Fluo-4 AM Research Articles

237 Diethylstilbestrol Activates Catsper and Disturbs Progesterone Actions in Human Sperm

Is the prototypical endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), able to evoke physiological changes in human spermatozoa and affects progesterone actions? DES promoted Ca2+ flux into human sperm by activating CatSper channel and suppressed progesterone-induced Ca2+ signaling, protein tyrosine phosphorylation and human sperm functions. Human sperm intracellular calcium concentrations ([Ca2+]i) were monitored with a plate reader following sperm loading with Ca2+ indicator Fluo-4 AM and further whole-cell patch-clamp technique was performed to record CatSper currents. Sperm viability and motility parameters were assessed by eosin-nigrosin staining kit and a computer-assisted semen analysis (CASA) system, respectively. The ability of sperm to penetrate into viscous media was detected by 1% methylcellulose. Sperm acrosome reaction was measured using chlortetracycline (CTC) staining. The level of protein tyrosine phosphorylation was determine by western blot analysis. DES rapidly increased human sperm [Ca2+]i even at environmentally relevant concentrations (100 pM). The elevation of [Ca2+]i was derived from extracellular Ca2+ influx and can be significantly inhibited by the CatSper blocker mibefradil, suggesting CatSper participate in this process. In fact, whole-cell patch-clamp recordings confirmed that DES could increase CatSper currents. DES did not affect sperm viability, motility, penetrate into viscous media, protein tyrosine phosphorylation and the acrosome reaction. However, it suppressed progesterone-stimulated Ca2+ signaling and tyrosine phosphorylation. Consequently, DES (1-100 μM) significantly inhibited progesterone-induced human sperm penetration into viscous medium and acrosome reaction.

Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow.

BackgroundBinding of P-selectin to P-selectin glycoprotein ligand-1 (PSGL-1) makes neutrophils roll on and adhere to inflammatory site. Intracellular calcium bursting of adhered neutrophils is a key event for subsequent arresting firmly at and migrating into the injured tissue. But, it remains unclear how the cytoplasmic calcium signaling of the cells were modulated by the fluid shear stress. Here, we focus on mechanical regulation of P-selectin-induced calcium signaling of neutrophil-like HL-60 cells under flow.MethodsHL-60 cells were loaded with Fluo-4 AM for fluorescent detection of intracellular calcium ion, and then perfused over P-selectin-coated bottom of parallel-plate flow chamber. The intracellular calcium concentration of firmly adhered cell under flow was observed in real time by fluorescence microscopy.ResultsForce triggered, enhanced and quickened cytoplasmic calcium bursting of HL-60 on P-selectin. This force-dependent calcium signaling was induced by the immobilized P-selectin coated on substrates in absence of chemokine. Increasing of both shear stress and P-selectin concentration made the calcium signaling intensive, through quickening the cytosolic calcium release and upregulating both probability and peak level of calcium signaling.ConclusionsImmobilized P-selectin-induced calcium signaling of HL-60 cells is P-selectin concentration- and mechanical force-dependent. The higher both the P-selectin concentration and the external force on cell, the more intensive the calcium signaling. It might provide a novel insight into the mechano-chemical regulation mechanism for intracellular signaling pathways induced by adhesion molecules.

Xanthohumol Modulates Calcium Signaling in Rat Ventricular Myocytes: Possible Antiarrhythmic Properties.

