title: Dynamics of intracellular oxygen in PC12 Cells upon stimulation of neurotransmission.
abstract: 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 approximately 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 approximately 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 approximately 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 d PC12 cells increased.
authors: Biochemistry Department, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland.
authors: Alexander V
authors: AV
authors: Zhdanov
authors: Manus W
authors: MW
authors: Ward
authors: Jochen H M
authors: JH
authors: Prehn
authors: Dmitri B
authors: DB
authors: Papkovsky
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques; D002118:Calcium / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D009473:Neuronal Plasticity / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects / Q000502:physiology*; D016716:PC12 Cells; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D013569:Synapses / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology*; D013744:Tetanus Toxin / Q000494:pharmacology; D013997:Time Factors; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: D009498:Neurotoxins; D013744:Tetanus Toxin; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D000255:Adenosine Triphosphate; D011188:Potassium; D010100:Oxygen; D002118:Calcium