Detoxification Mechanism of α,ß-Unsaturated Carbonyl Compounds in Cigarette Smoke Observed in Sheep Erythrocytes.

Highly reactive α,ß-unsaturated carbonyl compounds, such as acrolein (ACR), crotonaldehyde (CA) and methyl vinyl ketone (MVK), are environmental pollutants present in high concentrations in cigarette smoke. We have previously found that these carbonyl compounds in cigarette smoke extract (CSE) react with intracellular glutathione (GSH) to produce the corresponding GSH-ACR, GSH-CA and GSH-MVK adducts via Michael addition reaction. These adducts are then further reduced to the corresponding alcohol forms by intracellular aldo-keto reductases in highly metastatic mouse melanoma (B16-BL6) cells and then excreted into the extracellular fluid. This time, we conducted a similar study using sheep erythrocytes and found analogous changes in the sheep erythrocytes after exposure to CSE as those with B16-BL6 cells. This indicates similarity of the detoxification pathways of the α,ß-unsaturated carbonyl compounds in sheep blood cells and B16-BL6 cells. Also, we found that the GSH-MVK adduct was reduced by aldose reductase in a cell-free solution to generate its alcohol form, and its reduction reaction was completely suppressed by pretreatment with epalrestat, an aldose reductase inhibitor, a member of the aldo-keto reductase family. In the presence of sheep blood cells, however, reduction of the GSH-MVK adduct was partially inhibited by epalrestat. This revealed that some member of the aldo-keto reductase superfamily other than aldose reductase is involved in reduction of the GSH-MVK adduct in sheep blood. These results suggest that blood cells, mainly erythrocytes are involved in reducing the inhalation toxicity of cigarette smoke via an aldo-keto reductase pathway other than that of aldose reductase.

Mass Spectrometric Approaches to the Identification of Potential Ingredients in Cigarette Smoke Causing Cytotoxicity.

Cigarette smoke contains many harmful chemicals that contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer, and cardiovascular disease. Many studies have been done to identify cytotoxic chemicals in cigarette smoke and elucidate the onset of the above-mentioned diseases caused by smoking. However, definitive mechanisms for cigarette smoke toxicity remain unknown. As candidates for cytotoxic chemicals, we have recently found methyl vinyl ketone (MVK) and acetic anhydride in nicotine/tar-free cigarette smoke extract (CSE) using L-tyrosine (Tyr), an amino acid with highly reactive hydroxyl group. The presence of MVK and acetic anhydride in CSE was confirmed by gas chromatography-mass spectrometry (GC/MS). We also found new reaction products formed in B16-BL6 mouse melanoma (B16-BL6) cells treated with CSE using LC/MS. These were identified as glutathione (GSH) conjugates of α,ß-unsaturated carbonyl compounds, MVK, crotonaldehyde (CA), and acrolein (ACR), by the mass value and product ion spectra of these new products. ACR and MVK are type-2 alkenes, which are well known as electron acceptors and form Michael-type adducts to nucleophilic side chain of amino acids on peptides. These α,ß-unsaturated carbonyl compounds may have a key role in CSE-induced cell death.

Intracellular Metabolism of α,ß-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde-Ketone Sensitive Reductase Activity.

The major toxicants in cigarette smoke, α,ß-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,ß-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH-MVK adduct can be detected by LC/MS analysis while the GSH-CA adduct is hardly detected. In the present study, to clarify why the GSH-CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH-CA and GSH-ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH-MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,ß-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,ß-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,ß-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,ß-unsaturated aldehydes and ketones.

Methyl vinyl ketone, a toxic ingredient in cigarette smoke extract, modifies glutathione in mouse melanoma cells.

Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels.

Abnormal amounts of intracellular calcium regulatory proteins in SHRSP.Z-Lepr(fa)/IzmDmcr rats with metabolic syndrome and cardiac dysfunction.

