Glutamine synthetase expression in liver, muscle, stomach and intestine of Bostrichthys sinensis in response to exposure to a high exogenous ammonia concentration
1 Department of Biochemistry and Molecular Biology, University of Minnesota,
Duluth, Duluth, MN 55812, USA
2 Department of Biological Sciences, National University of Singapore,
Singapore 117543
3 Natural Sciences Academic Group, Nanyang Technological University,
National, Institute of Education, 1 Nanyang Walk, Singapore 637616
Accepted 23 April 2002
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Summary |
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Key words: Bostrichthys sinensis, teleost, sleeper, nitrogen excretion, enzyme induction, glutamine synthetase, exogenous ammonia exposure
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Introduction |
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Several recent studies have suggested, however, that ureotelism (i.e.
increased urea cycle pathway activity) is not the universal response of fish
to these kinds of environmental circumstances
(Ip et al., 2001a). Marble
goby (Oxyeleotris marmoratus) is a facultative fresh water
air-breather from Southeast Asia that can tolerate continuous air exposure for
up to a week and has low levels of a full complement of the urea cycle
enzymes, but does not increase urea excretion or the level of urea cycle
enzyme activity during long-term air exposure. Instead, GSase activity
increases and ammonia is converted to glutamine, which accumulates in the
tissues (Jow et al.,
1999
).
Bostrichthys sinensis (sleeper) inhabits brackish water near the
mouths of rivers in southeast Asia and is periodically subjected to air
exposure for variable periods of time. This species also synthesizes and
stores glutamine, primarily in the muscle, in the first few days of air
exposure, which is accompanied by an increase in GSase activity in liver
(Ip et al., 2001b). The level
of glutamine that accumulates during the first few days is equivalent to the
level of ammonia that would normally be excreted during this time. After 3
days of air exposure the fish adopt a reduced rate of ammonia production,
possibly via reduced rates of proteolysis or amino acid catabolism.
Although all urea cycle enzyme activities are present in liver, the levels of
activity are very low, considerably lower even than found in marble goby, and
are far too low to account for meaningful ammonia detoxification
(Ip et al., 2001b
).
Preliminary studies have indicated that the physiological and biochemical
responses of B. sinensis to air exposure and related environmental
circumstances that preclude ammonotelism are reproducible and readily
measurable, indicating that this fish is an excellent model for studying the
biochemical mechanisms of these adaptive responses.
The purpose of this study was to extend studies on the effects of air
exposure on GSase expression in B. sinensis by assessing the response
of B. sinensis to exogenous ammonia exposure, a strategy that has
been used to increase internal ammonia concentrations in fish, mimicking the
various environmental situations, such as air exposure, that preclude loss of
ammonia across the gills, thus triggering alternative mechanisms for ammonia
detoxification (see Kong et al.,
1998 and references therein). The sequence of GSase cDNA was
determined and changes in GSase activity, GSase protein and GSase mRNA levels,
as well as activities of some urea cycle enzymes, urea and ammonia
concentrations and excretion rates and amino acid levels, were measured in
response to exposure to NH4Cl. The results show that there are
relatively high levels of GSase activity in liver, and also, unexpectedly,
even higher levels in muscle, stomach and intestine, and that exposure to
ammonia results in significant increases in GSase activity, GSase protein and
GSase mRNA levels in all of these tissues except stomach.
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Materials and methods |
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Water samples were collected at time 0 and at 24 h and 48 h (days 1 and 2, respectively); the samples were acidified with 0.1 mol l-1 HCl to prevent loss of NH3 and stored at 4 °C for analysis of ammonia and urea concentration within 48 h. After 48 h the ammonia-exposed fish were returned to control conditions and the water was analyzed for ammonia and urea concentration 24 h later (day 3).
