CELL CYCLE CONTROL--THE ROLE OF MONOVALENT CATION FLUXES

细胞周期控制——单价阳离子通量的作用

• 批准号: 3172159
• 负责人: EDWARD A. ADELBERG
• 金额: $ 9.71万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-01-01 至 1986-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3172159
• 关键词: autoradiography; cations; cell cycle; cell growth regulation; cyclic AMP; ion transport; methane sulfonate; mutagen testing; platelet derived growth factor; scintillation counter; tissue /cell culture; vasopressins

The general goal of our project is to define the role of membrane ion transport systems in the hormonal regulation of cell growth. Our approach involves the selection of genetic mutants of established mammalian-cell lines altered in specific transport systems or in specific hormonal responses. In order to devise effective selection procedures, the cell lines must first be thoroughly characterized in terms of their ion transport activities, intracellular cation concentrations, and hormonal requirements for growth. We are particularly interested in the hormonal stimulation of serum-starved quiescent (GO) cells in relation to the activity of the Na+/H+ and Cl-/HCO3- ion exchange systems. During the past nine months, we have been analyzing the above parameters in Swiss mouse fibroblasts (3T3) cells and, more recently, in pig kidney epithelial (PK1) cells. We have obtained the following results for mouse fibroblasts (3T3 cells). An artificially induced decrease in intracellular K+ concentration (K+)i can inhibit the mitogenic stimulation of quiescent cells. At a (K+)i equal to that found in quiescent cells, however, mitogenic stimulation is only partially inhibited; furthermore, the stimulation of growth in the presence of normal extracellular K+ is not always preceded by a rise in (K+)i. Therefore, we have discarded our previous working hypothesis, namely, that an early rise in (K+)i is a necessary event in mitogenesis. Secondly, as part of our study on the role of K+ transport in growth control, we have demonstrated the presence of rapidly-growing 3T3 cells of a bumetanide-sensitive Na+, K+, Cl- co-transport system. This system disappears as cells become quiescent and appears in a time-\and dose-dependent manner upon stimulation with serum or insulin; it does not play an essential role in growth control, however, since the complete inhibition of transport activity does not inhibit mitogen-stimulation of quiescent cells. Finally, we have demonstrated the presence of an amiloride-sensitive Na+/H+ antiporter in 3T3 cells; it is stimulated by acid-loading, e.g., presence of nigericin. For pig kidney (PK) cells, SUI sub-line PK1 cells can be brought into a classical quiescent state by incubating subconfluent cultures with serum for 6 days. Quiescent cultures reenter S-phase with a lag of greater than 12 hrs, following restimulation by 10% serum and enter mitosis thereafter. PK1 cells contain an acid-stimulatable, amiloride-sensitive Na+/H+ antiporter and a Cl-/HCO3- exchanger. In HCO3- free media, the Na+/H+ antiporter becomes a significant factor in pH control. They undergo proton suicide, when Li+-loaded cells are placed in low-pH medium lacking Li+. (N)

我们项目的总体目标是确定膜离子的作用。 荷尔蒙调节细胞生长的运输系统。我们的方法 涉及已建立的哺乳动物细胞的遗传突变体的选择 在特定的运输系统或特定的激素中改变的线条 回应。为了制定有效的选择程序，该细胞 线路必须首先根据它们的离子进行彻底的表征 转运活性、细胞内阳离子浓度和激素 对增长的要求。我们对荷尔蒙特别感兴趣 血清饥饿静止期(GO)细胞的刺激与 Na+/H+和Cl-/HCO3-离子交换体系的活性。 在过去九个月，我们一直在分析上述参数 瑞士小鼠成纤维细胞(3T3)细胞以及最近在猪肾中发现的 上皮性(PK1)细胞。对于小鼠，我们得到了以下结果 成纤维细胞(3T3细胞)。一种人工诱导的细胞内下降 K+浓度(K+)i可抑制细胞有丝分裂刺激 静止的细胞。当(K+)i等于在静止单元中发现的值时， 然而，有丝分裂刺激只被部分抑制；此外， 在正常细胞外K+存在时对生长的刺激 并不总是在(K+)i上升之前。因此，我们已经放弃了我们的 先前的工作假设，即(K+)i的早期上升是一种 有丝分裂过程中的必要事件。第二，作为我们对角色研究的一部分 关于K+转运在生长控制中的作用，我们已经证明了 对钠、钾、氯敏感的布美他尼快速生长的3T3细胞 联运系统。当细胞静止时，这个系统就消失了 以时间和剂量依赖的方式出现在用血清或 胰岛素；然而，它在生长控制中并不起关键作用， 因为运输活动的完全抑制并不会抑制 有丝分裂原-刺激静止细胞。最后，我们演示了 3T3细胞对阿米洛利敏感的Na+/H+逆向转运蛋白的存在 由酸负荷刺激，例如，黑素的存在。对于猪肾来说 (PK)细胞，SUI亚系可将PK1细胞带入经典 用含血清的亚融合培养物孵育6天，使其处于静止状态。 静止的文化重新进入S阶段，滞后超过12小时， 再用10%血清刺激后进入有丝分裂。PK1 细胞含有酸刺激、阿米洛利敏感的Na+/H+ 反向转运体和Cl-/HCO3-交换剂。在无HCO3的介质中， Na+/H+逆向转运蛋白成为控制pH的一个重要因素。他们 当负载Li+的细胞被置于低pH介质中时，发生质子自杀 缺乏Li+。(N)