JEZ 0612 JOURNAL OF EXPERIMENTAL ZOOLOGY 286:107–113 (2000) Mitochondrial Contribution to Metabolic Changes in the Digestive Gland of Mytilus galloprovincialis During Anaerobiosis SIMONE BACCHIOCCHI, AND GIOVANNI PRINCIPATO* Istituto di Biologia e Genetica, Università di Ancona, 60100 Ancona, Italy ABSTRACT The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis . The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107–113, 2000. © 2000 Wiley-Liss, Inc. Exposition to intermittent severe hypoxia can be a frequent occurrence for mollusks as well as barnacles, sea anemones, and polychaete worms. In fact, several marine invertebrates are exposed to daily episodes of oxygen lack when the tides recede. Hypoxia as well as the restoration of oxygen supply can happen suddenly, thus cellular response must be fast and easily reverted. There is not enough time for large-scale reorganization of the amounts or types of cellular proteins. Thus biochemical modifications rather than gene expression modifications are the basis for survival in these harsh conditions (de Zwaan, ’77, ’83; Kreutzer et al., ’85; Storey, ’85). For intertidal marine invertebrates, resistance to severe hypoxia results as part of an adaptive strategy that maximize the survival time of the organism in the anoxic interval, ready to restart with respiration as soon as oxygen becomes available again. Adaptive strategy seems to involve: (1) metabolic rate depression; (2) activity modifications of some glycolytic enzymes; and (3) production of low toxicity metabolic wastes. Metabolic rate depression occurs by transition to a hypometabolic state throughout the coordinated reduction of the activity of some key regulatory enzymes. Facultative anaerobes do not show a Pasteur effect, but a “reverse Pasteur effect” occurs and the glycolytic rate in hypoxia is reduced below that seen in the normoxic situation (Ebberink and de Zwaan, ’80; © 2000 WILEY-LISS, INC. Lutz et al., ’84; Storey, ’85; Kelly and Storey, ’88; Storey et al., ’90). The mechanism involves covalent but reversible enzyme modifications, such as phosphorylation or dephosphorylation, and seems to be conserved in phylogenetic lines (Storey and Storey, ’90). Indeed, studies of the glycolytic response to the transition from normoxia to hypoxia show the same pattern in turtles, goldfish, and marine mollusks (Shoubridge and Hochachka, ’80; Kelly and Storey, ’88; Storey et al., ’90). Throughout the anoxic time, the metabolism of bivalve mollusks is sustained only by endogenous fermentable fuel reserves. Depression of the metabolic rate, when oxygen is depleted, allows energy demands to be met by the ATP output using alternative pathways of fermentative ATP production. Anaerobic ATP from substrate-level phosphorylations is greatly enhanced; instead of two ATP per glucose fermented to lactate, five/seven ATP per glucose fermented to succinate/propionate are produced (von Brandt, ’46; Hochachka, ’80; Brinkhoff et al., ’83; Hole et al., ’95). Besides the increase in ATP, these alternative pathways produce low toxicity Abbreviations used: ADH, alanopine dehydrogenase; DHAP, dihydroxyacetonephosphate, GI, glyoxalase I; MG, methylglyoxal; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PK, pyruvate kinase. *Correspondence to: Prof. Giovanni Principato, Istituto di Biologia e Genetica, Via Ranieri, Montedago, 60100 Ancona, Italy. E-mail: email@example.com Received 4 December 1998; Accepted 16 April 1999 108 S. BACCHIOCCHI AND G. PRINCIPATO metabolic wastes that are weak rather than strong acids as L-lactate. Gastropod and bivalve mollusks are generally lacking in lactic dehydrogenase. The redox equilibrium is ensured by alternative pathways for NADH oxidation that produce octopine or alanopine by a reductive condensation catalyzed by the corresponding dehydrogenases (Hochachka, ’80). MATERIALS AND METHODS Animals Specimens of the mussel Mytilus galloprovincialis were directly obtained by drawing from a cliff off the coast of Senigallia (Ancona, Italy) during September/October 1997. To impose severe hypoxia, mussels were simply exposed to air at room temperature (20–23°C) for intervals from 7 to 168 hr. Preparation of cytosolic and mitochondrial fractions Digestive glands were dissected from aerobic or anoxic mussels and quickly homogenized by Potter Elvehjem (1:2, w/v) in 100 mM Tris-HCl buffer, pH 8.0, containing 5 U/ml bacitracine, 0.009 TIU/ ml aprotinine and 0.1 mM phenylmethylsulfonyl fluoride. After centrifugation at 48,000g for 10 min at 4°C, supernatants were further centrifuged at 125,000g for 40 min at 4°C and stored at –20°C until used for enzyme assays. Some other glands were homogenized in 1:2 (w/v) 0.8 M perchloric acid and the mixture centrifuged at 38,000g for 15 min at 4°C. Supernatants were stored at –20°C until used for metabolite assays. Mitochondria were isolated in according to Ballantyne and Storey (’84), with the following modifications: digestive glands of five-six mussels were homogenized in 1:20 (w/v) ice-cold 5 mM TrisEDTA buffer, pH 7.4, containing 0.25 M sucrose. The homogenate was centrifuged at 1,500g for 8 min at 4°C, the supernatant was collected and recentrifuged at 9,000g for 15 min at 4°C. The mitochondrial pellet was then resuspended in 1:4 (w/v) ice-cold 24 mM Tris-HCl buffer, pH 7.6, containing 1 mM EDTA, 0.25 M sucrose and 0.5 mg/ml bovine serum albumin fatty acids-free, and recentrifuged at 9,000g for 15 min at 4°C. The final pellet was resuspended in a small volume of the latter solution at a mitochondrial protein content of 10–20 mg/ml. Enzyme assays Pyruvate kinase (PK) was measured by the continous coupled assay of Bergmeyer (’74) in the presence of final concentrations of 1 mM ADP, 9.4 mM magnesium chloride, and 94 mM potassium chloride. The phosphorylation reaction was evaluated by the NADH oxidized during the reduction of pyruvate by lactate dehydrogenase. NADH utilization was monitored at 340 nm on a Varian uv/ vis recording spectrophotometer attached to a circulating water bath at 25°C. The kinetics at different alanine concentrations in the presence of 1 mM ADP were not linear in the range of PEP 0.05–1.6 mM and alanine 0.05–15 mM. However, at the highest PEP concentrations double reciprocal plots approximate competitive kinetics and apparent Ki values were calculated from Dixon plots according to Plaxton and Storey (’84). Phosphoenholpyruvate carboxykinase (PEPCK) was assayed according to Urbina (’87). The carboxylation reaction was measured by coupling the formation of oxaloacetate to NADH oxidation by malate dehydrogenase. Alanopine dehydrogenase (ADH) activity was determined by following the NADH oxidation during the formation of alanopine from alanine and pyruvate (Fields et al., ’80). Glyoxalase I (GI) was determined according to Regoli and Principato, (’95), by following the formation of S-D-lactoylglutathione from the hemimercaptal adduct of methylglyoxal (MG) and glutathione at 240 nm (e = 3.37 mM–1 cm–1). Preliminary experiments indicate that enzymes activities were unchanged (e.g., Glyoxalase I) or decreased less than 20% (e.g., Pyruvate kinase) in supernatants stored at –20°C. Statistical evaluation (ANOVA) was carried out on the activities of at least three independent determinations for each hypoxia time. Metabolite assays Supernatants from perchloric acid precipitation were neutralized by addition of 1:1 (v/v) 1 M potassium carbonate solution. Metabolites were quantified by coupled enzyme assays (Bergmeyer, ’74), following the NADH oxidation in the assay conditions. Alanine and aspartate levels were evaluated in 10 mM sodium phosphate buffer, pH 6.8, containing 0.15 mM NADH and 20 mM α-ketoglutarate. Alanine was measured by the addition of 3 U/ml lactate dehydrogenase and 0.8 U/ml glutamicpyruvic transaminase. Aspartate was measured by addition of 12 U/ml malate dehydrogenase and 2 U/ml glutamic-oxalacetic transaminase. Succinate was determined in 100 mM triethanolamine buffer, pH 7.4, containing 100 mM magnesium chloride, 5 mM EDTA, 0.15 mM NADH and 25 MITOCHONDRIA AND ANAEROBIOSIS IN MUSSELS µl/ml of coenzyme A solution (5 mM coenzyme A, 50 mM PEP, 10 mM ATP). The reaction was started by adding 7.5 U/ml lactate dehydrogenase, 2 U/ml pyruvate kinase and 0.1 U/ml succinic thiokinase. D-and L-lactate were measured in 400 mM bicine buffer, pH 8.5, containing 4 mM Lglutamate and 6.8 mM NAD+. The reaction started by addition of 4.5 U/ml D- or L-lactate dehydrogenase and 1.12 U/ml glutamic-pyruvic transaminase. D- or L-lactate concentration were calculated by comparison with standard curves (0.4–6.4 mM). Mitochondrial pyruvate formation from D- and L-lactate was measured by adding 30 µg of protein from freshly prepared mitochondrial suspension (within 1 hr from isolation) to 1 ml of 20 mM HepesTris buffer, pH 7.3, containing 0.2 M sucrose, 10 mM potassium chloride, 3 mM magnesium chloride, 2 µg rotenone, 2 mM arsenite. Pyruvate formation was monitored by adding 3 U/ml L-lactate dehydrogenase, 0.2 mM NADH, 5 mM D- or L-lactate (final concentrations) and following NADH oxidation at 340 nm. Protein concentration was determined according to Lowry et al. (’51) by using bovine serum albumin as standard. RESULTS Table 1 shows the activities of PK, GI, PEPCK and ADH in the digestive gland of Mytilus galloprovincialis over a time course of hypoxia. All the enzymes show time-dependent activity variations with respect to control (zero time) values. For all the enzymes the activity changes in response to hypoxia are already evident in the first 7 hr. PK activity shows the strongest variation by decreasing to about 20% of the control value. GI and ADH TABLE 1. Activities of pyruvate kinase (PK), glyoxalase I (GI), phosphoenolpyruvate carboxykinase (PEPCK), and alanopine dehydrogenase (ADH) in digestive gland of mussel Mytilus galloprovincialis over a time course of anoxia1 (µmol/min/mg)* Severe hypoxia (hr) PK GI PEPCK ADH 0 7 24 48 72 96 168 0.671 a 0.153 b 0.108 b 0.082 c 0.087 c 0.096 c 0.121 b 0.179a 0.132 ab 0.140 b 0.106 b 0.125 b 0.062 c 0.037 c 0.0128a 0.0185 ac 0.0109 ad 0.0119 ad 0.0097 ad 0.0123 ad 0.0069 bd 0.0153 a 0.0126 a 0.0115 a 0.0074 b 0.0067 b 0.0072 b 0.0058 b 1 Enzyme activities were assayed spettrofotometrically at constant temperature (25°C) as described in Materials and Methods. Statistical evaluation (ANOVA) was carried out on the activities of at least three independent determinations for each hypoxia time. *Means followed by the same letters (within each column) are not significantly different at P < 0.05. 109 activity decrements are about 20–30%, whereas PEPCK shows an activity increase of approximately 40% in the same period of time. The activity values during the hypoxia exposure up to 168 hr show further variations for all the examined enzymes. Between 7 and 48 hr, PK activity decreases to about 10% of the initial value, then shows the tendency to increase so that its value after 168 hr is about 20% of the control activity. After the initial rise, PEPCK activity decreases rapidly after 24 hr, remains constant until 96 hr and then again decreases averaging 50% of the aerobic activity at 168 hr. GI activity slowly decreases until 72 hr and then falls at 96–168 hr averaging 20% of the control value. Km values of GI using hemimercaptal as substrate are 0.53 mM for the aerobic enzyme and 0.60 mM after 96 hr indicating that the decrease in activity is probably due to the decrease in amount of enzyme rather than to an intervention of a different enzymatic form. On the contrary, changes in kinetic parameters clearly result for PK. In Table 2 Km values of PK for the substrate PEP are reported together with apparent Ki values for the inhibitor alanine, calculated after 0-7-24 hr of anoxic exposure. Km values increase nearly 10-fold after 7 hr and double after 24 hr. Apparent Ki values decrease by more than 10-fold after 7 hr and by more than 20-fold after 24 hr. These two kinetic parameters show only small