Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death

M. Favaron, H. Manev, R. Siman, M. Bertolino, A. M. Szekely, G. Deerausquin, A. Guidotti, E. Costa

Research output: Contribution to journalArticlepeer-review

172 Scopus citations

Abstract

Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/pnosphatidylserine/ diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by ≈90% in the 100,000 × g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the β-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and a marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 μM glutamate for 15 min (no Mg2+) induces the translocation of supernatant PKC immunoreactivity to the pellet. Such translocation persists after glutamate withdrawal and is followed by a progressive increase in neuronal death, which begins 2 hr later. Neuronal death approaches completion in about 24 hr. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [3H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45Ca2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). Moreover, the immediate cell-body swelling and activation of spectrin proteolysis elicited by glutamate remain unchanged. On the other hand, PMA-induced PKC down-regulation reduces any increase in 45Ca2+ uptake or Ca2+-dependent proteolysis (measured as spectrin degradation) after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca2+ homeostasis and neuronal death.

Original languageEnglish (US)
Pages (from-to)1983-1987
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume87
Issue number5
DOIs
StatePublished - Mar 1990

Keywords

  • Ca
  • Calpain
  • Phorbol 12-myristate 13-acetate
  • Receptor abuse-dependent antagonism
  • c-fos

ASJC Scopus subject areas

  • General

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