Hypercholesterolemia and atherosclerosis lead to an impaired endothelium-dependent vasorelaxation both in humans and in animal models (1-4). Bioassay studies have suggested that the mechanism underlying this defect is related to a reduced activity of endothelium-derived nitric oxide (NO; 3-6). NO, which has been characterized as the active principle of the endothelium-derived relaxing factor (EDRF; 7), is formed from the amino acid precursor L-arginine by the enzyme NO synthase (NOS) in vascular endothelial cells (8). It induces smooth-muscle cell relaxation by activating the soluble guanylyl cyclase to form the intracellular second messenger cyclic guanosine monophosphate (cGMP; 9,10).
Endothelial cells not only synthesize the vasodilator NO, but they also produce the vasoconstrictor endothelin (ET), a 21-amino-acid peptide (11). The ET family comprises three related peptides; ET-1, ET-2, and ET-3 (12). ET-1 is the major isoform produced in endothelial cells by the action of ET-converting enzyme (ECE) and exerts potent vasoconstrictor effects by binding to ET-A receptors on vascular smooth-muscle cells (11,13). ET-1 is present in plasma and urine. It has been reported that plasma levels and tissue immunoreactivity of this peptide are increased in animal models as well as in hypercholesterolemic and atherosclerotic patients (14-16). In these diseases, ET-1 concentrations are positively correlated with the extent of arterial lesion formation (15).
Experimental evidence suggests that there is a mutual interaction between the L-arginine/NO and ET-1 pathways. The vasoconstrictor and pressor actions of ET-1 are enhanced in the presence of an NOS inhibitor (17,18). Thrombin-stimulated ET-1 formation by isolated porcine aorta is inhibited by EDRF/NO (19). Lüscher et al. (20) demonstrated that EDRF released by stimulating human arteries with acetylcholine (ACh) or bradykinin reverses ET-1-induced contractions. A disturbed balance between these endothelial autacoids may also underlie the impaired endothelium-dependent vascular relaxation in hypercholesterolemic arteries. Several experimental studies suggest a role for L-arginine in inhibiting atherosclerosis (3,4,21). L-Arginine improves endothelium-dependent relaxation by enhancing NO production and by reducing superoxide anion generation (3,4).
This study was designed to investigate whether the vascular contraction induced by ET-1 is augmented in cholesterol-fed rabbits as compared with normal controls. Moreover, we studied whether dietary supplementation with L-arginine may reverse the enhanced contractility of hypercholesterolemic vessel segments to ET-1. Whole-body ET-1 synthesis rates were assessed noninvasively by measuring urinary excretion rates of immunoreactive ET-1.
Animals and study design
Male New Zealand White rabbits (24) initially weighing 1.5-2 kg were used in this study, which conformed with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication 85-23, revised 1985) and had been approved by the Hannover supervisory committee for studies in animals. After 2 weeks of adaptation, the rabbits were randomly divided into three groups of eight animals each. Rabbits in the first group were fed normal rabbit chow (Nohrlin 20 ZH5; Eggersmann, Germany) and plain tap water throughout the study; they served as a control group. The other two groups of rabbits were fed a diet containing 1% cholesterol (Altromin, Lage, Germany) for the first 4 weeks followed by 0.5% cholesterol (cholesterol group), or 0.5% cholesterol supplemented with 2% L-arginine in drinking water (cholesterol + L-arginine group) for the next 12 weeks. Animals were housed individually. Food and water were allowed ad libitum. Body weight and food and water consumption were measured at weekly intervals.
The rabbits were placed in metabolic cages for the collection of 24-h urine samples before the start of the dietary intervention and every 4 weeks thereafter. At the same times, blood samples were obtained from the central ear artery. Plasma was removed after centrifugation (1,500 g, 10 min), and stored at −20°C before measuring plasma L-arginine and cholesterol concentrations. At the end of the experimental period, the rabbits were killed. The aorta was isolated and immediately placed in fresh ice-cold Krebs buffer for the measurement of vascular function.
