Cannabinoids
The following compounds were purchased from Cayman Chemical (Ann Arbor, MI, USA): (±) CP55,940, (+)-WIN 55,212-2 (mesylate), anandamide (AEA), Δ9-tetrahydrocannabinol (Δ9-THC), and SR141716A. All controlled substances were purchased through the cannabis safety program at the University of Saskatchewan (HS-002).
AtT20-SEPCB1 Cell Culture
The AtT20 pituitary cell line was obtained from ATCC (AtT-20/D16y-F2, CRL-1795) and grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (ATCC Gibco - Manassas, VA) and 1% Penicillin-Streptomycin (Pen-Strep) (Cytiva Hyclone – Vancouver, BC) for the GIRK channel assay. For the CB1R internalization assay, cells were grown in FluoroBrite media (Gibco) with 10% FBS (ATCC Gibco – Manassas, VA), 1% Pen-Strep (Cytiva Hyclone – Vancouver, BC), 2% Glutamax (ATCC Gibco – Manassas, VA), and 10 mM HEPES (Sigma – Oakville, ON). AtT20 cells were stably transfected with lentivirus vectors containing the human cannabinoid type-1 receptor (CB1R) tagged with a super-ecliptic pHluorin (AtT20SEP-CB1) (from Dr. Andrew Irving, University College Dublin) [22]. The tagged-CB1R displays a response similar to the unmodified receptor [25]. Cells were plated in poly-l-lysine-coated wells of black 96-well plates (Greiner Bio-One – Monroe, NC) (50,000 cells per well). AtT20-SEPCB1 cells were stored in an incubator at 37◦C (5% O2/95% CO2) and used 24 h (CB1R Internationalization assay) or 72 h (GIRK channel assay) after plating. CB1R internalization occurred 24 h after plating because the measurements depended on selection of individual AtT20-SEPCB1 cells in comparison to the GIRK channel assay, which measures the overall movement of MPSD molecules on across the AtT20-SEPCB1 cell monolayer.
GIRK Channel Assay and CB1R Internalization Assays
GIRK channel activation was monitored in the 96-well clear-bottom plates by recording cell membrane potential (MP) via fluorimetry as previously described [25, 26]. For the MP measurements, the AtT20-SEPCB1 cells were incubated for 30 min in a buffer solution consisting of 132 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2,5 mM dextrose, 5 mM HEPES, pH 7.4 (with NaOH), with a MP-sensitive fluorescent dye (MPSD) (FLIPR Membrane Potential kit RED; MolecularDevices). Prior to the fluorescence measurements, the cells were loaded with MPSD in buffer solution (as above) but containing 1 mM KCl and incubated for an additional 5 min. Fluorescent signals were recorded using a SynergyHT Cytation microplate reader (Biotek) [25, 26]. Cannabinoids were dissolved in DMSO at stock concentrations of 100 mM. All cannabinoids were diluted to working concentrations in 1 mM KCl buffer solution containing the MPSD. The cannabinoids or control solution (20 µL) were injected into each well (total volume = 220 µL) at time zero. Data were collected at 9 s intervals from 36 s before compound addition until 240 s after compound addition (Fig. 1) at excitation and emission wavelengths of 520 and 560 nm, respectively.
CB1R Imaging
CB1R internalization was recorded in 96-well plates by imaging AtT20-SEPCB1R expression on the cell surface. pHluorin is a pH-sensitive green fluorescent protein whose cell surface fluorescence can be visualized at 525 nm. Because FBS increases background fluorescence and decreases image clarity, the FluoroBrite media used for cell culture was replaced with 100µL FluoroBrite media containing 1% Pen-Strep, 2% Glutamax, 10 mM HEPES, and no FBS (Imaging media). Stock solutions of cannabinoids were diluted in imaging media to working concentrations. Images of AtT20-SEPCB1 cells were taken at 40x using a BioTek Cytation 5 microplate reader (Agilent) at excitation and emission wavelengths 469 and 525 nm, respectively. Cannabinoids or control were pipetted into each well (10 µL) (total volume = 110 µL) at time zero. Z-stack images were comprised of 20, 1 µm sections collected in each well before (baseline) and after post-drug injection for 35 minutes divided into 5-minute intervals.
Imaging Data Analysis
Z-stacks were compressed into 1 image, representing the average fluorescent intensity per time point using BioTek Gen5 3.1 (Agilent). Further analysis of images was conducted using ImageJ/FIJI, 2023 (National Institute of Health Image). Each set of images were then aligned across all time points, and the was background subtracted. Regions of interest (ROIs) were determined from cells in the baseline image (-5 minutes), then the mean fluorescent intensities (F) were measured within the ROIs for each time point (-5, 0 [time of compound addition], 5, 10, 15, 20, 25, and 30 minutes) (Fig. 1). For both GIRK channel response assays and CB1R internalization assays, change in fluorescent response (ΔF) post drug injection was normalized to the baseline fluorescent response values (Fo), then the fluorescent response values from the control wells were subtracted: ΔF = ((F/Fo) – control)
CB1R Image and Animation Generation
Visualization of CB1R internalization was conducted using ImageJ/FIJI software, 2023 (National Institute of Health Image). Z-stacks were compressed into 1 image per time point set to maximal fluorescence. Each set of images were then z-stacked and aligned across all time points. Images were background subtracted, and then a FIRE look-up table (LUT) was applied to represent change in fluorescent intensity. These images were not used for data analysis.
Statistical Analysis
Data from GIRK channel assays was fit to a one-site exponential decay curve in GraphPad Prism (v. 9.0) to estimate the rate of GIRK channel response (Suppl. Figure 1). Data from GIRK channel assays were also analyzed using the Area Under the Curve function with default settings in GraphPad Prism. Peak F/F0 readings at 240 s for each cannabinoid were plotted against compound concentration (Suppl. Figure 2). AUC and peak F/F0 data were then normalized to the (±)CP55,940 maximum and fit to the four-parameter, non-linear regression analysis in Graphpad Prism (v. 9.0): y = ymin + (ymax – ymin/1 + 10^ ((LogEC50-Log Concentration), where EC50 is the concentration producing a 50% increase in the maximal response ymax (Emax), and ymin is defined as a minimum fluorescent response. The same data analysis procedure was followed for CB1R internalization data using the one-site exponential decay curve for rate of internalization (Suppl. Figure 3), AUC analysis (Suppl. Figure 4) and subsequent concentration-response curve analyses. Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were one- or two-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s post-hoc test, respectively and as indicated in figure and table legends. P < 0.05 was considered statistically significant. In both assays, n values represent replicates from independent compound treatments.