Short-term hypoxic vasodilation in vivo is mediated by bioactive nitric oxide metabolites, rather...

J Physiol 592.5 (2014) pp 1061–1075 1061

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Short-term hypoxic vasodilation in vivo is mediated bybioactive nitric oxide metabolites, rather than free nitricoxide derived from haemoglobin-mediated nitritereduction

Michele Umbrello1,2, Alex Dyson1, Bernardo Bollen Pinto1, Bernadette O. Fernandez3, Verena Simon1,4,Martin Feelisch3 and Mervyn Singer1

1Bloomsbury Institute of Intensive Care Medicine, Division of Medicine, University College London, UK2Department of Pathophysiology & Transplantation, Universita degli Studi di Milano, Italy3University of Southampton School of Medicine, Southampton General Hospital, Tremona Road, Southampton, UK4Department of Anaesthesiology and Intensive Care, RWTH Aachen University, Aachen, Germany

Key points

� Hypoxia increases blood flow through vasodilation, an adaptive response that increases oxygenavailability to tissues.

� This response is probably mediated by nitric oxide (NO); different mechanisms have beenpostulated (increased de novo synthesis, release from preformed stores, reduced inactivation),but precise mechanisms remain controversial.

� In a short-term rodent model, hypoxaemia was associated with hypotension and lower plasmanitrite levels suggestive of nitrite bioactivation; this was only partially reversed by NO synthaseinhibition. Administration of sodium nitrite produced marked vasodilation and increasednitrosylation. Scavenging of NO had little effect.

� Early hypoxic vasodilation is mediated by a complex interaction of multiple NO-related species,rather than by NO from haemoglobin-mediated reduction of nitrite per se.

� NO and its metabolites play a pivotal role in the body’s initial adaptation to an acute decreasein oxygen supply, probably offering protection against a supply–demand mismatch.

Abstract Local increases in blood flow – ‘hypoxic vasodilation’ – confer cellular protectionin the face of reduced oxygen delivery. The physiological relevance of this response is wellestablished, yet ongoing controversy surrounds its underlying mechanisms. We sought to confirmthat early hypoxic vasodilation is a nitric oxide (NO)-mediated phenomenon and to study putativepathways for increased levels of NO, namely production from NO synthases, intravascular nitritereduction, release from preformed stores and reduced deactivation by cytochrome c oxidase.Experiments were performed on spontaneously breathing, anaesthetized, male Wistar rats under-going short-term systemic hypoxaemia, who received pharmacological inhibitors and activatorsof the various NO pathways. Arterial blood pressure, cardiac output, tissue oxygen tension andthe circulating pool of NO metabolites (oxidation, nitrosation and nitrosylation products) weremeasured in plasma and erythrocytes. Hypoxaemia caused a rapid and sustained vasodilation,which was only partially reversed by non-selective NO synthase inhibition. This was associatedwith significantly lower plasma nitrite, and marginally elevated nitrate levels, suggestive of nitritebioinactivation. Administration of sodium nitrite had little effect in normoxia, but producedsignificant vasodilation and increased nitrosylation during hypoxaemia that could not be reversed

M. Umbrello and A. Dyson have contributed equally to this work.

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society DOI: 10.1113/jphysiol.2013.255687

) at UCL Library Services on April 1, 2014jp.physoc.orgDownloaded from J Physiol (

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1062 M. Umbrello and others J Physiol 592.5

by NO scavenging. Methodological issues prevented assessment of the contribution, if any, ofreduced deactivation of NO by cytochrome c oxidase. In conclusion, acute hypoxic vasodilationis an adaptive NO-mediated response conferred through bioactive metabolites rather than freeNO from haemoglobin-mediated reduction of nitrite.

(Received 24 March 2013; accepted after revision 29 December 2013; first published online 6 January 2014)Corresponding author M. Singer: Bloomsbury Institute of Intensive Care Medicine, University College London,Cruciform Building, Gower Street, London WC1E 6BT, UK. Email: [email protected]

Abbreviation ABP, arterial blood pressure; C-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide;NOS, nitric oxide synthase; POE-Hb, pyridoxylated haemoglobin poly-oxoethylene conjugate; RNNO, N-nitrosamines;RSNO, S-nitrosothiols; RXNO, sum of nitrosation products; SEITU, S-ethyl-isothiourea; sGC, soluble guanylatecyclase; SVR, systemic vascular resistance

Introduction

Mammals crucially require oxygen (O2) as the terminalelectron acceptor in the mitochondrial respiratory chainto provide sufficient energy for cell metabolism, organfunction and survival (Brand, 2005). Any seriousimbalance between O2 delivery and consumption can leadto a state of energetic crisis (Taylor & Pouyssegur, 2007).This is a critical factor underlying cell death (Brunelle &Chandel, 2002) and organ failure (Rixen & Siegel, 2005).

Several complementary mechanisms work in concert toprotect the tissues in the face of reduced O2 delivery. Theseinclude measures to facilitate O2 availability such as localincreases in blood flow – ‘hypoxic vasodilation’ (Roy &Brown, 1880; Dreyer, 1926; Hackel et al. 1954; Heistad& Wheeler, 1970; Rowell & Blackmon, 1987, 1989),with flow redistribution to ‘vital’ organs and increasedrecruitment of perfused microvessels (Tsai et al. 2003). Inparallel, additional strategies are employed that includedecreasing metabolic requirements to match energysupply and demand, upregulating glycolytic generation ofATP and modifying substrate availability and utilization,for example through the hormonal response to acute stress(Semenza, 2007).

Nitric oxide (NO) is a key determinant of myocardialfunction and vascular tone. It is essential for both globalregulation and regional distribution of blood flow andpressure (Welch & Loscalzo, 1994). NO also plays acrucial role in energy regulation and metabolism throughits modulatory effects on mitochondrial activity, andon protein function through nitrosylation, nitrosationor nitration (Clementi et al. 1999; Jobgen et al. 2006;Cooper & Giulivi, 2007). NO levels in blood or tissuesrepresent a balance between production/release andmetabolism/bioinactivation.

