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Eur J Cardiothorac Surg 2006;30:464-471
© 2006 Elsevier Science NL

Effect of tetrahydrobiopterin on selective endothelial dysfunction of epicardial porcine coronary arteries induced by cardiopulmonary bypass

Louis-Mathieu Stevensa, Simon Fortiera, Marie-Claude Aubina, Ismail El-Hamamsya, Simon Maltaisa, Michel Carriera,b, Louis P. Perraulta,b,c,*

a Research Center, Montreal Heart Institute, 5000 Belanger Street, Montreal, Que. H1T 1C8, Canada
b Department of Surgery, Montreal Heart Institute, 5000 Belanger Street, Montreal, Que. H1T 1C8, Canada
c Department of Pharmacology, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montreal, Que. H3C 3J7, Canada

Received 17 January 2006; received in revised form 18 May 2006; accepted 2 June 2006.

* Corresponding author. Address: Department of Surgery, Montreal Heart Institute, 5000 Belanger Street, Montreal, Que. H1T 1C8, Canada. Tel.: +1 514 376 3330; fax: +1 514 376 1355. (Email: louis.perrault{at}icm-mhi.org).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Background: We hypothesized that cardiopulmonary bypass induces a selective alteration of the coronary arterial endothelial cell signal transduction which could be explained by a state of depletion and/or decreased activity of endogenous tetrahydrobiopterin (BH4). The aim of this study was to assess the effects of cardiopulmonary bypass and BH4 on the endothelial function of epicardial coronary arteries in a swine model of cardiopulmonary bypass. Methods: Swine underwent 90 min of cardiopulmonary bypass alone (N = 19) or in association with a brief cardioplegic arrest with (N = 6) or without (N = 5) in vivo BH4 administration, followed by a 60-min period following weaning from cardiopulmonary bypass and were compared to a control group (N = 7). Endothelium-dependent relaxations of epicardial coronary artery rings were studied using standard organ chamber experiments in the presence or absence of in vitro BH4 or superoxide dismutase (SOD) and catalase. Results: Cardiopulmonary bypass caused a statistically significant reduction of endothelium-dependent relaxations to serotonin (p < 0.0001), bradykinin (p < 0.001), UK14304 (p < 0.0001) and calcium ionophore (p < 0.01) in epicardial porcine coronary arteries. In vitro and in vivo BH4 supplementation improved endothelium-dependent relaxations to serotonin and bradykinin, which were left unchanged by SOD-catalase administration. Cardiopulmonary bypass was associated with a decrease in nitric oxide availability (p = 0.002) and increased oxidative stress (p < 0.001), which were both restored by in vivo BH4 administration (p < 0.001). Conclusion: Treatment with BH4 improves the endothelial dysfunction of porcine epicardial coronary arteries, restores nitric oxide availability and reduces the oxidative stress associated with cardiopulmonary bypass.

Key Words: Antioxidant • Cardiopulmonary bypass • Coronary artery • Endothelial dysfunction • Nitric oxide • Superoxide dismutase • Tetrahydrobiopterin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
The endothelium is a selective barrier responsible for maintaining a non-thrombogenic surface, ensures the metabolism of circulating substances, regulates smooth muscle proliferation and controls the vasomotor tone of vessels. Owing to its unique location at the blood–tissue interface, the endothelium is affected early in the systemic inflammatory response syndrome related to cardiopulmonary bypass (CPB) along with the contact system, complement, coagulation and fibrinolysis pathways. Activation of the endothelial cells, leukocytes and platelets leads to the massive release of cytokines and oxygen-derived free radicals, which contribute to the systemic activation of the endothelium [1,2].

Normal endothelial vasomotion results from a balance between endothelium-dependent relaxing factors (EDRF), such as nitric oxide (NO), prostacyclin and the endothelium-dependent hyperpolarizing factor (EDHF), and endothelium-dependent contracting factors, such as oxygen-derived free radicals, thromboxane A2 and endothelin. The release of EDRF can be activated by specific G-protein-linked receptors [3].

