Mechanisms of cell survival in hypoxia and hypothermia
Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK
*e-mail: rgb11{at}hermes.cam.ac.uk
Accepted July 2, 2001
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Summary |
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Key words: hypoxia, hypothermia, metabolic depression, ion homeostasis, channel arrest, ion-motive ATPase, neurone, muscle, hepatocyte.
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Introduction |
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Cellular responses to O2 lack |
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ATP-consuming processes in cellular respiration
At the cellular level, metabolic depression may be brought about (i) by decreasing energy-consuming processes and/or (ii) by increasing the efficiency of energy-producing pathways (Hochachka and Guppy, 1987; Storey and Storey, 1990; Hochachka et al., 1996; Brand et al., 2000; Boutilier and St-Pierre, 2000). Protein synthesis and ion-motive ATPases are the dominant energy-consuming processes of cells at standard metabolic rate (SMR) (Rolfe and Brown, 1997), so decreases in the ATP demand of these processes at minimal metabolic rate (MMR) are thought to be largely responsible for enabling the downregulation of energy turnover in metabolically depressed states (Guppy et al., 1994; Hand and Hardewig, 1996; Hochachka et al., 1996; Donohoe et al., 2000). Less is known about the metabolic efficiency of the processes that make up SMR in vertebrates. These involve stoichiometric efficiencies that maximize the molar ratios of ATP to O2 during hypoxia or of ATP to H+ during fermentation (Hochachka and Somero, 1984; Brand et al., 2000). Given that SMR represents the starting point against which metabolic reductions are compared, it is imperative that the reactions that constitute SMR are well defined. Approximately 20% of mammalian whole-animal O2 consumption can be attributed to mitochondrial proton leak and non-mitochondrial respiration (Brand et al., 2000). Of the 80% of SMR that is coupled to ATP synthesis, 2530% is used by protein synthesis, 1928% by the Na+/K+-ATPase, 48% by the Ca2+-ATPase, 28% by the actinomyosin-ATPase, 710% by gluconeogenesis, 3% by ureagenesis, with substrate cycling and mRNA synthesis making significant contributions (Rolfe and Brown, 1997). Thus, protein synthesis and ion-motive ATPases are the dominant ATP-consuming processes in cells, making up more than 90% of the oxidatively coupled ATP consumption of rat skeletal muscle (Rolfe and Brand, 1996) and as much as 66% of the ATP turnover in rat thymocytes (Buttgereit and Brand, 1995).
A number of cells and tissues of facultative anaerobes have become particularly useful models for the study of cellular energetics since they possess an intrinsic capacity for anoxia-induced hypometabolism. For example, the metabolic rates (measured as heat flow using micro-calorimetry) of turtle hepatocytes (Buck et al., 1993a), turtle brain cortical slices (Doll et al., 1994), frog skeletal muscle (West and Boutilier, 1998) and frog heart fall to approximately 25% of the normoxic rates after only 30min of exposure to anoxia (Fig.2). Reoxygenation reverses these effects rapidly and completely. Moreover, intracellular ATP concentrations remain stable throughout the cycles of anoxia and reoxygenation, consistent with the maintained energy balance observed in the tissues of animals exposed to O2 lack in vivo (Wegener and Krause, 1993; Donohoe and Boutilier, 1998; Donohoe at al., 1998). Since protein turnover and ion-motive ATPases are major ATP sinks in many cells and tissues at SMR (Rolfe and Brown, 1997), these energy-consuming processes represent primary cellular targets for downregulating ATP demands in response to O2 lack.