NO probably plays a key role in the body’s earlyadaptation to an acute energy supply–demand mismatch.However, considerable controversy persists with respectto its sources and mechanisms of action (Umbrello etal. 2013). Using a short-term rodent model of systemichypoxaemia, we sought to confirm that acute hypoxic

vasodilation is a NO-mediated phenomenon, and to studythe contribution of putative pathways known or suspectedto enhance NO bioactivity, namely increased productionfrom NO synthases (NOS), increased reduction of nitriteby red blood cells, increased release from preformedstorage forms of NO and reduced deactivation bymitochondrial cytochrome c oxidase.

Methods

Male Wistar rats of approximately 300 g body weight wereused in all experiments. Animals were purchased fromCharles River (Margate, Kent, UK) and housed in cagesof four on an alternating 12 h light–dark cycle, with freeaccess to chow (2018 rodent diet; Harlan Teklad, Madison,WI, USA) and tap water. Before instrumentation, animalswere allowed to acclimatize to the local environmentfor at least 1 week. All experiments were performedaccording to Home Office (UK) guidelines under the 1986Animal (Scientific Procedures) Act with University CollegeLondon Ethics Committee approval.

Instrumentation and monitoring

Spontaneously breathing animals were placed into a trans-parent plastic container and anaesthetized by 5% iso-flurane (Baxter Healthcare, Thetford, Norfolk, UK) inroom air. Following abolition of the righting reflex,animals were placed in the supine position on to a heatedmat to maintain rectal temperature at 37°C. Under 2–2.5%isoflurane, the left common carotid artery and right inter-nal jugular vein were located, isolated and cannulatedusing 0.96 mm outside diameter PVC tubing catheter(Biocorp Ltd, Huntingdale, WA, Australia). The arterialline was connected to a pressure transducer (Powerlab;AD Instruments, Chalgrove, Oxon, UK) for continuousmonitoring of arterial blood pressure (ABP). The venousline was used for subsequent administration of fluids anddrugs. A tracheostomy was sited using 2.08 mm externaldiameter polyethylene tubing (Portex Ltd, Hythe, Hants,

C© 2014 The Authors. The Journal of Physiology C© 2014 The Physiological Society




J Physiol 592.5 Nitric oxide and hypoxic vasodilation 1063

UK) to secure and suction the airways. This was connectedto a T-piece to maintain anaesthesia and to vary thefraction of inspired oxygen.

Continuous monitoring of muscle tissue PO2 (tPO2)levels was obtained by the use of Large Area Surface(LASTM) oxygen sensors (0.7 mm diameter) connected tothe OxyliteTM tissue monitoring system (Oxford Optronix,Abingdon, Oxon, UK), as described in detail elsewhere(Dyson et al. 2007). Briefly, the sensor sends shortpulses of light (475 nm) along a fibreoptic cable to aplatinum–complex fluorophore situated at the probe tip.This provides an 8 mm2 surface area in contact withthe tissue for tPO2 measurement. Upon interaction withoxygen, the fluorophore emits light (600 nm) back tothe detection unit, the lifetime of which is inverselyproportional to the local PO2 concentration withinthe tissue of interest. Unlike polarographic techniques,oxygen is not consumed during the measurement process;moreover, as the fluorescence decay is longer at lowervalues of PO2, highly accurate measurements can bemade in a hypoxic environment. A small incision wasmade at mid-thigh level, and the sensor inserted intothe vastus lateralis muscle to a depth of 10 mm usingan 18-gauge cannula for guidance (VenflonTM; BectonDickenson, Franklin Lakes, NJ, USA). The sensor was thenwithdrawn by 2 mm to prevent erroneous measurementsresulting from local haematoma. This was verified byseeing a rapid response in tPO2 following a change ininspired O2. ABP and muscle tPO2 measurements werecontinuously monitored and recorded on to a computerusing a Powerlab system and Chart 5.0 acquisition software(AD Instruments).

Isoflurane anaesthesia was reduced to 1.2% for theremainder of the experiment. Post-instrumentation, intra-vascular volume optimization was achieved by repeated0.5 ml intravenous challenges of 0.9% saline givenover 10 s every 5 min until ABP failed to increase>10%. A continuous infusion of 0.9% saline was thenadministered at a rate of 10 ml kg−1 h−1 for the durationof the experiment to ensure adequate filling conditionsthroughout. All animals were allowed to stabilize for atleast 30 min to achieve constant baseline physiologicalvariables before the beginning of the experiment.

At predefined time points, arterial blood samples(0.2 ml) were collected into heparinized capillary tubesfor blood gas analysis (ABL-625 analyser; Radiometer,Copenhagen, Denmark). Blood (0.5 ml) was also collectedinto tubes containing N-ethylmaleimide (10 mM) andEDTA (2.5 mM) to prevent artificial thiol nitrosation andto minimize transition metal-catalysed S-nitrosothiols(RSNO) degradation and trans-nitrosation reactions(Marley et al. 2000). Plasma and erythrocytes were thenobtained by centrifugation (800 g at 4°C for 10 min). Bothplasma and erythrocytes were immediately frozen in liquidnitrogen and stored at −80°C for subsequent analysis.

Transthoracic echocardiography was performed using aVivid 7 DimensionTM machine (GE Healthcare, Bedford,UK) with a 14 MHz probe recording at a depth of0–2 cm. Aortic blood flow velocities were determinedimmediately before the right carotid artery bifurcationusing pulsed-wave Doppler; the direction of blood flowwas confirmed by colour Doppler imaging. Stroke volumewas determined as the product of the velocity time integraland vessel cross-sectional area (� × [0.5 × diameter]2).Rats of this size have an aortic diameter of 0.26 cm (Slamaet al. 2003), thus a cross-sectional area of (0.13)2 × �was assumed for all animals studied. Heart rate (HR) wasdetermined by measuring the time between six consecutivecycles from the start of each Doppler trace to accountfor variation with respiration. Cardiac output (CO) wasdetermined as the product of stroke volume and HR.Systemic vascular resistance (SVR) was calculated as meanblood pressure divided by CO. The O2 content in thearterial blood and global O2 delivery were calculated usingstandard formulae.

