Effect of imidazole on renal gluconeogenesis.

The metabolic effects of imidazole were tested in rat renal cortex. Imidazole enhanced the activity of renal cortical phosphodiesterase in vitro. Imidazole inhibited glucose production in a dose-dependent fashion from a variety of substrates in the gluconeogenic pathway proximal to the triose phsophates. The stimulation in renal gluconeogenesis resulting from isoproterenol and parathyroid hormone was inhibited by imidazole. These changes correlated with an inhibition of the augmented levels of renal cortical cyclic AMP levels produced by these hormones. These studies indicate that imidazole is an effective activator of phosphodiesterase in intact renal cells and lend further support to the suggestion that the stimulation of renal gluconeogenesis produced by isoproterenol and parathyroid hormone is mediated by a release of cyclic AMP.     

Enhanced excitability of the uterus of the pregnant rabbit by imidazole stimulation of cyclic AMP phosphodiesterase

Imidazole, at concentrations between 10(-3) and 10(-2) M, exerts a profound stimulatory effect on rabbit uterine strips obtained during pregnancy and studied isometrically in vitro. The action is not duplicated by N-alkylimidazoles which have greater potency as inhibitors of thromboxane synthetase but the effect of imidazole was antagonized by isoproterenol or theophylline. Biochemical analysis indicated that imidazole at concentrations greater than 5 x 10(-4) M stimulated both high and low affinity forms of cyclic AMP phosphodiesterase. The uterus of pregnant rabbits is profoundly refractory to any kind of pharmacological stimulation and the effects of imidazole, acting to stimulate phosphodiesterase, suggest that the integrity of the adenyl cyclase-cyclic AMP-protein kinase system is a necessary requirement for this organ to remain quiescent during pregnancy.     

Effects of catecholamines on ammoniagenesis and gluconeogenesis by renal cortex in vitro

Norepinephrine (arterenol) and a synthetic catecholamine, isoproterenol, increase the production of ammonia and glucose from glutamine and glutamate by rat renal cortical slices in vitro. The stimulation of both ammonia and glucose production by isoproterenol was greater than that observed with identical molar concentrations of arterenol. Isoproterenol markedly increased the concentration of cyclic AMP in rat renal cortical slices. Addition of propranolol, a β-adrenergic blocking agent, prevented the increase of cyclic AMP levels induced by isoproterenol. Cyclic AMP increased both ammoniagenesis and gluconeogenesis by kidney cortex. Thehe increase in ammonia production produced by isoprotenol was blocked by the addition of propranolol. It is concluded that the increase in ammonia and glucose production caused by isoproterenol is mediated through the release of cyclic AMP.     

Effect of prostaglandin E on the adenyl cyclase-cyclic AMP system and gluconeogenesis in rat renal cortical slices.

Prostaglandin E was found to increase the formation of cyclic acdenosine 3',5'-monophosphate (cyclic AMP) by renal cortical slices. This increased release of cyclic AMP was not influenced by the absence of Ca2+ in the incubating media. The enhanced production of cyclic AMP was probably mediated by stimulation of membrane-bound adenylate cyclase activity. An increase in adenyl cyclase activity was observed with increasing concentrations of prostaglandin E. Furthermore, prostaglandin E augmented glucose production from alpha-ketoglutarate. This effect on gluconeogenesis was abolished by the removal of Ca2+ from the incubating medium. These effects are similar to those described for parathyroid hormone and suggest that the renal cortex is a prostaglandin-dependent system. Prostaglandin E decreased cyclic AMP production and glucose production (from alpha-ketoglutarate) in response to submaximal doses of parathyroid hormone, suggesting that prostaglandin may be important in modulating the intracelluar action of parathyroid hormone in the kidney cortex.     

The possible mediation by cyclic AMP of the stimulation of thymocyte proliferation by vasopressin and the inhibition of this mitogenic action by thyrocalcitonin

Lysine vasopressin (antidiuretic hormone), like cyclic adenosine 3',5'-monophosphate (cyclic AMP), rapidly (in less than 1 hour) stimulates the initiation of deoxyribonucleic acid synthesis and thereby increases the flow of cells into mitosis in rat thymic lymphocyte populations in vitro. This mitogenic action of vasopressin, again like that of cyclic AMP, is potentiated by caffeine, an inhibitor of the intracellular phosphodiesterase which catalyzes the degradation of cyclic AMP. On the other hand, vasopressin's mitogenic action (also like that of cyclic AMP) is blocked by imidazole, an activator of cyclic nucleotide phosphodiesterase activity. The hormone, thyrocalcitonin (calcitonin) which is known to block the cyclic AMP-mediated mitogenic effect of parathyroid hormone by interfering with cyclic AMP action, also blocks the mitogenic action of vasopressin. The inhibitory effects of imidazole and thyrocalcitonin on vasopressin's mitogenic action are both overcome by the phosphodiesterase inhibitor, caffeine. It is concluded from these observations that the mitogenic action of vasopressin is mediated by cyclic AMP.     

Activity of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by bovine heart and rat liver cyclic nucleotide phosphodiesterases.

The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10^?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10^?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10^?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca²⁺. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.     

Hormonal control of gluconeogenesis in tubule fragments from renal cortex of fed rats. Effects of α-adrenergic stimuli, glucagon, theophylline and papaverine

1. In incubated tubule fragments from renal cortex of fed rats gluconeogenesis from pyruvate was stimulated by adrenaline (1mum optimum) and by the selective alpha-adrenergic agonists oxymetazoline and amidephrine. The selective beta-agonists isoproterenol and salbutamol were ineffective at concentrations up to 10mum. 2. Stimulation of gluconeogenesis by 1mum-adrenaline was almost completely blocked by 10mum-phentolamine (alpha-antagonist), partially blocked by 10mum-phenoxybenzamine (alpha-antagonist) and unaffected by 10mum-propranolol (beta-antagonist). 3. Adrenaline stimulation of gluconeogenesis was rapid and was sustained for at least 1h. 4. Oxymetazoline (alpha-agonist) was extremely potent in stimulation of gluconeogenesis. This compound stimulated glucose production from pyruvate, lactate and glutamate, but not from succinate or glycerol. 5. In the absence of Ca(2+) oxymetazoline was ineffective, whereas some stimulatory effect of adrenaline on gluconeogenesis was still observed. 6. Glucagon had no effect on gluconeogenesis from pyruvate in the presence of 1.27mm-Ca(2+) and inhibited the process in the presence of 0.25mm-Ca(2+). Parathyrin (parathyroid hormone) stimulated gluconeogenesis at 1.27mm-Ca(2+). 7. In short incubations of tubule fragments glucagon, papaverine and adrenaline significantly increased 3':5'-cyclic AMP. Adrenaline also slightly decreased 3':5'-cyclic GMP. Oxymetazoline had no effect on the amount of either cyclic nucleotide. 8. At all concentrations tested, theophylline and papaverine decreased gluconeogenesis from pyruvate. 9. It is concluded that renal gluconeogenesis may be increased by alpha- but not beta-adrenergic stimuli and that this is probably independent of changes in 3':5'-cyclic AMP or 3':5'-cyclic GMP. An involvement of Ca(2+) in the action of oxymetazoline appears likely, but this is less certain with adrenaline.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

Introduction of cyclic AMP phosphodiesterase into rat submandibular acini prevents isoproterenol-stimulated cyclic AMP rise without affecting mucin secretion

Cyclic AMP phosphodiesterase has been incorporated into isolated rat submandibular acini by hypotonic swelling. This resulted in complete inhibition of the cyclic AMP rise stimulated by isoproterenol (10 microM), but had no effect on the stimulation of mucin secretion. Acini swollen in the absence of cyclic AMP phosphodiesterase showed similar cyclic AMP and mucin secretion responses to those of unswollen acini. The dissociation between cyclic AMP rise and mucin secretion was not due to stimulation of different beta-receptor subtypes since both responses to isoproterenol were inhibited by the beta 1 antagonist atenolol, but not by the beta 2 antagonist, butoxamine. The results are the first to directly demonstrate that a maximally effective concentration of isoproterenol can increase mucin secretion in the absence of a detectable increase in cyclic AMP.     

Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.     

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: Interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

The possible mediation by cyclic AMP of parathyroid hormone-induced stimulation of mitotic activity and deoxyribonucleic acid synthesis in rat thymic lymphocytes

Purified parathyroid hormone (PTH) strongly stimulates the initiation of deoxyribonucleic acid (DNA) synthesis and thereby raises the flow of cells into mitosis in rat thymic lymphocyte populations maintained in vitro. These actions of PTH are potentiated by caffeine and inhibited by imidazole which indicates that the hormonal action is mediated by cyclic adenosine 3′,5′,-monophosphate (cyclic AMP). The feasibility of cyclic AMP being the mediator of PTH action is established by the observation that a low concentration (10^?7 M) of dibutyryl cyclic AMP precisely mimics the stimulatory action of the hormone on DNA synthesis and cell proliferation.     

Effects of beta-adrenergic catecholamines on potassium transport in turkey erythrocytes.

Isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a 2- to 4-fold increase in bidirectional potassium fluxes in turkey erythrocytes. Ouabain, which does not alter catecholamine-stimulated cellular cyclic AMP, inhibits potassium influx by 50 to 70%, does not alter potassium outflux or isoproterenol-stimulated potassium influx, but increases isoproterenol-stimulated potassium outflux. Stimulation of potassium transport by isoproterenol can be reproduced by adding cyclic AMP to the medium and is inhibited by propranolol or dichloroisoproterenol but not by phentolamine. Theophylline at concentrations which inhibit cyclic nucleotide phosphodiesterase in isolated turkey erythrocyte plasma membranes by greater than 90%, does not augment isoproterenol stimulation of cellular cyclic AMP or of potassium transport but does potentiate stimulation of potassium influx produced by adding cyclic AMP to the medium. Isoproterenol-stimulated cellular cyclic AMP increases steadily for at least 2 hours. Potassium transport, however, increases rapidly, becomes maximal after 20 to 30 min of incubation, and thereafter decreases progressively so that after 2 hours of incubation potassium fluxes are only slightly greater than for the control. Ouabain prolongs the duration of catecholamine-stimulated potassium influx and potassium outflux, reflecting its ability to relieve the refractoriness developed by turkey erythroyctes to endogenous cyclic AMP.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase

When isolated diaphragms of hypophysectomized rats were incubated with bovine growth hormone in the presence of the cyclic nucleotide inhibitors theophylline, quinine and papaverine, the stimulatory effects of the hormone on leucine incorporation into protein, α-aminoisobutyric acid and 3-O-methylglucose transport were suppressed or abolished entirely. The degree of suppression of the hormone effects appeared to correlate with the extent of glycogenolysis caused by the drugs. Thoephylline also rapidly reversed the stimulation of protein synthesis and amino acid and sugar transport produced by growth hormone. When protein synthesis and transport were stimulated by preincubation of the diaphragm with growth hormone, the subsequent addition of theophylline to the medium inhibited the hormonal effects on protein synthesis and sugar transport within 15 min and the effect on amino acid transport within 60 min. These results may mean that the rapid in vitro effects of growth hormone on protein synthesis and membrane transport in rat diaphragm muscle are mediated by a reduction in the cellular level of cyclic AMP or some other nucleotide.Attempts to block the action of growth hormone on 3-O-methylglucose transport by preincubation of the diaphragm with high concentrations (10 mM) of cyclic GMP, cyclic UMP, cyclic TMP and cyclic CMP were unsuccessful. Also an effort was made to mimic the action of growth hormone on sugar transport by incubating the diaphragm with high concentrations of imidazole and histamine, agents known to activate cyclic nucleotide phosphodiesterase. Slight stimulatory effects were obtained, but they could not be correlated with any certainty to the actions of imidazole and histamine on phosphodiesterase.Like growth hormone, insulin also stimulates protein synthesis and amino acid and sugar transport in the isolated rat diaphragm. However, the actions of insulin on these processes were not abolished by theophylline, suggesting some basic difference in the mode of action of these two hormones on protein synthesis and membrane transport in muscle.     

Induction of refractoriness to isoproterenol by prior treatment of C6-2B rat astrocytoma cells with cholera toxin.

Rat C6-2B astrocytoma cells responded to cholera toxin treatment with an 8-fold increase in intracellular cyclic AMP concentrations. Cyclic AMP levels began to rise 60--90 minutes after addition of the toxin and reached maximal concentrations in 3 hours. Cells exposed to cholera toxin and the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX), displayed an increase in cyclic AMP of 15-fold. The peak isoproterenol response was reduced 80--90% in cells previously treated with cholera toxin. Cholera toxin-induced refractoriness was time dependent and was not altered by concurrent treatment with propranolol. Prolonged exposure of the cells to isoproterenol reduced the cyclic AMP response to cholera toxin by 80%. MIX augmented both cholera toxin-induced refractoriness and isoproterenol-induced refractoriness. Cycloheximide inhibited the full development of refractoriness to both cholera toxin and isoproterenol. These results indicate that C6-2B cell refractoriness to cholera toxin is mediated by cyclic AMP and requires new protein synthesis. Refractoriness in C6-2B cells does not appear to be agonist-specific and probably involves a common locus of action on adenylate cyclase beyond that of the membrane receptors for cholera toxin and isoproterenol.     

Inhibition of cardiac slow action potentials by 8-bromo-cyclic GMP occurs independent of changes in cyclic AMP levels

Cyclic GMP inhibits the slow inward Ca current of cardiac cells. This effect could be due to a cyclic GMP-mediated phosphorylation of the Ca channel (or some protein modifying Ca channel activity), or alternatively, to enhanced degradation of cyclic AMP owing to stimulation of a phosphodiesterase by cyclic GMP. To test the latter possibility, we examined the effect of extracellular 8-bromo-cyclic GMP on cyclic AMP levels in guinea pig papillary muscles, in parallel with electrophysiological experiments. Isoproterenol (10(-6) M) significantly increased the cyclic AMP levels and induced Ca-dependent slow action potentials. Superfusion with 8-bromo-cyclic GMP (10(-3) M) inhibited the slow action potentials induced by isoproterenol. However, muscles superfused with 8-bromo-cyclic GMP had cyclic AMP levels identical to those of muscles superfused with isoproterenol alone. Similarly, 8-bromo-cyclic GMP had no effect on the increase in cyclic AMP levels of muscles treated with forskolin (10(-6) M) or histamine (10(-6) M). We conclude that the inhibitory effect of cyclic GMP on slow Ca channels in guinea pig ventricular cells is not due to a decrease in the cyclic AMP levels. We hypothesize that a cyclic GMP-mediated phosphorylation is the most likely explanation for the Ca channel inhibition observed in this preparation.     

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.