Yoshitaka Kamegaya

Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol.

BACKGROUND & AIMS The precise pathogenic significance of oxidative injury in the evolution of alcohol-induced liver disease is still obscure. The present report was designed to investigate whether ethanol alters the production of active oxidants and biological activities of hepatocytes. METHODS The following parameters in rat hepatocytes were investigated by using fluorescence probes in vitro and ex vivo: (1) mitochondrial membrane potential and membrane permeability transition, (2) oxygen radicals generation, (3) membrane barrier function, and (4) glutathione level. RESULTS Ethanol (50 mmol/L) increased oxidative stress in hepatocytes and subsequently induced an increased mitochondrial permeability transition and a decreased membrane potential. These ethanol-induced alterations were attenuated by an inhibitor of alcohol dehydrogenase and an intracellular oxidant scavenger, whereas they were enhanced by diethyl maleic acid, a glutathione depletor. Ethanol plus diethyl maleic acid but not ethanol alone increased the number of hepatocytes with membrane barrier dysfunction. A continuous infusion of ethanol (50 mmol/L) increased oxidative stress and decreased mitochondrial membrane potential in the pericentral area of isolated perfused rat liver. CONCLUSIONS Active oxidants generated during ethanol metabolism increase mitochondrial permeability transition and modulate mitochondrial energy synthesis in hepatocytes. Reduction of glutathione level enhances mitochondrial dysfunction and impairs membrane barrier function of hepatocytes.

Superoxide anion release into the hepatic sinusoid after an acute ethanol challenge and its attenuation by Kupffer cell depletion.

Superoxide anion release into the hepatic sinusoids and subsequent damage to the endothelial cells of the hepatic sinusoids after ethanol challenge was examined. A 250 mg/kg body weight/hr dose of ethanol was given to rats for 3 hr, and superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome c method. Ethanol treatment resulted in superoxide anion release into the hepatic sinusoids (0.20 ± 0.01 vs. 0.12 ± 0.02 o.d., p< 0.05) and an increase in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate, a marker of damage to the endothelial cells of the hepatic sinusoids (0.003 ± 0.002 vs. 0.008 ± 0.002; p < 0.05). Tumor necrosis factor-alpha was not detectable in either group, and there were no significant differences in the population of hepatic macrophages, leukocytes, or Kupffer cells between the two groups. To clarify the role of Kupffer cells in the mechanism, 10 mg/kg of body weight of gadolinium chloride was given to rats twice, 24 hr apart, resulting in depletion of ED2-positive cells from the hepatic lobules. The superoxide anion release after the ethanol challenge was significantly attenuated in the Kupffer cell-depleted rats, compared with the controls (0.14 ±0.02;p <0.05, compared with ethanol alone). The change was associated with a significant decrease in the purine nucleoside phosphorylase/alanine aminotransferase ratio in the liver perfusate (0.004 ± 0.002; p < 0.05, compared with ethanol alone). Ethanol causes superoxide anion release into the hepatic sinusoid and subsequent damage to the sinusoidal endothelial cells. These changes were reduced by Kupffer cell depletion. This supports the view that Kupffer cell depletion has a protective effect on ethanol-induced liver injury.

Kupffer cell depletion attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide treatment

Background and Aim:  The mechanisms involved in the beneficial effect of gadolinium chloride against endotoxin‐induced liver damage were studied.

Splenectomy attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide challenge

BACKGROUND/AIMS The aim of this study was to determine whether the spleen contributes to superoxide anion release into the hepatic sinusoids and subsequent damage to endothelial cells of the hepatic sinusoids after lipopolysaccharide challenge. METHODS Rats were given 2 mg/kg body weight lipopolysaccharide. Three hours after the treatment, superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome C method. Damage to endothelial cells of the hepatic sinusoids was assessed from the purine nucleoside phosphorylase/glutamic-pyruvic transaminase ratio in the liver perfusate. To further characterize the mechanisms behind these changes, these studies were done in rats given superoxide dismutase or an anti-TNFalpha antibody. To study whether the spleen plays a role in the mechanisms, experiments with splenectomized rats were performed. RESULTS Lipopolysaccharide challenge resulted in superoxide anion release into the hepatic sinusoids and damage to endothelial cells of the hepatic sinusoids. These changes were significantly attenuated by the treatments with superoxide dismutase or an antibody against TNFalpha, as well as by splenectomy. The hepatic macrophage and Kupffer cell populations after lipopolysaccharide challenge were significantly smaller in the rats given splenectomy than in those given a sham operation. There were no significant differences in the neutrophil populations between the two groups. Levels of TNFalpha were significantly lower in the former than the latter, whereas there were no significant differences in levels of Interleukin-8 between the two groups. CONCLUSIONS Splenectomy reduced the superoxide anion release into the hepatic sinusoids caused by the lipopolysaccharide challenge and subsequent damage to endothelial cells of the hepatic sinusoids. This supports the view that splenectomy has a protective effect in lipopolysaccharide-induced liver injury.

