Actinomycin Phenobarbital

Induction involves increased synthesis of enzyme protein, which may be detected as an increase in total enzyme level as with phenobarbital induction or increase in a particular isoenzyme. Protein synthesis is increased, and this usually seems to be necessary as inhibition of protein synthesis results in inhibition of induction. The increased protein synthesis may involve increased mRNA synthesis and inhibitors of this, such as actinomycin D, block induction. For a simple diagram explaining the relationship of protein synthesis to DNA see Figure 6.38. 

On the basis of the early observation that the increases in microsomal enzyme activity produced by phenobarbital and 3-methylcholanthrene were blocked by actinomycin-D, it was suggested that enzyme induction resulted from the synthesis of new enzyme protein which was, in turn, dependent upon the DNA-directed synthesis of a messenger-like RNA. Treatment of rats with 3-methylcholanthrene causes an increase of about 40% in the level of RNA in rat liver nuclei and the nuclear RNA from 3-methylcholanthrene-treated rats is more active in directing protein synthesis than RNA from control animals. Moreover, the in vitro incorporation of radioactive precursors such as orotic acid or cytidine triphosphate into nuclear RNA is 50 to 100% greater in preparations from 3-methylcholanthrene-treated animals than controls. It is of interest that treatment of rats with phenobarbital has been recently reported to result in a marked suppression of endogenous hepatic ribonuclease activity. 

A stimulus which alters the steady-state level of an endogenous cellular component may do so by influencing its rate of synthesis, its rate of break-down, or both. When administered to intact animals, phenobarbital or 3-methylcholanthrene increase (20-50%) the steady-state level of microsomal protein. Similarly, micro-somes from animals pretreated with phenobarbital or 3-methylcholanthrene incorporate radioactive amino acids into protein more rapidly than microsomes from control animals and this effect is blocked by co-administration of actinomycin-D. It was therefore assumed that the increased levels of microsomal protein and enzyme activity after inducers were the result of enhanced synthesis. However, turnover studies have revealed that phenobarbital in particular has a profound effect upon microsomal protein catabolism. Proteins of the endoplasmic reticulum were labelled by injection of radioactive amino acids and the rate at which radioactivity disappeared from the microsomes was compared in control and phenobarbital-treated animals. Assuming a comparable degree of isotope re-utilization in the two groups, this approach provides a relative measure of microsomal-protein turnover. In control animals, radioactivity of total microsomal protein decreases with time with a half-time of about 3 days. In phenobarbital-treated animals, however, there is a marked stabilization of microsomal protein so that almost no radioactivity is lost over a S-day period. The reduced protein catabolism is observed both in total microsomes and in a purified microsomal protein, NADPH cytochrome c reductase. Thus, repeated administration of phenobarbital to animals evokes an increase in... 

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