低浓度的哇巴因引起豚鼠心室肌细胞内钙增高的可能途径

目的观察哇巴因(ouabain,OUA)对豚鼠心室肌细胞内游离钙浓度([Ca2+]i)的影响。方法酶解分离豚鼠心室肌细胞,负载Fluo3-AM,激光共聚焦显微镜术测定单个心室肌细胞[Ca2+]i的荧光密度,结果用相对荧光强度(FI-FI0)/FI0(%)表示,其中,FI0:给药前的荧光密度值,FI:给药后的荧光密度值。结果在正常台氏液及无钙台氏液中,OUA(1×10-9~1×10-6mol·L-1)浓度依赖性地升高细胞内钙浓度,在正常台氏液分别为16.7±6.8(P<0.01)、26.0±4.7(P<0.01)、183.0±101.0(P<0.01)、295.0±172.0(P<0.01),而在无钙台氏液中OUA升高[Ca2+]i不如在正常台氏液中,分别为9.19±4.73(P<0.05)、20.75±5.2(P<0.05)、85.79±64.7(P<0.05)、231.0±26.0(P<0.05)。肌浆网钙通道抑制剂理阿诺碱Ryanodine(1×10-5mol·L-1)可部分抑制正常台氏液时OUA的效应5.3±2.1(P<0.01)。在无钠无钾台氏液中,OUA(1×10-9~1×10-6mol·L-1)对心室肌细胞[Ca2+]i的影响分别为16.5±6.5、25.0±5.0、162.0±45.0、280.0±96.0与正常台氏液比较无差别(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,分别为17.5±3.1、14.2±8.9、0.8±7.6(P<0.05)、-1.9±6.7(P<0.01)。L-型钙通道激动剂BayK8644,肌浆网钙通道开放剂Ryanodine(1×10-7mol.L-1)在正常台氏液中均可提高[Ca2+]i为13.3±3.2(P<0.05)、6.4±5.6(P<0.05)。三羟异黄酮可取消其效应为-13.0±21.0(P<0.01)、-1.6±5.9(P<0.01)。结论低浓度的OUA升高豚鼠心室肌细胞内游离钙浓度,此作用与其开放钙通道及促进内钙释放有关,且信号转导通过此二途径起作用。

The probable pathway of low concentration of ouabain on intracellular calcium elevation in guinea ventricular mycytes

Aim To observe the effects of low concentration ouabain(OUA)on intracellular calcium concentration ([Ca2+]i) were investigated in guinea pig ventricular myocytes.Methods The guinea-pig ventricular myocytes were obtained by enzymatic dissociation technique, then were incubated with Fluo3-AM. The Fluo3-AM fluorescent signal was detected with confocal laser scanning microscopy. The change of [Ca2+]i was represented by the percentage of fluorescence intensity change [(FI-FI0)/FI0,%] (FI: fluorescent intensity after addimg OUA, FI0: control). Results In normal Tyrode,s solution and Ca2+-free Tyrode′s solution.OUA (1×10-9~1×10-6 mol·L-1)elevated [Ca2+]i in a concentration-dependent manner, in normal Tyrode′s solution by 16.7±6.8(P<0.01)、26.0±4.7(P<0.01)、183.0±101.0(P<0.01)、295.0±172.0(P<0.01)respectively,however,the level of[Ca2+]i elevation in Ca2+-free Tyrode′s solution was lower than that in normal Tyrode′s solution by 9.19±4.73、20.75±5.2、85.79±64.7、231.0±26.0(P<0.05)respectively. Ryanodine remarkably blocked the effects of OUA (10-8 mol·L-1) on [Ca2+]i elevation in normal Tyrode′s solution by 5.3±2.1(P<0.01), while completely blocked the elevation effects of OUA (10-8 mol·L-1) on [Ca2+]i in Ca2+-free Tyrode′s solution. In Na+,K+-free Tyrode′s solution OUA (1×10-9~1×10-6 mol·L-1)elveled [Ca2+]i by 16.5±6.5、25.0±5.0、162.0±45.0、280.0±96.0 respectively, to a similar as in normal Tyrode′s solution(P>0.05).Genistein (GST) (1、10、50、100 μmol·L-1)abolished the OUA-induced increases in[Ca2+]i in a concentration-dependent manner by 17.5±3.1、14.2±8.9、0.8±7.6(P<0.05)、-1.9±6.7(P<0.01)respectively. GST can also abolished the elevation effects of both ryanodine(10-7 mol·L-1)and BAY K8644 on [Ca2+]i in normal Tyrode′s solution.Conclusions Ca2+ elevation induced by OUA depends on both intracellular and extracellular Ca2+ stores, and tyrodine kinases are also responsible for OUA-induced increases [Ca2+]i.