Fluo-4 AM Research Articles

Enhanced calcium entry via activation of NOX/PKC underlies increased vasoconstriction induced by methylglyoxal

Advanced glycation end-products (AGEs) play a pivotal role in macro- and micro-vascular diabetic complications. We investigated the mechanism by which methylglyoxal (an endogenous generator of AGEs) affects vascular contractility using the isolated artery technique. Contractile responses to vasoconstrictors phenylephrine (PE), angiotensin II (Ang II), vasopressin (VP) and KCl were measured in the isolated rat aorta following one-our exposure to methylglyoxal (50–200 μM). The perfused rat kidney was employed to confirm the effect of methylglyoxal on microvessels. Methylglyoxal-induced changes in cytosolic calcium were measured in the smooth muscle layer of the aorta with the calcium-sensing fluorophore Fluo-4 AM. Methylglyoxal significantly increased maximal contraction of the rat aorta to PE, Ang II and VP. Similar results were seen in response to the depolarizing vasoconstrictor KCl in macro and micro vessels. The methylglyoxal-induced increases in aortic contraction mediated by the agonist and KCl were endothelium independent. Methylglyoxal-induced increases in KCl-dependent aortic contraction were abolished after the removal of extracellular calcium or in the presence of the calcium channel blocker nifedipine. Incubation with the antioxidant N-acetyl-l-cysteine (NAC), apocynin (a nonselective NADPH oxidase (NOX) inhibitor) or chelerythrine (a protein kinase C (PKC) inhibitor) prior to methylglyoxal pre-treatment reversed the methylglyoxal-induced increases in the rat aortic contractility. In conclusion, the formation of AGEs increases vasoconstriction of both macro- and micro-vessels by increasing the voltage-activated calcium entry in vascular smooth muscles in a NOX and PKC dependent manner.

3H-1,2-dithiole-3-thione protects PC12 cells against amyloid beta 1–42 (Aβ1–42) induced apoptosis via activation of the ERK1/2 pathway

AimsIncreasing evidence displays that deposition of aggregated β-amyloid (Aβ) leads to neuronal cell apoptosis, thus aggravates the pathological progression of Alzheimer's disease (AD). 3H-1,2-dithiole-3-thione (D3T) has been proved to exert neuroprotective effects. However, the effect of D3T on protecting against Aβ-induced apoptosis and the underlying mechanism are unknown. Main methodsMTT, DCFH-DA assay, LDH release assay, Fluo-3 AM assay, Flow cytometry and Western blot were used to examine cell viability, ROS level, LDH release, intracellular Ca2+ concentration, cell apoptosis and related proteins level respectively. Key findingsIn the present study, we found that D3T pretreatment significantly increased cell viability and decreased reactive oxygen species (ROS) levels, lactate dehydrogenase (LDH) levels and the intracellular calcium concentration of rat pheochromocytoma (PC12) cells after Aβ1–42 exposure. In addition, D3T pretreatment inhibited Aβ1–42 induced cell apoptosis as well as protein levels of Bax and Caspase-3 in PC12 cells. Further, D3T markedly activated extracellular regulated protein kinase 1/2 (p-ERK1/2) but not PI3K/Akt signaling. Moreover, the protective effect of D3T against Aβ1–42 induced apoptosis was abolished by the ERK1/2 pathway inhibitor PD98059 while PI3K inhibitor LY294002 had no significant effect. SignificanceTaken together, these findings suggest that D3T protects PC12 cells against Aβ1–42 induced apoptosis through activation of the ERK1/2 pathway.

Responses of intracellular Ca2+ and its sensors to Venturia nashicola infection in pear (Pyrus bretschneideri) with differing resistance

