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Colitis, induced by trinitrobenzene sulfonic acid (TNBS) in guinea pig, leads to decreased purinergic neuromuscular transmission resulting in a reduction in inhibitory junction potentials (IJPs) in colonic circular muscle. We explored possible mechanisms responsible for this inflammation-induced neurotransmitter plasticity. Previous studies have suggested that the deficit in inflamed tissue involves decreased ATP release. We therefore hypothesized that decreased purinergic transmission results from inflammation-induced free radical damage to mitochondria, leading to decreased purine synthesis and release. Stimulus-induced release of purines was measured using high-performance liquid chromatography, and quantities of all purines measured were significantly reduced in the inflamed colons as compared to controls. To test whether decreased mitochondrial function affects the IJP, colonic muscularis preparations were treated with the mitochondrial ATP synthase inhibitors oligomycin or dicyclohexylcarbodiimide, which resulted in a significant reduction of IJP amplitude. Induction of oxidative stress in vitro, by addition of H2O2 to the preparation, also significantly reduced IJP amplitude. Purinergic neuromuscular transmission was significantly restored in TNBS-inflamed guinea pigs, and in dextran sodium sulfate-inflamed mice, treated with a free radical scavenger. Furthermore, propulsive motility in the distal colons of guinea pigs with TNBS colitis was improved by in vivo treatment with the free radical scavenger. We conclude that oxidative stress contributes to the reduction in purinergic neuromuscular transmission measured in animal models of colitis, and that these changes can be prevented by treatment with a free radical scavenger, resulting in improved motility.

Enteric glia are increasingly recognized as important in the regulation of a variety of gastrointestinal functions.Here we tested the hypothesis that nicotinic signalling in the myenteric plexus results in the release of nitric oxide (NO) from neurons and enteric glia to modulate epithelial ion transport. Ion transport was assessed using full-thickness or muscle-stripped segments of mouse colon mounted in Ussing chambers. The cell-permeant NO-sensitive dye DAR-4M AM and amperometry were utilized to identify the cellular sites of NO production within the myenteric plexus and the contributions from specific NOS isoforms. Nicotinic receptors were localized using immunohistochemistry. Nicotinic cholinergic stimulation of colonic segments resulted in NO-dependent changes in epithelial active electrogenic ion transport that were TTX sensitive and significantly altered in the absence of the myenteric plexus. Nicotinic stimulation of the myenteric plexus resulted in NO production and release from neurons and enteric glia, which was completely blocked in the presence of nitric oxide synthase (NOS) I and NOS II inhibitors. Using the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), neuronal and enteric glial components of NO production were demonstrated. Nicotinic receptors were identified on enteric neurons, which express NOS I, and enteric glia, which express NOS II. These data identify a unique pathway in the mouse colon whereby nicotinic cholinergic signalling in myenteric ganglia mobilizes NO from NOS II in enteric glia, which in coordinated activity with neurons in the myenteric plexus modulates epithelial ion transport, a key component of homeostasis and innate immunity.

This study was undertaken to investigate neuromuscular transmission in regions of the inflamed colon in which motility is disrupted. Propulsive motility was evaluated in segments of control guinea pigs and those treated 6 days previously with trinitrobenzene sulfonic acid. Intracellular recordings were then obtained from circular muscle cells to examine excitatory and inhibitory junction potentials (EJPs and IJPs). In inflamed preparations, propulsion of fecal pellets was temporarily halted or obstructed at sites of mucosal damage, whereas the propulsive motility was linear in control colons. The amplitudes of evoked and spontaneous IJPs were significantly reduced in ulcerated regions of inflamed preparations, but EJPs were comparable to controls. Pharmacological dissection of the IJP revealed that the purinergic component was reduced, while the nitrergic IJP was slightly increased. Furthermore, the reduction in the purinergic IJP in inflamed preparations persisted in the presence of hexamethonium, suggesting that the deficit involved the inhibitory motor neuron and/or smooth muscle. Nerve fibre density was not altered in the circular muscle, and pre-contracted rings of inflamed colon relaxed normally to ATP, suggesting that the deficit involves altered ATP release and/or degradation. The P2Y1 receptor antagonist MRS2179 slowed propulsive motility indicating that decreased purinergic neuromuscular transmission could contribute to the inflammation-induced motor deficit. We conclude that purinergic inhibitory neuronal input to the circular muscle is selectively reduced in regions of the colon in experimental colitis where the mucosa is damaged, and this is likely to contribute to altered motility in colitis by diminishing downstream relaxation during the peristaltic reflex.

