Mitochondrial Ca(2+) signals in autophagy

Macroautophagy (autophagy) is a lysosomal degradation pathway that is conserved from yeast to humans that plays an important role in recycling cellular constituents in all cells. A number of protein complexes and signaling pathways impinge on the regulation of autophagy, with the mammalian target of rapamycin (mTOR) as the central player in the canonical pathway. Cytoplasmic Ca(2+) signaling also regulates autophagy, with both activating and inhibitory effects, mediated by the canonical as well as non-canonical pathways. Here we review this regulation, with a focus on the role of an mTOR-independent pathway that involves the inositol trisphosphate receptor (InsP(3)R) Ca(2+) release channel and Ca(2+) signaling to mitochondria. Constitutive InsP(3)R Ca(2+) transfer to mitochondria is required for autophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to insufficient production of reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer to mitochondria results in activation of AMPK, which activates mTOR-independent pro-survival autophagy. Constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration, maintenance of normal cell bioenergetics and suppression of autophagy.     

Uncoupling protein 3 adjusts mitochondrial Ca(2+) uptake to high and low Ca(2+) signals

Uncoupling proteins 2 and 3 (UCP2/3) are essential for mitochondrial Ca(2+) uptake but both proteins exhibit distinct activities in regard to the source and mode of Ca(2+) mobilization. In the present work, structural determinants of their contribution to mitochondrial Ca(2+) uptake were explored. Previous findings indicate the importance of the intermembrane loop 2 (IML2) for the contribution of UCP2/3. Thus, the IML2 of UCP2/3 was substituted by that of UCP1. These chimeras had no activity in mitochondrial uptake of intracellularly released Ca(2+), while they mimicked the wild-type proteins by potentiating mitochondrial sequestration of entering Ca(2+). Alignment of the IML2 sequences revealed that UCP1, UCP2 and UCP3 share a basic amino acid in positions 163, 164 and 167, while only UCP2 and UCP3 contain a second basic residue in positions 168 and 171, respectively. Accordingly, mutants of UCP3 in positions 167 and 171/172 were made. In permeabilized cells, these mutants exhibited distinct Ca(2+) sensitivities in regard to mitochondrial Ca(2+) sequestration. In intact cells, these mutants established different activities in mitochondrial uptake of either intracellularly released (UCP3(R171,E172)) or entering (UCP3(R167)) Ca(2+). Our data demonstrate that distinct sites in the IML2 of UCP3 effect mitochondrial uptake of high and low Ca(2+) signals.     

Mitochondrial Ca(2+) uptake depends on the spatial and temporal profile of cytosolic Ca(2+) signals

Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.     

Contribution of endoplasmic reticulum to Ca(2+) signals in Dictyostelium depends on extracellular Ca(2+)

We recently reported the first molecular genetic evidence that Dictyostelium Ca²⁺ responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) – responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca²⁺-binding proteins in the ER, even though the influx of Ca²⁺ into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30–70 nM additional Ca²⁺ to the responses. Here we report that this additional ER contribution to the cytosolic Ca²⁺ signal depends upon extracellular Ca²⁺– it does not occur in the absence of extracellular Ca²⁺, increases to a maximum as the extracellular Ca²⁺ levels rise to 10 μM and then remains constant at extracellular Ca²⁺ concentrations up to at least 250 μM. These results suggest that Ca²⁺ influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca²⁺-induced Ca²⁺ release from the ER. By way of contrast, we show that Ca²⁺ responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca²⁺. Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca²⁺.     

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.     

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.     

Attraction or repulsion by local Ca(2+) signals

Recent studies have shown that cytosolic Ca(2+) signals, generated on one side of a nerve growth cone, can induce turning either towards or away from the side of the Ca(2+) signal, depending on the global Ca(2+) level. The results indicate that local Ca(2+) signals may provide important directional cues for axon guidance.     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.     

Role of phospholamban in the modulation of arterial Ca(2+) sparks and Ca(2+)-activated K(+) channels by cAMP

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca²⁺-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca²⁺ release throughryanodine-sensitive Ca²⁺ release channels, measured asCa²⁺ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca²⁺ sparks were monitored opticallyusing the fluorescent Ca²⁺ indicator fluo 3 or electricallyby measuring transient large-conductance Ca²⁺-activatedK⁺ (BK) channel currents activated by Ca²⁺sparks. Basal Ca²⁺ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa²⁺ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca²⁺ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa²⁺ load, as measured by caffeine-induced Ca²⁺transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca²⁺ sparks and associated BKcurrents by PKA in smooth muscle.

     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Intracellular Ca(2+) and Zn(2+) signals during monochloramine-induced oxidative stress in isolated rat colon crypts

During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) and Zn(2+) accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca(2+) and Zn(2+) in the cytoplasm. Responses to different concentrations of NH(2)Cl (50, 100, and 200 microM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH(2)Cl-induced increases in Ca(2+) and Zn(2+) in the cytoplasm. Exposure to NH(2)Cl led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the range of 200-400 nM above baseline levels. Further studies indicated that NH(2)Cl-induced accumulation of Ca(2+) in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca(2+) through store-operated channels. In addition, exposure to NH(2)Cl resulted in dose-dependent and sustained increases in intracellular Zn(2+) concentration ([Zn(2+)](i)) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca(2+)- and Zn(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca(2+)](i) and [Zn(2+)](i). Under certain conditions, NH(2)Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.     

Adrenergic stimulation of rat resistance arteries affects Ca(2+) sparks, Ca(2+) waves, and Ca(2+) oscillations

Confocal laser scanning microscopy and fluo 4 were used to visualize local and whole cell Ca(2+) transients within individual smooth muscle cells (SMC) of intact, pressurized rat mesenteric small arteries during activation of alpha1-adrenoceptors. A method was developed to record the Ca(2+) transients within individual SMC during the changes in arterial diameter. Three distinct types of "Ca(2+) signals" were influenced by adrenergic activation (agonist: phenylephrine). First, asynchronous Ca(2+) transients were elicited by low levels of adrenergic stimulation. These propagated from a point of origin and then filled the cell. Second, synchronous, spatially uniform Ca(2+) transients, not reported previously, occurred at higher levels of adrenergic stimulation and continued for long periods during oscillatory vasomotion. Finally, Ca(2+) sparks slowly decreased in frequency of occurrence during exposure to adrenergic agonists. Thus adrenergic activation causes a decrease in the frequency of Ca(2+) sparks and an increase in the frequency of asynchronous wavelike Ca(2+) transients, both of which should tend to decrease arterial diameter. Oscillatory vasomotion is associated with spatially uniform synchronous oscillations of cellular [Ca(2+)] and may have a different mechanism than the asynchronous, propagating Ca(2+) transients.     

Characterisation of thapsigargin-releasable Ca(2+) from the Ca(2+)-ATPase of sarcoplasmic reticulum at limiting [Ca(2+)

The Ca(2+) binding sites of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using (45)Ca(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca(2+)-limited state, [Ca(2)](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca(2+)-ATPase, released a fraction of total Ca(2+) at [Ca(2+)](lim) that accumulated during active transport. Maximal Ca(2+) release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca(2+) resistant to TG. The amount of Ca(2+) released depended on the incubation time at [Ca(2+)](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0. 13 to 0.03 s(-1). The rapidly released early fraction declined with time and k=0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca(2+) followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 microM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca(2+) release mechanism. It is concluded that the Ca(2+) released by TG is from the occluded Ca(2+) fraction. The Ca(2+) occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca(2+) transport.     

Cannabinoid receptors regulate Ca(2+) signals and insulin secretion in pancreatic beta-cell

Insulin is the main hormone involved in the regulation of glycaemia, its impaired secretion is a hallmark of type I and type II diabetic individuals. Additionally, insulin is involved in lipogenesis and weight gain, provoking an anorexigenic action. The endocannabinoid system contributes to the physiological regulation of energy balance, food intake and lipid and glucose metabolisms. Despite that, an experimental link between the endocannabinoid system and the endocrine pancreas has not yet been described. Using quantitative real-time PCR and immunocytochemistry, we have demonstrated the existence of both CB1 and CB2 receptors in the endocrine pancreas. While the CB1 receptor is mainly expressed in non-beta-cells, the CB2 type exists in beta- and non-beta-cells within the islet. The endocannabinoid 2-arachidonylglycerol (2-AG) through CB2 receptors regulates [Ca(2+)](i) signals in beta-cells and as a consequence, it decreases insulin secretion. This effect may be a new component involved in the orexigenic effect of endocannabinoids and constitutes a potential target for pharmacologic manipulation of the energy balance.     

Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol

BACKGROUND: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.     

A new type of Ca(2+) channel blocker that targets Ca(2+) sensors and prevents Ca(2+)-mediated apoptosis.

Calmodulin (CaM), as well as other Ca(2+) binding motifs (i.e., EF hands), have been demonstrated to be Ca(2+) sensors for several ion channel types, usually resulting in an inactivation in a negative feedback manner. This provides a novel target for the regulation of such channels. We have designed peptides that interact with EF hands of CaM in a specific and productive manner. Here we have examined whether these peptides block certain Ca(2+)-permeant channels and inhibit biological activity that is dependent on the influx of Ca(2+). We found that these peptides are able to enter the cell and directly, as well as indirectly (through CaM), block the activity of glutamate receptor channels in cultured neocortical neurons and a nonselective cation channel in Jurkat T cells that is activated by HIV-1 gp120. As a consequence, apoptosis mediated by an influx of Ca(2+) through these channels was also dose-dependently inhibited by these novel peptides. Thus, this new type of Ca(2+) channel blocker may have utility in controlling apoptosis due to HIV infection or neuronal loss due to ischemia.     

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.     

Synchronous Ca(2+) oscillation emerges from voltage fluctuations of Ca(2+) stores

Synchronous Ca(2+) oscillation occurs in various cell types to regulate cellular functions. However, the mechanism for synchronization of Ca(2+) increases between cells remains unclear. Recently, synchronous oscillatory changes in the membrane potential of internal Ca(2+) stores were recorded using an organelle-specific voltage-sensitive dye [Yamashita et al. (2006) FEBS J273, 3585-3597], and an electrical coupling model of the synchronization of store potentials and Ca(2+) releases has been proposed [Yamashita (2006) FEBS Lett580, 4979-4983]. This model is based on capacitative coupling, by which transient voltage changes can be synchronized, but oscillatory slow potentials cannot be communicated. Another candidate mechanism is synchronization of action potentials and ensuing Ca(2+) influx through voltage-dependent Ca channels. The present study addresses the question of whether Ca(2+) increases are synchronized by action potentials, and how oscillatory store potentials are synchronized across the cells. Electrophysiological and Ca(2+)-sensitive fluorescence measurements in early embryonic chick retina showed that synchronous Ca(2+) oscillation was caused by releases of Ca(2+) from Ca(2+) stores without any evidence of action potentials in retinal neuroepithelial cells or newborn neurons. High-speed fluorescence measurement of store membrane potential surprisingly revealed that the synchronous oscillatory changes in the store potential were periodic repeats of a burst of high-frequency voltage fluctuations. The burst coincided with a Ca(2+) increase. The present study suggests that synchronization of Ca(2+) release is mediated by the high-frequency fluctuation in the store potential. Close apposition of the store membrane and plasma membrane in an epithelial structure would allow capacitative coupling across the cells.