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Extracellular Nitric Oxide Release Mediates Soluble Guanylate Cyclase-Independent Vasodilator Action of Spermine NONOate: Comparison with Other Nitric Oxide Donors in Isolated Rat Femoral Arteries

Miller, M. R. PhD*; Okubo, K. MD, PhD*; Roseberry, M. J. MBChB*; Webb, D. J. MD, DSc†; Megson, I. L. PhD*

Journal of Cardiovascular Pharmacology: March 2004 - Volume 43 - Issue 3 - pp 440-451
Original Article

Nitric oxide (NO) and NO donors exhibit actions that are not entirely mediated by soluble guanylate cyclase (sGC). The site of NO release may influence the involvement of sGC-independent effects. Here we use spermine NONOate (SPER/NO) to release NO extracellularly, compared with other NO donors. Isolated rat femoral arteries were perfused luminally and perfusion pressure monitored. Vessels were contracted with phenylephrine (2–14 μM) in the presence of an NO synthase inhibitor (Nω-nitro-L-arginine methyl ester; 20 μM). Vasodilator responses to NO donors were assessed before and after perfusion of an sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ; 20 μM), NO scavengers (hemoglobin; Hb & hydroquinone; HQ), and a superoxide generator (duroquinone; DQ). ODQ (20 μM) abolished the vasodilator responses to glyceryl trinitrate (10−8–10−3 M), and sodium nitroprusside (10−8–10−4 M), which release NO intracellularly. ODQ (20 μM) attenuated, but failed to abolish, the vasodilator responses to SPER/NO (10−6–10−3 M). ODQ abolished responses to S-nitrosoglutathione and S-nitroso-N-valeryl-D-penicillamine (10−8–10−4 M), but a small residual vasodilatation remained in response to 10−3 M. In the presence of ODQ, the remaining vasodilatation to SPER/NO was all but abolished by scavengers of extracellular NO (Hb; 10 μM, HQ; 100 μM). Superoxide generation (DQ; 100 μM) also attenuated ODQ-resistant vasodilatation. The data suggest that, in rat femoral arteries, NO donors that are capable of releasing extracellular NO cause vasodilatation that is only partially mediated by sGC. Lack of augmentation of sGC-independent effects by superoxide suggests that they are not mediated by peroxynitrite.

Nitric oxide (NO) is a powerful endogenous vasodilator that inhibits platelet aggregation, smooth muscle proliferation, and platelet and monocyte adhesion to the vascular wall. 1 NO synthesis and/or availability is diminished in a number of cardiovascular pathologies. 2 Delivery of exogenous NO is an attractive therapeutic option in the management of these conditions. Organic nitrates and sodium nitroprusside (SNP) are currently used for this purpose, but both have limitations. 1

The diazeniumdiolates (NONOates) constitute a novel class of NO donor drug that decomposes spontaneously to generate up to 2 molecules of NO at physiological pH and temperature. 3 Decomposition is not catalyzed by thiols or biologic tissue, but its rate is dependent on the nucleophile adduct. (Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropyl-ammonio)butyl]-amino}-diazen-1-ium-1,2-diolate (SPER/NO) contains spermine as the nucleophile and decomposes at a relatively slow rate (half life = 39 minutes; 37°C; phosphate buffer, pH 7.4). 4,5 SPER/NO-mediated vasodilatation 4 and inhibition of platelet aggregation 6 both correlate closely with the amount of NO generated.

It is generally accepted that the vasodilator effects of NO are mediated via activation of smooth muscle cell soluble guanylate cyclase (sGC) to generate cyclic guanosine-3`,5`-monophosphate (cGMP). 7 However, vasodilatation in response to high concentrations of NO is not exclusively cGMP mediated. 8,9 Using the selective sGC-inhibitor [1H-[1,2,4] oxadiazole [4,3-a]quinoxaline-1-one (ODQ), 10 an sGC-independent component of vasodilatation has been identified in response to several NO donors. 11,12 NONOates have also been shown to have a vasodilator effect in the presence of ODQ, despite complete abolition of cGMP generation. 13,14 The identity of the sGC-independent mechanism has not been fully elucidated, although it appears that the mode of NO generation from NO donors influences their actions. 12,15

