Nitric oxide (NO) signal transduction may involve at least two targets: the guanylyl cyclase-coupled NO receptor (NO GC R), which catalyzes cGMP formation, and cytochrome c oxidase, which is responsible for mitochondrial O 2 consumption and which is inhibited by NO in competition with O 2 . Current evidence indicates that the two targets may be similarly sensitive to NO, but quantitative comparison has been difficult because of an inability to administer NO in known, constant concentrations. We addressed this deficiency and found that purified NO GC R was about 100-fold more sensitive to NO than reported previously, 50% of maximal activity requiring only 4 nM NO. Conversely, at physiological O 2 concentrations (20 -30 M), mitochondrial respiration was 2-10-fold less sensitive to NO than estimated beforehand. The two concentration-response curves showed minimal overlap. Accordingly, an NO concentration maximally active on the NO GC R (20 nM) inhibited respiration only when the O 2 concentration was pathologically low (50% inhibition at 5 M O 2 ). Studies on brain slices under conditions of maximal stimulation of endogenous NO synthesis suggested that the local NO concentration did not rise above 4 nM. It is concluded that under physiological conditions, at least in brain, NO is constrained to target the NO GC R without inhibiting mitochondrial respiration. Nitric oxide (NO)1 is a diffusible biological messenger that subserves cell-to-cell signaling functions in most tissues. NO can also be cytotoxic and has been incriminated in many different pathologies, including atherosclerosis, septic shock, cancer, and neurodegenerative disorders (1). Although much has been learned about the mechanism of NO synthesis (2), the transduction pathways engaged by physiological NO signals to modify cell and tissue function remain to be clearly defined.The established target is the guanylyl cyclase-coupled receptor, or NO GC R, 2 which exists in at least two different heterodimeric isoforms (␣1␤1 and ␣2␤1). This is a metabotropic type of receptor equipped with a heme prosthetic group to which NO binds, triggering the formation of cGMP from GTP in the cyclase domain of the protein. Through this route, NO elicits many effects such as smooth muscle relaxation, inhibition of platelet aggregation, and synaptic plasticity (3, 4). Knowledge of the NO concentrations that engage the NO GC R is important for understanding the receptor kinetics, for informing on the physiological NO concentrations likely to exist in tissues, and for developing realistic models of NO signaling. Currently, however, the information on this issue is incoherent. Studies on the purified ␣1␤1 receptor protein have suggested that the NO concentration giving half-maximal activation (the EC 50 ) is 250 nM (5). More recently, an EC 50 of 1.6 M has been obtained for the enzyme in an extract of rat aorta (6). The validity of this range appears to be supported by several studies that have used the NONOate, diethylamine/NO adduct (DEA/NO), which degrades to release NO with ...

To function as such, biological signalling molecules need to be inactivated. In the case of nitric oxide (NO), which serves as an intercellular messenger throughout the body (Moncada et al. 1991), much has been learnt during the last decade about the synthetic pathway, but how the molecule is disposed of under physiological conditions remains unknown.NO is generated in cells from L-arginine and O 2 by NO synthases. Two isoforms, neuronal and endothelial (nNOS and eNOS) are constitutively expressed and typically become activated transiently as a result of a rise in cytosolic Ca 2+ . A third type, the inducible NO synthase (iNOS), can be expressed in many different cell types after exposure to inflammatory or proinflammatory mediators and this isoform manufactures NO continuously (Stuehr, 1999). Once produced, NO diffuses rapidly in three dimensions to elicit biological actions in neighbouring cells. Physiological NO signal transduction occurs through binding to the haem group of soluble guanylyl cyclase (sGC), leading to enzyme activation and cGMP accumulation (Waldman & Murad, 1987;Ignarro, 1991). However, NO can also contribute to tissue pathology by inhibiting mitochondrial respiration and promoting the generation of reactive free radicals (Gross & Wolin, 1995;Clementi et al. 1998;Heales et al. 1999). The rate of inactivation of NO will govern, inter alia, how far NO spreads within a tissue and at what concentrations (Wood & Garthwaite, 1994), and so is expected to be a critical determinant of whether NO acts as a physiological signal or as a toxin.The chemical reactivity of NO has been considered to be one means of disposal. NO can react with O 2 (a process termed autoxidation) but this is far too slow at the concentrations existing in vivo to be of relevance (Ford et al. 1993;Kharitonov et al. 1994). A much more rapid reaction is with superoxide ions, but the resulting peroxynitrite anion is highly toxic, making it unlikely that this would serve as the primary physiological pathway (Beckman & Koppenol, 1996 1. The functioning of nitric oxide (NO) as a biological messenger necessitates that there be an inactivation mechanism. Cell suspensions from a rat brain region rich in the NO signalling pathway (cerebellum) were used to investigate the existence of such a mechanism and to determine its properties.2. The cells consumed NO in a manner that could not be explained by reaction with O 2 , superoxide ions or contaminating red blood cells. Functionally, the mechanism was able to convert constant rates of NO formation into low steady-state NO concentrations. For example, with NO produced at 90 nM min _1, the cells (20 w 10 6 ml _1) held NO at 20 nM. Various other cell types behaved similarly.3. The influence of NO inactivation on the ability of NO to access its receptor, soluble guanylyl cyclase, was explored by measuring cGMP accumulation in response to the clamped NO concentrations. The extrapolated steady-state EC 50 for NO was 2 nM, a concentration readily achieved by low NO release rates, despite inactivatio...

