Human alpha-calcitonin gene-related peptide influences colonic secretion by acting on myenteric neurons

The effect of human alpha-calcitonin gene-related peptide (CGRP) on epithelial ion transport was investigated in guinea pig distal colon set up in Ussing flux chambers. Addition of CGRP to the serosal bathing solution evoked a dose-dependent increase in short-circuit current in whole-thickness tissues with intact myenteric and submucosal ganglia, but not in whole-thickness preparations when neural connections between myenteric and submucosal ganglia were severed, nor in sheets of submucosa/mucosa with intact submucosal ganglia. The effects of CGRP were nearly abolished in chloride-free solutions or after treatment with furosemide. Tetrodotoxin and hexamethonium abolished the effects of CGRP on basal short-circuit current whereas atropine did not. CGRP enhanced neurally evoked chloride secretion both in whole thickness and submucosa/mucosa preparations, but the effect in the latter was considerably smaller. These observations suggest that CGRP stimulates chloride secretion primarily by activating myenteric neurons that project either to submucosal ganglia or to the mucosa of the guinea pig distal colon. Furthermore, CGRP appears to have a greater effect on excitability of myenteric neurons than submucosal neurons.     

Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro

Glucagon-like peptide-1 (GLP-1) acts at the G protein-coupled receptor, GLP-1R, to stimulate secretion of insulin and to inhibit secretion of glucagon and gastric acid. Involvement in mucosal secretory physiology has received negligible attention. We aimed to study involvement of GLP-1 in mucosal chloride secretion in the small intestine. Ussing chamber methods, in concert with transmural electrical field stimulation (EFS), were used to study actions on neurogenic chloride secretion. ELISA was used to study GLP-1R effects on neural release of acetylcholine (ACh). Intramural localization of GLP-1R was assessed with immunohistochemistry. Application of GLP-1 to serosal or mucosal sides of flat-sheet preparations in Ussing chambers did not change baseline short-circuit current (I(sc)), which served as a marker for chloride secretion. Transmural EFS evoked neurally mediated biphasic increases in I(sc) that had an initial spike-like rising phase followed by a sustained plateau-like phase. Blockade of the EFS-evoked responses by tetrodotoxin indicated that the responses were neurally mediated. Application of GLP-1 reduced the EFS-evoked biphasic responses in a concentration-dependent manner. The GLP-1 receptor antagonist exendin-(9-39) suppressed this action of GLP-1. The GLP-1 inhibitory action on EFS-evoked responses persisted in the presence of nicotinic or vasoactive intestinal peptide receptor antagonists but not in the presence of a muscarinic receptor antagonist. GLP-1 significantly reduced EFS-evoked ACh release. In the submucosal plexus, GLP-1R immunoreactivity (IR) was expressed by choline acetyltransferase-IR neurons, neuropeptide Y-IR neurons, somatostatin-IR neurons, and vasoactive intestinal peptide-IR neurons. Our results suggest that GLP-1R is expressed in guinea pig submucosal neurons and that its activation leads to a decrease in neurally evoked chloride secretion by suppressing release of ACh at neuroepithelial junctions in the enteric neural networks that control secretomotor functions.     

Evidence for substance P as a functional neurotransmitter in guinea pig small intestinal mucosa

We showed previously that electrical transmural stimulation (TS) of guinea pig jejunal mucosa in vitro released neurotransmitters from submucosal plexus neurons which caused alterations in ion transport. The present studies were performed to obtain information regarding the identity of the neurotransmitters. The addition of exogenous substance P (SP) to the serosal side of the tissue caused a transient increase in short-circuit current (Isc) which closely mimicked the response to TS. Both TS and SP caused net secretion of Cl- ions by stimulating movement toward the luminal side. Tetrodotoxin abolished the response to TS, inhibited approximately 70% of the response to SP but did not affect the response to urecholine, a cholinergic muscarinic agonist. In the presence of the muscarinic antagonist, atropine, Isc responses to both TS and SP were reduced suggesting that a portion of both responses was due to action on enteric nerves causing release of acetylcholine. Following desensitization of the tissue with supramaximal doses of SP the response to TS was significantly reduced but the response to urecholine was unchanged. In the presence of atropine, SP desensitization reduced the nerve-stimulated response by approximately 65%; treatment of tissue with SP antibodies reduced the response by approximately 55%. Under the same conditions Isc responses to histamine were unaltered. Our results suggest that both SP (or a structurally analogous neurotransmitter) and acetylcholine as well as additional unidentified neurotransmitter(s) are functionally important in the regulation of intestinal ion transport in guinea pig jejunum.     

