Satoko Suzuki

Sphingosine 1‐phosphate protects rat liver sinusoidal endothelial cells from ethanol‐induced apoptosis: Role of intracellular calcium and nitric oxide

In alcoholic liver disease, ethanol‐induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol‐induced injury. To elucidate the role of sphingosine 1‐phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 μmol/L S1P attenuated ethanol‐induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P‐induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, NG‐nitro‐L‐arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca2+‐sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury. (HEPATOLOGY 2006;44:1278–1287.)

Dose-finding trial of tolvaptan in liver cirrhosis patients with hepatic edema: A randomized, double-blind, placebo-controlled trial

Liver cirrhosis represents the end stage of any chronic liver disease, and it is associated with hepatic edema such as ascites. Many patients with ascites do not respond to diuretic therapy or require administration of diuretics at high doses that can cause adverse events. This 7‐day, multicenter, double‐blind trial of tolvaptan was designed to determine the optimal dose of tolvaptan for producing the intended pharmacological effect in hepatic edema.

Burn injury sensitizes rat Kupffer cells via mechanisms dependent on gut-derived endotoxin

BackgroundBacterial translocation occurs after thermal injury in association with intestinal barrier loss. Recently, we found that sensitization of Kupffer cells involved gut-derived endotoxin; therefore, the purpose of this work was to study the mechanisms of sensitization of Kupffer cells in burn injury.MethodsRats received a 30% body surface area full-thickness steam burn 24 h before experiments. Serum alanine aminotransferase (ALT) was measured to assess liver damage, and plasma endotoxin in the portal vein were measured. Kupffer cells were isolated 24 h after the burn. Intracellular calcium ([Ca2+]i) in Kupffer cells was measured using a microspectrofluorometer with the fluorescent indicator, fura-2, and tumor necrosis factor (TNF)-α was measured by enzyme-linked immunosorbent assay (ELISA).ResultsLipopolysaccharide (LPS)-induced mortality was increased by burn treatment. This increase was blocked by gadolinium chloride, a Kupffer-cell toxicant. Accordingly, Kupffer cells were involved in this system. The LPS-induced increase of ALT was upregulated by the burn injury. This increase was blocked by pretreatment with antibiotics. Endotoxin levels were increased to almost 300 pg/ml (normal, <20 pg/ml) in the portal veins of rats that received a burn. This increase was blunted by antibiotics. In Kupffer cells isolated from untreated control rats, [Ca2+]i increased to 82 ± 7 nM after the addition of LPS (100 ng/ml). Levels were elevated twofold over control levels in the cells from rats with burn (174 ± 15 nM). In addition, TNF-α production by Kupffer cells isolated from rats with burn was increased fourfold over the based level. Sterilization of the gut with antibiotics completely blocked all effects of the burn on [Ca2+]i and TNF-α release.ConclusionsKupffer cells isolated from rats with burn exhibited sensitization to LPS, involving gut-derived endotoxin. It is concluded that burns sensitize Kupffer cells to LPS via mechanisms that are dependent on gut-derived endotoxin.

Effects of Wortmannin, a Novel Myosin Light-Chain Kinase Inhibitor, on Bile Canalicular Contraction in Vitro and in Vivo

BACKGROUND The cytoskeletal system is believed to play an important role in normal bile formation. The effects of wortmannin, a new myosin light-chain kinase inhibitor, on bile canalicular contraction and bile flow have been observed. METHODS The bile canalicular contraction of cultured hepatocyte doublets was investigated, using an image analyzer with a phase contrast microscope, and the intracellular Ca2+ concentration was measured, using microscopic fluorometry. We also investigated bile flow by in vivo intraportal infusion of the drug in rats. RESULTS Treatment with wortmannin inhibited norepinephrine-induced canalicular contraction and caused a decrease in bile flow without changing systematic and portal blood pressure. Morphologic examination of the electron microscopic study showed that most bile canaliculi were dilated, with loss of microvilli, but no other apparent damage was seen in parenchymal hepatocytes. CONCLUSIONS These data suggest that the integrity of the phosphorylation system of myosin is essential for normal bile flow.

Recurrent severe acute hepatitis caused by hypereosinophilic syndrome associated with elevated serum immunoglobulin G4 levels.

A 46-year-old male was admitted to our hospital with severe acute hepatitis, hypereosinophilia, and serum immunoglobulin G4 (IgG4) elevation. Plasma exchange was performed, and he was treated by prednisolone; however, his hepatitis recurred twice over the following twelve months. Transjuglar liver biopsy was performed at the third onset, which demonstrated extensive hepatocyte necrosis, congestion, and severe eosinophil infiltration. We diagnosed hypereosinophilic syndrome (HES)-related hepatitis. Although no cholangitis was detected by imaging and pathological diagnosis, IgG4-positive cells were detected in the liver and bone marrow. Furthermore, the elevation of serum IgG4 levels was associated with the eosinophil count and his clinical condition. After the addition of azathioprine to his treatment regimen, no reoccurrence was observed. IgG4-positive cells may have contributed to the severity and refractoriness of this recurrent acute HES-related hepatitis.

Integrity of myosin light chain kinase is essential for Ito cell contraction

Hepatic sinusoidal Ito cells (fat storing cells) are believed to play a regulatory role on hepatic sinusoidal blood flow through their contraction. The detailed mechanism of contraction of Ito cells, however, is still unknown. The present study was undertaken to clarify the effect of new myosin light chain kinase inhibitor, wortmannin, on Ito cell contraction. Ito cells prepared from rat liver were cultured for 4 days before the study. The contraction of Ito cells, which was monitored and analysed by time‐lapse video tape recording, was triggered by addition of endothelin‐1. Wortmannin pretreatment for 1 h inhibited endothelin‐induced Ito cell contraction dose‐dependently. Therefore, the integrity of the actomyosin system is essential for Ito cell contraction and normal sinusoidal blood flow.

460 Hepatic Steatosis Causes the Dysfunction of Autophagy in NAFLD Patients

triglycerides, and CARS microscopy. In contrast, GDC-0449 treatment significantly attenuated FFC diet-induced liver injury as manifest by reduced plasma ALT values and hepatic TUNEL positive cells. Consistent with a reduction in tissue injury, GDC-0449 normalized FFC diet-induced mRNA levels of TNF-alpha, IL-1beta, monocyte chemotactic protein-1 and a variety of macrophage markers to the levels of mice on chow diet and prevented accumulation of galectin-3 (a macrophage marker) positive cells in the liver. Furthermore, GDC-0449 therapy of FFC diet-fed mice abrogated mRNA upregulation of collagen 1a1, alpha-smooth muscle actin and osteopontin in the liver, and decreased hepatic collagen deposition to values of mice on chow diet as quantified by Sirius red staining and second harmonic generation microscopy of frozen sections using a two photon confocal microscope. GDC-0449 treatment also abolished FFC-diet induced hepatic expression of death receptor TRAIL-R2, but not FAS nor TNFR1. FCC diet fed mice, but not chow fed animals, underwent significant liver injury following treatment with a TRAIL agonist. TRAIL-mediated injury in the FCC diet fed mice was reduced by pre-treatment with GDC-0449. In conclusion, inhibition of hedgehog signaling with GDC-0449 appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition maybe salutary in human NASH.