Characterization of 3,5,3′-triiodothyronine receptors in primary cultures of hepatocytes and neurons from chick embryo

Abstract

We have detected the presence of nuclear 3,5,3′-triiodothyronine (T 3) receptors in primary cultures of chick embryo hepatocytes and neurons. Hepatocytes were isolated from livers of embryos of 12, 16 and 19 days by treatment with 0.2% collagenase and hyaluronidase. They were plated at a density of 3–4 × 10 5 35 -mm petri dish in Ham's F-10 medium containing fetal calf serum, tryptose phosphate, and antibiotics. Cells were used for the binding assay at Day 3 of culture. Neurons from 8-day-old embryo brains were cultured in a serum-free medium at a density of 1.2 × 10 6 cells/35-mm petri dish and used for the binding assay after 7 days of culture. Biological activity of hepatocytes was determined by measuring insulin binding, inositol phosphate formation, and 5′-monodeiodinase activity. Neurons or glial cells in culture were identified by immunostaining with anti-neurofilaments and anti-glial fibrillary acidic protein antisera. Binding assay was performed with isolated nuclei and 0.4 M NaCl nuclear extracts. With the latter preparation, the Scatchard analysis showed, in both cells, a single, high-affinity, low-capacity T 3 receptor. In the hepatocytes of 12-, 16-, and 19-day-old embryos association constants ( K a ) were, respectively, 0.93 ± 0.02, 0.74 ± 0.03, and 0.56 ± 0.04 n M −1, whereas the maximal binding capacities (MBC) were 2.26 ± 0.2, 2.72 ± 0.33, and 1.83 ± 0.19 fmol/μg DNA (mean ± SE, n = 3). In neurons K a was 1.25 ± 0.53 n M −1 and MBC 0.59 ± 0.14 fmol/μg DNA ( n = 3). The receptor had a sedimentation coefficient of 3.4 S, an estimated M r of 59 kDa, and the following relative affinity for thyroid hormone analogues: TRIAC l-T 3 > l-T 4. These data indicate that cultured hepatocytes and neurons of chick embryo contained T3 receptors with properties similar to those described in intact tissues from this and other species. Only the MBC of neurons was 50% lower than that observed in whole brain of embryo, but was comparable to values observed in cultured neurons from other species.