In vivo administration of d -arginine: effects on blood pressure and vascular function in angiotensin II-induced hypertensive rats

Atherosclerosis 176 (2004) 219–225

In vivo administration of d-arginine: effects on blood pressure and vascular function in angiotensin II-induced hypertensive rats Gerald Wölkart, Heike Stessel, Friedrich Brunner∗ Institut für Pharmakologie und Toxikologie, Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria Received 12 February 2004; received in revised form 14 May 2004; accepted 28 May 2004 Available online 28 July 2004

Abstract Objective: We tested the hypothesis that d-arginine (d-Arg), which is not a substrate for nitric oxide synthase but scavenges reactive oxygen in vitro, is protective in vivo. Methods: Rats were made hypertensive by administering angiotensin II (Ang II) (0.7 mg kg−1 per day) for 7 days (Ang II group). Two other groups additionally received either 3 mmol d-Arg (Ang II + d-Arg group) or vitamin C (1 g) (Ang II + Vit C group) per day. Sham-operated animals served as controls (n = 6–9). Systolic blood pressure was monitored daily and cardiovascular function determined ex vivo at 7 days. Results: Ang II raised systolic blood pressure to 184 mmHg, the increase was slightly attenuated by d-Arg treatment (−17 mmHg; P < 0.05 versus Ang II alone) and prevented by Vit C. Acetylcholine-induced coronary relaxation was impaired in the Ang II group (P < 0.05 versus sham), the impairment was no different in the Ang II + d-Arg group, but prevented by Vit C. Likewise, Vit C but not d-Arg ameliorated reperfusion endothelium-dependent relaxation. However, in aortic rings d-Arg slightly improved acetylcholine relaxation (P < 0.05). Oxidative stress load estimated in plasma with thiobarbituric acid reactive substance was higher in the Ang II than the sham group, Vit C abolished the increase, but d-Arg was without effect. Conclusion: d-Arg is weakly antihypertensive in vivo and ameliorates aortic, but not coronary endothelium-dependent relaxation ex vivo. Because d-Arg had no effect on plasma oxidant status, this protection appears to be independent of reactive oxygen scavenging activity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Arginine; Hypertension; Endothelium; Oxidation

1. Introduction l-Arginine is the substrate for the synthesis of nitric oxide (NO), the endothelium-derived relaxing factor essential for regulating vascular tone and haemodynamics [1]. This natural amino acid is classified as conditionally essential for adult humans and is a vascular protectant that alleviates endothelial injury and improves endothelial function. Enteral or parenteral administration of l-arginine was shown to reverse endothelial dysfunction associated with a hypercholesterolaemia, smoking, hypertension or diabetes and to ameliorate cardiovascular disorders such as coronary and peripheral arterial disease, ischaemia/reperfusion injury, and heart failure [2]. For this reason, there has been grow-

∗ Corresponding author. Tel.: +43-316-380-5559; fax: +43-316-380-9890. E-mail address: [email protected] (F. Brunner).

ing interest in the last years in using l-arginine to prevent and treat cardiovascular disorders [3,4]. The abnormal endothelial functions in many cardiovascular disease states appear to be related to a reduced ability of the endothelium to release biologically active NO, either due to reduced NO synthesis or increased oxidative degradation of NO [5]. Therefore, many of l-arginine’s beneficial effects may be mediated by an increased availability of NO in the vessel wall, both in experimental animals [6] and patients with hypercholesterolaemia [7], coronary artery disease [8], or hypertension [9]. Although most of these actions are usually attributed to enhanced NO formation, there is increasing evidence that l-arginine likely exerts NO-independent effects as well [3]. Because l-arginine supplementation reduced the production of superoxide anion in the vessel wall [6], some authors speculated that the amino acid might exert direct antioxidant effects in tissues. In a model of intestinal ischaemia/reperfusion, in which superoxide anion, NO and other reactive oxygen species were

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generated, l-arginine, but also d-Arg and NO synthase inhibitors, which do not serve as substrate for NO synthase, were effective in reducing oxidative damage [10], suggesting that besides serving as NO precursor, l-arginine may be an antioxidant by scavenging oxygen-derived free radicals. We recently investigated this question in isolated perfused rat hearts subjected to electrolysis-generated oxygen radicals and found that l-arginine prevented oxygen radical-induced cardiac contraction deficits by diminishing the generation of oxygen radicals, an effect that was also demonstrated using electron spin resonance spectroscopy [11]. In addition, we showed that NG -nitro-l-arginine and d-Arg exerted cardioprotective effects and reduced the generation of oxidants, strongly suggesting that the protection in this ex vivo model was due to scavenging of reactive oxygen species rather than an effect on cardiac NO synthesis [12]. In this study we investigated whether the scavenging activity of arginine is significant in vivo. Specifically, we asked whether arginine exerts antioxidant actions and tissue protection at free radical flux rates observed in vivo, and in the presence of other competing endogenous antioxidant systems. Rats were rendered hypertensive by infusing a pressor dose of angiotensin II (Ang II) [13], and the effects of concomitantly administered d-Arg or vitamin C (Vit C), a powerful antioxidant, on blood pressure and endothelial function ex vivo were assessed. d-Arg rather than l-arginine was used to exclude a NO-mediated effect.

2. Methods 2.1. Animals and experimental groups Male Sprague–Dawley rats weighting 270–350 g were trained to blood pressure measurements using the noninvasive tail cuff method (TSE Instruments, Bad Homburg, Germany) [14]. Rats were then divided into four groups: sham, angiotensin II (Ang II), Ang II plus d-Arg (Ang II + d-Arg) and Ang II plus Vit C (Ang II + Vit C). All animals were anaesthetized with diethyl ether, an incision was made in the midscapular region and osmotic minipumps (Alzet model 2ML1; Charles River, Germany), containing either saline (sham group) or Ang II, were implanted. The infused Ang II dose was 0.7 mg kg−1 per day for 7 days. 2.2. d-Arg and Vit C administration The Ang II + d-Arg group was treated with a daily subcutaneous injection of 3 mL d-Arg (1 M) solution starting 2 days before implantation of the osmotic pumps. This concentration was chosen based on pilot studies with [3 H]d-arginine which showed that this procedure reproducibly raised total d-Arg (i.e. [3 H]d-Arg + [1 H]d-Arg) plasma concentration up to ∼600 ␮M (see Fig. 1). The total amount of 3 H-label used per injection was 45 ␮Ci (radioactive concentration, 15 ␮Ci mL−1 or 200 nM). Plasma

Fig. 1. d-Arg concentration measured in plasma after daily subcutaneous injection for 7 days. The minipump containing Ang II was implanted 2 days after starting d-Arg injections, i.e. at day 0. Data are mean ± SEM of three determinations.

samples were collected and analyzed for [3 H]d-Arg 18 h after dosing using liquid scintillation counting. Vit C was administered at 1 g per day with the drinking water, also starting two days before implantation of the pumps. Blood pressure, heart rate and body weights were monitored for 7 days after implantation of the pumps. 2.3. Heart perfusion and experimental protocols Seven days after implantation of the osmotic pumps animals were anaesthetized with diethyl ether, a blood sample obtained, the hearts removed, mounted on a Langendorff apparatus and retrogradely perfused at 37 ◦ C with Krebs–Henseleit buffer at 10 mL min−1 g−1 [14]. Cardiac parameters were monitored continuously and included left-ventricular-developed pressure (LVDevP), left-ventricular end-diastolic pressure (LVEDP), heart rate, and coronary perfusion pressure. Hearts were equilibrated for 30 min, followed by a gradual increase in coronary flow up to 15 mL min−1 g−1 to obtain the desired perfusion pressure of ∼130 mmHg. Coronary endothelium-dependent relaxation was tested with acetylcholine given as bolus injections through a sideline, resulting in concentrations of ∼1–1000 nM (3–4 min per dose). After the last dose, acetylcholine was washed out, coronary flow was reduced to 10 mL min−1 g−1 and baseline re-established (perfusion pressure: ∼70 mmHg). Hearts were then subjected to 15 min no-flow ischaemia at 37 ◦ C, followed by 30 min of reperfusion and functional parameters were recorded [15]. Finally, the acetylcholine concentration–response curve was repeated. 2.4. Relaxation of aortic rings The thoracic aorta was cleaned of connective tissue and cut into rings of ∼3 mm length with endothelium present. The rings were suspended in tissue baths containing 5 mL Krebs–Henseleit solution maintained at 37 ◦ C,

