ULTRASTRUCTURAL AND IMMUNOCYTOCHEMICAL CHARACTERIZATION OF AUTOPHAGICVACUOLES IN ISOLATED HEPATOCYTES - EFFECTS OF VINBLASTINE AND ASPARAGINE ON VACUOLE DISTRIBUTIONS

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophag ic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepat ocytes, AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD) , Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly de graded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation . Lysosomes could be recognized by the presence of the lysosomal membr ane glycoprotein lgp120, which was absent from hepatocytic endosomes, Endocytic inputs into the AVs were detected by the presence of gold-co njugated bovine serum albumin (BSA-gold), taken up by fluid-phase endo cytosis, All vacuoles recognized morphologically as AVs were SOD-posit ive, as were essentially all of the lysosomes (96%), The majority (72% ) of the lysosomes also labeled positively for BSA within 2 h of endoc ytosis, The data are thus compatible with the notion that all lysosome s can engage in both autophagic and endocytic degradation, Lgp120 appe ared to distinguish well between lysosomes and nonlysosomal AVs: the l gp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to t hat of the cytosol, indicating that no degradation had occurred, In th e lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytos olic value, consistent with substantial CAIII degradation, Among the n onlysosomal AVs (about one-third of all AVs), one-half were BSA-positi ve, suggesting that early AVs (autophagosomes) and intermediary AVs (a mphisomes) that had fused with endosomes were equally abundant, These morphological data thus support previous biochemical evidence for a pr elysosomal meeting of the autophagic and endocytic pathways, The micro tubule inhibitor vinblastine inhibited the autophagic influx to the ly sosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size, Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive ly sosomes, High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acid otropic effect of ammonia generated by asparagine deamination, Asparag ine also caused an accumulation of autophagosomes, amphisomes, and BSA -negative lysosomes, presumably as a result of impaired fusion with th e swollen BSA-positive lysosomes, The two agents thus appear to pertur b the autophagic-endocytic-lysosomal vacuole dynamics by different mec hanisms, making them useful in the further study of these complex orga nelle interactions. (C) 1995 Academic Press, Inc.