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Journal of Applied Physiology
Vol. 81, No. 5, pp. 1973-1977, November 1996
METABOLISM

Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles

P. Granier, H. Dubouchaud, N. Eydoux, J. Mercier, and C. Préfaut

Laboratoire de Physiologie des Interactions, Service d'Exploration de la Fonction Respiratoire, Hôpital Arnaud de Villeneuve, 34295 Montpellier cedex 5, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Granier, P., H. Dubouchaud, N. Eydoux, J. Mercier, and C. Préfaut. Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles. J. Appl. Physiol. 81(5): 1973-1977, 1996.---2-Chloropropionate (2-CP) is a halogenated monocarboxylic acid generally used to decrease blood lactate concentration in various metabolic states. To investigate whether it has an inhibitory effect on sarcolemmal lactate transport, we compared the initial rate of lactate transport in sarcolemmal membrane vesicles purified from 20 male Wistar rats with and without 2-CP. Transport by these vesicles was measured as uptake of L-(+)-[U-14C]lactate under pH gradient-stimulated cis inhibition. The time courses of 1 mM L-(+)-lactate uptake into vesicles both with and without 10 mM 2-CP (L- or D-) displayed saturation kinetics. Lactate uptake values were lower with 10 mM L-2-CP and 10 mM D-2-CP in comparison to the control values. Both 10 mM L-2-CP and 10 mM D-2-CP significantly inhibited 1 mM L-(+)-lactate uptake (55.8 ± 9.1 and 53.5 ± 12.1%, respectively; P < 0.001), whereas a smaller inhibition was observed with a higher lactate concentration of 50 mM (40.2 ± 11.2 and 38.7 ± 12.4%; P < 0.001 and P < 0.05, respectively). However, a higher D-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mM L-(+)-lactate uptake (77.0 ± 9.4%; P < 0.001). D-2-CP had a trans-stimulation effect on the initial rate of lactate efflux of 1 mM L-(+)-lactate compared with baseline efflux (9.5 ± 0.8 vs. 5.1 ± 0.4 nmol · min-1 · mg protein-1; P < 0.05). 2-CP significantly inhibited the initial rate of lactate uptake in skeletal muscle sarcolemmal membrane vesicles. This result suggests that 2-CP is a nonstereoselective substrate of the lactate muscle carrier that impairs lactate transport.

trans stimulation

INTRODUCTION

THE TRANSPORT OF L-LACTATE across the plasma membrane is of fundamental importance to most mammalian cells (6, 11, 24). For tissues such as erythrocytes and tumor cells, lactate is produced in quantity as an end product of glycolysis and is then expelled from the cell (24). According to the "lactate shuttle" hypothesis (3), for other tissues such as skeletal muscle, lactate is an important intermediary metabolite rather than a simple metabolic end product. To characterize the functional and kinetic properties of skeletal muscle lactate transport and to eliminate the confounding problems of metabolism, capillary diffusional resistance and flow, interstitial concentration variabilities, and uncontrolled intracellular solute composition, different techniques of purified preparation of sarcolemmal membranes vesicles have been described (15, 17, 27). From these studies we know that lactate moves across the sarcolemmal membrane by a carrier-mediated transport characterized by stereospecificity for L-lactate; Michaelis-Menten saturation kinetics; pH and temperature sensitivity; H+ cotransport; competitive inhibition by monocarboxylates such as pyruvate, beta -hydroxybutyrate, and acetoacetate (27, 28); and trans-stimulation effect by L-(+)-lactate and pyruvate (4).

