221

Brain Research, 521 (1990) 221-228 Elsevier BRES 15667

Methylmercury uptake in rat primary astrocyte cultures: the role of the neutral amino acid transport system M. Aschner 1, N.B. Eberle 1, S. G o d e r i e 2 and H . K . K i m e l b e r g 2 1Department of Pharmacology and Toxicology and 2Division of Neurosurgery, and the Interdepartmental Neuroscience Training Program, Albany Medical College, Albany, NY 12208 (U.S.A.) (Accepted 24 April 1990) Key words: Methylmercury; L-Cysteine; Astrocyte; Transport; Rat

The significance of the dense labeling pattern of methylmercury (MeHg) over astrocytes in areas of damaged cortex remains obscure, and the extent to which individual neurons are altered by MeHg accumulation in astrocytes is unknown. As a first step in understanding the relationship between the astrocyte and the mechanisms of MeHg's neurotoxicity, studies were directed at how MeHg is transported into cultured astrocytes. Uptake of [2°3Hg]MeHg in primary astrocyte cultures from neonatal rat cerebral cortex following incubations with MeHgCI conformed to a simple diffusion process. Uptake of [2°3Hg]MeHgby astrocytes exhibited the kinetic criteria of a specific transport system when added to the media as the L-cysteine conjugate. Saturation kinetics, substrate specificity and inhibition, and trans-stimulation were demonstrated in the presence of this SH-containing amino acid. Cysteine-mediated uptake of MeHg was inhibited by the coadministration of L-methionine, and 2-aminobicyclo-[2,2,1]-heptane-2-carboxylicacid. 2-Methylaminoisobutyric acid was ineffective in inhibiting the uptake of the MeHg-cysteine conjugate. Preloading of the astrocytes with glutamate was moderately effective in trans-stimulating the uptake of MeHg-cysteine conjugates, while in the absence of cysteine, uptake of [2°3Hg]MeHg was unchanged. These results indicate the presence in astrocytes of a neutral amino acid carrier transport System L, capable of selectively mediating cysteine-MeHg uptake. The substrate specificity and high affinity of this transport system resemble the properties of the System L neutral amino acid transport across the blood-brain barrier in the rat. Cellular uptake of MeHg-cysteine conjugates was not inhibited by preincubation of astrocytes with 100/~M N-ethylmaleimide or NaF. Hence, endocytotic or pinocytotic mechanisms, and shuttling of MeHg via sequential sulfhydryl membrane ligand exchange do not appear to operate in the transport of MeHg into the astrocyte. INTRODUCTION M e H g is a particular threat to the CNS in humans, as evidenced by the tragic epidemics of MeHg poisoning in Japan and Iraq. Because methylation of inorganic mercury species to MeHg by micro-organisms is known to take place in waterways 19, resulting in its accumulation in the food chain, any source of environmental mercury represents a potential for M e H g poisoning. The acidification of freshwater streams and lakes in Northern Europe and North America, and the impoundment of water for large hydroelectric schemes has led to further increases in M e H g concentrations in fish 35, posing increasingly greater risk to human populations. Although it was previously assumed that MeHg formed lipid soluble compounds in the body, and therefore could passively diffuse across membranes 15, a lipid soluble form is only produced by the addition of concentrated hydrochloric acid to tissue homogenates in the process of MeHg extraction for analytical purposes 12, or by treatment with sodium selenide resulting in the formation of bis-dimethyl mercury selenide 22. All other

studies have found MeHg to be associated with water soluble molecules such as proteins or thiol-containing amino acids and peptides. The very high chemical affinity of the MeHg cation (CHaHg ÷) for sulfhydryl ( - S H ) groups provides the theoretical basis for these observations s. MeHg in the environment is produced by methylation of inorganic mercury by micro-organisms. An aerobic pathway involves methylation of homocysteine-bound inorganic Hg 2+ by those processes in the cell normally responsible for the formation of methionine 19. In other words, the Hg-cysteine complex is methylated by 'mistake'. Aschner and Clarkson 1'2 drew attention to the close structural similarity between MeHg-L-cysteine complexes and methionine, and demonstrated cysteinefacilitated transport of M e H g across the blood-brain barrier (BBB). This transport is inhibited by coadministration of neutral amino acids, both those that do and do not contain - S H groups. Recognition of MeHg as an important environment contaminant necessitates the development of experimental models to study its pathogenesis. Since in primate

