Exposure to air, but not seawater, increases the glutamine content and the glutamine synthetase activity in the marsh clam Polymesoda expansa
1 Department of Biological Sciences, National University of Singapore, 10
Kent Ridge Road, Singapore 117543, Republic of Singapore
2 Natural Sciences and Science Education, 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 5 October 2004
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Summary |
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Key words: evolution, alanine, glutamine, glutamine synthetase, clam, Polymesoda expansa
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Introduction |
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When exposed to air, lower intertidal and subtidal bivalves usually close
their shells and shift to anaerobic metabolic pathways
(Widdows et al., 1979;
de Zwaan, 1983
). In contrast,
P. expansa gapes and slightly exposes the mantle margins posteriorly
during aerial exposure (Morton,
1988
). Adduction of the valves immediately following emersion and
at periodic intervals helps to ventilate the mantle cavity
(McMahon, 1988
), and gas
exchange occurs largely across the surface of the mantle. Such an adaptation
sustains aerobic metabolism in the tissues of Polymesoda spp. during
long periods of aerial exposure. Thus, P. caroliniana is capable of
the same rate of O2 uptake whether in air or submerged in seawater
(Deaton, 1991
). The
air-breathing capacity of P. caroliniana is exceptional among bivalve
species found in high intertidal habitats; consequently, there is no marked
bradycardia and no evidence of accumulation of metabolites indicative of
anaerobic metabolism in this marsh clam during emersion
(Deaton, 1991
).
Although P. expansa is not confronted with hypoxia during
long-term aerial exposure, the mobilization of proteins and amino acids for
energy supply would lead to the release of ammonia
(Bishop et al., 1983). Indeed,
ammonia accumulates in the haemolymph and mantle cavity fluid of Mytilus
edulis exposed to air (Shick et al.,
1988
). Since ammonia is toxic, `terrestrial' bivalves like P.
expansa have to defend themselves against ammonia toxicity during
long-term emersion. Therefore, this study was undertaken to determine the
effects of aerial exposure (17 days) on nitrogen metabolism in P.
expansa. In order to confirm that the effects and phenomena observed
related specifically to aerial exposure, we also determined the effects of
salinity changes (from 10
to 30
) on this marsh clam for direct
comparison.
Ammonia is detoxified to urea as an excretory product in many vertebrates,
including a few fish species, the majority of amphibians, some reptiles and
all mammals (Campbell, 1973).
The synthesis of excretory urea in certain land planaria
(Campbell, 1965
), earthworms
(Bishop and Campbell, 1963
,
1965
) and snails
(Campbell and Bishop, 1970
;
Campbell and Speeg, 1968
;
Tramell and Campbell, 1972
)
via the arginineornithineurea cycle (OUC) indicates
that the modification of the basic nutritional pathway of arginine synthesis
for ammonia detoxification first took place among invertebrate animals
(Campbell, 1973
). However,
Andrews and Reid (1972
) were
unable to detect activities of carbamoyl phosphate synthetase III (CPS III), a
crucial enzyme of the OUC, in the tissues of several bivalve species
(Mytilus californianus, Anodonta kennerlgi, Saxidomus giganteus and
Compsomyax subdiaphana). At present, no bivalve is known to possess a
functional OUC (Bishop et al.,
1983
). Thus, the hypothesis tested in this study was that, like
other bivalves, CPS III was absent from the tissues of P. expansa,
and that this marsh clam was incapable of detoxifying ammonia to urea during
aerial exposure despite the harsh environment conditions in its natural
habitat.