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Channel arrest versus channel leak
The critical difference between anoxia-tolerant and anoxia-sensitive animals is that the latter show no reduction in the absolute ATP demand of the ion-motive ATPases in response to O2 lack. In contrast, cells of anoxia-tolerant animals exhibit large-scale reductions in absolute Na+/K+ activity during anoxia. Although this might normally be interpreted as pump failure in a mammalian cell, such decreases in Na+/K+-ATPase activity in the brain, liver and muscle cells of facultative anaerobes are brought about without any disruptions in electrochemical potentials, cellular ion levels or ATP concentrations (Buck et al., 1993b; Buck and Hochachka, 1993; Lutz and Nilsson, 1997; Bickler and Buck, 1998; West and Boutilier, 1998; Donohoe et al., 2000). Thus, quite apart from there being any failure of ionic homeostasis, the reduction in Na+ pump activity in anoxia-tolerant cells is part of a coordinated process of energy conservation wherein O2 lack initiates a generalised suppression of ion-channel densities and/or channel leak activities. The net result is that cell membrane permeabilities are reduced, thereby lowering the energetic costs of maintaining transmembrane ion gradients. This phenomenon, so-called channel arrest (Hochachka, 1986), serves as a potent mechanism for actively downregulating the ATP demands of cells in potentially energy-limited states. In contrast, anoxic mammalian cells, particularly in brain and heart, show all the hallmarks of a non-adaptive channel leak response. For example, hypoxia-induced increases in the persistent Na+ current in intact cardiac (Ju et al., 1996) and hippocampal (Hammarström and Gage, 1998) cells as well as corresponding increases in amiloride-sensitive Na+/H+ and Na+/Ca2+ exchange activity in nerve cells (Chidekel et al., 1997; Haddad and Jiang, 1997) lead to increases in transmembrane Na+ cycling. This evidently raises the ATP demand of ion-balancing ATPases to breaking point, which triggers abnormal accumulation of Na+, leading to deleterious Ca2+ overload and cell damage and/or death (Hammarström and Gage, 1998) (Fig.1).
Ion channel arrest has been studied most extensively in models of anoxia-tolerant brain, where it is considered to be one of the most important neuroprotective mechanisms against O2 lack (Lutz and Nilsson, 1997; Bickler and Buck, 1998). One of the tenets of the channel arrest theory is that ion-channel densities should be inherently lower in hypoxia-tolerant animals than in their hypoxia-sensitive counterparts (Hochachka, 1986). Although this appears to be the case for Na+ channel density both for turtle cerebellum (Xia and Haddad, 1991) and in isolated nerve endings of turtle brain (i.e. synaptosomes) (Edwards et al., 1989), it cannot in itself explain the 100-fold difference in anoxia-tolerance between turtle and rat brain. Unlike their mammalian counterparts, ATP levels and ionic gradients in turtle neurons are maintained perfectly homeostatic during anoxia, thereby avoiding the fatal consequences of energy failure (Fig.1). The remarkable anoxia-tolerance of the brain cells of these facultative anaerobes appears to be effected through decreased electrical activity (Sick et al., 1982; Feng et al., 1988) and reduced rates of transmembrane ion leakage (channel arrest) (Lutz et al., 1985; Hand and Hardewig, 1996). This downregulation of firing rates and synaptic transmission suppresses cellular metabolism by 5080% (Doll et al., 1994; Lutz and Nilsson, 1997). In turtle brain, anoxia-induced suppression of current flow through Na+ channels also leads to the elimination of action potentials (spike arrest), thereby reducing the energetic costs of neurotransmission and decreasing the ATP demands of the ion pumps involved in the maintenance of electrochemical gradients (Sick et al., 1993). Although decreases in neuronal Na+ channel density of turtle brain have been observed in response to O2 lack (Peréz-Pinzón et al., 1992a; Peréz-Pinzón et al., 1992b), the mechanisms of Na+ channel recycling from membrane to cytosol and of probable changes in leak activity at the level of the channel itself remain elusive (Bickler and Buck, 1998).
