Cytosolic Calcium Ion Research Articles

TOXIC GLOBALISATION OF SECRETAGOGUE-ELICITED ACINAR CELL CA2+ SIGNALING AND PREVENTION BY ATP OR REMOVAL OF CA2+ FROM THE CELL EXTERIOR

Background & Aims: We have previously demonstrated that low concentrations of the pancreatic acinar cell toxins taurolithocholic-3-sulphate (TLCS) or palmitoleic acid ethyl ester (POAEE) induce toxic globalisation of inositol trisphosphate (IP3)-mediated signaling increases in the cytosolic calcium ion concentration ([Ca2+]C, IP3 applied via patch pipette). We sought to determine whether toxic globalisation of [Ca2+]C can occur with physiological secretagogues, and how it can be prevented. Methods: Isolated mouse pancreatic acinar cells were examined by confocal microscopy to measure changes in [Ca2+]C (Fluo4-AM), mitochondrial function (NADH autofluorescence), ATP concentration (Mg Green) and cell fate (propidium iodide, PI). Cholecystokinin (CCK, 1-20 pM) or acetylcholine (ACh, 50 nM) were perfused extracellularly and low concentrations (10 mM) of TLCS or POAEE added, while whole-cell recordings of Ca2+-dependent Cl− currents were made. In some experiments, supplementary ATP was added to the internal pipette solution. Results: CCK and ACh elicited typical [Ca2+]C signals in the granular pole with stimulus-metabolism coupling (NADH). Extracellular TLCS or POAEE (10 mM) transformed these signals into prolonged (>30 s), global [Ca2+]C increases accompanied by NADH and ATP depletion, inducing necrosis. Supplementary ATP reduced the period of globalisation associated with each elevation of [Ca2+]C and prevented necrosis, as did removal of Ca2+ from the external medium, and in the case of POAEE, hydrolase inhibition, preventing POA formation. Conclusion: Physiological Ca2+ signals elicited by secretagogues can undergo toxic globalisation from low concentrations of acinar cell toxins, resulting in mitochondrial Ca2+ overload, inhibition of ATP production and cell death. Cell injury could be prevented by blockade of Ca2+ entry.

Calcium puffs in Xenopus oocytes.

The second messenger inositol 1,4,5-trisphosphate (InsP3) functions in large part by liberating calcium ions from intracellular stores. This release process is highly non-linear and shows a regenerative characteristic that allows production of all-or-none calcium spikes which propagate as waves. However, at low concentrations of InsP3 an additional mode of calcium liberation is seen in Xenopus oocytes, transient 'puffs' of cytosolic calcium that last for a few hundred milliseconds and are restricted to within a few micrometres. Puffs are generally of similar size and the amount of calcium released (about 3 x 10(-18) mol) suggests that they arise through the concerted opening of several InsP3-gated calcium release channels. Puff sites are present at a density of about one per 30 microns 2 in the animal hemisphere of the oocyte. Each site functions autonomously, producing puffs at largely random intervals. We conclude that calcium puffs represent 'quantal' units of InsP3-evoked calcium liberation, which may result from local regenerative feedback by cytosolic calcium ions at functionally discrete release sites.

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.

Modulation of the dynamics of mitochondrial calcium by buffer cytosolic proteins and plasma membrane fluxes

Buffer performance of mitochondria in intracellular calcium signaling is studied in relation to the amount of cytosolic calcium-binding proteins and calcium ion fluxes across the plasma membrane of the cell.

Coupling Diurnal Cytosolic Ca 2+ Oscillations to the CAS-IP 3 Pathway in Arabidopsis

Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.

BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional “Ca2+ nanodomain” where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Mechanisms involved in the cytosolic calcium ion elevation induced by activating factors in chicken and rat phagocytes

