Mercury, including its organic compounds, is a pervasive environmental and occupational neurotoxicant with multiple cellular targets of action. In addition to direct toxic effects on neurons, neurotoxicity results secondarily following impairment of other cell types that control the neuronal microenvironment, notably glia and the vascular endothelial cell layer that constitutes the blood-brain barrier. Astroglia and vascular endothelial cells exhibit mutual trophisms in the expression of differentiated blood-brain barrier properties. Many of these properties can be reproduced in vitro in cell culture. It is proposed to use primary cultures of mouse cerebral astrcyytes and bovine cerebral capillary endothelial cells as in vitro model systems. These widely studied cell cultures will provide accessibility for the kinetic analysis and characterization of membrane transporters, and for the quantitative analysis of cellular toxicity. The ultimate objective of the proposed research is a detailed understanding of the effects of mercury on the selective permeability and transport properties of the blood-brain barrier and its cellular components, which would serve as a basis for new therapeutic interventions aimed at compensating or reversing these effects of mercury intoxication, or aimed at manipulating rates of mercury entry into or egress from the brain. In pursuit of this objective, the Immediate goal of the research is to elucidate the functional properties and selective toxicity of the carriers that mediate blood-brain barrier transport of amino acids. This research project has demonstrated the high- affinity transport of essential amino acids In astrocytes and Its interaction with the glutamine cycle. Such Interactions are believed underlie the functional regulation by astrocytes of amino acid transport In cerebrovascular endothelium. Therefore, the finding that submicromolar concentrations of mercuric mercury suppress glutamine formation in astrocytes has direct consequences for the entry of essential amino acids to the brain. Coordinated studies of astrocytes and endothelial cells in isolation will allow a quantitative analysis of the comparative toxicology of amino acid carriers. Specifically, the proposed research will explore the selective impairment by mercuric chloride and methylmercury(II) chloride of characterized transport systems for anionic, neutral and cationic amino acids in endothelial cells and astrocytes. In the event of anomalies of transport in primary cerebrovascular cell cultures, alternative systems of bovine retinal or adrenal capillary endothelial cells will be available for comparative investigation. Impairment of transport will be correlated with cell content of mercury. The role of serum constituents in modifying fluxes of mercury in endothelial cells and astrocytes will be explored. Radioactive tracers will be used to measure transport of amino acids, and movement of mercury. High-performance liquid chromatography will be used to measure endogenous free amino acid content in the cell cultures, and to validate the identity of radiolabeled species.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003928-08
Application #
2153477
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1986-06-15
Project End
1996-06-30
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Pharmacology
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Nagaraja, T N; Brookes, N (1998) Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes. Am J Physiol 274:C883-91
Brookes, N (1997) Intracellular pH as a regulatory signal in astrocyte metabolism. Glia 21:64-73
Tsai, M J; Chang, Y F; Schwarcz, R et al. (1996) Characterization of L-alpha-aminoadipic acid transport in cultured rat astrocytes. Brain Res 741:166-73
Nagaraja, T N; Brookes, N (1996) Glutamine transport in mouse cerebral astrocytes. J Neurochem 66:1665-74
Nagaraja, T N; Brookes, N (1996) Mercuric chloride uncouples glutamate uptake from the countertransport of hydroxyl equivalents. Am J Physiol 271:C1487-93
Judd, M G; Nagaraja, T N; Brookes, N (1996) Potassium-induced stimulation of glutamate uptake in mouse cerebral astrocytes: the role of intracellular pH. J Neurochem 66:169-76
Brookes, N; Turner, R J (1994) K(+)-induced alkalinization in mouse cerebral astrocytes mediated by reversal of electrogenic Na(+)-HCO3- cotransport. Am J Physiol 267:C1633-40
Brookes, N (1993) Interaction between the glutamine cycle and the uptake of large neutral amino acids in astrocytes. J Neurochem 60:1923-8
Brookes, N; Turner, R J (1993) Extracellular potassium regulates the glutamine content of astrocytes: mediation by intracellular pH. Neurosci Lett 160:73-6
Brookes, N (1992) Regulation of the glutamine content of astrocytes by cAMP and hydrocortisone: effect of pH. Neurosci Lett 147:139-42

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