Fluo-4 AM Research Articles

Pterosin sesquiterpenoids from Pteris laeta Wall. ex Ettingsh. protect cells from glutamate excitotoxicity by modulating mitochondrial signals.

The genus Pteris (Pteridaceae) has been used as a traditional herb for a long time. In particular, Pteris laeta Wall. ex Ettingsh. has been widely used in traditional Chinese medicine to treat nervous system diseases and some pterosin sesquiterpenes from Pteris show neuroprotective activity, but their underlying molecular mechanisms remain elusive. Therefore, to investigate the neuroprotective activity and working mechanism of pterosin sesquiterpenes from P. laeta Wall. ex Ettingsh. will provide a better understanding and guidance in using P. laeta Wall. ex Ettingsh. as a traditional Chinese medicine. We aim to develop effective treatments for neurodegenerative diseases from pterosin sesquiterpenes by evaluating their neuroprotective activity and investigating their working mechanisms. Primary screening on the glutamate-induced excitotoxicity cell model was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Fluorescent-activated cell sorting (FACS) was used to analyze the activation level of glutamate receptors and mitochondria membrane potential after treatment. Transcriptomics and proteomics analysis was performed to identify possible targets of pterosin B. The key pathways were enriched by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The core targets were visualized by a protein-protein interaction network using STRING. The mRNA and protein expressions were evaluated using real-time quantitative polymerase chain reaction (Q-PCR) and western blot, respectively. Immunocytochemistry was performed to monitor mitochondrial and apoptotic proteins. Cellular reactive oxygen species (ROS) were measured by ROS assay, and Ca2+ was stained with Fluo-4 AM to quantify intracellular Ca2+ levels. We found pterosin B from Pteris laeta Wall. ex Ettingsh. showed significant neuroprotective activity against glutamate excitotoxicity, enhancing cell viability from 43.8% to 105% (p-value: <0.0001). We demonstrated that pterosin B worked on the downstream signaling pathways of glutamate excitotoxicity rather than directly blocking the activation of glutamate receptors. Pterosin B restored mitochondria membrane potentials, alleviated intracellular calcium overload from 107.4% to 95.47% (p-value: 0.0006), eliminated cellular ROS by 36.55% (p-value: 0.0143), and partially secured cells from LPS-induced inflammation by increasing cell survival from 46.75% to 58.5% (p-value: 0.0114). Notably, pterosin B enhanced the expression of nuclear factor-erythroid factor 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) by 2.86-fold (p-value: 0.0006) and 4.24-fold (p-value: 0.0012), and down-regulated Kelch-like ECH-associated protein 1 (KEAP1) expression by 2.5-fold (p-value: 0.0107), indicating that it possibly promotes mitochondrial biogenesis and mitophagy to maintain mitochondria quality control and homeostasis, and ultimately inhibits apoptotic cell death. Our work revealed that pterosin B protected cells from glutamate excitotoxicity by targeting the downstream mitochondrial signals, making it a valuable candidate for developing potential therapeutic agents in treating neurodegenerative diseases.

Beta-arrestin-biased agonism promotes TRPC6-mediated calcium influx in human podocytes

Background: Angiotensin Receptor blockers (ARBs) are the first-line treatment for hypertension and other chronic kidney diseases and act by inhibiting signaling through the angiotensin 1 receptor (AT1R). AT1R activation initiates signaling through G protein-coupled receptor (GPCR) effector signaling and β-arrestins. Recently, a novel AT1R agonist TRV120027 (TRV) that selectively activates the β-arrestin cascade without impacting the GCPR pathway has become available. Therefore, we hypothesized that β-arrestin and G-protein signaling pathways via AT1R have distinct downstream effects in podocytes. Furthermore, we propose that β-arrestin signaling initiates TRPC6-mediated Ca 2+ entry. Methods: We used a conditionally immortalized human podocyte cell line to determine β-arrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization. Intracellular Ca 2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV applications were analyzed with confocal microscopy. The role of TRPC channels was tested by single-channel electrophysiology and confocal imaging using a variety of commercially available inhibitors (AC1903 for TRPC5 and GSK283350A, SAR7334 for TRPC6). Western blotting for apoptosis-associated proteins, TUNEL staining to detect DNA cleavage, and actin cytoskeleton rearrangements were performed in podocytes exposed to TRV or Ang II treatment. Results: Our experiments determined that TRV-mediated β-arrestin pathway activation in podocytes promotes rapid elevation of intracellular Ca 2+ in a dose-dependent manner (IC 50 =15μM, ligand-receptor binding fit modeled with Levenberg–Marquardt algorithm, adj. R 2 =0.93). Interestingly, the amplitude of β-arrestin-mediated Ca 2+ influx was four times higher than the response to similar Ang II concentrations (t-test, n≥30, p&lt;0.05). Moreover, the TRV response was significantly increased under hyperglycemic stress conditions, and promoted rapid apoptosis in podocytes (one-way ANOVA, n≥21, p=0.003). The pharmacological blockade of TRPC6, but not TRPC5, significantly attenuated Ca 2+ influx in response to β-arrestin. In contrast to Ang II, TRV does not induce AT1R-mediated NO production, suggesting that β-arrestin activation only targets AT1R, and not AT2R. Single-channel analyses show rapid activation of TRPC activity in response to acute TRV application in podocytes (one-way ANOVA, n=8, p=0.004). Overall prolonged activation of the β-arrestin pathway in podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton distribution, and increased apoptotic cell markers. Conclusions: TRV-mediated β-arrestin signaling in podocytes promotes high ionotropic TRPC6 channel-mediated Ca 2+ influx, cytoskeleton rearrangement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment is controversial and requires further investigation. DK126720 (to OP), DK129227 (to AS and OP), and NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (to OP), endowed funds from the SC SmartState Centers of Excellence (to OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BS-452682-2 THE IMPAIRED INTRACELLULAR CALCIUM BUFFERING IN ATRIAL MYOCYTES OF PATIENTS WITH LONG-STANDING PERSISTENT ATRIAL FIBRILLATION IS MEDIATED BY REDUCED CARDIAC TROPONIN C LEVELS

Decreased levels of myofilament proteins and impaired atrial Ca2+ homeostasis contribute to atrial hypocontractility and thromboembolic events in long-standing persistent atrial fibrillation (AF). The myofilament protein cardiac troponin C (cTnC) is known to be the major cytosolic Ca2+ buffer, but its role in AF-associated Ca2+ handling abnormalities remains poorly investigated. This study aimed to clarify the influence of cTnC on impaired intracellular Ca2+ signalling in AF. Right atrial myocytes from sinus rhythm (Ctrl) and AF patients, as well as atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with knocked down cTnC levels (si-cTnC) were investigated by simultaneously measuring membrane currents (patch-clamp technique) and cytosolic Ca2+ (Fluo-3 AM and Fluo-4 AM). Myofilament protein levels were determined using immunoblotting. Cardiac troponin C protein levels were reduced in atrial tissues of AF patients. By integrating Na+-Ca2+-exchange current (INCX) during caffeine-induced Ca2+ release, SR Ca2+ content was determined and similar in both groups. Buffering parameters (maximum buffer capacity, Bmax, and dissociation constant of buffers, Kd) were obtained by fitting a hyperbolic function on buffering curves generated from INCX and the caffeine-induced Ca2+ transient. However, intracellular calcium buffering was reduced in AF, presumably due to the reduced cardiac troponin C. (Figures A and B). Interestingly, the reduction of cTnC levels using small interfering RNA (siRNA) caused the same alterations in intracellular Ca2+ buffering in atrial iPSC-CM. (Figure C). In these modelled cells, a higher Ca2+ spark frequency was observed as opposed to the control group (Figure D). Diminished cTnC levels underlie the reduced intracellular Ca2+ buffering observed in AF. By promoting spontaneous SR Ca2+ release events that could trigger delayed afterdepolarizations, reduced cTnC levels could contribute to the arrhythmogenic substrate promoting AF.

Dibazol-induced relaxation of ophthalmic artery in C57BL/6J mice is correlated with the potency to inhibit voltage-gated Ca2+ channels

We aimed to explore the effect of dibazol on the ophthalmic artery (OA) and ophthalmic artery smooth muscle cells (OASMCs) of C57BL/6J mice as well as the underlying mechanisms. The OA of C57BL/6J mice was isolated under a dissecting microscope for primary OASMCs culture and myogenic tests. OASMCs were identified through morphological and immunofluorescence analyses. Morphology changes in the OASMCs were examined by staining using rhodamine-phalloidin. We performed a collagen gel contraction assay to measure the contractile and relaxant activities of the OASMCs. The molecular probe Fluo-4 AM was used to examine intracellular free Ca2+ levels ([Ca2+]in). The myogenic effects of OA were examined using wire myography. Additionally, the whole-cell patch-clamp technique was used to investigate the mechanisms underlying the relaxant effect of dibazol on L-type voltage-gated Ca2+ channels (LVGC) in isolated cells. 10−5 M dibazol significantly inhibited the contraction of OASMCs and increased the [Ca2+]in response to 30 mM KCl in a concentration-dependent manner. Dizabol had a more significant relaxant effect than 10−5 M isosorbide dinitrate (ISDN). Similarly, dibazol showed a significant dose-dependent relaxant effect on OA contraction induced by 60 mM KCl or 0.3 μM 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619). The current-voltage (I–V) curve revealed that dibazol decreased Ca2+ currents in a concentration-dependent manner. In conclusion, dibazol exerted relaxant effects on the OA and OASMCs, which may involve the inhibition of the Ca2+ influx through LVGC in the cells.

