Cardiovascular Research

Cardiovascular Research 2002 53(2):405-413; doi:10.1016/S0008-6363(01)00472-2
© 2002 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Nitric oxide as a mediator of delayed pharmacological (A₁ receptor triggered) preconditioning; is eNOS masquerading as iNOS?

Robert M Bell, Christopher C.T Smith and Derek M Yellon^*

The Hatter Institute and Centre for Cardiology (Division of Medicine), University College London Medical School, Department of Cardiology, University College London Hospitals, Grafton Way, London WC1E 6DB, UK

^* Corresponding author, The Hatter Institute for Cardiovascular Studies, Department of Academic Cardiology, University College Hospital, Gower Street, London WC1E 6AU, UK. Tel.: +44-20-7380-9888; fax: +44-20-7388-5095 hatter-institute{at}ucl.ac.uk

Received 17 July 2001; accepted 6 September 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Background: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. Methods: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A₁ receptor agonist, 2-chloro N⁶ cyclopentyl adenosine (CCPA) (25 µg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. Results: WT and KO control hearts had identical infarct sizes of 37±3% and 37±2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22±2% and also, unexpectedly, in KO hearts (27±2%). This protection was abrogated with the non-specific NOS inhibitor, N nitro L-arginine methyl ester (L-NAME, 100 µM), and could be mimicked in naïve hearts with the NO donor, donor S-nitroso N-acetyl DL penicillamine (SNAP, 1 µM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO_x release in both WT and KO hearts. Conclusions: This is the first study to demonstrate a role for eNOS in delayed A₁ receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.

KEYWORDS Adenosine; Nitric oxide; Infarction; Preconditioning

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Preconditioning results in two temporally distinct phases of cardioprotection against lethal ischemic injury. The first, known as ‘classical preconditioning’, has immediate onset [1], but is of limited duration (2–3 h). The second phase, known as delayed preconditioning or ‘second window of protection’ (SWOP), results in cardioprotection 24 h after the preconditioning stimulus, with a duration of 2–3 days [2].

Whilst delayed preconditioning has been shown to be efficacious in reducing infarct size resulting from lethal ischemia, the mechanisms that trigger and mediate this protection remain unclear. Recent work by Bolli and co-workers has implicated the synthesis of nitric oxide (NO) as being an important mediator of delayed ischemic preconditioning [3,4]. Where NO production has been abrogated, the resistance to lethal ischemia is lost.

NO is synthesised by three isoforms of NO synthase (NOS) found in myocardium, catalysing the substrate, L-arginine, to L-citrulline and NO. Endothelial and neuronal NO synthases (eNOS and nNOS, respectively) are constitutively expressed NOS isoforms whose activity is regulated by cytosolic concentration of calcium and by the presence of cofactors such as tetra hydrobiopterin (BH₄), magnesium, and NADPH [5]. The inducible isoform of NO synthase (iNOS) is a calcium-independent synthase whose activity appears reliant upon protein transcription [6]. Whilst the iNOS protein is detectable at low levels in unstressed naïve myocardium [4,7] this observation is of unknown biological significance. However, iNOS is markedly induced following ischemic stress by signalling cascades resulting in the upregulation of transcription factors such as NF-B [8,9]. This protein transcription leads to upregulation of iNOS and thus NO synthesis has led many investigators to hypothesise that iNOS is the source of NO in delayed preconditioning. However, recent work by Vallance et al. in a cytokine model of vascular response to shock in man [10], has demonstrated that eNOS activity can be markedly upregulated by increased BH₄ availability and ‘masquerade’ as iNOS. The potential importance of this observation in the context of preconditioning has not been explored. Furthermore, it remains unclear whether the mechanisms that mediate delayed pharmacological preconditioning are the same as those triggered as a result of delayed ischemic preconditioning.

