Modulation of calcium balance in tilapia larvae (<Emphasis Type="Italic">Oreochromis mossambicus</Emphasis>) acclimated to low-calcium environments

This study examined how developing fish larvae regulate their Ca2+ balance for acclimation to low ambient Ca2+. Calcium balance in newly hatched larvae was examined individually. Developing larvae not only increased Ca2+ influx but also decreased Ca2+ efflux when they were acclimated to low-Ca2+ environments. After acclimation for 8 days, the influx and efflux of the low-Ca2+ (0.02 mM) group were about 106% and 43%, respectively, compared to those of the high-Ca2+ (1.0 mM) group. Sensitivity and response to low-Ca2+ environments are age-dependent. Upon acute exposure to low Ca2+. newly hatched (H0) larvae increased both Ca2+ influx (from 24% to 67% of high-Ca2+) and net uptake (from 5% to 69%) within 64 h, while 3-day-posthatching (H3) larvae managed to reach the levels of the control within 38 h. Declining Ca2+ efflux in H3 larvae occurred 14 h after exposure, much faster than those in H0 larvae (38 h). It is suggested that modulation of Ca2+-balance mechanisms in developing larvae is dependent upon the levels of Ca2+ in the larval body.     

Morphology and function of gill mitochondria-rich cells in fish acclimated to different environments

The objective of this study is to test the hypothesis that morphologically different mitochondria-rich (MR) cells may be responsible for the uptake of different ions in freshwater-adapted fish. Tilapia (Oreochromis mossambicus) were acclimated to high-Ca, mid-Ca, low-Ca, and low-NaCl artificial freshwater, respectively, for 2 wk. Cell densities of wavy-convex, shallow-basin, and deep-hole types of gill MR cells as well as whole-body Ca(2+), Na(+), and Cl(-) influxes were measured. Low-Ca fish developed more shallow-basin MR cells in the gills and a higher Ca(2+) influx than those acclimated to other media. However, fish acclimated to low-NaCl artificial freshwater predominantly developed wavy-convex cells, and this was accompanied by the highest Na(+) and Cl(-) influxes. Relative abundance of shallow-basin and wavy-convex MR cells appear to be associated with changes in Ca(2+) and Na(+)/Cl(-) influxes, suggesting that shallow-basin and wavy-convex MR cells are mainly responsible for the uptake of Ca(2+) and Na(+)/Cl(-), respectively.     

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.     

Reverse effect of mammalian hypocalcemic cortisol in fish: cortisol stimulates Ca2+ uptake via glucocorticoid receptor-mediated vitamin D3 metabolism

Cortisol was reported to downregulate body-fluid Ca(2+) levels in mammals but was proposed to show hypercalcemic effects in teleostean fish. Fish, unlike terrestrial vertebrates, obtain Ca(2+) from the environment mainly via the gills and skin rather than by dietary means, and have to regulate the Ca(2+) uptake functions to cope with fluctuating Ca(2+) levels in aquatic environments. Cortisol was previously found to regulate Ca(2+) uptake in fish; however, the molecular mechanism behind this is largely unclear. Zebrafish were used as a model to explore this issue. Acclimation to low-Ca(2+) fresh water stimulated Ca(2+) influx and expression of epithelial calcium channel (ecac), 11β-hydroxylase and the glucocorticoid receptor (gr). Exogenous cortisol increased Ca(2+) influx and the expressions of ecac and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), but downregulated 11β-hydroxylase and the gr with no effects on other Ca(2+) transporters or the mineralocorticoid receptor (mr). Morpholino knockdown of the GR, but not the MR, was found to impair zebrafish Ca(2+) uptake function by inhibiting the ecac expression. To further explore the regulatory mechanism of cortisol in Ca(2+) uptake, the involvement of vitamin D(3) was analyzed. Cortisol stimulated expressions of vitamin D-25hydroxylase (cyp27a1), cyp27a1 like (cyp27a1l), 1α-OHase (cyp27b1) at 3 dpf through GR, the first time to demonstrate the relationship between cortisol and vitamin D(3) in fish. In conclusion, cortisol stimulates ecac expression to enhance Ca(2+) uptake functions, and this control pathway is suggested to be mediated by the GR. Lastly, cortisol also could mediate vitamin D(3) signaling to stimulate Ca(2+) uptake in zebrafish.     

