Caspase-2 resides in the mitochondria and mediates apoptosis directly from the mitochondrial compartment.

Caspase-2 plays an important role in apoptosis induced by several stimuli, including oxidative stress. However, the subcellular localization of caspase-2, particularly its presence in the mitochondria, is unclear. It is also not known if cytosolic caspase-2 translocates to the mitochondria to trigger the intrinsic pathway of apoptosis or if caspase-2 is constitutively present in the mitochondria that then selectively mediates this apoptotic effect. Here, we demonstrate the presence of caspase-2 in purified mitochondrial fractions from in vitro-cultured cells and in liver hepatocytes using immunoblots and confocal microscopy. We show that mitochondrial caspase-2 is functionally active by performing fluorescence resonance energy transfer analyses using a mitochondrially targeted substrate flanked by donor and acceptor fluorophores. Cell-free apoptotic assays involving recombination of nuclear, cytosolic and mitochondrial fractions from the livers of wild type and Casp2-/- mice clearly point to a direct functional role for mitochondrial caspase-2 in apoptosis. Furthermore, cytochrome c release from Casp2-/- cells is decreased as compared with controls upon treatment with agents inducing mitochondrial dysfunction. Finally, we show that Casp2-/- primary skin fibroblasts are protected from oxidants that target the mitochondrial electron transport chain. Taken together, our results demonstrate that caspase-2 exists in the mitochondria and that it is essential for mitochondrial oxidative stress-induced apoptosis.

Control of IP(3)-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin.

1. Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP(3)Rs) at low concentrations of IP(3). Ca(2+) puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP(3) into the cell. However, cells appear to have sufficient concentrations of IP(3) (0.1-3.0 microM) to induce Ca2+ release under resting conditions. 2. Here, we investigated Ca2+ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca2+ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca2+ activity. 3. In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca(2+)-binding protein (CaBP), induced Ca2+ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP(3)R channel blocker, and by mutation of the Ca(2+)-binding sites in parvalbumin. 4. Ca2+ puff activity was also evoked by injection of low concentrations of the Ca2+ chelator EGTA, but not by calbindin D(28k), another member of the EF-hand CaBP superfamily. 5. BAPTA and the Ca2+ indicator dye Oregon Green 488 BAPTA-1 evoked Ca2+ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca(2+) buffer must be mobile in order to increase Ca2+ puff activity. 6. Together, the data indicate that some IP(3)Rs spontaneously release Ca2+ under resting concentrations of IP(3). These elementary Ca2+ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca2+ puffs by coordinating the activity of elementary IP(3)R channel openings. 7. We conclude that Ca2+ release can be evoked not only by hormone-induced increases in IP(3), but also by expression of mobile cytosolic CaBPs under resting concentrations of IP(3).

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b.

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.

Measurement of intracellular calcium.

To a certain extent, all cellular, physiological, and pathological phenomena that occur in cells are accompanied by ionic changes. The development of techniques allowing the measurement of such ion activities has contributed substantially to our understanding of normal and abnormal cellular function. Digital video microscopy, confocal laser scanning microscopy, and more recently multiphoton microscopy have allowed the precise spatial analysis of intracellular ion activity at the subcellular level in addition to measurement of its concentration. It is well known that Ca2+ regulates numerous physiological cellular phenomena as a second messenger as well as triggering pathological events such as cell injury and death. A number of methods have been developed to measure intracellular Ca2+. In this review, we summarize the advantages and pitfalls of a variety of Ca2+ indicators used in both optical and nonoptical techniques employed for measuring intracellular Ca2+ concentration.

Impact of mitochondrial Ca2+ cycling on pattern formation and stability.

Energization of mitochondria significantly alters the pattern of Ca2+ wave activity mediated by activation of the inositol (1,4,5) trisphosphate (IP3) receptor (IP3R) in Xenopus oocytes. The number of pulsatile foci is reduced and spiral Ca2+ waves are no longer observed. Rather, target patterns of Ca2+ release predominate, and when fragmented, fail to form spirals. Ca2+ wave velocity, amplitude, decay time, and periodicity are also increased. We have simulated these experimental findings by supplementing an existing mathematical model with a differential equation for mitochondrial Ca2+ uptake and release. Our calculations show that mitochondrial Ca2+ efflux plays a critical role in pattern formation by prolonging the recovery time of IP3Rs from a refractory state. We also show that under conditions of high energization of mitochondria, the Ca2+ dynamics can become bistable with a second stable stationary state of high resting Ca2+ concentration.

Differential modulation of SERCA2 isoforms by calreticulin.

In Xenopus laevis oocytes, overexpression of calreticulin suppresses inositol 1,4,5-trisphosphate-induced Ca2+ oscillations in a manner consistent with inhibition of Ca2+ uptake into the endoplasmic reticulum. Here we report that the alternatively spliced isoforms of the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA)2 gene display differential Ca2+ wave properties and sensitivity to modulation by calreticulin. We demonstrate by glucosidase inhibition and site-directed mutagenesis that a putative glycosylated residue (N1036) in SERCA2b is critical in determining both the selective targeting of calreticulin to SERCA2b and isoform functional differences. Calreticulin belongs to a novel class of lectin ER chaperones that modulate immature protein folding. In addition to this role, we suggest that these chaperones dynamically modulate the conformation of mature glycoproteins, thereby affecting their function.