The neurosecretory effect of ouabain on isolated rabbit ileal mucosa

Ouabain, when added to fluid bathing rabbit ileal mucosa mounted in a flux chamber, transiently increases short circuit current, implying a paradoxical secretory response. To determine the cause of this change, we studied unidirectional fluxes of 36Cl and 23Na and the effects of ion substitution, of reduced Ca concentration, verapamil, tetrodotoxin and atropine. Ouabain 0.1 mM, transiently increased the serosal to mucosal flux of Cl and Na, increased Isc and PD and reduced ion conductance. The Isc response to ouabain was diminished by reducing the bath fluid concentration of Cl, of Ca, and by adding verapamil. Tetrodotoxin both delayed and reduced the maximal Isc response; atropine had no effect. We conclude that ouabain acts by releasing a neurotransmitter of unknown identity and by increasing the serosal to mucosal flux of Cl.     

Neurokinin 1 receptors mediate substance P-induced changes in ion transport in guinea-pig ileum.

Tachykinin receptors mediating substance P-induced secretion were examined in muscle-stripped segments of guinea-pig ileum set up in flux chambers. Changes in the short-circuit current (Isc) served as an index of active, electrogenic ion transport. Substance P evoked a transient increase in Isc which was concentration-dependent. The maximal change in Isc occurred at 1 microM concentration. [Sar9,Met(O2)11]-substance P, a neurokinin 1 (NK-1) receptor agonist, evoked a similar concentration-dependent increase in Isc. [Nle10]NKA(4-10) (1 microM) or [Pro7]NKB (1 microM), selective NK2 and NK3 agonists, respectively, had minimal effects on Isc. CP-96,345 (5 microM), a nonpeptide NK-1 antagonist, and the peptide NK-1 antagonist, GR82334 (1 microM), reduced the secretory response to substance P (50 nM) in the presence and absence of tetrodotoxin (0.2 microM). The NK2 antagonist, [Tyr5,D-Trp6,8,9,Arg10]NKA(4-10) MEN 10207 had no effect on the substance P response. Tetrodotoxin (0.2 microM) significantly reduced, but did not abolish the Isc response to substance P (1 microM) and [Sar9,Met(O2)11]substance P (1 microM). The substance P response was unaltered by 5 microM atropine and 50 microM mecamylamine. Piroxicam (10 microM) or pyrilamine (10 microM) or a combination of both had no effect on the tetrodotoxin-resistant substance P response. Electrical field stimulation evoked a biphasic increase in Isc which was significantly reduced by 0.2 microM tetrodotoxin. Atropine (5 microM) reduced the first peak of the biphasic response and mecamylamine (50 microM) had no effect. Similarly, 5 microM CP-96,345 and 1 microM GR82334 did not alter the EFS-induced change Isc. The results suggest that substance P-evoked secretory responses are independent of histamine or prostaglandins. Substance P responses are mediated by an NK-1 receptor type on enteric neurons and possibly epithelial cells.     

Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current (Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.     

Differential effects of maturation on nicotinic- and muscarinic receptor-induced ion secretion in guinea pig distal colon

The incidence of constipation increases with age. This has been linked to age-related changes in the structure and function of myenteric neurons regulating intestinal motility; however, the role of submucous neurons is unknown. The aim of this study was to determine the effect of maturation on cholinergic receptor-induced ion secretion in guinea pig colon. Changes in the short-circuit current (Isc) and tissue conductance were monitored in muscle-stripped colonic segments from young (3-4-month-old) and mature (12-15-month-old) male guinea pigs. Thirty-one percent of colonic segments from young guinea pigs exhibited ongoing neural activity, which was absent in mature animals. Baseline Isc was significantly higher only in young guinea pig tissues with ongoing activity. Tissue conductance was similar in all tissues. Electrical field stimulation caused a biphasic increase in the Isc. At 15 V/10 Hz, only Peak 1 was attenuated, whereas both peaks were reduced in mature guinea pigs at 10 V/5Hz. 1,1, dimethyl-4-phenyl-piperazinium(DMPP)-induced ion secretion was blunted in mature guinea pigs. Atropine reduced the 1,1, dimethyl-4-phenyl-piperazinium response only in young guinea pigs. Carbachol-induced ion secretion was similar in tissues from both age groups. In conclusion, nicotinic receptor-induced secretion mediated by both cholinergic and noncholinergic secretomotor neurons was blunted; however, epithelial muscarinic receptor activity was unaltered during maturation.     

