Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Quantitative aspects of cyclic AMP and relaxation in the rabbit anterior mesenteric-portal vein.

Rabbit anterior mesenteric-portal vein was shown to exhibit a positive correlation between relaxation and cyclic AMP levels using different concentrations of isoproterenol for varying exposure times. No consistent relationship was found between cyclic AMP and relaxation using a series of phosphodiesterase inhibitors (papaverine, SC2964, or RA233). In sodium-free (Tris-substituted) Kreb's solution, the addition of isoproterenol still increased cyclic AMP levels, but there was no relaxation. Dibutyryl cyclic AMP was also ineffective in sodium-free solution. Under identical conditions, both relaxation and increases in cyclic AMP produced by papaverine and SC2964 were found to be independent of extracellular sodium. We conclude, therefore, that increased cyclic AMP may be involved in isoproterenol-induced relaxation possibly acting via increased Na+-Ca2+ exchange, but plays only a small role, if any, in smooth muscle relaxation produced by papaverine, RA233, or SC2964.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

Effects of glucagon, phosphodiesterase inhibitors, and trypsin treatment on cyclic 3',5' adenosine monophosphate levels in isolated hepatocytes.

Cyclic 3',5' adenosine monophosphate (cyclic AMP) levels were measured in isolated hepatocytes under several conditions. Following the addition of glucagon cyclic AMP levels increased rapidly with peak values occurring at three minutes. The increase in cyclic AMP was dose dependent. Significant increases were found with 10(-10)M glucagon and a maximum increase of twenty fold was produced by 10(-8) M glucagon. This action of glucagon was augmented by the phosphodiesterase inhibitors, theophylline, SQ 20,009, and papaverine. Treatment of the hepatocytes with trypsin markedly reduced the response to glucagon.     

Effects of potassium chloride and smooth muscle relaxants on tension and cyclic nucleotide levels in rat myometrium.

Ten minutes after KCl-depolarization of rat myometrial strips, at which time the muscles were in a state of sustained contracture, tissue levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) were increased by approximately 40% over relaxed controls, and levels of guanosine 3',5'-cyclic monophosphate (cyclic GMP) were decreased by 40%. At this point both nitroglycerin (4 X 10(-4) M) and papaverine (2 X 10(-5) M) were capable of relaxing the depolarized muscles without significantly increasing cyclic AMP levels. Isoproterenol, in concentrations from 5 X 10(-9) M to 10(-6) M, relaxed the depolarized muscles and significantly increased tissue levels of cyclic AMP. However, the magnitudes of the cyclic AMP increases seen after the lower concentrations of isoproterenol were small relative to the increases observed during KCl-contracture alone. For example, the 40% elevation of cyclic AMP seen 10 min after KCl-depolarization did not cause the muscles to relax, whereas 5 X 10(-9) M isoproterenol caused relaxation with an increase in cyclic AMP levels of only 16% over depolarized controls. It was concluded that changes in total tissue levels of cyclic AMP were not responsible for the uterine relaxation caused by nitroglycerin, papaverine or isoproterenol in these experiments. Cyclic GMP levels in the depolarized muscles were not significantly changed by isoproterenol or papaverine but were increased approximately 80% by nitroglycerin. The above results are not consistent with the previously suggested roles for cyclic GMP and cyclic AMP as mediators of smooth muscle contraction and relaxation, respectively.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase.

Adenosine, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phosphodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell surface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides had no effect on the accumulation of cyclic AMP. Among other adenine nucleotides we tested, adenosine 5'-monophosphoramidate, but not adenosine 5'-monosulfate significantly increased cyclic AMP especially with the addition of papaverine. Neither 2'- nor 3'-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase

Adenosie, AMP, ADP and ATP activated adenylate cyclase in pig skin (epidermis) slices resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of the cyclic AMP-phophodiesterase inhibitor, papaverine. But another inhibitor, theophylline, strongly blocked the activation of adenylate cyclase by adenosine and adenine nucleotides. Theophylline apparently competed with adenosine for the cell suface receptor. Like theophylline, the addition of adenine alone caused no accumulation of cyclic AMP, but it significantly inhibited the stimulatory effect of adenosine. Guanosine, or guanine, cytidine, uridine, or thymidine nucleotides has no effect on the accumulation of cyclic AMP. Among other adenine nucleotides was tested, adenosine 5′-monophosphoramidate, but not adenosine 5′-monosulfate, significantly increased cyclic AMP especially with the addition of papaverine. Neither 2′- nor 3′-adenylic acid were effective. Our data indicate that pig epidermis has four specific and independent adenylate cyclase systems for adenosine (and adenine nucleotides), histamine, epinephrine and prostaglandin E.     

