Separate roles for calcium and magnesium in their synergistic effect on uridine uptake by cultured cells: Significance for growth control

Abstract

The uptake of uridine by BALB/c3T3 cells is markedly inhibited by reducing the concentration of Mg2+ in medium containing only traces of Ca2+. When physiological [Ca2+J is present in the medium, omission of Mge+ has no effect on uridine uptake, and when Mg2+ is present, omission of Ca2+ has only a slight inhibitory effect. When both Ca2+ and Mg2+ are omitted, the concentration of Ca2+ in the cells is not reduced, but that of Mg2+ is reduced to about one-half in 3 hr. The concentration of K+ is also reduced, and that of Na+ is increased, suggesting increased membrane permeability to cations. 14; unpublished observation), a direct role for Mg2+ in regulating uridine uptake became highly plausible and a similar role for Ca2W unlikely. We decided to examine further the respective roles of the two ions in the regulation of uridine uptake because such a study promised to provide a clear-cut distinction between them. The basic strategy was to produce a powerful inhibition of uridine uptake by omitting both Ca2+ and Mg2+ from the medium and to observe the effect of restoring them singly or together. The effects of varying Na+ and K+ were also studied. The studies of uridine uptake were accompanied by measurements of the four major cations within the cells and of the cellular permeability to Lglucose. The results provide strong evidence for the direct role of Mg2+ in regulating uridine uptake and indicate the importance of Ca2+ in maintaining the permeability barrier of the cell. No evidence was found for a regulatory role of either Na+ or K+ in uridine uptake. MATERIALS AND METHODS Cell Culture and Labeling. BALB/c3T3 cells were maintained in modified Eagle's medium with 10% calf serum and labeled with [3H]thymidine as described (8). Except where noted, 60-mm polystyrene tissue culture dishes were used. After cultures had become confluent, they were switched to modified Eagle's medium with 1% serum overnight before use in experiments. In most experiments, cultures were labeled by addition of [3H]uridine (13.3 Ci/mmol, 1 Ci = 3.70 X 1010 Bq) for varying periods in the appropriate experimental medium and washed three times with ice-cold Tris-buffered saline. Acid-soluble material was extracted for 15 min with cold 5% trichloroacetic acid for scintillation counting. In the experiment of Fig. 1, however, cultures were washed five times by dipping in five successive beakers containing ice-cold 150 mM NaCl before extraction in trichloroacetic acid. The cultures were then dissolved in 0.1 M NaOH for subsequent measurement of protein content by the method of Lowry et al. (15) and, in the experiment of Fig. 6, of acid-insoluble radioactivity by scintillation counting.The uptake of L-glucose was measured in the various experimental media by addition of 5 ,uCi of L-[3H]glucose at tracer concentrations (10). After 20-40 min, the cultures were washed five times with Tris-buffered saline and material was extracted in cold 5% trichloroacetic acid for scintillation counting. Correction was made for extracellular label by sub...

Major intracellular cations and growth control: Correspondence among magnesium content, protein synthesis, and the onset of DNA synthesis in BALB/c3T3 cells

“…A similar conclusion was drawn in studying the effects of divalent cations on the uptake of uridine by 3T3 cells. The uptake of uridine is limited by the phosphorylation of uridine and is part of the coordinate response of 3T3 cells to serum (12). Uridine uptake is sharply inhibited by omission of Ca2+ and Mg2+ together, and can be restored to maximal rates by replacing Mg2+, but is unaffected by replacing Ca2+.…”

“…Uridine uptake is sharply inhibited by omission of Ca2+ and Mg2+ together, and can be restored to maximal rates by replacing Mg2+, but is unaffected by replacing Ca2+. One of the major effects of omitting Ca2+ is to increase the exchangeability of Mg2+ between cells and medium (12 (19) through initiating protein synthesis (20) and maintaining the conformation of ribosomes (21). These (24) and in heart muscle in a work jump (25).…”

“…The aggregate of these reactions has been designated the coordinate response (7,8), to distinguish it conceptually from the pleiotypic response (9) because the former embraces the differentiated functions of the cell whereas the latter explicitly excludes them (10). The diverse elements of the coordinate response can be modulated by varying the Mg2+ concentration of the medium, especially in cells made permeable to Mg2+ by withdrawing extracellular Ca2+ (11)(12)(13). We have proposed that Mg2+ plays an important role in mediating the coordinate response through its regulation of transphosphorylation and other Mg2+-dependent reactions (7,8).…”

Mg 2+ as activator of uridine phosphorylation in coordination with other cellular responses to growth factors

“…To achieve a wide range of Mg 2ϩ concentrations in cells, the Ca 2ϩ concentration of the medium was reduced to increase the permeability of the cells to Mg 2ϩ (16,17,27). Severe reduction in the Ca 2ϩ supply by itself decreases the trapping of uridine.…”

“…The activity of uridine kinase can also be assayed in cell-free extracts, where the concentrations of MgATP 2Ϫ and uridine can be controlled. The trapping of uridine, which increases within a few minutes of the application of growth factors to quiescent cells, is tightly linked to the extracellular and intracellular Mg 2ϩ concentrations (15)(16)(17). The increased uptake of uridine is gratuitous for the coordinate response and progression through G1 into S, as indicated by the absence of uridine in any of the media commonly used for cell culture.…”