title: Release and interconversion of P2 receptor agonists by human osteoblast-like cells.
abstract: Nucleotides, acting as agonists at P2 receptors, are important extracellular signaling molecules in many tissues. In bone they affect both bone-forming osteoblast and bone-resorbing osteoclast cell activity. The presence of nucleotides in the extracellular microenvironment is largely determined by their release from cells and metabolism by ecto-enzymes, both of which have scarcely been studied in bone. We have investigated adenosine 5'-triphosphate (ATP) release from SaOS-2 osteoblastic cells and the activities of cell surface ecto-enzymes on ATP metabolism. ATP, but not LDH, was detected in SaOS-2 cell conditioned medium, suggesting these cells were actively releasing ATP. Introduction of ADP resulted in increased ATP concentrations in the medium, which was found not to be receptor mediated. Nucleotide inhibition and substrate specificity studies revealed an ecto-nucleoside diphosphokinase (ecto-NDPK) was responsible for the ADP-->ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto-NDPK activity; confirming that the combined action of multiple osteoblast-expressed ecto-enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP-consuming, and for the first time, ATP-generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone.
authors.affiliation: Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, University of Liverpool, L69 3GE, UK. buckleyk@liv.ac.uk
authors.forename: K A
authors.initials: KA
authors.lastname: Buckley
authors.forename: S L
authors.initials: SL
authors.lastname: Golding
authors.forename: J M
authors.initials: JM
authors.lastname: Rice
authors.forename: J P
authors.initials: JP
authors.lastname: Dillon
authors.forename: J A
authors.initials: JA
authors.lastname: Gallagher
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D008565:Membrane Proteins; D020559:Monomeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000096:biosynthesis / Q000235:genetics; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics
chemical_list: D017077:Culture Media, Conditioned; D008565:Membrane Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; C546382:P2RY1 protein, human; C548576:P2RY12 protein, human; D058910:Purinergic P2 Receptor Agonists; D058919:Purinergic P2 Receptor Antagonists; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D014157:Transcription Factors; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins