Institute of Neuroscience, Laboratory for Translational Pain Medicine, Department of Neurobiology, Soochow University, Suzhou 215123, PR China.
Exp Neurol. 2013 Oct;248:275-85. doi: 10.1016/j.expneurol.2013.06.027. Epub 2013 Jul 6.
The 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.
Neonatal male rats (postnatal days 2-15) were exposed to a 3 hour 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 Na(V)1.8 was analyzed by Western blot and Immunofluorescence study. The endogenous H₂S producing enzyme CBS antagonist was injected intraperitoneally.
We showed that CBS was colocalized with Na(V)1.8 in colon-specific DRG neurons pre-labeled with DiI. Pretreatment of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, significantly reduced expression of Na(V)1.8 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.
These 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.
肠易激综合征（IBS）疼痛的发病机制尚不清楚，治疗仍然困难。我们之前的研究报告称，在由新生期母体分离（NMD）诱导的内脏高敏性大鼠模型中，结肠特异性背根神经节（DRG）神经元中的 TTX 抗性（TTX-R）钠离子通道被敏化，内源性硫化氢产生酶胱硫醚-β-合酶（CBS）的表达上调。然而，钠离子通道激活的详细分子机制尚不清楚。本研究旨在检查 CBS-H₂S 信号在先前验证的 IBS 大鼠模型中钠离子通道敏化中的作用。
雄性新生大鼠（生后第 2-15 天）接受每日 3 小时的母体分离，温度保持在~33°C。用 DiI 标记结肠特异性 DRG 神经元，并在全细胞膜片钳配置下急性分离以测量兴奋性和钠离子通道电流。通过 Western blot 和免疫荧光研究分析 Na(V)1.8 的表达。CBS 的内源性 H₂S 产生酶拮抗剂被腹腔内注射。
我们表明，CBS 与在 DiI 预标记的结肠特异性 DRG 神经元中 Na(V)1.8 共定位。CBS 抑制剂 O-（羧甲基）羟胺盐酸盐（AOAA）的预处理显著降低了 NMD 大鼠中 Na(V)1.8 的表达。AOAA 处理还抑制了 TTX-R 钠电流密度，右移激活曲线的 V₁/₂，并逆转了 NMD 大鼠结肠特异性 DRG 神经元的过度兴奋。相反，H₂S 的供体 NaHS 的添加大大增强了 TTX-R 钠电流密度，左移了激活曲线，并增强了年龄匹配的健康大鼠结肠 DRG 神经元的兴奋性。此外，蛋白激酶 A 抑制剂 H-89 的应用显著减弱了 NaHS 对 TTX-R 钠电流密度的增强作用。
这些数据表明，NMD 大鼠结肠 DRG 神经元钠离子通道的敏化很可能是由 CBS-H₂S 信号介导的，从而为 IBS 患者慢性内脏疼痛的治疗确定了一个潜在靶点。
