Brain Research, 530 (1990) 245-250 Elsevier
245
BRES 15937
Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling M. Aschner 1, N.B. Eberle 1, K. Miller I a n d H . K . K i m e l b e r g 2 1Department of Pharmacology and Toxicology and 2Division of Neurosurgery, and the Interdepartmental Neuroscience Training Program, Albany Medical College, Albany, NY 12208 (U.S.A.) (Accepted 24 april 1990)
Key words: Methylmercury; Rubidium; Glutamate; Na+,K÷-ATPase; Astrocyte
The ability of astrocytes to sequester MeHg may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10-5 M) significantly inhibited the initial rate (5 min) of uptake of 86RbC1, used as a tracer for K ÷. a6RbC! uptake was also sensitive to the omission of medium Na ÷. MeHg (10-5 M) also markedly inhibited the initial rate of uptake (1 min) of the Na+-dependent uptake of [3H]L-glutamate. A second neurotoxin, MnCI2 (0-5 x 10-4 M), did not alter [3H]glutamate or 86RbCI uptake. MeHg, but not MnC12, also stimulated the release of intracellular 8eRb+ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K + gradient. This would be expected to compromise the ability of astrocytes to control extracellular K ÷ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [t4C]3-O-methyl-o-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0-5 x 10-4 M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.
INTRODUCTION M e H g is a potent CNS toxin. A prominent feature of prenatal M e H g poisoning is the reduction in CNS mitotic activity 25, and the interference with neuronal cell migration 5. Astrocytes in the mammalian CNS are known targets of M e H g exposure and a predisposition of astrocytes for M e H g damage offers a possible explanation for the observed neurotoxicity in adult CNS, since it has been found that in human brain material, most of the M e H g appears to be localized within glial cells 21'29. The presence in astrocytes of a neutral amino acid carrier transport system L, capable of mediating MeHg-cysteine uptake was recently identified 1. It has long been noted that MeHg compounds can influence ion, water, and non-electrolyte transport in a variety of cells and tissues 16. The plasma membrane is believed to be the target organelle for these effects, although M e H g is also a potent inhibitor of intracellular enzymes and metabolic processes 6"3°. In recent years, primary cultures of astrocytes from neonatal rat brains have proven to be an excellent model for the study of in vitro differentiated cell functions and control mechanisms 7. Astroglial cells occupy approxi-
mately 20% of the total cell volume of the grey matter in the CNS, and their processes are found around synapses and in close association with nodes of Ranvier, axon tracts, and blood vessels 14. A m o n g the many roles proposed for astroglia are involvement in secretion of neurotrophic factors that regulate neuronal survival, biochemical maturation and morphological differentiation. These also include acid-base control, inactivation of extracellular glutamate, concentrative uptake and metabolism of neurotransmitters, expression of neurotransmitter receptors, CNS pathogenesis, and modulation of neuronal signals 14. Furthermore, astrocytes are now beginning to be appreciated as possible mediators of neurotoxic exposure to various chemical agents. For example, dense labeling of radioactive M e H g in astrocytes may implicate an astrocyte-mediated role in the neurotoxicity of M e H g 9. It has also been demonstrated that the astrocyte may represent the site of conversion of the Parkinsonism-inducing neurotoxin, MPTP, to its active metabolite MPP ÷ (see refs. 18,23). Here, we present studies on the effects of M e H g on ion and water transport in primary astrocyte cell cultures from neonatal rat cerebral cortex, as a first step in elucidating the relationship between the astrocyte and M e H g ' s toxicity.
Correspondence: M. Aschner, Department of Pharmacology and Toxicology, Albany Medical College, Albany, NY 12208, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
246 120
MATERIALS AND METHODS o
I00
Materials 86RbCI was purchased from Amersham Corporation (Arlington Heights, IL), and [3H]L-glutamate and [14C]3-O-methyl-D-glucose were purchased from New England Nuclear, (Boston, MA). Glutathione, N-methyl-D-glucamine, L-2-oxothiazolidine-4-carboxylate, N-ethylmaleimide, and diethyl maleate were of the highest analytical grade and were purchased from the Sigma Chemical Company (St. Louis, MI). Methylmercuric chloride was purchased from K & K Labs, ICN Pharmaceuticals (Plainview, NY). Cold vapor atomic absorption analysis by the method of Magos and Clarkson ~7 indicated that >99% of the total Hg was in the organic form.
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245
BRES 15937
Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling M. Aschner 1, N.B. Eberle 1, K. Miller I a n d H . K . K i m e l b e r g 2 1Department of Pharmacology and Toxicology and 2Division of Neurosurgery, and the Interdepartmental Neuroscience Training Program, Albany Medical College, Albany, NY 12208 (U.S.A.) (Accepted 24 april 1990)
Key words: Methylmercury; Rubidium; Glutamate; Na+,K÷-ATPase; Astrocyte
The ability of astrocytes to sequester MeHg may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10-5 M) significantly inhibited the initial rate (5 min) of uptake of 86RbC1, used as a tracer for K ÷. a6RbC! uptake was also sensitive to the omission of medium Na ÷. MeHg (10-5 M) also markedly inhibited the initial rate of uptake (1 min) of the Na+-dependent uptake of [3H]L-glutamate. A second neurotoxin, MnCI2 (0-5 x 10-4 M), did not alter [3H]glutamate or 86RbCI uptake. MeHg, but not MnC12, also stimulated the release of intracellular 8eRb+ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K + gradient. This would be expected to compromise the ability of astrocytes to control extracellular K ÷ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [t4C]3-O-methyl-o-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0-5 x 10-4 M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.
INTRODUCTION M e H g is a potent CNS toxin. A prominent feature of prenatal M e H g poisoning is the reduction in CNS mitotic activity 25, and the interference with neuronal cell migration 5. Astrocytes in the mammalian CNS are known targets of M e H g exposure and a predisposition of astrocytes for M e H g damage offers a possible explanation for the observed neurotoxicity in adult CNS, since it has been found that in human brain material, most of the M e H g appears to be localized within glial cells 21'29. The presence in astrocytes of a neutral amino acid carrier transport system L, capable of mediating MeHg-cysteine uptake was recently identified 1. It has long been noted that MeHg compounds can influence ion, water, and non-electrolyte transport in a variety of cells and tissues 16. The plasma membrane is believed to be the target organelle for these effects, although M e H g is also a potent inhibitor of intracellular enzymes and metabolic processes 6"3°. In recent years, primary cultures of astrocytes from neonatal rat brains have proven to be an excellent model for the study of in vitro differentiated cell functions and control mechanisms 7. Astroglial cells occupy approxi-
mately 20% of the total cell volume of the grey matter in the CNS, and their processes are found around synapses and in close association with nodes of Ranvier, axon tracts, and blood vessels 14. A m o n g the many roles proposed for astroglia are involvement in secretion of neurotrophic factors that regulate neuronal survival, biochemical maturation and morphological differentiation. These also include acid-base control, inactivation of extracellular glutamate, concentrative uptake and metabolism of neurotransmitters, expression of neurotransmitter receptors, CNS pathogenesis, and modulation of neuronal signals 14. Furthermore, astrocytes are now beginning to be appreciated as possible mediators of neurotoxic exposure to various chemical agents. For example, dense labeling of radioactive M e H g in astrocytes may implicate an astrocyte-mediated role in the neurotoxicity of M e H g 9. It has also been demonstrated that the astrocyte may represent the site of conversion of the Parkinsonism-inducing neurotoxin, MPTP, to its active metabolite MPP ÷ (see refs. 18,23). Here, we present studies on the effects of M e H g on ion and water transport in primary astrocyte cell cultures from neonatal rat cerebral cortex, as a first step in elucidating the relationship between the astrocyte and M e H g ' s toxicity.
Correspondence: M. Aschner, Department of Pharmacology and Toxicology, Albany Medical College, Albany, NY 12208, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
246 120
MATERIALS AND METHODS o
I00
Materials 86RbCI was purchased from Amersham Corporation (Arlington Heights, IL), and [3H]L-glutamate and [14C]3-O-methyl-D-glucose were purchased from New England Nuclear, (Boston, MA). Glutathione, N-methyl-D-glucamine, L-2-oxothiazolidine-4-carboxylate, N-ethylmaleimide, and diethyl maleate were of the highest analytical grade and were purchased from the Sigma Chemical Company (St. Louis, MI). Methylmercuric chloride was purchased from K & K Labs, ICN Pharmaceuticals (Plainview, NY). Cold vapor atomic absorption analysis by the method of Magos and Clarkson ~7 indicated that >99% of the total Hg was in the organic form.
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