Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land
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 24 November 2003
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Summary |
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Key words: aestivation, Protopterus dolloi, lungfish, nitrogen metabolism, ammonia excretion, urea excretion, mucus, cocoon
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Introduction |
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P. aethiopicus and P. annectens are ureogenic (Janssens
and Cohen, 1966,
1968a
;
Mommsen and Walsh, 1989
).
Similar to tetrapods, they possess in their livers mitochondrial carbamoyl
phosphate synthetase I (CPS I), which utilizes NH4+ as a
substrate, and an arginase that is present mainly in the cytosol
(Mommsen and Walsh, 1989
).
During 78129 days of aestivation in a nylon bag designed to replace
mud, P. aethiopicus accumulated urea in its body
(Janssens and Cohen, 1968a
).
However, it was reported that urea accumulation did not involve an increased
rate of urea synthesis (Janssens and
Cohen, 1968a
), even though the animals appeared to be in
continuous gluconeogenesis throughout aestivation
(Janssens and Cohen, 1968b
).
It was proposed that P. aethiopicus could undergo a profound
suppression of ammonia production under such conditions
(Janssens and Cohen, 1968a
).
However, it is difficult to envisage that the suppression of ammonia
production would occur instantly when the fish was out of water. During the
initial phase of aerial exposure, before the onset of a reduction in the rate
of ammonia production, the rate of urea synthesis ought to be increased to
detoxify the ammonia that is produced at a normal (or slightly sub-normal)
rate and retained within the body. To date, no such information is available
on African lungfishes.
It is important to point out that Janssens and Cohen
(1968a; their
table 3) also measured the
rates of incorporation of [14C]bicarbonate into urea during a 60-h
period at the very end of a long period (78129 days) of fasting or
aestivation in P. aethiopicus and obtained comparable results between
these two groups of specimens. These results were regarded as important
evidence in support of the conclusion on the lack of an increase in the rate
of urea synthesis in aestivating P. aethiopicus. However, later
findings have identified two problems with such a conclusion. Firstly,
decreases in metabolic rate in African lungfishes can actually be achieved
through progressive starvation and emaciation
(Fishman et al., 1987
).
Secondly, the lack of increased urea synthesis in specimens undergoing
prolonged aestivation does not necessarily imply that an increased rate of
urea synthesis would not occur during the initial period of (or short-term)
aestivation.
|
Found in Central Africa in the lower and middle Congo River basins is the
slender lungfish, P. dolloi, which can aestivate on land within a
layer of dried mucus (Brien,
1959; Poll, 1961
)
instead of inside a cocoon in the mud like P. aethiopicus and P.
annectens. Like elasmobranchs and some teleosts, P. dolloi
possesses carbamoyl phosphate synthetase III (CPS III), which uses glutamine
as a substrate, in the liver (Chew et al.,
2003
). Glutamine synthetase (GS) activity is present in both the
mitochondrial and cytosolic fractions of the liver of P. dolloi. In
the laboratory, P. dolloi can be induced to aestivate in a layer of
dried mucus in a plastic or glass aquarium containing only 1020 ml of
water in open air. The water would dry up in approximately 34 days, and
the specimen would enter a state of torpor in a layer of dried mucus on day 4
or day 5 (Fig. 1). By renewing
the small amount of water in the container daily to prevent the specimen from
entering into aestivation, Chew et al.
(2003
) indeed verified that
the rate of urea synthesis increased 10-fold in P. dolloi exposed to
air for 6 days. Aerial exposure also led to an increase in the hepatic
ornithine-urea cycle (OUC) capacity (Chew
et al., 2003
), with significant increases in activities of CPS III
(3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more
importantly, GS (2.2-fold), which produces glutamine, the substrate required
for CPS III activity.
|
However, there remain several fundamental questions. Would P. dolloi increase the rate of urea synthesis if it could enter into aestivation (i.e. after day 4) during the 6-day period? More importantly, would it sustain an increased rate of urea synthesis during a longer period (e.g. 40 days) of aestivation? Would there be a reduction in ammonia production, resulting from a reduction in catabolism of certain amino acids, in this lungfish during aestivation, and would the degree of reduction be constant throughout the 40-day period? The present study was therefore undertaken to examine nitrogen metabolism in P. dolloi that underwent a period of 6 or 40 days of aestivation in open air. The contents of ammonia, urea and free amino acids (FAAs) in various tissues and organs of the experimental specimens were determined. In addition, efforts were made to determine the activities of enzymes associated with the OUC in aestivating P. dolloi.
