Elevation Of Intracellular Free Ca2 Research Articles

Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway.

BACKGROUNDRecent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis (SAP). A stable intestinal mucosa barrier functions as a major anatomic and functional barrier, owing to the balance between intestinal epithelial cell (IEC) proliferation and apoptosis. There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling might play an important role in calcium-mediated apoptosis.AIMTo investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction (QYD) in SAP.METHODSA rat model of SAP was created via retrograde infusion of sodium deoxycholate. Serum levels of amylase, tumor necrosis factor (TNF-α), interleukin (IL)-6, D-lactic acid, and diamine oxidase (DAO); histological changes; and apoptosis of IECs were examined in rats with or without QYD treatment. The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative real-time polymerase chain reaction and western blotting. For in vitro studies, Caco-2 cells were treated with lipopolysaccharide (LPS) and QYD serum, and then cell viability and intracellular calcium levels were detected.RESULTSRetrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase, TNF-α, and IL-6. Both the indicators of intestinal mucosa damage (D-lactic acid and DAO) and the levels of IEC apoptosis were elevated in the SAP group. QYD treatment reduced the serum levels of amylase, TNF-α, IL-6, D-lactic acid, and DAO and attenuated the histological findings. IEC apoptosis associated with SAP was ameliorated under QYD treatment. In addition, the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group, and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group. QYD significantly restrained CaN and NFATc3 gene expression in the intestine, which was upregulated in the SAP group. Furthermore, QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca2+ levels and inhibited cell death.CONCLUSIONQYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated, at least partially, by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Endoplasmic Reticulum Stress Contributes to Gefitinib-Induced Apoptosis in Glioma.

Adequate stress on the Endoplasmic Reticulum (ER) with the Unfolded Protein Response (UPR) could maintain glioma malignancy. Uncontrolled ER stress, on the other hand, predisposes an apoptosis-dominant UPR program. We studied here the proapoptotic actions of the Epidermal Growth Factor Receptor (EGFR) inhibitor gefitinib, with the focus on ER stress. The study models were human H4 and U87 glioma cell lines. We found that the glioma cell-killing effects of gefitinib involved caspase 3 apoptotic cascades. Three branches of ER stress, namely Activating Transcription Factor-6 (ATF6), Protein Kinase R (PKR)-Like ER Kinase (PERK), and Inositol-Requiring Enzyme 1 (IRE1), were activated by gefitinib, along with the elevation of intracellular free Ca2+, Reactive Oxygen Species (ROS), and NADPH Oxidase2/4 (NOX2/4). Specifically, elevated IRE1 phosphorylation, Tumor Necrosis Factor (TNF) Receptor-Associated Factor-2 (TRAF2) expression, Apoptosis Signal-Regulating Kinase-1 (Ask1) phosphorylation, c-Jun N-Terminal Kinase (JNK) phosphorylation, and Noxa expression appeared in gefitinib-treated glioma cells. Genetic, pharmacological, and biochemical studies further indicated an active ROS/ER stress/Ask1/JNK/Noxa axis causing the glioma apoptosis induced by gefitinib. The findings suggest that ER-stress-based therapeutic targeting could be a promising option in EGFR inhibitor glioma therapy, and may ultimately achieve a better patient response.

A New Model Applied for Evaluating a Rhenium-diselenium Drug: Breast Cancer Cells Stimulated by Cytokines Induced from Polynuclear Cells by LPS.

New anticancer drugs are usually tested on cancer cells in culture in a standard medium. We stimulated immune polynuclear cells by lipopolysaccharides to obtain an enriched medium (EM) containing inflammatory cytokines more closely reflecting the tumor microenvironment and tested a rhenium-diselenium (Re-diSe) drug in this new model. Concentrations of cytokines were compared with a control medium (CM). Human-derived breast cancer cells were grown in culture either in CM or EM with or without Re-diSe. Assays of tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), intereukin 1 beta (IL1β), transforming growth factor-beta (TGFβ), insulin growth factor 1 (IGF1) and vascular epidermal growth factor A (VEGFA) were performed by enzyme-linked immunosorbent assays. The production of reactive oxygen species (ROS) was determined by 2,7-dichlorofluorescein test. The cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests. Concentrations of TNFα, IL6 and Il1β were observed to be significantly higher in EM than in CM. There was no difference for TGFβ, IGF1 and VEGFA. The cells were sensitive to Re-diSe, with reduced concentrations of TGFβ, IGF1, VEGFA and ROS, but the half-maximal inhibitory concentration was significantly higher in EM than in CM. The efficacy of the Re-diSe drug was confirmed in this model of aggressive cancer.

