The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism
1 Natural Sciences, National Institute of Education, Nanyang Technological
University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
2 Department of Biological Sciences, National University of Singapore, 10
Kent Ridge Road, Singapore 117543, Republic of Singapore
* Author for correspondence (e-mail: sfchew{at}nie.edu.sg)
Accepted 11 November 2002
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Summary |
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Key words: aerial exposure, alanine, amino acid, ammonia, Channa asiatica, glutamate dehydrogenase, proteolysis, snakehead
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Introduction |
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Three different major nitrogenous products accumulate in the tissues of
some tropical teleostean fishes during aerial exposure
(Ip et al., 2001a). These are
alanine (e.g. mudskipper; Ip et al.,
2001b
), glutamine (e.g. marble goby and sleeper;
Jow et al., 1999
;
Ip et al., 2001c
; Anderson et
al., 2001
,
2002
) and urea (e.g. Singhi
catfish; Saha and Ratha,
1998
). The weather loach (Chew
et al., 2001
) and the mangrove killifish
(Frick and Wright, 2002
)
accumulate both alanine and glutamine, although to a lesser extent than the
mudskipper and the sleeper, respectively, when exposed to terrestrial
conditions. These two fishes are uniquely capable of excreting ammonia through
volatilization of NH3 during aerial exposure
(Frick and Wright, 2002
;
Tsui et al., 2002
).
Urea production does not appear to be a common strategy adopted by teleosts
to detoxify endogenous ammonia during aerial exposure, probably because it is
energy expensive. 5 mol ATP are required for the formation of 1 mol urea
containing 2 nitrogen atoms (Ip et al.,
2001a). Glutamine formation is also energy dependent and appears
to be adopted mainly by fishes that remain completely quiescent on land. If
the reaction begins with glutamate and NH4+, 1 mol ATP
is utilized for every mol ammonia detoxified. If the starting point is
-ketoglutarate and NH4+, 4 mol ATP-equivalents (3
from NADH) would be needed to detoxify 2 mol ammonia (Ip et al., 2001). By
contrast, alanine formation through partial amino acid catabolism would lead
to the production of ATP without releasing ammonia
(Ip et al., 2001a
). Hence,
this pathway cannot be regarded as an ammonia-detoxifying mechanism, but
rather a way to use certain amino acids as energy sources without leading to
ammonia accumulation in the body. When the mudskipper Periophthalmodon
schlosseri was forced to exercise on land after 24 h of aerial exposure,
muscle alanine levels increased significantly, with no apparent change in
glycogen content (Ip et al.,
2001b
), indicating that the apparent increase in energy demand was
not met by an increase in the rate of fermentative glycolysis. Hence, Ip et
al. (2001b
) concluded that
P. schlosseri was capable of using certain amino acids as metabolic
fuels to support locomotory activities on land and avoided ammonia toxicity
through partial amino acid catabolism. They further suggested that such a
strategy may be widely adopted, especially by obligatory air-breathing fishes,
to avoid ammonia intoxication during aerial exposure
(Ip et al., 2001b
).
The aim of this study was to investigate the effects of aerial exposure on
the snakehead Channa asiatica, which is an obligatory air-breather,
and to test the hypothesis set forth by Ip et al.
(2001b). Channa
asiatica and other snakeheads are valued freshwater food fishes in
Southern China and Far East Asia. They command very high prices (S$10-20
kg-1) when fresh, because people there believe that consumption of
this fish aids the recovery of injuries (especially for post-surgical
patients) (Lim and Ng, 1990
;
Ng and Lim, 1990
). Channa
asiatica is a predaceous fish that resides in slow-flowing streams and in
crevices near riverbanks. In its natural habitat, it may encounter bouts of
aerial exposure during the dry seasons. While `walking' fishes like
Anabas sp. and Clarias sp. (see
Smith, 1945
) have frequently
been reported upon, `walking snakeheads' are less well known. Smith
(1945
) and Mohsin and Ambak
(1983
) all noted that
snakeheads could move across land. They can move a considerable distance on
all kinds of surfaces, squirming and skipping
(Ng and Lim, 1990
). However,
unlike mudskippers, C. asiatica is incapable of using its pectoral
fins to move actively on land, and does not exhibit any feeding, territorial
or courtship behaviour while out of water. When trapped in puddles of water,
it tried to struggle back to water with eel-like body movements, which could
last several minutes. After struggling for a while without managing to get
back to water, C. asiatica turned motionless and remained quiescent,
with only occasional eel-like movements, for an extended period. We therefore
hypothesized that C. asiatica was capable of undergoing partial amino
acid catabolism, leading to the formation of alanine to reduce the rate of
ammonia accumulation in its tissues during aerial exposure; however, it might
not be able to use amino acids to fuel locomotory activities on land as
observed in P. schlosseri (Ip et
al., 2001b
). Experiments were therefore undertaken to examine the
effects of aerial exposure or locomotory activities on the nitrogen metabolism
and excretion in this fish.
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Materials and methods |
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Exposure of C. asiatica to experimental conditions and
collection of samples
Effects of aerial exposure
All experiments performed in this study were done under a 12h:12h
dark:light regime unless stated otherwise. 24h before the experiment, a group
of specimens was separated individually and immersed completely in water with
continuous aeration. Food was withdrawn so that the fish had empty guts. At
the end of the 24h period (0h control), some of these specimens were
anaesthetized by the introduction of 3-aminobenzoic acid ethyl ester (MS-222)
at a final concentration of 0.02% and killed. Others were exposed to aerial
conditions individually in plastic aquaria (20.5 cmx14.5 cmx6 cm,
LxWxH) containing a thin film of 50 ml dechlorinated tapwater, for
a specific period (6 h, 12 h, 24 h or 48 h) at 25°C and 80-90% humidity.
Another group of fish was submerged in air-saturated, dechlorinated tapwater
at 25°C for 48 h as a control to eliminate the effects of fasting on the
fish.
Effects of locomotory activity
Specimens were submerged for 24 h in a 12h:12h dark:light regime. Some of
these specimens were then stimulated by mechanical disturbance to swim in
water for 3 min. A separate group of specimens was exposed to terrestrial
conditions for 24 h in the 12h:12h dark:light regime before being stimulated
to move on land for 3 min. This period was chosen because the fish became
refractory to stimulation after such a period of locomotory activity on land.
No anaesthesia was applied to the exercised fish, simply because recovery
would have taken place during the period of anaesthesia treatment
(approximately 5-7 min). Specimens were killed by a quick blow to the head
followed by pithing.
Effects of constant darkness
Specimens were kept in submerged conditions or exposed to air as stated
before but in constant darkness in a cabinet for 48 h, in order to minimize
visual stimulation and hence to reduce locomotory activities.
Collection of samples for further analyses
Anaesthetized or exercised fish were killed immediately by pithing. The
lateral muscle and the liver were quickly excised. No attempt was made to
separate the red and white muscle, but care was taken to sample muscles from
the same region of the body. The excised tissues and organs were immediately
freeze-clamped in liquid nitrogen with pre-cooled tongs. Frozen samples were
kept at -80°C until analysed (within a month). A separate group of fish
exposed to similar conditions was used for the collection of blood samples.
The caudal peduncle of the anaesthetized fish was severed, and blood exuding
from the caudal artery was collected in heparinized capillary tubes. The
collected blood was centrifuged at 4000 g at 4°C for 10
min to obtain the plasma. The plasma was deproteinized in an equal volume
(v/v) of ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 10 000
g at 4°C for 15 min. The resulting supernatant fluid was
kept at -80°C until analysed (within a month).
Ammonia and urea excretion rates in specimens in 12 h: 12 h
dark:light regime
Specimens were submerged individually in plastic aquaria tank (25
cmx14 cmx12 cm, LxWxH) containing 3.51 of water at
25°C with aeration. Preliminary experiments on the analysis of ammonia and
urea in the water sampled at 6 and 24 h showed that the ammonia and urea
excretion rates were linear for at least 24 h. Thus, subsequently, water was
sampled for ammonia and urea analysis after 24 h of exposure. The same
individuals were then exposed to terrestrial conditions in plastic tanks
containing 20 ml of water. After 24 h, the fish were sprayed thoroughly with
water. The water collected was used for ammonia and urea analyses. After
aerial exposure, the fish were resubmerged in water to study the rates of
ammonia and urea excretion upon recovery. Ammonia and urea concentrations were
determined as described below.
Analyses of ammonia and urea in tissue samples
The frozen sample was weighed, ground to a powder in liquid nitrogen, and
homogenized in 5 volumes (w/v) of 6% TCA for determinations of ammonia and
urea levels. For ammonia analysis, the pH of the deproteinized sample was
adjusted to between 5.5 and 6.0 with 2 mol l-1 KHCO3.
The ammonia content was determined as described previously
(Bergmeyer and Beutler, 1985).
Freshly prepared NH4Cl solution was used as the standard for
comparison.
Urea concentration was determined colorimetrically according to the method
of Jow et al. (1999). The
difference in absorbance obtained from the samples with and without urease
treatment was used for the estimation of the urea concentration in the sample.
Urea standards were processed by the same procedure for comparison. Results
were expressed as µmol g-1 wet mass tissue or µmol
ml-1 plasma.
Determination of activities of ornithineurea cycle
enzymes
The livers of experimental fishes were excised quickly for the isolation of
mitochondrial and cytosolic fractions, using a modification of the procedure
of Jow et al. (1999). The
excised livers were minced and suspended in 10 vol. (w/v) of ice-cold
mitochondria extraction buffer (285 mmol l-1 sucrose, 3 mmol
l-1 EDTA, 3 mmol l-1 Tris-HCl, pH 7.2), and homogenized
using three strokes of a Teflon-glass homogenizer. The homogenized sample was
centrifuged at 600 g for 15 min at 4°C to remove any
unbroken cells and nuclei. The supernatant obtained was further centrifuged
for 15 min at 10 000 g to obtain a mitochondrial pellet. The
mitochondrial pellet was washed twice with the extraction buffer, and
centrifuged for 15 min at 10 000 g. The final mitochondrial
pellet was suspended in 1 ml of suspension buffer (100 mmol l-1
Hepes, pH 7.6, 100 mmol l-1 KCl and 1 mmol l-1 EDTA). It
was then sonicated three times for 20 s with a 10 s break between each
sonication. The sonicated sample was centrifuged at 4000 g at
4°C for 3 min. After centrifugation, the supernatant was passed through a
10 ml Bio-Rad P-6DG column (Bio-Rad Laboratories; CA, USA) equilibrated with
suspension buffer.
To obtain the cytosolic fraction, the freshly excised liver was homogenized with 5 vol. (w/v) of mitochondria extraction buffer and centrifuged at 10 000 g for 15 min. The resulting supernatant was collected and passed through a 10 ml Bio-Rad column equilibrated with suspension buffer. The collected filtrates of the mitochondrial and cytosolic fractions were used for the subsequent enzyme analyses.
Carbamoyl phosphate synthetase (CPS; E.C. 2.7.2.5) activity was determined
in the mitochondrial fraction according to the method of Anderson and Walsh
(1995). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter (Bio Laboratories,
USA). The CPS activity was expressed as [14C]urea formed (µmol
min-1 mg-1 protein).
Ornithine transcarbamoylase (E.C. 2.1.3.3) activity was determined in the
mitochondrial fraction by combining the methods of Anderson and Walsh
(1995) and Xiong and Anderson
(1989
). Absorbance was
measured at 466 nm using a Shimadzu UV 160 UV VIS recording spectrophotometer.
The ornithine transcarbamoylase activity was expressed as citrulline formed
(µmol min-1 mg-1 protein).
Argininosuccinate synthetase (E.C. 6.3.4.5) and lyase (E.C. 4.3.2.1)
activities were determined together in the cytosolic fraction, 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. Argininosuccinate
synthetase and lyase activity was expressed as [14C]fumarate formed
(µmol min-1 mg-1 protein).
Arginase (E.C. 3.5.3.1) was assayed in both mitochondrial and cytosolic
fractions as described by Felskie et al.
