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PubMed1979

Alteration in testicular cyclic AMP system in the rat following vasectomy.

Endogenous cyclic AMP levels and the activities of adenylate cyclase, cyclic AMP-dependent and independent protein kinases were examined in testes of mature rats bilaterally vasectomized for one, three and seven months. Although no significant alteration in testicular cyclic AMP was detected one month following vasectomy, marked decreases (by 55% and 32%, respectively) were seen three and seven months postvasectomy. Likewise, vasectomy also resulted in a significant decrease (by 25%) in the activity of testicular adenylate cyclase three and seven months after vasectomy. Although soluble cyclic AMP-dependent protein kinase activity remained unaffected three months postvasectomy, the activity of the cyclic nucleotide-dependent enzyme was significantly increased (by 21%) when compared to the sham-operated controls. Furthermore, while the protein kinase ratio (--cyclic AMP/+cyclic AMP) was decreased in animals vasectomized for three months, the ability of the enzyme to bind (3H) cyclic AMP in vitro was significantly enhanced (18%). Rats vasectomized for seven months showed similar biochemical alterations but the effects of this procedure were more pronounced. Moreover, while short-term vasectomy increased the responsiveness of seminiferous tubular adenylate cyclase to in vitro stimulation by follicle stimulating hormone, the activity of the enzyme was also increased (by 100%) in the presence of luteinizing hormone in vasectomized rats. These data raise the possibility that changes in testicular function seen following vasectomy may be related to the alterations in cyclic AMP metabolism as well as in the sensitivity of testicular adenylate cyclase to regulation by gonadotropins.

Adenylyl Cyclases↗
PubMed1986

Induction of tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: effect of the inducing agents alone or in combination on the enzyme levels and rate of synthesis of tyrosine hydroxylase.

The enzymatic activity of tyrosine hydroxylase (EC 1.14.16.2) increases in rat pheochromocytoma PC18 cells exposed to either elevated levels of cyclic AMP or glucocorticoids. The cyclic AMP-mediated increase in activity is elicited by cyclic AMP analogs or by compounds which activate adenylate cyclase or inhibit phosphodiesterase. The glucocorticoid-mediated increase is elicited only by glucocorticoid steroid hormones; nonglucocorticoid steroid hormones have no effect on tyrosine hydroxylase. In PC18 cells exposed simultaneously to both cyclic AMP-elevating agents and glucocorticoids, the increase in tyrosine hydroxylase activity is greater than that observed in cells treated with optimal concentrations of either inducing agent alone. Immunochemical titration experiments demonstrate that the increases in tyrosine hydroxylase activity observed in cells treated with the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, are due to increases in enzyme protein. Time course studies show that in cells treated with either 8-bromocyclic AMP or dexamethasone, the enzyme level increases slowly to a level 5-7-fold greater than that observed in untreated cells after 4 days of treatment. In cells treated with both of these inducing agents simultaneously, the enzyme level increases to a level 10-12-fold greater than that observed in control cells after 4 days of treatment. This additive increase in activity in cells treated with both inducing agents is observed at all time points. The rates of synthesis and degradation of tyrosine hydroxylase have also been measured in PC18 cells, using an antiserum to tyrosine hydroxylase to rapidly isolate radiolabeled enzyme from cells that have been incubated in the presence of [3H]leucine. The apparent half-life of tyrosine hydroxylase in the PC18 cells is approximately 30 hr. In PC18 cells incubated in the presence of radiolabeled leucine for 60 min, 0.2-0.3% of the total soluble protein synthesized is identified as tyrosine hydroxylase. In cells treated with either 8-bromocyclic AMP or dexamethasone for 24 hr, there is a 6-8-fold increase in the rate of synthesis of the enzyme. In cells treated with both inducing agents simultaneously, there is a 10-12-fold increase in the rate of synthesis; thus, the additive increase in enzyme level observed in cells treated with both inducing agents is paralleled by an additive increase in the rate of synthesis of the enzyme in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Differential induction of c-fos and c-myc by cyclic AMP in Swiss 3T3 cells: significance for the mitogenic response.

