Biotransformation of organic nitrates to nitric oxide by vascular smooth muscle and endothelial cells
ABSTRACT The 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 function.
Full-textDOI: · Available from: Martin Feelisch, Aug 17, 2015
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- "A porphyrinic microsensor working electrode, platinum wire counter electrode and a miniature saturated silver/silver chloride reference electrode were connected to the ISO-NO MARK II potentiostat (WPI, USA). The measurement was practised using distilled water saturated with pure NO gas (according to the WPI manual, ). The injection of 1 µl of the NO-saturated water into the glass vial (final concentration of NO in the vial = 595 nM) caused the rapid increase (peak time = 15 ± 5 s) with a subsequent gradual decrease of an NO-induced signal until it reached the background current. "
ABSTRACT: H1-antihistamines are known to be important modulators of inflammatory response. However, the information about the influence of these drugs on reactive nitrogen species generation is still controversial. The main aim of the present study was to investigate the effects of selected H1-antihistamines on nitric oxide production by lipopolysaccharide-stimulated murine macrophages RAW 264.7, measured as changes in inducible nitric oxide synthase (iNOS) protein expression in cell lysates by Western blotting and nitrite formation in cell supernatants using the Griess reaction. In pharmacological non-toxic concentrations, H1-antihistamines significantly inhibited nitrite accumulation that was not caused by the scavenging ability of drugs against nitric oxide, measured amperometrically. The degree of inhibition of nitrite accumulation positively correlated with the degree of tested lipophilicity, measured by reversed-phase thin layer chromatography. Furthermore, H1-antihistamines differentially modulated the iNOS protein expression. In conclusion, as was shown in this study, the modulation of nitric oxide production could be caused by the downregulation of iNOS protein expression and/or the iNOS protein activity.International immunopharmacology 05/2009; 9(7-8):990-5. DOI:10.1016/j.intimp.2009.04.005 · 2.71 Impact Factor
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- "The measurement was practised using NO standard solutions prepared with pure NO gas. Preparation of 2 mM NO stock solution was described earlier (Feelisch and Kelm, 1991). 1 µl of NO stock solution was injected either into the 10 ml of PBS in a glass vial or into the 10 ml of PBS with the addition of 5×10 –5 M dithiaden and the signal was measured for 10 min. Then the integrals of control curve and sample curve were calculated and the scavenging activity of the dithiaden was evaluated. "
ABSTRACT: As reported in our previous studies, dithiaden (an antagonist of histamine H1-receptor, used clinically as an anti-allergic or anti-emetic drug) in a concentration range of 5×10−5–10−4 M decreased the production of reactive oxygen species by phagocytes. In this study we investigated the influence of dithiaden on nitric oxide (NO) production by LPS-stimulated macrophages. The cell viability in the presence of 10−4–5×10−5 M dithiaden was evaluated by an ATP-test. RAW 264.7 cells (2.5×106/well) were preincubated with dithiaden for 60 mins and subsequently stimulated with 0.1 µg/ml of bacterial lipopolysaccharide. After incubating for 24 hours the NO production was determined spectrophotometrically using Griess reaction as a concentration of nitrites (the end product of NO metabolism) accumulated in the cell supernatants. The expression of inducible nitric oxide synthase (iNOS) in cell-lysates was evaluated using Western blot analysis. Scavenging properties of dithiaden against NO were evaluated amperometrically. Our data demonstrate that dithiaden in the concentration of 5×10−5 M (approved by ATP test as non toxic) caused a significant decrease in the accumulation of nitrites, and in addition, this decline was followed by a marked reduction of iNOS protein expression. Amperometrical analysis did not show any scavenging properties of dithiaden against NO. From this data it can be suggested that the inhibition effect of dithiaden on macrophage NO production is caused exclusively by the suppression of iNOS protein expression.Interdisciplinary toxicology 12/2008; 1(3-4):214-7. DOI:10.2478/v10102-010-0044-9
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- "As explained by Huang et al. (2004) it is possible that the downstream effect of baicalein (on sodium nitroprusside-induced relaxation) was mediated through the inhibition of cGMP activity. It has previously been reported that sodium nitroprusside could induce relaxation by some nitric oxide-independent mechanism (Feelisch and Kelm, 1991). Therefore the possibility also exists for a non-nitric oxidedependent mechanism in explaining the effect of baicalein on sodium nitroprusside relaxation. "
ABSTRACT: Acute exposure to the flavonoid baicalein inhibited endothelium-dependent relaxation in physiological arteries, although the mechanisms are not fully understood. We investigated the effect of baicalein on vascular tone in Wistar-Kyoto (WKY) rat isolated aortic rings in the presence and absence of oxidative stress to further determine the underlying mechanisms. Exposure to baicalein (10 microM) completely abolished endothelium-dependent relaxation induced by acetylcholine and attenuated significantly the endothelium-independent relaxation induced by sodium nitroprusside. Baicalein, similar to Nomega-nitro-L-arginine methyl ester (L-NAME, 10 microM), potentiated significantly the contractile response of aortic rings to alpha1-adrenoceptor agonist phenylephrine. In the presence of L-NAME the baicalein effect on phenylphrine contraction or acetylcholine relaxation was unaltered, suggesting that these effects of baicalein are (like L-NAME effect) endothelial nitric oxide synthase (eNOS)/endothelium-derived nitric oxide-dependent. Inhibition of cyclooxygenase activity with indomethacin (10 microM) or scavenging of superoxide anions with superoxide dismutase (150 units/ml), but not scavenging of hydrogen peroxide with catalase (800 units/ml), enhanced significantly by an essentially similar extent the relaxation to acetylcholine in baicalein-pretreated aortic rings. Relaxant effect to acetylcholine was significantly attenuated in control aortic rings, but was completely abolished in baicalein-pretreated aortic rings in the presence of reduced form of beta-nicotinamide adenine di-nucleotide (beta-NADH, 300 microM). Baicalein blocked beta-NADH (300 microM)-induced transient contractions, suggesting that baicalein may have inhibited activity of NADH/NADPH-oxidase. Baicalein did not alter the failure of acetylcholine to induce relaxation in the presence of pyrogallol (300 microM). In summary, acute exposure to baicalein impairs eNOS/endothelium-derived nitric oxide-mediated vascular tone in rat aortas through the inhibition of endothelium-derived nitric oxide bioavailability coupled to reduced bioactivity of endothelium-derived nitric oxide and to cyclooxygenase-mediated release of superoxide anions.European Journal of Pharmacology 07/2007; 565(1-3):144-50. DOI:10.1016/j.ejphar.2007.03.009 · 2.68 Impact Factor