High-affinity Ca(2+) binding inhibits autoactivation of rat trypsinogen

The recent discovery that mutation Asn21 --> Ile in the human cationic trypsinogen (Tg) is associated with hereditary pancreatitis has brought into focus the functional role of amino acid 21 in mammalian Tgs. In the present paper, the effect of mutations Thr21 --> Asn and Thr21 --> Ile on the Ca(2+) dependence of zymogen activation was investigated, using the autolysis-resistant rat Tg mutant Arg117 --> His. In the absence of Ca(2+), rat Tg exhibited low but significant basal autoactivation, which was inhibited by micromolar concentrations of Ca(2+) (IC(50) 2.6 microM). Interestingly, basal autoactivation was diminished in both mutants, and no further inhibition by micromolar Ca(2+) was detectable. Millimolar Ca(2+) concentrations markedly and comparably stimulated autoactivation of wild-type and mutant zymogens (EC(50) 1.7-2.4 mM). The results indicate that rat Tg is subject to dual regulation by Ca(2+), allowing zymogen stabilization in a low-Ca(2+) environment and efficient activation in a high-Ca(2+) milieu.     

Responses to changes in Ca2+ supply in two Mediterranean evergreens, Phillyrea latifolia and Pistacia lentiscus, during salinity stress and subsequent relief

BACKGROUND AND AIMS: Changes in root-zone Ca(2+) concentration affect a plant's performance under high salinity, an issue poorly investigated for Mediterranean xerophytes, which may suffer from transient root-zone salinity stress in calcareous soils. It was hypothesized that high-Ca(2+) supply may affect differentially the response to salinity stress of species differing in their strategy of Na(+) allocation at organ level. Phillyrea latifolia and Pistacia lentiscus, which have been reported to greatly differ for Na(+) uptake and transport rates to the leaves, were studied. Methods In plants exposed to 0 mM or 200 mM NaCl and supplied with 2.0 mM or 8.0 mM Ca(2+), under 100 % solar irradiance, measurements were conducted of (a) gas exchange, PSII photochemistry and plant growth; (b) water and ionic relations; (c) the activity of superoxide dismutase and the lipid peroxidation; and (d) the concentration of individual polyphenols. Gas exchange and plant growth were also estimated during a period of relief from salinity stress. Key Results The performance of Pistacia lentiscus decreased to a significantly smaller degree than that of Phillyrea latifolia because of high salinity. Ameliorative effects of high-Ca(2+) supply were more evident in Phillyrea latifolia than in Pistacia lentiscus. High-Ca(2+) reduced steeply the Na(+) transport to the leaves in salt-treated Phillyrea latifolia, and allowed a faster recovery of gas exchange and growth rates as compared with low-Ca(2+) plants, during the period of relief from salinity. Salt-induced biochemical adjustments, mostly devoted to counter salt-induced oxidative damage, were greater in Phillyrea latifolia than in Pistacia lentiscus. CONCLUSIONS: An increased Ca(2+) : Na(+) ratio may be of greater benefit for Phillyrea latifolia than for Pistacia lentiscus, as in the former, adaptive mechanisms to high root-zone salinity are primarily devoted to restrict the accumulation of potentially toxic ions in sensitive shoot organs.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor

Two muscle thiol proteinases causing partial degradation of myofibrillar constituents were isolated and purified from skeletal muscle. The two proteinases that differ significantly in calcium requirements were designated respectively high- and low-Ca2+-requiring proteinase. Both are inhibited, in vitro, by a specific inhibitor which is a protein also isolated from skeletal muscle. Experiments using carboxymethylated monomeric proteinases and inhibitor-conjugated Sepharose were carried out in order to understand the mechanism of control of the proteinases by the inhibitor. The results using increasing inhibitor concentrations show a non-competitive inhibition for both enzymes. The Ki value for the low-Ca2+-requiring form was 0.3 microM, while the Ki value for the high-Ca2+-requiring form was 0.9 microM. Likewise, the low-Ca2+-requiring form needs about 3-fold more inhibitor than the high-Ca2+-requiring form for the same per cent inhibition.     