Urinary endothelin-1 and nitrate excretion rates
Urinary concentrations of immunoreactive (ir-) ET-1 were measured by using a specific radioimmunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA, U.S.A.). One milliliter of urine was acidified with 1.5 ml of 4% acetic acid. Extraction of ET-1 was accomplished by Sep-pak C-18 cartridge (Varian, Harbor City, CA, U.S.A.). ET-1 was eluted with 2 ml of 4% acetic acid in 86% ethanol. The eluates were evaporated to dryness and resuspended in 1 ml of radioimmunoassay buffer. Binding was initiated by adding 100 μl of anti-ET-1 and 100 μl of [125I]ET-1 into 200 μl of acetylated sample. Incubation was carried out in duplicate at 2°C for 18 h. A standard curve was prepared with various concentrations of standard ET-1 by using the same procedure as with the samples. Antibody-bound ET-1 was precipitated by using anti-rabbit coated cellulose, and the radioactivity of the pellet was counted in a γ counter (Berthold, Bad Wildbad, Germany). The detection limit of the assay was 1.48 fMol/ml; intra- and interassay variabilities were 4.5 and 6.8%, respectively. Cross-reactivity was 67% with ET-2, 84% with ET-3, and <3% with big-ET-1.
Urinary nitrate concentrations were determined by quantification of its pentafluorobenzyl (PFB) derivative by gas chromatography-mass spectrometry (GC-MS) as previously described (22). GC-MS analyses were performed on a MP 5890 Series II gas chromatograph interfaced with a MP 5989A MS engine (Hewlett Packard, Waldbronn, Germany) operating in the negative-ion chemical ionization (NICI) mode with methane as reagent gas (65 Pa). Helium was used as carrier gas at a pressure of 70 kPa. Quantitation was performed by selected ion monitoring at a mass/charge ratio (m/z) of 46 for endogenous nitrate and m/z 47 for the internal standard ([15N]NO3−). The detection limit of the method was 20 fmol nitrate/ml. Intra- and interassay variabilities were <3.8%.
Urinary and plasma creatinine concentrations were determined by using a commercially available creatinine reagent kit intended for use with the Beckman Creatinine Analyser (Beckman, Galway, Ireland). The urinary excretion rates of nitrate and ET-1 were expressed in relation to urinary creatinine concentration to limit variability due to changes in renal excretory function, as described previously (23). Urinary and plasma creatinine concentrations were used to calculate creatinine clearances.
Measurement of vascular function
The aortas were dissected free of adhering fat and connective tissue. The specimens were cut into rings ∼3 mm long, four rings per animal. During harvesting, special care was taken to avoid contact with the luminal surface of the rings to preserve the endothelium. The rings were placed into organ baths containing modified Krebs solution (pH 7.4), of the following composition (in mM): NaHCO3, 25; MgSO4, 0.6; NaCl, 120; KCl, 4.75; KH2PO4, 1.2; CaCl2, 1.28; glucose, 11; and EDTA, 0.026. The solution was maintained at 37°C and continuously aerated with a gas mixture consisting of O2/CO2 (95:5, vol/vol). The aortic rings were connected to force transducers (March, Germany) for the measurement of isometric tension. Each aortic ring was gradually stretched to a resting tension of 2 g and allowed to equilibrate for ≥60 min. The rings were then contracted with 1 μM noradrenaline (NA) and relaxed by 1 μM ACh for testing of endothelial integrity. Thereafter the vascular rings were repeatedly washed with Krebs buffer until the tension had returned to the previous baseline value. Aortic rings from the control group always showed >70% relaxation of the NA-induced contraction plateau in response to 1 μM ACh. Subsequently, cumulative concentration-response curves to ACh, sodium nitroprusside (SNP), NA, or ET-1 were obtained before and after incubation of aortic rings with the ET-A-receptor antagonist, BQ123 (1 μM; Saxon Biochemicals, Hannover, Germany), for 30 min. All contractions and relaxations were expressed as percentage of the contraction plateau induced by 1 μM NA. All drugs except BQ123 were purchased from Sigma (Munich, Germany).
Measurement of plasma cholesterol and L-arginine concentrations
Plasma total cholesterol concentrations were quantified by commercially available enzymatic methods (Boehringer-Mannheim, Mannheim, Germany). Plasma L-arginine levels were determined by high-performance liquid chromatography (HPLC) by using precolumn derivatization with o-phthalaldehyde (OPA; 24). The coefficients of variation of the method had previously been determined as 5.2% within assay and 5.5% between assay. The detection limit of the assay was 0.1 μM.
