Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes

Summary Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES¹ buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES² or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration.In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.1 PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - 2 HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid This study was supported by a Grant-in Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture, the Japanese Government (No. 57770012)     

A histochemical study about changes in rat liver plasma membrane enzyme activities after galactosamine administration

Summary In rats changes in plasma membrane enzyme activities due to Gal-N intoxication were studied by enzymehistochemical methods. The bile canalicular 5-nucleotidase and nucleoside polyphosphatase activities decreased; the sinusoidal 5-nucleotidase remained unchanged. The bile canalicular leucyl--naphthyl-amindase showed an increase in activity; the alkaline phosphatase activity remained unchanged. In contrast to the spotty necrosis, changes in plasma membrane enzyme activities were seen in all liver cells, suggesting that changes of these activities, occurring after Gal-N treatment, do not correlate with cell death. The conclusion was drawn that the deviations of the enzyme activities might be due to changes in the lipid environment of the enzyme proteins in the membrane.With the exception of alkaline phosphatase, partial hepatectomy caused the same changes in enzyme activities as did Gal-N intoxication. Nevertheless Gal-N administration to partial hepatectomized rats did not lead to hepatic necrosis. Galactose given simultaneously or within two hours after Gal-N prevented both changes in plasma membrane enzyme activities and hepatocellular damage. This suggests an important role of galactolipids and galactoproteins in the plasma membrane alterations.Dedicated to Prof. Dr. E. Havinga on the occasion of his 70th birthday     

Relocalization of membrane enzymes accompanies biliary atresia in lamprey liver

Summary Light- and electron-microscopic histochemical procedures were used to show the distribution of the membrane-bound enzymes alkaline phosphatase (Alp), adenosine triphosphatase (ATPase), and 5-nucleotidase (5-nuc) in the livers of lamprey, Petromyzon marinus, throughout the life cycle. In larvae, the three enzymes are located at the biliary pole on the canalicular membranes of microvilli. At metamorphosis the enzymes become localized at all lateral cell surfaces of hepatocytes as bile canaliculi degenerate in the programmed regression of the entire biliary tree. This latter pattern of enzyme distribution persists during the parasitic adult phase but no activity is evident in individuals in the spawning migration. As the timing of the relocalization of enzymatic activity correlates well with a build-up of bile products and iron during metamorphosis, it is suggested that the lateral surface may be the new site for transport of these products.Supported by NSERC of Canada grant no. A5945 to J.H.Y.     

5′-nucleotidase

Summary The distribution of 5-nucleotidase activity in rat liver shows a sexual dependence. In male liver the activity in the bile canalicular wall is most pronounced, whereas the activity at the sinusoidal border of the liver parenchymal cell is slightly more in the female rat. Castration and treatment with sex hormones change the distribution pattern. The greatest variations in enzyme activity are seen at the bile canalicular site of the liver cell. These changes are probably an expression of the altered functional state of the liver cell.     

A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol

Summary 5-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8m. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

Strain improvement of Aspergillus niger for phytase production

A strain improvement program was developed to increase extracellular phytase (E.C. 3.1.3.8.) production by Aspergillus niger (syn. A. ficuum) NRRL 3135. Ultraviolet (UV) radiation was used as the mutagen and resistance to 50 g/ml of hygromycin B as the selection method. Mutant 2DE, the product of two UV treatments, had phytase (PhytA) activity at pH 5.0 in the extracellular filtrate that was 3.3-fold higher than the wild-type activity. The activity of the non-specific acid phosphatase with a pH optimum of 6.0 (Pase) was one-fifth the activity of the wild type and the non-specific acid phosphatase with a pH optimum of 2.5 (PhytB) was not significantly different from the wild type. The mutant and wild-type PhytA, PhytB and Pase responsed similarly in inhibition studies. However, the wild-type enzymes were inhibited more by 1 mm sodium fluoride and 1 mm phosphomycin. PhytA production by the mutant was repressed 60% by inorganic phosphate concentrations of 0.006% (wt/vol) or above. The mutant had an extracellular protein concentration 3.2-fold higher than the wild type, which correlated with its higher phytase activity at pH 5.0, but not with phytase activity at pH 2.5 and acid phosphatase activities. The isolate may be a phytase catalytic mutant, as well as, on overproducer of phytase. In addition, a mutant with an apparent lack of activity of all three acid phosphatases was isolated.Correspondence to: R. J. Wodzinski     

Histochemical studies of some enzymes in the tissues of the schistosome vector snailBulinus truncatus (Audouin) with special reference to the effects of a molluscicide. II. Hydrolases

Summary Accomparative study of six hydrolases, acid and alkaline phosphatases, aryl sulphatase, -gluchronidase cholinesterase, and non-specific esterase, was carried out on the tissues of normal healthy and Frescon-treatedBulinus. The presence and activity of these enzymes in the tissues of normal animals were taken to indicate the probale functions of the tissues concerned. Frescon administration caused inhibition of acid phosphatase and also induced the release of cholinesterase and non-specific esterase in some tissue. It is concluded that the most important effects of Frescon on snail physiology are the disorganization of neuronal function and disturbance of olfactory activity.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Summary Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive sub-canalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per m of plasma membrane were 7.86 vs 2.46 (P<0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P<0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.This study was supported by the Swiss National Science Foundation grants 3.846.0.87 (to L.L.) and 3.992.0.87 (to P.J.M.)     

Cholestasis-induced alterations of the trans- and paracellular pathways in rat hepatocytes

Bile secretion depends on the vectorial transport of solutes from blood to bile and involves three different pathways: transcellular pathways mediated by transport proteins distributed asymmetrically in the basolateral and canalicular plasma membrane and by transcytotic vesicles, and a paracellular pathway allowing selective diffusion through tight junctions. All three pathways are impaired differentially by extrahepatic (bile duct ligation) or intrahepatic (ethinyloestradiol) cholestasis. Ethinyloestradiol treatment leads to tight junctional defects that are less severe than those induced by bile duct ligation. Junctional impairment is reflected functionally in increased permeability for horseradish peroxidase and structurally by decreased strand numbers and increased junctional length, but not by alterations at the level of the individual strands. The parallelism of physiophical and morphological perturbations indicates a structure-function relationship in hepatocellular tight junctions. In addition, impaired functional integrity of tight junctions following bile duct ligation is reflected in a partial loss of hepatocellular surface polarity owing to redistribution of some, but not all, domain-specific plasma membrane antigens, which might mimic the behaviour of transport systems. After ethinyloestradiol treatment no alterations of surface polarity were observed. Thus, immunohistochemistry supports the view that ethinyloestradiol results in less severe impairment of the tight junctions than bile duct ligation. Finally, bile duct ligation, but not ethinyloestradiol, affects the transcytotic vesicular pathway; severe impairment of this is reflected in the absence of a late horseradish peroxidase peak in bile and also in the accumulation of pericanalicular vesicles that are immunopositive for canalicular membrane proteins and accessible for bulk phase endocytic markers. This view is supported by the disappearance of the pericanalicular vesicles simultaneously with the resumption of transcytotic horseradish peroxidase transport following release of ligation, a finding that indicates rapid restoration of this defect.This paper was presented at the symposium Metabolic Zonation of the Liver: New Answers to Old Questions, held in honour of Prof. Dr. D. Sasse's 60th birthday, 26 August 1994, in Basel     

A histochemical study about changes in rat liver plasma membrane enzyme activities after galactosamine administration.

In rats changes in plasma membrane enzyme activities due to Gal-N intoxication were studied by enzymehistochemical methods. The bile canalicular 5'-nucleotidase and nucleoside polyphosphatase activities decreased; the sinusoidal 5'-nucleotidase remained unchanged. The bile canalicular leucyl-beta-naphthyl-amidase showed an increase in activity; the alkaline phosphatase activity remained unchanged. In contrast to the spotty necrosis, changes in plasma membrane enzyme activities were seen in all liver cells, suggesting that changes of these activities, occurring after Gal-N treatment, do not correlate with cell death. The conclusion was drawn that the deviations of the enzyme activities might be due to changes in the lipid environment of the enzyme proteins in the membrane. With the exception of alkaline phosphatase, partial hepatectomy caused the same changes in enzyme activities as did Gal-N intoxication. Nevertheless Gal-N administration to partial hepatectomized rats did not lead to hepatic necrosis. Galactose given simultaneously or within two hours after Gal-N prevented both changes in plasma membrane enzyme activities and hepatocellular damage. This suggests an important role of galactolipids and galactoproteins in the plasma membrane alterations.     

AKAP350 Is involved in the development of apical "canalicular" structures in hepatic cells HepG2

Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical "canalicular" membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.     

