The present study shows that the capacity of trout (Salmo trutta) red blood cells (RBCs) and freshly isolated hepatocytes to take up short-chain neutral amino acids changes according to a seasonal pattern. Maximal amino acid uptake rates in RBCs were obtained in winter and spring, while minima were seen in summer and autumn. In contrast, the maximal rates for the freshly isolated hepatocytes were obtained in autumn and winter, and the minima were seen in spring and summer.

In addition, by studying the uptake of glycine, evidence was found that the activities of the amino acids carriers ASC, asc and Gly in RBCs varied according to a seasonal rhythm. The activity of the ASC and asc systems changed in parallel with the global uptake of amino acids.

Moreover, the RBC:plasma concentration ratio for certain substrates of these carriers (alanine, serine and glycine) varied accordingly. In contrast, the activity of the Gly system was modified inversely with respect to the overall amino acid uptake.

The activity of the ASC system in freshly isolated hepatocytes was also seasonally modified, reaching a maximum in autumn, shortly before the reproductive period.

Poikilotherm organisms show seasonal changes in a variety of physiological parameters. Most studies have been carried out at the level of the whole organism; for example, by measuring changes in plasma hormone levels in various species of fish (Scott et al. 1980, 1983; Breton et al. 1983; Temma et al. 1990; Navarro et al. 1991; Rinchard et al. 1993), seasonal variation in their haematology (Dewilde and Houston, 1967; Haider, 1970; Denton and Yousef, 1975; Lane, 1979; Härding and Höglund; 1984; Pickering, 1986) and changes in membrane composition (Adams et al. 1973; Rady, 1993). However, a few studies have examined changes in various functional parameters, such as the activity of the Na+/H+ antiporter in trout red blood cells (Cossins and Kilbey, 1989) or the activity of Na+/K+-ATPase in carp (Rady, 1993) or trout (Gabbianelli et al. 1996) erythrocytes.

The transport of neutral amino acids into trout cells is effected by several distinct systems that have overlapping substrate specificities. Several of these carriers, including the ASC and Gly systems, are Na+-dependent (i.e. they use the Na+ transmembrane gradient as a driving force); others, such as the L and asc systems, are Na+-independent. Both the ASC and asc systems carry short-chain neutral amino acids, with L-cysteine being a good substrate for these carriers. The ASC system is the only Na+-dependent system involved in L-alanine uptake by trout hepatocytes; the A system is not present (Canals et al. 1992). Glycine and sarcosine are the substrates of the Gly system, since the β system does not carry glycine in trout RBCs (Canals et al. 1992; Gallardo et al. 1992, 1996a; Gallardo and Sánchez, 1993; Albi et al. 1994). The activities of some Na+-dependent uptake systems for short-chain neutral amino acids and the activities of Na+-independent systems were measured during two consecutive years in both RBCs and freshly isolated hepatocytes. The results indicate that the activities of both systems vary according to a seasonal pattern.

Animals and chemicals

Brown trout Salmo trutta L. (250–400 g) were obtained from fish farms (Medi Natural, Generalitat de Catalunya) in the Pyrenees, where they were maintained in open-water circuits, directly connected to a river. When fasting animals were used, they were deprived of food for 30 days before experiments were carried out.

All chemicals were of analytical grade. L-[U-14C]serine was obtained from Sigma Co. (St Louis, USA) while [2-3H]glycine and L-[3-3H]alanine were obtained from New England Nuclear (Dreieich, Germany).

Collection of red blood cells

Blood was obtained by caudal puncture from unanaesthetized fish and diluted with heparinized RPMI 1640 (Sigma Co., St Louis, USA), rinsed several times, and left at 4 °C to eliminate any possible catecholamine effect. Both white blood cells, which have a high metabolic activity, and thrombocytes were removed by centrifugation with Histopaque-1077 (Sigma Co., St Louis, USA), following the procedure suggested by the supplier with slight modifications to overcome the high viscosity of trout blood. Once separated, the RBCs were rinsed four times with slightly modified Cortland buffer (Houston et al. 1985): (in mmol l−1) NaCl, 141; KCl, 3.5; MgSO4, 1; NaH2PO4, 3; CaCl2, 1; pyruvic acid, 2;

Hepes, 10; glucose, 3; bovine serum albumin, 0.3 %; pH 7.4. The osmolality was adjusted to 305 mosmol kg−1 with a micro-osmometer (model 3MO, Advanced Instruments, Inc., USA). In some instances, K+ was used instead of Na+ as the main extracellular cation (all Na+ was replaced by K+); the entire rinsing procedure was performed using the final buffer.

Isolation of hepatocytes

Trout were anaesthetized in NaHCO3-buffered MS222 (in mmol l−1: NaHCO3, 2.4; MS222, 0.38), and cells were obtained essentially as described by French et al. (1981), except that hyaluronidase was omitted and 5 mmol l−1 rather than 25 mmol l−1 NaHCO3 was used, because of the difference in the gas mixture used to equilibrate all solutions (99.5 % O2:0.5 % CO2) before use. Final pH was adjusted to 7.6. The osmolality was adjusted to 305 mosmol kg−1. Cell viability was routinely assessed using the Trypan Blue exclusion test, and more than 95 % viability was obtained. Cell integrity over time was assessed by measuring lactate dehydrogenase (LDH) release, and less than 5 % of the initial intracellular LDH level was found in the medium after 6 h of incubation.

Following isolation, cells were suspended in Hanks’ solution (in mmol l−1): NaCl, 120; KCl, 5; MgSO4, 1; CaCl2, 2; Na2HPO4, 0.44; KH2PO4, 0.44; NaHCO3, 5; Hepes, 10; glucose, 5; fatty-acid-free bovine serum albumin, 2 %. Final pH was adjusted to 7.6. The osmolality was adjusted to 305 mosmol kg−1. When K+ was used instead of Na+ as the main extracellular cation, no loss of cell viability was observed.

Uptake of 14C-or 3H-labelled amino acids

For uptake experiments, both cells and solutions were pre-equilibrated at 15 °C. Experiments were started by mixing (1:1 v/v) the cell suspension with the labelled amino acid (14C, 1.5 Bq ml−1 cells; 3H, 11.1 Bq ml−1 cells) to obtain the desired concentrations. Incubations were performed in a shaking bath at 15 °C, using air as the atmosphere for red blood cells and, because of their high O2 requirements, a 99.5 % O2:0.5 % CO2 gas mixture for the hepatocytes. A transaminase inhibitor, amino-oxyacetic acid (2 mmol l−1), was used throughout the uptake experiments. The uptake of glycine and L-serine by RBCs and the uptake of L-alanine by freshly isolated hepatocytes was linear for 15 min (Canals et al. 1992; Gallardo et al. 1992; Gallardo and Sánchez, 1993), while the uptake of L-alanine by RBCs was linear for only 3 min (Albi et al. 1994). RBC suspensions were incubated for 10 min to measure glycine and L-serine uptake or for 2 min to measure L-alanine uptake before the incubation was stopped by dilution with Cortland buffer. Freshly isolated hepatocytes were incubated for 10 min before the uptake was stopped by dilution with Hanks’ solution. Cells were rinsed with these solutions and centrifuged (1000 g, 8 min, 4 °C) three times. Erythrocytes were deproteinized by adding a sufficient amount of ice-cold perchloric acid to obtain a final concentration of 6 %, while hepatocytes were lysed with 0.1 % Triton X-100. A clear supernatant was obtained by centrifugation (1825 g, 20 min, 4 °C). The activity of this supernatant was counted in a well-type liquid scintillation counter (Packard, UK).

RBC haemoglobin concentration was determined by means of Drabkin reagent (525-A, Sigma Co., St Louis, USA). Cell protein was determined by means of the Coomassie Blue technique (BioRad, Bradford reagent).

Nonlinear regression analysis was used to fit curves to the experimental data (SigmaPlot 2.1 and InPlot4). Parametric and non-parametric statistics were carried out by means of a computerized package (SPSS, Chicago, IL, USA).

Measurement of amino acid concentrations

Plasma and whole-blood amino acids were analyzed by ion-exchange chromatography, as described previously (Canals et al. 1992). The red blood cell amino acid concentration (RBC[aa]) was calculated according to the following relationship:
formula
,

where WB[aa] is the whole-blood amino acid concentration, Hct is the whole-blood haematocrit and P[aa] is the plasma amino acid concentration.

Fig. 1 shows the initial rates of glycine uptake by RBCs during the year, of L-alanine over a 6 month period and of L-serine over a 5 month period. Values varied according to a seasonal pattern, showing maximal activities in winter and spring and minima between summer and autumn. These data also show that Na+-dependent uptake of glycine accounts for most of the seasonal change in activity.

Fig. 1.

Seasonal variation in the initial rates of short-chain amino acid uptake by trout red blood cells (RBCs). Cells were suspended in buffers containing Na+ (open symbols) or K+ (filled symbols) as the main cation and were incubated for 10 min (glycine and L-serine) or for 2 min (L-alanine) before the uptake was stopped as described in Materials and methods. (○, •) 500 μmol l−1 glycine uptake. (□ ) 150 μmol l−1 L-serine uptake. (▿) 150 μmol l−1 L-alanine uptake. Each point is the mean ± S.D. of 3–6 individual experiments. Rough curves have been drawn to indicate the pattern of seasonal variation. Hb, haemoglobin.

Fig. 1.

Seasonal variation in the initial rates of short-chain amino acid uptake by trout red blood cells (RBCs). Cells were suspended in buffers containing Na+ (open symbols) or K+ (filled symbols) as the main cation and were incubated for 10 min (glycine and L-serine) or for 2 min (L-alanine) before the uptake was stopped as described in Materials and methods. (○, •) 500 μmol l−1 glycine uptake. (□ ) 150 μmol l−1 L-serine uptake. (▿) 150 μmol l−1 L-alanine uptake. Each point is the mean ± S.D. of 3–6 individual experiments. Rough curves have been drawn to indicate the pattern of seasonal variation. Hb, haemoglobin.

Fig. 2 shows the effect of season on the time course of the Na+-dependent uptake of 150 μmol l−1 L-alanine, indicating that there were differences in both the initial rates, as shown in Fig. 1, and the intracellular concentration at equilibrium at different times of the year. Furthermore, Fig. 3 and Table 1 show that the activity of the Na+-dependent ASC system, measured through the uptake of glycine, was modified according to a seasonal pattern and showed maximal activities in winter. In contrast, as shown in Fig. 4 and Table 1, the Gly system showed maximal activity in summer. Most of these changes can be attributed to the maximal capacity for uptake (Vmax) since there were no significant changes in the affinities of these carriers (Km values).

Table 1.

Seasonal variation in the kinetic constants for the ASC and Gly systems in trout red blood cells

Seasonal variation in the kinetic constants for the ASC and Gly systems in trout red blood cells
Seasonal variation in the kinetic constants for the ASC and Gly systems in trout red blood cells
Fig. 2.

Seasonal variation in the time course of 150 μmol l−1 L-alanine uptake through the ASC system in trout RBCs. The amino acid uptake was followed for different times at 15 °C in either a Na+-containing medium or a Na+-free medium in which Na+ was replaced by K+ to give a measurement of Na+-independent uptake. The uptake through the ASC system was measured as the Na+-dependent uptake minus the Na+-dependent uptake in the presence of 5 mmol l−1 of L-cysteine (used as inhibitor, Gallardo et al. 1992; Gallardo and Sánchez, 1993). The experiments were carried out in December (○), April (□) and July (▵). Each point is the mean ± S.D. of three individual experiments.

Fig. 2.

Seasonal variation in the time course of 150 μmol l−1 L-alanine uptake through the ASC system in trout RBCs. The amino acid uptake was followed for different times at 15 °C in either a Na+-containing medium or a Na+-free medium in which Na+ was replaced by K+ to give a measurement of Na+-independent uptake. The uptake through the ASC system was measured as the Na+-dependent uptake minus the Na+-dependent uptake in the presence of 5 mmol l−1 of L-cysteine (used as inhibitor, Gallardo et al. 1992; Gallardo and Sánchez, 1993). The experiments were carried out in December (○), April (□) and July (▵). Each point is the mean ± S.D. of three individual experiments.

Fig. 3.

Seasonal variation of ASC system activity in trout RBCs. The concentration-dependence of the rate of glycine uptake through the ASC system was followed in winter (○) and in summer (□). Cells were suspended in buffers containing Na+ or K+ as the main cation and were incubated for 10 min before the uptake was stopped as described in Materials and methods. The uptake through ASC system was measured as in Fig. 2. Each point is the mean ± S.D. of three individual experiments.

Fig. 3.

Seasonal variation of ASC system activity in trout RBCs. The concentration-dependence of the rate of glycine uptake through the ASC system was followed in winter (○) and in summer (□). Cells were suspended in buffers containing Na+ or K+ as the main cation and were incubated for 10 min before the uptake was stopped as described in Materials and methods. The uptake through ASC system was measured as in Fig. 2. Each point is the mean ± S.D. of three individual experiments.

Fig. 4.

Seasonal variation of Gly system activity in RBCs. The concentration-dependence of glycine uptake through the Gly system was followed in winter (○) and in summer (□ ). Initial rates were measured in the presence of buffers containing Na+ or K+ as the principal cation. Cells were incubated for 10 min before the uptake was stopped. The uptake through the Gly system was measured as the Na+-dependent uptake minus the Na+-dependent uptake in the presence of 5 mmol l−1 sarcosine used as a specific inhibitor of the Gly system (Gallardo and Sánchez, 1993). Each point is the mean ± S.D. of three individual experiments.

