Cellular and Molecular Neurobiology, Vol. 17, No. 4, 1997

Modulation of Glutamine Synthesis inCultured Astrocytes by Nitric Oxide

Maria-Dolores Minana,1 Elena Kosenko,2 GoizaneMarcaida,1 Carlos Hermenegildo,1 Carmina Montoliu,1Santiago Grisolia,1 and Vicente Felipo13

Recieved July 10, 1996; accepted August 30, 1996

KEY WORDS: glutamine; nitric oxide; nitroarginine; astrocytes; glutamine synthetase.

SUMMARY

1. Previous results suggest that glutamine synthesis in brain could bemodulated by nitrix oxide. The aim of this work was to assess this possibility.

2. As glutamine synthetase in brain is located mainly in astrocytes, we usedprimary cultures of astrocytes to assess the effects of increasing or decreasingnitrix oxide levels on glutamine synthesis in intact astrocytes.

3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor ofnitric oxide synthase. To increase nitric oxide we used 5-nitroso-N-acetylpenicillamine, a nitric oxide generating agent.

4. It is shown that 5-nitroso-N-acetylpenicillamine decreases glutaminesynthesis in intact astrocytes by =40-50%. Nitroarginine increases glutaminesynthesis slightly in intact astrocytes.

5. These results indicate that brain glutamine synthesis may be modulated invivo by nitric oxide.

INTRODUCTION

We have previously studied the role of nitric oxide in the mediation of acuteammonia toxicity. As a control for these experiments, we injected rats withnitroarginine, an inhibitor of nitric oxide synthase. Unexpectedly, we found that

1 Instituto de Investigaciones Citoldgicas de la Fundacion Valenciana de Investigaciones Biomedicas,Amadeo de Saboya 4, 46010 Valencia, Spain.

2 Institute of Theoretical and Experimental Biophysics, Pushchino, Russia.3 To whom correspondence should be addressed.

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0272.4340/97/0800.0433J12.50/0 © 1997 Plenum Publishing Corporation

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injection of 45 mg/kg nitroarginine results in a remarkable increase in glutaminecontent in brain (by 138%) and a significant decrease in glutamate content (by28%) (Kosenko et al., 1995). These results suggest that glutamine synthesis inbrain could be modulated by nitric oxide. Nitric oxide might act as an inhibitor ofglutamine synthesis and the decrease in nitric oxide after injection of nitroar-ginine would then allow increased synthesis of glutamine. The aim of this workwas to test this possibility.

As glutamine synthetase in brain is located mainly in astrocytes (Martinez-Hernandez et al., 1977), we used primary cultures of astrocytes to assess theeffects of decreasing or increasing the concentration of nitric oxide on thesynthesis of glutamine in intact astrocytes. To decrease the concentration of nitricoxide we used nitroarginine, an inhibitor of nitric oxide synthase. To increase theconcentration of nitric oxide we used S-nitroso-./V-acetylpenicillamine (SNAP), anitric oxide generating agent (Brown et al., 1993; Gopalakrishna et al., 1993). It isshown that nitroarginine increases, while SNAP decreases, the synthesis ofglutamine in cultured astrocytes. These results indicate that nitric oxide modu-lates glutamine synthesis in brain.

METHODS

Chemicals. Dulbecco's modified Eagle's medium (DMEM) and fetal bovineserum (FBS) were from GIBCO; gentamicin sulfate was from ICN. Theion-exchange resins, Amberlite CG-50, hydrogen form, and AG 50W-X8, H+

form, 200-400 mesh, and AG 1-X8, acetate form, 200-400 mesh, were obtainedfrom Sigma and Bio-Rad, respectively. S-Nitroso-N-acetylpenicillamine (SNAP)was from Molecular Probes. L-[U-14C]Glutamic acn was washed with 1 ml ofwater and 1 ml of 0.5 N HC1 and glutamate was eluted with 1 ml of 1 N HC1.After evaporation with a Rotavapor, [14C]glutamate was dissolved in water.

