Pre-clinical studies have identified nitroxyl (HNO), the reduced congener of nitric oxide (NO), as a potent vasodilator which is resistant to tolerance development. The present study explores the efficacy of HNO in human blood vessels and describes, for the first time, a vasodilator for humans that is not susceptible to tolerance. Human radial arteries and saphenous veins were obtained from patients undergoing coronary artery graft surgery and mounted in organ baths. Repeated vasodilator responses to the HNO donor Angeli's salt (AS) and NO donor glyceryl trinitrate (GTN) were determined. AS- and GTN-induced concentration-dependent vasorelaxation of both human radial arteries (AS pEC50: 6.5±0.2; −log M) and saphenous veins (pEC50: 6.7±0.1) with similar potency. In human radial arteries, GTN-induced relaxation was reduced by the NO scavenger hydroxocobalamin (HXC; P<0.05) but was unaffected by the HNO scavenger L-cysteine. Alternately, AS was unaffected by HXC but was reduced by L-cysteine (5-fold shift, P<0.05). The sGC (soluble guanylate cyclase) inhibitor ODQ abolished responses to both AS and GTN in arteries and veins (P<0.05). Inhibition of voltage-dependent potassium channels (Kv channels) with 4-AP also significantly reduced responses to AS (pEC50: 5.5) and GTN, suggesting that the relaxation to both redox congeners is cGMP- and Kv channel-dependent. Critically, a concentration-dependent development of tolerance to GTN (1 and 10 μM; P<0.05), but not to AS, was observed in both saphenous veins and radial arteries. Like GTN, the HNO donor AS causes vasorelaxation of human blood vessels via activation of a cGMP-dependent pathway. Unlike GTN, however, it does not develop tolerance in human blood vessels.

CLINICAL PERSPECTIVES

  • Pre-clincial studies have identified HNO, the reduced congener of NO, as a potent vasodilator which is resistant to tolerance development.

  • The present study explores the efficacy of HNO in human blood vessels and describes, for the first time, a vasodilator for humans that is not susceptible to tolerance with repeated use.

  • We thus conclude that HNO donors are a promising novel strategy in the treatment of cardiovascular disorders including angina and atherosclerosis.

INTRODUCTION

Nitrovasodilators such as glyceryl trinitrate (GTN) have been used for over 100 years for the treatment of vascular disorders, such as acute hypertensive crisis, heart failure and angina [1]. However, their therapeutic utility is limited due to the development of tolerance or diminished response with repeated use, their decreased efficacy under oxidative stress conditions and their potential cytotoxicity [2]. In the clinic, nitrate tolerance significantly limits the use of this class of nitrovasodilator and is managed by intermittent dosing or prolonged nitrate-free periods [3]. A well-studied phenomenon, tolerance is now known to be a multifactorial process attributed to impaired biotransformation of organic nitrates and sGC (soluble guanylate cyclase) signalling, increased reactive oxygen species (ROS) generation and activation of counter-regulatory pathways [3].

Donors of nitroxyl (HNO), the one-electron reduced and protonated congener of NO (nitric oxide), are novel entities with discernible advantages over NO donors [4,5]. HNO donors share many of the vasoprotective properties of NO donors including the ability to induce vasodilation, limit platelet aggregation, reduce ROS generation and inhibit vascular smooth muscle cell proliferation. Specifically, HNO serves as a vasodilator in rat, mouse, rabbit and bovine isolated large conduit arteries and in rat and mouse small resistance arteries [611]. The vasodilator activity of HNO is also apparent in vivo with HNO lowering mean arterial blood pressure in conscious dogs [12] and rats [13,14]. Interestingly, in vivo, HNO appears to serve as a preferential venodilator, yet in the setting of experimental heart failure, equivalent arterial and venous dilation in response to HNO is observed [12,15]. Like NO, HNO mediates vasorelaxation predominantly via stimulation of the sGC/cGMP signalling pathway [10]. However, HNO is also able to target vascular K+ channels to mediate vasorelaxation and hyperpolarization, including voltage-dependent potassium channels (Kv channels) in rat and mouse small mesenteric arteries [6] and ATP-sensitive potassium channels (KATP channels) in rat coronary arteries [16]. By contrast, NO has been shown to directly activate calcium-dependent potassium channels (KCa channels), as opposed to Kv channels in rat mesenteric arteries [17]. HNO is also resistant to scavenging by superoxide and does not develop tolerance either in vitro [18] or in vivo [13]. In addition, its vasoprotective actions are preserved in animal models of hypertension, hypercholesterolaemia, diabetes and heart failure [14,19]. These findings highlight the distinct pharmacology in the vascular actions between these two redox forms of NO, at least in animal experimental models of health and disease.

