Anandamide

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Sourced in United Kingdom

Anandamide is a lipid compound found naturally in the human body. It acts as a neurotransmitter and is involved in a variety of physiological processes. Anandamide is commonly used in research applications to study its role in the endocannabinoid system.

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Anandamide (AEA, (4 citations)

6 protocols using anandamide

Pharmacological Modulation of Sensory Signaling

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The following drugs were used in this study: capsaicin (500 nM or 100 nM, Tocris, UK); anandamide (1 mM to 30 μM, Tocris), rimonabant (200 nM, NIH, USA), mustard oil (50 μM; Sigma), NESS0327 (1nM and 100nM, Cayman Chamicals) and ionomycin (5 μM, Sigma). anandamide was dissolved in ethanol then the stock solution was prepared with Tocrisolve (Tocris). capsaicin, rimonabant, mustard oil, NESS0327 and ionomycin stock solutions were prepared in DMSO. The final DMSO dilution was equal or less than 1:2000. The minimum final Tocrisolve and ethanol dilutions were ~1:10⁶ and 1:500, respectively. In control experiments, none of the vehicles at their maximum concentration produced any responses (data not shown).

Chen J., Varga A., Selvarajah S., Jenes A., Dienes B., Sousa-Valente J., Kulik A., Veress G., Brain S.D., Baker D., Urban L., Mackie K, & Nagy I. (2016). Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their Crosstalk. Scientific Reports, 6, 33307.

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Bacterial Growth Conditions for EHEC and Salmonella

Cited 2 times

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Bacterial strains and plasmids used in this study are listed in Table S1. The isogenic mutants utilized in this study and their respective citations are listed in Table S1 and were generated using the lambda red recombinase method (Datsenko and Wanner, 2000 (link)) (Baba et al., 2006 ). EHEC and CR were subcultured into established LEE-inducing conditions as follows: DMEM medium with 1 g/L glucose and grown as standing cultures (microaerobic) or shaking at 250 rpm (aerobic) at 37°C (Abe et al., 2002 (link)) (Njoroge et al., 2012 (link)) (Carlson-Banning and Sperandio, 2016 (link)). S. Typhimurium strains were subcultured into LB broth for SPI-1-inducing conditions or subcultured into N9 minimal medium for SPI-2-inducing conditions and grown aerobically at 250 rpm at 37°C (Galán and Curtiss, 1989 (link)) (Lee et al., 2000 (link)) (Deiwick et al., 1999 (link)). Where indicated, bacteria were grown in the presence of 2-arachidonoyl glycerol, 2-AG (Tocris), 1-arachidonoyl glycerol, 1-AG (Cayman Chemical), anandamide (Tocris) or the vehicle control (methanol at a final concentration of 1:10000).

Ellermann M., Pacheco A.R., Jimenez A.G., Russell R., Cuesta S., Kumar A., Zhu W., Vale G.A., Martin S.A., Raj P., McDonald J., Winter S.E, & Sperandio V. (2020). Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens. Cell, 183(3), 650-665.e15.

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Endocannabinoid Signaling Modulators

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Anandamide, AM630, WIN55,212-2, 2-arachidonoyl
glycerol (2-AG), and d-[Trp7,9,10]-substance P were obtained
from Tocris Bioscience (R&D Systems, Minneapolis, MN). All other
chemicals were from Sigma (St. Louis, MO).

Brailoiu G.C., Deliu E., Marcu J., Hoffman N.E., Console-Bram L., Zhao P., Madesh M., Abood M.E, & Brailoiu E. (2014). Differential Activation of Intracellular versus Plasmalemmal CB2 Cannabinoid Receptors. Biochemistry, 53(30), 4990-4999.

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Endocannabinoid Modulation of ECFC Growth

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Three different ECFC donors were seeded in 6-well plates (Nalge Nunc, Rochester, NY) in EGM-2 at a density of 3,000 c/cm² and allowed to adhere for 24 hours. Subsequently, cells were subjected to growth factor reduced medium with or without AEA and different endocannabinoid receptor agonists and antagonists as indicated. The substances anandamide, CID16020046, SB366791 and Capsaicin were obtained by Tocris Bioscience (Northpoint, Avonmouth, Bristol, UK). SKM4-45-1, AM251, SR144528 (Cayman Chemical Europe, Tallinn, Estonia). After 48 hours treatment cells were harvested and cell numbers analyzed by a Casy cell counter (Roche, Mannheim, Germany).

Hofmann N.A., Barth S., Waldeck-Weiermair M., Klec C., Strunk D., Malli R, & Graier W.F. (2014). TRPV1 mediates cellular uptake of anandamide and thus promotes endothelial cell proliferation and network-formation. Biology Open, 3(12), 1164-1172.

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Anandamide Signaling Pathways in Osteoblasts

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Based on previous results, HOBs were treated with specific signalling inhibitors and receptor antagonists to identify any changes in the cell response to anandamide. HOBs were treated with media supplemented with 100nM AM251 (CB₁R antagonist), 100nM AM630 (CB₂R antagonist), 1μM capsazepine (TRPV1 antagonist), 500nM KT5720 (PKA inhibitor, PKA is upstream of CREB), 1μM PD98059 (MEK inhibitor) or 10μm SP600125 (JNK inhibitor) for 30 minutes before the addition of vehicle (0.1% ethanol) or 10μM anandamide (all Tocris, UK). HOBs were also treated with media supplemented with 10μM anandamide only and vehicle only (0.1% ethanol and 0.1% DMSO).

Smith M., Wilson R., O’Brien S., Tufarelli C., Anderson S.I, & O’Sullivan S.E. (2015). The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation. PLoS ONE, 10(9), e0136546.

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Endocannabinoid Regulation of Human Osteoblasts

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From the time of seeding, cells were treated with medium supplemented with 1nM, 10nM, 100nM, 1μM, 10μM anandamide (Tocris, UK) or 2-arachidonoylglycerol (Ascent, UK) or their vehicle, ethanol (0.1%). Cells were treated with the endocannabinoids for 24 hours, 4 days (to capture the proliferative phase of HOB growth and the early markers of differentiation) or 21 days (for later markers of differentiation such as matrix elaboration and calcification). After the treatment protocols, the media was removed and stored at -80°C, and the cells were lysed by 3 cycles of freeze-thawing in 1ml distilled water and stored at -80°C for later analysis.
To increase local levels of endocannabinoid, HOBs were treated for 24 hours with endocannabinoid degradation enzyme inhibitors, or vehicle (0.1% ethanol and 0.1% DMSO). Media was supplemented with 1μM URB597 (Sigma, UK), a fatty acid amide hydrolase inhibitor, an enzyme that degrades anandamide, or 1μM JZL184 hydrate (Sigma, UK), a monoacylglycerol lipase inhibitor, an enzyme that degrades 2-AG.

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