Parasporin-1, a novel cytotoxic protein from Bacillus thuringiensis, induces Ca2+ influx and a sustained elevation of the cytoplasmic Ca2+ concentration in toxin-sensitive cells

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxin-sensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca(2+) concentration; increases in the intracellular Ca(2+) levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca(2+) concentration. The toxin-induced elevation of the intracellular Ca(2+) concentration was markedly decreased in low-Ca(2+) buffer and was not observed in Ca(2+)-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca(2+) buffer and was restored by the addition of Ca(2+) to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca(2+) influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca(2+) level and that the Ca(2+) influx is the first step in the pathway that underlies parasporin-1 toxicity.     

Ca(2+) signalling in mitochondria: mechanism and role in physiology and pathology

Over recent years, a renewed interest in mitochondria in the field of Ca(2+) signalling has highlighted their central role in regulating important physiological and pathological events in animal cells. Mitochondria take up calcium through an uptake pathway that, due to its low-Ca(2+) affinity, demands high local calcium concentrations to work. In different cell systems high-Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of either plasma membrane or sarco/endoplasmic reticulum Ca(2+) channels. Mitochondrial Ca(2+) accumulation has a dual role, an universal one, which consists in satisfying energy demands by increasing the ATP production through the activation of mitochondrial enzymes, and a cell type specific one, which, through the modulation of the spatio-temporal dynamics of calcium signals, contributes to modulate specific cell functions. Recent work has revealed the central role of mitochondria dysfunction in determining both necrotic and apoptotic cell death. Evidence is also accumulating that suggests that alterations in mitochondrial function may act as predisposing factors in the pathogenesis of a number of neurodegenerative disorders. These include inherited disorders of the mitochondrial genome in which a defect in mitochondrial calcium accumulation has been shown to correlate with a defect in ATP production, thus suggesting a possible involvement of mitochondrial Ca(2+) dysfunction also for this group of diseases. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarise cell physiology and cell pathology aspects of the mitochondrial Ca(2+) transport machinery.     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Phosphatidic acid induces calcium influx in neutrophils via verapamil-sensitive calcium channels

Phosphatidic acid (PA) induces a biphasic Ca(2+) mobilization response in human neutrophils. The initial increase is due to the mobilization of Ca(2+) from intracellular stores, whereas the secondary increase is due to the influx of Ca(2+) from extracellular sources. The present investigation characterizes PA-induced Ca(2+) influx in neutrophils. Depolarization of neutrophils by 50 mM KCl enhanced PA-induced Ca(2+) influx, whereas verapamil, a Ca(2+) channel blocker, attenuated this response in a dose-dependent manner. These observations suggest that PA-induced Ca(2+) influx is mediated via verapamil-sensitive Ca(2+) channels. Stimulation of neutrophils with exogenous PA results in accumulation of endogenously generated PA with a time course similar to the effects of exogenous PA on Ca(2+) influx. Ethanol inhibited the accumulation of endogenous PA and calcium mobilization, indicating that activation of membrane phospholipase D plays a role in PA-mediated Ca(2+) influx. The results of this study suggest that exogenously added PA stimulates the generation of intracellular PA, which then mediates Ca(2+) influx through verapamil-sensitive Ca(2+) channels.     

Functional voltage-gated Ca2+ channels in muscle fibers of the platyhelminth Dugesia tigrina

The presence and function of voltage-gated Ca(2+) channels were examined in individual muscle fibers freshly dispersed from the triclad turbellarian Dugesia tigrina. Individual muscle fibers contracted in response to elevated extracellular K(+) in a concentration-dependent fashion. These depolarization-induced contractions were blocked by extracellular Co(2+) (2.5 mM), suggesting that they were dependent on depolarization-induced Ca(2+) influx across the sarcolemma. A voltage-gated inward current was apparent in whole cell recordings when the outward K(+) current was abolished by replacement of intracellular K(+) by Cs(+). This inward current was amplified with increasing concentration (相似文献   

The voltage-independent cation channel in the plasma membrane of wheat roots is permeable to divalent cations and may be involved in cytosolic Ca2+ homeostasis

