Fluo-4 AM Research Articles

Melatonin maintains mitochondrial membrane potential and decreases excessive intracellular Ca2+ levels in immature human oocytes

AimsPrevious reports have demonstrated that melatonin exists in multiple extrapineal sites, and higher amounts of melatonin are present in human follicular fluid than in serum, which indicates that it might play key roles in human oocyte maturation and subsequent embryonic development. Melatonin has been shown to be a potent antioxidant and might be beneficial to human oocytes during in vitro maturation (IVM). However, the underlying mechanisms of melatonin action during IVM have not been thoroughly investigated. Main methodsImmunofluorescence staining, western blotting, and ELISA were applied to investigate whether melatoninergic components are expressed in the cultured human ovarian cumulus cells. TMRE staining and Fluo-4 AM staining were performed to detect the mitochondrial membrane potential and intracellular Ca2+ levels of immature human oocytes respectively. Key findingsFirst, cultured human ovary cumulus cells synthesized melatonin in vitro, and it expressed serotonin (the precursor of melatonin) and the two key enzymes, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). Additionally, the results suggest that melatonin maintains the mitochondrial membrane potential and decrease excessive Ca2+ levels in immature human oocytes during IVM. SignificanceIn conclusion, we provide evidence that the melatoninergic components were expressed in cultured human ovarian cumulus cells, and melatonin might reduce oxidative stress of human oocytes by ameliorating mitochondrial function. In view of the significant clinical value that immature human oocytes have in assisted reproductive technology (ART), our findings highlight a potential treatment strategy of using melatonin to improve mitochondrial function and to enhance the quality of human oocytes during IVM.

Perfluorooctanoic acid-induced toxicities in chicken embryo primary cardiomyocytes: Roles of PPAR alpha and Wnt5a/Frizzled2

Perfluorooctanoic acid (PFOA) is a widespread persistent organic pollutant and may induce developmental toxicities, including developmental cardiotoxicity. To explore the potential mechanism of developmental cardiotoxicity induced by PFOA exposure, chicken embryo primary cardiomyocytes were extracted either from chicken embryos pretreated with PFOA (2 mg/kg), or from untreated embryos and then directly exposed cells to PFOA (1, 10, 30 or 100 μg/ml) in culture. Additionally, peroxisome proliferator activated receptor alpha (PPAR alpha) silencing lentivirus was applied to the embryos on embryonic day (ED2). Cell viability was measured with CCK-8 kit, morphology was assessed with hematoxylin and eosin staining, and intracellular Ca2+ concentrations were determined with Fluo-4 AM probe. Western blotting was utilized to confirm PPAR alpha silencing efficiency and the protein abundance of Wnt5a and Frizzled2. The results indicated that both PFOA pretreatment and direct exposure decreased primary cardiomyocyte viability, altered cell morphology and increased intracellular Ca2+ concentrations. While l-carnitine co-treatment effectively abolished such changes, PPAR alpha silencing only abolished most of the changes in PFOA pretreatment group, but not in cells directly exposed to relatively high doses of PFOA. The protein abundance of Wnt5a and Frizzled2 was increased by PFOA pretreatment, while direct exposure to PFOA increased Frizzled2 abundance but decreased Wnt5a abundance. PPAR alpha silencing resulted in over 50% decrease of PPAR alpha expression level, which abolished the Wnt5a/Frizzled2 expression alterations following PFOA exposure. In conclusion, PFOA-induced primary cardiomyocyte toxicity is associated with PPAR alpha and Wnt5a/Frizzled2, in which PPAR alpha seems to play regulatory roles towards Wnt5a/Frizzled2.

