ca is an ion involved in living processes in an atypical way : if other cations participate to enzyme activity without performing essential regulatory functions due to their abundance in all cell compartments , ca   has a peculiar distribution , being present at very low levels in the cytosol of eukaryotic cells ; this enables it to act as a messenger regulating cytosolic ca - dependent enzymes and functions , when and where its local concentration raises above the steady - state level . for many decades the research on ion ( and specifically ca ) roles in cell physiopathology was hampered by intrinsic technical difficulties . a big impulse came with the development of ca - sensitive fluorescent probes     [ 1 , 2 ] , that localize in specific cell compartments ( cytosol , er , and mitochondria ) allowing separately and specifically evaluating and quantifying ca   compartmentalization ; and with the diffusion of instrumentations performing kinetic analyses , which allowed performing accurate and quantitative analysis of ca   dynamics . an efficient ca signaling implies maintenance of ca  homeostasis , which requires mechanisms keeping cytosolic ca concentration ( [ ca]c ) low and stable . these include active pumping against gradient by ca atpases , enzymes present on the cytosolic side of plasma membrane and endoplasmic reticulum ( er ) performing the high energy - expensive task of pumping ca out of the cytosol against gradient , or by ion exchangers ( e.g. , the na / ca ) . instead , signaling is exerted by discrete and highly controlled er membrane channels , originating local ca rises possessing specific signaling roles , such as those regulated by the phospholipase c - inositol-3-phosphate pathway , or by cyclic ( adp - ribose ) . these local increases activate / deactivate ca - sensitive enzymes , eliciting signal transduction chains aimed at controlling many diverse cell functions such as mitosis , or activation , or motility or apoptosis . as a feedback mechanism , the channels rapidly close due to local high [ ca ] , which is rapidly extinguished by cytosolic buffering proteins , by the ca - atpases and by mitochondria , thus being in fact ca transients . to restore er homeostasis , the partially emptied er is replenished by ca entry from the extracellular space through controlled opening of plasma membrane channels ( capacitative ca entry , see below ) , or by ca released from mitochondria present in the vicinity of inositol-3-phosphate ( ip3 ) channels     . in the category of excitable cells , mainly neurons and myocytes , plasma membrane channels open following specific hormonal or physico - chemical stimulations , even in the absence of previous er emptying , and possess signaling meaning of their own . ca signaling requires the strict cooperation among the different cellular compartments and organelles , being in fact a highly sophisticated way of communication to maintain homeostasis and functionality of the whole cell . in particular , much attention is being given to the cooperation between er and mitochondria , which interact through highly dynamic physical connections   containing abundant ca - mediating transport systems . these mediate the controlled reciprocal exchange of ca   aimed at modulating and supporting each other functions in the guise of an interorganellar symbiotic relationship     [ 1113 ] . the important implication of the low cytosolic ca concentration ( [ ca]c ) for cell homeostasis is that it must be maintained low against gradient ( extracellular space and the internal ca stores such as er , have 10,000 times as much ca ) , since excess or deregulated [ ca]c pose cells in such a dramatic asset to be in fact cell - toxic . to this purpose , a wide range of mechanisms are displayed , including high capacity binding proteins , pumps and exchangers , and mitochondria , which possess a low affinity ca uniporter that sequesters cytosolic ca   when it reaches the dangerous threshold of 500  nm , thus being a major detoxifying mechanism against ca overload . failure of the mechanisms devoted to maintain ca homeostasis produces generalized ca   alterations , in turn producing rough cell damage , without involving specific signaling meaning ; when strong , ca    alterations cause cell death by necrosis     [ 1519 ] . what becomes clear after many years of intense studies about the role of ca    in cell stress response and death , is that pathological ca   alterations resulting from failure of homeostatic controls coexist with regulated ca    signaling . the former produces rough damage that leads to cell repair , or apoptosis , or necrotic cell death , according to the intensity of the damage , whereas the latter constitutes a controlled cell response participating in survival or apoptotic pathway . ca    deregulation is a consequence of many different insults that end up altering cahomeostasis , causing and increasing damage to cells ; for this reason , it may be defined as an  intrinsic stress ,    meaning that it is autoinduced by the cells as a consequence of an extrinsic stress of a different nature . the proteins controlling ca    homeostasis are so many and so diverse that it is quite likely that any insult or physico - chemical alterations end up deregulating some of them , producing a set of reactions that may not properly be defined as signaling ( since it has no physiological purposes ) , but it is nonetheless obliged by the presence of ca - sensitive determinants . what is lacking in such instances is the coordination between the multiple pathways , which are instead casually activated , overlapping and superimposing one another , leading to cell collapse . the intrinsic ca   stress may consist in either depletion of er ca , or increase of cytosolic ( or mitochondrial , or nuclear ) ca , or both . when stress leads to ca   overload , ca - produced damage may reach levels sufficient to cause necrotic cell death     [ 1519 ] . damage and death are due to excess stimulation of ca - sensitive targets , which are numerous and concern key cellular functions : many enzymes that control supramolecular assembly , or degrading nucleic acids , lipids or proteins are ca - sensitive . among them are m - calpains , activated by high ca   levels and implicated in cell death and in many neurological disturbances ; lipoxygenases , and a set of ca - activated enzymes modifying arachidonic acid ( aa ) that are major actors of the inflammatory response , and also involved in apoptotic pathways     ; phospholipases a2 , which liberate aa from phospholipids , thus favoring , in the presence of high [ ca]mt mitochondria stress or collapse     ; a set of dnases , one of which historical interest for being the enzyme responsible for apoptotic laddering . a major form of damage is caused by the intervention of mitochondria that , taking up the excess of cytosolic ca   for scavenging purposes , may be subjected to stress and even collapse if it exceeds a physiological threshold , therefore increasing cell damage ( see below ) . another form of damage comes from energy failure , which starves ca - atpases that stop pumping ca   against gradient from cytosol to er , or to the extra - cellular environment , thus simultaneously producing cytosolic ca    overload and er ca    depletion . excitotoxicity is a major cause of neuronal cell death ; it develops as a consequence of problems occurring during neurotransmission , in instances of excess of excitatory signals , such as those from the neurotransmitter glutamate     , or of deregulated signaling ; this ends up impairing the tight control of ca   channels , leading to ca   overload   and eventually cell death and neurodegeneration . ischemic and anoxic stress produce deep changes in cell metabolism that , upon reoxygenation / reperfusion converge into a dramatic , toxic increase of cytosolic ca    [ 26 , 27 ] . such changes include plasma membrane depolarization , which favors the opening of the plasma membrane ca   channels thus promoting ca   influx ; and acidification , which causes the inversion of the na / ca   plasma membrane exchanger , which begins pumping ca   within cells . mitochondria may buffer ca   and rescue cells in instances of mild reperfusion stress ; however , they paradoxically are the major cause of cell death in strong reperfusion stress , since the huge ca   overload stimulate ca   overcharging and collapse through phenomena of ca   cycling ( see below ) . due to the increasing evidence that most ( if not all ) pathologies involve , as etiological or concurrent agents , alterations of oxidative metabolism leading to oxidative stress , much attention was paid in the   80s and 90s to the mechanisms through which oxidative stress causes ca   derangements . oxidation and redox imbalance cause er and plasma membrane ca   channels malfunctions , since their oligomeric active form is controlled by disulfide bridges ; this increases [ ca]c and depletes [ ca]er . moreover , oxidative stress impairs the buffering capacity of mitochondria , lowering the internal ca   threshold level of ptp opening     [ 9 , 33 ] , thus depriving the cells of one of the major ca   detoxifying mechanisms . all of these derangements are especially critical for neurons , where ca   is crucial to neuronal functions , implying that they possess more controlling steps that can be altered . moreover , cell death is most devastating for tissues rich in post - mitotic cells , such as cardiomyocytes or neurons , which are difficult to replace ; indeed , most neurodegenerative conditions are characterized by neuronal death caused by ca   overload . the scenario is even more dramatic considering that the organs that mostly depend on post - mitotic , ca - sensitive cells are heart and brain , whose failure causes immediate death of the organism . mitochondria are very important for intracellular ca   homeostasis and signaling , acting in fact as pivot of intracellular ca   communications . mitochondria possess low affinity ( 500  nm ) ca uniporters that allow the accumulation of large amount of ca   within the mitochondrial matrix , which constitutes a high capacity ca   reservoir , allowing buffering [ ca]c increases over 500  nm     [ 9 , 14 ] . this mitochondria ability plays an important role in cell homeostasis and cell signaling , because it help extinguishing cytosolic ca   signals . the resulting [ ca]mt increase modulates mitochondrial activity ( i.e. , increases atp production     [ 9 , 11 , 12 ] ) ; moreover , overcharged mitochondria helps refilling er after physiological ca   emptying ( e.g. , after ip3-mediated signalling     ) . in instances of mild [ ca]c increases originating