Title:
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An investigation into the effects of endocannabinoids and the COX-2 metabolite of 2-Arachidonyl glycerol on bone cells
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The effects of endocannabinoids on human, mouse and rabbit bone cells were investigated. At high concentrations anandamide and 2-arachidonyl glycerol (2-AG) inhibited human osteoclast formation with no effects at lower concentrations. The inhibition was not attenuated by antagonists for the CB1, CB2 or TRPV1 receptors, indicating a non-receptor mediated effect. Conversely, anandamide and 2-AG increased mouse osteoclast formation. The effect of anandamide was enhanced in cells from fatty acid amide hydrolase (FAAH)-null mice and abolished in cells from CB1/2 knockout mice. The effect of 2-AG was not eliminated in CB1/2 knockout cells, indicating a non-CB1/CB2 action. The CB1 antagonist, AM251, and the CB2 antagonist, AM630, both inhibited mouse osteoclast formation. These effects were not rescued in the CB1/2-knockout mouse cells. Both anandamide and 2-AG stimulated actin ring formation and osteoclast activity in human and rabbit osteoclast. This was prevented in the presence of AM630 but not AM251, indicating a CB2-mediated response. The endocannabinoids and the cannabinoid receptor antagonists do not have a regulatory action on osteoblast activity. The effects of the novel cyclooxygenase-2 (COX-2) metabolite of 2-AG, prostaglandin E2-glycerol ester (PGE2-G), on human osteoclasts were examined. Treatment with PGE2-G inhibited formation and ERK phosphorylation of human osteoclasts. These effects were attenuated by a selective EP4 antagonist and mimicked by PGE2 alone, indicating that PGF2-G is rapidly metabolised into PGE2 in human osteoclast cultures. However, PGE2-G treatment elevated intracellular calcium levels in human osteoclasts, through a phospholipase C (PLC)- and IP3- dependent mechanism, indicative of a G-protein coupled receptor effect. This was not mimicked by PGE2, or prevented by the EP4 antagonist, but blocked by a putative PGE2-G receptor antagonist, PDA-94 indicating that PDA-94 may be a PGE2-G receptor antagonist.
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