Са2+-мобілізація у гладеньких міоцитах сечового міхура щура у відповідь на прикладання капсаїцину

According to the previous studies of the heat-activated TRPV1 ion channel, in the organism it could play not only the canonical role of the nociceptive temperature sensor (activation threshold >42 °С) but also be a polymodal receptor involved in numerous other processes due to the permeability of its selective pore for Ca 2+ ions. Prior studies conducted by other research groups provided indirect evidence obtained with immunochemistry and PCR approaches suggesting the presence of this ion channel in the smooth muscle cells (BSM) of the urinary bladder though the functional data is still absent. In this study we investigated the effect of capsaicin (CAP), a pungent component of hot chili pepper and an agonist of TRPV1, on the transmembrane currents and the Ca 2+ signaling of the bladder smooth muscle cells of the rat. CAP at the saturating concentration of 10 μM had no influence on the transmembrane currents of BSMs registered with the whole-cell patch clamp technique which suggests the absence of TRPV1 in the plasma membrane of these cells. Along with this negative data the control electrophysiological recordings on the dorsal root ganglion (DRG) neurons was conducted showing the presence of CAPS-evoked currents in this cells. On the opposite, the application of this pharmacological agent induced the apparent Ca 2+ response in calcium imaging experiments with the Ca 2+ -sensitive fluorescent dye Fluo-4 AM in 42 % of the BSMs examined. At the same time, the rest of these cells showed no calcium influx though their viability was validated with control cholinoreceptor agonist, carbachol (CCh), in the concentration of 100 μM. Therefore, our findings suggest the previously unreported expression of TRPV1 in the part of the BSMs and its subcellular localization in the sarcoplasmic reticulum of these cells.

Effects of hypoxia and ASIC3 on nucleus pulposus cells: From cell behavior to molecular mechanism

This study aimed to explore the effects of hypoxia and acid-sensing ion channel 3 (ASIC3) on nucleus pulposus cells from cell behavior to molecular mechanism. Primary rabbit nucleus pulposus cells were isolated and identified by HE, toluidine blue and immunohistochemical staining of collagen II. 2% O2 and 48 h were screened as optimal oxygen concentration and effect time, respectively, by determining cell apoptosis and mRNA expression of ASIC3, hypoxia inducible factor-1α (HIF-1α) and aquaporin 3. FLuo-3 AM labeling showed that the Ca2+ concentration in cells increased under hypoxia condition. shRNA-ASIC3 and ASIC3 expression vector were transfected into cells. Subsequently, cells were divided into six groups: Control, 2% O2, shRNA-NC+2% O2, shRNA-ASIC3 + 2% O2, Vector+2% O2 and ASIC3 + 2% O2. Flow cytometry, CCK-8 assay, transmission electron microscopy, immunofluorescent labeling, RT-PCR and western blot demonstrated that hypoxia and ASIC3 over-expression inhibited the proliferation, arrested cell cycle in G1 phase, promoted the apoptosis, initiated the autophagy and up-regulated the expression of ASIC3, HIF-1α, light chain 3, p-ERK1/2 and p-MAPK. However, ASIC3 silencing could significantly relieve these phenomena. Co-immunoprecipitation assay found ASIC3 was interacted with HIF-1α&ERK1/2. Evaluation of the effect of HIF-1αsilencing on ASIC3 expression showed that the high expression of ASIC3 induced by hypoxia was reduced significantly by HIF-1α silencing. In conclusion, hypoxia and ASIC3 changed the behavior of nucleus pulposus cells by activating the MAPK pathway. HIF-1α and ASIC3 could regulate each other in nucleus pulposus cells.

Membrane-permeabilized sonodynamic therapy enhances drug delivery into macrophages.

Macrophages play a pivotal role in the formation and development of atherosclerosis as a predominant inflammatory cell type present within atherosclerotic plaque. Promoting anti-atherosclerotic drug delivery into macrophages may provide a therapeutic potential on atherosclerotic plaque. In this study, we investigated whether membrane-permeabilized sonodynamic therapy (MP-SDT) enhances drug delivery into THP-1 macrophages. Images of confocal microscopy confirmed that the optimal plasma distribution of the sonosensitizer protoporphyrin IX (PpIX) was at 1 hour incubation. The non-lethal parameter of MP-SDT was determined by cell viability as measured by a CCK-8 assay. Bright field microscopy demonstrated plasma membrane deformation in response to MP-SDT. Using SYTOX Green, a model drug for cellular uptake, we found that MP-SDT significantly induced membrane permeabilization dependent on ultrasound intensity and exposure time. Using Fluo-3 AM, intracellular calcium elevation during MP-SDT was confirmed as a result of membrane permeabilization. Membrane perforation of MP-SDT-treated cells was observed by scanning electron microscopy and transmission electron microscopy. Moreover, MP-SDT-induced membrane permeabilization and perforation were remarkably prevented by scavenging reactive oxygen species (ROS) during MP-SDT. Furthermore, we assessed the therapeutic effect of MP-SDT in combination with anti-atherosclerotic drug atorvastatin. Our results showed that MP-SDT increased the therapeutic effect of atorvastatin on lipid-laden THP-1-derived foam cells, including decreasing lipid droplets, increasing the cholesterol efflux and the expression of PPARγ and ABCG1. In conclusion, MP-SDT might become a promising approach to facilitating the delivery of anti-atherosclerotic drugs into macrophages via membrane permeabilization.

Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII−/− mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE−/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE−/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE−/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

Bursopentin (BP5) induces G1 phase cell cycle arrest and endoplasmic reticulum stress/mitochondria-mediated caspase-dependent apoptosis in human colon cancer HCT116 cells

BackgroundBursopentin (BP5) is a multifunctional pentapeptide found in the chicken bursa of Fabricius. Recent study indicated that BP5 significantly stimulates expression of p53 protein in colon cancer HCT116 cells. However, the effects and underlying mechanisms of BP5 on HCT116 cell proliferation remain largely unclear.MethodsAnalyses of cell viability, cell cycle arrest as well as apoptosis were performed to study the actions of BP5 on HCT116 cells. Western blot analyse was assayed to measure the cell cycle-related and apoptosis-related proteins. Specific siRNAs targeting IRE1, ATF-6, and PERK were used for IRE1, ATF-6, and PERK knockdown, respectively. Cellular reactive oxygen species (ROS) were detected using a H2DCF-DA green fluorescence probe. Cytosolic free Ca2+ concentrations and mitochondrial membrane potential (ΔΨm) were measured using Fluo-3 AM and JC-1 stains, respectively.ResultsBP5 possessed strong inhibitory effects on the cell growth and induced apoptosis in HCT116 cells. Mechanistically, BP5 arrested the cell cycle at G1 phase by increasing p53 and p21 expression and decreasing cyclin E1-CDK2 complex expression. BP5 treatment dramatically activated the endoplasmic reticulum (ER) stress-mediated apoptotic pathway, as revealed by the significantly enhanced expression of unfolded protein response (UPR) sensors (IRE1α, ATF6, PERK) as well as downstream signaling molecules (XBP-1s, eIF2α, ATF4 and CHOP), and by the significantly altered the BP5-induced phenotypic changes in IRE1, ATF6, and PERK knockdown cells. Additionally, BP5-induced ER stress was accompanied by the accumulation of cytosolic free Ca2+ and intracellular ROS. Furthermore, BP5 treatment resulted in the increase of Bax expression, the decrease of Bcl-2 expression and the reduction of ΔΨm, subsequently causing a release of cytochrome c from the mitochondria into the cytoplasm and finally enhancing the activities of caspase-9 and -3. In addition, z-VAD-fmk, a pan-caspase inhibitor, markedly rescued BP5-induced cell viability reduction and reduced BP5-induced apoptosis.ConclusionsOur present results suggest that BP5 has an anticancer capacity to arrest cell cycle at G1 phase and to trigger ER stress/mitochondria-mediated caspase-dependent apoptosis in HCT116 cells. Therefore, our findings provide insight into further investigations of the anticancer activities of BP5.

Inhibitory effect of recombinant human CXCL8(3-72)K11R/G31P on atherosclerotic plaques in a mouse model of atherosclerosis

Context: Atherosclerosis is a chronic inflammatory disease in which the plaques were built up inside of the artery. Interleukin-8 (IL-8, CXCL8) is an inflammatory factor, known to play an important role in the development of atherosclerosis. G31P is an antagonist of the IL-8 receptor, which plays roles in vascular smooth muscle cell (VSMC) proliferation and migration. Objective: This study is to investigate the therapeutic effect of G31P on atherosclerosis through a mouse model.Materials and methods: A mouse model of atherosclerosis was generated through feeding the ApoE–/– mice with high fat diet for 12 weeks. G31P was injected subcutaneously into the mice. The levels of keratinocyte chemoattractant (KC), CXCR2, TNF-α, and IFN-γ were analyzed through ELISA. The expressions of MMP-2, MMP-9, PCNA, and Mef2a in aortic tissues were detected through RT-qPCR. In A7r5 cells, the levels of p-ERK, ROCK1, and ROCK2 were analyzed by western blot. Intracellular calcium levels were measured through Fluo-3 AM assay.Results and disccussion: G31P suppressed the abnormal lipid profile and decreased the levels of KC, MMP-2, MMP-9, PCNA, and Mef2a in a mouse model of atherosclerosis. In addition, G31P also inhibited the expressions of p-ERK, ROCK1, ROCK2, and decreased the calcium concentrations in A7r5 cells.Conclusions: These findings indicate the potential therapeutic effects of G31P in suppressing the development of atherosclerosis by antagonizing the IL-8 receptor. G31P inhibits the proliferation and migration of VSMCs through regulating the Rho-kinase, ERK, and calcium-dependent pathways.

Towards pharmacological treatment screening of cardiomyocyte cells using Si nanowire FETs

Silicon nanowires (Si NWs) are the most promising candidates for recording biological signals due to improved interfacing properties with cells and the possibility of high-speed transduction of biochemical signals into detectable electrical responses. The recording of extracellular action potentials (APs) from cardiac cells is important for fundamental studies of AP propagation features reflecting cell activity and the influence of pharmacological substances on the signal. We applied a novel approach of using fabricated Si NW field-effect transistors (FETs) in combination with fluorescent marker techniques to evaluate the functional activity of cardiac cells. Extracellular AP signal recording from HL-1 cardiomyocytes was demonstrated. This method was supplemented by studies of the pharmacological effects of stimulations using noradrenaline (NorA) as a modulator of functional activity on a cellular and subcellular levels, which were also tested using fluorescent marker techniques. The role of calcium alteration and membrane potential were revealed using Fluo-4 AM and tetramethylrhodamine, methyl ester, perchlorate (TMRM) fluorescent dyes. In addition, chemical treatment with sodium dodecyl sulfate (SDS) solutions was tested. The results obtained demonstrate positive prospects for AP monitoring in different treatments for studies related to a wide range of myocardial diseases using lab-on-chip technology.

Small molecules enhance neurogenic differentiation of dental-derived adult stem cells

ObjectiveDental-derived stem cells originate from the embryonic neural crest, and exhibit high neurogenic potential. This study aimed to investigate whether a cocktail of eight small molecules (Valproic acid, CHIR99021, Repsox, Forskolin, SP600125, GO6983, Y-27632 and Dorsomorphin) can enhance the in vitro neurogenic differentiation of dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAPs) and gingival mesenchymal stem cells (GMSCs), as a preliminary step towards clinical applications. Materials and MethodsNeural induction was carried out with a small molecule cocktail based two-step culture protocol, over a total duration of 14 days. At the 8 and 14 day timepoints, the cells were analyzed for expression of neural markers with immunocytochemistry, qRT-PCR and Western Blot. The Fluo 4-AM calcium flux assay was also performed after a further 14 days of neural maturation. ResultsMore pronounced morphological changes characteristic of the neural lineage (i.e. neuritogenesis) were observed in all three cell types treated with small molecules, as compared to the untreated controls. This was corroborated by the immunocytochemistry, qRT-PCR and western blot data, which showed upregulated expression of several early and mature neural markers in all three cell types treated with small molecules, versus the corresponding untreated controls. Finally, the Fluo-4 AM calcium flux assay showed consistently higher calcium transient (F/Fo) peaks for the small molecule-treated versus untreated control groups. ConclusionsSmall molecules can enhance the neurogenic differentiation of DPSCs, SCAPs and GMSCs, which offer much potential for therapeutic applications.

Calcium Signaling and Contractility in Cardiac Myocyte of Wolframin Deficient Rats.

