THAPSIGARGIN-RESISTANT INTRACELLULAR CALCIUM PUMPS - ROLE IN CALCIUM POOL FUNCTION AND GROWTH OF THAPSIGARGIN-RESISTANT CELLS

Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 mu M TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of Ca-45(2+) uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 mu M, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 mu M. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (K-m values similar or equal to 0.1 mu M) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP(3)) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP(3)-releasable. GTP induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-SF cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP(3); unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP(3) releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-SF cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP(3) receptors. It is concluded that the ability of TG-resistant DC-3F/TG2 TG2 cells to grow in the presence of 2 mu M TG results from expression of a distinct TG-resistant Ca2+ pump which serves to accumulate Ca2+ within an InsP(3),-releasable Ca2+ pool; this provides further evidence for the essential role of functional Ca2+ pools in progression of cells through the cell cycle and cell division.