Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.
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PMID:In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase. 200 28

1. Pulsatile application of serotonin (5-HT) leads to facilitation of excitatory postsynaptic potentials (EPSPs) in crayfish "opener" neuromuscular preparations. The facilitation resulting from a single application of serotonin shows two phases: an early, rapidly decaying phase, and a less intense, long-lasting phase of 1- to 2-h duration. A previous study implicated the phosphatidylinositol system as an essential component in serotonin-induced facilitation, especially the early phase. The present study was conducted to determine the roles of the adenylate cyclase and phosphatidylinositol systems in both phases of serotonin-induced facilitation. 2. Relatively brief applications of agents known to affect the intracellular concentration of cAMP (forskolin, 1 microM; and IBMX, 100 microM) cause an increase in EPSP amplitude, which persists for 1-2 h. 3. The duration of the less intense, long-lasting phase of serotonin-induced facilitation is prolonged in the presence of 1 microM IBMX. This concentration of IBMX does not affect EPSP amplitude by itself. A membrane-permeant analog of cAMP (applied in concentrations less than or equal to 1 mM) is also not effective in altering EPSP amplitude. However, when dibutyryl cAMP is applied in the presence of 1 microM IBMX, EPSP amplitude is increased (60-80%). 4. Localized presynaptic injection of the "Walsh Inhibitor" (PKI), which inhibits cAMP-activated protein kinase, blocks the less intense, long-lasting phase of serotonin-induced facilitation at synapses near the site of injection. Normal facilitation develops at synapses within the same preparation remote from the site of injection. Distribution of the injected inhibitor within the axon can be visualized by tagging PKI with a fluorescent marker.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conjoint action of phosphatidylinositol and adenylate cyclase systems in serotonin-induced facilitation at the crayfish neuromuscular junction. 248 Sep 94

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.
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PMID:Activation of tyrosine hydroxylase in PC12 cells by the cyclic GMP and cyclic AMP second messenger systems. 287 73

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.
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PMID:Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. 299 99

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposed to aldosterone. 2. The response to injected Gpp(NH)p is not only greater in size than in unexposed fibres but also sustained. 3. Injection of MgCl2 following peak stimulation causes a partial reversal of the response. 4. Injection of ATPNa2 (and 5'-App(NH)p) leads to a sustained stimulatory response which is not significantly greater than that seen in unexposed fibres. 5. MgCl2 injection causes complete reversal of this response. 6. The response of preexposed fibres to injected CaCl2 in varying concentration and to injected cholera toxin is not significantly different from that seen in unexposed fibres. 7. This is also true of Gpp(NH)p when it is injected after peak stimulation by cholera toxin. 8. Prior application of verapamil (10(-4)M) drastically reduces the response to injected Gpp(NH)p. 9. The residual response is sustained but markedly reduced by injected Mg2+, Fe or Zn. 10. Injection of PKI following Gpp(NH)p reduces the response, provided PKI is also injected before Gpp(NH)p. By contrast, injection of R11 subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine, when applied externally (5 X 10(-5)M), cause almost complete reversal of the response. 12. The suggestion is made that the response to injected Gpp(NH)p is mainly due to activation of Ca2+-channels resulting in activation of the calmodulin/Ca-dependent form of adenylate cyclase and that the primary site of aldosterone action is at the level of the calmodulin form of adenylate cyclase.
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PMID:Increased sensitivity to injected 5'-guanylylimidodiphosphate of the sodium efflux in barnacle muscle fibres preexposed to aldosterone. 613 62

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.
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PMID:Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP. 628 69

The effect of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activity on 4-aminopyridine (4-AP)-sensitive delayed rectifier current (IdK) in isolated rabbit portal vein smooth muscle cells was studied via whole cell voltage clamp (20-22 degrees C). A threefold increase in 4-AP-sensitive (5 mM) IdK was recorded after gaining cell access during dialysis with 5 mM intracellular ATP and internal Ca2+ buffered to a low level with 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Dialysis with the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate (5 mM) or the specific peptide inhibitor of PKA (PKI; 10 microM) reduced current runup by 50 and 70%, respectively. Delayed dialysis with PKI reversed runup and inhibited IdK to below initial levels. Forskolin (1 microM) caused a reversible increase in IdK, which was inhibited by 4-AP (5 mM). Isoproterenol (1 microM) reversibly enhanced IdK; the increase was sensitive to propranolol (2 microM) and 4-AP (5 mM) and was prevented by dialysis with PKI (10 microM). IdK was enhanced over the entire voltage range of activation and associated with a negative shift in reversal potential of net whole cell current, consistent with hyperpolarization of resting membrane potential. The data provide the first evidence for a signal transduction mechanism involving beta-adrenoceptors, adenylate cyclase, and a phosphotransferase reaction mediated by PKA for the regulation of delayed rectifier K+ channels in vascular smooth muscle.
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PMID:Phosphorylation by protein kinase A enhances delayed rectifier K+ current in rabbit vascular smooth muscle cells. 786 21

1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (ICa) in the absence of beta-adrenergic agonists. The increase occurred without effects on the peak current-voltage relation or voltage-dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (IK), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occurred without effects on voltage-dependent activation or reversal potential. 2. Analysis of the dose-response relations for OA and MC on ventricular cell ICa were best fitted with a single-site relationship with a K1/2 of 1.58 microM and 0.81 microM, respectively. These data suggest the predominant form of phosphatase active on ICa in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal ICa suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the ICa increase induced by OA or MC. This demonstrates that the effects of OA and MC on ICa are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished IK, suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was without effect on basal current levels of ICa or IK, suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of ICa to isoprenaline or forskolin, PKI does not affect the increase in ICa induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(Rp)-phosphothioate (Rp-cAMPS) also had no effect on the response to OA or MC. 5. Application of beta-adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK-A is not mediating the OA response and that phosphatase inhibition does not result in complete phosphorylation of PK-A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on ICa and IK, several types of protein kinase inhibitors were internally perfused.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes. 814 46

We studied the effects of prostaglandin E2 (PGE2) on hyaluronan synthesis in rabbit pericardial mesothelial cells, and the following results were obtained. (1) PGE2 (10-1000 ng/ml) stimulated hyaluronan synthesis and the level of hyaluronan synthase activity in a dose- and time-dependent manner, but PGF2 alpha did not. (2) Cyclic AMP (cAMP) levels in the cells peaked (about a 7-fold increase) at 5-10 min after adding PGE2 (1000 ng/ml). (3) Increased hyaluronan synthesis induced by PGE2 was significantly inhibited after pretreatment with either an adenylate cyclase inhibitor (2',5'-dideoxyadenosine) or a cAMP-dependent protein kinase inhibitor (PKI 5-24), but there was no inhibition with the protein kinase C inhibitor H-7. (4) When the intracellular cAMP level was raised by manipulating the levels of dibutyryl cyclic AMP or forskolin, hyaluronan synthesis and the level of hyaluronan synthase activity were also stimulated. These results suggest that PGE2 produced by cells stimulates hyaluronan synthesis in rabbit pericardial cells and that the stimulation mechanism involves the cAMP-mediated protein kinase signal transduction process.
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PMID:Prostaglandin E2 stimulates cyclic AMP-mediated hyaluronan synthesis in rabbit pericardial mesothelial cells. 838 37

The ultrarapid delayed rectifier K+ current (IKur) in human atrial cells appears to correspond to Kv1.5 cloned channels and to play an important role in human atrial repolarization. Kv1.5 channels have consensus sites for phosphorylation by protein kinase A and C, suggesting possible modulation by adrenergic stimulation. The present study was designed to assess the adrenergic regulation of IKur in human atrial myocytes. Isoproterenol increased IKur in a concentration-dependent manner, with significant effects at concentrations as low as 10 nmol/L. The effects of isoproterenol were reversible by washout or by the addition of propranolol (1 mumol/L). Isoproterenol's effects were mimicked by the direct adenylate cyclase stimulator, forskolin, and by the membrane-permeable form of cAMP, 8-bromo cAMP. Isoproterenol had no effect on IKur when the protein kinase A inhibitor peptide, PKI(6-22)amide, was included in the pipette solution; in a separate set of experiments in which isoproterenol alone increased IKur by 45 +/- 9% relative to control, subsequent superfusion with isoproterenol in the presence of the protein kinase inhibitor H-7 failed to alter IKur. In contrast to isoproterenol, phenylephrine (in the presence of propranolol to block beta-adrenegic effects) induced a concentration-dependent inhibition of IKur, with significant effects observed at concentrations as low as 10 mumol/L. The inhibitory actions of phenylephrine were reversed by the addition of prazosin and prevented by coadministration with a highly selective inhibitor of protein kinase C, bisindolylmaleimide. These results indicate that beta-adrenergic stimulation enhances, whereas alpha-adrenergic stimulation inhibits, IKur and suggest that these actions are mediated by protein kinase A and protein kinase C, respectively. The modulation of IKur by adrenergic influences is a potentially novel control mechanism for human atrial repolarization and arrhythmias.
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PMID:Adrenergic modulation of ultrarapid delayed rectifier K+ current in human atrial myocytes. 862 Jun 11


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