An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.     

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.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Chronic Exposure to Pertussis Toxin Alters Muscarinic Receptor-Mediated Regulation of Cyclic AMP Metabolism in Neuroblastoma × Glioma NG108–15 Hybrid Cells

Chronic pertussis toxin treatment (5 days) of NG108-15 neuroblastoma X glioma hybrid cells had no significant effect on basal cyclic AMP levels whereas it effectively blocked the inhibitory action of acute (10 min) exposure of carbachol (10(-4)M) on intracellular cyclic AMP accumulation, stimulated by prostaglandin E1. This action of pertussis toxin was found to be long lasting: exposure of the cells to pertussis toxin (100 ng/ml) for only 24 h followed by a 5-day withdrawal period still was shown effective on day 7 in abolishing the inhibitory action of carbachol on prostaglandin E1-stimulated cyclic AMP production. Chronic exposure (5 days) of NG108-15 cells to carbachol (10(-5)M) causes an increase in basal cyclic AMP levels by 98%, and a desensitization of the muscarinic inhibition of cyclic AMP accumulation, assessed after a 24-h withdrawal period. When carbachol treatment is carried out in the presence of pertussis toxin (100 ng/ml) both of these effects of carbachol are abolished.     

Adrenergic versus VIPergic control of cyclic AMP in human colonic crypts

The actions of catecholamines on VIP-induced cyclic AMP is studied in human colon. We show that: (1) Epinephrine in the 10(-7)-10(-3) M concentration range (ED50 = 11.10(-6) M) inhibits VIP-induced cyclic AMP rise in isolated colonic epithelial cells; the maximal inhibition reaches 30% of VIP effect; epinephrine alters the efficacy of the peptide and does not modify its potency; epinephrine also reduces the basal cyclic AMP level. (2) The inhibition is found with other alpha adrenergic agonists with the order of potencies epinephrine greater than norepinephrine greater than phenylephrine. Clonidine has a poor intrinsic activity but antagonizes the action of epinephrine. (3) The inhibition of VIP action by epinephrine is reversed by the alpha antagonists dihydroergotamine, phentolamine and the alpha 2 antagonist yohimbine, while unaffected by the beta antagonist propranolol and the alpha 1 antagonist prazosin, (4) Epinephrine inhibits VIP-stimulated adenylate cyclase activity in preparations of colonic plasma membranes. Thus catecholamines exert through an alpha 2 adrenoreceptor a negative control on basal and VIP-stimulated cyclic AMP formation in human colon. We suggest that colonic cyclic AMP metabolism undergoes a dual control: VIPergic, activator and adrenergic, inhibitor.     

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.     

Relationship between cyclic AMP production and lipolysis induced by forskolin in rat fat cells.

Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) induced both cyclic AMP production and lipolysis in intact fat cells, but stimulated lipolysis without increasing cyclic AMP at a concentration of 10(-5) M. Homogenization of fat cells elicited lipolysis without elevation of cyclic AMP. Forskolin did not stimulate lipolysis in the homogenate. Forskolin stimulated both cyclic AMP production and lipolysis in a cell-free system consisting of endogenous lipid droplets and a lipoprotein lipase-free lipase fraction prepared from fat cells. However, at a concentration of 10(-6) M, it induced lipolysis without increase in the cyclic AMP content in this cell-free system. In the cell-free system, homogenization of the lipid droplets resulted in marked increase in lipolysis to almost the same level as that with 10(-4) M forskolin without concomitant increase in cyclic AMP. Addition of forskolin to a cell-free system consisting of homogenized lipid droplets and lipase did not stimulate lipolysis further. Phosphodiesterase activities were found to be almost the same both in the presence and absence of forskolin in these reaction mixtures. Although 10(-3) M forskolin produced maximal concentrations of cyclic AMP: 6.7 x 10(-7) M in fat cells and 2.7 x 10(-7) M in the cell-free system, 10(-4) M cyclic AMP did not stimulate lipolysis in the cell-free system. In a cell-free system consisting of lipid droplets and the lipase, pyrophosphate inhibited forskolin-induced cyclic AMP production, but decreased forskolin-mediated lipolysis only slightly. Based on these results, mechanism of lipolytic action of forskolin was discussed.     

Cyclic AMP-induced enhancement of calcium accumulation by the sarcoplasmic reticulum with no modification of the sensitivity of the myofilaments to calcium in skinned fibres from a yeast skeletal muscle.

In the presence of low concentrations of total EGTA (5 . 10(-4) M) and free Mg2+ (3.16 . 10(-5) M) and in the presence of caffeine (8 . 10(-3) M), cyclic AMP (5 . 10(-6) M) produces a relaxation of the tension developed by skinned fibres from cat caudo-femoralis. The relaxation can be attributed to an enhancement of the Ca2+ accumulation by the sarcoplasmic reticulum, since cyclic AMP does not modify the sensitivity of the myofilaments of Ca2+. These results are similar to those previously reported for the effect of cyclic AMP on skinned cardiac cells in the presence of a higher free Mg2+ concentration and in the absence of caffeine. This similarity suggests that the mode of action of cyclic AMP on the sarcoplasmic reticulum is not fundamentally different in cardiac and fast skeletal muscles.     

Effect of secretin on protein phosphorylation in dispersed acini from rat pancreas

Dispersed acini from rat pancreas, incubated in the presence of KH2(32)PO4 to steady state 32P incorporation into cellular proteins, were exposed to secretin. 32P incorporated into selected proteins, separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, reached a plateau by 150 min. Effect of secretin on amylase release, cellular cyclic AMP levels and protein phosphorylation was then examined. Stimulation of amylase release was apparent with 10(-10)M and was maximal with 10(-7)M by 10 min incubation. Almost maximal increase in cellular cyclic AMP levels and 32P incorporation into selected proteins was also observed with 10(-7)M secretin by 10 min in the presence of 10 mM theophyllin. Both secretin (10(-8)M) and dibutyryl cyclic AMP (10(-3)M) induced the phosphorylation of similar proteins analyzed by counting 32P content in each peptide band after SDS gel electrophoresis. Addition of cyclic AMP (10(-6)M) to homogenates of acini also augmented 32P incorporation from [gamma-32P]ATP into similar proteins. These results indicate that secretin enhances protein phosphorylation in pancreatic acinar cells and cyclic AMP may mediate the action of secretin on protein phosphorylation.     

Antagonism by guanosine 3',5'-cyclic monophosphate derivatives against the action of adrenaline on the automaticity of isolated rat heart muscle cells in culture.

Spontaneously and rhythmically beating single cells from the ventricles of new-born rats were cultured on coverslips for 3 to 4 days and transferred to a small perfusion chamber for photoelectric recording of their contractions. The cells responded to 3 - 10(-7) M adrenaline with a pronounced increase and to 1.7 - 10(-5) M carbamylcholine with a mode-rate decrease in the rate of beat. Carbamylcholine nearly abolished the beat-accelerating effect of adrenaline. 8-Bromo cyclic GMP and N2-2'-O-'DIBUTYRYL CYCLIC GMP, in concentrations of 4.2 to 8.4 - 10(-4) M, exerted only very slight depressant effects on the basal rate of beat, but strongly antagonized, and in the majority of experiments with 8-bromo cyclic GMP completely suppressed, the positive chronotropic action of adrenaline. Cyclic GMP, 5'-GMP, and 5'-guanylylimidodiphosphate were without effect. 8-Bromo cyclic AMP caused a marked acceleration of beating. In view of the mediator roles that cyclic AMP and cyclic GMP have been assigned in the cardiac actions of beta-adrenergic and of cholinergic agents, respectively, the present results may be interpreted as being indicative of an adrenergic-cholinergic antagonism at the level of these two cyclic nucleotides.     

