Frederick W. Flitney

NO, nitrosonium ions, nitroxide ions, nitrosothiols and iron-nitrosyls in biology: a chemist's perspective

The multiplicity of biological functions thus far attributed to NO has led to suggestions that some effects might be mediated by other, related species instead. The radical nature of NO cannot account for its cytotoxicity, but its reaction with superoxide to form peroxynitite and highly reactive hydroxyl radicals may be important in this context. The ease with which NO can react with and destroy Fe-S clusters is also an important factor. Nitrosonium and nitroxide ions can be produced in vivo and will react under conditions that are physiologically relevant. Both could, in theory, serve in cell signalling or as cytotoxic agents. More direct experimental evidence for their involvement is needed before we can confidently assign them specific biological roles. In this article, Anthony Butler, Frederick Flitney and Lyn Williams discuss the chemistry of NO and related species.

Ruthenium complexes as nitric oxide scavengers: a potential therapeutic approach to nitric oxide-mediated diseases

1 Ruthenium(III) reacts with nitric oxide (NO) to form stable ruthenium(II) mononitrosyls. Several Ru(III) complexes were synthesized and a study made of their ability to bind NO, in vitro and also in several biological systems following expression of the inducible isoform of nitric oxide synthase (iNOS). Here we report on the properties of two, related polyaminocarboxylate‐ruthenium complexes: potassium chloro[hydrogen(ethylenedinitrilo)tetraacetato]ruthenate (=JM1226; CAS no.14741‐19‐6) and aqua[hydrogen(ethylenedinitrilo)tetraacetato]ruthenium (=JM6245; CAS no.15282‐93‐6). 2 Binding of authentic NO by aqueous solutions of JM1226 yielded a product with an infrared (IR) spectrum characteristic of an Ru(II)‐NO adduct. A compound with a similar IR spectrum was obtained after reacting JM1226 with S‐nitroso‐N‐acetylpenicillamine (SNAP). 3 The effect of JM1226 or JM6245 on nitrite (NO2−) accumulation in cultures of macrophages (RAW 264 line) 18 h after stimulating cells with lipolysaccharide (LPS) and interferon‐γ (IFNγ) was studied. Activation of RAW264 cells increased NO2− levels in the growth medium from (mean±1 s.e.mean) 4.9±0.5  μM to 20.9±0.4 μM. This was blocked by actinomycin D (10 μM) or cycloheximide (5 μM). The addition of JM1226 or JM6245 (both 100 μM) to activated RAW264 cells reduced NO2− levels to 7.6±0.2 μM and 8.8±0.6 μM, respectively. NG‐methyl‐L‐arginine (L‐NMMA; 250 μM) similarly reduced NO2− levels, to 6.1±0.2 μM. 4 The effect of JM1226 or JM6245 on NO‐mediated tumour cell killing by LPS+IFNγ‐activated macrophages (RAW 264) was studied in a co‐culture system, using a non‐adherent murine mastocytoma (P815) line as the ‘target’ cell. Addition of JM1226 or JM6245 (both 100 μM) to the culture medium afforded some protection from macrophage‐mediated cell killing: target cell viability increased from 54.5±3.3% to 93.2±7.1% and 80.0±4.6%, respectively (n=6). 5 Vasodilator responses of isolated, perfused, pre‐contracted rat tail arteries elicited by bolus injections (10 μl) of SNAP were attenuated by the addition of JM1226 or JM6245 (10−4 M) to the perfusate: the ED50 increased from 6.0 μM (Krebs only) to 1.8 mM (Krebs+JM6245) and from 7 μM (Krebs only) to 132 μM (Krebs+JM1226). Oxyhaemoglobin (5 μM) increased the ED50 value for SNAP from 8 μM to 200 μM. 6 Male Wistar rats were injected with bacterial LPS (4 mg kg−1; i.p.) to induce endotoxaemia. JM1226 and JM6245 (both 100 μM) fully reversed the hyporesponsiveness to phenylephrine of tail arteries isolated from animals previously (24 h earlier) injected with LPS. Blood pressure recordings were made in conscious LPS‐treated rats using a tail cuff apparatus. A single injection of JM1226 (100 mg kg−1, i.p.) administered 20 h after LPS (4 mg kg−1, i.p.) reversed the hypotension associated with endotoxaemia. 7 The results show that JM1226 and JM6245 are able to scavenge NO in biological systems and suggest a role for these compounds in novel therapeutic strategies aimed at alleviating NO‐mediated disease states.

Iron‐sulphur cluster nitrosyls, a novel class of nitric oxide generator: mechanism of vasodilator action on rat isolated tail artery

1 Two iron‐sulphur cluster nitrosyls have been investigated as potential nitric oxide (NO·) donor drugs (A: tetranitrosyltetra‐μ3‐sulphidotetrahedro‐tetrairon; and B: heptanitrosyltri‐μ3‐thioxotetraferrate(1‐)). Both compounds are shown to dilate precontracted, internally‐perfused rat tail arteries. 2 Bolus injections (10 μl) of compound A or B generate two kinds of vasodilator response. Doses below a critical threshold concentration (DT) evoke transient (or T‐type) responses, which resemble those seen with conventional nitrovasodilators. Doses > DT produce sustained (or S‐type) responses, comprising an initial, rapid drop of pressure, followed by incomplete recovery, resulting in a plateau of reduced tone which can persist for several hours. 3 T‐ and S‐type responses are attenuated by ferrohaemoglobin (Hb) and by methylene blue (MB), but not by inhibitors of endothelial NO· synthase. Addition of either Hb or MB to the internal perfusate can restore agonist‐induced tone when administered during the plateau phase of an S‐type response. Moreover, subsequent removal of Hb causes the artery to re‐dilate fully. 4 We conclude that T‐ and S‐type responses are both mediated by NO·. It is postulated that S‐type responses represent the sum of two vasodilator components: a reversible component, superimposed upon a non‐recoverable component. The former is attributed to free NO·, preformed in solution at the time of injection; and the latter to NO· generated by gradual decomposition of a ‘store’ of iron‐sulphur‐nitrosyl complexes within the tissue. This hypothesis is supported by histochemical studies which show that both clusters accumulate in endothelial cells.

Chemical mechanisms underlying the vasodilator and platelet anti-aggregating properties of S-nitroso-N-acetyl-DL-penicillamine and S-nitrosoglutathione

The chemistries of S-nitroso-DL-penicillamine (SNAP) and S-nitrosoglutathione (GSNO) in relation to their ability to relax vascular smooth muscle and prevent platelet aggregation have been investigated. Metal ion catalysis greatly accelerates the decomposition of SNAP, but has little effect on GSNO. Instead, NO release from GSNO is effected either by NO transfer to a free thiol (e.g. cysteine), or by enzymatic cleavage of the glutamyl-cystyl peptide bond. In both cases the resulting nitrosothiol (i.e. S-nitrosocysteine and S-nitrosocystylglycine, respectively) is susceptible to metal ion catalysed NO release. We conclude that transnitrosation or enzymatic cleavage are obligatory steps in the mechanism of NO release from GSNO, whereas SNAP needs only the presence of metal ions to effect this process. The different modes of NO production may go some way towards explaining the different physiological effectiveness of these S-nitrosothiols as vasodilators and inhibitors of platelet aggregation.

Laser diffraction studies of sarcomere dynamics during ‘isometric’ relaxation in isolated muscle fibres of the frog

1. A study has been made of changes in sarcomere length and tension which occur during relaxation from isometric (‘fixed ends’) tetani in isolated muscle fibres of the frog. Sarcomere lengths were calculated from measurements of the separation of the zero‐to‐first‐order intensity maxima in diffraction patterns generated by illuminating small segments of fibre with a He—Ne laser. Diffraction spectra were recorded continuously on cine‐film using the method of ‘streak’ photography.