Regional and global ischemia-reperfusion of the coronary circulation during CPB with aortic cross-clamping is associated with an impairment of EDRF release [4–6]. This dysfunction preferentially involves pertussis toxin sensitive G-proteins mediated relaxations (Gi-protein) [4]. During CPB, sanguinous cardioplegia generally provides better endothelial and myocardial protection than crystalloid cardioplegic solutions [6,7]. No study has shown an endothelial dysfunction of coronary arteries following CPB alone, without prolonged aortic cross-clamping [4,5]. On the other hand, a selective endothelial dysfunction of the pulmonary arterial tree following CPB involving the muscarinic receptor to acetylcholine (Gi-coupled) [8] with increased cyclic adenosine monophosphate (cAMP) levels [9] was demonstrated in our laboratory.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide biosynthesis. Decreased levels of BH4 lead to an uncoupling of NO synthase (NOS), with a shift towards production of peroxynitrites (OONO–) and hydrogen peroxide (H2O2), two oxygen free radicals with direct cytotoxic effects on the endothelium [10]. BH4 supplementation promotes NO production and other antioxidant effects known to improve endothelium-dependent vasomotion in a number of pathological states including coronary artery disease, hypercholesterolemia [11], ischemia-reperfusion [12], diabetes, arterial hypertension [13], smoking, and left ventricular hypertrophy [14].

Since CPB contributes to the systemic endothelial activation and dysfunction, we tested the hypothesis that: (1) CPB per se, without prolonged aortic cross-clamping, leads to specific alterations in the signal transduction pathways of coronary arterial endothelial cells and is associated with an impairment of NO expression and/or activity and (2) endogenous BH4 is decreased during CPB leading to an uncoupling of NOS which could be improved by BH4 administration.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
All experiments were performed using Landrace swine (McGill University, Montreal, Canada) of either gender weighing 28 ± 3 kg. Animals were maintained and tested in accordance with the guidelines issued by the Canadian Council on Animal Research. CPB protocol [8,9] and experimental group characteristics are summarized in Table 1 .


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  		Table 1. Experimental groups characteristics
 
2.1 Vascular reactivity studies protocol
Less than 10 min after heart excision, epicardial coronary arteries were carefully dissected free of the surrounding myocardium and epicardial fat, and were divided into rings 4 mm in length. A total of 16 coronary arterial rings were harvested. Rings from the left anterior descending, circumflex coronary and right coronary arteries were used randomly. The vascular reactivity was studied in organ chambers filled with 20 ml modified Krebs-bicarbonate solution at 37 °C with 95% O2 and 5% CO2 (composition in mmol/l: NaCl 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, glucose 11.1, CaCl2 2.5, NaHCO3 25 and ethylenediaminotetraacetic acid 0.026). The rings were suspended between two metal stirrups with the upper one connected to an isometric force transducer and then allowed to stabilize for 30 min. Epicardial coronary artery rings were stretched to the optimal point of their active length-tension curve (3.5–4.0 g) as determined by measuring the contraction to potassium chloride (KCl, 30 mmol/l) at different levels of stretch (data not shown). The studies were performed in the presence of ketanserin (10–6 mol/l, incubated 40 min before the addition of serotonin to competitively block direct vasoconstriction by serotonin 5HT2 receptors), indomethacin (10–5 mol/l, to exclude the production of endogenous prostanoids) and propranolol (10–7 mol/l, to prevent the activation of ß-adrenergic receptors). After 45 min of stabilization, prostaglandin F2{alpha} (range 2 x 10–6 to 3 x 10–5 mol/l) was added to obtain a contraction averaging 50% of the maximal contraction to KCl (60 mmol/l).

In each of the four groups, the NO-mediated relaxation pathway was studied by constructing concentration–response curves to serotonin (5-hydroxytryptamine (5HT) creatinine sulfate, 10–10 to 10–5 mol/l; an agonist coupled to a Gi-protein that binds to 5HT1D receptors) and to bradykinin (BK) (10–12 to 10–6 mol/l; an agonist coupled to Gq-proteins that binds to B2 receptors). At the completion of serotonin testing, the rings were left to recontract until a plateau was reached, then BK testing was initiated for each ring.

In the control and the CPB group, endothelial function was further studied with endothelium-dependent relaxation to the {alpha}2-adrenergic agonist UK14304 (10–10 to 10–5 mol/l; an agonist coupled to Gi-proteins that binds to {alpha}2 receptors), endothelium-dependent receptor-independent relaxations to calcium ionophore A23187 (10–10 to 10–6 mol/l), and endothelium derived hyperpolarizing factor (EDHF) pathway-mediated relaxations by adding L-nitroarginine 10–4 mol/l (a reversible NO pathway inhibitor).