Astrocyte Cultures. Rat cortical astrocyte cultures were prepared as de-scribed by Renau-Piqueras et al. (1989). The cerebral cortices from 1-day-oldWistar rat pups were dissected and the cortical tissue was finely minced andmechanically disrupted by pipetting in DMEM. The cell suspension was vortexedat maximum speed for 1 min and filtered through sterile nylon mesh of 90-/impore size. Dissociated cells were plated (80 cm2 per brain) on polystyrene 12-welltissue culture plates (Corning). Astrocytes were grown in DMEM containing 20%fetal bovine serum and 100 /Ag/ml gentamicin and maintained at 37°C in ahumidified atmosphere, 95% air/5% CO2. After 1 week, the serum content wasreduced to 10%. Culture medium was changed twice weekly. Cultures were used4 weeks after seeding.

Glutamate Metabolism. Astrocytes were rinsed three times with prewarmedsterile Hepes-buffered Hank's balanced solution (HBHS) (Martin and Shain,1979) and allowed to equilibrate for 15 min at 37°C. Astrocytes were incubated in500/xl of HBHS containing 60 /iM L-[14C]glutamate and SNAP (1 mM) or

Nitric Oxide and Glutamine Synthesis 435

nitroarginine (200 /iM) for up to 90 min. After 0, 10, 20, 30, 60, or 90 min,medium was removed and frozen at -70°C. This was considered the extracellularfraction. Intracellular fraction was prepared as described by Waniewski (1992).Briefly, the wells containing astrocytes were rinsed with cold HBHS solution andcells were collected in 1 nd buffered with a 1:1 mixture of 2.5 M KOH and2.4A/KHCO3 and cooled to 0-4°C. The KC1O4 precipitate was centrifuged at15,600g for 5 min at 0-4°C. The supernatants (intracellular fraction) were frozenat -70°C. Pelleted proteins were solubilized in 500 p\ of 0.5 N NaOH and proteincontent was measured by the BCA method (Pierce).

[14C]Glutamate and [HC]glutamine were measured in extracellular andintracellular fractions by passing the samples through two ion-exchange resins, asdescribed by Rothstein and Tabakoff (1984). Briefly, an aliquot of 100 ̂ 1 wasapplied to an AG 1-X8 acetate column (0.5 X 2 cm) mounted on top of anAmberlite CG-50 (H+) column (0.5 X 3.5 cm). Glutamine was recovered bywashing the columns with 2 ml of water. Glutamate was eluted by passing 2 ml of17VHC1 through the AG 1-X8 acetate column. Eluates containing glutamate orglutamine were collected and radioactivity was measured.

Assay of [l4C]Glutamate Uptake into Astrocytes, Uptake of glutamate byastrocytes was assayed as described by Waniewski and Martin (1986). Astrocytescultured in 24-well tissue culture plates were washed three times with HBHS andallowed to equilibrate in 250 yu.1 of the same medium for 15 min at 37°C. Sampleswere incubated with ['"Cjglutamate (0.15 /iCi/well) at concentrations rangingfrom 5 to 55 /nM. After 5 min, the medium was removed (extracellular fraction).Cells were washed rapidly four times with cold HBHS and resuspended in 250 p,\of 0.4 N NaOH (intracellular fraction). Protein was determined in this fractionand radioactivity was counted in both extracellular and intracellular fractions.Rates of [14C]glutamate uptake were calculated taking into account the radioac-tivity incorporated into the cells and the specific radioactivity of glutamateessentially as described by Marin et al. (1992). Cultured astrocytes were washedthree times at 5-min intervals with 2 ml of Krebs bicarbonate buffer (mA/: NaCl;124; KC1, 3.5; K2HPO4) 1.25; NaHCO3, 26.3; CaCl2, 0.75; MgSO4,1.2; glucose, 10;previously equilibrated with 95% O2/5% CO2) and preincubated in the samemedium for 20 min. 3-Isobutyl-l-methylxanthme (1 mAf) was added to inhibitphosphodiesterase and 1 mM SNAP or 200 /iM nitroarginine was added to somesamples. After 15 min of incubation at 37°C, the medium was removed and 1 mlof ethanol/formic acid (95/5) was added. Cells were resuspended and suspensionscentrifuged for 5 min at 10,000g. Pellets were used to measure protein. Thesupernatants were dried using a Rotavapor and cyclic GMP was measured usingthe cyclic GMP assay system from Amersham.

In vitro Assay of Glutamine Synthetase Activity. Glutamine synthetaseactivity was assayed in vitro in homogenates from cultured astrocytes. Astrocyteswere treated with SNAP (1 mM) or nitroarginine (200 /nM) as described abovefor glutamate metabolism experiments except that radioactively labeled glutamatewas omitted. After 90 min of incubation, cells were collected by centrifugation,resuspended in a final volume of 250 /zl of 50 mM imidazole-HCl, and disruptedby sonication at 4°C. Glutamine synthetase [14C]glutamate (0.5 ̂ Ci), 10 mM

436 Mifiana et al.

2-mercaptoethanol, 15 mM creatine phosphate, 0.25 U creatine kinase, and45-90/Ag cellular protein. Samples were incubated for 15 min at 37°C. Thereaction was stopped by adding 1 ml of ice-cold water and placing the tubesimmediately on ice. [14C]Glutamine formed was separated from [14C]glutamate byusing columns of Dowex AG 1x8 and Amberlite CG501 as described byHallermayer et al. (1981). Radioactivity incorporated into glutamine was countedin a liquid scintillation spectrometer.

The experimental protocols have been approved by the scientific committeeof the Institute and meet the guidelines of the European Union.

RESULTS AND DISCUSSION

As shown in Fig. 1, [14C]glutamate added to the culture medium was rapidlytaken up by astrocytes. In control astrocytes, about 75% of the glutamate addedwas taken up in the first 30 min of incubation. It seems that equilibrium wasreached then and the extracellular glutamate remained at the same level up to 90min. The intracellular level of [I4C]glutamate increased rapidly, reaching amaximum after =30 min of incubation (Fig. 2). Then intracellular glutamatedecreased, probably because of metabolism, mainly via glutamine synthesis.These kinetics are similar to those previously described by other groups (Farinelliand Nicklas, 1992; Waniewski and Martin, 1986; Waniewski, 1992).

Results shown in Figs. 1 and 2 suggest that SNAP seems to retard andnitroarginine to stimulate somewhat the transport of [14C]glutamate into astr-ocytes. We therefore studied the effects of SNAP or nitroarginine on the kineticsof the uptake of [14C]glutamate. As shown in Table I, SNAP markedly reducedthe Vmax for glutamate transport. Nitroarginine increased significantly both theVmax and the Km for glutamate transport. It seems therefore that, depending onthe extracellular concentration of glutamate, nitric oxide could modulate glutam-ate uptake by astrocytes. Nitric oxide could inhibit glutamate uptake as indicatedby the effect of SNAP (Table I); this is in agreement with a previous report byPogun et al. (1994). The effect of nitroarginine is unclear; since it increases boththe Vmax and the Km, the effect would depend on the glutamate concentration.Under our experimental conditions for the study of glutamate synthesis, it can beseen (Figs. 1 and 2) that at 10 or 20 min of incubation intracellular glutamate isincreased and extracellular glutamate decreased in samples treated with nitroar-ginine. This indicates that at these times, glutamate uptake is increased bynitroargnine. For longer times intracellular glutamate is not increased bynitroarginine. SNAP seems to retard somewhat glutamate uptake in the shortterm, but the effect is slight. The intracellular [14C]glutamate in astrocytes treatedwith SNAP was not different from that of controls at any time. In those treatedwith nitroarginine there was no significant difference at 30 min or later. Thisindicates that the effects of SNAP (and of nitroarginine after 20 min) on[14C]glutamine synthesis are unlikely to be a consequence of altered transport.