Metabolic syndrome is known to increase the risk of abnormal cardiac structure and function, which are considered to contribute to increased incidence of cardiovascular disease and mortality. We previously demonstrated that ventricular hypertrophy and diastolic dysfunction occur in SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP fatty) rats with metabolic syndrome. The aim of this study was to investigate the possible mechanisms underlying abnormal heart function in SHRSP fatty rats. The amount of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a, phospholamban (PLB) protein, and Ser(16)-phosphorylated PLB was decreased in cardiomyocytes from SHRSP fatty rats compared with those from control Wistar-Kyoto rats at 18 weeks of age, and the PLB-to-SERCA2a ratio was increased. Left ventricular developed pressure was unchanged, and coronary flow rate and maximum rate of left ventricular pressure decline (-dP/dt) was decreased in SHRSP fatty rats. Treatment with telmisartan reversed the abnormalities of PLB amount, coronary flow rate, and -dP/dt in SHRSP fatty rats. These results indicate that abnormal amounts of intracellular Ca(2+) regulatory proteins in cardiomyocytes, leading to reduced intracellular Ca(2+) reuptake into the sarcoplasmic reticulum, may play a role in the diastolic dysfunction in SHRSP fatty rats and that these effects are partially related to decreased coronary circulation. Telmisartan may be beneficial in protecting against disturbances in cardiac function associated with metabolic syndrome.

A chemical approach to searching for bioactive ingredients in cigarette smoke.

Cigarette smoke, a collection of many toxic chemicals, contributes to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease and cancer. Much work has been done on the chemical analysis of ingredients in cigarette smoke, but there are few reports on the active ingredients that can modify biomolecules. We used a sensitive liquid chromatography-mass spectrometry (LC/MS) and LC/MS/MS method to show that L-tyrosine (Tyr), an amino acid with a highly reactive hydroxyl group, readily reacts with cigarette smoke extract (CSE) at body temperature (37°C) to form various Tyr derivatives. Among these derivatives were N-(3-oxobutyl)-Tyr and two acetylated compounds, N-acetyl-Tyr and O-acetyl-Tyr, which were synthesized by reaction of Tyr with methyl vinyl ketone and acetic anhydride, respectively, at 37°C. The presence of methyl vinyl ketone and acetic anhydride in CSE was confirmed by gas chromatography-mass spectrometry (GC/MS). These results indicate that Tyr can easily react with active ingredients in CSE. The present analytical methods should aid the search for active ingredients in cigarette smoke.

Telmisartan provides protection against development of impaired vasodilation independently of metabolic effects in SHRSP.Z-Lepr(fa)/IzmDmcr rats with metabolic syndrome.

Metabolic syndrome is known to facilitate the development of cardiovascular disease. We have demonstrated that mesenteric arteries of SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP-fatty) rats with metabolic syndrome display an impaired vasorelaxation response mediated by nitric oxide. We examined whether the condition could be alleviated by treatment with telmisartan, an angiotensin II type 1 (AT1) receptor antagonist with PPAR-γ-activating properties and compared the results with those from pioglitazone, a PPAR-γ agonist. Telmisartan (5 mg·kg(-1)·day(-1)) or pioglitazone (2.5 mg·kg(-1)·day(-1)) was orally administered to male SHRSP-fatty rats for 8 weeks. Serum triglyceride and cholesterol levels were determined, and the oral glucose tolerance test was performed to evaluate insulin resistance. Vasodilations in response to acetylcholine and nitroprusside were determined by wire myographs under isometric tension conditions, protein expressions of soluble guanylyl cyclase in mesenteric arteries by Western blotting, and the contents of 3-nitrotyrosine in aortas by high-performance liquid chromatography with electrochemical detection. Telmisartan exerted antihypertensive effects, while pioglitazone ameliorated metabolic abnormalities in SHRSP-fatty rats. Telmisartan increased acetylcholine- and nitroprusside-induced relaxation and soluble guanylyl cyclase protein expression in mesenteric arteries and reduced 3-nitrotyrosine content in aortas. Pioglitazone displayed no such alleviating effects on vascular functions. These findings indicate that telmisartan protects against vasodilation disturbance through anti-oxidative and -nitrative stress independently of metabolic effects in SHRSP-fatty rats with metabolic syndrome.

Preventive effects of the extract of kinka-cha, a folk tea, on a rat model of metabolic syndrome.