Experimental protocols

Modulation of the various NO pathways was achievedthrough separate studies (Fig. 1). For each experimentalcondition, after recording of baseline haemodynamicvariables and blood sampling for NO metabolitedetermination (BL), animals were randomized to eitherbreathing room air (N) or a hypoxic mixture (H) at aninspired oxygen concentration (FiO2) of 0.125. They werethen allowed to stabilize for 10 min. Animals (six hypoxicand six normoxic per group) were then randomized intoone of the following groups:

i. sham, which only received the background infusion of0.9% saline;

ii. systemic vasoconstriction, using an intravenousinfusion of noradrenaline at a rate of 1.5 μg kg−1 min−1;

iii. NOS inhibition, where animals received a1.5 mg kg−1 min−1 infusion of the potent, non-selectiveNOS inhibitor S-ethyl-isothiourea (SEITU) (Southanet al. 1995);

iv. nitrite (NO2−) administration, where animals received

a 250 μg kg−1 min−1 infusion of sodium nitrite(NaNO2) (Badejo et al. 2010);

v. a combination of NOS inhibition with SEITU, andNaNO2 administration (doses as above);

vi. non-haem NO scavenging, using an infusion of thescavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO) (Yoshida et al.1994) running at 0.17 mg kg−1 min−1;

vii. haem-based NO scavenging, where animals received acontinuous infusion (50 mg kg−1 h−1) of pyridoxylatedhaemoglobin poly-oxoethylene (POE-Hb) (Kida et al.1995).






To avoid any possible confounding effect, drugconcentrations were calculated so that all animals receivedat total of 10 ml kg−1 h−1 of intravenous fluid. At the endof each experiment (30 min), another aliquot of blood wasdrawn for the determination of NO metabolites. All drugsused were purchased from Sigma-Aldrich (Gillingham,

Dorset, UK) with the exception of noradrenaline (AbbottLabs, Maidenhead, Berks, UK). POE-Hb was kindlydonated by Apex Bioscience (Chapel Hill, NC, USA), anddissolved in 0.9% saline.

Drug doses were selected after extensive exploratorydose–response experiments (supporting information

Figure 1. Sites of intervention and study protocolA, pathways of NO production with identification of sites of action of inhibitors and activators used in thestudy. For details about the pathways, please refer to the text. The bolt denotes increase in the targetconcentration; the dashed line denotes a decrease, through enzymatic inhibition (SEITU) or conversion/directscavenging (C-PTIO and POE-Hb). B, study design showing doses and timings of pharmacological inhibitors andactivators. ABP, arterial blood pressure; BGA, arterial blood gas analysis; BL, baseline measurement; C-PTIO, organicNO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; ECHO, echocardiography;L-NAME, non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester; N1/H1, measurement made 10 min afterrandomization to either hypoxia or normoxia; N2/H2, measurement made 20 min after the commencementof pharmacological inhibitors or activators; NaNO2, sodium nitrite; NO, nitric oxide; NO2

−, nitrite anion; NOS,nitric oxide synthase; POE-Hb, haem-based NO scavenger pyridoxylated haemoglobin poly-oxoethylene; RSNO,products of the nitrosation of critical thiol residues; SEITU, non-selective NOS inhibitor S-ethyl-isothiourea; sGC,soluble guanylate cyclase; tPO2, tissue (muscle) PO2.






Fig. S1). We chose a dose of SEITU for subsequentstudy that gave an approximate 40 mmHg rise in ABPin normoxic rats. Notably, no further increases in bloodpressure were observed above this dose in either normoxicor hypoxic animals. We also attempted to modulate NOmetabolism by altering the redox state of cytochromec oxidase through administration of either hydrogensulphide (given as NaHS) or sodium azide (both 10−8

to 10−2 g kg−1 min−1). Unfortunately, major side effects(severe haemodynamic instability leading to prematuredeath) precluded in vivo study of this pathway.

Figure 1B depicts the experimental schedule. Bloodpressure and muscle tissue oxygen tension were monitoredcontinuously. Echocardiography and blood gas analysiswas performed at baseline, 10 and 30 min withsampling for plasma and erythrocyte levels of NO-relatedcompounds (see below) at baseline and 30 min. In selectedexperiments, additional echocardiography and blood gasanalysis was performed at 20 min before administrationof NaNO2.

After analysis of the results, we performed two setsof additional experiments to understand better themechanisms underlying our findings. The hypotheses weset out to test were: (i) whether the vasodilation seenunder hypoxia was caused by increased levels of NO perse or if a NO-related compound could play a role, and(ii) similarly, whether the vasodilating effect of nitritein hypoxia was caused by increased levels of NO perse or if a NO-related compound could play a role. Wethus treated two groups of hypoxic animals (n = 6 each)with a combination of (i) NOS inhibition followed by acontinuous infusion of POE-Hb, and (ii) NOS inhibitionand NaNO2 administration followed by a continuousinfusion of POE-Hb; all drugs were used at the same dosesas previously described. To avoid POE-Hb oxidation, thelatter experiment required separate venous access for theadministration of both drugs: in this group, SEITU andNaNO2 were administered via the jugular line. The leftfemoral vein was also cannulated for administration ofPOE-Hb.

Biochemical analyses

Frozen sample aliquots were thawed just before use.Plasma was used undiluted while the erythrocytepellet was subjected to hypotonic lysis in coldaqueous N-ethylmaleimide/EDTA (10/2.5 mM; 1:4 v/v)immediately before analysis. The concentration of nitrosocompounds was assessed using group-specific reductivedenitrosation by iodine-iodide in glacial acetic acid,with subsequent detection of liberated NO into thegas phase by its chemiluminescent reaction with ozone(Feelisch et al. 2002). ‘RSNO’ signifies mercury-labileS-nitrosated species (including nitrosothiols), whereas

‘RNNO’ signifies mercury-resistant nitroso adducts andmay include N-nitrosamines and metal nitrosyls otherthan NO-haem species. ‘RXNO’ represents the totalpool of nitrosated species (i.e. the sum of RSNOs andRNNOs). NO-haem species in erythrocytes (nitrosylhaemoglobin) were determined by parallel injection ofreplicate aliquots of blood homogenates into a solutionof 0.05 M ferricyanide in phosphate-buffered saline at pH7.5 and 37°C (Bryan et al. 2004). This method employsone-electron oxidation rather than reduction to achievedenitrosation, with the liberated NO being quantified bygas-phase chemiluminescence (CLD 77 AM; Eco Physics,Ann Arbor, MI, USA). Nitrate and nitrite were quantifiedby ion chromatography with on-line reduction of nitrateto nitrite and post-column Griess diazotization (ENO20Analyser; Eicom, Kyoto, Japan) (Rassaf et al. 2002).

Statistical analysis

All data are presented as means ± S.E.M. (n = 6 per group)unless otherwise specified. Statistics were performed onraw data using a repeated-measures two-way ANOVAfollowed by Tukey’s post hoc test (SigmaStat 11.0; SystatSoftware Inc., San Jose, CA, USA) to compare multiplegroups at multiple time points or unpaired Student’s t testto compare two groups. For all comparisons, P < 0.05 wasconsidered statistically significant.

Results

Model characterization and effects of hypoxia

Hypoxia (12.5% O2) significantly reduced arterial PO2

and haemoglobin saturation (Table 1). Muscle tPO2 levelshalved. Despite an unchanged respiratory rate, hypoxiawas associated with reduced PaCO2, but similar arterialpH. Though tidal volumes were not formally measured,there was an obvious increase in chest wall excursion.

Blood pressure fell significantly during hypoxia, and thiswas maintained over time despite no significant change ineither stroke volume or HR (Fig. 2; Fig. S1). Consequently,SVR was significantly lower in the hypoxic group. Globaloxygen delivery was reduced by half, and blood lactatelevels increased significantly (Table 1).

Noradrenaline, titrated to increase mean ABP by40 mmHg above normoxic levels, failed to reach thistarget under hypoxia (Fig. 2 and Table 1). Whereas thisagent increased SVR in the normoxic group, it had littleeffect during hypoxia. HR was significantly increased bynoradrenaline in both groups to the same extent. Oxygendelivery increased in both groups, though failed to reachbaseline levels in the hypoxia group. Lactate levels werelower than during hypoxia alone, and similar to thatmeasured in normoxic sham animals.






Tab

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Hae

mo

dyn

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pir

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ryan

dm

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dat

a

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MN

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No

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117

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79±

9∗17

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104

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9±

11†

145

±13

∗‡99

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64±

15∗

140

±23

88±

28∗

137

±28

92±

8∗‡

119

±14

101

±13

‡

dA

BP

(mm

Hg

)67

±1

42±

4∗11

5±

17†

59±

16∗‡

122

±9†

108

±19

‡63

±12

43±

11∗

109

±17

†63

±23

∗‡10

1±

15†

63±

11∗‡

96±

12†

71±

13∗‡

HR

(1m

in–1

)39

6±

3935

8±

4545

9±

9†45

8±

27‡

370

±39

335

±39

397

±48

350

±29

373

±50

361

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474

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100

431

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388

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0.94

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0.72

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0.89

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120.

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46±

0.09

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83±

0.11

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0.34

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0.43

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0.93

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171.

07±

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83±

0.16

0.74

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08

SV(m

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26±

0.02

0.29

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0.23

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0.1

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0.16

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0.25

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32±

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16±

0.01

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25±

0.05

0.32

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020.

23±

0.04

0.22

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03

PaO

2(k

Pa)

10.8

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65.

6±

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9.6

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4.5

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11.4

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5±

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11.4

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8±

0.5

3.5

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4.7

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6±

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5.3

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64.

2±

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3.6

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3.9

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54.

7±

0.4

4.7

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84.

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4.4

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8

pH

7.43

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47±

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7.47

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58±

0.04

∗‡7.

46±

0.8

7.45

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057.

44±

0.05

7.47

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077.

53±

0.09

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49±

0.05

7.47

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027.

47±

0.8

7.48

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49±

0.08

SaO

2(%

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1.9

66.4

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7∗94

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4.6

62.5

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4.4

68.9

±19

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97.1

±2.

370

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11.6

∗97

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2.2

67.8

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96.3

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852

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1.8

63.2

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5∗

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(g10

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12.9

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112

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15.0

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13.8

±0.

814

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1.2‡

12.5

±1.

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.8±

0.4

13.1

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213

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13.1

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912

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13.4

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413

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RR

(1m

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±8

66±

1087

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86±

1672

±25

68±

2080

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59±

9∗11

6±

2966

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68±

1789

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76±

31

tPO

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Pa)

5.8

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73.

3±

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6±

1.4

2.1

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5.8

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42.

7±

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6±

0.6

3.3

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9±

1.9

2.4

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9±

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6.9

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11.3

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9∗18

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13.6

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1.3

11.7

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7∗18

±1.

712

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2.9∗

17.8

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9.4

±2.

2∗18

±0.

712

±0.

5∗

DO

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16.9

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49.

8±

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22.4

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613

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5±

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6.2

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116

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13.3

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6±

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8±

1.6

20±

29.

5±

2∗17

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3.7

9.5

±0.

9∗

Lac

(mM

)1.

7±

0.5

2.9

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8∗1.

2±

0.3

1.8

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84.

2±

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1.3

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33.

7±

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2.5

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83.

9±

0.5

1.6

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42.

3±

0.7

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rea;

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tro

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od

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0.05

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=6

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.