With the exception of studies with human platelets [20] and heart failure patients [21], the vasoprotective actions of HNO have not previously been examined in humans vessels. As such, the aim of the present study is to compare and contrast the vasorelaxant properties of the HNO donor Angeli's Salt (AS) to the clinically used nitrovasodilator GTN in human isolated radial arteries and saphenous veins.

MATERIALS AND METHODS

The Alfred Research and Ethics Unit (241/06) granted approval for all experimental procedures involving discarded human radial arteries and saphenous veins which were obtained from de-identified patients undergoing coronary artery graft surgery at the Alfred Hospital. Patient age, sex and medical histories were therefore not known.

Isolation and study of human radial arteries and saphenous veins

Vessels were placed in ice-cold Krebs solution containing 114 mM NaCl, 4.7 mM KCl, 0.8 mM KH2PO4, 1.2 mM MgCl2, 11 mM D-glucose, 25 mM NaHCO3, 2.5 mM CaCl2 and 0.026 mM EDTA, which was bubbled with carbogen (95% O2, 5% CO2). The buffer solution was changed once every hour for 3 h before they were stored at 4°C overnight. Vessels were cleared of all connective tissue and fat and cut into 3-mm rings. Each ring was suspended on two stainless steel wire hooks in 25 ml of jacketed glass organ baths containing Krebs solution gassed with carbogen. Changes in isometric tension were analysed using the Powerlab data acquisition system (Powerlab 8Sp, ADI Instruments Inc.) and recorded on a computer running chart (v4.0, ADI Instruments Inc.). The temperature was maintained at 37°C and the vessels were left to equilibrate under an initial force of 2 g for 1 h [2226].

Vascular reactivity experiments

Vessel viability was assessed using a K+ depolarizing salt solution (KPSS: 123 mM KCl, 1.17 mM MgSO4, 2.37 mM KH2PO4, 2.5 mM CaCl2, 11.1 mM D-glucose and 0.026 mM EDTA) to maximally constrict the rings. Each ring was then incubated either with a scavenger/inhibitor {i.e. L-cysteine, hydroxocobalamin (HXC), ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one), tetraethylammonium (TEA), 4-aminopyridine (4-AP)] or used as a time control. After this period each ring was constricted to approximately 50% of the KPSS value with endothelin-1 (ET-1; 1–10 nM) or noradrenaline (NA; 1–10 nM), for radial arteries and saphenous veins respectively. When responses to the constrictor plateaued, cumulative concentration–response curves (CRCs) to either the GTN or the HNO donor, AS (1 nM–10 μM) were constructed (0.5 log increments). Only one CRC was obtained in any one artery/vein.

Tolerance study

Each ring was incubated for 1 h with a concentration (0, 0.1, 1 or 10 μM) of either GTN or AS to induce tolerance in vitro [18,19,27,28]. The vessels were then washed with Krebs solution three times every 15 min for 1 h. After this period each artery/vein ring was constricted to approximately 50% of the KPSS value with ET-1/NA respectively. CRCs to either GTN or AS (1 nM–10 μM) were constructed using 0.5 log increment concentrations.

Data analysis and statistical procedures

Relaxation responses to GTN and AS were expressed as percentage reversal of the level of pre-contraction to NA or ET-1. The log EC50 and Rmax values were calculated as the concentration of relaxant required to cause 50% and the concentration required to produce the maximal relaxant response respectively. Individual relaxation curves were fitted using non-linear regression analysis (GraphPad Prism software, version 5.0) to provide an estimate of the pEC50 value (−log M). Log EC50 values were only calculated for each curve where the relaxation was near 100%, formed a maximum plateau response or was sigmoidal in nature. All results are expressed as means ± S.E.M., with the number of human subjects denoted by n and P<0.05 considered statistically significant. All statistical calculations were performed using GraphPad Prism version 5.0.

