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Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

L-Arginine treatment decreased superoxide generation by cNOS while increasing NO accumulation, leading to protection from constriction (microvessel area, 17.77+/-0.95 versus 11.66+/-2.21 microm2 untreated, P<.0005) and reduction of edema after reperfusion (interfiber area, 16.56+/-2.13% versus 27.68+/-7.70% untreated, P<.005).

Hyperleptinemia accompanying obesity affects endothelial nitric oxide (NO) and is a serious factor for vascular disorders. NO, superoxide (O 2 Ϫ ), and peroxynitrite (ONOO Ϫ ) nanosensors were placed near the surface (5 Ϯ 2 m) of a single human umbilical vein endothelial cell (HUVEC) exposed to leptin or aortic endothelium of obese C57BL/6J mice, and concentrations of calcium ionophore (CaI) (14,32). Endothelial dysfunction has been associated with several vascular diseases (32). Bioavailable nitric oxide (NO) production is diminished in dysfunctional endothelium, which hinders the L-arginine/NO pathway and increases vasoconstriction (13).-There have been several reports suggesting the potential mechanisms for diminished NO production in obesity. It has been proposed that the elevated free fatty acids levels observed in obesity may inhibit endothelial NO synthase (eNOS) (8). Also, the increased levels of cytokines IL-6 and TNF-␣ observed in obesity may affect the phosphorylation of tyrosine kinases and eNOS expression (16). It also has been suggested that obesity is associated with an increased production of reactive oxygen species in the cardiovascular system (31).Recent evidence indicates that adipose tissue-derived hormone leptin can be involved in vascular tone control (1, 7) and that leptin receptors (Ob-R) are expressed in endothelial cells (4). In a vascular ring model, leptin induced vasorelaxation in a dose-dependent manner, and this effect was abolished by eNOS inhibitors (18,19). The in vitro stimulatory effect of leptin on NO production in cultured endothelial cells was also tested by measuring nitrite and nitrate concentrations (30). The intravenous injection of leptin increased nitrite and nitrate concentrations in blood serum of normotensive Wistar rats but not in obese Zucker rats, which have a mutation in their leptin receptor gene (12). Intraperitoneal leptin administration also increased plasma concentration and urinary excretion of NO metabolites, as well as NO second messenger, guanosine 3Ј,5Ј-cyclic monophosphate (cGMP) (3). All this data indirectly indicate that leptin may increase NO production in endothelial cells and cause these hypotensive effects. It has also been shown that stimulation of endothelial cells with leptin leads to an increase in the production of reactive oxygen species, in particular superoxide (O 2 Ϫ ), which is known to react rapidly with NO to form peroxynitrite (ONOO Ϫ ) (5, 35). The leptin level increases in obesity (7), but the long-term effect of elevated leptin on the bioavailability of endothelial NO and cytotoxic ONOO Ϫ is not known. This work was designed to clarify the potential role of NO, O 2 Ϫ , and ONOO Ϫ in leptin-associated vascular disorders associated with obesity. A system of nanosensors was used for direct simultaneous measurements of the leptin-induced biosynthesis of NO, O 2 Ϫ , and ONOO Ϫ in human umbilical vein endothelial cell (HUVEC) or the endothelium of obese mice. METHODSAll materials were purchased from Sigma-Aldrich (St. Louis, MO), unl...

NO alters contractile and relaxant properties of the heart. However, it is not known whether changes in ventricular loading conditions affect cardiac NO synthesis. To understand this potential contractile-relaxant autoregulatory mechanism, production of cardiac NO in response to mechanical stimuli was measured in vivo using a porphyrinic sensor placed in the left ventricular myocardium. The beating rabbit heart exhibited cyclic changes in [NO], peaking at 2.7+/-0.1 micromol/L near the endocardium and 0.93+/-0.20 micromol/L in the midventricular myocardium (concentrations were 15+/-4% lower in the rat heart). In the present study, we demonstrate for the first time that increasing or decreasing ventricular preload in vivo is followed by parallel changes in [NO], which may represent a novel autoregulatory mechanism to adjust cardiac performance or perfusion on a beat-to-beat basis. To quantify the relationship between applied force and NO synthesis, intermittent compressive or distending forces applied to ex vivo nonbeating hearts were shown to cause bursts of NO synthesis, with peak [NO] linearly related to ventricular transmural pressure. Experiments in which denuding cardiac endothelial and endocardial cells abrogated the NO signal indicate that these cells transduce mechanical stimulation into NO production in the heart. Taken together, these studies may help explain load-dependent relaxation, cardiac memory for mechanical events of preceding beats, diseases associated with myocardial distension, autoregulation of myocardial perfusion, and protection from thrombosis in the turbulent flow environment within the beating heart.

Quercetin treatment mollified ischaemia-reperfusion injury to skeletal muscle by scavenging destructive superoxide and enhancing the cytoprotective nitric oxide concentration.

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