Roles of endothelin-1 and nitric oxide in the mechanism for ethanol-induced vasoconstriction in rat liver.

This study was designed to investigate the mechanism for ethanol-induced hepatic vasoconstriction in isolated perfused rat liver. Upon initiation of ethanol infusion into the portal vein at concentrations ranging from 25 to 100 mM, portal pressure began to increase in a concentration-dependent manner and reached maximal levels in 2-5 min (initial phase), followed by a gradual decrease over the period of ethanol infusion (escape phenomenon). Endothelin-1 antiserum significantly inhibited this ethanol-induced hepatic vasoconstriction by 45-80%. Cessation of infusion of endothelin-1 antiserum was followed by a subsequent increase in portal pressure. On the other hand, when a nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA), was infused into the portal vein simultaneously with ethanol, the initial phase of the response of portal pressure to ethanol was not altered and the peak values of portal pressure remained unchanged. However, after the peak increase in portal pressure, the rate of decrease was less than in the absence of L-NMMA. Thus, L-NMMA diminished the escape phenomenon and sustained the vasoconstriction. This study supports the hypothesis that two endothelium-derived vasoactive factors, endothelin-1 and nitric oxide, regulate hepatic vascular tone in the presence of ethanol.

Selective suppression of N-acetylglucosaminyltransferase III activity in a human hepatoblastoma cell line transfected with hepatitis B virus.

UDP-N-acetylglucosamine: beta-D-mannoside beta-1,4-N-acetylglucosaminyl-transferase III (GnT-III) is a key enzyme in the branching of asparagine-linked oligosaccharides, which are present in surface membrane proteins of various tissues and in secretory glycoproteins. The activity of GnT-III was assayed in 2 human hepatoblastoma cell lines, Huh6, which was the parental cell line, and HB611, which was established by transfection of 3 tandem copies of the hepatitis B virus genome into Huh6. A significant difference in GnT-III activity was found between Huh6 and HB611 (136 +/- 18.3 pmol/h/mg versus 6.7 +/- 2.4 pmol/h/mg; mean +/- SD, P < 0.001), whereas levels of the glycosyltransferases alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV, alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase-V, and beta-1,4-galactosyltransferase were almost the same in both cell lines. Northern blot analysis indicated that the decreased activity of GnT-III in HB611 was due to the decreased transcript. When HB611 was treated with interferon-alpha, expression of hepatitis B virus-related mRNA decreased, and the activity of GnT-III increased from 8.5 +/- 3.8 to 22.0 +/- 7.2 pmol/h/mg (mean +/- SD, P < 0.05). This increase was not found in Huh6. Binding capacity with erythrocyte phytohemagglutinin in these cells using fluorescence-activated cell sorter analysis was different, suggesting that the structure of sugar chain on the cell surface might be altered by suppression of GnT-III activity. This is the first report that hepatitis B virus selectively suppressed the GnT-III activity in hepatoblastoma cells.

N-acetylglucosaminyltransferase III and V messenger RNA levels in LEC rats during hepatocarcinogenesis.

The LEC (Long-Evans with a cinnamon-like color) rat is a mutant of the Long-Evans strain which develops hereditary hepatitis and hepatoma with age. Activities and mRNA levels of N-acetylglucosaminyltransferase III and V (GnT-III and GnT-V, respectively) were determined during hepatocarcinogenesis in this rat using a LEA (Long-Evans with an agouti color) rat as a control. GnT-III activity in LEC rat liver increased after 30 weeks of age, at the stage of chronic hepatitis, to about 2.5-11.5 times the level in LEC rats aged 1-9 weeks. GnT-V activity in the LEC rat liver increased after 20 weeks of age, at the stage of acute hepatitis, to about 1.5-2.5 times the level in LEC rats of 1-9 weeks of age and then remained elevated. Both enzymes showed more dramatic increases in males than in females. The mRNA levels of the enzymes increased in proportion with the enzyme activities. Furthermore, GnT-III and GnT-V mRNAs were highly expressed in both cancer lesion and adjacent tissues. In one case of hepatoma with lymph node metastasis, GnT-III and GnT-V mRNA expression was much higher in the metastatic lesion than in the original cancer. GnT-III and GnT-V levels in the original cancer lesions were similar to those in the cancer lesions of the other LEC rats. These results indicated that expression of GnT-III and GnT-V was induced by chronic liver damage and hepatocarcinogenic changes in the LEC rats.

In vivo gene transfer and expression in rat stomach by submucosal injection of plasmid DNA.

Gastrointestinal nonepithelial tissue is a useful target for in vivo gene transfer. The aim of this study was to investigate whether gene transfer into this organ could be achieved by submucosal injection of plasmid DNA. Plasmid DNA carrying either the firefly luciferase or Escherichia coli LacZ reporter gene was injected directly into the gastric submucosa of adult rats. Gene expression was characterized by quantitative luciferase assay and qualitative in situ beta-galactosidase (beta-Gal) staining. Luciferase activity was detected as early as 1 day after injection, increased markedly at 2 days, and then decreased. Some of the rats showed detectable levels of luciferase expression at 14 and 21 days postinjection. Histochemical staining for beta-Gal demonstrated that expression of the recombinant genes was localized in smooth muscle cells of the muscularis mucosae and the muscular layer and mesenchymal cells in the lamina propria. Our results indicate that gene transfer into the gastrointestinal tract can be achieved by simple needle insertion of naked plasmid DNA into the submucosa.

Heterogeneous activation of protein kinase C during rat liver regeneration induced by carbon tetrachloride administration.

During rat liver regeneration induced by carbon tetrachloride administration, the protein kinase C alpha subspecies was activated in a heterogeneous fashion, a higher number of hepatocytes expressing the protein kinase C alpha subspecies being detected in the pericentral zone than in the periportal zone. This zonal heterogeneity became maximal at 24 h after the treatment. The distribution of hepatocytes expressing the protein kinase C alpha subspecies was roughly coincident with that of hepatocytes exhibiting DNA synthesis. These results suggest that protein kinase C may play a crucial role in liver regeneration.

The F(ab')2 fragment of an anti-ICAM-1 monoclonal antibody attenuates liver injury after orthotopic liver transplantation.

Long-term storage of liver grafts results in increased adhesion of leukocytes onto the sinusoidal walls. This eventually leads to posttransplant graft damage through disturbances of hepatic microcirculation. Intracellular adhesion molecule-1 (ICAM-1) is known to be involved in attachment of leukocytes. This study was designed to examine whether ICAM-1 participated in the pathogenesis of posttransplant liver injury. Inbred Lewis rats were used as both donors and recipients to avoid immunoreactivity. Donor livers were stored for either 1 or 6 hr in ice-cold Euro-Collins solution and subsequently implanted. Expression of ICAM-1 was examined immunohistochemically. In some rats that received livers stored for 6 hr, the intact IgG (1.0 mg/kg) or the F(ab')2 fragment (0.5 mg/kg) of an anti-ICAM-1 mAb (1A29) was administered via the tail vein immediately after reperfusion of portal blood. In the group receiving livers stored for 6 hr, ICAM-1 began to be expressed on the sinusoidal endothelial cells as early as 15 min after reperfusion of the portal blood. Strong ICAM-1 expression was observed from 2 hr up to 24 hr after reperfusion. In contrast, expression of ICAM-1 was not evident at any time point after surgery in the 1-hr storage group as well as in untransplanted, normal livers. Serum alanine aminotransferase (ALT) levels were significantly higher in the 6-hr storage group compared with those of the 1-hr storage group (1-hr: 171 +/- 9 IU/L; 6-hr: 825 +/- 109 IU/L, P < 0.05; mean +/- SEM) 24 hr after transplantation. Serum ALT levels were markedly reduced by treatment with the F(ab')2 fragment of 1A29 (247 +/- 34 IU/L, P < 0.05 vs. 6-hr storage group). This was associated with reduced accumulation of leukocytes in the liver. In marked contrast, treatment with the intact IgG of 1A29 increased serum ALT levels dramatically (5297 +/- 634 IU/L, P < 0.05 vs. 6-hr storage group) and reduced serum complement. Histological examination revealed focal hepatocellular necrosis 24 hr after surgery in the 6-hr storage group. Treatment with the F(ab')2 fragment decreased the liver damage; in marked contrast, treatment with the intact IgG strikingly aggravated the injury, as characterized by massive necrosis throughout the liver. Liver damage caused by the intact IgG might be related to activation of the complement system by the Fc portion of the antibody. Taken together, these results indicate that ICAM-1 is involved in the mechanism of postoperative liver injury following liver transplantation.

Evaluation of effective portal venous flow in chronic liver diseases using echo-Doppler flowmetry combined with per jejunal portal scintigraphy.