In mammalian brains, ammonia toxicity can be ameliorated transiently
through the action of glutamine synthetase (GS), resulting in the synthesis of
glutamine. The accumulation of glutamine consequently leads to astrocyte
swelling and brain damage (Brusilow,
2002). The capacity for detoxification of ammonia to glutamine in
higher vertebrates can be traced back to fish. Several species of tropical
air-breathing fishes can detoxify ammonia to glutamine not only in the brain,
but also in liver, muscle, stomach and gut, during exposure to terrestrial
conditions or environmental ammonia (Peng
et al., 1998
; Jow et al.,
1999
; Ip et al.,
2001b
,
2004a
;
Chew et al., 2001
;
Anderson et al., 2002
;
Tay et al., 2003
;
Lim et al., 2004
). In
contrast, glutamine is not known to be accumulated as a product of ammonia
detoxification in tissues of invertebrates, although it functions as a minor
osmolyte in certain species (Livingstone,
1985
). Tracer studies using 14C-glucose with clams,
mussels, oysters, and pulmonate and prosobranch snails indicate that all these
molluscs have a strong capacity for rapid biosynthesis of glutamate, alanine
and aspartate, but the capacity for glutamine biosynthesis is weak or
nonexistent (see review by Bishop et al.,
1983
). Glutamine synthesis is energy dependent; one mole of ATP is
hydrolysed for each mole of amide-N formed. To date, no bivalve is known to
accumulate glutamine during emersion, because bivalves usually shift to
anaerobic energy metabolism and accumulate alanine, and sometimes alanopine,
during aerial exposure (Bishop et al.,
1983
).
P. expansa is capable of withstanding aerial exposure without undergoing anaerobiosis or a reduction in metabolic rate, so it is an ideal specimen to study whether the capacity for the detoxification of ammonia to glutamine first evolved among invertebrates. Therefore, in this study, we aimed to verify that glutamine accumulation occurred in association with increased ammonia levels in clams exposed to terrestrial conditions but not in those exposed to seawater, when ammonia could be excreted freely. We also aimed to demonstrate that glutamine accumulation occurred in association with an upregulation of GS activities in tissues of clams exposed to air.
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Materials and methods |
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Determination of wet mass of tissues
The mass of a clam (with shells) was obtained using a Libror EB-280M
balance (Shimadzu, Kyoto, Japan) to the nearest 0.01 g. It was then forced
open to dissect out the hepatopancreas, adductor muscle, foot muscle and
mantle. Results obtained from individual tissues and organs were expressed as
percentage of total clam mass (with shells). To determine the water content in
tissue samples, the wet masses were recorded to the nearest 0.001 g. They were
then dried in an oven at 95°C until constant mass and the dry mass
recorded. The water content of a sample was calculated as differences between
the wet mass and the dry mass, and expressed as percent of wet mass
tissue.
Experimental conditions
For exposure to seawater, groups of 10 clams were transferred from brackish
water directly to full strength seawater (30) in glass aquaria (43
cmx28 cmx30 cm, LxWxH). They were kept in seawater for
17 days, during which no food was supplied and water was changed daily. For
aerial exposure, groups of 10 clams were exposed to terrestrial conditions in
an uncovered dry glass aquarium for 17 days (8790% humidity). Clams
kept in brackish water and fasted for the same period served as controls. On
day 17, clams were forced open and the haemolymph was collected from the foot
muscle with a syringe and needle. After that, the adductor muscle was cut. The
hepatopancreas, mantle, adductor muscle and foot muscle were dissected and
freeze-clamped with liquid nitrogen-precooled aluminium tongs. Haemolymph
samples were deproteinized in two volumes of trichloroacetic acid (TCA) and
then centrifuged at 4000 g for 15 min to obtain the
supernatant. Samples collected were kept at 80°C until
analysis.
Determination of ammonia and urea excretion rates
To determine the rates of ammonia and urea excretion in P. expansa
in water, clams were weighed and kept individually in 120 ml of brackish water
(10) or seawater (30
) with slight aeration in cylindrical
containers (7.5 cmx8.5 cm, DxH), and the external media were
changed daily. On days 3, 6, 9, 12 and 17, water samples (2 ml) were collected
and acidified with 20 µl of 2 mol l1 HCl for ammonia and
urea analyses. Samples were kept at 4°C and analyses were done within a
week. Ammonia was determined according to the method of Anderson and Little
(1986
). Urea content was
analyzed as described by Jow et al.