Abstract 4863: PMCA inhibition induces cytotoxic calcium stress and suppression of Wnt signaling in refractory molecular subtype of gastric cancer

Abstract Background: Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide, but its genetic heterogeneity retards the development of novel therapeutics. Recently, tumor molecular classification identified clinically refractory SEM (i.e., Stem-like, EMT and Mesenchymal) subtype GC which exhibited over-expression of the plasma membrane Ca2+ ATPase (PMCA) whose role is circumventing calcium overload causing ER stress and apoptosis. Here, we showed that PMCA4 is associated with Wnt signaling activity by regulating Secreted Frizzled-Related Protein 2 (SFRP2) expression and demonstrated that PMCA4 inhibition might achieve dual mechanism-specific anti-cancer effect by inducing calcium-mediated apoptosis and restraining oncogenic Wnt activity in SEM subtype GC. Materials and Methods: GC cell lines and patient-derived organoids (PDOs) were categorized either SEM-subtype (SNU484, SNU668, SK4, MKN1; GA077T, GA265T, GA333T, GA352T) or non-SEM subtype (MKN45, NCIN87, SNU16, SNU601; GA200T, GA247T, GA215T) based on transcriptome signatures or subtype-specific biomarker genes. PMCA4 expression was confirmed using western blot and its calcium clearance function was examined using Fluo-4 AM fluorescent Ca2+ dye. Biological and molecular characteristics of GC cell lines and PDOs upon PMCA4 inhibition were investigated using RT-qPCR, western blot, and TOPflash/FOPflash reporter assay. The target engagement of novel PMCA inhibitor PM058 (bis-indole maleimide derivative) was evaluated by cellular thermal shift assay (CETSA), and the efficacy was determined by CCK8 cell viability assay for cell lines and by Cell Titer-Glo for PDOs.Results: PMCA4 was highly expressed in SEM subtype GC cell lines and PDOs. The siRNA-mediated PMCA4 knockdown (KD) or pharmaceutical inhibition of PMCA4 suppressed the calcium clearance, the cell viability, and decreased cell invasion/migration. TCF/LEF-responsive reporter assay revealed that pharmaceutical inhibition of PMCA4 suppressed Wnt/beta-catenin signaling activity, proven by decrease in beta-catenin and Wnt target gene expression. PMCA inhibition was mechanistically associated with the increased expression of SFRP2, a Wnt antagonist. Importantly, PM058, a PMCA inhibitor, treatment resulted in highly efficacious anti-cancer effect in PDOs at nano molar range, which correlated with PMCA4 protein expression.Discussion and Conclusion: Our results provide the proof of concept that PMCA4 can be a promising therapeutic target for refractory subtype cancer. The proposed mechanism of PMCA4 inhibition is calcium overload - induced apoptosis and interfering with Wnt signaling activity via SFRP2 in GC. Importantly, we demonstrated a novel potent inhibitor PM058 that blocks PMCA function in clinically intractable SEM subtype GC cancer. Citation Format: Saeli Ban, Jungmin Kim, Jonghwan Bae, Ki Cheong Park, Suji Lee, Moon Hwan Kim, Jae-Ho Cheong. PMCA inhibition induces cytotoxic calcium stress and suppression of Wnt signaling in refractory molecular subtype of gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4863.

The Mechanism of the Anti-Cardiac Hypertrophy Effect of Glycyrrhizic Acid Is Related to Reducing STIM1-Dependent Store-Operated Calcium Entry

We explored the anti-cardiac hypertrophy mechanism of glycyrrhizic acid from the perspective of calcium regulation under pathological conditions. For this purpose, we used a rat model of myocardial hypertrophy induced by pressure overload. The effect of glycyrrhizic acid on BP was measured non-invasively with a sphygmomanometer and recorded in PC. In rats with modeled cardiac hypertrophy, the effect of GA on expression of type 1 matrix interaction molecules was determined in horizontal tissues and cultured cardiomyocytes of the left ventricle. The laser confocal microscopy and calcium ion probe Fluo-4 AM were used to assess the effect of glycyrrhizic acid on stromal interaction molecule 1 (STIM1)-dependent store-operated calcium entry in cultured cardiomyocytes derived from the hypertrophic myocardium. Glycyrrhizic acid exerted the anti-hypertrophic effect in rats with hypertrophic myocardium by down-regulating STIM1 protein expression and reducing the intensity of STIM1-dependent store-operated calcium entry.

Rare-earth orthovanadate nanoparticles trigger Ca2+-dependent eryptosis

Introduction. Rare-earth orthovanadate nanoparticles (ReVO4:Eu3+, Re = Gd, Y or La) are promising agents for photodynamic therapy of cancer due to their modifiable redox properties. However, their toxicity limits their application. Objective. The aim of this research was to elucidate pro-eryptotic effects of GdVO4:Eu3+ and LaVO4:Eu3+ nanoparticles with identification of underlying mechanisms of eryptosis induction and to determine their pharmacological potential in eryptosis-related diseases. Methods. Blood samples (n = 9) were incubated for 24 h with 0–10–20–40–80 mg l−1 GdVO4:Eu3+ or LaVO4:Eu3+ nanoparticles, washed and used to prepare erythrocyte suspensions to analyze the cell membrane scrambling (annexin-V-FITC staining), cell shrinkage (forward scatter signaling), reactive oxygen species (ROS) generation through 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) staining and intracellular Ca2+ levels via FLUO4 AM staining by flow cytometry. Internalization of europium-enabled luminescent GdVO4:Eu3+ and LaVO4:Eu3+ nanoparticles was assessed by confocal laser scanning microscopy. Results. Both nanoparticles triggered eryptosis at concentrations of 80 mg l−1. ROS-mediated mechanisms were not involved in rare-earth orthovanadate nanoparticles-induced eryptosis. Elevated cytosolic Ca2+ concentrations were revealed even at subtoxic concentrations of nanoparticles. LaVO4:Eu3+ nanoparticles increased intracellular calcium levels in a more pronounced way compared with GdVO4:Eu3+ nanoparticles. Our data disclose that the small-sized (15 nm) GdVO4:Eu3+ nanoparticles were internalized after a 24 h incubation, while the large-sized (∼30 nm) LaVO4:Eu3+ nanoparticles were localized preferentially around erythrocytes. Conclusions. Both internalized GdVO4:Eu3+ and non-internalized LaVO4:Eu3+ nanoparticles (80 mg l−1) promote eryptosis of erythrocytes after a 24 h exposure in vitro via Ca2+ signaling without involvement of oxidative stress. Eryptosis is a promising model for assessing nanotoxicity.

Neuroprotective Effects of Ethanol Extract of Polyscias fruticosa (EEPF) against Glutamate-Mediated Neuronal Toxicity in HT22 Cells.

In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.

Baicalein facilitates gastric cancer cell apoptosis by triggering endoplasmic reticulum stress via repression of the PI3K/AKT pathway

ObjectiveGastric cancer (GC) remains a prevailing threat to life. Baicalein exhibits anti-cancer properties. This study estimated the mechanism of baicalein in GC cell apoptosis by mediating endoplasmic reticulum stress (ERS) through the PI3K/AKT pathway.MethodsAfter treatment with different concentrations of baicalein, GC cell (HGC-27 and AGS) viability was detected by MTT assay. AGS cells more sensitive to baicalein treatment were selected as study subjects. The IC50 of baicalein on AGS cells was determined. Colony formation, cell cycle, and apoptosis were detected using crystal violet staining and flow cytometry. Levels of ERS-related and BTG3/PI3K/AKT pathway-related proteins were determined by Western blot. Intracellular Ca2+ level was measured using Fluo-3 AM fluorescence working solution. GC mouse models were established by subcutaneously injecting AGS cells into the right rib and were intragastrically administrated with baicalein. Tumor volume and weight were recorded. Expression of Ki67 in tumor tissues and positive expression of apoptotic cells were detected by immunohistochemistry and TUNEL staining.ResultsBaicalein inhibited cell proliferation and induced G0/G1 arrest and apoptosis by regulating the cell cycle, and triggered ERS in GC cells. Baicalein impeded the PI3K/AKT pathway by activating BTG3, thereby triggering ERS and inducing apoptosis. BTG3 inhibition reversed baicalein-induced apoptosis and ERS. Baicalein regulated GC cells in a concentration-dependent manner. Moreover, in xenograft mice, baicalein prevented tumor growth, decreased Ki67-positive cells, activated BTG3, and inhibited the PI3K/AKT pathway, thus activating ERS and increasing apoptotic cells.ConclusionBaicalein facilitates GC cell apoptosis by triggering ERS via repression of the PI3K/AKT pathway.

Optimizing precision nanoparticle delivery for magneto-mechanically-based calcium modulation.

Modulating calcium influx events through mechanically actuated magnetic nanoparticles (MNPs) at the cell membrane or in the cytosol of neurons has been trending in the past few years. The mechanical force load is a critical parameter that can be fine-tuned through MNPs concentration and the incubation time. Commonly, MNPs get incubated for 24 h to improve the probability of neuronal uptake; however, critical cellular processes such as protein turnover also coincide during that time frame. Hence, we optimized the incubation time for MNPs to be below <24 h while showing similar calcium response events in neurons. To achieve this goal, we recorded transient calcium fluorescence (Fluo-4 AM, 10x Leica DMI-8, 4 fps) in dissociated primary rat cortical neurons (at 14 days in vitro) with starch-amine functionalized MNPs (incubated for 4 h and 24 h) over 12 min, where a magnetic field actuated 40-80 pN forces on the MNPs from 4 min to 8 min. We decomposed the mean somatic calcium signals into a resting concentration (lower envelope) and calcium events (magnitude and frequency) and correlated them with the Sørenson-Dice. All data was compared with the Mann-Whitney U test (N = 4 cultures, single neuron cells: control = 2262, 4 h = 2424, 24 = 1062). Our results showed that both incubation times provided increases in resting concentration and calcium event frequency and decreased event magnitude and synchronous correlation in comparison to the control. Although similar, we observed that the 4 h incubation time increased the calcium event response with higher frequency and decreased event magnitudes and synchronous correlation when compared to the 24 h incubation. Altogether, our results show that shorter MNPs incubation times can make calcium influx more precise and potentially overcome adverse effects stemming from protein turnover.

