Tillery Lakeisha C, Epperson Tenille A, Eguchi Satoru, Motley Evangeline D
Department of Microbiology and Immunology, Meharry Medical College, Nashville, TN 37208, USA.
Department of Physiology, School of Medicine, Meharry Medical College, Nashville, TN 37208, USA.
Exp Biol Med (Maywood). 2016 Mar;241(6):569-80. doi: 10.1177/1535370215622584. Epub 2016 Jan 4.
Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.
蛋白酶激活受体已被证明可通过对该酶特定位点的磷酸化来调节内皮型一氧化氮合酶。已证实PAR-2激活会使eNOS-Ser-1177磷酸化，并导致强效血管舒张剂一氧化氮的产生，而PAR-1激活会使eNOS-Thr-495磷酸化，并减少人脐静脉内皮细胞中一氧化氮的产生。在本研究中，我们假设蛋白酶激活受体与调节原代成人冠状动脉内皮细胞中内皮型一氧化氮合酶和一氧化氮产生的信号通路存在差异偶联。通过蛋白质免疫印迹分析，我们发现凝血酶和PAR-1激活肽TFLLR会导致人冠状动脉内皮细胞中eNOS-Ser-1177磷酸化，而PAR-1抑制剂SCH-79797（SCH）可阻断这种磷酸化。使用硝酸盐/亚硝酸盐测定法，我们还证明了凝血酶和TFLLR诱导的一氧化氮产生受到SCH和NOS抑制剂L-NAME的抑制。此外，我们观察到，与凝血酶不同，TFLLR会使eNOS-Thr-495显著磷酸化，这可能解释了与凝血酶相比观察到的一氧化氮产生延迟现象。PAR-2特异性配体SLIGRL激活PAR-2会导致两个催化位点的双重磷酸化，但主要调节eNOS-Thr-495磷酸化，而人冠状动脉内皮细胞中一氧化氮的产生没有变化。PAR-3被称为无信号传导受体，被PAR-3模拟肽TFRGAP激活，并显著诱导eNOS-Thr-495磷酸化，而eNOS-Ser-1177磷酸化极少，一氧化氮产生没有变化。此外，我们使用钙螯合剂BAPTA证实PAR介导的eNOS-Ser-1177磷酸化是钙依赖性的，而使用Rho激酶抑制剂Y-27632证实eNOS-Thr-495磷酸化是通过Rho激酶介导的，这表明蛋白酶激活受体分别与Gq和G12/13偶联。这些数据表明蛋白酶激活受体与调节内皮型一氧化氮合酶和一氧化氮产生的信号通路存在血管床特异性差异偶联，这可能是与心血管疾病相关的内皮功能障碍的原因。