(1998). Urea was determined as
described above. Arginase activity was expressed as urea formed (µmol
min-1 mg-1 protein).
Analysis of free amino acids
The frozen muscle and liver samples were weighed, ground to a powder in
liquid nitrogen, and homogenized in 5 vol. (w/v) of 6% TCA three times (20 s
each at 10 s intervals) using an Ultra-Turrax homogenizer at 24 000 revs
min-1. The homogenates were centrifuged at 10 000 g
at 4°C for 15 min to obtain the supernatant fluid for free amino acid
(FAA) analyses. The supernatant obtained or the deprotenized plasma 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. Free
amino acids were analyzed using a Shimadzu LC6A amino acid analysis system
(Kyoto, Japan) with a Shimpack ISC-07/S1504 Li-type column. The results of FAA
analyses are expressed as µmol g-1 wet mass tissue or µmol
ml-1 plasma.
Determination of activities associated with alanine or glutamine
formation
Alanine aminotransferase (ALT; E.C. 2.6.1.2) activities in both the
cytosolic and mitochondrial fractions in the direction of alanine degradation
were assayed according to Peng et al.
(1994) with some
modifications. The reaction mixture of 3.1 ml contained 50 mmol l-1
imidazole-HCl, pH 7.4, 10 mmol l-1
-ketoglutarate
(
-KG), 0.17 mmol l-1 NADH, 0.025 mmol l-1
pyridoxal phosphate, 6.2 i.u. lactate dehydrogenase (LDH) (Sigma Chemical Co.,
USA) and 0.2 ml of sample. The reaction was initiated with 0.4 ml of alanine
at a final concentration of 200 mmol l-1. ALT activity was
expressed as NADH utilized (µmol min-1 mg-1
protein).
The glutamate dehydrogenase (GDH; E.C. 1.4.1.3) activity in the
mitochondrial fraction was assayed in the aminating and deaminating directions
according to Ip et al. (1993),
with some modifications. For the amination reaction, the reaction mixture of 3
ml contained 200 mmol l-1 imidazole-HCl, pH 7.8, 10 mmol
l-1
-KG, 1 mmol l-1 ADP, 0.17 mmol l-1
NADH and 0.2 ml of sample. The reaction was initiated with 0.4 ml of ammonium
acetate at a final concentration of 250 mmol l-1. GDH activity was
expressed in NADH utilized (µmol min-1 mg-1 protein).
For the deamination reaction, the reaction mixture of 2.8 ml contained 200
mmol l-1 glycine-NaOH, pH 9, 1 mmol l-1 ADP, 0.1 mmol
l-1 nicotinamide adenine dinucleotide (NAD), 0.09 mmol
l-1 iodonitrotetrazolium chloride (INT), 0.28 i.u. diaphorase
(Sigma Chemical Co., USA) and 0.2 ml of sample. The reaction was initiated
with 0.2 ml of glutamate at a final concentration of 100 mmol l-1.
The change in absorbance was monitored at 492 nm and 25°C. The specific
activity was expressed as formazan formed (µmol min-1
mg-1 protein).
The activity of malic enzyme (E.C.1.1.1.40) was determined according to
Peng et al. (1994). The
reaction mixture of 1.45 ml contained 67 mmol l-1
triethanolamine-HCl (TEA), pH 7.4, 4 mmol l-1 MgCl2, 2
mmol l-1 NAD and 0.15 ml of sample. The reaction was initiated by
the addition of malate at a final concentration of 25 mmol l-1. The
change in absorbance was monitored at 340 nm at 25°C. The activity of
malic enzyme was expressed as NADH formed (µmol min-1
mg-1 protein).
Analyses of glycogen, ammonia, urea, alanine, lactate, ATP, ADP and
AMP in samples from the exercised specimens
For the determination of glycogen, frozen samples (0.5-1.0 g) were ground
to a powder and digested in 2 ml of 30% (w/v) KOH in a boiling water bath for
10 min. The glycogen was extracted according to the method of Lim and Ip
(1989), and was determined by
combining the methods of Bergmeyer et al.
(1974
) and Roehrig and Allred
(1974
). The glycogen content
was expressed as glycosyl units (µmol g-1 wet mass tissue).