Student's unpaired t test or one-way ANOVA was used to compare mean pEC50 values between two or between three or more experimental groups respectively. A Bonferroni post-hoc analysis was performed when the ANOVA indicated statistical differences. Where pEC50 values could not be determined, a two-way ANOVA with a Bonferroni post-hoc analysis was performed.

Materials

The following suppliers were used: GTN from David Bull Laboratories. AS from Sapphire Bioscience. ET-1 from Auspep. NA, ISO, L-cysteine, HXC, ODQ, TEA and 4-AP from Sigma–Aldrich. Aliquots of drugs were made in milliQ water and stored at −20°C, except ODQ aliquots which were made in 100% ethanol and AS which was made up fresh on the day of the experiment in 0.01 M NaOH.

RESULTS

HNO induces concentration-dependent vasorelaxation of human radial arteries and saphenous veins

The NO donor GTN (1 nM–10 μM) caused concentration-dependent relaxation (pEC50=7.1±0.1, Rmax=74±6%, n=4; Figure 1A) of human radial artery pre-constricted with ET−1 (1–10 nM). Incubation with the HNO scavenger L-cysteine (3 mM) had no effect on GTN-mediated vasorelaxation, but the NO scavenger HXC (100 μM) significantly attenuated the response (Rmax=28±14%, n=4, P<0.05). By contrast, HXC had no effect on AS-mediated vasorelaxation of human radial artery (control: pEC50=6.5±0.2, Rmax=97±1%, n=8; Figure 1B), yet L-cysteine caused a 5-fold rightward shift in the relaxation response curve (pEC50=5.8±0.1, n=7, P<0.05; Figure 1B).

Determination of the redox congeners involved in the vasorelaxation to GTN and AS

Figure 1
Determination of the redox congeners involved in the vasorelaxation to GTN and AS

CRCs in human radial arteries to (A) GTN and (B) AS and in human saphenous veins to (C) GTN and (D) AS in the absence (control) and presence of the NO scavenger HXC (100 μM) and the HNO scavenger L-cysteine (3 mM). Values are expressed as percentage reversal of pre-contraction to ET-1 (A and B) or NA (C and D) and given as means ± S.E.M. where n=number of human subjects. #P<0.05, ###P<0.001 compared with control (two-way ANOVA); *P<0.05 for pEC50 compared with control (one-way ANOVA).

Figure 1
Determination of the redox congeners involved in the vasorelaxation to GTN and AS

CRCs in human radial arteries to (A) GTN and (B) AS and in human saphenous veins to (C) GTN and (D) AS in the absence (control) and presence of the NO scavenger HXC (100 μM) and the HNO scavenger L-cysteine (3 mM). Values are expressed as percentage reversal of pre-contraction to ET-1 (A and B) or NA (C and D) and given as means ± S.E.M. where n=number of human subjects. #P<0.05, ###P<0.001 compared with control (two-way ANOVA); *P<0.05 for pEC50 compared with control (one-way ANOVA).

Similarly, in human saphenous veins, GTN caused a concentration-dependent relaxation (pEC50=7.3±0.6, Rmax=84±7%, n=4, Figure 1C), which was unchanged in the presence of L-cysteine, but significantly attenuated with HXC (n=4, P<0.05, two-way ANOVA). AS-induced relaxation in human saphenous veins (pEC50=6.7±0.1, Rmax=94±1%, n=9; Figure 1D) was also diminished by treatment with L-cysteine, evidenced by a 7-fold shift to the right in CRC to AS (P<0.01) but no change in maximum response (Rmax=87±4%, n=7). As in the radial artery, HXC had no effect on the relaxation to AS (pEC50=7.1±0.2, n=7, Rmax=93±3%, n=8) in saphenous veins.