The purpose of this study was to investigate the pre- and postsynaptic mechanisms that contribute to synaptic facilitation in the myenteric plexus of the trinitrobenzene sulphonic acid-inflamed guinea-pig distal colon. Intracellular recordings of evoked fast excitatory postsynaptic potentials (fEPSPs) in myenteric S neurons were evaluated, and the density of synaptic terminals was morphometrically analysed by transmission electron microscopy. In inflamed tissue, fEPSPs were reduced to control levels by the protein kinase A (PKA) inhibitor, H89, but H89 did not affect the fEPSPs in control tissue. This PKA activation in inflamed tissue did not appear to involve 5-HT4 receptors because the antagonist/inverse agonist, GR 125487, caused comparable decreases of fEPSPs in both tissues. Inhibition of BK channels with iberiotoxin did not alter the fEPSPs in inflamed tissue, but increased the fEPSPs in control tissue to the amplitude detected in inflamed tissue. During trains of stimuli, run-down of EPSPs was less extensive in inflamed tissue and there was a significant increase in the paired pulse ratio. Depolarizations in response to exogenous neurotransmitters were not altered in inflamed tissue. These inflammation-induced changes were not accompanied by alterations in the pharmacological profile of EPSPs, and no changes in synaptic density were detected by electron microscopy. Collectively, these data indicate that synaptic facilitation in the inflamed myenteric plexus involves a presynaptic increase in PKA activity, possibly involving an inhibition of BK channels, and an increase in the readily releasable pool of synaptic vesicles.

Intestinal secretion is regulated by submucosal neurones of the enteric nervous system. Inflammation of the intestines leads to aberrant secretory activity; therefore we hypothesized that the synaptic and electrical behaviours of submucosal neurones are altered during colitis. To test this hypothesis, we used intracellular microelectrode recording to compare the excitability and synaptic properties of submucosal neurones from normal and trinitrobenzene sulphonic acid (TNBS)-inflamed guinea-pig colons. Inflammation differentially affected the electrophysiological characteristics of the two functional classes of submucosal neurones. AH neurones from inflamed colons were more excitable, had shorter action potential durations and reduced afterhyperpolarizations. Stimulus-evoked fast and slow excitatory postsynaptic potentials (EPSPs) in S neurones were larger during colitis, and the incidence of spontaneous fast EPSPs was increased. In control preparations, fast EPSPs were almost completely blocked by the nicotinic receptor antagonist hexamethonium, whereas fast EPSPs in inflamed S neurones were only partially inhibited by hexamethonium. In inflamed tissues, components of the fast EPSP in S neurones were sensitive to blockade of P2X and 5-HT3 receptors while these antagonists had little effect in control preparations. Control and inflamed S neurones were equally sensitive to brief application of acetylcholine, ATP and 5-HT, suggesting that synaptic facilitation was due to a presynaptic mechanism. Immunoreactivity for 5-HT in the submucosal plexus was unchanged by inflammation; this indicates that altered synaptic transmission was not due to anatomical remodelling of submucosal nerve terminals. This is the first demonstration of alterations in synaptic pharmacology in the enteric nervous system during inflammation.

We have previously demonstrated that trinitrobenzene sulphonic acid (TNBS)-induced colitis in guinea pig is associated with hyperexcitability of myenteric AH neurones, enhanced synaptic activity in the myenteric plexus, increased serotonin (5-HT) availability in the mucosa, and decreased propulsive motor activity. The current study tested the hypothesis that the activation of cyclooxygenase (COX) contributes to these alterations in bowel functions. DFU inhibition of COX-2, but not SC-560 inhibition of COX-1, restored to normal levels the electrical properties of myenteric AH neurones, the proportion of S neurones exhibiting slow EPSPs, and the rate of propulsive motor activity. Neither inhibitor was effective in altering the level of inflammation, the increased availability of mucosal 5-HT, or the enhanced fast EPSPs in myenteric AH and S neurones. COX-2 expression is enhanced in the myenteric plexus and cells within the smooth muscle layers during colitis, possibly reflecting the site at which COX-2 inhibition acts to allow recovery of motor function. In support of this concept, COX-1, but not COX-2, inhibition was effective in restoring normal mucosal prostaglandin levels. These results indicate that the various changes that occur in the motor neural pathways of the distal colon in TNBS-induced colitis do not involve a single neuroimmune mechanism. COX-2 activation is a critical step in the enhanced excitability of AH neurones as well as diminished propulsive motility in TNBS colitis, whereas other yet to be resolved pathways, that do not involve COX-1 or COX-2 activation, lead to altered 5-HT content in the mucosa and an augmentation of fast EPSPs.

