Yukari Tada

Peroxynitrite is Involved in the dysfunction of vasorelaxation in SHR/NDmcr-cp rats, spontaneously hypertensive obese rats.

SHR/NDmcr-cp (SHR-cp) rats display typical symptoms and features of the metabolic syndrome. We previously reported that endothelium-dependent relaxation decreases in the thoracic aortas of SHR-cp rats, despite increased nitric oxide (NO) production from the endothelium. In the present study, to search for the reasons for this contradiction, we investigated whether vascular abnormality could be reduced by treatment of SHR-cp rats with antihypertensive drugs; a calcium channel blocker (amlodipine), an alpha 2 and imidazoline receptor agonist (moxonidine), and an angiotensin II type 1 (AT1) receptor antagonist (telmisartan). Telmisartan but not amlodipine and moxonidine ameliorated the impairment of relaxation in response to acetylcholine and the increased protein expression of endothelium NO synthase in thoracic aortas. All three drugs significantly lowered the blood pressure. Telmisartan decreased the serum levels of lipid peroxide and 8-hydroxy-2′-deoxyguanosine, oxidative stress markers, and also the aortic levels of the protein expression of gp91phox, a component of NADPH oxidase, and 3-nitrotyrosine, a biomarker of peroxynitrite. These findings suggest that NADPH oxidase-derived superoxide, probably produced due to stimulation of AT1 receptors, reacts with NO to form peroxynitrite, and consequently decreases active NO, leading to attenuation of endothelium-dependent relaxation. Angiotensin receptor antagonists may be effective for preventing endothelial dysfunction in metabolic syndrome.

Coronary vascular dysfunction promoted by oxidative-nitrative stress in SHRSP.Z-Leprfa/IzmDmcr rats with metabolic syndrome.

1. Metabolic syndrome is an independent risk factor for cardiovascular disease. SHRSP.Z‐Leprfa/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.

Chronic oxidative-nitrosative stress impairs coronary vasodilation in metabolic syndrome model rats

Metabolic syndrome (MetS) is a combination of clinical disorders that together increase the risk for cardiovascular disease and diabetes. SHRSP.Z-Lepr(fa)/IzmDmcr (SHRSP.ZF) rats with MetS show impaired nitric oxide-mediated relaxation in coronary and mesenteric arteries, and angiotensin II receptor type 1 blockers protect against dysfunction and oxidative-nitrosative stress independently of metabolic effects. We hypothesize that superoxide contributes to functional deterioration in SHRSP.ZF rats. To test our hypothesis, we studied effects of treatment with tempol, a membrane-permeable radical scavenger, on impaired vasodilation in SHRSP.ZF rats. Tempol did not alter body weight, high blood pressure, or metabolic abnormalities, but prevented impairment of acetylcholine-induced and nitroprusside-induced vasodilation in the coronary and mesenteric arteries. Furthermore, tempol reduced the levels of serum thiobarbituric acid reactive substance (TBARS) and 3-nitrotyrosine content in mesenteric arteries. Systemic administration of tempol elevated the expression of soluble guanylate cyclase (sGC) above basal levels in mesenteric arteries of SHRSP.ZF rats. However, acute treatment with tempol or ebselen, a peroxynitrite scavenger, did not ameliorate impaired relaxation of isolated mesenteric arteries. No nitration of tyrosine residues in sGC was observed; however, sGC mRNA expression levels in the arteries of SHRSP.ZF rats were lower than those in the arteries of Wistar-Kyoto rats. Levels of Thr(496)- and Ser(1177)-phosphorylated endothelial nitric oxide synthase (eNOS) were lower in arteries of SHRSP.ZF rats, and acetylcholine decreased Thr(496)-phosphorylated eNOS levels. These results indicated that prolonged superoxide production, leading to oxidative-nitrosative stress, was associated with impaired vasodilation in SHRSP.ZF rats with MetS. Down-regulated sGC expression may be linked to dysfunction, while reduced NO bioavailability/eNOS activity and modified sGC activity due to superoxide production were excluded as pivotal mechanisms.

Telmisartan provides protection against development of impaired vasodilation independently of metabolic effects in SHRSP.Z-Leprfa/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.

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 Ca2+ 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 µmol/L), gadolinium (100 µmol/L) and niflumic acid (100 µmol/L) all significantly inhibited ATP release from HT‐1080 cells (1 × 103 /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 µmol/L quinidine and 90 µmol/L verapamil) had any effect on ATP release from HT‐1080 cells. The gap junction hemichannel inhibitor Gap26 (300 µmol/L) slightly, but significantly, decreased ATP release by approximately 20%. The gap junction inhibitor 18‐α‐glycyrrhetinic acid (10 µmol/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.

