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Cardiovascular Research 2001 52(1):84-94; doi:10.1016/S0008-6363(01)00373-X
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Characteristics of L-aspartate transport and expression of EAAC-1 in sarcolemmal vesicles and isolated cells from rat heart

Nicola Kinga,*, Helen Williamsa, John D McGivanb and M.-Saadeh Suleimana

aBristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK
bDepartment of Biochemistry, University of Bristol, Bristol Royal Infirmary, Bristol BS2 8HW, UK

* Corresponding author. Tel.: +44-1179-283-587; fax: +44-1179-283-581 n.king{at}bristol.ac.uk

Received 3 January 2001; accepted 25 May 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: L-Aspartate is an important intermediary metabolite in the heart and has also been implicated in myocardial protection, but little is known about its transport across the cardiac sarcolemma. In this study we have tested the hypothesis that the high affinity sodium-dependent aspartate transporter, EAAC-1 is expressed in heart and have also characterised aspartate transport into the myocardium. Methods: Characteristics of L-[14C]aspartate uptake into rat heart were investigated using sarcolemmal vesicles and isolated myocytes. The expression of EAAC-1 in the two preparations was also investigated by western blotting. Results: The Km and Vmax of L-aspartate uptake was 9.78±0.7 µM and 1.17±0.27 pmol/mg/s in vesicles compared to 6.53±1.24 µM and 13.65±1.0 pmol/µl/s in cells. In vesicles, L-aspartate uptake was dependent on external sodium and internal potassium, and was rheogenic. In cells, L-aspartate uptake was also dependent on external sodium. Addition of unlabelled L- and D-aspartate and L-glutamate significantly inhibited L-[14C]aspartate uptake in both preparations but D-glutamate had no effect. An antibody to the aspartate transporter, EAAC-1 recognised a protein of appropriate size in both vesicles and cells. Conclusions: L-aspartate uptake in heart is mediated by a high affinity sodium-dependent transporter. This is accompanied by the expression in heart of EAAC-1. The physiological significance of this transporter with respect to aspartate utilisation in the heart is discussed.

KEYWORDS Cardioplegia; Membrane transport; Myocytes; Sarcolemma


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The dicarboxylic amino acid, aspartate plays an important role in intermediary metabolism in the heart. For example it is a major participant in the malate-aspartate shuttle, which is critical to the transfer of reducing equivalents produced in the cytoplasm through glycolysis and the oxidation of lactate into the mitochondria [1]. Another essential function of L-aspartate in heart is to act as an anaplerotic substrate to counteract disturbances in the concentration of TCA cycle intermediates [2]. This can happen as a result of loading with glucose, acetate, ketone bodies or fatty acids, or under pathological conditions such as on aerobic arrest and during ischaemia [3]. Indeed it is this potential for supporting cardiac metabolism that has led to the strategy of enriching crystalloid cardioplegia solutions with aspartate and other amino acids to provide myocardial protection during coronary artery bypass surgery [4].

The efficiency of L-aspartate to perform these tasks will be dependent upon its intracellular concentration in the heart. This in turn is likely to be influenced by the rate of L-aspartate transport across the cardiac sarcolemma. At present, there is little known about the characteristics of L-aspartate flux into and out of heart cells. It is known that the RNA for the glutamate aspartate transporter EAAC-1 is present in heart [5,6], but crucially this does not give any indication of the level of protein expression or the amount of any functional activity.

This study is based on the hypothesis that an aspartate transporter is present and active in rat cardiac sarcolemma. To test this the characteristics of L-aspartate transport were investigated using sarcolemmal vesicles and isolated cardiac myocytes, which were both prepared from the rat heart. This dual approach has the advantage that the cardiac sarcolemmal vesicles do not contain organelles [7,8] and therefore the measurements will not be affected by metabolism. Also, it is possible to control both the internal and external environment of the vesicles thereby simplifying experiments investigating the effects of specific ion gradients or of changing the membrane potential difference. These investigations are then complemented by the use of the isolated cardiac myocytes, where intracellular metabolism of aspartate was prevented by the addition of amino-oxyacetate. In addition and perhaps more importantly once the transport under normal conditions has been characterised, the isolated cardiac myocytes can then be used in future studies to examine the effects of different pathological conditions. Finally, with the knowledge gained from these experiments the expression of a candidate aspartate transporter, EAAC1 in the cells and vesicles was investigated.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials: chemicals and tissue
Radiochemicals were obtained from Amersham (Little Chalfont, Buckinghamshire, UK); collagenase, Worthington type I, was from Worthington Biochemical Corporation (Lakewood, NJ, USA); dinonylphthalate was from Fluka (Glossop, Derbyshire, UK) and N-glycosidase F was from Calbiochem (Beeston, Nottinghamshire, UK). Other chemicals were from either BDH or Sigma and were of analytical grade.

Tissue used in this study was obtained from male Wistar rats (250–300 g body weight). The rats were humanely killed by cervical dislocation, with the hearts either used for the preparation of cardiac sarcolemmal vesicles or the isolation of cardiac myocytes, and the kidneys used to prepare brush-border membrane vesicles (BBMV). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Preparation of cardiac sarcolemmal vesicles and measurement of L-[14C]aspartate uptake
Homogenisation and differential centrifugation was used to prepare the cardiac sarcolemmal vesicles [7,8]. In brief, the ventricles from eight rat hearts (wet weight 6.86±0.26 g, n=28±S.E.M.) were finely minced with scissors and then suspended 3–4 times weight for volume in a solution containing (in mM) 300 sucrose, 5 MgSO4, 0.1 phenylmethylsulphonyl fluoride (PMSF) and 10 imidazole–HCl (pH 7.0). The suspension was homogenised with 4x5-s bursts at full power using an Ystral X-1020. Then, 500 µl of the homogenate was immediately frozen for use in enzyme assays, whilst the sucrose concentration of the remaining homogenate was adjusted to 600 mM using a 2-M sucrose stock. The mix was stirred on ice for 15 min then centrifuged at 30 000xg and 4°C for 30 min. The supernatant was diluted 1.5 times volume for volume with a solution containing (in mM) 160 NaCl and 20 3-(N-morpholino)-propane sulphonic acid (MOPS)–Tris(hydroxymethyl)aminomethane (Tris) (pH 7.4), then centrifuged at 48 000xg and 4°C for 30 min. The pellet from this spin was suspended in approximately 5–6 ml of a solution containing (in mM) 300 mannitol and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid] (HEPES)–Tris (pH 7.4, solution 1) and re-spun at 48 000xg and 4°C for 30 min. The resultant pellet representing the purified sarcolemmal vesicles was suspended in 700 µl of solution 1. Finally, 200 µl of the sarcolemmal vesicles were frozen for use in enzyme assays, whilst the remaining sample was stored at –80°C until use.

The purity of the vesicle preparation was assessed from the activity, enrichment and percentage recovery of marker enzymes in comparison to samples of crude homogenate, as shown in Table 1 [9]. All the vesicle preparations which were used in experiments had a significantly greater enrichment and percentage recovery of the sarcolemmal marker, Na+,K+ATPase compared to the marker for the sarcoplasmic reticulum, Ca2+(K+)ATPase. The enrichment and percentage recovery of Na+,K+ATPase in the sarcolemmal vesicle preparations was also significantly greater than the marker for internal membranes, Mg2+ATPase, although some enrichment of Mg2+ATPase was observed (Table 1). This was consistent with previous measures of enzyme activities in cardiac sarcolemmal vesicles, which were prepared using this method [7,8].


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  		Table 1 Activity, enrichment and recovery of marker enzymes in cardiac sarcolemmal vesicles

 
Protein concentration was measured according to the method of Bradford [10]. The mean protein concentration of the crude homogenate and sarcolemmal vesicle samples was 30.21±0.59 and 5.11±0.3 mg/ml (n=28±S.E.M.), respectively.

