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(Circulation. 2000;102:2707.)
© 2000 American Heart Association, Inc.

Clinical Investigation and Reports

In Vivo and In Vitro Evidence for Impaired Arginine Transport in Human Heart Failure

David M. Kaye, MBBS, PhD; Belinda A. Ahlers, BSc; Dominic J. Autelitano, PhD; Jaye P. F. Chin-Dusting, PhD

From the Baker Medical Research Institute and Cardiovascular Medicine, Alfred Hospital, Melbourne, Australia.

Correspondence to Dr David M. Kaye, Molecular Neurocardiology Laboratory, Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia. E-mail david.kaye{at}baker.edu.au

	Abstract

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Background—The clinical features of congestive heart failure (CHF) result from a complex interaction between reduced ventricular function, neurohormonal activation, and impaired endothelial function. Although endothelial dysfunction has been well documented, the mechanisms that contribute to this abnormality remain unknown. Recent studies, however, indicate a potential therapeutic role for supplemental L-arginine, suggesting the presence of an underlying disorder of L-arginine metabolism.

Methods and Results—We used 2 complementary approaches to assess L-arginine transport in control subjects and patients with CHF. During a steady-state intra-arterial infusion of [³H]L-arginine (100 nCi/min), forearm clearance of [³H]L-arginine was significantly reduced in CHF patients compared with forearm kinetics in control subjects (64±2 versus 133±14 mL/min, P=0.002). In conjunction with this, [³H]L-arginine uptake by peripheral blood mononuclear cells (PBMCs) was also substantially reduced in heart failure patients compared with controls (V_max 10.1±1.3 versus 49.8±7.1 pmol/10⁵ cells per 5 minutes, P<0.001). In association with this finding, we observed a 76% (P<0.01) reduction in mRNA expression for the cationic amino acid transporter CAT-1, as assessed by ribonuclease protection assay.

Conclusions—These data document both in vivo and in vitro evidence for a marked depression of L-arginine transport in human CHF and therefore provide an explanation for the restorative actions of supplemental L-arginine on vascular function in CHF.

Key Words: amino acids • nitric oxide • radioisotopes • endothelium • cells

	Introduction

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The systemic vasoconstriction that occurs in heart failure is widely considered to represent the net effect of activation of neurohormonal vasoconstrictor systems in the presence of endothelial dysfunction. Numerous studies have consistently demonstrated the presence of an attenuated response to endothelium-dependent vasodilators, most typically acetylcholine, in the peripheral and coronary circulation in human and experimental models of heart failure.^{1 2 3 4 5 6} Furthermore, these observations have been confirmed by use of a more physiological stimulus such as exercise and the hyperemia induced by ischemia.^{7 8}

Although the evidence for reduced endothelial function in congestive heart failure (CHF) is compelling, the mechanisms responsible for this phenomenon remain unclear. In broad terms, potential mechanisms include reduced expression of muscarinic cholinergic receptors on endothelial cells, altered intracellular signaling, reduced nitric oxide (NO) production, increased NO degradation, or an attenuated response by the intracellular targets of NO or cGMP.⁹ Recently, several groups have demonstrated an improvement in endothelial function in patients with heart failure in response to supplemental oral or intravenous L-arginine.^{10 11} This strategy has also been shown to improve endothelial function in other clinical paradigms that are characterized by endothelial dysfunction, most notably atherosclerosis.^{12 13 14}

The observations that L-arginine supplementation can improve endothelial function may provide some insight into the mechanisms responsible for this phenomenon. One possible explanation is a reduction in the rate of transport of L-arginine by endothelial cells, leading to relative intracellular substrate depletion for NO synthase. In the present study, we aimed to test the hypothesis that L-arginine transport is reduced in human heart failure using a novel in vivo approach in the forearm, in conjunction with an in vitro assessment of the kinetics of L-arginine transport by isolated peripheral blood mononuclear cells (PBMCs).

	Methods

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Subjects
The study population included 16 patients with CHF (mean±SEM age 52±3 years) recruited from the Heart Failure Service, Alfred Hospital, Melbourne, Australia, and 21 healthy control volunteers (mean±SEM age 41±2 years). The cause of heart failure was nonischemic in 10 patients and myocardial ischemia in 6 patients. All patients were treated with an ACE inhibitor and diuretics, 10 received digoxin, and 1 received a ß-blocker. Left ventricular ejection fraction was 23±4% (mean±SEM), and all patients had New York Heart Association class III symptoms of CHF. All patients participated after giving informed consent, and the study was conducted with the approval of the Alfred Hospital Ethics Review Committee.

Forearm Vascular Function and [³H]L-Arginine Kinetics
In 7 healthy control subjects and 5 heart failure patients, a 3F cannula was inserted into the brachial artery of the nondominant arm under local anesthesia for the infusion of radiolabeled arginine and acetylcholine as indicated below. A 5F cannula was inserted percutaneously into a deep antecubital forearm vein, as previously described,¹⁵ for venous blood sampling. Forearm blood flow was measured by venous occlusion plethysmography, as previously described.¹⁶

After a stabilization period of 20 minutes, resting forearm blood flow was measured and intra-arterial infusion of radiolabeled arginine was begun. After an initial priming bolus of 1 µCi of [4,5-³H]L-arginine (ICN Pharmaceuticals, specific activity 98 to 106 Ci/mmol) in 2 mL of 0.9% NaCl, a continuous intra-arterial infusion of 100 nCi/min of [4,5-³H]L-arginine was begun. Deep venous blood samples were drawn at 10, 20, 40, and 80 minutes after commencement of the infusion and immediately transferred to ice-chilled tubes containing EGTA, which were stored on ice until completion of the study.

After deep venous sampling and forearm blood flow estimation at 80 minutes, acetylcholine (BDH Chemicals) was coinfused into the brachial artery at doses of 9.25 and 37 µg/min at a flow rate of 2 mL/min for 5 minutes, as previously described.¹⁶

After completion of the study, blood samples were centrifuged at 4°C, and plasma was stored at -70°C. The plasma concentration of [³H]L-arginine was determined by ion-exchange chromatography, essentially as described previously.¹⁷ In brief, plasma proteins were removed from 750 µL of plasma by the addition of 250 µL of 20% trichloroacetic acid, followed by cooling on ice and subsequent removal of the precipitated proteins by centrifugation. Samples were then extracted at least 5 times in ether to remove trichloroacetic acid and combined in equal volume with 20 mmol/L HEPES, pH 6. Samples were then applied to a Dowex 50W-X8 column that had been preequilibrated with 20 mmol/L HEPES, pH 6. After repeated washes with deionized water, [³H]L-arginine was eluted from the column with 1N NaOH. Radioactivity was determined by liquid scintillation spectroscopy. Recovery of [³H]L-arginine from standard plasma samples was typically 90% to 95%.

To obtain an index of arginine uptake in the forearm, the clearance rate of [³H]L-arginine was calculated when the plasma concentration of [³H]L-arginine in deep venous plasma had achieved steady state, according to the formula

[³H]L-Arginine Transport in PBMCs
To obtain a complementary index of arginine transport in healthy subjects and patients with heart failure, we investigated the kinetics of [³H]L-arginine transport by PBMCs. In 10 heart failure patients and 14 healthy control subjects, 30 mL of peripheral blood was collected into tubes containing EGTA and diluted in equal volume with PBS. PBMCs were then isolated by Ficoll Paque (Pharmacia) density gradient centrifugation according to the manufacturer’s instructions. The resultant lymphocyte-rich band typically contained at least 90% lymphocytes (as assessed by fluorescence-activated cell sorter analysis; data not shown), and 90% of the cells were viable (as assessed by trypan blue exclusion). The PBMC band was resuspended in 17 mL of Krebs-Henseleit buffer and divided into 32 aliquots of 500 µL. Cell numbers were determined manually by hemocytometer counting.

