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Am J Physiol Renal Physiol 291: F297-F304, 2006. First published March 28, 2006; doi:10.1152/ajprenal.00417.2005
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Amino acids as modulators of endothelium-derived nitric oxide

Masao Kakoki,1 Hyung-Suk Kim,1 Cora-Jean S. Edgell,1 Nobuyo Maeda,1 Oliver Smithies,1 and David L. Mattson2

1Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and 2Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 23 October 2005 ; accepted in final form 10 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To examine the mechanisms whereby amino acids modulate nitric oxide (NO) production and blood flow in the renal vasculature, chemiluminescence techniques were used to quantify NO in the renal venous effluent of the isolated, perfused rat kidney as different amino acids were added to the perfusate. The addition of 10–4 or 10–3 M cationic amino acids (L-ornithine, L-lysine, or L-homoarginine) or neutral amino acids (L-glutamine, L-leucine, or L-serine) to the perfusate decreased NO and increased renal vascular resistance. Perfusion with anionic amino acids (L-glutamate or L-aspartate) had no effect on either parameter. The effects of the cationic and neutral amino acids were reversed with 10–3 M L-arginine and prevented by deendothelialization or NO synthase inhibition. The effects of the neutral amino acids but not the cationic amino acids were dependent on extracellular sodium. Cationic and neutral amino acids also decreased calcimycin-induced NO, as assessed by DAF-FM-T fluorescence, in cultured EA.hy926 endothelial cells. Inhibition of system y+ or y+L by siRNA for the cationic amino acid transporter 1 or the CD98/4F2 heavy chain diminished the NO-depleting effects of these amino acids. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space stimulate efflux of L-arginine out of the cell. Thus the present experiments demonstrate that cationic and neutral amino acids can modulate NO production in endothelial cells by altering cellular L-arginine transport through y+ and y+L transport mechanisms.

kidney; L-arginine; flow cytometry

A LARGE BODY OF EVIDENCE indicates that the endogenous production of nitric oxide (NO) by nitric oxide synthase (NOS) in the kidney is partially dependent on the cellular uptake of NOS substrate L-arginine (L-Arg). Both in vitro and in vivo studies demonstrated that increasing extracellular L-Arg concentration can alter NO-dependent vasorelaxation in the kidney (9, 33). Cellular L-Arg uptake also appears necessary for NO-mediated effects in renal epithelial cells (20, 21, 30, 39, 41). Consistent with vasodilatory and/or natriuretic effects mediated by L-Arg uptake, supplementation of L-Arg increased sodium excretion (27, 28) and prevented or reversed the elevation of arterial blood pressure in a genetic rat model of sodium-sensitive hypertension (4, 18). Cellular L-Arg transport mechanisms therefore appear to be important to mediate NO-dependent function in the kidney.

Cationic amino acids, including L-Arg, are transported into cells by a number of differentially expressed transport systems (y+, bo,+, Bo,+, y+L) (10, 25). A y+ transport mechanism mediates cellular L-Arg uptake in renal epithelial cells (20, 21, 39, 41), whereas L-Arg uptake in endothelial cells is mediated by both y+ and y+L transport mechanisms (7, 35, 37). System y+ is a family of cationic amino acid transporters represented predominantly in the kidney by the CAT1 gene product (20, 41). System y+L mediates the cellular uptake of both cationic and neutral amino acids. The cellular uptake of cationic amino acids by y+ and y+L transporters is sodium independent, and uptake of neutral amino acids by the y+L transporter is sodium dependent. The y+L mechanism observed in endothelial cells has been identified to be associated with CD98/4F2 heavy chain (hc) (7, 35, 37). Both the y+ and y+L systems demonstrate competitive inhibition as well as trans-stimulation (exchange) (3, 5–7, 10, 35, 40). As such, it would be predicted that both cationic and neutral amino acids could influence intracellular L-Arg levels by decreasing cellular L-Arg uptake and/or by stimulating efflux of L-Arg from cells.

