Specific Potentiation of Endothelium-Dependent Contractions in SHR by Tetrahydrobiopterin
This study was designed to determine the effect of pteridines, R- and S-tetrahydrobiopterin, sepiapterin, and dihydrobiopterin on endothelium-dependent contractions to acetylcholine in isolated aortas from spontaneously hypertensive rat and normotensive Wistar-Kyoto rat. The noncumulative addition of redox-active pteridines R- and S-tetrahydrobiopterin (but not the oxidized analogues sepiapterin and dihydrobiopterin) produced a concentration-dependent transient contraction in isolated aortic rings from both normotensive and hypertensive rats. R- and S-tetrahydrobiopterin (but not sepiapterin or dihydrobiopterin) potentiated the endothelium-dependent contractions to acetylcholine but only in aortas from hypertensive rats and in the presence of NG-nitro-l-arginine. In these aortas, the generation of oxygen-derived free radicals by the combination of xanthine plus xanthine oxidase also potentiated the endothelium-dependent contractions to acetylcholine. The presence of R-tetrahydrobiopterin did not alter the characteristics of the endothelium-dependent contractions because they were inhibited by valeryl salicylate, an inhibitor of cyclooxygenase-1, by S18886, a TP-receptor antagonist or by Tiron, a cell permeable superoxide anion scavenger. However, the contractions to acetylcholine, which are unaffected by the combination of superoxide dismutase and catalase, become significantly inhibited by these two scavengers in the presence of R-tetrahydrobiopterin. In the presence of NG-nitro-l-arginine, R-tetrahydrobiopterin did not affect the contractions to phenylephrine, U 46619, or to oxygen-derived free radicals generated by xanthine plus xanthine oxidase. These results indicate that the production of superoxide by the autoxidation of tetrahydrobiopterin selectively enhances endothelium-dependent contractions in the spontaneously hypertensive rat when nitric oxide synthase is inhibited.
In the spontaneously hypertensive rat (SHR), endothelium-dependent relaxation to acetylcholine is impaired by the occurrence of a concomitant endothelium-dependent contraction attributed to the release of an endothelium-derived contracting factor(s) (EDCF).1 These contractions involve reactive oxygen species that could either scavenge nitric oxide or/and directly contract the vascular smooth muscle cells.2–5 In the presence of proper cofactors, endothelial nitric oxide synthase (NOS) produces l-citrulline and nitric oxide from l-arginine and molecular oxygen. A deficit in tetrahydrobiopterin (BH4), an essential cofactor for the activity of the NOS, uncouples l-arginine oxidation and oxygen reduction, causing an increased production of superoxide anions.6–7 Conversely, the supplementation with BH4 favors the production of nitric oxide from endothelial NOS.8 The administration of BH4 improves endothelial dysfunction not only in animal models of hypertension and diabetes but also in patients with atherosclerosis and hypercholesterolemia.9–12 Furthermore, exogenous BH4 attenuates the production of superoxide anions from endothelial NOS in aortas from prehypertensive as well as hypertensive rat.13,14 Taken in conjunction, these studies suggest a possible link between dysfunctional endothelial NOS, associated with the availability of BH4, and endothelium-dependent contractions. This study was designed to determine whether or not BH4 affects endothelium-dependent contractions to acetylcholine in the isolated aorta of the SHR.
Experiments were performed on thoracic aortas from 35-week-old male SHR and normotensive Wistar-Kyoto rats (WKY) of similar weight (352±10 and 364±8 g, n=58 and 14 for SHR and WKY, respectively). The rats were anesthetized with sodium pentobarbital (50 mg/kg IP), and the blood pressure was measured from the carotid artery (systolic blood pressure, 207±8 and 119±6 mm Hg, n=58 and 14 in SHR and WKY, respectively; P<0.05). The aorta was then dissected free, excised, and placed in cold modified Krebs-Ringer bicarbonate solution of the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2SO4 1.2, NaHCO3 25.0, and edetate calcium di-sodium 0.026; glucose 11.1 (control solution). The blood vessels were cut into rings (4 to 5 mm in length). In some preparations, the endothelium was removed by inserting the tip of a small forceps in the rings and rolling them back and forth on a wetted paper towel.1 The rings were suspended in organ chambers, which contained control solution (37°C) aerated with 95% O2 and 5% CO2, and were connected to a force transducer for the recording of isometric force. The rings were stretched progressively to reach the optimal point of their length–active tension relation (≈2g). The presence of an intact endothelium was confirmed by relaxation to thrombin (1 U/mL) in rings contracted with phenylephrine (10−7 mol/L). Drug incubation time was 45 minutes for most of the experiments. Oxygen-derived free radicals were generated by xanthine plus xanthine oxidase. Xanthine was added 10 minutes before xanthine oxidase. Concentration-response curves were obtained in a cumulative manner. Changes in tension were expressed as a percentage of the reference contraction to KCl (60 mmol/L) performed for each individual ring at the beginning of the experiment.
