Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration.
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 (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; 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 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, 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.
Siniscalchi
Anna
A
Department of Clinical and Experimental Medicine, Section of Pharmacology, University of Ferrara, 17, Via Fossato di Mortara, 44100 Ferrara, Italy. snn@unife.it
Marino
Silvia
S
Marani
Luca
L
Piubello
Carmela
C
Bianchi
Clementina
C
Selvatici
Rita
R
D000465:Algorithms; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles
D007527:Isoenzymes; D013778:Tetrazolium Salts; D013844:Thiazoles; D018698:Glutamic Acid; D011493:Protein Kinase C; C022616:thiazolyl blue; D002118:Calcium