Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II
Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II
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DOI：
10.1152/ajplung.1998.274.2.l278
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发表时间：
1998-02-01
影响因子：
4.9
通讯作者：
Murray, PA
中科院分区：
文献类型：
--
作者：
Damron, DS;Nadim, HS;Murray, PA
我们的目的是鉴定犬肺动脉平滑肌细胞（PASMCs）原代培养物中存在的蛋白激酶C （PKC）的异构体，并确定血管紧张素II （ATG II）是否会触发特定PKC异构体在细胞内离散位置的易位。利用异构体特异性抗体和Western blot分析鉴定PASMCs中PKC的异构体。使用间接免疫荧光和共聚焦显微镜检查PKC亚型的亚细胞分布。肌醇磷酸的产生被用来评估磷脂酶C的激活，fura 2被用来监测细胞内Ca2+浓度对ANG II的响应。Western blot分析发现PKC的α、δ、epsilon、zeta、iota/lambda和mu 6种亚型。对α、δ、zeta、iota/lambda和mu 5种异构体进行免疫定位，发现每个异构体都有独特的染色模式。ANG II引起pkc - α从细胞质转移到核膜，pkc - δ转移到肌丝。相比之下，胞浆PKC-zeta没有移位，但核PKC-I上调。ANG II型1受体拮抗剂氯沙坦可阻断pkc - α和pkc - δ的易位以及PKC-zeta的上调。此外，ANG II刺激肌醇磷酸产生和细胞内Ca2+浓度振荡，这被氯沙坦阻断。因此，ANG II型1受体的激活触发磷酸肌苷信号级联，导致细胞内离散位点特异性PKC异构体的易位或上调。a和zeta亚型可能调节核事件，而PKC-delta可能通过作用于肌丝参与调节收缩。
Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ATG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha, delta, epsilon, zeta, iota/lambda, and mu) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha, delta, zeta, iota/lambda, and mu) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-I was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The a and zeta isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.