Cell density-dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem 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 P2Y2 receptors. RT-PCR and immunohistochemical studies also showed the expression of P2Y2 receptors. [Ca2+]i response to UTP changed with cell density. The UTP-induced rise in [Ca2+]i was greatest at high density. Vmax (maximum Ca2+ response) and EC50 (agonist concentration that evokes 50% of Vmax) suggest that the amount and property of P2Y2 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 P2Y2 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. J. Cell. Physiol. 219: 372–381, 2009. © 2009 Wiley-Liss, Inc.