The complicated nature of NO generation from NO donors makes it difficult to draw conclusions about the nature of sGC-independent actions. In an effort to clarify the situation, the present study compared the vasodilatation to SPER/NO with that to a number of other NO donor drugs with different release mechanisms to test the hypothesis that sGC-independent activity of NO is exclusive to agents that generate NO outside target smooth muscle cells. Experiments were also designed to test the further hypothesis that sGC-independent vasodilatation is mediated by peroxynitrite.

From the Centre for Cardiovascular Science, *Hugh Robson Building and †Clinical Research Centre, University of Edinburgh, Edinburgh, Scotland, UK.

Received for publication January 6, 2003; accepted December 8, 2003.

Supported by the British Heart Foundation (PG/98123). Professor D.J. Webb was the recipient of a Research Leave Fellowship from the Wellcome Trust (WT 0526330).

Reprints: I.L. Megson, PhD, Centre for Cardiovascular Science, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, EH8 9XD, Mid-Lothian, UK (e-mail:

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Experiments were carried out on isolated segments of femoral artery from adult male Wistar rats (250–350 g; n = 93) in a perfusion system described previously. 16 Briefly, animals were killed by cervical dislocation and both femoral arteries were dissected free. Segments of artery (7–8 mm long) were cannulated immediately distal to the epigastric arterial branch. The vessels were transferred to Perspex organ chambers (1 mL volume) where they were perfused (0.6 mL min−1: Gilson miniplus 3; Anachem, Luton, UK) and superfused (1 mL min−1: Watson Marlow 302S; Watson Marlow, Falmouth, UK) with fresh oxygenated (95% O2, 5% CO2) Krebs buffer solution (composition mM: NaCl 118, NaHCO3 25, Glucose 5.7, KCl 4.7, MgSO4·7H2O 0.6, KH2PO4 1.2, CaCl2 2.5; dissolved in distilled and de-ionized water) at 37°C. The contractile state of the vessel was measured by monitoring perfusion pressure with a differential pressure transducer (T; Sensym SCX 15ANC; Farnell Electronic Components, Leeds, UK) located upstream of the artery. The apparatus permits exclusive drug delivery to the luminal surface of the vessel in the perfusate or by bolus microinjection (10 μL) through a resealable rubber septum into the perfusate immediately upstream of the vessel (transit time to artery ∼3 seconds, through lumen ∼300 milliseconds). Drugs were delivered intraluminally only, unless otherwise stated. All experiments were carried out in a darkened laboratory to protect photolabile drugs, prevent photorelaxation of vessels, 16 and minimize spontaneous superoxide generation in oxygenated Krebs buffer. 17

Vessels were preconstricted with phenylephrine (PE; 2–14 μM) in the presence of supramaximal concentrations of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 20 μM) 16 to exclude endothelium-derived NO in vasodilator responses. An l-NAME-induced increased in pressure of more than 50% of the existing PE-induced tone was indicative of an active endothelium. 16 Arteries were not denuded of endothelium to preserve the potential endothelium-dependent superoxide generating systems, 18,19 excluding superoxide generated by NO synthase, which would have been inhibited by l-NAME. 18,19

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Experimental Protocol

Sequential microinjections of increasing concentrations (10 μL; 10−8–10−3 M = 0.1 pmol–10 nmol) of NO donors were carried out before and after perfusion of modulators (1 NO donor and 1 modulator per vessel). Ferrohemoglobin (Hb, 10 μM) was used to investigate the role of extracellular NO release. 20 Hb can be S-nitrosated to form S-nitrosohemoglobin itself 21; and is also susceptible to oxidation to met Hb by ODQ. 22 For this reason, it was necessary to use an alternative NO scavenger to facilitate co-perfusion with ODQ. Hydroquinone (HQ; 100 μM) was also used to investigate extracellular NO release, as HQ was confirmed to be a direct NO scavenger in rat vascular tissue (see Results). An excess concentration (20 μM) of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used as a selective inhibitor of sGC. 10 Endogenous Cu/Zn SOD was inhibited using diethyldithiocarbamic acid(DETCA; 100 μM). 23 Duroquinone (DQ; 100 μM) 24 was perfused, following DETCA pretreatment, 25 to elevate superoxide levels further, confirmed by the NO electrode experiments.

ODQ was perfused for 20 minutes and then washed out; the irreversible nature 10,26 of inhibition of sGC with ODQ ensured activity throughout the experiment. DETCA was added to both the perfusate and superfusate for 30 minutes, and then washed out. Perfusion of Hb, HQ, and DQ began 20 to 30 minutes before administration of NO donors and was continued until the end of the experiment to ensure these drugs were present during application of NO donors. Where Hb, HQ or DQ, and ODQ were used together, ODQ was co-perfused only during the first 20 minutes of treatment and Hb, HQ, or DQ perfusion was continued alone (Fig. 1).

Following perfusion of modulators, PE concentrations were adjusted to re-establish the baseline pressure of the previous control dose-response curve, to eliminate pressure change as a possible cause of the subsequent changes in vasodilator amplitude. Following each concentration-response curve, a microinjection (10 μL) of the adenylate cyclase-activator isoprenaline (ISP; 10−3 M), was made before and after modulator perfusion, to investigate the effect of the modulator on NO:sGC-independent vasodilation.

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Nitric Oxide Electrode Measurements

Two-mL samples of Krebs buffer solution were prewarmed to 37°C in cuvettes stirred continuously at 1000 rpm. An isolated NO electrode (ISO-NO MARKII, World Precision Instruments, Stevenage, Hertfordshire, UK) was introduced into the cuvette and allowed to stabilize (10–30 minutes). Once a stable baseline was obtained, the electrode was calibrated using 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO; 100–800 nM) in phosphate buffer (pH 4). DEA/NO undergoes rapid, spontaneous decomposition when pH is less than 4. 27 Krebs buffer (pH 7.4) was used for subsequent protocols. SPER/NO (10 μM) was introduced into the cuvette and NO generation was allowed to reach plateau, before addition of either HQ (100 μM) or DQ (100 μM). Cu/Zn-SOD (250 U/ml) was added to establish the contribution of superoxide in HQ/DQ-induced changes in the NO signal. In a second set of experiments, 1-cm segments of rat aorta were homogenized in Krebs buffer (50 μL) using a micropestle. Supernatant (40 μL containing both particulate and cytosolic fractions) was removed and added to cuvettes, before addition of SPER/NO. In several experiments the aorta was pretreated with DETCA (100 μM) for 30 minutes and then thoroughly washed in normal Krebs buffer, before homogenization. Pilot experiments determined the limits for detection of NO in this system as ∼20 nM.

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Drugs and Reagents

D-SNVP was synthesized by a published method. 16 ODQ was obtained from Tocris Cookson (Langford, Bristol, UK). SPER/NO was obtained from Alexis Biochemicals (Bingham, Nottingham, UK). All other chemicals were obtained from Sigma Ltd. (Poole, Dorset, UK). Met-Hb was reduced to the Ferro-form with sodium dithionite (5-fold excess; 57.4 μM) as described previously. 16

All drugs were stored as solids and dissolved on the day of use with the exception of the NO donors and Hb, aliquots of which were stored at −70°C and used within 1 month. Isoprenaline was dissolved immediately before microinjection to prevent oxidation. All drugs were diluted in Krebs buffer or saline with the exception of ODQ and DQ, which were dissolved in dimethyl sulphoxide. The final concentration of dimethyl sulphoxide in the microinjection was less than 0.1%. SPER/NO was dissolved in 10 mM NaOH and diluted 100-fold with Krebs buffer solution to pH 8.05 immediately before microinjection. Preliminary experiments showed that, in all cases, microinjections of vehicle alone had no effect on vessel tone or NO electrode measurements.

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Analysis of Results

Signals from pressure transducers and the NO electrode were processed by a MacLab/4e analogue-digital converter and displayed through Chart™ software (AD Instruments, Sussex, UK) on a Macintosh Performa 630 microcomputer.

Vasodilator response amplitude was expressed as a percent of (PE + L-NAME)-induced pressure existing before drug delivery (% pressure change; positive values represent vasodilatation, where 100% represents complete abolition of agonist-induced tone). Preliminary experiments showed that peak dilatation correlated with the time course of vasodilatation (area under curve), regardless of the modulator used. Changes in tone induced by perfusion of modulators are expressed as a percent of perfusion pressure before drug perfusion. In the NO electrode experiments, maximum response (mV) to known concentrations of DEA/NO was used to calibrate the electrode. The effect of modulators on NO measurement was then expressed as a percent of the SPER/NO response. Mean values are given ± SEM.

P values in the text were obtained by two-factor, repeated measures analysis of variance (ANOVA), unless otherwise stated. Paired and unpaired Student t tests were all two-tailed. P < 0.05 was accepted as statistically significant.

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Preliminary Studies

Vessels were preconstricted with PE (5.0 ± 0.2 μM; n = 186) to give pressures of ∼50 mm Hg (49 ± 2 mm Hg; n = 186). l-NAME (20 μM) led to a 145 ± 8% increase in pre-existing PE-induced pressure (120 ± 3 mm Hg; n = 186).

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Vasodilator Responses to Bolus Injections of Nitric Oxide Donors

Microinjections of NO donors (10 μL; 10−8–10−3 M) produced concentration-dependent, transient vasodilatations in endothelium-intact vessels (Fig. 2). Following vasodilatation, perfusion pressure recovered fully and was not significantly different from pre-injection pressure (P > 0.05; paired Student t test; n = 6 for each NO donor). Sequential boluses of NO donors (10 μL; 10−8–10−3 M) had no effect on the magnitude of the response to subsequent microinjections. The maximum response to SNP (10−3 M) recovered slowly and, for this reason, a concentration range of 10−8 to 10−4 M was used. All NO donors exhibited a similar concentration response relationship, with the exception of SPER/NO, which was ineffective at concentrations of less than 10−6 M. Microinjections of spermine (10 μL; 10−6–10−3 M) had no vasodilator action (n = 4).

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Effect of Modulators on Baseline Pressure

There was no significant difference in perfusion pressure before and after Hb (10 μM) or ODQ (20 μM) perfusion, separately or when co-perfused (P > 0.05; n = 6, paired Student t test), confirming that the concentration of l-NAME was sufficient to abolish endothelial NO release in this system. Perfusion of DETCA (100 μM) produced a rapid increase in pressure of 26.3 ± 6.5%, which remained significantly greater than baseline after 30 minutes (P = 0.018; n = 6; paired Student t test). Washout of DETCA resulted in a fall in pressure, which often stabilized below baseline levels. HQ (100 μM) produced a significant increase in pressure of 17.5 ± 3.9% (P = 0.028; n = 6; paired Student t test). Preliminary experiments indicated that DQ caused sufficient increase in perfusion pressure to cause permanent damage to the vessel. As a precaution, PE concentration was reduced (× 0.5) before DQ perfusion, preventing a quantitative measurement from being made.

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Effect of Ferrihemoglobin on Responses to Vasodilators

Hb (10 μM) produced a significant rightwards shift in the concentration-response curve for SPER/NO (P < 0.0001; n = 6;Fig. 3A). Hb (10 μM) had no significant effect on the response to microinjections of GTN or SNP (P > 0.05; n = 6;Fig. 3C,D), but produced a significant rightwards shift in the concentration-response curves for GSNO and d-SNVP (P = 0.021 and P = 0.001; respectively; n = 6;Fig. 3E,F). Hb had no significant effect on the response to microinjection (10−3 M) of ISP (P = 0.52; n = 6; one-factor ANOVA;Fig. 3B).

