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.

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.

The effect of inositol 1,4,5-trisphosphate and GTP on calcium release from rat liver microsomes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP mobilized 8% and 90% of the ionophore-releaseable Ca2+ pool from rat liver microsomes, respectively. In contrast to GTP, which acted after a lag-time, the Ins(1,4,5)P3-induced Ca2+ release was immediate. Poly(ethylene glycol) inhibited the effect of Ins(1,4,5)P3 and enhanced that of GTP. Ins(1,4,5)P3 accelerated and enhanced the GTP-induced Ca2+ release. Guanylyl imidodiphosphate inhibited competitively the GTP stimulated Ca2+ release, but not the GTP-dependent phosphorylation of the Mr 17,000 and 38,000 protein bands.

Coupling between cytosolic and mitochondrial calcium oscillations: role in the regulation of hepatic metabolism.

Mitochondria are strategically localized at sites of Ca2+ release, such that increases in cytosolic free Ca2+ ([Ca2+]c) from either internal Ca2+ stores or Ca2+ influx across the plasma membrane can be rapidly transported into the mitochondrial matrix. The consequent elevation in mitochondrial Ca2+ ([Ca2+]m) stimulates the Ca2+-sensitive intramitochondrial dehydrogenases, resulting in elevation of NAD(P)H. The preferential coupling between increases in [Ca2+]c and [Ca2+]m is one proposed mechanism to coordinate mitochondrial ATP production with cellular energy demand. In liver cells, hormones that act through the second messenger inositol 1,4, 5-trisphosphate (IP3) generate oscillatory [Ca2+]c signals, which result from a periodic Ca2+- and IP3-mediated activation/deactivation of intracellular Ca2+ release channels. The [Ca2+]c spiking frequency increases with agonist dose, whereas the amplitude of each [Ca2+]c spike is constant. This frequency modulation of [Ca2+]c spiking encodes the signal from the extracellular agonist, which is then decoded by the internal Ca2+-sensitive proteins such as the Ca2+-sensitive intramitochondrial dehydrogenases. Our studies have investigated the relationship between IP3-dependent [Ca2+]c signals and [Ca2+]m in primary cultured hepatocytes. In addition, the changes in cellular [Ca2+] levels have been correlated with the regulation of intramitochondrial NAD(P)H levels, pyruvate dehydrogenase activity and the magnitude of the mitochondrial proton motive force.

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.

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.

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.

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.

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.

Propagation of cytosolic calcium waves into the nuclei of hepatocytes.

The temporal and spatial organization of [Ca2+] changes within the nucleus of Fura-2 loaded hepatocytes maintained in primary culture has been investigated. Vasopressin stimulation induced oscillatory waves of cytosolic free [Ca2+] increase, which propagated freely through the nuclear region. Based on the amplitude of the Fura-2 signals from this region, the morphology of the hepatocyte nucleus and the rapid penetration of the nucleus by injected Fura-2, it can be concluded that the nuclear Ca2+ responses reflect changes occurring within the nucleoplasm. Intranuclear Ca2+ increases occurred as waves that appear to be directed by the Ca2+ waves passing through the surrounding cytoplasm. The apparent velocity of Ca2+ waves was higher in the nucleoplasm than in the cytoplasm (19.5 +/- 2.9 versus 11.0 +/- 1.1 microns/s). The nucleoplasm does not contain vesicular Ca2+ stores that might be released by Ins(1,4,5)P3. However, the nuclear envelope functions as a Ca2+ store that is sensitive to mobilization by Ins(1,4,5)P3. We conclude that the [Ca2+] in the nucleoplasm of the hepatocyte is close to equilibrium with the cytosolic [Ca2+] and that oscillatory waves of cytosolic [Ca2+] are closely paralleled by similar [Ca2+] changes in the nucleoplasm. The nuclear envelope is a component of the intracellular Ins(1,4,5)P3-sensitive Ca2+ storage pool and may serve as a reservoir for [Ca2+] elevations within the nucleus.

The role of protein kinase-C in control of aldosterone production by rat adrenal glomerulosa cells: activation of protein kinase-C by stimulation with potassium.

The role of protein kinase-C (PKC) in control of the function of rat adrenal glomerulosa cells was studied. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited the stimulation of aldosterone production induced by K+ (5.4 mM) or ACTH (5 pM) in a dose-dependent manner. Phorbol 12,13-dibutyrate, another phorbol ester that activates PKC, also exerted an inhibitory effect, while the inactive 4 alpha-phorbol 12,13-didecanoate failed to affect aldosterone production. The inhibitory effect of PMA (5 nM) was reversed by preincubation of the cells with staurosporine (ST; 50 nM), an inhibitor of PKC. These data suggest that pharmacological activation of PKC initiates an inhibitory mechanism in rat glomerulosa cells. To elucidate whether PKC is activated by physiological stimuli, the effects of ST and down-regulation of PKC by prolonged pretreatment with PMA on stimulation of aldosterone production were studied. The effects of angiotensin-II (AII) and K+, but not that of ACTH, were enhanced by ST pretreatment. This potentiation was prompt and transient in the case of AII (2.5 nM), while it developed gradually when the cells were stimulated with K+ (5.4 or 18 mM). Long term pretreatment (6 h) of glomerulosa cells with PMA also enhanced the stimulatory effect of AII (300 pM) and K+ (5.4 mM). These data together suggest that the actions of AII and K+ on aldosterone production involve a PKC-mediated inhibition. Activation of PKC by AII is probably due to formation of diacylglycerol via receptor-mediated activation of phosphoinositide-specific phospholipase-C. Stimulation with K+ caused a moderate accumulation of [3H]inositol phosphate in a concentration-dependent manner. Since this effect was abolished by nifedipine, activation of phospholipase-C may have been secondary to Ca2+ entry. The concomitant formation of diacylglycerol may contribute to activation of PKC in K+ stimulated cells. In conclusion, our data support the view that PKC participates in the physiological control of aldosterone production by rat adrenal glomerulosa cells. In addition to AII, K+ may activate PKC. Regardless of whether the enzyme is activated by phorbol esters or physiological stimuli, it exerts an inhibitory, rather than stimulatory, action on steroid production.

Thapsigargin-induced increase in cytoplasmic Ca2+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and angiotensin-II.

Thapsigargin (Tg), a microsomal Ca2+ pump inhibitor, dose-dependently increases the cytoplasmic Ca2+ concentration and aldosterone production without having any striking effect on the formation of inositol phosphates in isolated rat adrenal glomerulosa cells. The interaction of Tg with the major Ca2(+)-mediated stimuli of glomerulosa cells on aldosterone production was also examined. The effects of Tg and the Ca2(+)-mobilizing angiotensin-II (AII) were additive. The aldosterone production stimulatory effect of potassium, which induces Ca2+ influx via voltage-operated Ca2+ channels, was potentiated by Tg. The positive interaction between Tg and potassium on aldosterone production raises the possibility that stimuli generating Ca2+ signal by depleting intracellular Ca2+ stores, such as Tg or AII, enhance the response of the cell to depolarization. Such an interaction between AII and potassium may have an important role in the physiological control of aldosterone production.

Angiotensin-II inhibits Na+/K+ pump in rat adrenal glomerulosa cells: possible contribution to stimulation of aldosterone production.

The control of Na+/K+ pump activity was studied in rat adrenal glomerulosa cells. Ninety percent of K+/86Rb accumulation was blocked by ouabain, and the dose-response curve of inhibition by ouabain was monophasic (IC50, approximately 80 microM), suggesting the role of a single type of Na+/K+ pump (alpha-isoenzyme) in 86Rb accumulation by rat glomerulosa cells. The basal activity of the Na+/K+ pump was much higher in glomerulosa cells than in adrenal fasciculata cells or hepatocytes, as judged by the ouabain-sensitive uptake of 86Rb. In contrast to the two other cell types, increasing Na+ influx with the Na+ ionophore monensin failed to significantly affect ouabain-sensitive 86Rb uptake in glomerulosa cells, suggesting that in glomerulosa cells even the resting intracellular Na+ concentration is sufficient for maximal activity of the Na+/K+ pump. Angiotensin-II (AII) inhibited the ouabain-sensitive 86Rb uptake by glomerulosa cells. The effect of AII was abolished by the selective antagonist of the AT1 type of AII receptors (DuP 753), while PD 123177, an AT2 antagonist was ineffective. AT1 receptors of glomerulosa cells coupled to phospholipase-C activation and, thus, to Ca2+ signal. The inhibitory effect of AII was dependent on the extracellular Ca2+ concentration, but an elevation of cytoplasmic Ca2+ by Ca2+ ionophore ionomycin failed to mimic the effect of AII. These data suggest that Ca2+ is required for but does not mediate the inhibitory effect of AII on the Na+/K+pump. Pharmacological activation of protein kinase-C by phorbol ester did not modify 86Rb accumulation by the cells. Ouabain induced a nifedipine-sensitive elevation in the cytoplasmic Ca2+ concentration and exerted a stimulatory effect on aldosterone production, suggesting participation of the inhibition of the Na+/K+ pump in the aldosterone stimulatory action of AII.

Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells.

Initial 45Ca uptake was measured in isolated rat glomerulosa cells. A small reduction in membrane potential produced by increasing the K+ concentration from 2 to 3.6 mmol/l stimulated 45Ca uptake by about 35%, while a bigger depolarization induced by 18.5 mmol K+/l increased the uptake by about 100%. Since Ca2+ influx was already activated at a calculated membrane potential below -70 mV, and was found to be sensitive to the dihydropyridine antagonist nifedipine (1 mumol/l), but insensitive to nickel ions (100 mumol/l), it does not meet the criteria established for T- or L-type voltage-dependent Ca2+ channels. Exposure of glomerulosa cells to angiotensin II (AII) for 10 min also enhanced the rate of 45Ca influx. The effect of AII was not sensitive to 1 mumol nifedipine/l, but was strongly inhibited by 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil (CBDMB, 30 mumol/l), an inhibitor of the Na+/Ca2+ antiporter. These observations suggest that during the sustained phase of stimulation with AII, a CBDMB-sensitive mechanism, rather than dihydropyridine-sensitive calcium channels, is involved in Ca2+ uptake in rat glomerulosa cells. The bulk Ca2+ influx did not correlate with aldosterone production; however, the maintained activity of different Ca2+ entry mechanisms seems to be essential for AII-induced aldosterone production.

Angiotensin II exerts its effect on aldosterone production and potassium permeability through receptor subtype AT1 in rat adrenal glomerulosa cells.

The stimulatory effect of angiotensin II (AT) on the accumulation of inositol phosphates and on aldosterone production is abolished by the AT1 selective receptor antagonist DuP753 while PD123177, an AT2 antagonist, is ineffective. Similarly, a depolarizing effect of AT (inhibition of K+/86Rb efflux) is prevented by DuP753. While mediators derived from phospholipase C activation have a central role in the stimulation of aldosterone production by AT, the effect of AT on K+ permeability is mimicked neither by elevation of cytoplasmic [Ca2+] by ionomycin nor by kinase C activation with phorbol ester. Our results suggest that AT stimulates phospholipase C and the subsequent steroid production by glomerulosa cells through AT1 receptors. In addition some events induced by the activation of AT1 receptors may not be mediated by the activation of phospholipase C.

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.

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.

The inositol trisphosphate calcium channel is inactivated by inositol trisphosphate.

Activation of intracellular Ca2+ channels by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) represents the initial Ca2+ mobilization step in response to many extracellular signals. Here we report that Ins(1,4,5)P3-induced channel activation in permeabilized hepatocytes is followed by a time-dependent inactivation, which is a direct consequence of ligand binding. The inactivation by Ins(1,4,5)P3 parallels the quantal character of channel opening, giving rise to a unique process of incremental inactivation whereby discrete channel populations are inhibited at each Ins(1,4,5)P3 dose. Ins(1,4,5)P3 can induce inactivation in the absence of stored Ca2+, but the inactivation rate is enhanced by increases of cytosolic Ca2+. The inhibitory effect of Ins(1,4,5)P3 can be reversed by Ins(1,4,5)P3 washout, or by chelation of cytosolic Ca2+. Thus, Ins(1,4,5)P3 and Ca2+ act as coinhibitors of the Ins(1,4,5)P3-sensitive Ca2+ channel. Inactivation is an inherent consequence of Ins(1,4,5)P3-induced channel opening which can terminate increases of cytosolic Ca2+.