Making the connection: How membrane contact sites have changed our view of organelle biology.

The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles in intracellular signaling, lipid metabolism, motor-protein-mediated membrane dynamics, organelle division, and organelle biogenesis. Here, we summarize the major conceptual changes that have occurred in cell biology as we have come to appreciate how contact sites integrate the activities of organelles.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Altered fusion dynamics underlie unique morphological changes in mitochondria during hypoxia-reoxygenation stress.

Functional states of mitochondria are often reflected in characteristic mitochondrial morphology. One of the most fundamental stress conditions, hypoxia-reoxygenation has been known to cause impaired mitochondrial function accompanied by structural abnormalities, but the underlying mechanisms need further investigation. Here, we monitored bioenergetics and mitochondrial fusion-fission in real time to determine how changes in mitochondrial dynamics contribute to structural abnormalities during hypoxia-reoxygenation. Hypoxia-reoxygenation resulted in the appearance of shorter mitochondria and a decrease in fusion activity. This fusion inhibition was a result of impaired ATP synthesis rather than Opa1 cleavage. A striking feature that appeared during hypoxia in glucose-free and during reoxygenation in glucose-containing medium was the formation of donut-shaped (toroidal) mitochondria. Donut formation was triggered by opening of the permeability transition pore or K(+) channels, which in turn caused mitochondrial swelling and partial detachment from the cytoskeleton. This then favored anomalous fusion events (autofusion and fusion at several sites among 2-3 mitochondria) to produce the characteristic donuts. Donuts effectively tolerate matrix volume increases and give rise to offspring that can regain ΔΨ(m). Thus, the metabolic stress during hypoxia-reoxygenation alters mitochondrial morphology by inducing distinct patterns of mitochondrial dynamics, which includes processes that could aid mitochondrial adaptation and functional recovery.

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Inhibition of collagen gene expression in systemic sclerosis dermal fibroblasts by mithramycin.

BACKGROUND: The anti-tumour antibiotic mithramycin is also a potent inhibitor of fibrosis after glaucoma surgery. This drug displays high affinity binding to GC-rich sequences in DNA, including those present in the promoter of the gene encoding the alpha1 chain of type I collagen (COL1A1). OBJECTIVE: To evaluate the effects of mithramycin on COL1A1 expression in systemic sclerosis fibroblasts. METHODS: Confluent cultures of dermal fibroblasts from patients with recent onset diffuse systemic sclerosis were treated with mithramycin in vitro. Cell viability and protein expression were examined by fluorescence and confocal imaging. Type I collagen production was analysed by confocal imaging and metabolic labelling. COL1A1 messenger RNA levels and stability were assessed by northern hybridisation, and COL1A1 transcription was examined by transient transfections. RESULTS: Treatment of systemic sclerosis fibroblasts with mithramycin (10-100 nmol/l) did not cause significant cytotoxicity. Type I collagen biosynthesis decreased by 33-40% and 50-70% in cells cultured with mithramycin at 10 nmol/l and 100 nmol/l, respectively. Mithramycin at 50 nmol/l decreased COL1A1 mRNA levels by 40-60%. The effects of mithramycin on collagen gene expression were mediated by transcriptional and post-transcriptional mechanisms as shown by the reduction of COL1A1 promoter activity and by a decrease in the stability of these transcripts, respectively. CONCLUSIONS: Mithramycin causes potent inhibition of collagen production and gene expression in systemic sclerosis dermal fibroblasts in vitro in the absence of cytotoxic effects. These results suggest that this drug may be an effective treatment for the fibrotic process which is the hallmark of systemic sclerosis.

Bacterial enterotoxins are associated with resistance to colon cancer.

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in under-developed countries. This geographic imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca(2+), or chelation of intracellular Ca(2+). In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca(2+) influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.

Old players in a new role: mitochondria-associated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria.

In many cell types, IP(3) and ryanodine receptor (IP(3)R/RyR)-mediated Ca(2+) mobilization from the sarcoendoplasmic reticulum (ER/SR) results in an elevation of mitochondrial matrix [Ca(2+)]. Although delivery of the released Ca(2+) to the mitochondria has been established as a fundamental signaling process, the molecular mechanism underlying mitochondrial Ca(2+) uptake remains a challenge for future studies. The Ca(2+) uptake can be divided into the following three steps: (1) Ca(2+) movement from the IP(3)R/RyR to the outer mitochondrial membrane (OMM); (2) Ca(2+) transport through the OMM; and (3) Ca(2+) transport through the inner mitochondrial membrane (IMM). Evidence has been presented that Ca(2+) delivery to the OMM is facilitated by a local coupling between closely apposed regions of the ER/SR and mitochondria. Recent studies of the dynamic changes in mitochondrial morphology and visualization of the subcellular pattern of the calcium signal provide important clues to the organization of the ER/SR-mitochondrial interface. Interestingly, key steps of phospholipid synthesis and transfer to the mitochondria have also been confined to subdomains of the ER tightly associated with the mitochondria, referred as mitochondria-associated membranes (MAMs). Through the OMM, the voltage-dependent anion channels (VDAC, porin) have been thought to permit free passage of ions and other small molecules. However, recent studies suggest that the VDAC may represent a regulated step in Ca(2+) transport from IP(3)R/RyR to the IMM. A novel proposal regarding the IMM Ca(2+) uptake site is a mitochondrial RyR that would mediate rapid Ca(2+) uptake by mitochondria in excitable cells. An overview of the progress in these directions is described in the present paper.

VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release.

Enhanced formation of reactive oxygen species (ROS), superoxide (O2*-), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2*- elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2*- and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2*--induced CCR. Furthermore, O2*- alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2*--induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2*- exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2*--induced CCR did not depend on Bax translocation to mitochondria. O2*--induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2*- triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.

Calcium signal transmission between ryanodine receptors and mitochondria in cardiac muscle.

Ryanodine receptor (RyR) mediated Ca(2)+ signals play a central role in excitation-contraction coupling in cardiac muscle. To support the rhythmic contractile activity there is a need for continuous tuning of cellular oxidative energy generation in the mitochondria to the actual work-load. Evidence has emerged that RyR-mediated cytosolic Ca(2)+ signals are efficiently transmitted to the mitochondria, providing a means for coupling cardiac muscle excitation to oxidative energy production, through activation of Ca(2)+ sensitive mitochondrial dehydrogenases. Recent data suggest that the Ca(2)+ signal transmission between RyR and mitochondria is dependent on local Ca(2)+ interactions between subdomains of sarcoplasmic reticulum (SR) and mitochondria. Here we give a short overview of the determinants and spatio-temporal organization of Ca(2)+ signal transmission between SR and mitochondria.

Propagation of the apoptotic signal by mitochondrial waves.

Generation of mitochondrial signals is believed to be important in the commitment to apoptosis, but the mechanisms coordinating the output of individual mitochondria remain elusive. We show that in cardiac myotubes exposed to apoptotic agents, Ca2+ spikes initiate depolarization of mitochondria in discrete subcellular regions, and these mitochondria initiate slow waves of depolarization and Ca2+ release propagating through the cell. Traveling mitochondrial waves are prevented by Bcl-x(L), involve permeability transition pore (PTP) opening, and yield cytochrome c release, caspase activation and nuclear apoptosis. Mitochondrial Ca2+ uptake is critical for wave propagation, and mitochondria at the origin of waves take up Ca2+ particularly effectively, providing a mechanism that may underlie selection of the initiation sites. Thus, apoptotic agents transform the mitochondria into an excitable state by sensitizing PTP to Ca2+. Expansion of the local excitation by mitochondrial waves propagating through the whole cell can be especially important in activation of the apoptotic machinery in large cells.

