Cellular and molecular biological study on the pathophysiological roles of receptors for lipid mediators on atharosclerosis

1)巨核细胞系脂质介质受体表达调控机制；MEG-01、血管平滑肌细胞、成纤维细胞和肾小球系膜细胞——我们发现血栓素（TX）A_2和前列腺素（PG）I_2受体的表达受蛋白激酶C的作用调节，并被它们自身的激动剂同源脱敏。在肾小球系膜细胞中，血小板活化因子（PAF）受体的表达受脂多糖（LPS）和cAMP的调控。从技术上讲，我们开发了一种新的RT-PCR系统，用于定量微量样品中的mRNA（参考文献5和11）。2)信号转导机制——我们对PGF_2 α受体进行了表征，并将其偶联的gtp结合蛋白鉴定为Gq（参考文献6）。我们还发现PGF_2 α受体介导的细胞组分酪氨酸磷酸化以细胞内Ca^<2+>依赖的方式发生，并且这种酪氨酸磷酸化可能在NIH-3T3细胞的有丝分裂发生中发挥生理作用。最近，我们进一步发现PGF_2 α受体介导的有丝分裂原活化蛋白（MAP）激酶的活化。3)串扰机制——我们分析了MEG-01细胞PGI_2和TXA_2受体信号通路之间的串扰机制。目前，我们正试图利用分子生物学技术开发针对脂质介质受体的特异性抗体，以进一步分析这些现象的分子机制。4)受体拮抗剂对脂质介质的作用——PAF受体拮抗剂（WEB2086）和TXA_2合成酶抑制剂（OKY046）的试验目前正在高胆固醇血症的动脉粥样硬化兔模型上进行试验。5)克隆——我们从豚鼠猪肺cDNA文库中克隆了多种gtp结合蛋白亚型（参考文献1）。

1)Mechanisms of regulation of expression of lipid mediator receptors in megakaryocytic cell line ; MEG-01, vascular smooth muscle cells, fibroblasts and glomerular mesangial cells--- We found that expression of thromboxane (TX)A_2 andprostaglandin (PG)I_2 receptors were regulated by the action of protein kinase C and homologously desensitized by their own agonist in MEG-01 cells. It was also found that platelet-activating factor (PAF) receptor expression was regulated by lipopolysacharide (LPS) and cAMP in glomerular mesangial cells. Technically, we developed a new RT-PCR system for quantitation of mRNA in minute samples (ref.5 and 11).2)Sinal transduction mechanisms--- We characterized the PGF_2 alpha receptor andidentified its coupled GTP-binding protein as Gq (ref.6). We also disclosed PGF_2 alpha receptor-mediated tyrosine phosphrylation of celular components in intracellular Ca^<2+>-dependent manner and that this tyrosine phosphorylation may play a physiological role in mitogenesis in NIH-3T3 cells. Recently, we further found PGF_2 alpha receptor-mediated activation of mitogen-activated protein (MAP) kinases.3)Mechanisms of Cross-talk--- We analyzed the mechanisms of cross-talks between signalling pathways of PGI_2 and TXA_2 receptors in MEG-01 cells. We are now trying to develop specific antibodies against lipid mediator receptors using molecular biological techniques for further analysis of molecular mechanisms of these phenomena.4)Effects of receptor antagonists for lipid mediator--- Trial of PAF receptor antagonist (WEB2086) and TXA_2 synthetase inhibitor (OKY046) are now being tested on a hyperchlesterolemic rabbit model for atherosclerosis.5)Cloning--- We have cloned various subtypes of GTP-binding proteins from aguinea piglung cDNA library (ref.1).