Biomedical subjects

I Kurose

Publications and source records attributed to I Kurose.

At least 37 records · Page 2Linked to original sources

PubMed1997

Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication.

The present study was designed to investigate whether acute ethanol intoxication increases the production of active oxidants, and subsequently promotes apoptosis of hepatocytes. Hepatocytes were isolated from male Wistar rats, and cultured in the presence or absence of ethanol. The fluorescence in situ nick end labeling method and an enzyme-linked immunosorbent assay (ELISA) system to quantify fragmented DNA were used to estimate apoptotic change in hepatocytes. Nuclear morphological alterations and membrane barrier dysfunction of hepatocytes were assessed by staining with Hoechst 33342 and propidium iodide (PI). Intracellular glutathione level was determined as the fluorescence of monochlorobimane (MCLB), which forms conjugate with glutathione to become fluorescent. Ethanol (100 mmol/L) increased the amount of fragmented DNA and the number of apoptotic hepatocytes in vivo as well as in vitro. These ethanol-induced alterations in hepatocytes were attenuated by simultaneous incubation with either 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, or dimethylthiourea, an intracellular oxidant scavenger. Diethyl maleic acid (DMA), a glutathione depletor, enhanced the induction of apoptotic change, and decreased membrane barrier function in ethanol-treated hepatocytes, whereas ethanol per se did not increase the number of PI-positive hepatocytes. Furthermore, combination of ethanol and DMA but not ethanol alone decreased the hepatocyte MCLB fluorescence. Taken together, the present study suggests that active oxidants produced during ethanol metabolism mediate fragmentation of DNA in hepatocytes, and that intracellular antioxidants such as glutathione play a critical role in the cytoprotective mechanisms of hepatocyte against lethal cell death, ie, apoptosis, induced by ethanol.

Animals↗

PubMed1997

Suppressed production of nitric oxide as a cause of irregular constriction of gastric venules induced by thermal injury in rats.

Microcirculatory disturbance is a crucial step in development of gastric mucosal lesions. In this study we investigated the influence of nitric oxide (NO) on the irregular constriction of gastric venules observed in rats subjected to thermal injury. Male Wistar rats were anesthetized and a 30% full skin thickness dorsal burn was inflicted. Microvascular images in the submucosal layer and basal region of the gastric mucosa were obtained through an intravital microscope. Distribution of constitutive-type nitric oxide synthase (cNOS) was observed by the immunofluorescence method. Venule walls were basically smooth in the control group (no injury), whereas irregular constrictions tended to be observed in the 5-h group (5 h after thermal injury). Venule diameter was significantly smaller in the 5-h group. In the control group, cNOS-associated fluorescence was found on the venule endothelium, whereas it was almost negligible after thermal injury. A NO donor, 3-morpholinosydnonimine hydrochloride, simultaneously diminished the thermal injury-associated venule constriction and irregularity of venule walls. Therefore, the present study suggests that venule constriction, which is observed 5 h after thermal injury, is related to decreased NO production by endothelial cNOS and is a cause of irregularity of the venule walls.

Animals↗

PubMed1997

Pathogenesis of alcoholic liver disease with particular emphasis on oxidative stress.

Oxidative stress is well recognized to be a key step in the pathogenesis of ethanol-associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species and/or by decreasing the level of endogenous antioxidants. Numerous experimental studies have emphasized the role of the ethanol-inducible cytochrome P450 in the microsomes and the molybdo-flavoenzyme xanthine oxidase in the cytosol. This review shows the putative role of ethanol-induced disturbances in iron metabolism in relation to iron as a pro-oxidant factor. Ethanol administration also affects the mitochondrial free radical generation. Many previous studies suggest a role for active oxygens in ethanol-induced mitochondrial dysfunction in hepatocytes. Recent studies in our laboratory in the Department of Internal Medicine, Keio University, using a confocal laser scanning microscopic system strongly suggest that active oxidants generated during ethanol metabolism produce mitochondrial membrane permeability transition in isolated and cultured hepatocytes. In addition, acetaldehyde, ethanol consumption-associated endotoxaemia and subsequent release of inflammatory mediators may cause hepatocyte injury via both oxyradical-dependent and -independent mechanisms. These cytotoxic processes may lead to lethal hepatocyte injury. Investigations further implicate the endogenous glutathione-glutathione peroxidase system and catalase as important antioxidants and cytoprotective machinery in the hepatocytes exposed to ethanol.

