Early and delayed glutamate effects in rat primary cortical neurons

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (α, β1, β2, γ, δ, ɛ, ζ and θ), performed 30 min after a 10 min treatment with 30 μM glutamate, revealed a decrease in the total β1 (−24%) and β2 (−40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while δ, ɛ, ζ and θ maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the ɛ isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C β1, δ, ɛ and ζ isoforms were mainly detected in the membrane compartment, while γ and θ signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24 h after a 10 min glutamate (10-100 μM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10 s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 μM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.