variations during the following exposure to hypoxia up to 168 hr. The activity of ADH continuously decreases by increasing the time of hypoxia exposure. After 168 hr the remaining activity is about 30% of the initial value. Figure 1A shows the effect of anoxia exposure on the levels of aspartate and alanine, Figure 1B shows the levels of D- and L-lactate and succinate in the digestive gland of Mytilus galloprovincialis. TABLE 2. Km and apparent Ki values of pyruvate kinase for the substrate phosphoenolpyruvate and the allosteric inhibitor alanine, respectively1 Anoxia (hr) 0 7 24 (mM)* Km Apparent Ki 0.05 a 0.49 b 0.84 c 14.4 a 1.19 b 0.54 b 1 Statistical evaluation (ANOVA) was carried out on the activities of at least three independent determinations for each hypoxia time. *Means followed by the same letters (within each column) are not significantly different at P < 0.05. 110 S. BACCHIOCCHI AND G. PRINCIPATO Fig. 1. Mean alanine and aspartate (A), succinate and DL-lactate (B) levels in the digestive gland of the mussel Mytilus galloprovincialis during anoxia exposure. Statistical evaluation (ANOVA) has been made for data relative to succinate (3–6 samples per experimental point). a: significantly different from the aerobic control value at P < 0.05; b: P < 0.01. Data relative to alanine were not compared by ANOVA since each experimental point was the mean of two samples. The concentrations of the end products alanine and succinate continuously increase with hypoxia exposure and their levels are almost doubled after 168 hr. The organ content of aspartate is unaffected even up to 168 hr of exposure to hypoxia, whereas D- and L-lactate levels are undetectable. In Figure 2 the amount of pyruvate that is formed by the mitochondrial oxidation of D-and L-lactate is shown. D-lactate is formed from methylglyoxal by means the glyoxalase enzyme system. DISCUSSION This paper deals with metabolic modifications in the digestive gland of Mytilus galloprovincialis during anaerobiosis. Mussels were exposed to air for intervals from 7 to 168 hr at room temperature (20–23°C). In these conditions M. gallo- Fig. 2. Possible contribution to the synthesis of alanopine by mitochondrial pyruvate formation from D-lactate in Mytilus galloprovincialis. (1) Formation of pyruvate through glycolysis. (2) MG is formed from an intermediate of glycolysis, DHAP, by means of triosephosphate isomerase (Richard, ’91). (3) D-lactate is formed from MG by means of the glyoxalase enzyme system (glyoxalase I and glyoxalase II) (Regoli and Principato, ’95). (4) The pyruvate production by D-lactate oxidation needs the presence of intact mitochondria. The enzyme involved is probably α-D-hydroxyacid dehydrogenase (Tubbs and Greville, ’61). (5) Formation of alanine from pyruvate by glutamic-pyruvic transaminase. (6) Reductive condensation of pyruvate and alanine with formation of alanopine by means of alanopine dehydrogenase (Fields et al., ’80). provincialis closes valves hermetically and can survive for several days. Variations of enzyme activities and metabolite levels in response to lack of oxygen are observed. These metabolic changes start at the beginning of hypoxia, after as soon as 7 hr. According to expectations, we found a notable drop in PK activity from 75 to 90% of the control value in the period from 7 to 48 hr of hypoxia. The kinetic properties of the enzyme are profoundly modified as demonstrated by the consistent decreases of Vmax (more than 10-fold) and apparent Ki for alanine (more than 20-fold). Km values for PEP instead are increased (about 20fold). These results are in agreement with those found in Mytilus edulis and in Busycotypus canaliculatum where the presence, in hypoxia, of a phosphorylated form of PK has been demonstrated (Siebenaller, ’79; Holwerda et al., ’83; Plaxton and Storey, ’84). Dephosphorylation occurs as soon as oxygen becomes available again with the complete restoration of PK activity typical of aerobiosis. It is possible that also