title: 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 Ca(2+)(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 Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(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 Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](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 Ca(2+)(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 [Ca(2+)](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 Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells.
authors.affiliation: Musculoskeletal Research Laboratory, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey 17033, USA. jxy118@psu.edu
authors.forename: Jun
authors.initials: J
authors.lastname: You
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Jacobs
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Steinberg
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Donahue
mesh_terms: D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D051379:Mice; D016376:Oligonucleotides, Antisense; D010006:Osteoblasts / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D016376:Oligonucleotides, Antisense; C546386:P2ry2 protein, mouse; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate