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Cardiovascular Research 2007 73(3):587-596; doi:10.1016/j.cardiores.2006.11.018
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Copyright © 2006, European Society of Cardiology

The nitroxyl anion (HNO) is a potent dilator of rat coronary vasculature

Joanne L. Favaloro* and Barbara K. Kemp-Harper

Department of Pharmacology and Centre for Vascular Health Initiative, School of Biomedical Sciences, Monash University, Clayton Vic 3800, Australia

* Corresponding author. Tel.: +61 3 9905 4674; fax: +61 3 9905 5851. Email address: joanne.favaloro{at}med.monash.edu.au

Received 26 September 2006; revised 12 November 2006; accepted 14 November 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary and conclusions
 References
 
Objective: The nitroxyl anion (HNO) is the one-electron reduction product of NO·. This redox variant has been shown to be endogenously produced and to have effects that are pharmacologically distinct from NO·. This study investigates the vasodilator and chronotropic effects of HNO in the rat isolated coronary vasculature.

Methods: Sprague-Dawley rat hearts were retrogradely perfused with Krebs' solution (8 ml/min) using the Langendorff technique. Perfusion pressure was raised using a combination of infusion of phenylephrine and bolus additions of the thromboxane mimetic U46619 [GenBank] to attain a baseline perfusion pressure of 100–120 mm Hg. The vasodilator effects of a nitroxyl anion donor, Angeli's salt, were examined in the absence and presence of HNO and NO· scavengers, K+ channel inhibition, and soluble guanylate cyclase (sGC) inhibition. In addition, the inotropic and chronotropic effects of Angeli's salt were examined in hearts at resting perfusion pressure (50–60 mm Hg) and compared to responses evoked by acetylcholine and isoprenaline.

Results: Angeli's salt causes a potent and reproducible vasodilatation in isolated perfused rat hearts. This response is unaffected by the NO· scavenger hydroxocobalamin (0.1 mM) but is significantly inhibited by the HNO scavenger N-acetyl-L-cysteine (4 mM), suggesting that HNO is the mediator of the observed responses. Vasodilatation responses to Angeli's salt were virtually abolished in the presence of the sGC inhibitor ODQ (10 µM). The magnitude of the vasodilatation response to Angeli's salt was significantly reduced in the presence of 30 mM K+, 10 µM glibenclamide and in the presence of the calcitonin gene-related peptide (CGRP) antagonist CGRP(8–37) (0.1 µM). Angeli's salt had little effect on heart rate or force of contraction, whilst isoprenaline and acetylcholine elicited significant positive and negative cardiotropic effects, respectively.

Conclusions: The HNO donor Angeli's salt elicits a potent and reproducible vasodilatation response. The results suggest that the response is elicited by HNO through sGC-mediated CGRP release and KATP channel activation.

KEYWORDS Nitroxyl anion; Vasoactive agents; Coronary circulation; Nitric oxide; Redox signaling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Summary and conclusions
 References
 
Nitric oxide (NO) is well known as an important mediator in the control of vascular tone and blood pressure. What is not so well understood is that NO can exist in several different redox forms; as the neutral free radical NO·, the positively charged nitrosonium cation (NO+) and as the nitroxyl anion (NO–). NO– is the one-electron reduction product of NO· which under physiological conditions is present predominantly as the conjugated weak acid (HNO), which is able to cross cellular membranes [1]. The nitroxyl anion can be formed endogenously by NO synthase (NOS) [2,3] particularly in the absence of the NOS cofactor tetrahydrobiopterin (BH4). [4] In addition, HNO is produced from the uncoupled NOS product N-hydroxy-L-arginine under oxidative stress, [5] from the reduction of NO· by mitochondrial cytochrome c [6] and non-enzymatically from the reaction of S-nitrosothiols with thiols. [7]. Thus HNO has potential as an important paracrine mediator which is independent of NO·.

