Michael Beyak

Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons

Intestinal inflammation induces hyperexcitability of dorsal root ganglia sensory neurons, which has been implicated in increased pain sensation. This study examined whether alteration of sodium (Na+) and/ or potassium (K+) currents underlies this hyperexcitability. Ileitis was induced in guinea pig ileum with trinitrobenzene sulphonic acid (TBNS) and dorsal root ganglion neurons innervating the site of inflammation were identified by Fast Blue or DiI fluorescence labelling. Whole cell recordings were made from acutely dissociated small‐sized neurons at 7–10 days. Neurons exhibited transient A‐type and sustained outward rectifier K+ currents. Compared to control, both A‐type and sustained K+ current densities were significantly reduced (42 and 34 %, respectively; P < 0.05) in labelled neurons from the inflamed intestine but not in non‐labelled neurons. A‐type current voltage dependence of inactivation was negatively shifted in labelled inflamed intestine neurons. Neurons also exhibited tetrodotoxin‐sensitive and resistant Na+ currents. Tetrodotoxin‐resistant sodium currents were increased by 37 % in labelled neurons from the inflamed intestine compared to control (P < 0.01), whereas unlabelled neurons were unaffected. The activation and inactivation curves of these currents were unchanged by inflammation. These data suggest ileitis increases excitability of intestinal sensory neurons by modulating multiple ionic channels. The lack of effect in non‐labelled neurons suggests signalling originated at the nerve terminal rather than through circulating mediators and, given that Na+ currents are enhanced whereas K+ currents are suppressed, one or more signalling pathways may be involved.

The gaseous mediator, hydrogen sulphide, inhibits in vitro motor patterns in the human, rat and mouse colon and jejunum

Abstract  Hydrogen sulphide (H2S) has been recently proposed as a transmitter in the brain and peripheral tissues. Its role in the gastrointestinal tract is still unknown despite some data which suggest an involvement mediating smooth muscle relaxation. The aim of this study was to investigate the effect of this gas on intestinal segments from mouse jejunum and colon, and muscular strips from the human and rat colon. In isolated segments of mouse colon and jejunum, bath applied sodium hydrogen sulphide (NaHS) (a H2S donor) caused a concentration‐dependent inhibition of spontaneous motor complexes (MCs) (IC50 121 μmol L−1 in the colon and 150 μmol L−1 in the jejunum). This inhibitory effect of NaHS on MCs was (i) unaffected by tetrodotoxin (TTX), capsaicin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate and N‐nitro‐l‐arginine suggesting a non‐neural effect and (ii) significantly reduced by apamin 3 μmol L−1. NaHS concentration‐dependently inhibited the spontaneous motility in strips from human colon (IC50 261 μmol L−1) and rat colon (IC50 31 μmol L−1). The inhibitory effect of NaHS on colonic strips was (i) unaffected by the neural blocker TTX (1 μmol L−1) with IC50 183 μmol L−1 for the human colon and of 26 μmol L−1 for the rat colon and (ii) significantly reduced by glybenclamide (10 μmol L−1), apamin (3 μmol L−1) and TEA (10 mmol L−1) with IC50 values of 2464, 1307 and 2421 μmol L−1 for human strips, and 80, 167 and 674 μmol L−1 for rat strips respectively. We conclude that H2S strongly inhibits in vitro intestinal and colonic motor patterns. This effect appears to be critically dependent on K channels particularly apamin‐sensitive SK channels and glybenclamide‐sensitive K (ATP) channels.

Inflammation-induced hyperexcitability of nociceptive gastrointestinal DRG neurones: the role of voltage-gated ion channels.

Abstract  Gastrointestinal (GI) inflammation modulates the intrinsic properties of nociceptive dorsal root ganglia neurones, which innervate the GI tract and these changes are important in the genesis of abdominal pain and visceral hyperalgesia. neurones exhibit hyperexcitability characterized by a decreased threshold for activation and increased firing rate, and changes in voltage‐gated Na+ and K+ channels play a major role in this plasticity. This review highlights emerging evidence that specific subsets of channels and signalling pathways are involved and their potential to provide novel selective therapeutic targets for the treatment of abdominal pain.

Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse

Non‐technical summary  Obesity is known to result from energy intake in excess of expenditure. What is not known is how individuals are able to eat in excess of their energy needs. We show that after chronic consumption of a high fat diet (which causes obesity), intestinal sensory nerves are less responsive to chemicals released from the gut during a meal (cholecystokinin and 5‐hydroxytryptamine) as well as to distension of the gut as might occur during a meal. This appears to be due to the fact that the ability of the nerve cells to be excited is impaired. This suggests that consumption of an unhealthy diet that leads to obesity causes decreased signalling from the intestine, which may lead to increased food intake and contribute to further weight gain, or allow the maintenance of excess weight and obesity.

Afferent hypersensitivity in a mouse model of post‐inflammatory gut dysfunction: role of altered serotonin metabolism

Visceral hypersensitivity is an important clinical feature associated with irritable bowel syndrome which in some patients has been linked to prior infection. Here we employ an animal model in which transient infection leads to persistent gut dysfunction to investigate the role of altered 5‐HT metabolism upon afferent mechanosensensitivity in the post‐infected gut. Jejunal segments isolated from Trichinella spiralis‐infected mice were used to assess 5‐HT metabolism whilst afferent activity in T. spiralis‐infected mice was studied by extracellular recordings from jejunal mesenteric afferent bundles and patch clamp recordings of isolated nodose ganglion neurons (NGNs). During acute infection, intestinal 5‐HT content and release increased, 5‐HT turnover decreased and afferent discharge in response to mechanical stimulation was attenuated. By day 28 post infection (PI), 5‐HT turnover had normalized, but 5‐HT content and release were still elevated. This was associated with afferent mechano‐hypersensitivity, which persisted for 8 weeks PI and was susceptible to 5‐HT3 receptor blockade. NGNs from post‐infected animals were more excitable than controls but their current densities in response to 2‐methyl‐5‐HT were lower. T. spiralis infection increased mucosal 5‐HT bioavailability and affected the spontaneous activity and mechanosensitivity of gastrointestinal sensory nerves. This involved an initial hyposensitivity occurring during acute infection followed by long‐term hypersensitivity in the post‐infectious period that was in part mediated by 5‐HT acting via 5‐HT3 receptors. Functional down‐regulation of 5‐HT3 receptors also occurs in the post‐infected animals, which may represent an adaptive response to increased mucosal 5‐HT bioavailability.

Sensitization of peripheral sensory nerves by mediators from colonic biopsies of diarrhea-predominant irritable bowel syndrome patients: a role for PAR2.

OBJECTIVES:This study examined whether mediators from biopsies of human irritable bowel syndrome (IBS) colons alter intrinsic excitability of colonic nociceptive dorsal root ganglion (DRG) neurons by a protease activated receptor 2 (PAR2)-mediated mechanism.METHODS:Colonic mucosal biopsies from IBS patients with constipation (IBS-C) or diarrhea (IBS-D) and from healthy controls were incubated in medium, and supernatants were collected. Small-diameter mouse colonic DRG neurons were incubated in supernatants overnight and perforated patch current-clamp recordings obtained. Measurements of rheobase and action potential discharge at twice rheobase were compared between IBS and controls to assess differences in intrinsic excitability.RESULTS:Supernatants from IBS-D patients elicited a marked increase in neuronal excitability compared with controls. These changes were consistent among individual patients but the relative contribution of rheobase and action potential discharge varied. In contrast, no differences in neuronal excitability were seen with IBS-C patient supernatants. The increased excitability seen with IBS-D supernatant was not observed in PAR2 knockout mice. A cysteine protease inhibitor, which had no effect on the pronociceptive actions of a serine protease, inhibited the proexcitatory actions of IBS-D supernatant.CONCLUSIONS:Soluble mediators from colonic biopsies from IBS-D but not IBS-C patients sensitized colonic nociceptive DRG neurons, suggesting differences between these two groups. PAR2 signaling plays a role in this action and this protease signaling pathway could provide novel biomarkers and therapeutic targets for treatment.

Histamine H1 and H3 vasodilator mechanisms in the guinea pig ileum

BACKGROUND/AIMS Histamine dilates gastrointestinal blood vessels. Whether this is caused by direct activation of vascular histamine receptors or by activation of enteric neurons is not known. The aim of this study was to determine which of these pathways is activated by histamine and to examine the cellular mechanisms involved. METHODS The effects of histamine were studied in in vitro submucosal preparations from the guinea pig ileum using videomicroscopy to monitor changes in submucosal arteriolar diameter. RESULTS Histamine caused a tetrodotoxin-insensitive dose-dependent dilation (median effective concentration [EC50], 1 mumol/L), showing direct activation of vascular histamine receptors. The H1 antagonist pyrilamine, but not the H2 blocker ranitidine, competitively inhibited the histamine dilatation. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) inhibited histamine vasodilations by 66%. Indomethacin alone did not alter histamine vasodilations but, when combined with L-NMMA, caused a significantly greater inhibition of the histamine response compared with L-NMMA alone. L-Arginine prevented the actions of L-NMMA. In the presence of both H1 and H2 antagonists, periarteriolar stimulation of sympathetic nerves evoked a tetrodotoxin-sensitive vasoconstriction, which was inhibited by histamine (EC50, 0.8 mumol/L). This histamine action was blocked by the H3 antagonist thioperamide. CONCLUSIONS Histamine can produce vasodilation of submucosal arterioles by two distinct mechanisms: activation of vascular H1 receptors resulting in release of nitric oxide from endothelium and activation of H3 receptors on sympathetic nerve terminals resulting in presynaptic inhibition of vasoconstrictor tone.

Glucagon-like peptide-1 inhibits voltage-gated potassium currents in mouse nodose ganglion neurons

Background  Glucagon‐like peptide‐1 (GLP‐1) is a major hormone known to regulate glucose homeostasis and gut function, and is an important satiety mediator. These actions are at least in part mediated via an action on vagal afferent neurons. However, the mechanism by which GLP‐1 activates vagal afferents remains unknown. We hypothesized that GLP‐1 acts on nodose ganglion neuron voltage‐gated potassium (KV) channels, increasing membrane excitability.

Visceral afferents - determinants and modulation of excitability.

An essential property of visceral sensory afferents is to be able to alter their firing properties in response to changes in the microenvironment at the level of the sensory ending. Significant progress has been made in recent years in understanding the ionic mechanisms of the regulation of afferent neuronal excitability, and in identifying the mechanisms by which this can be altered. This article will review some of the recent developments in the state of knowledge regarding mechanisms of increased excitability after inflammation, and pharmacological modulation of excitability, concentrating on afferent nerves innervating the GI tract and urinary bladder.

Effects of inflammation on the innervation of the colon.

Inflammatory bowel diseases (IBD) such as ulcerative colitis and Crohn’s disease lead to altered gastrointestinal (GI) function as a consequence of the effects of inflammation on the tissues that comprise the GI tract. Among these tissues are several types of neurons that detect the state of the GI tract, transmit pain, and regulate functions such as motility, secretion, and blood flow. This review article describes the structure and function of the enteric nervous system, which is embedded within the gut wall, the sympathetic motor innervation of the colon and the extrinsic afferent innervation of the colon, and considers the evidence that colitis alters these important sensory and motor systems. These alterations may contribute to the pain and altered bowel habits that accompany IBD.