store-operated calcium (SOC) channel

When MRS1845 (10 μM) is applied, the impact of β-glycerophosphate on store-sorbed Ca2+ entry (SOCE) is almost completely eliminated [2]. After placental growth factor (PlGF) therapy, MRS1845 (10 μM) virtually completely removes the increase in SOCE and greatly lowers it [3].

Certain DHPs appeared to cause an incomplete blockade of SOC channel-dependent elevations of calcium, suggesting the presence of more than one class of such channels in HL-60 cells. N-Methylnitrendipine (IC(50) 2.6 microM, MRS 1844) and N-propargylnifrendipine (IC(50) 1.7 microM, MRS 1845) represent possible lead compounds for the development of selective SOC channel inhibitors.[1]

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Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of β-glycerophosphate on SOCE. Moreover, the β-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing. [2]

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PlGF significantly increased store-operated Ca2+-entry following re-addition of extracellular Ca2+, an effect virtually abrogated by Orai1 inhibitor MRS1845 (10 μM). PlGF further increased HIF1α transcript and protein levels, an effect again significantly blunted by MRS1845 (10 μM). In conclusion, PlGF upregulates expression of both, Orai1 and STIM1 thus enhancing store-operated Ca2+-entry with subsequent upregulation of HIF1α.[3]

[1]. Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells. Biochem Pharmacol. 2003 Feb 1;65(3):329-38.

[2]. Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells. J Mol Med (Berl). 2019 Oct;97(10):1465-1475.

[3]. Upregulation of Orai1 and STIM1 expression as well as store-operated Ca2+ entry in ovary carcinoma cells by placental growth factor. Biochem Biophys Res Commun. 2019 May 7;512(3):467-472.

This study investigated the effects of a series of 1,4-dihydropyridine compounds (DHPs) as volumetric calcium influx inhibitors of reservoir-operated calcium (SOC) channels. These channels are activated in HL-60 leukemia cells following ATP-induced release of an inositol triphosphate (IP₃)-sensitive calcium reservoir. The most active DHPs were those with electron-withdrawing substituents at the 4-phenyl group, such as meta- or para-nitromethyl or meta-trifluoromethyl (IC₅₀ values: 3–6 μM). Benzyl esters corresponding to the ethyl/methyl esters of DHPs typically developed as L-type calcium channel blockers, as well as N-substituted DHPs, maintained their activity against SOC channels. N-methylation reduced the activity of DHPs against L-type channels by several orders of magnitude, resulting in DHPs with nearly identical activity against both SOC and L-type channels. Dihydropyridine compounds (DHPs) containing N-ethyl, N-allyl, and N-propynyl groups exhibit similar activities towards SOC and L-type channels. Replacing the common 6-methyl group in DHPs with a larger group (e.g., cyclobutyl or phenyl) eliminates their activity towards SOC channels; these DHPs instead induce the formation of inositol phosphate and the release of IP₃-sensitive calcium reservoirs. Other DHPs also induce calcium reservoir release, but typically at much higher concentrations than required to inhibit volumetric calcium influx. Some DHPs appear to have incomplete blocking effects on SOC channel-dependent calcium concentration elevations, suggesting the presence of more than one type of such channel in HL-60 cells. N-methylnifedipine (IC50 2.6 μM, MRS 1844) and N-propynylnifedipine (IC50 1.7 μM, MRS 1845) are potential lead compounds for the development of selective SOC channel inhibitors. [1]

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Improper renal phosphate clearance in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteogenic/chondrogenic signaling and vascular calcification in vascular smooth muscle cells (VSMCs). Osteogenic/chondrogenic transdifferentiation of VSMCs results in the upregulation of transcription factors MSX2, CBFA1, and SOX9, as well as tissue-nonspecific alkaline phosphatase (ALPL), which promotes calcification by degrading the calcification inhibitor pyrophosphate. Osteogenic/chondrogenic signaling in VSMCs involves serum and glucocorticoid-induced kinase SGK1. As with other cell types, SGK1 is a potent activator of ORAI1, a calcium channel that mediates the storage-operated calcium influx (SOCE). The calcium sensor STIM1 activates ORAI1 after depletion of the intracellular calcium storehouse. This study investigated whether phosphate regulates the expression of ORAI1 and/or STIM1 in VSMCs, thereby affecting SOCE. Therefore, we treated primary human aortic smooth muscle cells (HAoSMCs) with the phosphate donor β-glycerophosphate. Transcriptional levels were detected by qRT-PCR, protein abundance by Western blotting, ALPL activity by colorimetry, calcification by Alizarin Red S staining, and intracellular Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence assay. The increase in [Ca2+]i was assessed by replenishing extracellular Ca2+ after depletion of intracellular calcium stores with carotenoids, thus evaluating SOCE. The results showed that β-glycerophosphate treatment increased the transcriptional levels and protein abundance of ORAI1 and STIM1 in HAoSMCs and enhanced SOCE. Additional treatment with the ORAI1 inhibitor MRS1845 or the SGK1 inhibitor GSK650394 almost completely eliminated the effect of β-glycerophosphate on SOCE. Furthermore, in the presence of ORAI1 inhibitors or in the presence of ORAI1 gene silencing, the expression and activity of MSX2, CBFA1, SOX9, and ALPL mRNAs in β-glycerophosphate-induced HAoSMCs were all inhibited. In summary, elevated phosphate levels upregulate the expression of ORAI1 and STIM1, as well as intracellular calcium store-operated calcium ion influx (SOCE), thereby participating in the regulation of osteogenic/chondrogenic signaling pathways in vascular smooth muscle cells (VSMCs). Key information: • In aortic smooth muscle cells, the phosphate donor β-glycerophosphate upregulates the calcium channel ORAI1. • In aortic smooth muscle cells, β-glycerophosphate upregulates the ORAI1 activator STIM1. • In aortic smooth muscle cells, β-glycerophosphate upregulates intracellular calcium store-operated calcium ion influx (SOCE). • The ORAI1 inhibitor MRS1845 blocks the effect of β-glycerophosphate on SOCE. MRS1845 and ORAI1 silencing disrupt the stimulation of osteogenic signaling. [2] Placental growth factor (PlGF) is produced by tumor cells and stimulates tumor growth and metastasis in part by upregulating hypoxia-inducible factor HIF1α. Regulation of tumor cell proliferation and migration involves oscillations in cytoplasmic Ca2+ activity ([Ca2+]i). [Ca2+]i oscillations can be achieved by triggering intracellular Ca2+ release, followed by storage-operated Ca2+ influx (SOCE). The mechanism for achieving SOCE involves the pore-forming ion channel unit Orai1 and its regulator STIM1. This study investigated whether PlGF affects the expression of Orai1 and STIM1 and SOCE, and whether this effect affects the expression of HIF1α. To this end, ovarian cancer cells were cultured for 24 hours with or without PlGF (10 ng/ml). The transcriptional levels of Orai1, STIM1 and HIF1α were quantitatively analyzed by RT-PCR, and the protein levels of Orai1, STIM1 and HIF1α were quantitatively analyzed by Western blotting. Intracellular calcium ion concentration [Ca2+]i was estimated by Fura-2 fluorescence method, and the increase in intracellular calcium ion influx [SOCE]i was detected by adding calcium ions after extracellular calcium ion depletion. The experimental method for extracellular calcium ion depletion included the removal of extracellular calcium ions and the use of the sarcoplasmic reticulum calcium pump (SERCA) inhibitor thapsigargin (1 μM). The results showed that after PlGF treatment, the transcriptional and protein levels of Orai1 and STIM1 in ovarian cancer cells were significantly increased. PlGF significantly increased the intracellular calcium ion influx that occurred after the addition of extracellular calcium ions, while the Orai1 inhibitor MRS1845 (10 μM) almost completely blocked this effect. PlGF further increased the transcript and protein levels of HIF1α, while MRS1845 (10 μM) significantly inhibited this effect. In summary, PlGF upregulated the expression of Orai1 and STIM1, thereby enhancing the intracellular calcium pool-operated Ca2+ influx, and subsequently upregulated HIF1α. [3]

C₂₁H₂₂N₂O₆

398.40918

398.148

C, 63.31; H, 5.57; N, 7.03; O, 24.09

544478-19-5

11538542

Typically exists as Off-white to light yellow solids at room temperature

1.318g/cm3

530.8ºC at 760 mmHg

274.8ºC

1.595

3.372

0

7

7

29

798

0

CCOC(C1=C(C)N(CC#C)C(C)=C(C(OC)=O)C1C1C=CC=C([N+]([O-])=O)C=1)=O

BITHABUTZRAUGT-UHFFFAOYSA-N

InChI=1S/C21H22N2O6/c1-6-11-22-13(3)17(20(24)28-5)19(18(14(22)4)21(25)29-7-2)15-9-8-10-16(12-15)23(26)27/h1,8-10,12,19H,7,11H2,2-5H3

3-ethyl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1-(prop-2-yn-1-yl)-1,4-dihydropyridine-3,5-dicarboxylate

MRS 1845; MRS1845; MRS-1845; N-Propargylnitrendipene; N-Propargylnitrendipene; MRS-1845; 5-O-ethyl 3-O-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1-prop-2-ynyl-4H-pyridine-3,5-dicarboxylate; Lopac0_000763; MLS002172491;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 83.33 mg/mL (~209.16 mM)

Solubility in Formulation 1: ≥ 5 mg/mL (12.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (12.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 5 mg/mL (12.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)