In this study, we investigated the role of Ca2+ signaling in pear (Pyrus. bretschneideri Rehd) in response to pear scab (Venturia nashicola Tanak et Yamamota) infection. We analyzed the responses of intracellular [Ca2+] and expression changes of Ca2+ sensor-related genes in two pear cultivars of differing resistance (the resistant ‘Huangguan’ and the susceptible ‘Xuehua’) following V. nashicola infection by using fluorescence microscopy, microplate spectrophotometry, and real-time quantitative PCR assays. Over a 60 min treatment period, V. nashicola induced elevated Ca2+ fluorescence in cell protoplasts (loaded with Fluo-3 AM) from fruit callus cultures of both pear cultivars, but the Ca2+ change patterns differed. Both LaCl3 (a plasma membrane Ca2+ channel inhibitor) and EGTA (an extracellular Ca2+ chelator) inhibited [Ca2+]cyt in both cultivars, while BAPTA-AM (an intracellular Ca2+ chelator) showed no inhibitory effect. The relative expression levels of PbCaM1, PbCaM5, PbCBL1, PbCIPK2, PbCPK1, and PbCPK2 in ‘Huangguan’ pear were significantly upregulated following infection and peaked at 72 post-infection hour. PbCPK1 and PbCPK2 underwent the most intense expression changes of these six genes tested. In ‘Xuehua’ pear, only PbCBL1 and PbCIPK2 were significantly upregulated following infection, while the remaining genes were downregulated to varying degrees. In conclusion, V. nashicola infection induced Ca2+ signaling in pear cells, and changes in [Ca2+]cyt were mainly due to extracellular Ca2+ influx. In ‘Huangguan’ pear, all tested genes related to Ca2+ sensors responded to V. nashicola infection, and might participate in resistance to the scab pathogen.

Neutrophils injure gallbladder interstitial Cajal-like cells in a guinea pig model of acute cholecystitis.

Acute cholecystitis is a common disease with gallbladder dysmotility. Disease pathogenesis involves immune cell infiltration as well as changes in gallbladder interstitial Cajal-like cells (ICLCs). However, it remains unclear if or how the immune cells affect ICLC morphology, density, distribution, and function in gallbladder tissue during acute cholecystitis. In this study, we explored the acute cholecystitis-related alterations in gallbladder ICLCs in a guinea pig model, focusing on the effects of neighboring neutrophils. Adult guinea pigs were randomly divided into four groups (control, 24 hr common bile duct ligation [CBDL], 48-hr CBDL, and antipolymorphonuclear neutrophil [PMN] treated) and analyzed using methylene blue staining and immunofluorescence. Gallbladder contractility was also monitored. To culture gallbladder ICLCs, collagenase digestion was performed on tissue from 10- to 15-day-old guinea pigs. Neutrophils isolated from the peripheral blood of experimental animals 48-hr postsurgery were also cocultured with the gallbladder ICLCs. Intracellular calcium was detected with Fluo-4 AM dye. Our results showed that gallbladder ICLC density significantly declined during acute cholecystitis and was accompanied by shortening of the cellular processes and damage to their network-like structure. However, pretreatment with anti-PMN partially prevented these changes. Gallbladder contraction was also significantly decreased during acute cholecystitis, and this appeared to be mediated by the neutrophils. Moreover, ICLCs cocultured with neutrophils also had shortened and reduced processes and impaired network-like structure formation. Intracellular calcium transient was less sensitive to contraction agonists and inhibitors when cocultured with neutrophils. Taken together, neutrophils greatly affect gallbladder ICLCs and dysmotility during acute cholecystitis.

A new pH-sensitive fluorescent probe for visualization of endoplasmic reticulum acidification during stress

A novel pH-sensitive fluorescent probe termed ER-H for visualization of ER acidification during ER stress in live cells and in vivo was presented. ER-H consists of a piperazine-linked 1,8-naphthalimide as a fluorescence turn-on signaling unit, a 4-methyl benzenesulphonamide moiety for ER anchoring, and a thiol reactive benzyl chloride subunit for biomacromolecules fixation at the ER. ER-H displays sensitive fluorescence enhancement under acidic condition, thus enabling it to monitor ER acidification. Meanwhile, ER-H shows negligible response to common species, high photostability and low cytotoxicity. In biological experiments, the probe ER-H can solely accumulate into ER and track the acidification during ER stress. By combining ER-H with calcium ion sensor Fluo-3 AM, we achieved the simultaneous fluorescence imaging of decrease of ER pH and increase of cytoplasmic calcium ion. Additionally, the probe was also utilized for fluorescence imaging of the acidification in zebrafish and mice model successfully. We thus believe that ER-H has great potential as a practical tool for real-time monitoring of the acidification of ER, contributing to revealing the pathogenesis of ER stress associated diseases.