HNO-induced vasorelaxation is sGC- and Kv channel-dependent

Both the Kv channel inhibitor 4-AP (1 mM) and the KCa channel inhibitor TEA (1 mM) affected GTN-induced vasorelaxation, whereas the KATP channel inhibitor glibenclamide (10 μM) did not; 4-AP caused a 7-fold shift to the right in the CRC to GTN (pEC50=6.2±0.1, n=5, P<0.05; Figure 2A) and TEA reduced the maximum relaxation at 10 μM (Rmax=46±7%, n=4; P<0.001). The sGC inhibitor ODQ (10 μM) completely abolished the response to GTN (n=3; P<0.001). Although TEA and glibenclamide had no effect on AS-induced vasorelaxation in human radial arteries, 4-AP significantly reduced the sensitivity to AS (pEC50=5.5±0.3, n=11, P<0.05; Figure 2B) without affecting the maximum response (Rmax=85±6%, n=11, P>0.05). ODQ markedly attenuated the response to AS reducing the maximal response to 22±8% (n=11, P<0.001).

Contribution of K+ channels to the vasorelaxation to GTN and AS

Figure 2
Contribution of K+ channels to the vasorelaxation to GTN and AS

CRCs in human radial arteries to (A) GTN and (B) AS in the absence (control) and presence of the KATP channel inhibitor glibenclamide (10 μM), the Kv channel inhibitor 4-AP (1 mM), the BKCa channel inhibitor TEA (10 mM) or the sGC inhibitor ODQ (10 μM). Values are expressed as percentage reversal of pre-contraction to ET-1 and given as means ± S.E.M. where n=number of human subjects. *P<0.05 for pEC50 compared with control (one-way ANOVA); ###P<0.001 compared with control (two-way ANOVA).

Figure 2
Contribution of K+ channels to the vasorelaxation to GTN and AS

CRCs in human radial arteries to (A) GTN and (B) AS in the absence (control) and presence of the KATP channel inhibitor glibenclamide (10 μM), the Kv channel inhibitor 4-AP (1 mM), the BKCa channel inhibitor TEA (10 mM) or the sGC inhibitor ODQ (10 μM). Values are expressed as percentage reversal of pre-contraction to ET-1 and given as means ± S.E.M. where n=number of human subjects. *P<0.05 for pEC50 compared with control (one-way ANOVA); ###P<0.001 compared with control (two-way ANOVA).

Comparable results were also observed in saphenous veins pre-constricted with NA (Table 1). Kv channel inhibition with 4-AP significantly shifted the CRC to GTN and AS to the right without affecting the response to 10 μM GTN or AS. Furthermore, ODQ attenuated responses to both GTN and AS (P<0.001).

Table 1
Effect of modulators on AS- and GTN-induced vasorelaxation in human saphenous veins

pEC5O values are expressed as −log M and Rmax values as percentage reversal of the level of pre-contraction in response to 10 μM of the nitrovasodilator. NC, not calculated. Values are given as means ± S.E.M. n=4–7 per group. *P<0.05 for pEC50 compared with control.

VasodilatorGTNAS
Treatment pEC50 Rmax pEC50 Rmax 
Control 7.9±0.3 89±6 6.7±0.1 95±1 
4-AP (1 mM) 6.7±0.3* 68±8 6.0±0.3* 77±8 
ODQ (10 μM) NC 8±5 NC 43±18 
VasodilatorGTNAS
Treatment pEC50 Rmax pEC50 Rmax 
Control 7.9±0.3 89±6 6.7±0.1 95±1 
4-AP (1 mM) 6.7±0.3* 68±8 6.0±0.3* 77±8 
ODQ (10 μM) NC 8±5 NC 43±18 

Vasorelaxation to HNO is not susceptible to the development of tolerance

CRCs to GTN were constructed in radial arteries and saphenous veins after exposure to vehicle or increasing concentrations of GTN (0.1, 1 and 10 μM) for 1 h followed by a 1 h washout period. Previous exposure to 1 and 10 μM GTN in radial arteries and 10 μM GTN in saphenous veins caused concentration-dependent shifts to the right of the CRC to GTN (n=6–7, P<0.05; Figures 3A and 3B) without attenuating the maximum response.