Depolarization-evoked accumulation of cyclic AMP in brain slices: inhibition by exogenous adenosine deaminase

In guinea pig cerebral cortical slices labeled during a prior incubation with radioactive adenine, electrical stimulation or the presence of depolarizing agents such as veratridine, ouabain, and high concentrations of K⁺ elicit a marked accumulation of radioactive cyclic AMP. This accumulation is reduced in all cases by the presence of theophylline, a compound that antagonizes the stimulatory effects of adenosine on cyclic AMP accumulation in brain slices. Exogenous adenosine deaminase also reduced the accumulation of cyclic AMP elicited by electrical stimulation, veratridine, and high concentrations of K⁺. Thus, adenosine formed in neuronal compartments under depolarizing conditions appears to be released into the extracellular medium as a prerequisite to stimulation of the cyclic AMP-generating system. Adenosine deaminase does not prevent the reduction in levels of ATP under depolarizing conditions, nor does it antagonize the accumulation of cyclic AMP elicited by a combination and norepinephrine. Adenosine deaminase does not, however, prevent the accumulations of cyclic AMP elicited by the depolarizing agent, ouabain.     

The mechanism by which 2-deoxyglucose inhibits glucose-induced activation of Pilobolus longipes spores

Sporangiospores of Pilobolus longipes were activated by either glucose, 6-deoxyglucose, or derivatives of cyclic AMP. Cyclic AMP content increased after the addition of either glucose or 6-deoxyglucose and the increase preceded spore activation, indicating that glucose triggers germination via cyclic AMP. Activation, whether induced by glucose, 6-deoxyglucose, or cyclic nucleotides was inhibited by 2-deoxyglucose. However, cyclic AMP levels also increased after the addition of 2-deoxyglucose. Radioactive 2-deoxyglucose was recovered from spores mainly as 2-deoxyglucose 6-phosphate, suggesting that phosphorylation of 2-deoxyglucose may inhibit spore activation by trapping ATP. Support for the hypothesis came from ATP assays which showed that 2-deoxyglucose reduced intracellular ATP to undetectable levels. Moreover, when ATP levels were restored with exogenous fructose, 2-deoxyglucose was no longer inhibitory but was then an effective germination trigger.     

Assessment of selective inhibition of rat cerebral cortical calcium-independent and calcium-dependent phosphodiesterases in crude extracts using deoxycyclic AMP and potassium ions

The effects of various inhibitors on the activity of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex were examined. While the agents varied greatly in their relative potency, each was found to be approximately equipotent in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP. In contrast, the inhibitors displayed a marked substrate specificity for the calcium-independent enzyme with ratios of IC₅₀ values for inhibition of cyclic GMP hydrolysis when compared to cyclic AMP hydrolysis in decreasing order being: ZK 62711 (? 100) > Ro 20–1724 (?>25) papaverine (13) > 7-benzyl IBMX (4) > quercetin and kaempferol (2). The differential selectivity of the inhibitors for the two enzymes was most pronounced for ZK 62711 and Ro 20–1724 which were at least 25–100-times more potent in inhibiting the calcium-independent hydrolysis of cyclic AMP when compared to the calcium-dependent hydrolysis of cyclic AMP. In contrast, 7-benzyl IBMX, kaempferol and quercetin were 8–100-times more effective as inhibitors of cycluc GMP hydrolysis by the calcium-dependent phosphodiesterase while 7-benzyl IBMX and trimazosin displayed a similar enzyme selectivity using cyclic AMP as substrate. With the exception of papaverine, all agents were competitive inhibitors of the calcium-dependent phosphodiesterase. The type of inhibition observed with the calcium-independent enzyme was dependent on the substrate employed. The specificity of potassium ions in inhibiting the activity of the calcium-dependent phosphodiesterase and deoxycyclic AMP in inhibiting the calcium-independent enzyme was found to provide a convenient means to assess the effects of agents on these activities in crude extracts of cerebral cortex.     

