Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells
Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells
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DOI：
10.1161/circulationaha.108.764571
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发表时间：
2009-02-17
期刊：
影响因子：
37.8
通讯作者：
Kaczmarek, Elzbieta
中科院分区：
文献类型：
--
作者：
da Silva, Cleide Goncalves;Specht, Anke;Kaczmarek, Elzbieta
背景-内皮型一氧化氮合酶（eNOS）活性和NO生成的降低是许多疾病（包括糖尿病）中观察到的内皮功能障碍和血管并发症的关键因素。细胞外核苷酸激活eNOS和增加NO的产生，然而，这种观察的机制是不完全cleared.Methods和结果阐明的信号通路（S）导致核苷酸介导的eNOS磷酸化在Ser-1177，人脐静脉内皮细胞处理与几种核苷酸，包括ATP，UTP，和ADP，在存在或不存在的选择性抑制剂。这些实验鉴定了P2 Y1、P2 Y2和可能的P2 Y 4作为参与eNOS磷酸化的嘌呤能受体，并证明该过程是腺苷非依赖性的。核苷酸诱导的eNOS磷酸化和活性被BAPTA-AM（细胞内游离钙螯合剂）、rottlerin（蛋白激酶C δ抑制剂）和蛋白激酶C δ siRNA抑制。相反，阻断AMP活化蛋白激酶、钙/钙调蛋白依赖性激酶II、钙/钙调蛋白依赖性激酶激酶、丝氨酸/苏氨酸蛋白激酶B、蛋白激酶A、细胞外信号调节激酶1/2、和p38丝裂原活化蛋白激酶不影响核苷酸介导的eNOS磷酸化。介导的eNOS磷酸化是钙和蛋白激酶C δ依赖性的。这种新发现的信号通路为治疗内皮功能障碍开辟了新的治疗途径。（循环。2009;119：871-879）。
Background-Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified.Methods and Results-To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase C delta inhibitor), and protein kinase C delta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation.Conclusions-The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase C delta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction. (Circulation. 2009;119:871-879.)