Calcium ionophore

Streptomyces conglobatus produces the antibiotic and calcium ionophore Ionomycin [1]. When 2 μM Ionomycin was added to LCLC 103H cells, the intracellular Ca2+ concentration temporarily increased from 50 to 180 nM. In ionomycin-treated cultures, DNA and protein analysis showed fragmentation of DNA and PARP probes for an 85-kDa fragment characteristic of caspase-mediated cell disinfection. About 1–5% of the cells treated with ionomycin had Taipei. The increase in anti-Ac-DEVD-amc activity after ionomycin treatment was observed after caspase activation in entire cells [2]. Interstitial and exosome release mediated by ionomycin. The quantity of exosomes carrying L1-32 slices in the SKOV3ip cells' conditioned media increased following ionomycin treatment [4]. Additionally, ionomycin phosphorylates p38 MAPK via SOCE influx of Ca2+, which prevents NF-κB phosphorylation produced by TNF-α [5].

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The ionophorous properties of a new antibiotic, Ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.[1]

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We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore Ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.[2]

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Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined Ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.[3]

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Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by Ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.[4]

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Calcium influx via store-operated calcium entry (SOCE) has an important role for regulation of vast majority of cellular physiological events. MAPK signalling is also another pivotal modulator of many cellular functions. However, the relationship between SOCE and MAPK is not well understood. In this study, we elucidated the involvement of SOCE in Gαq/11 protein-mediated activation of p38 MAPK in an intestinal epithelial cell line HT-29/B6. In this cell line, we previously showed that the stimulation of M3 muscarinic acetylcholine receptor (M3-mAChR) but not histamine H1 receptor (H1R) led to phosphorylation of p38 MAPK which suppressed tumor necrosis factor-α (TNF-α)-induced NF-κB signalling through ADAM17 protease-mediated shedding of TNF receptor-1 (TNFR1). First, we found that stimulation of M3-mAChR and protease-activated receptor-2 (PAR-2) but not H1R induced persistent upregulation of cytosolic Ca2+ concentration through SOCE. Activation of M3-mAChR or PAR-2 also suppressed TNF-α-induced NF-κB phosphorylation, which was dependent on the p38 MAPK activity. Time course experiments revealed that M3-mAChR stimulation evoked intracellular Ca2+-dependent early phase p38 MAPK phosphorylation and extracellular Ca2+-dependent later phase p38 MAPK phosphorylation. This later phase p38 MAPK phosphorylation, evoked by M3-mAChRs or PAR-2, was abolished by inhibition of SOCE. Thapsigargin or Ionomycin also phosphorylate p38 MAPK by Ca2+ influx through SOCE, leading to suppression of TNF-α-induced NF-κB phosphorylation. Finally, we showed that p38 MAPK was essential for thapsigargin-induced cleavage of TNFR1 and suppression of TNF-α-induced NF-κB phosphorylation. In conclusion, SOCE is important for p38 MAPK phosphorylation and is involved in TNF-α signalling suppression[5].

Ionomycin treatment rescued osteoporosis in BMSC-specific conditional alpl knockout mice [6]
To further determine whether ALPL deficiency in BMSCs caused altered osteogenesis and adipogenesis in vivo, we assessed BV/TV and Tb.N in 3-month-old Prrx1-alpl−/− mice, and we found that they were markedly decreased compared with that of their control alplf/f littermates (Fig. 7a, b). Floxed alpl littermates (alplf/f) were used as controls. MicroCT and histological analyses showed that, BMD, BV/TV, and Tb.N in 3-month-old Prrx1-alpl−/− mice treated with Ionomycin were markedly increased compared with what was observed in the Prrx1-alpl−/− mice (Fig. 7a, b). To further detect changes in osteogenic/adipogenic lineage differentiation in BMSCs, we examined the number of adipocytes in the bone marrow of alplf/f, Prrx1-alpl−/− mice, and Prrx1-alpl−/− mice treated with ionomycin. Oil red O staining showed that the number of adipocytes in Prrx1-alpl−/− bone marrow was markedly increased compared with that in the control alplf/f littermates (Fig. 7c). However, the number of adipocytes in Prrx1-alpl−/− bone marrow after ionomycin treatment was markedly decreased compared with that of the Prrx1-alpl−/− mice (Fig. 7c). Calcein double labeling analysis showed a decreased bone formation rate in Prrx1-alpl−/− mice relative to that of control alplf/f mice (Fig. 7d). Ionomycin treatment reversed the impaired osteogenesis in Prrx1-alpl−/− mice. Moreover, the serum levels of RANKL and OPG were not significantly changed, as assessed by ELISA (Fig. S7c, d), suggesting that osteoclasts may not be altered in Prrx1-alpl−/− mice. The intracellular level of Ca2+ in alpl−/− BMSCs was decreased compared with that in the control BMSCs, and ionomycin treatment elevated the intracellular level of Ca2+ in alpl−/− BMSCs (Fig. 7e). In addition, BMSCs from Prrx1-alpl−/− mice showed decreased osteogenic differentiation and increased adipogenic differentiation compared with BMSCs from alplf/f mice (Fig. 7f–i). BMSCs from Prrx1-alpl−/− mice treated with ionomycin showed increased osteogenic and decreased adipogenic differentiation (Fig. 7f–i). These results indicate that ALPL deficiency in BMSCs induces an age-related osteoporosis phenotype and that ionomycin treatment reversed this phenotype.

Protease Activity in Living Cells [3]
Calpain- or caspase-like activities in whole cells were measured with the fluorogenic substrates Suc-LLVY-amc (160 μm) or Ac-DEVD-amc (200 μm), respectively. The substrates Ionomycin, etoposide, and AC27P were mixed in the appropriate HEPES-buffered serum-free growth media. LCLC 103H cells were plated on 24-well plates (105 cells/well) and preincubated with the substrate for 30 min at 37 °C in a humidified 5% CO2 incubator. Substrate hydrolysis at 37 °C was monitored using a fluorescence reading system (Fluoroskan ascent) set to 355 ± 20 nm for excitation and 460 ± 20 nm for emission. Fluorescence readings were collected every 5 min (up to 400 min), before and after addition of ionomycin, and after preincubation of the cells for 1 h at 37 °C with 50 μm AC27P or 20 μm of Z-DEVD-fmk.

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FACS Analysis of Apoptotic Cells [3]
Cell viability was assessed by flow cytometry simultaneously monitoring annexin V binding and propidium iodide uptake. LCLC 103H cells were plated on inner diameter 10-cm culture plates and treated at 37 °C with the indicated inhibitor/insult, resuspended in 2 ml of annexin V binding buffer, and finally treated with annexin V-fluorescein and propidium iodide (9:1) for 5 min at room temperature. Fluorescence of 20000 cells was measured with a FACS-Calibur Sort through a 530/30 bandpass filter to monitor annexin-fluorescein-phosphatidylserine binding and through a 585/42 filter to monitor propidium iodide uptake.

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Measurement of Free Calcium Concentrations [3]
Free calcium concentrations in LCLC 103H cells were determined before and after adding 2 μm Ionomycin. These measurements and the calibration of Fura-2 fluorescence was performed according to standard protocols (31). Briefly, cells were loaded for 1 h with 5 μm Fura-2 AM diluted in FluoronicR F-127. Coverslips were rinsed with a solution containing 20 mmHEPES, 5.6 mm glucose, 137 mm NaCl, 0.8 mm KCl, 0.5 mm CaCl2, 1.0 mm MgCl2, 2 mm EDTA, pH 7.4, placed in an imaging chamber, and mounted in a platform at 37 °C on the stage of a Nikon Diaphot. Fura-2 was excited at alternating wavelengths of 340- and 380-nm using a 75-watt xenon light source and a filter wheel (Ludl). Emitted wavelengths passed through a 510-nm filter cube set before detection by an enhanced CCD camera. Saturating calcium concentrations were measured after rinsing and preincubation of the cells with a solution of 10 mm CaCl2 in 20 mm HEPES, pH 7.4, containing 5.6 mm glucose, 137 mm NaCl, 0.8 mm KCl, 1.0 mmMgCl2, 2 mm EDTA, and two subsequent additions of 2 μm Ionomycin with 5-min interval between them. After that, absence of calcium was measured by adding 2 ml of 80 mm EGTA, pH 8.0. Data were stored and processed using the IonWizard software.

