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Insulin was employed as a stimulant in our continuing investigations of the molecular mechanisms involved in the coordinate control of cellular metabolism and growth. Incubation of chicken embryo fibroblasts for 16 hours in media containing 0-0.1 U insulin/ml resulted in a 17-fold increase in the rate of 3H-thymidine incorporation into DNA. Concomitantly, there were graded increases in intracellular K+ (14%) AND Mg2+ (22%) and no significant change in Ca2+. These changes in cation content occurred within 10 to 30 minutes and preceded the changes in 3H-thymidine incorporation. Insulin produced a consistent graded decrease in externally bound Mg2+ and Ca2+ and a concomitant increase in bound Na+ and K+ with no significant change in the rates of K+ and Mg2+ efflux. The results are consistent with the concept of Mg2+ as a second messenger for insulin action, as well as with the more general hypothesis that Mg2+ is the centtral agent in the coordinate control of metabolism and growth in animal cells.

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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of K+ is also reduced, and that of Na+ is increased, suggesting increased membrane permeability to cations. 14; unpublished observation), a direct role for Mg2+ in regulating uridine uptake became highly plausible and a similar role for Ca2W unlikely.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...

Omission of Ca2+ from the medium of confluent BALB/c3T3 cells for a period of 17 hr causes a large de-