Inhibition of regulatory volume decrease in swollen rat primary astrocyte cultures by methylmercury is due to increased amiloride-sensitive Na+ uptake.

Primary astrocyte cultures from neonatal rats were swollen by exposure to hypotonic buffer with and without 10 microM methylmercury (MeHg). We investigated the effects of MeHg on K+ (using 86Rb), taurine, D-aspartate (a non metabolizable analogue of glutamate) and Na+ fluxes during regulatory volume decrease (RVD), with an electrical impedance method for determination of cell volume, coupled with on-line measurements of efflux of radioactive ions and amino acids. Addition of 10 microM MeHg completely inhibited RVD in swollen astrocytes, increased the uptake of 22Na+, increased 86Rb release, and decreased 3H-taurine release. There was no effect on the rate of release of 3H-D-aspartate from swollen astrocytes. 0.5 mM amiloride completely inhibited MeHg-induced increased Na+ influx during RVD, while 1 mM furosemide had no effect. When Na+ in the hypotonic buffer was replaced with N-methyl-D-glucamine (NMDG), RVD in the presence of MeHg was indistinguishable from controls. These results indicate that MeHg increases cellular permeability to ions such as Na+ and K+, and that an increase in Na+ permeability via Na+/H+ exchange, offsetting K+ loss, is the primary mechanism in its inhibition of RVD in swollen astrocytes.

Metallothionein induction protects swollen rat primary astrocyte cultures from methylmercury-induced inhibition of regulatory volume decrease.

Metallothionein (MT) proteins have been postulated to play a role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, and MT-1 and MT-2 are astrocyte-specific MT isoforms, we investigated the ability of MTs to attenuate MeHg-induced cytotoxicity. The toxic effects of MeHg on astrocytes were investigated in a model of regulatory volume decrease (RVD) in which the cells are swollen by exposure to a hypotonic buffer. Preexposure to CdCl2 (1 microM) for 72, 96 or 120 h, prior to acute exposure to hypotonic buffer and MeHg (10 microM) led to a time-dependent increase in the intracellular levels of astrocyte MT proteins. The acute MeHg-induced inhibition of RVD was significantly, and almost fully reversed by preexposure to CdCl2. This reversal was time-dependent, 120-h preexposure to CdCl2 producing the greatest reversibility. Furthermore, the ability of astrocytes to efficiently volume regulate in the presence of MeHg-containing hypotonic buffer was highly correlated (r = 0.99) with the intracellular levels of MT proteins. The release of [3H]taurine, an osmolyte involved in the RVD process was also measured. The inhibitory effect of MeHg on [3H]taurine in swollen cells was significantly, and fully reversed by CdCl2 preexposure. The study suggests that astrocytes induced to express high levels of MT proteins are resistant to the acute inhibitory effect of MeHg on RVD.

Cadmium chloride (CdCl2)-induced metallothionein (MT) expression in neonatal rat primary astrocyte cultures.

Metallothionein (MT) protein and mRNA levels were studied following exposure of rat neonatal primary astrocyte cultures to cadmium chloride (CdCl2). MT mRNA was probed on Northern blots with a 32P labeled synthetic cDNA probe specific for rat MT mRNA. The probe hybridizes to a single mRNA with a size appropriate for MT, approximately 550 bases. Expression of MT-I mRNA in astrocyte monolayers exposed to 2 x 10(-6) M CdCl2 for 6 h was increased approximately 5-fold (9.7 fg/micrograms total RNA) over MT-I mRNA levels in controls (2 fg/micrograms total RNA). MT-I mRNA could also be detected in untreated cells, suggesting constitutive MT expression in these cells. Western-blot analysis revealed a marked increase in MT protein levels upon exposure to CdCl2 (1 x 10(-6) M; 96 h). Consistent with the constitutive expression of MTs both at the mRNA level and protein level, we have also demonstrated a time-dependent increase in MT-immunoreactivity in astrocytes exposed to CdCl2. The present study suggests that astrocytes constitutively express MTs, and that MT-induction by CdCl2 may be an example of a generalized increase in MTs in response to heavy metal exposure, thus protecting astrocytes, and perhaps also indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.

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.

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.

Lead increases inositol 1,4,5-trisphosphate levels but does not interfere with calcium transients in primary rat astrocytes.