Modulation of colonic motility by substance P, cholecystokinin and neuropeptide Y.

The effects of substance P, cholecystokinin and neuropeptide Y were examined on rabbit distal colonic motility. All three agents produced increased contractile activity but the mechanisms responsible differed depending on the agent tested. In the intact animal, peptide effects were measured under basal conditions and following exposure to atropine, tetrodotoxin and the alpha-adrenergic antagonist phentolamine. Administration of all three peptides resulted in a stimulation of colonic motility. Phentolamine did not significantly effect substance P-, cholecystokinin- or neuropeptide Y-induced activity. By contrast, the in vivo activity induced by cholecystokinin and neuropeptide Y, but not substance P, was nearly eliminated by tetrodotoxin. Only the neuropeptide Y response was partially atropine sensitive. In isolated colonic strips, cholecystokinin-induced activity, but not that produced by neuropeptide Y or substance P, was blocked by tetrodotoxin. Atropine did not significantly inhibit any of the hormone-induced contractions.     

Differences in the distribution and chemical coding between neurons in the inferior mesenteric ganglion supplying the colon and rectum in the pig

The distribution and chemical coding of neurons in the porcine left and right inferior mesenteric ganglion projecting to the ascending colon and rectum have been investigated by using combined retrograde tracing and double-labelling immunohistochemistry. The ganglion contained many neurons supplying both gut regions. The colon-projecting neurons (CPN) occurred exclusively in the cranial part of the ganglia where they formed a large cluster distributed along the dorso-lateral ganglionic border and a smaller cluster located close to the caudal colonic nerve output. The rectum-projecting neurons (RPN) formed a long stripe along the entire length of the lateral ganglionic border and, within the right ganglion only, a small cluster located close to the caudal colonic nerve output. Immunohistochemistry revealed that the vast majority of the CPN and RPN were noradrenergic (tyrosine-hydroxylase-positive). Many noradrenergic neurons supplying the colon contained somatostatin or, less frequently, neuropeptide Y. In contrast, a significant subpopulation of the noradrenergic RPN expressed neuropeptide Y, whereas only a small proportion contained somatostatin. A small number of the non-adrenergic RPN were cholinergic (choline-acetyltransferase-positive) and a much larger subpopulation of the nerve cells supplying both the colon and rectum were non-adrenergic and non-cholinergic. Many cholinergic neurons contained neuropeptide Y. The non-adrenergic non-cholinergic neurons expressed mostly somatostatin or neuropeptide Y and some of those projecting to the rectum contained nitric oxide synthase, galanin or vasoactive intestinal polypeptide. Many of both the CPN and RPN were supplied with varicose nerve fibres exhibiting immunoreactivity against Leu5-enkephalin, somatostatin, choline-acetyltransferase, vasoactive intestinal polypeptide or nitric oxide synthase The somatotopic and neurochemical organization of this relatively large population of differently coded inferior mesenteric ganglion neurons projecting to the large bowel indicates that these cells are probably involved in intestino-intestinal reflexes controlling peristaltic and secretory activities.     

Oxytocin is expressed by both intrinsic sensory and secretomotor neurons in the enteric nervous system of guinea pig

Single- and double-immunostaining techniques were used systematically to study the distribution pattern and neurochemical density of oxytocin-immunoreactive (-ir) neurons in the digestive tract of the guinea pig. Oxytocin immunoreactivity was distributed widely in the guinea pig gastrointestinal tract; 3%, 13%, 17%, 15%, and 10% of ganglion neurons were immunoreactive for oxytocin in the myenteric plexuses of the gastric corpus, jejunum, ileum, proximal colon, and distal colon, respectively, and 36%, 40%, 52%, and 56% of ganglion neurons were immunoreactive for oxytocin in the submucosal plexuses of the jejunum, ileum, proximal colon, and distal colon, respectively. In the myenteric plexus, oxytocin was expressed exclusively in the intrinsic enteric afferent neurons, as identified by calbindin 28 K. In the submucosal plexuses, oxytocin was expressed in non-cholinergic secretomotor neurons, as identified by vasoactive intestinal polypeptide. Oxytocin-ir nerve fibers in the inner circular muscle layer possibly arose from the myenteric oxytocin-ir neurons, and oxytocin-ir nerve fibers in the mucosa possibly arose from both the myenteric and submucosal oxytocin-ir neurons. Thus, oxytocin in the digestive tract might be involved in gastrointestinal tract motility mainly via the regulation of the inner circular muscle and the balance of the absorption and secretion of water and electrolytes.     