Wolframin (Wfs1) is a membrane protein of the sarco/endoplasmic reticulum. Wfs1 mutations are responsible for the Wolfram syndrome, characterized by diabetic and neurological symptoms. Although Wfs1 is expressed in cardiac muscle, its role in this tissue is not clear. We have characterized the effect of invalidation of Wfs1 on calcium signaling-related processes in isolated ventricular myocytes of exon5-Wfs1 deficient rats (Wfs1-e5/-e5) before the onset of overt disease. Calcium transients and contraction were measured in field-stimulated isolated myocytes using confocal microscopy with calcium indicator fluo-3 AM and sarcomere length detection. Calcium currents and their calcium release-dependent inactivation were characterized in whole-cell patch-clamp experiments. At 4 months, Wfs1-e5/-e5 animals were euglycemic, and echocardiographic examination revealed fully compensated cardiac function. In field-stimulated isolated ventricular myocytes, both the amplitude and the duration of contraction of Wfs1-e5/-e5 animals were elevated relative to control Wfs1+/+ littermates. Increased contractility of myocytes resulted largely from prolonged cytosolic calcium transients. Neither the amplitude of calcium currents nor their voltage dependence of activation differed between the two groups. Calcium currents in Wfs1-e5/-e5 myocytes showed a larger extent of inactivation by short voltage prepulses applied to selectively induce calcium release-dependent inactivation of calcium current. Neither the calcium content of the sarcoplasmic reticulum, measured by application of 20 mmol/l caffeine, nor the expression of SERCA2, determined from Western blots, differed significantly in myocytes of Wfs1-e5/-e5 animals compared to control ones. These experiments point to increased duration of calcium release in ventricular myocytes of Wfs1-e5/-e5 animals. We speculate that the lack of functional wolframin might cause changes leading to upregulation of RyR2 channels resulting in prolongation of channel openings and/or a delay in termination of calcium release.

Effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin-treated microenvironment of A549 cell line

Objective To investigate the effects of ultrasound combined with microbubbles on intracellular Ca2+ homeostasis in carboplatin(CBP)-treated A549 cell and its possible mechanisms of inhibiting A549 cell line activity. Methods According to whether SonoVue was used or not, and the different dose of CBP, the groups A-F were arranged as the ultrasound(US) group(group A), the ultrasound combined with microbubbles (USMB) group(group B), the low dose CBP (100 μg/ml)+ US group(group C), the low dose CBP+ USMB group(group D), the high dose CBP (200 μg/ml)+ US group (group E) and the high dose CBP+ USMB group(group F). A549 cells were bathed and washed by a calcium-free buffer, loaded with Ca2+ indicator fluo-4 AM. Real-time images were acquired using laser confocal microscopy. The fluorescence intensity of intracellular calcium ion concentration ([Ca2+ ]i) in individual living cell was observed and the calcium overload was analyzed. Results After ultrasound irradiation, the normalized fluorescence intensity of [Ca2+ ]i increased rapidly, then returned to a new homeostasis (selected cells in groups A, B, E, F) or experienced a second calcium oscillation (some cells in group C and D). All the selected cells in group B and some cells in group C and D exhibited superimposed oscillations. The calcium overloading time in group D was longer than those of any other groups. Four cells in group A experienced delayed calcium oscillations. Compared with group A, the selected cells in other groups exhibited a larger amplitude of calcium oscillation(all P<0.05) and the selected cells in group B and D exhibited calcium oscillation for a longer period of time(all P<0.05). Conclusions In the calcium-free buffer, US, USMB, CBP+ US, CBP+ USMB are direct stimuli of calcium overload in A549 cells. SonoVue, CBP, CBP+ SonoVue are all synergistic stimuli of calcium overload in A549 cells irradiated by ultrasound.US, USMB and CBP may synergistically induce calcium release from intracellular store sites in A549 cells. Calcium overload is a possible mechanism of ultrasound combined with microbubbles in assisting CBP chemotherapy. Key words: Sonication; Microbubbles; Carboplatin; A549 cell; Calcium overload; Cavitation effect

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

Real-time Microwave Exposure Induces Calcium Efflux in Primary Hippocampal Neurons and Primary Cardiomyocytes

ObjectiveTo detect the effects of microwave on calcium levels in primary hippocampal neurons and primary cardiomyocytes by the real-time microwave exposure combined with laser scanning confocal microscopy. MethodsThe primary hippocampal neurons and primary cardiomyocytes were cultured and labeled with probes, including Fluo-4 AM, Mag-Fluo-AM, and Rhod-2, to reflect the levels of whole calcium [Ca2+], endoplasmic reticulum calcium [Ca2+]ER, and mitochondrial calcium [Ca2+]MIT, respectively. Then, the cells were exposed to a pulsed microwave of 2.856 GHz with specific absorption rate (SAR) values of 0, 4, and 40 W/kg for 6 min to observe the changes in calcium levels. ResultsThe results showed that the 4 and 40 W/kg microwave radiation caused a significant decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT in primary hippocampal neurons. In the primary cardiomyocytes, only the 40 W/kg microwave radiation caused the decrease in the levels of [Ca2+], [Ca2+]ER, and [Ca2+]MIT. Primary hippocampal neurons were more sensitive to microwave exposure than primary cardiomyocytes. The mitochondria were more sensitive to microwave exposure than the endoplasmic reticulum. ConclusionThe calcium efflux was occurred during microwave exposure in primary hippocampal neurons and primary cardiomyocytes. Additionally, neurons and mitochondria were sensitive cells and organelle respectively.

Myocardial Rad Deletion Modulates L-Type Calcium Channel Current

Rad inhibits L-type calcium channel current (ICa,L), and in vivo deletion of Rad results in elevated ICa,L density. The aim of this study is to test the hypothesis that Rad-deletion modulates L-type calcium channel current (ICa,L). We engineered a cardiac-restricted inducible Rad knockout mouse (cRadKO). We examined ICa,L using the whole cell configuration and the cell attached configuration of the patch clamp technique. We assessed global and local cellular calcium handling using Fura-2 AM and Fluo-4 AM, respectively. cRadKO displayed an increase in ICa,L compared to WT (maximal conductance: cRadKO = 254 ± 19 pS/pF, n=15; WT= 144 ± 12 pS/pF, n=18; p<0.0001). ICa,L activated at more negative voltages (activation midpoint: cRadKO = −18.3 ± 1.0 mV, n=15; WT= −8.1 ± 1.9 mV, n=18; p<10−4). WT cells treated with forskolin and IBMX demonstrated a ceiling effect of modulation in WT ICa,L. ICa,L kinetics are accelerated in cRadKO concordant with the upstroke velocity of Ca2+ (transient velocity 48.7 ± 2.4 AU/s, n=49; WT=38.1 ± 3.5 AU/s, n=37; p=.014). cRadKO ICa,L was no different when treated with isoproterenol, consistent with saturable modulation of the LTCC. Myocardial Rad deletion increases ICa,L and activates ICa,L at more negative potentials that is beyond PKA induced modulation of the L-type calcium channel; cRadKO ICa,L remains unchanged when PKA is activated. This unmasks a new form of modulation of ICa,L, demonstrating that Rad plays a critical role in how the L-type calcium channel responds to β-Adrenergic Receptor stimulation.

Potential mechanisms of cellular injury following exposure to a physiologically relevant species of inorganic mercury

Mercury is a toxic metal that is found ubiquitously in the environment. Humans are exposed to different forms of mercury via ingestion, inhalation, and/or dermal absorption. Following exposure, mercuric ions may gain access to target cells and subsequently lead to cellular intoxication. The mechanisms by which mercury accumulation leads to cellular injury and death are not understood fully. Therefore, purpose of this study was to identify the specific intracellular mechanisms that are altered by exposure to inorganic mercury (Hg2+). Normal rat kidney (NRK) cells were exposed to a physiologically relevant form of Hg2+, as a conjugate of cysteine (10 μM or 50 μM). Alterations in oxidative stress were estimated by measuring lipid peroxidation and mitochondrial oxidative stress. Alterations in actin and tubulin were measured using specific fluorescent dyes. Calcium levels were measured using Fluo-3 AM Calcium Indicator while autophagy was identified with Premo™ Autophagy Sensor LC3B-GFP. The current findings show that exposure to Hg2+ leads to enhanced oxidative stress, alterations in cytoskeletal structure, increases in intracellular calcium, and enhanced autophagy. We have established a more complete understanding of intoxication and cellular injury induced by a relevant form of Hg2+ in proximal tubule cells.

Involvement of Orai1 in tunicamycin-induced endothelial dysfunction.

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca2+ homeostasis. The store-operated calcium (SOC) channel is the primary Ca2+ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca2+ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca2+ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

Endocytosis-mediated mitochondrial transplantation: Transferring normal human astrocytic mitochondria into glioma cells rescues aerobic respiration and enhances radiosensitivity.

Emerging evidence indicates that reprogramming of energy metabolism involving disturbances in energy production from a defect in cellular respiration with a shift to glycolysis is a core hallmark of cancer. Alterations in cancer cell energy metabolism are linked to abnormalities in mitochondrial function. Mitochondrial dysfunction of cancer cells includes increased glycolysis, decreased apoptosis, and resistance to radiotherapy. The study was designed for two main points: firstly, to investigate whether exogenous functional mitochondria can transfer into glioma cells and explore the underlying molecular mechanisms from the perspective of endocytosis; secondly, to further verify whether the mitochondrial transplantation is able to rescue aerobic respiration, attenuate the Warburg effect and enhance the radiosensitivity of gliomas.Methods: Mitochondria were isolated from normal human astrocytes (HA) and immediately co-incubated with starved human glioma cells (U87). Confocal microscopy and gene sequencing were performed to evaluate the ability of isolated mitochondria internalization into U87 cells. The interaction between endocytosis and isolated mitochondria transfer were captured by 3D tomographic microscopy and transmission electron microscopy. NAD+, CD38, cADPR and Ca2+ release were determined by commercial kits, western blot, HLPC-MS and Fluo-3 AM respectively. PCR array expression profiling and Seahorse XF analysis were used to evaluate the effect of mitochondrial transplantation on energy phenotypes of U87 cells. U87 cells and U87 xenografts were both treated with mitochondrial transplantation, radiation, or a combination of mitochondrial transplantation and radiation. Apoptosis in vitro and in vivo were detected by cytochrome C, cleaved caspase 9 and TUNEL staining.Results: We found that mitochondria from HA could be transferred into starved U87 cells by simple co-incubation. Starvation treatment slowed the rate of glycolysis and decreased the transformation of NAD+ to NADH in U87 cells. A large amount of accumulated NAD+ was released into the extracellular space. CD38 is a member of the NAD+ glycohydrolase family that catalyzes the cyclization of extracellular NAD+ to intracellular cADPR. cADPR triggered release of Ca2+ to promote cytoskeleton remodeling and plasma membrane invagination. Thus, endocytosis involving isolated mitochondria internalization was mediated by NAD+-CD38-cADPR-Ca2+ signaling. Mitochondrial transfer enhanced gene and protein expression related to the tricarboxylic acid (TCA) cycle, increased aerobic respiration, attenuated glycolysis, reactivated the mitochondrial apoptotic pathway, inhibited malignant proliferation of U87 cells. Isolated mitochondria injected into U87 xenograft tumors also entered cells, and inhibited glioma growth in nude mice. Mitochondrial transplantation could enhance the radiosensitivity of gliomas in vitro and in vivo.Conclusion: These findings suggested that starvation-induced endocytosis via NAD+-CD38-cADPR-Ca2+ signaling could be a new mechanism of mitochondrial transplantation to rescue aerobic respiration and attenuate the Warburg effect. This mechanism could be a promising approach for radiosensitization.

