MECHANISMS OF MEMBRANE FUNCTION IN CELL GROWTH

本项目的长期目标是阐明 生长因子激活阿米洛利敏感的钠转运途径， 培养的人成纤维细胞，并评估生长的作用， 因子刺激的钠流调节有丝分裂。 的具体目标 这项研究计划主要是为了证明我们的假设 生长因子通过升高细胞内Ca ~（2+）调节Na ~+内流 活性，导致钙调素依赖性激活的Na 运输途径 我们建议研究细胞内 Ca对激活Na内流的治疗的反应，并研究 两种可能的机制（转运蛋白的磷酸化和 微管的破坏）用于钙调素调节Na通量。 的 血清刺激的Na内流快速失活的机制 途径将进行调查，以确定它是否导致生长 因子受体下调或来自负反馈信号， 细胞内钠浓度升高。 我们将决定 lys-bradykinin，我们已经证明是一个有效的刺激钠 内流，也刺激人成纤维细胞的DNA合成， Na内流与DNA合成的相关性 肽（EGF、血管加压素、赖氨缓激肽）的组合可 演示。 我们将研究质子参与Na 流入过程和细胞生长调节。 将试图 从中国人卵巢（CHO）细胞中选择Na转运突变体， 证明了在人类中Na转运途径的上调 成纤维细胞在低Na培养基中生长。 旨在解决这些问题的实验 具体目的包括测量穿过细胞膜的离子通量， 使用荧光指示剂测量细胞内Ca活性， 生长因子与膜受体结合的测定 使用3 H-Thd掺入的DNA合成和蛋白质的测量 在培养的人成纤维细胞中的磷酸化。 此外，“氚 自杀”技术将用于选择转运阴性突变体 在CHO细胞中。

The long term goals of this project are to elucidate the mechanism by which growth factors activate the amiloride-sensitive Na transport pathway in cultured human fibro-blasts and to assess the role of the growth factor-stimulated Na flux in regulating mitogenesis. The specific aims of this research proposal are directed principally attesting our hypothesis that growth factors regulate Na influx by elevating intracellular Ca activity which results in a calmodulin-dependent activation of the Na transport pathway. We propose to study the mobilization of intracellular Ca in response to treatments which activate Na influx and to investigate two possible mechanisms (phosphorylation of the transport protein and disruption of microtubules) for calmodulin regulation of Na flux. The mechanism for the rapid inactivation of the serums-stimulated Na influx pathway will be investigated to determine whether it results from growth factor-receptor down regulation or from negative feedback signals due to elevated intracellular Na concentration. We will determine whether lys-bradykinin, which we have demonstrated to be a potent stimulator of Na influx, also stimulates DNA synthesis in human fibroblasts and whether a correlation between Na influx and DNA synthesis stimulated by various combinations of peptides (EGF, vasopressin, lysbradykinin) can be demonstrated. We will investigate the involvement of protons in the Na influx process and in cell growth regulation. Attempts will be made to select Na transport mutants from Chinese Hamster Ovary (CHO) cells and to demonstrate an up-regulation of the Na transport pathway in human fibroblasts grown in low Na medium. Experiments designed to address these specific aims involve measurement of ion fluxes across cellular membranes, measurement of intracellular Ca activity using a fluorescence indicator, measurement of binding of growth factors to membrane receptors, measurement of DNA synthesis using 3H-Thd incorporation, and measurement of protein phosphorylation in cultured human fibroblasts. In addition, the "tritium suicide" technique will be used for selection of transport negative mutants in CHO cells.