FM1-43 Dye Memorizes Piezo1 Activation in the Trigeminal Nociceptive System Implicated in Migraine Pain.

It has been proposed that mechanosensitive Piezo1 channels trigger migraine pain in trigeminal nociceptive neurons, but the mechanosensitivity of satellite glial cells (SGCs) supporting neuronal sensitization has not been tested before. Moreover, tools to monitor previous Piezo1 activation are not available. Therefore, by using live calcium imaging with Fluo-4 AM and labeling with FM1-43 dye, we explored a new strategy to identify Piezo channels' activity in mouse trigeminal neurons, SGCs, and isolated meninges. The specific Piezo1 agonist Yoda1 induced calcium transients in both neurons and SGCs, suggesting the functional expression of Piezo1 channels in both types of cells. In Piezo1-transfected HEK cells, FM1-43 produced only a transient fluorescent response, whereas co-application with Yoda1 provided higher transient signals and a remarkable long-lasting FM1-43 'tail response'. A similar Piezo1-related FM1-43 trapping was observed in neurons and SGCs. The non-specific Piezo channel blocker, Gadolinium, inhibited the transient peak, confirming the involvement of Piezo1 receptors. Finally, FM1-43 labeling demonstrated previous activity in meningeal tissues 3.5 h after Yoda1 washout. Our data indicated that trigeminal neurons and SGCs express functional Piezo channels, and their activation provides sustained labeling with FM1-43. This long-lasting labelling can be used to monitor the ongoing and previous activation of Piezo1 channels in the trigeminal nociceptive system, which is implicated in migraine pain.

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations &gt; 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

Capsazepine antagonizes TRPV1 activation induced by thermal and osmotic stimuli in human odontoblast-like cells.

Dental pain, which is the main reason for patients consulting dentists, is classified as a public health concern. The study of cellular and molecular mechanisms contributing to pain is a fundamental element for developing new analgesics. By using a selective antagonist in an in vitro model, this study aimed to establish the role of TRPV-1 in human odontoblast-like cells (OLCs) as a therapeutic target for dental pain mediated by noxious thermal and osmotic stimuli. OLCs were differentiated from dental pulp mesenchymal cells and TRPV1 expression was evaluated. Activation of TRPV-1 was determined by evaluating changes in calcium concentration after stimulation with mannitol and xylitol hyperosmotic solutions or DMEM heated at 45°C, using the fluorescent calcium probe Fluo-4 AM. In addition, changes in fluorescence (F/F0) due to calcium flux were evaluated using fluorometry and flow cytometry. Simultaneously, the cells were co-stimulated with the selective antagonist capsazepine (CZP). OLCs expressed DSPP and DMP-1, confirming their cellular phenotype. TRPV1 was expressed, and its activation by different stimuli produced an increase in cytosolic Ca2+ which was reduced by the antagonist. Both methods used to evaluate TRPV1 activation through the measurement of calcium probe fluorescence showed similar patterns. These results suggest that TRPV-1 modulation using an antagonist can be implemented as a pharmacological strategy for managing dental pain mediated by hyperosmotic and thermal stimuli.

Impaired Platelet Function and Thrombus Formation in PDE5A-Deficient Mice.

Intracellular cyclic GMP (cGMP) inhibits platelet function. Platelet cGMP levels are controlled by phosphodiesterase 5A (PDE5A)-mediated degradation. However, the exact role of PDE5A in platelet function and thrombus formation remains poorly understood. In this study, we characterized the role of PDE5A in platelet activation and function. Platelets were isolated from wild type or PDE5A-/- mice to measure platelet aggregation, activation, phosphatidylserine exposure (annexin-V binding), reactive oxygen species (ROS) generation, platelet spreading as well as clot retraction. Cytosolic calcium mobilization was measured using Fluo-4 AM by a microplate reader. Western blot was used to measure the phosphorylation of VASP, ERK1/2, p38, JNK, and AKT. FeCl3-induced arterial thrombosis and venous thrombosis were assessed to evaluate the in vivo hemostatic function and thrombus formation. Additionally, in vitro thrombus formation was assessed in a microfluidic whole-blood perfusion assay. PDE5A-deficient mice presented significantly prolonged tail bleeding time and delayed arterial and venous thrombus formation. PDE5A deficiency significantly inhibited platelet aggregation, ATP release, P-selectin expression, and integrin aIIbb3 activation. In addition, an impaired spreading on collagen or fibrinogen and clot retraction was observed in PDE5A-deficient platelets. Moreover, PDE5A deficiency reduced phosphatidylserine exposure, calcium mobilization, ROS production, and increased intracellular cGMP level along with elevated VASP phosphorylation and reduced phosphorylation of ERK1/2, p38, JNK, and AKT. In conclusion, PDE5A modulates platelet activation and function and thrombus formation, indicating that therapeutically targeting it might be beneficial for the treatment of thrombotic diseases.

