Role Of Cyclic AMP Research Articles

The role of cyclic AMP in modulating cytotoxic T lymphocytes. II. Sequential changes during culture in responsiveness of cytotoxic lymphocytes to cyclic AMP-active agents.

Agents that increase cyclic AMP (cAMP) levels inhibited the activity of cytotoxic T lymphocytes (CTL) obtained from spleens of mice immunized with allogeneic cells. Cultured CTL, however, were desensitized to cAMP-active agents, in that the capacity of these agents to inhibit the activity of cultured CTL was markedly reduced. The capacity to inhibit CTL activity was reduced more rapidly for some agents than for others; the percent inhibition by histamine and PGE2 was reduced after 4 hr and was reduced more than 20% after 24 hr, whereas the percent inhibition by dibutyryl cAMP, theophylline, and cholera enterotoxin was reduced less than 6% after 24 hr, and was reduced significantly only after 48 hr. Culture in the presence of antigen accelerated desensitization to the latter three agents. CTL populations were also tested for their capacity to increase cAMP levels in response to agonists. The capacity of histamine to increase cAMP levels of the CTL was lost within 4 hr (i.e., as rapidly as its capacity to inhibit CTL activity) and was never restored, whereas the capacity of PGE2 to increase cAMP levels persisted throughout culture. These results suggest that culture induces multiple alterations in cAMP metabolism of CTL. These alterations, which result in dissociation of CTL activity from cAMP-mediated regulatory steps, may include loss of histamine receptors and/or histamine receptor-adenylate cyclase coupling, and also loss of one or more biochemical reactions that link elevated cAMP levels to inhibition of lysis.

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells.

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP.

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.

Early Events in the Interaction of Melanotropin with Cloudman S91 Melanoma Cells

Melanotropin stimulates melanin synthesis in Cloudman S91 melanoma cells, the accumulation of melanin being preceded by a number of events which are understood to various degrees. In this review I will discuss the control of melanogenesis by melanotropin; emphasis will be on the early events of the process, although other aspects will be discussed. The probes (fluorescent and radioactive labels) used to examine the binding of the hormone to the cells will be described, as will be the results of initial binding studies. One of the events immediately following the binding of the hormone is the activation of adenylate cyclase: the evidence supporting this, plus the role of cyclic AMP in stimulating melanin synthesis, will be discussed. Also examined is the role which internalization of the melanotropin-receptor-adenylate cyclase complex may play in mediating hormone action.

The role of cyclic AMP, calcium, and prostaglandins in the induction of osteoclastic bone resorption associated with experimental tooth movement.

Administration of imidazole and verapamil inhibited the appearance of osteoclasts and bone resorption induced by experimental tooth movement in rats. On the other hand, administration of theophylline and ouabain enhanced the appearance of osteoclasts. A mixed solution of DB cyclic AMP and A-23187 injected in gingiva near the orthodontically-treated teeth caused enhancement of the appearance of osteoclasts and bone resorption.

Changes in cellular levels of cyclic AMP in rat mast cells during secretion of histamine induced by immunoglobulin E decapeptide and ACTH(1–24) peptide: Comparison with immunological and ionophore triggers

The role of cyclic AMP in the secretory mechanism of mast cells has been investigated by comparing the time course of changes in cellular levels of this cyclic nucleotide with the kinetics of secretion induced by basic peptides, antigen, anti-IgE and calcium ionophore. ACTH(1–24) peptide and a synthetic decapeptide representative of the sequence 497–506 within the Cε4 domain of human IgE induced a transient rise in cyclic AMP which reached approx. 150% of the resting levels by 10 s. Peptide-induced secretion of histamine was also rapid, reaching a maximum after 5–10 s. Immunological triggering of mast cells with antigen and anti-IgE raised levels of cyclic AMP to 150% of resting levels within 15 s, accompanying secretion of histamine which reached a maximum after 30 s. A relatively slower release of histamine induced by the calcium ionophore A23187 was paralleled by a significant reduction in cyclic AMP to 50% of the resting levels after 300 s. These data suggest a relationship between the accumulation of cyclic AMP in mast cells and secretion of histamine mediated by the Cε4 decapeptide and the ACTH(1–24) peptide as well as by IgE-dependent mechanisms. However, the simultaneous increase in cyclic AMP and secretion of histamine suggests that the two events may not be causally related.

Forskolin: Effects on mouse parotid gland function

Forskolin stimulates cyclic AMP accumulation, cyclic-AMP dependent protein kinase activation and amylase release from mouse parotid acini in a dose-dependent manner. In addition, forskolin augments β-adrenergic (isoproterenol)-stimulated cyclic AMP accumulation and amylase release. These results suggest that forskolin may be a valuable tool in examining the role of cyclic AMP in salivary gland secretion from intact cell preparations.

Catecholamine stimulation of steroidogenesis in Leydig cells.

Although p-adrenergic control of another steroidogenic tissue, the ovary, has been established (Godkin et al., 1977; Jordan et al, 1978; Hunzicker-Dunn, 1980; Hunzicker-Dunn et al., 1979; Kliachko & Zor, 1981), there have been no reports that catecholamines can directly stimulate steroidogenesis in Leydig cells. During the course of our investigations on factors controlling Leydig activity in culture we found that catecholamines do stimulate testosterone production. Because of the possible physiological implications and also potential use in studying hormonal control of adenylate cyclase in Leydig cells (Dix & Cooke, 1981) more detailed investigations have been carried out. The time and dose-responses of the p-adrenergic hormone agonist, isoprenaline and adrenaline and the pantagonist, propranolol, on testosterone production have been determined and the role of cyclic AMP has been investigated. The results obtained indicate that physiological amounts of catecholamines stimulate testosterone production via cyclic AMP in mouse Leydig cells and that this response ‘develops’ in the Leydig cells after 1 day in culture and is then maintained for approx. 2 days. Adult mouse Leydig cells were isolated by mechanical dissociation of the testes followed by purification on a &90% Percoll gradient as described previously (Schumacher et al., 1978; Hunter et al., 1982). The Leydig cells (100000 cells/well) were cultured in Dulbecco’s modified Eagle’s medium (1 ml) with Kanamycin (0.1 ng/ml) (Gibco) and 10% foetal-calf serum (Gibco). The medium was changed daily and the cells were stimulated on day 2 of culture with catecholamines or lutropin as indicated for 2h in the presence of isobutylmethylxanthine (0.2 mM), unless otherwise stated. In a series of experiments carried out it was found that isoprenaline (10-0.1 PM) added to freshly prepared Leydig cells 2h after being plated out in the culture wells had little or no effect on testosterone production. However, addition of this catecholamine analogue on days 1, 2 and 3 of culture consistently stimulated testosterone production. The results from a representative experiment are shown in Fig. 1. In this experiment two preparations of Leydig cells were made each from 10 mice killed either by cervical dislocation or by gassing with CO,. There were no detectable differences in the results obtained from these two preparations. In contrast with testosterone production, isoprenaline did stimulate cyclic AMP production on day 0 of culture, which increased on each subsequent day of culture. The capacity of the Leydig cells to respond to isoprenaline in the stimulation of testosterone production varied with different preparations from 20 to 90% of that obtained with lutropin. Further experiments were carried out to determine if inclusion of foetal-calf serum in the culture medium influenced the response to catecholamines. The results are given in Table 1. In agreement with the results in Fig. 1, little or no stimulation by isoprenaline of testosterone production was obtained on day 0 of culture in the presence of either foetal-calf serum or 1.5% (w/v) albumin. However, stimulation of cyclic AMP levels on day 0 was approx. three times higher in the cells cultured with foetal-calf serum compared with albumin. On subsequent days, stimulation by isoprenaline of both testosterone and cyclic AMP was obtained with albumin in the culture medium but much higher levels were attained in the presence of foetal-calf serum. Higher lutropin-stimulated testosterone but not cyclic-AMP levels were obtained in the presence of foetal-calf serum compared with albumin on all days of culture. Dose-response experiments for isoprenaline showed that a small increase in testosterone production was obtained with 10nwisoprenaline and reached a maximum with approx. 1 PM. A parallel dose-response curve for isoprenaline-stimulated cyclic AMP production was obtained, the only difference being that a stimulation was not observed with IOnM. Isoprenaline stimulation of testosterone production was inhibited by the

Regulatory peptide-controlled release of prolactin from GH 3 pituitary tumour cells: The role of cyclic AMP

Regulatory peptide-controlled release of prolactin from GH 3 pituitary tumour cells: The role of cyclic AMP

CRF stimulates α-MSH secretion and cyclic AMP accumulation in rat pars intermedia cells

Ovine corticotropin-releasing factor (CRF) stimulates α-MSH release and cyclic AMP accumulation in rat pars intermedia cells in culture at ED 50 values of 1 and 6 nM, respectively. The stimulatory effect of CRF on both parameters is inhibited by the dopaminergic agonist 2-bromo-α-ergocryptine (CB-154). The present data show that CRF is a potent stimulator of peptide secretion in the intermediate lobe of the pituitary gland and suggest a role of this pituitary lobe in the response to stress. In addition, the present data clearly indicate a role of cyclic AMP as mediator of the action of CRF in pars cells.

