溶血磷脂酰胆碱对心室肌细胞T型钙离子通道电流的影响及机制探讨

The effect of lysophosphatidylcholine on cardiac T-type Ca^(2+) channel current and its mechanism

目的研究溶血磷脂酰胆碱(LPC)对心肌细胞T型钙通道电流(ICa,T)的影响并探讨其机制。方法新生大鼠心室肌细胞由酶法消化分离获得后,使用全细胞膜片钳技术记录LPC(10μmol/L)处理前后的搏动频率及ICa,T。表达人类心脏T型钙通道CaV3.1和Cav3.2亚基的人胚肾(HEK)293细胞通过传代培养获得。各种蛋白激酶抑制剂或蛋白激酶C(PKC)亚型特异抑制剂预处理1 h后,用全细胞膜片钳测定分别记录LPC处理前后的CaV3.1和Cav3.2 T型钙离子通道电流。结果 LPC显著提高了新生大鼠心室肌细胞的自律性(61±7次/分vs 40±6次/分,n=6)和ICa,T(4.4±0.5 pA/pF,n=12 vs 3.7±0.4 pA/pF,n=15)。在HEK293细胞中,LPC对ICaV3.1无显著影响;但显著增加了ICav3.2[LPC处理前:36.7±2.2 pA/pF(n=20);10μmol/L时:42.3±3.0 pA/pF(n=12);50μmol/L:47.5±3.8 pA/pF(n=8)]。只有PKC抑制剂(白屈菜红碱,5μmol/L)显著减弱了LPC的作用[LPC组:增大58.5%±14.2%;PKC预处理+LPC组:增大20.4%±10.4%(P<0.05)];PKCα特异性抑制剂Ro-32-0432(30 nmol/L)模拟了白屈菜红碱对ICav3.2的作用[白屈菜红碱:12.7±2.6 pA/pF(n=8);Ro-32-0432:12.9±2.3pA/pF(n=9)],而PKCβI、PKCβII特异性抑制剂并没有影响LPC对ICav3.2的作用。结论 LPC可能通过激活PKCα途径增强ICa,T,从而增加心室肌细胞的自律性。

Objective To study the effects of lysophosphatidylcholine （LPC） on T-type calcium channel currents （Ⅰ_Ca,T） in cardiomyocytes and underlying intracellular signaling pathways. Methods Neonatal rat cardiomyocytes by u- sing of enzyme digestion methods were prepared for recording the T-type Ca2± channel current with or without LPC （ 10 μmoL/L）. Human cardiac T-type calcium channel α1-subunits, Ca_v3.1 and Ca_v3.2 were stablely expressed in human em- bryonic kidney （HEK） 293 cells. Ⅰ_Ca,T was recorded by whale-cell patch clamp technique in pretreated with several kinds of kinase inhibitors for lh followed by 10 μmoL/L LPC purfusing for 10 min. Results ① LPC markedly accelerated the spontaneous beating rates of neonatal rat cardiomyocytes （40 ±6 bpm vs 61 ±7 bpm, n =6）, and augmented the Ⅰ_Ca,T（3. 7 ±0.4 pA/pF,n = 15 vs 4.4 ±0.5 pA/pF,n = 12） ;② LPC exerted no effect on the Ca_v3.1 T-type Ca2± channel current （Ⅰ_Cav3 1）. In contrast, LPC significantly up-regulated the Car3.2 T-type Ca2± channel current （Icav3.2） [47.5 ± 3.8 pA/ pF（50 μmol/L, n = 8 ） vs 42.3 ± 3.0 pA/pF（ 10μmol/L, n = 12 ） vs 36.7 ± 2.2 pA/pF（ n = 20 ） ] ; ③ Pre-treated withRo-32-0432 （30 nmol/L）, a specific inhibitor of protein kinase Ca, the up-regulated Ⅰ_Cav3.2 by LPC was completely blocked, which mimiced the role of pan-PKC inhibitor （chelerythrine, 5μmol/L） in the modulation of LPC on Ⅰ_Cav3 2 （ chelerythrine ： 12.7 ± 2.6 pAZpF, n = 8 ; Ro-32- 0432：12.9 ± 2.3 pA/pF, n = 9）. However, specific inhibitors of PKCβⅡ, PKCβⅡ did not interfered the effect of LPC on Ⅰ_Cav3.2. Conclusion LPC stimulates IC.,T viaPKCα activation, which may accelerate the pathophysiological cardiac automaticity.