Portal circulation changes due to the progression of chronic liver disease and portal venous flow are also affected by pharmacotherapy. Thus, noninvasive measurement of effective portal venous flow (EPVF) is highly desirable. We evaluated EPVF under steady-state conditions using echo-Doppler flowmetry combined with per jejunal portal scintigraphy in 32 patients with chronic liver disease. After introduodenal administration of 37 MBq (1 mCi) of 123I-iodoamphetamine, scintigraphy of the pulmonary and hepatic regions was performed and a portosystemic shunt index (SI) calculated. EPVF was calculated as follows: EPVF = PVFx (1-SI/100). EPVF in chronic hepatitis, compensated cirrhosis and decompensated cirrhosis was 12.0 +/- 1.8 ml/min/kg, 10.3 +/- 1.6 ml/min/kg and 8.0 +/- 2.5 ml/min/kg, respectively. There were significant differences in EPVF between all groups, although PVF was similar in each group. EPVF correlated with liver function tests and was a better indicator of liver function than PVF. Measurement of EPVF may provide useful information in the management of patients with chronic liver disease.

Ammonia: a possible promotor in Helicobacter pylori-related gastric carcinogenesis.

Helicobacter pylori (HP) has been shown to possibly be a pathogen of gastric carcinoma. HP has urease activity and produces ammonia in the stomach. In this study, the role of ammonia on gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were investigated in rats. After 24 weeks pretreatment with MNNG (83 mg/l), 0.01% ammonia or tap water as a drinking water was administered for 24 weeks. The ammonia-treated rats showed a significantly higher incidence of gastric cancer (percent of animals with tumors and number of tumors per rat). Ammonia would thus appear to have an important role in HP-related human gastric carcinogenesis.

Helicobacter pylori extract stimulates inflammatory nitric oxide production.

This study examined whether an extract of Helicobacter pylori had the ability to stimulate an inflammatory synthesis of nitric oxide, a mutagen and precursor of nitrosocompounds. Macrophages and neutrophils were prepared from rat and incubated with the Helicobacter pylori extract. L-Arginine-dependent nitric oxide production in these cells was significantly stimulated by the co-incubation with the Helicobacter pylori extract. This ability of the extract was strongly attenuated by protease digestion or heating. These results indicate that Helicobacter pylori induces production of nitric oxide and participates in development of gastritis and gastric carcinogenesis.

Evidences for involvement of cyclooxygenase-2 in proliferation of two gastrointestinal cancer cell lines.

Cyclooxygenase (COX) consists of two isozymes, COX-1 and COX-2. The roles of these isozymes in the gastrointestinal tract are unknown. We investigated messenger RNA expression of the COX-1 and COX-2 genes in the gastrointestinal cancer cell lines MKN28, MKN45, KATO III CACO-2, DLD-1 and LoVo. These cell lines expressed comparable levels of COX-1 mRNA, although their expression of COX-2 varied. Therefore, we studied the effects of NS-398 and indomethacin, specific and non-specific inhibitors for COX-2, on proliferation of the cell lines. Both of the inhibitors suppressed proliferation of the two cell lines that highly expressed COX-2 (MKN45 and CACO-2). However, these inhibitors exerted minimal effects on proliferation of the other cell lines, which expressed significantly lower levels of COX-2. Therefore, it was proposed that COX-2 participates in proliferation of cancer cells because of over expression of the COX-2 gene.

Effects of low-dose simvastatin therapy on serum lipid levels in patients with moderate hypercholesterolemia: a 12-month study. The Simvastatin Study Group.

The aim of this study was to investigate the safety and long-term effects on serum lipid levels of low-dose simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, in Japanese patients with moderate primary hypercholesterolemia. We assigned 201 patients (68 men and 133 women; mean +/- SD age, 61.3 +/- 10.2 years) with serum total cholesterol levels > or = 220 mg/dL to receive simvastatin 5 mg each evening; the treatment period was 1 year. Serum total cholesterol, triglycerides, and low-density lipoprotein (LDL) cholesterol levels decreased significantly in response to simvastatin therapy, and the changes were maintained throughout the treatment period. Mean total cholesterol decreased from 269.9 +/- 35.4 mg/dL to 215.2 +/- 34.5 mg/dL (20.3%), triglycerides decreased from 183.0 +/- 110.2 mg/dL to 155.5 +/- 88.5 mg/dL (15.0%), and LDL cholesterol decreased from 180.0 +/- 33.1 mg/dL to 130.1 +/- 35.1 mg/dL (27.7%). Total cholesterol, triglycerides, and LDL cholesterol tended to decline when the pretreatment values were higher; the critical values and the bidirectional changes of the serum lipid levels were 188.1, 109.5, and 91.6 mg/dL, respectively. Although the serum level of high-density lipoprotein cholesterol did not change significantly, it tended to increase more when the pretreatment values were lower; the "critical value" was 70 mg/dL. Nine patients experienced mild adverse events, but none discontinued simvastatin during the 12-month treatment period. We found that low-dose simvastatin therapy is effective in achieving long-term decreases in serum lipid levels and is well tolerated by patients with moderate hypercholesterolemia. Simvastatin therapy may result in normalization of serum lipid levels.

Effects of proton pump inhibitor on gastric mucosa hemodynamics and tissue oxygenation in anesthetized rats.

Proton pump inhibitors have been reported to have a cytoprotective action in addition to the anti-secretory action of acid. The precise mechanism, however, remains obscure. In this study, the effects of proton pump inhibitors (omeprazole and NC-1300) on gastric mucosa hemodynamics and tissue oxygenation were investigated using organ reflectance spectrophotometry in a hemorrhagic shock-reperfusion model involving anesthetized rats. Neither drug affected gastric mucosa hemodynamics nor tissue oxygenation in the basal state before hemorrhage. During the hemorrhagic shock state, however, these drugs maintained tissue oxygenation and reduced ulcer formation, although they did not show a significant effect on gastric mucosa blood volume. The results suggest that both proton pump inhibitors have an anti-ulcer action by maintaining mucosal oxygenation in addition to the anti-secretory activity of acid.

Impaired gastric mucosal energy metabolism in congestive gastropathy in cirrhotic patients.

To clarify the characteristics of congestive gastropathy, we investigated gastric mucosal hemodynamics and energy metabolism in cirrhotic patients, using a reflectance spectrophotometry system and high performance liquid chromatography. The index of the gastric mucosal blood volume of cirrhotic patients with esophageal varices was significantly higher, and the index of gastric mucosal blood oxygenation significantly lower, than those in controls, thus indicating congestion and hypoxia in the gastric mucosa. Energy charge levels in the gastric mucosa of cirrhotic patients with esophageal varices were also significantly decreased. The energy charge level showed a strong linear correlation with the index of mucosal blood oxygenation in the antral (r = 0.996, P < 0.01) and body (r = 0.994, P < 0.01) mucosa of the stomach. These findings suggest that congestive gastropathy in a portal hypertensive state causes hypoxia in the gastric mucosa, leading to a mucosal energy deficit that may increase mucosal susceptibility to aggressive factors.

Colonic mucosal hemodynamics and tissue oxygenation in patients with ulcerative colitis: investigation by organ reflectance spectrophotometry.

Colonic mucosal hemodynamics were investigated at the rectosigmoidal region of the colon in 46 patients with ulcerative colitis and in 18 normal subjects by organ reflectance spectrophotometry under colonoscopy. The value for the index of mucosal hemoglobin concentration (IHb) was significantly higher, and value for the index of mucosal hemoglobin oxygen saturation (ISO2) was significantly lower in patients with active ulcerative colitis than values in the normal controls or in patients with inactive ulcerative colitis. The results indicate mucosal congestion and hypoxemia in patients with active ulcerative colitis. The changes in IHb and ISO2 correlated well with the severity of ulcerative colitis scored by endoscopic findings and with the number of infiltrating inflammatory cells in the mucosa analyzed histologically in biopsy samples. In conclusion, the colonic mucosal microcirculation in patients with active ulcerative colitis was disturbed and showed congestion and hypoxemia. The analysis of hemodynamic changes may be helpful for assessing the activity of ulcerative colitis.

Dependence of ultrasonic attenuation of liver on pathologic fat and fibrosis: examination with experimental fatty liver and liver fibrosis models.

To clarify the effect of the pathological state of the liver on ultrasonic attenuation, we produced two experimental rabbit models. The influence of fat on ultrasonic attenuation was examined using a fatty liver model without liver fibrosis, and that of fibrosis on attenuation using a liver fibrosis model without fatty infiltration. Ultrasonic data were obtained in vivo directly from the liver, and an acoustic attenuation coefficient slope was obtained by the spectral difference method. Tissue components of the liver, namely the total lipid, hydroxyproline and water contents, were measured precisely by quantitative methods. We revealed that ultrasonic attenuation depends mainly on fatty infiltration of the liver and to a lesser extent on fibrosis, but not on the water content.

Hepatic hemodynamics in alcoholic liver injuries assessed by reflectance spectrophotometry.

We have investigated the hepatic hemodynamics by reflectance spectrophotometry in patients with alcoholic liver disease. The analysis of 32 cases has shown that the estimated regional hepatic tissue blood hemoglobin concentration, expressed as a difference in absorbance between 569 and 650 nm (delta Er569-650), decreased significantly with progress of fibrosis in the liver, suggesting the relative compression of the vascular compartment due to the progress of alcoholic liver disease. The estimated hepatic oxygen consumption also decreased with progress of fibrosis in the liver. The estimated hepatic oxygen consumption correlated positively with prothrombin time and serum albumin level, and negatively with the fifteen minute retention rate of indocyanine green. Thus, it is concluded that the imbalance between supply and utilization of oxygen in the liver may have an important role in the progress of alcoholic liver disease.

