Signaling Pathways from Cannabinoid Receptor-1 Activation to Inhibition of N-Methyl-D-Aspartic Acid Mediated Calcium Influx and Neurotoxicity in Dorsal Root Ganglion Neurons

Although the activation of cannabinoid receptor-1 (CB1) receptors by cannabinoids is known to inhibit neuronal hyperexcitability and reduce excitotoxic cell death, the mechanistic links between these two actions remain elusive. We tested the hypothesis that activation of CB1 receptors inhibits N-methyl-D-aspartic acid (NMDA)-mediated calcium influx and cell death via the inositol triphosphate (IP3) signaling pathway in both primary dorsal root ganglia neurons and a cultured neuronal cell line (F-11 cells). These cells were pretreated with the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (R-(+)-WIN 55,212-2; WIN) before exposure to NMDA. Concentrations of cytosolic calcium were measured with the ratiometric calcium indicator, Fura-2, and cell death was determined by a cell viability test. WIN dose-dependently attenuated both the calcium influx and cell death induced by NMDA. These effects were blocked by selective cannabinoid CB1 receptor antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) or N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), but not CB2 receptor antagonist N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide (SR144528). It is interesting to note that a transient Ca2+ signal was observed after the acute application of WIN. This Ca2+ increase was blocked by a CB1 receptor antagonist AM251, IP3 receptor antagonist 2- aminoethyl diphenylborinate, or by depleting intracellular Ca2+ stores with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin. Removal of extracellular Ca2+, on the other hand, had no effect on the CB1 receptor-induced Ca2+ increase. These data suggest that WIN triggers a cascade of events: it activates the CB1 receptor and the IP3 signaling pathway, stimulates the release of Ca2+ from intracellular stores, raises the cytosolic Ca2+ levels, and inhibits the NMDA-mediated Ca2+ influx and cell death through a process that remains to be determined.