PROSTAGLANDINS, THE KIDNEY AND HYPERTENSION

我们提出了研究肾小球系膜细胞的实验，在 培养，以评估膜受体和信号 血管紧张剂、内皮素激活的信号转导通路 (ET)和血栓素A2(TXA2)。我们将对受体进行量化 数量、亲和力和特异性及其相关受体 细胞对这些配体的反应的特性。我们会 也评估GTP结合蛋白在连接受体中的作用 对质膜酶，如腺苷环化酶和 磷脂酶A、C和D。将测量G蛋白的作用 在完整细胞和使用稳定GTP的细胞膜中 类似物。磷脂酶D介导细胞内信号转导的能力 将研究对ET和TXA2的反应以及可能的情况 此外，磷脂酶C还将使用磷脂底物 到多聚磷脂酰肌醇。在随后的研究中，我们将评估 这些信号转导通路在调节 收缩和增殖的细胞反应。具体来说， 我们将尝试模仿细胞的增殖和收缩行为 ET和TXA2通过细胞内肌醇磷酸酶的变化， (Ca~(2+))i·phi和蛋白激酶C活性。我们还将尝试 使磷脂酶C的激活与膜受体分离 通过EJ-ras基因转染系膜细胞。这些 操纵将允许解剖相对重要的 这些信号转导事件可以单独发生，也可以组合在一起 以调节系膜的收缩和增殖。最后，我们 将寻找系膜和血管顺畅的遗传缺陷 遗传性高血压菌株中的肌肉细胞。我们建议 磷脂酶C信号通路的高反应性可能 介导血管收缩增强和肾小球减少 过滤。这一理论将通过培养的血管进行验证。 平滑肌和系膜细胞不仅评估了 它们对收缩的收缩和增殖反应 但也比较了磷脂酶C的激活与 测定(Ca~(2+))i、p~(2+)、肌醇磷酸盐和 蛋白激酶C的刺激作用。

We propose experiments to study glomerular mesangial cells, in culture, in order to evaluate membrane receptors and signal transduction pathways activated by the vasoconstrictors, endothelin (ET) and thromboxane A2 (TxA2). We will quantitate receptor number, affinity and specificity and correlate receptor characteristics with cellular responses to these ligands. We will also evaluate the role of GTP binding proteins to link receptors to plasma membrane enzymes such as adenylate cyclase and phospholipases A, C and D. The role of G proteins will be measured in both intact cells and with cell membranes using stable GTP analogues. The capacity of phospholipase D to mediate cellular responses to ET and TxA2 will be studied as will the possibility that phospholipase C will use phospholipid substrates in addition to polyphosphoinositides. In subsequent studies, we will evaluate the importance of these signal transduction pathways to mediate the cellular responses of contraction and proliferation. Specifically, we will attempt to mimic proliferative and contractile actions of ET and TxA2 through changes of cellular inositol phosphates, (Ca2+)i pHi and protein kinase C activity. We will also attempt to dissociate phospholipase C activation from the membrane receptor through EJ-ras transfection of the mesangial cells. These manipulations will allow dissection of the relative importance of these signal transduction events either singly or in combination to mediate mesangial contraction and proliferation. Finally, we will search for a genetic defect of mesangial and vascular smooth muscle cells in genetically hypertensive strains. We propose that hyperresponsiveness of the phospholipase C signalling pathways may mediate enhanced vasoconstriction and reduced glomerular filtration. This theory will be tested using cultured vascular smooth muscle and mesangial cells with an assessment not only of their contractile and proliferative responses to contractile agonists but also a comparison of phospholipase C activation with measurements of (Ca2+)i, pHi, release of inositol phosphates and stimulation of protein kinase C.