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 O 2 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 ϳ10 min, during which average intracellular O 2 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 O 2 consumption coincided with a transient increase in cellular ATP by ϳ60%, which was provided largely by oxidative phosphorylation and by glycolysis. The increase of respiration was orchestrated mainly by Ca 2؉ release from the endoplasmic reticulum, whereas the influx of extracellular Ca 2؉ contributed ϳ20%. Depletion of Ca 2؉ 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 d PC12 cells increased.

PGC-1α is coupled to HIF-1α-dependent gene expression by increasing mitochondrial oxygen consumption in skeletal muscle cells

“…3B). In separate experiments, C2C12 cells were cotransfected with an intracellular oxygen probe (34) and either a PGC-1␣ expression vector or an empty vector and intracellular oxygen levels were measured under oil (to prevent oxygen diffusion). Cells overexpressing PGC-1␣ had decreased intracellular oxygen levels, reflecting increased oxygen consumption compared with control cells (Fig.…”

“…At 50% confluence, C2C12 cells were transfected with expression vectors (0.1 g per well of 96-well plate) with Lipofectamine 2000. On day 1 of differentiation, cells were transfected with 1.2 M MitoXpress O2 probe (Luxcell Biosciences)/6 M Endo-Porter (Gene Tools) as described (34). Cells were washed and analyzed on a fluorescent plate reader in 100 L of RPMI medium 1640 without serum or phenol red.…”

Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells

“…For O 2 consumption assay, we used the phosphorescent oxygen-sensing probe MitoXpress (Luxcel Biosciences) as previously described (Zhdanov et al, 2008). The extracellular acidification assay was performed as previously described (Hynes et al, 2009).…”

Krüppel-like factor 6 regulates mitochondrial function in the kidney

“…An assay using the phosphorescent oxygen-sensing probe MitoXpress (Cayman Chemical) was performed to measure extracellular oxygen consumption as previously described (43). Briefly, human podocytes were initially treated with serum-free medium containing either control vehicle or ADR.…”