glutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes. Am. J. Physiol. 270 (Heart Circ. PhysioZ. 39): Hl825-H1832,. 1996.-The.
‘Ransport and metabolism of L-glutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes LUDGER M. DINKELBORG, ROLF K. H. KINNE, AND MANFRED K. GRIESHABER Institut fir Zoophysiologie, Lehrstuhl fir Stoffwechselphysiologie, Heinrich-Heine-Universittit, 40225 Diisseldorfi and Max-Planck Institut fir molekulare Physiologie, 44026 Dortmund, Dinkelborg, Ludger M., Rolf K. H. Kinne, and Manfred K. Grieshaber. Transport and metabolism of Lglutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes. Am. J. Physiol. 270 (Heart Circ. PhysioZ. 39): Hl825-H1832, 1996.-The intracellular glutamate concentration of oxygenated, isolated adult rat heart cells incubated with 0.15 mM glutamate amounts to 2.89 t 0.6 mM. Under these conditions the velocity of glutamate transport was 24.3 t 1.6 pmol=min-l*rng protein-l and occurs via a high-affinity carrier characterized by an apparent affinity (Km) value of 0.18 t 0.03 mM. At high glutamate concentrations (> 1 mM) this high-affinity transport system is superimposed by additional uptake processes of a low affinity but a high capacity for glutamate. The 1.6-fold increased uptake of glutamate observed during 30 min of anoxic incubation of cardiomyocytes does not prevent an intracellular decrease in this amino acid to a concentration of 0.49 mM. After 15 min reoxygenation of cardiomyocytes the intracellular glutamate content increases to the control values of oxygenated cells. Only 2.4% of the glutamate increase after reoxygenation is due to the transport of glutamate from the incubation medium. The competitive inhibitor of transaminases, aminooxyacetate, prevents both the observed intracellular decrease in glutamate during anoxia and the increase in intracellular glutamate after reoxygenation of cardiomyocytes. Half of the amino groups needed for the synthesis of glutamate originate from intracellular alanine, which increases during anoxia and is metabolized during reoxygenation of cardiomyocytes. The velocity of the glutamate uptake of cardiomyocytes incubated in a medium containing 10 mM L-glutamate amounted to 728 ? 140 pmol*min-l l mg protein-? During anoxic incubation of cardiomyocytes at this high extracellular glutamate concentration, the intracellular glutamate breakdown may be compensated by a simultaneous uptake of this amino acid via the transport processes characterized by a high capacity. cardiomyocytes; hypoxia; aminooxyacetate; tricarboxylic acid cycle; transsarcolemmal amino acid gradients; cardioplegia; protective effect of glutamate; isolated adult rat heart myocytes
of aerobic metabolism of the physically arrested human heart, free fatty acid, lactate, and glucose are taken up from the blood and oxidized in equal parts (12). Less than 5% of the oxygen consumed by the healthy heart is used for the oxidation of amino acids (12). Although amino acids seem to play a minor role for the substrate supply of the heart, both the oxygen consumption and the oxidation of glucose in isolated cardiomyocytes are decreased by half if amino acids are missing from the inc ubation medium (5) Hence, the substrate metabolism of the heart seem.s td
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be affected, if not regulated, by the content of amino acids in the blood. L-Aspartate, L-glutamate, L-alanine, and L-glutamine in particular have a potential role in myocardial energy metabolism. Aspartate and glutamate, as well as their corresponding 2-oxoacids, oxaloacetate and 2-oxoglutarate, are involved in the malateaspartate cycle to transport reducing equivalents originating from glycolysis and oxidation of exogenous lactate into the mitochondrial compartment (25). Moreover, transamination of these amino acids acts as an anaplerotic reaction to dampen fluctuations in the concentrations of tricarboxylic acid (TCA) intermediates, which depend on the substrate supply to the heart. The pool size of TCA cycle intermediates varies in diabetes, on aerobic arrest of the heart, in cardiac ischemia, or within minutes after the addition of glucose, acetate, ketone bodies, or fatty acids to the perfusion medium of an isolated heart (18). In ischemia, glutamate seems to be of additional importance, since the provision of glutamate improves the mechanical function of the ischemic or hypoxic myocardium (3, 20, 21). Heart surgery has taken advantage of this beneficial effect by using cardioplegic solutions enriched with this amino acid (23). The enhanced extraction of glutamate from the blood of patients suffering from coronary artery disease may be the explanation for the possibility of imaging ischemic regions using L-[13N]glutamate in positron scintigraphy (32). Glutamate transport has been characterized in sarcolemma1 vesicles from rat hearts (7). In this investigation we were able to demonstrate that the high-affinity glutamate carrier is Na+- and proton-dependent and that it is inhibited by L-aspartic and L-cysteic acid. The aim of this study was to determine the rate of glutamate uptake against the existing high concentration gradient across the sarcolemma of cardiomyocytes and to elucidate the mechanism of increased glutamate uptake during hypoxia. The investigation of both the glutamate supply from the extracellular compartment via glutamate transport and the intracellular metabolism of this amino acid under normoxic and anoxic conditions should lead to a better understanding of the observed protection of the ischemic heart during glutamate infusion. MATERIALS
AND
METHODS
Amino acids, vitamins, and enzymes were purchased from Boehringer Mannheim (Mannheim, Germany) and Worthington collagenase from Biochrom (Berlin, Germany). L-[U14C]glutamate and D- [l-“H(N)] sorbitol were obtained from DuPont-New England Nuclear (Dreieich, Germany). All other
1996 the American
Physiological
Society
Hl825
H1826
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chemicals were purchased from Merck (Darmstadt, Germany). Isolation and incubation of heart cells. Myocytes were prepared from adult male Wistar rats (300 g body wt) by a collagenase digestion procedure described by Hohl et al. (11). Cells were incubated in a gyratory water-bath shaker at 37°C in physiological, phosphate-buffered saline (pH 7.4) containing 11 mM D-ghOSe and a complete mixture of amino acids and vitamins (11). Oxygenated cells were incubated in open siliconized Erlenmeyer flasks (25 ml) in a humid chamber under an atmosphere of 100% 02 throughout the experiment. Anoxic cells were incubated in stoppered flasks flushed with humidified argon for 5 min. Before the beginning of an uptake study myocytes were preincubated for 30 min under either an oxygen or an argon atmosphere. Reoxygenation of anoxic cardiomyocytes was achieved by opening the stoppered flasks, pumping oxygen (100%) into the flasks for 1 min, and incubating the opened flasks with oxygen in the humidified chamber. The uptake studies were initiated by adding tracer concentrations of L-[14C]glutamate to the medium. The cell titer was -360,000 cells/ml, equal to -3.2 mg of cellular protein/ml. Fractionation and extraction of cells. Cells were simultaneously separated from the incubation medium and extracted by centrifugation of 1 ml of cell suspension through 350 ul of bromododecane into 100 ul of 2 M HC104, using a Heraeus microfuge (Osterode, Germany). For calculation of intracellular amino acid concentrations as well as glutamate uptake, the amount of extracellular glutamate in the perchloric acid extracts was quantitated by two different approaches. In a first attempt, the amount of extracellular fluid present in the cell extracts was measured by adding [3H]sorbitol to the incubation medium. Samples of the medium as well as perchloric acid extracts were cornbusted (model 306, Packard Oxidizer, IL) and the resulting 3H20 and 14C02 were counted separately in a liquid scintillation counter. In a second attempt, we measured the amount of extracellular glutamate by incubating myocytes on ice and then adding labeled glutamate and immediately centrifuging the myocytes into the perchloric acid. The extracellular space determined with [3H]sorbitol amounted to 1.3 * 0.2 ul/mg protein (n = 8). By incubating myocytes with [14C]glutamate on ice, a value of 0.9 t 0.1 ul/mg protein (n = 20) was determined. Because sorbitol yields higher values than other extracellular space markers, a result that has been demonstrated by other investigators (29), the second procedure was used in further experiments. The intracellular amino acid concentrations were calculated from the determined amino acid content and an intracellular volume of rat heart myocytes of 3.7 ul/mg protein (31). They were corrected for the contamination by amino acids from the incubation medium. Uptake assay. Transport of L-glutamate into myocytes was measured using 18.5 kBq ~+~C]glutamate and, unless otherwise stated, the L-glutamate concentration in the medium was 0.15 mM. The initial rates of glutamate uptake were calculated from data derived in the first 3 min of the individual uptake experiment when uptake of glutamate was found to be linear with time. Determination of metabolites, amino acids, and protein. Extracts and deproteinized incubation media were neutralized using KZC03 (2 M) in triethanolamine (0.5 M). ATP, phosphocreatine, lactate, and glutamate were measured by standard enzymatic methods (2), either by spectrophotometry (Kontron, Eching, Germany) or fluorometry (Foci A 4, Farrand, NY). Amino acids were measured using an automated amino acid analyzer (Liquimat III, Kontron, Eching, Germany), and the specific radioactivity of glutamate was
