Neurochemical Research, Vol. 23, No. 6, 1998, pp. 869-873

Ammonia and Manganese Increase Arginine Uptake inCultured Astrocytes

Alan S. Hazell1,2 and Michael D. Norenberg1-4

(Accepted October 13, 1997)

Recent work has suggested a possible role for nitric oxide (NO) in the development of hepaticencephalopathy (HE). In this study, we examined the effect of ammonia and manganese, factorsimplicated in the pathogenesis of HE, on the transport of arginine (a precursor of NO) into primarycultures of astrocytes. Treatment with 5 mM ammonia for 1-4 days produced a maximal (53%)increase in L-arginine uptake at 3 days when compared to untreated cells. Kinetic analysis follow-ing 4-day treatment with 5 mM ammonia revealed an 82% increase in the Vmax and a 61% increasein the Km, value. Similar analysis with 100 uM manganese showed a 101% increase in Vmax and a131% increase in the Km value. These results suggest that both manganese and ammonia alter L-arginine uptake by modifying the transporter for arginine. A decrease of 32% in the non-saturablecomponent of L-arginine transport was also observed following treatment with ammonia. Whencultures were treated separately with 5 mM ammonia and 100 |O.M manganese for 2 days, theuptake of L-arginine increased by 41% and 57%, respectively. Combined exposure led to no furtherincrease in uptake. Our results suggest that ammonia and manganese may contribute to the patho-genesis of HE by influencing arginine transport and thus possibly NO synthesis in astrocytes.

KEY WORDS: Arginine transport; manganese; ammonia; hepatic encephalopathy; astrocyte.

INTRODUCTION

The pathogenesis of hepatic encephalopathy (HE)is incompletely understood. Although current evidencestrongly favors a functional role for ammonia in the de-velopment of this condition (1,2), our understanding ofhow it leads to CNS dysfunction remains uncertain. Re-cently, a role for manganese has been proposed since an

1 Laboratory of Neuropathology, Veterans Administration MedicalCenter.

2 Department of Pathology, University of Miami School of Medicine,Miami, FL 33101.

3 Department of Biochemistry & Molecular Biology, University of Mi-ami School of Medicine, Miami FL 33101.

4 Address reprint requests to Dr. M.D. Norenberg, Department of Pa-thology (D-33), P.O. Box 016960, University of Miami School ofMedicine, Miami, FL 33101 U.S.A. Tel. (305) 585-7017, FAX (305)545-6735.

8690364-3190/98/0600-0869$ 15.00/0 C 1998 Plenum Publishing Corporation

accumulation of this metal in the brains of patients withchronic HE has been identified (3,4). These findings areconsistent with magnetic resonance imaging data show-ing elevated signal intensities in affected subjects (5,6).

Astrocytes play an important role in HE (7). Whileacute HE is associated with swelling of these cells lead-ing to raised intracranial pressure, a characteristic mor-phological finding in chronically affected patients is thepresence of Alzheimer type II astrocytosis. Chronictreatment of cultured astrocytes with high doses of am-monia produces alterations that also resemble this latterchange (8,9). Previous studies have also demonstratedthat astrocytes are able to accumulate manganese to lev-els far in excess of that present in the external medium(10,11). The presence of similar Alzheimer type IIchanges is a feature of manganese toxicity (12). How-ever, the precise involvement of manganese in the de-velopment of HE is currently unknown.

870 Hazell and Norenberg

Fig. 1. Effect of ammonia (5 mM) on L-arginine uptake in culturedastrocytes examined during a 4 day period. Uptake was studied at anL-arginine concentration of 30 |O.M. Error bars represent SEM of 3separate experiments each performed in duplicate. *p < 0.05, **p <0.001 compared with control cells.

Recent studies have indicated a possible involve-ment of nitric oxide (NO) in HE. Rao et al. (13) showedincreased nitric oxide synthase (NOS) activity in thebrains of portacaval-shunted (PCS) rats, a model ofchronic HE. Additionally, we have shown increasedNOS activities in both hyperammonemic mice and micewith acute liver failure produced by thioacetamide (14),while Kosenko et al. (15) found that L-nitroarginine, anNOS inhibitor, reduced ammonia toxicity.

The basic amino acid arginine is considered the di-rect precursor of NO (16-18) and displays a high affinityfor the y+ transporter (19,20). Indications are that thiscarrier may be synonymous with a basic amino acidtransporter that was originally identified as a cell-surfaceretroviral receptor in the mouse (21,22), since renamedMCAT-1 (23). In view of the potential role of NO inthe pathogenesis of HE, we examined the uptake of L-arginine (a precursor of NO) following treatment withammonia and manganese in cultured astrocytes. A por-tion of this work has been presented in preliminary form(24).

EXPERIMENTAL PROCEDURE

Cell Culture Preparation. Primary astrocyte cultures from 1-2day old rats were grown as described by Booher and Sensenbrenner(25). Briefly, the cerebral cortex was removed and the tissue dissoci-ated and plated in 35 mm dishes. Cultures were maintained in serum-containing Dulbecco's modified Eagle medium, and the cells supple-mented with dibutyryl cyclic AMP after 2 weeks. Cells were grownfor a total of 3—5 weeks, during which the culture medium was

changed twice a week. At least 95% of cells were determined to beastrocytes, based on GFAP immunohistochemistry.

