Influence of environmental hypertonicity on the induction of ureogenesis and amino acid metabolism in air-breathing walking catfish (Clarias batrachus, Bloch)

Indian Journal of Experimental Biology

Vol. 52, July 2014, pp. 728-738

Influence of environmental hypertonicity on the induction of ureogenesis

and amino acid metabolism in air-breathing walking catfish

(Clarias batrachus, Bloch)

Bodhisattwa Banerjee, Gitalee Bhuyan & Nirmalendu Saha*

Biochemical Adaptation Lab., Department of Zoology, North-Eastern Hill University, Shillong 793 022, India

Received 11 November 2013; revised 28 April 2014

Effect of environmental hypertonicity, due to exposure to 300 mM mannitol solution for 7 days, on the induction of

ureogenesis and also on amino acid metabolism was studied in the air-breathing walking catfish, C. batrachus, which is

already known to have the capacity to face the problem of osmolarity stress in addition to other environmental stresses in its

natural habitats. Exposure to hypertonic mannitol solution led to reduction of ammonia excretion rate by about 2-fold with a

concomitant increase of urea-N excretion rate by about 2-fold. This was accompanied by significant increase in the levels of

both ammonia and urea in different tissues and also in plasma. Further, the environmental hypertonicity also led to

significant accumulation of different non-essential free amino acids (FAAs) and to some extent the essential FAAs, thereby

causing a total increase of non-essential FAA pool by 2-3-fold and essential FAA pool by 1.5-2.0-fold in most of the tissues

studied including the plasma. The activities of three ornithine-urea cycle (OUC) enzymes such as carbamoyl phosphate

synthetase, argininosuccinate synthetase and argininosuccinate lyase in liver and kidney tissues, and four key amino acid

metabolism-related enzymes such as glutamine synthetase, glutamate dehydrogenase (reductive amination), alanine

aminotransaminase and aspartate aminotransaminase were also significantly up-regulated in different tissues of the fish

while exposing to hypertonic environment. Thus, more accumulation and excretion of urea-N observed during hypertonic

exposure were probably associated with the induction of ureogenesis through the induced OUC, and the increase of amino

acid pool was probably mainly associated with the up-regulation of amino acid synthesizing machineries in this catfish in

hypertonic environment. These might have helped the walking catfish in defending the osmotic stress and to acclimatize

better under hypertonic environment, which is very much uncommon among freshwater teleosts.

Keywords: Amino acid metabolism, Ammonia, Clarias batrachus, Environmental hypertonicity, Mannitol, Ornithine-urea

cycle, Urea, Walking catfish

The air-breathing walking catfish (Clarias batrachus, Bloch), found predominantly in tropical Southeast Asia, is reported to be more resistant to various environmental challenges such as high environmental ammonia, hypoxic and desiccation stresses

1,2. Further,

it is reported to be euryhaline, inhabiting fresh and brackish waters as well as muddy marshes, thus facing wide variations of external osmolarity changes

3; it frequently encounters the problem of

osmolarity changes in the same habitat during different seasons of the year, especially in summer when the ponds and lakes dry up, thus compelling this fish to migrate inside the mud peat to avoid total dehydration, and during the monsoon season due to rainfall the water in the same habitat gets diluted.

Several unique physiological and biochemical adaptations have already been reported in this air-breathing catfish with relation to nitrogen, carbohydrate and protein metabolism. These include the presence of a unique functional and regulatory ornithine-urea cycle (OUC) with the capacity of induction of ureogenesis during hyper-ammonia and desiccation stresses

4–8. It has recently been

demonstrated that the cell volume changes due to osmotic stress can affect the glycogenesis, glycogenolysis

9 and gluconeogenesis

10, hexose

monophosphate pathway11

, autophagic proteolysis12

and also protein synthesis

13 in the perfused liver of

this air-breathing catfish. More recently, it has been reported that the walking catfish can survive up to 300 mM mannitol under the laboratory conditions for months without having any mortality, and in situ exposure to higher environmental salinity causes induction of gluconeogenesis

14. However, no

information is available on how the air-breathing

——————

*Correspondent author

Telephone: +91 364 27722322 (Off); +91 9436100836 (M)

Fax: +91 364 2550076

E-mail: [email protected]; [email protected]

BANERJEE et al.: INFLUENCE OF ENVIRONMENTAL HYPERTONICITY

729

catfish osmoregulate during exposure to hypertonic environment. Thus, looking at its enormous capacity in challenging the external osmolarity changes, the present study has been undertaken to elucidate the possible induction of ureogenesis with an intension to synthesize and accumulate more urea as an osmolyte, and also the possible changes of amino acid pool by changing the activities of certain key amino acid metabolism-related enzymes in the walking catfish during exposure to hypertonic environment of 300 mM mannitol (equivalent to 300 mOsmol L

-1)

for 7 days.

Materials and Methods

Chemicals―Enzymes, co-enzymes, substrates

and mixture of physiological FAA standard, and

OPA were purchased from Sigma Chemicals

(St. Louis, USA). Other chemicals were of

analytical grades and obtained from local sources.

Deionized double distilled water was used in

all preparations.

Experimental animals―C. batrachus,

weighing 150±15 g body mass) were purchased from

a single source that are bred and cultured in

selected commercial ponds. Fish were acclimatized

in the laboratory approximately for 1 month at

28±2 ºC with 12 h:12 h light and dark photoperiods

before experiments when food consumption

became normal. No sex differentiation of the fish

was done while performing these studies. Minced

dry fish and rice bran (5% of body wt) were given

as food every day, and the water, collected

from a natural stream, was changed on alternate

days. Food was withdrawn 24 h prior to

experiments.

Experimental set up―Ten fishes (pre-weighed)

were placed individually in plastic buckets containing

2 L of 300 mM mannitol solution prepared in

bacteria-free filtered stream water (pH 7.10 ± 0.05)

for 7 days. Another 10 fishes were kept individually

in plastic buckets containing 2 L of bacteria-free

filtered stream water (pH 7.02±0.06) for 7 days and

served as controls. Both the mannitol solution and

water from each bucket were replaced with a fresh

medium every day at a fixed time after collecting

some samples from each bucket for analysis of

ammonia and urea concentrations. After 3 and 7 days,

five fish each from control and treated buckets were

anesthetized in neutralized 3-aminobenzoic acid ethyl

ester (MS-222, 0.2 g L-1

) for 5 min, blood samples

were collected from the caudal vasculature with a

heparinized syringe, tissues such as liver, kidney,

muscle and brain were dissected out, plunged

into liquid nitrogen and stored at -80 oC. Blood

collected from each fish was centrifuged at 10,000 g

for 10 min, and plasma were processed4 for further

analysis. All analyses were completed within 2 weeks

of collecting the tissues.

Analyses of ammonia, urea-N and free amino acids

(FAAs)―Amounts of ammonia and urea-N excreted

by both control and mannitol-treated fish were

measured enzymatically15

. Ammonia and urea-N

concentrations in different tissues and in blood plasma

were also measured by the same enzymatic methods

after processing the tissue as described by Saha

and Ratha4.

The concentrations of different physiological free

amino acids (FAAs) in tissues and plasma were

analyzed in a Shimadzu HPLC (Model LC 20AD)

with a post-column derivatization method using o-

phthaldehyde (OPA) reagent as a fluorescent dye

following the method of Fujiwara et al.16

with certain

modifications as detailed in Saha et al17

. In brief, a

strong cation-exchange column (Shim-Pack ISC –07

Li, 10 cm long) was used for separation of FAAs. The

detector (Shimadzu RF-535 fluorescent detector) was

set at an excitation of 365 nm and an emission of 455

nm, and coupled to a data integrator (Shimadzu

CR6A) for quantification of the eluted peak areas.

The eluting mobile phase was a gradient of buffer A

(0.16 N lithium citrate containing 7% methyl

cellusolve, pH 2.5) and buffer B (0.32 N lithium

citrate containing 0.62% of boric acid, pH 10.0),

starting with 100% mobile phase A; the flow rate was

0.4 mL min at 0 to 53 min, followed by 0.3 mL/min

until the end of the run; the column temperature was

40 ºC at 0-40 min, and 50 ºC thereafter to 240 min. In

the first 40 min the linear gradient progressed to 4%

mobile phase B, followed by a linear increase to 10%

in 93 min, 30% in 106.7 min, changed to 40% in

106.7 min and was held there until 123 min. The

gradient was then increased linearly to 53% mobile

phase B in 135 min and held there until 170 min, and

finally increased linearly to 100% mobile phase B

from 170 to 190 min and held there until 240 min.

Hypochloride reagent for on-line oxidation was

prepared by adding 0.4 mL of the commercial sodium

hypochlorite solution to 1000 mL of the buffer

solution (pH 10) containing sodium carbonate (0.384

M), boric acid (0.216 M) and potassium sulphate

(0.108 M). The fluorescence reagent was prepared by

INDIAN J EXP BIOL, JULY 2014

730

adding 2.0 g OPA (dissolved in 1 mL of ethanol),

4 mL of 10% aqueous Brij 35, and 2 mL

2-mercaptoethanol to 980 mL of the above alkaline

buffer.

Extraction procedure―For the assay of enzymes,

a 10% homogenate (w/v) of different tissues was

prepared in a homogenizing buffer containing 100 mM

Tris-HCl buffer (pH 7.5), 50 mM KCl, 1 mM ethylene

diamine tetra acetic acid (EDTA), 1 mM dithiothreitol

(DTT) and a cocktail of protease inhibitor (Roche,

Germany) using a motor-driven Potter-Elvehjem glass

homogenizer with a Teflon pestle. The homogenate

was treated with 0.5% Triton X-100 in 1:1 ratio for

30 min. The homogenate was then subjected to mild

sonication for proper breakage of mitochondria and

centrifuged at 10,000 g for 10 min. The supernatant

was used for assaying the enzymes. All steps were

carried out at 4 oC.

For the analysis of FAAs, a 10% homogenate (w/v)

of different tissues was prepared in HPLC grade

water17

. Proteins were immediately precipitated out

from the homogenate by adding 2 M perchloric acid

(PCA) in 1:1 ratio, followed by centrifugation at

10,000 g for 10 min. The plasma was also treated with

2 M PCA in a 1:1 ratio to precipitate out the proteins,

and further processed as above. All these steps were

performed at 4 ºC. The pH of the resultant supernatant

was adjusted to 2.2 by adding a known volume of

0.4 N Li-hydroxide. The supernatant was passed

through a Millipore micro filter (0.45 µM pore size)

before analysis.

Enzyme assay―The enzymes of OUC, viz.,

carbamoyl phosphate synthetase (CPS), ornithine

transcarbamylase (OTC), argininosuccinate synthetase

(ASS), argininosuccinate lyase (ASL), and arginase

(ARG) were assayed following the method described by

Saha et al18

. However, for the assay of OUC-related

CPS activity, 1 mM of uridine-5'-triphosphate (UTP)

was also added in the reaction mixture to inhibit the

pyrimidine synthesis-related CPS II activity19

. It should

be noted that the assay method used here for CPS

activity does not distinguish between the two different

forms of urea synthesis-related enzymes namely CPS I

(ammonia- and N-acetyl-L-glutamate-dependent,

mitochondrial) and CPS III (glutamine- and N-acetyl-L-

glutamate-dependent, mitochondrial). The reaction for

all the enzymes was stopped by adding 0.5 mL of 10%

perchloric acid mL-1

of reaction mixture after a specific

time of reaction, followed by centrifugation to

precipitate out the proteins. Citrulline formed in the

case of CPS and OTC, citrulline used in the case of

ASS, and urea formed in the case of ASL and ARG

were measured spectrophotometrically (Varian, Cary

50) in the supernatant20

and expressed as enzyme

activity. All the enzyme assays were carried out at

30 oC. One unit of enzyme activity was defined as that

amount that catalyzed 1 µmole of product formed or

substrate used h-1 at 30

oC.

