PROSTAGLANDINS--THE KIDNEY AND HYPERTENSION

我们建议实验研究肾小球系膜细胞， 培养，以评估膜受体和信号 由血管收缩剂内皮素激活的转导通路 (ET)和血栓素A2（TxA 2）。 我们将定量受体 数量、亲和力和特异性以及相关受体 这些特征与细胞对这些配体的反应有关。 我们将 还评估GTP结合蛋白连接受体的作用 质膜酶如腺苷酸环化酶和 磷脂酶A、C和D。 G蛋白的作用将被测量 在完整细胞和细胞膜中使用稳定的GTP 类似物 磷脂酶D介导细胞凋亡的能力 将研究对ET和TxA 2的反应， 磷脂酶C将使用磷脂底物， 到聚磷酸肌醇。 在接下来的研究中，我们将评估 这些信号转导通路的重要性，以介导 收缩和增殖的细胞反应。 具体地说， 我们将尝试模拟 ET和TxA 2通过改变细胞磷酸肌醇， (Ca2+）i pHi和蛋白激酶C活性。 我们还将尝试 使磷脂酶C活化与膜受体分离 通过EJ-ras转染系膜细胞。 这些 操作将允许解剖的相对重要性， 这些信号转导事件或者单独地或者组合地 介导系膜收缩和增殖。 最后我们 将寻找系膜和血管平滑的遗传缺陷 肌肉细胞的遗传性高血压菌株。 我们建议 磷脂酶C信号通路的高反应性可能 介导增强血管收缩和减少肾小球 过滤 这一理论将用培养的血管 平滑肌和系膜细胞，不仅评估 它们的收缩和增殖反应， 激动剂，但也比较磷脂酶C激活与 测量（Ca 2+）i、pHi、肌醇磷酸的释放和 刺激蛋白激酶C。

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.