Ca(2+)-regulated nitric oxide generation in rabbit parotid acinar cells.

In rabbit parotid acinar cells, the muscarinic cholinergic agonist methacholine induced an increase in the intracellular Ca(2+) concentration and provoked nitric oxide (NO) generation. Ca(2+)-mobilizing reagents such as thapsigargin and the Ca(2+) ionophore A23187 mimicked the effect of methacholine on NO generation. Methacholine-induced NO generation was inhibited by the removal of extracellular Ca(2+). Immunoblot analysis indicated that the antibody against the neuronal type of nitric oxide synthase (NOS) cross-reacted with NOS in the cytosol of rabbit parotid gland cells. Immunofluorescence testing showed that neuronal NOS is present in the cytosol of acinar cells but less in the ductal cells. NOS was purified approximately 8100-fold from the cytosolic fraction of rabbit parotid glands by chromatography on Sephacryl S-200, DEAE-Sephacel, and 29,59-ADP-Sepharose. The purified NOS was a NADPH- and tetrahydroxybiopterin-dependent enzyme and was activated by Ca(2+) within the physiological range in the presence of calmodulin. These results suggest that NO is generated by the activation of the neuronal type of NOS, which is regulated in rabbit parotid acinar cells by the increase in intracellular Ca(2+) levels induced by the activation of muscarinic receptors.

Ca2+-nitric oxide-cGMP signaling in rabbit parotid acinar cells.

Guanosine 3',5'-cyclic monophosphate (cGMP) is a second messenger generated in response to hormones or neurotransmitters in various tissues and cells. In parotid acinar cells, the activation of muscarinic cholinergic and beta-adrenergic receptors induces an increase in intracellular cGMP. However, the mechanism of cGMP production in parotid acinar cells has not been well elucidated. cGMP production is induced by the activation of guanylyl cyclases, which are directly activated by nitric oxide (NO). NO plays an important role as an inter- and intracellular signal molecule in various organs and cells. Biosynthesis of NO is catalyzed by NO synthase (NOS), and NO generation is controlled by the regulation of NOS activity, for example by Ca2+. We have studied the regulation of NOS activity, NO generation and cGMP production in rabbit parotid acinar cells, and have demonstrated a functional Ca2+-NO-cGMP signaling pathway.

cGMP production is coupled to Ca(2+)-dependent nitric oxide generation in rabbit parotid acinar cells.

We investigated the mechanism of guanosine 3',5'-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.