To investigate the comparative importance of the NOS isoforms in the mediation of delayed pharmacological preconditioning, we used the adenosine A₁ receptor agonist, 2-chloro N⁶ cyclopentyl adenosine (CCPA) as a trigger of delayed preconditioning in both mice with targeted disruption of the iNOS gene and their wild-types.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Animals
All experiments were performed on adult female B6,129 mice (3–4 months, 20–25 g). Wild type mice (WT) were compared to the iNOS knockout (KO) strain, with a targeted mutation of the iNOS gene (Jackson labs: bred under licence at University College London). Studies were undertaken in accordance with guidelines on the operation of the Animals (Scientific Procedures) Act 1986.

2.2 Drugs and chemicals
Constituents for the Krebs Henseleit buffer were purchased from BDH Laboratory supplies. CCPA, N nitro L-arginine methyl ester (L-NAME), S-nitroso N-acetyl penicillamine (SNAP) and triphenyl tetrazolium chloride (TTC) were purchased from Sigma Chemical Co.

2.3 Langendorff perfusion
Mouse heart Langendorff perfusion and protocols have been previously described [11]. In brief, mice anaesthetised and anticoagulated with 60 mg/kg sodium pentobarbitone i.p. and 100 IU heparin i.p., respectively, underwent parasternotomy. The heart and lungs were excised en-bloc and immersed in ice cold Krebs Henseleit buffer (NaCl 118 mM, NaCO₃ 24 mM, D-Glucose 10 mM, KCl 4 mM, NaH₂PO₄ 1.0, Na₂EDTA 0.5 mM, MgCl₂ 1.2 mM, CaCl₂ 2.5 mM) dissected and the aorta cannulated with a 22 gauge cannula. The hearts were then rapidly transferred to the Langendorff apparatus, and perfused retrogradely at constant pressure (110 cmH₂O). The perfusate was oxygenated with 95% O₂/5% CO₂ gas mixture (perfusate pH 7.4). A temperature probe was inserted into the right ventricle to enable the maintenance of normothermia (37°C) throughout the experiment, and a pacing electrode placed onto the left ventricle. The aortic cannula is employed as the earth electrode. Hearts are paced at 600 beats/min throughout the stabilisation and for the latter 20 min of reperfusion.

Global ischemia was achieved by the cessation of coronary flow. Normothermia was maintained by submerging the hearts in warmed (37°C) non-oxygenated Krebs Henseleit buffer. Pacing was discontinued after 5 min of ischemia.

Contractile function and resting tension were monitored with a linear force transducer (Scame model GM3), with a 3/0 silk tie through the apex, recorded on a Gould WindoGraf chart recorder.

2.4 Experimental protocols
The study consisted of five groups (summarised in Fig. 1). In groups A, B, C and E, WT and KO mice were randomly allocated to i.v. CCPA or control (0.9% saline vehicle) groups. In group C, L-NAME (100 µM) was dissolved in the perfusate and present throughout the ischemia/reperfusion protocol. In group D, naïve WT hearts were randomly assigned to normal buffer perfusion, or to perfusion with buffer containing 1 µM SNAP.

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Experimental protocols. (A) Determination of CCPA dose: 25 or 100 µg/kg i.v. administered 24 h prior to index ischemia compared to control group. (B) Saline vehicle treatment versus CCPA preconditioning in WT and KO hearts. (C) As for protocol B, with the co-infusion of L-NAME (100 µM) throughout the perfusion protocol. (D) Effect of SNAP 1 µM to iNOS KO hearts, present throughout the perfusion protocol. (E) Animals were treated with vehicle or CCPA as previously described. The hearts were isolated and Langendorff perfused for 20 min and then snap frozen for later Western blot analysis.

 
2.5 Determination of infarct size
TTC staining of hearts have previously been described [11]. The risk volume is the total ventricular volume, minus the cavity spaces. Infarct size for each heart is expressed as a percentage of risk volume.

2.6 Determination of nitric oxide activity
Nitric oxide synthase activity in the Langendorff perfused hearts were determined by measurement of the oxidised products of nitric oxide, nitrite and nitrate (NO_x), in the coronary effluent by high pressure liquid chromatography (HPLC) as described by Smith et al. [12].