Cardiac arrhythmia is a major cause of mortality in cardiovascular pathologies. A host of drugs targeted to sarcolemmal Na+, Ca2+, and K+ channels has had limited success clinically. Recently, Ca2+ signaling has been target of pharmacotherapy based on finding that leaky ryanodine receptors elevate local Ca2+ concentrations causing membrane depolarizations that trigger arrhythmias. In this study, we report that xanthohumol, an antioxidant extracted from hops showing therapeutic effects in other pathologies, suppresses aberrant ryanodine receptor Ca2+ release. The effects of xanthohumol (5-1000 nM) on Ca2+ signaling pathways were probed in isolated rat ventricular myocytes incubated with Fluo-4 AM using the perforated patch-clamp technique. We found that 5-50 nM xanthohumol reduced the frequency of spontaneously occurring Ca2+ sparks (>threefold) and Ca2+ waves in control myocytes and in cells subjected to Ca2+ overload caused by the following: 1) exposure to low K+ solutions, 2) periods of high frequency electrical stimulation, 3) exposures to isoproterenol, or 4) caffeine. At room temperatures, 50-100 nM xanthohumol reduced the rate of relaxation of electrically- or caffeine-triggered Ca2+transients, without suppressing ICa, but this effect was small and reversed by isoproterenol at physiologic temperatures. Xanthohumol also suppressed the Ca2+ content of the SR and its rate of recirculation. The stabilizing effects of xanthohumol on the frequency of spontaneously triggered Ca2+ sparks and waves combined with its antioxidant properties, and lack of significant effects on Na+ and Ca2+ channels, may provide this compound with clinically desirable antiarrhythmic properties.

Effect of nitric oxide donor SNAP on GABA release from rat brain nerve terminals.

In this work we investigated the effect of nanomolar concentrations of nitric oxide on the release of gamma-aminobutyric acid (GABA) from rat brain nerve terminals using a radioisotope method with [3H]GABA and a spectrofluorimetric method with Ca2+-sensitive probe Fluo-4 AM. It was shown that in the presen­ce of dithiothreitol (DTT), nitric oxide donor SNAP at concentration, in which it produces NO in the nanomolar range, caused Ca2+-independent [3H]GABA release from nerve terminals. The applications of 4-aminopyridine (4-AP) and nipecotic acid (NA), as the inducers of GABA release from vesicular and cytoplasmic pools, showed that the maximum of SNAP/+DTT-induced [3H]GABA release was registered at 10th min of incubation and coincided in time with significant increase (almost double) in NA-induced [3H]GABA release. At this time point, 4-AP-induced release of [3H]GABA was drastically reduced. At the 15th min of incubation of nerve terminals with SNAP/+DTT, the opposite picture was observed: the decrease in NA- and increase in 4-AP-induced [3H]GABA release. Thus, nitric oxide in the form of S-nitrosothiols at nanomolar concentrations causes Ca2+-independent GABA leakage from synaptic vesicles into cytosol with subsequent release from nerve terminals. The reuptake of the neurotransmitter and its re-accumulation in synaptic vesicles occur later.

Калікс[4]арени як модулятори енергозалежної акумуляції Са2+ та функціонування електронно-транспортного ланцюга в мітохондріях гладенького м’яза

The influence of supramolecular macrocyclic compounds calix[4]arenes (C-97, C-99, C-107) at a concentration of 100 nM in the process of energy-dependent Ca²⁺-transport in isolated mitochondria of smooth muscle, as well as autofluorescence mitochondrial coenzyme NADH, FAD and hydrodynamic diameter of these organelles was investigated. Using Ca²⁺-sensitive fluorescent dye Fluo-4 AM it was shown that the selected calix[4]arenes can suppress energy-dependent accumulation of Ca²⁺ by mitochondria. Accumulation of Ca²⁺ (80 jiM in the medium) accompanied by the growth of the fluorescent probe response from a conventional unit to a value of 1,57±0,04 (n=5). Calix[4]arenes C-97, C-99, C-107 falls fluorescent signal below the 0,88±0,08, 0,92±0,08 and 0,78±0,04 respectively. Thus, the selected calix[4]arenas lead to release of previously accumulated Ca2+ from mitochondria. Under the influence of C-97 and C-99 fluorescent signal from NADH reduced to -0,11±0,02 and -0,12±0,02, respectively, in relation to the reference value - -0,05±0,01 (n=5). Analysis of fluorescence response NADH and FAD in a suspension of isolated mitochondria suggests that the effects of test compounds on the functional activity of the electron transport chain is associated with the initial stimulation of its 1-th complex and subsequent inhibition of Ca²⁺-dependent NAD- containing Krebs cycle dehydrogenases. Along with this, the use of photon correlation spectroscopy to assess changes in the volume of mitochondria (their hydrodynamic diameter) under the action of selected calix[4]arenes has shown that interference with the electron transport chain leads to changes in the osmotic balance between the matrix of the mitochondria and the external environment. The result is the growth of isolated organelles volume. In particular, the hydrodynamic diameter of mitochondria increased by 22±6 % and 34±8 % (n=5) in presence of C-97 or C-99. The conclusion was done about the advisability of further studies of the calyx[4]arenes effect on smooth muscle Ca²⁺-homeostase and mitochondrial bioenergetics in order to find effective modifiers of their func- tional activity.

