NADPH-diaphorase-positive nerve fibers in smooth muscle layers of opossum esophagus: gradients in density.

Nitric-oxide-releasing nerves regulate esophageal smooth muscle function. The density of such nerve fibers may differ in the different functional parts of the esophagus. We used both inspection and gray-scale analysis of digitized images to seek differences in density of such nerve fibers, stained for reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase), between esophageal body and esophago-gastric sphincter and between smooth muscle layers in the opossum esophagus. Sections of Swiss roll preparations of the entire organ were stained for NADPH-diaphorase and for immunoreactivity to vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP) and constitutive nitric oxide synthase (cNOS). In the circular muscle layer, NADPH-diaphorase-positive fibers were most abundant at the cephalic end of the esophageal body with a significant decline toward and through the esophago-gastric sphincter. In the longitudinal muscle layer and the longitudinally-oriented muscularis mucosae, NADPH-diaphorase-positive nerve fibers were most abundant at the esophago-gastric sphincter with a significant decline toward and through the striated-smooth muscle junction. cNOS immunoreactivity co-localized with NADPH-diaphorase activity. Fibers stained for CGRP immunoreactivity were distributed like the NADPH-diaphorase-positive fibers. Fibers stained for immunoreactivity to the other peptides (VIP, GAL, SP) showed no clear differences in distribution along the esophagus in any of the muscle layers.