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Fish Physiol Biochem (2010) 36:1727

DOI 10.1007/s10695-008-9275-5

Influence of cell volume changes on protein synthesis


in isolated hepatocytes of air-breathing walking
catfish (Clarias batrachus)
Kuheli Biswas Lucy M. Jyrwa
Dieter Haussinger Nirmalendu Saha

Received: 5 June 2008 / Accepted: 30 September 2008 / Published online: 7 November 2008
Springer Science+Business Media B.V. 2008

Abstract The present study aimed at determining


the effect of cell volume changes on protein synthesis,
measured as the incorporation of [3H]leucine into acidprecipitable protein, in isolated hepatocytes of airbreathing walking catfish (Clarias batrachus). The
rate of protein synthesis, which was recorded to be
10.02 0.10 (n = 25) nmoles mg-1 cell protein h-1
in isotonic incubation conditions, increased/decreased
significantly by 18 and 48%, respectively, following
hypo- (-80 mOsmol l-1)/hypertonic (?80 mOsmol l-1) incubation conditions (adjusted with
NaCl), with an accompanying increase/decrease of
hepatic cell volume by 12 and 20%, respectively.
Similar cell volume-sensitive changes of protein
synthesis were also observed when the anisotonicity
of incubation medium was adjusted with mannitol.
Increase of hepatic cell volume by 9%, due to addition
of glutamine plus glycine (5 mM each) to the isotonic
control incubation medium, led to a significant
increase of protein synthesis by 14%. Decrease of
hepatic cell volume by 15 and 18%, due to addition of
dibutyl-cAMP and adenosine in isotonic control
K. Biswas  L. M. Jyrwa  N. Saha (&)
Biochemical Adaptation Lab, Department of Zoology,
North Eastern Hill University, Shillong 793 022, India
e-mail: nsaha@nehu.ac.in; nsahanehu@hotmail.com
D. Haussinger
Department of Internal Medicine, Division of
Gastroenterology, Hepatology and Infectiology, HeinrichHeine University, 40225 Dusseldorf, Germany

incubation medium, led to a significant decrease of


protein synthesis by 30 and 34%, respectively. Thus, it
appears that the increase/decrease of hepatic cell
volume, caused either by changing the extracellular
osmolarity or by the presence of amino acids or certain
other metabolites, leads to increase/decrease of protein
synthesis, respectively, and shows a direct correction
(r = 0.99) between the hepatic cell volume and
protein synthesis in walking catfish. These cell
volume-sensitive changes of protein synthesis probably help this walking catfish in fine tuning the different
metabolic pathways for better adaptation during cell
volume changes and also to avoid the adverse affects
of osmotic stress. This is the first report of cell volumesensitive changes of protein synthesis in hepatic cells
of any teleosts.
Keywords Adenosine  Anisotonicity  Cell
volume  Clarias batrachus  Dibutyl-cAMP 
Glutamine  Glycine  Intracellular water space 
Protein turnover  Protein synthesis

Introduction
Hepatic cell volume may be challenged by a variety of
factors, which distort the osmotic equilibrium between
the intra- and extra-cellular spaces primarily during
intestinal absorption of water, various amino acids,
and metabolites, or by the exposure to different
osmotic environments especially in the case of aquatic

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animals. Although most of the cells possess various


volume regulatory mechanisms to maintain the constancy of cell volume and also the hydration status of
cells via the regulatory volume decrease (RVD) and
regulatory volume increase (RVI) mechanisms, largely by changing the permeability of various ions such
as K?, Na?, H?, Cl- and HCO3-, and certain organic
osmolytes, it has been noticed in many cell types that
they remain in either a slightly swollen or shrunken
state for the duration of anisotonic exposure (for
review, see Haussinger and Lang 1991). These minute
changes of cell volume critically contribute to the
regulation of hepatocellular metabolism and gene
expression (Saha et al. 1992; Haussinger et al. 1994;
Goswami and Saha 1998; Schliess and Haussinger
2003; Saha and Goswami 2004).
In comparison with mammals, teleost fishes face
more problems of osmotic stress primarily due to
osmolarity changes in their external environment, and
also due to intestinal absorption of amino acids and
other metabolites. Looking at the enormous importance of osmoregulation in fishes, some reports on
this line are already available concerning the cell
volume regulatory mechanism in red blood cells
(Fugelli and Thoroed 1986; Motais et al. 1991;
Haynes and Goldstein 1993; Cossins et al. 1994),
renal cells (Kanli and Terreros 1997; Kanli and
Norderhus 1998), intestinal cells (Lionetto et al.
2001, 2002, 2005; Trischitta et al. 2004), and few
reports in fish liver cells (Bianchini et al. 1988;
Ballatori and Boyer 1992; Fossat et al. 1997; Espelt
et al. 2003). More recently, it has been demonstrated
that the liver cells of air-breathing walking catfish
(Clarias batrachus) possess an efficient volume regulatory mechanism, but remain in a partly swollen or
shrunken state as long as they are exposed to
anisotonicity (Goswami and Saha 2006). These
minute changes of cell volume due to anisotonicity
have been reported to cause changes of glucose,
pyruvate, and lactate efflux along with the changes of
glycogen metabolism (Goswami and Saha 1998),
hexose monophosphate pathway (HMP) (Saha and
Goswami 2004), and on gluconeogenesis (Goswami
et al. 2004) in the intact perfused liver of walking
catfish. Hallgren et al. (2003) also reported similar
effects of cell volume changes at least on glycogen
metabolism in the hepatocytes of three fish species.
In mammalian liver, hepatocellular hydration has
been recognized as an important physiological