The biological actions of HNO can be studied using the HNO-donor compound Angeli's salt. Of interest are numerous reports that HNO dilates both large [8] and small resistance-like arteries [9], and furthermore that endogenously produced HNO contributes to endothelium-derived relaxing factor (EDRF) responses in rat [8] and mouse [10] aorta. In addition, HNO has been shown to dilate the pulmonary vasculature [11] and cause a reduction of blood pressure in vivo. [12] These studies indicate that the nitroxyl anion plays an important role in modulating vascular tone, and that this role may be exploited for useful therapeutic potential.

Exogenous HNO has been shown to cause vasorelaxation via cGMP activation [8,10] as responses to HNO are largely abolished by the sGC inhibitor ODQ, and application of NO– induces cGMP production in vascular tissues [13]. Importantly, we have recently shown that HNO can relax small-resistance-like vessels, via a cGMP- and KV channel-dependent mechanism [9] thus providing evidence for a mechanism of action of HNO that may be pharmacologically distinct from that of NO·.

Interestingly, there have been a number of reports of orthogonal effects of HNO and NO· [14,15]. For example, HNO responses are sensitive to scavenging by thiols, whereas NO· is not [16], and HNO has been shown to activate heme-proteins in the ferric (Fe3+) state, whilst NO· activates ferrous (Fe2+) proteins [17]. In addition, differing effects of HNO and NO· are marked in the cardiovascular system, particularly during cardiovascular disease. Exogenous NO· applied following ischaemic myocardial infarction has been shown to improve cardiac function [12]. Additionally, HNO has been shown to be a particularly effective vasodilator in heart failure since it increases myocardial contractility and enhances relaxation in an in vivo model of heart failure [18]. In contrast, there are limitations in the use of NO· donors for this condition. [18] The beneficial effects of HNO reported in that study were independent of cGMP, and mediated via calcitonin gene-related peptide (CGRP). This was in contrast to the actions of NO·, which acted via cGMP but not CGRP. This is further evidence that HNO and NO· have unique effects, and it has been suggested that HNO donors may represent a new class of inodilators for the treatment of heart failure [19]. However, not all reports of the vascular effects of HNO are beneficial. Exogenous HNO is reported to exacerbate cardiac damage and contribute to endothelial dysfunction in ischaemia/reperfusion injury [12].

Given that HNO donors could be a useful therapy for heart failure and hypertension, there is a need to characterise the effects of HNO when administered to the coronary circulation. The aim of this study was to specifically examine the pharmacology of the direct coronary vascular effects of the HNO donor Angeli's salt, and to determine the mechanism of these effects in the isolated-perfused rat heart preparation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Summary and conclusions
 References
 
2.1. Ethics statement
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85–23, revised 1996) and with the NHMRC (Australia) animal ethics standards. This study was approved by the Monash University Pharmacology Animal Ethics Committee.

2.2. Isolation and perfusion of rat hearts
A total of 65 male Sprague-Dawley rats, 300–400 g, were used in these studies. Following stunning and cervical dislocation the hearts (including the aortic arch) were quickly excised into ice-cold Krebs' solution (Composition in mM: NaCl 119, KCl 4.7, MgSO4.7H2O 1.17, KH2PO4 1.18, CaCl2 2.5, NaHCO3 25 and glucose 11). The aorta was then cannulated and the heart retrogradely perfused at constant flow (8 ml/min) with oxygenated Krebs solution at 37 °C, according to the method of Langendorff [20], as modified by Broadley [21]. Perfusion pressure was measured using a pressure transducer (Gould–Statham pressure transducer, USA). The hearts were then left to equilibrate for 15 min. Perfusion pressure data were collected by using the CVMS data acquisition system (Version 2.0, World Precision Instruments, USA).