2.7 In vivo measurement of blood pressure
To determine the effect of CCPA upon blood pressure, arterial pressure lines were inserted into the right common carotid artery of halothane anaesthetised, spontaneous respiring WT mice. Pressure traces were recorded on a PowerLab chart recorder via a Medex transducer dome. Pressures were recorded for 15 min prior to drug injection, and then for a subsequent 150 min.

2.8 Western blot analysis
Hearts of WT and KO mice were harvested 24 h after vehicle or CCPA as described in Fig. 1E. Total cellular protein was extracted and separated by 8% SDS/PAGE (protein loading 60 µg per well) and electrotransferred to nitrocellulose membranes (Amersham). eNOS and iNOS were detected using rabbit polyclonal antibodies (New England Biolabs) and visualised with an Enhanced ChemiLuminescence (ECL) detection kit (Amersham). Each NOS signal was normalised to the corresponding Ponceau signal [13] and presented as a proportion of WT control NOS content.

2.9 Statistical analysis
All data are expressed as means±standard error of the mean (S.E.M.). Infarct and NO_x data were compared with factorial ANOVA and Fisher's PLSD. Differences between groups were considered significant if P<0.05. Where measures were compared over a period of time, statistical significance was determined by ANOVA for repeated measures and the Bonferroni/Dunn post hoc test.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Determination of optimal CCPA dose
Whilst adenosine A₁ receptor activation has been demonstrated to elicit delayed protection against infarction in mammalian species such as rabbit [14], until recently it was unknown whether A₁ receptor agonists such as 2-chloro N⁶ cyclopentyl adenosine (CCPA) triggered delayed preconditioning in mice. Therefore, WT hearts were subjected to ischemia/reperfusion as outlined in Fig. 1A, comparing i.v. saline (vehicle), 25 µg/kg and 100 µg/kg CCPA treatment. The mice were observed during and following drug administration, their physical activity noted and recorded. Animals administered with 100 µg/kg CCPA displayed physical evidence of shock (piloerection, haunched posture and minimal provoked behaviour) lasting over 3 h. Animals receiving 25 µg/kg CCPA demonstrated none of these features, although exploratory behaviour was subdued until returning to normal within 2 h.

Both doses of CCPA resulted in equivalent significant infarct size reduction (Fig. 2A). Of note however, the hearts of animals treated with 100 µg/kg CCPA had significantly lower baseline coronary flow rates compared to vehicle and 25 µg/kg CCPA-treated animals, as indicated in Table 1. The behaviour of the high dose CCPA-treated mice suggested shock, and to confirm this diagnosis, the arterial blood pressure was measured in control, 25 µg/kg and 100 µg/kg CCPA-treated animals. The results are summarised in Fig. 2B. Both concentrations of CCPA resulted in a significant and rapid drop in mean arterial blood pressure. In the low 25 µg/kg CCPA-treated group, there was rapid blood pressure recovery (at least 80% recovery within 30 min of administration). This pressure response appears to be equivalent to the pressure trace associated with 100 µg/kg CCPA in rabbit [14]. In contrast however, the higher 100 µg/kg concentration of CCPA resulted in a markedly and significantly prolonged depression of blood pressure, requiring up to 2 h to recover a similar level of blood pressure recovery.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Comparison of low and high dose CCPA in mice. (A) CCPA infarct size dose–response curve in wild type mice. Both 25 µg/kg and 100 µg/kg resulted in equivalent infarct size reduction. **P<0.001 and *P<0.05 versus control. (B) Hemodynamic response in WT mice to i.v. 25 and 100 µg/kg CCPA, in-vivo. The higher concentration of CCPA resulted in significantly greater prolongation of mean arterial blood pressure depression. P<0.0001 100 versus 25 µg/kg treatment group.

 

View this table: [in this window] [in a new window]   Table 1 Baseline morphometric data

 
Therefore, to avoid systemic hemodynamic depression (and therefore potential unwanted cytokine induction) or coronary hemodynamic sequelae, the lower 25 µg/kg dose of CCPA was employed for the remainder of this study.

