TROPHIC EFFECTS OF DEPOLARIZATION ON CULTURED CEREBELLAR GRANULE NEURONS

MARIA CLARA GRAVIELLE; MARIA DEL VALLE ARMANINO; LAURA BORODINSKY; MONICA FISZMAN

Tandil

Congreso; XXXIV Reunión Anual de la Asociación Argentina de Farmacología Experimental; 2002

Asociación Argentina de Farmacología Experimental

Our previous results demonstrate that depolarizing KCl concentrations (25 mM, 25K) promote cerebellar granule cell (CGC) survival. The aim of this study was to investigate the molecular mechanism of this trophic effect. CGC were obtained from 6-8-day old rats and grown in free-serum medium in the presence of 5 mM KCl (5K, basal conditions). Treatments with 25K or NMDA were performed two hours after plating and survival was measured 7 days later by the MTT assay. In order to analyze the intracellular pathways activated by elevated KCl concentrations, different channel blockers, protein kinase inhibitors and receptor antagonists were added 1 hour before 25K incubation. Addition of 25K induced a significant increase in CGC survival (55 ± 8 %) compared to control cultures, grown under basal conditions during 7 days. This stimulation was completely prevented by pre-incubation with 10 mM MgCl2, 10 mM nifedipine and a CaMKII inhibitor, KN93 (1-30 mM). These results indicate that depolarization evokes a calcium influx through L-type voltage-gated calcium channels (VGCCL) and this leads to CaMKII activation. The partial inhibition of 25K-stimulated CGC survival obtained in the presence of 20-75 mM PD98059, a MEK1 inhibitor, and 1-10 mM SB202190, a p38 kinase cascade blocker, suggests that activation of these kinases is involved. The addition of a non-competitive NMDA receptor antagonist (MK-801, 10 mM) partially blocked 25K actions. Moreover, 100 mM NMDA was as effective as 25K in increasing CGC survival. In conclusion, results presented here demonstrate that depolarization-stimulated CGC survival is mediated by different protein kinase pathways and this effect involves VGCCL and NMDA receptor activation.