L-Arginine Uptake Assay. Uptake studies were carried out bypreincubating cells for 30 min to allow equilibrium in a solution con-taining the following (in mmol/L): 120 NaCl, 2.7 K.C1, 0.9 CaCl2, 0.5MgCl2, 6 glucose, 10 phosphate buffer, pH 7.4. This was followed byincubation in a similar media containing in addition 30 (iM L-arginine(approximating the Km for L-arginine uptake) and 0.2 |lCi/ml of [3H]L-arginine at 37°C. Uptake was stopped by rapid washing of cultureswith ice-cold buffer. Cells were then solubilized in 0.5 ml of 1 MNaOH. Sample aliquots were then measured for incorporated radio-activity (liquid scintillation counting) and protein content (26) for cal-culation of uptake rates.

Studies on the effect of ammonium chloride and manganese (II)chloride on L-arginine uptake were carried out for periods of up to 4days. The concentration of manganese used in this study (100 |O.M)was based on brain concentrations found in patients with HE (3,4).The incubation time for most experiments was 2 min as linearity ofuptake was maintained for at least 5 min at all concentrations of L-arginine studied (data not shown). Kinetic determinations were madefollowing incubation of cells for 2 min with L-arginine concentrationsof 1-1000 |iM, with correction of the resulting uptake rates for "zerotime" uptake at 0-4°C. Kinetic parameters were obtained by non-linear regression analysis utilizing the program Kaleidagraph (Abel-beck software) for the Macintosh. The resulting curves consisted of asaturable component following Michaelis-Menten kinetics and a non-saturable component, which most probably represents uptake by sim-ple diffusion and, therefore, will be described by kdiff.

Statistical Analysis. Comparisons of the effects of different treat-ments with ammonia were made using one-sample t-tests. Effects ofdifferent treatments between groups of cells were examined using ei-ther ANOVA with post hoc Bonferroni correction or the Mann-Whit-ney (7-test.

RESULTS

Chronic Effects of Ammonia and Manganese onL-Arginine Uptake. The effect of ammonia (5 mM) overa 4-day treatment period is shown in Fig. 1. In the pres-ence of 30 |J,M L-arginine, a maximal increase in L-arginine uptake of 53% was observed followingexposure to ammonia for three days (p < 0.001). Con-centration dependence studies were performed to deter-mine kinetic parameters following 4 days of treatmentwith 5 mM ammonia (Fig. 2), and after 2 days exposureto 100 uM manganese (Fig. 3). As shown in Table I,ammonia produced an 82% increase in Vmax comparedwith controls (p < 0.001). This effect was associatedwith a 61% increase in the Km (p < 0.05). In addition,a 32% decrease in the diffusion coefficient (kdiff) wasobserved (p < 0.001). Treatment with manganese re-sulted in a 101% increase in Vmax (p < 0.005) and a131% increase in the Km value (p < 0.05, Table I) withno change in kdiff.

Ammonia and Arginine Uptake 871

Fig. 2. Effect of ammonia on the concentration dependence of L-ar-ginine uptake in cultured astrocytes. Cells were exposed to varyingconcentrations of L-arginine ranging from 1 to 1000 uM following a4 day treatment with ammonia (5 mM) and L-arginine uptake wasmeasured. Results show the means ± SEM of 3 separate experimentseach performed in duplicate. Inset: Eadie-Hofstee plots from a repre-sentative experiment.

Combination of Ammonia and Manganese on L-Ar-ginine Uptake. The effect of a 2-day exposure to 5 mMammonia and 100 uM manganese, alone or in combi-nation, on L-arginine uptake was examined in the pres-ence of 30 |aM L-arginine (Fig. 4). Treatment withammonia and manganese led to a 41% (p < 0.05) and57% (p < 0.01) increase in L-arginine uptake comparedwith controls, respectively. Following exposure to a

combination of manganese and ammonia, uptake of L-arginine increased by 57% relative to control values (p< 0.01).

DISCUSSION

NO has been implicated in the pathophysiology ofseveral neurological disorders, including multiple scle-rosis, bacterial and viral infections, trauma, and ischemia(for review, see 27). Recent studies have indicated thatNO may also be involved in HE. Rao et al. (13) showedincreased NOS activity in the brains of PCS rats. Wehave also demonstrated increased NOS activities in bothhyperammonemic mice and mice with acute liver failureproduced by thioacetamide (14), while a reduction inammonia toxicity was reported following NOS inhibi-tion (15). Since the production of NO requires arginineas substrate, we chose to examine whether arginine up-take might be altered in response to chronic ammoniaand manganese treatment.