Correspondence: M. Aschner, Department of Pharmacology and Toxicology, Albany Medical College, Albany, NY 12208, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

222 brain m a t e r i a l m o s t o f t h e M e H g a p p e a r s to be localized within glial cells 13"27"36, astrocytic p r e d i s p o s i t i o n for M e H g d a m a g e m a y o f f e r a possible e x p l a n a t i o n for its n e u r o t o x i c i t y . A t p r e s e n t , the f u n c t i o n a l significance o f the dense

labeling pattern

remains obscure.

of M e H g

o v e r astrocytes

In this p a p e r we p r e s e n t studies on

t r a n s p o r t m e c h a n i s m s for M e H g into p r i m a r y a s t r o c y t e cell c u l t u r e s f r o m n e o n a t a l rat c e r e b r a l c o r t e x , as a first step in e l u c i d a t i n g the r e l a t i o n s h i p b e t w e e n the a s t r o c y t e and M e H g ' s toxicity.

MATERIALS AND METHODS

Materials Radiolabeled MeHg was produced by methylation of radioactive mercuric chloride ([:°3Hg]MeHgClz; New England Nuclear) with tetramethyl tin, followed by two benzene extractions with sodium thiosulfate which separates the MeHg into the aqueous phase 38. The specific radioactivity of the [2°3Hg]CH3HgCI was 0.965/~Ci//~gHg; 137 ~Ci/ml. Cold vapor atomic absorption analysis by the method of Magos and Clarkson 23 indicated that >99% of the total Hg was in the organic form. Radioactive MeHg was synthesized and kindly supplied by Dr. T. Toribara according to his method. All other chemicals were from the highest analytical grade, and were obtained from the Sigma Chemical Company.

Cell culture Astrocytes were grown as primary cultures from neonatal rat brains u. Briefly, the cerebral cortices of newborn rats were removed, the meninges were carefully dissected off, and the tissue was dissociated using a neutral protease (Dispase, Boehringer Mannheim). The dissociated cells were seeded at 8 × 104 viable cells/ml in 12-well trays (1 ml of cell suspension and 4 cm 2 growing area per well) and grown for 3-5 weeks at 37 °C in a 5% CO2/95% O z atmosphere in Eagle's minimum essential medium with 10% horse serum (GIBCO) plus 100 units penicillin and 100 /~g streptomycin per ml. About 2-3% of the seeded cells attach to the culture dishes and attain a saturation density of 2-3 × I04 viable cells/cme by 3-4 weeks. These cultures show >95% staining of the cells for the astrocyte marker, glial fibrillary acidic protein (GFAP).

MeHg uptake determinations A description of the uptake of [2°3Hg]MeHgCI will serve as an example. In brief, the growth medium was removed and the astrocytes were rinsed rapidly 4 times with 1 ml of a N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)buffered Ringer's solution containing (in mM): NaCI 122, KCI 3.3, KH2PO 4 1.2, CaCI 2 1.3, MgSO4 0.4, o-glucose 10 and HEPES 25; acid adjusted to pH 7.4 with NaOH (total [Na ÷] was 140 mM). The total osmolality is approximately 300 osmol/kg. Cells were equilibrated in 1 ml of this medium at 37 °C for 30 min in a Wedco CO 2 incubator prior to the actual transport measurements. Uptake was initiated by aspirating the medium and adding 0.5 ml of prewarmed buffer containing radiolabeled [2°3Hg]MeHgCl. When uptake measurements were carried at 5 min or longer, the cells were returned to the incubator. The total radioactivity added was 0.2/aCi. After the appropriate times, uptake was terminated by rapid aspiration of the medium from the wells, followed by 4 × 1 ml washes with ice-cold (4 °C) 0.29 M mannitol solution buffered with 10 mM TRIS/TRIS nitrate, and also containing 0.5 mM calcium nitrate to maintain cell adhesion to the substrate. The solution was titrated to a pH 7.4 with HNO 3. Subsequently, the cells were dissolved in 1 ml t N NaOH. Aliquots (800 /A) were removed for counting in a Clini-Gamma LKB 1272 counter (Pharmacia), and for protein determination (100 /~1) according to the rapid method of Goldschmidt and Kimelberf 4. For the purpose of studies on the uptake

of MeHg-cysteine conjugates, cultures were allowed to equilibrate at 37 °C in HEPES-buffer under identical conditions. After 30 min of equilibration the astrocytes were treated with a similar HEPESbuffer containing [2°3Hg]MeHgCI (10 ~M) and c-cysteine at a final concentration between 0.02 and 2 raM. Initial rates of MeHg uptake by the astrocytes were calculated by using the cpm of accumulated [e°3Hg]MeHg and the specific radioactivity of MeHg at each substrate concentration, and expressed as the mean (_+_S.E.M.) ng Hg/mg protein/rain. While we have not measured the rate of conversion of cysteine to cystine, considering the structure and properties of these amino acids, it is implausible that cystine would affect the rate of uptake of MeHg-cysteine conjugates. The substratc specificity of the MeHg-cysteine conjugate for System L of the neutral amino acid carrier transport system and competition by related substances was also examined. After washing, as described above, warmed media containing 0.5 ml of [Z°3Hg]MeHgCI (10/~M) and ~-cysteine (20/~M) along with 50 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) 9. 1 mM c-methionine, or 10 mM 2-methylaminoisobutyric acid (MeAIB), was added to the cultures. At various times during the initial phase of uptake (up to 10 min), the wells were washed with ice-cold mannitol solution, and the amount of Hg (ng Hg/mg protein) in the astrocytes was determined, as described above. The role of Na ÷ in the transport of the MeHg-cysteine conjugate was studied by replacing the Na + in the HEPES-buffer with N-methyl-D-glucamine (NMDG) (final concentration 122 mM), both during the equilibration period, and at the time of treatment with [2°3Hg]MeHgC1 (10 #M) and L-cysteine (20/tM). Preloading of the astrocytes with u-glutamate was carried out by allowing the astrocytes to equilibrate for 30 min at 37 °C in identical media to that described for the uptake experiments, but with the addition of 10 I~M glutamate. Uptake of 10/~M [2°SHg]MeHgC1 was then measured as described above.

Transport of MeHg via membrane -SH ligand exchange, pinocytosis and endocytosis In order to assess the role of membrane - S H ligand exchange from MeHg-thiol complexes into the astrocyte, and the role of pinocytosis and endocytosis in MeHg transport, astrocytes were equilibrated for 30 min at 37 °C with 100 ktM of N-ethylmaleimide (NEM) and NaF, respectively. Uptake of [2°3Hg}MeHgCl (10 klM) as the cysteine (20 /~M) conjugate was carried out as previously described.

RESULTS

Uptake of MeHg by astrocytes T h e u p t a k e of [ 2 ° 3 H g ] M e H g C l i n t o c u l t u r e d a s t r o c y t e s i n c r e a s e s as a f u n c t i o n of t i m e for 10 m i n and subseq u e n t l y p l a t e a u s (Fig. 1A). Initial r a t e s (1 rain) of M e H g uptake show a linear dependence on the media concentration

of

MeHgC1.

