Adenylate cyclase and cAMP-dependent protein kinase activity in the adrenal glands of animals after chronic administration of ACTH

ACTH, a prolonged action hormone, in a dose of 2.5 mu. was injected into guinea pigs daily for 5-35 days. The adenylate cyclase activity of the crude adrenal membrane fraction and the activity of cAMP-dependent protein kinases in the cytoplasmic fraction were determined. Cyclic changes in the basal and stimulated adenylate cyclase activities occurring with 15-20-day intervals have been established for the first time. The sensitivity of adenylate cyclase to ACTH, NaF and GTP did not change in the course of two cycles. The activity of cAMP-dependent protein kinases increased during the first few days after ACTH administration and decreased after further injections of the hormone. The role of cyclic changes of the enzyme activity in the mechanism of proliferative effect of ACTH is discussed.     

Clonal variation in adrenocorticotropin-induced desensitization of adenylate cyclase in Y1 adrenocortical tumor cells. Association with a 68,000-dalton protein

Adrenocorticotropin(ACTH)-induced desensitization of adenylate cyclase was examined in subclones derived from the ACTH-responsive, Y1 mouse adrenocortical tumor cell line. This report describes clonal variation in ACTH-induced desensitization of adenylate cyclase and an associated variation in the level of a 68,000-dalton protein, p68. A subclone of Y1 cells with a low level of p68 (0.8% of total protein) exhibited a faster rate of desensitization and a slower rate of recovery from desensitization when compared with a clone containing a high level of p68 (10% of total protein). In three clones with low levels of p68, ACTH desensitized adenylate cyclase with ED50 values from 0.3 to 0.5 nM. In several clones with high levels of p68, the adenylate cyclase system was more resistant to ACTH-induced desensitization; the ED50 values for ACTH in these clones ranged from 2 to 12 nM. Among 11 ACTH-responsive subclones, the level of p68 correlated significantly (p less than 0.001, r = 0.87) with resistance to the desensitization induced by 1 nM ACTH. These results suggest that p68 may function in the maintenance of an ACTH-responsive adenylate cyclase system, or that the level of p68 and responsiveness to ACTH are coordinately regulated.     

Pertussis toxin prevents homologous desensitization of adenylate cyclase in cultured renal epithelial cells

The biochemical mechanisms of adenylate cyclase desensitization in arginine vasopressin-responsive epithelial cells remain unclear. Preincubation of cultured rabbit renal cortical collecting tubular cells with arginine vasopressin leads to a 30-100% decline in arginine vasopressin-stimulated adenylate cyclase activity. This loss of adenylate cyclase activity is time- and arginine vasopressin concentration-dependent. Preincubation with arginine vasopressin does not result in significant changes in basal, NaF-, forskolin-, isoproterenol- or cholera toxin-stimulated adenylate cyclase activity. Preincubation of cells with chlorophenylthio-cAMP, forskolin, and cholera toxin does not result in loss of arginine vasopressin-stimulated adenylate cyclase activity. Since products of cyclo-oxygenase inhibit arginine vasopressin action, cells were preincubated with indomethacin. Arginine vasopressin-induced adenylate cyclase desensitization is not reversed by indomethacin. By contrast, incubation with pertussis toxin prevents arginine vasopressin-induced adenylate cycle desensitization. These data demonstrate that arginine vasopressin induces homologous desensitization in membranes from cultured rabbit cortical collecting tubular cells and suggest that this desensitization is mediated, at least in part, by pertussis toxin substrate. These observations provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened alpha-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochrome P-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native beta-subunit and alpha-subunits enzymatically shortened by 2-3 amino acid residues, were used for studying the influence of hCG on the content of microsomal progesterone-binding cytochrome P-450 in rat testis. When 2-3 residues have been removed from the alpha-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88-92)-alpha chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-alpha or des-(90-92)-alpha, respectively, were found to have induced a decrease in microsomal cytochrome P-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochrome P-450 system had persisted in case of the hCG species containing a des-(90-92)-alpha chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-alpha and a native beta-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochrome P-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Intrahypothalamic action of corticotrophin-releasing factor (CRF) to inhibit growth hormone and LH release in the rat

The effects of intravenous or intraventricular injection of synthetic ovine corticotrophin-releasing factor (oCRF) on plasma levels of anterior pituitary hormones were studied in conscious, ovariectomized (OVX) female rats and compared with the actions of the peptide on dispersed anterior pituitary cells from OVX female rats incubated in the presence of CRF. Third ventricular injection of oCRF in freely moving rats caused a significant increase in plasma levels of ACTH in a dose-related manner with a minimal effective dose of less than 0.5 micrograms (0.1 nmol). The effect was observable at 5 min after injection and persisted for the 60 min duration of the experiment. In contrast, growth hormone levels were significantly depressed within 15 min with a minimal effective intraventricular dose of 0.5 micrograms. The suppression persisted for the duration of the experiment but there was no additional effect of the higher dose of 5 micrograms. Plasma LH levels were also lowered by the highest dose of 5 micrograms (1.0 nmol) of oCRF, with the first significant lowering at 30 min. Lower doses had no effect on plasma LH. Plasma TSH levels were not significantly altered. Control injections of the 0.9% NaCl diluent were without effect on the levels of any of the hormones. Intravenous injection of similar doses of oCRF had no effect on plasma levels of GH or LH. The ACTH-releasing action of the oCRF preparation was confirmed by in vitro incubation of the peptide with dispersed anterior pituitary cells for 2 h. A dose-related release of ACTH occurred in doses ranging from 0.1-10 nM, but there were no effects on the release of the other anterior pituitary hormones. The results suggest that oCRF may act within the hypothalamus to suppress the release of GH and to a lesser extent LH. The stimulation of ACTH release following intraventricular CRF is presumably related to its uptake by portal blood vessels with delivery to the pituitary and stimulation of the corticotrophs.     

The role of periodic changes in cyclase and protein kinase activity in the mechanism of the proliferative effect of ACTH

A prolonged effect of ACTH on the state of adenylate and guanylate cyclase systems in the adrenal glands of experimental animals was investigated. It was found that in guinea pigs injected with ACTH (4 units daily for 1-50 days) the weight of adrenal glands and the DNA content in these organs increased 2.0-2.5-fold by the end of experiment; the increase in both values was stepwise. The corticosteroid level in the blood varied throughout the experiment: the changes in the DNA content in adrenals and in the corticosteroid content in the blood were oppositely directed. This was accompanied by cyclic changes in the basal and stimulated activities of adenylate and guanylate cyclases and proteinases in the adrenal glands occurring with a periodicity of 6-15 days. The activity peaks for cyclases and protein kinases preceded the rise in the DNA content in the adrenals. A clearcut correlation between the changes in the enzyme activity and the hormone dose was observed. The changes in the basal and stimulated activities of guanylate cyclase seem to be due to the control of cAMP level in the cell (stimulation of cGMP-dependent cAMP phosphodiesterase). Apparently, the periodic changes in the activity of cAMP-dependent protein kinases in the cytoplasmic and nuclear fractions and a relatively high activation of nuclear protein kinases (by 30-60%) in comparison of cytoplasmic ones (8-10%) are related to stimulation of DNA synthesis. It is concluded that the changes in the activity of cyclases and protein kinases play a role in the mechanism of proliferative effect of ACTH.     

The inhibitory guanine nucleotide-binding regulatory subunit of adenylate cyclase has an adenylate cyclase-independent modulatory effect on ACTH secretion from mouse pituitary tumor cells

Somatostatin inhibits agonist-stimulated cAMP synthesis and ACTH secretion from mouse pituitary tumor cells. It also decreases basal hormone release without affecting cAMP levels and inhibits ACTH secretion in response to agonists whose action is independent of prior cAMP synthesis. These inhibitory effects are attenuated by pertussis toxin, suggesting that the inhibitory guanine nucleotide-binding regulatory subunit of adenylate cyclase modulates effectors, other than adenylate cyclase, during transduction of negative hormonal signals.     

Gonadotropin-induced loss of hormone receptors and desensitization of adenylate cyclase in the ovary.

Gonadotropin receptor sites and adenylate cyclase activity were analyzed in luteinized rat ovaries following injection of human chorionic gonadotropin (hCG). Gonadotropin binding capacity and hormonal stimulation of adenylate cyclase declined rapidly to a minimum at 6 to 12 h, remained depressed for 4 days, and returned to the control level between 5 and 7 days. Total adenylate cyclase activity measured in the presence of fluoride fell by 50% within a few hours but returned to normal by 24 h. A close correlation was observed between the number of gonadotropin receptors and the ability of adenylate cyclase to be stimulated by hormone. Assay of tissue-bound hormone showed that the initial loss of hormone sensitivity and binding capacity was associated with occupancy of luteinizing hormone receptor sites, but that the prolonged changes in these activities were not attributable to receptor occupancy. These studies have demonstrated that induction of a refractory or desensitized state in ovarian adenylate cyclase by gonadotropin results from the loss of specific hormone receptor sites.     

Osteosarcoma cytosol factor promotes parathyroid hormone stimulation of adenylate cyclase independent of GTP

The adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1)-stimulating factor from rat osteosarcoma cytosol was purified 600-fold by ion-exchange chromatography. The factor has an apparent M_r of 20 000, is cold-labile, but retains activity at ?20°C in 10% glycerol.The factor enhanced parathyroid hormone stimulation of adenylate cyclase and restored hormone responsiveness to membranes washed with 0.5 M NaCl. These ‘GTP-like’ effects were not inhibited by 100 μM GDP-β-S, which completely abolished the GTP enhancement of both basal and hormone-stimulated adenylate cyclase.Adenylate cyclase activity in the presence of the stimulating factor was linear with time, and showed hyperbolic dependence on factor concentration. The factor also linearized (in double reciprocal plots) the downward-concave Mg²⁺-dependence of adenylate cyclase, increasing the apparent affinity of the enzyme for Mg²⁺.The presence of the factor in two clonal osteosarcoma cell lines correlated with parathyroid hormone-stimulatable adenylate cyclase. Factor stimulation was absent while GTP stimulation was retained in the hormone-nonresponsive clone. Factor and hormone sensitivity were restored by in vivo passage. This factor thus may represent a guanyl nucleotide-independent path for cellular regulation of hormone response.     

Effect of long-term and short-term diabetes on the parathyroid hormone sensitive rat renal adenylate cyclase: correlation with vitamin D metabolism

In short-term experiments, male Wistar rats were made diabetic for 10 days with a single injection of streptozotocin (65 mg/kg body weight). One group of diabetic rats was treated with insulin for 3 days prior to sacrifice. In long-term experiments, vitamin D replete or vitamin D depleted rats were made diabetic for 6 weeks. Criteria for diabetes were loss of weight, glycosuria (Tes-Tape), and hyperglycemia. In long-term diabetic rats the activity of renal mitochondrial 25-hydroxyvitamin D3 (25-(OH)D3) 1 alpha-hydroxylase was significantly decreased and that of 25-(OH)D3 24-hydroxylase increased. However, the parathyroid hormone (PTH) sensitive renal adenylate cyclase activity of diabetic rats was not different from that of the nondiabetic rats in either the vitamin D replete group or the vitamin D depleted group. On the other hand, the PTH-sensitive renal adenylate cyclase activity was significantly higher in short-term diabetic rats than in control and insulin-treated rats. These differences were observed at doses of 10(-8) to 10(-5) M of PTH. This study has demonstrated for the first time that there are differences in the PTH-sensitive adenylate cyclase response between long-term and short-term diabetic rats. The hypersensitivity to PTH of the renal adenylate cyclase observed in short-term diabetic rats probably represents a response to insulin deficiency during the early development of diabetes mellitus in the rats.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened α-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochromeP-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native β-subunit and α-subunits enzymatically shortened by 2–3 amino acid residues, were used for studying influence of hCG on the content of microsomal progesterone-binding cytochromeP-450 in rat tests. When 2–3 residues have been renuwed from the α-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88–92)-α chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-α or des-(90–92)-α, respectively, were found to have induced a decrease in microsomal cytochromeP-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochromeP-450 system had persisted in case of the hCG species containing a des-(90–92)-α chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-α and a native β-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochromeP-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells

A novel neuropeptide which stimulates adenylate cyclase in rat anterior pituitary cell cultures was isolated from ovine hypothalamic tissues. Its amino acid sequence was revealed as: His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln- Met-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys - NH2. The N-terminal sequence shows 68% homology with vasoactive intestinal polypeptide (VIP) but its adenylate cyclase stimulating activity was at least 1000 times greater than that of VIP. It increased release of growth hormone (GH), prolactin (PRL), corticotropin (ACTH) and luteinizing hormone (LH) from superfused rat pituitary cells at as small a dose as 10(-10)M (GH, PRL, ACTH) or 10(-9)M (LH). Whether these hypophysiotropic effects are the primary actions of the peptide or what physiological action in the pituitary is linked with the stimulation of adenylate cyclase by this peptide remains to be determined.     

