Biomedical subjects

M Aschner

Publications and source records attributed to M Aschner.

At least 55 records · Page 3Linked to original sources

PubMed1998

Acidosis-induced metallothionein (MT) mRNA expression in neonatal rat primary astrocyte cultures.

Metallothionein (MT) mRNA levels were determined following exposure of neonatal rat primary astrocyte cultures to acidosis. Astrocyte total RNA was probed on northern blots with [alpha 32 P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. The probe for MT-I mRNA hybridized to a single mRNA with a size appropriate for MT, approximately 550 nucleotides. MT-I mRNA levels in astrocyte monolayers exposed to pH 6.5 and 6.0 for 3 or 6 hours were unchanged compared with MT-I mRNA levels in control cultures exposed to pH 7.4. In contrast, 9 hour exposure of astrocytes to pH 6.5 and 6.0 led to a significant increase in MT-I mRNA transcripts compared with controls maintained at pH 7.4 (p < 0.001 and p < 0.02, respectively). A probe for MT-II mRNA that hybridizes to a single mRNA (450 nucleotides) was also used to determine the effect of acidosis on astrocyte MT-II mRNA transcripts. Although statistical significance was not attained, a similar trend was noted, with a 9 hour exposure to pH of 6.5 and 6.0 resulting in increased astrocytic expression MT-II mRNA compared with control cells maintained at pH 7.4. Acidosis was also associated with a pH-dependent increase in astrocytic volume. Accordingly, acidosis is invoked as an added stimulus to stress factors associated with the induction of astrocytic MT mRNA transcripts.

Acidosis↗

PubMed1998

Astrocytes as mediators of immune and inflammatory responses in the CNS.

The long-standing view that the brain is isolated from the effects of the immune system has recently been challenged, with experimental evidence suggesting that in response to invasion by microorganisms, the CNS can mount its own defense by resident cells, such as the microglia and astrocytes. Both cell types produce and secrete a number of cytokines and therefore can potentially modulate and integrate the communication between hematogenous cells and resident cells of the CNS. This manuscript will commence with a brief overview of astrocytic functions in the CNS, and proceed to discuss astrocytic responses that may regulate CNS inflammation. Specifically, it will address (1) the function of astrocytes as the antigen presenting cells (APCs) of the CNS, and (2) the role afforded by astrocyte-derived cytokines, and astrocytic responses to cytokines secreted elsewhere, in mediating and sustaining immune responses. Finally, some recent experimental evidence on the possibility that astroglial impairment by pathogens may contribute to the etiology of neurologic diseases will be highlighted.

Animals↗

PubMed1997

Metallothionein induction in fetal rat brain and neonatal primary astrocyte cultures by in utero exposure to elemental mercury vapor (Hg0).

Brain metallothionein (MT) protein and mRNA levels were determined in the fetal rat following in utero (gestational days 7-21) exposure to elemental mercury vapor (Hg0; 300 microg Hg/m3; 4 h/day). Total RNA was probed on Northern blots with [alpha-32P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. The probes for MT-I and MT-II mRNA hybridized to a single band of approximately 550 and 450 nucleotides, respectively. Expression of whole brain MT-I mRNA in full-term fetal rats (day 21) was significantly increased (P < 0.03) by in utero exposure to Hg0 compared to nonexposed controls. This corresponded to a 14-fold increase (P < 0.001) in fetal brain Hg concentration after in utero Hg0 exposure. In addition, astrocytes from both control and in utero Hg0-exposed fetuses were isolated, and neonatal primary astrocyte cultures were established and maintained in vitro for up to 3 weeks without additional experimental intervention. Astrocyte monolayers derived from in utero Hg0-exposed fetuses consistently expressed increased abundance of MT-I mRNA transcripts after 1, 2, and 3 weeks in culture (P < 0.03, P < 0.01, and P < 0.03, respectively) compared with controls. The abundance of astrocyte MT-II mRNA was unchanged at 1 and 2 weeks in culture, but was significantly increased at 3 weeks in cultures derived from brains of Hg0-exposed fetuses (P < 0.04). Consistent with the increase in MT mRNA, an increase in astrocytic levels of MT proteins was noted by Western blot analysis and MT-immunoreactivity. These studies suggest that in utero exposure to Hg0 induces brain MT gene expression, and that MT mRNAs and their respective proteins are useful quantitative biochemical markers of intrauterine exposure to Hg0, a potentially cytotoxic challenge to astrocytes in the developing brain. It is concluded that induction of MT by fetal/neonatal astrocytes represents an attempt by these glial cells to protect against Hg cytotoxicity in maintaining cerebral homeostasis.

