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
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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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