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Notizen: 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 (〈15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, α- difluoromethylornithine (DFMO, 10 m 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.

Notizen: Abstract: An early increase in ornithine decarboxylase (ODC) activity and polyamine levels in rat cerebral capillaries was previously implicated in the mediation of blood–brain barrier (BBB) breakdown in cold-injured brain. A time course study in rat cerebrum indicated that cold injury evokes a biphasic increase in ODC activity and polyamine levels in perilesional cortex. ODC activity rose sharply (fourfold) within 1 min, remained elevated for 5 min, and then returned to the basal level by 10 min. A transient rise in polyamine concentration followed in the rank order of putrescine 〉 spermidine 〉 spermine. A secondary rise in ODC activity commenced in perilesional tissue at 2–6 h and peaked (8.8-fold) at 48 h. Major increases in the content of putrescine (330%), spermidine (103%), and spermine (50%) developed at 48–72 h. α-Difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, suppressed the evoked increase in ODC activity and abolished the associated increase in content of polyamines, findings indicating that the accumulation of polyamines in cryoinjured brain reflects enhanced synthesis resulting from an ODC-mediated increase in putrescine content. Cycloheximide and actinomycin D were without effect on the early increase in ODC activity but inhibited the delayed increase in ODC activity, an observation suggesting that the initial increase in activity reflects an activation of a cryptic ODC via a posttranslational process, whereas the delayed increase in activity results from ODC synthesis mainly under transcriptional control. Because membrane phospholipid degradation, release of diacylglycerol and free arachidonic acid, and prostaglandin formation are early events in cold-injured brain, we assessed the effects of verapamil (a calcium channel blocker), dexamethasone (which inhibits arachidonic acid release), and aspirin (a cyclooxygenase inhibitor). These agents resembled DFMO in that they inhibited the early (2-min) and delayed (24-h) increase in ODC activity and polyamine concentrations and concurrently attenuated BBB breakdown in the perilesional cortex, as monitored by fluorescein transport. Exogenous putrescine nullified the protective effect of verapamil, dexamethasone, and aspirin on BBB breakdown following cryogenic injury. These results implicate Ca2+ influx via calcium channels, phospholipid hydrolysis, and prostaglandin synthesis in cryogenically induced stimulation of ODC activity and further strengthen the evidence linking polyamines to BBB breakdown. Changes in ODC-regulated polyamine synthesis in brain cells may play an important role in other aspects of the pathophysiology of cerebral injury.

Notizen: Abstract: We investigated the role of polyamines and their regulatory enzyme ornithine decarboxylase in N-Methyl-D-aspartate-induced excitotoxicity in embryonic chick retina. N-Methyl-D-aspartate (200 μM) produced an early increase in ornithine decarboxylase activity, putrescine concentration, and Ca2+ entry, leading to selective neuronal death by 30 min. This response was attenuated by the ornithine decarboxylase inhibitor α-difluoromethylornithine and the N-methyl-D-aspartate receptor antagonist 5-aminophosphonovaleric acid. Exogenous putrescine increased intracellular putrescine and spermine levels and reversed neuroprotection by α-difluoromethylornithine, but not by 5-aminophosphonovaleric acid. N-Methyl-D-aspartate-receptor stimulation of putrescine/polyamine synthesis mediates abnormal Ca2+ entry and acute excitotoxic neuronal death. Postreceptor inhibition of the ornithine decar-boxylase/polyamine cascade by α-difluoromethylornithine may provide neuroprotection against N-methyl-D-aspartate-induced excitotoxicity.