Roles of intestinal glutamate dehydrogenase and glutamine synthetase in environmental ammonia detoxification in the euryhaline four-eyed sleeper, Bostrychus sinensis

Aquatic Toxicology 98 (2010) 91–98

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Roles of intestinal glutamate dehydrogenase and glutamine synthetase in environmental ammonia detoxification in the euryhaline four-eyed sleeper, Bostrychus sinensis W.Y.X. Peh a , S.F. Chew b , B.Y. Ching a , A.M. Loong a , Y.K. Ip a,∗ a b

Department of Biological Science, National University of Singapore, Kent Ridge, Singapore 117543, Republic of Singapore Natural Sciences and Science Education, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore

a r t i c l e

i n f o

Article history: Received 28 September 2009 Received in revised form 16 December 2009 Accepted 25 January 2010 Keywords: Ammonia Bostrychus sinensis Glutamate dehydrogenase Glutamine Glutamine synthetase Intestine

a b s t r a c t This study aimed to examine the hypothesis that intestinal glutamate dehydrogenase (GDH) and glutamine synthetase (GS) could be involved in ammonia detoxification in the euryhaline Bostrychus sinensis exposed to ammonia in a hyperosmotic environment, whereby drinking was essential for osmoregulation. Our results indicate that there was a significant increase in ammonia content in the intestine of B. sinensis exposed to 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. There were also significant increases in the amination and deamination activities and protein abundance of intestinal GDH. The GDH amination/deamination ratio remained unchanged, indicating that there could be increases in the turnover of glutamate. However, the difference between the amination and deamination activities increased 2-fold, implying that there could be an increase in glutamate formation in the intestine. Since the intestinal glutamate content remained unchanged, excess glutamate formed might have been channeled into other amino acids and/or transported to other organs. Indeed, the intestinal glutamine content increased significantly by 2-fold, with a significant increase in the activity and protein abundance of intestinal GS. Since the magnitude of glutamine accumulation in the intestine was lower than those in liver and muscle, which lacked changes in GDH activities, intestinal glutamate could have been shuttled to liver and muscle to facilitate increased synthesis of glutamine therein. By contrast, when fish were exposed to a much higher concentration (30 mmol l−1 ) of NH4 Cl in 5‰ water (pH. 7.0), the magnitude of increase in ammonia content in the intestine was less prominent, and there were no changes in activities and kinetic properties of intestinal GDH. Therefore, it can be concluded that the intestine of B. sinensis was involved in the defense against ammonia toxicity during exposure to ammonia in a hyperosmotic medium. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Ammonia is produced by and toxic to animals; therefore, it has to be excreted and/or detoxified (see reviews by Cooper and Plum, 1987; Ip et al., 2001a; Chew et al., 2006). Teleostean fishes are generally ammonotelic in water, excreting nitrogenous wastes mainly as ammonia, but ammonia excretion can be impeded in alkaline water, during emersion, or when exposed to environmental ammonia. In general, ammonia is more toxic to marine than freshwater teleosts, and the average acute toxicity (1.86 mg NH3 l−1 ) for 17 marine fishes is lower than that (2.79 mg NH3 l−1 ) for 32 freshwater species (Ip et al., 2001a). Such a phenomenon has been attributed to a greater influx of NH4 + through the gills of marine fishes (Ip et al., 2001a), whose branchial epithelia are more permeable to ions than those of freshwater fishes (Evans, 1984). However, there

∗ Corresponding author. Tel.: +65 6516 2702; fax: +65 6779 2486. E-mail address: [email protected] (Y.K. Ip). 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.01.018

could be another reason—the regulation of drinking for osmoregulatory purposes—which has been largely neglected. For freshwater fishes, drinking rates are low since the osmoregulatory processes focus on ion uptake and water removal (Perrot et al., 1992). By contrast, marine fishes imbibe the external medium at high rates in order to prevent dehydration in a hyperosmotic environment (Shehadeh and Gordon, 1969; Kirsch et al., 1985). Intestines of marine teleosts have a crucial role in absorbing water, which occurs in conjunction with active absorption of ions, e.g. Na+ , K+ and Cl− (Skadhauge, 1974; Mackay and Lahlou, 1980; Usher et al., 1991). Since NH4 + is known to substitute for K+ to act as substrates for Na+ /K+ -ATPase and Na+ :K+ :2Cl− co-transporter (see reviews by Ip et al., 2001a; Chew et al., 2006), it is highly probable that intestines would act as an additional route of ammonia influx in fish exposed to seawater, and intestinal cells would undergo ammonia-loading after ingestion of the external medium that contains ammonia. Surprisingly, no information is available on the involvement and ability of fish intestines to detoxify environmental ammonia at present.