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Materials and methods |
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Determination of contents of ammonia, urea and FAAs in various tissues
Specimens were allowed to enter into a state of aestivation individually in
plastic tanks (29 cmx19 cmx17.5 cm, length x width x
height) containing a thin film of 10 ml dechlorinated tap water. For those
that were allowed to aestivate for 40 days, 12 ml of water was sprayed
on the surface of the brown cocoon covering the body surface every 6 days. A
preliminary experiment indicated that this was essential to reduce the rate of
dehydration of the specimens in laboratory conditions (humidity, 80%),
which are likely to be drier than the natural habitat of P. dolloi.
Another group of fish was placed in dechlorinated tap water at 25°C for
the same period of time (i.e. 6 or 40 days) to serve as controls to evaluate
the effects of fasting alone. At the end of 6 or 40 days, specimens were
killed with a strong blow to the head.
The lateral muscle, liver, gut and brain were quickly excised. No attempt was made to separate the red and white muscle. 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.
It was essential to show that any change in the contents of ammonia, urea
or FAAs in the tissues was not a result of a loss of water due to dehydration.
Since the muscle comprises the bulk of the body, an attempt was made to
determine the water content of muscle samples by estimating the difference in
wet mass and dry mass. The wet mass of the muscle was determined to the
nearest milligram using a Sartorius analytical balance. The dry mass was
determined after the muscle had been dried in an oven at 90°C for 24 h
until it reached constant mass.
The frozen samples were weighed, ground to a powder in liquid nitrogen and homogenized three times in five volumes (w/v) of 6% trichloroacetic acid (TCA) at 24 000 revs min1 for 20 s each using an Ultra-Turrax homogenizer, with intervals of 10 s between each homogenization. The homogenate was centrifuged at 10 000 g at 4°C for 15 min, and the supernatant obtained was kept at 80°C until analysed.
For ammonia analysis, the pH of the deproteinized sample was adjusted to
between 5.5 and 6.0 with 2 mol l1 KHCO3. The
ammonia content was determined using the method of Bergmeyer and Beutler
(1985). The change in
absorbance at 25°C and 340 nm was monitored using a Shimadzu UV-160A
spectrophotometer. Freshly prepared NH4Cl solution was used as the
standard for comparison.
Urea contents in 0.2 ml of the neutralised sample were analyzed
colorimetrically according to the method of Anderson and Little
(1986), as modified by Jow et
al. (1999
). The difference in
absorbance obtained from the sample in the presence and absence of urease
(#U7127; Sigma Chemical Co., St Louis, MO, USA) was used for the estimation of
urea content in the sample. Urea obtained from Sigma Chemical Co. was used as
a standard for comparison. Results were expressed as µmol
g1 wet mass tissue.
For FAA analysis in muscle, liver and brain samples, the supernatant obtained was adjusted to pH 2.2 with 4 mol l1 lithium hydroxide and diluted appropriately with 0.2 mol l1 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 for FAA analyses were expressed as µmol g1 wet mass or µmol ml1 plasma.
Determination of activities of OUC enzymes from the liver of experimental specimens
The liver was homogenized in five volumes (w/v) of ice-cold extraction
buffer containing 50 mmol l1 Hepes (pH 7.6), 50 mmol
l1 KCl and 0.5 mmol l1 EDTA. The
homogenate was sonicated three times for 20 s each, with a 10 s break between
each sonication. The sonicated sample was centrifuged at 10 000
g and 4°C for 15 min. After centrifugation, the
supernatant was passed through a Bio-Rad P-6DG column (Bio-Rad Laboratories;
Hercules, CA, USA) equilibrated with the extraction buffer without EDTA. The
filtrate obtained was used directly for enzyme assay. Preliminary results
indicated that CPS III activity was present only in the liver of P.
dolloi.
