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.