The potential function of SP1 and CPPED1 in restenosis after percutaneous coronary intervention.

Impacts of molecular pathways have been discussed recently on restenosis after percutaneous coronary intervention (PCI). Hence, this study aimed to explore the impact of calcineurin-like phosphoesterase domain containing 1 (CPPED1) and specificity protein 1 (SP1) on restenosis after PCI. A carotid balloon injury rat model was established, followed by western blot analysis of SP1 and CPPED1 expression in carotid artery (CA) tissues. After SP1 and CPPED1 were overexpressed, the neointimal hyperplasia and luminal stenosis were assessed. In addition, EPC underwent hypoxia/reoxygenation (H/R) treatment to construct an endothelial injury cell model. Then, cell proliferation, apoptosis, intracellular reactive oxygen species (ROS), and Ca2+ concentration were detected with cell counting kit-8 (CCK-8), flow cytometry, Chloromethyl-2'7'-dichlorofluorescein diacetate (CM-H2DCFDA) penetrant, and Fluo-4 AM staining, respectively. The binding relationship between SP1 and CPPED1 was verified by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. SP1 and CPPED1 were lowly expressed in the model rats with carotid balloon injury. Mechanistically, SP1 bound to the promoter region of CPPED1 to activate CPPED1 expression. Overexpressing SP1 or CPPED1 lowered neointimal formation and restenosis rate, thus promoting the recovery of carotid balloon injury in rats. Meanwhile, SP1 and CPPED1 upregulation reduced ROS levels, Ca2+ concentration, and apoptosis of EPCs, accompanied by accelerated EPC viability. SP1 or CPPED1 overexpression reduced neointimal formation and restenosis rate in carotid balloon injury.

Abstract 11976: Functional Characterization of Calcium Handling Kinetics in Patient-Derived Ipsc-Cm Models of Pkp2-Mediated Arrhythmogenic Right Ventricular Cardiomyopathy

Introduction: Pathogenic variants in PKP2 -encoded plakophilin 2 cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Electrocardiographically, PKP2 -mediated ARVC may mimic RYR2 -mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). We compare the underlying mechanism of arrhythmia in ARVC and CPVT using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods: iPSC-CMs were generated from 2 ARVC patients with different PKP2 variants [E149X (PKP2-1) and c.2146-1G&gt;C (PKP2-2)], 1 patient with a CPVT variant [RyR2-R176Q (CPVT1)], and a wild-type (WT) control. Fluo-4 AM calcium dye was used to assess calcium kinetics in iPSC-CMs before and during treatment with 100 nM isoproterenol (ISO) at 8-10 min. Results: Compared to WT, PKP2 and RYR2 iPSC-CMs had a greater amplitude before (WT: 0.331±0.031, PKP2-1: 1.048±0.074, PKP2-2: 1.076±0.095, CPVT1: 0.966±0.061; P&lt;0.0001) and during ISO (WT: 0.277±0.022, PKP2-1: 0.971±0.069, PKP2-2: 0.622±0.041, CPVT1: 0.756±0.051; P&lt;0.0001). The Ca 2+ transient duration (CTD100) was longer in PKP2 and RYR2 iPSC-CMs compared to WT before (WT: 1.01±0.05 s, PKP2-1: 1.84±0.01 s, PKP2-2: 1.76±0.02 s, CPVT1: 1.81±0.02 s; P&lt;0.0001) and during ISO (WT: 1.38±0.05 s, PKP2-1: 1.65±0.03 s, PKP2-2: 1.71±0.03 s, CPVT1: 1.61±0.05 s; P&lt;0.001). Before (WT: 259±13 ms, PKP2-1: 538±23 ms, PKP2-2: 493±28 ms, CPVT1: 586±24 ms; P&lt;0.0001) and during ISO (WT: 311±19 ms, PKP2-1: 406±21 ms, P=0.0172, PKP2-2: 487±28 ms, P&lt;0.0001, CPVT1: 453±21 ms, P=0.0001), peak to 50% decay time was slower in PKP2 and RYR2 iPSC-CMs compared with WT. Upstroke time was longer in PKP2 and RYR2 iPSC-CMs compared to WT before (WT: 205±5 ms, PKP2-1: 539±13 ms, PKP2-2: 429±19 ms, CPVT1: 455±12 ms; P&lt;0.0001) and during ISO (WT: 213±7 ms, PKP2-1: 375±16 ms, PKP2-2: 416±20 ms, CPVT1: 389±10 ms; P&lt;0.0001). Conclusion: Here, iPSC-CMs, derived from patients with either PKP2 -mediated ARVC or RYR2 -mediated CPVT, display similar abnormal calcium handling properties such as greater amplitude, longer CTD100, and slower peak to 50% decay time and upstroke time compared to WT iPSC-CMs before and during ISO treatment. This may explain why patients with PKP2 -mediated ARVC often mimic the treadmill stress test seen in patients with CPVT1.

