Quantitative aspects of cellular autophagocytosis. Morphometric and cell fractionation studies

The mode of action of agents and treatments known to induce the appearance of autophagic vacuoles (AV) were studied in mouse liver, pancreatic and seminal vesicle cells by morphometric evaluation of the time-dependent changes in the volume fraction of AVs following suppression of sequestration by cycloheximide. Rapid decrease of the AV compartment was observed in cells of animals pretreated with agents including Triton X-100, pilocarpine, leupeptin and estron acetate. The half-life of AVs, estimated from the decay, range between 5.3 to 8.7 min, which does not deviates from normal steady state values. In contrast, no regression or very slow decay was seen in cells of animals pretreated with methylamine, chloroquine and vinblastine (VBL). We think that the main mechanisms responsible for the enlargement of the AV compartment in these experiments are: stimulation of the sequestration under the effect of agents which maintain the half-life of AVs within the physiologic range of 5-10 min and accumulation of AVs due to their prolonged lifetime under the effect of acidotropic agents and VBL. However, our data suggest than in addition to the accumulatory effect on AVs, stimulation of sequestration may also play a role in the enlargement of the AV compartment after treatment with VBL. While it was not possible to isolate a relatively pure fraction of early AVs from the liver, a light and a dense AV fraction was successfully purified in our laboratory from the pancreas treated with either VBL or neutral red. Preliminary enzymological and morphological evidence is presented that the light AVs are of autophagosomal, whereas the dense ones are of autolysosomal nature.