Ionomycin-induced calcium influx induces neurite degeneration in mouse neuroblastoma cells: analysis of a time-lapse live cell imaging system

Reactive oxygen species induce neuronal cell death. However, the detailed mechanisms of cell death have not yet been elucidated. Previously, we reported neurite degeneration before the induction of cell death. Here, we attempted to elucidate the mechanisms of neurite degeneration before the induction of cell death using the neuroblastoma N1E-115 cell line and a time-lapse live cell imaging system. Treatment with the calcium ionophore ionomycin induced cell death and neurite degeneration in a concentration- and time-dependent manner. Treatment with a low concentration of ionomycin immediately produced a significant calcium influx into the intracellular region in N1E-115 cells. After 1-h incubation with ionomycin, the fluorescence emission of MitoSOXTM increased significantly compared to the control. Finally, analysis using a new mitochondrial specific fluorescence dye, MitoPeDPP, indicated that treatment with ionomycin significantly increased the mitochondrial lipid hydroperoxide production in N1E-115 cells. The fluorescence emissions of Fluo-4 AM and MitoPeDPP were detected in the cell soma and neurite regions in ionomycin-treated N1E-115 cells. However, the emissions of neurites were much lower than those of the cell soma. TBARS values of ionomycin-treated cells significantly increased compared to the control. These results indicate that ionomycin induces calcium influx into the intracellular region and reactive oxygen species production in N1E-115 cells. Lipid hydroperoxide production was induced in ionomycin-treated N1E-115 cells. Calcium influx into the intracellular region is a possible activator of neurite degeneration.

Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

The possible association of intracellular Ca2+ with metastasis in human cancer cells is poorly understood. We have studied Ca2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca2+ was measured using a membrane-permeant fluorescent Ca2+-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca2+ level could be induced by applying a high-K+ solution. Various parameters of the oscillations depended on extracellular Ca2+ and voltage-gated Na+ channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na+ channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca2+ oscillations. It is concluded that the functional voltage-gated Na+ channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca2+ activity. Possible mechanisms and consequences of the Ca2+ oscillations are discussed.

Abstract 3519: Paroxetine induces apoptosis in human breast cancer MCF-7 cells through mitochondrial dysfunction and modulation of K+ channels

Abstract Purpose: Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of patients with depression. Approximately 60% of women with breast cancer suffer from depression. This study was performed to investigate the effect of SSRIs on cell viability of human breast cancer MCF-7 cells. Methods: Cell viability, ROS generation, and Ca2+ increase were quantified using MTT, H2DCFDA, and Fluo-3 AM. Apoptotic signaling pathways were analyzed by immuno-blotting and -staining. Plasma and mitochondrial membrane potentials were determined by DiO PMP and JC-1 dyes, respectively. K+ channel activity was analyzed by whole-cell and single-channel recordings. Results: Of the antidepressants tested in this study, paroxetine most reduced the viability of MCF-7 cells. Paroxetine (10 and 30 μM) treatment resulted in a significant increase in the number of apoptotic cells, an increase in Bax/Bcl-2 ratio, an activation of caspase-8, 9, and PARP, and a release of cytochrome c from mitochondria. In addition, paroxetine markedly increased ROS generation and intracellular Ca2+ levels. Moreover, paroxetine blocked K+ channels, such as large conductance Ca2+-activated K+ channel and TASK-3 channel, which are present in plasma membrane and mitochondria. The paroxetine-induced apoptosis was reduced by antioxidants, in particular NAC. Combination with SB202190 (a p38 MAPK inhibitor) prevented cells from undergoing apoptosis. Conclusion: Our findings show that apoptotic effect of paroxetine is mediated by mitochondrial dysfunction and modulation of K+ channels in MCF-7 cells. We suggest that paroxetine could be an agent for anti-tumor therapy in breast cancer. Citation Format: Kee R. Kang, Young-Woo Cho, Ji Hyun Ryu, Eun-Jin Kim, Jaehee Han, Dawon Kang. Paroxetine induces apoptosis in human breast cancer MCF-7 cells through mitochondrial dysfunction and modulation of K+ channels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3519.

Effects of taurochenodeoxycholic acid on Ca2+/CaM signalling mediated by the TGR5 signalling pathway.

Taurochenodeoxycholic acid (TCDCA), a natural bioactive substance in animal bile, has anti-inflammatory and immunoregulatory effects. This study evaluates the effects of TCDCA on calcium/calmodulin (Ca2+/CaM) signalling mediated by G Protein Coupled Bile Acid Receptor 1 (TGR5) to provide preliminary information on the mechanism of TCDCA in immune regulation and also to benefit future research. After treatment of NR8383 and high TGR5 expression cell (TGR5-NR8383) with TCDCA (10-6 mol/L, 10-5 mol/L, and 10-4 mol/L) for 1 h, we measured TGR5 and CaM gene and protein levels by quantitative reverse transcription-polymerase chain reaction (qPCR) and western blotting, respectively. The inositol triphosphate (IP3) concentration was measured by Enzyme-linked Immunosorbent Assay (ELISA), and the Ca2+ concentration was measured by calcium fluorescent probe (Fluo-3 AM). The present study showed that the expression levels of IP3, Ca2+, and CaM in NR8383 cells were increased by TCDCA at concentrations ranging from 10-6 mol/L to 10-4 mol/L. TCDCA (10-4 mol/L) increased both the gene and protein expression of IP3and CaM through TGR5. TCDCA (10-4 mol/L or 10-5 mol/L) also increased the Ca2+ concentration via the TGR5 receptor. Our data suggest TCDCA activates Ca2+/CaM signalling via the TGR5 signalling pathway.

Lidocaine inhibits the invasion and migration of TRPV6-expressing cancer cells by TRPV6 downregulation.

It is well known that local anesthetics have a broad spectrum of pharmacological actions, acting as nerve blocks, and treating pain and cardiac arrhythmias via blocking of the sodium channel. The use of local anesthetics could reduce the possibility of cancer metastasis and recurrence following surgical tumor excision. The purpose of the present study was to investigate the inhibitory effect of lidocaine upon the invasion and migration of transient receptor potential cation channel subfamily V member 6 (TRPV6)-expressing cancer cells. Human breast cancer MDA-MB-231 cells, prostatic cancer PC-3 cells and ovarian cancer ES-2 cells were treated with lidocaine. Cell viability was quantitatively determined by MTT assay. The migration of the cells was evaluated using the wound healing assay, and the invasion of the cells was assessed using a Transwell assay. Calcium (Ca2+) measurements were performed using a Fluo-3 AM fluorescence kit. The expression of TRPV6 mRNA and protein in the cells was determined by quantitative-polymerase chain reaction and western blot analysis, respectively. The results suggested that lidocaine inhibits the cell invasion and migration of MDA-MB-231, PC-3 and ES-2 cells at lower than clinical concentrations. The inhibitory effect of lidocaine on TRPV6-expressing cancer cells was associated with a reduced rate of calcium influx, and could occur partly as a result of the downregulation of TRPV6 expression. The use of appropriate local anesthetics may confer potential benefits in clinical practice for the treatment of patients with TRPV6-expressing cancer.

Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

LicochalconeA (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER)stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamineB assay. Apoptosis was detected by flow cytometry with AnnexinV/propidium iodide staining, and by fluorescent microscopy with Hoechst33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be considered an important mechanism by which LCA induces apoptosis of T24 bladder cancer cells.

Dependence of damage within 10 min to myocardial cells by a photodynamic reaction with a high concentration of talaporfin sodium outside cells in vitro on parameters of laser irradiation

BackgroundTo investigate the immediate occurrence of irreparable severe damage to myocardial cells up to 10min after a photodynamic reaction with a high concentration of photosensitizer outside cells, we measured the damage response time and the parameters that govern the response time via rat myocardial Ca2+ concentration. In our proposed method for catheter ablation of tachyarrhythmia by photodynamic reaction, there are two components to the electrical conduction block: an immediate electrical conduction block of several tens of seconds to several minutes, and a permanent electrical conduction block. MethodsRat myocardial intracellular Ca2+ concentration changes before, during and after the photodynamic reaction with a high concentration of photosensitizer outside myocardial cells were continuously observed using a Fluo-4 AM Ca2+ probe. Talaporfin sodium with 663-nm excitation was used to induce the photodynamic reaction. Talaporfin concentration was 10–30μg/ml, radiant exposure was 10–40J/cm2, and irradiance was 30–290mW/cm2. We evaluated the response time of irreparable severe damage to myocardial cells, according to Ca2+ concentration. ResultsThe response time of the defined severe damage occurrence to myocardial cells ranged from 200 to 500s. The response time decreased with increasing irradiance and photosensitizer concentration, but exhibited no significant change with total radiant exposure. ConclusionsWe found that severe myocardial cell damage caused by a photodynamic reaction with a high concentration of photosensitizer outside cells occurred within a few minutes, which might be useful for catheter ablation for tachyarrhythmia that needs immediate response during the ablation procedure.

Organic two-photon nanoparticles modulate reactive oxygen species, intracellular calcium concentration, and mitochondrial membrane potential during apoptosis of human gastric carcinoma SGC-7901 cells

To investigate the biocompatibility of human gastric carcinoma cells (SGC-7901) with organic two-photon nanoparticles (NPs). Different concentrations of NPs were incubated with SGC-7901 cells for different times. The levels of cell apoptosis, reactive oxygen species (ROS), intracellular calcium, and mitochondrial membrane potential (MMP) were measured by staining the SGC-7901 cells with Annexin V-FITC/PI, 2',7'-dichlorofluorescin diacetate, Fluo-3 AM, and Rhodamine 123, followed by the flow cytometry assay. NPs at <4µg/ml, did not have any significant effect on apoptosis, necrosis, generation of ROS, increase of intracellular Ca(2+) concentration or decrease of MMP in SGC-7901 cells, but >4µg/ml had a major effects on all the above mentioned parameters. 2,5,2',5'-Tetra(4-N,N-diphenylamine styryl) biphenyl NPs can be used at an appropriate concentration as a safe drug carrier or imaging marker and may serve as an effective tool for developing a photodynamic cancer therapy.

Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result.

OC-08 - Multiple functional defects in platelets from thrombocytopenic cancer patients undergoing chemotherapy

Severe thrombocytopenia (≤50×10(9) platelets/L) is often the consequence of hematological malignancies and intensive chemotherapy. The risk of clinically significant bleeding is increased in these patients, despite the use of prophylactic platelet transfusions. The fact that there is no clear correlation between the platelet count and the risk of hemorrhage, suggests that there are other contributing factors. The contribution of impairments in platelet and coagulant function remains poorly understood. In patients with chemotherapy-induced thrombocytopenia due to hematological malignancies, we evaluate platelet and coagulant functions and determine the effects of platelet transfusion. Ultimately, we can identify specific hemostatic factors that aid in the prediction of bleeding. In total 58 patients were included and blood was collected before and, if indicated (≤10×10(9) platelets/L), 1 hour after transfusion with platelet concentrate. Platelet function was assessed using flow cytometry by determining: 1) integrin αIIbβ3 activation (PAC-1 antibody), 2) P-selectin expression (anti-P-selectin antibody), 3) phosphatidylserine exposure (Annexin-V) and 4) intracellular calcium (Fluo-4 AM). Factor levels were determined in plasma. Thrombus and fibrin formation was assessed by perfusion of whole blood over a collagen-tissue factor surface at a shear rate of 1,000 s-1. Platelets from the thrombocytopenic patients before transfusion showed markedly reduced integrin αIIbβ3 activation and P-selectin expression in response to thrombin, collagen-related peptide and ADP, compared to healthy donor platelets. Also, agonist-induced intracellular calcium fluxes were greatly reduced. However, calcium fluxes with thapsigargin, a SERCA pump inhibitor, were similar in patient and control platelets, suggesting a normal calcium store content in the patient platelets. Furthermore, phosphatidylserine exposure was increased in unstimulated patient platelets compared to control platelets (8.2 vs. 1.8%, p<0.0001). Coagulation factor levels were within the normal range, with the exception of von Willebrand factor and fibrinogen levels, which were elevated. Platelet transfusion partly recovered the platelet integrin αIIbβ3 activation and P-selectin expression induced by agonists. Platelet deposition (6.7 vs. 1.7%, p<0.0001) and fibrin formation (7.6 vs. 0.9%, p=0.0005) under flow conditions were substantially improved after platelet transfusion. Platelets from cancer patients undergoing chemotherapy appear to display impaired functional responses to activating stimuli. Platelet transfusion partly restores these functional defects, resulting in improved thrombus and fibrin formation.

Altered cellular localization and hemichannel activities of KID syndrome associated connexin26 I30N and D50Y mutations.

BackgroundGap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells.ResultsImmunostaining experiments showed that Cx26I30N and D50Y failed to form gap junction plaques at cell-cell contact sites. Further, these mutations resulted in the retention of Cx26 protein in the Golgi apparatus. Examination of hemichannel function by fluorescent dye uptake assays revealed that cells with Cx26I30N and D50Y mutations had increased dye uptake compared to Cx26WT (wild-type) containing cells, indicating abnormal hemichannel activities. Cells with mutant proteins had elevated intracellular calcium levels compared to Cx26WT transfected cells, which were abolished by a hemichannel blocker, carbenoxolone (CBX), as measured by Fluo-3 AM loading and flow cytometry.ConclusionsHere, we demonstrated that Cx26I30N and D50Y mutations resulted in the formation of aberrant hemichannels that might result in elevated intracellular calcium levels, a process which may contribute to the hyperproliferative epidermal phenotypes of KID syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0081-0) contains supplementary material, which is available to authorized users.

Using Action Potential Clamp Data to Determine the Calcium Fluxes and Contributions in Excitation-Contraction Coupling in Vivo in Cardiomyocytes

Aim: The significance of the different calcium influx pathways like L-type Ca2+-channels (LTCC), reverse Na+/Ca2+-exchange (NCXrev) or Ca2+-induced Ca2+-release (CICR) varies with species and the state of a cell. For instance, in case of heart failure, CICR decreases and influx via NCXrev increases. The aim of this study is to develop a method to quantify calcium fluxes in cardiac excitation-contraction coupling under physiological conditions.Methods: We used action potential clamp to measure ILTCC and INCX currents in rainbow trout ventricular myocytes. Fluorescence changes induced by Ca2+-transients were recorded using 1μM Fluo-4 AM. To measure ILTCC and/or INCX, we used 10μM nifedipine and 1 or 5mM NiCl2 in extracellular solution. Ryanodine receptors and SR calcium ATPase were inhibited by incubating cells in the presence of 10μM ryanodine and 2μM thapsigargin. We used 240μg/ml amphotericin B in pipette solution for the perforated-patch clamp configuration. Also, we composed a simplified mathematical model of Ca2+-dynamics. By fitting model solution to the recorded experimental data, we determined contribution of LTCC, NCX and CICR currents in trout.Results: From experiments where CICR was inhibited we determined the calcium buffering capacity in the cells that is 55μM (+/- 8 μM). With this method we quantify the contribution and kinetics of LTCC (10-20%), NCX (20-40%) and CICR (50-70%).Conclusions: We have developed a method to quantify calcium fluxes under physiological conditions in cardiomyocytes. Furthermore, we found that SR plays a significant role in trout EC coupling. This is in sharp contrast with previous estimates. Also, the method is not specific to trout and is easily applicable to different species.

