CELL CYCLE CONTROL--THE ROLE OF MONOVALENT CATION FLUXES

我们项目的总体目标是确定膜离子的作用 运输系统中的激素调节细胞生长。 我们的方法 包括选择已建立的哺乳动物细胞的遗传突变体 在特定的运输系统或特定的激素 应答 为了制定有效的甄选程序，小组 线必须首先彻底的特点，在其离子 转运活动、细胞内阳离子浓度和激素 增长的要求。 我们对荷尔蒙特别感兴趣 刺激血清饥饿静止（GO）细胞与 钠离子活度+/小时+ 和Cl-/HCO3- 离子交换系统 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（3 T3）细胞，最近在猪肾中 上皮细胞（PK1）细胞。 我们获得了以下小鼠结果 成纤维细胞（3 T3细胞）。 一种人工诱导的细胞内 K+ 浓度（K+）我可以抑制促有丝分裂刺激 静止细胞 在（K+i等于在静止细胞中发现的， 然而，促有丝分裂刺激仅被部分抑制;此外， 在正常细胞外钾离子存在下的生长刺激+ 是 并不总是在（K）的上升之前+）i. 因此，我们放弃了我们的 先前的工作假设，即，早期上升（K+）我是一个 有丝分裂的必要事件。 其次，作为我们研究的一部分， 的K+ 运输在生长控制，我们已经证明了存在的 快速生长的布美他尼敏感性Na +3 T3细胞+，K+得双曲正切值.- 共同运输系统。 当细胞变得静止时， 在用血清刺激后以时间和剂量依赖性方式出现，或 胰岛素;它在生长控制中不起重要作用，然而， 因为完全抑制转运活性并不抑制 有丝分裂原刺激静止细胞。 最后，我们展示了 存在阿米洛利敏感的Na+/小时+ 3 T3细胞中的反向转运蛋白， 受酸负荷刺激，例如，尼日利亚的存在。 用于猪肾 (PK)细胞，SUI亚系PK1 细胞可以被带入一个经典的 通过将亚融合培养物与血清孵育6天来保持静止状态。 静止培养物以大于12小时的滞后重新进入S期， 然后用10%血清再刺激，然后进入有丝分裂。 PK1 细胞含有酸刺激的阿米洛利敏感的Na+，+/小时+ 反向转运蛋白和Cl-/HCO3- 交换器 在HCO3- 自由媒体 Na+/小时+ 反向转运蛋白成为pH控制中的重要因素。 他们 进行质子自杀，当Li+- 将负载的细胞置于低pH培养基中 缺锂+. （N）个

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)