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Neuroscience. 1993 Nov;57(1):9-20.

Promotion of granule cell survival by high K+ or excitatory amino acid treatment and Ca2+/calmodulin-dependent protein kinase activity.

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Netherlands Institute for Brain Research, Amsterdam.


Cerebellar granule cells in culture develop survival requirements which can be met either by chronic membrane depolarization (25 mM K+) or by stimulation of ionotropic excitatory amino acid receptors. We observed previously that this trophic effect is mediated via Ca2+ influx, either through dihydropyridine-sensitive, voltage-dependent calcium channels (activated directly by high K+ or indirectly by kainate) or through N-methyl-D-aspartate receptor-linked ion channels. Steps after Ca2+ entry in the transduction cascade mediating the survival-supporting effect of high K+ and excitatory amino acids have now been examined. Using protein kinase inhibitors (H-7, polymixin B and gangliosides), and modulating protein kinase C activity by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, we obtained evidence against the involvement of protein kinase C and cyclic nucleotide-dependent protein kinases in the transduction cascade. On the other hand, calmidazolium (employed as a calmodulin inhibitor) counteracted the trophic effect of elevated K+ with high potency (IC50 0.3 microM), which exceeded by approximately 10-fold the potency for the blockade by the drug of voltage-sensitive calcium channels. The potency of calmidazolium in interfering with the N-methyl-D-aspartate rescue of cells was also much higher in comparison with the inhibition of 45Ca2+ influx through N-methyl-D-aspartate receptor-linked channels. Our results indicated that after calmodulin the next step in the trophic effects involves Ca2+/calmodulin-dependent protein kinase II activity. KN-62, a fairly specific antagonist of this enzyme, compromised elevated K+ or excitatory amino acid-supported cell survival with high potency (IC50 2.5 microM). In the relevant concentration range, KN-62 had little or no effect on Ca2+ entry through either voltage- or N-methyl-D-aspartate receptor-gated channels. Combining information on the toxic action of glutamate in "mature" granule cells with the trophic effect of either excitatory amino acids or high K+ treatment on "young" cells, we conclude that after the initial steps involving calcium in both cases the respective transduction pathways diverge. The toxic action of glutamate seems to be mediated through protein kinase C [Favaron et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1983-1987 whereas a Ca2+/calmodulin-dependent protein kinase, which can be inhibited by KN-62 (but is resistant to gangliosides and to inhibitors whose potency is higher for protein kinase C than for Ca2+ calmodulin-dependent protein kinases, such as H-7 and polymixin B), is involved critically in the trophic effect.

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