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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

基本信息

批准号：
3172160
负责人：
EDWARD A. ADELBERG
金额：
$ 10.08万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/3172160
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

项目摘要

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定义膜离子的作用细胞生长激素调节中的运输系统。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运输系统或特定激素中改变的线路回应。为了设计有效的选择程序，细胞首先必须根据离子线彻底表征运输活动、细胞内阳离子浓度和激素成长的要求。我们对荷尔蒙特别感兴趣刺激血清饥饿的静止细胞（G O ）与 Na + /H + 和 Cl - /HCO 3 - 离子交换系统的活性。在过去的九个月里，我们一直在分析上述参数瑞士小鼠成纤维细胞 (3T3) 细胞以及最近的猪肾细胞上皮（PK 1 ）细胞。我们得到了以下小鼠结果成纤维细胞（3T3 细胞）。人为诱导的细胞内减少 K + 浓度 (K + )i 可抑制有丝分裂刺激静止细胞。当 (K + )i 等于静止细胞中的值时，然而，有丝分裂刺激仅被部分抑制；此外，在正常细胞外 K + 存在的情况下刺激生长是并不总是先于 (K + )i 的上升。因此，我们放弃了我们的先前的工作假设，即 (K + )i 的早期上升是有丝分裂发生中的必要事件。其次，作为我们角色研究的一部分 K + 运输在生长控制中的作用，我们已经证明了布美他尼敏感的 Na + 、K + 、Cl - 快速生长的 3T3 细胞协同运输系统。当细胞变得静止时，这个系统就会消失在用血清或刺激后以时间和剂量依赖性方式出现胰岛素;然而，它在生长控制中并不发挥重要作用，因为运输活动的完全抑制不会抑制静止细胞的有丝分裂原刺激。最后，我们展示了 3T3 细胞中存在阿米洛利敏感的 Na + /H + 逆向转运蛋白；它受到酸负荷的刺激，例如尼日利亚菌素的存在。猪肾用 (PK)细胞，SUI亚系PK 1细胞可带入经典通过将亚汇合培养物与血清一起孵育 6 天来达到静止状态。静止培养物重新进入 S 期，滞后时间大于 12 小时， 10%血清再刺激后进入有丝分裂。 PK 1 细胞含有酸刺激的、阿米洛利敏感的 Na + /H + 反向转运蛋白和 Cl - /HCO 3 - 交换器。在 HCO 3 - 自由培养基中， Na + /H + 逆向转运蛋白成为 pH 控制的重要因素。他们当Li + 负载细胞置于低pH 介质中时，会发生质子自杀缺少Li + 。 (N)