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J. Biol. Chem., Vol. 283, Issue 9, 5650-5661, February 29, 2008

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Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission^*

Alexander V. Zhdanov, Manus W. Ward, Jochen H. M. Prehn, and Dmitri B. Papkovsky^¶1

From the Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland, the Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, Ireland, and the ^¶Luxcel Biosciences, BioInnovation Centre, University College Cork, Cork, Ireland

Neurotransmission, synaptic plasticity, and maintenance of membrane excitability require high mitochondrial activity in neurosecretory cells. Using a fluorescence-based intracellular O₂ sensing technique, we investigated the respiration of differentiated PC12 cells upon depolarization with 100 mM K⁺. Single cell confocal analysis identified a significant depolarization of the plasma membrane potential and a relatively minor depolarization of the mitochondrial membrane potential following K⁺ exposure. We observed a two-phase respiratory response: a first intense spike lasting 10 min, during which average intracellular O₂ was reduced from 85–90% of air saturation to 55–65%, followed by a second wave of smaller amplitude and longer duration. The fast rise in O₂ consumption coincided with a transient increase in cellular ATP by 60%, which was provided largely by oxidative phosphorylation and by glycolysis. The increase of respiration was orchestrated mainly by Ca²⁺ release from the endoplasmic reticulum, whereas the influx of extracellular Ca²⁺ contributed 20%. Depletion of Ca²⁺ stores by ryanodine, thapsigargin, and 4-chloro-m-cresol reduced the amplitude of respiratory spike by 45, 63, and 71%, respectively, whereas chelation of intracellular Ca²⁺ abolished the response. Uncoupling of the mitochondria with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone amplified the responses to K⁺; elevated respiration induced a profound deoxygenation without increasing the cellular ATP levels reduced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Cleavage of synaptobrevin 2 by tetanus toxin, known to reduce neurotransmission, did not affect the respiratory response to K⁺, whereas the general excitability of _dPC12 cells increased.

Received for publication, August 3, 2007 , and in revised form, December 14, 2007.

^* The work was supported by the European Commission, FP6 Project LSHM-CT-2005-018725, and the Science Foundation of Ireland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ To whom correspondence should be addressed: Biochemistry Dept., University College Cork, Cavanagh Pharmacy Bldg., Cork, Ireland. Tel.: 353-21-4901698; E-mail: d.papkovsky{at}ucc.ie.

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