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gassed with carbogen and tension was recorded isometrically. All details have been reported previously [16]. Tissues were precontracted with 9,11-dideoxy-11a,9aepoxymethanoprostaglandin F2␣ (U 46619) to ∼5 g, and the relaxation response to acetylcholine (1 nM–10 ␮M) was recorded, followed by wash-out, reapplication of U 46619 and addition of nitroprusside (1 nM–100 ␮M). The contractile force corresponding to each agonist concentration was recorded and expressed as percent of precontraction (=baseline). 2.5. Oxidant status The oxidant status in the rats was determined after 7 days of dosing [17]. Total antioxidant status of plasma was measured with a commercial kit (Randox, Crumlin, UK). The assay relies on the ability of antioxidants in the plasma to inhibit oxidation of 2,2 azino-bis-[3-ethylbenz-diazoline6-sulfonic acid] (ABTS) to ABTS+ by metmyoglobin. This assay uses 6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (TROLOX) as reference. Results were expressed as TROLOX equivalents (nM). Levels of thiobarbituric acid reactive substance (TBARS) in plasma were determined using another kit (Oxitek, ZeptoMetrix Corporation, Buffalo, NY). Thiobarbituric acid reacts with malondialdehyde resulting from lipid peroxidation, and the reaction product was measured sprectrofluorometrically. Lipoprotein was precipitated from the sample to minimize interfering soluble reactants. TBARS levels were expressed as nmol mL−1 malondialdehyde [18]. 2.6. Materials d-[4,5-3 H]Arginine was obtained from American Radiolabelled Chemicals (St. Louis, MO). All other chemicals were from Sigma (Vienna). 2.7. Statistics Data are expressed as mean and standard error of the mean (SEM). Effects of test compounds were compared using unpaired Student’s t-test. EC50 and Emax values were determined via non-linear curve-fitting using standard methods. Statistical significance was assumed at P < 0.05.

Fig. 2. Systolic blood pressure in rats treated with Ang II via minipump for 7 days in absence (solid circles; n = 9) or presence of d-Arg (diamonds; n = 7) or Vit C (squares; n = 6). Sham-operated animals served as controls (open circles; n = 7). Data are mean ± SEM. ∗ P < 0.05 for d-Arg versus Ang II group. The significant blood pressure lowering effect of Vit C (P < 0.05 versus Ang II group) is not indicated.

3.2. Haemodynamic measurements in vivo All animals used in the study were normotensive before pumps were implanted (mean arterial pressure: 126 ± 1 mmHg; n = 29). Ang II infusion (n = 9) increased systolic blood pressure up to 184 ± 3 mmHg at day 7. d-Arg treatment (n = 7) significantly attenuated the blood pressure rise starting at day 2. The reduction was small but highly reproducible and reached statistical significance on days 4–6 (∼11 mmHg), (Fig. 2). Vit C totally prevented the Ang II-induced increase in blood pressure (134 ± 3 mmHg at day 7; n = 6). As expected, no increase in blood pressure was observed in the sham-operated group (126 ± 2 mmHg; n = 7). In animals not treated with Ang II, d-arginine had no significant effect on blood pressure (n = 3, data not shown). Heart rate was not altered by Ang II infusion and was not different between groups (391 ± 5 beats min−1 at the beginning and 387 ± 7 beats min−1 at the end of treatment; mean of all groups; n = 29). Surgery led to a slight loss of body weight (−5%) in all four experimental groups within the first 3 days, without significant differences between groups. 3.3. Effects of acetylcholine on heart function in normoxic perfusion

3. Results 3.1. Plasma levels The daily application of d-Arg resulted in plasma levels of ∼600 ␮M (Fig. 1). This concentration is several times higher than the physiological l-arginine concentration (range: 100–300 ␮M) [4]. Vit C levels were not determined, as the current dose was previously shown to be effective as an antioxidant in another hypertension model [19].

Acetylcholine dose-dependently reduced coronary perfusion pressure indicating vasodilatation (Fig. 3). In shamoperated hearts (n = 5), pressure was reduced to 39 ± 3% of baseline (control), in the Ang II group (n = 7) to 69 ± 4%, indicating substantial endothelial dysfunction (P < 0.05). Vit C (n = 6) restored dilatation, but d-Arg (n = 5) was without effect, both in terms of EC50 and Emax values, respectively (P = NS versus Ang II). Acetylcholine dosedependently reduced LVDevP (−21% in sham hearts) and

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Fig. 5. Effects of ischaemia/reperfusion on left-ventricular end-diastolic pressure. Pressure rose as expected and regained baseline at the end of reperfusion. There were no significant differences between experimental groups. Data are mean ± SEM, n = 6–7. NS = nonsignificant. Fig. 3. Effects of acetylcholine on coronary perfusion pressure in hearts perfused ex vivo at constant flow. The agonist-induced pressure reduction (vasodilatory effect) was significantly impaired in the Ang II group (not indicated), and Vit C but not d-Arg antagonized the impairment. Data are mean ± SEM, n = 5–7. NS = nonsignificant.

