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)

1. The monoamine dopamine and the amino acid glutamate are major neurotransmitters in the basal ganglia implicated in the normal functions of the striatum and in extrapyramidal disease states. To study the effects of these neurotransmitters on gene transcription in striatal neurons, we treated rats with dopamine (monoamine) agonists and with glutamate agonists and monitored the induction of Fos-like protein in striatal neurons. We administered the indirect monoamine agonists cocaine and amphetamine intraperitoneally and gave the glutamate agonist quinolinic acid by direct intrastriatal injection. We identified the phenotypes of the responsive neurons by immunohistochemistry and by enzyme histochemistry in double staining protocols. 2. Both the indirect monoamine agonists and the glutamate receptor agonist stimulated rapid nuclear expression of Fos-like protein in specific classes of striatal neurons. The induction by cocaine and amphetamine was blocked by pretreatment with the dopamine D1-like receptor antagonist SCH23390, and the induction by quinolinic acid was blocked by pretreatment with MK-801, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) glutamate receptor. 3. The monoamine and glutamate agonists both induced Fos-like protein exclusively in striatal neurons that constitutively expressed the protein phosphatase inhibitor DARPP-32 (dopamine and cAMP-regulated phosphoprotein). 4. The dopamine agonists failed to induce detectable Fos-like protein in striatal neurons expressing enkephalin, even though many such neurons expressed DARPP-32. By contrast, many enkephalinergic neurons did express Fos-like protein in response to glutamatergic stimulation. 5. Glutamate agonist stimulation, but not dopamine agonist stimulation, induced Fos-like protein in a subpopulation of striatal interneurons, namely, a group of neurons exhibiting NADPH-diaphorase activity. 6. These findings suggest that stimulation of dopamine D1-like receptors (or related monoamine receptors) and glutamate NMDA receptors activates neuron-specific programs of immediate-early gene expression in the striatum. Our findings further suggest that monoamine and glutamate may act cooperatively at the transcriptional level on a functionally defined subset of striatal neurons.
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PMID:Dopamine and glutamate agonists stimulate neuron-specific expression of Fos-like protein in the striatum. 135 24

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthesized by bovine aortic endothelial cells and subcellular fractions thereof was assayed by its stimulating effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells). The release of EDRF/NO by intact endothelial cells could be stimulated with bradykinin, thrombin, or ADP and was abolished in Ca2(+)-free medium. When subcellular fractions were analyzed, some EDRF/NO-synthesizing activity was found in the cytosolic fraction, but most of the activity was associated with the particulate fraction. Both enzyme activities required L-arginine and NADPH for EDRF/NO synthesis, both were inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine, and hemoglobin or methylene blue abolished the effect of the EDRF/NO produced by both enzymes. Both enzymes were highly sensitive to Ca2+; the major increase in activity occurred between 100 and 500 nM free Ca2+. Exposure of the particulate enzyme activity to 1 M KCl removed 39% of the protein and reduced total activity by 46%, but the activity was restored when exogenous calmodulin (CaM) was added. Further KCl washes caused little further loss of protein or EDRF/NO synthase activity. The KCl-washed particulate enzyme could be solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The CaM antagonists calmidazolium and trifluoperazine as well as the CaM-binding protein calcineurin inhibited the EDRF/NO synthesis by both the cytosolic and the particulate enzyme. These effects were partially reversed with exogenous CaM. Partial purification of the cytosolic and solubilized particulate enzymes by affinity chromatography on adenosine 2',5'-bisphosphate-Sepharose resulted in EDRF/NO synthase activities dependent on exogenous CaM. We conclude that endothelial cells contain both cytosolic and particulate enzymes that synthesize EDRF/NO. Both enzymes are regulated by free Ca2+ and, at least in part, by CaM.
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PMID:Calmodulin-dependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. 170 8

We investigated the molecular mechanisms whereby Ca2+ enters the endothelial cytosol and regulates endothelial nitric oxide synthesis L-arginine-dependent nitric oxide synthesis by isolated endothelial cytosol as quantified by activation of a purified soluble guanylate cyclase was concentration-dependently enhanced by free Ca2+ (EC50 0.3 microM). The Ca(2+)-dependent activation was inhibited by the calmodulin antagonists mastoparan, melittin, and calcineurin (IC50 450, 350, and 60 nM, respectively) in a calmodulin-reversible manner. After removal of endogenous calmodulin the Ca(2+)-dependency of endothelial NO synthase was lost, but could be reconstituted with exogenous calmodulin. The results indicate that Ca(2+)-calmodulin directly activates the endothelial nitric oxide synthase, thereby transducing agonist-induced increases in intracellular free Ca2+ concentration to nitric oxide formation from L-arginine, K(+)-induced depolarization of the endothelial cells markedly inhibited the sustained, but not initial phase of the intracellular Ca2+ response to bradykinin, indicating that K(+)-induced depolarization depresses the transmembrane Ca2+ influx. On the contrary, the K+ channel activator Hoe 234 which elicits hyperpolarization of the endothelial cell membrane, augmented the sustained phase of the agonist-induced intracellular Ca2+ signal, but not the resting intracellular Ca2+ level. The effects of K+ and Hoe 234 on the agonist-induced Ca(2+)-response were reflected by corresponding changes in agonist-induced EDRF/NO release. From these data, we suggest that the endothelial membrane potential may play an important role for the extent of agonist-induced Ca2+ influx and, thereby, the endothelial EDRF/NO synthesis.
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PMID:Cellular mechanisms controlling EDRF/NO formation in endothelial cells. 171 54