Kinka-cha (dried leaf of Camellia nitidissima) is used as a folk tea for detoxication, diuresis and antihypertension. In the present study, we evaluated the extract of kinka-cha on metabolic, vascular and oxidative stress parameters in a model of metabolic syndrome, SHR/NDmcr-cp/cp (SHR/cp) rats that manifest hypertension, obesity, glucose intolerance and hyperlipidemia. Treatment with the extract of kinka-cha alleviated the increase in blood pressure, decrease in tail blood flow and elevated serum oxidative stress marker levels including lipid peroxides, 8-hydroxy-2'-deoxyguanosine, 3-nitrotyrosine and 3-chlorotyrosine. However, kinka-cha did not affect weight gain, hyperglycemia and hyperlipidemia, nor the relaxation responses of the aorta mesenteric artery, thoracic aortas and tail vein, and blood clotting and platelet aggregation. These results suggest that kinka-cha can help reduce the risk of developing metabolic syndrome, possibly due to the presence of antioxidants.

Inhibitory effect of Cordyceps sinensis on experimental hepatic metastasis of melanoma by suppressing tumor cell invasion.

We investigated the anti-metastatic activity of a water extract of Cordyceps sinensis (WECS) using a model of mice injected with B16-F0 mouse melanoma cells into the spleen. WECS administered intraperitoneally reduced the number of metastatic surface nodules of B16-F0 cells in the liver of C57BL/6Cr mice in a dose-dependent manner, and significantly prolonged their survival. To identify the mechanism of the anti-metastatic effect of WECS, we examined its effects on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. As a result, WECS reduced HGF-accelerated B16-F0 cell invasion in a concentration-dependent manner. These findings suggest that WECS exerts an anti-metastatic action, in part by inhibiting the HGF-accelerated tumor invasiveness of mouse melanoma cells.

An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma.

BACKGROUND: Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs) could be a sensitive marker to predict future cardiovascular events. However, a method to evaluate oxidized forms of LDLs systemically in human plasma is not yet established. In this study, we developed a novel and convenient high-performance liquid chromatography (HPLC) method for measuring ox-LDL levels in humans. METHODS: Human plasma lipoproteins were separated by a modified HPLC method using a diethylaminoethyl-type anion-exchange gel column with stepwise elution. Ox-LDLs were detected by postcolumn reaction with a reagent containing cholesterol esterase and cholesterol oxidase. Particle size of each LDL fraction separated by HPLC was determined in 61 healthy subjects. RESULTS: Our HPLC method separated LDLs into three fractions, which were designated as LDL-1, LDL-2 and LDL-3, on the basis of their negative charges, with LDL-3 the most strongly retained fraction migrating fastest in the anodic direction, a property that reflects the net negative charge of the molecule. Western blot analysis revealed that apolipoprotein B100 in LDL-3 fraction was the most fragmented and oxidatively modified. When LDLs were oxidized in vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modified by various aldehydes, all of the LDL fractions migrated at the position of LDL-3. Further, among three fractions, particle size was smallest in LDL-3 fraction. CONCLUSION: Here, we developed a convenient HPLC method and identified LDL-3 as oxidized LDL fractions, although ox-LDLs were present in LDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction. Measuring ox-LDL levels in human plasma by this method may be useful to evaluate atherosclerotic disorders.

Coronary vascular dysfunction promoted by oxidative-nitrative stress in SHRSP.Z-Lepr(fa) /IzmDmcr rats with metabolic syndrome.

1. Metabolic syndrome is an independent risk factor for cardiovascular disease. SHRSP.Z-Lepr(fa) /IzmDmcr (SHRSP fatty) rat, established as a new rat model of metabolic syndrome, spontaneously develops obesity, severe hypertension and shows hypertriglyceridaemia, hypercholesterolaemia and abnormal glucose tolerance. Using SHRSP fatty rats, we examined whether or not oxidative stress was correlated with vascular dysfunction in small and large calibre coronary arteries in ex vivo beating hearts, isolated mesenteric arteries and aortas in comparison with normal rats, Wistar-Kyoto rats (WKY). Vasodilation of coronary arteries was determined by microangiography of the Langendorff heart. 2. Compared with WKY, acetylcholine (ACh) and sodium nitroprusside (SNP)-induced relaxations were impaired in the coronary arteries of SHRSP fatty rats. The mesenteric arteries and aorta of SHRSP fatty rats showed impaired relaxation responses to ACh and SNP, decreased 3',5'-monophosphate (cGMP) production, and reduced soluble guanylyl cyclase protein expression. Superoxide release, angiotensin II and 3-nitrotyrosine contents were increased. 3. SHRSP fatty rats were orally administered olmesartan, an angiotensin II receptor type 1 (AT(1) ) antagonist, and amlodipine, a calcium channel blocker, at doses of 5 and 8mg/kg per day, respectively, for 8weeks. Both olmesartan and amlodipine reduced blood pressure, but only olmesartan prevented the development of abnormal vascular and biochemical parameters in the SHRSP fatty rats. 4. The results showed that in the SHRSP fatty rats, the impaired nitric oxide- and cGMP-mediated relaxation of vascular smooth muscle cells were linked to AT(1) receptor-induced oxidative-nitrative stress, which occurred concurrently with severe hypertension and metabolic abnormalities in vivo.