Tonic activity of submucosal neurons influences basal ion transport

The influence of tonically active submucosal neurons on basal ion transport was studied using sheets of guinea pig ileum set up in flux chambers. Tetrodotoxin evoked an immediate and sustained decrease in short-circuit current that was sustained for 60 minutes compared with control tissues in which basal currents gradually decreased over time. Time-dependent changes in basal short-circuit currents in tissues treated with atropine were not significantly different from control tissues. The decrease in short-circuit current after tetrodotoxin resulted from a greater increase in net chloride absorption than sodium absorption. Changes in net sodium and chloride transport were due to an increase in the mucosal-to-serosal fluxes of these ions. The results suggest that tonic activity of submucosal neurons limits the absorptive capacity of the guinea pig ileum.     

Actions of neuropeptide Y on basal, cyclic AMP-induced and neurally evoked ion transport in porcine distal jejunum

Neuropeptide Y (NPY) and its homolog, peptide YY, are present respectively in neurons and endocrine cells within the mammalian small intestine. In this study, we examined the actions of NPY on ion transport in the porcine distal jejunum mucosa-submucosa in vitro. Peptide YY and NPY were equieffective in producing rapid and sustained decreases in basal short-circuit current (Isc), a bioelectrical measure of active ion transport, eliciting half-maximal decreases at respective serosal concentrations of 0.8 and 30 nmol/l. NPY-induced changes in Isc were due to increased mucosa-to-serosa and net Cl fluxes and were not affected by the absence of extracellular HCO3 ions. NPY activity was correlated with the magnitude of the basal Isc and appeared to depend on the spontaneous production of eicosanoids. The peptide also decreased Isc stimulated by forskolin and 8-bromo-cyclic AMP, but the ionic bases for this effect were complex and differed from those determined under basal conditions. NPY attenuated increases in Isc produced by electrical stimulation of enteric neurons with an IC50 = 5 nmol/l. The actions of the peptide on basal and cyclic AMP-induced ion transport were abolished by the neuronal conduction blocker tetrodotoxin, but not by the opiate antagonist naloxone. The alpha-adrenoceptor blocker phentolamine diminished the effects of NPY on basal, but not cyclic AMP-induced Isc. These results indicate that NPY is capable of modulating NaCl transport in the porcine jejunal mucosa under several different conditions. Furthermore, the effects of the peptide are mediated in part through noradrenergic nerves as well as enteric neurons of unknown chemical identity.     

Electrogenic ion secretion in proximal,mid and distal colon from fed and starved mice

1. Electrogenic ion transport was monitored in vitro as the short-circuit current (Isc in μA/cm²) across proximal, mid and distal colon removed from fed and 48 hr-starved Swiss albino mice (Mus muscaris).2. Electrogenic secretion was induced either with serosal bethanechol (muscarinic agonist), DMPP (nicotinic agonist) or dibutyryl-cyclic AMP (DbcAMP). Proximal and distal colon from starved mice showed greater electrogenic secretion in response to bethanechol than those from the fed controls while DMPP and DbcAMP did not activate the hypersecretion.3. In the distal colon, starvation induced a large increase in the basal Isc that was unaffected by mucosal amiloride but was inhibited by tetrodotoxin (TTX) and by diphenylamine-2-carboxylic acid (DPC) unlike the fed basal Isc. Bethanechol activated a biphasic response consisting of a transient decrease in the Isc followed by a sustained increase both of which were significantly greater in the starved than the fed tissue and were inhibited by TTX, DPC and atropine but not hexamethonium.4. Starvation enhances the secretory response to muscarinic activation in proximal and distal colon and induces an increased basal electrogenic (Cl ⁻) secretion in the distal colon stimulated by an augmented neural tone.     

