INTRODUCTION

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SKELETAL MUSCLE is the major tissue that produces lactic acid during intense exercise. At physiological pH, lactic acid is dissociated, and both the lactate anion and the associated change in pH are known to contribute to fatigue of the exercising muscle fibers (3). Consequently, it has been argued that export of lactate, together with its proton, out of the working muscle and into the blood may prolong the muscle's capacity for anaerobic work, and the activity of lactate transporters is therefore an important regulatory site for prevention of fatigue. From plasma, lactate is further transported into noncontracting tissues (1, 6) in which lactate is actively metabolized or into red blood cells (RBC; see Ref. 6), which provide an additional space for lactate. This redistribution of lactate thus enhances the capacity of plasma to receive more lactate from the working muscles. The importance of lactate transport into the noncontracting tissues and also to the RBC has been demonstrated by Lindinger et al. (6), who in their recent review calculated that, in human subjects lacking such transport, after four 30-s bouts of high-intensity exercise, the concentration of lactate in plasma would reach 162 mM.

Lactic acid may diffuse across cell membranes, but, for the transport of lactate anion, a carrier protein is needed. The RBC membrane contains at least two distinct carriers as follows (14): the monocarboxylate transporter (MCT), which cotransports lactate anion with a proton, and the inorganic anion exchange system (band 3 protein), which is an antiport carrier that exchanges lactate for Cl and .

The role of different carriers and the rate of total lactate influx vary both among tissues and also among animal species. RBC of rats, rabbits, and guinea pigs possess higher MCT activity than the RBC of human subjects and especially that of cattle, which appear to be devoid of MCT (2, 13, 14). Skelton et al. (18), comparing total activity of lactate influx into the RBC in several species, found such activity to decrease in the following order: dogs  horses  cattle  goats. In the RBC of athletic animals like dogs and horses, the MCT was the predominant pathway for lactate transport, and the importance of the inorganic anion exchange system was small. On the other hand, in the nonathletic cattle and goats, inorganic anion exchange plays a more important role. Variation in lactate transport within a single species has not been studied, but it has been found that the muscle-plasma lactate concentration gradients in human subjects (19) and the plasma-RBC lactate gradients in horses (15) vary widely between individual subjects. In our previous study on the distribution of lactate in equine blood (15), we measured only postexercise lactate concentrations in plasma and RBC; the aim of this study was therefore to examine whether the rate of lactate influx into the RBC exhibits similar interindividual variation.

	MATERIALS AND METHODS

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The experimental protocol was approved by the Ethics Committee for Animal Experiments of the Faculty of Veterinary Medicine, University of Helsinki.

Horses. For the 89 clinically healthy standardbred horses used, age and sex are shown in Table 1. All of the young horses from suckling foals to 2-yr-olds were bred and raised at the same breeding establishment.

Blood samples. Blood samples were taken from the jugular vein into tubes that contained EDTA as anticoagulant. All samples were taken in the morning before the daily exercise. The samples were taken one day before or on the day they were transported to the laboratory. Those samples taken on the previous day were stored in a refrigerator overnight.

Methods. Preparation of RBC and the lactate influx measurements were performed according to Skelton et al. (18). Briefly, RBC were collected by centrifugation at room temperature (2,000 g, 15 min), mixed with 30 vol of a chloride buffer (150 mM NaCl and 10 mM sodium tricine, pH 8.0 at 37°C), and incubated in a water bath for 30 min at 37°C. The cells were then washed three times with four volumes of the same buffer. After the final wash, the RBC were suspended in a volume of a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (90 mM NaCl and 50 mM HEPES, pH 7.4 at 37°C) to reach a hematocrit of 30%. For measurement of lactate influx, the suspension of RBC was divided into three parts. The first portion contained 5 mM -cyano-4-hydroxycinnamic acid (CHC; Sigma Chemical, St. Louis, MO), which inhibits both the MCT and the band 3 protein (14). The second portion contained 0.2 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Sigma) to inhibit the inorganic anion exchange system (7, 18). The last portion contained no inhibitors. Influx measurements were made at lactate concentrations of 10 and 30 mM. The specific radioactivity of lactate (L-[U-¹⁴C]lactic acid sodium salt, 5.62 GBe/mmol; Amersham, Buckinghamshire, UK) in these solutions was 40 and 13 counts · min¹ · nmol lactate¹, respectively. To measure the lactate influx, the RBC were incubated in the HEPES buffer for 20 s at 37°C. The incubation was stopped with 5 ml of ice-cold stop solution [150 mM NaCl and 10 mM Na-3-(N-morpholino)propanesulfonic acid, pH 6.5]. The cells were collected by centrifugation for 15 min at 2,000 g and +4°C and were washed two times with the stop buffer. After the final centrifugation, the RBC were hemolyzed with 4.2% perchloric acid and centrifuged, and a liquid scintillation cocktail (Ultima Gold; Packard Instruments, Groningen, The Netherlands) was added to measure the radioactivity of the supernatant with a liquid scintillation counter (Rack-Beta Scintillation Counter, model 1217; Wallac, Turku, Finland). All measurements were made in triplicate. Blank assays in which the stop solution was mixed with the lactate-containing incubation buffer before the addition of the RBC were run to test the residual extracellular radioactivity as well as the binding of radioactive lactate to the carrier protein. This residual radioactivity was 0.08% of the total radioactivity. Hemoglobin concentrations were measured by an automatic counter (Coulter Counter, model T 850; Coulter Electronics, Hialeah, FL) and used to calculate protein concentration of the RBC (18). The results are expressed as nanomole lactate per milligram RBC protein per minute.

Lactate influx through the different transport systems was estimated as follows. The difference between uninhibited flux and the DIDS-inhibited flux was taken to represent the transport through the band 3 system. Accordingly, the difference between the uninhibited flux and the CHC-inhibited flux was taken to estimate the flux through the MCT. Because CHC is not specific to the MCT only, this value somewhat overestimates the role of the MCT. The rest of the flux is supposed to represent the nonionic diffusion. Due to the nonspecific nature of the CHC, the role of nonionic diffusion is somewhat underestimated.

Statistical analysis. Linear regression analysis was used to calculate correlations. Analysis of variance was used to compare the lactate influx rates in different age groups, and the effect of sex and the inhibitors was tested by t-test. The differences were regarded significant at P < 0.05.

	RESULTS

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To test the viability of the washed RBC, the rate of lactate influx was measured on six successive days. We found that, when the cells were stored at +4°C, the rate of lactate influx remained unchanged for this time period. In the following experiments, the cells were washed on the same day or the day after the blood sample was taken, and the influx measurements were performed on the following day.

The effect of lactate concentration on the rate of influx is shown in Fig. 1. The following two concentrations were chosen for further experiments: 10 mM, which represents values after moderate-intensity exercise, and 30 mM, which is close to the values seen after trotting races. The role of the MCT and the band 3 system in the lactate influx was tested with the inhibitors CHC and DIDS, respectively, as described in MATERIALS AND METHODS (Fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of lactate concentration and transport inhibitors on the rate of lactate influx into red blood cells (RBC) of standardbred horses. Values are means ± SD of 4 separate experiments. Blood was taken from 2 adult horses. , No inhibitors; , 5 mM -cyano-4-hydroxycinnamic acid (CHC); , 0.2 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS).

Mean values for the rate of lactate influx in all age groups without and with inhibitors are shown in Table 2. Both in the absence and presence of inhibitors, as indicated by the range values, interindividual variation in the lactate influx was large in all age groups; due to this variation, the mean values did not differ significantly from each other. To demonstrate this interindividual variation, the distribution of total lactate influx, i.e., the uninhibited influx at lactate concentrations of 10 and 30 mM in all horses (n = 89), is shown as a histogram (Fig. 2). Based on their total lactate influx, the horses could be divided into two separate groups. For example, at a lactate concentration of 30 mM, in 30% of the horses, the rate of lactate influx was <4 nmol · mg protein¹ · min¹; in the majority of the horses (62%), the flux varied from 6 to 11 nmol · mg protein¹ · min¹; and, in a few individual horses (8%), an exceptionally high activity was apparent. These separate groups also existed when the samples from the same horses were assayed at the lower lactate concentration.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Frequency distribution of total lactate influx in 89 standardbred horses at 10 (A) and 30 (B) mM lactate concentration.

To test whether the interindividual variation was due to either one of the two transport mechanisms, the MCT or the band 3 system, the data from the assays without inhibitors were plotted against the data obtained from the incubations in which the inhibitors CHC or DIDS were present (Fig. 3). In all conditions, the group of horses (30%) in which the rate of uninhibited lactate influx into the RBC was low remained clearly separated. In these horses, CHC did not inhibit the lactate influx (Table 2), and the average inhibition by DIDS at lactate concentrations of 10 and 30 mM was 18 to 34%, respectively, which in absolute mean values was 0.21 and 0.42 nmol · mg protein¹ · min¹ at those two lactate concentrations. The remaining horses (70%) in which the rate of total lactate influx varied from medium to high values could be further divided into two groups according to their sensitivity to CHC. At the lactate concentration of 10 mM in a group of 12 horses, the inhibitory action of CHC was markedly low (39%), whereas, in the rest of the horses (n = 50), the lactate influx was inhibited by CHC even more significantly (74%). At the lactate concentration of 30 mM, these two groups were not as clearly separate, although a similar tendency was apparent. In the presence of DIDS, these subgroups were not distinguistable. The average inhibition by DIDS at lactate concentrations of 10 and 30 mM was 31 and 39%, respectively.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Roles of lactate carriers in RBC membranes of 89 standardbred horses as estimated with inhibitors of monocarboxylate-specific transporter and the band 3 system. , Horses with low lactate transport; , horses with high total lactate transport in which the transport was insensitive to CHC; , horses with high, CHC-sensitive lactate transport. A: lactate concentration 10 mM, CHC 5 mM; B: lactate concentration 30 mM, CHC 5 mM; C: lactate concentration 10 mM, DIDS 0.2 mM; D: lactate concentration 30 mM, DIDS 0.2 mM.

In Fig. 4, data on the total lactate influx are grouped according to sex. When all age groups are combined, the mares at both lactate concentrations had a significantly (P < 0.05) higher influx activity than did the stallions. This result was, at least partly, due to the fact that, in 21% of mares, the total lactate influx was low, whereas this group included 35% of the stallions. When the lactate transport activity in geldings was compared with the respective values in adult mares and stallions, statistically significant differences were not found.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Rate of lactate influx into RBC in mares (crosshatched bars), stallions (open bars), and geldings (filled bar) from suckling foals to adults. Results are means + SE.

	DISCUSSION

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This is the first study to show large interindividual differences in total rate of lactate influx into the RBC of standardbred horses. In the majority of these horses, the variation followed a normal distribution pattern, but a substantial number of horses in which the rate of lactate influx was low formed a clearly distinguishable subgroup. This suggests that the horses of our study belong to two separate populations with a different number or type of lactate transporters on their RBC membranes. Because the mean rate of lactate influx and the interindividual variation in the different age groups were similar, the lactate influx activity appears to be inherited rather than influenced by age or training.

In all horses, total flux was higher at a lactate concentration of 30 mM than at the concentration of 10 mM. Although part of this increase is evidently due to the increase in the nonionic diffusion at the higher lactate concentration, this result also suggests that the carrier-mediated flux was not saturated at 10 mM. If the results shown in Fig. 1 are used to calculate Michaelis constant values, a value for uninhibited influx will be 24.1. This value is the same as the value of 24.4 mM obtained for equine RBC by Skelton et al. (18) and also close to the Michaelis constant values for human subjects and rats (14).

In this study, the role of different transporters was studied with inhibitors of the MCT and the band 3 protein. CHC is a noncompetitive inhibitor of both transporters, whereas DIDS, at the concentration used in this study, irreversibly inhibits mainly the band 3 protein (2, 14). In a recent study, Skelton et al. (18) observed that, in athletic animal species such as dogs and horses, the activity of band 3 represents <10% of the total lactate influx, and the MCT is the predominant transporter. According to our results based on a number of horses 20 times larger, the role of the band 3 system appears to be slightly more important (20-40%). Our results from 89 horses, in accordance with an earlier finding (18), show that, in the majority of horses, the MCT represents 60-70% of the total lactate influx; however, in a significant number of horses, MCT appears to be inactive or nonexistent. This suggests that the large interindividual variation in lactate transport activity is due to variation in the number or type of transporters on the RBC membrane.

The horses could be divided into separate groups according to the rate of total influx and to their sensitivity to inhibitors. Most uniform was the group in which the total influx rate was low. In these horses, CHC did not inhibit lactate influx, which suggests that the activity of MCT on their RBC membranes was low. Because in absolute mean values the inhibitory effect of DIDS was also low in these horses, the most important mechanism to transport lactate into the RBC was the nonionic diffusion. The second group consists of horses that had moderate or high total influx of lactate into the RBC. In the majority of these horses, the inhibition of lactate transport by CHC was 56-67% and by DIDS 31-39%. The most interesting subgroup comprised 12 horses in which the effect of CHC was remarkably low, but the activity of their band 3 system did not differ from that of the rest of the horses. On the basis of these results only, we cannot conclude whether in these horses the rate of nonionic diffusion was exceptionally high or whether their RBC possess a variant of MCT protein that is insensitive to CHC. Together, these results suggest that variation in the lactate influx activity is very complex and that individual horses have different amounts of lactate carriers on their RBC membranes. In some horses, the activity of both carriers is high; in some individual horses, the activity of MCT is high, and the activity of the band 3 system low; and finally, in a substantial number of horses the nonionic diffusion overwhelms the activity of both carriers.

Our present findings are in accordance with those of our previous study (15) in which the distribution of lactate between plasma and RBC after exercise was shown to be highly variable interindividually. High variation in the distribution between muscle and plasma has previously been found in studies on human subjects (5, 19). Moreover, we have shown that, after trotting races, horses with high individual performance indexes have the highest concentration of lactate in their RBC (16). Although the lactate influx and the interindividual variation were similar in all age groups, we cannot, however, exclude the possible effects of training on the activity of carriers. Earlier studies on human subjects have found that training either has no influence (17) or enhances the transport of lactate from exercising muscle fibers (8, 9, 11, 12).

When all age groups were combined, the rate of lactate influx in mares was higher than in stallions. In the presence of the inhibitors CHC and DIDS, the situation was the same. On the basis of these results, it is not possible to explain the difference, but it can be speculated that either androgenic or estrogenic hormones may have an effect on lactate influx capacity. Previously, it has been shown that chronic estrogen administration to male rats can alter carbohydrate metabolism, including lactate production, and, further, it has been suggested that the transport of lactate could also be affected (10).

In summary, in standardbred horses, interindividual differences in the rate of lactate influx into the RBC are very large, and the role of different carriers is also variable. In the majority of the horses in our study, the main carrier of lactate on the RBC membrane is the MCT, the role of the band 3 system varies between 20 and 40%, and in a substantial number of horses the activity of both carriers is low. Individual variation is apparent in all age groups, and total transport activity is higher in mares than stallions.

Perspectives