Postnatal Development of T-Tubules in Sheep Atrial Myocytes

Background: Cardiac t-tubules are essential in ensuring systolic calcium rises synchronously to enable contraction. The t-tubule network is absent or very sparse at birth in the ventricles of small mammals and develops postnatally with subsequent changes in calcium handling. Nothing is currently known about the development of t-tubules in the atria.Methods and Results: Newborn, 1 month, 3 month, 6 month and adult sheep (∼18 months) were euthanased and cells isolated from the left atrial appendage. T-tubule density assessed using di-4-ANEPPS and confocal microscopy showed partially developed t-tubules were present at birth in sheep atrial myocytes but at 30±9% of adult density (p<0.001). T-tubule density increased at 1 month and by 3 months t-tubules appeared fully developed despite decreased cell width compared to adult (p<0.01). In current clamp experiments, performed at 37°C with fluo-3 AM loaded cells, APD10, 25 and 50 were shorter in newborn, 1 month and 3 months compared to adult (p<0.01). There was no difference in calcium transient amplitude between newborn, 1 month and 3 month cells but the rate of decay of systolic calcium was slower in newborn compared to both 1 month and 3 months (p<0.05).Conclusion: T-tubules are present at birth in sheep atrial myocytes and develop over the first 3 months of life. Both action potential duration and rate of calcium transient decay increase during development. We hypothesise that this may be due to increased L-type calcium current as t-tubules develop and increased SERCA function as the sarcoplasmic reticulum matures. Further investigation is required to determine how the expression of t-tubule regulatory proteins and activity of calcium handling proteins changes throughout development.

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Epigallocatechin-3-Gallate Attenuates the Effects of TNF-α in Vascular Endothelial Cells by Causing Ectodomain Shedding of TNF Receptor 1

Background/Aims: We investigated the mechanism underlying anti-tumor necrosis factor-α (TNF-α) effects of epigallocatechin-3-gallate (EGCG) in human aortic endothelial cells. Methods: Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Cytosolic Ca2+ was measured using Fluo-4 AM. A disintegrin and metalloprotease 10 (ADAM10) was localized by immunofluorescence staining. Results: EGCG caused ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced endothelin-1 (ET-1) expression. EGCG-induced TNFR1 ectodomain shedding was prevented by BAPTA-AM (intracellular Ca2+ chelator), but not by the absence of extracellular Ca2+. In physiologic extracellular Ca2+ concentration, EGCG markedly increased cytosolic Ca2+. Even in the absence of extracellular Ca2+, EGCG raised cytosolic Ca2+, though less potently. siRNA depletion of ADAM10 prevented EGCG-induced ectodomain shedding of TNFR1 and also diminished the inhibitory effect of EGCG on TNF-α-induced ET-1 expression. EGCG caused translocation of ADAM10 to the plasma membrane, and this effect was prevented by BAPTA-AM. Besides extracellular Ca2+ influx, release of intracellular stored Ca2+ caused ADAM10-dependent ectodomain shedding of TNFR1. Conclusion: EGCG decreases the responsiveness of cells to TNF-α by causing ADAM10-dependent ectodomain shedding of TNFR1. This effect was attributed to its property to increase cytosolic Ca2+ through both extracellular Ca2+ influx and release of stored Ca2+.