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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) has been shown to potentiate the stimulatory effect of ethanol on the hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Following an initial 20-min period, the main product of PtdEtn degradation in cells treated with TPA plus ethanol was ethanolamine phosphate. Here, we have examined the regulatory role of PKC and the possible catalytic role of phospholipase C in the formation of ethanolamine phosphate. TPA, bryostatin, and bombesin, direct or indirect activators of PKC, had similar potentiating effects on ethanol-induced formation of [14C]ethanolamine phosphate from [14C]PtdEtn in [14C]ethanolamine-prelabelled NIH 3T3 fibroblasts. At lower concentrations of ethanol (40-80 mM), significant stimulation of ethanolamine phosphate formation required longer treatments (2 h or longer). The combined effects of TPA (100 nM) and ethanol (50-200 mM) on ethanolamine phosphate formation were not inhibited by the PKC inhibitors staurosporine or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7). In contrast, these inhibitors significantly inhibited TPA-induced formation of ethanolamine, catalyzed by a phospholipase-D-type enzyme. In membranes isolated from TPA+ethanol-treated cells, enhanced formation of ethanolamine phosphate was maintained for at least 20 min. Down-regulation of PKC by prolonged (24-h) treatment of NIH 3T3 fibroblasts by 300 nM TPA enhanced, while overexpression of alpha-PKC in Balb/c fibroblasts diminished, the stimulatory effect of ethanol on the formation of ethanolamine phosphate. Finally, addition of the protein phosphatase inhibitor okadaic acid (2 microM) to fibroblasts inhibited TPA+ethanol-induced formation of ethanolamine phosphate. These results suggest that alpha-PKC-mediated protein phosphorylation may negatively regulate PtdEtn hydrolysis and that the potentiating effect of TPA may result, at least partly, from increased degradation of this PKC isoform.
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PMID:The long-term combined stimulatory effects of ethanol and phorbol ester on phosphatidylethanolamine hydrolysis are mediated by a phospholipase C and prevented by overexpressed alpha-protein kinase C in fibroblasts. 132 80

We have investigated the role of protracted phosphatase inhibition and the consecutive protracted protein phosphorylation on neuronal viability. We found that in primary cultures of cerebellar granule neurons, the protracted (24-h) inhibition of the serine/threonine protein phosphatases 1 and 2A (EC 3.1.3.16) by treatment of the cultures with okadaic acid (OKA; 5-20 nM) caused neurotoxicity that could be inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) or by the previous down-regulation of the neuronal protein kinase C (PKC; ATP:protein phosphotransferase; EC 2.7.1.37). PKC was down-regulated by exposure of the cultures for 24 h to 100 nM phorbol 12-myristate 13-acetate (TPA). The effect of the drugs used in the viability studies on the pattern of protein phosphorylation was measured by quantitative autoradiography. In particular, the 50- and 80-kDa protein bands showed dramatic changes in the degree of phosphorylation: increase by OKA and brief TPA treatment; decrease by H7 or 24 h of TPA treatment; and inhibition of the OKA-induced increase by H7 or 24 h of TPA treatment. The results suggest that the protracted phosphorylation, in particular that mediated by PKC, may lead to neuronal death and are in line with our previous suggestion that prolonged PKC translocation is operative in glutamate neurotoxicity.
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PMID:Pathological phosphorylation causes neuronal death: effect of okadaic acid in primary culture of cerebellar granule cells. 140 5

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.
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PMID:Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators. 165 30

Okadaic acid, a potent inhibitor of Type 1 and Type 2A protein phosphatases, was used to investigate the mechanism of insulin action on membrane-bound low Km cAMP phosphodiesterase in rat adipocytes. Upon incubation of cells with 1 microM okadaic acid for 20 min, phosphodiesterase was stimulated 3.7- to 3.9-fold. This stimulation was larger than that elicited by insulin (2.5- to 3.0-fold). Although okadaic acid enhanced the effect of insulin, the maximum effects of the two agents were not additive. When cells were pretreated with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), the level of phosphodiesterase stimulation by okadaic acid was rendered smaller, similar to that attained by insulin. In cells that had been treated with 2 mM KCN, okadaic acid (like insulin) failed to stimulate phosphodiesterase, suggesting that ATP was essential. Also, as reported previously, the effect of insulin on phosphodiesterase was reversed upon exposure of hormone-treated cells to KCN. This deactivation of previously-stimulated phosphodiesterase was blocked by okadaic acid, but not by insulin. The above KCN experiments were carried out with cells in which A-kinase activity was minimized by pretreatment with H-7. Okadaic acid mildly stimulated basal glucose transport and, at the same time, strongly inhibited the action of insulin thereon. It is suggested that insulin may stimulate phosphodiesterase by promoting its phosphorylation and that the hormonal effect may be reversed by a protein phosphatase which is sensitive to okadaic acid. The hypothetical protein kinase thought to be involved in the insulin-dependent stimulation of phosphodiesterase appears to be more H-7-resistant than A-kinase.
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PMID:Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation. 165 32

IL-1 treatment of human endothelial cells leads to the rapid phosphorylation of a Mr = 29,000 (P29) set of proteins to 18 times that of control cultures. Approximately 80% of the phosphorylated P29 (pP29) disappeared within 60 min although the remaining component was stable and remained for at least another 2 h. IL-1R antagonist protein blocked phosphorylation completely. Secondary treatment of IL-1 failed to increase the level of pP29 above that remaining after 1 h although other unrelated agonists that stimulated pP29 generation could. Removal of the cytokine and incubation of the cells in agonist-free medium for 2 h resulted in the total loss of the remaining pP29. Readdition of IL-1 2 h after washout restimulated P29 phosphorylation but only back to the lower level. Maximum rephosphorylation could not be attained until 16 h after IL-1 removal. Protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and staurosporine, the calcium chelators bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester and EGTA, and the calmodulin inhibitor N-(6-aminohexyl)-1-naphthalene-sulfonamide had no effect on IL-I-induced phosphorylation. However, when cultures were treated with the protein phosphatase inhibitor okadaic acid alone, the level of pP29 increased after 1 h and the presence of okadaic acid during prolonged IL-1 treatment blocked the decline in pP29. The protein synthesis inhibitors puromycin, emetine, and cycloheximide also blocked the decline in pP29 during IL-1 treatment. These data suggest that IL-1-stimulated P29 phosphorylation is made up of two components, one susceptible to prolonged down-regulation even in the absence of the cytokine and one refractory to desensitization but that remains active only in the presence of IL-1. IL-1-induced changes in pP29 levels may be dependent on the relative activities of protein kinase and protein phosphatase activities.
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PMID:Phosphorylation of an Mr = 29,000 protein by IL-1 is susceptible to partial down-regulation after endothelial cell activation. 203 50

The effects of okadaic acid, a phosphoprotein phosphatase inhibitor, on the contractile response and on myosin light chain phosphorylation were studied in intact lamb tracheal smooth muscle. The effects of okadaic acid were compared to the response of the same fibers stimulated with 1 microM methacholine, a concentration that induces 90% of maximal force. Okadaic acid (50 microM) produced a slow but maximal contraction that was accompanied by an increase in phosphorylation of the 20 kDa light chain of myosin. The myosin light chain phosphorylation pattern induced by okadaic acid, however, differed from that induced by methacholine. Ca2+ depletion, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, blocked or attenuated methacholine-induced contractions but had no significant effect on force development or myosin light chain phosphorylation induced by okadaic acid. These results suggest that phosphorylation of the 20 kDa light chain of myosin is essential for smooth muscle contraction; they also suggest that okadaic acid either uncovers or activates an apparently Ca2+ and calmodulin-independent protein kinase activity that phosphorylates the 20 kDa light chain of myosin at multiple sites.
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PMID:Okadaic acid, a phosphatase inhibitor, produces a Ca2+ and calmodulin-independent contraction of smooth muscle. 254 93

The characteristics of ATP release evoked by forskolin and ouabain from atrial segments of guinea-pig were evaluated under electrical stimulation. Forskolin (1 microM) produced a massive release of ATP together with a positive inotropic response. Both 30 microM W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.HCI), a calmodulin antagonist, and 30 microM vinblastine, a mitotic inhibitor, markedly inhibited the evoked release of ATP without affecting the evoked contraction. However, 100 microM N-ethylmaleimide abolished completely the basal and drug-evoked ATP release and further the evoked contraction. Both the ATP release and contraction evoked by ouabain (3 microM) were similarly affected by W-7, vinblastine and n-ethylmaleimide. The release of ATP, but not the contraction, evoked by forskolin was strongly suppressed by 10 microM okadaic acid, a protein phosphatase inhibitor. The suppression by okadaic acid of the evoked release was thoroughly antagonized in the presence of 0.01 microM PMA (phorbol 12-myristate 13-acetate), but not 10 microM H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine). These results suggest that forskolin, like ouabain, may dominantly cause the neuronal release of ATP from cardiac adrenergic nerves, although the possible participation of release from muscular sources cannot be ignored.
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PMID:Possible neuronal origin of ATP release evoked by forskolin and ouabain from guinea-pig atrial segments. 749 79

The mechanism by which norepinephrine (NE) down-regulates alpha 1B-adrenergic receptor (alpha-AR) mRNA was studied in rabbit aortic smooth muscle cells. NE, phorbol esters, and bradykinin each decreased alpha-AR mRNA levels by 70-80%. The protein kinase C inhibitor (+)-1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) abolished the effects of phorbol esters and NE and decreased basal mRNA levels by 52 +/- 3%. Neither ryanodine nor EGTA inhibited down-regulation of alpha-AR mRNA by NE. Actinomycin D caused alpha-AR mRNA level to decrease with a half-life of 3.2 +/- 0.4 h and blocked the effect of H-7 to decrease basal alpha-AR mRNA level. Both NE and phorbol esters increased the rate of alpha-AR mRNA degradation. In NE-desensitized cells, phorbol esters and bradykinin each caused the expected down-regulation of alpha-AR mRNA. The protein phosphatase inhibitor okadaic acid prolonged the normally transient effect of NE for at least 24 h. We conclude that protein kinase C exerts two opposing effects on alpha-AR mRNA levels, 1) a decrease in the stability of the mRNA that requires the sustained phosphorylation of a protein kinase C substrate and 2) a permissive effect on alpha-AR gene transcription.
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PMID:Phorbol esters and norepinephrine destabilize alpha 1B-adrenergic receptor mRNA in vascular smooth muscle cells. 829 18

Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 microM 8-bromo-cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13-dibutyrate to activate protein kinase C. During repeated 6-s stimulation at 5-min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8-bromo-cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13-dibutyrate, desensitization is blocked. Okadaic acid-treated cells stimulated in the presence of 8-bromo-cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated K+ as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin cells.
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PMID:Protein phosphorylation at a postreceptor site can block desensitization and induce potentiation of secretion in chromaffin cells. 838 51

The effect of hyperosmolarity on cloned Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 was studied in stably transfected PS120 fibroblasts. Na+/H+ exchanger activity was determined spectrofluorometrically in acidified cells that were exposed to isosmolar (300 mosmol/kg) or hyperosmolar (450 mosmol/kg) media, in which the only difference is the presence or absence of 150 mM mannitol. Hyperosmolar solution reversibly inhibited NHE2 and NHE3 with a delay of approximately 15 s. Hyperosmolarity significantly reduced their maximal reaction velocity compared with isosmolar medium but did not alter their Michaelis-Menten constant for intracellular H+. The Michaelis-Menten constant of the exchangers for extracellular Na+ in hyperosmolar medium was not different from that in isosmolar medium. Pretreatment of PS120/NHE3 cells with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the tyrosine kinase inhibitor genistein, and the serine/threonine protein phosphatase inhibitor okadaic acid did not affect the hyperosmolar inhibition of NHE3. Hyperosmolar inhibition of Na+/H+ exchanger activity was also observed in PS120 cells transfected with truncated NHE3 cDNAs (E3/585, E3/543, E3509, and E3/475) and NHE2 cDNA (E2/499). We conclude that 1) hyperosmolarity inhibits NHE2 and NHE3, in contrast to the stimulatory effect on the housekeeping isoform NHE1, 2) this inhibition is reversible, and 3) the COOH termini of NHE2 and NHE3 are not necessary for hyperosmolar inhibition of NHE2 and NHE3.
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PMID:Hyperosmolarity inhibits the Na+/H+ exchanger isoforms NHE2 and NHE3: an effect opposite to that on NHE1. 863 9


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