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First published online August 9, 2007
Journal of Experimental Biology 210, 2905-2911 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003905
Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia
Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2W1 Canada
* Author for correspondence (e-mail: patwrigh{at}uoguelph.ca)
Accepted 5 June 2007
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Summary |
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Key words: ammonia toxicity, liver, enzyme activity, gene expression, mRNA, nitrogen metabolism, glutamine, sublethal ammonia exposure
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The major end product of hepatic protein and amino acid
catabolism, ammonia is normally eliminated by passive diffusion across the
gills of fish down the blood-to-water gradient
(Evans et al., 2005). Fish may
experience environments in the wild where ammonia is elevated as a result of
agricultural, industrial or sewage wastes or in culture settings where
stocking density is high and/or water turnover is low. Fish vary considerably
in their ability to tolerate elevated external ammonia, for example the total
ammonia (NH3+NH4+) 96 h LC50 value
for adult freshwater rainbow trout is 0.89 mmol l-1, pH 8.2
(Thurston et al., 1981) and
for the marine toadfish Opsanus beta is 
9.75 mmol l-1
(Wang and Walsh, 2000).
Elevated external ammonia reverses the branchial diffusion gradient, resulting
in the uptake of ammonia and accumulation in the extra- and intracellular
compartments and, if severe, can result in convulsions, coma and eventually
death (Randall and Tsui,
2002). Ammonium can be combined with 
-ketoglutarate to form
glutamate, catalyzed by glutamate dehydrogenase (GDH; EC1.4.1.3). A second
ammonium ion can then be added to glutamate to form glutamine, catalyzed by
glutamine synthetase (GSase; EC6.3.1.2). GSase is a multifunctional enzyme
that is involved in amino acid balance, nucleotide biosynthesis,
neurotransmitter metabolism, as well as ammonia detoxification
(Walsh and Mommsen, 2001).
GSase activity in fish brain is several orders of magnitude higher relative
to other tissues, including liver (Webb
and Brown, 1976; Chamberlin et
al., 1991; Wang and Walsh,
2000; Wicks and Randall,
2002; Mommsen et al.,
2003; Essex-Fraser et al.,
2005). Cerebral glutamine levels increase in fish in response to
exposure to elevated levels of ammonia in the environment
(Levi et al., 1974;
Arillo et al., 1981;
Iwata, 1988;
Vedel et al., 1998;
Peng et al., 1998;
Wang and Walsh, 2000;
Veauvy et al., 2005) or
feeding (Wicks and Randall,
2002). Brain GSase activity in three batrachoidid fish species
(O. beta, O. tau, Porichthys notatus) was positively correlated with
ability to tolerate ammonia (Wang and
Walsh, 2000). Moreover, pretreatment with methionine sulfoximine
(MSO), an inhibitor of GSase, reduced survival in toadfish subjected to
sub-lethal levels of external ammonia, suggesting that GSase is critical to
the ammonia stress response (Veauvy et
al., 2005). In other studies, induction of brain GSase in fish
exposed to sub-lethal ammonia concentrations was relatively small or absent
(Peng et al., 1998;
Wang and Walsh, 2000;
Wicks and Randall, 2002).
Our understanding of the role of GSase in the ammonia stress response in
fish should be enhanced by new molecular information. In mammals, GSase is
encoded by one gene that is expressed at relatively high levels in brain
tissue (Mearow et al., 1989).
Gene sequences for GSase have been reported in several fish species, including
O. beta (Walsh et al.,
1999; Walsh et al.,
2003), Oreochromis niloticus
(Mommsen et al., 2003),
Danio rerio (see Murray et al.,
2003) and elasmobranchs (Laud
and Campbell, 1994). In rainbow trout, four GSase isoforms have
been identified (Onmy-GS01-GS04)
(Murray et al., 2003), and
mRNA levels for each isoform have been measured in early stages of development
(Essex-Fraser et al., 2005). An
initial induction of Onmy-GS01 and -GS03 prior to hatching
in trout embryos was correlated with rising levels of ammonia. In adult trout,
only Onmy-GS01 and -GS02 were expressed at appreciable
levels in liver and expression was generally very low in skeletal muscle
tissue (Essex-Fraser et al.,
2005). Little information is available on mRNA levels in the
brain, where GSase is thought to play a very important role.
In the present study, two hypotheses were tested. First, we hypothesized
that the full complement of GSase isoforms (Onmy-GS01-GS04)
are expressed in cerebral tissues of the rainbow trout, where GSase activity
is 200-500 times higher than other tissues
(Essex-Fraser et al., 2005).
Second, we hypothesized that GSase activity and mRNA levels are upregulated in
the brain of the ammonia-intolerant rainbow trout during ammonia stress. In
previous studies, GSase activity measurements on whole brain homogenates in
fish may have masked more subtle changes in specific regions, therefore we
subdivided the brain into its three primary areas (fore-, mid- and hindbrain).
Trout were exposed to control water or 670 µmol l-1
NH4Cl for 9 h or 48 h. Ammonia concentrations were measured in
plasma, brain (fore-, mid- and hindbrain) and liver samples. GSase activity
and mRNA levels for Onmy-GS01-GS04 were determined in brain
(fore-, mid- and hindbrain) and liver tissue.
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Materials and methods |
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Experimental protocol
For each exposure period, fish were divided into four groups. Two tanks of
fish were randomly assigned to the control group (no NH4Cl added,
pH 8.2) while the remaining two tanks were assigned to the 670 µmol
l-1 NH4Cl exposure group (pH 8.2; NH3
concentration 31 µmol l-1; ammonia N, 12 mg
l-1). This level of ammonia was 75% of the 96 h-LC50
value (0.89 mmol l-1, pH 8.2)
(Thurston et al., 1981). This
sublethal level of ammonia was used because in preliminary experiments, where
trout were exposed to lower ammonia levels under the same conditions, there
was no significant change in brain GSase activity. At the start of the
experiment, the water in each of the ammonia exposure tanks was spiked with
5.24 g of NH4Cl dissolved in 2 l of tank water in order to
immediately bring the tank concentration up to 670 µmol l-1
NH4Cl. To maintain this concentration in a flow-through system for
the duration of the 48 h exposure period, a 1.2 mol l-1
NH4Cl solution was added to each ammonia exposure tank at a rate of
2.5 ml min-1 using a peristaltic pump (Minipuls 3, Villiers Le Bel,
France). The freshwater flow rate was 4 l min-1. Water samples
(5 ml) were collected 5 min after the initiation of the experiment (time 0 h)
and every 6 h thereafter. Water samples were stored at -20°C for later
analysis (within 1 week).
At the end of the 9 h or 48 h exposure period, all fish were terminally
anaesthetized in 2 ml l-1 2-phenoxyethanol. Blood samples (1 ml)
were collected by caudal venipuncture in 0.5 mol l-1
Na2EDTA-coated syringes (to prevent coagulation), placed on ice,
then centrifuged at 11 000 g for 5 min. Plasma was decanted
and stored at -80°C for later analysis (within 1 week). Whole brains were
removed and dissected into three regions: (i) the telencephalon (forebrain),
(ii) the preoptic area, optic tectum, hypothalamus and midbrain (midbrain) and
(iii) the cerebellum and hindbrain (hindbrain), according to the figure
published elsewhere (Doyon et al.,
2003). Although it would have been ideal to measure GSase activity
in each of the nine distinct brain tissues
(Doyon et al., 2003), tissues
were pooled in the midbrain and hindbrain in order to obtain sufficient mass
to detect GSase activity. The olfactory bulbs, optic nerves, pituitary gland
and spinal cord were not included in these brain sections. Liver tissue was
also collected for comparison with brain values. Livers were removed, and
tissue samples were immediately frozen in liquid nitrogen and stored at
-80°C for later analysis (within 2 weeks).
Analyses
Ammonia
Ammonia concentration in water samples was determined using a colorimetric
assay (Verdouw et al., 1978).
Brain regions and liver samples were ground to a fine powder in liquid
nitrogen using a mortar and pestle and deproteinized by adding 0.5 w/v 8%
perchloric acid (PCA). Plasma (500 µl) was deproteinized in 250 µl 8%
PCA. Samples were centrifuged at 16 000 g (4°C) for 5 min.
The resulting supernatant was neutralized using 0.5 v/v saturated potassium
bicarbonate and centrifuged at 16 000 g (4°C) for 5 min.
Ammonia levels in the resulting supernatant were analyzed according to the
enzymatic method described by Kun and Kearney
(Kun and Kearney, 1974). All
spectophotometric measurements were performed on a SpectraMax 190 microplate
spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
Glutamine synthetase activity
Brain regions and liver samples were prepared for enzyme analysis as
previously described (Steele et al.,
2001), with the exception that all samples were homogenized in
approximately 70 volumes of ice-cold homogenization buffer. After
centrifugation, endogenous substrates were removed from the homogenate by
passage through Sephadex columns as described
(Felskie et al., 1998).