For the other metabolites, samples were homogenized as stated above except
in 5 vol. 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 K2CO3. The ammonia
and urea contents were determined as stated before. The alanine content was
determined according to the method of Williamson
(1974). Lactate was determined
using the method of Gutmann and Wahlefeld
(1974
) while ATP, ADP and AMP
were determined spectrophotometrically by the procedures of Scheibel et al.
(1968
). Results were expressed
as µmol g-1 wet mass tissue or µmol ml-1
plasma.
Statistical analysis
Results are presented as means ± the standard error of the mean
(S.E.M.). 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 where
P<0.05 were regarded as statistically significant.
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Results |
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There were significant increases in concentrations of ammonia in the muscle, liver and plasma after exposure to terrestrial conditions for 48 h (Fig. 2A). The urea level increased slightly after 12 h of aerial exposure and returned to the normal value thereafter (Fig. 2B). Aerial exposure had no effect on the urea content in the liver (Fig. 2B). There was a significant increase in the urea concentration in the plasma of specimens exposed to 24 or 48 h of terrestrial conditions (Fig. 2B), but the change was minor compared to that for ammonia (Fig. 2A).
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In the absence or presence of glutamine and N-acetylglutamate in the assay medium, no carbamoylphosphate synthetase-III activity could be detected from liver mitochondria isolated from specimens kept under control conditions or exposed to terrestrial conditions for 48 h. In addition, no argininosuccinate synthetase + lyase activity could be detected in the cytosol of the liver of these specimens. Ornithine transcarbamoylase and arginase activities were present in the liver mitochondria (0.0022±0.0004 and 0.093±0.023 µmol min-1 mg-1 protein, respectively, N=3) from control specimens. The former was unaffected by aerial exposure, but the latter increased significantly to 0.39±0.04 µmol min-1 mg-1 protein (N=3) after exposure to terrestrial conditions for 48 h. Arginase activity could also be detected in the cytosol, though at a much lower level (0.0072±0.0036 µmol min-1 mg-1 protein), and was unaffected by 48 h of aerial exposure.
Levels of alanine, asparagine, glutamine, histidine, serine and tyrosine in the muscle of C. asiatica increased significantly after 48 h of aerial exposure (Table 1). The alanine level (3.7 µmol g-1) rose to 6.4 and 12.6 µmol g-1 after 24 h and 48 h of exposure to terrestrial conditions, respectively. The TFAA level in the muscle of specimen exposed to 48 h of aerial exposure was significantly higher than that of the 0 h submerged control, but was comparable to the submerged control fasted for 48 h (Table 1). There were significant changes to some of the FAAs in the liver of specimens exposed to terrestrial conditions for 48 h, as compared to the 0 h submerged control (Table 2). However, the majority of these changes were also detected in the specimens kept in the submerged conditions and fasted for the same period (Table 2). Aerial exposure induced only minor changes to some of the amino acids in the plasma of C. asiatica (Table 3).
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Activities of ALT in the cytosolic and mitochondrial fractions of the muscle and liver were unaffected by aerial exposure (Table 4). However, there were significant decreases in the aminating activity of GDH from the muscle and liver of specimens exposed to terrestrial conditions, leading to significant decreases in the amination:deamination ratio (Table 5). Malic enzyme is present in the muscle of C. asiatica (0.0030±0.0007 µmol min-1 mg-1, N=3) and was unaffected by aerial exposure (0.0042±0.0007).
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Effects of short burst of locomotory activity
Exercise in water for 3 min after 24 h of submergence had no significant
effect on the energy charge and content of ATP, ADP, AMP, alanine, glutamate,
glutamine, ammonia and glycogen in the muscle of C. asiatica
(Table 6). However, there was a
significant increase in lactate levels in the muscle of these exercised
specimens (Table 6). Similar
results were obtained for fish that were exercised on land after 24 h of
aerial exposure, except that the increase in lactate in the muscle was
accompanied by a significant decrease in the glycogen content
(Table 6).
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Effects of constant darkness
The contents of ammonia, urea, alanine and total free amino acids (TFAA) in
the muscle of specimens exposed to terrestrial conditions in constant darkness
and those of specimens in a 12 h:12 h dark:light regime were comparable
(Fig. 3).
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Discussion |
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There was no change in the rate of urea excretion in C. asiatica
exposed to terrestrial conditions compared to submerged controls. In addition,
there was no major change in the urea content of muscle, liver and plasma
after 48 h of aerial exposure. C. asiatica did not therefore detoxify
ammonia to urea when out of water, contrary to the belief that there is a
tendency towards predominance of ureotelism in amphibious species
(Wright, 1995;
Mommsen and Walsh, 1992
;
Walsh, 1997
;
Wright and Land, 1998
), which
are found mainly in the tropics. Furthermore, carbamoylphosphate synthetase
and argininosuccinate synthetase + lyase activities remained below the
detection limits of the methods adopted in this study, although ornithine
transcarbamoylase and arginase activities were detected in the hepatic
mitochondria, so it is unlikely that C. asiatica possesses a
`functional' ornithineurea cycle due to the low activities of some of
the enzymes.
Ramaswamy and Reddy (1983)
concluded that another snakehead Channa gachua shifted toward
ureotelism during aerial exposure. This has been cited to support the
hypothesis of urea formation as a means of detoxifying ammonia in
air-breathing teleosts exposed to land
(Anderson, 1995
;
Graham, 1997
;
Saha and Ratha, 1998
;
Frick and Wright, 2002
).
However, we wish to point out that there were apparent discrepancies in
results reported by Ramaswamy and Reddy
(1983
). Taking the normal
ammonia excretion rate over 5 h in water from their results as 7.13 mg N
h-1 kg-1, and assuming that no ammonia excretion took
place when C. gachua was exposed to terrestrial conditions for 10 h,
the total amount of ammonia excreted in the subsequent 5 h (a total of 15 h)
of resubmergence should have been 7.13x15=106.95 mg N kg-1.
Since the reported result obtained for ammonia excretion during this
subsequent 5 h period was only 14.03x5= 70.15 mg N kg-1,
there was a deficit of 106.95-70.15= 36.8 mg ammonia-N kg-1.
However, Ramaswamy and Reddy
(1983
) reported the urea
excretion rate in this subsequent 5 h period, during which the urea and
ammonia levels in the blood and liver returned back to normal, to be 50.14 mg
N h-1 kg-1. This represents a total of 50.14x5 or
250.7 mg N kg-1, or a surplus of 250.7-(9.91x15) or 102.05 mg
urea-N kg-1, which was much greater than the deficit of 36.8 mg
ammonia-N kg-1 in ammonia excretion. Actually, the only possible
explanation for such results is that increases in proteolysis and amino acid
catabolism had taken place, but such an adaptation is physiologically
unfitting during aerial exposure and was unlikely to have occurred.
Alternatively, the discrepancy might have arisen from the high variability and
low sensitivity of the method of ammonia and urea assay (urease treatment plus
Nesslerization) adopted in their study. There may be physiological differences
between two species of fishes, even if they are grouped under one single genus
(in this case, Channa). However, Ramaswamy and Reddy
(1983
) did not examine urea
and ammonia contents in the muscle, which constitutes the bulk of the fish,
nor assay for ornithineurea cycle enzymes in C. gachua. Since
our results on C. asiatica do not support urea formation as the major
strategy for detoxifying ammonia during aerial exposure, whether C.
gachua would turn ureotelic on land is an issue that needs to be
re-examined.
Ip et al.
