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Journal of Physiology (1999), 516.1, pp. 1—17


Topical Review
Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death
Michael R. Duchen
Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK
(Received 6 January 1999; accepted after revision 10 February 1999)

Over recent years, it has become clear that mitochondria play a central role in many key aspects of animal physiology and pathophysiology. Their central and ubiquitous task is clearly the production of ATP. Nevertheless, they also play subtle roles in glucose homeostasis, acting as the sensor for substrate supply in the transduction pathway that promotes insulin secretion by the pancreatic â_cell and that modulates the excitability of the hypothalamic glucose-sensitive neurons involved in appetite control. Mitochondria may also act as sensors of availability of oxygen, the other major mitochondrial substrate, in the regulation of respiration. Mitochondria take up calcium, and the high capacity mitochondrial calcium uptake pathway provides a mechanism that couples energy demand to increased ATP production through the calcium-dependent upregulation of mitochondrial enzyme activity. Mitochondrial calcium accumulation may also have a substantial impact on the spatiotemporal dynamics of cellular calcium signals, with subtle differences of detail in different cell types. Recent work has also revealed the centrality of mitochondrial dysfunction as an irreversible step in the pathway to both necrotic and apoptotic cell death. This review looks at recent developments in these rapidly evolving areas of cell physiology in an attempt to draw together disparate areas of research into a common theme.
Since the award of the 1978 Nobel prize for Chemistry to Peter Mitchell for his formulation of the chemiosmotic theory of oxidative phosphorylation (Mitchell & Moyle, 1967), most physiologists have regarded mitochondria as ‘solved’. They have been perceived as divorced from those aspects of physiology that are deemed exciting and significant, and dismissed as boring structures whose sole function is to manufacture ATP unobtrusively and on demand, subservient to the energetic demands of the cell. This essay was unwittingly prompted by a colleague who sat down next to me at coffee one morning, after giving an undergraduate lecture on basic cell biology and commented ‘You work on mitochondria, don’t you? Why are they interesting? What should I tell the students?’ Over the last few years, mitochondria have re-emerged into the spotlight of scientific scrutiny after many years in the wings, as unfamiliar roles are attributed to them that even Peter Mitchell would probably never have imagined (see also Miller, 1998). relationship with their eukaryotic hosts (for review, see Margulis, 1996 and see Gray, 1998), mitochondria are our primary consumers of oxygen, and it could be argued that the mitochondrial requirement for oxygen delivery has driven the evolution of the respiratory and cardiovascular systems. Mitochondria, despite all that follows here, are primarily ATP generators. This is far from trivial: ATP is the major currency in the cellular economy, required to drive by far the majority of energy-requiring reactions in all living things, from bacteria and plants to man. It is necessary for the phosphorylation reactions that modulate so many fundamental cellular processes. It may be stored and used as a (neuro)transmitter, and it controls the activity of several classes of ion channel (such as the ATP-sensitive K¤ (KATP) channel, the calcium release channel of sarcoplasmic reticulum and voltage-gated calcium channels). Mitochondria also take up calcium, and are functionally tightly integrated into mechanisms of cellular calcium signalling, and this will provide a major focal point below. It is also often forgotten that mitochondria house a number of critically important biochemical processes quite distinct and separate from energy metabolism — key enzymes involved in haemoglobin and steroid synthesis, for example, and even the carbonic anhydrase needed to make gastric acid.

‘The biggest picture?’

Oxygen is necessary for life — but only because our mitochondria use it to generate ATP. Thought to be derived from prokaryotic micro-organisms which evolved a symbiotic

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1998). Apart from ATP synthesis. while the debilitating mitochondrial encephalomyopathies result from a range of genetic disorders of mitochondrial enzyme pathways (for reviews. insidious forms of mitochondrial dysfunction may contribute to the pathophysiology of neurodegenerative disorders such as Parkinson’s disease (for review. Heritable disorders of the haem synthetic pathway cause the porphyrias. Several points shown in this scheme may be manipulated pharmacologically (a few are indicated in blue in Fig. Given that mitochondria play such a major role in the life of the cell. The channel is thought to arise from an alternate function of the adenine nucleotide translocase. diverted from the business of ATP synthesis into the generation of heat through the expression of catecholamine-regulated uncoupling proteins in brown fat in neonates and in hibernating mammals (Palou et al. 1998). 1998). apparently concentrated at contact sites between the mitochondrial inner and outer membranes (Fig. as the ion concentration gradient is small) and uncouple respiration from phosphorylation. The latter is maintained by efflux pathways — carriers which exchange Ca¥ for protons or Na¤ ions. The scheme also introduces several variables which are accessible for measurement and which can be used to inform us about the behaviour of mitochondria in cells. see Morgan-Hughes et al. 1992. 1995). perhaps in regulating the production of free radical species by the respiratory chain (see Boss et al. which may contribute specifically to the mitochondrial responses to brief physiological stimuli (see Gunter et al.g. this phenomenon was intensively studied for many years in a small number of laboratories before leaping into prominence recently through the proposal that it may play a role in apoptosis (reviewed by Kroemer et al. quite possibly a channel opened when [Ca¥]c is high. others inhibit the ATP synthase. Acute mitochondrial dysfunction causes acute energy failure and necrotic cell death. Downloaded from J Physiol (jp.physoc. 1998 for review). if speculative. The pathway is provided by a uniporter. More recently. a rise in inorganic Pé. 1998).1 Mitochondrial energy production may also be harnessed in different ways. but most notably by a rise in intramitochondrial [Ca¥] ([Ca¥]m). but with multiple subconductance states. 1998). A reasonable understanding of mitochondrial physiology requires a little background knowledge of basic biophysics and an appreciation of the actions of a few pharmacological agents. while protonophores (such as carbonyl cyanide m_ chlorophenyl hydrazone (CCCP) and carbonyl cyanide p_trifluromethoxy-phenylhydrazone (FCCP)) short circuit the electrochemical potential for protons (primarily expressed as a mitochondrial membrane potential (ÄØm). This is a pore with a large maximal conductance (•1200 pS). and the mitochondrially generated protoporphyrin IX accounts for the photosensitivity that is so disabling in porphyria cutanea tarda. see Crompton & Andreeva. Recent developments suggest other more subtle. and motoneuron disease (or amyotrophic lateral sclerosis. careful use of these agents permits a systematic dissection of mitochondrial function under many conditions. The mitochondrial permeability transition (MPT) or megachannel. 1994). which is blocked by Ruthenium Red (RuR) and various RuR derivatives and analogues (e. 516. These are revisited in Fig. Green & Reed. More subtle. for example during ischaemia or anoxia. Duchen J. actions of these proteins in cell physiology. Most recently. which shows a cartoon representing the key elements of oxidative phosphorylation (see figure legend. an electrogenic mechanism. with some subtle differences. 1972). a phenomenon exploited in the design of photodynamic therapies for cancer. and an estimated pore diameter of •2 nm (Crompton & Costi. the role of mitochondria in apoptotic cell death may well underpin the pathological changes seen in the neurodegenerative disorders listed above. and Nicholls & Ferguson. 2011 . see Kong & Xu. and see below). for a comprehensive account and see also Noji et al.2 M. Physiol. 1998. The fundamental principles outlined here govern mitochondrial function and inevitably dictate the approach adopted to investigate mitochondrial function in cells. Graeber & Muller.b. The MPT is triggered by a host of different conditions (see the extensive review by Zoratti & Szabo. Calcium uptake is driven by the electrochemical potential gradient generated by the combination of the mitochondrial inner membrane potential and the low concentration of Ca¥ in the matrix. perhaps it is not so surprising to find that mitochondrial dysfunction plays a crucial role in disease and cell death. 1979a. 1998 and see below). Agents are available that block respiration at each of the complexes of the respiratory chain. 1990). This has has recently been recognized as a A little background bioenergetics: some basic principles and definitions central participant in several important pathological and possibly physiological processes. Indeed. evidence has emerged suggesting that a rapid higher-affinity uptake pathway may also be expressed in heart and. 1990.c). and needs to be introduced. 1A). see Schapira et al. 1994. most particularly mitochondrial potential and redox state (Fig. 1A). 1A. First discovered by the observation of Ca¥-induced mitochondrial swelling (Haworth & Hunter. accompanied by a fall in ATPÏADP ratio. in liver. mitochondrial Ca¥ uptake represents a major function of mitochondria which is also driven by chemiosmotic principles. 1998). it has become clear that mitochondria play a crucial role in apoptosis (for recent reviews see Kroemer et al. R. 1997). by guest on July 3. a state of oxidative stress (excessive generation of reactive oxygen species coupled with depletion of cellular antioxidant defences) and resulting oxidation of critical thiol groups that appear to govern the channel properties (Petronilli et al. Matlib et al. which plays so fundamental a role both in shaping organ systems during development and as a route to delayed cell death following subacute injury or reperfusion after ischaemia (Kerr et al. 1B and D). As the functional consequences for each is distinct.

It was very difficult to gather data about the specific properties of mitochondria in discrete cell types — in cerebellar granule cells as opposed to Purkinje cells or hippocampal pyramidal neurons for example. it now consumes ATP and pumps protons outwards. can sustain the potential in some cells until ATP is depleted (e. ATP may be consumed in minutes in response to an uncoupler (e. Perhaps most significantly. In both the pancreatic â_cell and the glucose-sensing neurons of the hypothalamus. In the hypothalamus. the power play between the interests of mitochondrial and cell requirements is revealed. It is Until recently. see Leyssens et al. seem appropriate that mitochondria are used as substrate sensors in the major biological systems involved in the homeostatic regulation of these fundamental variables — in the pancreatic â-cell (Ashcroft & Gribble. while its possible physiological and pathophysiological roles will be discussed below. Under these conditions. some expressing a KATP channel and depolarizing in response to an increased supply of glucose. heart or liver. methyl-valine CsA was made available with a more selective action on the channel. 1988). The consequence then depends on the leakiness of the inner mitochondrial membrane and on the activity of the ATPase in reverse mode. the situation is even more complex: two cell types have been described. and the resulting increase in ATPÏADP.J. Studies of mitochondrial function in single cells Causes and consequences of mitochondrial depolarization. 1996). Ichas & Mazat. the enzyme may be regarded simply as an ATPase which is driven as an ATP synthase by virtue of the mitochondrial potential. Influx of cations such as Ca¥ or K¤ will carry a current and depolarize the membrane. delivery of substrate stimulates mitochondrial respiration.e. in a futile. and Downloaded from J Physiol (jp. Removal of substrate — either of oxygen or of carbon sources — or exposure to inhibitors of the respiratory chain stops respiration. Indeed. can show channel-like properties in the presence of high [Ca¥] (Brustovetsky & Klingenberg. as interpretation of the fluorescence signals from some of the indicators used remains somewhat ambiguous.physoc.b). such as FCCP or CCCP. Indeed. the process that sustains the potential. Measurements of mitochondrial potential. this promotes insulin secretion. and ATP may be consumed as the F1F0-ATP synthase runs in reverse mode: the equilibrium of the ATP synthase is determined both by ÄØm and by the ATPÏADP. The pharmacology is unsatisfactory as CsA has many sites of action. 1B—D). and the reader is referred to a number of recent reviews (Zoratti & Szabo. this means brain. Physiol. 1A). the insulin lowers plasma [glucose] and closes the feedback loop. Recently. The growth of our knowledge about the behaviour of mitochondria in living cells owes much to the development of fluorescence measurement and imaging technologies (see Fig. mitochondria as ATP consumers. by acting as a proton pump. therefore. see Duchen et al. of intramitochondrial [Ca¥] ([Ca¥]m) and of mitochondrial redox state in single cells has made it possible to explore the properties of mitochondria within identifiable cell types and their responses to physiological and pathophysiological stimuli in great detail. In response to mitochondrial depolarization. which. or the action of the protonophoric uncouplers. Is it not remarkable that the ‘powerhouse of the cell’ may provide a primary mechanism of ATP depletion and may precipitate cell death when function is compromised? This activity can be ascribed to the relative independence of mitochondria: it is useful for the mitochondrion to consume ATP and conserve ÄØm as this prevents mitochondrial swelling. there is no driving force for ATP synthesis. The cell membrane potential depolarizes. while another class of cells express an ATP-activated K¤ channel. The intact respiratory chain responds to the depolarization by consuming oxygen at a maximal rate. 1996). However. a component of the ATP synthase complex. perhaps giving a reminder of their evolutionary origins. Indeed. short circuits ÄØm. It might. a mitochondrial protein that mediates channel opening (Fig. Mitochondrial contributions to ‘macrophysiology’: mitochondria play a central role in sensing glucose and oxygen The primary energy supply for mitochondrial metabolism is derived from glucose and by guest on July 3. Opening of a channel such as the large conductance pathway provided by the MPT. regulation of neuronal excitability by ATP-dependent K¤ channels also appears to play a role in the control of total body glucose through the regulation of appetite. 1993a). 1992a. Role of mitochondria in glucose homeostasis. Under these conditions. 1998). which inhibits mitochondrial ATP consumption. 1998). i. in the glucosesensing neurons of the hypothalamus (Ashford et al. and express a protein known as IF1 (the inhibitory protein — see Walker. opening voltage-gated Ca¥ channels. and many cells have evolved a process to impose their own interests over and above that of the mitochondrial pool. the enzyme activity reverts to its ATPase activity. interacting with a whole family of cyclophilins whose properties remain poorly characterized. In the â_cell. A detailed review of the channel is out of place here. 516. perhaps important here to emphasise that mitochondrial depolarization is not specific to any single process. Crompton et al.Pé ratio closes a class of K¤ channels in the plasmalemma (KATP channels). which shuttle protons across the membrane.1 Mitochondrial physiology 3 which in isolation. raising [Ca¥]é. just as an inward current depolarizes the plasma membrane. which binds to cyclophilin D (cyp D).Pé ratio. 1990) and in the oxygen-sensing carotid body (Duchen & Biscoe. Presumably there is still much to be learned about the specializations of mitochondrial function in different cell types.g. the channel is closed by cyclosporin A (CsA. although it has to be said that these measurements are not without pitfalls.g. 2011 . energy-consuming cycle. it is not in the best interests of the cell. almost all the literature concerning mitochondrial function was derived from studies of isolated mitochondria in suspension. Typically. 1995. 1994).

