Alan E. Lomax

The participation of the sympathetic innervation of the gastrointestinal tract in disease states.

Abstract  Knowledge of neural circuits, neurotransmitters and receptors involved in the sympathetic regulation of gastrointestinal (GI) function is well established. However, it is only recently that the interaction of sympathetic neurons, and of sympathetic transmitters, with the GI immune system and with gut flora has begun to be explored. Changes in the behaviour of sympathetic nerves when gut function is compromised, for example in ileus and in inflammation, have been observed, but the roles of the sympathetic innervation in these and other pathologies are not adequately understood. In this article, we first review the principal roles of the sympathetic innervation of the GI tract in controlling motility, fluid exchange and gut blood flow in healthy individuals. We then discuss the evidence that there are important interactions of sympathetic transmitters with the gut immune system and enteric glia, and evidence that inflammation has substantial effects on sympathetic neurons. These reciprocal interactions contribute to pathological changes in ways that are not yet clarified. Finally, we focus on inflammation, diabetes and postoperative ileus as conditions in which there is sympathetic involvement in compromised gut function.

Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits

Inflammation of the gastrointestinal (GI) tract has pronounced effects on GI function. Many of the functions of the GI tract are subject to neural regulation by the enteric nervous system (ENS) and its extrinsic connections. Therefore, it is possible that inflammatory effects on the ENS contribute to altered function during GI inflammation. The reflex circuitry of the ENS is comprised of sensory transducers in the mucosa (enteroendocrine cells), afferent neurons, interneurons and motor neurons. This review focuses on recent data that describe inflammation-induced changes to the ENS and mucosal enteroendocrine cells. Studies of tissues from patients with inflammatory bowel disease (IBD) and from animal models of IBD have demonstrated marked changes in mucosal enteroendocrine cell signaling. These changes, which have been studied most intensely in 5-HT-containing enterochromaffin cells, involve changes in the number of cells, their signaling molecule content or their means of signal termination. Morphological evidence of enteric neuropathy during inflammation has been obtained from human samples and animal models of IBD. The neuropathy can reduce the number of enteric neurons in the inflamed region and is often accompanied by a change in the neurochemical coding of enteric neurons, both in the inflamed region and at distant sites. Electrophysiological recordings have been made from enteric neurons in inflamed regions of the colon of animal models of IBD. These studies have consistently found that inflammation increases excitability of intrinsic primary afferent neurons and alters synaptic transmission to interneurons and motor neurons. These data set the stage for a comprehensive examination of the role of altered neuronal and enteroendocrine cell signaling in symptom generation during GI inflammation.

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Abstract Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.

Ileitis alters neuronal and enteroendocrine signalling in guinea pig distal colon

Background and aims: Intestinal inflammation alters neuronal and enteroendocrine signalling, leading to functional adaptations in the inflamed bowel. Human studies have reported functional alterations at sites distant from active inflammation. Our aims were to determine whether neuronal and enteroendocrine signalling are altered in the uninflamed colon during ileitis. Methods: We used neurophysiological, immunohistochemical, biochemical and Ussing chamber techniques to examine the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced ileitis on the properties of submucosal neurones, enteroendocrine cells and epithelial physiology of the distal colon of guinea pigs. Results: Three days after TNBS administration, when inflammation was restricted to the ileum, the properties of colonic enteric neurones were altered. Submucosal AH neurones were hyperexcitable and had reduced afterhyperpolarisations. S neurones received larger fast and slow excitatory postsynaptic potentials, due to an increase in non-cholinergic synaptic transmission. Despite the absence of inflammation in the colon, we found increased colonic prostaglandin E2 content in animals with ileitis. Ileitis also increased the number of colonic 5-hydroxytryptamine (5-HT)- and GLP-2-immunoreactive enteroendocrine cells. This was accompanied by an increase in stimulated 5-HT release. Functional alterations in epithelial physiology occurred such that basal short circuit current was increased and veratridine-stimulated ion transport was reduced in the colon of animals with ileitis. Conclusion: Our data suggest that inflammation at one site in the gut alters the cellular components of enteric reflex circuits in non-inflamed regions in ways similar to those at sites of active inflammation. These changes underlie altered function in non-involved regions during episodes of intestinal inflammation.

