Effects of ATP on the intracellular calcium level in the osteoblastic TBR31-2 cell line

We investigated the effects of extracellular ATP on TBR31-2 cells established from the bone marrow of transgenic mice harboring the temperature-sensitive simian virus (SV) 40 T-antigen gene. These cells showed the capacity to differentiate toward osteoblasts and could be enhanced by bone morphogenetic protein (BMP)-2, an inducer of osteoblasts. The intracellular calcium ion level ([Ca(2+)](i)) in differentiating TBR31-2 cells was measured by fluorescence confocal microscopic imaging using the Ca(2+)-sensitive probe, Calcium Green 1/AM. P2 receptor agonists, such as ATP (1 microM), uridine 5'-triphosphate (1 microM), and ADP (1 microM), significantly increased the [Ca(2+)](i) of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, alpha,beta-methylene ATP (10 microM), did not increase [Ca(2+)](i). The increase in [Ca(2+)](i) induced by ATP in the 2-d culture tended to be higher than in the 5-d culture. The increase in [Ca(2+)](i) of both cultures was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2 receptor antagonist. However, in an external Ca(2+)-free condition ATP-induced increase in [Ca(2+)](i) was unchanged at either stage. U73122, phospholipase C inhibitor and Thapsigargin, a calcium-pump inhibitor, significantly inhibited the increase in [Ca(2+)](i) at both stages. Reverse transcription-polymerase chain reaction analysis showed that the expression of P2Y receptor mRNA was higher in the 2-d culture than in the 5-d culture. These results indicate that ATP induces the increase in [Ca(2+)](i) from the calcium store through activating P2Y receptors in TBR31-2 cells and that the 2-d culture can respond to ATP more than the 5-d culture due to the higher expression of P2Y receptors. This suggests that the physiological role of ATP in osteoblasts is altered during differentiation.