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Fish Physiol Biochem (2010) 36:1727

determinant of protein synthesis, since hypertonic


cell shrinkage and hypotonic cell swelling are
reported to cause inhibition and stimulation of protein
synthesis, respectively (Stoll et al. 1992; Meijer et al.
1993). Further, amino acids are known to stimulate
hepatic protein synthesis (Munro 1968; Jefferson and
Korner 1969; Seglen 1978), whereas hormones like
glucagon and vasopressin inhibit (Woodside et al.
1974; Kimball and Jefferson 1990); this was considered later to be mainly due to changes of hepatic cell
volume (for review, see Haussinger et al. 1994).
However, hormone effects on liver protein synthesis
cannot consistently be explained on the basis of cell
volume changes; here, other mechanisms such as
volume-independent hormone signaling toward gene
expression come into play.
However, compared with mammals, no information is available about the interaction between cell
volume changes and hepatic protein synthesis in
teleost fishes, in spite of the fact that they are more
challenged with osmolarity changes in the external
environment. The freshwater air-breathing walking
catfish, found predominantly in the Indian subcontinent, is reported to be more resistant against
various environmental changes such as high environmental ammonia and hypoxic and desiccation stresses
(for reviews, see Saha and Ratha 1998, 2007).
Moreover, in addition to routine changes of internal
osmolarity due to intestinal absorption of amino acids
(Goswami et al. 2004) and various other metabolites,
they frequently encounter problems of osmolarity
changes during different seasons of the year, especially in the summer when the ponds and lakes dry
up, and in the monsoon when the water in the same
habitat gets diluted (for review, see Saha and Ratha
2007). Thus, looking at their extreme habitat, the
present study was undertaken in order to gain insight
into the effects of hepatic cell volume changes or
hydration status, caused by exposure to anisotonicity
and other effectors, in the regulation of hepatic
protein synthesis in walking catfish.

Materials and methods


Animals
The walking catfish (150 15 g body mass) were
purchased from commercial sources and acclimatized

Fish Physiol Biochem (2010) 36:1727

in the laboratory for ca. 1 month at a room temperature of 28 2C in plastic aquaria with 12:12 h
light and dark photoperiod before being used for
experiments. No sex differentiation of the fish was
done while performing these studies. Minced pork
liver and rice bran (5% of the body wt.) were given as
food, and the water was changed on alternate days.
Food was withdrawn 24 h prior to experiments.
Isolation and incubation of isolated hepatocytes
The walking catfish hepatocytes were isolated by
perfusion technique with collagenase-treatment following the method of Mommsen et al. (1991) with
certain modifications made by Saha et al. (1995).
Fishes were anesthetized in neutralized 3-aminobenzoic acid ethyl ester (MS222, 0.2 g l-1) for 5 min
before the operation for liver perfusion. Livers were
initially perfused for 15 min via the portal vein in a
non-circulating manner with hemoglobin-free medium having a pH of 7.6 and contained 119 mM NaCl,
5 mM NaHCO3, 5.4 mM KCl, 0.35 mM Na2HPO4,
and 0.44 mM KH2PO4 (buffer A), followed by
perfusion of livers for another 15 min with the same
buffer, but containing 500 units collagenase per
100 ml of perfusion medium and 1% bovine serum
albumin (BSA) (buffer B). The perfusate was saturated with O2/CO2 (99:1 v/v) before infusing into the
liver. The livers were perfused at a flow rate of 4
5 ml g-1 liver min-1 at a temperature of 30C. The
membrane capsule outside the liver was slowly
removed and the cells were flushed in a glass beaker
with pre-oxygenated buffer A where extra 1.25 mM
CaCl2 and 0.81 mM MgSO4 were added (buffer C).
The cells were filtered through a fine mesh in a
beaker on an ice bath. The cells were then collected
by centrifugation at 150g for 24 min and the
supernatant was discarded. The pellet was washed
twice in buffer C before performing experiments with
these isolated cells.
The isolated hepatocytes at a density of
6 9 106 ml-1 were then incubated in buffer C
containing 5 mM glucose in a shaker water bath at
30C in a volume of 3 ml. The medium also contained
amino acids at about twice the physiological concentrations as found in vivo (Saha et al. 2002) in order to
provide enough substrates for protein synthesis, i.e.,
alanine (0.5 mM), proline (0.10 mM), serine