Role of cyclic AMP in the inhibition of human platelet aggregation by quercetin, a flavonoid that potentiates the effect of prostacyclin

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) has previously been shown to inhibit cyclic nucleotide phosphodiesterases prepared from various cell homogenates and the function of intact human platelets. We now report that (1) high concentrations of quercetin raise platelet cAMP levels; and (2) quercetin potentiates the inhibitory effect of prostacyclin (PGI 2) on ADP-induced washed human platelet aggregation and the elevation of platelet cAMP levels elicited by PGI 2. These results suggest a role for cAMP in the mechanism of action of quercetin on blood platelets.

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone.

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.

Effect of caffeine on glucose-induced inactivation of gluconeogenetic enzymes in Saccharomyces cerevisiae. A possible role of cyclic AMP.

The mechanism of catabolite inactivation of three gluconeogenetic enzymes, fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase, has been studied in the yeast Saccharomyces cerevisiae. The glucose-induced inactivation of the three enzymes is remarkably retarded by preincubation of the cells with different caffeine concentrations; however, a full conservation of activity has never been obtained, even at the highest drug concentration. Caffeine modifies the metabolic effects produced in the yeast cell by exposure to glucose. It reduces the consumption rate of glucose; changes the glycolytic intermediate pattern, giving rise to a crossover point at the level of the phosphofructokinase/fructose-bisphosphatase cycle; and increases the ATP level and the energy charge. Moreover, it substantially reduces the peak of intracellular cAMP content that immediately follows glucose entry; the magnitude of this effect is dependent on the drug concentration. The effect on the change of intracellular cAMP level appears, among all metabolic effects determined by caffeine, the only plausible one to explain the interference with catabolite inactivation of enzymes. Actually a strong negative correlation between residual activity of each of the three investigated enzymes and intracellular cAMP level has been demonstrated. The existence of a common mechanism of action of cAMP, as the mediating factor for catabolite inactivation of all three enzymes, is proposed.

Effects of cyclic AMP analogues and phosphodiesterase inhibitors on K+-induced [3H]noradrenaline release from rat brain slices and on its presynaptic alpha-adrenergic modulation.

The possible role of cyclic AMP in the presynaptic alpha-adrenoceptor-mediated modulation of [3H]noradrenaline (NA) release induced by 13 mM K+ from superfused rat cerebral cortex slices was investigated. Both dibutyryl-cyclic AMP (db-cAMP) and 8-bromo-cyclic AMP (8-Br-cAMP) dose-dependently (10(-4) - 10(-2) M) enhanced K+-induced (3H]NA release, maximally to about 160% of control. In contrast, db-cAMP had no effect on calcium-induced [3H]NA release in the presence of the calcium ionophore A 23187. Surprisingly, the phosphodiesterase (PDE) inhibitors 3-isobutyl-1-methylxanthine (IBMX). 7-benzyl-IBMX, 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771), and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) appeared to inhibit K+-induced [3H]NA release in a dose-dependent (10(-5) - 10(-3) M) manner. At a concentration of 10(-4) M, AK 62771 caused an inhibition of [3H]NA release by 30%, and this inhibitory effect was not affected by 10(-6) M phentolamine nor by 10(-3) M db-cAMP or 10(-4) M theophylline. Theophylline by itself enhanced [3H]NA release to about 135% of control. The inhibitor effect of the alpha-adrenoceptor agonist oxymetazoline (1 micro M) and the enhancing effect of the antagonist phentolamine (1 micro M) on [3H]NA release were significantly decreased in the presence of 10(-3) M db-cAMP or 8-Br-cAMP, whereas 10(-4) M ZK 62771 had no effect. In the presence of 10(-2) M NaF, a potent activator of adenylate cyclase, the inhibitory effect of oxymetazoline (1 micro M) on [3H]NA release was significantly decreased. The data obtained with the cyclic AMP analogues support the hypothesis that activation of presynaptic alpha-receptors modulating NA release results in an inhibition of a presynaptic adenylate cyclase. Possible causes for the anomalous effects of th PDE inhibitors are discussed.

Pathological physiology and general pathology role of cyclic AMP in regulation of platelet shape

Pathological physiology and general pathology role of cyclic AMP in regulation of platelet shape

Investigation of the role of cyclic AMP and prostaglandin E2 in the mechanism of the inhibitory action of nicotinic acid on platelet aggregation

Investigation of the role of cyclic AMP and prostaglandin E2 in the mechanism of the inhibitory action of nicotinic acid on platelet aggregation

The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells

The role of cyclic AMP in the induction of enzymes involved in estrogen and progestin biosynthesis in undifferentiated granulosa cells was investigated. When granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2 days in serum-free medium with aromatase substrate (10 −7M androstenedione) together with graded doses of FSH, prostaglandin E 2 (PGE 2), cholera toxin (CT), or dibutyryl cyclic AMP (Bu 2cAMP), there was a dose-related increase in estrogen (E) production. The induction of E production by saturating doses of FSH, PGE 2, CT and Bu 2cAMP required a lag phase of ∼24 h, after which the E response increased sharply to maximum levels at day 3 and then declined gradually to day 5. Treatment for 24 h (day 0–1) with FSH, together with 1 μg/ml of either actinomycin D or cycloheximide, completely abolished the stimulatory action of FSH on E production. When the inhibitors were removed, the FSH-induced increases in E returned to near normal levels after a 24-h lag period. Similar effects of the inhibitors upon E production by CT, PGE 2 and Bu 2cAMP were observed. As with E, the production of progesterone and 20 α-dihydroprogesterone was markedly stimulated by FSH, PGE 2, CT and Bu 2cAMP and the results of the time course, dose response and inhibitor experiments were similar to those for E production. These results indicate that FSH induces the de novo synthesis of enzymes required for both estrogen and progestin biosynthesis by undifferentiated granulosa cells and suggest that this action is mediated by cyclic AMP.

Stimulation of alpha-amylase release and cyclic AMP accumulation by catecholamine in rat parotid slices in vitro.

The role of cyclic AMP in α-amylase release by catecholamines was studied in rat parotid slices. The order of potency required for catecholamines to induce α-amylase release as well as cyclic AMP accumulation was as follows: isoproterenol>norepinephrine=epinephrine>> phenylephrine. At the lowest concentration tested, all of the catecholamines stimulated α-α-mylase release without causing a detectable accumulation of cyclic AMP. High doses of phenylephrine and other derivatives of catecholamines such as salbutamol and soterenol stimulated α-amylase release, but they did not cause accumulation of cyclic AMP and their maximum effects on α-amylase release were significantly lesser than those of norepinephrine. High concentrations of these derivatives decreased the effect of norepinephrine on α-amylase release, which may be attributed to be the cause of this smaller maximum effect. The stimulation of cyclic AMP accumulation by norepinephrine was much more sensitive to inhibition by β-adrenergic antagonists than was the stimulation of α-amylase release by norepinephrine. The effect of norepinephrine on cyclic AMP accumulation was potentiated by isobutylmethylxanthine, which correlated with the degree of inhibition of phosphodiesterase. The effect of low doses of norepinephrine on α-amylase release was increased by isobutyl-methylxanthine, but there was no such effect when either high doses of norepinephrine or dibutyryl cyclic AMP was used. The stimulation of α-amylase release by 1 μm norepinephrine was much greater than that by 0.2 μM norepinephrine plus 0.5 mm isobutylmethylxanthine, while the reverse was took place in cyclic AMP accumulation. Thus, there was a marked dissociation of cyclic AMP accumulation and α-amylase release. The bearing of these results on the role of cyclic AMP in α-amylase release was discussed.

Proving a role for cyclic AMP in synaptic transmission

Proving a role for cyclic AMP in synaptic transmission

Involvement of cyclic amp in the regulation of lymphokine induced glia cell stimulation

T and B lymphocytes of human or murine origin were found to secrete a factor which increases the DNA and RNA synthesis of cultured glia cells. This factor, termed glia cell stimulating factor (GSF), is released upon stimulation of such immune cells by mitogen or antigen qualifying it as a lymphokine. In this communication we report on the role of cyclic AMP (cAMP) in regulating the effect of GSF on glia cells. Prostaglandin E 1 (PGE 1), isoproterenol and theophylline were effective in suppressing the GSF-induced increase of the glia cell proliferation. No inhibition of DNA synthesis and no decrease in cell number was observed when testing these substances on glia cells not being activated by GSF. The drugs were found to induce an increase in cAMP concentrations of glia cells. A partial desensitization of the glia cells to these drug induced elevations of cAMP was detected after pretreatment of the glia cell cultures with GSF. It is suggested that stimulated lymphocytes not only release GSF but also low molecular weight proteins such as PGE 1 which regulate the effects of GSF on glia cells by activating their adenylate cyclase.