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)

Okadaic acid completely inhibits phosphatase 2A at nanomolar concentrations, while complete inhibition of type 1 phosphatases occurs at 1 microM. Phosphatase 2B is significantly inhibited only at concentrations > 1 microM. In rat pancreatic acini, 1 microM okadaic acid shifted the cholecystokinin (CCK) dose-response curve for stimulating amylase release to the right without reducing maximal secretion. At 3 microM, okadaic acid inhibited maximal CCK-induced amylase release to 78 +/- 7% of control, whereas the inactive analogue 1-Nor-okadaone had no effect. Three lines of evidence indicate that this inhibition by okadaic acid occurs at a late step in stimulus-secretion coupling: 1) intracellular Ca2+ signaling in response to agonist stimulation was not appreciably altered by okadaic acid; 2) stimulation with phorbol ester plus thapsigargin (thus by-passing receptor activation), which gave 85 +/- 4% of maximal CCK-induced amylase release, was inhibited 66 +/- 4% by 3 microM okadaic acid; and 3) Ca(2+)-induced amylase secretion in streptolysin O-permeabilized cells was also reduced by 85 +/- 7%. Two-dimensional polyacrylamide gel electrophoresis of 32P-labeled acini and autoradiography demonstrated that okadaic acid dose dependently increased overall protein phosphorylation. Correspondingly, okadaic acid also led to an inhibition of CCK-induced dephosphorylation. These results show that okadaic acid inhibits pancreatic acinar secretion at a step after generation of intracellular messengers and indicate a role for protein dephosphorylation in stimulus-secretion coupling.
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PMID:Effects of okadaic acid indicate a role for dephosphorylation in pancreatic stimulus-secretion coupling. 128 97

The immunosuppressant cyclosporin A (CsA) was utilized as a highly specific inhibitor of the Ca2+/calmodulin-dependent protein phosphatase, PP2B in rat pancreatic acinar cells. Treatment of cells with CsA for 20 min resulted in a concentration-dependent inhibition of PP2B that was maximal (> 90%) at 1 microM and exhibited an IC50 of 65 nM. CsA also inhibited cholecystokinin-, 100 pM, or carbamylcholine-, 10 microM, induced amylase release in a concentration-dependent manner. A maximal inhibition to 55% of stimulated control cells occurred at 1 microM CsA with half-maximal inhibition occurring at approximately 200 nM. Secretion in response to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) was uneffected by CsA treatment. Conversely, amylase release stimulated by the Ca2+ mobilizing agent, thapsigargin, when added alone at 2 microM or in combination with TPA, was inhibited by CsA to 66 and 61% of control cells, respectively. These data indicate that CsA-mediated inhibition occurs only when stimulation involves an increase in intracellular Ca2+. In addition, analogues of CsA, 6-methyl-alanine-CsA, and 11-methyl-leucine-CsA had no effect on PP2B activity or amylase secretion. The chemically distinct immunosuppressant, FK506, produced only partial inhibition of PP2B activity and did not significantly inhibit amylase secretion at concentrations up to 1 microM. Two-dimensional gel electrophoresis of proteins from 32P-labeled acinar cells revealed that CsA specifically blocked the cholecystokinin-stimulated dephosphorylation of a 24-kDa protein in a concentration range similar to that seen for inhibition of secretion. Using 32P-labeled cytosol and purified calcineurin, a Ca(2+)- and calmodulin-dependent dephosphorylation of the 24-kDa protein was also demonstrated in vitro. Collectively, these data indicate that the Ca2+/calmodulin-dependent protein phosphatase, PP2B, plays a significant role in stimulus-secretion coupling in pancreatic acinar cells.
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PMID:Cyclosporin A inhibits Ca2+/calmodulin-dependent protein phosphatase and secretion in pancreatic acinar cells. 751 49

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

Despite physiological evidence that cholecystokinin (CCK) is an excitatory neurotransmitter in the brain, little is known about its mechanism of action. CCK immunoreactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phosphorylation of a dopamine- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) in striatal neurons. The effect of CCK-8S is prevented by antagonists of CCKB and N-methyl-D-aspartate receptors. Our results support a model in which CCK-8S, originating from CCK or CCK/glutamate corticostriatal neurons, promotes the release of an excitatory neurotransmitter that causes the dephosphorylation and inactivation of DARPP-32, a potent protein phosphatase inhibitor, thereby modulating neuronal excitability.
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PMID:Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum. 824 41