19

(0.15 mM), glycine (0.4 mM), aspartate (0.3 mM),


asparagine (0.1 mM), valine (0.15 mM), isoleucine
(0.2 mM), methionine (0.05 mM), tyrosine (0.1 mM),
phenylalanine (0.5 mM), histidine (0.05 mM), arginine
(0.1 mM), threonine (0.15 mM), lysine (0.05 mM),
tryptophan (0.05 mM), cysteine (0.02 mM), glutamate
(0.2 mM), and glutamine (0.25 mM). Additionally,
4 mM [3H]leucine was also added in the incubation
medium while measuring the rates of protein synthesis
by the isolated hepatocytes under various treatments.
The incubations were continuously gassed with water
vapor-saturated O2/CO2 (99:1, v/v). In isotonic control
incubations, the osmolarity was 280 mOsmol l-1 (i.e.,
265 mOsmol l-1, plus the osmolarity due to addition of
amino acids). Hypotonic medium (200 mOsmol l-1)
and hypertonic medium (360 mOsmol l-1) were
prepared by removing/adding NaCl (40 mM), respectively, from/to the standard isotonic incubation
medium. Additional experiments were also performed
with different ranges of anisotonicity adjusted mainly
by changing the NaCl concentration in the incubation
medium to find a correlation between cell volume
changes and protein synthesis in isolated hepatocytes.
In another set of experiments, the anisotonicity (hypotonicity and hypertonicity) of the medium was adjusted
with mannitol (80 mM) instead of NaCl, where 40 mM
NaCl was initially replaced with 80 mM mannitol from
the isotonic medium. Hypotonicity of the medium was
adjusted by removing mannitol from the isotonic
medium without changing the NaCl concentration,
and the hypertonicity of the medium was adjusted by
adding extra 80 mM mannitol to the above pre-adjusted
isotonic medium. Glutamine and glycine (5 mM each)
were added in the isotonic control incubation medium
in a separate set of experiments to elucidate their effects
on hepatic cell volume and the corresponding changes
of protein synthesis. Dibutyl-cAMP and adenosine,
which are known to cause shrinkage of liver cells
(Haussinger et al. 1994), were added separately to the
isotonic control incubation medium at the beginning of
incubation in amounts so that the final concentration of
these two effectors in the medium became 50 and
100 lM, respectively, in two different sets of experiments. Hepatocyte viability, assessed by Trypan Blue
exclusion before and after the end of incubations for
each set of experiment, was found to be always more
than 95%. The protein content in the incubations ranged
between 6 and 10 mg ml-1.

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Determination of hepatocyte cell volume


The intracellular water space (as a measure of cell
volume) of isolated hepatocytes of walking catfish
was determined following Stoll et al. (1992), where
[3H]inulin was used as a marker for extracellular
space and [14C]urea as a marker for intra- plus extracellular spaces. In brief, hepatocytes were incubated
both in isotonic and anisotonic media in a volume of
3 ml with different combinations as mentioned above
for 45 min, followed by addition of [3H]inulin
(15 kBq) and [14C]urea (15 kBq) to the incubations.
After 5 min, the cell suspensions were centrifuged at
500g for 30 s. The 3H and 14C radioactivities were
determined by liquid-scintillation spectrophotometry
in the supernatant and the pellet, respectively. The
intra-cellular water space (cell volume) was calculated from the 14C radioactivity found in the pellet
after correction for residual extra-cellular water in the
pellet by using the specific radioactivity in the
supernatant. Data on intra-cellular water space are
expressed as ll mg-1 of cell protein.
Determination of [3H]leucine incorporation
into protein
[3H]Leucine (125 kBq ml-1) at a concentration of
4 mM was present in all incubations while measuring
the protein synthesis. The rate of incorporation of
[3H]leucine into acid-precipitable protein, as a measure of the rate of protein synthesis by the isolated
hepatocytes of walking catfish, was determined
following the method of Stoll et al. (1992). During
the 2-h incubation period, samples (0.1 ml) were
taken out from the flask at 20-min intervals for
immediate protein precipitation with 0.15 ml of 1 M
PCA, followed by subsequent centrifugation at
12,000g in a microcentrifuge for 10 min. The
precipitate was washed three times with 0.25 ml of
0.6 M PCA in each washing step, followed by
centrifugation at each step. The pellet obtained after
the last washing step was assayed for 3H radioactivity
by dissolving it in 2 ml of scintillation cocktail. The
3
H radioactivity was determined in a liquid scintillation spectrophotometer (Packard, 1600 TR) and the
[3H]leucine incorporation was taken as the newly
synthesized protein. In another set of experiments,
5 lM cycloheximide was added in the isotonic
control incubation medium to validate our results of

