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

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.

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.

Polymyositis, pulmonary fibrosis and malignant lymphoma associated with hepatitis C virus infection.

Polymyositis has been associated with various viral infections, and a spectrum of immune-related diseases may occur with hepatitis C (HCV) infection. Both polymyositis and HCV infection may be accompanied by pulmonary fibrosis. An association between polymyositis and malignancy has also been reported. We report a 55-year-old woman accompanied cryoglobulinemia with HCV infection and manifesting polymyositis, pulmonary fibrosis and malignant lymphoma. Steroid therapy was effective to improve interstitial pneumonia, polymyositis, and liver function.

Local regulators of hepatic sinusoidal microcirculation: recent advances.

This article reviews our recent studies on the local regulation of hepatic microcirculation with special reference to the inlet sphincter-like structures, the roles of sinusoidal endothelial cells and the mechanism of dynamic changes in the sinusoidal endothelial fenestrae (SEF) as well as in the terminal portal venules and the terminal hepatic arterioles induced by the potent vasoconstrictor endothelin (ET)-1. There are two types of sphincter-like structures at the entering sites of hepatic sinusoids. One is located at the junction between the terminal portal venule and the sinusoid, and is characterized by the large endothelial cells surrounded with Ito cells (hepatic stellate cells: HSCs). The other is located at the junction between the terminal hepatic arteriole and the sinusoid, and corresponds to the precapillary sphincter since our enzymohistochemical demonstration of arterial capillaries in close association with the sinusoids combined with intravital microscopy has revealed that the terminal hepatic arteriole directly terminates in the sinusoid. It is essential for the local control of hepatic sinusoidal blood flow that the dynamic contracting and relaxing changes not only in these inlet sphincter-like structures but also in the SEF correspond with those of the HSCs, both of which are mediated by the sinusoidal endothelium-derived vasoconstrictor endothelins (ETs) and vasodilator nitric oxide (NO). The contractility of the SEF and HSCs depends on the intracellular Ca++-calmodulin-actomyosin system.

Bile canalicular contraction and dilatation in primary culture of rat hepatocytes--possible involvement of two different types of plasma membrane Ca(2+)-Mg(2+)-ATPase and Ca(2+)-pump-ATPase.

Increasing evidence has indicated that bile canalicular contraction is mediated by the nonmuscular Ca(2+)-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 Ca(2+)-ATPase, i.e., Ca(2+)-Mg(2+)-ATPase and Ca(2+)-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 Ca(2+)-Mg(2+)-ATPase activity was identified by the electron cytochemical method of Ando. Ultrastructural localization of Ca(2+)-pump-ATPase was examined by immunogold electron microscopy. We found that cytochemical reaction products showing the presence of Ca(2+)-Mg(2+)-ATPase activity were localized on the luminal side of the bile canalicular membranes. Immunogold particles, indicating the presence of Ca(2+)-pump-ATPase, were located mainly on the cytoplasmic side of the bile canalicular membranes. The expression of both Ca(2+)-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 Ca(2+)-ATPase may be involved in the regulation of canalicular contractility to control the extrusion of intracytoplasmic free calcium ions into the canalicular lumen.

Hepatic sinusoidal endothelial fenestrae express plasma membrane Ca++pump and Ca++Mg++-ATPase.

BACKGROUND/AIM: In general, intracytoplasmic free calcium ions (Ca++) are maintained at a very low concentration in mammalian tissue by extruding Ca++ against a high concentration of extracellular Ca++, mainly through the activity of the plasma membrane Ca++pump-ATPase. The aim of the present study was to demonstrate by electron cytochemical and immunogold methods the ultrastructural localization of two different types of plasma membrane Ca++-ATPase, i.e. Ca++Mg++-ATPase and Ca++pump-ATPase in the hepatic sinusoidal endothelium. METHODS: Liver tissues and the isolated hepatic sinusoidal endothelial cell (SEC)s were subjected to the following procedures. The ultrastructural localizations of Ca++Mg++-ATPase were examined by an electron cytochemical method. The ultrastructural localization of Ca++pump-ATPase was identified by an electron immunogold method. RESULTS: The cytochemical reaction of Ca++Mg++-ATPase was found to be localized on the outer sites of the plasma membrane of sinusoidal endothelial fenestrae (SEF). The immunogold particles indicating the presence of Ca++pump-ATPase were identified on the inner sites (cytoplasmic) of the invaginated plasma membrane of SEF CONCLUSIONS: Both Ca++Mg++-ATPase and Ca++pump-ATPase demonstrated on the SEF plasma membrane may be involved in the regulation of intracytoplasmic Ca++ concentration.

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 Ando's 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.

Signal detection of endothelin receptor subtypes in human cirrhotic liver by a new in situ hybridization method.

Endothelin (ET) has been implicated in the regulation of hepatic microcirculation and development of portal hypertension, but the role of ET in cirrhosis of the liver has not yet been elucidated. We present here the application of peptide nucleic acid (PNA) probes in a fast and sensitive in situ hybridization (ISH) method for localizing the mRNA of endothelin receptor subtypes in formalin-fixed, paraffin-embedded sections of normal and cirrhotic human liver. The results of ISH using synthetic FITC-labeled PNA probes combined with the catalyzed signal amplification (CSA) system were compared with those using the standard detection system. It was indicated that the CSA-ISH protocol is more sensitive for the detection of mRNA target than the standard ISH protocol. Our results with CSA-ISH showed that the expression of mRNA for the endothelin B receptor was significantly upregulated in hepatic sinusoidal lining cells in cirrhotic human liver tissues compared to control normal liver tissue. Therefore, the CSA detection system may facilitate and enhance the use of in situ hybridization protocols, and CSA-ISH will be used as an important diagnostic technique in the future.

Enhanced expression of endothelin receptor subtypes in cirrhotic rat liver.

BACKGROUND/AIMS: A number of vasoactive substances have been implicated as potential mediators of intrahepatic portal hypertension. Endothelin (ET)-1 has been suggested to be involved in the regulation of hepatic microcirculation and development of portal hypertension. The aim of this study was to clarify the localization of two subtypes of ET receptors, ET A (ETAR) and B receptors (ETBR), in normal rat liver, and how the receptor expressions are altered in CCl4-induced cirrhotic rat liver. METHODS: Liver specimens were examined immunohistochemically after reacting with anti-ETAR and anti-ETBR rabbit polyclonal antibodies. Immunogold staining was also performed using the same antibodies, and examined under light and electron microscopy. RESULTS: In normal rat liver, immunohistochemistry revealed expression of ETAR and ETBR on the hepatic sinusoidal lining cells. By immunogold electron microscopy, electron-dense gold particles indicating the presence of ETARs were localized mainly on hepatic stellate cells (HSCs) and to a lesser extent on sinusoidal endothelial cells (SECs), while ETBRs were expressed equally intensely on HSCs and SECs. In cirrhotic animals, both ETAR and ETBR increased significantly on HSCs, while there were no significant increases in either receptor on SECs. CONCLUSIONS: In the normal state, HSCs possess both ETARs and ETBRs, while SECs mainly possess ETBRs. In cirrhosis, endothelins may exert more intense effects on HSCs via the enhanced ETARs and ETBRs, causing an increase in hepatic sinusoidal microvascular tone.

Endothelial nitric oxide synthase and caveolin-1 are co-localized in sinusoidal endothelial fenestrae.

BACKGROUND/AIMS: Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases (NOS). Caveolin, the principal structural protein in caveolae, interacts with endothelial NOS leading to enzyme inhibition in a reversible process modulated by Ca++-calmodulin. The aim of the present study was to clarify the ultrastructural localization of eNOS and caveolin-1 in hepatic sinusoidal endothelium by an electron immunogold method. METHODS: Male Wistar rats were used. Liver tissues and hepatic sinusoidal endothelial cells isolated from rat livers by collagenase infusion were studied. For immunohistochemistry, liver specimens were reacted with anti-eNOS or anti-caveolin-1 antibody. The ultrastructural localization of eNOS or caveolin-1 was identified by electron microscopy using an immunogold post-embedding method. RESULTS: Immunohistochemical studies using liver tissues localized endothelial NOS in hepatic sinusoidal lining cells, portal veins and hepatic arteries; and caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein and hepatic arteries. Immunogold particles indicating the presence of eNOS and caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae in liver tissue and also in isolated sinusoidal endothelial cells. CONCLUSION: Endothelial NOS and caveolin are co-localized on sinusoidal endothelial fenestrae, suggesting that interaction of the two may modulate cellular regulation of NO synthesis.

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.

[2 surgically treated cases of adrenocortical carcinoma producing steroid hormones without endocrinological symptoms--case report and a review of cases in the Japanese literature].

We have experienced two male cases of an adrenocortical carcinoma that have produced steroid hormones without endocrinological symptoms. Both men were admitted to hospital because of an abdominal pain and left hypochondral tumors. In each case, an abdominal CT, an ultrasonogram and an angiogram demonstrated a left adrenal tumor, and levels of blood dehydroepiandrosterone sulfate, etiocholanolone, pregnenolone sulfate and urine 17 KS were elevated. The tumors were resected and the histology revealed that they were adrenocortical carcinomas. Postoperatively, the levels of their urine hormones returned to normal values, though in one man recurrence occurred a year later. Only 8 cases of an adrenocortical carcinoma that has produced steroids without specific endocrinological symptoms have been reported in Japan, and we also report on two cases herein.