Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

摘要

pDegradation of cytochrome iP/i-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome iP/i-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome iP/i-450 iin vivo/i. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin iin vitro/i, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome iP/i-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome iP/i-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal sup14/supC-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome iP/i-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome iP/i-450 degradation iin vivo/i. After administration of bromobenzene, the degradation of the haem moiety of cytochrome iP/i-450 was accelerated iin vivo/i in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome iP/i-450. However, the drug failed to affect degradation of haem not associated with cytochrome iP/i-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome iP/i-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state iin vivo/i, as stimulated by bromobenzene iin vivo/i, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring./p