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)

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

The phosphorylation of one receptor that occurs as a result of the stimulation of a different receptor on a cell is a common mechanism for heterologous regulation or "cross-talk," which has been implicated in desensitization. In this work, we focus on the mechanisms of phosphorylation of the rat pancreatic acinar cell cholecystokinin (CCK) receptor that occur upon stimulation of this cell by various agonists. Phosphorylation was allowed to occur in dispersed intact acinar cells in response to the experimental manipulation, and the phosphoreceptor was subsequently purified and quantified as an indication of response. Agonists such as vasoactive intestinal polypeptide and secretin, which act via activation of adenylate cyclase, had no effect on CCK receptor phosphorylation, whereas carbamylcholine and bombesin stimulated increased phosphorylation of the CCK receptor. Because these agents would be expected to activate protein kinase C (PKC) as well as a number of calcium-sensitive kinases and phosphatases, these activities were further dissociated by using more direct activators and inhibitors acting intracellularly. Manipulation of calcium independent of PKC by using a calcium ionophore, inhibition of calcium/calmodulin-dependent kinase II, and inhibition of calcium-dependent protein phosphatase type 2B had no effect on the state of CCK receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanisms of heterologous agonist-stimulated phosphorylation of cholecystokinin receptor. 817 63

In C6-2B rat glioma cells, agonist-stimulated cAMP accumulation is potently inhibited after the stimulation of endogenous bradykinin receptors or stably transfected substance K receptors, coupled to phosphatidylinositol hydrolysis. In the present report, pharmacological tools were used to selectively stimulate either protein kinase C or Ca2+, the two final effectors activated upon phosphatidylinositol hydrolysis, and their role in the inhibition of the C6-2B cell cAMP signaling pathway was investigated. Activation of protein kinase C by an acute treatment with phorbol 12-myristate 13-acetate or L-alpha-1-oleoyl-2-acetyl-sn-3-glycerol did not reduce, but rather enhanced, the cAMP accumulation elicited by forskolin, a direct activator of adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. This effect was antagonized by the protein kinase inhibitor H-7 and mimicked by the protein phosphatase inhibitor okadaic acid. Thapsigargin, a selective microsomal Ca(2+)-ATPase inhibitor, evoked a sustained increase in the intracellular free Ca2+ concentration, with an EC50 of 24.8 +/- 4.3 nM, and inhibited the cAMP accumulation induced by the beta-adrenergic receptor agonist isoproterenol with comparable potency (IC50 = 19.3 +/- 0.2 nM), strongly suggesting a causal relationship between the two phenomena. The inhibition by thapsigargin of isoproterenol- or forskolin-stimulated cAMP accumulation was not affected by pertussis toxin or down-regulation or inhibition of protein kinase C. Dantrolene, a blocker of Ca2+ release from intracellular stores, antagonized 1) the Ca2+ transient in response to thapsigargin and substance K and 2) the inhibitory effect of these compounds on isoproterenol- or forskolin-induced cAMP accumulation. Moreover, sequestration of intracellular Ca2+ with the cell-permeable Ca2+ chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester abolished the cAMP inhibition mediated by thapsigargin. Finally, isoproterenol- or forskolin-stimulated adenylyl cyclase activity in digitonin-permeabilized cells was not affected by either thapsigargin or substance K. These data provide compelling evidence that increases in intracellular free Ca2+ concentration without activation of protein kinase C suffice and are responsible for the inhibition of cAMP accumulation in C6-2B cells.
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PMID:Ca2+ inhibition of beta-adrenergic receptor- and forskolin-stimulated cAMP accumulation in C6-2B rat glioma cells is independent of protein kinase C. 838 3

Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period. When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine. Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid. Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA. Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA. Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity. Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells. It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells. The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.
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PMID:Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C. 855 18

