MEMBRANE ESTROGEN RECEPTOR-ENRICHED GH(3)/B6 CELLS HAVE AN ENHANCED NON-GENOMIC RESPONSE TO ESTROGEN

We immunoselected CH3/B6 cells for a membrane estrogen receptor (mER) using antibodies generated against the rat intracellular ER (iER). Immunocytochemistry with anti-ER antibodies revealed bright fluorescence distributed in patches over the surface of mER-enriched cells, while cells immune-depleted for mER showed only low-level membrane immunofluorescence. Quantitation via digital image analysis confirmed that immuno-enriched populations show increases in both stained cell numbers and intensity of staining. Short-term culturing with serum reversibly decreased the intensity of immunostaining in mER-enriched cells to immune-depleted cell levels. The mER-enriched populations initially contained similar to 85% immunopositive cells in defined medium, but when cultured continuously with serum gradually decline to similar to 22% immunopositive cells by 10 weeks. Cells enriched for mER showed a significant increase in rapid (after 2 or 5 min) prolactin release when treated with 17 beta-estradiol, while mER-depleted cells lacked this response. Immunoprecipitable membrane proteins isolated from mER-enriched cells were 60,000, 74,000 and similar to 200,000 MW, compared to an iER size of 67,000. Therefore, the presence and level of an mER that is antigenically related to iER is correlated with the ability of GH(3)/B6 cells to mediate a rapid action of estrogen.