Abstract 15915: Ultrasound Targeted Microbubble Cavitation-Induced Endothelial Hyperpermeability is Calcium-Dependent and Regulated by Enos Signaling

Introduction: Ultrasound ( US )-targeted microbubble ( MB ) cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier; mechanisms are poorly understood. We reported that UTMC causes inter-endothelial gaps in cultured endothelial cell ( EC ) monolayers, which could increase barrier permeability, and that UTMC increases Ca 2+ influx into ECs. Given that increased intracellular Ca 2+ activates eNOS, and nitric oxide (NO) release promotes microvascular permeability, we hypothesized that UTMC increases intracellular Ca 2+ through activation of mechanosensitive Piezo-1 Ca 2+ channels, leading to NO synthesis and endothelial barrier hyperpermeability. Methods: Transwells were endothelialized with human coronary artery ECs. Lipid MBs were added, and US was delivered for 10 s (1 MHz, 250 kPa, 10 cycles, 10 ms interval). Permeability was measured using dextran transfer and transendothelial electrical resistance ( TEER ). GsMTx4 and L-NAME were used to inhibit Piezo1 and eNOS, respectively; Fluo-4 AM to visualize Ca 2+ influx; DAF-FM to detect NO. S-nitrosylation was measured with biotin switch assay. Results: UTMC reduced TEER (1.7 fold, p&lt;0.05 ) and increased dextran flux across the transwell (2-fold, p&lt;0.01 for 70 kDa dextran, 1.6 fold, p&lt;0.05 for 10 kDa dextran), which was abrogated by L-NAME. UTMC significantly increased Ca 2+ influx into ECs and tripled NO ( p&lt;0.05 ), but these increases and UTMC-mediated hyperpermeability were blunted by Piezo1 channel inhibition ( p&lt;0.0001) . UTMC increased S-nitrosylation of proteins (1.7 fold, p&lt;0.01 ) and was associated with s-nitrosylation of VE-cadherin, its redistribution into focal adhesion junctions, and increased stress fiber formation, which were abolished by L-NAME. Conclusions: UTMC causes endothelial barrier hyperpermeability through a Ca 2+ -dependent mechanism, where Ca 2+ influx into ECs, mediated in part by mechanosensitive Piezo1 channels, modulates eNOS signaling. Our data suggest that eNOS regulates UTMC-induced hyperpermeability, possibly through s-nitrosylation of VE-cadherin. Further studies to examine mechanisms regulating UTMC bioeffects will aid in clinical translation and optimization of UTMC for delivery of cell-impermeant drugs.

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A2A receptors (A2A R) coupled to Gs/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A2A R in the modulation of free cytosolic Ca2+ concentration ([Ca2+]i) involving IP3, cAMP and PKA was investigated in HEK 293-A2A R. The levels of IP3 and cAMP were observed by enzyme immunoassay detection method and [Ca2+]i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A2A R agonist NECA showed increased levels of cAMP, PKA, IP3 and [Ca2+]i levels. However, the reverse effect was observed with A2A R antagonists (ZM241385 and caffeine). Blocking the Gαq/PLC/DAG/IP3 pathway with neomycin, a PLC inhibitor did not affect the modulation of IP3 and [Ca2+]i levels in HEK 293-A2A R cells. To investigate the Gαi/AC/cAMP/PKA, HEK 293-A2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A2A R agonist (NECA) showed no effect on cAMP levels. Further, Gαs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP3 mediated [Ca2+]i modulation. In the HEK 293-A2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP3 and [Ca2+]i levels. The study distinctly demonstrated that A2A R modulates IP3 levels to release the [Ca2+]i via cAMP-dependent PKA. The role of A2A R mediated Gαs pathway inducing IP3 mediated [Ca2+]i release may open new avenues in the therapy of neurodegenerative disorder.