Only a subpopulation of mouse sperm displays a rapid increase in intracellular calcium during capacitation.

Mammalian sperm must undergo a functionally defined process called capacitation to be able to fertilize oocytes. They become capacitated in vivo by interacting with the female reproductive tract or in vitro in a defined capacitation medium that contains bovine serum albumin, calcium (Ca2+ ), and bicarbonate (HCO3- ). In this work, sperm were double stained with propidium iodide and the Ca2+ dye Fluo-4 AM and analyzed by flow cytometry to determine changes in intracellular Ca2+ concentration ([Ca2+ ]i ) in individual live sperm. An increase in [Ca2+ ]i was observed in a subpopulation of capacitated live sperm when compared with noncapacitated ones. Sperm exposed to the capacitating medium displayed a rapid increase in [Ca2+ ]i within 1 min of incubation, which remained sustained for 90 min. These rise in [Ca2+ ]i after 90 min of incubation in the capacitating medium was evidenced by an increase in the normalized median fluorescence intensity. This increase was dependent on the presence of extracellular Ca2+ and, at least in part, reflected the contribution of a new subpopulation of sperm with higher [Ca2+ ]i . In addition, it was determined that the capacitation-associated [Ca2+ ]i increase was dependent of CatSper channels, as sperm derived from CatSper knockout (CatSper KO) or incubated in the presence of CatSper inhibitors failed to increase [Ca2+ ]i . Surprisingly, a minimum increase in [Ca2+ ]i was also observed in CatSper KO sperm suggesting the existence of other Ca2+ transport systems. Altogether, these results indicate that a subpopulation of sperm increases [Ca2+ ]i very rapidly during capacitation mainly due to a CatSper-mediated influx of extracellular Ca2+ .

Nanosecond pulsed electric field induces calcium mobilization in osteoblasts

Nanosecond pulsed electric field (nsPEF) has the ability to induce a host of intracellular biochemical processes in living cells as a function of parameter setting. In vitro experiments proved that nsPEF stimulation could remarkably promote biomineralization processes and new bone formation. However, the impact of nsPEF parameter settings on the calcium flux of osteoblasts, as well as the intracellular mechanisms underlying those observations have yet to be elucidated. In this study, live osteoblast-like MG63 cells were loaded with fluorescence indicator dye fluo-4 AM. nsPEF stimulation could induce intracellular calcium mobilization in MG63 cells with no refractory period. We confirmed that moderate output voltage (4–8 KV) and large pulse number (25) were required for efficient nsPEF irritation of MG63 cells. Additionally, nsPEF stimulation-induced calcium flux in MG63 cells was dramatically reduced with the treatment by ethylene glycol tetraacetic acid (EGTA), proving that the intake of extracellular calcium ions is crucial for increasing intracellular calcium concentration of nsPEF-treated MG63 cells. Our preliminary study investigated the mechanisms underlying nsPEF stimulation-induced calcium mobilization in osteoblast-like cells, and it has the potential to accelerate the application of nsPEF stimulation in new bone formation.

Nanosecond pulsed electric fields promoting the proliferation of porcine iliac endothelial cells: An in vitro study.

Currently, nanosecond pulsed electric fields (nsPEFs) with short pulse duration and non-thermal effects have various potential applications in medicine and biology, especially in tumor ablation. Additionally, there are a few investigations on its proliferative effects in the normal cell. Clinically, proliferation of endothelial cells can perhaps accelerate the stent endothelialization and reduce the risk of acute thrombosis. To explore the feasibility using nsPEFs to induce proliferation of endothelial cells, in this study, porcine iliac endothelial (PIEC) cell line was cultured and tested by CCK-8 assay after nsPEFs treatment. The results reflected that nsPEFs with low field strength (100ns, 5 kV/cm, 10 pulses) had a significant proliferative effect with an increase in the PIEC cell growth of 16% after a 48 hour’ post-treatment. To further understand the mechanism of cell proliferation, intracellular Ca2+ concentration was measured through fluo-4 AM and reactive oxygen species assay was applied to estimate the level of intracellular reactive oxygen species (ROS). Finally, the total nitric oxide assay for NO production in the cultured medium was evaluated. An enhanced concentration of intracellular Ca2+ and ROS were observed, while the concentration of extracellular NO also increased after nsPEFs treatment. Such experimental results demonstrated that nsPEFs with appropriate pulse parameters could effectively enhance cell proliferation on PIEC cells, and the cell proliferation associated strongly with the changes of intracellular Ca2+ concertation, ROS and NO production induced by nsPEFs treatment. This in vitro preliminary study indicates that as a novel physical doping, the nsPEFs have potential in stimulating endothelial cells to accelerate stent endothelialization.

Effects of nanosecond pulsed electric fields (nsPEFs) on the human fungal pathogen Candida albicans: an in vitro study

Candida albicans is the leading human fungal pathogen that causes many life-threatening infections. Notably, the current clinical trial data indicate that Candida species shows the emerging resistance to anti-fungal drugs. The aim of this study was to evaluate the antifungal effects of nanosecond pulsed electric fields (nsPEFs) as a novel drug-free strategy in vitro. In this study, we investigated the inactivation and permeabilization effects of C. albicans under different nsPEFs exposure conditions (100 pulses, 100 ns in duration, intensities of 20, 40 kV cm−1). Cell death was studied by annexin-V and propidium iodide staining. The changes of intracellular Ca2+ concentration after nsPEFs treatment were observed using Fluo-4 AM. Results show that C. albicans cells and biofilms were both obviously inhibited and destroyed after nsPEFs treatment. Furthermore, C. albicans cells were significantly permeabilized after nsPEFs treatment. Additionally, nsPEFs exposure led to a large amount of DNA and protein leakage. Importantly, nsPEFs induced a field strength-dependent apoptosis in C. albicans cells. Further experiments revealed that Ca2+ involved in nsPEFs induced C. albicans apoptosis. In conclusion, this proof-of-concept study provides a potential alternative drug-free strategy for killing pathogenic Candida species.

Islet protection and amelioration of type 2 diabetes mellitus by treatment with quercetin from the flowers of <em>Edgeworthia gardneri</em>

Background and purposeThe traditional Chinese medicine – the flower of Edgeworthia gardneri – is reported as an effective therapeutic for type 2 diabetes mellitus (T2DM). Nevertheless, most constituents of the flowers of E. gardneri have not yet been studied. This study was conducted to investigate the effect of quercetin extracted from the flowers of E. gardneri on islet protection and amelioration in T2DM and explore its mechanism.MethodQuercetin was extracted from the flowers of E. gardneri and verified by high-performance liquid chromatography. Quercetin or crude extract’s effect on insulin secretion was investigated. ERK1/2 and phospho-ERK1/2 were detected by Western blot analysis, and fluo-3 AM was used to detect intracellular Ca2+. The anti-apoptosis effect of quercetin or crude extract on MIN-6 cells was investigated by thiazolyl blue tetrazolium bromide (MTT) assay and flow cytometry analysis. Activation of caspases and expression of Bcl-2 and BAX were tested by Western blot analysis. In addition, the mitochondrial membrane potential was determined by JC-1 probe. Moreover, in vivo activity was also tested in db/db mice.ResultsA quercetin level of >10 μmol/L could induce insulin secretion. Intracellular Ca2+ and ERK1/2 were involved in the signaling pathway of quercetin-induced insulin secretion. We also observed that quercetin could inhibit palmitic acid-induced cell apoptosis via suppressing the activation of caspase-3, -9, -12; increasing the ratio of Bcl-2/BAX and reversing the impaired mitochondrial membrane potential. Crude extract’s effect on insulin secretion was similar to that of pure extracted quercetin, while it possessed higher anti-apoptosis activity. Additionally, intraperitoneal glucose tolerance, plasma insulin level, hepatic triglyceride, hepatic glycogen and the pathological histology of both pancreatic islet and liver in db/db mice were significantly improved by the administration of the extracted quercetin.ConclusionOur study indicated that quercetin extracted from the flowers of E. gardneri exerted excellent properties in islet protection and amelioration.

