Cell Density-Dependent Changes in Intracellular Ca2+ Mobilization via the P2Y2 Receptor in Rat Bone Marrow Stromal Cells

We investigated intracellular Ca2+ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca2+ levels ([Ca2+](i)). The order of potency followed ATP = UTP > ADP = UDP. ATP-induced rise in [Ca2+](i) was suppressed by U73122 and suramin, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suggesting the functional expression of G protein-coupled P2Y(2) receptors. RT-PCR and immunohistochemical studies also showed the expression of P2Y(2) receptors. [Ca2+](i) response to UTP changed with cell density. The UTP-induced rise in [Ca2+](i) was greatest at high density. V-max (maximum Ca2+ response) and EC50 (agonist concentration that evokes 50% of V-max) suggest that the amount and property of P2Y(2) receptors were changed by cell density. Note that UTP induced Ca2+ oscillation at only medium cell density. Pharmacological studies indicated that UTP-induced Ca2+ oscillation required Ca2+ influx by store-operated Ca2+ entry. Carbenoxolone, a gap junction blocker, enhanced Ca2+ oscillation. Immunohistochemical and quantitative real-time PCR Studies revealed that proliferating cell nuclear antigen (PCNA)-positive cells declined but the mRNA expression level of the P2Y(2) receptor increased as cell density increased. Co-application of fetal calf serum with UTP induced Ca2+ oscillation at high cell density. These results suggest that the different patterns observed for [Ca2+](i) mobilization with respect to cell density may be associated with cell cycle progression.