Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.
Cells from the fetal uterus of the guinea pig have been grown as monolayer cell cultures both as primary cultures and through several passages. The cells have a fibroblast-like morphology and ultrastructure, and the subcultures are estrogen responsive. Estradiol induced a 2- to 3-fold increase in specific binding of [3H]R5020 by 9 days in culture, with no effect on proliferation. This binding has the characteristics of the progesterone receptor from the fetal guinea pig uterus (saturable, high affinity, specific for progestins). The increase in progesterone receptor depended on the dose of estradiol, with a half-maximal response at about 5 X 10(-11) M. Progesterone receptor concentrations were inhibited to below basal levels by progesterone and R5020 and the nonsteroidal antiestrogens, tamoxifen, and 4-hydroxytamoxifen. Both progestins and antiestrogens antagonized the stimulatory effect of estradiol. None of these compounds had any effect on cell growth. On the other hand, insulin and epidermal growth factor caused a great increase in cell proliferation. Insulin alone had no effect on progesterone receptor concentrations, but epidermal growth factor stimulated the progesterone receptor about as much as estradiol. Furthermore, coincubation of insulin with estradiol produced a synergistic effect. Estrogen receptor levels were low or undetectable at any time in either the primary culture or the subcultures. It is concluded that fetal uterine cells in culture can serve as a good in vitro model for study of the control of the progesterone receptor in a fetal target tissue.
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