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.
Highlights
Elevation of iCa2ϩ induces fast NT release via activation of the vesicular Ca2ϩ sensor synaptotagmin 1 [19]
Considering the strong effect of extracellular Kϩ (eKϩ) on Differentiated PC12 (dPC12) cell respiration, we investigated whether Kϩ ionophore valinomycin can mimic it (Fig. 2D)
We found that the respiratory response of dPC12 to FCCP had a characteristic profile, with a fast strong initial spike followed by a slower second wave in O2 consumption reaching maximum after ϳ20 min
Summary
Oxygen Consumption in Stimulated PC12 Cells neurotransmission driven by the ER [20, 21] and mitochondrial [22] Ca2ϩ stores. Derived from rat adrenal pheochromocytoma, PC12 cells have rather heterogeneous pool of Ca2ϩ stores and express L, N, T, and P/Q types of VGCC [25, 26]. Such architecture provides diverse mechanisms of regulation of iCa2ϩ, neurotransmission, and mitochondrial activity. Oxygen supply and consumption within the cell are informative markers of OxPhos, cell energetics, and signaling Far these parameters were not amenable to routine analysis. We applied this technique to examine the respiratory responses of dPC12 cells to sustained membrane depolarization by high eKϩ and to metabolic and Ca2ϩ effectors. Hampered NT exocytosis was shown not to alter significantly the respiratory response of dPC12 cells to membrane depolarization
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