Indian Journal of Experimental Biology Vol.50, September 2012 pp.652-659
Functional ureogenesis and changes of amino acid metabolism in amphihaline shad hilsa (Tenualosa ilisha, Hamilton-Buchanan) while inhabiting in estuary and freshwater habitats Umesh C Goswami1, Makibur Rahman1 & Nirmalendu Saha2 * 1
Department of Zoology, Gauhati University, Guwahati 781 014, India Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793 022, India
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Received 4 January 2012; revised 8 June 2012 The possible occurrence of a functional ornithine-urea cycle (OUC) and changes of activity of key amino acid metabolism-related enzymes were studied in the amphihaline shad hilsa (Tenualosa ilisha) that were collected from estuarine water of Kakdwip and from freshwater river basin of Bhrahmaputra during the breeding season. Very high concentration of urea was detected in different tissues and plasma of shad hilsa collected from estuarine water compared to the one collected from freshwater river basin. This observation clearly suggests that the shad hilsa has the potential of synthesizing and retaining urea inside the body for the purpose of osmoregulation while living in hypertonic saline environment of estuary. This was accompanied by the presence of high activity of all the five OUC enzymes in hepatic and in certain non-hepatic tissues such as the kidney and muscle of shad hilsa in support of its potential ureogenic capacity while inhabiting in estuarine water. The activities of different key amino acid metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase, alanine aminotransaminase and aspartate aminotransaminase were also found to be significantly higher in shad hilsa of estuarine water compared to the one collected from freshwater habitat. Thus the adjustment to amino acid metabolism in shad hilsa in different environmental salinities appears to play significant roles for osmotic balance and also for proper energy supply in addition to the presence of a functional OUC while migrating between marine and freshwater habitats throughout their life cycle. Keywords: Amino acid metabolism, Ammonia, Environmental salinity, Migratory fish, Ornithine-urea cycle, Osmotic stress, Urea
Although the majority of fish species excrete predominantly ammonia as a nitrogenous waste, a small amount of urea, which is usually around 10-15% of the total nitrogenous wastes, is also found to be excreted by most teleosts1,2 . The formation of urea in most teleosts is thought to result from the breakdown of dietary arginine and/or uric acid3-5, but not from the conventional ornithine-urea cycle (OUC), the primary source of urea synthesis in mammals, adult amphibians and elasmobranchs. The existence of a very active and functional OUC in ureosmotic elasmobranchs and chimaeras has been well established6. The genes for OUC enzymes are either absent or not expressed in all teleosts7,8. In recent years, however, there has been renewed interest on the studies of OUC in teleosts after reports on expression of high levels of OUC enzymes in several teleost species primarily as an adaptation to —————— *Correspondent author Telephone: +91 364 2722322 E-mail:
[email protected];
[email protected]
survive in unique circumstances. Examples include the marine toad fish (Opsanus tau and O. beta)9, two Indian air-breathing catfish (Heteropneustes fossilis and Clarias batrachus)10-12, and the gobiid fish (Mugilogobius abei)13. Further, some of these species excrete significant amount of urea in response to adverse environmental conditions such as confinement (stress), alkaline water, ammonia loading, and exposure to air or while living inside the mud peat under water restricted conditions2,7,8,14. Other than all the mentioned environmental constraints, certain fish species, especially the migratory fish, face the problem of vast changes of environmental salinity while migrating from marine to brackish water and finally to freshwater river basin mainly for breeding purpose. Again the new offspring return back to marine habitat after attaining certain stages of development. Fish that migrate between salt and freshwater habitats are known as amphihaline and the examples include the salmon, hilsa and eel. The osmoregulatory problem in amphihaline fish is
GOSWAMI et al.: UREOGENESIS & AMINO ACID METABOLISM IN SHAD HILSA
quite different compared to the fish living either in marine or freshwater habitat, and hence the ability of these fish to adjust to both habitats during their migration is of special interest. Nitrogenous compounds such as urea, trimethyl amine oxide (TMAO) and certain amino acids play important roles as osmolytes in marine fish15-17. In elasmobranchs, the role of urea as a good osmolyte is well established and is known to accumulate urea in the plasma as high as 0.4-0.8 M6. However, the role of urea for osmotic balance, its possible synthesis through the functional OUC, and also the pattern of amino acid metabolism are not well established in amphihaline fish. The Indian shad hilsa (Tenualosa ilisha) is one of the preferred edible fish in south east Asian countries. The shad hilsa is a widely distributed amphihaline fish species mainly inhabiting the coastal waters starting from the Arabian Sea to the Bay of Bengal and ascending most of the major estuaries, rivers and brackish water bodies of Indo-Pacific faunistic regions. Of late, the abundance of shad hilsa has drastically dwindled in all river systems. It has been observed that various factors like heavy siltation of riverine system, increase of water pollution due to input of industrial wastes into river water basins etc. are responsible for declining the shad hilsa fishery in river system. Therefore, there is an urgent need for conservation of this species. To achieve this objective, mass scale production as well as rearing of seeds may be considered as one of the possible approaches. Several workers have reported the successful breeding and rearing of shad hilsa fry18. Considering the culture prospects of shad hilsa, it is essential to know some vital physiological and biochemical adaptational strategies adopted by this fish to cope with the wide variations in environmental salinities, which they face during migration mainly for breeding purpose. Thus, in the present study, it is aimed at to find out the possible occurrence of a functional OUC and the levels of activity of certain key amino acid metabolism-related enzymes in this amphihaline fish while inhabiting the estuary of Bay of Bengal and the freshwater basin of river Brahmaputra. Materials and Methods Animals—The hilsa fish (Tenualosa ilisha), weighing 500-750 g, were collected from the estuarine water of Kakdwip with water osmolarity of 710 mOsmol L-1 and from the freshwater basin of river Brahmaputra with a water osmolarity of 105
