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Biomedical subjects

M Aschner

Publications and source records attributed to M Aschner.

At least 19 recordsLinked to original sources
PubMed1999

Transfection and overexpression of metallothionein-I in neonatal rat primary astrocyte cultures and in astrocytoma cells increases their resistance to methylmercury-induced cytotoxicity.

Metallothionein-I (MT-I) was expressed in neonatal rat primary astrocyte cultures and an astrocytoma cell line by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. Following transient transfection of the pGFAP-MT-I plasmid, MT-I mRNA and MT-I protein levels were determined by northern blot and immunoprecipitation analyses, respectively. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na251CrO4, an indicator of membrane integrity. Transfection with the pGFAP-MT-I plasmid led to increased mRNA (2. 5-fold in astrocytes and 7.4-fold in astrocytomas) and MT-I protein (2.4-fold in astrocytes and 4.0-fold in astrocytomas) levels compared with their respective controls. Increased expression of MT-I was associated with attenuated release of Na251CrO4 upon MeHg (5 microM) treatment. These results demonstrate that MT-I can be highly expressed both in primary astrocyte cultures and astrocytomas by pGFAP-MT-I plasmid transfection, and lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg. Taken together, the data suggest that MT offer effective cellular adaptation to MeHg cytotoxicity.

Animals↗
PubMed1998

Immune and inflammatory responses in the CNS: modulation by astrocytes.

Because the skull bones, the cerebrospinal fluid, the blood-brain barrier (BBB), and the meninges effectively shield the central nervous system from other tissues, it was proposed that the brain is an 'immunologically privileged' organ. However, with recent evidence that in response to invasion by microorganisms, resident cells, such as astrocytes and microglia can fully mount an immune response, this long-standing view has been rethought and revised. Over the last two decades, both astrocytes and microglia have been shown to secrete numerous cytokines, and, therefore, it is presently widely accepted that these cells actively participate in an integrative communicative pathway between resident immune cells of the CNS and those of the periphery. While clearly implicated in the initiation, maintenance, and suppression of immune responses, cytokines produced by these cells (e.g. astrocytes and microglia), as well as the responses of these cells to cytokines produced elsewhere, has also been shown to propagate CNS damage. Therefore the potential involvement of these cells in neurodegenerative disorders has been raised and subjected to intense experimentation. The objective of this synopsis is to review the role played by astrocytes in the initiation and modulation of immune responses.

Animals↗
PubMed1998

Induction of astrocyte metallothioneins (MTs) by zinc confers resistance against the acute cytotoxic effects of methylmercury on cell swelling, Na+ uptake, and K+ release.

Metallothionein (MT) proteins play an important role in the detoxification of heavy metals. Since methylmercury (MeHg) preferentially accumulates in astrocytes, we investigated the ability of the astrocyte-specific MT isoform, MT-I, to attenuate MeHg-induced cytotoxicity. Increased astrocytic MT expression was achieved by 24-h pretreatment of neonatal rat primary astrocyte cultures with 100 microM zinc (ZnSO4). Subsequently, the astrocytes were treated with MeHg (10 microM), and its toxic effects on cell volume, Na+ uptake, and K+ release were investigated and compared to cells treated with or without MeHg, but in the absence of Zn pretreatment. Pretreatment of astrocytes with Zn was associated with a 2.9-fold increase in MT protein levels (P<0.02), and a 5.6-fold increase in MT mRNA levels (p<0.002) compared to control astrocytes. Astrocytes expressing increased MT protein levels were resistant to MeHg-induced swelling. In isotonic buffer the effect of MeHg on swelling was abolished (p<0.01) by 24-h Zn pretreatment, in such a way that volume profiles in these cells did not differ from controls. Zn-induced increased expression of MTs was also associated with significant attenuation of astrocytic Na+ uptake (p<0.01) and Rb+ (a marker for K+) release (p<0.001) in response to treatment with MeHg. These results demonstrate (1) that astrocytes can be induced to express high levels of MT proteins by pretreatment with Zn, and (2) that Zn confers resistance against the acute effect of MeHg on astrocytic swelling and the associated changes in ion (Na+ and K+) transport. Taken together, the data suggest that astrocytic MT induction offers effective cellular adaptation to MeHg cytotoxicity.

Animals↗
PubMed1998

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.

Amiloride↗
PubMed1998

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.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1998

Dimethyl sulfoxide, but not acidosis-induced metallothionein mRNA expression in neonatal rat primary astrocyte cultures is inhibited by the bioflavonoid, quercetin.

Metallothionein (MT) mRNA levels were analyzed following exposure of neonatal rat primary astrocyte cultures to physiologic pH (7.4), acidosis (pH 6.5 and 6.0), and dimethyl sulfoxide (DMSO). Treatments were carried out both in the presence and absence of the bioflavonoid, quercetin. Total RNA was probed on northern blots with [alpha32P]dCTP-labeled synthetic cDNA probes specific for rat MT isoform mRNAs. MT-I and MT-II mRNA levels in astrocytes exposed to pH 6.5 or pH 6.0 were increased compared to controls (pH 7.4). Treatment with DMSO in the presence and absence of acidosis, also increased MT-I and MT-II mRNA levels compared to controls (pH 7.4). The DMSO-induced increase in MT mRNA expression was reversed by treatment of astrocytes with quercetin, such that MT-I and MT-II mRNA levels in DMSO plus quercetin-treated astrocytes were indistinguishable from mRNA levels in their respective controls at pH 7.4, pH 6.5, and pH 6.0. These findings suggest that both acidosis and DMSO exposure are associated with increased astrocytic MT synthesis at the mRNA level, and that quercetin, effectively blocks MT mRNA induction by DMSO.

Acidosis↗
PubMed1998

Role of branched-chain aminotransferase isoenzymes and gabapentin in neurotransmitter metabolism.

Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with alpha-ketoglutarate to form the branched-chain alpha-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-14C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to alpha-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an alpha-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from alpha-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures.

During stroke or head trauma, extracellular K+ concentration increases, which can cause astrocytes to swell. In vitro, such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which such anionic organic cellular osmolytes can be released. In the present study, we sought to identify the swelling-activated channel(s) responsible for D-[3H]aspartate release from primary cultured astrocytes exposed to either KCl or hypotonic medium. KCl-induced D-[3H]aspartate release was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, and tamoxifen but not by cAMP. The cell swelling caused by raised KCl was not inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects on D-[3H]aspartate release from cells swollen by hypotonic medium, but such release was inhibited by NPPB, dideoxyforskolin, and tamoxifen. Of the swelling-activated channels so far identified, our data suggest that a volume-sensitive outwardly rectifying channel is responsible for D-[3H]aspartate release from primary cultured astrocytes during raised extracellular K+ and possibly during hypotonic medium-induced release.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

Astrocytic functions and physiological reactions to injury: the potential to induce and/or exacerbate neuronal dysfunction--a forum position paper.

This forum highlights the wide diversity of astrocytic functions which maintain CNS homeostasis, well beyond the originally proposed passive cytoskeletal support role for these cells. Astrocytic potential in modulating damage and repair is also reflected in this forum. While astrocytes may potentially play a primary role in epilepsy, and in neurodegenerative disorders such as Huntington's diseases, HIV, and demyelination, one needs to keep in mind that to a large extent evidence supporting involvement of astrocytes in these diseases is derived from in vitro studies. Observations on regional heterogeneity and functional specialization of astrocytes also suggest that astrocytes have adapted to perform functions specific to their respective residence site. Therefore, it is necessary to identify potentially damaging consequences of astrocytic functions in vivo, although these analyses will be undoubtedly extremely complex. Expanded investigations on astrocytic involvement in neurotoxicity and neurodegeneration is clearly warranted, and as new experimental tools are developed it is likely that further strides will be made in our understanding of astrocyte functions, both in health and disease.

Animals↗
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↗