Eur Respir J 2010; 36: 323–330 DOI: 10.1183/09031936.00188809 CopyrightßERS 2010 Tetrahydrobiopterin and the regulation of hypoxic pulmonary vasoconstriction B.N. Francis, M.R. Wilkins and L. Zhao ABSTRACT: Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthases (NOS). AFFILIATIONS Experimental Medicine and This study investigated the effect of increasing BH4 levels on hypoxia-induced pulmonary Toxicology, Imperial College London, vasoconstriction (HPV). Hammersmith Hospital, London, UK. Sprague Dawley rats and hph-1 (BH4 deficient) mice were given BH4 before and during HPV in an isolated perfused lung preparation. BH4 inhibited HPV in a concentration-dependent manner CORRESPONDENCE and increased NO metabolites in the perfusate. Bradykinin-induced reductions in HPV were L. Zhao Experimental Medicine and blunted in hph-1 mice and pre-administration of BH4 restored the response. The effect of BH4 was Toxicology, Imperial College London attenuated by L-NAME (NOS inhibitor), PTIO (NO scavenger), and catalase (H2O2 catalyser) Hammersmith Hospital administered prior to HPV but enhanced by MnTMPyP (superoxide dismutase mimetic). The effect Ducane Road London of BH4 on HPV was partially recapitulated by NH4, a stereoisomer that shares antioxidant W12 ONN properties with BH4 but is not a NOS cofactor. UK The bioavailability of BH4 is an important determinant of the pulmonary vascular response to E-mail: [email protected] hypoxia. Its effects are mediated via nitric oxide, hydrogen peroxide and its antioxidant properties, Received: and are attenuated by oxidant stress. Pharmacological administration of BH4 may have Nov 26 2009 therapeutic potential in pulmonary hypertension. Accepted after revision: Feb 28 2010 KEYWORDS: Animal models, hypoxia, pulmonary vasoconstriction, pulmonary hypertension, First published online: nitric oxide, superoxide, tetrahydrobiopterin March 11 2010 itric oxide synthases (NOS), particularly analysis of tissue homogenates from hph-1 mice endothelial NOS (eNOS), play a major shows increased local pulmonary vascular super- N role in maintaining normal pulmonary oxide production [7]. Vascular superoxide produc- vascular tone and structure through the produc- tion has a number of important actions on vascular tion of nitric oxide (NO) from L-arginine [1–3]. tissue, such as scavenging of NO, peroxynitrite Critical to NO synthesis by NOS is the bioavail- formation and modulation of redox-sensitive ability of tetrahydrobiopterin (BH4). When BH4 is signalling pathways [9], which may adversely deficient through inadequate synthesis or oxida- affect vascular structure. tion, NOS becomes uncoupled and generates superoxide instead of NO [4]. There is increasing evidence of oxidative stress in pulmonary hypertension [10, 11], including The hph-1 mouse, generated by N-ethyl-N-nitroso- reports of nitrotyrosine formation (a marker of urea (ENU) mutagenesis, has low tissue BH4 levels peroxynitrite production) in the lungs of patients because of constitutively reduced expression of with the disease [12]. Superoxide and peroxy- GTP cyclohydrolase-1, the rate-limiting enzyme in nitrite oxidise BH4, and enhanced oxidative BH4 biosynthesis [5]. Congenital deficiency of BH4 degradation is thought to be a major cause of in the hph-1 mouse leads to pulmonary hyperten- reduced BH4 bioavailability and eNOS uncou- sion, distal pulmonary vessel muscularisation and pling in endothelial dysfunction states [13–15]. right ventricular hypertrophy [6, 7]. The pulmon- As a result, there is interest in using exogenous ary vascular pathology in hph-1 mice is more BH4 therapeutically in pulmonary hypertension marked than that reported in NOS-deficient mice to restore tissue levels and promote NO produc- and suggests that loss of NO production alone is tion from NOS. Recent studies suggest that BH4 not the sole reason for the vascular pathology [1, may also act independently of NO, generating 8]. In addition to reduced NO synthesis, and hydrogen peroxide, another molecule with vasor- consistent with uncoupling of NOS, biochemical elaxant properties [16, 17]. European Respiratory Journal For editorial comments see page 234. Print ISSN 0903-1936 c This article has supplementary material available from www.erj.ersjournals.com Online ISSN 1399-3003 EUROPEAN RESPIRATORY JOURNAL VOLUME 36 NUMBER 2 323 PULMONARY VASCULAR DISEASE B.N. FRANCIS ET AL. The purpose of this study was to investigate the pharmacological BH4 (calculated to produce perfusate concentrations of 0.3 or -1 effects of BH4 on pulmonary vascular tone, specifically hypoxia- 1 mg?mL ) added to the perfusate (fig. 1). Perfusate samples induced vasoconstriction (HPV) in an in situ isolated perfused (100 mL) were collected after passage through the lungs 1 min lung preparation. Employing the BH4 deficient hph-1 mouse, we prior to and 4 min after BH4 administration and before the explored further the underlying mechanism of BH4 in balancing hypoxic challenge for measurement of nitrate/nitrite (NOx) NO and superoxide production in pulmonary vasculature. concentration. The effect of BH4 was