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L.N. Franks et al. / European Journal of Pharmacology 737 (2014) 140–148
Eagle's Medium (DMEM; Cellgro, Manassas, VA) containing 10%
FetalPlex™ animal serum complex (Gemini Bio Products, West
Sacramento, CA) and 1% penicillin/streptomycin (10,000 IU/ml
2.7. Measurement of intracellular cAMP levels in intact cells
Adenylyl cyclase assays were conducted similar to experiments
reported in Rajasekaran et al. (2013). Briefly, cells cultured
between passages 8 and 18 were seeded into 24-well plates
(6.5 ꢁ 106 cells per plate) and incubated overnight in a humidified
incubator maintained at 37 1C and 5% CO2. After culturing over-
night, growth media in each well was removed and replaced with
0.5 ml of an incubation media containing DMEM with 0.9 g/L NaCl,
penicillin, 10,000 g/ml streptomycin; Cellgro, Manassas, VA).
μ
Culture media for CHO-hCB2 and CHO-hMOR cells also contained
0.5 mg/ml of the selection antibiotic geniticin (G418; Sigma-
Aldrich, St. Louis, MO) to maintain stable expression of human
cannabinoid type-2 receptors and human mu-opioid receptors.
Cells were cultured at 371 C under 5% CO2 in a humidified
incubator and harvested with PBS (10 mM)/EDTA (1 mM) when
culture flasks were approximately 90–100% confluent. Cell pellets
were stored at ꢀ801 C for future preparation of membrane
homogenates, reseeded into flasks for continued cell line culturing
or seeded into 24-well plates for adenylyl cyclase experiments (see
Section 2.7).
2.5
Ci/ml [3H]adenine and 0.5 mM isobutyl-methyl-xanthine
μ
(IBMX) for 3 h. The incubation media was then removed and
plates were floated briefly on an ice-water bath only long enough
to add 0.5 ml of test compounds in a Krebs-Ringer–HEPES solution
(10 mM HEPES, 110 mM NaCl, 25 mM Glucose, 55 mM Sucrose,
5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2, pH 7.4) containing
0.5 mM IBMX and 10
transferred to a 37 1C water bath for 15 min and all reactions then
terminated by addition of 50
L of 2.2 N HCl to each well. [3H]
μ
M forskolin. Plates were immediately
2.5. Membrane preparation
μ
Crude membrane homogenates of mouse brain (for cannabi-
noid type-1) or CHO-hCB2 cells (for cannabinoid type-2) receptors
were prepared with minor modifications as described previously
(Madadi et al., 2013). In short, on the day of membrane prepara-
tion, frozen whole brains or CHO-hCB2 cell pellets were thawed on
ice and homogenized in 20 ml of ice-cold homogenization buffer
(50 mM HEPES at pH 7.4, 3 mM MgCl2, and 1 mM EGTA) by
employing a 40 ml Dounce homogenizer. First, 10 strokes with a
fine grinding pestle “A” were performed on ice, followed by
centrifugation for 10 min at 40,000g at 41 C. Pellets were then
resuspended in 20 ml of homogenization buffer and the homo-
genization and centrifugation steps were repeated two more
times. A final homogenization step using a course grinding pestle
“B” was conducted to evenly suspend the homogenates prior to
aliquoting and storage at ꢀ801 C for future use. Protein concen-
tration was determined using the BCA™ Protein Assay kit (Thermo
Scientific, Rockford, IL).
cAMP was isolated by employing alumina column chromatogra-
phy. [3H]cAMP present in 4 ml of the final eluate was quantified
following addition of 10 ml of scintiverseTM BD cocktail scintilla-
tion fluid (Fisher Scientific, Pittsburg, PA) by liquid scintillation
spectrophotometry (Tri Carb 2100TR Liquid Scintillation Analyzer,
Packard Instrument Company, Meriden, CT).
2.8. Statistical analysis
Curve-fitting and statistical analyses were conducted utilizing
GraphPad Prisms v6.0b (GraphPad Software, Inc.; San Diego, CA).
Non-linear regression for one-site competition was used to deter-
mine the IC50 for competition receptor binding. The Cheng and
Prusoff (1973) equation was employed to convert the experimental
IC50 values obtained from competition receptor binding experi-
ments to Ki values (a quantitative measure of receptor affinity).
Curve fitting of concentration–effect curves via non-linear regres-
sion was also employed to determine the ED50 (a measure of
potency) and Emax (a measure of efficacy) for the adenylyl cyclase
experiments. A one-sample t-test was used to determine whether
the modulation of adenylyl cyclase activity produced by a 10 mM
concentration of test compounds was significantly different than
basal levels in CHO-hMOR cells. Data obtained from three or more
experimental groups were analyzed by a one-way ANOVA, fol-
lowed by either Dunnett's or Tukey's post-hoc comparisons of
individual groups. A non-paired Student's t-test was employed to
statistically compare data obtained from two experimental groups.
2.6. Competition receptor binding
Receptor binding assays were conducted essentially as detailed
previously in Madadi et al. (2013). Each binding sample contained
50
μ
g (mouse brain) or 25 g (CHO-hCB2 cells) of membrane
μ
homogenates, 0.2 nM of the high affinity non-selective cannabi-
noid type-1/type-2 agonist [3H]-CP-55,940, 5 mM MgCl2, and
increasing concentrations (0.1 nM–10 M) of the non-radioactive
μ
competitive ligands in an incubation mixture containing 50 mM
Tris–HCl buffer (pH 7.4) with 0.05% bovine serum albumin (BSA).
Assays were performed in triplicate in a final volume of 1 ml of
incubation mixture. Total binding was defined as the amount of
radioactivity observed when 0.2 nM [3H]CP-55,940 was incubated
in the absence of any competitor. Non-specific binding was
defined as the amount of radioligand binding remaining in the
presence of a single 1 mM concentration of non-radioactive WIN-
55,212-2, a high affinity non-selective cannabinoid type-1/type-2
agonist. Specific binding was calculated by subtracting non-
specific from total binding. Reaction mixtures were mixed and
binding allowed to reach equilibrium during an incubation at
room temperature for 90 min. Termination of the reactions was
achieved by rapid vacuum filtration through Whatman GF/B glass
fiber filters followed by four 1 ml washes with ice cold filtration
buffer (50 mM Tris at pH 7.4 and 0.05% BSA). Filters were then
immediately placed into scintillation vials with 4 ml of Scinti-
verseTM BD cocktail scintillation fluid (Fisher Scientific, Pittsburg,
PA). After overnight incubation in scintillation fluid, bound reac-
tivity was determined by liquid scintillation spectrophotometry
(Tri Carb 2100TR Liquid Scintillation Analyzer, Packard Instrument
Company, Meriden, CT).
3. Results
3.1. Determination of intrinsic activity of novel indole quinuclidine
analogs at cannabinoid type-1 receptors
3.1.1. Screen for cannabinoid type-1 receptor modulation of adenylyl
cyclase activity by indole quinuclidine analogs
Cannabinoid receptors activate Gi/Go-proteins that then pro-
ceed to inhibit activity of the downstream intracellular effector
adenylyl cyclase (Dalton et al., 2009). As such, cannabinoid
agonists reduce, neutral antagonists have no effect upon, and
inverse agonists increase levels of intracellular cAMP (Pertwee,
2005). Therefore, as a measure of intrinsic activity at cannabinoid
type-1 receptors, the ability of eleven previously reported indole
quinuclidine analogs (Fig. 1, Table 1) (Madadi et al., 2013) to
modulate forskolin-stimulated adenylyl cyclase activity via canna-
binoid type-1 receptors endogenously expressed in intact Neuro2A
cells (He et al., 2006) was examined (Fig. 2). For these studies, a
receptor saturating concentration of each compound (e.g., 410
Franks, Lirit N.