Glycogenolytic responsiveness to glucagon, epinephrine, vasopressin and angiotensin II in the liver of developing hypothyroid rats. A comparative study of in vitro hormonal binding and in vivo biological response

Both dose-response curves and time-courses of plasma glucose levels after single maximal doses showed that in vivo glycogenolytic responsiveness to glucagon and epinephrine was significantly higher in developing hypothyroid rats, whereas it remained unchanged after vasopressin and angiotensin II injections. In contrast with the decreased basal activity of phosphorylase(a), the glucagon-stimulated activity increased in hypothyroid rats, whereas it was only slightly modified under vasopressin stimulation. Daily thyroxine treatment abolished these abnormalities. Thus, there is a close correlation between glucose output and enzyme activation. The maximal binding capacity of [3H]vasopressin and [125I]glucagon was significantly decreased in hypothyroid rats, without changes in the apparent dissociation constant of hormone from its specific receptor. Daily thyroxine treatment also abolished this deficit, which moreover appeared to be independent of possible changes in plasma hormone levels. With respect to glucagon action, neither basal nor Gpp(NH)p-stimulated adenylate cyclase activities were affected in hypothyroid rats. Glucagon-sensitive adenylate cyclase activity and the apparent activation constant appeared to be unaffected. The apparent discrepancy between the results obtained from in vivo and in vitro experiments is discussed on the basis of different membrane transducing phenomena and related intracellular mechanisms underlying the biological response to hormonal stimulation.     

Hormonal control of the adenyl cyclase activity of adipose cell membranes prepared from badger, rabbit, fox and rat adipose tissues]

1. We have shown differences in hormonal regulation of adenylate cyclase activity in fat cell ghosts prepared from rat, rabbit, fox and badger adipose tissue, under the influence of catecholamines, ACTH and insulin. a) In the rat, catecholamines induced a large stimulation (+315%) of adenylate cyclase. b) In the rabbit, ACTH was the most effective hormone. c) In the fox and the badger, only catecholamines could stimulate adenylate cyclase. d) In both rat and rabbit, insulin did not reduce spontaneous enzymatic activity. Moreover, the activation of adenylate cyclase by ACTH in the rabbit was not altered by insulin, while in the rat, this hormone slightly decreased epinephrine stimulation. 2. Hormonal regulation of adenylate cyclase correlated with the lipolytic response.     

Cell-free lutropin-dependent desensitization of the lutropin-sensitive adenylate cyclase of pig ovarian follicles is dependent on ATP.

Experiments were conducted to clarify the nucleotide requirements for lutropin (LH)-dependent adenylate cyclase desensitization in a cell-free membrane preparation derived from a thecal-cell-enriched component of preovulatory pig ovarian follicles. The follicular membranes were extensively washed in 2M-urea to remove endogenously bound GTP, and ATP devoid of GTP was utilized. Results conducted in the presence of 60 microM-GTP and various concentrations of ATP confirm the dependence of LH-stimulated adenylate cyclase activation and desensitization on millimolar concentrations of ATP. In experiments in which adenylate cyclase activation was supported by Mg2+, LH and adenosine 5'-[beta, gamma-imido]triphosphate, GTP did not support the desensitization response. Moreover, although GTP increased both basal and LH-stimulable adenylate cyclase activities in a concentration-dependent manner, the percentage desensitization was not significantly modified by the presence of 10nM-10mM-GTP. These results demonstrate that, even in the presence of exogenous GTP and Mg2+, activation of adenylate cyclase by saturating concentrations of LH in the presence of adenosine 5'-[beta, gamma-imido]triphosphate is not sufficient to initiate desensitization; millimolar concentrations of ATP are also required for the adenylate cyclase desensitization response.     

Treatment of intact hepatocytes with synthetic diacyl glycerols mimics the ability of glucagon to cause the desensitization of adenylate cyclase

Incubation of intact hepatocytes with either of the synthetic diacyl glycerols 1-oleoyl-2-acetyl glycerol (OAG) or dihexanoyl glycerol (DHG) caused the transient uncoupling of the ability of glucagon to stimulate adenylate cyclase in membranes prepared from those cells. No change occurred in either the activity of the catalytic unit of adenylate cyclase or the coupling of Gs to adenylate cyclase. Diacyl glycerol action appeared to mimic glucagon-mediated desensitization of adenylate cyclase, suggesting that protein kinase C activation may provide the molecular trigger for glucagon desensitization.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

Effect of the "memory peptide" ACTH 5-10 on the self-stimulation reaction of rabbits

Experiments on rabbits were made to study variation in the frequency of the self-stimulation reaction from the lateral hypothalamus under the effect of the corticotropin fragment ACTH5-10. Intraperitoneal administration of the peptide in a dose of 50 micrograms/kg that causes the improved training in different behavioral models produces no significant effect on the mechanism of intracranial positive reinforcement. Intraventricular injection of 5 microliters of 0.9% NaCl leads to a short-term suppression of the self-stimulation reaction. Administration of 50 pcM/kg ACTH5-10 in the same volume of physiological saline completely abolishes the inhibitory action of the intraventricular injection itself.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.