Acetylcholine hydrochloride, catalase, dithiothreitol (DTT), NG-nitro-l-arginine, NG-nitro-l-arginine methyl ester, papaverine, phenylephrine, superoxide dismutase, thrombin, Tiron (4,5-dihydroxy-1,3-benzene-disulfonic acid), xanthine, and xanthine oxidase were purchased from Sigma Chemical Company. S 18886 (3-[(6-amino-(4-chlorobenzensulfonyl)-2-methyl-5,6,7,8-tetrahydronapht]-1-yl)propionic acid) was synthesized at the Institut de Recherches Servier. Valeryl salicylate was purchased from Cayman Chemical Company. Sepiapterin, (6R)-tetrahydrobiopterin, (6S)-tetrahydrobiopterin (R-BH4 and S-BH4), 7,8-dihydrobiopterin (BH2) and U46619 (9,11-dideoxy-9α,11α-epoxymethano prostaglandin F2α) were purchased from Alexis Biochemicals. Drug concentrations are expressed as final molar concentrations in the bath solution.
Data are expressed as mean±SEM; n refers to the number of rats from which the aortas were taken. Statistical analysis was performed by 2-tailed Student t test for control and treatment comparisons and by ANOVA1 or ANOVA2 analysis for multiple comparisons followed by a Newman-Keuls or a Bonferroni post hoc test, respectively, where appropriate. Differences were considered to be statistically significant at a value of P<0.05.
Intrinsic Effects of Pteridines
In quiescent isolated aortic rings with endothelium taken from SHR, the noncumulative addition of R-BH4 (10−5, 10−4, and 5×10−4 mol/L) caused a transient contraction, with a return to basal tension in <40 minutes (Figure 1). This effect was mimicked by S-BH4 (10−4 mol/L) but not by sepiapterin or BH2 (10−4 mol/L). The presence of NG-nitro-l-arginine (10−4 mol/L) did not significantly affect this transient increase in tension; however, removal of the endothelium increased the amplitude of the transient contraction produced by either R- or S-BH4 (Table 1).
The contractions to R-BH4 (10−4 mol/L) were significantly smaller in aortas from WKY when compared with SHR (changes in tension in percentage of KCl: 60 mmol/L, rings with endothelium: 6.8±0.1.0% and 12.2±0.2.3% in the absence and presence of NG-nitro-l-arginine, respectively; rings without endothelium: 12.8±3.8% and 9.1±1.1% in the absence and presence of NG-nitro-l-arginine, respectively; n=6, P<0.05 when compared with SHR rings).
In SHR, the contractions to R-BH4 (10−4 mol/L) were significantly reduced by either indomethacin (5×10−6 mol/L), valeryl salicylate (3×10−3 mol/L), or S18886 (10−7 mol/L) (Table 1).
Pteridines and Endothelium-Dependent Contractions
Acetylcholine (10−8 to 10−4 mol/L) evoked endothelium-dependent contractions in aortas of SHR but not WKY. Incubation of the isolated rings with R-BH4 (10−4 mol/L) for 40 minutes before the addition of acetylcholine did not significantly affect the response in either SHR or WKY aortas (Figure 2).
In the presence of NG-nitro-l-arginine (or NG-nitro-l-arginine methyl ester: 10−4 mol/L, data not shown), the endothelium-dependent contractions to acetylcholine were augmented significantly in aortas from SHR and unmasked in those of WKY (Figure 2). Under these conditions, the presence of R-BH4 (10−4 mol/L) further potentiated the acetylcholine-dependent contractions in aortas of SHR but not in those of WKY (Figure 2). However, this potentiation produced by R-BH4 was not concentration-dependent, as at a lower concentration R-BH4 (10−5 mol/L) did not significantly influence acetylcholine-induced endothelium-dependent contractions, whereas at the highest concentration tested R-BH4 (5×10−4 mol/L) significantly inhibited the contraction produced by acetylcholine (Figure 3). This inhibitory effect was not specific, as the contraction to phenylephrine (10−9 to 10−4 mol/L) was also significantly inhibited by R-BH4 at this high concentration (maximal response to phenylephrine: 153±7% and 105±17% of the reference contraction to KCl for control and R-BH4-treated rings, respectively; n=4, P<0.05, ANOVA2 followed by a Bonferroni post hoc test).
Similarly, in the SHR, S-BH4 (10−4 mol/L) produced a significant potentiation of the endothelium-dependent contractions in response to acetylcholine in the presence of NG-nitro-l-arginine. In contrast, sepiapterin (10−4 mol/L) produced only a small but significant increase in the maximal amplitude of the endothelium-dependent contraction to acetylcholine without producing a shift in the concentration-response curve (maximal response to acetylcholine: 61.3±8.5% and 76.0±9.6% of the reference contraction to KCl for control and sepiapterin-treated rings, respectively; n=8, P<0.05, ANOVA2 followed by a Bonferroni post hoc test), whereas BH2 (10−4 mol/L) did not affect the endothelium-dependent contractions to acetylcholine in the presence or the absence of NG-nitro-l-arginine (Figure 4).
Characteristics of R-BH4–Induced Potentiation of Endothelium-Dependent Contraction to Acetylcholine in SHR
Experiments were performed in SHR aortas with endothelium in the presence of NG-nitro-l-arginine (10−4 mol/L).
The endothelium-dependent contractions to acetylcholine were abolished by valeryl salicylate (3×10−3 mol/L) or S18886 (10−7 mol/L). The inhibitory effect of the preferential cycloxygenase-1 inhibitor (or indomethacin: 5 μmol/L, data not shown) or of the antagonist of TP receptor was not affected by the presence of R-BH4 (10−4 mol/L) (Figure 5).
The endothelium-dependent contractions to acetylcholine were not significantly affected by the combination of superoxide dismutase (120 U/mL) and catalase (1200 U/mL) but were partially inhibited by Tiron (10−2 mol/L). In the presence of R-BH4 (10−4 mol/L), both Tiron and the combination of superoxide dismutase plus catalase produced a significant inhibition of the endothelium-dependent contraction to acetylcholine (Figure 6).
Specificity of R-BH4–Induced Potentiation of Endothelium-Dependent Contractions
In rings with or without endothelium taken from SHR and studied in the presence of NG-nitro-l-arginine (10−4 mol/L), the addition of R-BH4 (10−4 mol/L) did not significantly affect the concentration-dependent contractions to phenylephrine (10−9 to 10−4 mol/L), U46619 (10−10 to 10−6 mol/L), or to oxygen-derived free radicals generated by the combination of xanthine (10−4 mol/L) plus xanthine oxidase (0.001 to 0.03 U/mL) (Table 2).
Reducing Agent, Free Radical Production, and Endothelium-Dependent Contractions
In rings with endothelium [from SHR and in the presence of NG-nitro-l-arginine (100 μmol/L)] dithiothreitol (3×10−6, 3×10−5, and 3×10−4 mol/L) did not significantly influence the basal tone (data not shown). Dithiothreitol (3×10−6 and 3×10−5 mol/L) did not significantly affect the endothelium-dependent contraction to acetylcholine, whereas at the highest concentration tested (3×10−4 mol/L), it produced a significant inhibition (Figure 3). This elevated concentration of the reducing agent also produced an inhibition of phenylephrine-induced contraction (maximal response to phenylephrine: 162±8% and 83±14% of the reference contraction to KCl for control and dithiothreitol-treated rings, respectively; n=5, P<0.05, ANOVA2 followed by a Bonferroni post hoc test).
In aortas of SHR with endothelium, in the presence of NG-nitro-l-arginine (10−4 mol/L), oxygen-derived free radicals generated from xanthine (10−4 mol/L) plus xanthine oxidase (0.003 U/mL) produced a transient increase in tension (36.8±7.0% of the reference contraction to KCl, n=5). In the presence of xanthine plus xanthine oxidase, the concentration response-curve to acetylcholine was significantly shifted to the left (Figure 6), whereas that to phenylephrine was not affected (data not shown).
R-BH4 and Endothelium-Dependent Relaxation
In rings contracted with phenylephrine from both SHR and WKY, acetylcholine (10−9 to 10−4 mol/L) induced a biphasic response: relaxation at the lower concentrations of acetylcholine (up to 3×10−7 mol/L) and contractions for higher concentrations. The relaxation observed in SHR was significantly smaller than in WKY, whereas the delayed contraction was significantly larger (ANOVA2 followed by a Bonferroni post hoc test, Figure 7). R-BH4 (10−4 mol/L) did not significantly affect the relaxation to acetylcholine in aortas from either WKY or SHR. However, the delayed contraction observed in SHR was significantly inhibited by R-BH4 (ANOVA2 followed by a Bonferroni post hoc test, Figure 7).
This study showed that in the presence of l-arginine derivatives, inhibitors of NOS, the acute addition of BH4 potentiates the endothelium-dependent contractions to acetylcholine in the aorta of the SHR but not in that from normotensive animals.
Endothelium-dependent contractions in response to acetylcholine are observed in SHR but not in WKY, except at an advanced age.1,15 However, in the presence of a NOS inhibitor, the response was observed in both strains, although the contractions remained significantly larger in SHR aortas when compared with WKY. These observations are consistent with the interpretation that nitric oxide inhibits and/or inactivates the putative EDCF.16 In conditions of substrate (l-arginine) or cofactor deficiency (BH4), the function of the NOS is altered, and the production of superoxide anion is favored.17,18 The acute or chronic administration of BH4 corrects endothelial dysfunction in various animal models of hypertension and diabetes but also in patients with atherosclerosis and hypercholesterolemia.9–12 This beneficial effect of BH4 is attributed to the attenuation of the production of superoxide anion from eNOS and therefore to an increase the availability of NO.13,14,19,20 However, in the SHR, in the presence of inhibitors of the NOS, BH4 did not decrease the endothelium-dependent contractions but produce a marked potentiation. Several interpretations may underlie these apparently divergent findings.
Reducing Properties of BH4
BH4 is a potent reducing agent.20,21 However, the intrinsic effect of BH4, a concentration-dependent contraction, is not mimicked by another powerful reducing agent dithiothreitol. Furthermore, the effects of BH4 on the endothelium-dependent contractions in the SHR aorta are also significantly different than the effects of dithiothreitol. Indeed, dithiothreitol did not potentiate the acetylcholine-induced contractions. However, at the highest concentration tested, both compounds produced a significant but nonselective inhibition of the contractile responses. The mechanism of this nonspecific inhibition has not been explored in the current study but could be linked to the reducing properties of both compounds. However, the obvious conclusion reached from comparing the effects of BH4 and dithiothreitol is that the potentiation of the endothelium-dependent contractions seen with the former is unlikely to be due to its reducing properties.
Superoxide Generation by eNOS
One possible paradoxical explanation for the effect of BH4 could have been an increased production of superoxide by the eNOS itself. In the presence of inhibitors of eNOS, such as l-arginine analog (but not in presence of heme iron ligands), the isolated eNOS enzyme theoretically still has the ability to produce superoxide anions,17,22 although several studies involving isolated blood vessels conclude that l-arginine analogs inhibit the eNOS-dependent production of superoxide anion.14,23 However, presuming that under the present experimental conditions the eNOS of the SHR was able to produce superoxide anion in the presence of l-arginine analogs, the increase in the acetylcholine-induced endothelium-dependent contraction by the presence of BH4 would still be unexpected since this cofactor favors the production of nitric oxide by eNOS instead of superoxide anion.17 One possible explanation could be that the autoxidation of BH4 in the oxygenated Krebs-Ringer solution yields BH2.24 This stable oxidation product of BH4 acts as a competitive antagonist of BH4 at the eNOS level25 and therefore could shift the balance toward an increase in superoxide production.22 Similarly, sepiapterin not only is a precursor for BH4 synthesis, through the so-called salvage pathway, but can also antagonize the effects of BH4 and increase superoxide formation by eNOS.22 However, neither BH2 nor sepiaterin mimicked the effects of BH4, ruling out that the potentiating effect of BH4 involves an augmented generation of superoxide anions by eNOS.
Superoxide Production by the Autoxidation of BH4
The autoxidation of BH4 in oxygenated buffers is a source of superoxide anions.24,26,27 Production of superoxide anion by xanthine plus xanthine oxidase causes contractions in SHR in vessels with and without endothelium,2,5 and the production of superoxide linked to BH4 autoxidation produces endothelium-dependent contractions in the canine basilar artery.26 In the current study, exogenous BH4 per se caused a transient increase in tension in quiescent rings with or without endothelium from both SHR and WKY. These contractions are produced only by pteridines susceptible to autoxidation (R- and S-BH4) but not by the redox-inactive, oxidized BH4 analogues BH2 and sepiapterin. Furthermore, these contractions had the same pharmacological characteristics as those produced by oxygen-derived free radicals,2,5,28 as they were inhibited by indomethacin, valeryl salicylate, a preferential inhibitor of cyclooxygenase-1,29 or S 18886, a selective TP receptor antagonist.30 Interestingly, the potentiation of the endothelium-dependent contraction was observed with the two pteridines susceptible of autoxidation, R- and S-BH4, but not with the oxidized BH4 analogs BH2 and sepiapterin. Furthermore, the production of superoxide anions by the combination of xanthine plus xanthine oxidase mimics the effects BH4 as it produced a contraction per se and potentiated the endothelium-dependent contractions. Finally, the endothelium-dependent contractions observed in the SHR in the presence of both NG-nitro-l-arginine and BH4 are sensitive to Tiron (as the response observed in the absence of BH4) but are also sensitive to the combination of the scavengers of the oxygen-derived free radical scavengers superoxide dismutase and catalase, whereas the endothelium-dependent contractions observed in the absence of BH4 are not.2,5 Altogether, these results indicate that in the SHR aorta, superoxide anions, produced by the autoxidation of BH4, potentiate the endothelium-dependent contraction to acetylcholine.
Importance of the Presence of NG-Nitro-l-Arginine
In the SHR aorta, the potentiation produced by BH4 is observed only after inhibition of NOS. This is probably because, beside autoxidation, the acute addition of the pteridine also improves eNOS function. In the SHR, the endothelium-dependent relaxation to acetylcholine is significantly increased by BH4, especially for the highest concentrations of the muscarininic agonist, those that also provoke endothelium-dependent contractions. In WKY, the endothelium-dependent relaxation to acetylcholine is not affected by the presence of BH4, confirming earlier studies.9,12 Therefore, in the SHR aorta, in the absence of l-arginine analogs, the beneficial effect of BH4 on eNOS compensates for the detrimental production of superoxide anion resulting from the autoxidation of the pteridine.
Selectivity for the SHR
The aortas of WKY are less sensitive to oxygen-derived free radicals than are those of SHR. The contractions to BH4, as those caused by free radical generation by xanthine and xanthine oxidase, are smaller in the aorta from the WKY than in that from the SHR.2,15,28 Although the molecular mechanism linked to the higher resistance of WKY has not been directly assessed in the present study, this most certainly explains why BH4, at the concentration tested, potentiates endothelium-dependent contractions in SHR but not in WKY aorta.
This study confirms the important role of redox phenomena in the control of vascular tone and its potential role in vascular diseases. The potentiating effect of BH4 was fully endothelium-dependent and not observed during responses to various endothelium-independent vasoconstrictors (an α1-adrenergic agonist, a TP receptor agonist, and oxygen-derived free radicals). Furthermore, the augmented contractions observed in the presence of BH4 conserved the characteristics of EDCF-mediated responses because they were abolished by valeryl salicylate, a preferential inhibitor of cyclooxygenase-1 or S 18886, a selective TP receptor antagonist, and partially inhibited by Tiron, a scavenger of superoxide anions. Thus, BH4 must selectively facilitate the release of EDCF in the SHR aorta.
The potentiating effect of BH4 on the endothelium-dependent contraction of SHR aorta may be helpful to elucidate the nature of EDCF, for instance by facilitating its bioassay.
This work was supported by an international educational grant from Institut de Recherches Servier.
- Received September 19, 2002.
- Revision received October 24, 2002.
- Accepted November 7, 2002.
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