Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay.

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the ß-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The ß-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid's GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.

Molecular signaling of synthetic cannabinoids: Comparison of CB1 receptor and TRPV1 channel activation.

Recreational use of synthetic cannabinoids (SCs) is associated with desirable euphoric and relaxation effects as well as adverse effects including anxiety, agitation and psychosis. These SC-mediated actions represent a combination of potentiated cannabinoid receptor signaling and "off-target" receptor activity. The goal of this study was to compare the efficacy of various classes of SCs in stimulating CB1 receptors and activating "off-target" transient receptor potential (TRP) channels. Cannabinoid-type 1 (CB1) receptor activity was determined by measuring SC activation of G protein-gated inward rectifier K+ (GIRK) channels using a membrane potential-sensitive fluorescent dye assay. SC opening of vanilloid type-1 (TRPV1) channels was measured by recording intracellular Ca2+ transients. All of the SCs tested activated the GIRK channel with an efficacy of 4-fluoro MDMB-BUTINACA > 5-fluoro MDMB-PICA > MDMB-4en-PINACA ≈ WIN 55,212-2 > AB-FUBINACA > AM1220 ≈ JWH-122 N-(5-chloropentyl) > AM1248 > JWH-018 ≈ XLR-11 ≈ UR-144. The potency of the SCs at the CB1 receptor was 5-fluoro MDMB-PICA ≈ 4-fluoro MDMB-BUTINACA > AB-FUBINACA ≈ MDMB-4en-PINACA > JWH-018 > AM1220 > XLR-11 > JWH-122 N-(5-chloropentyl) > WIN 55,212-2 ≈ UR-144 > AM1248. In contrast, when tested at a SC concentration that produced a maximal effect on the Gi/GIRK channel, only XLR-11, UR-144 and AM1220 caused a significant activation of the TRPV1 channels. The TRPV1 channel/Ca2+ signal measured during application of 10 µM XLR-11 was similar to the signal induced by the endocannabinoid N-arachidonoylethanolamine (AEA). Thus, while various SCs share the ability to stimulate CB1 receptor/Gi signaling, they display limited efficacy in opening TRPV1 channels.

Molecular Pharmacology of Synthetic Cannabinoids: Delineating CB1 Receptor-Mediated Cell Signaling.

Synthetic cannabinoids (SCs) are a class of new psychoactive substances (NPSs) that exhibit high affinity binding to the cannabinoid CB1 and CB2 receptors and display a pharmacological profile similar to the phytocannabinoid (-)-trans-Δ9-tetrahydrocannabinol (THC). SCs are marketed under brand names such as K2 and Spice and are popular drugs of abuse among male teenagers and young adults. Since their introduction in the early 2000s, SCs have grown in number and evolved in structural diversity to evade forensic detection and drug scheduling. In addition to their desirable euphoric and antinociceptive effects, SCs can cause severe toxicity including seizures, respiratory depression, cardiac arrhythmias, stroke and psychosis. Binding of SCs to the CB1 receptor, expressed in the central and peripheral nervous systems, stimulates pertussis toxin-sensitive G proteins (Gi/Go) resulting in the inhibition of adenylyl cyclase, a decreased opening of N-type Ca2+ channels and the activation of G protein-gated inward rectifier (GIRK) channels. This combination of signaling effects dampens neuronal activity in both CNS excitatory and inhibitory pathways by decreasing action potential formation and neurotransmitter release. Despite this knowledge, the relationship between the chemical structure of the SCs and their CB1 receptor-mediated molecular actions is not well understood. In addition, the potency and efficacy of newer SC structural groups has not been determined. To address these limitations, various cell-based assay technologies are being utilized to develop structure versus activity relationships (SAR) for the SCs and to explore the effects of these compounds on noncannabinoid receptor targets. This review focuses on describing and evaluating these assays and summarizes our current knowledge of SC molecular pharmacology.

A real time screening assay for cannabinoid CB1 receptor-mediated signaling.

The cannabinoid CB1 receptor is expressed throughout the central nervous system where it functions to regulate neurotransmitter release and synaptic plasticity. While the CB1 receptor has been identified as a target for both natural and synthetic cannabinoids, the specific downstream signaling pathways activated by these various ligands have not been fully described. In this study, we developed a real-time membrane potential fluorescent assay for cannabinoids using pituitary AtT20 cells that endogenously express G protein-gated inward rectifier K+ (GIRK) channels and were stably transfected with the CB1 receptor using a recombinant lentivirus. In whole-cell patch clamp experiments application of the cannabinoid agonist WIN 55,212-2 to AtT20 cells expressing the CB1 receptor (AtT20/CB1) activated GIRK currents that were blocked by BaCl2. WIN 55,212-2 activation of the GIRK channels was associated with a time- and concentration-dependent (EC50 = 309 nM) hyperpolarization of the membrane potential in the AtT20/CB1 cells when monitored using a fluorescent membrane potential-sensitive dye. The WIN 55,212-2-induced fluorescent signal was inhibited by pretreatment of the cells with either the GIRK channel blocker tertiapin-Q or the CB1 receptor antagonist SR141716. The cannabinoids displayed a response of WIN 55,212-2 ≈ anandamide (AEA) > CP 55,940 > Δ9-tetrahydrocannabinol (THC) when maximal concentrations of the four ligands were tested in the assay. Thus, the AtT20/CB1 cell fluorescent assay will provide a straightforward and efficient methodology for examining cannabinoid-stimulated Gi signaling.

Behavioral History of Withdrawal Influences Regulation of Cocaine Seeking by Glutamate Re-Uptake.

Withdrawal from cocaine regulates expression of distinct glutamate re-uptake transporters in the nucleus accumbens (NAc). In this study, we examined the cumulative effect of glutamate re-uptake by multiple excitatory amino acid transporters (EAATs) on drug-seeking at two different stages of withdrawal from self-administered cocaine. Rats were trained on fixed ratio 1 (FR1), progressing to FR5 schedule of reinforcement. After one day of withdrawal, microinfusion of a broad non-transportable EAAT antagonist, DL-threo-beta-benzyloxyaspartate (DL-TBOA), into the NAc shell dose-dependently attenuated self-administration of cocaine. Sucrose self-administration was not affected by DL-TBOA, indicating an effect specific to reinforcing properties of cocaine. The attenuating effect on cocaine seeking was not due to suppression of locomotor response, as DL-TBOA was found to transiently increase spontaneous locomotor activity. Previous studies have established a role for EAAT2-mediated re-uptake on reinstatement of cocaine seeking following extended withdrawal and extinction training. We found that blockade of NAc shell EAATs did not affect cocaine-primed reinstatement of cocaine seeking. These results indicate that behavioral history of withdrawal influences the effect of re-uptake mediated glutamate clearance on cocaine seeking. Dynamic regulation of glutamate availability by re-uptake mechanisms may impact other glutamate signaling pathways to account for such differences.