Invited Symposium: Cerebral Artery Pharmacology and Physiology
During the last two decades it has become widely accepted that neuronal damage in acute neurodegenerative disease is due to a perturbation of cellular calcium metabolism (for recent reviews see Choi 1995, KristiŠn and SiesjŲ, 1996; KristiŠn and SiesjŲ, 1998). Insults, like ischemia, which compromise cell bioenergetic status, lead to cell depolarisation and to a rise in intracellular calcium concentration (Ca2+i). When ischemia is terminated by reperfusion the bioenergetic potential usually recovers and the ion gradients are re-established. However, in spite of that neuronal damage is observed following hours or days of recirculation (Kirino, 1982, Pulsinelli et al., 1982).
A marked and prolonged increase in Ca2+i is harmful to cells because it leads to activation of calcium-dependent enzymes having a potentially adverse effects, such as lipases, proteases, endonucleases and phosphatases. However, cell calcium overload can also cause mitochondrial failure, if this becomes irreversible it leads to cell death. Recently, the mitochondria have became the main focus of interest in cell death pathways known as apoptosis (for reviews see Kroemer et al., 1998; Green and Reed, 1998) and mitochondria probably also play a key role in delayed postischemic cell death (Siesjo et al., 1998a; 1998b). This is because during recovery re-energized mitochondria take up most of the calcium which has entered the cell during the insult. The uptake of calcium activates mitochondrial phospholipases, and seems to trigger increased production of free radicals (Dugan et al., 1995; Reynolds and Hastings, 1995; Stout et al., 1998) as well as release of mitochondrial proteins, some of which are proapoptogenic (Ouyang et al., 1998). The proapoptotic factors seem to be released when the mitochondrial membrane permeability is increased by the opening of a large conductance channel, the mitochondrial permeability transition (MPT) pore (Zamzami et al., 1996; Scarlett and Murphy, 1997). Thus, gradually raising Ca2+i followed by mitochondrial calcium accumulation and increased free radical production finally leads to irreversible damage to mitochondria, and to the triggering of a cell death program. In this review we will focus on some aspects of ischemia-induced and calcium-dependent mitochondrial dysfunction leading to the assembling of a mitochondrial transition pore.
1. Cell calcium metabolism and ischemia
The calcium ion plays a key role as a regulator of numerous cellular functions. Therefore, cells tightly control the free intracellular calcium concentration (Ca2+i)(Carafoli, 1987; Miller, 1991; Berridge, 1997, 1998). Since the extracellular calcium concentration (Ca2+e) is several-fold higher (1mM) than the intracellular one (about 100 nM) even a small increase in the permeability of cell membranes to calcium ions lead to significant rise in Ca2+i. As fig. 1 shows calcium can enter cells via voltage- and agonistĖoperated calcium channels and can be released from intracellular stores, i.e. the endoplasmic reticulum (ER) or so called calciosomes. Calcium stores of ER origin are depleted either when inositol trisphophate (IP3) is formed by activated phospholipase C (PLC) or when a rise in Ca2+i triggers a release of calcium from IP3 non-sensitive stores via calcium induced calcium release (CICR) mechanisms. The mitochondria also represent a potential calcium source; however, during normal physiological conditions mitochondrial calcium content is low (abour 200 nM) in resting cells (Rizzuto et al. 1994; Babcock et al. 1997).
Mitochondria modulate the free cytosolic calcium concentration during and following intense activation of calcium conductances in plasma membranes (Friel and Tsien, 1994; White and Reynolds, 1996; White and Reynolds, 1997; Wang and Thayer, 1996). At steady state, there is a balance between influx and efflux of Ca2+ across the mitochondrial membrane. Mitochondria start to accumulate calcium when the cytosolic calcium concentration rises over a "set point" (about 500 nM). Ca2+ uptake by the mitochondria occurs via an uniporter (channel), and is driven by the mitochondrial membrane potential (*Ym). The rate of uptake is proportional to Ca2+i (for review see Gunter and Pfeiffer, 1990).
The efflux of Ca2+ from mitochondria can take place by to different mechanisms. After moderate calcium accumulation the Ca2+ ions are transported out from mitochondria via Ca2+/2Na+ exchange, with coupled Na+/H+ exchange to normalizing the Na+ gradient. However, when mitochondria are exposed to a sustained elevation of extramitochondrial calcium concentration a mitochondrial permeability transition (MPT) pore is apt to be opened. The MPT pore is a high conductance one since it is open to molecules with a molecular size up to 1500 Daltons. As a result the accumulated calcium is released, the mitochondria depolarize, and a large amplitude swelling of mitochondrial matrix occurs (Gunter and Pfeiffer, 1990; Zoratti and Szabů, 1995; Bernardi and Petronilli, 1996).
Fig 1 Schematic diagram illustrating calcium fluxes associated with the plasma membrane, and endoplasmic reticulum, as well as with mitochondria. Influx across the plasma membrane occurs through voltage- and agonist operated calcium channels. Agonists acting on surface receptors coupled to phospholipase C (PLC) trigger the production of inositol trisphosphate (IP3) and diacylglyceride (DG). IP3 activates receptors on ER, triggering Ca2+ release. A rise in Ca2+i can then activate a calcium-induced calcium release from calciosomes. The lower part of figure shows the calcium fluxes across mitochondrial membranes. Mitochondria take up calcium via an electrogenic uniporter, while efflux occurs by Ca2+/Na+ exchange, with H+/Na+ exchange restoring the Na+ gradient. When the mitochondrial permeability transition (MPT) pore is opened solutes of molecular weight up to 1.5 kDa can be released from mitochondrial matrix. Slightly modified after SiesjŲ et al. 1999..
As mentioned above ischemia is accompanied by bioenergetical failure causing massive cell depolarisation, with efflux of K+ and uptake of Na+, Cl-, and Ca2+ (for review see KristiŠn and SiesjŲ, 1997). Fig 2 shows that the depolarisation causing downhill ion fluxes occurs after a short delay (about 1 min) following the onset of ischemia. During this period cells are using the available energy stores for fueling the membrane pumps. At the time of ischemic depolarisation extracellular calcium concentration (Ca2+e) is reduced from a baseline level of about 1.2 mM to about 0.1 mM.
Changes in Ca2+i during ischemia mirror the alterations in Ca2+e (Silver and Erecinska, 1990; Silver and Erecinska, 1992). During the first min of ischemia there is a small rise in Ca2+i (from about 70 nM to 150 nM) which is then followed by a marked increase to about 30 mMif ischemia is followed by optimal recirculation the phosphorylation potential recovers after about two min, and ion gradients are restored. Normalisation of Ca2+e takes place in two phases. First, a partial rapid recovery of Ca2+e occurs during the cell membrane repolarisation (up to about 70% of control values). This is followed by a slow, gradual increase towards the preischemic baseline level. The later probably reflects the extrusion of calcium which is secondarily released from ER or mitochondria.
Even if cells seem to survive the ischemic insult, cell death occurs after hours or days. In this reperfusion period there are sings of a perturbed cell calcium metabolism. For example, the total cell calcium content increases (Dienel, 1984; Deshpande et al., 1987; Young et al., 1986; KristiŠn et al., 1998). Also recording of Ca2+i during the recovery period revealed a delayed secondary rise in Ca2+i (Silver and Erecinska 1992). The intracellularly accumulated calcium progressive uptake of Ca2+ by the mitochondria (Dux et al., 1987; Zaidan and Sims, 1994). A high intramitochondrial calcium increases the probability of an MPT pore opening (see above). The immunosuppressant cyclosporin A (CsA) which efficiently blocks the pore, protects against the neuronal damage which is associated with forebrain ischemic insult (Uchino et al., 1995, 1998), suggesting that CsA blocks Ca2+ mediated pore opening.
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Fig 2 Schematic illustration of changes in extra-, intracellular Ca2+ concentration. (Ca2+e, Ca2+i ), and mitochondrial Ca2+ content (Ca2+m ) during ischemia and reperfusion period..
2. Mitochondria and ischemic cell death
Under normal conditions electron transport in the mitochondrial respiratory chain creates both a H+ gradient across the inner mitochondrial membrane and an electrical potential, the inside of the mitochondria being negative. During this process reactive oxygen species (ROS) are produced (see fig.3). Up to 5 % of the oxygen reduced is converted by complex I to superoxide (oO2-)(Cadenas, 1989). It is known that the MPT pore can be induced by ROS probably due to dithiol cross-linking (Kowaltowski , 1998; Zamzami, 1998). Thus, increased production of superoxide favors the activation of a mitochondrial MPT pore. It appears that this pore can act at least in two different levels of conductance and reversibility. At low level of conductance the MPT pore opening is reversible and does not entail a large amplitude swelling of mitochondrial matrix, although it does cause a collapse of the *Ym (Ichas and Mazat, 1998). At high level of conductance, the MPT pore opening is irreversible and leads to large amplitude swelling of the mitochondrial matrix. During this event cytochrome c and the apoptosis inducing factor (AIF) are released from mitochondria (Kantrow and Piantadosi, 1997; Susin et al., 1997; Zamzami et al., 1997; Kluck et al., 1997). However, it is not clear if increased production of ROS precedes the MPT pore opening, or if the rise in ROS production is the result of the permeability change of the mitochondrial membranes.