A voltage-independent cation (VIC) channel has been identified in the plasma membrane of wheat (Triticum aestivum) root cells (P.J. White [1999] Trends Plant Sci 4: 245-246). Several physiological functions have been proposed for this channel, including roles in cation nutrition, osmotic adjustment, and charge compensation. Here, we observe that Ca(2+) permeates this VIC channel when assayed in artificial, planar lipid bilayers, and, using an energy barrier model to describe cation fluxes, predict that it catalyzes Ca(2+) influx under physiological ionic conditions. Thus, this channel could participate in Ca(2+) signaling or cytosolic Ca(2+) homeostasis. The pharmacology of (45)Ca(2+) influx to excised wheat roots and inward cation currents through the VIC channel are similar: Both are insensitive to 20 microM verapamil or 1 mM tetraethylammonium, but inhibited by 0.5 mM Ba(2+) or 0.5 mM Gd(3+). The weak voltage dependency of the VIC channel (and its lack of modulation by physiological effectors) suggest that it will provide perpetual Ca(2+) influx to root cells. Thus, it may effect cytosolic Ca(2+) homeostasis by contributing to the basal Ca(2+) influx required to balance Ca(2+) efflux from the cytoplasm through ATP- and proton-coupled Ca(2+) transporters under steady-state conditions.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.     

Mechanisms of enhanced taurine release under Ca2+ depletion

The sulfur-containing amino acid taurine is an inhibitory neuromodulator in the brain of mammals, as well as a key substance in the regulation of cell volumes. The effect of Ca(2+) on extracellular taurine concentrations is of special interest in the context of the regulatory mechanisms of taurine release. The aim of this study was to characterize the basal release of taurine in Ca(2+)-free medium using in vivo microdialysis of the striatum of anesthetized rats. Perfusion of Ca(2+)-free medium via a microdialysis probe evoked a sustained release of taurine (up to 180 % compared to the basal levels). The Ca(2+) chelator EGTA (1mM) potentiated Ca(2+) depletion-evoked taurine release. The substitution of CaCl(2) by choline chloride did not alter the observed effect. Ca(2+)-free solution did not significantly evoke release of taurine from tissue loaded with the competitive inhibitor of taurine transporter guanidinoethanesulfonate (1mM), suggesting that in Ca(2+) depletion taurine is released by the transporter operating in the outward direction. The volume-sensitive chloride channel blocker diisothiocyanostilbene-2,2'-disulfonate (1mM) did not attenuate the taurine release evoked by Ca(2+) depletion. The non-specific blocker of voltage-sensitive Ca(2+) channels NiCl(2) (0.65 mM) enhanced taurine release in the presence of Ca(2+). CdCl(2) (0.25 mM) had no effect under these conditions. However, both CdCl(2) and NiCl(2) attenuated the effect of Ca(2+)-free medium on the release of taurine. The data obtained imply the involvement of both decreased influx of Ca(2+) and increased non-specific influx of Na(+) through voltage-sensitive calcium channels in the regulation of transporter-mediated taurine release in Ca(2+) depletion.     

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.     

Copper elicits an increase in cytosolic free calcium in cultured tobacco cells

At concentrations greater than 0.1 mM, CuSO(4) provoked a rapid and sustained increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), in tobacco suspension culture cells expressing apoaequorin, a Ca(2+)-sensitive photoprotein. The increase was suppressed by treatment with LaCl(3), indicating that the increase is due to an influx of Ca(2+) from the apoplast through plasma membrane Ca(2+) channels. Although stimulation of H(2)O(2) production upon the CuSO(4) treatment (0.1 mM) was observed, treatment with catalase did not inhibit the increase in [Ca(2+)](cyt), and treatment with H(2)O(2) dose-dependently suppressed or delayed the increase. These results suggested that active oxygen species generated through copper-mediated reactions, or copper-mediated oxidative damages to plasma membrane, are not responsible for the increase. Treatment with sulfhydryl reagents, which alkylate or oxidize thiol groups, or acidification of the culture medium suppressed the increase in [Ca(2+)](cyt). These results demonstrated that copper causes an influx of Ca(2+) through plasma membrane Ca(2+) channels, and that plasma membrane thiol groups play an important role in activating the Ca(2+) channels.