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apoptosis and signalling?

Alternatively, as suggested above, changes in cytosolic calcium might be largely irrelevant in the regulation of apoptosis. Instead alterations in free calcium concentration of other cellular compartments, such as the mitochondria could effect the calcium-dependency of some forms o f apoptosis. Thus changes in cellular calcium fluxes across a range of membranes could lead to, for example, mitochondrial calcium loading from the endoplasmic reticular (ER) stores or the extracellular compartment, perhaps via the ER. Slow transfer of calcium through the cytosol, or transfer between compartments by close membrane apposition, as recently suggested for mitichondrial and ER membranes (Simpson et al., 1997), could mean that such changes were not necessarily accompanied by raised cytosolic free calcium as measured by Fura-2. However chelation of intracellular free calcium with B APTA-AM, or inhibition of calcium flux across the plasma-membrane with blockers or EGTA, could still all affect such a change in any intracellular compartment, and thus inhibit apoptosis as shown above.

Changes in mitochondrial calcium uptake and content may be of particular relevance to apoptosis. As described above, changes in mitochondrial function, in particular the mitochondrial permeability transition and cytochrome C release are thought to be cmcial to apoptosis. Furthermore, earlier mitochondrial events have also been shown to occur during apoptosis; recent work has shown that a fall in mitochondrial membrane potential (A'F) is

such an early event in apoptosis (Petit et al., 1995; Zamzami et al., 1995; Zamzami et al., 1995). Such a drop in A Y , which results in a temporary inability to continue ATP production, if extended, may indicate impending terminal mitochondrial dysfunction and perhaps leads to the permeabihty transition, which is a point of no-retum in apoptosis; a cell with disrupted mitochondria cannot survive.

A role for mitochondrial calcium uptake in apoptosis is suggested by the abihty of ruthenium red, an inhibitor of mitochondrial Ca^^ uptake, to block TNF-induced apoptosis in L929 fibroblasts, and to inhibit the progression o f apoptosis in glucocorticoid-treated splenocytes (Hennet et al., 1993). However since it is AY that drives mitochondrial calcium uptake, such calcium uptake may in fact preceed, and perhaps precipitate, the drop in AY seen during apoptosis. Indeed the dependence of AY on mitochondrial Ca^^ flux can be demonstrated under conditions of oxidative stress, where mitochondrial Ca^^ cycling can reach critical levels, leading to a dramatic fall in AY (Richter, 1993). It is thus possible that an early calcium-dependent step in apoptosis in response to some stimuh may be the loading o f mitochondria with calcium; compounds that inhibit plasma-membrane entry of calcium into the cytosol, a likely source o f such for mitochondrial calcium loading, or chelate cytosohc calcium might inhibit this step and thus the apoptotic program as shown in section 5.11.

The identities of the channels responsible for calcium influx into the mitochondria are unknown (section 1.6 ), though a voltage-dependent calcium uniporter has been

described. It is dependent on AY, with conductance of 20 pS (Mironova et al., 1994), and is activated by cytosohc ATP and free Ca^^ (Litsky and Pfeiffer, 1997). The voltage- dependent annexin V Ca^^ channel activity, which may also have a similar conductance (Burger et al., 1994), may be enhanced by ATP (Arispe et al., 1996), and has been locahsed to mitochondria, amongst other sub-cellular compartments (Diakonova et al., 1997), is thus a good candidate for such a channel. Furthermore the observations of H . Haigler that following injection of purified annexin V, but not annexin I, into HeLa cells, the earliest sign of damage was the breakdown o f the mitochondria (personal communication), also implicates annexin V in mitochondrial function during apoptosis. While yeast, which may not encode annexins, may also have mitochondrial uniporters, they are activated, instead o f inhibited, by ruthenium red, and may thus have a different molecular identity.

Other additional possible candidates for channels involved in apoptotic mitochondrial signalling, are members of the bcl- 2 family, also involved in apoptosis,

which may have the ability to insert pH-dependently into membranes to form pores (Reed JC 97 Nature), possibly in a similar way to annexin V. Thus Bcl-2 and bcl-X^ GFP fusion proteins colocalise with mitochondria, while bax-GFP translocates to the mitochondria during staurosporine-induced apoptosis (Wolter et al., 1997), and bax promotes the mitochondrial permeability transition, while bcl-2 inhibts this event (Kluck et al., 1997;

Yang et al., 1997). Furthermore, over-expression of bcl-X^ suppresses the apoptotic change in A'F (Vander Heiden et al., 1997). The crucial role of members o f the bcl-2 family in apoptosis, and their localisation to, and effects on mitochondria during this process, show how such a protein, involved in mitochondrial apoptotic function, may affect the entire cellular apoptotic program. Such activity may thus be a ‘blue-print’ for a mitochondrial role of annexin V in the process.

That mitochondrial calcium fluxes may be important in many forms of calcium signalling, as well as during apoptosis, is also becoming clear (Babcock and Hille, 1998). The model now emerging is that mitochondria, particularly where they are in close apposition to sites of ER Ca^^ release, can act as transient calcium stores, taking up calcium early on during a response, and releasing it gradually subsequently, thus prolonging a response (Babcock et al., 1997). This raises the interesting possibility that if annexin V does mediate mitochondrial calcium entry, it may be the reduction of this buffering activity in annexin V -/- cells that leads to the changes in the H2 0 2-induced calcium réponse in these

cells.

Thus while in wild-type cells a significant proportion of the influx of extracellular calcium that accompanies the response may enter the mitochondria, to be released subsequently over a prolonged period, in annexin V -/- cells the mitochondria may fail to take up calcium entering the cell, and thus contribute nothing to the late phase of the response. This could result in the loss of the late-phcise calcium response in annexin V -/- cells as observed (figure 5.9), and could also enhance the initial peak response.

If the majority o f the calcium entering mitochondria entered across the plasma- membrane, this would also account for the dependency of the observed differences on extracellular calcium. Furthermore given that mitochondrial calcium flux may be especially active under conditions o f oxidative stress (Richter, 1993), it is possible that such fluxes would be most apparent during signalling induced by oxidative stress and not other stimuh (section 5.6). In addition under reducing intracellular conditions when cells are grown in the presence of 0.1 mM |3-Me, such mitochondrial calcium fluxes may instead be generally inhibited leading to the reduction of the late-phase H2O2 calcium response in

wild-type, and even annexin V -/- cells (section 6.4).

It is thus possible that a role for annexin V in the mitochondria, perhaps as a calcium channel, could explain both the inhibition of calcium-dependent apoptosis, and the loss of the late-phase H2 0 2-induced calcium response.