Changes in lactate dehydrogenase and malate dehydrogenase activities during hypoxia and after temperature acclimation in the armored fish, Rhinelepis strigosa (Siluriformes, Loricariidae)

Mudanças na atividade da lactato desidrogenase e malato desidrogenase durante hipóxia e após aclimatação a diferentes temperaturas no cascudo, Rhinelepis strigosa (Siluriformes, Loricariidae)

Abstracts

Foram estudadas a lactato desidrogenase (LDH) e malato desidrogenase (MDH) de músculo branco de peixes aclimatados a 20, 25 e 30°C em diferentes concentrações de substrato e submetidos à hipóxia. Diferenças significativas em atividade enzimática para LDH e MDH são descritas aqui pela primeira vez em Rhinelepis strigosa em peixes aclimatados a 20°C e submetidos à hipóxia por seis horas. A LDH apresentou uma diminuição significativa na afinidade enzimática ao piruvato em hipóxia severa de peixes aclimatados a 20°C e um aumento significativo na afinidade enzimática ao piruvato em peixes aclimatados a 30°C.

hipóxia; aclimatação à temperatura; lactato desidrogenase; malato desidrogenase; cascudo; Rhinelepis strigosa

Changes in lactate dehydrogenase and malate dehydrogenase activities during hypoxia and after temperature acclimation in the armored fish, Rhinelepis strigosa (Siluriformes, Loricariidae)

PANEPUCCI, L., FERNANDES, M. N., SANCHES, J. R. and RANTIN, F. T.

Universidade Federal de São Carlos, Departamento de Ciências Fisiológicas, C.P. 676, CEP 13565-905, São Carlos, SP, Brazil

Correspondence to: Lucia Panepucci, Universidade Federal de São Carlos, Departamento de Ciências Fisiológicas, C.P. 676, CEP 13565-905, São Carlos, SP, Brazil, e-mail: [e-mail]

Received November 30, 1998 – Accepted June 28, 1999 – Distributed May 31, 2000

(With 1 figure)

ABSTRACT

Lactate (LDH) and malate dehydrogenase (MDH) of white skeletal muscle of fishes acclimated to 20, 25 and 30°C and thereafter submitted to hypoxia were studied in different substrate concentrations. Significant differences for LDH and MDH of white muscle enzyme activities are described here for the first time in Rhinelepis strigosa of fishes acclimated to 20°C and submitted to hypoxia for six hours. LDH presented a significant decrease in enzyme affinity for pyruvate in acute hypoxia, for fishes acclimated to 20°C and an increase for fishes acclimated to 30°C.

Key words: hypoxia, acclimation temperature, lactate dehydrogenase, malate dehydrogenase, armored fish, Rhinelepis strigosa.

RESUMO

Mudanças na atividade da lactato desidrogenase e malato desidrogenase durante hipóxia e após aclimatação a diferentes temperaturas no cascudo, Rhinelepis strigosa (Siluriformes, Loricariidae)

Foram estudadas a lactato desidrogenase (LDH) e malato desidrogenase (MDH) de músculo branco de peixes aclimatados a 20, 25 e 30°C em diferentes concentrações de substrato e submetidos à hipóxia. Diferenças significativas em atividade enzimática para LDH e MDH são descritas aqui pela primeira vez em Rhinelepis strigosa em peixes aclimatados a 20°C e submetidos à hipóxia por seis horas. A LDH apresentou uma diminuição significativa na afinidade enzimática ao piruvato em hipóxia severa de peixes aclimatados a 20^oC e um aumento significativo na afinidade enzimática ao piruvato em peixes aclimatados a 30°C.

Palavras-chave: hipóxia, aclimatação à temperatura, lactato desidrogenase, malato desidrogenase, cascudo, Rhinelepis strigosa.

