Facilitation of dopamine and acetylcholine release by intermittent hypoxia in PC12 cells: involvement of calcium and reactive oxygen species

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Abstract
We have investigated the effects of preconditioning pheochromocytoma (PC12) cells with intermittent hypoxia (IH) on transmitter release during acute hypoxia. Cell cultures were exposed to either alternating cycles of hypoxia (1% O2+ 5% CO2; 30 s/cycle) and normoxia (21% O2+ 5% CO2; 3 min/cycle) for 15 or 60 cycles or normoxia alone (control) for similar durations. Control and IH cells were challenged with either hyperoxia (basal release) or acute hypoxia (Po2of ∼35 Torr) for 5 min, and the amounts of dopamine (DA) and acetylcholine (ACh) released in the medium were determined by HPLC combined with electrochemical detection. Hypoxia augmented DA (∼80%) but not ACh release in naive cells, whereas, in IH-conditioned cells, it further enhanced DA release (ranging from 120 to ∼145%) and facilitated ACh release (∼30%). Hypoxia-evoked augmentation of transmitter release was not seen in cells conditioned with sustained hypoxia. IH-induced increase in DA but not IH-induced ACh release during hypoxia was partially inhibited by cadmium chloride (100 μM), a voltage-gated Ca2+channel blocker. By contrast, 2-aminoethoxydiphenylborate (75 μM), a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, and N-acetyl-l-cysteine (300 μM), a potent scavenger of reactive oxygen species, either attenuated or abolished IH-evoked augmentation of transmitter release during hypoxia. Together, the above results demonstrate that IH conditioning increases hypoxia-evoked neurotransmitter release from PC12 cells via mechanisms involving mobilization of Ca2+from intracellular stores through activation of IP3receptors. Our findings also suggest that oxidative stress plays a central role in IH-induced augmentation of transmitter release from PC12 cells during acute hypoxia.