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Am J Physiol Heart Circ Physiol 287: H395-H400, 2004. First published March 11, 2004; doi:10.1152/ajpheart.01094.2003
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In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Melinda M. Parnell, Jaye P. F. Chin-Dusting, Jennifer Starr, and David M. Kaye

Wynn Department of Metabolic Cardiology, Baker Heart Research Institute, Melbourne, Victoria 8008, Australia

Submitted 20 November 2003 ; accepted in final form 4 March 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y+ transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [3H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 µg/min), Iso (25–50 and 200 µg/min), and sodium nitroprusside (SNP) (1–2 and 8 µg/min) on forearm plasma flow (FPF), L-[3H]arginine uptake, and L-[3H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[3H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = –0.50; P < 0.005). At a similar FPF (ACh 56.82 ± 9.25, Iso 58.49 ± 5.56, SNP 57.92 ± 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[3H]arginine (54,655 ± 8,018 dpm/min), compared with that observed with either Iso (40,517.23 ± 6,841 dpm/min; P = 0.01) or SNP (36,816 ± 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[3H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[3H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 107 ± 1.83 x 107 vs. control 6.67 x 107 ± 1.16 x 107 vs. Iso 7.35 x 107 ± 1.63 x 107 vs. SNP 6.01 x 107 ± 1.11 x 107 fmol·min–1·mg–1 at 300 µmol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.

endothelial function; membrane transport; nitric oxide; substrate availability

CONSIDERABLE WORK HAS IDENTIFIED the presence of endothelial dysfunction in a broad range of cardiovascular diseases, including heart failure and hypertension, as well as in the setting of cardiovascular risk factors, including hyperlipidemia, smoking, and diabetes (3). Whereas many have pursued the mechanism(s) responsible for endothelial dysfunction, commonly characterized by reduced nitric oxide (NO) bioavailability, causative processes are not well defined. Of interest, supplemental L-arginine has been demonstrated to augment endothelial function in many cardiovascular disease states (9, 31, 34), suggesting, in part, that endothelial dysfunction may involve defective L-arginine transport.

Although the intracellular concentration of L-arginine is reported to be far in excess of the Michaelis-Menten constant (Km) for NO synthase (NOS) (32), extracellular supplementation of L-arginine is reported to augment endothelial function in cardiovascular disease states (11, 12, 20, 26). This "arginine paradox" may be partially explained by the colocalization of y+ transporter and endothelial NOS (eNOS) in the membrane-bound caveolae (28). Furthermore, it has been suggested that caveolae-associated eNOS is more active in producing NO than cytosolic eNOS (15), placing further importance on extracellular substrate availability for NO synthesis. Although ~80% of all cationic amino acid transport (CAT) occurs via system y+ (30), little information on its acute regulation is available. In addition to y+-mediated transport in endothelial cells, a second transporter, y+L (36), appears to play a role in the cellular uptake of L-arginine. However, the precise extent of its role in endothelial L-arginine transport remains somewhat controversial (27).

One of the more commonly used experimental measures of endothelial dysfunction has been the vasodilatory response to ACh exposure. Imaizumi and co-workers (22) determined that in a healthy forearm, L-arginine but not D-arginine increased blood flow and that L-arginine increased forearm blood flow (FBF) responses to ACh but not sodium nitroprusside (SNP). These findings suggest that FBF responses to ACh may be partially dependent on the conversion of L-arginine to NO. In addition, although it is known that ACh activates eNOS by increasing intracellular Ca2+ (14), little is known about its ability to alter substrate availability.

To address these issues, the goal of the present study was to investigate the role of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and cultured bovine aortic endothelial cells (BAEC). We also aimed to determine the effects of blood flow on forearm L-arginine uptake in humans with the use of SNP.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
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Sixteen healthy male volunteers were recruited from the local community through advertisement. Procedures were followed in accordance to institutional guidelines, and, before experimentation, subjects gave written informed consent, which was performed with the approval of The Alfred Healthcare Group Ethics Committee in accordance with the Declaration of Helsinki (1989).

