Oxygen Consumption Rates and Oxygen Concentration in Molt-4 Cells and Their mtDNA Depleted (ρ0) Mutants

Abstract

Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondria and to understand the pathophysiology of mitochondrial genetic disease. We have investigated the oxygen consumption rates and oxygen concentration in wild-type (WT) and mitochondrial DNA (mtDNA) depleted (rho(0)) Molt-4 cells. Wild-type Molt-4 cells have moderate oxygen consumption rates, which were significantly reduced in the rho(0) cells. PCMB (p-chloromercurobenzoate) inhibited the oxygen consumption rates in both WT and rho(0) cells, whereas potassium cyanide decreased the oxygen consumption rates only in WT Molt-4 cells. Menadione sodium bisulfite (MSB) increased the oxygen consumption rates in both cell lines, whereas CCCP (carbonyl cyanide m-chlorophenylhydrazone) stimulated the oxygen consumption rates only in WT Molt-4 cells. Superoxide radical adducts were observed in both WT and rho(0) cells when stimulated with MSB. The formation of this adduct was inhibited by PCMB but not by potassium cyanide. These results suggest that the reactive oxygen species (ROS) induced by MSB were at least in part produced via a mitochondrial independent pathway. An oxygen gradient between the extra- and intracellular compartments was observed in WT Molt-4 cells, which further increased when cells were stimulated by CCCP and MSB. The results are consistent with our earlier findings suggesting that such oxygen gradients may be a general phenomenon found in most or all cell systems under appropriate conditions.

HUVECs from newborns with a strong family history of diabetes show diminished ROS synthesis in the presence of high glucose concentrations

“…We found an inhibition in ROS synthesis in the presence of CCCP independently of the HUVECs background, but interestingly the inhibition was more severe in experimental HUVECs. CCCP stimulates oxygen consumption [38], but in hepatic mitochondrias from diabetic rats it decreases respiration [39]. Our results suggest that mitochondrias from HUVEC from newborns with DM background have a diminished cellular respiratory capacity thus corroborating the results of Nishikawa et al and Peterside et al results [39,40].…”

Theory, Instrumentation, and Applications of Electron Paramagnetic Resonance Oximetry

“…In the open-chamber model, the oxygen consumption rate was quantified by measuring the amount of oxygen diffusing through an oxygen permeable membrane, such as Teflon or methyl pentene polymer152, separating the sample from the surroundings. Recently, EPR oximetry has also been shown to successfully monitor the intracellular and extracellular oxygen simultaneously by using two different spin probes174,200. One spin probe was internalized in the cells and reported the intracellular pO 2 , while other particulate spin probe stayed outsides and reported the extracellular pO 2 values.…”

Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate

“…Since a functional ETC depends on 13 proteins encoded by the mitochondrial genome [11], cells depleted of mitochondrial DNA (ρ 0 cells) cannot use molecular oxygen as the final electron acceptor for cellular respiration, providing a unique model to study cellular adaptation to defective oxygen utilization [12]. Interestingly, the ETC-defective ρ 0 cells absolutely depend on exogenously supplemented pyruvate for survival and proliferation [13], indicating that exogenous pyruvate, in addition to pyruvate endogenously generated from glycolysis, plays a critical role in cells that cannot use molecular oxygen.…”