Fig. 3. Morphine-induced OPRM1 desensitization was resulted from loss of OPRM1 activity.

A, Morphine or DAMGO pretreatment did not affect ADP-mediated response. HEK293 cells expressing wild type OPRM1 were pretreated with 1µM morphine(), DAMGO() or HBSS(▲)respectively. In second injection, various concentration of ADP together with 30µM naloxone was injected. Then the concentration-response curves of ADP were determined as described in materials and methods. B, Morphine or DAMGO pretreatment did not alter [Ca²⁺]_i store availability. HEK293 cells expressing wild type OPRM1 were cultured and seeded as described in materials and methods. After 1 hr incubation of fluorescence dye, cells were washed with Ca²⁺ free HBSS buffer. Then cells were incubated in Ca²⁺ free HBSS buffer with 1mM EGTA, and were treated with agonists. After cells were pretreated with 1µM morphine, DAMGO, 10µM ADP or HBSS respectively, 1µM thapsigargin was added. Thapsigargin-induced total fluorescence change in second injection was calculated as described in materials and methods; the data were expressed as the raw fluorescence units in bar graph. Student t-test was used to compare the data in treated groups and control group. ** denotes p< 0.01.