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本研究旨在观察氯离子通道阻断剂尼氟灭酸(niflumic acid,NFA)引起豚鼠耳蜗螺旋动脉平滑肌细胞产生超极化的机制。以豚鼠为实验动物,运用细胞内微电极和全细胞膜片钳记录技术,观察NFA和其它药物对急性分离的耳蜗螺旋动脉平滑肌细胞的作用。结果显示:NFA、indanyloxyacetic acid94(IAA-94)和disodium4,4’-diisothiocyanatostilbene-2,2’-disulfonate(DIDS)可使低静息膜电位的细胞产生超极化,但对高静息膜电位的细胞无明显作用。低静息膜电位细胞的平均静息电位为(-42.47±1.38)mV(n=24),100μmol/LNFA、10μmol/LIAA-94和200μmol/LDIDS分别使细胞超极化至(13.7±4.3)mV(n=9,P<0.01),(11.4±4.2)mV(n=7,P<0.01)和(12.3±3.7)mV(n=8,P<0.01),这种氯离子通道阻断剂引起细胞超极化反应的效应呈浓度依赖性。NFA引起的超极化和外向电流几乎完全被100nmol/L iberiotoxin、100nmol/L charybdotoxin、10mmol/L tetraethylammonium、50μmol/LBAPTA-AM、10μmol/L ryanodine和0.1～10mmol/L caffeine阻断,但不能被100μmol/L nifedipine、100μmol/L CdCl2和无Ca2+灌流外液阻断。结果提示:氯离子通道的阻断剂NFA可通过平滑肌细胞内钙库的钙释放增加细胞内钙,进而激活钙依赖的钾通道,产生耳蜗螺旋动脉平滑肌细胞的超极化反应。
The purpose of this study was to investigate the mechanism of hyperpolarization induced by niflumic acid (NFA), a chloride channel blocker, in the cochlear spiral artery smooth muscle cells of guinea pigs. Guinea pigs were used as experimental animals, and intracellular microelectrode and whole-cell patch clamp recording technique was used to observe the effect of NFA and other drugs on acutely isolated cochlear spiral artery smooth muscle cells. The results showed that NFA, indanyloxyacetic acid94 (IAA-94) and disodium4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS) could hyperpolarize cells with low resting membrane potential, No significant effect on the cells. The mean resting potential of cells with low resting membrane potential was (-42.47 ± 1.38) mV (n = 24). The cells were hyperpolarized to (13.7 ± 4.3) μmol / L with 10μmol / L IAA-94 and 200μmol / mV (n = 9, P <0.01), (11.4 ± 4.2) mV (n = 7, P <0.01) and (12.3 ± 3.7) mV The effect of agent on cell hyperpolarization was concentration dependent. NFA induced hyperpolarization and outward currents were almost completely blocked by 100nmol / L iberiotoxin, 100nmol / L charybdotoxin, 10mmol / L tetraethylammonium, 50μmol / LBAPTA-AM, 10μmol / L ryanodine and 0.1-10mmol / L caffeine, 100μmol / L nifedipine, 100μmol / L CdCl2 and no Ca2 + perfusion. The results suggest that NFA, a blocker of the chloride channel, can increase intracellular calcium through the calcium release from the smooth muscle intracellular calcium stores, which in turn activate the calcium-dependent potassium channel and produce hyperpolarization of the cochlear spiral artery smooth muscle cells.