Abstract

Recent studies have demonstrated that the majority of endogenous cannabinoid type 1 (CB 1 ) receptors do not reach the cell surface but are instead associated with endosomal and lysosomal compartments. Using calcium imaging and intracellular microinjection in CB 1 receptor-transfected HEK293 cells and NG108-15 neuroblastoma × glioma cells, we provide evidence that anandamide acting on CB 1 receptors increases intracellular calcium concentration when administered intracellularly but not extracellularly. The calcium-mobilizing effect of intracellular anandamide was dose-dependent and abolished by pretreatment with SR141716A, a CB 1 receptor antagonist. The anandamide-induced calcium increase was reduced by blocking nicotinic acid-adenine dinucleotide phosphate- or inositol 1,4,5-trisphosphate-dependent calcium release and abolished when both lysosomal and endoplasmic reticulum calcium release pathways were blocked. Taken together, our results indicate that, in CB 1 receptor-transfected HEK293 cells, intracellular CB 1 receptors are functional; they are located in acid-filled calcium stores (endolysosomes). Activation of intracellular CB 1 receptors releases calcium from endoplasmic reticulum and lysosomal calcium stores. In addition, our results support a novel role for nicotinic acid-adenine dinucleotide phosphate in cannabinoid-induced calcium signaling.