The swamp eel Monopterus albus reduces endogenous ammonia production and detoxifies ammonia to glutamine during 144 h of aerial exposure
1 Department of Biological Science, National University of Singapore, Kent
Ridge, Singapore 117543, Republic of Singapore
2 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 22 April 2003
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Summary |
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Key words: ammonia, amino acid, Monopterus albus, glutamate, glutamine, glutamine synthetase, swamp eel, urea
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Introduction |
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In this report, NH3 represents un-ionized molecular ammonia,
NH4+ represents ammonium ions, and ammonia refers to
both NH3 and NH4+. Ammonia is usually
excreted as NH3 across the body surface, usually the gills, of fish
into the surrounding water (Wilkie,
1997). Under acidic environmental conditions, NH3
diffusing across the gills is converted to NH4+ and
trapped in the water. Thus, acidic conditions in the environment augment
ammonia excretion. The diffusion of ammonia into the environment is inhibited
if the pH of the environment is high, and ammonia subsequently accumulates in
the body. Decomposition of organic matter or the use of fertilizers will
increase ammonia levels in the water and this will result in elevated ammonia
levels in the fish. Finally, excretion is also reduced if the fish moves out
of water (reviewed by Ip et al.,
2001a
).
The swamp eel Monopterus albus (Zuiew 1793) is a bony fish (family
Synbranchidae; order Synbranchiformes; class Actinopterygii). It is not really
an eel because it does not belong to the family Anguillidae of the order
Anguilliformes. M. albus can be found in the tropics (34° N to
6° S) from India to southern China, Malaysia and Indonesia. It has an
anguilliform body reaching a maximal length of 100 cm at maturity, with no
scale and no pectoral and pelvic fins, and the dorsal, caudal and anal fins
are confluent and reduced to a skin fold. M. albus lives in muddy
ponds, swamps, canals and rice fields
(Rainboth, 1996), where it
burrows in moist earth in dry season, surviving for long periods without water
during summer (Shih, 1940
;
Davidson, 1975
). During
prolonged drought, it burrows deep into the mud to remain in contact with the
water table (Liem, 1987
).
While M. albus can tolerate the seasonal draining of rice patties in
the mud, it can survive only several days in the market without water
(Wu and Kung, 1940
). At
present, the reason behind this discrepancy in the capability of M.
albus to survive in mud and in air is not clear. Preliminary observations
made in our laboratory confirmed that the mortality of specimens exposed to
air rose from 0% after 6 days to 30% after 8 days. Therefore, this study was
undertaken to examine the strategies adopted by M. albus to defend
against ammonia toxicity during 144 h (6 days) of aerial exposure.
Like many other tropical air-breathing fish, when M. albus moves on or hides in the mud in the dry seasons it encounters a lack of water. This would lead to difficulties in excreting ammonia through its gills and cutaneous surfaces. Hence, one of the objectives of this study was to examine if aerial exposure would cause the accumulation of ammonia in the body and if M. albus could tolerate high levels of ammonia at the tissue and cellular levels.
Ammonia is toxic and affects various cellular processes
(Ip et al., 2001a). Therefore,
many tropical species have evolved mechanisms to deal with the increased body
ammonia loads resulting from reduction in ammonia excretion associated with
aerial exposure. These mechanisms include: (1) reducing ammonia production, as
in the mudskippers Periophthalmodon schlosseri and Boleophthalmus
boddaerti, the four-eyed sleeper Bostrichyths sinensis, the
weather loach Misgurnus anguillicaudatus and the mangrove killifish
Rivulus marmoratus (Ip et al.,
2001a
,b
,2001c
;
Lim et al., 2001
;
Chew et al., 2001
;
Frick and Wright, 2002a
); (2)
undergoing partial amino acid catabolism leading to the accumulation of
alanine, as in the giant mudskipper P. schlosseri and the snakehead
Channa asiatica; (3) detoxifying ammonia to urea, as in the African
lungfishes Protopterus aethiopicus
(Janssens and Cohen, 1968
)
and, possibly, the Indian catfishes Heteropneustes fossilis and
Clarias batrachus (Saha and
Ratha, 1998
; Anderson,
2001
; but see review by Chew
et al., in press
, for a different view); (4) detoxifying ammonia
to glutamine, as in the marble goby Oxyeleotris marmoratus and the
four-eyed sleeper (Jow et al.,
1999
; Ip et al.,
2001a
,b
);
(5) actively excreting NH4+ into water trapped between
secondary lamellae of the gills, as in the giant mudskipper
(Randall et al., 1999
;
Chew et al., 2003a
); and (6)
excreting NH3 into air, as in the blenny Alticus kirki,
the weather loach and the mangrove killifish
(Rozemeijer and Plaut, 1993
;
Tsui et al., 2002
;
Frick and Wright, 2002b
). The
responses of tropical fish to aerial exposure are many and varied, determined
by the behaviour of the fish and the nature of the environment in which it
lives. Thus, the additional aims of this study were to examine whether M.