For uptake studies, PBMCs were incubated in balanced salt solution containing L-arginine in concentrations ranging from 1 to 300 µmol/L, which included 100 nmol/L [³H]L-arginine, for a period of 5 minutes at 37°C. Uptake studies were performed in duplicate. For each subject, additional parallel uptake studies were performed in the presence of 10 mmol/L L-lysine, a specific competitor for transport by the cationic amino acid transport system. At the conclusion of the incubation period, the incubation tubes were rapidly cooled on ice. Cells were washed twice in ice-cold Krebs-Henseleit buffer and then lysed in 0.1% Triton X for subsequent liquid scintillation spectroscopy. Arginine transport by cationic acid transporter(s) was calculated as the difference between uptake in the absence and presence of 10 mmol/L lysine in the uptake solution.

Molecular Biology
In conjunction with the above protocol, PBMCs were isolated from peripheral blood as described above for studies of cationic amino acid transporter (CAT) mRNA expression in 7 healthy subjects and 5 patients with heart failure. After collection of PBMCs from 30 mL of peripheral blood, total cellular RNA was isolated according to the method of Chomczynski and Sacchi.¹⁸ Reverse transcription–polymerase chain reaction (RT-PCR) amplification of total RNA from mononuclear cells was used to construct cDNA fragments of CAT-1 and CAT-2B, with species- and isoform-specific sequences as follows: CAT-1 sense 5'-CCCCCGGCGTGCTGGCTGAAAA-3' and antisense 5'-TTCATCGCCTACTTTGGGGTGTCG-3' (GenBank accession number x59155); CAT-2B sense 5'-GATCCATTTTCCCAATGCCTCG-3' and antisense 5'-GGTGCAGTGGCAGCTTTGAT-3' (GenBank accession number u76369). PCR amplification was performed for 40 cycles, 30 seconds at 95°C denaturation, 30 seconds at 63°C annealing, and 1 minute at 72°C extension with Perkin-Elmer Taq polymerase and 1.5 mmol/L MgCl₂. The amplified cDNA was subsequently cloned into pGEM-T vector (Promega). The identities of the cloned PCR products were confirmed by sequencing.

The cloned CAT-1 and CAT-2B fragments were then used as templates to generate ³²P-labeled riboprobes for use in ribonuclease protection assays, as previously described.¹⁹ RNA loading was assessed with a riboprobe specific for human GAPDH. The protected fragments were then separated and analyzed on a 5% denaturing polyacrylamide/urea gel. Radioactive signals were measured with a PhosphorImager (Fuji BAS-1000).

Statistical Analysis
Data are presented as mean±SEM. Between-group comparisons were performed by Student’s t test. ANCOVA was also performed to determine the potential contribution of differences in blood flow to the between-group differences in [³H]arginine clearance. The Mann-Whitney test was used for comparison of group data that were not normally distributed. A P value <0.05 was considered significant.

	Results

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Forearm Blood Flow and Arginine Transport
In response to a continuous intra-arterial infusion of [³H]L-arginine, there was a progressive increase in the deep venous concentration of the radiotracer, which reached steady-state levels in plasma after 40 minutes (Figure 1, top). At 80 minutes, the deep venous plasma concentration of [³H]L-arginine was consistently higher in patients with CHF (Figure 1, bottom). Consistent with previous studies, the resting forearm blood flow of patients with CHF was somewhat lower than that in control subjects, although the difference was not statistically significant (Figure 2, top). Mean arterial pressures were significantly lower in CHF patients (72±3 versus 90±3 mm Hg, P<0.01), with similar forearm vascular resistance in the 2 groups (CHF versus controls: 35±3 versus 35±6 resistance units, P=NS).

View larger version (16K): [in this window] [in a new window]   Figure 1. Top, Time-dependent increase to steady state in plasma [3H]L-arginine concentration (conc.) in CHF patient (•) and healthy volunteer (). Bottom, Group data for plasma [3H]L-arginine concentration at steady state. dpm indicates disintegrations per minute.

View larger version (12K): [in this window] [in a new window]   Figure 2. Top, Forearm blood flow (BF) in control (n=7) and CHF patients (n=5). Bottom, Forearm [3H]L-arginine clearance rate in control and CHF patients. ** P<0.01.

As reflected by the higher plasma [³H]L-arginine concentration in deep venous blood samples from patients with heart failure, calculation of the [³H]L-arginine clearance rate from the forearm revealed a significant reduction in the rate of uptake of [³H]L-arginine across the forearm in heart failure (Figure 2, bottom). Although the forearm blood flow was lower, albeit nonsignificantly, we further examined the role of this difference as a potential confounding factor. By ANCOVA, the difference in forearm arginine clearance between heart failure patients and healthy volunteers remained statistically significant (P<0.01) when forearm blood flow was included as a covariate. In conjunction with the findings of reduced arginine clearance, we also observed a significant depression in the forearm vascular response to the endothelium-dependent vasodilator acetylcholine (Figure 3). A modest positive relationship between the maximal vasodilator response to acetylcholine and the forearm arginine clearance was observed (r=0.58, P=0.06).

View larger version (13K): [in this window] [in a new window]   Figure 3. Change in forearm blood flow (FBF) in response to intra-arterial acetylcholine (ACh) in control (•) and CHF patients (). *P<0.05.

Mononuclear Cell Arginine Transport in Heart Failure
Arginine transport mediated by CAT(s) was examined in PBMCs obtained from patients with heart failure and healthy control subjects. In preliminary experiments (Figure 4A), incubation times of up to 10 minutes were observed to fall within the period of maximal transport velocity, and accordingly incubation times of 5 minutes were chosen for the remainder of the study.

View larger version (19K): [in this window] [in a new window]   Figure 4. A, Relationship between incubation time and [3H]L-arginine uptake by PBMCs at substrate concentration of 30 µmol/L. B, [3H]L-arginine uptake kinetics in PBMCs obtained from control subjects (n=14) and CHF patients (n=10).

In both healthy volunteers and heart failure patients, accumulation of [³H]L-arginine was readily detectable over the physiological range (Figure 4B). However, the rate of accumulation of arginine by PBMCs obtained from individuals with heart failure was consistently substantially lower than that in healthy subjects. Furthermore, in heart failure patients, the maximal rate of transport (V_max) was significantly lower than that seen in healthy individuals (10.1±1.3 versus 49.8±7.1 pmol/10⁵ cells per 5 minutes, P<0.001), whereas no significant change in K_m was apparent (CHF patients versus controls, 107±43 versus 185±37 µmol/L).

CAT mRNA Expression
To examine the molecular basis for the reduction in the rate of arginine accumulation by PBMCs in patients with heart failure, we used ribonuclease protection analysis to examine the abundance of mRNA for CAT-1 and CAT-2B. We did not specifically examine the expression of CAT-2A, because preliminary studies using RT-PCR were barely able to detect CAT-2A mRNA expression in PBMCs. As shown in Figure 5, we observed a significant (76%) reduction in mRNA expression for CAT-1 that was readily detected by ribonuclease protection assay. Expression of CAT-2B in mononuclear cells, however, was negligible in both control subjects and heart failure patients (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 5. A, Ribonuclease protection assay for CAT-1 and GAPDH mRNA abundance in PBMCs from control and CHF subjects. B, CAT-1:GAPDH mRNA ratio from control (n=7) and CHF (n=5) subjects. **P<0.01.