Quiescent Swiss 3T3 cells can be stimulated to reinitiate DNA synthesis through specific intracellular signalling pathways which include an increase in cyclic AMP levels and the activation of protein kinase C. In the present study we have compared the effect of these two pathways on the expression of c-fos and c-myc in Swiss 3T3 cells. In cells treated with the cyclic AMP-elevating agents forskolin or prostaglandin E1 or with the permeable cyclic AMP analogue, 8-bromo cyclic AMP, the levels of c-fos messenger RNA were 5 to 10% of those induced by bombesin, which activates protein kinase C. In contrast, a dramatic and comparable increase in the expression of c-myc messenger RNA was observed when cells were treated either with the cyclic AMP-increasing agents or with bombesin. Thus an increase in cyclic AMP leads to the differential induction of c-fos and c-myc messenger RNA in Swiss 3T3 cells. Significantly, we obtained comparable results when cells were treated with a mitogenic combination of cyclic AMP and insulin. Furthermore, these findings have been confirmed at the protein level. We concluded that the large induction of c-fos can be dissociated from cyclic AMP-mediated mitogenesis in Swiss 3T3 cells.

1-Methyl-3-isobutylxanthine↗
PubMed1989

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

5'-Nucleotidase↗
PubMed1988

[Levels of plasma cyclic AMP in cardiac surgery].

The actions of hormones such as catecholamines, vasopressin and growth hormones are mediated by a common intracellular second messenger, cyclic AMP (adenosine 3',5'-monophosphate). The effects of cardiac surgery on plasma cyclic AMP were studied in 32 adults patients with aorta-coronary bypass or with valvular disease. Blood specimens were obtained before operation, at the beginning and at the end of the cardiopulmonary bypass, 1, 3, 6, 9, 12, 24, 48, 72, 168 hours after surgery. The plasma cyclic AMP level during cardiac operation was elevated above the preoperative level. High levels of plasma cyclic AMP were found both in the aorta-coronary bypass and in the valvular disease immediately after the end of cardiopulmonary bypass. The plasma cyclic AMP level in patients undergoing aorta-coronary bypass with aortic clamping time more than 60 minutes was 38.6 +/- 11.7 pmol/ml, compared to 25.6 +/- 6.6 pmol/ml with aortic clamping time less than 60 minutes immediately after the end of cardiopulmonary bypass. In patients undergoing valve replacement and/or commissurotomy with aortic clamping time more than 60 minutes, the plasma cyclic AMP level immediately after the end of cardiopulmonary bypass was 113 +/- 63.3 pmol/ml, compared to 45.4 +/- 10.3 pmol/ml with aortic clamping time less than 60 minutes (p less than 0.01, Student's t test). During 24 hours after cardiac surgery, the plasma cyclic AMP concentration returned to normal range. It is considered that the plasma cyclic AMP level reflects the risk of cardiac surgery in response to homeostatic derangement.

Cardiac Surgical Procedures↗
PubMed1988

Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

Adenylyl Cyclases↗
PubMed1986

Induction of mRNA for tyrosine hydroxylase by cyclic AMP and glucocorticoids in a rat pheochromocytoma cell line: evidence for the regulation of tyrosine hydroxylase synthesis by multiple mechanisms in cells exposed to elevated levels of both inducing agents.