Susceptibility to tolerance to GTN and AS

Figure 3
Susceptibility to tolerance to GTN and AS

CRCs to GTN (A and B) and AS (C and D) in human radial arteries (A and C) and saphenous veins (B and D) after 1-h incubation in either the absence (control) or the presence of GTN (A and B) or AS (C and D). Values are expressed as percentage reversal of pre-contraction to ET-1 (A and C) or NA (B and D) and given as means ± S.E.M. where n=number of human subjects. *P<0.05, **P<0.01 for pEC50 compared with control (one-way ANOVA); ###P<0.001 compared with control (two-way ANOVA).

Figure 3
Susceptibility to tolerance to GTN and AS

CRCs to GTN (A and B) and AS (C and D) in human radial arteries (A and C) and saphenous veins (B and D) after 1-h incubation in either the absence (control) or the presence of GTN (A and B) or AS (C and D). Values are expressed as percentage reversal of pre-contraction to ET-1 (A and C) or NA (B and D) and given as means ± S.E.M. where n=number of human subjects. *P<0.05, **P<0.01 for pEC50 compared with control (one-way ANOVA); ###P<0.001 compared with control (two-way ANOVA).

In contrast with the tolerance observed with GTN (Figure 3A), AS was resistant to tolerance development. Thus pre-treatment of both radial arteries and saphenous veins with increasing concentrations of AS (0.1, 1 and 10 μM) did not alter the subsequent CRCs to AS (n=3–6, P>0.05; Figures 3C and 3D).

To determine whether there was cross-tolerance between the vasodilators, saphenous veins were pre-incubated with either AS (10 μM) or GTN (10 μM) followed by a 1 h washout period. Subsequently a CRC to the opposite vasodilator was performed. There was no apparent cross-tolerance since both GTN and AS were unaffected (Figure 4A) with pre-incubation of AS and GTN respectively (Figures 4A and 4B).

Susceptibility of cross tolerance between GTN and AS

Figure 4
Susceptibility of cross tolerance between GTN and AS

CRCs to GTN (A) and AS (B) in human radial arteries after 1-h incubation in either the absence (control) or the presence of AS (A) or GTN (B). Values are expressed as percentage reversal of pre-contraction to ET-1 and given as means ± S.E.M. where n=number of human subjects.

Figure 4
Susceptibility of cross tolerance between GTN and AS

CRCs to GTN (A) and AS (B) in human radial arteries after 1-h incubation in either the absence (control) or the presence of AS (A) or GTN (B). Values are expressed as percentage reversal of pre-contraction to ET-1 and given as means ± S.E.M. where n=number of human subjects.

DISCUSSION

The present study demonstrates for the first time that the HNO donor AS causes vasorelaxation in human arteries and veins, predominantly via activation of a sGC/cGMP-dependent pathway and in part via Kv channels. Importantly, unlike GTN, it is neither susceptible to the development of tolerance, nor does it induce cross-tolerance to organic nitrates. Together these findings suggest that HNO donors may provide an improved, rational, clinical alternative to GTN.

It is well established that HNO mediates vasorelaxation of animal large conduit [7,11,18] and small resistance-like [6,10,16] arteries in vitro and in vivo [13]. However, its ability to cause relaxation in human vessels and the underlying mechanism of action had not yet been examined. Our study shows that the HNO donor AS induces concentration-dependent relaxation, with a potency similar to GTN, of human radial arteries and human saphenous veins. It has been reported that HNO preferentially dilates veins in conscious dogs [12]. However, in the present in vitro study, the potency was similar in both arteries and veins. This is supported by studies using GTN, where venous dilation is thought to account for the majority of vasodilation and the reduction in preload to the heart in patients given clinically therapeutic doses. There are limited in vitro studies assessing the effects of GTN in both arteries and veins in the same study. For those that have there was either no appreciable difference in the potency of GTN in arteries and veins [25,29] or GTN was more potent in the radial artery than the saphenous vein [30].

