Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes.

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70---80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80---85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstatin, bestatin and aprotinin (Trasylol) produced minor inhibitory effects (possibly on both degradation pathways), whereas bacitracin and soybean trypsin inhibitor were ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their known actions only sosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptin and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosomes. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.