11,12-epoxyeicosatrienoic acid stimulates endogenous mono-ADP-ribosylation in bovine coronary arterial smooth muscle
11,12-epoxyeicosatrienoic acid stimulates endogenous mono-ADP-ribosylation in bovine coronary arterial smooth muscle
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DOI：
10.1161/01.res.85.4.349
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发表时间：
1999-08-20
影响因子：
20.1
通讯作者：
Campbell, WB
中科院分区：
文献类型：
--
作者：
Li, PL;Chen, CL;Campbell, WB
内源性ADP-核糖基化在介导Ca ~（2+）激活的K ~+通道激活中的作用在牛冠状动脉中测定。通过将冠状动脉匀浆或培养的冠状动脉平滑肌细胞的裂解物与[腺苷酸-P-32]NAD一起孵育来检查内源性ADP核糖基化。在匀浆和裂解物中观察到四种P-32标记的蛋白质，分子量分别为51、52、80和124 kDa。该反应通过向孵育中加入11，12-环氧二十碳三烯酸（11，12-EET）（细胞色素P450衍生的类二十烷酸）和GTP而增强。通过Western印迹分析，42-和70-kDa的蛋白质被识别的冠状动脉匀浆和平滑肌细胞裂解物中的ADP-核糖基转移酶的特异性抗体，但不是在内皮细胞裂解物。内源性ADP-核糖基化的52-kDa受体蛋白与被霍乱毒素ADP-核糖基化的蛋白质共迁移，并被抗G（S）α抗体识别和免疫沉淀。这些结果表明，G（S）α是ADP-核糖部分的几种受体之一。膜片钳技术显示，11，12-EET可刺激平滑肌细胞K+通道的激活，这种激活可被内源性单ADP核糖基转移酶抑制剂新生霉素、维生素K-1、3-氨基苯甲酰胺和间碘苄胍完全阻断。我们的结论是内源性单ADP核糖基转移酶存在于牛冠状动脉平滑肌。这些酶将ADP-核糖转移到细胞蛋白如G（S）α，并可能介导冠状动脉血管平滑肌中的细胞内信号转导。在冠状循环中，ADP-核糖基化信号通路可能在介导11，12-EET激活K+通道中起重要作用。
The role of endogenous ADP-ribosylation in mediating the activation of the Ca2+-activated K+ channels was determined in bovine coronary arteries. Endogenous ADP-ribosylation was examined by incubating coronary arterial homogenates or lysates of cultured coronary arterial smooth muscle cells with [adenylate-P-32]NAD. Four P-32-labeled proteins were observed at 51, 52, 80, and 124 kDa in the homogenates and lysates. This reaction was enhanced by the addition of 11,12-epoxyeicosatrienoic acid (11,12-EET), a cytochrome P450-derived eicosanoid, and GTP to the incubation. By Western blot analysis, 42- and 70-kDa proteins were recognized by specific antibodies against ADP-ribosyltransferase in the coronary arterial homogenates and smooth muscle cell lysate but not in the lysate of endothelial cells. The 52-kDa acceptor protein of endogenous ADP-ribosylation comigrated with a protein ADP-ribosylated by cholera toxin and was recognized and immunoprecipitated by an anti-G(S)alpha antibody. These results suggest that G(S)alpha is one of several accepters of the ADP-ribose moiety. As shown by the patch-clamp technique, 11,12-EET stimulated the activation of the K+ channels in the smooth muscle cells, and this activation was completely blocked by novobiocin, vitamin K-1, 3-aminobenzamide, and m-iodobenzylguanidine, inhibitors of endogenous mono-ADP-ribosyltransferases. We conclude that endogenous mono-ADP-ribosyltransferases are present in smooth muscle from bovine coronary arteries. These enzymes transfer ADP-ribose to the cellular proteins such as G(S)alpha and may mediate intracellular signal transduction in coronary vascular smooth muscle. In the coronary circulation, the ADP-ribosylation signaling pathway may play an important role in mediating the activation of the K+ channels induced by 11,12-EET.