in our case the modified PK is phosphorylated. The small MITOCHONDRIA AND ANAEROBIOSIS IN MUSSELS increases of PK activity in the final hr of hypoxia could be due to dephosphorylation linked to cell damage as demonstrated in other organisms in similar conditions (Storey and Storey, ’90). The decreased velocity together with the decreased affinity for the substrate PEP and the increased inhibition by alanine (that increases), indicates that the effective intracellular PK activity must be very low, much less than 10% with respect to control activity, with consequent PEP accumulation. Because of PK inhibition the glycolytic flux in anoxia could be channeled in alternative pathways (metabolic branches). We found a measurable amount of PEPCK in Mytilus galloprovincialis, thus PEP can become substrate of this enzyme and go into succinate-producing pathways that yield additional ATP from substrate-level phosphorylations (Storey and Storey, ’90). This metabolic shunt is of special importance in the adaptation to severe hypoxia of helminth worms (Hochachka, ’80), but seemed not to be as important in mollusks such as oysters, where PEPCK activity is undetectable (Hochachka, ’80). Another glycolytic shunt that can be increased by slowing down the carbon flux through PK leads to increased methylglyoxal formation. In conditions of reduced glycolytic flux, Richard demonstrated that DHAP could be transformed into MG by an elimination reaction catalyzed by triosephosphate isomerase (Richard, ’91). Methylglyoxal formation from glycolytic intermediates can also occur non-enzymatically (Phillips and Thornalley, ’93). Methylglyoxal formation is then unavoidable. This α-ketoaldehyde is a known toxic metabolite that is efficiently inactivated to D-lactate in the presence of GSH and glyoxalase I. The activity of this enzyme protects cells from damage as long as its activity is maintained. GI activity is physiologically high in M. galloprovincialis and apparently is not greatly modified over anaerobiosis up to 72 hr. Activity values are higher than that of the inhibited activity of pyruvate kinase. The observed small decrease in GI activity in the last hr of anoxia is probably not due to enzyme modification, because of the constant Km. Lack of GSH can also be excluded because its concentration is constant throughout anoxia time (data not shown). The large amount of glyoxalase II activity present in M. galloprovincialis (Regoli and Principato, ’95) indicates the presence for an active glyoxalase metabolism. The importance of the glyoxalase shunt is probably high in hypoxia since the ratio PK/GI is 3.75 111 before hypoxia but is 1.19 after 7 hr of hypoxia and decrease to 0.70 after 72 hr. Therefore there are the right conditions for D-lactate formation in hypoxia. Contrary to expectations, however, D-lactate is undetectable and does not accumulate in the digestive gland of M. galloprovincialis, suggesting a possible further metabolization. Indeed our data demonstrate that D-lactate can be transformed into pyruvate, probably by means of α-D-hydroxyacid dehydrogenase (EC 220.127.116.11) (Tubbs and Greville, ’61) or another unknown enzyme. In our experimental conditions, in vitro, L-lactate was used as well as D-lactate for mitochondrial pyruvate formation. However since we found neither lactic dehydrogenase nor lactic acid in the digestive gland of M. galloprovincialis, it seems that L-lactate cannot be formed and that D-lactate is formed by the glyoxalase enzyme system. The reaction catalyzed by glyoxalases is stereospecific and only D-lactate is produced from methylglyoxal (Ekwall and Mannervik, ’73). The relative changes of PK and GI activities in hypoxia indicate that the glycolytic carbon flux after dihydroxyacetone phosphate could be channeled in two similar branches, one giving phosphoenolpyruvate and the other giving methylglyoxal (Fig. 2). Both branches can be a source of pyruvate necessary for alanine and alanopine formation. By comparing the