Role of endothelin receptors in endothelin-1-induced morphological changes of hepatic sinusoidal endothelial fenestrae: morphometric evaluation with scanning electron microscopy

Endothelin (ET)-1, a potent vasoconstrictor, is involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF) through ET-1 receptors. To clarify the role of each receptor (R) in ET-1 induced contraction of SEF, we studied the size of hepatic SEF under various experimental conditions. Scanning electron microscopy was used for morphometric analysis of the fenestrae of sinusoidal endothelial cells isolated from male Wistar rats under the following conditions: (1) control, (2) ET-1, (3) Bosentan (ET(A)-R+ET(B)-R antagonist)right arrowET-1, (4) BQ485 (ET(A)-R antagonist)right arrowET-1, (5) BQ788 (ET(B)-R antagonist)right arrowET-1. Each experiment was based on the observations of 200--205 fenestrae (15--20 fenestrae per cell, two cells per dish and six dishes). The diameter of the endothelial pores of the isolated sinusoidal endothelial cells was 123plus minus35 nm in group (1), 46plus minus21 nm in group (2), 130plus minus40 nm in group (3), 72plus minus28 nm in group (4), and 130plus minus27 nm in group (5). The differences between groups (2) and (4), and between groups (2) and (5), were statistically significant (P<0.05, P<0.01, respectively). Endothelin B receptor (ET(B)-R) antagonist pretreatment abolished the ET-1-induced contraction of SEF, whereas endothelin A receptor (ET(A)-R) antagonist pretreatment appeared to partially block this contraction. The present findings indicate that ET(B)-R plays a primary role in endothelin-1 induced SEF morphological changes, while ET(A)-R plays a subsidiary role.

Bile canalicular contraction and dilatation in primary culture of rat hepatocytes – possible involvement of two different types of plasma membrane Ca2+-Mg2+-ATPase and Ca2+-pump-ATPase

Increasing evidence has indicated that bile canalicular contraction is mediated by the nonmuscular Ca2+-calmodulin-actomyosin system, and the contraction facilitates canalicular bile flow. The aim of the present study was to examine, by electron cytochemistry, how the expression of two types of plasma membrane Ca2+-ATPase, i.e., Ca2+-Mg2+-ATPase and Ca2+ pump-ATPase, is related to the dynamic changes of bile canalicular contraction. Hepatocytes isolated from male Wistar rat liver by collagenase perfusion were cultured to form a primary monolayer. The canalicular dynamics in the couplets and triplets were analyzed by time-lapse cinematography. The Ca2+-Mg2+-ATPase activity was identified by the electron cytochemical method of Ando. Ultrastructural localization of Ca2+ pump-ATPase was examined by immunogold electron microscopy. We found that cytochemical reaction products showing the presence of Ca2+-Mg2+-ATPase activity were localized on the luminal side of the bile canalicular membranes. Immunogold particles, indicating the presence of Ca2+ pump-ATPase, were located mainly on the cytoplasmic side of the bile canalicular membranes. The expression of both Ca2+-ATPases on the canalicular membranes was enhanced during the contracting stage of bile canaliculi, whereas their expression was diminished in the dilating stage. We conclude that two different types of bile canalicular Ca2+-ATPase may be involved in the regulation of canalicular contractility to control the extrusion of intracytoplasmic free calcium ions into the canalicular lumen.