INTRODUCTION

The first responses of fishes to environmental hypoxia are related to respiratory and circulatory changes. Many studies have been conducted by submitting organisms to hypoxia in order to study intermediary metabolites and enzymes (Shoubridge & Hochachka, 1983; Claireaux & Dutil, 1992; Sébert et al., 1993; Almeida-Val et al., 1995) but none of them focused on the effects of acute hypoxia on enzymes of fish acclimated to different temperatures. Hochachka & Somero (1973, 1984) proposed that ectothermic organisms, particularly fish, use adaptive biochemical strategies to obtain metabolic homeostasis during oscillations in dissolved oxygen, in temperature and in some other water physicochemical parameters. Studies on exposure of fishes acclimated to different dissolved oxygen concentrations did not give a single answer for enzyme responses (Shaklee et al., 1977; Almeida-Val & Hochachka, 1993; Almeida-Val et al., 1995). There is an extensive background of work in general and specific properties of lactate dehydrogenases (LDH) (Wilson 1977; Graves & Somero, 1982; Panepucci et al., 1984, 1987; Coppes & Somero, 1990) and in the soluble form of malate dehydrogenases (cMDH) (Shaklee et al., 1977; Schwantes & Schwantes, 1982a, b; Farias & Almeida-Val, 1992; Lin & Somero, 1995a, b). Lactate dehydrogenase (LDH, lactate; NAD-oxidoredutase, EC 1.1.1.27) is among the most extensively studied glycolitic enzyme. In fishes it is usually encoded by three loci, one expressed principally in skeletal muscle (LDH-A), another in heart muscle (LDH-B) and a third one in the eye (LDH-C). Malate dehydrogenase (L-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) catalyzes the reversible oxidation of malate to oxalacetate requiring NAD⁺ as a cofactor. It is involved in gluconeogenesis and lipogenesis, and in the malate-aspartate shuttle during aerobic glycolysis. The mitochondrial form (mMDH) acts in the Krebs cycle (Zink & Shaw, 1968). The present work aimed at understanding how fish enzymes respond to acute hypoxia at different acclimation temperatures.

MATERIAL AND METHODS

The armored fish, Rhinelepis strigosa, a facultative air-breather, found in the Mogi-Guaçu River basin, Brazil, is a stenothermal, detritivore-herbivore sedentary fish with moderate economic importance. The habitat temperature in the Mogi-Guaçu River varies from 20 to 30^oC during the year. Low temperatures occur only within a short period (June and July) and high temperatures in the middle of summer (January and February). Adult fishes, "Cascudos pretos", Rhinelepis strigosa, (wt 200 g) were net fished in the Mogi-Guaçu River, São Paulo State, Brazil. Fish were kept for at least 30 days at acclimation temperatures of 20, 25 and 30^oC ± 1^oC in 250 L tanks with water circulation and continuous aeration (P_WO2 > 130 mm Hg). The tanks were illuminated with natural light and fish fed on lettuce and aquatic plants "ad libitum". Feeding was stopped 24 hours before experiments. After acclimation to the experimental temperatures, six fishes were placed in a special aquarium for 24 hours with proper aeration (P_WO2 > 130 mm Hg). Oxygen tensions of inlet and outlet water were measured continuously by O₂ electrodes connected to a O₂ analyzer. The water oxygen tensions (Po₂) inside the experimental chamber were gradually decreased until critical oxygen tensions were reached as already determined by Fernandes et al. (1995) and Fernandes (personal comunication) and kept at stable levels by bubbling N₂. Fishes were kept in hypoxia during 6 h, then killed with a blow to the head. Tissues were excised and saved frozen at –20^oC until needed for use.