Fig. 4.

Seasonal variation of Gly system activity in RBCs. The concentration-dependence of glycine uptake through the Gly system was followed in winter (○) and in summer (□ ). Initial rates were measured in the presence of buffers containing Na+ or K+ as the principal cation. Cells were incubated for 10 min before the uptake was stopped. The uptake through the Gly system was measured as the Na+-dependent uptake minus the Na+-dependent uptake in the presence of 5 mmol l−1 sarcosine used as a specific inhibitor of the Gly system (Gallardo and Sánchez, 1993). Each point is the mean ± S.D. of three individual experiments.

As shown in Fig. 1, the Na+-independent uptake of glycine also seemed to be influenced by season. However, the actual amount of amino acid taken up was low, and a clear pattern did not emerge. However, as shown in Fig. 5 for the Na+-independent mediated uptake of L-alanine, differences did exist.

Fig. 5.

Seasonal variation in the capacity of trout RBCs to concentrate L-alanine through the asc system. Cells were suspended in a Na+-free buffer and incubated for different times in the presence of 150 μmol l−1 L-alanine. The uptake through the asc system was measured as the Na+-independent uptake minus the Na+-independent uptake in the presence of 5 mmol l−1 of L-cysteine. The experiments were carried out in December (○), April (□) and July (▵). Each point is the mean ± S.D. of three individual experiments.

Fig. 5.

Seasonal variation in the capacity of trout RBCs to concentrate L-alanine through the asc system. Cells were suspended in a Na+-free buffer and incubated for different times in the presence of 150 μmol l−1 L-alanine. The uptake through the asc system was measured as the Na+-independent uptake minus the Na+-independent uptake in the presence of 5 mmol l−1 of L-cysteine. The experiments were carried out in December (○), April (□) and July (▵). Each point is the mean ± S.D. of three individual experiments.

To determine the relationship between these seasonal rhythms in amino acid concentration in RBCs and the in vivo amino acid levels in plasma, the plasma concentrations of L-alanine, L-serine and glycine were measured. The plasma levels of all three amino acids remained almost constant through the year except in the reproductive season (late autumn), when there was a drop in their concentration (P⩽0.01; data not shown). However, the ratio between amino acid concentrations in RBCs and in plasma followed a seasonal pattern similar to that obtained for the activities of the ASC system and probably also to the activity of the asc system (Fig. 6).

Fig. 6.

Seasonal variation in values for the RBC:plasma concentration ratio of L-alanine (A), L-serine (B) and glycine (C). Amino acid concentrations in RBCs and plasma were calculated as described in Materials and methods. Results are expressed as a percentage of the maximum value for RBC amino acid concentration:plasma amino acid concentration. Each point is the mean ± S.E.M. of 3–6 individuals.

Fig. 6.

Seasonal variation in values for the RBC:plasma concentration ratio of L-alanine (A), L-serine (B) and glycine (C). Amino acid concentrations in RBCs and plasma were calculated as described in Materials and methods. Results are expressed as a percentage of the maximum value for RBC amino acid concentration:plasma amino acid concentration. Each point is the mean ± S.E.M. of 3–6 individuals.

To assess whether the changes shown above were restricted to RBCs or could be found in other cells, the initial rates of alanine uptake by isolated hepatocytes were measured throughout the year. The results shown in Fig. 7 indicate that there was a seasonally dependent change, but the pattern appeared to be different from that obtained for RBCs. Maximal values for the initial uptake rates were obtained in September, while minimum values were obtained during March and April.

Fig. 7.

Seasonal variation in the initial rates of L-alanine uptake through the ASC system by freshly isolated hepatocytes. The amino acid uptake was followed for 10 min at 15 °C in either a Na+-containing or a Na+-free medium. The rate of uptake by the ASC system was measured as described in Fig. 2. Results are expressed as the mean ± S.D. for N=3–6 individual experiments. A rough curve hase been drawn to indicate the pattern of seasonal variation.

Fig. 7.

Seasonal variation in the initial rates of L-alanine uptake through the ASC system by freshly isolated hepatocytes. The amino acid uptake was followed for 10 min at 15 °C in either a Na+-containing or a Na+-free medium. The rate of uptake by the ASC system was measured as described in Fig. 2. Results are expressed as the mean ± S.D. for N=3–6 individual experiments. A rough curve hase been drawn to indicate the pattern of seasonal variation.