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In order to elucidate the functional role of fish intestine in ammonia detoxification, it would be essential to work with an euryhaline fish that has a relatively high tolerance of environmental ammonia. The four-eyed sleeper, Bostrychus sinensis (Lacepède), is an appropriate specimen as it is a facultative air-breathing teleost that inhabits brackish (Rainboth, 1996) and marine (Ni and Kwok, 1999; Huang, 2001) environments in the Indo-Pacific. It can be found occasionally in freshwater (Hwang et al., 1988; Kottelat et al., 1993), but it also inhabits marine ecosystems like the coral reefs (Nguyen and Nguyen, 2006). In estuaries, it would be confronted with salinity stress during tidal changes. Recently, Peh et al. (2009) reported that acclimation of B. sinensis from 5‰ water to seawater (30‰) involved both branchial and intestinal osmoregulatory responses. There were significant increases in protein abundance of branchial Na+ /K+ -ATPase ␣-subunit and Na+ :K+ :2Cl− co-transporter, and in the activity and protein abundance of intestinal Na+ /K+ -ATPase from B. sinensis acclimated to seawater, which indicate indirectly that fish exposed to seawater imbibed the external medium and increased Na+ and water absorption through the intestine (Peh et al., 2009). On the other hand, B. sinensis can be confronted with environmental ammonia toxicity when trapped in crevices along the intertidal zone of estuaries. The continual excretion of ammonia into a small volume of external medium would result in high concentrations of environmental ammonia which would in turn reduce the rate of ammonia excretion. Previous works reveal that B. sinensis up-regulates liver and muscle glutamine synthetase (GS) activities and detoxify ammonia to glutamine during environmental ammonia exposure (Anderson et al., 2002) or emersion (Ip et al., 2001b). In addition, it has been reported that B. sinensis possesses high levels of GS activity in the digestive tract (Anderson et al., 2002), but the functional role of intestinal GS in B. sinensis is unclear at present. Therefore, this study was undertaken to examine the hypothesis that the intestine of B. sinensis exposed to 15 mmol l−1 NH4 Cl in seawater (with pH adjusted to 7.0) could be involved in environmental ammonia detoxification. We hypothesized that exposure to ammonia in a hyperosmotic medium would lead to an increase in the GS activity from the intestine of B. sinensis. Since glutamine synthesis requires glutamate as a substrate, and since glutamate dehydrogenase (GDH) is essential to glutamate metabolism, we further hypothesized that there could be an increase in the amination activity of intestinal GDH in fish exposed to 15 mmol l−1 NH4 Cl in seawater. Western blotting was performed to examine whether protein abundance of GS and GDH would be up-regulated by exposure to 15 mmol l−1 NH4 Cl in seawater. In addition, efforts were made to determine whether accumulation of free amino acids (FAAs), especially glutamate and glutamine, would occur in the intestine. In order to elucidate whether cooperation existed between the intestine and other tissues/organs in ammonia detoxification, we also examined activities of GDH and GS, and contents of glutamate and glutamine in liver and muscle of fish exposed to 15 mmol l−1 NH4 Cl in seawater. Preliminary results obtained indicated that B. sinensis could survive in 30 mmol l−1 NH4 Cl in 5‰ water for at least 6 days, although it succumbed after 1–2 days of exposure to 30 mmol l−1 NH4 Cl in sea water. Since it is well established that osmoregulation in a hypoosmotic environment involves a much lower rate of drinking than in a hyperosmotic environment (Perrot et al., 1992; Fuentes and Eddy, 1997), and since the gastrointestinal tract of fish exposed to a hyperosmotic environment would act to increase ion uptake to facilitate water absorption (Mackay and Lahlou, 1980; Usher et al., 1991), it was essential to determine the intestinal ammonia content instead of the concentrations of ammonia in the intestinal lumen, which would have no direct effects on intracellular enzyme activities and nitrogen metabolism. Therefore, an attempt was made to confirm that exposure of B. sinensis to a 2-fold higher concentra-

tion of ammonia in a hypoosmotic medium (30 mmol l−1 NH4 Cl in 5‰water) would result in a less prominent build up of ammonia in the intestinal tissues and thus have no significant effects on the intestinal GDH activity. 2. Materials and methods 2.1. Fish B. sinensis (50–130 g) were purchased from Yuen Long wet market in Hong Kong and transferred by air to the National University of Singapore (NUS). Fishmongers in the wet market collected them from the Pearl River Delta, a naturally brackish environment, and kept them in 5–7‰ water. In NUS, groups of 30 individuals or less were kept submerged in aerated water of 5‰ at 25–28 ◦ C, in fiberglass containers (L 72 cm × W 55 cm × H 56 cm), under a 12 h:12 h dark:light regime. Waters of 5‰ salinity was prepared by mixing aerated freshwater with aerated natural seawater. Seawater (30–31‰) was collected from the sea 2 km away from the Singapore main island and transferred back to NUS. No attempt was made to differentiate the sexes. Fish were acclimated to laboratory conditions in 5‰ water for at least 2 weeks before experimentation. During the acclimation period, water was changed every 2 days and fish were fed cod fish fillet every 4 days. Fish were fasted 48 h prior to experiments to empty the intestine. Wet mass of the fish was obtained to the nearest 0.1 g using a Shimadzu animal balance (Shimadzu, Kyoto, Japan). Experimental protocols of this study were approved by the NUS Institutional Animal Care and Use Committee (Permit S 050/08). 2.2. Exposure of fish to 15 mmol l−1 NH4 Cl in seawater and collection of samples Preliminary experiments indicated that B. sinensis could not withstand 6 days of exposure to 30 mmol l−1 NH4 Cl, but could survive well in 15 mmol l−1 NH4 Cl, in seawater (pH 7.0). Thus, fish were kept individually in plastic aquaria tanks (L 23.5 cm × W 13 cm × H 13 cm) containing 15–20 volumes (w/v) of aerated external medium, and exposed to daily increases in salinity through a 5-day period from 5‰ to 10‰, followed by 15‰, 20‰, 25‰ and finally to seawater (30‰; pH 8.3). They were then exposed to seawater with pH adjusted to 7.0 for another 3 days, followed with 1 or 6 days of exposure to 15 mmol l−1 NH4 Cl in seawater (pH 7.0). No mortality of experimental fish was recorded. Control fish were kept in seawater (pH 7.0) for a total of 6 days. Control and experimental fish were not fed during the experimental period. Water was changed daily, and water samples were collected randomly before the change of water to confirm the ammonia concentration to be at 15 ± 1 mmol l−1 24 h after the introduction of NH4 Cl. As for the control fish the ammonia concentration in the external medium never exceeded 0.2 mmol l−1 at the end of the 24-h period. After 6 days, experimental fish (N = 7) and control fish (N = 7) were killed by a strong blow to the head. The intestine, liver and lateral muscle were excised. The intestine was flushed quickly with saline solution and divided into halves longitudinally. Tissue samples were immediately freeze-clamped with tongs pre-cooled in liquid nitrogen and stored at −80 ◦ C for future analysis. Some fish were killed after 1 day of exposure to 15 mmol l−1 NH4 Cl in seawater, and the intestines were sampled for ammonia assay only. 2.3. Determination of ammonia content Frozen intestine samples were ground to powder, weighed, and homogenized three times for 20 s with 10 s intervals at 24,000 r.p.m. in 5 volumes (w/v) of ice-cold 6% trichloroacetic acid

