虎杖苷对休克大鼠微血管平滑肌细胞内钙、pH和膜电位的影响

The effects of polydatin on intracellular free calcium, pH and membrane potential of vascular smooth muscle cells after hemorrhagic shock in rats

目的探讨虎杖苷（ＰＤ）改善休克动物微循环的作用机制。方法股动脉放血复制休克模型，取肠系膜微动脉用链霉蛋白酶Ｅ消化以分离单个血管平滑肌细胞（ＶＳＭＣ），再用不同荧光染料分别标记细胞，在激光共聚焦显微镜上测定细胞内钙（［Ｃａ２＋］ｉ）、ｐＨ和膜电位的变化。结果ＰＤ（０４ｍｍｏｌ·Ｌ－１）使休克大鼠ＶＳＭＣ［Ｃａ２＋］ｉ浓度在１０ｍｉｎ内显著下降至加药前的７８４％±５６％、ｐＨ下降至原来的７２８％±８２％；而正常对照组ＶＳＭＣ［Ｃａ２＋］ｉ升高１７５％±６３％、ｐＨ上升３４％±１０１％。当ＰＤ加入前１０ｍｉｎ用钙通道阻断剂维拉帕米（５０μｍｏｌ·Ｌ－１）预处理后，则动物不论休克与否，其［Ｃａ２＋］ｉ、ｐＨ都显著下降。另外ＰＤ使正常及休克大鼠ＶＳＭＣ膜电位负值下降，出现去极化反应。加入ＥＧＴＡ和维拉帕米预处理不能阻断ＰＤ作用，但加入钠通道阻断剂河豚毒素（１μｍｏｌ·Ｌ－１）则可完全阻断ＰＤ的去极化作用。结论ＰＤ对细胞内钙、ｐＨ有双向调节作用，正常情况下ＰＤ增加细胞内游离钙及升高ｐＨ以提高血管张力，休克时ＰＤ降低细胞内钙浓度及降低细胞内ｐＨ以降低血管张力，使血管扩张。此外ＰＤ还可能通过促进细胞外钠离子?

AIM To elucidate the mechanism of Polydatins effects on small blood vessels. METHODS The changes of cytosolic free calcium concentration [Ca 2+ ] i, intracellular pH and cell membrane potential (MP) kinetically were determined in a single vascular smooth muscle cell (VSMC) isolated immediately with 20～30 min pronase E (1 g·L -1 )digestion from mesenteric arterioles of rats. Animals were divided into two groups: control group and shock group. The hemorrhagic shock model was prepared by withdrawal of blood from femoral arteries and maintained mean arterial pressure at 5 32 kPa for 90 min. VSMC were labeled with specific fluorescent probe of Fluo 3 AM, Snarf and DiBAC 4 respectively to measure [Ca 2+ ] i, pH and MP with a adherent cell analysis and sorting system(ACAS 570). RESULTS It was found that polydatin(0 4 mmol·L -1 ) caused a significant decrease of the VSMC [Ca 2+ ] i in shock group (78 4%±5 6% vs baseline), but had a increase of the VSMC [Ca 2+ ] i in control group (117 5%±6 3% vs baseline). The pH changes after adding polydatin was similar to that of [Ca 2+ ] i. When pretreated with verapamil (50 μmol·L -1 ) 10 min before giving polydatin, the VSMC [Ca 2+ ] i was decreased remarkably both in control and shock group. It was also found that polydatin might cause the VSMC MP elevated (depolarization) about 15%±2 1% in control group and 23%±3 0% in shock group respectively. When pretreated with verapamil and EGTA (2 mmol·L -1 ), the MP was still elevated 28%±5 7% vs baseline; But when pretreated with tetrodotoxin (1 μmol·L -1 , a kind of sodium channel blocker), the depolarization of polydatin was inhibited completely. CONCLUSION These data suggested that polydatin had up and down regulation effects on [Ca 2+ ] i and pH of VSMC, and polydatin also induce the entry of extracellular sodium ion to make the cell depolarization.