Alteration of receptor-mediated signal transduction pathways by inorganic lead (Pb) has been postulated to contribute to the neurotoxicity of this environmental toxicant, some of these effects involving astrocytes. As Pb is known to mimic Ca2+ in various biological systems or alter Ca(2+)-mediated cellular processes, we analyzed the effect of Pb exposure on alpha 1 receptor activated astrocytic phosphoinositide metabolism and Ca2+ responses in primary astrocyte cultures prepared from cerebral cortex of 1-day-old rats. Exposure to norepinephrine (NE; 10-100 microM) resulted in a significant increase in astrocytic inositol 1,4,5-trisphosphate levels, concomitant with an increase in intracellular Ca2+ levels. Fifteen minute exposure to Pb (10 microM lead acetate) significantly increased inositol 1,4,5-trisphosphate generation compared with controls, both in the presence and absence of NE. However, the inositol 1,4,5-trisphosphate-mediated Ca2+ transients following NE stimulation was unaltered in the presence of Pb (1-100 microM). NE-evoked intracellular Ca2+ responses, both in the presence and absence of extracellular Ca2+ did not differ between control and Pb-treated astrocytes. Additional studies failed to demonstrate the occurrence of Pb influx into astrocytes within the first 12 min of exposure such that Ca2+ responses would be directly affected. It therefore appears unlikely that astrotoxic effects of Pb are mediated via direct changes in intracellular Ca2+ transients.

Methylmercury-induced astrocytic swelling is associated with activation of the Na+/H+ antiporter, and is fully reversed by amiloride.

Astrocytes are a known 'sink' for brain methylmercury (MeHg) deposition. Yet, the significance of the preferential accumulation of MeHg within these cells is imprecisely defined. To determine whether MeHg in isotonic buffer has the potential to interfere with homeostatic functions, we measured its effect on astrocytic volume using an electrical impedance method [E.R. O'Connor, H.K. Kimelberg, C.R. Keese, I. Giaever, Electrical impedance method for measuring volume changes in astrocytes, Am. J. Physiol. 264 (1993) C471-C478.]. In addition, we have characterized the alterations in astrocytic ion permeability associated with exposure to this organometal. The results show that MeHg rapidly induces astrocytic swelling, and that this effect is secondary to increased astrocytic Na+ uptake. Furthermore, the effect of MeHg on astrocytic swelling is completely inhibited by amiloride, but not by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), furosemide, or bumetanide. Accordingly, increased cellular permeability to Na+ via the Na+/H+ antiporter is invoked as the primary mechanism of MeHg-induced astrocytic swelling.

Methylmercury-induced inhibition of regulatory volume decrease in astrocytes: characterization of osmoregulator efflux and its reversal by amiloride.

Swelling of neonatal rat primary astrocyte cultures by hypotonic media leads to regulatory volume decrease (RVD) and the resumption of resting cell volume. RVD is associated with activation of conductive K+ and Cl- channels, allowing for the escape of KCl, as well as the release of osmoregulators, such as taurine and myoinositol. As we have previously shown [D. Vitarella, H.K. Kimelberg, M. Aschner, Inhibition of RVD in swollen rat primary astrocyte cultures by methylmercury (MeHg) is due to increase amiloride-sensitive Na+ uptake, Brain Res. 732 (1996) 169-178.], MeHg, when added to hypotonic buffer inhibits RVD, primarily due to increased cellular permeability to Na+ via the Na+/H+ antiporter. The present study was, therefore, undertaken to assess the ability of cation-anion cotransport blockers to reverse the inhibitory effect of MeHg on RVD in swollen astrocytes, and to further characterize MeHg-induced changes in astrocytic osmoregulatory release processes. The studies demonstrate the following: (1) MeHg-induced inhibition of RVD is partially inhibited by the Na+/H+ antiporter blocker, amiloride, but not SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid), furosemide or bumetanide; (2) exposure of swollen astrocytes to MeHg is associated with specific effects on osmoregulatory release, leading to significant inhibition of taurine release and a significant increase in potassium and myoinositol release compared with release in hypotonic conditions.

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.

Regulatory volume decrease in primary astrocyte cultures: relevance to methylmercury neurotoxicity.

