Control of respiration and oxidative phosphorylation in isolated rat liver cells

Abstract

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats.Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply).From the response of respiration to the mitochondrial membrane potential (dyM) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate dyM, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e. g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.The short-term control of cellular respiration and oxidative phosphorylation has not been well characterized in any cell type (for review see [l]). However, many mechanisms of control have been demonstrated with isolated enzymes and isolated mitochondria, and this has led to several hypotheses as to how respiration may be controlled in cells. The simplest hypothesis, arising from the work of Chance and Williams on isolated mitochondria [2, 31, is that mitochondrial respiration in cells is controlled almost exclusively by ATP usage acting via phosphorylation potential on the rate of oxidative phosphorylation and thus via proton-motive force on respiration rate. In accordance with this hypothesis several different means by which phosphorylation potential may control phosphorylation have been proposed, including thermodynamic control (assuming that phosphorylation is close to equilibrium) [4] or kinetic control of the adenine-nucleotide carrier by ADP or the ADP/ATP ratio [5].A second group of hypotheses suggests that mitochondrial respiration and phosphorylation are also controlled by NADH supply acting via mitochondrial NADH on the respiratory chain (see for example . Several different agents have been proposed to increase respiration by increasing mitochondrial NADH supply including (a) respiratory substrates such as fatty acids, lactate ...

Control of respiration and ATP synthesis in mammalian mitochondria and cells

“…A decreased Ap elicited by ATP turnover or more directly by proton ionophores has been shown to stimulate the respiratory chain in isolated liver mitochondria (Nicholls, 1974) and cells (see Fig. 6; Brown et al, 1990c). Decreased ARf or ApH stimulates cytochrome oxidase (Moroney et al, 1984;Brown & Brand, 1985;Capitanio et al, 1990), the cytochrome bcl complex (Papa et al, 1981;Brown & Brand, 1985) and probably the NADH-ubiqinone oxidoreductase (complex I) (Rottenberg & Gutman, 1977;.…”

“…6; Brown et al, 1990c). A decreased Ap elicited by ATP turnover or more directly by proton ionophores has been shown to stimulate the respiratory chain in isolated liver mitochondria (Nicholls, 1974) and cells (see Fig.…”

“…In all the above cases the changes in substrate concentration are large compared to the changes in respiration rate, implying that, although substrate supply can control respiration in isolated mitochondria and some tissues, the control is small relative to that by ATP utilization (as shown in isolated liver cells by Brown et al, 1990c). Changes in glucose concentration around the physiological level do not normally significantly change tissue respiration rates (e.g.…”

“…3; Brown et al, 1990c;Nobes et al, 1990b), in rat brain synaptosomes (Kauppinen & Nicholls, 1986a), and in rabbit proximal tubules (Balaban & Mandel, 1988). However, Kim et al (1991) found that although increased NADH supply increased the ATP/ADP ratio in rat heart in vivo it did not increase oxygen consumption.…”

“…This method has been used to locate where in energy metabolism the changes occur when respiration is stimulated by: glucagon , and thyroid hormone (Hafner et al, 1990a) in isolated mitochondria, and fatty acids (Nobes et al, 1990b) and vasopressin (Brown et al, 1990c;see Fig. 3) in liver cells.…”