Ionomycin induces prostaglandin E2 formation in murine osteoblastic MC3T3-E1 cells via mechanisms independent of its ionophoric nature

Ionomycin and A23187 are divalent cation ionophores with a marked preference for calcium. Studies using these ionophores have almost exclusively interpreted their results in the light of calcium elevation. It was the aim of this study to investigate the effects of ionomycin in osteoblatic MC3T3-E1 cells that are not attributable to its ionophoric properties. Thus, we have found that in contrast to A23187, ionomycin shows similar effects on prostaglandin E2 formation as bradykinin and endothelin-1, being potentiated by extracellular nickel and inhibited by cholera toxin and pertussis toxin. Our data strongly suggest that inomycin, at least in part, exerts its effects via specific binding to a G-protein coupled receptor, thereby evoking downstream cellular events like arachidonate release with subsequent prostaglandin formation.

17β-Estradiol Stimulates Arachidonate Release from Human Amnion-Like WISH Cells through a Rapid Mechanism Involving a Membrane Receptor

17β-Estradiol (17β-E2) greatly and dose-dependently stimulates [3H]arachidonic acid (AA) release from the human amnion-like Wistar Institute Susan Hayflick (WISH) cells. This action is abolished by the phospholipase A2 inhibitor AACOCF3, significantly reduced by the estrogen receptor (ER) antagonist ICI 182,780, and uninfluenced by cycloheximide. The estradiol-BSA conjugate E2coBSA, which binds putative membrane ERs and is unable to enter the cell, also highly stimulates [3H]AA release from WISH cells, although to a lesser extent compared with 17β-E2. The fluorescent conjugate E2coBSA-FITC specifically binds to the surface of a subset of intact WISH cells, and labeling intensity appears dose and time dependent. Cell permeabilization results in a dense intracellular staining, mainly in the peripheral cytoplasm. H-150, an antibody against the N terminus of human ERβ, also labels the plasma membrane of intact WISH cells and the cytoplasm of permeabilized cells. Almost no labeling is observed using ER-21, an antibody against the N terminus of human ERα. RT-PCR evidences the presence of mRNA for ERβ, not for ERα. Our data suggest that 17β-E2 stimulates [3H]AA release from WISH cells through an apparently nongenomic pathway and interaction with membrane binding sites. These last are, at least in part, similar if not identical to ERβ.

Arachidonate release and prostaglandin production by group IVC phospholipase A2 (cytosolic phospholipase A2gamma)

While the role of the group IVA Ca2+-dependent cytosolic phospholipase A2α (cPLA2α) in arachidonic acid (AA) metabolism has been well documented, that of its paralogue, Ca2+-independent group IVC PLA2 (cPLA2γ), has remained uncertain. Here we show, using a transfection strategy, that cPLA2γ has the ability to increase the spontaneous and stimulus-induced release of cellular fatty acids. The AA released by cPLA2γ was metabolized further to prostaglandin E2 via cyclo-oxygenase-1 (COX-1) in the immediate response, and via COX-2 in the delayed response. Mutation of the putative catalytic-centre residue Ser82 abrogated the AA-releasing function of cPLA2γ both in vitro and in vivo. Confocal microscopy revealed that cPLA2γ was distributed in the perinuclear endoplasmic reticulum membranes. Mutating the C-terminal prenylation site of cPLA2γ abrogated its intracellular membrane localization and cellular AA-releasing function, without reducing its enzyme activity in vitro. Our results indicate that cPLA2γ is the second cPLA2 enzyme that contributes to cellular AA metabolism and phospholipid remodelling under appropriate conditions.