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Substrate utilization by Rana ridibunda erythrocytes
Martha Kaloyianni and Katerina Moutou Laboratory of Animal Physiology, Zoology Department, Science School, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece
Various monosaccharides, including ribose, mannose, galactose, and urea, in combination with glucose, were studied to determine their efficacy in supporting the formation of pyruvate, lactate, 2,3-diphosphoglycerate and ATP in Rana ridibunda erythrocytes. Lactate formation was found to increase during the course of incubation in the presence of all the substrates. None of the studied substrates maintained cellular ATP levels. About 0.36 pmole of lactic acid per hour was produced for each/tmole of ribose that was metabolized. The presence of 1 mM Na-iodoacetate accelerated the loss of ATP and lactate in the presence of either glucose or ribose. Additionally, ouabain suppressed lactate formation from ribose alone, as well as in combination with glucose. From the metabolic substrates studied, ribose was shown to be the most efficient substrate to support Rana ridibunda erythrocyte metabolism. Mannose, galactose and urea may also be used as alternative metabolic substrates by Rana ridibunda erythrocytes.
Introduction It is well-established that mature mam- malian erythrocytes, for the most part, are glycolytically dependent structures and their survival depends upon a continuously available source of glucose (Rapoport, 1968; Kim and McManus, 1971; Harvey and Kaneko, 1976). However, it would be an oversimplification to suggest that erythrocytes are totally dependent upon glucose to maintain an adequately balanced intracellular pool of ATP. In addition to glucose the glycolytic pathway of the mam- malian erythrocyte can utilize alternative substrates including adenosine, inosine, ribose, deoxyribose, dihydroxyacetone,
Correspondence to: M. Kaloyianni, Laboratory of Animal Physiology, Zoology Department, Science School, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece.
Received 2 November 1993; accepted 2 February 1994.
glyceraldehyde and xylitol (Kim, 1990). Human red cells can also use galactose (Grimes, 1980) and mannose (Rapoport, 1968). On the other hand, adenine nucleo- sides have been found to constitute an alternative source of energy for pig erythro- cytes and they also activate glycolysis in human and cow erythrocytes (Kim, 1983; Kim, 1985). Erythrocytes from the pig, unlike other mammalian red cells, are unable to metabolize glucose and seem to rely on inosine as their main metabolic sub- strate (McManus and Kim, 1968; Watts et al., 1979; Young et al., 1986). Erythro- cytes of several fish, such as rainbow and brown trout and carp, can utilize other substrates as glycolytic intermediates, such as monocarboxylic acids, amino acids and substrates of the pentose-phosphate path- way via the TCA cycle for energy produc- tion (Bolis et al., 1971; Tse and Young, 1990;
Tiihonen and Nikinmaa, 1991). Also, urea, as an end-product of nitrogen metabolism, plays a significant role in the osmoregula- tion of erythrocytes in a number of fresh- water species (Janssens, 1964). However, the extent to which alternative energy sub- strates play a physiological role in vivo is not clear.
The mature nucleated avian erythrocyte seems to consume little glucose (Rosa et al., 1983; Kalomenopoulou and Beis, 1990), while the available information concerning the amphibian Rana ridibunda erythrocytes indicates that they also depend on glucose as well as on adenosine as predominant glycolytic substrates to maintain energy needs (Kaloyianni-Dimitriades and Beis, 1984; Kaloyianni et al., 1993). 2,3-DPG is found in the red cells of the frog but, unlike in mammalian erythrocytes, its concen- tration (0.3 mM) is 10 times less than that of ATP (Kaloyianni-Dimitriades and Beis, 1984). Additionally, there are no available data on the use of either monosaccharides or urea as possible alternative sources of energy in frog erythrocytes.
The present study reports the results of our investigation on the metabolic behaviour of frog erythrocytes suspended in various metabolic substrates. The efficacy of several monosaccharides, nucleosides and urea in supporting pyruvate and lactic production and ATP and 2,3-DPG for- mation in Rana ridibunda red cells was determined. In order to gain more insight into the metabolic fate of the metabolic substrate used, the effects of Na-iodo- acetate and ouabain were also studied. These alternative substrates could be used by frog red cells as "emergency substrates" during water stress or exercise.
