Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood‐Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

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Abstract
We recently presented evidence that the reversible opening of the blood‐brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15‐fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (M). Mannitol (1 M), as well as 2 M urea, evoked a two‐ to fivefold increase in the temperature‐sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2‐deoxy‐D‐[1‐3H]glucose (DG), but not α‐[1‐14C]aminoisobutyrate, during a 2‐min incubation. DFMO (10 mM) abolished 1 M mannitol‐mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)‐induced stimulation of ODC activity and membrane transport processes was Ca2+‐dependent and verapamil‐ and nisoldipine‐sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two‐ to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC‐controlled polyamine synthesis. The effects of 10 nM PMA and 1 M mannitol were additive, suggesting that hyperosmolal stimulation of ODC‐activated polyamine synthesis does not involve protein kinase C. These data support the hypothesis that ODC‐activated polyamine synthesis and Ca2+ influx (via Ca2+ channels) play a key role in mediating the effects of hyperosmolality on BBB permeability.