Inhibition Of Calcium Entry Research Articles

CD38 in SLE CD4 T cells promotes Ca2+ flux and suppresses interleukin-2 production by enhancing the expression of GM2 on the surface membrane.

CD38 has emerged as a potential therapeutic target for patients with systemic lupus erythematosus (SLE) but it is not known whether CD38 alters CD4+ T cell function. Using primary human T cells and CD38-sufficient and CD38-deficient Jurkat T cells, we demonstrate that CD38 shifts the T cell lipid profile of gangliosides from GM3 to GM2 by upregulating B4GALNT1 in a Sirtuin 1-dependent manner. Enhanced expression of GM2 causes ER stress by enhancing Ca2+ flux through the PLCγ1-IP3 pathway. Interestingly, correction of the calcium overload by an IP3 receptor inhibitor, but not by a store-operated calcium entry (SOCE) inhibitor, improves IL-2 production by CD4+ T cells in SLE. This study demonstrates that CD38 affects calcium homeostasis in CD4+ T cells by controlling cell membrane lipid composition that results in suppressed IL-2 production. CD38 inhibition with biologics or small drugs should be expected to benefit patients with SLE.

SKF96365 Inhibits Tumor Proliferation by Inducing Apoptosis and Autophagy in Human Esophageal Squamous Cell Carcinoma.

Calcium channel blockers are emerging as a new generation of attractive anticancer drugs. SKF96365, originally thought to be a store-operated calcium entry (SOCE) inhibitor, is now often used as a TRPC channel blocker and is widely used in medical diagnostics. SKF96365 has shown antitumor effects on a variety of cancer cell lines. The objective of this study was to investigate the anticancer effect of SKF96365 on esophageal cancer in vivo and in vitro. Cell Counting Kit-8 (CCK-8) and colony formation were used to test the proliferation inhibition of SKF96365 on cell lines. Western blot and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to detect cell apoptosis rates. In addition, we demonstrated the antitumor effect of SKF96365 in vivo in xenografted mice. As a result, SKF96365 significantly inhibited the proliferation of K510, K30, and EC9706 in vitro. SKF96365 induces apoptosis in three cell lines through the poly(adenosine diphosphate-ribose) polymerase (PARP), caspase-9, and BCL-2 pathways in a dose-dependent and time-dependent manner. Moreover, SKF96365 treatment also induced apoptosis and inhibited tumor growth in nude mice. The calcium channel TRPC1 was significantly downregulated by SKF96365. Autophagy was also induced during the treatment of SKF96365. In summary, SKF96365 induces apoptosis (PARP, caspase-9, and BCL-2) and autophagy (LC3-A/B) by inhibiting TRPC1 in esophageal cancer cells, thereby inhibiting tumor growth.

Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.

The new quinazoline derivative (N2-methyl-N4-[(thiophen-2-yl)methyl]quinazoline-2,4-diamine) vasodilates isolated mesenteric arteries through endothelium-independent mechanisms and has acute hypotensive effects in Wistar rats

During the screening of new N2,N4-disubstituted quinazoline 2,4-diamines as phosphodiesterase-5 inhibitors and pulmonary artery vasodilators, one N2-methyl-N4-[(thiophen-2-yl)methyl]quinazoline-2,4-diamine (compound 8) presented a greater selectivity for systemic than pulmonary vasculature. The present study aimed to characterize its vasorelaxant and hypotensive effects in Wistar rats. Vasorelaxant effects of compound 8 and underlying mechanisms were evaluated on isolated mesenteric arteries. Acute hypotensive effect was evaluated in anesthetized rats. Additionally, cell viability and cytochrome P450 (CYP) activities were studied in rat isolated hepatocytes. Nifedipine was used as a comparator. Compound 8 induced a strong vasorelaxant effect, similar to nifedipine. This was unaffected by endothelium removal but was decreased by inhibitors of guanylate cyclase (ODQ) and KCa channel (iberiotoxin). Compound 8 enhanced sodium nitroprusside-induced relaxation, but inhibited vasoconstriction evoked by α1-adrenergic receptor activation and extracellular Ca2+ influx via receptor-operated Ca2+ channels. Acute intravenous infusion of compound 8 (0.05 and 0.1 mg/kg) produced hypotension. It showed similar potency to nifedipine for lowering diastolic and mean arterial blood pressure, but less so for the effect on systolic blood pressure. Compound 8 had no effect on hepatocyte viability and CYP activities except at high concentration (10 μM) at which a weak inhibitory effect on CYP1A and 3A was observed. In conclusion, this study identified a N2-methyl-N4-[(thiophen-2-yl)methyl]quinazoline-2,4-diamine with a potent vasodilator effect on resistance vessels, leading to an acute hypotensive effect and a low risk of liver toxicity or drug-drug interactions. These vascular effects were mediated mainly through sGC/cGMP pathway, opening of KCa channels, and inhibition of calcium entry.

22. The Role of Orai1 in Osteogenic Differentiation Induced by Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

PURPOSE: Development of synthetic regenerative materials inspired by the extracellular matrix (ECM) has been a topic of significant interest due to the known properties of the ECM to direct cell fate determination via tunable material composition and biomechanical properties. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) is a low density, open-cell foam that has been evaluated in our laboratory to induce osteogenic differentiation of primary osteoprogenitors in vitro as well as regeneration of rabbit skull defects in vivo without pre-seeding with progenitor cells or addition of exogenous growth factors, which was not observed in non-mineralized collagen glycosaminoglycan (Col-GAG) materials. Given that the critical difference between MC-GAG and Col-GAG is the mineral content, the spatial concentration of inorganic ions on MC-GAG may be critical to its osteogenic properties. This is supported by prior research demonstrating that high calcium concentrations in the bone microenvironment stimulate osteoblastic differentiation. How these biochemical cues are transmitted to cells and activate downstream osteogenic signaling pathways should be further elucidated. Orai1 is a critical subunit of store-operated calcium release-activated calcium channels (CRAC) that has been implicated to be involved in bone homeostasis. In this work, we evaluated the contribution of Orai1 to MC-GAG-mediated osteogenesis. METHODS: To examine the role of extracellular calcium signaling through store-operated calcium channels, such as Orai1, on osteogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs), we treated 2D cell cultures with a small molecule inhibitor of store-operated calcium entry (SOCE), MRS1845, for 14 days. hMSCs were subsequently seeded on MC-GAG or Col-GAG for 7 days with or without MRS1845. Baseline Orai1 gene and protein expression was evaluated. Next, small interfering RNAs (siRNAs) targeting ORAI1 and a scrambled control were used to specifically knockdown ORAI1 expression in hMSCs cultured on Col-GAG and MC-GAG. Osteogenic gene and protein expression were measured using quantitative RT-PCR and western blot analyses, respectively. Mineralization was evaluated by Alizarin Red staining. RESULTS: To understand whether store-operated calcium entry was essential to osteogenic differentiation, we treated hMSCs in 2D cultures with a small molecule inhibitor of SOCE, MRS1845, and found a reduction in calcium depositions visualized with Alizarin Red staining when compared to untreated hMSCs. In 3D cultures, MRS1845 downregulated the gene expression of early osteogenic marker ALP and protein expression of the intracellular osteogenic mediator Runx2 specifically on MC-GAG compared to controls. As small molecule inhibitors lack channel specificity, we knocked down ORAI1 expression through siRNA transfection. When MC-GAG was compared with Col-GAG, baseline ORAI1 gene expression was 1.8-fold higher (p=0.02) and Orai1 protein expression was upregulated (p=0.003). ORAI1 knockdown reduced the protein expression of Runx2 and gene expression of late osteogenic marker BSP2 specifically on MC-GAG compared to scrambled control siRNA. Mineralization significantly diminished on MC-GAG with ORAI1 knockdown, whereas no effects were evident on Col-GAG as demonstrated by Alizarin Red staining. CONCLUSIONS: ORAI1-mediated calcium ion signaling was required for MC-GAG-induced osteogenic differentiation. Further elucidation of the osteogenic mechanisms and refinement of calcium levels on MC-GAG may facilitate its clinical application in the reconstruction of osseous defects.

Store-operated calcium entry inhibition ameliorates Ang II induced podocyte apoptosis and mitochondria respiratory dysfunction

Purpose and hypothesis: Podocyte injury induced by Angiotensin II (Ang II) is the key factor contributing to proteinuria and glomerulopathy in many kidney diseases including diabetic nephropathy and hypertensive kidney disease. Recent studies suggest that mitochondria dysfunction mediates Ang II-induced podocyte injury. However, the mechanism for Ang II-induced podocyte mitochondria damage is poorly understood. Store-operated Ca2+ entry (SOCE) has multiple functions in both excitable and non-excitable cells. Ang II can activate SOCE by releasing endoplasmic reticulum Ca2+ through generation of IP3. In addition, Ang II also activates Transient Receptor Potential Conventional Member 6 (TRPC6) channel through diacylglycerol (DAG). Our previous study demonstrated that enhanced SOCE mediated high glucose-induced podocyte cytoskeleton remodeling. However, the role of SOCE in podocyte injury by Ang II is not clear. The present study was carried out to test the hypothesis that enhanced SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiration dysfunction. Methods: All experiments were carried out using cultured immortalized human podocytes. BTP2 (4 μM) was used to inhibit SOCE. SAR 7334 (100 nM) was used to block TRPC6 channel. Podocyte apoptosis was determined by flow cytometry using Annexin V/Propidium iodide (PI) staining. The conventional whole-cell patch-clamp was performed to measure store-operated Ca2+ channel (SOC) currents. Mitochondrial respiration function and mitochondria ATP production were evaluated by oxygen consumption rate (OCR) using seahorse analysis. Results: Ang II (1 μM) evoked inward currents, which were significantly blunted by BTP2 (10 μM), an SOC channel blocker. Ang II (1 μM) treatments for 24 hours significantly increased podocyte apoptosis and reduced podocyte basal OCR, maximal OCR, spare capacity and mitochondria ATP production. All these responses were significantly blunted by BTP2. However, Ang II treatment for 30 min did not induce a significant response of OCR, but significantly reduced maximal respiration and spare capacity. These responses were also significantly inhibited by BTP2 (10 μM), but not by SAR 7334 (100 nM). Conclusion: SOCE contributed to Ang II-induced podocyte apoptosis and mitochondria respiratory dysfunction. Translational Project Award from American Heart Association (20TPA35500045, to RM); National Institute of Diabetes and Digestive and Kidney Disease (DK115424-01, to RM); Predoctoral Fellowship from American Heart Association (903925, to YT). 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.

Dithiadiazole derivative 3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxide – Novel modulator of store-operated calcium entry

Pathological calcium homeostasis accompanies the development of a large number of different diseases, therefore, the search for new modulators of calcium signaling remains highly actual. Last decades store-operated calcium channels have been repeatedly postulated as a therapeutic target, so the compounds acting on them can be considered promising drug prototypes. Here, we tested several derivatives of 1,2,3,4-dithiadiazole, 1,3-thiazine, pyrazolopyrimidine and thiohydrazides for the ability to affect the thapsigargin-induced calcium response. Using calcium imaging and the patch-clamp technique we found that dithiadiazole derivative3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxidehad a strong inhibitory effect on store-operated calcium entry at the micromolar concentration in HEK293 cells. Moreover, incubation of the cells with this compound also resulted in the decrease of ER calcium content. Thus, we have postulated 3-(4-nitrophenyl)-5-phenyl-3H-1,2,3,4-dithiadiazole-2-oxide as a novel inhibitor of store-operated calcium entry and suggested the derivatives of 1,2,3,4-dithiadiazole as a prospective class of compounds for searching new calcium modulators.

