Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.     

Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues.

Three agents that activate guanylate cyclase, sodium nitroprusside, nitroglycerin and sodium axide, were examined for their effects on cyclic GMP and cyclic AMP accumulation and muscle motility with several tissues. All of these agents, except nitroglycerin with ventricle preparations, increased cyclic GMP levels and did not alter cyclic AMP in incubations of preparations of bovine tracheal smooth muscle, guinea pig tracheal chains, taenia cecum, atria and ventricle, and rat liver and cerebral cortex. Increases in cyclic GMP with these agents occurred with relaxation of smooth muscle preparations and without alteration in the contractility of atrial preparations. These observations support the hypothesis that cyclic GMP accumulation in smooth muscle may be related to relaxation rather than contraction as proposed previously. Relaxation with these agents is not associated with alterations in cyclic AMP levels. Increases in cyclic GMP levels in atrial preparations can also occur without changes in contractile force or rate of contraction.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

Effects of LY83583, nordihydroguaiaretic acid, and quinacrine on cyclic GMP elevation and inhibition of tension by muscarinic agonists in rabbit aorta and left atrium

Elevation of cyclic GMP by muscarinic agonists has been suggested to be responsible for the negative inotropic effects of these agents in cardiac muscle, and for the endothelium-dependent relaxation caused by these agents in vascular smooth muscle. These relationships were studied by monitoring the effects of muscarinic agonists on tension and cyclic GMP levels in rabbit left atrial strips and aortic rings, in the presence and absence of the cyclic GMP lowering agent, LY83583. LY83583 completely blocked both the cyclic GMP increase and the relaxation caused by acetylcholine in rabbit aortic rings with intact endothelial cells. Acetylcholine-induced cyclic GMP elevation and relaxation in these preparations were also blocked by quinacrine and nordihydroguaiaretic acid (NDGA), but neither response was blocked by the 5-lipoxygenase inhibitor U-60257. In the experiments with rabbit left atrium, LY83583 blocked the acetylcholine-induced cyclic GMP elevation but did not block the negative inotropic effects of the drug. Quinacrine, NDGA, and a guanylate cyclase inhibitor, methylene blue, failed to block either the cyclic GMP increase or the decrease in contractile force caused by carbachol in atrial strips. These results support the suggestion that an increase in cyclic GMP may be responsible for the endothelium-dependent relaxation of rabbit aorta by muscarinic agonists, but not for the direct negative inotropic effects of these drugs in rabbit atrium. Muscarinic agents appear to increase cyclic GMP levels in rabbit atrium and aorta by different mechanisms. Although both are blocked by LY83583, they differ not only in their requirements for endothelial cells, but also in their susceptibility to other blocking agents.     

Bidirectional regulation of lysosomal enzyme secretion and phagocytosis in human neutrophils by guanosine 3',5'-monophosphate and adenosine 3',5'-monophosphate.

The biologic roles of guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neurtrophils were studied. Contact between neurophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular caclium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects of cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.     

Ascorbic acid modulation of splenic cell cyclic GMP metabolism.

Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells $\text{in vitro}$ restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H₂O₂ production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component.     

The involvement of prostaglandin endoperoxide formation in the elevation of cyclic GMP levels during platelet aggregation.

Arachidonic acid- or collagen-induced aggregation was accompanied by a progressive elevation in the level of cyclic GMP in washed human platelets with no significant alteration in the concentration of cyclic AMP. The extent of the increase in cyclic GMP was proportional to the concentration of arachidonic acid added. Enhanced accumulation of cyclic GMP produced by arachidonic or collagen was prevented by prior exposure of platelets to aspirin or indomethacin. Prostaglandin endoperoxide G2 caused platelet aggregation and an increase in cyclic GMP concentration; neither event was blocked by prostaglandin synthesis inhibitors. These results indicate that the generation of prostaglandin endoperoxides is a step in the sequence of events in platelet aggregation leading to the enhanced accumulation of cyclic GMP.     

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

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.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

Histamine release from human leukocytes: relationships between cyclic nucleotide, calcium, and antigen concentrations.

The antigen-induced IgE-mediated release of histamine from human basophils has previously been shown to require calcium, to be inhibited by agents which raise cyclic AMP levels and by high antigen levels, and to be unaffected by cyclic GMP. The interrelationship between these phenomena has been studied. The major findings are: 1) in the region of antigen-excess inhibition dibutyryl cyclic AMP potentiates release; 2) antigen-excess inhibition is seen at lower antigen concentrations when the calcium concentration is reduced from 0.6 to 0.1 mM; and 3) cyclic GMP modestly potentiates release when the calcium concentration is 0.1 mM.     

Adrenocortical cyclic GMP-dependent protein kinase: purification, characterization, and modification of its activity by calmodulin, and its relationship with steroidogenesis

Cyclic GMP-dependent protein kinase has been purified to apparent homogeneity from bovine adrenal cortex and its presence in the rat adrenal cortex has been demonstrated. Sucrose density sedimentation studies indicated that the M_r of the enzyme was 145,000. This protein was composed to two identical subunits each with M_r of 75,000. The enzyme molecule was asymmetric with a frictional coefficient of 1.54, Stokes radius of 53.5 Å and a sedimentation coefficient of 6.5. The enzyme self-phosphorylated and the stoichiometry of cyclic GMP binding was two molecules per holoenzyme. Calmodulin or troponin C markedly stimulated the apparent maximal velocity of cyclic GMP-dependent protein kinase without affecting its basal activity. This effect of protein modulators was independent of calcium. Sucrose density gradient studies indicated that the stimulatory effect of calmodulin was due to its interaction with histones. An interaction of calmodulin with the enzyme was not observed. The steroidogenic potential of cyclic GMP and its analogs correlated closely with their ability to stimulate cyclic GMP-dependent protein kinase; the order of potency for both activities was 8-bromocylic GMP > cyclic GMP > N²-monobutyryl cyclic GMP > N², O²-dibutyryl cyclic GMP. In each case, calmodulin enhanced the cyclic GMP-dependent protein kinase activity for histone phosphorylation. These results indicate that although cyclic GMP is the primary regulator of cyclic GMP-dependent protein kinase, other modulator proteins such as calmodulin could act as additional regulators of the phosphorylation of substrate proteins. In addition, the demonstration of cyclic GMP-dependent protein kinase in rat adrenal glands, and the results with cyclic GMP and its analogs relating to their activation of protein kinase and steroidogenesis are consistant with the concept that cyclic GMP is one of the mediators of adrenal steroidogenesis.     

Atrial natriuretic factor-induced egression of cyclic guanosine 3':5'-monophosphate in cultured vascular smooth muscle and endothelial cells

The induction of cyclic GMP formation in target tissues, i.e. vascular smooth muscle and endothelial cells, by atrial natriuretic factor is followed by its egression into plasma and urine. Since the extracellular appearance of this cyclic nucleotide is used as a marker of atrial natriuretic factor's biological activity, the present study was designed to investigate the characteristics of its egression to the extracellular fluid. In contrast to cyclic AMP, whose profile of egression in time closely follows its intracellular levels, cyclic GMP egression begins with the intracellular decline but continues for a prolonged period, even accumulating for up to 24 h in the extracellular medium. The relative egression of cyclic GMP decreases slightly in the presence of phosphodiesterase inhibitors. On the other hand, the process is sensitive to temperature, inhibited by such agents as probenecid, and occurs against a gradient of 7 orders of magnitude. Large increases of cyclic AMP, as induced by forskolin, can effectively compete for the cyclic GMP transport system, resulting in a 3-fold rise in intracellular cyclic GMP levels, which corresponds to a 3-fold decline of extracellular accumulation. Although the biological significance of cyclic GMP egression is unknown, the results of this study suggest that the process may be one of the significant contributors to the control of cyclic GMP levels in the cell with potential physiological consequences.     

A possible role for guanosine 3',5'-monophosphate in the stimulus-secretion coupling in exocrine pancreas.

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, CL. and Krishna, G. (1977) Science 196, 1003--1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium. There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 micrometer concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

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.     

Regulation by beta-adrenergic receptor and muscarinic cholinergic receptor activation of intracellular cyclic AMP and cyclic GMP levels in rat lung slices

The effects of adrenergic and cholinergic agents, present singly or in combination, on the levels of cyclic AMP and cyclic GMP in slices of rat lung were studied. It was found that isoproterenol increased pulmonary cyclic AMP levels about 3-fold, and this increase was abolished by propranolol, but not by phenoxybenzamine. Acetylcholine increased the cyclic GMP levels also about 3-fold (thus raising its tissue content above that of cyclic AMP), and this increment was largely reduced by atropine, but not by hexamethonium. While without effects on the cyclic GMP levels when present alone, isoproterenol antagonized acetylcholine in increasing cyclic GMP levels. Acetylcholine, while lacking effects on the basal levels of cyclic AMP, on the other hand, depressed the augmented levels caused by isoproterenol.The data presented indicate that cyclic GMP may mediate the cholinergic action in lung and that the pulmonary cyclic GMP levels are also closely regulated by β-adrenergic receptor activation.