2.3. Vasodilatation experiments
Following the equilibration period, baseline perfusion pressure (BPP) was 50–60 mm Hg. Perfusion pressure was then raised using a combination of infusion of phenylephrine (PE, 0.1 mM 0.5–2 ml/min) and bolus additions of the thromboxane mimetic U46619 [GenBank] (U4, 0.1–10 nmol) to attain a constricted perfusion pressure (CPP) of 100–120 mm Hg. This was necessary to generate vascular tone and thus to amplify and observe vasodilator responses. The amounts of U4 and PE were titrated to obtain a stable CPP of 100–120 mm Hg, which was matched between the control and the treatment groups. Following this, concentration-response curves to the NO– donor Angeli's salt (1 pmol–100 nmol) were constructed by adding the Angeli's salt as a bolus in log unit increments.

Angeli's salt dissociates at physiological pH as per the following equation:

N2O32–+H+->HNO+NO2–

Thus concentration response curves to sodium nitrite (NaNO2) were also carried out.

The response to Angeli's salt was then repeated in the presence of the NO· scavenger hydroxocobalamin (HXC, 0.1 mM), the NO– scavenger N-acetyl-L-cysteine (NAC, 4 mM), the soluble guanylate cyclase inhibitor ODQ (10 µM), high K+ (30 mM), the KV channel inhibitor 4-aminopyridine (4-AP, 1 mM), the KATP channel inhibitor glibenclamide (10 µM), the KIR channel inhibitor Ba2+ (30 µM), the KCa channel inhibitor combination of charybdotoxin and apamin (ChTX 10 nM and Apa 100 nM) or the CGRP receptor antagonist CGRP(8–37) (0.1 µM). All these agents were added to the Krebs solution and perfused for 15 min (30 min for ODQ and 4-AP) prior to confirming blockade with the appropriate control (i.e. sodium nitroprusside (SNP, 100 nmol), levcromakalim (LKM, 100 nmol), 1-EBIO (1 nmol) or CGRP (1 pmol)) then re-assessing the response to Angeli's salt. Only 1 inhibitor was tested in each experiment and control responses were compared with the responses obtained with the inhibitor within each heart. Appropriate time control and vehicle control experiments were also carried out. At the end of each experiment the dilator response to nifedipine (100 nmol) was obtained as an estimate of the maximum dilatation of the coronary vasculature.

2.4. Heart rate and force of contraction experiments
Direct effects of Angeli's salt (1 pmol–100 nmol) on heart rate and force of contraction were examined in a separate experimental group at baseline perfusion pressure (BPP; 50–60 mm Hg). In these experiments a hook was attached to the apex of the heart and connected to a force transducer (Grass FT03, USA) via a pulley as a measure of force of contraction and to measure heart rate. Responses to isoprenaline (Iso, 1 fmol–1 nmol) and acetylcholine (ACh, 10 pmol–10 nmol) were also measured as positive and negative controls, respectively, for cardiotropic effects. Heart rate and force of contraction data were collected by using the CVMS data acquisition system (Version 2.0, World Precision Instruments, USA).

2.5. Data analysis and statistics
Vasodilator responses to Angeli's salt, NaNO2 and CGRP were recorded as the decrease in perfusion pressure (mm Hg) from the CPP recorded immediately prior to drug addition. Individual vasodilatation curves were fitted to a sigmoidal logistic equation (Graphpad Prism®, version 4.0) and pEC50 values (concentration of agonist causing a 50% dilatation) calculated and expressed as –log M. The curve maximum (Vmax expressed in mm Hg) was also calculated. Where curves could not be fitted vasodilator responses were compared using 2-way RM ANOVA or a Student's t-test on the maximum attained vasodilatation, as indicated. Vasodilator responses to nifedipine are expressed as a percentage, normalised to the BPP following the control phase of the experiment and prior to the addition of the inhibitor. Data from the treatment phase were compared to their controls using Student's t-test or 2-way RM ANOVA, and responses to nifedipine were compared using 1-way ANOVA. These analyses were carried out using Graphpad Prism®4.0 or Sigmastat®3.1 (for the 2-way ANOVA). Heart rate and force data were compared using Student's t-test. P<0.05 was considered statistically significant. Results given in the text are mean±SEM.

