.-The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N -nitro-Larginine methyl ester (L-NAME; 120 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] ϭ 1.6-5.5 M) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] ϭ 5.7 M). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method. nitric oxide; hemoglobin-nitric oxide adduct; electron paramagnetic resonance; temocapril NITRIC OXIDE (NO), a free radical molecule, has numerous roles in various physiological functions, such as the regulation of the cardiovascular, immune, and nervous systems. An imbalance of NO results in many diseases such as hypertension, atherosclerosis, septic and hemorrhage shock, ischemia-reperfusion injury, diabetes, and neurodegenerative disorders (18, 40,70).Because of increasing interest in the effect of NO, an accurate method is required for the measurement of this radical. Several methods for the quantitation of NO such as chemiluminescence (46), methemoglobin formation (23), and electron paramagnetic resonance (EPR) spectroscopy of nitrosyl-metal complexes (15) have been developed (3). The Griess and cGMP methods have been widely used to measure NO production in vivo (35, 62) and in vitro (34, 61), but these values do not always reflect the concentration of NO, because of their poor specificities for blood NO. EPR spectroscopy is a technique used for the detection and measurement of free radical species. EPR methods using endogenous and exogenous spin-trapping agents, which stabilize NO as a polyatomic spin adduct, have been developed (1,4,29, 41,72).In the circulation, NO exists as relatively stable hemoglobin (Hb)-NO adduct (HbNO) (48), which means that the amount of HbNO may reflect the blood NO ...

Adiponectin, an adipocyte-derived hormone, has been proposed to show antiatherogenic properties through the inhibitory effects against various growth factors. Insulin-like growth factor-1 (IGF-1) is one of the potent mitogens, which has been considered to play important roles in both atherogenesis and plaque stabilization in accordance to the phase of atherosclerosis. The aim of this study is to elucidate the adiponectin effects on IGF-1-induced cell migration and its intracellular signaling pathways in vascular smooth muscle cells (VSMCs). In this study, we assessed cell migration and several kinase activities in cultured rat aortic smooth muscle cells (RASMCs). Adiponectin pretreatment suppressed IGF-1-induced cell migration and extracellular signal-regulated kinase (ERK)1/2 activation, which is one of the major mediators for IGF-1-induced cell migration. In RASMCs, adiponectin and 5-aminoimidazole-4-carboxamide riboside (AICAR), a 5¢-AMP-activated protein kinase (AMPK) activator, stimulated AMPK activation. AMPK activation by AICAR inhibited IGF-1-induced ERK1/2 activation and cell migration in RASMCs. On the other hand, phosphorylation of Akt and Bad, proapoptotic molecules of the Bcl-2 family, which were increased by IGF-1 stimulation, was not diminished by the pretreatment with adiponectin. It was shown that adiponectin inhibited IGF-1-induced VSMC migration through suppression of ERK1/2 activation, which might be implicated in AMPK activation. Furthermore, adiponectin selectively inhibited ERK1/2 pathway, not Akt-Bad pathway, stimulated by IGF-1. From these findings, it was implied that adiponectin suppressed IGF-1-induced VSMC migration and its signaling selectivity.

Many epidemiologic studies have reported that dietary flavonoids provide protection against cardiovascular disease. Quercetin, a member of the bioflavonoids family, has been proposed to have anti-inflammatory, anti-atherogenic, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. Recent studies demonstrated that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore, we examined the effect of chemically synthesized quercetin glucuronide on platelet-derived growth factor (PDGF)-induced cell migration and kinase activation in cultured rat aortic smooth muscle cells (RASMCs). PDGF-induced RASMC migration was inhibited by quercetin 3-O-beta-D-glucuronide (Q3GA). Q3GA also attenuated PDGF-induced cell proliferation in RASMCs. PDGF activated extracellular-signal regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and Akt in RASMCs. PDGF-induced JNK and Akt activations were suppressed by Q3GA, whereas ERK1/2 and p38 MAP kinase activations were not affected. We also confirmed that PDGF-induced JNK and Akt activations were inhibited by antioxidants, N-acetylcysteine and diphenyleneiodonium chloride, in RASMCs. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may possess preventing effects for cardiovascular diseases relevant to vascular smooth muscle cell disorders.

We have reported that pharmacological doses of oral nitrite increase circulating nitric oxide (NO) and exert hypotensive effects in N -nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats. In this study, we examined the effect of a chronic dietary dose of nitrite on the hypertension and renal damage induced by chronic L-NAME administration in rats. The animals were administered tap water containing L-NAME (1 g/l) or L-NAME ϩ nitrite (low dose: 0.1 mg/l, medium dose: 1 mg/l, high dose: 10 mg/l) for 8 wk. We evaluated blood NO levels as hemoglobin-NO adducts (ironnitrosyl-hemoglobin), using an electron paramagnetic resonance method. Chronic administration of L-NAME for 8 wk induced hypertension and renal injury and reduced the blood iron-nitrosyl-hemoglobin level (control 38.8 Ϯ 8.9 vs. L-NAME 6.0 Ϯ 3.1 arbitrary units). Coadministration of a low dose of nitrite with L-NAME did not change the reduced iron-nitrosyl-hemoglobin signal and did not improve the L-NAMEinduced renal injury. The blood iron-nitrosyl-hemoglobin signals of the medium dose and high dose of nitrite were significantly higher than that of L-NAME alone. Chronic administration of a medium dose of nitrite attenuated L-NAME-induced renal histological changes and proteinuria. A high dose of nitrite also attenuated L-NAME-induced renal injury. These findings suggest that dietary doses of nitrite that protect the kidney are associated with significant increase in iron-nitrosyl-hemoglobin levels. We conclude that dietary nitrite-derived NO generation may serve as a backup system when the nitric oxide synthase/L-arginine-dependent NO generation system is compromised.vegetables; N -nitro-L-arginine methyl ester; kidney NITRIC OXIDE (NO) is a gaseous signaling molecule and exerts a variety of biological actions under physiological and pathophysiological conditions, such as regulation of the cardiovascular, inflammation, immune, and neuronal systems. NO also has numerous important functions in the kidney, such as the regulation of renal hemodynamics and glomerular microcirculation, the regulation of salt balance, the blunting of tubuloglomerular feedback, and the modulation of renal sympathetic nerve activity (18,26). Chronic blockade of nitric oxide synthases (NOSs) with N -nitro-L-arginine methyl ester (L-NAME) in rats results in severe hypertension and progressive kidney damage (3, 11). On the other hand, NO production seems to be low in chronic kidney disease (CKD) patients, and NO deficiency may play a role in CKD progression (5, 32). Treatment strategies to maintain the circulating NO level in healthy young people may be useful in preventing or slowing the progression of renal diseases.It is believed that NO is synthesized from L-arginine, NADPH, tetrahydrobiopterin, and the molecular oxygen catalyzed by NOSs in endothelial and other cells. The biological activity of NO is terminated by oxidation into nitrite and nitrate. Therefore, nitrite and nitrate in the blood are recognized as waste forms of NO. However, this dogma is now being challe...

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