Analysis of real-time serotonin (5-HT) availability during experimental colitis in mouse

Serotonin (5-HT)-containing enterochromaffin (EC) cells of the intestine transduce chemical and mechanical stimuli from the intestinal lumen by releasing 5-HT on to afferent nerve terminals. Dysfunctional mucosal 5-HT signaling has been implicated in heightened visceral sensitivity and altered motility in patients with inflammatory bowel disease and in animal models. Our aim was to characterize the release and uptake of 5-HT in the mouse dextran sulfate sodium (DSS; 5% wt/vol) model of colitis. We made electrochemical recordings and used an ELISA assay to determine mucosal 5-HT release and uptake in untreated mice and mice with DSS-induced colitis. Peak and steady-state 5-HT concentrations were measured before and during blockade of the serotonin reuptake transporter (SERT) with 1 microM fluoxetine. Electrochemical recordings showed that colons from DSS-treated mice had roughly twice the steady-state levels of extracellular 5-HT and compression-evoked 5-HT release compared with untreated mice. Fluoxetine doubled the compression-evoked and steady-state 5-HT levels in control and DSS mice. These data were supported by ELISA assays, which showed enhanced 5-HT release during colitis, by immunohistochemical analyses, which showed increases in EC cell numbers, and by real-time PCR, which identified a decrease in SERT mRNA expression in the mucosa during colitis. These data are the first to demonstrate 5-HT release close to its release site and near its site of action during DSS-colitis. We conclude that DSS-colitis increases 5-HT availability primarily by an increase in the numbers of EC cells and/or of content of 5-HT in these EC cells.

Sympathetic vasoconstrictor regulation of mouse colonic submucosal arterioles is altered in experimental colitis

Recent studies suggest that altered neural regulation of the gastrointestinal microvasculature contributes to the pathogenesis of inflammatory bowel disease. Therefore, we employed video microscopy techniques to monitor nerve‐evoked vasoconstrictor responses in mouse colonic submucosal arterioles in vitro and examined the effect of 2,4,6‐trinitrobenzene sulphonic acid (TNBS) colitis. Nerve stimulation (2–20 Hz) caused frequency‐dependent vasoconstrictor responses that were abolished by tetrodotoxin (300 nm) and guanethidine (10 μm). The P2 receptor antagonist suramin (100 μm) or the α1‐adrenoceptor antagonist prazosin (100 nm) reduced the vasoconstriction and the combination of suramin and prazosin completely abolished responses. Nerve‐evoked constrictions of submucosal arterioles from mice with TNBS colitis were inhibited by prazosin but not suramin. Superfusion of ATP (10 μm) resulted in large vasoconstrictions in control mice but had no effect in mice with colitis whereas constrictions to phenylephrine (3 μm) were unaffected. P2X1 receptor immunohistochemistry did not suggest any alteration in receptor expression following colitis. However, Western blotting revealed that submucosal P2X1 receptor expression was increased during colitis. In contrast to ATP, αβ‐methylene‐ATP (1 μm), which is resistant to catabolism by nucleotidases, constricted control and TNBS arterioles. This indicates that reduced purinergic transmission to submucosal arterioles may be due to increased degradation of ATP during colitis. These data comprise the first description of the neural regulation of mouse submucosal arterioles and identify a defect in sympathetic regulation of the GI vasculature during colitis due to reduced purinergic neurotransmission.

Inhibition of sympathetic N-type voltage-gated Ca2+ current underlies the reduction in norepinephrine release during colitis

Inflammatory bowel diseases (IBD) are associated with altered neuronal regulation of the gastrointestinal (GI) tract and impairment of norepinephrine release from sympathetic varicosities. The sympathetic innervation of the GI tract modulates motility, blood flow, and secretion, and therefore defective norepinephrine release may contribute to symptom generation in IBD. Accordingly, our aim here was to utilize the mouse model of dextran sulfate sodium (DSS; 5% wt/vol) of IBD to determine how norepinephrine release is reduced during GI inflammation. We hypothesized that the inflammation-induced reduction in norepinephrine release was due to inhibition of voltage-gated Ca(2+) current (I(Ca)) in prevertebral sympathetic neurons. We compared [(3)H]norepinephrine release in the colon and jejunum and I(Ca) amplitude in superior mesenteric ganglion (SMG) neurons from control mice and mice with DSS-induced colitis. Changes to voltage-gated Ca(2+) channels were investigated by fura 2-AM Ca(2+) imaging, perforated patch-clamp electrophysiology, and real-time PCR. Depolarization-induced norepinephrine release from the inflamed colon and uninflamed jejunum was significantly impaired in mice treated with DSS, as was depolarization-induced Ca(2+) influx in SMG neurons. Colitis reduced I(Ca) in SMG neurons by inhibiting omega-conotoxin GVIA (300 nM)-sensitive N-type Ca(2+) channels. The omega-conotoxin GVIA-sensitive component of norepinephrine release was significantly smaller in the colon during colitis. The inhibition of I(Ca) was accompanied by a decrease in mRNA encoding the Ca(2+) channel alpha subunit (CaV 2.2) and a rightward shift in the voltage dependence of activation of I(Ca). These findings suggest that DSS-induced colitis attenuates norepinephrine release in the colon and jejunum due to inhibition of N-type voltage-gated Ca(2+) channels. This may contribute to functional alterations in both inflamed and uninflamed regions of the GI tract during inflammation.

LOSS OF PURINERGIC VASCULAR REGULATION IN THE COLON DURING COLITIS IS ASSOCIATED WITH UPREGULATION OF CD39

Evidence from patients with inflammatory bowel disease (IBD) and animal models suggests that inflammation alters blood flow to the mucosa, which precipitates mucosal barrier dysfunction. Impaired purinergic sympathetic regulation of submucosal arterioles, the resistance vessels of the splanchnic vasculature, is one of the defects identified during IBD and in mouse models of IBD. We hypothesized that this may be a consequence of upregulated catabolism of ATP during colitis. In vivo and in vitro video microscopy techniques were employed to measure the effects of purinergic agonists and inhibitors of CD39, an enzyme responsible for extracellular ATP catabolism, on the diameter of colonic submucosal arterioles from control mice and mice with dextran sodium sulfate [DSS, 5% (wt/vol)] colitis. Using a luciferase-based ATP assay, we examined the degradation of ATP and utilized real-time PCR, Western blotting, and immunohistochemistry to examine the expression and localization of CD39 during colitis. Arterioles from mice with DSS colitis did not constrict in response to ATP (10 microM) but did constrict in the presence of its nonhydrolyzable analog alpha,beta-methylene ATP (1 microM). alpha,beta-Methylene ADP (100 microM), an inhibitor of CD39, restored ATP-induced vasoconstriction in arterioles from mice with DSS-induced colitis. CD39 protein and mRNA expression was markedly increased during colitis. Immunohistochemical analysis demonstrated that, in addition to vascular CD39, F4/80-immunoreactive macrophages accounted for a large proportion of submucosal CD39 staining during colitis. These data implicate upregulation of CD39 in impaired sympathetic regulation of gastrointestinal blood flow during colitis.

Tumour necrosis factor α activates nuclear factor κB signalling to reduce N‐type voltage‐gated Ca2+ current in postganglionic sympathetic neurons

Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca2+ influx through voltage‐gated Ca2+ channels (VGCCs) is a critical determinant of excitation–secretion coupling in nerve terminals, the aim of this study was to characterize the effect of overnight incubation in the inflammatory mediator tumour necrosis factor α (TNFα; 1 nm) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage‐gated Ca2+ currents (ICa) were measured using the perforated patch clamp technique and the VGCC subtypes present in SMG neurons were estimated based on inhibition by selective VGCC blockers: ω‐conotoxin GVIA (300 nm; N‐type), nifedipine (10 μm; L‐type), and ω‐conotoxin MVIIC (300 nm; N‐, P/Q‐type). We used intracellular Ca2+ imaging with Fura‐2 AM to compare Ca2+ influx during depolarizations in control and TNFα‐treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel α subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFα significantly decreased ICa amplitude and depolarization‐induced Ca2+ influx. The reduction in ICa was limited to ω‐conotoxin GVIA‐sensitive N‐type Ca2+ channels. Depletion of glial cells by incubation in cytosine arabinoside (5 μm) did not affect ICa inhibition by TNFα. Preincubation of neurons with SC‐514 (20 μm) or BAY 11‐7082 (1 μm), which both inhibit nuclear factor κB signalling, prevented the reduction in ICa by TNFα. Inhibition of N‐type VGCCs following TNFα incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFα inhibits ICa in SMG neurons and identify a novel role for NF‐κB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFα, such as Crohns disease and Guillain‐Barré syndrome.

Clinical and experimental evidence of sympathetic neural dysfunction during inflammatory bowel disease

1. Inflammatory bowel diseases (IBD) alter the function of the enteric nervous system and the sensory innervation of the gastrointestinal (GI) tract. Less is known about whether IBD also affects the sympathetic nervous system (SNS). Given the importance of the SNS in regulating GI function and possibly immune system activation, the present review examines the evidence of sympathetic dysfunction during IBD and its possible consequences.