Putative modulators of anoxia-induced channel arrest
Little is known about the signalling pathways involved either in channel arrest or in the transition to hypometabolic states during anoxia. Although there has been some speculation that decreased intracellular pH and/or increased CO2 partial pressures may trigger metabolic rate depression (Lutz, 1989; Wasser et al., 1990), the mechanisms remain elusive. While there are many other possible systemic co-mediators of metabolic rate (e.g. cortisosteroids, insulin, glucagon, thyroid hormones), there has been intense interest of late in more local chemical triggers. As the initial end-product of ATP degradation, adenosine could offer a direct measure of cell metabolic status through its receptor systems (Belardinelli and Shryock, 1992). For example, concentrations of adenosine are inversely proportional to energy charge in the isolated heart of the frog Rana ridibunda (Lazou and Beis, 1986). In such a case, adenosine would be an obvious candidate for signalling when rates of ATP synthesis fall below rates of ATP usage. Indeed, marked increases in extracellular adenosine concentration are thought to play an important neuroprotective role during the initial stages of energy failure in anoxic and ischaemic mammalian brain (Hagberg et al., 1987; Newby et al., 1990). Adenosine is also released into the extracellular space of turtle brain shortly after the onset of anoxia (Nilsson and Lutz, 1992; Lutz and Nilsson, 1993; Pék and Lutz, 1997). Moreover, when anoxic isolated turtle cerebellum is superfused with adenosine receptor blockers, rates of K+ efflux increase and thereby hasten the onset of membrane depolarisation (Pék and Lutz, 1997). That adenosine itself may be involved in coordinating metabolic and ion-channel arrest seems well worth pursuing since, at least in brain, it is known to reduce cell excitability, to stimulate glycogenolysis and to increase cerebral blood flow (Magistretti et al., 1986; Morii et al., 1987; Lutz and Nilsson, 1997).
Because the Na+/K+-ATPase ion pump is the primary ATP-consuming process in neurons, its failure as a result of anoxia-induced energy shortages in largely aerobic animals leads to a rapid dissipation of the pre-existing Na+ gradient. This causes the Na+/glutamate cotransporter to reverse direction, so that instead of the cell taking up glutamate following its synaptic release, glutamate accumulates in the extracellular space. Because glutamate is a major excitatory neurotransmitter, its extracellular accumulation in anoxia-sensitive brain hyperactivates glutamate receptors and leads to excitotoxic injury. In contrast, glutamate is not released by the turtle brain during anoxia (Nilsson and Lutz, 1991). Moreover, glutamate receptor activity decreases when turtle brain is exposed to hypoxia, stabilising intracellular [Ca2+] and thereby promoting cell survival (Bickler and Buck, 1998). However, increases in levels of the inhibitory neurotransmitter -aminobutyric acid (GABA) and in GABA receptor numbers in anoxic turtle brain (Nilsson and Lutz, 1991; Nilsson and Lutz, 1993) may facilitate the suppression of neurotransmission and the overall depression of brain electrical activity that accompanies metabolic depression.
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Mitochondrial responses to O2 lack: cellular treason |
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Because the rate of ATP use by the F1Fo-ATPase decreases to a new steady state when frog skeletal muscle mitochondria are exposed to anoxia, this leads to the maintenance of a lower mitochondrial membrane potential (St-Pierre et al., 2000). Although this probably means that anoxic mitochondria have a reduced capacity to transport substrates compared with their normoxic counterparts, the energy saved by reducing the membrane potential (to lower but biologically viable values, rather than maintaining it at pre-existing levels) may be critical to the preservation of cell membrane potential (Fig.3). Mitochondrial membrane potential cannot be abolished completely during anoxia since it is crucially needed to import proteins that ensure mitochondrial maintenance. Moreover, the disruption of mitochondrial membrane potential during anoxia would lead to a series of events culminating eventually in cell death by apoptosis (Kroemer et al., 1998).
It is important to note that backwards operation of the ATP synthase occurs only during anoxia and/or ischaemia since even trace amounts of oxygen can make a vital energetic contribution to aerobic ATP production. Indeed, recent studies show that mitochondrial oxidative phosphorylation is more efficient at low O2 tensions (01.1kPa) than at air saturation as a result of depressed proton leak and uncoupled respiration (Gnaiger et al., 2000).
