Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity--formation of crinosomes.
The number of rat liver autophagic vacuoles (AVs) was increased by separate injection of three different inhibitors--vinblastine, leupeptin, and chloroquine--of lysosomal protein degradation. The different mechanisms of action of the agents correlated to the ultrastructure of the AVs. Accumulation of the base chloroquine with ensuing influx of water into AVs caused a significant swelling. The leupeptin-induced AVs were processed into residual-body-like structures within a few hours of exposure in line with the presence of a leupeptinase in liver tissue. Vinblastine was the most efficient agent in increasing the occurrence of AVs. The effect of vinblastine lasted for the entire study period (36 hr) with continuous formation of nascent AVs. In addition, vinblastine caused the appearance of a subpopulation of AVs laden with VLDL particles. The term crinosomes was suggested for these hybrid organelles, since they seemed to evolve by fusion between secretory granules and lysosomes. In addition to sequestered cell organelles, the AVs harbored cytosolic enzyme activities (LDH and aldolase). Leupeptin was the only agent that caused a decrease in cathepsin B and L activities. Similarly, leupeptin impeded protein breakdown in isolated AVs, whereas vinblastine and chloroquine evoked an increase. In vivo, chloroquine and vinblastine block protein degradation. The reason for this discrepancy is probably that during in vivo exposure the substrate (cytoplasmic proteins) is built up in the AVs because degradation is retarded. Upon isolation of the AVs the inhibitor block is released, and proteolysis proceeds at enhanced rates over control due to excess of substrates. Leupeptin, on the other hand, caused a substantial inhibition of thiol proteinases; this block remained in the isolated AVs. Accordingly, leupeptin-induced AVs displayed decreased protein degradation following shorter exposure times. Later, when leupeptin was metabolized, catch-up proteolysis was noted. The differing mechanisms of action of the inhibitors were also apparent as regards lipid contents and lipolysis. Whereas chloroquine and vinblastine increased the amounts of cholesterol and triglycerides parallel to proteins, leupeptin had no such effect. Lipolysis proceeded at normal rate following leupeptin administration, which was not the case after vinblastine and chloroquine exposure. Leupeptin has no effect on acid lipases; therefore lipids do not accumulate in AVs of hepatocytes that are exposed to leupeptin.
Glaumann
H
H
Department of Pathology, Karolinska Institutet, Huddinge University Hospital, Sweden.
Ahlberg
J
J
D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D050356:Lipid Metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D008854:Microscopy, Electron; D010587:Phagocytosis / Q000187:drug effects; D011506:Proteins / Q000378:metabolism; D051381:Rats; D014617:Vacuoles / Q000648:ultrastructure; D014747:Vinblastine / Q000494:pharmacology
D007976:Leupeptins; D011506:Proteins; D014747:Vinblastine; D002738:Chloroquine; C032854:leupeptin