Effects of ATP on the Intracellular Calcium Level in the Osteoblastic TBR31-2 Cell Line(Biochemistry)

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概要

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• 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 (1μM), uridine 5'-triphosphate (1μM), and ADP (1μM), significantly increased the [Ca^<2+>]_i of TBR31-2 cells in 2-d and 5-d cultures, but a potent P2X receptor agonist, α,β-methylene ATP (10μM), 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.

• 2009-01-01

著者

• YANAI Nobuaki

  Department of Cell Biology, Institute of Development, Aging and Cancer

• Yanai Nobuaki

  武庫川女子大学 薬学部生化学i

• Yanai Nobuaki

  Department Of Food And Nutritional Science Faculty Of Liberal Arts Miyagi Gakuin Women's Colleg

• Yanai Nobuaki

  Department Of Biological Science Tohoku University

• SHINOZUKA Kazumasa

  Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women's University

• NEJIME Namie

  Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's Univers

• Shinozuka Kazumasa

  Department Of Pharmacology School Of Pharmacy And Pharmaceutical Sciences Mukogawa Women's Univ

• Shinozuka Kazumasa

  Dep. Of Pharmacology School Of Pharmacy And Pharmaceutical Sciences Mukogawa Women's Univ. Jpn

• Shinozuka Kazumasa

  Department Of Pharmacology Shizuoka College Of Pharmaceutical Sciences

• Shinozuka Kazumasa

  Department Of Pharmacology Faculty Of Pharmaceutical Science Mukogawa Women's University

• NISHII Naomi

  First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's U

• YAMAUCHI Chisako

  First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's U

• NAKABAYASHI Toshikatsu

  First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's U

• Nishii Naomi

  First Department Of Biochemistry School Of Pharmaceutical Sciences Mukogawa Women's University

• Nakabayashi Toshikatsu

  First Department Of Biochemistry School Of Pharmaceutical Sciences Mukogawa Women's University

• Yamauchi Chisako

  First Department Of Biochemistry School Of Pharmacy And Pharmaceutical Sciences Mukogawa Women'

• Yanai Nobuaki

  Department of Food and Nutritional Science, Faculty of Liberal Arts, Miyagi Gakuin Women's College

• Nejime Namie

  Department of Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University

• NAKABAYASHI Toshikatsu

  First Department of Biochemistry, School of Pharmaceutical Sciences, Mukogawa Women's University

• Shinozuka Kazumasa

  Department of Applied Chemistry, Faculty of Engineering, Yokohama National University

• Nejime Namie

  Department of Pharmacology, School of Pharmaceutical Sciences, Mukogawa Women's University

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