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Oxygen sensing and molecular adaptation to hypoxia |
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There are now many examples of hypoxia-induced induction of genes, including those for (i) erythropoietin (EPO) and vascular endothelial growth factor (VEGF), both of which are involved in enhancing O2 delivery to tissues, (ii) glycolytic enzymes involved in anaerobic ATP supply pathways, and (iii) heat-stress proteins (HSPs), whose expression constitutes a ubiquitous intracellular protective response to stress. There is circumstantial evidence that the universal sensor is a haem protein embodied within a multi-subunit assembly that contains a NAD(P)H oxidase capable of producing reactive oxygen species (ROS). The ROS act as signalling molecules in a cascade that normally inhibits the activation of hypoxia-inducible factor 1 (HIF-1) by mediating the degradation of HIF-1a subunits in the proteosome. The model predicts that a decrease in ROS production occurs during hypoxia, or when cobalthaem binding mimics hypoxia, and that this decrease in levels of ROS leads to the activation of an HIF-1 heterodimer that is required for the induction of hypoxia-responsive gene expression (Bunn et al., 1998). Although there have been intense efforts to identify the various response elements of this putative O2-sensing pathway, the underlying mechanisms of cellular O2 sensing and regulation of the activity of HIF remain elusive (Zhu and Bunn, 2001). Others suggest that mitochondria themselves can act as O2 sensors by increasing ROS production during hypoxia since cells depleted of their mitochondrial DNA not only fail to produce ROS in response to hypoxia but lose the ability of their wild-type counterparts for hypoxia-induced gene expression (Chandel et al., 1998). Clearly, full elucidation of the cellular O2-sensing and signal-transduction pathways represents a major challenge for the future.
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Cellular responses to hypothermia |
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Ionic integrity of cells during hypothermia
To understand why many mammalian tissues and cells are so cold-sensitive, we must again turn to the ion-regulatory mechanisms at the cell membrane. As with hypoxia-induced membrane destabilisation (Fig.1), deep hypothermia in non-hibernating mammals leads to marked disturbances in cellular ion homeostasis that may be further influenced in the cold by alterations in membrane fluidity (Kruuv et al., 1983; Zachariassen, 1991; Stefanovich et al., 1995). For example, rat ventricular cells at 10°C show a greater than 50% increase in [Ca2+]i compared with those at 30°C, supporting the idea that intracellular Ca2+ overload takes place in cardiac myocytes of non-hibernating mammals during deep hypothermia (Wang and Zhou, 1999). Wang and co-workers (Wang et al., 1997) also determined the relationship between cardiac hypothermia-tolerance and the sources of activator Ca2+ by modifying sarcoplasmic reticulum function in papillary muscles from the cold-sensitive rat and the cold-tolerant hibernating ground squirrel. Rat muscle preparations showed a marked increase in resting tension and aftercontraction and became inexcitable as temperature was lowered from 25 to 712°C. In contrast, muscle preparations from the ground squirrel maintained contractility down to 35°C without aftercontraction or increased resting tension. Depressed Ca2+ influx evidently helps to prevent hypothermic Ca2+ overload of the cardiac cells of the hibernating mammal (Wang et al., 1997).
Early recognition of the deleterious effects of cold on the ionic integrity of cells from non-hibernating mammals came from studies on dogs (Thauer and Brendel, 1962) showing that deep hypothermia (Tb<15°C) causes massive brain swelling, with accumulation of Na+ in brain parenchyma and cellular depletion of K+. This so-called cold swelling of brain may result from a mismatch between active and passive membrane transport processes, leading to membrane depolarization and irreversible injury to cells (Hochachka, 1986; Singer and Bretschneider, 1990). If, for example, the temperature coefficient for K+ channel efflux were significantly lower than that for ATP-dependent active accumulation by the Na+/K+ pump, passive diffusion processes would dominate at low temperatures. Alternatively, cellular swelling and membrane destabilisation during deep hypothermia could be caused by differential permeabilities of the major ions themselves, with little or no direct involvement of the Na+/K+-ATPase (see below) (Plesnila et al., 2000). Ionic disruptions of the kind seen in brain can lead to dissipation of the membrane potential in other cell types of non-hibernating mammals (Wang and Wojowyk, 1988). For example, when vascular smooth muscle cells from the rat were incubated for 48h at 7°C, intracellular [K+] decreased by 138mmoll-1 and cytosolic [Na+] increased by 148mmoll-1, with half-times of 14 and 11h respectively. In contrast, smooth muscle cells from hibernating ground squirrels were able to maintain their ionic integrity when incubated under identical conditions (Kamm et al., 1979). Even so, the depth of hypothermia exposure is critical, since the vascular smooth muscle of both the rat and ground squirrel was able to maintain K+ content at 17°C (Kamm et al., 1979).
