Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

Abstract

The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4‐II‐E cells was investigated. Hepatoma cells were cultured for 24–72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 μM) and calmodulin (2.5 μg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, inhibited the effect of calcium (10 μM) addition in increasing NO synthase activity, indicating the existence of Ca²⁺/calmodulin‐dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10⁻⁸ or 10⁻⁷ M) in the reaction mixture without or with Ca²⁺/calmodulin addition. The effect of regucalcin (10⁻⁷ M) in decreasing NO synthase activity was also seen in the presence of TFP (50 μM) or EGTA (1 mM). The presence of anti‐regucalcin monoclonal antibody (10–50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti‐regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca²⁺/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild‐type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4‐II‐E cells. J. Cell. Biochem. 89: 800–807, 2003.