Canine myenteric, deep muscular, and submucosal plexus preparations of purified nerve varicosities: content and chromatographic forms of certain neuropeptides.

Partially purified nerve varicosities prepared from canine small intestinal myenteric, deep muscular and submucosal plexuses were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P, Leu-enkephalin, Met-enkephalin, vasoactive intestinal polypeptide (VIP) and neurokinin A, but did not contain detectable amounts of neurokinin B. In all three plexus preparations, VIP was present in the highest concentration. In contrast to other species, GRP and the enkephalins were found to be present in relatively high concentrations in the submucosal plexus and GRP was present in low concentrations in the deep muscular plexus. Equal concentrations of substance P and neurokinin A were found in the myenteric and deep muscular plexus preparations but greater concentrations of substance P relative to neurokinin A were found in the submucosal plexus preparations. On reverse phase HPLC, a major peak of immunoreactivity occurred at the retention times of standard preparations for all six neuropeptides measured. Significant heterogeneity was found for GRP- and VIP-like immunoreactivity, especially in the submucosal plexus preparations. These partially purified canine small intestine nerve varicosity preparations may prove of value in studying release mechanisms for, and the posttranslational processing of, neuropeptides.

Galanin-like immunoreactivity in enteric neurons of the human colon.

The distribution of galanin was investigated at the light microscopic level in the human distal colon using immunocytochemical techniques. Galanin-like immunoreactivity was seen in nerve cell bodies in ganglia of the myenteric and submucous plexuses and in nerve fibres innervating all the muscle layers of the colon, the lamina propria, and epithelial cells at the base of crypts or lining the colonic lumen. Immunoreactivity was more intense in the circular muscle than in the longitudinal muscle or the muscularis mucosae. Immunoreactive nerve cell bodies were much scarcer in the myenteric than in the submucous plexus. Within subdivisions of the submucous plexus, galanin-like immunoreactivity was heterogeneously distributed. In Henle's plexus and Meissner's plexus 82-83% of galanin-positive cell bodies were up to 360 microns 2 in profile-area, but in the intermediate plexus nearly all (99.8%) were below 360 microns 2. The frequency-distribution of cell body area of galanin-containing nerve cell bodies was similar for Henle's plexus and Meissner's plexus but these two plexuses contain different size-populations of neurons when stained for NADH-diaphorase activity. Galanin-like immunoreactive nerve fibres were found in the plexus entericus (submucosus) extremus, and this is the first report of neuropeptide in this location.

Localization of calcitonin gene-related peptide in the small intestine of various vertebrate species.

Calcitonin gene-related peptide (CGRP) was found extensively in the small intestine of both non-mammalian and mammalian vertebrates using radioimmunoassay and immunocytochemistry. By radioimmunoassay, the levels of CGRP in rats, mice, chickens, bullfrogs and rainbow trout were found to range from 91.5 to 419.1 ng/g tissue. To localize CGRP in the small intestine, we used three different tissue preparations for immunocytochemistry: whole-mount preparations, and frozen and Paraplast sections. The combination of three tissue preparations made it easier to visualize the three-dimensional structure and reduced the possibility of missing the immunoreaction. Immunoreactive cell bodies were found in the plexi in the mammalian species. Dense and regular networks of CGRP fibers were observed in the smooth muscle layers, when examined in whole-mount preparations. In non-mammalian species, however, immunoreactive cell bodies could not be detected, although immunoreactive fibers were present, forming less dense and regular networks. Our results indicate that CGRP-immunoreactive fibers are present in the smooth muscle layers of the intestine from fish to mammals, suggesting that CGRP may be involved in regulating gastrointestinal smooth muscles in vertebrates.

Immunohistochemical detection of GTP-binding regulatory protein (Go) in the autonomic nervous system including the enteric nervous system, superior cervical ganglion and adrenal medulla.

The localization of a GTP-binding regulatory protein, Go, in the autonomic nervous system including the enteric nervous system, superior cervical ganglion, and adrenal medulla, has been immunohistochemically examined by use of affinity-purified antibody against the alpha-subunit of Go. In the small intestine, dense Go-immunoreactive products were localized on the enteric nervous system, i.e. the myenteric plexus of Auerbach and the submucosal plexus of Meissner. In the superior cervical ganglion, presynaptic terminals were strongly immunoreactive to the Go antibody. The adrenal medulla was stained with this antibody, but the adrenal cortex was not immunoreactive to this antibody. Thus, the present study strongly suggests that Go is localized in the autonomic nervous system and plays its role in transmembrane signal transmission in this system.