activities of PEPCK and PK it is possible to determine that about 10% of PEP could be carboxylated to oxaloacetate and be a source of the increasing succinate. Then PEPCK shunt decreases the glycolytic pyruvate and the high intracellular alanine levels strongly inhibits PK to activity values lower than those measured in vitro. In anoxic mitochondria D-lactate formed from methylglyoxal is reduced to pyruvate (Fig. 3). This pathway can play an important role for the increased alanine and alanopine biosynthesis. In terms of energy, glycolysis yields ATP but the metabolism of methylglyoxal throughout glyoxalase I produces the high-energy thioester S-D-lactoylglutathione. In addition, the mitochondrial D-lactate → pyruvate reaction liberates 2H that represent an additional and potentially important reducing power available. Our results indicate the metabolic role of the glyoxalase pathway are: (1) contributing to respiration in the presence of oxygen by forming pyruvate, and (2) contributing with glycolysis in anaerobiosis to form the pyruvate that is necessary to produce alanine and alanopine in M. 112 S. BACCHIOCCHI AND G. PRINCIPATO Fig. 3. Possible contribution of mitochondrial D-lactate oxidation in the formation of succinate in the digestive gland of Mytilus galloprovincialis. galloprovincialis. In fact, in the digestive gland of M. galloprovincialis, as in the oysters, NADH reoxidation for the maintenance of the cytoplasmic redox balance is associated with the reductive condensation of pyruvate and alanine into alanopine catalyzed by alanopine dehydrogenase. Therefore in catalyzing NADH reoxidation this reaction is formally analogous to that catalyzed by lactic dehydrogenase in vertebrate anaerobic glycolysis. In M. galloprovincialis L-lactic acid production and consequent acidification is avoided since alanine and alanopine are weak acids. Their accumulation produces a reduced cytoplasmic acidification and, at the same time, stores pyruvate for the restart of respiration as soon as oxygen becomes available again. A consistent increase in both alanine and succinate levels is found in M. galloprovincialis during severe hypoxia. Similar results have been found in oysters, where the rise of these end products correlates with the decrease of aspartate levels (Hochachka, ’80). On the contrary, aspartate levels in the digestive glands of M. galloprovincialis remain constant throughout exposure to hypoxia. However, the other possibility, the decrease of glutamate, seems to be excluded by preliminary data (not shown). The existence of an unknown aspartate and/or glutamate supply is possible. The source of amino groups could be ammonia formed during protein degradation and fixed by glutamic dehydrogenase. In Figure 3 the forward and reverse flows of carbons in Krebs cycle that might contribute to succinate accumulation in anaerobiosis are shown. NADH and CO2 are produced by the oxidation of α-ketoglutarate to succinyl-CoA, while NADH is consumed by the oxaloacetate reduction to malate. The source of two H equivalents necessary to re- duce fumarate to succinate is unknown, but it probably could derive from D-lactate oxidation to pyruvate, at least in M. galloprovincialis. The observed increase of succinate (Krebs cyclelinked), and the involvement in producing pyruvate, suggests that mitochondria are involved in the anaerobic metabolism of M. galloprovincialis. The mitochondrial production of pyruvate is possible only in the presence of intact mitochondria. In conclusion our hypothesis is that in Mytilus galloprovincialis both glucose (deviation at the PEP branchpoint) and aspartate contribute in raising the amount of succinate. Furthermore the forward flow of carbons from α-ketoglutarate or metabolites convertible to a-ketoglutarate (e.g., glutamate) supplies another possible route for succinate formation. By these reactions mitochondria are maintained metabolically active in anaerobiosis. 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