As activation of the opioid receptors decreases Ca2+ uptake (36, 37) and inhibits voltage-dependent calcium channels (13), measuring intracellular Ca2+ levels is an important assay for characterizing MOR signaling. Fluorescent Ca2+ detection was developed in the late eighties (33). This approach allows for optical detection and quantification analysis of intracellular calcium levels. Due to the inhibitory nature of GPCRs coupled to Gi, Ca2+ levels are assayed in cells with artificially (capsaicin) elevated Ca2+ levels (13, 34, 35). Since not all cell types express capsaicin receptor TRPV1, the cells are transfected with TRPV1 expressing construct. In the experiment described below, the HEK293 cells that do not express either MOR or TRPV1 are transfected with these receptors prior to the experiments.