Reciprocal

plots

(Lineweaver-

B u r k e ) of t h e u p t a k e p r o f i l e s h a v e a z e r o i n t e r c e p t o n the 1/V axis (Fig. 1B, inset), s u g g e s t i n g a s i m p l e diffusion process. Since at h i g h e r c o n c e n t r a t i o n s , M e H g C I e x e r t s c y t o t o x i c e f f e c t on v a r i o u s cell f u n c t i o n s , t h e t e s t e d r a n g e of M e H g c o n c e n t r a t i o n s was l i m i t e d to 5 × 10 -7 to 2 × 10 5 M. T h e c o n c l u s i o n t h a t M e H g C I e n t e r s by diffusion is b a s e d

on

the

linear

concentration

dependence

of

u p t a k e at low c o n c e n t r a t i o n s ( 0 . 5 - 2 0 / ~ M ) . A d m i t t e d l y , this is t h e s a m e b e h a v i o r e x p e c t e d if a s a t u r a b l e u p t a k e system w e r e o p e r a t i n g well b e l o w its Km. T h i s a p p e a r s unlikely because the linear concentration dependence

223

A

600 "

500 "

400 " ,m

60

om *M ¢D

300

m • []

50

~ 40

g~

200

~

30

~

20

.5 p M M e H g C I 5 pM MeHgC! 20 p M M e H g C I

~2 10 100

0

T

0 m

m "1"

0

2000

I

3"

1 2 I/s, pM MeHgCI

3

t

4000

6000

8000

time (sec.)

Fig. 1. A: rates of uptake of [2°3Hg]MeHgCI in astrocytes as a function of time. Uptake of [2°3Hg]MeHgC1 was measured as described in Materials and Methods in HEPES medium and is expressed as ngHg/mg protein (mean + S.E.M.). B: rate of uptake of [2°3Hg]MeHgCl (V) as a function of external [2°3Hg]MeHgCIconcentration(s). The mean uptake data shown in A are plotted as double reciprocal plots (l/V, l/s). The lines shown are the best fit of the data to the Michaelis-Menten equation. Uptake of [2°3Hg]MeHgC1is expressed as ngHg/mg protein/min.

appears to be maintained even at correspondingly higher concentrations of MeHgCl than those used in the presence of cysteine (see Fig. 2). Nonetheless, lack of saturation of the initial velocity over a limited concentration range does not exclude other uptake mechanisms, as for example, that uptake is limited by the rate of protein unfolding which uncovers new binding sites for MeHg. Fig. 2 shows the uptake of 10/tM [2°3Hg]MeHg in the presence of excess L-cysteine (20 /~M-2 mM) in the media. The initial rates of MeHg uptake show a nonlinear dependence on media L-cysteine concentrations conforming to saturation kinetics. Fig. 3A demonstrates the uptake of 10 /~M [2°3Hg]MeHg in the presence of 20 /+M o-cysteine as compared to L-cysteine. The reduced uptake of [2°3Hg]MeHg uptake in the presence of the D-enantiomorph of cysteine emphasizes the specificity of this transport system, indicating stereoselectivity in the recognition of the MeHg-L-cysteine conjugate. Since it seems likely that the MeHg-cysteine conjugate

is being transported on the L-neutral amino acid carrier we examined the effects of amino acids associated with Na+-independent System L on the uptake of MeHg as the cysteine conjugate. B C H (Fig. 3B) the model substrate for System I, and L-methionine (Fig. 3C) inhibited the initial rate of [2°3Hg]MeHgCl-L-cysteine uptake to various degrees, and also slowed the rate of approach to a steady state. M e A I B , an N-methylated model substrate for System A, was ineffective in inhibiting MeHg uptake (data not shown). MeHg-cysteine uptake was compared in N a t - c o n taining H E P E S buffer and in Na+-free buffer. Removal of medium Na ÷ caused a small inhibition of [2°3Hg]MeHg uptake (Fig. 3D). On calculating the cumulative results of 4 experiments, it became evident that the uptake of MeHg in the presence of Na t , expressed as a fraction of uptake in N a t - f r e e buffer increased as the incubation time increased. Decreased levels of MeHg uptake in astrocytes in the absence of medium Na t are likely to result from transport inhibition of a minor component of MeHg-cysteine conjugates on the N a t - d e p e n d e n t A and

224

120

A

100'

80 e~ °~

m.

• •

60 J

2 mM 0.5 mM 20 luM

¢m e~

4O o

20

•i 0,0

I

I

200

I

400

600

800

t i m e (sec.)

