Differential effects of tri- n-butylin chloride on macromolecular synthesis and ATP levels of rat thymocyte subpopulations obtained by centrifugal elutriation

Abstract

Using centrifugal elutration, rat thymocytes were separated into three fractions by size. Fraction 1 (F1) consisted of a large population (>88% of all cells) of small, nonproliferating thymocytes. Fraction 2 (F2; 7% of all cells) was enriched in medium-sized thymocytes and showed an increased macromolecular synthesis. In the small fraction 3 (F3; <5% of all cells), large thymocytes, monocytes, granulocytes and cells in mitosis were concentrated. F3 demonstrated the highest proliferative activity. Fractions were characterized by size, cell morphology, DNA, RNA and protein synthesis and steady state ATP levels. The effects of the inhibitor of oxidative phosphorylaiton, tri- n-butyltin chloride (TBTC) on ATP levels and the incorporation of DNA, RNA and protein precursors were investigated for each thymocyte fraction. Although ATP levels increased with increasing thymocyte volume, TBTC reduced ATP levels in each subfraction proportionally. The incorporation of thymidine and leucine was markedly reduced in all fractions by TBTC, but not to the same extend. Dependent on the TBTC concentration, the rapidly proliferating cells of F2 and F3 were less affected compared to the noncycling cells of F1. The incorporation of uridine in the unfractionated cells and in F2 and F3 was also decreased by TBTC. However, at concentrations between 0.1 and 1 μM, TBTB stimulated uridine incorporation in the small thymocytes of F1. Therefore, fractionation of thymocyte suspensions was necessary to defect differential effects of TBTC on subpopulations. Centrifugal elutriation was found to be a useful toll with which to obtain subfractions of isolated rat thymocytes.