Suppressive role of endogenous regucalcin in the regulation of nitric oxide synthase activity in heart muscle cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of nitric oxide (NO) synthase activity in the cytosol of rat heart muscle was investigated. The addition of calcium chloride (5-20 µM) into the enzyme reaction mixture containing the heart cytosolic protein caused a significant increase in NO synthase activity. The Ca2+ effect was significantly inhibited by trifluoperazine (TFP; 20 or 50 µM), an antagonist of calmodulin, indicating the existence of Ca2+/calmodulin-dependent NO synthase activity in rat heart muscle cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10-9 or 10-8 M). This effect was also seen in the presence of calcium chloride (10 µM), TFP (50 µM) or EGTA (1 mM), a chelator of Ca2+. Meanwhile, the effect of regucalcin (10-8 M) in decreasing NO synthase activity was not seen in the presence of Nw-nitro-L-arginine methylester (NAME; 10-6 or 10-5 M), an inhibitor of the enzyme. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This effect was completely abolished by the addition of regucalcin (10-7 M). NO synthase activity was not significantly changed in the heart muscle cytosol of transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium (10 µM) addition in increasing NO synthase activity was significantly weakened in the heart muscle cytosol of regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the heart muscle cytosol of rats.