In the CPB group, endothelium-dependent relaxation to serotonin and bradykinin were also studied in the presence or absence of: (1) in vitro BH4 (150 U/ml) added in the organ chamber 45 min before exposure to PGF2{alpha} (N = 5), or (2) in vitro superoxide dismutase (SOD, 150 U/ml) and catalase (CAT, 1200 U/ml) added in the organ chamber 5 min before exposure to PGF2{alpha} (N = 4).

At the end of each experiment, endothelium-independent relaxations were studied with sodium nitroprusside (SNP, 10–5 mol/l, a nitric oxide donor) to study the integrity of vascular smooth muscle cells.

2.1.1 Epicardial coronary artery cyclic guanosine monophosphate (cGMP) levels
Basal cGMP level in epicardial coronary arteries of the four groups was measured to assess NO bioavailability. Segments were collected after sacrifice, frozen in liquid nitrogen, stored at –70 °C, pulverized, resuspended in trichloracetic acid solution (6.25%, w/v), centrifuged, washed with diethylether and heat dried with nitrogen to obtain purified cGMP. cGMP levels were measured using a non-acetylation enzyme immunoassay system based on rabbit anti-cGMP antibody (Amersham Pharmacia Biotech, Baie d’Urfé, Que., Canada) which were adjusted to the quantity of proteins measured in the tissue using the Bradford microassay technique (Bio-Rad Laboratories, Hercules, CA, USA).

2.2 Epicardial coronary artery protein carbonyl levels
Protein carbonyl content of the four groups was measured as a marker of protein oxidation and oxidative stress. Vessels were homogenized in cold buffer, centrifuged, the supernatant removed, frozen and stored. Protein carbonyl content was measured with an assay kit (Cayman Chemicals, Ann Arbor, MI, USA), which was adjusted to the quantity of proteins as with cGMP levels.

2.3 Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM). For each ring, ‘N’ refers to the number of animals from which blood vessels were taken. ANOVA studies were performed to compare concentration–response curves. E max represent the maximal relaxation achieved. Student's t-test was used for the comparison of the cGMP content. Differences were considered to be statistically significant when p < 0.05. The statistical analyses were performed using SAS (SAS Institute, Cary, NC).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 Vascular reactivity studies
3.1.1 Contraction
There was no difference in the amplitude of the contraction to potassium chloride (60 mmol/l) between the groups (Table 2 ). The amplitude of contraction to PGF2{alpha} was significantly higher in the control group (p = 0.03) and lower in the CPB with in vivo BH4 group (p = 0.04), but there was no difference in the concentration of PGF2{alpha} needed to achieve the target level of contraction.


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  		Table 2. Contraction to potassium chloride and prostaglandin F2{alpha} of porcine coronary arteries after cardiopulmonary bypass
 
3.1.2 Relaxations
There was a statistically significant decrease in endothelium-dependent relaxation to serotonin (E max [5HT]: 53 ± 4% versus 28 ± 4% for control, p < 0.0001) (Fig. 1A–C) and to UK14304 (p < 0.0001) (Fig. 2A) in coronary rings following CPB compared with controls. Endothelium-dependent relaxation to bradykinin was decreased in the CPB group compared with control (p < 0.001) (Fig. 1B–D). No difference in the maximal relaxation to bradykinin was observed between groups, but the CPB group reached maximal relaxation at a higher bradykinin concentration compared with control. There was no significant difference in the endothelium-dependent relaxation to serotonin and bradykinin in coronary rings following CPB with or without brief cardioplegic arrest (Fig. 1).


Figure 1
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  		Fig. 1. (A and B) Cumulative concentration–response curves to the endothelium-dependent vasodilator serotonin (5HT) or bradykinin (BK) in rings of porcine coronary arteries following CPB with or without in vitro BH4. (C and D) Cumulative concentration–response curves to the endothelium-dependent vasodilator 5HT or BK in rings of porcine coronary arteries following CPB and brief cardioplegic arrest with or without in vivo BH4. Responses are given as a percentage of maximal contraction to prostaglandin F2{alpha} or 5HT (% E max). Results are presented as mean ± SEM. CPB: cardiopulmonary bypass; BH4: tetrahydrobiopterin.

