nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and Ca2+-handling proteins?

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Abstract
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+(BKCa) channels were located in discrete peripheral sites, some with Cav. Immunoreactivites 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 BKCachannels, 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.