During ischemic stroke, massive neural damage occurs due to excess release of glutamate which acts mainly through N-methyl-D-aspartate (NMDA) receptors. Activation of the NMDA receptor stimulates nitric oxide (NO) production by NO synthase (NOS). NO mediates glutamate neurotoxicity as inhibitors of NOS prevent neuronal death. FK506, an immunosuppressant drug, binds to FK506 binding protein (FKBP). One target of the FK506/FKBP complex is the calcium/calmodulin-dependent protein phosphatase calcineurin, whose activity is inhibited upon interaction with FK506/FKBP. FK506 treatment increases phosphorylation level of calcinurin substrates including NOS. As a potent neuroprotective agent in vitro and in vivo, FK506 increases NOS phosphorylation and decreases NO production. NO activates poly(ADP-ribose) synthetase (PARS), a nuclear enzyme that synthesizes poly(ADP-ribose) from NAD. Prolonged activation of PARS depletes NAD and lowers cellular energy levels. Inhibition of PARS also prevents NO toxicity. NOS inhibitors, immunosuppressants and PARS inhibitors may be useful agents to prevent neuronal damage during stroke.
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PMID:Nitric oxide synthase, immunophilins and poly(ADP-ribose) synthetase: novel targets for the development of neuroprotective drugs. 747 44

The objective of these investigations was to study the regulatory properties of brain constitutive NO synthase. NOS activity was determined in 18,000 X g supernatant by conversion of 3H-L-arginine to 3H-L-citrulline in the presence of NADPH. The expression of catalytic activity of NOS required the presence of calcium ion and calmodulin. The preincubation of enzyme preparations at 37 degrees C in standard reaction mixture led to time-dependent inhibition of L-citrulline formation. This inhibition also required the presence of calcium ion during preincubation phase, and the enzyme remained calmodulin-dependent as exhibited by sensitivity to calmodulin antagonists trifluoperazine (TFP) and calcineurin. The modified enzyme showed significant decrease in the Vmax with NADPH and L-arginine without any change in apparent Km. Inclusion of protease inhibitors, leupeptin, pepstatin A, PMSF and soyabean trypsin inhibitor to the preparations did not alter preincubation-dependent inhibition of NO synthase. Thus, the calcium-dependent inhibitory phenomenon was not due to either the denaturation or proteolysis or the loss of calmodulin sensitivity of NO synthase. These observations indicate that cytosolic isoform of constitutive NO synthase undergoes dual regulation by physiological concentrations of calcium ion.
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PMID:Calcium-dependent inhibition of constitutive nitric oxide synthase. 752 Nov 66

A calmodulin-like protein, which is identical in size and 85% identical to vertebrate calmodulin, was recently identified by 'subtractive hybridization' comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of calmodulin, calmodulin-like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, calmodulin-like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare calmodulin-like protein functions with those of calmodulin. We find that calmodulin-like protein activation of multifunctional Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II) is equivalent to activation by calmodulin, but that four other calmodulin-dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric-oxide synthase, and myosin-light-chain kinase, display much weaker activation by calmodulin-like protein than by calmodulin. In the case of myosin-light-chain kinase, calmodulin-like protein competitively inhibits calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, calmodulin-like protein may have evolved to function as a specific agonist of certain calmodulin-dependent enzymes, and/or as a specific competitive antagonist of other calmodulin-dependent enzymes.
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PMID:Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells. 752 42

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme's catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.
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PMID:Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. 823 9

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-kappaB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-alpha also depends on the activation of NF-kappaB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-kappaB and production of TNF-alpha in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-kappaB and production of TNF-alpha. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-kappaB activation; however, activation of NF-kappaB is not sufficient for the induction of iNOS in macrophages and that apart from NF-kappaB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-kappaB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.
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PMID:Inhibitors of protein phosphatase 1 and 2A differentially regulate the expression of inducible nitric-oxide synthase in rat astrocytes and macrophages. 957 70

We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006-17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at -104 to -90 and PEA3 sites at -40 to -24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity.
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PMID:Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine. 961 91

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-gamma caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-gamma-induced expression of iNOS, but not interferon regulatory factor-1 (IRF-1) mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by protein synthesis inhibitor cycloheximide (CHX). FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action.
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PMID:Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line. 987 97


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