A quantitative cytochemical investigation of osteoclasts and multinucleate giant cells

Summary Quantitative cytochemical, immunocytochemical, autoradiographic and electron cytochemical investigations have been used to compare osteoclasts with multinucleate giant cells that had been freshly obtained from the same animal. The levels of -acid galactosidase activity, the DNA in individual nuclei and the cellular protein content were similar in both cell types. However, osteoclasts generally possessed greater acid phosphatase and NADH dehydrogenase activity but lower levels of fluoride-inhibited non-specific esterase activity than multinucleate giant cells. The acid phosphatase activity in multinucleate giant cells was completely inhibited by 100 mM tartrate, but in osteoclasts only a 20% reduction in activity was observed. Formation of multinucleate giant cells in a bone microenvironment (thin bone slices) did not increase their content of tartrate-resistant acid phosphatase activity. Moreover, in osteoclasts, endogenous peroxidase activity was undetectable but present in several granules within the cytoplasm of multinucleate giant cells. Osteoclasts and multinucleate giant cells displayed a similar microtubular distribution, but calcitonin, which induced rearrangement of microtubules and cellular contraction in osteoclasts, had no effect on multinucleate giant cells. Thus, these investigations reveal both similarities and differences between these two syncytia and support the hypothesis that osteoclasts and multinucleate giant cells are related. Possibly osteoclasts arise from monocyte progenitors before commitment to a macrophage lineage has occurred.     

Quantitative topography of organelles in the liver

Summary After seven days of feeding fructose the liver of Wistar rats showed enormous accumulations of glycogen, which completely altered the original pattern of distribution of organelles. A quantitative morphological method was used to analyze these changes.The cytoplasm was mapped into arbitrary distance classes corresponding to concentric rings beginning at the outer nuclear membrane. This allowed the density of organelles in a given zone to be estimated.In cells filled with glycogen as a result of the fructose feeding, the following rearrangements were found: in the intermediate zone of both cellular poles (i.e., bile canalicular pole and sinusoidal pole) the mitochondria disappeared, being replaced by glycogen.The endoplasmic reticulum was accumulated in the perinuclear zone of both cellular poles, as in control animals, but was reduced throughout the rest of cytoplasm. It showed a peripheral density maximum at the biliary canalicular pole, in contrast to the cells of control animals.These changes in the distribution of the organelles and cellular compartments correspond to histochemical findings and demonstrate an adaptive reaction in the liver parenchyma to fructose ingestion, the organelles arranging themselves in cytoplasmic regions which still show a metabolic activity.Supported by a grant from the Deutsche Forschungsgemeinschaft Az Ri 271/6-5     

Liver cell plasma membrane lipids and the origin of biliary phospholipid.

The liver cell plasma membranes of fed male Wistar rats were separated into a fraction rich in bile canaliculi and the remainder of the plasma membrane. Electron-microscopically, the bile canalicular fraction consisted almost exclusively of intact bile canaliculi with thier contiguous membranes. The remaining plasma membrane fraction consisted primarily of vesicles and sheets of membranes essentially free from the bile canaliculi. The bile canalicular membrane fraction contained relatively more total lipid, cholesterol, and phospholipid, and relatively less protein. Although the phospholipid composition of the two fractions was the same, the specific activity of the bile canalicular membrane phosholipids, up to 12 h following in vivo administration of [2-3H]glycerol, was always significantly greater than that of the remaining plasma membranes, and showed a biphasic response not found in the latter. The specific activity of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membranes rose to a peak within 40 min after administration of the label, fell sharply and then rose to a second peak after 120 min. The specific activity of the sphingomyelin and phosphatidylserine plus phosphatidylinositol of the bile canalicular membranes and of all the phospholipids of the remaining plasma membranes diphasic pattern but increased steadily to reach a maximum at 120 min. The specific activity of biliary phosphatidylcholine followed a pattern identical to that of the phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine of the bile canalicular membrane fraction. These results show that the average rate of turnover of phospholipid in the bile canalicular membranes is considerably greater than that in the remaining plasma membrane and other cell membrane fractions; they indicate that the phospholipid of the bile canalicular membranes exists in two or more pools, turning over a different rates; and they support the concept that biliary phospholipid is derived from the bile canalicular membrane. The results also suggest that bile canalicular phospholipid may be derived from two different sources, in contrast to the remainong plasma membrane.     

Isolation and partial characterization of a bile canalicular plasma membrane fraction from normal and regenerating rat liver.

A bile canalicular membrane fraction was isolated from 24-hour regenerating rat livers, and its properties were compared to those of homologous fractions prepared from the livers of sham-operated and unoperated controls. These canalicular membrane fractions were found to be closely related in terms of their morphology, their purity, their yield, and their qualitative protein banding profiles on sodium dodecyl sulfate-polyacrylamide gels. However, when a rigorous examination of plasma membrane enzyme marker activities was made, the regenerating liver membranes were shown to possess an increased specific activity of alkaline phosphatase and lower levels of Mg2+ ATPase and 5'-nucleotidase in comparison with control specific activity values.