For example, superoxide is produced by mitochondria due to a switch from the normal four-electron reduction of O2 to a one-electron reduction even when cytochrome c is released from mitochondria (Cai and Jones, 1998; see also Skulachev, 1998). As shown in fig 3 cytochrome c participates in electron transport from complex III (bc1 complex) to complex IV (cytochrome oxidase). However, it can also act as an antioxidant (see Skulachev, 1998). In this case the superoxide is converted back to oxygen and cytochrome c is reduced. The reduced from of cytochrome c is then oxidized either by complex IV or cytochrome c peroxidase localized in the intermembrane space (Gilmour et al., 1994). Following the release of cytochrome c the electron transport by respiratory complexes localized in the inner membrane is inhibited however, the respiration and electron transport can continue via respiratory enzymes located at external membrane sites(Skulachev, 1998; La Piana et al., 1998; see fig 3).
Thus, cytochrome c located on the external side of the outer membrane can activate an electron transport chain which promotes the transfer of electrons from substrate present in the cytosol to cytochrome oxidase inside the mitochondria. Therefore the mitochondrial membrane potential can be upheld and ATP can still be formed even though part of the pool of cytochrome c has been released. However, if the MPT pore is ireversibly opened the respiration is inhibited due to loss of pyridine nucleotides and substrates from mitochondria, blocking in turn also the citric acid cycle metabolism.
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Fig 3 Schematic illustration of the respiratory chain complexes under normal physiological conditions (upper panel) and after cytochrome c release into the cytosol (lower panel). A H+ gradient generated by complex I (NADH dehydrogenase), III (ubiquinol-cytochrome c reductase), and IV (cytochrome c oxidase) is used by complex V (ATP synthase) to generate ATP from ADP. During this process a superoxide (oO2-) is produced by complex I (Q = ubiquinone, C = cytochrome c). The lower panel illustrates possible mechanisms of continued respiration after cytochrome c is released from the intermembrane space to the cytosol. Tentatively, cytochrome c localized on the outer membrane can receive electrons from an external respiratory chain (Fp5 NADH cytochrome b5 reductase), bypassing complex I and III. The H+ gradient would then be generated by complex IV.
Released cytochrome c and AIF activate the proapoptotic proteases-caspases whose activation leads to apoptotic cell death. However, activated caspases can also cause damage to mitochondrial membranes accompanied by irreversible swelling of mitochondria (Marzo et al., 1998). Furthermore, changes in energy metabolism can shift the balance between apoptosis and necrosis (Richter et al., 1996; Hirsch et al., 1997).
Fig 4 attempts to summary events which are triggered by mitochondrial dysfunction, involving a decrease in *Ym, probably also an enhanced production of ROS. The decrease in *Ym can trigger a transient MPT of low coductance which causes release of cytochrome c, activation of caspase 3, and apoptotic cell death. Caspase-3 activation may feed back to the mitochondria and cause further depolarisation and dysfunction, If the bioenergetic state is compromised, or massive amount of cytochrome c is released, the electrochemical potential for H+ is collapsed and ATP production ceases. The result is necrotic cell death. However, as pointed out by MacMannus and Linnik (1997) ischemic cell death may be an entity per se since it does not exclusively conform to either necrosis or apoptosis.
...as can be seen in Figure 4.
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Fig 4 Schematic diagram illustrating the cascade of events triggered by mitochondria which can lead to apoptotic/necrotic cell death following ischemic insult. A decrease of mitochondrial membrane potential due to calcium accumulation rises the production of ROS and triggers activation of an MPT and the release of cytochrome c. Activated caspases can act as positive feedback leading to further damage to mitochondrial membranes and to an irreversible MPT pore opening accompanied by mitochondrial matrix swelling. Extensive bionergetical failure due to dysfunction of a large part of the mitochondrial population within the cell can activate necrotic processes which do not require ATP. Modified after SiesjŲ et al. (1999).
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|Kristian, T; Siesjo, BK; (1998). Calcium and Mitochondria in Ischemic Cell Death. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/laher/kristian0829/index.html|
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