Cellular L-Arg uptake, mediated by a y+ mechanism, influences NO production in renal epithelial cells (20, 21, 39, 41). The importance of y+ and/or y+L-mediated L-Arg uptake mechanisms in the regulation of NO production in the renal vasculature and the regulation of renal vascular resistance has not been examined. In the present study, we tested the hypothesis that both cationic and neutral amino acids modulate NO and NO-dependent function in the renal vasculature through y+ and/or y+L-mediated L-Arg transport. Experiments were performed in the isolated, perfused rat kidney and in cultured endothelial cells to determine the importance of L-Arg transport in the regulation of NO production and vascular resistance.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male Sprague-Dawley rats were used in this study. The animals were obtained from Harlan Sprague Dawley (Madison, WI) and housed in the Animal Resource Center at the Medical College of Wisconsin. Food and water were available ad libitum. All experiments were conducted in accordance with the Medical College of Wisconsin Institutional Animal Care and Use Committee's guidelines.

Isolated, Perfused Kidney

Rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and the right kidney was isolated and perfused as described previously (19, 23). The renal artery was cannulated via the aorta to ensure constant perfusion. The kidneys were perfused with Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25.0 mM NaHCO3, and 10 mM glucose) saturated with 95% O2-5% CO2. The buffer contained phenylephrine (10–7 M) to maintain renal perfusion pressure (RPP) at ~100 mmHg with a constant perfusion rate of 5 ml/min using a roller pump. The renal venous effluent was collected continuously (2 ml/min), mixed with a chemiluminescence probe (0.5 ml/min; 2 mM H2O2, 18 µM luminol, 2 mM potassium carbonate, and 150 mM desferrioxamine), and forwarded into a flow-cell type chemiluminescence analyzer (CL-1525, JASCO, Tokyo, Japan) to quantify NO in the effluent. The experimental protocols were performed following a 60-min equilibration period.

Validation experiments demonstrated that this system responded with a decrease in renal vascular resistance and an increase in chemiluminescent signal when acetylcholine was added to the perfusate (10–7-10–6 M). Endothelial disruption was performed by serial introduction of seven individual air boli (0.5 ml) into the renal arterial perfusate. The functional absence of the endothelium was confirmed by the lack of the vascular response to 10–7 M acetylcholine.

Cell Culture Experiments

The EA.hy926 human vascular endothelial cell line was used in the present study; these cells were established from human umbilical vein endothelial cells as a continuous cell line (11). The cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 2 mM L-glutamine.

For flow cytometry analysis, the EA.hy926 cells were trypsinized and resuspended in Locke's solution (154 mM NaCl, 5.6 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 10 mM HEPES, 3.6 mM NaHCO3, and 5.6 mM glucose). After a 60-min equilibration period, calcimycin (10 µM) and amino acids (100 µM) were added and the cells were incubated for 60 min at 37°C in a CO2 incubator. The cells were then incubated for 30 min with 2 µM DAF-FM diacetate (Molecular Probes). Flow cytometry for the fluorescence of the benzotriazole derivative of DAF-FM (DAF-FM-T) was performed with a FACScan Cytometer equipped with an argon laser for excitation at 488 nm and a 530/30-nm bandpass filter (Becton Dickinson). We observed in preliminary experiments that the DAF-fluorescence was very low in the absence of calcimycin, and no differences were detected between the cells with vehicle and those treated with 1-mM concentrations of cationic amino acids, neutral amino acids, or L-NAME; the NO response to calcimycin stimulation was therefore compared when the cells were treated with different amino acids. A minimum of 2,500 events was collected for each analysis. Data acquisition and analysis were made with Summit 3.1 software (DakoCytomation).

Generation and Transfection of Small Interference RNA

Small interference RNAs (siRNAs) (12) for CAT1 and CD98/4F2hc were prepared with a Dicer siRNA Generation Kit (Gene Therapy Systems) according to the manufacturer's instructions. The sequences of the T7 promoter (lower case)-tagged primers used for RT-PCR are 5'-GCGtaatacgactcactatagggagaGTTGGTCTTACGGTACCAGC-3' (CAT1 Fwd; Genbank gi 4507046), 5'-GCGtaatacgactcactatagggagaTGCAGTGAGGGTGTGGACG-3' (CAT1 Rev), 5'-GCGtaatacgactcactatagggagaTCTCCACGACCGTCCTGTG-3' (CD98 Fwd; Genbank gi 21361343), and 5'-GCGtaatacgactcactatagggagaCCACTCTGCAAACCCTAAGG-3' (CD98 Rev). The delivery of siRNAs into the cells was confirmed using siRNAs labeled with Cy3 using Silencer siRNA Labeling Kit (Ambion). The siRNA (100 nmol) was transfected into 50% confluent EA.hy926 cells with Oligofectamine Reagent (Invitrogen), and the cells were harvested 48 h after the beginning of the 3-h incubation.

