This continuation application proposes to pursue the long-term goal of elucidating the molecular and cellular mechanisms, of blood- brain barrier (BBB) breakdown, and to initiate new studies of BBB regulation (stimulus-transport coupling) by vasoactive neurotransmitters.
The specific aims are to continue studies on two experimental models of BBB breakdown, hyperosmolal stimulation and cold injury, using intact rat brain, isolated rat cerebral capillaries, and primary monolayer cultures of brain capillary endothelial cells. We also wish to investigate receptor-mediated brain capillary transport processes using putative physiological agonists, e.g., beta-adrenergic and cholinergic agonists, adenosine, serotonin, histamine. These studies will focus on molecular events coupling endothelial cell stimulation (hyperosmolal, injury-induced, receptor-mediated) to enhancement of ODC activity and polyamine synthesis. This will include possible involvement of: (a) guanine nucleotide regulatory (G) proteins; (b) phosphodiesteratic cleavage of phosphatidylinositol- 4,5-bisphosphate and the putative messenger roles of diacylglycerol and inositol 1,4,5-trisphosphate (IP3); (c) derivatives of the arachidonate cascade, including prostaglandins; and (d) O2-derived free radicals. Stimulus-transport coupling will be a second major topic for investigation and will emphasize Ca2+- and polyamine- sensitive processes involved in the regulation of: (a) Ca2+ fluxes and cytosolic Ca2+ levels; (b) transcytosis and paracellular transport; (c) participation of cytoskeletal elements, including F-actin-containing stress fibers. These studies have a direct impact on the basic mechanisms underlying vasogenic brain edema and its treatment. 1.6 M mannitol is infused into the right carotid artery of rats to induce reversible BBB opening. Focal cold injury is induced by applying a steel rod precooled to -186 degrees C to the rat skull. In vitro experiments are performed on capillary preparations isolated from normal and injured rat cerebra, and on primary monocell cultures of rat cerebral capillary endothelium. Transendothelial transport will be quantitated in vivo and in vitro in isolated capillaries and cultured capillary endothelium using 45Ca, horseradish, peroxidase (HRP), (3H)sucrose, and alpha(3H)aminoisobutyrate (AIB). Transport pathways will be characterized ultrastructurally with HRP. Cytosolic Ca2+ concentration will be monitored spectrofluorometrically with the Ca2+-sensitive fluorescent dye fura 2.
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