Recent studies on brain glial cells from 1- to 3-day-old rat pups show that the primary cause of cellular swelling during both mild (32°C) and moderate (27°C) hypothermia is intracellular accumulation of Na+ (Plesnila et al., 2000)(Fig.4), the effects of which can be reversed completely by incubation of hypothermic cells in a Na+-free medium (i.e. choline chloride substituted for NaCl). The Na+/K+ pump is thought to be largely inhibited by the cold either as a result of the direct thermodynamic effects of decreased ATP production (Singer and Bretschneider, 1990) or of the progressive development of a hypothermia-induced mismatch between ATP supply and ATP demand pathways (Hochachka, 1986). In glial cells from newborn rat pups, the Na+/K+ pump is thought to have no direct involvement in the cold-induced rise in intracellular [Na+]. For example, blockade of the Na+/K+-ATPase with the drug strophanthidin, which mimics its hypothermia-induced inhibition, has no effect on the membrane potential of glial cells or astrocytes at 37°C or during hypothermia (Plesnila et al., 2000). Instead, the cold-induced accumulation of Na+ is evidently caused by a differential effect of cooling on the passive movements of Na+ and K+ across the cell membrane (Fig.4). Cell volume and membrane potential are unaffected during inhibition of the Na+/K+-ATPase with ouabain since the compensatory changes in Na+ influx and K+ efflux occur in a 1:1 ratio. During strophanthidin- or hypothermia-induced inhibition of the Na+ pump, however, the differential effects on Na+ and K+ permeability favour Na+ influx (Dipolo and Latorre, 1972; Plesnila et al., 2000). Cold-induced acceleration of Na+/H+ exchange is an additional route for Na+ influx (Fig.4) since inhibition of the antiporter with amiloride effectively attenuates the hypothermia-induced swelling of glial cells (Plesnila et al., 2000). However, whereas this antiporter is normally activated by intracellular acidification, decreases in temperature are well known to raise rather than lower intracellular pH in most animal cells (Aickin and Thomas, 1977; Boutilier et al., 1987). A possible explanation of this apparent paradox comes from studies by Marjanovic et al. (Marjanovic et al., 1998) showing that the activation curve of the Na+/H+ antiporter in frog skeletal muscle is shifted to more alkaline values in the cold.
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Little is known about the prolonged effects of cold exposure on cellular ion homeostasis in cold-tolerant animals. However, recent studies on frogs hibernating for up to 4 months reveal that these animals suppress their aerobic metabolic rate to 50% of that seen in the resting air-breathing animal at 3°C (Boutilier et al., 1997; Donohoe et al., 1998). During this time, a 30% decrease in skeletal muscle Na+/K+ pump activity is accompanied by reduced Na+ influx and K+ efflux across the sarcolemma, the latter being mediated by ATP-sensitive K+ (KATP) channels (Donohoe et al., 2000). The lowered rates of passive ion flux are coincident with reduced transmembrane ion gradients for [Na+] and [K+], which may also lower the energy costs of the skeletal muscle Na+/K+-ATPase. The ability of the skeletal muscle to maintain its resting membrane potential, coincident with decreased Na+/K+ pump activity and lowered membrane permeability, provides evidence of functional channel arrest as an energy-sparing strategy during hibernation in the cold-submerged frog (Donohoe et al., 2000).
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Acknowledgments |
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