2.6. Drugs and reagents
Angeli's salt, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), 9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy prostaglandin F2{alpha} (U46619 [GenBank] ), charybdotoxin (ChTX) and apamin (Apa) were all obtained from Sapphire Bioscience, Australia. Calcitonin gene-related peptide (CGRP), CGRP(8–37), 1-ethyl-2-benzimidazolinone (1-EBIO) and LKM were obtained from Tocris, Australia. NaNO2 was from Ajax Chemicals, Australia. HXC, NAC, nifedipine, PE, Iso, ACh, SNP, Glib and 4-AP were all obtained from the Sigma Chemical Co. Angeli's salt was dissolved and diluted in 0.1 M NaOH. ODQ and nifedipine were dissolved in ethanol, Glib in methanol, and U46619 [GenBank] was diluted in water. HXC, Iso, ACh, SNP, Apa, CGRP and CGRP(8–37) and 4-AP were dissolved in water and diluted in Krebs' solution if needed. NAC and PE were dissolved in Krebs' solution. ChTX was dissolved in ChTX dilution buffer, composition in mM: NaCl 100, Tris base 10, EDTA 1 in bovine serum albumin 0.1% (w/v).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Summary and conclusions
 References
 
Rats used in this study were 388±11 g body weight, 2.5±0.1 g heart weight and had a cardiac weight to body weight ratio of 0.65±0.01 (n=65). The baseline perfusion pressure (BPP) was recorded and the perfusion pressure in the presence of the constrictor agents U4 and PE (CPP) was carefully matched between the control and treatment for each group. These data are provided in Table 1.


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  		Table 1 Mean perfusion pressure values in the absence and presence of inhibitors

 
3.1. Vasodilatation to Angeli's salt in coronary vasculature
The HNO donor, Angeli's salt (10 pmol–100 nmol) caused a concentration-dependent vasodilatation (Fig. 1) which was reproducible after 30 min (n=4, Fig. 2, Tables 1 and 2Go). The vehicle for Angeli's salt (0.01 M NaOH) had no effect on the perfusion pressure (data not shown).


Figure 1
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  		Fig. 1 Reproduction of a typical trace showing the change in perfusion pressure in response to Angeli's salt. Baseline perfusion pressure (BPP) of approximately 65 mm Hg was raised to approximately 100 mm Hg (the constricted perfusion pressure (CPP)) by addition of phenylephrine (PE, 0.1 M, 1 ml/min) and U46619 (U4, 0.1 nmol bolus). The dilatation response to bradykinin (BK, 0.1 nmol bolus) indicates that the endothelium is functional. Discrete, bolus additions of Angeli's salt are indicated by the dots. Concentrations are given as log(mol).

 

Figure 2
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  		Fig. 2 Concentration response curve to Angeli's salt before ({circ}) and after (bullet) a 30 min time control period (n=4). Values are expressed as mean±SEM.

 

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  		Table 2 Effect of inhibitors on the vasodilatation response to Angeli's salt and nifedipine

 
3.2. Redox species of NO responsible for the effects of Angeli's salt
Addition of the NO· scavenger HXC (0.1 mM) or the HNO scavenger NAC (4 mM) had no effect on the BPP (Table 1). The vasodilator response to Angeli's salt was unaffected by HXC (n=4, Fig. 3A and Table 2), but was significantly inhibited by NAC (n=6, P<0.05, 2-way RM ANOVA, Fig. 3B and Table 2). The vasodilator response to Angeli's salt was also compared to that of NaNO2, which was found to be significantly weaker than Angeli's Salt (Vmax (mm Hg) Angeli's salt: 23.7±6.7, NaNO2 7.0±2.2, n=5, P<0.05, Student's paired t-test, Fig. 3C).


Figure 3
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  		Fig. 3 Concentration response curve to Angeli's salt in the absence ({circ}) and presence of (A) hydroxocobalamin (HXC, 0.1 mM, n=4, {blacktriangleup}) or (B) N-acetyl-L-cysteine (NAC, 4 mM, n=6, bullet). (C) Comparison of the concentration response curve to Angeli's salt ({circ}, n=5) and sodium nitrite ({square}, n=5). {Phi} = P<0.05, 2-way RM ANOVA, * =P <0.05, paired t-test on the response at the 0.1 µmol concentration. Values are expressed as mean±SEM.