heart rate (−16%), without differences between experimental groups (n = 5–7, data not shown). 3.4. Effects of ischaemia/reperfusion Because ischaemia/reperfusion is known to generate a burst of oxygen radicals that are partly responsible for the post-ischaemic myocardial depression [20], we verified the potential of d-Arg to protect against reperfusion injury. In all groups, reperfusion myocardial function was similar, with LVDevP remaining depressed to ∼80% of preischaemic pressure (P < 0.05 versus the corresponding preischaemic value in each group) (Fig. 4). Heart rate totally recovered to preischaemic rate (286 ± 10 min−1 ). LVEDP increased during ischaemia and returned to preischaemic values at the end of reperfusion in all experimental groups (Fig. 5). Coronary perfusion pressure increased 1.9-fold (P < 0.05 ver-

Fig. 4. Effects of 15 min of ischaemia followed by reperfusion on left-ventricular-developed pressure (LVDevP) in hearts perfused ex vivo. There were no significant differences between groups. Data are mean ± SEM, n = 6–7. NS = nonsignificant.

sus preischaemic level), with no differences between groups. When applied post-reperfusion, the coronary relaxant effect of acetylcholine (1–1000 nM) was attenuated, reflecting reperfusion endothelial dysfunction, but the differences between experimental groups were unchanged (n = 5–7, data not shown). 3.5. Macrovascular studies We also tested the reactivity of aorta, a conduit vessel, to acetylcholine and nitroprusside (n = 29–33 tissues). Acetylcholine reduced aortic tone to 73 ± 2% in the sham group and 81 ± 2% in the Ang II group (P < 0.05) (Fig. 6). Vit C completely and d-Arg partly antagonized the impairment as evident from the reduction in the EC50 value (Ang II, 1100 ± 230 nM; Ang II + d-Arg, 460 ± 79 nM (P < 0.05)). However, the Ang II-induced reduction in Emax was antagonized only by Vit C (P < 0.05 versus Ang II).

Fig. 6. Effects of acetylcholine on tone of aortic rings superfused ex vivo. Tissues were precontracted to 4.9 ± 0.1 g (baseline) and the agonist was added non-cumulatively. Relaxation was significantly impaired in the Ang II group (not indicated) and Vit C completely (not indicated) and d-Arg partly antagonized the impairment (indicated by ∗). Data are mean ± SEM of 29–33 aortic tissues derived from 4 to 5 animals.

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Fig. 7. Effect of nitroprusside on tone of aortic rings. Tissues were precontracted to 4.8 ± 0.1 g (baseline) and the agonist was added non-cumulatively. Relaxation was significantly impaired in the Ang II group (not indicated). d-Arg was without effect, whereas Vit C improved relaxation over and above the level in sham hearts (indicated by †). Data are mean ± SEM of 29–33 aortic tissues derived from 4 to 5 animals.

The relaxant effect of nitroprusside is shown in Fig. 7. There was no difference in Emax between groups. The respective mean EC50 values (nM) were 120 ± 16 (sham), 710 ± 270 (Ang II), 460 ± 82 (Ang + d-Arg) and 30 ± 6 (Ang II + Vit C), indicating a highly significant protective effect of Vit C (P < 0.05 versus Ang II), but no influence of d-Arg (P = NS versus Ang II). Interestingly, Vit C also improved relaxation compared to vessels from sham animals (P < 0.05). 3.6. Oxidative stress To determine the oxidant stress load in animals of the different groups, total antioxidant status (TROLOX equivalents) and systemic levels of TBARS were determined. The levels of quantitative TROLOX equivalents were 0.55 ± 0.05 mM (sham group), 0.63 ± 0.06 mM (Ang II group), 0.59 ± 0.7 mM (Ang II + d-Arg) and 0.74 ± 0.04 mM (Ang II + Vit C). Only Vit C increased the antioxidant status significantly (P < 0.05 versus sham group), whereas dArg was without effect. With respect to TBARS, the Ang II group had higher systemic levels compared to those of controls. TBARS were 2.8 ± 0.3 nmol mL−1 versus 2.0 ± 0.1 nmol mL−1 (P < 0.05, n = 8–9). Vit C abolished the increase (1.6 ± 0.2 nmol mL−1 , n = 6), whereas d-Arg was without effect (3.2 ± 0.3 nmol mL−1 , n = 7) (P = NS versus Ang II group).