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Isolation of membrane vesicles [4]
Cells were cultivated overnight in a serum-free medium and then treated with or without Ionomycin (1 μM), APMA (50 μM), MCD (10 mM) or PMA (50 ng/ml) for the indicated length of time. Tissue culture supernatants were collected and centrifuged for 10 min at 300 g and for 20 min at 10000 g to remove cellular debris. Membrane vesicles were collected by centrifugation at 100000 g for 2 h at 4 °C using a Beckman SW40 rotor. Vesicles were directly dissolved in SDS-sample buffer [30% sucrose, 80 mM Tris/HCl (pH 8.8), 3% SDS and 0.01 mg/ml Bromphenol Blue] or processed further for gradient centrifugation (see below).

Ionomycin treatment [6]
Ionomycin was dissolved in DMSO. For in vivo treatment, ionomycin was intraperitoneally administered to 12-week-old alpl+/− mice and alpl−/− CKO mice at a dose of 1 mg·kg−1 per day for 28 days. The control mice were treated with only the vehicle. After ionomycin treatment, all groups of mice were healthy.

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In vivo oil red O staining [6]
To assess adipose tissue in trabecular areas, femurs were fixed in 4% paraformaldehyde and were decalcified with 5% EDTA (pH 7.4), which was followed by cryosectioning. Sections were stained with oil red O, and positive areas were quantified under microscopy and are shown as a percentage of the total area. Briefly, sections were washed with 60% isopropanol and then were incubated in fresh oil red O staining solution for 15 min at room temperature before being counterstained with hematoxylin. All reagents for oil red O staining were purchased from Sigma-Aldrich.

mouse LD50 subcutaneous 28 mg/kg Journal of Antibiotics., 31(815), 1978 [PMID:711623]
6446270 mouse LD50 oral 650 mg/kg Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed., Grayson, M., and D. Eckroth, eds. New York, John Wiley & Sons, Inc., 1978, 3(47), 1978
6446270 mouse LD50 intraperitoneal 12 mg/kg Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed., Grayson, M., and D. Eckroth, eds. New York, John Wiley & Sons, Inc., 1978, 3(47), 1978

[1]. Characterization of ionomycin as a calcium ionophore. J Biol Chem. 1978 Sep 10;253(17):5892-4.

[2]. Mechanisms of T cell activation by the calcium ionophore ionomycin. J Immunol. 1989 Aug 15;143(4):1283-9.

[3]. Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members. J Biol Chem. 2002 Jul 26;277(30):27217-26.

[4]. A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44. Biochem J. 2006 Feb 1;393(Pt 3):609-18.

[5]. Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells. Cell Signal. 2019 Nov;63:109358.

[6]. Ionomycin ameliorates hypophosphatasia via rescuing alkaline phosphatase deficiency-mediated L-type Ca2+ channel internalization in mesenchymal stem cells. Bone Res. 2020 Apr 26:8:19.

Ionomycin is a very long-chain fatty acid that is docosa-10,16-dienoic acid which is substituted by methyl groups at positions 4, 6, 8, 12, 14, 18 and 20, by hydroxy groups at positions 11, 19 and 21, and by a (2',5-dimethyloctahydro-2,2'-bifuran-5-yl)ethanol group at position 21. An ionophore produced by Streptomyces conglobatus, it is used in research to raise the intracellular level of Ca(2+) and as a research tool to understand Ca(2+) transport across biological membranes. It has a role as a metabolite and a calcium ionophore. It is an enol, a cyclic ether, a very long-chain fatty acid and a polyunsaturated fatty acid.
Ionomycin has been reported in Streptomyces conglobatus with data available.
Ionomycin is a polyether antibiotic isolated from Streptomyces conglobatus sp. nov. Trejo with antineoplastic activity. Ionomycin is a calcium ionophore that increases intracellular Ca++ levels, possibly relating to endonuclease activation of lymphocytes and decreased ratio of Bcl-2 to Bax and ultimately apoptosis. In addition, this agent is used to investigate the role of intracellular calcium in cellular processes. (NCI)
A divalent calcium ionophore that is widely used as a tool to investigate the role of intracellular calcium in cellular processes.

