nNOS in canine lower esophageal sphincter: colocalized with Cav-1 and [Ca.sup.2+]-handling proteins?

Immunochemical studies with light microscopy, confocal microscopy, and electron microscopy were used to examine proteins associated with caveolin (Cay) 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 [NH.sub.2]-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-[Ca.sup.2+] channels, the PM [Ca.sup.2+] pump, and, in part, calreticulin and calsequestrin. The largeconductance [Ca.sup.2+]-activated [K.sup.+] ([BK.sup.Ca]) channels were located in discrete peripheral sites, some with Cav. Immunoreactivites not fully colocalized with nNOS were to the sarcoplasmic reticulum [Ca.sup.2]+ pump, connexins 43, 40, and 45, and vinculin. In patch-clamp studies, NO-driven outward currents, mainly through [BK.sub.Ca] channels, were inhibited by antibodies to Cav-1 and not by calmodulin and were restored by an NO donor. Several [Ca.sup.2+] -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. PM organization; cellular calcium compartments; caveolin association; neuronal nitric oxide synthase