The role of -SH groups in methylmercuric chloride-induced D-aspartate and rubidium release from rat primary astrocyte cultures.

Methylmercuric chloride (MeHgCl) was shown to increase D-aspartate and rubidium (Rb; a marker for potassium) release from preloaded astrocytes in a dose- and time-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 MeHgCl on the efflux of radiolabeled D-aspartate as well as Rb. MeHgCl-induced D-aspartate and Rb release was completely inhibited by the addition of 1 mM DTT or GSH during the actual 5 min perfusion period with MeHgCl (10 microM). However, when added after MeHgCl treatment, this inhibition could not be fully sustained by GSH, while DTT fully inhibited the MeHgCl-induced release of D-aspartate. Neither DTT or GSH alone had any effect on the rate of astrocytic D-aspartate release. Accordingly, it is postulated that the stimulatory effect exerted by MeHgCl on astrocytic D-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 MeHgCl-induced D-aspartate release, suggesting that reversal of the D-aspartate carrier cannot be invoked to explain MeHgCl-induced D-aspartate release. Omission of Ca2+ from the perfusion solution increased the time-dependent MeHgCl-induced D-aspartate release.

Intracellular glutathione (GSH) levels modulate mercuric chloride (MC)- and methylmercuric chloride (MeHgCl)-induced amino acid release from neonatal rat primary astrocytes cultures.

Mercuric chloride (MC) and methylmercury (MeHg) were found to increase amino acid release from astrocytes. This suggests interaction with sulfhydryl (-SH) groups which are controlled by glutathione [GSH] levels. In the present study, we evaluated the effects of alterations in intracellular glutathione concentrations [GSH]i on the outcome of MC and MeHg treatment. [GSH]i were increased in a time-dependent fashion by incubating the astrocytes with 1 mM L-2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor. OTC attenuated the release of [2,3-3H]D-aspartic acid from astrocytes exposed to MC- (5 microM) and MeHg-(10 microM). MeHg-induced [3H]D-taurine release was also reduced by pretreatment of astrocytes with OTC. Treatment with BSO (50 microM) decreased [GSH]i in astrocytes, and increased [2,3-3H]D-aspartate release from MC- and MeHg-treated astrocytes, and [3H]D-taurine release from MeHg-treated cells. Neither OTC nor BSO when added to cultures in the absence of MC or MeHg had an effect on amino acid release by astrocytes. The current study underscores both the sensitivity of astrocytes to mercurials in terms of amino acid release and the relationship of these effects of astrocytic [GSH]i.

The role of sulfhydryl groups in D-aspartate and rubidium release from neonatal rat primary astrocyte cultures.

We have recently demonstrated that both methylmercury (MeHg) and mercuric chloride (MC) induce D-aspartate release from neonatal rat primary astrocyte cultures maintained in isotonic conditions. In the present study, we compare several other sulfhydryl-(-SH) selective alkylating reagents [methyl methanethiosulfonate (MMTS), N-ethylmaleimide (NEM), and iodoacetamide (IA)] in isotonic, as well as hypotonic conditions to discern the functional importance of -SH groups in [3H]D-aspartate and 86rubidium (86Rb) release from astrocytes. Treatment of astrocytes (5 min) in isotonic buffer with the hydrophobic reagent NEM (10 microM) caused a marked increase in 86Rb release but had no effect on [3H]D-aspartate release. Neither IA-, nor MMTS-treatment (both at 10 microM) induced increase in [3H]D-aspartate or 86Rb release in isotonic buffer. In hypotonic condition (-50 mM Na+), astrocytes were most sensitive to MC exposure (5 microM), exhibiting an increase in both [3H]D-aspartate and 86Rb efflux. The hydrophobic compounds MMTS and NEM, and the hydrophilic -SH modifying reagent, IA, attenuated the hypotonic-induced efflux of [3H]D-aspartate, in the absence of an effect on 86Rb release. These observations are consistent with a critical role for -SH groups both in basal (i.e. isotonic) and hypotonic-induced release of D-aspartate and Rb from astrocytes. Lack of uniformity of these effects may be attributed to site-specificity, related to the physicochemical properties of these -SH alkylating reagents.

Adenosine modulates methylmercuric chloride (MeHgCl)-induced D-aspartate release from neonatal rat primary astrocyte cultures.

The effects of adenosine, and selective adenosine receptor agonists and antagonists on methylmercury (MeHg)-induced aspartate release were studied in neonatal rat primary astrocyte cultures. Whereas basal levels of D-[3H]aspartate release were unchanged upon treatment with adenosine or the selective A1 receptor agonists, N6-cyclopentyladenosine (CPA), cyclohexyladenosine (CHA), and R-phenylisopropyladenosine (R-PIA), all partially reversed the MeHg-induced release of D-aspartate. Treatment of astrocytes with the xanthine derivative, theophylline, an adenosine antagonist, reversed the inhibitory effect of adenosine on MeHg-induced D-[3H]aspartate release. Since the effect of MeHg on D-[3H]aspartate release is known to be associated with sulfhydryl (-SH) groups which are controlled by intracellular glutathione concentrations [GSH]i, we also evaluated the effects of adenosine, the A1 agonists CPA and CHP, and the adenosine antagonist, theophylline, on astrocytic [GSH]i. Attenuation of the stimulatory effect of MeHg on D-[3H]aspartate release by adenosine and its agonists occurred in the presence of reduced astrocytic [GSH]i, suggesting that other mechanisms must be invoked for this protective effect. Whilst the mechanism of MeHg-induced D-[3H]aspartate release is not known, the data suggest a role for adenosine in its regulation.

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

Mercuric chloride (HgCl2; MC) was shown to increase D-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 D-aspartate. MC-induced D-aspartate release was completely inhibited by the addition of 1 mM DTT or GSH during the actual 5 min perfusion period with MC (5 microM). However, when added after MC treatment, this inhibition could not be sustained by GSH, while DTT fully inhibited the MC-induced release of D-aspartate. Neither DTT nor GSH alone had any effect on the rate of astrocytic D-aspartate release. Accordingly, it is postulated that the stimulatory effect exerted by MC on astrocytic D-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 D-aspartate release, suggesting that reversal of the D-aspartate carrier can not be invoked to explain MC-induced D-aspartate release. Furthermore, MC did not appear to be associated with astrocytic swelling.

Differential sensitivity of neonatal rat astrocyte cultures to mercuric chloride (MC) and methylmercury (MeHg): studies on K+ and amino acid transport and metallothionein (MT) induction.

Mercuric chloride (MC, Hg2+) and methylmercury (MeHg, CH3Hg+) significantly inhibited the initial rates of uptake of 86Rb (a tracer for K+), as well as the Na(+)-dependent uptake of [3H]-L-glutamate. Both mercury species were also found to increase [3H]-D-aspartate and 86Rb release from cultured astrocytes. Astrocytes were more sensitive to the effects of MC with IC50's for glutamate and Rb uptake an order of magnitude lower than those noted for the organic species (MeHg). Increased potency, and irreversibility relative to MeHg, were also noted for MC induced astrocytic D-aspartate and Rb release. These observations support the hypothesis that the astrocyte plasma membrane is an important target for mercurials and specifically that low concentrations of MC and MeHg inhibit the ability of astrocytes to maintain transmembrane ion gradients. The propensity of MC to interfere with astrocytic functions, relative to MeHg, was also corroborated by measurements on the inducibility of the astrocytic metalloprotein, metallothionein (MT). Whilst a dose-dependent increase in MT protein synthesis occurred upon exposure to either MC or MeHg, MC was shown to be the more potent of the mercurials. The greater susceptibility of astrocytes to MC compared with MeHg lends support, at the cellular level, to the hypothesis that accumulation of inorganic mercury (MC) at an order of magnitude lower concentration than MeHg, may be equally neurotoxic.

Astrocytes as mediators of methylmercury neurotoxicity: effects on D-aspartate and serotonin uptake.

In this study we address the effects of methylmercuric chloride (MeHgCl), a metal that is preferentially sequestered in astrocytes, on 5-HT and glutamate/aspartate uptake by rat primary astrocyte cultures. Quantitative autoradiography (ARG) combined with glial acidic fibrillary protein (GFAP) immunocytochemistry, as well as intact-cell (bulk) measurements of radiolabel uptake of these neurotransmitters were performed in 7- and 21-day-old primary astrocyte cultures. MeHg (10 microM for 30 min) treatment of astrocytes (21 days in culture) significantly inhibited the Na(+)-dependent and fluoxetine-sensitive [3H]5-HT uptake. D-aspartate uptake in 7- and 21-day-old cultures was even more sensitive to MeHg, leading to > 99% inhibition of D-aspartate uptake by astrocytes (30 min; 10 microM MeHg). These results imply that the Na(+)-dependent and fluoxetine-sensitive 5-HT uptake, as well as the Na(+)-dependent L-glutamate/D-aspartate uptake systems in primary astrocyte cultures are sensitive to low concentrations of MeHg. Since astrocytic removal of glutamate (and aspartate) and 5-HT from the extracellular space in situ is crucial to the maintenance of chemical homeostasis, MeHg-induced uptake inhibition of 5-HT and aspartate could have cytotoxic effects on neighboring neurons.

Astrocytes as potential modulators of mercuric chloride neurotoxicity.

1. MC has been shown to inhibit the uptake of L-glutamate and increase D-aspartate release from preloaded astrocytes in a dose-dependent fashion. 2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-induced D-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5 microM); when added after MC treatment, DTT fully inhibits the MC-induced D-aspartate release, while GSH does not. 3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocytic D-aspartate release. Other studies demonstrate that although MC treatment (5 microM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate. 4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocytic D-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca(2+)-channel antagonist nimodipine (10 microM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.