from stressing events , potentially toxic ca   is sequestered within mitochondria , and then released after the stress is over : in this instance mitochondria play a prosurvival role . however , if the amount of sequestered ca   exceeds mitochondrial capacity , it leads to collapse through opening of the permeability transition pores ( ptp , formerly referred to as megachannel )     [ 18 , 19 ] . since ptp is a multi - ion channel , the consequence is that the captured ca   ions are dissipated , creating new cytosolic ca   increase , which can be in turn taken up by new intact mitochondria . this creates cycles of ca   uptake and dissipation , recruiting more mitochondria , up to a sort of mitochondrial suicide cascade . this phenomenon was named ca   cycling , and raised much interest in the 80s ; the interest then declined because it did not support a clear physiological role , being rather considered a futile cycle , because it does not help cells to survive . nowadays , a re - evaluation of this mechanism suggests that ca   cycling provides a physiological advantage : ptp opening by itself causes release of cytochrome c ( even in the absence of an upstream canonical apoptotic signaling such as bax translocation     [ 19 , 36 , 37 ] ) , which in turn may activate caspases and promote apoptosis , thus transforming a necrotic cell death into a more physio - compatible apoptosis . it seems worth mentioning here that localized phenomena of mitochondria ca cycling may have a pro - apoptotic signaling meaning since local and controlled small episodes of cytochrome c release act as initiators of the intrinsic pathway of apoptosis      ( see below ) . mitochondria can adjust their cellular localization by moving around the microtubular network ; it is tempting to hypothesize that they reach positions required to perform ca   detoxification , or to modulate specific signaling events , that is , extinguish some and exacerbating others , according to need . as an example , acute oxidative stress induces the reorganization of mitochondrial pattern from pan - cytoplasmatic , to peri - nuclear ( ghibelli , unpublished observation ) , possibly buffering excess er ca   leakage due to oxidations . this scenario suggests that local ca   increases of a stress nature , even of a small extent , may trigger an apoptotic signaling via recruitment of mitochondria . when referring to intracellular ca   depletion , the emphasis goes to emptying of er , which elicits what was recently recognized as er stress     . er stress is caused by different disturbances affecting er homeostasis , such as protein malfolding , glucose starvation , disturbance of membrane turnover / synthesis , or of protein trafficking , which all lead to er vesiculation and loss of function     . ca   plays a key role in maintaining er structure , since the flat shape of the cisternae is actively kept by bridges constituted by high capacity ca   binding proteins such as calreticulin , calsequestrin , and calnexin ; upon er ca   emptying , ca   binding is lost , the bridges weaken and er resumes the low energy spherical shape of lipids droplets in acqueous solution , thus losing function . er stress , as any other stress , can evolve into repair or apoptosis . in the first instance , the stress response implies up - regulation of stress proteins such as grp78 , a major luminal er protein      that plays a central role as er stress sensor displaying multiple functions . it coordinates the activation of other proteins implicated in er stress , such as atf6     , a transcription factor transactivating prosurvival genes whose promoters containing er stress response elements ( erses ) . grp78 also promotes removal by autophagy of the altered portions of er by controlling the correct formation of autophagosome     . multifaceted is its ability to prevent apoptosis     [ 42 , 46 ] : a fraction of grp78 is present as a transmembrane er protein , exposing a cytoplasmic domain able to directly interact and form an inhibitory complex with caspase-7 and/or caspase-12     [ 42 , 47 ] ; grp78 limits the pro - apoptotic activation of c - jun n - terminal kinase ( jnk ) , normally acting as a transducer of er stress     ; in addition , cell - free studies suggest a direct ability of grp78 to control mitochondria , by inhibiting cytochrome c release     . big emphasis was given to caspase-12 , which is activated in er membranes in instances of disruption of er ca   homeostasis or accumulation of unfolded proteins in the er lumen of mice cell models . caspase-12 initiates apoptosis either in a mitochondrial - independent fashion   or recruiting and activating mediators of the intrinsic pathway of apoptosis . however , caspase 12 is present only in rodents , and to - date no functional caspase-12 was identified in human cells ( which in fact possess only a pseudo - gene ) , nor a functionally equivalent protein . while the search for a human equivalent of caspase 12 is still active ( especially concerning a possible role for caspase-4 ) , other caspase - independent scenarios , have been explored to describe the transduction of the er stress to apoptosis via mitochondria . recently , klee et al .       