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). Imm unocytochemistry with anti-ER antibodies revealed bright fluorescence distributed in patches over the surface of mER-enriched cells, while c ells immune-depleted for mER showed only low-level membrane immunofluo rescence. Quantitation via digital image analysis confirmed that immun o-enriched populations show increases in both stained cell numbers and intensity of staining. Short-term culturing with serum reversibly dec reased the intensity of immunostaining in mER-enriched cells to immune -depleted cell levels. The mER-enriched populations initially containe d similar to 85% immunopositive cells in defined medium, but when cult ured continuously with serum gradually decline to similar to 22% immun opositive cells by 10 weeks. Cells enriched for mER showed a significa nt 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 cell s were 60,000, 74,000 and similar to 200,000 MW, compared to an iER si ze of 67,000. Therefore, the presence and level of an mER that is anti genically related to iER is correlated with the ability of GH(3)/B6 ce lls to mediate a rapid action of estrogen.