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Nitric Oxide Electrode Measurements

SPER/NO (10 μM) generated NO reaching a plateau of ∼490 nM in 12 to 16 minutes. In the presence of tissue, the amplitude of the NO signal for SPER/NO was reduced to ∼170 μM. HQ (100 μM) all but abolished the NO signal in both the absence and presence of the aortic homogenate (n = 6–8). This attenuation was not reversed by SOD (250 U/ml). DQ (100 μM) produced a small attenuation (15.3 ± 3.4%; n = 9) of the NO signal from SPER/NO in the absence of tissue. However, in the presence of tissue, DQ significantly reduced the NO signal to 34.3 ± 5.6% (P < 0.001; unpaired t test; n = 10). This effect was reversed by SOD (250 U/ml;Fig. 4). SOD had no effect on the NO signal in the absence of HQ/DQ, irrespective of the presence or absence of homogenate (n = 4). Similar results were found using homogenate from DETCA-pretreated aortae (n = 8).

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Effect of DETCA and HQ on Responses to Vasodilators

DETCA (100 μM) alone had no effect on the response to microinjections of SPER/NO, GTN, SNP, GSNO, or ISP (P > 0.05; n = 6–7). However, DETCA alone produced a rightwards shift in the dose-response curve to d-SNVP (P = 0.005; n = 6). HQ (100 μM) had a similar inhibitory effect on SPER/NO-induced vasodilatation in both untreated and DETCA-treated vessels (P = 0.20; two-way factorial ANOVA; n = 6–7). Therefore, responses to vasodilators in the presence of HQ in DETCA-pretreated vessels were compared with those in vessels pretreated with DETCA alone (two-way, factorial ANOVA).

On account of the results from experiments using the NO electrode (see above), HQ (100 μM) was used as an alternative NO scavenger. Addition of HQ produced a rightward shift in the concentration-response curves for SPER/NO, GSNO, and d-SNVP (P < 0.02; n = 6–7;Fig. 5). In the presence of HQ, responses to high concentrations (>10−5 M) of GTN were significantly attenuated (P < 0.001; n = 6) with a reduction in maximal vasodilatation. DETCA and HQ perfusion had no effect on the response to SNP (P = 0.14; n = 6), or microinjections (10−3 M) of ISP (P = 0.48; one-factor ANOVA; n = 11;Fig. 5).

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Effect of DQ on Responses to Vasodilators

DQ (100 μM) produced a large attenuation of the responses to all the NO donors tested (P < 0.0001; n = 6–7;Fig. 6) and greater than the attenuation produced by HQ. DQ also produced an inhibition of isoprenaline relaxation, reducing vasodilatation from 46.8 ± 2.5% to 25.1 ± 3.0%, although this difference was not significant (10−3 M; P > 0.05; Dunnett post hoc test, following one-factor ANOVA; n = 25). NO donor relaxations of a similar amplitude showed a greater sensitivity to DQ, reducing vasodilatation to less than 15%. In addition, DQ perfusion without DETCA pretreatment still exerted a potent inhibitory effect on vasodilatations to SPER/NO, but had no effect on the vasodilator action of ISP (10−3 M; P > 0.05; Dunnett post hoc test, following one-factor ANOVA; n = 5).

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Effect of ODQ on Responses to Vasodilators

ODQ (20 μM) abolished the response to lower concentrations (< 10−5 M) of SPER/NO, GSNO, and d-SNVP. Higher concentrations of these agents produced vasodilatations that were greatly attenuated compared with control (P < 0.004; n = 6–7 for all). Responses to the maximum concentration tested (10−3 M) were reduced from 75.0 ± 1.3% to 48.2 ± 4.7% for SPER/NO, 68.9 ± 8.1% to 22.4 ± 5.8% for GSNO, and 59.0 ± 5.6% to 28.9 ± 6.7% for d-SNVP (shown on Figs. 3, 5, and 6). ODQ had no effect on the response to microinjection (10−3 M) of ISP (P = 0.53; paired t test; n = 6).

