Cyclic GMP-dependent protein kinase Iα inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells
Cyclic GMP-dependent protein kinase Iα inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells
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DOI：
10.1074/jbc.m512770200
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发表时间：
2006-03-31
影响因子：
4.8
通讯作者：
Mendelsohn, ME
中科院分区：
文献类型：
--
作者：
Christensen, EN;Mendelsohn, ME
血管平滑肌的收缩状态受细胞内钙离子水平的调节。一氧化氮通过刺激产生环GMP引起血管舒张，环GMP激活血管平滑肌细胞（VSMC）中的I型cGMP依赖性蛋白激酶（PKGI），抑制激动剂诱导的细胞内Ca 2+动员（[Ca 2 +]（i））。两种PKGI同工酶PKGI α和PKGI β在VSMC中环GMP介导的[Ca 2 +]（i）抑制中的相对作用尚不清楚。在这里，我们研究了PKGI亚型抑制[Ca 2 +]（i）对VSMC活化的响应的能力。建立了表达PKGI α或PKGI β的稳定的中国仓鼠卵巢细胞系，并检查了PKGI同种型响应凝血酶受体刺激抑制[Ca 2 +]（i）的能力。在稳定表达PKGI α或PKGI β的中国仓鼠卵巢细胞中，8-Br-cGMP激活抑制凝血酶受体激活肽（TRAP）的[Ca 2 +]（i），分别为98 +/- 1和42 +/-5%（p < 0.002）。使用PKGI α和PKGI β特异性抗体对来自多个部位的培养的人VSMC细胞进行免疫印迹研究，结果显示PKGI α是主要的VSMC PKGI同种型。[Ca2+]（i）在人冠状动脉VSMC细胞（Co 403）中，在不存在或存在环GMP的情况下，在凝血酶受体刺激后检查。8-Br-cGMP显著抑制TRAP诱导的Co 403 [Ca ~（2+）]（i），导致[Ca ~（2+）]（i）的EC_（50）增加4倍。在不存在8-Br-cGMP的情况下，与对照RNAi处理的Co 403细胞相比，通过RNA干扰（RNAi）抑制PKGI α水平导致显著更大的TRAP刺激的[Ca-1（2+）]升高。在存在8-Br-cGMP的情况下，通过RNAi抑制PKGI α表达导致cGMP介导的[Ca 2 +]抑制的完全丧失（i）。在通过RNAi抑制PKGI α表达之前或之后，Co 403细胞中PKGI β的腺病毒过表达不能改变TRAP刺激的Ca 2+动员。这些结果支持PKGI α是介导通过一氧化氮/环GMP途径抑制VSMC活化的主要cGMP依赖性蛋白激酶亚型。
Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP- dependent protein kinase ( PKGI) in vascular smooth muscle cells ( VSMC), inhibiting agonist- induced intracellular Ca2+ mobilization ([Ca2+] (i)). The relative roles of the two PKGI isozymes, PKGI alpha and PKGI beta, in cyclic GMP- mediated inhibition of [Ca2+] (i) in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+](i) in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGI alpha or PKGI beta were created, and the ability of PKGI isoforms to inhibit [Ca2+](i) in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGI alpha or PKGI beta, 8- Br- cGMP activation suppressed [ Ca2+](i) by thrombin receptor activation peptide ( TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively ( p < 0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGI alpha- and PKGI beta- specific antibodies showed PKGI alpha is the predominant VSMC PKGI isoform. [Ca2+](i) following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells ( Co403). 8- Br- cGMP significantly inhibited TRAP- induced [Ca2+](i) in Co403, causing a 4- fold increase in the EC50 for [Ca2+](i). In the absence of 8- Br- cGMP, suppression of PKGI alpha levels by RNA interference ( RNAi) led to a significantly greater TRAP- stimulated rise in [Ca-i(2+]) as compared with control RNAi- treated Co403 cells. In the presence of 8- Br- cGMP, the suppression of PKGI alpha expression by RNAi led to the complete loss of cGMP- mediated inhibition of [Ca2+](i). Adenoviral overexpression of PKGI beta in Co403 cells was unable to alter TRAP- stimulated Ca2+ mobilization either before or after suppression of PKGI alpha expression by RNAi. These results support that PKGI alpha is the principal cGMP- dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/ cyclic GMP pathway.