Salt stress-induced programmed cell death via Ca<Superscript>2+</Superscript>-mediated mitochondrial permeability transition in tobacco protoplasts

The change in cytosolic free concentration of calcium ([Ca²⁺]_cyt) plays a key role in regulating apoptosis in animal cells. In our experiment, we tried to investigate the function of Ca²⁺ in programmed cell death (PCD) in tobacco (Nicotiana tobacum, cultivar BY-2) protoplasts induced by salt stress. An obvious increase in [Ca²⁺]_cyt was observed a few minutes after treatment and the onset of a decrease in mitochondrial membrane potential (ΔΨ_m) was also observed before the appearance of PCD, pre-treatment of protoplasts with EGTA or LaCl₃ effectively retarded the increase in [Ca²⁺]_cyt, which was concomitant with the decrease in the percentage of cell death and higher ΔΨ_m, pre-treatment with cyclosporine A (CsA) also effectively retarded the increase in [Ca²⁺]_cyt, the decrease in ΔΨ_m and the onset of PCD. All these results suggest that Ca²⁺ is a necessary element in regulating PCD and the increase in [Ca²⁺]_cyt and the opening of mitochondrial permeability transition pore (MPTP) could promote each other in regulating PCD in tobacco protoplasts induced by salt stress.Jiusheng Lin and Yuan Wang-These authors contributed equally for this work.     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

Osmotin induces cold protection in olive trees by affecting programmed cell death and cytoskeleton organization

Osmotin is a pathogenesis-related protein exhibiting cryoprotective functions. Our aim was to understand whether it is involved in the cold acclimation of the olive tree (Olea europaea L.), a frost-sensitive species lacking dormancy. We exposed olive trees expressing tobacco osmotin gene under the 35S promoter (35S:osm) [in the same manner as wild type (wt) plants] to cold shocks in the presence/absence of cold acclimation, and monitored changes in programmed cell death (PCD), cytoskeleton, and calcium ([Ca²⁺]_c) signalling. In the wt, osmotin was immunolocalized only in cold-acclimated plants, and in the tissues showing PCD. In the 35S:osm clones, the protein was detected also in the non-acclimated plants, and always in the tissues exhibiting PCD. In the non-acclimated wt protoplasts exposed to cold shock, a transient decrease in phallotoxin signal suggests a temporary disassembly of F-actin, a transient increase occurred instead in 35S:osm protoplasts exposed to the same shock. Transient increases in [Ca²⁺]_c were observed only in the wt protoplasts. However, when F-actin was depolymerized by cytochalasin or latrunculin, and microtubules by colchicine, increase in [Ca²⁺]_c also occurred in the 35S:osm protoplasts. Successive cold shocks caused transient rises in [Ca²⁺]_c and transient decreases in the phallotoxin signal in wt protoplasts. No change occurred in [Ca²⁺]_c occurred in the 35S:osm protoplasts. The phallotoxin signal transiently increased at the first shock, but did not change after the subsequent shocks, and an overall signal reduction occurred with shock repetition. Following acclimation, no cold shock-induced change in [Ca²⁺]_c levels and F-actin signal occurred either in wt or 35S:osm protoplasts. The results show that osmotin is positively involved in the acclimation-related PCD, in blocking the cold-induced calcium signalling, and in affecting cytoskeleton in response to cold stimuli.     

Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells

Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd²⁺). Cd²⁺ is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd²⁺-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 μM CdCl₂ underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd²⁺ concentration was measured subsequently. SNP led more Cd²⁺ content than Cd²⁺ treatment alone. By contrast, the prevention of NO by l-NAME decreased Cd²⁺ accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd²⁺ fluxes. This analysis revealed the promotion of Cd²⁺ influxes into cells by application of SNP, while l-NAME and cPTIO reduced the rate of Cd²⁺ uptake or even resulted in net Cd²⁺ efflux. Based on these founding, we concluded that NO played a positive role in CdCl₂-induced PCD by modulating Cd²⁺ uptake and thus promoting Cd²⁺ accumulation in BY-2 cells.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

An ATP signalling pathway in plant cells: extracellular ATP triggers programmed cell death in Populus euphratica

We elucidated the extracellular ATP (eATP) signalling cascade active in programmed cell death (PCD) using cell cultures of Populus euphratica. Millimolar amounts of eATP induced a dose‐ and time‐dependent reduction in viability, and the agonist‐treated cells displayed hallmark features of PCD. eATP caused an elevation of cytosolic Ca²⁺ levels, resulting in Ca²⁺ uptake by the mitochondria and subsequent H₂O₂ accumulation. P. euphratica exhibited an increased mitochondrial transmembrane potential, and cytochrome c was released without opening of the permeability transition pore over the period of ATP stimulation. Moreover, the eATP‐induced increase of intracellular ATP, essential for the activation of caspase‐like proteases and subsequent PCD, was found to be related to increased mitochondrial transmembrane potential. NO is implicated as a downstream component of the cytosolic Ca²⁺ concentration but plays a negligible role in eATP‐stimulated cell death. We speculate that ATP binds purinoceptors in the plasma membrane, leading to the induction of downstream intermediate signals, as the proposed sequence of events in PCD signalling was terminated by the animal P2 receptor antagonist suramin.     

Mechanisms of Extracellular NO and Ca<Superscript>2+</Superscript> Regulating the Growth of Wheat Seedling Roots

Our previous studies suggested the cross talk of nitric oxide (NO) with Ca²⁺ in regulating stomatal movement. However, its mechanism of action is not well defined in plant roots. In this study, sodium nitroprusside (SNP, a NO donor) showed an inhibitory effect on the growth of wheat seedling roots in a dose-dependent manner, which was alleviated through reducing extracellular Ca²⁺ concentration. Analyzing the content of Ca²⁺ and K⁺ in wheat seedling roots showed that SNP significantly promoted Ca²⁺ accumulation and inhibited K⁺ accumulation at a higher concentration of extracellular Ca²⁺, but SNP promoted K⁺ accumulation in the absence of extracellular Ca²⁺. To gain further insights into Ca²⁺ function in the NO-regulated growth of wheat seedling roots, we conducted the patch-clamped protoplasts of wheat seedling roots in a whole cell configuration. In the absence of extracellular Ca²⁺, NO activated inward-rectifying K⁺ channels, but had little effects on outward-rectifying K⁺ channels. In the presence of 2 mmol L⁻¹ CaCl₂ in the bath solution, NO significantly activated outward-rectifying K⁺ channels, which was partially alleviated by LaCl₃ (a Ca²⁺ channel inhibitor). In contrast, 2 mmol L⁻¹ CaCl₂ alone had little effect on inward or outward-rectifying K⁺ channels. Thus, NO inhibits the growth of wheat seedling roots likely by promoting extracellular Ca²⁺ influx excessively. The increase in cytosolic Ca²⁺ appears to inhibit K⁺ influx, promotes K⁺ outflux across the plasma membrane, and finally reduces the content of K⁺ in root cells.     

Thapsigargin,a selective inhibitor of sarco-endoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPases,modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca²⁺] _i ) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca²⁺] _i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca²⁺-ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 μmol/L) for 0–24 or 24–48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca²⁺] _i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0–24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 μmol/L produced secondary necrosis. During 24–48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 μmol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24–48 h. Our data suggest that the extent of the [Ca²⁺] _i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.     

Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca²⁺ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca²⁺ uptake and Ca²⁺ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca²⁺ levels ([Ca²⁺]_mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca²⁺ uptake, whereas inhibition of mtNOS with 10 µM L-N⁵-(1-iminoethyl)ornithine resulted in a significant increase of [Ca²⁺]_mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca²⁺-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca²⁺ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca²⁺ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca²⁺ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium     

Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts

The effects of gibberellic acid (GA₃) and Ca²⁺ on the synthesis and secretion of α-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA₃, CaCl₂, or both. Incubation of protoplasts in medium containing both GA₃ and Ca²⁺, however, causes an increase in the α-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA₃ or Ca²⁺ alone. Isoelectric focusing shows that adding Ca²⁺ to incubation media containing GA₃ increases the levels of α-amylase isozymes having high isoelectric points (pI). In the presence of GA₃ alone, only isozymes with low pIs accumulate. The increase in α-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca²⁺ to maintain elevated levels of α-amylase release. Immunoelectrophoresis shows that Ca²⁺ stimulates the release of low-pI α-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA₃ alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl₂. The response is not specific for Ca²⁺ since Sr²⁺ can substitute, although less effectively than Ca²⁺. Pulse-labeling experiments show that α-amylase isozymes produced by aleurone protoplasts in response to GA₃ and Ca²⁺ are newly synthesized. The effects of Ca²⁺ on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on α-amylase stability or on protoplast viability. We conclude that Ca²⁺ directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca²⁺-stimulated enzyme release.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

β-Amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons

β-Amyloid, a 39–43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble β-amyloid_1–42 on the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5–20 mM), evoked dose-dependent increases in intracellular [Ca²⁺]_i that were mediated by the entry of extracellular Ca²⁺ via nicotinic acetylcholine receptors, and the release of intracellular Ca²⁺ from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 ± 12% (P < 0.05) while β-amyloid_1–42 (100 nM) was present for ∼5 min. This potentiation became larger during the subsequent washout of β-amyloid_1–42, which was associated with a gradual rise in baseline [Ca²⁺]_i. Application of β-amyloid_1–42 by itself did not alter [Ca²⁺]_i, and β-amyloid_1–42 also had no significant effect on the response to repeated KCl challenges. Therefore, β-amyloid_1–42 caused neither gross disturbance of cellular Ca²⁺ homeostasis nor enhancement of voltage-gated Ca²⁺ channels. Interestingly, β-amyloid_1–42 transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca²⁺]_i. β-amyloid_1–42 potentiation of nicotine-evoked rises in [Ca²⁺]_i was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca²⁺]_i by β-amyloid_1–42 during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca²⁺ efflux from the mitochondria.     

Calcium Mediates the NO-induced Potassium Current in Toad and Rat Olfactory Receptor Neurons

Nitric oxide (NO) activates a K⁺ current in dissociated amphibian olfactory receptor neurons. Using the patch-clamp technique in its whole-cell mode and stimulation with puffs of the NO-donor sodium nitroprusside, we further studied this effect and show that it was sensitive to the K⁺-channel blockers tetraethylammonium and iberiotoxin, indicating the activation of a Ca²⁺-dependent K⁺ conductance. The Ca²⁺-channel blockers nifedipine and cadmium abolished the NO-induced current, and lowering external Ca²⁺ reduced it significantly. Ca²⁺ imaging showed a transient fluorescence increase upon stimulation with NO, and after blockade of K⁺ currents, an NO-induced inward current could be measured, suggesting that the activation of the Ca²⁺-dependent K⁺ conductance is mediated by Ca²⁺ influx. LY83583, a blocker of the ciliary cAMP-gated channels, did not affect the current, and experiments with focal stimulation indicated that the effect is present in the soma, therefore Ca²⁺ is unlikely to enter via the transduction channels. Finally, we show that NO exerts an effect with similar characteristics on olfactory receptor neurons from the rat. These data represent the first evidence that NO activates a Ca²⁺-dependent K⁺ conductance by causing a Ca²⁺ influx in a sensory system, and suggest that NO signaling plays a role in the physiology of vertebrate olfactory receptor neurons. Received: 25 October 1999/Revised: 2 March 2000     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ efflux rates through Na⁺-dependent and Na⁺-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca²⁺]_i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca²⁺. Maximal level of Ca²⁺, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A₂ and/or Ca²⁺/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca²⁺]_i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca²⁺-sparks and Ca²⁺-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca²⁺-microdomain formation, where the “vicious circle” of Ca²⁺-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca²⁺-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na⁺ and Ca²⁺ (lipid pore).     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

Intracellular mobilization of Ca by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca²⁺ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca²⁺ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist, and an L- or T-type Ca²⁺ channel blocker. The T-type Ca²⁺ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca²⁺-free medium, indicating that the source of Ca²⁺ is an intracellular reservoir. The IP₃R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca²⁺ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca²⁺ by activating IP₃R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP₃.