(1999
). Rates of excretion are
presented as µmol day1 g1 clam (with
shell). No attempt was made to determine the rates of ammonia and urea
excretion in clams exposed to air. Although certain amounts of ammonia and
urea could be excreted into the mantle cavity fluid during aerial exposure,
the fluid was kept within the animal and was in direct contact with various
tissues.
Enzyme assays
The hepatopancreas, mantle, adductor muscle and foot muscle were
homogenized three times in 5 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, using an
Ultra Turrax (Janke and Kundel, Staufeni, Staufen, Germany) homogeniser at 24
000 revs min1 for 20 s each separated by intervals of 10 s
off. The homogenate was centrifuged at 10 000 g and 4°C
for 15 min to obtain the supernatant, which was subsequently passed through a
10 ml Bio-Rad P-6DG column (Bio-Rad Laboratories, Hercules, CA, USA)
equilibrated with ice-cold extraction buffer without EDTA. The filtrate was
used directly for enzyme assay.
CPS III (E.C. 2.7.2.5) activity was determined in the presence of
glutamine, N-acetylglutamate and uridine triphosphate as described by
Anderson and Walsh (1995).
Radioactivity was measured using a Wallac 1414 liquid scintillation counter
(Wallac, Oy, Finland). The CPS III activity was expressed as µmol
[14C]urea formed min1 g1 wet
mass. GS (E.C. 6.3.1.2) transferase activity was assayed according to the
method of Shankar and Anderson
(1985
). The formation of
-glutamylhydroxymate was determined at 500 nm using a Shimadzu UV 160
recording spectrophotometer. The GS activity was expressed as µmol
-glutamylhydroxymate formed min1 g1
wet mass.
Determination of ammonia, urea and free amino acids (FAAs)
The frozen sample was weighed, ground to a powder in liquid nitrogen, and
homogenized three times in 5 volumes (w/v) of 6% TCA using an Ultra-Turrax
homogenizer at 24 000 revs min1 for 20 s each separated by
intervals of 10 s off. The homogenate was centrifuged at 10 000
g and 4°C for 15 min to obtain the supernatant.
For ammonia determination, the pH of the supernatant was adjusted to
5.56.0 with 2 mol l1 KHCO3. Ammonia was
determined according to the methods of Bergmeyer and Beutler
(1985). Urea was determined as
described by Jow et al.
(1999
). The difference in
absorbance of the sample with and without urease treatment was used to
estimate the urea concentration in the sample. For FAA analysis, 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. Although a complete free amino acid analysis was performed on
every sample, only the contents of alanine, glutamate, glutamine, glycine,
taurine and total FAA (TFAA) are presented in this report. Results are
expressed as µmol g1 wet mass tissue or µmol
ml1 haemolymph, as appropriate.
Statistical analyses
Results are presented as means ± the standard error of the mean
(S.E.M.). Student's t-test or analysis of variance (ANOVA)
followed by multiple comparisons using Duncan's procedure was used to evaluate
differences between means in groups where appropriate. Arsine transformation
was applied to percentage results before statistical analyses. Differences
where P<0.05 were regarded as statistically significant.
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Results |
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Ammonia was excreted by P. expansa during the 17 days of exposure to brackish water or seawater, but no urea was detected in the external media (Fig. 1). On day 17, there was a significant increase in the rate of ammonia excretion in clams exposed to brackish water (Fig. 1). For clams exposed to seawater, there were significant increases in rates of ammonia excretion on day 15 and day 17, and these rates were significantly greater than those of the corresponding control in brackish water (Fig. 1).
|
There were no detectable CPS III activities (detection limit=0.001 µmol min1 g1 tissue) in hepatopancreas, mantle, adductor muscle and foot muscle of P. expansa kept in brackish water, seawater or terrestrial conditions for 17 days. In spite of high background absorbance readings, treatments of samples with urease revealed that no urea was present in these tissues.