Endoscopic appearances of hemorrhagic peptic ulcers and efficacy of H2-receptor antagonists.

BACKGROUND/AIMS: In this retrospective study, we compared the effects of histamine H2-receptor antagonists to those of antacids and anticholinergics in patients with hemorrhagic ulcers with various endoscopic appearances of bleeding. PATIENTS AND METHODS: Patients with hemorrhagic ulcers (n = 376) were examined by emergency endoscopy and were treated with 1) antacids and anticholinergic drugs or 2) H2-receptor antagonists. RESULTS: In ulcer patients with oozing or fresh red coagulation, H2-receptor antagonists ceased further hemorrhage more effectively (65.9% of the cases) than antacids and anticholinergic drugs (46.7%). In patients with projectile bleeding, both of the treatments failed to stop hemorrhage. There were no significant differences in favorable outcome in the patients only with old black coagulation between antacid and anticholinergic drugs-treated group and H2-receptor antagonists-treated group (94.4% and 93.8%, respectively). CONCLUSIONS: The results suggest that H2-receptor antagonists are more effective than antacids and anticholinergic drugs in patents with peptic ulcer with fresh coagulation or oozing, but not with projectile bleeding or old black coagulation. The results also indicate that endoscopic appearances of peptic ulcer bleeding are good predictors for the effects of medication.

Binding cells of 125I-iodoamphetamine in rat liver.

We recently reported that transrectal or intestinal portal scintigraphy with 123I-iodoamphetamine (IMP) could be a useful method for the non-invasive and quantitative evaluation of the portosystemic shunt in portal hypertension, but what cells in the liver trap IMP has not been clarified. This study was aimed at elucidating whether IMP was extracted by parenchymal cells, sinusoidal endothelial cells, Kupffer cells or fat storing cells. Each type of liver cell was isolated from rats and cultured. The cells were incubated with 125I-IMP and the radioactivity of the lysate was determined. Nonspecific binding was assessed in the presence of an excess of unlabeled IMP, and specific binding was determined by subtracting the nonspecific from total binding. Specific binding observed in parenchymal cells, endothelial cells and Kupffer cells was 70.2 +/- 0.4, 4.2 +/- 1.4 and 2.3 +/- 0.8 pmol/well, respectively, but no specific binding was observed in fat storing cells. The binding in parenchymal cells was much higher than that in endothelial cells or Kupffer cells (p < 0.005). In addition, the binding to parenchymal cells reached equilibrium within 20 min and was not saturable over the concentration range tested (0.5-10 microM). These findings indicate that IMP is mostly extracted by parenchymal cells in the liver.

Obesity and liver disease: evaluation of fatty infiltration of the liver using ultrasonic attenuation.

We developed an in vivo ultrasonic attenuation measurement system with which we attempted to evaluate the degree of fatty infiltration in the liver. In an animal study, fatty liver was induced in rabbits, and ultrasonic radiofrequency waveforms from the liver were obtained using a 10 MHz A mode transducer. Frequency-dependent attenuation of the ultrasound, which was correlated with total lipid content, was calculated using a spectral difference method. In a human study, ultrasonic waveforms were obtained using a 3.5 MHz transducer. Frequency-dependent attenuation also showed a significant correlation with the grading of fatty infiltration of the liver. These results suggested that fatty infiltration of the liver could be evaluated quantitatively and noninvasively using frequency-dependent attenuation of the ultrasound.

Organ assimilation of peptides.

Dipeptides injected intravenously or added to liver perfusion medium were hydrolyzed rapidly to amino acids. The clearance volumes per min of plasma Gly-Phe and Gly-Lys were 63% and 224%, respectively, of the total plasma volume. These values far exceed the blood flow in any single organ, suggesting that several organs must be involved in peptide assimilation. Intravenous administration of peptides increased the levels of their constituent amino acids in organs. Two possible explanations for this were assimilation of the peptides by the organs, and transport into the organs of the amino acids generated by extracellular hydrolysis of the peptides. The former possibility was tested by eliminating plasma lysine by enzymic degradation, so that the amino acid would accumulate only in the organs that assimilate lysine-containing peptides. Results showed that all organs tested, except the brain, had an intrinsic ability to assimilate peptides.