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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. REFERENCES 1. Denek S.M., Fanburg B.L. (1989) Am. J. Physiol., 257, 163-173. 2. Pyke S., Lew H., Quintanilha A. (1986) Biochem. Biophys. Res. Commun., 139, 926-931. 3. Simmons H.F., James R.C., Roberts S.M. (1990) Toxicology, 61, 59-71. 4. Rao A.M., Drake M.R., Stipanuk M.H. (1990) J. Nutr., 120, 837-845. 5. Estrela J.M., Gil F., Vila J.M., Vina J. (1988) Am. J. Physiol., 255, E801-E805. 6. Reese J.A., Byard J.L. (1981) In vitro, 17, 935940. 7. Kreamer B.L., Stacker J.L., Sawada N., Sattler G.L., Hsia M.T., Pitot H.C. (1986) In Vitro Cell Develop. Biol., 22, 201-211. 8. Saville B. (1958) Analyst., 83, 670-672. 9. Atmaca M., Fry J.R., (1996) Biochem. Pharmacol., 52, 1423-1428. 10. 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