Furthermore, it will shift the extracellular Pi/PPi ratio in favour of Pi, as nucleotides will preferentially be degraded by apyrase to produce Pi rather than by NPP1 to produce PPi. The most significant effect of the removal of endogenous ATP by apyrase was the strikingly increased formation of mineralised bone nodules. The lack of effect of apyrase treatment on collagen production indicates that this osteogenic effect was due primarily to enhanced mineralisation. This finding is consistent with 3PO supplier earlier observations that exogenous extracellular nucleotides selectively inhibit 410536-97-9 mineralisation in vitro. This effect occurs via dual mechanisms firstly, ATP acts via the P2Y2, P2X1 and P2X7 receptors to inhibit TNAP expression and activity and, secondly, it can be directly hydrolysed by NPP1 to increase the local concentration of the physicochemical mineralisation inhibitor, PPi. Selective P2X1 and P2X7 receptor antagonists were used to study the role of these receptors in the regulation of bone mineralisation by endogenous ATP. At present, there are no selective P2Y2 receptor antagonists available and so a pharmacological approach to studying this receptor was not possible. Since many of these selective antagonists are likely to have some effects on other P2 receptor subtypes, we tested a number of different compounds. Our data showing that three different P2X1 and P2X7 receptor antagonists increased bone mineralisation suggest that locally released ATP acts via these receptors to regulate bone mineralisation. The extent to which individual antagonists promoted bone mineralisation was variable, most probably reflecting differences in potency, selectivity and/or binding. One P2X7 receptor antagonist, AZ10606120, caused a reduction in mineralisation. This inhibition was not seen with any of the other P2X7 receptor antagonists and might therefore reflect non-selective cell toxicity rather than specific effects on P2X7 receptor signalling. The ability of the abovementioned P2 antagonists to promote bone mineralisation is consistent with our earlier findings implicating the P2X1 and P2X7 receptors in the regulation of bone mineralisation by extracellular nucleotides. Whilst signalling via the P2X1 receptor appears to regulate bone mineralisation directly, the role of the P2X7 receptor may be more complex. This is because ATP release from osteoblasts involves efflux via the P2X7 receptor; thus, the effects of P2X7 receptor inhibition on bone mineralisation could be due to a direct inhibition of receptor-mediated signalling and/or a secondary effect due to reduced ATP release.
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