Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death.
Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/phosphatidylserine/diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by approximately 90% in the 100,000 x g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the beta-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 microM 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.
Fidia Georgetown Institute for the Neurosciences, Georgetown University Medical School, Washington, DC 20007.
M
M
Favaron
H
H
Manev
R
R
Siman
M
M
Bertolino
A M
AM
Szekely
G
G
DeErausquin
A
A
Guidotti
E
E
Costa
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism / Q000494:pharmacology*; D018698:Glutamic Acid; D006706:Homeostasis; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D016760:Proto-Oncogene Proteins c-fos; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D005971:Glutamates; D007473:Ion Channels; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D018698:Glutamic Acid; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium