Abstract
Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05–0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1–100 μM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1–10 μM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1–10 μM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone.
Highlights
The idea that purines act as extracellular signaling molecules was first suggested in 1929, yet it was not until 1972 that the concept of purinergic neurotransmission was proposed (Burnstock, 1972)
P2 receptors respond to a range of adenine and uridine-containing nucleotides including adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate (UTP), and uridine diphosphate (UDP)
We demonstrated that osteoclasts contain extensive intracellular ATP stores and constitutively release ATP in the range 0.05–0.5 pmol/ml/cell
Summary
The idea that purines act as extracellular signaling molecules was first suggested in 1929, yet it was not until 1972 that the concept of purinergic neurotransmission was proposed (Burnstock, 1972). It has become evident that extracellular nucleotides play a significant role in bone biology modulating both osteoblast and osteoclast function (see reviews by Grol et al, 2009; Orriss et al, 2010). Expression of multiple P2 receptor subtypes by osteoblasts (Maier et al, 1997; Hoebertz et al, 2000; Nakamura et al, 2000; Gartland et al, 2001; Ke et al, 2003; Ihara et al, 2005; Orriss et al, 2006, 2010; Alqallaf et al, 2009) and osteoclasts has been reported (Bowler et al, 1995; Naemsch et al, 1999; Buckley et al, 2002; Gartland et al, 2003a; Korcok et al, 2005; Orriss et al, 2010, 2011b). The important role of P2 receptors in bone homeostasis is further highlighted by the recent studies of several knockout mouse models (P2Y1, P2Y2, P2Y6, P2X7), all www.frontiersin.org
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