Characterization of nitric oxide synthase in the rat parotid gland

Nitric oxide (NO) acts as an inter- and intracellular signalling molecule of various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from l-arginine. Here the characteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For full activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca2+ and calmodulin were needed as cofactors, because the activity was clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca2+, or in the absence of calmodulin and presence of trifluoperazine, a calmodulin antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca2+, and activated by them in a dose-dependent manner; EC50 for calmodulin and Ca2+ were 10 and 340 nM, respectively. The Km for l-arginine was 1.57 μM. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cytosolic fraction cross-reacted with a rabbit polyclonal antibody against human brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concentrations of calmodulin and Ca2+.