Sensitization of sodium channels by cystathionine β-synthetase activation in colon sensory neurons in adult rats with neonatal maternal deprivation

Abstract

BackgroundThe pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood and treatment remains difficult. We have previously reported that TTX-resistant (TTX-R) sodium channels in colon-specific dorsal root ganglion (DRG) neurons were sensitized and the expression of the endogenous hydrogen sulfide producing enzyme cystathionine β-synthetase (CBS) was upregulated in a rat model of visceral hypersensitivity induced by neonatal maternal deprivation (NMD). However, the detailed molecular mechanism for activation of sodium channels remains unknown. This study was designed to examine roles for CBS–H2S signaling in sensitization of sodium channels in a previously validated rat model of IBS. MethodsNeonatal male rats (postnatal days 2–15) were exposed to a 3hour period of daily maternal separation with temperature maintained at ~33°C. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch clamp configurations. The expression of NaV1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H2S producing enzyme CBS antagonist was injected intraperitoneally. ResultsWe showed that CBS was colocalized with NaV1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of NaV1.8 in NMD rats. AOAA treatment also inhibited the TTX-R sodium current density, right-shifted the V1/2 of activation curve, and reversed hyperexcitability of colon-specific DRG neurons in NMD rats. Conversely, addition of NaHS, a donor of H2S, greatly enhanced TTX-R sodium current density, left shifted the activation curve and enhanced excitability of colon DRG neurons in age-matched healthy rats. Furthermore, application of H-89, an inhibitor of protein kinase A, markedly attenuated the potentiation of TTX-R sodium current density by NaHS. ConclusionThese data suggest that sensitization of sodium channels of colon DRG neurons in NMD rats is most likely mediated by CBS–H2S signaling, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.