to beta-Endorphin (B-EP), ACTH and alpha-MSH were evaluated in pituitary homogenates using specific RIAs. The bromocriptine-treated rabbits showed a significant increase in the percentage of the adrenal zona reticularis (21.5 +/- 3.9% of total cortex vs. 12.7 +/- 1.3% in controls, p less than 0.05, mean +/- SE), and a decrease of the zona fasciculata (57.6 +/- 3.13% vs. 67.7 +/- 2.05% in controls, p less than 0.05). No significant changes were observed in the relative percentage of the zona glomerulosa.(ABSTRACT TRUNCATED AT 250 WORDS)