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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS018047-10
Application #
3398101
Study Section
Pathology A Study Section (PTHA)
Project Start
1981-12-01
Project End
1992-11-30
Budget Start
1990-12-01
Budget End
1992-11-30
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Northwestern University at Chicago
Department
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Trout, J J; Lu, C Y; Goldstone, A D et al. (1994) Polyamines mediate coronary transcapillary macromolecular transport in the calcium paradox. J Mol Cell Cardiol 26:369-77
Trout, J J; Koenig, H; Goldstone, A D et al. (1993) N-methyl-D-aspartate receptor excitotoxicity involves activation of polyamine synthesis: protection by alpha-difluoromethylornithine. J Neurochem 60:352-5
Koenig, H; Trout, J J; Goldstone, A D et al. (1992) Capillary NMDA receptors regulate blood-brain barrier function and breakdown. Brain Res 588:297-303
Koenig, H; Goldstone, A D; Lu, C Y et al. (1990) Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation. Stroke 21:III98-102
Koenig, H; Fan, C C; Goldstone, A D et al. (1989) Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes. Circ Res 64:415-26
Koenig, H; Goldstone, A D; Lu, C Y et al. (1989) Polyamines and Ca2+ mediate hyperosmolal opening of the blood-brain barrier: in vitro studies in isolated rat cerebral capillaries. J Neurochem 52:1135-42
Koenig, H; Goldstone, A D; Lu, C Y (1989) Polyamines mediate the reversible opening of the blood-brain barrier by the intracarotid infusion of hyperosmolal mannitol. Brain Res 483:110-6
Koenig, H; Goldstone, A D; Lu, C Y (1989) Blood-brain barrier breakdown in cold-injured brain is linked to a biphasic stimulation of ornithine decarboxylase activity and polyamine synthesis: both are coordinately inhibited by verapamil, dexamethasone, and aspirin. J Neurochem 52:101-9
Koenig, H; Goldstone, A D; Lu, C Y (1988) Polyamines are intracellular messengers in the beta-adrenergic regulation of Ca2+ fluxes, [Ca2+]i and membrane transport in rat heart myocytes. Biochem Biophys Res Commun 153:1179-85
Fan, C C; Koenig, H (1988) The role of polyamines in beta-adrenergic stimulation of calcium influx and membrane transport in rat heart. J Mol Cell Cardiol 20:789-99

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