Low Reserve of Cytochrome c Oxidase Capacity in Vivo in the Respiratory Chain of a Variety of Human Cell Types

Abstract

The question of whether and to what extent the in vivo cytochrome c oxidase (COX) capacity in mammalian cells exceeds that required to support respiration is still unresolved. In the present work, to address this question, a newly developed approach for measuring the rate of COX activity, either as an isolated step or as a respiratory chain-integrated step, has been applied to a variety of human cell types, including several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. KCN titration assays, carried out on intact uncoupled cells, have clearly shown that the COX capacity is in low excess (16 -40%) with respect to that required to support the endogenous respiration rate. Furthermore, measurements of O 2 consumption rate supported by 0.4 mM tetramethyl-p-phenylenediamine in antimycin-inhibited uncoupled intact cells have given results that are fully consistent with those obtained in the KCN titration experiments. Similarly, KCN titration assays on digitonin-permeabilized cells have revealed a COX capacity that is nearly limiting (7-22% excess) for ADP ؉ glutamate/malate-dependent respiration. The present observations, therefore, substantiate the conclusion that the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria. This conclusion has important implications for understanding the role of physiological or pathological factors in affecting the COX threshold.In recent years, the metabolic control of oxidative phosphorylation has received growing attention, and the approach based on the "control of flux" theory (1, 2) has been increasingly applied to the study of mitochondrial metabolism since its first use for such purpose (3). The discovery that mitochondrial DNA mutations can cause diseases in humans (4, 5), affecting either components of the translation apparatus or subunits of various respiratory complexes, and the increasing evidence that a reduction in the activities of some respiratory chain complexes is associated with aging or neurodegenerative diseases have raised fundamental questions as to the degree of control that the individual steps of oxidative phosphorylation exert on the rate of mitochondrial respiration. Most of the experimental work aimed at answering these questions has been carried out by inhibitor titration experiments on isolated mitochondria and has led to the conclusion that the activity of the various components of the respiratory chain is in excess, sometimes a large excess (2-4-fold), with evidence for a tissue-specific pattern, over the rate required to support the endogenous respiration rate (6 -11). However, the issue has been recently raised as to how accurately a metabolic control analysis applied to isolated mitochondria can reflect the in vivo situation, considering the possible loss of essential metabolites during organelle isolation and the disruption of the normal interactions of mitochondria with the cytoskeleton, which may b...

Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes

“…The observation mentioned above of a slight, although apparently significant, delay in the kinetics of decrease in the endogenous respiration rate relative to the kinetics of decrease in TMPD-dependent respiration rate could reflect an excess of COX capacity over that required to support the normal endogenous respiration. In fact, recent studies have demonstrated that, in a variety of human cell types analyzed, including fibroblasts and myoblasts, there is in vivo a relatively low excess of COX capacity (19,21). In the present work, the TMPD-dependent respiration rate had been measured in naive uncoupled Jurkat cells, and found to be ϳ33% higher than the endogenous uncoupled respiration rate (Fig.…”

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a metabolic regulator of intestinal epithelial cell fate

“…1C). In situ measurement of COX activity by histochemical staining is an indication for respiratory capacity (11)(12)(13). Furthermore, ROS accumulation (Fig.…”

A Pathogenic 15-Base Pair Deletion in Mitochondrial DNA-encoded Cytochrome c Oxidase Subunit III Results in the Absence of Functional Cytochrome c Oxidase

“…Inhibitor titrations of COX activity have shown that in some cell types a 50 -60% reduction in COX activity does not significantly inhibit metabolic respiratory function or ATP synthesis, but larger reductions in COX activity are associated with a sharp decline in respiratory function (47,48). The amount of control that COX exerts on respiratory function is dependent on the type of cell lines and tissues (47,49,50). The relatively high rates of oxygen consumption in the 97% mutant cells suggest that, in these cell lines, COX may exert a low control strength on respiratory function.…”