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Effect of Modulators on ODQ-Treated Vessels

To investigate the role of NO in sGC-independent effects, ODQ was co-perfused with Hb (Fig. 3), HQ (Fig. 5), or DQ following DETCA pretreatment (Fig. 6). Co-perfusion of Hb, HQ, or DQ in ODQ-treated vessels almost abolished vasodilator responses to SPER/NO, with the maximum response (10−3 M) reduced to 12.1 ± 2.1%, 8.8 ± 2.4%, 7.7 ± 2.6% respectively (all P < 0.001; n = 6). The remaining vasodilatation was not significantly different from responses to SPER/NO in vessels pretreated with DETCA, ODQ, and HQ (P = 0.46; two-way, factorial ANOVA; n = 6).

HQ and ODQ together produced a small but statistically significant attenuation of the vasodilatation produced by microinjections of GSNO and d-SNVP, compared with ODQ perfusion alone (P < 0.03; two-way, factorial ANOVA; n = 6–7;Fig. 5). The response to GSNO (10−3 M) was reduced from 22.4 ± 5.8% to 14.2 ± 3.1% and d-SNVP reduced from 28.9 ± 6.7% to 24.2 ± 4.1%.

Microinjections (10−3 M) of ISP in the presence of ODQ caused vasodilatations that were not significantly different from those to ISP in the presence of Hb, HQ, or DQ alone (10−3 M; P > 0.05; Dunnett post hoc test, following one-factor ANOVA; n = 11–25).

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Here, we show that NO donors that release NO intracellularly, GTN and SNP, cause vasodilatation of isolated rat femoral arteries exclusively via activation of sGC. In contrast, NO donors that release NO extracellularly, such as SPER/NO, induce a vasodilatation that is partially sGC independent. The sGC-independent effect is mediated by NO and attenuated by superoxide, suggesting that it is unlikely to require prior generation of peroxynitrite.

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sGC-Independent Vasodilatation and Site of Nitric Oxide Generation

SPER/NO is a diazeniumdiolate (NONOate) 4,5 and decomposes spontaneously in physiological solution to generate NO, independent of tissue factors. 3 SPER/NO is a large molecule and is unlikely to gain intracellular access, suggesting that its vasodilator action is primarily through extracellular NO release. Similarly, ferrihemoglobin is an NO scavenger that will not penetrate cell membranes, 28 but hydroquinone is a much smaller NO scavenger. Contrary to some studies, 25,29,30 but in support of others, 24,31–33 our NO electrode data (Fig. 4) show that HQ acts as a direct NO scavenger, without prior generation of superoxide. In perfused femoral arteries, the effect of HQ was similar to that of Hb, even following DETCA pretreatment, suggesting that HQ does not generate significant amounts of superoxide. Hb and HQ did not abolish the vasodilator action of SPER/NO, suggesting that a proportion of SPER/NO-derived NO reacts with extracellular factors, such as superoxide or molecular oxygen, before scavenging can occur. However, we cannot exclude the possibility that a proportion of the NO is generated intracellularly and is therefore inaccessible to Hb and HQ.

Experiments using the selective sGC inhibitor ODQ showed that SPER/NO has sGC-independent actions, which are not mediated by the parent molecules and metabolite, spermine, as spermine itself did not cause vasodilatation in this preparation. Previous studies have shown that NO donors are still able to cause vasodilatation 11,14,34 when increases in cGMP levels have been abolished by ODQ at lower concentrations (1–10 μM) than those used here (20 μM).

Alternative mechanisms of action of endogenous NO and different NO donors in blood vessels have been addressed recently 15 in a study that focused on the role of other redox forms of NO (NO, NO+) in vasodilatation. The authors concluded that redox status was not the main determinant of sGC involvement. Homer et al 12 found that NO donors requiring intracellular metabolism to generate NO (GTN, isosorbide dinitrate, and SNP), induce vasodilatation that is abolished by ODQ, whereas NO donors that do not require tissue activation to generate NO (linsidomine, NONOates) exhibit sGC-independent effects. Our findings are consistent with this view, but we go a step further and suggest that the site of NO generation is more likely to influence sGC-independent mechanisms than requirement for metabolism.