Materials and Methods Animals
Frogs (Rana ridibunda) were supplied by a local dealer and were kept in con- tainers in the laboratory in fresh water; no signs of any pathological conditions were noticed. The animals were used a week after arrival.
Chemicals and enzymes
The substrates, enzymes and coenzymes were purchased from Sigma Chemical
Co. (St Louis, MO). All other chemicals were purchased either from Serva (Heidel- berg, Germany) or Merck (Darmstadt, Germany).
Sampling of red cells
Frogs were anaesthetized by immersion in 0.1 g/1 MS-222 for 5 min. Blood samples were obtained by heart puncture from anaesthetized frogs using heparinized syringes. Immediately after collection, the blood was centrifuged at 500g for 10 min and the plasma and the surface layer of white cells removed by aspiration. After- wards, the erythrocytes were suspended in 10 vol 0.1 M NaC1 three times. The osmo- larity of NaC1 used was the same as that of the amphibian plasma (200 mOsm/kg H20). Osmolarity was measured in a Fiske OS TM
osmometer.
Metabolic studies
All metabolic studies were performed with washed red cells drawn from three to five animals to give one pool of cells. The washed erythrocytes were subsequently suspended at 12-18% haematocrit, in an imidazole-glycylglycine buffer, pH 7.4, containing 100mM NaC1, 5 .9mM CaC12, 2.4 mM MgSO4, 4.2 mM imidazole, 7.6 mM glycylglycine, 1.2 mM KH2PO4 and 5 mM substrate. Previous results have shown that pH 7.4 promotes Rana ridibunda erythro- cyte glycolysis (Kaloyianni-Dimitriades, 1983) and so the pH of the incubation mixture was initially adjusted to 7.4 and no changes were noted during the incubation time. Two millilitres of erythrocyte suspen- sion were gassed with a mixture of 95% O2 and 5% CO2 and incubated in Erlenmeyer flasks (25 ml) fitted with a rubber cap. The flasks were incubated in a 25°C shaking water bath for 4 hr. Elapsed time between blood removal and time zero of incubation was about 1 hr. Aliquots were removed at the beginning and the end of the incubation time and immediately thereafter aliquots were assayed for adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), pyruvate and lactate contents.
Preparation of perchloric acid (PCA) extracts
Red cell metabolites were assayed in neutralized perchloric acid extracts. The
extracts were prepared by addition to the packed red cells of an equal volume of 70% perchloric acid followed by immediate mixing. After centrifugation at 4000g for 10min, the supernatant was neutralized with 0.5 M Tris-C1 in 0.5 M KOH. The precipitate of potassium perchlorate was removed by centrifugation at 4000g for 5 min. The supernatant was used for metab- olite determination.
Separation of intra- and extracellular phase
For the determination of intra- and extracellular ribose and glucose concen- trations, 0.7 ml of the incubated cell suspen- sion was withdrawn from the flasks at the beginning and the end of the incubation time and was gently layered over 0.35 ml of silicon oil (density 1.035g/cm 3) that had been layered on top of 0.15ml of 4% PCA in a 1.5-ml microcentrifuge tube. Tubes were immediately centrifuged for 1 min in an Eppendorf microcentrifuge. The top layer was subsequently withdrawn and deproteinized and was considered to be the extracellular fluid and contents (Kaloyi- anni and Freedland, 1990). The acid layer represented the intracellular contents of the cells with some (less than 5%) extracellular contamination (Zuurendonk and Tager, 1974).
Determination of intermediates, adenos&e triphosphate, lactate, ribose, mannose and urea
Glucose was estimated by the method of Krebs et al. (1964); 2,3-diphosphoglycerate (2,3-DPG) was estimated by the method of Michal (1974) whereas pyruvate was estimated by the method of Bucher et al. (1963); adenosine triphosphate (ATP) was estimated by the method of Lamprecht and Trautschold (1963); lactate was estimated by the method of Hohorst (1965); ribose by the method of Ceriotti (1955) and mannose by the method of Gawehn (1963). Urea was measured by the Biocon Diag- nostik Kit, based on the urease (Barthelot) reaction.
All metabolites were measured spectro- photometrically by using a Hitachi record- mg spectrophotometer and following the change in optical density at 340 nm caused by t h e oxidation or the reduction of NAD(H) or NADP(H). When necessary,
enzymatic systems coupled to NAD(H) or NADP(H) were used.
