Stimulation of d -aspartate efflux by mercuric chloride from rat primary astrocyte cultures

261

Developmental Brain Research, 75 (1993) 261-268 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

BRESD 51697

Stimulation of D-aspartate efflux by mercuric chloride from rat primary astrocyte cultures K.J. Mullaney

a,

D. Vitarella

a,

J. Albrecht

c,

H.K. Kimelberg band M. Aschner

a

Department ofa Pharmacology and Toxicology, and b Dicision of Neurosurgery, Albany Medical College, Albany, NY 12208 (USA) and C Department of Neuropathology, Medical Research Centre, Polish Academy of Sciences, Warsaw (Poland)

(Accepted 25 May 1993)

Key words: Mercuric chloride; o-Aspartate; Astrocyte

Mercuric chloride (HgCI 2 ; MC) was shown to increase o-aspartate release from preloaded astrocytes in a dose-dependent fashion. Two sulfhydryl (-SH) protecting agents, a cell membrane non-penetrating compound, reduced glutathione (GSH), and the membrane-permeable dithiothreitol (DTT), were found to inhibit the stimulatory action of MC on the efflux of radiolabeled o-aspartate. MC-induced o-aspartate release was completely inhibited by the addition of 1 mM DTI or GSH during the actual 5 min perfusion period with MC (5 I'M). However, when added after MC treatment, this inhibition could not be sustained by GSH, while DTT fully inhibited the MC-induced release of o-aspartate. Neither DTI nor GSH alone had any effect on the rate of astrocytic o-aspartate release. Accordingly, it is postulated that the stimulatory effect exerted by MC on astrocytic o-aspartate release is associated with vulnerable -SH groups located within, but not on the surface of the cell membrane. Omission of Na + from the perfusion solution did not accelerate MC-induced o-aspartate release, suggesting that reversal of the o-aspartate carrier can not be invoked to explain MC-induced o-aspartate release. Furthermore, MC did not appear to be associated with astrocytic swelling.

INTRODUCTION

Exposure to mercury vapor (HgO) is known to exert neurotoxic effects, which are believed to be subsequent to the biotransformation of HgO to inorganic mercury (Hg2+) within the CNS 20,29. However, the biochemical mechanisms underlying Hg 2 + neurotoxicity have not been clearly defined. Recently, it was observed that treatment with low micromolar concentrations of Mercuric chloride (HgCI 2 , MC) inhibits the uptake of the excitotoxic neurotransmitter, L-glutamate, into cultured mouse and rat cerebral cortical astrocytes 1,8,9. Since astrocytes are a major site of glutamate inactivation I5 ,22,38, their dysfunction may indirectly contribute to MC-induced damage to juxtaposed neurons l - 3 . To further test whether astrocytic homeostasis may be affected by MC, we studied its effect on D-aspartate (a non metabolizable substrate for the Na +-dependent L-glutamate/L-aspartate carriers) efflux from preloaded astrocytes. Exposure of astrocytes to MC lead to a dose-dependent increase in the release of D-

aspartate. To ascertain whether stimulation of Daspartate release by MC results from its interaction with -SH groups on the surface and/or within the astrocytic membrane, the ability of reduced glutathione (GSH) and dithiothreitol (DTT) to inhibit the MC-induced D-aspartate release was evaluated. MATERIALS AND METHODS Materials 0-[2,3- 'HjAspartic acid and Na~l Cr04 were purchased from

Amersham Corp. (Arlington Heights, IL). All other chemicals were purchased from the Sigma Chemical Co. (St. Louis, MO). Cell culture

The cerebral hemispheres of newborn rats (Sprague-Dawley) were removed, the meninges were carefully dissected off, and the tissue was dissociated using Dispase II (Boehringer-Mannheim Biochemicals; neutral protease, Dispase Grade II). Cultures were prepared as described by Frangakis and Kimelberg\6 and grown on a plastic cell support film (Belko Biotechnology, Vineland, NJ). They were used after approximately 3-4 weeks when cells had reached a confluent monolayer. Immunocytochemically, ~ 95% of the cells stained positively for the astrocytic marker, glial fibrillary acidic protein (GFAP), using a previously reported procedure l6 . Cell viabil-

Correspondence: M. Aschner, Department of Pharmacology/Toxicology A-136, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA. Fax: (l) (518) 262-5799.