ABSTRACTThe aim of the present study was to determine the mechanism of cytosolic calcium ion concentration [Ca2+]i elevation in chicken and rat phagocytes stimulated with phorbol myristate acetate (PMA), leukotriene B4 (LTB4), formyl‐methionyl‐leucyl‐phenylananine (fMLP) and Saccaromyces cerevisiae culture supernatant (SCS). Pretreatment with EGTA completely suppressed the PMA‐induced [Ca2+]i elevation in rat and chicken phagocytes, suggesting that all the [Ca2+]i elevation induced in the PMA‐stimulated rat and chicken phagocytes was attributable to the influx of extracellular Ca2+. On the other hand, the elevation of LTB4‐, FMLP‐ and SCS‐induced [Ca2+]i was only partially suppressed by ethyleneglycol‐bis (β‐aminoethyl)‐N,N,N′,N′‐tetraacetic acid ethylene (EGTA) pretreatment of phagocytes. The results indicated that two pathways of [Ca2+]i elevation, recruitment from the intracellular Ca2+ store and influx of extracellular Ca2+, are involved in the [Ca2+]i elevation of LTB4‐, fMLP‐ and SCS‐stimulated phagocytes. In fMLP‐stimulated rat neutrophils, [Ca2+]i elevation showed a two‐phase pattern in which the time lag between the first and second phase was approximately 1 min. The EGTA treatment of the fMLP‐stimulated cells induced a reduction of the first phase level and a disappearance of the second phase. The reason for the special influence of EGTA observed in fMLP‐stimulated cells is unknown, but the disappearance of the second phase of the [Ca2+]i may be elicited by the EGTA‐induced decrease of the first phase [Ca2+]i elevation that depends on IP3 and diacylglycerol induced by fMLP.

BK Ca -Cav Channel Complexes Mediate Rapid and Localized Ca 2+ -Activated K + Signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.

Dynamic quantification of the tyrosine phosphorylation of the sperm surface proteins during capacitation

Spermatozoa acquire active fertilizing competence only after deposition in the female tract and subsequent capacitation. Recent studies on the cellular location of major sperm phosphoproteins suggest that capacitation is associated with tyrosine phosphorylation of proteins exposed on the sperm surface. However, these changes have not yet been quantified objectively. A calcium influx seems to be required for the completion of tyrosine phosphorylation in some species; however, the exact temporal coordination between these processes is still poorly understood. Flow cytometry was used to quantify the degree of phosphorylation of the sperm surface proteins by probing with fluorescein isothiocyanate-conjugated anti-phosphotyrosine (pY) antibody raised in mouse. Dynamic changes in other sperm parameters (calcium influx, membrane integrity, and spontaneous acrosome reaction) were assessed to analyze their temporal coordination. : The changes in specific phosphotyrosine (pY) fluorescence signal detected in live, nonpermeabilized boar cell suspensions were biphasic during incubation under capacitating conditions. After 120 min of incubation, the degree of pY fluorescence increased threefold, indicating the changes in proteins exposed on sperm surface. At the same time there was a gradual increase in cytosolic calcium ion levels with the maximal rate at 60 min of incubation. This rate slowed immediately before the onset of the massive rise in tyrosine phosphorylation and decreased by 90% after its completion. The integrity of plasma and acrosome membranes decreased only slowly, illustrating that the changes observed were not due to the process of spontaneous acrosome reaction. These data provide quantitative evidence for the appearance of tyrosine-phosphorylated proteins on the surface of live boar spermatozoa during capacitation. An exact temporal coordination exists between cytosolic calcium ion content and protein tyrosine phosphorylation under these conditions. This novel approach has the advantage of making possible a precise quantification and kinetic comparison of molecular processes in different cell subpopulations.

P1-063: Deregulation of NMDA-receptor function and signaling in APP[V717O] transgenic mice

We previously demonstrated the causal role of amyloid peptides in the deficit in LTP in our APP[V717I] mice. However, the mechanisms causing impaired cognitive performance and defects in synaptic plasticity in APP transgenic mice remain to be identified. We aimed to obtain evidence for the hypothesis that the deficit in LTP depends on the NMDA–receptors, to analyse the mechanisms of NMDA–dependent defects in synaptic functioning by oligomeric amyloid peptides and to demonstrate an impairment in their downstream signalling pathways. We analysed NMDA–dependent LTP, and signalling pathways involved in cognition in APP[V717I] transgenic mice, in combination with the analysis of the modulation of NMDA–receptor functioning by oligomeric Aβ42 peptides in primary neuronal cultures and xenopus oocytes. The induction of c–fos as a major downstream signal that becomes activated by learning in the fear–conditioning task, was significantly reduced in APP[V717I] as compared to non–transgenic mice. To define the primary defect, we examined and confirmed that oligomeric Aβ42 peptides bind to synapses formed by hippocampal and cortical neurons in culture, and mainly to synapses that express NMDA–receptors. Moreover, pre–incubation of primary cortical and hippocampal neurons with oligomeric Aβ42 significantly attenuated the NMDA–R dependent increase in cytosolic calcium ion levels. However, in autaptic hippocampal cultures, the direct application of oligomeric Aβ42 did not affect the NMDA–receptor dependent currents. This finding was further corroborated in xenopus oocytes that express functional NMDA–receptors. Oligomeric Aβ42 binds to synapses that contain NMDA–receptors, but affect NMDA–receptor function indirectly. We further demonstrate that amyloid peptides deregulate signalling pathways downstream of the NMDA–receptor and their involvement in defective cognition in APP[V717I] transgenic mice.

