NPP activity in an attempt to return the Pi/PPi ratio to normal. The question of whether apyrase treatment influences the expression and activity of other potentially important ATPdegrading enzymes, such as ecto-5′-nucleotidase, will need to be examined in a future study. The major source of extracellular ATP is normally controlled release from cells ; cell culture medium ATP levels are typically measured in the nanomolar range. All three types of bone cell, osteoblasts, osteoclasts and MLO-Y4 osteocyte-like cells release ATP in a constitutive manner. ATP release from osteoblasts occurs primarily via vesicular exocytosis, although the P2X7 receptor is also involved. Blocking ATP release with inhibitors of vesicular exocytosis provides another method for studying the effects of reduced extracellular ATP on osteoblast function. We found that both NEM, which inhibits fusion of vesicles with the plasma membrane, and brefeldin A, which disrupts protein transport between the endoplasmic reticulum and the Golgi apparatus, increased bone mineralisation in osteoblast cultures. Interestingly, the concentrations at which these inhibitors increased bone mineralisation were significantly lower than the levels which acutely inhibit ATP release. Prolonged culture with ��10��M NEM and brefeldin A and ��10nM SCH727965 monensin was toxic to osteoblasts and resulted in significant cell death, possibly due to the intracellular accumulation of ATP. Thus, the lower concentration of NEM and brefeldin A may reduce ATP release enough to influence bone formation but, given that ATP levels are measured in several ml of media, not enough to be detected via the luciferin-luciferase assay. Previous work showed that ATP stimulates the proliferation of osteoblast-like cells. In agreement, we found that elimination of extracellular ATP by apyrase resulted in small decreases in osteoblast numbers during the early, proliferative stages of culture. No differences in cell number were observed by day 7, suggesting that the removal of extracellular ATP retards cell growth, rather than inducing apoptosis. Thus as growth rates slow, which is commonly seen in these osteoblast cultures from ~ day 7, the apyrase-treated cells effectively catch up. Recent studies have implicated extracellular nucleotides and purinergic signalling in the control of mesenchymal stem cell differentiation into osteoblasts or adipocytes. We found that removal of endogenous extracellular nucleotides by apyrase did not affect the level of adipocyte formation or PPAR�� expression. This indicates that ATP is not a significant regulator of osteogenic/adipogenic differentiation in the rat calvarial osteoblast model. It should be noted that because the calvarial cells are treated with dexamethasone to promote the formation of osteoblasts the basal adipocyte formation in these cultures is relatively low. Therefore, the apparent lack of effect of extracellular nucleotides on differentiation could be because the cells used here were more committed to the osteoblast lineage than mesenchymal stem cells. There is increasing interest in the potential roles of adenosine, AMP and P1 receptor-mediated signalling in the regulation of bone cell function. For example, it has been R428 1037624-75-1 reported that adenosine is mitogenic to osteoblast-like cells and may influence the differentiation of osteoprogenitor cells in vitro. Given that apyrase treatment would be expected to cause increased levels of extracellular adenosine, it is plausible that some of the effects we observed here were due to altered P1 receptor signalling. However, we have previously shown that adenosine and AMP have no effects on the function of rat calvarial osteoblasts. This suggests that the effects of apyrase on mineralisation are unlikely to be due to increased adenosine or AMP levels following the rapid hydrolysis of ATP. Thus our data indicate that the increased bone mineralisation seen in apyrase-treated cultures is probably because the reduction in extracellular ATP decreases both P2 receptor-mediated signalling and alters the extracellular Pi/PPi concentration. In summary, the work presented here shows that ATP released from osteoblasts acts via P2 receptors or degradation by NPP1 to produce PPi, so as to function as an endogenous restraint on bone mineralisation. Our findings also raise the interesting question of whether ATP released from osteocytes could be hydrolysed to PPi and thus act to prevent hypermineralisation within bone.