Streptozotocin-induced diabetes caused a profound increase in the steady-state level of phosphorylation of the alpha-subunit of the adenylate cyclase inhibitory protein Gi2 in hepatocytes. Unlike hepatocytes from control animals, those from streptozotocin-diabetic animals showed no increase in the phosphorylation of Gi2 alpha in response to a challenge with the protein kinase C activator phorbol myristate acetate. However, a stimulatory effect of 8-bromo-cAMP on Gi2 alpha phosphorylation was evident in hepatocytes from diabetic animals but this was severely reduced compared with that observed in hepatocytes from normal animals. Two-dimensional tryptic phosphopeptide mapping showed that Gi2 alpha in resting hepatocytes from diabetic animals was phosphorylated exclusively at the protein kinase C site (C-site) but no labelling was evident at the protein kinase A-regulated site (AN-site). Treatment of hepatocytes from diabetic animals with phorbol myristate acetate did not change this pattern of labelling. In contrast, challenge of hepatocytes from diabetic animals with 8-bromo-cAMP led to the appearance of a new labelled phosphopeptide that was consistent with labelling at the AN-site. Analysis of the C-site and AN-site phosphopeptides from hepatocytes of diabetic animals treated with 8-bromo-cAMP showed that the increase in labelling of Gi2 alpha caused by this ligand could be attributed almost entirely to labelling at the AN-site. Thus streptozotocin diabetes appears to cause enhanced labelling of hepatocyte Gi2 alpha by exclusively increasing phosphorylation at the C-site. It is suggested that the increased labelling at the C-site reflects an augmentation of the protein kinase C signalling system in hepatocytes from streptozotocin-induced diabetic animals. This may have wide-spread functional consequences for these cells and may result either from an increased protein kinase C activity and/or a reduction in protein phosphatase 1 and/or 2A activity.
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PMID:Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2 alpha at the protein kinase C site but not at the protein kinase A-controlled site. 861 8

Neurotransmitters and hormones such as somatostatin, galanin, and adrenalin reduce insulin secretion. Their inhibitory action involves direct interference with the exocytotic machinery. We have examined the molecular processes underlying this effect using high resolution measurements of cell capacitance. Suppression of exocytosis was maximal at concentrations that did not cause complete inhibition of glucose-stimulated electrical activity. This action was dependent on activation of G proteins but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. The molecular processes initiated by the agonists culminate in the activation of the Ca(2+)-dependent protein phosphatase calcineurin, and suppression of the activity of this enzyme abolishes their action on exocytosis. We propose that mechanisms similar to those we report here may contribute to adrenergic and peptidergic inhibition of secretion in other neuroendocrine cells and in nerve terminals.
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PMID:Neurotransmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin. 881 14

Many of the hormone-regulated ion transport processes in distal nephron involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate molecular mechanisms underlying the ionic permeability of the distal tubule apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused onto a planar lipid bilayer. These experiments led to the identification of several ionic channels including a Cl(-)-permeable channel of 14 pS with a Na+ over Cl- permeability ratio, PNa/PCl < 0.09. The open channel probability (Po) showed a weak voltage dependency with Po increasing slightly at negative potential values (intracellular (trans) relative to extracellular (cis) for right-side-out vesicles). Channel activity was inhibited by NPPB at high concentrations (> 100 microM) and by DIDS (300 microM). A small inhibitory effect was also observed in the presence of DPC at concentrations ranging from 200 microM to 500 microM. The presence of SO4(2-) (32 mmol/l) in the trans solution caused a complete inhibition of channel activity, but no modification of channel behaviour was observed with the non-selective channel blocking agent gadolinium (Gd3+) at 100 microM. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to an increase in channel activity characterized by a greater number of active channels coupled to an increase of the individual channel open probability. The action of the protein kinase A could be cancelled by the addition of a non specific protein phosphatase, such as alkaline phosphatase. Our results suggest that the apical membrane of the rabbit distal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones linked to the adenylate cyclase pathway.
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PMID:Evidence from incorporation experiments for an anionic channel of small conductance at the apical membrane of the rabbit distal tubule. 897 99

