Diurnal variation in glycogen phosphorylase activity in rat liver. A quantitative histochemical study.

The diurnal variations of the glycogen content and of glycogen phosphorylase activity in periportal and pericentral areas of rat liver parenchyma have been analyzed in periodic acid Schiff (PAS)-stained cryostat sections using quantitative microdensitometry. Glycogen content and phosphorylase activity were always higher in periportal areas than in pericentral areas throughout the daily cycle. The glycogen content was highest at the end of the active period during darkness and lowest at the end of the resting period. Phosphorylase activity appeared to be inversely correlated with the glycogen content in both areas. It is concluded that the glycogen content is regulated by phosphorylase activity, which may be due to local cAMP concentration.

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.

Growth pattern of individual liver cells after partial hepatectomy in the rat.

The growth (i.e. changes in protein content) of isolated parenchymal cells of rat liver after partial hepatectomy has been investigated cytophotometrically. 3, 14 and 56 days after operation in rats of different age, no statistically significant differences could be measured in the Naphthol Yellow S-protein content of the most commonly found types of parenchymal cells in comparison with controls of the same age. An exaggeration of the hyperplasia of binuclear diploid (BD) and mononuclear tetraploid (MT) cells, characteristic for normal postnatal growth, could not be detected in any of the age groups investigated (30, 65 and 99 at operation). Consequently, it appears that the accelerated growth after partial hepatectomy mainly takes place by a growth mechanism primarily based on multiplication of cells (hypertrophy) as usually found in the embryonic period.

Localization of superoxide dismutase activity in rat tissues.

Superoxide anion radicals have been implicated in a variety of pathological processes. Under physiological conditions, superoxide dismutase (SOD) is effectively able to disproportionate superoxide anions into hydrogen peroxide and dioxygen. Until now, no techniques have been available to localize SOD activity within tissues. In the present study, SOD activity was detected in different rat tissues using a thin film of xanthine oxidase between the glass slide and the unfixed cryostat section and a medium containing hypoxanthine as a source of electrons for the production of superoxide anions. The incubation medium also contained cerium ions to precipitate the hydrogen peroxide product and polyvinyl alcohol to prevent leakage of soluble and/or loosely bound enzymes from the sections into the incubation medium. The cerium perhydroxides that are formed were visualized for the light microscope in a second step using an incubation medium consisting of 3,3'-diaminobenzidine, cobalt ions, and hydrogen peroxide, which results in oxidation of the diaminobenzidine to the final insoluble blue reaction product. By this methodology, high enzyme activity was found not only in endothelial cells of liver and kidney but also in hepatocytes of liver, myocytes of heart, smooth and striated cells of muscle, acinar cells of pancreas, epithelial cells of kidney ducts, and epithelial cells of the small intestine and colon. These findings were largely in agreement with immunohistochemical data obtained using antibodies against the Cu/Zn- and Mn-SODs. However, high activity was also detected extra-cellularly at the surface of epithelia of trachea, esophagus, small intestine, and colon and at the extracellular matrices, cartilage, and connective tissues. We conclude from these latter data that the activity of the extracellular form of the dismutase is localized. The present method allows the analysis of all three types of known SOD activity (Cu/Zn, Mn, and extracellular) in different tissues and cell compartments.

Quantitative in situ analysis of xanthine oxidoreductase activity in rat liver.

The tetrazolium salt method previously developed for the detection of xanthine oxidoreductase activity in unfixed cryostat sections has been validated for quantitative purposes. The specificity of the enzyme reaction was studied by incubating unfixed cryostat sections of rat liver in test medium containing the substrate hypoxanthine, in control medium that lacked the substrate, and in medium containing substrate and allopurinol, a specific inhibitor of xanthine oxidoreductase activity. The specific reaction rate was determined cytophotometrically by subtracting the amount of final reaction product generated in the control reaction from that formed in the test reaction. Highest specific enzyme activity in rat liver was found when the incubation medium contained 18% (w/v) polyvinyl alcohol, 100 mM phosphate buffer, pH 7.8, 0.45 mM 1-methoxyphenazine methosulfate, 5 mM tetranitro BT, and 0.5 mM hypoxanthine. Enzyme activity was present in liver parenchymal cells and in sinusoidal cells (endothelial and Kupffer cells) and was completely inhibited by allopurinol. A linear relationship was observed between the specific amount of final reaction product generated at 37 degrees C and incubation time at least up to 21 min, as well as section thickness up to 12 microns. Xanthine oxidoreductase activity, expressed as mumoles substrate converted per cm3 tissue/min, was 1.61 +/- 0.34 in pericentral areas and 1.24 +/- 0.16 in periportal areas. These values are similar to biochemical data reported in the literature. In conclusion, the tetrazolium method to detect xanthine oxidoreductase activity in unfixed cryostat sections of rat liver gives a reliable reflection of in situ activity.

A quantitative histochemical study of xanthine oxidase activity in rat liver using the cerium capture method in the presence of polyvinyl alcohol.

A recently developed histochemical technique to demonstrate xanthine oxidase activity in milk globules of bovine mammary gland and in epithelial cells of rat small intestine using cerium ions and a semipermeable membrane was slightly modified. The semipermeable membrane method was replaced by the addition of 10% (w/v) polyvinyl alcohol to the incubation medium. This technically more simple procedure enabled detection of xanthine oxidase activity in unfixed cryostat sections of rat liver. Both methods gave qualitatively and quantitatively similar results. Activity was found in sinusoidal cells and in liver parenchymal cells, with 50% higher activity in pericentral than in periportal areas. The specificity of the reaction was proven by the generation of only small amounts of final reaction product on incubation either in the absence of the substrates hypoxanthine or oxygen or in the presence of hypoxanthine and allopurinol. Allopurinol is a specific inhibitor of xanthine oxidase activity. The amount of final reaction product, as measured cytophotometrically in rat liver, increased linearly with incubation time (15-90 min) and with section thickness (up to 12 microns). By varying the hypoxanthine concentrations, a Km value of 0.05 mM was found. Addition of dithiothreitol to the incubation medium reduced the amount of final reaction product by 85%, which was caused by conversion of reversible xanthine oxidase into xanthine dehydrogenase. This histochemical method can be used for quantitative analysis of in situ xanthine oxidase activity.

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium.

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.

The role of xanthine oxidase in ischemia/reperfusion damage of rat liver.

Oxygen radicals have been proposed to be involved in the induction of liver cell damage during reperfusion after ischemia. The role of xanthine oxidase in this process and the potential of the antioxidant system have been studied in a model of in vivo ischemia of rat liver followed by 1 h reperfusion by the use of enzyme histochemistry. Based on decreased lactate dehydrogenase activity in certain areas of liver parenchyma, cell damage could already be detected at 1 h reperfusion after ischemia. Incubations performed on serial sections showed that the same areas contained decreased activities of xanthine oxidoreductase, xanthine oxidase, catalase and glucose-6-phosphate dehydrogenase. Some individual cells in the undamaged liver parenchyma expressed a very high glucose-6-phosphate dehydrogenase, which suggests that these cells have a good defence against oxidative stress. It is concluded that oxygen radicals derived from xanthine oxidase do not play a decisive role in the induction of cell damage immediately at reperfusion after ischemia. However, it cannot be excluded that xanthine oxidase present in the blood stream can give rise to the development of additional damage later on.

Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes.

When 2 inbred rat strains, the Brown-Norway rat and the Lewis rat were exposed to the same amount of CdCl2 for 15 days, a completely different immunological reaction pattern could be demonstrated. Despite the same amount of intrathymic cadmium in both strains, the Brown-Norway rat showed a significant decrease in thymocytes in the S-phase and a significant increase of thymocytes in the G2 phase and mitosis, in contrast with findings in the Lewis rats. A new method for estimating subtle forms of thymus atrophy showed a slight decrease in the number of the smallest thymocytes in the Brown-Norway rat after exposure to cadmium, in contrast with that in the Lewis rat. Evidence is presented that the approximately 1.7 times larger number of thymocytes/mg thymus in the Lewis rat, compared to the Brown-Norway rat, as well as the approximately 2.5 times lower proliferation rate of the thymocytes, and an approximately 1.5 times higher metallothionein content of the thymus medulla epithelial cells in the Lewis rat, might be responsible for the observed difference in toxicity. The zinc content of the thymus was not significantly decreased by exposure to CdCl2, and did not differ significantly between both strains.

Application of keratin immunocytochemistry and sirius red staining in evaluating intrahepatic changes with acute extrahepatic cholestasis due to hepatic duct carcinoma.

A series of 10 cases of biliary obstruction due to primary cholangiocarcinoma has been studied with histological and immunocytochemical means. The total duration of cholestasis (as manifested by jaundice) was between 2 and 11 weeks with variable period of preoperative drainage. Liver biopsy specimens taken during surgery for cholangiocarcinoma were investigated for the presence of ductular proliferation and the development of fibrosis, as demonstrated by Sirius Red F3BA collagen staining. The differentiation of epithelial components was evaluated by AEC-immunostaining with chain-specific monoclonal antibodies specifically directed against human keratins type 7, 18 and 19. Keratin 7, normally occurring only in the ductular system, was expressed in hepatocytes at the periphery of the hepatic lobule (zone I) following about 4 weeks' cholestasis, when an increase of ductular profiles in the enlarged portal areas had become manifest. Such keratin 7 positive cells, however, still retained all morphological aspects of hepatocytes. Keratin 19, normally also restricted to the ductular system in liver, is not expressed by zone I hepatocytes even after longer duration (up to 11 weeks) of cholestasis. It is concluded that the increase in ductular profiles during the first week is mainly due do proliferation of pre-existing ductules, while ductular metaplasia occurs in more chronic cholestasis. Development of fibrosis, not always strictly paralleling the multiplication of ductular profiles in sections through a portal tract, represents an early change, and is clearly apparent after 2 weeks of obstruction.