A tissue culture ischemic device to study eicosanoid release by pheochromocytoma PC12 cultures.
In an attempt to search for neuronal models to investigate the molecular 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 treatment with nerve growth factor (NGF), differentiate to a sympathetic phenotype expressing neurites and excitability. To measure eicosanoid production, undifferentiated or NGF-treated PC12 cultures have been exposed for 1 h to a mixture of N2/CO2 (95:5%), resulting in hypoxia (5 +/- 1% O2), followed by 1 h reoxygenation (21% O2) using a special ischemic device. Hypoxia, up to 2 h, was not followed by significant cytotoxicity or significant production of prostaglandin PGE2. However, upon reoxygenation, a specific release of PGE2 (2-3 fold over control) was measured. A similar PGE2-enhanced release could be induced by 'chemical hypoxia' using 2-deoxyglucose and oligomycin to reduce cellular adenosine triphosphate (ATP). Anoxia (0.1-1% O2, 1 h) achieved by a reduction of culture incubation volume and the reduction in ATP level have been found as critical parameters leading to PC12 cells cytotoxicity. These results emphasize the simplicity and applicability of the tissue culture ischemic device proposed to investigate hypoxia and ischemia at a cellular level.
Abu Raya
S
S
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Israel.
Trembovler
V
V
Shohami
E
E
Lazarovici
P
P
D000310:Adrenal Gland Neoplasms / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia; D046508:Culture Techniques / Q000295:instrumentation / Q000379:methods; D015777:Eicosanoids / Q000378:metabolism*; D007511:Ischemia; D008959:Models, Neurological; D009414:Nerve Growth Factors / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009840:Oligomycins / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000378:metabolism*; D051381:Rats
D015777:Eicosanoids; D009414:Nerve Growth Factors; D009840:Oligomycins; D010100:Oxygen