G. Malmfors

A novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), in human intestine: evidence for reduced content in Hirschsprung's disease

SummaryA novel neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exhibits sequence homology with vasoactive intestinal polypeptide (VIP) and occurs in the mammalian brain, lung and gut. The distribution of PACAP in ganglionic and aganglionic portions of the large intestine of patients with Hirschsprungs disease was examined by immunohistochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were distributed in all layers of the ganglionic and aganglionic segments of the intestine, although they were less numerous in the latter, and PACAP-immunoreactive nerve cell bodies were seen in the ganglionic portion of the intestine. The concentration of immunoreactive PACAP was lower in the aganglionic than in the ganglionic segment of the intestinal wall. PACAP and VIP were found to coexist in both ganglionic and aganglionic segments of the intestine. Apparently, PACAP participates in the regulation of gut motility. The scarcer PACAP innervation of the aganglionic segment may contribute to the defect in intestinal relaxation seen in patients with Hirschsprungs disease.

Peptide-containing neurons intrinsic to the gut wall

SummaryNerve fibers containing substance P, VIP, enkephalin or somatostatin are numerous in the porcine gut wall. They are particularly numerous in the submucosal and myenteric plexuses where peptide-containing cell bodies are also observed. Peptide-containing nerve fibers occur also in the vagus nerves, suggesting that the gut receives an extrinsic supply of peptidergic nerves. The extrinsic contribution to the peptide-containing nerve supply of the gut wall has not yet been quantitatively assessed. In an attempt to clarify this question pigs were subjected to bilateral subdiaphragmatic vagotomy. Another group of animals was subjected to complete extrinsic denervation by autotransplantation of a jejunal segment. The pigs were killed at various time intervals after the operations; the longest time interval studied was four months. Following vagotomy the innervation pattern of the jejunum appeared completely unaffected. Following complete extrinsic denervation the adrenergic nerve fibers disappeared, while peptide-containing and acetylcholinesterase-positive nerve fibers remained apparently unaltered. This was confirmed chemically in the case of substance P.The motor activity of smooth muscle from the jejunum was studied in vitro. At low stimulation frequencies the smooth muscle from control jejunum responded by relaxation; upon cessation of stimulation a contraction occurred. With increasing stimulation frequencies the duration of the relaxation decreased; at high frequency stimulation only a contraction was recorded. In the autotransplant low frequency stimulation induced no or only a weak relaxation; high frequency stimulation induced contraction. After cholinergic and adrenergic blockade, the muscle responded with relaxation at all frequencies; the response was similar in innervated and denervated specimens. On the whole, the effects of extrinsic denervation on the motor activity of smooth muscle from porcine jejunum were minor, possibly reflecting the high degree of autonomy of the gut.

Neuropeptide Y, calcitonin gene-related peptide, and galanin in Hirschsprung's disease: an immunocytochemical study

The aganglionic intestinal segment in Hirschsprungs disease is known to contain a reduced number of nerve fibers storing vasoactive intestinal peptide (VIP), substance P (SP), enkephalin, and gastrin-releasing peptide (GRP). In this study, nerves containing three newly described neuropeptides: neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and galanin were examined using immunocytochemistry. Nerve fibers displaying NPY immunoreactivity were found to be more frequent in the aganglionic than in nonafflicted ganglionic intestine. Nerve fibers storing CGRP and galanin on the other hand were roughly equally frequent but the distribution pattern differed in that the bulk of fibers in the aganglionic intestine was localized to large nerve trunks not seen in the ganglionic segment. The functional significance of these changes has yet to be defined.

Ontogeny of peptide-containing neurones in human gut—an immunocytochemical study

Neurons containing enkephalin, substance P (SP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), galanin, calcitonin gene-related peptide (CGRP), peptide histidine isoleucine (PHI) or gastrin-releasing peptide (GRP) are known to occur in the human intestinal tract. The knowledge of the ontogeny of these neurones is, however, limited. Intestinal specimens from 24 human foetuses with gestational ages varying between 8 and 40 weeks were examined by immunocytochemistry. No peptide-containing neurones could be detected before the 14th week of gestation after which a rapid development was seen. Generally, peptide immunoreactivity was first noted in the myenteric ganglia and somewhat later in the other layers of the intestinal wall. There was no major difference between the peptides studied or between different parts of the intestinal tract with respect to time of appearance.

Peptidergic Nerves Persist After Jejunal Autotransplantation: An Experimental Study in the Piglet

The gut has been shown to contain four types of peptidergic nerves besides the adrenergic and cholinergic ones. They store substance P, enkephalin, somatostatin and VIP (vasoactive intestinal peptide) respectively. It is not previously known which of the neurons are intrinsic or extrinsic to the gut wall. Jejunal autotransplantation was performed in 10 piglets. This procedure implies degeneration of the extrinsic neurons (extrinsic denervation). The transplants were examined 1 to 4 mo postoperatively with histochemical techniques for demonstration of the intramural adrenergic and the different peptidergic nerves. The distribution of the peptidergic nerves was not changed indicating that they all originate within the gut. Following extrinsic denervation adrenergic nerves on the contrary were completely missing.

Hirschsprung's disease: A comparison of the nervous control of ganglionic and aganglionic smooth muscle in vitro

Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine patients with Hirschsprungs disease. Transmural nerve stimulation of ganglionic smooth muscle in vitro evoked an initial relaxation followed by a contraction. This contraction was reduced but not abolished by atropine and it was further reduced by substance P antagonists. Guanethidine did not affect the electrically evoked responses. In aganglionic smooth muscle, an atropine-sensitive contraction but no initial relaxation was registered. Tetrodotoxin abolished all responses to electrical stimulation in both ganglionic and aganglionic specimens. Application of carbachol or substance P produced contraction and the adrenergic agonist isoprenaline or vasoactive intestinal peptide produced relaxation in ganglionic as well as aganglionic specimens. Two other gut neuropeptides, neuropeptide Y and galanin, were without effect. The results do not indicate a different receptor set up in ganglionic v aganglionic gut. The results are compatible with a lack of noncholinergic nonadrenergic inhibitory neurons in the aganglionic gut.

NPY hyperinnervation in Hirschsprung's disease: Both adrenergic and nonadrenergic fibers contribute

In Hirschsprungs disease, the aganglionic bowel is characterized by an absence of ganglion cells and an increased number of adrenergic and presumed cholinergic nerve fibers. In addition, a severe derangement of peptide-containing nerve fibers is encountered including a hyperinnervation of neuropeptide Y (NPY)-containing fibers. Using immunochemical and immunocytochemical methods, we examined the nature of the NPY-containing nerve fibers contributing to the hyperinnervation. The concentration of NPY was markedly increased in the aganglionic segment. Coexistence of NPY, vasoactive intestinal peptide (VIP), and the adrenergic enzyme tyrosine hydroxylase (TH) showed small populations of nerve fibers containing NPY/TH, NPY/VIP, or TH alone in ganglionic intestine. Numerous nerve fibers stored VIP but lacked NPY. These fibers did not contain TH, indicating that all VIP-containing fibers are nonadrenergic. In the aganglionic intestine there was a marked increase in the number of nerve fibers storing NPY/TH and NPY/VIP, whereas the fibers storing VIP alone were reduced in number. A small number of nerve fibers storing NPY alone occurred in the hypertrophic nerve bundles. NPY/VIP-containing nerve fibers were particularly numerous in the mucosa in aganglionic intestine, which may be of interest in the diagnosis of Hirschsprungs disease allowing the use of mucosal biopsy specimens. Thus, the proliferating NPY-containing nerve fibers in the aganglionic intestine seem to comprise three different populations, one adrenergic and two nonadrenergic, one of which contains in addition VIP.

Defects in peptidergic innervation in Hirschsprung's disease

Aganglionic and ganglionic intestinal specimens from 14 patients with Hirschsprungs disease were examined by immunocytochemistry. The study revealed a reduction in nerve fibers storing vasoactive intestinal peptide (VIP), substance P (SP), and a total depletion of nerves storing enkephalin and gastrin-releasing peptide in aganglionic intestine. Nerve fibers storing peptide histidine isoleucine (PHI) or neurokinin A (NKA) were also examined. It was shown that VIP coexisted with PHI and SP with NKA and that the number of nerve fibers containing these substances was markedly reduced in the smooth-muscle layers in aganglionic bowel. In the mucosa, on the other hand, the innervation was virtually unchanged. Thus, it appears that the mucosa secretory and sensory innervation is not reduced and that the defects in peptidergic innervation in Hirschsprungs disease mainly involve motor neurons.

Chromogranin A and B in neuronal elements in Hirschsprung's disease: An immunocytochemical and radioimmunoassay study☆

Chromogranin A and B (CAB) occur in several peptide hormone-producing cells and in neurons of the brain. The aim of the present study was to investigate the possible neuronal localization of these chromogranins in the ganglionic and aganglionic bowel in Hirschsprungs disease by immunocytochemistry and radioimmunoassay, using antibodies recognizing either chromogranin A or both chromogranin A and B. Further, the coexistence of chromogranins and other neuronal constituents was studied. CAB were found in nerve fibers and occasionally in nerve cell bodies of submucous and myenteric ganglia in the ganglionic bowel, indicating that at least a population of chromogranin-immunoreactive nerve fibers is intrinsic in origin. CAB-immunoreactive fibers were numerous in the muscle layers of the aganglionic segment. These fibers contained tyrosine hydroxylase (TH), which indicates that they are adrenergic, in both ganglionic and aganglionic bowel. In the muscle layers of aganglionic (but not ganglionic) bowel, chromogranin A coexisted with galanin, neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). The concentration of CAB in smooth muscle specimens was higher in the aganglionic bowel than in the ganglionic bowel. Thus, chromogranins are present in the human enteric gut hyperinnervating the aganglionic bowel of Hirschsprungs disease.