The contributions of several Ca2+-dependent processes to neurotoxicity
were examined in primary cultures of rat cortical neurons. The Ca2+ i
onophore ionomycin induced a rapid loss of axonal morphology and conco
mitant release of inositol phosphates that preceded morphological alte
rations of neuronal cell bodies, choline and arachidonate release, and
protein degradation. These events were followed by a degree of neuron
al lysis proportional to the external Ca2+ concentration and exposure
time. The phospholipase inhibitor neomycin decreased both arachidonate
release and the phospholipid hydrolysis catalysed by phospholipases C
and D. Proteolysis was abated by the protease inhibitor leupeptin, bu
t not by lysosomal proteolysis inhibitors. Neuronal lysis was inhibite
d partially by either leupeptin or neomycin and almost completely by b
oth in combination. However, neither agent, alone or in combination, a
ffected the morphological derangements. The diacylglycerol lipase inhi
bitor RHC-80267 reduced arachidonate release, but not neuronal lysis.
Phospholipase A(2) inhibitors had no effect on either arachidonate rel
ease or lysis. Treatment of mixed cultures of neurons and glia with a
Ca2+-dependent glutamate challenge caused similar morphological change
s and a delayed neuronal lysis that was also diminished by leupeptin a
nd neomycin, but not by inhibitors of lysosomal proteolysis. These dat
a describe several distinct stages of Ca2+-dependent injury to cortica
l neurons, a key feature of which is the stimulation of protease, and
phospholipase C and D activities. The initial stage is characterized b
y a rapid loss of axonal morphology and increased phosphatidylinositol
hydrolysis. An intermediate stage involves changes in cell body morph
ology plus the degradation of neuronal protein and phosphatidylcholine
. In a later stage, the loss of plasma membrane integrity denotes neur
onal death. (C) 1998 IBRO. Published by Elsevier Science Ltd.