Because of the multiple and varied roles of astrocytes in brain homeostasis, primary cultures of astrocytes from neonatal rat brains have proven to be an excellent model for the study of in vitro cell functions and control mechanisms. In addition, their ability to preferentially sequester a number of heavy metals, such as methylmercury, has lead to intense research on their potential to modulate heavy metal-induced dysfunction. In the present review we briefly discuss the mechanisms associated with astrocytic swelling, an early and prominent event in brain injury, followed by a description on cellular mechanisms associated with regulatory volume decrease (RVD) processes, and specifically those likely to represent sensitive sites for MeHg-induced cytotoxicity.

Pulmonary clearance of manganese phosphate, manganese sulfate, and manganese tetraoxide by CD rats following intratracheal instillation.

Manganese (Mn) is ubiquitous in ambient air due to both industrial and crustal sources. It is also a component of the octane-enhancing fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT). The combustion of MMT by the automobile engine results in the formation of Mn particulates including phosphate, sulfate, and oxide forms. The objectives of this study were to determine the contribution of particle dissolution on pulmonary clearance rates of Mn sulfate (MnSO(4)), Mn phosphate, and Mn tetraoxide (Mn(3)O(4)) in CD rats following an intratracheal instillation exposure. In addition, brain (striatal) Mn concentrations were evaluated following exposure. Adult CD rats were intratracheally instilled with 0, 0.04, 0.08, or 0.16 microg Mn/g of either MnSO(4), Mn phosphate, or Mn(3)O(4). Rats were euthanized at 0, 1, 3, or 14 days after instillation. Lung and striatal Mn concentrations were measured by neutron activation analysis. Pulmonary clearance following single intratracheal instillation of MnSO(4), Mn phosphate, or Mn(3)O(4) was similar for each of the three compounds at each of the three doses used. All pulmonary clearance half-times were less than 0.5 day. At the concentrations used, striatal Mn levels were unaffected, and lung pathology was unremarkable. The dissolution rate constant of the Mn particles was determined in vitro using lung simulant fluids. The solubility of the Mn compounds was in general 20 to 40 times greater in Hatch artificial lung lining fluid than in Gamble lung simulant fluid. The dissolution rate constant of the water-soluble MnSO(4) particles in Hatch artificial lung fluid containing protein was 7.5 x 10(-4) g (Mn)/cm(2)/day, which was 54 times that of relatively water-insoluble Mn phosphate and 3600 times that of Mn(3)O(4). The dissolution rate constants for these compounds were sevenfold slower in Gamble lung fluid simulant. For both solutions, the time for half the material to go into solution differed only by factors of 1/83 to 1/17 to 1 for MnSO(4), Mn phosphate, and Mn(3)O(4), respectively, consistent with measured differences in size distribution, specific surface, and dissolution rate constant. These data suggest that dissolution mechanisms only played a role in the pulmonary clearance of MnSO(4), while nonabsorptive (e.g., mechanical transport) mechanisms predominate for the less soluble phosphate and oxide forms of Mn.

Metallothionein induction in neonatal rat primary astrocyte cultures protects against methylmercury cytotoxicity.

Metallothionein (MT) protein and mRNA levels were monitored following exposure of rat neonatal primary astrocyte cultures to methylmercury (MeHg). MT-I and MT-II mRNAs were probed on northern blots with an [alpha-32P]dCTP-labeled synthetic cDNA probe specific for rat MT mRNA. MT-I and MT-II mRNAs were detected in untreated cells, suggesting constitutive MT expression in these cells. The probes hybridize to a single mRNA with a size appropriate for MT, approximately 550 and 350 bp for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 x 10(-6) M MeHg for 6 h was increased over MT-I and MT-II mRNA levels in controls. Western blot analysis revealed a time-dependent increase in MT protein synthesis through 96 h of exposure to MeHg. Consistent with the constitutive expression of MTs at both the mRNA level and the protein level, we have also demonstrated a time-dependent increase in MT immunoreactivity in astrocytes exposed to MeHg. The cytotoxic effects of MeHg were measured by the rate of astrocytic D-[3H]aspartate uptake. Preexposure of astrocytes to CdCl2, a potent inducer of MTs, completely reversed the inhibitory effect of MeHg on D-[3H]aspartate uptake that occurs in MeHg-treated astrocytes with constitutive MT levels. Associated with CdCl2 treatment was a time-dependent increase in astrocytic MT levels. In summary, astrocytes constitutively express MTs; treatment with MeHg increases astrocytic MT expression, and increased MT levels (by means of CdCl2 pretreatment) attenuate MeHg-induced toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of inhaled manganese phosphate in male Sprague-Dawley rats following subacute (14-day) exposure.