Enzyme preparation and assay of LDH and MDH activity

White muscle, heart and brain tissues from fishes acclimated to 20, 25, and 30^oC, were weighed and homogenized at ice-temperature with a 9-fold volume of Imidazol 5 mM, KCN 1 mM, pH 7.4 (at 25 ^oC) buffer. The homogenate was centrifuged at 17,000 g at 5^oC for 30 min. The supernatant was used directly as an LDH and MDH source in the kinetic study. LDH and MDH activities were determined by following the oxidation of NADH at 340 nm in a circulating thermobath at 25^oC. The reaction mixture was contained in a total volume of 1 ml, 50 mM Imidazol, 1 mM KCN buffer pH 7.4 at 25^oC, 0.13 mM of NADH and different concentrations of pyruvate for LDH saturation plots. Substrate saturation plots for oxalacetate were determined for MDH by following the oxidation of NADH at 340 nm. The reaction mixture was contained in a total volume of 1 ml, 50 mM Imidazol, 1 mM KCN, 100 mM KCl buffer pH 7.2 at 25^oC, 0.12 mM of NADH and different concentrations of oxalacetate. NADH saturation plots were determined for MDH activity with 0.3 mM oxalacetate and different concentrations of NADH. For obtaining K_M values, mathematical analyses using the Michaelis-Menten model were used with the aid of a computer program, Origin version 4.1. Activity of enzymes were expressed as U/gwt (Unit per gram of wet tissue). One unit of enzyme activity is defined as the amount of enzyme utilizing 1 mmole of substrate per minute at 25^oC. Non-parametric Mann-Whitney test was used to estimate differences between experiments with fishes submitted to both hypoxic and normoxic conditions. Rates of MDH/LDH activity were calculated in concentrations of 0.3 mM oxalacetate for MDH and in 1 mM pyruvate for LDH and 0.13 mM NADH for white muscle, heart muscle and brain tissue. Low and high ratios of LDH activity (L/H) were calculated in 1 mM and 10 mM pyruvate respectively for white muscle tissues.

RESULTS AND DISCUSSION

Experiments on fishes submitted to hypoxia showed significant differences in enzyme activity from fishes in normoxia at 20^oC. LDH pyruvate saturation plots of white muscle showed significant differences (P < 0.05) between hypoxia and normoxia (Fig. 1a). MDH oxalacetate saturation plots of white muscle submitted to hypoxia also showed significant differences P < 0.01) in all substrate concentrations from fishes in normoxia (Fig. 1b). MDH saturation plots of white muscle submitted to hypoxia using NADH as a substrate showed significant differences (P < 0.01) in all substrate concentrations (Fig. 1c). The fact that MDH using oxalacetate as a substrate and MDH using NADH as a substrate differed in normoxia and hypoxia may reflect its dual role in both aerobic and anaerobic energy metabolism at low temperature in this case, as pointed out by Hochachka & Somero (1984).

Table 1 shows K_M values for hypoxia and normoxia from the above experiments for all temperatures. Except for LDH of fishes acclimated to 20 and 30^oC, K_Ms did not reveal significant differences between fishes submitted to hypoxia. Table 2 shows enzyme activities for muscle, heart and brain tissues in normoxia and hypoxia. Significant differences between normoxia and hypoxia were found for white muscle LDH and MDH_[OAA], for fishes acclimated to 20^oC and, also, for heart muscle MDH_[OAA] of fishes acclimated to 25^oC. Brain tissue did not show significant differences for enzymes tested. In fishes acclimated to 25^oC significantly higher values during hypoxia suggest that MDH has a role in redox regulation during hypoxic stress. Table 3 shows the ratios of MDH/LDH activity which demonstrate the oxidative capacity of the tissues at all temperatures of acclimation (high rates denote high oxidative capacity). These ratios are extremely high (up to 280 times higher than white muscle) for heart muscle of fishes acclimated to all temperatures, showing the importance of this organ for the survival of fish in critical hypoxia situations and at the extreme temperatures found in their habitat. Brain tissue also presented a high ratio (11 times higher than white muscle) at all temperatures of acclimation. Short term hypoxia seems to be more stressful for heart muscle and brain because they need oxygen for their metabolism in order to avoid excessive metabolite accumulation. A high MDH/LDH ratio may cause an attenuated pyruvate to lactate flux and as a consequence carbohydrate metabolism will be largely channeled toward complete oxidation (Almeida-Val & Hochachka, 1995). This will benefit hypoxia situations like in heart muscle acclimated to 20 and 30^oC.

