Kayo Nagashima

Functional diversity between Rho-kinase- and MLCK-mediated cytoskeletal actions in a myofibroblast-like hepatic stellate cell line.

Using a rat myofibroblast-like hepatic stellate cell line, we studied the actomyosin-based cytoskeletal actions mediated by Rho-kinase and/or myosin light chain kinase (MLCK). Calmodulin/MLCK inhibitors W-7 and ML-7 attenuated cell migration dose-relatedly at concentrations from 10(-6) to 10(-4)M and collagen gel-contraction by the cells at 10(-4)M, respectively. Rho-kinase inhibitors Y-27632 and HA1077 attenuated the gel-contraction at concentrations from 10(-6) to 10(-4) M, respectively. These Rho-kinase inhibitors attenuated cell migration at 10(-7)M but enhanced the migration at 10(-4)M, respectively. They altered cell morphology showing prominent peripheral actin bundles and sparse central stress fibers, in comparison with the calmodulin/MLCK inhibitors. Both ML-7 and Y-27632 attenuated phosphorylation of myosin regulatory light chain and cell attachment to extracellular substrate. ML-7 attenuated the activation of GTP-binding protein Rac, while Y-27632 did not. These findings suggest that the actomyosin-based cytoskeletal actions can be functionally diverse depending on the Rho-kinase-mediated pathway and the MLCK-mediated pathway.

Leucine stimulates the secretion of hepatocyte growth factor by hepatic stellate cells

Branched-chain amino acids (BCAAs) modulate various cellular functions, in addition to providing substrates for the production of proteins. In this study, we examined the effect of BCAAs on the secretion of hepatocyte growth factor (HGF) by hepatic stellate cells. A hepatic stellate cell clone was cultured in medium supplemented with various concentrations of valine, leucine, or isoleucine. Of these BCAAs, leucine markedly induced an increase in the levels of HGF in the medium in a dose-dependent manner. The addition of valine or isoleucine had no significant effect on HGF levels in the medium. The difference in levels of HGF in the medium between leucine-treated and non-treated cells was enhanced by the incubation period. These results demonstrate that, among BCAAs, leucine stimulates the secretion of HGF by cultured hepatic stellate cells.

Ischemic preconditioning in liver pathophysiology

Brief periods of tissue ischemia produced tissue resistance to prolonged ischemia and reperfusion, a phenomenon called ischemic preconditioning. The mechanisms of ischemic preconditioning were examined in a rat warm ischemia–reperfusion model as well as the effect of ischemic preconditioning on liver regeneration. Ischemic preconditioning decreased liver injury after warm ischemia–reperfusion, which was reversed by Kupffer cell depletion. Ischemic preconditioning stimulated Kupffer cells to produce reactive oxygen species. Scavengers of reactive oxygen species reversed the effect of ischemic preconditioning, and pretreatment with sublethal dose of hydrogen peroxide mimicked ischemic preconditioning effect. Rat livers were preconditioned by ischemia and subjected to 70% partial hepatectomy. Liver regeneration was then evaluated serially. Ischemic preconditioning promoted liver regeneration, which was reversed by adenosine A2 receptor antagonism and mimicked by adenosine A2 receptor agonism. Promotion of liver regeneration by ischemic preconditioning and adenosine A2 receptor agonism were reversed by Kupffer cell depletion. In conclusion, ischemic preconditioning stimulates Kupffer cells to produce reactive oxygen species, leading to hepatocyte protection against warm ischemia–reperfusion injury; and ischemic preconditioning promoted liver regeneration via adenosine A2 receptor pathway in Kupffer cells.

Effect of Rapamycin on Hepatocyte Function and Proliferation Induced by Growth Factors

Background: Rapamycin is a specific inhibitor of the mammalian target of rapamycin (mTOR). The effect of rapamycin on proliferation and cellular function was studied in hepatocytes stimulated by hepatocyte growth factor (HGF) or transforming growth factor-α (TGFα). Methods and Results: When isolated rat hepatocytes were cultured at low density, the addition of HGF or TGFα increased DNA synthesis but did not affect albumin or fibrinogen concentrations in the medium. In contrast, in hepatocytes cultured at high density, the albumin and fibrinogen concentrations, but not DNA synthesis, were increased by HGF or TGFα. The HGF- or TGFα-induced increase in DNA synthesis and in albumin or fibrinogen concentrations was suppressed by the addition of rapamycin, as well as wortmannin, a phosphatidylinositol-3 kinase inhibitor. Conclusion: HGF and TGFα stimulate proliferation and function of hepatocytes depending upon the conditions, and rapamycin inhibited these stimulatory effects, possibly by inhibiting the mTOR-dependent signaling pathway.

HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins

Department of Internal Medicine, Kawakita General Hospital, Tokyo, JapanEmail: Mikio Yanase* - yanase-1IM@h.u-tokyo.ac.jp; Hitoshi Ikeda- ikeda-1IM@h.u-tokyo.ac.jp; Atsushi Matsui - atsushim@saitama-med.ac.jp; Eisei Noiri - noiri-1IM@h.u-tokyo.ac.jp; Tomoaki Tomiya -tomiya-1IM@h.u-tokyo.ac.jp; Masahiro Arai - arai-1IM@h.u-tokyo.ac.jp; Yukiko Inoue - yanase-1IM@h.u-tokyo.ac.jp; Kazuaki Tejima - yanase-1I M@h.u-tokyo.ac.jp; Kayo Nagashima- yanase-1IM@h.u-tokyo.ac.jp; Takako Nishikawa - yanase-1IM@h.u-tokyo.ac.jp; Satoshi Kimura - yanase-1IM@h.u-tokyo.ac.jp; Kenji Fujiwara - yanase-1IM@h.u-tokyo.ac.jp; Marcos Rojkind - marcos.rojkind@na.amedd.army.mil; Itsuro Ogata - itsuro@viola.ocn.ne.jp* Corresponding author