The overall objective of this research proposal is the investigation of the regulation of nutrient transport across the blood-brain-barrier (BBB). The functional site of the BBB is the microvascular endothelial cell and transport of any substance into, or out of, the brain occurs at the luminal (blood-facing) and abluminal (brain-facing) membranes of this polarized cell. The central hypothesis to be tested is that nutrient transport across the BBB is regulated through the cooperative activity and interaction between the specific luminal and abluminal transport systems, combined with a dynamic intracellular pool of transport proteins. We will test this hypothesis for transport systems vital to cerebral metabolism: glucose transport via the facilitative transporter protein, GLUT1, and iron transport via the transferrin receptor and the divalent metal transporter, DMT1. The polarity of transport across cells in the periphery has been described for these systems and will be used to predict potential mechanisms in the brain. We will extend the well-characterized bovine microvessel fractionation procedure for the isolation of luminal, abluminal, and intracellular membrane fractions to determine levels and activities of the respective transporters in each fraction to support/refute the predictions.
The Specific Aims are: 1) the complete characterization of the microvessel fractionation with specific attention to the fractions of the intracellular compartment; 2) the determination of structure/function relationships for GLUT1 in luminal, abluminal, and endosomal fractions; 3) the mechanism of regulation of iron transport across the BBB; and 4) the adaptation of these studies to the rat for the analysis of the effects of diabetes, hypoglycemia, as well as the genetically determined defect in iron transport in the Belgrade rat. The strength of this proposal is the availability of sufficient tissue from the bovine microvessel preparation to reliably determine activity and structure/function relationships for both of these transport systems within each compartment. The application of this information to studies in the rat, both in a miniaturized microvessel fractionation and in situ, will provide further information on the response to situations of altered metabolism, such as diabetes and hypoglycemia. Taken together these studies will provide new information on the regulation of transport across the BBB and will further our understanding of nutrient and drug delivery to the brain ? ?
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