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目的:探讨低渗溶液通过激活KATP通道对大鼠缺血心肌的保护作用。方法:用RT-PCR法检测大鼠心肌细胞上KATP通道内向整流性亚单位基因的表达。用膜片钳单通道记录方法,观察急性分离的大鼠心肌细胞在低渗溶液刺激下,钾离子电流的变化及电生理学特征。将大鼠心脏随机分为5组:缺血对照(CON)组、格列苯脲(glibenclamide,Glib)组、缺血预适应(IPC)组、低渗处理(HYPO)组和HYPO+Glib组,用Langendorff方法灌流离体大鼠心脏,分别记录各组左心室形成压(LVDP)和左心室等容期压力最大变化速率(±LVdP/dtmax)。观察低渗溶液对缺血心肌的保护作用。结果:①RT-PCR检测证实,大鼠心肌细胞上KATP通道的Kir亚单位存在两种类型:Kir6.1与Kir6.2。②膜片钳单通道记录证实,低渗溶液的刺激可以激活电导为(30.34±1.76)pS的KATP通道电流(-80 mV),开放概率(Po)明显增加(等渗溶液时为:0.01±0.01;低渗溶液时为:0.51±0.12),该电流可被10μmol/L的Glib阻断(Po降低为0.03±0.02,P<0.05)。③灌流实验证实,在一定条件下,与CON组相比,再灌注前给予缺血心脏低渗溶液灌流5 min可明显改善心脏的功能[再灌注60 min时,CON组的LVDP为缺血前的(45.02±1.70)%;而HYPO组的LVDP则升高至(58.43±0.86)%;+LVdP/dtmax由(42.85±4.45)%升高至(61.96±2.98)%;-LVdP/dtmax由(53.94±2.52)%升高至(66.60±3.86)%,均P<0.05],且该保护作用可部分被10μmol/LGlib阻断(P<0.05)。结论:大鼠心肌细胞上的KATP通道具有容积敏感性,低渗溶液对大鼠缺血心肌的保护作用可能是通过激活K通道发挥作用。
Objective: To investigate the protective effect of hypotonic solution on ischemic myocardium in rats by activating KATP channel. Methods: The expression of inward rectifier subunit gene of KATP channel in rat cardiomyocytes was detected by RT-PCR. The patch clamp method was used to observe the change of potassium current and the electrophysiological characteristics of acutely isolated rat cardiomyocytes stimulated by hypotonic solution. The hearts of rats were randomly divided into 5 groups: ischemia control (CON) group, Glibenclamide (Glib) group, IPC group, HYPO group and HYPO + Glib group The left ventricular pressure (LVDP) and left ventricular pressure (LVdP / dtmax) were recorded by Langendorff perfusion in isolated rat heart. To observe the protective effect of hypotonic solution on ischemic myocardium. Results: ①RT-PCR confirmed that there are two types of Kir subunits of KATP channels on rat cardiomyocytes: Kir6.1 and Kir6.2. (2) Patch clamp test showed that stimulation of hypotonic solution could activate KATP channel current (-80 mV) with conductivity of (30.34 ± 1.76) pS and significantly increase open probability (0.01 ± 0.01; 0.51 ± 0.12 for hypotonic solution). This current was blocked by Glib at 10 μmol / L (Po decrease to 0.03 ± 0.02, P <0.05). (3) Perfusion experiments confirmed that, under certain conditions, perfusion of hypotonic solution of ischemic heart for 5 min before reperfusion could significantly improve cardiac function compared with CON group. [At 60 min after reperfusion, LVDP in CON group was (45.02 ± 1.70)%, while the LVDP in HYPO group increased to (58.43 ± 0.86)%; the LVdP / dtmax increased from (42.85 ± 4.45)% to (61.96 ± 2.98)%; (53.94 ± 2.52)% to (66.60 ± 3.86)%, respectively, all P <0.05], and the protective effect was partly blocked by 10μmol / LGlib (P <0.05). CONCLUSION: KATP channels on rat cardiomyocytes are volume sensitive. The protective effect of hypotonic solution on ischemic myocardium may be through activation of K channels.