Effects of Cardiotoxins from Naja oxiana Cobra Venom on Rat Heart Muscle and Aorta: A Comparative Study of Toxin-Induced Contraction Mechanisms.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na+/Ca2+ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca2+ influx.

Inhibition of Calcium Signaling Prevents Exhaustion and Enhances Anti-Leukemia Efficacy of CAR-T Cells via SOCE-Calcineurin-NFAT and Glycolysis Pathways.

Chimeric antigen receptor (CAR) T cells are potent agents for recognizing and eliminating tumors, and have achieved remarkable success in the treatment of patients with refractory leukemia and lymphoma. However, dysfunction of T cells, including exhaustion, is an inevitable obstacle for persistent curative effects. Here, the authors initially found that calcium signaling is hyperactivated via sustained tonic signaling in CAR‐T cells. Next, it is revealed that the store‐operated calcium entry (SOCE) inhibitor BTP‐2, but not the calcium chelator BAPTA‐AM, markedly diminishes CAR‐T cell exhaustion and terminal differentiation of CAR‐T cells in both tonic signaling and tumor antigen exposure models. Furthermore, BTP‐2 pretreated CAR‐T cells show improved antitumor potency and prolonged survival in vivo. Mechanistically, transcriptome and metabolite analyses reveal that treatment with BTP‐2 significantly downregulate SOCE‐calcineurin‐nuclear factor of activated T‐cells (NFAT) and glycolysis pathways. Together, the results indicate that modulating the SOCE‐calcineurin‐NFAT pathway in CAR‐T cells renders them resistant to exhaustion, thereby yielding CAR products with enhanced antitumor potency.

Роль рианодиновых и IP 3 -рецепторов в генерации кальциевых ответов, вызываемых трициклическими антидепрессантами в нейронах неокортекса крысы

Tricyclic antidepressants, in particular amitriptyline (ATL) and desipramine (DES), are currently used to treat depression and chronic pain of various origins, in which NMDA receptor dysfunctions play an important role. The effect of therapeutic concentrations of ATL on calcium-dependent desensitization of NMDA receptors, driven by the level of free calcium in the cytoplasm, is well-known. In addition, in cardiomyocytes, ATL and DES can cause the release of calcium into the cytoplasm from intracellular stores by opening inositol-3-phosphate (IP3R) and / or ryanodine receptors (RyR) channels. The aspect of the effect of these drugs on neurons remains poorly understood. We studied the dependence of the calcium response to DES and ATL on the activation of IP3R and RyR of the endoplasmic reticulum and mitochondria using rat neocortex neurons in primary culture. Short-term (30 s) paired (5 min interval) applications of 200 μM DES or 200 μM ATL induce similar magnitude calcium responses in cortical neurons. The use of RyR and IP3R antagonists showed that responses to ATL are blocked by the IP3R antagonist 2-APB (100 μM), while responses to DES are blocked by ryanodine, the RyR antagonist (100 nM). Since intracellular distribution of RyR and IP3R is not homogenous, it can be assumed that DES and ATL stimulate calcium release from different calcium depots, representing segments of the reticulum or mitochondria. In addition, ATL and DES, being channel blockers of NMDA receptors, inhibited calcium entry from outside the cell via activated NMDA receptors. Considering the high concentrations of DES and ATL (more than 100 μM) required to stimulate the release of deposited calcium in neurons, it seems unlikely that such effects participate their therapeutic action. However, the found specificity of DES and ATL for RyR and IP3R, respectively, can be used as a tool for experimental purposes.

Synthesis and Characterization of Store-Operated Calcium Entry Inhibitors Active in the Submicromolar Range.

The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.

Repurposing calcium channel blockers as antiviral drugs.

The current pandemic caused by the SARS-CoV-2 has claimed over a half a million lives within a very short span of time. A therapeutic drug which could prevent the entry and propagation of the virus is the need of the hour. Several lines of evidence collected from experimental studies older than three decades have pointed out the fact that inhibiting calcium entry into cells can affect vital steps in the lifecycle of viruses. Hence, calcium channel blockers may be considered as an effective measure in the containment of the viruses. This commentary throws light two scientific papers although with divergent facts converging at a point by suggesting a promising treatment option for CoVID-19 (Fang et al. Lancet Respir Med 8:e21, 2020; Straus et al. J Virol 94:e00426, 2020).

IGF-1 Deficiency Rescue and Intracellular Calcium Blockade Improves Survival and Corresponding Mechanisms in a Mouse Model of Acute Kidney Injury.

This study was undertaken to test two therapies for acute kidney injury (AKI) prevention, IGF-1, which is renal protective, and BTP-2, which is a calcium entry (SOCE) inhibitor. We utilized lipopolysaccharide (LPS) IP, as a systemic model of AKI and studied in five groups of animals. Three experiments showed that at 7 days: (1) LPS significantly reduced serum IGF-1 and intramuscular IGF-I in vivo gene therapy rescued this deficiency. (2) Next, at the 7-day time point, our combination therapy, compared to the untreated group, caused a significant increase in survival, which was noteworthy because all of the untreated animals died in 72 h. (3) The four pathways associated with inflammation, including (A) increase in cytosolic calcium, (B) elaboration of proinflammatory cytokines, (C) impairment of vascular integrity, and (D) cell injury, were adversely affected in renal tissue by LPS, using a sublethal dose of LPS. The expression of several genes was measured in each of the above pathways. The combined therapy of IGF-1 and BTP-2 caused a favorable gene expression response in all four pathways. Our current study was an AKI study, but these pathways are also involved in other types of severe inflammation, including sepsis, acute respiratory distress syndrome, and probably severe coronavirus infection.

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca2+ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K+ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition

Context Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. Objective This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. Materials and methods Male Sprague-Dawley rats (170–180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. Results Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51–100 and 101–150 μm o.d. Conclusion Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Inhibiting store-operated calcium entry attenuates white matter secondary degeneration following SCI

Axonal degeneration plays a key role in the pathogenesis of numerous neurological disorders including spinal cord injury. After the irreversible destruction of the white matter elements during the primary (mechanical) injury, spared axons and their supporting glial cells begin to breakdown causing an expansion of the lesion site. Here we mechanistically link external sources of calcium entry through axoplasmic reticulum calcium store depletion that contributes to secondary axonal degeneration through a process called store-operated calcium entry. There is increasing evidence suggesting that store-operated calcium entry impairment is responsible for numerous disorders. Nevertheless, its role following spinal cord injury remains poorly understood. We hypothesize that store-operated calcium entry mediates secondary white matter degeneration after spinal cord injury. We used our previously published model of laser-induced spinal cord injury to focally transect mid cervical dorsal column axons from live 6–8-week-old heterozygous CNPaseGFP/+: Thy1YFP+ double transgenic murine spinal cord preparations (five treated, eight controls) and documented the dynamic changes in axons over time using two-photon excitation microscopy. We report that 1 hour delayed treatment with YM-58483, a potent inhibitor of store-operated calcium entry, significantly decreased intra-axonal calcium accumulation, axonal dieback both proximal and distal to the lesion site, reduced secondary axonal “bystander” damage acutely after injury, and promoted greater oligodendrocyte survival compared to controls. We also targeted store-operated calcium entry following a clinically relevant contusion spinal cord injury model in vivo. Adult, 6–8-week-old Advillin-Cre: Ai9 mice were subjected to a mild 30 kdyn contusion and imaged to observe secondary axonal degeneration in live animals. We found that delayed treatment with YM-58483 increased axonal survival and reduced axonal spheroid formation compared to controls (n = 5 mice per group). These findings suggest that blocking store-operated calcium entry acutely is neuroprotective and introduces a novel target to prevent pathological calcium entry following spinal cord injury using a clinically relevant model.