B—D. B shows a confocal image to illustrate the distribution of endogenous or ‘auto’fluorescence derived from NADH in a single rat cardiomyocyte illuminated with UV light (at 350 nm). generating a potential gradient of approximately −150 to −200 mV (referred to as ÄØm). unpublished observation). R. ATP synthesis takes place at a separate site. producing ATP. but other substrates — ketones and fatty acids — are also used by some cells) to the tricarboxylic acid cycle (TCA or Krebs’ cycle) promotes the reduction of NAD¤ to NADH and of FAD to FADHµ. Those electrons are transferred through the enzyme complexes of the respiratory chain to oxygen. which is transported out of the mitochondria by the adenine nucleotide translocase (ANT. cartoon of a mitochondrion. The mitochondria within these cells run longitudinally with the axis of the cell (M. Imaging mitochondrial function phosphorylation. R. Duchen. The supply of substrate (only glucose is shown. with the production of water. In C. Duchen J.1 Figure 1. illustrating the basic principles of the chemiosmotic basis for oxidative . images of living mitochondria within single cells. which also imports ADP).org) by guest on July 3. they supply electrons to the respiratory chain. the ATP synthase. Physiol.physoc. 2011 A. which consists of a proton channel (the F0 subunit) and an ATP synthase (the F1 subunit).4 M. the mitochondria of a rat cortical astrocyte Downloaded from J Physiol (jp. The sites of action of a number of commonly used reagents are indicated in blue. The ANT forms part of the mitochondrial permeability transition pore (MPTP) but has been shown separately for clarity. As these are re-oxidized. protons are translocated across the inner mitochondrial membrane. 516. In the process. The enzyme is driven by the downhill movement of protons through the F0 channel and phosphorylates ADP.

1994. 1997) also are regulated by leptin. changing at POµ values of 20—30 mmHg. There is an attractive symmetry in a scheme whereby the organelles that require oxygen and glucose are also responsible for ensuring the homeostasis of those very substrates. 1993b. but there is a case to be addressed for a specialized role of mitochondria in these cells coupled with evidence for expression of a specialized cytochrome aa3 (Mills & Jobsis. although these are probably mainly regulated by transmitter (rather than metabolic) activity. agents that have no structural pharmacokinetic features in common (Anichkov & Belen’kii. if only briefly. for review. mitochondria release a (still unidentified) factor which facilitates insulin secretion at a permissive [Ca¥]c (Maechler et al. Mitochondrial calcium uptake and physiological calcium signalling Apart from the production of ATP. with permission). although the mechanism for this process remains unknown. 1998. 1972. This drives the hyperpolarization of the mitochondrial inner membrane which is the basis for the increased rate of ATP production (Duchen et al. the type I (or ‘glomus’) cells of the structure depolarize. 1970. The image was obtained on a confocal system and shows a reconstruction of a series of confocal slices through the ‘z’ plane (M. In response to a fall in oxygen tension. 1997). the â_cell KATP channels (at least. one of the remarkable features of the carotid body is that it is excited by all agents known to impair mitochondrial oxidative phosphorylation. Duchen. The jury is still out. Role of the mitochondria in oxygen sensing. It is the excitation of the carotid body that makes you feel breathless in the rarefied atmosphere at high altitude and that contributes to the sense of breathlessness of the chronic bronchitic. see also Lahiri et al. 1996). which have no effect on mitochondrial function in any other cell that we have ever studied. D shows a 3-dimensional reconstruction of digital fluorescent images of mitochondria expressing targeted green fluorescent protein (GFP) in a HeLa cell. 1996) and for modulation of Ca¥-dependent K¤ channels (Peers. 1997. but see also Lahiri et al. The modulation of these conductances by leptin. 516. unpublished observations). 1992a. 1997). 1998). The role of the mitochondria in oxygen sensing is far more contentious. increases the provision of NADH and FADHµ to the respiratory chain. but stress that this mechanism is by no means widely accepted (for counter views. These authors suggested that a reduction in the mitochondrial [Ca¥] load could account for the reduction in insulin secretion. 1997). Nevertheless. may involve mitochondrial dysfunction.b. Lopez Barneo. see also Buckler & Vaughan-Jones. Downloaded from J Physiol (jp. 1989). 1997). 1998). Biscoe & Duchen. Excitation increases the frequency of action potentials conveyed by the sinus nerve to the respiratory centres. but the key difference between responses was a decrease in associated mitochondrial Ca¥ accumulation. following mitochondrial Ca¥ uptake. the observations emphasize the pivotal role of mitochondria in the regulation of insulin secretion (see also. Buckler. Duchen & Biscoe. and given an appropriate carbon substrate for the TCA cycle. T. 2011 . and it is tempting to speculate that this is another example in which mitochondrial substrate supply plays a central role in a cellular transduction process. (Spanswick et al. Even local oxygen delivery to tissues is regulated by the energy-transducing KATP channels expressed in vascular smooth muscle cells (see Quayle et al. In the â_cell. for K¤channels that directly sense oxygen (Lopez-Barneo. These authors have also suggested that the modulation of mitochondrial Ca¥ uptake may be involved in the desensitization of insulin secretion in response to repeated exposures to metabolic substrate: a dissection of the metabolic pathway (Maechler et al. and abnormalities in the genetically obese mouse seem to place their role firmly in the realm of appetite control (and see Friedman & Halaas.1 Mitochondrial physiology 5 therefore hyperpolarize on the supply of glucose (Rowe et al. and so appetite control and glucose homeostasis appear closely linked. the supply of glucose to the cell increases substrate supply to the TCA cycle. A most surprising story to emerge recently in the â_cell literature suggests that. 1998 and Donnelly. 1996. R. see Acker.physoc. the modulator of appetite. The carotid body senses a fall in arterial oxygen tension. in which the mitochondria appear to form a contiguous but labile network (reproduced from Rizzuto et al. in an insulin-secreting cell line. a disorder responsible for such widespread morbidity and mortality. encouraging mitochondrial Ca¥ accumulation. Harvey et al. which partitions between the mitochondria and cytosol in response to ÄØm by virtue of the positive charge on the by guest on July 3. The basis for the transduction mechanism is still troublesome — how do the cells sense subtle changes in oxygen tension that would be insignificant for most other cells? There is evidence for a specialized NADPH oxidase in the plasma membrane (Acker. Adding a little flavour of integrative physiology to the story. 1998). While this factor remains mysterious. and increases respiratory rate. 1997). Isolated mitochondria will accumulate have been stained with the potentiometric dye. the major contribution of mitochondria to cell physiology must lie in their impact on Ca¥ signalling. [Ca¥]é rises and catecholamines are secreted. The central role of mitochondria in the transduction pathway has led to suggestions that at least some forms of late onset diabetes mellitus. I will rehearse it. 1990. Kennedy & Wollheim. 1994). This sequence results in a high [Ca¥]c in the presence of hyperpolarized mitochondria. tetramethyl rhodamine ethyl ester (TMRE). Biscoe and I found that mitochondrial membrane potential and redox state of the Type I cells show a unique sensitivity to moderately low oxygen tensions. Physiol.J. see Ashcroft & Rorsman. 1998) revealed that ATP production was equivalent for each exposure. As I have a personal interest in this story. 1963. 1996).