 

Figure 2
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  		Fig. 2. (A) Cumulative concentration–response curves to the endothelium-dependent vasodilator UK14304 in rings of porcine coronary arteries. (B) Cumulative concentration–response curves to the endothelium-dependent receptor-independent vasodilator calcium ionophore (A23187) in rings of porcine coronary arteries. (C) Cumulative concentration–response curves to bradykinin (BK) with L-nitroarginine (L-NA) (EDHF pathway). Responses are given as a percentage of maximal contraction to prostaglandin F2{alpha} (% E max). Results are presented as mean ± SEM. CPB: cardiopulmonary bypass; BH4: tetrahydrobiopterin.

 
There was a statistically significant decrease in endothelium-dependent relaxation to the receptor-independent agonist calcium ionophore A23187 in the CPB group (p < 0.01) with no significant difference in maximal relaxations (Fig. 2B).

There was a statistically significant shift to the right of the concentration–response curve to BK with L-nitroarginine and indomethacin (EDHF pathway) in the control group and no statistically significant difference in the endothelium-dependent relaxation to BK with L-nitroarginine and indomethacin in the CPB group (Fig. 2C). There was a statistically significant increase in EDHF-mediated endothelium-dependent relaxation in the CPB group when compared to the control group (p < 0.0001).

Finally, there was no statistically significant difference in the endothelium-independent relaxation to SNP between groups with all rings achieving 100% relaxation (data not shown). Furthermore, histological examination demonstrated preservation of the endothelial coverage after CPB.

3.2 Effect of tetrahydrobiopterin (BH4)
In vitro and in vivo BH4 supplementation significantly improved endothelium-dependent relaxation to serotonin following CPB (E max [5HT]: 45 ± 3%, p = 0.0001) (Fig. 1A–C). There was a statistically significant difference between the CPB groups with BH4 and the control group (p < 0.0001). In vitro BH4 supplementation also improved endothelium-dependent relaxation to bradykinin (p = 0.005) in the CPB group with no significant difference between the CPB group with BH4 and the control group (Fig. 1B). However, this difference was less apparent with in vivo BH4 administration (Fig. 1D). There was no statistically significant difference between in vitro and in vivo BH4 administration groups for endothelium-dependent relaxations to serotonin and to bradykinin.

3.3 Effect of in vitro SOD-CAT
There was no significant difference in the maximal relaxation achieved between CPB group with SOD-CAT and CPB group without SOD-CAT (Fig. 3A and B).


Figure 3
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  		Fig. 3. Cumulative concentration–response curves to the endothelium-dependent vasodilator serotonin (5HT) or bradykinin (BK) in rings of porcine coronary arteries with or without in vitro superoxide dismutase (SOD) and catalase (CAT). Responses are given as a percentage of maximal contraction to prostaglandin F2{alpha} or 5HT (% E max). Results are presented as mean ± SEM. CPB: cardiopulmonary bypass.

 
3.4 Epicardial coronary artery cyclic guanosine monophosphate (cGMP) levels
Bioavailability of NO, assessed by cGMP levels, was significantly decreased in the CPB group with or without brief cardioplegic arrest compared with the control group (Fig. 4A; p = 0.002). In vivo BH4 administration restored vascular cGMP content (p < 0.001 compared with the untreated CPB groups) with no difference between the CPB group with in vivo BH4 and the control group.


Figure 4
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  		Fig. 4. (A) Epicardial coronary artery cyclic guanosine monophosphate levels, a marker of nitric oxide biodisponibility, in the control group, CPB group, CPB group with brief cardioplegic arrest and CPB group with brief cardioplegic arrest and in vivo BH4. (B) Epicardial coronary artery protein carbonyl levels, a marker of oxidative stress, in the same four groups. Results are presented as mean ± SEM. CPB: cardiopulmonary bypass; BH4: tetrahydrobiopterin.

 
3.5 Epicardial coronary artery protein carbonyl levels
Protein carboxyl levels, a marker of oxidative stress, were significantly increased following CPB with or without brief cardioplegic arrest compared with the control group (Fig. 4B; p < 0.001). In vivo BH4 administration significantly decreased the protein oxidation levels (p < 0.001 compared with the untreated CPB groups) with no difference between the CPB group with in vivo BH4 and the control group.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
The major findings of the present study are the following: (1) Gi- and Gq-protein-mediated endothelium-dependent relaxations to serotonin, bradykinin and UK14304, as well as receptor-independent endothelium-dependent relaxations to calcium ionophore A23187 and EDHF pathway-mediated relaxations were significantly altered following CPB with preserved endothelial coverage and vascular smooth muscle integrity; (2) cGMP content, a marker of nitric oxide bioavailability, was decreased and oxidative stress was increased following CPB; (3) BH4 treatment improved the Gi- and Gq-protein-mediated endothelium-dependent relaxations to serotonin and bradykinin, restored the cGMP levels and decreased the oxidative stress.

4.1 Cardiopulmonary bypass induced endothelial dysfunction
The vasoregulatory function of the endothelium could be affected in many ways following CPB: (1) impairment of G-proteins and/or their respective receptors, (2) decreased release or production of vasodilators, such as NO, prostacyclin and/or EDHF, (3) increased destruction or scavenging of NO, (4) increased production of vasoconstrictors, including endoperoxides, reactive oxygen species and/or endothelin-1, (5) resistance of the vascular smooth muscle to vasodilators and (6) increased sensitivity of the vascular smooth muscle to vasoconstrictors [3].

4.1.1 Relaxation—NO pathway
In this swine model of CPB, endothelium-dependent relaxations mediated by G-proteins were impaired although those mediated by Gi-protein (serotonin, UK14304) were altered to a lesser degree than those mediated by pertussis toxin sensitive Gq-proteins (bradykinin). This preferential involvement of the Gi-protein mediated pathway is similar to that observed in CPB with prolonged cross-clamping (ischemia-reperfusion model) in the canine model [4,5] and other porcine cardiovascular pathology models, such as regenerated endothelium, hypercholesterolemia, chronic atherosclerosis [17] and rejection after heart transplantation [18] in which there is an early selective involvement of endothelium-dependent relaxations mediated by Gi-proteins with later alteration of the Gq-protein mediated pathway as the endothelial dysfunction progresses.

Endothelium-dependent relaxation to the receptor-independent agonist calcium ionophore A23187, which represents the global capacity for eNOS to release NO, was significantly lower, suggesting an alteration of intracellular calcium release mechanisms, but the global relaxation capacity was maintained.

Our results differ from the only two studies we found on the endothelial function of coronary arteries with CPB alone, without aortic cross-clamping [4,5]. These experiments were performed using canine models and the exact CPB duration was not specified (between 45 and 150 min). No significant impairment of endothelium-dependent relaxations to serotonin, adenosine diphosphate (coupled with Gq-protein), calcium ionophore A23187 or sodium fluoride (a direct activator of G-proteins) were demonstrated. One possible explanation for these differences could be the increased coronary collateralization present in the canine model compared with the porcine model and an increased susceptibility to injury of the latter [19].

4.1.2 Relaxation—EDHF pathway
EDHF is a diffusible factor different from NO which causes endothelium-dependent relaxations by hyperpolarization of smooth muscle cells mediated by an increase influx of K+ ions through ATP dependent K+ channels [3]. The exact role of EDHF remains hypothetical but this pathway may become upregulated and serve as a backup mechanism to maintain endothelium-dependent vasodilator function under condition of impaired NO release or bioavailability [20]. The results of the present study show that the EDHF pathway became predominant in the non-physiological conditions associated with CPB perfusion.

4.2 Effects of tetrahydrobiopterin
BH4 helps to stabilize the dimeric active form of NO synthase by increasing its affinity to L-arginine and is a kinetically privileged source of electrons for this reaction [21]. Decreased concentration of endogenous BH4 leads to uncoupling of NO synthase with its substrate L-arginine, which in turn leads to decreased NO production and a shift towards increased production of hydrogen peroxide and other reactive oxygen species by NO synthase [10]. Endothelial cells activation and disruption by reactive oxygen species eventually trigger direct smooth muscle vasoconstriction. Increased levels of oxygen free radicals may also alter the involvement of BH4 in the oxidation process of NO synthesis from L-arginine and affect BH4 biosynthesis and recycling by depletion of NADPH. The antioxidants properties of BH4 are explained by: (1) coupling/activation of NO synthase and inhibition of NO negative retroaction on NO synthase, (2) dose-dependent direct inhibition of superoxide anion production by hypoxanthine/xanthine oxidase [22], and (3) scavenging of free radicals by the direct redox effect of BH4 [23].

In this study, the tested hypothesis was that BH4 endogenous concentration is decreased during CPB leading to an uncoupling of NOS and increased oxidative stress, and that supplementation of BH4 improved the endothelial dysfunction of coronary arteries secondary to CPB (Fig. 5 ). Both in vitro and in vivo BH4 administration partially restored the endothelium-dependent relaxations mediated by Gi-proteins, shown by a significant improvement of maximal relaxations to serotonin, and completely restored those mediated by Gq-proteins. Incomplete reversal of the endothelial dysfunction could be explained by impairment at the level of receptor, Gi-proteins or intracellular cascade, which are not directly addressed by BH4 supplementation.


Figure 5
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  		Fig. 5. eNOS uncoupling caused by endogenous tetrahydrobiopterin (BH4) depletion and increased oxidative stress following cardiopulmonary bypass and their effect on vasomotor tone. 5HT: serotonin; (+): stimulate; (–): inhibition; BH4: tetrahydrobiopterin; BK: bradykinin; CPB: cardiopulmonary bypass; eNOS: endothelial NO synthase; Gi/Gq: Gi/Gq protein complex and their respective receptors; H2O2: hydrogen peroxide; L-Arg: L-arginine; L-Cit: L-citrulline; NO: nitric oxide; O2: oxygen; OONO–: peroxynitrites.

 
There was no demonstrable effect of the antioxidant SOD-CAT for the endothelium-dependent relaxations mediated by Gi- and Gq-proteins. In this CPB model, SOD-CAT may have opposing effects as with spontaneous hypertensive rats [24] or become ineffective with endogenous BH4 depletion.

4.3 Nitric oxide bioavailability and oxidative stress
The coronary artery cGMP content, a marker of NO bioavailability, was lower after CPB which could be due to: (1) increased catabolism of NO by free radicals generated during CPB, as shown by the increase in protein oxidation, (2) decreased biosynthesis of NO by negative feedback of NO on NO synthase or cofactor depletion, such as L-arginine or endogenous BH4, as shown with the improvement in both NO bioavailability and oxidative stress with in vivo BH4 administration. Shiraishi et al. [25] have shown that cGMP inhibit GTP cyclohydrolase I activity, the rate-limiting enzyme for the biosynthesis of BH4. The decreased cGMP levels following CPB would normally lead to stimulation of GTP cyclohydrolase I activity and BH4 biosynthesis. However, BH4 is only active in its reduced state and is autooxided in the presence of high concentrations of oxygen free radicals, rendering endogenous BH4 insufficient in marked oxidative states.

4.4 Etiologic hypothesis
Global ischemia without reperfusion is not associated with a significant impairment of endothelium-dependent relaxations of coronary arteries [1,6]. However, reperfusion following ischemia impairs vasorelaxation responses in coronary arteries. Since no prolonged aortic cross-clamp was applied, there was no reperfusion except from the pulmonary vasculature and distal less perfused vascular beds. We therefore speculate that CPB perfusion per se produces toxic metabolites, cytokines and oxygen-derived free-radicals, which are involved in a systemic endothelial activation leading to significant alterations of receptors, G-proteins and intracellular enzymes such as NOS.

4.5 Limitations
The following limitations to this study need to be addressed. First, this is an acute healthy animal study and although earlier experiments suggest a close relationship between porcine and human endothelial responses, these data should be interpreted with the proper caution. Second, in order to specifically assess the response to CPB, no prolonged cardioplegic arrest or heparin reversal with protamine were used. Other experiments are needed to assess the impact of these interventions. Third, a sham group and a control group with BH4 supplementation were not included in our protocol since our previous studies have shown no difference between concentration–response curves of the control and sham [9] or control with BH4 [14] groups in the same swine model.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
In summary, CPB is associated with a selective dysfunction of porcine coronary endothelium-dependent relaxations mediated by pertussis-toxin sensitive Gi-proteins, and to a lesser extent, to pertussis-toxin insensitive Gq-proteins. In vitro and in vivo BH4 administration improves the endothelium-dependent relaxations mediated by Gi-proteins and Gq-protein, restores nitric oxide bioavailability and reduces the oxidative stress. In vivo administration of BH4 in the circuit priming or cardioplegia may prevent endothelial dysfunction caused by toxic metabolites and oxygen-derived free-radicals secondary to CPB perfusion.


   Acknowledgments
 
This work was supported by the ‘Fondation de l’Institut de Cardiologie de Montréal’ (FICM), and Department of Surgery of the Université de Montréal. Dr Louis P. Perrault is a Research Scholar Junior 2 from the ‘Fonds de la recherche en santé du Québec’. The authors would like to thank Mme Marie-Pierre Mathieu for her skilful technical assistance and Mme Annik Fortier for her statistical expertise.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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