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First published online October 7, 2008
Journal of Experimental Biology 211, 3226-3236 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.020396
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The effects of CO2 and external buffering on ammonia excretion and Rhesus glycoprotein mRNA expression in rainbow trout
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
* Author for correspondence (e-mail: nawatacm{at}mcmaster.ca)
Accepted 18 August 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: hypercapnia, Rhesus glycoproteins, Oncorhynchus mykiss, gills, skin, cortisol, Hepes, ammonia transport
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Several non-erythrocytic Rh proteins have now been
identified in a variety of species ranging from bacteria to mammals (e.g.
Chain et al., 2003;
Huang and Peng, 2005;
Weihrauch, 2006). Structural
and functional studies have revealed that Rh proteins are transporters of
ammonia, a property shared with AmtB, their Escherichia coli
homologue (for reviews, see Planelles,
2007; Van Kim et al.,
2006; Weiner and Hamm,
2007). There is some conflict as to whether the primary substrate
is NH3 or NH4+, but most evidence points to
NH3 (Javelle et al.,
2007).
A number of different Rh orthologues have been characterized in fish
(Huang and Peng, 2005;
Hung et al., 2007;
Nakada et al., 2007a;
Nakada et al., 2007b;
Nawata et al., 2007). While
Rhag appears to be limited to erythrocytes in mammals
(Huang and Peng, 2005), it
occurs in both erythrocytes and pillar cells in the pufferfish Takifugu
rubripes (Nakada et al.,
2007b). Additionally, Nakada and colleagues showed in pufferfish
that while Rhbg and Rhcg2 are situated basolaterally and apically,
respectively, in the pavement cells, Rhcg1 is located apically in
mitochondria-rich cells (Nakada et al.,
2007b). Exactly how these Rh proteins function in ammonia
transport in fish is not yet clear; however, Rh mRNA expression levels do
change in response to high external ammonia in some fish species
(Hung et al., 2007;
Nawata et al., 2007), but not
all (Nakada et al.,
2007a).
The bulk of studies implicate Rh proteins in NH3 transport,
although there remains the possibility that they also facilitate transfer of
CO2, another readily hydrated gas of similar size
(Endeward et al., 2006;
Kustu and Inwood, 2006).
Soupene and colleagues reported that the Rh1 protein of the green alga
Chlamydomonas reinhardtii was upregulated by high CO2 and
that the absence of Rh1 impaired the growth of this alga under high
CO2, a condition that normally promotes rapid growth
(Soupene et al., 2002;
Soupene et al., 2004).
Recently it was demonstrated, using 18O and mass spectrometry, that
both NH3 and CO2 pass through the RhAG protein of human
erythrocytes (Endeward et al.,
2006). These two substrates compete for entrance and passage
through the RhAG channel, although the affinity appears to be greater for
NH3 than CO2. Furthermore, structural studies performed
on the Rh1 protein of the ammonia-oxidizing bacterium Nitrosomonas
europaea revealed that it may have a much lower ammonium affinity than
the Amt proteins (Lupo et al.,
2007) and a possible CO2 binding site at the
COOH-terminal end has been described (Li
et al., 2007).
A few studies suggest, however, that CO2 is not transported
through the Rh channel. Ripoche and colleagues, for example, found no
difference in the permeability to CO2 in human erythrocyte
membranes lacking RhAG, using a stopped-flow method
(Ripoche et al., 2006). More
recently it was shown that both wild-type
(Weidinger et al., 2007) and
Rh1 knockout mutants
(Cherif-Zahar et al., 2007) of
N. europaea were unresponsive to CO2.
Like ammonia, CO2 in fish is believed to pass through membranes
mainly in its gaseous form (Henry and
Heming, 1998), while the transport of CO2 occurs in the
plasma in its hydrated form, HCO –3
(Perry and Gilmour, 2002). A
small amount of CO2 exits the apical gill epithelium as HCO
–3 in exchange for Cl– after
being hydrated in the gill by carbonic anhydrase and thus serves a role in
acid–base regulation (Claiborne et
al., 2002; Perry,
1986; Perry and Gilmour,
2006).
In the present study, our goal was to determine whether exposure to high
external CO2 levels could elicit changes in Rh mRNA expression in
rainbow trout gill, skin and erythrocytes, which would indicate a possible
dual role for the Rh glycoproteins as NH3 and CO2
channels. However, studying the effects of high CO2 on Rh mRNA
expression in fish is problematic in that hypercapnia causes an increase in
endogenous ammonia production (Claiborne
and Heisler, 1986). Increased ammonia excretion rates have also
been associated with exposure to external hypercapnia
(Claiborne and Heisler, 1986)
and this may be due to the favourable plasma to water ammonia gradient created
by the lowered water pH as a result of the hydration of CO2 in the
water (Claiborne and Heisler,
1986; Larsen and Jensen,
1997; Lloyd and Herbert,
1960; Wright et al.,
1989). Buffering minimizes acidification of the water during
hypercapnia; however, this introduces the confounding effect of abolishing the
acidified boundary layer at the gill that normally facilitates ammonia
excretion (Salama et al.,
1999; Wilson et al.,
1994; Wright et al.,
1986; Wright et al.,
1989).
We therefore examined the effect of three experimental conditions on the
mRNA expression of Rh glycoproteins in trout: hypercapnia alone, hypercapnia
with Hepes buffering, and normocapnia with Hepes buffering. The hypercapnia
treatment chosen (1% CO2 for 12 h) directly duplicated a previous
study in our lab in identical water quality where internal acid–base and
ion status (both extracellular and intracellular) were monitored
(Wood and LeMoigne, 1991).
Additionally we analysed the ammonia excretion rates, plasma cortisol levels
and the mRNA expression of other gill transporters possibly involved in
ammonia excretion in each experimental condition. Finally, we isolated three
full-length Rhbg2 cDNA variants and assessed the mRNA expression of
these in the gill and skin.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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8.0; hardness 140 mg
l–1 as CaCO3 equivalents; temperature
12–16°C) and fed ad libitum until 1 week before
experimentation, during which time food was withheld. Fish were transferred to
individual, opaque boxes supplied with aerated, flowing dechlorinated tap
water (15±0.5°C) and allowed to recover overnight. All procedures
used were approved by the McMaster University Animal Research Ethics Board and
are in accordance with the Guidelines of the Canadian Council on Animal
Care.