L-[14C]aspartate uptake into the sarcolemmal vesicles was measured by rapid filtration at room temperature, as described previously [12]. Initially, the vesicles were thawed on ice then centrifuged at 48 000xg for 40 min. The resulting pellet was pre-equilibrated with either (in mM) 300 mannitol, 10 HEPES–Tris (pH 7.4) or 100 K gluconate, 100 mannitol, 10 HEPES–Tris (pH 7.4) ± 100 µg valinomycin per mg of vesicle protein using freeze thaw fractionation [11]. Experiments were then started by the addition of vesicles to a tube containing the transport medium and L-[14C]aspartate. The tube contents were mixed then sucked up into the tip of a semi-automatic pipette (Eppendorff Response 4850). At appropriate time points, 60-µl samples in triplicate were dispensed directly onto nitro-cellulose filters (0.45 µm Sartorius, Göttingen, Germany) maintained on Millipore Manifolds under vacuum. Filters were immediately washed with 3x3 ml of an ice cold solution containing 150 mM NaCl and 10 mM HEPES (pH 7.4 with Tris) before being processed for liquid scintillation counting. Controls were taken for zero time and non-specific binding of the isotope to the filters.

In most cases, the transport media contained (in mM) 100 NaCl or choline Cl, 100 mannitol, 10 HEPES/Tris (pH 7.4) and 10 µM L-[14C]aspartate. The only exceptions to this were experiments investigating the kinetics of L-[14C]aspartate uptake, where the substrate concentration was varied over the range 2–300 µM, and those investigating the sensitivity of L-[14C]aspartate uptake to amino acid inhibitors, where 100 µM of each putative inhibitor was added (equivalent to a ten-fold excess when compared to the L-[14C]aspartate concentration).

Initial rate measurements were made at 1 s. To do this, the basic method [12] was modified, so that 940 µl of an ice cold stop solution containing (in mM) 150 Na-gluconate, 10 HEPES–Tris (pH 7.4) was added directly to tubes containing 60 µl of the vesicles and transport solution. The tubes were then placed on ice before being filtered and washed with an additional 2x3 ml ice-cold stop solution.

2.3 Isolation of cardiac myocytes and measurement of L-[14C]aspartate uptake
A combination of enzyme digestion and mechanical dispersion was used to isolate the cardiac ventricular myocytes [13]. The cells were then transferred into transport media containing (in mM) 158 NaCl or choline Cl, 5 KCl, 1.2 MgSO4, 1 CaCl2, 0.5 amino-oxyacetate and 10 HEPES–Tris (pH 7.4), by three rounds of centrifugation at 900xg and room temperature followed by suspension of the pellet in fresh media. When this was completed cell viability and morphology was examined by light microscopy and the measurement of Trypan blue exclusion. This indicated that >80% of the cells were rod shaped and able to exclude Trypan blue (not shown).

The basic protocol used to measure the uptake of 2–1000 µM L-[14C]aspartate into the cells was an oil filtration technique carried out at room temperature as described previously [12]. In brief, 10 µl of the transport medium containing the appropriate L-[14C]aspartate concentration and 3H2O was mixed with 100 µl of the cell suspension. The mixture was pipetted into a small Eppendorff tube preloaded with 75 µl of a silicone/dinonylphthalate mix at a ratio of 70:30 (v/v), layered over 50 µl of 10% (v/v) HClO4 containing 25% (w/v) glycerol. The tube was placed into a refrigerated centrifuge (Eppendorff 5402), and, at the appropriate time transport was terminated, by spinning at 15 800xg and 4°C for 0.5 min.

Then, 20 µl of the supernatant was added to 780 µl 2% HClO4, whilst the remaining sample was snap frozen in liquid nitrogen. The tip of the tube containing the frozen cell pellet in the HClO4/glycerol phase was cut into a scintillation vial preloaded with 780 µl H2O using a special tool (BDH). Each vial was thoroughly mixed by vortex before being processed for liquid scintillation counting.