Abnormal amounts of intracellular calcium regulatory proteins in SHRSP.Z-Leprfa/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 novel method using confocal laser scanning microscopy for sensitive measurement of P‐glycoprotein‐mediated transport activity in Caco‐2 cells

Objectives  The aim of this study was to use time‐lapse confocal laser scanning microscopy to establish a more sensitive and specific method for evaluating P‐glycoprotein activity in Caco‐2 cells.

GINKGO BILOBA EXTRACT CAUSES DECREASE IN HEART RATE IN AGED SPONTANEOUSLY HYPERTENSIVE RATS

1 We previously reported that Ginkgo biloba extract (GBE) improves cardiovascular function in young spontaneously hypertensive rats (SHR). In the present study, changes in the cardiovascular parameters of aged SHR were examined following a 4‐week diet of GBE. 2 Feeding with GBE significantly decreased the heart rate and blood flow velocity in the tails of aged SHR. The contractile and relaxation responses were unchanged in isolated aortas and mesenteric arteries of aged SHR fed the GBE diet. The GBE diet did not influence the protein levels of endothelial nitric oxide synthase or soluble guanylyl cyclase in the aortas. 3 These findings indicate that in aged SHR, the ingestion of GBE may cause bradycardia without a beneficial effect on the vascular relaxation response. Intake of GBE as a supplement in elderly hypertensive patients should be carefully monitored.

Impairment of vasodilation and effects of perivascular adipose tissue in metabolic syndrome

生 活 習 慣 病 と 循 環 器 疾 患 研 究 の 新 展 開 2 要約:内臓脂肪型肥満を基盤とするメタボリックシン ドロームは,心血管病の発症リスクが相乗的に増加す ることから,重要な予防ターゲットとして注目されて いる.血管の内側を覆う内皮細胞は,種々の因子を産 生・放出し,血管の恒常性維持に重要な役割を担って いる.血管緊張性を抑制的に制御する因子として発見 された一酸化窒素(NO)は,これまでに様々な心血管 病の発症における意義が報告されている.我々は, NOによる血管弛緩機能に注目し,モデルラットを用 いて,メタボリックシンドロームでは,動脈は酸化ス トレスに慢性的に曝露されることによって NOに対す る反応性が低下すること,冠動脈に生じる拡張機能低 下が心機能の低下を引き起こす要因となることを見出 した.さらに,動脈に拡張障害が生じている場合,代 償的に血管周囲の脂肪組織は NOに対する血管弛緩作 用を亢進させ,血管緊張性維持に寄与している可能性 を見出した.このことは,動脈とその周囲脂肪組織と の間にクロストークが存在することを示唆するもので あり,肥満と心血管病とを結びつける臓器間ネット ワークとして注目される.

878 CHRONIC OXIDATIVE-NITROSATIVE STRESS VIA ANGIOTENSIN II TYPE 1 RECEPTOR ACTIVATION INVOLVES IN DYSFUNCTION OF CORONARY VASODILATION IN METABOLIC SYNDROME RATS, SHRSP.Z-Leprf1/IzmDmcr RATS (SHRSP.ZF)

Objectives: Metabolic syndrome (MetS) is facilitate the development of cardiovascular disease. In SHRSP.ZF with MetS, we have demonstrated that oxidative-nitrosative stress was increased, and vasodilation was impaired in the coronary vasculature. To investigate whether superoxide production via angiotensin II activation is linked to the functional deterioration, we studied the effects of administered olmesartan, an angiotensin II receptor type 1 (AT1) antagonist, and tempol, a radical scavenger, on impaired vasodilations in SHRSP.ZF. Methods: Olmesartan (5 mg/kg/day) was administered orally once daily to male 10 weeks of age SHRSP.ZF for a period of 8 weeks. Tempol was administered as a 2 mM solution in drinking water to the same rats for 8 weeks. Vasodilations of coronary, and mesenteric arteries were measured by coronary microangiography of ex vivo beating hearts and myograph methods, respectively. Results: Treatment with olmesartan and tempol reversed the impaired acetylcholine and nitroprusside-induced vasodilations in the coronary and mesenteric arteries of the SHRSP.ZF. Olmesartan and tempol reduced content of 3-nytrotyrosine, a footprint of peroxynitrite, in mesenteric arteries. In contrast to the results in vivo, impaired relaxation in coronary and mesenteric arteries was not normalized by addition of tempol to the organ perfusion solutions and myograph tissue baths, even though the superoxide level in mesenteric arteries was almost abolished in vitro. Conclusions: These results indicate that reduced NO bioavailability is not critical to the vascular dysfunction in SHRSP.ZF with MetS in vivo, but chronic exposure to oxidative-nitrosative stress induced by AT1 activation is closely linked to development of the functional deterioration.