OF
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either measured using an on-line radioactivity monitor (Ramona, Isomess, Essen, Germany) before ninhydrin derivatization or by counting the radioactivity of extracts and media in a liquid scintillation counter (Beckmann, Irvine, CA). Total cellular protein was determined after dissolving the perchloric acid pellet in NaOH (1 M) by the method of Bradford (4) using bovine serum albumin as standard. Statistical analysis was performed with the use of the Student’s t-test. Values were considered significant at P < 0.05; n refers to the number of myocyte preparations. RESULTS
Viability, high-energy phosphate content, and lactate production. The percentage of rod-shaped cells, which was 85 t 5% (n = 25), as well as the content of high-energy phosphates, are good indicators of the viability of isolated cardiac myocytes. The ATP and phosphocreatine contents of isolated myocytes were 18.8 t 2.0 and 50.0 t 4.0 nmolmg protein-l, respectively (a = 4), which is in good agreement with the results of Dow et al. (8). No significant decreases in the percentage of rod-shaped cells and ATP content were observed during anoxic incubation, oxygenation, or reoxygenation of cardiomyocytes. Lactate production was linear with time in anoxic incubations and amounted to 4.3 t 0.3 pmolhl l mg protein (n = 6). This production rate is comparable to the maximal glycolytic flux measured in Langendorff-perfused rat hearts (24). Sarcolemmal amino acid gradients. To determine the sarcolemmal gradients of some amino acids, rat heart myocytes were incubated for 30 min under oxygenated conditions in a medium containing the amino acids specified in Table 1. In the right column of Table 1 the cell-to-medium ratios of amino acids are shown. Note that the sarcolemmal gradient of glutamate, which amounts to 19, is far above all other amino acid gradients listed in Table 1. L-Glutamate uptake during oxygenation. Figure 1 shows the accumulation of the amino acid L-glutamate in cardiomyocytes incubated with 0.15 mM glutamate under oxygenated conditions. The initial uptake of L-glutamate in the first 3 min of incubation was linear with time and reached a plateau after 5 min of incubation. The initial rate of glutamate uptake amounted to 24.3 IT 1.6 pmolminl~mg protein-l (Table 2). During the first 3 min of either oxygenation, reoxygenation, or anoxic incubation, more than 98% of the intracellular radioactivity represented labeled glutamate. Therefore, in all experiments the radioactivity found in cell extracts during the initial phase of incubation (3 min) was attributed to glutamate, and these values were used to calculate initial rates of uptake. Figure 2 shows the dependence of the initial rate of glutamate uptake on the extracellular glutamate concentration. Cardiomyocytes were incubated under oxygenated conditions with glutamate concentrations between 0.06 and IO mM. Raising of the extracellular L-glutamate concentration up to 0.33 mM resulted in saturation of the initial uptake rate. At high concentrations of L-glutamate (up to 10 mM) this high-affinity glutamate transport was superimposed by unsaturable
TRANSPORT
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Table 1. Danssarcolemmal amino acid gradients oxygenated isolated adult rat heart myocytes with specified concentrations of amino acids
OF
of
GLUTAMATE
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IN HEART
Table 2. Initial rates of L-glutamate uptake in oxygenated isolated adult rat heart myocytes V,, pmol-min
Incubation Cell Concn., mM
System Leu Ile Phe Val TYr
0.34 0.23 0.22 0.40 0.21
+ 0.09 + 0.05 t 0.03 -+: 0.11 t 0.04
Thr Ser
0.69 + 0.13 0.34 + 0.07
Ala GlY
0.70 + 0.14 0.28 + 0.07
Glu
2.89 t 0.60 0.20 + 0.04
System
System
System ASP
System Gln
1.68 t 0.56
Cell
Medium Concn., mM
L (Na+ 0.42 0.412 0.21 0.41 0.22 ASC
Medium
Oxygenation Anoxic incubation Reoxygenation
Concn.
independent) + 0.06 0.08 t 0.03 t 0.05 t 0.02
(Na+
A (Na+
dependent) 1.7 2.8
dependent)
0.12 + 0.03 0.11 t 0.02 XiG (Na+
5.8 2.5
dependent)
0.15 + 0.01 0.08 t 0.01
19.3 2.5
dependent)
0.66 + 0.07
l.rng-l
24.3 + 1.6 38.12 3.8 31.9 + 2.5
Values are means + SD; n = 4 myocyte preparations. L-glutamate uptake rate. Initial uptake rates differ significantly,
0.8 0.6 1.0 1.0 1.0
0.41+ 0.05 0.12 + 0.01
N (Na+
Concn.
2.8
Values are means + SD; n = 4 myocyte preparations. Intracellular amino acid concentrations were calculated using an intracellular water volume of myocytes of 3.7 ul/mg protein (31) and corrected for contamination from incubation medium (see MATERIALS and METHODS). For amino acid transport systems the nomenclature proposed by Christensen is used (5a).
uptake processes directly proportional to the extracellular glutamate concentration (see Fig. 2, inset). Representation of these data in a Lineweaver-Burk plot leads to an apparent affinity (I&) value of the high-affinity glutamate carrier of 0.18 t 0.03 mM and an apparent maximal initial uptake rate (V,,,) of 44 t 12 pmol. min-l . mg protein -l (Fig. 3, line A).
Vi, initial P < 0.05.
The effect of increasing glutamate concentrations in the incubation medium on the intracellular glutamate content of oxygenated cardiomyocytes is summarized in Fig. 4. Myocytes were incubated for 30 min with 0.15, 1, 5, or 10 mM L-glutamate. With the exception of cardiomyocytes, which were incubated with 0.15 mM L-glutamate, the intracellular glutamate content reached a new equilibrium after 10 min. A rise in the extracellular concentration of L-glutamate was accompanied by a rise in the intracellular glutamate content (Fig. 4). The incubation of cardiomyocytes with 10 mM L-glutamate (100 times the physiological rat blood concentration) led to a 1.8-fold increase in the glutamate content. Anoxic incubation of cardiomyocytes. During 30 min of anoxic incubation of cardiomyocytes, glutamate content decreased from 10.4 2 2.6 to 2.3 t 0.4 nmol/mg protein (Fig. 5). Prolongation of the anoxic incubation did not lead to a significant change in intracellular glutamate content. The initial rate of glutamate uptake that amounted to 24.3 % 1.6 pmol min. mg protein during oxygenation increased significantly to 38.1 t 3.8 pmol minl l mg protein-l under anoxia (Table 2). Reoxygenation of cardiomyocytes. Changes in glutamate content after reoxygenation of cardiomyocytes are l
l
30
25
/
L-glutamate
15 time
20 (min)
Fig. 1. Time course of L-glutamate uptake (in pmol/mg protein) oxygenated isolated rat heart myocytes (means + SD, n = 3).
of
0
concentration
2
4
6
(mM
8
10
)
Fig. 2. Initial L-glutamate uptake rates (pmol*min-lsmg-l) vs. L-glutamate concentration of oxygenated isolated rat heart myocytes (means + SD, n = 5). Initial rates of L-glutamate uptake at L-glutamate concentrations are from 0.06 to 0.33 mM (main graph) and from 0.06 to 10 mM (inset).
HI828
TRANSPORT
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METABOLISM
OF GLUTAMATE
IN HEART
-c
80 0
60
40
\
~
---t---I---[
I
-8
-4
0
4
8
12
16
,
0 1 L-glutamate
,
,
,
10
20
30
40
50
(mM -I)
concentration
time
Fig. 3. Lineweaver-Burk diagram of initial rate of L-glutamate uptake of oxygenated isolated adult rat heart myocytes (means + SD, n = 3). Data are recalculated from initial L-glutamate uptake as shown in Fig. 2. A: 0.06 - 0.33 mM L-glutamate, apparent affinity (K,) = 0.18 + 0.03 mM, apparent maximal uptake rate (V,,,,) = 44 + 12 pm01 min+ mg protein -I; B: 0.33 - 10 mM, K, = infinite. l
i
0
l
summarized in Fig. 6. Cardiomyocytes were incubated without oxygen for 30 min to deplete the intracellular glutamate content. Reoxygenation of cardiomyocytes led to a rapid increase in the intracellular glutamate content that reached control values of oxygenated cells after 15 min. An initial velocity of glutamate repletion of 1,329 t 160 pmol .rninl* mg protein-l was calculated from these data. Glutamate extraction from the
incubation medium was measured simultaneously and amounted to only 31.9 t 2.5 pmol=minl.mg protein-l (Table 2). During 30 min of anoxia, the alanine content of cardiomyocytes increased, whereas reoxygenation of cardiomyocytes was accompanied by a rapid decrease in intracellular alanine (Fig. 7). Metabolism and intracellularprovision ofglutamate. The intracellular glutamate depletion during anoxia as well as its provision after reoxygenation of cardiomyocytes was studied using aminooxyacetate, a specific and competitive inhibitor of transaminases (28). The inhibition of glutamate depletion in anoxic cardiomyocytes incubated in the presence of aminooxyacetate is summa-
T
I’
I
1
I
I
I
0
2
4
6
8
10
(min)
Fig. 5. L-Glutamate content (in nmol/mg protein) of isolated adult rat heart myocytes during 50 min of anoxic incubation (means + SD, n = 5).
reoxygenation I
mw extracellular
L-glutamate
concentration
Fig. 4. Intracellular vs. extracellular L-glutamate concentration (mM) in incubation medium of oxygenated adult rat heart myocytes (means + SD, n = 5, the intracellular contents of L-glutamate differ significantly, P < 0.05). Myocytes were incubated for 10 min with specified glutamate concentrations. Dotted line represents identical intra- and extracellular concentrations.
time Fig. 6. L-Glutamate content (in nmol/mg heart myocytes during 30 min of anoxic reoxygenation (means ? SD, n = 4).
(min)
protein) of isolated adult rat incubation and subsequent
TRANSPORT
AND
METABOLISM
OF
GLUTAMATE
. T
reoxygenation
H1829
IN HEART
reoxygenation I
1
+ 5 mM aminooxyacetate
0
I
I
I
I
I
30
35
40
45
50
time Fig. 7. Time isolated adult and subsequent
40
45
50
55
60
Time (min)
(min)
course of L-alanine content (in nmol/mg rat heart myocytes during 30 min of anoxic reoxygenation (means + SD, YL = 4).
protein) of incubation
rized in Fig. 8. The initial velocity of glutamate decrease in anoxic cardiomyocytes amounted to 1,063 t 112 pm010 minl mg protein and was inhibited to a value of 95.7 ? 32.3 pmolminl~mg protein when 5 mM aminooxyacetate was present in the incubation medium (Fig. 8). Further investigations elucidated whether the observed glutamate repletion after reoxygenation of heart muscle cells was inhibited by suppressing transaminase reactions. The intracellular glutamate content was therefore decreased by incubating cardiomyocytes for 30 min in the absence of oxygen (Fig. 9). Afterwards 5 mM of aminooxyacetate was added to the incubation medium, and the cells were l
1T
0
0
Fig. 9. Time course of L-glutamate content (in nmol/mg protein) of isolated adult rat heart myocytes during 40 min of anoxic incubation and subsequent reoxygenation (means + SD, y1 = 6). After 30 min of anoxia incubation medium was supplemented with 5 mM aminooxyacetate.
incubated for another 10 min under an argon atmosphere to ensure entry of aminooxyacetate into the cardiomyocytes. After reoxygenation of cardiomyocytes, the initial velocity of glutamate increase was inhibited and amounted to only 57.3 ? 29 pmol min-l mg protein (Fig. 9). Aminooxyacetate abolished both the intracellular increase of alanine observed during anoxia and its metabolization after reoxygenation of cardiomyocytes. Hence, the increase in intracellular glutamate after reoxygenation of cardiomyocytes is provided from intracellular sources and catalyzed by aminotransferases. l
l
DISCUSSION
10
20
30 time
(min)
Fig. 8. Time course of L-glutamate content (in nmol/mg protein) of isolated adult rat heart myocytes during 30 min of anoxic incubation either incubated without (triangles) or in the presence (circles) of 5 mM aminooxyacetate (means + SD, n = 4).