2-Chloropropionate (2-CP), a halogenated monocarboxylic acid, exerts multiple effects on pathways of intermediary metabolism. One of the most important effects of 2-CP is the activation of pyruvate dehydrogenase via the inhibition of pyruvate dehydrogenase kinase (5). Thus the reduced blood lactate concentration observed with 2-CP in the resting state in animals (5, 25, 32, 33) has been attributed to a greater flux of pyruvate from the glycolytic pathway to the tricarboxylic acid cycle. Consequently, 2-CP is an important tool used against the lactic acidosis induced by various metabolic disorders such as diabetes, sepsis, and hepatic insufficiency (22) as well as by intense exercise in humans (20). However, two recent studies (12, 16) concerning the effects of dichloroacetate (DCA), an analogue of 2-CP but with greater toxicity, showed that the decrease in lactate concentration cannot be explained solely by an enhanced rate of lactate oxidation. Moreover, Mazer et al. (19) demonstrated that, during myocardial ischemia, intracoronary DCA increases carbohydrate uptake and decreases lactate release. Furthermore, from in vitro experiments, it has been shown that 2-CP inhibits L-lactate transport in rat erythrocytes and pyruvate transport in guinea pig cardiac myocytes (23). Comparison of the affinities for the two transporters reveals a higher affinity for 2-CP (L- and D-) than for L-lactate.

To explain the great decrease in blood lactate concentration observed with 2-CP, we hypothesized that 2-CP could have a direct effect on lactate transport in muscles by an inhibition of the sarcolemmal lactate transport. In an attempt to verify this hypothesis, we investigated the effect of 2-CP on initial lactate uptake by skeletal muscle sarcolemmal vesicles in rats. We also performed trans-stimulation experiments to determine whether 2-CP is actually transported into the vesicle or merely binds to the transporter.

MATERIALS AND METHODS

Animals. Twenty male Wistar rats (321.5 ± 9.9 g), kept in a room (24-26°C) with a 12:12-h inversed light-dark cycle and housed in individual cages with food and drink ad libitum, were killed at 17 wk of age after an overnight fast.

Sarcolemmal isolation. Purification was achieved according to the method of Grimditch et al. (10) with slight modification. Briefly, a single animal was killed. The hindlimbs were skinned and plunged into an ice-cold medium with 250 mM sucrose, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 1 mM EDTA, pH 7.4, at 37°C. All subsequent steps were carried out at 4°C. Mixed muscles (15-20 g) were carefully dissected to avoid fat and connective tissue, thus ensuring highly homogeneous muscular tissues. The pieces were cut in sucrose medium and homogenized with two bursts of Ultra-Turrax T25. The homogenate was filtered and termed crude homogenate (CH). A 1-ml aliquot was partitioned and saved at 4°C for subsequent analysis. The CH was centrifuged twice at 900 g in a Sorvall RC-28S with an SA-600 rotor. Supernatants were diluted with 3 M KCl/250 mM pyrophosphate to solubilize contractile proteins and pelleted by ultracentrifugation in a Beckman 60 Ti rotor (200,000 g for 45 min at 4°C). Pellets were resuspended by using Teflon pestle homogenization in 30 ml of sucrose medium. The suspension was centrifuged twice, and supernatants were collected and spun down (200,000 g for 45 min at 4°C). Pellets were homogenized with a glass tissue homogenizer in 7-8 ml of 40% sucrose. A discontinuous density gradient was constructed on prepurified sarcolemma by addition of the following four fractions of sucrose: 38, 32, 27, and 12%. Tubes were centrifuged overnight at 130,000 g at 4°C in an SW 27 rotor. The F2 band (27% sucrose) was harvested, diluted with a Krebs-Ringer-HEPES (KRH) buffer (118 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, and 50 mM HEPES, pH 7.5) and pelleted in a 60 Ti rotor (200,000 g for 80 min). The vesicles were resuspended in KRH buffer up to 4 mg/ml and stored at -80°C until used for the transport experiments.

Sarcolemmal characterization. For sarcolemmal characterization, K+-stimulated p-nitrophenylphosphatase (K+pNPPase) was used as the sarcolemmal marker (1) and assayed for its total activity, as described by Grimditch et al. (10) in 40 mM HEPES, 0.8 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 4 mM MgCl2, 20 mM KCl, and 5 mM p-nitrophenylphosphate, pH 7.4. The absorbance of the resulting p-nitrophenol was read at 412 nm. Nonspecific K+-pNPPase activity was determined in a KCl-free medium, subtracted from the total activity to give the specific K+-pNPPase activity, and expressed in micromoles per milligram per hour. The purification index (PI) was defined as the ratio of the specific activity of the F2 fraction to the specific activity measured in the CH. Proteins were estimated by the Coomassie brilliant blue method by using bovine gamma -globulin as a standard (Bio-Rad protein assay), as described by Bradford (2). Skeletal muscle sarcolemmal yield was the ratio of milligrams of sarcolemmal proteins obtained in F2 to the muscle weight in grams after the trimming process (wet weight).