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(TCA) using an Ultra-Turrax T25 homogenizer (Ika-Labortechnik, Staufen, Germany). The homogenates were centrifuged at 10,000 × g and 4 ◦ C for 15 min to obtain the supernatant. The pH of the supernatant was adjusted to 5.5–6.0 with 2 mol l−1 KHCO3 . Ammonia content was determined according to the method of Bergmeyer and Beutler (1985). Results were expressed as ␮mol g−1 tissue. 2.4. Determination of GS and GDH activities Tissue samples were homogenized three times in 5 volumes (w/v) of ice-cold extraction buffer containing 50 mmol l−1 imidazole-HCl (pH 7.0), 50 mmol l−1 sodium fluoride, 3 mmol l−1 EGTA and 3 mmol l−1 EDTA at 24,000 r.p.m. for 20 s each with a 10 s off interval using an Ultra-Turrax homogenizer. The homogenates were centrifuged at 10,000 × g and 4 ◦ C for 20 min. A portion of the supernatant obtained from intestine samples was diluted to 2 ␮g protein ␮l−1 in Laemmli’s buffer (Laemmli, 1970) and denatured before they were used for subsequent Western blotting. For GDH assay, the supernatant was passed through a 5 ml Econo-Pac 10DG desalting column (Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with 50 mmol l−1 imidazole-HCl (pH 7.0) and eluted with the same buffer. As for GS assay, the column was equilibrated and sample eluted with a buffer composed of 50 mmol l−1 imidazole-HCl (pH 7.6), 50 mmol l−1 KCl and 1 mmol l−1 EDTA (Anderson et al., 2002). The resulting eluents were used for the determination of GDH or GS activities. The dilution involved was corrected by monitoring the change in protein concentrations of the sample before and after passing through the column. Protein was determined according to the method of Bradford (1976). Bovine gamma globulin dissolved in 25% glycerol was used as a standard for comparison. GS activity was determined by the method of Tay et al. (2003), and expressed as ␮mol ␥glutamylhydroxamate formed min−1 g−1 tissue. Freshly prepared glutamic acid monohydroxamate solution was used as a standard for comparison. The amination and deamination activities of GDH were determined according to Peng et al. (1994). Amination activity was expressed as ␮mol NADH oxidized min−1 g−1 tissue while deamination activity was expressed as ␮mol formazan formed min−1 g−1 tissue. Vmax (␮mol NADH oxidized min−1 tissue) and Km (mmol l−1 ) of GDH in the amination direction from the intestine of fish exposed to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. 2.5. SDS-Page and Western blotting Proteins from intestine samples were separated by SDS-PAGE (10% acrylamide for resolving gel, 4% acrylamide for stacking gel) under conditions as described by Laemmli (1970) using a vertical mini-slab apparatus (Bio-Rad Laboratories). Proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes using a transfer apparatus (Bio-Rad Laboratories). After transfer, membranes were blocked overnight with 10% skim milk in TTBS (0.05% Tween 20 in Tris-buffered saline: 20 mmol l−1 Tris–HCl; 500 mmol l−1 NaCl, pH 7.6). The blocked membranes were then incubated at room temperature for 1 h with either rabbit anti-B. sinenesis GS primary antibody generated by Anderson et al. (2002) (1:4000, Quality Controlled Biochemicals, Hopkinton, MA, USA), or rabbit anti-bovine GDH primary antibody (1:4000, US Biological, Swampscott, MA, USA). For internal and loading controls, membranes loaded with the same samples were blocked for 1 h at room temperature in the blocking buffer and incubated overnight at 4 ◦ C with rabbit antihuman glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH)

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antibody (1:1000, Cell Signaling Technology, Inc.). All primary antibodies were diluted in 1% BSA in TTBS. Membranes were then rinsed and incubated with goat anti-rabbit horseradishperoxidase-conjugated antibody (1:10,000 dilution; Santa Cruz Biotechnology, CA, USA). Bands were visualized by chemiluminescence with the enhanced chemiluminescence (ECL) system (Amersham Biosciences, Piscataway, NJ, USA) with exposure to Konica Minolta film and processed using Kodak X-Omat 3000 RA processor (Kodak, Rochester, NY, USA). Since anti-bovine GDH primary antibody was involved in this study, we used mouse liver as a positive control and confirmed the 57 kDa band, as indicated by molecular markers, to be the GDH band as has been reported for other fishes (Chew et al., in press; Ip et al., 2009). Densitometric quantification of band intensities were performed using ImageJ (version 1.40, NIH), with a calibrated 37 step reflection scanner scale (1 × 8 ; Stouffer #R3705-1C) as a reference for comparison. Results are presented as relative protein abundance of GS or GDH normalized with GAPDH. 2.6. Determination of FAAs To determine FAA contents, the intestine, liver and muscle samples were weighed and ground to a powder in liquid nitrogen. Five volumes of ice-cold 6% TCA were added to each sample and the mixture was homogenized three times for 20 s each (with 10 s off intervals) with an Ika-werk Staufen Ultra-Turrax homogenizer (Janke and Kundel, Stanfeni, Germany) at 24,000 r.p.m. The samples were then centrifuged for 20 min at 10,000 × g, 4 ◦ C. The supernatant obtained was stored at −20 ◦ C until analysis. For FAAs analysis, the pH of the supernatant (N = 4) obtained above was adjusted to pH 2.2 with 4 mol l−1 lithium hydroxide and diluted appropriately with 0.2 mol l−1 lithium citrate buffer (pH 2.2). The level of FAAs was analyzed using a Shimadzu LC-10ATVP amino acid analysis system with a Shim-pack-07/Amino Li-type column. A complete FAA profile was reported for the intestine, but for liver and muscle, only contents of glutamate and glutamine were presented. The total FAA (TFAA) content was calculated by the summation of all FAAs, while the content of total essential free amino acid (TEFAA) was calculated as the sum of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine contents. Results for FAA analyses were expressed as ␮mol g−1 tissue. 2.7. Exposure of fish to 30 mmol l−1 NH4 Cl in 5‰ water and determination of intestinal GS and GDH activities Fish were kept individually in plastic aquaria tanks (L 23.5 cm × W 13 cm × H 13 cm) containing 15–20 volumes (w/v) of aerated water of 5‰ salinity, adjusted to pH 7.0, at 25–28 ◦ C for 8 days. Then, the fish were exposed to 30 mmol l−1 NH4 Cl in 5‰ water with pH adjusted to 7.0 for a total of 6 days. There was no mortality of experimental fish exposed to 30 mmol l−1 NH4 Cl in 5‰ water although B. sinensis could not withstand 6 days of exposure to 30 mmol l−1 NH4 Cl in seawater (pH 7.0). Control fish was kept in 5‰ water (pH 7.0) without NH4 Cl for the same length of time. Water was changed daily throughout the experimental period. Some fish were killed after 1 day of exposure to ammonia, and the intestines were sampled for ammonia assay. Others were killed at the end of day 6 and the intestine sampled for the determination of ammonia content and activities of GS and GDH as described above. 2.8. Statistics Results are presented as mean ± standard error of the mean (S.E.M.). Two-tail Student’s t-test was used to evaluate differences between means. Differences where P <0.05 were regarded as statistically significant.