The ability of HXC to scavenge NO and not HNO is well established [6,11,31,32]. Likewise, the sensitivity of HNO to thiols, such as L-cysteine, has been reported extensively in the literature [6,7,10]. Consistent with this, HXC was found to reduce the sensitivity to GTN while having no significant effect on AS-mediated responses in the present study. As well, L-cysteine did not affect GTN-induced responses but attenuated the responses to AS. Therefore, through the use of these scavengers, we were able to differentiate between the actions of NO and HNO in the human vasculature and infer that GTN-mediated responses were due to NO and that AS-mediated responses were due to HNO. Since decomposition of AS releases HNO, as well as the vasorelaxant nitrite, it could be argued that nitrite is causing part of the physiological response. However, nitrite displays a potency 15000-fold less than that of AS [10] and thus is unlikely to contribute to the vasorelaxation to AS in this case. This is further supported by our observation that decomposed AS did not induce vasorelaxation in radial arteries (results not shown).

We and others have previously shown that the sGC inhibitor ODQ markedly attenuates relaxation to GTN and AS in animal arteries [6,18,33]. Consistent with this, in the present study, ODQ virtually abolished responses to GTN and AS suggesting that vasorelaxation was dependent on the sGC/cGMP pathway. Currently, there is robust debate surrounding the mechanism via which HNO activates sGC and whether it targets the ferrous (Fe2+) or NO-insensitive ferric (Fe3+) redox form of the enzyme. It was originally reported that HNO required oxidation to NO prior to sGC activation [34]. In previous studies, performed using isolated and purified sGC, Miller et al. [35] found that AS activates sGC in the absence of oxidizing agents that convert HNO into NO. However, Zeller et al. [36] found HNO needed initial conversion to NO, via superoxide dismutase, before activation of sGC. Although further work is required to determine the precise mechanism via which HNO activates sGC, these studies have indicated that HNO targets the Fe2+ form of sGC [35,36], despite its preference for Fe3+ compared with Fe2+ haem proteins [37]. Our study demonstrating that the response to AS is diminished in intact human blood vessels following pre-treatment with ODQ (which oxidizes sGC to its Fe3+ state) and in the presence of EDTA (a Cu2+ chelator which inhibits extracellular conversion of HNO to NO) is further proof that HNO may directly target the Fe2+ form of the enzyme.

HNO also causes vasorelaxation in rodent vessels through activation of Kv [6,8,10,16] and KATP [8] channels. To examine the possible involvement of Kv channels in human blood vessels, treatment with the Kv channel inhibitor 4-AP was used. Kv channel inhibition attenuated the response to AS in both vessel types. However, unlike in rodent conduit and resistance arteries, we report the novel finding that 4-AP significantly attenuates the response to GTN in human radial arteries and saphenous veins. This finding indicates that Kv channels play a role in the vasorelaxation response to both NO and HNO and suggest a common mechanism of action for these redox congeners in human blood vessels. Given that we have previously shown in rodent electrophysiological studies that HNO induces relaxation via a cGMP-dependent mechanism [16] and since the responses to both GTN and AS were abolished in the presence of ODQ, we speculate that both HNO and NO target Kv channels in a cGMP-dependent manner. The involvement of potassium channels in NO-mediated responses in animal studies has been widely reported and it appears that the type of potassium channel activated is largely species- and vessel-dependent. Furthermore, there are limited studies investigating the nature of potassium channels involved in NO-mediated relaxations in human blood vessels. However, in human saphenous veins and thoracic, placental and radial arteries, the large-conductance calcium activated potassium (BKCa) channel is thought to play a predominant role via cGMP-dependent [38] and -independent [3840] actions. Kv and KATP channel currents have also been shown to contribute to resting membrane potential in radial arteries [40] but not to hyperpolarization induced by the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP). In the present study, we show for the first time that GTN-induced vasorelaxation was dependent on both KCa and Kv channels but not KATP channels.

The NO donor GTN has been in clinical use for the treatment of angina for over a century, based primarily on its ability to cause vasodilation of arteries and veins. An intermittent dosing regimen can be implemented in patients with angina pectoris in order to avoid nitrate tolerance development to GTN [41]. Although this dosing regimen is preferred over continuous treatment, it is subject to rebound myocardial ischaemia during the nitrate-free period [42]. Rodent studies performed in our laboratory have suggested that HNO does not develop tolerance with long-term use [13]. Using a well-established acute in vitro model of nitrate tolerance [18], we demonstrated tolerance development to GTN in human radial arteries and saphenous veins, such that a concentration-dependent decrease in sensitivity to GTN was observed following pre-treatment with increasing doses of GTN. Although concentrations used to induce tolerance in the present study were markedly lower than in previous studies [18], they are clinically relevant.

