Download GLUTAMINE CONCENTRATION MAY LIMIT GLUTATHIONE

GLUTAMINE CONCENTRATION MAY LIMIT
GLUTATHIONE SYNTHESIS IN THE PRESENCE OF
α-ADRENOCEPTOR AGONISTS AND GLUCAGON
M. Atmaca1,2, J.R. Fry2
University of Dicle, School of Medicine, Diyarbakir, Turkey1
School of Biomedical Sciences, Medical School, Queen’s Medical Centre, Nottingham,
U.K.2
ABSTRACT
Glutathione (GSH) is an ubiquits tripeptide which is composed of glutamine-cysteine-glycine. In general, methionine is the preffered precursor of cysteine in cell culture.It has
been indicated that glucagon, adrenaline and phenylephrine inhibit hepatocellular glutathione synthesis in the presence of methionine. Therefore, cysteine is considered rate-limiting for GSH synthesis. To examine whether changes in glutamine concentration in the
medium are also rate-limiting for GSH synthesis, short term cultured hepatocytes were
treated with different concentrations of glutamine in the presence of the other two precursors. The inhibitory effects of glucagon were observed when lower concentrations of glutamine (0.2 or 0.5mM) were used. However, this effect was attenuated when hepatocytes
were incubated with 2mM concentrations of glutamine. The effect of phenylephrine on
GSH synthesis was measured in the presence of different concentrations of methionine (2,
0.5, 0.2 and 0.1mM), given as a precursor of cysteine, with 2mM glutamine and 2mM glycine. The inhibitory effect of phenylephrine was not apparent at any concentration of methionine. Our results suggest that, under physiological conditions, the availability of glutamine in the plasma, and possible changes in L-glutamate concentration, appear to be
important for maintaining GSH synthesis in the presence of glucagon and the α-adrenergic agents, adrenaline and phenylephrine.
Introduction
patocellular glutathione synthesis in the
presence of methionine. In the light of
these observations cysteine is considered
rate-limiting for GSH synthesis. We wondered if a similar response will be obtained
in the presence of a mixture of glutathione
precursors (methionine, glutamate, glycine)
when hepatocytes are treated with adrenaline, phenylephrine and glucagon in short
term culture. Estrela and co-workers (5)
suggested that under physiological conditions, changes in glutamate concentration
in hepatocytes may limit glutathione synthesis in the presence of phenylephrine.
The present study suggests that in short
term cultured hepatocytes, the availability
Glutathione (GSH) is a tripeptide that plays
a central role in metabolic pathways, detoxification and in the antioxidant system
of most aerobic cells (1). It has been well
established that hepatic GSH levels fall
after stressful experimental conditions such
as severe exercise and cold strain (2,3), and
this has been associated with the increased
sympathetic stimulation response to stress.
The intracellular cysteine concentration
is normally very low therefore the majority
of cysteine required for GSH synthesis in
hepatocytes is derived from methionine (4).
It has been indicated that glucagon,
adrenaline and phenylephrine inhibit heBiotechnol. & Biotechnol. Eq. 19/2005/2
144
of glutamine in cell culture may be ratelimiting.
Materials and Methods
Adult male Wistar rats (150-180g) were
obtained from the University of Nottingham Biomedical Services Unit. They were
housed at a constant room temperature of
22 ºC and had free access to standard laboratory diet and tap water.
Six well cluster dishes were purchased
from Fahrenheit Laboratory Supplies (Nottingham, UK). Culture medium, adrenaline, glucagon, phenylephrine, Trinder glucose assay kit, collagen and collagenase
were purchased from Sigma Chemicals Co.
(Poole, UK).
Hepatocytes were isolated by perfusion
of liver lobes with collagenase essentially
as described by Reese and Byard (6). The
hepatocyte suspension was washed with
Hanks buffer, pH 7.4 (140mM NaCl, 5mM
KCI, 0.4mM KH2PO4, 0.3mM Na2HPO4,
20mM HEPES, 50µM phenol red). Cells
were resuspended in L-15 medium and
centrifuged (50g for 10 min) in a 90% percoll solution to improve the separation of
viable and nonviable cells, as described by
Kreamer et al. (7). Cell viability as detected
by trypan blue exclusion was typically
greater than 95%. Hepatocytes were suspended in L-15 medium containing 10%
v/v calf serum and dispensed into collagencoated six-well plates at a density of 1x106
cells per well. After 2 hours the culture
medium was replaced with 2 ml of Hanks
buffer (without serum) including appropriate concentrations of glucagon, adrenaline
and phenylephrine and incubated for 2
hours. After this period, either the medium
was replaced with 1 ml of sulphosalicyclic
acid (SSA) and the glutathione content
analysed immediately, or glutathione synthesis was determined in the following
manner. Cultures were incubated with
0.6mM diethylmaleate (DEM) in Hanks
buffer for 30 minutes to deplete cellular
GSH. The cells were then washed and in145
cubated with appropriate concentrations of
glucagon, adrenaline and phenylephrine in
Hanks buffer (without serum) containing
2mM methionine for 2 hours to allow GSH
synthesis. After that period, the medium
was removed and 1 ml SSA added to each
well. Cellular GSH was immediately measured by the method of Saville (8), which
detects total non-protein thiols. Glucose
levels in the medium were assessed as described by the manufacturer of the assay
kit.
Data are presented as mean ± SEM of at
least three separate experiments, each value
representing the mean of a minimum of
three measurements. Statistical analysis
was undertaken using unpaired t-tests or
analysis of variance (ANOVA)/ Dunnett’s
test as appropriate. A value of P<0.05 was
considered significant.
Results and Discussion
In preliminary experiments, it was established that membrane receptors are capable
of responding to glucagon, adrenaline and
phenylephrine with an increase in glucose
output (data not shown). Adrenaline, phenylephrine and glucagon all decreased cellular glutathione levels significantly in isolated rat hepatocytes (Fig. 1).
The concentration dependence of glucagon, adrenaline and phenylephrine on glutathione synthesis was examined. Hepatocytes were treated for 30 min with 0.6mM
DEM to deplete cellular GSH about 70%,
which then recovered in a time-dependent
manner during incubation with simple salt
solution supplemented with methionine as
a cysteine donor as described previously
(9). The increase in cellular GSH following
DEM treatment was taken to represent
GSH synthesis. Glucagon, adrenaline and
phenylephrine inhibited hepatocellular
glutathione synthesis 10-5M concentration
in the presence of 2mM methionine (Fig. 2).
The inhibitory effect of adrenaline, phenylephrine and glucagon at concentration
(10-5M) was not observed when hepatoBiotechnol. & Biotechnol. Eq. 19/2005/2
GSH Level (nmol/well)
40
**
30
**
**
20
10
Phenylephrine(10µM)
Control
Adrenaline(10µM)
Control
Glucagon(10µM)
Control
0
Fig. 1. Effect of glucagon,adrenaline and phenylephrine on GSH level. Results are mean ± SEM of 5-6 seperate
experiments.Where indicated, values are significantly different from control at **P<0.01.
GSH synthesis rate (nmol/well/2hrs)
30
20
**
**
**
10
Phenylephrine(10µM)
Control
Adrenaline(10µM)
Control
Glucagon(10µM)
Control
0
Fig. 2. Effect of glucagon adrenaline and phenylephrine on GSH synthesis in the presence of methionine
(2mM). Results are mean ± SEM of 5-6 seperate experiments. The rate of GSH synthesis from methionine over
a 2 hrs period was measured in rat hepatocytes (after an initial depletion with DEM) in absence or presence of
glucagon,adrenalin and phenylephrine. Where indicated, values are significantly different from control at
**P<0.01.
cytes were incubated in the presence of all
three constituent amino acids of glutathione
(methionine, L-glutamine and cysteine, all
at 2mM). In the presence of these three
amino acids re-synthesis following DEMtreatment restored GSH to the initial level
after 2 hours (Fig. 3). Combination of meBiotechnol. & Biotechnol. Eq. 19/2005/2
thionine with glycine did not abolish the
inhibitory effect of phenylephrine whereas
the inhibitory effect of phenylephrine was
abolished when cells were incubated with
the combination of methionine and glutamine(Table 1). However, the rate of
synthesis varied with methionine.
146
20
Phenylephrine(10µM)
Control
Adrenaline(10µM)
Control
0
Control
10
Glucagon(10µM)
GSH synthesis rate (nmol/well/2hrs)
30
Fig. 3. Effect of glucagon, adrenaline and phenylephrine on the rate of GSH synthesis in the presence of equimolar concentrations of methionine, glutamine and glycine (2mM each). Results are mean ± SEM of 7 separate
experiments.
0.5mM, 0.2mM and 0.1mM concentrations) and with the other two precursors
glutamine and glycine, both at 2mM, in the
presence and absence of phenylephrine at
10-5M. However, the lack of effect of
phenylephrine on glutathione synthesis was
not abolished with the inclusion of decreased concentrations of methionine (2,
0.5, 0.2 and 0.1mM) in the incubation medium (Table 2). This suggests that limitations in intracellular cysteine may not be
responsible for the effects for phenylephrine on GSH synthesis.
To investigate whether changes in Lglutamine concentration in the medium
may limit glutathione synthesis, hepatocytes were incubated with different concentrations of glutamine (2, 0.5 and
0.1mM) and 2mM methionine and glycine
in the presence of 10-5M glucagon (Table
3). Under these conditions, addition of glucagon promoted a decrease in intracellular
glutathione synthesis. The inhibitory effects of glucagon were observed with 0.5
and 0.1mM glutamine.
Similar results were obtained by Estrela
et al. (5) who indicated that phenylephrine
TABLE 1
Effect of phenylephrine on rate of GSH synthesis
in the presence of glutathione precursors
Rate of GSH synthesis
(nmol/well/2hrs)
Precursors (2mM)
- Phenylephrine +Phenylephrine
(10–5M)
Methionine
15.8±2.2
9.9±1.9**
Methionine+Glycine
17.9±2.3
11.5±2.3**
Methionine+Glutamine 20.7±2.4
19.2±2.4
24.3±3.6
Methionine+Glycine+ 25.3±4.2
Glutamine
Results are mean ± SEM of 7 separate experiments.
**P<0.01
The failure of phenylephrine to inhibit
glutathione synthesis in the presence of
methionine and glutamine was examined in
greater detail by decreasing individually the
concentrations of these amino acids in the
incubation medium. It is known that the
main source of intracellular L-cysteine is
by intracellular synthesis from L-methionine through the cystathionine pathway
(4). In the light of these observations cells
were incubated with methionine (at 2mM,
147
Biotechnol. & Biotechnol. Eq. 19/2005/2
TABLE 2
Effectt of phenylephrine (10-5 M) on GSH synthesis in medium containing different concentration
of methionine
TABLE 3
Effect of glucagon(10-5 M) on GSH synthesis in
medium containing different concentration of
glutamine
Additions
(mM)
Rate of GSH
synthesis
nmol/well/2hrs
Met(0.1)+Gly(2)+Gln(2)
10.9±0.3
Met(0.1)+Gly(2)+Gln(2)+Phenylephrine
9.1±0.1
Met(0.2)+Gly(2)+Gln(2)
13.5±0.08
Met(0.2)+Gly(2)+Gln (2)+
12.2±0.3
Phenylephrine
Met(0.5)+Gly(2)+Gln(2)
21.3±0.6
Met(0.5)+Gly(2)+Gln(2)+Phenylephrine 19.8±0.6
Met(2)+Gly(2)+Gln(2)
31.8±0.8
Met(2)+Gly(2)+Gln(2)+Phenylephrine
31.4±0.7
Additions
(mM)
Met(2)
Met(2)+ Glucagon
Met(2)+Gly(2)+ +Gln(0.1)
Met(2)+Gly(2)+
+Gln(0.1)+Glucagon
Met(2)+Gly(2)+ +Gln(0.5)
Met(2)+Gly(2)+ +Gln(0.5)+Glucagon
Met(2)+Gly(2)+ +Gln(2)
Met(2)+Gly(2)+ +Gln(2)+Glucagon
GSH Synthesis
Rate
(nmol/well/2hrs)
17.8 ±0.9
13.4±1.4**
17.1±1.2
13.1±0.9**
25.4±1.6
17.2±0.7**
35.1±1.1
35.6±1.3
Results are mean±SEM of 4-6 separate experiments.
Met (Metionine), Gly (Glycine), Gln(Glutamine).
Results are mean±SEM of 4-6 separate experiments.
Met (Metionine), Gly (Glycine), Gln(Glutamine).
Where indicated, values are significantly different
from appropriate control at **P<0.01
decreased the glutathione synthesis level in
cells, incubated with a mixture of amino
acids containing 2mM L-glutamine, to
~50% of the controls. They also indicated
that an increased concentration of glutamine prevents the inhibitory effect of
phenylephrine. Since the Km of γ-glutamylcysteinyl synthetase for glutamate is
~2mM, a lower concentration of L-glutamate in cells cannot overcome phenylephrine inhibition on GSH synthesis. In
the present study cellular glutamate levels
were not measured, hence the extent to
which hormones contribute to glutamate
levels is unknown. Therefore, the possibility of decreased glutamate levels by glucagon or adrenergic agonists cannot be rejected.
It has been reported (10) that rat hepatocytes concentrate glutamine from the extracellular fluid to a constant value of ~8 between 0.5-5mM and cellular glutamine concentration was found to be approximately
5mM.
Haussinger et al. (11) indicated that the
physiological portal glutamine concentration is 0.6mM which would give an inter-
nal glutamine level of ~4.8mM which fits
well with the 3.02mM concentration for rat
liver in vivo cited by Haussinger et al (11).
The cellular glutamate concentration is
lower than the glutamine level (being
~2.58mM) which is slightly greater than
the Km value for γ-glutamylcysteinyl synthetase (~2mM).
Perfusion of rat liver with a simple salt
solution without glutamine considerably
decreased cellular glutamine concentration
from 3.1mM to 0.38mM, indicating that
hepatic glutamine concentration is dependent on portal glutamine supply. In the
same experimental conditions the cellular
glutamate level was also markedly decreased from 2.58 to 0.89mM, which is
lower than Km value for the synthetase.
Inclusion of 0.6mM glutamine in the perfusate dramatically increased the cellular
glutamine level (11).
According to the constant value (~8)
which is suggested by Fafournoux et al.
(10) inclusion of low level of glutamine
(0.1 or 0.5mM) should give cellular glutamine levels of 0.8mM and 4.0mM respectively, whereas inclusion of 2mM
Biotechnol. & Biotechnol. Eq. 19/2005/2
148
would give 16mM.
It has been well established that L-glutamine metabolism is stimulated in isolated
hepatocytes by a variety of hormones, including glucagon (12), α-adrenergic agonists, vasopressin and angiotensin II (13).
In the absence of glutamine in the incubation medium, cysteine is the rate-limiting
precursor of GSH synthesis and in the
presence of 10-5M glucagon cellular GSH
was lowered significantly. Inclusion of
0.1mM and 0.5mM glutamine in the incubation medium presumably increased the
cellular glutamate level, however this concentration was not high enough, being
lower than the Km value for synthesising
enzyme, to abolish the inhibition of glucagon. At a concentration of 2mM the level
of glutamine presumably reached or exceeded the Km value of the enzyme and
overcame the inhibitory effect of glucagon.
The first step in glutathione synthesis, the
reaction catalysed by γ-glutamylcysteine
synthetase is rate limiting. The hepatocellular L-cysteine concentration is very low,
around 2x10-4M (0.21µmol/g liver), since
it is rapidly incorporated into GSH. The
glutathione-synthesising system, γ-glutamylcysteine synthetase, has a relative
high Km value (2.5x10-3M) for cysteine.
Therefore cysteine has generally been considered limiting for glutathione synthesis,
and the ability of glutamine was considered
less important than cysteine. Although un-
der most conditions the availability of Lcysteine has generally been considered the
rate-limiting step in glutathione synthesis,
the present study suggests that under
physiological conditions, the availability of
glutamine in the plasma may be rate-limiting.
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