A comparison of the concentration gradient of glutamate across the sarcolemma with the transsarcolemma1 gradients of all other amino acids (Table 1) clearly demonstrates that glutamate is accumulated inside the cardiomyocyte in a unique manner. Furthermore, the positive arteriovenous difference of glutamate in the healthy heart as well as its increased uptake in the hearts of patients suffering from coronary artery disease (17) support a particular transport mechanism for this amino acid in the heart. The aim of the present study was to elucidate both the transport pathways of glutamate across the sarcolemma and the effect of glutamate metabolism on the intracellular concentration of this amino acid. Isolated adult rat heart myocytes are a suitable model for these investigations. The ATP content of the cells used in this study was stable throughout the experiments in oxygenated, reoxygenated, as well as in anoxic incubations, where anaerobic glycolysis is sufficient for ATP regeneration. Furthermore, the model allows a distinction to be made between glutamate
H1830
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uptake by cardiomyocytes and that by other cell types of the heart, e.g., endothelial or smooth muscle cells. Glutamate transport during oxygenation. The incubation of cardiomyocytes with glutamate concentrations from 0.06 to 10 mM shows that this amino acid is transported into the cell by transport processes, which differ in their affinity and their capacity. The transport mechanism characterized with an apparent K, value of 0.18 mM and an apparent Vmax value of 44 pmol. min l l mg protein-l (Figs. 2 and 3) represents a highaffinity glutamate carrier. This affinity of glutamate transport observed in isolated myocytes is in good agreement with the reported apparent K, value of the same carrier investigated in sarcolemmal vesicles from rat heart (7). At high substrate concentrations (> 1 mM) this high-affinity glutamate transport is superimposed by transport processes characterized by nonsaturation kinetics and high transport capacity. The glutamate concentration in rat blood amounts to 0.1 zt 0.02 mM (n = 3). Hence, under physiological glutamate concentrations only the high-affinity glutamate carrier is active. In comparison with transport system A for neutral amino acids, which was likewise investigated in cardiomyocytes, the high-affinity glutamate carrier is characterized by a sevenfold higher substrate affinity and a sixfold lower transport capacity (29). Similar to the investigated glutamate transport in rat heart, the transport of this amino acid in other cell types, e.g., human fibroblasts (6), is characterized by nonsaturation kinetics. We conclude that the transport mechanisms with low affinity and high capacity are due to an uptake of glutamate via other transport systems. Indeed, transport of glutamate could be demonstrated via transport systems ASC, XC, and L (1,6, 10). The initial rate of glutamate uptake in oxygenated cardiomyocytes amounts to 24.3 t 1.6 pmol=minl l mg protein-l (Table 2). The glutamate concentration in rat blood amounts to 0.1 mM, whereas the calculated intracellular glutamate concentration of cardiomyocytes amounts to 2.9 t 0.6 mM (Table 1). Therefore, at physiological glutamate concentrations, cardiomyocytes take up this amino acid against a high concentration gradient, and the underlying mechanism for glutamate transport into the heart muscle cell must be active. Studies with sarcolemmal vesicles of rat heart have shown that glutamate transport is Na+ dependent and inhibited by L-aspartic and L-cysteic acid (7). If cardiomyocytes are incubated with glutamate concentrations higher than their physiological intracellular glutamate content of 2.9 mM (Table 1), transport occurs with an inwardly directed concentration gradient. Under these conditions intracellular glutamate rises linearly with the concentration of glutamate in the medium due to the high capacity of the underlying transport processes (Fig. 4). At high extracellular concentrations of glutamate above 4 mM a balance between intracellular and extracellular glutamate concentration is not achieved, since glutamate has to be transported into the cardiomyocyte against the existing negative electrical potential.
