Effects of some autonomic drugs and neuropeptides on the mechanical activity of longitudinal and circular muscle strips isolated from the carp intestinal bulb (Cyprinus carpio)

1. The mechanical responses to some autonomic drugs and neuropeptides of longitudinal muscle (LM) and circular muscle (CM) strips isolated from the carp intestinal bulb were investigated in vitro. 2. Acetylcholine and carbamylcholine caused concentration-dependent transient contraction of both LM and CM strips. Tetrodotoxin had no effect, but atropine selectively decreased the contractile responses to acetylcholine and carbamylcholine. 3. Excitatory alpha-2 and inhibitory beta adrenoceptors were present in both LM and CM strips. 4. 5-Hydroxytryptamine (5-HT) caused concentration-dependent contraction of both LM and CM strips. Tetrodotoxin, atropine and methysergide decreased the contractile responses to 5-HT. 5. Some neuropeptides (angiotensin I, angiotensin II, bombesin, bradykinin, neurotensin, somatostatin and vasoactive intestinal polypeptide) did not cause any mechanical response (contraction or relaxation) in either smooth muscle strip. 6. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) caused contraction of both LM and CM strips. However, the time course of the contraction in LM was different from that in CM. The order of potency was NKA greater than SP greater than NKB in LM strips and NKA greater than SP much greater than NKB in CM strips. In LM strips, the contractile responses to tachykinins were unaffected by spantide and methysergide, but partly decreased by tetrodotoxin and atropine. On the other hand, the contractile responses of CM strips were unaffected by tetrodotoxin, atropine, methysergide and spantide. 7. Dynorphin (1-13) (DYN), leucine-enkephalin (L-Enk) and methionine-enkephalin (M-Enk) caused concentration-dependent contraction of both LM and CM strips. The order of potency was DYN greater than M-Enk greater than L-Enk. Naloxone selectively decreased the responses to opiate peptides. 8. The present results indicate that acetylcholine, carbamylcholine, catecholamines, 5-HT, tachykinins (SP, NKA and NKB) and opiate peptides (DYN, L-Enk and M-Enk) affect the mechanical activity of LM and CM strips isolated from the carp intestinal bulb through their specific receptors.     

The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

Catecholamine-synthesizing enzymes and neuropeptides in rat heart epicardial ganglia; an immunohistochemical study

Summary The subepicardial atrial ganglia of rat hearts were examined using immunohistochemical techniques and antibodies against the catecholamine-synthetic enzymes tyrosine hydroxylase (TH) and dopamine--hydroxylase (DBH), and the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and met-5-enkephalin (ENK). Some of the ganglion cells present in the ganglia exhibited DBH-like immunoreactivity (LI) and NPY-LI, whilst these cells nerver exhibited TH-, VIP-, CGRP-, SP- or ENK-LI. Groups of small cells exhibiting an intense TH-LI, corresponding to cells referred to as catecholamine-containing cells and sometimes small intensely fluorescent cells in the literature, were observed in the ganglia. A subpopulation of these cells exhibited immunoreactivity to one of the neuropeptides tested, namely SP. Only a few of the cells showing TH-LI displayed DBH-LI. Nerve fibres showing SP-, CGRP-, DBH- and TH-LI were present in the ganglia; some of these fibres being closely associated with the ganglion cells or with the cells showing TH-LI. The observations provide new information on the catecholaminesynthetic enzyme/neuropeptide expression of the ganglion and catecholamine-containing cells and of the associated nerve fibres of rat heart subepicardial ganglia.     

Neuropeptide expression in the airways of COPD patients and smokers with normal lung function

Neurogenic mechanisms seem to play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD), as suggested by a number of in vitro data. However, few studies have investigated the presence of neuropeptides in the airways of patients with COPD, and they have yielded conflicting results. The aim of this study is to compare the expression of the neuropeptide substance P (SP), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) in the airways of smokers with and without COPD. Surgical lung samples were obtained from 15 smokers with COPD and 16 smokers with normal lung function, who underwent lobectomy for a solitary lung carcinoma. Airway expression and distribution of SP, VIP, and NPY were identified by immunohistochemistry and analyzed by a computerized image analysis system. Compared to smokers with normal lung function, COPD patients exhibited an increased immunoreactivity for SP and VIP, paralleled by a decreased NPY expression in the epithelium and glands, and a decreased expression of all these three neuropeptides in the smooth muscle layer. Therefore, in the present study we have documented a different expression and distribution of the neuropeptides SP, VIP, and NPY in the airways of smokers with and without COPD. These findings suggest a possible involvement of such neuropeptides in the pathogenesis of some changes occurring in COPD.     