262 calculated by summing the effluxed radioactivity to that point including the remaining radioactivity counted in the cell support film. A computer program (Microsoft Excel) was adopted for the calculations.

ity was also routinely measured by the Trypan blue exclusion method (20% v Iv of 0.4% staining solution). D-[2,3- 3 H]Aspartate and Na~lCr04 efflux measurements The control bathing medium for all experiments, unless otherwise noted, consisted of the following: 122 mM NaCl, 3.3 mM KCI, 0.4 mM MgS04, 1.3 mM CaCl z, 1.2 mM KH Z P04 , 10 mM D-glucose, and 25 mM HEPES (N-2-hydroxyethylpiperazine N'-2-etahesulfonic acid). HEPES-buffered solutions were maintained at pH 7.4 by the addition of 1 N NaOH. The osmolality of the solutions was approximately 300 mosmol as measured by a freezing point osmometer (Advanced Instruments Inc., Needham Heights, MA). Astrocytes grown on the cell support film were transferred to a 60 mm dish and loaded overnight by the addition of 5 ml of warmed minimum essential medium (MEM) containing 10% horse serum, 40 /LCi of Na~ICr04 (radioactive concentration 1 /LCijml; specific activity 50 mCijmg Cr) and 20 /LCi D-[2,3- 3H]aspartic acid (radioactive concentration 1 /LCijml; specific activity 300 mCi/mg D-aspartate). Radiolabeled D-aspartate was used as a marker for intra-astroeytic glutamate and aspartate. All of these amino acids are likely to be transported on the same carrier protein and, if there are no major compartmentalization problems, the radioactive probe used at low concentrations but high specific activity should equilibrate with the entire pool of glutamate and aspartate and label it uniformly. Prior to actual efflux measurements the cell support film was washed (x 2) with 5 ml of HEPES buffer and carefully rolled into a cylinder shape, with the cells facing inward. This was then inserted into a 1 ml plastic tuberculin syringe, cut-off at the plunger end. The cells were continuously perfused at a constant rate of 1 mljmin. All the perfusion solutions were brought up to 37°C by heating the inflow tubing and syringe with a metal coil twisted around the outside surface and connected to a power source. The collected fractions (I ml each) were first counted in a Clini-Gamma LKB 1272 (Pharmacia, Gaithersburg, MD) to determine S1Cr radioactivity. Subsequently, Ecoscint (National Diagnostics, Manville, NJ) was added to each fraction and the f3 activity was determined by a Beckman LS 3801 Liquid Scintillation Analyzer (Beckman Instruments, Irvine, CAl. Results were expressed as fractional release of the respective emitter contained in the astrocytes at each time point. This was