Fig. 2. Rates of uptake of 10/~M [2°3Hg]MeHgCl as the L-cysteine (20/~M-2 mM) conjugate in astrocytes as a function of time. Uptake of [2°3Hg]MeHgCl was measured as described in Materials and Methods in HEPES medium and is expressed as ngHg/mg protein (mean + S.E.M.). Uptake of [2°3Hg]MeHgC! is expressed as ngHg/mg proteirdmin.

A S C systems 1°. Nonetheless, M e H g uptake as the cysteine conjugate may be described as Na+-independent in the conventional way of not requiring Na ÷. Equilibration of astrocytes with glutamate (Fig. 4),

TABLE I Effects of 30 rain equilibration of primary astrocytes with NaF and NEM on the uptake of [2°3Hg]MeHgCl (10 tiM) and c-cysteine (20 I~M) conjugates Time (s)

30 60 120 300 600

Percent uptake vs controls (means + S. E. M. ) NaF (IO01~M)

NEM (IO01~M)*

100.3 + 13.6 114.0 + 3.4 114.0 + 0.3 105.4 + 2.7 106.7 + 2.7

120.0 + 5.0 131.4 + 7.0 125.1 + 2.0 121.6 + 3.0 130.9 + 3.1

* Astrocytes treated with NEM exhibited a statistically significant increase in the uptake of the MeHg-cysteine conjugate at all the time points. Statistical analysis was carried out by one-way ANOVA, followed by a post hoc Neuman-Keuls multiple comparison test at P < 0.0l.

followed by removal of the glutamate-containing media, and treatment with M e H g - c y s t e i n e resulted in an increase in the rate of [2°3Hg]MeHg uptake. Thirty minute equilibration of astrocytes with glutamate, and subsequent exposure to [2°3Hg]MeHg as the chloride salt (in the absence of L-cysteine) had no discernible effect on the rate of [2°3Hg]MeHg uptake (data not shown). Uptake o f methylmercury after pretreatment with N E M and N a F The data in Table I demonstrate that equilibration of astrocytes for 30 min in the presence of 100/~M N E M , an - S H alkylating agent, did not inhibit the initial rate of [2°3Hg]MeHg uptake. U p o n further analysis of the data by one-way analysis of variance (P < 0.05) followed by N e u m a n - K e u l s analysis, a significant stimulatory effect by N E M on the uptake of [2°3Hg]MeHg was noted (P < 0.001). Equilibration of astrocytes for 30 min with 100 /~M NaF, an inhibitor of pinocytosis, did not significantly affect the initial rate of M e H g - c y s t e i n e uptake.

225 60"

120

B

A

i n~ uo gl

I00

50"

80'

40"

60

30"

40

20-

B

B a

20

• 0~, 0

L-cysteine D-cysteine

I

!

!

200

400

600

0 800

time (see) 80 rn

C

control

a control * BCH

10" I

I

!

I

0

200

400

600

,oj

methionine

i ca

~o

60

60

40

4O

20

D

control

*

NMDG ( S o d i u m - f r e e )

t B

rn

0

time (see.)

[]

2O []

0

800

[] i

i

I

200

400

600

time (see.)

0 800

i

0

200

I

400

i

600

800

time (see.)

Fig. 3. A: rates of uptake of 10/~M [2°3Hg]MeHgCl as the L- and D-cysteine (20/~M) conjugate in astrocytes as a function of time. Uptake of [2°3Hg]MeHgCl in the presence of the o-enantiomorph of cysteine is greatly reduced, emphasizing the specificity of the transport system to the L-cysteine-MeHg conjugate. B: inhibition of [2°3Hg]MeHgCluptake in astrocytes by 50 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH). C: inhibition of [2°3Hg]MeHgCluptake in astrocytes by 1 mM L-methionine. D: [2°3Hg]MeHgCIuptake in astrocytes is reduced by substitution of Na + in HEPES medium with N-methyI-D-glucamine.[2°3Hg]MeHgCl(10/~M) was added to the media with 20/~M L-cysteine (A-D) or D-cysteine (A). Uptake of [2°3Hg]MeHgClwas measured as described in Materials and Methods in HEPES medium and is expressed as ngHg/mg protein (mean + S.E.M.). DISCUSSION A predisposition of astrocytes for MeHg damage offers a possible explanation for the observed neurotoxicity. Neuropathological evidence depicting degenerating neurons juxtaposed to densely 2°3MeHg-radiolabeled astrocytes ~3 may indicate an astrocyte-rnediated role in MeHg's toxicity on neurons. A direct toxic effect on astrocytes, themselves, would lead to failure of astrocyte homeostatic functions, resulting in neuronal impairment, injury and death. One possible example of such an indirect effect would be an 'excitotoxic' mechanism resulting from failure of glutamate uptake by the damaged astrocytes 4,25. Alternatively, the dense labeling of MeHg may constitute a reservoir for the continuous