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Table 1 Activities of glutamine synthetase (GS; ␮mol min−1 g−1 tissue) and glutamate dehydrogenase (GDH; ␮mol min−1 g−1 tissue), in the amination and deamination directions, the difference between GDH amination and deamination activities (␮mol min−1 g−1 tissue), and the GDH amination:deamination ratio (which has no unit), from the intestine of Bostrychus sinensis exposed to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. Intestine

Seawater (pH 7.0) Controla

GS GDH amination GDH deamination GDH amination minus deamination GDH amination:deamination ratio a *

1.47 20.7 1.11 19.6 19.1

± ± ± ± ±

0.21 2.6 0.14 2.5 1.4

NH4 Cl (15 mmol l−1 )a 3.44 39.8 2.38 37.4 16.6

± ± ± ± ±

0.36* 5.4* 0.29* 5.1* 1.2

Values are means ± S.E.M., N = 5. Significantly different from the corresponding control, P < 0.05.

3. Results 3.1. Fish exposed to 15 mmol l−1 NH4 Cl in seawater 3.1.1. Ammonia content in intestine The ammonia content (␮mol g−1 tissue) in the intestine of fish exposed to 15 mmol l−1 NH4 Cl in seawater for 1 and 6 days were 8.75 ± 2.5 and 9.73 ± 0.94, respectively, which were significantly greater than that of the control fish (1.35 ± 0.93) kept in seawater. 3.1.2. GS and GDH activities in intestine There was a significant increase in the GS activity from the intestine of fish exposed to 15 mmol l−1 NH4 Cl in seawater (Table 1). Additionally, there were significant increases in intestinal GDH amination activities, at various concentrations of ␣-KG or NH4 + , in fish exposed to 15 mmol l−1 NH4 Cl in seawater (Fig. 1 and Table 1), resulting in a significant change in Vmax , but there were no significant changes in Km values for ␣-KG and NH4 + (Table 2). The intestinal GDH deamination activity also increased significantly (2.1-fold) in fish exposed to 15 mmol l−1 NH4 Cl in seawater. However, the GDH amination/deamination ratio remained unchanged, although the difference between the amination and deamination GDH activities from the intestine of the experimental fish was ∼2fold greater than that of the control (Table 1). 3.1.3. Protein abundance of GDH and GS in intestine Intestinal GDH was detectable as a single band at around 57 kDa and intestinal GS was detected at ∼45 kDa with 50 ␮g of protein (Fig. 2). There were significant increases in the normalized protein abundance of intestinal GDH and GS in fish exposed to

Table 2 Vmax (␮mol NADH oxidized min−1 g−1 tissue) and Km (mmol l−1 ) of glutamate dehydrogenase (GDH), in the amination direction, from the intestine of Bostrychus sinensis exposed to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. Intestinal GDH

Seawater (pH 7.0) Controla

NH4 Cl (15 mmol l−1 )a

[␣-KG] Vmax Km

22.7 ± 2.9 0.52 ± 0.04

43.3 ± 5.8* 0.51 ± 0.03

[NH4 + ] Vmax Km

26.9 ± 3.4 70.6 ± 2.0

51.3 ± 6.6* 70.5 ± 3.1

a *

Values are means ± S.E.M., N = 5. Significantly different from the corresponding control, P < 0.05.

Fig. 1. Effects of varying concentrations of (A) ␣-ketoglutarate (␣-KG) and (B) NH4 + on the activity (␮mol NADH oxidized min−1 g−1 tissue) of glutamate dehydrogenase (GDH), in the amination direction, from the intestine of Bostrychus sinensis exposed to seawater (30‰, pH 7.0; closed circle) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0; open circle) for 6 days. Values are means ± S.E.M., N = 5. *Significantly different from the corresponding control (P < 0.05).

15 mmol l−1 NH4 Cl in seawater as compared with the control (Fig. 2). 3.1.4. Contents of FAA, TEFAA and TFAA in intestine There was a significant increase in the glutamine content, with the glutamate content remained unchanged, in the intestine of fish exposed to 15 mmol l−1 NH4 Cl in seawater. In addition, there were only minor changes in contents of several FAAs (Table 3). Overall, there were no significant changes in the intestinal TEFAA and TFAA contents in fish exposed to 15 mmol l−1 NH4 Cl in seawater (Table 3). 3.1.5. Contents of glutamate and glutamine, and activities of GDH and GS from liver and muscle There were significant increases in contents of the glutamine in liver and muscle of fish exposed to 15 mmol l−1 NH4 Cl in seawater (Table 4). On the other hand, exposure to 15 mmol l−1 NH4 Cl in seawater led to a significant decrease in the muscle glutamate content with the liver glutamate content remained unchanged (Table 4). In contrast to intestine, there were no significant changes in activities of GDH, in both amination and deamination directions, although the GS activities increased significantly, from liver and muscle of fish exposed to 15 mmol l−1 NH4 Cl in seawater (Table 5).

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Table 3 Contents (␮mol g−1 tissue) of various free amino acids (FAA), total FAA (TFAA) and total essential FAA (TEFAA) in the intestine of Bostrychus sinensis exposed to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. Intestinal FAA

Seawater (pH 7.0) NH4 Cl (15 mmol l−1 )a

Controla Alanine Arginine Asparagine Aspartate Glutamate Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Taurine Threonine Tryptophan Tyrosine Valine TFAA TEFAA a *

0.52 0.091 0.078 0.96 1.4 0.43 0.53 0.059 0.037 0.13 0.21 0.025 0.056 0.065 0.11 14 0.13 0.021 0.062 0.087

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.006 0.010 0.26 0.1 0.04 0.08 0.006 0.004 0.01 0.04 0.002 0.007 0.008 0.00 1 0.02 0.001 0.010 0.007

0.94 0.084 0.096 0.25 1.6 0.93 0.35 0.058 0.053 0.27 0.19 0.038 0.091 0.13 0.16 14 0.13 0.030 0.090 0.13

19 ± 1 0.84 ± 0.06

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.26 0.006 0.030 0.06 0.2 0.17* 0.03 0.011 0.007 0.04* 0.02 0.007 0.008* 0.03 0.02 1 0.02 0.001* 0.010 0.02

20 ± 1 1.1 ± 0.1

Values are means ± S.E.M., N = 4. Significantly different from the corresponding control, P < 0.05.

Table 4 Contents (␮mol g−1 tissue) of glutamate and glutamine in the liver and muscle of Bostrychus sinensis exposed to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. Seawater (pH 7.0) NH4 Cl (15 mmol l−1 )a

Controla 5.7 ± 0.2 1.9 ± 0.1

8.5 ± 1.5 4.4 ± 0.6*

Muscle Glutamate Glutamine

0.44 ± 0.07 0.63 ± 0.13

0.23 ± 0.04* 8.2 ± 1.0*

*

Table 6 Activities of glutamine synthetase (GS; ␮mol min−1 g−1 tissue) and glutamate dehydrogenase (GDH; ␮mol min−1 g−1 tissue), in the amination and deamination directions, the difference between GDH amination and deamination activities (␮mol min−1 g−1 tissue), and the GDH amination:deamination ratio (which has no unit), from the intestine of Bostrychus sinensis after 6 days of exposure to 5‰ water (pH 7.0; control) or 30 mmol l−1 NH4 Cl in 5‰ water (pH 7.0). Intestine

5‰ water (pH 7.0) Controla

Liver Glutamate Glutamine

a

Fig. 2. (A) Western blots of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and (B) relative protein abundance (arbitrary densitometric unit) of GS (white bars) and GDH (black bars) normalized with GAPDH from intestine of Bostrychus sinensis exposed to seawater (pH 7.0) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0) for 6 days. Values are means ± S.E.M., N = 4. *Significantly different from the corresponding control (P < 0.05).

Values are means ± S.E.M., N = 4. Significantly different from the corresponding control, P < 0.05.

GS GDH amination GDH deamination GDH amination minus deamination GDH amination:deamination ratio a *

1.47 34.7 2.00 32.7 20.2

± ± ± ± ±

0.18 6.6 0.47 6.2 3.7

NH4 Cl (30 mmol l−1 )a 2.84 32.4 2.07 30.4 15.9

± ± ± ± ±

0.26* 3.9 0.31 3.6 0.5

Values are means ± S.E.M., N = 5. Significantly different from the corresponding control (P < 0.05).

3.2. Fish exposed to 30 mmol l−1 NH4 Cl in 5‰ water Table 5 Activities of glutamine synthetase (GS; ␮mol min−1 g−1 tissue) and glutamate dehydrogenase (GDH; ␮mol min−1 g−1 tissue), in the amination and deamination directions, the difference between GDH amination and deamination activities (␮mol min−1 g−1 tissue), and the GDH amination:deamination ratio (which has no unit), from the liver and muscle of Bostrychus sinensis after 6 days of exposure to seawater (pH 7.0; control) or 15 mmol l−1 NH4 Cl in seawater (pH 7.0). Seawater (pH 7.0) Controla

NH4 Cl (15 mmol l−1 )a

Liver GS GDH amination GDH deamination GDH amination minus deamination GDH amination:deamination ratio

0.35 ± 0.09 26.4 ± 2.3 1.11 ± 0.13 25.3 ± 2.2 24.5 ± 1.7

1.46 ± 0.33* 28.7 ± 2.9 0.99 ± 0.15 27.7 ± 2.7 30.0 ± 2.0

Muscle GS GDH amination GDH deamination

0.24 ± 0.08 n.d. n.d.

0.87 ± 0.11* 0.19 ± 0.02 n.d.

n.d.: not detectable. a Values are means ± S.E.M., N = 5. * Significantly different from the corresponding control (P < 0.05).

3.2.1. Ammonia content in intestine The ammonia content in the intestine of fish exposed to 30 mmol l−1 NH4 Cl in 5‰ water for 1 day was 2.25 ± 1.5 ␮mol g−1 which was comparable with that of the control fish kept in 5‰ water (1.45 ± 0.45 ␮mol g−1 ). By contrast, after 6 days of exposure to 30 mmol l−1 NH4 Cl in 5‰ water, the intestinal ammonia content increased significantly to 6.53 ± 1.34 ␮mol g−1 . 3.2.2. Activities of GS and GDH in intestine There was a significant increase in GS activities, but amination and deamination activities of GDH remained unchanged, in the intestine of B. sinensis exposed to 30 mmol l−1 NH4 Cl in 5‰ water (Table 6). In contrast with fish exposed to 15 mmol l−1 NH4 Cl in seawater, exposure to 30 mmol l−1 NH4 Cl in 5‰ water had no significant effects on intestinal GDH amination activities, determined at various concentrations of ␣-KG or NH4 + (Fig. 3). 4. Discussion The gastrointestinal tracts of marine teleosts play an important role in osmo- and iono-regulation (Mackay and Lahlou, 1980; Usher

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4.1. Intestinal GS had a minor role in ammonia detoxification in fish exposed to 15 mmol l−1 NH4 Cl in seawater In agreement with the report of Anderson et al. (2002), there were up-regulation of intestinal GS activity and protein abundance in B. sinensis exposed to 15 mmol l−1 NH4 Cl in seawater for 6 days. Anderson et al. (2002) first demonstrated that high GS activity was present in the gastrointestinal tract of B. sinensis. Subsequently, Mommsen et al. (2003) reported the presence of high level of GS activity in the gastrointestinal tract of the tilapia, Oreochromis mossambicus, and hypothesized that GS was concentrated towards the posterior regions of the intestine for the efficient trapping of ammonia generated upstream in the intestinal tract as a by-product of digestion or through microbial activities. However, while the marble goby, Oxyeleostris marmorata, also possesses high activity of intestinal GS (Tng et al., 2008), accumulation of glutamine in its intestine does not occur during the 24 h post-feeding period despite a significant increase in the intestinal GS activity at hour 12 (Tng et al., 2008). Instead, there were significant increases in GDH activities, in both amination and deamination directions, and glutamate content in the intestine of O. marmorata after feeding. Therefore, Tng et al. (2008) concluded that intestinal GDH had a more important role than intestinal GS in the overall defense against postprandial ammonia toxicity in juvenile O. marmorata. Results obtained in this study corroborate the conclusion of Tng et al. (2008), since the quantity of glutamine accumulated in the intestine of B. sinensis exposed to 15 mmol l−1 NH4 Cl in seawater was relatively minor (∼0.5 ␮mol g−1 ).

4.2. Up-regulation of intestinal GDH activity and protein abundance in fish exposed to 15 mmol l−1 NH4 Cl in seawater

Fig. 3. Effects of varying concentrations of (A) ␣-ketoglutarate (␣-KG) and (B) NH4 + on the activity (␮mol NADH oxidized min−1 g−1 tissue) of glutamate dehydrogenase (GDH), in the amination direction, from the intestine of Bostrychus sinensis exposed to 5‰ water (pH 7.0) or 30 mmol l−1 NH4 Cl in 5‰ water (pH 7.0) for 6 days. Values are means ± S.E.M., N = 5.

et al., 1991; Marshall and Grosell, 2006). Drinking rates in freshwater fishes are low, and there is no obvious physiological role (Perrot et al., 1992), except in larvae where there may be links with calcium uptake or feeding (Tytler et al., 1990). By contrast, marine teleosts have to imbibe seawater in order to prevent dehydration (Shehadeh and Gordon, 1969; Kirsch et al., 1985). As imbibed seawater passes down the gastrointestinal tract, ions are absorbed across the intestinal epithelium to facilitate water absorption (Loretz, 1995; Grosell et al., 2001). Therefore, regulation of drinking is an essential part of the osmoregulatory process in teleosts moving from a hypoosmotic medium to a hyperosmotic medium and vice versa. The transfer of euryhaline flounder, eels and juvenile Atlantic salmon from freshwater to higher salinities results in a 10–20-fold increase in drinking (Balment and Carrick, 1985; Tierny et al., 1995; Fuentes and Eddy, 1997). Hence, we proposed that the ammonia content of intestinal tissues in B. sinensis exposed to seawater containing 15 mmol l−1 NH4 Cl would be greater than that of control fish kept in seawater since the former had to imbibe the external medium to compensate for dehydration. Indeed, our results support such a proposition, and suggest that the intestine of B. sinensis could take part in environmental ammonia detoxification in cooperation with other organs.

We demonstrated for the first time that exposure to 15 mmol l−1 NH4 Cl in seawater for 6 days resulted in increases in amination and deamination activities (Vmax ) of GDH from the intestine of B. sinensis. Western blotting results indicate that the increase in GDH activity was attributable to an increase in GDH protein expression. Induction of GDH activity has been reported for brains and livers of several teleosts, e.g. the scale-less carp, Gymnocypris przewalskii (Wang et al., 2003), the air-breathing catfish, Clarias batrachus (Saha et al., 2000), and the largemouth bass, Micropterus salmoides (Kong et al., 1998), exposed to ammonia. However, there is no record of increases in activity and protein expression of GDH in fish intestine in response to environmental ammonia exposure in the literature. Isoforms of GDH have been described for zebrafish (D. rerio; NM 212576.1, NM 199545.3), rainbow trout (Oncorhynchus mykiss; AF427344.1, AF427342.1) and Atlantic salmon (Salmo salar; AJ556995.1, NM 001123636.1, AJ532826.1, AJ532827.1). Two distinct forms of GDH, with different affinities for glutamate, ammonia and ␣-ketoglutarate, have been reported in hibernating Richardson’s ground squirrel (Thatcher and Storey, 2001). Recently, Loong et al. (2008) reported that aestivation led to a significant change in the amination, but not the deamination, activity of GDH from liver of the African lungfish Protopterus annectens, and suggested that variations in GDH isoforms could be involved. Since the affinity of the intestinal GDH to ␣-KG and NH4 + , and the GDH amination/deamination ratio, remained unchanged in B. sinensis exposed to 15 mmol l−1 NH4 Cl in seawater, it can be deduced that a change in expression of GDH isoforms might not be involved. However, whether intestinal GDH of B. sinensis was regulated by ADP-ribosylation (Herrero-Yraola et al., 2001) is unclear at present.

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4.3. Effects of exposure to 15 mmol l−1 NH4 Cl in seawater on intestinal FAAs and their implications

4.5. The roles of intestinal GDH and hepatic GDH in amino acid metabolism and ammonia detoxification

Our results indicate that the turnover of glutamate could be upregulated in the intestine of B. sinenesis exposed to 15 mmol l−1 NH4 Cl in seawater since there were increases in GDH amination and deamination activities without a change in the amination/deamination. Furthermore, there could be an increase in the formation of glutamate through the GDH reaction because of the 2-fold increase in the difference between the amination and the deamination activities. However, the glutamate content and contents of other non-essential FAAs, except glutamine, remained unchanged in the intestine. Since the increase in glutamine content was relative minor, it can be concluded that increased glutamate synthesis in the intestine, if occurred, was not utilized solely for ammonia detoxification through increased glutamine synthesis therein. Indeed, exposure of B. sinensis to 15 mmol l−1 NH4 Cl in seawater led to significant increases in GS activities and glutamine contents in liver and muscle, but the liver glutamate content remained unchanged and the muscle glutamate content decreased only slightly (∼0.22 ␮mol g−1 ). More importantly, unlike intestinal GDH, there were no changes in the amination and deamination activities of GDH from the liver, while the muscle GDH activity was low or undetectable. Hence, it is probable that the excess glutamate formed as a result of increased GDH activity in the intestine of B. sinensis was transported to liver and muscle to support increased GS synthesis for ammonia detoxification. Taken together, these results confirm that the mucosal surface of the intestine could act as an additional route of ammonia entry in B. sinensis exposed to seawater. They also indicate that the intestine of B. sinensis acted more than a digestive, absorptive and osmoregulatory organ, as it could take part in the detoxification of ammonia.

The intestines of carnivorous fishes, like B. sinensis, are adapted to process diets that are high in protein and low in carbohydrate (Buddington et al., 1997). Karlsson et al. (2006) determined changes in plasma concentrations of FAAs and their metabolites in preand post-hepatic blood following a single meal in rainbow trout (O. mykiss), and confirmed that amino acids could be metabolized in the intestine before reaching the liver. The plasma ammonia level in the hepatic portal vein was higher than that in the dorsal aorta, and the difference between the two blood sampling sites increased during FAA absorption after a meal. Thus, Karlsson et al. (2006) concluded that deamination of certain amino acids occurred in the intestine of the rainbow trout after feeding. Subsequently, Tng et al. (2008) reported that postprandial amino acid metabolism indeed occurred in the intestine of juvenile O. marmorata. The major amino acid accumulated in the intestine and liver of juvenile O. marmorata after feeding was glutamate, and feeding led to a significant increase in GDH activities in the intestine and liver of O. marmorata, which resulted in a high retention of the ingested nitrogen for somatic growth. Consequently, only 33% of the ingested nitrogen was excreted during the 24 h post-feeding period, and the brain was effectively prevented from exposure to postprandial ammonia toxicity (Tng et al., 2008). From these results, it can be concluded that intestinal GDH is pivotal to effective postprandial nitrogen retention and defense against postprandial ammonia toxicity in fish, and it is therefore logical that intestinal GDH was involved in ammonia detoxification in B. sinensis exposed to environmental 15 mmol l−1 NH4 Cl in seawater. B. sinensis is able to detoxify ammonia through increased glutamine synthesis in liver and muscle during emersion (Ip et al., 2001b) or environmental ammonia exposure (Anderson et al., 2002). Increased glutamine synthesis requires a supply of glutamate, but there is low or undetectable activity of GDH in fish muscle while fish liver is the main site of glutamate catabolism (Campbell, 1991). Excess dietary free amino acids that are not used for protein synthesis and other essential functions are catabolized to ammonia and ␣-keto acids through transdeamination in the liver (Campbell, 1991). Since transdeamination involves glutamate degradation, hepatic GDH would have to operate in the deamination direction. Thus, it would be essential for the intestine to supply glutamate through increased GDH amination activity to the liver and muscle to support increased glutamine synthesis therein.

4.4. Exposure to 30 mmol l−1 NH4 Cl in 5‰ water had no significant effect on intestinal GDH There could be several reasons why B. sinensis succumbed to 30 mmol l−1 NH4 Cl in seawater (pH 7.0), but survived 6 days of exposure to 30 mmol l−1 NH4 Cl in 5‰ water (pH 7.0). Firstly, the branchial epithelia of fish exposed to seawater could be more “leaky” to NH3 and NH4 + than those of fish exposed to 5‰ water (Ip et al., 2001a; Chew et al., 2006). Secondly, fish exposed to ammonia in a hyperosmotic medium could have a higher drinking rate than those exposed to ammonia in a hypoosmotic medium (Perrot et al., 1992; Fuentes and Eddy, 1997). Thirdly, there could be adaptive changes in transporters along the intestinal mucosal surface of fish to increase ion and water absorption in a hyperosmotic environment (Mackay and Lahlou, 1980; Usher et al., 1991), leading to an increase in NH4 + uptake. Hence, it was essential to determine the intestinal ammonia content instead of the concentrations of ammonia in the intestinal lumen, which would not exert direct effects on intracellular enzyme activities and nitrogen metabolism. Indeed, the intestinal ammonia content remained unchanged after 1 day of exposure to 30 mmol l−1 NH4 Cl in 5‰ water. Since ammonia could enter intestinal cells through the blood, there was a significant increase in the ammonia content in the intestine of fish exposed to 30 mmol l−1 NH4 Cl in 5‰ water for 6 days, but the magnitude of increase was smaller than that of fish exposed to 15 mmol l−1 NH4 Cl in seawater. Consequently, unlike fish exposed to 15 mmol l−1 NH4 Cl in seawater, there were no significant changes in intestinal GDH amination and deamination activities in fish exposed to 30 mmol l−1 NH4 Cl in 5‰ water.

5. Conclusion The intestine is a complex multifunctional organ; besides digestion and absorption, it is crucial for water and electrolyte balance, endocrine regulation of digestion and metabolism, and immunity. Furthermore, our results indicate for the first time that the intestine of B. sinensis could function cooperatively with other organs to detoxify ammonia during exposure to environmental ammonia. Since the anatomy of the intestine is complex, efforts are being made in the authors’ laboratory to elucidate possible zonation, i.e. proximal versus distal or mucosal versus serosal, of the intestine of B. sinenesis in relation to the ammonia detoxification capacity.

Acknowledgement This project is supported by the Ministry of Education of the Republic of Singapore through a grant R-154-000-409-112.

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References Anderson, P.M., Broderius, M.A., Fong, K.C., Tsui, K.N.T., Chew, S.F., Ip, Y.K., 2002. Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichyths sinensis in response to exposure to a high exogenous ammonia concentration. J. Exp. Biol. 205, 2053–2065. Balment, R.J., Carrick, S., 1985. Endogenous renin–angiotensin system and drinking behavior in flounder. Am. J. Physiol. 248, R157–R160. Bergmeyer, H.U., Beutler, H.O., 1985. Ammonia. In: Bergmeyer, H.U., Bergmeyer, J., Grassl, M. (Eds.), Methods of Enzymatic Analysis, vol. VIII. Academic Press, New York, pp. 454–461. Bradford, M.M., 1976. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Buddington, R.K., Krogdahl, A., Bakke-McKellep, A.M., 1997. The intestines of carnivorous fish: structure and functions and the relations with diet. Acta Physiol. Scand. Suppl. 638, 67–80. Campbell, J.W., 1991. Excretory nitrogen metabolic. In: Prosser, C.L. (Ed.), Comparative Animal Physiology Environmental and Metabolic Animal Physiology, 4th ed. Wiley-Liss Inc, New York, pp. 277–324. Chew, S.F., Tng, Y.Y.M., Wee, N.L.J., Tok, C.Y., Wilson, J.M., Ip, Y.K. Intestinal osmoregulatory acclimation and nitrogen metabolism in the freshwater marble goby, Oxyeleotris marmorata, exposed to seawater. J. Comp. Physiol. B, in press. Chew, S.F., Wilson, J.M., Ip, Y.K., Randall, D.J., 2006. Nitrogenous excretion and defense against ammonia toxicity. In: Val, A., Almedia-Val, V., Randall, D.J. (Eds.), Fish Physiology, vol. 21. The Physiology of Tropical Fishes. Academic Press, New York, pp. 307–395. Cooper, J.L., Plum, F., 1987. Biochemistry and physiology of brain ammonia. Physiol. Rev. 67, 440–519. Evans, D.H., 1984. The role of gill permeability and transport mechanisms in euryhalinity. In: Hoar, W.S., Randall, D. (Eds.), Fish Physiology, vol. X. Academic Press, Orlando, pp. 239–283. Fuentes, J., Eddy, F.B., 1997. Effect of manipulation of the rennin–angiotensin system in control of drinking in juvenile Atlantic salmon (Salmo salar L) in freshwater and after transfer to sea water. J. Comp. Physiol. B 167, 438–443. Grosell, M., Laliberte, C.N., Wood, S., Jensen, F.B., Wood, C.M., 2001. Intestinal HCO3 − secretion in marine teleost fish: evidence for an apical rather than a basolateral Cl− /HCO3 − exchanger. Fish Physiol. Biochem. 24, 81–95. Herrero-Yraola, A., Bakhit, S.M.A., Franke, P., Weise, C., Schweiger, M., Jorcke, D., Ziegler, M., 2001. Regulation of glutamate dehydrogenase by reversible ADPribosylation in mitochondria. EMBO J. 20, 2404–2412. Huang, Z., 2001. Marine species and their distribution in China’s seas. In: Vertebrata. Smithsonian Institution, Florida, pp. 404–463. Hwang, H.C., Chen, I.Y., Yueh, P.C., 1988. The Freshwater Fishes of China in Colored Illustrations, vol. 2. Shanghai Sciences and Technology Press, Shanghai, p. 201. Ip, Y.K., Chew, S.F., Leong, I.W.A., Jin, Y., Wu, R.S.S., Lim, C.B., 2001b. The sleeper Bostrichyths sinensis (Teleost) stores glutamine and reduces ammonia production during aerial exposure. J. Comp. Physiol. B 171, 357–367. Ip, Y.K., Chew, S.F., Randall, D.J., 2001a. Ammonia toxicity, tolerance and excretion. In: Wright, P.A., Anderson, P.M. (Eds.), Fish Physiology. Vol. 20. Nitrogen Excretion. Academic Press, New York, pp. 109–140. Ip, Y.K., Loong, A.M., Ching, B., Tham, G.H.Y., Wong, W.P., Chew, S.F., 2009. The freshwater Amazonian stingray. Potamotrygon motoro, up-regulated glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15‰) water. J. Exp. Biol. 212, 3828–3836. Karlsson, A., Eliason, E.J., Mydland, T., Farrell, A.P., Kiessling, A., 2006. Postprandial changes in plasma free amino acid levels obtained simultaneously form the hepatic portal vein and the dorsal aorta in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 209, 4885–4894. Kirsch, R., Humbert, W., Simmoneaux, V., 1985. The gut as an osmoregulatory organ: comparative aspects and special references to fishes. In: Gilles, R., Gilles-Bailen, M. (Eds.), Transport Processes, Iono- and Osmoregulation. Springer, New York, pp. 265–277. Kong, H., Edberg, D.D., Korte, J.J., Salo, W.L., Wright, P.A., Anderson, P.M., 1998. Nitrogen excretion and expression of carbamoyl-phosphate synthetase III activity and

mRNA in extrahepatic tissues or largemouth bass (Micropterus salmoides). Arch. Biochem. Biophys. 350, 157–168. Kottelat, M., Whitten, A.J., Kartikasari, S.N., Wirjoatmodjo, S., 1993. Freshwater Fishes of Western Indonesia and Sulawesi. Periplus Editions, Hong Kong, p. 221. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680–685. Loong, A.M., Ang, S.F., Wong, W.P., Pörtner, H.O., Bock, C., Wittig, R., Bridges, C.R., Chew, S.F., Ip, Y.K., 2008. Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon. J. Comp. Physiol. B 178, 853–865. Loretz, C.A., 1995. Electrophysiology of ion transport in the teleost intestinal cells. In: Wood, C.M., Shuttleworth, T.J. (Eds.), Fish Physiology, vol. 14. Cellular and Molecular Approaches to Fish Ionic Regulation. Academic Press, New York, pp. 25–56. Mackay, W.C., Lahlou, B., 1980. Relationships between Na+ and Cl− fluxes in the intestine of the European flounder, Platichthys flesus. Epithelial Transp. Lower Vertebr., 151–162. Marshall, W.S., Grosell, M., 2006. Ion transport, osmoregulation and acid-base balance. In: Evans, D.H., Claiborne, J.B. (Eds.), The Physiology of Fishes, 3rd ed. Taylor and Francis, London, pp. 177–230. Mommsen, T.P., Busby, E.R., von Schalburg, K.R., Evans, J.C., Booth, H.L., Elliot, M.E., 2003. Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol. J. Comp. Physiol. 173B, 419–427. Nguyen, N.T., Nguyen, V.Q., 2006. Biodiversity and Living Resources of the Coral Reef Fishes in Vietnam Marine Waters. Science and Technology Publishing House, Hanoi. Ni, I.H., Kwok, K.Y., 1999. Marine fish fauna in Hong Kong waters. Zool. Stud. 38, 130–152. Peh, W.Y.X., Chew, S.F., Wilson, J.M., Ip, Y.K., 2009. Branchial and intestinal osmoregulatory acclimation in the four-eyed sleeper. Bostrychus sinensis (Lacepède), exposed to seawater. Mar. Biol. 156, 1751–1764. Peng, K.W., Chew, S.F., Ip, Y.K., 1994. Free amino acids and cell volume regulation in the sipunculid Phascolosoma arcuatum. Physiol. Zool. 67, 580–597. Perrot, M.N., Grierson, C.E., Haxon, N., Balment, R.J., 1992. Drinking behavior in sea water and fresh water teleosts, the role of the renin–angiotensin system. Fish Physiol. Biochem. 10, 161–168. Rainboth, W.J., 1996. Fishes of the Cambodian Mekong. FAO Species Identification Field Guide for Fishery Purposes. FAO, Rome. Saha, N., Dutta, S., Haussinger, D., 2000. Changes in free amino acid synthesis in the perfused liver of an air-breathing walking catfish. Clarias batrachus infused with ammonium chloride: a strategy to adapt under hyperammonia stress. J. Exp. Zool. 286, 13–23. Skadhauge, E., 1974. Coupling of transmural flows of NaCl and water in the intestine of the eel (Anguilla anguilla). J. Exp. Biol. 60, 535–546. Shehadeh, Z.H., Gordon, M.S., 1969. The role of the intestine in salinity adaptation in the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 30, 397–418. Tay, S.L.A., Chew, S.F., Ip, Y.K., 2003. The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during aerial exposure. J. Exp. Biol. 206, 2473–2486. Thatcher, B.J., Storey, K.B., 2001. Glutamate dehydrogenase from liver of euthermic and hibernating Richardson’s ground squirrels: evidence for two distinct enzyme forms. Biochem. Cell Biol. 79, 11–19. Tierny, M.L., Luke, G., Cramb, G., Hazon, N., 1995. The role of the renin–angiotensin system in the control of blood pressure and drinking in the European eel, Anguilla anguilla. Gen. Comp. Endocrinol. 100, 39–48. Tng, Y.Y.M., Wee, N.L.J., Ip, Y.K., Chew, S.F., 2008. Postprandial nitrogen metabolism and excretion in juvenile marble goby. Oxyeleotris marmorata (Bleeker, 1852). Aquaculture 284, 260–267. Tytler, P., Tatner, M., Findlay, C., 1990. The ontogeny of drinking in the rainbow trout Oncorhynchus mykiss (Walbaum). J. Fish Biol. 36, 867–875. Usher, M.L., Talbot, C., Eddy, F.B., 1991. Intestinal water transport in juvenile Atlantic salmon (Salmo salar L.) during smolting and following transfer to seawater. Comp. Biochem. Physiol. A 100, 813–818. Wang, Y.S., Gonzalez, R.J., Marjorie, L.P., Grosell, M., Zhang, C.G., Feng, Q., Du, J.Z., Walsh, P.J., Wood, C.M., 2003. Unusual physiology of scale-less carp. Gymnocypris przewalskii, in Lake Qinghai: a high altitude alkaline saline lake. Comp. Biochem. Physiol. A 134, 409–421.