Canine jejunal submucosa cultures: characterization and release of neural somatostatin

A primary culture of the canine jejunal submucosa has been established and used to investigate neuronal somatostatin release. Immunocytochemical characterization of the cultures demonstrated the presence of the following peptidergic neurons: neurotensin (30%), somatostatin (27%), vasoactive intestinal polypeptide (14%), neuropeptide Y (10%), and substance P (5%). No immunoreactive neurons were observed with the available antisera to galanin, gastrin-releasing peptide, and motilin. The concentration of somatostatin-like immunoreactivity, as determined by radioimmunoassay of cell extracts, was 358 +/- 105 pmol/well. Basal release of somatostatin was 4.4 +/- 0.9% total cell content and was significantly inhibited by the addition of substance P at 1 and 100 nM. The addition of the calcium ionophore, A23187, with phorbol 12-myristate 13-acetate stimulated somatostatin release in a concentration-dependent manner. These data indicate that short-term cultures of the jejunal submucosal plexus will be an excellent model for determination of the factors influencing the release of neural somatostatin.     

Mechanisms underlying distension-evoked peristalsis in guinea pig distal colon: is there a role for enterochromaffin cells?

The mechanisms underlying distension-evoked peristalsis in the colon are incompletely understood. It is well known that, following colonic distension, 5-hydroxytryptamine (5-HT) is released from enterochromaffin (EC) cells in the intestinal mucosa. It is also known that exogenous 5-HT can stimulate peristalsis. These observations have led some investigators to propose that endogenous 5-HT release from EC cells might be involved in the initiation of colonic peristalsis, following distension. However, because no direct evidence exists to support this hypothesis, the aim of this study was to determine directly whether release of 5-HT from EC cells was required for distension-evoked colonic peristalsis. Real-time amperometric recordings of 5-HT release and video imaging of colonic wall movements were performed on isolated segments of guinea pig distal colon, during distension-evoked peristalsis. Amperometric recordings revealed basal and transient release of 5-HT from EC cells before and during the initiation of peristalsis, respectively. However, removal of mucosa (and submucosal plexus) abolished 5-HT release but did not inhibit the initiation of peristalsis nor prevent the propagation of fecal pellets or intraluminal fluid. Maintained colonic distension by fecal pellets induced repetitive peristaltic waves, whose intrinsic frequency was also unaffected by removal of the submucosal plexus and mucosa, although their propagation velocities were slower. In conclusion, the mechanoreceptors and sensory neurons activated by radial distension to initiate peristalsis lie in the myenteric plexus and/or muscularis externa, and their activation does not require the submucosal plexus, release of 5-HT from EC cells, nor the presence of the mucosa. The propagation of peristalsis and propulsion of liquid or solid content along the colon is entrained by activity within the myenteric plexus and/or muscularis externa and does not require sensory feedback from the mucosa, nor neural inputs arising from submucosal ganglia.     

Cholinergic neurons are involved in the effect of substance P on the circular muscle of the guinea pig small intestine.

The mode of action of the excitatory neuropeptide substance P was studied on the circular muscle of the guinea pig ileum in vitro. Atropine or tetrodotoxin strongly inhibited substance P-induced phasic contractions. The atropine-resistant part of the circular response was blocked by tetrodotoxin. A newly-developed method for quantitative evaluation revealed a rightward displacement of the substance P concentration-response curve, as well as a strong depression of the maximum effect, in the presence of atropine. These results indicate that cholinergic (and probably also non-cholinergic) excitatory neurons mediate phasic contractions due to substance P. The tonic component of the substance P-induced contraction was slightly reduced by atropine.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Mucosal stimulation activates secretomotor neurons via long myenteric pathways in guinea pig ileum