(2001a,b
)
suggested that fishes such as the mudskipper P. schlosseri, which are
active on land with impeded excretion of ammonia, would partially catabolize
certain amino acids to alanine. This would allow amino acids to be used as an
energy source without polluting the internal environment. Partial amino acid
catabolism coupled with a reduction in amino acid catabolism in general
constitutes the most cost-effective way to minimize endogenous ammonia
build-up. Alanine may be more suitable for accumulation because it has fewer
effects than other amino acids on the kinetics of many enzymes (e.g.
versus arginine; Bowlus and
Somero, 1979
). Indeed, alanine levels increased fourfold, from 3.7
to 12.6 µmol g-1, in the muscle of C. asiatica after 48
h of aerial exposure. The accumulated alanine accounted for 70% of the deficit
in ammonia excretion during that period. This would allow the utilization of
certain amino acids as energy sources and, at the same time, minimize ammonia
accumulation. However, in contrast to mudskippers
(Ip et al., 2001b
), the
reduction in nitrogenous excretion during 48 h of aerial exposure was
completely balanced by nitrogenous accumulation in the tissues
(Table 7). Hence, it is
unlikely that the rates of proteolysis and amino acid catabolism were reduced
during aerial exposure, as in the case of mudskippers
(Ip et al., 2001b
), which
implies that C. asiatica does not have the mudskippers' capability of
surviving on land. Actually, the ability to reduce rates of amino acid
catabolism during aerial exposure may not be a common phenomenon among
teleostean fishes. For example, when the marble goby Oxyeleotris
marmoratus, which remains inactive on land, is exposed to terrestrial
conditions for 72 h, it does not undergo a reduction in amino acid catabolism.
Instead, it appears that protein and/or amino acid catabolism may increase
during aerial exposure because glutamine accumulates to levels far in excess
of those needed to detoxify the ammonia produced during that period
(Jow et al., 1999
).
|
The presence of malic enzyme and ALT in the tissues of C. asiatica
supports the proposition that this fish is capable of undergoing partial amino
acid catabolism. In this pathway, malate is channelled out of the Krebs cycle
through malic enzyme to form pyruvate. Pyruvate then undergoes transamination
through the ALT reaction to form alanine. For this mechanism to work,
-KG produced must be channelled into the Krebs cycle to maintain a
supply of pyruvate for transamination so that no endogenous ammonia is
released in the process (Fig.
4). However,
-KG also acts as a substrate for the GDH
reaction, in the aminating direction, which is also occurring in the
mitochondria. If
-KG were converted into glutamate, partial amino acid
catabolism would not proceed, ATP would not be produced, and endogenous
ammonia would start to accumulate in the terrestrial conditions at a higher
rate (Fig. 4). Indeed, aerial
exposure significantly decreased the aminating activities of GDH from the
muscle and liver of C. asiatica. This can be seen as an important
adaptation to facilitate the formation of alanine through partial amino acid
catabolism in order to reduce the rate of endogenous ammonia accumulation in
adverse conditions. However, suppression of the GDH aminating activity would
not be a good strategy for handling exogenous ammonia during ammonia-loading,
when ammonia has to be detoxified through glutamate and glutamine formation
(Fig. 4)
(Ip et al., 2001a
). At this
moment, it is uncertain if C. asiatica can upregulate the aminating
activity of GDH in response to exogenous ammonia, as in the mudskipper
(Peng et al., 1998
).
|
For P. schlosseri exposed to 24 h of terrestrial conditions,
alanine accumulates in the body (3-4 µmol g-1 muscle; Ip et al.,
1993,
2001b
), but the aminating GDH
activity in the muscle is unchanged (Ip et
al., 1993
). The activity (both aminating and deaminating) in the
liver decreases, although there is an apparent increase in the
amination:deamination ratio (Ip et al.,
1993
). This may represent a compromise between adaptations to
aerial exposure (to decrease endogenous ammonia production) during an
excursion on land at low tides and adaptations to ammonia loading (to detoxify
exogenous ammonia) during a stay in the burrow at high tide or during the
breeding seasons.
On the contrary, Iwata et al.
(1981) reported that the
aminating GDH activities in the muscle and liver of the mudskipper
Periophthalmus modestus (as P. cantonensis) increased
significantly after aerial exposure. Iwata et al. interpreted the results as
ammonia (endogenous) being detoxified to amino acids through the amination of
-KG. However, it would be futile if GDH acts, on the one hand, to
release ammonia through transdeamination
(Mommsen and Walsh, 1992
), and
on the other hand, to detoxify the released ammonia back to glutamate
simultaneously, as suggested by Iwata et al.
(1981
). If indeed alanine is
formed through partial amino acid catabolism, the amino groups of various
amino acids (e.g. glutamate, valine and leucine) are not physically released,
and are reconverted into amino acids (Ip et al.,
2001a
,b
).
Their amino groups are channelled to glutamate, which acts as a substrate for
the transamination of pyruvate. Thus, the glutamate involved in this reaction
does not arise from the GDH aminating reaction
(Fig. 4).
Effects of constant darkness
To slow down the build-up of ammonia internally, it would be necessary to
decrease the rate of ammonia production through amino acid catabolism. The
steady-state concentration of amino acids in the tissues depends on the rates
of their degradation and production. Lim et al.
(2001) observed decreases in
TFAA contents in some of the tissues of two mudskipper species exposed to
terrestrial conditions in constant darkness, and there was no accumulation of
alanine at all. Lim et al.
(2001
) suggested that
simultaneous decreases in the rates of proteolysis and amino acid catabolism
would have occurred, and the decrease in proteolytic rate was greater than the
decrease in the rate of amino acid catabolism. This would lead to decreases in
the steady-state concentrations of various FAAs and, consequently, lowering
the TFAA concentrations. This is an effective strategy for slowing down the
internal build-up of endogenous ammonia. However, such a phenomenon was not
observed in C. asiatica exposed to terrestrial conditions in constant
darkness. There was no change in the TFAA contents in all the tissues and
organs examined. In addition, alanine accumulated to the same extent in the
muscle of these specimens compared to those exposed to terrestrial conditions
in a dark:light regime. This would imply that, with respect to nitrogen
metabolism, the adaptation of C. asiatica to aerial exposure is less
effective than that of the mudskippers.
Effects of short burst of locomotory activity
Exercise in water led to a decrease in glycogen content, and increases in
the levels of lactate in the muscle of C. asiatica. However, it had
no significant effect on the ammonia and alanine contents in the muscle. It
can be deduced that glycogen was mobilized in this situation, and there was an
increase in glycolytic rate to supply ATP for locomotory activities,
maintaining a relatively constant energy charge in this tissue. Similar
results were obtained when specimens were forced to exercise on land after 24
h exposure to terrestrial conditions. Hence, unlike P. schlosseri
(Ip et al., 2001b), C.
asiatica is incapable of increasing the rate of partial amino acid
catabolism to sustain locomotory activities on land, although it can undergo
partial amino acid catabolism to reduce ammonia production during aerial
exposure. In the case of P. schlosseri, exercise on land leads to
increases in muscle ammonia and alanine levels with the glycogen content
unaffected (Ip et al., 2001b
).
This difference may explain, at least partially, why P. schlosseri
can maintain long periods of locomotory activity on land, but under similar
conditions C. asiatica can only afford short bursts of muscular
activities to try to get back to water.
A comparative perspective
On land, C. asiatica exhibits activity that is intermediate
between that of the marble goby (totally quiescent) and the mudskipper (very
active). In relation to this, it is important to note that C.
asiatica accumulates both alanine and glutamine in the muscle, with
glutamine accounting for only 20% of the deficit in reduction in ammonia
excretion. In contrast to alanine formation, glutamine synthesis represents an
ammonia detoxification mechanism that, in effect, removes the accumulating
ammonia. Production of glutamine is energy expensive
(Ip et al., 2001a), and
appears to be adopted by fishes that remain relatively inactive on land such
as the marble goby (Jow et al.,
1999
) and sleeper Bostrichthys sinensis
(Ip et al., 2001c
;
Anderson et al., 2001
). As for
the amphibious mudskippers Boleophthalmus boddaerti and P.
schlosseri, they do not accumulate glutamine during aerial exposure
(Lim et al., 2001
;
Ip et al., 2001b
). Instead,
glutamine formation is only used as a means of detoxifying exogenous (and
endogenous) ammonia when confronted with sublethal concentrations of external
ammonia (Peng et al., 1998
).
Hence, it can be concluded that teleostean fishes exhibit a variety of
strategies to survive aerial exposure, and they represent a range of specimens
available for us to examine various biochemical adaptations that would have
facilitated their invasion of the terrestrial habitat during evolution.
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