Isolation and behavior of plasma membrane vesicles made from cerebral capillary endothelial cells

Abstract

Introduction The endothelial cells of cerebral capillaries are joined by tight junctions that impede paracellular movement of solutes. Therefore, nutrients and ions must be transported across the respective luminal (blood-facing) and abluminal (brain-facing) plasma membranes (Brightman and Reese, 1969; Pardridge, 1983). These membranes are structurally and functionally different, resulting in a polarized configuration typical of epithelial cells (Betz and Goldstein, 1978; Betz et al ., 1980). Such an arrangement favors net unidirectional transport of certain solutes across the barrier, and implies that transendothelial flux is a balance of events occurring at each membrane domain (Ennis et al ., 1996). Clearly, the properties of both membranes must be defined and distinguished to characterize the blood–brain barrier properly. A technique is here in described for the isolation of enriched luminal and abluminal plasma membranes. Because these membranes spontaneously form tightly sealed vesicles, they can be used to study the transport and enzymatic properties of each side of the blood–brain barrier separately. Isolation of luminal and abluminal plasma membrane vesicles from the blood–brain barrier Plasma membrane vesicles from bovine cerebral capillary endothelial cells are isolated in two stages. During the first phase, brain microvessels are isolated as described by Pardridge in Chapter 6.