Volume Fraction Of Autophagic Vacuoles Research Articles

Influence of X-ray on the autophagic-lysosomal system in rat pancreatic acini

Lysosomes have an important role in radiation injury of cells and tissues. Activation of autophagy is frequently observed in different types of pathological tissue degeneration. In radiation response it increases in some cases, and lysosomes are responsible for regulated degradation of the autophagic vacuoles. Lysosomes are also involved in ionizing radiation induced cell death. In apoptosis lysosomes degrade content of the phagocytotic vacuoles derived from engulfed apoptotic blebs. On the other hand lysosomal enzymes discharged from disintegrated cells have a key role in induction of necrotic changes. In this work we investigate autophagy and lysosomal protein degradation in the relatively radiation insensitive exocrine pancreatic acini in vivo and in vitro. Type of cell death induced by X-ray was also examined in relation to the changes of the lysosomal processes. In 5 h after 16 Gy in vivo whole body irradiation we observed significant increase in the cytoplasmic volume fraction of autophagic vacuoles and in the number of apoptotic cells in vivo. But in the acini isolated from irradiated rats we could not detect a change in the lysosomal degradation of intracellular proteins. Therefore irradiation probably influences the autophagy in an earlier step than lysosomal degradation. Extended necrotic lesions were not observed in vivo as long as 48 h. Isolated pancreatic acini usually contain more autophagic vacuoles than in vivo, but we could not observe additional increase in autophagy after 8 Gy, in vitro irradiation. Lysosomal degradation of intracellular proteins was also unaltered after 8 Gy, in vitro irradiation. Other biochemical functional parameters of the isolated pancreatic acini, like protein synthesis and amylase secretion were not changed either after 8 Gy, in vitro X-ray treatment. These results indicate that pancreatic acinar cells in vitro have a high tolerance to irradiation. The observed in vivo radiation induced changes of the exocrine pancreas are possibly indirectly induced by injuries of more sensitive mechanisms.

Regression of autophagic vacuoles in house pancreatic cells: A morphometric study of the effect of methylamine and chloroquine followed by cycloheximide treatment

Treatment of mice with methylamine and chloroquine for two hours markedly increased the volume fraction of autophagic vacuoles in the pancreatic acinar cells of the mouse. The autophagic vacuoles disappeared, from the cells within 20 min after the administration of a suppressor of autophagic sequestration cycloheximide to animals pretreated with 0,25 mg b.w. dose of methylamine. In contrast, no regression or very slow decay was seen in cells of mice pretreated with higher doses of methylamine (0,50-0,70 mg/g b.w.) and chloroquine (0,08 mg/g b.w.). Our data show that the two drugs retard the disintegration of autophagic vacuole content. It is concluded, that accumulation of autophagic vacuoles due to their slow turnover is an important mechanism of the expansion of autophagic vacuole compartment under the effect of methylamine and chloroquine.

Combined effects of fasting and vinblastine treatment on serum insulin level, the size of autophagic-lysosomal compartment, protein content and lysosomal enzyme activities of liver and exocrine pancreatic cells of the mouse

1. 1. The volume fraction of autophagic vacuoles in liver parenchymal and exocrine pancreatic cells was smallest and the serum insulin level highest in the 24 hr prestarved mouse immediatly after 3 hr feeding period. 2. 2. The size of the autophagic vacuole and lysosome (donse body) compartments increased in both types of cells during 2–72 hr fasting parallel with decreasing serum insulin levels. 3. 3. The protein content of the cells decreased and the DNA-based activity of acid phosphatase showed little change throughout fasting. The activity of cathepsin D increased during days 2 and 3 of food deprivation. 4. 4. Vinblastine (50 mg/kg body wt) applied for the last 2 hr of different periods (2, 12, 24, 48 and 72 hr) of fasting decreased serum insulin level and increased the fractional cytoplasmic volume of autophagic vacuoles and dense bodies. This increase was smaller when the drug was applied shortly after feeding and much larger after prolonged fasting. The increase was more pronounced in the pancreatic than in the liver cells. 5. 5. Our data show that the effect of vinblastine on the size of the autophagic-lysosomal compartment depends on the feeding status of the animals.

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: A comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.

Short-term inhibition of cellular autophagy by isoproterenol in the submandibular gland.

The present study examines the short-term (i.e. 10 min after injection) influence of isoproterenol on cellular autophagy in the rat submandibular gland. The volume fraction of autophagic vacuoles was significantly reduced, suggesting that an anticatabolic reaction, namely inhibition of cellular autophagy, is an early and significant event in the growth of the submandibular gland which is known to occur with long-term isoproterenol treatment.

Short-term inhibition of cardiac cellular autophagy by isoproterenol

It is shown in the present study by means of quantitative electron microscopy that the volume fraction of autophagic vacuoles is significantly reduced to about half the control value within a short time interval (about 10 min) after low dose administration of isoproterenol. The data suggest that stimulation of cardiac work is accompanied by an immediate anticatabolic reaction which can be assumed to represent an early contribution to the adaptative increase in myocardial mass known to result from long-term stimulation of cardiac work.

Inhibited autophagic degradation during ACTH-stimulated growth of rat adrenal zona fasciculata.

Autophagic vacuoles (AVs) in adrenal parenchymal cells of the outer part of the zona fasciculata were investigated by means of electron microscopic morphometry in 40 adult male Sprague Dawley rats. Four test groups and four control groups received twice daily subcutaneous injections of ACTH (12.5 U/kg body weight) or physiological saline, respectively, for 3, 6, 9 and 12 days. At 12 days the total dry weight of the adrenals was increased in ACTH-treated animals from 17.2 to 71.0 mg (about fourfold) and the average volume of zona fasciculata cells from 1,500 to 5,100 microns3 (about threefold). The relatively larger increase in total mass reflects mitotic activity in the growing adrenal tissue. The calculated growth rate of total tissue expressed as the percentage increase in dry weight per day was 12% at day 3 and decreased to 4.6% at day 12 of ACTH stimulation. During the time interval investigated the cytoplasmic volume fraction of autophagic vacuoles was significantly reduced from 10 X 10(-5) in controls to 1 X 10(-5) in ACTH-treated animals (p less than 0.001). Assuming a half-life for AVs of 7.9 min (Papadopoulos and Pfeifer 1986), autophagic degradation of cytoplasm can be calculated as 1.3%/day in controls, but only 0.15%/day in ACTH-treated animals. Thus growth of 1.15%/day can be attributed to inhibition of autophagic degradation. Although this is only part of total growth, it is evident from the present results that anticatabolic growth induction is utilized nearly to its maximum in adrenocortical fasciculata cells under the influence of ACTH.

Ultrastructure and morphometric analysis of preimplantation mouse embryos

During cleavage, both qualitative and quantitative morphologic characters of mouse ova change. Up to the 8-cell stage, the volume density of mitochondria remains nearly the same although it increases during early and late blastocyst stages. While a rise of the volume fraction of granular endoplasmic reticulum is noticed during cleavage, the volume density of agranular endoplasmic reticulum diminishes gradually from the 1-cell stage onwards. An increase in the volume fraction of autophagic vacuoles is found, the maximum being reached in early blastocysts. The volume fraction of crystalloid inclusions slightly increases after the 4-cell stage, but this increase is statistically insignificant. The volume density of filamentous material (plaques) conspicuously decreases in all studied embryos from the beginning of cleavage. Starting with the 2-cell stage, the volume fraction of lipid droplets remains practically unchanged. No differences in volume densities of the Golgi apparatus, multivesicular and residual bodies, and large cytoplasmic vesicles with medium electron-dense content are found between the respective cleavage stages.

Morphometric study of the effect of leupeptin, vinblastine, estron acetate and cycloheximide on the autophagic vacuole-lysosomal compartments in mouse seminal vesicle cells.

