Correlates of cognitive impairment in elderly residents of long term care institutions in the metropolitan area of Guadalajara, Mexico.

AIM: To determine the prevalence of cognitive impairment and to examine the correlation between demographic and social factors, the activities of daily living (ADL), and depression with cognitive function in elderly Mexicans living in nursing homes. DESIGN: Cross-sectional. SETTING: Fourteen nursing homes in Guadalajara. PARTICIPANTS: Data were drawn from a random sample of 451 elderly, aged 60-104 years. MEASUREMENTS: The Spanish versions of MMSE (Folstein), ADL index (Barthel), and the Geriatric Depression Screening (GDS) (Yesavage); demographic factors were obtained. RESULTS: The prevalence of cognitive impairment was 52.3%, with a cut-off of 19/20 (Bohnstedt). Cognitive impairment was significantly related to gender, educational level, activity participation, pension, ADL, and depression. CONCLUSION: The results indicate a higher prevalence of cognitive impairment than in other Mexican studies. The data are consistent with previous findings that cognitive impairment in the elderly is more common among females, those with a low level of education, the lack of participation in social/leisure activities, ADL dependencies, and depression.

Morphological aspects of ionizing radiation response of small intestine.

Knowledge of the acute and late ionizing radiation exposure damage to the gastrointestinal tract, particularly injury of the small intestine, is of great significance in radiotherapy, as is management of accidental radiation exposure. Irradiation (X-ray, neutron, cobalt gamma) induces a series of events in this rapidly renewing tissue resulting in the well-known symptoms of the gastrointestinal (GI) radiation syndrome, such as GI haemorrhage, endotoxemia, bacterial infection, anorexia, nausea, vomiting, diarrhoea, and loss of electrolytes and fluid. In spite of the significant advances that have occurred in research on underlying mechanisms over the last two decades, the overall etiology and pathogenesis of the GI-syndrome still remains unclear. Currently, to our knowledge, these symptoms are probably due to a rapid modification of the intestinal motility and to the structural alteration of the intestinal mucosa (cell loss and altered crypt integrity). Several evidences suggest that radiation-induced dysfunctions and structural changes of this organ (either changes in subcellular, cellular, and histological structure) are mediated by concerted and interrelated changes of a plethora of various extracellular mediators and their intracellular messengers. The aim of this review is to summarize our current knowledge about the pathomorphology and cell biology of the ionizing radiation response of the GI tract with a focus on the small intestine.

Ubiquitin cytochemical changes during azaserine-initiated pancreatic carcinogenesis.

The ubiquitin (Ub)-proteasome proteolytic system is highly selective, and the specific proteins involved in cell division, growth, activation, signaling and transcription are degraded at different rate depending on the physio-pathological state of the cell. Ubiquitination serves first of all as a signal for protein degradation of short-lived and abnormal proteins under several stressful conditions. The immunocytochemical localization of Ub in some malignant tumours has recently been presented and differences in Ub expression has been observed during malignant transformation. Change in the level of Ub and Ub-conjugated proteins might reflect a higher metabolic-catabolic ratio in neoplastic cells. Most studies have been focused on the malignant stage of tumour progression, and only a few papers have dealt with the change in Ub and Ub-protein conjugates level during the whole progression. To address this problem, we applied an azaserine-induced pancreatic carcinogenesis model, in which premalignant and malignant stages were investigated throughout the progression. The level of Ub immunoreactivity was measured in nucleus and cytoplasm by electron microscopic immunocytochemical and morphometrical methods. We found a significant increase of Ub level in the nucleus and the cytoplasmic area in premalignant atypical acinar cell nodule (AACN) cells and in malignant adenocarcinoma in situ (CIS) cells at month 20 after initiation.

Changes in cellular autophagic capacity during azaserine-initiated pancreatic carcinogenesis.

