神经肽S受体过表达及单核苷酸位点突变对μ阿片受体功能的影响OA

OBJECTIVE To investigate the effects of overexpression of the neuropeptide S recep-tor(NPSR)and its single-nucleotide variant(N107I)on the function of the μ-opioid receptor(OPRM).METHODS HEK293T cell lines were established via lentiviral infection:one stably expressing the OPRM(OPRM),one stably co-expressing the OPRM and NPSR N107(OPRM/NPSR N107),and one stably co-expressing the OPRM and NPSR I107(OPRM/NPSR I107).The mRNA and total protein levels of OPRM and NPSR in these cells were determined by RT-qPCR and Western blotting,respec-tively.For cAMP assays,cells were acutely co-treated with morphine(0.01,0.1,1,and 10 μmol·L-1)and the adenylyl cyclase activator forskolin(1 μmol·L-1)for 30 min before levels of intracellular cyclic adenosine monophosphate(cAMP)were measured by homogeneous time-resolved fluorescence assay.For ERK phosphorylation assays,cells were acutely treated with morphine(0.1,1,and 10 μmol·L-1)for 5 min,and levels of extracellular signal-regulated kinase(ERK)phosphorylation were determined by Western blotting.RESULTS Compared with OPRM cells,intracellular cAMP levels were significantly increased by forskolin(1 μmol·L-1)in both OPRM/NPSR N107 and OPRM/NPSR I107 cells,especially OPRM/NPSR N107 cells.Intracellular cAMP levels were significantly decreased by morphine(1 and 10 μmol·L-1)in OPRM cells and In OPRM/NPSR N107 cells(0.1,1,and 10 μmol·L-1),but only signifi-cantly decreased by the high concentration of morphine(10 μmol·L-1)in OPRM/NPSR I107 cells.Compared with OPRM cells,morphine-induced ERK phosphorylation was higher in OPRM/NPSR N107 cells but lower in OPRM/NPSR I107 cells.CONCLUSION The overexpression of NPSR can enhance intracellular adenylyl cyclase activity while the NPSR N107I variant can attenuate the morphine-induced decrease in cAMP levels and ERK phosphorylation following acute morphine treatment.