Calculations and statistical analyses
All values are given as mean ± standard error of the mean (SEM). Statistical analysis was performed by using one-way analysis of variance (ANOVA) followed by Fisher's protected least-significant difference test. Maximum responses and EC50 values of relaxations or contractions induced by ET, NA, ACh, or SNP were used for statistical comparison of differences in contraction or relaxation response of isolated aortic rings. EC50 values were calculated according to the method of Hafner et al. (25). A p value of <0.05 was considered significant.
Plasma cholesterol and L-arginine concentrations and creatinine clearances
Table 1 shows the lipid profiles and plasma L-arginine concentrations in the three experimental groups at baseline and at monthly intervals. There were no significant differences in plasma total cholesterol concentrations at baseline between the groups. High-cholesterol diet resulted in a significant increase in plasma total cholesterol levels. Supplementation with L-arginine had no effect on plasma total cholesterol concentrations.
The mean baseline plasma L-arginine concentration was 132.2 ± 4.6 μM with no significant differences between the groups. Plasma L-arginine levels increased about twofold during L-arginine supplementation (p < 0.05 vs. control and cholesterol groups), whereas they remained unchanged in the other two groups.
Mean creatinine clearances varied between 8 and 12 ml/min in the three groups of animals during the entire study period. No statistically significant differences were observed between any of the groups at any time (Table 1).
Urinary ir-ET-1 and nitrate excretion rates
At baseline, urinary excretion of ir-ET-1 was 1.6 ± 0.1 pmol/mmol creatinine with no significant differences between the three study groups (Fig. 1). Urinary ir-ET-1 excretion was significantly increased in the cholesterol-fed rabbits after 12 and 16 weeks (2.1 ± 0.3 and 2.0 ± 0.2 pmol/mmol creatinine, respectively; p < 0.05 vs. control). In the group supplemented with L-arginine, this increase in urinary ir-ET-1 was completely prevented (1.3 ± 0.1 and 1.5 ± 0.1 pmol/mmol creatinine at 12 and 16 weeks, respectively; each p < 0.05 vs. cholesterol). Urinary ir-ET-1 excretion in the cholesterol + L-arginine group was not significantly different from the control group at any time.
At baseline, mean urinary nitrate excretion was 767 ± 28 μmol/mmol creatinine with no significant differences between the groups (Fig. 2). After 4 weeks of high-cholesterol feeding, urinary nitrate excretion significantly decreased to ∼50% of baseline values (p < 0.05), whereas urinary nitrate excretion remained relatively unchanged in control rabbits. Urinary nitrate excretion remained on this reduced level in the cholesterol group throughout the study, whereas in the group supplemented with L-arginine from weeks 5-16, the decrement in urinary nitrate excretion was partially reversed. However, this effect of L-arginine was short-term but not long-term by nature (p < 0.05 vs. cholesterol at 12 and 16 weeks). In week 16, urinary nitrate excretion in the cholesterol + L-arginine group was ∼22% lower than in the control group, but some 43% higher than in the cholesterol group (Fig. 2).
The maximal vasoconstrictor response of isolated aortic rings to ET-1 was significantly increased in the cholesterol group as compared with the control group (p < 0.05; Fig. 3). Supplementation with L-arginine reduced the ET-1-induced constrictor response to control level (p = NS vs. control; p < 0.05 vs. cholesterol). Preincubation with BQ123 almost completely abolished ET-1-induced aortic contraction of rabbits in all groups. By contrast, there was no significant difference in the vasoconstrictor effect of NA among the three groups of animals (Table 2).
Endothelium-dependent relaxation of isolated rabbit aortic rings to ACh was severely impaired after cholesterol feeding, with the maximal ACh-induced relaxation reduced from 87.4 ± 2.4% (control) to 31.8 ± 5.1% (cholesterol). Relaxation to ACh was partly restored by supplementation with L-arginine (50.5 ± 2.5%; p < 0.05 vs. cholesterol; Fig. 4A). After preincubation with BQ123, the maximal vasorelaxant response to ACh of aortic rings from cholesterol-fed rabbits was increased (p < 0.05). By contrast, addition of BQ123 had no significant effect on the maximal vasorelaxant responses of isolated aortic rings from rabbits in the control and cholesterol + L-arginine groups (p = NS before and after incubation with BQ123). Endothelium-independent relaxation in response to SNP was not significantly different between any of the groups (Fig. 4B). The EC50 value for endothelium-dependent relaxation in response to ACh was significantly greater in the cholesterol group than in the control group; L-arginine supplementation reversed this difference (Table 2). The EC50 value for SNP-induced, endothelium-independent relaxation was not significantly different among any of the groups.
