Triiodothyronine binding in adult rat brain: compartmentation of receptor populations in purified neuronal and glial nuclei.

Abstract

In order to investigate the localization of nuclear T3 receptors in adult rat brain, we have developed a technique for separating neuronal and glial nuclei, and compared nuclear T3 binding in both fractions. Glial and neuronal nuclear populations were prepared by homogenization of the whole brain except the cerebellum, followed by discontinuous density gradient centrifugation in 2.4 and 2.2 M sucrose. For purposes of comparison, liver nuclei were prepared by centrifugation in 2.4 M sucrose. Microscopic examination of the nuclei confirmed the purity of the two nuclear fractions to be above 90% with minimal extranuclear contamination. T3 binding studies were performed on the washed nuclear fractions using saturation analysis techniques. Maximum binding (Bmax) and dissociation constant (Kd) were obtained by nonlinear least-squares regression analysis. After 30 min incubation at 37 C, Bmax in neuronal nuclei was 1203 +/- 118 fmol/mg DNA (mean +/- SE) (n = 6), in comparison with 196 +/- 14 fmol/mg DNA in glial nuclei (n = 7). The corrected value for Bmax in glial nuclei, assuming 8% neuronal nuclear contamination, was 100 fmol/mg DNA. Bmax in liver nuclei was contamination, was 100 fmol/mg DNA. Bmax in liver nuclei was 299 +/- 38 fmol/mg DNA (n = 6). The Kd values were 0.38 +/- 0.04 nM, 0.34 +/- 0.05 nM, and 0.33 +/- 0.04 nM in neuronal, glial, and liver nuclei, respectively. The specificity of T3 binding to the nuclear receptor in both neuronal and glial nuclei was studied in competition studies, and revealed closely similar relative binding affinities (T3 greater than T4 greater than rT3) in both. We conclude that the higher T3 binding capacity of neuronal nuclei may be related to their higher protein synthetic ability, suggesting that T3 may be of importance in a rapid structural turnover in these cells. The effect of T3 on myelination in glial cells may be mediated through the nucleus.