Arachidonic acid for loading induced prostacyclin and prostaglandin E2 release from osteoblasts and osteocytes is derived from the activities of different forms of phospholipase A2

Mechanical loading of bone stimulates resident bone cells to produce prostacyclin (PGI2) and prostaglandin (PG)E2 by a mechanism that can be differentially regulated by ion channel blockers. We have investigated differences in the loading-related PG production mechanisms in rat ulnae explants loaded ex vivo. Loading and aluminium fluoride (AlF3, a nonselective activator of G-proteins) both increased PGI2 and PGE2 release into culture medium. Pertussis toxin (PTX) blocked loading-related release of PGE2, but not PGI2, while isotetrandrine, an inhibitor of G-protein-mediated activation of phospholipase (PL)A2, abolished the loading-related release of both PGs. This suggests both PTX-sensitive and -insensitive G-protein-dependent, PLA2-mediated mechanisms for loading-related PG production. Blockade of secretory (s)PLA2 activity prevented loading-related release of PGE2 and PGI2, whereas inhibition of cytosolic (c)PLA2 activity blocked loading-related release of PGE2 alone. cPLA2 was localized immuno-cytochemically to osteoblasts, but not to osteocytes. sPLA2 was localized to osteocytes and osteoblasts. Exogenous type-IA sPLA2 and type-IB sPLA2 stimulated significant increases in PGE2 and PGI2 release. PTX reduced the release of both PGs stimulated by type IA PLA2, but not type IB. Furthermore, inhibition of protein kinase C (PKC) activity blocked loading-related release of PGE2, but not that of PGI2. These data suggest that loading-related release of PGI2 and PGE2 utilizes arachidonic acid derived from the activity of different PLA2s. In osteocytes and osteoblasts, arachidonic acid for PGI2 synthesis is liberated by PTX-insensitive G-protein-dependent sPLA2 alone. In osteoblasts, arachidonic acid for PGE2 synthesis is released by PTX-sensitive, G-protein-dependent, cPLA2-mediated activity, which also requires upstream sPLA2 and PKC activities.