Hb and HQ caused relatively little inhibition of vasodilatation in response to GTN compared with the other NO donors, supporting the concept of intracellular activation of GTN in target smooth muscle cells. 35–37 Release of NO from SNP is often considered to be spontaneous but SNP does not decompose at physiological pH unless light, thiols, other reducing agents, or biologic tissue is present. 38 The resistance of SNP to Hb and HQ inhibition also implies intracellular decomposition. The vasodilator response to both GTN and SNP was abolished by the selective sGC-inhibitor, ODQ, suggesting that NO released intracellularly by these agents induces vasodilatation exclusively via sGC. Clearly the close proximity of sGC might account for all the NO generated, but it is possible that the intracellular milieu also lacks critical substituents necessary to mediate sGC-independent actions.

A number of factors are involved in the decomposition of S-nitrosothiols, including metal ions, thiols, and enzymes. 1 The inhibitory effect of Hb and HQ on the vasodilator actions of both GSNO and d-SNVP suggest that a proportion of NO is released at an extracellular site. GSNO and d-SNVP are less susceptible to Cu(I)-mediated decomposition in Krebs buffer than conventional S-nitrosothiols 39 and it is more likely that tissue components, such as proteins in the plasma membrane, 40,41 mediate NO release. ODQ abolished the vasodilator actions of both S-nitrosothiols at low concentrations, although a small resistant response remained at high concentrations, adding further weight to the suggestion that only a small proportion of NO is released extracellularly.

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Reactions of Nitric Oxide in Extracellular Space

There are 2 possible extracellular reactions of NO. Firstly, it can react with superoxide (O2) to form peroxynitrite (ONOO). 42 EQUATION

The concentration of superoxide is believed to be negligible in healthy vascular tissue due to the activity of antioxidant systems. 43 However, Krebs buffer contains trace metal ions and superoxide may be generated due to the continuous bubbling of oxygen. 17 Secondly, NO reacts with molecular oxygen to form higher nitrogen oxides (eg, N2O3). EQUATION

The reaction of NO with molecular oxygen is slower and second order with respect to NO, 44,45 although the reaction could be significant in oxygen-saturated buffers. 17 Therefore, both reactions are plausible, especially at the high concentrations of NO donor required to produce sGC-independent vasodilatation.

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Role of Peroxynitrite in Vasodilatation to Nitric Oxide Donors

Superoxide levels were raised to investigate the involvement of peroxynitrite in the vasodilator activity of NO donors. High levels of superoxide could affect the activity of NO-releasing compounds in several ways. Firstly, superoxide will scavenge free NO and prevent it from reacting with sGC. Secondly, the reaction of NO with superoxide will generate significant amounts of peroxynitrite, which in turn is believed to regulate cellular function through the oxidation of biologic molecules or through the nitration of tyrosine-containing proteins. 17 Thirdly, the rapid reaction rate of NO with superoxide will compete with the reaction of NO with molecular oxygen, limiting the production of higher oxides of nitrogen.

DETCA was used to enhance superoxide levels by inactivating Cu/Zn-SOD, the enzyme that dismutates superoxide in the cytoplasm. 23 The concentration and period of DETCA perfusion were minimized to limit indirect actions. 24,33 The lack of effect of DETCA on the vasodilator response to the adenylate cyclase-activator ISP suggests that its actions are restricted to the NO:sGC pathway. Superoxide levels were further enhanced using DQ as an exogenous superoxide generator. DQ auto-oxidation to generate superoxide is greatly enhanced in the presence of tissue factors. 46 Indeed, our electrode experiments showed that DQ could only reduce the amount of detectable SPER/NO-derived NO in the presence of tissue, an effect that was reversed by SOD (Fig. 3). In femoral arteries, the vasodilator response to SPER/NO was attenuated by DQ perfusion, especially in DETCA-pretreated arteries, suggesting that DQ generates superoxide in the close vicinity of tissue, which then gains access to the intracellular environment.

