Ca2+-regulated nitric oxide generation in rabbit parotid acinar cells

in rabbit parotid acinar cells, the muscarinic cholinergic agonist methacho line induced an increase in the intracellular Ca2+ concentration and provok ed nitric oxide (NO) generation. Ca2+-mobilizing reagents such as thapsigar gin and the Ca2+ ionophore A23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the remova l of extracellular Ca2+. Immunoblot analysis indicated that the antibody ag ainst the neuronal type of nitric oxide synthase (NOS) cross-reacted with N OS in the cytosol of rabbit parotid gland cells. Immunofluorescence testing showed that neuronal NOS is present in the cytosol of acinar cells but les s in the ductal cells. NOS was purified approximately 8100-fold from the cy tosolic fraction of rabbit parotid glands by chromatography on Sephacryl S- 200, DEAE-Sephacel, and 2',5'-ADP-Sepharose. The purified NOS was a NADPH- and tetrahydroxybiopterin-dependent enzyme and was activated by Ca2+ within the physiological range in the presence of calmodulin. These results sugge st that NO is generated by the activation of the neuronal type of NOS, whic h is regulated in rabbit parotid acinar cells by the increase in intracellu lar Ca2+ levels induced by the activation of mus.carinic receptors. (C) 200 1 Harcourt Publishers Ltd.