Ca2+ wave dispersion and spiral wave entrainment in Xenopus laevis oocytes overexpressing Ca2+ ATPases.

Complex, spatiotemporal patterns of intracellular Ca2+ release in Xenopus oocytes can be accounted for by the operation of two fundamental processes: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor (IP3R) with its inherent dependency on cytosolic Ca2+, and Ca2+ uptake via Ca2+ ATPases. Overexpression of sarco-endoplasmic reticulum Ca2+ ATPases (SERCAs) in Xenopus oocytes, increases IP3-induced Ca2+ wave frequency and amplitude [1-3]. This effect can be attributed to an increased removal of cytoplasmic Ca2+ and more efficient refilling of Ca2+ stores. By overexpressing SERCA isoforms, we report here that Ca2+ waves exhibit dispersion [4-6]. At wavelengths greater than 60 microns, wave velocity is constant. However, wave velocity and amplitude progressively decreases at smaller wavelengths. Below gamma approximately 20 microns, Ca2+ waves disperse and fail to propagate. In oocytes exhibiting both spiral and target patterns of Ca2+ release, spiral waves had higher frequencies and showed entrainment of the surrounding regions. These properties are characteristic of a classical excitable medium [4-6].

Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes.

In Xenopus oocytes, as well as other cells, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release is an excitable process that generates propagating Ca2+ waves that annihilate upon collision. The fundamental property responsible for excitability appears to be the Ca2+ dependency of the Ins(1,4,5)P3 receptor. Here we report that Ins(1,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD). Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(1,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Calreticulin inhibits repetitive intracellular Ca2+ waves.

Inositol 1,4,5-trisphosphate (IP3)-mediated calcium (Ca2+) signaling is subject to cytosolic and luminal regulatory mechanisms. In Xenopus oocytes, Ca(2+)-sensitive gating of the IP3 receptor (IP3R) produces repetitive waves of Ca2+ release. We examined the role of the luminal Ca(2+)-binding protein calreticulin (CRT) in IP3-mediated Ca2+ signaling by using Ca2+ wave activity as a sensitive Ca2+ release assay. Overexpression of CRT inhibited repetitive IP3-induced Ca2+ waves. Deletion mutagenesis demonstrated that CRT inhibition was mediated by the high affinity-low capacity Ca(2+)-binding domain, which contributes little to Ca2+ storage. This novel function of CRT in intracellular Ca2+ signaling may be regulated by Ca2+ occupancy of the high affinity binding site.

Spiral calcium waves: implications for signalling.

Spiral patterns of intracellular Ca2+ release demonstrate a direct relationship between increasing wavefront curvature and increasing propagation velocity. An equally important phenomenon is the annihilation of colliding Ca2+ waves, which reveals an underlying refractory period during which further Ca2+ release is temporarily inhibited. Treatment of intracellular Ca2+ release as an excitable medium accounts for both observations. This theoretical framework is analogous to the more familiar concept of electrical excitability in neuronal membranes. In this analogy, the inositol 1,4,5-trisphosphate receptor ion channel plays a role analogous to that of Na+ channels while Ca(2+)-induced Ca2+ release provides the mechanism for excitation. Furthermore, Ca(2+)-ATPases play a role similar to that of the K+ channels in neuronal excitation, that is, they return the system to rest. We demonstrated that overexpression of a sarco/endoplasmic reticulum Ca(2+)-ATPase increases the frequency of Ca2+ wave activity. More recent experiments reveal a strong dependence of the propagation velocity on wavelength as predicted by the dispersion relation of excitability. This important result accounts for an observed correlation between wave frequency and spatial dominance of Ca2+ foci and suggests a new mechanism for the encoding of signal information.

Increased frequency of calcium waves in Xenopus laevis oocytes that express a calcium-ATPase.

When inositol 1,4,5-triphosphate (IP3) receptors are activated, calcium is released from intracellular stores in excitatory propagating waves that annihilate each other upon collision. The annihilation phenomenon suggests the presence of an underlying refractory period that controls excitability. Enhanced calcium-adenosine triphosphatase (ATPase) activity might alter the refractory period of calcium release. Expression of messenger RNA encoding the avian calcium-ATPase (SERCA1) in Xenopus laevis oocytes increased the frequency of IP3-induced calcium waves and narrowed the width of individual calcium waves. The effect of SERCA1 expression on calcium wave frequency was dependent on the concentration of IP3 and was larger at higher (1 microM) than at lower (0.1 microM) concentrations of IP3. The results demonstrate that calcium pump activity can control IP3-mediated calcium signaling.

Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.