Similar to the findings of others, 32,47 GTN was more resistant to superoxide than other NO donors. In particular, superoxide produced a reduction in the maximum response to GTN. This profile of inhibition is unlikely to arise from competitive scavenging of NO by superoxide, and may instead reflect inhibition of the bioconversion of GTN to NO, as suggested previously. 48 Bioconversion of GTN and subsequent release of NO both occur intracellularly, suggesting that a significant proportion of the action of DQ occurs at an intracellular site. The DQ-induced increase in perfusion pressure and the small inhibitory effect on ISP, suggest that DQ has some nonspecific actions. However, increasing superoxide levels in the absence of SOD will unavoidably affect other antioxidant systems, including those related to glutathione. Interestingly, thiols like glutathione have been shown to be involved in ISP-induced vasodilatation. 49

DQ further attenuated the sGC-independent vasodilatation of SPER/NO, GSNO, and d-SNVP in ODQ-treated vessels. Under these conditions, superoxide will rapidly react with NO to form peroxynitrite. 17 Since superoxide generation did not potentiate the responses to NO donors in the presence of ODQ, it is unlikely that peroxynitrite mediates sGC-independent vasodilatation.

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Potential Nitric Oxide-Mediated sGC-Independent Mechanisms

The sGC-independent actions of SPER/NO were almost abolished by extracellular NO scavengers (Hb, HQ, DQ-derived superoxide). We hypothesize that at high concentrations of SPER/NO, sufficient NO is released extracellularly to react with molecular oxygen. This reaction can form nitrosating species that interact with sulfhydryl-containing molecules. 44,50 Nitrosation of thiol-containing residues in enzymes has been shown to regulate their function. 51–56 Therefore, S-nitrosation of thiol-containing enzymes and ion channels, including voltage-sensitive calcium channels 57 and calcium-dependent potassium channels, 14,34,58 represents a plausible sGC-independent mechanism.

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Physiological Relevance of Data

Further work is needed to ascertain the relative importance of these mechanisms in vivo. Indeed, the non-equilibrium nature of our bolus injection experiments does not preclude the possibility that the differences in cGMP-dependence that we see with SPER/NO are partly due to differences in pharmacokinetic properties of the drugs. However, parallel experiments in isolated perfused rat femoral arteries and conventional myography, in which GTN and SPER/NO were allowed to reach equilibrium, have since confirmed this fundamental finding (n = 6 for both, data not shown).

Another consideration is whether physiological concentrations of oxygen and NO are high enough to react together to produce sGC-independent effects. Levels of circulating S-nitrosothiols have been estimated to be in the nanomolar 59,60 to micromolar range. 61 However, the sGC-independent vasodilatation induced by S-nitrosothiols is small even at high concentrations and, therefore, is unlikely to significantly influence vessel tone. A far more dramatic sGC-independent component of S-nitrosothiol-mediated inhibition of platelet aggregation has been linked to the proportion of NO released extracellularly by these agents. 62 It is possible that the sizeable sGC-independent actions of S-nitrosothiols account for the platelet selectivity of these compounds. 63

The sGC-independent vasodilatation seen with SPER/NO may be mirrored by endothelium-derived NO in healthy blood vessels or in NO therapy. It should also be noted that the contribution of NO-related sGC-independent pathways may be up-regulated in cardiovascular conditions, such as hypercholesterolemia 64 or inflammatory conditions where iNOS is expressed. 65

In summary, we show that SPER/NO releases NO extracellularly and induces vasodilatation that is entirely dependent on NO, but not exclusively mediated by sGC. The sGC-independent vasodilatation to SPER/NO is not augmented—indeed it is attenuated—by superoxide generation, suggesting that these effects are unlikely to be mediated by peroxynitrite. Instead, we suggest that sGC-independent vasodilatation is mediated through the reaction of NO with molecular oxygen, forming higher nitrogen oxides that can regulate the function of thiol-containing proteins through S-nitrosation (Fig. 7).

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D-SNVP was kindly synthesized by Dr. F.A. Mazzei and Dr A.R. Butler, University of St. Andrews, St. Andrews, Fife, UK.

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guanylate cyclase; nitric oxide; NONOates; organic nitrates; S-nitrosothiols; sodium nitroprusside

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