Glial-Cell Cultures from Brains of Carbonic Anhydrase II-Deficient Mutant Mice: Delay in Oligodendrocyte Maturation

Carbonic anhydrase II (CAII) is a multifunctional enzyme found in oligodendrocytes and astrocytes in normal mouse brains. We have begun to compare the glial cells in primary cultures from neonatal genetically CAII-deficient (Car) mice to those from normal (con) mice in order to detect developmental defects, if any, in Car glial cells. In con cultures intensely CAII-positive cells costained with antibodies against the oligodendrocytic markers, O4 and myelin basic protein (MBP), respectively. Most (82%) of the CAII-positive cells were O4-positive, but only 60% were MBP-positive. Some clumps of GFAP-positive cells were CAII-positive. At each respective number of days in vitro (DIV) total numbers of O4-positive cells were similar in Car and con cultures, and total numbers of galactocerebroside-positive cells also were similar in Car and con cultures. However, compared to cells in con cultures at 7 DIV, a lower percent of Car cells in the oligodendrocyte lineage expressed MBP, and morphological differentiation also was subnormal in that the Car cells showed fewer processes and membrane sheets. Car and con cultures expressed similar numbers of MBP-positive cells by 10 DIV. The results suggest a temporary delay in the maturation of Car oligodendrocytes.     

Purification of Glycogen Phosphorylase from Bovine Brain and Immunocytochemical Examination of Rat Glial Primary Cultures Using Monoclonal Antibodies Raised Against This Enzyme

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

High-Affinity Uptake of [3H]Norepinephrine by Primary Astrocyte Cultures and Its Inhibition by Tricyclic Antidepressants

Abstract: Primary astrocyte cultures from neonatal rat brains show uptake of [³H]norepinephrine ([³H]NE). This uptake has a high-affinity component with an apparent K_m of approximately 3 × 10^?7 M. At 10^?7 M [³H]NE both the initial rate of uptake and steady-state content of [³H]NE is inhibited by up to 95% by omission of external Na⁺. The Na⁺-dependent component of this uptake is totally inhibited by the tricyclic antidepressants desipramine (DMI) and amitryptyline with IC₅₀ values of 2 × 10^?9 and 4 × 10^?8 M, respectively. Inhibition of [³H]NE uptake by DMI shows competitive kinetics. These characteristics are essentially identical to those found for high-affinity uptake of NE in total membrane or synaptosome fractions from rodent brains and suggests that such uptake in neural tissue is not exclusively neuronal.     

Effects of Melatonin on Carbonic Anhydrase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo

The in vitro effects of melatonin (N-acetyl-5-methoxytryptamine) on human carbonic anhydrase isozymes (HCA-I and HCA-II) from human erythrocytes and in vivo effects on rat erythrocytes carbonic anhydrase (CA) were determined. Human erythrocyte carbonic anhydrase isozymes were purified by haemolysate preparation and Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. The HCA-I enzyme, having a specific activity of 7337.5?EU/mg protein, was purified 843-fold with a yield of 60% and the HCA-II enzyme, having a specific activity of 17067?EU/mg protein, was purified 1962-fold with a yield of 22.7%. For in vitro experiments, the enzyme activity was minimal at 2×10-4?M melatonin concentration and increased above this concentration. Ten mg?kg-1 melatonin was administered intraperitoneally and showed a stimulatory effect on the enzyme. Time-dependent in vivo studies were conducted for melatonin in Sprague–Dawley type rats. It was found that CA activity in the rat erythrocytes was decreased by the melatonin after 1 and 3 hours to 2500±500.0 and 1875±239.4 respectively which were statistically significant (p<0.05) differences to the control (2660±235.8). However, CA activity was restored to its normal level after 6?h (2666±235.7) (p>0.05) probably due to metabolism of the melatonin. The findings indicate that melatonin may be pharmacologically useful in some diseases.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

Effects of Maturation on the Phospholipid and Phospholipid Fatty Acid Compositions in Primary Rat Cortical Astrocyte Cell Cultures

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.     

Immunocytochemical Examination of Neural Rat and Mouse Primary Cultures Using Monoclonal Antibodies Raised Against Pyruvate Carboxylase

Abstract: Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO₂ to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the ∼10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain.     

The Effect of Carbonic Anhydrase Inhibition on Leptin Secretion by Rat Adipose Tissue

It is well known that the role of leptin in the body is to regulate food intake and energy expenditure but the process of leptin secretion by adipose tissue and the components involved in this process are still obscure. Carbonic anhydrase III (CA III) is the most abundant protein of the rat adipose tissue and its amount decreases with obesity. The effect of the inhibition of CA III on leptin secretion by rat epididymal adipose tissue was examined. Dorzolamide, a CA inhibitor, caused a decrease in dexamethasone and insulin-induced leptin secretion suggesting a possible role for CA III in the mechanism of leptin secretion.     

Up-Regulation of Carbonic Anhydrase Isozyme IV in CNS Myelin of Mice Genetically Deficient in Carbonic Anhydrase II

Abstract: Carbonic anhydrase (CA) II is the major CA isozyme in the brain, where it participates in acid-base homeostasis, fluid transport, and myelin synthesis. The CA II deficiency [CA(II)D] mutation in the mouse results in structural changes in the glial cells in the CNS and in decreased susceptibility to seizures, but no detectable changes in myelin yield and ultrastructure. We compared the CA isozymes in brain and spinal cord fractions, as well as in purified myelin, between CA(II)D and control mice. CA(II)D resulted in a much lower total CA specific activity in all tissues examined but in higher CA IV specific activities in soluble and membrane-associated fractions and pure myelin. Western blots of purified myelin showed a band corresponding to CA IV in CA(II)D mice. This band was weak or undetectable in myelin samples from normal mice. Immunocytochemical staining demonstrated CA IV in oligodendrocytes and myelinated tracts in normal mouse brains and stronger staining of the same structures in brains of CA(II)D mutants. We conclude that CA(II)D mutation in the mouse up-regulates CNS CA IV. We speculate that this up-regulation could mitigate the effect of CA(II)D on myelin formation and maintenance.     

Two Types of Clathrin-Coated Vesicles Isolated from Rat Brain: Analysis of Biochemical Properties and Cellular Origin

Two major fractions rich in clathrin-coated vesicles (CVs) (fraction I, rho = 1.140 g/cm3; fraction II, rho = 1.113 g/cm3) were separated from rat brain using a sucrose gradient and compared for their cellular origins and Cl- translocation systems. Electron micrographs showed that both fractions contained CVs of different size distributions (fraction I, 85 +/- 9.5 nm in diameter; fraction II, 72 +/- 6.8 nm in diameter). Fraction II contained potent ouabain-sensitive ATPase activity, whereas fraction I contained only a little activity. Immunoblot analysis for the Na+,K(+)-ATPase catalytic subunit, alpha and alpha(+), demonstrated that fraction II exhibited predominantly alpha(+), whose proportion to alpha was analogous to that observed in the extracts of primary cultured neuronal cells. Furthermore, on a sucrose density gradient, cultured neuronal cells yielded fraction II but not fraction I, whereas primary cultured glial cells yielded fraction I but not fraction II. Labeling-chase experiments using 125I-transferrin in cultured neuronal cells showed the internalized ligand in fraction II and the surface-bound ligand in the fraction with lower density (rho = 1.090 g/cm3), a result suggesting that the involvement of Na+,K(+)-ATPase in fraction II is attributable to endocytic vesicles. Cl- uptake in fraction II was approximately threefold higher than that in fraction I. N-Ethylmaleimide (100 microM) completely inhibited the Cl- uptake in fraction I but partially (approximately 50%) inhibited that in fraction II. These findings suggest that the two CV fractions isolated from rat brain originate from different cell types--glial and neuronal cells--and differ in size distribution of CVs, content of Na+,K(+)-ATPase, and mechanism for Cl- uptake.     

Neonatal Hypothyroidism Affects the Adenine Nucleotides Metabolism in Astrocyte Cultures from Rat Brain

Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.Elizandra Braganhol and Alessandra Nejar Bruno are first authors.     

Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox State and Carbonic Anhydrase Isozyme IX Expression in Human Renal Cell Carcinoma: Biochemical and Morphological Investigations

Clear renal cell carcinomas (RCC) frequently express carbonic anydrase IX (CA IX) because of non-functional mutation of von Hippel Lindau (VHL) tumor suppressor gene. CA IX is a tumor-associated transmembrane antigen, which catalyzes the extracellular, reversible hydration of carbon dioxide to bicarbonate and proton and thereby contributes to acidification of extracellular milieu. Extracellular acidic pH facilitates tumor growth and progression. CA IX expression is upregulated by Hypoxia Inducible Factor-1 (HIF-1), which is negatively controlled by oxygen via wild type VHL protein and is also regulated by the cell redox state. We investigated the immunohistochemical pattern of distribution of CA IX in a small series (14 cases) of RCCs. CA IX expression was matched with the redox state of RCC, stratifying our series in relation to clinical and histopathological parameters, such as Fuhrman grade, staging, proliferation markers expression, and particularly, the presence of necrosis. Our results show for the first time the existence of a perivascular pattern of CA IX distribution in RCC. We also found a significant relationship between CA IX expression and the presence of necrosis. Tumors with higher CA IX expression exhibited higher degree of necrosis [Formula: See Text] Notably, an almost significant relationship between the redox state and CA IX expression was detected in RCC patients with 5 years disease-free survival, most of them showing organ-confined disease. Tumors with lower redox state showed an algebraically higher degree of CA IX expression. On the contrary, tumors with higher redox state exhibited an algebraically lower CA IX expression [Formula: See Text] The observed relationship of CA IX expression and necrosis suggests a role for CA IX in RCC. Further investigations are necessary to further establish the role of the redox state in regulation of CA IX expression in RCC.     

Purification of Cytosolic Malic Enzyme from Bovine Brain, Generation of Monoclonal Antibodies, and Immunocytochemical Localization of the Enzyme in Glial Cells of Neural Primary Cultures

Abstract: Cytosolic malic enzyme (EC 1.1.1.40) was purified from bovine brain 5,600-fold to a specific activity of 47 U/mg. The enzyme is a homotetramer with a subunit molecular mass of 60 kDa and an isoelectric point of 6.2. Mouse monoclonal antibodies raised against this enzyme were purified and shown to be monospecific, as indicated by immunoblotting. Immunocytochemical examination of rat astroglia-rich primary cultures at the light microscopic level revealed colocalization of cytosolic malic enzyme with the astroglial marker glial fibrillary acidic protein. Also, a colocalization with the oligodendroglial marker myelin basic protein was found. Neurons in rat neuron-rich primary cultures did not show positive staining. The data suggest that cytosolic malic enzyme is a glial enzyme and is lacking in neurons.     

Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract: Increased production of amyloid β peptide (Aβ) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because Aβ deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa Aβ exceeds that of nonamyloidogenic 3-kDa Aβ in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable Aβ. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Carbonic Anhydrase Inhibitors. Arylsulfonylureido-and Arylureido-Substituted Aromatic and Heterocyclic Sulfonamides: Towards Selective Inhibitors of Carbonic Anhydrase Isozyme I

Abstract

Reaction of twenty aromatic/heterocyclic sulfonamides containing a free amino, imino, hydra-zino or hydroxyl group, with tosyl isocyanate or 3,4-dichlorophenyl isocyanate afforded two series of derivatives containing arylsulfonylureido or diarylureido moieties in their molecule respectively. The new derivatives were assayed as inhibitors of three carbonic anhydrase (CA) isozymes, CA I, II (cytosolic forms) and IV (membrane-bound form). Potent inhibition was observed against all three isozymes but especially against CA I, which is generally 10-75 times less susceptible to inhibition by the classical sulfonamides in clinical use as compared to the other major red cell isozyme, CA II, or the membrane-bound one, CA IV. The derivatives obtained from tosyl isocyanate were generally more potent than the corresponding ones obtained from 3,4-dichlorophenyl isocyanate. This is the first reported example of selective inhibition of CA I and might lead to more selective drugs/diagnostic agents from this class of pharmacologically relevant compounds.     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metallothionein Induction in Neonatal Rat Primary Astrocyte Cultures Protects Against Methylmercury Cytotoxicity

Abstract: 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 [α-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, ∼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 × 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. Increased MT expression may represent a generalized response to heavy metal exposure, thus protecting astrocytes and perhaps also, indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.