Neuropeptide-containing nerve fibres in the human parotid gland: a semiquantitative analysis using an antibody against protein gene product 9.5

The occurrence and distribution of neuropeptide-containing fibres in the human parotid gland were examined by the peroxidase--antiperoxidase method with attention to the quality of fixation and the condition of patients. Many fibres immunoreactive for neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) and a moderate number of galanin- positive (GAL) fibres were distributed around the acini. A moderate number of NPY and VIP fibres were distributed around the intercalated ducts. The semiquantitative mean densities (_SD) of periacinar NPY, VIP and GAL fibres expressed as a percentage of the total protein gene product (PGP) 9.5 immunoreactive fibres were 75.62 _ 7.25%, 70.52 _ 9.33% and 41.76 _ 5.45%, respectively, whereas those of substance P (SP), calcitonin gene-related peptide (CGRP) and FMRF amide (FMRF) fibres were below 10%. The mean densities of NPY and VIP fibres around the intercalated ducts expressed as the percentage of PGP 9.5 fibres associated with these ducts were 52.37 _ 6.19% and 59.62 _ 7.02% respectively. Those of SP, CGRP, GAL, and FMRF fibres were below 10%. The densities of NPY, VIP, SP, CGRP, GAL and FMRF fibres around the striated and excretory ducts were also below 10%. In the vasculature, NPY fibres were the most prominent. Similarly, the mean density of perivascular NPY fibres was 93.76 _ 2.03%. No somatostatin or leucine or methionine enkephalin immunoreactivity was detected around the acini, duct system or blood vessels. These findings suggest that, in this gland, the periacinar NPY, VIP and GAL fibres may participate in regulating the synthesis of saliva and its secretion and that perivascular peptidergic fibres, especially NPY fibres, may be involved in controlling local blood flow This revised version was published online in November 2006 with corrections to the Cover Date.     

Regional Distribution of Methionine-Enkephalin-Arg6-Phe7 in the Rat Brain: Comparative Study with the Distribution of Other Opioid Peptides

The distribution of the opioid peptide methionine-enkephalin-arginine6-phenylalanine7 (M-Enk-Arg6-Phe7) has been investigated in various structures of the rat brain by using a highly specific radioimmunoassay (RIA). Immunoreactive M-Enk-Arg6-Phe7 has been further characterized by high performance liquid chromatography. The levels of M-Enk-Arg6-Phe7 in various structures of the rat brain were compared with the levels of several other opioid peptides, including methionine-enkephalin (M-Enk), leucine-enkephalin (L-Enk), dynorphin 1-13, and alpha-neoendorphin, which were also measured by RIA. There was a close relationship between the distribution of M-Enk-Arg6-Phe7 immunoreactive material (ir), M-Enk ir, and L-Enk ir. The distribution of dynorphin 1-13 ir and alpha-neoendorphin ir appeared to be distinct from that of the enkephalin group. These results are in agreement with recent reports on the cloning and sequencing of the c-DNA coding for the prohormones, in which it has been hypothesized that M-Enk-Arg6-Phe7 and M-Enk are synthesized by the same precursor, called proenkephalin, and that dynorphin-related peptides and alpha-neoendorphin arise from a separate precursor, prodynorphin.     

