oligomycins and Pheochromocytoma

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.

Topics: Adrenal Gland Neoplasms; Animals; Cell Differentiation; Cell Hypoxia; Culture Techniques; Eicosanoids; Ischemia; Models, Neurological; Nerve Growth Factors; Neurites; Neurons; Oligomycins; Oxygen; PC12 Cells; Pheochromocytoma; Rats

PC12 is a clonal line of rat pheochromocytoma. The cells secrete dopamine and acetylcholine by a Ca2+-dependent process. The requirement for adenosine 5'-triphosphate (ATP) in secretion from PC12 was investigated. Cellular levels of ATP were decreased by brief treatments with inhibitors of glycolysis or oxidative phosphorylation. In order to deplete ATP stores below 40% of control values during a 5-min treatment with these compounds, it was necessary to inhibit both glycolysis and oxidative phosphorylation. Depletion of ATP pools to as little as 10% of control values did not result in any inhibition of dopamine or acetylcholine release evoked by 55 mM K+ or by carbachol. Rather, in many cases spontaneous and evoked releases were significantly increased under these conditions. The release observed in ATP-depleted cells was Ca2+ dependent. The data indicate that, unlike the situation with most other secretory cells, secretion from PC12 is independent of ATP. In PC12 some proteins involved in the secretion process may be altered so as to make the process independent of ATP; this may be due to the fact that PC12 is a tumor cell line. Phosphotyrosine-containing proteins, which are present in some other tumor cells, were not found in PC12.

Topics: Acetylcholine; Adenosine Triphosphate; Adrenal Gland Neoplasms; Animals; Cell Line; Deoxyglucose; Dopamine; Energy Metabolism; Iodoacetates; Iodoacetic Acid; Neoplasm Proteins; Neoplasms, Experimental; Oligomycins; Pheochromocytoma; Rats