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.
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