calculated as the per cent change in HPV response and NOx levels after BH4 adminis- METHODS tration compared to the HPV response and NOx levels before Animals BH4 administration in the same animal. Male Sprague-Dawley rats (250–360 g) from Charles River (Margate, UK) and hph-1 mice aged 3–5 months generated by Isolated perfused mouse lung preparation ENU mutagenesis were used for experiments. Wild-type (WT) Animals were anaesthetised and the lungs ventilated with air animals, hph-1 heterozygous (+/-) and hph-1 homozygous (volume controlled ventilation, 230 mLtidalvolume,80breaths? (hph-1) littermates on a C57BL/6 background were obtained -1 min and 2 cmH2O positive end-expiratory pressure) via the by interbreeding hph-1 heterozygotes [5]. All studies were trachea. The lungs were perfused through the main pulmonary conducted in accordance with UK Home Office Animals artery accessed via the right ventricle and perfusate collected (Scientific Procedures) Act 1986. from the left atrium for recycling in a closed circuit (type 839; Hugo Sachs Electronik-Harvard Apparatus GmbH, March- Reagents Huggstetten, Germany). Using a nonperistaltic pump, the 6R-BH4 (sapropterin dihydochloride or BH4) was supplied by perfusate (DMEM with 4% Ficol, HEPES-buffered, pH 7.4) was BioMarin Pharmaceuticals, Inc. (Novato, CA, USA). U46619 (9, delivered at an initial flow of 0.2 mL?min-1, increasing in a a -1 11-dideoxy-11 ,9 -epoxymethanoprostaglandin F2a), L-NAME increments after 10 min to 2 mL?min . The reservoir volume (Nv-nitro-L-arginine methyl ester hydrochloride), Catalase, was set at 10 mL and pH monitored by the pH sensor placed in MnTMPyP (Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin pen- the reservoir. Ppa was measured using a pressure transducer tachloride), PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1- connected to the inflow catheter. Left atrial pressure was oxyl 3-oxide), bradykinin (BK) and chemical components of the perfused solutions were obtained from Sigma-Aldrich a) 30 (Poole, UK). NH4 (6R, S-5,6,7,8-tetrahydro-D-neopterin) was from Schircks Laboratories (Jona, Switzerland). All drugs were μ -1 μ -1 -1 Vehicle BH4 0.3 g·mL BH4 1 g·mL prepared fresh every day before experiments. 10 mg?uL BH4 or NH4 were dissolved in HEPES buffered saline (composition: 20 NaCl 137 mM, KCl 4.0 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 10 mM, glucose 10 mM; pH adjusted to 7.4 with NaOH). mmHg pa Isolated perfused rat lung preparation P 10 The pulmonary vascular response to BH4 was studied using the isolated rat lung preparation previously described [18]. Briefly 21% O 2% O 21% O 2% O 21% O 2% O 21% O rats were anaesthetised with Hypnorm (fentany-fluanisone). 2 2 2 2 2 2 2 The lungs were left in situ after the trachea had been 0 0 10 20 30 40 50 60 70 80 90 cannulated, ventilated with air (21% O2,5%CO2 balanced N2) Time min at constant end expiration pressure of 4 mmHg with 60 breaths?min-1; and perfused with a mixture (1:1) of DMEM b) 80 (Dubelcco’s Modified Eagles Medium) with 4% Ficoll, and blood withdrawn from the experimental animal (pH 7.4) at a 60 flow rate of 18 mL?min-1 using a non-pulsatile pump 40 (Masterflex model 7519; Cole-Parmer Instrument Co. Ltd, London, UK). Pulmonary artery pressure (Ppa) was measured 20 using a pressure transducer connected to PowerLab Data Acquisition system (ADInstruments Limited, Chalgrove, UK). Change % 0 HPV was produced by ventilating the lung with 2% O2,5%CO2 and 93% N2 for 15 min. The ventilation was then returned to air -20 for 10 min to resume the PAP baseline before the next hypoxic challenge. Successive hypoxic challenges (usually 3) were -40 μ -1 μ -1 repeated until the HPV response was consistent before vehicle BH4 0.3 g·mL BH4 1 g·mL or BH4 treatment was given to examine the effect on HPV. FIGURE 1. a) Representative pulmonary arterial pressure (Ppa) trace illustrat- BH4 ing the protocol for tetrahydrobiopterin (BH4) administration prior to induction of -1 BH4 (final perfusate concentration 0.3 mg?mL ) was added to hypoxia-induced pulmonary vasoconstriction (HPV) in the rat isolated perfused the perfusate ,4 min before the hypoxic challenge. Once the lung. b) The effect of BH4 on HPV response (reduction expressed as a percentage pressor response had returned to baseline, the hypoxic of HPV before BH4 administration in the same animal, &) and percentage increase challenge was repeated with or without a second aliquot of in perfusate NOx levels (&) following BH4 administration (n55). 324 VOLUME 36 NUMBER 2 EUROPEAN RESPIRATORY JOURNAL B.N. FRANCIS ET AL. PULMONARY VASCULAR DISEASE measured using a pressure transducer connected to the out- a) 40 Vehicle Normoxia flow catheter. Pressure tracings were collected and analysed using PowerLab Data Acquisition system. U46619 (thrombox- ane analogue; 5 mM) was added to reservoir to induce basal 30 pulmonary vasoconstriction. 2 min after U46619 administra- Hypoxia tion, a stable Ppa was achieved and recording was continued U46619 20 for a further 6 min before HPV was induced by ventilating mmHg pa Flow the lung with 2% O ,5%CO and 93% N .