Intracellular Free Calcium Levels Research Articles

Bone marrow mesenchymal stem cells protected post-infarcted myocardium against arrhythmias via reversing potassium channels remodelling.

Bone marrow mesenchymal stem cells (BMSCs) emerge as a promising approach for treating heart diseases. However, the effects of BMSCs-based therapy on cardiac electrophysiology disorders after myocardial infarction were largely unclear. This study was aimed to investigate whether BMSCs transplantation prevents cardiac arrhythmias and reverses potassium channels remodelling in post-infarcted hearts. Myocardial infarction was established in male SD rats, and BMSCs were then intramyocardially transplanted into the infarcted hearts after 3 days. Cardiac electrophysiological properties in the border zone were evaluated by western blotting and whole-cell patch clamp technique after 2 weeks. We found that BMSCs transplantation ameliorated the increased heart weight index and the impaired LV function. The survival of infarcted rats was also improved after BMSCs transplantation. Importantly, electrical stimulation-induced arrhythmias were less observed in BMSCs-transplanted infarcted rats compared with rats without BMSCs treatment. Furthermore, BMSCs transplantation effectively inhibited the prolongation of action potential duration and the reduction of transient and sustained outward potassium currents in ventricular myocytes in post-infarcted rats. Consistently, BMSCs-transplanted infarcted hearts exhibited the increased expression of KV4.2, KV4.3, KV1.5 and KV2.1 proteins when compared to infarcted hearts. Moreover, intracellular free calcium level, calcineurin and nuclear NFATc3 protein expression were shown to be increased in infarcted hearts, which was inhibited by BMSCs transplantation. Collectively, BMSCs transplantation prevented ventricular arrhythmias by reversing cardiac potassium channels remodelling in post-infarcted hearts.

Sodium and potassium currents influence Wallerian degeneration of injured Drosophila axons.

Axons degenerate after injury and in neuropathies and disease via a self-destruction program whose mechanism is poorly understood. Axons that have lost connection to their cell bodies have altered electrical and synaptic activities, but whether such changes play a role in the axonal degeneration process is not clear. We have used a Drosophila model to study the Wallerian degeneration of motoneuron axons and their neuromuscular junction synapses. We found that degeneration of the distal nerve stump after a nerve crush is greatly delayed when there is increased potassium channel activity (by overexpression of two different potassium channels, Kir2.1 and dORKΔ-C) or decreased voltage-gated sodium channel activity (using mutations in the para sodium channel). Conversely, degeneration is accelerated when potassium channel activity is decreased (by expressing a dominant-negative mutation of Shaker). Despite the effect of altering voltage-gated sodium and potassium channel activity, recordings made after nerve crush demonstrated that the distal stump does not fire action potentials. Rather, a variety of lines of evidence suggest that the sodium and potassium channels manifest their effects upon degeneration through changes in the resting membrane potential, which in turn regulates the level of intracellular free calcium within the isolated distal axon.

BH3-mimetic ABT-737 induces strong mitochondrial membrane depolarization in platelets but only weakly stimulates apoptotic morphological changes, platelet shrinkage and microparticle formation

Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation. To elucidate this relationship in human platelets. Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis. We found that at optimal treatment conditions (90min, 37°C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P<0.0001). These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis.

S100B protein serum level as a biomarker of minor head injury

S100B is a small protein selectively synthesized by cerebral astroglial cells. S100B participates physiologically in the regulation of intracellular free calcium levels, and exerts a neurotrophic activity on cerebral cells. The interest of S100B protein in clinical biology results from its physiological presence in biological fluids (cerebrospinal fluid, blood, urine) and from significant increased levels when an acute brain injury occurred, from vascular (intracranial hemorrhage, ischemic stroke) or traumatic (traumatic brain injury) origins. Thus, elevated plasma concentrations of S100B were significantly increased in patients with a minor, moderate and of course severe traumatic brain injury. By contrast, serum S100B levels remained unchanged in patients with negative craniocerebral tomography results, confirming the diagnostic value of this biomarker. A prognostic value of the biomarker in the context of minor head injury is also reported.