albus would (1) volatilize NH3, (2) detoxify ammonia to urea
when exposed to terrestrial conditions, (3) be capable of suppressing
endogenous ammonia production by reductions in proteolysis and amino acid
catabolism, (4) be capable of undergoing partial amino acid catabolism leading
to the formation of alanine without releasing ammonia, and (5) be capable of
detoxifying ammonia to glutamine while it is out of water. It was hypothesized
that M. albus had adopted one or more of these strategies to survive
144 h of aerial exposure.
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Materials and methods |
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Experiment 1: does M. albus volatilize
NH3 during aerial exposure?
The set-up used by Tsui et al.
(2002) for the weather loach
was adopted for this experiment, except that a sealed plastic container with a
total volume of 1.5 litres was used to house M. albus. Specimens
(N=8) were kept individually in plastic containers with 1 litre of
freshwater (pH 7.0) at 25°C. After 24 h, the water in the container and
the acid in the two NH3 traps were analyzed for ammonia
concentration. The containers were then rinsed. The same eels were immediately
exposed to air for 24 h in the same container but with only 50 ml of
freshwater (pH. 7.0). At the end of the second 24 h period, the water in the
container and the acid in the NH3 traps were collected for ammonia
assay. Ammonia concentration was determined colorimetrically according to the
method of Anderson and Little
(1986
).
Experiment 2: effects of aerial exposure on the rates of ammonia
and urea excretion in M. albus
For the control (N=4), M. albus were exposed to 20
volumes (w/v) of freshwater (pH. 7.0) in plastic containers (50 cmx30
cmx20 cm, length x width x height). Water samples (3 ml)
were collected at 6 h and 24 h, acidified with 0.07 ml of 1 mol l-1
HCl to prevent the loss of NH3 and stored at 4°C. Ammonia and
urea assays were performed within 48 h of sample collection. Preliminary
results indicated that the rates of ammonia and urea excretion were linear
within the 24 h period. Water was changed daily after the collection of the 24
h sample. The experiment lasted 144 h.
For experimental specimens (N=4) exposed to air, M. albus
were kept in similar plastic containers but with only 200 ml of freshwater (pH
7.0). Water was sampled at 24 h, after which the container was rinsed and new
freshwater (200 ml) was added. After 144 h of aerial exposure, the fish was
immersed in 20 volumes (w/v) of freshwater to study the rates of ammonia and
urea excretion upon recovery from aerial exposure. Analyses of ammonia and
urea concentration were performed within 48 h using the methods described
above. Ammonia concentration was determined colorimetrically according to the
method of Anderson and Little
(1986). Urea was assayed
according to the method of Jow et al.
(1999
).
Experiment 3: effects of aerial exposure on ammonia, urea and
free amino acid (FAAs) concentration and enzyme activity in M. albus
Tissue preparation
A group of specimens (N=6) were killed at the start of the
experiment to act as 0 h controls. Another group (N=2-5) was kept in
20 volumes (w/v) of freshwater (pH 7.0) for 6 days and served as the 144 h
controls. Other specimens (N=20) were exposed to air in plastic
containers (50 cmx30 cmx20 cm, length x width x
height) with only a thin film of freshwater (200 ml; pH 7.0). Specimens were
killed after 24 h, 72 h or 144 h of air exposure. The water was changed daily
in all cases.