The signaling molecule nitric oxide (NO) could engage multiple pathways to influence cellular function. Unraveling their relative biological importance has been difficult because it has not been possible to administer NO under the steady-state conditions that are normally axiomatic for analyzing ligand-receptor interactions and downstream signal transduction. To address this problem, we devised a chemical method for generating constant NO concentrations, derived from balancing NO release from a NONOate donor with NO consumption by a sink. On theoretical grounds, 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) was selected as the sink. The mixture additionally contained urate to convert an unwanted product of the reaction (NO 2 ) into nitrite ions. The method enabled NO concentrations covering the physiological range (0 -100 nM) to be formed within approximately 1 s. Moreover, the concentrations were sufficiently stable over at least several minutes to be useful for biological purposes. When applied to the activation of guanylyl cyclase-coupled NO receptors, the method gave an EC 50 of 1.7 nM NO for the protein purified from bovine lung, which is lower than estimated previously using a biological NO sink (red blood cells). The corresponding values for the ␣1␤1 and ␣2␤1 isoforms were 0.9 nM and 0.5 nM, respectively. The slopes of the concentrationresponse curves were more shallow than before (Hill coefficient of 1 rather than 2), questioning the need to consider the binding of more than one NO molecule for receptor activation. The discrepancies are ascribable to limitations of the earlier method. Other biological problems can readily be addressed by adaptations of the new method.

IntroductionInflammation and dysregulated immune responses play a crucial role in atherosclerosis, underlying ischaemic heart disease (IHD) and acute coronary syndromes (ACSs). Immune responses are also major determinants of the postischaemic injury in myocardial infarction. Regulatory T cells (CD4+CD25+FOXP3+; Treg) induce immune tolerance and preserve immune homeostasis. Recent in vivo studies suggested that low-dose interleukin-2 (IL-2) can increase Treg cell numbers. Aldesleukin is a human recombinant form of IL-2 that has been used therapeutically in several autoimmune diseases. However, its safety and efficacy is unknown in the setting of coronary artery disease.Method and analysisLow-dose interleukin-2 in patients with stable ischaemic heart disease and acute coronary syndromes is a single-centre, first-in-class, dose-escalation, two-part clinical trial. Patients with stable IHD (part A) and ACS (part B) will be randomised to receive either IL-2 (aldesleukin; dose range 0.3–3×106IU) or placebo once daily, given subcutaneously, for five consecutive days. Part A will have five dose levels with five patients in each group. Group 1 will receive a dose of 0.3×106IU, while the dose for the remaining four groups will be determined on completion of the preceding group. Part B will have four dose levels with eight patients in each group. The dose of the first group will be based on part A. Doses for each of the subsequent three groups will similarly be determined after completion of the previous group. The primary endpoint is safety and tolerability of aldesleukin and to determine the dose that increases mean circulating Treg levels by at least 75%.Ethics and disseminationThe study received a favourable opinion by the Greater Manchester Central Research Ethics Committee, UK (17/NW/0012). The results of this study will be reported through peer-reviewed journals, conference presentations and an internal organisational report.Trial registration numberNCT03113773; Pre-results.

NO functions ubiquitously as a biological messenger but has also been implicated in various pathologies, a role supported by many reports that exogenous or endogenous NO can kill cells in tissue culture. In the course of experiments aimed at examining the toxicity of exogenous NO towards cultured cells, we found that most of the NO delivered using a NONOate (diazeniumdiolate) donor was removed by reaction with the tissue-culture medium. Two NO-consuming ingredients were identified : Hepes buffer and, under laboratory lighting, the vitamin riboflavin. In each case, the loss of NO was reversed by the addition of superoxide dismutase. The effect of Hepes was observed over a range of NONOate concentrations (producing up to 1 µM NO). Furthermore, from measurements of soluble guanylate cyclase activity, Hepes-dependent NO consumption remained significant at the low nanomolar NO concentrations relevant to physiological NO