Regression of rat liver autophagic vacuoles by locally applied cycloheximide

Defined areas of the intact liver surface of male Sprague-Dawley rats were exposed by means of small plastic rings to cycloheximide (1.8 X 10(-6) M to 1.8 X 10(-2) M). The incorporation of systemically applied [3H]leucine in a subcapsular tissue layer of 40 in micron thickness, was completely abolished at 1.8 X 10(-2) M and was reduced to 20 to 30% by concentrations between 1.8 X 10(-4) M and 1.8 X 10(-6) M cycloheximide as assessed by autoradiography. After fixation in situ with OsO4 this tissue layer was used for electron microscopy. Early autophagic vacuoles (AVs) whose contents could clearly be identified as being derived from cytoplasm were evaluated morphometrically. Their volume fraction in hepatocyte cytoplasm was reduced by cycloheximide within 10 minutes to about 50% of control tissue exposed to Ringer solution only. Maximal reduction was already observed with concentrations as low as 1.8 X 10(-5) M. When exposing the test areas to 1.8 X 10(-4) M cycloheximide for time intervals of 10, 20, 30, and 60 minutes, an exponential decrease in the AV volume fraction was seen in each individual animal over 20 minutes without further decrease between 30 and 60 minutes. The half-life of AV contents which could be calculated from this exponential decrease, was different for the different AV types, as distinguished according to their contents. Glycogen sequestered within AVs showed the largest morphological half-life of about 45 minutes, whereas, the half-life of the remaining components of AV contents, i.e., mitochondria, microbodies and endoplasmic reticulum together with cytoplasmic ground substance, was 10.1, 5.7, and 5.2 minutes, respectively. The average half-life of total AVs excluding those containing glycogen was 7.9 minutes. (first order rate constant: 0.088 min-1). The coefficient of variation (s/x), as calculated from single animal values, was lower for AV half-life (0.21) than for control AV volume fraction (0.67).