Stearoylethanolamide binds to specific binding sites (SBS) on C6 cell membranes, distinct from CB1, CB2, or vanilloid receptors.
Binding affinity: dissociation constant Kd = 264 ± 27 pM.
Maximum binding Bmax = 343 ± 11 fmol·mg protein⁻¹. [1]

Similar amounts of stearoyl ethanolamide (SEA) and the endocannabinoid anandamide (arachidonoyl ethanolamide; AEA) are found in the brains of rats, mice, and humans. In contrast to AEA, stearoyl ethanolamide, an endocannabinoid-like substance with pro-apoptotic action, is regulated by NO in a different way [1].

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Stearoylethanolamide (0.1–1 μM) dose-dependently inhibited nitric oxide synthase (NOS) activity in C6 cells (to 70±7%, 55±5%, and 35±4% of control at 0.1, 0.5, and 1 μM respectively), but did not affect adenylate cyclase (AC) activity. The NOS inhibition was not prevented by the CB1 antagonist SR141716 (10 μM) or by pertussis toxin (PTX, 5 μg/ml), but was partly (~50%) prevented by the vanilloid receptor antagonist capsazepine (Caps, 10 μM). In contrast, 2-arachidonoylglycerol (2-AG, 1 μM) enhanced NOS activity (to 250±25% of control) and inhibited AC (to 30±3% of control); these effects were counteracted by SR141716 (0.1 μM) or PTX, but not by Caps. [1]
Stearoylethanolamide (2.5 μM) alone did not decrease forskolin-induced cAMP levels in C6 cells, whereas AEA (2.5 μM) reduced cAMP from 10±1 to 6.0±0.5 pmol/10⁶ cells. Co-incubation of SEA (2.5 μM) with AEA further reduced cAMP to 3.0±0.3 pmol/10⁶ cells. [1]
Stearoylethanolamide (0.1–1 μM) induced apoptosis in C6 cells in a dose- and time-dependent manner: after 48 h, apoptotic bodies increased 2.0±0.2-fold (0.1 μM), 3.2±0.3-fold (0.5 μM), and 4.4±0.5-fold (1 μM) over control. The CB1 agonist HU-210 (1 μM) almost doubled SEA (0.1 μM)-induced apoptosis (to 3.8±0.4-fold), which was prevented by the NOS inhibitor L-NAME (500 μM). The peroxynitrite donor SIN-1 (1 mM) also doubled SEA-induced apoptosis (to 3.8±0.4-fold). Caps (10 μM) reduced SEA-induced apoptosis by ~50% (to 1.5±0.1-fold). The lipoxygenase inhibitor ETYA (10 μM) and cyclooxygenase inhibitor indomethacin (10 μM) fully prevented SEA-induced apoptosis, whereas MAP kinase inhibitor PD98059 (10 μM) and phosphoinositide 3’-kinase inhibitor wortmannin (10 μM) had no effect. [1]
Stearoylethanolamide (0.1–1 μM) caused dose-dependent mitochondrial uncoupling (measured by JC-1 fluorescence) after 6 h: 2.3±0.3-fold (0.1 μM), 4.5±0.5-fold (0.5 μM), and 6.3±0.6-fold (1 μM) over control. It also caused a rapid (within 6 min) increase in intracellular calcium (Fluo-3 AM): 2.4±0.3-fold (0.1 μM), 2.6±0.3-fold (0.5 μM), and 3.0±0.3-fold (1 μM) over control. HU-210 (1 μM) or SIN-1 (1 mM) further enhanced these effects, which were counteracted by L-NAME (500 μM) or partly by Caps (10 μM). [1]

Binding assay for [³H]SEA: C6 cell membranes (200×10⁶ cells/test) were incubated with [³H]SEA (0–1000 pM) for 30 min at 37°C in a final volume of 0.5 ml, then rapidly filtered through glass fibre filters. Non-specific binding was determined in the presence of 1 μM unlabelled SEA. Binding data were analysed by non-linear regression to calculate Kd and Bmax. Displacement studies used 500 pM [³H]SEA and 1 μM of various test compounds. [1]
Resinferatoxin binding assay: Binding of 100 pM [³H]resinferatoxin to C6 cells was evaluated by rapid filtration assays. Non-specific binding was determined with 1 μM unlabelled agonist. [1]
Nitric oxide synthase (NOS) assay: C6 cells (5×10⁶/test) were incubated with SEA or 2-AG for 15 min at 37°C, then washed, homogenised, and incubated with [³H]arginine. The product [³H]citrulline was measured. NOS activity was expressed as pmol citrulline released per min per mg protein. For PTX treatment, cells were preincubated with 5 μg/ml PTX for 3 h at 37°C before compound addition. [1]
Adenylate cyclase (AC) assay: AC activity was determined by measuring cAMP in cell extracts using a cAMP enzyme immunoassay. Cells (5×10⁶/test) were treated with 1 μM forskolin plus test compounds for 15 min, then homogenised, and cAMP levels were quantified. AC activity was expressed as pmol cAMP per min per mg protein. [1]
SEA uptake (SMT) assay: C6 cells (2×10⁶/test) were incubated with 300 nM [³H]SEA at 37°C or 4°C for various times, then washed with PBS containing 1% BSA. Lipids were extracted and radioactivity measured. Uptake at 4°C (non-carrier-mediated) was subtracted from that at 37°C to obtain carrier-mediated transport. For kinetic analysis, cells were incubated with 0–1000 nM [³H]SEA for 15 min. Apparent Km and Vmax were calculated by non-linear regression. Q10 was calculated as ratio of uptake at 30°C vs 20°C. For inhibitor studies, compounds were added directly to assay buffer. For CCCP, cells were preincubated with 50 μM CCCP for 15 min at 37°C before [³H]SEA addition. [1]
FAAH hydrolysis assay: C6 cell extracts were incubated with [³H]SEA at pH 9.0 for 15 min at 37°C. The reaction was stopped, and [³H]stearic acid released was measured by reversed-phase HPLC. FAAH activity was expressed as pmol stearate released per min per mg protein. Apparent Km and Vmax were calculated by non-linear regression. For inhibitor studies, compounds were added directly to assay buffer. Competitive inhibition between SEA and AEA was studied using varying substrate concentrations. Hydrolysis at pH 5.0 was also tested. [1]

Cell culture and apoptosis determination: Rat C6 glioma cells were cultured in Ham’s F-12 medium with 10% fetal calf serum at 37°C in 5% CO₂. After 48 h treatment with compounds, floating and detached cells were collected by centrifugation at 200g for 5 min. Viability was estimated by Trypan Blue exclusion. Apoptosis was quantified using a cell-death detection ELISA kit that measures histone-associated DNA fragments in cytoplasm. This was validated by flow cytometry with propidium iodide staining (50 μg/ml) to quantify apoptotic bodies (control cells had <4.0±1.0 apoptotic bodies per 100 cells). [1]
Mitochondrial uncoupling measurement: C6 cells were treated with compounds, then incubated with 20 μM JC-1 probe for 20 min at 37°C, washed, and analysed by flow cytometry (FL1/FL2 dot plot at 530/570 nm) gating on morphologically normal cells. [1]
Intracellular calcium measurement: C6 cells were washed, incubated with 10 μM Fluo-3 AM for 40 min at 37°C in the dark, then resuspended in medium without serum. Fluorescence was recorded at 530 nm (bandwidth 30 nm) at ~1000 cells/s; mean fluorescence for 3000 events was registered every 10 s. [1]
SEA and AEA uptake comparison: Uptake of [³H]AEA (200 nM) by C6 cells was measured similarly to [³H]SEA, and the effect of SEA (up to 1 μM) on AEA uptake was tested. [1]

Uptake (SMT) kinetics: Stearoylethanolamide uptake by C6 cells was temperature-dependent (Q₁₀ ≈ 1.5), time-dependent (t₁/₂ ≈ 5 min), and saturable. Apparent Km = 398 ± 58 nM, Vmax = 82 ± 5 pmol·min⁻¹·mg protein⁻¹. Uptake at 4°C was subtracted from 37°C to determine carrier-mediated transport. CCCP (50 μM) did not affect SMT activity, indicating energy independence. SMT was significantly inhibited by NO donors (SNP, SNAP) and peroxynitrite donor SIN-1, but not by AEA transporter inhibitors (VDM11, N-4-hydroxyphenyl arachidonoylamide), excess AEA or 2-AG (up to 1 μM), or Caps (10 μM). [1]
Hydrolysis (FAAH) kinetics: C6 cells hydrolysed [³H]SEA with Michaelis-Menten kinetics: apparent Km = 6 ± 1 μM, Vmax = 300 ± 23 pmol·min⁻¹·mg protein⁻¹. Hydrolysis at pH 5.0 was hardly detectable. The catalytic efficiency (Vmax/Km) for SEA was ~8-fold lower than that for AEA. FAAH inhibitors (PMSF, arachidonoyl trifluoromethyl ketone, 10 μM) fully inhibited SEA hydrolysis, as did excess AEA or 2-AG (10 μM). NO donors or SIN-1 had no effect on FAAH activity. AEA competitively inhibited SEA hydrolysis (Ki = 7 ± 1 μM), and SEA competitively inhibited AEA hydrolysis (Ki = 5 ± 1 μM). [1]

[1]. Binding, degradation and apoptotic activity of stearoylethanolamide in rat C6 glioma cells. Biochem J. 2002 Aug 15;366(Pt 1):137-44.

N-(octadecanoyl)ethanolamine is an N-acylethanolamine 18:0, which is the ethanolamide of octadecanoic acid. It is functionally related to octadecanoic acid.

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Stearoylethanolamide is an endocannabinoid-like compound that is present in rat, mouse and human brain at levels higher than anandamide. It does not bind to CB1 or CB2 receptors (failed to displace [³H]CP55,940) nor to vanilloid VR1 receptors (failed to displace [³H]resinferatoxin). Its pro-apoptotic activity is mediated by specific binding sites (SBS) distinct from cannabinoid and vanilloid receptors. SEA potentiates AEA activity (entourage effect) by inhibiting AEA degradation via FAAH. The apoptotic pathway triggered by SEA involves increased intracellular calcium, activation of lipoxygenase and cyclooxygenase, mitochondrial uncoupling, and does not involve MAP kinase or PI3-kinase. In contrast, CB1 receptor activation enhances SEA-induced apoptosis by increasing NO production, which inhibits SMT (SEA uptake) thus prolonging SEA extracellular half-life. This regulation is opposite to that of AEA, where NO increases AEA uptake and reduces its extracellular concentration. [1]

C20H41NO2

327.54504

327.313

111-57-9

27902

White to light yellow solid powder

0.9±0.1 g/cm3

486.0±28.0 °C at 760 mmHg

98.5 °C

247.7±24.0 °C

0.0±2.8 mmHg at 25°C

1.464

6.88

2

2

18

23

244

0

OTGQIQQTPXJQRG-UHFFFAOYSA-N

InChI=1S/C20H41NO2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-20(23)21-18-19-22/h22H,2-19H2,1H3,(H,21,23)

N-(2-hydroxyethyl)octadecanamide

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 : ~33.33 mg/mL (~101.76 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.63 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 (7.63 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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 (Please use freshly prepared in vivo formulations for optimal results.)