Mitochondrial ca(2+) signaling and cardiac apoptosis.

The broad significance of apoptosis in the cardiovascular system only began to be recognized more widely recently. Apoptotic cell death is a normal component of postnatal morphogenesis of the human cardiac conduction system and may also be involved in the pathogenesis of a variety of cardiovascular diseases, including heart failure, myocardial infarction and atherosclerosis. Recently, it has become evident that mitochondria play important role in the signaling machinery of apoptotic cell death by releasing several apoptotic factors such as cytochrome c, apoptosis-inducing factor and procaspases. Furthermore, calcium signals have been identified as one of the major signals that converge on mitochondria to trigger the mitochondrion-dependent pathway of the apoptotic cell death. Calcium signals are also important in the physiological control of mitochondrial energy metabolism and it has not yet been explored how Ca(2+) turns from a signal for life to a signal for death. Since large elevations of cytosolic [Ca(2+)] ([Ca(2+)](c)) occur during each heartbeat in cardiac myocytes and these [Ca(2+)](c) signals may efficiently propagate to the mitochondria, the Ca(2+)-dependent mitochondrial pathways of apoptosis can be particularly important in the heart. This review is concerned with the role of mitochondrial Ca(2+) signaling in the control of cardiac apoptosis.

Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria.

Calcium signal transmission between endoplasmic reticulum (ER) and mitochondria is supported by a local [Ca(2+)] control that operates between IP(3)receptor Ca(2+)release channels (IP(3)R) and mitochondrial Ca(2+)uptake sites, and displays functional similarities to synaptic transmission. Activation of IP(3)R by IP(3)is known to evoke quantal Ca(2+)mobilization that is associated with incremental elevations of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)). Here we report that activation of IP(3)R by adenophostin-A (AP) yields non-quantal Ca(2+)mobilization in mast cells. We also show that the AP-induced continuous Ca(2+)release causes relatively small [Ca(2+)](m)responses, in particular, the sustained phase of Ca(2+)release is not sensed by the mitochondria. Inhibition of ER Ca(2+)pumps by thapsigargin slightly increases IP(3)-induced [Ca(2+)](m)responses, but augments AP-induced [Ca(2+)](m)responses in a large extent. In adherent permeabilized cells exposed to elevated [Ca(2+)], ER Ca(2+)uptake fails to affect global cytosolic [Ca(2+)], but attenuates [Ca(2+)](m)responses. Moreover, almost every mitochondrion exhibits a region very close to ER Ca(2+)pumps visualized by BODIPY-FL-thapsigargin or SERCA antibody. Thus, at the ER-mitochondrial junctions, localized ER Ca(2+)uptake provides a mechanism to attenuate the mitochondrial response during continuous Ca(2+)release through the IP(3)R or during gradual Ca(2+)influx to the junction between ER and mitochondria.

Spatio-temporal organization of the mitochondrial phase of apoptosis.

Signaling cascades often utilize highly ordered interactions between intracellular organelles to propagate a particular signal throughout the cell. In many apoptotic paradigms, the stress signal is delivered to the mitochondria, and mitochondria release to the cytosol several apoptotic factors, which in turn, trigger execution of the program of cell suicide. Although intermitochondrial communication has been shown to give rise to regenerative phenomena such as Ca2+ release waves and depolarization waves, it has not been elucidated whether mitochondria interact with each other during apoptosis and whether the postmitochondrial phase of apoptosis displays a coordinated response throughout the cell. The objective of this review is to draw attention to recent observations that shed some light onto the subcellular spatio-temporal organization of the apoptotic machinery.

Quantification of calcium signal transmission from sarco-endoplasmic reticulum to the mitochondria.

Recent studies have shown that ryanodine and IP3 receptor (RyR/IP3R)-mediated cytosolic Ca2+ signals propagate to the mitochondria, initiating chains of events vital in the regulation of different cellular functions. However, the fraction of released Ca2+ utilized by the mitochondria during these processes has not been quantified. To measure the amount of Ca2+ taken up by the mitochondria, we used a novel approach that involves simultaneous fluorescence imaging of mitochondrial and cytosolic [Ca2+] in permeabilized H9c2 myotubes and RBL-2H3 mast cells. Communication between sarco-endoplasmic reticulum (SR/ER) and mitochondria is maintained in these permeabilized cells, as evidenced by the large RyR/IP3R-driven mitochondrial matrix [Ca2+] and NAD(P)H signals and also by preservation of the morphology of the SR/ER-mitochondrial junctions. Ca2+ was released from the SR/ER by addition of saturating caffeine or IP3 and subsequently thapsigargin (Tg), an inhibitor of SR/ER Ca2+ pumps. The amount of Ca2+ transmitted to the mitochondria was determined by measuring increases of global [Ca2+] in the incubation medium (cytosolic [Ca2+] ([Ca2+]c)). Mitochondrial Ca2+ uptake was calculated from the difference between [Ca2+]c responses recorded in the absence and presence of uncoupler or from [Ca2+]c elevations evoked by uncoupler or ionophore applied after complete Ca2+ mobilization from the SR/ER. [Ca2+]c increases were calibrated by adding Ca2+ pulses to the permeabilized cells. In H9c2 cells, caffeine induced partial mobilization of SR Ca2+ and mitochondria accumulated 26% of the released Ca2+. Sequential application of caffeine and Tg elicited complete discharge of SR Ca2+ without further increase in mitochondrial Ca2+ uptake. In RBL-2H3 mast cells, IP3 by itself elicited complete discharge of the ER Ca2+ store and the increase of the ionophore-releasable mitochondrial Ca2+ content reached 50% of the Ca2+ amount mobilized by IP3 + Tg. Thus, RyR/IP3R direct a substantial fraction of released Ca2+ to the mitochondria.

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals.

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.

The machinery of local Ca2+ signalling between sarco-endoplasmic reticulum and mitochondria.

Growing evidence suggests that propagation of cytosolic [Ca2+] ([Ca2+]c) spikes and oscillations to the mitochondria is important for the control of fundamental cellular functions. Delivery of [Ca2+]c spikes to the mitochondria may utilize activation of the mitochondrial Ca2+ uptake sites by the large local [Ca2+]c rise occurring in the vicinity of activated sarco-endoplasmic reticulum (SR/ER) Ca2+ release channels. Although direct measurement of the local [Ca2+]c sensed by the mitochondria has been difficult, recent studies shed some light onto the molecular mechanism of local Ca2+ communication between SR/ER and mitochondria. Subdomains of the SR/ER are in close contact with mitochondria and display a concentration of Ca2+ release sites, providing the conditions for an effective delivery of released Ca2+ to the mitochondrial targets. Furthermore, many functional properties of the signalling between SR/ER Ca2+ release sites and mitochondrial Ca2+ uptake sites, including transient microdomains of high [Ca2+], saturation of mitochondrial Ca2+ uptake sites by released Ca2+, connection of multiple release sites to each uptake site and quantal transmission, are analogous to the features of the coupling between neurotransmitter release sites and postsynaptic receptors in synaptic transmission. As such, Ca2+ signal transmission between SR/ER and mitochondria may utilize discrete communication sites and a closely related functional architecture to that used for synaptic signal propagation between cells.

Sustained down-regulation of the epidermal growth factor receptor by decorin. A mechanism for controlling tumor growth in vivo.

The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by approximately 40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent.

