CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.

Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells.

Clostridium difficile toxin A-induced microvascular dysfunction. Role of histamine.

Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

Role of H1 receptors and P-selectin in histamine-induced leukocyte rolling and adhesion in postcapillary venules.

The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.

Nitric oxide mediates Kupffer cell-induced reduction of mitochondrial energization in hepatoma cells: a comparison with oxidative burst.

The metabolic changes in rat hepatoma cell line, AH70 cells, after coculturing with Kupffer cells were visualized using a silicon-intensified target camera and subsequent processing with a computer-assisted digital imaging processor. In cocultured tumor cells, nonactivated Kupffer cells reduced mitochondrial energization as indicated by the decrease in the fluorescence intensity of rhodamine 123 (Rh123) and induced lipid peroxidation as shown by the dichlorofluorescein (DCF) activation. The reduction in Rh123 could be eliminated by addition of an analogue of L-arginine (NG-monomethyl-L-arginine), suggesting the involvement of nitric oxide (NO.) in the decrease in mitochondrial energization. Superoxide dismutase did not inhibit the reduction in Rh123 but significantly inhibited DCF activation. These findings indicate that the latter reaction was mediated by superoxide anion. Two h after the cells were cocultured, propidium iodide-positive, severely injured tumor cells significantly increased in number. This increase was significantly attenuated by addition of NG-monomethyl-L-arginine but not by superoxide dismutase, suggesting that NO. may be greatly involved in Kupffer cell-mediated injury of AH70 cells. In another set of experiments, the culture medium of Kupffer cells caused no significant alteration of Rh123, DCF, and propidium iodide-associated fluorescences in AH70 cells. In addition, ultrastructural observation revealed that the membrane-to-membrane attachment between Kupffer cells and tumor cells occurred within 30 min after coculturing. These results suggest that Kupffer cell-derived NO. release, triggered by the close contact with tumor cells, may induce damage to tumor cells via inhibition of mitochondrial energization.

P-selectin-dependent leukocyte recruitment and intestinal mucosal injury induced by lactoferrin.

Plasma concentrations of lactoferrin relevant to an inflammatory response are known to elicit leukocyte-endothelial cell adhesion in mesenteric venules. The objectives of this study were (1) to determine whether exogenously administered lactoferrin causes microvascular and mucosal injury in rat intestine and (2) to assess the contribution of adherent leukocytes to a lactoferrin-mediated injury process. Mucosal myeloperoxidase (MPO) activity and vascular protein clearance were monitored in the distal intestine of male Sprague-Dawley rats. Macroscopic erosive lesions of the mucosa and increases in mucosal MPO and intestinal vascular protein were observed 2 h following the lactoferrin infusion, results consistent with granulocyte accumulation and microvascular protein leakage. These lactoferrin-induced alterations were significantly attenuated in animals pretreated with a monoclonal antibody (mAb) directed against P-selectin but not by an E-selectin-specific mAb. In another series of experiments, leukocyte adherence/emigration and leakage of fluorescein isothiocyanate (FITC)-labeled albumin were measured in rat mesenteric venules using intravital video microscopy. Lactoferrin elicited increases in both leukocyte adhesion/emigration and albumin extravasation, which were attenuated by mAbs directed against P-selectin but not E-selectin. These observations indicate that (1) the lactoferrin released by activated neutrophils may lead to significant microvascular and mucosal injury or dysfunction and (2) the lactoferrin-induced injury is related to P-selectin-mediated adhesion of leukocytes to microvascular endothelium. Our results raise the possibility that neutrophil-derived lactoferrin contributes to the inflammatory response by promoting further granulocyte accumulation and activation and that mAbs to P-selectin may be therapeutically beneficial in inflammatory disorders.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches.

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

Microvascular responses to ischemia/reperfusion in normotensive and hypertensive rats.

The objective of the present study was to determine whether long-term arterial hypertension renders the microvasculature more vulnerable to the deleterious inflammatory responses elicited by ischemia and reperfusion (I/R). Intravital fluorescence microscopy was used to monitor leukocyte adherence and emigration, platelet-leukocyte aggregation, and albumin extravasation in mesenteric postcapillary venules of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) after 10 minutes of ischemia and subsequent reperfusion. Significant and comparable increases in leukocyte adherence/emigration and the formation of platelet aggregates were elicited by I/R in both WKY and SHR. Albumin extravasation was enhanced after I/R in SHR, but not in WKY. Monoclonal antibodies directed against the adhesion glycoproteins CD18, P-selectin, or ICAM-1 showed similar patterns of protection against the I/R-induced inflammatory responses in WKY and SHR. The enhanced albumin extravasation noted in postischemic venules of SHR was prevented by immunoneutralization of either CD18 on leukocytes or ICAM-1 on endothelial cells. These results suggest that, whereas long-term arterial hypertension does not significantly modify the leukocyte and platelet recruitment normally elicited in venules by I/R, it does result in an exaggerated albumin leakage response, which is mediated by an interaction between beta(2) (CD18) integrins on leukocytes and ICAM-1 on endothelial cells.