Nitric Oxide and Glutamine Synthesis 437

Fig. 1. Effect of SNAP and nitroarginine on extracellular [14C]glutamate concentration.Astrocytes were incubated in the presence of 1 mM SNAP or nitroarginine (200 fj,M)and [14C]glutamate (60/tM, 850,000 cpm/well) in Hanks-Hepes solution. At theindicated times, incubation medium was taken and [14C]glutamate was measured asindicated under Materials and Methods. Each value is the mean ± standard deviation ofthree experiments (each in triplicate). Glutamate levels are expressed as percentage ofinitial radioactivity added to each well. Values that are significantly different fromcontrols are indicated by asterisks (*/"«; 0.05; **/)«0.01).

[14C]Glutamine is synthesized by glutamine synthetase within the astrocytesbut much of the glutamine synthesized is released into the medium. Figures 3 and4 show the time courses of the intracellular and extracellular contents of[14C]glutamine. For control astrocytes, the level of intracellular glutamineincreased with time (Fig. 3). Under the conditions used, =20% of the radioac-tively labeled glutamate is metabolized to glutamine (Fig. 5). This quantity is lowcompared to the values (=80%) quoted by McKenna et al. (1996) but is moresimilar to the values (25-30%) reported by Waniewski and Martin (1986) and byWaniewski (1992), using culture conditions similar to those used in the presentwork. The reasons for this discrepancy are not clear, although it has been shown

438 Mifiana et at.

Fig. 2. Effects of SNAP and nitroarginine on intracellular [14C]glutamateaccumulation. Astrocytes were incubated in the presence of SNAP ornitroarginine and [14C]glutamate as indicated in the legend to Fig. 1. At theindicated times, incubation medium was removed and cells were resuspendedin perchloric acid. Intracellular radioactive glutamate was measured asindicated under Materials and Methods. Each point is the mean ± standarddeviation of three experiments (each in triplicate).

Table I. Effects of SNAP and of Nitroarginine on[14C]Glutamate Uptake by Astrocytes"

Treatment

ControlSNAPNitroarginine

vKmax

(nmol/minX mg prot.)

3.1 ± 0.51.3 ±0.37.4 ± 1.2

Km(»M)

23 ±418±382 ±14

" Astrocytes were cultured in 24-well plates. SNAP (1 mM ) ornitroarginine (200 pM) were added after 15 min of prein-cubation with HBHS. [14C]Glutamate was added immediatelyand its uptake after 5 min of incubation was determined asdescribed under Materials and Methods. Values are the meanof duplicate samples from three experiments using 5 to 55 /j,Mglutamate.

Nitric Oxide and Glutamine Synthesis 439

Fie. 3. Effects of SNAP and nitroarginine on intracellular levels of['X^glutamine. Astrocytes were incubated in the presence of SNAP ornitroarginine and [14C]glutamate as indicated in the legend to Fig. 1. Atthe indicated times, incubation medium was removed and cells wereresuspended in perchloric acid. Intracellular radioactive glutamine wasmeasured as indicated under Materials and Methods. Each point is themean ± standard deviation of three experiments (each in triplicate). Valuesthat are significantly different from controls are indicated (* P =50.01; */>« 0.001).

that metabolism of glutamate to glutamine is influenced by the culture conditionsand area of the brain used.

For astrocytes treated with nitroarginine, intracellular glutamine was sig-nificantly higher than in controls at 20 min but not at the other times. In contrast,in astrocytes treated with SNAP, intracellular glutamine increased more slowlythan in controls (Fig. 3). The intracellular content of [14C]glutamine is sig-nificantly lower at all times of incubation. This suggests that glutamine synthesis isdecreased in astrocytes treated with SNAP.

The effects of the treatments on the extracellular content of [14C]glutamineare shown in Fig. 4. For control astrocytes, the release of newly synthesizedglutamine into the medium was low until 30 min and then increased rapidly andlinearly up to 90 min. For astrocytes treated with SNAP the extracellular

440 Minana et al.

Fig. 4. Effects of SNAP and nitroarginine on [14C]glutamine release.Astrocytes were incubated in the presence of 1 mM SNAP or nitroarginine(200/xM) and [14C]gIutamate in Hanks-Hepes solution. At the indicatedtimes, incubation medium was taken and [ C]glutamine was measured asindicated under Materials and Methods. Each point is the mean ± standarddeviation of three experiments (each in triplicate). Values that aresignificantly different from controls are indicated (* P=s0.01; ** P*s 0.005;*/>*s 0.001).

concentration of glutamine was significantly lower than in controls at all timestested. In contrast, extracellular glutamine concentration was significantly higherafter 20 and 30 min in astrocytes treated with 200 fj,M nitroarginine than incontrols (but not at other time points).

Figure 5 shows the total amount (intracellular plus extracellular) of[14C]glutamine synthesized by astrocytes subjected to the different treatments.The synthesis of glutamine in astrocytes was linear with time up to 90 min. Assuggested by the above results, the synthesis of glutamine was significantlydecreased in astrocytes treated with SNAP. The reduction was by 39, 36, 53, 49,and 59% at 10, 20, 30, 60, and 90 min, respectively. Treatment of astrocytes with200 p,M nitroarginine slightly increased the synthesis of glutamine. The synthesiswas increased by 14, 63, 26, 19, and 16% at 10, 20, 30, 60, and 90 min,respectively.

Nitric Oxide and Glutamine Synthesis 441

Fig. 5. Time course of the synthesis of [14C]glutamine. Experiments werecarried out as described in the legends to Figs. 3 and 4. For each time, theradioactivity incorporated in glutamine in the extracellular (incubationmedium) and intracellular (perchloric acid extracts) fractions was deter-mined. The sum of intracellular plus extracellular [14C]glutamine is shown.Values are a combination of the data from Figs. 3 and 4. Values that aresignificantly different from controls are indicated (* P^sO.Ol; ** />« 0.005;* P« 0.001).

Under the conditions used, =85% of the initial radioactivity is recovered asglutamate or glutamine (Figs. 1-5). Some glutamate may be metabolized tointermediates of the tricarboxylic acid (McKenna et ai, 1996). Although it ispossible that the activity of some enzymes associated with the tricarboxylic acidcycle might be affected by nitric oxide, such an action is unlikely to explain theeffect of nitric oxide on glutamine synthesis.

To check that nitroarginine and SNAP were affecting the nitric oxidecontent, we measured the effects of these treatments on cGMP levels. Guanylatecyclase is activated by nitric oxide and formation of cyclic GMP is used as ameasure of nitric oxide levels (Garthwaite et ai, 1989; Miki et al., 1977). Wedetermined the content of cGMP in control astrocytes and in those treated for 15min with 1 mM SNAP or 200 ̂ M nitroarginine. For controls, the cGMP contentwas 2.4 ± 0.3 pmol/mg protein. For astrocytes treated with SNAP or nitroar-ginine, the cGMP contents were 12.6 ±3.3 and 1.9 ± 0.4 pmol/mg protein. These

442 Minana et al.

results indicate that SNAP actually increases the content of nitric oxidesignificantly, while nitroarginine decreases it slightly.

The above results indicate that nitric oxide modulates glutamine synthesis inintact astrocytes. Increasing the concentration of nitric oxide by using SNAPsignificantly reduces glutamine synthesis, clearly supporting the conclusion thatincreased nitric oxide levels produce a decrease in glutamine synthesis in vivo.

We assayed the in vitro activity of glutamine synthetase in homogenates ofastrocytes treated with SNAP or nitroarginine for 90 min. For homogenates fromcontrol astrocytes, glutamine synthetase activity was 302 ± 39 nmol/mg proteinper hr. For homogenates from astrocytes treated with 1 mM SNAP or 200 ^Mnitroarginine, the activities were 252 ± 32 and 329 ± 35 nmol/mg protein per hr,respectively. The slight reduction of the activity induced by treatment with SNAPwas statistically significant (p =£ 0.05).

The above results show that SNAP, a nitric oxide generating agent, reducesthe synthesis of glutamine markedly (by 36-59%). This indicates that nitric oxidewould modulate glutamine synthesis in vivo. This would explain the increase inbrain glutamine content in rats injected with nitroarginine reported previously(Kosenko et al., 1995). It should be noted that i.p. injection of nitroarginineincreased the glutamine content in whole brain by ^ISS^o (Kosenko et al., 1995).However, in the present work nitroarginine increased glutamine synthesis onlyslightly (Figs. 3-5). Under the conditions used, nitroarginine also reduced onlyslightly and nonsignificantly the formation of nitric oxide in astrocytes, asreflected by the small decrease in cGMP content. This could be explained if thebasal activity of nitric oxide synthase (NOS) in astrocytes is low. The large effectof nitroarginine on whole-brain glutamine content could be explained if glutaminesynthetase in astrocytes is modulated by nitric oxide synthesized in other celltypes. Nitric oxide is freely diffusible and it is known that nitric oxide synthesizedwithin a cell can affect neighboring cells. In brain, there are different types ofNOS in different cell types: neurons, epithelial cells, and astrocytes. However,nitric oxide synthase is located mainly in neurons (Murphy et al., 1993; Vincentand Kimura, 1992). It is then possible that the activity of glutamine synthetasewithin astrocytes could be modulated by nitric oxide synthesized within neighbor-ing neurons, which would maintain glutamine synthetase only partially active.Inhibition of neuronal NOS by nitroarginine could lead to release of inhibition ofGS by nitric oxide and would explain the remarkable increase in glutamineinduced by i.p. injection of nitroarginine (Kosenko et al., 1995).

From the above results it is not possible to discern whether nitric oxideaffects glutamine synthetase directly or indirectly via an intermediary whichwould affect glutamine synthetase.

Glutamine synthetase is a highly oxidatively sensitive enzyme. Oxidativeinactivation of glutamine synthetase subunits has been studied extensively inEscherichia coll (e.g., Cervera and Levine, 1988; Nakamura and Stadtman, 1984,and references therein). It has been shown that, for bacterial glutaminesynthetase, oxidative inactivation precedes proteolysis (Levine et al., 1981; Rivett,1985) and that inactivation is due to alterations of one histidine and one arginineresidue (Climent and Levine, 1991; Levine, 1983). Modulation of mammalian

Nitric Oxide and Glutamine Synthesis 443

glutamine synthetase activity by oxidation has not been as extensively studied. Ithas been shown that ischemia/reperfusion in gerbil brain results in loss ofglutamine synthetase activity, and it has been suggested that oxidative inactiva-tion of glutamine synthetase may be a critical factor in the neurotoxicity producedafter cerebral ischemia/reperfusion (Oliver et al., 1990). Moreover, the spin-trapping compound N-t-butyl-a-phenylnitrone decreases the loss of glutaminesynthetase (Carney et al., 1991), indicating that it is mediated by free radicals.These and other reports (e.g., Dicker and Cederbaum, 1993; Floyd and Carney,1992; Hensley et al., 1994; Lam et al., 1994) indicate that mammalian glutaminesynthetase can be inactivated in vivo under conditions of oxidative stress.

The modulation of glutamine synthetase by nitric oxide has been suggestedby the results reported by McBean et al. (1995). These authors have shown, usingcoronal slices of rat brain, that neurotoxic concentrations of N-methyl-D-aspartateinhibit the activity of glutamine synthetase by 21%. Preincubation of the sliceswith nitroarginine, to inhibit nitric oxide synthase, prevented the NMDA-inducedreduction in glutamine synthetase activity, suggesting that this enzyme isinactivated by nitric oxide synthesized by NOS following its activation by calciumentering through the ionic channel associated with the NMDA receptor.

The results presented here also support that glutamine synthetase activitycould be modulated in vivo by nitric oxide (a free radical), so that increased nitricoxide would decrease glutamine synthetase activity. This modulation could haveimportant physiological or pathological implications, especially in situations inwhich the production of nitric oxide is increased.

ACKNOWLEDGMENTS

This work was supported in part by a grant from the Fondo de Inves-tigaciones Sanitarias (93/0187). Elena Kosenko received a visiting fellowship fromthe Generalitat Valenciana. Goizane Marcaida was a research fellow of theConsellaria de Education y Ciencia de la Generalitat Valenciana. CarlosHermenegildo received the "Carmen y Severo Ochoa" fellowship of theAyuntamiento de Valencia.

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