Formation of lead-induced inclusion bodies in primary rat kidney epithelial cell cultures: Effect of actinomycin D and cycloheximide

Abstract

Cells were isolated from adult rat kidney by mild trypsin digestion and maintained in Eagles MEM- d-valine medium for 3 days to form a monolayer primary culture of epithelial cells. When these cells in monolayer were exposed to a medium containing lead nitrate (4.5 × 10 −4 m) complexed with glutamic acid (5 × 10 −4 m), inclusion bodies with characteristic ultrastructure were detected morphologically in the cytoplasm within 4 hr. At 24 hr after lead exposure the inclusion bodies were localized mainly in the nucleus. Addition of actinomycin D (0.05 and 5 μg/ml) or cycloheximide (5 and 10 μg/ml) directly to cultures prior to lead exposure resulted in the absence of inclusion bodies at high concentrations and in fewer inclusions at low concentrations. These inhibitors did not affect the uptake of lead by the cell; but they markedly inhibited incorporation of [ 3H]leucine into cellular proteins. The initial appearance of inclusion bodies in cytoplasm after lead exposure, its subsequent relocalization in the nucleus and the effect of inhibitors in these processes were also confirmed by quantitation of inclusion bodies ultrastructurally in randomly selected cells in each experiment. These results indicate that active protein synthesis is required for the formation of lead-induced inclusion bodies.