Clinical procedures. Each study was performed in the afternoon, in a quiet, temperature controlled room (22°C), after a standardized light lunch. Under local anesthesia (1% lignocaine, Astra; Ryde, Australia), a 3-Fr cannula (Cook; Eight-Mile Plains, Australia) was inserted into the brachial artery of the left arm to allow intra-arterial blood pressure recordings and drug infusions. A retrograde, percutaneous 5-Fr cannula (Cook) was inserted into an antecubital forearm vein for venous blood sampling. After an initial priming bolus of 1 µCi of L-[4,5-3H]arginine in 2 ml of 0.9% NaCl, a continuous intra-arterial infusion of 100 nCi/min L-[4,5-3H]arginine (ICN Pharmaceuticals; Seven Hills, Australia, specific activity 92 nCi/mmol) was commenced and continued throughout the study. In each of the 16 subjects, 10, 20, 30, and 40 min after the commencement of the infusion, deep venous blood samples were drawn, placed in tubes containing EGTA, and stored on ice. Two doses of ACh (9.25 and 37 µg/min Calbiochem-Novabiochem; Alexandria, Australia), Iso (25–50 and 200 µg/min; Abbott; Kurnell, Australia), and SNP (1–2 and 8 µg/min; Bull Laboratory; Mulgrave, Australia) were then infused into the brachial artery at a flow rate of 2 ml/min for 3 min each, with rest periods of at least 5 min between drug concentrations and of 15 min between each drug to allow complete washout of the previous infusion. FBF was measured using venous occlusion plethysmography (8) and was measured before and during the third minute of each infusion. Venous blood samples were drawn in the third minute of each infusion for the determination of L-[3H]arginine uptake, as described below.

To eliminate any effect of the order of the endothelium-dependent vasodilator drug infusions, in 4 of 16 subjects, the order of the drugs was reversed. It was established that there was no effect of the drug infusion order on FBF or L-arginine uptake responses.

Assessment of forearm plasma flow. FBF was measured using strain-gauge venous occlusion plethysmography, as previously described (8). Forearm plasma flow (FPF) was calculated as FPF (ml/min) = FBF x (1 – hematocrit) x forearm volume.

Assessment of forearm L-arginine uptake. On completion of the study, blood samples were centrifuged at 4°C and plasma was stored at –80°C. Plasma concentrations of L-[3H]arginine and L-[3H]citrulline were determined with the use of anion-exchange chromatography, as previously described (7, 25). Briefly, plasma proteins were removed from 750 µl of plasma by the addition of 250 µl of 20% TCA (BDH; Poole, UK), followed by cooling on ice and subsequent removal of the precipitated proteins by centrifugation. The samples were then extracted five times in diethyl ether to remove the TCA and combined in equal volume with 20 mmol/l pH 6 HEPES (Boehringer Mannheim). Samples were then applied to a Dowex 50-X8 column (Bio-Rad; Hercules, CA) that had been preequilibrated with 20 mmol/l HEPES. L-[3H]citrulline was identified in the initial column flow through and water elution, whereas L-[3H]arginine was eluted from the column using 1 mol/l NaOH. Radioactivity was determined by liquid scintillation spectroscopy. Recovery of L-[3H]arginine from standard plasma samples was typically 90–95%. To obtain an index of L-arginine transport in the forearm, the rate of L-[3H]arginine uptake was calculated according to the following formula: L-[3H]arginine uptake (dpm/min) = FPF x L-[3H]ArgA x FEX, where FEX is the fractional extraction of L-[3H]arginine across the forearm, calculated as

(1)
and L-[3H]ArgA/V are arterial and venous L-[3H]arginine concentrations, respectively.

Assessment of L-arginine uptake in BAEC. Consistent with previous studies (1, 29) that have reported positive results in response to ACh in BAEC passages 2–6, we used cultures of BAEC (passages 3–8). BAECs were prepared in 24-well plates and grown in DMEM (GIBCO; Grand Island, NY) + 10% FCS (Commonwealth Serum Laboratory; Lenexa, KS) until confluent. BAEC were FCS deprived 24 h before experimentation. BAECs were then incubated for 1 h in the presence or absence of (in µmol/l) 200 ACh, 200 Iso, or 100 SNP before L-[3H]arginine uptake experiments were performed. A solution containing 100 nmol/l L-[3H]arginine (New England Nuclear, Geneworks Australia), 300 µM of unlabeled L-arginine (Sigma; St. Louis, MO), and transport buffer (1 mol/l PBS, 1 mmol/l MgCl2, and 1 µmol/l CaCl2) was added to BAEC for 5 min. All studies were performed in triplicate. For each L-arginine concentration, additional parallel uptake studies were performed in the presence of 10 mmol/l L-lysine (Sigma), a specific competitor for transport by the CAT system. The uptake of L-[3H]arginine was terminated by two washes of 500 µl ice-cold PBS buffer. The cells were then lysed with 0.1% Triton X-100 (Sigma) for subsequent liquid scintillation spectroscopy. Protein content was calculated using the Lowry protein assay protocol (Bio-Rad). L-Arginine uptake by CAT(s) was calculated as the difference between uptake (in fmol·mg–1·min–1) in the absence and presence of 10 mmol/l L-lysine.