Radioisotopes and monocarboxylates. L-(+)-[U-14C]lactate was purchased from Amersham Life Science (Buckinghamshire, UK), which certified the radiochemical purity to 98.5%. Stock isotope solutions had specific activity of 157 mCi/mmol.

L-alpha -Chloropropionic acid (L-2-CP) and D-alpha -chloropropionic acid (D-2-CP) were purchased from Sigma Chemical (L'Isle d'Abeau Chesnes, France).

Transport assays. Sarcolemmal membranes were defrosted at room temperature (25°C); tested again for protein concentration; and resuspended in 280 mM sucrose and 50 mM HEPES, pH 7.4, to a protein concentration of 2 mg/ml. Transport assays were performed under cis-inhibition conditions. For these experiments L-2-CP or D-2-CP was added directly to the external medium (EM; on the cis side of the membrane) from stock solutions buffered in 10 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.9, adjusted with tris(hydroxymethyl)aminomethane (Tris) to a final concentration of 10 or 50 mM, with EM consisting of 280 mM sucrose, pH 5.9. Uptakes were assayed in pH gradient-stimulated conditions, with an inwardly directed pH gradient from EM (pH 5.9) to internal medium (pH 7.4) (28). Vesicles (50 µg) from rats were incubated by using the method of Roth and Brooks (27) with two external lactate concentrations (1 and 50 mM). Reactions were stopped at appropriate time intervals by vacuum filtration on nitrocellulose filters (average pore size of 0.45 µm; Whatman WCN). Filters were then rinsed three times with an ice-cold isosmotic medium consisting of KRH buffer with 3 mM HgCl2, pH 7.4. Each measurement was in duplicate. Filters were then dissolved with 600 µl of ethylene glycol monomethyl ether, and the radioactivity was counted in a scintillation analyzer (Packard 2200 CA). Nonspecific transport activity was determined by incubation of vesicles in a tracer-containing medium with HgCl2 medium. Values were subtracted from those of the total transport activities. To determine initial lactate uptake, the slopes of curves were determined by computer linear-regression analysis by using the least-squares method. The initial rates were determined by measuring transport at 5, 10, and 20 s on each membrane preparation in duplicate and expressed in nanomoles per minute per milligram protein. The inhibition of pH gradient-stimulated L-(+)-lactate uptake was defined as the ratio of the difference between initial uptake rate with and without inhibitor present to the initial rate without inhibitor, multiplied by 100 (i.e., Delta %).

Additional trans-stimulation experiments were conducted according to the method of Brown and Brooks (4). These consisted of the incubation of sarcolemmal vesicles previously loaded with L-(+)-[U-14C]lactate diluted with unlabeled L-(+)-lactate to a final concentration of 1 mM in 280 mM sucrose and 50 mM Tris, pH 7.4, with different EM. Baseline efflux was measured by incubation of 50 µg of loaded vesicles in 280 mM sucrose and 50 mM Tris, pH 7.4. Trans stimulation was achieved with an EM composed of either 100 mM unlabeled lactate, 180 mM sucrose, and 50 mM Tris, pH 7.4; or 50 mM lactate, 50 mM D-2-CP, 180 mM sucrose, and 50 mM Tris, pH 7.4. Efflux was stopped by the addition of HgCl2 medium at the 5-, 10-, and 20-s time points. Initial rates were determined as described in Transport assays. Blank measurments were done by incubation of previously loaded vesicles in HgCl2 medium. Experiments were done in duplicate.