In experiments where the rate of L-[14C]aspartate uptake into the cells was measured this was carried out using first order rate plots [14]. These were calculated according to the equation:

Formula
Where aspmax was the maximal L-[14C]aspartate uptake measured (taken as the uptake at 30 min, which was 248.28±8.5 pmol/µl in NaCl containing media and 68.25±8.5 pmol/µl for the sodium dependent component, all n=5±S.E.M.) and aspt was L-[14C]aspartate uptake at time t min, as shown in Fig. 4. Thus in subsequent experiments investigating the kinetics of L-[14C]aspartate uptake these rates were evaluated from uptakes measured at intervals over the first 0.5 min using plots similar to that shown in Fig. 4.


Figure 4
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  		Fig. 4 Time-course of L-aspartate uptake into isolated cardiac myocytes. First-order rate plot of the first minute of 10 µM L-[14C]aspartate uptake into the cells in the presence of an inward NaCl gradient ({circ}) or as that represented by the sodium-dependent component (sodium-choline, {square}). Least-squares analysis was used to fit the best straight lines to the data. Aspmax: L-[14C]aspartate uptake at equilibrium (taken as 248.28 pmol/µl with an inward NaCl gradient and 68.25 pmol/µl for the sodium-dependent component). Aspt: L-[14C]aspartate uptake at time t s. Data shown were means±S.E.M. of n=5 cell isolations.

 
Results from the isolated cardiac myocytes are expressed as pmol/µl (i.e in terms of the cell space or volume). To determine the intracellular space or volume the uptake of 1 mM [14C]sucrose (impermeable) and 3H2O (fully permeable) were measured. This yielded the total (intra- and extracellular space) and extracellular space. The difference between the two values equalled the intracellular space (volume). This value was then used to calculate aspartate uptake in pmol/µl from the amount of aspartate present in each space. The mean cell volume from 21 cell isolations was 1.32±0.12 nl in sodium media compared to 1.33±0.14 nl in sodium-free media. There was no significant difference between these values. Results from cell experiments were also calculated per mg protein, however these are not shown because the sample contains dead cells.

2.4 Preparation of kidney brush border membrane vesicles (BBMV)
BBMV were prepared by a dual stage magnesium precipitation technique [15]. Completed BBMV were suspended in 1 ml (in mM) 300 mannitol, 10 HEPES–Tris (pH 7.4) and stored at –80°C until use. The mean protein concentration of the BBMV was 5.31±0.44 mg/ml (n=9±S.E.M.).

2.5 Western blotting
Isolated myocytes, sarcolemmal vesicles and kidney BBMV containing 50 µg protein per sample were added to a tube containing 150 mM Tris–HCl (pH 6.8), 6% sodium dodecyl sulphate (SDS), 0.3% bromophenol blue, 30% glycerol and 15% β-mercaptoethanol (loading solution) at a ratio of two parts sample to every one part loading solution. The tubes were then heated to 90°C for 10 min. After this the tube contents were transferred onto 10% (w/v) polyacrylamide gels together with standardised molecular weight markers (RainbowTM coloured protein molecular weight markers from Amersham, used according to the manufacturer’s instructions) and electrophoresed at 90 V for 1–1.5 h. The proteins were then transferred onto nitrocellulose membranes, prior to an overnight incubation with a monoclonal mouse anti-EAAC-1 antibody (Chemicon, Temecula, CA, USA) at 4°C. This was followed by a 1-h incubation at room temperature with HRP-conjugated rabbit anti-mouse IgG. Positive samples were detected with ECL Western blotting detection reagents used according to the manufacturer’s instructions. X-ray films were visualised on a BioRad scanning densitometer (BioRad Laboratories, Hemel Hempstead, UK) with incorporation into Molecular Analyst.

2.6 Deglycosylation of EAAC-1
N-glycosidase F treatment of the cardiac myocytes, cardiac sarcolemmal vesicles and BBMV was carried out according to the method of Tarentino et al. [16]. In brief, samples containing 100 µg protein were added to a tube containing 50 mM sodium phosphate (pH 7.5), 0.1 mM PMSF, 10% Nonidet P-40, and 16.6 mU/ml N-glycosidase F and incubated at 30°C for 1.5 h. The tubes were then centrifuged at 15 800xg for 1 min with the resulting pellet suspended in 50 mM sodium phosphate solution (pH 7.5). Then, 20 µl of this suspension was then added to 10 µl loading solution and used directly for western blotting as described above.

2.7 Statistics
Results are expressed as means±S.E.M. of n experiments. All measurements within experiments for the vesicles were made in triplicate and for the cells in quadruplicate. Data was fitted to curves or the best straight line using FigP version 6.0c. Comparisons between datasets were made using either the Student’s t-test or ANOVA with an appropriate post-test available in Instat.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characteristics of L-[14C]aspartate uptake into cardiac sarcolemmal vesicles
Fig. 1a shows a time course of L-[14C]aspartate uptake measured with either an inward 100 mM NaCl or choline Cl gradient into potassium-loaded vesicles. The inward NaCl gradient stimulated a significantly greater level of L-[14C]aspartate uptake for all points prior to equilibrium at 3600 s (P<0.05, ANOVA). This included an overshoot (where the maximal uptake is greater than the uptake at equilibrium) of 2.47±0.48-fold (n=5±S.E.M.). In contrast with an inward choline Cl gradient L-[14C]aspartate merely equilibrated into the vesicles with no comparable overshoot.


Figure 1
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  		Fig. 1 L-Aspartate uptake into cardiac sarcolemmal vesicles is dependent on external sodium and internal potassium. (a) Time-course of 10 µM L-[14C]aspartate uptake into potassium-loaded vesicles in the presence of either an inward 100 mM NaCl ({star}, —) or choline Cl ({circ}, - - -) gradient. (b) Time-course of 10 µM L-[14C]aspartate uptake measured with an inward sodium gradient into vesicles loaded with either 100 mM K-gluconate ({star}, —) or no internal ions ({circ}, - - -). Data shown were means±S.E.M. of n=5 vesicle preparations.

 
Fig. 1b shows another time course where L-[14C]aspartate uptake was measured with an inward NaCl gradient. For this experiment the vesicles were either preloaded with 100 mM K-gluconate, or had no ions in their internal media. In the absence of any internal ions, L-[14C]aspartate slowly equilibrated into the vesicles and there was no overshoot. On the other hand the inclusion of 100 mM K-gluconate within the vesicles promoted a significantly greater uptake and overshoot.

The possibility that L-[14C]aspartate uptake into the cardiac sarcolemmal vesicles was rheogenic was investigated by measuring the initial rate in the presence or absence of valinomycin. Fig. 2 shows that the initial rate of L-[14C]aspartate uptake measured in the presence of 100 µg valinomycin per mg vesicle protein was significantly faster compared to that measured without valinomycin. It should be noted that in the absence of a potassium gradient valinomycin had no effect on L-[14C]aspartate uptake (not shown).


Figure 2
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  		Fig. 2 L-Aspartate uptake into cardiac sarcolemmal vesicles is rheogenic. Initial rate of the sodium-dependent component of L-[14C]aspartate uptake into potassium-loaded vesicles measured in the presence (solid bars) or absence (open bars) of 100 µg valinomycin per mg protein. * P<0.01 vs. the uptake in the presence of valinomycin (Student’s t-test). Data shown were means±S.E.M. of n=5 vesicle preparations.

 
Fig. 3 shows the kinetics of the sodium dependent component (calculated as the uptake in NaCl minus the uptake in choline Cl) of L-[14C]aspartate uptake into potassium loaded vesicles. In the main graph the initial rate of uptake for a range of L-[14C]aspartate concentrations between 2 and 300 µM is shown. The initial rate of uptake increased linearly in proportion to the substrate concentration until 30 µM when saturation occurred. This data represented a good fit to the Michaelis–Menten equation described by: Y=Vmax·X/(Km+X)+NSB·X (the constant NSB was not significantly different from zero), from which the Km of 9.78±0.7 µM and Vmax of 1.17±0.27 pmol/mg/s were calculated.


Figure 3
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  		Fig. 3 Kinetics of L-aspartate uptake into cardiac sarcolemmal vesicles. Main graph: initial rate of the sodium-dependent component of L-[14C]aspartate uptake into potassium-loaded vesicles for substrate concentrations between 0 and 300 µM. Least-squares analysis was used to fit the data to the Michaelis–Menten equation: Y=VmaxxX/(Km+X)+NSB·X. Inset: Lineweaver–Burk plot of the same data. Least squares analysis was used to fit the best straight line. Data shown were mean±S.E.M. of n=5 vesicle preparations.

 
The inset shows the same data represented as a Lineweaver–Burke plot. The interception on the x-axis is –0.081 and the interception on the y-axis is 0.393. This corresponds to a Km and Vmax of 12.35 µM and 2.54 pmol/mg/s, respectively. These values were similar to those calculated from the computer fit of the line in the main graph.

The effect of adding 0.1 mM (10-fold excess) of various putative inhibitors to the external solution upon L-[14C]aspartate uptake was investigated. Table 2 shows that the sodium-dependent component of L-[14C]aspartate uptake in potassium-loaded vesicles was significantly reduced by all of the agents tested except D-glutamate.


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  		Table 2 Specificity of the sodium-dependent component of L-aspartate uptake into the cardiac sarcolemmal vesicles and isolated cardiac myocytes

 
3.2 Characteristics of L-[14C]aspartate uptake into isolated cardiac myocytes
Fig. 4 shows a first-order rate plot (see Section 2) of the first minute of 0.01 mM L-[14C]aspartate uptake into the isolated cardiac myocytes, which was measured in sodium-containing media and as that represented by the sodium-dependent component, i.e. sodium-choline. During this time the uptake of L-[14C]aspartate in both conditions exhibited apparent first order kinetics. At 0.01 mM L-[14C]aspartate the apparent rate constant in sodium-containing media was 1.49/min and the rate of L-[14C]aspartate uptake was 318.86±15.79 pmol/µl/min (n=5±S.E.M.), whilst for the sodium-dependent component the apparent rate constant was 1.3/min and the rate of L-[14C]aspartate uptake was 68.5±13.78 pmol/µl/min (n=5±S.E.M.).

Fig. 5 shows the kinetics of the sodium-dependent component of L-[14C]aspartate uptake into the cells, which was measured over a concentration range of 2–1000 µM. The initial rate of L-[14C]aspartate increased in proportion to the concentration up to 20 µM. After this a plateau was reached, where the rate of L-[14C]aspartate uptake did not significantly change for concentrations up to 1000 µM (uptakes for concentrations between 300 and 1000 µM are not shown for reasons of clarity). From this a Km of 6.53±1.24 µM and a Vmax of 13.65±1 pmol/µl/s were calculated by fitting the data to the Michaelis–Menten equation: Y=Vmax·X/(Km+X)+NSB·X (NSB was not significantly different from zero). These figures were in agreement with those obtained from a Lineweaver–Burk plot (data not shown).


Figure 5
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  		Fig. 5 Kinetics of L-aspartate uptake into isolated cardiac myocytes. Initial rate (at 30 s) of the sodium-dependent component of L-[14C]aspartate at concentrations between 2 and 300 µM. Least-squares analysis was used to fit the data to the Michaelis–Menten equation: Y=VmaxxX/(Km+X)+NSB·X. Data shown were means±S.E.M. of n=8 cell isolations.

 
Table 2 shows the effect of adding 0.1 mM of various dicarboxylic amino acids to the sodium-dependent component of L-[14C]aspartate uptake in isolated cardiac myocytes. The addition of unlabelled L-aspartate, D-aspartate and L-glutamate all inhibited the uptake, whilst D-glutamate had no effect.

3.3 Is EAAC-1 expressed in cardiac sarcolemmal vesicles and isolated heart cells?
Fig. 6 shows the result of two representative western blots that were carried out using an antibody to the C-terminal portion of EAAC-1. The upper panel shows untreated samples and the lower panel shows the same samples following treatment with N-glycosidase F. In both panels, a single positive band corresponding to the expression of an approximately 66 kDa protein (by comparison to rainbow molecular weight markers run on the same gel, not shown) was observed for both cells and vesicles. The bands for cells and vesicles were comparable to a similar band observed in a sample of kidney brush border membrane vesicles used as a positive control, because this tissue is known to express high levels of EAAC-1 [5,6,17–19].


Figure 6
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  		Fig. 6 Expression of the aspartate transporter, EAAC-1, in cardiac sarcolemmal vesicles and isolated cardiac myocytes. Western Blots carried out using an antibody to the C-terminal portion of EAAC-1. Upper panel: Western blot of native samples. Lower panel: Western blot of the same samples following treatment with N-glycosidase F. Equal amounts of protein was loaded for each sample. The band on the left indicates a molecular weight of 66 kDa determined from a molecular weight ladder, which was run on the same gel. BBMV: rat kidney brush border membrane vesicles (positive control). Data shown in each panel is from a single Western blot representative of five such blots.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In this study we have investigated the hypothesis that there is an active L-aspartate transporter in the rat heart. To do this we have characterised L-[14C]aspartate uptake using sarcolemmal vesicles and isolated cardiac myocytes and have investigated the expression of a candidate aspartate transporter, namely EAAC-1 in both preparations.

In vesicles L-aspartate uptake was dependent on an inward sodium gradient and an outward potassium gradient. This was apparent from the greater magnitude of L-aspartate uptake under these conditions compared to no inward sodium gradient and no outward potassium gradient and the occurrence of an overshoot. At equilibrium, the level of uptake reflects an equal movement of L-[14C]aspartate into and out of the vesicle. The greater level of uptake at earlier time points indicates that L-[14C]aspartate accumulation into the vesicles has occurred against a concentration gradient. Eventually, the dissipation of the inward sodium gradient removes the driving force for L-aspartate uptake and the L-aspartate equilibrates. This dependence upon external sodium and internal potassium is one of the defining characteristics of the family of high affinity glutamate aspartate transporters of which EAAC-1 is a member [5,6,17–19].

L-Aspartate uptake into the sarcolemmal vesicles was of high affinity and low capacity. The exact conditions of the experiment combined with the dependence of the transporter upon an inward sodium gradient and an outward potassium gradient would prevent any L-aspartate uptake by vesicles with an inside out orientation. On the assumption that the sample contained both right side out and inside out vesicles it is possible that the Vmax may have been underestimated, but the Km would be unaltered. In fact, the Km for L-aspartate uptake into the vesicles is very similar to values reported for the high affinity glutamate aspartate transporters [5,18,19].

The dicarboxylic amino acids, L-aspartate, D-aspartate, L-glutamate all significantly inhibited the uptake, whilst D-glutamate had no effect. L-Aspartate uptake was also reduced by the pharmacological agents D,L-threo-β-hydroxyaspartate and L-{alpha}-aminoadipate, with D,L-threo-β-hydroxyaspartate as the more effective substance. This pattern of sensitivity to D-aspartate but insensitivity to D-glutamate together with the greater potency of D,L-threo-β-hydroxyaspartate compared to L-{alpha}-aminoadipate has also been described for the high affinity glutamate aspartate transporters and EAAC-1 in particular [5,18,19].

There was a strong similarity between these characteristics of L-aspartate uptake in vesicles compared to cells. In cells L-aspartate uptake was also sodium-dependent, showed the identical sensitivity to dicarboxylic amino acids, and had a Km of 6.53±1.24 µM. Taken together the results from the cells and the vesicles suggest that a high-affinity sodium dependent L-aspartate transporter is present in the rat cardiac sarcolemma. Indeed all of the transport characteristics described here for the heart cells and vesicles are very consistent with the transport properties, which have been reported for EAAC-1 [5,6,17–19].

The expression of the system Xag– transporter, EAAC-1 [5,6] in both cells and vesicles may suggest that this transporter is responsible for L-aspartate transport in heart. It should be noted however that this does not exclude the possibility that (a) other system Xag– transporters may be present in heart and (b) that these other transporters may also participate in L-aspartate transport [17]. The most likely explanation for the slightly smaller molecular weight of EAAC-1 in the native cardiac sarcolemmal vesicles compared to the native isolated cardiac myocytes (Fig. 6, upper panel) was differences in the glycosylation pattern of the two samples. This was apparent from the similarity in the molecular weight of EAAC-1 in the cells and vesicles following treatment with N-glycosidase F (Fig. 6, lower panel). During vesicle preparation outer and inner cell membranes were disrupted [7,8], which most likely caused the release of various enzymes including glycosylases. These intracellular enzymes are not liberated during cell isolation [13].

The dependence of L-aspartate flux across the cardiac sarcolemma on an inward sodium gradient and an outward potassium gradient is important, because this will permit the inward flux of L-aspartate into heart cells against the large tissue to plasma concentration gradient that exists in vivo. The intracellular myocardial concentration of aspartate is usually around 100-fold greater than the concentration found in the cardiac circulation [20]. Under normal conditions, the energy required to maintain such a concentration gradient would be 12.5 kJ/mol (calculated using the equation Formula , where R=8.314 J/K/mol and is the gas constant, T=310°C and is the absolute temperature and [AA]i and [AA]o are intracellular and extracellular concentrations, respectively). This could easily be met by the energy available from the sodium electrochemical gradient which is 28 kJ/mol assuming that the stoichiometry in heart is similar to that reported for L-aspartate transport in other tissues [5,6,16–18], i.e. 2 sodium to 1 aspartate.

Unlike L-aspartate, where little was known about its transport in the myocardium, the uptake of another dicarboxylic amino acid L-glutamate uptake has been studied in cardiac sarcolemmal vesicles. In one study L-glutamate uptake did not overshoot; was not stimulated by internal potassium and had a Km of 149±40 µM [21]. Another study, which was reported as an abstract only, showed a Km of 240±40 µM for L-glutamate uptake into pig cardiac sarcolemmal vesicles and inhibition by D-glutamate [22]. Clearly these characteristics of L-glutamate uptake are quite distinct to those described here for L-aspartate uptake. However, whether these differences are due to species variations or represent the expression of additional glutamate transporters in heart (e.g. the glutamate/cystine exchanger [17]) could not be determined without further experimentation.

In a similar fashion L-glutamate transport has also been studied in isolated cardiac myocytes. It is more difficult to compare these studies with L-aspartate uptake into heart cells, because of differences in experimental protocol. Thus in the study of Dinkelborg et al. [23] sodium dependence was not determined, therefore the Km of 180 µM was for total L-glutamate uptake. In another abstract L-glutamate uptake in heart cells is reported with a Km of 40 µM but with significant inhibition by D-glutamate [24]. These characteristics are consistent neither with system Xag– nor with L-aspartate uptake as reported here.

Recently expression of the ionotropic and metabotropic glutamate receptors have been reported in heart, where they have been implicated in cardiac excitation, rhythmicity, and the autonomic control of cardiac function [25–27]. It is possible to speculate that if there was a close association between these receptors and EAAC-1, then a possible role of EAAC-1 in the heart could be to control the local neurotransmitter concentration. This would parallel the function of the glutamate aspartate family of transporters in the CNS [6,19]. Certainly if EAAC-1 operates in the same fashion in heart as it does in the CNS, where changes to ion gradients during ischaemia cause aspartate and glutamate to be exported out of the neurons [17], then this could provide a possible explanation for the significant loss of these amino acids from the myocardium that occurs during cardiac insults and upon aerobic arrest [20,28,29].

In conclusion, these results suggest that L-aspartate flux across the cardiac sarcolemma is mediated through a high affinity sodium dependent transporter. This is accompanied by the expression of EAAC-1 and possibly other glutamate aspartate transporters in the heart. It is now intended to use the isolated cardiac myocytes to investigate the important question of how pathological conditions might affect L-aspartate uptake.

Time for primary review 43 days.


   Acknowledgements
 
We would like to thank V. Buswell for her excellent technical assistance. Dr Nicola King is a British Heart Foundation Junior Research Fellow.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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