Changes in the cytoplasmic volume fractions of autophagic vacuoles and lysosomes in mouse seminal vesicle cells were evaluated by morphometric analysis following administration of either one of the drugs estrone acetate, vinblastine sulphate, leupeptin, cycloheximide, or their different combinations. The treatments lasted for 2 h. Leupeptin caused a significant increase in the cytoplasmic volume fractions of lysosomes (defined as dense bodies limited by a single membrane) and autophagic vacuoles (defined as membrane-limited bodies containing recognizable fragments of cytoplasm) which became about 6 and 40 times larger, respectively, than controls. This was accompanied by a decrease in the ratio of surface to volume of lysosomes. An increase in the sizes of autophagic vacuole and lysosomal compartments (by about 3 and 10 times, respectively, as compared with controls) was observed in the estrone acetate — treated group. The shape of lysosomes became highly irregular in these animals and their surface densities did not differ significantly from those of controls. The cytoplasmic volume fraction of autophagic vacuoles was largest in the vinblastine treated animals (an increase by about 80 times, as compared with controls) in which the autophagic vacuole compartment became almost two times larger than the lysosomal one. The size of the latter did not differ significantly from that of controls.

Acute effects of isoproterenol on cellular autophagy. Inhibition in myocardium but stimulation in liver parenchyma.

The influence of isoproterenol (IPR) on cellular autophagy was examined in left ventricular myocardium and in liver parenchyma of rats two hours after a subcutaneous injection of a low dose (3 mg/kg body weight). 4 animals were treated with IPR, 4 controls received Ringer solution. The average cytoplasmic volume fraction of the autophagic vacuoles (AV) was 1.6 X 10(-4) in the heart muscle of the controls. After treatment with IPR this value was reduced by 70% to 0.5 X 10(-4). This inhibition of cellular autophagy is interpreted as an initial anticatabolic reaction which might be responsible for the myocardial hypertrophy after chronic administration of IPR. An opposite influence of IPR was observed in the hepatocytes. The volume fraction of AV's increased twofold to 8.7 X 10(-4) after IPR, compared to 4.0 X 10(-4) in control animals. In the controls, the volume fraction of AV's in heart muscle was 57% of the value found in the liver. Comparing liver tissue after fixation by immersion and by perfusion, no statistically significant differences in the volume fractions and in the numerical densities of AV's were observed.

Autophagic vacuoles in heart muscle and liver. A comparative morphometric study including circadian variations in meal-fed rats

Large test areas of 36 000 to 38 000 μm 2 of scctioned cytoplasm/animal and organ were morphometrically evaluated for autophagic vacuoles (AVs) in heart muscle and in liver parenchymal cells. Rats were kept in a nutritional steady state by feeding a timely, i.e. shortly before the onset of darkness, single daily meal (10 g of a standard diet). Circadian variations in volume fraction as well as in numerical density of AVs were found. The values reached a maximum in the late light period, whereas minimal values were obtained in the early dark period. The variations were synchronous in heart muscle and in liver. The average cytoplasmic volume fraction of AVs in heart muscle was 0.79 × 10 −4, that is less than half the value found in liver 1.74 × 10 −4. The differences were less pronounced in the segregated fraction of mitochondria which amounted to 1.68 × 10 −4 in heart muscle and 2.85 × 10 −4 in the liver. These data suggest that in heart muscle cytoplasmic components are degraded by cellular autophagy to a similar, perhaps some lower rate than in liver. The absence of myofibrils in the AVs does not exclude a possible lysosomal degradation of myofibrillar proteins.

The Evaluation of Large Test Fields for Morphometric Studies in Electron Microscopy

The surface area of test fields required for morphometric evaluation of sectioned particles increases with the square of the mean particle diameter, but is inversely proportional to the volume fraction of the particles. A method is presented for an economical evaluation of large test fields. Factors giving rise to variation in the sampling procedure are analyzed. The volume fraction of autophagic vacuoles, for instance, shows a rather broad variation between animals, probably because of the short half-life of these particles. Other applications of the method include bile capillaries under different experimental conditions, and human biopsy specimens, e.g. liver tissue containing very few profiles resembling small peroxisomes in a case of Zellweger syndrome.