Statistical analysis. Results are expressed as means ± SE. Statistical significance was tested by Student's t-test to examine the effect of 2-CP on the variables studied. A probability level of P < 0.05 was used throughout the study.

RESULTS

Sarcolemmal characterization. Characterization of membrane fractions isolated from rat hindlimb skeletal muscles revealed that the highest PI K+-pNPPase was observed in the F2 band, which appeared within the 27% layer of the sucrose gradient (Table 1). K+-pNPPase in the F2 band was purified 17.2 ± 2.9-fold over that of CH. The F2 band was thus defined as the sarcolemmal fraction and used for all subsequent transport experiments.

Table 1. Characterization of membrane fractions isolated from rat hindlimb skeletal muscles

Fraction Protein Yield, mg/g tissue wet wt K+-pNPPase Specific Activity, µmol · mg-1 · h-1
Crude homogenate 141.1 ± 8.2  0.146 ± 0.017 
Plasma membrane (F2: 27% fraction) 0.44 ± 0.05  2.510 ± 0.150
Values are means ± SE. K+-pNPPase, K+-stimulated p-nitrophenylphosphatase.

Lactate transporter kinetics. Figure 1A shows the time course of 1 mM L-(+)-lactate uptake under pH-stimulated gradient into vesicles with or without 10 mM L-2-CP. Figure 1B shows the time course of 1 mM L-(+)-lactate uptake under pH-stimulated gradient into vesicles with or without 10 mM D-2-CP. The three patterns of lactate uptake were similar, with a sharp increase up to 20 s and a leveling off at ~60 s. Lactate uptake values were lower with 10 mM L-2-CP and 10 mM D-2-CP compared with the control experiment, but the results obtained with L-2-CP and D-2-CP did not differ from each other.
Fig. 1. A: time course of 1 mM L-(+)-lactate uptake into vesicles with L-alpha -chloropropionic acid (L-2-CP; open circle ) and without L-2-CP (bullet ). B: time course of 1 mM L-(+)-lactate uptake into vesicles with D-alpha -chloropropionic acid (D-2-CP; down-triangle) and without D-2-CP (bullet ). L-2-CP or D-2-CP was added directly to external medium from stock solutions buffered in 10 mM MES, pH 5.9, and adjusted with Tris to a final concentration of 10 mM. External medium consisted of 280 mM sucrose, pH 5.9. Vesicles were loaded with 280 mM sucrose, pH 7.4. All assays were performed in duplicate. Each experiment was performed with 4-5 different membrane preparations. Values are means ± SE.
[View Larger Version of this Image (38K GIF file)]

Inhibitions of pH-stimulated 1 mM L-(+)-lactate and 50 mM L-(+)-lactate uptake by 10 mM L-2-CP and D-2-CP are reported in Table 2. Both 10 mM L-2-CP and 10 mM D-2-CP significantly inhibited 1 mM L-(+)-lactate uptake (55.8 ± 9.1 and 53.5 ± 12.1%, respectively; P < 0.001). A smaller inhibition was observed with a higher external lactate concentration of 50 mM (40.2 ± 11.2 and 38.7 ± 12.4%; P < 0.001 and P < 0.05, respectively).

Table 2. L-2-CP and D-2-CP cis inhibition of initial rate of 1 and 50 mM L-(+)-lactate uptake