Growth regulation is a crucial event in tumour progression. Surprisingly, relatively few papers have dealt with the catabolic side of regulation, and there are practically no data regarding the autophagic process during tumour development. We approach this problem by morphometrical investigation into the possible changes of autophagic activity during the progression of rat pancreatic adenocarcinoma induced by azaserine. In the present study, autophagic capacity of the azaserine-induced premalignant and malignant cells were characterised and compared to the respective host tissue cells of the rat pancreas and to the acinar cells in other stages of tumour development. Using vinblastine (VBL) as an enhancer, and cycloheximide (CHI) as an inhibitor of autophagic segregation we observed that autophagic capacity of premalignant cells (month 6 and 10 after initiation) is much higher than in the host tissue cells. We found a sharp decrease in self-digesting capacity in adenocarcinoma cells (month 20) where VBL induced a minimal accumulation of autophagic vacuoles which was, surprisingly, not inhibited by CHI, i.e. the CHI-sensitive regulatory step was lost. The changes in autophagic capacity are probably associated to specific steps of tumour progression in our system.

Quantitative microvascular changes during azaserine-initiated pancreatic carcinogenesis.

Although angiogenesis is considered to be indispensable for continuous tumour growth, only very few studies have been published performing microvessel quantification during tumour progression. We measured the tumour vascularity in different stages of rat pancreatic carcinogenesis induced by azaserine and promoted by raw soya flour-containing pancreatotrophic diet. Besides the tumour samples taken at 6 (atypical acinar cell nodules), 15 (adenomas) and 20 (localised adenocarcinomas) months after carcinogen initiation, we also investigated 3 control groups: tumour-bearing host tissue of azaserine-treated rats and normal tissue of untreated rats kept on standard or pancreatotrophic diet. In contrast with the usual microvessel counting on hot spots, we determined microvascular surface density (Sv) and volume density (Vv) by electron microscopic morphometry. There was no significant difference in these respect between the control groups. At month 6 after the azaserine induction Sv and Vv showed slight, nonsignificant decrease as compared to the host control. Both values remained unchanged until the 15th month and increased significantly by the 20th month. These results may indicate comparable growth rate of tumour and new microvessels in the premalignant stages of carcinogenesis while a more intense angiogenesis than tumour growth afterwards.

Tight junctional changes upon microwave and x-ray irradiation.

Tight junctions (zonulae occludentes, ZO) are cellularly regulated dynamic structures sensitive to environmental stress agents including ionizing radiation. Radiation induced pathological alterations of the small intestine (gastrointestinal radiation syndrome) are related to altered ZO-mediated paracellular transport. We carried out a quantitative morphological evaluation of the murine jejunal epithelial tight junctional structure in freeze fracture replicas as changed upon whole body X-ray irradiation and low energy microwave exposition. X-ray treatment (4 Gy, 1, 24 h) brought about a partial dearrangement of the ZO strand network which regenerated only partially by 24 h. This observation is in line with data on paracellular permeability increases and ZO-bound calcium drop caused by X-ray irradiation. On the other hand, microwave treatment (16 Hz-modulated 2.45 GHz wave, 1 mW/cm2 power density, I h exposition, samples at I and 3 h after exposition) did not cause dearrangement but, rather an increase in the integration of thight junctional structure, which is in agreement with an increase in cytochemically detectable ZO-bound calcium.

Autophagy in the epithelial cells of murine seminal vesicle in vitro. Formation of large sheets of nascent isolation membranes, sequestration of the nucleus and inhibition by wortmannin and 3-ethyladenine.

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.

Cellular autophagic capacity is highly increased in azaserine-induced premalignant atypical acinar nodule cells.

Although cellular autophagy is recognized as a major pathway of macromolecular catabolism, little data are available regarding its activity or regulation in tumor cells. We approach this problem by morphometrical investigation into the possible changes in autophagic activity during progression of rat pancreatic adenocarcinoma induced by azaserine and promoted by a raw soya flour-containing pancreatotrophic diet. In the present study, the autophagic capacity of the carcinogen-induced premalignant atypical acinar nodule cells was characterized and compared with controls (normal tissue of rats kept on standard laboratory or pancreatotrophic diet and host tissue of the premalignant nodules of the azaserine-treated rats). Given for 90 min, vinblastine, an enhancer of autophagic segregation (i.e. formation of autophagic vacuoles), caused a one to two orders of magnitude larger expansion of the autophagic compartment in atypical nodule cells than in the controls. Then a 20 min blockade of segregation by cycloheximide led to regression of the autophagic compartment, which was barely measurable or moderate in the controls but exceeded 50% in the premalignant cells. At the same time, the cytoplasmic volume fraction of early autophagic vacuoles regressed to a near zero value in each cell type. Expansion and regression rates of these nascent vacuoles showed that both segregation and degradation were 6-20 times faster in the nodule than in normal tissue cells. These results show that the autophagic capacity of the premalignant cells in our system is greatly increased, possibly making these cells unusually sensitive to up-regulation of their self-digesting activity in response to different extracellular signals or drugs.