Abstract 008: Bone Marrow Mesenchymal Stem Cells Prevented Cardiac Arrhythmias Of Infarcted Heart Via Regulating Calcineurin And Potassium Channels Expression

Bone marrow mesenchymal stem cells (BMSCs) emerge as a new promising approach for treating heart diseases. However, the effects of BMSCs-based therapy on cardiac electrophysiological remodeling following myocardial infarction were largely unclear. This study was aimed to investigate whether BMSCs transplantation prevented cardiac arrhythmias in post-infarcted hearts. Myocardial infarction was established in male SD rats, and BMSCs were then intramyocardially transplanted into the infarcted hearts after 3 days. Cardiac electrophysiological remodeling in the border zone was evaluated by western blotting and whole-cell path clamp technique after 2 weeks. We found that BMSCs transplantation significantly ameliorated the increased heart weight index, the impaired left ventricular functions, and the enhanced cardiac fibrosis in infarcted hearts. The survival ratio of infarcted rats was also improved after BMSCs transplantation. Importantly, electrical stimulation-induced arrhythmias were seldom observed in BMSCs-transplanted infarcted rats compared with rats without BMSCs treatment. Furthermore, BMSCs transplantation effectively inhibited the prolongation of action potential duration and the reduction of transient and sustained outward potassium currents in ventricular myocytes from post-infarcted rats. Consistently, BMSCs-transplanted infarcted hearts exhibited the more expression of K V 4.2, K V 4.3, K V 1.5 and K V 2.1 proteins than infarcted hearts. Moreover, intracellular free calcium level, calcineurin and nuclear NFATc3 protein expression was shown activated in infarcted hearts, which was inhibited after BMSCs transplantation. Collectively, BMSCs transplantation prevented ventricular arrhythmias by reversing the reduction of cardiac potassium channels in post-infarcted heats, which involves attenuated intracellular calcium overload and calcineurin/NFATc3 signal pathway.

A variety of mild stresses upregulate stanniocalcin-1 (STC-1) and induce mitohormesis in neural crest-derived cells

We induced upregulation of stanniocalcin-1 (STC-1) by various mild and long lasting stresses and assayed its influence on mitochondrial membrane potential (MMP) in the neural crest-derived cell line Paju. The obtained data showed that starvation (24–96h), exposure to 10nM TPA, and low concentrations (0.05–1μM) of As2O3 significantly (3–5 times) upregulated Paju cell STC-1 RNA and stabilized the mitochondrial membrane potential (MMP). However, high concentrations of As2O3 (2.5–5.0μM) increased intracellular ROS and free calcium levels and, consequently, suppressed STC-1 and MMP. The results show that cells preconditioned by various mild stresses expressed more STC-1 and their MMP were more resistant to a secondary exposure to As2O3 (2.5–5μM, 96h) demonstrating mitohormesis. We suggest that MMP deviation from control levels, to an extent innocuous to cell viability, is a general signal for STC-1-induction and MMP-protection. Our findings of Paju cell MMP-regulation may be of great importance for inventing new ways to prevent neurodegenerative diseases and unravel the mechanisms behind drug resistance.

Protein tyrosine kinase signaling in the mouse oocyte cortex during sperm–egg interactions and anaphase resumption

