Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Keynes, Robert G.; Griffiths, Charmaine; Garthwaite, John

doi:10.1042/bj20020933

Cited by 49 publications

(44 citation statements)

References 38 publications

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“…Although the lower amounts of NO detected could be due to the partial release or formation of alternative nitrogen oxide species such as NO -or NO 2 , nitroxyl formation would be unlikely for the secondary amine-derived substrates involved and significant oxidation would be unexpected at the concentration levels employed. Keynes et al have reported that NO delivered via a diazeniumdiolate was consumed by reaction with ingredients, Hepes buffer and riboflavin, present in a tissue-culture medium [32]. A mechanism involving formation of superoxide radical (O 2 ▪ -) through oxidation of Hepes followed by the rapid reaction of superoxide with NO to form peroxynitrite was proposed.…”

Section: Discussionmentioning

confidence: 99%

Diazeniumdiolate reactivity in model membrane systems

et al. 2008

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The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R 1 R 2 N[N(O)NO] -, [1: R 1 = H 2 N(CH 2 ) 3 -, R 2 = H 2 N(CH 2 ) 3 NH (CH 2 ) 4 -; 2: R 1 = R 2 = H 2 N(CH 2 ) 3 -; 3: R 1 = n-butyl-, R 2 = n-butyl-NH 2 + (CH 2 ) 6 -; 4: R 1 = R 2 = n Pr-] has been examined at pH 7.4 and 37 °C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/ DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010M phosphate buffer, pH 7.4, 37 °C) at 10 mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudophase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG = 1,2

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Section: Discussionmentioning

confidence: 99%

Diazeniumdiolate reactivity in model membrane systems

et al. 2008

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“…The result is the formation of peroxynitrite, a reactive oxidizing species causing direct or radical-mediated covalent modifications to proteins and other molecules (24) and the generation of chemicals containing releasable NO (25,26). The inclusion of superoxide dismutase and urate at appropriate concentrations (22), guarded against peroxynitrite formation in our experiments. Even so, active secondary NO sources were formed when high NO concentrations were applied, particularly in Hepes buffer (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The use of Hepes buffer (used at 50 mM in the experiments of Cary et al) is already known to be hazardous. Hepes catalyses the production of superoxide anions, which rapidly combine with NO, even at the low NO concentrations activating GC-coupled receptors (22,23). The result is the formation of peroxynitrite, a reactive oxidizing species causing direct or radical-mediated covalent modifications to proteins and other molecules (24) and the generation of chemicals containing releasable NO (25,26).…”

Section: Discussionmentioning

confidence: 99%

Nitric oxide activation of guanylyl cyclase in cells revisited

Roy

Garthwaite²

2006

Proc. Natl. Acad. Sci. U.S.A.

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It was concluded that GC in vivo will display a dual regulation by NO: a long-lasting tonic activity (10 -20% of maximum) due to persistent occupation by NO of the heme binding site and phasic activity due to engagement of another unidentified, lower affinity site. The hypothesis was first tested by monitoring GC activity in rat platelets maintained in vitro and exposed to calibrated NO transients. The kinetics was as expected for a single binding site for NO (EC50 ‫؍‬ 10 nM), with activation and deactivation of enzyme activity conforming to the predictions of a simple receptor model. No tonic GC activity attributable to long-term NO binding was detected after exposure to the full range of active NO concentrations (peaking at 2-500 nM). Comparable results were obtained by using neural cells isolated from the cerebellum. After exposure to high NO concentrations, persistent GC activity could be recorded, but this activity was caused artifactually by secondary NO sources being formed in the medium. The new scheme for regulation of GC activity by NO is of doubtful relevance to cells.cerebellum ͉ cyclic GMP ͉ platelet N itric oxide (NO) functions as an intercellular signaling molecule in most tissues and participates in a diverse range of phenomena, including the regulation of blood flow, neurotransmission, and the immune response (1). Although NO may engage other mechanisms, many of the physiological effects of NO are exerted through specialized receptors possessing intrinsic guanylyl cyclase (GC) activity. In this way, NO causes the accumulation of cGMP in target cells, leading to activation of one or more downstream targets, including kinases, phosphodiesterases (PDEs), and ion channels. The best characterized GC-coupled NO receptors exist as heterodimers of ␣-and ␤-subunits. According to the conventional scheme, NO binds to a prosthetic heme group associated with the ␤-subunit, after which the bond between the heme and a nearby histidine residue breaks, causing a conformational change that propagates to the catalytic domain, greatly speeding the conversion of GTP into cGMP (2-4). Upon the removal of NO, the receptor in cells deactivates within a few hundred milliseconds (5), although when the purified protein is used, deactivation is somewhat slower (6). Nevertheless, in both cases, the existing mechanism is analogous to classical receptor activation, in which agonist binding reversibly triggers a conformational change that transduces the signal in a way that is graded with agonist concentration (7,8).Recently, a radical revision of the mechanism of GC activation by NO in vivo has been proposed on the basis of spectroscopic and enzymatic studies of purified recombinant ␣1␤1 protein (9). According to this hypothesis, NO remains firmly bound to the heme in the presence of physiological concentrations of ATP and GTP, resulting in a tonic level of GC activity amounting to 10-20% of the maximum. Rapidly reversible GC activity of the type that has been found in cells (5) and which is likely to underlie the transient fu...

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“…NO levels in the superfusate were measured continuously in a small, closed fluid reservoir immediately upstream from the recording chamber. Uric acid (30 M) was added to the superfusate to reduce the levels of NO 2 resulting from NO oxidation (Keynes et al, 2003).…”

Section: Nitric Oxide Rampmentioning

confidence: 99%

Glial Cells Dilate and Constrict Blood Vessels: A Mechanism of Neurovascular Coupling

Metea¹,

Newman²

2006

J. Neurosci.

558

584

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Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of -hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.

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Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

Cited by 49 publications

References 38 publications

Diazeniumdiolate reactivity in model membrane systems

Diazeniumdiolate reactivity in model membrane systems

Nitric oxide activation of guanylyl cyclase in cells revisited

Glial Cells Dilate and Constrict Blood Vessels: A Mechanism of Neurovascular Coupling

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