Ammonia contents in the adductor muscle, foot muscle, hepatopancreas, mantle and haemolymph of P. expansa kept in seawater for 17 days were comparable to those of clams kept in brackish water (control) for the same period (Fig. 2). In contrast, there were significantly greater levels of ammonia in all these tissues in clams after 17 days of aerial exposure compared with those exposed to brackish water or seawater (Fig. 2).
|
The contents of alanine in the adductor muscle, foot muscle, hepatopancreas and mantle (Fig. 3) were greater than those of glycine (Fig. 4), glutamate (Fig. 5) and glutamine (Fig. 6) in P. expansa exposed to all three experimental conditions. Taurine content in the adductor muscle, foot muscle, hepatopancreas and mantle of clams in brackish water as 2.67±0.31, 2.34±0.55, 2.03±0.48, 0.76±0.05, respectively. Thus, alanine was a major contributor to the TFAA pool (Fig. 7) in P. expansa. The contents of alanine (Fig. 3) and glycine (Fig. 4) in the adductor muscle, foot muscle, hepatopancreas and mantle in clams exposed to seawater for 17 days were significantly greater than those in clams exposed to brackish water or terrestrial conditions for a similar period. Exposure to seawater induced greater glutamate content only in the hepatopancreas and mantle (Fig. 5), and had no effect on the glutamine content of any of the tissues studied (Fig. 6). In contrast, aerial exposure for 17 days resulted in significantly greater glutamine content in the adductor muscle, foot muscle, hepatopancreas and mantle of P. expansa (Fig. 6). Both seawater and air exposure had only minor effects on taurine content in these tissues (results not shown). Overall, exposure to seawater, but not to air, led to significantly greater TFAA levels in all the tissues of P. expansa examined as compared with exposure to brackish water (Fig. 7).
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GS (transferase) activities were detected in the adductor muscle, foot muscle, hepatopancreas and mantle of P. expansa (Fig. 8). The activities of GS in these tissues in P. expansa were unaffected by exposure to seawater for 17 days (Fig. 8), but aerial exposure led to significant greater activities of GS in the adductor muscle and hepatopancreas (Fig. 8).
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Discussion |
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FAAs are substrates for energy metabolism, protein synthesis and
osmoregulation. Changes in salinity and/or exposure to environmental
contaminants may affect the concentration and composition of FAAs in bivalves
(Bayne et al.,
1976a,b
;
Livingstone, 1985
). It has
been reported that the FAA levels in the tissues of brackish water bivalves
(e.g. P. caroliniana and Rangia cuneata) contribute
2530% of the intracellular solute at 1020
salinity
(Allen, 1961
;
Fyhn, 1976
; Gainey,
1978a
,b
;
Henry et al., 1980
), and
alanine is the major contributor to the FAA pool
(Virkar and Webb, 1970
;
Pierce, 1971
;
Gainey, 1978b
;
Henry et al., 1980
;
Matsushima et al., 1984
).
Indeed, similar to other bivalves, alanine (4348%) was the major FAA in
P. expansa in brackish water (also in seawater and in air).
More importantly, our results confirm that glutamine contributed
significantly (8.516.1%) to the TFAA pool of P. expansa in
brackish water. This contribution was greater than that of glycine
(1.68.8%) and comparable to that of glutamate (9.313.5%). The
high levels of glutamine in the tissues of P. expansa indicate that
this marsh clam was capable of synthesizing glutamine. Indeed, we verified the
presence of GS (transferase) activities in all the tissues examined, with the
greatest activity in the hepatopancreas. Due to the paucity of information in
this area, generalizations about glutamine biosynthetic capabilities of
bivalves cannot be made. However, studies with 14C-labelled
precursor molecules (glutamate, Krebs cycle intermediates and glucose) in
M. edulis and other bivalve species confirm the absence of any
glutamine biosynthetic capacity (Wijsman
et al., 1977; Baginski and
Pierce, 1978
; Collicutt and
Hochachka, 1977
). In contrast, it has been reported that giant
clams that harbour symbiotic zooxanthellae are capable of glutamine synthesis
(Rees et al., 1994
), and that
a reasonably rapid glutamine synthesis and turnover occurs in species that
excrete reasonable amounts of purines (terrestrial pulmonate snails and some
prosobranch gastropods; see review by
Bishop et al., 1983
). Although
GS has been found in tissues of several gastropod species
(Campbell and Bishop, 1970
;
Reddy and Swami, 1975
;
Horne, 1977
), there has been
no report of the activity in tissues of non-symbiotic bivalves. Thus, this is
the first report of the presence of glutamine synthetic capacity in a
non-symbiotic intertidal bivalve, and therefore we made an effort to elucidate
its functional role in P. expansa (see below).
Since food was withheld for 20 days (3 days prior to and 17 days during the experiment), the rate of proteolysis was likely to be greater than the rate of protein synthesis, and net protein degradation would have occurred. Protein degradation together with increased amino acid catabolism would lead to an increase in the production of ammonia. Indeed, there was a significant increase in the rate of ammonia excretion in clams maintained in brackish water for 17 days. These results indicate that P. expansa could effectively excrete the excess ammonia produced during this period of fasting in brackish water.
P. expansa in seawater
Exposure to seawater for 17 days did not induce CPS III activity or
accumulation of urea in the hepatopancreas, mantle, adductor muscle and foot
muscle of P. expansa. Thus, urea did not act as an osmolyte in this
marsh clam.
In invertebrates, FAAs are major intracellular osmolytes, contributing
effectively to cell volume regulation
(Pierce and Greenberg, 1972).
Concentrations of intracellular FAAs of marine or brackish water bivalves
fluctuate in response to changes in the ambient salinity
(Pierce, 1971
; Gainey,
1978a
,b
).
In response to hypo-osmotic stress, intracellular levels of FAAs decrease as a
result of their efflux to extracellular compartments (Pierce and Greenberg,
1972
,
1973
) and/or transformation to
other substances inside cells (Matsushima
et al., 1986
). Conversely, in response to hyperosmotic stress,
intracellular FAA concentrations are elevated as a result of protein
degradation (Baginski and Pierce,
1978
; Livingstone et al.,
1979
; Henry et al.,
1980
). Indeed, the TFAA content in the adductor muscle, foot
muscle, hepatopancreas and mantle increased significantly by 2.6-, 2.3-, 4.2-
and 3.6-fold, respectively, in P. expansa kept in seawater for 17
days. These changes were not a result of changes in water contents of the
tissues. Simultaneously, the alanine content increased by 3.1- to 6.4-fold,
and that of glycine by 3.0- to 5.9-fold. However, in terms of absolute
quantity, alanine was the major contributor, because its percentage
contribution to the TFAA pool increased from 43.048.0% to
61.773.1% in various tissues. Thus, as suggested before for other
brackish and marine bivalves (Bayne et al.,
1976a
; Bishop et al.,
1983
), the functional role of alanine in P. expansa is
mainly connected with intracellular osmoregulation.
The steady-state concentrations of FAAs in tissues are determined and
maintained by the rates of their degradation and production (through
proteolysis and/or synthesis). Alteration of these two rates would lead to
changes in FAA concentration. The supply of FAAs for osmoregulatory purposes
through protein degradation in clams in high salinity is confirmed by studies
on enzymes involved in amino acid metabolism e.g. transaminase
(Greenwalt and Bishop, 1980)
and peptidase/proteinase (Bayne et al.,
1981
; Deaton et al.,
1984
). Since the ammonia excretion rates in P. expansa
exposed to seawater were significantly greater than in clams exposed to
brackish water on day 15 and day 17, it can be deduced that the rates of amino
acid catabolism in the former were greater than those in the latter. In
addition, since there were greater TFAA contents in tissues of the former than
the latter, it can be concluded that a greater rate of protein degradation had
occurred in clams exposed to seawater compared with those exposed to brackish
water.
Protein degradation leads to the release of FAAs. FAAs can be further
catabolized, releasing ammonia. Increases in ammonia concentration would push
reactions catalysed by glutamate dehydrogenase and alanine aminotransferase
towards the synthesis of glutamate and alanine
(Newsholme and Leech, 1983),
leading to their accumulation. Indeed, there were minor but significant
increases in levels of glutamate in the hepatopancreas and mantle of P.
expansa, indicating that the glutamate dehydrogenase reaction had been
perturbed. Furthermore, certain amino acids (e.g. arginine, glutamine,
histidine and proline) can be converted to glutamate. Glutamate can undergo
transamination with pyruvate, catalyzed by alanine aminotransferase, producing
-ketoglutarate without the release of ammonia (Ip et al.,
2001a
,c
;
Chew et al., 2003
).
-Ketoglutarate is then channelled into the tricarboxylic acid (TCA)
cycle for partial metabolism. The removal of malate from the TCA cycle can
give a continuous supply of pyruvate. Transamination of pyruvate would produce
alanine continuously, facilitating the oxidation of carbon chains of certain
amino acids without releasing ammonia.
In contrast to alanine, there were no significant increases in glutamine levels in various tissues of P. expansa in seawater. As a result, there were decreases in the percentages contribution of glutamine to the TFAA pools in the adductor muscle, foot muscle, hepatopancreas and mantle of clams exposed to seawater (6.0, 9.1, 6.8 and 5.5, respectively) compared with those exposed to brackish water (10.4, 14.7, 8.5 and 16.1, respectively). Thus, it can be concluded that the physiological significance of glutamine synthesis in P. expansa is unrelated to osmoregulation.
P. expansa in air
Emersion inhibits several metabolic functions, such as feeding, respiration
and excretion, in bivalves. In many cases, oxygen consumption rates are
reduced during air exposure. In response to a lack of oxygen, the energy
requirements of clams are satisfied through anaerobic glucose catabolism, for
which alanine is one of the major end-products
(de Zwaan, 1977;
Widdows et al., 1979
;
Henry et al., 1980
;
Zurburg and de Zwaan, 1981
).
However, P. expansa was exceptional because there were no significant
increases in alanine content in any of the tissues studied after 17 days of
aerial exposure. This could be related to the unique capability of P.
expansa to maintain a normal O2 utilization rate during
emersion (see Introduction). In spite of maintaining aerobic respiration, the
tissues of P. expansa were not exposed to dehydration during
emersion.
Although P. expansa does not encounter a lack of oxygen in air, it
is confronted with problems associated with ammonia excretion when water is
lacking. During aerial exposure, ammonia can be excreted into the mantle
cavity fluid (Shick et al.,
1988), but ammonia concentrated therein would build up and lead to
accumulation of ammonia in its tissues. Indeed, the ammonia contents in
various tissues increased between 2.1-fold and 3.0-fold in P. expansa
exposed to air for 17 days. Because there was no detectable CPS III activity
and no accumulation of urea in various tissues of P. expansa exposed
to air for 17 days, it can be concluded that, unlike in some gastropods (see
review by Bishop et al.,
1983
), ammonia accumulated during emersion was not detoxified to
urea in this marsh clam.
Since reactions catalyzed by transaminases and glutamate dehydrogenase are
near equilibrium in vivo
(Newsholme and Leech, 1983),
substrate concentrations (e.g. ammonia) determine whether transamination and
deamination of amino acids or amination of
-keto acids occurs. An
increase in tissue ammonia concentration would push the equilibrium towards
amination of
-keto acids through the reaction catalyzed by glutamate
dehydrogenase. This would theoretically result in an accumulation of glutamate
and transaminable non-essential FAAs, including alanine (see above), in the
tissues. However, there were no increases in alanine content in any of the
tissues studied in P. expansa exposed to air for 17 days. Hence,
alanine was not a product ammonia detoxification in P. expansa. Our
results confirm that ammonia was detoxified to glutamine in P.
expansa during 17 days of aerial exposure. That means glutamate formed
from NH4+ and
-ketoglutarate reacted further with
ammonia, in the presence of ATP, to produce glutamine. The contents of
glutamine in the adductor muscle, foot muscle, hepatopancreas and mantle
increased 2.9-, 2.5-, 4.5- and 3.4-fold, respectively. Simultaneously, there
were significant increases in GS activities in the adductor muscle (1.56-fold)
and hepatopancreas (3.8-fold). This is the first report on the upregulation of
GS and accumulation of glutamine in a clam in response to aerial exposure.
Such a phenomenon may be peculiar to those bivalves that can maintain aerobic
energy metabolism during emersion, because synthesis of glutamine is
ATP-dependent and production of glutamate requires NAD(P)H. It would be
energetically uneconomical for an animal to increase glutamine and glutamate
syntheses while undergoing anaerobic energy metabolism due to a lack of oxygen
supply, because there would be a decrease in ATP production and problems
associated with redox balance. Indeed, it has been reported recently that the
swamp eel Monopterus albus does not accumulate glutamine after 40
days of aestivation in hypoxic mud, but accumulates glutamine to high levels
in its tissues during 6 days of aerial exposure
(Chew et al., 2005a
).
Sokolowski et al. (2003)
determined FAA contents in the clam Macoma balthica L. from brackish
waters of the southern Baltic Sea, and reported that the overall temporal
pattern of variations in the concentration of glutamine in the period analysed
resembled, in general, that of alanine, with high values in the winter and low
in spring. Sokolowski et al.
(2003
) suggested that the
physiological roles of these two amino acids were similar within a seasonal
cycle, despite the lack of evidence for such metabolic connections. Contrary
to the proposition of Sokilowski et al. (2003), our results reveal that the
functional role of glutamine was distinctly different from that of alanine in
P. expansa. However, how alanine formation and glutamine synthesis,
both involving glutamate as a substrate, are regulated in P. expansa
in response to different environmental conditions (exposure to seawater
vs exposure to air) is uncertain at present.
Conclusion
Returning to the initial impetus of this study, the results obtained with
P. expansa demonstrate that the adoption of glutamine synthesis,
similar to the adoption of urea synthesis, for ammonia detoxification first
took place among invertebrates. Similar to other bivalve species, P.
expansa is incapable of urea synthesis de novo, but is capable
of detoxifying ammonia to glutamine during emersion. The capacity for
glutamine synthesis was present in a variety of tissues, including the
hepatopancreas, mantle, adductor muscle and foot muscle, in P.
expansa. This capacity apparently extends to the lower vertebrates,
because some air-breathing fishes are capable of synthesizing glutamine in
their liver, muscles, and digestive tracts as a major strategy to defend
against ammonia toxicity during aerial
(Jow et al., 1999;
Ip et al., 2001b
;
Chew et al., 2001
;
Tay et al., 2003
) or
environmental (Peng et al.,
1998
; Anderson et al.,
2002
; Ip et al.,
2004a
; Lim et al.,
2004
) ammonia exposure. Brains of vertebrates, from fish
(Campbell and Anderson, 1991
;
Ip et al., 2001a
,
2004b
;
Chew et al., 2005b
) to mammals
(Cooper and Plum, 1987
;
Brusilow, 2002
;
Felipo and Butterworth, 2002
),
are capable of detoxifying ammonia to glutamine. However, higher vertebrates
such as mammals do not adopt glutamine synthesis as a major strategy to deal
with ammonia toxicity in extra-cranial tissues, because they have evolved to
depend mainly on urea synthesis through the hepatic OUC to detoxify ammonia
(Cooper and Plum, 1987
).
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References |
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