Respiratory carbon fluxes in leaves

Leaf respiration is a major metabolic process that drives energy production and growth. Earlier works in this field were focused on the measurement of respiration rates in relation to carbohydrate content, photosynthesis, enzymatic activities or nitrogen content. Recently, several studies have shed light on the mechanisms describing the regulation of respiration in the light and in the dark and on associated metabolic flux patterns. This review will highlight advances made into characterizing respiratory fluxes and provide a discussion of metabolic respiration dynamics in relation to important biological functions.     

Mitochondrial respiratory control in the myocardium

The heart muscle has proved to be a practical model for studying respiratory control in intact tissues. It also demonstrates that control at the level of the respiratory chain is augmented by metabolic control at the substrate level as exemplified by the very narrow range of changes in the redox state of the mitochondrial NADH/NAD couple even during extensive changes in ATP and oxygen consumption. The behaviour of mitochondria when isolated can largely be duplicated in the intact myocardium. Moreover, the high intracellular concentrations of enzymes, coenzymes and adenine nucleotides create conditions of high reaction rates, enabling the formation of a near equilibrium network of certain main pathways. This equilibrium network in connection with metabolic regulation of the hydrogen pressure upon the matrix NADH/NAD pool is a prerequisite for the regulation of cellular respiration at a high efficiency of energy transfer. Experimentation on the intact myocardium also seems to be capable of resolving some of the uncertainties about prevailing mechanisms for the regulation of cellular respiration.     

The role of mitochondrial respiration in physiological and evolutionary adaptation

Aerobic mitochondria serve as the power sources of eukaryotes by producing ATP through oxidative phosphorylation (OXPHOS). The enzymes involved in OXPHOS are multisubunit complexes encoded by both nuclear and mitochondrial DNA. Thus, regulation of respiration is necessarily a highly coordinated process that must organize production, assembly and function of mitochondria to meet an organism's energetic needs. Here I review the role of OXPHOS in metabolic adaptation and diversification of higher animals. On a physiological timescale, endocrine-initiated signaling pathways allow organisms to modulate respiratory enzyme concentration and function under changing environmental conditions. On an evolutionary timescale, mitochondrial enzymes are targets of natural selection, balancing cytonuclear coevolutionary constraints against physiological innovation. By synthesizing our knowledge of biochemistry, physiology and evolution of respiratory regulation, I propose that we can now explore questions at the interface of these fields, from molecular translation of environmental cues to selection on mitochondrial haplotype variation.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.     

The contribution of sensory inputs to the pattern generation of breathing

Current concepts of the basic neural control system and its modulation by afferent inputs are reviewed. It is emphasized that, in analogy with locomotion, the central pattern generator (CPG) for automatic metabolic respiration does not depend on any afferent feedback from receptors sensitive to the movements of the "pump," or the streams of pumped air, for its production of a rhythmic motor output provides the CPG receives some "drive" inputs above threshold and adequate bias. The operation for a variety of reflexes and feedback loops is of fundamental importance, however, for adapting the breathing pattern to the varying requirements for gas exchange to the many behavioural, nonmetabolic demands on the breathing apparatus which are competing with its primary metabolic control functions. The presentation is focussed also on available evidence that the respiratory CPG exerts powerful modulations on the transmission in these reflex pathways controlling the pattern of breathing and adjusting it to the various metabolic and behavioural demands. Mechanisms for "gating," "phasic gain changes," and "phase-dependent reflex reversal" are exemplified.     

Determination of normal ranges of mitochondrial respiratory activities by mtDNA transfer from 54 Human subjects to mtDNA-less HeLa cells for identification of the pathogenicities of mutated mtDNAs

To determine the pathogenicities of mutated mtDNAs in patients with respiration defects, the possible involvement of nuclear DNA mutations has to be excluded, since respiratory function is controlled by both nuclear DNA and mtDNA. This was achieved by showing that the mutated mtDNAs and respiration defects were co-transferred from patients to mtDNA-less human cells, and the resultant cybrid clones carrying mutated mtDNAs expressed respiration defects. To decide whether the cybrid clones expressed respiration defects, in this study the lowest limits of normal respiratory function were evaluated by transfer of mtDNAs from 54 normal subjects to mtDNA-less HeLa cells. The resultant cybrid clones showed that 71% respiratory function was the lowest limit of mtDNAs from normal subjects. On the other hand, cybrid clones carrying pathogenic mtDNAs from patients with mitochondrial diseases showed 0-64% respiratory function, suggesting that less than 71% respiratory function in cybrid clones should be a reliable indicator of whether the mutated mtDNAs of the patients were pathogenic.     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.     

A flux-sensing mechanism could regulate the switch between respiration and fermentation

The yeast Saccharomyces cerevisiae can show different metabolic phenotypes (e.g. fermentation and respiration). Based on data from the literature, we argue that the substrate uptake rate is the core variable in the system that controls the global metabolic phenotype. Consequently the metabolic phenotype that the cell expresses is not dependent on the type of the sugar or its concentration, but only on the rate at which the sugar enters the cell. As this requires the cells to 'measure' metabolic flux, we discuss the existing clues toward a flux-sensing mechanism in this organism and also outline several aspects of the involved flux-dependent regulation system. It becomes clear that the sensing and regulation system that divides the taken up carbon flux into the respiratory or fermentative pathways is complex with many molecular components interacting on multiple levels. To obtain a true understanding about how the global metabolic phenotype of S. cerevisiae is controlled by the glucose uptake rate, different tools and approaches from systems biology will be required.     

Tissue Respiration and Mitochondrial Oxidative Phosphorylation of NaCl-Treated Pea Seedlings

The effect of sodium chloride added to root medium of pea seedlings on respiratory activity of tissue segments and on isolated mitochondria was studied. Salinization enhances the respiration of leaves about one-third on a fresh weight, dry weight or protein basis. Roots and stems show only 10 to 15% respiratory stimulation. The onset of respiratory increase in leaves roughly parallels the increase in NaCl content and the decrease in growth rate. At a later stage the elevated respiration is apparent in treated plants even though the concentration of NaCl reaches a plateau and osmotic adjustment is being reached. Stimulation of respiration was found in both etiolated and green plants. Experiments with DNP show that simple uncoupling by salt is not involved; the respiratory increase in control and treated tissue is proportionally the same.

In accordance with increased respiration rates observed in vivo, mitochondria from salt-treated plants show higher rates of oxygen uptake on several substrates. The effect of NaCl added during growth is long term and is distinct from the effect of NaCl added to mitochondria isolated from control plants. Since P/O ratios are not affected by NaCl, the potential for oxidative phosphorylation in salt-affected tissue appears to increase. It is postulated that this increase may lead to changes in ADP and ATP content, and in turn, affect regulation of metabolic pathways.

     

Control of plant mitochondrial respiration

Plant mitochondria are characterised by the presence of both phosphorylating (cytochrome) and non-phosphorylating (alternative) respiratory pathways, the relative activities of which directly affect the efficiency of mitochondrial energy conservation. Different approaches to study the regulation of the partitioning of reducing equivalents between these routes are critically reviewed. Furthermore, an updated view is provided regarding the understanding of plant mitochondrial respiration in terms of metabolic control. We emphasise the extent to which kinetic modelling and 'top-down' metabolic control analysis improve the insight in phenomena related to plant mitochondrial respiration. This is illustrated with an example regarding the affinity of the plant alternative oxidase for oxygen.     

Transitions in insect respiratory patterns are controlled by changes in metabolic rate

We examined the respiratory patterns of Rhodnius prolixus and Gromphadorhina portentosa as metabolic rates varied with temperature to determine whether insects transition from discontinuous (DGC), cyclical and continuous respiration as a response to increasing aerobic demand. Using flow through respirometry we: (1) determined the effects of temperature on metabolic rate; (2) objectively defined periods of spiracular closure; (3) observed whether there was a correlation between metabolic rate and length of spiracular closure. At low temperatures both species exhibit lengthy periods of spiracular closure reflecting a discontinuous respiratory pattern. As metabolic rate increased, periods of spiracular closure decreased and insects displayed a more cyclical pattern of respiration. As metabolic rates increased even further under the highest experimental temperatures, periods of spiracular closure decreased even more and a continuous respiratory pattern was employed by both species. Our results suggest that the three described respiratory patterns in insects are not distinct but are instead a continuum of respiratory responses driven by the metabolic demand experienced by the insect.     

Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

Behavioural and cardiovascular responses to electrical stimulation of the medulla oblongata of the cat

Effects of stimulation of the nucleus tractus solitarii, the dorsal motor nucleus of the vagus, the nucleus reticularis paramedianus, and the nucleus cuneatus were studied in free-moving cats. Stimulation of the medullary nuclei that are known to be involved in the central nervous control of cardiovascular functions might activate preprogrammed motor responses such as licking and sniffing, and induce complex behavioural response patterns such as sleep or flight reaction. Moreover, both lever-pressing for rewarding brain stimulation, and eating in food deprived cats might be modulated by these stimulations. In a shuttle box the cats showed no tendency toward shuttling during stimulation, except the stimulation of the nucleus reticularis paramedianus which produced aversion. The cardiovascular and respiratory effects varied parallel with the behavioural responses. It is concluded that the medullary nuclei related to visceral functions are capable of affecting somatomotor behaviour either directly on the motor system, or by inducing complex response patterns in which somatomotor and visceral responses are integrated.     

The mitochondrial uncoupling protein-2: current status

In eukaryotic cells ATP is generated by oxidative phosphorylation, an energetic coupling at the mitochondrial level. The oxidative reactions occurring in the respiratory chain generate an electrochemical proton gradient on both sides of the inner membrane. This gradient is used by the ATPsynthase to phosphorylate ADP into ATP. The coupling between respiration and ADP phosphorylation is only partial in brown adipose tissue (BAT) mitochondria, where the uncoupling protein UCP1 causes a reentry of protons into the matrix and abolishes the electrochemical proton gradient. The liberated energy is then dissipated as heat and ATP synthesis is reduced. This property was for a long time considered as an exception and specific to the non-shivering thermogenesis found in BAT. The recent cloning of new UCPs expressed in other tissues revealed the importance of this kind of regulation of respiratory control in metabolism and energy expenditure. The newly characterised UCPs are potential targets for obesity treatment drugs which could favour energy expenditure and diminish the metabolic efficiency. In 1997, we cloned UCP2 and proposed a role for this new uncoupling protein in diet-induced thermogenesis, obesity, hyperinsulinemia, fever and resting metabolic rate. Currently, an abundant literature deals with UCP2, but its biochemical and physiological functions and regulation remain unclear. The present review reports the status of our knowledge of this mitochondrial carrier in terms of sequence, activity, tissue distribution and regulation of expression. The putative physiological roles of UCP2 will be introduced and discussed.     

Parenchyma cell respiration and survival in secondary xylem: does metabolic activity decline with cell age?

Sapwood respiration often declines towards the sapwood/heartwood boundary, but it is not known if parenchyma metabolic activity declines with cell age. We measured sapwood respiration in five temperate species (sapwood age range of 5-64 years) and expressed respiration on a live cell basis by quantifying living parenchyma. We found no effect of parenchyma age on respiration in two conifers (Pinus strobus, Tsuga canadensis), both of which had significant amounts of dead parenchyma in the sapwood. In angiosperms (Acer rubrum, Fraxinus americana, Quercus rubra), both bulk tissue and live cell respiration were reduced by about one-half in the oldest relative to the youngest sapwood, and all sapwood parenchyma remained alive. Conifers and angiosperms had similar bulk tissue respiration despite a smaller proportion of parenchyma in conifers (5% versus 15-25% in angiosperms), such that conifer parenchyma respired at rates about three times those of angiosperms. The fact that 5-year-old parenchyma cells respired at the same rate as 25-year-old cells in conifers suggests that there is no inherent or intrinsic decline in respiration as a result of cellular ageing. In contrast, it is not known whether differences observed in cellular respiration rates of angiosperms are a function of age per se, or whether active regulation of metabolic rate or positional effects (e.g. proximity to resources and/or hormones) could be the cause of reduced respiration in older sapwood.     

The response of individual polypeptides of the mammalian respiratory chain to thyroid hormone

The effects of thyroid hormone on the accumulation of inner membrane polypeptides in rat liver mitochondria have been investigated using Western blot analysis. Respiration and mitochondrial protein synthesis were also measured. Levels of the subunits of cytochrome oxidase, the cytochrome bc1 complex, and the beta-subunit of F1-ATPase increase relatively late, requiring 3-6 days of treatment and high doses of hormone. In contrast, respiration increases under conditions in which no significant accumulation of individual subunits is observed. Our results indicate that increased oxidative capacity of mitochondria can be divided into an early response which probably involves metabolic regulation of mitochondrial respiration by hormone and a later response which is due to elevated mitochondrial protein synthesis and the accumulation of polypeptides of the respiratory chain.