Time course of vinblastine-induced autophagocytosis and changes in the endoplasmic reticulum in murine pancreatic acinar cells: a morphometric and biochemical study.

The time course of the vinblastine(-sulfate; 10 mg/kg body weight, single injection)-induced enlargement and subsequent regression of the autolysosomal compartment was studied by electron microscopic morphometrical and cell biochemical methods in order to gain information concerning some key problems of this major route of intralysosomal degradation of the cell's endogenous macromolecules and structures. Detailed analysis of the dynamics of the total autophagic vacuole (AV) compartment and its different subcompartments (early, advanced, late, and fused AVs), as well as of changes of rough-surfaced endoplasmic reticulum (RER) showed: 1. Pancreatic acinar cells react to vinblastine biphasically, i.e. two expansion phases of the AV compartment, the first in the 0 to 90 min and the second in the 2 to 8 h post-injectional periods, were detected. 2. Fusions of AVs are not inhibited by vinblastine, at least during the second expansion phase when cytoplasmic volume fraction (CVF) of fused AVs steadily increased until the 12th h. Fusion of early, advanced and late AVs or composition of fused complex vacuoles (AVc) are somehow regulated, as the proportion of the three AV stages from the CVF of AVc, was maintained constant throughout the second expansion phase. 3. Stimulation of autophagosome formation and resulting substrate overload seems to be the primary mode of action by which vinblastine causes the enormous expansion of the autolysosomal compartment. 4. Degranulation of the rough-surfaced endoplasmic reticulum (RER) membranes occurs in a biphasic fashion, similarly to the volume and surface changes of the AV compartment, thus supporting our previous hypothesis, that labilization or change of RER may have a role in the formation of autophagosomes. 5. Vinblastine-induced autophagocytosis is a selective process, as mitochondria, Golgi elements and zymogen granules are very much underrepresented, whereas RER is more than twice overrepresented in the volume of early AVs, when compared to their volume fraction in the whole cytoplasm. 6. Immunogold electron microscopy revealed the presence of ubiquitinylated proteins in advanced and late, but not in early AVs.

Dynamics of vinblastine-induced autophagocytosis in murine pancreatic acinar cells: influence of cycloheximide post-treatments.

Accumulation of autophagic vacuoles (AVs) was monitored by electron microscopic morphometry in murine pancreatic acinar cells during the 5-hr period after a single injection of vinblastine (VBL). The expansion of the autophagic compartment (AC) occurred in two waves. AVs accumulated in the first 90 min and regressed in the next hour, but thereafter AC expanded again, and 5 hr following the VBL injection, as much as 5.3% of the cytoplasmic volume was found sequestered into the AC. The high rates of accumulation of AVs indicated that VBL stimulated AV formation (segregation) during both expansion phases. To have a deeper insight into the dynamics of the process segregational inhibitor cycloheximide (CHI) was given 1 and 3 hr after VBL and the subsequent regression of the AC and its subcompartments (i.e., early, advanced, and late AVs) were measured during the next 90 min. We found that regression of AVs was fast in the first expansion and slowed down in the second expansion phase during which only early AVs regressed. CHI proved to be a fast and effective inhibitor of autophagic segregation, whether it was given before, simultaneously, or after the VBL injection. The aforementioned results argue for a dual mode of action of VBL (i.e., a prompt stimulation of segregation and a delayed retardation of AV maturation). The two effects of the alkaloid prevail differently along the time course. A further analysis of the behavior of the AC subcompartments showed that CHI perhaps inhibits segregational step(s) occurring prior to the actual formation of the autolysosomes.

Time course of vinblastine-induced cellular autophagy in the murine pancreatic acinar cells in vivo. Different regression rates of the autophagic compartment caused by cycloheximide given different times after the vinca alkaloid. A morphometric study.

Autophagy is a three-step process in which parts of cytoplasm are segregated by membranes to form autophagosomes gaining acid hydrolases later, being converted this way into autolysosomes in which lysosomal degradation takes place. The actual size of the autophagic vacuole compartment (AVC) is obviously dependent on the velocity of these main steps. According to our morphometric measurements, a single dose (10 mg/kg b.wt.) of vinblastine (VBL) caused a conspicuous expansion of the AVC in pancreatic acinar cells, occurring in two waves: it expanded in the first 90 min but regressed in the next hour. This was followed by a second expansion monitored until the 5th post-injectional hour. The expansion rates indicate the existence of stimulation of autophagic segregation in both expansion phases. To take a further look, into the dynamics of the process, we blocked segregation by giving cycloheximide (CHI 0.2 mg/g b.wt.) 1 and 3 h after VBL and the subsequent regression of the AVC was followed by morphometry in the next 90 min. At the height of the first wave (1-2 h after VBL) the regression of AVC was not retarded, but rather, degradation rate seemed elevated. When CHI was given 1 h after VBL, 92% of the cytoplasmic volume fraction (CVF) of AVC regressed within the next 30 min. The main factor causing the expansion of AVC might be enhanced segregation in the first wave. Contrarily, at the beginning of the second wave, the turnover of AVs is dramatically slowed down. When CHI was given 3 h after VBL, only 27% of CVF of AVC regressed in the next 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of different protein synthesis inhibitors on the exocrine pancreatocytic autophagocytosis in vivo.

The translational inhibitor cycloheximide is also used as an inhibitor of cellular autophagy and intracellular degradation of endogenous cellular proteins. Some evidence for a similar effect of other inhibitors of protein biosynthesis is also available (largely from in vitro systems). In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the fact that when the formation of autophagosomes is inhibited, a regression of the autophagolysosomal compartment can be measured, provided intralysosomal degradation in the pre-existing autophagic vacuoles continues at an unchanged rate. To make the measurements easier, autophagolysosomal compartment of the cells was enlarged by administering vinblastine (10 mg/kg b.wt.) for 2 h when the inhibitors were given for an additional 30 min. During this time cycloheximide (0.2 mg/g b.wt.), emetine (0.12 mg/g b.wt.) and puromycin (0.2 mg/g b.wt.), respectively caused 35, 25 and 52% regression of the pancreatocytic autophagolysosomal compartment. Since all the above translational inhibitors inhibited autophagocytosis as well, the possibility of a coupling between the regulation of synthesis and inhibition of proteins arises.

Occurrence and inhibition by cycloheximide of apoptosis in vinblastine-treated murine pancreas. A role for autophagy?

While investigating the time course of the influence of autophagy-inducer vinblastine in pancreatic acinar cells, we discovered that about 24 h after the single injection of the drug (10 mg/kg b.wt.) when most of the cells were already recovering from the autophagic wave, dying cells with apoptotic nuclei and peculiar morphology of their cytoplasm appeared in the exocrine pancreas. Apoptotic blebs (ABs) separating from apoptotic cells or already phagocytized by neighbouring cells were also also observed. A large number of autophagic vacuoles (AVs) were seen in these cells termed apoptotic cell (AC). They are most probably derived from cells with morphologically normal nuclei but with unusually high number of AVs in their cytoplasm. We termed these cells highly autophagic cells (HAC). Our morphometric measurements show that the partial volume of both HAC and AC increased to 7.5 and 9.5%, respectively, of total cellular volume in the 25th h after vinblastine treatment. This increase could be inhibited by a treatment at the 24th h with cycloheximide (0.2 mg/g b.wt.) an inhibitor of both translation and autophagic segregation. Thus, synthesis of proteins or an enhanced autophagy may be indispensable step(s) in the apoptotic process in this system.

Mechanism and dynamics of macroautophagy in murine exocrine pancreatic cells. A review of vinblastine-induced changes.

Autophagy is a major pathway of lysosomal degradation of cellular macromolecules. The paper summarizes the results obtained in the studies on macroautophagy using the exocrine pancreatic acinar cell as model system and vinblastine as inducer. Current knowledge about the origin and properties of the limiting membranes of autophagic vacuoles, and the results of quantitative morphological studies into the dynamics and kinetics of vinblastine-induced autophagocytosis, as well as recent achievements in isolation and characterization of subclasses of autophagic vacuoles (autophagosomes and autolysosomes) are reviewed.

Translational inhibitors cycloheximide, emetine, and puromycin inhibit cellular autophagy in mouse liver parenchymal and pancreatic acinar cells in vivo.

The protein synthesis inhibitor cycloheximide is widely used (in vitro or in vivo) to inhibit the autophagic degradation of endogenous cellular proteins. Circumstantial evidence has been obtained largely from in vitro experiments for a similar effect of other translational inhibitors. In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the assumption that the autophagic compartment will regress if the formation of the vacuoles is blocked while degradation in the pre-existing vacuoles goes on. To make the measurements easier, autophagic compartment of the cells was greatly enlarged in both cell types by administering vinblastine (10 mg/kg b. wt.) for 2 h when the inhibitors were set on for an additional 30 min. During this half-an-hour, cycloheximide (0.2 mg/g b. wt.), emetine (0.12 mg/g b. wt.) and puromycin (0.2 mg/g b. wt.), respectively caused 58.5, 35.6, and 69.5% regression of the pancreatic and 46.7, 64.2, and 54.2% of the hepatocytic autophagic vacuole compartment. Thus, similarly to cycloheximide, both emetine and puromycin have proved to be inhibitors of autophagy in vivo. The results argue for a possible relationship between the synthesis and degradation of endogenous cellular proteins.

Time course of neutral red-induced autophagy in murine pancreatic acinar cells. A morphometrical and cytochemical study.

The knowledge of the time course of the influences of chemicals on autophagy is of great importance in the study of their modes of action and hence provides information relating the mechanism and dynamics of this catabolic process. Neutral red (NR) treatment has long been used to produce an accumulation of autolysosomes in different cell types. In the present study early (AV1), advanced (AV2) and late (AV3), as well as complex (fused) AVs (AVc) were distinguished. In our morphometrical measurements, we found all these AV subcompartments significantly expanded as early as 30 min after the injection of NR (0.4 mg/g b.wt.), i.e. a large number of AVs accumulated in the cells. Since cytoplasmic volume fraction (CVF) of AV increased 3-fold during this early period we conclude that, unlike vinblastine, NR is not a fusion inhibitor. Accumulation of AV1 (3-fold) in the presence of fusions possibly indicates that NR stimulates formation of AVs in this early period, after the accumulation of AVs continued. The maximal CVF of AVs were measured at 4 h, when 7.6% of the cytoplasmic volume was sequestered into the AV compartment, two third of which came from AV3. This finding indicates that NR is probably an inhibitor of intravacuolar degradation. However, the high rate of accumulation of AV2, AV3, and total AVs including a slower but still pronounced accumulation of AV1 cannot be explained solely from inhibition of degradation, but indicates a stimulated segregation (AV formation). Our results therefore argue for a possible coupling of the regulation of autophagic segregation and degradation since vinblastine and possibly some other degradation inhibitors were also found to stimulate AV formation in other studies. Another goal of this study was to follow the time course of changes in distribution of certain lysosomal enzymes after NR treatment. According to our enzyme cytochemical studies, acid phosphatase (AP) of untreated cells is mainly located in large and small lysosomal elements of the Golgi zone, aryl sulfatase B (AS) in trans-Golgi elements including pre-secretory granules and trimetaphosphatase (TP) in basal lysosomes. After NR injection TP seemed to appear first in AV1 whereas AP activity was characteristic of more advanced AVs. AS activity only occasionally appeared in AV3 and exclusively at late times after NR injection.

Time course of quantitative morphological changes of the autophagic-lysosomal compartment of murine seminal vesicle epithelial cells under the influence of vinblastine.

Changes of the autophagic-lysosomal compartment (ALC) of the murine seminal vesicle epithelial cells were monitored by electron microscopic morphometry during 36 h following a single 10 mg/kg bw dose of vinblastine sulfate (VBL), a widely used tool to cause an accumulation of the autophagic vacuoles (AVs). Three morphologically distinct subcompartments of the ALC, i.e. early autophagic vacuoles (AV1) being presumably prelysosomal autophagosomes, advanced AVs (AV2) containing material under degradation and dense bodies (DB) were defined. The ALC and its subcompartments expanded after VBL in a two-phase reaction. The first subcompartments to react significantly were AV1 and AV2 (at 30 min) followed by DBs with a 30 min delay. The ALC then ceased to grow until the 90th min when a second expansion phase started peaking around 8 h with a cytoplasmic volume fraction 15 times larger than in the untreated control. This second growth was entirely brought about by the expansion of the two AV subcompartments. After 8 h the volume fraction of both AV1 and AV2 decreased to cause a gradual regression of the ALC. AV1, however, already ceased to expand as early as after 6 h, i.e. during the last 2 h of the expansion phase of the ALC. Comparison of this time curve with the one we previously measured in mouse liver shows considerable differences between the two cell types. The growth curves of the AV subcompartments in our experiment along with others' kinetic data obtained in steady state cells not treated with VBL show that segregation (= formation of AV1) is possibly stimulated by VBL in our system.

Isolation of pancreatic autophagic vacuoles induced by vinblastine or neutral red. Separation of autophagosomes and autolysosomes by Percoll density gradient centrifugation.

Our purpose was to elaborate a cell fractionation method for the preparation and purification of macroautophagic vacuoles (AVs) and their subfractions: autophagosomes and autolysosomes. To overcome the difficulties caused in liver and some other cell types by the overlapping buoyant densities and sizes of different subclasses of lysosomes and other subcellular particles, we chose the murine pancreatic acinar cell as experimental system in which enormous numbers of large-sized AVs are readily accumulated upon certain treatments. As we measured by electron microscopic morphometry, cytoplasmic volume fraction of AVs was as small as 0.31% in the untreated cells, while it was elevated to 8.1% 4 h after the injection of 50 mg/kg body weight vinblastine sulfate (a widely used inducer of macroautophagy). From vinblastine-treated pancreas, a 5500g sediment containing AVs and mitochondria (AV-M fraction) was obtained by differential centrifugation. This fraction was resolved in a 50% Percoll gradient (15 min, 92,000g) into three distinct particle populations. Mitochondria were localized near the upper boundary of the gradient at a buoyant density of 1.075 to 1.08, whereas directly under them light AVs (1.085-1.09) were banded. Heavy AVs (1.13-1.14) formed a broad layer near the bottom of the tube. Electron microscopic comparison of the morphology of these fractions and AVs in situ showed that light AVs correspond to AVs in early, whereas heavy AVs to AVs in advanced and late stages of degradation of the segregated material. The activity of lysosomal enzymes were found low in both fractions, being several times higher in the heavy than in the light one.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of the quantitative changes in the autophagic-lysosomal and secretory granule compartments of murine liver cells under the influence of vinblastine.

The dynamics of the transient expansion of the autophagic-lysosomal (ALC) and secretory granule (SGC) compartments in mouse liver cells were monitored by electron microscopic morphometry after a single injection of 10 mg/kg b.w. vinblastine sulfate (VBL). Initially (first phase) the cytoplasmic volume fractions of the total ALC and its subcompartments, as well as of the SGC increased by an order of magnitude and peaked at the second h. In the second phase, all the aforementioned compartments regressed gradually, approaching their normal size between 12 and 36 h after VBL injection. Analysis of the dynamic changes in fractional volumes of subcompartments of the ALC showed that early autophagic vacuoles (AV1) were the first to enlarge. Advanced AVs (AV2) reacted 30 min later and a further 30 min time lag was required before late autolysosomes, appearing as dense bodies (DB), started to expand. We regard these data as kinetic proof that the bodies of the later reacting subcompartments developed from the earlier reacting ones. The time lag between the expansion of AV1 and AV2 subcompartments may be explained by a period of retardation of conversion of nascent autophagosomes (AV1) to autolysosomes (AV2) which is known to occur normally by fusion of AV1 with enzyme-carrying lysosomes. However, transformation of AV1 to AV2 and later to DB resumed after the respective time lags. Moreover, our quantitative data lend support to the view that segregation of cytoplasmic portions into newly-formed autophagosomes was stimulated by VBL, at least in the first 2 h of treatment. The expansion of ALC accelerated during this period and led to an obvious overload of the lysosomal apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

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.