Endothelial Nitric Oxide Synthase (eNOS): CORM-401-induced NO production is completely abolished by L-NAME, confirming NO is produced by eNOS. [1]
Ryanodine Receptors (RyR): CORM-401-induced calcium signals are completely blocked by dantrolene, an inhibitor of RyR. [1]
Mitochondrial large-conductance calcium-regulated potassium channels (mitoBKCa): CORM-401 directly activates mitoBKCa channels in mitoplasts, even in low-calcium conditions. [3]
Pentose Phosphate Pathway (PPP): CORM-401 accelerates the PPP, leading to increased NADPH concentration. [1]
NADPH Oxidase (NOX): CORM-401 reduces TNF-α/CHX-induced total cellular ROS, which is partially derived from NOX. [2]

Endothelial EA.hy926 cells are stimulated to produce NO by CORM-401 (100 μM; 1 h)[1]. Endoplasmic reticulum and plasma membrane pool-operated calcium channels are better coupled when CORM-401 (30 μM) is added, inducing peak calcium signaling [1]. CORM-401 (50 μM; 1h) dramatically lowers ROS generation and cell death caused by TNF-α/CHX and H2O2 [2]. The oxygen consumption rate of endothelial EA.hy926 cells is sustainedly increased by CORM-401 (0.5, 1 mM) [3]. While decreasing ECAR, CORM-401 (10, 30, and 100 μM) causes an increase in OCR that is concentration-dependent [3].

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In human endothelial EA.hy926 cells, CORM-401 (100 μM, 1h) induced a 5.2-fold increase in NO production compared to untreated cells, and a 5.7-fold increase compared to cells treated with inactive CORM-401. This effect was completely abolished by pre-incubation with L-NAME (100 μM). [1]
CORM-401 (30 μM) induced two types of cytosolic calcium signals in EA.hy926 cells: a slow/gradual increase and peak-like increases/oscillations. These signals were not induced by iCORM-401. [1]
CORM-401-induced peak-like calcium signals originate from the endoplasmic reticulum, as they were reduced by thapsigargin (SERCA inhibitor) and completely blocked by dantrolene (RyR inhibitor), but not by U73122 (PLC inhibitor). [1]
CORM-401-induced slow increase in cytosolic calcium was dependent on store-operated calcium (SOC) entrance. In Ca2+-free medium, CORM-401 augmented ER coupling with plasmalemmal SOC channels, leading to a two-fold increase in [Ca2+]c upon Ca2+ re-addition compared to control. [1]
In EA.hy926 cells, CORM-401 accelerated the pentose phosphate pathway (PPP), as evidenced by a significant increase in the R5P/6PG ratio, increased NADPH concentration, and decreased GSH/GSSG ratio. The increase in NO was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN). [1]
In murine intestinal epithelial MODE-K cells, CORM-401 (50 μM, pre-incubation 1h + co-incubation 6h) significantly reduced TNF-α/CHX-induced intracellular total ROS production and cell death. However, it did not influence TNF-α/CHX-induced mitochondrial superoxide production. [2]
In MODE-K cells, CORM-401 (50 μM) did not reduce rotenone-induced mitochondrial superoxide production but did reduce antimycin-A-induced mitochondrial superoxide production. [2]
In MODE-K cells, CORM-401 (50 μM, pre-incubation 1h + co-incubation 40 min) significantly attenuated H2O2 (1 mM)-induced intracellular total ROS production. [2]
In EA.hy926 cells, CORM-401 (10-100 μM) induced a concentration-dependent increase in oxygen consumption rate (OCR) and a simultaneous decrease in extracellular acidification rate (ECAR), indicating uncoupling of mitochondrial respiration and inhibition of glycolysis. [3]
In EA.hy926 cells, CORM-401 (30 μM) increased proton leak and non-mitochondrial respiration, while decreasing ATP-linked respiration, maximal respiration, and reserve capacity. [3]
In patch-clamp experiments on mitoplasts from EA.hy926 cells, CORM-401 (30 μM) reactivated mitoBKCa channels in a low-calcium (1 μM) solution, which were otherwise inactive. This effect was reversed by paxilline (1 μM). [3]

Mitochondrial large-conductance calcium-regulated potassium channel (mitoBKCa) activity was assessed using patch-clamp technique in mitoplasts. Mitochondria were isolated from EA.hy926 cells, and the outer mitochondrial membrane was disrupted by changing the isotonic solution to a hypotonic one (5 mM HEPES, 100 μM CaCl2, pH 7.2), followed by restoration with a hypertonic solution (750 mM KCl, 30 mM HEPES, 100 μM CaCl2, pH 7.2) to obtain mitoplasts. Experiments were carried out in inside-out mode. The bath and pipette solutions were symmetrical (150/150 mM KCl). The effect of CORM-401 (30 μM) was tested in a low-calcium solution (1 μM Ca2+), and channel activity was recorded at various voltages (from -60 mV to +60 mV). The probability of channel opening (Po) and mean lifetime of closure and opening were calculated. Currents were recorded using a patch-clamp amplifier, low-pass filtered at 1 kHz, and sampled at 100 kHz. [3]

