Ca2+, calmodulin and phospholipids regulate nitricoxide synthase activity in the rabbit submandibular gland

Nitric oxide (NO) plays an important role as an intra- and intercellular signaling molecule in mammalian tissues. In the submandibular gland, NO has been suggested to be involved in the regulation of secretion and in blood flow. NO is produced by activation of NO synthase (NOS). Here, we have investigated the regulation of NOS activity in the rabbit submandibular gland. NOS activity was detected in both the cytosolic and membrane fractions. Characteristics of NOS in the cytosolic and partially purified membrane fractions, such as Km values for l-arginine and EC(50) values for calmodulin and Ca(2+), were similar. A protein band that cross-reacted with anti-nNOS antibody was detected in both the cytosolic and membrane fractions. The membrane-fraction NOS activity increased 1.82-fold with treatment of Triton X-100, but the cytosolic-fraction NOS activity did not. The NOS activity was inhibited by phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP(2)). The inhibitory effects of phospholipids on the NOS activity were relieved by an increase in Ca(2+) concentrations. These results suggest that the Ca(2+)- and calmodulin-regulating enzyme nNOS occurs in cytosolic and membrane fractions, and PA and PIP(2) regulate the NOS activity in the membrane site by regulating the effect of Ca(2+) in the rabbit submandibular gland.