	Discussion

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Over the past decade, there has been an exponential accumulation of data highlighting the crucial role that the endothelium plays in regulating vascular function in both health and disease. In this context, a key finding in a wide range of conditions, including heart failure, atherosclerosis, diabetes, and hypertension, has been a significant reduction in the response to endothelium-dependent vasodilators such as acetylcholine. In human heart failure, depressed vasodilator responses to acetylcholine have been observed in multiple vascular beds, including the forearm,^{5 16 20 21} lower limb,⁵ coronary circulation,^{4 22} and isolated resistance arteries.²³

The finding of a beneficial effect of L-arginine supplementation^{10 11 24} on endothelial function suggests that a relative deficiency of L-arginine, the key substrate for NO synthesis, may exist in heart failure. However, these observations do not distinguish whether the deficiency is extracellular or intracellular. In the present study, we tested the hypothesis that an impairment of cellular arginine transport was present in human heart failure. Using a novel in vivo approach in the human forearm, we have demonstrated for the first time a substantial reduction in the rate of clearance of [³H]L-arginine from the circulation, consistent with reduced endothelial arginine transport. In conjunction, we have also demonstrated the presence of endothelial dysfunction in our heart failure cohort, as reflected by the presence of a significantly attenuated response to intra-arterial infusions of acetylcholine. To provide a more detailed evaluation of arginine transport in the context of human heart failure, we also examined the kinetics of [³H]L-arginine uptake by freshly isolated PBMCs from healthy subjects and CHF patients. In keeping with the findings of the in vivo radiotracer study, we observed a substantial diminution in the rate of [³H]L-arginine accumulation by mononuclear cells over a range of physiologically relevant concentrations.

Our findings would therefore suggest that in heart failure, a reduction in arginine transport by endothelial cells could lead to a relative deficiency of intracellular arginine, thereby affecting NO synthesis. Supplementation with L-arginine would be expected to deliver increased substrate intracellularly, given that the normal plasma concentration of L-arginine is well below the saturating range for CAT-mediated transport, and therefore an increase in the L-arginine concentration would be expected to result in an increased rate of transport. Other workers have investigated the possibility that the beneficial actions of L-arginine supplementation represent the expected response to a deficiency in the plasma concentration of this amino acid, although this remains controversial.^{25 26} Alternatively, other investigators have examined the potential role of circulating inhibitors of L-arginine transport, such as asymmetric dimethyl arginine (ADMA) and N^G-monomethyl-L-arginine (L-NMMA). Recently, studies in human and experimental heart failure have reported low, micromolar-range plasma concentrations for both ADMA and L-NMMA. However, it appears unlikely that either compound would exert a significant effect on either L-arginine transport or endothelial function in the range of reported concentrations.^{25 27} Furthermore, in the present study, the abnormality of L-arginine transport was detected in both the forearm circulation and isolated PBMCs, making it unlikely that accumulation of potential transport inhibitors provides a major mechanism for this apparent defect. Alternatively, our in vivo findings could possibly be explained by the presence of antifailure medications, although such therapy has generally been shown to improve endothelial function.²⁸

Despite the intracellular concentration of L-arginine being far in excess of the K_m for NO synthase,^{29 30 31} limitation of L-arginine availability has been shown to reduce NO generation by both NOS2 and NOS3.^{32 33} Furthermore, the term "L-arginine paradox" has been applied to the converse observation in which L-arginine supplementation augments NO production.^{13 34 35} On the basis of these observations, it might be expected that the attenuation of arginine transport observed in the present study could result in a diminution of substrate delivery to NO synthase sufficient to reduce NO generation. To the best of our knowledge, the intracellular concentration of L-arginine in the setting of heart failure has only been examined in erythrocytes and surprisingly was found to be 100 µmol/L, whereas it was undetectable in red cells from healthy volunteers.²⁵ The significance of this observation is uncertain given that in the same study, the intracellular concentration of another cationic amino acid, L-lysine, was not different from that in control subjects, and also that erythrocytes do not express NO synthase.²⁵

At physiological concentrations of L-arginine, a single functional transport system (y⁺) accounts for 60% to 80% of the total carrier-mediated uptake activity.^{36 37 38} Cloning studies have identified 4 CAT proteins.^{39 40} CAT-1 and CAT-2B are both high-affinity, low-capacity transporters, thus resembling system y⁺. In contrast, CAT-2A is a low-affinity, high-capacity transporter with limited distribution.⁴⁰ CAT-3, a recently identified high-affinity transporter, has limited distribution, confined largely to the brain.⁴¹ To identify the mechanism responsible for the observed reduction in both the V_max for transport in isolated PBMCs and forearm clearance of [³H]L-arginine, we examined the mRNA expression of CAT-1 and CAT-2B in PBMCs. Using RNase protection analysis, we observed a significant reduction in the expression of CAT-1 mRNA, whereas expression of CAT-2B was barely detectable. Although the precise mechanism responsible for the downregulation of CAT-1 mRNA expression remains unclear, our findings could be explained by elevated levels of circulating cytokines that are known to be present in heart failure.^{42 43} In particular, it has recently been shown that inflammatory mediators may downregulate CAT-1 mRNA in macrophages.⁴⁴ In the present study, we did not discontinue antifailure medication because of concerns about the severity of the patients’ degree of left ventricular dysfunction. Therefore, we cannot exclude the possibility that our observations were influenced by the presence of antifailure medications, although these drugs have not been shown to influence CAT expression.

In the present study, we did not aim to pharmacologically characterize the precise nature of the L-arginine–mediated transport in PBMCs, other than to identify the L-lysine–sensitive component. Although the y⁺ system accounts for the major proportion of arginine transport, it is possible that changes in expression of other arginine transporting systems, including the b^o,+ system, could explain our findings.⁴⁰ However, this would seem unlikely given the fact that heart failure was associated with up to an 80% reduction in arginine transport, suggesting the involvement of the predominant arginine transport system.

In summary, for the first time, we demonstrate an impairment of L-arginine transport in patients with CHF. This observation provides one explanation for the well-documented impairment of endothelial function in human heart failure and for the observed beneficial actions of supplemental L-arginine.

	Acknowledgments

 

An institute grant to the Baker Medical Research Institute from the National Health and Medical Research Council of Australia supported this study. Dr Kaye is the recipient of a Wellcome Trust Senior Research Fellowship. The excellent technical assistance of Janice Fulton is acknowledged.

Received April 20, 2000; revision received July 12, 2000; accepted July 14, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kaiser L, Spickard RC, Olivier NB. Heart failure depresses endothelium-dependent responses in canine femoral artery. Am J Physiol. 1989;256:H962–H967.[Abstract/Free Full Text]
   
2. Ontekan M, Gay R, Greenberg B. Diminished endothelium-derived relaxing factor activity in an experimental model of chronic heart failure. Circ Res. 1991;69:1088–1096.[Abstract/Free Full Text]
   
3. Drexler H, Hayoz D, Munzel T, et al. Endothelial function in chronic congestive heart failure. Am J Cardiol. 1992;69:1595–1601.
   
4. Treasure CB, Vita JA, Cox DA, et al. Endothelium-dependent dilation of the coronary microvasculature is impaired in dilated cardiomyopathy. Circulation. 1990;81:772–779.[Abstract/Free Full Text]
   
5. Katz SD, Biasucci L, Sabba C, et al. Impaired endothelium-mediated vasodilation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol. 1992;19:918–925.[Abstract]
   
6. Kaye DM, Jennings G, Angus JA. Evidence for impaired endothelium dependent vasodilation in experimental left ventricular dysfunction. Clin Exp Pharmacol Physiol. 1994;21:709–719.[Medline] [Order article via Infotrieve]
   
7. Zelis R, Mason DT, Braunwald E. A comparison of the effects of vasodilator stimuli on peripheral resistance vessels in normal subjects and in patients with congestive heart failure. J Clin Invest. 1968;47:960–970.
   
8. Zelis R, Longhurst J, Capone RJ, et al. A comparison of regional blood flow and oxygen utilization during dynamic forearm exercise in normal subjects and patients with congestive heart failure. Circulation. 1974;50:137–143.[Abstract/Free Full Text]
   
9. Chin-Dusting JPF, Kaye DM. Restoration of nitric oxide function in human hyperlipidaemia, congestive heart failure and liver cirrhosis. Clin Exp Pharmacol Physiol. 1998;25:645–652.[Medline] [Order article via Infotrieve]
   
10. Hirooka Y, Imaizumi T, Tagawa T, et al. Effects of L-arginine on impaired acetylcholine-induced and ischemic vasodilation of the forearm in patients with heart failure. Circulation. 1994;90:658–668.[Abstract/Free Full Text]
    
11. Rector TS, Bank AJ, Mullen KA, et al. Randomized, double-blind, placebo-controlled study of supplemental oral L-arginine in patients with heart failure. Circulation. 1996;93:2135–2141.[Abstract/Free Full Text]
    
12. Lerman A, Burnett JC Jr, Higano ST, et al. Long-term L-arginine supplementation improves small-vessel coronary endothelial function in humans. Circulation. 1998;97:2123–2128.[Abstract/Free Full Text]
    
13. Creager MA, Gallagher SJ, Girerd XJ, et al. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248–1253.
    
14. Girerd XJ, Hirsch AT, Cooke JP, et al. L-Arginine augments endothelium-dependent vasodilation in cholesterol-fed rabbits. Circ Res. 1990;67:1301–1308.[Abstract/Free Full Text]
    
15. Bruning TA, Kemme MJ, Chang PC, et al. Calculation of plasma concentrations of intra-arterially infused compounds in forearm plethysmography. Cardiovasc Res. 1998;37:210–215.[Abstract/Free Full Text]
    
16. Chin-Dusting JPF, Kaye DM, Lefkovits J, et al. Dietary supplementation with L-arginine fails to restore endothelial function in forearm resistance arteries of patients with severe heart failure. J Am Coll Cardiol. 1996;27:1207–1213.[Abstract]
    
17. Bredt DS, Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989;86:9030–9033.[Abstract/Free Full Text]
    
18. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
    
19. Autelitano DJ. Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors. Biochem Biophys Res Commun. 1998;250:689–693.[Medline] [Order article via Infotrieve]
    
20. Kubo SH, Rector TS, Bank AJ, et al. Endothelium-dependent vasodilation is attenuated in patients with heart failure. Circulation. 1991;84:1589–1596.[Abstract/Free Full Text]
    
21. Hirooka Y, Imaizumi T, Harada S. Endothelium dependent forearm vasodilation to acetylcholine but not substance P is impaired in patients with heart failure. J Cardiovasc Pharmacol. 1992;20:S221–S225.
    
22. Inoue T, Sakai Y, Morooka S, et al. Vasodilatory capacity of coronary resistance vessels in dilated cardiomyopathy. Am Heart J. 1994;127:376–381.[Medline] [Order article via Infotrieve]
    
23. Angus JA, Ferrier CP, Sudhir K, et al. Impaired contraction and relaxation in skin resistance arteries from patients with congestive heart failure. Cardiovasc Res. 1993;27:204–210.[Abstract/Free Full Text]
    
24. Kubota T, Imaizumi T, Oyama J, et al. L-arginine increases exercise-induced vasodilation of the forearm in patients with heart failure. Jpn Circ J. 1997;61:471–480.[Medline] [Order article via Infotrieve]
    
25. Hanssen H, Brunini TM, Conway M, et al. Increased L-arginine transport in human erythrocytes in chronic heart failure. Clin Sci. 1998;94:43–48.[Medline] [Order article via Infotrieve]
    
