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Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells
No full textThe vasodilator action of organic nitrates is thought to be mediated by an increase in the level of cGMP following stimulation of the cytosolic enzyme guanylate cyclase in the vascular smooth muscle cell. However, direct evidence for the formation of the putative active metabolite, nitric oxide (NO) within the different compartments of the vascular wall is still missing. We here demonstrate for the first time that cultured vascular smooth muscle cells as well as endothelial cells from different species actively metabolize organic nitrates to NO. We furthermore present evidence for an outward transport of cGMP from both cell types following stimulation of soluble guanylate cyclase. The rate of NO release closely correlated with the rate of cGMP egression. Biotransformation of organic nitrates to NO appeared to comprise at least two different components, a heat-sensitive enzymatic pathway which is short-lived and prone to rapid desensitization and a second non-enzymatic component which is apparently unsaturable and longer lasting. The marked decrease in the release of NO and cGMP upon the repeated administration of organic nitrates suggests that the phenomenon of "nitrate tolerance" is mainly due to an impaired biotransformation. We propose that the metabolism of nitrates to NO may have important implications for the prevention of atherosclerosis and the therapeutic modulation of blood cell functio
Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues
No full textThe formation of nitric oxide (NO) has been linked to many regulatory functions in mammalian cells. With the appreciation that NO-mediated nitrosation reactions are involved in cell signaling and pathology there is a need to elucidate and better characterize the different biochemical pathways of NO in vivo. Despite significant methodological advances over the years one major obstacle in assessing the significance of nitrosated species and other NO-related metabolites remains: their reliable measurement in complex biological matrices. In this review we briefly discuss the major routes of NO metabolism and transport in the mammalian circulation, considering plasma, red blood cell, and tissue compartments separately. In addition, we attempt to give a recommendation as to the most appropriate analytical technique and sample processing procedures for the reliable quantification of either species
Concomitant presence of N-nitroso and S-nitroso proteins in human plasma
No full textNitric oxide (NO)-mediated nitrosation reactions are involved in cell signaling and pathology. Recent efforts have focused on elucidating the role of S-nitrosothiols (RSNO) in different biological systems, including human plasma, where they are believed to represent a transport and buffer system that controls intercellular NO exchange. Although RSNOs have been implicated in cardiovascular disease processes, it is yet unclear what their true physiological concentration is, whether a change in plasma concentration is causally related to the underlying pathology or purely epiphenomenological, and to what extent other nitrosyl adducts may be formed under the same conditions. Therefore, using gas phase chemiluminescence and liquid chromatography we sought to quantify the basal plasma levels of NO-related metabolites in 18 healthy volunteers. We find that in addition to the oxidative products of NO metabolism, nitrite (0.20 +/- 0.02 micromol/l nitrite) and nitrate (14.4 +/- 1.7 micromol/l), on average human plasma contains an approximately 5-fold higher concentration of N-nitroso species (32.3 +/- 5.0 nmol/l) than RSNOs (7.2 +/- 1.1 nmol/l). Both N- and S-nitroso moieties appear to be associated with the albumin fraction. This is the first report on the constitutive presence of a high-molecular-weight N-nitroso compound in the human circulation, raising the question as to its origin and potential physiological role. Our findings may not only have important implications for the transport of NO in vivo, but also for cardiovascular disease diagnostics and the risk assessment of nitrosamine-related carcinogenesis in man
The nitric oxide/superoxide assay. Insights into the biological chemistry of the NO/O-2 interaction.
No full textNitric oxide (NO) is a widespread signaling molecule involved in the regulation of an impressive spectrum of diverse cellular functions. Superoxide anions (O-2) not only contribute to the localization of NO action by rapid inactivation, but also give rise to the formation of the potentially toxic species peroxynitrite (ONOO-) and other reactive nitrogen oxide species. The chemistry and biological effect of ONOO- depend on the relative rates of formation of NO and O-2. However, the simultaneous quantification of NO and O-2 has not been achieved yet due to their high rate of interaction, which is almost diffusion-controlled. A sensitive spectrophotometric assay was developed for the simultaneous quantification of NO and O-2 in aqueous solution that is based on the NO-induced oxidation of oxyhemoglobin (oxyHb) to methemoglobin and the O-2-mediated reduction of ferricytochrome c. Using a photodiode array photometer, spectral changes of either reaction were analyzed, and appropriate wavelengths were identified for the simultaneous monitoring of absorbance changes of the individual reactions. oxyHb oxidation was followed at 541.2 nm (isosbestic wavelength for the conversion of ferri- to ferrocytochrome c), and ferricytochrome c reduction was followed at 465 nm (wavelength at which absorbance changes during oxyHb to methemoglobin conversion were negligible), using 525 nm as the isosbestic point for both reactions. At final concentrations of 20 microM ferricytochrome c and 5 microM oxyHb, the molar extinction coefficients were determined to be epsilon465-525 = 7.3 mM-1 cm-1 and epsilon541.2-525 = 6.6 mM-1 cm-1, respectively. The rates of formation of either NO or O-2 determined with the combined assay were virtually identical to those measured with the classical oxyhemoglobin and cytochrome c assays, respectively. The assay was successfully adapted to either kinetic or end point determination in a cuvette or continuous on-line measurement of both radicals in a flow-through system. Maximal assay sensitivity was approximately 25 nM for NO and O-2. Cross-reactivity with ONOO- was controlled for by the presence of L-methionine. Generation of NO from the NO donor spermine diazeniumdiolate could be reliably quantified in the presence and absence of low, equimolar, and high flux rates of O-2. Likewise, O-2 enzymatically generated from hypoxanthine/xanthine oxidase could be specifically quantified with no difference in absolute rates in the presence or absence of concomitant NO generation at different flux rates. Nonenzymatic decomposition of 3-morpholinosydnonimine hydrochloride (100 microM) in phosphate buffer, pH 7.4 (37 degrees C), was found to be associated with almost stoichiometric production of NO and O-2 (1.24 microM NO/min and 1.12 microM O-2/min). Assay selectivity and applicability to biological systems were demonstrated in cultured endothelial cells and isolated aortic tissue using calcium ionophore and NADH for stimulation of NO and O-2 formation, respectively. Based on these data, a computer model was elaborated that successfully predicts the reaction of NO and O-2 with hemoprotein and may thus help to further elucidate these reactions. In conclusion, the nitric oxide/superoxide assay allows the specific, sensitive, and simultaneous detection of NO and O-2. The simulation model developed also allows the reliable prediction of the reaction between NO and O-2 as well as their kinetic interaction with other biomolecules. These new analytical tools will help to gain further insight into the physiological and pathophysiological significance of the formation of these radicals in cell homeostasis
Impaired effectiveness of nitric oxide-donors in resistance arteries of patients with arterial hypertension
No full textDilation of resistance arteries in response to infusion of nitric oxide donors is impaired in hypertensive patients and the degree of this impairment depends critically on the severity of arterial hypertension. The reduced effectiveness of nitric oxide appears to be independent of the class of nitric oxide donor and thus of the mode of intravascular nitric oxide generation. These findings are likely to have important implications not only for our understanding of the pathophysiological mechanisms of endothelial dysfunction but also for nitric oxide donor therapy in arterial hypertension
Release of endothelium derived nitric oxide in relation to pressure and flow
Full text link(1) The rate of NO release from endothelial cells increases when flow is enhanced. (2) Endothelial cells possess a high capacity for NO production, permitting a rapid adjustment of NO release to changes in flow. (3) The rate of NO release is not causally related to changes in perfusion pressure
Role of nitric oxide in the regulation of coronary vascular tone in hearts from hypertensive rats. Maintenance of nitric oxide-forming capacity and increased basal production of nitric oxide
Full text linkIn arterial hypertension, coronary flow reserve, expressed by the difference between autoregulated and maximal coronary flow, is frequently impaired. Previous experimental and clinical investigations using acetylcholine as a stimulus for the production of endothelium-derived relaxing factor suggested that an impaired endothelium-dependent vasodilation, presumably caused by a decreased formation of nitric oxide (NO), may account for this microvascular dysfunction. However, so far no study has been performed that quantifies the formation of NO within the coronary circulation of hypertensive hearts to assess its role in setting coronary vascular tone in the hypertensive heart. We therefore quantified NO formation within the coronary circulation of constant flow-perfused, isolated hearts from spontaneously hypertensive rats (SHR, 16th to 26th week), as a model for hypertensive heart disease, and from the normotensive control strain (Wistar-Kyoto, WKY) using the oxyhemoglobin technique. Coronary perfusion pressure and vascular resistance were almost 30% higher in SHR compared with WKY hearts. Intracoronarily applied NO decreased coronary vascular resistance by maximally 45% of resting values in a concentration-dependent manner in both groups. The bradykinin-induced decrease in coronary vascular resistance and the parallel increase in NO release were comparable in SHR and WKY hearts and fell within the vasodilator range of exogenously applied NO. Moreover, basal release of NO normalized to heart wet weight was 50% higher in SHR compared with WKY hearts. Rates of basal NO release were correlated inversely with changes in coronary perfusion pressure and vascular resistance in both groups (r = -.85 and -.84, respectively, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS
Mechanisms of histamine-induced coronary vasodilatation: H1-receptor-mediated release of endothelium-derived nitric oxide
No full textAlthough the content of histamine in myocardial tissue is high, its contribution to the regulation of coronary blood flow has not been clearly defined. The aim of the present study was to investigate whether or not nitric oxide (NO), an important modulator of coronary vascular tone, is involved in histamine-induced coronary vasomotion and to characterize which histaminergic receptor subtype mediates this process. Isolated, constant-flow-perfused guinea pig hearts were challenged with histamine, the H1-receptor agonist pyridylethylamine (PYR) and the H2-receptor agonist dimaprit (DIM). Apart from coronary perfusion pressure (CPP), left ventricular pressure (LVP) and the development of contractile force (dp/dt), the release of NO and cyclic GMP (cGMP) were continuously measured. Histamine and DIM induced concentration dependently a coronary vasodilatation with an almost 50% decrease in CPP paralleled by an enhancement of LVP and dp/dt by more than 80%. PYR selectively reduced CPP by 47% without affecting LVP and dp/dt. Histamine- and PYR-induced coronary vasodilatation were paralleled by a more-than-twofold increase in basal cGMP release from isolated hearts, whereas DIM exerted no effects on cGMP release. Oxyhemoglobin (4 µM), an effective scavenger of NO, shifted the concentration-response curve for histamine- and PYR-induced changes in CPP significantly to the right and in parallel inhibited the increase in cGMP release. Histamine and PYR rapidly (within 2 s) decreased CPP, while the onset of DIM-induced coronary vasodilatation followed changes in LVP with a lag period of 10 s. Histamine increased basal NO release concentration dependently by a maximum of 351 ± 21 pmol/min
Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action
No full textThe plasma level of NO(x), i.e., the sum of NO(2)- and NO(3)-, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO(x) concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO(2)- and NO(3)- concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO(2)- or NO(3)-. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1-10 microg/min) dose-dependently augmented venous NO(2)- levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO(2)-. Intraarterial infusion of NO(2)- had no effect on FBF. NOS inhibition (N(G)-monomethyl-l-arginine; 4-12 micromol/min) dose-dependently reduced basal NO(2)- and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO(3)- and total NO(x) remained unchanged as did systemic arterial NO(2)- and NO(3)- levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P < 0.001). These results contradict the current paradigm that plasma NO(3)- and/or total NO(x) are generally useful markers of endogenous NO production and demonstrate that only NO(2)- reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive
Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells
Full text linkEndothelial cells (EC) contribute to the control of local vascular diameter by formation of an endothelium derived relaxant factor (EDRF) (1). Whether nitric oxide (NO) is identical with (EDRF) or might represent only one species of several EDRFs has not been decided as yet (2-5). Therefore, we have directly compared in cultured EC the kinetics of NO formation determined in a photometric assay with the vasodilatory effect of EDRF and NO in a bioassay. Basal release of NO was 16, 4 pmol/min/ml packed EC column. After stimulation with bradykinin (BK) and ATP onset of endothelial NO release and maximal response preceded the EDRF-mediated relaxation. Concentrations of NO formed by stimulated EC were quantitatively sufficient to fully explain the smooth muscle relaxation determined in the bioassay. Our data provide convincing evidence that under basal, BK and ATP-stimulated conditions 1. endothelial cells release nitric oxide as free radical, 2. nitric oxide is solely responsible for the vasodilatory properties of EDRF
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