Animals↗

PubMed1997

Astrocyte metallothioneins (MTs) and their neuroprotective role.

I have briefly detailed in this review the role of astrocytes in MeHg neurotoxicity, emphasizing the mechanisms and significance of astrocytic swelling in neuropathological conditions. I have also described the functions of brain MTs and have reported recent observations on their propensity to attenuate cytotoxicity. While it is unclear why three different MT genes are expressed in the brain, this redundancy should allow for greater accumulation of MTs under stressful conditions compared to its accumulation if only a single gene was present. Another explanation may be that genes encoding functionally identical MTs might be regulated independently, thus permitting cell-specific MT expression. Finally, each of the three MT isoforms may have distinct functions. As discussed herein, astrocytic MTs afford protection from the acute cytotoxic effects of MeHg, reversing the effect of this organometal on RVD and inhibition of taurine release. Whether other vital cellular functions are protected by MTs will have to await future studies, as will the mechanisms associated with MT-induced cellular protection. That the resistance to heavy metal toxicity is closely related to the cellular ability to synthesize MTs, raises interesting questions regarding the potential involvement of heavy metals in neurodegenerating (amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease) under conditions of compromised MT synthesis. Future studies on the expression and regulation of MT genes are likely to culminate in novel strategies for manipulating intracellular MT levels, providing insight to their role in both health and disease.

Animals↗

PubMed1997

Induction of metallothionein-I (MT-I) mRNA in primary astrocyte cultures is mediated by hypotonicity and not ethanol (EtOH) per se.

Metallothionein (MT) mRNA was determined in rat astrocyte cultures in response to ethanol (EtOH). MT-I mRNA was significantly increased after 6 h exposure to isosmotic EtOH, but not hyperosmotic EtOH. Exposure to a hyposmotic/hypotonic solution also led to a significant increase in the expression of astrocytic MT-I mRNA. The large increase in MT-I mRNA was not due to removal of extracellular NaCl, because this effect was reversed by replacement of NaCl with N-methyl D-glucamine chloride. A significant decrease in MT-I mRNA was also noted in astrocytes exposed to an EtOH-free hyperosmotic/hypertonic solution. These results suggest (1) that EtOH per se does not directly induce MT-I mRNA expression, (2) that the induction by EtOH of MT-I mRNA is secondary to hypotonicity, and (3) that hyperosmotic/hypertonic exposure is associated with reduced expression of MT-I mRNA in astrocyte cultures.

Animals↗

PubMed1997

Metallothioneins in brain--the role in physiology and pathology.

A symposium on the role of brain metallothioneins (MTs) in physiology and pathology was held at the 1996 Annual Society of Toxicology Meeting in Anaheim, California. The objectives of this symposium were to: (1) review the physiologic function of MTs, (2) examine the distribution of brain MTs with particular emphasis on cell-specific localization (neurons vs neuroglia), (3) discuss MT gene responsiveness upon toxic insult with metals, and (4) discuss the potential role of MTs in the etiology of neurodegenerative disorders. Dr. Cherian discussed the biochemical properties of the MTs, emphasizing structural similarities and differences between the MTs. Dr. Klaassen addressed the expression and distribution of the MTs in brains with special reference to the cell-specific localization of MTs. Dr. Aschner provided data illustrating a potential role for MTs in attenuating the cytotoxicity caused by methylmercury (MeHg) in cultured neonatal astrocytes. Dr. Palmiter discussed the properties of MT-III and the increased sensitivity of MT-III knockout mice to kainate-induced seizures. Cerebral zinc metabolism, its relationship to MT homeostasis, and its pathogenic potential in Alzheimer's disease was addressed by Dr. Bush.

Animals↗

PubMed1996

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.

Analysis of Variance↗

PubMed1996

Metallothionein induction by bismuth in neonatal rat primary astrocyte cultures.

Metallothionein (MT) proteins and mRNA levels were determined following exposure of neonatal rat primary astrocyte cultures to bismuth. MT mRNA were probed on Northern blots with [alpha-32P]dCTP labeled synthetic cDNA probes specific for rat MT-I and MT-II mRNA. The probes for MT-I and MT-II mRNA hybridize to a single mRNA with a size appropriate for MT, approximately 550 and 450 base pairs for MT-I and MT-II, respectively. Expression of MT-I and MT-II mRNA in astrocyte monolayers exposed to 2 and 10 microM bismuth for 6 h was increased in a dose-dependent manner over MT-I and MT-II mRNA levels in control cells. Western-blot analysis revealed a time-dependent increase in MT protein synthesis through 72 h of exposure to bismuth (2-10 microM). Consistent with the bismuth-induced increase in MTs at the mRNA and protein levels, a time (24-72 h) and dose-dependent (2-10 microM) increase in astrocytic MT-immunoreactivity was noted.

Animals↗

PubMed1996

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.

Amiloride↗

PubMed1996

The functional significance of brain metallothioneins.

Metallothioneins (MTs) are ubiquitous low molecular weight proteins characterized by their abundant content of cysteines. Two MT isoforms, MT-I and MT-II, are expressed coordinately in all mammalian tissues. In the CNS, MT-I and MT-II are conspicuously absent from neuronal populations, yet abundant in fibrous and protoplasmic astrocytes. A newly identified brain-specific MT gene, MT-III, is predominantly expressed in zinc-containing neurons of the hippocampus and absent from glial elements. MTs have been implicated as regulator molecules in gene expression, homeostatic control of cellular metabolism of metals, and cellular adaptation to stress. MTs store and release essential metals, such as zinc and copper, maintaining the low intracellular concentration of free essential metals. Thus, MTs fulfill a regulatory capacity and influence transcription, replication, protein synthesis, metabolism, as well as other zinc-dependent biological processes. Because MT-III is particularly abundant in zinc-containing neurons of the hippocampus, it is likely to play an important role in neuromodulation by zinc-containing neurons and to act as a sink for free zinc. It may also play an etiologic role in various pathophysiological conditions associated with increased extracellular zinc. Studies demonstrating that MT-III prevents neuronal sprouting in vitro, appears to be down-regulated in Alzheimer's disease, and that MT-III "knockout" mice appear highly sensitive to kainateinduced seizures have focused growing attention on the etiologic role of MT-III in neurodegeneration.-Aschner, M. The functional significance of brain metallothioneins.

Alzheimer Disease↗

PubMed1996

Methylmercury in astrocytes--what possible significance?

This manuscript starts with a brief review of recent research on the role of astrocytes in homeostatic mechanisms within the CNS. Astrocytes in primary culture have been found to contain the whole pantheon of receptors and also some of the uptake systems for CNS transmitters; properties which were formerly thought to be exclusively neuronal. The uptake of transmitters, usually by Na(+)-dependent mechanism, and their subsequent inactivation by metabolism is then briefly described. The effects of methylmercury (MeHg) on astrocytic function are discussed, and the relatively small amount of information currently available of how MeHg may interfere with astrocytic functions is also reviewed. The potential role of astrocytes in MeHg sequestration is also discussed, particularly in terms of the ability of this heavy metal to induce the expression and synthesis of metallothionein (MT).

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Adenosine↗

PubMed1995

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.

Animals↗

PubMed1995

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)

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Alkylating Agents↗