nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?
Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cav) in canine lower esophageal sphincter. The main Cav was Cav-1. It appeared to be colocalized at the cell periphery, in punctate sites, with immunoreactivity to antibodies against different COOH- and NH2-terminal epitopes of neuronal nitric oxide (NO) synthase (nNOS). One COOH-terminal-directed antibody, made in guinea pig, was used to colocalize other immunoreactivities. Those that apparently colocalized with nNOS were L-Ca2+ channels, the PM Ca2+ pump, and, in part, calreticulin and calsequestrin. The large-conductance Ca2+-activated K+ (BK(Ca)) channels were located in discrete peripheral sites, some with Cav. Immunoreactivities not fully colocalized with nNOS were to the sarcoplasmic reticulum Ca2+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through BK(Ca) channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several Ca2+-handling molecules are localized at the PM with and/or near Cav. This may allow intracellular calcium concentration levels to be controlled differently than those in the cytosol near caveolae.
Daniel
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Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, Ontario L8N 3Z5, Canada. edaniel@ualberta.ca
Jury
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Wang
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YF
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis*; D021941:Caveolae / Q000737:chemistry; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis*; D018031:Connexin 43 / Q000032:analysis; D004285:Dogs; D004943:Esophagogastric Junction / Q000737:chemistry* / Q000166:cytology; D007150:Immunohistochemistry; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology; D019001:Nitric Oxide Synthase / Q000032:analysis*; D052248:Nitric Oxide Synthase Type I; D018408:Patch-Clamp Techniques; D016596:Vinculin / Q000032:analysis
D002135:Calcium-Binding Proteins; D051242:Caveolin 1; D022461:Caveolins; D018031:Connexin 43; D016596:Vinculin; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I