in the past years , bcl-2 , the mammalian homologue of ced-9 , the antiapoptotic gene initially described in caenorhabditis elegans , has emerged as a major checkpoint controlling the escape of cells from their death fate ( kroemer 1997 ) . in mammals , bcl-2 is a member of a broad family of related gene products , with various degrees of sequence identity and different functions . while bcl-2 and some members of the family ( e.g. , bcl - x ) protect the cells from apoptosis , others ( e.g. , bax ) have the opposite effect . however , while there is a general consensus on the role of bcl-2 , its mechanism of action remains elusive .
on one hand , the various members of the family have been shown to dimerize and to interact with cofactors of caspases , key executors of the apoptotic machinery ( li and yuan 1999 ) ; on the other , the specific localization of the protein on the er and outer mitochondrial membrane ( lithgow et al .
1994 ) suggests that these proteins may interfere with some specific functions localized in these organelles .
in particular , bcl-2 has been shown to prevent the release of cytochrome c and aif from mitochondria ( kluck et al .
1997 ) , events that are considered essential in triggering apoptosis , whereas expression of bax has opposite effects ( jurgensmeier et al .
thus , the intriguing possibility has emerged that members of the bcl-2 family affect the apoptotic process by modulating ion fluxes in these organelles ( minn et al .
in particular , recent work in cell clones stably expressing the oncogene suggests that bcl-2 causes an overload of ca within the er ( lam et al .
. however , this effect appears in contrast with the observation that bcl-2 insertion in artificial lipid bilayers results in the formation of ionic channels of low selectivity ( minn et al .
such property should in fact cause a reduction in the er ca content ( due to an increased leak ) rather than excess accumulation . in this report , we have undertaken a detailed investigation of ca homeostasis at the subcellular level using the recombinant aequorin ( aeq ) approach developed in our laboratory ( rizzuto et al .
we conclude that bcl-2 overexpression affects ca handling by reducing the state of filling of the intracellular ca stores .
the implications of these results for the protective action of bcl-2 on apoptosis are discussed .
hela cells were grown in dme supplemented with 10% fcs in 75-cm falcon flasks . before transfection , cells were seeded onto 13-mm glass coverslips and allowed to grow to 50% confluence . at this stage ,
transfection with 4 g of dna ( 3 g bcl-2 + 1 g aeq ) was carried out as previously described ( rizzuto et al .
for fura-2 measurements , the cells were seeded onto 24-mm coverslips and transfection was carried out with 8 g plasmid dna ( 6 g bcl-2 + 2 g mtgfp ) . for cytosolic aeq ( cytaeq ) and
mitochondrial aeq ( mtaeq ) , the coverslip with the cells was incubated with 5 m coelenterazine for 12 h in dme supplemented with 1% fcs , and then transferred to the perfusion chamber . for reconstituting with high efficiency , the aeq chimeras targeted to the golgi apparatus and the er ( goaeq and eraeq , respectively )
this was obtained by incubating the cells for 1 h at 4c in krb ( krebs - ringer modified buffer : 125 mm nacl , 5 mm kcl , 1 mm na3po4 , 1 mm mgso4 , 5.5 mm glucose , and 20 mm hepes , ph 7.4 , at 37c ) supplemented with coelenterazine 5 m , the ca ionophore ionomycin , and 600 m egta . after this incubation
, the cells were extensively washed with krb supplemented with 2% bsa and 1 mm egta , and then aeq reconstitution was carried out as described .
agonists and other drugs were added to the same medium . in experiments with permeabilized cells , a buffer mimicking the cytosolic ionic composition ( intracellular buffer , ib : 140 mm kcl , 10 mm nacl , 1 mm k3po4 , 5.5 mm glucose , 2 mm mgso4 , 1 mm atp , 2 mm sodium succinate , 20 mm hepes , ph 7.05 , at 37c ) was employed .
this solution was supplemented with either 100 m egta or a fixed [ ca ] ( buffered with 2 mm egta ) .
the experiments were terminated by lysing the cells with 100 m digitonin in a hypotonic ca - rich solution ( 10 mm cacl2 in h2o ) , thus discharging the remaining aeq pool . the light signal was collected and calibrated into [ ca ] values as previously described ( brini et al .
, a 13-mm round coverslip with the transfected cells was placed in a perfused , thermostated chamber located in the close proximity of a low - noise photomultiplier , with built - in amplifier discriminator .
the output of the discriminator was captured by a thorn - emi photon counting board and stored in an ibm - compatible computer for further analyses .
the aeq luminescence data were calibrated off - line into [ ca ] values , using a computer algorithm based on the ca response curve of wild - type and mutant aeqs , as previously described ( brini et al .
1997 ) . in some experiments using eraeq , after reaching the plateau level , an artifactual slow decrease in [ ca ] was observed .
such an artifact is due to a small fraction of missorted aeq in a low [ ca ] compartment and a simple practical cure for it has been described by maechler et al .
4 , cells transfected with eraeq were used to determine the steady [ ca]er obtained by incubating the cells in low ca medium .
two protocols were adopted that gave identical results . in the first , cells incubated in normal medium were depleted according to the standard protocol , but the ca concentration during the refilling was reduced to 100 m . in the second protocol
, the cells were preincubated in 100 m cacl2 for 16 h , depleted , and finally refilled with an extracellular ca concentration of 100 m .
the coverslip with the cells was incubated with 5 m fura-2 ( added to dme + 1% fcs ) at 37c for 30 min , and after a brief washout was then transferred to the thermostated stage of a zeiss axiovert inverted microscope equipped with a sutter filterwheel and 340/380 excitation filters .
the fluorescence data were collected with a princeton instruments back - illuminated camera and expressed as emission ratios , using metafluor software ( universal imaging ) . within the microscope field ,
transfected and untransfected cells were identified before carrying out the fura-2 monitoring by revealing mitochondrial - targeted green fluorescent protein ( mtgfp ) fluorescence ( excitation 480 nm , emission 510 nm ) .
hela cells were grown in dme supplemented with 10% fcs in 75-cm falcon flasks . before transfection , cells were seeded onto 13-mm glass coverslips and allowed to grow to 50% confluence . at this stage ,
transfection with 4 g of dna ( 3 g bcl-2 + 1 g aeq ) was carried out as previously described ( rizzuto et al .
for fura-2 measurements , the cells were seeded onto 24-mm coverslips and transfection was carried out with 8 g plasmid dna ( 6 g bcl-2 + 2 g mtgfp ) .
for cytosolic aeq ( cytaeq ) and mitochondrial aeq ( mtaeq ) , the coverslip with the cells was incubated with 5 m coelenterazine for 12 h in dme supplemented with 1% fcs , and then transferred to the perfusion chamber . for reconstituting with high efficiency , the aeq chimeras targeted to the golgi apparatus and the er ( goaeq and eraeq , respectively ) , the luminal [ ca ] of these compartments first must be reduced .
this was obtained by incubating the cells for 1 h at 4c in krb ( krebs - ringer modified buffer : 125 mm nacl , 5 mm kcl , 1 mm na3po4 , 1 mm mgso4 , 5.5 mm glucose , and 20 mm hepes , ph 7.4 , at 37c ) supplemented with coelenterazine 5 m , the ca ionophore ionomycin , and 600 m egta . after this incubation
, the cells were extensively washed with krb supplemented with 2% bsa and 1 mm egta , and then aeq reconstitution was carried out as described .
agonists and other drugs were added to the same medium . in experiments with permeabilized cells , a buffer mimicking the cytosolic ionic composition ( intracellular buffer , ib : 140 mm kcl , 10 mm nacl , 1 mm k3po4 , 5.5 mm glucose , 2 mm mgso4 , 1 mm atp , 2 mm sodium succinate , 20 mm hepes , ph 7.05 , at 37c ) was employed .
this solution was supplemented with either 100 m egta or a fixed [ ca ] ( buffered with 2 mm egta ) .
the experiments were terminated by lysing the cells with 100 m digitonin in a hypotonic ca - rich solution ( 10 mm cacl2 in h2o ) , thus discharging the remaining aeq pool . the light signal was collected and calibrated into [ ca ] values as previously described ( brini et al .
