Masao Yoshitake

Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPases, inhibits the receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

BACKGROUND/METHODS The role of vacuolar type H(+)-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of asialoglycoproteins was morphologically investigated in isolated rat hepatocytes using bafilomycin A1, a specific inhibitor of v-ATPases. RESULTS Fluorescent staining by acridine orange showed that bafilomycin A2 inhibited the acidification of the endocytic compartments. Uptake of gold-conjugated asialofetuin was significantly inhibited by bafilomycin A1. However, bafilomycin A1 did not significantly inhibit uptake of a fluid phase marker, horseradish peroxidase. The number of autophagic vacuoles increased after the bafilomycin A1 treatment. However, materials in the autophagic vacuoles were rapidly degraded after the removal of bafilomycin A1. CONCLUSIONS Results suggest that: (a) v-ATPases are necessary for acidification of the endocytic compartments; (b) the pH gradient between the endocytic compartments and the cytoplasm which is generated by v-ATPases is necessary for the receptor-mediated endocytosis of asialoglycoproteins, and (c) v-ATPases may contribute to the degradation of the materials in autophagic vacuoles.

Ultrastructure of the intracellular membranous system of rat hepatocytes in intrahepatic cholestasis induced by phalloidin

To investigate the effect of a thickened pericanalicular ectoplasm in tubulovesicular transport and biliary excretion, we examined the ultrastructure of the intracellular membranous system in rat hepatocytes with and without phalloidin treatment, by transmission electron microscopy and scanning electron microscopy combined with the Aldehyde prefix Osmium-Dimethyl Sulfoxide-Osmium method. Hepatocytes possessed elaborate networks of tubules around bile canaliculi, and some of them extended to the bile canaliculi in control rats. Vesicles were also present around the bile canaliculus. Treatment of rats with phalloidin produced a thick pericanalicular ectoplasm around the bile canaliculus visualized by transmission electron microscopy, and the density of vesicles (p < 0.001) and tubules (p < 0.001) within 0.5 microns around the bile canaliculus significantly decreased in phalloidin-treated rats. The number of lysosomes in hepatocytes apparently increased in phalloidin-treated rats; however, they were rarely observed around the bile canaliculus. The Aldehyde prefix Osmium-Dimethyl Sulfoxide-Osmium method produced an organelle-free space around the bile canaliculus by removing the thick pericanalicular ectoplasm in scanning electron microscopic examination, and the thickened pericanalicular ectoplasm inhibited the approach of intracellular membranes to the canalicular membrane in the transmission electron microscopic examination. In some pathological cholestatic conditions, the thickened pericanalicular ectoplasm may inhibit not only bile canalicular contraction but also biliary excretion of substances, which is mediated by the tubulovesicular transport system.

Involvement of activated polymorphonuclear leukocytes in galactosamine-induced hepatic injury in rats.

This study attempted to elucidate the pathological role of peripheral blood polymorphonuclear leukocytes (PMNs) in the damage to hepatic sinusoidal endothelial cells (HSECs) in hepatic injury . Wistar male rats weighing about 200 g received a single injection of 1 g/kg body weight of galactosamine (GalN) intraperitoneally for the induction of hepatitis. The level of serum glutamic pyruvate transaminase activity increased time-dependently concomitantly with serum endotoxin level and hepatic lipid peroxide content. Histological studies using light and electron microscopy showed that a large number of PMNs infiltrated the midzonal area of the liver and that HSECs in contact with the PMNs were injured 6 h after GalN injection. Superoxide anion production by PMNs isolated from the peripheral blood of rats treated with GalN was increased significantly compared with that from control rats, as estimated by the reduction of exogenously added cytochrome c in the presence of phorbol myristate acetate. A cytotoxicity study using the 51Cr release assay revealed that PMNs isolated from GalN-treated rats caused more serious injury to the HSECs than those from control rats. These results suggest that the oxygen-derived free radicals released from the activated PMNs directly injure HSECs and lead to a disturbance of sinusoidal microcirculation, causing an extended liver cell necrosis in GalN hepatitis.

Serum type III procollagen N paptide(PIIIP) and laminin levels and hepatic immunohistochemical study in patients with various liver diseases.

各種肝疾患95例における血清タイプIIIプロコラーゲンNペプチド(PIIIP)やラミニン値の変動と肝生検組織における肝線維化,細胞浸潤および巣状壊死の程度,タイプIIIコラーゲンやラミニンの分布およびその産生細胞を観察し,さらに血清PIIIP,ラミニン値が肝線維化のマーカーになり得るか否かを検討した.血清PIIIPは活動性の肝病変を呈する疾患で高値を呈し,肝細胞障害に伴なうタイプIIIコラーゲンの形成を反映するものと思われた.また,血清ラミニンは肝線維化の高度な肝疾患例で高値を呈し,基底膜形成の増加をよく反映するものと思われ,血清PIIIPとラミニンを同時に測定することは,肝疾患の活動性,線維化の形成および程度の判定に有用と思われた.また,タイプIIIコラーゲンは伊東細胞,肝細胞,内皮細胞や線維芽細胞,ラミニンは内皮細胞,伊東細胞,肝細胞および胆管上皮細胞などにより産生されることが示唆された.