Haemodynamic effects of the modulation of the nitricoxide system

Non-selective NOS inhibition with SEITU increasedABP largely in normoxic compared to hypoxic animals.A similar pattern was observed with the structurallydistinct NOS inhibitor, N(G)-nitro-L-arginine methylester (Fig. S2). However, while SEITU significantly reducedCO, mainly through its effect on stroke volume, hypoxicanimals showed a lesser fall. Oxygen delivery was sub-stantially reduced but did not differ between the twogroups. Lactate levels increased largely in hypoxic animalstreated with SEITU compared to that seen during hypoxiaalone.

Although nitrite administration had only a mild hypo-tensive effect in either normoxia or hypoxia, stroke volume

increased in the hypoxia group and vascular resistancefell significantly, indicating further vasodilatation. Thisresulted in an overall oxygen delivery that was similar to thenormoxic group treated with nitrite, although lactataemiawas significantly greater. When nitrite was given afternon-selective NOS inhibition with SEITU, the hypotensiveeffect was significantly more pronounced under hypoxicconditions.

NO scavenging with C-PTIO had a greater effect on bloodpressure in the normoxic group. CO also significantlyincreased with C-PTIO in normoxia, mainly through anincrease in HR. On the other hand, the haem-based NOscavenger POE-Hb produced similar changes in bloodpressure in both groups, though ABP values did not reachthe levels obtained with NOS blockade or noradrenaline.

Figure 2. Haemodynamic effects of modulation of NO bioavailabilityC-PTIO, organic NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide group; NaNO2,sodium nitrite group; NEPI, noradrenaline (norepinephrine) group; NO, nitric oxide; POE-Hb, haem-basedNO scavenger pyridoxylated haemoglobin poly-oxoethylene group; SEITU, non-selective NO synthase inhibitorS-ethyl-isothiourea group. BL, H1, N1, H2, N2 refer to the time points shown in Figure 1.






When an NO scavenger was added after NOS inhibitionin the hypoxic group, ABP failed to increase further (meanchange +10.7 mmHg, P = 0.799), with no changes seen ineither CO (mean increase 11.1 ml min−1, P = 0.478) norSVR (mean increase 0.60 mmHg/(ml min−1), P = 0.247).Similarly, when an NO scavenger was added after NOSinhibition and nitrite administration in the hypoxic group,ABP failed to increase (mean change +14.3 mmHg,P = 0.154), with no changes in either CO (mean increase11.7 ml min−1, P = 0.424) or SVR [mean increase0.09 mmHg/(ml min−1), P = 0.982] (Fig. S3).

Plasma nitric oxide metabolites (Fig. 3, Table 2)

Despite similar concentrations of circulating total NOoxidation products, 30 min of hypoxia significantly

reduced plasma nitrite levels. Nitrate levels did notchange significantly at this time point. Administration ofnoradrenaline resulted in lower plasma levels of nitriteand nitrate compared to the sham-treated groups, with nodifferences between normoxia and hypoxia. On the otherhand, NOS inhibition reduced plasma nitrite levels duringnormoxia, but without any further reduction duringhypoxia, whereas the concentration of total nitrosationproducts (RXNO) increased. RSNOs accounted typically,for less than half of the RXNO signal, and that this ratiodid not change much under any of the experimentalconditions applied.

Administration of nitrite increased plasma nitrite byapproximately 20-fold, with a near doubling of plasmanitrate and an increase in plasma total nitrosationproducts. However, no differences were seen between

Figure 3. Plasma metabolites related to oxidation of and nitrosation by NOC-PTIO, organic NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide group; Hypo,hypoxia; NaNO2, nitrite-donor sodium nitrite group; NEPI, noradrenaline group; NO, nitric oxide; Norm, normoxia;NOx, total oxidation products (sum of nitrite and nitrate); POE-Hb, haem-based NO scavenger pyridoxylatedhaemoglobin poly-oxoethylene group; RNNO, N-nitrosamines; RSNO, S-nitrosothiols; RXNO, total nitrosationproducts (sum of RSNO and RNNO); SEITU, non-selective NO synthase inhibitor S-ethyl-isothiourea group. Bluebars denote the hypoxic groups. ∗P < 0.05 vs. normoxia; #P < 0.05 vs. sham normoxia; °P < 0.05 vs. shamhypoxia. n = 6 animals per group.






Tab

le2.

Rel

ativ

eco

mp

osi

tio

no

fo

xid

ativ

ean

dn

itro

sati

veN

Om

etab

olit

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sma

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s

SHA

MN

EPI

SEIT

UN

aNO

2SE

ITU

+N

aNO

2C

-PTI

OPO

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b

No

rmo

xia

Hyp

oxi

aN

orm

oxi

aH

ypo

xia

No

rmo

xia

Hyp

oxi

aN

orm

oxi

aH

ypo

xia

No

rmo

xia

Hyp

oxi

aN

orm

oxi

aH

ypo

xia

No

rmo

xia

Hyp

oxi

a

PLA

SMA

NO

2–/N

Ox

0.15

±0.

100.

05±

0.04

∗0.

02±

0.01

†0.

03±

0.02

0.04

±0.

02†

0.03

±0.

010.

66±

0.04

†0.

63±

0.06

‡0.

69±

0.05

†0.

67±

0.08

‡0.

02±

0.01

†0.

02±

0.02

0.02

±0.

01†

0.02

±0.

01

RSN

O/R

XN

O0.

28±

0.10

0.22

±0.

140.

14±

0.15

0.22

±0.

150.

41±

0.15

0.29

±0.

170.

40±

0.17

0.37

±0.

160.

44±

0.22

0.36

±0.

170.

34±

0.18

0.18

±0.

120.

22±

0.14

0.20

±0.

09

ERY

THR

OC

YTE

S

NO

2–/N

Ox

0.05

±0.

030.

07±

0.03

0.02

±0.

020.

01±

0.01

‡0.

05±

0.04

0.04

±0.