CPS (E.C. 2.7.2.5) activity was determined according to the method of
Anderson and Walsh (1995).
Radioactivity was measured using a Wallac 1414 liquid scintillation counter
(Wallac Oy, Turku, Finland). CPS activity was expressed as µmol
[14C]urea formed min1 g1 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 UV 160 UV VIS recording spectrophotometer.
OTC activity was expressed as µmol citrulline formed min1
g1 wet mass.
Argininosuccinate synthetase (ASS; E.C. 6.3.4.5) and lyase (L; E.C.
4.3.2.1) activities were determined together (ASS + L), 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. The ASS + L
activity was expressed as µmol [14C] fumarate formed
min1 g1 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
min1 g1 wet mass.
Glutamine synthetase (GS; E.C. 6.3.1.2) activity was measured according to
the method described by Shankar and Anderson
(1985). The formation of
-glutamylhydroxymate was determined at 500 nm using a Shimadzu UV 160
UV VIS recording spectrophotometer. GS activity was expressed as µmol
-glutamylhydroxymate formed min1 g1
wet mass.
Determination of ammonia and urea excretion rates of specimens
After 48 h of fasting, specimens (N=5) were kept individually in a
plastic tank (20.5 cmx14.5 cmx6 cm, length x width x
height) containing 2 litres of water at 25°C for 24 h (regarded as day 0
control). Preliminary experiments on the analysis of ammonia and urea in the
water sampled at 6 h and 24 h showed that the ammonia and urea excretion rates
were linear up to at least 24 h. Thus, 3 ml of water was sampled for ammonia
and urea analysis after 24 h of exposure. The same individuals were then kept
in plastic tanks containing 10 ml of water, i.e. experimental conditions that
would induce aestivation (see above). At the end of day 3, only a very small
amount of water was left. In order to find out the amounts of ammonia and urea
excreted during this period before the onset of aestivation, a small volume of
water was sprayed on the fish and the side of the tank. The water was then
collected, made up to a known volume and used for ammonia and urea analyses.
Ammonia and urea in water samples were determined according to the methods of
Jow et al. (1999).
A preliminary study was performed to demonstrate that the rates of ammonia and urea excretion were not affected by bacterial actions. Small volumes (200 ml; N=4) of the external medium in which the control fish had been exposed for 24 h were set aside at 25°C. Water samples were collected 24 h later. The concentrations of ammonia and urea before and after this 24-h period of incubation were compared and were confirmed not to be significantly different from each other.
Statistical analyses
Results were presented as means ± S.E.M. Student's
t-test and one-way analysis of variance (ANOVA) followed by
StudentNeumanKeul's multiple range test were used to evaluate
differences between means where applicable. Differences were regarded as
statistically significant at P<0.05.
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Results |
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Effects of aestivation on ammonia levels
There were no significant increases in ammonia content in the muscle,
liver, gut or brain of P. dolloi after 6 days of aestivation compared
with the control fasted for 6 days(Fig.
2). Surprisingly, ammonia levels in the muscle, liver and gut of
specimens aestivated for 40 days were significantly lower than those of the
40-days fasted controls (Fig.
2). The specimens kept in water but fasted for 40 days had
significantly higher ammonia levels in their liver, gut and brain compared
with those fasted for 6 days only.
|
Effects of aestivation on urea levels
Urea contents in the muscle, liver, gut and brain of specimens aestivated
for 6 days were 4.1-, 4.3-, 5.5- and 6.4-fold greater than the corresponding
value of specimens fasted for the same period in water
(Fig. 3). After aestivating for
40 days, the urea contents in the muscle, liver, gut and brain increased by
9.5-, 11.3-, 10.9- and 9.9-fold when compared with the corresponding values of
specimens kept in water and fasted for 40 days. Again, the specimens fasted
for 40 days had significantly higher levels of urea in their tissues than
those fasted for 6 days only (Fig.
3).
|
Effects of aestivation on FAA levels
The content of total FAA (TFAA) remained unchanged in the muscle of P.
dolloi aestivated for 6 or 40 days
(Table 1). There was only a
significant increase in isoleucine content in the muscle of specimens
aestivated for 6 days. On the other hand, fish aestivated for 40 days showed
significant increases in alanine, aspartate and glutamate levels in the
muscle. By contrast, there was a significant decrease in the TFAA content in
the livers of specimens that underwent 40 days of aestivation
(Table 2), which was attributed
mainly to decreases in contents of proline and glutamate. The arginine content
in the liver of P. dolloi aestivated for 6 days was significantly
higher than that of the control fasted for 6 days
(Table 2). However, the hepatic
arginine content became undetectable on day 40 of fasting or aestivation
(Table 2). In addition, there
was a significant decrease in the arginine content in the muscle of these
specimens (Table 1). In the
brain, there was a significantly higher TFAA content in specimens aestivated
for 40 days, which was attributed mainly to an increase in glutamine
(Table 3). Forty days of
aestivation also led to a 57% decrease in the content of tryptophan in the
brain compared with the 40-day fasted control
(Table 3).
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Effects of aestivation on OUC cycle enzymes
The activities of OUC enzymes, inclusive of CPS III, in the liver of P.
dolloi were unaffected by 6 days of aestivation or 40 days of fasting in
water (Table 4). There was a
significant increase in hepatic GS activity in specimens aestivated for 6
days. Forty days of aestivation led to significant increases in the activities
of GS, CPS III, OTC and ASS + L (Table
4).
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Rates of ammonia and urea excretion
The rates of ammonia and urea excretion in specimens (N=5) kept in
water at day 0 were 6.35±0.87 µmol day1
g1 fish and 0.25±0.03 µmol day1
g1 fish, respectively. The averaged rates of ammonia and
urea excretion in the first 3 days of the aestivation period were
0.70±0.11 µmol day1 g1 fish and
0.21±0.02 µmol day1 g1 fish,
respectively.
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Discussion |
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Fasting for 40 days led to significantly higher levels of ammonia in
various tissues and organs of P. dolloi. This is probably due to the
mobilization of protein and amino acids to sustain energy production during
fasting, which led to an increase in the rate of ammonia production. However,
the interesting observation here is that ammonia accumulation occurred in
these fasted animals despite their being kept in water. When NH4Cl
was infused peritoneally into P. dolloi, >80% of the infused
ammonia could be excreted to the external medium within the first 4 h, which
indicates that P. dolloi has high capacity in ammonia excretion (Y.
K. Ip and S. F. Chew, unpublished results), despite its gills being degenerate
(Graham, 1997). Therefore, it
would appear that P. dolloi was regulating the rate of ammonia (and
urea) excretion during fasting with the aim of retaining it. Since fasting had
been proposed as one of the initiating factors of aestivation
(Fishman et al., 1987
), it is
possible that an initial accumulation of ammonia, leading to subsequent urea
synthesis and its accumulation (see below), is essential for initiating those
changes.
Compared with the controls fasted for 40 days, there were significant decreases in concentrations of ammonia in the muscle, liver and gut of specimens aestivated for 40 days. These results suggest that a decrease in the rate of ammonia production had occurred in specimens undergoing aestivation, especially considering the fact that ammonia excretion would have been completely impeded for 36 days.
P. dolloi detoxified endogenous ammonia to urea with increased rates of urea synthesis during aestivation
It has been suggested that the capacity to synthesize urea during periods
of restricted water availability, as demonstrated by African lungfishes, would
have pre-adapted the early vertebrates for their transition to the land
(Campbell, 1973;
Graham, 1997
). Indeed, P.
dolloi detoxified ammonia to urea during 40 days of aestivation on land,
and the excess urea formed was mainly stored in the body. Urea excretion would
not have occurred during aestivation due to a lack of water. Consequently, the
accumulated urea could fulfil a secondary function of facilitating water
retention through a reduction in vapour pressure, since aestivation prescribes
desiccation.
Fasting also led to significant increases in the urea content in the body
of P. dolloi. As mentioned above, this could be due to an increase in
the degradation of protein, leading to increased ammonia production. However,
why was urea retained in the body instead of being excreted despite the fish
being immersed in water? For a long time, it was accepted that urea permeates
biomembranes by diffusion. To date, five urea transporters have been
identified, which aid in urea transport across biomembranes
(Sands et al., 1997). It is
uncertain if urea transporters exist in P. dolloi, but our results
suggest that increased urea production was not accompanied immediately by an
upregulation of urea transport when the fish was fasted in water.
Alternatively, these results may suggest a physiological role of urea in
initiating and/or perpetuating aestivation in P. dolloi, because
fasting is known to affect the metabolic, circulatory and respiratory changes
in ways similar to aestivation in African lungfishes
(Fishman et al., 1987
).
In a submerged specimen, the steady-state level of urea in the body is
maintained through a balance of urea production and urea loss (through
excretion). Therefore, it can be deduced that the rate of urea synthesis in a
submerged P. dolloi (day 0 control) was 0.25 µmol
day1 g1. The amount of urea synthesized in
a 100 g specimen during the 6-day period of aestivation is equal to the
summation of urea excreted in the first 3 days and the urea stored in the
body. The amount excreted was equal to 0.2 µmolx3 daysx100 g=60
µmol. The excess amount of urea accumulated in the body of a 100 g
specimen, which consists of 55 g muscle, 2 g liver, 0.3 gbrain and 3 g gut,
can be calculated (from Fig. 3)
as [(6.431.58) µmol x 55 g+(7.331.71) µmol x 2
g+(8.991.40) µmolx0.3 g+(7.391.34) µmolx3 g],
or 298.91 µmol. This is equivalent to an averaged urea synthesis rate of
(60+298.91) µmol/(100 gx6 days) or 0.598 µmol
day1 g1 during this 6-day period, which is
2.39-fold greater than the value of 0.25 µmol day1
g1 of the day 0 control in water. Chew et al.
(2003) estimated the averaged
urea synthesis rate for P. dolloi exposed to air for 6 days without
undergoing aestivation as 2.21 µmol day1
g1. They concluded that the rate of urea synthesis was
upregulated 8.8-fold in order to detoxify the endogenous ammonia that could
not be excreted as NH3 during aerial exposure. The differences in
results obtained in these two studies suggest that endogenous ammonia
production was suppressed to a much greater extent when P. dolloi
progressively entered the state of aestivation (especially on days 46)
but there was still a substantial increase in the rate of urea synthesis. The
latter was apparently necessary to maintain the ammonia contents in the body
at low levels.
Janssens and Cohen (1968b)
induced P. aethiopicus to aestivate for 78129 days (but the
exact duration was not given) in the laboratory. They reported that the rate
of urea synthesis and the activity of OUC enzymes in these experimental
specimens were comparable with those of the fasted control. Since then, it has
been generally accepted that the accumulation of urea in African lungfishes
during aestivation does not involve an increased rate of urea synthesis
(Graham, 1997
). In the present
study, we have verified that this is not the case for P. dolloi
during the first 6 days of aestivation on land nor is it the case for
specimens aestivated on land for a 40-day period.
For specimens of P. dolloi that underwent aestivation for 40 days, the urea contents in the muscle, liver, brain and gut increased 9.48-, 11.3-, 9.96- and 10.89-fold, respectively, compared with fasted controls. The excess urea accumulated between day 6 and day 40 (a total of 34 days) of aestivation in a 100 g fish amounted to [(60.96.43) µmolx55 g]+ [(777.33) µmolx2 g]+[(61.18.99) µmolx0.3 g]+[(58.37.39) µmolx3 g], or 3303.25 µmol. This would give an averaged rate of 3303.25/(100 gx34 days) or 0.97 µmol day1 g1 for urea synthesis during the latter 34 days of aestivation, which is 3.8-fold greater than the value of 0.25 µmol day1 g1 for the day 0 control in water, and 1.62-fold greater than the rate (0.598 µmol day1 g1) obtained for specimens aestivated for 6 days only.
It might be argued that results would be different if the experiment for
P. dolloi was prolonged to 78 days, as Janssens and Cohen
(1968a) did for P.
aethiopicus. However, that does not seem to be the case, because even if
we made the assumption that absolutely no urea synthesis took place in the
subsequent 38-day period (7840 days), the averaged rate of urea
synthesis for a total period of 38 days + 34 days (or72 days) based simply on
the urea accumulated by day 40 can be calculated as 3303.25 µmol/(100
gx72 days), or 0.46 µmol day1 g1.
This is still 1.8-fold greater than the day 0 control value (0.25 µmol
day1 g1).
The OUC capacity for urea synthesis remained unchanged during the first 6 days of aestivation but increased by day 40
A full complement of OUC enzymes was detected in vitro from the
liver of P. dolloi, suggesting the occurrence of urea synthesis
de novo in this lungfish. Chew et al.
(2003) reported increases in
activities of some OUC enzymes, including CPS III, in the liver of P.
dolloi after 6 days of aerial exposure, which represents the initial
phase that the lungfish has to go through before aestivation occurs. The
maximal level of CPS III activity determined in vitro from the liver
of the day 0 control fish was unable to sustain the rate of urea synthesis
(2.21 µmol day1 g1) during these 6
days. By contrast, no induction of CPS activity was observed in P.
dolloi aestivated for 6 days in this study. The likely reason is that
aestivation led to a greater reduction in the rate of ammonia production,
which eventually exerted a smaller demand on the OUC and could be adequately
handled by the control level of CPS III activity.
For specimens aestivated for 40 days, the activities of GS, CPS III, OTC and ASS + L were significantly greater than the 6 days and 40 days fasted control in water. This is in agreement with the above analysis that the rate of urea synthesis in the latter 34 days was greater than in the first 6 days of aestivation.
P. dolloi suppressed ammonia production during aestivation
Since the ammonia and urea excretion rates of P. dolloi in water
on day 0 were 6.35 µmol day1 g1 and
0.25 µmol day1 g1, respectively, the
total amount of nitrogen excreted theoretically by a 100 g specimen during a
6-day period in water was equal to [6.35+(0.25x2)] µmolx6
daysx100 g, or 4110 µmol N. However, since only 2.1 µmol
g1 and 0.6 µmol g1 of ammonia and urea,
respectively, were excreted in the first 3 days before the external medium
completely dried up, the deficit in nitrogen (N) excretion in a 100 gspecimen
during this period amounts to 4110 µmol [2.1+(0.6x2)]
µmolx100 g=3780 µmol. The excess amount of urea accumulated in the
body of a 100 g specimen was equal to 298.9 µmol, which is equivalent to
298.9x2, or 597.8 µmol N. The deficit of 3780597.8, or 3182.2
µmol N, indicates that a reduction in the rate of production of endogenous
ammonia must have occurred, and this reduction is indeed much greater than
that obtained for specimens exposed to air without undergoing aestivation for
6 days (1060 µmol; Chew et al.,
2003). The deficit of 3182.2 µmol N corresponds to a reduction
of 5.30 µmol day1 g1 in the ammonia
production rate, which is equivalent to 77% (5.30x100/6.85) of the rate
of ammonia + urea production (6.85 µmol N day1
g1) in the day 0 control kept in water.
For the period between 6 and 40 days of aestivation, the reduction in ammonia excretion would theoretically amount to 6.85 µmolx34 daysx100 g, or 23 290 µmol, for a 100 g fish, assuming that the rate of ammonia + urea production remained constant at 6.85 µmol N day1 g1 as in the day 0 control. However, the excess amount of urea accumulated during these 34 days was only 3303.25 µmol, or 6606.5 µmol N. The deficit of 23 2906606.5 µmol N, or 16 683.5 µmol N, in 34 days implies a suppression of 4.91 µmol ammonia day1 g1 or 72% (4.91x100/6.85). This is in close approximation to the value of 77% obtained for the first 6 days of aestivation, indicating that ammonia production remained reduced during the 40-day period. These results confirm the validity of including the first 34 days of aerial exposure in the aestivation period.
Reduction in the rate of amino acid catabolism and changes in arginine and tryptophan contents during aestivation
In specimens aestivated for 40 days, there were significant increases in
alanine, aspartate and glutamate content in the muscle. To slow down the
build-up of ammonia internally (see above), it was necessary to decrease the
rate of amino acid catabolism. The steady-state concentration of amino acids
in the tissues depends on the rates of their degradation and production. In
the case of the experimental subjects in this study, FAAs would be produced
mainly through proteolysis because the specimen was undergoing aestivation
(and fasting simultaneously). Hence, these results support the proposition
that amino acid catabolism, specifically for alanine, aspartate and glutamate,
in the muscle had been suppressed.
In the liver of specimens aestivated for 40 days, there was a significant decrease in the glutamate level. This suggests that glutamate was channelled into glutamine, which acted as a substrate for urea synthesis via CPS III. For specimens aestivated for 6 or 40 days, or those fasted for 40 days,increases in urea synthesis and urea accumulation were accompanied by a significant decrease in the glutamine content in the liver. Again, this is in support of the proposition that hepatic CPS III, which utilizes glutamine as a substrate, was involved in urea synthesis in P. dolloi.
Arginine is a powerful activator of N-acetylglutamate synthetase
(Ka=510 µmol l1), and it
increases the Vmax of the enzyme with no effect on the
Km value for the substrates
(Shigesada and Tatibana,
1978). N-acetylglutamate is the product of the reaction
catalysed by N-acetylglutamate synthetase, and CPS III has an
absolute requirement for it (Campbell and
Anderson, 1991
). The arginine content in the liver of P.
dolloi aestivated for 6 days was significantly higher than that of the
control fasted for 6 days. This is in support of the proposition that urea
synthesis rate increased during this period. By contrast, the hepatic arginine
content became undetectable on day 40 of fasting or aestivation. Therefore,
despite an increase in the OUC capacity in the fish on day 40 of aestivation,
hepatic CPS III activity in vivo might be activated to a lesser
extent after long-term aestivation (i.e.
40 days). These results are also
in agreement with the observations that fasting can initiate physiological and
biochemical changes similar to aestivation
(Fishman et al., 1987
).
Aestivation for 6 or 40 days led to a slight but significant increase in
glutamine content in the brain of P. dolloi, indicating that a small
amount of ammonia was detoxified through glutamine formation. Forty days of
aestivation also led to a 57% decrease in the content of tryptophan, which is
the amino acid precursor of serotonin (5-hydroxytryptamine), a
neurotransmitter in the brain. The rate of serotonin synthesis is normally
restricted by tryptophan availability in mammals
(Boadle-Biber, 1982). In
rainbow trout, increased dietary tryptophan increases brain serotonin levels
(Johnston et al., 1990
),
indicating that the rate of serotonin synthesis can also be dependent upon
tryptophan availability in fish. Interestingly, stress has been reported to
increase brain tryptophan concentrations in mammals
(Neckers and Sze, 1975
;
Dunn, 1988
;
Dunn and Welch, 1991
). In
mice, stress leads to an increase in serotonin release that depletes the
existing serotonin stores (Dunn,
1988
), and Dunn and Welch
(1991
) argued that the
increase in brain tryptophan concentration during stress could counteract this
depletion. From this information, it can be deduced that the decrease in the
concentration of tryptophan in the brain of aestivating P. dolloi may
indicate a decrease in brain serotonin, which can be an important aspect of
the aestivation process in African lungfishes.
A comparative perspective
It was reported that there was no change in the rate of urea synthesis in
African lungfishes (e.g. P. aethiopicus) that aestivate naturally in
a subterranean mud cocoon or artificially in nylon bags
(Janssens and Cohen, 1968a).
Urea synthesis is energy intensive and it is possible that previous
observations made on those African lungfishes were related to a limited energy
supply in hypoxia during aestivation in mud or in an artificial aestivation
apparatus. By contrast, P. dolloi aestivates in a thin layer of dried
mucus on land, where the O2 tension is high. It is probably because
of this that P. dolloi is able to sustain a high rate of urea
production throughout the 40 days of aestivation. Our results indicate that
P. dolloi reduces ammonia production during 40 days of aestivation,
but this is not necessarily equivalent to a reduction in metabolic (catabolic
and anabolic) rate. Whether P. dolloi suppresses its metabolic rate
during aestivation awaits future investigation, but it is obvious from this
study that its rate of urea synthesis (anabolic) is increased during this
period, although it is energy intensive to do so.
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