The electrical and synaptic properties of myenteric neurones in normal and inflamed guinea-pig distal colons were evaluated by intracellular microelectrode recording. Chronic inflammation was established 6 days following administration of trinitrobenzene sulfonic acid (TNBS). In S neurones, inflammation only altered synaptic inputs as the amplitude of fast excitatory postsynaptic potentials were significantly larger (31 +/- 2 mV compared to 20 +/- 1 mV) and they were more likely to receive slow excitatory synaptic input (85% compared to 55%). AH neurones displayed altered electrical properties in colitis compared to control tissues: they generated more action potentials during a maximal depolarising current pulse (7 +/- 1 compared to 1.6 +/- 0.2); they had a smaller after hyperpolarisation (9 +/- 2 mV s compared to 20 +/- 2 mV s); and they were more likely to receive fast excitatory synaptic input (74% compared to 17%), possess spontaneous activity (46% compared to 3%), and generate anodal break action potentials (58% compared to 19%). Although the resting membrane potential, input resistance and action potential characteristics were unaltered in AH neurones from inflamed tissues, they exhibited an enhanced Cs+-sensitive rectification of the current-voltage relationship. This suggests that the increase in excitability of AH neurones may involve a colitis-induced augmentation of the hyperpolarisation-activated cation current (Ih) in these cells. An increased excitability, selectively in AH neurones, suggests that the afferent limb of intrinsic motor reflexes is disrupted in the inflamed colon and this may contribute to dysmotility associated with inflammatory diseases.

Colitis, induced by trinitrobenzene sulfonic acid (TNBS) in guinea pig, leads to decreased purinergic neuromuscular transmission resulting in a reduction in inhibitory junction potentials (IJPs) in colonic circular muscle. We explored possible mechanisms responsible for this inflammation-induced neurotransmitter plasticity. Previous studies have suggested that the deficit in inflamed tissue involves decreased ATP release. We therefore hypothesized that decreased purinergic transmission results from inflammation-induced free radical damage to mitochondria, leading to decreased purine synthesis and release. Stimulus-induced release of purines was measured using high-performance liquid chromatography, and quantities of all purines measured were significantly reduced in the inflamed colons as compared to controls. To test whether decreased mitochondrial function affects the IJP, colonic muscularis preparations were treated with the mitochondrial ATP synthase inhibitors oligomycin or dicyclohexylcarbodiimide, which resulted in a significant reduction of IJP amplitude. Induction of oxidative stress in vitro, by addition of H2O2 to the preparation, also significantly reduced IJP amplitude. Purinergic neuromuscular transmission was significantly restored in TNBS-inflamed guinea pigs, and in dextran sodium sulfate-inflamed mice, treated with a free radical scavenger. Furthermore, propulsive motility in the distal colons of guinea pigs with TNBS colitis was improved by in vivo treatment with the free radical scavenger. We conclude that oxidative stress contributes to the reduction in purinergic neuromuscular transmission measured in animal models of colitis, and that these changes can be prevented by treatment with a free radical scavenger, resulting in improved motility.

