Pneumocystis pneumonia in patients with giant cell arteritis treated with high dose steroids: is there an indication for prophylaxis?

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).