Response of autophagic protein degradation to physiologic and pathologic stimuli in rat hepatocyte monolayer cultures

Abstract

The lysosomes of hepatocytes increase in numbers and size during acute cell injury in vivo. To elucidate the mechanism of this change, we have studied in vitro the response of the autophagic lysosomal system to several physiologic mediators of autophagy, and to agents known to induce injury and/or the accumulation of lysosomes in vivo. To this end, monolayer cultures of rat hepatocytes were labeled with [14C]leucine to measure hepatocyte protein degradation; ultrastructural analyses were carried out to measure the volume fraction of lysosomes in the hepatocytes. Dibutyryl cyclic AMP increased protein degradation in the hepatocytes either in the presence or absence of serum and insulin. Deprivation of serum and insulin also increased hepatocyte protein degradation. Morphometric analysis indicated parallel increases in the volume fraction of lysosomes in the hepatocytes. The calcium ionophore ionomycin (5 microM), in the presence of 1.3 mM extracellular calcium, increased protein degradation (but not the volume fraction of lysosomes), and this increase was abolished by the addition of ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. On the other hand, vasopressin (5 nM) caused an increase in protein degradation coupled with an increase in volume fraction of lysosomes. The microtubule depolymerizer vinblastine decreased protein degradation. The microtubule stabilizer taxol did not prevent the inhibitory effects caused by vinblastine. Parallel decreases in the lysosomal compartment were found in the hepatocytes exposed to vinblastine or taxol. Dimethylnitrosamine inhibited protein degradation as well as decreased the volume fraction of lysosomes. Finally, carbon tetrachloride also decreased protein degradation. These data indicate that in physiologic conditions, increases in numbers of hepatocyte lysosomes reflect increased sequestration and degradation of cytoplasmic proteins in response to changes in the levels of hormones, serum factors and nutrients as well as cyclic AMP. The induction of acute cell injury in vitro by calcium ionophore, microtubule active agents, and hepatotoxins inhibits lysosomal proteolysis and causes a decrease in the volume fraction of lysosomes. We conclude that the increase in lysosomal size and numbers occurring in acutely injured hepatocytes in vivo is induced primarily by altered levels of nutritional and hormonal regulators of lysosomal protein degradation.