H2O2 modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H2O2 and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H2O2 induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H2O2 doses. The H2O2-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H2O2 failed to evoke calcium signals and millimolar H2O2 induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H2O2 up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y2 cells, stably expressing the P2Y2 receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H2O2 induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y2 cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y2 receptor.