ABSTRACT. The maximal in vitro activities of eight enzymes involved with the metabolism ofL-lactate were measured in hepatopancreas, gills, heart, and dark ...
BULLETIN OF MARINE SCIENCE, 48(3): 665~69,
1991
METABOLIC POTENTIAL IN TISSUES OF THE BLUE CRAB, CALL/NECTES SAP/DUS Fram;ois H, Lallier and Patrick J. Walsh ABSTRACT The maximal in vitro activities of eight enzymes involved with the metabolism ofL-lactate were measured in hepatopancreas, gills, heart, and dark and light levator muscles of the blue crab, Ca/linectes sapidus. The activities found in both muscle types were similar for all enzymes except for citrate synthase, where dark muscle showed a much greater potential for aerobic metabolism than light muscle, as expected from their structure. Heart tissue showed good potential for both aerobiosis and anaerobiosis. Gills demonstrated relatively low activities for these enzymes of energy metabolism. Hepatopancreas completely lacked LDH activity, although we found activities for other enzymes of glycolytic and gluconeogenic pathways. We conclude that lactate produced by muscles (including the heart) during anaerobiosis may be reoxidized in situ and that the gluconeogenic potential of hepatopancreas and gills appears to be low.
Numerous studies have now established that L-lactate is the almost exclusive end-product of anaerobiosis in Crustacea (reviewed in Giide and Grieshaber, 1986) and that it accumulates during both muscular exercise (or functional anaerobiosis, e.g., Booth et al., 1982; Milligan et al., 1989) and severe hypoxia or anoxia (or environmental anaerobiosis, e.g., Bridges and Brand, 1980; Albert and Ellington, 1985; Lallier et al., 1987). Lactate is then released to the hemolymph and subsequently removed from it following return to rest and normoxia, but complete removal of the lactate load is a relatively slow process (4-24 h, e.g., Phillips et al., 1977; McDonald et al., 1979; Bridges and Brand, 1980; Booth et al., 1984; Gade, 1984; Albert and Ellington, 1985; Milligan et al., 1989). However, the actual fate of lactate during recovery-i.e., how and where is it disposed of?remains an ambiguous and controversial point in crustaceans (Ellington, 1983; Gade and Grieshaber, 1986). Lactate could be eliminated by excretion, oxidation to CO2 or reconversion
into aerobic substrates (i.e., gluco- or glyconeogenesis) (Ellington, 1983). Excretion of lactate into the external medium does not seem to occur (Bridges and Brand, 1980; Gade et al., 1986). Minimum prerequisites for the oxidation of lactate are the presence oflactate dehydrogenase (LDH) functioning in the "lactate oxidase," or "reverse," direction (as opposed to the "pyruvate reductase" or "forward" direction) together with an active tricarboxylic acid cycle. Finally, gluconeogenesis from lactate also requires LDH and the key enzymes phosphoenolpyruvate carboxykinase and fructose-1,6-bisphosphatase. The hepatopancreas (Munday and Poat, 1971), the gills (Thabrew et al., 1971) and the hemocytes (Johnston et al., 1971) have all been proposed as possible sites of gluconeogenesis in crustaceans. The study of Phillips et al. (1977) on the crayfish Cherax destructor reported in vivo incorporation oflabelled carbon from lactate into glucose in the hemolymph but failed to demonstrate in vitro incorporation in preparations ofhepatopancreas, gill or hemolymph. More recently data from the crabs Menippe mercenaria (Gade et al" 1986) and Potamonautes warreni (van Aardt, 1988) indicate that lactate may be metabolized into CO2, glycogen and glucose but results did not identify any particular tissue as sites of lactate conversion. In order to identify potential sites and routes of lactate elimination, we have investigated the activity of eight key enzymes of lactate metabolism in five tissues of the blue crab Callinectes sapidus. 665
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MATERIAL AND METHODS Adult blue crabs, Ca/linectes sapidus (Rathburn) (6 females and 1 male; fresh weight range 70-170 g), were purchased from Gulf Specimen Company (panacea, Florida) in October 1989 and maintained in aquaria with running seawater (23-26°C, 36%0). Animals were fed three times a week with frozen shrimp until 2~8 h prior to sacrifice, and all crabs were used by the end of November 1989. After cooling the animal on crushed ice for 30-45 min (depending on size) to ensure complete torpor, five tissues were dissected as completely and quickly as possible. Selected tissues were the heart, the hepatopancreas, the gills on both sides, and the light and dark levator muscles of both swimming legs (White and Spirito, 1973). Tissues were blotted, weighed and homogenized in 5: 1 to 7: 1 volumes ice-cold buffer with a hand-held glass tissue grinder with glass pestle for muscles and gills and a Potter-Elvehjem tissue grinder with motor-driven teflon pestle for hepatopancreas and heart. The homogenization buffer contained 50 mmol·L-' imidazole, pH 7.8, plus 0.1 mmol·L-1 phenylmethylsulfonyl fluoride (PMSF)-added as lIlL·mL-1 ofa 0.1 mol·L-' solution of PM SF in isopropanol to avoid deactivation of this protease inhibitor (James, 1978). Homogenates were centrifuged at 10,000 g for 20 min in a Sorvall SS34 rotor and supernatants were used directly in enzyme assays or diluted further with homogenization buffer. Enzymes were assayed spectrophotometrically with an LKB Ultrospec 4050 spectrophotometer using slight modifications of assays previously published for fish tissues (Mommsen et aI., 1980). Except for citrate synthase (see below), assays were buffered with 50 mmol· L -I imidazole, pH 7.4 and oxidation/reduction ofNAD(P)H/NAD(P)+ was followed at 340 nm (extinction coefficient E = 6.22 L·mmol-'·cm-'). All assays were performed at 24°C and specific assay conditions were as follows. Alanine Amino Transferase (AlaA T, EC 2.6.1.2) : 50 ilL homogenate, 0.12 mmol' L -I NADH, 200 mmol·L-1 alanine, 0.02 mmol·L-1 pyridoxal-5'-phosphate, 12 units L-LDH and 10.5 mmol·L-' a-ketoglutarate. Hexokinase (HK, EC 2.7.1.1): 50 ilL homogenate, 0.16 mmol' L-I NADP+, 1 mmol·L-1 glucose,S mmol·L-' MgCl" 2 units glucose-6-phosphate dehydrogenase and 1 mmol·L-' ATP. Phosphofructokinase (PFK, EC 2.7.1.11): 50 ilL homogenate, 0.12 mmol· L-' NADH, 2 mmol·L-' ATP, 50 mmol·L-' KCl, 10 mmol·L-' MgCl" 1 unit a-glycerophosphate dehydrogenase,S units aldolase, 3 units triose phosphate isomerase and 3 mmol· L -I fructose-6-phosphate. Lactate dehydrogenase (LDH, EC 1.1.1.27) : forward: 0.12 mmol· L -I NADH, 2 mmol· L -I pyruvate and 10 ilL homogenate; reverse: 2 mmol· L-I NAD+, 250 mmol· L -I L-lactate and 10 ilL homogenate, pH = 7.1. Pyruvate kinase (pK, EC 2.7.1.40): 10 ilL homogenate, 0.12 mmol·L-' NADH, 2.5 mmol·L-' ADP, 30 mmol·L-' KCI, 10 mmol·L-' MgCl" 20 units L-LDH and 0.5 mmol·L-' phosphoenolpyruvate. Fructose-l ,6-bisphosphatase (FBPase, EC 3.1.3.11): 50 ilL homogenate, 0.2 mmol· L-I NADP+, IS mmol· L-I MgCI" 10 units phosphoglucose isomerase, 2 units glucose-6-phosphate dehydrogenase and 0.1 mmol·L-1 fructose-1,6-bisphosphate. Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32): 50 ilL homogenate, 0.12 mmol·L-' NADH, 0.5 mmol·L-' phosphoenolpyruvate, 20 mmol·L-' NaHC03, I mmol·L-1 MnCI" 8 units malate dehydrogenase and 0.2 mmol·L-' deoxyguanosine diphosphate. Citrate synthase (CS, EC 4.1.3.7) : 50 ilL homogenate, 0.1 mmol· L -I 5,5' -dithiobis-(2 nitrobenzoic acid) (DTNB), 0.3 mmol·L-' acetylcoenzyme A and 0.5 mmol·L-' oxaloacetate. This assay was buffered with 50 mmol·L-1 HEPES, pH 8.1 and the reduction ofDTNB was followed at 409 nm (E = 14.15 L'mmol-I'cm-I). For each assay, 50 itL of homogenate was added to 1 mL of buffer containing all reagents except the last one as listed above. Controls were recorded where appropriate before adding a small volume (typically 50 ilL, total volume 1.1 mL) of the last item. Control activity, which was less than 5% in all cases, was subtracted from the activity with substrate and relative values of activity are given as itmoles of product/substrate produced/consumed per minute, per gram fresh weight tissue. REsULTS AND DISCUSSION
As stressed for fishes by Somero and Childress (1980), LDH (fwd) activity may be considered as an index ofthe anaerobic potential of a tissue whereas CS reflects aerobic capabilities. In the blue crab all tissues tested showed CS activity (Table 1), and the highest activities were found in heart and dark muscle, which is not surprising considering the higher density of mitochondria found in these two tissues (Johnson, 1980). In contrast, light muscle shows only 27% of the CS activity of dark muscle (4.2 ± 1.5 JLmol·min-I.g-l vs. 15.7 ± 2.5, P < 0.01), but their LDH activities, in either forward or reverse direction, are similar (Table 1, P >
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LALLIER AND WALSH: BLUE CRAB METABOLIC POTENTIAL
Table I. Maximal in vitro enzyme activities in various tissues of the blue crab Callinectes sapidus (values are expressed in Itmoles·min-'·g-' wet weight at 24°C, and presented as mean values ± SEM. NO: not detectable) Tissues Hepatopancreas
Metabolic function Enzyme· N
Heart
Gills
Activities (mean ± SEM, Itmol·min-'·g-'
Tricarboxylic acid cycle CS 7 2.39 ± 0.57
0.83 ± 0.12
Glycolysis HK PFK PK LOH fwd
1.51 0.42 3.03 3,22
7 4 4 7
Gluconeogenesis LOH rev 7 PEPCK 5 FBPase 7
Dark muscle
0.96 0.20 0.21 0.02
± ± ± ±
0.37 0.20 0.10 0.02
± ± ± ±
0.31 0.16 1.42 0.49
13.66 ± 1.38 2.48 3.32 14.79 17.97
± ± ± ±
0,39 2.00 1.21 1.79
wet weight)
15,66 ± 2.46 1.77 6.08 19.46 41.06
Light muscle
± ± ± ±
0.16 4.12 4.99 4.97
4.16 ± 1.47 0.47 8.64 14.85 39.83
± ± ± ±
0,14 2.35 4.67 6.96
NO 0.16 ± 0.09 0,64 ± 0,05
1.53 ± 0.37 0,04 ± 0,04 0,36 ± 0,07
5.47 ± 1.40 NO 0.27 ± 0,06
12.01 ± 2.56 NO 0.29 ± 0,12
10.52 ± 2.65 0.55 ± 0.55 0.14 ± 0.06
Amino acid metabolism 7 3,75 ± 0.49 AlaAT
3.65 ± 0.38
12.22 ± 0.63
20.28 ± 5.60
13.48 ± 1.74
• cs:
citrate synthase; HK: hexokinase; PFK: phosphofructokinase. I; PK: pyruvate kinase; LDH lactate dehydrogenase, fwd: forward = pyruvate to lactate, rev: reverse = lactate to pyruvate; PEPCK: phosphoenolpyruvate carboxy kinase; FBPase: fructose· [ ,6bisphosphatase; AlaA T: alanine aminotransferase.