26. Mehta S, Stewart DJ, Langleben D, et al. Short-term pulmonary vasodilation with L-arginine in pulmonary hypertension. Circulation. 1995;92:1539–1545.[Abstract/Free Full Text]
    
27. Feng Q, Lu X, Fortin AJ, et al. Elevation of an endogenous inhibitor of nitric oxide synthesis in experimental congestive heart failure. Cardiovasc Res. 1998;37:667–675.[Abstract/Free Full Text]
    
28. Nakamura M, Funakoshi T, Arakawa N, et al. Effect of angiotensin converting enzyme inhibitors on endothelium-dependent peripheral vasodilation in patients with chronic heart failure. J Am Coll Cardiol. 1994;24:1321–1327.[Abstract]
    
29. Liu J, Garcia-Cardena G, Sessa WC. Palmitoylation of endothelial nitric oxide synthase is necessary for optimal stimulated release of nitric oxide: implications for caveolae localization. Biochemistry. 1996;35:13277–13281.[Medline] [Order article via Infotrieve]
    
30. Palmer RM, Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun. 1989;158:348–352.[Medline] [Order article via Infotrieve]
    
31. Pollock JS, Forstermann U, Mitchell JA, et al. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A. 1991;88:10480–10484.[Abstract/Free Full Text]
    
32. Hibbs JB, Vavrin Z, Taintor RR. L-Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells. J Immunol. 1987;138:550–555.[Abstract]
    
33. Gold ME, Bush PA, Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun. 1989;164:714–721.[Medline] [Order article via Infotrieve]
    
34. Aisaka K, Gross SS, Griffith OW, et al. L-Arginine availability determines the duration of acetylcholine- induced systemic vasodilation in vivo. Biochem Biophys Res Commun. 1989;163:710–717.[Medline] [Order article via Infotrieve]
    
35. Eddahibi S, Adnot S, Carville C, et al. L-Arginine restores endothelium-dependent relaxation in pulmonary circulation of chronically hypoxic rats. Am J Physiol. 1992;263:L194–L200.[Abstract/Free Full Text]
    
36. Macleod CL, Kakuda DK. Regulation of CAT: cationic amino acid transporter gene expression. Amino Acids. 1996;11:171–191.
    
37. Greene B, Pacitti AJ, Souba WW. Characterization of L-arginine transport by pulmonary artery endothelial cells. Am J Physiol. 1993;264:L351–L356.[Abstract/Free Full Text]
    
38. McDonald KK, Rouhani R, Handlogten ME, et al. Inhibition of endothelial cell amino acid transport system y+ by arginine analogs that inhibit nitric oxide synthase. Biochim Biophys Acta. 1997;1324:133–141.[Medline] [Order article via Infotrieve]
    
39. Closs EI. CATs, a family of three distinct mammalian cationic amino acid transporters. Amino Acids. 1996;11:193–208.
    
40. Closs EI, Graf P, Habermeier A, et al. Human cationic amino acid transporters hCAT-1, hCAT-2A, and hCAT-2B: three related carriers with distinct transport properties. Biochemistry. 1997;36:6462–6468.[Medline] [Order article via Infotrieve]
    
41. Hosokawa H, Sawamura T, Kobayashi S, et al. Cloning and characterization of a brain-specific cationic amino acid transporter. J Biol Chem. 1997;272:8717–8722.[Abstract/Free Full Text]
    
42. Levine B, Kalman J, Mayer L, et al. Elevated circulating levels of tumor necrosis factor-alpha in severe chronic heart failure. N Engl J Med. 1990;323:236–241.[Abstract]
    
43. Mohler ER, Sorensen LC, Ghali JK, et al. Role of cytokines in the mechanism of action of amlodipine: the PRAISE heart failure trial. J Am Coll Cardiol. 1997;30:35–41.[Abstract]
    
44. Kakuda DK, Sweet MJ, MacLeod CL, et al. CAT-2 mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J. 1999;340:549–553.
    

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