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Abstract. 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.

Abstract. We investigated the effects of P2-receptor agonists on cell size, intracellular calcium levels ( ] i by 2meS-ATP were blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, P2Y 1 -antagonist), thapsigargin (Ca 2+-pump inhibitor), and U73122 (phospholipase C inhibitor). Furthermore, 2meS-ATP (P2Y 1 -receptor agonist) enhanced permeation of FD-4 through the endothelial cell monolayer. The 2meS-ATP-induced enhancement of the permeation was also prevented by PPADS, thapsigargin, and U73122. These results indicate that activation of P2Y receptors induces a decrease in cell size, an increase in [Ca 2+ ] i , and may participate in facilitating macromolecular permeability in HUVEC.

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 gp91, 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.

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.

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.

Lifestyle-related diseases-visceral obesity, hypertension, dyslipidemia and glucose intolerance-are known to appear in a cluster referred to as metabolic syndrome. As the number of disease components in metabolic syndrome increases in a patient, the structure and function of the heart becomes more compromised. 1) For example, metabolic syndrome increases the risk of atrial enlargement and fibrillation 2,3) and left ventricular hypertrophy and diastolic dysfunction. 1,4) These structural and functional abnormalities are considered to contribute to the increased cardiovascular morbidity and mortality associated with metabolic syndrome. However, the mechanisms underlying these abnormalities are not well understood.SHRSP.Z-Lepr fa /IzmDmcr rats (SHRSP-fatty) were established by crossing stroke-prone SHR (SHRSP/Izm) with Zucker obese rats to create a new animal model of metabolic syndrome.5) These rats develop spontaneous severe hypertension and obesity, and exhibit metabolic abnormalities (dyslipidemia, hyperinsulinemia and hyperglycemia), which are similar to those found in human metabolic syndrome. To date SHRSP-fatty have been used in studies that evaluated dysfunction of vasodilation mechanisms, cardiovascular remodeling, and atherogenic dyslipidemia in metabolic syndrome. [6][7][8] The aim of the present study was to characterize the cardiac structure and function in SHRSP-fatty. Increased cardiac stiffness induced by disturbed myocardial collagen turnover is suggested as one of the mechanisms of cardiac abnormalities in hypertension and diabetes.9,10) Therefore, as structural parameters, heart weight and a major myocardial collagen, collagen type I, as an index of fibrosis, were measured. Additionally, collagen type III, which contributes to tissue elasticity, was measured and the ratio of type I to type III was calculated as an index of myocardial stiffness. To assess cardiac function in SHRSP-fatty, heart rate, systolic blood pressure, cardiac output, blood flow and stroke volume were measured by tail-cuff and plethysmography, and compared to those in hypertensive lean SHRSP and normotensive lean WistarKyoto rats (WKY). Systolic and diastolic cardiac functions were also determined by echocardiography. The results may inform us whether this model would be a useful animal model to evaluate cardiac complications of metabolic syndrome. MATERIALS AND METHODS Experimental AnimalsMale SHRSP-fatty (nϭ18), SHRSP (nϭ18) and normotensive control WKY (nϭ18) were purchased at 16 weeks of age from Japan SLC, Inc. (Hamamatsu, Japan). The rats were established by the Disease Model Cooperative Research Association (Hamamatsu, Japan). Standard chow (CE-2; Clea Japan Inc., Tokyo, Japan) and water were available ad libitum during the experimental period. The study protocols were performed according to the Guideline for the Care and Use of Laboratory Animals approved by Mukogawa Women's University.Tail-Cuff Method and Plethysmography Systolic blood pressure and heart rate of conscious rats were meas- Cardiac structural and funct...

We demonstrated previously that stimulation of the P2Y receptor enhanced the macromolecular permeability of cultured endothelial cell monolayers via the paracellular pathway. To determine whether the P2Y receptor participates in the regulation of permeability in intact microvessels, we have examined the effects of exogenous and endogenous ATP on the permeation of the surface tissue of perfused rat tail caudal artery using a fluorescein isothiocyanate-dextran (FD-4; MW 4400; 1.0 mg mL(-1)). The permeation of FD-4 was assessed by a confocal fluorescence imaging system. We found that 2-methylthioadenosine 5'-triphosphate, a P2Y receptor agonist, enhanced the fluorescence intensity of FD-4 in the surface of the rat caudal artery tissue and that it was inhibited by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, a P2 receptor antagonist. Also, noradrenaline, a sympathetic neurotransmitter, and bradykinin, an inflammatory autacoid, enhanced the fluorescence intensity of FD-4 in the surface tissue of the rat caudal artery. The enhancement by noradrenaline was significantly inhibited by the P2 receptor antagonist. In addition, noradrenaline and bradykinin caused the release of ATP, ADP, AMP and adenosine from the endothelium of the rat caudal artery. These results indicated that the exogenous and endogenous ATP increased the macromolecular permeability of blood capillaries via the P2Y receptor. Such purinergic regulation of endothelial permeability may function in physiological and pathophysiological conditions.