Aims/hypothesis Nitric oxide (NO) is synthesised not only from L-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, but whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency would induce the metabolic syndrome in mice. Methods To this end, we prepared a low-nitrite/nitrate diet (LND) consisting of an amino acid-based low-nitrite/nitrate chow, in which the contents of L-arginine, fat, carbohydrates, protein and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. Results Three months of the LND did not affect food or water intake in wild-type C57BL/6J mice compared with a regular diet (RD). However, in comparison with the RD, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia and glucose intolerance. Eighteen months of the LND significantly provoked increased body weight, hypertension, insulin resistance and impaired endothelium-dependent relaxations to acetylcholine, while 22 months of the LND significantly led to death mainly due to cardiovascular disease, including acute Mika Kina-Tanada and Mayuko Sakanashi contributed equally to this work.

Results demonstrated that tetrahydrobiopterin lessens ischemia-reperfusion injury in isolated perfused rat hearts, probably independent of its intrinsic radical scavenging action. The cardioprotective effect of tetrahydrobiopterin implies that tetrahydrobiopterin could be a novel and effective therapeutic option in the treatment of ischemia-reperfusion injury.

Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

Changes in splenic diameter measured by sonomicrometry in response to various adrenergic stimulants were estimated together with simultaneously measured arterial haemoglobin content (HGB) and haematocrit (HCT) in anaesthetized dogs. Splenic diameter decreased following intravenous injections (i.v.) of adrenaline, noradrenaline and phenylephrine and splenic nerve stimulation associated with increases in arterial HGB and HCT, which were significantly attenuated by prazosin i.v. After prazosin i.v., adrenaline i.v. increased splenic diameter significantly, but noradrenaline i.v. did not. Isoprenaline i.v. increased splenic diameter transiently, followed by a decrease that was abolished by prazosin i.v. During occlusion of splenic arteries and veins, adrenaline i.v. and phenylephrine i.v. did not cause any change in arterial HGB and HCT. Injection to splenic artery (i.a.) of phenylephrine induced a significant decrease in splenic diameter that was attenuated by prazosin i.a. but not by yohimbine i.a. Clonidine i.a. did not change splenic diameter. The present results indicate that splenic contraction, which is mediated through alpha 1-adrenoceptor activation, causes a significant increase in arterial HGB and HCT.

The aim of this study was to determine whether the medicinal herbs growing in Okinawa and possessing a radical-scavenging activity would exert cardioprotective effects against ischemia-reperfusion injury using isolated perfused rat hearts. Effects of the aqueous extracts from Psidium guajava L. and Limonium wrightii at concentrations having an equipotent radical-scavenging activity on myocardial injury produced by global ischemia followed by reperfusion were tested and were further compared with those of quercetin and gallic acid, major antioxidative components of P. guajava L. and L. wrightii, respectively. Both extracts significantly attenuated ischemic contracture during ischemia and improved myocardial dysfunction after reperfusion. Decreases in high-energy phosphates and increases in malondialdehyde in the reperfused hearts were significantly lessened with both plant extracts. Quercetin and gallic acid also exerted similar beneficial effects. These results indicate that P. guajava L. and L. wrightii both have cardioprotective effects against myocardial ischemia-reperfusion injury in isolated rat hearts, primarily through their radical-scavenging actions.

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

1 Chronic administration of erythropoietin (EPO) is often associated with hypertension in animals and humans. The aim of this study was to estimate whether 1-week treatment with EPO can aect the vascular endothelial function. 2 Rabbits were given with EPO (400 iu kg 71 s.c.) or saline each other day for 1 week. Hypotensive responses to intravenously given acetylcholine (ACh), endothelium-independent nitric oxide donors (NOC7, nitroprusside and nitroglycerin) and prostaglandin I 2 were tested before and after administration of N G -nitro-L-arginine methyl ester (L-NAME), a speci®c nitric oxide synthase inhibitor, under pentobarbitone anaesthesia. 3 Blood haemoglobin concentration in EPO group was signi®cantly higher than that in control group, whereas baseline values of aortic pressure, heart rate and femoral vascular resistance were similar. The dose of ACh (172 ng kg 71) requiring for a 15 mmHg hypotension from the baseline in EPO group was apparently higher than that (55 ng kg 71) in control group. On the contrary, hypotensive responses to NOC7, nitroprusside, nitroglycerin and prostaglandin I 2 were comparable between two groups. The extent of ACh-induced hypotension did not correlate with haemoglobin concentration. 4 L-NAME signi®cantly inhibited the ACh-induced vasodilating response in control group but did not in EPO group. 5 In another set of rabbits, the same treatment with EPO also decreased vasodilating responses to carbachol, bradykinin and substance P besides ACh as compared with control group. 6 These results indicate that 1-week treatment with EPO selectively attenuates depressor responses to endothelium-dependent vasodilators in anaesthetized rabbits, most likely due to inhibition of endothelial nitric oxide synthase.

Abstract. The role of nitric oxide (NO) derived from all three NO synthases (NOSs) in renal lesion formation remains to be fully elucidated. We addressed this point in mice lacking all NOSs. Renal injury was induced by unilateral ureteral obstruction (UUO). UUO caused significant renal lesion formation (tubular apoptosis, interstitial fibrosis, and glomerulosclerosis) in wild-type, singly, and triply NOS −/− mice. However, the extents of renal lesion formation were markedly and most accelerated in the triply NOS −/− genotype. UUO also elicited the infiltration of inflammatory macrophages, up-regulation of transforming growth factor (TGF)-β1, and induction of epithelial mesenchymal transition (EMT) in all of the genotypes; however, the extents were again largest by far in the triply NOS −/− genotype. Importantly, long-term treatment with the angiotensin II type 1 (AT 1 )-receptor blocker olmesartan significantly prevented the exacerbation of those renal structural changes after UUO in the triply NOS −/− genotype, along with amelioration of the macrophage infiltration, TGF-β1 levels, and EMT. These results provide the first evidence that the complete disruption of all NOS genes results in markedly accelerated renal lesion formation in response to UUO in mice in vivo through the AT 1 -receptor pathway, demonstrating the critical renoprotective role of all NOSs-derived NO against pathological renal remodeling.