Cell density‐dependent changes in intracellular Ca 2+ mobilization via the P2Y 2 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 Ca 2+ signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca 2+ levels ([Ca 2+ ] i ). The order of potency followed ATP = UTP > ADP = UDP. ATP‐induced rise in [Ca 2+ ] 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. [Ca 2+ ] i response to UTP changed with cell density. The UTP‐induced rise in [Ca 2+ ] i was greatest at high density. V max (maximum Ca 2+ response) and EC 50 (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 Ca 2+ oscillation at only medium cell density. Pharmacological studies indicated that UTP‐induced Ca 2+ oscillation required Ca 2+ influx by store‐operated Ca 2+ entry. Carbenoxolone, a gap junction blocker, enhanced Ca 2+ 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 Ca 2+ oscillation at high cell density. These results suggest that the different patterns observed for [Ca 2+ ] 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.