Quantitative ultrastructural analysis of enkephalin‐, substance P‐, and VIP‐immunoreactive nerve fibers in the circular muscle of the guinea pig small intestine

The present work was undertaken to determine what proportion of all nerve fibers in the circular muscle of the guinea pig small intestine contain the neuropeptides enkephalin, substance P, and vasoactive intestinal peptide and in which combinations these peptides occur in the fibers. It was envisaged that such an analysis would provide insights into the chemical identity of excitatory and inhibitory nerve fibers that innervate the muscle. Whole‐mount preparations from normal and extrinsically denervated gut were labelled with antiserum to the individual peptides or with combinations of antipeptide antisera and processed for electron microscopy. Reactive and nonreactive vesicle‐containing nerve fiber profiles were examined and counted in ultrathin sections. Vesicle‐containing nerve fiber profiles immunoreactive for enkephalin, substance P, or vasoactive intestinal peptide had similar morphologies in that they all contained variable proportions of small clear and large granular vesicles. In all samples stained for single peptides or combinations of peptides, a small proportion of immunoreactive profiles approached smooth muscle cells to within 15‐20 nm with no intervening basal lamina. A total of 14,694 vesiculated nerve fiber profiles from three control and three extrinsically denervated animals were scored for the presence of immunoreactivity to enkephalin, substance P, vasoactive intestinal peptide, or combinations of these peptides. Analysis of variance showed that the number of profiles labelled for substance P was not different from the number of profiles labelled for vasoactive intestinal peptide and that the number labelled with the substance P and vasoactive intestinal peptide antisera simultaneously were not different from the sum of the numbers obtained with each alone. The number of profiles labelled for substance P plus enkephalin was greater than the number labelled for substance P alone and the number labelled with vasoactive intestinal peptide plus enkephalin was greater than that with vasoactive intestinal peptide alone. Simultaneous labelling for substance P and vasoactive intestinal peptide resulted in immunoreactivity in the same number of profiles as did reaction for all three peptides at the same time. In both cases, about 95% of the profiles were labelled. The results from extrinsically denervated muscle were not different from control circular muscle. These results indicate that nearly all the intrinsic nerve fibers supplying the circular muscle of the guinea pig small intestine contain either substance P or vasoactive intestinal peptide but not both. Enkephalin is not found alone in intrinsic nerve fibers in the circular muscle but always occurs with either substance P or vasoactive intestinal peptide. Enkephalin is absent from about one‐third of the nerve fibers that contain substance P and a similar number that contain vasoactive intestinal peptide. These findings, along with data from physiological experiments, imply that both excitatory nerve fibers to the circular muscle, which are cholinergic, and inhibitory nerve fibers, which are neither noradrenergic nor cholinergic, contain one or more neuropeptides. Substance P is probably present in some, possibly all, of the excitatory nerve fibers whereas vasoactive intestinal peptide is probably present in some, if not all, of the inhibitory nerve fibers.