 
3.3. Mechanism of vasodilator response to HNO
3.3.1. Role of soluble guanylate cyclase
Perfusion of the hearts with the sGC inhibitor ODQ (10 µM) caused a transient contraction in 1 of 5 experiments and slight arrhythmia in another experiment. ODQ had no effect on BPP (Table 1). The vasodilator response to Angeli's salt was virtually abolished by ODQ (n=5, P<0.01, Student's paired t-test comparing the response at 100 nmol AS, Fig. 4A, Table 2). Similarly the vasodilatation response to SNP (100 nmol) was also inhibited by ODQ (n=5, Student's paired t-test, Fig. 4B).


Figure 4
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  		Fig. 4 Concentration response curves to Angeli's salt in the absence ({circ}) and presence of (A) ODQ (10 µM, n=5, {diamondsuit}), (C) CGRP(8–37) (0.1 µM, n=6, {blacksquare}), or (E) isotonic K+ (30 mM, n=6, {blacktriangledown}). Panels on the right show the appropriate control data for the inhibitors used; (B) the response to sodium nitroprusside (SNP, 100 nmol) in the absence (open bars) and presence (shaded bars) of ODQ (10 µM, n=5) (D) the response to CGRP (100 pmol) in the absence and presence of CGRP(8–37) (0.1 µM, n=6) and (F) the response to levcromakalim (LKM, 100 nmol) in the absence and presence of isotonic K+ (30 mM, n=6). * = P<0.05, ** = P<0.01, paired t-test at the concentration indicated. {psi} = P<0.05, paired t-test on Vmax. Values are expressed as mean±SEM.

 
3.3.2. Role of CGRP
CGRP caused a potent and concentration-dependent vasodilatation of the coronary vasculature (pEC50: 10.27±0.67, Vmax: 22.1±4.7 mm Hg, n=4) which was not significantly different to the vasodilator response to Angeli's salt (pEC50: 9.02±0.16, Vmax: 36.7±12.5 mm Hg, n=4). The vasorelaxation response to CGRP (100 pmol) was inhibited by ~50% in the presence of CGRP(8–37) (0.1 µM, P<0.05, Paired t-test, Fig. 4D). In addition, the maximum vasodilator response to Angeli's salt was suppressed by ~30% in the presence of 0.1 µM CGRP(8–37) (n=5, P <0.05, Paired t-test on Vmax, Fig. 4C), but the potency of Angeli's salt-mediated vasodilatation was unaffected (Table 2).

3.3.3. Role of K+ channels
Perfusion of the hearts with isotonic 30 mM K+ solution tended to increase perfusion pressure (Table 1) although this was not statistically significant. In addition, perfusion of 30 mM K+ caused the development of spontaneous contractile activity in the coronary vasculature. The maximum vasodilator response to Angeli's salt was also inhibited in the presence of 30 mM K+, although it's potency was unaffected (n=6, P<0.05, paired t-test on Vmax, Fig. 4E, Table 2) In addition 30 mM K+ caused a significant inhibition of the vasodilation response to 100 nmol LKM (n=6, P<0.05, paired t-test, Fig. 4F).

Perfusion of the selective K+ channel inhibitors 4-AP (1 mM), Ba2+ (30 µM), the combination of charybdotoxin and apamin (ChTX 10 nM and Apa 100 nM), or glibenclamide (Glib 10 µM) each caused a significant increase in perfusion pressure (Table 1). In the presence of the KATP channel inhibitor Glib (10 µM) the vasodilator response to 100 nmol LKM was significantly attenuated (n=6, P<0.05, Paired t-test) as was the maximum vasodilatation in response to Angeli's salt (n=6, P<0.05, Paired t-test on Vmax, Fig. 5A, Table 2). 4-AP, Ba2+and the combination of ChTX and Apa each failed to affect the vasodilator response to Angeli's salt (Fig. 5B,C,D, Table 2), although ChTX and Apa inhibited the vasodilator response to the BKCa channel opener 1-EBIO (1 nmol), by 61% (n=5).