Fertilization triggers activation of a series of pre-programmed signal transduction pathways in the oocyte that establish a block to polyspermy, induce meiotic resumption, and initiate zygotic development. Fusion between sperm and oocyte results in rapid changes in oocyte intracellular free-calcium levels, which in turn activate multiple protein kinase cascades in the ooplasm. The present study examined the possibility that sperm-oocyte interaction involves localized activation of oocyte protein tyrosine kinases, which could provide an alternative signaling mechanism to that triggered by the fertilizing sperm. Confocal immunofluorescence analysis with antibodies to phosphotyrosine and phosphorylated protein tyrosine kinases allowed detection of minute signaling events localized to the site of sperm-oocyte interaction that were not amenable to biochemical analysis. The results provide evidence for localized accumulation of phosphotyrosine at the site of sperm contact, binding, or fusion, which suggests active protein tyrosine kinase signaling prior to and during sperm incorporation. The PYK2 kinase was found to be concentrated and activated at the site of sperm-oocyte interaction, and likely participates in this response. Widespread activation of PYK2 and FAK kinases was subsequently observed within the oocyte cortex, indicating that sperm incorporation is followed by more global signaling via these kinases during meiotic resumption. The results demonstrate an alternate signaling pathway triggered in mammalian oocytes by sperm contact, binding, or fusion with the oocyte.

Telekin Induces Apoptosis Associated with the Mitochondria-Mediated Pathway in Human Hepatocellular Carcinoma Cells

Telekin, a eudesmane-type sesquiterpene lactone compound isolated from Chinese folk medicine Carpesium divaricatum, has been reported to strongly inhibit the proliferation of cancer cells. In this study, the involvement of a mitochondria-mediated pathway in the pro-apoptotic action of telekin was investigated in human hepatocellular carcinoma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that telekin exhibited excellent anti-proliferation activity in hepatocellular carcinoma cells and low cytotoxicity to normal hepatocyte cells. Telekin-induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the extracellular surface, as revealed by 4,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry. Flow cytometry analysis showed that telekin induced the loss of mitochondrial membrane potential (MMP), as well as increased the levels of intracellular free calcium and reactive oxygen species (ROS). Additionally, Western blot results demonstrated that telekin induced the decrease in Apaf-1 and Bcl-2 expression, increase in Bax expression, release of cytochrome C, and activation of caspase-9 and caspase-3 in HepG-2 cells. These findings indicate that telekin activates the mitochondria-mediated apoptotic pathway in hepatocellular carcinoma cells and may merit further investigation as a potential therapeutic agent for the treatment of hepatocellular carcinoma.

328 – Fuction of grp78 in hippocampus in a rat model of posttraumatic stress disorder

The mechanism of PTSD is not fully understood until now. Previous studies showed PTSD induced endoplasmic reticulum stress (ERS). Grp 78 plays important role in ERS. Objectives To explore changes of Grp78 and Ca2+-CaM-CaMKIIα in hippocampus of PTSD rats may reveal part of the pathogensis. Methods The models of PTSD were created by SPS, which is an established animal model for PTSD. Wistar rats were selected for this study and randomly divided into a normal control group and SPS groups of 1d, 4d and 7d. The expression of GRP78 was examined by immunofluorescence, western blotting and RT-PCR. The intracellular free calcium level was examined by fluorescence spectrophotometer. CaM and CaMKIIα were examined by RT-PCR. Apoptosis was examined by TUNEL and TEM. Results The results showed the intracellular free calcium level reached the peak 1 day after SPS stimulation, then gradually decreased to normal level. The expression of CaM 1 day after SPS is the most and then decreased. CaMKIIα expression showed a significant down-regulation 1 day after SPS throughout and then gradually increased to normal level. Grp 78 reached the peak 4 day after SPS stimulation. TUNEL-positive cells significantly more than the normal control group and peaked at 7d after SPS stimuli, then gradually decreased to normal level. Furthermore, some cells had a characteristics change, including chromatin condensation, appearance of chromatin crescents, and nucleus fragmentation. Conclusions The results suggest that Grp 78 and changes of Ca2+-CaM-CaMKIIα in hippocampal might play an important role in the pathology of PTSD.

