钙敏感受体参与乳鼠心肌细胞缺氧/复氧损伤的机制研究

目的 观察钙敏感受体(CaSR)对缺氧/复氧(A/R)乳鼠心肌细胞内钙的影响,探讨CaSR参与缺氧/复氧乳鼠心肌细胞损伤的机制及信号传导途径.方法 原代培养乳鼠的心肌细胞,建立A/R乳鼠心肌细胞模型(采用95%N2+5%CO2饱和2 h的低糖、无血清DMEM液孵育60 min,再更换成正常氧合细胞培养液孵育30 min的方法).心肌细胞随机分为6组:(1)正常对照组;(2)A/R组;(3)A/R+GdCl3组:在复氧开始时给予GdCl3(CaSR激动剂)300 μmol/L;(4)A/R+NiCl2+CdCl2+GdCl3组:在复氧时细胞培养液内加入10 mmol/L NiCl2(Na+-Ca2+交换阻断剂)和200 μmol/L CaCl2(L-Ca2+阻断剂)孵育10min,再加入300/μmol/L GdCl3;(5)A/R+U73122+GdCl3组:在复氧时细胞培养液内加入10μmol/L磷脂酶C(PLC)阻断剂U73122孵育10 min,再加入300μmol/L GdCl3;(6)A/R+thapsigargin+GdCl3组:在复氧时细胞培养液内加入10μmol/L肌浆网三磷酸肌醇(IP3)敏感Ca2+泵阻断剂thapsigargin孵育10 min,再加入300μmol/L GdCl3.应用Fluo-3/AM荧光指示剂负载心肌细胞,激光共聚焦显微镜连续观察细胞内钙荧光强度的动态变化.结果 A/R使心肌细胞内钙离子浓度(Ca2+];)增加,GdCl3使A/R乳鼠心肌细胞Ca2+]?I进一步增加;NiCl2和CdCl2对GdCl3引起的心肌细胞Cd2+]I增加无影响;而U73122和thapsigargin则抑制了GdCl3引起的心肌细胞Ca2+]I增加.结论 CaSR通过磷脂酶C-三磷酸肌醇途径参与了A/R所致心肌细胞钙超载.

Calcium-sensing receptors Induce calcium overload in cultured neonatal rat ventricular cardiomyocytes during anoxia/reoxygenation

Objective To observe the influence of calciurn-sensing receptor (CaSR) on intracellular calcium and to investigate the mechanism of CaSR induced injury of cultured neonatal rat cardiomyocytes under anoxia/reoxygenation(A/R). Methods We incubated primarily neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 60 min, and re-incubated cell in normal culture medium for 30 min to establish a model of A/R. The cultured cardiomyocytes were randomly divided into six groups: (1) control group; (2) A/ R group;(3) A/R + GdCl3 group:300/μmol/L GdCl3(a specific CaSR activator) was added to the culture medium at the beginning of reoxygenation; (4) A/R + NiCl2 + CdCl2 + GdCl3 group:at the beginning of reoxygenation, NiCl2 ( Na+/Ca2+ exchanger inhibitor) 10 mmol/L and CdCl2 ( L-typecalcium channel blocker) 200 μmol/L were added to the culture medium prior to GdCl3 (300μmol/L) ; (5) A/R + U73122 + GdCl3 group:at the beginning of reoxygenation, cardiomyocytes were preincubated with U73122 10 μmol/L(phospholipase C inhibitor) 10 min prior to GdCl3 (300 μmol/L);(6) A/R + thapsigargin (TG) + GdCl3 group:at the beginning of reoxygenation, cardiornyocytes were preincubated with thapsigargin (inositolt riphosphate inhibitor) 10 μmol/L 10 min prior to GdCl3 (300μmol/L). The changes of fluorescent intensities in Ca2+ ]I were recorded continuously with laser scanning confocal microscopy in different treatments. Results The results showed that A/R increased the intracellular calcium of cardiomyocytes. GdCl3, a specific activator of CaSR, further enhanced cardiomyocytes Ca2+ ]I during A/R. NiCl2 and CdCl2 did not affect the increase in Ca2+ ]I induced by GdCl3. However, U73122 and thapsigargin inhibited the increase in Ca2+ ]I. Conclusion CaSR is involved in A/R-induced calcium overload of neonatal rat cardiornyocytes through PLC-IP3 signaling pathway.