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The loss-of-function mutations in the ALPL result in hypophosphatasia (HPP), an inborn metabolic disorder that causes skeletal mineralization defects. In adults, the main clinical features are early loss of primary or secondary teeth, osteoporosis, bone pain, chondrocalcinosis, and fractures. However, guidelines for the treatment of adults with HPP are not available. Here, we show that ALPL deficiency caused a reduction in intracellular Ca2+ influx, resulting in an osteoporotic phenotype due to downregulated osteogenic differentiation and upregulated adipogenic differentiation in both human and mouse bone marrow mesenchymal stem cells (BMSCs). Increasing the intracellular level of calcium in BMSCs by ionomycin treatment rescued the osteoporotic phenotype in alpl+/- mice and BMSC-specific (Prrx1-alpl-/-) conditional alpl knockout mice. Mechanistically, ALPL was found to be required for the maintenance of intracellular Ca2+ influx, which it achieves by regulating L-type Ca2+ channel trafficking via binding to the α2δ subunits to regulate the internalization of the L-type Ca2+ channel. Decreased Ca2+ flux inactivates the Akt/GSK3β/β-catenin signaling pathway, which regulates lineage differentiation of BMSCs. This study identifies a previously unknown role of the ectoenzyme ALPL in the maintenance of calcium channel trafficking to regulate stem cell lineage differentiation and bone homeostasis. Accelerating Ca2+ flux through L-type Ca2+ channels by ionomycin treatment may be a promising therapeutic approach for adult patients with HPP.[6]

C41H72O9

709.0050

708.518

C, 69.46; H, 10.24; O, 20.31

56092-81-0

Ionomycin calcium;56092-82-1

6912226

Colorless to light yellow liquid

1.072 g/cm3

817.2ºC at 760 mmHg

235.2ºC

1.512

7.799

5

9

22

50

1120

14

O1[C@](C([H])([H])[H])([C@@]([H])(C([H])([H])[H])O[H])C([H])([H])C([H])([H])[C@]1([H])[C@]1(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[C@@]([H])([C@]([H])(C([H])([H])[H])[C@@]([H])([C@@]([H])(/C(/[H])=C(\[H])/C([H])([H])[C@@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])(C([H])([H])[H])/C(=C(\[H])/C([C@@]([H])(C([H])([H])[H])C([H])([H])[C@@]([H])(C([H])([H])[H])C([H])([H])[C@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])C(=O)O[H])=O)/O[H])C([H])([H])[H])O[H])O[H])O1

PGHMRUGBZOYCAA-OJFQOPKISA-N

InChI=1S/C41H72O9/c1-25(21-29(5)34(43)24-35(44)30(6)22-27(3)20-26(2)14-15-38(46)47)12-11-13-28(4)39(48)31(7)36(45)23-33-16-18-41(10,49-33)37-17-19-40(9,50-37)32(8)42/h11,13,24-33,36-37,39,42-43,45,48H,12,14-23H2,1-10H3,(H,46,47)/b13-11+,34-24-/t25-,26-,27+,28-,29-,30+,31+,32-,33?,36+,37?,39-,40+,41+/m1/s1

(4R,6S,8S,10Z,12R,14R,16E,18R,19R,20S,21S)-11,19,21-trihydroxy-22-((2S,5'S)-5'-((R)-1-hydroxyethyl)-2,5'-dimethyloctahydro-[2,2'-bifuran]-5-yl)-4,6,8,12,14,18,20-heptamethyl-9-oxodocosa-10,16-dienoic acid

SQ23377; ionomycin; 56092-81-0; Ionomycin free acid; SQ-23377; CHEBI:63954; 54V905V6AT; (4R,6S,8S,10Z,12R,14R,16E,18R,19R,20S,21S)-11,19,21-trihydroxy-22-[(2S,5S)-5-[(2R,5S)-5-[(1R)-1-hydroxyethyl]-5-methyloxolan-2-yl]-5-methyloxolan-2-yl]-4,6,8,12,14,18,20-heptamethyl-9-oxodocosa-10,16-dienoic acid; UNII-54V905V6AT; SQ-23377; SQ 23377

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 : ~100 mg/mL (~141.04 mM)
Ethanol : ~100 mg/mL (~141.04 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.53 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 25.0 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: ≥ 2.5 mg/mL (3.53 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 25.0 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: ≥ 2.5 mg/mL (3.53 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (3.53 mM) (saturation unknown) in 10% EtOH + 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 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix well.

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