3',5'-cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na+-channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine-induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP.     

Platelet aggregation. IV. Platelet phosphodiesterase and its inhibition by vasodilators

Platelet cyclic AMP phosphodiesterase (PDE) has been partially purified, its properties studied and inhibition by certain vasodilators observed. Quazodine, dipyridamole, papaverine, Paveril^® and other agents inhibit platelet adenosine uptake, potentiate both the inhibition of aggregation and PGE₁ stimulated cyclic AMP synthesis and inhibit PDE. The mechanism of action of vasodilators as inhibitors of aggregation is discussed.     

Regulation of adenosine 3':5'-monophosphate efflux from animal cells.

Cyclic AMP efflux was measured following hormonal stimulation of adenylate cyclase in a variety of animal cells including C-6 rat glioma cells, WI-38 human fibroblasts, and avian erythrocytes. Using a variety inhibitors of mitochondrial function and glycolysis, a correlation was noted between cellular ATP levels and the rate of cyclic AMP efflux in all cells examined. A relationship between the efflux rate and the magnitude of the membrane potential was not observed. Pharmacological agents which inhibited cyclic AMP egress in these cells without reducing ATP levels included several prostaglandins (A greater than B greater than E greater than F) and probenecid. The characteristics of the cyclic AMP efflux system resemble those of the organic anion transport system.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

3′,5′‐Cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na⁺‐channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine‐induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 281–288, 2001     

Dual control of pupal diapause by cyclic nucleotides in the bertha armyworm,Mamestra configurata Wlk.

Treatment of diapausing pupae of M. configurata with dibutyryl cyclic AMP or 8-(4-chlorophenylthio) cyclic AMP (CPT cyclic AMP) reduced the incidence of eclosion to zero compared to about 15% for controls, whereas treatment with cyclic GMP increased eclosion to more than 90%. Treatment with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) resulted in a high incidence (79.8%) of eclosion, but treatment with dibutyryl cyclic AMP + IMBX or CPT cyclic AMP + IBMX gave low incidences (<9.1%) of eclosion. Other methylxanthines (theophylline, 8-phenyltheophylline, caffeine) and papaverine had relatively little effect on eclosion even at high doses.Treatment of post-diapause pupae with dibutyryl cyclic AMP or CPT cyclic AMP resulted in a low incidence (<5.0%) of eclosion compared to 98.8% eclosion in controls. Suppression of eclosion was more effective if dibutyryl cyclic AMP was given within the first 2 days of pupal-adult development at 20°C and became less effective as development progressed, indicating that dibutyryl cyclic AMP inhibits endocrine events initiating development rather than inhibiting subsequent metamorphic development. Treatment of post-diapausing pupae with cyclic GMP, IBMX, other methylxanthines or papaverine did not affect eclosion. These results are consistent with a dual control of pupal diapause in M. configurata by cyclic nucleotides, with cyclic AMP acting to maintain diapause and cyclic GMP acting to terminate it.     

On the mechanism by which antiadrenergic drugs increase survival of critical skin flaps

In order to asses the possibility that degeneration release of noradrenaline influences the survival of critical skin flaps, we studied the effect of various antiadrenergic drugs on skin-flap levels of noradrenaline, ATP, and cyclic AMP. Reserpine treatment depleted the skin flaps of noradrenaline and counteracted the fall in ATP and the cyclic AMP accumulation. Guanethidine had similar but less pronounced effects. Propranolol did not affect noradrenaline levels or depletion rate, but reduced the metabolic stimulation, as assessed by cyclic AMP levels in the flap. Phentolamine had no effect on basal noradrenaline levels, but tended to accelerate its disappearance and reduce lactate accumulation, a measure of hypoxia. All these drugs are known to increase skin-flap survival. It is suggested that they do so by, respectively, depleting the flap of its content of noradrenaline prior to operation or preventing the vasoconstriction and metabolic stimulation caused by released noradrenaline.     

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland.

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E1 and E2. Prostaglandin F1alpha and F2alpha have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE1 is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE1. Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

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.