Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5′-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of ¹²⁵I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells.We conclude that the corticotropin and choleragen receptors are different.     

Rat pancreas adenylate cyclase VII. Effect of extracellular calcium on pancreozymin-induced cyclic AMP formation

1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.

2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.

3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.

4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.

5. 5. It suffices to lower the Ca²⁺-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.

6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca²⁺-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.

Localization of the metabolic processes affected by calcium during corticotropin action

To define the role of calcium during corticotropin-induced steroidogenesis, adrenal sections were incubated under conditions of varying degrees of calcium depletion. Corticosterone production, [¹⁴C]leucine incorporation into protein, and tissue cyclic AMP levels were measured concomitantly. Omitting calcium from the incubation inhibited all three processes to variable extents, thus limiting conclusions regarding which process is most dependent on calcium.While calcium was required during the early phase of corticotropin action, it was not required during later phases; rapid induction of calcium defiency did not diminish the heightened rate of steroidogenesis previously induced by corticotropin in the presence of calcium. Thus, while calcium is required for induction of steroidogenesis factor(s), the operation of the latter is not dependent upon calcium in the extracellular fluid.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Adenylate cyclase in sea anemone implication for chemoreception

Adenylate cyclase of the sea anemoneAnthopleura elegantissima was found to be associated with the heavy particulate fraction of the cell and to be activated by NaF and 2-mercaptoethanol. Reduced glutathione, which elicits the ciliary swallowing response during feeding, also activated adenylate cyclase in particles from the oral disc and pharynx. The GSH effect was dependent on homogenization procedure, whereas the NaF and 2-mercaptoethanol activation was not. The activation of adenylate cyclase from the oral disc and pharynx by GSH was correlated with increased Ca²⁺ binding to the particulate fraction. When activation by GSH was abolished by mechanical homogenization, no increasea in Ca²⁺ binding was observed in the presence of GSH. It is suggested that chemoreception for the swallowing response of this organism is mediated by cyclic AMP control of Ca²⁺ distribution in the cell.     

Cyclic AMP-Elevating Agents Prevent Oligodendroglial Excitotoxicity

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca²⁺ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca²⁺ influx (～5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca²⁺ influx, leading to a less prominent response of intracellular Ca²⁺ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca²⁺ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca²⁺ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca²⁺ influx.     

Control of Ca2+ efflux and cyclic AMP by 5-hydroxytryptamine and dopamine in abalone gill.

5-Hydroxytrptamine increased the rate of Ca²⁺ efflux and the concentration of endogenous cyclic AMP in abalone gill in both 10 mM and 50 mM CaCl₂ concentrations externally. Dopamine decreased the rate of Ca²⁺ efflux in 50 mM CaCl₂ but slightly increased the efflux rate in 10 mM CaCl₂. At both external Ca²⁺ concentrations, dopamine increased the endogenous cyclic AMP concentration in the gill. 5-Hydroxytryptamine but not dopamine was found to activate adenylate cyclase in broken cell preparations of abalone gill. Cyclic AMP-dependent protein kinase activity was also demonstrated in homogenate fractions of abalone gill. It is suggested that both Ca²⁺ and cyclic AMP act as second messengers in the response of abalone gill to 5-hydroxytryptamine and dopamine.     

Inbition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1 by the ionophore A23187 Role of protein biosynthesis

Addition of the ionophore A123187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 · 10^?7 M). inhibition is rapid in onset and is not overcome by addition of external Ca²⁺. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effects and this effect is not overcome by high concentrations of Ca²⁺. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steriod synthesis after entry of cholesteriol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca²⁺.     

Effects of serotonin,dopamine and prostaglandin E2 on adenylate cyclase activity and cyclic AMP levels in different ganglia of the freshwater snail Planorbis corneus L.

The effects of different neuroactive agents on cyclic AMP level of selected ganglia of Planorbis corneus were studied. Serotonin, dopamine and prostaglandin E₂ were capable of increasing significantly cyclic AMP synthesis in all the preparations. When such substances were tested in pairs, supra-additive effects were always observed. In high Ca²⁺-high Mg²⁺ solutions dopamine action was blocked, meanwhile serotonin and prostaglandin E₂ were still effective in stimulating cyclic AMP synthesis. In the same experimental condition the supra-additive increases of the nucleotide level by drug combinations disappeared. Serotonin, but not dopamine, significantly stimulated adenylate cyclase activity in all the preparations, while prostaglandin E₂ was effective only in the Viscero-Parietal Complex. The presence of the adenylate cyclase activity in the nervous tissue of Planaorbis was substained by histochemical studies.These results demonstrating that in the nervous system of Planorbis cyclic AMP level is affected by neurotransmitters and neuromodulators, might support the idea of the crucial role of the cyclic nuclotide in the modulation of synaptic transmission.     

Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain

—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [³H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [³²P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg²⁺ and Ca²⁺, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)₂-ZnSO₄ mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain.     

Cyclic AMP modulation of calcium accumulation by sarcoplasmic reticulum from fast skeletal muscle

The role of cyclic 3′,5?AMP in modulating sarcoplasmic reticulum from fast skeletal muscle was studied. The rate of Ca²⁺ uptake was stimulated in the presence of protein kinase plus 1 μM cyclic AMP. The stimulation was absent when denatured protein kinase was used. When an adenylate cyclase inhibitor was added, the uptake rates fell to 55% of control. This decrease in rate was partially overcome by 1 μM cyclic AMP. A modulating role for cyclic AMP in fast skeletal muscle is proposed.     

Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors.

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5'-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not, during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of 125I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells. We conclude that the corticotropin and choleragen receptors are different.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca2+ uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of ⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

Adrenocorticotropic hormone and dibutyryl adenosine cyclic monophosphate-mediated Ca uptake by rat adrenal glands

The effect of adrenocorticotropic hormone and dibutyryl cyclic AMP on the uptake of⁴⁵Ca²⁺ by the rat adrenal gland has been investigated. After injection of ⁴⁵Ca²⁺ and adrenocorticotropic hormone into rats, the adrenal ⁴⁵Ca²⁺ concentration was significantly enhanced 90 to 180 min following hormone administration. The rise in adrenal ⁴⁵Ca²⁺ content was accompanied by a marked increase of the serum corticosterone levels. During incubation of rat adrenal glands in the presence of ⁴⁵Ca²⁺, adrenocorticotropic hormone and dibutyryl cyclic AMP caused significant accumulation of adrenal ⁴⁵Ca²⁺ and increased corticosterone synthesis. The degree of stimulation of both adrenal ⁴⁵Ca²⁺ uptake and corticosterone synthesis by adrenocorticotropic hormone or dibutyryl cyclic AMP was dependent upon the concentration of calcium in the incubation medium and upon the amount of adrenocorticotropic hormone or dibutyryl cyclic AMP added. Theophylline mimicked the stimulatory effect of adrenocorticotropic hormone and dibutyryl cyclic AMP and increased the uptake of ⁴⁵Ca²⁺ by rat adrenal glands in vitro. Determination of calcium by atomic absorption spectroscopy showed that the adrenocorticotropic hormone-mediated adrenal ⁴⁵Ca²⁺ uptake was due to a net accumulation of calcium in the tissue and not only to an increased rate of exchange of extracellular ⁴⁵Ca²⁺ with the intracellular calcium pool. Adrenocorticotropic hormone-stimulated adrenal ⁴⁵Ca²⁺ uptake was not observed when steroidogenesis was inhibited with elipten. Both adrenocorticotropic hormone-mediated corticosterone synthesis and adrenal ⁴⁵Ca²⁺ uptake were abolished after treatment of rats with cycloheximide but not after treatment with actinomycin D, indicating that adrenal ⁴⁵Ca²⁺ uptake and steroidogenesis have similar requirements for de novo protein synthesis, but not RNA synthesis.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Calcium ion effects on guanylate cyclase of brain.

Soluble guanylate cyclase activity of brain is stimulated by Ca²⁺ in the presence of low concentrations of Mn²⁺. Unlike Ca²⁺ stimulation of adenylate cyclase, the effect does not depend upon interaction of guanylate cyclase with a specific high-affinity Ca²⁺-binding protein. In the presence of Mg²⁺, Ca²⁺ inhibits soluble guanylate cyclase as well as the particulate enzyme. The concept that stimulation of brain cells results in increased cyclic GMP concentration secondary to Ca²⁺ influx merits additional critical study.     

Inhibitory effect of clonidine upon adenylate cyclase activity,cyclic AMP production,and insulin release in rat pancreatic islets

Conflicting opinions were recently expressed concerning the possible effect of ₂-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca²⁺), NaF, GTP,, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable , in part at least, to inhibition of adenylate cyclase.     

Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn²⁺ for activity, though Mg²⁺ gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.

Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5′-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.

Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.