The purpose of this study was to investigate the pre- and postsynaptic mechanisms that contribute to synaptic facilitation in the myenteric plexus of the trinitrobenzene sulphonic acid-inflamed guinea-pig distal colon. Intracellular recordings of evoked fast excitatory postsynaptic potentials (fEPSPs) in myenteric S neurons were evaluated, and the density of synaptic terminals was morphometrically analysed by transmission electron microscopy. In inflamed tissue, fEPSPs were reduced to control levels by the protein kinase A (PKA) inhibitor, H89, but H89 did not affect the fEPSPs in control tissue. This PKA activation in inflamed tissue did not appear to involve 5-HT(4) receptors because the antagonist/inverse agonist, GR 125487, caused comparable decreases of fEPSPs in both tissues. Inhibition of BK channels with iberiotoxin did not alter the fEPSPs in inflamed tissue, but increased the fEPSPs in control tissue to the amplitude detected in inflamed tissue. During trains of stimuli, run-down of EPSPs was less extensive in inflamed tissue and there was a significant increase in the paired pulse ratio. Depolarizations in response to exogenous neurotransmitters were not altered in inflamed tissue. These inflammation-induced changes were not accompanied by alterations in the pharmacological profile of EPSPs, and no changes in synaptic density were detected by electron microscopy. Collectively, these data indicate that synaptic facilitation in the inflamed myenteric plexus involves a presynaptic increase in PKA activity, possibly involving an inhibition of BK channels, and an increase in the readily releasable pool of synaptic vesicles.

Intestinal secretion is regulated by submucosal neurones of the enteric nervous system. Inflammation of the intestines leads to aberrant secretory activity; therefore we hypothesized that the synaptic and electrical behaviours of submucosal neurones are altered during colitis. To test this hypothesis, we used intracellular microelectrode recording to compare the excitability and synaptic properties of submucosal neurones from normal and trinitrobenzene sulphonic acid (TNBS)-inflamed guinea-pig colons. Inflammation differentially affected the electrophysiological characteristics of the two functional classes of submucosal neurones. AH neurones from inflamed colons were more excitable, had shorter action potential durations and reduced afterhyperpolarizations. Stimulus-evoked fast and slow excitatory postsynaptic potentials (EPSPs) in S neurones were larger during colitis, and the incidence of spontaneous fast EPSPs was increased. In control preparations, fast EPSPs were almost completely blocked by the nicotinic receptor antagonist hexamethonium, whereas fast EPSPs in inflamed S neurones were only partially inhibited by hexamethonium. In inflamed tissues, components of the fast EPSP in S neurones were sensitive to blockade of P2(X) and 5-HT(3) receptors while these antagonists had little effect in control preparations. Control and inflamed S neurones were equally sensitive to brief application of acetylcholine, ATP and 5-HT, suggesting that synaptic facilitation was due to a presynaptic mechanism. Immunoreactivity for 5-HT in the submucosal plexus was unchanged by inflammation; this indicates that altered synaptic transmission was not due to anatomical remodelling of submucosal nerve terminals. This is the first demonstration of alterations in synaptic pharmacology in the enteric nervous system during inflammation.

We have previously demonstrated that trinitrobenzene sulphonic acid (TNBS)-induced colitis in guinea pig is associated with hyperexcitability of myenteric AH neurones, enhanced synaptic activity in the myenteric plexus, increased serotonin (5-HT) availability in the mucosa, and decreased propulsive motor activity. The current study tested the hypothesis that the activation of cyclooxygenase (COX) contributes to these alterations in bowel functions. DFU inhibition of COX-2, but not SC-560 inhibition of COX-1, restored to normal levels the electrical properties of myenteric AH neurones, the proportion of S neurones exhibiting slow EPSPs, and the rate of propulsive motor activity. Neither inhibitor was effective in altering the level of inflammation, the increased availability of mucosal 5-HT, or the enhanced fast EPSPs in myenteric AH and S neurones. COX-2 expression is enhanced in the myenteric plexus and cells within the smooth muscle layers during colitis, possibly reflecting the site at which COX-2 inhibition acts to allow recovery of motor function. In support of this concept, COX-1, but not COX-2, inhibition was effective in restoring normal mucosal prostaglandin levels. These results indicate that the various changes that occur in the motor neural pathways of the distal colon in TNBS-induced colitis do not involve a single neuroimmune mechanism. COX-2 activation is a critical step in the enhanced excitability of AH neurones as well as diminished propulsive motility in TNBS colitis, whereas other yet to be resolved pathways, that do not involve COX-1 or COX-2 activation, lead to altered 5-HT content in the mucosa and an augmentation of fast EPSPs.