Memantine ameliorates pulmonary inflammation in a mice model of COPD induced by cigarette smoke combined with LPS

An enhanced chronic inflammatory response in the airways has been regarded as a critical characteristic of chronic obstructive pulmonary disease (COPD). Memantine, an N-methyl-d-aspartate (NMDA) receptors antagonist, has been reported to alleviate lung inflammation. In this study, we investigated the effect and mechanism of memantine on the COPD model induced by cigarette smoke (CS) combined with LPS. Mice and RAW264.7 cells were treated with LPS in the presence or absence of CS. We performed H&E staining to analysis the lung histopathological characteristics. Cytokines (IL-6, TNF-α, and IFN-γ) levels in bronchoalveolar lavage fluid (BALF), lung tissue homogenates and RAW264.7 cell culture medium were determined. Glutamate levels in plasma and culture medium of RAW264.7 were determined. The intracellular Ca2+ flux in RAW264.7 cells was measured by fluo-3 AM staining. The protein levels of NR-1, xCT, ERK1/2, and AKT signaling in the lung tissue and cells were investigated. The result showed that CS and LPS stimulation caused inflammation response, a significant increase in the release of cytokines, including TNF-α, IL-6, and IFN-γ, the elevated release of glutamate and protein levels of NR-1 and xCT, increased Ca2+ influx, and the activation of the ERK1/2 pathway in vitro and in vivo. The above effects of CS and LPS stimulation could be significantly attenuated by memantine treatment. In conclusion, memantine can effectively ameliorate pulmonary inflammation in CS + LPS-induced COPD in mice via reducing NR-1 and xCT expression, glutamate release, Ca2+ influx, and the phosphorylation of Erk1/2. We provided a possible mechanism by which memantine ameliorates COPD in mice.

Ethanolic extract of Cordyceps cicadae exerts antitumor effect on human gastric cancer SGC-7901 cells by inducing apoptosis, cell cycle arrest and endoplasmic reticulum stress

Ethnopharmacological relevanceCordyceps cicadae (Miq.) Massee is a traditional Chinese medicine that has been used for approximately 1600 years in China. C. cicadae, a member of the Cordyceps genus, exerts a therapeutic effect on many diseases, such as cancer. ObjectiveThis study aimed to evaluate the antineoplasmic activity of C. cicadae and to identify its molecular mechanism of cell death. Materials and methodsThe toxicity of the ethanolic extract of C. cicadae (EEC) against different cancer cell lines was determined through MTT assay. Human gastric cancer SGC-7901 cells were treated with EEC for 48 h. Cell morphology was examined by using an Olympus phase-contrast microscope. The cell apoptosis was quantified through Annexin V-FITC/PI staining. Cells were stained with PI and then subjected to flow cytometry for the investigation of cell cycle status. Cells were subjected to mitochondrial membrane potential (MMP) assay after incubation with JC-1 probes and to intracellular Ca2+ measurement through flow cytometry after incubation with Fluo-3 AM fluorescent probes. Western blot analysis was conducted to quantify the expression of proteins related to apoptosis, cell cycle and endoplasmic reticulum stress. High-performance liquid chromatography (HPLC) analysis was performed to analyse the biological activity components of EEC. ResultsEEC suppressed the proliferation of SGC-7901 cells and induced the development of abnormal morphological features in a dose-dependent manner. Flow cytometry results indicated that EEC treatment caused cell apoptosis and arrested the cell cycle in the S phase. In addition, EEC treatment triggered MMP depolarization and Ca2+ overloading in the cytosol of SGC-7901 cells. Western blot analysis demonstrated that EEC increased Bax, AIF, caspase-8, caspase-6 and caspase-3 activities and decreased Bcl-2 activity. The release of cytochrome c from mitochondria was associated with mitochondrial dysfunction, which was caused by the activation of the cell surface receptor Fas and the cleavage of PARP. EEC-induced S phase arrest was associated with the up-regulation of E2F1, cyclin A2, cyclin E and p53 expression levels and the down-regulation of CDK2 expression. In addition, EEC increased the expression of endoplasmic reticulum stress-related proteins, such as calpain-1, caspase-12 and caspase-9. HPLC assay results suggested that EEC contained adenine, uridine, adenosine and N6-(2-Hydroxyethyl)-adenosine. ConclusionEEC inhibited the proliferation of SGC-7901 cells by inducing caspase-dependent apoptosis, arresting the cell cycle in the S phase and increasing endoplasmic reticulum stress. This study revealed that C. cicadae is a potential natural source of anticancer drugs.