Neocuproine, a selective Cu(I) chelator, and the relaxation of rat vascular smooth muscle by S-nitrosothiols

A study has been made of the effect of neocuproine, a specific Cu(I) chelator, on vasodilator responses of rat isolated perfused tail artery to two nitrosothiols: S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) and S‐nitroso‐glutathione (GSNO). Bolus injections (10μl) of SNAP or GSNO (10−7–10−3m) were delivered into the lumen of perfused vessels pre‐contracted with sufficient phenylephrine (1–7μm) to develop pressures of 100–120mmHg. Two kinds of experiment were made: SNAP and GSNO were either (a) pre‐mixed with neocuproine (10−4m) and then injected into arteries; or (b) vessels were continuously perfused with neocuproine (10−5m) and then injected with either pure SNAP or GSNO. In each case, neocuproine significantly attenuated vasodilator responses to both nitrosothiols, although the nature of the inhibitory effect differed in the two types of experiment. We conclude that the ability of exogenous nitrosothiols to relax vascular smooth muscle in our ex vivo model is dependent upon a Cu(I) catalyzed process. Evidence is presented which suggests that a similar Cu(I)‐dependent mechanism is responsible for the release of NO from endogenous nitrosothiols and that this process may assist in maintaining vasodilator tone in vivo.

Selective modifiers of glutathione biosynthesis and ‘repriming’ of vascular smooth muscle photorelaxation

Photorelaxation of vascular smooth muscle (VSM) is caused by the release of nitric oxide (NO) from a finite molecular store that can be depleted by irradiating pre‐contracted arteries with visible light. The ability of an ‘exhausted’ vessel to respond to a further period of illumination is lost temporarily but then recovers slowly as the photosensitive store is reconstituted in the dark. The recovery process, termed repriming, displays an absolute requirement for endothelium‐derived NO and is inhibited by pre‐treating arteries with ethacrynic acid, a thiol‐alkylating agent. Here we demonstrate that agents that up‐ or down‐regulate glutathione (GSH) biosynthesis influence the extent to which the store is regenerated in the dark. Isolated rat tail arteries (RTAs) were perfused internally with Krebs solution containing phenylephrine (PE; mean [PE]±s.e.mean: 5.78±0.46 μM) and periodically exposed to laser light (λ=514.5 nm, 6.3 mW cm−2 for 6 min). Photorelaxations of control RTAs were compared with those from either (a) vessels taken from animals previously injected i.p. with buthionine sulphoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase (three injections, 100 mg kg−1 at 8 h intervals); or (b) isolated RTAs that were perfused ex vivo with oxothiazolidine (OXO), a precursor of cysteine (10−4 M OXO for 60 min). RTAs from BSO‐treated animals exhibited attenuated photorelaxations: the mean (±s.e.mean) amplitude of the response recorded after 72 min recovery in the dark was 12.4±1.6% versus 21.4±2.9% for control arteries (n=5; P<0.01). Conversely RTAs treated with OXO and allowed to recover for a similar period showed enhanced photorelaxations, 32.6±6.3% as compared to 21.4±2.9% for control arteries (n=5; P<0.01). A hyperbolic curve fit to repriming curves for BSO‐treated and control arteries returned asymptote values (maximum photorelaxations) of (mean±s.e.mean) 24.2±3.2% and 55.2±8.5%, respectively. The level of GSH in RTA extracts was measured by high‐pressure liquid chromatography (HPLC). Injecting animals with BSO decreased GSH to 85% of control levels (P<0.05) while treatment of isolated vessels with OXO resulted in a 31% increase above control levels (P<0.05). Thus, drug‐induced changes in RTA GSH levels were positively correlated with altered photorelaxations. The results lead us to postulate that the photosensitive store in VSM is generated, at least in part, from intracellular GSH which becomes converted to S‐nitrosoglutathione (GSNO) by nitrosating species that are formed ultimately from endothelium‐derived NO. The possible physiological significance of a photolabile store of NO in VSM is discussed briefly.