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mOsmol l-1 in the month of June. The temperature of estuarine and freshwater, from where the fish were collected, ranged between 28 to 32 ºC. Immediately after collection, five fish from each habitat were killed by decapitation after collecting blood from the caudal vein with a heparinized syringe; different tissues such as liver, kidney, muscle, gills and brain were dissected out, dipped into liquid nitrogen and transported to the laboratory for further analyses. Estimation of ammonia-N and urea-N— Concentrations of ammonia-N and urea-N in different tissues and in plasma of shad hilsa were measured enzymatically19 after processing the tissue and plasma following the method of Saha and Ratha11. Enzyme assay—A 10% homogenate (w/v) of individual tissues was prepared in a homogenizing buffer containing 100 mM Tris-HCl buffer (pH 7.5), 50 mM KCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 1 mM dithiothreitol (DTT) and a cocktail protease inhibitor, which inhibits a broad range of proteases (Roche, Germany, Cat. No. 11697498001), using a motor-driven Potter-Elvehjem glass homogenizer with a Teflon pestle. The homogenate was treated with 0.5% Triton X-100 in 1:1 ratio for 30 min. The homogenate was then subjected to mild sonication for proper breakage of mitochondria, followed by centrifugation at 10,000 g for 10 min. The supernatant was used for assaying the enzymes. All steps were carried out at 4 °C. All the five ornithine-urea cycle (OUC) enzymes, viz., carbamyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase (ARG) were assayed following the method of Saha et al20. The reaction for all the enzymes was stopped by adding 0.5 mL of 10% perchloric acid per 1 mL of reaction mixture after a specific time of reaction, followed by centrifugation to precipitate out the protein. Citrulline formed by CPS and OTC, citrulline used by ASS and urea formed by ASL and ARG were measured spectrophotometrically (Varian, Cary 50) in the supernatant21 and expressed as enzyme activity. The assay method used here for CPS activity does not distinguish between the two different isoforms of urea synthesis-related enzymes namely CPS I (ammonia-and N-acetyl-Lglutamate-dependent, mitochondrial) and CPS III (glutamine- and N-acetyl-L-glutamate-dependent, mitochondrial). Glutamine synthetase (GS) was
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assayed by the γ-glutamyl transferase reaction as described by Webb and Brown22. Glutamate dehydrogenase (GDH, both reductive amination and oxidative deamination) activity was assayed following the method of Olson and Anfinsen23 with modifications of substrate (optimal) concentrations24. The alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) activities were assayed following the method of Foster and Moon25 with modifications in the substrate (optimal) 24 concentrations . All the enzyme assays were carried out at 30 °C. In case of all the OUC enzymes, one unit of enzyme activity was defined as that amount which catalyzed the formation of 1 µ mole of product or substrate used h-1 at 30 °C. For GS, one unit of enzyme activity was expressed as that amount which catalyzed the formation of 1 µ mole of γ-glutamyl hydroxamate h-1 at 30 °C. In case of GDH, AST and ALT, one unit of enzyme activity was expressed as that amount which catalyzed the oxidation of 1 µmole of NADH or reduction of 1 µ mole of NAD h-1 at 30 °C Measurement of blood and water osmolarity, and tissue water content—The blood was collected with a heparinized syringe from the caudal vein immediately after capturing the fish and centrifuged at 10,000 g for 10 min at 0± 2 °C for separating out the plasma from blood cells. The plasma and water (collected from two catching sites) osmolarity was measured with a Camlab (Model 200) osmometer using the freezing point depression method. The water content in different tissues of both estuarine and freshwater fish was determined by oven drying method following Goswami and Saha26.
Results Tissue ammonia-N and urea-N levels—The urea concentration was found to be about 1.6-4.7 fold higher in different tissues and about 7 fold higher in plasma of fish collected from estuary than that of fish collected from freshwater habitat (Fig. 1). Other than in plasma, highest urea concentration was recorded in liver, followed by kidney, muscle, gills and brain of estuarine fish. Whereas, the ammonia concentration in different tissues and in plasma of fish collected from both the habitats did not show any significant differences. The ammonia concentration was found to be highest in liver, followed by kidney, muscle, gills, brain and plasma of fish collected from both the habitats. OUC and amino acid metabolism-related enzymes activity—A full complement of OUC enzymes was detected in liver, kidney and muscle of shad hilsa collected from both estuary and freshwater habitats (Table 1). However, the levels of activity of most of the enzymes (except the ARG) were found to be 1.6-3.0 fold higher in all the three ureogenic tissues of estuarine fish compared to freshwater habitat fish. Overall, the activities of all the OUC enzymes were highest in liver, followed by kidney and muscle. Relatively high levels of activity of some key amino acid metabolism-related enzymes such as GS, GDH (both in reductive amination and oxidative
Chemicals—All the enzymes, co-enzymes and substrates were purchased from Sigma Chemicals, St. Louis, U.S.A. Other chemicals used were of analytical grades and obtained locally. Deionized double-glass distilled water was used in all preparations. Statistical analysis—The data collected from different replicates were statistically analyzed and presented as mean ± SE (n), where n equals the number of animals in the sample. Comparisons of the unpaired mean values between the experimental and respective controls were made using Student’s t-test and differences with P