Figure 5
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  		Fig. 5 Concentration response curves to Angeli's salt in the absence ({circ}) and presence of (A) glibenclamide (Glib, 10 µM, n=6, bullet), (B) charybdotoxin (ChTX, 10 nM) in combination with apamin (Apa, 100nM, n=5, {blacktriangledown}), (C) 4-aminopyridine (4-AP, 1 mM, n=5, {blacksquare}) or (D) Ba2+ (30 µM, n=5, {blacktriangleup}). {psi} = P<0.05, paired t-test on Vmax. Values are expressed as mean±SEM.

 
3.4. Vasodilatation response to nifedipine
At the end of every vasodilatation experiment the response to 100 nmol nifedipine was recorded and expressed as a percentage of the BPP prior to inhibitor infusion. Nifedipine caused a vasodilatation of 103.8±5.7% (n=4) in the time control experiment. The response to nifedipine was unaffected in the presence of any of the inhibitors employed in this study (P>0.05, 1-way ANOVA, compared to the time control).

3.5. Effect of Angeli's salt on heart rate and force of contraction
In hearts at resting perfusion pressure (60±4 mm Hg) Angeli's salt (1 fmol–100 nmol) had no effect on heart rate (control heart rate 306±23 beats/min, {delta} heart rate with Angeli's salt (100 nmol) 7±4 beats/min, n=4, Fig. 6A), or mean force of contraction (control force 1.8±0.3 gm, {delta} force with Angeli's salt (100 nmol): 0.08±0.4 gm, n=4, Fig. 6B). However, Iso (0.1 pmol–1 nmol) caused a significant increase in heart rate (baseline heart rate 307±15 beats/min, {delta} heart rate with 1 nmol Iso: 99±14 beats/min, P<0.01, paired t-test, n=4) and force of contraction (mean force: 1.79±0.25 gm, {delta} force with Iso (0.1 nmol): 1.58±0.19 gm, P<0.05, paired t-test, n=4). ACh (10 pmol–1 nmol) caused a decrease in heart rate (baseline heart rate 312±22, {delta} heart rate with 1 nmol ACh: –46±20 beats/min) and mean force of contraction (mean force: 2.03±0.22, {delta} force with ACh (1 nmol): –0.36±0.15, n=4). The addition of higher ACh concentrations (10 nmol) caused cardiac arrest (data not shown).


Figure 6
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  		Fig. 6 Concentration response curves to Angeli's salt (bullet), isoprenaline (Iso, {triangleup}) and acetylcholine (ACh, {square}) showing (A) change in heart rate and (B) change in mean force of contraction. * = P<0.05, paired t-test at the concentration indicated. Values are expressed as mean±SEM.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Summary and conclusions
 References
 
This study is the first report of a vasodilator effect of Angeli's salt in the rat coronary resistance vasculature. The HNO donor, Angeli's salt causes a concentration-dependent vasodilatation that is reproducible and does not have inotropic or chronotropic effects. Our data indicates that the response is mediated via HNO and not Angeli's salt derived NO2– and is abolished by the sGC inhibitor ODQ. We also provide evidence that HNO induces CGRP release and KATP channel activation, most likely through a cGMP-dependent mechanism.

4.1. Redox species of NO responsible for Angeli's salt mediated effects
In this study Angeli's salt is employed as an HNO donor. Angeli's salt breaks down at physiological pH to produce equimolar amounts of HNO and NO2–, however the present study showed that NO2– had little vasodilator effect in comparison to Angeli's salt. In addition, we have previously shown that NO2– is 15,000 fold less effective that Angeli's salt as a dilator in resistance vessels [9], thus in the present study NO2– can be considered as having little contribution to the vasodilator effect of Angeli's salt.

