Cholesterol‐rich lipid rafts are involved in neuropeptide FF anti‐nociceptin/orphanin FQ effect

The participation of a signaling platform to the anti‐nociceptin/orphanin FQ (N/ OFQ ) effect of neuropeptide FF ( NPFF ) receptors was investigated in both acutely dissociated neurons and SH ‐ SY 5Y human neuroblastoma cells. The NPFF anti‐N/ OFQ , not anti‐μ opioid effect, on the Ca 2+ transient triggered by depolarization was reversed by methyl‐β‐cyclodextrin which depletes cholesterol from cell membranes. While the inactive α‐cyclodextrin had no effect. By using [ 35 S] GTP γS binding assay, a significant 20% decrease in the activity of nociceptin/orphanin FQ peptide receptors induced by the NPFF analog 1 DM e was observed in detergent‐resistant membranes, but not in total membranes of SH ‐ SY 5Y cells. Moreover, si RNA knock‐down of G‐protein‐coupled receptor kinase 2 indicated that G‐protein‐coupled receptor kinase 2, but not protein kinase C, acted as the mediator in the NPFF anti‐N/ OFQ process. These data indicate that cholesterol‐rich lipid rafts play an important role in the anti‐N/ OFQ effect of NPFF receptors.We proposed the participation of a signaling platform to the anti‐Nociceptin/Orphanin FQ (N/OFQ) effect of Neuropeptide FF (NPFF) receptors both in mouse neurons and SH‐SY5Y cells, with GRK2 protein acting as the mediator in this process. These findings should provide a more precise way to understand the anti‐opioid effect of NPFF. NOP, Nociceptin/Orphanin FQ peptide.