Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl- d-aspartate receptors and glucocorticoids

Abstract

Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl- d-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl- d-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an up-regulation of protein kinase Cδ and ζ messenger RNA at 4 and 8 h, whereas protein kinase Cα, β, γ and ϵ did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cδ-immunoreactive neurons. Protein kinase Cδ and ζ inductions were inhibited by pretreatment with an N-methyl- d-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A 2 inhibitor, reduced only protein kinase Cζ induction. In addition, N G-nitro- l-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cδ or ζ induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca 2+-independent protein kinase C subspecies δ and ζ, but not Ca 2+-dependent subspecies, through activation of N-methyl- d-aspartate receptors and phospholipase A 2. Even though the signal pathway is similar to the induction described previously in ischaemia for genes implicated in delayed neuronal death, the gene inductions observed here are not necessarily pathogenetic, but may represent a general reaction to metabolic stress.