Statistical analysis. Statistical analysis was performed using SigmaStat version 2.03. The significance level employed was P < 0.05. Basal data were compared with the data after intervention using ANOVA with repeated measures, with post hoc analysis by Student-Newman-Keuls method. Multivariate ANOVA was employed to compare agonist responses in cell culture. All results, including those shown in figures, are presented as means ± SE.


   RESULTS
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DISCUSSION
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In vivo L-arginine uptake. All volunteers were within the healthy range in terms of body mass index lipid and blood pressure profile (Table 1). Venous L-arginine concentration reached steady state during the initial 40-min infusion period (Table 1). At this time, the basal FPF was 20.27 ± 3.10 ml/min and forearm uptake of L-arginine was 57,061 ± 6,845 dpm/min Intra-arterial infusions of ACh, Iso, and SNP all induced a dose-dependent increase in FPF (Fig. 1). Over a range of SNP-induced increases in FPF, we observed that the forearm uptake of L-arginine was reduced (r = –0.44; P < 0.005; Fig. 2). Hence, in the presence of the increased flow, L-arginine uptake was significantly diminished. Accordingly, matched agonist-induced FPF were selected to compare L-arginine uptake during agonist infusions. Under these conditions, intra-arterial infusion of ACh-stimulated forearm uptake of L-[3H]arginine compared with that observed with both Iso (P = 0.01) and SNP (P = 0.011; Fig. 3).


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  		Table 1. Baseline characteristics

 


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  		Fig. 1. Dose-dependent increases in forearm plasma flow (FPF) in response to infusions of vasodilator drugs Each volunteer received an intraarterial infusion of each of the following: ACh doses of 9.25 and 37 µg/min, isoprenaline (Iso) doses of 25–50 and 200 µg/min, and sodium nitroprusside (SNP) doses of 1–2 and 8 µg/min. FPF was measured before and during the third minute of each drug infusion via venous occlusion plethysmography, as described in MATERIALS AND METHODS. Data are expressed as means ± SE; n = 16 for each data point. *P < 0.01, significant increase in FPF from basal in all conditions.

 


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  		Fig. 2. Scatterplot demonstrating an inverse correlation between FPF induced by SNP and L-arginine uptake. Once a steady forearm plasma concentration of L-[3H]arginine was established, FPF and blood samples were obtained under resting conditions (n = 16) and then after 2 min of intra-arterial infusion of 2 doses of SNP [1–2 (n = 16) and 8 µg/min (n = 13)] (r = –0.48; P = 0.001).

 


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  		Fig. 3. Forearm venous blood samples were obtained for the determination of L-[3H]arginine uptake at basal (FPF: 20.27 ± 3.10 ml/min) and after intra-arterial infusions of ACh (37 µg/min), Iso (25–50 µg/min), and SNP (1–2 µg/min). ACh, Iso, and SNP all induced a similar increase in FPF above basal FPF (ACh: 56.82 ± 9.24 ml/min, Iso: 58.49 ± 5.56 ml/min, and SNP 57.92 ± 4.96 ml/min). ACh stimulated L-arginine uptake above that of Iso (*P = 0.01) and SNP ({dagger}P = 0.011; n = 16).

 
In addition, venous plasma L-[3H]citrulline levels were significantly elevated during ACh infusion (217.46 ± 30.12 dpm/ml) compared with those at baseline (175.43 ± 18.81 dpm/ml; P = 0.046) and SNP (176.88 ± 20.71 dpm/ml; P = 0.049; Fig. 4). Of interest, whereas L-[3H]citrulline concentrations during Iso infusion (192.27 ± 21.51 dpm/ml; P = 0.07) tended to be lower than ACh, it appeared to be intermediate between ACh and SNP.