The results of this study demonstrate that (a) cholesterol feeding reduces NO formation and increases ET-1 production (as assessed by decreased urinary excretion of the NO metabolite, nitrate, and increased urinary excretion of ir-ET-1); (b) aortic rings isolated from rabbits fed a cholesterol-rich diet show impaired endothelium-dependent relaxation to ACh and enhanced vascular contraction induced by exogenous ET-1; (c) supplementation with dietary L-arginine improves endothelium-dependent relaxation and normalizes the vasoconstrictor response to ET-1; and (d) these effects of L-arginine are found in association with an increased NO formation and a decreased ET-1 production during L-arginine supplementation.
Under physiological conditions, circulating levels of ET-1 are low (12). In addition to plasma ET-1 levels, measurement of immunoreactive ET-1 in urine is useful as an indicator to assess whole-body ET production (26,27), although kidney epithelial cells can also produce ET-1 with only minimal daily variability (27). Increased levels of ET-1 have been found in hypercholesterolemia and atherosclerosis (14-16) and in patients with acute or chronic renal failure (28). However, it remains undetermined from these cross-sectional studies at which stage of the atherogenic process increased ET-1 formation occurs. In our study, quantitation of urinary ir-ET-1 excretion allowed repeated, noninvasive determination of 24-h integrated whole-body ET-1 synthesis rates. We found that urinary excretion of ir-ET-1 in rabbits was increased after 12 weeks of cholesterol feeding in spite of normal renal excretory function, as indicated by unaltered creatinine clearance in the three groups, indicating that increased ET-1 formation occurs relatively late during the pathogenesis of atherosclerosis. At 12 weeks of cholesterol feeding, rabbits have already developed well-defined atherosclerotic lesions throughout the thoracic and abdominal aorta, with marked intimal thickening (3-5,29). The mechanism by which increased ET-1 formation is induced during hypercholesterolemia has not yet been fully elucidated. Because ET-1 is not stored intracellularly, agents that affect its secretion likely do so by modulating the transcription, translation, or posttranslational processing of the peptide (12,13), or some combination of these. A variety of growth factors and vasoactive mediators are known to modulate the transcription of the ET-1 gene (30,31). It has been shown that oxidized low-density lipoprotein (LDL) stimulates the expression of prepro-ET-1 messenger RNA (mRNA) and ET-1 release by cultured endothelial cells (32,33). ECE activity is present within serum lipoproteins; this activity has been shown to correlate with serum total cholesterol levels (34). By these mechanisms, hypercholesterolemia may have increased ET-1 formation in our rabbit model.
The presence of a defect in the L-arginine/NO pathway is a well-characterized phenomenon of hypercholesterolemia and atherosclerosis. Impaired responses to endothelium-dependent vasodilators have previously been shown in arteries and arterioles from cholesterol-fed animals (2-4) and from humans with hypercholesterolemia and atherosclerosis (1,5). In addition to increased inactivation of NO by oxygen-derived free radicals, which are present in the atherosclerotic vascular wall (3,29), decreased NO synthesis also contributes to its impaired biological activity (21). Our data show decreased urinary nitrate excretion as early as 4 weeks after the induction of hypercholesterolemia. This finding supports our earlier data in a similar rabbit model (3,35). It indicates that defective NO formation occurs at a very early stage during hypercholesterolemia. Long-term supplementation with L-arginine has been shown to reverse endothelial dysfunction in cholesterol-fed rabbits and to increase urinary nitrate excretion (3,36,37). In this study, urinary nitrate excretion was significantly increased by long-term L-arginine supplementation as compared with the cholesterol group, and relaxation to acetylcholine was partly restored.