1067 – Effect of calreticulin on hippocampus of post-traumatic stress disorder rats

Objective To detect molecular chaperone calreticulin(CRT) expression on the hippocampus in the rat model of post-traumatic stress disorder(PTSD), and discuss the regulation of CRT on Ca2+ in hippocampus of PTSD rats, further provide the experiment basis for pathogenesis of memory anomaly in PTSD rats. Methods The single-prolonged stress(SPS) is one of the animal models was used to set up the rat PTSD models. Male Wistar rats were randomly divided into 1, 4, 7 days groups after exposure to SPS and a normal control group. The expression of CRT was detected by using immunohistochemistry, Western blotting and RT-PCR. The intracellular free calcium was examined by fluorescence spectrophotometer. Results The expression of CRT in the hippocampus obviously increased after SPS stimulation, and reached the peak at SPS 4d. The intracellular free calcium level in the hippocampus obviously increased, and reached the peak at SPS 1d, then gradually decreased. Conclusions PTSD caused endoplasmic reticulum stress(ERS), calcium overload, up-regulated expression of CRT, activation of unfolded protein response(UPR), which maybe result in cell apoptosis and maybe the pathogenesis basis on memory anomaly in PTSD rats.

1070 – Expression of caspase-12 and GRP94 in medial prefrontal cortex of PTSD rats

Introduction Post-traumatic stress disorder is an important manifestation of mental and behavioral disorders after the disaste Single-prolonged stress (SPS) is an received established animal model for post-traumatic stress disorder. Objectives To investigate endoplasmic reticulum apoptosis pathway and unfolded protein reaction plays an important role in medial Prefrontal Cortex of PTSD rats by Single-prolonged stress (SPS). Aims Determined by the change of free intracellular Ca2+ the glucose-regulated Protein (GRP)94 and apoptosis-related cacaspase-12 expression. Methods A total of 60 healthy, male Wistar rats were selected for this study,randomly divided into a normal control group and SPS groups of 1d,4d,7d,14day and 28day. Behavioral of learning and memory capabilities of rats was observed by using Morris water maze. The expression of, GRP94 and cacaspase-12 was detected using immunohistochemical,Western Blotting and reverse transcription-polymerase chain reaction. Results In this study compared with control groups the intracellular free calcium level in mPFC was increased 1 day after SPS exposure (P Conclusions In SPS-PTSD rats the learning and memory capabilities of the rats decline;mPFC free intracellular Ca2+ may relate to endoplasmic reticulum stress;Endoplasmic reticulum stress launch unfolded protein reaction Endoplasmic reticulum apoptotic process which may relate to the pathogenesis of medial prefrontal cortex abnormal function in PTSD.

Protective effects of hydrogen sulfide on oxidative stress and fibrosis in hepatic stellate cells

In hepatic fibrosis, hepatic stellate cells (HSCs) are activated and change into myofibroblast-like cells which are characterized by increased proliferation and extracellular matrix (ECM) synthesis. In this study, we investigated the regulatory effects of hydrogen sulfide (H2S) on hepatic fibrosis. We detected the proliferation, cell cycle progression, apoptosis, intracellular reactive oxygen species (ROS) and free calcium levels in ferric nitrilotriacetate (Fe-NTA)-activated HSCs treated with sodium hydrogen sulphide (NaHS), an H2S-releasing molecule. We also evaluated the effects of NaHS on fibrosis and ECM synthesis in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). MTT assay revealed that NaHS (500µmol/l) suppressed the Fe-NTA-induced proliferation of HSC-T6 cells in a dose-dependent manner. NaHS induced G1 phase cell cycle arrest and apoptosis in the Fe-NTA-treated HSC-T6 cells. Furthermore, in the Fe-NTA-treated HSC-T6 cells, NaHS reduced intracellular levels of ROS at 1, 3 and 6h and reduced intracellular free calcium levels at 3 and 6h. H2S administration attenuated hepatic fibrosis and collagenⅠ protein expression in the rats with CCl4-induced hepatic fibrosis. In conclusion, exogenous H2S inhibits proliferation and induces cell cycle arrest and apoptosis in activated HSCs and attenuates CCl4-induced hepatic fibrosis and ECM expression.

Screening acute cytotoxicity biomarkers using a microalga as test organism

The present study checked the suitability of the integration of flow cytometry (FCM) as technique and a freshwater microalga (Chlamydomonas moewusii) as cell system model for ecotoxicological studies, looking for sensitive biomarkers of acute cytotoxicity of potential contaminants in aquatic systems. The detection of the potential acute toxicity of a pollutant is of interest because pulse discharges of contaminants to natural waters could lead to high concentrations of these substances that are only present for short periods of time but can affect aquatic organisms such as microalgae. Physiological alterations in C. moewusii cells were analysed after 1h of exposure to different concentrations of the herbicide paraquat. Cell viability was not affected, but the acute toxicity of paraquat was evident at other levels of cell physiology. Herbicide-treated cells showed lower autofluorescence and higher size and internal complexity, lower esterase activity and lower mitochondrial membrane potential. Paraquat induced the depolarisation of the plasma membrane and the increase of intracellular free calcium level and cytosolic pH in a concentration-dependent percentage of cells. All these effects can be related to the oxidative stress induced by the herbicide, as revealed the significantly increased intracellular levels of reactive oxygen species in cultures exposed to paraquat concentrations which induced the physiological alterations mentioned above. Excluding cell viability and mitochondrial membrane potential, these cytotoxicity endpoints could be considered sensitive biomarkers for the short-term exposure to pollutants such as herbicides.

Overexpression of Guanylate Cyclase Activating Protein 2 in Rod Photoreceptors In Vivo Leads to Morphological Changes at the Synaptic Ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

On Ca2+ signalling research

As a highly versatile intracellular signal, calcium (Ca) regulates many different cellular processes in both animal and plant systems. Disruption of Ca homeostasis contributes to several human diseases. Owing to the importance of Ca signalling, its research is now an active field in life science. There are numerous Ca signalling systems, consisting of a diverse array of signalling units that deliver Ca signals with different spatial and temporal properties [1,2], playing roles in ubiquitous biological processes including gene regulation, fuel generation, substance transport, hormone and neurotransmitter secretion, cell motility and muscle contraction [3]. Consequently, exquisite homeostasis of Ca cycling is the key for health of humans, the disruption of which is related to many human diseases such as heart failure, neuron-degeneration, and diabetes [46]. Many remarkable achievements have greatly enhanced our understanding of Ca signaling, including those from Chinese scientists [710]. The 17th International Symposium on Ca-binding Proteins and Ca Function in Health and Disease was held in Beijing, China, on July 16–20, 2011 [11], accompany which, a special issue of Science China Life Sciences was published for transducing Ca signals to effectors. The first part focused on the mechanisms in maintaining a low cytosolic level of Ca, with two articles reviewing the properties of the plasma membrane calcium ATPases (PMCA) in ejecting Ca into the extracellular space. First, Carafoli [12] reviewed the role of the plasma membrane calcium pump, PMCA2, in the hearing process. As an important plasma membrane Ca ATPases, PMCA2 plays an important role in maintaining intracellular Ca homeostasis in the inner ear. It is involved in the maintenance of intracellular free calcium levels and the dysfunction of which leads to deafness. Recent studies show that another important isoform of PMCA, PMCA4, has a number of roles for heart functioning [13,14]. Cartwright et al. [15] then reviewed the roles of plasma membrane calcium pumps related to cardiovascular disease, focusing on PMCA4, including its function in heart and vasculature, linking PMCA4 to contractile function, cardiac hypertrophy, cardiac rhythm, blood pressure control and hypertension. Undoubtedly, further understanding of the roles of PMCAs will facilitate the development of new treatment strategies for related diseases. As a versatile intracellular messenger, Ca performs roles in many different ways to regulate cellular processes [16]. The second part of this special issue focuses on mechanisms for spatiotemporally-specific increases in cytosolic Ca. First, Lee [17] reviewed studies on the fraternal twin messengers for calcium signaling, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Since the initial finding by Lee and his colleagues that cADPR and NAADP could liberate stored Ca, they have been shown to be active in mobilizing Ca in a wide range of cells spanning three biological kingdoms. He summarized the cADPR/NAADP/CD38signalling pathway and also described the structure and function of its components. Kimlicka and van Petegem [18] then reviewed the structural biology aspect of ryanodine receptors (RyRs) which are high-conductance ion channels

