Complexes of inorganic pyrophosphate, orthophosphate, and calcium as stimulants of 3T3 cell multiplication

Abstract

Addition of 0.1-0.5 mM sodium PPi [inorganic pyrophosphate] for 17 h to confluent cultures of [mouse] BALB/c 3T3 cells in low serum concentrations stimulated the incorporation of [3H]thymidine into DNA to an extent equal to that produced by high serum concentration. PPi prevented much but not all of the cell detachment that accompanies decreasing the serum concentration of confluent cultures and it increased the saturation density of cultures in high serum concentrations. The stimulation had a sharp concentration depdendence and was associated with the appearance in the medium of a flocculent precipitate. Stimulation and precipitate formation were dependent on Ca2+ and inorganic orthophosphate (HPO42-) and were inhibited by Mg2+. More than half the Ca2+ requirement could be met with Sr2+. In the absence of PPi, supranormal concentrations of either Ca2+ or HPO42- caused graded increased in [3H]thymidine incorporation and total cell yield. The effect of supranormal [Ca2+] depended on [HPO42-] and vice versa, and the Ca2+ requirement could be partially met by Sr2+. The stimulation was associated with increasing turbidity of the medium. Various other complexing agents of Ca2+, including the divalent cation inonophore A 23187, failed to produce stimulation of 3T3 cells. The water insoluble complexes of PPi, HPO42- and Ca2+ or, at much higher concentrations, the latter 2 together, stimulate 3T3 cells and this is probably brought about by the association of these complexes with the cell membrane.