Ichiro Wakabayashi

Wogonin inhibits inducible prostaglandin E2 production in macrophages

Effects of 5,7-dihydroxy-8-methoxyflavone (wogonin) on cyclooxygenase-2 (COX-2)-mediated prostaglandin E(2) production in macrophages were investigated. Stimulation with lipopolysaccharide (LPS; 1 microg/ml) greatly increased prostaglandin E(2) production in RAW 264.7 murine macrophages. The stimulated prostaglandin E(2) production was abolished in the presence of indomethacin (1 microM) or cycloheximide (2 microM), suggesting that the increased production of prostaglandin E(2) by LPS reflects the inducible synthesis of prostaglandin E(2) by COX-2. Wogonin (0.1-50 microM) concentration-dependently inhibited inducible prostaglandin E(2) production. Wogonin at concentrations as low as 0.5 microM directly attenuated enzymatic activity of COX-2. The protein expression of COX-2 was depressed by wogonin at concentrations of 10 microM and more. These results suggest that wogonin decreases inducible prostaglandin E(2) production in macrophages by inhibiting both COX-2 activity and COX-2 expression. The former action requires much lower doses of wogonin. These wogonin actions may explain, in part, its anti-inflammatory action.

Extracellular pH Affects Platelet Aggregation Associated with Modulation of Store-Operated Ca2+ Entry

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.

Associations of alcohol drinking and cigarette smoking with serum lipid levels in healthy middle-aged men

AIMS The aim of this study is to determine whether influences of drinking alcohol on serum lipid levels are different in smokers and non-smokers. METHODS Subjects were 25,689 healthy male workers aged 40 to 59 years. Serum total and HDL cholesterol and triglyceride concentrations were measured and LDL cholesterol concentrations were estimated by using the Friedewald formula. The subjects were divided into three groups by average daily consumption of cigarettes (non-smokers; light smokers, less than 20 cigarettes per day; heavy smokers, 20 or more cigarettes per day) and by average daily alcohol consumption (non-drinkers; light drinkers, less than 30 g of ethanol per day; heavy drinkers, 30 g or more of ethanol per day). RESULTS In overall subjects, serum HDL, LDL and total cholesterol were significantly lower and triglyceride was significantly higher in heavy smokers than in non-smokers. In the smoker groups, serum total cholesterol was significantly lower in heavy drinkers than in non-drinkers, while no difference in total cholesterol was observed in non- and heavy drinkers of the non-smoker group. Both in the smoker and non-smoker groups, HDL cholesterol was higher and LDL cholesterol was lower in drinkers than in non-drinkers. The difference in LDL cholesterol between non-drinkers and drinkers was more prominent in smokers than in non-smokers. The above associations were not altered after the adjustment for age, body weight and alcohol intake. CONCLUSIONS The results suggest that smoking increases the lowering effect of alcohol drinking on LDL cholesterol, but does not affect the relationship of alcohol drinking with HDL cholesterol.

Intracellular pH as a Determinant of Vascular Smooth Muscle Function

Intracellular pH (pH_i) is a physiological parameter that is intimately linked to contractility, growth and proliferation of vascular smooth muscle (VSM). Regarding contractility, no general unifying concept of pH_i regulation but a rather complex relation between pH_i signals and vascular tone has been revealed so far. The modulation of vasotone by pH_i depends on the type of blood vessel as well as on the pattern of regulatory input signals. In addition, changes in pH_i have been recognized as an important cellular signal to determine the fate of cells in terms of proliferation or apoptosis. Cellular sensors for pH_i include a variety of ion transport systems which control intracellular Ca²⁺ gradients and are likely to serve as a link between pH_i and cell functions. Here we provide an overview on the potential targets and mechanisms that transduce pH_i signals in VSM. The role of pH_i-sensing signaling complexes and localized pH_i signaling as the basis of diversity of pH_i regulation of VSM function is discussed.

