title: Effects of ATP on the intracellular calcium level in the osteoblastic TBR31-2 cell line.
abstract: 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.
authors.affiliation: First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Hyogo, Japan.
authors.forename: Naomi
authors.initials: N
authors.lastname: Nishii
authors.forename: Namie
authors.initials: N
authors.lastname: Nejime
authors.forename: Chisako
authors.initials: C
authors.lastname: Yamauchi
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Yanai
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Shinozuka
authors.forename: Toshikatsu
authors.initials: T
authors.lastname: Nakabayashi
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D000880:Anthraquinones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002461:Cell Line, Transformed; D004187:Disaccharides / Q000378:metabolism; D045604:Extracellular Fluid / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects*; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D013997:Time Factors; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D000880:Anthraquinones; D004187:Disaccharides; C546383:P2ry1 protein, mouse; C546386:P2ry2 protein, mouse; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; C064333:ruberythric acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium; D014544:Uridine Triphosphate