3.2 The role of iNOS in mediating delayed preconditioning
Following ischemia and reperfusion, both WT and KO hearts of animals pretreated with the saline vehicle had equivalent infarct sizes — 37±3% and 37±2%, respectively (Fig. 3). Prior CCPA administration significantly reduced infarct size in WT hearts to 22±2% and also, unexpectedly, in KO hearts to 27±2%, possibly implying that iNOS is not essential for delayed pharmacological preconditioning.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The effect of delayed pharmacological preconditioning with CCPA in both iNOS WT and KO hearts. Infarct size in control iNOS WT and KO hearts were identical. Prior administration of CCPA led to the attenuation of infarction in WT hearts and unexpectedly in KO hearts. **P<0.001, *P<0.05 versus respective WT and KO vehicle control.

 
3.3 NOS activity assessment — compensatory NOS upregulation?
The coronary effluent NO_x concentrations at the end of the 20-min stabilisation period are summarised in Fig. 4. Prior administration of CCPA resulted in a significantly increased basal release of NO_x compared to the release measured in control hearts. Mean NO_x concentrations in iNOS WT and KO hearts after 20 min of normal Langendorff perfusion were 4.84±0.68 µM and 3.72±1.94 µM, respectively, whilst in control hearts, NO_x was barely detectable. Therefore whilst iNOS may not be pivotal for delayed pharmacological preconditioning, it may be mediated by nitric oxide generated from an upregulated constitutive isoform of NOS.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Nitric oxide release into the coronary effluent. Basal NO release in control hearts is minimal, but is significantly augmented by the prior administration of CCPA 24 h earlier. **P<0.001, *P<0.05 versus respective WT and KO control. Each group represents 6 separate experiments.

 
3.4 Nitric oxide — cardioprotective mediator or epi-phenomenon?
To determine the importance of nitric oxide synthesis to this form of delayed preconditioning, two approaches were taken: (i) to determine whether attenuation of NO generation abrogated delayed preconditioning and (ii), to investigate whether exogenous NO could mimic preconditioning infarct limitation.

To abrogate all NOS activity during ischemia and reperfusion, a non-specific NOS inhibitor, L-NAME (100 µM) was added to the Krebs Henseleit buffer (protocol, Fig. 1C). Consistent with the hypothesis that NOS activity is essential for infarct sparing effect of delayed pharmacological preconditioning, L-NAME abrogated CCPA-triggered delayed protection (Fig. 5).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 The effect of L-NAME upon delayed preconditioning with CCPA. L-NAME did not increase infarct size in control hearts when administered throughout the perfusion protocol. The NOS inhibitor completely abrogated the protection conferred by prior CCPA administration implicating a role for NO in mediating delayed pharmacological preconditioning.

 
In the second phase, using naive iNOS KO hearts, the spontaneous NO donor, S-nitroso N-acetyl penicillamine (SNAP, 1 µM) was added to the perfusion buffer (protocol, Fig. 1D). This concentration was derived from the measured NO_x content of CCPA pretreated WT hearts (4.84±0.68 µM), the known coronary flow rate of iNOS knockout hearts (3.5±0.2 ml/min) and upon previously published data on the NO release properties of SNAP. SNAP donates NO with 1:1 stoichiometry at a high rate of degradation; 100 µM SNAP releases 1.4 µM NO/min at 37°C, linear over a wide temperature range [15].

Compared to control hearts perfused with the unmodified Krebs Henseleit buffer, exogenous NO administered to iNOS KO hearts reduced infarction to 21±1% from 37±3% (Fig. 6) — equivalent to that observed in CCPA-treated WT and KO hearts.

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 The effect of the administration of exogenous NO upon infarct size; 1 µM SNAP administered throughout the perfusion protocol leads to significant infarct size limitation equivalent to that observed with delayed preconditioning with CCPA. **P<0.001. The number within the bars signifies the number of animals used.

