title: Calcium signalling via multiple P2 purinoceptor subtypes in rat osteoclasts.
abstract: Extracellular nucleotides bind to P2 purinoceptors in many tissues. P2X purinoceptors are intrinsic ion channels that mediate depolarization and influx of Ca(2+), whereas P2Y purinoceptors are coupled through G-proteins to mobilization of intracellular Ca(2+). Previous studies have yielded conflicting information on the responses of osteoclasts to nucleotides. The purpose of this study was to investigate the pathways underlying purinoceptor-mediated Ca(2+) signalling in authentic mammalian osteoclasts. Osteoclasts, isolated from the long bones of neonatal rats, were loaded with the Ca(2+)-sensitive probe fura-2 and [Ca(2+)](i) was monitored by microspectrofluorimetry. ATP (10-100 microM) induced transient elevation of [Ca(2+)](i) in 74% of osteoclasts tested. Similar responses were observed in Ca(2+)-free media, consistent with release of Ca(2+) from intracellular stores. Oscillations in [Ca(2+)](i) were observed only in osteoclasts that had a 'rounded' morphology. Responses to selective P2 agonists were consistent with the presence of multiple purinoceptor subtypes, including members of both the P2Y and P2X families. Alendronate, a bisphosphonate with structural similarities to methylene ATP analogues, neither activated nor blocked the Ca(2+) response mediated by osteoclast purinoceptors. Mechanical stimulation of osteoclasts elicited transient elevation of [Ca(2+)](i) which involved Ca(2+) influx and, in some cases, release from stores. The nucleotidase apyrase did not inhibit deformation-induced elevation of [Ca(2+)](i) in the presence of extracellular Ca(2+), indicating that nucleotide release is not essential for mechanically induced Ca(2+) influx. These findings indicate that osteoclasts exhibit multiple P2 purinoceptor subtypes, linked to elevation of [Ca(2+)](i).
authors.affiliation: Department of Physiology, School of Dentistry, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.
authors.forename: S H
authors.initials: SH
authors.lastname: Wiebe
authors.forename: S M
authors.initials: SM
authors.lastname: Sims
authors.forename: S J
authors.initials: SJ
authors.lastname: Dixon
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000502:physiology*; D048429:Cell Size; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010812:Physical Stimulation; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018048:Receptors, Purinergic P2; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D002118:Calcium