Abstract C045: RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML

Abstract Background: Store-operated calcium entry (SOCE) through calcium release activated calcium (CRAC) channels contribute to calcium influx in non-excitable cells. Elevation of cytosolic calcium through SOCE mediates an array of cellular responses including metabolism and gene expression, cell growth, and proliferation. Aberrant CRAC activity has been linked to various auto-immune disorders and certain cancers via the NFAT pathway. Calcium signaling via CRAC channels also mediates several physiological processes including cell proliferation and apoptosis. RP4010 is a potent inhibitor of CRAC channel activity (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. The objective of this study was to evaluate the single agent activity and the combination of RP4010 and standard of care drugs in AML models in vitro and in vivo. Methods: Cell growth following incubation with RP4010 (5 μM), Gilteritinib (0.25-0.75 μM), or a combination of both agents was determined in the AML lines: THP-1, HL-60, KG-1, and U-937. Apoptosis was evaluated following incubation of cell lines (THP-1 and U-937) with compounds for 72 h, subsequent staining with Annexin-V-PE and 7-AAD, and analysis by flow cytometry. For cell cycle, cells were incubated with compounds for 72 h, fixed with 70% ethanol, incubated at 4°C for 3 h, and stained with Propidium Iodide prior to analysis by flow cytometry. AKT and Stat-5 phosphorylation was determined by Western blotting. In vivo efficacy was evaluated in a MV-4-11 mouse xenograft model. Results: Store-operated calcium entry with RP4010 potentiated Gliteritinib activity manifested by a significant (P<0.05) reduction in cell growth when compared to the activity of the individual agents. Similarly, the combination (3 µM RP4010 + 1 or 2 µM Gilteritinib) led to a 13% increase in the number of apoptotic cells besides increasing cells in the G0/G1 phase by 17%. Combining RP4010 (1 µM) and Gilteritinib (0.1 µM) reduced AKT and Stat-5 phosphorylation by 45 and 57%, respectively, when compared to Gilteritinib in the THP-1 cell line. In the MV-4-11 mouse xenograft model, RP4010 when administered orally at 30 mg/kg/BID for 21 days demonstrated significant (P<0.001) anti-tumor activity both as a single agent and in combination with Cytarabine with tumor growth inhibitions of 42 and 68 %, respectively. Both RP4010, as well as the combination of RP4010 + Cytarabine, were well tolerated throughout the study period with no appreciable change in body weight. Conclusions: RP4010, a potent SOCE inhibitor, attenuated AML tumor growth as a single agent besides demonstrating remarkable potentiation in activity of approved/standard of care drugs for treating AML. Findings provide a rationale for use of this molecule in clinical trials involving naïve or relapsed/refractory AML patients. RP4010 is currently being evaluated in a Phase-1 dose-escalation study in patients with relapsed/refractory lymphomas. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. RP4010, a small molecule inhibitor of Store-Operated Calcium Entry (SOCE), potentiates activity of Gilteritinib and Cytarabine in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C045. doi:10.1158/1535-7163.TARG-19-C045

Ethyl Acetate Fraction of Lannea microcarpa Engl. and K. Krause (Anacardiaceae) Trunk Barks Corrects Angiotensin II-Induced Hypertension and Endothelial Dysfunction in Mice.