4. Discussion We recently reported that arginine and arginine derivatives have reactive oxygen scavenging activity in vitro. In the present study we showed that d-Arg, which is not a substrate for NO synthase, reduces the hypertensive response in rats dosed with Ang II and improves acetylcholine-induced aor-

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tic relaxation ex vivo. However, d-Arg had no discernable effect on coronary endothelium-dependent relaxation in normoxic or ischaemia/reperfused hearts or on plasma antioxidant status. On the other hand, Vit C completely prevented the hypertensive effect of Ang II, ameliorated coronary and aortic vascular function and reduced plasma oxidant load. The Ang II-induced hypertension model is well established. Infusion of Ang II via osmotic minipump raised blood pressure gradually up to ∼180 mmHg, identical to previous observations [13]. This hypertensive response is usually paralleled by an increase in reactive oxygen species in the systemic circulation and in tissues, as measured with oxidative stress markers such as isoprostanes [18]. Other studies have shown that oxidative stress per se can induce hypertension, and antioxidants are hypotensive in several models of raised arterial pressure [18,21]. Therefore, we expected that quenching Ang II-induced oxidant stress would curtail the hypertensive response. This was the case with Vit C which prevented the rise in blood pressure, along with significantly reduced pro-oxidant metabolites in plasma, resulting in lower TBARS levels than in the group receiving Ang II alone. The much lower, yet significant, antihypertensive action of d-Arg was not paralleled by increased antioxidant (TROLOX) or reduced TBARS plasma levels, suggesting that in vivo the antioxidant activity of d-Arg is rather weak or nonexistent. This implies that the observed antihypertensive action (Fig. 2) is based on some other unknown mechanism. A limitation to this interpretation is the nonspecific nature of the thiobarbituric acid test as a lipid peroxidation marker which results from the reaction of several compounds of serum other than malondialdehyde with thiobarbituric acid [22]. Therefore, small anti-oxidant effects of d-arginine may have been missed in the TBARS test (see also below). Endothelial function in the vasculature is determined by the bioavailability of NO which promotes vasorelaxation by the activation of guanylyl cyclase in smooth muscle cells. The availability of NO is affected by both its rate of production and degradation, and these processes are altered as a result of impaired antioxidant defences [23,24]. Our data confirm that Ang II-induced hypertension is associated with a significant degree of endothelial dysfunction in coronary vessels and aorta. The mechanism and signalling processes whereby Ang II might cause endothelial dysfunction have been studied in some detail. Ang II was found to activate an NADH/NADPH oxidase in cultured vascular smooth muscle cells [25] and isolated vessels [13], resulting in increased superoxide anion production. The increased production of superoxide anion by NAD(P)H oxidase activation, together with subsequent uncoupling of endothelial NO synthase can result in a reduction of vascular NO bioavailability, reduced cGMP-dependent protein kinase activity and impaired vasorelaxation [26]. Because the oxidant stress appears to originate largely in the smooth muscle [13] or adventitial layer of the vessels, the effectiveness of Vit C to restore acetylcholine-induced relaxation (Figs. 3 and 6) could be due to its rapid penetration to the

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subendothelial space, thus preventing the local inactivation of NO. As in the case of blood pressure, the protective effect of d-Arg on endothelium-dependent relaxation was small, yet significant in aorta, but nonexistent in coronary vessels. Thus, the antioxidant effect of d-Arg that we previously observed in vitro [11] hardly translated into measurable endothelium protection and actually might not be expected given unchanged TBARS levels in plasma. One reason for the latter may be an insufficient d-Arg dose level in plasma. This is unlikely as the plasma level was ∼600 ␮M, i.e. some 3–5 times the physiological level of l-arginine in animals and humans [4]. More important may be the fact that in the present in vivo situation, d-Arg competes with a host of other exogenous and endogenous antioxidant systems, both located in the plasma and within cells, so that its radical scavenging effect would not show in the TBARS test. In any case, because d-Arg is unlikely to cross endothelial cells because there is no suitable carrier for it [27], the antioxidant action largely would be located in the vascular lumen. Hence, in view of the great differences in antihypertensive activity between Vit C and d-Arg and unchanged oxidant status of the plasma after d-Arg it is not likely that the small protection observed with the amino acid is due to oxygen radical scavenging in our model. Our results complement previous studies on the role of l-arginine and the NO/cGMP pathway in endothelial dysfunction. Numerous reports have attributed impairments in endothelium-dependent relaxation to l-arginine deficiency, the presence of endogenous inhibitors, or other factors because exogenous l-arginine improved vascular function and the interaction between vessel wall and components of blood [2]. Accordingly, systemic or oral l-arginine was shown to reduce blood pressure and renal vascular resistance in hypertensive patients and to increase NO synthesis or NO availability as implied from higher rates of urinary nitrate/nitrite excretion [9]. However, l-arginine potentially exerts many other, NO-independent effects, including regulation of intracellular pH or and the release of various hormones with pleiotropic actions. l-Arginine also functions as a precursor for the synthesis of protein and amino acids, inhibits leucocyte adhesion to non-endothelial matrix (cotton wool), independently of NO production [28] and scavenges superoxide anions [11,29]. In view of its small, but consistent antihypertensive in vivo effects (this study, Fig. 2) and the small effect on saline-superfused aorta (Fig. 7), the present study with d-Arg strongly supports the notion of arginine as a multi-functional amino acid with physiologically relevant protective effects independent of NO generation. Nonetheless, by implication our data suggest that nutritional arginine supplementation to improve cardiovascular functions in humans [3] should be done with l-arginine, from which greater positive effects can be expected than from d-arginine. A final aspect of the present study concerns the endothelium-independent relaxation induced by nitroprusside. d-Arg was ineffective in improving nitroprusside-

mediated relaxation both of the coronary vessels and aorta, whereas Vit C significantly enhanced it over and above the level in the control group. This relaxation-potentiating effect of Vit C was observed previously and explained by a mechanism that could involve the release of NO from nitrosothiols [30]. Thus, although Vit C undoubtedly is a strong antioxidant, the cardiovascular protection observed in our study may actually partly result from influences independent of its oxygen radical scavenging activities. In conclusion, our study showed that d-Arg partly antagonizes the rise in systemic arterial pressure in rats treated with Ang II and slightly improves endothelium-dependent relaxation in aorta ex vivo without significantly affecting the antioxidant status of the plasma. By implication, our findings suggest that dietary arginine supplementation as a novel nutritional strategy for preventing and treating cardiovascular disease might merit further study. Acknowledgements This work was supported by the Austrian Research Council (FWF), project 17339. References [1] Palmer RMJ, Rees DD, Ashton DS, Moncada S. l-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988;153:1251–6. [2] Goumas G, Tentolouris C, Tousoulis D, Stefanadis C, Toutouzas P. Therapeutic modification of l-arginine-eNOS pathway in cardiovascular diseases. Atherosclerosis 2001;154:255–67. [3] Wu G, Meininger CJ. Arginine nutrition and cardiovascular function. J Nutr 2000;130:2626–9. [4] Tapiero H, Mathé G, Couvreur P, Tew KD. Free amino acids in human health and pathologies. I. Arginine. Biomed Pharmacother 2002;56:439–45. [5] Li H, Wallerath T, Münzel T, Förstermann U. Regulation of endothelial-type NO synthase expression in pathophysiology and in response to drugs. Nitric Oxide 2002;7:149–64. [6] Böger RH, Bode-Böger SM, Mügge A, et al. Supplementation of hypercholesterolaemic rabbits with l-arginine reduces the vascular release of superoxide anions and restores NO production. Atherosclerosis 1995;117:273–84. [7] Clarkson P, Adams MR, Powe AJ, et al. Oral l-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults. J Clin Invest 1996;97:1989–94. [8] Adams MR, McCredie R, Jessup W, et al. Oral l-arginine improves endothelium-dependent dilatation and reduces monocyte adhesion to endothelial cells in young men with coronary artery disease. Atherosclerosis 1997;129:261–9. [9] Hishikawa K, Nakaki T, Suzuki H, Kato R, Saruta T. Role of l-arginine nitric oxide pathway in hypertension. J Hypertens 1993;11:639–45. [10] Haklar G, Ulukaya-Durakbasa Ç, Yüksel M, Dagh T, Yalçin AS. Oxygen radicals and nitric oxide in rat mesenteric ischaemia-reperfusion: modulation by l-arginine and NG-nitro-l-arginine methyl ester. Clin Exp Pharmacol Physiol 1998;25:908–12. [11] Lass A, Suessenbacher A, Wölkart G, Mayer B, Brunner F. Functional and analytical evidence for scavenging of oxygen radicals by l-arginine. Mol Pharmacol 2002;61:1081–8.

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