Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[14C]lysine uptake experiments were conducted within a 103-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies. Two Na+-independent, N-ethylmaleimide-sensitive uptake components were found, one with high apparent affinity (Km=222±71 μM) and low transport capacity (Vmax=121±36 pmol/min per mg of vesicle protein) and the other with low apparent affinity (Km=16±4 mM) and high capacity (Vmax=4.0±0.4 nmol/min per mg of vesicle protein). L-Lysine uptake mediated by both components was stimulated by the presence of intravesicular L-lysine as well as by valinomycin-induced membrane hyperpolarization. Altogether, this behaviour is consistent with the functional properties of the CAT-1 and CAT-2A members of the system y+ family of cationic amino acid transporters. Furthermore, mRNA transcripts for these two carrier proteins were identified in freshly isolated rat cardiac myocytes, the amount of CAT-1 mRNA, relative to β-actin, being 33-fold larger than that of CAT-2A. These two transporters appear to function simultaneously as a homoeostatic device that supplies cardiac-muscle cells with cationic amino acids under a variety of metabolic conditions. Analysis of two carriers acting in parallel with such an array of kinetic parameters shows significant activity of the low-affinity component even at amino acid plasma levels far below its Km.
The L-isomers of lysine (L-lysine) and arginine (L-arginine) play central roles in a number of metabolic pathways. L-Lysine is an essential dietary amino acid for all vertebrates, with a primary role as substrate for protein synthesis and also as a precursor in the synthesis of 5-OH-lysine, a constituent of collagen, and of carnitine, involved in β-oxidation of fatty acids. L-Arginine, on the other hand, is a conditionally essential amino acid derived from both endogenous and dietary sources, which becomes essential during physiological body growth and in catabolic states such as trauma, severe burns, stress, sepsis and starvation. Among its many functions, L-arginine stimulates the release of peptide hormones, and is a precursor for the synthesis of urea, creatine, agmatine and polyamines [1,2]. L-Arginine is also the substrate for nitric oxide (NO) and citrulline production via nitric oxide synthase. NO is a major regulator of the nervous, immune and cardiovascular systems . In the heart, ventricular myocyte-generated NO affects myocardial contractile performance through modulation of β-adrenergic inotropic responses , and changes in myocardial relaxation [5,6], force–frequency relationship  and the Frank–Starling response .
The enzymes required for endogenous synthesis of L-arginine or those involved in L-arginine recycling from citrulline are not significantly expressed in cardiac-muscle cells [9,10]. Thus L-arginine (and the essential L-lysine) must enter cardiac myocytes from the circulation through a carrier-mediated process. In this regard, large L-Arg-activated electrical currents representing L-arginine transport were recently detected in whole-cell voltage-clamped cardiac ventricular myocytes . These inwardly directed currents were activated by L-arginine in an Na+-independent manner, with apparent affinity in the millimolar range, and the process showed high transport capacity and selectivity for the L-enantiomers of cationic amino acids. In fact, L-lysine and L-ornithine (but not D-arginine) elicited inward currents of magnitude and Δψ (membrane potential) dependence similar to those displayed by L-arginine. In addition, currents were irreversibly blocked by briefly exposing the cells to low concentrations of the thiol reagent NEM (N-ethylmaleimide).
Among the five transport systems described so far for cationic amino acids, only the so-called system y+ includes a low-affinity member (CAT-2A), is highly selective for dibasic amino acids and the transport process is both Na+ independent and highly sensitive to NEM . Therefore L-Arg-activated currents in acutely isolated rat cardiomyocytes are compatible with amino acid transport through the low-affinity, high-capacity CAT-2A. However, the functional presence of this transporter under physiological conditions in ventricular myocytes remains controversial. To the best of our knowledge, functional expression of CAT-2A has been reported only in neonatal rat cardiac myocytes on extensive cytokine treatment . On the other hand, the CAT-1 member of system y+, reported to be constitutively expressed in rat cardiac-muscle cells [12–14], was not detected with electrophysiological techniques . CAT-1 transports cationic amino acids with relatively high apparent affinity (K0.5=0.1–0.25 mM) and low capacity . Interestingly, such a concentration range for half-maximal activation of transport matches that of L-arginine and L-lysine plasma levels in healthy humans and rats [15,16].
The present study was thus aimed at addressing the significance of a low-affinity transporter in this context as well as investigating additional components of cationic amino acid transport in cardiac-muscle cells. Radiolabelled L-lysine uptake experiments were performed in giant sarcolemmal vesicles prepared from adult rat cardiac ventricles by modifying a technique originally described to measure glucose and fatty acid transport in the absence of metabolism . We found two uptake components with strikingly different apparent affinities and transport capacities, whose properties are compatible with the simultaneous activity of CAT-1 and CAT-2A. Because of its high capacity, the low-affinity component accounts for half of the total cationic amino acid transport activity at physiological plasma levels of L-arginine and L-lysine.
Isolation of giant vesicles
For each experiment, three adult Sprague–Dawley rats were injected with Nembutal (100 mg· kg−1; intraperitoneal) in accordance with institutional guidelines, and hearts (1.2–1.4 g) were removed under complete anaesthesia. Preparation of giant sarcolemmal vesicles followed the procedure described by Koonen et al. , with some modifications. In brief, cardiac ventricles were dissected in a Petri dish (placed on ice) containing 140 mM KCl and 20 mM Mops (pH 7.40) at 23°C with KOH (KCl/Mops solution) and 1 mM PMSF, and cut into 1–3 mm thick layers. Tissue layers were then washed with KCl/Mops solution and incubated for 90 min at 34°C (in a shaking water bath) with a vesicle preparation medium (2.5 ml for up to 8 g of tissue) containing KCl/Mops solution, 0.8 mM CaCl2, 1 mg/ml aprotinin and 0.3% (w/v) collagenase type II. After incubation, the supernatant was transferred to another tube and the remaining tissue was washed with ∼1 ml of KCl/Mops solution containing 10 mM EDTA. The supernatant plus the 1 ml wash were thoroughly mixed with Percoll (final concentration: 16%, v/v) and aprotinin (1 mg/ml). The resulting suspension was set as the bottom layer of a density gradient that also included a 3 ml middle layer of 4% (w/v) Nycodenz and a 1 ml KCl/Mops upper layer. This sample was centrifuged at 50 g for 50 min at room temperature (22–24°C) and the vesicles were harvested from the fraction just above the Nycodenz layer in a 0.5–1 ml volume. Vesicles were centrifuged at 900 g for 10 min at room temperature and resuspended in the appropriate volume of KCl/Mops solution. The preparation was kept on ice until used for uptake assays, usually within 15–60 min depending on the experimental design. After saving an aliquot for protein quantification (see below), each vesicle preparation yielded enough material to test two to three experimental conditions (uptake plus background, in triplicate).
L-[14C]lysine uptake experiments
Vesicles suspended in KCl/Mops solution (90 μl) were placed in glass tubes containing 10 μl of the same KCl/Mops solution in the absence or presence of 4 mM NEM. After incubating the samples for at least 10 min at 37°C, transport assays were started by adding 100 μl of a prewarmed solution containing L-[U-14C]lysine (0.5–1 μCi per tube) plus twice the selected final concentration of non-radioactive L-lysine in KCl/Mops solution. KCl concentration in this solution was adjusted to satisfy the following equation: [KCl]+[L-lysine]=140 mM. The concentration of L-[U-14C]lysine (20–40 μM) was only included in the calculation of total [L-lysine] for experiments in the range 50–500 μM. Uptake was allowed to proceed for the desired time at 37°C and stopped by quenching the reaction mixture in 5 ml of an ice-cold solution that contained (in mM) 100 L-lysine, 40 KCl and 20 Mops (pH 7.40 with KOH) (23°C). The suspension was immediately filtered through 0.45 μm Millipore filters (presoaked in 0.3% polyethylenimine in water) and the collected material was washed three times with 5 ml of ice-cold KCl/Mops solution. Filters were dried and placed in scintillation vials containing 10 ml of Scintiverse I cocktail (Fisher Scientific) for liquid-scintillation counting. Due to the large isotopic dilution when testing the L-lysine concentrations 10–50 mM, liquid-scintillation counting was allowed to proceed for longer periods of time to detect radioactivity levels statistically significantly larger than background.
RNA isolation and RT (reverse transcriptase) reaction
RNA was isolated from freshly prepared rat cardiac ventricular myocytes and hepatocytes (positive control for CAT-2A) by using a Qiagen RNeasy® Mini kit according to the manufacturer's protocol. DNA-free™ (Ambion) was used to remove DNA contamination from total RNA samples (50 μg of total RNA, 1×DNase I buffer and 0.08 unit of DNase I). The quality and integrity of the DNase-treated RNA was confirmed by running 2 μg of total RNA on a 1.2% TBE (Tris/borate/EDTA; 1×TBE=45 mM Tris/borate and 1 mM EDTA) agarose gel. To set up the RT reaction, 5 μg of DNase-treated total RNA and 0.3 μg of random hexamers (Roche) were combined. The mixture was denatured by heating at 65°C for 5 min and quick chilling in ice slurry. After the addition of 1× first-strand Superscript III RT buffer (Invitrogen), the denatured samples were annealed at room temperature for 5 min. To initiate cDNA synthesis, 0.5 mM dNTPs, 10 mM dithiothreitol, 1 unit of SUPERase In (Ambion), and 10 units of Superscript III RT buffer (Invitrogen) were added and the reaction was incubated at 48°C for 1 h. The reaction was stopped by heat inactivation at 70°C for 15 min.
Quantitative real-time PCR was performed with a Light Cycler (Roche) in the SYBR® Green format using primers and conditions described previously . Briefly, the final 20 μl PCR reaction contained: one-tenth RT reaction, 0.4 μM custom primers (UMDNJ Molecular Resource Facility), 0.2 mM dNTPs (Invitrogen), 0.25 μg of BSA (Idaho Technology), 1.6 μl of Enzyme Diluent (10 mM Tris, pH 8.3, and 2.5 mg/ml BSA) (Idaho Technology), 0.075× SYBR® Green I, 1× Advantage 2 PCR buffer and 1× Advantage 2 PCR Polymerase Mix (Clontech).
The primer pairs used in the present study were: sense CAT-1, 5′-ACGGTGATGATAAGAACGGC-3′; antisense CAT-1, 5′-GCTTACTCTTTGGTGGCTGC-3′; sense CAT-2A, 5′-CTTTGGTGATGGTCATTTGC-3′; antisense CAT-2A, 5′-TTCATGGTCCCGTTCTTACC-3′; sense β-actin, 5′-GAGGCTCTCTTCCAGCCTTCCTTCCT-3′; antisense β-actin, 5′-CCTGCTTGCTGATCCACATCTGCTGG-3′.
Optimal PCR amplification conditions for individual primer pairs were determined experimentally as reported previously . CAT-1 and CAT-2A amplification conditions were: denaturation 94°C for 2 s; annealing 60°C for 2 s; and extension 68°C for 12 s. β-Actin amplification conditions were: denaturation 94°C for 2 s; annealing 63°C for 2 s; and extension 68°C for 14 s. Two negative controls were incorporated into each RT–PCR (reverse transcription–PCR) run, including replacement of PCR templates with water, and running the PCR with products from an RT reaction in which the Superscript III RT enzyme was omitted.
Vesicles were washed twice with 140 mM NaCl and 20 mM Mops (pH 7.40 with NaOH, 23°C) to remove external KCl. To measure enzymatic activity in inside-out vesicles, 800 μl of a K+-free reaction medium containing (final concentration, in mM) 140 NaCl, 0.2 EGTA and 20 Mops/Na were added to glass tubes that contained 100 μl of the washed vesicles (6–8 μg of protein per tube). Total ATPase activity was determined in a similar medium except for the inclusion of (final concentrations) 20 mM KCl and 0.2 mg/ml of the detergent C12E8 (octaethylene glycol monododecyl ether) to open the vesicles . The reaction was started by adding 100 μl of an ice-cold solution containing (final concentration, in mM) 3 ATP-Mg salt, 1.5 MgCl2 and 20 Mops/Na to samples previously incubated at 37°C for 2 min. After 10 min at 37°C, the reaction was stopped with 0.5 ml of a solution containing (in mM) 160.7 ascorbic acid, 3.8 ammonium heptamolybdate and 472 HCl, and the release of phosphate was measured as described by Baginski et al.  as modified by Cornelius , against an Na2HPO4 standard curve. Na,K-ATPase activity was evaluated by subtracting the amount of phosphate released in parallel assays that included 1 mM ouabain. In the case of inside-out vesicles, ouabain was added to inhibit Na,K-ATPase activity from contaminating membrane fragments.
The amount of vesicle protein was measured with a BCA (bicinchoninic acid) assay kit (Sigma–Aldrich), using BSA as a standard. Samples (25 μl) were processed in duplicate using a microwell-plate protocol with a final volume of 225 μl. Attenuance was measured at 562 nm in a SpectraMax Plus spectrophotometer.
Results are expressed as means±S.E.M. for the indicated number of experiments. Propagation of errors was performed assuming normal distribution. Statistical significance was determined using Student's t tests (P<0.05). Curve fitting was carried out with statistical weights proportional to (S.E.M.)−1 using nonlinear least-squares routines included in SigmaPlot v10.0 (Systat Software).
L-[U-14C]lysine hydrochloride (specific radioactivity 228 mCi/mmol) was from Sigma. L-Lysine-hydrochloride salt, ATP-Mg salt, C12E8, PMSF, aprotinin (from bovine lung), Nycodenz (Histodenz™) and Percoll were from Sigma–Aldrich. Valinomycin and NEM were from Fluka. Polyethylenimine (branched) was from Aldrich, ouabain octahydrate was from Calbiochem and collagenase type II was from Worthington Biochemical. Salts and reagents were of analytical reagent grade.