Noradrenaline-induced increases in calcium and tension in skeletal muscle conductance and resistance arteries from rats with post-infarction heart failure

We tested the hypothesis that arterial reactivity to noradrenaline is augmented in congestive heart failure (CHF), which could contribute to the deleterious changes in peripheral vascular resistance and compliance in this condition. From male Wistar rats with post-infarction CHF and sham-operated rats, skeletal muscle conductance and resistance arteries (mean lumen diameters: 514 and 186 μm) were isolated and mounted on wire myographs, and wall tension was recorded in response to cumulative application of acetylcholine and noradrenaline to the vessel segments. In a subset of experiments, wall tension and cytosolic free calcium ion concentration [Ca 2+] i were recorded simultaneously during noradrenaline application, using wire myography and the FURA-2 technique. No significant differences were found in the arterial baseline levels of [Ca 2+] i or tension between CHF and sham rats. In the resistance arteries of CHF rats, the noradrenaline-induced increases in [Ca 2+] i were significantly enhanced ( P = 0.003). Despite the augmented [Ca 2+] i levels, the tension responses to noradrenaline were unaltered in these arteries. In the conductance arteries, there were no significant differences in noradrenaline-induced [Ca 2+] i or tension responses between CHF and control rats. CHF did not alter vascular morphology or change vascular relaxations to acetylcholine in either type of artery. In conclusion, these results do not support the contention that arterial reactivity to noradrenaline is augmented in the skeletal muscle vascular bed in CHF. On the contrary, the unchanged contractile responsiveness in the resistance arteries despite the enhanced levels of [Ca 2+] i during noradrenaline application suggests that the contractile function of these vessels is compromised in CHF. Neither vascular remodeling, endothelial dysfunction nor changes in baseline vascular tone could be demonstrated in the skeletal muscle vascular bed of this animal model of heart failure.

Fatty Acid Ethyl Esters Cause Pancreatic Calcium Toxicity via Inositol Trisphosphate Receptors and Loss of ATP Synthesis

Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca(2+)](C)) implicated in pancreatitis. We sought to define the mechanisms of this elevation. Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca(2+)](C) (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca(2+)](ER), Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10-100 micromol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. Intracellular delivery of ester induced oscillatory increases of [Ca(2+)](C) and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca(2+)](ER), not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca(2+)](C), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. Fatty acid ethyl ester increases [Ca(2+)](C) through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.

Mathematical modeling of calcium homeostasis in yeast cells

In this study, based on currently available experimental observations on protein level, we constructed a mathematical model to describe calcium homeostasis in normally growing yeast cells ( Saccharomyces cerevisiae). Simulation results show that tightly controlled low cytosolic calcium ion level can be a natural result under the general mechanism of gene expression feedback control. The calmodulin (a sensor protein) behavior in our model cell agrees well with relevant observations in real cells. Moreover, our model can qualitatively reproduce the experimentally observed response curve of real yeast cell responding to step-like disturbance in extracellular calcium ion concentration. Further investigations show that the feedback control mechanism in our model is as robust as it is in real cells.

Butyl benzyl phthalate blocks Ca2+ signaling coupled with purinoceptor in rat PC12 cells

