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Research ArticleCELLULAR AND MOLECULAR

Activation of Endogenous Nitric Oxide Synthase Coupled with Methacholine-Induced Exocytosis in Rat Parotid Acinar Cells

Yasuko Ishikawa, Hirokazu Iida, Mariusz T. Skowronski and Hajime Ishida
Journal of Pharmacology and Experimental Therapeutics April 2002, 301 (1) 355-363; DOI: https://doi.org/10.1124/jpet.301.1.355
Yasuko Ishikawa
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Hirokazu Iida
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Mariusz T. Skowronski
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Hajime Ishida
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Abstract

Methacholine (MCh) interacted with M3 muscarinic receptors in rat parotid tissue slices and induced amylase secretion. MCh- and calcimycin-induced exocytosis was completely inhibited byN-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide,NG-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 μM) induced a fast increase in 4,5-diaminofluorescein fluorescence, corresponding to an increase in the NO synthesis in the presence of extracellular Ca2+ 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 Ca2+ 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.

Footnotes

  • This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

  • Abbreviations:
    NO
    nitric oxide
    NOS
    nitric oxide synthase
    nNOS
    neuronal NOS
    eNOS
    endothelial NOS
    iNOS
    inducible NOS
    A23187
    calcimycin
    CaM
    calmodulin
    CCh
    carbachol
    [Ca2+]i
    intracellular concentration of Ca2+
    IP3
    inositol 1,4,5-trisphosphate
    GC
    guanyl cyclase
    GC-S
    soluble GC
    MCh
    methacholine
    M3 receptors
    muscarinic receptors
    PLC
    phospholipase C
    PKG
    cGMP-dependent protein kinase
    KN-93
    (N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl) phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide
    l-NAME
    NG-nitro-l-arginine methylester
    ODQ
    1H-(1,2,4)-oxadiazolo [4,3-a]quinoxaline-1-one
    carboxy-PTIO
    2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide
    DAF-2/DA
    4,5-diaminofluorescein diacetate
    DAF-2
    4,5-diaminofluorescein
    BAPTA-AM
    2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester)
    PMA
    phorbol 12-myristate 13-acetate
    TMB-8
    3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester
    KRT
    Krebs-Ringer Tris
    IPR
    isoproterenol
    • Received August 14, 2001.
    • Accepted December 20, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 301 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 301, Issue 1
1 Apr 2002
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Research ArticleCELLULAR AND MOLECULAR

Activation of Endogenous Nitric Oxide Synthase Coupled with Methacholine-Induced Exocytosis in Rat Parotid Acinar Cells

Yasuko Ishikawa, Hirokazu Iida, Mariusz T. Skowronski and Hajime Ishida
Journal of Pharmacology and Experimental Therapeutics April 1, 2002, 301 (1) 355-363; DOI: https://doi.org/10.1124/jpet.301.1.355

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Research ArticleCELLULAR AND MOLECULAR

Activation of Endogenous Nitric Oxide Synthase Coupled with Methacholine-Induced Exocytosis in Rat Parotid Acinar Cells

Yasuko Ishikawa, Hirokazu Iida, Mariusz T. Skowronski and Hajime Ishida
Journal of Pharmacology and Experimental Therapeutics April 1, 2002, 301 (1) 355-363; DOI: https://doi.org/10.1124/jpet.301.1.355
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