provided evidence that ca    mobilization from the er is required to initiate the mitochondrial death pathway , by cooperating with the effectors of er stress surveillance machinery irea / traf2 ; according to this model , ca   promotes the jnk pro - apoptotic pathway through a complex set of steps involving the bcl-2 family . when the regulated , physiological mode of cell death , apoptosis , came into the general interest , the involvement of de - regulated ca rises as causative agent of apoptosis was sought for , in the view that apoptosis was a sort of  petit necrosis ;  in particular , a ca / mg - dependent dnase was hypothesized , considering that dna laddering was the earliest apoptotic marker of a biochemical nature to be widely accepted . the issue however never came to a definite picture , because if an increase of [ ca]c was indeed occurring in some examples of apoptosis , such as the paradigmatic model of rat thymocytes treated with corticosteroids , in many other instances it was a drop in overall ca   level the event that promotes apoptosis . the paradox of this dual , opposite role of ca   in apoptosis occupied several years of research . thapsigargin ( thg ) , a ( still ) popular inducer of apoptosis , is an irreversible poison of er ca - atpases ( serca ) , thereby inducing a transient increase in cytosolic ca   and a sustained depletion of the er ca pool     ; since the two events occur simultaneously , it is quite difficult to assess the specific contribution of one or the other to apoptosis . if the early studies were taking for granted that the apoptogenic event was the increase of [ ca]c , it was later shown that in many instances intracellular ca   buffering did not abrogate , but even increased , thg - induced apoptosis , demonstrating that also er ca   depletion was an apoptogenic event     [ 56 , 57 ] . in fact , in most instances [ ca]c rises in apoptosis are not rough event , but precise signaling steps , such as ,   for example ,   the ca - dependent proteases calpains   or calmodulin . the notion of apoptotic ca   signaling evolved , with not small effort , together with the awareness that apoptosis is not a  petit necrosis  but a regular signal transduction chain , occurring in functioning cells , ending up with coordinate cell demise instead of activation , or mitosis , or transcription .   to - date , the issue of the actual role of ca   in cell death is still debated ; the different contributions of the different cells ( e.g. , excitable versus   non - excitable ; dividing versus post - mitotic ; tumor versus   normal ) and of the different apoptogenic treatments ( stress or physiologic ; allowing or not protein neosynthesis , etc . ) produce a whole continuum of variations , and render it hard to interpret the thousands of studies on the topic . however , some generalizations can be attempted : er ca   depletion may elicit apoptosis through the er stress pathway     [ 57 , 60 ] ; rough , stress - induced ca   overloads produce necrosis     [ 17 , 19 , 61 ] ; regulated ca   increases play a role as signaling events in the intrinsic apoptotic pathway     [ 6265 ] . the poor knowledge of the role of ca   in apoptosis , which is perhaps surprising considering that ca   dynamics were among the first alterations proposed as causative of apoptosis , is also due to other , more intrinsic problems . among them the fact that ca   transients are very much localized in terms of space ( i.e. , cytosolic micro - domains ) and time ( seconds ) , and it is very easy to miss them even with sophisticated technologies . also the asynchrony of the apoptotic process hampers the analyses : even homogeneous cultured cells initiate apoptosis at different moments after stimulation , overlapping different phases and rendering inappropriate any biochemical analysis performed in bulk . these problems were overcome with technological approaches allowing analysis at the single cell level     [ 1 , 2 ] , that is , living cell imaging and flow cytometry , which are beginning to shed light on the process , helping to separate different phases and different subregions of ca   signaling     [ 1 , 2 , 64 , 65 ] . it is now well established that the intracellular apoptotic signaling evolves through at least two different pathways , triggered by ligand stimulation of death receptor ( extrinsic pathway ) , or by cell damage ( intrinsic pathway ) . the extrinsic pathway is a typical signal transduction consisting of protein - protein interaction and conformational changes from the very beginning , being induced by a molecular event such as ligand - receptor interaction and culminating with caspase activation and cell dismantling . the intrinsic pathway is instead induced not by molecular , but by physicochemical events , implying that ( a ) sensor(s ) of micro - environmental alterations or cell damage must be activated to promote the apoptotic signal ; afterwards , a molecular signal transduction chain of events similar to the extrinsic pathway is activated , also culminating with caspase activation . sensors are proteins that are modified by physico - chemical alterations such as ph , redox equilibrium , or ca   levels , thus acquiring the ability to trigger a molecular signal cascade . the most upstream molecular event of the intrinsic pathway is the translocation of bax , which moves to mitochondria and induces mitochondrial outer membrane permeabilization ( momp ) . the difficulty of finding molecular events upstream of bax activation suggested that bax itself might be a sensor of physico - chemical alterations . indeed , recent reports indicate that bax activation can occur via direct oxidation of cysteines     [ 68 , 69 ] , or via proteolytical activation by calpains     . it is emerging a pre- or early - commitment phase of the intrinsic apoptotic pathway , occurring before momp , during which potential apoptotic signals , mostly relying on ca   messages , are selected and amplified by cross - talk between er and mitochondria   ( figure 1 ) . momp is a set of different phenomena allowing release ( or leakage , see below ) of apoptogenic factors such as cytochrome c , smac / diablo , aif , through mitochondrial membrane pores . cytochrome c received most attention for its ability to nucleate the apoptosome and to initiate the caspase cascade ; its release occurs through at least two different mechanisms , the apoptosis - specific bax - based pore , and the ptp channel , both of which can be modulated by ca   in a very different way . the relation between ca   and cytochrome c release via bax consists of a feed - forward amplification loop between er and mitochondria : local high concentrations of ca   ( such as those created by the ca   efflux from ip3 channels ) favor the release of cytochrome c from mitochondria through bax pores     [ 62 , 71 ] on the one side ; on the other , cytosolic cytochrome c increases ca   levels in the vicinity of ip3 channels on er      by fixing them in the open configuration after a signaling stimulus , thus transforming a transient flux into a sustained one     . as a result of this interplay , small cytosolic cytochrome c leakage may promote secondary and massive releases ( i.e. , that required for apoptosome nucleation ) , via local ca   messages     . this provides a rationale to previous reports indicating that small amounts of cytochrome c are released from mitochondria as a very early step of apoptosis , with the goal of expanding the signal . intriguingly , the ca   sensitivity of bax mitochondrial pores seems to be limited to the intrinsic pathway : when bax is activated by the extrinsic pathway via t - bid      ( i.e. , death receptor stimulation in type 2 cell ) , the bax pores are insensitive to ca   modulation . this indicates that active bax is different according to the route of activation ( i.e. , damage versus   t - bid ) , suggesting that multiple , alternative mechanisms for bax activation may exist , possibly leading to different effects on the folding and functions of the protein     [ 68 , 70 , 73 ] . ptp opening is an automatic response to excess [ ca]mt , which causes the interaction between the inner mitochondrial membrane complex adenine nucleotide translocator ( ant ) and the outer mitochondrial membrane complex voltage - dependent anion channels ( vdac ) , leading to the formation of the membrane - spanning ptp pore . cyclophilin d ( cyp - d ) is a component of ptp resident in the mitochondrial matrix , which is activated by high [ ca]mt , favoring ptp opening by lowering the ca   threshold required for ant - vdac interaction     [ 7476 ] . in instances of mitochondrial ca overload , inhibitors of cyp - d activation , such as cyclosporins , contrast ptp opening and the eventual cell death , therefore exerting a net cell protective effect , which is often used in therapies to limit immune deficiencies or neurodegenerations     [ 76 , 77 ] . the mechanism of cytochrome c release via ptp opening , which was historically the first mechanism proposed , is still unclear from the molecular and functional point of view ( figure 1 ) . is liberated from its natural position on the outer side of the internal mitochondrial membrane to the intermembrane space ; thus , it requires pores in the outer membrane to be released , whereas pores that span the two membranes , such as ptp , would lead to the release of molecules residing in the mitochondrial matrix . to explain cytochrome c release via ptp , it may be hypothesized that ptp may cause mitochondrial membrane perturbations that allow cytochrome c ( and other factors ) to leak rather than be specifically released . in such instances , the gross alterations caused by ptp - mediated ionic redistribution will be necrogenic , even if cytosolic re - localization of cytochrome c may circumstantially activate caspases . conceivably , the extent of ptp may determine the final outcome , and apoptosis or necrosis may follow according to the strength of ptp . indeed , cyp - d inhibitors are often reported to prevent cell death by necrosis . as an alternative mechanism of cytochrome c relase via ptp , it was suggested that a vdac - only channel may form on the outer membrane , with the help , but without the physical participation , of ant , thus connecting the cytosol not with the matrix , as in the canonical ptp , but with the intermembrane space , thus allowing cytochrome c release . this model is supported by experiments performed in liver mitochondria from mice knock out for ant isoforms     , where the release of cytochrome c following an apoptogenic stimulus still occurred , but the susceptibility to ca alterations in the mitochondrial matrix was reduced . in this instance ant , a sensor of ca through its interaction with cyp - d , plays the regulatory function to transduce ca alterations to vdac , promoting its oligomerization and the formation of pores mediating release of cytochrome c. bax pores and ptp are different in molecular , mechanistic and functional term . this implies physical interaction between bax and ptp components     , such as cyp - d or ant . the two mechanisms of cytochrome c release also coparticipates in the same induction pathway in a different temporal relationship , that is , a mild stress - induced ptp opening first causes a small cytochrome c leakage , which stimulates via ca   modulation ( see above ) a second intense bax - mediated release sufficient for caspase activation . the co - operation between the two pores ( and the two pathways ) also provides a mechanistic explanation to the established but still unexplained finding that bcl-2 , though not modulating ptp directly     , all the same provides protection to cells against necrosis . the pro - apoptotic protein bax exerts its functions by inserting into membranes and forming pores . very well described is the anchoring to mitochondrial membrane , where bax forms , perhaps with adjuvant proteins , pores of a size large enough to allow passage of diffusible pro - apoptotic proteins such as cytochrome c or smac     [ 8385 ] ; the anti - apoptotic role of the cognate bcl-2 , which integrates into mitochondrial membranes also in healthy cells , is believed to be the prevention of bax pore forming , perhaps due to the extra protein domain ( bh4 ) shared by all anti - apoptotic members of the family . it is now emerging that bax and bcl-2 play a similar role also in the er membranes , where they would prevent or favor , respectively , ca leakage to the cytosol . bcl-2 is found within er membranes of healthy cells , where it prevents ca leaking from er     [ 63 , 86 ] ; as the mechanism involved , it was proposed that bcl-2 may work as a pump additional to the sercas , and/or to prevent ip3 channels opening .   in apoptosis , bax translocates not only to mitochondria , but also to er membranes     [ 63 , 88 ] , where it favors ca release   from the   er   lumen     [ 62 , 63 ] , possibly after oligomerization     ; similar evidence was shown for the cognate pro - apoptotic bak     [ 63 , 88 ] . the mechanism through which bax ( and bak ) favors er ca release is currently debated . recent cell - free studies have shown that bax forms small pores , compatible with multi - ion passage , on membranes , thus possibly directly allowing ca leakage . other studies indicate an indirect role , that is , favoring ip3 channels opening     [ 90 , 91 ] . ca release from er in turn favors the recruitment of more bax molecules from the cytosol to er membranes     , thus amplifying the ca - dependent apoptotic signal     . thus , the pro - apoptotic functions of bax and bak are not limited to mitochondria , but consist of a dual concerted role played at an earlier pre - commitment step at the level of er membranes , promoting ca movements ; and at a later step in mitochondria to promote momp and the release of the apoptotic factors . it remains to be clarified if the mechanism of bax translocation to er in pre - apoptosis occurs with the same mechanism as mitochondrial translocation , that is , if there is a role for t - bid or other bh3-only proteins of the bcl-2 family ; moreover , it is still unclear if bax domains involved in promoting er ca leakage coincide with those required for releasing the apoptotic factors from mitochondria . a role for the cysteine proteases calpains , which are activated by ca increase , was investigated since the earlier studies of apoptotic signaling , considering that ( a ) cytosolic ca overload was then considered as the main mediator of apoptosis , and ( b ) apoptosis might be conceivably executed by coordinate protein dismantling ( which was later demonstrated for caspases )     [ 58 , 93 ] . the focus was placed on the known calpain target fodrin , the protein bridging plasma membrane with the cortical actin cytoskeleton ; it was hypothesized that fodrin degradation might destabilize the cytoskeleton - membrane asset and promote plasma membrane blebbing , one of the earliest apoptotic features described ; however , such evidences have not been confirmed ; actually , calpain activation seems rather inhibiting plasma membrane blebbing ( de nicola et . many pieces of evidence show that calpains are required for apoptosis in some systems     [ 97100 ] , being dispensable ( or not involved ) in others ; when calpains are involved , they act at a very early step , upstream of caspases     [ 100 , 102 ] , thus participating to the commitment phase of signaling rather than to the execution . the notion that the form of calpain involved in apoptosis is m - calpain     , the one also involved in cell stress   and that requires high ( mm ) ca levels ( as opposed to -calpain , involved in cell signaling , and requiring lower , m doses ) was very important because it allowed linking environmental alterations to apoptosis via ca overload . perhaps the most clear - cut hint is the calpain - mediated proteolytic bax activation     [ 70 , 104 ] , one of the few mechanisms so far proposed for direct bax activation by cell damage     [ 104106 ] . two mitochondrial calpains co - operate in the release of a truncated active form of aif ( taif ) thus promoting