L-2-CP D-2-CP
%Inhibition of pH-stimulated 1 mM L-(+)-lactate uptake 55.8 ± 9.1* 53.5 ± 12.1*
%Inhibition of pH-stimulated 50 mM L-(+)-lactate uptake 40.2 ± 11.2* 38.7 ± 12.4dagger
Values are means ± SE. Each experiment was performed with 4 different membrane preparations. All monocarboxylates were added directly to external medium from stock solutions buffered in 10 mM MES, pH 5.9, and adjusted with Tris to a final concentration of 10 mM. External medium consisted of 280 mM sucrose, pH 5.9. Vesicles were loaded with 280 mM sucrose, pH 7.4. All assays were performed in duplicate. Initial rates of 1 mM L-(+)-lactate uptake: 6.14 ± 0.61 nmol · min-1 · mg protein-1 [without 2-chloropropionate (2-CP)], 2.72 ± 0.10 nmol · min-1 · mg protein-1 [with 10 mM L-alpha -chloropropionic acid (L-2-CP)], 2.85 ± 0.21 nmol · min-1 · mg protein-1 [with 10 mM D-alpha -chloropropionic acid (D-2-CP)]. Initial rates of 50 mM L-(+)-lactate uptake: 240.97 ± 36.97 nmol · min-1 · mg protein-1 (without 2-CP), 143.66 ± 12.49 nmol · min-1 · mg protein-1 (with 10 mM L-2-CP), 145.10 ± 20.52 nmol · min-1 · mg protein-1 (with 10 mM D-2-CP). * P < 0.001.  dagger P < 0.05.

An increased D-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mM L-(+)-lactate uptake (77.0 ± 9.4%; P < 0.001).

Table 3 shows the trans-acceleration effect of high external lactate and D-2-CP concentrations on the initial rate of lactate efflux. A concentration of 100 mM external L-(+)-lactate increased the labeled lactate efflux by 51% (P < 0.05) while 50 mM D-2-CP and 50 mM L-(+)-lactate caused stimulation of ~84% compared with baseline efflux (P < 0.05). There was no significant difference between the effect of lactate alone and lactate plus 2-CP on lactate efflux.

Table 3. Trans stimulation of 1 mM L-(+)-lactate efflux from rat sarcolemmal vesicles under different conditions

Condition External Medium 1 mM L-(+)-Lactate Efflux
Baseline efflux experiments 280 mM sucrose and 50 mM Tris, pH 7.4  5.15 ± 0.37 
Trans-stimulation efflux experiments 100 mM L-(+)-lactate, 180 mM sucrose, and 50 mM Tris, pH 7.4  7.80 ± 0.95*
50 mM L-(+)-lactate, 50 mM D-2-CP, 180 mM sucrose, and 50 mM Tris, pH 7.4  9.48 ± 0.82*
Values are means ± SE of duplicate experiments done with 5 different sarcolemmal preparations. Internal medium was composed of L-(+)-[U-14C]lactate diluted with unlabeled L-(+)-lactate up to 1 mM in 280 mM sucrose and 50 mM Tris, pH 7.4. All experiments were done at room temperature (25°C). * Significantly different from baseline efflux experiments, P < 0.05.

DISCUSSION

Initial lactate uptake by rat skeletal muscle sarcolemmal vesicles was significantly decreased with L- and D-2-CP under pH-stimulated gradient; there was no difference between L- and D-2-CP. Moreover, the inhibition was higher with 50 mM D-2-CP than with 10 mM D-2-CP.

A recent study from our laboratory (9) demonstrated that rat skeletal muscle sarcolemmal vesicles are suitable for investigating the lactate exchanges; we thus selected the membrane vesicle model to determine the effect of 2-CP on lactate transport. Indeed, compared with experiments on whole muscle systems, this technique offers two major advantages. First, it allows elimination of concurrent influences of intracellular metabolism, one of several complicating factors that directly or indirectly affects interpretation of results from studies of lactate flux in vivo and in situ (13, 14, 18, 26). Second, the use of purified vesicles also affords the unique ability to control the solution composition on both sides of the membrane, an important advantage for testing drug effects.

We chose to test the effect of 2-CP on lactate uptake rather than the effect of DCA, another halogenated monocarboxylic acid often used to decrease plasma lactate concentration in vivo (12, 16, 21, 29, 31), because of its lower toxicity; indeed this drug may become a useful tool for metabolic research (33), especially in humans (5). 2-CP is as effective as DCA as an activator of the pyruvate dehydrogenase complex with the perfused rat heart and isolated mitochondria (5). However, although it has been proposed that DCA affects various enzyme activities and transport systems between cytoplasm and mitochondria (30), nothing is known concerning the other pharmacological effects of 2-CP.

This study shows for the first time that 2-CP also affects lactate transport. Indeed, 2-CP significantly decreased the initial L-lactate uptake into rat sarcolemmal vesicles under pH gradient-stimulated conditions. The percentage of inhibition of pH gradient-stimulated L-lactate uptake was compared with zero percent inhibition and not with D-lactate inhibition as in the studies of Roth and Brooks (27, 28). However, because of the high percentage of inhibition, our result would probably remain significant even if compared with D-lactate inhibition. Compared with the significant effect of 10 mM beta -hydroxybutyrate and acetoacetate seen by Roth and Brooks (27 and 32% of inhibition, respectively), the inhibition of 1 mM lactate uptake in rat sarcolemmal vesicles was higher with L- and D-2-CP (55 and 53% inhibition, respectively). However, when inhibitions of L- and D-2-CP were compared, no difference appeared. This indicates that the lactate muscle carrier is not stereoselective for the two isomers of 2-CP. Because this carrier is almost completely stereoselective for L- over D-lactate (4, 27), our result suggests that lactate transport may involve the hydrogen of the hydroxy group. Moreover, this result reveals similarities in the substrate specificities of various monocarboxylate transporters in mammalian plasma membranes because Poole et al. (23) also found that monocarboxylate transport into guinea pig cardiac myocytes and rat erythrocytes was not stereoselective for D- or L-2-CP.

In accordance with the studies of Roth and Brooks (27, 28), the cis inhibition of 1 mM L-lactate transport was studied by adding 10 mM 2-CP to the external medium. To test the effect of increasing drug concentration, 50 mM 2-CP was also tested. Because there is no stereoselectivity, only the D-2-CP isomer was tested. Our results showed that the inhibition was higher with a higher 2-CP concentration. This suggests that a higher proportion of monocarboxylate binds the transporter at the same site as lactate (23), thus decreasing lactate transport by the carrier. This result was supported by the smaller inhibition of 10 mM 2-CP with an increasing 50 mM L-lactate uptake. Indeed, the higher lactate concentration may, in this case, decrease the competitive cis inhibition of 2-CP on lactate transport. To determine whether 2-CP is actually transported across the sarcolemmal or merely binds to the transporter, we performed trans-stimulation experiments. As reported by Brown and Brooks (4), our results showed a stimulatory effect of a high external L-(+)-lactate concentration on the lactate efflux. This effect was also seen with D-2-CP. This suggests that 2-CP was also transported by the carrier. It appears that 2-CP is a putative substrate for the lactate transporter, and our findings confirm the hypothesis of a competitive inhibition of lactate transport by 2-CP.

Moreover, 2-CP likely enters the muscle cell via the lactate transporter, based on the finding that substrates with a chloro substitution at C2 are compatible with monocarboxylate transport in erythrocytes (7, 8, 23). We can thus speculate that in vivo the increasing uptake of 2-CP in muscle may impair lactate release from muscle, as recently observed by Mazer et al. (19). Thus the decreased plasma lactate concentration observed in vivo with 2-CP may be due to not only an inhibition of the pyruvate dehydrogenase kinase but also an alteration of the lactate muscle-blood exchanges.

In conclusion, 2-CP significantly inhibits the initial rate of lactate uptake by skeletal muscle sarcolemmal vesicles: this effect is not stereoselective but is dependent on the 2-CP concentration. Moreover, D-2-CP significantly inhibits the initial rate of lactate efflux. This result suggests that 2-CP is a specific substrate of lactate muscle carrier that impairs lactate transport.

FOOTNOTES

Address for reprint requests: P. Granier, Laboratoire de Physiologie des Interactions, Service d'Exploration de la Fonction Respiratoire, Hôpital Arnaud de Villeneuve, 371, Ave. du Doyen G. Giraud, 34295 Montpellier cedex 5, France.

Received 18 July 1995; accepted in final form 11 July 1996.

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H. Dubouchaud, N. Eydoux, P. Granier, C. Prefaut, and J. Mercier
Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise
J Appl Physiol, September 1, 1999; 87(3): 955 - 961.
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