OF
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Glutamate content and glutamate transport during anoxia. Isolated cardiomyocytes react to anoxia with a rapid decrease in intracellular glutamate (Fig. 5) and increase in glutamate uptake to 38.1 t 3.8 pmol *min.-l l mg protein (Table 2). This 1.6-fold increase in glutamate uptake compared with oxygenated cardiomyocytes is remarkably similar to the reported increase in glutamate uptake by patients suffering from coronary artery disease (17). Therefore, the mechanisms for glutamate uptake estimated by determination of arteriovenous differences in patients with coronary disease can be observed even on the cellular level of anoxic cardiomyocytes. The intracellular metabolization of glutamate during anoxia reduces the transsarcolemmal concentration gradient of this amino acid. This decrease leads to an increase in the driving force of glutamate entry and might contribute to the accelerated. transport of this amino acid during anoxia. In sarcolemmal vesicles from rat heart we were able to show that transport of glutamate is increased if either the intravesicular or the extravesicular space has been acidified (7). In addition to the increased driving force of glutamate entry, acidosis may also increase glutamate transport during anoxia. Although the depletion of the intracellular glutamate pool is counteracted by 1.6-fold increase in uptake rate (Table 2), it cannot compensate for the rapid utilization of glutamate during anoxia. Thus the high-affinity glutamate carrier because of its low capacity acts more as a means of steady-state regulation of the transsarcolemma1 glutamate gradient than of short-term regulation of changes in the intracellular glutamate concentration, which occur dependent on the oxygen supply to the heart. Glutamate content and glutamate transport during reoxygenation. Reoxygenated cardiomyocytes reestablish the intracellular glutamate content rapidly with a calculated initial rate of 1,329 t 160 pmol .min-l .mg protein-l (Fig. 6). The simultaneously measured initial uptake rate of glutamate after reoxygenation (31.9 t 2.5 pmol*mir+ ‘mg protein, Table 2) does not explain this observed replenishment. Cardiomyocytes must, therefore, replenish their glutamate content primarily by endogenous processes. While the intracellular alanine content of rat heart muscle cells increased after anoxia, a rapid decrease in alanine could be observed after reoxygenation (Fig. 7). Metabolism of glutamate. Investigations using isolated heart preparations have shown that intracellular aspartate and glutamate decrease during hypoxia, whereas alanine and succinate are produced simultaneously (27,28,31). During ischemia the decline of aspartate and glutamate and the production of alanine and succinate are more gradual. Although intracellular aspartate decreases and production of alanine and succinate increases during early ischemia (15, 19), the decrease in intracellular glutamate is not significant (16, 19). Prolonged ischemia leads to a significant decrease in
TRANSPORT
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glutamate (9, 30) and leads to further production of succinate (30) but not of alanine (15). Taegtmeyer et al. (28) demonstrated that glutamate consumption and alanine production observed during hypoxic incubation of isolated rabbit right ventricular papillary muscle were abolished in the presence of the transamination inhibitor aminooxyacetate. Likewise, in anoxic cardiomyocytes the intracellular decrease in glutamate was inhibited by 91% (Fig. S), and the increase in alanine was abolished when aminooxyaceate (5 mM) was added to the incubation medium. Furthermore, the velocity of glutamate replenishment after reoxygenation of cardiomyocytes was decreased to 57.3 t 29 pm01 . min+ mg protein if aminooxyacetate was present in the incubation medium (Fig. 9). This reduced rate lies within the range of the glutamate uptake velocity determined after reoxygenation of heart muscle cells (Table 2). Because transaminase reactions participate in intracellular glutamate provision, the origin of amino groups as well as the carbon skeleton for glutamate synthesis have to be discussed. Half of the amino groups required for transamination originate from intracellular alanine, which is rapidly degraded after reoxygenation of heart muscle cells (Fig. 7). The source of the remaining amino groups was not investigated further. Some of the amino groups may originate from intracellular ammonium, which accumulates during ischemic (19) or hypoxic (20) perfusion of rat hearts, while reoxygenation leads to a rapid decrease in intracellular