Duchen J. Pretreatment with FCCP had no significant effect on the amplitude of the response but significantly slowed the decay phase. measurements of mitochondrial [Ca¥] (i) and cytosolic [Ca¥] (ii) in a rat adrenal chromaffin cell . Note the very large increase in [Ca¥]m which was abolished by pretreatment with FCCP (blue trace).org) by guest on July 3.) C shows digital fluorescence images of rat Downloaded from J Physiol (jp.physoc. 1998. 2011 A. Direct measurements of mitochondrial Ca¥ uptake following depolarization of the plasma membrane and Ca¥ influx through voltage_gated channels. The red trace shows the rise in [Ca¥]m revealed with aequorin targeted to the mitochondrial matrix. 1997. consistent with a role of mitochondrial uptake in removing [Ca¥]c. The brisk [Ca¥]c increase and early recovery (ii) were followed by a prolonged slower decay phase (seen as the dotted and enlarged trace) which coincided with the time course of the slow decline in [Ca¥]m revealed with rhod_2 fluorescence (i) (modified from Babcock et al.6 M. from Rizzuto et al. (These data were obtained from populations of cells. 516. with permission. B: (i) stimulation of a HeLa cell with histamine mobilizes Ca¥ from IP×-sensitive ER stores. with permission).1 Figure 2. (ii). Physiol. preventing mitochondrial Ca¥ uptake. an aequorin construct targeted to the mitochondrial intermembrane space (mims) to sense local changes in [Ca¥]c showed a brisk but smaller rise in [Ca¥] in response to the same stimulus. R.

site-directed expression of the Ca¥-sensitive photoprotein. 1993). a rise in [Ca¥]c in response to voltagegated Ca¥ influx was associated with a modest transient mitochondrial depolarization and a prolonged increase in NADH autofluorescence (an increase in NADHÏNAD¤ ratio. IP×-dependent oscillations of [Ca¥]c in hepatocytes. The functional importance of this mechanism is illustrated nicely by studies of the cardiomyopathic hamster (Di Lisa et al. In contrast. which would then drive an increase in ATP production. 1998). Gunter et al. Babcock et al. These apparent differences in signal need to be explored further to establish the degree and origin of variability in mitochondrial function between different cell types. 1995). Robb-Gaspers et al. or more objectively. 2) or using mitochondrially localized fluorescent indicators. measured as an intensity profile along a line selected along the axis of the cell and displayed as a surface plot in (i) and as a ‘line image’. a more teleological question — what is that uptake for. More recent studies have extended these observations to explore the metabolic consequences of Ca¥ mobilization from IP×-dependent internal stores (Pralong et al. Thus. In the hepatocytes. what is its functional significance? More recently.g. Duchen & E. 1994. This makes good sense — oxidative phosphorylation is modulated in relation to energy demand by the relayed Ca¥ signal. 1998. Until recently. Thus. 1998). 516. high capacity pathway (see Rizzuto et al. It now seems likely that the major consequence of the Ca¥ uptake pathway in terms of mitochondrial function is the upregulation of energy by guest on July 3. Babcock & Hille. and net Ca¥ uptake is rather a kinetic balance between uniporter activity and the efflux pathways (Fig. There is now clear evidence that this process is significant during physiological [Ca¥]c signalling. Ca¥ influx is driven primarily by the mitochondrial inner membrane potential. seen as if from above in (ii). although it must be said that the concept of a Kd may be misleading. 1998). This is illustrated further by the images in D. A substantial impetus was given to attempts to address these issues by the discovery that the key rate-limiting enzymes of the citric acid cycle are upregulated by Ca¥. 1994. 1997. in which an impaired [Ca¥]c signal in cardiac muscle is associated with impaired energy metabolism. [Ca¥]c oscillations in pancreatic â_cells and adrenal glomerulosa cells were associated with oscillatory changes in NADH (Pralong et al. 1A). 1994. these questions have been extended to ask about the impact of mitochondrial Ca¥ uptake on the quantitative and spatiotemporal characteristics of Ca¥ signals (see also recent reviews by Miller. Fig. as increased energy demand is almost always accompanied by a rise in [Ca¥]c — for secretion or contraction for example. that permits specific measurement of [Ca¥] within unambiguously identifiable compartments (see Brini et al. Physiol. Downloaded from J Physiol (jp. see Hajnoczky et al. 1998. Indeed. 1990). thereby acting as an integrator of oscillatory [Ca¥]ésignalling activity. The rise in [Ca¥]m was shown to upregulate pyruvate dehydrogenase. Not only has this body of work demonstrated the functional significance of mitochondrial Ca¥ uptake. Hajnoczky et al. consistent with upregulation of the TCA cycle) which was blocked by inhibition of the mitochondrial uniporter by microinjection of RuR (Duchen. Simpson & Russell. 2). the Ca¥dependent metabolic activation clearly outlasted the [Ca¥]m signal.physoc. but it also showed that physiological Ca¥ signalling is associated with significant mitochondrial Ca¥ uptake. Most intriguingly. see McCormack et al. The first direct study in intact cells was probably that of Fein & Tsacopoulos (1988). and. Boitier. perhaps most dramatically. although communicated to mitochondria as oscillatory changes in [Ca¥]m (Hajnoczky et al. through the introduction of genetically engineered. aequorin. the driving force for Ca¥ movement will favour uptake into the matrix once the concentration of Ca¥ close to the mitochondria rises into the range of the affinity of the uniporter. In sensory neurons. 1995 and Fig. perhaps reflecting differences in [Na¤]é availability for the mitochondrial cortical astrocytes loaded with rhod_2. 1995. followed later by a large and sustained increase over the mitochondria. either directly or through the regulation of phosphorylation reactions (for review. 1998). R. 1998. two major questions remained: firstly. best seen over the nucleus (*. The kinetics of mitochondrial Ca¥ uptake and release appear to differ between cell types (Fig. increase NADH autofluorescence and then to initiate a slower increase in mitochondrial potential (Robb-Gaspers et al. were translated as graded but sustained changes in the reduction of mitochondrial NAD¤ to NADH (Pralong et al. such as rhod-2 (e. The relatively fast and transient increase over the nucleus (*) contrasts with the slower but sustained increase in signal over the mitochondria (downward-pointing arrowhead) (M. is mitochondrial Ca¥ uptake significant during physiological Ca¥ signalling? Secondly. Hajnoczky et al. 1995. 2). which show the evolution of the signal with time.1 Mitochondrial physiology 7 huge amounts of Ca¥ in the presence of inorganic phosphate. especially through improvements in confocal microscopy. normalized with respect to the first image to reveal relative changes in signal. Robb-Gaspers et al. 1994). 1995). 2011 . typically described as a low affinity (Kd 1—5 ìÒ). Stimulation with ATP (100 ìÒ) to mobilize Ca¥ from ER stores raised [Ca¥]c first. 1992). The technology required to answer these questions has developed largely through improvements in fluorescence microscopy and imaging techniques. at 13 s into the sequence). it is now clear that mitochondria do indeed take up Ca¥ during physiological signalling in most cells that have been studied. unpublished observations). who showed that a rise of [Ca¥]c following a flash of light increased oxygen consumption in Limulus photoreceptors. there appear to be subtle differences in the responses of different cell types.

Our own imaging study of mitochondrial Ca¥ uptake in adult rat cortical astrocytes using the mitochondrially localized Ca¥ indicator rhod_2 suggests that a brief transient rise in [Ca¥]c was followed by a sustained increase in [Ca¥]m that could last for many minutes (Boitier et al. and the local accumulation of [Ca¥]c close to the channel mouth will close the channels. David et al. High [Ca¥] reduces the open probability of the channel that carries the Ca¥ flux in response to emptying of ER stores (ICRAC — the calcium release-activated calcium current). 3A and B. 1994. 1999. during measurements of capacitative Ca¥ signalling in populations of T lymphocytes (Hoth et al. loading conditions etc. For example. Does this mean that Ca¥ could be taken up in one part of the cell and released at some distant site from another part of the same mitochondrial network — a notion of Ca¥ tunnelling through an organelle structure akin to that described for the ER network of pancreatic acinar cells (Mogami et al. 1995. achieved most simply by dissipating ÄØm using an uncoupler (Thayer & Miller. promoting rapid recovery from a peak of high [Ca¥]c followed by a sustained plateau of raised [Ca¥]c as the mitochondrial Ca¥ homeostatic systems re-equilibrate. Werth & Thayer. see Fig. In some other models. 1997. the ICRAC channels stay open. It is not yet clear whether these are real differences in [Ca¥]m homeostasis between cell types.physoc. and changes due to ATP depletion that may follow a loss of ÄØm (see also Landolfi et al. sensory neurons and central neurons when mitochondrial Ca¥ uptake was disabled.1 Na¤—Ca¥ exchanger. 1998). 2011 . The slow phase is also impaired by inhibition of the mitochondrial Na¤—Ca¥ exchange by agents such as diltiazem or CGP-37157 (White & Reynolds. more recently combined with GFP targeted to ER Downloaded from J Physiol (jp. (1996) described oscillations of [Ca¥]m associated with [Ca¥]c oscillations in a neonatal astrocytic cell line. the kinetics are important and have a number of fascinating functional consequences. Physiol. Stout et al. 1D) and even the more conventional confocal reconstruction shown in Fig. Further stimulation during that period therefore raises [Ca¥]c more than would otherwise be expected and potentiates transmitter release — in other words. with the exception of the measurements by Rizzuto et al.) of the indicators used. Khodorov et al. and therefore limits store refilling. Inhibition of mitochondrial Ca¥ uptake generally increases the amplitude of the transient response and removes the slow recovery phase (Fig. (1998) in hepatocytes. Herrington et al. 1C shows a substantial network structure. a rapid early phase followed by a long slow phase that may last for minutes (Fig. the plateau phase of sustained Ca¥ influx seen in measurements from cell populations was impaired by the uncoupler CCCP (Fig. recovery of [Ca¥]m appeared relatively fast despite a sustained rise in [Ca¥]c. These considerations might prompt speculation about the spatial organization of mitochondrial structures: the observations of Rizzuto et al. In both these studies. On this point it is crucial to differentiate between changes in [Ca¥]c signal due specifically to reduced mitochondrial Ca¥ uptake. 2C and D). revealing profound alterations in shaping global [Ca¥]c responses to depolarization in adrenal chromaffin cells. or technical issues related to the properties (consumption. In many excitable cells. Remove mitochondrial Ca¥ uptake. 1997). they may act as a fixed buffering system. Most obviously. 1998. the relative volume of mitochondria in the cell and the mitochondrial membrane potential. for example. Mitochondrial Ca¥ export through a Na¤—Ca¥ exchange mechanism in turn apparently maintains the high plateau global [Ca¥]c signal during the activation of the Ca¥ influx pathway. Werth & Thayer. Friel & Tsien. not least the balance of Ca¥-regulating systems expressed by the cell. 516. 1996). Duchen J. affinity. although Jou et al. if mitochondria take up Ca¥. 1997)? Modulation of spatiotemporal [Ca¥]c signals by mitochondria The elegant experiments of Rizzuto et al. 1998). the total Ca¥ flux is greater and ER store refilling is more effective. The consequences of mitochondrial Ca¥ uptake for [Ca¥]c signalling has now been widely studied. Almost all reports to date describe a rapid uptake followed by much slower re-equilibration. the temporal buffering of [Ca¥]c by mitochondrial Ca¥ uptake plays a central role in post-tetanic potentiation (Tang & by guest on July 3. The impact that this has on cellular [Ca¥]c signalling will depend on a number of factors.8 M. (1998) in HeLa cells transfected with a mitochondrially localized green fluorescent protein (GFP) suggested that. However. 3C). or the model could not work — mitochondrial efflux would simply sustain the high local [Ca¥]c and cause channel inactivation. Melamed-Book & Rahamimoff. depolarization causes Ca¥ influx through voltage gated channels and a fast [Ca¥]c transient which shows a biphasic recovery. 1994) in HeLa cells. at least in these cells. suggesting Ca¥ release from Ca¥-loaded mitochondria. (1998) using sitedirected transfection of the Ca¥-sensitive photoprotein aequorin. Mitochondrial depolarization during the slow recovery phase causes a large [Ca¥]c transient (Fig. mitochondria may form a contiguous network (Fig. 1994. 1997). as depletion of ATP will clearly alter the ability of cells to extrude or sequester a Ca¥ load quite independently of mitochondrial Ca¥ uptake. 1990. (1992. 3A and B). 1996. using site-directed aequorin and by Robb-Gaspers et al. 1998). This mechanism thus shapes the global [Ca¥]c transient following periods of depolarization. R. the functional consequences of mitochondrial Ca¥ uptake are rather more complex and subtle. Herrington et al. White & Reynolds. At nerve terminals. 1996. 3B). These authors therefore proposed that the spatial buffering of Ca¥ by mitochondrial Ca¥ uptake limits the rise in [Ca¥]c close to the channel mouth. using rhod-2. 1994. This whole process is perhaps more complex than it initially seems — it must of necessity require some spatial separation of the mitochondrial efflux pathway from the focal high [Ca¥]c near the channel mouth responsible for channel inactivation. the slow release of the Ca¥ that accumulates within mitochondria during periods of rapid repeated stimulation maintains an elevated [Ca¥]c in the terminal for a substantial period of time.

suggesting Ca¥ release from Ca¥-loaded mitochondria. a challenge with ionomycin revealed that an internal store.physoc. Downloaded from J Physiol (jp. Prior application of CCCP abolished the plateau phase completely (modified from Werth & Thayer. with permission). Fig. a brief depolarization caused a transient rise and rapid recovery of [Ca¥]c which was followed by a prolonged ‘tail’ phase of slow recovery (cf. Physiol. (ii) greatly slowed recovery.J. probably mitochondrial. 2011 A. along with other data from the study. 2A). Reintroduction of [Ca¥]ï caused a large sustained [Ca¥]c signal.1 Mitochondrial physiology 9 Figure 3. the sustained component of the capacitative influx signal was much reduced. Impact of mitochondrial Ca¥ uptake on global [Ca¥]c signalling in a variety of cell types mitochondrial Ca¥ uptake by prior depolarization of ÄØm with CCCP: (i) increased the amplitude of the response. If Ca¥ was then again removed. and the final ionomycin challenge confirmed the lack of mitochondrial Ca¥ accumulation. capacitative Ca¥ influx was studied thus: internal stores were emptied by inhibition of the ER Ca¥ ATPase with thapsigargin (TG) in the absence of external Ca¥. with permission). B. Preventing . a similar pattern of [Ca¥]c signal was seen in sensory neurons: a large [Ca¥]c load following prolonged depolarization showed a biphasic recovery — a rapid phase followed by a prolonged secondary plateau. by guest on July 3. 516. 1997. Depolarization of ÄØm by the application of CCCP during the plateau caused a massive increase in [Ca¥]c. in a bovine adrenal chromaffin cell. C. With mitochondrial uptake disabled with CCCP (dotted line). in a population of T lymphocytes. 1994. with permission). had now been filled. 1996. and (iii) abolished the slow recovery phase (modified from Herrington et al. showed that mitochondrial uptake helps to define the characteristics of capacitative Ca¥ influx (modified from Hoth et al.

1991). Intuitively. It is proposed that a rise in [Ca¥]m and a resultant small fall in intramitochondrial pH might suffice to trigger transient Downloaded from J Physiol (jp. Brind & M. acting as a spatial buffer. with type 2 receptors dominant. For example.e. during which mitochondrial potential will dissipate. S. (ii) the regulation of local [Ca¥]c by strategically placed mitochondria may regulate local Ca¥ flux through Ca¥-sensitive Ca¥ release channels and thus modulate [Ca¥]c signalling.10 M. enhanced at low [Ca¥] (< •500 nÒ). the IP× (type 1) Ca¥ release channel is sensitive to [Ca¥]. while the spatial orientation of mitochondria may support the dispersal of the Ca¥ in the cytosol away from the mouth of open Ca¥-sensitive IP×-sensitive channels. The peak of the Ca¥ releaseÏ[Ca¥]c curve lies around 400—500 nÒ. or MPT (see above). Observations from our own laboratory also point to a tight local coupling of ERÏSR Ca¥ release and Ca¥ uptake by nearby mitochondria. 1990. 1998. giving rise to a bell-shaped [Ca¥]cÏCa¥ release curve (Iino. the consequence of this focal interaction between organelles may vary between cells. Why should these similar experimental models yield different answers? One possibility is simply that. 4B). type 3 receptors also show a monotonic [Ca¥]c sensitivity (Hagar et al. and have found the opposite — [Ca¥]c wave propagation was clearly significantly faster when mitochondrial Ca¥ uptake was impaired (Boitier et al. i. However. Alternatively. R. 2011 . mitochondria could regulate the rate of efflux from the ER Ca¥ release channels. Thus. mitochondria act effectively as a spatial buffer. 516. in the astrocytes. We have measured local changes in mitochondrial potential in cardiomyocytes and. Astrocytes predominantly express type 2 IP×-dependent ER Ca¥ release channels (Shepherd et al. they are more likely to lie close to the plasmalemma in excitable cells. in which the rate of propagation of [Ca¥]c waves was modulated by varying mitochondrial potential — the larger the mitochondrial potential. 1998). the value at which mitochondrial Ca¥ uptake becomes significant. The functional consequences of the local juxtaposition of mitochondria and ERÏSR for [Ca¥]c signalling are not so easy to predict. E. the answer may lie in the properties of different classes of IP× receptors. This might seem counter-intuitive — with a larger potential. Boitier. Duchen J. but inhibited as the Ca¥ concentration rises into the micromolar range. (1996) suggest that while mitochondria may be juxtaposed to ER in inexcitable cells. Removal of local Ca¥ by mitochondria may thus serve to keep the channels open and therefore help sustain ER Ca¥ efflux. see Ichas & Mazat. 1997.physoc. 1998). 1998). channel opening is inhibited. 1999. limiting the rate and extent of spread of a [Ca¥]c signal. This may have significant implications for the propagation of Ca¥ signals during periods of anoxiaÏischaemia in the CNS. have revealed a tight apposition of mitochondria with ER membranes and a functional exchange of ER Ca¥ release and mitochondrial Ca¥ uptake. as it implies that mitochondria within the cell may be exposed in some sites to a local [Ca¥]c far higher than the averaged [Ca¥]c measured across the cell. This is the mechanism proposed by Jouaville et al. Duchen et al. As far as I am aware.1 membranes and a separate aequorin construct targeted to the outer leaflet of the inner mitochondrial membrane (Rizzuto et by guest on July 3. 1998). 4A. by buffering microdomains of [Ca¥]c. in astrocytes in culture. for review). unpublished observations) which do not show the same bell-shaped [Ca¥]c sensitivity as the type 1 receptors (Ramos-Franco et al. Bezprozvanny et al. local [Ca¥]c may not have reached levels at which ER Ca¥ release is impaired. While most of the relevant literature deals with pathological consequences of the MPT — with proposed roles for initiation of apoptosis. As [Ca¥]c rises to levels closer to 1 ìÒ. and noted highly localized transient mitochondrial depolarizations which were dependent on local Ca¥ release from internal stores and subsequent mitochondrial Ca¥ uptake (Fig. and becomes particularly interesting in the light of recent demonstration that ‘death’ signals may also propagate through cellular networks (Lin et al. pulling the [Ca¥]c into a region in which the [Ca¥]c dependence of Ca¥ release lay at the rising phase of the bell-shaped curve. there are no clues to tell us how this organization is controlled. the faster and more co-ordinated the wave (Fig. more recently. Physiol. depending on the relative expression of these proteins. (1995) to account for observations in Xenopus oocytes. ryanodinesensitive Ca¥-induced Ca¥ release from SR). 4A). mitochondrial Ca¥ uptake is more efficient and should serve as a more powerful buffer. Ultrastructural studies also suggest that the cellular organization of mitochondria may be arranged in relation to cell function: EM studies described by Lawrie et al. two quite opposing consequences suggest themselves: (i) mitochondria take up Ca¥ as it is released and therefore limit the spatial spread of the [Ca¥]c signal. This means that in cells that depend primarily on IP×mediated Ca¥ signalling from ER (or perhaps. 1998. This is important. Thus the local buffering of [Ca¥]c by mitochondria may have a major impact on ER Ca¥ release. In the astrocyte. The mechanism that underpins this story is the mitochondrial permeability transition. 2B). (1997) suggested that the MPT may open transiently at a low conductance state during normal [Ca¥]c signalling. A most intriguing contribution to this story has evolved recently. the anatomical organization of mitochondria results in the exposure of mitochondria to microdomains of high [Ca¥]c close to ER Ca¥ release sites (Fig. R. growing out of a number of related observations. It has been suggested that mitochondria may participate in the active propagation of a [Ca¥]c signal across the cell through a process of Ca¥-induced Ca¥ release (CICR. Jacobson & Duchen. ATP depletion and reperfusion injury — Ichas et al. Thus attempts to extrapolate from studies of Ca¥ handling by isolated mitochondria in order to predict the functional consequences of mitochondrial Ca¥ uptake must consider the local not the global Ca¥ concentrations. much as described above for ICRAC. Indeed. Fig. 1998). We have recently examined the role of mitochondrial Ca¥ uptake as a determinant of the rate of Ca¥ wave propagation in mammalian astrocytes. Duchen.