Inhibition of type I and III IP(3)Rs by TGF-beta is associated with impaired calcium release in mesangial cells.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) mediate cytosolic free calcium concentration ([Ca(2+)](c)) signals in response to a variety of agonists that stimulate mesangial cell contraction and proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP(3)Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-beta1 (TGF-beta1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP(3)Rs as measured by immunoblot and confocal analysis. TGF-beta1 treatment does not affect IP(3) levels, and downregulation of type I IP(3)R is not due to enhanced degradation of the protein, as the half-life of type I IP(3)R is unchanged in the presence or absence of TGF-beta1. Functional effects of TGF-beta1-induced downregulation of the IP(3)Rs were evaluated by measuring [Ca(2+)](c) changes in response to epidermal growth factor (EGF) in intact cells and sensitivity of [Ca(2+)](c) release to IP(3) in permeabilized cells. TGF-beta1 pretreatment led to a significant decrease of [Ca(2+)](c) release induced by EGF in intact cells and by submaximal IP(3) (400 nm) in permeabilized cells. Total IP(3)-sensitive [Ca(2+)](c) stores were not changed, as assessed by stimulation with maximal doses of IP(3) (10.5 microm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-beta1 leads to downregulation of both type I and III IP(3)Rs in mesangial cells and this is associated with impaired sensitivity to IP(3).

Calcium signal transmission between ryanodine receptors and mitochondria.

Control of energy metabolism by increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) may represent a fundamental mechanism to meet the ATP demand imposed by heart contractions, but the machinery underlying propagation of [Ca(2+)] signals from ryanodine receptor Ca(2+) release channels (RyR) to the mitochondria remains elusive. Using permeabilized cardiac (H9c2) cells we investigated the cytosolic [Ca(2+)] ([Ca(2+)](c)) and [Ca(2+)](m) signals elicited by activation of RyR. Caffeine, Ca(2+), and ryanodine evoked [Ca(2+)](c) spikes that often appeared as frequency-modulated [Ca(2+)](c) oscillations in these permeabilized cells. Rapid increases in [Ca(2+)](m) and activation of the Ca(2+)-sensitive mitochondrial dehydrogenases were synchronized to the rising phase of the [Ca(2+)](c) spikes. The RyR-mediated elevations of global [Ca(2+)](c) were in the submicromolar range, but the rate of [Ca(2+)](m) increases was as large as it was in the presence of 30 microm global [Ca(2+)](c). Furthermore, RyR-dependent increases of [Ca(2+)](m) were relatively insensitive to buffering of [Ca(2+)](c) by EGTA. Therefore, RyR-driven rises of [Ca(2+)](m) appear to result from large and rapid increases of perimitochondrial [Ca(2+)]. The falling phase of [Ca(2+)](c) spikes was followed by a rapid decay of [Ca(2+)](m). CGP37157 slowed down relaxation of [Ca(2+)](m) spikes, whereas cyclosporin A had no effect, suggesting that activation of the mitochondrial Ca(2+) exchangers accounts for rapid reversal of the [Ca(2+)](m) response with little contribution from the permeability transition pore. Thus, rapid activation of Ca(2+) uptake sites and Ca(2+) exchangers evoked by RyR-mediated local [Ca(2+)](c) signals allow mitochondria to respond rapidly to single [Ca(2+)](c) spikes in cardiac cells.

Mitochondrial calcium signaling driven by the IP3 receptor.

Many agonists bring about their effects on cellular functions through a rise in cytosolic [Ca2+] ([Ca2+]c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP3). Imaging studies of single cells have demonstrated that [Ca2+]c signals display cell specific spatiotemporal organization that is established by coordinated activation of IP3 receptor Ca2+ channels. Evidence emerges that cytosolic calcium signals elicited by activation of the IP3 receptors are efficiently transmitted to the mitochondria. An important function of mitochondrial calcium signals is to activate the Ca2+-sensitive mitochondrial dehydrogenases, and thereby to meet demands for increased energy in stimulated cells. Activation of the permeability transition pore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death. Furthermore, mitochondrial Ca2+ transport appears to modulate the spatiotemporal organization of [Ca2+]c responses evoked by IP3 and so mitochondria may be important in cytosolic calcium signaling as well. This paper summarizes recent research to elucidate the mechanisms and significance of IP3-dependent mitochondrial calcium signaling.