The ratio of LDH activity at low to that at high pyruvate concentrations (L/H) is often used as an index of the kinetic poise of LDH (Kaplan & Goodfriend, 1964). L/H LDH ratios for white muscle at different temperatures in normoxia and hypoxia suggest anaerobic organization (Table 4). These values are higher in normoxic than in hypoxic conditions, indicating that an increase exists in the reduction of pyruvate to sustain glycolisis under anaerobic conditions.

The results of K_M values obtained for LDH and MDH of fishes acclimated to 20^oC and submitted to hypoxia suggest that naturally intense fluctuations in dissolved environmental oxygen may result in significant changes in enzyme activity, such as the ability of enzymes to respond to acute hypoxia. Lushchak et al. (1997) found differences in enzyme activity throughout anaerobiosis and recovery of a sea mussel. Shaklee et al. (1977) found significant differences in enzymatic activity for liver LDH and white muscle aldolases in fishes acclimated to different oxygen concentrations.

This ability to respond to hypoxia may have been acquired in times of oxygen deficiency. Experiments at 25 and 30^oC did not result in significant changes in enzyme activities and K_M for acute hypoxia except for white muscle of fishes acclimated to 30^oC.

It is interesting to notice that these temperatures are those encountered in the environment of the fish almost all year round.

A number of studies with many species have shown that fishes frequently respond to change in environmental oxygen levels with changes within hematological parameters and alteration in physiological responses (Randall, 1993; Fernandes et al., 1995). Furthermore, fishes are known to avoid low oxygen concentrations (Reynolds & Thomson, 1974). According to M. N. Fernandes & J. R. Sanches (personal communication) no differences were found in Rhinelepis strigosa acclimated at different temperatures and submitted to hypoxia for the oxygen carrying capacity of the blood mesured by changes in hematocrit, hemoglobin concentration and red cell count, although changes were found in cardiac frequency, metabolic rate, oxygen uptake, ventilation rate and volume. Probably, hematological changes are subtle while physiological and biochemical adjustments provide the strategy used to deal with changes in oxygen concentrations in natural environments.

Changes in K_M are difficult to explain in short term periods of acute hypoxia. Changes in K_M of pyruvate and NADH for M₄-LDH from shallow and deep sea living species were related with changes in pressure in fishes (Siebenaller & Somero, 1979). According to Greaney & Somero (1980) studies of NADH binding suggest that for M₄-LDHs and other dehydrogenases NADH (NAD) binding sites should remain cofactor-saturated, so that the direction of dehydrogenases function is established by the redox state, i.e. the NADH/NAD ratio of the cell.

Differences in K_M of enzymes have also been attributed to a modulation resulting from changes in the pH milieu (Wilson, 1977; Yancey & Somero, 1978; Walsh & Somero, 1982; Somero, 1983; Coppes et al., 1992).

This would explain the higher affinity (lower K_M) of muscle LDH in hypoxia and the lower affinity (higher K_M) of muscle LDH at 20^oC in cascudo preto. These hidden strategies such as the ability of enzymes to respond to acute hypoxia may explain differential responses to hypoxia situations which fishes encounter in different environments.

Acknowledgments — We wish to thank Mr. N. S. A. Matos for fishing assistance. We wish to thank FAPESP and CNPq for grants that made this work possible. Lucia L. L. de Panepucci was supported by a CNPq fellowship.

• ALMEIDA-VAL, V. M. F. & HOCHACHKA, P. W., 1993, Hypoxia tolerance in Amazon fishes: Status of an under-explored biological "goldmine". In: P. W. Hochachka, P. L. Lutz, T. Sick, M. Rosenthal & G. Van den Thillart (eds.), Surviving hypoxia: Mechanisms of Control and Adaptation, pp. 435-445. CRC Press, Boca Raton.
• ALMEIDA-VAL, V. M. F. & HOCHACHKA, P. W., 1995, Air breathing fishes: Metabolic biochemistry of the first diving vertebrates. In: P. W. Hochachka & T. P. Mommsen (eds.), Biochemistry and Molecular Biology of Fishes. Vol. 5. Springer-Verlag, Berlin, Heidelberg, New York.
• ALMEIDA-VAL, V. M. F., FARIAS, I. P., SILVA, M. N. P., DUNCAN, W. P. & VAL, A. L., 1995, Biochemical adjustments to hypoxia by Amazon cichlids. Braz. J. Med. Biol. Res., 28: 1257-1263.
• CLAIREAUX, G. & DUTIL, J. D., 1992, Physiological response of the Atlantic cod (Gadus morrhua) to hypoxia at various environmental salinities. J. Exp. Biol., 163: 97-118.
• COPPES, Z. L. & SOMERO, G. N., 1990, Temperature differences between the M_{4 l} lactate dehydrogenase of stenothermal and eurythermal Sciaenid fishes. J. Exp. Zool., 254: 127-131.
• COPPES, Z., MARTINEZ, G. & HIRSCHHORN, M., 1992, pH and temperature effects on the K_M values of muscle lactate dehydrogenase isozyme LDH-A₄ from fishes of the family Scianidae (Perciformes). Comp. Biochem. Physiol, 103B: 869-874.
• FARIAS, I. P. & ALMEIDA-VAL, V. M. F., 1992, Malate dehydrogenase (sMDH) in Amazon cichlid fishes: Evolutionary features. Comp. Biochem. Physiol., 103B: 939-943.
• FERNANDES, M. N., SANCHES, J. R. & RANTIN, F. T., 1995, Effects of long and short-term changes in water temperature on the cardiac and respiratory responses to hypoxia of the air-breathing fish, Hypostomus regani. Proceedings of the International Congress of Comparative Physiology and Biochemistry. Physiol. Zool., 68(4): 67.
• GRAVES, J. E. & SOMERO, G. N., 1982, Electrophoretic and functional enzymic evolution in four species of Eastern Pacific barracudas from different thermal environments. Evolution, 36: 97-106.
• GREANEY, G. S. & SOMERO, G. N., 1980, Contributions of binding and catalytic rate constants to evolutionary modifications in K_m of NADH for muscle-type (M₄) lactate dehydrogenases. J. Comp. Physiol, 137: 115-121.
• HOCHACHKA, P. W. & SOMERO, G. N., 1973, Strategies of Biochemical adaptation. W. B. Saunders Company. Philadelphia, London, Toronto.
• HOCHACHKA, P. W. & SOMERO, G. N., 1984, Biochemical adaptation Princeton University Press, Princeton, NJ.
• KAPLAN, N. O. & GOODFRIEND, T. L., 1964, Role of two types of lactic dehydrogenases. Adv. Enzyme Regul, 2: 203-212.
• LIN, J. & SOMERO, G. N., 1995a, Thermal adaptation of cytoplasmic malate dehydrogenase of eastern pacific barracuda (Sphyraena ssp.): The role of differential isoenzyme expression. J. Exp. Biol., 198: 551-560.
• LIN, J. & SOMERO, G. N., 1995b, Temperature-dependent changes in expression of thermostable and thermolabile isozymes of cytosolic malate dehydrogenase in the eurythermal goby fish Gillichthys mirabilis. Physiol. Zool., 68(1): 114-128.
• LUSHCHAK, V. I., BAHNJUKOVA, T. V. & SPICHENKOV, A. V., 1997, Modification of pyruvate kinase and lactate dehydrogenase in foot muscle of the sea mussel Mytilus galloprovincialis under anaerobiosis and recovery. Braz. J. Bed. Biol. Res., 30(3): 381-385.
• PANEPUCCI, L., SCHWANTES, M. L. & SCHWANTES, A. R., 1984, Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the Orders Cypriniformes, Siluriformes and Perciformes: Adaptative Features. Comp. Biochem. Physiol., 77B: 867-876.
• PANEPUCCI, L., SCHWANTES, M. L. & SCHWANTES, A. R., 1987, Biochemical and physiological properties of the lactate dehydrogenase allozymes of the brazilian Teleost, Leporinus friderici, Anostomidae, Cypriniformes. Comp. Biochem. Physiol., 87B: 199-206.
• RANDALL, D. J., 1993, The regulation of breathing in aquatic vertebrates. In: J. E. P. W. Bicudo (ed.), The Vertebrate Gas Transport Cascade. Adaptations to Environment and Mode of Life, pp. 54-59. Boca Raton, FL: CRC Press.
• REYNOLDS, W. W. & THOMSON, D. A., 1974, Responses of young gulf grunion, Leuresthes sardina, to gradients of temperature, light, turbulence, and oxygen. Copeia, pp. 747-758.
• SCHWANTES, M. L. B. & SCHWANTES, A. R., 1982a, Adaptative features of ectothermic enzymes-I. Temperature effects on the malate dehydrogenase from a temperate fish Leiostomus xanthurus. Comp. Biochem. Physiol., 72B: 49-58.
• SCHWANTES, M. L. B. & SCHWANTES, A. R., 1982b, Adaptative features of ectothermic enzymes-II. The effects of acclimation temperature on the malate dehydrogenase of the Spot, Leiostomus xanthurus. Comp. Biochem. Physiol., 72B: 59-64.
• SÉBERT, P., SIMON, B. & BARTHELEMY, 1993, Hidrostatic pressure induces a state resembling histotoxic hypoxia in anguilla-anguilla. Comp. Biochem. Physiol., 105B: 255-258.
• SHAKLEE, J. B., CHRISTIANSEN, J. A., SIDELL, B. D., PROSSER, C. L. & WHITT, G. S., 1977, Molecular aspects of temperature acclimation in fish: Conributions of changes in enzyme activities and isozyme patterns to metabolic reorganization in the green sunfish. J. Exp. Zool., 201: 1-20.
• SHOUBRIDGE, E. A. & HOCHACHKA, P., 1983, The integration and control of metabolism in the anoxic goldfish. Molecular Physiology, 4: 165-195.
• SIEBENALLER, J. F. & SOMERO, G. N., 1979, Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M₄) lactate dehydrogenases of shallow and deep-living marine fishes. J. Comp. Physiol., 129: 295-300.
• SOMERO, G. N., 1983, Environmental adaptation of proteins: Strategies for the conservation of critical functional and structural traits. Comp. Biochem. Physiol., 76A: 621-633.
• WALSH, P. J. & SOMERO, G. N., 1982, Interactions among pyruvate concentration, pH, and K_m of pyruvate in determining in vivo Q₁₀ values of the lactate dehydrogenase reaction. Can. J. Zool, 60: 1293-1299.
• WILSON, T. L., 1977, Interrelations between pH and temperature for the catalytic rate of the M₄ isozyme of lactate dehydrogenase (EC 1.1.1.27) from goldfish (Carassius auratus). Arch. Biochem. Biophys., 179: 378-390.
• YANCEY, P. H. & SOMERO, G. N., 1978, Temperature dependence of intracellular pH: its role in the conservation of pyruvate apparent K_m values of vertebrate lactate dehydrogenases. J. Comp. Physiol, 125: 129-134.
• ZINK, M. W. & SHAW, D. A., 1968, Regulation of malic isozymes and malic dehydrogenase in Neurospora crassa. Can. J. Microbiol., 14: 907-912.

Publication Dates

• Publication in this collection
  21 Aug 2000
• Date of issue
  May 2000

History

• Accepted
  28 June 1998
• Received
  30 Nov 1998