17-Estradiol (17-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 17-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 ER, 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 ER.RT-PCRevidences the presence ofmRNAforER, not for ER. Our data suggest that 17-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 ER.
File in questo prodotto:
Non ci sono file associati a questo prodotto.