Axon reflexes evoked by transient receptor potential vanilloid 1 activation are mediated by tetrodotoxin-resistant voltage-gated Na+ channels in intestinal afferent nerves.

Capsaicin-sensitive nerves mediate axon vasodilator reflexes in the intestine, but the ion channels underlying action potential (AP) propagation are poorly understood. To examine the role of voltage-gated Na(+) channels underlying these reflexes, we measured vasomotor and electrophysiological responses elicited by capsaicin in guinea pig and mouse dorsal root ganglia (DRG) neurons, submucosal arterioles, and mesenteric arteries in vitro. Transient receptor potential vanilloid 1 (TRPV1) agonists dilated guinea pig ileal submucosal arterioles and were blocked by capsazepine and ruthenium red. In double-chamber baths, capsaicin-evoked activation of TRPV1 on proximal perivascular nerves in the left chamber evoked dilations of the distal segment of the submucosal arteriole in the right chamber. Dilations were tetrodotoxin (TTX) (1 microM)-resistant, but reducing extracellular Na(+) (10% solution) or applying the Na(v) 1.8 antagonist A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide] (1 microM) in the proximal chamber blocked capsaicin-evoked dilations in the distal chamber (88%; P = 0.01 and 75% and P < 0.02, respectively). In mouse mesenteric arteries, electrical field stimulation and capsaicin (2 microM) evoked dilations that were also TTX-resistant. In perforated patch-clamp recordings, APs in mouse and guinea pig capsaicin-sensitive DRG neurons were TTX-resistant but blocked by 10% extracellular Na(+). When capsaicin-evoked AP conduction was studied in in vitro ileal multiunit afferent nerve preparations, capsaicin responses were elicited in the presence of TTX, whereas distention-evoked responses were almost completely blocked by TTX. Together, these data provide evidence for TTX-resistant AP conduction in extrinsic sensory neurons that innervate guinea pig and mouse intestine and suggest this neural propagation is sufficient to mediate axon reflexes in the intestine.

Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons.

The three most common Cys-loop receptors expressed by myenteric neurons are nACh, 5-HT3 and GABAA . To investigate the function of these proteins researchers have used channel inhibitors such as hexamethonium (antagonist of nACh receptors), ondansetron (antagonist of 5-HT3 receptors), picrotoxin and bicuculline (both antagonists of GABAA receptors). The aim of this study was to investigate the specificity of these inhibitors on Cys-loop receptors of primary cultured neurons obtained from the guinea-pig small intestine. The whole-cell configuration of the patch clamp techniques was used to record membrane currents induced by ACh (IACh ), 5-HT (I5-HT ) and GABA (IGABA ) in the absence and the presence of various concentrations of hexamethonium, ondansetron, picrotoxin or bicuculline. The three Cys-loop receptors present in enteric neurons are expressed independently and they do not cross-desensitized. Hexamethonium inhibited IACh without affecting I5-HT and IGABA . Ondansetron inhibited I5-HT and also IACh but did not affect IGABA . Picrotoxin and bicuculline inhibited I5-HT , IACh and IGABA with different potency, being the lowest potency on 5-HT3 receptors. All these inhibitory effects were concentration dependent and reversible. Our observations showed that except for hexamethonium, all other inhibitors used here show different degrees of selectivity, which has to be considered when these antagonists are used in experimental studies aimed to investigate the functions of these receptors. In particular, in tissues expressing nACh receptors because these are the targets of all other inhibitors used here. The low potency of picrotoxin and bicuculline to inhibit 5-HT3 receptors suggests that these receptors are heteromeric proteins.

Release of endogenous opioids during a chronic IBD model suppresses the excitability of colonic DRG neurons.

BACKGROUND: Endogenous opioids are implicated in pain-regulation in chronic inflammatory bowel disease (IBD). We sought to examine whether endogenous opioids suppress the excitability of colonic nociceptive dorsal root ganglia (DRG) neurons during chronic IBD, and if so, whether modulation of underlying voltage-gated K(+) currents was involved. METHODS: The effects of chronic dextran sulfate sodium (DSS) colitis on afferent signaling in mice was studied using patch clamp recordings. Colonic DRG neurons were identified using Fast Blue retrograde labeling and recordings obtained from small DRG neurons (<40 pF). KEY RESULTS: In current-clamp recordings, the rheobase of neurons was increased 47% (P < 0.01) and action potential discharge at twice rheobase decreased 23% (P < 0.05) following incubation in colonic supernatants from chronic DSS mice. ß-endorphin increased 14-fold, and tissue opioid immunoreactivity and expression in CD4+ cells observed by flow cytometry increased in chronic DSS colons. Incubation of naïve neurons in the µ-opioid receptor agonist D-Ala(2), N- MePhe(4), Gly-ol (DAMGO) (10 nM) partially recapitulated the effects of supernatants from DSS mice on rheobase. Supernatant effects were blocked by the µ-opioid receptor antagonist naloxone. In voltage clamp, chronic DSS supernatants and DAMGO increased I(A) K(+) currents. CONCLUSIONS & INFERENCES: The release of endogenous opioids during chronic inflammation in mice suppresses the excitability of nociceptive DRG neurons. Targeting immune cells may provide a novel means of modulating IBD pain.