MECHANISMS OF MEMBRANE FUNCTION IN CELL GROWTH

这个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 培养人成纤维细胞并评估其生长的作用 调节有丝分裂的因子刺激的钠流通量。的具体目标 这项研究建议主要是为了证明我们的假设 生长因子通过升高细胞内钙调节钠离子内流 导致钙调素依赖的钠激活的活性 运输途径。我们建议研究细胞内的动员 CA对激活Na内流的处理的响应和研究 两种可能的机制(运输蛋白的磷酸化和 微管的破坏)钙调素对钠通量的调节。这个 血清刺激的钠内流快速失活的机制 将对途径进行调查以确定它是否是生长的结果 因子-受体下调或来自负反馈信号 细胞内钠离子浓度升高。我们将确定是否 Lys-Bradykinin，我们已经证明它是一种有效的钠刺激物 也刺激人成纤维细胞的DNA合成，以及一种 不同药物刺激的钠离子内流与DNA合成的相关性 多肽(EGF、加压素、溶舒缓激肽)的组合可以 演示了。我们将研究质子在钠原子中的作用 内流过程和细胞生长调节。我们将尝试 从中国仓鼠卵巢(CHO)细胞中筛选钠转运突变体 证实人类体内钠转运途径的上调 成纤维细胞在低钠条件下生长。旨在解决这些问题的实验 具体目标包括测量跨细胞膜的离子通量， 使用荧光指示剂测量细胞内钙活度， 生长因子与膜受体结合的测定 用~3H-THD掺入的DNA合成和蛋白质的测量 培养的人成纤维细胞中的磷酸化。此外，“氚” “自杀”技术将被用于筛选运输阴性突变体 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.