Acetaldehyde↗

PubMed1997

Vasoactive intestinal peptide modulates T lymphocyte migration in Peyer's patches of rat small intestine.

Although vasoactive intestinal peptide (VIP) has been postulated to function in modulation of T cell trafficking, the exact mechanism has not been elucidated in vivo. In the present study, the effects of VIP on T lymphocyte migration were examined in rat Peyer's patches. T lymphocytes collected from intestinal lymph of rats were labeled with carboxyfluorescein diacetate succinimidyl ester and injected into the jugular vein. Peyer's patches of the recipient rats were observed with intravital fluorescence microscopy. In vivo intra-arterial infusion of or in vitro incubation with VIP did not affect the initial lymphocyte interaction with postcapillary venules of Peyer's patches. However, these treatments with VIP significantly inhibited transendothelial migration and also significantly blocked the interstitial migration of T cells and inhibited their subsequent appearance in the interfollicular lymphatics. Treatment with adenosine 3',5'-cyclic monophosphate (cAMP)-inducing agents resulted in similar inhibitory effect on T lymphocyte migration in Peyer's patches. In conclusion, VIP has significant inhibitory effects on T lymphocyte migration in Peyer's patches, possibly mediated by elevation of the intracellular cAMP concentrations.

Animals↗

PubMed1997

Ischemia/reperfusion-induced microvascular dysfunction: role of oxidants and lipid mediators.

The objective of this study was to define the role of oxidants and lipid mediators in the leukocyte-endothelial cell adhesion and albumin leakage elicited in rat mesenteric venules by ischemia-reperfusion (I/R). Intravital fluorescence microscopy was used to monitor leukocyte adherence and emigration, platelet-leukocyte aggregation, mast cell degranulation, and albumin leakage after release of a 20-min arterial occlusion. I/R elicited large increases in leukocyte-endothelial cell adhesion and albumin leakage. These responses were significantly attenuated in venules treated with either superoxide dismutase, oxypurinol (an inhibitor of xanthine oxidase), lodoxamide (a mast cell stabilizer), WEB-2086 (a platelet-activating factor antagonist), or SC-41930 (a leukotriene B4-receptor antagonist) but not by U-74006F (an inhibitor of lipid peroxidation). Platelet-leukocyte aggregates and mast cell degranulation induced by I/R were also attenuated by administration of either superoxide dismutase or lodoxamide. These results support the hypothesis that oxidants produced, in part, by xanthine oxidase promote the formation (by mast cells and endothelial cells) of platelet-activating factor and leukotriene B4, which recruit and activate leukocytes in postischemic venules. The adherent and emigrated leukocytes then mediate the increased albumin extravasation observed in the postcapillary venules.

Animals↗

PubMed1997

Reperfusion-induced leukocyte adhesion and vascular protein leakage in normal and hypercholesterolemic rats.

The objective of this study was to define the influence of hypercholesterolemia on ischemia-reperfusion (I/R)-induced leukocyte-endothelial cell adhesion and albumin leakage in rat mesenteric venules. The microvascular alterations normally elicited by I/R (leukocyte adherence and emigration, albumin leakage, and platelet aggregation) were more pronounced in hypercholesterolemic rats (compared with control rats). Monoclonal antibodies against the adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 attenuated the I/R-induced leukocyte adherence and emigration and albumin leakage. Leukocyte adherence, but not albumin leakage, was diminished in animals pretreated with a P-selectin-specific antibody. Platelet aggregation was reduced by antibodies directed against either P-selectin, CD18, or intercellular adhesion molecule-1, as well as a GPIIb-IIIa antagonist. These results indicate that the enhanced reperfusion-induced albumin leakage in hypercholesterolemic rats is dependent on leukocyte-endothelial cell adhesion. Furthermore, P-selectin- and CD11/CD18-dependent heterotypic and GPIIb-IIIa-mediated homotypic platelet aggregation appear to influence the extravasation of both leukocytes and albumin in postischemic venules of hypercholesterolemic rats.

Animals↗

PubMed1997

Effects of fluvastatin on leukocyte-endothelial cell adhesion in hypercholesterolemic rats.

The overall objective of this study was to determine whether peroral treatment with the 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor fluvastatin influences the leukocyte-endothelial cell adhesion (LECA) observed in postcapillary venules of hypercholesterolemic rats. Rats were fed either normal chow or a chow supplemented with 1% cholesterol for 10 days. Leukocyte adherence and extravasation, leukocyte rolling velocity, red blood cell velocity, and vessel diameter were monitored in mesenteric venules superfused with either 100 nmol/L platelet-activating factor (PAF) or 20 nmol/L leukotriene B4 (LTB4). Hypercholesterolemic rats exhibited an exaggerated LECA response compared with their normocholesterolemic counterparts. In hypercholesterolemic rats, treatment with fluvastatin significantly attenuated the leukocyte-adherence responses to PAF and LTB4 as well as the leukocyte emigration response to LTB4. Fluvastatin treatment also inhibited the PAF- and LTB4-induced reductions in leukocyte rolling velocity. These findings indicate that fluvastatin blunts the inflammatory responses elicited in post-capillary venules by lipid mediators.

Animals↗

PubMed1997

Oxidant-regulation of gene expression in the chronically inflamed intestine.

It is becoming increasingly apparent that the chronic gut inflammation observed in the idiopathic inflammatory bowel diseases (e.g. ulcerative colitis, Crohn's disease) is associated with enhanced production of leukocyte-derived oxidants. Oxidants such as hydrogen peroxide are known to activate certain transcription factors such as nuclear transcription factor kappa beta. Nuclear transcription factor kB (NF-kappa B) is a ubiquitous transcription factor and pleiotropic regulator of numerous genes involved in the immune and inflammatory responses. This transcription factor is activated via the selective phosphorylation, ubiquination and degradation of its inhibitor protein I-kB thereby allowing translocation of NF-kappa B into the nucleus where it upregulates the transcription of a variety of adhesion molecules (e.g. ICAM-1, VCAM-1), cytokines (TNF, IL-1, IL-6) and enzymes (iNOS). The proteolytic degradation of the post-translationally modified I-kappa B is known to be mediated by the 26S proteasome complex. Based upon work from our laboratory, we propose that inhibition of NF-kappa B activation produces significant anti inflammatory activity which may be mediated by the inhibition of transcription of certain pro-inflammatory mediators and adhesion molecules.

Animals↗

PubMed1996

Diabetes exacerbates inflammatory responses to ischemia-reperfusion.

BACKGROUND: Diabetes is associated with an increased incidence of ischemic organ damage. The objectives of present study were to compare the leukocyte-endothelial cell adhesive interactions and albumin leakage response of mesenteric venules to ischemia-reperfusion between control rats, rats with streptozotocin-induced diabetes, and rats with hyperglycemia induced by glucose infusion and to define the molecular determinants of the leukocyte accumulation elicited by ischemia-reperfusion in diabetic rats. METHODS AND RESULTS: Under baseline conditions, lower venular shear rates and an increased number of rolling leukocytes were noted in diabetic rats, whereas the number of adherent and emigrated leukocytes did not differ from that in control rats. Spontaneous albumin leakage from mesenteric venules was markedly increased in diabetic rats but not in hyperglycemic nondiabetic rats. Ischemia-reperfusion elicited significantly larger increases in leukocyte adhesion and emigration and albumin leakage in diabetic rats. Acute elevation of glucose levels did not modify the microvascular responses to ischemia-reperfusion compared with control rats. Antibodies directed against CD11/CD18, intercellular adhesion molecule-1 (ICAM-1), or P-selectin but not L-selectin significantly decreased the number of adherent and emigrated leukocytes after ischemia-reperfusion in diabetic rats. However, none of the antibodies significantly attenuated the increased albumin leakage response to ischemia-reperfusion in diabetic rats. CONCLUSIONS: These results indicate that diabetes mellitus is associated with exaggerated leukocyte-endothelial cell adhesion and albumin leakage responses to ischemia-reperfusion. The enhanced leukocyte accumulation in response to ischemia-reperfusion is mediated by CD11/CD18-ICAM-1 interactions (firm adhesion) and P-selectin (rolling). The exaggerated albumin leakage response to ischemia-reperfusion in diabetics is not mediated by the recruited inflammatory cells.

Albumins↗

PubMed1996

Increased nitric oxide synthase activity as a cause of mitochondrial dysfunction in rat hepatocytes: roles for tumor necrosis factor alpha.

Kupffer cells have been implicated in playing an important role in the pathogenesis of endotoxemia-associated liver injury. The present study was designed to investigate whether Kupffer cell-derived mediators alter the mitochondrial oxidative phosphorylation of hepatocytes in the endotoxemic condition. Liver cells were isolated from male Wistar rats. Oxidative phosphorylation was monitored as the fluorescence of rhodamine 123 (Rh123), which is the fluorescent cationic dye used to indicate mitochondrial energy synthesis. Two hours after coculture of hepatocytes with lipopolysaccharide (LPS)-pretreated Kupffer cells, a marked decrease in hepatocyte rhodamine 123 fluorescence was observed. The hepatocyte mitochondrial dysfunction was attenuated by the addition of either N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis, or aminoguanidine, an inducible-type of NO synthase inhibitor, to the culture medium of cocultures, to the pretreatment of LPS-activated Kupffer cells with antisense oligodeoxynucleotides against iNOS messenger RNA (mRNA), or to tumor necrosis factor alpha (TNF-alpha) mRNA. Four hours after the coculture, hepatocyte Rh123 fluorescence further decreased, and an iNOS induction as well as an increased NO production were observed in hepatocytes that were cocultured with LPS-pretreated Kupffer cells. The membrane barrier dysfunction of hepatocytes, indicated by propidium iodide staining, was also induced by a 4-hour coculture with LPS-pretreated Kupffer cells. These late-phase changes were inhibited either by the pretreatment of hepatocytes with antisense oligodeoxynucleotides against iNOS mRNA or by treatments that are effective in the early phase (within 2 hours). Incubation with recombinant rat TNF-alpha decreased hepatocyte Rh123 fluorescence within 2 hours. Thus, the present study suggests that NO and TNF-alpha released from LPS-pretreated Kupffer cells directly inhibit the hepatocyte mitochondrial function in the early phase, and then NO synthesized by TNF-alpha-induced hepatocyte iNOS causes lethal hepatocyte injury, characterized by diminished mitochondrial energization and membrane barrier function in the late phase.

Animals↗

PubMed1996

IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage.

Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1alpha levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.

Acrylates↗

PubMed1996

Rat Kupffer cell-derived nitric oxide suppresses proliferation and induces apoptosis of syngeneic hepatoma cells.

BACKGROUND & AIMS: Evidence increasingly indicates that nitric oxide plays an important role in antitumor mechanisms. The aim of this study was to investigate the role of NO in the mechanisms regulating the proliferation and death of hepatoma cells cocultured with Kupffer cells. METHODS: Kupffer cells were isolated from male Wistar rats and cocultured with rat hepatoma AH70 cells. Proliferation was determined by calculating the number of total and 5-bromodeoxyuridine-positive AH70 cells. Apoptosis was assessed by electron-microscopic and fluorescence-microscopic observations and in situ nick end labeling method. Immunofluorescence and in situ hybridization studies were performed to investigate the induction of inducible NO synthase (iNOS). RESULTS: Kupffer cells reduced proliferation and induced apoptosis of AH70 cells, which were attenuated by the NO synthesis inhibitors NG-monomethyl-L-arginine and aminoguanidine. Increased inductions of iNOS messenger RNA and iNOS were observed in Kupffer cells cocultured with AH70 cells. Addition of monoclonal antibody directed against either rat CD18 or intercellular adhesion molecule 1 also attenuated the increased NO production of Kupffer cells and the alterations of AH70 cells. CONCLUSIONS: Kupffer cell-derived NO suppresses proliferation and induces apoptosis of hepatoma cells. The CD18 intercellular adhesion molecule 1-dependent adhesive interaction with hepatoma cells triggers NO production by Kupffer cells.

Animals↗

PubMed1996

Role in nitric oxide in Kupffer cell-mediated hepatoma cell cytotoxicity in vitro and ex vivo.

The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG-monomethyl-L-arginine (L-NMMA), an analogue of L-arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L-NMMA, suggesting that Kupffer cell-mediated injury of tumor cells largely depends on NO. The concentrations of NO-2 + NO-3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell-induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time-dependent manner within 2 hours. The number of PI-positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega-L-nitroarginine-methylester (L-NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.

Animals↗

PubMed1996

alpha 4 integrin plays a critical role in early stages of T lymphocyte migration in Peyer's patches of rats.

Lymphocyte recirculation through the blood flow circuit and lymphoid organs is important for the maintenance of immune defense, and is defined as lymphocyte homing. During the homing process, several adhesion molecules have been postulated to play an important role in lymphocyte recruitment from the vascular space. In the present study, we investigated the effect of a novel mAb against rat alpha 4 integrin (MR alpha 4-1) on the interaction of T lymphocytes with postcapillary venules (PCV) and their subsequent migration into the interstitium of Peyer's patches, using intravital video microscopy. T lymphocytes collected from intestinal lymph were labeled with a fluorochrome carboxyfluorescein diacetate succinimidyl ester and were than injected into the jugular vein of recipient rats. The microvasculature in the ileal Peyer's patches of recipient rats was observed sequentially by intravital fluorescence microscopy. In controls, lymphocytes exhibited rolling behavior which was followed by firm adhesion to the endothelium of PCV. The density of sticking lymphocytes gradually increased during the first 30 min. These initial interactions of lymphocytes with the PCV (rolling and adherence) were drastically inhibited by treatment with MR alpha 4-1, both when MR alpha 4-1 was preinfused into rats and when T cells were preincubated in vitro with MR alpha 4-1 before administration. MR alpha 4-1 also significantly inhibited the transendothelial migration of T lymphocytes, associated by the ratio of migration to adherence. However, once T lymphocytes migrated into the interstitium, treatment with MR alpha 4-1 did not affect the pattern of travel of these lymphocytes in the interstitium or their transport into the microlymphatics in the parafollicular area. Therefore, we conclude that alpha 4 integrins play a critical role in the rolling and sticking of T cells and their transendothelial migration in PCV of Peyer's patches.

Animals↗

PubMed1996

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18.

Rat Kupffer cell (KC)-mediated cytotoxicity against both the syngeneic hepatoma cell line AH70 and hepatocytes was evaluated by changes in mitochondrial function, and the possible role of ICAM-1/CD18 in the interaction between the cells was studied. Rhodamine 123 fluorescence, a marker of the mitochondrial membrane potential, decreased in AH70 cells after co-culture with CK, while that in hepatocytes was unchanged by co-culture. This decrease was blocked by anti-ICAM-1 anti-CD18 and the inhibition of nitric oxide synthesis. Cytometric studies demonstrated that ICAM-1 expression on AH70 cells increased after addition of IFN-gamma, IL-1beta, tumor necrosis factor (TNF)-alpha or KC, while in hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatment inhibited the increase in ICAM-1 expression and the decrease in rhodamine 123 fluorescence on AH70 cells after co-culture with KC. CD18 on KC was increased only after co-culture with AH70. TNF-alpha but not IFN-gamma was detected in the supernatant of co-culture between KC and AH70 cells, and this production was partially inhibited by anti-ICAM-1 and anti-CD18. The activity of inducible nitric oxide synthase in Kupffer cells and the levels of nitrites and nitrates in the co-culture supernatant increased over time, and this increase was attenuated either by addition of NO synthesis inhibitors, anti-ICAM-1 or anti-CD18. These results indicate that the rat KC causes mitochondrial dysfunction in cancer cells via the production of NO and cell-to-cell adhesion via ICAM-1/CD18 has an important role in this cytotoxic process.

Animals↗

PubMed1996

Role of endothelin-1 in repeated electrical stimulation-induced microcirculatory disturbance and mucosal damage in rat stomach.