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): interaction with pentobarbital in oxygen consumption and cyclic AMP formation of rat cerebral cortex slices

Effects of TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L- prolinamide ) and pentobarbital, alone or in combination, on oxygen consumption and cyclic AMP formation in rat cerebral cortex slices were investigated. The oxygen consumption of rat cerebral cortex slices as measured with a Warburg apparatus, increased linearly over time (0 to 60-min incubation at 37C). Addition of pentobarbital (1 to 7 x 10-4M) inhibited oxygen consumption, in a concentration-dependent manner, up to 45% of control. A concomitant application of DN-1417 (10-5M) or TRH (10-4M) and pentobarbital (5 x 10-4M) led to a partial recovery of the pentobarbital effect. The similar anti-pentobarbital effects were observed with the addition of carbachol (10-4M) or dibutyryl cyclic AMP (10-3M), but not norepinephrine (10-4M) or dopamine (10-4M). DN-1417, TRH, carbachol, norepinephrine or dopamine at 10-4M stimulated cyclic AMP formation in the cerebral cortex slices. Addition of pentobarbital (1 to 7 x 10-4M) inhibited the cyclic AMP formation, in a concentration-dependent manner. DN-1417, TRH or carbachol at 10-4M but not norepinephrine or dopamine at 10-4M significantly reversed the reduction of cyclic AMP formation induced by pentobarbital (5 x 10-4M). Atropine (10-4M) almost completely abolished DN-1417-, TRH- and carbachol-induced cyclic AMP formation in the presence and absence of pentobarbital.     

Galanin Inhibits Noradrenaline-Induced Accumulation of Cyclic AMP in the Rat Cerebral Cortex

The effect of galanin on noradrenaline (NA)-induced accumulation of cyclic AMP was investigated in slices of rat cerebral cortex. NA (10(-4)M) increased cyclic AMP levels during a 20-min observation period. Galanin (3 X 10(-7)M) significantly inhibited this response at all time points examined, although it did not change the basal levels of cyclic AMP. Galanin (10(-8)-3 X 10(-6)M) inhibited the cyclic AMP response to NA (10(-4)M) in a dose-dependent manner, with an IC50 of approximately 5.6 X 10(-8)M and a maximum inhibition of 59%. These results suggest that galanin, devoid of any detectable effects by itself, modulates the cyclic AMP response to NA in the rat cerebral cortex.     

Corticotropin-Peptide Regulation of Intracellular Cyclic AMP Production in Cortical Neurons in Primary Culture

Previous studies have provided evidence for adrenocorticotropic hormone (ACTH) effects on a wide variety of behaviors. However, the precise sites of action and the mechanisms by which these effects may be mediated have yet to be clearly elucidated. Although ACTH was shown to augment cyclic AMP levels in glial cells isolated from whole brain, other studies found little or no effect of ACTH peptides on cyclic nucleotide metabolism in slices of cerebral cortex or homogenates of whole brain. In the present study, our objective was to determine whether ACTH peptides regulate intracellular cyclic AMP levels in neurons of the cerebral cortex in primary culture. ACTH peptides stimulated cyclic AMP synthesis up to threefold in a dose-dependent manner; stimulation was complete within 5-10 min of exposure to agonists. Neurohormone efficacy was augmented by 0.1 microM forskolin (which was virtually ineffective alone); potency was unaffected. The order of potency (EC50) for increasing intracellular cyclic AMP levels was as follows: ACTH (1-24), ACTH (1-17) (10 nM) greater than alpha-melanocyte stimulating hormone, beta-melanocyte stimulating hormone (alpha-MSH, beta-MSH) (100 nM) greater than ACTH (1-10) (1 microM) greater than ACTH (4-10) (5 microM). The hexapeptide ACTH (4-9) as well as ACTH (11-24) were inactive at concentrations as high as 10 microM. Other neuropeptides derived from proopiocortin, such as beta-endorphin and Met- and Leu-enkephalin were without effect on basal or hormonally stimulated cyclic AMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

Diffusion modeling system of a neuron's molecular calculating machine

Intracellular injection of cyclic nucleotides through 3-4 barrel microelectrodes usually results in depolarization in case of cyclic AMP and in hyperpolarization in case of cyclic GMP. But sometimes the neuron response is more complex and changes with time. Phosphodiesterase inhibitors, papaverine, 3-isobutyl-1-methylxantine, SQ-20009 increase and prolong the effects of cyclic nucleotides and the complex effect of cyclic GMP is transformed into simple hyperpolarization. Large neurons respond to cyclic AMP with a delay (1-3 sec) after the beginning of iontophoresis. The solution of diffusion equation presents the distance from the microelectrode tip to the point of cyclic AMP action as a function of the delay (100-160 microns). The maximum value of concentration that may be reached at this point after a prolonged injection (10(-5) M) is calculated as well. The system producing and destroying cyclic nucleotides is supposed to be a diffusion analog input of the cell molecular computer. This system can solve various equations of mathematical physics. For this reason guanylatecyclase is supposed to be connected with special biochemical system which realizes harmonic analysis.     

Altered regulation of cyclic AMP-dependent protein kinase in a mouse lymphoma cell line.

The ability of cyclic AMP to inhibit growth, cause cytolysis and induce synthesis of cyclic AMP-phosphodiesterase in S49.1 mouse lymphoma cells is deficient in cells selected on the basis of their resistance to killing by 2 mM dibutyryl cyclic AMP. The properties of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in the cyclic AMP-sensitive (S) and cyclic AMP-resistant (R) lymphoma cells were comparatively studied. The cyclic AMP-dependent protein kinase activity or R cells cytosol exhibits an apparent Ka for activation by cyclic AMP 100-fold greater than that of the enzyme from the parental S cells. The free regulatory and catalytic subunits from both S and R kinase are thermolabile, when associated in the holoenzyme the two subunits are more stable to heat inactivation in R kinase than in S kinase. The increased heat stability of R kinase is observed however only for the enzyme in which the catalytic and cyclic AMP-binding activities are expressed at high cyclic AMP concentrations (10(-5)--10(-4) M), the activities expressed at low cyclic AMP concentrations (10(-9)--10(-6) M) being thermolabile. The regulatory subunit of S kinase can be stabilized against heat inactivation by cyclic AMP binding both at 2-10(-7) and 10(-5) M cyclic AMP concentrations. In contrast, the regulatory subunit-cyclic AMP complex from R kinase is stable to heat inactivation only when formed in the presence of high cyclic AMP concentrations (10(-5)M). The findings indicate that the transition from a cyclic AMP-sensitive to a cyclic AMP-resistant lymphoma cell phenotype is related to a structural alteration in the regulatory subunit of the cyclic AMP-dependent protein kinase which has affected the protein's affinity for cyclic AMP and its interaction with the catalytic subunit.     

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.     

Cyclic AMP binding proteins in early embryos of Drosophila melanogaster.

A variety of effects of cyclic AMP on cellular and subcellular phenomena suggest that there may be other modes of action of cyclic AMP then activation of protein kinase. It is also known that developing embryos contain cyclic AMP and its related enzymes. In order to explore the role of cyclic AMP in embryogenesis, a survey of proteins capable of binding cyclic AMP in the embryonic supernatant of Drosophila melanogaster was carried out. As the result, two cyclic AMP-binding proteins were found and characterized. The one (L) is, as expected, associated with protein kinase and has a dissociation constant of about 10(-9) M. Its molecular weight of 21 000 daltons is extremely small when compared with similar proteins in other organisms. The other (H), whose function is yet to be found, has a molecular weight of about 200 000 daltons and has a dissociation constant of about 10-7 M. Some laxity in binding specificity of the latter protein among adenosine nucleotides was observed, but cyclic AMP is the strongest ligand among them.     

P2-purinergic control of liver glycogenolysis.

Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10(-7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity.