Hepatic transport of serum bilirubin, bromsulfophthalein, and indocyanine green in patients with congenital non-hemolytic hyperbilirubinemia and patients with constitutional indocyanine green excretory defect.

We carried out a retrospective study of 71 patients with congenital non-hemolytic hyperbilirubinemia who had been treated at our institution over the 25 years from 1965 to 1990. Twenty patients had Gilbert's syndrome, 1 had Crigler-Najjar syndrome, 1 had new type unconjugated hyperbilirubinemia, 21 had Dubin-Johnson syndrome, and 28 had Rotor's syndrome. We also reviewed 20 patients with constitutional indocyanine green (ICG) excretory defect. The study focused on the hepatic transport of serum bilirubin, bromsulfophthalein (BSP), and ICG. In Dubin-Johnson syndrome, a defect appeared in late-stage transport, while uptake and storage capacity were normal. In Rotor's syndrome, defects were found in the early stage, and storage capacity was reduced, while excretion into bile was slightly suppressed. A secondary rise in serum ICG was seen in 5 of the 10 patients with Dubin-Johnson syndrome. The transport defect in Gilbert's syndrome was unclear. It could not be considered to be homogeneous, but it may exist at multiple sites, from the conjugation with serum proteins to excretion into bile. Following phenobarbital administration, the ICG secondary rise in the 5 patients with Dubin-Johnson syndrome disappeared, and ICG was rapidly cleared from blood. However, in patients with Dubin-Johnson syndrome, BSP clearance in serum did not show any change before and after phenobarbital administration. ICG excretion in patients with constitutional ICG excretory defect was due only to the impairment o ICG transport, and the defect was suggested to be hepatic uptake. These results indicate that studies of the hepatic transport of bilirubin, BSP, and ICG are useful for determining the etiological factors involved in congenital hyperbilirubinemia and constitutional ICG excretory defect.

A multi-center double-blind controlled trial of ursodeoxycholic acid for primary biliary cirrhosis.

A multi-center double-blind controlled trial of ursodeoxycholic acid (UDCA) for treatment of primary biliary cirrhosis (PBC) was carried out. Twenty two and 23 patients were treated with 600 mg/day UDCA and placebo, respectively, for 24 weeks. In UDCA-treated patients, fall of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma-glutamyltranspeptidase activities started within 4 weeks after start of the trial and continued throughout the trial period. The serum IgM level fell in 7 UDCA-treated patients examined but not in 10 placebo-treated patients examined. Serum bilirubin concentration showed no significant change at the end of the study in either of UDCA- and placebo-treated group of patients. There was no significant difference between these two groups with respect to the frequency of improvement of pruritus. In UDCA-treated patients, serum bile acid composition changed markedly, though its concentration showed no significant change. The percentage of total bile acid which ursodeoxycholic acid took up increased, whereas those which cholic acid, chenodeoxycholic acid and deoxycholic acid took up were decreased.

Integrity of the cytoskeletal system is important for phagocytosis by Kupffer cells.

The mechanism of phagocytosis by Kupffer cells is believed to involve the Ca2(+)-calmodulin system. However, the role of myosin in this system is still unknown. In this study, we found that myosin light chain kinase inhibitor (ML-9) inhibited phagocytosis by cultured Kupffer cells using polystyrene beads, a time-lapse VTR system and fluorescent staining techniques. The inhibitory effects of ML-9 suggest that myosin may be involved in this complex cellular function and that the integrity of the cytoskeletal system is essential for normal phagocytosis.

Phalloidin-induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation.

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.

[The effect of smoking and drinking habit on the process from liver cirrhosis to liver cancer].

A retrospective cohort study of liver cirrhosis cases was carried out. 270 cases of liver cirrhosis which were confirmed histologically from 1972 to 1981 at Juntendo University Hospital, were followed up until the end of 1988. The average observation period for these cases was 74 months or 6.2 years, and we found 141 death cases including 46 of primary liver cancer (PLC). The analysis of survival rate revealed that the smoking group had a slightly higher death rate of PLC than non-smokers, and the ex-smoker group had a much higher survival rate than the continuous smoking group. On the other hand, it was found that the alcohol-drinking group had a much lower death rate of PLC than the non-drinkers group, and that there was no difference of the survival rate among drinkers, non-drinkers and ex-drinkers.

Isolation of tryptic fragment of antigen from mitochondrial inner membrane proteins reacting with antimitochondrial antibody in sera of patients with primary biliary cirrhosis.

Most of the sera from patients with primary biliary cirrhosis contains antimitochondrial antibodies, which react with four proteins of the mitochondrial inner membrane. We reported in a previous paper that when beef heart mitochondrial inner membrane proteins were digested by trypsin, a new reactive 36 kDa fragment with antimitochondrial antibody was obtained. This 36 kDa fragment derives from original 70 kDa protein because the monoclonal antibody specific to 70 kDa protein reacts with the 36 kDa band equivalent to 70 kDa band. The 36 kDa fragment was purified using an affinity column conjugated with an immunoglobulin-rich fraction of primary biliary cirrhosis serum containing antimitochondrial antibody, preparative electrophoresis and high-performance liquid chromatography using a reverse phase column. The final preparation showed a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Its amino acid composition is in good agreement with that of the subunit binding domain of the pyruvate dehydrogenase complex E2 from bovine heart.

Immunocytochemical localization of myosin in normal and phalloidin-treated rat hepatocytes.

To clarify the intracellular distribution of myosin in normal rat hepatocytes and its alterations in phal-loidin-treated rat hepatocytes as a morphologic basis for the dysfunction of microfilaments, we performed indirect immunofluorescence using monospecific antibody raised against rat hepatocyte myosin. Cryostat rat liver sections analyzed by the use of this antibody showed a characteristic polygonal staining pattern, indicating that myosin is localized close to the plasma membrane including the region of bile canaliculi. The observed myosin staining pattern of normal liver coincides with the pattern of actin distribution as demonstrated by double-staining on the same liver section with antimyosin antibody and rhodamine-phalloidin. Upon administration of phalloidin to rats, the following changes in the myosin staining pattern were observed. (a) Peripheral fluorescence along the plasma membrane, especially around the bile canaliculi and sinusoids, was greatly enhanced. (b) Numbers of small fluorescent dots appeared in the cytoplasm of hepatocytes. These changes in the localization of myosin are shown to overlap with those of actin filament distribution. Accompanying these changes of localization, cellular myosin content appears to be increased, as the myosin marker-enzyme NH4+(-)ethylenediaminetetraacetic acid-adenosine triphosphatase activity in hepatocyte extracts was elevated threefold after 7 days of phalloidin treatment. This increase of myosin may be due to the previously observed stabilizing effect on microfilaments of phalloidin against cellular proteases. Thus, phalloidin, which primarily alters actin filament distribution, induces the changes in myosin localization and the increase in cellular myosin content without causing dissociation of myosin from actin in the hepatocyte.

Electrophoretic studies of antimitochondrial antibodies: identification of mitochondrial antigens specific to primary biliary cirrhosis.

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular masses of 70 kDa, 50 kDa, 47 kDa and 40 kDa, as defined by their relative mobility (Rf) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All sera of 114 PBC patients were positive with at least one and as many as four of the mitochondrial proteins. The major antigenic proteins of mitochondrial inner membrane to which AMA reacts were the 70 kDa and 47 kDa proteins. All PBC sera containing antibodies to the 50 kDa and/or 40 kDa proteins reacted with 70 kDa as well. The isolation of antigen reacting with AMA of PBC is important to warrant further study of AMA and the cause of the disease. The isolation of responsible antigens had been difficult because the four antigens were insoluble. However, the antigen newly found by us, the 36 kDa fragment, obtained by partial trypsin digestion, is soluble. Using several procedures, the antigenic protein target of AMA was purified from mitochondria for the first time. We determined the N-terminal sequence of the soluble 36 kDa fragment, 25 residues in length. Until now the N-terminal sequence of the 36 kDa protein has not shown significant homology with any known protein. The present results of antigen purification would contribute to the elucidation of the epitopes of AMA antigen.

[A long-term follow-up study of the histologically confirmed chronic liver diseases in the Juntedo University Hospital].

In order to calculate the survival rate and elucidate the risk factors of liver cancer, 1,034 patients of the chronic liver diseases were followed up for 7.1 years of average. All patients were confirmed histologically from 1973 to 1982 in Juntendo University Hospital as liver cirrhosis (367), liver fibrosis (27), and chronic hepatitis (640). Until the end of 1986, 244 cases were died including 52 cases of liver cancer. The 10 years survival rate by Kaplan-Meier's method ranged from 39.85 for male liver cirrhosis to 80.64 for male chronic hepatitis. The results of proportional hazard model for male liver cirrhosis revealed that drinking alcohol affected negatively to the death from liver cancer, and that cigarette smoking and no history of operation due to portal hypertension affected slightly to it and that HBs antigen, history of blood transfusion and history of acute hepatitis almost have no relation to it.

Calmodulin antagonists inhibit the phagocytic activity of cultured Kupffer cells.

The mechanism of phagocytosis by Kupffer cells is still unknown. In this study we found that trifluoperazine, chlorpromazine, and W-7, drugs which bind to Ca2+-calmodulin and inhibit its interaction with other proteins, inhibit phagocytosis by cultured Kupffer cells using polystyrene beads, time-lapse VTR systems, and fluorescent staining techniques. Inhibitory effects of these drugs on phagocytosis suggests that the Ca2+-calmodulin system may be involved in this complex cellular function and the integrity of the cytoskeletal system of Kupffer cells is essential to this phenomenon.

Immunocytochemical localization of myosin in rabbit liver cell.

It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.

Bile canalicular contraction in the isolated hepatocyte doublet is related to an increase in cytosolic free calcium ion concentration.

Dynamic contractions of bile canaliculi have been observed in cultured doublet hepatocytes by means of time-lapse cinephotomicrography, and this contractile movement plays an important role in bile secretion. Although details of the mechanism are still unknown, the Ca2+-calmodulin system is believed to play a main role in this mechanism. In this study we measured the intracellular Ca2+ concentration of individual doublet hepatocytes using the Ca2+ indicator "fura 2" and microscopic fluorometry. We also observed the effects of A23187, norepinephrine and epinephrine on bile canalicular contraction and intracellular Ca2+ concentration. After loading 1 mumol/l fura 2 in doublet cells, we added A23187, epinephrine or norepinephrine and then measured the Ca2+ concentration in a given small area in the cytoplasm of individual doublet cell. A23187, norepinephrine and epinephrine caused a prompt increase of the intracellular Ca2+ concentration and also caused bile canalicular contraction. The present study indicates that the sudden increase of intracellular Ca2+ concentration causes bile canalicular contraction through the Ca2+-calmodulin system.

Reticular meshwork of the spleen in rats studied by electron microscopy.

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.

Distribution of Ito cells in experimental hepatic fibrosis.

Ito cells (FSC) have been thought to be collagen-producing cells with regard to hepatic fibrosis. We have found that desmin, a structural protein of muscular intermediate filaments, is found in the cytoplasm of FSC, even in negative vitamin A-autofluorescence FSC, by using immunocytochemical techniques. In the present study, numbers of FSC produced by the administration of heterogeneic serum or carbon tetrachloride were detected in fibrous and non-fibrous areas of hepatic lobules by using a morphometrical analyser. After 8 weeks of intraperitoneal injections of heterogeneic serum into rats, FSC in fibrous septa increased in number and the increase continued to 12 weeks-although the numbers of FSC in non-fibrous areas and in controls given homologous serum did not change during the experiment. In the case of rats administered CCl4 once, FSC in fibrous areas increased in numbers after 48 h through 72 h, although the number of FSC in non-fibrous areas and in controls did not change. The results, with an apparent increase in numbers of FSC in fibrous areas and no change in non-fibrous areas, showed the possibility of hyperplasia due to cell division of FSC in the new fibroplastic areas.

Microscopic fluorometric analysis of Na-fluorescein transport in the smallest secretory unit (couplet hepatocytes) and the effects of cytochalasin B.

The uptake and secretion of sodium fluorescein by couplet hepatocytes was examined in primary culture. When sodium fluorescein was added at an early stage of primary couplet hepatocytes culture, this resulted in a rapid uptake of the dye and its subsequent accumulation in bile canaliculi. From microscopic fluorometry observations, cytochalasin B pretreatment of the couplet hepatocytes caused much more rapid uptake and secretion of the dye in bile canaliculi. The present study indicates that the cholestatic agent cytochalasin B causes choleresis at the bile canalicular level of cultured couplet hepatocytes and also indicates that a defect in canalicular secretion plays no role in the cytochalasin B-induced impairment of bile flow.