release of MeHg from astrocytes, affecting adjacent neurons, and would be consistent with the original suggestion of Garman et al. 13, that astrocytes 'continue to take up or concentrate MeHg after the neurons have disappeared', i.e. after the neurons have been killed by MeHg released from the astrocytic reservoir. Hughes la was the first author to draw attention to the remarkable affinity of MeHg for the anionic form o f - S H groups. The principal chemical reaction of MeHg is with thiols; variations in distribution and effect of MeHg are dependent upon this reaction. The affinity of MeHg for the anionic form o f - S H groups (log K, where k is the affinity constant is in the order of 15-23), whereas its affinity constants for oxygen-, chloride-, or nitrogencontaining ligands such as carboxyl or amino groups are

226 80

60

qa, 40

20

glutamate * 0

i

0

200

control

i

i

400

600

800

time ( s e c . )

Fig. 4. [2°3Hg]MeHgCIuptake is enhanced by 30 min preincubation of astrocytes with 10/aM L-glutamate. [2°3Hg]MeHgCl(10/aM) was added to the media as the L-cysteine(20/aM) conjugate. Uptake of [2°3Hg]MeHgCl was measured as described in Materials and Methods in HEPES medium and is expressed as ngHg/mg protein (mean _+ S.E.M.).

about 10 orders of magnitude lower 8. Indeed, wherever a MeHg compound has been identified in biological media, it has been complexed to -SH-containing ligands; complexes with cysteine and glutathione have been identified in blood 24'3°, and complexes with glutathione have been identified in brain 37, liver26, and bile 31. Thus, any evaluation of the membrane transport of MeHg must recognize the functional importance of this reaction. To date, mechanisms of membrane transport of MeHg have been addressed only with regard to the transport of MeHg from blood to brain and from liver to bile. In both, transport of MeHg is closely linked to the transport of thiol-containing amino acids or peptides 1-3"16A7. We show that MeHgCl readily crosses into the astrocyte in the absence of L-cysteine in the media, corroborating observations by Lackowicz and Anderson 2~ that the MeHgCl salt can diffuse across lipid bilayers. Furthermore, in view of a concentration ratio of 2 × 10 6 to 1 of the MeHg-thiol complex to the chloride complex in v i v o TM, and the affinity constant of MeHg to - S H groups, it seems unlikely that a MeHg-chloride complex can contribute significantly to transport in vivo. We have found that when L-cysteine is present in the medium, [2°3Hg]MeHgCl is transported on the Na +independent neutral amino acid carrier transport System L, presumably as the MeHg-cysteine conjugate. A similar mechanism for the uptake of MeHg-cysteine conjugates has been described for the BBB endothelial capillary cell 1"2. Hence, the new finding in the present study is that the L-preferring system has the same specificity for MeHg-cysteine transport in astrocyte