Measurements of astrocyte volume changes Astrocytic volume was measured by substituting CI- with the non-permeable ion gluconate, and a novel dynamic method which measures electrical impedance z8 . Isotonic buffer composition is as follows: 22 mM NaCl, 200 mM mannitol, 3.3 mM KCl, 0.4 mM MgS04, 1.3 mM CaCl z, 1.2 mM KH Z P0 4 , 10 mM D-glucose, and 25 mM HEPES. 200 mM mannitol are omitted in hypotonic buffer ( - 200 mM). Briefly, astrocytes grown on coverslips are placed in a perfusion chamber containing a channel bridged between two 1 x 1 cm gold-plated electrodes (channel height is 100 /Lm). The gold electrodes are made by evaporating gold through a mask, at 10- 4 Torr, onto the bottom of the electrode. Leads from the gold electrodes are made of insulated copper wire, soldered to the gold with pure indium and the connections are covered with wax. The gold electrodes are connected through a large resistance (l Mil) to a lock-in amplifier that supplies a 500 Hz, 5 V signal to the system. The solutions used in the experiments are balanced to the same conductivity to assure that differences in impedance measurements are truly representative of changes in cell volume. As cell volume increases, the volume of the solution within the channel available for current flow decreases proportionally, resulting in an increase in measured resistance in the channel above the cells. Since V = IR (where V is voltage, I is current and R is resistance) and I is constant (500 Hz) changes in V are directly proportional to changes in R. The calculated initial height of the cell monolayer is approximately 5 /Lm. A 1% change in the voltage (or resistance) translates to a 1 /Lm change in the average astrocytic cell height of the monolayer. The paradigm of astrocytic cell volume measurement was identical to the one described above for efflux measurements. Twenty min perfusion with HEPES-buffer at 37°C followed by 5 min exposure to MC (5 /LM) followed by perfusion with HEPES (25 min). Swelling measurements were also combined with on-line measurements of radiolabeled D-aspartate and Cr release from astrocytes by collecting the effluent in 1 ml fractions.

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TIME (min.) Fig. 1. Effects of MC on the D-[2,3- 3H]aspartate and Na~1Cr04 release from astroeytes. Five minute {20-25 min).perfusion of astrocytes with MC (5 /LM) enhanced the release of D-[3H]aspartate above control levels. MC-induced release of D-[3H]aspartate was not accompanied by cell lysis or sloughing as indicated by measurements of Na~lCr04' Astroeytes were preloaded overnight with 20 /L Ci of D-[3H]aspartate and 40 /LCi of Na~1Cr04 prior to the actual efflux measurements. Cultures were then washed, and at time zero, they were perfused with HEPES-acid buffer. MC was added at 20-25 min. The perfusate was collected as described in Materials and Methods. Values shown are means of triplicate experiments (S.E.M. < 5%).

263 tate release with methyl methanethiosulfonate (MMTS; 50 and 100 ,uM) the Na~lCr04 method was found to be a valid indicator of non-specific efflux (results not shown).) Treatment of astrocytes with MC (5 ,uM; 20-25 min) simultaneously with either 1 mM GSH, an -SHprotecting agent that does not penetrate the astrocytic cell membrane (for references on this subject see ref. 19), or the membrane-permeable -SH-reagent, DTT (l mM), produced a complete inhibition of MC-induced o-aspartate release (Fig. 3A), presumably via complexation of MC with -SH groups (-S-Hg-S-). The inhibitory effect of GSH was not, however, observed when added 5 min after MC perfusion, whereas subsequent addition of 1 mM DTT completely inhibited the MC-induced release of o-aspartate (Fig. 3B). GSH or DTT added alone did not affect the rate of release of o-aspartate (results not shown). To determine whether the stimulatory effect of MC on astrocytic release of o-aspartate is related to the reversal of the o-aspartate carrier, as has been shown to occur for L-glutamate 5, Na + was omitted from the perfusion media and replaced with N-methyl-oglucamine (NMDG) at time O. As shown in Fig. 4, perfusion of astrocytes with Na+-free HEPES buffer led to increased release of o-aspartate (0-15 min) compared with controls. However, o-aspartate release upon MC treatment in Na +-free HEPES buffer was unchanged compared to its release in Na +-containing buffer. Since in several tissues mercurials are known to

