Journal of Biological Chemistry
Volume 277, Issue 50, 13 December 2002, Pages 48724-48729
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MECHANISMS OF SIGNAL TRANSDUCTION
P2Y Purinoceptors Are Responsible for Oscillatory Fluid Flow-induced Intracellular Calcium Mobilization in Osteoblastic Cells*

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We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca2+imobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca2+i mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca2+]i in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5′ nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca2+]i in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca2+]i, suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca2+]i. Furthermore, adenosine, ADP, UDP, or adenosine 5′-O-(3-thiotriphosphate) did not induce Ca2+i mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4′-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca2+i response, whereas pertussis toxin, a Gi/o-protein inhibitor, inhibited fluid flow-induced increases in [Ca2+]i in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca2+i response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca2+]i, and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca2+imobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca2+i mobilization in osteoblastic cells.

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Published, JBC Papers in Press, October 9, 2002, DOI 10.1074/jbc.M209245200

*

This work was supported by National Institutes of Health Grants AG13087 (to H. J. D.) and AR49129 (to J. Y.) and United States Army Medical Research and Materiel Command Award DAMD 17-98-1-8509 (to H. J. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.