05‡

0.02

±0.

010.

02±

0.02

‡0.

03±

0.02

0.02

±0.

01‡

0.03

±0.

020.

03±

0.03

0.01

±0.

010.

02±

0.01

‡

RSN

O/R

SNO

0.15

±0.

100.

24±

0.10

0.22

±0.

160.

38±

0.14

0.40

±0.

25†

0.30

±0.

100.

27±

0.28

0.23

±0.

150.

38±

0.23

†0.

23±

0.16

0.31

±0.

230.

25±

0.10

0.30

±0.

09†

0.32

±0.

29

C-P

TIO

,N

Osc

aven

ger

2-(4

-car

bo

xyp

hen

yl)-

4,4,

5,5-

tetr

amet

hyl

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idaz

olin

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oxy

l-3-

oxi

de;

NaN

O2,

sod

ium

nit

rite

;N

EPI,

no

rad

ren

alin

e;N

O,

nit

ric

oxi

de;

NO

2–,

nit

rite

;N

Ox,

tota

lo

xid

atio

n(n

itri

te+

nit

rate

)sp

ecie

s;PO

E-H

b,

hae

mN

O

scav

eng

erp

yrid

oxy

late

dh

aem

og

lob

inp

oly

-oxo

eth

ylen

e;R

SNO

,S-n

itro

soth

iols

;R

XN

O,t

ota

ln

itro

sati

on

spec

ies

(S-n

itro

soth

iols

+N

-nit

rosa

min

es);

SEIT

U,n

on

-sel

ecti

veN

Osy

nth

ase

inh

ibit

or

S-et

hyl

-iso

thio

ure

a.∗ P

<0.

05vs

.n

orm

oxi

a;† P

<0.

05vs

.no

rmo

xia

sham

;‡P

<0.

05vs

.hyp

oxi

ash

am.n

=6

anim

als

per

gro

up

.

hypoxic and normoxic groups. Addition of NOS inhibitionto nitrite did not change the plasma NO metabolite profile.

Scavenging NO with C-PTIO reduced plasma nitritelevels to virtually zero, whereas nitrate levels wereconserved. Plasma nitrosation products were also reduced,though no differences were seen between hypoxia andnormoxia. Utilization of POE-Hb as the NO scavengerwas associated with a similar profile of plasma oxidationproducts. However, in stark contrast to C-PTIO, plasmanitrosation products increased compared to sham in bothnormoxic and hypoxic animals. Table 2 shows the relativecontribution of nitrite to total oxidation products, andthat of RSNO to total nitrosation products.

Erythrocyte nitric oxide metabolites (Fig. 4, Table 2)and nitrosyl haemoglobin (Fig. 5)

In sham animals, erythrocyte nitrite and nitrate levelswere conserved during hypoxia, with no differences seenin nitrosation products. Noradrenaline reduced intra-cellular nitrite levels in normoxic animals but RSNO andRNNO remained largely unchanged. NOS inhibition bySEITU had no impact upon erythrocyte NO oxidationand nitrosation products, and no differences were seenbetween hypoxia and normoxia states.

Notably, nitrite administration did not modify intra-erythrocytic nitrite levels. However, nitrate concentrationswere higher than in sham animals, particularly underhypoxic conditions. Nitrosyl haemoglobin levels werealso higher during hypoxia. Similar results were seenwhen nitrite was co-administered with the NOS inhibitor,SEITU. NO scavenging was associated with lower intra-cellular nitrite levels, yet most other NO metabolites wereunchanged. Of note, the NO metabolite profile observedin the presence of C-PTIO was different from that of atypical NO scavenger; besides the expected reductions inblood nitrite concentrations under normoxic and hypo-xic conditions due to oxygen-independent conversion ofNO to NO2, hypoxia was associated with increases innitrosation products other than RSNOs and increasesin nitrosyl haemoglobin. This suggests preferentialformation of N-nitrosated species in red blood cells thatact as potential NO donors with this particular compound.Thus, care should be taken when C-PTIO is used in in vivostudies, emphasizing the need to compare mechanisticallydistinct NO scavengers before reaching conclusions aboutthe possible involvement of NO in a given physiologicalprocess.

Discussion

Several complementary mechanisms protect the body’stissues in the face of a reduced O2 delivery. These includeredistribution of blood flow to ‘vital’ organs, and increased






recruitment of perfused microvessels to facilitate O2

availability (Tsai et al. 2003). At the systemic level, the mostevident adaptation to hypoxaemia involves an increase inblood flow – ‘hypoxic vasodilation’ (Roy & Brown, 1880;Dreyer, 1926; Hackel et al. 1954; Heistad & Wheeler, 1970;Rowell & Blackmon, 1987, 1989). This aims to compensatefor the reduction in arterial O2 content by attempting tomaintain an acceptable O2 supply–demand balance. Thesympathetic nervous system is activated with increasedcatecholamine levels, albeit with a reduced response toexogenous vasopressors (vascular hyporeactivity).

Although the physiological relevance of hypoxic vaso-dilation is well established, ongoing controversy surroundsits underlying mechanisms. An increase in NO is stronglyimplicated. This key signalling and effector moleculeis a major determinant of vascular tone, an important

modulator of myocardial function, and essential for bothglobal regulation and regional distribution of blood flowand pressure (Welch & Loscalzo, 1994).

NO levels in blood and tissues represent abalance between production/release and metabolism/bioinactivation. Its best-recognized biosynthetic route isthe L-arginine–NO pathway (Moncada & Higgs, 1993;Boucher et al. 1999). However, alternative pathways areproposed, involving (i) nitrite and nitrate, oxidative endproducts traditionally considered to be inert (Modin etal. 2001), and (ii) RSNO, the reaction products betweenreactive NO species and thiol residues (Diesen et al. 2008).

Particularly under conditions of hypoxia, proteins fromthe haem-globin family (Gladwin & Kim-Shapiro, 2008)or from pteryn-based molybdenum enzymes (Li et al.2008) may catalyse the reduction of nitrite to NO, beyond

Figure 4. Erythrocytic metabolites related to oxidation of and nitrosation by NOC-PTIO, organic NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide group; Hypo,hypoxia; NaNO2, nitrite-donor sodium nitrite group; NEPI, noradrenaline group; NO, nitric oxide; Norm, normoxia;NOx, total oxidation products (sum of nitrite and nitrate); POE-Hb, haem-based NO scavenger pyridoxylatedhaemoglobin poly-oxoethylene group; RNNO, N-nitrosamines; RSNO, S-nitrosothiols; RXNO, total nitrosationproducts (sum of RSNO and RNNO); SEITU, non-selective NO synthase inhibitor S-ethyl-isothiourea group. Dashedbars denote the hypoxic groups. ∗P < 0.05 vs. normoxia; #P < 0.05 vs. sham normoxia; °P < 0.05 vs. shamhypoxia. n = 6 animals per group






their conventional physiological functions. Release of NOfrom RSNO storage pools may also be enhanced underlow oxygen conditions (Jia et al. 1996). These pathwaysmay provide important alternative sources of NO, whereincreased blood flow may be beneficial, such as duringhypoxia, particularly recognizing the critical requirementfor O2 as a cofactor for NOS. However, to date, manyof these mechanisms have only been demonstrated innon-physiological conditions, either in vitro or ex vivo,or indirectly implicated by utilization of pharmacologicaltools. Thus, an open question remains as to their in vivo(patho)physiological relevance.

The in vivo fate of NO is also highly complex; severalcatabolic pathways exist with varying relevance in differentbody compartments. The reaction with haemoglobin toform NO3

− constitutes the major catabolic pathway.However, in plasma in the presence of O2, the principalreaction is the formation of NO2

− (Ignarro et al.1993) occurring through oxidation by mitochondrialcytochrome c oxidase (Shiva et al. 2001; Pearce et al.2002). An alternative theory for the hypoxia-inducedincrease in NO is reduced elimination. Using an invitro model, Moncada and colleagues demonstrated thatcytochrome c oxidase in its oxidized state constantlyinactivates NO, contributing to its intracellular regulation(Palacios-Callender et al. 2007; Unitt et al. 2010). However,in the reduced state typical of hypoxia, an impairedinactivation would account for the witnessed increase inNO.

NO acts at multiple sites within the body(Martinez-Ruiz et al. 2011). It promotes vasorelaxationeither via activation of soluble guanylate cyclase (sGC) andsubsequent generation of cyclic GMP (cGMP), or throughS-nitrosation of critical thiols sited on ion channels. NOalso inhibits the mitochondrial electron transport chainthrough direct competition with oxygen at Complex IV(cytochrome c oxidase), and via nitrosation/nitration of

other respiratory chain complexes (Frost et al. 2005),ultimately causing a decrease in O2 consumption.

As the vasodilating (Movahed et al. 2003) and metabolic(Frost et al. 2005) effects of NO are both enhanced duringhypoxia, it probably plays a major role in re-equilibratingany O2 supply–demand mismatch (Umbrello et al. 2013).Hypoxia-induced vasorelaxation may be reversed byscavenging NO (Pohl & Busse, 1989), or inhibiting sGCwith methylene blue (Iwamoto et al. 1992). Whereasincreased NO synthesis by NOS may be a potential causeof hypoxic vasodilation (Edmunds & Marshall, 2001;Hunter et al. 2003), others report an ineffectiveness ofNOS inhibition on decreased hindlimb resistance (Valletet al. 1994) and dilation of small coronary arteries (Liu &Flavahan, 1997) induced by hypoxia.

We set out to study mechanisms underlying hypoxicvasodilation in vivo using a well-established short-termrodent model of systemic hypoxia (Dyson et al. 2007,2009). We chose FiO2 at 0.125 as this concentration wasassociated with the largest fall in PaO2 that did not carryshort-term lethality (Dyson et al. 2007). Our findingssupport the role of NO as a key mediator of hypoxic vaso-dilation.

Hypoxaemia caused an immediate and sustainedhypotension associated with muscle tissue hypoxia andincreased glycolytic flux. Vasopressor hyporesponsivenesswas confirmed by the limited increase in ABP withnoradrenaline. Notably, hypoxia reduced ABP and SVRbut had little effect on CO: this may be explained bythe balanced effect of an increased perfusion due tovasodilation and some degree of hypoxic myocardialdepression, possibly NO-mediated (Brady et al. 1993;Mohan et al. 1996).

Non-selective NOS inhibition with SEITU reversedhypoxic vasodilation but failed to achieve ABP valuesobtained from normoxic, SEITU-treated animals; weobserved a similar pattern using the non-selective NOS

Figure 5. Haem nitrosylation as marker ofNO availability in erythrocytesC-PTIO, organic NO scavenger2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide group; NaNO2,nitrite-donor sodium nitrite group; NEPI,noradrenaline group; NO, nitric oxide; NO-Hb,nitrosyl haemoglobin; POE-Hb, haem-basedNO scavenger pyridoxylated haemoglobinpoly-oxoethylene group; SEITU, non-selectiveNO synthase inhibitor S-ethyl-isothioureagroup. ∗P < 0.05 vs. normoxia; #P < 0.05 vs.sham normoxia; °P < 0.05 vs. sham hypoxia.n = 6 animals per group






inhibitor N(G)-nitro-L-arginine methyl ester and infer thedifference in blood pressure between normoxic and hypo-xic rats is due to NO not generated by NOS but derivedfrom other pathways (nitrite reduction or release frompreformed nitroso pools). Hypoxia was associated withsignificantly lower plasma levels of nitrite, supportingthe concept of increased NO production originatingfrom nitrite reduction. When NOS was blocked bySEITU, plasma nitrite was lowered even under normoxicconditions. Most probably, this change is unrelated tonitrite reduction but a direct consequence of reducedNO production from NOS. Administration of nitritecaused little hypotensive effect in normoxia, and onlysmall reductions of ABP under hypoxia. However, strokevolume and CO, and thus SVR, were significantlyhigher in the hypoxic group, suggesting a stronger vaso-dilating effect of nitrite administration under hypo-xia. The failure of blood pressure to fall any furthermay be explained by being already low due to hypo-xia alone. However, nitrite administration significantlyincreased nitrosyl haemoglobin generation during hypo-xia, consistent with higher levels of NO production. Whengiven after non-selective NOS blockade with SEITU, nitritecaused a significantly greater hypotensive effect underconditions of hypoxia, accompanied by a further increasein nitrosyl haemoglobin levels.

Experiments with NO scavengers could distinguishwhether the vasodilating factor acting in hypoxia was dueto intravascular generation of NO per se or one or moreNO-related compound(s). Both C-PTIO and POE-Hb arespecific extracellular NO scavengers and both increasedABP irrespective of the presence of hypoxia. A possibleexplanation is that NO produced by endothelial cellsexerts its vasorelaxing activity by a paracrine effect onsurrounding vascular smooth cells, with no transit intothe bloodstream. However, the changes in the circulatingpool of NO oxidation and nitration products during NOscavenging suggest that a portion of the NO generatedin vascular tissue is transferred to the bloodstream.Alternatively, NO does not exert its vasodilatory actiondirectly, but rather through the action of other down-stream mediators. A study performed under physiologicalconditions found that nitrite was not directly reducedto NO but instead modulated several different signallingpathways (Bryan et al. 2005). In addition to sGC activation,post-translational modifications typically associated withNO occur, such as the formation of nitrosated, nitrosylatedand nitrated species (Bryan et al. 2005; Perlman et al.2009). Thus, nitrite may exert its signalling functionsdirectly, without the need for intermediary formationof free NO. Hypoxia markedly potentiates tissue NOproduction from nitrite in a dose-dependent manner(Feelisch et al. 2008). This occurs particularly inheart, liver and vascular tissue, with multiple haem,iron–sulphur cluster and molybdenum-based reductases

distributed among distinct subcellular compartmentsacting in a multifactorial and cooperative mannerto catalyse the reaction. Acute hypoxia also reducednitrite concentrations yet simultaneously enhanced theformation of NO metabolites such as RSNOs andRNNOs in an NO-independent manner, again consistentwith a pathway that generates bioactive NO metaboliteswithout the intermediacy of free NO. In this paradigm,conversion of nitrite to NO and the storage of NObioactivity as RSNOs may both be constituents of a morecomplex regulatory mechanism of interaction of multipleNO-related species. To further test this hypothesis, and itsin vivo relevance, we designed experiments whereby NOwas scavenged after hypoxia and NOS inhibition ± nitriteadministration. Notably, in both cases, blood pressurefailed to increase upon NO scavenging, further supportingthe hypothesis that actions of NO in hypoxia are mediatedby NO metabolite signalling pathways rather than by NOdirectly.

Our study has several limitations: first, we focusedon the acute hypoxic response, which may presentdifferent mechanisms from the response to chronichypoxia. However, 10-fold higher levels of circulatingNO products were found in Tibetan plateau residentscompared to sea-level dwellers (Erzurum et al. 2007).This was associated with increased resting forearm bloodflow, suggesting a NO-mediated adaptive mechanism thatoffsets the O2 lack caused by high altitude and persistsover time. Our rats were anaesthetized and spontaneouslybreathing, and responded to hypoxia with an increasein their ventilatory drive. The subsequent reduction inPaCO2 (compared to group-matched normoxic rats) was,however, limited, and probably more representative of thesituation that occurs in vivo. The (patho)physiology ofvascular adaptation to hypoxia is indeed complex. Studieshave demonstrated that NO is a major, though not thesole, contributor to this response (Liu & Flavahan, 1997),or that it acts within a network of synergistic factors (vonBeckerath et al. 1991; Park et al. 1992). Indeed, some ofour results may be explained by concomitant activationof other signalling pathways. Another limitation of ourstudy is that changes in NO metabolite status have onlybeen assessed in blood and not in the vasculature itself.While a contribution by NO generated in the vascular wallcan thus not be entirely excluded, this is unlikely to haveoccurred under our experimental conditions as the highconcentrations of the two NO scavengers used shouldhave created a sink for NO that would be efficient enoughto show clear reversal of the effects of nitrite; however, thisis not what we observed. Regrettably, we were unable toexplore the possibility that NO levels may be increased inhypoxia because of a reduced metabolism/bioinactivation.Although we tried several strategies to modulate theredox state of cytochrome c oxidase (namely azide andhydrogen sulphide administration), unavoidable side






effects prevented the in vivo construction of such a model.Owing to these methodological constraints we cannotexclude that reduced inactivation of NO by cytochromec oxidase may be an additional contributory factor.

In conclusion, we demonstrated that short-term hypo-xic vasodilation in vivo is an adaptive response mediatedby NO. The mediator seems to act through bioactivemetabolites, in a complex regulatory network of inter-action of multiple NO-related species, rather than directlyby NO itself.

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Additional information

Competing interests

The authors declare no competing financial interests.






Author contributions

Experiments were performed at University College London.M.U., A.D., M.F. and M.S. conceived the study and designedthe experiments; M.U., A.D., B.B.P. and V.S. performed theexperiments and collected the physiological data. M.U. and V.S.undertook the data analysis. B.O.F. performed the biochemicalmeasurements. M.U. and M.S. drafted the first version of themanuscript; A.D., B.B.P., B.F. and M.F. revised it critically. Allauthors approved the final version of the manuscript.

Funding

This work was undertaken at UCLH/UCL who received aproportion of funding from the Department of Health’sNIHR Biomedical Research Centres funding scheme. M.U. wassupported by a Physiology Award from the Accademia Nazionaledei Lincei (Roma)/Royal Society (London).

Acknowledgement

None.





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