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Fish Physiol Biochem (2010) 36:1727

[3H]leucine incorporation into newly synthesized


protein. For each time point under different treatments, [3H]leucine incorporation into acidprecipitable protein was calculated on the basis of
the specific radioactivity of leucine in the medium at
the beginning of incubation and was expressed as
nmoles mg-1 of liver cell proteins h-1. Proteins were
determined by the dye-binding method (Bradford
1976) using BSA as the standard.
Chemicals
[3H]Inulin, [14C]urea and [3H]leucine were obtained
from the Board of Radiation and Isotope Technology,
BARC, Mumbai. Collagenase (Type VIII), cycloheximide and BSA were obtained from Sigma
Chemicals, St. Louis, USA. The other chemicals
used were of analytical grades and obtained from
local sources. Deionized and double glass distilled
water was used in all preparations.
Statistical analysis
The data collected from different replicates were
statistically analyzed and presented as mean SEM
(n = number of independent cell preparations to
perform independent experiments). Students t test
followed by multiple comparisons of means by
StudentNewmanKeuls multiple range tests were
used to evaluate differences between means where
applicable. Differences with P \ 0.05 were regarded
as statistically significant. Linear regression analysis
was also done to find a correlation between cell
volume changes and protein synthesis.

Results
Effects of anisotonicity, amino acids,
dibutyl-cAMP, and adenosine on cell volume
As shown in Table 1, the intracellular water space (as a
measure of cell volume) of isolated hepatocytes of
walking catfish in isotonic solution containing 5 mM
glucose was found to be 2.35 0.08 (n = 10) nmoles mg-1 protein, which increased to 2.57 0.07
nmoles mg-1 protein (9%) when leucine (4 mM) and
other amino acids at about twice the physiological
concentrations were added to the isotonic medium

Fish Physiol Biochem (2010) 36:1727

21

Table 1 Cell volume changes of isolated hepatocytes of walking catfish (Clarias batrachus) following anisotonic exposures and due
to the presence of other effectors
Condition

Intracellular water space


(ll mg-1 of cell protein) [%]

Isotonic

2.35 0.11 (10)

Isotonic (with amino acid mixture) (Isotonic control)

2.57 0.07 (15) [?9]

Hypotonic (-80 mOsmol l-1)

2.82 0.16* (4) [?12]

Hypertonic (-80 mOsmol l-1)

2.01 0.08* (4) [-20]

Isotonic control

2.74 0.13* (4) [?9]

? Glutamine (5 mM)
? Glycine (5 mM)
Isotonic control ? Dibutyl-cAMP (50 lM)

2.15 0.10* (4) [-15]

Isotonic control ? Adenosine (100 lM)

2.06 0.09* (4) [-18]

Values are presented as mean SEM (n = number of independent cell preparations to perform independent experiments)
Cell volume was determined as intracellular water space. Initially the intracellular water space of hepatocytes was determined in
isotonic incubation medium with 5 mM glucose. The hepatic cell volume was also determined in presence of amino acids at about
twice the physiological concentrations (considered as isotonic control) in the incubation medium. All other incubation media also
contained amino acids at about twice the physiological concentrations. Anisotonic exposures were performed by adding or removing
corresponding amounts of NaCl to/from the incubation medium. Glutamine plus glycine, dibutyl-cAMP and adenosine were added in
the isotonic control incubation medium at the beginning of treatments. Percentage increase (?)/decrease (-) of cell volume in
isotonic control incubation compared to isotonic incubation but without amino acid mixture, and also other experimental conditions
compared to isotonic control incubation are given within square brackets
*Significant at P \ 0.05 level compared to isotonic control

(considered as isotonic control). During hypo(-80 mOsmol l-1) and hypertonic (?80 mOsmol l-1)
exposures along with the amino acid mixture, the cell
volume increased and decreased significantly
(P \ 0.05) by 12 and 20%, respectively, compared to
isotonic control. Addition of glutamine plus glycine
(5 mM each) in isotonic control medium also resulted in
a 9% increase of hepatic cell volume. Addition of
dibutyl-cAMP (50 lM) and adenosine (100 lM) to the
isotonic control medium, however, significantly
(P \ 0.05) decreased the hepatic cell volume by 15
and 18%, respectively.
Effects of anisotonicity and cycloheximide
on protein synthesis
Protein synthesis in isolated hepatocytes of walking
catfish was assessed by measuring the incorporation
of [3H]leucine from the incubation medium into acidprecipitable protein, which increased at a constant
rate over 120140 min of incubation period under all
experimental conditions (Figs. 13). Amino acid
mixture at about twice the physiological concentration (for composition, see Materials and methods)
was present in order to provide enough substrates for

protein synthesis. [3H]Leucine was present in excess


(4 mM) to minimize label dilution by leucine derived
from proteolysis during the 2-h incubation period,
since cell volume changes due to anisotonic exposure
have recently been reported to influence the autophagic proteolysis in the intact perfused liver of
walking catfish (Biswas et al. 2008). The [3H]leucine
incorporation was inhibited by about 90% when the
hepatocytes were incubated in isotonic control
medium containing cycloheximide (5 lM), suggesting that 3H incorporation into acid-precipitable
protein was due to new protein synthesis (Fig. 1).
In isotonic control (280 mOsmol l-1) incubation
conditions, the rate of protein synthesis by the isolated
hepatic cells was recorded to be 10.02 0.10 nmoles mg-1 protein h-1 (n = 25) and remained constant
over a time period (Table 2, Fig. 1). During incubation
with hypotonic (200 mOsmol l-1) medium, where the
hypotonicity was adjusted by withdrawing equivalent
amount of NaCl, the hepatic protein synthetic rate
increased significantly (P \ 0.05) by 18%. In contrast,
in hypertonic (360 mOsmol l-1) medium the hepatic
protein synthetic rate decreased significantly (P \
0.01) by 48% (Fig. 1, Table 2). However, restoration
of isotonic incubation conditions from hypertonicity

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Fish Physiol Biochem (2010) 36:1727

[3H]Leucine incorporation (nmoles mg-1 protein)

24

Hypotonic
(200 mOsmol l -1 )

20

Isotonic
(280 mOsmol l -1 )

16

12
Hypertonic
(360 mOsmol l -1 )

4
Isotonic (280 mOsmol l -1 )
+ Cycloheximide (5 M)

0
0

20

40

60

80

100

Further, when glutamine plus glycine (5 mM each)


was added in the hypertonically pre-shrunken cells,
there was a significant increase (P \ 0.05) of protein
synthesis compared to the hypertonic incubation
conditions, but the linearity in increasing rate was
seen only after 50 min of incubation, probably owing
to progressive cell swelling resulting from a timedependent intracellular accumulation of glutamine
and glycine (Fig. 4). Addition of dibutyl-cAMP
(50 lM) and adenosine (100 lM) separately to the
isotonic control incubation medium caused significant (P \ 0.05) decrease of protein synthesis by 30
and 34%, respectively, compared to isotonic incubation conditions with a constant increase over a time
period (Table 2; graphs not shown).

Incubation time (min)

Fig. 1 Effects of anisotonicity and cycloheximide on protein


synthesis, as estimated from the rates of incorporation of
[3H]leucine into acid-precipitable protein, in isolated hepatocytes of walking catfish (Clarias batrachus). Values are plotted
as mean SEM of independent cell preparations from four
different isolations

reverted back the protein synthetic rate almost to


control value, i.e., from 3.96 0.20 to 9.26 0.45
nmoles mg-1 protein h-1 (Fig. 2). This reversibility
within individual incubation indicated that inhibition
of protein synthesis during hypertonic exposure was
not due to loss of cell viability.
In another set of experiments, while measuring the
rate of protein synthesis by the isolated hepatocytes
under anisotonic conditions, the hypo- and hypertonicity of the extracellular medium was adjusted with
mannitol without changing the NaCl concentration
(Fig. 3, Table 2). In hypotonic (200 mOsmol l-1)
and hypertonic (360 mOsmol l-1) incubation conditions, the rates of protein synthesis significantly
increased by 16% (P \ 0.05) and decreased by 49%
(P \ 0.01), respectively.
Effects of glutamine plus glycine, dibutyl-cAMP
and adenosine on protein synthesis
Addition of extra glutamine plus glycine (5 mM
each) to the isotonic control incubation medium
significantly (P \ 0.05) increased the protein synthesis by 14% (Fig. 4, Table 2). However, under this
condition, the linearity in the rate of protein synthesis
was achieved only after 4050 min of incubation.

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Cell volume changes and protein synthesis


in isolated hepatocytes
As shown in Fig. 5, the rates of protein synthesis by
isolated catfish hepatocytes progressively increased/
decreased with the increase/decrease of cell volume,
respectively, irrespective of cell volume changes
which were evoked by changing the osmolarity of the
incubation medium or by addition of glutamine plus
glycine, dibutyl-cAMP or adenosine. Linear regression analysis of the data presented revealed the
following relationship between the cell volume
changes and protein synthesis by the isolated hepatocytes: y = - 9.84 ? 7.68x (n = 12; r = 0.99).

Discussion
Using an intact organ model of perfused liver, it has
already been demonstrated that the hepatocytes of
walking catfish remain in partly swollen and partly
shrunken states during hypo- and hypertonic exposures, respectively, although they possess a very
efficient volume regulatory mechanisms (Goswami
and Saha 1998, 2006). Further, the slight changes of
cell volume, due to alterations of hydration status,
were reported to cause various metabolic changes
related to carbohydrate and oxidative metabolism
(Goswami and Saha 1998; Saha and Goswami 2004;
Goswami et al. 2004), and also the autophagic proteolysis (Biswas et al. 2008) in an intact model of
perfused walking catfish liver. The data obtained in the

Fish Physiol Biochem (2010) 36:1727

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Table 2 Effects of anisotonicity and other effectors on protein synthesis in the isolated hepatocytes of walking catfish
Condition

Rates of protein synthesis


(nmoles mg-1 of cell protein h-1) [%]

Isotonic control

10.02 0.10 (25)

Isotonic control ? Cycloheximide (5 lM)

1.32 0.09*** (4) [-87]

Hypotonic (adjusted with NaCl) (-80 mOsmol l-1)

11.84 0.44* (4) [?18]

Hypertonic (adjusted with NaCl) (?80 mOsmol l-1)

5.24 0.20** (4) [-48]

Hypotonic (adjusted with mannitol) (-80 mOsmol l-1)

11.64 0.47* (4) [?16]

Hypertonic (adjusted with mannitol) (?80 mOsmol l-1)

5.10 0.24** (4) [-49]

Isotonic control

11.44 0.41* (4) [?14]

? Glutamine (5 mM)
? Glycine (5 mM)
Isotonic control ? Dibutyl-cAMP (50 lM)

7.02 0.24** (5) [-30]

Isotonic control ? Adenosine (100 lM)

6.61 0.29** (4) [-34]

Hypertonic (?80 mOsmol l-1)

8.96 0.38* (5) [-11]

? Glutamine (5 mM)
? Glycine (5 mM)
Values are presented as mean SEM (n = number of independent cell preparations to perform independent experiments)
Hepatocytes were incubated as described in Materials and methods with a medium containing amino acids at about twice the
physiological concentrations in vivo plus [3H]leucine (4 mM). Anisotonicity of the incubation media were adjusted either with NaCl
or mannitol. Glutamine plus glycine, dibutyl-cAMP and adenosine were added in the isotonic control incubation medium at the
beginning of incubations. In the individual incubation, the rate of protein synthesis was determined between 60120 min of
incubation in all sets of experiments. Percentage increase (?)/decrease (-) of protein synthesis compared to isotonic control are
given within square brackets
*P \ 0.05; **P \ 0.01; ***P \ 0.001; significant compared to isotonic control

present investigation further suggest that anisotonic


exposures also lead to changes of hydration status of
isolated hepatocytes of walking catfish, thus causing a
significant increase and decrease of cell volume during
hypo- and hypertonic exposures, respectively
(Table 1). These changes of cell volume were accompanied with modifications in the rates of protein
synthesis in isolated hepatocytes (Table 2). The great
inhibition of protein synthesis (about 50%) in isolated
hepatocytes due to hypertonic (?80 mOsmol l-1) cell
shrinkage, observed in the present study, could not be
due to loss of cell viability in hypertonic environment
since the rates of protein synthesis returned almost to
the basal level when the hepatocytes were reimmersed back to isotonic control incubation conditions (Fig. 2); also, the cell viability, checked by
Trypan Blue exclusion technique, was found to be
more than 95% after 2 h of incubation in hypertonic
medium. However, it could possibly be an effect of
decreased cell volume as reported in rat hepatocytes
(Stoll et al. 1992) and other cell types, such as in
cultured mouse myeloma (MPC 11) cells (Nuss and
Koch 1976; Kruppa and Clemens 1984) or HeLa cells

(Saborio et al. 1974). On the other hand, cell swelling


due to hypotonic exposure (-80 mOsmol l-1), stimulated the protein synthesis in isolated fish
hepatocytes. However, the levels of cell volume
increase and subsequently the protein synthesis were
not so prominent compared to the levels of cell
shrinkage and inhibition of protein synthesis as
observed during ?80 mOsmol l-1 hypertonic exposure. Apparently, the presence of amino acid mixture
at about twice the physiological concentrations in the
incubation medium already caused some swelling of
hepatocytes (Table 1), possibly by cumulative uptake
of amino acids, which might have already stimulated
the protein synthesis to a significant level. It is also
seen that the incorporation of [3H]leucine into acidprecipitable protein is greatly inhibited in the presence
of cycloheximide, a known inhibitor of protein
synthesis (Fig. 1), thus indicating that the [3H]leucine
incorporation into acid-precipitable protein is due to
new protein synthesis, and this inhibition appeared to
occur at the initiation level of peptide synthesis.
Protein synthesis was also stimulated or inhibited
by decreasing or increasing the extracellular

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Fish Physiol Biochem (2010) 36:1727


24

20

Isotonic
(280 mOsmol l-1)

16

12
Isotonic
(280 mOsmol l-1)

[3 H]Leucine incorporation (nmoles mg-1 protein)

[3H]Leucine incorporation (nmoles mg-1protein)

24

Hypotonic

20

(200 mOsmol l-1)


Isotonic (280 mOsmol l-1)
- 40 mM NaCl
+ 80 mM Mannitol

16

12

Hypertonic (360 mOsmol l-1)


with 80 mM Mannitol

0
0

20

40

60

80

100

Incubation time (min)


Hypertonic
(380 mOsmol l-1)

0
0

20

40

60

80

100

120

140

Incubation time (min)

Fig. 2 Reversal of hypertonically-induced inhibition of protein synthesis by re-exposing the isolated hepatocytes of
walking catfish in isotonic medium. Values are plotted as
mean SEM of independent cell preparations from four
different isolations. Isolated fish hepatocytes were initially
incubated for 60 min in isotonic control (280 mOsmol l-1; j)
or hypertonic (360 mOsmol l-1; m) medium (3 ml incubation-1). Hypertonicity was achieved by the addition of 40 mM
NaCl. After removal of 0.1 ml sample from the incubations at
0, 20, 40, and 60 min, 2 ml of pre-warmed and pre-gassed
isotonic control (280 mOsmol l-1) medium was added to the
isotonic incubation conditions (arrow), where as 2 ml of
hypotonic (160 mOsmol l-1 owing to removal of 60 mM
NaCl) medium was added to the hypertonic incubation
conditions. Thus, isotonic incubation conditions (280 mOsmol
l-1) were created in both types of incubations from 60 to
140 min

osmolarity, respectively, with mannitol instead of


adjusting with NaCl (Fig. 3), further suggesting that
external osmolarity-induced cell volume changes
appear to be one of the critical factors of changing
the protein synthesis in isolated hepatocytes of
walking catfish, and not due to changes of Na? or
Cl- activity such as the changes of membrane
potential as reported in rat hepatocytes (Graf et al.
1988).
Other than anisotonicity, certain amino acids (glutamine and glycine) and metabolites are known to
influence the cell volume changes in the intact liver
organ of walking catfish (Saha and Goswami 2004),

123

Fig. 3 Effects of mannitol and anisotonicity on protein


synthesis in isolated hepatocytes of walking catfish. Values
are plotted as mean SEM of independent cell preparations
from four different isolations. In isotonic (280 mOsmol l-1)
control incubation medium, 40 mM NaCl was replaced with
80 mM mannitol (r). Under hypotonic incubation conditions,
80 mM mannitol was withdrawn from the isotonic medium
without changing the NaCl concentration (j), and under
hypertonic incubation conditions, extra 80 mM mannitol was
added to the isotonic control incubation medium (.)

and also in mammals (Wettstein et al. 1990; Haussinger et al. 1994). Similarly, addition of extra
glutamine plus glycine (5 mM each) to the isotonic
control incubation medium also caused further swelling of isolated hepatocytes as a result of concentrative
uptake of these two amino acids via the Na?-dependent amino acid transporter present in the cell
membrane of hepatocytes (Wettstein et al. 1990).
Additionally, the inhibition of protein synthesis during
hypertonic cell shrinkage was largely abolished by
glutamine plus glycine-induced cell swelling of preshrunken cells (Fig. 4). Thus, the stimulation of
protein synthesis reported here after the addition of
these two amino acids in the isotonic control incubation medium appears to be an effect of cell swelling
due to concentrative uptake of these two amino acids.
Other than the extra-cellular osmolarity changes and
accumulation of amino acids, certain metabolites such
as dibutyl-cAMP and adenosine are known to influence the cell volume changes in mammalian
hepatocytes (Haussinger et al. 1994). Similarly, in
our present study, addition of dibutyl-cAMP and
adenosine to the isotonic control incubation medium

Fish Physiol Biochem (2010) 36:1727

25
Isotonic control
Anisotonicity by altering [NaCl]
Anisotonicity without altering [NaCl]
Gln + Gly
cAMP
Adenosine

Isotonic (280 mOsmol l-1)


+ Glutamine (5 mM)
+ Glycince (5 mM)

24

20

Isotonic
(280 mOsmol l-1)

16

Hypertonic (360 mOsmol l-1)


+ Glutamine (5 mM)
+ Glycine (5 mM)

12
Hypertonic
(360 mOsmol l-1)

0
0

20

40

60

80

100

120

140

Incubation time (min)

Fig. 4 Effects of glutamine plus glycine (5 mM each) on


protein synthesis in the isolated hepatocytes of walking catfish
under isotonic (280 mOsmol l-1) and hypertonic (360 mOsmol
l-1) incubation conditions. Values are plotted as mean SEM
of independent cell preparations from five different isolations.
Hepatocytes were incubated in isotonic control incubation
medium with (j) or without (h) glutamine plus glycine
(5 mM each). Hypertonic incubation conditions were achieved
by adding 40 mM NaCl to the isotonic control medium (m).
Hypertonically induced inhibition of protein synthesis reverted
back when extra glutamine plus glycine (5 mM each) was
added to the hypertonic incubation medium (D)

caused significant shrinkage of isolated hepatocytes of


walking catfish (Table 1), thereby suggesting that the
decrease of protein synthesis by these two compounds
is probably an effect of cell shrinkage. Krumschnabel
et al. (2000) also reported the significant inhibition of
protein synthesis by adenosine in isolated hepatocytes
of goldfish (Carassius auratus) and trout (Oncorhynchus mykiss) which, they suggested, acted via a
membrane receptor-mediated pathway. Although they
did not evaluate the effect of adenosine on hepatic cell
volume, it seems that the inhibition of protein synthesis by adenosine is a widespread phenomenon in
teleosts and probably occurs mainly through adenosine-induced cell shrinkage. It was also noted that the
rates of protein synthesis by the isolated hepatocytes
varied with the changes of hepatic cell volume, caused
either due to changes of extracellular osmolarity or due
to addition of amino acids or certain metabolites
(Fig. 5), thus showing a direct correlation (r = 0.99)
between the protein synthesis and the accompanying
hepatic cell volume changes. However, the present

Protein synthesis (nmoles mg-1 cell protein h-1)

[3 H]Leucine incorporation (nmoles mg-1 protein)

28

12

10

6
Y = -9.84 + 7.68x (n = 12; r = 0.99)

4
1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

Intracellular water space (l mg-1 cell protein)

Fig. 5 Relationship between cell volume changes and protein


synthesis in isolated hepatocytes of walking catfish during
anisotonicity, and also in the presence of amino acids
(glutamine and glycine), dibutyl-cAMP and adenosine. Values
are plotted as mean SEM of independent cell preparations
from 4 to 5 different isolations

data do not explain the probable molecular mechanisms of cell volume-sensitive changes of protein
synthesis, which require a detailed investigation.
In summary, the present data suggest that cell
swelling and cell shrinkage, caused by changing the
extracellular osmolarity by addition of amino acids or
other metabolites, stimulates and inhibits protein
synthesis, respectively, in isolated hepatocytes of
walking catfish. This is the opposite effect reported
recently with respect to hepatic proteolysis under
anisotonicity in an intact liver organ of this catfish
(Biswas et al. 2008). Thus, with respect to protein
turnover, cell swelling may represent an anabolic
signal, and cell shrinkage a catabolic signal.
However, these considerations apply only for those
proteins that are sensitive to cell volume changes and
not for all types of proteins. These changes of protein
turnover probably help this walking catfish in fine
tuning the different metabolic pathways for a better
adaptation during cell volume changes and also to

123

26

avoid the adverse affects of osmotic stress both


during intra- and extra-cellular osmolarity changes.
This is the first report of cell volume-sensitive
changes of protein synthesis in hepatocytes of any
teleost.
Acknowledgments This study was supported by a project
sanctioned to N.S. by the Department of Science and
Technology, 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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