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 220.127.116.11) 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 language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Mar 1990|
- Phorbol 12-myristate 13-acetate
- Receptor abuse-dependent antagonism
ASJC Scopus subject areas