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.     

The effects of calcium ionophore A23187 on interstitial cell steroidogenesis

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.     

Calcium-dependent regulation of progesterone production by isolated rat granulosa cells: effects of the calcium ionophore A23187, prostaglandin E2, dl-isoproterenol and cholera toxin

The role of calcium in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various calcium channel blockers (verapamil, cobalt or manganese) and a calcium chelator (EGTA) resulted in marked decreases in progesterone production in response to follicle-stimulating hormone (FSH), cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cyclic AMP (Bt2cAMP). Cyclic AMP production, however, was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogenesis (0.1-1.0 mM and 125 microM, respectively). Two inhibitors of the calcium-dependent regulatory protein, calmodulin [trifluoperazine, 40 microM and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy )-phenethyl]imidazolium chloride, ( R24571 ) 20 microM] significantly inhibited both cyclic AMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml-1.0 micrograms/ml, the calcium ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at approximately 0.14 microgram/ml. Maximal steroidogenic response to the calcium ionophore (1 microgram/ml) however, was only 50% of that evoked by FSH (0.33 microgram/ml). A23187 (0.5 microgram/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 microgram/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 microgram/ml). These findings support our earlier suggestion that the calcium-calmodulin system plays a central role in the gonadotropic regulation of ovarian steroidogenesis and suggest that a transmembrane flux of extracellular calcium may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.     

Effect of calcium ionophore A23187 on pregnenolone metabolism to progesterone in rat granulosa cells

The effect of calcium ionophore A23187 on the metabolism of pregnenolone to progesterone was examined in rat granulosa cells during a 24-h culture period. Granulosa cells harvested from pregnant mare's serum gonadotropin treated immature rats were incubated in the presence and absence of the divalent cation ionophore A23187. The ionophore induced progesterone synthesis from both endogenous sterol substrate and exogenous pregnenolone in a time- and concentration-dependent manner. Pregnenolone metabolism was examined in the presence of aminoglutethimide phosphate, an inhibitor of endogenous pregnenolone production. Steroid secretion resulting from metabolism of endogenous substrate was more sensitive to A23187 in that a lower concentration of the ionophore was required to induce a significant increase than that noted for exogenous pregnenolone metabolism. In addition, progesterone production from endogenous sterol occurred 6 h earlier than the observed increase in the conversion of pregnenolone to progesterone. These results indicate that A23187 and therefore possibly enhanced calcium influx may play a significant role in the regulation of pregnenolone metabolism in granulosa cells depending on the duration of incubation. The earlier steroidogenic response from endogenous substrate may be a reflection of an acute effect of A23187 on certain steroidogenic steps proximal to pregnenolone production.     

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells.

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol.

In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.     

Insulin secretion. Interrelationships of glucose, cyclic adenosine 3:5-monophosphate, and calcium.

Glucose elevates both cyclic adenosine 3:5-monophosphate (cyclic AMP) and insulin secretion rapidly and in a parallel dose-dependent fashion in perifused rat islets. Theophylline stimulates cyclic AMP much more than glucose, yet secretion is much less. When the two agents are combined, cyclic AMP is similar to theophylline alone yet secretion is augmented synergistically. Glucose-induced cyclic AMP generation and insulin secretion are dependent on extracellular calcium. Theophylline-induced insulin secretion is also extracellular calcium-dependent; however, theophylline-induced cyclic AMP elevation is independent of extracellular calcium. Thus, extracellular calcium has multiple effects on insulin secretion, some of which appear unrelated to a terminal secretory process. When glucose is combined with theophylline at physiologic levels of extracellular calcium, both the first and second phases of secretion are prominent. At extracellular calcium levels of 0.05 mM, only the second phase is prominent whereas at 10 nM extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,-tetraacetic acid) only the first phase is prominent. A divalent cation ionophore (a23187, Eli Lilly), which transports calcium and magnesium ions across biological membranes, was used to elucidate further the role of calcium and magnesium. If the ionophore (10 muM) is perifused for 5 min at low extracellular calcium and magnesium, and physiologic calcium is then added, a sudden spike of insulin release occurs in the absence of cyclic AMP generation. Similar results were obtained with magnesium. When the ionophore is perifused for 30 min at low calcium and magnesium, insulin secretion again occurs in the absence of cyclic AMP generation. Electron microscopic examination of the B cells following perifusion with the ionophore shows no specific alterations. These observations suggest that: (a) glucose elevates cyclic AMP, but the latter acts primarily as a positive feed-forward modulator of glucose-induced insulin release; and (b) extracellular calcium has multiple effects on insulin secretion both upon, and independent of, the cyclic AMP system.     

The effect of ionophore A23187, verapamil, and dibutyryl cyclic AMP on bile acid synthesis in isolated rat hepatocytes

The effect of the divalent cation ionophore A23187 and the calcium channel-blocker verapamil on bile acid synthesis in isolated hepatocytes in the presence and absence of dibutyryl cyclic AMP was studied. Both A23187 (1 microM) and verapamil (0.04 mM) caused a small (approximately 15-20%) but consistent decrease in total bile acid synthesis in the cells. When hepatocytes were incubated with dibutyryl cyclic AMP (1 mM) production of total bile acid was increased by about 25%, and this effect was unchanged by A23187 but abolished by verapamil. The relative proportions of the individual bile acids produced were not affected by either A23187 or verapamil. Dibutyryl cyclic AMP (1 mM) lowered the ratio of the amount of conjugated cholic acid to conjugated chenodeoxycholic + beta-muricholic acid formed in the cells by about 50%. Neither A23187 nor verapamil was able to prevent this change. These results suggest that the stimulatory effect of dibutyryl cyclic AMP on total bile acid synthesis is dependent on mobilisation of calcium from intracellular stores, but its effect on the relative proportions of bile acid formed via the cholic acid versus the chenodeoxycholic acid pathway is independent of calcium movement.     

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)     

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

Addition of the ionophore A23187 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 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 steroid synthesis after entry of cholesterol 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+.     

Role of calcium in the regulation of theca cell androstenedione production in the domestic hen

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.     

Effects of divalent cation ionophore A23187 on potassium permeability of rat erythrocytes.

A23187 transports calcium rapidly into rat erythrocytes, apparently by an electroneutral exchange for intracellular magnesium and protons. When red cells are incubated in the absence of any added divalent cations, A23187 transports internal magnesium out of the cells, in exchange for extracellular protons. Magnesium uptake into erythrocytes is produced by A23187, providing the extracellular concentration of this cation exceeds intracellular levels, and the ionophore also transports strontium, but not barium, into red cells. A23187 produces a rapid and extensive loss of intracellular potassium from erythrocytes during uptake of calcium or strontium, but not magnesium. When red cells are incubated in the absence of any exogenous divalent cations, A23187 still produces a potassium efflux and this is inhibited completely by small amounts of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and restored by the addition of calcium in excess of the chelator. Although EDTA enhances the extent of magnesium release from erythrocytes incubated with A23187, it prevents the potassium efflux. Dipyridamole and 4-acetamid-4'-isothiocyano-stilbene-2,5'-disulfonic acid, which decrease chloride premeability of erythrocytes, inhibit the A23187-induced potassium loss from red cells. Rutamycin, peliomycin, venturicidin, and A23668B also inhibit potassium efflux from intact cells incubated with A23187, but this effect is not correlated with their abilities to inhibit various ATPases in red cell membrane preparations. It is concluded that A23187 does not transport potassium directly across the erythrocyte plasma membrane, but permits small amounts of endogenous calcium to interact with some membrane component to enhance potassium permeability of the cell.     

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Effect of Forskolin and Prostaglandin E1 on Stimulus Secretion Coupling in Cultured Bovine Adrenal Chromaffin Cells

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.     

Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore.

Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.