RESULTS

D-[2,3- 3H]Aspartate and Na~lCr04 efflux measurements To assess the effect of MC on the glutamate / aspartate carrier, astrocytes were preloaded overnight with both o-[3H]aspartate and Nail Cr04" An inherent difficulty with continuous perfusion methods is that loss or lysis of cells will also contribute to radioactivity in the perfusate. To distinguish efflux from intact cells of preloaded o-[3H]aspartate from MC-induced cell lysis or sloughing we compared the astrocytic release of o-[3H]aspartate and Nail CrOll. As shown in Fig. 1, 5lCr release from preloaded astrocytes exposed to MC was negligible (range 0.00-0.11) at 5 ,uM MC while a progressive release of o-[3H]aspartate was observed. In the absence of MC, the loss of radioactive o-aspartate from the astrocytes exceeded that of 5lCr (range 0.220.30), attesting to net efflux of o-aspartate from preloaded astrocytes. The dose-dependency of MC treatment on astrocytic o-aspartate release is illustrated in Fig. 2. MC caused a dose-dependent increase in astrocytic release of o-aspartate. MC concentrations exceeding 2 ,uM caused a marked increase in o-aspartate release, with no effect observed at lower MC concentrations. MC-induced release of o-aspartate was somewhat delayed (4 min), even when accounting for 'dead space' within the system (approximately 90 s). Na~lCr04 was not detectable in the perfusate at 0-10 ,uM MC concentrations indicative of the absence of cell lysis or sloughing. (Using the same experimental paradigm for o-aspar-

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TIME (min.) Fig. 2. Dose-dependency of o-[2,3- 3H]aspartate efflux from primary astrocytes incubated with Me. Astrocytes were preloaded overnight with 20 /LCi of D-[3H)aspartate prior to the actual efflux measurements. Cultures were then washed, and at time zero on the x-axis, they were perfused with HEPES-acid buffer. MC was added at 20-25 min. The perfusate was collected as described in Materials and Methods. Values shown are means of triplicate experiments (S.E.M. < 5%).

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TIME (min.) Fig. 3. Effects of the addition of GSH (J mM) and DTT (J mM) on D-[2,3- 3 Hlaspartate release in cultured astrocytes treated with 5 p,M Me. A: when added simultaneously with MC, both DTT and GSH completely inhibited the effect of Me. B: when added after MC treatment (25-50 min) GSH could not inhibit the effect of MC, however MC-induced release of D-aspartate was fully inhibited by treatment with 1 mM DDT. Values shown are means of triplicate experiments (S.E.M. < 5%).

cause swelling 3,34 and a number of anion-exchange and co-transport inhibitors have been shown to inhibit hypotonic-induced release of o-aspartate 24, we examined their effectiveness in blocking the MC-induced release of o-aspartate. As shown in Fig. 5, 1 mM 4-acetamido4'-isothiocyanatostilbene-2,2'-disulfonic acid 10 (SITS), 1 mM bumetanide l7 , and 5 mM furosemide 6 , when added together with 5 J.LM MC (20-25 min) were completely effective in inhibiting the MC-induced release of o-aspartate throughout the efflux measurement (Fig. 5). However, when we examined whether MC-induced astrocytic o-aspartate release is a result of swelling-induced efflux we could not ascertain any change in cell volume. Utilizing the direct method of electrical impedance there was no evidence for MC-induced changes in cell volume (Fig. 6). A positive control,

exposure to a hypotonic HEPES buffer (- 200 mM mannitol; n = 6), caused marked astrocytic swelling (mean 5.6% increase in voltage, which translates to approximately doubling of the cell volume) followed by regulatory volume decrease 24 (RVD). Furthermore, substitution of CI - in the perfusion solution with the non-permeable anion, gluconate, did not alter the release of o-aspartate in MC-treated cells (results not shown). DISCUSSION

The present study suggests that astrocytic o-aspartate transport mechanisms are highly sensitive to inorganic mercury, supporting the concept that accelerated release of o-aspartate by astrocytes may mediate inorganic mercury neurotoxicity. MC is also known to in-

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Fig. 4. Effect of Na + on MC-induced D-[2,3- 3H]aspartate efflux in primary astrocytes incubated with MC (5 ,uM) in the presence and absence of Na +. Astrocytes were preloaded overnight with 20 ,uCi of D-[3H]aspartate. Cultures were then washed, and at time zero they were perfused with HEPES-acid buffer ± Na +. MC was added at 20-25 min. Omission of Na resulted in increased D-aspartate efflux prior to the addition of MC (20-25 min), with no appreciable differences at subsequent time points. The perfusate was collected as described in Materials and Methods. Values shown are means of triplicate experiments (S.E.M. < 5%).