Immunoblot Analysis

Cells were lysed with 3 vol of lysis buffer containing 10 mM Tris (pH 7.5), 10 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P40, and 1 mM phenylmethylsulfonyl fluoride (16). Following a 10-min low-speed centrifugation (800 g), the supernatant was treated with N-glycosidase F (10 U/100 µg protein) for 1 h at 37°C. Protein samples (100 µg/lane) were separated under reducing conditions on a 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinyl difluoride membrane by wet blotting. The membranes were incubated with a rabbit polyclonal anti-CAT1 antibody (1:1,000) or mouse monoclonal anti-hCD98 antibody (1:50; Cymbus/Chemicon) as the primary antibody and with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibody (1:5,000; DakoCytomation) as the secondary antibody. Visualization of the bands was performed by chemiluminescence (Amersham).

L-Arg Efflux

Transport experiments were performed using the cluster-tray method for the measurement of solute flux in adherent cells (13). The serum-depleted (60 min) EA.hy926 cells were incubated with Locke's buffer with or without 1 mM N-ethylmaleimide (NEM) containing 0.5 µCi/ml of [14C]L-Arg for 60 min. After the incubation, the cells were washed twice and incubated with the buffer with L-amino acids (100 µM). Arginine efflux was determined by the ratio [Rsup/(Rsup + Rcell)] where Rsup is the radioactivity in the supernatant and Rcell is the radioactivity in the cells. Cell extracts were obtained by incubating the cells with 10% (wt/vol) trichloroacetic acid for 60 min. The radioactivity was determined by liquid scintillation counting.

Experimental Protocols

Protocol 1: influence of cationic, neutral, and anionic amino acids on vascular resistance and NO release in the isolated, perfused rat kidney. The isolated, perfused rat kidney was prepared as described above. The changes in renal vascular resistance and renal venous NO were measured during a 30-min control period and during two 30-min experimental periods in which 10–4 and 10–3 M concentrations of the following amino acids were successively added to the perfusate: L-ornithine (L-Orn), L-lysine (L-Lys), L-homoarginine (L-Harg), L-glutamine (L-Gln), L-leucine (L-Leu), L-serine (L-Ser), L-glutamate (L-Glu), and L-aspartate (L-Asp). The control and experimental periods were each 30 min in duration.

Protocol 2: influence of L-NAME, L-Arg, sodium substitution, or endothelial cell disruption on renal vascular resistance and NO release in isolated, perfused rat kidneys administered cationic or neutral amino acids. The isolated, perfused rat kidney was prepared as described above. Throughout the control and experimental portion of the experiment, the following substances were included in the perfusate: vehicle, 10–4 M L-NAME, or 10–4 M L-Arg. Additional experiments were performed in kidneys in which the endothelial cell layer was damaged by injection of air bubbles into the arterial perfusate until the renal vasculature no longer dilated in response to 10–7 M acetylcholine. A final group of kidneys was studied in which NaCl in the perfusate was replaced with N-methyl-glucamine (NMG). The changes in renal vascular resistance and chemiluminescent signal in each of these groups were measured during a 30-min control period and during a 30-min period following the addition of 10–4 M of the following amino acids to the perfusate: L-Orn, L-Lys, L-Harg, L-Gln, L-Leu, and L-Ser.

Protocol 3: influence of cationic, neutral, and anionic amino acids on NO in cultured endothelial cells. Experiments were performed to determine whether cationic, neutral, and anionic amino acids influenced NO, as measured by DAF fluorescence, in calcimycin-stimulated EA.hy926 cells. The cells were prepared as described above and incubated with vehicle plus 10–4 M L-Arg, L-NAME, L-Orn, L-Lys,L-Harg, L-Gln, L-Ser, L-Glu, or L-Asp. Additional experiments were performed in which the cells were incubated with excess L-Arg (10–3 M) along with 10–4 M of each of the cationic or neutral amino acids.

