Cytosolic and nuclear estrogen receptors in the isolated perfused rat liver.

The isolated perfused rat liver was investigated as a potential model to analyze binding of 17 beta-[3H]estradiol (E2) to cytosolic and nuclear estrogen receptors. Viability of the isolated perfused liver was monitored by measuring leakage of cytosolic enzymes into the perfusate. These studies indicated that the liver remained viable for at least a 90-min perfusion period although significant decreases in cytosolic estrogen receptor concentrations occurred during this perfusion period. Estrogen receptor loss was minimized by supplementing the red blood cell-free perfusion medium with 5 micrograms of insulin per ml. Uptake of [3H]E2 by hepatic cytosolic estrogen receptors of the isolated perfused liver was rapid as measured by partial purification of radiolabeled ligand receptor complexes after varying times of perfusion. Peak liver concentrations of receptor-bound E2 were achieved 15 min after the onset of perfusion when using livers from either male or female rats. After 15 min, radiolabeled cytosolic ligand receptor complexes decreased rapidly, reaching lowest concentrations at 60 min. Radiolabeled salt-extractable nuclear-binding sites increased up to 30 min and then decreased slightly between 30 and 90 min. Both 4S and 5S forms of nuclear binding sites were detected in the isolated perfused livers as evaluated by sedimentation analysis on 5 to 20% sucrose density gradients. Concentrations of radiolabeled cytosolic and nuclear receptors were greater in females than males at all perfusion periods examined when the initial concentration of [3H]E2 was 4 nm. Sex differences in receptor uptake were not as great when higher concentrations of [3H]E2 were added to the perfusion medium. These studies suggest that the isolated perfused liver is a suitable model to investigate short-term uptake of estrogens by cytosolic and nuclear receptors.

Effect of gonadectomy on the ontogeny of estrogen-binding components in rat liver cytosol.

These studies elucidate the ontogeny of two classes of estrogen-binding sites present in rat liver cytosol. The first class of sites is precipitated from whole cytosol fractions by ammonium sulfate (30% saturation) and exhibits characteristics of specific estrogen receptors. Detectable levels of receptors are attained during the third postnatal week. During days 30--40, receptors reach maximum concentration and remain relatively constant thereafter. The second class of sites, detected in whole cytosol fractions, possess a high binding capacity for estrogens and are present in similar amounts in male and female liver before day 34. However, between days 34--40 male levels increase dramatically while female levels remain constant. This sex difference is maintained throughout the duration of the study (160 days). Specific estrogen receptors from immature (26 days) and mature (70--80 days) rat liver have similar characteristics in terms of sedimentation properties in sucrose gradients, ligand binding specificity, and heat and pronase susceptibility. After prepuberal (19--21 days) gonadectomy, levels of receptor in subsequent adult animals of both sexes are increased approximately 40%. No alterations in receptor levels are seen after neonatal (day 1) castration of males. Prepuberal (day 19) gonadectomy does not alter the normal development of sexual differentiation of high capacity estrogen-binding sites. However, after neonatal (day 1) castration male rats, levels of those sites do not undergo sexual differentiation, and neonatally castrated adult males exhibit female levels of high capacity estrogen-binding site. These studies suggest that sexual differentiation of high capacity estrogen-binding sites may be programmed at birth by testicular androgens.

Sex differences in hepatic oestrogen-binding proteins.

Gel-filtration (Sephadex G-75) analysis of hepatic cytosol reveals both qualitative and quantitative sex differences in oestrogen-binding proteins. The elution profile of [(3)H]oestradiol-labelled cytosol shows four species of oestrogen-binding proteins (peaks I, II, IV and V) common to both sexes. The amount of [(3)H]oestradiol binding in peak I is equivalent in both males and females and corresponds quantitatively to the specific oestrogen receptor. The amount of binding in the remaining three peaks is greater in males than females. In addition, an oestrogen-binding protein (peak III) is present that is unique to male cytosol. Proteinase-inhibition studies demonstrate that the observed multiplicity of oestrogen-binding proteins is not an artefact of proteolytic breakdown. Sex differences in oestrogen-binding proteins are absent in immature male and female animals; the oestrogen-binding protein profile in immature rats resembles that of an adult female. Gonadectomy of adult animals does not affect the oestrogen-binding-protein profile. In contrast, neonatal (day 1) castration results in partial feminization of the characteristic oestrogen-binding protein profile seen in the adult male; the appearance of Peak III is suppressed and marked decreases in the amount of oestradiol binding occurs in the remaining peaks. Hypophysectomy of adult animals results in near abolishment of the observed sex differences; the male oestrogen-binding protein profile is partially feminized and the female profile is partially masculinized, as characterized by the appearance of [(3)H]oestradiol binding in the region of peak III and increased amounts of binding in peaks IV and V. The present studies demonstrate a multiplicity of oestrogen-binding proteins in liver cytosol and raise the possibility that the presence of some of these proteins may be imprinted at birth through the hypothalamic-pituitary axis, by a mechanism requiring neonatal androgen exposure.

Influence of anti-oestrogens on the specific binding in vitro of (3H)oestradiol by cytosol of rat mammary tumours and human breast carcinomata.

The anti-oestrogenic potential of two nitrogen-mustard-containing compounds, I.C.I. 79792 and I.C.I. 85966, was studied. I.C.I. 85966 usually did not decrease specific binding of [3H]oestradiol by breast-tumour cytoplasmic proteins. I.C.I. 79792 decreased specific [3H]oestradiol binding, but not to the same extent as similar concentrations of I.C.I. 46474, diethylstilboestrol or dibutyldihydrostilboestrol.