Phosphorylation of cell surface receptors regulates the physiological response to many hormones and neurotransmitters. We have demonstrated that the receptor for cholecystokinin (CCK), the major secretagogue for the exocrine pancreas, is phosphorylated on serines in response to CCK (Klueppelberg, U.G., Gates, L.K., Gorelick, F.S., and Miller, L.J. (1991) J. Biol. Chem. 266, 2403-2408). Receptor phosphorylation was transient, even in the continued presence of agonist, and suggested a role for pancreatic protein phosphatases (PP) in regulating receptor phosphorylation in the intact cell. Treatment of acinar cells with okadaic acid increased the extent and duration of receptor phosphorylation induced by CCK. Receptor phosphorylated in the intact cell in response to CCK was used as substrate to analyze protein phosphatases in pancreatic acinar cell extracts. The predominant CCK receptor phosphatase activity was found in the cytosol and was potently inhibited by okadaic acid (IC50 = 0.2 nM). This phosphatase activity was unaffected by inhibitor-2, Ca2+, or Mg2+, suggesting that the major receptor phosphatase was PP-2A. Stimulation of cells with CCK resulted in a 3-fold increase in protein phosphatase activity toward the CCK receptor, at the same time that phosphorylase a phosphatase activity was unchanged. This increase in receptor phosphatase activity was reflected only in the cytosol, with the particulate activity unchanged. Consistent with this subcellular localization, the hormone-regulated receptor phosphatase activity was PP-2A, with chromatographic separation demonstrating activity in both PP-2A1 and PP-2A2 forms. These data suggest that CCK coordinates the activity of both protein kinases and phosphatases acting on the CCK receptor to control the extent and duration of receptor phosphorylation in the pancreatic acinar cell.
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PMID:Control of cholecystokinin receptor dephosphorylation in pancreatic acinar cells. 838 71

In cholera toxin-treated gastric chief cells, incubation with a cholinergic agonist (carbamylcholine), a regulatory peptide (cholecystokinin), or a calcium ionophore (A23187) causes a dose- and time-dependent potentiation of cAMP levels. Because this augmented response is calcium/calmodulin-dependent, we hypothesized that it was mediated by calcineurin (protein phosphatase 2B). To test this hypothesis, we examined the actions of calcineurin inhibitors on secretagogue-induced potentiation of cAMP levels in guinea pig chief cells. Preincubation of cells with 0.1 microM FK-506 completely prevented carbachol-induced augmentation of cAMP levels and pepsinogen secretion from cholera toxin-treated cells. Cyclosporin-A, another calcineurin inhibitor, also prevented the augmented cAMP response. FK-506 and cyclosporin inhibited augmentation of cAMP levels following treatment with cholecystokinin(26-33) and A23187, but not the smaller increase in cAMP following treatment with a phorbol ester that activates protein kinase C. Hence, the actions of calcineurin inhibitors were limited to secretagogues that increase cellular calcium. Rapamycin, an agent that competes with FK-506 for the immunophilin, FK binding protein 12, does not inhibit calcineurin. In the present study, preincubation with rapamycin did not prevent carbachol-induced augmentation of cAMP levels in cholera toxin-treated chief cells. However, a molar excess of rapamycin reversed the inhibitory actions of FK-506. These experiments provide further evidence that the actions of FK-506 on cholera toxin-treated gastric chief cells are caused by its inhibitory actions on calcineurin. FK-506 also inhibited potentiation of cAMP levels when carbachol was added to cells that were preincubated with forskolin, an agent that directly activates adenylyl cyclase. We conclude that, in gastric chief cells, calcineurin mediates cross-talk between the calcium/calmodulin and adenylyl cyclase signaling pathways.
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PMID:Calcineurin mediates calcium-induced potentiation of adenylyl cyclase activity in dispersed chief cells from guinea pig stomach. Further evidence for cross-talk between signal transduction pathways that regulate pepsinogen secretion. 870 99

Cholecystokinin (CCK) is known to rapidly and transiently increase both [Ca2+]i and autonomous CaM kinase II activity in rat pancreatic acini. Because induction of autonomous activity may involve intramolecular autophosphorylation, the effects of protein phosphatase inhibitors were examined. None of the inhibitors tested (okadaic acid, calyculin A, and cyclosporin A) affected basal activity. Okadaic acid, a potent inhibitor of PP2A and weaker inhibitor of PP1, increased the peak autonomous activity by 30% over the level normally induced by CCK alone, while calyculin A, a potent inhibitor of both PP1 and PP2A, showed an even greater increase of 97%. Both inhibitors also delayed the decline of autonomous activity and calyculin A had a more potent effect than okadaic acid. Cyclosporin A, an inhibitor of PP2B, had no effect. The data indicate that PP1 may be involved in the dephosphorylation of CaMK II and decline of autonomous activity.
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PMID:Protein phosphatase inhibitors potentiate Ca2+/calmodulin-dependent protein kinase II activity in rat pancreatic acinar cells. 875 94

Effects of cholecystokinin octapeptide (CCK-8) were investigated in CA1 pyramidal neurons of rat hippocampal slice cultures using the whole-cell patch-clamp technique. In the current-clamp mode, CCK-8 (100 nM) produced slight depolarizaton (2.1 +/- 0.3 mV) and reduced the amplitude of afterhyperpolarization following a train of spikes. CCK-8 (10 nM-1 microM) concentration-dependently reduced the amplitude of afterhyperpolarization. CCK-4, a selective agonist for CCK(B) receptors, also attenuated the amplitude of afterhyperpolarization. CCK-8-induced suppression was completely abolished by (+)L-365,260, a selective CCK(B) receptor antagonist, but not by (-)L-364,718, a selective CCK(A) receptor antagonist. Similarly, CCK-8 reduced the tail currents following a depolarizing pulse. The tail currents were characterized as Ca2+-activated K+ currents. When neurons were held at a holding potential of -40 mV, CCK-8 elicited inward currents with a reduction of membrane conductance. This current had a relatively linear current voltage relationship and was reversed in polarity at membrane potentials close to the K+ equilibrium potential, suggesting that CCK-8 decreases leak K+ currents. Moreover, voltage-activated Ca2+ currents were partially blocked by CCK-8, and this effect was enhanced by intracellular application of GTPgammaS (300 microM) or a protein phosphatase inhibitor, okadaic acid (100 nM), and attenuated by GDPbetaS (300 microM) or a protein kinase inhibitor, staurosporin (400 nM). In acutely-prepared hippocampal slices from neonatal rats, CCK-8 also depolarized CA1 pyramidal neurons and suppressed afterhyperpolarization following a train of action potentials. These results indicate that CCK-8 increases neuronal excitability by suppressing leak K+ currents and Ca2+-activated K+ currents in CA1 pyramidal neurons of the hippocampus through activation of CCK(B) receptors.
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PMID:Electrophysiological changes in rat hippocampal pyramidal neurons produced by cholecystokinin octapeptide. 917 69

The molecular mechanisms whereby calcium stimulates secretion are uncertain. In the present study, we used streptolysin O (SLO)-permeabilized chief cells from guinea pig stomach to investigate whether protein phosphatase-2B (calcineurin), a calcium/calmodulin-dependent, serine/threonine phosphatase plays a role in mediating calcium-induced pepsinogen secretion. Preincubation of cells with alpha-naphthylphosphate, a non-specific phosphatase inhibitor, decreased calcium-induced secretion. Likewise, specific inhibitors of protein phosphatase-2B (cyclosporin-A and FK-506) caused a dose-dependent reduction in calcium-induced pepsinogen secretion. Moreover, in intact cells, cyclosporin-A and FK-506 inhibited pepsinogen secretion caused by cholecystokinin, carbamylcholine and A23187, agonists known to increase chief cell cytosolic calcium. Okadaic acid, an inhibitor of protein phosphatase-1 and -2A, had no effect on secretion caused by these agonists. Chief cell calcium-dependent phosphatase activity, measured using radiolabeled casein as substrate, was reduced selectively by inhibitors of protein phosphatase-2B. Endogenous substrates for calcium/calmodulin-dependent phosphatase activity were identified by analyzing chief cell lysates using 2-dimensional gel electrophoresis. Increasing the cytosolic calcium concentration resulted in dephosphorylation of a 55-kDa, acidic cytoskeletal protein. FK-506 inhibited dephosphorylation of this protein. Thus, in permeabilized chief cells, specific inhibitors of protein phosphatase-2B inhibit calcium-induced pepsinogen secretion, calcium/calmodulin-dependent phosphatase activity and calcium-induced dephosphorylation of a 55-kDa, acidic cytoskeletal protein. These results support the hypothesis that protein phosphatase-2B (calcineurin) plays an important role in mediating calcium-induced exocytosis.
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PMID:Regulation of calcium-induced exocytosis from gastric chief cells by protein phosphatase-2B (calcineurin). 920 77

We aimed to clarify the topology and immunohistochemistry of CO2/H+-sensitive neurons in the ventral medullary surface (VMS), the central chemoreceptor area in rats. Inhalation of 3 and 7% CO2 in air significantly decreased pH in arterial blood and increased paCO2, which caused hyperpneic and tachypneic responses. Following inhalation of 3 and 7% CO2 in air for 5 min, the density of c-Fos-immunoreactive (IR) neurons increased stepwise not only in the 3rd-5th divisions of the VMS (between the caudal end of the nucleus corporis trapezoidei and the caudal end of the area postrema), but also in the rostroventromedial medulla (RVMM). Following inhalation of 7% CO2 in air for 5 min, glutamate-, glutamic acid decarboxylase (GAD)-, calcineurin- and cAMP-IR neurons were found not only in the VMS, but also in the RVMM. The topology of these neurons was similar to that of the c-Fos-IR neurons. No immunoreactivity was found for serotonin, substance P, somatostatin, cholecystokinin-octapeptide, methionine-enkephalin, choline acetyltransferase, tyrosine hydroxylase, phenylethanolamine N-methyltransferase, NO-synthase, S-100, calbindin-D, calmodulin, or parvalbumin. The densities of c-Fos-, glutamate-, GAD-, calcineurin- and cAMP-IR neurons were almost zero in the 1st division of the VMS, but became higher along the 2nd-4th divisions of the VMS. Regression lines of the density against the 1st-4th divisions of the VMS were significantly linear. These results indicate that H+-sensitive neurons are common in the 4th-5th divisions of the VMS, and that they are glutamatergic, GABAergic, and containing calcineurin and cAMP.
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PMID:Topology and immunohistochemistry of proton-sensitive neurons in the ventral medullary surface of rats. 947 76


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