Calcium signalling via multiple P2 purinoceptor subtypes in rat osteoclasts

Extracellular nucleotides bind to P2 purinoceptors in many tissues. P2X purinoceptors are intrinsic ion channels that mediate depolarization and influx of Ca2+, whereas P2Y purinoceptors are coupled through G-proteins to mobilization of intracellular Ca2+. 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 Ca2+ signalling in authentic mammalian osteoclasts, Osteoclasts, isolated from the long bones of neonatal rats, were loaded with the Ca2+-sensitive probe fura-2 and [Ca2+](i) was monitored by microspectrofluorimetry. ATP (10-100 mu M) induced transient elevation of [Ca2+](i) in 74% of osteoclasts tested. Similar responses were observed in Ca2+-free media, consistent with release of Ca2+ from intracellular stores. Oscillations in [Ca2+](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 Ca2+ response mediated by osteoclast purinoceptors. Mechanical stimulation of osteoclasts elicited transient elevation of [Ca2+](i) which involved Ca2+ influx and, in some cases, release from stores. The nucleotidase apyrase did not inhibit deformation-induced elevation of [Ca2+](i) in the presence of extracellular Ca2+ indicating that nucleotide release is not essential for mechanically induced Ca2+ influx. These findings indicate that osteoclasts exhibit multiple P2 purinoceptor subtypes, linked to elevation of [Ca2+](i). Copyright (C) 1999 S. Karger AG. Basel.