低浓度哇巴因升高豚鼠心室肌细胞内游离钙浓度可能的信号转导途径  

Low concentration of ouabain elevates intracellular free calcium in guinea pig ventricular myocytes:the possible signal transduction pathway

作　　者：熊晨[1] 武彦昭[2] 张哲[1] 王永利[1] 

机构地区：[1]河北医科大学药理学教研室,石家庄050017 [2]河北医科大学附属四院五官科,石家庄050017

出　　处：《第二军医大学学报》2007年第8期850-853,共4页Academic Journal of Second Military Medical University

基　　金：河北省自然科学基金(301360)~~

摘　　要：目的:观察哇巴因(ouabain,OUA)对豚鼠心室肌细胞内游离钙浓度([Ca^(2+)])的影响,并探讨低浓度OUA是否可通过Na^+,K^+-ATPase的信号转导途径升高[Ca^(2+)]。方法:分离豚鼠心室肌细胞,激光共聚焦显微镜术测定单个心室肌细胞[Ca^(2+)]的荧光密度,另用不同浓度OUA孵育急性分离的豚鼠心室肌细胞后,分别取其沉淀用Western印迹检测观察OUA对酪氨酸蛋白(Src)磷酸化的影响。结果:在正常台氏液及无钙台氏液中,OUA(10^(-9),10^(-8),10^(-7),10^(-6)mol·L^(-1))均可浓度依赖性地升高细胞内钙浓度(P<0.05或0.01),且在正常台氏液中升高更为显著(P<0.05)。蛋白酪氨酸激酶抑制剂三羟异黄酮(genistein,GST)(1、10、50、100μmol·L^(-1))可浓度依赖性地抑制正常台氏液中的OUA效应。磷酸化Src包括相对分子质量120 000和70 000两个条带,各剂量OUA(10^(-4),10^(-6),10^(-8)mol·L^(-1))均能使Src的两个磷酸化条带的密度较阴性对照组显著升高(P<0.05),而GST(100μmol·L^(-1))可显著抑制OUA的升高效应。结论:低浓度OUA可能通过使酪氨酸蛋白磷酸化,激活OUA/Na^+,K^+ATPase/Src信号转导通路,促使钙通道开放及内钙释放,从而升高豚鼠心室肌细胞内游离钙浓度。Objective： To study the effect of low concentration of ouabain （OUA） on intracellular calcium concentration （[Ca^2＋ ]） in guinea pig ventricular myocytes and to understand whether low concentration of OUA can increase [Ca^2＋ ]. through Na^＋ , K^＋ -ATPase channel. Methods： The guinea pig ventricular myocytes were obtained by enzymatic digestion and the [Ca^2＋ ] fluorescent density of individual myocytes was observed under confocal laser scanning microscope. The isolated ventricular myocytes were then incubated with different concentrations of ouabain（ 10^-9 , 10^-8, 10^-7, 10^-6 mol · L^-1 ）. The sediment was subjected to Western blot analysis to assess the phosphorylation of Src by OUA. Results： In normal Tyrode＇s solution and Ca^2＋ -free Tyrode＇s solution, OUA （1 × 10^-9 -1 × 10^-6 ）mol · L^-1 elevated [Ca^2＋ ] in a concentration-dependent manner, with the elevation in normal Tyrode＇s solution more obvious（P〈0. 05）. Genistein （GST） （1, 10, 50, and 100 μmol ·L^-1） abolished the OUA-induced increases of [Ca^2＋ ]1 in a concentration dependent manner. There were two tyrosine-phosphorylated bands, with the molecular weights being 120 000 and 70 000. Compared with control group, the densities of the 2 bands in all OUA groups were significantly higher （P〈0.05） and GST could obviously inhibit the elevating effect of OUA. Conclusion： Low concentration of OUA may promote opening of Ca^2＋ channel and release of intracellular Ca^2＋ , and subsequently elevate intracellular free calcuim through phosphorylation of tyrosine and activition of OUA/Na^＋ , k^＋-ATPase/Src signal transduction pathway.

关 键 词：NA^+ -K^+ -交换ATP酶 心室肌细胞 哇巴因 钙 

分 类 号：R972.1[医药卫生—药品]