When rat pheochromocytoma PC18 cells are exposed to the cyclic AMP analog, 8-bromocyclic AMP, and/or the synthetic glucocorticoid, dexamethasone, there is a marked increase in the level of a single RNA species that hybridizes to the recombinant plasmid pTH.4, which contains sequences complementary to the RNA coding for tyrosine hydroxylase. This RNA species is 1800-1900 nucleotides in length and is presumably identical to an RNA species of similar size, isolated from rat pheochromocytoma PC8b cells and shown to code for tyrosine hydroxylase. Using RNA dot hybridization to quantitate the relative level of this tyrosine mRNA species, time course studies show that this mRNA increases relatively rapidly in PC18 cells treated with either 8-bromocyclic AMP or dexamethasone. A new steady state level of tyrosine hydroxylase mRNA is achieved after 6 hr or 12-24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, respectively. The changes in the level of the mRNA slightly precede the changes in the rate of synthesis of tyrosine hydroxylase in cells treated with these inducing agents. After 24 hr of treatment with either 8-bromocyclic AMP or dexamethasone, the increases in the level of tyrosine hydroxylase mRNA are identical to the increases in the rate of synthesis of the enzyme in the cells. In cells treated simultaneously with both 8-bromocyclic AMP and dexamethasone, the increases in the enzyme level and rate of synthesis of tyrosine hydroxylase are approximately equal to the sum of the increases in these parameters observed in cells treated with either inducing agent alone. In contrast, there is not an additive increase in the level of tyrosine hydroxylase mRNA in cells treated with both inducing agents. This lack of an additive increase in mRNA for tyrosine hydroxylase is observed in total cellular RNA samples or in cytoplasmic RNA samples. Our results suggest that in cells exposed to elevated levels of either cyclic AMP or glucocorticoids, tyrosine hydroxylase is induced by a mechanism which increases the level of its mRNA, resulting in an increased rate of synthesis of the enzyme. However, in cells exposed to elevated levels of both cyclic AMP and dexamethasone, tyrosine hydroxylase enzyme levels are regulated by multiple mechanisms, one of which regulates the rate of synthesis of the enzyme without affecting the level of its mRNA.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Action of the cardiac alpha 1-adrenergic receptor. Activation of cyclic AMP degradation.

Using purified rat ventricular myocytes and membranes prepared from them, we have previously found that alpha 1-adrenergic stimulation causes decreased cyclic AMP accumulation and decreased activation of cyclic AMP-dependent protein kinase. We have now analyzed the mechanism by which alpha 1 stimulation is linked to cyclic AMP metabolism. In an adenylate cyclase assay in which carbachol inhibits the stimulatory effect of norepinephrine, the addition of prazosin (alpha 1-antagonist) has no effect on the response to norepinephrine. In membranes prepared from myocytes treated with pertussis toxin, norepinephrine competes for alpha 1-receptors (assessed by [3H]prazosin binding) with two components, binding to the high affinity component being sensitive to exogenous GTP, exactly as in membranes prepared from control myocytes. In intact cells labeled with [3H]adenine in which carbachol antagonizes the norepinephrine response, prazosin enhances accumulation of [3H]cyclic AMP due to norepinephrine. Treatment of cells with pertussis toxin eliminates inhibition by carbachol but does not alter prazosin's capacity to enhance the norepinephrine response. Addition of phosphodiesterase inhibitors eliminates this effect of alpha 1 blockade. In [3H]adenine-labeled cells loaded with [3H]cyclic AMP by prior treatment with isoproterenol, alpha 1-adrenergic stimulation enhances disappearance of [3H]cyclic AMP. Measurements of cellular cyclic AMP give results similar to those obtained with the adenine labeling technic. We conclude that occupation of the myocyte alpha 1-receptor results in stimulation of cyclic AMP phosphodiesterase activity.

Adenine↗
PubMed1985

D2-dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture.

