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First published online February 13, 2009
Journal of Experimental Biology 212, 704-712 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.015875
Urea transporter and glutamine synthetase regulation and localization in gulf toadfish gill
1 Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600
Rickenbacker Causeway, Miami, FL 33149 USA
2 Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N
6N5
* Author for correspondence (e-mail: dmcdonald{at}rsmas.miami.edu)
Accepted 11 December 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: facilitated diffusion, urea production, urea excretion, stress, cortisol, glutamine production, Opsanus beta
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Hopkins et al.,
1999; Barimo and Walsh,
2005). When removed from nature and placed under stressful
conditions in the laboratory (crowding, confinement or exposure to air or
ammonia), toadfish switch to excreting predominantly urea
(Walsh et al., 1990;
Walsh et al., 1994;
Walsh and Milligan, 1995) in a
pulsatile manner (1–2 distinct events of urea excretion per 24 h)
(Wood et al., 1995;
Wood et al., 1997). The
transition to ureotelism is marked by several events, the first being a surge
in circulating levels of the stress hormone cortisol
(Walsh et al., 1994;
Walsh and Milligan, 1995;
Hopkins et al., 1995). Second
is the induction of glutamine synthetase (GS) activity in the liver, an
important enzyme that shuttles ammonia as glutamine into the piscine O-UC,
ultimately resulting in an increase in urea production and circulating urea
levels (Walsh et al., 1994;
Walsh and Milligan, 1995;
Hopkins et al., 1995). The
final two events marking the transition to ureotelism involve the reduction in
ammonia-N excretion (Walsh and Milligan,
1995) and the initiation of pulsatile excretion of urea-N
(Wood et al., 1995;
Wood et al., 1997).
A potential factor in reducing branchial ammonia excretion is the existence
of two GS isoforms in the toadfish gill: a ubiquitous isoform that was
initially purified and characterized from the liver (hence called LGS)
(Walsh, 1996) and is found in
various tissues throughout the body including the liver and the gill, and a
second isoform that, to date, has been found only in the gill (GGS)
(Walsh et al., 2003). Although
the gill is the only organ in toadfish expressing both GS isoforms, total GS
activity in the gill contributes only a small percentage (3.3%) to total body
GS activity, suggesting a minor role in terms of whole-body glutamine/urea
production (Walsh et al.,
2003). However, the multiple GS isoforms in the gill could explain
the reduction measured in ammonia-N excretion during the switch to ureotely
(Walsh and Milligan, 1995),
with these isoforms participating in the shuttling of ammonia into glutamine,
greatly reducing the amount of ammonia that crosses the gill during stressful
situations. Very little is known about the gill GS isoforms in terms of their
cortisol sensitivity or, furthermore, the cell type (i.e. pavement cell or
mitochondria-rich cell, MRC) (see Evans et
al., 2005) in which either GS isoform is expressed. However, with
respect to the latter, it has been speculated that GGS may be found in
pavement cells, with LGS in MRCs (Walsh et
al., 2003).
Extensive research over the past decade has focused on pulsatile urea
excretion in toadfish (reviewed by Wood et
al., 2003). Excretion is facilitated by a urea transport protein
(tUT) that shows greater than 60% identity at the amino acid level to
mammalian UT-A2 facilitated diffusion urea transporters
(Smith et al., 1998;
Walsh et al., 2000) and it is
hypothesized that the pulsatile aspect of urea excretion is due to the
periodic insertion or activation of tUT. The cellular location of tUT has not
been firmly established although changes in pavement cell morphology and
increased vesicular trafficking within these cells during urea pulsing led to
speculation that tUT and GGS may be localized mainly in pavement cells
(Laurent et al., 2001).
Circulating cortisol is an important regulatory component of tUT function
that is not completely understood. While it might be beneficial for the surge
in cortisol measured in conjunction with the transition to ureotely to prompt
an upregulation in tUT transcription as a way to facilitate urea excretion
across the gill, high levels of cortisol have consistently been shown to
inhibit tUT function on both an acute and a chronic level
(Hopkins et al., 1995;
Wood et al., 1997;
Wood et al., 2001;
McDonald et al., 2004). On the
acute time course, urea pulses appear only to occur when the normally elevated
circulating cortisol concentrations of ureotelic toadfish periodically drop,
suggesting inhibition of tUT function at high cortisol concentrations and the
reduction in cortisol acting in a permissive manner for tUT activation
(Hopkins et al., 1995;
Wood et al., 1997;
Wood et al., 2001). On a more
chronic time course, continuous cortisol infusion (as a way to prevent the
pre-pulse drop) results in a significant reduction in the size of the urea
pulse (McDonald et al., 2004).
