Characterization of nitric oxide synthase in the rat parotid gland

Nitric oxide (NO) acts as an inter- and intracellular signalling molecule o f various cells such as vascular endothelium, macrophages, and neurones. NO is produced by nitric oxide synthase (NOS) from L-arginine. Here the chara cteristics of NOS in the rat parotid gland were investigated. Approximately 74% of total activity of NOS was present in the cytosolic fraction. For fu ll activation of the NOS in the cytosolic fraction, tetrahydroxybiopterin, NADPH, Ca2+ and calmodulin were needed as cofactors. because the activity w as clearly reduced in the absence of tetrahydroxybiopterin, NADPH, or Ca2or in the absence of calmodulin and presence of trifluoperazine, a calmodul in antagonist, in the reaction mixture. The partially purified NOS activity was completely abolished in the absence of calmodulin or Ca2+. and activat ed by them in a dose-dependent manner; EC50 for calmodulin and Ca2+ were 10 and 340 nM, respectively. The K-m for L-arginine was 1.57 mu M. Immunoblot analysis revealed that a 165-kDa protein band in the rat parotid gland cyt osolic fraction cross-reacted with a rabbit polyclonal antibody against hum an brain NOS. These results suggest that NOS of the rat parotid gland is a neuronal isoform and that its activity is regulated by physiological concen trations of calmodulin and Ca2+. (C) 2000 Elsevier Science Ltd. All rights reserved.