Butyl benzyl phthalate (BBP) is a plasticizer and causes public concern because of its genomic estrogenic effects via estrogen receptors. We previously found that BBP has non-genomic effects, exerting inhibitory effects on the functional activities of nicotinic acetylcholine receptors (nAChR) in bovine adrenal chromaffin cells. nAChR belongs to the superfamily of neurotransmitter-gated channels, so does P2X purinoceptor that is widely distributed in the nervous system and play a role in pain reactions. In this study, we investigated the effects of BBP on the change of [Ca2+]c (cytosolic calcium ion concentration) under the stimulation of purinoceptors in PC12 cells and found that BBP inhibited ATP-induced [Ca2+]c rise (IC50=8.3 microM). The inhibitory rate of BBP remained under the increase of ATP concentration; therefore, the possibility of competitive inhibition was excluded. The inhibition of BBP on P2Y was excluded because its inhibition on ATP-induced [Ca2+]c rise was not found in the absence of extracellular Ca2+. BBP might have some actions on voltage-operated Ca2+ channels (VOCCs) since BBP inhibited the Ca2+ signaling responding to high K+ stimulation (IC50=1.2 microM). We suggest that BBP inhibits the ATP-induced [Ca2+]c rise via its non-competitive inhibition on P2X purinoceptors and VOCCs in the plasma membrane.

In vitro responses of rat alveolar macrophages to particle suspensions and water-soluble components of dust storm PM 2.5

A study was conducted to investigate the in vitro toxicities of dust storm particulate matter with an aerodynamic diameter ⩽ 2.5 μm (PM 2.5) on rat alveolar macrophages (AM). This was based on the ambient PM 2.5 collected in March 2004 from Baotou city, Inner Mongolia Autonomous Region, China. The particles were classified as normal (from sunshiny and clean days) and dust storm samples according to the dust storm classification, and the local weather and air quality monitoring data. The cell viability, levels of cellular thiobarbituric acid-reactive species (TBARS), glutathione (GSH), and cytosolic free calcium ions (Ca 2+), and the plasma membrane ATPase activities and membrane lipid fluidity were determined 4 h following the in vitro treatment of AM with differing dosages of the collected samples. Results revealed that dust storm PM 2.5 and their water-soluble fractions at high dosages generated oxidant stress on AM, induced leakage of lactate dehydrogenase (LDH), significantly decreased activities of plasma membrane Na +K +-ATPase, increased intracellular Ca 2+ levels, and led to significant alterations in membrane lipid fluidity, finally resulting in cytotoxicity. A two-way ANOVA showed that there was no significant difference in the above indices measured between the normal and dust storm PM 2.5, suggesting the deleterious effects of ambient PM 2.5 from Baotou city were based on the dose used rather than the type of particles. But due to a higher concentration of airborne PM 2.5 mass concentration in dust storm days than that in normal days, it concluded that during dust storm episodes, a much more serious effect on AM was imminent.

Protective Effect of Diltiazem on Hepatic Ischemia-Reperfusion Injury in Rats by Improving Liver Tissue Blood Flow

Background Cytosolic calcium ions are known to play an important role in ischemia-reperfusion (IR) injury. However, the protective effect of calcium channel blockers remains controversial in liver IR injury. Moreover, calcium channel blockers improve hepatic IR injury not due to blocking an increase in hepatic calcium concentration. Therefore, we hypothesized that calcium antagonists protected a liver from IR injury by a vasodilatory action rather than by the inhibition of an increase in Ca 2+ within parenchymal cells. This study evaluated the effects of diltiazem, a calcium channel blocker, on liver energy metabolism and blood flow after IR injury. Methods Twenty-seven rats underwent hepatic ischemia for 30 minutes followed by 60 minutes of reperfusion. The animals were allocated into group C (without drug); group D5 (diltiazem, 5 μg/kg per min); or group D10 (diltiazem, 10 μg/kg per min). Diltiazem was infused before laparotomy and then throughout the experiment. Results After 60 minutes of reperfusion, liver tissue blood flow and ATP concentrations were significantly higher in group D10 than the other animals (both, P < .05). Changes in ATP values strongly correlated those observed in blood flow (R = 0.80, P < .001). Conclusion Diltiazem improved ATP-generating capacity during reperfusion by improving liver tissue blood flow. An improvement in hepatic tissue perfusion may be a therapeutic strategy for liver IR injury.

Glycogen metabolism in rat ependymal primary cultures: Regulation by serotonin

Ependymal primary cultures are a model for studying ependymal energy metabolism. Intracellular glycogen is built up in the cultures dependent on culture age and the presence of glucose and glutamate. This energy store is mobilized upon glucose withdrawal, stimulation with isoproterenol, forskolin or serotonin and after uncoupling of oxidative phosphorylation from ATP production. Serotonin regulates ependymal glycogen metabolism predominantly via 5-HT receptor (5-HTR) 7, which elicits an increase in the level of ependymal cyclic AMP. Although the most abundant mRNAs for serotonin receptors are those of 5-HTR 2B and 5-HTR 3A, ependymal cells in primary culture do not respond to serotonin with an increase in their concentration of cytosolic calcium ions. The mRNAs of 5-HTRs 1A, 6, 1B, 5B, 7, 1/2C and 5A are also detectable in order of decreasing abundance. The mRNAs for 5-HTRs 1D, 1F, 3B and 4 are absent from the cultured cells. The ability of serotonin to mobilize ependymal glycogen depends on the culture age and the time allowed for glycogen buildup. During glycogen buildup time, glutamate is consumed by the cells. An increased ability of 5-HT to mobilize ependymal glycogen stores is noticed after the depletion of glutamate from the glycogen buildup medium. In ependymal primary cultures, cilia are colocalized with glycogen phosphorylase isozyme BB, while the MM isoform is not expressed. It is known from the literature that an increase in the concentration of cytosolic cAMP in ependymal cells leads to a decrease in ciliary beat frequency. Therefore, the present data point towards a function for ependymal glycogen other than supplying energy for the movement of cilia.

Ca 2+ signalling and Ca 2+-activated ion channels in exocrine acinar cells

The development of the calcium signalling field, from its early beginnings some 40 years ago to the present, is described. Calcium signalling in exocrine gland acinar cells and the effects of neurotransmitter- or hormone-elicited rises in the cytosolic calcium ion concentration on ion channel gating are reviewed. The highly polarized arrangement of the organelle systems in living acinar cells is described as well as its importance for the physiologically relevant local and polarized calcium signalling events.

Trypsin and thrombin accelerate aggregation of human endocrine pancreas precursor cells

Human islet-derived precursor cells (hIPCs) and human pancreatic ductal carcinoma (PANC-1) cells can be induced to form aggregates that subsequently differentiate into hormone-expressing islet-like cell aggregates (ICAs). We show that challenge of hIPCs or PANC-1 cells with thrombin or trypsin resulted in stimulation of signaling via the inositol-tris-phosphate second messenger pathway leading to rapid, transient increases in cytosolic calcium ion concentration in the majority of the cells. Because we found that hIPCs, PANC-1 cells, human fetal pancreas, and human adult islets express two protease-activated receptors (PARs), PAR-1 and PAR-2, we tested whether the effects of thrombin and trypsin were mediated, at least in part, by these receptors. Peptide agonists that are relatively specific for PAR-1 (SFLLRN-amide) or PAR-2 (SLIGRL-amide) stimulated increases in inositol phosphates and cytosolic calcium ion concentration, and increased the phosphorylation of Rho, a small G-protein associated with cytoskeletal changes affecting cellular morphology and migration. Most importantly, we show that these agonists increased the rate of hIPC aggregation leading to the formation of more viable, smaller ICAs. Our data show that thrombin and trypsin accelerate aggregation, an early stage of hIPC differentiation in vitro, and imply that pancreatic trypsin and thrombin may be involved in islet development in vivo.

Mitochondrial response and calcium ion change in apoptotic insect cells induced by SfaMNPV

Mitochondrial responses and changes of calcium ions in apoptotic insect SL-1 cells induced by Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) are reported in this paper. By using Rhodamine 123 as a fluorescent labeling probe, flow cytometry analysis and confocal laser scanning microscope observation we observed that the mitochondrial transmembrane potential (ΔΨm) began to decrease in SL-1 cells at 4 h post infection and Δψm reduced continuously with the extension of virus infection. Western blotting indicated that the Bcl-2 level in the mitochondria gradually declined and was downregulated. Cells undergoing apoptosis were found to have an elevation of cytochrome c in the cytosol and a corresponding decrease in the mitochondria, which indicated that cytochrome c was released from mitochondria into cytosol. These results suggest that mitochondrionmediated apoptotic signal transduction pathway exists in apoptotic insect cell induced by SfaMNPV. Cytosolic free calcium ([Ca2+]i) concentration rapidly increased after SfaMNPV infection and the elevated calcium was tested to come partly from extracelllular calcium ion influx. Flow cytometry analysis indicated that the apoptosis in SL-1 cells was not influenced by established cytosolic calcium clamped conditions and the EGTA inhibiting calcium influx. Therefore, neither the elevation of cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of ER Ca2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV.