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The role of microtubules on membrane fluidity has been investigated on freshly isolated whole rat hepatocytes prepared by the perfusion method and exposed either to the microtubule-depolymerizing drugs colchicine and vincristine or to beta-lumicolchicine, a colchicine analog deprived of biological activity. Exposure of hepatocytes to 6.3 microM colchicine or to 3.0 microM vincristine led to a significant decrease of membrane fluidity as measured by fluorescence polarization of trimethylammoniodiphenylhexatriene (TMA-DPH). No changes were observed in cells exposed to 10.0 microM beta-lumicolchicine. These observations support the hypothesis that the microtubular system plays a role in the modulations of physico-chemical properties of the plasma membrane.

The study of membrane fluidity is a rapidly expanding field of research and its interest in hepatology has been stressed recently. The present study is the first report concerned with the determination of membrane fluidity of isolated rat hepatocytes. The data have been compared with those obtained in plasma membrane fractions and subfractions (basolateral or canalicular) derived from homogenates. The fluorescent probes used to measure the fluidity were diphenylhexatriene (DPH) a 'classical' probe, and its derivative trimethylammoniodiphenylhexatriene (TMA-DPH) at 25 degrees C and at 37 degrees C. The values obtained with DPH were lower than those with TMA-DPH, probably due to the localization of the probes in different regions of the phospholipid bilayer. In addition, DPH revealed significant differences in the fluorescence polarization values obtained in isolated hepatocytes compared with membrane fractions, which was in contrast to TMA-DPH, where the respective values were of the same order of magnitude. This behaviour is probably due to the mobility of DPH in the membrane core and its rapid internalization into the cell, whereas TMA-DPH remains anchored for a long time on the cell surface. These findings suggest that TMA-DPH is a better probe than DPH for measuring the fluorescence polarization of whole isolated hepatocytes and that the use of different probes might be of help in exploring different zones of the membrane bilayer.

The livers of rats given ethanol for 5 weeks showed marked structural alterations of hepatocytes of acinar zone 3 including mitochondrial pleomorphism, increased smooth endoplasmic reticulum and deposition of small (less than 0.5 micron) lipid droplets. In addition, apoptotic bodies involving altered parenchymal cells were frequently observed, together with prominent mononuclear infiltrates adjacent to the terminal hepatic veins. It is suggested that 'age' of liver cells may play a role in the preferential perivenular localization of early ethanol-induced liver damage.

The aim of the present investigation was to study the number and acinar distribution of apoptotic bodies in normal liver as an approach to a better understanding of cell kinetics in the hepatic parenchyma. The material included 20 normal human needle liver biopsies and 20 normal male Sprague-Dawley rats. For each liver sample, the following parameters were measured: number of apoptotic bodies in the lobule, topographical localization, and distance from terminal hepatic veins (THV), i.e., the row of hepatocytes concerned, H1 being the closest to the THV. The results were strikingly similar in human and in animal material, showing that apoptotic bodies are rare in the normal liver and, when present, are always observed in zone 3, next to the THV. In fact, the first two rows of hepatocytes (H1 and H2) contained 80% of the apoptotic bodies in human liver, and 95% in rat liver. These data show that apoptotic bodies are not randomly dispersed in normal liver tissue but show a preferential acinar distribution. In addition, the vast majority of apoptotic bodies are located in the row of liver cells immediately adjacent to the THV. If apoptosis is indeed an expression of physiological cell renewal or 'programmed cell death', these findings support the concept of an aging gradient of liver cells, with zone 3 containing older hepatocytes.

The present study is concerned with the early events associated with the development of cirrhosis induced by dimethylnitrosamine (DMN). The antigenic expression of MHC class II components (Ia) and of some intermediate filament proteins (vimentin and desmin) have been studied by immunohistochemistry and the findings correlated with ultrastructural data. Micronodular cirrhosis developed after 3 weeks of treatment with DMN but enhanced expression of Ia antigen on macrophages and on infiltrating lymphocytes was observed after 1 week, before the formation of septa, suggesting that immune-mediated mechanisms are involved in the response to DMN-induced liver injury. The expression of vimentin and of desmin also increased at an early stage and at 3 weeks the septa were outlined by cellular elements showing positivity for both intermediate filament proteins. In keeping with these observations, ultrastructural data showed active division of macrophages in situ, infiltration of the parenchyma by T and B lymphocytes, activation of lipocytes (Ito cells) showing evidence of mitosis, and the presence of transitional elements between lipocytes, myofibroblasts and fibroblasts. This experimental model may be helpful in understanding the relationship between immune-mediated response to liver injury and development of hepatic fibrosis.

The distribution of the endoplasmic reticulum in human hepatocytes is defined in quantitative terms using the techniques of morphometry. The subjects of the study are liver biopsies from normal, untreated subjects and patients being treated with various drugs. In contrast to rat hepatocytes, the amount of smooth endoplasmic reticulum (SER) in man exceeds that of the rough endoplasmic reticulum (RER) and accounts for 76.3% of the total endoplasmic reticulum. This is to be taken into consideration in pharmacological or toxicological studies. In addition, two components of the SER have been identified: more prominent is the type 1 or vesicular which has a regular honeycomb pattern, made up of cisternae with patent lumina and a mean width of 1500 A; the type 2, or non-vesicular, occurs in discrete foci of densely packed smooth membranes with a spacing of about 140 A. In subjects under short-term treatment with Benzodiazepin (diazepam) the RER remained unchanged but the SER membranes were significantly increased with a remarkable, two-to threefold increase of the SER type 2 in three out of four patients. A rise in incorporation of (14)C-acetate into digitonin-precipitable sterols as measured in liver biopsy material was also noted in these three patients. The suggestion is made that the SER 2 represents the newly formed membranes whereas the SER 1 would represent ;adult' membranes. No changes were observed in two patients under short-term treatment with phenobarbital or Dilantin.

Hepatic fibrosis has been induced in rats by low doses of dimethylnitrosamine (DMN) and special attention has been paid to early morphological events. DMN (10 microliter/kg body wt., i.p.) was given 3 days a week for 3 weeks to Sprague-Dawley rats. Liver samples were taken on days 7, 14, 21, 28 and 35 and examined by light and electron microscopy. Hemorrhagic necrosis, mainly centrolobular, was evident on day 7, with disruption of the sinusoidal lining, and widening or disappearance of the spaces of Disse invaded by erythrocytes and lymphocytes. Large granular lymphocytes similar to pit cells were also present in close contact with hepatocytes. At day 14, fibrotic septa were associated with cells bearing 'transitional' features between those of lipocytes, myofibroblasts and fibroblasts. Hepatocytes showed foci of increased smooth endoplasmic reticulum, and altered sinusoidal and canalicular membranes. At day 21, all animals showed nodularity of the parenchyma, with evidence of micronodular cirrhosis associated with ascites in two animals. At day 35 (19 days after cessation of treatment) there was little residual inflammation, but well-defined micronodules were still present in all animals. This shows that, in the rat, 3-week treatment with a low dose of DMN produces micronodular cirrhosis following diffuse hemorrhagic necrosis without steatosis. The response of the animals was uniform and reproducible. Lesions of the sinusoidal wall and of membranes of liver cells associated with the inflammatory reaction appeared prominent.

The influence of sex and age on membrane fluidity, has been investigated in 6, 12, 18 weeks old Sprague-Dawley rats. Fluorescence polarization (P) was determined at 37 degrees C with a Perkin Elmer MPF 44A fluorescence spectrophotometer. The fluorescent probe TMA-DPH was added to isolated hepatocytes prepared by collagenase method. The membrane fluidity was constantly lower in males than in females, but the difference was statistically significant only in the 12 weeks old group. Major differences appeared related to aging with a significant age-related decrease in fluidity in all animals.