title: Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate.
abstract: Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.
authors.affiliation: Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Isabel R
authors.initials: IR
authors.lastname: Orriss
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Utting
authors.forename: Andrea
authors.initials: A
authors.lastname: Brandao-Burch
authors.forename: Kay
authors.initials: K
authors.lastname: Colston
authors.forename: Barbara R
authors.initials: BR
authors.lastname: Grubb
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Burnstock
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Arnett
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D003570:Cytidine Triphosphate / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D011756:Diphosphates / Q000494:pharmacology*; D045604:Extracellular Fluid / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006706:Homeostasis / Q000502:physiology; D009596:Nitrophenols / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology / Q000502:physiology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058473:Receptors, Purinergic P2Y2
chemical_list: D018076:DNA, Complementary; D011756:Diphosphates; D009596:Nitrophenols; D009711:Nucleotides; D009943:Organophosphorus Compounds; C546386:P2ry2 protein, mouse; C546387:P2ry2 protein, rat; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C082483:O-ethyl O-4-nitrophenyl phosphoramidate; D012313:RNA; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D003094:Collagen; D000469:Alkaline Phosphatase; D011755:Pyrophosphatases