Morrison, Matthew Sam; (1999) Osteoclast function: Role of extracellular pH and ATP. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Osteoclasts are multinucleated cells responsible for the resporption of both the organic and inorganic components of bone. The molecular mechanisms by which these cells are activated to resorb bone are still poorly understood. Previous work has shown that mature rat osteoclasts in short term cultures are extremely sensitive to small shifts in extracellular pH (H+ out) and are strongly stimulated in acidified conditions (pH 6.8 - 7.0). The aim of the work presented in this thesis was to further investigate the actions of [H+ out] on osteoclast function and to study the interactions of ([H+ out]) with other stimulators of resorption. Experiments with mature rat osteoclasts indicated that the acid-activation effect does not abate over time but may become even more pronounced. Conversely, osteoclast formation in long-term mouse marrow cultures was inhibited at low pH and stimulated in more alkaline conditions; however, after formation in marrow cultures, the mature mouse osteoclasts exhibited the same acid-activation characteristics as freshly isolated mature rat osteoclasts. Experiments with cultured mouse calarial bones showed similar effects: osteoclastic resorption was strongly activated below pH 7.0, but acidified conditions resulted in a reduction in the number of osteoclasts visible in bones. Acid-stimulated resorption in calvaria occurred with HCO3- rather than CO2 acidosis and was blocked by inhibitors of prostaglandin and leukotriene synthesis. In contrast, prostaglandin inhibitors stimulated pit formation by cultured rat osteoclasts. I also found that resorption pit formation by isolated chick osteoclasts was very sensitive to small changes in although maximum pH sensitivity occurs over a more alkaline pH range than is the case for rodent osteoclasts. The data from these diverse systems provide strong support for the critical role of acid-base balance in modulating osteoclast function, despite apparent differences in the role of prostaglandins. The results show how pH can be manipulated to optimise resorption assays, and emphasise the importance of controlling this key variable. I also discovered that extracellular ATP, now recognised as an important signalling molecule in many tissues, stimulated both the activation and formation of rodent osteoclasts. There was a marked synergy between the stimulatory effects of [H+ out] and low dose ATP on the resorptive activity of rat osteoclasts. Acid-activated resorption was blocked by apyrase (an ecto-ATPase) and by suramin (an ATP antagonist). Thus, extracellular ATP and low pH both appear to be necessary for osteoclast activation. The findings suggest an important new mechanism for the local control of osteoclast function which may be relevant to, for example, the bone loss associated with inflammation.

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