For calcium imaging, EA.hy926 cells were loaded with 5 μM fura-2 AM or fluo-4 AM and 0.005% Pluronic for 30 minutes at room temperature. Fluorescence measurements were obtained on an epifluorescence inverted microscope. Cytosolic calcium concentration ([Ca2+]c) was monitored in single cells using excitation at 340 and 380 nm for fura-2. For confocal imaging, a 488 nm Argon laser line was used to excite fluo-4, which was measured at 505-550 nm. CORM-401 (30 μM) was added to the cells to induce calcium signals. [1]
NO production by EA.hy926 cells was measured using electron paramagnetic resonance (EPR) spectroscopy with the spin trap colloidal Fe2+(DETC)2. Cells were incubated with CORM-401 (100 μM) or iCORM-401 for 1 hour at 37°C. The cells were then collected, snap-frozen in liquid nitrogen, and the NO-Fe2+(DETC)2 signal was measured in a finger dewar using an EPR spectrometer with settings: microwave power, 10 mW; modulation amplitude, 0.8 mT; scan width, 11.5 mT; scan time, 61.44 s; number of scans, four. [1]
For intracellular ROS and cell death measurement in MODE-K cells, cells were treated with CORM-401 (40 or 50 μM) and oxidative stress stimuli (TNF-α/CHX, H2O2, rotenone, antimycin-A). Cells were loaded with 10 μM carboxy-H2DCFDA for 40 minutes or 5 μM MitoSOX Red for 30 minutes before the end of the treatment. Cells were then stained with Sytox Red (2.5 nM) or Sytox Green (2 nM) and analyzed by flow cytometry using 488 nm excitation. ROS production was measured in gated viable (Sytox-negative) cells. [2]
Mitochondrial dysfunction in MODE-K cells was assessed by double staining with 200 nM MitoTracker Green FM and 25 nM MitoTracker Deep Red FM for 30 minutes. The percentage of cells with non-respiring (dysfunctional) mitochondria (MitoTracker Green-positive/MitoTracker Deep Red-negative) was determined by flow cytometry. [2]
Mitochondrial membrane potential (Ψm) in MODE-K cells was measured using 200 nM tetramethylrhodamine methyl ester (TMRM) for 30 minutes, followed by staining with 2 nM Sytox Green. The percentage of cells with depolarized mitochondria was analyzed in gated viable (Sytox Green-negative) cells by flow cytometry. [2]
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in EA.hy926 cells were measured using a Seahorse XF24 Analyzer. Cells were seeded at 30,000 cells per well. For the mitochondrial function assay, CORM-401 was injected, followed by sequential injections of oligomycin (1 μg/ml), FCCP (0.7 μM), and rotenone/antimycin A (1 μM/1 μM). For the glycolysis stress test, sequential injections of glucose (10 mM), CORM-401, oligomycin (1 μg/ml), and 2-deoxy-glucose (100 mM) were performed. [3]

CORM-401 is a water-soluble CO-releasing molecule. [1][2]
It releases up to three equivalents of CO per mole of compound. [2][3]
In cell-free in vitro systems, the half-life (t1/2) of CORM-401 is reported as < 4 minutes (Crook et al., 2011) and approximately 15 minutes (Fayad-Kobeissi et al., 2016). [2]
The rate of CO release from CORM-401 is accelerated in the presence of biologically relevant oxidants, such as hydrogen peroxide (H2O2). [2]
The compound is dissolved in phosphate buffered saline (PBS) for experimental use. [3]

In EA.hy926 endothelial cells, CORM-401 at concentrations of 10, 30, and 100 μM did not show evident cytotoxicity. However, at 300 μM, it caused a rapid initial increase in OCR followed by a profound decrease after ~45 min of incubation, indicating toxicity. [3]
In MODE-K intestinal epithelial cells, CORM-401 at 50 μM had no effect per se on cell viability when incubated for 12 hours. This concentration was selected as the highest without an effect on cell viability. [2]

[1]. CORM-401 induces calcium signalling, NO increase and activation of pentose phosphate pathway in endothelial cells. FEBS J. 2018 Apr;285(7):1346-1358.

[2]. Differential Effects of CORM-2 and CORM-401 in Murine Intestinal Epithelial MODE-K Cells under Oxidative Stress. Front Pharmacol. 2017 Feb 8;8:31.

[3]. Carbon monoxide released by CORM-401 uncouples mitochondrial respiration and inhibits glycolysis in endothelial cells: A role for mitoBKCa channels. Biochim Biophys Acta. 2015 Oct;1847(10):1297-309.

CORM-401 is a manganese-based metal carbonyl. [2][3]
The compound is light-sensitive and was protected from light in all experiments. [3]
An inactive form, iCORM-401, which does not liberate CO, was used as a negative control. iCORM-401 is composed of MnSO4 and the ligand for CORM-401. In contrast to other CO-RMs, no true iCORM-401 can be prepared as its solutions are stable. [1][2]
CO liberated from CORM-401 induces a two-component metabolic response in endothelial cells: uncoupling of mitochondrial respiration (dependent on mitoBKCa channel activation) and inhibition of glycolysis (independent of mitoBKCa channels). [3]
CORM-401-induced NO production and calcium signaling are interlinked, with NO production upstream of calcium signaling. This process is dependent on the pentose phosphate pathway (PPP) and NADPH. [1]

330.902

1001015-18-4

1001040-67-0 (cation);1001015-18-4;

168430661

Light yellow to yellow solid powder

0.016

1

8

2

18

151

0

[H+].[O+]#C[Mn+]1([SH-]C(N(CC([O-])=O)C)=S1)(C#[O+])(C#[O+])C#[O+]

BYBYPEYFKXEVIY-UHFFFAOYSA-M

InChI=1S/C4H7NO2S2.4CO.Mn/c1-5(4(8)9)2-3(6)7;4*1-2;/h2H2,1H3,(H,6,7)(H,8,9);;;;;/p-1

carbon monoxide;N-(carboxymethyl)-N-methylcarbamodithioate;manganese

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 : ~25 mg/mL (~75.48 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.55 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (7.55 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)