Significant Increase In Cyclic AMP Research Articles

Interactive inhibitory effects of formoterol and montelukast on activated human neutrophils

The research question addressed in the current study was: do formoterol (1 and 10 nM) and montelukast (2 μM) possess interactive inhibitory effects on activated human neutrophils, particularly in relation to alterations in cyclic AMP and cytosolic Ca²(+) fluxes? Isolated human blood neutrophils were activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 μM) in combination with cytochalasin B (CB; 3 μM). Fura-2-acetoxymethyl ester-based spectrofluorimetry, lucigenin-enhanced chemiluminescence, colorimetric and flow cytometric procedures were used to measure cytosolic Ca²(+) fluxes, production of superoxide, elastase release and beta-2 integrin (CR3) expression, respectively, while cyclic AMP and leukotriene (LT)B₄ were assayed using competitive binding ELISA procedures. Activation of the cells with fMLP/CB resulted in abrupt and sustained increases in cytosolic Ca²(+), as well as release of elastase and production of superoxide and LTB₄, and expression of CR3, all of which were attenuated by formoterol and montelukast individually, and especially by the combination of these agents. These anti-inflammatory effects of each agent, as well as the combination, were associated with significant increases in cyclic AMP. The findings of the current study may explain the efficacy of montelukast and formoterol when used in combination with inhaled corticosteroids in the treatment of severe asthma, possibly by controlling neutrophil-driven inflammation of the airways.

Possible Existence of Novel Endothelium-Derived Relaxing Factor in the Endothelium of Rat Mesenteric Arterial Bed

Both acetylcholine (ACh) and cyclopiazonic acid (CPA) caused vasodilation of the mesenteric arterial bed in a concentration-dependent manner. When the mesenteric arterial bed was perfused with 0.1% Triton X-100 for 30 s, ACh- or CPA-induced vasodilation was almost abolished. ACh-induced vasodilation was significantly attenuated in isotonic high K+ (60 mM) solution and significantly decreased by treatment with methylene blue (MB) with NG-nitro-L-arginine (L-NNA) in isotonic high K+ (60 mM) solution, whereas CPA-induced vasodilation of the mesentery was not affected by these treatments. ACh- or CPA-induced vasodilation was not affected by indomethacin. ACh caused significant increase in cyclic GMP levels and cyclic AMP in effluents from the perfused mesentery, whereas CPA could not increase cyclic GMP. CPA caused significant increase in cyclic AMP in a concentration-dependent manner, and CPA-induced increase in cyclic AMP was completely inhibited by removal of the endothelium. These results suggest that one or more endothelium-derived relaxing factor (EDRF) or factors should exist other than endothelium-derived nitric oxide (EDNO) or endothelium-derived hyperpolarizing factor (EDHF) in the endothelium of the rat mesenteric arterial bed. The novel EDRF may relax the mesenteric arterial bed through production of cyclic AMP but not cyclic GMP.

Effects of timolol, terbutaline and forskolin on IOP, aqueous humour formation and ciliary cyclic AMP levels in the bovine eye.

