Molecular expression and pharmacological evidence for a functional role of kv7 channel subtypes in Guinea pig urinary bladder smooth muscle.
Molecular expression and pharmacological evidence for a functional role of kv7 channel subtypes in Guinea pig urinary bladder smooth muscle.
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DOI：
10.1371/journal.pone.0075875
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Petkov GV
中科院分区：
文献类型：
--
作者：
Afeli SA;Malysz J;Petkov GV
电压门控Kv7（KCNQ）通道正逐渐成为平滑肌兴奋性和收缩性的重要调节因子。然而，它们在逼尿肌平滑肌（DSM）中的生理作用仍有待阐明。在此，我们通过逆转录聚合酶链反应（RT - PCR）、实时定量逆转录聚合酶链反应（qRT - PCR）、免疫组织化学、电生理学以及等长张力记录等方法，探究了豚鼠DSM中Kv7通道亚型的分子表达及功能。在整个DSM组织中，所有Kv7通道亚型的mRNA按以下顺序被检测到：Kv7.1≈Kv7.2＞Kv7.3≈Kv7.5＞Kv7.4。相比之下，新鲜分离的DSM细胞显示出以下mRNA表达情况：Kv7.1≈Kv7.2＞Kv7.5＞Kv7.3≈Kv7.4。通过使用Kv7通道亚型特异性抗体和α - 平滑肌肌动蛋白共同标记对DSM进行的免疫组织化学共聚焦显微镜分析，在DSM细胞中检测到了除Kv7.4之外所有Kv7通道亚型的蛋白质表达。Kv7.1通道激活剂L - 364373（R - L3）以及Kv7.2 - 7.5通道激活剂瑞替加滨，抑制了DSM离体肌条的自发性阶段性收缩以及10Hz电场刺激（EFS）诱导的收缩。两种泛Kv7（对Kv7.1 - Kv7.5亚型均有效）通道抑制剂利诺吡啶和XE991具有相反的作用，它们增加了DSM的自发性阶段性收缩以及10Hz EFS诱导的收缩。由多种刺激频率产生的EFS诱导的DSM收缩，被L - 364373（10μM）或瑞替加滨（10μM）减弱，被XE991（10μM）增强。瑞替加滨（10μM）在新鲜分离的DSM细胞中诱导超极化并抑制自发性动作电位。总之，Kv7通道亚型在豚鼠DSM细胞的mRNA和蛋白质水平均有表达。它们的药理学调节能够控制DSM的收缩性和兴奋性；因此，Kv7通道亚型为膀胱功能障碍提供了潜在的新型治疗靶点。
Voltage-gated Kv7 (KCNQ) channels are emerging as essential regulators of smooth muscle excitability and contractility. However, their physiological role in detrusor smooth muscle (DSM) remains to be elucidated. Here, we explored the molecular expression and function of Kv7 channel subtypes in guinea pig DSM by RT-PCR, qRT-PCR, immunohistochemistry, electrophysiology, and isometric tension recordings. In whole DSM tissue, mRNAs for all Kv7 channel subtypes were detected in a rank order: Kv7.1~Kv7.2Kv7.3~Kv7.5Kv7.4. In contrast, freshly-isolated DSM cells showed mRNA expression of: Kv7.1~Kv7.2Kv7.5Kv7.3~Kv7.4. Immunohistochemical confocal microscopy analyses of DSM, conducted by using co-labeling of Kv7 channel subtype-specific antibodies and α-smooth muscle actin, detected protein expression for all Kv7 channel subtypes, except for the Kv7.4, in DSM cells. L-364373 (R-L3), a Kv7.1 channel activator, and retigabine, a Kv7.2-7.5 channel activator, inhibited spontaneous phasic contractions and the 10-Hz electrical field stimulation (EFS)-induced contractions of DSM isolated strips. Linopiridine and XE991, two pan-Kv7 (effective at Kv7.1-Kv7.5 subtypes) channel inhibitors, had opposite effects increasing DSM spontaneous phasic and 10 Hz EFS-induced contractions. EFS-induced DSM contractions generated by a wide range of stimulation frequencies were decreased by L-364373 (10 µM) or retigabine (10 µM), and increased by XE991 (10 µM). Retigabine (10 µM) induced hyperpolarization and inhibited spontaneous action potentials in freshly-isolated DSM cells. In summary, Kv7 channel subtypes are expressed at mRNA and protein levels in guinea pig DSM cells. Their pharmacological modulation can control DSM contractility and excitability; therefore, Kv7 channel subtypes provide potential novel therapeutic targets for urinary bladder dysfunction.
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