Notably, using the same protocol that induced tolerance to GTN, tolerance to AS did not develop in either human radial arteries or saphenous veins. This is consistent with previous findings in the rodent vasculature both in vitro [18] and in vivo [13]. Findings from previous studies suggest that tolerance appears to be specific to GTN, as it is not evident with other NO donors such as DEA/NO (diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) [13]. Indeed, as reported in several animal studies, it appears that the impaired biotransformation of GTN to NO, by mitochondrial ALDH-2 (aldehyde dehydrogenase 2), may play a pivotal role in its tolerance development in human blood vessels [43]. Both DEA/NO and AS donate NO and HNO respectively, via spontaneous release at physiological pH and do not require enzymatic biotransformation, and this may underlie their desirable inability to develop tolerance. Although spontaneous NO donors, such as DEA/NO, do not develop tolerance, their use is still limited due to their ability to induce rebound hypertension/myocardial ischaemia and their decreased efficacy under oxidative stress conditions whereby NO is scavenged by superoxide. Thus HNO donors with a lack of tolerance development and resistance to scavenging by superoxide may prove superior over both organic nitrates and spontaneous NO donors. Further, cross-tolerance to other vasodilators has been widely reported with GTN treatment [13,44] which is thought to be due to its ability to reduce ALDH-2 activity. However, in vitro and in vivo rodent studies suggest that cross-tolerance does not occur with HNO treatment [13,18] and indeed this is also true in human radial arteries. As such, HNO donors could be used in patients who exhibit resistance to organic nitrates either alone or in conjunction with GTN, allowing the use of lower doses of GTN which may reduce the development of tolerance. Furthermore, with an ability to induce vasodilation and increase myocardial contractility, HNO donors represent a novel therapeutic strategy in the treatment of heart failure. This is further supported by the recent report that the HNO donor CXL-1020 reduced heart filling pressure and systemic vascular resistance and increased cardiac and stroke volume in patients with heart failure [21].

The present study demonstrates that, like GTN, the HNO donor AS has the ability to cause vasorelaxation in human arteries and veins. In contrast with GTN, however, AS was shown to be resistant to tolerance.

AUTHOR CONTRIBUTION

Natalie Lumsden, Julie Farry, Ann-Maree Jefferis and Karen Andrews performed the research and analysed the data. Karen Andrews, Barbara Kemp-Harper and Jaye Chin-Dusting designed the research study, interpreted the data and prepared and wrote the paper.

We gratefully acknowledge and thank Emma Harris and Margaret Vincent for their technical assistance during the study.

FUNDING

This work was supported by a programme grant from the National Health and Medical Research Council of Australia Program Grant [grant number 1036352]; and the Victorian Government's OIS (Operational Infrastructure Support) Program.

Abbreviations

     
  • 4-AP

    4-aminopyridine

  •  
  • ALDH-2

    aldehyde dehydrogenase

  •  
  • AS

    Angeli's salt

  •  
  • BKCa channel

    large-conductance calcium-activated potassium channel

  •  
  • CRC

    concentration–response curve

  •  
  • ET-1

    endothelin-1

  •  
  • GTN

    glyceryl trinitrate

  •  
  • HNO

    nitroxyl

  •  
  • HXC

    hydroxocobalamin

  •  
  • KATP channel

    ATP-sensitive potassium channel

  •  
  • KCa channel

    calcium-dependent potassium channel

  •  
  • Kv channel

    voltage-dependent potassium channel

  •  
  • NA

    noradrenaline

  •  
  • ODQ

    1H-[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one

  •  
  • ROS

    reactive oxygen species

  •  
  • sGC

    soluble guanylate cyclase

  •  
  • TEA

    tetraethylammonium

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Author notes

1

Joint senior authorship.