For the collection of plasma, the caudal peduncle of the fish was severed, and blood exuding from the caudal artery was collected in sodium heparin-coated Eppendorf tubes. The plasma obtained after centrifugation at 5000 g at 4°C for 5 min was deproteinized by adding 2 volumes (v/v) of ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 10 000 g at 4°C for 10 min. The resulting supernatant was kept at -80°C for analysis of ammonia, urea and FAAs. The brain, liver, lateral muscle and gut (flushed thoroughly with saline) were excised and immediately freeze-clamped with tongs pre-cooled in liquid nitrogen. Samples were stored at -80°C until analysis.
Analysis of ammonia, urea and FAA concentration
For ammonia, urea and FAA analysis, the frozen liver, muscle and gut tissue
samples were weighed and pulverised to a powder at -80°C. Five volumes of
ice-cold 6% TCA were added and the mixture was homogenized three times for 20
s each (with 10 s intervals) with an Ultra-Turrax homogenizer (Janke &
Kunkel GmBH and Co., Staufen, Germany) at 24 000 revs min-1. Frozen
brain samples were weighed and then hand-homogenized in 10 volumes (w/v) of 6%
TCA using a glass-pestle homogenizer (Wheaton Science Products, Millville, NJ,
USA). The samples were then centrifuged at 10 000 g at 4°C
for 10 min, and the supernatants were stored at -80°C for subsequent
analysis. Ammonia and urea assays were performed within 2 weeks, and FAA
analysis was completed within one month.
For ammonia analysis, the pH of the deproteinized sample was adjusted to
6.0-6.5 with 2 mol l-1 KHCO3. Ammonia concentration was
determined as described by Kun and Kearney
(1974). Freshly prepared
NH4Cl solution was used as the standard for comparison. Urea
concentration in a 0.2 ml deproteinized sample was analyzed colorimetrically
according to the method of Jow et al.
(1999
). Urea (Sigma, St Louis,
MO, USA) was used as a standard. Results are expressed as µmol
g-1 wet mass tissue or µmol ml-1 plasma.
For FAA analysis, deproteinized muscle, liver, brain, gut and plasma samples were thawed and diluted with an equal volume of 2 mol l-1 lithium citrate buffer and adjusted to pH 2.2 with 4 mol l-1 LiOH. These samples were then analyzed for FAA concentration using an LC-6A Amino Acid Analysis System with a Shim-pack ISC-07/S1504 Li-type column (Shimadzu, Nakagyo-ku, Kyoto, Japan). The concentrations of FAAs are expressed as µmol g-1 wet mass for brain, liver, muscle and gut samples and as µmol ml-1 for plasma samples; the total FAA (TFAA) concentration is expressed as the sum of the FAAs.
Analysis of enzyme activity
For enzyme assays, the frozen muscle, liver and gut samples were weighed
and homogenized three times in 5 volumes (w/v) of ice-cold extraction buffer,
containing 50 mmol l-1 imidazole-HCl (pH 7.0), 50 mmol
l-1 NaF and 3 mmol l-1 EGTA, at 24 000 revs
min-1 for 20 s with 10 s intervals. The homogenate was sonicated
three times for 20 s with 10 sintervals and then centrifuged at 10 000
g at 4°C for 20 min. The supernatant was then passed
through a 10 ml Econo-Pac 10DG desalting column (Bio-Rad Laboratories, Inc.,
Hercules, CA, USA) equilibrated with 50 mmol l-1 imidazole-HCl (pH
7.0). The resulting eluent was used for enzymatic analysis. The protein
concentration of the extract was measured before and after filtration to
calculate the dilution factor involved. The frozen brain sample was weighed
and then homogenized in 5 volumes (w/v) of ice-cold extraction buffer using a
Wheaton glass-pestle homogenizer. The homogenate was centrifuged at 10 000
g for 15 min at 4°C. The supernatant was dialyzed against
a buffer containing 50 mmol l-1 imidazole-HCl (pH 7.0) using
Microdialyzer System 100 (Pierce, Rockford, IL, USA). The dialysate was used
for enzyme analyses. Enzyme analyses were recorded with a Shimadzu UV-1601
UV-VIS recording spectrophotometer at 25°C. All chemicals and coupling
enzymes were obtained from Sigma.
Glutamine synthetase (GS; EC 6.3.1.2) activity was determined
colorimetrically according to the method of Shankar and Anderson
(1985). GS activity was
expressed as µmol
-glutamyl hydroxamate formed min-1
g-1 wet mass tissue. Freshly prepared glutamic acid monohydroxamate
solution was used as a standard for comparison.
The activities of alanine aminotransferase (ALT; EC 2.6.1.2), which
catalyses alanine degradation, and aspartate aminotransferase (AST; EC
2.6.1.1), which catalyses aspartate degradation, were determined according to
Peng et al. (1994). Glutamate
dehydrogenase (GDH; EC 1.4.1.3) activity, which catalyses amination, was
assayed according to Ip et al.
(1993
). ALT, AST and GDH
activities were monitored at 340 nm. Enzyme activities were expressed in
µmol NADH utilized min-1 g-1 wet mass.
Statistical analyses
Results were presented as means ± S.E.M. Student's
t-test and one-way analysis of variance (ANOVA) followed by
Student-Newman-Keuls multiple range test were used to compare differences
between means where applicable. Differences with P<0.05 were
regarded as statistically significant.
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Results |
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Ammonia levels in the muscle (Fig. 2A), liver (Fig. 2B), brain (Fig. 2C), gut (Fig. 2D) and plasma (Fig. 2E) of M. albus increased significantly during aerial exposure. After 72 h of aerial exposure, ammonia concentration in the liver, brain and plasma were 3-fold, 3.5-fold and 5-fold, respectively, those of the control values. In the muscle and gut, the ammonia concentration reached the highest level of 6.9 µmol g-1 and 4.5 µmol g-1, respectively, after 144 h of aerial exposure. These were approximately 3.5 times that of the control values (Fig. 2A,D).
|
Urea concentration did not increase significantly in the muscle, liver and plasma of eels after 144 h of aerial exposure compared with the 144 h water controls (Fig. 3A,B,E). The urea concentration in the brain of the specimens exposed to air for 144 h was 1.5-fold that of the 144 h control (Fig. 3C). In the gut, the urea concentration rose after 24 h of aerial exposure but that of specimens exposed to air for 144 h was comparable with that of the 144 h control in water (Fig. 3D).
|
In the muscle, glutamine concentration peaked after 72 h of aerial exposure and was 4.5-fold higher than that of the 0 h control value by 144 h (Table 2). Significant increases were also observed in the concentrations of alanine, histidine, isoleucine, leucine, methionine, serine, taurine, threonine, tyrosine and valine after 72 h of air exposure compared to 0 h control (Table 2). However, by 144 h, these values had either returned to control levels (histidine, isoleucine, leucine, taurine, threonine, tyrosine and valine) or fallen to levels lower than those of the 144 h control (alanine, methionine and serine) (Table 2). The total free amino acid (TFAA) content in specimens exposed to air for 144 h was comparable with that of the 144 h control in water (Table 2).
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In the liver, the glutamine concentration compared with the 0 h water control increased 36- and >29-fold by 72 h and144 h, respectively (Table 3). At 144 h, the levels of alanine, histidine and tyrosine were markedly lower than the corresponding control values (Table 3). However, the TFAA content of specimens exposed to 144 h terrestrial conditions was comparable with that of the 144 h control.
|
In the brain, the glutamine level increased significantly to 4-fold the 0 h control values after 72 h of aerial exposure (Table 4). The concentrations of leucine, lysine, proline, serine and tyrosine were also affected after exposure to air for 144 h compared with the 144 h water control (Table 4). Similar large increases in glutamine levels were also observed in the gut and plasma of air-exposed specimens (Tables 5, 6).
|
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After 144 h of aerial exposure, GS and GDH (which catalyse amination) activities were halved in the muscle of M. albus, reaching 1.09±0.17 µmol min-1 g-1 and 0.06±0.01 µmol min-1 g-1, respectively (Table 7). In the liver, there were marked increases in the activities of GS and AST after 144 h of aerial exposure, approaching 1.4- and 1.3-fold the corresponding control value (Table 7). In the brain, significant decreases in ALT and AST activities were observed (Table 7). In the gut, ALT activity rose significantly from 1.00±0.05 µmol min-1 g-1 to 1.47±0.05 µmol min-1 g-1 after 144 h of aerial exposure (Table 7).
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Discussion |
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In a number of terrestrial ammonotelic invertebrates, NH3
volatilization contributes significantly to total ammonia elimination and also
water conservation (e.g. Wright et al.,
1993; Greenaway and Nakamura,
1991
). Amongst vertebrates, however, terrestrial ammonotely is
uncommon and examples of significant NH3 volatilization are rare.
In teleost fish, the first report of ammonia volatilization was in the
temperate intertidal blenny (Blennius pholis) but it only accounted
for 8% of the total ammonia excreted during emersion
(Davenport and Sayer, 1986
).
However, there are a few studies on ammonotelic tropical fish capable of
enduring emersion and volatilizing significant amounts of ammonia
(Rozemeijer and Plaut, 1993
;
Frick and Wright, 2002b
;
Tsui et al., 2002
). High
temperatures and humidity, characteristic of the tropical climates and the
experimental conditions in this study, are factors that increase the
likelihood of the ammonia excreted into the film of water covering the body
surface being volatilized in significant quantities. The most important factor
is the effect of temperature on evaporation rate. Higher temperatures will
also decrease the ammonia equilibrium constant (pKamm), resulting
in a higher fraction of NH3 at a given pH. Although M.
albus is a tropical fish living in environments of high temperature
(25-34°C), the low percentage of total ammonia excreted as ammonia gas
showed that it was unable to eliminate significant amounts of ammonia in
gaseous form during aerial exposure (Table
1).
Ammonia is not detoxified to urea during aerial exposure
There was an apparent increase in the percentage of total-N excreted as
urea when M. albus was exposed to terrestrial conditions
(Fig. 1; Table 8). However, this was
mainly due to the drastic decrease of the ammonia excretion rate during aerial
exposure. In fact, aerial exposure also affected the rate of urea excretion,
which decreased to 25% of the control value
(Fig. 1B). However, in contrast
to ammonia, the changes in urea concentrations in various tissues and organs
examined were relatively minor (Fig.
3A-E). After 24 h of aerial exposure, the urea concentration in
the muscle decreased significantly, producing a deficit of-134 µmol urea-N
per 200 g fish (Table 8). It is
uncertain of the fate of this amount of urea because the rate of urea
excretion actually decreased and urease is known to be absent from tissues of
vertebrates. Nonetheless, these results suggest that M. albus does
not adopt urea synthesis as a strategy to defend against endogenous ammonia
toxicity during 144 h of aerial exposure.
Contrary to the belief that there is a tendency towards predominance of
ureotelism in amphibious species (Mommsen and Walsh,
1989,
1992
;
Wright, 1995
;
Saha and Ratha, 1998
;
Wright and Land, 1998
), only a
few teleosts are ureotelic, and the majority of adult tropical fish studied so
far do not adopt ureogenesis as a major strategy to detoxify endogenously
produced ammonia during aerial exposure. These include the mudskippers P.
schlosseri and B. boddaerti
(Lim et al., 2001
), the marble
goby O. marmoratus (Jow et al.,
1999
), the four-eyed sleeper B. sinensis
(Ip et al., 2001c
), the
weather loach M. anguillicaudatus
(Chew et al., 2001
), the
snakehead C. asiatica (Chew et
al., 2003b
) and the mangrove killifish R. marmoratus
(Frick and Wright, 2002b
)
exposed to terrestrial conditions for various periods. Through the present
study, one more species can be added to this list - M. albus. The
synthesis of urea de novo in fish 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 urea synthesis via the ornithine
urea cycle is rare in adult fish (Ip et
al., 2001a
).
Endogenous ammonia is detoxified to glutamine during aerial
exposure
Ammonia toxicity can be avoided by converting ammonia to glutamine.
Glutamine is produced from glutamate and NH4+, the
reaction catalyzed by GS in the muscle and/or liver. Glutamate may in turn be
produced from -ketoglutarate (
-KG) and
NH4+, catalysed by GDH, or from
-KG and other
amino acids, catalysed by various transaminases. In other words, formation of
one glutamine molecule allows the uptake of two ammonia molecules
(Campbell, 1973
). One mole of
ATP is required for the production of every amide group of glutamine
via GS. If the reaction begins with ammonia and
-KG, every
mole of ammonia detoxified would result in the hydrolysis of 2 moles of
ATP-equivalent (Ip et al.,
2001a
). Hence, glutamine formation would be more effective than
ureogenesis (2.5 moles of ATP for every mole of nitrogen detoxified) with
respect to energy expenditure. More importantly, glutamine is stored within
the body after being synthesized, and can be used for other anabolic processes
(e.g. purine, pyrimidine and mucopolysaccharides) when the environmental
conditions become more favourable. Urea, being a small and uncharged molecule
permeable to biomembranes, can be excreted easily upon its synthesis, and
ureotely actually represents a loss of both nitrogen and carbon to the
environment.
Indeed, M. albus appears to have adopted glutamine formation as a major strategy to defend against ammonia toxicity during 144 h of aerial exposure. The glutamine concentrations in the muscle of M. albus increased by 6-fold (to 10.11 µmol g-1) and 4.5-fold (to 7.62 µmol g-1) after 72 h and 144 h exposure to terrestrial conditions, respectively (Table 2). In the liver, the increase in glutamine level (39-fold and 31-fold after 72 h and 144 h, respectively) was even more drastic (Table 3).
Sleepers (O. marmoratus and B. sinensis) belonging to the
family Eleotridae can detoxify endogenous ammonia to glutamine in non-cerebral
tissues during aerial exposure (Jow et
al., 1999; Ip et al.,
2001b
). Since sleepers remain quiescent on land, the reduction in
energy demand for muscular activity may provide them with the opportunity to
exploit glutamine formation as a means to detoxify ammonia. Results obtained
in the present study proved that glutamine synthesis and accumulation as a
strategy to defend against endogenous ammonia exposure is not exclusive to the
sleepers. M. albus, being an eel, can move on land more effectively
than the sleepers. However, it usually remains motionless if undisturbed,
especially if it has access to a muddy substratum, in which it will bury
itself. Presumably, this behaviour of M. albus facilitates its
adoption of glutamine synthesis as a major strategy to handle ammonia
toxicity.
The concentrations of glutamine in the tissues and organs of specimens exposed to terrestrial conditions for 144 h were generally lower than those of specimens exposed for 72 h. Assuming that endogenous ammonia production was reduced through suppression of proteolysis and amino acid catabolism (see below), glutamine that had been synthesized and accumulated should at least remain at the same level. This indicates that glutamine might not be an end product for accumulation. Rather, in M. albus, it could be mobilized to other compounds yet to be identified for storage under long-term exposure to land. It is essential to elucidate this possibility in future studies and to differentiate the metabolic fates of glutamine during long-term aerial exposure and during subsequent recovery in water.
The brain, liver and gut of M. albus have a high
capacity for GS, which is enhanced by aerial exposure
The brain of M. albus has a very high level of GS activity
(Table 7), probably the highest
known in fish. In the brain, the ALT and AST activities decreased
(Table 7), probably to avoid
consuming glutamate in order to save it for the synthesis of glutamine.
Elevated plasma ammonia concentration is associated with an increase in
glutamine concentration in the brain of M. albus and other
vertebrates (Ip et al., 2001a;
Brusilow, 2002
). It has also
been suggested, however, that high glutamine levels can contribute to toxicity
(Brusilow, 2002
). It is
proposed that increased glutamine production and accumulation cause increased
astrocyte cell volume, leading to cellular dysfunction, brain edema and death.
L-Methionine S-sulfoximine inhibits glutamine synthetase, reduces
edema, attenuates ammonia-induced increases in brain extracellular
K+ and ameliorates ammonia toxicity
(Brusilow, 2002
). Thus,
glutamine formation may either exacerbate or ameliorate ammonia toxicity,
depending on the site of formation and the species in question. At present, it
is unclear how M. albus solves the problems associated with increased
glutamine formation when exposed to terrestrial conditions.
Besides the brain, GS activity was also present in the liver, gut and muscle of M. albus (Table 7), with decreasing maximal activity (close to Vmax) in that order. There was a significant increase in the activity of GS in the liver after specimens were exposed to terrestrial conditions for 144 h. Hence, it would appear that the liver is the major site of endogenous ammonia detoxification in M albus. This contrasts with exposure to environmental ammonia, during which other tissues and organs are involved (Y. K. Ip, S. L. A. Tay, K. H. Lee and S. F. Chew, manuscript submitted).
M. albus does not undergo partial amino acid catabolism when
exposed to land
Certain amino acids (e.g. arginine, glutamine, histidine and proline) can
be converted to glutamate. Glutamate can undergo deamination catalyzed by GDH,
producing NH4+ and -KG
(Campbell, 1991
). The latter is
then fed into the Krebs cycle. Glutamate can also undergo transamination with
pyruvate, catalyzed by ALT, producing
-KG and alanine without the
release of ammonia (Ip et al.,
2001a
,b
;
Chew et al., 2003b
). If there
were a continuous supply of pyruvate, transamination leading to the formation
of alanine would facilitate the oxidation of carbon chains of some amino acids
without polluting the internal environment with ammonia. Available information
indicates that it is a major strategy adopted mainly by fish that are
relatively active on land but not by those that have to deal with ammonia
loading situations. In the case of M. albus exposed to terrestrial
conditions, no accumulation of alanine in the muscle and other tissues was
observed (Tables 2,
3,
4,
5,
6). Also, there was no change
in the activity of ALT in the muscle and liver
(Table 7). Hence, it can be
concluded that alanine formation was not adopted as a strategy to slow down
the release of ammonia under such conditions. In the gut, there was an
increase in ALT activity upon aerial exposure, the meaning of which is not
clear at this moment (Table 7).
Whether M. albus would adopt partial amino acid catabolism to
facilitate the utilization of amino acid as an energy source during locomotion
on land awaits future studies. If it does not adopt such a strategy, then it
is probably related to its possible exposure to environmental ammonia in its
natural habitat.
M. albus reduces the rates of proteolysis and amino acid
catabolism, and hence the rate of ammonia production, during aerial
exposure
In order to slow down the build up of ammonia internally, fish can augment
excretion by decreasing the rate of ammonia production through amino acid
catabolism. The steady-state concentrations of FAAs in tissues depend on the
rates of degradation and production (through proteolysis or digestion),
alteration of which would lead to changes in concentrations of various amino
acids. Being able to alter these rates is a valuable strategy to a fish that
has to endure short periods of water shortage, because it would slow down the
build up of endogenous ammonia.
From the results obtained, a balance sheet on the reduction in nitrogenous
excretion and the increase in nitrogenous accumulation (as ammonia, urea and
glutamine) was constructed for a 200 g specimen of M. albus exposed
to various periods (24 h, 72 h and 144 h) of aerial exposure
(Table 8). After the first 24 h
of aerial exposure, the deficit involved was -1073 + 742 = -331 µmol N.
After 72 h of aerial exposure, the discrepancy between the reduction in
nitrogenous excretion (3204 µmol N) and the retention of nitrogen (2615
µmol N) in the body of a 200 g M. albus became larger: -589
µmol N (Table 8). The
deficit continued to increase with time, reaching -3516 (-5735 + 2219) µmol
N at 144 h (Table 8). It is
therefore logical to deduce that reductions in proteolysis and amino acid
catabolism occurred when M. albus was exposed to long periods of
terrestrial conditions. In this regard, M. albus is different from
O. marmoratus (Jow et al.,
1999) and C. asiatica
(Chew et al., 2003b
) but
similar to M. anguillicaudatus
(Chew et al., 2001
) and B.
sinensis (Ip et al.,
2001b
). O. marmoratus
(Jow et al., 1999
) and C.
asiatica (Chew et al.,
2003b
) are apparently incapable of reducing proteolysis and amino
acid catabolism during aerial exposure.
Reductions in proteolysis and amino acid catabolism constitute an effective strategy to slow down the internal accumulation of ammonia. If the rate of amino acid catabolism decreased while the rate of proteolysis remained unchanged, the steady-state concentrations of FAAs would increase. However, if the rate of proteolysis decreased to a greater extent than the rate of amino acid catabolism, the steady-state levels of FAAs would decrease. In the case of M. albus, it would appear that the rate of proteolysis and the rate of amino acid catabolism were reduced proportionally because aerial exposure exhibited no significant effect on the TFAA content in the muscle, which is the bulk of the fish by mass, and other tissues.
The cells and tissues of M. albus have high ammonia
tolerance
It is surprising that the ammonia concentrations in the tissues of M.
albus built up to very high levels upon aerial exposure, reaching >7
µmol g-1, >14 µmol g-1 and >3 µmol
g-1 in the muscle, liver and brain, respectively
(Fig. 2A-C). For mammals, a
brain ammonia level of >1 µmol g-1 leads to encephalopathy.
In mice, high ammonia levels in the brain induce an increase in extracellular
glutamate, due to increased neuronal release, decreased reuptake or both
(Hilgier et al., 1991;
Rao et al., 1992
;
Bosman et al., 1992
;
Schmidt et al., 1993
;
Felipo et al., 1994
). It has
been proposed that ammonia toxicity is mediated by excessive activation of
N-methyl-D-aspartate (NMDA)-type glutamate receptors in the brain
(Marcaida et al., 1992
),
leading to cerebral ATP depletion
(Marcaida et al., 1992
;
Felipo et al., 1994
) and
increases in intracellular Ca2+, with subsequent increases in
extracellular K+ and cell death. Hermenegildo et al.
(2000
), however, showed that
activation of NMDA receptors preceded the increase in extracellular glutamate.
It had been suggested much earlier that NH4+ can
substitute for K+ and affect the membrane potential in the squid
(Loligo pealei) giant axon
(Binstock and Lecar, 1969
). In
addition, Beaumont et al.
(2000
) reported measured levels
of depolarisation of muscle fibers in trout (Salmo trutta) 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
ammonium ions across the cell membranes. Thus, ammonia toxicity may be due to
membrane depolarization and a rise in extracellular K+ in the
brain, exacerbated by NMDA receptor activation, glutamine-mediated astrocyte
swelling, depletion of Krebs cycle intermediates and disruption of redox
balance. These mechanisms are not mutually exclusive and could be additive in
their effects. Besides M. albus, there are few fish that accumulate
ammonia in their bodies to tolerate aerial exposure, as seen in the Indian
catfishes (Saha and Ratha,
1998
) and the weather loach
(Chew et al., 2001
;
Tsui et al., 2002
). The
ammonia levels are not always evenly distributed within the fish; some exhibit
much higher levels in the muscle compared with the brain whereas others can
tolerate very high levels in the brain. How the cells and tissues of these
animals tolerate these high ammonia levels is not clear at present. It is
possible that they evolved to have special NMDA receptors,
K+-specific channels and K+-specific
Na+/K+-ATPase.
Conclusion
It can be concluded that the major strategies adopted by M. albus
to deal with ammonia toxicity during 144 h of aerial exposure are (1)
tolerance of ammonia at the cellular and subcellular levels, (2)
detoxification of ammonia to glutamine and (3) reduction in ammonia
production. The fact that ammonia and glutamine built up to high levels in the
bodies of the experimental specimens by 144 h indicates that the fish might
have already been stressed to a limit. Hence, it is unlikely that the same
strategies would serve effectively to facilitate those M. albus that
have to burrow into the mud and survive therein for long periods during
drought. How M. albus deals with ammonia toxicity after burrowing
into the mud awaits future studies.
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