The ability of fasting fish to modify their amino acid uptake capacity also changed during the year. Fig. 8 shows the concentration-dependence of the initial rate of L-alanine uptake by hepatocytes in experiments carried out in spring and in autumn. In autumn, fasting fish showed both a higher rate of uptake of L-alanine than in spring and a greatly enhanced amino acid uptake ability compared with fed animals. Unlike the variation in alanine uptake seen in RBCs, seasonal changes in hepatocyte alanine uptake seem to involve changes in both Km and Vmax (P ⩽0.01; Table 2).

Table 2.

Influence of seasonal variation and food deprivation on the kinetic constants for the ASC system in freshly isolated hepatocytes

Influence of seasonal variation and food deprivation on the kinetic constants for the ASC system in freshly isolated hepatocytes
Influence of seasonal variation and food deprivation on the kinetic constants for the ASC system in freshly isolated hepatocytes
Fig. 8.

Seasonal variation of the influence of food deprivation on the activity of the ASC system in freshly isolated trout hepatocytes. The concentration-dependence of L-alanine uptake through the ASC system in fed (○) and fasting (□ ) animals in spring (A) and in autumn (B) was measured. Note the change in scale. The rate of the ASC system was measured as described in Fig. 2. Each point is the mean ± S.D. of three individual experiments.

Fig. 8.

Seasonal variation of the influence of food deprivation on the activity of the ASC system in freshly isolated trout hepatocytes. The concentration-dependence of L-alanine uptake through the ASC system in fed (○) and fasting (□ ) animals in spring (A) and in autumn (B) was measured. Note the change in scale. The rate of the ASC system was measured as described in Fig. 2. Each point is the mean ± S.D. of three individual experiments.

The considerable variation in environmental conditions to which fish are subjected during the year modifies both their behaviour and their physiology. From the present data, it can be concluded that the amino acid uptake ability of at least two carrier systems in trout red cells is also modified according to a seasonal pattern. Whether these changes are due to modifications in the physico-chemical environment of the carriers in the cell membrane, as has been suggested for the Na+/K+-ATPase (Gabbianelli et al. 1996), or whether they are related to biological variables, such as the reproductive cycle, requires further clarification.

The results from hepatocytes can be interpreted through the key role played by these cells in the overall metabolism of the animal. The spawning period for brown trout takes place in November and December and is preceded by an increase in the work of the liver through its involvement in protein synthesis for gonadal maturation. Thus, the up-regulation of the ASC system in hepatocytes can be correlated with the onset of anabolic activity for reproduction. Moreover, the increase in this activity and the greater response to fasting in the experiments carried out in autumn than in those conducted in spring may be related either to the natural fasting period experienced by these animals at this time or to the different hormonal environment at different times of the year.

It has been shown that insulin stimulates the activity of the ASC system in trout hepatocytes (Canals et al. 1995), while glucagon has a complex effect, showing either a stimulatory or an inhibitory action according to a seasonal pattern (Gallardo et al. 1996b). It is known that plasma steroid levels also change seasonally in this species (Scott et al. 1980, 1983; Breton et al. 1983; Rinchard et al. 1993), leaving open the possibility that these hormones may modify the activity of amino acid carriers on hepatocytes.

A mechanistic explanation for the results obtained in RBCs is more difficult because of the apparently low metabolic activity of these cells. Changes in erythropoiesis have been suggested as a possible explanation for the seasonal variations observed in Na+/H+ antiporter activity in trout red blood cells (Cossins and Kilbey, 1989), but this has yet to be verified. It is possible that our results might be matched with changes in erythropoietic activity, at least for the ASC system. If the formation of new cells stops in winter, the fish would have an older population of the cells at the end of winter/start of spring followed by a sudden increase in the number of newly formed cells. In the fish studied here, haematocrit was fairly constant, except in spring when a significant rise occurred (34 % in February versus 41 % in April/May; results not shown). If this rise in haematocrit is due to the release of a new cell population, then the variation in the rate of uptake of amino acids could be correlated with the age of circulating RBCs.

This work was supported in part by grants from the DGICYT (PB86-0054 and PB91-0235) of the Spanish Government and by grants from the CIRIT (AR90-3.3394 and AR91-21). M.A.G. is the recipient of a fellowship from the Generalitat de Catalunya. Free plasma amino acid separations were carried out by Pilar Fernández and Isidre Casals from the Serveis Científico-Tècnics de la Universitat de Barcelona. We would like to express our sincere thanks to Mrs Rosa Marçol and to Mr Antonino Clemente (Medi Natural, Generalitat de Catalunya) for their help and logistical assistance and to Robin Rycroft for his editorial help.

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