We have studied the effects of terbutaline, timolol, forskolin and 8-bromo cyclic AMP on aqueous humour formation, intraocular pressure and on ciliary epithelial cyclic AMP levels, either in presence or in absence of IBMX, using the bovine isolated arterially perfused eye, excised ciliary processes and cultured ciliary epithelium. Both terbutaline, a beta-adrenoceptor agonist, and timolol, a beta-adrenoceptor antagonist, caused significant reduction in aqueous humour formation and intraocular pressure but produced no effect on ciliary epithelial cyclic AMP content in the absence of IBMX. Even a three times higher dose of terbutaline was entirely ineffective in producing any effect on ciliary cyclic AMP in the perfused eye. On the other hand, terbutaline at the IOP-reducing dose, produced a significant increase in cyclic AMP when injected after 30 min perfusion with IBMX. Incubation of excised ciliary processes or cultured ciliary epithelial cells with terbutaline (10(-6) to 10(-4) M) produced concentration-dependent increases in cyclic AMP, in both tissues, even in the absence of IBMX. Forskolin, which stimulates cyclic AMP synthesis without interacting with cell surface receptors, was found to produce highly significant increases in ciliary cyclic AMP content both in presence and in absence of IBMX but had no effect on aqueous humour formation in the isolated eye. IBMX perfused at concentrations of 1 mM or 10 microM had no effect on basal levels of ciliary cyclic AMP but the 1 mM concentration produced a marked and significant reduction in IOP. Direct application of 8-bromo cyclic AMP, a cell permeable analogue, more resistant to hydrolysis by phosphodiesterases, had also no effect on aqueous humour formation in the perfused eye. It is concluded that in the bovine arterially perfused eye, the correlation between the aqueous humour formation rate and ciliary epithelial cyclic AMP content is unclear.

Effect of flosequinan on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content in the anaesthetized rat.

1. Flosequinan, milrinone, isoprenaline and forskolin given intravenously at similarly hypotensive doses have been evaluated in separate studies for their effect on ischaemia-induced arrhythmias and on ventricular cyclic nucleotide content following coronary artery ligation in the pentobarbitone anaesthetized rat. 2. Flosequinan did not affect mortality or arrhythmias following coronary artery ligation in either study and no change in ventricular cyclic nucleotide content was observed. 3. Isoprenaline caused a significant increase in mortality (P < 0.05) in both studies whereas milrinone and forskolin caused a significant increase in mortality in only one of the two studies conducted. All three agents caused significant increases in cyclic AMP which were associated with increased incidence of arrhythmias. 4. When compared at similarly hypotensive doses, flosequinan, in contrast to milrinone, isoprenaline and forskolin, did not influence ischaemia-induced arrhythmias or raise ventricular cyclic nucleotide levels in the anesthetized rat.

Studies on the mechanism of cytotoxicities of polyacetylenes against L1210 cell

This study was performed to investigate the mechanism ofin vitro cytotoxic actions of polyacetylenes which are panaxydol, panaxynol and panaxytriol isolated fromPanax ginseng C.A. Meyer. DNA synthesis of L1210 cells was significantly inhibited with dose-dependent pattern when L1210 cells were treated for 1 hour with over 5 μg/ml of polyacetylenes. Panaxydol which had the most potent cytotoxicity among three polyacetylenes showed also the strongest inhibitory effect on DNA synthesis. Intracellular cyclic AMP levels of L1210 cells treated with 2.5 μg/ml of panaxydol or panaxytriol were significantly elevated on the incubation duration. The elevation of cyclic AMP levels by panaxytriol was higher than that by panaxydol, but no significant increase in cyclic AMP by panaxynol was observed. All three polyacetylenes had no effect on glycolysis of L1210 cells. Electron microscopic observations revealed that polyacetylenes caused damage to plasma membranes of L1210 cells in proportion to their cytotoxicities at each ED50 value (panaxydol>panaxynol>panaxytriol). These results suggest that cytotoxicities of polyacetylenes against L1210 cells might be mediated by elevated cyclic AMP level, even though the relationship among their cytotoxicities, inhibitory effect on DNA synthesis and ability to elevation of cyclic AMP level are not fully agreed, and might be also related to membrane damage.

The effects of aluminum on agonist-induced alterations in cyclic AMP and cyclic GMP concentrations in rat brain regions in vivo

Aluminum administered intracerebroventricularly (icv) (1 μmol) caused a significant decrease in cyclic GMP in the cortex after 3 days and a significant increase in cyclic AMP in the cortex after 14 days. Pilocarpine administration to untreated rats elevated cyclic AMP and cyclic GMP levels in specific brain regions. These pilocarpine-induced increases in the cyclic nucleotide concentrations were significantly attenuated in rats that had been treated with aluminum 14 days previously. Isoproterenol administration to control rats did not alter cyclic AMP concentrations; however, cyclic AMP concentrations were significantly reduced in the cortex of aluminum-treated animals after isoproterenol administration. Apomorphine elevated cyclic GMP concentrations in the cerebellum, hippocampus, and striatum of naive rats. This apomorphine-induced elevation in cyclic GMP concentrations was significantly potentiated in aluminum-treated rats.These results indicate that: (1) cyclic AMP synthesis in the cortex is most sensitive to aluminum; (2) agonist-stimulated changes in cyclic nucleotide concentrations can be altered by pretreatment with aluminum; (3) effects of aluminum persist for at least 2 weeks after central administration; and (4) modulation of the metabolism of cyclic nucleotides may play a role in the neurotoxic effects of aluminum.

Effects of prenalterol on beta adrenergic responsiveness and receptors in the cerebral cortex of the rat

The activity of the beta-adrenoceptor agonist, prenalterol, at beta adrenoceptors in the cerebral cortex of the rat and the effect of chronic intraperitoneal infusion of prenalterol on the biochemical responsiveness and density of cerebral cortical beta adrenoceptors was studied. Whereas isoproterenol caused a four-fold rise in the content of cyclic AMP in slices of cerebral cortex, prenalterol did not produce a significant increase in cyclic AMP. However, prenalterol inhibited the isoproterenol-stimulated increase in cyclic AMP in cortical slices in a concentration-dependent manner. Using an in vivo binding technique, prenalterol (3.8 mg/kg/hr) infused intraperitoneally through osmotic minipumps, penetrated the brain and significantly inhibited the binding of the beta adrenoceptor antagonist, [ 125I]iodopindolol ( 125I-IPIN), to cortical beta adrenoceptors. Infusion of prenalterol (3.8 mg/kg/hr) for 7 days resulted in a small (20%), but significant, reduction in the ability of isoproterenol to stimulate maximally the accumulation of cyclic AMP in slices of cerebral cortex. No alteration in the B max or K D of the binding of [ 125I]iodopindolol was observed in homogenates of cortex obtained from prenalterol-treated rats. Furthermore, no change was observed in the binding of the hydrophilic ligand [ 3H]CGP-12177 in homogenates of cortex. In this study, then, prenalterol exhibited properties in vitro of a beta adrenoceptor antagonist, but did cause modest desensitization of beta adrenoceptor responsiveness when administered continuously in vivo.

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A series of xanthine derivatives and papaverine have been studied to determine their abilities to alter tissue levels of cyclic AMP and cyclic GMP, inhibit phosphodiesterase activities, and cause relaxation of pig coronary arteries. The agents selected for this study exhibited a wide range of potencies to inhibit phosphodiesterase activities in the coronary artery supernatant fraction. In addition, some of these agents were up to 10 times more potent as inhibitors of cyclic GMP hydrolysis than of cyclic AMP hydrolysis, while others were 2-4 times more potent as inhibitors of cyclic AMP than of cyclic GMP hydrolysis. The rank order of potencies of these agents to cause relaxation of coronary artery strips was similar to the rank order of potencies to inhibit cyclic nucleotide phosphodiesterase activities. There were, however, some notable exceptions to the correlation between inhibition of cyclic nucleotide phosphodiesterase activities and relaxation. 1-Isoamyl-3-isobutylxanthine was a more potent relaxing agent than might be expected from its relatively low potency to inhibit cyclic nucleotide hydrolysis in tissue extracts. On the other hand, 1-methyl-3-isobutyl-7-(3-chlorobenzyl)-xanthine was one of the more potent inhibitors of cyclic nucleotide hydrolysis but was not as potent in causing relaxation as might have been expected. Exposure of the coronary artery strips to inhibitors caused increases in tissue levels of cyclic AMP and cyclic GMP and there was a statistically significant multiple linear regression of cyclic AMP and cyclic GMP levels on percent relaxation after 5 min of exposure to the agents. Cyclic AMP and cyclic GMP levels made approximately equal contributions to the regression of changes in percent relaxation, as determined by analysis of variance methods. While 1-isoamyl-3-isobutylxanthine did not fit the correlation between phosphodiesterase inhibition and potency to relax the arterial strips as well as the other agents, this agent also was found to cause unexpectedly large increases in cyclic AMP levels. Some agents caused relaxation accompanied by significant elevation of cyclic GMP levels and no significant change in cyclic AMP levels while other agents caused relaxation accompanied by significant increases in cyclic AMP but not cyclic GMP. These data offer some support for a hypothesis that both cyclic AMP and cyclic GMP are involved in the relaxation processes of pig coronary arteries.

The effects of VIP on cyclic AMP and glycogen levels in vertebrate retina

The effects of VIP and related-peptides (PHI, secretin, glucagon) on cyclic AMP formation were investigated in intact pieces of rabbit retina. VIP and PHI increased cyclic AMP levels with EC 50 of 160 nM and 300 nM respectively. At 5 μM the peptides increased cyclic AMP 46 fold (VIP) and 38 fold (PHI). Secretin was much less potent and glucagon was totally inactive. VIP was also tested for its effects on glycogen levels under similar experimental conditions. In contrast to its pronounced glycogenolytic action in mouse cerebral cortical slices, VIP at 1 μM decreased only moderately (38.3%) 3H-glycogen newly synthesized from 3H-glucose by pieces of rabbit retina. Furthermore a discrepancy between the efficacy of VIP in increasing cyclic AMP and in promoting glycogenolysis appears to exist. A similar dissociation between these two cellular events was also observed with other neuroactive substances. Thus the pronounced increase in cyclic AMP induced by dopamine and forskolin was accompanied by only a moderate decrease in 3H-glycogen levels. Conversely 50 mM potassium induced a 79.9% decrease in 3H-glycogen levels without any significant increase in cyclic AMP.

The activation of Na+-dependent efflux of Ca2+ from liver mitochondria by glucagon and beta-adrenergic agonists.

The Na+-induced efflux of Ca2+ from liver mitochondria was activated by tissue pretreatment with 1 microM-adrenaline, 1 microM-isoprenaline, 10 nM-glucagon and 100 microM-cyclic AMP when 10 mM-lactate plus 1 mM-pyruvate were present in the perfusion medium. Infusion of the alpha 1-adrenergic agonist, phenylephrine (10 microM), was ineffective. The activation induced by the beta-adrenergic agonist, isoprenaline, was maximal after infusion of agonist for 2 min. The isoprenaline-induced activation was very marked (120-220%), with about 7 nmol of intramitochondrial Ca2+/mg of protein, but was not evident with greater than 15 nmol of Ca2+/mg. Ca2+ efflux in the absence of Na+ and in the presence of the Ca2+ ionophore A23187 was not affected by isoprenaline pretreatment over the range 6-23 nmol of internal Ca2+/mg. With 10 mM-lactate plus 1 mM-pyruvate in the perfusion medium, glucagon and isoprenaline infusion increased tissue cyclic AMP content about 8-fold and 3-fold respectively. With 10 mM-pyruvate alone, neither glucagon nor isoprenaline caused a significant increase in cyclic AMP. Omission of lactate also abolished the ability of glucagon, but not of isoprenaline, to activate the Na+-induced efflux of Ca2+. The data indicate that cyclic AMP may mediate the activation caused by glucagon, but provide no evidence that cyclic AMP is an obligatory link in the beta-adrenergic-induced activation.

Different effects of selenium on cyclic AMP metabolism in hepatoma cells and normal liver cells

Sodium selenite administered to normal and hepatoma (HA)-bearing mice sc (2 mg/kg) or ip (1 mg/kg) led to a significant increase in cyclic AMP (cAMP) level and a decrease in cAMP phosphodiesterase (PDE) activity in HA cells. In contrast, in tumor host liver and normal liver the cAMP level was reduced, and the PDE activity was slightly elevated, whereas the cAMP adenylate cyclase (AC) was little affected by Na2SeO3, if at all. These results imply that selenite-induced changes in PDE activity play a decisive role in regulating intracellular cAMP level. Kinetic studies revealed that there were different forms of PDE. TheK m value of PDE isozymes in normal liver and host liver were identical, but differed from those of HA. It seems likely that the selective responsiveness of PDE to selenite may be related to the difference in PDE isozyme patterns. Addition of DBcAMP to the culture medium resulted in an inhibition of(3)H-thymidine incorporation into hepatoma cells, indicating that the inhibition of HA cell proliferation was cAMP-mediated. Thus, selenium has been shown to exert a selective effect on cAMP metabolism of hepatoma cells, which may account for its inhibitory effects on cancer cells.

Rabbit atrial histamine receptors and cyclic AMP.

The present study confirms the presence of H2 receptors in rabbit right and left atrium by measuring changes in cyclic AMP. Histamine produced significant increases in cyclic AMP in both atria although the increases were less in left atria. Since H2 receptors are always associated with the adenylate cyclase--cyclic AMP system, the data provide further support for the suggestion that H2 receptors exit in rabbit right and left atria.

Radiation Effects on Cyclic AMP, Cyclic GMP, and Amino Acid Levels in the CSF of the Primate

Silastic Pudenz catheters were chronically implanted in the fourth ventricle of cynomolgus monkeys and were connected to compressible polyethylene reservoirs placed subcutaneously over the occiput. Cerebrospinal fluid (CSF) could be aseptically aspirated from the reservoir in the awake animal. Prior to irradiation, baseline samples were taken after repeated reservoir pumping to ensure good mixing with the CSF in the ventricle. The heads of the animals were then exposed to bremsstrahlung radiation and CSF samples were taken at predetermined times postirradiation. Significant increases in cyclic AMP and cyclic GMP levels were observed following irradiation. An analysis of brain areas obtained by biopsy of irradiated animals showed significant decreases in only the cerebellar cyclic AMP and cyclic GMP. No appreciable changes were found in the CSF amino acid composition.

Effect of histamine on adenosine 3', 5'-Cyclic monophosphate levels in granulation tissue

The effect of histamine on cyclic AMP levels in rat granulation tissue was examined in vivo and in vitro. Granulomas formed by subcutaneous implantation of formalin-soaked filter paper disks were used as granulation tissue. Histamine injection (5 mg/kg, i.p.) produced a significant increase in the cyclic AMP levels for 10–30 min after injection. Administration of either burimamide or mepyramine at 1 mg/kg, s.c., 30 min before histamine administration blocked the cyclic AMP increase induced by 5 mg/kg histamine. Burimamide was more effective than mepyramine. Neither of these histamine antagonists at 1 mg/kg blocked the cyclic AMP increase induced by adrenaline (1 mg/kg, i.p.). Propranolol at 1 mg/kg, s.c., reversed the cyclic AMP increase due to adrenaline but partially blocked a similar effect of histamine. Histamine (1 mM and less), added alone to the incubation medium in which the chopped granulation tissue was immersed, had little or no effect on cyclic AMP levels in the granulation tissue. However, when histamine (1 and 10 mM) was added concomitantly with 3-isobutyl-1-methylxanthine (IBMX) (1 mM), a significant increase in cyclic AMP occured, compared to the addition of IBMX alone. IBMX (1 mM) alone caused a significant increase in cyclic AMP compared to controls in which the drug was omitted. 2-Methylhistamine (5 mM) induced a cyclic AMP increase in the presence of IBMX (1 mM). Metiamide (0.01 mM) partially blocked the cyclic AMP increase caused by histamine (1 mM) in the presence of IBMX (1 mM). Mepyramine (0.01 mM) completely blocked this histamine effect. Propranolol (0.01 mM) markedly inhibited the effect of histamine(1 mM). These data suggest that histamine causes the cyclic AMP increase partly by the activation of H 2-receptors on the cells of granulation tissue and partly through the β-receptor action of catecholamines released, although the involvement of H 1-receptors cannot be excluded.

In Vitro Studies of the Testis: The Effect of LH on Cyclic Nucleotides

This study evaluated the relationship between LH, cyclic AMP, cyclic GMP, and testosterone using in vitro incubation of decapsulated rat testes and sampling incubation medium. With added LH (1.0, 5.0, 100, and 500 mIU/ml) there were statistically significant increases in cyclic AMP at 5 mIU/ml or more LH, and progressively greater titers of this nucleotide were produced as LH was increased. For cyclic GMP all levels of added LH caused significant increments in titers of nucleotide; however, peak cyclic GMP concentrations occurred with 5 mIU/ml of LH. The addition of 10(-3) and 10-(4)M 8-bromo-cyclic AMP caused significant increases in testosterone production, while no changes in production of this androgen were found with 10(-3), 10(-4), or 10(-5)M 8-bromo-cyclic GMP. Neither cyclic AMP nor cyclic GMP titers were altered by the addition of 1 to 50 micrograms/ml of testosterone to medium bathing the rat testes. The dose response curves of cyclic AMP and cyclic GMP to LH are different. Progressive increments in added LH cause parallel increases of cyclic AMP and a biphasic change of cyclic GMP, 8-bromo-cyclic GMP does not cause testosterone generation, suggesting that cyclic GMP does not result in androgen synthesis. However, cyclic GMP may be involved in other Leydig cell functions.

Pharmacological characterization of the dopamine-mediated accumulation of cyclic AMP in intact retina of rabbit

Exposure of intact retinae of rabbit to dopamine, epinephrine and norepinephrine led to dose-related accumulations of cyclic AMP. Dopamine appears to be more potent than the two other catecholamines, since at 10 −6M it still induced a significant increase in cyclic AMP, whereas the two latter drugs were ineffective. Pure α- or β-adrenergic agonists such as phenylephrine or isoproterenol, as well as other drugs such as clonidine, DPI, (+)- and (±)-amphetamine, used at 10 −4M, were also devoid of agonist activity. In contrast a dopamine-analogue (epinine) and a dopamine-like drug (apomorphine) were as potent as dopamine. Blockade of the dopamine- or norepinephrine-elicited accumulation of cyclic AMP was achieved by antipsychotics such as fluphenazine, (+)-butaclamol and lithium, whereas propranolol (a β-adrenergic antagonist), phentolamine (an α-adrenergic antagonist) and (−)-butaclamol (an inactive compound), at 10 −4 to 5 × 10 −4M concentrations, showed no antagonist activity. The results indicate that the cyclic AMP production induced by catecholamines in intact retina of rabbit is a result of an activation of relatively pure dopamine receptors.

Cyclic nucleotide levels in the perfused rat heart subjected to hypoxia

Isolated rat hearts were subjected to hypoxic perfusion on a recirculating Langendorff apparatus. Following a 30-min-period of aerobic stabilization the hearts were perfused for 30 min with media equilibrated with 84% N2, 12%O2 and 4% CO2. At the end of the hypoxic period myocardial concentrations of cyclic AMP and cyclic GMP were determined by radioimmunoassay. Exposure to hypoxia resulted in a significant increase in cyclic AMP (p less than 0.01) and a decrease in cyclic GMP (p less than 0.05) as compared to hearts perfused for 60 min with media gassed with 96% O2. 4% CO2.

Evaluation of a new beta-adrenergic blocking agent, carteolol, based on metabolic responses in rats—II: Blockade by carteolol of the epinephrine- and isoproterenol-induced increases of tissue and blood cyclic AMP in vivo

Intraperitoneal injection of epinephrine caused a slight but significant increase in cyclic AMP in the liver and skeletal muscle in 5–10 min. Likewise, there was a significant increase in cyclic AMP in the muscle and adipose tissue 5 min after the injection of isoproterenol. However, no change was detected in the cardiac muscle. Pretreatment of rats with theophylline was so effective in enhancing responses of tissue cyclic AMP as to make it possible to detect a 2- to 3-fold increase of cyclic AMP even in the heart. The maximal increases in tissue cyclic AMP induced by 500 μg/kg of isoproterenol or epinephrine were blocked by carteolol, a new beta-adrenergic blocking agent, at doses of 10–100 μg/kg, regardless of whether or not the rats had been treated with theophylline. In contrast, neither the increase of liver cyclic AMP by glucagon nor the increase of adrenal cyclic AMP by adrenocorticotropic hormone (ACTH) was affected by this agent. Blood cyclic AMP rose sharply after isoproterenol, the maximal response attaining 20- to 30-fold of the preinjection level. Based on this tremendous increase of blood cyclic AMP, the dose of isoproterenol and carteolol required for the half-saturation of the beta-adrenergic receptor was calculated as 20–30 and 0.5 μg/kg of body wt respectively. These values are in the same order as the respective values obtained from the response of carbohydrate and lipid metabolites in blood.

Functional incorporation of ganglioside into intact cells: induction of choleragen responsiveness.

NCTC 2071 cells are unable to synthesize the monosialoganglioside GM1. When grown in chemically defined medium these cells contained no detectable GM1 and did not accumulate 3': 5'-cyclic AMP in response to choleragen. Incubation of the cells with [3H]GM1 permitted quantification of ganglioside uptake which was dependent on time and concentration of [3H]GM1 in the medium. Responsiveness to choleragen was demonstrated with binding of as few as 17,000 molecules of [3H]GM1 per cell; a maximal response was observed with 10(5) molecules per cell. With increasing cellular content of GM1, the rate of rise in intracellular cyclic AMP in response to choleragen was increased. With greater than 1 X 10(5) molecules of GM1 per cell, the delay between addition of choleragen and the cyclic AMP response was inversely proportional to choleragen concentration; less than 250 molecules of choleragen per cell caused a significant increase in cyclic AMP after 8 hr of incubation. Although the responsiveness of intact cells to choleragen was dependent on GM1, choleragen activation of adenylate cyclase in homogenates with 0.6 mM NAD was independent of added ganglioside. These observations are consistent with the view that exogenous ganglioside GM1 can be functionally integrated into the surface membrane of intact cells and serve as the choleragen receptor. Furthermore, although exogenous GM1 is required for choleragen responsiveness in intact cells, the ganglioside does not play an obligatory role in cell homogenates, where the surface receptor can presumably be bypassed.

RELATIONSHIP BETWEEN THE LEVELS OF INTRACELLULAR CYCLIC NUCLEOTIDES AND MECHANICAL RESPONSES INDUCED BY DRUGS

The tracheal smooth muscle dissected free from connective tissues was used as a test preparation, since isoprenaline caused a decrease in cyclic G MP in the muscle while it caused an increase in cyclic G MP in the remaining tracheal tissue. Acetylcholine, histamine and papaverine increased both cyclic AMP and cyclic GMP levels in the tracheal muscle. Isoprenaline increased the cyclic AMP level but decreased the cyclic GMP level. However, when tracheal muscle was incubated with isoprenaline in the presence of acetylcholine, isoprenaline did not cause a decrease in cyclic GMP but rather a significant increase in cyclic AMP after only 1 min incubation. These results indicate that the relaxation of the tracheal muscle by isoprenaline is initiated by the increase in cyclic AMP but is not associated with the change in cyclic GMP.