title: Regulatory effect of regucalcin on nitric oxide synthase activity in rat kidney cortex cytosol: Role of endogenous regucalcin in transgenic rats.
abstract: The effect of regucalcin, a regulatory protein in Ca2+ signaling, on nitric oxid (NO) synthase activity in the cytosol of kidney cortex of rats was investigated. The presence of calcium chloride (10 micro M) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This increase was significantly prevented by the addition of trifluoperazine (TFP; 20 or 50 micro M), an antagonist of calmodulin, supporting the existence of Ca2+/calmodulin-dependent NO synthase in rat kidney cortex cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-10)-10(-8) M) in the reaction mixture in the absence or presence of calcium chloride (10 micro M). The regucalcin (10(-8) M) effect was not seen in the presence of Nw-nitro-L-argine metylester (NAME; 10(-6) or 10(-5) M), an inhibitor of NO synthase. Regucalcin significantly reduced NO synthase activity in the presence of TFP (50 micro micro M) or EGTA (1 mM) which has a significant inhibitory effect on the enzyme activity. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in NO synthase activity. This increase was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the kidney cortex cytosol of regucalcin transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium chloride (10 micro M) in increasing NO synthase activity in the kidney cortex cytosol of wild-type rats was significantly weakened in regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the kidney cortex cytosol of rats.
authors.affiliation: Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Zhong Jie
authors.initials: ZJ
authors.lastname: Ma
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000494:pharmacology / Q000502:physiology*; D002265:Carboxylic Ester Hydrolases; D003600:Cytosol / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D014268:Trifluoperazine; D004533:Egtazic Acid; D019001:Nitric Oxide Synthase; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D002122:Calcium Chloride; D019331:NG-Nitroarginine Methyl Ester