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

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.
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PMID:Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. 169 82

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 calmodulin-binding polypeptide neuromodulin (GAP-43) was tested in vitro for its ability to modulate a typical calmodulin target, the enzyme nitric oxide synthase. The titration of enzyme with increasing neuromodulin concentrations demonstrated a concentration-dependent decrease in enzyme activity. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was tested in the presence or absence of neuromodulin. The effect of neuromodulin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 6 microM calcium. Treatment of the neuromodulin polypeptide with protein kinase C eliminated its ability to inhibit nitric oxide synthase activation. Subsequent treatment of the phosphorylated neuromodulin with calcineurin (phosphatase 2b) caused it to regain its inhibitory action on the enzyme. The results from these in vitro studies have indicated that neuromodulin has the ability to affect the activation of a calmodulin-dependent enzyme at levels of the polypeptide that exist in neurons. They also demonstrated that the regulation occurred within a physiological range of calcium concentrations. Since the inhibition of enzyme activity appeared to be occurring through the interaction of neuromodulin with calmodulin, it seems likely that neuromodulin has a general ability to impede activation of calmodulin-dependent targets.
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PMID:Neuromodulin (GAP-43) can regulate a calmodulin-dependent target in vitro. 751 37

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

Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 1 microM calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.
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PMID:The dendritic peptide neurogranin can regulate a calmodulin-dependent target. 752 68

The effect of cyclosporin A on induction of nitric oxide synthase in rat aortic smooth muscle cells was examined. A combination of interleukin-1 alpha (100 U/mL) and tumor necrosis factor--alpha (5000 U/mL) induced accumulation of nitrite/nitrate, the stable end products of nitric oxide, in culture media within 48 hours. Cyclosporin A inhibited this nitrite/nitrate accumulation in a concentration-dependent manner with an IC50 of 4 x 10(-7) mol/L when applied simultaneously with the cytokines. The expression of inducible nitric oxide synthase messenger RNA (mRNA) induced by the combination of interleukin-1 alpha and tumor necrosis factor-alpha was inhibited by the cyclosporin A cotreatment. Cyclosporin A did not decrease inducible nitric oxide synthase mRNA stability in the presence of transcription inhibitor actinomycin D (5 micrograms/mL). Induction of nitrite/nitrate production by the combination of tumor necrosis factor-alpha and bacterial lipopolysaccharide or that of interleukin-1 alpha and interferon gamma (100 U/mL) was also inhibited by cyclosporin A cotreatment. Another inhibitor of calcineurin, FK506 (up to 10(-6) mol/L), had no effect on the induction of nitrite/nitrate production, suggesting the possibility that the inhibitory effect of cyclosporin A may be exerted by means of a novel pathway other than inhibition of calcineurin. These results indicate that cyclosporin A inhibits inducible nitric oxide synthase induction at the mRNA level and that inducible nitric oxide synthase in vascular smooth muscle cells can be a target for cyclosporin A, providing a possible mechanism for the interference of the drug with the balance of vasoactive substances.
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PMID:Cyclosporin A inhibits nitric oxide synthase induction in vascular smooth muscle cells. 753 14

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


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