Giant vesicles from cardiac ventricular sarcolemma
A low-affinity L-arginine transport process was recently detected in voltage-clamped cardiac ventricular myocytes . However, the activity of CAT-1, a high-affinity cationic amino acid carrier previously described in this cell type , was not observed. Considering that CAT-1 half-maximal activation of transport takes place at physiological plasma levels of L-arginine and L-lysine, the previous observations called for in-depth transport studies. Thus detailed cationic amino acid uptake experiments were performed in giant vesicles prepared by collagenase treatment of rat heart ventricles in a high-potassium medium (see the Experimental section). This procedure yielded a population of spherical vesicles heterogeneous in size (Figure 1), with an average diameter of 20±1 μm (n=65).
Giant sarcolemmal vesicles from rat cardiac ventricle
Vesicles obtained with this method were reported to be largely, if not completely, of sarcolemmal origin . To determine vesicle orientation we took advantage of the sidedness in substrates required for Na,K-ATPase activity. Since vesicles were loaded with 140 mM KCl, only those inside-out oriented will hydrolyse ATP in the presence of extravesicular nucleotide, sodium ions and magnesium. Total Na,K-ATPase activity was estimated by measuring the ouabain-sensitive release of Pi in media containing 0.2 mg/ml of the non-ionic surfactant C12E8. This concentration of C12E8 has been reported to effectively open vesicles with no effect on ATPase activity . Results in Table 1 indicate that vesicles were on average 94% outside-out oriented. Thus kinetic parameters reported here describe cationic amino acid transport through the extracellularly facing form of the pertinent protein(s).
L-Lysine uptake in giant cardiac vesicles
Equivalent concentrations of L-lysine and L-arginine generate inward currents of similar magnitude and Δψ dependence in rat ventricular myocytes . With this in mind, the more stable L-lysine uniformly labelled with 14C was selected as the radioligand for these assays as an alternative to L-arginine labelled with 3H in the more reactive guanidinium group. To determine the time course of L-lysine influx, the uptake of 10 mM L-lysine was followed at 37°C in an Na+-free extravesicular medium containing 140 mM KCl and 20 mM Mops (pH 7.4). Results of Figure 2(A) (filled circles) show that uptake was linear for at least 3 min and thus this incubation time was used in all further experiments unless otherwise indicated. The initial rate of L-lysine uptake, calculated from the slope of the straight line that best fitted the first four data points, was 1.84±0.09 nmol/min per mg of vesicle protein. Vesicles boiled for 10 min in the presence of 1% (w/v) SDS, used as a control for non-specific radiolabelling, showed low levels of background radioactivity (Figure 2A, open triangles).
L-Lysine uptake in giant vesicles
Members of system y+ are the only cationic amino acid transporters reported to be fully inhibited by brief exposure to low concentrations of NEM [11,12]. Thus the effect of NEM on L-lysine uptake was investigated to assess the participation of these proteins in cationic amino acid transport into giant sarcolemmal vesicles. We found that vesicle incubation with 0.2 mM NEM for 10 min at 37°C reduced L-lysine uptake to levels similar to those of the boiling/SDS treatment (Figure 2A, filled triangles). These results strongly suggest that L-lysine uptake in cardiac vesicles is entirely mediated by system-y+ transporters.
Some experiments required the vesicles to undergo incubations, centrifugations and washes, all of which are treatments that added to the intrinsic variability in the amount of vesicular protein obtained from different preparations. Thus it was relevant to study the behaviour of the uptake reaction within a wide range of protein concentrations. To this end, the rate of 10 mM L-lysine uptake from ten randomly selected experiments was plotted against the corresponding amount of vesicular protein. Figure 2(B) shows that the rate of uptake remained linear within the 10-fold range of protein amounts studied. Furthermore, the slope of the best-fitting straight line in Figure 2(B) yielded a value of 1.68±0.19 nmol/min per mg of vesicle protein for the initial rate of L-lysine uptake, a value not significantly different from that obtained with the time course in Figure 2(A).