Dopamine (DA) regulation of intracellular cyclic AMP formation in purified, intact striatal neurons in primary culture was examined. DA (EC50, 3 microM) and vasoactive intestinal polypeptide (VIP; EC50, 10 nM) stimulated cyclic AMP formation by 2- and 5-fold, respectively. In the presence of 0.1 microM forskolin (which was virtually ineffective alone), neurohormone efficacy was augmented; potency was unaffected. In the presence of 0.1 microM SCH 23390, a selective D1 antagonist, the DA dose-response curve was shifted rightward in a competitive manner. At low concentrations (0.01-1.0 microM), however, DA inhibited basal cyclic AMP formation. The inhibitory effect, but not the shift of the dose-response curve, was blocked by 5 microM l-sulpiride, a selective D2 antagonist. At saturating concentrations of VIP (0.1-1.0 microM), no other neurohormone can further augment cyclic AMP formation. Under these conditions, increasing concentrations of DA resulted in a dose-dependent (IC50, 0.5 microM) inhibition of VIP-stimulated cyclic AMP synthesis. This effect was augmented in the presence of 0.1 microM SCH 23390 and blocked by 5 microM l-sulpiride. Sulpiride antagonism was stereospecific, with the l-isomer being 30-fold more potent than the d-isomer. The rank order of potency for a series of dopaminergic agonists and antagonists at the receptor mediating attenuation of cyclic AMP formation suggests that it is of the D2 type. Furthermore, both DA and Met-enkephalin inhibition of cyclic AMP formation is lost after exposure of striatal neurons to islet activator protein. These findings suggest that a D2 receptor mediates the inhibition of intracellular cyclic AMP formation by DA in striatal neurons in primary culture, and may do so by an interaction with the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1985

Calmodulin plus cyclic AMP-dependent phosphorylation of a Mr 22,000 pituitary protein.

Protein phosphorylation was examined in cytosolic extracts of adult rat anterior pituitary. In the presence of both cyclic AMP and calmodulin, the phosphorylation of a Mr 22,000 protein was markedly stimulated. Cyclic AMP and calmodulin must both be present in order for this effect to be observed; cyclic GMP does not substitute for cyclic AMP, and the effect is abolished by either trifluoperazine or the heat-stable inhibitor of cyclic AMP-dependent protein kinase. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that there are three molecular species of the Mr 22,000 phosphoprotein, with pI values ranging from 6.8 to 8.1. Phosphorylation of this protein is maximally stimulated by 5 microM cyclic AMP and 5.7 microM calmodulin. The effect of cyclic AMP plus calmodulin is enhanced by preincubation and requires a divalent cation; maximal phosphorylation takes place at 100 microM Mn2+, although higher concentrations of Mg2+ and Co2+ support an equivalent degree of phosphorylation. Cyclic AMP plus calmodulin-dependent protein phosphorylation was not detected in other rat tissues surveyed, including brain, testes, adrenal, kidney, liver, spleen, skeletal muscle, pineal, or posterior pituitary. These results help to explain the previous findings of Brattin and Portanova (Brattin, W.J., Jr., and Portanova, R. (1981) Mol. Cell. Endocr. 23, 77-90) of in vivo but not in vitro phosphorylation of three Mr 20,000 anterior pituitary proteins and indicate a possible point of convergence for calcium and cyclic AMP actions in the anterior pituitary.

Animals↗
PubMed1980

Inhibition of cyclic AMP accumulation in hamster adipocytes with phosphatidic acid: differences and similarities with alpha adrenergic effects.