Expression of plasma membrane Ca2+-ATPase on hepatic sinusoidal endothelial fenestrae: modification of the one-step method

The intracytoplasmic free calcium ion (Ca2+) concentration is maintained at a low level in mammalian tissues by extruding Ca2+ against a high extracellular Ca2+ concentration, mainly through the activity of the plasma membrane Ca2+-ATPase pump. The objective of the present study was to localize the plasma membrane Ca2+-ATPase activity on hepatic sinusoidal endothelial cells (SECs) by electron microscopic cytochemistry. The ultrastructural localization of Ca2+-ATPase activity on ultrathin sections of liver tissue and cultured SEC monolayer was examined by the electron microscopic cytochemical method of Ando (method A: original method) and by our modified method (method B: shortened fixation method). By method A, scanty cytochemical reaction products of Ca2+-ATPase were found in the SECs. By method B, Ca2+-ATPase activity was clearly localized on the outer surface of the plasma membrane of sinusoidal endothelial fenestrae (SEF). Our modification of Andos method by shortening the incubation time of liver tissue or isolated SEC sections in the substrate allowed clear demonstration of Ca2+-ATPase activity on the SEF membrane. Use of tangential sections of primary cultures of SEC provided excellent localization results. The cytochemically reactive Ca2+-ATPase expressed on the SEF plasma membrane may be involved in regulation of the intracytoplasmic Ca2+ concentration.

Elevation of serum eosinophil cationic protein in primary biliary cirrhosis

Abstract It has recently been reported that in patients with primary biliary cirrhosis (PBC), eosinophils are not only increased in the peripheral blood, but also infiltrted in the liver portal tracts. There is general agreement that eosinophils and eosinophil cationic protein (ECP) released from eosinophils contribute to cellular damage, particularly in allergic inflammation. In the present study, ECP was measured by the radioimmunoassay in sera of patients with a variety of liver diseases including PBC. Serum ECP levels were significantly higher in patients with PBC than in those with chronic viral hepatitis, in those with liver cirrhosis, and in healthy subjects. There were no significant differences in serum ECP levels between symptomatic and asymptomatic PBC. The present study suggests that high levels of serum ECP may reflect high grades of eosinophil infiltration around the septal and interlobular bile ducts characterized by chronic non-suppurative destructive cholangitis, particularly in the early stages of PBC.

A novel immunocytochemical staining method that preserves cell membranes: Application for demonstrating Ca2+ pump ATPase in the liver

We have devised a method for immunogold staining of unosmicated, plastic-embedded cells that gives high levels of specific staining without sacrificing cell ultrastucture. Important conditions include PLP (periodate-lysine-paraformaldehyde) fixation, postfixation with uranyl acetate to preserve membrane phospholipids, dehydration with acetone, low-temperature embedding in LR gold resin, and use of osmium tetroxide to stain thin sections after immunogold labeling. We developed this method to localize plasma membrane calcium pump ATPase in rat hepatocytes and hepatic sinusoidal endothelial cells. Most gold particles of Ca2+ pump ATPase were easily assigned to bile canalicular membranes in rat hepatocytes, and the gold particles of Ca2− pump ATPase were located on the labyrinthlike structures of the endothelial sinusoidal fenestrae in rat hepatic sinusoidal endothelial cells. In these studies, it was useful to preserve the cell membrane for postembedding methods.

Formation of superoxide anion in the hepatic sinusoid after lipopolysaccharide challenge

Using the cytochrome c method, superoxide anion that is released into the hepatic sinusoid was measured after a lipopolysaccharide challenge in a liver perfusion system. Moreover, damages of epithelial cells of the hepatic sinusoid were estimated with scanning electron microscopic analysis and levels of purine nucleoside phosphorylase/GPT ratio. Lipopolysaccharide administration increased the conversion of oxidized cytochrome c into reduced cytochrome c in the perfusate, indicating that superoxide anion was formed in the hepatic sinusoid. This change was associated with increase in levels of portal tumor necrosis factor-alpha and attenuated by the simultaneous administration of superoxide dismutase. Scanning electron microscope analysis revealed that diameters of sinusoidal fenestrae increased in rats treated with lipopolysaccharide, compared with controls. Moreover, levels of purine nucleoside phosphorylase/GPT ratio was significantly increased in the liver perfusate in lipopolysaccharide-treated rats, compared with controls. Superoxide anion in hepatic sinusoid may be one of the pathogenic factors behind damages of epithelial cells of the hepatic sinusoid caused by lipopolysaccharide.