There is evidence that the L-arginine/NO pathway contributes to the regulation of ET-1 synthesis: inhibition of NO formation by the NOS inhibitors, Nω-monomethyl-L-arginine (L-NMMA) or Nω-nitro-L-arginine methyl ester (L-NAME), has been found to increase circulating ir-ET-1 levels (18,19). However, decreased NO formation may not have been the major cause for increased ir-ET-1 excretion in our study, because there was an interval of 8 weeks between the occurrence of reduced nitrate excretion (week 4) and increased ir-ET-1 excretion (week 12) in the cholesterol group. On the other hand, restoration of NO production with L-arginine resulted in a concomitant reduction of urinary ir-ET-1 excretion to normal levels without affecting renal excretory function (creatinine clearance), indicating that these changes were not brought about by changes in renal function.
Although it is not possible directly to link urinary ir-ET-1 excretion with vascular reactivity of the aorta because other vascular areas like the microcirculation will contribute to the majority of vascular ET-1 production, our data show that besides changes in the production rates of these vascular autacoids, hypercholesterolemia and L-arginine supplementation also affected their biologic functions. Aortic rings from cholesterol-fed animals showed reduced relaxation to ACh and increased contraction induced by exogenous ET-1. ET-1-induced vasoconstriction is mediated by activation of phospholipase C, which causes hydrolysis of phosphatidylinositol and in turn increases intracellular calcium in vascular smooth muscle (38). Because LDL increases phosphatidylinositol turnover and intracellular calcium in cultured vascular smooth-muscle cells (39), this may sensitize vascular smooth muscle to the vasoconstrictor actions of ET-1. By contrast, NO increases smooth-muscle cGMP, thereby reducing smooth-muscle intracellular calcium and inducing relaxation (9,40). Thus besides interfering with ET-1 secretion, NO may functionally antagonize ET-1-induced vascular contractions. Indeed, our data point to a mutual interaction between ET-1-induced contraction and NO-mediated relaxation in hypercholesterolemic rabbit aorta: selective inhibition of ET-1 binding to its ET-A receptors on vascular smooth-muscle cells with BQ123 ameliorated the endothelium-dependent response to ACh in aortic rings isolated from cholesterol-fed rabbits but not from normal controls. This suggests that the endothelium-dependent dilator effect of ACh is counteracted by the enhanced release and vasoconstrictor activity of ET-1 in hypercholesterolemia. In contrast, in aortic rings from cholesterol-fed rabbits supplemented with L-arginine, the ET-A-receptor antagonist BQ123 did not affect ACh-induced relaxation, indicating that the biologic function of ET-1 was normalized in this group. A recent study of Wang et al. (41) showed that L-arginine administration prevented the increase in myocardial ET-1-like immunoreactivity induced by ischemia/reperfusion. Mathew et al. (42) recently demonstrated concomitant enhanced ET-mediated vasoconstriction and attenuated basal NO activity in atherosclerotic pig coronary arteries.
The mechanism(s) by which long-term L-arginine administration normalized the vascular reactivity to ET-1 remains undetermined. An increased number of ET receptors have been identified in atherosclerotic lesions (43,44). These results, along with the study of Kurata et al. (45), who reported that binding of ET-1 in the injured rabbit aorta increased immediately after deendothelialization, suggest that there may be an increased number of binding sites for ET-1 at the vascular wall in hypercholesterolemia/atherosclerosis, contributing to the increased vascular reactivity to ET-1. The L-arginine may downregulate the number of ET receptors in the hypercholesterolemic vascular wall. Moreover, L-arginine-derived NO has been found to decrease the vascular release of superoxide radicals (3) and to inhibit LDL oxidation (46). Because oxidized LDL (oxLDL) is a strong stimulus to induce the expression of prepro-ET-1 mrnA and to stimulate ET-1 release in endothelial cells (32,33), one possible mechanism by which L-arginine inhibits ET-1 release or production or both may be mediated by decreasing oxLDL formation. Further studies will be needed to elucidate the nature of the interaction between L-arginine/NO and ET-1 in hypercholesterolemia.
In conclusion, our results emphasize that the vasoconstrictor activity of ET-1 is enhanced in hypercholesterolemic rabbits. This effect is completely reversed by dietary L-arginine supplementation. The L-arginine changes the balance between the vascular effects of NO and ET in favor of vasodilation. This phenomenon may contribute to the favorable cardiovascular effects of L-arginine in hypercholesterolemic rabbits.
Acknowledgment: L. Phivthong-ngam is the recipient of a postgraduate grant from the Konrad-Adenauer Foundation. The excellent technical assistance of M.-T. Suchy and F.-M. Gutzki is gratefully acknowledged.
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