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藜芦定增强反向Na+/Ca2+交换电流诱导家兔心室肌细胞内Ca2+超载和动作电位异常延长

孔令浩, 马季骅^*, 张培华, 罗岸涛, 张硕, 任志强, 冯娟, 陈玖玲

武汉科技大学医学院，武汉 430065

摘要

本文应用全细胞膜片钳记录技术、可视化动缘探测系统及细胞内钙测定系统和多道生理信号采集处理系统，研究藜芦定(veratridine, VER)对家兔心室肌细胞持续钠电流(persistent sodium current, I_Na.P)、Na⁺/Ca²⁺交换电流(Na⁺/Ca²⁺ exchange current, I_NCX)、钙瞬变及动作电位(action potential, AP)的影响，并探讨其引起细胞内Ca²⁺超载和增强心肌收缩的发生机制。结果显示，给予心室肌细胞10、20 μmol/L VER后，INa.P和反向INCX电流密度均增大，再加入4 μmol/L河豚毒素(tetrodotoxin, TTX)后，I_Na.P和反向INCX电流密度均又明显减小。在另一组实验中，特异性I_Na.P阻断剂雷喏嗪(ranolazine, RAN)也明显抑制由VER诱导增大的I_Na.P和反向I_NCX。加入2.5 μmol/L VER后，心室肌细胞最大收缩速率、收缩幅度和钙瞬变基线(舒张期Ca²⁺浓度)均增大，加入2 μmol/L TTX后上述三项指标均明显减小。VER (20 μmol/L)可使心室肌细胞AP复极至50% (action potential duration at 50% repolarization, APD₅₀)和90%的时间(action potential duration at 90% repolarization, APD₉₀)延长，其中3例出现早期后除极(early afterdepolarizations, EADs)，加入4 μmol/L TTX后APD₅₀、APD₉₀均明显缩短，3例EADs均消失。实验结果提示：(1) VER作为特异性I_Na.P的开放剂，可引起心室肌细胞I_Na.P增大而产生细胞内Na⁺超载，继而通过反向I_NCX的增大导致细胞内Ca²⁺超载。(2)由VER诱导增大的心室肌细胞I_Na.P还可引起心室肌细胞APD延长，诱发EADs。(3) TTX对由VER引起的上述指标的异常变化均有明显抑制作用，说明上述指标的异常变化是由I_Na.P增大所引起。以上结果表明，VER作为I_Na.P的开放剂，通过增大I_Na.P而引起反向INCX增强，最终导致细胞内Ca²⁺超载和APD异常延长。

关键词： 藜芦定; 钠钙交换; 动作电位; 膜片钳; 钙超载; 心律失常

分类号：R33

[Involvement of veratridine-induced increase of reverse Na(+)/Ca(2+) exchange current in intracellular Ca(2+) overload and extension of action potential duration in rabbit ventricular myocytes.] [Article in Chinese]

KONG Ling-Hao, MA Ji-Hua^*, ZHANG Pei-Hua, LUO An-Tao, ZHANG Shuo, REN Zhi-Qiang, FENG Juan, CHEN Jiu-Ling

Medical College of Wuhan University of Science and Technology, Wuhan 430065, China

Abstract

The objectives of this study were to investigate the effects of veratridine (VER) on persistent sodium current (I(Na.P)), Na(+)/Ca(2+) exchange current (I(NCX)), calcium transients and the action potential (AP) in rabbit ventricular myocytes, and to explore the mechanism in intracellular calcium overload and myocardial contraction enhancement by using whole-cell patch clamp recording technique, visual motion edge detection system, intracellular calcium measurement system and multi-channel physiological signal acquisition and processing system. The results showed that I(Na.P) and reverse I(NCX) in ventricular myocytes were obviously increased after giving 10, 20 μmol/L VER, with the current density of I(Na.P) increasing from (-0.22 ± 0.12) to (-0.61 ± 0.13) and (-2.15 ± 0.14) pA/pF (P < 0.01, n = 10) at -20 mV, and that of reverse I(NCX) increasing from (1.62 ± 0.12) to (2.19 ± 0.09) and (2.58 ± 0.11) pA/pF (P < 0.05, n = 10) at +50 mV. After adding 4 μmol/L tetrodotoxin (TTX), current density of I(Na.P) and reverse I(NCX) returned to (-0.07 ± 0.14) and (1.69 ± 0.15) pA/pF (P < 0.05, n = 10). Another specific blocker of I(Na.P), ranolazine (RAN), could obviously inhibit VER-increased I(Na.P) and reverse I(NCX). After giving 2.5 μmol/L VER, the maximal contraction rate of ventricular myocytes increased from (-0.91 ± 0.29) to (-1.53 ± 0.29) μm/s (P < 0.01, n = 7), the amplitude of contraction increased from (0.10 ± 0.04) to (0.16 ± 0.04) μm (P < 0.05, n = 7), and the baseline of calcium transients (diastolic calcium concentration) increased from (1.21 ± 0.08) to (1.37 ± 0.12) (P < 0.05, n = 7). After adding 2 μmol/L TTX, the maximal contraction rate and amplitude of ventricular myocytes decreased to (-0.86 ± 0.24) μm/s and (0.09 ± 0.03) μm (P < 0.01, n = 7) respectively. And the baseline of calcium transients reduced to (1.17 ± 0.09) (P < 0.05, n = 7). VER (20 μmol/L) could extend action potential duration at 50% repolarization (APD(50)) and at 90% repolarization (APD(90)) in ventricular myocytes from (123.18 ± 23.70) to (271.90 ± 32.81) and from (146.94 ± 24.15) to (429.79 ± 32.04) ms (P < 0.01, n = 6) respectively. Early afterdepolarizations (EADs) appeared in 3 out of the 6 cases. After adding 4 μmol/L TTX, APD(50) and APD(90) were reduced to (99.07 ± 22.81) and (163.84 ± 26.06) ms (P < 0.01, n = 6) respectively, and EADs disappeared accordingly in 3 cases. It could be suggested that: (1) As a specific agonist of the I(Na.P), VER could result in I(Na.P) increase and intracellular Na(+) overload, and subsequently intracellular Ca(2+) overload with the increase of reverse I(NCX). (2) The VER-increased I(Na.P) could further extend the action potential duration (APD) and induce EADs. (3) TTX could restrain the abnormal VER-induced changes of the above-mentioned indexes, indicating that these abnormal changes were caused by the increase of I(Na.P). Based on this study, it is concluded that as the I(Na.P) agonist, VER can enhance reverse I(NCX) by increasing I(Na.P), leading to intracellular Ca(2+) overload and APD abnormal extension.

Key words: veratridine; sodium-calcium exchanger; action potentials; patch-clamp techniques; calcium overloading; arrhythmias

收稿日期：2012-01-20　　录用日期：2012-04-06

通讯作者：马季骅　　E-mail: mjhua@wust.edu.cn

引用本文：

孔令浩, 马季骅, 张培华, 罗岸涛, 张硕, 任志强, 冯娟, 陈玖玲. 藜芦定增强反向Na+/Ca2+交换电流诱导家兔心室肌细胞内Ca2+超载和动作电位异常延长[J]. 生理学报 2012; 64 (4): 433-443.

KONG Ling-Hao, MA Ji-Hua, ZHANG Pei-Hua, LUO An-Tao, ZHANG Shuo, REN Zhi-Qiang, FENG Juan, CHEN Jiu-Ling. [Involvement of veratridine-induced increase of reverse Na(+)/Ca(2+) exchange current in intracellular Ca(2+) overload and extension of action potential duration in rabbit ventricular myocytes.] [Article in Chinese] . Acta Physiol Sin 2012; 64 (4): 433-443 (in Chinese with English abstract).