This was. a wave of [Ca¥]m. the image series was differentiated (a process of sequential subtractions that reveals a signal only in pixels in which the signal changed). unpublished observations). 516. and a plot of amplitude with time for a single point in the cell is shown in (iii) (modified from Jouaville et al. Physiol. a wave of Ca¥ through the mitochondrial lawn was generated in the absence of any other components of the cytosol. B (i) shows a plot of intensity with time at a series of equidistant points through a single cell. simply in response to a microdomain of [Ca¥]c generated at one corner of the lawn. B. 1995. R. R. and the rate of propagation increased. causing a local microdomain of high [Ca¥]c. 2011 .e. A(i) shows examples of the waves before (90 s) and after (at 240 and 480 s) injection of the substrates. A(ii) shows a line image to illustrate the characteristics of the waves with time. with permission). The rate of propagation of the wave clearly increased in cells in which mitochondrial Ca¥ accumulation was abolished by dissipating ÄØm (iv).1 Mitochondrial physiology 11 flickering openings of the channel in a low conductance state. to limit mitochondrial ATP consumption (M. non-hydrolysable analogue of IP× were initially chaotic and small in amplitude.J. revealing the wave front as a diagonal band as it progressed across the cell with time. The authors were able to create a propagating. i. To obtain the images shown in (iii) and (iv). larger in amplitude. not the propagation of a cytosolic wave. Boitier. [Ca¥]c waves in a Xenopus oocyte triggered by flash photolytic release of a by guest on July 3. of course. using antimycin A1 combined with oligomycin. Evidence for the role of this mechanism in cellular [Ca¥]c signalling was based on the observations that the Figure 4. A line was then selected along the axis of the cell and the colour-coded intensity profile along that line was plotted as a function of time. Duchen. once open. This may trigger local CICR from ER or may be taken up by the next bank of quiescent mitochondria which will in turn release the Ca¥. Downloaded from J Physiol (jp. which illustrate the propagating wave front. CsA-sensitive [Ca¥] wave through a ‘lawn’ of isolated mitochondria seeded onto a coverslip. Impact of mitochondrial Ca¥ uptake on propagated [Ca¥]c waves A shows the effect of mitochondrial energization on the properties of [Ca¥]c waves in Xenopus oocytes. mitochondrial potential increased (measured separately) and the waves became increasingly co-ordinated. mitochondrial Ca¥ uptake has opposite effects on the propagation of [Ca¥]c waves in rat cortical astrocytes in culture. Rea & E. the accumulated Ca¥ is released. Following microinjection of the mitochondrial substrates pyruvate and malate (at the time indicated by the arrows). The images in B (ii) illustrate the spread of the [Ca¥]c signal through the cell with time.physoc.

org) by guest on July 3. A [Ca¥]é wave in these cells causes a contractile wave which was associated here with a wave of mitochondrial depolarization. The contraction can be seen at the edges of the line image (arrows) as the line extended beyond the edges of the cell (modified from Duchen et al. tetramethyl rhodamine ethyl ester (TMRE). The line image (ii) illustrates the evolution of the signal with time along a line selected along the axis of the cell (shown in (i). (i). also raised [Ca¥]c but Downloaded from J Physiol (jp. C shows simultaneous measurements of [Ca¥]c and ÄØm made in rat hippocampal neurons in culture using fura_2 (0) and rhodamine 123 (continuous lines). 2011 A. R.1 Figure 5. illustrated during a [Ca¥]é wave in a cardiomyocyte (also loaded with TMRE).physoc. involving either clusters of mitochondria or even a single mitochondrion.12 M. with permission). The time course of the event is illustrated in (iii) in which the intensity profile along a line selected through the mitochondrion is shown with time. as shown in (ii) in a neonatal cardiomyocyte in culture. Duchen J. Two spontaneous transient events of similar amplitude as the wave were seen later (*). Physiol. seen as an increase in fluorescence signal. 1998. depolarization for 10 min with glutamate under conditions that caused 75% cell death 24 h later. These events were abolished by ryanodine or intracellular calcium chelation. These events were seen in single cells loaded with the potentiometric probe. 516. microdomains of high [Ca¥]c due to release from SR or ER cause brief transient depolarizations of . B. (ii). depolarization of the plasma membrane with 50 mÒ KCl for 10 min raised [Ca¥]c but caused only a small transient mitochondrial depolarization. a similar process operates during co-ordinated [Ca¥]c signalling. The contraction started first at the upper margin (the top left in (i) and propagated along the length of the cell. Impact of mitochondrial Ca¥ uptake on mitochondrial potential mitochondria. white line). The mean intensity plotted with time is superimposed.

R. 1996. it should be clear from the arguments presented that mitochondria play a significant. hippocampal neurons in slices (Bindokas et al. Perhaps the most actively studied model that bears an obvious relationship to cell death in a common human pathology involves glutamate toxicity. as the uniporter is electrogenic. 1998). Consequences of mitochondrial Ca¥ uptake for mitochondrial function: from cell signalling to cell death The latter may be driven by the release of apoptosis-inducing factor (AIF. However. role in shaping cellular [Ca¥]é signals. the details of this process remain to be clarified. At the very least. but the possibility that mitochondria may play an active role in [Ca¥]c signalling. 1994). a functional consequence of the close apposition of ER Ca¥ release sites and mitochondria. Duchen. The loss of mitochondrial potential in this model is clearly linked to cell death. but see Keelan et al. remains highly intriguing. but the evidence for a role caused a progressive and very nearly complete dissipation of ÄØm (note that the response could barely be increased further by application of FCCP. and more broadly. The toxicity of glutamate under these conditions has been recognized and attributed to a cellular Ca¥ overload for many years (see Choi. 1998. This is most probably simply the consequence of the flow of a Ca¥ current. There is some controversy at present about the exact sequence — perhaps even by the time this essay has appeared on paper the answer will be known. 1998 and see by guest on July 3. Fig. either through ATP consumption. and so this remains an area in which it is necessary to tread carefully. energetic collapse and necrotic cell death. It is also perhaps worth remarking on the paradox embedded in this story: apoptosis is an ATPdependent process and yet opening of the megachannel is likely to lead rapidly to ATP depletion. such as cyclosporin A. which inhibits the MPT. O. Physiol. pore opening).J. Kroemer et al. 5A). 2011 . Perhaps the full opening of the megachannel would lead rather to ATP depletion. 1992). Green & Reed. Keelan. Duchen. and have also been seen in inexcitable cells in response to mobilization of ER Ca¥ (Peuchen et al. 5C) and in response to a propagating [Ca¥]c wave in cardiomyocytes (Fig. leading to caspase (cysteine protease) activation and a suicidal (apoptotic) cascade. submitted). 1998). and to measurements of ÄØm which are fraught with inaccuracies and misinterpretations. [Ca¥]é signalling is so central to all normal physiological functions in all cell types that any disturbances in the subtleties of [Ca¥]é signalling that result from mitochondrial dysfuction will inevitably have a substantial impact on cell function. It has been clear for many years that mitochondrial Ca¥ accumulation during a state of oxidative stress may precipitate opening of the megachannel or mitochondrial permeability transition pore MPT (see above. and may result from MPT. which itself is not specific. recent evidence suggests that a key process that links the rise in [Ca¥]c to toxicity is mitochondrial Ca¥ accumulation and resultant mitochondrial depolarization. so fast is this field evolving (for recent reviews.physoc. on the integrative function of more complex organ systems. Recently we have also described focal mitochondrial depolarizations in relation to focal SRÏER Ca¥ release (Duchen et al. Clearly. which in turn triggers the permeability transition (i. acute energy failure and necrosis or through the initiation of apoptosis.e. at least in some cells. It is seen as a response to the addition of Ca¥ to preparations of isolated mitochondria. and is seen in intact cells as a reponse to physiological [Ca¥]c signals. Transient mitochondrial depolarizations have been described in excitable cells in response to depolarization in sensory neurons (Duchen. and Lemasters et al. and drives a relentless progression to cell death. glutamate accumulates in the extracellular space. although varied. Perhaps apoptosis will only result from MPT if mitochondrial ATP consumption is somehow limited. How then is the MPT compatible with a role in apoptosis? Perhaps initiation of apoptosis requires only flickering transient openings of the megachannel sufficient to release some cytochrome c. Duchen et al.1 Mitochondrial physiology 13 amplitude of cytosolic [Ca¥]c signals were modulated by CsA. 1996) or both. unpublished observations. calcineurin. Vergun & M. Downloaded from J Physiol (jp. 1998). 1998). 516. It is also important to emphasise that the interpretation of experimental evidence in favour of a role of the MPT in apoptotic cell death relies heavily on the use of pharmacological agents. It would be nice to be completely confident that the CsA was doing nothing else in the cell. during the evolution of a stroke or following neonatal asphyxia. initiating a positive feedback cycle in which caspase activation releases more cytochrome c or AIF. Rae & M. for example. 1997). 1998) or of cytochrome c (see Liu et al. It seems increasingly likely that a key step in several routes to programmed cell death involves the release of mitochondrial cytochrome c from the intermembrane space and activation of caspases. R. Boitier. The depolarization of ÄØm in response to a rise in [Ca¥]c lies at the borderlines between mitochondrial physiology and mitochondrial pathology. 5B. Most of the models of apoptotic cell death involve the application of toxic agents to or the withdrawal of growth factors from cell lines in culture. see Kroemer et al. J. much as seen in response to an uncoupler (see above). 1998. the MPT generally represents a catastrophe for the cell and will lead inexorably to cell death. as CsA is also a potent inhibitor of the Ca¥dependent phosphatase. 1998) or in culture (Keelan et al. The most immediate consequence of mitochondrial Ca¥ uptake is a mitochondrial depolarization. While flickering subconductance states may play roles in [Ca¥]c signalling (Ichas et al. During periods of anoxia and ischaemia in the CNS.

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A similar process may take place in the heart following reperfusion after an ischaemic episode — again conditions in which mitochondrial Ca¥ loading will be combined with oxidative stress. and other processes — notably inhibition of the respiratory chain — could produce similar consequences (Fig. (1998). S.physoc. Biscoe. (1998). MacMillan. Pozzan. Cyclophilin D and the benzodiazepine receptor are also attached to this complex and appear to regulate pore opening (Crompton et al. P. Changes in mitochondrial function resulting from CODA Mitochondria have become recognized as fascinating structures that have a major impact on the physiology and pathophysiology of the organism that they inhabit at many levels. R. J. Chong. V. 479—483. (1998). T. (1996). why do mitochondrial structures cytosolic Ca¥ concentration ([Ca¥]c). It seems likely that the antiapoptotic proteins bcl-2 and bcl-xL also act as mitochondrial pores or regulators (Vander Heiden et al. & Giacobino. Schinder et al. 288—294. J. Is it not remarkable that if you wish to express bax in bacteria you must also express bcl-2 or the bacteria die? Why cytochrome c should have become a major trigger to cell death is harder (for me) to understand but perhaps its presence in the cytosol informs the cell that its mitochondria are damaged. Biophysical Journal 76. is the proapoptic protein bax (Marzo et al. 4570—4587. Ca¥ signaling. (1997). Castilho. a channel-forming protein reminiscent of the bacterial pore-forming colicins. Griffiths & Halestrap. 21—27.1 of the MPT in this model is still weak.

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