Clark et al (Journal of Cyclic Nucleotide Research 6:37 (1980)) demonstrated phosphatidic acid inhibition of cyclic AMP formation in WI-38 fibroblasts and suggested the hypothesis that cholinergic inhibition of adenylate cyclase is mediated through accumulation of this phospholipid. In view of these data, we tested the hypothesis that phosphatidic acid is involved in alpha-adrenergic inhibition of cyclic AMP formation in hamster adipocytes. The effects of phosphatidic acid on hormone and methyl xanthine stimulated cyclic AMP accumulation and lipolysis were studied. Phosphatidic acid inhibited 3-isobutyl-1-methyl xanthine (IBMX) stimulated cyclic AMP formation. The maximum inhibition (85% to 100%) of IBMX stimulated cyclic AMP accumulation was detected at 1.0 microM phosphatidic acid. When lipolysis was measured, however, inhibitory effects of phosphatidic acid were not evident until the concentration of phospholipid was increased to 300 microM. The cyclic AMP lowering and antilipolytic effects were detected using egg yolk phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidic acid, distearoyl phosphatidic acid and palmitoyl lysophosphatidic acid but not with phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine or 1,2- diglyceride. In contrast to phosphatidic acid, clonidine or N6-phenylisopropyl adenosine inhibited lipolysis in concert with inhibition of methyl xanthine stimulated cyclic AMP accumulation. Cyclic AMP accumulation increased by isoproterenol in combination with IBMX was partially blocked by clonidine but not by phosphatidic acid. These results show similarities as well as differences between clonidine and phosphatidic acid actions on hamster fat cells and do not, therefore, provide unequivocal support for the possibility that phosphatidic acid is intimately involved in alpha-adrenergic effects on hamster adipocytes. However, the high selectivity of phosphatidic acid suggests a physiological role of this agent in regulation of adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Cyclic AMP as a mediator of prostaglandin E-induced suppression of human natural killer cell activity.

The role of cyclic AMP in mediating the prostaglandin (PG) E2-induced suppression of natural killer (NK) cell function was studied. Highly purified preparations of human large granular lymphocytes (LGL; which have been shown to be closely associated with human NK activity) and T lymphocytes were obtained by using Percoll gradients. Basal cyclic AMP was similar in both cell populations. With LGL, PGE2 (but not PGF2 alpha) suppressed NK activity (75%) and enhanced cellular cycle AMP (600%). In contrast, in small, high density T lymphocytes, PGE2 caused very small increases in cyclic AMP (20%). Phosphodiesterase inhibitors (PDEI) such as isobutyl methylxanthine and theophylline also increased cyclic AMP and suppressed NK activity in LGL. The effect of PGE2 and PDEI, in combination on cellular cyclic AMP and NK activity, was greater than the effect of each agent alone. Finally, exogenous derivatives of cyclic AMP also suppressed NK activity. These results indicate that in LGL, increased cellular cyclic AMP mediates the action of PGE2 on the suppression of NK activity.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Forskolin-stimulated cyclic AMP accumulation mediates protein synthesis-dependent refractoriness in C6-2B rat glioma cells.

We have examined the roles that cyclic AMP and protein synthesis play in the development of refractoriness in C6-2B rat glioma cells using the diterpene, forskolin, a general activator of cyclic AMP-generating systems. Forskolin-stimulated cyclic AMP accumulation peaked at 30 min and declined thereafter to 10% of peak levels by 3 hr despite the continued presence of sufficient forskolin to produce 98% of the control response when the incubation medium was transferred to naive cells. C6-2B cells treated for 3 hr with forskolin were refractory to a subsequent challenge with forskolin or isoproterenol. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) increased the degree of refractoriness developed after forskolin treatment. In the presence of IBMX, the induction of refractoriness by forskolin and forskolin-stimulated cyclic AMP accumulation were similarly dependent on forskolin concentration. Pre-treatment with isoproterenol or the cyclic AMP analogue, dibutyryl cyclic AMP, induced refractoriness to forskolin. When C6-2B cells were pre-treated with forskolin plus the protein synthesis inhibitor, cycloheximide, the development of refractoriness to forskolin or isoproterenol was attenuated. Cycloheximide prevented isoproterenol- or dibutyryl cyclic AMP-induced refractoriness to forskolin. These data provide further evidence that the onset of the refractory state in C6-2B cells is mediated by cyclic AMP and is a protein synthesis-requiring process.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Desensitization of catecholamine-stimulated adenylate cyclase and down-regulation of beta-adrenergic receptors in rat glioma C6 cells. Role of cyclic AMP and protein synthesis.

