Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats

Abstract

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1–20 μM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 μM). NO synthase activity was significantly increased by the addition of calcium (5–20 μM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10–50 μM), an antagonist of Ca²⁺/calmodulin. The addition of regucalcin (0.1–0.5 μM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 μM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA, 1 mM) or TFP (20 μM), indicating that regucalcin acts independent on Ca²⁺/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 μM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats. J. Cell. Biochem. 88: 1226–1234, 2003.