apoptosis : a matrix m - calpain cleaves aif ; and a transmembrane -calpain cleaves vdac , promoting the formation of bax - vdac pores on the outer mitochondrial membrane and the release of taif . calpain have been proposed to proteolytically activate some caspases     [ 100 , 109112 ] ; paradoxically , caspases may also be degraded by calpains     , which in such instances would act to prevent , rather than promote , apoptosis ; the factors influencing this discrepant behavior have not been clarified . stress consists of any physico - chemical alteration of cell environment that interferes with cell functioning , potentially or actually producing damage . stress elicits active cell responses that , according to cell type , and to type and extent of damage , aim at cell survival ( cell - protective and/or cell - reparative stress response , such as the heat shock response ) or cell death ( apoptosis ) . stress responses are specific for a given type of alteration / damage : heat , oxidation , hypoxia , starvation , all trigger the synthesis or activation of molecular determinants adequate to cope with the specific type of damage ; for example , heat shock will induce synthesis of molecular chaperones to cope with exposure of hydrophobic residues of proteins ; oxidative stress will induce the synthesis / activation of anti - oxidant enzymes or molecules ; hypoxia promotes anaerobic metabolism     ; starvation promotes the disassembly of whole cell areas that are digested by autophagy , in order to recycle the building blocks for housekeeping purposes . this specificity of response limits the cross - resistance between different stress , even though a partial overlapping exist . a brief / mild insult is often sufficient to trigger protective responses but not to produce damage . this protects the cells from a second , more severe insult of the same type , thus producing transient tolerance to further stress , as occurring , for example , during thermotolerance . treatments with important clinical relevance , such as ischemia preconditioning , that is a short anoxic treatment that is protective towards a more severe ischemia , and whose mechanisms are still to be elucidated at the molecular level , seem to depend on ca signaling     . ca participates as second messenger to such defensive , reparative , or survival pathways , propagating the cell - protective signals . high [ ca]c is involved in the stimulation of the autophagic response through the activation of calcium / calmodulin - dependent kinase - b that inhibits mtor     , the main negative regulator of authophagic processes in mammals , with the goal of eliminating cellular areas that may be damaged by ca overload . in addition to these specific stress responses , cells are capable to build up survival pathways that render them less prone to apoptosis , thus promoting cell survival whatever the type of damage ; this especially occurs in cells that reside in highly stressing environments , such as inflammatory or immune cells while exerting their functions , or transformed cells undergoing tumor progression , process in which cells carrying apoptosis - resistant mutations are favored by natural selection . ca is involved in pro - survival or anti - apoptotic pathways , such as the activation of protein kinase c , whose many isoforms play pivot roles in coordinating survival cell responses . capacitative ca entry ( cce ) is ca influx from the extracellular environment through specific and tightly controlled plasma membrane channels     [ 4 , 5 ] . cce only transiently crosses the cytosol , its aims being rather the replenishment of er , after it was partially emptied by signaling events such as cyclic adp - ribose-   or ip3-mediated opening of er ca channels . cce poorly alters cytosolic homeostasis , but prevents er vesiculation due to ca emptying , thus being a net cell - protective event . recently , another mechanism of ca influx is being considered , namely the noncapacitative ca influx ( ncce )     , a non - store - operated mechanism that allows ca entry through plasma membrane channels that are different from those of cce from the molecular and regulative point of view . as depicted in figure 2 , ncce , as cce , occurs as a response to receptor stimulation implying g - protein and phospholipase c ( plc ) but , unlike cce , it does not respond to ip3-induced er emptying ; instead , it results from the processing of diacylglycerol ( the other product of inositol - bis - phosphate cleavage by plc ) , which promotes a signal transduction chain culminating with ncce assembly . interestingly , ncce occurs also in non - excitable cells . even though mechanisms and functions are still poorly characterized , it is clear that ca entry via ncce has a signaling function , possibly implying the control of survival pathways . interestingly , it requires production of no , a molecule that is involved in many survival pathways , including a strict interrelationship with protein kinase c     . agents promoting cell survival such as magnetic fields reduce stress - induced apoptosis by increasing ca influx     [ 126 , 127 ] , involving ncce rather than cce ( cerella and ghibelli , in preparation ) . the decision between cell repair or demise of damaged cells is a choice between the risk of mal - repair , leading to stabilize mutations and potentially preserve precancerous cells , versus loss of viable cells performing useful functions , which must be expensively replaced . even though the former risk is definitely worse than the latter , mechanisms aiming at avoiding unnecessary loss of precious cells have evolved . to this purpose , it is important that damaged cells do not initiate apoptosis before attempting to repair the damage : this is actively achieved by damaged cells via the set - up of reversible standby scenarios , during which apoptotic signaling is transiently kept at bay . one of such standby mechanisms implies that potentially apoptogenic stress conditions such as h2o2 treatment cause the transient inhibition of glycolysis mediated by the reversible adp - ribosylation of glyceraldehyde-3-phosphate - dehydrogenase ( gapdh )     ; this inactivates er ca - atpases , which are fed by glycolytic atp , thus decreasing er ca while increasing [ ca]c and impairing ca - mediated cell signaling . many pieces of evidence from the literature show that cells with partial ca - depleted er can not initiate apoptosis     [ 130132 ] , and indeed , during the standby period of glycolysis block , apoptosis can not initiate     . starting at around 90 minutes after recovering from h2o2 stress , glycolysis resumes     , er ca   increases , and [ ca]c is reduced ; only then h2o2-induced apoptosis begins     ; ( cerella et al . , in preparation ) . similar findings were reported also for other , oxidation - unrelated apoptogenic agents , strongly supporting the scenario according to which ( a ) ca signaling is required for stress - induced apoptosis , and ( b ) er is the initiator of the apoptotic signaling , since the standby phase seems to prevent er from amplifying apoptotic signal and mitochondrial recruitment . a model representing the relationship between apoptogenic , repair and standby signals in damaged cells is shown in figure 3 . the relationship between ca and cell death has a long and complex story . it was a reasonably simple task when the goal was describing how strongly deregulated intracellular ca may cause the passive cell death by necrosis . the scenario became very much complex when the increasing information of the mechanisms of ca - mediated cell signaling in general , and apoptotic signaling in particular , begun to merge . figure 4 depicts the different roles that ca alterations , as an intrinsic stressor , play in the survival or death of damaged cells , aiming at separating ca deregulation from pro - apoptotic ca signaling . perhaps the hottest topic to - date in this field is the role that er , and the ca messages it exchanges with mitochondria , plays in the amplification of the apoptotic signal , ending up with the promotion of momp and the trigger of the commitment phase of the intrinsic apoptotic signaling . the amplification loops created by the concerted action of er , bax , ip3 channels and cytochrome c with ca signals , spanning er and mitochondria via cytosol , as depicted in figure 1 , are beginning to define a novel precommitment phase of apoptosis . this is a very important issue because , unlike the extrinsic apoptotic pathway , which has been very well characterized at the molecular level since many years , the molecular events of the intrinsic pathway upstream of momp are poorly understood . from the functional point of view , a precommitment phase might have the role of selecting , among the pro - apoptotic signals deriving from cell damage , the ones that have to be finalized in cell death , thus avoiding unnecessary cell loss . unlike receptor - induced apoptosis , before commitment to damage - induced apoptosis cells must check the extent of the damage , and the possibility to repair it , before engaging the apoptotic signaling and commit suicide . to this purpose , different signals reporting the nature and the extent of the damage must merge into a mainstream signal that actually allows the onset of apoptosis , which in molecular terms coincides with momp . many evidences allow proposing the fascinating scenario according to which er plays as a pivot that receives the damage signals and select those that actually deserve ending up in apoptosis . the acknowledgement of a ca - dependent pre - commitment apoptotic phase would place ca - related events among the earliest of apoptosis , which would make the closing of a circle that begun almost 20 years ago , when ca as an intrinsic stressor was considered as
ca2 + is an important second messenger participating in many cellular activities ; when physicochemical insults deregulate its delicate homeostasis , it acts as an intrinsic stressor , producing / increasing cell damage . 
 damage elicits both repair and death responses ; intriguingly , in those responses ca2 + also participates as second messenger . 
 this delineates a dual role for ca2 + in cell stress , making difficult to separate the different and multiple mechanisms required for ca2 + -mediated control of cell survival and apoptosis . 
 here we attempt to disentangle the two scenarios , examining on the one side , the events implicated in deregulated ca2 + toxicity and the mechanisms through which this elicits reparative or death pathways ; on the other , reviewing the role of ca2 + as a messenger in the transduction of these same signaling events .