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TRANSLATIONAL PHYSIOLOGY
Department of Cardiology, Karolinska Hospital, S-171 76 Stockholm, Sweden
Submitted 14 May 2003 ; accepted in final form 15 August 2003
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ABSTRACT |
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endothelium; nitric oxide; ischemia; reperfusion
Previous experimental animal studies have demonstrated that ischemia-reperfusion injury can be limited by means of pharmacological interventions (24). Administration of the NO substrate L-arginine or NO donors reduces myocardial infarct size in animal models in vivo (15, 21) and improves myocardial performance in rat hearts in vitro (12). In addition, postischemic coronary endothelial function in the rat in vitro is preserved after administration of L-arginine (12). However, data concerning pharmacological treatment of ischemia-reperfusion injury aiming at preserving NO bioavailability in humans are sparse. One previous study described no protective effect of L-arginine on pulmonary arterial endothelial function in patients after cardiopulmonary bypass (2). The lack of effect may have been related to the timing and method of drug administration. Because endothelial dysfunction develops rapidly after reperfusion, the protective agent may have to be present in the jeopardized area at the onset of reperfusion.
The aim of the present study was to test the hypothesis that local administration of L-arginine into the jeopardized vasculature at the end of the ischemic period and during early reperfusion prevents the development of ischemia-reperfusion-induced endothelial dysfunction in humans in vivo by using a recently described ischemia-reperfusion protocol in the forearm (10).
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METHODS |
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Forearm blood flow measurements. After administration of local anesthetics, a percutaneous catheter was inserted into the brachial artery of the nondominant arm for drug infusion and determination of blood pressure. Forearm blood flow was measured simultaneously in both arms with venous occlusion plethysmography by using the mercury-in-Silastic straingauge technique (3, 25). A venous occlusion cuff placed around the upper arm was inflated to 40 mmHg for 10 s, followed by deflation for 5 s to obtain recordings of arterial inflow. During recordings of blood flow, the circulation of the hand was occluded by a cuff inflated to 30 mmHg above systolic blood pressure. Heart rate was determined from an ECG recording.
Study protocol. Using a crossover protocol, each subject received vehicle and L-arginine on the two separate study occasions. The order of administration was randomized. Basal forearm blood flow was determined during a 2-min infusion of 0.9% NaCl at a rate of 2.5 ml/min. Endothelium-dependent vasodilatation was assessed by intra-arterial infusion of ACh (3, 10, and 30 µg/min). Each dose was given for 2 min at a rate of 2.5 ml/min. The NO-dependent property of the vasodilatation induced by ACh has been validated in this model previously (3). After determination of basal endothelium-dependent vasodilatation, forearm ischemia was induced by a blood pressure cuff proximal to the arterial catheter inflated to 200 mmHg. The ischemia was maintained for 20 min. At 15 min of ischemia, an intra-arterial infusion of L-arginine (20 mg/min) or vehicle was started at a rate of 1 ml/min. The infusion was stopped after 15 min, i.e., at 10 min of reperfusion. This dose did not affect basal endothelium-dependent vasodilatation in two pilot experiments on healthy subjects, in accordance with previous observations (4). Saline was given as vehicle to 12 subjects. Four additional subjects were given D-arginine (20 mg/min). Endothelium-dependent vasodilatation was assessed again at 15, 30, and 60 min of reperfusion. Endothelium-independent vasodilatation was determined by infusion of sodium nitroprusside (SNP; 1, 3, and 10 µg/min) at 30 and 60 min of reperfusion. SNP was not given before ischemia due to the possible preconditioning-like effect of NO donors given briefly before ischemia (6). Blood pressure and heart rate were measured before and after each infusion of ACh.
Drugs. The following drugs were used: ACh (Miochol, OMJ Pharmaceuticals, San German, PR), SNP (Abbott Laboratories), and L-arginine and D-arginine (Clinalfa, Läufelfingen, Switzerland). All drugs were diluted in sterile saline immediately before use.
Calculations and statistics. Basal forearm blood flow was calculated as the mean of eight recordings during the 2-min infusion of saline. Blood flow during infusion of ACh and SNP was calculated as the mean of the four highest flow recordings during each infusion. The changes in forearm blood flow are expressed in absolute values by using the noninfusion arm as the control arm (25) and as percent recovery of the preischemic response to ACh. Because no infusions affected blood pressure, all hemodynamic effects are expressed as blood flow changes. A two-way ANOVA was used to compare the dose-response curves for ACh at the different time points. A probability (P) <0.05 was regarded as statistically different. All data are given as means ± SE.
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RESULTS |
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There was no difference in preischemic forearm blood flow between the two study occasions (Table 1). Basal blood flow increased significantly after ischemia, but there was no difference between the two study occasions (Table 1). Blood flow in the control arm remained unchanged throughout the study protocol, and it was not affected by any of the infusions. ACh increased forearm blood flow by similar magnitudes before ischemia on the two study occasions. Intra-arterial blood pressure and heart rate did not change significantly during the study occasions.
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The increase in forearm blood flow evoked by ACh was impaired during the 60-min reperfusion period after ischemia on the saline study occasion (P < 0.001). This was due to a significant attenuation of the vasodilator response to 3 and 10 µg/min ACh at 15 and 30 min of reperfusion (Fig. 1). The vasodilator effect of ACh was restored 60 min after the onset of reperfusion. The response to ACh after ischemia was better preserved on the L-arginine study occasion. Thus the vasodilator response to the three doses of ACh was not significantly impaired after ischemia (Fig. 2). The vasodilator effect of ACh at 15 and 30 min of reperfusion was significantly greater after administration of L-arginine compared with saline when expressed as percent recovery of the preischemic vasodilatation (Fig. 3). Administration of D-arginine did not affect the recovery of the response to ACh after ischemia. The recovery of the increase in forearm blood flow evoked by 3 and 10 µg/min ACh at 30 min of reperfusion after D-arginine administration was 44 ± 21 and 68 ± 7% of the preischemic response vs. 35 ± 6 and 66 ± 8% after saline, respectively. There was no difference in the forearm vasodilator response to SNP at 30 and 60 min of reperfusion on either the saline (Fig. 4) or the L-arginine study occasion.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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It is well known that endothelial dysfunction occurs early during reperfusion of a previously ischemic tissue (11). The endothelial dysfunction is characterized by reduced endothelium-dependent vasodilatation, expression of adhesion molecules, adherence of inflammatory cells, transmigration of white blood cells, and production of oxygen free radicals (11). These events will eventually contribute to the development of myocardial necrosis. Thus endothelial dysfunction may be a key factor in the development of the reperfusion injury.
The vasodilator response to ACh, which, to a large part, is dependent on endogenous NO production in the forearm (3), was markedly attenuated during reperfusion after a 20-min episode of ischemia, in accordance with a previous report (10). On the other hand, the response to the endothelium-independent vasodilator SNP was not affected at 30 min of reperfusion (when the response to ACh was reduced) compared with 60 min of reperfusion (when the response to ACh was normal), suggesting that the ability of the smooth muscle to relax was unaffected by ischemia-reperfusion. These observations indicate that endothelial dysfunction occurred after ischemia-reperfusion. It cannot be excluded that mediators other than NO were affected by the ischemia-reperfusion, because it is known that other mediators, including endothelium-derived hyperpolarizing factor, are involved in endothelium-dependent vasodilatation in the human forearm (7, 9). The important finding, however, is that the development of endothelial dysfunction after ischemia was prevented by administration of L-arginine. Administration of D-arginine, which is not a substrate for NO synthesis, did not modify the endothelium-dependent vasodilatation after ischemia, which indicates that the protective effect of L-arginine was related to NO formation.
In a previous study on patients undergoing heart surgery, administration of L-arginine failed to inhibit the attenuation of ACh-induced pulmonary vasodilatation after cardiopulmonary bypass (2). The reason behind the lack of effect may be related to the fact that L-arginine was given systemically during and after the cardiopulmonary bypass and did, therefore, not reach the jeopardized pulmonary vascular bed during cardiopulmonary bypass, but only after reperfusion was established (2). Timing of the administration seems to be a crucial factor, because reperfusion injury develops rapidly after the onset of reperfusion (11), and it may be important that a protective substance is present in the jeopardized vascular region when reperfusion is initiated (24). In the present study, a protocol was chosen that allowed administration of L-arginine into the ischemic vascular bed before the onset of reperfusion.
Several possible mechanisms responsible for the protective effect of L-arginine/NO on endothelial function may exist. It is known that the intracellular concentration of L-arginine is sufficient to saturate NO synthase (17). Thus the binding kinetics do not explain the improvement in endothelial function demonstrated by intravascular and oral supplementation of L-arginine (4, 5, 18, 19). Under conditions of ischemia and reperfusion, the activity of NO synthase is markedly suppressed (23). Supplementation with L-arginine enhances both the activity of NO synthase and postischemic endothelial function, suggesting that a relative lack of substrate exists in this situation (12, 23). It is known from several studies that NO donors and L-arginine attenuate neutrophil accumulation in the reperfused myocardium via an effect that may involve inhibition of the expression of adhesion molecules (8, 11). Another mechanism may be inactivation of superoxide radicals from neutrophils and other sources during ischemia and reperfusion. Because superoxide is regarded as contributing to endothelial dysfunction (13), increased production of NO may attenuate its harmful effect. Whether these effects contribute to the presently observed protective effect of L-arginine remains to be elucidated.
The present observation may have important therapeutic implications, because it suggests that L-arginine may be beneficial in the treatment of ischemia-reperfusion injury in humans. Previous studies have shown that there is a good correlation between endothelial function in the forearm and the coronary arteries, suggesting that the forearm is a suitable model to study effects of drugs on endothelial function (25). It is of importance to establish therapeutic strategies to limit ischemia-reperfusion injury in patients with acute myocardial infarction. It has previously been suggested that administration of adenosine may attenuate reperfusion injury when given as adjunct to reperfusion therapy to patients with acute myocardial infarction (14). Adenosine reduced infarct size in patients with anterior myocardial infarction but was without effect in patients with other infarct locations, however, and there was a trend toward excess clinical events in the adenosine group. Studies with the Na+/H+ exchange inhibitor cariporide have demonstrated inconsistent results (20, 22). The present data demonstrating improved endothelial function during reperfusion after administration of L-arginine suggest that L-arginine may be a promising therapeutic agent in the setting of ischemia-reperfusion.
A limitation of the present study is that it was performed on healthy individuals and in the forearm vasculature. On the other hand, endothelial dysfunction in the forearm correlates with coronary endothelial dysfunction and with prognosis in patients with cardiovascular disease (16, 25). Thus the forearm vasculature may be used as a surrogate for the coronary vasculature (25). It will be important to evaluate the protective effect of L-arginine during ischemia-reperfusion in a patient group with atherosclerotic coronary artery disease. Another limitation is that endothelial function was tested only by stimulation of endothelium-dependent vasodilatation with ACh. The study protocol did not permit evaluation of basal NO production after ischemia, which would require infusion of a NO synthase inhibitor. Such a procedure could not be combined with administration of ACh, because the NO synthase inhibitor would block the vasodilator response to ACh. Nevertheless, the data obtained by using the present protocol suggest that endothelial dysfunction, as determined by administration of ACh, induced by ischemia-reperfusion, is attenuated by L-arginine.
In conclusion, this study demonstrates that local administration of L-arginine results in improved endothelial function during reperfusion after ischemia in the human forearm in vivo. L-Arginine administration may thus represent an important therapeutic strategy to limit ischemia-reperfusion injury by enhancing NO bioavailability.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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This article has been cited by other articles:
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  Y. Bai, L. Sun, T. Yang, K. Sun, J. Chen, and R. Hui Increase in fasting vascular endothelial function after short-term oral L-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials Am. J. Clinical Nutrition, January 1, 2009; 89(1): 77 - 84. [Abstract] [Full Text] [PDF]
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 L. A. Holowatz, C. S. Thompson, and W. L. Kenney L-Arginine supplementation or arginase inhibition augments reflex cutaneous vasodilatation in aged human skin J. Physiol., July 15, 2006; 574(2): 573 - 581. [Abstract] [Full Text] [PDF]
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 H. L. Gornik and M. A. Creager Arginine and Endothelial and Vascular Health J. Nutr., October 1, 2004; 134(10): 2880S - 2887S. [Abstract] [Full Text] [PDF]
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