This study examined whether mucosal stimulation activates long secretomotor neural reflexes and, if so, how they are organized. The submucosa of in vitro full thickness guinea pig ileal preparations was exposed in the distal portion and intracellular recordings were obtained from electrophysiologically identified secretomotor neurons. Axons in the intact mucosa of the oral segment were stimulated by a large bipolar stimulating electrode. In control preparations, a single stimulus pulse evoked a fast excitatory postsynaptic potential (EPSP) in 86% of neurons located 0.7-1.0 cm anal to the stimulus site. A stimulus train evoked multiple fast EPSPs, but slow EPSPs were not observed. To examine whether mucosal stimulation specifically activated mucosal sensory nerve terminals, the mucosa/submucosa was severed from the underlying layers and repositioned. In these preparations, fast EPSPs could not be elicited in 89% of cells. Superfusion with phorbol dibutyrate enhanced excitability of sensory neurons and pressure-pulse application of serotonin to the mucosa increased the fast EPSPs evoked by mucosal stimulation, providing further evidence that sensory neurons were involved. To determine whether these reflexes projected through the myenteric plexus, this plexus was surgically lesioned between the stimulus site and the impaled neuron. No fast EPSPs were recorded in these preparations following mucosal stimulation whereas lesioning the submucosal plexus had no effect. These results demonstrate that mucosal stimulation triggers a long myenteric pathway that activates submucosal secretomotor neurons. This pathway projects in parallel with motor and vasodilator reflexes, and this common pathway may enable coordination of intestinal secretion, blood flow, and motility.     

Platelet-activating factor in the enteric nervous system of the guinea pig small intestine

Platelet-activating factor (PAF) is a proinflammatory mediator that may influence neuronal activity in the enteric nervous system (ENS). Electrophysiology, immunofluorescence, Western blot analysis, and RT-PCR were used to study the action of PAF and the expression of PAF receptor (PAFR) in the ENS. PAFR immunoreactivity (IR) was expressed by 6.9% of the neurons in the myenteric plexus and 14.5% of the neurons in the submucosal plexus in all segments of the guinea pig intestinal tract as determined by double staining with anti-human neuronal protein antibody. PAFR IR was found in 6.1% of the neurons with IR for calbindin, 35.8% of the neurons with IR for neuropeptide Y (NPY), 30.6% of the neurons with IR for choline acetyltransferase (ChAT), and 1.96% of the neurons with IR for vasoactive intestinal peptide (VIP) in the submucosal plexus. PAFR IR was also found in 1.5% of the neurons with IR for calbindin, 51.1% of the neurons with IR for NPY, and 32.9% of the neurons with IR for ChAT in the myenteric plexus. In the submucosal plexus, exposure to PAF (200-600 nM) evoked depolarizing responses (8.2 +/- 3.8 mV) in 12.4% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.5% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology, whereas in the myenteric preparations, depolarizing responses were elicited by a similar concentration of PAF in 9.5% of the neurons with S-type electrophysiological behavior and uniaxonal morphology and in 12.0% of the neurons with AH-type electrophysiological behavior and Dogiel II morphology. The results suggest that subgroups of secreto- and musculomotor neurons in the submucosal and myenteric plexuses express PAFR. Coexpression of PAFR IR with ChAT IR in the myenteric plexus and ChAT IR and VIP IR in the submucosal plexus suggests that PAF, after release in the inflamed bowel, might act to elevate the excitability of submucosal secretomotor and myenteric musculomotor neurons. Enhanced excitability of motor neurons might lead to a state of neurogenic secretory diarrhea.     

Regulation of epithelial transport in the jejunal mucosa of the guinea pig by neurokinins

This study characterizes the actions of the neurokinins and calcitonin-gene related peptide (CGRP) on electrolyte transport across the mucosa of the guinea pig jejunum in vitro in a modified Ussing chamber. By following changes in short circuit current (Isc) induced by substance P (SP) and neurokinins A & B (NKA & NKB) in the presence and absence of tetrodotoxin (TTX) and atropine, it was established that two distinct neurokinin receptors are involved in the regulation of electrolyte transport. NKA preferentially activates a neuronal receptor since the actions of this neurokinin were inhibited by both TTX and atropine. SP, whose actions were reduced to a lesser extent by TTX and atropine, is considered to activate preferentially a receptor on the epithelial cells. The third neurokinin, NKB, appears to act non-selectively on both the neuronal and epithelial receptors. CGRP, which per se did not affect Isc, markedly potentiated the increases in Isc induced by SP and NKB, and thus acts synergistically with the epithelial neurokinin receptor. These results suggest that two distinct neurokinin receptors (the NK-1 and the NK-2) regulate epithelial transport in the jejunal mucosa of the guinea pig, and furthermore indicate that at least one of the peptides found in enteric nerves (i.e. CGRP) modulates the actions of neurokinins on epithelial cells.