Traditional remedies prepared from Lannea microcarpa leaves, barks, roots, and fruits are used to treat many diseases including hypertension. This study investigated whether oral administration of the ethyl acetate fraction of Lannea microcarpa trunk barks (LMAE) corrects angiotensin (Ang) II-induced hypertension in mice. Its effects on vascular function were specifically investigated. Experiments explored hemodynamic and echocardiographic parameters in vivo and vascular reactivity to acetylcholine (ACh) and CaCl2 ex vivo on isolated aortas. Mice received LMAE for 3 weeks (50 mg/kg/day) by oral gavage. In the last two weeks of treatment, mice were implanted with osmotic minipumps delivering NaCl (0.9%) or Ang II (0.5 mg/kg/day). LMAE completely prevented the increase in systolic and diastolic blood pressure induced by Ang II. Echocardiographic and kidney parameters were not affected by the different conditions. LMAE abrogated Ang II-induced impairment of ACh-induced relaxation without affecting that of sodium nitroprusside. LMAE also completely prevented CaCl2-induced contraction in KCl-exposed aorta ex vivo. The extract alone did not modify superoxide (O2 −) and nitric oxide (NO·) production in femoral arteries from control mice but significantly limited Ang II-induced O2 − production. These effects were associated with reduced expression of inducible isoform of cyclooxygenase- (COX-) 2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase isoform NOX-2 in aortas. Finally, phytochemical analysis showed that LMAE contains sterols, triterpenes, coumarins, and anthraquinone. These results showed that LMAE prevents Ang II-induced hypertension and vascular dysfunction through a reduction of oxidative stress linked to COX-2 and NOX-2 pathway and inhibition of calcium entry. This study provides pharmacological basis of the empirical use of Lannea microcarpa trunk bark extract against hypertension.

Antispasmodic Effects of <i>Salvia officinalis</i> in Isolated Ileum of Rabbit

Salvia officinalis has been commonly used in Syria and other Middle East countries in popular medicine for many medical purposes. One of the strong claims about its properties is the spasmolytic effect. The aim of this research was to investigate the spasmolytic effect of ethanolic extract of Salvia officinalis in isolated rabbit ileum preparations. Male rabbits were sacrificed and portions of ileum were isolated and mounted in Tyrode’s solution. Afterwards, the experiments were performed according to different groups with different spasmogenic agents: the first group was only with spontaneous contraction, the second was KCl-induced contractions and the third was Carbachol-induced contractions. The examined extract was added cumulatively in all previous groups. Verapamil was used as a reference. The results showed profound relaxing effect of S. officinalis extract in mentioned groups, especially in the group of KCl-induced contraction. These findings suggest that the main mechanism of the spasmolytic action is related to the inhibition of calcium entry through cell membrane as well as via internal stores.

Pharmacological inhibition of store-operated calcium entry in MDA-MB-468 basal A breast cancer cells: consequences on calcium signalling, cell migration and proliferation.

Store-operated Ca2+ entry is a pathway that is remodelled in a variety of cancers, and altered expression of the components of store-operated Ca2+ entry is a feature of breast cancer cells of the basal molecular subtype. Studies of store-operated Ca2+ entry in breast cancer cells have used non-specific pharmacological inhibitors, complete depletion of intracellular Ca2+ stores and have mostly focused on MDA-MB-231 cells (a basal B breast cancer cell line). These studies compared the effects of the selective store-operated Ca2+ entry inhibitors Synta66 and YM58483 (also known as BTP2) on global cytosolic free Ca2+ ([Ca2+]CYT) changes induced by physiological stimuli in a different breast cancer basal cell line model, MDA-MB-468. The effects of these agents on proliferation as well as serum and epidermal growth factor (EGF) induced migration were also assessed. Activation with the purinergic receptor activator adenosine triphosphate, produced a sustained increase in [Ca2+]CYT that was entirely dependent on store-operated Ca2+ entry. The protease activated receptor 2 activator, trypsin, and EGF also produced Ca2+ influx that was sensitive to both Synta66 and YM58483. Serum-activated migration of MDA-MB-468 breast cancer cells was sensitive to both store-operated Ca2+ inhibitors. However, proliferation and EGF-activated migration was differentially affected by Synta66 and YM58483. These studies highlight the need to define the exact mechanisms of action of different store-operated calcium entry inhibitors and the impact of such differences in the control of tumour progression pathways.