The vasodilator response to Angeli's salt was unaffected by the NO· scavenger hydroxocobalamin, but significantly inhibited by the HNO scavenger NAC. Both these results suggest that the coronary vasodilator response to Angeli's salt is due to the HNO species. HNO can be oxidized to NO· by Cu2+-containing enzymes [16,22,23], and this was avoided in the extracellular space in the present experiments by including a Cu2+-chelator (EDTA) in the Krebs' perfusion solution. Under these conditions we have previously shown that the extracellular conversion of HNO to NO· is minimal [9]. Hydroxocobalamin only has access to the extracellular space and has no effect on Angeli's salt-mediated vasodilatation, suggesting that extracellular oxidation of HNO to NO· is not occurring. NAC, on the other hand, can access the cellular interior and would be expected to scavenge any intracellular or extracellular HNO and attenuate HNO-induced responses. Currently there is no assay for detecting HNO in vivo, or in intact cells, either directly or indirectly, nor are there any agents that prevent intracellular conversion of HNO to NO·. Thus the possibility that intracellular oxidation of HNO is occurring in the present study cannot be discounted.

4.2. Mechanism of vasodilatation elicited by Angeli's salt
The vasodilator response to Angeli's salt was profoundly attenuated by the sGC inhibitor ODQ, and partially reduced by high K+ solution and CGRP(8–37) indicating a role for sGC, K+ channels and CGRP in the vasodilator response to HNO. Whilst these agents inhibited the response to Angeli's salt they had no effect on the vasodilatation response to nifedipine. Nifedipine was chosen as a positive control for vasodilatation in these experiments as it has potent and long-lasting dilator effects via L-type Ca2+ channels, which are independent of effects at the other vasodilation pathways examined. The vasodilatation to nifedipine was unaffected by any of the inhibitor compounds used in this study, indicating that there was no non-specific impairment of vasodilatation that would interfere with our interpretation of the data.

4.2.1. Role of sGC
We have previously reported that HNO causes relaxation of non-coronary vessels via sGC and KV channel activation, [9]. HNO has also been reported to cause vasorelaxation in large conduit arteries from rat [8] and mouse [10] via a mechanism involving sGC activation and cGMP production. However, whether or not HNO can itself directly activate sGC is a matter of contention. Numerous studies have reported that ODQ attenuates the response to Angeli's salt in both vascular and non-vascular tissues, implicating a role for sGC [8,10,24,25]. The present results are consistent with this, as ODQ virtually abolished the vasodilatation response to Angeli's salt. However, it has been reported that NO· is the only form of NO that can activate sGC. [26] This suggests that HNO is intracellularly oxidized to NO· prior to activation of sGC [13]. On the other hand, the study reporting exclusive activation of cGC by NO· has been criticized [8] as the experiments examining HNO activation of sGC were carried out in the presence of thiols, which would be expected to scavenge the HNO and therefore reduce or eliminate it's effects. Thus, to date, a definite answer as to which redox state(s) of NO can activate sGC is still unavailable.

4.2.2. Role of K+ channels
In addition to the sGC-mediated effect, the vasodilator response to Angeli's salt was also attenuated by K+ channel blockade. There was a similar level of inhibition with either blockade of K+ channel with high K+ solution or with the KATP channel blocker, glibenclamide. The selective blockers of Kir, KCa and KV channels (Ba2+, ChTX in combination with Apa and 4-AP, respectively) had no effect on the Angeli's salt vasodilatation, although all three of these subtypes exist on coronary vascular smooth muscle cells [27,28]. The exclusive involvement of KATP channels in the present study is in contrast to our previous findings that HNO causes cGMP-dependent KV channel activation in rat mesenteric arteries [9]. This could be due to KV channel subtype and/or expression differences between the coronary and mesenteric vasculature.

KATP channels are well known to be involved in coronary vascular tone regulation and are present on coronary vascular smooth muscle cells [29]. In the present study sGC inhibition virtually abolishes vasodilatation to Angeli's salt, whereas glibenclamide only suppresses the maximum response, implying that the KATP channel activation is a cGMP-mediated effect.

4.2.3. Role of CGRP
CGRP is a small neuropeptide found in the heart and peri-adventitial nerve fibres throughout the coronary and peripheral vascular system [30,31]. It has prominent cardiovascular effects including vasodilatation and positive inotropy [32]. Vascular relaxation to CGRP is mediated via the endothelial CGRP1 receptors through NO-release and sGC stimulation [32] and interestingly via vascular smooth muscle cell CGRP1 receptors via cAMP and KATP channel activation [33]. Thus, given that both the CGRP antagonist CGRP(8–37) and glibenclamide inhibited the vasodilator response to Angeli's salt it is possible that the KATP channel activation occurred subsequent to CGRP release, and is cAMP-mediated, although if this was the case we would not expect the observed abolition of the response with ODQ. This could be alternative to or in addition to the cGMP-mediated KATP channel activation discussed above and at this time it is not possible to determine which mechanism(s) are responsible.

Indeed there is evidence that NO· can cause release of CGRP from NANC nerves in rat heart [34], moreover it is reported that HNO donors caused significantly greater release of CGRP than NO· donors [35]. However the exact mechanism of this NO·/HNO evoked release of CGRP is currently unknown. Considering that ODQ abolished the response to Angeli's salt, yet CGRP receptor and KATP channel blockade only suppressed the maximal effect it would seem likely that CGRP release is dependent on sGC activation. This is an important point which requires further investigation.

4.3. Cardiac effects of Angeli's salt
This study also showed that Angeli's salt had no direct inotropic or chronotropic effect on the isolated rat heart. Although our preparation could be criticised as it was not a "working heart" model, we still observed that isoprenaline caused a significant rise in heart rate and mean force of contraction, and conversely that ACh caused a decrease in rate and force. However, Angeli's salt, at concentrations that caused vasodilatation, had no effect. This is in agreement with a previous report that infusion of Angeli's salt was without effect in the isolated perfused rat heart [36], but is in contrast to another report which indicates that Angeli's salt is a positive inotrope [18,37]. This latter report was from an in vivo preparation, in a different species and this discrepancy could be explained in that our isolated preparation lacks reflex changes in heart rate or force that may occur in vivo by systemic administration of Angeli's salt.

There is considerable interest in this redox variant of NO, since there is mounting evidence that HNO acts via a pharmacological pathway that is distinct from NO·; an intriguing concept with the potential to give rise to a new class of vasodilator agents. Indeed, the potential advantage of HNO over NO· donors in heart failure has been documented [19]. Currently used dilators of coronary resistance vessels (e.g. dipyridamole or nifedipine) are limited in that they precipitate a vascular "steal", preferentially dilating non-ischaemic regions of the coronary vasculature. The effect of HNO donors under these conditions is yet to be fully determined. Further investigation of the vasodilator effects of HNO is warranted, under both physiological and pathological conditions.


   5. Summary and conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary and conclusions
References
 
The primary novel finding of this study is that HNO, the one electron reduced form of NO·, derived from Angeli's salt, causes a potent vasodilatation elicited via cGMP, CGRP release and KATP channels without any cardiotropic effects in the isolated rat heart. This is the first study to investigate the basic pharmacology of HNO-induced vasodilatation in the coronary vasculature. The data presented here further clarify the pharmacological effects of this redox variant of NO in an isolated organ preparation and support the concept that HNO donors may have potential as a new class of therapeutic vasodilators.


   Acknowledgements
 
Dr. Favaloro is an NHMRC (Australia) Peter Doherty Fellow. The authors are grateful to Assoc. Prof. Chris Sobey, Assoc Prof Robert Widdop, Dr. Rebecca Ritchie and Assoc. Prof. Owen Woodman for their assistance with this project.


   Notes
 
Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Summary and conclusions
 References
 

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