cultures as the ~,-preferring system in capillary endothelial cells 7~29. The structural similarity of the cysteine conjugate of MeHg to methionine provides the theoretical basis for these findings. An important characteristic of a carrier-mediated transport system is the phenomenon of counter transport or accelerated exchange diffusion. For example, Cangiano et al. 6 observed that Na+-dependent accumulation of glutamine in brain microvessels trans-stimulated the uptake of neutral amino acids. Astrocytes represent a compartment in which exogenous glutamate is rapidly accumulated and extensively metabolized to glutamine, aspartate and 2-0xyglutarate. At low glutamate concentrations (10 gM) to preferred pathway is to glutamine. Within 15-30 min after addition of glutamate to the incubation media, and in the presence of NH4C1, the cellular content of glutamine is doubled; within 60-120 min virtually all the glutamine is transported into the media, presumably by the L-system39. The implication of the trans-stimulatory effect of glutamate on the uptake of MeHg-cysteine conjugates described here, extends beyond the general economy of large amino acid transport in the CNS. Since astrocytes are characterized by a high content of endogenous glutamine 39, its countertransport may offer a driving force for the concentrative mechanism of MeHg in these cells, and its preferential accumulation within astrocytes as evidenced by autoradiographic labeling 13. Despite the high thermodynamic stability of the M e H g - S H bond, very rapid exchange of MeHg between - S H groups is known to occur 3°. Recently, Snyder et al. 34 have postulated an - S H shuttle mechanism for the cellular uptake of auranofin, an orally active antiarthritic agent. This mechanism differs from traditional concepts of cellular uptake. It is not based on simple diffusion, it is not driven by a transmembrane concentration gradient, and it is not a specific facilitated diffusion or active transport process because no specific membrane-localized carrier system is necessary. The cellular uptake of auranofin is postulated to occur via a ligand exchange shuttle, shuttling-SH-bound auranofin via sequential membrane - S H groups into the cytoso134. However, this mechanism does not appear to apply to uptake of MeHg-cysteine conjugates by astrocytes since such uptake was not diminished by alkylation of membrane - S H groups by NEM. Rather, a stimulatory effect on the initial rate of uptake of MeHg, presumably, via dissociation of - S -CH3Hg + bonds and the formation of the lipid soluble MeHgCI salt, was seen. Absence of an inhibitory effect of NaF indicates that uptake of MeHg into astrocytes is not related to a stimulus-triggered pinocytosis or endocytosis 33. In summary, the pattern and inhibition of uptake of

227 M e H g - c y s t e i n e c o n j u g a t e s by p r i m a r y astrocyte cultures suggests that system L o f the neutral a m i n o acid t r a n s p o r t is p r e s e n t in the astrocytes, c o r r o b o r a t i n g recent findings by B r o o k e s 5. U p t a k e of M e H g as the L-cysteine adduct occurs p r e d o m i n a n t l y on the neutral a m i n o acid carrier L t r a n s p o r t system with s o m e c o n t r i b u t i o n from the A and A S C systems, and as discussed a b o v e , the M e H g cysteine c o n j u g a t e is likely to overwhelmingly r e p r e s e n t the f o r m o f M e H g in vivo. F u r t h e r m o r e , u p t a k e of M e H g into astrocytes does not a p p e a r to be r e l a t e d to endocytosis o r pinocytosis, or a sequential - S H ligand exchange. U p t a k e of M e H g in astrocytes could serve as a reservoir of, o r b e a site of action for the neurotoxicity of M e H g . R e c e n t l y , it has b e e n shown astrocytes derived from different b r a i n regions vary c o n s i d e r a b l y in the kinetic p a r a m e t e r s of g l u t a m a t e t r a n s p o r t 32, serotonin and catec h o l a m i n e u p t a k e 2°, and g l u t a m i n e synthetase activity28. O n e interesting extension of these findings afforded by

use of p r i m a r y cultures is e v a l u a t i o n of possible differences in the u p t a k e , efflux a n d toxicity of M e H g in astrocyte cultures from different regions of the CNS. Such differences m a y r e p r e s e n t possible d e t e r m i n a n t s of the regional toxicity associated with e x p o s u r e to this metal. It seems likely that future studies could be profitably d i r e c t e d at regional differences in the u p t a k e , m e t a b o l i s m , and release of M e H g by astrocytes, as a possible e x p l a n a t i o n for the differential n e u r o t o x i c i t y of this o r g a n o m e t a l .

REFERENCES

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