权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study on Regulation of Ca^<2+> transport systems in vasculas smooth muscle cells.

血管平滑肌细胞Ca^2转运系统调控的研究。

基本信息

批准号：
63570420
负责人：
SHIGEKAWA Munekazu
金额：
$ 1.28万
依托单位：
National Cardiovascular center
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

We have devised a method by which we estimated quantitativelv activities of the sarcolemmal Ca pump and NalCa exchanger in intact cultured rat aotic smooth muscle cells. Using this method, we examined the effect of the activation of various protein kinases on activity of sarcolemmal Ca pump. We found that the Ca pump activity was stimulated by agents, which increased the intracellular cGMP level, and TPA, an activator of protein kinase C (PKC), whereas it was not affected by the cAMP-producing agents. We then examined the biochemical basis for the stimulatory effect of PKC. We found that PKC treatment resulted in phosphorylation of the Ca-ATPase to a level of about 1 mol per mol of the enzyme. Since there was good parallelity between the ATPase phosphorylation and the extent of enzyme activation, we concluded that the sarcolemmal Ca pump is regulated through its direct phosphorylation. In the next series of experiments, in which we used the same experimental system, we examined the effect of membrane potential (E_m) on the activity of the sarcolemmal Ca pump. Inside-negative K diffusion potential higher or lower than the resting E_m was artificially imposed with various concentrations of extracellular K and valinomycin. We found that the Ca pump was accelerated by depolarizing E_m, whereas it was retarded by hyperpolarizing E_m. These results therefore strongly suggested that the Ca pump is electrogenic and that alteration in E_m can modulate the intracellular Ca concentration in intact smooth muscle cells by changing the rate of Ca extrusion by the Ca pump. In the third series of experiments, we tested the effect of a rise in the intracellular cAMP concentration on the ATP-induced Ca mobilization in cultured aotic smooth muscle cells. We found that cAMP potentiated the ATP-induced intracellular Ca transient and that this vas due to the enhancement of IP_3-induced Ca release from the intracellular Ca store.

我们设计了一种定量测定大鼠主动脉平滑肌细胞膜钙泵和钠钙交换器活性的方法。用这种方法，我们研究了各种蛋白激酶的激活对肌膜钙泵活性的影响。我们发现，钙泵活性的刺激剂，这增加了细胞内的cGMP水平，TPA，蛋白激酶C（PKC）的激活剂，而它不受cAMP产生剂。然后，我们研究了PKC的刺激作用的生化基础。我们发现，PKC治疗导致磷酸化的Ca-ATP酶的水平约为1摩尔每摩尔的酶。由于ATP酶磷酸化和酶激活程度之间存在良好的平行性，我们得出结论，肌膜钙泵是通过其直接磷酸化来调节的。在接下来的一系列实验中，我们使用相同的实验系统，我们检查了膜电位（E_m）对肌膜钙泵活性的影响。用不同浓度的细胞外钾和缬氨霉素人工施加高于或低于静息E_m的内负钾扩散势。我们发现去极化的E_m对Ca泵有促进作用，而超极化的E_m则使Ca泵阻滞。这些结果表明，平滑肌细胞内钙泵是生电性的，E_m的改变可通过改变钙泵的钙排出速率来调节细胞内钙浓度。在第三个系列的实验中，我们测试了在培养的主动脉平滑肌细胞的ATP诱导的Ca动员的细胞内cAMP浓度的上升的效果。我们发现cAMP增强ATP诱导的细胞内Ca瞬变，这是由于cAMP增强了IP_3诱导的细胞内Ca库的Ca释放。