Dynamics of intracellular oxygen in PC12 cells upon stimulation of neurotransmission

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 100mMK⁺. 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 Ca ²⁺ 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.