Sa. Raya et al., A TISSUE-CULTURE ISCHEMIC DEVICE TO STUDY EICOSANOID RELEASE BY PHEOCHROMOCYTOMA PC12 CULTURES, Journal of neuroscience methods, 50(2), 1993, pp. 197-203
In an attempt to search for neuronal models to investigate the molecul
ar pharmacology of central nervous system ischemia, we have focused on
PC12 pheochromocytoma cultures which are now popular in neuroscience
research. These chromaffinergic transformed cells, originary from the
adrenal medulla, synthesize and release catecholamines and, upon treat
ment with nerve growth factor (NGF), differentiate to a sympathetic ph
enotype expressing neurites and excitability. To measure eicosanoid pr
oduction, undifferentiated or NGF-treated PC12 cultures have been expo
sed for 1 h to a mixture of N-2/ CO2 (95.5%), resulting in hypoxia (5
+/- 1% O-2), followed by 1 h reoxygenation (21% O-2) using a special i
schemic device. Hypoxia, up to 2 h, was not followed by significant cy
totoxicity or significant production of prostaglandin PGE(2). However,
upon reoxygenation, a specific release of PGE, (2-3 fold over control
) was measured. A similar PGE(2)-enhanced release could be induced by
'chemical hypoxia' using 2-deoxyglucose and oligomycin to reduce cellu
lar adenosine triphosphate (ATP). Anoxia (0.1-1% O-2, 1 h) achieved by
a reduction of culture incubation volume and the reduction in ATP lev
el have been found as critical parameters leading to PC12 cells cytoto
xicity. These results emphasize the simplicity and applicability of th
e tissue culture ischemic device proposed to investigate hypoxia and i
schemia at a cellular level.