Electrical Stimulation Activates Fibroblasts through the Elevation of Intracellular Free Ca2+: Potential Mechanism of Pelvic Electrical Stimulation Therapy.

Ca2+ is an important ion in response to electrical stimulation (ES) and acts as second messenger in the regulation of various physiological processes. Pelvic floor electrical stimulation (PES) is a low-voltage clinical application, available for urinary incontinence (UI) treatment. Fibroblasts, as the main cellular component of vaginal wall and pelvic ligament, play an important role in the maintenance of pelvic health. We studied the effect of ES on fibroblasts in this study. ES was conducted with electrotaxis chambers on L929 fibroblast and the ES parameter was 100 mV/mm×2h. The results showed that ES increased intracellular Ca2+ concentration, promoted the expression of PCNA, CyclinB1, and CyclinD1, and increased the proportion of cells in S and G2 phages. After ES, fibroblasts get activated and proliferated. Besides, BAPTA-AM, a membrane permeated chelator for intracellular free Ca2+, partially inhibited the effect of ES on fibroblasts activation and proliferation promotion. Furthermore, we elucidated that Ca2+, as a second messenger and upstream signal for Smads and Akt signaling, regulated ES-induced nuclear translocation of smad2/3, phosphorylation of smad2/3, Akt, and GSK3β. Finally, we validated the effect of ES on PES mouse model. The results indicated that PES promoted the activation and proliferation of fibroblasts in vivo. In conclusion, we verify that ES can elevate the concentration of intracellular Ca2+ and activate its downstream signaling and then promote the activation of fibroblasts, which may be one of the mechanisms of PES therapy.

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

Regulation of islet glucagon secretion: Beyond calcium.

The islet of Langerhans plays a key role in glucose homeostasis through regulated secretion of the hormones insulin and glucagon. Islet research has focused on the insulin-secreting β-cells, even though aberrant glucagon secretion from α-cells also contributes to the aetiology of diabetes. Despite its importance, the mechanisms controlling glucagon secretion remain controversial. Proper α-cell function requires the islet milieu, where β- and δ-cells drive and constrain α-cell dynamics. The response of glucagon to glucose is similar between isolated islets and that measured in vivo, so it appears that the glucose dependence requires only islet-intrinsic factors and not input from blood flow or the nervous system. Elevated intracellular free Ca2+ is needed for α-cell exocytosis, but interpreting Ca2+ data is tricky since it is heterogeneous among α-cells at all physiological glucose levels. Total Ca2+ activity in α-cells increases slightly with glucose, so Ca2+ may serve a permissive, rather than regulatory, role in glucagon secretion. On the other hand, cAMP is a more promising candidate for controlling glucagon secretion and is itself driven by paracrine signalling from β- and δ-cells. Another pathway, juxtacrine signalling through the α-cell EphA receptors, stimulated by β-cell ephrin ligands, leads to a tonic inhibition of glucagon secretion. We discuss potential combinations of Ca2+ , cAMP, paracrine and juxtacrine factors in the regulation of glucagon secretion, focusing on recent data in the literature that might unify the field towards a quantitative understanding of α-cell function.

Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia.

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P< 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.

Effects of Photodynamic Therapy Using Yellow LED-light with Concomitant Hypocrellin B on Apoptotic Signaling in Keloid Fibroblasts

Keloid is a common and refractory disease characterized by abnormal fibroblast proliferation and excessive deposition of extracellular matrix components. Hypocrellin B (HB) is a natural perylene quinone photosensitizer. In this experiment, we studied the effects of photodynamic therapy (PDT) using yellow light from light-emitting diode (LED) combined with HB on keloid fibroblasts (KFB) in vitro. Our results showed that HB-LED PDT treatment induced significant KFB apoptosis and decreased KFB cell viability. HB-LED PDT treatment lead to significant BAX upregulation and BCL-2 downregulation in KFB cells, which led to elevation of intracellular free Ca2+ and activation of caspase-3. Our data provides preliminary evidence for the potential of HB-LED PDT as a therapeutic strategy for keloid.

Relationship between ketamine-induced developmental neurotoxicity and NMDA receptor-mediated calcium influx in neural stem cell-derived neurons

Ketamine, a noncompetitive NMDA receptor antagonist, is used as a general anesthetic and recent data suggest that general anesthetics can cause neuronal damage when exposure occurs during early brain development. To elucidate the underlying mechanisms associated with ketamine-induced neurotoxicity, stem cell-derived models, such as rodent neural stem cells harvested from rat fetuses and/or neural stem cells derived from human induced pluripotent stem cells (iPSC) can be utilized. Prolonged exposure of rodent neural stem cells to clinically-relevant concentrations of ketamine resulted in elevated NMDA receptor levels as indicated by NR1subunit over-expression in neurons. This was associated with enhanced damage in neurons. In contrast, the viability and proliferation rate of undifferentiated neural stem cells were not significantly affected after ketamine exposure. Calcium imaging data indicated that 50μM NMDA did not cause a significant influx of calcium in typical undifferentiated neural stem cells; however, it did produce an immediate elevation of intracellular free Ca2+ [Ca2+]i in differentiated neurons derived from the same neural stem cells.This paper reviews the literature on this subject and previous findings suggest that prolonged exposure of developing neurons to ketamine produces an increase in NMDA receptor expression (compensatory up-regulation) which allows for a higher/toxic influx of calcium into neurons once ketamine is removed from the system, leading to neuronal cell death likely due to elevated reactive oxygen species generation. The absence of functional NMDA receptors in cultured neural stem cells likely explains why clinically-relevant concentrations of ketamine did not affect undifferentiated neural stem cell viability.

SERCA1 overexpression minimizes skeletal muscle damage in dystrophic mouse models

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting secondary to repeated muscle damage and inadequate repair. Elevations in intracellular free Ca²⁺ have been implicated in disease progression, and sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1 (SERCA1) overexpression has been shown to ameliorate the dystrophic phenotype in mdx mice. The purpose of this study was to assess the effects of SERCA1 overexpression in the more severe mdx/Utr(-/-) mouse model of DMD. Mice overexpressing SERCA1 were crossed with mdx/Utr ± mice to generate mdx/Utr(-/-)/+SERCA1 mice and compared with wild-type (WT), WT/+SERCA1, mdx/+SERCA1, and genotype controls. Mice were assessed at ∼12 wk of age for changes in Ca²⁺ handling, muscle mass, quadriceps torque, markers of muscle damage, and response to repeated eccentric contractions. SERCA1-overexpressing mice had a two- to threefold increase in maximal sarcoplasmic reticulum Ca²⁺-ATPase activity compared with WT which was associated with normalization in body mass for both mdx/+SERCA1 and mdx/Utr(-/-)/+SERCA1. Torque deficit in the quadriceps after eccentric injury was 2.7-fold greater in mdx/Utr(-/-) vs. WT mice, but only 1.5-fold greater in mdx/Utr(-/-)/+SERCA1 vs. WT mice, an attenuation of 44%. Markers of muscle damage (% centrally nucleated fibers, necrotic area, and serum creatine kinase levels) were higher in both mdx and mdx/Utr(-/-) vs. WT, and all were attenuated by overexpression of SERCA1. These data indicate that SERCA1 overexpression ameliorates functional impairments and cellular markers of damage in a more severe mouse model of DMD. These findings support targeting intracellular Ca²⁺ control as a therapeutic approach for DMD.

The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells

Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca2+ levels ([Ca2+]i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca2+]i rises which were reduced by removing extracellular Ca2+. Eugenol-induced [Ca2+]i rises were not altered by store-operated Ca2+ channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca2+]i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca2+]i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca2+]i rises by inducing PLC-dependent release of Ca2+ from the endoplasmic reticulum and caused Ca2+ influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.

Nobiletin, a polymethoxy flavonoid, exerts anti-allergic effect by suppressing activation of phosphoinositide 3-kinase

Nobiletin is a polymethoxy flavonoid present in fruit tissues of citrus species and has been reported to suppress degranulation of RBL-2H3 cells. However, the detailed mechanism is still unknown. In this study the mechanism of suppressive effect of nobiletin on degranulation of RBL-2H3 cells was examined. Nobiletin suppressed degranulation of both RBL-2H3 cells and bone marrow-derived mast cells induced by antigen stimulation. Nobiletin also suppressed degranulation stimulated by the calcium ionophore A23187 and thapsigargin, a specific inhibitor of the Ca2+-dependent ATPase. Elevation of intracellular free Ca2+ concentration was also suppressed by nobiletin when RBL-2H3 cells were stimulated by either antigen or A23187. Immunoblot analysis revealed that the inhibitory effect results from down-regulated phosphorylation of both phosphatidylinositol 3-kinase (PI3K) and phospholipase Cγ1 in the signaling pathways related to degranulation stimulated by antigen. Furthermore, oral administration of nobiletin suppressed vascular permeability in passive cutaneous anaphylaxis model mice. The tendency in all experiments obtained by treating with nobiletin was highly similar to that obtained by wortmannin, a specific inhibitor of PI3K. These findings suggest that nobiletin suppresses Ca2+ influx and consequently exerts the anti-allergy activity in a similar way to wortmannin.

A novel chenodeoxycholic acid-verticinone ester induces apoptosis and cell cycle arrest in HepG2 cells

In this study, the in vitro antitumor activity of chenodeoxycholic acid-verticinone ester (CDCA-Ver), a novel compound and its underlying mechanisms were evaluated. Results showed that CDCA-Ver significantly inhibited HepG2 cell viability in a both dose- and time-dependent manner, moreover CDCA-Ver induced apoptotic cell death and G0/G1 cell cycle arrest in HepG2 cells. ROS generation, loss of balance of Bax/Bcl-2 ratio, loss of mitochondrial transmembrane potential, activation of caspases and elevation of intracellular free Ca2+ concentration were involved in the CDCA-Ver induced apoptosis pathway in HepG2 cells. We concluded that CDCA-Ver may be a potential candidate for the therapy of cancer.

Rhodiola rosea Extract Protects Human Cortical Neurons against Glutamate and Hydrogen Peroxide‐induced Cell Death Through Reduction in the Accumulation of Intracellular Calcium

The aim of this study was to investigate the neuroprotective effects of a titolated extract from Rhodiola rosea L. (RrE) and of salidroside (Sa), one of the major biologically active compounds extracted from this medicinal plant, against oxidative stressor hydrogen peroxide (H₂O₂) and glutamate (GLU)-induced cell apoptosis in a human cortical cell line (HCN 1-A) maintained in culture. The results obtained indicate that exposure of differentiated HCN 1-A neurons to GLU or H₂O₂ resulted in concentration-dependent cell death. A 24 h pre-treatment with RrE significantly increased cell survival and significantly prevented the plasma membrane damage and the morphological disruption caused by GLU or H₂O₂, indicating that neurons treated with RrE were protected from the neurotoxicity induced by the oxidative stressor used. In addition, RrE significantly reduced H₂O₂ or GLU-induced elevation of intracellular free Ca²⁺ concentration. The results obtained have also shown that Sa caused similar effects in all experimental models used; however, the potency of the action was lower than that of the extract containing corresponding quantities of Sa. These findings indicate that RrE has a neuroprotective effect in cortical neurons and suggest that the antioxidant activity of the RrE, due to the structural features of the synergic active principles they contain, may be responsible for its ability to stabilize cellular Ca²⁺ homeostasis.

Chronic exposure to high fatty acids impedes receptor agonist-induced nitric oxide production and increments of cytosolic Ca2+ levels in endothelial cells

Dyslipidemia is a common metabolic disorder in diabetes. Nitric oxide (NO) production from endothelium plays the primary role in endothelium-mediated vascular relaxation and other endothelial functions. Therefore, we investigated the effects of elevated free fatty acids (FFA) on the stimulation of NO production by phospholipase C (PLC)-activating receptor agonists (potent physiological endothelium-dependent vasodilators) and defined the possible alterations of signaling pathways implicated in this scenario. Exposure of bovine aortic endothelial cells (BAECs) to high concentrations of a mixture of fatty acids (oleate and palmitate) for 5 or 10 days significantly reduced NO production evoked by receptor agonists (bradykinin or ATP) in a time- and dose-dependent manner. Such defects were not associated with alterations of either endothelial NO synthase mass or inositol phospholipid contents but were probably due to reduced elevations of intracellular free Ca(2)(+) levels ([Ca(2)(+)](i)) under these conditions. Exposure of BAECs to FFA significantly attenuated agonist-induced [Ca(2)(+)](i) increases by up to 54% in a dose- and time-dependent manner. Moreover, bradykinin receptor affinity on the cell surface was significantly decreased by high concentrations of FFA. The morphology of BAECs was altered after 10-day culture with high FFA. Co-culture with protein kinase C (PKC) inhibitors or antioxidants was able to reverse the impairments of receptor agonist-induced NO production and [Ca(2)(+)](i) rises as well as the alteration of receptor affinity in BAECs exposed to FFA. These data indicate that chronic exposure to high FFA reduces NO generation in endothelial cells probably by impairing PLC-mediated Ca(2)(+) signaling pathway through activation of PKC and excess generation of oxidants.

Role of the Serine/Threonine Phosphatases During the Progesterone-Induced Acrosome Reaction and Increase in Intracellular Free Calcium Concentration in Human Sperm.

The acrosome reaction (AR) is an exocytotic event that takes place by fusion of the plasma membrane and the outer acrosomal membrane in sperm previously capacitated. It is widely accepted that progesterone stimulates human sperm AR and that acrosomal exocytosis is accompanied by a transient elevation of intracellular free Ca2+ concentration ([Ca2+]I), which is a consequence of a Ca2+ influx. It is known that during capacitation changes occur in the phosphorylation state of proteins, mediated by protein kinases (PKs) and phosphatase (PPs). However, it is not yet clear whether something similar takes place during the AR. The aim of this work was to study the role of serine/threonine PPs during the progesterone-induced AR and increase of [Ca2+]L, using specific inhibitors. Motile human sperm, free of seminal plasma, were obtained with a Percoll gradient and then incubated in Tyrode medium (2.6% BSA, 37 °C, 5% CO2). After 18 hr, aliquots were treated with different inhibitors of PPs by 15 min. The inhibitors used were 0.1 nM deltamethrin, 10 nM FK-506, and 10 nM cyclosporine A for PP2B; 90 nM endothal and 0.1 nM okadaic acid for PP2A; and 10 nM okadaic acid for PP1. Then, 7 µM progesterone was added and incubated for extra 15 minutes. Acrosomal status and viability were evaluated using PSA-FITC and Hoechst 33258, respectively. To assess the effect of the PPs inhibitors on [Ca2+]I, capacitated sperm were incubated for 30 minutes with 3 µM fura 2AM; after 15 min inhibitors were added. Calcium influx was stimulated with 7 µM progesterone. The fluorescence was monitored with a spectrofluorometer at an excitation wavelength pair of 340/380 nm and emission wavelength of 510 nm. The results indicated that most PPs inhibitors do not affect the induction of the AR by progesterone or the increase in [Ca2+]I. However, two inhibitors of PP2B, FK506 and cyclosporine A, inhibited the progesterone-induced AR but they did not have an effect on the increase of [Ca2+]I. These results suggest that PP2B (calcineurin) may be involved in the exocytosis of the acrosome induced by progesterone but not in the Ca2+ influx that precedes it. The mechanism of this inhibition is still unknown. Fondecyt 1080028. (poster)

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu) on antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells

We found that two distinct flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu), isoquercitrin (Isq) and hyperin (Hyp), are capable of inhibiting antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells. In order to elucidate the underlying mechanisms, we examined effects of Isq and Hyp on cellular responses induced by antigen stimulation. Treatment with both Isq and Hyp markedly inhibited antigen-stimulated elevation of intracellular free Ca2+ concentration and reactive oxygen species (ROS). Isq and Hyp did not affect NADPH oxidase (NOX) activity, but they possessed DPPH radical-scavenging activity similar to that of epigallocatechin gallate, a potent anti-oxidant, Finally, Isq and Hyp showed little or no effects on Ag-stimulated Syk activation or phosphorylation of signalling molecules. These results indicate that inhibition of antigen-stimulated degranulation by Isq and Hyp is mainly due to suppression of intracellular Ca2+ elevation, which is caused by direct scavenging of ROS that are generated by NOX. Our findings suggest that Isq and Hyp, isolated from the peel of persimmon, would be beneficial for alleviating symptoms of type I allergy.

Monitoring the [ATP]/[ADP] Ratio in Beta-Cells During Glucose Stimulated Insulin Secretion Using the Genetically Encoded Fluorescent Reporter Perceval

Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Glucose stimulated insulin secretion depends on glucose metabolism that produces ATP. The resulting increase in [ATP]/[ADP] ratio closes ATP-sensitive potassium (KATP) channels, which leads to membrane depolarization and opening of voltage-dependent Ca2+ channels. This causes an elevation of intracellular free Ca2+ and insulin exocytosis. Insulin is secreted in a pulsatile manner, which is thought to be regulated in part by oscillations in glucose metabolism. Such metabolic oscillations would also lead to oscillations in the [ATP]/[ADP] ratio and hence regulate KATP channel activity.Oscillations in [ATP]/[ADP] ratio have been demonstrated using biochemical and luciferase assays, but neither approach allows measurements of such oscillations in single cells. Perceval is a recently developed fluorescent protein biosensor for [ATP]/[ADP] ratio, and it permits direct measurement of [ATP]/[ADP] ratios inside living cells. We use Perceval in combination with quantitative confocal and two-photon excitation microscopy for direct measurement of the [ATP]/[ADP] ratio in beta-cells during glucose stimulated insulin secretion. For this purpose we have developed an adenoviral vector to express Perceval specifically in the beta-cells of intact mouse islets. Dynamic changes in [ATP]/[ADP] ratio can be correlated with glucose metabolism (by simultaneous imaging of Perceval fluorescence and NAD(P)H autofluorescence) and with intracellular free Ca2+ levels (by simultaneous imaging of Perceval fluorescence and the calcium sensor, FuraRed). This data allows us to test hypotheses regarding the role of localized subcellular signaling complexes and putative microdomains of glucose metabolism, [ATP]/[ADP] ratio, and Ca2+ dynamics in the regulation of glucose stimulated insulin secretion.

Expression and functionality of proteinase-activated receptors (PARs) on primary human lung mast cells

Introduction: Mast cell (MC) activation is known to promote anaphylactic reactions as well as the development of allergic and non-allergic inflammatory diseases. Apart from reacting to cross-linking of IgE-receptors by allergen, MC can be activated through a variety of alternative mechanisms. There is initial evidence that also proteinase-activated receptors (PARs) could have a role. PARs belong to the G protein-coupled receptor family and comprise four members (PAR-1 to 4). While the expression of functional PAR-2 on human skin MC as well as mRNA expression for PAR-1, PAR-3 and PAR-4 has been reported recently, so far nothing is known about the expression and the potential function(s) of PARs on primary human lung MC. We, therefore, isolated primary MC from human lung tissue and examined the expression and functionality of PARs. Methods: Human primary lung MC were isolated by a three-step procedure including enzymatic lung tissue digestion, density gradient centrifugation, and immunoaffinity purification by using anti-CD117 and anti-CD203c antibodies. Results: Using &gt;95% pure MC we could show mRNA expression for PAR-1, PAR-2, PAR-3 and PAR-4. PAR-specific antibodies revealed the expression of PAR-1 on the cell membrane and of PAR-2 being located intracellularly, whereas PAR-3 and PAR-4 were not detected by the antibodies used. PAR-activating peptides (PAR-APs) selective for PAR-1, PAR-2 and PAR-4 were able to induce elevation of intracellular free Ca2+-ion concentration ([Ca2+]i) in these cells. Moreover, PAR-1-AP and PAR-4-AP, but not PAR-2-AP stimulated MC degranulation as determined by the release of β-hexosaminidase. Stimulation with PAR-1-selective ligands led to downregulation of PAR-1. Looking for the effect of natural PAR-ligands, we tested proteinases from blood (thrombin, factor Xa, plasmin), from leukocytes (elastase, cathepsin G, proteinase 3), trypsin, and also MC-specific proteinases (chymase, tryptase). Out of these, only thrombin (PAR-1, and -4-ligand) and trypsin (PAR-2-ligand) induced Ca2+-signalling whereas none induced degranulation. Discussion: Our results demonstrate the functional expression of PAR-1, PAR-2 and PAR-4 in primary human lung MC. However, the induction of Ca2+-signalling in the absence of a degranulation response to thrombin and trypsin suggests, that natural ligands for these receptors may act differently from synthetic APs and moreover are likely to stimulate alternative functions in human primary lung MC.

Evidence for calcium-mediated perception of plant symbiotic signals in aequorin-expressing Mesorhizobium loti

BackgroundDuring the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca2+ changes are an integral part of the signalling mechanism. In rhizobia it is not yet known whether Ca2+ can act as a transducer of symbiotic signals.ResultsA plasmid encoding the bioluminescent Ca2+ probe aequorin was introduced into Mesorhizobium loti USDA 3147T strain to investigate whether a Ca2+ response is activated in rhizobia upon perception of plant root exudates. We find that M. loti cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca2+ concentration. Only root exudates from the homologous host Lotus japonicus induce Ca2+ signalling and downstream activation of nodulation genes. The extracellular Ca2+ chelator EGTA inhibits both transient intracellular Ca2+ increase and inducible nod gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible.ConclusionThese findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis.