Urea synthesis in the African lungfish Protopterus dolloi - hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure
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 21 July 2003
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Summary |
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Key words: amino acid, ammonia, arginase, carbamoyl phosphate synthetase, dipnoan, lungfish, glutamine synthetase, ornithine-urea cycle, Protopterus dolloi, urea, urea transporter
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Introduction |
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Unlike their Australian (Neoceratodus forsteri) and South American
(Lepidosiren paradoxa) counterparts, the African lungfish
Protopterus aethiopicus and Protopterus annectens can
aestivate in subterranean mud cocoons for long periods of time
(Smith, 1935; Janssens and
Cohen,
1968a
,b
).
On land, there would be a lack of water to flush the branchial and cutaneous
surfaces, impeding the excretion of ammonia and consequently leading to the
accumulation of ammonia in the body. Ammonia is toxic
(Ip et al., 2001a
), and
therefore African lungfish have to avoid ammonia intoxication when out of
water. Previous work on P. aethiopicus and P. annectens
revealed that they are ureogenic (Janssens
and Cohen, 1966
; Mommsen and
Walsh, 1989
). Similar to tetrapods, they possess mitochondrial
carbamoyl phosphate synthetase I (CPS I), which utilizes
NH4+ as a substrate, and an arginase that is present
mainly in the cytosol of the liver
(Mommsen and Walsh, 1989
). By
contrast, coelacanths, marine elasmobranchs and some teleosts are known to
have carbamoyl phosphate synthetase III (CPS III;
Mommsen and Walsh, 1989
;
Anderson, 1980
;
Randall et al., 1989
), which
utilizes glutamine as a substrate, and arginase in the hepatic mitochondria.
It was suspected that the replacement of CPS III with CPS I, and that of
mitochondrial arginase with cytosolic arginase, occurred before the evolution
of the lungfish (Mommsen and Walsh,
1989
).
Found in Central Africa in the lower and middle Congo River Basins, the
slender lungfish Protopterus dolloi aestivates on land within a dry
layer of mucus (Brien, 1959;
Poll, 1961
) instead of in a
cocoon inside the mud like P. aethiopicus and P. annectens.
It is likely that African lungfish evolved through a sequence of events, i.e.
air breathing, migration to land and then burrowing into mud. Aestivation
could occur on land or in mud, but the latter must have certain advantages,
such as predator avoidance, over the former. This led us to suspect that
burrowing into the mud could be a more advanced development during evolution
and to hypothesize that P. dolloi might be a more-primitive extant
lungfish that bore some of the characteristics and traits of its piscine
ancestors. Therefore, the first objective of this study was to determine what
type of CPS was present in the liver and to elucidate the compartmentation of
hepatic arginase in P. dolloi. Furthermore, it has been reported that
CPS III is present in extra-hepatic tissues of some teleosts (see review by
Anderson, 2001
), but no such
information is available for lungfish. Hence, attempts were made to examine
whether the muscle or gut of P. dolloi would also have CPS I or CPS
III activity.
To date, no glutamine synthetase (GS) activity has been detected in the
liver of African lungfish (Campbell and
Anderson, 1991), probably because the African lungfish examined
possess CPS I and not CPS III. If indeed P. dolloi possessed CPS III,
then it would be essential for it to have GS in the hepatic mitochondria to
supply the glutamine needed for urea synthesis de novo. Hence, the
second objective of this study was to verify whether GS activity was present
in the liver of P. dolloi.
During 78-129 days of aestivation out of water, P. aethiopicus
accumulates 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 appear to be in continuous gluconeogenesis throughout
aestivation (Janssens and Cohen,
1968b
). This apparent controversy arose because of two
counteracting factors: (1) increase in the rate of urea production and (2)
decrease in the rate of ammonia production. During the initial phase of aerial
exposure before the onset of a reduction in the rate of ammonia production,
the rate of urea synthesis de novo theoretically has to be increased
to detoxify ammonia that is produced at a normal (or slightly sub-normal) rate
and cannot be excreted. After entering into aestivation for a relatively long
period, ammonia production rate would have been suppressed
(Smith, 1935
;
Janssens, 1964
). This would
subsequently result in a decrease in the rate of urea synthesis de
novo, leading to those observations made in previous studies (Janssens
and Cohen,
1968a
,b
).
