P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells

Abstract

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca2+i mobilization 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+i mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca2+i mobilization in osteoblastic cells.