Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Bioenergetic and antiapoptotic properties of mitochondria from cultured human prostate cancer cell lines PC-3, DU145 and LNCaP.

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia

Determination of the Michaelis constants for NADH in the submitochondrial particles from normal human PrEC and HLB cells, human prostate cancer cell lines PC-3, DU145, LNCaP and human fibrobsarcoma HT1080 cells.

<p>Incubation conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072078#pone-0072078-g002" target="_blank">Figure 2</a>.</p

Mitochondrial yields from cultured normal human prostate PrEC cells, prostate cancer cells PC-3, DU145, LNCaP, human fibrosarcoma cells HT1080C, and human lymphoblastoid cells (HLB).

<p>Mitochondria were prepared as described in Methods. The results are presented as Mean ± SE, n = 5-7 (separate isolations from cells). Values are expressed as mg mitochondrial protein per 1 gram of wet cells. Statistics: ** <i>p</i> < 0.05; *** <i>p</i> < 0.001. Values for prostate cancer cells PC-3, DU145 and LNCaP were compared with normal prostate cells PrEC.</p

Dependence of Complex I activity on concentration of Decylubiquinone (DB) in submitochondrial particles from normal human PrEC and HLB cells, human prostate cancer cell lines PC-3, DU145, LNCaP and human fibrobsarcoma HT1080 cells.

<div><p>Incubation conditions are described in Methods. SMP (0.15 mg) were incubated with various concentrations of DB for 5 min at 30^oC; the reaction was started by addition of 1 mM NADH.</p> <p>Panel <b>A</b>: the rate of DB reduction by SMP from LNCaP (■), PC-3 (●), and DU145 (▲) cells.</p> <p>Panel <b>B</b>: the rate of DB reduction by SMP from PrEC (■), HLB (▲), and HFS (●) cells.</p></div

Changes in the membrane potential (red) and medium pH (blue) during titration of mitochondria with calcium.

<p>(<b>A</b>) Human lymphoblast mitochondria. (<b>B</b>) Mitochondria from PC-3 prostate cancer cells. <b>Additions</b>: TPP⁺ was added in 0.5 µM aliquots, final concentration 1.5 µM; Ca²⁺ 20 nmol/ml, HCl 125 nmol/ml caused ΔpH of 0.07.</p

Respiratory activities and respiratory control ratios of mitochondria isolated from normal human prostate PrEC cells, human lymphoblastoid cells (HLB), prostate cancer cells PC-3, DU145, LNCaP, and human fibrosarcoma cells HT1080C.

<p><b>Incubation conditions</b> are described in Methods. <b>Substrates</b>: succinate 10 mM; glutamate 10 mM + malate 2 mM; citrate 10 mM + malate 2 mM. Oxidative phosphorylation (State 3) respiration was stimulated by addition of 150 µM ADP; uncoupled respiration (State 3U) was stimulated by addition of 0.5 µM cyanide-<i>m</i>-chlorophenylhydrazone (CCCP). Respiratory controls were calculated as the ratios of the State 3 respiration rate to the respiration rate in State 4₀ (before addition of ADP).</p

Swelling of mitochondria from rat liver, human lymphoblastoid and PC-3 cells.

<p><b>Incubation conditions</b> (<b>Medium B</b>) sucrose 210 mM, KCl 20 mM, glycyl-glycine 3mM, pH 7.2, KH₂PO₄ 1 mM, succinate 10 mM, mitochondria 0.5 mg, final volume 1.0 ml. Mitochondrial swelling was recorded as optical density (OD) at 520 nm using Shimadzu Multispec-1501 model spectrophotometer. Additions: Ca²⁺ 50 nmol/ml, alamethicin 4 µg/ml.</p

Membrane potential (ΔΨ) of mitochondria from cultured normal human prostate PrEC cells, prostate cancer cells PC-3, DU145, LNCaP, human fibrosarcoma cells HT1080C, and HLB oxidizing succinate in the metabolic State 4.

<p>Incubation conditions and calculation of ΔΨ as -mV are described in Methods. Values are expressed as Mean (-mV) ± SE. Statistics: ** <i>p</i> < 0.05; *** <i>p</i> < 0.001. Values for prostate cancer cells PC-3, DU145 and LNCaP were compared with normal prostate cells PrEC.</p