Experimental conditions
Effect of external hypercapnia
Water flow was closed off to each box and the box water volume was set to 4
l. Fish were exposed for 12 h to a 1% CO2 in air mixture
(hypercapnia), provided by a Wösthoff gas-mixing pump (Calibrated
Instruments, Ardsley, NY, USA), bubbled into the water. Control fish were
treated identically with a 12 h exposure to 100% air (normocapnia).
Effect of Hepes buffering
Bubbling of 1% CO2 in air into the external water reduced the pH
by approximately 1.22 pH units, a similar reduction to that reported earlier
by other investigators (Larsen and Jensen,
1997; Wright et al.,
1988a). In order to minimize acidification of the water while fish
were exposed to hypercapnia, Hepes (Sigma, St Louis, MO, USA) was added to the
external water to a nominal concentration of 10 mmol l–1
using a 1 mol l–1 stock solution adjusted to pH 8.0 with KOH.
This maintained the pH within the range of 7.76–7.39 during the course
of 12 h. To examine the effects of Hepes buffering alone on ammonia excretion
and mRNA expression, another set of fish were exposed to normocapnia for 12 h
in the presence of 10 mmol l–1 Hepes, as above.
Effect of high environmental ammonia
In order to check some particular parameters highlighted in the results of
the hypercapnia and Hepes-buffering exposures, a high environmental ammonia
(HEA) exposure protocol identical to that used by Nawata and colleagues
(Nawata et al., 2007) was
repeated using an ammonia concentration similar to that used by previous
investigators (Cameron, 1986;
Cameron and Heisler, 1983;
Claiborne and Evans, 1988;
Wilson and Taylor, 1992;
Wilson et al., 1994). Trout
were exposed to a nominal total waterborne ammonia (TAmm)
concentration of 1.5 mmol l–1 NH4HCO3
(pH 7.95±0.05) for 12 h. During this period the water flow was stopped
and the box volume was set to 4 l. Water samples were removed every 3 h and
total water ammonia concentration remained virtually constant at
1.41±0.03 mmol l–1 over the 12 h period.
Analyses
Water samples (10 ml) were removed at 3 h intervals, stored at
–20°C and later analysed in triplicate for total ammonia
(TAmm) using a modified salicylate-hypochlorite method
(Verdouw et al., 1978).
Because Hepes altered colour generation in this assay, additional ammonia
standards containing 10 mmol l–1 Hepes were prepared and
analysed in conjunction with the samples from the Hepes experiments. At the
end of each experiment, fish were anaesthetized with 0.1 g
l–1 MS222 (Sigma) and caudal blood samples were collected
into a heparinized syringe. Blood cells were separated from plasma by
centrifugation and both blood fractions were snap frozen in liquid nitrogen
and stored at –70°C.
Net flux rates of total ammonia (JAmm; µmol kg–1 h–1) into the water were calculated as: JAmm=(TAmm,i–TAmm,f)xV/(txM) where i and f refer to the initial and final concentration (µmol l–1), V is the box water volume (l), t is the time elapsed (h) and M is the fish mass (kg). A negative JAmm indicates net excretion into the water. Plasma total ammonia was measured enzymatically (Raichem, Hemagen Diagnostics, San Diego, CA, USA) and reported in µmol l–1.
External water pH was monitored with a Radiometer GK2401C (Copenhagen, Denmark) low ionic strength combination electrode thermostatically set to the experimental water temperature.
Tissue sampling
Prior to tissue extraction, fish were perfused free of blood using Cortland
saline (Wolf, 1963) as
previously described (Nawata et al.,
2007). Samples of gill, skin and blood were removed, snap frozen
in liquid nitrogen and stored at –70°C until later processing. Gill
samples from control fish were used for cloning of Rhbg2 and
additional samples of brain, gill, intestine, kidney, liver, muscle and skin
were removed for screening of Rhbg2 mRNA expression.
Total RNA isolation and reverse-transcriptase PCR amplification
Total RNA was extracted from blood and tissues using Trizol (Invitrogen,
Burlington, ON, Canada) and concentrations were quantified and checked for
quality with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies,
Wilmington, DE, USA) and electrophoresed on 1% agarose gels stained with
ethidium bromide, to verify integrity. First strand cDNA synthesis was
performed using 1µg of DNaseI-treated (Invitrogen) total RNA with
oligo(dT17) primer and Superscript II reverse transcriptase
(Invitrogen).
Full-length cDNA of Rhbg2 was obtained by 5'- and
3'-rapid amplification of cDNA ends (Smart RACE cDNA amplification kit,
BD Bioscience Clontech, Mississauga, ON, Canada) using the RACE primers listed
in Table 1 and the protocol
described previously (Nawata et al.,
2007). Sequence analyses were performed with BioEdit
(Hall, 1999) and CLUSTAL W
(Thompson et al., 1994).
Hydropathy profile and N-linked glycosylation site predictions were
made using SPLIT 4.0 (Juretic et al.,
2002), and ScanProsite (de
Castro et al., 2006), respectively.
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Tissue distribution of Rhbg2 was determined by performing reverse
transcriptase PCR (PCR) on the above-mentioned cDNA with the primer set listed
in Table 1 at 35 cycles for
Rhbg2 and 25 cycles for elongation factor EF-1
.
Products were electrophoresed on 1.5% agarose gels stained with ethidium
bromide and sequenced to confirm identity.
mRNA expression
Quantitative real-time PCR (qPCR) was performed on the cDNA described above
using the primers listed in Table
1. Rh mRNA expression was assessed in the gill, skin and
erythrocytes and the expression of carbonic anhydrase (CA2;
cytoplasmic), H+-ATPase (v-type, B-subunit),
Na+/H+ exchanger (NHE) and
Na+/K+-ATPase (NKA; 1a-subunit) was
analysed in the gill. Each 20 µl reaction contained 4 µl of cDNA, 8 pmol
of each primer and 10 µl of RT2 Real-Time SYBR Green/ROX PCR
Master Mix (SuperArray, Bioscience Corp., Frederick, MD, USA). Analyses were
performed at 95°C for 2 min, followed by 40 cycles of 95°C for 30 s
and 60°C for 1 min. Melt-curve analysis and gel electrophoresis verified
the presence of a single product. No-template controls and
non-reverse-transcribed controls were run in parallel. Data were extrapolated
from standard curves generated by serial dilution of one randomly selected
control sample. Three housekeeping genes encoding β-actin, EF-1
and 18S rRNA proved to be unstable across treatments and data were therefore
normalized to ng total RNA, another acceptable method of normalization
(Bustin, 2000;
Bustin, 2002;
Nolan et al., 2006), as
further elaborated in the Discussion.
