A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro
1 Department of Biological Science, National University of Singapore, Kent
Ridge, Singapore 117543, Republic of Singapore
2 Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG
2W1
3 Natural Sciences, National Institute of Education, Nanyang Technological
University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
* Author for correspondence (e-mail: dbsipyk{at}nus.edu.sg)
Accepted 15 July 2003
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Summary |
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Key words: ammonia, ammonia detoxification, ammonia excretion, amino acid, carbamoyl phosphate synthetase, elasmobranch, Himantura signifer, nitrogen metabolism, ornithine-urea cycle, osmoregulation, stingray, urea, urea excretion
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Introduction |
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While synthesizing urea for both water retention (osmoregulation) and
ammonia detoxification prescribes a high rate of urea synthesis de
novo, the former dictates the majority of the urea synthesized to be
retained within the body while the latter requires urea to be excreted
instead. Hence, during evolution, there must be a dichotomy in the development
of control for urea transport to either facilitate the retention of urea for
osmoregulation or to remove urea for nitrogenous excretion. To date, at least
five urea transporters are known to be present in the mammalian kidney,
facilitating the excretion of urea (see review by
Sands et al., 1997). This
capability can be traced back to amphibians, whose kidneys can excrete urea
actively (Foster, 1954
;
Balinsky, 1970
). However, fully
aquatic amphibians lack the power of active urea secretion
(Balinsky 1970
), while marine
elasmobranchs actively re-absorb urea instead
(Fines et al., 2001
;
Smith and Wright, 1999
).
Marine elasmobranchs are ureosmotic, synthesizing urea through the OUC with
carbamoyl phosphate synthetase III (CPS III; Anderson,
1980,
1991
,
1995
,
2001
;
Campbell and Anderson, 1991
),
primarily for osmoregulation (Anderson,
2001
; Ballantyne,
1997
; Perlman and Goldstein,
1998
). Urea is retained at high concentrations (300-600 mmol
l-1) in the tissues. This is accomplished by the low permeability
of the gills to urea (Fines et al.,
2001
) and by the re-absorption of urea via secondary
active (Na+-coupled) urea transporters in the gills
(Smith and Wright, 1999
) and
kidney (see review by Walsh and Smith,
2001
). Despite the decrease in effective urea permeabilities
(Fines et al., 2001
), marine
elasmobranchs (sharks, skates and rays) are ureotelic, excreting the majority
of their nitrogenous wastes as urea via the gills
(Perlman and Goldstein, 1998
;
Shuttleworth, 1988
; Wood,
1993; Wood et al., 1995
). For
elasmobranchs living in freshwater, they must evolve mechanisms to suppress
urea production, urea retention (including active urea reabsorption) or both.
Hence, they are ideal models for the unravelling of the intricate relationship
between urea synthesis de novo and urea excretion in response to high
concentrations of environmental ammonia.
In tropical waters in Southeast Asia (Thailand, Indonesia and Papua New
Guinea) and South America (Amazon River basin), a number of elasmobranch
species migrate into low salinity waters where they reduce plasma salt, urea
and trimethylamine oxide (TMAO) levels. The stenohaline Amazonian stingray
Potamotrygon spp., being permanently adapted to freshwater, is
primarily ammonotelic like other teleosts
(Barcellos et al., 1997;
Goldstein and Forster, 1971
)
and cannot accumulate urea in laboratory salinity stress
(Gerst and Thorson, 1977
;
Thorson et al., 1967
). In the
present study, an attempt was made to use environmental ammonia as a probe to
elucidate whether ammonia exposure (10 µmol ml-1
NH4Cl in freshwater at pH 7.0) would induce the synthesis and
accumulation of urea in Potamotrygon motoro because no such
information is available. Results obtained subsequently revealed that it was
unable to do so and therefore P. motoro was not an appropriate
organism to evaluate the evolutionary role of urea, i.e. whether it was
designed for osmoregulatory or ammonia detoxification purposes.
