Lanthanum: inhibition of ACTH-stimulated cyclic AMP and corticosterone synthesis in isolated rat adrenocortical cells

Lanthanum (La+++) is a well-known Ca++ antagonist in a number of biological systems. It was used in the present study to examine the role of Ca++ in the regulation of adenyl cyclase of the adrenal cortex by ACTH. In micromolar concentrations, .La+++ inhibited both cyclic AMP and corticosterone response of isolated adrenal cortex cells to ACTH. However, a number of intracellular processes were not affected by La+++. These include the stimulation of steroidogenesis by dibutyryl cyclic AMP, conversion of several steroid precursors into corticosterone, and stimulation of the latter by glucose. Thus, inhibition of steroidogenesis by La+++ appears to be solely due to an inhibition of ACTH-stimulated cyclic AMP formation. Electron microscope examination showed that La+++ was localized on plasma membrane of the cells and did not appear to penetrate beyond this region. Since La+++ is believed to replace Ca++ at superficial binding sites on the cell membrane, it is proposed that Ca++ at these sites plays an important role in the regulation of adenyl cyclase by ACTH. Similarities in the role of Ca++ in "excitation-contraction" coupling and in the ACTH-adenyl cyclase system raise the possibility that a contractile protein may be involved in the regulation of adenyl cyclase by those hormones which are known to require Ca++ in the process.     

Direct effects of IL-15 on corticosterone secretion by rat adrenocortical cells

Cytokines might regulate the function of the hypothalamic-pituitary-adrenal axis. IL-15 is a potent non-T-cell-derived cytokine with IL-2-like activities. It has been shown that IL-15 can reverse the inhibition of glucocorticoids on PBMC. In vitro experiments were designed to assess the direct effect of IL-15 on corticosterone (CORT) secretion in the adrenal zona fasciculata-reticularis (ZFR) cells of male rats. Administration of IL-15 dose dependently decreased the basal and adrenocorticotropin-stimulated release of CORT and production of cAMP in ZFR cells. The stimulatory effect of forskolin (an adenylate cyclase activator) on CORT secretion and accumulation of cAMP in ZFR cells was attenuated by the administration of IL-15. However, 8-Br-cAMP (a cAMP analogue)-stimulated release of CORT was not affected by IL-15. Exogenous administration of IL-15 (10(-7) mol/L) significantly attenuated the pregnenolone (the substrate of 3beta-hydroxysteroid dehydrogenase)- or deoxycorticosterone (the substrate of 11beta-hydroxylase)-induced release of CORT. The results indicate that decrease of CORT secretion by IL-15 is in part because of (i) the decrease of adenylate cyclase activity and cAMP production and (ii) the inhibition of 3beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase activities in rat ZFR cells.     

Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells

Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others.     

Steroidogenic refractoriness of bovine adrenocortical cells to dibutyryl cyclic AMP

The long-term effects of dibutyryl cyclic AMP [(Bu)₂-cAMP] on steroidogenesis in bovine adrenocortical cells maintained in primary culture were investigated. During the first 36 h, total steroid production by cells incubated with (Bu)₂-cAMP increased progressively. Thereafter, however, there was a marked fall in steroid output. During the first 36 h adrenocortical cells incubated in the presence of (Bu)₂-cAMP produced substantially more C₁₉-steroids and 17α-hydroxylated C₂₁ -steroids than did cells incubated in the absence of (Bu)₂-cAMP. By 48 h, however, such steroid secretion by cells incubated in the continued presence of (Bu)₂-cAMP declined toward control levels. By contrast, the secretion of corticosterone and 11-deoxycorti-costerone was consistently less by cells maintained in the presence of (Bu)₂-cAMP than by cells maintained in its absence. These results suggest that refractoriness results, at least in part, from events which occur distal to the formation of cAMP. The action of ACTH and (Bu)₂cAMP to promote the secretion of 17α-hydroxylated C₂₁-steroids and C₁₉-steroids, on the other hand, appears to reflect an increase in the rate of cholesterol side-chain cleavage, as well as an increase in 17α-hydroxylase and possibly also 17,20-lyase activities.     

Effects of ethanol on gastric acid secretion and gastric mucosal cyclic AMP in the rat.

The effect of ethanol on the secretion of gastric acid and the content of cyclic AMP of the gastric mucosa was studied in rats. Intravenously, ethanol (10 to 800 mg/kg) had no effect on the output of acid. Upon local application into the stomach, ethanol (1 to 10%) caused a concentration-dependent inhibition of the output of gastric acid. The effect was evident within 5 min. At the concentration of 1 %,ethanol decreased the rate of acid secretion maximally by about 30%. At the concentration of 3 %, the maximal inhibition was about 70 %. At the concentration of 10 %, ethanol caused a total cessation of the output of acid within 20 to 60 min.Five and 25 min after the administration of 10 % ethanol into the stomach, the gastric mucosal content of cyclic AMP was decreased by approximately 50 %. Also in vitro, the mucosal content of cyclic AMP was decreased by ethanol within 5 min. The decrease was about 30 % with 2.5 % ethanol, approximately 60 % with 10 % ethanol, and approximately 45 % with 20 % ethanol. Alcohol inhibited the activity of the cyclic AMP phosphodiesterase of the gastric mucosa in a competitive manner. The K_i-value was 0.16 M which would correspond to an alcohol concentration of 9.1 % (v/v). Ethanol caused a concentration-dependent inhibition of the activity of the gastric mucosal adenyl cyclase. By 0.166 M (9.4 %) alcohol the inhibition was nearly 100 %.It is concluded that the ethanol-induced decrease of cyclic AMP in the gastric mucosa is due to a decreased formation of the nucleotide. The accompanying inhibition of the output of acid by ethanol is consistent with the view that cyclic AMP is an intracellular regulator of the gastric acid secretion. In view of the role of cyclic AMP in the control of the integrity of the cells, it is suggested that the ethanol-induced damage of gastric mucosa might also be, at least partly, due to the decreased mucosal content of cyclic AMP.     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat

Injection of LH (2 and 10 μg) into proestrus rats increased ovarian cyclic AMP levels and concomitantly decreased the levels of cyclic GMP. When injected into diestrus rats, cyclic AMP increases were even greater, whereas cyclic GMP levels were not significantly different from controls receiving saline injections. Ovarian cyclic nucleotide levels were also examined on different days of the cycle. On the afternoon of proestrus (1700 h), the time when circulating levels of LH are at their maximum, the concentration of cyclic AMP showed a moderate but insignificant increase. At the same time, cyclic GMP levels were significantly decreased. An inverse relation between cyclic AMP and cyclic GMP levels was seen on each day of the cycle. When rats were injected with pentobarbital (35 mg/kg) on the afternoon of proestrus (1300 h) to block the LH surge, the expected increases in ovarian cyclic AMP and decreases in cyclic GMP were effectively blocked. These results indicate that ovarian cyclic AMP and cyclic GMP levels are regulated by circulating LH. The apparent differences in direction of nucleotide response to LH, suggest divergent roles for the nucleotides in ovarian function.     

Effects of water deprivation on corticosterone production in the male Brattleboro rat genetically deprived of vasopressin

Effects of 72 h water-deprivation on plasma corticosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE), as controls. To determine the global effect of water deprivation, drinking water deprived rats were compared with hydrated animals. Because water deprived rats showed a depressed food intake, to elucidate the specific effect of dehydration alone, drinking water deprived rats were compared with similar food-restricted but water supplied animals. Increases in adrenal weights and in plasma corticosterone content, following 72 h water-deprivation, were greater in DI than in LE rats. In LE rats, they seemed to be the result of both dehydration and denutrition. Conversely in DI rats lacking vasopressin, dehydration alone increased neither adrenal weights nor plasma concentration of corticosterone; the whole plasma corticosterone content was reduced. So, in DI rats, the global response to drinking water deprivation was essentially due to food restriction, whose effect was partly suppressed by dehydration. Whatever the circumstances, plasma concentrations of corticosterone were higher in DI than in LE rats. Interrelationships between water deprivation, stress, vasopressin and glucocorticoids are discussed.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

Inhibition of dibutyryl cyclic AMP induced steroidogenesis in rat adrenocortical cells by the putative calcium antagonist TMB-8

A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced a smaller but significant reduction of EGTA-facilitated 45Ca2+ efflux from a mitochondrial-enriched fraction. We interpret these results to mean that TMB-8 blocks the steroidogenic effect of dibutyryl cyclic AMP by interfering with the mobilization of a cellular pool of calcium that is probably localized to the endoplasmic reticulum. The physiological implications of these findings in relation to the complex interactions between calcium and cyclic AMP in adrenal steroidogenesis are discussed.     

Effects of ACTH (corticotropin) analogues on steroidogenesis and cyclic AMP in rat adrenocortical cells. Evidence for two different steroidogenically responsive receptors.

Comparative studies on the mechanism of action of ACTH1-39 and ACTH5-24 [corticotropin-(1-39)- and corticotropin-(5-24)-peptides] on isolated rat adrenal cells were performed. The relationship between stimulated steroidogenesis and cyclic AMP was very different, suggesting that cyclic AMP does not play the same role in mediating the action of the two agonists. Data from three separate experiments showed that the competitive antagonist ACTH6-24 [corticotropin-(6-24)-peptide] had mean inhibitor constants of 13.4 +/- 3.1 nM against ACTH1-39 and 3.4 +/- 1.0 nM against ACTH5-24, indicating that the steroidogenic actions of these two agonists are mediated by two different receptor types. We conclude that there are two possible mechanisms for corticotropin action, only one of which involves the necessary production of cyclic AMP.     

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.     

Forskolin-induced cyclic AMP production and gastric acid secretion in dispersed rabbit parietal cells: novel evidence for a major role of cyclic AMP in acid release

Forskolin, a unique diterpine which is a direct activator of cyclic AMP-generating systems, stimulated both cyclic AMP and acid production in dispersed rabbit parietal cells. This agent was also capable of augmenting the action of histamine on both cyclic AMP and acid production at a low concentration. These findings provided novel evidence for a major role of cyclic AMP in gastric acid secretion.     

Effects of chronic exercise on the ultrastructure of the adrenocortical cells in the rat

Summary Four groups of male rats were exercised for periods of 2, 4, 6, and 8 weeks with controls in each group. As a result of chronic exercise there was an increase in the width of the zona fasciculata of the adrenal cortex. Also, there was an increase in the number and size of the mitochondria, and an increase in the quantity of smooth endoplasmic reticulum, and during the first 4 weeks of exercise an increase in the number of lipid droplets in the zona fasciculata. The close relationship between the smooth endoplasmic reticulum and the mitochondria, and the relationship between the smooth endoplasmic reticulum and the lipid droplets suggests a possible means for a transport mechanism for movement of precursors between these organelles.This research was supported in part by a Public Health Research Career Development Award KO4 GM42,355 from the National Institute of General Medical Sciences     

Influence of A23187 and dibutyryl cyclic AMP on progestagen production by rat granulosa cells in vitro

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)