Calcium Signalling via Multiple P2 Purinoceptor Subtypes in Rat Osteoclasts

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Abstract
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 μM) 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.

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