Plasma cortisol
Plasma cortisol levels were measured in duplicate on 25 µl samples by
radioimmunoassay (Cortisol 125I RIA Kit, MP Biomedicals,
Orangeburg, NY, USA) and values are reported in ng ml–1.
Statistical analysis
Data are presented as means ± s.e.m. (N, number of fish).
One-way analysis of variance (ANOVA) followed by Fisher's least significant
difference post-hoc test was used to analyse both the effects of the
experimental treatments and the relative abundance of Rhbg2 mRNA in
the skin. One-way repeated measures ANOVA was used to analyse ammonia
excretion rates. Significance was set at =0.05.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Tissue distribution and expression of Rhbg2
PCR analysis of the various trout tissues from control fish revealed that
Rhbg2 is expressed in the brain, gill, intestine, skin and possibly
in the liver and muscle (Fig.
2). The truncated form, Rhbg2c was only detected in the
gill (data not shown). qPCR analysis performed on control samples of gill and
skin showed that the 5'-UTR variants were present in equal abundance in
the gill. In the skin, however, variants 1 and 3 were significantly more
abundant than the other two. To examine whether or not these variants respond
differentially, we analysed the expression in the skin of fish exposed to
hypercapnia in Hepes-buffered water. Expression of all four variants decreased
such that they were no longer differentially expressed
(Fig. 3).
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External hypercapnia with Hepes buffering
Throughout the 12 h exposure to hypercapnia in Hepes-buffered water, trout
exhibited ammonia excretion rates that were significantly lower than those in
fish exposed to hypercapnia alone, with a 57% reduction at 3 h and a 42%
reduction at 12 h. At 9 h, however, the rates were significantly increased
above the 3 h control rates and by 12 h they were not different from the 12 h
control rates (Fig. 4). Plasma
ammonia levels were significantly increased 2.3-fold above the control value
after 12 h of exposure (Fig.
5). In the gill, CA2 and NHE mRNA levels were
significantly increased (Fig.
9) and Rhcg2 mRNA was a significantly decreased by 60%
(Fig. 6). No significant
changes were noted in the mRNA expression of Rhag or
Rh30-like2 in the erythrocytes, although expression of Rhag
was 2-fold higher than the control (Fig.
8). In the skin, there was a 67% reduction in Rhbg2 but a
30-fold increase in Rhcg2. Rhcg1 was decreased, but not significantly
(Fig. 7). Plasma cortisol was
not significantly different from the control value at 12 h
(Fig. 10).
External normocapnia with Hepes buffering
The ammonia excretion rate in fish exposed to normocapnia in Hepes-buffered
water was not significantly different from the 3 h control rate, throughout
the 12 h exposure. However, there was a trend towards decreased excretion and
by 12 h the rate was reduced to 71% of the 12 h control value
(Fig. 4). Plasma ammonia was
significantly elevated over the control value at 12 h
(Fig. 5). Rhcg2 mRNA
levels were significantly reduced in the gill to about half of the control
level (Fig. 6), but no changes
were noted in the other gill mRNAs (CA2, H+-ATPase, NHE2,
NKA; Fig. 9). In the skin,
while there was a 2-fold reduction in Rhbg1 and a 4-fold reduction in
Rhbg2 mRNA, Rhcg1 and Rhcg2 mRNA expression was
increased by 4- and 58-fold, respectively
(Fig. 7). In the erythrocytes,
there was a 3-fold reduction in both Rhag and Rh30-like2
mRNA expression (Fig. 8) and
plasma cortisol levels were low, but not significantly different from control
values (Fig. 10).
High environmental ammonia
Because of the high nucleotide sequence identity between the Rhbg1
and the Rhbg2 cDNAs (the highest being 91.5%, between the
Rhbg1a and Rhbg2a variants), we designed a primer set more
specific for Rhbg1 than that used previously by Nawata and colleagues
(Nawata et al., 2007), and
assessed the expression of Rhbg1 and Rhbg2 in the gill and
skin of HEA-exposed fish. Both Rhbg1 and Rhbg2 mRNA
expression levels remained unchanged in the gill during HEA
(Fig. 6). In the skin,
Rhbg1 mRNA levels were not significantly different from that of the
control, but Rhbg2 mRNA expression was significantly downregulated by
about 4-fold (Fig. 7).
Expression of Rhcg1 and Rhcg2 mRNA in the gill and skin,
Rhag and Rh30-like2 mRNA in the erythrocytes, and CA2,
H+-ATPase, NHE2 and NKA mRNA in the gill was
also measured and confirmed the results reported earlier by Nawata and
colleagues (Nawata et al.,
2007) and are therefore not reported here. Plasma ammonia and
cortisol levels were both significantly elevated above the control values by
10- and 4-fold, respectively (Figs
5 and
10).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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) are frequently used as internal controls to
normalize expression levels of target genes in real-time PCR studies
(Guilietti et al., 2001; Nolan et al.,
2006), as in our previous studies on Rh proteins
(Hung et al., 2007;
Nawata et al., 2007). Several
recent reports, however, have shown that reference genes themselves may be
modulated by experimental conditions, which in turn may result in inaccurate
and misleading interpretations of data
(Gibbs et al., 2003;
Huggett et al., 2005;
Ings and Van der Kraak, 2006;
Kubista et al., 2006;
Tricarico et al., 2002).
Indeed, our initial experiments revealed that the expression levels of three
reference genes (encoding β-actin, EF-1 and 18S rRNA) were
elevated in tissues after exposure to Hepes. Currently, no universal method of
normalization exists that accounts for all possible variables; however, an
accepted method commonly used (e.g.
Luqueti et al., 2005;
Serrano et al., 2007;
Tipsmark et al., 2008;
Vera Cruz et al., 2006) is
normalization against total RNA (Bustin,
2000; Bustin, 2002;
Huggett et al., 2005;
Nolan et al., 2006).
Therefore, to eliminate any artificial bias in the data due to reference gene
variability, we chose to normalize quantitative expression data to total RNA.
In this regard it is important to note that our approach is validated by the
target gene responses to HEA normalized against total RNA reported in the
present study, which were qualitatively and quantitatively similar to those
reported earlier by Nawata and colleagues
(Nawata et al., 2007) in
parallel but separate HEA experiments on trout where β-actin or
EF-1 was used for normalization.
Recently, we established a link between Rh proteins and ammonia by showing
that rainbow trout exposed to high environmental ammonia (HEA) responded with
upregulated Rhcg2 mRNA expression in the gill
(Nawata et al., 2007). This
response coincided with the re-establishment and enhancement of ammonia
excretion that was initially inhibited by the reversed plasma to water ammonia
gradient. In the present study, we used hypercapnia as a tool to assess the
response of Rh mRNA transcription levels to high CO2 in the rainbow
trout. This proved problematic in that, like the study by Claiborne and
Heisler (Claiborne and Heisler,
1986), hypercapnia caused an increase in internal ammonia
production as evidenced by the elevated rate of ammonia excretion
(Fig. 4) without alteration of
plasma total ammonia concentration (Fig.
5). Bubbling of CO2 into the water lowered the pH,
creating a sink for NH3, thus enhancing the rate of excretion. This
increased excretion rate was sufficient to maintain plasma ammonia at control
levels (Fig. 5) and may explain
why changes in Rh mRNA levels were not observed. Any effects that elevated
CO2 may have had were not reflected as changes in mRNA
transcription levels in the gill or skin.
Buffering of the external water effectively reduced the acidification
caused by the CO2, but it also probably abolished the acidified
gill boundary layer. It has been well documented that the boundary layer,
whether acidified by hydration of CO2 by carbonic anhydrase in the
mucus (Wright et al., 1986;
Wright et al., 1989) or by
release of protons from an apical H+-ATPase
(Lin et al., 1994;
Lin and Randall, 1990),
normally facilitates ammonia excretion. Indeed, the present study confirms
previous reports (Salama et al.,
1999; Wright et al.,
1989) showing that there was a decline in the ammonia excretion
rate when fish were exposed to normocapnia in Hepes-buffered water, with the
rate reduced to a third of the control rate by 12 h
(Fig. 4). As a consequence of
the reduced excretion, plasma ammonia levels in these fish were elevated after
12 h of treatment (Fig. 5).
Despite this elevation, and unlike HEA-exposed fish, which also have high
plasma ammonia (Nawata et al.,
2007), the Rhcg2 mRNA transcription level was decreased
in the gill (Fig. 6).
Upregulated or enhanced ammonia transport capacity at the gill would probably
prove futile in the presence of Hepes since any protons formed or released at
the apical gill surface would be rapidly removed by the buffer, effectively
eliminating a favourable ammonia gradient.
However, in the skin of fish exposed to Hepes in the presence of
normocapnia, both Rhcg1 and Rhcg2 mRNA levels were highly
elevated (Fig. 7). Rhcg2
exhibited the greatest fold increase in mRNA expression, suggesting that this
protein may have a dominant role in the skin, but this requires further
investigation because only mRNA level and not protein function was measured.
Upregulation of Rhcg2 mRNA expression in the skin also occurred when
excretion was blocked at the gill after 12 h of exposure to HEA
(Nawata et al., 2007). Fish
skin is generally thought to have low permeability
(Fromm, 1968) and therefore
its role in gas and ion exchange has been largely ignored. A few studies have
measured ammonia excretion from fish skin (for a review, see
Wood, 1993), but it appears
that the skin has a secondary role to the gill, if any, in freshwater fish.
This may be true especially under normal circumstances, but under conditions
in which excretion is blocked at the gill, the skin may become an important
alternative site of ammonia release and this possibility needs to be explored
further in freshwater species. Indeed the skin becomes a site of ammonia
excretion via volatilization during aerial exposure in the mangrove
killifish Kryptolebias marmoratus, and skin Rh mRNA levels are also
upregulated during this time (Hung et al.,
2007).
The acid–base disturbance in response to hypercapnia has been well
described in catheterized fish (for reviews, see
Claiborne, 1998;
Heisler, 1993).
Catheterization was avoided in the present study, as our initial trials
indicated that catheterization alone may alter Rh expression (C.M.N. and
C.M.W., unpublished data). However, we used an exposure regime in which the
acid–base effects had been documented previously. In an identical 1%
CO2 exposure protocol in Hamilton tap water, chronically
catheterized rainbow trout exhibited a 0.3 unit depression in arterial pH at 3
h with partial recovery by 12 h [see figure
7 of Wood and LeMoigne (Wood
and LeMoigne, 1991)]. Although gill cell intracellular ion levels
(Na+ and Cl–) declined, gill intracellular pH did
not change.
The associated increase in acid excretion which helps correct internal pH
by building up internal HCO –3 levels during
hypercapnia involves the hydration of CO2 by carbonic anhydrase
within the gill (Henry and Heming, 1988;
Perry, 1986;
Perry and Gilmour, 2006).
Apically located H+-ATPase and/or NHE may then be involved in the
release of protons into the water (Edwards
et al., 2005). Although buffering during hypercapnia would cause
an internal elevation of both CO2 and ammonia, the results from our
study suggest that fish in this treatment group were responding to an acidosis
induced by the high CO2. The upregulation of NHE2 mRNA
levels in the gill corresponds well with the findings of Edwards and
colleagues, who reported an increase in NHE2-like protein expression in
Fundulus heteroclitus exposed to external hypercapnia
(Edwards et al., 2005). This
suggests that, although H+-ATPase is believed to play the major
role in acid secretion in freshwater teleosts
(Edwards et al., 2005), NHE2
may also have a role during acidosis in rainbow trout, especially in Hamilton
tap water where Na+ levels are relatively high (700 µmol
l–1). H+-ATPase mRNA expression levels
were not upregulated at 12 h (Fig.
9), but this does not necessarily mean that the corresponding
protein is non-functional during acidosis. The expression levels of
H+-ATPase mRNA may in fact peak as early as 2 h after
hypercapnia exposure (Perry et al.,
2000), a response that could then result in a sufficient quantity
of protein by 12 h. The increased CA2 mRNA levels that occurred are
also in agreement with previous studies that reported increased carbonic
anhydrase activity (Dimberg and Hoglund,
1987) and increased CA2 mRNA and protein expression
(Georgalis et al., 2006) in
the gills of hypercapnia-exposed trout.
Fish exposed to hypercapnia in Hepes-buffered water also experienced inhibition of ammonia excretion at the gill as evidenced by the elevated plasma ammonia (Fig. 5) as well as excretion rates that were significantly lower than those of the hypercapnia-exposed fish in unbuffered water (Fig. 4). Although the excretion rates started to rise at 9 h (Fig. 4), the downregulation of Rhcg2 and H+-ATPase mRNA levels in the gill indicate that these fish were probably not using the same mechanism to enhance ammonia excretion as that used during HEA. Both treatments resulted in blockage of ammonia excretion from the gill, but it appears that in hypercapnic fish, the primary mRNA response in the gill was to CO2 rather than high ammonia. Similar to the fish exposed to normocapnia in Hepes-buffered water, there was an upregulation of Rhcg2 mRNA in the skin (Fig. 7), again reinforcing the idea that this may be an alternative route for ammonia excretion.
Previously we reported that Rhag as well as the Rh30-like mRNAs
were downregulated during HEA, when plasma ammonia levels were high
(Nawata et al., 2007). In the
present study, both groups of fish exposed to Hepes-buffered water had
elevated plasma ammonia levels, albeit lower than HEA-exposed fish
(Fig. 5), so we would have
expected a similar downregulation of Rhag expression in these two
groups. Fish exposed to normocapnia did indeed exhibit lowered Rhag
mRNA levels; however, the hypercapnia-exposed fish did not. Instead, the
Rhag mRNA expression in the hypercapnia-exposed fish was increased
2-fold over that of the control fish, a response similar to that of the
hypercapnia-exposed fish in unbuffered water
(Fig. 8). This suggests that
Rhag was responding to both high CO2 and high ammonia, but
in a differential fashion.
Ammonia accumulates in trout erythrocytes during hypercapnia
(Wright et al., 1988b). If
Rhag allows passage of NH3, then downregulation when plasma ammonia
levels are elevated may be a protective response. Transport of NH3
with CO2 and H2O into the erythrocyte would produce
NH4+ and HCO3– and once
HCO3– is exchanged for Cl–, the
formation of NH4Cl would lead to H2O uptake and result
in swelling (Bruce, 2008).
Also, the buffering effect of NH3 could interfere with the Bohr and
Haldane effects, and therefore with O2 and CO2
transport. In human erythrocyte membranes, RhAG forms a macrocomplex with a
number of proteins including the band-3 anion exchanger (AE1) and carbonic
anhydrase, and this complex is thought to function in
CO2/O2 gas exchange
(Bruce et al., 2003). Trout
erythrocytes also have AE1 present (Michel
and Rudloff, 1989), but whether or not it forms a similar complex
with Rhag is unknown. However, if Rhag functions as a CO2 channel,
then upregulation along with AE1 could be beneficial during hypercapnia when
plasma HCO –3 is high.
It has also been speculated that the Rh30 proteins could allow
CO2 passage (Callebaut et al.,
2006), although Endeward and colleagues found no difference in
CO2 uptake in erythrocytes lacking the RhD (Rh30) protein (Endeward
et al., 2007). Data from our study also suggest that the Rh30-like proteins
are not involved in CO2 transport as there were no notable changes
in mRNA expression in erythrocytes from either of the two groups exposed to
hypercapnia (Fig. 8).
Rh30-like2 mRNA expression, however, was downregulated in the fish
exposed to normocapnia and Hepes, like HEA-exposed fish
(Nawata et al., 2007),
suggesting a response to high ammonia only.
So far, studies have indicated that fish have multiple Rh genes
(Huang and Peng, 2005;
Hung et al., 2007;
Nakada et al., 2007a;
Nakada et al., 2007b;
Nawata et al., 2007) but the
significance of this seeming redundancy is unclear. Nakada and colleagues
(Nakada et al., 2007b)
proposed that Rhag in the pillar cells of pufferfish may work in conjunction
with the basolaterally located Rhbg and apically situated Rhcg2 in the
lamellae to keep plasma ammonia levels low, with additional excretion aided by
Rhcg1 in the mitochondria-rich cells. Others have shown that some Rh proteins
may be dispensable like Rhbg in the mouse
(Chambrey et al., 2005) and
Rh1 in the slime mould (Benghezal et al.,
2001). Likewise, the physiological significance of the multiple
variants of Rhbg2 (Fig.
1) is puzzling, but the presence of uORFs and splicing in the
5'- and 3'-UTRs of the mRNAs suggests complex regulation of this
protein. uORFs have the potential to impact gene expression and some may serve
as cis-acting regulatory elements modulating translation of the main
ORF (Meijer and Thomas, 2002;
Morris and Geballe, 2000).
Similarly, the UTRs are involved in many post-transcriptional pathways that
control the localization, stability and translation efficiency of mRNAs
(Pesole et al., 2001). The
existence of a truncated version of the Rhbg2 protein suggests yet another
level of complexity. Truncation alters function and/or intracellular location
of some transporters while some alternative-splice products negatively
regulate the wild-type protein (Kitayama
et al., 1999; Mangravite et
al., 2003). Whether the truncated version of Rhbg2 functionally
interacts with the full form or whether it has a completely different function
has yet to be analysed.
Interestingly, the same 5'-UTR Rhbg2 variants are present in
both the skin and gill, although in the gill they are expressed equally while
in the skin they are expressed differentially
(Fig. 3). The downregulation of
Rhbg2 mRNA expression that occurred in the skin with Hepes treatment
during both hypercapnia and normocapnia appears to be in response to high
plasma ammonia rather than high CO2 as suggested by the unchanged
expression in the hypercapnia-treated fish in unbuffered water. To support
this idea, we found that HEA-exposed fish exhibited a similar downregulation
(Fig. 7). Rhbg was reported to
be non-essential during acidosis in knockout mice
(Chambrey et al., 2005). An
abundance of Rhbg2 in fish skin may similarly be unnecessary during
hypercapnia and HEA, and instead Rhbg2 could have a role other than ammonia
transport.
Glucocorticoids are known for their role in regulating gene expression in
mammals and the same is likely to be true for fish
(Mommsen et al., 1999). Data
from this study, however, showed no correlation between Rh mRNA abundance and
cortisol levels. Plasma cortisol and gill Rhcg2 mRNA levels were both
elevated during HEA but in the two Hepes-exposed groups Rhcg2 mRNA
was elevated in the skin while the cortisol remained at control levels
(Fig. 10). A lack of
correlation, however, does not exclude the possibility that cortisol could be
involved either directly or indirectly in the control of Rh protein
expression. Indeed, cortisol levels need not be elevated in order to have an
influence on physiological processes (e.g.
Wood et al., 2001) and other
factors such as the clearance rate and non-genomic actions of cortisol need to
be taken into consideration (Mommsen et
al., 1999).
We have evaluated the expression of Rh protein mRNAs in rainbow trout
exposed to hypercapnic and normocapnic conditions in the presence and absence
of Hepes buffer. Previously, we showed that at the mRNA level, Rh proteins
clearly respond to high ammonia conditions in rainbow trout tissues
(Nawata et al., 2007). A
similar response to high CO2 would suggest that Rh proteins may
have a dual role as CO2 and NH3 gas channels in trout
tissues. Data from our present study revealed, however, that high
CO2 did not directly elicit changes in Rh mRNA transcription levels
in the gill and skin. Instead, the changes that did occur probably reflected
responses to high plasma ammonia, and thus reinforce the connection between
the Rh proteins and ammonia. As such, a dual role for Rh proteins in the
rainbow trout is not apparent from this study. There does remain, however, the
possibility that Rhag interacts with both CO2 and ammonia in the
erythrocyte and further work is needed to clarify this.
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Acknowledgments |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Avent, N. D., Madgett, T. E., Lee, Z. E., Head, D. J., Maddocks, D. G. and Skinner, L. H. (2006). Molecular biology of Rh proteins and relevance to molecular medicine. Expert Rev. Mol. Med. 8,1 -20.[Medline]
Benghezal, M., Gotthardt, D., Cornillon, S. and Cosson, P. (2001). Localization of the Rh50-like protein to the contractile vacuole in Dictylostelium. Immunogenetics 52,284 -288.[CrossRef][Medline]
Bruce, L. J. (2008). Red cell membrane transport abnormalities. Curr. Opin. Hematol. 15,184 -190.[CrossRef][Medline]
Bruce, L. J., Beckmann, R., Ribeiro, M. L., Peters, L. L.,
Chasis, J. A., Delaunay, J., Mohandas, N., Anstee, D. J. and Tanner, M. J.
A. (2003). A band 3-based macrocomplex of integral and
peripheral proteins in the RBC membrane. Blood
101,4180
-4187.
Bustin, S. A. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25,169 -193.[Abstract]
Bustin, S. A. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29,23 -39.[Abstract]
Callebaut, I., Dulin, F., Bertrand, O., Ripoche, P., Mouro, I., Colin, Y., J,-P. Mornon, and J. P. Cartron. (2006). Hydrophobic cluster analysis and modeling of the human Rh protein three-dimensional structures. Transfus. Clin. Biol. 13, 70-84.[CrossRef][Medline]
Cameron, J. N. (1986). Responses to reversed NH3 and NH +4 gradients in a teleost (Ictalurus punctatus), an elasmobranch (Raja erinacea), and a crustacean (Callinectes sapidus): evidence for NH +4/H+ exchange in the teleost and the elasmobranch. J. Exp. Zool. 239,183 -195.[CrossRef][Medline]
Cameron, J. N. and Heisler, N. (1983). Studies
of ammonia in the trout: physico-chemical parameters, acid–base
behaviour and respiratory clearance. J. Exp. Biol.
105,107
-125.
Chain, P., Lamerdin, J., Larimer, F., Regala, W., Lao, V., Land,
M., Hauser, L., Hooper, A., Klotz, M., Norton, J. et al.
(2003). Complete genome sequence of the ammonia-oxidizing
bacterium and obligate chemolithoautotroph Nitrosomonas europaea.J. Bacteriol. 185,2759
-2773.
Chambrey, R., Goossens, D., Bourgeois, S., Picard, N.,
Bloch-Faure, M., Leviel, F., Geoffroy, V., Cambillau, M., Colin, Y., Paillard,
M. et al. (2005). Genetic ablation of Rhbg in the mouse does
not impair renal ammonium excretion. Am. J. Physiol. Renal
Physiol. 289,F1281
-F1290.
Cherif-Zahar, B., Durand, A., Schmidt, I., Hamdaoui, N., Matic,
I., Merrick, M. and Matassi, G. (2007). Evolution and
functional characterization of the RH50 gene from the ammonia-oxidizing
bacterium Nitrosomonas europaea. J. Bacteriol.
189,9090
-9100.
Claiborne, J. B. (1998). Acid-base regulation. In The Physiology of Fishes (ed. D. H. Evans), pp.179 -200. Boca Raton: CRC Press.
Claiborne, J. B. and Evans, D. H. (1988).
Ammonia and acid-base balance during high ammonia exposure in a marine teleost
(Myoxocephalus octodecimspinosus). J. Exp.
Biol. 140,89
-105.
Claiborne, J. B. and Heisler, N. (1986).
Acid-base regulation and ion transfers in the carp (Cyprinus carpio):
pH compensation during graded long- and short-term environmental hypercapnia,
and the effect of bicarbonate infusion. J. Exp. Biol.
126, 41-61.
Claiborne, J. B., Edwards, S. L. and Morrison-Shetlar, A. I. (2002). Acid-base regulation in fishes: cellular and molecular mechanisms. J. Exp. Zool. 293,302 -319.[CrossRef][Medline]
de Castro, E., Sigrist, C. J. A., Gattiker, A., Bulliard, V.,
Petra, S., Langendijk-Genevaux, P. S., Gasteiger, E., Bairoch, A. and Hulo,
N. (2006). ScanProsite: detection of PROSITE signature
matches and ProRule-associated functional and structural residues in proteins.
Nucleic Acids Res. 34,W362
-W365.
Dimberg, K. and Hoglund, L. B. (1987). Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard, bicarbonate-rich freshwater. J. Comp. Physiol. 157B,405 -412.[CrossRef]
Edwards, S. L., Wall, B. P., Morrison-Sheltar, A., Sligh, S., Weakley, J. C. and Claiborne, J. B. (2005). The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus). J. Exp. Zool. 303A,464 -475.
Endeward, V., Cartron, J.-P., Ripoche, G. and Gros, G. (2006). Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS. Transfus. Clin. Biol. 13,123 -127.[CrossRef][Medline]
Fromm, P. O. (1968). Some quantitative aspects of ion regulation in teleosts. Comp. Biochem. Physiol. 27,865 -869.
Georgalis, T., Perry, S. F. and Gilmour, K. M.
(2006). The role of branchial carbonic anhydrase in acid-base
regulation in rainbow trout (Oncorhynchus mykiss). J. Exp.
Biol. 209,518
-530.
Gibbs, P. J., Cameron, C., Tan, L. C., Sadek, S. A. and Howell, W. M. (2003). House keeping genes and gene expression analysis in transplant recipients: a note of caution. Transpl. Immunol. 12,89 -97.[CrossRef][Medline]
Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R. and Mathieu, C. (2001). An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25,386 -401.[CrossRef][Medline]
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95-98.
Heisler, N. (1993). Acid-base regulation. In The Physiology of Fishes (ed. D. H. Evans) pp.343 -378. Boca Raton: CRC Press.
Henry, R. P. and Heming, T. A. (1998). Carbonic anhydrase and respiratory gas exchange. In Fish Respiration (ed. S. F. Perry and B. L. Tufts), pp.75 -111. San Diego: Academic Press.
Huang, C.-H. and Peng, J. (2005). Evolutionary
conservation and diversification of Rh family genes and proteins.
Proc. Natl. Acad. Sci. USA
102,15512
-15517.
Huggett, J., Dheda, K., Bustin, S. and Zumla, A. (2005). Real-time RT-PCR normalization: strategies and considerations. Genes Immun. 6, 279-284.[CrossRef][Medline]
Hung, C. Y. C., Tsui, K. N. T., Wilson, J. M., Nawata, C. M.,
Wood, C. M. and Wright, P. A. (2007). Rhesus glycoprotein
gene expression in the mangrove killifish Kryptolebias marmoratus
exposed to elevated environmental ammonia levels and air. J. Exp.
Biol. 210,2419
-2429.
Ings, J. S. and Van der Kraak, G. J. (2006). Characterization of the mRNA expression of StAR and steroidogenic enzymes in zebrafish ovarian follicles. Mol. Reprod. Dev. 73,943 -954.[CrossRef][Medline]
Javelle, A., Lupo, D., Li, X.-D., Merrick, M., Chami, M., Ripoche, P. and Winkler, F. K. (2007). Structural and mechanistic aspects of Amt/Rh proteins. J. Struct. Biol. 158,472 -481.[CrossRef][Medline]
Juretic, D., Zoranic, L. and Zucic, D. (2002). Basic charge clusters and prediction of membrane protein topology. J. Chem. Inf. Comput. Sci. 42,620 -632.[CrossRef][Medline]
Kitayama, S., Ikeda, T., Mitsuhata, C., Sato, T., Morita, K. and
Dohi, T. (1999). Dominant negative isoform of rat
norepinephrine transporter produced by alternative RNA splicing. J.
Biol. Chem. 274,10731
-10736.
Kubista, M., Andrade, J. M., Bengtsson, M., Forootan, A., Jonak, J., Lind, K., Sindelka, R., Sjoback, R., Sjogreen, B., Strombom, L. et al. (2006). The real-time polymerase chain reaction. Mol. Aspects Med. 27,95 -125.[CrossRef][Medline]
Kustu, S. and Inwood, W. (2006). Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus) proteins are CO2 channels. Transfus. Clin. Biol. 13,103 -110.[CrossRef][Medline]
Larsen, B. K. and Jensen, F. B. (1997). Influence of ionic composition on acid-base regulation in rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia. Fish Physiol. Biochem. 16,157 -170.[CrossRef]
Li, X., Jayachandran, S., Nguyen, H.-H. T. and Chan, M.
(2007). Structure of the Nitrosomonas europaea Rh
protein. Proc. Natl. Acad. Sci. USA
104,19279
-19284.
Lin, H. and Randall, D. J. (1990). The effect of varying water pH on the acidification of expired water in rainbow trout. J. Exp. Biol. 141,14 -160.
Lin, H., Pfeiffer, D. C., Wayne, V. A., Pan, J. and Randall, D. J. (1994). Immunolocalization of H+-ATPase in the gill epithelia of rainbow trout. J. Exp. Biol. 195,169 -183.[Abstract]
Lloyd, R. and Herbert, D. W. M. (1960). The influence of carbon dioxide on the toxicity of un-ionized ammonia to rainbow trout (Salmo gairdnerii Richardson). Ann. Appl. Biol. 48,399 -404.[CrossRef]
Lupo, D., Li, X. D., Curand, A., Tomikazi, T., Cherif-Zahar, B.,
Matassi, G., Merrick, M. and Winkler, F. K. (2007). The
1.3-Å resolution structure of Nitrosomonas europaea Rh50 and
mechanistic implications for NH3 transport by Rhesus family
proteins. Proc. Natl. Acad. Sci. USA
104,19303
-19308.
Luqueti, C. M., Weihrauch, D., Senek, M. and Towle, D. W.
(2005). Induction of branchial ion transporter mRNA expression
during acclimation to salinity change in the euryhaline crab Chasmaganthus
granulatus. J. Exp. Biol.
208,3627
-3636.
Mangravite, L. M., Xiao, G., and Giacomini, K. M. (2003). Localization of human equilibrative nucleoside transporters, hENT1 and hENT2 in renal epithelial cells. Am. J. Physiol. 284,F902 -F910.
Meijer, H. A. and Thomas, A. A. M. (2002). Control of eukaryotic protein synthesis by upstream open reading frames in the 5'-untranslated region of an mRNA. Biochem. J. 367, 1-11.[CrossRef][Medline]
Michel, F. and Rudloff, V. (1989). Isolation and characterization of the rainbow trout erythrocyte band-3 protein. Eur. J. Biochem. 181,182 -187.
Mommsen, T. P., Vijayan, M. M. and Moon, T. W. (1999). Cortisol in teleosts: dyanamics, mechanisms of action, and metabolic regulation. Rev. Fish Biol. Fish. 9, 211-268.[CrossRef]
Morris, D. R. and Geballe, A. P. (2000).
Upstream open reading frames as regulators of mRNA translation.
Mol. Cell. Biol. 20,8635
-8642.
Nakada, T., Hoshijima, K., Esaki, M., Nagayoshi, S., Kawakami, K. and Hirose, S. (2007a). Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression. Am. J. Physiol. 293,R1743 -R1753.
Nakada, T., Westhoff, C. M., Kato, A. and Hirose, S.
(2007b). Ammonia secretion from fish gill depends on a set of Rh
proteins. FASEB J. 21,1
-8.
Nawata, C. M., Hung, C. C. Y., Tsui, T. K. N., Wilson, J. M.,
Wright, P. A. and Wood, C. M. (2007). Ammonia excretion in
rainbow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein and
H+-ATPase involvement. Physiol. Genomics
31,463
-474.
Nolan, T., Hands, R. E. and Bustin, S. A. (2006). Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 3,1559 -1582.
Perry, S. F. (1986). Carbon dioxide excretion in fishes. Can. J. Zool. 64,565 -572.[CrossRef]
Perry, S. F. and Gilmour, K. M. (2002). Sensing and transfer of respiratory gases at the fish gill. J. Exp. Zool. 293,249 -263.[CrossRef][Medline]
Perry, S. F. and Gilmour, K. M. (2006). Acid-base balance and CO2 excretion in fish: unanswered questions and emerging models. Respir. Physiol. Neurobiol. 154,199 -215.[CrossRef][Medline]
Perry, S. F., Beyers, M. L. and Johnson, D. A. (2000). Cloning and molecular characterization of the trout (Oncorhynchus mykiss) vacuolar H+-ATPase B subunit. J. Exp. Biol. 203,459 -470.[Abstract]
Pesole, G., Mignone, F., Gissi, C., Grillo, G., Licciulli, F. and Liuni, S. (2001). Structural and functional features of eukaryotic mRNA untranslated regions. Gene 276, 73-81.[CrossRef][Medline]
Planelles, G. (2007). Ammonium homeostasis and human Rhesus glycoproteins. Nephron Physiol. 105, 11-17.[CrossRef]
Ripoche, P., Goossens, D., Devuyst, O., Gane, P., Colin, Y., Verkman, A. S. and Cartron, J.-P. (2006). Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis. Transfus. Clin. Biol. 13,117 -122.[CrossRef][Medline]
Salama, A., Morgan, I. J. and Wood, C. M. (1999). The linkage between sodium uptake and ammonia excretion in rainbow trout-kinetic analysis, the effects of (NH4)2 SO4 and NH4 HCO3 infusion, and the influence of gill boundary layer pH. J. Exp. Biol. 202,697 -709.[Abstract]
Serrano, L., Halanych, K. M. and Henry, R. P.
(2007). Salinity-stimulated changes in expression and activity of
two carbonic anhydrase isoforms in the blue crab Callinectes sapidus.J. Exp. Biol. 210,2320
-2332.
Soupene, E., King, N., Field, E., Liu, P., Niyogi, K. K., Huang,
C.-H. and Kustu, S. (2002). Rhesus expression in a green alga
is regulated by CO2. Proc. Natl. Acad. Sci.
USA 99,7769
-7773.
Soupene, E., Inwood, W. and Kustu, S. (2004).
Lack of the Rhesus protein Rh1 impairs growth of the green alga
Chlamydomonas reinhardtii at high CO2. Proc.
Natl. Acad. Sci. USA 101,7787
-7792.
Thompson, J. D., Higgins, D. G. and Gibson, T. J.
(1994). CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res.
22,4673
-4680.
Tipsmark, C. K., Baltzegar, D. A., Ozden, O., Grubb, B. J. and Borski, R. J. (2008). Salinity regulates mRNA and protein expression in the teleost gill. Am. J. Physiol. 294,R1004 -R1014.[CrossRef]
Tricarico, C., Pinzani, P., Bianchi, S., Paglierani, M., Distante, V., Pazzagli, M., Bustin, S. A. and Orlando, C. (2002). Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal. Biochem. 309,293 -300.[CrossRef][Medline]
Van Kim, C. L., Colin, Y. and Cartron, J.-P. (2006). Rh proteins: key structural and functional components of the red cell membrane. Blood Rev. 20, 93-110.[CrossRef][Medline]
Vera Cruz, E. M., Brown, C. L., Luckenbach, J. A., Picha, M. E., Bolivar, R. B. and Borski, R. J. (2006). Insulin-like growth factor-I cDNA cloning, gene expression and potential use as a growth rate indicator in Nile tilapia, Oreochromis niloticus.Aquaculture 251,585 -595.[CrossRef]
Verdouw, H., van Echteld, C. J. A. and Dekkers, E. M. J. (1978). Ammonia determinations based on indophenol formation with sodium salicylate. Water Res. 12,399 -402.
Weidinger, K., Neuhauser, B., Gilch, S., Ludewig, U., Meyer, O. and Schmidt, I. (2007). Functional and physiological evidence for a Rhesus-type ammonia transporter in Nitrosomonas europaea.FEMS Microbiol. Lett. 273,260 -267.[CrossRef][Medline]
Weihrauch, D. (2006). Active ammonia absorption in the midgut of the Tobacco hornworm Manduca sexta L: transport studies and the mRNA expression analysis of a Rhesus-like ammonia transporter. Insect Biochem. Mol. Biol. 36,808 -821.[CrossRef][Medline]
Weiner, I. D. and Hamm, L. L. (2007). Molecular mechanisms of renal ammonia transport. Annu. Rev. Physiol. 69,317 -340.[CrossRef][Medline]
Wilson, R. W. and Taylor, E. W. (1992).
Transbranchial ammonia gradients and acid-base responses to high external
ammonia concentration in rainbow trout (Oncorhynchus mykiss)
acclimated to different salinities. J. Exp. Biol.
166,95
-112.
Wilson, R. W., Wright, P. M., Munger, S. and Wood, C. M. (1994). Ammonia excretion in fresh water rainbow trout (Oncorhynchus mykiss) and the importance of gill boundary layer acidification: lack of evidence for Na+/NH +4 exchange. J. Exp. Biol. 191, 37-58.[Abstract]
Wolf, K. (1963). Physiological salines for freshwater teleosts. Prog. Fish-Cult. 25,135 -140.
Wood, C. M. (1993). Ammonia and urea metabolism and excretion. In The Physiology of Fishes (ed. D. H. Evans), pp. 379-425. Boca Raton: CRC Press.
Wood, C. M. and LeMoigne, J. (1991). Intracellular acid-base responses to environmental hyperoxia and normoxic recovery in rainbow trout. Respir. Physiol. 86, 91-113.[CrossRef][Medline]
Wood, C. M., Warne, J. M., Wang, Y., McDonald, M. D., Balment, R. J., Laurent, P. and Walsh, P. (2001). Do circulating plasma AVT and/or cortisol levels control pulsatile urea excretion in the gulf toadfish (Opsanus beta)? Comp. Biochem. Physiol. 129A,859 -872.
Wright, P. A., Heming, T. and Randall, D. J.
(1986). Downstream pH changes in water flowing over the gills of
rainbow trout. J. Exp. Biol.
126,499
-512.
Wright, P. A., Randall, D. J. and Wood, C. M.
(1988a). The distribution of ammonia and H+ between
tissue compartment in lemon sole (Parophrys vetulus) at rest, during
hypercapnia and following exercise. J. Exp. Biol.
136,149
-175.
Wright, P. A., Wood, C. M. and Randall, D. J.
(1988b). An in vitro and in vivo study of the
distribution of ammonia between plasma and red cells of rainbow trout
(Salmo gairdneri). J. Exp. Biol.
134,423
-428.
Wright, P. A., Randall, D. J. and Perry, S. F. (1989). Fish gill boundary layer: a site of linkage between carbon dioxide and ammonia excretion. J. Comp. Physiol. 158,627 -635.[CrossRef]
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