Fig. 1. Activation of OPRM1 induced [Ca²⁺]_i release in HEK293 cells.

A, OPRM1-mediated [Ca²⁺]_i release required P₂Y receptor co-activation. Data shows the real time intracellular fluorescence change in raw fluorescence unit (RFU). HEK293 cells were cultured as described in materials and methods. Fluorescence dye to detect free [Ca²⁺]_i was added 1 hour before compound injection. After 30 second baseline reading, as indicated by the arrow, HEK293 cells expressing OPRM1 were injected with 200nM ADP (), 1µM DAMGO (□), 200nM ADP with 1µM DAMGO (), 200nM ADP, 1µM DAMGO and 30µM Naloxone () respectively. B, The concentration-response curves of DAMGO (■) and morphine (○) were determined in the presence of 200nM ADP. Total fluorescence response change induced by ADP or by ADP with OPRM1 ligands was quantified by analyzing the areas under the curves with Prism program. ADP response was then subtracted from the response in the presence of ADP and OPRM1 ligands to obtain the DAMGO and morphine responses.