title: Activation of endogenous nitric oxide synthase coupled with methacholine-induced exocytosis in rat parotid acinar cells.
abstract: Methacholine (MCh) interacted with M(3) muscarinic receptors in rat parotid tissue slices and induced amylase secretion. MCh- and calcimycin-induced exocytosis was completely inhibited by N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide, N(G)-nitro-L-arginine methylester (L-NAME), 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxaline-1-one, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, suggesting that activations of calmodulin (CaM) kinase II, nitric oxide synthase (NOS), and cGMP-dependent protein kinase (PKG) were coupled with the exocytosis. These suggestions were supported by the results that exposure of the slices to MCh induced a rapid increase in these enzyme activities. Western blot analysis showed that neuronal NOS (nNOS) was expressed in isolated parotid acinar cells of rats. To measure nitric oxide (NO) production in response to the stimulation with MCh in real time, the isolated parotid acinar cells had been preloaded with 4,5-diaminofluorescein diacetate and incubated with the agonist. MCh (1 microM) induced a fast increase in 4,5-diaminofluorescein fluorescence, corresponding to an increase in the NO synthesis in the presence of extracellular Ca(2+) but not in the absence of it. When the isolated parotid acinar cells preloaded with L-NAME or 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) were treated simultaneously with MCh, the increase in the fluorescence also was not observed. The MCh-induced increase in the fluorescence was not observed in the cells incubated in the absence of extracellular calcium, showing the importance of Ca(2+) entry from extracellular sites for MCh-induced NOS activation. These results indicate that nNOS is endogenously present in rat parotid acinar cells and that the rapid activation of this enzyme together with those of CaM kinase II and PKG contributes to MCh-induced amylase secretion.
authors.affiliation: Department of Pharmacology, Tokushima University School of Dentistry, Tokushima, Japan. isikawa@dent.tokushima-u.ac.jp
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Ishikawa
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Iida
authors.forename: Mariusz T
authors.initials: MT
authors.lastname: Skowronski
authors.forename: Hajime
authors.initials: H
authors.lastname: Ishida
mesh_terms: D000681:Amylases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D005456:Fluorescent Dyes; D016210:Methacholine Chloride / Q000494:pharmacology*; D018721:Muscarinic Agonists / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010306:Parotid Gland / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D018721:Muscarinic Agonists; D016210:Methacholine Chloride; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000681:Amylases; D002118:Calcium