By contrast, the freshwater white-edge whip ray Himantura signifer
(Family: Dasyatidae), a stingray found in the Batang Hari Basin in Jambi,
Sumatra, retains the capability for urea synthesis de novo but has a
reduced capacity to retain urea in freshwater
(Tam et al., 2003). Unlike
potamotrygonid rays living in the Amazon River, H. signifer might
have invaded the freshwater environment only recently. It is currently unclear
whether H. signifer returns to brackish water to reproduce, as does
the bull shark Carcharhinus leucas of Lake Nicaragua
(Thorson, 1976
). Although
H. signifer can be found in Batang Hari as far as 400 km from the
South China Sea, there is still the possibility that it may re-enter estuarine
and marine environments. Therefore, it would be essential for H.
signifer to retain the ureosmotic osmoregulatory mechanisms to survive in
higher salinities (Tam et al.,
2003
). It is because of this that H. signifer represents
an ideal species for studies on the effects of ammonia loading on the capacity
of urea synthesis and capacity of urea retention in a primarily freshwater
elasmobranch. In the present study, we aimed to elucidate whether the capacity
of H. signifer to synthesize urea de novo could be
upregulated and, more importantly, whether urea excretion would be enhanced
during exposure to environmental ammonia in freshwater. We hypothesized that
it was capable of doing so. The rate of urea excretion in specimens being
exposed to 10 mmol l-1 NH4Cl in freshwater at pH 7.0 was
determined. The contents of ammonia, urea and free amino acids (FAAs) in
various tissues and organs of the specimens were measured. In addition,
activities of OUC enzymes were assayed.
We also aimed to evaluate whether urea synthesis alone would be an
effective measure to defend against environmental ammonia toxicity. The
formation of urea in fishes is highly energy dependent. A total of 5 moles of
ATP are hydrolyzed to ADP for each mole of urea synthesized, corresponding to
2.5 moles of ATP used for each mole of nitrogen assimilated. This may be the
major reason why the majority of tropical teleosts studied so far do not adopt
ureogenesis as a major strategy to detoxify exogenous and endogenous ammonia
during ammonia loading (Ip et al.,
2001). However, if the animal had high ammonia tolerance at the
cellular and subcellular levels, it could allow ammonia to build up in its
tissues and plasma during the early phase of exposure to environmental
ammonia. In effect, this would reduce or impede the net influx of exogenous
ammonia, and urea synthesis de novo could be reserved to detoxify the
endogenously produced ammonia, maintaining the newly established steady-state
concentration of ammonia in the body. Therefore, we hypothesized that an
increase in ammonia contents in the body of H. signifer would occur
during environmental ammonia exposure, despite its being ureogenic and
ureotelic (Tam et al.,
2003
).
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Materials and methods |
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Exposure of specimens to experimental conditions and collection of
water samples and tissues
H. signifer and P. motoro were exposed to freshwater
(0.7) containing 10 mmol l-1 NH4Cl at pH 7 for 4
days. Water samples (3 ml) were collected daily, acidified with 70 µl of 1
mol l-1 HCl and kept at 4°C until analysed. Urea was determined
according to the method of Felskie et al.
(1998
). No ammonia assays were
performed on these water samples. Control specimens were exposed to ordinary
freshwater (0.7
). In order to calculate the deficit in ammonia
excretion in H. signifer exposed to NH4Cl, water samples
were collected daily for the control specimen, and ammonia was determined
according to the methods of Anderson and Little
(1986
), as modified by Jow et
al. (1999
). The rates of urea
and ammonia excretion were expressed as µmol day-1
g-1 fish.
Anaesthetized fish (0.12% ethyl-3-aminobenzoate methanesulfonate) were rinsed thoroughly several times with freshwater to avoid environmental contamination. The caudal peduncle of the experimental specimen was severed, and blood was collected from the caudal vessels into heparinized capillary tubes. The blood sample was centrifuged at 4000 g at 4°C for 10 min to obtain the plasma. A portion of the plasma was used for analyses of osmolality and concentrations of Na+ and Cl-. Another portion was deproteinized in 2 volumes (v/v) of ice-cold 6% HClO4 and centrifuged at 10 000 g at 4°C for 15 min. The resulting supernatant was kept at -80°C until analysed. The liver, stomach and muscle were quickly excised, with the stomach flushed with ice-cold saline solution (0.9% NaCl). The excised tissues and organs were immediately freeze-clamped with tongs pre-cooled in liquid nitrogen. Samples were kept at -80°C until analysed.
Determinations of plasma osmolality, concentrations of Na+
and Cl- and blood pH
Plasma osmolality was analysed using a Wescor 5500 vapour pressure
osmometer. Na+ and Cl- concentrations were determined
using a Corning 410 flame photometer and Corning 925 chloride analyzer,
respectively (Corning Ltd, Halstead, Essex, UK).
Analysis of free FAAs
The frozen muscle and liver samples were weighed, ground to powder in
liquid nitrogen and homogenized three times in 5 volumes (w/v) of 6%
trichloroacetic acid using an Ultra-Turrax homogenizer at 24 000 revs
min-1 for 20 s each with 10 s off intervals. The homogenate was
centrifuged at 10 000 g at 4°C for 15 min to obtain the
supernatant for FAA analyses. The plasma was deproteinized in 2 volumes (v/v)
of ice-cold 6% trichloroacetic acid and centrifuged at 10 000
g at 4°C for 15 min.
For the analysis of FAA, the supernatant obtained was adjusted to pH 2.2 with 4 mol l-1 lithium hydroxide and diluted appropriately with 0.2 mol l-1 lithium citrate buffer (pH 2.2). FAAs were analyzed using a Shimadzu LC-6A amino acid analysis system (Kyoto, Japan) with a Shim-pack ISC-07/S1504 Li-type column. Results are expressed as µmol g-1 wet mass tissue or mmol l-1 plasma.
Analyses of ammonia and urea
Samples of muscle and liver were homogenized as stated above except in 5
volumes of 6% HClO4. After centrifugation at 10 000
g for 15 min, the supernatant was decanted and the pH adjusted
to 5.5-6.0 with 2 mol l-1 KHCO3. The pH of the plasma
sample was also adjusted to 5.5-6.0 after deprotenization. The ammonia and
urea contents were determined according to the methods of Bergmeyer and
Beutler (1985) and Felskie et
al. (1998
), respectively.
Results were expressed as µmol g-1 wet mass tissue or mmol
l-1 plasma.
Determination of activities of OUC enzymes
The liver and the stomach were minced and suspended in 10 volumes (w/v) of
ice-cold extraction buffer (285 mmol l-1 sucrose, 3 mmol
l-1 EDTA and 3 mmol l-1 Tris-HCl, pH 7.2), homogenized
using an Ultra-Turrax homogenizer at 24 000 revs min-1 and
sonicated three times for 20 s with a 10 s break between each sonication. The
homogenate was centrifuged at 10 000 g at 4°C for 15 min
to obtain the supernatant, which was subsequently passed through a 10 ml
Bio-Rad P-6DG column (Bio-Rad Laboratories, Hercules, CA, USA) equilibrated
with cold suspension buffer. The collected filtrates were used for the
subsequent enzyme analyses.
CPS III (E.C. 2.7.2.5) activity was determined as described by Anderson and
Walsh (1995). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter (Wallac Oy, Turku,
Finland). The CPS activity was expressed as µmol [14C]urea
formed min-1 g-1 wet mass.
Ornithine transcarbamoylase (OTC; E.C. 2.1.3.3) activity was determined by
combining the methods of Anderson and Walsh
(1995) and Xiong and Anderson
(1989
). Absorbance was
measured at 466 nm using a Shimadzu 160 UV VIS recording spectrophotometer.
The OTC activity was expressed as µmol citrulline formed min-1
g-1 wet mass.
Argininosuccinate synthetase (E.C. 6.3.4.5) and lyase (E.C. 4.3.2.1) (ASS +
L) activities were determined together, assuming that both were present, by
measuring the formation of [14C]fumarate from
[14C]aspartate using the method of Cao et al.
(1991). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter. ASS + L activity
was expressed as µmol [14C]fumarate formed min-1
g-1 wet mass.
Arginase (E.C. 3.5.3.1) was assayed as described by Felskie et al.
(1998). Urea was determined as
described above. Arginase activity was expressed as µmol urea formed
min-1 g-1 wet mass.
Glutamine synthetase (GS; E.C. 6.3.1.2) activity was measured according to
the method of Shankar and Anderson
(1985). The formation of
-glutamylhydroxymate was determined at 500 nm using a Shimadzu 160 UV
VIS recording spectrophotometer. The GS activity was expressed as µmol
-glutamylhydroxymate formed min-1 g-1 wet
mass.
Statistical analyses
Results are presented as means ± S.E.M. Two-tail
Student's t-test or analysis of variance (ANOVA) followed by multiple
comparisons of means by Duncan's procedure was used to evaluate differences
between means in groups where appropriate. Differences of P<0.05
were regarded as statistically significant.
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Results |
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The ammonia excretion rates of control H. signifer on days 1, 2, 3 and 4 were 5.5±0.8 µmol day-1 g-1, 5.8±1.2 µmol day-1 g-1, 5.3±0.7 µmol day-1 g-1 and 6.1±1.2 µmol day-1 g-1, respectively. The rate of urea excretion in specimens exposed to ammonia for the first 2 days was comparable with that of the control (Fig. 1). However, on days 3 and 4, there was a significant increase in the rate of urea excretion in the experimental specimens (Fig. 1). Ammonia accumulated in the muscle and plasma, but not in the liver, of H. signifer exposed to 10 mmol l-1 NH4Cl at pH 7 (Table 3). However, there was no change in the urea content of the muscle or liver. A significant increase in urea level occurred only in the plasma (Table 3). In addition, there was no change in plasma osmolality and plasma Na+ and Cl- concentrations in H. signifer exposed to ammonia (Table 4).
|
Ammonia exposure induced a higher OUC capacity in the liver of H. signifer. The activities of hepatic GS, CPS III, OTC, ASS + L and arginase increased approximately 3-, 4-, 3-, 4- and 7-fold, respectively (Table 5). A complete OUC was detected in the stomach (Table 6). However, there was no significant increase in the activities of any OUC enzymes in the stomach during exposure to environmental ammonia (Table 6).
|
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In general, the concentrations of various FAAs and total FAA (TFAA) of H. signifer exposed to ammonia were comparable with those of the control (Table 7). In particular, there was no accumulation of glutamine during ammonia loading.
|
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Discussion |
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H. signifer upregulates the capacity to synthesize urea de
novo when exposed to environmental ammonia
Similar to marine elasmobranchs, freshwater H. signifer is
ureogenic and has a functional OUC with CPS III in the liver. In addition, a
full complement of OUC enzymes was detected in the stomach of H.
signifer. In order to maintain the concentration of urea at a steady
state under a certain environmental condition, the rate of urea excretion must
be balanced with the rate of urea production. In freshwater, the rate of urea
excretion was 2.2 µmol day-1. In a 100 g specimen, there was
approximately 50 g muscle, 3 g liver and 2 ml plasma (Y.K.I. and S.F.C.,
unpublished results). Therefore, for a 100 g fish, the rate of urea excretion
was 2.2 µmol day-1x100 g=220 µmol day-1 or
0.15 µmol min-1. This implies that the liver must be
synthesizing urea at a rate of 0.05 µmol min-1 g-1
(in a 3 g liver) to sustain the steady-state level of urea in the body of
H. signifer in freshwater. This is much lower than the maximal
capacity of OUC based on CPS activity present in the liver (0.13 µmol
min-1 g-1).
Unlike P. motoro, H. signifer increased urea excretion after exposure to 10 mmol l-1 NH4Cl for 3 days, confirming its capability to upregulate urea synthesis in response to environmental ammonia. By day 4, the urea excretion rate increased 2.6-fold to 5.4 µmol day-1 g-1. Since this was not accompanied by an increase in urea content in the muscle, which is the bulk of the animal, the rate of urea production in the liver of the experimental animal can be estimated to be 0.125 µmol min-1 g-1, which is almost equivalent to the limit of synthetic capacity in the liver of a control specimen (maximal CPS III activities of 0.13 µmol min-1 g-1). This could be the reason why inductions of higher activities of CPS III and related enzymes (GS, OTC, ASS + L and arginase) were necessary in specimens being exposed to environmental ammonia.
Exposure to brackish (20) water led to an increase in the activity
of CPS III in the stomach of H. signifer, based on which Tam et al.
(2003
) concluded that the
stomach of H. signifer (and another marine stingray, Taeniura
lymma) could be involved in producing urea for osmoregulation in addition
to its digestive function. Results from this study revealed that the CPS III
activity in the stomach of H. signifer could not be induced by
exposure to environmental ammonia, indicating that osmotic stress was the
primary inductive factor. How the stomach OUC and hepatic OUC in H.
signifer respond differentially to two different environmental stresses
is unclear at present.
Increase in urea excretion in H. signifer in response to
ammonia loading
When being exposed to environmental ammonia, H. signifer was able
to release the excess urea without creating a problem for osmoregulation. The
traditional view of urea transport is that urea crosses all cell membranes by
lipid-phase permeation. To date, there is evidence for active or facilitated
urea transport across various tissues in a number of vertebrates. Five urea
transporters have been identified so far
(Sands et al., 1997), and an
Na+-dependent urea transporter (for urea re-absorption) has been
found in the gills of marine elasmobranchs
(Fines et al., 2001
). In
higher salinities (20
), H. signifer was able to retain urea
for osmoregulation, albeit with limited capacity
(Tam et al., 2003
). By
contrast, during exposure to environmental ammonia in freshwater, the excess
urea synthesized was not retained but instead excreted to the external medium.
Contrary to those exposed to 20
water
(Tam et al., 2003
),
experimental animals in the present study showed no change in plasma
osmolality and tissue (except plasma) urea content despite similar increases
in the rate of urea synthesis and the capacity of the OUC. These results imply
that there must be a further reduction in urea re-absorption to facilitate the
excretion of the excess urea formed during exposure to environmental ammonia
in a freshwater environment. Hence, H. signifer is an ideal organism
for future studies on the regulation of urea transport in response to osmotic
or ammonia stress. In future studies, it would be meaningful to find out if
urea excretion was facilitated or impeded in specimens exposed to
environmental ammonia in brackish water instead of freshwater.
Both H. signifer and P. motoro can tolerate
high levels of ammonia in the body
Despite being ureogenic, ammonia accumulated in the body of H.
signifer during environmental exposure, albeit at a lower level than that
accumulated in the ammonotelic P. motoro. It had been suggested much
earlier that NH4+ could substitute for K+ and
affect the membrane potential in the squid giant axon
(Binstock and Lecar, 1969). In
addition, Beaumont et al.
(2000
) reported depolarisation
of muscle fibres in trout with elevated levels of ammonia in their tissues
(from -87 mV to -52 mV) that matched the effect predicted on the basis of the
measured gradient for NH4+ across the cell membranes.
How the cells and tissues, especially those in the brain and the heart, of
H. signifer and P. motoro tolerate these high ammonia levels
awaits future study.
Since H. signifer was ureogenic, and there was an upregulation of the capacity of urea synthesis through OUC, what would be the advantage of allowing the ammonia levels in its tissues to build up when confronted with environmental ammonia? Apparently, the development of the capability to tolerate ammonia at the cellular and subcellular levels facilitates the development of a very effective strategy in defending toxicity of ammonia of exogenous origin: a relatively higher concentration of ammonia can be accommodated in the plasma, which would decrease the NH3 partial pressure gradient across the branchial and body surfaces and would reduce the net influx of NH3 during ammonia loading.
Increase in plasma ammonia concentration would reduce the net
influx of ammonia in H. signifer (and P. motoro) during
ammonia loading
Detoxification of the accumulating ammonia did not occur in H.
signifer, at least in the first day of exposure to environmental ammonia.
This was reflected by the unaltered urea excretion rate (2.2 µmol
day-1 g-1) in specimens exposed to ammonia for the first
day, during which ammonia excretion (5.5 µmol day-1
g-1) was presumably impeded totally. At pH 7.0 and a total ammonia
concentration of 10 mmol l-1, the concentration of NH3
in the external medium was calculated as 0.042 mmol l-1 according
to the Henderson-Hasselbalch equation (pKamm=9.18;
Boutilier et al., 1984). At
the beginning of the experiment, the plasma of H. signifer contained
0.33 mmol l-1 total ammonia and had a pH of 7.521 (Y.K.I. and
S.F.C., unpublished results), producing an NH3 concentration of
0.0073 mmol l-1 (pKamm=9.34;
Boutilier et al., 1984
). This
was much lower than that in the external medium, resulting in a steep
NH3 gradient impeding ammonia excretion and driving NH3
inwards to the specimen. During this period, both exogenous and endogenous
ammonia contributed to the increase in ammonia concentration in the body of
the experimental specimens. By the time the plasma ammonia concentration
increased to 2.15 mmol l-1 in the experimental specimen, which had
a blood pH of 7.498 (Y.K.I. and S.F.C., unpublished results) on day 4, the
NH3 concentration increased to 0.047 mmol l-1, which was
more than adequate to oppose any net influx of NH3 from the
external medium. This implies that, despite the exchange of endogenous and
exogenous ammonia during ammonia loading, the specimens were, in effect,
detoxifying endogenous ammonia to urea because the net influx of ammonia
(exogenous) would be very small (or closed to zero) after the build-up of the
plasma ammonia concentration.
Synthesis of urea de novo in fish is energetically intensive. The
detoxification of any net influx of exogenous ammonia to urea and excreting it
would result in a high expenditure of energy and the maintenance of an
inwardly driven NH3 partial pressure gradient. It is probably
because of this that ureogenesis is not commonly adopted by teleosts
confronted with high environmental ammonia concentrations
(Ip et al., 2001).
For P. motoro exposed to environmental ammonia, ammonia also accumulated in the plasma, increasing by 6.3-fold. Basically, this would slow down the influx of exogenous ammonia. However, since P. motoro was unable to detoxify ammonia into urea, the primary strategy adopted was to simply tolerate ammonia at the cellular and subcellular levels.
Reduction in rates of proteolysis and/or amino acid catabolism
in H. signifer during ammonia loading
Ignoring momentarily the net influx of exogenous ammonia, which presumably
occurred mainly at the beginning of the experiment, the deficit in ammonia
excretion due to its being totally impeded in a 100 g specimen is estimated to
be (5.5+5.8+5.3+6.1) µmolesx100 g=2270 µmoles (based on results
from the present study). The excess amount of ammonia accumulated in the
muscle, liver and plasma during such a period was 148.6 µmoles (calculated
from Table 3). The excess
amount of nitrogen excreted as urea during this 4-day period amounted to only
{[0+(3.7-2.2)+(4.3-2.2)+ (5.4-2.2)]x100}x2=1360 µmoles N
(calculated from Fig. 1). Hence, the sum of ammonia equivalents accumulated and excreted (as urea) was
only 148.6+1360=1508.6 µmoles. The deficit of 2270-1508.6 µmoles (761.4
µmoles) suggests indirectly that there was a decrease in endogenous ammonia
production in specimens during the 4 days of exposure to NH4Cl,
which was essential to maintaining the newly established internal ammonia
level.
In addition, there was a significant decrease in the content of TFAA in the liver. This implies that both proteolysis and amino acid catabolism decreased in the liver, but the decrease in the former was greater than in the latter. This might be a strategy that marine elasmobranchs cannot afford; being ureosmotic, marine elasmobranchs are committed to carnivory or high rates of proteolysis and amino acid catabolism during fasting because large amounts of nitrogen are needed to synthesize urea to maintain the internal steady-state concentration of urea. Taken together, these results indirectly indicate that the net influx of ammonia into the experimental animal during the 4 days of ammonia exposure was unlikely to be great and that the bulk of the ammonia detoxified to urea was mainly produced endogenously.
There was no significant increase in the TFAA in the muscle of specimens
exposed to 10 mmol l-1 NH4Cl. This implies that the
process of urea synthesis in H. signifer was so effective that it did
not have to resort to `fixing' the endogenous or exogenous ammonia as FAAs, as
suggested elsewhere for teleosts (Iwata,
1988). More importantly, the glutamine levels in the muscle, liver
and plasma remained relatively unchanged in specimens exposed to 10 mmol
l-1 NH4Cl despite the 3-fold increase in hepatic GS
activity. Hence, the excess glutamine formed in the experimental specimens
must have been channelled completely into urea formation.
Conclusion
Although both P. motoro and H. signifer are freshwater
stingrays, only the latter possesses a functional OUC in the liver and
stomach. The capacity of urea synthesis through OUC in the liver of H.
signifer could be upregulated by exposure to environmental ammonia.
Unlike specimens exposed to brackish water, the excess urea produced by H.
signifer exposed to environmental ammonia is excreted to the external
medium instead of being retained in the body. These results suggest that urea
has the dual functions of osmoregulation and ammonia detoxification in
elasmobranchs living in a freshwater environment, the success of which depends
primarily on the regulation of the direction and rate of urea transport.
Hence, it can be concluded that the freshwater H. signifer is an
ideal species for future studies on signals and mechanisms involved in
regulating the rate of urea synthesis (in response to salinity changes or
ammonia loading) and in controlling the direction and rate of urea transport
(for urea retention or urea excretion).
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