P2X7-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells

Grol MW, Zelner I, Dixon SJ. P2X(7)-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells. Am J Physiol Endocrinol Metab 302: E561-E575, 2012. First published December 20, 2011; doi:10.1152/ajpendo.00209.2011.-The P2X(7) receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X(7) function exhibit altered bone formation. Moreover, activation of P2X(7) in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X(7) agonist 2', 3'-O-(4-benzoylbenzoyl) ATP (BzATP; 300 mu M) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X(7) receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 mu M) caused a dramatic increase in proton efflux from MC3T3-E1 cells (similar to 2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 mu M), which activate P2 receptors other than P2X(7), failed to elicit a sustained increase in proton efflux. Specific P2X(7) receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca2+ was required during P2X(7) receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X(7) receptors on osteoblast-like cells triggers a dramatic, Ca2+ -dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.