Journal: Annals of Dermatology

Article Title: Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

doi: 10.5021/ad.2016.28.1.22

Figure Lengend Snippet: Newly synthesized cannabinoids and the cAMP assay screening for cannabinoid receptor modulators. (A) The structure of α-oleoyl oleoylamine ethanolamine (α-OOE) and α-oleoyl oleoylamine serinol (α-OOS). (B, C) Synthetic compounds were screened in stable CHO cell lines overexpressing cannabinoid receptor-1 (CB1R) or CB2R. Pre-treatment with CBR agonists significantly diminished the cytosolic levels of cAMP induced by 10 µM forskolin (B). N-arachidonoylethanolamine (AEA) and Hu308 were used as controls for CB1R and CB2R, respectively. All data are presented as the mean±standard error, t -test and analysis of variance. * p <0.01.

Article Snippet: To establish stable cell lines expressing CBR, CHO-K1 cells were transfected with human CB1R (Cat. RC210397) and CB2R (Cat. SC118984) cDNA constructs (Origene, Rockville, MD, USA); the following day, media were replaced with F12 standard medium containing G418 (100 μg/ml) to select for stable clones.

Techniques: Synthesized, cAMP Assay

Journal: Annals of Dermatology

Article Title: Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

doi: 10.5021/ad.2016.28.1.22

Figure Lengend Snippet: Effects of cannabinoid receptor-1 (CB1R) agonists on the degranulation of rat basophilic leukemia cell line (RBL-2H3) mast cells. (A, B) The inhibitory effects of α-oleoyl oleoylamine ethanolamine (α-OOE), α-oleoyl oleoylamine serinol (α-OOS), and N-arachidonoylethanolamine (AEA) were determined on ionomycin (2 µM)-induced histamine (A) or β-hexosaminidase release (B) from rat mast cells. (C, D) RBL-2H3 cells sensitized with anti-dinitropheno-immunoglobulin E (anti-DNP-IgE) (0.5 µg/ml) for 12 hr were pretreated with the CB1R agonists. Histamine (C) or β-hexosaminidase release (D) from mast cells was measured. (E) Bone marrow derived-cultured mast cells from mouse bone marrow was sensitized with anti-DNP-IgE for 12 hr and histamine release from cells was measured. All data are presented as the mean±standard error, t-test and analysis of variance. NON: non-stimulated, CON: control (stimulated), IgE: Immunoglobulin E, DNP: dinitrophenol, BSA: bovine serum albumin. * p <0.01 and ** p <0.05.

Article Snippet: To establish stable cell lines expressing CBR, CHO-K1 cells were transfected with human CB1R (Cat. RC210397) and CB2R (Cat. SC118984) cDNA constructs (Origene, Rockville, MD, USA); the following day, media were replaced with F12 standard medium containing G418 (100 μg/ml) to select for stable clones.

Techniques: Derivative Assay, Cell Culture, Control

Journal: Annals of Dermatology

Article Title: Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

doi: 10.5021/ad.2016.28.1.22

Figure Lengend Snippet: Inhibition of proliferation of rat basophilic leukemia cell line (RBL-2H3) cells by cannabinoid receptor-1 (CB1R) agonists. RBL-2H3 cells were untreated or treated with the indicated concentrations of CB1R agonists for 24 hr (A) or with 25 µM agonists for the indicated time periods (B). (C) RBL-2H3 cell proliferation was analyzed by counting cell numbers following CB1R agonist treatment for 48 hr. α-OOE: α-oleoyl oleoylamine ethanolamine, α-OOS: α-oleoyl oleoylamine serinol, AEA: N-arachidonoylethanolamine. Significant difference ( * p <0.01 in Student's t -test) as compared with control samples.

Article Snippet: To establish stable cell lines expressing CBR, CHO-K1 cells were transfected with human CB1R (Cat. RC210397) and CB2R (Cat. SC118984) cDNA constructs (Origene, Rockville, MD, USA); the following day, media were replaced with F12 standard medium containing G418 (100 μg/ml) to select for stable clones.

Techniques: Inhibition, Control

Journal: Annals of Dermatology

Article Title: Selective Cannabinoid Receptor-1 Agonists Regulate Mast Cell Activation in an Oxazolone-Induced Atopic Dermatitis Model

doi: 10.5021/ad.2016.28.1.22

Figure Lengend Snippet: Effects of cannabinoid receptor-1 agonists in oxazolone-induced atopic dermatitis mice models. (A) Histology of dorsal skin lesions. The dorsal skin of each mouse was removed and fixed for toluidine blue staining (×400). Images are representative of five mice. (B) Change of skin fold thickness in oxazolone model. α-oleoyl oleoylamine ethanolamine (α-OOE), α-oleoyl oleoylamine serinol (α-OOS), and N-arachidonoylethanolamine (AEA) treatment prevented the increase of skin fold thickness. (C) Quantitative histomorphometry of the number of mast cells. Mast cells were counted in the toluidine blue-stained slide. (D) Total blood histamine levels in serum. The histamine concentration of each mouse was measured according to a previously described method. Each point represents the mean±standard error. Data are from five to six mice. VEH: vehicle, DEXA: dexamethasone. * p <0.01, ** p <0.05 significantly different from the vehicle group (Student's t-test).

Article Snippet: To establish stable cell lines expressing CBR, CHO-K1 cells were transfected with human CB1R (Cat. RC210397) and CB2R (Cat. SC118984) cDNA constructs (Origene, Rockville, MD, USA); the following day, media were replaced with F12 standard medium containing G418 (100 μg/ml) to select for stable clones.

Techniques: Staining, Concentration Assay

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Structure of representative CB1 allosteric modulators

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: PAM activity of fluoro-analogs of GAT211

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Activity Assay

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: PAM activity of aza-analogs of GAT211

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Activity Assay, Solubility

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of 6r, 9j, and 6s on the binding of a) 1 nM [3H]SR141716A or b) [3H]CP55,940 to membranes obtained from CHO cells stably-expressing human CB1R; or the binding of [35S]GTPγS to G protein in membranes obtained from CHO cells stably-expressing human CB1R and treated with c) CP55,940 and 1 μM GAT compound, or d) GAT compound alone. Ki (nM) and EC50 (nM) are mean with 95% CI. EMax data are mean ± SEM. Data were fit to a variable slope (4 parameter) nonlinear regression model, mean ± SEM, n = 4–6 independent experiments performed in duplicate. ***P < 0.001 Student’s one sample t-test.

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Binding Assay, Stable Transfection, Expressing

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: (a-c) CHO cells stably-expressing hCB1R were treated with 0.10 nM – 10 μM GAT compounds alone for 90 min and cAMP inhibition (a) or βarrestin2 recruitment was measured (b). Compound agonist bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in c. (d-f) CHO cells stably-expressing hCB1R were treated with 100 nM CP55,940 + 0.10 nM – 10 μM GAT compounds for 90 min and cAMP inhibition (d) or βarrestin2 recruitment was measured (e). Compound PAM bias between cAMP inhibition and βarrestin2 recruitment (ΔΔLogR (cAMP - βarr2)] is shown in f. cAMP data are expressed as % Forskolin response within compound treatment. βarrestin2 recruitment data are expressed as % CP55,940 response. Data were fit to a nonlinear regression (4 parameter model, GraphPad v. 7) for statistics in Table 5; or fit to the operational model (eq. 1) to calculate bias (c,f). Data are mean ± S.E.M. or 95% CI (c,f), n ≥ 6 independent experiments performed in triplicate.

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Stable Transfection, Expressing, Inhibition

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: The CB1 positive allosteric modulators 6r and 6s suppress CFA-Induced mechanical allodynia. (A, B) Both 6r (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) and 6s (0.1, 0.3, 1, 3, 10 and 30 mg/kg i.p.) increase mechanical paw withdrawal thresholds in the paw ipsilateral (A) but not contralateral (B) to CFA injection. (C, D) Both 6r (30 mg/kg i.p.) and 6s (30 mg/kg i.p.) elevated mechanical paw withdrawal thresholds in the paw ipsilateral (C) but not contralateral (D) to CFA injection with efficacy observed over at least 2.5 h post injection. (E, F) Neither 6r nor 6s altered mechanical paw withdrawal thresholds in CB1 KO mice in the paw ipsilateral (E) or contralateral (F) to CFA injection.

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Injection

Journal: Journal of medicinal chemistry

Article Title: Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of CB1 Receptor Positive Allosteric Modulators

doi: 10.1021/acs.jmedchem.9b01142

Figure Lengend Snippet: Effect of key CB1R ago-PAMs on catalepsy, hypothermia and tail flick test. Male C57BL/6 mice treated with 0.1 – 10 mg/kg compound or vehicle (1:1:18 ethanol:cremaphor:saline) and physiological assessments of catalepsy (5 min, % MPE 60 sec) (a), body temperature relative to vehicle (15 min, 100%) (b), and nociception in the tail flick assay (52°C) (20 min, % MPE 20 sec) (c). n = 5–6, data are mean ± SEM. *P < 0.05, **P < 0.01 6r compared to vehicle; ^P < 0.05, ^^^P < 0.001 6s compared to vehicle as determined via two-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: PathHunter® CB1R βarrestin2 assay: βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter® assay (DiscoveRx®).

Techniques: Tail Flick Test, Saline

Journal: bioRxiv

Article Title: A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

doi: 10.1101/2020.10.08.329169

Figure Lengend Snippet: a , Schematic diagram depicting the design and principle of the GRAB eCB sensor, consisting of the CB1 receptor and circular-permutated GFP. Ligand binding activates the sensor, inducing a change in fluorescence. b , Screening and optimization steps of GRAB eCB sensors and the normalized fluorescence response to 10 μM 2-AG. eCBmut was generated by introducing the F177 2.64 A mutation in eCB2.0. c , Expression and fluorescence change in response to 100 μM 2-AG and AEA in HEK293T cells expressing eCB2.0. Scale bar, 30 μm. d , Dose-response curves measured in HEK293T cells expressing eCB2.0 or eCBmut, with the corresponding EC 50 values for 2-AG and AEA shown; n = 3 wells each. e , Normalized fluorescence change in response to the indicated compounds (each at 10 μM) measured in cells expressing eCB2.0; n = 3–4 well each. Where indicated, the CB1R inverse agonist AM251 was also added. LPA, lysophosphatidic acid; S1P, sphingosine-1-phosphate; ACh, acetylcholine; DA, dopamine; GABA, gamma-aminobutyric acid; Glu, glutamate; Gly, glycine; NE, norepinephrine; 5-HT, 5-hydroxytryptamine; His, histamine; Epi, epinephrine; Ado, adenosine; Tyr, tyramine. f , Illustration of the localized puffing system using a glass pipette containing 100 μM 2-AG and/or AM251 positioned above an eCB2.0-expressing cell. The dotted black line indicates the region of interest for line scanning. Scale bar, 30 μm. g , Change in eCB2.0 fluorescence was measured in an eCB2.0-expressing cell using line scanning; where indicated, 2-AG and AM251 were puffed on the cell. The graph at the right summarizes the on and off time constants measured upon application of 2-AG and upon application of AM251, respectively; n = 11 ( t on ) and 4 (τoff) cells. h , G protein coupling was measured using a BRET G βγ sensor in cells expressing CB1R, eCB2.0, or eCBmut. i , β-arrestin coupling was measured using the Tango assay in cells expressing CB1R, eCB2.0, or eCBmut. Student’s t tests were performed in e and h : ***p < 0.001; n.s., not significant.

Article Snippet: For the G βγ sensor assay, the human CB1R was cloned into the pCI vector (Promega), and eCB2.0 and eCBmut were cloned into the peGFP-C1 vector (Takara), replacing the eGFP open reading frame.

Techniques: Ligand Binding Assay, Fluorescence, Generated, Mutagenesis, Expressing, Transferring

Journal: bioRxiv

Article Title: A fluorescent sensor for spatiotemporally resolved endocannabinoid dynamics in vitro and in vivo

doi: 10.1101/2020.10.08.329169

Figure Lengend Snippet: a , Schematic diagram depicting the structure of the GRAB eCB2.0 sensor. The IgK leader sequence and the sequence derived from GRAB NE are shown. b , Amino acids sequence of the eCB2.0 sensor. The phenylalanine residue at position 177 2.64 in the CB1 receptor was mutated to an alanine to generate the eCB mutant sensor (indicated by the gray box). Note that the numbering used in the figure corresponds to the start of the IgK leader sequence.

Article Snippet: For the G βγ sensor assay, the human CB1R was cloned into the pCI vector (Promega), and eCB2.0 and eCBmut were cloned into the peGFP-C1 vector (Takara), replacing the eGFP open reading frame.

Techniques: Sequencing, Derivative Assay, Mutagenesis

Journal: American journal of kidney diseases : the official journal of the National Kidney Foundation

Article Title: Cannabinoid Signaling in the Diabetic Proximal Tubule: Of Mice and Men

doi: 10.1053/j.ajkd.2022.09.004

Figure Lengend Snippet: Proximal tubule (PT) cells and diabetic kidney disease (DKD). (A) Schematic of glucose transporters in the initial segments of the kidney PT. SGLT2 transports glucose from the PT lumen into the cytoplasm against its concentration gradient owing to the energetically favorable electrochemical gradient for sodium. The reabsorbed glucose is largely transported out of the cell down its concentration gradient by GLUT2, a high-capacity facilitative glucose transporter found on the basolateral surface. Relatively small numbers of GLUT2 molecules can be found on the luminal (apical) surface of the PT cells, where they transport glucose down its concentration gradient either in or out of the cell. Under usual physiologic conditions such transport would be out of the cell, since SGLT2 action increases glucose concentrations inside the cell relative to the concentration in the lumen, which falls substantially along the length of the proximal tubule. (B) Comparison between the cell-specific transcript expression of CNR1 (transcript coding for CB 1 R) and SLC5A2 (SGLT2) in PT cells from healthy controls (n = 18; 4,100 cells; left panel) or patients with DKD (n = 12; 5,300 cells; right panel). Data for healthy controls were adapted from Menon et al 23 while data for the patients with DKD were from the Kidney Precision Medicine Project and obtained from Lake et al. 24 At the protein level, CB 1 R is not detectable in glomeruli and tubules in human kidneys (sample image from Human Protein Atlas, available at www.proteinatlas.org/ENSG00000118432-CNR1/tissue/kidney#img ).

Article Snippet: Similarly, there is no detectable CB 1 R protein in the PT or kidney cortex of any of the samples (with or without disease) assessed by the Human Protein Atlas.

Techniques: Concentration Assay, Comparison, Expressing, Clinical Proteomics