Effects of Age on the Relationship between Drinking and Atherosclerotic Risk Factors

Background: Drinking modulates the progress of atherosclerotic cardiovascular diseases by affecting atherosclerotic risk factors. However, age-dependent effects of drinking on atherosclerotic risk factors have not been clarified in detail. Objective: In this cross-sectional study, we investigated whether the relationship between drinking and atherosclerotic risk factors is influenced by age in male workers (12,386 men aged from 20 to 69 years) in Yamagata, a district of Japan. Methods: The subjects were divided into five age groups, and each group was further divided into three subgroups according to ethanol consumption. The mean levels of each atherosclerotic risk factor were compared among the groups. Results: Neither body mass index nor fasting blood glucose levels were significantly affected by drinking at any age. In the heavy drinkers (ethanol consumption of 30 g per day or more) in all age groups, blood pressure, serum triglyceride and HDL cholesterol levels were significantly higher and serum LDL cholesterol level and the atherogenic index were significantly lower than in the nondrinkers. In the light drinkers (ethanol consumption of less than 30 g per day) in all age groups, serum HDL cholesterol level and the atherogenic index were also higher and lower, respectively, than in the nondrinkers. However, light drinking significantly increased blood pressure only in the middle aged and relatively elderly groups (40–49, 50–59, 60–69 years of age) and significantly decreased the serum LDL cholesterol level only in relatively young and middle aged groups (30–39, 40–49, 50–59 years of age). Thus, the effects of light drinking on blood pressure and serum LDL cholesterol are dependent on age. The serum triglyceride level was not significantly affected by light drinking in any age group. Conclusions: Our results suggest that light drinking increases blood pressure in the middle-aged and the elderly but not in the young, while its beneficial effects on serum HDL cholesterol and atherogenic index are not changed with age.

Serum albumin induces iNOS expression and NO production in RAW 267.4 macrophages

We investigated the effects of serum albumin on inducible nitric oxide synthase (iNOS) expression in RAW 267.4 macrophages. Crude fraction‐V type albumin as well as bovine serum albumin filtrated for endotoxin induced concentration‐dependent iNOS expression in macrophages. Accordingly, NO production (estimated by supernatant nitrite) was markedly (up to 10‐fold) increased in the presence of albumin. Albumin‐induced expression of iNOS protein was inhibited by cycloheximide and NO production was abolished after incubation of the cells with an iNOS inhibitor, NG‐monomethyl‐L‐arginine (LNMMA). An inhibitor of the NF‐κB pathway, pyrrolidine dithiocarbamate (PDTC), as well as inhibitors of JAK2/STAT and ERK, AG490 and U0126, respectively, significantly reduced albumin‐induced iNOS expression and NO production, while an inhibitor of the p38 pathway, SB203580, did not significantly affect NO production induced by albumin. Both types of serum albumin were contaminated with traces of endotoxin. The endotoxin levels were found not to be sufficient for the observed induction of nitrite production in RAW 267.4 cells. In addition, the albumin‐stimulated induction of iNOS was not reduced by preincubation of albumin‐containing media with polymyxin B, a LPS inhibitor. Polymerised albumin fractions were detected in the commercially available albumin tested in this study. A monomeric albumin‐rich fraction, separated by ultrafiltration, showed a potent inducing effect on iNOS expression and NO production, while a polymer‐rich fraction showed a smaller effect. Advanced glycation endproducts (AGE) of albumin were not formed by interaction with glucose in incubation medium, as AGE was not increased even after long‐time (4 weeks) incubation in albumin‐containing media [3.2–4.4 μg ml−1 (basal) vs 4.8–5.6 μg ml−1 (in glucose‐containing media)]. However, the duration of albumin exposure to glucose influenced the basal stimulatory properties of albumin. Our results suggest that serum albumin fractions, as gained by cold alcoholic extraction, may include determinants that stimulate or further enhance stimulation of RAW 267.4 cells and are different from endotoxin, polymeric albumin and AGE.

Modification of the association between alcohol drinking and non-HDL cholesterol by gender

BACKGROUND Serum non-HDL cholesterol is a strong predictor of cardiovascular diseases. We studied the relationship between habitual alcohol drinking and non-HDL cholesterol. METHODS Healthy male subjects (n = 27,005) and female subjects (n = 16,805) were divided into 5 groups by average daily ethanol intake. Serum non-HDL cholesterol level and prevalence of serum high non-HDL cholesterol (> or = 170 mg/dl) were compared among the groups. RESULTS Non-HDL cholesterol level and prevalence of high non-HDL cholesterol became lower as alcohol intake increased. The threshold alcohol intake in the drinker groups showing significantly lower non-HDL cholesterol level and significantly lower prevalence of high non-HDL cholesterol, compared with those in non-drinkers, was lower in women (<10 g/d) than in men (> or = 10 and <20 g/d). Odds ratios of each drinker group vs. the non-drinker group for high non-HDL cholesterol became lower as alcohol intake increased. The odds ratio of each drinker group vs. the non-drinker group for high non-HDL cholesterol tended to be lower in women than in men. CONCLUSIONS The results suggest that even light drinking is sufficient to significantly lower serum non-HDL cholesterol and that this effect of alcohol drinking on non-HDL cholesterol is more pronounced in women than in men.

5-Hydroxytryptamine as a potent migration enhancer of human aortic endothelial cells.

The purpose of the present study was to investigate whether 5‐hydroxytryptamine (5‐HT, serotonin) affects migration of vascular endothelial cells. 5‐HT significantly enhanced migration of human aortic endothelial cells (HAECs), and this enhancement was completely inhibited by GR 55562, a 5‐HT1 receptor antagonist, and fluoxetine, a 5‐HT transporter inhibitor, but was not affected by ketanserin, a 5‐HT2 receptor antagonist. 5‐HT stimulation increased RhoA and ERK activity of HAECs, and inhibitors of RhoA (Y‐27632 and H‐1152) and inhibitors of MEK (U0126 and PD98059) abolished the 5‐HT‐induced increase in migration velocity. Inhibition of Rho kinase by Y‐27632 blocked stress fiber formation and rear release of HAECs. Thus, 5‐HT has a potent enhancing action on migration of HAECs through activating the RhoA and ERK pathways following 5‐HT1 receptor stimulation.

Modification of the association of alcohol drinking with blood pressure by cigarette smoking

The purpose of this study was to investigate whether the association of alcohol drinking with blood pressure was modified by cigarette smoking. The subjects were healthy male workers aged 40–59 years and were divided into three different groups by average daily consumption of alcohol (non‐drinkers; light drinkers, less than 30 g ethanol per day; heavy drinkers, 30 g or more ethanol per day) and cigarettes (non‐smokers; light smokers, less than 20 cigarettes per day; heavy smokers, 20 cigarettes or more per day). The mean levels of both systolic and diastolic blood pressures were significantly lower in the light and heavy smoker groups than in the non‐smoker group. In the light and heavy smoker groups, systolic blood pressure was higher in the light drinker subgroup than in the non‐drinker subgroup, while there was no significant difference between systolic blood pressures in the non‐ and light drinker subgroups of non‐smokers. In the non‐, light and heavy smoker groups, systolic and diastolic blood pressures were significantly higher in the heavy drinker subgroup than in the non‐drinker subgroup, and these differences tended to be greater in light and heavy smokers than in non‐smokers. The above differences in the relationships of alcohol drinking with blood pressure in non‐, light and heavy smokers were also observed when age and body mass index were adjusted and when alcohol intake‐matched groups were used. These results suggest that the association of alcohol drinking with blood pressure is stronger in smokers than in non‐smokers, independently of age, body mass index and alcohol intake.

Influence of Gender on the Association of Alcohol Drinking With Blood Pressure

BACKGROUND The purpose of this study was to determine whether gender influences the association of alcohol drinking with blood pressure. METHODS The subjects (43,810 healthy men and women at ages of 35-54 years) were divided into five groups by average daily ethanol intake (non-, very light (<10 g per day), light (> or =10 g and <20 g per day), moderate (> or =20 g and <30 g per day), and heavy (> or =30 g per day) drinkers). The means of each variable after adjustment for age, body weight, and history of smoking were compared among the groups. RESULTS Systolic blood pressure of men was significantly higher in moderate and heavy drinkers than in nondrinkers, and systolic blood pressure of women was significantly higher in heavy drinkers but not in moderate drinkers than in nondrinkers. Diastolic blood pressure of men and women was significantly higher in light, moderate and heavy drinkers than in nondrinkers. The differences in systolic and diastolic blood pressure between drinkers and nondrinkers were greater in men than in women. Both in men and women, serum HDL cholesterol was significantly higher in all four drinker groups than in the nondrinker group, and the difference between drinkers and nondrinkers was greater in women than in men. The above findings were not altered when age- and alcohol intake-matched groups of subjects were used. CONCLUSIONS The results suggest that blood pressure is more prone to be elevated by alcohol drinking in men than in women.