 
3.5 Protein regulation of NOS following CCPA administration
The significant synthesis of NO in the iNOS KO group that is associated with infarct size reduction implies the upregulation of a constitutively expressed NOS isoform. As eNOS isoform is widely expressed in the myocardium [16] and eNOS activity appears to be significantly upregulated in the absence of iNOS activity [10], the expression of the eNOS and iNOS isoforms were quantified. Consistent with the knockout status, no iNOS protein was expressed in either vehicle control or CCPA-treated iNOS KO groups (Fig. 7A). In the iNOS WT group, CCPA administration resulted in significant upregulation of iNOS protein content (231±17%). Significantly however, CCPA resulted in upregulation of eNOS content in both iNOS WT and iNOS KO groups (210±35% and 241±22% of iNOS WT control eNOS expression, respectively). Comparing iNOS WT and iNOS KO control animals, baseline eNOS content was marginally higher in the control iNOS KO hearts (138±19%) than that found in iNOS WT groups (100±18%), although the trend was not significant (P=0.297). Therefore, delayed pharmacological preconditioning with the adenosine A₁ agonist, CCPA, at a dose of 25 µg/kg, appears to be mediated via the upregulation of eNOS and iNOS. Thus in the absence of iNOS the eNOS may become pivotal to the protection observed.

View larger version (38K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Western blot analysis. (A) In iNOS WT hearts, the administration of CCPA 24 h earlier significantly augmented the expression of the protein (P=0.002). No iNOS protein was detectable in the iNOS KO hearts. (B) In both iNOS WT and KO hearts, the administration of CCPA significantly increased the myocardial eNOS protein content (WT, P=0.013; KO, P=0.018). The number within the bars signifies the number of animals used in the respective Western blot analysis.

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Consistent with the published literature on ischemic and pharmacological delayed preconditioning, we have demonstrated that delayed pharmacological preconditioning with CCPA appears dependent upon the generation of NO. However, where previous investigations have implicated the inducible isoform of NOS as being the obligatory source of NO, our results suggest that another isoform of NOS may have an important role in mediating protection.

The NO hypothesis for delayed preconditioning as proposed by Bolli et al. is attractive [17]. A number of recent studies, using pharmacological inhibitors of iNOS [3,18] or transgenic mice with targeted deletion of iNOS [4,19], have implicated the potential of NO in delayed preconditioning. Moreover, Bolli's group have demonstrated that transient ischemia results in protein kinase C (PKC) activation and translocation [20], activation of the transcription factor NF-B and ultimately iNOS induction and expression [8], providing further support for the hypothesis.

With this evidence in mind, we anticipated that pharmacologically triggered delayed preconditioning would also be mediated via a similar iNOS-dependent mechanism. Adenosine A₁ receptor activation has been shown to trigger delayed preconditioning in a number of animal species including rabbit [14] and rat [21]. In this study, we demonstrate that CCPA administration 24 h prior to a lethal ischemic insult does indeed result in marked reduction of infarction resulting from a lethal ischemic insult in mice, and is consistent with recently published work by Kukreja et al. [22]. However, whereas in species such as rabbit, 100 µg/kg results in a short duration depression of blood pressure [14], the time course with this concentration in mouse was found to be significantly prolonged. To emulate the short duration hemodynamic changes found in rabbit, 25 µg/kg of CCPA was found to be optimal and still trigger identical protection.

Using the lower 25 µg/kg CCPA dose, we have demonstrated that the delayed protection imbued upon the heart of WT animals is associated with increased NO_x release from the heart. This is entirely consistent with the previously proposed ‘NO' hypothesis. Remarkably however, iNOS knockout mice (with no demonstrable iNOS protein) display a similar degree of infarct limitation following CCPA administration 24 h earlier. One potential explanation for this observation is that there is a NO-independent signalling mediating protection. However, the NOS activity assay we present here appears to allude to another possibility. In hearts of saline-treated WT and KO mice there was negligible NO_x release into the coronary effluent. The NO_x release from hearts of CCPA-treated animals was significantly greater however, and similar in both WT and KO groups. Therefore we hypothesised that NO is the mediator of delayed pharmacological preconditioning, irrespective of enzymatic source — be it iNOS or another NOS isoform. That NO is the mediator of delayed protection is supported by the demonstration that CCPA-triggered protection is attenuated by the non-specific NOS inhibitor, L-NAME, and emulated by the NO donor, SNAP in naïve hearts.

If NO is a mediator of delayed CCPA-triggered preconditioning in iNOS KO hearts, then a constitutive NOS isoform is responsible for its synthesis: eNOS being the most likely candidate. Whilst eNOS may have the lowest intrinsic levels of catalytic activity relative to nNOS and iNOS [6], it nonetheless has significant capacity to be upregulated. Several mechanisms exist to augment eNOS activity, which include receptor-mediated translocation from the sarcolemmal caveoli [23,24], eNOS serine 1177 phosphorylation by Akt [25,26] and cyclic adenosine monophosphate-activated protein kinase [27,28], and HSP90 interaction with eNOS [29,30]. The potential for eNOS activity to be significantly increased following a stressful stimulus has been demonstrated by Bhagat et al. in a human vascular model of cytokine-triggered septic shock [10]. They demonstrated that eNOS was capable of generating significant quantities of NO through the induction of GTP cyclohydrolase-1 expression and activity and thus increasing the bioavailability of the co-factor, tetra hydrobiopterin (BH₄). Therefore it appears to be feasible for eNOS to emulate iNOS under certain circumstances. It is therefore attractive to postulate that the data presented here is an example of eNOS ‘masquerading’ as iNOS in delayed pharmacological preconditioning. This hypothesis appears to be supported by Western blot analysis of the protein content of hearts 24 h following CCPA administration. Whilst there is a clear increase in iNOS protein over basal expression in hearts of WT CCPA-treated mice, no iNOS protein was found in either of the iNOS KO groups. Interestingly, in both WT and KO hearts, there was also a marked and significant increase of eNOS protein content. Thus there appears to be evidence to support the hypothesis that eNOS does have a role to play in the mediation of delayed pharmacological protection.

The present study appears to contradict three recent investigations of delayed preconditioning in mice with targeted disruption of the iNOS gene. In an in vivo model using ischemia to precondition the myocardium, Bolli et al. demonstrated that delayed protection was absent in the iNOS KO [4]. Kukreja and co-workers have had similar results using both monophosphoryl lipid A (MLA) and 100 µg/kg CCPA as a trigger of delayed pharmacological preconditioning [19,22], in which ischemia/reperfusion protocol was performed in-vitro 24 h after the i.p. drug administration. There are two potential explanations for the observed difference between these studies and the data presented here. One would be the sex of the mice used in the studies where we have used female mice. However, from our observations, there is no difference between the sexes in either WT or KO mice in their infarct limiting cardioprotective response to CCPA administration (data not shown). The other explanation involves the manner in which the protection has been triggered. Both transient ischemia and MLA will have multifarious effects upon the whole animal. Both are associated with significant cytokine induction [31,32], and thus neither stimulus will be acting via a specific signalling pathway, and cytokines are classically associated with iNOS induction [33,34]. Similarly we have found 100 µg/kg CCPA to result in profound hemodynamic compromise, and could therefore be associated with non-specific activation of signalling pathways that mask the putative protective eNOS pathway in iNOS KO animals that we propose. Therefore, depending upon patterns of receptor activation, there may be differing activation of signalling cascades regulating eNOS and iNOS protein expression; these differences warrant further investigation.

In summary, we have demonstrated a model of pharmacological delayed preconditioning that is mediated by the generation of NO. From the evidence presented, we would suggest a modification to the NO hypothesis of delayed preconditioning to include the possibility of NO generation from other isoforms of NOS. The implication that eNOS NO generation may be upregulated sufficiently to provide protection against infarction may be of clinical importance, as well as providing a potential target for future drug development.

Time for primary review 28 days.

	Acknowledgements

 
This work was supported by a grant from the British Heart Foundation. We would like to thank Valerie Taylor from Patrick Vallence's Laboratory for her technical assistance in the measurement of in-vivo blood pressure monitoring.

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