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.
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PMID:cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression. 901 60

The infection of cultured endothelial cells with human cytomegalovirus (HCMV) is generally limited to less than 10% of the cells in contrast to HCMV infection of fibroblasts, where essentially all cells can be infected. It is known that HCMV infection influences a number of signal transduction pathways of infected cells. We therefore questioned whether, conversely, the infectivity of human umbilical vein endothelial cells could be influenced by the deliberate activation of these pathways. When endothelial cells were treated prior to infection with phorbol myristoyl acetate, an activator of protein kinase C, the number of HCMV-positive cells increased two to three times. On the other hand, pretreatment of the cells with RO 31-8220, a specific protein kinase C inhibitor, or with staurosporine, a general protein kinase inhibitor, resulted in a decreased infection level and in abolishment of the PMA-induced effect. Pretreatment with the protein phosphatase inhibitor, okadaic acid, caused a slight increase in infectivity, whereas pretreatment with the protein tyrosine kinase inhibitor, genistein, was without effect. Furthermore, neither forskolin and ilomedine, compounds known to activate the endothelial adenylate cyclase, nor the calcium ionophore A23187 were able to influence HCMV infectivity. It is concluded that: (a) the HCMV infection level of unstimulated endothelial cells is influenced by the basal level of protein kinase C; and (b) stimulation of protein kinase C prior to infection results in an increase of infection by HCMV.
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PMID:Activation of protein kinase C enhances the infection of endothelial cells by human cytomegalovirus. 917 59

Treatment of rat hepatocytes with the phosphatase inhibitors okadaic acid or ortho-vanadate had led to an 80% decrease in the bacterial mutagenicity of several aromatic amines metabolically activated by these hepatocytes. This is the most dramatic change yet demonstrated in mutagenicity by phosphorylation modulation. However, incorporation of phosphate into and catalytic activity of cytochromes P450 (CYP) 1A1 and 1A2, the major catalysts for the first step in the toxication of aromatic amines, were unchanged. We therefore investigated whether changes in the phosphorylation status would influence the activities of the N-acetyltransferases NAT1 and/or NAT2, being responsible for one of the two major pathways leading to the ultimate mutagens, the reactive esters which are derived from the N-hydroxylated metabolites of aromatic amines. Hepatocytes were derived from the livers of rats pretreated with CYP1A1/1A2 inducers and from untreated rats using conditions under which the phosphorylation-dependent drastic decrease of the arylamine mutagenicity was observed. Treatments were exposure to 1 mM dibutyryl-cAMP (protein kinase A stimulator), 100 nM okadaic acid or 20 nM calyculin A (preferential inhibitors of serine/threonine phosphatases PP2A and PP1, respectively), 2 mM ortho-vanadate (inhibitor of tyrosine phosphatases), and 50 mM NaF (stimulator of adenylate cyclase and non-specific inhibitor of protein phosphatases). None of the phosphorylation modulators led to a significant change in NAT1 or NAT2 activities. This was true for hepatocytes from rats which had been pretreated with inducers for CYP1A1 and CYP1A2 as well as from untreated rats. The inducers led to the expected increases in CYP1A1 and CYP1A2 but the NAT1 and NAT2 activities remained unchanged. Our study shows that the N-acetyl transferases NAT1 or NAT2, the catalysts responsible for the formation of the highly reactive N-acetoxy derivatives of N-hydroxylated aromatic amines, are not responsible for the drastic decrease in arylamine genotoxicity after treatment of the metabolizing system with protein phosphatase inhibitors. The data also show that NAT1 and NAT2 are not regulated by the classical xenobiotic metabolizing enzyme inducers nor by any of the phosphorylation modulators used.
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PMID:Control of the mutagenicity of arylamines by protein kinases and phosphatases: II. Lack of response of rat liver N-acetyl transferases to phosphorylation modulators. 933 4


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