Neurotensin-containing neurons in the canine enteric innervation.

The intrinsic innervation of the gastrointestinal tract has been demonstrated to contain numerous peptidergic neurons. Neurotensin, originally isolated from bovine hypothalamus, has been localized in intestinal epithelial endocrine cells but not convincingly in the enteric innervation. The present study demonstrates the presence of neurotensin-immunoreactive neurons and nerve fibers in the canine submucous and myenteric ganglia. The peptide was characterized as neurotensin 1-13 by high pressure liquid chromatography and there was a mean concentration of 18.4 +/- 3.9 pmol (+/- S.E.M., n = 3) per g wet weight of submucosal extract. These neurons were a separate population from the vasoactive intestinal peptide- and somatostatin-immunoreactive cell bodies. These results demonstrate that neurotensin is present in significant amounts in the canine submucous plexus.

mu-Opioid receptors and alpha 2-adrenoceptors coexist on myenteric but not on submucous neurones.

Intracellular recordings were made from neurones in the myenteric and submucous plexuses of the guinea-pig ileum. All myenteric neurones that were hyperpolarized by [Met5]enkephalin (or normorphine) were also hyperpolarized by noradrenaline (or clonidine); neurones unaffected by opioids were unaffected by noradrenaline. The hyperpolarizations resulted from an increase in potassium conductance of the membrane and were blocked by the respective antagonists naloxone and idazoxan. Neurones of the submucous plexus were hyperpolarized by noradrenaline but not by normorphine. The results suggest that myenteric neurones possesses both mu-opioid receptors and alpha 2-adrenoceptors whereas submucous neurones have alpha 2-adrenoceptors but not mu-opioid receptors.

Autoradiographic localisation of muscarinic receptors in guinea-pig intestine: distribution of high and low affinity agonist binding sites.

Muscarinic receptors mediate a variety of intestinal functions including smooth muscle contraction, ganglionic transmission and water and electrolyte secretion. In this study, we have used [3H]quinuclidinyl benzilate ([3H]QNB) in an in vitro autoradiographic method to map the distribution of muscarinic receptors in guinea-pig ileum, colon and caecum. In addition, the relative distribution of low and high affinity agonist binding sites was assessed by the addition of the muscarinic agonist, carbachol, to selectively inhibit the binding of [3H]QNB to the high affinity sites. Although quantitative differences existed, the overall distribution of muscarinic receptors was similar in the 3 regions of intestine examined. Autoradiograph grains were found distributed over the myenteric and sub-mucous plexuses, the longitudinal and circular muscle layers and in the case of the colon, the muscularis mucosa. The inclusion of carbachol demonstrated that a greater proportion of high affinity sites were associated with the musculature than with the enteric plexuses. These findings are discussed in relation to the role of muscarinic mechanisms in intestinal motility and secretion.

Immunohistochemical studies on the gastrointestinal tract using antisera to Met-enkephalin and Met-enkephalin Arg6Phe7.

Antisera specific for the C-terminus of Met-enkephalin and two of its variants isolated from adrenal medulla and brain, namely Met-enk Arg6 and Met-enk Arg6Phe7, have been used in immunohistochemical studies of the gastrointestinal tract in rat, mouse and guinea pig. Met-enk and Met-enk Arg6Phe7-like immunoreactivities were found with similar distribution in nerve cell bodies of the myenteric plexus, and in fibers that were particularly dense in the myenteric plexus and circular smooth muscle. The Met-enk Arg6 antiserum showed weak staining of nerve cells and fibers. In rat and mouse, the antiserum to Met-enk Arg6Phe7, but not those to Met-enk or Met-enk Arg6, also stained numerous endocrine-like cells of the antral mucosa. These were identified as gastrin cells by elution and re-staining experiments with C-terminal gastrin antisera. The rat and mouse gastrin cells might conceivably express the enkephalin gene, but fail to process it to yield Met-enkephalin; alternatively, the Met-enk Arg6Phe7-like immunoreactivity in gastrin cells could be due to a cross-reacting peptide that does not belong to the opioid series.

Immunocytochemical localization of substance P in mammalian intestine.

In mammalian intestine immunoreactive Substance P is localized not only in the plexuses of Auerbach and Meissner, as could be anticipated, but also in a number of basally situated, often basigranular, endocrine cells which have been identified tentatively as enterochromaffin. The presence of a neurohormone in cells of this type confirms their close association with the nervous system, noted by Masson (1924), and suggests that their postulated origin from the nervous system (Danisch, 1924) may well be correct.