Involvement of Leukemia Inhibitory Factor in the Increases in Galanin and Vasoactive Intestinal Peptide mRNA and the Decreases in Neuropeptide Y and Tyrosine Hydroxylase mRNA in Sympathetic Neurons After Axotomy

Abstract: In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy-induced down-regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF-blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down-regulation of peptides/proteins involved in normal neurotransmission and the up-regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

Neuropeptides in sensory perikarya projecting to the rat ovary

Afferent perikarya in dorsal root ganglia (DRG) at the T13 and L1 segmental levels projecting to the rat ovary were identified by utilizing the fluorescent retrograde tracer true blue (TB). Subsequently, TB-labeled ovarian afferent perikarya in DRG were examined for vasoactive intestinal polypeptide (VIP), substance P (SP), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and somatostatin (SOM) immunoreactivity and for the presence of fluoride-resistant acid phosphatase (FRAP) enzyme activity. Of the ovarian afferent perikarya at the T13 and L1 segmental levels, 20.5% displayed VIP immunoreactivity, 23.8% displayed SP immunoreactivity, and 43.1% were immunoreactive for CCK-8. No ovarian afferent perikarya contained SOM or NYP immunoreactivity or FRAP activity. It is suggested that different neuropeptides may participate in modulation of specific ovarian functions.     

Cholinergic innervation of the pancreas in the sheep

Antibodies raised against vesicular acetylcholine transporter (VAChT) were applied to study the cholinergic innervation pattern of the pancreas of the sheep. To determine whether the cholinergic pancreatic neuronal elements contain tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) or substance P (SP) double immunocytochemistry was used. A moderate number of VAChT-immunoreactive (IR) nerve terminals were distributed between the acini, whereas only single cholinergic nerve fibres innervated the interlobular connective tissue. VAChT-positive nerve fibres supplying the endocrine pancreas were found only occasionally. The pancreatic blood vessels and ducts system were devoid of VAChT-containing nerve endings. All intrapancreatic neurons studied showed immunoreactivity to VAChT, but intrapancreatic ganglia were not innervated with cholinergic nerve fibres. The colocalization of VAChT and TH or VAChT and SP was detected in distinct populations of nerve fibres localized amongst the acini, but not within the islet nor in the connective tissue. Single VAChT-IR nerve terminals co-expressing NPY were distributed around the acini, islets as well as in the connective tissue septa. A moderate number of VAChT-IR/VIP-IR nerve endings were located in the exocrine pancreas, whereas the islets and connective tissue were innervated with VAChT/VIP-containing nerve fibres only occasionally. In the vast majority of VAChT-positive intrapancreatic perikarya the presence of TH was additionally found. A moderate number of VAChT-IR intrapancreatic perikarya co-expressed NPY, SP or VIP. The results of the present study demonstrate species-dependent cholinergic innervation pattern of the pancreas of the sheep. The co-localization of VAChT with the neuropeptides suggests the existence of functional interactions influencing the ovine pancreas (mainly exocrine) activity.     

Quantitative comparison of growth-associated protein GAP-43, neuron-specific enolase, and protein gene product 9.5 as neuronal markers in mature human intestine.

This study was performed to compare GAP-43, PGP 9.5, synaptophysin, and NSE as neuronal markers in the human intestine. GAP-43-immunoreactive nerve fibers were abundant in all layers of the ileum and colon. GAP-43 partially co-localized partially with every neuropeptide (VIP, substance P, galanin, enkephalin) studied. All neuropeptide-immunoreactive fibers also showed GAP-43 reactivity. By blind visual estimation, the numbers of GAP-43-immunoreactive fibers in the lamina propria were greater than those of PGP 9.5, synaptophysin, or NSE. In the muscle layer, visual estimation indicated that the density of GAP-43-immunoreactive fiber profiles was slightly greater than that of the others. The number and intensity of GAP-43-, PGP 9.5-, and NSE-immunoreactive fibers were estimated in sections of normal human colon and ileum using computerized morphometry. In the colon, the numbers of GAP-43-immunoreactive nerve profiles per unit area and their size and intensity were significantly greater than the values for PGP and NSE. A similar trend was observed in the ileum. Neuronal somata lacked or showed only weak GAP-43 immunoreactivity, variable PGP 9.5 immunoreactivity, no synaptophysin immunoreactivity, and moderate to strong NSE immunoreactivity. We conclude that GAP-43 is the superior marker of nerve fibers in the human intestine, whereas NSE is the marker of choice for neuronal somata. (J Histochem Cytochem 47:1405-1415, 1999)     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.     

Modulation of the enkephalinergic phenotype of rat sympathetic neurons by hormonal and transynaptic mechanisms

Most sympathetic neurons contain one or more neuropeptides in addition to catecholamines. Although the regulation of catecholamines has been studied extensively, comparatively little is known about the regulation of neuropeptides. Since glucocorticoids and preganglionic innervation regulate catecholaminergic properties in chromaffin cells, we examined the effects of these factors on a co-localized neuropeptide, leucine enkephalin (L-Enk), in adult rat sympathetic neurons in vivo. Lowered serum glucocorticoid levels as a consequence of bilateral adrenalectomy resulted in a reduction of ganglionic L-Enk content that was reversed by exposure of adrenalectomized animals to the synthetic glucocorticoid, dexamethasone. Surgical denervation of the SCG eliminated L-Enk-IR preganglionic fibers and caused a dramatic increase in the number of L-Enk-IR neurons. Inhibition of the enkephalinergic component of the preganglionic innervation by chronic exposure to the opiate receptor antagonist naloxone increases the number of L-Enk-IR cell bodies and total ganglionic L-Enk content. None of the experimental manipulations noticeably altered the number or distribution of NPY-IR neurons, suggesting that the effects of glucocorticoids and the innervation on ganglionic L-Enk levels were specific and not simply an alteration of the biosynthetic state of the cells. These results demonstrate that circulating glucocorticoids and the preganglionic innervation regulate L-Enk levels in sympathetic neurons. Since both glucocorticoid levels and preganglionic activity are known to be altered by stressful stimuli, acute regulation of sympathetic L-Enk levels may constitute an important component of the autonomic response to stress. © 1993 John Wiley & Sons, Inc.     

Peptidergic inputs to sympathetic preganglionic neurons

This paper presents data showing that the sympathetic autonomic areas of the cat thoracolumbar spinal cord contain nerve terminals and fibres with immunoreactivity for at least seven neuropeptides. The distribution in the intermediolateral cell column of the terminals and fibres which contain enkephalin-, neuropeptide Y-, neurotensin-, substance P-, and neurophysin II-like immunoreactivity (ENK, NPY, NT, SP, and NP2, respectively) suggests that these peptides are involved in more generalized functions of the autonomic nervous system. On the other hand, peaks in density of immunoreactivity at certain levels suggest that different levels of influence of sympathetic preganglionic neurons by the various peptides may occur along the length of the thoracolumbar cord. The distribution of terminals and fibres containing somatostatin- and oxytocin-like immunoreactivity (SS and OXY) suggests that these peptides may be part of specific pathways to particular sympathetic preganglionic neurons. The possible sources of the terminals and fibres containing ENK, NPY, NT, SS, and SP include the spinal cord and supraspinal areas, whereas the source of these structures with OXY and NP2 is most likely supraspinal. The data suggest that coexistence of peptides and interactions between structures containing different neuropeptides occur in the spinal autonomic areas. It is speculated that neuropeptides have an important role to play in the regulation of the cardiovascular division of the autonomic nervous system.     

Light- and electron-microscopic immunochemical analysis of nerve fibre types innervating the taenia of the guinea-pig caecum

Summary The present work was undertaken to determine by immunocytochemical methods which of the putative enteric neurotransmitters are contained in axons supplying the guinea-pig taenia coli and what proportion of axons is accounted for by the presence of these substances. Numerous fibres displayed immunoreactivity for dynorphin (DYN), enkephalin (ENK), -aminobutyric acid (GABA), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP), but, in contrast to other gut regions, fibres showing immunoreactivity for gastrin-releasing peptide, galanin and neuropeptide Y were rare in the taenia. Fibres reactive for calbindin, calcitonin gene-related peptide, cholecystokinin, 5-hydroxytryptamine and somatostatin were also rare. Tyrosine hydroxylase-like immunoreactivity (TH-LI) was present in numerous fibres that disappeared after extrinsic denervation, a procedure that did not detectably affect any of the other major groups of fibres. Simultaneous staining of extrinsically denervated preparations revealed that SP-LI and VIP-LI were located in separate fibres, and ultrastructural studies showed these to be 58% and 33% of intrinsic fibres supplying the muscle. Immunoreactivity for the general marker, neuron-specific enolase, was located in 95–98% of axons. ENK-LI and DYN-LI were in the same axons, and similar proportions of the fibres with either SP-LI or VIP-LI, about 85%, contained immunoreactivity for ENK and DYN. All VIP-LI fibres, but no SP-LI fibres, were reactive for NOS. The results imply that the taenia of the guinea-pig caecum is innervated by two major groups of enteric neurons: (i) excitatory neurons that contain ACh, SP, other tachykinins, and, in most cases, DYN-LI and ENK-LI; and (ii) inhibitory neurons that contain NOS-LI, VIP-LI, in most cases, the two opioids and, quite probably, ATP as a transmitter. GABA-LI is contained in a smaller population of intrinsic axons. Even though the taenia represents one of the simplest tissues for examining transmission from enteric neurons to intestinal muscle, it shares some of the complexity of other regions, in that four major axon types supply the muscle and both the enteric excitatory and enteric inhibitory neurons contain multiple transmitters.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

Nitric oxide synthase in the gastrointestinal tract of the estuarine crocodile, Crocodylus porosus

The aim of this study was to investigate the distribution of nitric oxide synthase (NOS)-containing nerve cells in the gastrointestinal tract of a reptile and to compare it with the pattern in other vertebrate classes. In the estuarine crocodile, Crocodylus porosus, NOS-positive nerve cell bodies and fibres were found in all regions of the gut examined. Most myenteric microganglia contained one or several NOS-immunoreactive neurons together with unlabelled neurons. The majority of the neurons were multipolar, ranging from 10 to 25 microns in diameter. Both the circular and the longitudinal muscle layers were innervated by NOS-immunoreactive nerve fibres, which mostly ran parallel to the muscle fibres. In addition, small blood vessels in the submucosa and on the serosal surface of the gut were innervated by NOS-immunoreactive fibres. Double labelling with antisera to NOS and vasoactive intestinal peptide (VIP) revealed three neuronal subpopulations. A small proportion of the NOS-immunoreactive cells also contained immunoreactivity to VIP while a majority of the VIP-immunoreactive cells were NOS immunoreactive. There were more nerve fibres showing VIP immunoreactivity than fibres with NOS immunoreactivity, although most of the latter also contained immunoreactivity to VIP. VIP-immunoreactive fibres often surrounded the NOS-immunoreactive nerve cells. These results suggest that neuronally released nitric oxide is likely to be involved in the control of gastrointestinal motility in the crocodile as in most other vertebrate species.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.     

Expression of neuron-specific enolase, Leu-7, and neuropeptides in human fetal salivary gland epithelium

The immunoreactivity of anti-neuron-specific enolase (NSE) and anti-Leu-7 on formalin-fixed sections of human fetal salivary gland epithelium was determined by the avidin-biotin-peroxidase complex (ABC) method. In addition, expression of some neuropeptides such as vasoactive intestinal polypeptide (VIP), somatostatin (SRIF), and substance P in the human salivary gland epithelium during the gestational period was observed, whereas the other polypeptides examined, including glucagon, cholecystokinin (CCK), Leu-enkephalin, and calcitonin were absent. NSE and Leu-7 immunoreactivity in the fetal salivary gland epithelium was observed solitarily or in groups commonly restricted to the developing duct epithelium. Positive immunoreactivity was observed in 46 cases with NSE (73%) and 44 cases with Leu-7 (70%) in 63 fetal salivary glands examined. In contrast, the incidence of positive cases stained with neuropeptides was lower than those of NSE and Leu-7 immunoreactivity in the human fetal salivary gland epithelium. These findings indicate that certain neuropolypeptides, as well as VIP, SRIF, and substance P present in the human fetal salivary gland epithelium may play a significant role in the development of the gland.