CD18/ICAM-1-dependent nitric oxide production of Kupffer cells as a cause of mitochondrial dysfunction in hepatoma cells: influence of chronic alcohol feeding.

The present study was designed to monitor the process for hepatoma cell injury induced by Kupffer cells. The non-activated Kupffer cells isolated from male Wistar rats reduced the mitochondrial membrane potential in the cocultured AH70 cells, which was indicated by the decreased rhodamine 123 (Rh123) fluorescence. Increased level of nitrite and nitrate in the medium and induction of iNOS in Kupffer cells were observed after coculture with AH70 cells. Incubation with either NG-monomethyl-L-arginine or aminoguanidine attenuated the increased nitric oxide (NO) production of Kupffer cells and the decreased Rh123 fluorescence of AH70 cells. Fluo-3, a calcium-sensitive probe, fluorescence in Kupffer cells increased after coculture with AH70 cells. Addition of TMB-8, a calcium inhibitor, or monoclonal antibody directed against ICAM-1 or CD18 prevented the increases in fluo-3 fluorescence and NO production of Kupffer cells and Kupffer cell-induced mitochondrial dysfunction in AH70 cells, suggesting the involvement of calcium mobilization and CD18/ICAM-1. It is therefore suggested that the Kupffer cell-mediated mitochondrial dysfunction of hepatoma cells largely depends on NO production by iNOS, and that the NO production by Kupffer cells is triggered by CD18/ICAM-1-dependent interaction with hepatoma cells and subsequent calcium mobilization. In other series of experiments, male Wistar rats fed ethanol for 4 weeks were used. The NO production and calcium mobilization of Kupffer cells and reduction of the mitochondrial membrane potential in cocultured hepatoma cells were diminished in the case of Kupffer cells isolated from chronically ethanol-fed rats, while CD18 and ICAM-1 expression was still observed. Thus, the present study further suggests that NO-dependent anti-hepatoma cell activity of Kupffer cells is suppressed in chronically ethanol-fed animals.

Attenuating effect of antithrombin III on the fibrinolytic activation and microvascular derangement in rat gastric mucosa.

The roles for the fibrinolytic activation and disorder of coagulation in formation of gastric ulcer induced by microvascular derangement were investigated. The rat stomach was exposed and repeated electrical stimuli (RES) were applied on the small arterial wall close to the lesser curvature to induce mucosal microcirculatory disturbances. The level of tissue-type plasminogen activator (t-PA), a key enzyme for fibrinolytic activity, in the regional blood of the stomach was significantly elevated immediately after RES. At 5 min after RES, the leakage of FITC-labeled albumin and thrombus formation in the mucosal microvasculature were visually demonstrated by using an intravital microscopic system. At 30 min, hemorrhagic erosions and linear ulcers were observed in the gastric mucosa. Pretreatment with human antithrombin-III (AT-III) in the range of 0.1-10 U/kg dose-dependently attenuated both the fibrinolytic activation and microvascular alteration promoted by RES. Human AT-III also prevented RES-induced gastric mucosal injury. Thrombin inhibitory activity in the gastric vein decreased (69.0 +/- 2.1%) just after RES, and further reduced at 30 min (47.7 +/- 5.3%). The present study suggests a hypothesis that human AT-III has a preventive effect on the gastric mucosal hemorrhagic changes via attenuating the fibrinolytic activation and subsequent microcirculatory disturbances.

Kupffer cell-mediated oxidative stress on colon cancer cell line visualized by digital imaging fluorescence microscopy.

Temporal and spatial changes of lipid peroxides in a cultured colon cancer cell line, Colo-205 cells, were investigated after culturing with Kupffer cells by using 2',7'-dichlorofluorescein diacetate and a digital imaging processor equipped with an inverted microscope. By this method, we successfully visualized the alteration of lipid peroxides in the individual cancer cell. Without any prior activation, Kupffer cells isolated from an intact rat liver caused rapid increase in the intensity of dichlorofluorescein in tumor cells in a time-dependent manner. The increase of the fluorescent intensity was significantly attenuated by pretreatment with superoxide dismutase. In ex vivo study using isolated perfused rat liver, dichlorofluorescein-preloaded cancer cells, which were transportally injected, were found to adhere to hepatic sinusoids and then to enhance their fluorescence. The present study suggested that the resident Kupffer cell-derived oxidative stress participates in the cytotoxic process against cancer cells by inducing intracellular lipid peroxidation. It may be sustained that Kupffer cells play a role in the host defence mechanisms against the liver metastasis of colon cancer cells.

Antisense oligodeoxynucleotide against c-myc mRNA induces differentiation of human hepatocellular carcinoma cells.

We have previously demonstrated that the human liver-specific antigen (HLSA) expression was enhanced and c-myc levels were reduced during sodium butyrate-induced differentiation in human hepatoma cells. To further elucidate a linkage between the reduction of c-myc levels and an increase in the HLSA expression, antisense oligodeoxynucleotide against c-myc mRNA was transferred into human hepatoma cells. Human hepatoma cell lines, HCC-M, HCC-T and PLC/PRF/5 were transfected with antisense oligodeoxynucleotide and changes in the cell cycle, expression of the HLSA, albumin, and alpha-fetoprotein were examined. Antisense oligodeoxynucleotide was successfully induced into cells visualized by a confocal microscope using fluorescein-labeled oligodeoxynucleotides, and Northern blot analysis revealed that c-myc expression was reduced three and six hours after the transfection. Following these changes, cell proliferation was inhibited and flow cytometric analysis showed that cell number in the G1 phase significantly increased. Increased expression of the HLSA and albumin, and decreased expression of alpha-fetoprotein was observed by flow cytometry in accordance with those changes. These results showed similar changes to those induced by butyrate-treatment obtained in our previous studies. The present study indicates that the reduction of c-myc transcription increases HLSA expression levels through intracellular changes similar to those induced by butyrate, a differentiation inducer.

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.

Quantitative analysis of mesenteric microcirculatory disturbances induced by autonomic nervous irritation.

In this study, we have demonstrated that repeated electrical stimuli to the artery of the mesenteric pedicle can produce mesenteric microcirculatory disturbance by autonomic nervous irritation in rats. The parameters to demonstrate microcirculatory damage were observed and quantitatively analyzed using the intravital microscopy after electrical stimulation for 40 minutes. The blood flow of arterioles and venules in the mesentery showed ischemia-reperfusion pattern during the repeated electrical stimulations. The diameter of arterioles did not show significant change, while RBC velocity of arterioles was significantly decreased at 30 min after the irritation. The RBC velocity in venules was decreased from the early period to about 20%. But this values were not significantly dropped during the later observation period, suggesting the formation of short circuit flow by passing the collapsed capillary beds. The number of rolling WBC in the venules was notably increased at the time immediately after irritation, and thereafter the number of rolling WBC number was rather reduced. The number of sticking WBC in venules was time-dependently increased and reached its maximum at 30 min. When permeability of venular wall was determined by the injection of pontamine sky blue, significant increase in permeability was already shown immediately after irritation, suggesting that the integrity of microvascular wall was disturbed in this early period. The permeated area expanded thereafter in parallel with the increase in sticking WBC number. From these observations, it is suggested that endothelial cell damage and following leukocyte-endothelium interaction induced by autonomic nerve irritation appear to be an important factor in microcirculatory disturbances.

In vivo visualization of oxyradical-dependent photoemission during endothelium-granulocyte interaction in microvascular beds treated with platelet-activating factor.

Oxyradical-dependent chemiluminescence from granulocytes sticking to venular endothelium was successfully visualized in rat mesenteric microvascular beds treated with platelet-activating factor (PAF). Intravital photonic image intensifier microscopy revealed that topical application of PAF-acether (100 nM) caused remarkable granulocyte adherence on endothelial walls and the subsequent activation of a luminol-dependent photonic burst. Chemilumigenic sites clearly corresponded to the spatial distribution of sticking cells in post-capillary venules. The present findings thus serve as the first demonstration of an intravital oxidative burst of granulocytes on venular endothelium in PAF-induced microcirculatory disturbances.

Mechanisms of endothelin-induced macromolecular leakage in microvascular beds of rat mesentery.

