Submitted on August 19, 2003
Accepted on November 24, 2003
Facilitation of Dopamine and Acetylcholine Release by Intermittent Hypoxia in PC12 cells: Involvement of Calcium and Reactive Oxygen Species
Dong-Kyu Kim1, Niranjana Natarajan1, Nanduri R Prabhakar2, and Ganesh K Kumar1*
1 Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA
2 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA
* To whom correspondence should be addressed. E-mail: kgk{at}po.cwru.edu.
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 sec/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 (pO2 of ~35 mmHg) for 5 min and the amount of dopamine (DA) and acetylcholine (ACh) released in the medium was determined by HPLC combined with electrochemical detection. Hypoxia augmented DA (~80%) but not ACh release in control 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 ACh release during hypoxia was partially inhibited by cadmium chloride (100 µM), a voltage-gated calcium channel blocker. By contrast, 2-aminoethoxydiphenylborate (75 µM), a blocker of inositol 1,4,5-triphosphate (InosP3) 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 mobilization of calcium from intracellular stores through activation of InosP3 receptors. Our findings also suggest that reactive oxygen species play a central role in IH-induced augmentation of transmitter release from PC12 cells during acute hypoxia.
