Regenerating rat liver: correlations between estrogen receptor localization and deoxyribonucleic acid synthesis

Abstract

Estrogen receptor activity was quantitated in the cytosol and nucleus of normal rat liver and in regenerating rat liver at several time intervals after 75% hepatectomy. Cytosolic estradiol binding in regenerating liver decreases at 12, 24, and 48 h after hepatectomy and at 48 h is 30% of that in normal rat liver. Nuclear estrogen binding 48 h after surgery is elevated fivefold over normal values. No alterations in affinity of the receptor for estrogen have been observed. Specificity studies indicate that the estrogen receptors from both normal and regenerating liver were similar and are highly specific for estrogens. These changes in cellular distribution of receptors parallel increases in nuclear deoxyribonucleic acid synthesis and mitotic indices in the liver.

Figure 1

A. Specific binding of [³H]E₂ by cytosol of normal and regenerating male rat liver. Two-hundred microliter aliquots of cytosol (5 mg/ml) from normal or regenerating rat liver 48 h posthepatectomy precipitated with protamine sulfate were incubated with seven different concentrations of [³H]E₂ (0.15–5 nM) for 18 h at 0°C in the absence (total binding) and presence (nonspecific binding) of 100-fold unlabeled E₂. Specific binding was calculated by subtracting nonspecific binding from total binding. Each point is the average of triplicate determinations. B. Scatchard plot analysis of the specific [³H]E₂ binding data obtained using normal and regenerating male rat liver. The specific binding data illustrated in A was replotted according to the method of Scatchard. Closed circles represent normal liver; open circles represent regenerating liver in both A and B.

Figure 2

Cytosolic and nuclear specific [³H]E₂ binding, DNA synthesis, and mitotic index in normal and regenerating livers at different days posthepatectomy. A. Two-hundred microliter aliquots of cytosol (5 mg/ml) precipitated with protamine sulfate were incubated with 1.5 nM E₂ for 18 h at 0°C in the presence and absence of 100-fold unlabeled E₂. Each point is the mean ± SEM of six experiments. B. Two-hundred microliter aliquots of nuclear suspension were incubated at 30°C for 60 min with 10 nM of [³H]E₂ in the presence and absence of 100-fold excess concentration of unlabeled DES (exchange assay). Each point is the mean ± SEM of 5 animals; each assay is performed in triplicate. C. Deoxyribonucleic acid synthesis was determined as described in Materials and Methods for 1 mg of DNA at different times after partial hepatectomy. D. Mitotic index as an expression of the number of hepatocytes in metaphase for 1000 cells was determined as outlined in Materials and Methods.

Figure 3

Specificity of binding to cytosolic proteins in normal and regenerating male rat liver cytosol. Two-hundred microliter aliquots of male rat liver cytosol were precipitated with protamine sulfate and were incubated with 1.5 nM [³H]E₂ in the presence and absence of 100-fold excess of competing ligand for 18 h at 0°C. Each bar represents the mean results of triplicate determinations for each competing ligand. The solid bars represent data obtained using normal liver; the hatched bars represent data obtained using regenerating liver. Progesterone (PROG), testosterone (TEST), cortisone (CORT).