Thyroidal stimulation of tubulin and actin in primary cultures of neuronal and glial cells of rat brain

Abstract

The influence of triiodothyronine (T₃) on the level of tubulin and other proteins in primary cultures of neuronal (N) and glial (G) cells from rat brain has been investigated. Quantitation of tubulin by [³H]colchicine binding assay revealed that when cells from 1 day rat brain were cultured for 18 hr with physiological doses (0.5-5 nM) of T₃, the hormone elicited 35-40% increase in the soluble (30,000 g supernatant) tubulin content of G cells only. This stimulation was age-dependent and occurred neonatally at a time corresponding to the onset of synaptogenesis. In mouse and chick brain also, [³H]colchicine binding assay showed a similar selective stimulation of the soluble tubulin content of G cells by T₃ with virtually no effect on N cells. However, SDS-polyacrylamide gel electrophoresis of the total proteins in the 30,000 g supernatants from N and C cells of rat brain, labeled for 18 hr with [¹⁴C]leucine in the presence of T₃, revealed that T₃ elicited 2-3-fold enhancement of radiolabeled tubulin in the N cells which is relatively greater than the 1.5-fold increase seen in the G cells. Analysis of the autoradiograms of these labeled proteins also revealed that in addition to tubulin, T₃ stimulated the accumulation of radiolabeled actin by 1.5- and 2-fold in N cells and G cells respectively. Similar electro-phoretic analysis of the solubilized labeled proteins in the 30,000 g pellets from N and G cells indicated that the failure to detect the stimulation of tubulin in the 30,000 g supernatants from N cells by [³H]colchicine binding assay could be at least partly due to rapid translocation of the dimeric soluble tubulin into insoluble membrane fractions or due to presence of higher oligometric forms of tubulin which are insensitive to [³H]colchicine binding assay.