Malathion/Oxon and Lead Acetate Increase Gene Expression and Protein Levels of Transient Receptor Potential Canonical Channel Subunits TRPC1 and TRPC4 in Rat Endothelial Cells of the Blood–Brain Barrier

This study examined the effects of malathion and lead on transient receptor potential canonical channel TRPC1/TRPC4 channels in rat brain endothelial cells as a mechanism to explain previously noted blood-brain barrier (BBB) permeability induced by these compounds. Lead, malathion, malaoxon and combinations of these were assessed for protein levels and gene expression of TRPC1/C4 at 2, 4, 8, 16, and 24 hours after exposure. Changes in intracellular free calcium dynamics were also assessed. Compounds increased TRPC1 and TRPC4 protein levels as well as gene expression within 4 hours after exposure. Basal levels of intracellular free calcium were also elevated. Increases in gene and protein expression may be associated with an increase in the numbers of TRP channels, and the increases in intracellular calcium may be associated with activation of such channels. Therefore, upregulation and activation of the TRPC1/TRPC4 may be a mechanism by which these neurotoxicants affect BBB permeability.

Arsenite promotes apoptosis and dysfunction in microvascular endothelial cells via an alteration of intracellular calcium homeostasis

Vascular endothelium has been considered as a target for arsenic-induced cardiovascular toxicity. The present study demonstrated that arsenite caused slow and sustained elevation of intracellular free calcium levels ([Ca2+]i) in HMEC-1, a human microvessel-derived endothelial cell line, in a concentration-dependent manner. Pretreatment with U-73122 (a specific PLC inhibitor) or 2-APB (a specific IP3 receptor antagonist) attenuated this effect, suggesting that PLC/IP3 signaling cascade is involved in arsenite-induced elevation of [Ca2+]i. Cytotoxic concentrations of arsenite (5 and 10μM) significantly enhanced endothelial nitric oxide synthase (eNOS) phosphorylation, nitric oxide (NO) production and apoptosis after 24-h exposure. Additionally, 2-APB attenuated eNOS phosphorylation and apoptosis induced by arsenite, indicating that Ca2+-mediated eNOS activation participates in arsenite-induced endothelial cell apoptosis. Moreover, we also found that non-apoptotic concentrations of arsenite (0.5 and 1μM) dramatically mitigated thrombin-induced rapid transient rise of [Ca2+]i, eNOS phosphorylation and NO production, suggesting functional disruption of endothelial by arsenite, and these effects occurred without an alteration of PLC-β1 and thrombin receptor levels. Altogether, the results reveal that arsenite induces apoptotic cell death and endothelial dysfunction as indicated by the reduction of thrombin responses, particularly related to an alteration of intracellular Ca2+ homeostasis.

Altered calcium signaling following traumatic brain injury.

Cell death and dysfunction after traumatic brain injury (TBI) is caused by a primary phase, related to direct mechanical disruption of the brain, and a secondary phase which consists of delayed events initiated at the time of the physical insult. Arguably, the calcium ion contributes greatly to the delayed cell damage and death after TBI. A large, sustained influx of calcium into cells can initiate cell death signaling cascades, through activation of several degradative enzymes, such as proteases and endonucleases. However, a sustained level of intracellular free calcium is not necessarily lethal, but the specific route of calcium entry may couple calcium directly to cell death pathways. Other sources of calcium, such as intracellular calcium stores, can also contribute to cell damage. In addition, calcium-mediated signal transduction pathways in neurons may be perturbed following injury. These latter types of alterations may contribute to abnormal physiology in neurons that do not necessarily die after a traumatic episode. This review provides an overview of experimental evidence that has led to our current understanding of the role of calcium signaling in death and dysfunction following TBI.

184 INHIBITION OF CALCIUM EFFLUX EXTENDS THE DURATION OF CALCIUM SIGNALS IN PIG OOCYTES

Calcium signaling involves the transient elevation in the intracellular free-calcium concentration, which is responsible for controlling a great number of biological functions. In many cell types, a signal is generated when calcium stored in the endoplasmic reticulum is released into the cytoplasm, followed by an influx of calcium across the plasma membrane. At the same time, calcium is removed from the cytosol by ATPases, which pump it back into the intracellular store or out of the cell. The size of the calcium signal is thus determined by the amount of calcium moving into and out of the cytoplasm. In the present study, we investigated the effect of inhibiting the movement of calcium across the plasma membrane on a calcium-signal that was artificially induced in pig oocytes. In vitro-matured pig oocytes were loaded with the calcium-indicator dye, fura-2. The release of calcium from the cytoplasmic stores was stimulated by adding ethanol at a final concentration of 7% and changes in the intracellular free-calcium levels were monitored by using InCyt Im2, a dual-wavelength fluorescence imaging system. In the control group, fluorescent measurements were performed in the presence of extracellular calcium. In additional treatment groups, the ethanol treatment was performed in (1) a calcium-free medium (prevents calcium influx); (2) the presence of 1 mM gadolinium (limits calcium fluxes across the plasma membrane in both directions); and (3) the presence of gadolinium in a calcium-free medium. In each group, 15 oocytes were measured; the data were subjected to 1-way ANOVA and differences between treatment means were compared by the Tukey's test. We found that in control oocytes, 7% ethanol induced a rise of 722.1 ± 49.2 nM in the cytoplasmic calcium levels and the average duration of the calcium transient was 309.1 ± 11.3 s. In the calcium-free medium, this increase was significantly smaller (only 117.9 ± 4.8 nM; P &lt; 0.01), probably because the calcium release was not followed by an influx of calcium across the plasma membrane. In the presence of 1 mM gadolinium in the regular calcium-containing medium, the intracellular calcium levels climbed by 278.8 ± 45.4 nM and dropped to baseline levels only after 773.3 ± 79.9 s. Finally, in the calcium-free medium and in the presence of 1 mM gadolinium, 7% ethanol induced only a small rise (64.5 ± 6.3 nM) in the cytoplasmic calcium levels. These latter increases were both significantly lower (P &lt; 0.01) compared to that in the control group. The results indicate that (1) intracellular calcium signals are sustained by a calcium influx from the extracellular medium and (2) preventing calcium efflux extends the duration of the cytoplasmic calcium elevation in the oocyte. This may have relevance in the manipulation of intracellular calcium levels in oocytes for the development of novel parthenogenetic activation methods.

Melatonin elicits protein kinase C-mediated calcium response in immortalized GT1–7 GnRH neurons

Melatonin is suggested to have effects on hypothalamic–pituitary–gonadal (HPG) axis. The pulsatile pattern of GnRH release, which results in the intermittent release of gonadotropic hormones from the pituitary, has a critical importance for reproductive function but the factors responsible from this release pattern are not known. Calcium is a second messenger involved in hormone release. Therefore, investigation of the effects of melatonin on intracellular free calcium levels ([Ca2+]i) would provide critical information on hormone release in immortalized GnRH neurons. The pattern of melatonin-induced intracellular calcium signaling was investigated by fluorescence calcium imaging using the immortalized GnRH-secreting GT1–7 hypothalamic neurons. Melatonin caused a significant increase in [Ca2+]i, which was greatly blocked by luzindole, a melatonin antagonist, or attenuated by pre-treatment with protein kinase C inhibitor. This study suggests that melatonin seems to have a direct effect on GnRH neurons.