Measurement of cytoplasmic Ca2+ concentration in Saccharomyces cerevisiae induced by air cold plasma

In this study, a novel approach to measure the absolute cytoplasmic Ca2+ concentration ([Ca2+]cyt) using the Ca2+ indicator fluo-3 AM was established. The parameters associated with the probe fluo-3 AM were optimized to accurately determine fluorescence intensity from the Ca2+-bound probe. Using three optimized parameters (final concentration of 6 mM probe, incubation time of 135 min, loading probe before plasma treatment), the maximum fluorescence intensity (Fmax = 527.8 a.u.) and the minimum fluorescence intensity (Fmin = 63.8 a.u.) were obtained in a saturated Ca2+ solution or a solution of lacking Ca2+. Correspondingly, the maximum [Ca2+]cyt induced by cold plasma was 1232.5 nM. Therefore, the Ca2+ indicator fluo-3 AM was successfully applied to measure the absolute [Ca2+]cyt in Saccharomyces cerevisiae stimulated by cold plasma at atmospheric air pressure.

High-Content Screening of the Medicines for Malaria Venture Pathogen Box for Plasmodium falciparum Digestive Vacuole-Disrupting Molecules Reveals Valuable Starting Points for Drug Discovery.

Plasmodium falciparum infections leading to malaria have severe clinical manifestations and high mortality rates. Chloroquine (CQ), a former mainstay of malaria chemotherapy, has been rendered ineffective due to the emergence of widespread resistance. Recent studies, however, have unveiled a novel mode of action in which low-micromolar levels of CQ permeabilized the parasite's digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization, and DNA degradation. These phenotypes implicate the DV as an alternative target of CQ and suggest that DV disruption is an attractive target for exploitation by DV-disruptive antimalarials. In the current study, high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box (2015) was performed to select compounds which disrupt the DV membrane, as measured by the leakage of intravacuolar Ca2+ using the calcium probe Fluo-4 AM. The hits were further characterized by hemozoin biocrystallization inhibition assays and dose-response half-maximal (50%) inhibitory concentration (IC50) assays across resistant and sensitive strains. Three hits, MMV676380, MMV085071, and MMV687812, were shown to demonstrate a lack of CQ cross-resistance in parasite strains and field isolates. Through systematic analyses, MMV085071 emerged as the top hit due to its rapid parasiticidal effect, low-nanomolar IC50, and good efficacy in triggering DV disruption, mitochondrial degradation, and DNA fragmentation in P. falciparum These programmed cell death (PCD)-like phenotypes following permeabilization of the DV suggests that these compounds kill the parasite by a PCD-like mechanism. From the drug development perspective, MMV085071, which was identified to be a potent DV disruptor, offers a promising starting point for subsequent hit-to-lead generation and optimization through structure-activity relationships.

The Bright Calbryte 520 Enables the Probenecid-Free Intracellular Calcium Assays

The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein-coupled receptors (GPCRs) and calcium channel targets. Followed by Fluo-3, Fluo-4 was developed with improved signal/background ratio, and became one of the most widely used Ca2+ indicators. However, there are still a few severe problems with Fluo-4. For example, the use of probenecid with Fluo-4-based calcium assays compromise the assay results since probenecid is well-documented to have a variety of complicated cellular effects. CalBryte 520, which has the identical excitation and emission wavelength as Fluo-4, overcomes this problem. Like other AM cell loading principles, CalBryte 520 AM ester is non-fluorescent and once gets inside the cell, it is hydrolyzed by intracellular esterase and activated to a polar molecule that is well trapped inside the cell. Upon binding Ca2+ ions, CalBryte 520 produces bright signal, extremely high signal to noise ratio, and also demonstartes greatly improved intracellular retention. In this study, the performance of CalBryte 520 AM was evaluated with different receptor signaling pathways using several cell lines including HEK-293, CHO-M1 and CHO-K1 cells in microscope imaging and microplate assays. The microscope imaging data showed that brightness of CalBryte 520 is 5 to 10 times brighter than that of Fluo-4 AM upon binding Ca2+, and requires and no organic-anion transporter inhibitors (such as probenecid) present in the assay system and has around ∼1000 % of response over background signal in microplate assays. In conclusion, CalBryte 520 AM is a new generation of fluorescent indicator for the measurement of intracellular calcium with high signal-to-noise ratio and intracellular retention properties, is a superior indicator for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.

Effects of sevoflurane pretreatment on the apoptosis of rat H9c2 cardiomyocytes and the expression of GRP78.

The protective effect of sevoflurane on apoptosis of rat H9c2 cardiomyocytes induced by H2O2 and the effect on the expression of glucose-regulated protein 78 (GRP78) were investigated. H9c2 cells were routinely cultured and divided into the control, model and sevoflurane groups. Cells in the model group were treated with 400 µM H2O2, and cells in the sevoflurane group were pretreated with sevoflurane prior to treatment with 400 µM H2O2. MTT assay was used to assess cell viability. Annexin V-propidium iodide (AV-PI) double staining flow cytometry was used to detect apoptosis. The intracellular free Ca2+ concentration was measured by the fluorescence-based assay using Fluo-3 AM as a calcium ion fluorescence probe. The mRNA expression level of GRP78 and protein expression levels of GRP78, CHOP and caspase-12 were measured using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The assays showed that after sevoflurane pretreatment the H9c2 cell viability was significantly increased, whereas the H2O2-induced apoptosis, intracellular Ca2+ concentration, mRNA expression of GRP78, and the protein expression of GRP78, CHOP and caspase-12 were all reduced. The results show that pretreatment with sevoflurane inhibited H2O2-induced apoptosis in H9c2 cells. The mechanism may be related to inhibition of the stress-related protein GRP78 expression in endoplasmic reticulum, resulting in decreased intracellular Ca2+ concentration and the downregulation of CHOP and caspase-12 expression levels.

Effects of the Calcium-Activated Chloride Channel Inhibitors T16Ainh-A01 and CaCCinh-A01 on Cardiac Fibroblast Function

Background/Aims: Calcium-activated chloride channels (CaCCs) regulate numerous physiological processes including cell proliferation, migration, and extracellular matrix secretion. T16Ainh-A01 and CaCCinh-A01 are selective inhibitors of CaCCs. But it is unknown whether these two compounds have functional effects on cardiac fibroblasts (CFs). Methods: Primary CFs were obtained by enzymatic dissociation of cardiomyocytes from neonatal rat hearts. Intracellular Ca²⁺ ([Ca²⁺]_i) and Cl^- ([Cl^-]_i) were measured using the fluorescent calcium indicators (Fluo-4 AM) and N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide respectively. The expression of anoctamin-1 (ANO1) and α-smooth muscle actin (α-SMA) was detected by quantitative RT-PCR, immunofluorescence, and western blotting. A hydroxyproline assay was used to examine collagen secretion. Cell proliferation, cell cycle distribution, and cell migration were assessed by Cell Counting Kit-8, flow cytometry, and Transwell assays, respectively. Results: ANO1 was preferentially expressed on the nuclear membrane and partially within intracellular compartments around the nucleus. T16Ainh-A01 and CaCCinh-A01 displayed different inhibitory effects on [Cl^-]_i in CFs. T16Ainh-A01 considerably decreased [Cl^-]_i in the nucleus, whereas CaCCinh-A01 reduced [Cl^-]_i in intracellular compartments around the nucleus, and both inhibitors exhibited a minimal effect on [Ca²⁺]_i in CFs. ANO1 and α-SMA expression levels were significantly repressed by CaCCinh-A01. T16Ainh-A01 showed a marked inhibitory effect on the mRNA levels of ANO1 and α-SMA, but had a negligible effect on ANO1 at the protein level. T16Ainh-A01 and CaCCinh-A01 led to the significant repression of cell proliferation, cell migration, and collagen secretion in CFs. Conclusion: Our findings indicate that T16Ainh-A01 and CaCCinh-A01 have the potential to inhibit the proliferation and collagen secretion of CFs and may serve as novel anti-fibrotic therapeutic drugs in the future.

Antiarrhythmic Effects of Carvedilol and Flecainide in Cardiomyocytes Derived from Catecholaminergic Polymorphic Ventricular Tachycardia Patients.

Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for catecholaminergic polymorphic ventricular tachycardia (CPVT). In this study, we evaluated antiarrhythmic efficacy of carvedilol and flecainide in CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying different mutations in RYR2. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion and L4115 or V4653F mutation in RYR2 and of a healthy individual. Ca2+ kinetics and drug effects were studied with Fluo-4 AM indicator. Carvedilol abolished Ca2+ abnormalities in 31% of L4115F, 36% of V4653F, and 46% of exon 3 deletion carrying CPVT cardiomyocytes and flecainide 33%, 30%, and 52%, respectively. Both drugs lowered the intracellular Ca2+ level and beating rate of the cardiomyocytes significantly. Moreover, flecainide caused abnormal Ca2+ transients in 61% of controls compared to 26% of those with carvedilol. Carvedilol and flecainide were equally effective in CPVT iPSC-CMs. However, flecainide induced arrhythmias in 61% of control cells. CPVT cardiomyocytes carrying the exon 3 deletion had the most severe Ca2+ abnormalities, but they had the best response to drug therapies. According to this study, the arrhythmia-abolishing effect of neither of the drugs is optimal. iPSC-CMs provide a unique platform for testing drugs for CPVT.

Bubbles Induce Endothelial Microparticle Formation via a Calcium-Dependent Pathway Involving Flippase Inactivation and Rho Kinase Activation

Background/Aims: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. Methods: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry. Cytoplasmic calcium (Ca²⁺) was measured by the Ca²⁺ binding dyes Fluo-3 AM and flippase activity was assessed by translocation rate of fluorescent phosphatidylserine (PS) analogue NBD-PS. Protein levels of phospho-myosin light chain (MLC, a Rho kinase substrate) and phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were determined by western blotting. The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy. Results: EMPs level markedly increased after bubble stimulus. Cytoplasmic calcium (Ca²⁺) significantly elevated (P< 0.05), flippase activity decreased (P< 0.05), protein levels of phospho- MLC and phospho- ERK1/2 significantly increased (P< 0.05, P < 0.05), and the score of actin colocalization markedly reduced (P< 0.05) in bubble-stimulated HUVECs. All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺ channel blocker LaCl₃ (P< 0.05). Additionally, MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor pretreatment and more importantly, bubble-induced EMPs markedly decreased. Conclusions: These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺ elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization. Simultaneously, cytoplasmic Ca²⁺ inhibits the flippase activity and subsequently increases phosphatidylserine exposure, which also contributes to EMPs formation.

Calcium Imaging of Nerve-Mast Cell Signaling in the Human Intestine.

Introduction: It is suggested that an altered microenvironment in the gut wall alters communication along a mast cell nerve axis. We aimed to record for the first time signaling between mast cells and neurons in intact human submucous preparations.Methods: We used the Ca2+ sensitive dye Fluo-4 AM to simultaneously image changes in intracellular calcium [Ca+2]i (%ΔF/F) in neurons and mast cells. Data are presented as median with interquartile ranges (25/75%).Results: We recorded nerve responses in 29 samples upon selective activation of 223 mast cells by IgE receptor cross linking with the antibody mAb22E7. Mast cells responded to mAb22E7 with a median [Ca+2]i increase of 20% (11/39) peaking 90 s (64/144) after the application. Only very few neurons responded and the median percentage of responding neuronal area was 0% (0/5.9). Mast cell activation remained in the presence of the fast sodium channel blocker tetrodotoxin. Specific neuronal activation by transmural electrical field stimulation (EFS) in 34 samples evoked instantaneously [Ca+2]i signals in submucous neurons. This was followed by a [Ca+2]i peak response of 8%ΔF/F (4/15) in 33% of 168 mast cells in the field of view. The mast cell response was abolished by the nerve blocker tetrododoxin, reduced by the Calcitonin Gene-Related Peptide receptor 1 antagonist BIBN-4096 and the Vasoactive Intestinal Peptide receptor antagonist PG97-269, but not by blockade of the neurokinin receptors 1–3.Conclusion: The findings revealed bidirectional signaling between mast cells and submucous neurons in human gut. In our macroscopically normal preparations a nerve to mast cell signaling was very prominent whereas a mast cell to nerve signaling was rather rare.