, a 13-mm round coverslip with the transfected cells was placed in a perfused , thermostated chamber located in the close proximity of a low - noise photomultiplier , with built - in amplifier discriminator .
the output of the discriminator was captured by a thorn - emi photon counting board and stored in an ibm - compatible computer for further analyses .
the aeq luminescence data were calibrated off - line into [ ca ] values , using a computer algorithm based on the ca response curve of wild - type and mutant aeqs , as previously described ( brini et al .
1997 ) . in some experiments using eraeq , after reaching the plateau level , an artifactual slow decrease in [ ca ] was observed .
such an artifact is due to a small fraction of missorted aeq in a low [ ca ] compartment and a simple practical cure for it has been described by maechler et al .
4 , cells transfected with eraeq were used to determine the steady [ ca]er obtained by incubating the cells in low ca medium .
two protocols were adopted that gave identical results . in the first , cells incubated in normal medium were depleted according to the standard protocol , but the ca concentration during the refilling was reduced to 100 m . in the second protocol
, the cells were preincubated in 100 m cacl2 for 16 h , depleted , and finally refilled with an extracellular ca concentration of 100 m .
the coverslip with the cells was incubated with 5 m fura-2 ( added to dme + 1% fcs ) at 37c for 30 min , and after a brief washout was then transferred to the thermostated stage of a zeiss axiovert inverted microscope equipped with a sutter filterwheel and 340/380 excitation filters .
the fluorescence data were collected with a princeton instruments back - illuminated camera and expressed as emission ratios , using metafluor software ( universal imaging ) . within the microscope field , transfected and untransfected cells were identified before carrying out the fura-2 monitoring by revealing mitochondrial - targeted green fluorescent protein ( mtgfp ) fluorescence ( excitation 480 nm , emission 510 nm ) .
to directly investigate the role of bcl-2 in ca homeostasis , we have selectively measured the [ ca ] in different cell compartments , i.e. , the cytoplasm and the organelles acting as sources ( er and golgi apparatus ) or targets ( mitochondria ) of the ca signal .
1992 ; brini et al . 1995 ; montero et al . 1995 ; pinton et al .
the first is the high subcellular specificity of the probes , which allows to clearly identify the effects of bcl-2 expression on the signaling properties of the various compartments ; the second is that the ca probe can be cotransfected with bcl-2 .
we and others have previously shown that in transient cotransfection , the two recombinant proteins are expressed by the same subset of cells , thus avoiding the risks associated with the use of stable clones or single cells ( brini et al .
1995 ) . the effect of bcl-2 expression on the cytosolic ca signal elicited by an agonist , atp , acting on receptors coupled , through gq proteins , to the production of inositol 1,4,5 trisphosphate ( ip3 ) was first investigated . in the experiment shown in fig .
1 , hela cells , either coexpressing bcl-2 and cytaeq ( bcl-2transfected ) or expressing only cytaeq ( controls ) were challenged with atp , which acts on a gq - coupled p2y receptor . both in control and bcl-2transfected cells
, atp stimulation causes a rapid rise in cytoplasmic ca concentration ( [ ca]c ) , followed by a gradually declining sustained plateau . in bcl-2transfected cells , the [ ca ] increases evoked by stimulation with atp are significantly smaller than in controls ( peak amplitude 1.53  0.22 vs. 2.06  0.13 m , n = 10 ; fig .
1 a ) . since bcl-2 is localized in the mitochondria also , and thus could , at least in principle , enhance ca uptake in this organelle , a simple explanation for this result would be that cytosolic ca is more rapidly cleared by mitochondria , acting as an immobile buffer , placed in closed proximity to ip3-gated channels .
indeed , bcl-2 overexpression does not increase , but rather reduces , the mitochondrial [ ca ] rise induced by atp ( peak amplitude 6.56  0.72 in controls vs. 4.42  0.46 m in bcl-2expressing cells ; n = 10 ) .
interestingly , the reduction caused by bcl-2 overexpression appears larger in the mitochondria than in the cytosol , both in absolute terms and as percent ( 26 vs. 33% reduction , respectively ) . while not responsible for the alteration of the cytosolic ca signal , the effect of bcl-2 on mitochondrial ca homeostasis appears of major functional relevance , given that key events occurring in the mitochondrial matrix , such as the stimulation of atp production ( jouaville et al .
1999 ) and possibly the opening of the permeability transition pore , ptp ( bernardi 1999 ) , are regulated by [ ca]m .
a second possible explanation for the reduction in the agonist - dependent [ ca]c ( and [ ca]m ) increases was next considered , i.e. , a decrease in the amount of ca released by the agonist - sensitive ca stores . to investigate this possibility , the [ ca ] in the lumen of the er was measured in cells cotransfected with bcl-2 and an eraeq chimera ( montero et al .
in addition , since we recently have demonstrated that the golgi apparatus shares many of the ca homeostatic properties of the er ( pinton et al . 1998 ) , this latter organelle was also investigated .
2 shows the calibrated [ ca ] values in the two compartments . to obtain reliable quantitative estimates of the [ ca ] in the lumen of these two organelles , their [ ca ] needs to be decreased during both the reconstitution of aeq with coelenterazine and the subsequent initial phase of perfusion with krb / egta in the luminometer ( see materials and methods ) . under those conditions , the [ ca ] was < 10 m in both organelles .
when the [ ca ] in the perfusion medium was switched to 1 mm , the [ ca ] in the lumen of the two compartments gradually increased , reaching in control cells plateau values of 452  46 m ( n = 7 ) in the er and 262  47 m ( n = 7 ) in the golgi apparatus , respectively . in bcl-2 transfected cells , lower steady state values were attained in both compartments ( 304  38 m , n = 7 for the er , and 186  34 m , n = 7 for the golgi apparatus , respectively ) . when the cells were treated with atp , rapid decreases in the [ ca ] of the two compartments
the decrease of [ ca ] was larger and faster in controls compared with bcl-2expressing cells , reflecting the higher filling state and thus the more rapid flow through the ip3-gated channels .
the data reported above differ substantially from those reported in wehi7.2 clones , where bcl-2 overexpression results in an augmentation of the er ca content ( lam et al .
this discrepancy may be due to the use of transient vs. stable transfections , in which the results might reflect unique properties of the selected clone and/or the long - term expression of a foreign gene may result in adaptation phenomena that are only indirect consequences of the expressed protein .
this interpretation might also explain the results obtained by kuo and coworkers in clones of mcf10a cells overexpressing bcl-2 , in which an increase in serca2 ( sarcoendoplasmic reticulum ca atpases 2 ) protein leads to accelerated ca uptake and enhanced ca loading ( kuo et al .
1998 ) . on the other hand , it is possible that , although the duration of these experiments is compatible with the time course of the apoptotic process , the events observed in transient experiments could include only some of the relevant effects of bcl-2 on the mechanisms controlling programed cell death .
given that the steady - state [ ca ] in both organelles depends on the equilibrium between active accumulation and passive leak , the effect of bcl-2 could be on either process .
on one hand , bcl-2 could reduce ca uptake by the serca , either by a direct effect on the pump or by reducing the resting cytosolic [ ca ] . on the other , it could increase the passive diffusion of ca from the er . to clarify this critical issue , we investigated these possibilities independently . at first , the possibility that bcl-2 affected primarily cytosolic [ ca ] ( and hence [ ca]er only indirectly ) was verified . given that aeq is not accurate enough to reveal small differences of [ ca ] 10 m , the resting [ ca]c was measured with the fluorescent indicator fura-2 ( grynkiewicz et al .
to identify bcl-2overexpressing cells in single - cell imaging experiments , the cells were cotransfected with mitochondrially targeted gfp , mtgfp(s65 t ) ( rizzuto et al .
gfp - positive cells were distinguished from controls by the typical fluorescence emitted upon illumination with blue light .
no difference in the resting cytoplasmic [ ca ] between bcl-2transfected and control cells was observed ( data not shown ) .
then , we investigated whether bcl-2 overexpression had any direct effect on the activity of the serca pump .
for this purpose , the kinetics of ca accumulation were studied in permeabilized cells ( fig .
2 , bcl-2transfected and control cells were transferred to the luminometer chamber and perfused with a buffer ( ib , see materials and methods ) mimicking intracellular ionic conditions , supplemented with 100 m egta ( ib / egta ) .
after permeabilization with 100 m digitonin added to the same medium and wash in ib / egta , the medium was switched to ib , containing 200 nm [ ca ] ( ib / ca ) . under those conditions ,
[ ca]er gradually increased , reaching , in control cells , a plateau value of 728  60 , n = 5 .
in bcl-2transfected cells , the maximal rate of ca accumulation was the same as in control cells ( 13  2 vs. 12  3 m / s , n = 5 ) , arguing that the ca pumping activity is not modified .
however , the steady - state value eventually attained was distinctly lower ( 504  52 m , n = 5 ) .
therefore , these data exclude that the alteration of er ca homeostasis is due to a reduction in the activity of the sercas or to soluble cytosolic factors ( that are released into the medium after permeabilization with digitonin ) .
rather , by demonstrating a difference only when er refilling approaches the high [ ca]er values of intact resting cells , i.e. , in conditions in which passive leak becomes significant , they suggest that bcl-2 acts on the latter process .
we sought direct evidence for this interpretation by measuring the ca leak rate from the er of bcl-2expressing and control cells .
after the depletion protocol , the er was first refilled by exposing the cells to extracellular [ ca ] ranging from 0.23 mm , thus resulting in different levels of steady - state [ ca]er . the serca blocker , 2,5-di-(tert - butyl)-1,4-benzohydroquinone ( tbubhq ) , was then added that initiates the release of stored ca .
given that , by definition , the rates of ca uptake and leak in steady state are equal , the rate of [ ca]er decrease upon blockade of the serca must reflect the rate of ca cycling across the er membrane and , thus , of the leak rate at any given steady - state [ ca]er .
b shows the relationship between [ ca]er and leak rate , which can be fitted by a power equation
. in bcl-2overexpressing cells , the data are more scattered , but for each [ ca]er value , the rate of ca efflux is faster than in control cells .
this result is consistent with the evidence that bcl-2 can induce cation channels of low selectivity in artificial lipid bilayers ( minn et al .
previously , we have demonstrated that a reduction of 30% in steady - state [ ca]er results in a substantial activation ( > 50% ) of the so - called capacitative ca influx ( hofer et al .
the reduction in steady - state [ ca]er caused by bcl-2 overexpression is thus expected to cause an activation of this pathway .
however , indirect evidence ( namely , the observation that the resting cytosolic ca level is indistinguishable from controls ) suggests that this is not the case . to further investigate the problem , we measured the capacitative ca influx in bcl-2transfected and control hela cells expressing cytaeq ( fig .
the cells were treated with the serca blocker ( 10 m tbubhq ) while perfused with ca - free medium ( krb / egta ) .
this procedure first evoked a small transient increase in [ ca]c due to release of ca from the stores .
when the release of stored ca was complete ( 3 min ) , ca was readded to the medium .
this maneuver evoked a second , larger [ ca]c increase via the capacitative pathway , which was markedly lower in bcl-2transfected than in controls cells ( 1.19  0.11 in controls vs. 0.68  0.13 m in bcl-2expressing cells , n = 6 ) .
the question then arises as to the mechanism(s ) through which bcl-2 downregulates capacitative ca influx .
two possibilities were considered : one was that the reduction of this ca influx pathway is an adaptive mechanism to the long lasting reduction in steady - state [ ca]er ; the second was that the reduction is a direct consequence of bcl-2 overexpression .
to distinguish between these two possibilities , we verified whether a long - term reduction of [ ca]er , obtained independently of bcl-2 , could cause a comparable downregulation of the capacitative ca influx .
capacitative ca influx was thus compared in controls and in cells whose [ ca]er was reduced to values similar to those of bcl-2overexpressing cells by prolonged incubation with a low ( 100 m ) extracellular [ ca ] . in cells that had been maintained at low external [ ca ] , and thus experienced a long - term reduction in the resting [ ca]er levels , the [ ca]c peak evoked by activation of the capacitative ca influx was markedly lower than in controls ( 0.59  0.11 m , n = 5 ) . of interest , the level of steady - state [ ca]er in cells incubated in low extracellular ca was very similar ( 269  46 m ,
n = 4 ) to that measured in bcl-2overexpressing cells incubated in normal ( 1 mm ) extracellular ca concentration .
