Characterisation Of Cannabinoid CB1 Receptor-Mediated MAP Kinase Responses Reveal Functionally Selective Early And Late Phase ERK Responses In Transfected CHO Cells

RS Priestley, SPH Alexander, DA Kendall. University of Nottingham, Nottingham, UK

The cannabinoid CB1 receptor is a G protein-coupled receptor which has been shown to activate several intracellular signalling pathways, including the phosphorylation of several members of the mitogen activated kinase (MAP) superfamily - extracellular signal-regulated (ERK 1/2), p38 and c-JUN N-terminal (JNK) kinases. Activation of CB1 receptor-dependent pathways can occur in an agonist-dependent manner, including the activation of specific MAP kinases.

The aim of the present study was to compare the activation of MAP kinase signalling pathways by several structurally distinct cannabinoid receptor agonists in Chinese Hamster Ovary (CHO) cells stably transfected with the human cannabinoid CB1 receptor. Cells were cultured in 96-well plates, 2 days prior to experimentation when they were treated with different concentrations of cannabinoid agonists. Levels of MAP kinase phosphorylation, relative to total MAP kinase levels were determined using an optimised In-Cell Western assay technique.

The cannabinoid agonists HU-210, CP 55940 (CP), WIN-55212 (WIN), Δ9-THC and methanandamide (all 10 µM) produced significant increases in phospho-ERK levels between 2 and 8 min after agonist addition (max response ˜250% basal phospho-ERK levels; p<0.05), returning to near basal levels after 20 min. HU-210, WIN and foetal bovine serum (10% v/v) did not produce any significant change in phospho-p38 and JNK levels over the same time-course, indicating that these pathways are not CB1 receptor-coupled in these cells. Stimulation with CP (10 uM) over a 6 hour period revealed a second, late-phase ERK response (peak 60 – 90 min) of approximately half the magnitude of the initial response (˜180 % of basal phospho-ERK). In complete contrast, WIN produced a significant sustained decrease in phosphorylated ERK with a profile distinct from the CP response, reaching a plateau at 4 hours (˜35% basal phospho-ERK). Overnight pre-treatment of cells with pertussis toxin (100 ng/ml) completely abolished both phases of the CP response and the initial phase of the WIN response while the second phase WIN response remained unaffected. Both initial and late phase CP responses were concentration-dependent; however, the concentration-response curves for 60min, but not 5min, stimulation exhibited both high and low potency components (5 min EC50; 0.21 nM, 60 min EC501; 0.10 nM; EC502, 1.72 µM). The CB1 selective antagonist AM 251 produced a significant decrease in CP potency at 5 min and at the high potency component at 60 min, indicating that these responses are CB1 receptor-mediated. AM 251 had no effect on the low potency response at 60 min indicating that this response is CB1-independent.

In conclusion, CP and WIN exhibit agonist bias when comparing late and early-phase ERK MAP kinase responses, providing an interesting example of functional selectivity at the CB1 receptor. These profiles might have important implications in relation to agonist-directed downstream functional responses.