Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat.

Expression of the calcitonin gene-related peptide, alpha-calcitonin gene-related peptide (CGRP), and the homologous beta-CGRP were compared in sensory and enteric nerves of the rat. Analysis of CGRP-like immunoreactivity by cation exchange chromatography and radioimmunoassay showed that in the dorsal root ganglia, dorsal spinal cord and in those peripheral tissues where CGRP-like immunoreactivity is primarily localized to sensory fibres, alpha-CGRP concentrations were three to six times greater than beta-CGRP concentrations. In the intestine, however, beta-CGRP concentrations were up to seven times greater than alpha-CGRP concentrations. Only beta-CGRP was detected in the intestines of capsaicin-treated rats. Northern blot and in situ hybridization to alpha-CGRP- and beta-CGRP-specific probes showed that while both alpha-CGRP and beta-CGRP messenger ribonucleic acids occurred in the dorsal root ganglia, only beta-CGRP messenger ribonucleic acid occurred in the intestine, where it was localized to enteric neurons. Receptor binding sites on membranes of rat heart and colon had approximately equal affinities for alpha-CGRP and beta-CGRP. The two peptides were equipotent in increasing the rate and force of atrial contractions but alpha-CGRP was slightly (2.6 times) more potent than beta-CGRP in relaxing colonic smooth muscle. Thus, both alpha-CGRP and beta-CGRP occur in the rat nervous system and are both biologically active. Sensory neurons and enteric neurons have been identified as populations which preferentially express alpha-CGRP and beta-CGRP, respectively.

Simultaneous measurement of plasma protein extravasation and carotid vascular resistance in the rat.

Stimulation of the right trigeminal ganglion in pentobarbital anaesthetised rats increased mean arterial blood pressure and decreased right carotid vascular resistance but had no effect on left carotid vascular resistance. Sumatriptan (0.3 mg/kg i.v.) pretreatment did not significantly affect basal levels or stimulation induced changes in blood pressure or carotid vascular resistance. Trigeminal stimulation produced plasma protein extravasation (measured using a fluorescent marker) into the dura mater on the ipsilateral side which was significantly reduced by sumatriptan. These studies show that sumatriptan can reduce plasma protein extravasation while having no measurable effect on total carotid blood flow.

Comparison of neuromedin-N and neurotensin on net fluid flux across rat small intestine.

Neuromedin-N, a hexapeptide recently isolated and purified from porcine spinal cord, has close sequence homology with the C-terminal region of the tridecapeptide neurotensin. Both peptides have a remarkably similar peripheral distribution. Little is known of the biological activity of neuromedin-N. Neurotensin and peptide histidine methionine are known to stimulate net fluid secretion into rat small intestine. We have therefore tested the effect of neuromedin-N and the hexapeptide neurotensin-(8-13), the smallest fully active analogue of neurotensin in this system, compared with that of neurotensin and peptide histidine methionine. All four peptides reduced net absorption in low doses and caused net secretion in larger doses. However, whereas peptide histidine methionine was active in all areas of the small intestine, neurotensin, neurotensin-(8-13) and neuromedin-N were inactive in the duodenum. In the post-duodenal areas neurotensin was approximately 7 times more active than peptide histidine methionine, 21 times more potent than neuromedin-N and 33 times more potent than neurotensin-(8-13).

Increased pressor effect of repeated doses of rat or porcine endothelin in the anaesthetised rat.

Rat and porcine endothelin differ by 6 amino acids. Porcine and rat endothelin were equiactive at producing the transient fall in blood pressure in the anaesthetised rat but porcine endothelin was three times more potent at producing the subsequent sustained rise. Repeated doses of either peptide in the same rat produced a larger pressor response with each repetition, the peak response being reached more rapidly. The magnitude of the blood pressure fall did not change significantly but the first response in each rat was of longer duration than subsequent responses, especially with rat endothelin.

Studies with endothelin-3 and endothelin-1 on rat blood pressure and isolated tissues: evidence for multiple endothelin receptor subtypes.

In an anesthetized rat, endothelin-1 (ET-1) causes a rapid, transient fall in blood pressure followed by a large, sustained rise. Rat endothelin-3 (ET-3) differs from porcine endothelin-1 (ET-1) by six amino acids and is 20 times less potent at contracting rat aortic strips. The comparative effects of ET-3 and ET-1 on blood pressure were studied in anesthetized rats in which the arterial blood pressure was continually monitored. Dose-response curves for the contraction of rat stomach strips, guinea pig ileum, and guinea pig trachea were also constructed. In the anesthetized rat, ET-3 was slightly (but not significantly) less potent than ET-1 at producing the transient blood pressure fall but was three times less potent at causing the subsequent rise. The two peptides were equipotent at contracting rat stomach strips but ET-3 was 10 times less potent at contracting the guinea pig ileum. Rat ET-3 was less potent at contracting the guinea pig trachea and had a lower maximal effect. These results suggest the existence of multiple receptor subtypes for ET.

Effects of prepro-vasoactive intestinal polypeptide-derived peptides on net fluid flux in small intestine of anesthetized rats.

Intravenous infusion of low doses of vasoactive intestinal polypeptide, peptide histidine valine-42, and peptide histidine methionine (and the rat equivalent, peptide histidine isoleucine) into anesthetized rats caused a reduction in net absorption of fluid from the small intestine. Larger doses caused a net fluid secretion. At the same nominal infusion rates, peptide histidine valine-42 appeared to be the most potent. In terms of plasma concentrations achieved, however, vasoactive intestinal polypeptide was approximately six times more active than the other two human peptides. If confirmed in humans, these results would suggest that peptide histidine methionine and peptide histidine valine may be as important as vasoactive intestinal polypeptide in causing the watery diarrhea seen in the Verner-Morrison syndrome in which plasma levels of all three peptides are raised.

Isolation, characterization, and pharmacological actions of peptide histidine valine 42, a novel prepro-vasoactive intestinal peptide-derived peptide.

The primary structure and biological activity of a novel prepro-vasoactive intestinal peptide (prepro-VIP)-derived peptide has been determined from an adrenal pheochromocytoma. The peptide was purified sufficiently for characterization by fast atom bombardment mapping after cation-exchange and reverse-phase fast protein liquid chromatography. The sequence of this novel peptide corresponds exactly to prepro-VIP-81-122 and has been designated peptide histidine valine 42 (PHV-42). Synthetic PHV-42 reduced both the force and frequency of spontaneous contractions of isolated rat uterus and was at least 12 times more potent than peptide histidine methionine (prepro-VIP-81-107), and over a hundred times more potent than noradrenaline. PHV-42 was also more potent than peptide histidine methionine in relaxing smooth muscle preparations of rat stomach and guinea pig trachea, but was approximately 4-fold less potent in reducing blood pressure than VIP. PHV-42 thus forms a separate subsystem in the VIP family of peptides and may be the most biologically active product of prepro-VIP in certain tissues such as the uterus and trachea.

The distribution, purification, and pharmacological action of an amphibian neuromedin U.

The distribution, primary structure, and relative biological activity of neuromedin U has been determined from the frog Rana temporaria. Following sequential column chromatography of a gastrointestinal extract, the peptide was sufficiently pure to enable characterization by micro-sequence analysis. The entire sequence was found to be an icosapentapeptide which displays marked sequence similarity to both porcine and rat neuromedin U. The sequence of the biologically active, COOH-terminal region is almost completely conserved across all species. Synthetic, COOH-terminally amidated amphibian neuromedin U, like the porcine and rat peptides, stimulates rat uterine contraction in vitro thereby fulfilling the criterion upon which the nomenclature of this peptide family is based. In addition, the peptide demonstrates parallel pressor effects when infused systemically into rats. The high degree of amino acid sequence conservation is indicative of strong evolutionary pressure acting to retain the presence of this possibly physiologically important peptide across the vertebrate subphylum.

Endothelin in the gastrointestinal tract. Presence of endothelinlike immunoreactivity, endothelin-1 messenger RNA, endothelin receptors, and pharmacological effect.

The possible production and role of endothelin in the gastrointestinal tract was investigated in rats by radioimmunoassay, Northern-blot hybridization, receptor assay using membrane preparations, and pharmacological study using gut strips. Endothelinlike immunoreactivity was detected in all regions (from stomach to colon) of the rat gastrointestinal tract (13-48 fmol/g wet tissue) including the mucosal layer of the ileum and colon (8.4 +/- 2.0 fmol/g wet tissue and 18.4 +/- 2.1 fmol/g wet tissue, respectively, mean +/- SEM; n = 5). Fast protein liquid chromatographic analysis of the endothelinlike immunoreactivity in jejunum, ileum, colon, and colon mucosa extracts showed peaks in the positions of endothelin-1 and endothelin-3. The presence of endothelin-1 messenger RNA was demonstrated by Northern-blot hybridization in the whole colon and pooled ileal and colonic mucosa, but not in the whole jejunum. Specific binding in the rat gastrointestinal tract was particularly high in the fundus of stomach, jejunum, ileum, and colon. In the ileum, many binding sites were found in the circular and longitudinal muscle layers, but few in the mucosal layer. Endothelin-1 and endothelin-3 caused contraction of rat stomach strips, rat colon, and guinea pig ileum. These findings indicate that endothelin is present in the rat gastrointestinal tract, perhaps produced by both vascular endothelial cells and mucosal epithelial cells, and can cause contraction of gastrointestinal smooth muscle. Thus, endothelin may have a physiological role in the control of gastrointestinal function.