A non-paired Student’s test). Table 1 Comparison of Cannabinoid type-1 Receptor affinity with potency and efficacy for Adenylyl Cyclase Inhibition produced by Indole Quinuclidine compounds 1C11. test; Reported as mean Goat polyclonal to IgG (H+L) S.E.M., n=3C4 NT = Not Tested 3.1.2. agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. have been utilized historically for a variety of medicinal purposes, including use as analgesics, anti-bacterials, anti-migraines and anti-inflammatory agents (Russo, 2007). Discovery of type-1 (Matsuda et al., 1990) and type-2 (Munro et al., 1993) cannabinoid receptors in the 1990’s spurred increased research for additional therapeutic uses of products and analogues derived from these natural compounds (Grotenhermen and Muller-Vahl, 2012). Cannabinoid type-1 receptors are present in greatest abundance in the CNS (Herkenham et al., 1990), but also are found in the periphery (Kress and Kuner, 2009; Nogueiras et al., 2008). In contrast, cannabinoid type-2 receptors are most prevalent in immune cells (McCarberg and Barkin, 2007), although also observed in the brain (Van Sickle et al., 2005; Xi et al., 2011). Both receptors are linked to inhibitory G-proteins (Gi/o) that inhibit downstream cAMP production and activate the MAP-kinase cascade (Dalton et al., 2009). Cannabinoid type-1, but not type-2 receptors, also modulate the activity of voltage-gated Ca2+ and inward rectifying K+ ion channels (Mackie et al., 1995). The major psychoactive cannabinoid isolated from experiments. All other drugs were obtained from Tocris Bioscience (Ellisville, MO). [3H]CP-55,950 (168 Ci/mmol) was purchased from Perkin Elmer (Boston, MA) and [3H]adenine (26 Ci/mmol) was obtained from (Vitrax; Placenia, CA). All other reagents were purchased from Fisher Scientific Inc. (Pittsburgh, PA). 2.2. Animals The University of Arkansas for Medical Sciences institutional animal care and use committee (at 4C. Pellets were then resuspended in 20 ml of homogenization buffer and the homogenization 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 ?80C for future use. Protein concentration was determined using the BCA? Protein Assay kit (Thermo Scientific, Rockford, IL). 2.6. Competition Receptor Binding Receptor binding assays were conducted essentially as detailed previously in (Madadi et al., 2013). Each binding sample contained 50 g (mouse brain) or 25 g (CHO-hCB2 cells) of Cefpiramide sodium membrane homogenates, 0.2 nM of the high affinity non-selective cannabinoid type-1/type-2 agonist [3H]-CP-55,940, 5 mM MgCl2, and increasing concentrations (0.1 nM C 10 M) of the non-radioactive 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 1ml 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 M concentration of non-radioactive WIN-55,212C2, 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 Cefpiramide sodium ml of ScintiverseTM BD cocktail scintillation fluid (Fisher Scientific, Pittsburg, PA). After overnight incubation in scintillation fluid, bound reactivity was determined by liquid scintillation spectrophotometry (Tri Carb 2100 TR Liquid Scintillation Analyzer, Packard Instrument Company, Meriden, CT). 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 37C and 5% CO2. After culturing overnight, growth media in each well was removed and replaced with 0.5 ml of an incubation media containing DMEM with 0.9g/L NaCl, 2.5 Ci/ml [3H]adenine and 0.5 mM isobutyl-methyl-xanthine (IBMX) for 3 hr. The incubation media.After overnight incubation in scintillation fluid, bound reactivity was determined by liquid scintillation spectrophotometry (Tri Carb 2100 TR Liquid Scintillation Analyzer, Packard Instrument Company, Meriden, CT). 2.7. cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. have been utilized historically for a variety of medicinal purposes, including use as analgesics, anti-bacterials, anti-migraines and anti-inflammatory agents (Russo, 2007). Discovery of type-1 (Matsuda et al., 1990) and type-2 (Munro et al., 1993) cannabinoid receptors in the 1990’s spurred increased research for additional therapeutic uses of products and analogues derived from these natural compounds (Grotenhermen and Muller-Vahl, 2012). Cannabinoid type-1 receptors are present in greatest abundance in the CNS (Herkenham et al., 1990), but also are found in the periphery (Kress and Kuner, 2009; Nogueiras et al., 2008). In contrast, cannabinoid type-2 receptors are most prevalent in immune cells (McCarberg and Barkin, 2007), although also observed in the brain (Van Sickle et al., 2005; Xi et al., 2011). Both receptors are linked to inhibitory G-proteins (Gi/o) that inhibit downstream cAMP production and activate the MAP-kinase cascade (Dalton et al., 2009). Cannabinoid type-1, but not type-2 receptors, also modulate the activity of voltage-gated Cefpiramide sodium Ca2+ and inward rectifying K+ ion channels (Mackie et al., 1995). The major psychoactive cannabinoid isolated from experiments. All other drugs were obtained from Tocris Bioscience (Ellisville, MO). [3H]CP-55,950 (168 Ci/mmol) was purchased from Perkin Elmer (Boston, MA) and [3H]adenine (26 Ci/mmol) was obtained from (Vitrax; Placenia, CA). All other reagents were purchased from Fisher Scientific Inc. (Pittsburgh, PA). 2.2. Animals The University of Arkansas for Medical Sciences institutional animal care and use committee (at 4C. Pellets were then resuspended in 20 ml of homogenization buffer and the homogenization 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 ?80C for future use. Protein concentration was determined using the BCA? Protein Assay kit (Thermo Scientific, Rockford, IL). 2.6. Competition Receptor Binding Receptor binding assays were conducted essentially as detailed previously in (Madadi et al., 2013). Each binding sample contained 50 g (mouse brain) or 25 g (CHO-hCB2 cells) of membrane homogenates, 0.2 nM of the high affinity non-selective cannabinoid type-1/type-2 agonist [3H]-CP-55,940, 5 mM MgCl2, and increasing concentrations (0.1 nM C 10 M) of the non-radioactive 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 level of 1ml of incubation blend. Total binding was thought as the quantity of radioactivity noticed Cefpiramide sodium when 0.2 nM [3H]CP-55,940 was incubated in the lack of any rival. nonspecific binding was thought as the quantity of radioligand binding staying in the current presence of an individual 1 M focus of nonradioactive WIN-55,212C2, a higher affinity nonselective cannabinoid type-1/type-2 agonist. Particular binding was determined by subtracting nonspecific from total binding. Response mixtures had been combined and binding permitted to reach equilibrium during an incubation at space temp for 90 min. Termination.