Fluo-3 Fluorescence Research Articles

Omega-3 fatty acids improve flow-induced vasodilation by enhancing TRPV4 in arteries from diet-induced obese mice.

Previous studies have shown the intake of omega-3 polyunsaturated fatty acids is associated with low rates of obesity and ischaemic pathologies. Omega-3 also have anti-inflammatory and plaque-stabilization effects and regulate vasodilation and constriction. However, there are few studies of the role of omega-3 in flow-induced vasodilation involving Ca2+-permeable ion channel TRPV4 in high-fat diet-induced obese (DIO) mouse. Here, we determined whether omega-3 protect against vascular dysfunction induced by a high-fat diet by enhancing TRPV4 activity and subsequently improving flow-mediated vasodilation. Flow-mediated vasodilation in second-order mesenteric arteries from mice was measured using a pressure myograph. The intracellular Ca2+ concentration in response to flow and GSK1016790A (a TRPV4 agonist) was measured by Fluo-4 fluorescence. Whole-cell current was measured by patch clamp. Cell membrane tether force was measured by atomic force microscopy. Impairment of flow-mediated vasodilation in arteries and Ca2+ influx in endothelial cells from DIO mice was restored by omega-3 treatment. The improved flow-induced vasodilation was inhibited by the TRPV4 antagonist HC067047 and in TRPV4-/- mice. Omega-3 treatment enhanced endothelial TRPV4 activity and altered cell membrane mechanic property, as indicated by enhanced GSK1016790A-induced Ca2+ influx and whole-cell current and altered membrane mean tether force in endothelial cells from DIO mice. Omega-3 improve vascular function by improving flow-induced vasodilation via enhancing TRPV4 activity in the endothelium of obese mice which may be related to improved cell membrane physical property. Activation of TRPV4 in endothelium plays an important role in the protective mechanisms of omega-3 against vascular dysfunction in obesity by improving flow-mediated vasodilation.

Adapting techniques for calcium imaging in muscles of adult Brugia malayi.

Brugia malayi is a human filarial nematode parasite that causes lymphatic filariasis or 'elephantiasis' a disfiguring neglected tropical disease. This parasite is a more tractable nematode parasite for the experimental study of anthelmintic drugs and has been studied with patch-clamp and RNAi techniques. Unlike in C. elegans however, calcium signaling in B. malayi or other nematode parasites has not been achieved, limiting the studies of the mode of action of anthelmintic drugs. We describe here the development of calcium imaging methods that allow us to characterize changes in cellular calcium in the muscles of B. malayi. This is a powerful technique that can help in elucidating the mode of action of selected anthelmintics. We developed two approaches that allow the recording of calcium signals in the muscles of adult B. malayi: (a) soaking the muscles with Fluo-3AM, promoting large-scale imaging of multiple cells simultaneously and, (b) direct insertion of Fluo-3 using microinjection, providing the possibility of performing dual calcium and electrophysiological recordings. Here, we describe the techniques used to optimize dye entry into the muscle cells and demonstrate that detectable increases in Fluo-3 fluorescence to elevated calcium concentrations can be achieved in B. malayi using both techniques.

Direct 3D bioprinting of cardiac micro-tissues mimicking native myocardium

The heart possesses a complex three-dimensional (3D) laminar myofiber organization; however, because engineering physiologically relevant 3D tissues remains a technical challenge, the effects of cardiomyocyte alignment on excitation-contraction coupling, shortening and force development have not been systematically studied. Cellular shape and orientations in 3D can be controlled by engineering scaffold microstructures and encapsulating cells near these geometric cues. Here, we show that a novel method of cell encapsulation in 3D methacrylated gelatin (GelMA) scaffolds patterned via Microscale Continuous Optical Printing (μCOP) can rapidly micropattern neonatal mouse ventricular cardiomyocytes (NMVCMs) in photocrosslinkable hydrogels. Encapsulated cardiomyocytes preferentially align with the engineered microarchitecture and can display morphology and myofibril alignment phenotypic of myocardium in vivo. Utilizing the μCOP system, an asymmetric, multi-material, cantilever-based scaffold was directly printed, so that the force produced by the microtissue was transmitted onto a single deformable pillar. Aligned 3D encapsulated NMVCM scaffolds produced nearly 2 times the force compared to aligned 2D seeded samples. To further highlight the flexibility of μCOP, NMVCMs were encapsulated in several patterns to compare the effects of varying degrees of alignment on tissue displacement and synchronicity. Well aligned myofiber cultured patterns generated 4–10 times the contractile force of less anisotropically patterned constructs. Finally, normalized fluo-4 fluorescence of NMVCM-encapsulated structures showed characteristic calcium transient waveforms that increased in magnitude and rate of decline during treatment with 100 nM isoproterenol. This novel instrumented 3D cardiac microtissue serves as a physiologically relevant in vitro model system with great potential for use in cardiac disease modeling and drug screening.

Thiopental Elevates Steady-State Levels of Intracellular Ca2+ and Zn2+ in Rat Thymic Lymphocytes

Thiopental is an ultra-short-acting barbiturate and has been used commonly in the induction phase of general anesthesia. However, the toxic effect of thiopental is not completely clear. The effect of thiopental on intracellular Ca2+ ([Ca2+]i) levels was investigated in non-excitable cells. Experiments were carried out using a flow-cytometric technique, rat thymic lymphocytes (as non-excitable cells), and appropriate fluorescent probes. Treatment of cells with 300 µM thiopental increased Fluo-3 fluorescence intensity, indicating elevation of [Ca2+]i. This increase was partially attenuated by a chelator of intracellular Zn2+. Thus, thiopental elevated both [Ca2+]i and intracellular Zn2+ ([Zn2+]i) levels. Under intracellular Zn2+-free conditions, 100–300 µM thiopental was still able to induce a statistically significant increase in [Ca2+]i, whereas removal of extracellular Ca2+ greatly reduced the increase in [Ca2+]i induced by this dose of thiopental. Therefore, the thiopental-induced increase in [Ca2+]i was mainly due to an increased influx of Ca2+. Treatment of cells with 300 µM thiopental increased FluoZin-3 fluorescence intensity, indicating the presence of [Zn2+]i, both in the presence and absence of extracellular Zn2+. The thiopental-induced elevation of [Zn2+]i was due to an increase in both influx of Zn2+ and intracellular Zn2+ release. Concanavalin A (10 µg/mL) augmented Fluo-3 fluorescence in the presence of an intracellular Zn2+ chelator. The combination of concanavalin A and 100–300 µM thiopental synergistically increased [Ca2+]i. Results suggest that thiopental increases [Ca2+]i in both quiescent and activated lymphocytes, possibly resulting in modulation of immune system function.

Trimethylamine-N-oxide acutely increases cardiac muscle contractility.

Cardiovascular disease is a major cause of morbidity and mortality among patients with chronic kidney disease (CKD). Trimethylamine-N-oxide (TMAO), a uremic metabolite that is elevated in the setting of CKD, has been implicated as a nontraditional risk factor for cardiovascular disease. While association studies have linked elevated plasma levels of TMAO to adverse cardiovascular outcomes, its direct effect on cardiac and smooth muscle function remains to be fully elucidated. We hypothesized that pathological concentrations of TMAO would acutely increase cardiac and smooth muscle contractility. These effects may ultimately contribute to cardiac dysfunction during CKD. High levels of TMAO significantly increased paced, ex vivo human cardiac muscle biopsy contractility (P < 0.05). Similarly, TMAO augmented contractility in isolated mouse hearts (P < 0.05). Reverse perfusion of TMAO through the coronary arteries via a Langendorff apparatus also enhanced cardiac contractility (P < 0.05). In contrast, the precursor molecule, trimethylamine (TMA), did not alter contractility (P > 0.05). Multiphoton microscopy, used to capture changes in intracellular calcium in paced, adult mouse hearts ex vivo, showed that TMAO significantly increased intracellular calcium fluorescence (P < 0.05). Interestingly, acute administration of TMAO did not have a statistically significant influence on isolated aortic ring contractility (P > 0.05). We conclude that TMAO directly increases the force of cardiac contractility, which corresponds with TMAO-induced increases in intracellular calcium but does not acutely affect vascular smooth muscle or endothelial function of the aorta. It remains to be determined if this acute inotropic action on cardiac muscle is ultimately beneficial or harmful in the setting of CKD.NEW & NOTEWORTHY We demonstrate for the first time that elevated concentrations of TMAO acutely augment myocardial contractile force ex vivo in both murine and human cardiac tissue. To gain mechanistic insight into the processes that led to this potentiation in cardiac contraction, we used two-photon microscopy to evaluate intracellular calcium in ex vivo whole hearts loaded with the calcium indicator dye Fluo-4. Acute treatment with TMAO resulted in increased Fluo-4 fluorescence, indicating that augmented cytosolic calcium plays a role in the effects of TMAO on force production. Lastly, TMAO did not show an effect on aortic smooth muscle contraction or relaxation properties. Our results demonstrate novel, acute, and direct actions of TMAO on cardiac function and help lay the groundwork for future translational studies investigating the complex multiorgan interplay involved in cardiovascular pathogenesis during CKD.

Stretch synchronizes sarcomere shortening in isolated cardiomyocytes

According to the Frank-Starling mechanism, stretching cardiac muscle augments contractile force. However, it is unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres and their local Ca2+ signals. To investigate this issue, we simultaneously monitored cytosolic Ca2+ and sarcomere striations in isolated rat cardiomyocytes by recording Fluo-4 fluorescence (515–560 nm) and brightfield signals respectively, using a high-speed sCMOS camera. Cells were affixed to glass rods (CellTester, World Precision Instruments) to allow control of cell length and measurement of force production during electrical stimulation at 1 Hz. At resting sarcomere length (SL), differential sarcomere deformation was observed despite uniform Ca2+ transients corresponding to individual sarcomeres. Specifically, while most sarcomeres contracted during the stimulus, a minority of sarcomeres (~13%) were observed to be stretched or were stationary. Sarcomeres which exhibited stretch during the Ca2+ transient tended to be shorter than those that contracted, and were significantly shorter than their neighbours. Step-wise stretch of the cell generated an expected increase in force development, but also revealed that more sarcomeres exhibited contraction, reaching nearly 100% of sarcomeres at 20% stretch. During cell lengthening, resting SL increased in all sarcomeres, suggesting that the shortest sarcomeres surpassed a critical length which enabled sufficient force generation and shortening. SL variability in unstretched cells was not caused by regional differences in Ca2+, as it remained apparent during permeabilization in Ca2+-free conditions. Rather, electron microscopy analysis traced differences in SL to the I-band, where dimensions are regulated by titin. We postulate, therefore, that slackened titin in unstretched cells allows greater variability in I-band length, cross-bridge formation, and active force generation between sarcomeres. When the cell is lengthened, sarcomere shortening is synchronized, which may contribute to augmented force production defined by the Frank-Starling mechanism.

Supramaximal calcium signaling triggers procoagulant platelet formation

AbstractProcoagulant platelets promote thrombin generation during thrombosis. Platelets become procoagulant in an all-or-nothing manner. We investigated how distinct Ca2+ signaling between platelet subpopulations commits some platelets to become procoagulant, using the high-affinity Ca2+ indicator Fluo-4, which may become saturated during platelet stimulation, or low-affinity Fluo-5N, which reports only very high cytosolic Ca2+ concentrations. All activated platelets had high Fluo-4 fluorescence. However, in Fluo-5N–loaded platelets, only the procoagulant platelets had high fluorescence, indicating very high cytosolic Ca2+. This finding indicates a novel, “supramaximal” Ca2+ signal in procoagulant platelets (ie, much higher than normally considered maximal). Supramaximal Ca2+ signaling and the percentage of procoagulant platelets were inhibited by cyclosporin A, a mitochondrial permeability transition pore blocker, and Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, with no effect on Fluo-4 fluorescence. In contrast, Synta-66, an Orai1 blocker, reduced Fluo-4 fluorescence but did not directly inhibit generation of the supramaximal Ca2+ signal. Our findings show a distinct pattern of Ca2+ signaling in procoagulant platelets and provide a new framework to interpret the role of platelet signaling pathways in procoagulant platelets. This requires reassessment of the role of different Ca2+ channels and may provide new targets to prevent formation of procoagulant platelets and limit thrombosis.

Triggering of eryptosis, the suicidal erythrocyte death, by phenoxodiol.

Phenoxodiol is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. At least in theory, phenoxodiol could similarly stimulate suicidal erythrocyte death or eryptosis. Eryptosis is characterized by cell shrinkage and breakdown of cell membrane asymmetry with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), formation of reactive oxygen species (ROS), and increase of ceramide abundance at the cell surface. The present study explored whether phenoxodiol induces eryptosis and whether it modifies Ca2+ entry, ROS, and ceramide. Using flow cytometry, phosphatidylserine exposure at the cell surface was quantified from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, ROS from DCFDA-dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48-h exposure of human erythrocytes to phenoxodiol (100μg/ml [416μM]) significantly increased the percentage of annexin V binding cells, significantly decreased average forward scatter and Fluo3 fluorescence and significantly increased ceramide abundance, but did not significantly modify DCFDA fluorescence. The effect of phenoxodiol on annexin V binding tended to decrease following removal of extracellular Ca2+, an effect, however, not reaching statistical significance. In conclusion, phenoxodiol triggers eryptosis, an effect paralleled by increase of ceramide abundance.

Garcinol A Novel Inhibitor of Platelet Activation and Apoptosis.

Garcinol, an anti-inflammatory and anti-carcinogenic polyisoprenylated benzophenone isolated from Garcinia plants, stimulates tumor cell apoptosis and suicidal erythrocyte death, but supports the survival of hepatocytes and neurons. The present study explored whether the substance influences platelet function and/or apoptosis. To this end, we exposed murine blood platelets to garcinol (33 µM, 30 min) without and with activation by collagen-related peptide (CRP) (2–5 µg/mL) or thrombin (0.01 U/mL); flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, and aggregation utilizing staining with CD9-APC and CD9-PE. As a result, in the absence of CRP and thrombin, the exposure of the platelets to garcinol did not significantly modify [Ca2+]i, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity, and aggregation. Exposure of platelets to CRP or thrombin was followed by a significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as significant cell shrinkage. All effects of CRP were strong and significant; those of thrombin were only partially and slightly blunted in the presence of garcinol. In conclusion, garcinol blunts CRP-induced platelet activity, apoptosis and aggregation.

Dopamine induces release of calcium from internal stores in layer II lateral entorhinal cortex fan cells.

The entorhinal cortex plays an important role in temporal lobe processes including learning and memory, object recognition, and contextual information processing. The alteration of the strength of synaptic inputs to the lateral entorhinal cortex may therefore contribute substantially to sensory and mnemonic functions. The neuromodulatory transmitter dopamine exerts powerful effects on excitatory glutamatergic synaptic transmission in the entorhinal cortex. Interestingly, inputs from midbrain dopamine neurons appear to specifically target clusters of excitatory cells located in the superficial layers of the entorhinal cortex. We have previously demonstrated that dopamine facilitates synaptic transmission through the activation of D1-like receptors. This facilitation of synaptic transmission is dependent on both activation of classical D1-like-receptors, and upon activation of dopamine receptors linked to increases in phospholipase C, inositol triphosphate (IP3), and intracellular calcium. In the present study we combined electrophysiological recordings of evoked excitatory postsynaptic currents with imaging of intracellular calcium using the fluorescent indicator fluo-4 to monitor calcium transients evoked by dopamine in electrophysiologically identified putative fan and pyramidal cells of the lateral entorhinal cortex. Bath application of dopamine (1 μM), or the phosphatidylinositol (PI)-linked D1-like-receptor agonist SKF83959 (5 μM), induced reliable and reversible increases in fluo-4 fluorescence and excitatory postsynaptic currents in fan cells, but not in pyramidal cells. In contrast, application of the classical D1-like-receptor agonist SKF38393 (10 μM) did not result in significant increases in fluorescence. Blocking release of calcium from internal stores by loading cells with the IP3 receptor blocker heparin (1 mM) or the ryanodine receptor blocker dantrolene (20 μM) abolished both the calcium transients and the facilitation of evoked synaptic currents induced by dopamine. Dopamine also induced calcium transients in fan cells when calcium was excluded from the extracellular medium, further indicating that the calcium transients are linked to release from internal stores. These results indicate that following D1-like-receptor binding, dopamine selectively induces transient elevations in intracellular calcium via activation of IP3 and ryanodine receptors, and that these elevations are linked to the facilitation of synaptic responses in putative layer II entorhinal cortex fan cells.

Identification of critical stage responding to consecutive monoculture obstacle in Rehmannia glutinosa L.

Rehmannia glutinosa L. (R. glutinosa) is one of the most common traditional Chinese medicines, whose productivity and quality, however, are seriously impacted by replanting disease, also known as consecutive monoculture obstacle. Little is known about R. glutinosa’s critical responsive stage to consecutive monoculture. There were two parts to this study: first, we analyzed different gene expression profiles in root of R. glutinosa between first-year planting (FP) and second-year replanting (SP) in five development stages (stage I to V) using Illumina sequencing to interpret the stage that most dramatically responding to consecutive monoculture; second, applying tissue culture technique, the harm of consecutive monoculture in a sterile condition was simulated and the medium was added with the soil extracts from FP and SP soil. The results showed that a significant difference in gene expression appeared at stage I under consecutive monoculture. Using a calcium indicator, the fluo-3 fluorescence to detect calcium distribution in the root tip of seedlings cultured in medium, the results revealed that after being cultured for 10 days, the SP fluorescence intensity of the seedlings was significantly higher than that of FP while crown position was particularly prominent. The evidence from gene expression profiling and tissue culture reached agreement that early stage of R. glutinosa growth was the critical stage to perceive consecutive monoculture stress. The results are of great significance to study the molecular mechanism of consecutive monoculture, which is expected to await a quick and accurate evaluation of land feasibility to plant R. glutinosa.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET)

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1–5 mM (0.02%–0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

324 Characterization and functional roles of Kv7 Channels in corpus cavernosum smooth muscle

KCNQ-encoded voltage-gated potassium channels (Kv7) have recently been identified as key regulator of vascular and non-vascular smooth muscle tone. Kv7 channel subtypes (Kv7.1-Kv7.5) have a specific tissue distribution and pathophysiological role. Loss of function mutations in four of the five KV7 genes lead to distinct inherited diseases, such as cardiac arrhythmias, epilepsy and sensorineuronal deafness. However, their physiological role in corporal smooth muscle (CSM) remains to be fully elucidated. In this study, we examined the molecular expression and functional role of Kv7 channels in corporal smooth muscle. Expression of KCNQ isoforms in human corporal smooth muscle (hCSM) cells was examined using RT-PCR. Functional responses to Kv7 channel modulators were evaluated in normal and diabetic (DM) rabbit corporal smooth muscle (CSM) tissue. Isolated CSM strips were mounted in an organ-bath system, and the relaxation effects of the following Kv7 channel subtype selective activators: ML213 (Kv7.2/Kv7.4channels), ML277 (Kv7.1) and ICA 069673 (Kv7.2/7.3), Flupirtine (Kv7.2–7.5 channels) were evaluated by cumulative addition to strips pre-contracted with10-5 M phenylephrine (PE). Fluo-4 fluorescence techniques were used to monitor changes in [Ca2+]cyt.

A Novel Tetramethylpyrazine Derivative Prophylactically Protects against Glutamate-Induced Excitotoxicity in Primary Neurons through the Blockage of N-Methyl-D-aspartate Receptor.

The over-activation of NMDA receptor via the excessive glutamate is believed to one of the most causal factors associated with Alzheimer’s disease (AD), a progressive neurodegenerative brain disorder. Molecules that could protect against glutamate-induced neurotoxicity may hold therapeutic values for treating AD. Herein, the neuroprotective mechanisms of dimeric DT-010, a novel derivative of naturally occurring danshensu and tetramethylpyrazine, were investigated using primary rat cerebellar granule neurons (CGNs) and hippocampal neurons. It was found that DT-010 (3–30 μM) markedly prevented excitotoxicity of CGNs caused by glutamate, as evidenced by the promotion of neuronal viability as well as the reversal of abnormal morphological changes. While its parent molecules did not show any protective effects even when their concentration reached 50 μM. Additionally, DT-010 almost fully blocked intracellular accumulation of reactive oxygen species caused by glutamate and exogenous oxidative stimulus. Moreover, Western blot results demonstrated that DT-010 remarkably attenuated the inhibition of pro-survival PI3K/Akt/GSK3β pathway caused by glutamate. Ca2+ imaging with Fluo-4 fluorescence analysis further revealed that DT-010 greatly declined glutamate-induced increase in intracellular Ca2+. Most importantly, with the use of whole-cell patch clamp electrophysiology, DT-010 directly inhibited NMDA-activated whole-cell currents in primary hippocampal neurons. Molecular docking simulation analysis further revealed a possible binding mode that inhibited NMDA receptor at the ion channel, showing that DT-010 favorably binds to Asn602 of NMDA receptor via arene hydrogen bond. These results suggest that DT-010 could be served as a novel NMDA receptor antagonist and protect against glutamate-induced excitotoxicity from blocking the upstream NMDA receptors to the subsequent Ca2+ influx and to the downstream GSK3β cascade.

Inhibition of Suicidal Erythrocyte Death by Indirubin-3’-Monoxime

Background/Aims: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3’-monoxime (IDM), have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. Methods: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC). The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca²⁺ activity, reactive oxygen species (ROS) formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. Results: The 24-h exposure of human erythrocytes to IDM (12 µM) significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca²⁺]_i). IDM (3-12 µM) did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours) significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM). Moreover, the Ca²⁺ ionophore ionomycin (1 µM, 60 min) and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP) similarly triggered eryptosis, which was also significantly suppressed by IDM. Conclusions: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca²⁺ entry into erythrocytes.

Inhibition of Collagen Related Peptide Induced Platelet Activation and Apoptosis by Ceritinib

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca²⁺ channel Orai1, increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 – 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 fluorescence, ROS abundance from 2’,7’-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α_IIbβ₃ integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca²⁺]_i without significantly modifying the other measured parameters. CRP significantly increased [Ca²⁺]_i, ROS abundance, P-selectin abundance, activated α_IIbβ₃ integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

TRPC6-Mediated Ca2+ Signaling is Required for Hypoxia-Induced Autophagy in Human Podocytes

Background/Aims: Intracellular Ca²⁺ signaling plays an important role in the regulation of autophagy. However, very little is known about the role of Ca²⁺ influx, which is induced by plasma membrane Ca²⁺ channels. Our previous study showed that transient receptor potential canonical channel-6 (TRPC6), a major Ca²⁺ influx pathway in podocytes, was activated by hypoxia. Here, we investigated whether TRPC6 is involved in hypoxia-induced autophagy in cultured human podocytes. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i), and western blotting was used to measure autophagy and protein expression. Results: We found that blockade TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker attenuated hypoxia-induced autophagy, while enhancement of TRPC6 activity with a TRPC6 activator enhanced hypoxia-induced autophagy. Furthermore, TRPC6-dependent Ca²⁺ signaling is responsible for hypoxia-induced autophagy since both an intracellular and extracellular Ca²⁺ chelator abolished hypoxia-induced autophagy. Moreover, we found that blockade of TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker decreased the expression of adenosine monophosphate-activated protein kinase (AMPK), an important signaling molecule in Ca²⁺-dependent autophagy activation, which is activated under hypoxic conditions. These data suggest that the involvement of TRPC6 in hypoxia-induced autophagy is associated with AMPK signaling. Conclusion: TRPC6 is essential for hypoxia-induced autophagy in podocytes.

N-acetyl-l-cysteine and Mn2+ attenuate Cd2+-induced disturbance of the intracellular free calcium homeostasis in cultured cerebellar granule neurons

Cadmium is a highly toxic heavy metal that is capable of accumulating in the body via direct exposure or through the alimentary and respiratory tract, leading to neurodegeneration. In this article, we show that the application of CdCl2 (0.001–0.005mM) for 48h induced high dose-dependent death rate of cultured cerebellar granule neurons (CGNs). Unlike Trolox or vitamin E, antioxidant N-acetyl-l-cysteine (NAC, 1mM) and Mn2+ (0.0025–0.005mM) significantly protected CGNs from this toxic effect. Using Fluo-4 AM, measurements of intracellular calcium ions demonstrated that 24h-exposure to Cd2+ induced intensive increase of Fluo-4 fluorescence in neurons accompanied by mitochondria swelling. These data imply that the cadmium-induced Ca2+ increase is an important element in the death of neurons due to toxic effect of cadmium and the mechanism of protective action of manganese and NAC is mediated by the prevention of increase in calcium levels.

Streptozotocin causes neurotoxic effect in cultured cerebellar granule neurons.

Streptozotocin (STZ) is a glucosamine-nitrosourea compound used for experimental simulation of sporadic Alzheimer's disease at intracerebroventricular administration in vivo. The studies of STZ influence on neurons of central nervous system performed on the primary cultures are practically absent. We have shown the application of STZ (1-5mM) in primary culture for 48h induced strong dose-dependent death in cultured cerebellar granule neurons. This toxic effect was decreased by pyruvate, insulin partially. Using the indicator Fluo-4 AM for measurements of intracellular calcium ions and tetramethylrhodamine ethyl ester (TMRE) for detection of changes of mitochondrial membrane potential in live cells we have shown that 5 h-exposure to STZ induced intensive increase of Fluo-4 and decrease TMRE fluorescence in neurons. STZ exposure caused considerable ultrastructural alterations in granule neurons: chromatin clumping, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. Probably, STZ significantly impaired glucose metabolism and mitochondrial function that, in turn, resulted in mitochondrial membrane potential damage, excessive calcium overload and neuronal death.

Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α_IIbβ₃ integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca²⁺]_i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated α_IIbβ₃ integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca²⁺]_i, P-selectin abundance, α_IIbβ₃ integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.