Glutamine synthetase activity was determined as the production of
-glutamyl hydroxamate from 0 to 3 min at 26°C as previously
described (Shankar and Anderson,
1985), using a Ultrospec 3100 pro UV/Visible spectrophotometer
(Biochrom Ltd, Cambridge, UK).
RNA extraction and cDNA synthesis
Total RNA was isolated from brain regions and liver using TRIzol Reagent
(Invitrogen, Carlsbad, CA, US). Samples (<100 mg) were homogenized in 1 ml
TRIzol by repeatedly drawing the mixture into a sterile syringe (3 ml) fitted
with a 20 G needle. After following the manufacturer's instructions, RNA
pellets were reconstituted in 30 µl water and stored at -80°C. To
eliminate possible genomic DNA contamination, total RNA (3 µg) was treated
with Deoxyribonuclease (DNase) I, amplification grade (Invitrogen, Carlsbad,
CA, USA). The DNase-treated total RNA samples were reverse transcribed using
SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and
primer, Poly-T. Non-reverse transcribed controls were synthesized using the
same reaction but substituting water for the SuperScript enzyme.
Real-time PCR
mRNA expression of Onmy-GS01-GS04 was quantified from the
above cDNA products using the ABI Prism 7000 sequence detection system
(Applied Biosystems, Forster City, CA, USA). The coding sequences of these
isoforms are very similar while the 3' UTRs are more diverged. The
nucleotide sequences of the 3' UTR for Onmy-GS01 and
-GS03 were 81% homologous, whereas Onmy-GS02 and
-GS04 were 79% homologous. The low level of variation in the coding
sequence of the isoforms made probe and primer design for mRNA analysis more
difficult. The approach we selected was real-time PCR using a gene-specific
probe and set of primers that matched unique 3' UTR sequences in each
isoform. In preliminary trials, one PCR reaction from each primer set was
purified using a QIAquick PCR purification kit (Qiagen Inc., Hilden, Germany)
and sequenced to ensure that each primer set was only amplifying the target
sequence.
Primers and dual-labeled probes (Table
1) were designed for each gene using PrimerExpress software (v.
2.0, Applied Biosystems). All probes were dual-labeled with 6-FAM fluorescent
reporter at the 3' end and TAMRA quencher at the 5' end. Each PCR
reaction contained 5 µl template, 12.5 µl Taqman Universal PCR Master
Mix (no AmpErase UNG, Applied Biosystems, Foster City, CA, USA), and 2.5 µl
each of forward and reverse primers (9 µmol l-1) and probe (2.5
µmol l-1). The following conditions were used: 10 min at
95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. To
correct for variability in amplification efficiency between different cDNAs, a
standard curve was performed (Giulietti et
al., 2001) for each glutamine synthetase gene using serial
dilutions of cDNA samples from whole trout brain. The relative dilution of a
given sample was extrapolated by linear regression using the threshold cycle
of each unknown. To account for differences in cDNA loading and RNA reverse
transcriptase efficiency, each sample was normalized to the expression level
of the control gene ß-actin. The expression of ß-actin was not
significantly different between any of the tissues nor between control and
ammonia exposed individuals (data not shown).
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Samples were assayed in triplicate with only one target gene assayed per well. Non-reverse transcribed RNA and water-only controls were run to ensure that no genomic DNA was being amplified and that reagents were not contaminated.
Statistical analysis
Statistical analyses of GSase activity and ammonia concentrations were
performed using Sigma Stat (Version 3.0, SPSS Inc., Chicago, IL, USA). For
GSase activity and ammonia concentrations, differences between control and
ammonia treated groups as well as differences within groups were analyzed
using one-way analysis of variance (ANOVA). General Linear Model (GLM)
analysis was used to compare mRNA levels of individual GSase genes
(Onmy-GS01-GS04) between control and ammonia-exposed groups,
as well as to compare control levels of individual genes between tissues and
between genes within a tissue type. A Tukey post-hoc test was applied
if statistical differences were detected with the above analysis. Results were
declared to be significant if P<0.05.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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1.4-fold) compared to brain levels in most regions at both the 9 h and
48 h fish (Fig. 1).
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GSase activity
In control fish, GSase activity in brain tissue was, on average, 270-fold
higher relative to liver values (Fig.
2). The mid- and hindbrain had significantly higher GSase
activities relative to the forebrain in both control groups of fish (9 h and
48 h).
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GSase gene expression
All four GSase isoforms, Onmy-GS01-GS04, were expressed in brain
tissue of control fish (Fig.
3A,B). Each isoform was also detected in the liver, but the level
of expression of Onmy-GS04 was very low relative to the brain
(Fig. 3A,B). In control fish (9
h experiment), Onmy-GS03 mRNA levels were significantly higher
(Fig. 3A) relative to
Onmy-GS02 in all three brain regions and Onmy-GS04 in the
forebrain and hindbrain, but these differences were not apparent in the 48 h
experiment (Fig. 3B).
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Brain GSase mRNA expression was induced relative to control fish following
9 h of exposure to external ammonia (Fig.
3A), but by 48 h mRNA levels had returned to control values
(Fig. 3B). There were no
significant changes in liver GSase expression at 9 h
(Fig. 3A) or at 48 h
(Fig. 3B). Onmy-GS01
and -GS02 mRNA levels were significantly increased (twofold) in
ammonia-exposed fish in all three brain regions
(Fig. 3A). Onmy-GS03
mRNA levels were fourfold higher in the forebrain and twofold higher in the
hindbrain of ammonia-exposed fish relative to control fish
(Fig. 3A). Onmy-GS04
mRNA levels were also increased by >twofold in the mid- and hindbrain of
the experimental (9 h ammonia exposure) compared to control fish
(Fig. 3A).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The GSase gene pairs in each lineage are thought to be the
result of the ancient salmonid tetraploidization event that occurred between
25 and 100 mya (Allendorf and Thorgaard,
1984). We have recently reported that of the four GSase isoforms,
only Onmy-GS01 and -GS03 are initially induced during early
developmental stages of rainbow trout
(Essex-Fraser et al., 2005).
After the yolk is absorbed (80 days post-fertilization), the full expression
of all four GSase isoforms is evident in whole trout homogenates. We show here
that all four GSase isoforms are co-expressed in three trout brain regions,
with much lower levels detected in the liver using real time PCR. These
results agree with our preliminary findings using northern analysis
(Murray et al., 2003). It was
surprising that Onmy-GS03 mRNA levels in the brain were higher than
other GSase isoforms in both control and ammonia-exposed fish in the 9 h
experiment, but not the 48 h experiment. Further work is necessary to
determine if the four GSase isoforms have specific functions at the enzyme
level.
We hypothesized that GSase is induced in the brain of rainbow trout during
ammonia stress. With exposure to sub-lethal NH4Cl in the external
water, ammonia concentrations and GSase activities and mRNA levels were
significantly enhanced in each brain region. The time course of these changes
indicates that tissue and plasma ammonia concentrations reached their peak
(9-10 µmol g-1) in 9 h or less. High levels of body ammonia
directly or indirectly stimulated the induction of brain GSase mRNA levels,
indicated by the twofold higher Onmy-GS01-GS04 levels in
almost all brain regions after 9 h of ammonia exposure. In addition, there was
a significant elevation of brain GSase activity in two of the three brain
regions by 9 h. A further step increase in brain GS activity was observed
after 48 h of high external ammonia, but by this time mRNA levels had returned
to control levels. Hence, the response to elevated external ammonia in trout
involves a relatively rapid induction of four GSase isoforms and upregulation
of the functional protein in all regions of the brain. It may not be
surprising that harsher ammonia exposures in more ammonia-tolerant species
resulted in relatively modest increases (11-26%) or no change in brain GSase
activities; however, baseline GSase activities in mudskippers,
Periophthalmodon schlosseri and Boleophthalmus boddaerti
(Peng et al., 1998), and
marine toadfishes, O. beta and O. tau
(Wang and Walsh, 2000), were
several fold higher than rainbow trout
(Fig. 2).
GSase in mammals is expressed in astrocytes, but in goldfish and amphibians
it is thought to be localized in the ependymoglial cells that line the
ventricles and spinal canal (Norenberg,
1983). Although the ventricular system is fairly extensive in the
teleost brain (Meek and Nieuwenhuys,
1998), the higher GSase expression in the midbrain and hindbrain
is possibly due to a higher population of ependymoglial cells or to the
expression of GSase in other neural cells. In future studies, in situ
hybridization would be invaluable in localizing GSase isoforms to specific
cells in the trout brain.
External ammonia exposure resulted in an elevation of hepatic ammonia
content and GSase activities but not mRNA levels. Liver ammonia content was
twofold higher after 9 h of external ammonia treatment and remained high for
the 48 h exposure. Although the absolute level of GSase activity in liver
tissue is very low relative to brain, activities were significantly elevated
at both 9 and 48 h of ammonia treatment. These small changes in liver GSase
activity were not correlated with changes in the level of mRNA at 9 or 48 h
and therefore may be due to post transcriptional regulation
(Labow et al., 1999) or
possibly changes in transcription occurred very quickly and had returned to
control levels by 9 h. Anderson et al.
(Anderson et al., 2002) found a
5- to 20-fold elevation of hepatic GSase mRNA, protein and activity in the
sleeper (Bostrichthys sinensis) exposed to 15 mmol l-1
NH4Cl for 48 h. Higher concentrations of external ammonia (25 mmol
l-1 NH4Cl) induced hepatic GSase by threefold in
the air-breathing catfish (Clarias batrachus)
(Saha et al., 2002), but liver
GSase of other ammonia-tolerant species does not respond to relatively high
levels of external NH4Cl (Peng
et al., 1998; Wang and Walsh,
2000; Ip et al.,
2004). The increase in liver GSase activity in response to ammonia
exposure in the present study is in agreement with findings in trout under
similar conditions (Wicks and Randall,
2002).
Plasma and tissue ammonia content reached a steady state by 9 h of ammonia
exposure because there was no further change after 48 h of exposure. The total
ammonia content in a fish exposed continuously to elevated external ammonia
will depend on four parameters: (1) the rate of influx across the gills, (2)
the rate of ammonia production by the liver, (3) the rate of ammonia efflux
(against the gradient) and (4) the rate of conversion of ammonia to another
compound (e.g. glutamine). Brain ammonia content remained elevated at 48 h
despite a significantly higher GSase activity at this time period. As more and
more ammonia enters the brain, glutamine accumulates over glutamate levels
(Vedel et al., 1998;
Veauvy et al., 2005),
presumably because of the relatively high activity of brain GSase compared to
GDH activity (see Introduction). The extra- to intracellular ammonia gradient
in the brain would be maintained by the incorporation of ammonia into
glutamine and ammonia would continue to move into brain cells. The key role of
GSase, therefore may not be to lower brain ammonia levels but rather to
prevent a rise in glutamate, a major excitatory neurotransmitter. Indeed, in
studies on O. beta exposed to sublethal water ammonia, brain GSase
activity was reduced by 80% by pretreatment with the inhibitor MSO, brain
glutamine:glutamate levels were significantly reduced relative to the ammonia
treatment minus MSO, and fish died 40 h into the experiment. These experiments
underline the importance of brain GSase activity in the overall response to
elevated external ammonia in O. beta and comparative studies in
rainbow trout are warranted.
Our results provide an interesting contrast to studies on the mammalian
brain response to an ammonia load. It should be noted that arterial ammonia
levels in mammals are typically well below 100 µmol l-1
(Felipo and Butterworth, 2002)
and hyperventilation and convulsions were observed in rats with experimentally
manipulated brain ammonia concentrations of 3-4 µmol g-1
(Kensenko et al., 1994; Kensenko et al., 1995), levels comparable to control
brain ammonia concentrations in rainbow trout
(Fig. 1). Thus, mammalian brain
tissue is far more sensitive to elevated ammonia. In mammals, cerebral GSase
activity is an order of magnitude lower relative to trout
(Kosenko et al., 1995) and is
inhibited in acute ammonia exposure (Felipo
and Butterworth, 2002; Kosenko
et al., 2003). High plasma ammonia activates NMDA glutamate
receptors in the brain and the subsequent release of nitric oxide is thought
to induce a reversible covalent modification of GSase, which in turn reduces
enzyme activity (Miñana et al.,
1997; Monfort et al.,
2002; Kosenko et al.,
2003). Blocking NMDA receptors with antagonists such as MK-801 has
been shown to prevent death, as well as to increase cerebral GSase activity in
hyperammonemic rats (e.g. Hermenegildo et
al., 1996; Monfort et al.,
2002). MK-801 had a protective effect in plainfin midshipman
Porichtys notatus (Walsh et al.,
2007) and loach Misgurnus anguillicaudatus in vivo
(Randall and Tsui, 2002) given
an ammonia load. It is not clear how NMDA receptors are involved in the
brain's response to elevated ammonia in fish; however, both in vitro
and in vivo experiments are required to understand the mechanisms
more thoroughly.
In conclusion, all four genes coding for trout GSase are expressed in fore-, mid- and hindbrain of rainbow trout. Exposure to sublethal ammonia results in a relatively rapid, but transient increase in the transcription of the four GSase genes in the brain and a corresponding increase in brain GSase activity. The fact that brain GSase is induced in response to hyperammonemia in the ammonia intolerant rainbow trout indicates that GSase plays a key role in the cerebral response to ammonia stress.
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Acknowledgments |
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