The regulation of bone cell function by extracellular nucleotides
Extracellular nucleotides, acting via P2 receptors, modulate bone remodelling by inhibiting osteoblast and stimulating osteoclast activity. The aim of this thesis was to investigate further the effects of extracellular nucleotides on bone cell function under both normal and stress situations. Nucleotide agonists were shown to evoke intracellular Ca2+ responses in rat osteoblasts from > 0.2 uM. The approximate order of potency was ATP > UTP = ATPyS > ADP > UDP > 2-MeSATP = BzATP > a0- meATP, consistent with the expression of functional P2X2, P2X5, P2X7, P2Yi, P2Y2, P2Y4 and P2Y6 receptors. A dramatic increase in intracellular Ca responses to ATP or UTP was observed in osteoblasts cultured for 8-10 days compared to 4 days, indicating that osteoblast responsiveness to nucleotides increases with cell differentiation. P2 receptor mRNA and protein expression in osteoblasts was shown to be differentiation dependent and was characterised by a shift from P2X to P2Y expression, with mature osteoblasts strongly expressing P2Y2, P2Y4 and P2Y6 receptors. Closer investigation revealed ATP and UTP decrease alkaline phosphatase expression and activity, indicating that extracellular nucleotides primarily inhibit mineralisation rather than organic matrix synthesis. Taken together these data suggest the P2Y2 receptor, and possibly the P2Y4 receptor could function as "off-switches" for mineralised bone formation. Constitutive ATP release from primary rat osteoblasts was found to occur via vesicular exocytosis in a differentiation dependent manner. Moreover, transient exposure to hypoxia (2% 02) or hyperthermia (40 C) induced a rapid, significant increase in ATP release. In contrast, continuous culture at 2% 02, 34 C or 40 C caused marked impairment of osteoblast function and ATP release. Given the negative effects of extracellular nucleotides on bone cell function, locally increased concentrations following stress situations may contribute to the bone loss associated with hypoxia or inflammation in vivo. A bone densitometer was used to screen mice deficient in selected P2 receptors. Bone mineral content and density were increased up to 18% in P2X27 mice, decreased up to 10% in P2YfA and P2X2/3dbl '' mice but unaffected in P2X3"A animals. In summary, the work described here provides further evidence for the role of extracellular nucleotides and their receptors in the regulation of bone cell function in health and disease.