Glutamate dehydrogenase (GDH, both reductive

amination and oxidative deamination) activity was

assayed following the method of Olson and

Anfinsen21

with modifications of substrate (optimal)

concentrations17

. The alanine aminotransaminase

(ALT) and aspartate aminotransaminase (AST)

activities were assayed following the method of

Foster and Moon22

with modifications in substrate

(optimal) concentration17

. All these enzymes were

assayed at 30 oC in a UV-visible spectrophotometer

fitted with a peltier temperature-controlled unit

(Varian, Cary 50) at 340 nm (Em M340 = 6.22).

Enzyme activities were expressed as units g-1

wet wt

of tissue and corrected for any non-specific activity in

the absence of substrate. One unit of enzyme activity

was expressed as that amount which oxidized 1 µmole

of NADH or reduced 1 µmole of NAD+ h

-1 at 30

oC.

Glutamine synthetase (GS) was assayed by the γ-

glutamyl transferase reaction as described by Webb

and Brown23

. One unit of GS activity was expressed

as that amount which catalyzed the formation of

1 µmole of γ-glutamyl hydroxamate h-1 at 30

oC.

Blood sampling and osmolarity measurement―The

blood from each fish was collected with a heparinized

syringe from the caudal vein and centrifuged at 10,000 g

for 10 min at 0±2 °C for separating out the plasma from

blood cells and the plasma osmolarity was measured

with a Camlab (Model 200) osmometer using the

freezing point depression method.

Analysis of water content in different tissues―The

water content in cells of different tissues of both

control and mannitol–treated fish was determined by

oven drying method following Goswami and Saha9.

Statistical analyses―The data collected from

different replicates, were statistically analyzed and

presented as mean±SE. Student’s t-test, followed by

multiple comparisons of means by Student-Newman-

Keuls Multiple Range Test was performed to evaluate

differences between means where applicable.

Differences with P<0.05 were regarded as statistically

significant.


731

Results Excretion pattern of ammonia and urea-N by the

fish in hypertonic environment―As shown in Fig. 1,

the rate of ammonia excretion averaged to

308 µmoles kg-1 body wt h

-1 by the control fish during

the period of 7 days. Exposure to hypertonic

environment (300 mM mannitol, which is equivalent

to 300 mOsmol L-1

) led to a decrease of ammonia

excretion by 2.1-fold within the first day. The

decreasing pattern of ammonia excretion rates were

maintained in hypertonic environment over the period

of 7 days by the catfish with a maximum decrease by

3.3-fold after 5 days.

In contrast, the rate of urea-N excretion, which

was averaged to 86 µmoles kg-1

body wt h-1 by the

control fish during the period of 7 days,

increased significantly by 1.5-fold after the first day

of exposure to hypertonic environment with a

maximum increase by about 1.8-fold after 6

days (Fig. 1). The ratio of ammonia/urea-N

excretion decreased initially from 3.5 to 1.2 within

2 days of exposure, followed by further decrease

to about 0.65 from third day onwards and

was maintained till 7 days of experimental periods.

Changes of tissue levels of ammonia, urea-N and

FAAs in hypertonic environment―The changes in

concentrations of ammonia and urea-N in different

tissues and plasma of C. batrachus during exposure to

hypertonic environment for 7 days are shown in

Fig. 2. The concentration of ammonia increased

significantly in all the tissues studied (except in

brain), and in plasma of fish within 3 days of

exposure to hypertonic environment, followed by

further increase to about 1.5 to 1.75-fold after 7 days

of exposure. Similarly, the concentration of urea-N

in different tissues and plasma also increased

significantly by about 1.3 to 2.3-fold after 3 days,

Fig. 2―Changes in concentrations of ammonia and urea-N in

different tissues (µmoles g-1 wet wt) and in plasma (µmoles mL-1)

of following exposure to hypertonic mannitol solution. Values are

mean ± S.E. from 5 observations each. P values: a <0.05, b <0.01,

c <0.001 (Student’s t-test)

Fig. 1―Changes in the rates of excretion of ammonia and urea-N

(µmoles kg-1 body wt h-1) by C. batrachus following exposure

to hypertonic mannitol solution. Values are mean ± S.E. from

5 observations each. *P <0.001 (Student’s t-test)


732

followed by further increase by 1.5 to 3.1-fold after 7

days of hypertonic exposure. Hypertonic exposure was recorded to cause

significant increase in the concentration of total non-essential and essential FAAs in different tissues and plasma of C. batrachus (Fig. 3 A-E). In liver, a significant increase in concentrations of different

non-essential FAAs was seen after 3 days of exposure to hypertonic environment, followed by further increase of approximately 2.0-fold after 7 days (Fig. 3A). This was mainly attributable to the increase of concentrations of Asp, Gly, Ala, Glu, Gln and Tau. The essential FAAs also increased significantly in liver by 1.3-fold after 7 days of exposure, which was

Fig. 3―Changes in concentrations

of different FAAs (µmoles g-1wet

wt) in (A) liver (B) kidney (C)

muscle (D) brain and (E) plasma

of C. batrachus following

exposure to hypertonic

mannitol solution. Values are

mean ± S.E. from 5 observations

each. P values: a <0.05,

b <0.01, c <0.001 (Student’s t-test)


733

mainly attributable to the increase of Thr, Mat, Leu and Arg.

In kidney, the concentration of total non-essential

FAAs increased significantly by 1.75-fold after 3 days

and by 1.9-fold after 7 days of exposure, which was

mainly attributable to the increase of Asp, Ala and

Glu, Gly and Gln (Fig. 3B). The essential FAAs also

significantly increased by 1.5-fold within 3 days of

exposure but with not much of changes after 7 days.

In muscle, the concentration of total non-essential

FAAs increased by 1.75-fold after 3 days and by

2.3-fold after 7 days of exposure (Fig. 3C). It was

mainly due to increase of Asp, Gly, Ala, Glu, Gln, Pro

and Tau. The essential FAAs also increased significantly

by 1.50-fold after 7 days of exposure, which was mainly

attributable to the increase of Thr, Val, Met and Phe.

In brain, the total non-essential FAAs

concentration significantly increased by 1.7-fold

after 3 days with a further increase by 1.90-fold after

7 days of hypertonic exposure, which was mainly

attributable to the increase of Asp, Gly, Glu, Tau and

Gln (Fig. 3D). Likewise, the levels of essential FAAs

also increased by 1.95-fold after 3 days of exposure

with no further changes after 7 days.

A significant increase in the concentration of total

non-essential FAAs was also seen in plasma by 2.6-

and 3.9-fold after 3 and 7 days of exposure to

hypertonic exposure, respectively (Fig. 3E). This was

mainly attributable to the increase of Asp and Gly,

Ala, Glu, Gln and Tau. The levels of essential FAAs

also increased significantly by 1.90-fold after 3 days

with no further changes after 7 days.

The changes in the activities of OUC enzymes in

hypertonic environment―The changes in the

activities of OUC enzymes in C. batrachus due to

exposure to hypertonic environment were studied in

liver and kidney (two ureogenic tissues) (Fig. 4). Both

in liver and kidney tissues significant increases of

activities of CPS, ASS and ASL were observed within

3 days of exposure to hypertonic environment,

followed by further increases of activities after 7 days.

In liver, the CPS, ASS and ASL activities increased

maximally by 2, 1.8 and 7.5-fold, respectively, after

7 days of exposure. In case of kidney, the activities

of CPS, ASS and ASL increased maximally by 1.8,

1.9 and 1.67-fold, respectively.

The changes in the activities of certain key amino

acid metabolism-related enzymes in hypertonic

environment―The activities of 4 key amino acid

metabolism-related enzymes, viz., GS, GDH, AST and

ALT also increased significantly in different tissues of

C. batrachus after 3 days of exposure to hypertonic

environment with a further increase after 7 days (Fig. 5).

The activities of GS, GDH, AST, and ALT increased

maximally by 1.6 to 1.8-fold, 1.6 to 2.0-fold, 1.65 to 1.8-fold and 1.5 to 2.0-fold, respectively, in different tissues.

Changes in plasma osmolarity and tissue water

content due to environmental hypertonicity―In situ

exposure of C. batrachus in hypertonic environment

(300 mM mannitol) led to significant increase of plasma

osmolarity by 23 and 30%, respectively, after 3 and 7

days of hypertonic exposure (Table 1). Hypertonic

exposure also led to a significant decrease of water

content in different tissues within 3 days, followed by

further decrease in most of the cases after 7 days.

Fig. 4―Changes in the activity (units g-1wet wt) of different OUC

enzymes in the liver and kidney of C. batrachus following

exposure to hypertonic mannitol solution. Values are mean ± S.E.

from 5 observations each. P values: a <0.05, b <0.01 (Student’s t-

test) CPS – carbamoyl phosphate synthetase; OTC – ornithine

transcarbamylase; ASS – argininosuccinate synthetase; ASL –

argininosuccinate lyase; ARG – arginase


734

Discussion The role of urea for osmoregulation is well

established in marine elasmobranchs24

, and to certain

extents in marine teleosts25

. The crab-eating frog

(Rana cancrivora), which is one of only a handful

amphibians worldwide that tolerate saline waters, is

also reported to synthesize and retain urea inside the

body by up-regulating the hepatic urea synthetic

capacity in hypertonic saline environment26

.

However, till date no report is available on the role of

urea for osmoregulation in freshwater teleosts and

more specifically in air-breathing teleosts especially

when they are submerged in brackish or saline waters.

As reported recently, the C. batrachus has a capacity

to acclimatize well in high environmental salinity14

. In

the present study, when C. batrachus was exposed to

hypertonic environment (300 mM mannitol), the

ammonia excretion rate reduced to half with a

concomitant increase of urea-N excretion rate by

2-fold, thus causing a reduction in the ammonia:urea-

N excretion rate from 3.5 to 0.65. The initial decrease

of ammonia excretion in this fish was accompanied

by the increase in concentrations of ammonia in

different tissues. However, the toxic ammonia was not

allowed to accumulate in body tissues to a lethal

concentration in fish exposed to hypertonic

environment possibly due to possessing various

biochemical adaptational strategies to nitrogen

metabolism that have been reported in this fish while

exposing to different environmental constraints2,1

.

One of the unique adaptations that it possesses is the

presence of a functional and regulatory OUC, and

reported to help in detoxification of ammonia to urea

under hyper-ammonia stress4,6,8

. Further, since

mannitol fails to enter into the animal body, it caused

dehydration stress to different tissues by exosmosis,

Fig. 5―Changes in the activity (units g-1wet wt) of certain key

amino acid metabolism-related enzymes in different tissues of

C. batrachus following exposure to hypertonic mannitol solution.

Values are mean ± S.E. from 5 observations each. P values: a

<0.05, b <0.01, c <0.001 (Student’s t-test) GS – glutamine

synthetase; GDH (R.A.) – glutamate dehydrogenase (reductive

amination); GDH (O.D.) – glutamate dehydrogenase (oxidative

deamination); AST – aspartate aminotransaminase; ALT – alanine

aminotransaminase

Table 1—Changes of plasma osmolarity and tissue water content in C. batrachus following exposure to hypertonic environment

(300 mM mannitol). [Values are mean ± S.E. from 5 observations each]

3 days 7 days

Control Treated Control Treated

Plasma osmolarity (mOsmol l-1) 265 ± 3 325 ± 5* (+23) 266 ± 4 345 ± 6* (+30)

Tissue water content

Liver 78 ± 1.5 70 ± 1.2* (-10) 77 ± 1.8 65 ± 1.3* (-15)

Kidney 79 ± 2.0 72 ± 1.8* (-9) 78 ± 2.1 68 ± 1.6* (-13)

Muscle 76 ± 2.1 69 ± 1.7* (-9) 77 ± 1.9 64 ± 1.8* (-14)

Brain 77 ± 1.9 72 ± 1.8 (-6) 77 ± 2.2 70 ± 1.7* (-9)

Tissue water contents are presented as percentage of wet mass weight of individual tissue. Percentage increase (+) and decrease (-)

of values corresponding to respective controls are given in parentheses.

* P value significant at <0.05 level compared to respective controls (Student’s t-test)


735

thereby resulting in significant decrease of cellular

water content in different tissues. The decrease of cell

volume/hydration status due to hypertonicity has

recently been reported to cause the stimulation of

proteolysis in perfused liver of C. batrachus12

. Thus,

the increase of ammonia levels in body tissues

appeared to be mainly through protein degradation

and amino acid catabolism primarily to meet the high

energy demand for the osmoregulalory acclimation.

Further, the increase of urea excretion rate and higher

accumulation of tissue urea levels, observed in

hypertonically-treated fish, were mostly associated

with the induction of activities of certain key OUC

enzymes, viz., CPS, ASS and ASL in liver and kidney

tissues of this fish. A 5-fold increase of CPS activity

during exposure to 0.9% sodium chloride solution for

14 days27

and during exposure to hypertonic saline

environment28

was reported in the liver of aquatic frog

(Xenopus laevis). Increase of both ammonia and urea

excretion rates were reported in another air-breathing

fish, the climbing perch (Anabas testudineus) while

acclimating to 30% seawater29

. However, the source

of urea in this fish was suggested to be either through

arginolysis and/or purine degradation pathway, since

the functional OUC was reported to be non-functional

in this fish4. Similarly, in swamp eel (Monopterus

albus) decrease of both ammonia and urea excretion

rates have been reported during exposure to brackish

water30

. Increase of urea level has also been reported

in the plasma of carp (Cyprinus carpio)31

and in anura

(Rana cancrivora)32

while exposing to hyper- and

hypotonic mannitol. The decrease in ammonia

excretion rate in C. batrachus, observed during

exposure to hypertonic environment, was probably

associated with the enhanced synthesis of urea from

ammonia via the induced OUC, thereby leading to

stimulation of urea excretion rate and more

accumulation of urea in body tissues. Thus, it appears

that urea, which is also known to be a good osmolyte,

plays a critical role in this fish for cell volume

regulation and also to avoid excessive water loss

under hypertonic stress.

Liver is the most important organ with relation to

nitrogen metabolism and also it is the most important

organ governing amino acid homeostasis in fish33

.

Exposure to hypertonic mannitol solution led to

significant increase in the levels of various non-

essential FAAs (Asp, Gly, Ala, Asn, Glu, Gln and

Tau) and also certain essential FAAs (Met, Leu, Trp,

and Phe) in the liver of walking catfish. Similar

increasing patterns of different FAAs were also

observed with minor variations in kidney, muscle,

brain and plasma of C. batrachus while exposing to

hypertonic mannitol solution. One reason of

increasing the amino acid pool in different tissues

could be associated with the stimulation of protein

degradation, which was suggested earlier at least in

the liver of this fish under hypertonic stress12

.

The adjustment of intracellular FAA concentrations

in response to osmotic stress has widely been

documented among crustaceans as well as other

marine invertebrates34–36

. However, reports on the role

of amino acid pool in osmoregulation in freshwater

teleosts are scanty. More recently, role of FAAs in

osmoregulation has been emphasized in the climbing

perch (A. testudineus)29

, swamp eel (M. albus)30

and

in freshwater marble goby (Oxyeleotris marmorata)37

during acclimation to either sea or brackish water.

The basal levels of activity of some of the key

enzymes related to amino acid metabolism such as the

GS, GDH (reductive amination), AST and ALT were

found to be quite high in C. batrachus compared to

many ammoniotelic teleosts including the gobiid

fish38-43

, which got further stimulated in different

tissues (except for AST in liver and brain) under

hypertonic stress. Thus, the increasing activity of

most of the enzymes, observed in different tissues of

mannitol-exposed fish, might have helped in extra

synthesis of various non-essential FAAs from

ammonia, generated due to protein degradation and

amino acid catabolism through the involvement of

coupled GDH (reductive amination), which

synthesizes Glu by assimilating ammonia with

α-ketoglutarate, and GS enzymes to incorporate

another molecule of ammonia to Glu forming Gln,

and also to other amino acids by transamination

reactions. Stimulation of activity of these enzymes

was probably associated with higher accumulation of

ammonia in different tissues as reported earlier in the

same fish under hyper-ammonia stress17,7

. Thus, it is

very much evident that the up-regulation of

non-essential FAAs synthetic pathways would be

another source of increasing the amino acid pool in

this fish under hypertonic stress. Further, the increase

of amino acid pool along with the increase of urea

concentration, observed in the present study, was

associated with the increase of plasma osmolarity,

thus counteracting the higher external osmolarity to

some extent mainly to avoid the water loss under

hypertonic stress.


736

Interestingly, out of different FAAs, which got

accumulated in different tissues of C. batrachus, Tau

increased maximally during exposure to hypertonic

mannitol solution. Tau is a relatively inert molecule,

which even at high concentration is not only

compatible with the function and organization of

macromolecules, but may in addition act as an

osmoprotectant counteracting the deleterious effects

of denaturing solutes as inorganic ions. The

occurrence of Tau synthetic pathway, which

constitutes the most predominant amine in most

teleosts including C. batrachus, is not very clear in

fish44

. Similar increase of Tau concentration was also

noticed in this fish under hyper-ammonia stress17,7

.

Therefore, it is necessary to investigate the occurrence

of the Tau synthetic pathway with its physiological

significance in this fish.

The mechanism(s) by which different amino acid

metabolism-related enzymes and three key OUC

cycle enzymes are stimulated in this fish under

hypertonic stress is difficult to explain with the

available data. More recently, the increase in the

abundance of GS and GDH enzyme proteins and

mRNA for GDH in mud eel45,30

, and GS and GDH

enzyme proteins in the juvenile of freshwater marble

goby37

have been reported while exposing to

seawater. The more abundance of GS and CPS

enzyme proteins and different isoforms of GS

mRNAs have also been reported in C. batrachus

while adapting to high environmental ammonia46,47

.

Therefore, the transcriptional and translational

regulation of these enzymes cannot be ruled out in

this fish under hypertonic stress. Another strategy of

enzymatic induction could be through changes in the

concentration of various effector molecules, and/or

by changing the phosphorylation status of the

pre-existing enzymes as observed in the case of

glycogen phosphorylase and glycogen synthase

enzymes in C. batrachus9, and glycogen phosphorylase

enzyme in another catfish, Amereiurus nebulosus48

under aniso-osmotic conditions. Numerous protein

kinases have been implicated in metabolic alterations

with volume changes in mammalian hepatocytes49

and

in fish chloride cells50

, and several osmosensing

receptors associated with the activation of various

kinases have been identified51

. Molecular crowding

may also exert long-term effects, such as cAMP-

mediated changes in gene transcription under osmotic

stress52,53

. However, a thorough investigation requires

to be performed to understand better about the

regulation of enzymatic activities under osmotic stress

in this fish.

In conclusion, the capacity of up-regulating the

urea synthesis via the induced OUC resulting to more

accumulation of urea in body tissues to defend the

osmotic stress in hypertonic environment and also

more excretion of urea under hyper-osmotic stress,

observed in C. batrachus, is uncommon among

freshwater teleosts. It resembled more with

some aquatic frogs and elasmobranchs. Further

C. batrachus has the capacity to increase the amino

acid pool, by up-regulating the activities of enzymes

responsible for synthesis of different non-essential

FAAs and probably also by stimulating the protein

degradation, is another unique biochemical adaptation

that has been observed to acclimatize better under

hypertonic environment. Thus, due to possessing

these adaptational capabilities C. batrachus is able to

survive successfully in wide ranges of osmolarity

changes in its natural environment.

Acknowledgement This study was supported by a project sanctioned to

NS by the Department of Biotechnology, New Delhi,

the DSA programme to the Department of Zoology

and the UPE-Bioscience project to the North-Eastern

Hill University, Shillong by the University Grants

Commission, New Delhi.

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Influence of environmental hypertonicity on the induction of ureogenesis and amino acid metabolism in air-breathing walking catfish (Clarias batrachus, Bloch)

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