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‐SY5Y human neuroblastoma cells. The NPFF anti‐N/OFQ, not anti‐μ opioid effect, on the Ca2+ 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 [35S]GTPγS binding assay, a significant 20% decrease in the activity of nociceptin/orphanin FQ peptide receptors induced by the NPFF analog 1DMe was observed in detergent‐resistant membranes, but not in total membranes of SH‐SY5Y cells. Moreover, siRNA 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.