Treatment of astrocytes with either ammonia ormanganese led to an increase in both the Vmax and K™for arginine uptake at physiological concentrations ofthis amino acid. These results obtained following am-monia treatment are consistent with those of Rao et al.(13,28) who reported an increase in arginine uptake intocerebellar synaptosomes from PCS rats. Although somedifferences were observed in the kinetic parameters ofour study compared with those of Rao et al. (13,28),these can be accounted for by differences in preparations(cultured astrocytes vs. synaptosomes) and by variationsin brain regions examined. In addition, we also observeda pronounced decrease in the diffusional component ofuptake. Such findings suggest a complex interaction ofboth agents with processes related to the transport ofarginine into the cell. An increase in Km and/or decreasein diffusion coefficient tend to reduce the uptake of ar-ginine. The observed increases in Vmax may be represen-tative of an increase in transporter synthesis. Increasedarginine transporter synthesis has been shown bySchmidlin and Wiesinger (29,30) following treatmentwith bacterial lipopolysaccharide (LPS). Alternatively, amigration of additional preformed transporters from thecytosol into the plasma membrane may provide the basisfor these findings. Such a process has previously beenreported for the taurine transporter in renal cells (31).

An increase in Vmax with ammonia treatment mayalso be related to the ability of ammonia to increase thefluidity of biological membranes (32). This physical ef-fect has been associated with conformational changes in

Fig. 3. Effect of manganese (II) chloride on the concentration de-pendence of L-arginine uptake in cultured astrocytes. Cells were ex-posed to varying concentrations of L-arginine ranging from 1 to 1000U.M following a 2 day treatment with manganese (100 (J.M) and L-arginine uptake was measured. Results show the means ± SEM of 3separate experiments each performed in triplicate. Inset: Eadie-Hofsteeplots from a representative experiment.

872 Hazell and Norenberg

Fig. 4. Effect of manganese (II) chloride on the L-arginine uptakeresponse of astrocytes to ammonia. Cells were treated with 5 mMammonia for 2 days in the absence or presence of manganese (100(iM) and L-arginine uptake was measured. Uptake was studied at anL-arginine concentration of 30 \lM. Error bars represent SEM of 3separate experiments each performed in duplicate. *p < 0.05, **p <0.01 compared with control cells.

ported (41). Increased activities of the enzymesassociated with the arginine-glutamate shunt have alsobeen demonstrated in synaptosomes (42), suggesting arole for arginine as a precursor for glutamate, an aminoacid known to be depleted in HE/hyperammonemia (43).Also consistent with this possibility is the recent findingof decreased levels of arginine in brain during acute HE(44). The present results showing increased arginine up-take in astrocytes following ammonia treatment may in-dicate the existence of a similar pathway in astrocytesfor replenishing glutamate following its conversion toglutamine.

In conclusion, our results show that ammonia andmanganese increased arginine uptake into cultured astro-cytes. The findings provide evidence for the potentialinvolvement of arginine and possibly NO in the patho-physiology of HE.

ACKNOWLEDGMENTS

membrane bound proteins (33). Such alterations in mem-brane fluidity may influence the function of these pro-teins, leading to increased mobility and disorder withinthe lipid bilayer and possibly contribute to the decreasein diffusional uptake. Furthermore, the uptake of cal-cium into brain mitochondria is reduced by manganese(34), which may produce an increase in the intracellularfree calcium load resulting in phosphorylation of the ar-ginine transporter by calcium-dependent protein kinases.Indeed, activities of the GLT-1 (35,36) and EAAC1 (37)subtypes of glutamate transporter appear to be increasedby the activity of protein kinase C. Additionally, directactivation of protein kinases by manganese has previ-ously been demonstrated (38,39). Recently, Kanamoriand Ross (40) have reported an increase in intracellularpH in glial cells during severe hyperammonemia. Whileit is possible that such an ammonia-induced alkaliniza-tion may influence arginine transporter activity, this is-sue remains currently unresolved.

In thioacetamide-induced HE, an increase in argi-nine uptake in synaptosomal preparations has been re-

The technical assistance of Ms. Hongying Xi and Ms. HuapingLiu is gratefully acknowledged. This work was supported by researchgrants from the Department of Veterans Affairs and the National In-stitutes of Health (NS-30291 and DK-38153).

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Table I. Effect of Ammonia and Manganese on the Kinetic Parameters of L-Arginine Uptake in Cultured Astrocytes

Kinetic parameters

Kn.OiM)Vmax (nmol.miir-1.mg-1 protein)kdiff(nL.min-1.mg-1 protein)

Control

26.0 ± 4.60.73 ± 0.001.78 ± 0.06

NH4

42.0 ± 5.3°1.33 ± 0.01*1.20 ± 0.01*

Control

26.3 ± 0.21.16 ± 0.023.70 ± 0.50

Mn2+

60.8 ± 11.7a

2.34 ± 0.21b

2.20 ± 0.71

Astrocytes were treated with either 5 mM ammonia for 4 days or 100 |iM manganese for 2 days and then incubated with [3H]L-arginine for 2 minat 37°C. Data are mean ± SEM from 3 separate experiments each performed either in duplicate (ammonia) or triplicate (manganese). a p < 0.05;b p < 0.005 compared with control values.

Ammonia and Arginine Uptake 873

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