Acetylcholine activates protein kinase C-α in pulmonary venous smooth muscle
Acetylcholine activates protein kinase C-α in pulmonary venous smooth muscle
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DOI：
10.1097/00000542-200703000-00015
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发表时间：
2007-03-01
期刊：
影响因子：
8.8
通讯作者：
Murray, Paul A.
中科院分区：
文献类型：
--
作者：
Ding, Xueqin;Murray, Paul A.
背景资料：作者研究了乙酰胆碱诱导的肺静脉平滑肌（PVSM）收缩是否与特异性蛋白激酶C（PKC）亚型的激活有关。方法：将犬离体去内皮肺静脉环悬于改良Krebs-Ringer缓冲液中，测定等长收缩张力。评估了非特异性PKC抑制（双吲哚马来酰亚胺I; 3 x 10 - 6 m）和常规PKC亚型抑制（Go 7936 10-6 m）对乙酰胆碱剂量-反应关系的影响。通过蛋白质印迹分析测量PVSM细胞中常规PKC亚型（α、β、γ）、新型PKC亚型（δ、β、θ）和非典型PKC亚型（ζ、μ、μ）的表达。应用免疫荧光技术和共聚焦显微镜对乙酰胆碱作用前后PKC亚型的分布进行了定位。结果：乙酰胆碱引起E肺静脉收缩呈剂量依赖性。用双吲哚马来酰亚胺I或Go 7936预处理减弱乙酰胆碱收缩。在PVSM细胞中表达PKC-α、-i、-mu和-zeta，而不表达PKC-β、-γ、-δ、-β和-θ。PKC亚型的免疫荧光染色显示，在未刺激的。细胞中，仅在细胞质中检测到PKC-α和PKC-μ。PKC-iota和PKC-zeta也表现出胞质免疫荧光模式，其在核周区中特别丰富。乙酰胆碱激活诱导PKC-α从细胞质易位到细胞膜，而乙酰胆碱对其他PKC亚型没有影响。乙酰胆碱引起的PKC-α转位可被毒蕈碱受体拮抗剂阿托品阻断。结论：抑制PKC可减弱PVSM的乙酰胆碱收缩。乙酰胆碱诱导PVSM中PKC-α从细胞质易位到膜。这些结果表明PVSM中PKC依赖性乙酰胆碱收缩可能涉及PKC-α的激活和易位。
Background: The authors investigated whether acetylcholine-induced contraction in pulmonary venous smooth muscle (PVSM) is associated with the activation of specific protein kinase C (PKC) isoforms.Methods: isolated canine pulmonary venous rings without endothelium were suspended in modified Krebs-Ringer's buffer for measurement of isometric tension. The effects of nonspecific PKC inhibition (bisindolylmaleimide I; 3 x 10(-6) m) and conventional PKC isoform inhibition (Go7936 10-6 m) on the acetylcholine dose-response relation were assessed. The expression of conventional PKC isoforms (alpha, beta, gamma), novel PKC isoforms; (delta, epsilon, theta), and atypical PKC isoforms (zeta, iota, mu) was measured in PVSM cells by Western blot analysis. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of PKC isoforms before and after the addition of acetylcholine.Results: Acetylcholine caused dose-dependent contraction in E-pulmonary veins. Pretreatment with bisindolylmaleimide I or Go7936 attenuated acetylcholine contraction. PKC-alpha, -iota, -mu, and -zeta were expressed, whereas PKC-beta, -gamma, -delta, -epsilon, and -theta were not expressed in PVSM cells. Immunofluorescence staining for PKC isoforms showed that in unstimulated. cells, PKC-alpha and PKC-mu were detected only in the cytoplasm. PKC-iota and PKC-zeta also exhibited a cytoplasmic immunofluorescence pattern, which was especially abundant in the perinuclear zone. Activation with acetylcholine induced translocation of PKC-alpha from cytoplasm to membrane, whereas acetylcholine had no effect on the other PKC isoforms. Translocation of PKC-alpha in response to acetylcholine was blocked by the muscarinic receptor antagonist, atropine.Conclusion: Acetylcholine contraction is attenuated by PKC inhibition in PVSM. Acetylcholine induces translocation of PKC-alpha from cytoplasm to membrane in PVSM. These results suggest that PKC-dependent acetylcholine contraction in PVSM may involve activation and translocation of PKC-alpha.