For the collection of plasma and tissues, the fish were killed by a sharp blow to the head. The caudal peduncle was severed and blood from the caudal artery was collected in heparinized micro-hematocrit capillary tubes. The plasma obtained after centrifugation at 5000 g for 5 min was deproteinized by addition of an equal volume of 6 % trichloroacetic acid (TCA) and centrifugation at 5000 g for 10 min; the supernatant was stored at -80 °C for subsequent analysis of ammonia, urea and amino acids. The liver, a large section of lateral muscle, stomach and intestine were excised, immediately freeze-clamped in liquid nitrogen, wrapped in aluminum foil and stored at -80 °C until analysis (stomach and intestine were removed together, flushed well with deionized water, and cut into two sections, corresponding to stomach and intestine; where indicated the latter was cut into two halves, foregut and hindgut). For ammonia, urea and amino acid analysis, weighed frozen liver and muscle tissue samples were powdered at -80 °C and added to 5 volumes of 6 % TCA. After homogenizing for three 20 s intervals (cooling between intervals) with an Ika-werk Staufen Ultra-Turrax homogenizer at 24 000 revs min-1 the sample was centrifuged for 10 min at 10000 g at 4 °C; the supernatant was stored at -80 °C for subsequent analysis.
Ammonia and urea analysis
Acidified water samples were thawed and neutralized with 2 mol
l-1 KHCO3 to pH 5.5-6.0; acidified liver, muscle and
plasma samples were similarly neutralized with 5 mol l-1
K2CO3. Ammonia concentration was measured as described
by Kun and Kearney (1974), by
conversion to glutamate in the presence of
-ketoglutarate, NADH and
glutamate dehydrogenase and measuring the decrease in absorbance at 340 nm
accompanying the stoichiometric conversion of NADH to NAD+. The
reaction mixtures contained 115 mmol l-1 ethanolamine, pH 8, 11
mmol l-1
-ketoglutarate, 0.12 mmol l-1 NADH, 0.6
mmol l-1 ADP, axcess units of glutamate dehydrogenase, and 0.45 ml
of sample in a final volume of 1.55 ml. For urea analysis, 0.5 ml of the
neutralized samples was mixed with 0.5 ml of 20 mmol l-1 imidazole
buffer, pH 7.2; a second 0.5 ml of sample was mixed with 0.5 ml of this same
buffer containing 2 units of urease to break down all urea and serve as a
blank to correct for any other colour-contributing components. After 15 min
incubation at 30 °C, the urea concentration in each of the 1 ml solutions
was determined as described by Kong et al.
(1998
).
Amino acid analysis
Acidified liver, muscle and plasma samples were thawed and diluted with an
equal volume of 0.2 mol l-1 lithium citrate buffer and the pH
adjusted to 2.2 with 4 mol l-1 LiOH. These samples were then
analyzed for free amino acids using a Shimadzu LC-6A Amino Acid Analysis
System with a Shimpack ISC-07/SI504 Li type column. The concentrations of free
amino acids are expressed as µmol g-1 wet mass for muscle and
liver samples and as µmol ml-1 for plasma samples; the total
free amino acid concentrations are expressed as the sum of the free amino
acids The detection limit of the assay was 0.002 µmol g-1.
Enzyme assays
CPSase III activity was measured as previously described, with appropriate
precautions taken to assure that CPSase II, which is related to pyrimidine
nucleotide biosynthesis and normally also present in tissues
(Anderson, 1995b), was not the
primary CPSase activity being measured
(Korte et al., 1997
; Anderson,
1995a
,
2001
). GSase
(Shankar and Anderson, 1985
),
ornithine carbamoyltransferase (Xiong and
Anderson, 1989
), and argininosuccinate synthetase and lyase
(Cao et al., 1991
) were
measured as previously described (Korte et
al., 1997
; Anderson,
1995a
,
2001
) generally following the
protocols described by Anderson and Walsh
(1995
).