hibit L-glutamate and o-aspartate uptake mechanisms 1,8. o-aspartate transport (both uptake and efflux) is a Na +-dependent, carrier-mediated, electrogenic process, with the Na + gradient plus the membrane potential providing the driving force 4,7,13,24. The finding that omission of extracellular sodium leads to accelerated basal release of o-aspartate (Fig. 4) supports the concept that o-aspartate transport is reversible and 'symmetric' in the sense that the o-aspartate carrier

mediates both uptake and efflux of o-aspartate-Na + complexes 13. Additional evidence is provided by the observation that depolarization stimulates o-aspartate efflux via the same Na+-dependent system I8 ,27,35. Therefore, activation of o-aspartate release by Me may be due to one, or any combination, of the following events: (a) cell membrane depolarization; (b) inhibition of the Na +/K+-ATPase activity, which keeps intracellular [Na +l low, (c) direct activation of the

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Fig. 5. The effects of SITS (l mM), bumetanide (l mM), and furosemide (5 mM) on MC (5 ,uM) induced D-[2,3- 3H]aspartic acid efflux in primary astrocytes. Astrocytes were preloaded overnight with 20 ,uCi of D-[ 3H]aspartate. They were then washed, and at time zero on the x-axis, cultures were perfused with HEPES-acid buffer. MC was added at 20-25 min. SITS, bumetanide and furosemide, added together with MC, were fully effective in inhibiting the MC-induced D-aspartate release. The perfusate was collected as described in Materials and Methods. Values shown are means of triplicate experiments (S.E.M. < 5%).

266 D-aspartate carrier, and (d) increased membrane permeability to D-aspartate. One can argue against possibility (b), because it takes about 1 h before inhibition of the Na +/K +-ATPase by ouabain results in dissipation of the astrocytic transmembraneous Na + gradient 23 • Although a decrease in the concentration of Na + in the medium (Fig. 4) enhances baseline Daspartate efflux corroborating previous observations 13 ,24, Na+-free media has no effect on MC-induced D-aspartate release, arguing against reversal of the D-aspartate carrier by MC treatment (c). It should be noted however that in the absence of sodium, MC may still be capable of activating the carrier while transporting D-aspartate in the reverse direction. Since mercurials, such as methylmercury (MeHg), are known to cause cellular swelling 3,34 , the possibility that MC-induced D-aspartate release is associated with astrocytic swelling was explored. Two lines of evidence refute this possibility. First, D-aspartate release from MC-treated astrocytes is not inhibited by replacing CIwith the impermeant anion, gluconate, a condition preventing astrocytic swelling. Secondly, direct measurements using an electrical impedance method for measuring volume changes combined with on-line radiolabel release measurements show marked release of D-aspartate upon MC treatment in the absence of changes in the measured resistance through the channel above the cells (Fig. 6). A positive control, namely, hypotonic-induced swelling ( - 200 mM mannitol), can be seen by the method of electrical impedance to induce marked astrocytic swelling and RVD (Fig. 6) as

well as D-aspartate release 37 • Accordingly, MC-induced swelling leading to non-specific opening of membrane channels can not be implicated as a mechanism for the augmented release of D-aspartate. The inability of MC vis-a-vi MeHg to induce astrocytic swelling can perhaps be attributed to the hydrophilic nature of MC, which restricts its permeability across the astrocytic membrane. Hughes 19 was the first author to draw attention to the remarkable affinity of mercurials for the anionic form of -SH groups. The principal chemical reaction of MC is with thiols; variations in distribution and effect of mercurials are dependent upon this reaction. The affinity of MC for the anionic form of -SH groups (log k, where k is the affinity constant and is in the order of 15-23), whereas its affinity constants for oxygen-, chloride-, or nitrogen-containing ligands such as carboxyl or amino groups are about 10 orders of magnitude lower. The MC-accessible -SH groups involved in D-aspartate transport could be assumed to be located (a) on the external surface of the cell membrane, (b) within the membrane bilayer, and/or (c) on the inner membrane surface. To distinguish between these possibilities, we compared the ability of two -SH-reducing agents, GSH and DTT, to reverse MC-induced Daspartate release in astrocytes. GSH is unlikely to penetrate beyond the external surface of the astrocytic cell membrane, which has not been reported to possess an effective GSH transport system and is rich in yglutamyl transpeptidase, a GSH-degrading enzyme 32,36. The astrocytic transport of DTT has not been studied;

TIME (min.)

Fig. 6. Volume changes in astrocytes upon exposure to MC (5 ,uM, 20-25 min) and hypotonic HEPES buffer ( - 200 mM mannitol; 15-45 min). As described in the text, the changes in voltage are related to changes in average volume and height of the astrocytic monolayer. No change in voltage was ascertained upon exposure to MC, while exposure to hypotonic solution resulted in a peak volume change around 3 minutes with a return to normal volume within 30 min, i.e. demonstrating RVD.

267 however, intramembraneous and intracellular effects of this compound in other tissues have been reported 11,21 • The observation that MC-induced D-aspartate release was fully inhibited by DTT, but not at all by GSH when added after MC treatment, was consistent with MC modifying the critical -SH groups associated with Daspartate transport, either within the membranes or on their internal surface. Since small water-soluble mercurials such as MC do not readily penetrate hydrophobic regions of the membrane 2,19 the former possibility appears most likely. Since anion-exchange and co-transport systems have been implicated in the induction of D-aspartate release, and since hypotonic media swelling-induced effluxes of amino acids are inhibited by anion-exchange and co-transport inhibitors 24, their effectiveness in inhibiting MC-induced amino acid release was tested. Furosemide (5 mM), SITS (l mM), and bumetanide (l mM) were all completely effective in inhibiting MC-induced D-aspartate release (Fig. 5). However, si9nce these agents are not known to complex with MC and MC-induced astrocytic swelling is absent, the mechanisms associated with such inhibition remain obscure. It would appear, however, that MC- and hypotonic-induced release of D-aspartate may share common functional groups because they are both inhibited by furosemide, SITS, and bumetanide. In summary, we have demonstrated for the first time that MC affects the efflux of D-aspartate from preloaded astrocytes. This effect is mediated via -SHsensitive sites, and it can be completely inhibited with permeable -SH reagents, such as DTT. This effect of MC is not surprising because it is known that MC will disturb many membrane functions, such as ion and electrolyte transport, and various enzyme activities 2•25 ,26. The simultaneous occurrence of decreased uptake!, and increased efflux of excitatory amino acids in MC-treated astrocytes, persuasively supports the argument that Me alters specific sites in the membrane. Given the complex inter-relationships between proteins and lipids, and the ability of MC to interact with both, its effect on the cell membrane is extremely complex, reflecting the varied roles played by sensitive -SH membrane components 25 .33 • Regardless of the mechanism by which MC promotes the efflux of D-aspartate, this study shows that MCdamaged astrocytes can serve as a source for the release of excitatory amino acids, further increasing their extracellular concentrations. Abnormally high levels of excitatory amino acids causing exaggerat'ed stimulation of excitatory amino acid receptors on the surface of adjacent neurons can trigger a destructive cascade of events that can damage neurons en masse 12 ,14,30. It

seems likely that future studies could be profitably directed at the role of astrocytes in MC-induced neurotoxicity via these mechanisms. Acknowledgments. J.A.'s visit to the Department of Pharmacology and Toxicology at the Albany Medical College was partly supported by the Jurzykowski Foundation. The study was also supported in part by PHS Grants NIEHS 05223 awarded to M.A., NS 23750 and NS 30303 awarded to H.K.K., and R-8I921O awarded to M.A by the USEPA

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