L-Lysine concentration dependence of uptake
To study the occurrence of multiple components of cationic amino acid transport, the rate of NEM-sensitive uptake was measured in giant vesicles incubated with media containing L-lysine concentrations in the range 0.05–50 mM. When plotted as a function of L-lysine concentration, uptake appears as a saturable, low-affinity process (Figure 3A). However, a closer examination of the initial portion of the curve (Figure 3B) shows that, at micromolar concentrations, the data points deviate significantly from a single hyperbolic behaviour (dotted line). The presence of two uptake components is more clearly seen with an Eadie–Hofstee linear transformation of the data (Figure 3A, inset). In fact, the continuous line in Figure 3 represents the best-fit solution of the following sum of two hyperbolic functions:
where the subscripts ‘h’ and ‘l’ denote high- and low-affinity components respectively. Best-fit values for all four parameters were Km,h=0.222±0.071 mM, Vmax,h=0.121±0.036 nmol/min per mg of vesicle protein, Km,l=16.0±3.6 mM and Vmax,l=4.07±0.36 nmol/min per mg of vesicle protein. Inspection of these kinetic data shows three distinct features. First, apparent affinities for the two cationic amino acid transport processes were 72-fold apart. Secondly, the maximal turnover rate for the high-affinity component was only 3% that of the low-affinity component. Finally, the value of Km,l was not significantly different from the K0.5=11.1±1.4 mM obtained for L-arginine activation of current at zero Δψ in voltage-clamped cardiomyocytes .
L-Lysine concentration dependence of uptake
Similar experiments performed at selected L-lysine concentrations after replacing KCl with equimolar NaCl in the extravesicular solution yielded kinetic parameters for both uptake components that were not significantly different from those reported above (results not shown). These results indicate that Na+ was not required for L-lysine uptake in giant vesicles.
L-[U-14C]Lysine uptake experiments were also conducted with the cluster-tray method [13,21] using rat cardiac ventricular myocytes plated on laminin-coated coverslips. Differential attachment of rod-shaped cardiomyocytes to these coverslips resulted, after extensive washing with Dulbecco's modified Eagle's medium, in an enriched myocyte population of ∼98%, as evaluated by hemacytometry. These experiments also yielded high- and low-affinity uptake components with Km and Vmax parameter values comparable, on average, with those from giant vesicles, although with larger S.E.M. (results not shown). In particular, Vmax for the high-affinity uptake component was found to be ∼5% that of the low-affinity component.
Altogether, these results suggest that the two uptake components are the kinetic expression of two carriers acting in parallel on the myocyte plasma membrane. Since L-lysine uptake was Na+-independent and fully sensitive to brief incubations with 0.2 mM NEM, both carrier proteins are probably members of system y+.
Are these two
L-lysine uptake components independent?
An alternative interpretation of the results of Figure 3 is that L-lysine uptake in cardiac sarcolemmal vesicles is mediated by one transporter with negatively interacting binding sites. In fact, a Hill equation with the following best-fit parameters: Km=22.6±9.0 mM, Vmax=4.6±0.7 nmol/min per mg of vesicle protein and nH=0.79±0.06 also reasonably describes the results of Figure 3 (results not shown). However, an evaluation of the goodness of fit using the second-order Akaike Information Criterion and the evidence ratio  indicates that equation (1) is twice as likely to be the best-fitting equation than a Hill equation. Thus the two uptake components appear to correlate with the activity of two different molecular entities.
To test whether these uptake processes function in parallel and independently, we took advantage of the large difference in apparent affinities with the idea that high-affinity uptake could be almost entirely blocked without affecting the low-affinity component. Towards this end, the L-lysine concentration dependence of uptake was determined in the simultaneous presence of 1 mM D-arginine. D-Enantiomers of cationic amino acids bind to system y+ members but are not transported and thus are effective blockers of transport [11,12]. Results of Figure 4(A) show that the high-affinity L-lysine uptake component practically disappeared in the presence of 1 mM D-arginine as the data are well described by the initial portion of a single hyperbolic function (dotted line). On the other hand, no detectable inhibition of the low-affinity component was observed with this concentration of D-arginine (results not shown). An Eadie–Hofstee transformation of these data confirmed the presence of a single uptake component (Figure 4B). Therefore high-affinity uptake is not required for L-lysine to be transported with low apparent affinity, suggesting two independent uptake processes.
D-arginine on the L-lysine concentration dependence of uptake
Trans-stimulation of L-lysine uptake
Cationic amino acid transport mediated by members of system y+ is accelerated by the presence of unlabelled substrate on the other side of the membrane, a feature called trans-stimulation . Interestingly, the low-affinity CAT-2A has been reported to be much less amenable to trans-stimulation than the high-affinity CAT-1 [23,24]. Therefore, if CAT-1 and CAT-2A are the two transporters present in cardiac vesicles, the prediction can be made that trans-stimulation of uptake will decrease as the concentration of extravesicular L-lysine is increased.
To study the effect of trans-substrate on L-[14C]lysine uptake, giant vesicles were incubated for 30 min at 37°C with a solution containing 10 mM L-lysine, 130 mM KCl and 20 mM Mops (pH 7.4). Lysine-loaded vesicles were washed and resuspended with KCl/Mops solution and kept in a water-ice bath to minimize L-lysine efflux. Samples were brought to 37°C for 3 min and uptake of L-[14C]lysine was measured in vesicles loaded with L-lysine (trans-stimulated uptake) and compared with the rate of uptake in vesicles that were not incubated with the amino acid (control uptake). Results of Figure 5(A) show that uptake levels in the presence of intravesicular L-lysine (open bars) were significantly higher than control uptake levels (filled bars) at all extravesicular L-lysine concentrations tested. More importantly, the level of trans-stimulation decreased from 9-fold at 0.1 mM L-lysine (a concentration where uptake is dominated by the high-affinity component) to 2.7-fold at 50 mM extravesicular L-lysine (Figure 5B). It must be pointed out that, when evaluating trans-stimulation by the low-affinity component, the ratios calculated at 5, 10 and 50 mM L-lysine were overestimated (due to Vmax,h) by 15, 10 and 7% respectively. These results are consistent with the differential stimulatory effect of trans-substrate reported for high- and low-affinity members of system y+.
Effect of intravesicular
L-lysine on the rate of L-lysine uptake
CAT-1 and CAT-2A mRNA levels in cardiac ventricular myocytes
At the present time, there are neither specific blockers  nor reliable commercially available antibodies for members of system y+. Thus, as an approach to identifying the molecular entities responsible for cationic amino acid uptake, RT–PCR techniques were used to investigate the expression of CAT-1 and CAT-2A mRNA transcripts in freshly isolated rat cardiac myocytes. Experiments were carried out as described in the Experimental section, using freshly isolated rat hepatocytes as a CAT-2A positive control  and β-actin as an internal control. mRNA levels for both CAT isoforms, determined using crossing-point analysis as described previously , are displayed in Figure 6 relative to β-actin mRNA levels. Results show a small but significant number of CAT-2A mRNA transcripts in cardiac myocytes, compared with the previously described CAT-1 (CAT-1/CAT-2A ratio≈33). On the other hand, CAT-2A was found to be profusely expressed in hepatocytes where, in agreement with previous reports , it is the only detectable member of system y+ (CAT-2A/CAT-1 ratio≈400). Agarose-gel electrophoresis of the respective cDNA products showed bands at the expected locations and relative intensities (Figure 6, insets). Finally, mRNA transcript identities were unequivocally established by sequencing the corresponding cDNA products. Sequence alignment returned a 100% match with CAT-1 and CAT-2A from Rattus norvegicus. It must be pointed out that, since the sum of two hyperbolic functions (eqn 1) produced the best fit of the experimental data, the presence of mRNA transcripts for a second high-affinity isoform, the inducible CAT-2B [12,13], was not investigated.
Expression of CAT-1 and CAT-2A mRNA transcripts
Effect of hyperpolarization on the kinetics of
The low-affinity cationic amino acid transporter from cardiac myocytes  and the high-affinity CAT-1 overexpressed in Xenopus oocytes  show a significant increase in turnover rates and apparent affinities at hyperpolarizing Δψ. To test whether hyperpolarization affected the kinetics of the two-component L-lysine uptake in cardiac sarcolemmal vesicles, samples were incubated for 1 h with a solution containing 50 μM valinomycin (added from a 20 mM stock in ethanol), 3.3 mM KCl, 138.5 mM NaCl and 20 mM Mops/NaOH (pH 7.4). The K+ ionophore valinomycin has been reported to interact with the cell plasma membrane at micromolar concentrations producing hyperpolarization at low extracellular potassium . Thus the incubation medium above results in a calculated vesicle Δψ=−100 mV at 37°C, assuming that vesicles are impermeable to sodium ions (but see the Discussion section). L-[14C]lysine uptake experiments performed in this medium showed increased uptake levels compared with untreated vesicles that have a Δψ≈0 mV in symmetrical KCl media (Figure 7). The L-lysine concentration dependence of uptake in valinomycin-treated vesicles was fitted with eqn (1), yielding the following parameters: Km,h=0.080±0.029 mM, Vmax,h=0.188±0.025 nmol/min per mg of vesicle protein, Km,l=6.9±2.2 mM, and Vmax,l=6.84±0.71 nmol/min per mg of vesicle protein. Comparison of these values with those of the control curve shows a 2.8- and 2.3-fold reduction in Km, and a 1.6- and 1.7-fold increase in Vmax for the high- and low-affinity uptake components respectively. Therefore hyperpolarization increased apparent affinities and turnover rates in a manner consistent with the Δψ dependence described for members of the y+ system of cationic amino acid transporters.
Effect of a hyperpolarizing transmembrane potential on
The present study reports, for the first time, two coexisting cationic amino acid transport processes in giant sarcolemmal vesicles from cardiac ventricles. Previous voltage-clamp experiments on intact cardiac myocytes revealed the presence of a low-affinity, high-capacity component of L-arginine import with functional properties consistent with the activity of the CAT-2A member of the y+ family of dibasic amino acid carriers . Because, instead, a high-affinity member of this family, CAT-1, has been previously described in this cell type , radioligand influx experiments were aimed at carefully investigating transport within a wide range of cationic amino acid concentrations.
Sarcolemmal vesicles were largely right-side out and had an average diameter of 20 μm. Neglecting the membrane thickness, the volume of a spherical vesicle of this diameter is ∼4 pl, which represents one-third of the volume of a typical cardiac ventricular myocyte as described by a rectangular block of dimensions (100×20×6) μm3 . Thus the large intravesicular compartment and a surface-to-volume ratio comparable with that of an epithelial cell make these vesicles an excellent system for detailed uptake studies.
The influx of L-lysine was Na+ independent, fully sensitive to NEM and enhanced by intravesicular unlabelled substrate and by membrane hyperpolarization, all of which are signature features of system y+ . In the absence of specific inhibitors and good available antibodies, these functional characteristics remain the only biochemical markers for these transporters. The use of siRNA (small interfering RNA) technology to knock down transporter expression in the cardiomyocyte plasma membrane did not offer a valid alternative to identify the proteins responsible for the two L-lysine uptake components, because, after just 72 h in culture, control voltage-clamped myocytes showed a substantial decrease in cationic amino acid-activated current levels (R. D. Peluffo, unpublished work).
L-Lysine uptake displayed two distinct components of transport: one with high apparent affinity for the amino acid and low transport capacity and the other with low apparent affinity and high transport capacity. The low capacity of the high-affinity component may explain our inability to detect its presence in voltage-clamp experiments . Biphasic concentration curves with two saturable components have not been reported for any given member of system y+ [12,28]. Early work reported cationic amino acid uptake–concentration curves described by a hyperbola (Km=0.03–0.2 mM) plus a non-saturable component in cultured fibroblasts, reticulocytes and hepatoma cell lines [29–31]. The similarity between uptake curves in these three reports and those shown in Figures 3(B) and 7(B) of the present study raises the possibility that the linear component was indeed the initial portion of a second hyperbola.
The two components of L-lysine uptake show a 34-fold difference in Vmax values. Considering that 1 mM D-arginine completely blocked high-affinity uptake with no effect on the low-affinity component, these uptake components likely represent the simultaneous activity of two transporters. The difference in Vmax values may thus result from the number of transporters of each class on the sarcolemmal membrane or, alternatively, from actual kinetic differences in their transport capacity. In this regard, mRNA levels detected for system y+ members, although not a direct indicator of the actual number of functional transporters on the membrane, strongly suggest a larger expression of high-affinity CAT-1 transporters on the sarcolemmal membrane. It seems indeed unlikely that after producing a 33-fold larger number of CAT-1 mRNA transcripts, cardiac myocytes will go on to express more CAT-2A functional transporters. Instead, this observation points to real differences in turnover rates as responsible for the differences in Vmax values. Thus the high capacity would appear to overcome (or result in) the low expression levels measured for the low-affinity CAT-2A. A similar pattern has been reported for the expression levels of high- and low-affinity monocarboxylate transporters in butyrate-resistant colon cancer cells . Collagenase-induced differential damage of the protein responsible for high-affinity L-lysine uptake seems unlikely because all functional features, namely NEM-sensitivity, trans-stimulation and Δψ-dependent shift in kinetic parameters, were conserved in this uptake component.
The finding that Km values were more than 70-fold apart suggests that these transporters are active at different ranges of cationic amino acid concentrations. However, the 34-fold difference in Vmax results in a significant uptake through the low-affinity component even at amino acid concentrations far below its Km, as is the case with plasma levels of L-lysine, L-arginine and L-ornithine, which in humans and rats range from 100 to 250 μM [15,16]. To clarify this point, high- and low-affinity uptake components, calculated with the corresponding best-fit parameters from eqn (1), were plotted separately as a function of cationic amino acid concentration (Figure 8). This analysis indicated that 40–50% of the total transport activity already occurs via the low-affinity component at normal plasma levels of cationic amino acids. Furthermore, L-arginine, L-lysine and L-ornithine are equally efficiently transported by members of system y+  and thus carriers are probably sensing an effective plasma concentration of ∼0.6 mM for these ligands . At this concentration, the low-affinity component accounts for 62% of total cationic amino acid transport. Finally, amino acid plasma levels can transiently increase at least 5-fold due to peripheral protein degradation during catabolic states such as starvation, severe burns, cancer, sepsis and trauma . At a hypothetical concentration of 1.2 mM, as much as 74% of the total transport proceeds through the low-affinity carrier even though this cationic amino acid concentration represents only one-tenth of its Km. In light of these calculations, the previously accepted idea that significant CAT-2A-mediated uptake occurs only at amino acid concentrations that exceed systemic plasma levels  should be re-evaluated.
Concentration dependence of cationic amino acid (CAA) uptake through two carriers acting in parallel
The occurrence of trans-stimulation and the finding that its effect on uptake was significantly more pronounced at low concentrations of external L-lysine are in line with our detection of CAT-1 and CAT-2A mRNA transcripts in intact cells. The high-affinity CAT-1 has been reported to be highly sensitive to trans-stimulation, whereas CAT-2A appears to be less responsive to the presence of substrate at the other side of the membrane [23,24,35]. This differential behaviour can be explained through the kinetics of binding site translocation from one face of the membrane to the other, which depends on substrate occupancy [23,36]. Thus the finding that uptake stimulation by 10 mM intravesicular L-lysine decreased from 9- to 2.7-fold as the concentration of external L-lysine increased from 0.1 to 50 mM also points to CAT-1 and CAT-2A as the two cationic amino acid transporters present in cardiac sarcolemmal vesicles. Interestingly, the increase in uptake levels displayed by the low-affinity transporter at 50 mM L-lysine in the presence of trans-substrate was substantially larger than that reported for CAT-2A heterologously expressed in Xenopus oocytes (1.1–1.5-fold; ).
Although adult rat ventricular cardiomyocytes have a resting Δψ in the region of −80 mV , no Δψ was expected to develop in giant vesicles loaded and incubated with the same solution. A remarkable agreement was found, in this regard, between the Km for L-lysine uptake through the low-affinity component and the K0.5 for L-arginine-activated current obtained at zero Δψ in voltage-clamped rat cardiac myocytes . Hyperpolarization brought about by the K+ ionophore valinomycin resulted in a decrease in Km and an increase in Vmax for both uptake components. The direction of the shift for these kinetic parameters was similar to that previously reported for the Δψ dependence of CAT-1 expressed in Xenopus oocytes , and the low-affinity cationic amino acid transport process in cardiac myocytes . Furthermore, hyperpolarization had a proportional effect on both uptake components. Accordingly, the substrate permeability ratio (F), defined as the ratio of initial uptake rates for two transporters acting in parallel , F=Vmax,h×Km,l/(Km,h×Vmax,l), was calculated to be 2.1±1.0 and 2.4±1.1 in the absence and presence of valinomycin respectively. Therefore the relative contribution of these two transport components at low cationic amino acid levels is independent of Δψ.
The magnitude of the valinomycin-induced hyperpolarization could not be calculated with Nernst's equation despite the equilibrium between intra- and extra-vesicular K+ concentrations because of slow but significant permeation of Na+ ions. In fact, extending valinomycin incubation for an additional hour resulted in uptake levels similar to those of the control curve (results not shown), indicating equilibration of Na+ ions and the consequent dissipation of Δψ. The shift in Km and Vmax values produced by valinomycin treatment is compatible with an average shift of −50 mV in Δψ (from Δψ=0), according to published K0.5=f(Δψ) and Imax=f(Δψ) curves for CAT-1  and the cardiac CAT-2A-like L-arginine transport process . When this Δψ is replaced in the Goldman–Hodgkin–Katz equation, a permeability ratio for Na+ and K+ ions, PNa/PK=0.13, results under our experimental conditions. In any case, the observation that kinetic parameters were shifted by hyperpolarization in a manner consistent with the Δψ dependence of these transporters is, for the purpose of this analysis, more important than the actual Δψ value.
In summary, L-lysine uptake assays identified two components of cationic amino acid transport with markedly different kinetic properties in cardiac sarcolemmal vesicles. Such an array of two transporters acting in parallel, one with high affinity and low capacity, the other with low affinity and high capacity, is likely to function as a homoeostatic device to supply cardiac-muscle cells with cationic amino acids under a wide variety of metabolic conditions. Particularly, the low-affinity carrier is expected to transport substrates in vivo at a rate proportional to their concentration, because of a Km value substantially higher than plasma levels of cationic amino acids. Dual carrier systems that combine the parallel functioning of high- and low-affinity transporters have been described for butyrate  and glutamate uptake . However, in the present case, the activity of the low-affinity transporter is predicted to be physiologically relevant at normal systemic levels of L-arginine and L-lysine, because of its high transport capacity.
We thank Dr Andrew Thomas and Dr Andrew Harris, who critically read earlier versions of this paper and provided useful suggestions and comments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health.
This work was supported by the National Heart, Lung, and Blood Institute [grant number R01HL076392] (to R. D. P.).