Inhibitory role of regucalcin in the regulation of nitric oxide synthase activity in rat brain cytosol: involvement of aging

Abstract

Regucalcin is shown to play a regulatory role in intracellular signaling system in many cells. The protein has been reported to be present in rat brain neurons. The role of regucalcin in the regulation of nitric oxide (NO) synthase activity in the brain cytosol of female young and aged rats was investigated. The presence of regucalcin (10 −9 and 10 −8 M) in the enzyme reaction mixture caused a significant decrease in NO synthase activity in the absence or presence of both calcium chloride (10 μM) and calmodulin (2.5 μg/ml). The effect of regucalcin (10 −8 M) in decreasing brain cytosolic NO synthase activity was not seen in the presence of Nw-nitro- l-argine methyl ester (NAME) (10 −4 M), trifluoperazine (20 μM) or EGTA (1 mM). Regucalcin protein levels were significantly lowered in the brain cytosol of aged (50 weeks old) rats as compared with that of young (5 weeks old) rats. Brain cytosolic NO synthase activity was significantly reduced with increasing age. In aged rat brain cytosol, regucalcin caused a significant decrease in NO synthase activity. The presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) caused a significant increase in NO synthase activity in the brain cytosol of young and aged rats. This elevation was completely blocked by the addition of regucalcin (10 −8 M). The present study demonstrates that endogenous regucalcin has an inhibitory effect on NO synthase activity in the brain cytosol of young and aged rats.