Mariko Ogi

Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells

Abstract: Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and vascular permeability. Hepatic sinusoidal endothelial cells (SECs) possess sieve‐like pores that form an anastomosing labyrinth structure by the deeply invaginated plasma membrane. Caveolin is the principal structural protein in caveolae. In this study, we examined the role of VEGF on the fenestration and permeability of SECs and the relation with caveolin‐1. SECs isolated from rat livers by collagenase infusion method were cultured for 24 h with (10 or 100 ng/ml) or without VEGF. The cells were then examined by transmission and scanning electron microscopy (EM). The expression of caveolin was investigated by confocal immunofluorescence, immunogold EM, and Western blot. Endocytosis and intracellular traffic was studied using horseradish peroxidase (HRP) reaction as a marker of fluid phase transport in SECs.

Enhanced Expression of Endothelin B Receptor at Protein and Gene Levels in Human Cirrhotic Liver

Endothelin (ET) has been implicated in the regulation of hepatic microcirculation and development of portal hypertension. This study examined the localization of ETA receptor (ETAR) and ETB receptor (ETBR) in cirrhotic liver tissues from patients with hepatocellular carcinoma with hepatitis C-related cirrhosis, and normal liver samples from patients with metastatic liver carcinoma. Anti-ETAR and ETBR antibodies were used for immunohistochemistry and Western blot. Immunoelectron microscopy was conducted using immunoglobulin-gold and silver staining. For in situ hybridization (ISH), human ETAR and ETBR peptide nucleic acid probes were used with the catalyzed signal amplification system. In normal liver tissue, immunohistochemistry revealed that ETBR was predominantly expressed on hepatic sinusoidal lining cells, particularly on sinusoidal endothelial (SECs) and hepatic stellate cells (HSCs), and ETAR was scantily expressed. These findings were confirmed by Western blot and ISH. In cirrhotic liver tissue, overexpression of ETBR was demonstrated by Western blot and ISH. Morphometric analysis showed significant increase of ETBR expression on HSCs and SECs in cirrhotic liver, particularly on HSCs. ETAR expression was increased but remained low. Enhanced ETBR expression in cirrhosis may intensify the effect of endothelin on HSCs and increase hepatic microvascular tone.

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver

 Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Endothelin-1 suppresses plasma membrane Ca++-ATPase, concomitant with contraction of hepatic sinusoidal endothelial fenestrae.

Intracytoplasmic free calcium ions (Ca ++ ) are maintained at a very low concentration in mammalian tissue by extruding Ca ++ from the cytoplasm against a steep extracellular Ca ++ concentration gradient, mainly through the activity of plasma membrane Ca ++ pump-ATPase. The present study aimed to elucidate how endothelin-1 (ET-1) affects the morphology of sinusoidal endothelial fenestrae and ultrastructural distribution of plasma membrane ATPases and intracytoplasmic free Ca ++ in isolated rat hepatic sinusoidal endothelial cells. Sinusoidal endothelial fenestrae were observed by scanning electron microscope. Andos electron cytochemical method was used for ultrastructural localization of Ca ++ -Mg ++ -ATPase activity, electron immunogold postembedding method for Ca ++ pump-ATPase immunoactivity, and antimonate method for intracytoplasmic free Ca ++ . Addition of ET-1 to sinusoidal endothelial cells significantly decreased Ca ++ -Mg ++ -ATPase activity and Ca ++ pump-ATPase expression and increased intracytoplasmic free Ca ++ concentration, concomitant with a decrease in diameter of sinusoidal endothelial fenestrae. Co-treatment with Bosentan abolished the actions of ET-1. These results suggest that ET-1 suppresses Ca ++ -Mg ++ -ATPase activity and Ca ++ pump-ATPase expression on the plasma membrane of sinusoidal endothelial fenestrae, thereby attenuating the extrusion of intracytoplasmic free Ca ++ into the extracellular space, leading to an increased concentration of intracytoplasmic free calcium ions and contraction of sinusoidal endothelial fenestrae.

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.

Hepatic stellate cells express Ca2+ pump-ATPase and Ca2+-Mg2+-ATPase in plasma membrane of caveolae

Abstract: Intracytoplasmic free calcium ions (Ca2+) are maintained at a very low concentration in mammalian tissue by the extrusion of Ca2+ across a steep extracellular Ca2+ gradient, mainly through the activity of plasma membrane Ca2+ pump-ATPase. The present study aimed to identify, by electron cytochemical and electron immunogold methods, the ultrastructural localizations of two types of plasma membrane Ca2+-ATPase; Ca2+-Mg2+-ATPase and Ca2+ pump-ATPase, in hepatic stellate cells. Liver tissues and isolated hepatic stellate cells (HSCs) were studied. The ultrastructural localization of Ca2+-Mg2+-ATPase activity was examined by the electron cytochemical method of Ando. The localization of Ca2+ pump-ATPase was identified by immunofluorescence. The ultrastructural localization of Ca2+ pump-ATPase was identified by the electron immunogold method. The cytochemical reaction products of Ca2+-Mg2+-ATPase activity were localized on the outer (cavity) side of the plasma membrane of caveolae. Immunofluorescence of Ca2+ pump-ATPase was seen as small dots along the cell edge in HSCs. Immunogold particles indicating the presence of Ca2+ pump-ATPase were identified on the inner (cytoplasmic) side of the plasma membrane of caveolae. We localized Ca2+ pump-ATPase on the inner side of the plasma membrane caveolae and Ca2+-Mg2+-ATPase on the outer leaflet of the caveolar plasma membrane in stellate cells, suggesting that Ca2+ pump-ATPase may play a key role in the Ca2+ reflux.

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.

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.

NG-L monomethyl arginine (L-NMMA) enhances bile canalicular contractions in cultured rat hepatocytes

Time lapse cinematographic analysis revealed that the frequency of canalicular contractions in primary monolayer-cultured hepatocytes (couplets or triplets) isolated from rat livers increased in the presence of monolayer-sinusoidal endothelial cells by the NG-L monomethyl arginine (L-NMMA)-treatment as compared to that in a control. Transmission electron microscopy revealed an enhanced vesicular transport to the canalicular lumens at the contracted bile canaliculi in the L-NMMA-treated couplets or triplets.

Endothelin-1 (ET-1) Enhances Bile Canalicular Contractions in Cultured Rat Hepatocytes : A Time Lapse Cinematographic and Electron Microscopic Study

Time lapse cinematographic analysis revealed that the frequency of canalicular contractions in primary monolayer-cultured hepatocytes (couples and triplets) isolated from a rat liver was increased by endothelin-1 (ET-1)-treatment as compared with that of a control group. By transmission electron microscopy, vesicles were more frequently observed to be opened to the canalicular lumens during the contracting stage of the bile canaliculi in the ET-1-treated couplets and triplets than in the control group.