title: Agonist-regulated internalization and desensitization of the human nociceptin receptor expressed in CHO cells.
abstract: In this study we examined agonist-induced internalization of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by receptor binding assay on viable cells and confocal microscopy. The agonists nociceptin/orphanin FQ (NC), NC-NH(2), NC(1-13)-NH(2), [(pF)Phe(4)]NC-NH(2) and RO 64-6198 promote a rapid, concentration-dependent internalization of the hNOP receptor. Under the same conditions, [Phe(1),psi(CH(2)NH)Gly(2)]NC(1-13)-NH(2) and [Phe(1), psi(CH(2)NH)Gly(2),Arg(14),Lys(15)]NC(1-13)-NH(2) failed to induce significant, concentration-dependent NOP receptor endocytosis; even when present at high concentrations (up to 1 mM) they promoted only an approximately 25-30% internalization of hNOP receptors. We also investigated hNOP receptor desensitization upon agonist challenge: ligand efficacy to inhibit forskolin-stimulated cAMP production. After 1 h exposure to NC, NC-NH(2), NC(1-13)-NH(2), [(pF)Phe(4)]NC-NH(2) and RO 64-6198 (5 microM) = 20 to 30% of receptor desensitization was observed. Moreover, we found that the blockade of hNOP receptor recycling by monensin would cause a more prolonged and relevant desensitization of this receptor. The non-internalizing agonists [Phe(1),psi(CH(2)NH)Gly(2)]NC(1-13)-NH(2) and [Phe(1), psi(CH(2)NH)Gly(2),Arg(14),Lys(15)]NC(1-13)-NH(2) (100 microM) resulted in a strong (67 and 74 %, respectively) receptor desensitization which was not influenced by monensin. Finally, CHO-hNOP cells exposed to the receptor-internalizing agonists for 24 h resulted in a significantly higher cAMP accumulation (defined supersensitization) compared with the non-internalizing agonists. In addition, blocking of receptor recycling by monensin led to a decrease of the cAMP accumulation only in cells exposed to internalizing agonists. These data show that prolonged receptor signaling mediated by receptor endocytosis and recycling/reactivation might reduce the development of tolerance but can enhance compensatory mechanisms that lead to supersensitivity of specific signaling pathways.
authors.affiliation: Department of Pharmacology, University of Bologna, Irnerio 48, 40126 Bologna, Italy. spampi@biocfarm.unibo.it
authors.forename: S
authors.initials: S
authors.lastname: Spampinato
authors.forename: M
authors.initials: M
authors.lastname: Baiula
authors.forename: M
authors.initials: M
authors.lastname: Calienni
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011957:Receptors, Opioid / Q000819:agonists* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D000094942:Nociceptin Receptor
chemical_list: D011957:Receptors, Opioid; D000094942:Nociceptin Receptor