Inhibitory effect of cordycepin on hematogenic metastasis of B16-F1 mouse melanoma cells accelerated by adenosine-5'-diphosphate.

Platelet aggregation induced by cancer cells is an indispensable step for hematogenic metastasis. In these studies, we investigated whether platelet aggregation accelerates hematogenic metastasis of cancer cells in mice and the effect of cordycepin (3'-deoxyadenosine), a component of Cordyceps sinensis, on hematogenic metastasis accelerated by adenosine-5'-diphosphate (ADP). ADP significantly increased the number of metastatic lung nodules in mice injected intravenously with B16-F1 mouse melanoma cells (B16-F1 cells) in a dose-dependent manner and cordycepin significantly reduced the number of metastatic nodules of B16-F1 cells formed in the lung accelerated by ADP injected simultaneously with B16-F1 cells. These results suggest that ADP accelerated hematogenic metastasis and cordycepin has an inhibitory effect on hematogenic metastasis of B16-F1 melanoma cells via blocking of ADP-induced platelet aggregation in vivo.

Biochemical evidence of atherosclerosis progression mediated by increased oxidative stress in apolipoprotein E-deficient spontaneously hyperlipidemic mice exposed to chronic cigarette smoke.

Cigarette smoking is a major risk factor for cardiovascular disease. The induction of oxidative stress by smoking plays a key role in the progression of atherosclerosis. However, the underlying mechanisms are not fully understood. In the present study, we investigated whether long-term smoking can accelerate the progression of atherosclerosis and whether oxidative stress is implicated in its pathogenesis. Apolipoprotein E-deficient spontaneously hyperlipidemic mice, a model of atherosclerosis, were exposed to the gas-phase of smoke, from which tar and nicotine had been removed, for 15 min a day, 6 days a week, for 16 weeks. Exposure to cigarette smoke significantly increased the serum levels of oxidative stress markers such as thiobarbituric acid-reactive substances, oxidatively modified low-density lipoproteins, and 3-nitrotyrosine, but it did not affect serum cholesterol and triglyceride levels. Exposure to smoke also accelerated the accumulation of total cholesterol levels in the aorta that was accompanied by an increase in 3-nitrotyrosine levels of the atherosclerotic mice. These changes in the serum and aorta that progressed with exposure to smoke were prevented by vitamin E administration. Our data suggest that chronic cigarette smoking promotes and aggravates atherosclerosis and that the antioxidant vitamin E exerts an anti-atherogenic effect via reduction of oxidative stress.

Chronic production of peroxynitrite in the vascular wall impairs vasorelaxation function in SHR/NDmcr-cp rats, an animal model of metabolic syndrome.

We have previously reported that peroxynitrite is involved in dysfunction of nitric oxide (NO)-mediated vasorelaxation in SHR/NDmcr-cp rats (SHR-cp), which display typical symptoms of metabolic syndrome. This study investigated whether peroxynitrite is actually generated in the vascular wall with angiotensin II-induced NADPH-oxidase activation, thus contributing to the dysfunction. In isolated mesenteric arteries of male 18-week-old SHR-cp, relaxations in response to acetylcholine and sodium nitroprusside were impaired compared with that in Wistar-Kyoto rats. This impaired relaxation was not restored by treatment with apocynin, an NADPH-oxidase inhibitor. Protein expression of endothelial NO synthase increased while that of soluble guanylyl cyclase (sGC) decreased in the artery. We observed increased production of superoxide anions and peroxynitrite from the artery and their inhibition by apocynin, and also increased contents of nitrotyrosine, a biomarker of peroxynitrite, in mesenteric arteries and angiotensin II in aortas. Long-term (8 weeks) administration of telmisartan, an angiotensin II type 1-receptor antagonist, prevented the impaired vasorelaxation, decreased sGC expression and increased nitrotyrosine content in mesenteric arteries. These findings suggest that in the vascular wall of SHR-cp, peroxynitrite is continually produced by the reaction of NO with NADPH oxidase-derived superoxide via angiotensin II and gradually denatures sGC protein, leading to vasorelaxation dysfunction.

Possible participation of chloride ion channels in ATP release from cancer cells in suspension.

1. Cancer cells must detach from the primary focus to initiate the process of metastasis. Previously, we demonstrated that intracellular Ca(2+) levels are increased in endothelial cells in the presence of cancer cells and that ATP derived from these cells causes this increase. The present study clarifies the mechanism of ATP release from cancer cells by investigating the effects of Cl(-) channel inhibitors and other drugs on ATP release from human fibrosarcoma cells (HT-1080 cells). 2. Levels of extracellular ATP and its metabolites were measured using high-performance liquid chromatography (HPLC) with fluorescent detection. 3. Significantly more extracellular ATP was released by suspended than by adherent HT-1080 cells. The Cl(-) channel inhibitors 5-nitro-2-(3-phenylpropylamino) benzoic acid (100 micromol/L), gadolinium (100 micromol/L) and niflumic acid (100 micromol/L) all significantly inhibited ATP release from HT-1080 cells (1 x 10(3) /mL) to 39.7 +/- 6.5, 28.5 +/- 2.5 and 82.5 +/- 4.1% of control, respectively. 4. Neither of the p-glycoprotein inhibitors (i.e. 50 micromol/L quinidine and 90 micromol/L verapamil) had any effect on ATP release from HT-1080 cells. The gap junction hemichannel inhibitor Gap26 (300 micromol/L) slightly, but significantly, decreased ATP release by approximately 20%. The gap junction inhibitor 18-alpha-glycyrrhetinic acid (10 micromol/L) tended to inhibit ATP release from HT-1080 cells, but the difference did not reach statistical significance. 5. These findings indicate that Cl(-) channels play the most important role in ATP release from detached cancer cells and that gap junction hemichannels are also associated with ATP release.

Beneficial effect of coenzyme Q10 on increased oxidative and nitrative stress and inflammation and individual metabolic components developing in a rat model of metabolic syndrome.

Metabolic syndrome (MetS) is a group of cardiovascular risk factors, including visceral obesity, glucose intolerance, hypertension, and dyslipidemia. Increased oxidative and nitrative stress and inflammation and decreased endothelial function occur in an animal model of metabolic syndrome, SHR/NDmcr-cp (SHR/cp) rats. The present study investigated the effects of coenzyme Q10 (CoQ10), one of the important antioxidants, on the abnormal oxidative condition and characteristic components of metabolic syndrome in SHR/cp rats by maintaining them on a diet supplemented with 0.07% - 0.7% CoQ10 for 26 weeks. We determined serum levels of oxidatively modified low-density lipoprotein (Ox-LDL) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) as oxidative stress markers, 3-nitrotyrosine as a nitrative stress marker, 3-chlorotyrosine as a marker of myeloperoxidase (MPO)-catalyzed oxidation and high-sensitivity C-reactive protein (hsCRP) as an inflammatory marker. The administration of CoQ10 significantly attenuated the increase of oxidative and nitrative stress markers and inflammatory markers in a dose-dependent manner. CoQ10 prevented the elevated serum insulin levels, although it did not affect the elevated glucose level and dyslipidemia. CoQ10 also reduced elevated blood pressure, but did not affect body weight gain. In addition, CoQ10 improved endothelial dysfunction in the mesenteric arteries. These findings suggest that the antioxidant properties of CoQ10 can be effective for ameliorating cardiovascular risk in MetS.

Dietary corosolic acid ameliorates obesity and hepatic steatosis in KK-Ay mice.

Corosolic acid (CRA), a constituent of Banaba leaves, has been reported to exert anti-hypertension, anti-hyperinsulinemia, anti-hyperglycemia, and anti-hyperlipidemia effects as well as to induce anti-inflammatory and anti-oxidative activities. The aim of this study was to investigate the inhibitory effects of CRA on the development of obesity and hepatic steatosis in KK-Ay mice, a genetically obese mouse model. Six-week-old KK-Ay mice were fed a high fat diet for 9 weeks with or without 0.023% CRA. Nine-week CRA treatment resulted in 10% lower body weight and 15% lower total fat (visceral plus subcutaneous fat) mass than in control mice. CRA treatment reduced fasting plasma levels of glucose, insulin, and triglyceride by 23%, 41%, and 22%, respectively. The improved insulin sensitivity in CRA-treated mice may be due on part to the increased plasma adiponectin and white adipose tissue (WAT) AdipoR1 levels. In addition, CRA treatment increased the expression of peroxisome proliferator-activated receptor (PPAR) alpha in liver and PPAR gamma in WAT. This is the first study to show that CRA treatment can contribute to reduced body weight and amelioration of hepatic steatosis in mice fed a high fat diet, due in part to increased expression of PPAR alpha in liver and PPAR gamma in WAT.

Effect of P2 receptor on the intracellular calcium increase by cancer cells in human umbilical vein endothelial cells.

One of the important functions of vascular endothelial cells is as a barrier between blood and vascular tissue. This led us to speculate that cancer cells affect endothelial cells during metastasis. In the present study, we investigated the influence of human fibrosarcoma cells (HT-1080) on human umbilical vein endothelial cells (HUVEC), particularly intracellular calcium ion levels ([Ca2+]i), which are known to be an important intracellular signal transduction factor. HUVEC were treated with a fluorescent marker, and the fluorescence intensity of [Ca2+]i was then measured by phase contrast microscopic imaging. Extracellular adenosine triphosphate (ATP) release was measured using the chemiluminescence of luciferin-luciferase and a photon counting imaging system. HT-1080 (5x10(4) cells per dish) was found to increase [Ca2+]i in HUVEC. This [Ca2+]i rise was significantly reduced by U-73122 (phospholipase C inhibitor, 1 microM) and thapsigargin (calcium pump inhibitor, 1 microM). Interestingly, the [Ca2+]i rise in HUVEC was also significantly reduced by pyridoxalphosphare-6-azophenyl-2', 4'-disulfonic acid, a P2Y receptor antagonist (100 microM) and apyrase, a nucleotidase inhibitor (2 U/ml). In addition, we observed ATP release from HT-1080. These results suggest that [Ca2+]i in HUVEC was increased through the phospholipase C-IP3 pathway via ATP release from cancer cells. We previously reported that extracellular ATP increased [Ca2+]i and enhanced macromolecular permeability via the P2Y receptor. In tumor metastasis, cancer cells may exploit these regulatory mechanisms in the endothelial cell layer.

Long-term feeding of Ginkgo biloba extract impairs peripheral circulation and hepatic function in aged spontaneously hypertensive rats.

We previously demonstrated that Ginkgo biloba extract (GBE) produces an anti-hypertensive effect via enhanced vasodilation responses in young spontaneously hypertensive rats (SHR) and hepatic hypertrophy occurs with increased cytochrome P450 (CYP) mRNA expression in young rats. In the present study, to clarify whether these actions of GBE are observed in older rats, we investigated cardiovascular functions and hepatic CYP protein expressions in aged SHR fed a control diet or a diet containing 0.5% GBE for 4 weeks. In aged SHR, GBE feeding significantly increased liver weight per 100 g body weight without changing the body weight. Furthermore, significant increases in alanine aminotransferase level in serum and marked increase in CYP2B protein expression in the liver were observed in aged SHR fed GBE. On the other hand, GBE feeding did not affect blood pressure, but significantly reduced heart rate and blood flow velocity in tail arteries of aged SHR. Furthermore, GBE feeding did not affect contractile response to phenylephrine, relaxation responses to not only sodium nitroprusside but also acetylcholine, and protein levels of endothelium nitric oxide synthase and soluble guanylate cyclase in aortas of aged SHR. These results suggested that long-term GBE feeding impairs peripheral circulation due to bradycardia and hepatic function in aged SHR. Thus, in the elderly population with hypertension, the use of GBE may need to be assessed for effects on heart rate and liver function.

Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.