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Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca 2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H 2 O 2 , 10-100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H 2 O 2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca 2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca 2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear., Methods: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1 R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery., Results: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn 2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca 2+ signaling alterations (increased SOCE, decreased [Ca 2+ ] i transients amplitude and decay rate, lower SR Ca 2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult., Conclusions: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Calcium (Ca 2+ ) and its relevant transmembrane and intracellular calcium channels were previously thought to be chiefly associated with the regulation of cardiovascular and neuronal systems. Nowadays, an increasing evidence shows those calcium channels are also responsible for tumorigenesis and progression. However, the general underlying mechanisms and the involving signaling transduction pathways still remain unclear. Therefore, in this mini-review, we are mainly focusing on the linkage between calcium channels and major characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, evasion of immune surveillance, and the alterations of tumor microenvironment. We will shed light on the possible therapeutic approaches to counteract tumors regarding the intervention of calcium channel., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit + progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca 2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit + progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca 2+ ( Ca i 2 + ) by triggering a transient Ca 2+ release from ER IP3Rs followed by sustained Ca 2+ influx through store-operated Ca 2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca 2+ release and Ca 2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Ca i 2 + via ER-IP3R3 Ca 2+ release followed by Ca 2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit + progenitor cells., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The role of Ca 2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca 2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca 2+ , while both cell lines showed marked reduced levels of Ca 2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca 2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca 2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca 2+ ], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca 2+ homeostasis and that such changes favor viral replication., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin., Objective and Methods: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE., Results: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1., Conclusions: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca 2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca 2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca 2+ levels, respectively. Histamine release and Ca 2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca 2+ increase. FcεRI-induced Ca 2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca 2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca 2+ influx enhanced the process. Furthermore, cytosolic Ca 2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca 2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca 2+ signaling in regulating histamine degranulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calcium release-activated calcium modulator 1(ORAI1) is an integral component of the calcium release-activated calcium channel (CRAC) channel complex and plays a central role in regulating Ca 2 +  concentrations in T-lymphocytes. It is critical for many physiological processes, including cell-proliferation, cytokine production and activation of the immune system. Loss of ORAI1 function is linked with rheumatoid arthritis (RA) and hence pharmacological blockers of ORAI1 could be potential therapeutic agents for the treatment of RA. In this study, we have used a high-throughput screening approach to inhibit the binding of Ca 2+  toward ORAI1 and the interactions are verified through induced fit docking. The results hint that these compounds act by possibly binding with, and thereby blocking Ca 2+ -binding with ORAI1 (E106). The molecular dynamics (MD) simulations shows strong support toward the hit compounds by showing the ligand potency throughout the simulation timescale of 30 ns. We have thus identified a novel class of highly stable, potential lead compounds that directly bind with the selectivity filter region E106 and block Ca 2+ binding on ORAI1. This resulting alteration in the pore geometry of ORAI1 due to the strong blocking mechanism of lead compounds will greatly diminish its function and the downstream activities that result from the same including decreased production of cytokines in autoimmune disorders. This study may lay the foundation for finding novel lead compounds for clinical trials that could positively modulate the course of autoimmune disorders with ORAI1 as its specific target.

Evidence supports an important role of Ca 2+ release-activated Ca 2+ channel protein 1 (Orai1)-mediated Ca 2+ entry in the development of renal fibrosis, a common pathologic feature of CKDs that lead to ESRD, but the molecular mechanisms remain unclear. We determined the role of Orai1 calcium channel in renal fibrosis induced by high-fat diet and by unilateral ureteral obstruction. Mouse kidneys with fibrosis had higher levels of Orai1 protein expression than did kidneys without fibrosis. In vivo knockdown of Orai1 with adenovirus harboring Orai1-short hairpin RNA or inhibition of Orai1 with SKF96365 dramatically prevented renal fibrosis and significantly decreased protein expression of fibronectin, α‑smooth muscle actin, and TGF‑β1 in the kidney cortex of ApoE -/- mice on a high-fat diet and in the obstructed kidneys of mice with unilateral ureteral obstruction. Compared with kidney biopsy specimens of patients with glomerular minimal change disease, those of patients with fibrotic nephropathy had higher expression levels of Orai1. In cultured human proximal tubule epithelial cells (HK2), knockdown of Orai1 Ca 2+ channel with adenovirus-Orai1-short hairpin RNA markedly inhibited TGF-β1-induced intracellular Ca 2+ influx and phosphorylation of smad2/3. Knockdown or blockade of the Orai1 Ca 2+ channel in HK2 cells also prevented epithelial-to-mesenchymal transition induced by TGF‑β1. In conclusion, blockade of the Orai1 Ca 2+ channel prevented progression of renal fibrosis in mice, likely by suppressing smad2/3 phosphorylation and TGF-β1-induced epithelial-to-mesenchymal transition. These results render the Orai1 Ca 2+ channel a potential therapeutic target against renal fibrosis., (Copyright © 2016 by the American Society of Nephrology.)