the simplest explanation of these results is that the decrease in capacitative ca influx is an adaptive consequence to the prolonged reduction in steady - state [ ca]er , though , at the moment , it can not be excluded that other factors ( e.g. , a reduction in plasma membrane potential ) contribute to this effect .
this reduction in capacitative ca influx , on one hand , appears critical to maintain a resting [ ca]c indistinguishable from that of control cells , despite a major reduction of [ ca ] in the er ; on the other hand , it could account , at least in part , for a higher resistance of cells expressing bcl-2 to apoptotic cell death , as the reduction of this ubiquitous ca influx pathway may prevent the overload of ca , considered one of the key events in apoptosis . in agreement with this view
, ma and coworkers recently demonstrated that serca overexpression , by causing er ca overload , increases spontaneous apoptosis ( ma et al .
finally , our data are not in contrast with previous results showing that the serca inhibitor , thapsigargin , activates programed cell death ( bian et al . 1997 ) . in this latter case ,
in fact , the ca depletion is acute ( 12 min ) and complete , whereas in bcl-2transfected cells , the drop in [ ca]er is modest and develops slowly .
thus , thapsigargin not only causes a massive activation of the capacitative ca influx pathway , but also such a drastic reduction in the level of [ ca]er to interfere with the basic activity of er chaperonins .
we have demonstrated that overexpression of bcl-2 causes a reduction of the steady - state [ ca ] levels , both in the er and in the golgi apparatus , the two main agonist - sensitive ca stores .
we also showed that this alteration affects the ca signal to which the effector systems are exposed , in particular those located in local environments highly dependent on the filling state of the intracellular stores ( such as the mitochondria ) .
the chronic reduction in [ ca ] of the er results in a downregulation of the capacitative ca influx .
the alterations in ca handling provoked by bcl-2 overexpression could be part of the mechanisms through which bcl-2 exerts its role in the control of cell growth .
targets of bcl-2 effects on ca homeostasis could be : the er itself , where sorting and trafficking of proteins , e.g. , enzymes or membrane receptors , are modulated by luminal [ ca ] ( booth and koch 1989 ) ; the mitochondria , where ca accumulation regulates the production of atp ( robbgaspers et al .
1998 ; the concentration of which is a key determinant of the apoptosis vs. necrosis choice , leist et al .
1997 ) , and , possibly , the release of the apoptotic cofactor cytochrome c ( krajewski et al .
1999 ) ; or the cytoplasm , where bulk or local [ ca ] increases activate enzymatic effectors , such as the ca - dependent protease , calpain ( carafoli and molinari 1998 ; squier et al .
1999 ) , or the phosphatase , calcineurin ( wang et al . 1999 ) . whether , and how , these routes interact , and how they are affected by the alteration in ca homeostasis caused by bcl-2 , awaits further investigation .
 the mechanism of action of the oncogene bcl-2 , a key regulator of the apoptotic process , is still debated . 
 we have employed organelle - targeted chimeras of the ca2 + -sensitive photoprotein , aequorin , to investigate in detail the effect of bcl-2 overexpression on intracellular ca2 + homeostasis . in the er and the golgi apparatus , bcl-2 overexpression increases the ca2 + leak ( while leaving ca2 + accumulation unaffected ) , hence reducing the steady - state [ ca2 + ] levels . as a direct consequence , 
 the [ ca2 + ] increases caused by inositol 1,4,5 trisphosphate ( ip3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria . 
 bcl-2 overexpression also reduced the rate of ca2 + influx activated by ca2 + store depletion , possibly by an adaptive downregulation of this pathway . by interfering with ca2 + -dependent events at multiple intracellular sites 
 , these effects of bcl-2 on intracellular ca2 + homeostasis may contribute to the protective role of this oncogene against programed cell death . 
Introduction
Materials and Methods
Cell Culture and Transfection
Aequorin Measurements
Fura-2 Measurements
Results and Discussion