Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

Neuropeptides in the gastrointestinal canal of Necturus maculosus

Summary The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut.Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum.Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa.The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity.It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.     

Ontogeny of immunoreactive CCK and VIP in pig brain and gut

The concentrations and hormonal forms of CCK and VIP have been determined in extracts of the brain and duodenum of the developing and adult pig. In methanol extracts of the brain cortex, the single hormone form, CCK8, increased from 130 +/- 20 (Mean +/- SEM) pmol/g at birth to an adult level of 300 +/- 50 pmol/g. In acid extracts of brain, the predominant immunoreactive form had N-terminal immunoreactivity and increased from 240 +/- 20 pmol/g at birth to an adult level 490 +/- 30 pmol/g; the C-terminal immunoreactivity was about 10-fold lower. The concentrations and hormonal forms of immunoreactive CCK in duodenal extracts did not appear to be age-related. C-terminal immunoreactivity in methanol extracts averaged 140 +/- 20 pmol/g and in acid extracts 240 +/- 60 pmol/g. The concentration of N-terminal immunoreactivity in acid extracts averaged 490 +/- 70 pmol/g. The VIP concentrations in acid extracts of the brain cortex was 13.5 +/- 2 pmol/g at birth and rose gradually to 30 +/- 9 pmol/g in the adult; in duodenal extracts it was 240 +/- 18 pmol/g at birth and 195 +/- 38 pmol/g in the adult. These results are in marked contrast with the ontogeny of these hormones in the rat in which brain concentrations of CCK and VIP in the neonate are less than 10% of adult levels and in which there are age-related changes in the content of these hormones in the duodenum as well.     

VIP-, Bombesin- and Neurotensin-Like Immunoreactivity in Neurons of the Gut of the Holostean Fish,Lepisosteus platyrhincus

VIP-like immunoreactivity was found in nerve fibres in all layers of the gut wall in both stomach and intestine, and was abundant in the myenteric and submucous plexuses. A few fibres were associated with blood vessels. Nerve cells showing VIP-like immunoreactivity were found in the myenteric plexus. Neurotensin-like immunoreactivity was found in nerve cells and numerous nerve fibres in the myenteric plexus of both stomach and intestine and in nerve fibres of the circular muscle layer, while bombesin-like immunoreactivity was confined to a low number of nerve fibres in the myenteric plexus of the stomach. The results indicate that a VIP-like, a neurotensin-like and a bombesin-like peptide are present in neurons of the gut of Lepisosteus.     

Autoradiographic localization of binding sites for galanin and VIP in small intestine

The distribution of the binding sites for [125I]galanin and [125I]iodotyrosyl VIP in sections of small intestine from rat, rabbit, guinea pig and man was investigated using in vitro receptor autoradiography. The specific binding sites for [125I]galanin were most concentrated in the myenteric plexus and longitudinal muscle layers. The distribution of [125I]VIP binding was similar, with the highest level of binding in the myenteric plexus. Some VIP binding was also seen in the mucosa. The abundance of binding sites for both peptides in the myenteric plexus suggests that this is the anatomical site of the functional antagonism between these endogenous peptides in the mammalian intestine.     

VIP-, substance P-, gastrin/CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout,Salmo gairdneri

Summary The presence of peptides in the gastrointestinal tract of the rainbow trout, Salmo gairdneri, was investigated immunocytochemically. VIP-like immunoreactivity was demonstrated in nerves in all layers of the stomach and the intestine, whereas substance P-like immunoreactivity was localized to endocrine cells, predominantly in the mucosa of the stomach, and to nerves mainly concentrated in the myenteric plexus throughout the gut. Endocrine cells reactive to gastrin/CCK antiserum were demonstrated in the intestinal mucosa, while no immunoreactivity was found in the stomach. Bombesin-immunoreactive and somatostatin-immunoreactive cells were localized in the stomach mucosa, and cells reactive to glucagon antiserum in the intestinal mucosa. Radioimmunoassay of stomach mucosa and muscle confirmed the presence of VIP-like and substance P-like immunoreactivity in these tissues, while gastrin/CCK-like immunoreactivity was low and bombesin-like immuno-reactivity was insignificant. In conclusion, molecules resembling the mammalian brain-gut peptides may be involved in the neuronal and hormonal control of gut function in fish.     

Distribution of vasoactive intestinal peptide, bombesin, and gastrin-cholecystokinin like peptides in the avian intestinal tract and brain

The distribution of vasoactive intestinal peptide (VIP), bombesin and gastrin-cholecystokinin in the chicken was studied by radioimmunoassay of tissue extracts. VIP was present in high concentrations in colon (186 +/- 29 pmol/g), cloaca (116 +/- 27 pmol/g), jejunum (97 +/- 14 pmol/g) and pancreas (15 +/- 3 pmol/g) but not detected in lung, liver or thymus. The highest concentration of bombesin was in the proventriculus (92 +/- 13 pmol/g), negligible in remaining gut but found in brain. Gel chromatography indicated two forms of bombesin: one form eluting with bombesin-14 and the other with gastrin releasing peptide. Gastrin-like immunoreactivity was found in low levels in the gut and brain. The concentrations were higher with an antiserum which cross reacted with the carboxy terminus common to gastrin-17 and CCK compared to a gastrin specific antisera (P less than 0.01). This suggests that the carboxy terminal region has been conserved during evolution. Each distribution pattern of bombesin, VIP and gastrin CCK is different, and distinct from that found in mammals, suggesting specific roles for these peptides in birds.     

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.     

The use of region-specific antibodies in the characterization and localization of vasoactive intestinal polypeptide-like substances in the rat gastrointestinal tract

Antisera specific for different regions of porcine VIP have been used in radioimmunoassay and immunohistochemical studies of immunoreactive VIP in rat small and large intestine. Cation exchange chromatography of intestinal extracts separated two major and one minor peak of immunoreactivity. One major peak eluted in a similar position to natural porcine VIP and was read equally by NH₂-terminal-specific, and mid- and COOH-terminal-specific antisera. A second major peak, and the minor peak, eluted earlier than porcine VIP, and were read significantly less well with mid- and COOH-terminal antisera compared with NH₂-terminal-specific antisera. All forms of VIP occurred mainly in extracts of muscle layers of the gut, and no antiserum revealed more than trace amounts of immunoreactivity in mucosal extracts. In immunohistochemical studies all antisera demonstrated fluorescent nerve fibres in the enteric plexuses, circular smooth muscle and lamina propria; some antisera demonstrated nerve cell bodies predominantly in the submucous plexus. NH₂-terminal-specific antisera also demonstrated a sparse population of mucosal endocrine-like cells in the ileum and colon that were not seen with other antisera. It is concluded that VIPergic neurons of the rat gut contain a peptide closely resembling porcine VIP and at least two less basic variants with similar NH₂-terminal antigenic determinants. VIP-like peptides may also occur in endocrine cells, but since these peptides appearto fact that the majority of neuronal VIP in rat gut exists in a form that is both chromatographically and immunochemically distinct from porcine VIP, and may well possess different biological properties.     

Mapping, quantitative distribution and origin of substance p- and VIP-containing nerves in the uvea of guinea pig eye

VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3 +/- 4.8 pmol/g, mean +/- SEM; substance P:11.1 +/- 1.8 pmol/g) in the uveal portion of the guinea pig eye. Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p less than 0.0005) than those immunoreactive for substance P (95 +/- 7 nm and 82 +/- 9 nm respectively; mean +/- SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1 +/- 1.5 pmol/g, mean +/- SEM, control; 5.3 +/- 1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.     

Effect of cholecystokinin octapeptide and somatostatin on the motility of guinea pig and canine gallbladder

Species differences have been observed in the effect of cholecystokinin octapeptide (CCK OP) on the canine and guinea pig gallbladder smooth muscle motility. 1. CCK OP was more potent stimulant in canine than in guinea pig gallbladder smooth muscles. Its pD2 values were 10 and 9.2, respectively. 2. The acetylcholine (10(-4) M)-induced maximum contractions in canine gallbladder muscle strips were by 50% lower as compared to the CCK OP (10(-8) M) maximum responses while in guinea pig gallbladder muscle strips the acetylcholine (ACh) maximum responses were by 20% lower than the CCK OP maximum responses. 3. CCK OP increased [3H]ACh release by 27% in canine gallbladder and by 40% in guinea pig gallbladder. 4. Somatostatin (SOM) had not any direct myogenic effect in guinea pig and canine gallbladder but it decreased [3H]ACh release from gallbladder intrinsic cholinergic neurons.     

Distribution and ontogeny of VIP-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris

Summary The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.     

Mapping,quantitative distribution and origin of substance P- and VIP-containing nerves in the Uvea of guinea pig eye

Summary VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3±4.8 pmol/g, mean ± SEM; substance P:11.1±1.8 pmol/g) in the uveal portion of the guinea pig eye.d Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p<0.0005) than those immunoreactive for substance P (95±7 nm and 82±9 nm respectively; mean ± SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1±1.5 pmol/g, mean ± SEM, control; 5.3±1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.To whom offprint requests should be sent     

Distribution and heterogeneity of immunoreactive cholecystokinin (CCK) in the mucosa of the porcine gastrointestinal tract

The concentration and molecular nature of cholecystokinin-like immunoreactivity (CCK-LI) in extracts of porcine intestinal mucosa were determined using sequence-specific radioimmunoassays. Highest CCK concentrations were measured in duodenal mucosa (258 +/- 60 pmol/g in the distal duodenum) followed by jejunal mucosa (204 +/- 36 pmol/g in the proximal jejunum) and pylorus (51 +/- 9 pmol/g). All other gastrointestinal regions proximal to the pylorus and distal to the jejunum contained less than 20 pmol/g. Pancreas contained less than 1 pmol/g. Gel chromatography in 6 M urea revealed four immunoreactive forms and this was confirmed by reverse-phase high-pressure liquid chromatography (HPLC). The predominant molecular form in acid extracts of duodenal mucosa resembled CCK-33 although high concentrations of the larger CCK form ('CCK-58') and of the form intermediate in size between CCK-33 and CCK-8 were measured. A molecular form resembling CCK-8 was the principal form in neutral extracts of the duodenum.     

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

The distribution and cellular origin of vasoactive intestinal polypeptide in the avian gastrointestinal tract and pancreas

Summary The distribution and origins of vasoactive intestinal peptide (VIP) in the gut and pancreas of the turkey were studied by radioimmunoassay of tissue extracts and by immunocytochemistry. Several antisera were used that vary in their specificity for different regions of porcine or chicken VIP. Radioimmunoassays using NH₂-terminal specific antisera that react almost equally with porcine and chicken VIP's revealed significant amounts of immunoreactive VIP in extracts of pancreas, brain and all regions of the gastrointestinal tract from crop to colon. Highest concentrations (300pmol/g) were found in the colon muscle, and concentrations were generally low (< 20 pmol/g) in the mucosal layers of the small intestine. After ion exchange chromatography of extracts on CM-Sephadex three immunoreactive forms of VIP were separated corresponding to the three molecular forms previously found in mammalian gut extracts. In immunocytochemical studies nerve fibres were found throughout the gut, and in the pancreas. Immunoreactive nerve cell bodies were also identified in the submucous plexus throughout the gut, but were particularly prominent in the oesophagus and pancreas. It has previously been shown that VIP is a strong stimulant of the flow of pancreatic juice in birds whereas the structurally related hormone secretin, which is known to control the flow of pancreatic juice in mammals, is a weak stimulant. It is proposed that in birds VIP might regulate the pancreas, and other aspects of gut function, as a neurotransmitter or neurohormone.     

Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine

The axons of neurons that innervate the longitudinal muscle of the small intestine in small mammals such as rabbit, rat, guinea pig and mouse form a network, the tertiary plexus, against the inner surface of the muscle. In general, because of their substantial overlap, it has not been possible to follow the ramifications of individual axons in the tertiary plexus. In the present work, the longitudinal muscle motor neurons were filled with marker dyes through an intracellular microelectrode, and their morphologies and projections were examined in whole-mount preparations of longitudinal muscle and myenteric plexus. Most neurons that were examined were in the small intestine (ileum and duodenum), but a few were examined in the distal colon. Neurons in all regions had similar morphologies and projections. The cell bodies were amongst the smallest in myenteric ganglia, with major and minor axes of 14 microns and 25 microns (mean, n = 40) in the plane of the myenteric plexus. Each neuron had a single axon that branched extensively in the tertiary plexus, most had multiple lamellar dendrites and a few had filamentous dendrites or a mixture of filamentous and lamellar dendrites. The mean area of muscle covered by an axon and its branches extended 1.6 mm orally to anally and 1.7 mm circumferentially. The area covered was 2.8 +/- 1.9 mm2 (mean +/- SD, n = 23). From the density of occurrence of cell bodies, it can be calculated that each point in the longitudinal muscle is innervated by the processes of about 100 motor neurons and is influenced by electrotonic conduction of signals through the muscle by about 300 motor neurons.     

Characterization of a monoclonal antibody to human sex hormone binding globulin

The occurrence and distribution of PHI-like immunoreactivity in the guinea pig gallbladder has been analysed by radioimmunoassay and immunocytochemistry. Chromatography of gallbladder extracts by gel permeation and high-performance liquid chromatography revealed that guinea pig PHI-like immunoreactivity is of a similar size to that of porcine PHI but may differ in its amino acid sequence. Immunocytochemistry showed PHI-immunoreactivity to be localised to nerves found predominantly in the ganglionated plexus and the mucosal plexus of the gallbladder. Pure natural porcine PHI induced a dose-dependent relaxation of the isolated guinea pig gallbladder muscle which was not blocked by antagonists to acetylcholine, catecholamines, histamine, and 5-hydroxytryptamine. PHI may thus be one of the local factors involved in controlling gallbladder function.