When exposed to the beta-agonist (-)-isoproterenol, rat glioma C6 cells exhibited a time-and concentration-dependent reduction in isoproterenol responsiveness (desensitization) and a loss of beta-adrenergic receptors (down-regulation). Other agents, such as dibutyryl cyclic AMP, isobutylmethylxanthine, and cholera toxin, all of which elevate intracellular cyclic AMP levels, also induced receptor down-regulation but at a much slower rate than isoproterenol. Loss of beta-receptors was detected with intact cells, cell lysates, and cell membranes. Receptor loss was accompanied by a reduction in isoproterenol-stimulated cyclic AMP production and adenylate cyclase activity. For a given amount of receptor loss, this reduction was much greater with isoproterenol than with other agents. In addition, the concentration of isoproterenol required for half-maximal stimulation of cyclic AMP production was increased in cells treated with isoproterenol but not with isobutylmethylxanthine or dibutyryl cyclic AMP. The affinity of beta-receptors for the agonist was also lower in membranes from cells treated with isoproterenol but not the other agents. Prior treatment of the cells with cycloheximide inhibited receptor loss by isoproterenol but did not prevent desensitization or reduced affinity of beta-receptors for the agonist. Cycloheximide also blocked the loss of receptors induced by dibutyryl cyclic AMP and, in addition, prevented a reduction in agonist-stimulated adenylate cyclase activity. We propose that desensitization is mediated in rat glioma C6 cells only by agonists and is not dependent on either cyclic AMP or protein synthesis. Down-regulation can be induced both by agonists and by cyclic AMP and does depend on protein synthesis. Thus, desensitization and down-regulation can occur independently.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Dependence of ATP-citrate lyase kinase activity on the phosphorylation of ATP-citrate lyase by cyclic AMP-dependent protein kinase.

ATP-citrate lyase from rat liver and adipose tissue is phosphorylated by either ATP-citrate lyase kinase or catalytic subunit of cyclic AMP-dependent protein kinase to 0.5-0.6 mol/subunit. We previously demonstrated that the site phosphorylated by ATP-citrate lyase kinase (peptide B) is different from that phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase (peptide A) (Ramakrishna, S., Pucci, D. L., and Benjamin, W.B. (1981) J. Biol. Chem. 256, 10213-10216). ATP-citrate lyase phosphorylation by both protein kinases added simultaneously was increased synergistically. When ATP-citrate lyase was first phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase, the net phosphorylation of the fragments subsequently phosphorylated by lyase kinase increased about 6-fold. However, when ATP-citrate lyase was first phosphorylated by lyase kinase, there was no effect on the subsequent phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Alkaline phosphatase-dephosphorylated ATP-citrate lyase was phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase to 0.9-1.0 mol/subunit. However, dephospho-ATP-citrate lyase was not phosphorylated by lyase kinase. The addition of both protein kinases simultaneously phosphorylated ATP-citrate lyase up to 2 mol/subunit. Phosphorylation of dephospho-ATP-citrate lyase first by catalytic subunit of cyclic AMP-dependent protein kinase and ATP enabled the lyase to be phosphorylated by lyase kinase. Peptide mapping and phosphoamino acid analysis of dephospho-ATP-citrate lyase phosphorylated by catalytic subunit of cyclic AMP-dependent protein kinase and/or lyase kinase conclusively showed that phosphorylation of ATP-citrate lyase by ATP-citrate lyase kinase was completely dependent on peptide A phosphorylation by cyclic AMP-dependent protein kinase. Furthermore, increased phosphorylation when both protein kinases were added simultaneously was due to increased phosphorylation at peptide B.

ATP Citrate (pro-S)-Lyase↗
PubMed1995

Nitric oxide synthesis in rat peritoneal macrophages is induced by IgE/DNP complexes and cyclic AMP analogues. Evidence in favor of a common signaling mechanism.

