Adrenal Cells in Tissue Culture. VII. Effect of Inhibitors of Protein Synthesis on Steroidogenesis and Glycolysis

Abstract

We report our experience with 2 inhibitors of protein synthesis, cycloheximide and puromycin, and their effects on the metabolism of ACTH-responsive monolayer cultures of mouse adrenal tumor cells. At high concentrations, these agents promptly inhibit both protein and steroid synthesis. Protein synthesis can be inhibited 60% before any lowering of steroidogenesis occurs. When precautions are taken to remove preformed steroids, increasing concentrations of either inhibitor progressively inhibit not only ACTH-responsive but also resting steroid output. Steroid synthesis can be virtually obliterated with 1.0 mM puromycin, but with cycloheximide at concentrations of 10 5g/ml or higher, there is still a small but measurable steroidogenic response to ACTH. Cultures can be incubated with cycloheximide for as long as 5 hr, with complete and immediate reversal of its inhibitory effects on steroidogenesis upon its removal. Removal of ACTH from ACTHstimulated cells results in a rapid decline in steroidogenic activity following a lag of several minutes. This effect can be duplicated by simply adding cycloheximide to the ACTH-stimulated cultures. Cycloheximide blocks the incorporation of ¹⁴C-acetate into steroids without inhibiting its incorporation into cholesterol. Similarly, there is no effect on the metabolism of progesterone or pregnenolone, localizing the action of the inhibitor in the intact cell to a step between cholesterol and pregnenolone. This is due neither to a direct interaction with the steroidogenic enzymes nor to an inhibition of their synthesis and turnover. Cell-free homogenates prepared from cycloheximide-inhibited or ACTH-stimulated cells show no difference from comparable incubated controls in their capability to synthesize ³H-pregnenolone and other steroids from ³H-cholesterol in the presence of a TPNHgenerating system. Cycloheximide added directly to these homogenates also has no effect on this conversion. Although cycloheximide can lower glycolytic activity in the intact cell (0-50%), it does not block the stimulatory action of ACTH on glycolysis. These studies suggest that agents which inhibit protein synthesis decrease the steroidogenic response to ACTH by lowering the steroidogenic potential of the cell, and do not interfere with other stimulatory effects of ACTH on adrenal cell metabolism. (Endocrinology87: 951, 1970)