Following receptor activation in Xenopus oocytes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca2+ excitability. The patterns of Ca2+ release produced by GTP-gamma-S and by inositol 1,4,5-trisphosphate (IP3) are indistinguishable from receptor-induced Ca2+ patterns. The regenerative Ca2+ activity is critically dependent on the presence of IP3 and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+.

Spiral waves and intracellular calcium signalling.

Confocal imaging of intracellular Ca2+ brings a new level of resolution to the study of hormonal control of intracellular Ca2+ release. This approach has demonstrated the existence of pulsatile circular and spiral waves of Ca+ release induced by receptor activation. The data obtained by confocal imaging support a new framework for understanding intracellular Ca2+ signalling. The goal of this chapter is to review our data on the complexity of intracellular Ca2+ release in Xenopus oocytes, introduce the concept of Ca2+ excitability as a model for Ca2+ release and discuss the implications for encoding intracellular signal information.

Intracellular regulation of ion channels in cell membranes.

Cells communicate with their environment through receptor proteins on the cell membrane. Some ion channels are receptors, whereas others are linked to receptors through guanine nucleotide-binding proteins (G proteins). Ion channels control intracellular concentrations of ions such as calcium, and these concentrations control cell functions such as secretion and cell division. This review summarizes the current state of knowledge about the control of ion channels.

Elevation of [Ca2+]i and the activation of ion channels and fluxes by extracellular ATP and phospholipase C-linked agonists in rat parotid acinar cells.

Extracellular ATP initiates a variety of changes in the parotid acinar cell. The initial effect appears to be the entry of Ca2+ (and perhaps Na+), and a series of ion transport events result from the subsequent elevation of [Ca2+]i. Agonists of phospholipase C-linked receptors elevate [Ca2+]i by a different pathway, involving the generation of inositol polyphosphate compounds, but share in the subsequent initiation of the ion transport events. Although the maintenance of the physiological changes may depend on specific inositol polyphosphate intermediates, the critical initiating factor is the elevation of [Ca2+]i. Fluid secretion by the parotid gland is triggered by the action of neurotransmitters, which alter the membrane permeability of the acinar cell. The similarities between the two receptor-mediated activation pathways suggests that ATP may act as a neurotransmitter and play a role in the control of fluid secretion. Basing our analysis on the purinoceptor characteristics outlined by Gordon, we suggest that the parotid receptor belongs to the P2Z class, which is highly sensitive to ATP4-. Basing his analysis on the earlier report by Gallacher of the effects of ATP on mouse parotid cells, Gordon placed the parotid purinoceptor in a different P2 subclass (P2Y). However, our findings of an increased potency of ATP in the absence of Mg2+, as well as the potency order of different nucleotides, indicate that the P2Z class is a more appropriate category.

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.

Vasoactive intestinal peptide activates Ca2(+)-dependent K+ channels through a cAMP pathway in mouse lacrimal cells.

The action of vasoactive intestinal peptide (VIP) on Ca2(+)-dependent K+ currents, in dissociated mouse lacrimal cells, was investigated using patch clamp techniques. In whole cell recordings, VIP (10-100 pM) increased the magnitude of the Ca2(+)-dependent K+ current. In single channel recordings, VIP increased the fraction of time the large charybdotoxin-sensitive Ca2(+)-activated K+ channel spent in the open state. The activity of this channel was also increased by adding forskolin or 8-bromo cAMP to the bath. Additionally, application of either cAMP or catalytic subunit of cAMP-dependent protein kinase directly to the cytoplasmic surface of excised inside out patches reversibly lengthened the time Ca2(+)-activated K+ channels spent in the open state. These data suggest that VIP stimulates Ca2(+)-activated K+ channels by a cAMP-dependent pathway in mouse lacrimal acinar cells.

Single channel properties of newly synthesized acetylcholine receptors following denervation of mammalian skeletal muscle.

We have examined the single channel properties of newly synthesized acetylcholine (ACh) receptors in denervated adult mouse muscle. Patch-clamp recordings were made on freshly isolated fibers from flexor digitorum brevis (fdb) muscles that had been denervated in vivo for periods up to 3 wk. Muscles were treated with alpha-bungarotoxin (alpha-BTX), immediately before denervation, in order to block pre-existing receptors. Denervated fibers exhibited two types of ACh receptor channels, which differed in terms of single channel conductance (45 and 70 pS) and mean channel open time (approximately 7 and 2.5 ms, respectively). In contrast to innervated muscle, where only 3% of the total openings were contributed by the low-conductance channel type, greater than 80% of the openings in the nonsynaptic membrane of denervated muscle were of this type. Importantly, a similar increase in the proportion of low-conductance channels was observed for recordings from synaptic membrane after denervation. These data argue against the proposal that, in denervated muscle, the low-conductance channels undergo continued conversion to the high-conductance type focally at the site of former synaptic contact. Rather, our findings provide additional support for the idea that the functional properties of ACh receptors are governed uniformly by the state of innervation of the fiber and not by proximity to the site of synaptic contact.