Combined, this evidence suggests that elevations in cortisol may affect the
number of functional urea transporters, perhaps through transcriptional or
post-transcriptional downregulation; however, regulation of tUT could also be
an indirect result of cortisol. As mentioned previously, a surge in cortisol
results in the switch to ureotelism and a consequent increase in urea
production by the liver. Even when already ureotelic, a further increase in
circulating cortisol by infusion results in an additional increase in plasma
urea concentrations (McDonald et al.,
2004). While urea concentration changes have been ruled out as
triggering the actual pulse event (Wood et
al., 1997), there is a precedent in the mammalian literature for
potential urea effects on the transcription of UT message
(Klein et al., 1999).
Therefore, the goal of the present study was to investigate in more detail
the potential for transcriptional regulation of GS, involved in the
incorporation of ammonia into glutamine, and tUT, involved in urea excretion
in the gill of the gulf toadfish. The central hypothesis is that, in response
to stress, the toadfish gill exhibits increased expression of GS and tUT so as
to conserve ammonia yet excrete urea. An alternative hypothesis is that tUT
mRNA expression is actually downregulated by elevated plasma cortisol and/or
plasma urea levels. To test these hypotheses, GGS, LGS and tUT mRNA expression
and activity were measured in toadfish exposed to treatments to induce
variable stress responses. To determine the cell type(s) involved in glutamine
production and urea excretion, we attempted to localize GGS, LGS and tUT mRNA
expression using in situ hybridization. The role of circulating urea
in tUT regulation was also investigated by infusing toadfish with urea alone
or in combination with intraperitoneal injection of RU486, a corticosteroid
type II receptor antagonist (Bertagna et
al., 1984; Gaillard et al.,
1985) that has been shown to prevent cortisol-induced urea
excretion effects in toadfish (McDonald et
al., 2004).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Initially the fish were kept in 50 l glass aquaria with
flowing, aerated seawater (24–26°C) and fed once weekly with
previously frozen squid.
Experimental protocol
Series i: endogenous cortisol elevation by crowding
Uncrowded toadfish (N=6) were kept individually in large, mesocosm
tanks (1 m3) that simulated the natural toadfish environment, for 1
week prior to sampling. Crowded toadfish (N=8) were maintained
together in 10 l plastic containers for 1 week prior to sampling. After the
acclimation period, toadfish were removed from either the mesocosm or the
crowding tanks, wrapped in wet paper towels and the blood quickly sampled by
caudal puncture. Fish were then anesthetized with a lethal dose of MS-222 (3 g
l–1) and gill tissue dissected. Blood samples were
centrifuged at 10,000g for 1 min and the plasma decanted.
Plasma and gill samples were frozen immediately in liquid nitrogen and stored
at –80°C for no longer than 1 month before analysis of plasma urea
and cortisol, gill tUT and GS mRNA expression, and GS activity.
Series ii: exogenous cortisol loading through arterial infusion
Caudal arterial catheterizations and recovery were performed as described
previously (McDonald et al.,
2000). In a protocol similar to that described before
(McDonald et al., 2004), the
arterial catheter was connected to one channel of a Gilson 8-channel
peristaltic pump and fish were infused for 48 h with isosmotic NaCl (150 mmol
l–1) at an infusion rate of 3 ml kg–1
h–1; the rate was checked by periodic measurement of the mass
of each individual infusion reservoir. After the 48 h infusion with NaCl, a
blood sample was taken. Toadfish were then separated into two treatment
groups. Saline-infused fish (mean mass ± s.e.m. 0.065±0.006 kg,
N=10) continued to be infused with isosmotic NaCl at an infusion rate
of 3 ml kg–1 h–1 for a second 48 h. Fish
treated with cortisol (mean mass 0.060±0.003 kg, N=10) were
infused with cortisol (0.19 mmol l–1; hydrocortisone
hemisuccinate salt; Sigma-Aldrich Chemicals, St Louis, MO, USA) in isosmotic
NaCl at a rate of 0.56 µmol cortisol kg–1
h–1 for 48 h. In both groups, after the second 48 h infusion,
a blood sample was taken through the arterial catheter; fish were then
anesthetized with a lethal dose of MS-222 (3 g l–1) and gill
tissue was dissected. Blood samples were centrifuged at 10 000
g for 1 min and the plasma decanted. Plasma and gill samples
were frozen immediately in liquid nitrogen and stored as described above. The
concentration of infused cortisol was chosen to raise circulating cortisol
levels to approximately 5-fold higher than in a typical ureotelic toadfish;
levels which have been shown in a previous study to result in an inhibition of
pulsatile urea excretion in toadfish
(McDonald et al., 2004).
Series iii: exogenous urea loading through arterial infusion
Arterial catheter implantation was as described above. At the same time,
intraperitoneal (IP) catheters (Clay-Adams PE160, Franklin Lakes, NJ, USA)
filled with peanut oil were inserted through a small ventral incision and
threaded approximately 4 cm inside the body cavity as described by McDonald
and Walsh (McDonald and Walsh,
2004). The fish were left to recover undisturbed for 24 h, water
flow to the fish box was stopped, set to a known volume and an initial water
sample was taken for the measurement of urea concentration. Water samples were
continued as described above.
The arterial catheter was connected to one channel of a Gilson 8-channel peristaltic pump and fish were infused for 48 h with an isosmotic load of NaCl (150 mmol l–1) at an infusion rate of 3 ml kg–1 h–1. After the 48 h infusion with NaCl, a blood sample was taken and toadfish were then separated into two treatment groups. Fish treated with urea+peanut oil (mean mass 0.066±0.005 kg, N=10) were infused with urea (100 mmol l–1) in isosmotic NaCl at a rate of 300µmol urea kg–1 h–1 for 48 h during which the toadfish were injected through the IP catheter with 0.4 ml of peanut oil starting immediately before the urea infusion. Injections through the IP catheter were repeated every 12 h for the remainder of the experiment while fish were continuously infused with urea. This treatment group served as a vehicle and IP injection control for fish infused with urea+RU486 (mean mass 0.067±0.008 kg, N=11). Fish in this group were infused with urea during which they were injected intraperitoneally with 1.5 mg RU486, a glucocorticoid receptor antagonist (mifepristone, 11β–[4-dimethylamino]phenyl-17β-hydroxy-17[1-propynyl]estra-4,9-dien-3-one; Sigma-Aldrich Chemicals) in 0.1 ml peanut oil followed by 0.3 ml of peanut oil, starting immediately before the urea infusion. In both groups, after the second 48 h infusion, a blood sample was taken through the arterial catheter; fish were then anesthetized with a lethal dose of MS-222 (3 g l–1) and gill tissue was dissected. Blood samples were centrifuged at 10000g for 1 min and the plasma decanted. Plasma and gill samples were frozen immediately in liquid nitrogen and stored as described above.
The concentration of infused urea was chosen to raise circulating urea
levels to approximately 5-fold higher than in a typical ureotelic toadfish as
described previously (McDonald et al.,
2003). The amount of RU486 injected was chosen such that
circulating levels of antagonist were 10-fold greater than circulating
cortisol concentrations. This concentration had also been shown previously to
inhibit a cortisol-induced reduction in urea excretion in toadfish
(McDonald et al., 2004). The
IP injection of lipophilic compounds (such as RU486) in oil vehicles (i.e.
peanut oil, coconut oil, corn oil) mediates the slow release of these
substances into the circulation (Vijayan
and Leatherland, 1989;
Christensen et al., 1999;
McDonald et al., 2004).
Quantitative PCR
Total RNA was isolated from whole gill within 1 month of obtaining the
sample following the protocol provided with Trizol reagent (Invitrogen,
Carlsbad, CA, USA) and treated with DNAse to remove potential residual genomic
DNA (TurboDNA-free kit; Ambion, Austin, TX, USA). cDNA synthesis using random
hexamers was performed according to the protocol provided with the SuperScript
first-strand synthesis system for RT-PCR (Invitrogen). Quantitative PCR (qPCR)
was performed for tUT, GGS and LGS (genes of interest; GOI)
using a Mx4000 multiple quantitative PCR system (Stratagene, La Jolla, CA,
USA) with SYBR Green. For elongation factor 1
(EF1; forward
primer 5'-GTT GGT GTC ATC AAG GCT GTT A-3', reverse primer
5'-TGA ACT CTG CCT TGA AGA TGA A-3'), tUT (forward primer
5'-CAT CAT CTC CCT CTT CAT CTC C-3', reverse primer 5'-GTA
TCC CCA CAA GCC AAA ATA A-3'), GGS (forward primer 5'-AAA CCC AGG
TCA CCT ACA TCT G-3', reverse primer 5'-GCA CAC TGG GAT GAG GTA
CAT A-3') and LGS (forward primer 5'-TTG AGT AAA GCT GTC AAG AAG
CA-3', reverse primer 5'-AAC CAA GTA CAT GTC GCT GTT G-3'),
primers were designed based on published toadfish sequences (GenBank accession
nos AF165893, AF532312 and AF118103 for tUT, GGS and LGS, respectively). The
amount of cDNA for the GOI was expressed relative to the amount of cDNA from a
normalizer gene (18S for tUT and GGS, and
EF1 for LGS). Two different normalizers were used
because the abundance of the normalizer gene should be similar to that of the
GOI as evidenced by similar Ct values. The stability of normalizer
gene expression with treatment was tested by comparing normalizer gene
expression in fish from different treatment groups, using cDNA obtained from
normalized quantities of total RNA as described in Schmittgen and Zakrajsek
(Schmittgen and Zakrajsek,
2000). To determine whether the amplification/detection
efficiencies of the GOI and the normalizer gene were similar, a standard curve
was generated with known quantities of cDNA for the GOIs and the normalizer
gene plotted versus their Ct values. The standard curves of
the GOIs and normalizer genes in the present studies gave PCR efficiencies of
100% (tUT), 100% (GGS), 99.8% (LGS), 80.6% (18S) and 74.2% (EF1). To
ensure that the amplification of only one PCR product was contributing to the
measured Ct value, a dissociation curve was established for each
product, which revealed only a single peak signifying only one amplified
product. No-template controls were also run to ensure that primer
concentrations were optimized and primer-dimers were not contributing to the
fluorescence. To verify that the correct product for each primer pair was
being amplified, the size of the PCR product for a subset of samples was
determined using gel electrophoresis. Gel-extracted PCR products (with Qiaex
II from Qiagen, Valencia, CA, USA) were then cloned and amplified in
Escherichia coli according to the protocol provided with the TOPO TA
cloning kit for sequencing using TOP10 chemically competent one shot cells
(Invitrogen). The plasmid cDNA was isolated (Qiagen miniprep) and the clones
sequenced and identified (Geneway LLC, CA, USA).
Tissue preservation
Gill filaments were removed from freshly dissected gill arches collected
from uncrowded and crowded fish as described in series i. The filaments were
placed in ice-cold 4% paraformaldehyde (pH 7.4) and kept at 4°C overnight.
They were then transferred to phosphate-buffered saline (PBS) containing 15%
sucrose for 2 h at 4°C and, finally, transferred to PBS containing 30%
sucrose. Tissue samples were embedded in Shandon Cryomatrix embedding medium
(Fisher Scientific, Pittsburgh, PA, USA), and sections (10 µm) were
prepared using a Leica CM 1850 cryostat at –22°C. Sections were
placed on SuperFrost++ (Fisher Scientific) microscope slides, air
dried for 30 min, and stored at –20°C until use.
Immunocytochemistry
Sections were washed in situ (3x5 min) with a washing buffer
containing 0.1 mol l–1 PBS, and 0.9% Triton X-100. They were
then incubated for 2 h at 37°C, in a humidified chamber, with primary
antibodies diluted in the buffer: 5, a mouse monoclonal antibody
against the 1-subunit of chicken
Na+/K+-ATPase (1:100; University of Iowa Hybridoma
Bank). For negative controls, sections were incubated with washing buffer
lacking primary antibodies. The 5 antibody has been used in numerous
previous studies to localize Na+/K+-ATPase in fish
tissues (e.g. Wilson et al.,
2000). The slides were then washed (3x5 min) in 0.1 mol
l–1 PBS. The 5 antibody was detected with a 1:400
dilution of Alexa 546-coupled goat anti-mouse IgG (Fisher Scientific). Slides
were incubated in a humid chamber for 1 h at room temperature. They were then
washed (3x5 min) in 0.1 mol l–1 PBS and mounted with a
mounting medium (Vector Laboratories, Burlingame, MA, USA) with or without
4',6'-diamidino-2-phenylindole (DAPI) to stain nuclei.
In situ hybridization
For in situ studies, digoxigenin-labeled RNA probes were prepared
by in vitro transcription using linearized plasmid cDNA and SP6 RNA
polymerase (for antisense) or T7 RNA polymerase (for sense). To generate a
homologous probe for Na+/K+-ATPase, primers were
designed against conserved regions of the 1 subunit. These
primers (Na+/K+-ATPase forward 5'-TAC TAC CAA GAR
GCC AAG AGC T-3'; Na+/K+-ATPase reverse
5'-GTT CTG GGT CAG GGT GC-3') corresponded to nucleotides
487–508 and 1181–1197 of the 1a isoform of
rainbow trout (Oncorhynchus mykiss)
Na+/K+-ATPase (GenBank accession no. AY319391.1). The
resultant 587 bp PCR product was ligated into PCR II vector (Invitrogen) and
transformed into competent DH5 E. coli cells. Purified
plasmids were sequenced to confirm that the cloned PCR product was homologous
to Na+/K+-ATPase. For tUT (forward primer 5'-ATC
ACA CGG CAC AAA GG AT-3', reverse primer 5'-ATG AAC AGC TTG GGC
AAA T-3'), GGS (forward primer 5'-CGC TGT TTG GTA CAG ATG
GA-3', reverse primer 5'-GTA CGG GTC ACA GTT TGC AG-3') and
LGS (forward primer 5'-TCT TCC GGA ATG GAA CTT TG-3', reverse
primer 5'-CTT CTC CTG GCC GAC ACT AC-3'), primers were designed
based on published toadfish sequences.
These primers were used to amplify selected regions of full-length cDNAs from previously prepared plasmids. The PCR products (tUT 783 bp, GGS 635 bp and LGS 832 bp) were cloned and sequenced as described above. Sections on slides were hydrated (2x15 min) in 1x PBST (PBS with 0.1% Tween 20). Proteinase K (20 µgml–1 in 1x PBST; Gibco-BRL, Orand Island, NY, USA) was used to deproteinate samples for 20 min at room temperature. Following deproteination, samples were fixed in 4% formaldehyde (in PBS) for 5 min. Fixed tissues were subsequently rinsed twice (10 min per wash) with 1x PBST and air dried at 60°C for 15 min.
Probes (approximately 200 ng per reaction) were denatured for 3 min at 94°C in a solution containing 250 µgml–1 salmon sperm DNA, 250 µg poly(A)+, topped up to 12.5 µl with DEPC (diethyl pyrocarbonate) H2O. Probes were then quickly chilled on ice and centrifuged (7500 g) for 1 min. Hybridization buffer (100 µl of 4x SSC, 20% dextran sulfate, 50% formamide, 250 µgml–1 poly(A)+, 250 µgml–1 ssDNA, 0.1 mol l–1 DTT, 250 µgml–1 tRNA, 0.5x Denhardt's solution) was added to each probe. Each probe was then mixed well by vortexing and placed onto sections. Hybridization was performed for 48 h at 57°C in a humid chamber. Following overnight hybridization, sections were washed twice (15 min per wash, 58°C) with 2x SSC and twice (15 min per wash, 58°C) with 0.2x SSC, followed by one wash in 0.1x SSC for 10 min at room temperature and two washes in 0.1x PBS (10 min per wash, room temperature). To detect hybridization, sections were incubated for 1 h at room temperature with 1% goat serum, 2 mg ml–1 BSA in 0.1 mol l–1 PBS with 0.3% Triton X-100, followed by overnight incubation at 4°C in anti-digoxigenin antibody conjugated to alkaline phosphatase (1:1000 dilution; Roche Molecular Biochemicals, Temecula, CA, USA). Slides were washed at room temperature in 0.1 mol l–1 PB for 15 min and then briefly rinsed in water. The slides were next washed twice (5 min per wash) in coloration buffer (100 mmol l–1 Tris pH 9.5, 50 mmol l–1 MgCl2, 100 mmol l–1 NaCl, 0.1% Tween 20). Nitroblue tetrazolium (NBT) and a single 5-bromocresyl-3-indolyl phosphate (BCIP) tablet (Sigma-Aldrich Chemicals) were dissolved in 10 ml of H2O and layered over the sections. Color was allowed to develop in a humid chamber at room temperature for at least 4 h or until satisfactory coloration was observed. The slides were then washed twice with 0.1 mol l–1 PBS (15 min per wash). Coverslips were placed on the slides using 60% glycerol as mounting medium.
Once prepared, all specimens were observed and photographed using a Zeiss Axiophot microscope (Zeiss, Jena, Germany) equipped with a Hamamatsu C5985 chilled CCD camera, using Metamorph imaging software 4.01 (Molecular Devices, Dowingtown, PA, USA).
Assays
Urea concentrations in plasma and water were measured using the diacetyl
monoxime method of Rahmatullah and Boyde
(Rahmatullah and Boyde, 1980)
with appropriate adjustments of reagent strength for the different urea
concentration ranges in water and blood plasma. Total urea excretion was
measured and calculated as described previously
(McDonald et al., 2004). GS
activity in gill tissue was measured using the transferase assay as described
by Walsh (Walsh, 1996). Plasma
cortisol concentrations were measured using a commercial 125I
radioimmunoassay kit (MP Biomedical, Solon, OH, USA) with standards diluted to
the same protein range as toadfish plasma.
Statistics
Data are reported as means ± 1 s.e.m. (N=number of fish).
The significance of differences between means was evaluated using Student's
unpaired two-tailed t-test (P<0.05). When data were still
not normally distributed upon log transformation, a Mann–Whitney rank
sum test was used.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Total GS activity was significantly higher in all treatment groups compared with that in the uncrowded, control fish, with the greatest elevation being measured in the crowded toadfish (Fig. 2A). The relative expression of GGS mRNA did not show the same level of sensitivity to treatment as GS activity (Fig. 2B). Specifically, GGS mRNA expression in crowded, saline-infused or urea-infused ±RU486 fish was not significantly different from that measured in uncrowded fish (Fig. 2B). However, a 4.8-fold elevation in GGS expression was measured in fish that were infused with cortisol (Fig. 2B). The mRNA expression of the LGS isoform showed slightly more sensitivity to treatment than that of the GGS isoform. Similar to GGS, crowding had no effect on LGS mRNA expression; however, saline infusion resulted in a significant 4.7-fold increase in LGS mRNA expression, which remained elevated in the other treatments (Fig. 2C). Nonetheless, a rough comparison of normalized GGS and LGS transcript abundance indicates that the GGS isoform was approximately 20-fold more abundant in the gill under all conditions tested than the LGS isoform (data not shown).
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The relative expression of tUT mRNA showed a 1.9-fold and 1.7-fold elevation in crowded and saline-infused fish, respectively, compared with fish that were held in uncrowded conditions (Fig. 3). Fish that were cortisol infused had tUT mRNA levels that were 3.3-fold higher than those of uncrowded fish and significantly greater than those of both crowded and saline-infused fish. Interestingly, a 6.0-fold increase was seen in urea-infused fish compared with uncrowded fish, which was reduced to a 3.2-fold increase in fish that were treated with urea+RU486 (Fig. 3).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Gill GS activity and mRNA expression
An endogenous elevation in circulating cortisol levels in response to
crowding under laboratory conditions has been shown to result in a significant
increase in the activity and mRNA expression of GS in the liver of toadfish
(Hopkins et al., 1995;
Walsh et al., 2003). In
correspondence with these documented changes in urea production by the liver,
toadfish in the present study showed a significant elevation in the total GS
activity within the gill (a measure that includes the activity of both LGS and
GGS isoforms). Sensitivity of non-hepatic GS to cortisol has been demonstrated
in tilapia gastrointestinal tract, stomach and muscle
(Mommsen et al., 2003) as well
as in mammalian astrocytes (O'Banion et
al., 1994) and intestine
(Sarantos et al., 1994). In
toadfish, the increase in gill total GS activity in response to crowding alone
is in contrast to previous findings, which showed no change
(Walsh et al., 2003); however,
the inconsistency between the two studies could be explained by protocol
differences; toadfish in the previous study were only crowded for 48 h (and
levels of stress were not estimated by measurement of cortisol levels)
compared with the present 1 week crowding protocol. While crowding resulted in
an increase in the total GS activity in the gill, there was no corresponding
increase in the mRNA expression of either LGS or GGS with crowding. Induction
of the GS enzyme without changes in transcription has been measured in the
mammalian jejunum; in this case it was postulated that glucocorticoids
increased GS levels by accelerating protein translation
(Sarantos et al., 1994).
In contrast to fish that were simply crowded, an increase in the
transcription of both LGS and GGS isoforms was measured in fish that were
infused with saline or cortisol, respectively. This result is unlikely to be
explained by measured differences in plasma cortisol concentrations amongst
the three groups (the crowded and cortisol-treated fish had similar levels of
circulating cortisol). The upregulation of LGS mRNA expression could be in
response to the volume loading experienced by infused fish; however, this
would not explain the upregulation measured in GGS, which appears only to be
sensitive to cortisol infusion. Another difference between crowded,
saline-infused and cortisol-infused fish is the stressor involved; the social
stress experienced by crowded fish is very intense and, in addition to
cortisol elevation, could result in changes in many other physiological
parameters that may interfere with the changes in GGS gene expression measured
in fish infused with cortisol alone
(Sloman et al., 2005). It also
cannot be ruled out that the varied exposure to different acute stressors
experienced by saline- and cortisol-infused fish (i.e. surgery) may have
resulted in periods of much greater cortisol levels that were not captured by
the single post-infusion blood sample. Nevertheless, the pattern of
transcriptional upregulation measured in GGS and LGS suggests that a more
chronic stressor may be required to increase GGS transcription than is
necessary for the upregulation of LGS. Interestingly, total GS enzyme activity
was not different between saline- and cortisol-infused fish, which is
reflected in similar branchial ammonia excretion rates between the two groups
(data not shown); however, this may be a consequence of relatively small
transcriptional changes not translating to detectable changes in protein
activity.
The significantly higher GGS compared with LGS transcript levels suggests
that GGS probably makes up a greater proportion of the total GS activity of
the gill, providing functional significance to this gill-specific isoform and
putting into context the differences measured in the apparent cortisol
sensitivity of the two isoforms. Walsh and colleagues
(Walsh et al., 2003)
determined that the cellular compartmentation of GGS differs from that of LGS,
because GGS is missing the mitochondrial leader sequence that would target it
to the mitrochondrial compartment, and it had an exclusively soluble/cytosolic
distribution, potentially increasing its direct contact with ammonia. The
demonstrated cytosolic location of GGS combined with the higher transcript
levels of GGS compared with those of LGS in the gill supports an
ammonia-trapping function of the gill itself that differs from the function of
GS in other organs. That higher cortisol concentrations are required to
increase GGS transcription than are necessary for the upregulation of LGS
suggests that glutamine production may occur secondary to the increase in urea
production in response to stress by liver-bound LGS, as the pattern of mRNA
expression measured in LGS probably reflects what is going on in the liver.
Gill-bound LGS probably does not play a major role in gill glutamine
production as indicated by the low transcript levels and lack of LGS signal
using in situ hybridization.
tUT activity and mRNA expression
The findings of the present study suggest that both cortisol and urea have
potential regulatory effects on toadfish gill tUT activity and mRNA
expression. Previous studies investigating the role of cortisol in the
regulation of toadfish pulsatile urea excretion have consistently demonstrated
an inhibitory effect of both acute and chronic elevations of cortisol on tUT
function (Hopkins et al., 1995;
Wood et al., 1997;
Wood et al., 2001;
McDonald et al., 2004). These
past results were further supported by the present study, in which exogenous
cortisol loading through infusion resulted in a significant decrease in urea
excretion, suggesting a potential downregulation of tUT mRNA expression or
function. A decrease in mRNA abundance in response to glucocorticoid treatment
was measured in several mammalian facilitated diffusion urea transporters,
namely UT-A1, UT-A3 and UT-A3b found in the inner medullary collecting duct
(IMCD); glucocorticoids suppressing the activity via the promoter
region responsible for transcription
(Knepper et al., 1975;
Naruse et al., 1997;
Peng et al., 2002). However,
Peng and colleagues (Peng et al.,
2002) did not find any change in the transcription of UT-A2 in
response to glucocorticoids, which is the mammalian isoform that most closely
resembles toadfish tUT (Walsh et al.,
2000). In contrast to the hypothesized downregulation or potential
insensitivity measured by Peng and colleagues
(Peng et al., 2002), the mRNA
abundance of tUT in fish with either endogenous (through crowding) or
exogenous (through infusion) elevations in cortisol was significantly
increased compared with that of uncrowded fish, revealing a clear distinction
between the transcription of tUT and the capacity of the fish to excrete urea,
a measure of tUT protein function.
An increase in tUT mRNA abundance in association with the surge in cortisol
during the transition to ureotely would be adaptive for timing the increase in
urea production with an increased ability to excrete urea across the gill.
What has become apparent, however, is that cortisol may have two roles in tUT
regulation (Hopkins et al.,
1995; Wood et al.,
1997; Wood et al.,
2001; McDonald et al.,
2004). It appears that a chronic elevation in circulating cortisol
serves to increase tUT mRNA expression, which may in fact allow more urea
transporter to be translated. However, elevated cortisol levels measured in
ureotelic toadfish also appear to prevent urea from being excreted. When
circulating cortisol concentrations periodically drop, a pulse of urea occurs,
suggesting that the cortisol drop is permissive to the post-transcriptional
modification of tUT (Hopkins et al.,
1995; Wood et al.,
1997; Wood et al.,
2001). Without the periodic, natural drop in cortisol, as observed
in toadfish infused with cortisol, the newly transcribed tUTs might never be
utilized. Alternatively, maybe the increase in tUT mRNA abundance is simply a
(failed) attempt to offset an increased degradation of tUT protein that may be
occurring in response to elevated cortisol concentrations, as suggested by
Kong and colleagues (Kong et al.,
2000) in the case of toadfish carbamoyl phosphate synthetase III.
Then again, there could be a second urea transporter in the gill, as evidenced
by Walsh and colleagues (Walsh et al.,
2000) using northern analysis, that responds to cortisol with a
downregulation in transcription which overwhelms the upregulation measured in
tUT, resulting in the measured overall decrease in urea excretion.
The differing transcriptional responses of tUT and the closely related
mammalian UT-A2 isoform to cortisol begged the question of whether cortisol
was acting directly on tUT or whether its apparent sensitivity to cortisol was
instead due to the elevation in circulating urea concentrations that is often
measured in stressed toadfish and other teleosts
(Vijayan et al., 1996;
McDonald and Wood, 2004). An
upregulation of mammalian UT-A protein in response to uremia, a pathological
condition resulting in higher circulating urea concentrations, was originally
documented in rats subjected to nephrectomy
(Klein et al., 1999). However,
a later study by Klein and colleagues
(Klein et al., 2002)
determined that it was the acidosis that occurred in response to nephrectomy
that directly resulted in the increase in UT-A2 abundance and not the
elevation in urea concentrations that was a byproduct of the acidosis
(Klein et al., 2002). In the
present study, there did appear to be a urea-sensitive component to tUT mRNA
expression; however, blood pH in toadfish infused with urea was not measured
and thus it cannot be conclusively ruled out that acidosis was a contributing
factor.
An increased expression of UT-A2 transporters has also been shown to occur
in response to the hydration state of the animal (Smith et al., 1995), with
the increase in transcription resulting from the activation of the UT-A
promoter by cAMP-dependent pathways
(Nakayama et al., 2001).
Changes in hydration status based on the volume loading experienced by both
cortisol-infused and urea-infused fish would not explain the significant
difference in tUT transcription measured between these two groups.
Furthermore, a significant difference in tUT mRNA expression is not observed
between crowded toadfish and those that are saline infused, two groups that
have similar plasma cortisol and urea levels but differ in that the latter is
volume loaded. Thus, it appears that the hydration status of toadfish is
probably not a regulatory component of tUT transcription.
In situ hybridization and colocalization of GGS and tUT
Because of the lack of a mitochondrial leader sequence in GGS, Walsh and
colleagues (Walsh et al.,
2003) speculated that the gill and ubiquitous forms of GS might be
expressed in different cell types; GGS in pavement cells and LGS in MRCs. In
marked contrast to their speculation, GGS showed a similar pattern of staining
to Na+/K+-ATPase, which is expressed in MRCs, while we
were unable to determine the location of LGS, probably due to its markedly
lower transcript abundance as demonstrated by qPCR measurements. Similar to
GGS, and in contrast to speculation by Laurent and colleagues
(Laurent et al., 2001), tUT
also showed a similar pattern of staining to
Na+/K+-ATPase, suggesting MRC localization. Changes in
pavement cell morphology and increased vesicular trafficking during urea
pulsing suggested that tUT may be found in gill pavement cells
(Laurent et al., 2001).
However, the present findings are in agreement with immunohistochemistry on a
largely ammoniotelic species, the Japanese eel (Anguilla japonica)
urea transporter (eUT), which was localized to the basolateral membrane of
MRCs (Mistry et al., 2001).
Thus, the toadfish MRCs express both GGS and tUT and these cells probably have
a combined function to reduce ammonia excretion by producing glutamine while
at the same time excreting urea in times of stress. Interestingly, the
elaboration of the pavement cells suggests excretion of an electron-dense
material to the apical surface via vesicles
(Laurent et al., 2001) and, in
the light of recent behavioral studies in toadfish
(Sloman et al., 2005;
Barimo and Walsh, 2006), it
would be of interest to examine these materials for molecules involved in
communication that might accompany urea and ammonia excretion as regulated by
the chloride cells.
Recent evidence has outlined an important role for the combined reduction
of ammonia excretion and elevation in urea excretion in toadfish survival.
Toadfish have been shown to excrete a combination of urea and ammonia in the
wild (Hopkins et al., 1997;
Hopkins et al., 1999). A recent
study has revealed ammonia waste to be an important chemical attractant in the
aquatic environment which becomes undetectable if excreted in combination with
urea (Barimo and Walsh, 2006).
Having the combined control to upregulate urea-N production in the liver, and
upregulate urea-N excretion across the gill while decreasing ammonia-N
excretion across the gill when under stressful conditions ensures that the
fish will be excreting the appropriate mix of ammonia:urea as a
predator-avoidance tactic. When under conditions of very high stress, such as
in a laboratory environment, evidence shows that fish almost shut down ammonia
excretion entirely, becoming predominantly ureotelic
(Walsh, 1997;
Wood et al., 1995;
Wood et al., 1997,
Wood et al., 1998;
Wood et al., 2001). In terms
of predator avoidance where a natural analog to the high stress laboratory
model might be a fish under repeated attack by predators, this `full ureotely'
response is not disadvantageous; Barimo and Walsh
(Barimo and Walsh, 2006) did
not find a significant difference in the ability of ammonia+urea or urea alone
to attract/avoid predators.
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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P<0.05, significantly
different from control infusion period.