Protocol 4: influence of siRNA for CAT1 or CD98/4F2hc on NO release in cultured endothelial cells treated with cationic or neutral amino acids. Experiments in this protocol were performed to determine the role of the y+ transporter CAT1 and the y+L transporter CD98/4F2hc in the NO-suppressive response to cationic or neutral amino acids in cultured cells. Cells were treated with siRNA for either CAT1 or CD98/4F2hc for 48 h. Control cells were transfected with siRNA for green fluorescent protein (GFP). The NO response to calcimycin stimulation during the incubation with the cationic amino acid L-Orn or the neutral amino acid L-Gln was then examined in the absence of extracellular L-Arg in control and siRNA-treated cells.

Protocol 5: influence of cationic, neutral, and anionic amino acids on L-Arg efflux from cultured endothelial cells. Transport experiments were performed to quantify L-Arg efflux from cultured cells as described above in untreated cells and those incubated with 10–4 M L-Arg, L-Orn, L-Lys, L-Harg, L-Ser, L-Gln, L-Leu, L-Glu, or L-Asp (n = 4/group). L-Arg efflux was determined at 5, 10, 20, and 40 min after the incubation was begun. Experiments were performed in the presence and absence of 1 mM N-ethyl-maleimide (NEM), an agent which inhibits y+ but not y+L transport.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Protocol 1: Influence of Cationic, Neutral, and Anionic Amino Acids on NO Release and Vascular Resistance in the Isolated, Perfused Rat Kidney

The effects of cationic, neutral, and anionic amino acids on vascular resistance and NO release in the effluent of the isolated, perfused kidney are illustrated in Fig. 1 (n = 4/group). The addition of 10–4 and 10–3 M of the cationic amino acids L-Orn, L-Lys, or L-Harg led to a significant decrease in NO release in the venous effluent with an accompanying increase in renal vascular resistance. These vasoconstrictive responses took ~10 min to reach a stable level. The addition of the neutral amino acids L-Gln, L-Leu, or L-Ser to the perfusate also led to a decrease in NO and an elevation of vascular resistance in the isolated, perfused kidney. Neither the 10–4 or 10–3 M concentration of the anionic amino acids L-Glu or L-Asp altered NO or vascular resistance in this preparation. The addition of 10–4 M L-NAME, the NOS inhibitor, decreased the NO in the perfusate and increased vascular resistance; the L-NAME data (n = 8) were obtained following the amino acid infusion from the kidneys perfused with L-Glu or L-Asp.


Figure 1
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  		Fig. 1. Influence of L-ornithine (L-Orn), L-lysine (L-Lys), L-homoarginine (L-Harg), L-glutamine (L-Gln), L-leucine (L-Leu), L-serine (L-Ser), L-aspartate (L-Asp), L-glutamate (L-Glu), and Nw-nitro-L-arginine methyl ester (L-NAME) on vascular resistance and nitric oxide (NO) release from the isolated, perfused rat kidney. *P < 0.05 compared with the control value. {dagger}P < 0.05 compared with the 10–4 M dose.

 
Protocol 2: Influence of L-NAME, L-Arg, Sodium Substitution, or Endothelial Cell Disruption on Renal Vascular Resistance and NO Release in Isolated, Perfused Rat Kidneys Administered Cationic or Neutral Amino Acids

A summary of data from experiments to examine the mechanism of the changes in vascular resistance and NO release in kidneys in which cationic or neutral amino acids were added to the perfusate is illustrated in Fig. 2; each bar represents the value from an individual group (n = 4/group). The addition of 10–4 M of each cationic or neutral amino acid led to a significant decrease in NO in the renal venous effluent and an increase in renal vascular resistance. None of the amino acids affected vascular resistance or NO release in kidneys in which air boli were used to disrupt the endothelium or in kidneys pretreated with L-NAME. These experiments indicated that the effects of the cationic and neutral amino acids are endothelium dependent and NOS dependent. The addition of 10–4 M L-Arg to the perfusate in addition to the 10–4 M concentration of each amino acid significantly blunted the increase in renal vascular resistance and the decrease in NO in the renal venous effluent, indicating that the effects of the cationic and neutral amino acids are mediated through competition for L-Arg uptake. Finally, to test whether the effects of the amino acids are sodium dependent, NaCl in the perfusate was replaced with NMG. The substitution for NaCl with NMG did not affect the change in NO or vascular resistance associated with infusion of the cationic amino acids L-Orn, L-Lys, or L-Harg. The addition of NMG did, however, significantly blunt the changes in vascular resistance and NO in the groups administered the neutral amino acids L-Gln, L-Leu, and L-Ser. These results indicate that the effects of the cationic amino acids are sodium independent, whereas the effects of the neutral amino acids are sodium dependent.


Figure 2
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  		Fig. 2. Influence of 10–4 M L-Orn, L-Lys, L-Harg, L-Gln, L-Leu, and L-Ser on vascular resistance and NO release from the isolated, perfused rat kidney. Experiments were performed on groups of vehicle-treated control kidneys or those in which 10–4 M L-NAME or L-arginine (L-Arg) was added to the perfusate throughout the control and experimental portion of the experiment. Additional experiments were performed in kidneys in which the endothelial layer was damaged (endo–) by continuous injection of air until the renal vasculature no longer dilated to 10–7 M acetylcholine. A final group of kidneys was studied for each amino acid in which the NaCl in the perfusate was replaced with N-methyl-D-glucamine (NMG). *P < 0.05 compared with the control value. {dagger}P < 0.05 compared with the vehicle treatment for that amino acid.

 
Protocol 3: Influence of Cationic, Neutral, and Anionic Amino Acids on NO in Cultured Endothelial Cells

To elucidate the mechanisms through which both cationic and neutral amino acids can decrease NO release and increase renal vascular resistance, further experiments were performed in cultured endothelial cells (EA.hy926). The NO production in calcimycin-stimulated cells subjected to various treatments was assessed by DAF fluorescence (Fig. 3; >1,000 cells/group). Compared with vehicle-treated cells, 10–4 M L-Arg increased DAF fluorescence while the NOS inhibitor L-NAME significantly decreased calcimycin-stimulated NO in the cultured cell preparation. The addition of 10–4 M of the cationic amino acids L-Orn, L-Lys, and L-Harg or the neutral amino acids L-Gln and L-Ser led to a significant blunting of NO production in the cells compared with vehicle-treated cells. The reduction in the NO signal was attenuated when excess L-Arg (10–3 M) was added along with the cationic or neutral amino acids. Consistent with the observation in the isolated, perfused kidney, neither of the anionic amino acids, L-Glu or L-Asp, altered calcimycin-stimulated DAF fluorescence in the cultured cells. The changes in NO observed in the calcimycin-stimulated endothelial cells are therefore similar to those observed in the isolated, perfused kidney.


Figure 3
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  		Fig. 3. Intensity of calcimycin-stimulated DAF-FM-T fluorescence in cultured human EA.hy926 endothelial cells incubated with 10–4 M L-Arg, L-NAME, L-Orn, L-Lys, L-Harg, L-Gln, L-Ser, L-Glu, or L-Asp. Additional experiments were performed in which excess L-Arg (10–3 M) was added in addition to the cationic and neutral amino acids. *P < 0.05 vs. control cells. {dagger}P < 0.05 compared with the individual cationic or neutral amino acid in the absence of excess L-Arg.

 
Protocol 4: Influence of siRNA for CAT1 or CD98/4F2hc on NO Release in Cultured Endothelial Cells Treated with Cationic or Neutral Amino Acids

To determine the role of the y+ transporter CAT1 and the y+L transporter CD98/4F2hc in the NO-suppressive response to cationic amino acids in cultured EA.hy926 cells, cells were treated with siRNA inhibition before stimulation with calcimycin. Photomicrographs demonstrating the fluorescence of Cy3-labeled siRNA for GFP or CAT1 48 h after the transfection in cultured human EA.hy926 endothelial cells indicate that the siRNA is taken up by most cells and located mainly in the perinuclear cytoplasm (Fig. 4). Representative immunoblots for CAT1 or CD98/4F2hc in the cells incubated with the siRNAs for GFP (negative control) or with those for CAT1 or CD98/4F2hc demonstrate a reduction of the siRNA-targeted protein. Densitometric analysis indicated that CAT1-immunoreactive protein was significantly reduced by greater than 80% by CAT1 siRNA treatment compared with cells treated with nonspecific siRNA for GFP (n = 4). Similarly, siRNA for CD98/4F2hc protein significantly reduced CD98/4F2hc protein by greater than 90% (n = 4).