The Janus-faced role of TRPM2-S in retroperitoneal liposarcoma via increasing ROS levels

BackgroundRetroperitoneal liposarcoma (RPLS) is a specific soft tissue sarcoma with a high recurrence rate. The short isoform of transient receptor potential cation channel subfamily M member 2 (TRPM2-S) plays an important role in the regulation of reactive oxygen species (ROS). However, the association between TRPM2-S and RPLS and its underlying mechanisms remains unclear.MethodsThe expression of both TRPM2-S and TRPM2-L in RPLS tissues was verified by kimmunohistochemistry (IHC). The regulation on Ca2+ influx by TRPM2-S was evaluated by Fluo-4 AM staining. The effect of TRPM2-S on cell proliferation and apoptosis was tested by 5-Ethynyl-2′-deoxyuridine (EdU) staining and Flow cytometry respectively. The level of cellular ROS was assessed by the DCFH-DA probe. Different concentrations of H2O2 were used to provide oxidative stress on RPLS cells. The underlying mechanisms were further explored by Western blotting.ResultsThe IHC assays showed that TRPM2-S, but not TRPM2-L, was prognostic in RPLS. Low TRPM2-S level was associated with poor disease-free survival (DFS). Calcium influx signal intensity was significantly decreased under TRPM2-S overexpression, which resulted in a decrease in the levels of FOXO3a and PTEN. Correspondingly, the levels of pERK, pAKT, pP65, pGSK-3β, Bcl-2, and β-catenin were upregulated, and cellular ROS was gently increased under TRPM2-S overexpression. Moreover, TRPM2-S slightly promoted cell proliferation and inhibited apoptosis of RPLS cell lines under normoxia, but largely increased apoptosis rates under oxidative stress. The cleaved caspase3 was significantly upregulated by TRPM2-S overexpression under oxidative stress. N-Acetyl-l-cysteine (NAC), a small molecule antioxidant, could largely rescue RPLS cells from the apoptosis induced by H2O2.ConclusionTRPM2-S exerts Janus-faced effects in RPLS by increasing the ROS levels via inhibition on FOXO3a, which promotes cell proliferation under normoxia but induces apoptosis under oxidative stress.FQgJQSptf854GdLd9y3hLjVideo abstract

Activated TRPA1 plays a therapeutic role in TMZ resistance in glioblastoma by altering mitochondrial dynamics

BackgroundGlioblastoma (GBM) represents nearly one-half of primary brain tumors, and the median survival of patients with GBM is only 14.6 months. Surgery followed by radiation with concomitant temozolomide (TMZ) therapy is currently the standard of care. However, an increasing body of evidence suggests that GBM acquires resistance to TMZ, compromising the effect of the drug. Thus, further exploration into the mechanism underlying this resistance is urgently needed. Studies have demonstrated that TMZ resistance is associated with DNA damage, followed by altered reactive oxygen species (ROS) production in mitochondria. Studies have also showed that Ca2+-related transient receptor potential (TRP) channels participate in GBM cell proliferation and metastasis, but the detailed mechanism of their involvement remain to be studied. The present study demonstrates the role played by TRPA1 in TMZ resistance in GBM and elucidates the mechanism of resistance.MethodsU251 and SHG-44 cells were analyzed in vitro. A CCK-8 assay was performed to verify the effect of TMZ toxicity on GBM cells. Intracellular ROS levels were detected by DCFH-DA assay. A MitoSOX Red assay was performed to determine the mitochondrial ROS levels. Intracellular Ca2+ levels in the cells were determined with a Fluo-4 AM calcium assay kit. Intracellular GSH levels were determined with GSH and GSSG Assay Kit. MGMT protein, Mitochondrial fission- and fusion-, apoptosis- and motility-related protein expression was detected by western blot assay. A recombinant lentiviral vector was used to infect human U251 cells to overexpress shRNA and generate TRPA1+/+ and negative control cells. All experiments were repeated.ResultsIn the U251 and SHG-44 cells, TMZ induced a small increase in the apoptosis rate and intracellular and mitochondrial ROS levels. The expression of antioxidant genes and antioxidants in these cells was also increased by TMZ. However, pretreatment with a TRPA1 agonist significantly decreased the level of antioxidant gene and antioxidants expression and enhanced intracellular and mitochondrial ROS levels. Also TMZ induced the level of MGMT protein increased, and pretreatment with a TRPA1 agonist decreased the MGMT expression. Moreover, Ca2+ influx, mitochondrial damage and cell apoptosis were promoted, and the balance between mitochondrial fission and fusion protein expression was disrupted in these GBM cells. Pretreatment with a TRPA1 inhibitor slightly enhanced the level of antioxidant gene expression and reduced the apoptosis rate. TRPA1 gene overexpression in the U251 cells was similar to that after inhibitor intervention, confirming the aforementioned experimental results.ConclusionThe present study proved that activating TRPA1 in glioma cells, which leads to mitochondrial damage and dysfunction and ultimately to apoptosis, may decrease the TMZ resistance of GBM cells.