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  		Fig. 4. Venous plasma L-[3H]citrulline concentration at basal (FPF: 20.27 ± 3.10 ml/min) and after intra-arterial infusions of ACh (37 µg/min), Iso (25–50 µg/min), and SNP (1–2 µg/min). ACh, Iso, and SNP all induced a similar increase in FPF above basal FPF (ACh: 56.82 ± 9.24 ml/min, Iso: 58.49 ± 5.56 ml/min, SNP: 57.92 ± 4.96 ml/min). ACh stimulated L-[3H]citrulline production above basal (*P = 0.046), Iso (P = 0.07), and SNP infusion ({dagger}P = 0.049, n = 16).

 
L-Arginine uptake in BAEC. In a complementary manner to the human in vivo forearm studies, ACh significantly increased L-[3H]arginine uptake compared with either control, Iso, or SNP (ACh 12.0 x 107 ± 1.83 x 107 vs. control 6.67 x 107 ± 1.16 x 107 vs. Iso 7.35 x 107 ± 1.63 x 107 vs. SNP 6.01 x 107 ± 1.11 x 107 fmol·min–1·mg–1 at 300 µmol/l L-arginine; Fig. 5; P = 0.043).



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  		Fig. 5. Effect of ACh (200 µM), Iso (200 µM), and SNP (100 µM) on L-[3H]arginine uptake in bovine aortic endothelial cells (BAEC; passages 3–8). After 1-h preincubation with the above treatments, BAEC were incubated with uptake buffer containing L-[3H]arginine or 300 µM L-arginine ± 10 mM L-lysine (system y+ competitive inhibitor) for 5 min, as described in MATERIALS AND METHODS. ACh stimulated L-arginine uptake above control, SNP, and Iso (*P = 0.043; n = 7–8 for each treatment).

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to investigate the regulation of L-[3H]arginine uptake in the human forearm, in particular during exposure to vasodilator drugs that are commonly used in research practice. Whereas the present study is based on the principle of forearm L-arginine uptake as a measure of forearm endothelial cell L-arginine transport (25), clearly other cell types (both circulating and tissue based) probably contribute to overall uptake of the radiolabeled arginine. Given previous studies (13) that demonstrate an effect of flow on radiotracer uptake, we first established the effect of increasing FBF on L-arginine uptake. In this component of our study, we observed a flow-dependent decrease in L-arginine uptake during incremental SNP infusion. In addition, under conditions of similar FPF, the forearm uptake of L-arginine was increased significantly by the endothelium-dependent vasodilator ACh, but not by Iso. These findings corresponded with an increased production of L-[3H]citrulline induced by ACh, indicating enhanced NO production. In a complementary manner, we further demonstrated that ACh stimulate L-arginine uptake above that of Iso, SNP, and control conditions in BAEC culture. Taken together, these data indicate that the uptake of L-arginine, both in vivo and in vitro, can be acutely regulated.

In the context of our findings of the effect of SNP on forearm L-arginine uptake, it has been suggested that NO may directly influence L-arginine uptake in a dose-dependent manner. Cell culture studies have documented an inhibition of L-arginine transport after high concentrations of NO donor (S-nitroso-N-acetyl penicillamine) incubation (30), whereas others have shown an acute increase in L-arginine transport (15 min), followed by a sustained inhibition (1–5 h) with S-nitroso-N-acetyl-penicillamine (29) and glyceryl trinitrate (1) incubation. A range of potential mechanisms of action for the putative direct effect of NO on L-arginine transport has been proposed. These include membrane hyperpolarization (6, 24) and superoxide-mediated modulation of L-arginine transport (30). Alternatively, potential actions of NO on arginine transport have also been explained by a more "classic" cGMP-mediated process (37). In vitro findings in the present study support other similar studies (5), demonstrating no change in L-arginine uptake after administration of incremental concentrations of SNP. It may therefore be suggested that the decrease in L-arginine uptake after an acute 3-min forearm SNP infusion may be a result of increased and/or a redistribution of flow, rather than the SNP per se.

Over the past decade, the biochemical aspect of the regulation of NOS activity and NO generation have been extensively characterized. Of relevance to the present study, it has been clearly shown that extracellular L-arginine availability plays a key role in NO production (23). This observation occurs despite the demonstration that the intracellular L-arginine concentration is in excess of the Km for NOS (17); this is termed the L-arginine paradox. This finding has led to the discovery of a caveolae-based microdomain (10, 19) that incorporates eNOS and CAT, CAT-1 (28), giving preferential substrate delivery to eNOS. In this model, it is therefore evident that processes that regulate CAT affinity could exert considerable influence on the generation of NO by eNOS.

By virtue of the emerging evidence for the importance of CAT-mediated transport of L-arginine in NO production, we aimed to characterize the potential for rapid modification of L-arginine uptake in endothelial cells. With our chosen method, while likely to be negligible, L-arginine uptake into circulating cells such as macrophages, erthyrocytes, and leucocytes cannot be excluded, nor can passive movement into smooth muscle cells. System y+-mediated L-arginine transport accounts for ~80% of the total endothelial cellular L-arginine transport (30). In addition to system y+-mediated transport of L-arginine, other transport systems for arginine have been well characterized (27). In particular, the y+L transporter may play an important role in certain tissues, although in the vasculature it seems unlikely that y+L plays a major role based upon the somewhat higher Km and maximum velocity for y+-mediated arginine transport and the greater sensitivity of y+L to inhibition by neutral amino acids (27). We showed that L-arginine uptake can be stimulated by ACh, but not by Iso or SNP. Importantly, we further demonstrated elevated plasma L-[3H]citrulline concentration after ACh stimulation, above that of Iso and SNP, supporting the notion that extracellular substrate delivery plays a key role in the generation of NO. As a corollary of our findings, the current data suggests that the vasodilatory action of ACh may depend on the uptake of L-arginine underscoring an important role for the acute regulation of L-arginine transport.

The vasodilator actions of ACh are not only mediated by release of NO, but may also be mediated via endothelium-dependent hyperpolarizing factor. Regulation of L-arginine transport appears to involve cellular membrane potential, and exposure of endothelial cells to hyperpolarizing agents such as ATP and bradykinin increases L-arginine uptake (6), whereas subsequent depolarization can cause a decrease in L-arginine uptake (4, 39). Because ACh has previously been shown to induce cellular hyperpolarization (2, 35), the increase in y+ transporter activity after ACh incubation in the present study may have occurred by a similar mechanism. However, the present study did not directly assess this component. In conjunction with this potential mechanism, Posch et al. (33) described that shear stress induced but not agonist-induced NO production is dependent on L-arginine uptake. Cell culture studies have also indicated that NOS agonists also stimulate system y+ (29) given they are substrates for this system.

It is of interest that the addition of Iso elicited no significant increase in L-arginine uptake or [3H]L-citrulline production in the present study. That the ACh-induced increased in L-[3H]citrulline production was not statistically significant from Iso (P = 0.07) suggests that the vasodilatory effects of Iso may occur, in a minor role, via production of NO. Our findings are of interest, as previous work (16, 38) has suggested that Iso has a vasodilatory effect that is mediated via {beta}-adrenergic and cAMP activation of the L-arginine/NO system. Others, however, have found no direct synthesis of NO after the addition of Iso to porcine aortic endothelial cells (18). Furthermore, it appears that vascular responses to Iso vary between both species and vascular beds (21). Whereas researchers are beginning to recognize that vasodilation to {beta}-adrenergic agonists such as Iso is mediated in part by NO, the endothelium-independent actions of Iso via direct elevation of cAMP in vascular smooth muscle remains of importance.

The present study demonstrates for the first time that agonists such as ACh can acutely modulate L-arginine transport in humans. This was associated with acute changes in L-citrulline production, indicating substrate delivery to eNOS and subsequent NO production. These findings suggest a dependence of NO production on L-arginine transport by agonists other than shear. Given evidence for the presence of impaired endothelial function in a range of cardiovascular diseases, altered L-arginine transport provides a potential explanation for these findings, particularly when the responses to ACh are used as a pharmacological test.


   GRANTS
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The Wynn Department of Metabolic Cardiology is supported by a grant from the Atherosclerosis Research Trust. M. M. Parnell was a recipient of a Monash Postgraduate Research Scholarship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. M. Kaye, Wynn Dept. of Metabolic Cardiology, Baker Heart Research Institute, PO Box 6492, St. Kilda Rd. Central, Melbourne, Victoria 8008, Australia (E-mail: david.kaye{at}baker.edu.au).

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.


   REFERENCES
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