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Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis. alcoholic liver disease; mitochondria; oxidative stress; cytochrome c

Bile acids transactivate the EGF receptor (EGFR) in cholangiocytes. However, the mechanisms by which bile acids transactivate the EGFR remain unknown. Our aims were to examine the effects of bile acids on EGFR activation in human cholangiocyte cell lines KMBC and H-69. Bile acids stimulated cell growth and induced EGFR phosphorylation in a ligand-dependent manner. Although cells constitutively expressed several EGFR ligands, only transforming growth factor-[alpha] (TGF-[alpha]) antisera effectively blocked bile acid-induced EGFR phosphorylation. Consistent with the concept that matrix metalloproteinase (MMP) activity is requisite for TGF-[alpha] membrane release and ligand function, bile acid transactivation of EGFR and cell growth was blocked by an MMP inhibitor. In conclusion, bile acids activate EGFR via a TGF-[alpha]-dependent mechanism, and this EGFR activation promotes cellular growth. c-Src kinase; matrix metalloproteinase; ligand dependent; transactivation

Toxic bile acids facilitate Fas and tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) death-receptor oligomerization and activation. Bile acid modulation of death-receptor signaling is multifactorial and includes trafficking of Fas to the cell surface, enhancing TRAIL-R2/DR5 expression, and suppression of function of cFLIP, an antiapoptotic protein modulating death-receptor function. Because bile acid-associated death receptor-mediated apoptosis is a common mechanism for cholestatic hepatocyte injury, inhibition of death receptors and their cascades may prove useful in attenuating liver injury during cholestasis. Fas; tumor necrosis factor-associated apoptosis-inducing ligand; Bid

Endothelins are powerful vasoconstrictor peptides with 21 amino acid residues. A study was conducted to explore the possibility that intestinal epithelial cells express and release endothelins. Results showed that a potent cytokine termed IL-2 stimulated the release from intestinal cells of ET isopeptide ET-1 which, in turn, may be involved in regulating the growth of these cells. The findings suggest that ET can facilitate a coordinated response by epithelial cells and cellular constituents of the local immune system that is present in the intestinal mucosa.

The Peyers patches of rat small intestines were analyzed by intravital microscope and fluorochrome carboxyfluorescein diacetate succinimidyl ester (CFDASE) to determine the dynamics of intestinal T lymphocyte migration. CFDASE analysis of rat ileal Peyers patches indicated the inhibitory role of vasoactive intestinal polypeptides (VIP) on the transendothelial migration of T lymphocytes through the ileal postcapillary venules. Furthermore, VIP-induced attenuation of ileal T cell migration involves the relaxation of vascular smooth muscles which alters the permeability of the capillaries.

Endothelin (ET) is a peptide produced by the endothelium and is suggested to participate in the maintenance of gastrointestinal integrity. To determine the role of ET1, red blood cell (RBC) velocity in arterioles and venules from the rat ileum were examined after treatment with endotoxin, a substance which causes mucosal damage. Intravital microscopy revealed a marked increase in leukocytes and significant decrease in the platelet-activating factor and nitric oxide synthase activity. Treatment with an ET(sub A) receptor antagonist led to a further decrease in leukocytes, attenuated the increase in RBC, and prevented mucosal injury. These results suggest that ET1 via ET(sub A) receptors causes endotoxin-induced leukocyte accumulation.

Endotoxins promote the interactions between lymphocytes and endothelium in the intestinal Peyer's patches and villus mucosa of rats. Lymphocytes from the blood are utilized by the rat intestinal tissues in the presence of a constant antigen stimuli. T lymphocytes show rolling and adhesive behavior in microvessels of both Peyer's patches and villi. These behaviors are regulated by alpha-4-integrins, and partly by beta-2-integrins. In the capillaries of the intestinal mucosa of lipopolysaccharide-treated rats, adhesion occurs without rolling and is alpha-4-integrin-independent.

Chronic ethanol consumption exacerbates microcirculatory damage in rat mesentery after reperfusion. Am J Physiol Gastrointest Liver Physiol 280: G939-G948, 2001.--Although the negative effect of excessive alcohol consumption on later stressful events has long been recognized, pathophysiological mechanisms are incompletely understood. We examined possible roles of oxygen radicals and glutathione content in mesenteric venules of chronically ethanol-fed rats exposed to ischemia-reperfusion. Changes in microvascular hemodynamics, such as red blood cell (RBC) velocity, leukocyte adherence, and albumin extravasation, were monitored in postcapillary venules by intravital fluorescence microscopy. Chronic ethanol feeding significantly exaggerated the magnitude of the decrease in RBC velocity, the increased number of adherent leukocytes, and increased albumin leakage elicited by 10 min of ischemia followed by 30 min of reperfusion. Oxidative stress in the endothelium of venules monitored by dihydrorhodamine 123 (DHR) fluorescence was more severe in rats fed ethanol chronically. Both superoxide dismutase and N-acetyl-L-cysteine, which is known to increase glutathione content, reduced the ischemia-reperfusion-induced decrease in RBC velocity, the number of adherent leukocytes, and the increase in albumin leakage, as well as oxidative activation of DHR. This suggests that the increased reperfusion-induced microvascular disturbances in the mesenteric venules of rats fed ethanol chronically are significantly correlated with excessive production of oxygen-derived free radicals and decreased glutathione synthesis. leukocyte adherence; vascular permeability; mast cell degranulation; oxygen radicals; glutathione

Hokari, Ryota, Soichiro Miura, Hitoshi Fujimori, Seiichiro Koseki, Yoshikazu Tsuzuki, Hiroyuki Kimura, Hajime Higuchi, Hiroshi Serizawa, D. Neil Granger, and Hiromasa Ishii. Altered migration of gut-derived T lymphocytes after activation with concanavalin A. Am. J. Physiol. 277 (Gastrointest. Liver Physiol. 40): G763-G772, 1999.--Although activation of lymphocytes is known to be associated with profound changes in homing behavior, it remains unclear how activation alters migration of gut-derived lymphocytes in lymphoid and nonlymphoid organs. The objectives of this study were 1) to compare migration of naive and concanavalin A (ConA)-activated T lymphocytes into the gut mucosa, spleen, and liver and 2) to define the role of specific adhesion molecules in this homing process. Fluorescently labeled T lymphocytes collected from rat intestinal lymph were injected into the jugular vein, and the kinetics of appearance of the infused lymphocytes were monitored in ileal Peyer's patches, spleen, and liver. The migration of naive and ConA-activated T lymphocytes into microvessels were compared using an intravital microscope. ConA stimulation significantly increased the rolling velocity of T lymphocytes in postcapillary venules of Peyer's patches, and ConA-stimulated lymphocytes exhibited a loss of the selective adherence properties in Peyer's patches that is normally observed with naive T cells. ConA activation also suppressed the accumulation of T cells in the spleen. On the other hand, the adherence of T cells to hepatic sinusoidal endothelium was significantly increased after ConA activation, especially in the periportal area, and this increase was attenuated by an anti-intercellular adhesion molecule (ICAM)-1 antibody. Flow cytometry analysis revealed a decline in L-selectin expression and an increase in CD11a expression and ICAM-1 on the surface of ConA-treated T cells. In conclusion, activation of gut-derived T lymphocytes with ConA significantly alters their migration path, with a diminished localization to Peyer's patches and spleen and a preferential accumulation in hepatic sinusoids. This altered migration pattern likely results from changes in the expression of leukocyte adhesion molecules such as L-selectin and CD11a. Peyer's patches; spleen; adhesion molecules; intestinal lymph; CD11a; intercellular adhesion molecule-1