The production of nitric oxide was studied in rat adherent peritoneal cells stimulated with preformed IgE/DNP-BSA complexes made of IgE obtained from a secreting hybridoma. Stimulation with complexes at equivalence induced both the production of NO and an increased expression of the mRNA of the inducible isoform of NO synthase (iNOS). Production of NO was also elicited by a rabbit polyclonal F(ab')2 anti-CD23 cross-reacting with rat CD23. Because IgE/DNP-BSA complexes did not elicit Ca2+ mobilization and genistein did not influence the production of NO, cyclic AMP was considered as an alternative signaling molecule. Combination of a suboptimal concentration of dibutyryl cyclic AMP and IgE/DNP-BSA complexes showed an additive effect on NO production, whereas this was not observed when the agonists were used at supraoptimal doses. The inhibitor of cyclic AMP-specific phosphodiesterase IV, rolipram, which acts on the enzyme isoform predominantly expressed in inflammatory cells, also induced the production of NO. Furthermore, IgE/DNP-BSA complexes increased intracellular levels of cyclic AMP. Taken together, these data indicate that stimulation of mononuclear phagocytes via the low-affinity receptor Fc epsilon RII or rising intracellular concentrations of cyclic AMP leads to an enhanced expression of iNOS. Evidence in favor of the involvement of cyclic AMP in the signaling pathway linked to Fc epsilon RII is provided by the effect of IgE/DNP-BSA complexes on intracellular cyclic AMP levels and by the additive effect produced by dibutyryl cyclic AMP on NO production elicited by IgE/DNP-BSA complexes.

Amino Acid Oxidoreductases↗
PubMedDate not supplied

Modulation of the forskolin-induced cyclic AMP accumulation by corticosterone.

Experiments were undertaken to examine the influence of corticosterone on forskolin-stimulated cyclic AMP accumulation in rat cerebral cortical slices. Incubation in vitro of cerebral cortical slices with increasing concentrations of corticosterone (1 nM-100 microM) did not influence basal cyclic AMP response. Despite the lack of effect when used alone, corticosterone attenuated the effect of forskolin (10 microM) on cyclic AMP accumulation with IC50 = 24.1 +/- 5.1 microM. Corticosterone (10 microM) added to the incubation medium, for 10 min, reduced the cyclic AMP accumulation in response to increasing concentrations of forskolin (1 microM-100 microM), the concentration-response curve was shifted to the right by about one order of magnitude. In order to compare the in vitro effect of corticosterone with its effects in vivo in the next experiment the forskolin-induced cyclic AMP accumulation was measured in cerebral cortical slices from rats which were treated with corticosterone or vehicle. Similarly as in in vitro model, single dose of corticosterone (10 mg/kg sc) given to rats 2 h before sacrifice inhibited forskolin-stimulated cyclic AMP accumulation when compared with vehicle-treated control animals. In contrast, prolonged administration of corticosterone (10 mg/kg sc, twice daily for 4 and 7 days) increased forskolin-stimulated cyclic AMP accumulation in rat cerebral cortical slices when measured 2, 24 and 48 h after the administration of the last dose. These findings suggest that glucocorticoids exert multiple actions on the adenylate cyclase-coupled cyclic AMP generating system in the brain, with the ultimate effect being dependent upon amount and duration of exposure to these hormones.

Adenylyl Cyclases↗
PubMed1988

Cyclic AMP induces differentiation in vitro of human melanoma cells.

Treating human melanoma lines with dibutyryl adenosine 3':5'-cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and 11 monoclonal antibodies defining eight different melanoma-associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)-ABC antigens and beta-2-microglobulin in five of five melanoma lines. In the two HLA-DR-positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA-DR or HLA-DC antigens was observed in the Class II negative cell lines. Furthermore, dbc-AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90-kilodalton (KD) antigen, which has been found to be upregulated by interferon-gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan-associated antigen (220-240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma-associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody-defined melanoma-associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.

Antigens, Neoplasm↗