All data are expressed as m e a n + SEM. Statistical significance (P < 0.05) was assessed with Student's t-test applied to unpaired grouped samples. Changes in initial values or 0-4 hr values, as well as effects of additions after the 4-hr incubation time, were also statistically examined.
Results
The effect of monosaccharides on Rana ridibunda erythrocyte glycolysis was examined in Table 1. Ribose, mannose or galactose, and these monosaccharides in combination with glucose, were studied to determine their efficacy in supporting the formation of pyruvate and lactate, as well as ATP and 2,3-DPG in red cells. Both ribose and mannose have been detected in Rana ridibunda plasma. The levels of mannose (0.08 mM) are much lower than those of ribose (0.24 mM). Mannose and galactose appear to inhibit glucose metab- olism as measured by lactate production. Glucose seems to take part in erythrocyte metabolism but at a lower level when another monosaccharide is present at the same time. The highest production of lactate is that observed when glucose and ribose are present together in the incu- bation medium (Table 1). From the data of Table 1 it is evident that frog red cells contain 3/zmol/ml RBC of ATP, but are unable to maintain this level in the presence of any substrate tested. On the other hand, all monosaccharides significantly enhanced 2,3-DPG levels after the 4-hr incubation time.
In Table 2 the effect of the nitrogenous compounds, including nucleosides and urea on Rana ridibunda erythrocyte glycolysis, is shown. In the course of incubation in the absence of substrate, lactate was produced at a rate of 1.01 #mol/ml cells/hr. Inosine resulted in a similar lactate production as glucose alone (Table 2). Lactate formation of erythrocytes suspended in adenosine and glucose together is significantly higher (P < 0.05) than the lactate produced from adenosine alone (Table 2). Unlike the results seen for monosaccharides, incu- bation with adenosine resulted in a net synthesis of ATP (Table 2). A maximum
360 Martha Kaloyianni and Katerina Moutou
Table 1. Effect of monosaecharides on ATP 2,3-DPG, pyruvate and lactate levels in Rana ridibunda erythrocytes
Erythrocytes were incubated for 4 hr in the presence of 5 mmol/1 of the indicated substrates. The values correspond to the contents of the metabolites #mol/mt RBCs ± SEM at the end of incubation time, in 10 different experiments. Each experiment was conducted with three to five animals to give one pool of cells (N = 40). The initial contents were: 3.08 ± 0.04#mol/ml RBCs, ATP; 0.34 ___ 0.01 #mol/ml RBCs, 2,3-DPG; 0.14± 0.01/~mol/ml RBCs, pyruvate; 0.81 ± 0.04/~mol/ml medium, lactate.
ND corresponds to non-detectable value.
net synthesis of ATP, which can occur by adenosine either with or without glucose, was at least 40% higher than the ATP content seen in glucose alone. These findings are in accordance with those found in human erythrocytes after adenosine infusion (Kim, 1990). However, inosine, alone or together with glucose, did not result in the maintenance of ATP content.
Urea levels in Rana ridibunda plasma are considered high (16 mM) and possibly play a significant role in the osmoregulation of blood, as is the case in other aquatic species (Janssens, 1964). Nevertheless, urea does not seem to maintain ATP levels or lactate production by frog erythrocytes,
since ATP and lactate levels are similar to those produced in the absence of substrates (Table 2).
In order to determine the number of moles of lactate produced per mole of sugar metabolized, the loss of ribose was measured (Table 3). Table 3 shows pentose consumption and lactate production from Rana ridibunda erythrocytes. The methods used for sugar estimation did not dis- tinguish between the free sugar and sugar in the form of an ester or a nucleoside. The loss of ribose represents the difference between the total amount of sugar before (0.037mmol ribose/ml RBC) and after incubation in the extracellular compart- ment. The molar ratios of the lactate
Table 2. Effect of nucleosides and urea on ATP, 2,3-DPG, pyruvate and lactate levels in Rana ridibunda erythrocytes
Erythrocytes were incubated for 4 hr in the presence of 5 mmol/l of the indicated substrates. The values correspond to the contents of the metabolites/~mol/mi RBCs _ SEM at the end of incubation time, in 10 different experiments. Each experiment was conducted with three to five animals to give one pool of cells (N = 40).
ND corresponds to non-detectable value. *Results taken from Kaloyianni et al. (1993).
Erythrocytes were incubated for 4 hr in the presence of 5 mmol/I of the indicated substrates. Three experiments were conducted with three to five animals to give one pool of cells (N = 9).
361
produced to sugar metabolized show that almost 1.43 moles of lactate were produced for each mole of ribose metabolized, the ratio being 1.86 for inosine and 2.4 for adenosine. The theoretical yield of lactate from ribose is 1.6 (Prankerd, 1956). Note that free ribose is consumed more rapidly than ribose bound in the nucleoside. On the other hand, the ratio of lactate produced to ribose consumed is lower for free ribose than for bound. The findings are in accordance with the suggestion that the greater part of ribose arising from adenosine may be metabolized via the glycolytic pathway while the greater part of free ribose seems to produce other com- pounds such as hexose phosphates and 2,3-DPG (Prankerd, 1956). The increased intracellular concentration of ribose in the presence of either inosine or adenosine (data not shown) is an indication of the permeability of Rana ridibunda erythrocyte membrane to the latter nucleosides which,
in turn, may serve to support erythrocyte glycolysis.
Na-iodoacetate is known to inhibit the glycolytic pathway by inhibiting glyceralde- hyde 3-phosphate dehydrogenase (Lew and Ferreira, 1978). The presence of Na- iodoacetate in the medium accelerated the loss of ATP in the presence of glucose, while lactate production from glucose was one-fifth of that produced in the absence of the inhibitor (Table 4). These results corroborate the findings that glucose is a significant metabolic substrate for Rana ridibunda erythrocyte glycolysis. Ribose, together with Na-iodoacetate, on the other hand, significantly reduced the production of lactate and ATP from Rana ridibunda erythrocytes to a much lower extent when compared with glucose.
Table 5 shows the effect of ouabain on ATP, 2,3-DPG, pyruvate and lactate production of Rana ridibunda erythrocytes. Ouabain inhibits active cation transport
Table 4. Effect of Na- iodoaceta te on ATP, 2 ,3-DPG, pyruvate and lactate levels in Rana ridibunda erythrocytes
Erythrocytes were incubated for 4 hr in the presence of 5 mmol/1 of the indicated substrates and 1 mmol/1 Na-iodoacetate . The values cor respond to the contents of the metaboli tes /~mol/ml RBCs + SEM at the end of the incuba t ion t ime in five different experiments. Each experiment was conducted with three to five animals to give one pool of cells (N = 20).
N D corresponds to non-detectable value.
CBPB 108/3~G
362 Martha Kaloyianni and Katerina Moutou
Table 5. Effect of ouaba in on ATP, 2,3-DPG, pyruvate and lactate levels in Rana ridibunda erythrocytes
Erythrocytes were incubated for 4 hr in the presence of 5 mmol/! of the indicated substrates and I mmol/ l ouabain. The values correspond to the contents of the metabol i tes /~mol /ml RBCs _ SEM at the end of the incubat ion t ime in five different experiments. Each experiment was conducted with three to five animals to give one pool of cells (N = 20).
(Schatzmann, 1953) and affects the activity of the glycolytic enzyme complex (Fossel and Solomon, 1978). The results show (Table 5) that ouabain suppressed lactate formation from glucose and ribose as well as from ribose and glucose together. How- ever, the levels of ATP and 2,3-DPG are not significantly (P < 0.05) affected by the presence of ouabain (Table 5).
Discussion
The findings reported herein support the theory that Rana ridibunda erythrocytes utilize a variety of metabolic substrates. The studied substrates could play a signifi- cant role in the energetics of Rana ridibunda erythrocytes, since they could be utilized in stress conditions such as during exercise or water stress. Previous studies indi- cated that Rana ridibunda erythrocytes metabolize glucose through the Embden- Meyerhof pathway (EMP) while they prob- ably are deprived of the oxidative enzymes of Krebs cycle. Electron microscopy obser- vations showed that frog erythrocytes have only one or two mitochondria per cell and there was no detectable activity of any of the TCA cycle enzymes measured (Kaloyianni-Dimitriades and Beis, 1984; Kaloyianni and Kalomenopoulou, 1990).
In mammals, the mature red cells are solely dependent upon glycolytic carbon flow for their energy metabolism, yet glucose-impermeable cells are still present. The pig is a case in point (Watts et al., 1979). However, this peculiarity relates to glucose in permeability as the characteristic feature confined only to the adult stage (Zeidler et al., 1976). The nucleated
erythrocytes of teleost fish demonstrate a wide range of diversification in glucose consumption. Erythrocytes of yellow perch Perca flavens (Bachand and Leray, 1975), rainbow trout Oncorhynchus mykiss (Ferguson and Storey, 1991), lungfish and electric eel (Kim and Isaacks, 1978) and Anguilla japonica (Tse and Young, 1990) are able to take up and utilize glucose. On the other hand, red cells of Salmo gairdneri (Tse and Young, 1990), brown trout Salmo trutta (Bolis et aL, 1971) and carp Cyprinus carpio (Tiihonen and Nikinmaa, 1991), are barely permeable to glucose and probably utilize a variety of alternative substrates as metabolic fuels.
Since frog red cells have a nucleus they may utilize compounds that could act either singularly or synergistically as meta- bolic substrates for the red cells. It should be recalled that glucose alone, despite a consumption rate of 0.76pmol/ml red cell/hr (Kaloyianni and Kalomenopoulou, 1990), cannot fully support cellular ATP levels in the frog erythrocyte (Table 1). The combination of glucose and ribose yielded lactate production of 12.7 pmol/ml cells which is higher than that produced by either ribose (8.00) or glucose alone (9.00), yet ATP levels in glucose plus ribose media were less than those found in either substrate alone. The reason for this inconsistency is not known. A possible explanation could be that the presence of both substrates may stimulate ion move- ments across the red cell membrane, e.g. Na-K-ATPase and, as a result, the con- sumption of ATP. Also, the experimental conditions could influence ATP levels as, for example, in the erythrocytes of
Metabolism of frog erythrocytes 363
the opossum, where ATP levels are not maintained unless the incubations are held in an N2 atmosphere (Bethlenfalvay et aL, 1990).
Neither mannose nor galactose main- tained the ATP level in frog erythrocytes (Table 1). Lactate production from the latter monosaccharide did not seem to result from glycolysis since the lactate pro- duced was almost the same amount as that produced in the absence of substrate. How- ever, the detection of mannose (0.08 mM) in Rana ridibunda plasma suggests a poss- ible physiological role of this sub- strate under stress conditions. Additionally, mannose isomerase has been detected in Rana ridibunda erythrocytes (Kaloyianni- Dimitriades and Beis, 1984). Galactose in mammalian erythrocytes, other than hu- man, is not metabolizable due to the fact that it is not a proper substrate for hexo- kinase (Kaneko, 1974). A similar expla- nation could be made in the case of Rana ridibunda erythrocytes.
Lactate production from cells incubated with ribose, together with glucose, was found to be 1.5 times greater than lactate production in the presence of glucose alone, but none of the monosaccharides main- tained the levels of ATP (Table 1). Rana ridibunda red cells may utilize ribose; its role as a metabolic substrate in vivo may be significant. Ribose has been detected in frog plasma (0.28 mM). Therefore, in Rana ridibunda erythrocytes, a critical balance must exist between supply and utilization of energy substrate and the erythrocytes must be able to take up substrate from the circulating blood. A similar suggestion has been made for pig erythrocytes where ribose concentration in plasma is unable to sustain ATP levels (Kim and McManus, 1971). In addition, Gercken and Hinzen (1965) have also confirmed that ribose can maintain ATP levels in rabbit red cells. Pirarucu and pig erythrocytes are per- meable to ribose, unlike glucose, and utilize ribose to form lactate (Kim and Isaacks, 1978). Of mammalian species, guinea-pig red cells have the highest affinity for ribose, whereas human red cells utilize little ribose as a metabolic substrate. (McManus, 1974). In hurnan red cells, it has also been reported (Jaffe, 1959) that r ibose promotes the reduction of methemoglobin.
The fact that, in the presence of ribose together with glucose, the lactate produced is several-fold greater than that produced in the presence of either mannose or galactose together with glucose, could be attributed to the greater permeability of the cells to ribose compared to mannose and galactose and this permeability difference could ac- count for the different structure of ribose (pyranose ring) in relation to the other monosaccharides tested (Table 1). Simi- larly, in human erythrocytes, monosacchar- ides are transported by diffusion, facilitated by a cell-membrane protein exhibiting a high degree of specificity for pentoses and hexoses, whose structure contains a pyra- nose ring (Le Fevre, 1961). Furthermore, the 20-times higher concentration of ribose in Rana ridibunda plasma than that of mannose may indicate the high demands of erythrocytes for ribose.
Ribose, upon its entry in the frog ery- throcyte, may follow either the glycolytic pathway or, alternatively, may be metab- olized through the pentose-phosphate pathway either at the fructose-6-P or the triose step. Similarly, it has been suggested for pig erythrocytes that the pentoses are initially phosphorylated by ATP under the influence of ribokinase (McManus and Kim, 1968). The fact that the percentage inhibition of lactate and ATP production after incubation of Rana ridibunda ery- throcytes with ribose, together with the inhibitor Na-iodoacetate, is 37 and 17% respectively, while they are inhibited 87 and 37%, respectively, when glucose is also present (Table 4), may indicate that the greater part of ribose upon its entry to the erythrocyte may be metabolized through the pentose-phosphate pathway.
Small polar molecules such as urea are believed to share a common leak pathway in the red cells. The red cells of the giant salamander, Amphiuma means, were found to have a high urea permeability (Wieth and Brahm, 1977) and the urea transport system can be very tight to water. Kaplan et al. (1974) observed the presence of both a simple diffusion and a facilitated diffusion across the red cells in vertebrates from hagfish to man, and suggested that the facilitated diffusion of urea across the red cell appears to have developed with the emergence to a terrestrial life. Additionally,
364 Martha Kaloyianni and Katerina Moutou
the red cells of the Amazon fish, with a high urea permeability, play a key role in the adaptation of the lungfish to the hostile environment (Kim and Isaacks, 1978). In frog red cells, pyruvate, lactate production and ATP maintenance in the presence of urea are similar to those produced in the absence of substrate (Table 2). Further- more, the significantly increased lactate production in the presence of both glucose and urea may be attributed to glucose rather than to urea. The high urea levels in frog plasma could serve either as an emergency substrate, during diving or stress conditions, or as an osmoregulator of the blood cells, as is the case in other aquatic species (Kim and Isaacks, 1978).
F rom the increase in lactate and pyruvate production and the synthesis of ATP when the red cells of Rana ridibunda were incubated in the presence of adenosine (Table 2), we could suggest that adenosine plays a significant role in frog erythrocyte metabolism (Kaloyianni et al., 1993). Fur- thermore, frog erythrocytes, like those of the chicken (Espinet et al., 1989) possibly possess an enzyme system for the conver- sion of adenosine to inosine and, sub- sequently, the cleavage of inosine to hypoxanthine and ribose 1-P which, in turn, would provide glycolytic intermediates. In point of fact, adenosine has already been cast for a number of possible physiolog- ical roles such as a direct precursor of erythrocyte adenine nucleotides (Lerner and Lowy, 1974) and as a modula tor serv- ing to enhance coronary blood flow (Berne, 1963).
Regulation of metabolism may be related to active ion transport. Whit tam and Agar (1965) suggested that part of glyco- lysis and respiration is regulated by active ion transport at a percentage of 25-75%, depending on the cell type. Energy pro- duction by glycolysis may be closely coupled to energy consumption by the sodium pump. In Rana ridibunda erythro- cytes, glucose and ribose uptake and metab- olism may also be coupled to active ion influx or N a - K - A T P a s e since lactate pro- duction in the presence of ribose and glu- cose is inhibited by ouabain. However, the levels of 2 ,3-DPG were unchanged by ouabain, suggesting that this pathway is unaffected by the inhibitor (Table 5).
In conclusion, the results of the present study corroborate that glucose is a possible substrate for frog Rana ridibunda erythro- cyte glycolysis. It is shown that ribose, adenosine and, to a lesser extent, inosine, mannose and urea may be alternative sub- strates for the erythrocytes, indicating a metabolic flexibility which is consistent with the occasionally increased needs of the erythrocytes to respond either to the oxi- dant challenge or to environmental stress conditions.
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