Methylcyclopentadienyl manganese tricarbonyl (MMT) is used as a gasoline octane enhancer. Manganese phosphate is the primary respirable (PM(2.5)) MMT-combustion product emitted from the automobile tailpipe. The goal of this study was to determine the exposure-response relationship for inhaled manganese phosphate in adult male CD rats. Rats were exposed 6-h/day for either 5 days/week (10 exposures) or 7 days/week (14 exposures) to manganese phosphate at 0, 0.03, 0.3, or 3 mg Mn/m(3) (MMAD congruent with 1.5 micrometer). The following tissues collected at the end of the 2-week exposure: plasma, erythrocytes, olfactory bulb, striatum, cerebellum, lung, liver, femur, and skeletal muscle (n = 6 rats/exposure group) were analyzed for manganese content by neutron activation analysis. Intravenous (54)MnCl(2) tracer studies were also conducted following the 14th exposure (n = 6 rats/concentration), and whole-body gamma spectrometry was performed immediately after injection and at 1, 2, 4, 8, 12, and 16 weeks after (54)MnCl(2) administration. Increased manganese concentrations were observed in olfactory bulb, lung, femur, and skeletal muscle following exposure to 3 mg Mn/m(3) (10 or 14 exposures). Increased manganese concentrations were also observed in olfactory bulb, striatum, and lung following exposure to 0.3 mg Mn/m(3) (14 exposures only). Red blood cell and plasma manganese concentrations were increased only in rats exposed to 3 mg Mn/m(3) (10 exposures). Rats exposed to 3 mg Mn/m(3) also had an increased whole-body manganese clearance rate when compared to air-exposed control animals. Our results suggest that the rat olfactory bulb may accumulate more manganese than other brain regions following inhalation exposure.

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.

Potassium and taurine release are highly correlated with regulatory volume decrease in neonatal primary rat astrocyte cultures.

Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K+ (using 86Rb), taurine, and D-aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 mM quinine, 10 microM nimodipine, 100 microM BAPTA-AM, 10 microM trifluoperazine, or a calcium-free buffer significantly (p < 0.0001) inhibited RVD. This was accompanied by inhibition of 86Rb release but an increase in D-[3H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K+ channels, and inhibition of calmodulin all inhibit RVD. Because D-[3H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that D-aspartate release is a main determinant of RVD. In contrast, [3H]taurine release was increased by 1 mM quinine and inhibited by 10 microM trifluoperazine. The net release of K+ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K+ and taurine release in RVD.

Neurotoxicity of manganese chloride in neonatal and adult CD rats following subchronic (21-day) high-dose oral exposure.

The purpose of this study was to evaluate the relative sensitivity of neonatal and adult CD rats to manganese-induced neurotoxicity. Identical oral manganese chloride (MnCl(2)) doses (0, 25, or 50 mg kg(-1) body wt. day(-1)) were given to neonatal rats throughout lactation (i.e. from postnatal day (PND) 1 through 21) and to adult male rats for 21 consecutive days. The MnCl(2) doses administered to neonates were ca. 100-fold higher than those resulting from the consumption of an equivalent volume of rat's milk. Rats were assessed using similar behavioral and neurochemical evaluations. Several statistically significant changes occurred in Mn-exposed rats relative to control animals. Neonates given the high dose of MnCl(2) had reduced body weight gain. An increased pulse-elicited acoustic startle response amplitude was observed in neonates from both MnCl(2) treatment groups on PND 21. Increased striatal, hippocampal, hindbrain and cortical Mn concentrations were observed in all Mn-exposed neonates on PND 21. Increased hypothalamic and cerebellar Mn concentrations were also observed on PND 21 in neonates from the high-dose group only. Increased striatal, cerebellar and brain residue Mn concentrations were observed in adult rats from the high-dose group. Increased striatal dopamine and 3,4-dihydroxyphenylacetic acid levels were observed only in PND 21 neonates from the high-dose group. No treatment-related changes were observed in clinical signs, motor activity (assessed in neonates on PND 13, 17, 21 +/- 1 and in adults), passive avoidance (assessed in neonates on PND 20 +/- 1 and in adults) or neuropathology (assessed in PND 21 neonates only). The results of our experiment suggest that neonates may be at greater risk for Mn-induced neurotoxicity when compared to adults receiving similar high oral levels of Mn.