Hypoxia stimulates vesicular ATP release from rat osteoblasts

Abstract

Many neuronal and non-neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y(1) receptor, stimulate osteoclast formation and activity, whilst P2Y(2) receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation-dependent manner, with mature, bone-forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N-ethylmaleimide (1-1,000 microM) inhibited basal ATP release by up to 99%. The presence of granular ATP-filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O(2)) for up to 3 min increased ATP release from osteoblasts up to 2.5-fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 microM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N-ethylmaleimide (100 microM) attenuated the hypoxia-induced ATP release by up to 80%. Depletion of quinacrine-stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O(2) could influence local purinergic signaling and affect the balance between bone formation and bone resorption.

The role of purinergic signalling in the musculoskeletal system

“…Controlled ATP release has been demonstrated from numerous excitatory and non-excitatory cells. In the bone microenvironment, osteoblasts (Buckley et al, 2003;Genetos et al, 2005;Orriss et al, 2009;Romanello et al, 2001;Rumney et al, 2012), osteoclasts and MLO-Y4 osteocyte-like cells (Genetos et al, 2007;Kringelbach et al, 2014) have all been shown to constitutively release ATP.…”

“…Since then several studies have indicated that the primary method of ATP release from osteoblasts is by vesicular exocytosis (Genetos et al, 2005;Orriss et al, 2009;Romanello et al, 2005). However, it has been suggested that the P2X7 receptor may also be involved (BrandaoBurch et al, 2012).…”

Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

“…Osteoblasts release ATP constitutively [44,45] and can generate low micromolar concentrations of adenosine in vitro [21,27]. Therefore, the possibility remains that endogenous adenosine exerts effects on osteoblasts that are not enhanced further by the addition of exogenous adenosine.…”

The touching story of purinergic signaling in epithelial and endothelial cells

“…There are substantial evidence for vesicular release from neuronal [22] and endocrine tissues [23]. Vesicular release has likewise been suggested to occur in non-excitable cells such as endothelial cells [24] and osteoblasts [25], but non-vesicular release is thought to contribute in a major way to ATP release in many cell types. The molecular identity of the ATP release channel is unknown, but potential candidates include large anion channels, cystic fibrosis transmembrane conductance regulator (CFTR), and hemichannels like connexins and pannexin-1, which have been suggested to form ATP-releasing units together with the ATP-gated ion channel P2X7 [26][27][28].…”