Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells.

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA. These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

The effect of cyclic GMP on phosphofructokinase from rat tissues.

In view of the recently proposed hypothesis of biologic regulation through opposing influences of cyclic AMP and cyclic GMP, and since cyclic AMP is a well-known allosteric activator of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the effect of cyclic GMP on the activity of this enzyme from several rat tissues was investigated. It was found that cyclic GMP exerted an inhibitory effect on the activity of rat heart and skeletal muscle phosphofructokinase. This effect was most pronounced under conditions in which the enzyme was partially inhibited by ATP or by citrate. Cyclic GMP also antagonized the deinhibitory action of cyclic AMP and other allosteric activators, such as glucose 1,6-bisphosphate or AMP, on the ATP or citrate-inhibited heart or muscle phosphofructokinase. In contrast to the heart and skeletal muscle phosphofructokinase, the adipose-tissue enzyme was not affected by cyclic GMP to any significant degree. The antagonistic action of cyclic GMP to the activation of heart-phosphofructokinase, may suggest a mechanism by which the activity of phosphofructokinase is synchronized with the activity of glycogen phosphorylase, as a result of acetylcholine action in heart, to achieve a decrease in total glycogenolysis and glycolysis.     

Glutamate release is involved in PAF-increased cyclic GMP levels in hippocampus

The involvement of glutamate in PAF-increased cyclic GMP levels was studied. Glutamate treatment caused a dose-response increase of cyclic GMP levels in hippocampal slices. The presence of 1 mM glutamate did not modify the effect caused by 10(-7)M PAF. To elucidate the involvement of glutamate in this action, slices were treated with PAF in the presence of MK-801, a NMDA receptor antagonist. Results indicate that PAF-increased cyclic GMP levels were obtained by NMDA receptors activation. Finally, results obtained from the experiments performed with PAF in the presence of riluzole, to inhibit the glutamate release, demonstrated that glutamate release is a stage in the PAF-induced increase of cyclic GMP levels in hippocampus.     

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.     

Mepacrine-induced inhibition of human platelet cyclic-GMP phosphodiesterase

The effect of mepacrine (DL-quinacrine-HCI), a specific inhibitor of phospholipase C, on cyclic-GMP levels in human platelets was investigated. The concentrations of mepacrine producing 50% inhibition of human platelet aggregation induced by 5 microM ADP and 3 micrograms/ml of collagen were 50 +/- 8 and 70 +/- 15 microM, respectively. Addition of mepacrine to human platelet suspension resulted in increases in cyclic GMP. In contrast to cyclic-GMP levels, cyclic-AMP content was not affected by mepacrine. Mepacrine did not stimulate guanylate cyclase, but did specifically inhibit human platelet cyclic-GMP phosphodiesterase, separated from cyclic-AMP phosphodiesterase or other forms of phosphodiesterase on DEAE-cellulose columns. Stimulation by cyclic GMP of human platelet cyclic-GMP-stimulated cyclic-AMP phosphodiesterase activity was not inhibited by mepacrine. The IC50 value of the drug for cyclic-GMP phosphodiesterase was 40 microM, and IC50 for cyclic-AMP phosphodiesterase was 1.2 mM. Mepacrine was 30-times more potent as an inhibitor of human platelet cyclic GMP than of cyclic-AMP phosphodiesterase. Mepacrine blocks arachidonate release from human platelets by inhibiting phosphatidylinositol-specific phospholipase C. The increase in cyclic-GMP levels produced by addition of mepacrine will explain part of the pharmacological action of this drug.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Cyclic GMP inhibits protein kinase C-mediated secretion in rat pancreatic acini

Dibutyryl cyclic GMP (Bu2cGMP) inhibited agonist-induced secretion of amylase from isolated rat pancreatic acini. In contrast to previous studies, this inhibitory action was not confined to butyryl derivatives of cyclic GMP, since the membrane-permeant cyclic GMP analogues Bu2cGMP and cyclic 8-bromo-GMP (8-Br-cGMP) were equipotent (IC50 2 nM) in their inhibition of amylase secretion stimulated by cholecystokinin-(26-33)-octapeptide (CCK8): at extracellular concentrations up to 1 mM, cyclic GMP itself was devoid of inhibitory activity. Both Bu2cGMP and 8-Br-cGMP also potently inhibited secretion stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) (IC50 6 nM), but only partially inhibited responses elicited by bethanechol or bombesin and were without effect on A23187-evoked secretion. Furthermore, agents that are known to raise intracellular cyclic GMP levels (MB22948 (2-o-propoxyphenyl-8-azapurin-6-one) or nitroprusside) or antagonize the actions of protein kinase C (4 alpha-PMA or staurosporine), also inhibited CCK8- or PMA-stimulated secretion but not secretion elicited by bombesin, bethanechol, or A23187. It is concluded from these and other observations reported here that protein kinase C is the major intracellular mediator of amylase secretion stimulated by CCK8 and that this pathway may be regulated by cyclic GMP at a step that follows protein kinase C activation.     

A cholera toxin substrate regulates cyclic GMP content of rat pinealocytes

The adrenergic regulation of cyclic GMP in isolated pinealocytes was investigated. In this cell, norepinephrine stimulates cyclic GMP and cyclic AMP greater than 100-fold by activating both alpha 1- and beta-adrenoceptors. beta-Adrenergic activation is a requisite event and is potentiated by alpha 1-adrenergic activation (Vanecek, J., Sugden, D., Weller, J. L., and Klein, D. C. (1985) Endocrinology 116, 2167-2173). The current study found that cholera toxin could substitute for beta-adrenergic agonists in stimulating pinealocyte cyclic GMP content, as has been found to be the case for cyclic AMP. Treatment with cholera toxin alone (1 microgram/ml for 90 min) had a small effect (2- to 4-fold increase) on cyclic GMP; addition of the alpha 1-adrenergic agonists, phenylephrine, cirazoline, or methoxamine to cholera toxin-treated cells rapidly (peak at 5 min) caused a further 30- to 300-fold increase. The alpha 1-adrenergic agonists had little effect by themselves at concentrations which potentiated the effects of cholera toxin. The potentiating effect of phenylephrine was inhibited nearly completely by an alpha 1-adrenergic antagonist, but not by either an alpha 2- or beta-adrenergic antagonist. The purified cholera toxin subunits A and B did not stimulate cyclic GMP either alone or in the presence of phenylephrine. Furthermore, the potentiating action of phenylephrine was observed following 90 min but not 20 min of cholera toxin pretreatment. these results suggest that the regulation of cyclic GMP levels in the pineal gland involves an Ns-like GTP-binding regulatory protein. This is of interest because it is the first indication that cyclic GMP is regulated by such a GTP-binding protein in nonretinal tissue. It remains to be determined whether the mechanisms involved in the transmembrane regulation of cyclic AMP and cyclic GMP in any other tissue are similar.     

8-Bromo-cyclic GMP inhibits the calcium channel current in embryonic chick ventricular myocytes

Superfusion with 8-bromo-cyclic GMP or intracellular injection of cyclic GMP inhibits calcium-dependent slow action potentials in embryonic chick or guinea pig ventricular cells, suggesting that cyclic GMP inhibits calcium currents. Recently, cyclic GMP has been shown to reduce cyclic AMP-stimulated calcium currents in voltage-clamped ventricular myocytes. Since earlier results in intact cells had suggested that cyclic GMP might inhibit basal (i.e., unstimulated by cyclic AMP) calcium currents, we directly investigated the effect of 8-bromo-cyclic GMP on basal calcium channel currents (using barium as the charge carrier) in voltage-clamped ventricular myocytes isolated from embryonic chick hearts. Superfusion with 1 mM 8-bromo-cyclic GMP (without prior cyclic AMP elevation) progressively decreased peak calcium channel currents (-68% at 15 min after the onset of drug exposure). In contrast, the currents were unchanged during 15 min superfusion with control solution, or 1 mM 8-bromo-GMP (the noncyclic inactive analog of 8-bromo-cyclic GMP). The present results in voltage-clamped embryonic chick heart cells indicate that cyclic GMP can inhibit basal calcium channel currents, apparently through a cyclic AMP-independent mechanism.     

In vitro immune response of human peripheral lymphocytes. III. Effect of anti-mu or anti-delta antibody on PWM-induced increase of cyclic nucleotides in human B lymphocytes.

Stimulation of human peripheral blood lymphocytes (PBL) with pokeweed mitogen (PWM) induced consistent increases of intracellular levels of cyclic AMP and cyclic GMP within 15 min. Increases of cyclic AMP were observed in both B and T lymphocyte populations, but increase of cyclic GMP was observed only in the B lymphocyte population. The addition of anti-mu antibody to B cells abolished PWM-induced increase of cyclic GMP without any effect on cyclic AMP response. Anti-delta antibody did not show any inhibitory or stimulatory effect on PWM-induced increase of cyclic GMP or cyclic AMP. Pretreatment of B cells with anti-mu antibody at 37 degrees C for 1 hr inhibited PWM-induced increase of cyclic GMP, whereas pretreatment with anti-mu antibody at 4 degrees C did not show any inhibitory effect on PWM-induced increase of cyclic GMP. The effect of anti-mu-pretreatment was reversible and pretreated cells were recovered from the inhibitory effect of anti-mu antibody after 36 hr culture.     

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.     

Effects of pyruvate and other metabolites on cyclic GMP levels in incubations of rat hepatocytes and kidney cortex

Pyruvate increased cyclic GMP levels in rat hepatocytes. The effects were observed without or with 1-methyl-3-isobutylxanthine. Lactate, acetate, oxaloacetate, alpha-ketoglutarate, succinate, acetoacetate and beta-hydroxybutyrate also increased cyclic GMP levels. Some compounds increased cyclic GMP in kidney cortex slices. The effects were dependent upon Ca2+ in the medium. Cyclic AMP was increased 30-50% by some of these substances with 2.6 mM Ca2+. Rotenone, oligomycin, antimycin, dinitrophenol, KCN, and arsenate decreased GTP and ATP, basal cyclic GMP and the pyruvate effect, but did not alter cyclic AMP. Although fluoroacetate alone had no effect on cyclic nucleotides, GTP, or ATP, it potentiated the pyruvate effect on cyclic GMP. Adenosine and guanosine increased cyclic GMP and GTP to a similar extent of 30-50%. Aminooxyacetate, cycloserine, pentenoic acid and mepacrine decreased the pyruvate effect while cycloserine or mepacrine alone increased cyclic GMP. Citrate and mepacrine inhibited soluble and particulate guanylate cyclase from rat liver while cycloserine and acetoacetate increased guanylate cyclase activity. None of the other compounds altered guanylate cyclase activity. These results indicate that various metabolites and inhibitors can alter cyclic GMP accumulation in hepatocytes and renal cortex slices. Several mechanisms may be involved in these effects.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Mechanisms of Cyclic AMP Regulation in Cerebral Anoxia and Their Relationship to Glycogenolysis

Abstract: Anoxia elevates levels of cyclic AMP and depresses levels of cyclic GMP in cerebral cortex of mice. Similar effects are also observed in other regions of the brain. Aminophylline inhibits accumulation of cyclic AMP about 50% in hippocampus and cerebellum, but not in cerebral cortex and striaturn; however, this effect requires high doses (²⁵⁰ mgikg). Pretreatment of animals with reserpine, which depletes brain stores of norepinephrine, dopamine, and serotonin, and also produces sedation and mild hypothermia, markedly inhibits accumulation of cyclic AMP in all regions of anoxic brain. Destruction of norepinephrine terminals by treatment of neonatal animals with ⁶-OH- dopamine, which does not sedate or produce hypothermia, has an effect on cyclic AMP levels similar to that of reserpine. None of the above treatments modifies the effect of anoxia on cyclic GMP levels. These data indicate that norepinephrine is a major regulator of cyclic AMP levels in anoxic brain and that adenosine and, perhaps, other unidentified substances have lesser roles in this process. In contrast, biogenic amines and adenosine appear to have no effect on cyclic GMP regulation in anoxic brain. Reserpine slows the activation of phosphorylase and the utilization of ATP, and slightly attenuates the breakdown of glycogen caused by anoxia, but has no effect on the changes in glucose, lactate, or phosphocreatine. In contrast, ⁶-OH-dopamine has no effect on any of these anoxiainduced changes. It is concluded that the effect of reserpine on phosphorylase, glycogen, and ATP is most likely related to the hypothermic and sedative effect of the drug, and that either cyclic AMP is not responsible for initiating glycogenolysis in anoxic brain or only a small rise in cyclic AMP levels is necessary for this process.     

Cyclic guanosine 3':5'-monophosphate and the regulation of lipolysis in rat fat cells.

The addition of the divalent cation ionophore A23187, carbachol, norepinephrine or insulin to rat fat cells elevated cyclic GMP. The increase in cyclic GMP due to these agents was greater at 4 than at 2 minutes after their addition. Cyclic GMP accumulation was also elevated by the addition of 0.1 to 0.5 mM sodium oleate in the presence of 0.1% albumin and by albumin containing added palmitate with an FFA/albumin molar ratio of 6.7. The rise in cyclic GMP due to all agents was markedly reduced in calcium-free buffer. The effects of the various agents on cyclic GMP accumulation in rat fat cells had little correlation with lipolysis. Insulin was an effective anti-lipolytic agent in both the presence and absence of calcium while neither A23187 nor carbachol had any effect on fat cell lipolysis.     

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.     

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart.

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.     

Atriopeptin II elevates cyclic GMP, activates cyclic GMP-dependent protein kinase and causes relaxation in rat thoracic aorta

Synthetic atriopeptin II, an atrial natriuretic factor with potent vasodilatory effects, was studied in isolated strips of rat thoracic aorta to determine its actions on contractility, cyclic nucleotide concentrations and endogenous activity of cyclic nucleotide-dependent protein kinases. Atriopeptin II was found to relax aortic strips precontracted with 0.3 microM norepinephrine whether or not the endothelial layer was present. Relaxation to atriopeptin II was closely correlated in a time- and concentration-dependent manner with increases in cyclic GMP concentrations and activation of cyclic GMP-dependent protein kinase (cyclic GMP-kinase). The threshold concentration for all three effects was 1 nM. Atriopeptin II (10 nM for 10 min) produced an 80% relaxation, an 8-fold increase in cyclic GMP concentrations and a 2-fold increase in cyclic GMP-kinase activity ratios. Atriopeptin II did not significantly alter cyclic AMP concentrations or cyclic AMP-dependent protein kinase activity. These data suggest that cyclic GMP and cyclic GMP-kinase may mediate vascular relaxation to a new class of vasoactive agents, the atrial natriuretic factors. Similar effects have been observed with the nitrovasodilator, sodium nitroprusside, and the endothelium-dependent vasodilator, acetylcholine. Therefore, a common biochemical mechanism of action that includes cyclic GMP accumulation and activation of cyclic GMP-kinase may be involved in vascular relaxation to nitrovasodilators, endothelium-dependent vasodilators and atrial natriuretic factors.