The aim of the present study was to clarify the involvement of endogenous endothelin in the pathogenesis of gastric mucosal damage. The rat stomach was exposed and repeated electrical stimulation (RES) was applied to the small arterial wall close to the lesser curvature. Significant mucosal haemorrhagic lesions (ulcer and erosion) were noted within 30 min after RES. Intravital microscopic observations revealed that an arteriolar constriction occurred in the submucosal layer of the rat stomach approximately 5 min after the completion of RES. Following the arteriolar constriction, the mucosal blood flow of the rat stomach, which was monitored by using a laser Doppler velocimeter, decreased to approximately 30% of the control value. The plasma immunoreactive endothelin-1 level in the regional blood of the stomach was significantly increased immediately after RES preceding the decrease in mucosal blood flow. Immunohistochemical studies revealed that endothelin-1 and big-endothelin-1 were detectable in the arteriolar endothelium around the muscularis mucosa, supporting the involvement of endothelin-1 in RES-induced mucosal ischaemia. In addition, BQ-123, a specific antagonist of the endothelin A (ETA) receptor, attenuated the reduction of blood flow and the development of haemorrhagic lesions observed in gastric mucosa subjected to RES. The results of the present study suggest that an excessive production of endothelin-1 in the arteriolar endothelium leads to microvascular derangements accompanied by haemorrhagic alterations of the gastric mucosa.

Animals↗

PubMed1996

Effect of chronic ethanol feeding on Kupffer cell-mediated antitumor cell activity.

We have previously reported that the Kupffer cell has antitumor activity through mitochondrial damage to tumor cells by nitric oxide production. In this study, the effect of chronic ethanol feeding on antihepatoma cell activity of the Kupffer cell was examined in rats. Male rats of the Wistar strain were fed ethanol chronically for 8 weeks by liquid diets. Kupffer cells were isolated from the control rat or the ethanol-fed rat, and cocultured with AH 70 cells, a rat hepatoma cell line. Fluorescence of rhodamine 123 or propidium iodide was observed as indicators of the mitochondrial damage or cell membrane injury, respectively, by a laser scanning confocal microscopy. Mitochondrial damage of AH 70 cells as indicated by reduction of rhodamine 123 fluorescence was smaller by the coculture with Kupffer cell from the ethanol rat than that from the control. Cell membrane barrier dysfunction of AH 70 cell was less frequently observed with the Kupffer cell from ethanol-fed rats. A metabolite of nitric oxide (nitrite and nitrate) was less in the cultured medium with the ethanol Kupffer cell than with the control Kupffer cell. Ca2+ mobilization, which induces inducible nitric oxide synthase and observed by the fluorescence of fluo-3, in Kupffer cells cocultured with AH 70 cells was suppressed in ethanol-fed rats. These result suggests that chronic ethanol feeding suppresses antitumor cell activity of Kupffer cell through the impairment of Ca2+ mobilization and nitric oxide production.

Animals↗

PubMed1996

Effect of chronic ethanol feeding on nitric oxide synthesis by rat Kupffer cells.

Kupffer cells contribute to the important role of the liver defense mechanism through nitric oxide (NO) production. In this study, the effect of chronic ethanol administration on the ability of Kupffer cells to synthesize and release NO was investigated after stimulation with lipopolysaccharide (LPS). Male Wistar rats were chronically fed ethanol for 8 weeks according to the method described by DeCarli and Lieber et al. (J Nutr.91:331-336, 1967). Kupffer cells were isolated and cultured with LPS (1 micrograms/ml) for 24 hr. The levels of nitrite and nitrate, metabolites of NO, were determined in the culture medium, NO synthase (NOS) activity in Kupffer cells was determined by the method that measures conversion of [14C]arginine into [14C]citrulline. In control rats, a significant increase of nitrite and nitrate levels in culture medium was observed after LPS treatment. The magnitude of this increase was significantly smaller in chronic ethanol-fed rats. When the activity of NOS was determined, inducible NOS (iNOS) activity was higher than that of constitutive NOS, and LPS administration produced a significant elevation of iNOS activity in both control and chronic ethanol-fed rats. However, the elevation of iNOS activity by LPS stimulation was diminished by chronic ethanol administration. Distribution of iNOS in Kupffer cells as determined by an immunofluorescence method using a laser scanning confocal image system showed a lower expression of iNOS in chronic ethanol-fed rats even in the presence of LPS. These results demonstrate that the excessive production of NO by increased iNOS activity in Kupffer cells is diminished by chronic ethanol administration.

Animals↗