Microvascular responses to endothelin-3 were investigated in the rat mesentery under fluorescence microscopy. Endothelin-3 in a range of 0.1-100 pM induced arteriolar constriction in a dose-dependent manner, and stimulated Ca2+ mobilization, demonstrated by fura-2-associated fluorography, in both arterioles and venules. Cyclo(-D-Trp-D-Asp-Pro-D-Val-Leu-) (BQ123), and endothelin ETA receptor antagonist, at a concentration of 10 microM inhibited the endothelin-3 (100 pM)-induced arteriolar constriction and Ca2+ mobilization in arterioles but not in venules. In venules, an early onset leakage of FITC (fluorescein isothiocyanate)-labeled albumin and subsequent reduction of red blood cell velocity without arteriolar constriction were observed after the superfusion of endothelin-3 with BQ123, suggesting that a non-endothelin ETA receptor mediates macromolecular leakage followed by a decrease in blood flow. Endothelin-3 with BQ123 neither stimulated leukocyte adhesion nor activated luminol-dependent chemiluminescence in venules, showing that endothelin-3-increased permeability may be induced by leukocyte-independent and oxyradical-independent mechanisms. These microvascular alterations of permeability and red blood cell velocity were significantly attenuated by the addition of phalloidin, an F-actin stabilizer, suggesting the involvement of endothelial cell contraction. Nicardipine (1,4-dihydro-2,6-dimethyl-4-[3-nitrophenyl]methyl-2- [methyl(phenylmethyl)amino]-3,5-pyridinedicarboxylic acid ethyl ester), a dihydropyridine-type Ca2+ channel antagonist, eliminated endothelin-3-induced arteriolar constriction; however, it did not affect albumin leakage promoted by endothelin-3 with BQ123, suggesting that a non-voltage-dependent Ca2+ channel(s) is involved in non-endothelin ETA receptor-mediated Ca2+ mobilization and contraction of venular endothelial cells. Overall, it is conceivable that endothelin ETA receptor and voltage-dependent Ca2+ channel are involved in endothelin-3-induced arteriolar constriction. In addition, the present results suggest that Ca2+ mobilization in venular endothelium, which is mediated by a non-endothelin ETA receptor, possibly endothelin ETB receptor and regulated by non-voltage-dependent Ca2+ channel(s), may cause endothelial cell contraction and subsequently increase macromolecular permeability in microvascular beds treated with endothelin-3.

Xanthine oxidase-mediated intracellular oxidative stress in response to cerulein in rat pancreatic acinar cells.

Intralobular oxygen radical formation was examined in cerulein-stimulated rat pancreatic acinar cells by digital imaging microscopic fluorography using a hydroperoxide-sensitive fluorescent probe, dichlorofluorescin (DCFH) diacetate. The isolated pancreatic acinar cells loaded with DCFH diacetate were microscopically observed, and the dichlorofluorescein (DCF) fluorescence yielded by DCFH oxidation via hydroperoxides was digitally processed. Within the initial 20 min after the application of cerulein (10 microM), intracellular oxidative stress was observed as indicated by the increase in DCF fluorescence intensity and reached its maximum at 60 min. DCF fluorescence intensity was then gradually decreased until 80 min, followed by a marked increase in propidium iodide (PI) fluorescence, suggesting irreversible cell death. Allopurinol (1 microM), a xanthine oxidase inhibitor, significantly attenuated the early increase of DCF fluorescence intensity as well as the late cell damage. Treatment with hyperbaric oxygen (PO2 300 mm Hg) also significantly attenuated both the increase of DCF fluorescence and the number of PI-positive cells. The results suggest that xanthine oxidase-mediated oxygen radicals may play an important role in cerulein-induced intracellular oxidative stress in pancreatic acinar cells of rats.

Involvement of superoxide anion and platelet-activating factor in increased tissue-type plasminogen activator during rat gastric microvascular damages.

The role of tissue-type plasminogen activator (t-PA) was investigated in the gastric ulcer formation induced by microvascular derangement. The rat stomach was exposed and repeated electrical stimuli (irritation) were applied on the small arterial wall close to the lesser curvature to induce mucosal ischemia followed by hyperemia. The t-PA activity in the regional blood of the stomach was significantly elevated as early as 5 min after the irritation. Immunohistochemical study using anti-t-PA monoclonal antibody revealed that t-PA was detectable in the endothelial cells of capillaries and collecting venules, suggesting the involvement of endothelium-mediated fibrinolytic activity in the irritation-induced ulcer formation. Pretreatment of SOD or allopurinol significantly attenuated the irritation-induced t-PA activation, suggesting that the t-PA activity was modulated by xanthine oxidase-associated superoxide anions. CV-6209, a selective antagonist of platelet-activating factor (PAF), also prevented the activation of t-PA as well as ulcer formation, providing a concept that PAF may be associated with the local fibrinolytic activation which may cause hemorrhagic changes in the gastric mucosal microvasculature. The present study supports the hypothesis that increased t-PA activity may reflect the microvascular endothelial damages caused by vasomotor derangement and suggests that oxygen-derived free radicals may participate in the regulation of endothelium-derived fibrinolytic activities in the mucosal microvasculature.

Oxidative stress in gastric mucosal injury: role of platelet-activating factor-activated granulocytes.

Temporal and spatial changes due to oxidative stress in the rat gastric mucosa were visualized and quantified during the process of mucosal hemorrhagic change. The fluorescence associated with dichlorofluorescein (DCF), a hydroperoxide-sensitive fluorochrome, increased 30 min after repeated electrical stimuli to the gastric artery. The increase in the fluorescence was enhanced in the area between two adjacent collecting venules. The content of platelet-activating factor (PAF), the activity of myeloperoxidase (MPO) in the gastric mucosa, the area of mucosal lesions, and the luminol-dependent chemiluminescence activity in zymosan-treated blood samples, obtained from the gastric vein, were measured and found to increase significantly 30 min after the stimuli. The intravenous injection of CV-6209, a PAF antagonist, 5 min prior to the stimuli significantly inhibited the DCF activation, the increases in PAF level and MPO activity, the mucosal hemorrhagic change, and the elevation in chemiluminescence activity. In addition, continuous infusion of superoxide dismutase also inhibited all these changes, except for chemiluminescence activity. These results suggest that oxygen radicals derived from PAF-activated granulocytes induce oxidative stress, and that oxidative changes are actually implicated in the pathogenesis of gastric mucosal injury.

Wound healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin, with special reference to prolylhydroxylase and collagenase enzyme activity.

The healing of acetic acid-induced gastric ulcer in rats and the effects of cimetidine and calcitonin were investigated with reference to the enzyme activity of both prolylhydroxylase and collagenase as related to histological findings. The rats were observed by endoscopy on the 3rd day after the subserosal injection of acetic acid; rats with ulcers were divided into three groups: non-treated, and cimetidine- and calcitonin-treated. The latter two groups were treated for 7 days. Prolylhydroxylase activity in active ulcers in the non-treated group was slightly higher on the 3rd day and significantly higher on the 10th day than the activity in control rats that had received subserosal injections of physiological saline solution on the respective days. In non-treated rats, the healed ulcer on the 10th day showed lower prolylhydroxylase activity than that in the active ulcer on the same day. Cimetidine did not affect prolylhydroxylase activity, but, with calcitonin, there was higher prolylhydroxylase activity in the healed than in the active ulcer, although the difference was not significant. Interstitial collagenase showed the highest activity on the 3rd day and decreased on the 10th day in non-treated rats. Collagenase activity was higher in the cimetidine-treated group, than that in the non-treated group, and numerous peroxidase-positive granulocytes were seen in the mucosa and submucosa. Calcitonin did not affect collagenase activity. The participation of both enzymes is indispensable in the healing process and the effects of anti-ulcer agents on these enzymes must be considered.

Effect of lipopolysaccharides on erythrocyte flow velocity in rat liver.

Although endotoxin exacerbates hepatic microcirculatory disturbance, little is known of the way in which it acts on the hepatic microcirculation. We measured endotoxin-induced changes in hepatic microcirculation and investigated the effect of endotoxin on hepatic microcirculation in rats. After male Wistar rats were anesthetized, a lobe of the liver was observed with an inverted intravital microscope. Erythrocytes (RBC) were labeled with fluorescein isothiocyanate (FITC) and injected. The flow velocity (FV) of FITC-RBC in sinusoids was measured with an off-line velocimeter. Portal pressure (PP) and mean arterial pressure (MAP) were measured with a catheter cannulated in the portal vein and the left carotid artery, respectively. After a small dose (1 mg/kg) of endotoxin had been administered intravenously, FV decreased and PP increased gradually after 30 min. MAP showed no significant change, except for an initial decrease. However, when 5 mg/kg of endotoxin was administered, FV and PP increased, with a peak at 10 min, which was not observed with the small dose. In the late phase, FV decreased and PP increased, as was seen with the small dose. Endotoxin increased serum aspartate aminotransferase and lactate dehydrogenase activities. These results suggest that endotoxin induces hepatic microcirculatory disturbance, which may cause liver injury.