Abstract

A method was developed to study simultaneously 1) release of ACh from nerve varicosities and terminals in and 2) motility of the myenteric plexus-free circular muscle of the canine ileum. Tissues were incubated with 3H-choline. Release of 3H was analyzed before and during electrical field stimulation of nerves in the presence and absence of choline oxidase. The major components released into the superfusate during field stimulation were (in the absence of choline oxidase) 3H-ACh and 3H-choline as determined by column chromatography and (in the presence of choline oxidase) the choline metabolite 3H-betaine as isolated by tetraphenylboronbutyronitrile extraction. Addition of tetrodotoxin (1 x 10(-6)M) or superfusion with Ca(++)-free Krebs reduced motor activity and the release of 3H-ACh, which confirmed that release was mediated from nerve varicosities. Addition of Nw-nitro L-arginine methyl ester or Nw-nitro L-arginine increased field-stimulated 3H-ACh release. This effect was reduced in the presence of L-arginine but not D-arginine. Motility was also increased by the addition of Nw-nitro-L-arginine methyl ester and Nw-nitro L-arginine. In contrast, the addition of L-arginine did not restore motor activity to control levels, and D-arginine had no effect. Our findings that inhibition of nitric oxide synthesis amplifies ACh release during field stimulation suggest that field stimulation releases both ACh and nitric oxide from nerve terminals of the deep muscular plexus and that this nitric oxide inhibits field-stimulated ACh release and circular muscle contractility.