Figure 4
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  		Fig. 4. Photomicrographs of the fluorescence of Cy3-labeled small interference RNAs (siRNAs) for cationic amino acid transporter 1 (CAT1) 48 h after the transfection in cultured human EA.hy926 endothelial cells. The siRNA is located mainly in the perinuclear cytoplasm. A: phase contrast. B: Cy3 fluorescence. C and D: immunoblot for CAT1 (C) or CD98/4F2hc (D) in the cells incubated with the siRNAs for green fluorescence protein (GFP; negative control) or with those for CAT1 or CD98/4F2hc.

 
Experiments were performed in calcimycin-stimulated cells to examine the influence of L-Orn and L-Gln on NO production in control cells and cells with siRNA inhibition of CAT-1 or CD98/4F2hc. Compared with control cells incubated with 10–4 M L-Orn, calcimycin-stimulated cells subjected to siRNA inhibition of CAT-1 or CD98/4F2hc had a significantly greater NO than observed in the control cells (Fig. 5, top). These results indicate that both the y+ and y+L mechanisms are important for the L-Orn-mediated reduction in NO in these cultured cells.


Figure 5
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  		Fig. 5. Intensity of calcimycin-stimulated DAF-FM-T fluorescence in cultured human EA.hy926 endothelial cells treated with vehicle, CAT1 siRNA, or CD98/4F2hc siRNA. Fluorescence was measured under control conditions or following addition of 10–4 M L-Orn or L-Gln. *P < 0.05 vs. control cells. {dagger}P < 0.05 compared with L-Gln alone.

 
Further experiments examined the NO responses in calcimycin-stimulated cells incubated with the neutral amino acid L-Gln (Fig. 5, bottom). The siRNA inhibition of CAT-1 did not affect NO production in cells incubated with 10–4 M L-Gln compared with NO production in control cells exposed to L-Gln. In contrast, calcimycin-stimulated NO production during incubation with L-Gln was significantly greater in cells in which CD98/4F2hc was inhibited when compared with control cells. These results demonstrate that the effects of L-Gln are not mediated by a y+ mechanism, but the y+L mechanism is important in the reduction of NO in response to this neutral amino acid.

Protocol 5: Influence of Cationic, Neutral, and Anionic Amino Acids on L-Arg Efflux from Cultured Endothelial Cells

To test whether cationic and neutral amino acids lead to trans-stimulation of L-Arg, the influence of different amino acids (n = 4/group) on efflux of 14C-L-Arg from cultured endothelial cells was determined. Radiolabeled L-Arg moved out of cells subjected to each treatment during the 40-min experiment. Compared with untreated cells, the addition of the cationic amino acids (L-Arg, L-Orn, L-Lys, and L-Harg) increased L-Arg efflux (Fig. 6A ). The addition of the neutral amino acids L-Ser, L-Gln, and L-Leu also stimulated L-Arg efflux from the cultured cells (Fig. 6B). Incubation of the cells with the anionic amino acids L-Glu and L-Asp did not affect L-Arg movement from the cells compared with the untreated group of cells (Fig. 6C). There were no significant differences in L-Arg efflux between the cells treated with the four different cationic amino acids, between the cells incubated with the three different neutral amino acids, or between the cells treated with the two different anionic amino acids. For purposes of statistical comparison, the data from the different cationic amino acids (n = 16), neutral amino acids (n = 12), and anionic amino acids (n = 8) were combined. Although both cationic and neutral amino acids stimulated L-Arg movement out of the cultured endothelial cells, the cationic amino acids had a significantly greater stimulatory effect on L-Arg efflux than the neutral amino acids. Compared with control, the anionic amino acids did not stimulate L-Arg efflux from the cells.


Figure 6
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  		Fig. 6. Efflux of intracellular L-Arg from EA.hy926 cells. Data are expressed as the ratio of the [14C]L-Arg in the supernatant (Rsup) to the total radioactivity in the well (Rtot). A: L-Arg efflux in control cells (Blank) and cells incubated with 10–4 M of the cationic amino acids L-Arg, L-Orn, L-Lys, or L-Harg. B: L-Arg efflux in control cells (Blank) and cells incubated with 10–4 M of the neutral amino acids L-Ser, L-Gln, or L-Leu. C: L-Arg efflux in control cells (Blank) and cells incubated with 10–4 M of the anionic amino acids L-Glu or L-Asp. D: pooled effects of the cationic and neutral amino acids on L-Arg efflux; L-Arg efflux after 10 min was compared in the presence and absence of 1 mM N-ethyl-maleimide (NEM). *P < 0.05 vs. blank for same treatment (NEM + or –). #P < 0.05 vs. NEM(–) for the same amino acid. {dagger}P < 0.05 vs. cationic amino acids for same treatment (NEM + or –).

 
The effects of the cationic and neutral amino acids on L-Arg efflux following 10 min of incubation in control cells and in cells treated with NEM, a blocker of y+ transport, are illustrated in Fig. 6D. Both cationic and neutral amino acids stimulated L-Arg efflux, although the cationic amino acids had a significantly greater effect than the neutral amino acids. The addition of NEM did not alter the efflux of L-Arg in the control cells or in those treated with neutral amino acids. In contrast, the addition of NEM significantly decreased cationic amino acid-mediated L-Arg efflux by ~40% to a level not significantly different from that observed with neutral amino acids. The addition of NEM did not alter the efflux of L-Arg in control cells or in those incubated with neutral amino acids.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present experiments demonstrate that the cationic amino acids L-Orn, L-Lys, and L-Harg and the neutral amino acids L-Gln, L-Leu, and L-Ser decrease NO and increase renal vascular resistance in the isolated, perfused kidney. The effects of both the cationic and neutral amino acids are endothelium dependent, NOS dependent, and reversed by coadministration of excess NOS substrate. Moreover, the effects of the cationic amino acids are sodium independent, the effects of the neutral amino acids are sodium dependent, and the anionic amino acids have no influence on vascular resistance or NO release. Further studies in cultured cells confirmed the effects of the cationic, neutral, and anionic amino acids on NO production. It was also demonstrated that siRNA inhibition of the y+ transporter CAT-1 attenuated the NO-depleting influence of the cationic amino acid L-Orn, whereas siRNA inhibition of the y+L transporter CD98/4F2hc attenuated the NO-depleting effects of L-Orn and the neutral amino acid L-Gln in cultured cells. Finally, transport studies in cultured cells demonstrated that cationic or neutral amino acids in the extracellular space can lead to a depletion of cellular L-Arg. Together, these data are consistent with the hypothesis that y+ and y+L transporters are present in the endothelial layer of the rat renal vasculature and that manipulation of L-Arg transport by either mechanism can alter NO and renal vascular resistance.

These experiments demonstrate that a number of cationic and neutral amino acids can have a potent influence on L-Arg efflux, NO production, and vascular resistance. Although the in vitro nature of these studies necessitates a limited interpretation of these data, the concentrations of the amino acids used in the present study (10–4-10–3 M) are in the range of the physiological concentrations of these amino acids in the extracellular fluid (15). Moreover, increasing extracellular L-Arg concentration increases NO-dependent vasorelaxation in the renal and systemic vasculature (1, 9, 33); and cellular L-Arg uptake appears necessary for NO-mediated effects in the thick ascending loop of Henle (30), the macula densa (39), and the inner medullary collecting duct (41). In addition, physiological concentrations of L-Gln, but not L-Glu, markedly impaired endothelium-dependent vasorelaxation in the thoracic rat aorta and decreased intracellular concentration of L-Arg in cultured vascular endothelial cells (36). The manipulation of cellular L-Arg uptake can therefore have a potent effect on vascular NO and NO-dependent function.

Results of the present experiments are consistent with both competitive inhibition and trans-stimulation mechanisms influencing L-Arg in the renal vasculature. The observations that L-Arg increased NO in the cultured cells while excess or equimolar amounts of L-Arg blocked the NO-depleting effects of the cationic and neutral amino acids in the cultured cells or the isolated, perfused kidney are consistent with competitive inhibition. The other cationic amino acids and the neutral amino acids thus compete with L-Arg for cellular uptake, likely through y+ and y+L mechanisms. Interestingly, addition of the cationic and neutral amino acids to the perfusate, in the absence of L-Arg, also led to a decrease in NO in the isolated, perfused kidney. This observation is consistent with trans-stimulation of systems y+ and y+L which mediates the transmembrane exchange of extracellular and intracellular amino acids (3, 5–7, 10, 35, 40). This is a well-described mechanism whereby cationic or neutral amino acids in the extracellular fluid stimulate the efflux of L-Arg from the cells. As evidence of trans-stimulation, we observed that the addition of cationic or neutral amino acids, but not anionic amino acids, to the extracellular fluid enhanced the efflux of L-Arg from the cultured cells. The present data therefore indicate that cationic or neutral amino acids in the extracellular space can lead to a decrease in intracellular NOS substrate by both decreasing the amount of L-Arg taken into cells and also by increasing the movement of L-Arg out of endothelial cells.

The present data may help explain the discrepancy, known as the "arginine paradox," between the results of in vivo and in vitro studies which examined extracellular L-Arg and NO production. Because intracellular L-Arg (100–3,800 µM) (2) is much greater than the Km of NOS for L-Arg (<5 µM) (31), it has been thought that L-Arg levels are not rate limiting. A number of in vivo studies, however, have indicated that L-Arg supplementation improves endothelial function in humans and experimental animals with hypertension (17, 24), diabetes mellitus (29, 32), or hypercholesterolemia (8, 32, 34). Moreover, NO metabolites in plasma were shown to be decreased in a patient with lysinuric protein intolerance in whom the concentration of L-Arg in plasma is subnormal; in addition, the circulating level of NO metabolites increased in this patient following L-Arg supplementation (22). Finally, we recently demonstrated in anesthetized rats that supplementation of L-Arg in the renal medullary interstitial space leads to an increase in NO while administration of L-Lys, L-Orn, or L-Harg decreases NO and results in a reduction in blood flow in this portion of the kidney (20). These observations in humans and experimental animals support the hypothesis that extracellular L-Arg and/or the ratio of L-Arg to other cationic or neutral amino acids are important in the production of NO in the vasculature.

A number of explanations have been proposed for the arginine paradox. One possibility is that endogenous inhibitors of NOS, such as NG-monomethyl-L-arginine and asymmetric NG,NG-dimethyl-L-arginine, increase the functional Km of L-Arg in vivo (38). Another potential explanation is spatial sequestration of L-Arg in intracellular pools, some of which are inaccessible to eNOS (37). In addition, the immunolocalization of CAT1 with endothelial NOS (26) suggests that the close association of the L-Arg transporter and NOS could lead to L-Arg uptake-dependent NO production. The present results, which indicate that cationic and neutral amino acids are competitors of L-Arg for cellular uptake and can also lead to L-Arg depletion, are consistent with each of these possibilities. The cellular and molecular mechanisms which mediate the observed dependence of NO on L-Arg transport remain to be fully resolved.

In summary, the present experiments indicate that renal vascular NO and resistance can be modulated by manipulation of cellular y+ and y+L amino acid transport mechanisms. Physiological levels of several different cationic and neutral amino acids decreased endothelium-derived NO and increased vascular resistance in the isolated, perfused kidney. Further studies in cultured cells indicated that cationic and neutral amino acids can modulate NO production in endothelial cells by depletion of intracellular L-Arg through a combination of competitive inhibition which decreased cellular L-Arg uptake and trans-stimulation which increased the efflux of L-Arg. Further study will be required to determine the importance of these mechanisms in the physiological control of vascular resistance and to determine whether abnormalities in y+ or y+L transport in the endothelium or whether an imbalance between L-Arg and other amino acids in the extracellular fluid are important in disease.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was partially supported by National Institutes of Health Grants HL-29587 and DK-62803 to D. Mattson and a fellowship program of the Japanese Heart Foundation and of Sankyo Life Science Foundation to M. Kakoki.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. L. Mattson, Dept. of Physiology, Medical College of Wisconsin, PO Box 26509, 8701 Watertown Plank Road, Milwaukee, WI 53226-26509 (e-mail: dmattson{at}mcw.edu)

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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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