CELL CYCLE CONTROL--THE ROLE OF MONOVALENT CATION FLUXES

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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)