Glyceraldehyde-3-phosphate dehydrogenase
(Low et al., 1993
), citrate
dehydrogenase (Moon and Ouellet, 1979) and glutamate dehydrogenase (amination
direction) (Ip et al., 1993
)
activities were assayed essentially as described (in the indicated references
for each enzyme). Extracts for measuring the levels of GSase and CPSase III in
frozen tissues stored at -80 °C were prepared by homogenizing tissue
(powdered at -80 °C) with 5 volumes of extract buffer (3 mmol
l-1 EGTA, 3 mmol l-1 EDTA, 50 mmol l-1 NaF,
50 mmol l-1 KCl, 50 mmol l-1 Hepes, pH 7.6, 50 mmol
l-1 glutamine, 2 mmol l-1 dithiothreitol, plus 0.2 mmol
l-1 phenylmethanesulfonyl fluoride (PMSF) added just before
homogenization in a small volume of ethanol) for 60 s using the homogenizer
described above at 24 000 revs min-1. The homogenate was sonicated
for 20 s and then centrifuged at 10 000 g for 10 min. A 3 ml sample
of supernatant was then added to a 10 ml column of Sephadex G-25 equilibrated
with 50 mmol l-1 Hepes, pH 7.6, 50 mmol l-1 KCl, 1 mmol
l-1 EDTA, 50 mmol l-1 glutamine and 1 mmol
l-1 dithiothreitol, and eluted. The first 1 ml of eluate was
discarded and the next 2.8 ml, which contained the majority of the protein
well separated from lower molecular weight components in the homogenate, was
collected; correction for dilution was made by measuring the protein
concentration in the homogenate and the eluate. All steps were carried out at
4 °C and activities were measured immediately. The unusual buffer (i.e.
presence of NaF, high concentrations of EDTA and EGTA) used for homogenization
was based on the efforts of another study to prevent possible
dephosphorylation of a putative covalently modified GSase; control studies
using the buffer employed in the gel-filtration column indicated that these
components had no effect on the results.
For measurement of all urea cycle enzyme activity in liver samples, extracts were made from freshly excised liver and the extract fractionated into cytosolic and mitochondrial fractions. The sample was homogenized with 10 volumes of mitochondrial extraction buffer (285 mmol l-1 sucrose, 3 mmol l-1 Tris-HCl, pH 7.2, 3 mmol l-1 EDTA) using a Potter-Elvejhem-type glass homogenizer with a Teflon pestle and a variable speed motor using 1-3 strokes at moderate revs min. The homogenate was centrifuged at 600 g for 15 min and the resulting supernatant at 10 000 g for 15 min, three times. After each of the first two centrifugations the supernatant was discarded and the pellet gently resuspended in the same volume of mitochondrial extraction buffer. After the third centrifugation the pellet (mitochondrial fraction) was suspended in 1 ml of buffer containing 50 mmol l-1 Hepes, pH 7.6, 50 mmol l-1 KCl, 0.5 mmol l-1 EDTA, 1 mmol l-1 dithiothreitol, sonicated for 1 min, centrifuged at 10 000 g for 10 min, and the supernatant passed through a gel-filtration column equilibrated with the same buffer. A cytosolic fraction was prepared by homogenizing with 5 volumes of mitochondrial extraction buffer and centrifugation at 10 000 g for 15 min; the supernatant (cytosolic fraction) was also passed through a gel-filtration column equilibrated with the same buffer. Enzyme assays were performed immediately.
Subcellular localization
The following fractionation method was used to determine if GSase was
localized in the mitochondrial or cytosolic fractions and if the increase in
GSase activity observed in liver occurred primarily in the cytosolic or the
mitochondrial fraction. Liver of freshly killed control fish was cut into
small pieces and homogenized as described above in 10 volumes of
homogenization buffer (50 mmol l-1 Tris-HCl, pH 7.6, 100 mmol
l-1 sucrose, 3 mmol l-1 EDTA, 3 mmol l-1
EGTA, 50 mmol l-1 NaF, 20 mmol l-1 KCl, plus 0.5 mmol
l-1 PMSF, added as described above). The homogenized sample was
passed through four layers of coarse cheesecloth and centrifuged at 42
g for 15 min at 4 °C to remove unbroken cells and debris. The
supernatant, regarded as the homogenate, was centrifuged at 1000 g
for 15 min at 4 °C, and the pellet, regarded as the nuclear fraction, was
resuspended in homogenization buffer. The supernatant was centrifuged at 8000
g for 15 min to give a further pellet and supernatant (cytosolic
fraction). This pellet was gently resuspended in homogenization buffer and
centrifuged at 8000 g for 15 min; the resulting pellet was
resuspended in homogenization buffer (mitochondrial fraction). The three
fractions (nuclear, mitochondrial and cytosolic) were each sonicated for 60 s
and then passed through 10-ml columns of Sephadex G-25 (as described above)
equilibrated with 50 mmol l-1 Tris-HCl, pH 7.2, containing 50 mmol
l-1 KCl, and the following enzyme activities were assayed
immediately: glyceraldehyde-3-phosphate dehydrogenase as a cytosolic marker,
glutamate dehydrogenase as a mitochondrial marker, and GSase. The results of
trial experiments showed that (1) only 1 % of glyceraldehyde-3-phosphate
dehydrogenase activity was present in the mitochondrial fraction, indicating
that mitochondria were quite free of cytosolic components, and (2) more than
70 % of the glutamate dehydrogenase activity was present in the mitochondrial
fraction, indicating that the mitochondrial fraction represented a high
percentage of the mitochondrial enzymes (the majority of the remainder was in
the nuclear fraction, suggesting that there had been some loss of mitochondria
to this fraction, but some was also found in the soluble fraction, suggesting
some breakage of mitochondria had occurred).
GSase cDNA sequence
mRNA was isolated from stomach and liver tissue (stored at -80 °C)
using Poly (A) Pure mRNA kit 1915 from Ambion, Inc. (Austin, TX, USA). First-
and second-strand cDNA was prepared from this mRNA and ligated to the
kit-supplied adapter oligonucleotide using Marathon cDNA Amplification kit
K1802-1 from Clontech (Palo Alto, CA, USA). Two overlapping fragments of GSase
cDNA were amplified by the polymerase chain reaction (PCR) using AdvanTAge
cDNA Polymerase Mix (Clontech 8417-1). The primers used for the amplification
were adapter-specific AP1 from the kit and one (right primer) or the other
(left primer) of two different consensus primers (see
Fig. 1), designed on the basis
of identifying highly conserved regions of several aligned mammalian and fish
Gsases. The two primers were TGRCASCCDGCHCCRTTCCAGTT (RcGS1, right primer,
giving a 5' fragment of the cDNA that overlaps the 3' fragment)
and CCDTGGTTTGGVATGGARCARGA (LcGS1, left primer, giving a 3' fragment of
the cDNA that overlaps the 5' fragment). PCR conditions were varied to
maximize the yield and purity of the band of the correct size as estimated by
agarose gel electrophoresis (to obtain the product of interest, touchdown PCR
cycling parameters were used initially, followed by a two-step PCR procedure
using a higher annealing temperature of 64-68 °C to increase specificity
and stringency). After conditions were maximized, the product from the first
amplification was re-amplified using the nested internal primer
adapter-specific AP2 from the kit in place of AP1. Clontech AdvanTAge PCR
Cloning kit K1901-1 was used to ligate the amplified products into the
kit-supplied pT-Adv Vector and to clone this fragment-containing Vector by
transformation into kit-supplied TOP10F' competent Escherichi
coli cells and growing on LB/ampicillin/X-gal/IPTG agar plates. White
colonies were selected and grown on LB/ampicillin cultures and plasmid DNA was
isolated using QIAprep Spin Miniprep kit 27104 (QIAGEN, Valencia, CA, USA).
The presence of the inserted fragment of correct size was confirmed by cutting
with EcoRI to release the insert, followed by electrophoresis on 1 %
agarose and comparison of the migration distance with that of fragments from a
DNA ladder of known sizes and the uncut plasmid as control.
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The insert in the plasmid was sequenced by primer walking, beginning with AP2 and/or the consensus primer as the sequencing primers, respectively; the sequence for the entire 5' UTR region through the coding region and into the 3' UTR was obtained for both strands. Automatic sequencing was carried out by the University of Minnesota Advanced Genetic Analysis Center. Primers were purchased from Integrated DNA Technologies (Coraville, IA, USA). PCRs were carried out with a Perkin Elmer Gene Amp 2400 PCR instrument. The Advantage cDNA polymerase mix from Clontech was used for all PCR amplifications. The nucleotide and deduced amino acid GSase sequences are available from GenBank under Accession numbers AY071837 (liver) and AY071838 (stomach). Multiple sequence alignments were performed using the program ClustalW.
Partial sequence of ß-actin cDNA
In another project, the partial sequence of ß-actin cDNA from liver of
marble goby had been determined, and a probe prepared from this cDNA was used
for measuring ß-actin mRNA by ribonuclease protection assays as described
below. This was possible because this portion of the ß-actin sequence is
very highly conserved (Kong et al.,
2000). Liver cDNA from marble goby was used as template in the
PCRs carried out for generating the ß-actin DNA fragment; primers used
for PCR and for sequencing were the same as previously described
(Kong et al., 2000
). The base
sequence of the portion sequenced is:
ATCCGTAAGGACCTGTATGCCAACACTGTGCTGTCTGGAGGTACCACCATGTACCCTGGCATTGCTGACAGGATGCAGAAGGAGATCACAGCCCTGGCTCCATCCACCATGAAGATCAAGATCATTGCCCCTCCAGAGCGTAAATACTCTGTCTGGATCGGAGGCTCCATCCTGGCCTCTCTGTCCACCTTCCAACAAATGTGGATC.
The deduced amino acid sequence based on the reading frame beginning at the first codon (ATC) is:
IRKDLYANTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWI.
Measurement of GSase mRNA by ribonuclease protection assays
Templates for preparing RNA probes for measuring GSase and ß-actin
mRNA levels using specific GSase primers (see
Fig. 1; left or sense primer =
GGCATGGAACAAGAGTACACGATT; right or antisense primer = GCTTGGGGTCAAATGAGGCAAC)
and specific ß-actin primers (left or sense primer =
ATCCGTAAGGACCTGTATGCCAAC; right or antisense primer =
GATCCACATTTGTTGGAAGGTGGA), respectively, with a T7 promoter appended to the
5' end of the antisense primers, were made from liver cDNA from B.
sinensis and marble goby, respectively, using MAXIscript T7 kit 1312 from
Ambion, Inc., as previously described
(Kong et al., 2000). The
probes were isolated and labeled with psoralen-biotin using BrightStar
Psoralen-Biotin labeling kit 1480 from Ambion, Inc., as previously described
(Kong et al., 2000
). GSase and
ß-actin mRNA levels in total RNA isolated from B. sinensis were
measured by ribonuclease protection assays using these probes with RPA III
Ribonuclease Protection Assay kit 1414 and BrightStar BioDetect detection kit
1930 from Ambion, Inc., following the instructions provided with the kits and
as previously described (Kong et al.,
2000
). Total RNA was isolated from approximately 0.5 g tissue
using RNAwizTM from Ambion, Inc. (9736) according to the instructions
provided. The relative intensities of the GSase and ß-actin bands were
determined using a FluorChemTM 8000 Advanced Imaging system (Alpha
Innotech Corp., San Leandro, CA, USA) for quantification of the
chemiluminescence signal.
GSase antibody and western blotting
Affinity-purified rabbit antibodies against the KLH-conjugated highly
conserved oligopepetide GSase sequence
(acetylcysteinyl-CPRSVGQEKKGYFEDRRPS-amide) identified in
Fig. 1 were purchased from
Quality Controlled Biochemicals (Hopkinton, MA, USA) and stored at -20°C.
Immunoblotting was carried out by standard protocols using SDS-PAGE [7.5%
acrylamide/bisacrylamide (19:1)] and electroblotting apparatus and reagents
from Bio-Rad Laboratories (Hercules, CA, USA). Immunodetection of the GSase on
the blotted nitrocellulose membrane was carried out using western blotting
detection reagents (RPN21OT) from Amersham Pharmacia Biotech (Piscataway, NJ,
USA) according to the manufacturers directions, except 5% dry milk powder in
TBST (50 mmol l-1 Tris, pH 8.0, 0.9% NaCl, 0.1% Tween 20) was used
as the blocking agent instead of the kit reagent. After exposure to X-ray film
for an optimal period of time, the intensity of the images was quantified as
described above.
Statistical analyses
Results are presented as means ± standard errors (S.E.M.). Student's
t-test or analysis of variance (ANOVA) followed by multiple
comparisons of means by Duncan's procedure were used to evaluate differences
between means where applicable. Arcsine transformation was performed on the
`percentage' data before statistical analyses. Differences were regarded as
statistically significant at P<0.05.
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Results |
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Increased expression of GSase
As shown in Table 1,
exposure to 15 mmol l-1 NH4Cl for 2 days results in a
significant increase in GSase activity (units g-1 tissue and units
mg-1 protein) in liver, and a smaller but significant increase in
intestine and muscle, but no increase in stomach levels. The levels of GSase
activity were found to be unexpectedly high in muscle and intestine and,
especially, in stomach. The increases in GSase activity are accompanied by a
correspondingly significant increase in GSase protein levels in liver and a
small increase in intestine and muscle
(Fig. 2 and
Table 2) and a significant
increase in GSase mRNA concentration in all tissues
(Fig. 3, Table 2); although GSase
activity and protein levels did not increase in stomach, it is notable that
there was a small increase in GSase mRNA. As shown in
Table 3, virtually all GSase
activity in liver both before and after exposure to NH4Cl is
localized in the cytosol.
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Effect on CPSase III activity and other urea cycle enzymes
The levels of CPSase III activity were found to be very low in freshly
excised liver tissue, i.e. < 1 nmol min-1 g-1 liver,
as compared, for example, to 500, 60 and 10 nmol min-1
g-1 liver in Gulf toadfish
(Anderson and Walsh, 1995), the
alkaline lake tilapia (Lindley et al.,
1999
) and largemouth bass Micropterus salmoides
(Cao et al., 1991
), and even
lower or not detectable in muscle, stomach and intestine; an increase in
CPSase III activity upon ammonia exposure was not observed for any of these
tissues (data not shown). Ornithine carbamyltransferase (mitochondrial) and
argininosuccinate synthetase and lyase activities (cytosolic) in liver were
also very low (300 and 0.4 nmol min-1 g-1 liver,
respectively) and did not increase in fish exposed to ammonia (data not
shown).
Effect on various metabolite levels
As expected, exposure to ammonia resulted in a net influx of ammonia, which
was reversed by removal of ammonia from the external medium, and a dramatic
increase in ammonia concentration in liver, plasma and muscle
(Table 4). There was no
significant change in the rate of urea excretion and little change in the urea
concentration in these three tissues (Table
5). However, there was a significant increase in the concentration
of glutamine in these three tissues after 48 h of exposure to ammonia, which
was accompanied by a decrease and increase in glutamate concentration in
muscle and liver, respectively (Table
6). Total free amino acids increased only in muscle, which may be
significant given the large muscle mass of fish relative to total body mass
(>50 %). The concentrations of several other amino acids changed, as noted
in Table 6, including an
increase in alanine in muscle, but none were as dramatic and represented much
lower levels of total nitrogen than glutamine. This result is consistent with
the noted increase in GSase activity.
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Discussion |
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In contrast to these two fish species (S. acanthias and Gulf toadfish, as noted above), the sequences of the GSase cDNAs derived from mRNA from liver and stomach, respectively, of B. sinensis reported here are characterized by a single start codon for translation. This would be consistent with the observation that the liver GSase appears to be localized exclusively in the cytosol and that B. sinensis is not ureotelic. GSase cDNA from stomach was sequenced as well as GSase cDNA from liver because of the unexpectedly high levels of GSase activity found to be present in stomach. The results suggest that the mRNAs for the two tissues are coded for by the same gene; the significance of the extra 13 bases on the 5' end of the stomach cDNA is not known, perhaps reflecting differential splicing during transcription related to specific tissue expression or an artifact of experimental reverse transcription of the mRNA and subsequent adapter ligation. The sequence of the 5' untranslated region of the GSase cDNA from stomach bears little relationship to the 5' untranslated region of Gulf toadfish cDNA upstream from the second start codon. As noted in the Results and in Fig. 1, the derived amino acid sequences of the open reading frame of both cDNAs are very similar to the sequences of GSases from Gulf toadfish and S. acanthias, as well as to GSases from many mammalian species. These results do not exclude the possibility that additional GSase genes are expressed in each of these tissues in B. sinensis, since only one cDNA may have been amplified in each tissue; multiple GSase genes have recently been identified in rainbow trout (Oncorhynchus mykiss) (B. Murray, E. Busby, T. Mommsen, and P. Wright, personal communication) and Gulf toadfish (P. Walsh, personal communication).
A major effect seen when B. sinensis is exposed to air for 2 days
is increased GSase activity in liver and increased levels of glutamine in
several tissues, muscle in particular (Ip
et al., 2001b). The increase in glutamine levels in muscle and
other tissues is equivalent to the amount of ammonia that would have been
excreted, but could not be excreted due to air exposure; it has been proposed
that this increased glutamine synthesis capability and glutamine accumulation
represents the normal short-term adaptive response as an alternative pathway
for ammonia detoxification during air exposure in this fish
(Ip et al., 2001b
). The
results reported here when the fish is exposed to exogenous ammonia are
similar, but more dramatic. This may not be unexpected if increased ammonia
concentration is the primary regulatory signal in both situations, since
ammonia exposure leads to considerably higher tissue ammonia levels than occur
as a result of air exposure. GSase activity increased 5.4-fold in liver and
glutamine concentration in muscle increased 3.1-fold compared to 1.8-fold and
1.7-fold, respectively, when exposed to air. The increase in GSase activity in
liver was accompanied by a 5.9-fold increase in GSase protein and a 20-fold
increase in GSase mRNA, suggesting that the increased activity is due
primarily to transcriptional regulation. These effects are similar to results
obtained when GSase protein and mRNA are measured in Gulf toadfish subjected
to conditions that promote induced GSase activity
(Kong et al., 2000
).
Brain tissue of virtually all fish has high GSase activity, which functions
to detoxify ammonia (Webb and Brown,
1976; Webb, 1980
).
In contrast, the level of GSase activity in other fish tissues is variable
(Webb, 1980
;
Campbell and Anderson, 1991
);
it is high in liver of ureo-osmotic elasmobranchs and a few teleosts reported
to be ureotelic, low or non-detectable in liver, somewhat higher in intestine,
and very low or non-detectable in muscle of non-ureotelic/non-ureogenic fish
(Table 7). The level of GSase
activity in B. sinensis liver is comparable to measurable levels of
GSase activity reported in liver of several other non-ureotelic/non-ureogenic
fishes (Table 7). However, the
level of GSase activity in muscle is about tenfold higher than in muscle of
most of these other species and is as high as the levels in B.
sinensis liver; the level of GSase in intestine (posterior and anterior)
is also higher than in the intestine of these other species
(Table 7). Exposure to ammonia
resulted in increased GSase activity, GSase protein and GSase mRNA levels in
these tissues (intestine and muscle), as well as in liver. The total amino
acid concentration increased significantly only in muscle, due primarily to
the large increase in glutamine concentration. Given that muscle represents
>50 % of the body mass (Ip et al.,
2001b
), muscle GSase in B. sinensis is likely to play the
major role in ammonia detoxification in B. sinensis under conditions
that limit ammonotelism. This function may, in fact, explain the uniquely high
levels of GSase in muscle of this species and might partially explain the
capability of B. sinensis to survive exposure to high concentrations
of environmental ammonia (up to 50 mmol 1-1 NH4Cl at pH
7; S. F. Chew and Y. K. Ip, unpublished results) and extended periods of air
exposure (Ip et al., 2001b
).
The observed relatively large increase in GSase mRNA with little increase in
GSase protein in muscle (as well as intestine) is worth noting, perhaps
reflecting additional regulatory control mechanisms, such as
post-translational covalent modification.
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Unexpectedly high levels of GSase activity were found in stomach tissue
and, unlike liver, intestine and muscle, the levels of GSase activity and
protein were not affected by exposure to ammonia, although a small increase in
GSase mRNA was observed. As noted in Table
7, P. J. Walsh (personal communication) has observed high levels
of GSase in stomach of Gulf toadfish and this may be characteristic of fish in
general, reflecting a role unrelated to that in liver and muscle. As noted
above, the GSase sequence is essentially identical to that from liver, but the
observations that the mRNA appears to be 13 bases longer on the 5' end
and that GSase activity is not induced during ammonia exposure could reflect
unique stomach transcriptional regulatory factors. Further study aimed at
elucidating the physiological role of this high GSase activity in stomach of
this fish, and perhaps other fish, is clearly needed. Measurement and
characterization of the properties of the biosynthetic activity, as opposed to
the transferase activity assay used here and by most investigators, may be
especially important; the ratio of transferase to biosynthetic activity has
been reported to vary with different GSase isozymes
(Walsh, 1996), and it is not
impossible that stomach may have unique glutamine-related transferase
activities that are not actually due to GSase.
The brain is often the organ undergoing the largest increases in glutamine
concentration in fish exposed to ammonia, hence the high levels of GSase in
the brain. Increases in glutamine concentrations in brain of more than tenfold
resulting from ammonia exposure have been reported in rainbow trout Salmo
gairdneri (Arillo et al.,
1981), goldfish Carassius auratus
(Levi et al., 1974
) and common
carp Cyprinus carpio (Dabrowska
and Wlasow, 1986
). For mudskippers exposed to sublethal
concentrations of ammonia, the glutamine levels in the brains increase from
2.46 to 28 µmol g-1 (Periophthalmodon schlosseri) and
2.77 to 15 µmol g-1 (Boleophthalmus boddaerti);
although accumulation of glutamine in the liver also occurs, the levels
attained were much lower than in the brains
(Peng et al., 1998
). Thus,
Korsgaard et al. (1995
)
proposed glutamine formation in the brain as one of the strategies available
to fishes to deal with increasing concentrations of internal ammonia. Ammonia
presumably exerts its toxic effects in the brain, and it is essential to have
mechanisms to protect the brain against ammonia toxicity. For fishes that
adopt this strategy, there is usually high ammonia tolerance in non-cerebral
tissues (Korsgaard et al.,
1995
). Not unexpectedly, ammonia is also detoxified to glutamine
in the brain of B. sinensis (Y. K. Ip and S. F. Chew, unpublished
data). The results reported here with B. sinensis, however, represent
the first report of a non-ureotelic teleost fish responding to environmental
ammonia by increasing the expression of GSase in non-cerebral tissues.
Previously, it was believed that only cerebral GSase was inducible by
sublethal concentrations of environmental ammonia
(Korsgaard et al., 1995
;
Peng et al., 1998
).
The changes in levels of amino acids other than glutamine in muscle, liver
and plasma, and the observed lack of urea cycle activity after exposure to
ammonia reported here, are also similar to the changes observed when B.
sinensis is exposed to air (Ip et
al., 2001b). In addition to the large increase in glutamine
concentration in muscle, there was also a significant increase in the total
free amino acid content in this tissue; 72% of this increase is attributable
to the increase in glutamine content, 18% to glycine and 10% to alanine. As
with exposure to air, this indicates a possible decrease in the rate of amino
acid catabolism (Lim et al.,
2001
), reducing the rate of endogenous ammonia accumulation, or an
increase in the synthesis of certain amino acids (e.g. glutamine), or both.
There might also be a slight increase in partial amino acid catabolism,
leading to the formation of alanine (Ip et
al., 2001c
). The glutamine concentration in liver of B.
sinensis exposed to ammonia increased from 0 to 3.3 µmol
g-1. In comparison, less than a 20% increase in hepatic glutamine
concentration, with a corresponding decrease in glutamate concentration, was
observed in ranbow trout exposed to ammonia
(Arillo et al., 1981
). For
goldfish exposed to 0.75 mmol l-1 NH4Cl, the liver
glutamine concentration increased only slightly
(Levi et al., 1974
).
The high levels of GSase in liver, intestine and, particularly, muscle, and
the resulting significant increases in the level of GSase activity in these
tissues accompanied by significant increases in tissue glutamine
concentrations in B. sinensis upon exposure to exogenous ammonia
appear to reflect a unique strategy to handle ammonia detoxification when
tissue levels of ammonia increase. This appears to be the same strategy as
that used by this fish to detoxify the increased endogenous ammonia
concentrations that occur during air exposure
(Ip et al., 2001b). Presumably
the underlying cause of the effects induced by both exposure to exogenous
ammonia and extended periods of air exposure is increased ammonia
concentrations in the tissues. Since ammonia exposure results in higher tissue
levels of ammonia than air exposure and the effects are greater and more
reproducible than air exposure, ammonia exposure of B. sinensis
represents an excellent model for investigating the underlying molecular
mechanisms that regulate detoxification of ammonia to glutamine in
non-cerebral tissues.
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Acknowledgments |
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References |
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