Abstract 20953: M1 and M2 Specific Purinergic Receptor Repertoire Alters Response to Damage Associated Molecular Patterns

Introduction: Dying cells release damage associated molecular patterns (DAMPs) like nucleotides into the extracellular fluid, orchestrating a profound immune response. Purinergic receptors decrypt the composition of extracellular nucleotides and translate it to distinct cellular reactions. M1 and M2 macrophages are immune cells involved in many diseases which react in a completely different way. Nevertheless, how the microenvironment exactly triggers their behavior is not fully understood. Hypothesis: We hypothesize a distinct expression pattern of purinergic receptors on M1 and M2 macrophages which leads to an unique microenvironment recognition and behavior. Methods: Macrophages were generated by bone marrow isolation and cultivation with macrophage colony stimulating factor (M-CSF). Bone marrow derived macrophages (BMDMs) were differentiated to M1 via addition of IFNγ and LPS. M2 differentiation was initiated by the addition of IL-4. Gene transcription quantification was assessed by real-time PCR. Intracellular Ca 2+ signaling was quantified by Fluo-4 AM. Results: Differentiation of BMDMs with either IFNγ/LPS or IL-4 leads to the upregulation of M1 or M2 specific gene transcription. Subsequent real-time PCR analysis of purinergic receptors revealed a strong relationship between the expression pattern and the differentiation to M1 or M2 macrophages. Whereas M1 macrophages upregulate predominantly G αi coupled metabotropic P2Y receptors, M2 macrophages preferentially upregulate G αq coupled P2Y receptors. Comparison of the two ADP-dependent P2Y receptors (P2Y 1 and P2Y 13 ) showed a M1 and M2 specific expression pattern with M1 macrophages solely expressing the G αi coupled P2Y 13 and M2 macrophages exclusively expressing the G αq coupled P2Y 1 receptor. In agreement with the specific expression pattern, ADP stimulation evokes a significant Ca 2+ flux in M2 macrophages but failed to induce it in M1 macrophages. Conclusion: Our experiments revealed for the first time that M1 and M2 macrophages express a unique purinergic receptor repertoire and sense the nucleotide based microenvironment differently. With this knowledge we could interfere with one macorphage subtype without disturbing the others.

Peptide YY (3–36) modulates intracellular calcium through activation of the phosphatidylinositol pathway in hippocampal neurons

Peptide YY (PYY) belongs to the neuropeptide Y (NPY) family, which also includes the pancreatic polypeptide (PP) and NPY. PYY is secreted by the intestinal L cells, being present in the blood stream in two active forms capable of crossing the blood brain barrier, PYY (1–36) and its cleavage product, PYY (3–36). PYY is a selective agonist for the Y2 receptor (Y2R) and these receptors are abundant in the hippocampus. Here we investigated the mechanisms by which PYY (3–36) regulates intracellular Ca2+ concentrations ([Ca2+]i) in hippocampal neurons by employing a calcium imaging technique in hippocampal cultures. Alterations in [Ca2+]i were detected by changes in the Fluo-4 AM reagent emission. PYY (3–36) significantly increased [Ca2+] from the concentration of 10−11M as compared to the controls (infusion of HEPES-buffered solution (HBS) solution alone). The PYY (3–36)-increase in [Ca2+]i remained unchanged even in Ca2+-free extracellular solutions. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pump (SERCA pump) inhibition partially prevent the PYY (3–36)-increase of [Ca2+]i and inositol 1,4,5-triphosphate receptor (IP3R) inhibition also decreased the PYY (3–36)-increase of [Ca2+]i. Taken together, our data strongly suggest that PYY (3–36) mobilizes calcium from the neuronal endoplasmic reticulum (ER) stores towards the cytoplasm. Next, we showed that PYY (3–36) inhibited high K+-induced increases of [Ca2+]i, suggesting that PYY (3–36) could also act by activating G-protein coupled inwardly rectifying potassium K+ channels. Finally, the co-infusion of the Y2 receptor (Y2R) antagonist BIIE0246 with PYY (3–36) abolished the [Ca2+]i increase induced by the peptide, suggesting that PYY (3–36)-induced [Ca2+]i increase in hippocampal neurons occurs via Y2Rs.