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)     

Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism

We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of [3H]glycerol and [14C]palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.     

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)     

Expression and regulation of adenylyl cyclase isoforms in the human adrenal gland

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the betagamma-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.     

The role of endothelin in the control of adrenocortical function: stimulation of endothelin release by ACTH and the effects of endothelin-1 and endothelin-3 on steroidogenesis in rat and human adrenocortical cells.

The rate of blood flow through the intact adrenal gland is closely linked to steroid hormone secretion, and although the mechanism involved is unknown, it is thought to involve secretory products of the vascular endothelium. In dispersed cell preparations, endothelin-1 and -3 both caused a dose-dependent and highly sensitive increase in steroid secretion by zona glomerulosa and zona fasciculata cells of the rat and human adrenal cortex. In addition, when the perfused rat adrenal was stimulated with ACTH, significant increases in steroid secretion and perfusion medium flow rate were accompanied by significantly increased secretion of immunoreactive endothelin into the adrenal vein. It is proposed that endothelin has a role in mediating the adrenocortical response to ACTH stimulation.     

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.     

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.     

Excitation-secretion coupling: involvement of potassium channels in ACTH-stimulated rat adrenocortical cells

The mechanism by which ACTH stimulates calcium influx and steroid secretion was studied using rat adrenal glomerulosa cells, which were either freshly isolated or maintained in primary culture for 3 days. The potassium channel blocker tetraethylammonium chloride (TEA) stimulated twofold both corticosterone and aldosterone secretion; this stimulation was lower than that induced by ACTH at low concentrations (10 pmol/l). However, TEA and ACTH induced similar increases in Ca2+ influx and inositol phosphate accumulation. The three responses (steroid secretion, calcium influx and inositol phosphate accumulation) induced by TEA or low concentrations of ACTH were blocked by CoCl2. The greater stimulatory effect on steroid secretion of 10 nmol ACTH/l was decreased but not blocked by CoCl2. These data further document the complex mechanism of action of ACTH. It is postulated that, at low concentrations, ACTH binds preferentially to the high-affinity site of its receptor, leading to calcium influx by depolarization of the membrane potential, and to steroid secretion predominantly through an inositol phosphate- and Ca2+-stimulated pathway and also a cyclic AMP pathway. At higher concentrations, the hormone also binds to the low-affinity site of its receptor, largely stimulating cyclic AMP production and further increasing steroid secretion.     

Divergent differentiation of rat adrenocortical cells is associated with an interruption of angiotensin II-mediated signal transduction.

The zones of the adrenal cortex contain distinct populations of cells which share a common developmental origin and steroidogenic template. In the rat, zona glomerulosa cells respond to angiotensin II (Ang II) with increased steroidogenesis while zona fasciculata/reticularis cells do not. We have examined Ang II-mediated signal transduction in homogeneous cellular sub-populations derived from either the zona glomerulosa (GLOM) or the zona fasciculata (FASC). In both of these sub-populations Ang II treatment significantly increased the levels of 3H-labelled inositol phosphates as well as the total mass of inositol 1,4,5-triphosphate. In contrast, the two cell types exhibited very different Ang II-mediated changes in free intracellular calcium ([Ca2+]i). Ang II (10 nM), induced [Ca2+]i increases of > 50 nM in 90% of individual GLOM cells (53/58), but in only 28% of FASC cells (11/39). These [Ca2+]i responses occurred after a transient Ang II stimulation ( < 1 min), in the presence of verapamil and in the absence of extracellular calcium, indicating an intracellular release. In small groups of 10-30 cells, stimulation with 1, 10 and 100 nM Ang II induced [Ca2+]i increases of 78, 178 and 215 nM respectively in GLOM cultures compared to only 35, 64, and 65 nM in FASC cultures. Thapsigargin treatment, which releases intracellular calcium in an inositol phosphate independent manner, elicited [Ca2+]i increases in both populations. Importantly, a calcium ionophore induced elevation of [Ca2+]i increased steroidogenesis in both cell types. These results suggest that an interruption of the signaling cascade at the level of intracellular calcium release contributes to the lack of a steroidogenic response to Ang II by the FASC cells. Therefore, in the rat adrenal cortex, divergent differentiation of related cell types may involve alterations within signal transduction pathways distal to initial receptor-mediated events (i.e. inositol phosphate production) and proximal to downstream effector events (i.e. steroidogenesis).     

Hypophysectomy results in a loss of connexin gap junction protein from the adrenal cortex

To test the hypothesis that gap junctions are dependent on the tropic state of the adrenal gland, the effect of hypophysectomy on connexin 43 (alpha1-Cx43) gap junction protein occurrence and distribution was examined in mice. Gap junction protein occurrence was assessed with immunohistochemical techniques. In the adrenal gland from intact animals, alpha1-Cx43 gap junction protein was detected in the zonae fasciculata(ZF) and reticularis (ZR) while only a few alpha1-Cx43 gap junction plaques were found connecting zona glomerulosa(ZG) cells. Hypophysectomy led to a profound atrophy of the cortex which was more marked in the inner zones (zonae fasciculata and reticularis) than in the zona glomerulosa. There was a time dependent loss of alpha1-Cx43 gap junction protein in the adrenal cortex after hypophysectomy. At 33 day following hypophysectomy there was a two fold decrease in gap junctions in the zona fasciculata while the average gap junction plaque size was not different than the size seen in control animal adrenal glands.. ACTH (1U/gm body weight) treatment in hypophysectomized animals increased the number of gap junction plaques in the zona fasciculata. Hypophysectomy led to diminished alpha1-Cx43 gap junction expression in the zona fasciculata which could be restored by ACTH treatment. Because altering the tropic state of the adrenal glands via hypophysectomy leads to a reduction in gap junction number, it can be suggested that control of gap junction expression in the adrenal gland is hormone dependent and linked to adrenal gland function.     

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.     

Studies on the dynamics of adrenocortical responses to ACTH in the rat

The transient dynamics of plasma ACTH, adrenal cyclic AMP, adrenal corticosterone and plasma corticosterone were evaluated in male Sprague-Dawley rats, whose endogenous release of ACTH had been blocked by dexamethasone: (1) 40 min after single injections of ACTH ranging from 2 to 300 ng ACTH/100 g B.W., i.V.; (2) at time intervals after single injections of 9, 37 and 300 ng ACTH/100 g B.W.; (3) during and after prolonged infusion of 4 ng ACTH/min/100 g B.W. Plasma corticosterone concentration was still at a nearly maximal level 40 min after the injection of ACTH at a dose level for which the adrenal cyclic AMP content had fallen back to a value that was scarcely above the control one; a narrow window, defined by a 2-fold increase in the dose of ACTH, represents the transition between a minimal and a maximal adrenal cyclic AMP content. The adrenal cyclic AMP transient response after injection of graded doses of ACTH increased rapidly to a peak whose amplitude was dose-dependent; the duration of the cyclic nucleotide response, however, appeared to be independent of the ACTH dose level. The adrenal corticosterone content rose rapidly, and the eventual fall was delayed by increasing doses of ACTH. The time course of the early plasma corticosterone concentrations exhibited a similar rate of increase after any dose of ACTH; in any case, a steady state whose duration was dose-dependent was eventually reached and the ensuing fall therefrom occurred at a time when the adrenal cyclic AMP had fallen to very low levels. The adrenal cyclic AMP content showed an overshoot at a time when ACTH and corticosterone had reached a constant steady state, during a prolonged infusion of ACTH; adrenal cyclic AMP stabilized during the later phase of the infusion. After removal of the infusion, plasma ACTH levels fell relatively slowly as compared with adrenal cyclic AMP, whereas corticosterone remained at a maximal level for at least 120 min.Our results, derived from experiments in vivo, support the recent proposal by Bristow et al. (1980), derived from studies in vitro, that ACTH can act via either of two types of receptor: binding to one receptor elicits steroidogenesis via cyclic AMP production whereas binding to the other receptor elicits steroidogenesis through some other mechanism.     

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.     

Differential suppression of the outer and inner zones of the adrenal cortex of the guinea pig

To examine the regulation of the zona fasciculata and the zona reticularis of the guinea pig adrenal cortex, animals were placed on a chronic regimen of dexamethasone. Changes in adrenal zonal weight, cholesterol side-chain cleavage activity, and tissue and serum steroid concentrations were measured after 1 month of dexamethasone administration. With dexamethasone treatment, the weight of the outer zone (glomerulosa/fasciculata) decreased significantly, while the weight of the inner zone (reticularis) did not change. Cholesterol side-chain cleavage activity in mitochondria isolated from the outer zone also declined significantly, while a similar activity in the inner zone did not change. The serum cortisol concentration in response to dexamethasone administration decreased by 85%, as did the concentrations of cortisol and progesterone in the outer zone; the concentration of cortisol in the inner zone decreased by only one third, while the concentration of progesterone did not change. These results in association with previous reports indicate that the zona reticularis of the guinea pig adrenal cortex (in contrast to the zona fasciculata) is not regulated by ACTH in terms of either steroidogenesis or maintenance of cell growth.     

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.     

Cytological maturity of zona fasciculata cells in the fetal sheep adrenal following ACTH infusion: an electron microscope study.

Electron microscopy was used to assess the cytological maturity of the zona fasciculata cells in the adrenal cortex of fetal sheep at 105 days of gestation, following several ACTH infusion regimes. The aim of this study was to correlate the morphological appearance of the fetal adrenal zona fasciculata cells with the expression of the steroid hydroxylase genes and the fetal plasma cortisol concentrations in a parallel study. Immediately following infusion of ACTH for 24 or 72 h, the zona fasciculata cells at the cortico-medullary junction were more mature than those in the saline-infused controls. When ACTH infusions were withdrawn for 24-72 h prior to the termination of the experiment, the deep cortical cells appeared less mature than those in fetuses which had received ACTH right up until the time of tissue collection. Following ACTH administration, mitochondrial changes preceded changes in the smooth endoplasmic reticulum, and when ACTH was withdrawn, the smooth endoplasmic reticulum responded before the mitochondria. The study demonstrated a correlation between the cytological maturity of the deep zona fasciculata cells and the expression of the genes for the steroidogenic enzymes P-450(17)alpha and P-450scc in the 105-day fetal sheep adrenal following ACTH infusion.     

Hormone-sensitive lipase deficiency in mice causes lipid storage in the adrenal cortex and impaired corticosterone response to corticotropin stimulation

Hormone-sensitive lipase (HSL, E.C.3.1.1.3, gene designation Lipe) is reportedly the major cholesteryl esterase of adrenal cortex. Because of the potential importance of cholesteryl ester hydrolysis in steroidogenesis, gene-targeted HSL-deficient mice were assessed for adrenal cortical morphology and function. Compared with control animals, HSL deficiency results in a marked accumulation of lipid droplets both in zona glomerulosa and zona fasciculata. In the zona fasciculata, lipid accumulation was observed progressively from the outer to the inner regions, culminating near the corticomedullary junction with the formation of syncytial-lipoid structures having the appearance of degenerative cells. These morphological changes did not significantly alter the basal levels of circulating corticosterone, but following ACTH stimulation, corticosterone levels were decreased (P < 0.001). The observation of normal basal corticosterone and aldosterone levels demonstrates that some free cholesterol for steroid synthesis can be produced independently of HSL. Taken together, these results indicate that HSL-deficient mice accumulate lipid droplets in such a way as to impair acute ACTH stimulation of corticosterone secretion. Such observations are also found in some forms of congenital adrenal hyperplasia. By extension, HSL deficiency may be a cause of hereditary adrenocortical hypofunction in humans.     

Potassium fluxes in bovine adrenal cells during adrenocorticotropin stimulation

Active (ouabain-sensitive) and passive (ouabain-insensitive) fluxes of 43K were measured in isolated bovine zona fasciculata/reticularis cells. ACTH inhibited passive influx in a dose-dependent manner and, at a concentration that maximally stimulated steroidogenesis (10(-8) M), also inhibited passive efflux. ACTH did not affect active potassium transport. Angiotensin II (10(-6) M) and cAMP (10(-2) M) inhibited passive influx to the same extent as did 10(-8) M ACTH. Angiotensin II (10(-6) M) also reduced active potassium uptake, although this did not appear to be related to changes in cortisol biosynthesis. Increases in cortisol synthesis by ouabain-treated cells in response to angiotensin II and ACTH were proportional to decreases in potassium influx. The addition of cortisol (1 microgram/ml) to cells slightly reduced passive potassium uptake, but not to the same extent as did 10(-8) M ACTH. Frusemide, an inhibitor of passive sodium/potassium cotransport, did not wholly abolish the effects of ACTH on potassium influx. These changes in flux are probably related to electrophysiological changes in membrane potential. Their significance in the regulation of ACTH actions has yet to be determined.