Therefore, the third objective of the present study was to elucidate whether
there was actually a large increase in the rate of urea production in P.
dolloi and whether increases in the hepatic CPS III and GS activities
would occur during 6 days of aerial exposure without undergoing
aestivation.
Detoxification of ammonia to urea does not appear to be a common strategy
adopted by adult teleosts exposed to air, probably because it is energy
intensive, having a stoichiometry of 4 moles (via CPS I) to 5 moles
(via CPS III) of ATP per mole of urea
(Ip et al., 2001a). Instead,
several tropical teleosts accumulate alanine [e.g. mudskipper
(Periophthalmodon schlosseri), Ip
et al., 2001c
; snakehead (Channa asiatica),
Chew et al., 2003
] and
glutamine [e.g. marble goby (Oxyeleotris marmoratus),
Jow et al., 1999
; sleeper
(Bostrichthyes sinensis), Ip et
al., 2001b
; mangrove killifish (Rivulus marmoratus),
Frick and Wright, 2002
; swamp
eel (Monopterus albus), Tay et
al., 2003
]. The formation of glutamine from glutamate (only 1 mole
of ATP per mole of ammonia detoxified) is also energy dependent and appears to
be adopted mainly by teleosts that remain completely quiescent on land. By
contrast, alanine formation through the partial catabolism of certain amino
acids would lead to the production of ATP, which can support locomotory
activities on land (Ip et al.,
2001b
) without releasing ammonia
(Ip et al., 2001a
). Unlike
amphibious teleosts (e.g. P. schlosseri), P. dolloi is
completely inactive on land. Therefore, we hypothesized that P.
dolloi would not adopt the strategy of partial amino acid catabolism and
would not accumulate alanine when exposed to air. Also, we hypothesized that
it would not accumulate glutamine during aerial exposure, because glutamine is
channelled into the OUC for urea synthesis via CPS III.
It has been suggested that suppression of proteolysis and/or amino acid
catabolism may be a fundamental strategy adopted by some tropical teleost fish
to decrease endogenous ammonia production during aerial exposure
(Lim et al., 2001; Ip et al.,
2001b
,c
;
Chew et al., 2001
). Since
P. dolloi can tolerate aerial exposure better than any teleost, the
fourth objective of this study was to verify that P. dolloi could
indeed suppress ammonia production on land even before aestivation took
place.
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Materials and methods |
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Verification of the presence of CPS III and GS
The liver, muscle and gut of P. dolloi in the control condition
were excised quickly and homogenized in 5 volumes (w/v) of ice-cold extraction
buffer containing 50 mmol l-1 Hepes (pH 7.6), 50 mmol
l-1 KCl and 0.5 mmol l-1 EDTA. The homogenate was
sonicated (110 W, 20 kHz; Misonix Incorporated, Farmingdale, NY, USA) 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 OUC enzymes were present only
in the liver of P. dolloi. For comparison, the livers excised from
the marine blue-spotted fan-tailed ray Taeniura lymma (obtained from
the local wet market) and the mouse Mus musculus (obtained through
the Animal Holding Unit of the National University of Singapore) were
processed at the same time with those from P. dolloi and the CPS
activities assayed by the same batch of chemicals.
CPS (E.C. 2.7.2.5) activity was determined according to the method of
Anderson and Walsh (1995), as
applied to the mudskipper (Lim et al.,
2001
). Radioactivity was measured using a Wallac 1414 liquid
scintillation counter (Wallac Oy, Turku, Finland). Enzyme activity was
expressed as µmol [14C]urea formed min-1
g-1 wet mass.
Ornithine transcarbamoylase (OTC; E.C. 2.1.3.3) activity was determined by
combining the methods of Anderson and Walsh
(1995) and Xiong and Anderson
(1989
). Absorbance was
measured at 466 nm using a Shimadzu 160 UV VIS recording spectrophotometer
(Shimadzu Co., Kyoto, Japan). Enzyme activity was expressed as µmol
citrulline formed min-1 g-1 wet mass.
Argininosuccinate synthetase (E.C. 6.3.4.5) and lyase (E.C. 4.3.2.1) (ASS +
L) activities were determined together, assuming that both were present, by
measuring the formation of [14C]fumarate from
[14C]aspartate using the method of Cao et al.
(1991). Radioactivity was
measured using a Wallac 1414 liquid scintillation counter. ASS + L activity
was expressed as µmol [14C]fumarate formed min-1
g-1 wet mass.
Arginase (E.C. 3.5.3.1) was assayed as described by Felskie et al.
(1998). Urea was determined as
described above. Arginase activity was expressed as µmol urea formed
min-1 g-1 wet mass.
GS (E.C. 6.3.1.2) was assayed as transferase activity according to the
method of Shankar and Anderson
(1985). Its activity was
expressed as µmol
-glutamylhydroxymate formed min-1
g-1 wet mass.
Cellular fractionation of liver from P. dolloi was performed
according to the methods of Anderson et al.
(2002). Lactate dehydrogenase
and cytochrome c oxidase were used as markers for cytosol and
mitochondria, respectively.
Evaluation of the effects of 6 days aerial exposure on nitrogenous
excretion and accumulation
Specimens were immersed individually in 2 litres of water at 25°C with
aeration in separate plastic tanks (20.5 cmx14.5 cmx6 cm, length
x width x height). 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. Subsequently, 3 ml
of 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. The process
was repeated for 6 days. The disturbance created by the daily collection and
introduction of water prevented the experimental subject from initiating
aestivation during this period. After 6 days of aerial exposure, specimens
were reimmersed in water for 24 h to study the rates of ammonia and urea
excretion upon recovery. A separate group of fish submerged in water for the
same period of time served as the control. 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) 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.
At the end of 6 days, specimens were killed with a strong blow to the head. The lateral muscle and liver were quickly excised. The excised tissues and organs were immediately freeze-clamped in liquid nitrogen with pre-cooled tongs. Frozen samples were kept at -80°C. A separate group of fish exposed to similar conditions was used for the collection of blood samples. The blood was collected in heparinized capillary tubes by caudal puncture. The collected blood was centrifuged at 4000 g at 4°C for 10 min to obtain the plasma. The plasma was deproteinized in 2 volumes (v/v) of ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 10 000 g at 4°C for 15 min. The resulting supernatant was kept at -80°C until analyzed.
The frozen samples were weighed, ground to a powder in liquid nitrogen and homogenized three times in 5 volumes (w/v) of 6% TCA at 24 000 revs min-1 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 analyzed.
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 using the method of Bergmeyer and Beutler
(1985). The reaction mixture,
in a total volume of 1.55 ml, consisted of 115 mmol l-1
triethanolamine-HCl (pH 8.0), 11 mmol l-1
-ketoglutarate,
0.56 mmol l-1 ADP, 0.19 mmol l-1 reduced NADH, 7.4 i.u.
ml-1 glutamate dehydrogenase (Sigma Chemical Co., St Louis, MO,
USA) and an aliquot part of sample. Glutamate dehydrogenase was added last to
initiate the reaction. 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.
The urea content in 0.2 ml of the neutralized sample was 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 was
used for the estimation of urea content. Urea (Sigma Chemical Co.) was used as
a standard for comparison. Results were expressed as µmol g-1
wet mass tissue or mmol l-1 plasma.
For FAA analysis, the supernatant obtained was adjusted to pH 2.2 with 4 mol l-1 lithium hydroxide and diluted appropriately with 0.2 mol l-1 lithium citrate buffer (pH 2.2). FAAs were analyzed using a Shimadzu LC-6A amino acid analysis system with a Shim-pack ISC-07/S1504 Li-type column. Results for FAA analyses were expressed as µmol g-1 wet mass or mmol l-1 plasma.
Elucidation of whether the OUC capacity would be enhanced by aerial
exposure
Specimens were exposed to the control (immersed) or terrestrial conditions
individually in plastic aquaria as described above. OUC enzyme and GS activity
in the liver was assayed according to the above-mentioned methods.
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 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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Only 0.8% of lactate dehydrogenase activity was present in the mitochondrial fraction, indicating that mitochondria were quite free of cytosolic components. Eighty-five percent of the cytochrome c oxidase activity was present in the mitochondrial fraction, with 12% present in the nuclear fraction, indicating that the mitochondrial fraction represented a high percentage of the mitochondrial enzymes. CPS III was present exclusively in the liver mitochondria, but 90.8±4.6% (N=5) of the arginase was present in the cytosol. GS activity was detected in both the mitochondrial (0.042±0.006 µmol min-1 mg-1 protein; N=4) and cytosolic (0.018±0.003 µmol min-1 mg-1 protein; N=4) fractions from the liver of P. dolloi.
Aerial exposure led to significant increases in the activities of CPS III (3.8-fold), ASS + L (1.8-fold) and GS (2.2-fold) in P. dolloi (Table 1).
Rates of ammonia and urea excretion
Aerial exposure significantly decreased the rate of ammonia excretion in
P. dolloi (Fig. 1).
During the 6 days of aerial exposure, the rate of ammonia excretion was
approximately 8-16% of the control (immersed) value. Upon reimmersion, the
ammonia excretion rate was still significantly lower than that of the immersed
control (Fig. 1). The urea
excretion rate was not affected during the first 3 days of aerial exposure
(Fig. 2). However, there was a
3-fold and 2.8-fold increase in the rate of urea excretion on day 4 and day 5,
respectively. Upon re-immersion after 6 days of aerial exposure, there was a
22-fold increase in the urea excretion rate
(Fig. 2).
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Ammonia and urea content in the tissues
There was no significant change in the ammonia content in the muscle, liver
or plasma of P. dolloi exposed to terrestrial conditions for 6 days
(Table 3). However, the urea
content in the muscle, liver and plasma increased by 8-, 10.5- and 12.6-fold,
respectively (Table 3).
|
FAAs in the tissues
There was no significant change in the contents of FAAs and total FFA
(TFAA) in the muscle of fish exposed to terrestrial conditions for 6 days
(Table 4). However, there were
significant decreases in the glutamate, glutamine and lysine levels in the
liver of these experimental specimens
(Table 5). In addition, the
TFAA in the liver decreased significantly. There were slight increases in the
concentrations of leucine and tryptophan, but a slight decrease in the
concentration of threonine, in the plasma
(Table 6). Six days of aerial
exposure had no significant effect on the concentration of TFAA in the plasma
of P. dolloi.
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Discussion |
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Urea synthesis in P. dolloi involved mainly the OUC, as indicated
by the presence of the full complement of OUC enzymes in the liver. Unlike
P. aethiopicus and P. annectens, which possess CPS I
(Janssens and Cohen, 1966;
Mommsen and Walsh, 1989
), the
CPS activity from the liver of P. dolloi had characteristics
comparable with that of T. lymma but different from that of M.
musculus. Thus, P. dolloi evidently possesses CPS III, which is
known to be present in coelacanths, marine elasmobranchs and some teleosts
(Mommsen and Walsh, 1989
;
Anderson, 1980
;
Randall et al., 1989
) but not
in lungfish (Janssens and Cohen,
1966
; Mommsen and Walsh,
1989
). Cellular fractionation studies revealed that CPS III was
present exclusively in the mitochondria. However, similar to other African
lungfish and amphibians (Mommsen and
Walsh, 1989
), the majority of arginase was present in the cytosol.
To date, there is no report on the presence of GS activity in the livers of
African lungfish. However, GS activity, which is essential to the supply of
glutamine for the reaction catalyzed by CPS III, was detected in both the
mitochondrial and cytosolic fractions from the liver of P. dolloi,
with the specific activity in the former greater than that in the latter.
Taken together, these results indicate that the evolution of CPS from type
III in fish to type I in tetrapods occurred within the Sarcopterygii,
specifically within dipnoans. Aerial exposure could be an important factor
leading to the substitution of NH4+ for glutamine as the
substrate for CPS during evolution. These results also suggest P.
dolloi as the more-primitive extant African lungfish, which is
intermediate between aquatic fish (having mitochondrial CPS III and GS) and
terrestrial tetrapods (having cytosolic arginase). Other extant African
lungfish are likely to be more advanced (having CPS I and no detectable GS)
and evolved later to aestivate in subterranean mud cocoons instead. An
analysis of the relatedness of mitochondrial DNA in the coelacanth, lungfish
and tetrapods (Zardoya and Meyer,
1996) supports the hypothesis that lungfish are the closest living
relatives of terrestrial vertebrates. However, a re-analysis of the data led
Rasmussen et al. (1998
) to
conclude that lungfish occupy a basal position among gnathostome fish as the
sister-group to all other bony fish. Our results obtained from P.
dolloi indeed support the proposition made by Rasmussen et al.
(1998
).
Ammonia excretion was impeded on land but did not lead to its
accumulation in the body
In terrestrial conditions, no water current is available to take away the
excreted ammonia from the gills; the partial pressure of NH3
(PNH3) increases quickly in the boundary layer, leading to
the reduction of the blood-to-boundary-water NH3 gradient. Thus,
branchial ammonia excretion by diffusion is repressed. Although the role of
the branchial epithelium in NH3 excretion in P. dolloi is
unclear, the ammonia excretion rate in specimens exposed to terrestrial
conditions decreased significantly. Theoretically, this would imply that
ammonia was accumulated in these experimental specimens. However, there was no
change in the ammonia content in the muscle, liver and plasma of these
specimens after 6 days of aerial exposure. This is an extraordinary adaptation
exhibited by P. dolloi, which apparently cannot be surpassed by any
other teleosts (see review by Ip et al.,
2001a; Chew et al., in
press
).
Ammonia was detoxified to urea during 6 days of exposure to
terrestrial conditions
It has been established that P. aethiopicus and P.
annectens have a full complement of OUC enzymes in the liver and are able
to synthesize urea from ammonia and bicarbonate in vitro
(Janssens and Cohen, 1968a;
Mommsen and Walsh, 1989
). In
addition, it has been suggested that the capacity to synthesize urea during
periods of restricted water availability, as demonstrated by African lungfish,
would have pre-adapted the early vertebrates for their transition to land
(Campbell, 1973
;
Graham, 1997
). Indeed, P.
dolloi detoxified ammonia to urea during aerial exposure, and the excess
urea was mainly stored in the body. There was only a slight increase in urea
excretion in specimens exposed to terrestrial conditions for
3 days.
Although no information on the role of the kidneys of lungfish in urea
excretion is available at present, it would be impractical for P.
dolloi to excrete urea through its kidney due to the lack of water. Urea
excretion might take place across the branchial/opercular epithelium or the
skin; but without water to flush away the excreted urea, the excretion process
would not be effective to compensate for the increased rate of urea
production. Consequently, urea accumulates in the body and serves the
secondary function of facilitating water retention through vapour pressure
depression during desiccation.
Ammonia production was suppressed during aerial exposure
The deficit in ammonia excretion in a 100 g specimen during the 6 days of
aerial exposure amounted to [(6.4-0.7)+
(6.8-0.6)+(6.0-0.7)+(4.9-0.8)+(5-0.7)+(5.7-0.7)] µmol g-1x
100 g, or 3060 µmoles. From Table
3, the excess amount of urea accumulated in the body of a 100 g
specimen, which contains approximately 55 g muscle, 2 g liver and 1 ml plasma
(Y.K.I. and S.F.C., unpublished data), can be calculated as [(18.6-2.25)
µmol g-1x55 g]+[(19.2-1.83) µmol g-1x2
g]+ [(32.7-2.58) mmol l-1x1 ml], or 964 µmoles. Since
there are two moles of N in one mole of urea, this is equivalent to
964x2=1928 µmoles of ammonia. The deficit (3060-1928=1132 µmoles)
indicates indirectly the occurrence of a reduction in the rate of endogenous
ammonia production in these experimental animals.
This proposition is further supported by the fact that the ammonia excretion rate of specimens re-immersed in water after 6 days of aerial exposure remained low. If we take the rate of ammonia production in an immersed specimen (control) to be the summation of the rate of ammonia and urea excretion (from Figs 1, 2), this amounts to [6.3+(0.27x2)], or 6.84 µmol N day-1 g-1. During the 24-h of subsequent reimmersion, the rate of ammonia production is equal to the summation of the rate of ammonia excretion (from Fig. 1) and the normal rate of urea excretion (from Fig. 2), assuming that the increased rate of urea synthesis (see below) had returned to a normal level, which amounts to [1+(0.27x2)], or 1.54 µmol N day-1 g-1. This estimated rate of ammonia production during the 24 h of re-immersion is only 22% of the control value.
The rate of urea synthesis increased >8-fold and the OUC capacity
was enhanced during aerial exposure
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). Hence, it can be deduced that the rate of urea synthesis in a
submerged P. dolloi was approximately 0.25 µmol day-1
g-1. The amount of urea synthesized in a 100 g specimen during the
6-day period of aerial exposure is equal to the summation of urea excreted and
stored, or [(0.25+0.4+0.5+0.9+0.8+0.45) µmol g-1x100 g]+
964 µmol=1294 µmoles. This is equivalent to a rate of 1294
µmoles/(100 gx6 days), or 2.16 µmol day-1
g-1. In other words, in order to detoxify the endogenous ammonia,
which could not be excreted as NH3 during aerial exposure, the rate
of urea synthesis was upregulated 8.6-fold. The normal rate of urea synthesis
(0.25 µmol day-1 g-1) was definitely inadequate to
detoxify the amount of ammonia formed, even after a suppression of the rate of
ammonia production to 0.7-1.0 µmol day-1 g-1 by day 6
of the experimental period.
Janssens and Cohen (1968a)
induced P. aethiopicus to aestivation for 78-129 days 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
unfed control. Since then, it has been a general belief that the accumulation
of urea in African lungfish during aestivation does not involve an increased
rate of urea synthesis (Graham,
1997
). In the present study, we confirmed that this was not the
case for P. dolloi during 6 days of aerial exposure. Aerial exposure
is a phase that P. dolloi (and presumably also other African
lungfish) has to naturally go through before aestivation. Presumably, during
aestivation, ammonia production decreases further (<0.7 µmol
day-1 g-1) so that the normal rate of urea synthesis (as
in the submerged control) can adequately detoxify the ammonia produced,
thereby preventing ammonia from reaching toxic levels. Indeed, 6 days of
aerial exposure without aestivation led to significant increases in the
activities of GS (2.2-fold), CPS III (3.8-fold) and ASS + L (1.8-fold) in
P. dolloi (Table 1).
Previous work on aestivating P. aethiopicus (Janssens and Cohen,
1968a
,b
)
revealed a rate of urea synthesis comparable with that of the immersed control
because the aestivating specimen had entered a profound metabolic rate
reduction. Hence, this is the first report on the induction of OUC enzyme
activities in the liver of an African lungfish.
The rate of urea excretion increased 22-fold during subsequent
recovery in freshwater
Despite the lack of capability to excrete urea on land, the rate of urea
excretion in P. dolloi increased 22-fold, which is probably the
greatest increase known amongst fish, upon reimmersion. This suggests that,
unlike marine elasmobranchs (Fines et al.,
2001) and coelacanths (Yancey,
2001
), which retain urea for osmoregulation, P. dolloi
possessed transporters to facilitate urea excretion in freshwater, as observed
in some teleosts [e.g. gulf toadfish (Opsanus beta),
Wood et al., 1995
;
Walsh et al., 2000
; Lake
Magadi tilapia (Alcolakia grahami),
Wood et al., 1994
;
Walsh et al., 2001
]. It was
known for a long time that the amphibian kidney can secrete urea actively
(Balinsky, 1970
). In addition,
phloretin-sensitive and/or sodium-independent active urea transporters have
been reported in the skin of several amphibians (see
Sands et al., 1997
for a
review). At present, the nature of these transporters and their location in
the body tissues of P. dolloi is not clear, and therefore P.
dolloi appears to be an ideal specimen for future studies on the
regulation of urea transport.
Aerial exposure affected the contents of FAAs and TFAA in the
liver
There were significant decreases in the TFAA content in the liver of
specimens exposed to terrestrial conditions for 6 days. To slow down the
build-up of ammonia internally (see above), it would be necessary to decrease
the rate of amino acid catabolism. The steady-state concentrations of amino
acids in the tissues depend on the rates of their degradation and production.
In the case of the experimental subjects in this study, amino acids would be
produced mainly through proteolysis because food was withdrawn 48 h before,
and during, experiments. Under such conditions, it is logical to assume that
the rate of protein degradation was higher than the rate of protein synthesis,
which led to a net proteolysis. If the rate of proteolysis remained relatively
constant and was unaffected by aerial exposure, there would be accumulations
of FAAs, leading to an increase in the internal TFAA content. Therefore, the
decrease in TFAA content in the liver of P. dolloi exposed to air
indicates that simultaneous decreases in the rates of proteolysis and amino
acid catabolism would have occurred. Furthermore, the decrease in proteolytic
rate must be greater than the decrease in the rate of amino acid catabolism,
subsequently leading to decreases in the steady-state concentrations of some
FAAs and, consequently, lowering the TFAA concentration.
Despite the 2.2-fold increase in GS activity in the liver, there was a significant decrease in the hepatic glutamine content in specimens exposed to air for 6 days. This suggests that the excess glutamine formed was completely channelled into urea synthesis via CPS III. Simultaneously, there was a significant decrease in the content of glutamate in the liver of these specimens, indicating that the utilization of glutamate for glutamine formation out-paced the formation of glutamate through glutamate dehydrogenase or its release via proteolysis. This would suggest that the decrease in the rate of ammonia production was achieved through the regulation of hepatic glutamate dehydrogenase activity in P. dolloi during aerial exposure.
Ip et al. (2001c) reported
that the mudskipper P. schlosseri was capable of using certain amino
acids as a metabolic fuel and avoided ammonia toxicity through partial amino
acid catabolism during an excursion on land. However, a similar phenomenon was
not observed in P. dolloi. In contrast to the mudskipper, the
pectoral and pelvic fins of P. dolloi are filamentous and incapable
of sustaining locomotion on land. On land, P. dolloi remains
quiescent and is relatively motionless. It is probably because of this that
partial amino acid catabolism was not adopted as a strategy by P.
dolloi to survive aerial exposure.
Conclusion
P. dolloi possesses GS and CPS III in the liver, and not CPS I as
has been shown previously in other African lungfish. Hence, in this regard,
P. dolloi is a more-primitive extant African lungfish intermediate
between aquatic fish and terrestrial tetrapods. Six days of exposure to
terrestrial conditions without aestivation led to an increase in urea content
in the body, accompanied by an increase in hepatic OUC capacity, in P
dolloi. The accumulated urea was released to the external medium during
the subsequent 24 h of re-immersion, during which the rate of urea excretion
increased 22-fold.
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