In situ Ca2+ dependence for activation of Ca2+/calmodulin-independent protein kinase II in vascular smooth muscle cells
In situ Ca2+ dependence for activation of Ca2+/calmodulin-independent protein kinase II in vascular smooth muscle cells
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DOI：
10.1074/jbc.271.5.2506
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发表时间：
1996-02-02
影响因子：
4.8
通讯作者：
Singer, HA
中科院分区：
文献类型：
--
作者：
Abraham, ST;Benscoter, H;Singer, HA
Ca 2 +/钙调蛋白（CaM）依赖性蛋白激酶II的激活（CaM激酶II）和Ca 2 +/CaM非依赖性在培养的血管平滑肌（VSM）细胞中研究了激酶的（自主）形式，在离子霉素（1 μ M）暴露的15秒内，52.7 +/-4.4%的激酶变成自主的，这种反应部分维持至少10分钟，这与P-32磷酸化的钙调素激酶II δ亚基在原位，并取消了预处理与钙调素激酶II抑制剂KN-93，在原位钙离子依赖性产生自主钙调素激酶II测定细胞中选择性透化Ca ~（2+）和消耗肌浆网Ca ~（2+）预处理毒胡萝卜素。对所得曲线的分析显示EC（50）（产生50%最大反应的浓度）为692 +/-28 nM [Ca 2 +]（i），总活性的最大值68 +/-2%变成自主的，反映CaM激酶II几乎完全活化，Hill斜率为3，表明高度协同的过程，基于这种依赖性和完整细胞中测量的[Ca 2 +]（i）反应，血管紧张素II、加压素和血小板衍生生长因子-BB刺激的自主活动增加（分别为4.6、2和1.7倍）出乎意料地高。在离子霉素刺激的完整细胞中，自主活性和[Ca 2 +]（i）之间的相关性导致EC（50）为304 +/-23 nM [Ca 2 +]（i）的平行曲线。在完整的VSM细胞中产生自主活性的Ca 2+敏感性的这种明显增加被毒胡萝卜素预处理消除。我们的结论是，在生理范围内改变[Ca 2 +]（i）激活VSM中的CaM激酶II，并且该过程通过从细胞内池释放Ca 2+来促进，所述细胞内池启动合作性自磷酸化并随后产生自主CaM激酶II活性。
Activation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM kinase II) and development of the Ca2+/CaM-independent (autonomous) form of the kinase was investigated in cultured vascular smooth muscle (VSM) cells, Within 15 s of ionomycin (1 mu M) exposure 52.7 +/- 4.4% of the kinase became autonomous, a response that was partially maintained for at least 10 min, This correlated with P-32 phosphorylation of CaM kinase II delta-subunits in situ and was abolished by pretreatment with the CaM kinase II inhibitor KN-93, The in situ Ca2+ dependence for generating autonomous CaM kinase II was determined in cells selectively permeabilized to Ca2+ and depleted of sarcoplasmic reticulum Ca2+ by pretreatment with thapsigargin. Analysis of the resulting curve revealed an EC(50) (concentration producing 50% of maximal response) of 692 +/- 28 nM [Ca2+](i), a maximum of 68 +/- 2% of the total activity becoming autonomous reflecting nearly complete activation of CaM kinase II and a Hill slope of 3, indicating a highly cooperative process, Based on this dependence and measured [Ca2+](i) responses in intact cells, increases in autonomous activity stimulated by angiotensin II, vasopressin and platelet-derived growth factor-BB (4.6-, 2-, and 1.7-fold, respectively) were unexpectedly high, In intact cells stimulated by ionomycin, the correlation between autonomous activity and [Ca2+](i) resulted in a parallel curve with an EC(50) of 304 +/- 23 nM [Ca2+](i), This apparent increase in Ca2+ sensitivity for generating autonomous activity in intact VSM cells was eliminated by thapsigargin pretreatment. We conclude that alteration of [Ca2+](i) over a physiological range activates CaM kinase II in VSM and that this process is facilitated by release of Ca2+ from intracellular pools which initiates cooperative autophosphorylation and consequent generation of autonomous CaM kinase II activity.