0.5), indicating good anaerobic capacities and potential to metabolize lactate in both types of muscle. Additionally, measurable activities of PEPCK and FBPase in light muscle indicate that this tissue (but not dark muscle) may be capable of gluconeogenesis. The high CS activity in heart tissue is not surprising considering that it is perfused directly with oxygenated postbranchial blood. Overall, cardiac muscle seems very comparable to dark muscle since the heart also exhibits a high LDH activity, a pattern matching that found in Pachygrapsus crassipes (Shatzein et aI., 1973). However, in the land crab Birgus latro, heart LDH is thought to be functionally similar to the H4 isozyme from vertebrate heart, as opposed to a M4 type for leg muscle LDH (Morris and Greenaway, 1989). Gluconeogenesis in the cardiac muscle is not likely since the tissue lacks PEPCK activity, but the heart may have a role in the oxidation of lactate. In all muscle tissues, HK activity is lower than PFK activity (although the difference is significant for light muscle only, P < 0.01), possibly indicating a preferred glycolytic pathway as opposed to the pentose phosphate shunt (Change and O'Connor, 1983). Despite low activities, possibly due in part to the fact that the cuticle was not removed before the homogenization phase during tissue preparation, gills may be a good site for lactate oxidation. This tissue possesses LDH activity and the ratio of reverse: forward activities (0.48) is higher than found in muscles (0.27). Also, this tissue has an abundant supply of hemolymph and the best oxygen availability. Gill tissue is known to oxidize lactate and acetate to CO2 in aquatic crabs (Burnett, pers. comm. in Morris and Greenaway, 1989). It has been suggested as a site for gluconeogenesis by Thabrew et aI. (1971), but considering its very low PEPCK activity we find this unlikely. Finally PFK activity is lower than HK (Table 1, P < 0.05), and this would be consistent with a predominance of the pentose phosphate pathway over glycolysis in this tissue (Chang and O'Connor, 1983).
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The net flux of glucose in the hepatopancreas may also be directed toward the pentose phosphate shunt although PFK. activity is not significantly lower than HK activity (Table 1, P > 0.3). But the most striking result is the absence ofLOH activity in the hepatopancreas. The reported value of 0.02 ± 0.02 ~mol' min-I. g-I (Table 1) actually results from a very small activity found in only one individual out of seven and might be due to tissue contamination in the preparative phase (possibly hemocytes). Previous studies have reported such a lack ofLOH enzyme activity (Shatzlein et aI., 1973) but since it was not restricted to LOH, it was attributed to the action of proteinases during and after tissue preparation. However, in our study we have been able to measure activities for all other enzymes in the same homogenates used for LOH determination (Table 1). Furthermore, since all other tissues were homogenized in the same buffer containing PMSF, it is unlikely that this particular serine protease inhibitor exerts a selective inhibitory action on hepatopancreas LOH activity. In agreement with this interpretation, PMSF has no effect on purified rabbit muscle LOH (Sigma, type XXXIX) activity. To assess the possibility that our homogenization procedure may impair LOH activity in hepatopancreas we added purified LDH to hepatopancreas samples and analyzed them as indicated above. The recovery ofLOH activity was complete with a mean 106 ± 11% (N = 5) of standard activity. Walsh and Henry (1990) also found very low, non-significant LOH activity values in the hepatopancreas of C. sapidus and two species of deep-sea crabs. Since LOH is an obligatory first step in lactate oxidation and gluconeogenesis, our findings would suggest that the hepatopancreas is not capable of metabolizing lactate. However, the presence of PEPCK and FBPase activities argue in favor of gluconeogenic capacity in this tissue. This pathway could be fueled directly with circulating pyruvate, derived from lactate oxidation in an other tissue (e.g., muscles, hemocytes). However, it is more likely that gluconeogenic enzymes are present in this tissue to utilize amino-acids such as alanine as precursors; in this regard we found significant activity of AlaA Tin hepatopancreas (Table 1). Finally, PEPCK activity may not reflect only gluconeogenic potential since it may be involved in anaplerotic processes (e.g., generating oxaloacetate from pyruvate to replenish tricarboxylic acid cycle intermediates). Similarly, FBPase could also be involved in other metabolic reactions (Suarez and Mommsen, 1987). This study, focused on maximum enzyme activities in tissue homogenates, was not designed to establish the actual fate of lactate in crustaceans. However, our results do suggest that, unlike vertebrate liver, crustacean hepatopancreas may not be the most important organ for lactate reprocessing following functional or environmental anaerobiosis. Light muscle may be a suitable alternative, considering its gluconeogenic potential and its important mass in the body. Clearly, further studies on the regulation of lactate metabolism, for example in isolated or cultured crustacean cells, are needed. ACKNOWLEDGMENTS This research was supported by an NSF grant to PJW (DCB 8608728). We are grateful to Dr. T. P. Mommsen for his comments on an earlier draft of this manuscript. LITERATURE
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