ammonium. Furthermore, intracellular proteolysis (19) and transamination of extracellular alanine after transport into the cardiomyocyte (7) may contribute to the supply of amino groups after reoxygenation. Based on the described anaplerotic reactions in metabolism (13), only the carboxylation of pyruvate via the NADP-dependent malic enzyme can be responsible for the synthesis of the glutamate carbon skeleton (26). This carboxylation of pyruvate via NADP-dependent malic enzyme should also be possible in the hypoxic or ischemic myocardium, since the NADPIUNADP quotient is increased under these conditions. Protective effect of glutamate. Because of the massive decrease in glutamate and aspartate and the transport of alanine and succinate into the blood, cataplerotic reactions prevail during hypoxia and prolonged ischemia leading to a decrease in TCA cycle metabolites (19,20). Although myocardial uptake of glucose is enhanced about threefold after reperfusion, it could be demonstrated that only 30-40% of the glucose taken up from the perfusion medium is oxidized by the heart (22). After reoxygenation, aspartate and glutamate first have to be replenished before the contents of the corresponding oxoacids, oxaloacetate and 2-oxoglutarate, can be increased to their normoxic values, since transaminase reactions are catalyzed near equilibrium. As long as glutamate and aspartate concentrations, which in the heart are very high compared with the other amino acids, do not reach their normoxic control values. the TCA cvcle and oxidative phosshorvl
OF GLUTAMATE
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lation cannot work at maximum capacity. Furthermore, redox equivalents derived from glycolysis and the oxidation of exogenous lactate cannot be transported into the mitochondrium, because the malate-aspartate cycle depends on high concentrations of these amino acids due to the low affinity of the glutamate-aspartateantiporter to its substrates (14). Thus the observed inhibition of glucose oxidation after reperfusion could be due to the loss of TCA cycle metabolites during the preceding period of decreased perfusion. A protective effect of glutamate cou Id only be observed if exogenous concentrations of 2 mM (3) $5 mM (21), or 26 mM (23) L-glutamate were provided. This corresponds to 20-260 times the physiological glutamate concentration in the blood. At these high glutamate concentrations the lion’s share of glutamate is transported into the cell via ‘the uptake processes characterized by a high capacity (see Figs. 2 and 3). If the heart is perfused with glutamate concentrations higher than the intracellular glutamate concentration (> 2.9 mM, see Table 1), the glutamate transport occurs inwardly. Normoxic incubation of cardiomyocytes in an extracel lular glutamate concentration of 10 mM nearly doubles the intracellular glutamate concentration (see Fig. 4). Likewise, hypoxic perfusion of the heart with 10 mM glutamate increases the intracellular glutamate content (20). From the calculation of the data concerning the substrate affinity of the glutamate transport in cardiomyocytes (see Figs. 2 and 3) a velocity of glutamate uptake in the presence of 10 mM of 728 pmol 0min. mg protein can be deduced. This rate of glutamate uptake lies within the range of the decrease in intracellular glutamate in anoxically incubated cardiomyocytes (1,063 pmol 0min. mg protein-l, Fig. 5) and may therefore prevent glutamate decrease during anoxia and reoxygenation. At higher intracellular glutamate concentrations, the loss of TCA metabolites, leaving the cell with succinate and alanine during hypoxia, is not as crucial because glutamate can provide sufficient precursors for the synthesis of these metabolites. Similarly, after reoxygenation, cardiomyocytes are able to increase TCA cycle metabolites more quickly. Interestingly enough, the transaminase inhibitor aminooxyacetate inhibits not only the synthesis of succinate (27) and alanine (28) but also the observed protective effect of glutamate in the hypoxic myocardium (20). A larger intracellular amount of TCA metabolites during reoxygenation should improve ATP synthesis from the aerobic oxidation of substrates. This enhanced ATP supply could be of utmost importance to the high-energy demand of the heart after reoxygenation. Hence, the observed protective effect of glutamate could be due to the anaplerotic role of this amino acid. Address for reprint Berlin, Germany Received
16 Mav
requests:
1995: accented
L. Dinkelborg, in final
form
Schering 6 November
AG, 1995.
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AND
METABOLISM
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