Early manifestations of vitamin D effects in rat osteogenic sarcoma cells

Summary

We have recently demonstrated that 48 hour exposure of ROS 17/2 cells to low concentrations of 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) (1.0 pg/ml) stimulated the cellular accumulation of⁴⁵Ca, and exposure to high concentrations (160 pg/ml) inhibited such accumulation. In the present study, short-term (15 min) effects of the sterol on⁴⁵Ca accumulation in ROS 17/2 cells were compared with the long-term (48 hours) effects in order to clarify mechanisms responsible for 1,25(OH)₂D₃ control of calcium metabolisms in ROS 17/2 cells. ROS 17/2 cells were grown for 48 hours in the presence and absence of 1,25(OH)₂D₃ and then incubated for an additional 15 min in the presence and absence of 1,25(OH)₂D₃ immediately before measuring⁴⁵Ca accumulation. Cellular⁴⁵Ca was measured after incubating the cells in the medium containing 0.5 μCi/ml of⁴⁵CaCl₂ for 4 min at 25°C. The effect of actinomycin D was determined by preincubating the cells in 0.1 μg/ml of actinomycin D for 45 min at 25°C. Exposure to low concentrations (1.0 pg/ml) of 1,25(OH)₂D₃ for either 48 hours or 15 min increased⁴⁵Ca in the cells by 10–20%. An additional 15 min exposure following 48 hour exposure yielded an increase in the cellular⁴⁵Ca similar to that after 48 hours or 15 min exposure. Exposure to high concentrations (160 pg/ml) for either 48 hour or 15 min decreased cell⁴⁵Ca by approximately 20%.An additional 15 min exposure to the high concentrations did not change the 48 hour effect. Actinomycin D reversed early inhibitory effects of high concentrations, but had no effect on the early stimulatory effects of low concentrations. These results suggest that the mechanisms underlying the 15 min and 48 hour effects of 1,25(OH)₂D₃ are the same. Moreover, the mechanism responsible for the inhibitory effect of high concentrations is dependent onde novo protein synthesis whereas that for the stimulatory effects of low concentrations is not.