Activation of NF-κB nuclear transcription factor by flow in human endothelial cells

The tractive force generated by blood flow, called fluid shear stress, is an important regulator of endothelial cell gene expression. Several transcription factors are activated by shear stress, including members of the NF-kappaB/Rel family. The nature of the upstream-signaling components involved in the activation of NF-kappaB by flow has been studied in human endothelial cells. Flow rapidly increased endogenous IKK1/2 activity and transiently degraded IkappaBalpha and IkappaBbeta1, but not p105/p50. Nuclear translocation of the p65 subunit was induced by flow in wild-type (w/t) cells and in cells overexpressing w/t NIK, IKK1 or IKK2, but not in cells transiently transfected with kinase-inactive mutants of these enzymes. Nuclear translocation of p65 in response to flow was not affected by overexpressing a dominant-negative mutant of a MAPKKK related to NIK, called TPL2 kinase, nor by pretreating cells with the selective PKC inhibitor bisindoylmaleimide-1. Gel shift assays showed that the binding of p50/p65 heterodimer to radiolabeled oligonucleotide containing a shear-stress response element was increased by flow. The activity of a 3kappaB conA-luciferase reporter was also increased, confirming that NF-kappaB activated by flow was transcriptionally active. We conclude that shear stress induces gene transactivation by NF-kappaB (p50/p65) via the NIK-IKK1/2 pathway and proteosome-dependent degradation of IkappaB and that induction by flow does not involve TPL-2 kinase or PKC.

Evidence that Cyclic GMP may Regulate Cyclic AMP Metabolism in the Isolated Frog Ventricle

Abstract Both acetylcholine and 8-bromo cyclic GMP depress the contractile response of the isolated frog ventricle. An investigation has been made of the effects of both substances on the metabolism of endogenous 3,5 cyclic nucleotides. The levels of adenosine 3′, 5′ cyclic monophosphate (cyclic AMP) and guanosine 3′, 5′ cyclic monophosphate (cyclic GMP) were measured after superfusing preparations with varying concentrations (10 −10 to 10 −4 m ) of acetylcholine and 8-bromo cyclic GMP. The decline in contractile force was found to be accompanied by a progressive fall in intracellular cyclic AMP and a rise in cyclic GMP levels. Both the decline in contractility and the reduction in endogenous cyclic AMP are attenuated by 10 −4 theophylline. The decline in isometric twitch tension was paralleled, under all conditions, by a quantitatively equivalent reduction in the ratio cyclic AMP: cyclic GMP. The possibility that endogenous cyclic GMP may accelerate the conversion of cyclic AMP to 5 AMP, by stimulating a cyclic GMP-sensitive form of cyclic AMP phosphodiesterase, is discussed.

Adenosine depresses contractility and stimulates 3′,5′ cyclic nucleotide metabolism in the isolated frog ventricle

Adenosine depresses the contractile response of the isolated frog ventricle. An investigation has been made of its effects on the metabolism of endogenous 3′,5′ cyclic nucleotides. The levels of adenosine 3′,5′ cyclic monophosphate (3′,5′ cyclic AMP) and guanosine 3′,5′ cyclic monophosphate (3′,5′ cyclic GMP) were measured at different times during exposure of the ventricle to adenosine (10−3m). The decline in isometric twitch tension is found to be accompanied by a progressive fall in intracellular 3′,5′ cyclic AMP and a concomitant rise in 3′,5′ cyclic GMP. These effects are dose related. It is suggested that the ability of adenosine to stimulate 3′,5′ cyclic nucleotide turnover is the result of a fall in intracellular Ca2+, acting via the Ca2+-binding protein modulator, calmodulin. Interestingly, the extent to which the contractile response is depressed is found to be paralleled closely by a quantitatively equivalent reduction in the ratio 3′,5′ cyclic AMP: 3′,5′ cyclic GMP. A possible biochemical basis for the antagonistic regulatory effects of 3′,5′ cyclic AMP and 3′,5′ cyclic GMP on ventricular contractility, implied by these (and other) results, is discussed briefly.