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Oxidative stress is an important pathogenetic factor in cerebral ischemia, which occupies one of the leading places among various forms of cerebral pathology in mortality and disability of the working-age population and is recognized as an actual problem of experimental and clinical neurology. One way to reduce damage and mortality from strokes is to study the mechanisms of ischemia by modeling its damaging factors and in vitro protection methods. To reveal the influence of chemical preconditioning induced by transient inhibition of Na+/K+-ATPase activity on tolerance of cultured cerebellar granule neurons to oxidative stress at different stages of their differentiation in vitro. The activity of Na+/K+-ATPase was inhibited with ouabain, which was added at 3–4 and 7–8 days in vitro to cerebellar cell cultures of 7-day rats at a concentration of 0.1 mM for 24 hours before induction of oxidative stress by hydrogen peroxide (0.05 and 0.075 mM, 4 hours) or paraquat (0.15 and 0.2 mM, 24 hours). Oxidative stress induced by paraquat caused the most pronounced death of cultured granule neurons in immature (3–4 days) cultures, in which survival was 44 ± 2.5% of neurons, compared to mature (7–8 days) cultures, in which survival was 61 ± 5.4%. Pretreatment of cultures with ouabain had a protective effect, the most significant in mature cultures. The exposure of mature cultures to hydrogen peroxide killed more than 90% of neurons, whereas pretreatment with ouabain increases the viability by 44%. At the same time, in immature cultures the damaging effect of Н2О2 and the protective effect of ouabain were less pronounced. The increased tolerance of cultured cerebellar granule cells to oxidative stress after transient inhibition of Na+/K+-ATPase activity by ouabain is shown. The direct dependence of the efficiency of the ouabain protection on the degree of neuronal morphochemical differentiation in vitro is revealed. [ABSTRACT FROM AUTHOR]

The synthesis and pharmacological characterisation of (S)-CPW 399 as a novel, potent and subtype-selective agonist of the AMPA receptor was recently reported. Studies have been extended to investigate its excitotoxic action in primary cultures of mouse cerebellar granule cells. (S)-CPW 399 induced neuronal cell death in a time- and concentration-dependent manner (EC50 ∼70 μM) at 24-h exposure. (S)-CPW-induced neuronal death could be prevented by co-administration with either of the AMPA/kainate selective receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) or by the 2,3-benzodiazepine, GYKI 53655 (a selective AMPA receptor antagonist); while no protection was afforded by either the NMDA receptor antagonist d,l(±)-2-amino-5-phosphonopentanoate (APV) or by nifedipine (an L-type calcium channel antagonist) when used alone or in combination. Cyclothiazide, which blocks AMPA receptor desensitisation, caused minimal potentiation of (S)-CPW 399-induced neuronal death, supporting accumulating evidence that (S)-CPW 399 is a full AMPA receptor agonist that markedly prevents a receptor desensitised conformation. (S)-AMPA, (S)-willardiine (a naturally-occurring heterocyclic excitatory amino acid) and its halogenated derivative, (S)-5-fluorowillardiine, had no deleterious effect on neuronal viability when used alone but each, in the presence of cyclothiazide, induced a concentration-dependent excitotoxic cell death with a rank order of potency (fluorowillardiine≫AMPA=willardiine). (S)-CPW 399 stimulated an increase in intracellular free-calcium levels ([Ca2+]i) in a concentration-dependent fashion (EC50 ∼5 μM) attaining a value of six-fold that of ‘resting’ cells at maximum stimulation; achieved at ∼100 μM (S)-CPW 399. The (S)-CPW 399-stimulated increase in [Ca2+]i was virtually abolished by GYKI 53655, NBQX, CNQX and by cobalt ions; markedly inhibited by nifedipine and marginally affected by d-APV. These results suggest that (S)-CPW 399 may be used as a pharmacological tool to aid in the investigation of the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitisation. [Copyright &y& Elsevier]