title: P2X₇-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells.
abstract: The P2X₇ receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X₇ function exhibit altered bone formation. Moreover, activation of P2X₇ 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 P2X₇ agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X₇ receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X₇, failed to elicit a sustained increase in proton efflux. Specific P2X₇ receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²⁺ was required during P2X₇ 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 P2X₇ 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.
authors.affiliation: Dept. of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, Univ. of Western Ontario, London, ON, Canada.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Grol
authors.forename: Irene
authors.initials: I
authors.lastname: Zelner
authors.forename: S Jeffrey
authors.initials: SJ
authors.lastname: Dixon
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016723:Bone Remodeling / Q000187:drug effects; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D008024:Ligands; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D058911:Purinergic P2X Receptor Agonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D058486:Receptors, Purinergic P2X7 / Q000737:chemistry / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; D004791:Enzyme Inhibitors; D005947:Glucose; D019344:Lactic Acid; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D014544:Uridine Triphosphate; C546469:P2rx7 protein, mouse