NOCICEPTIN (ORL-1) AND MU-OPIOID RECEPTORS MEDIATE MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVATION IN CHO CELLS THROUGH A G(I)-COUPLED SIGNALING PATHWAY - EVIDENCE FOR DISTINCT MECHANISMS OF AGONIST-MEDIATED DESENSITIZATION

The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) recep tor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 r eceptor (OFQR) may be to modulate opioid-induced analgesia. The molecu lar mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid -stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction , indicating these receptors are capable of coupling to G(i)/G(o) prot eins. This study determines that nociceptin stimulates an intracellula r signaling pathway, leading to activation of mitogen-activated protei n (MAP) kinase in CHO cells expressing ORL-1 receptor(OFQR). Nocicepti n (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by ex pression of the carboxyl terminus of P-adrenergic receptor kinase (bet a ARKct), which specifically blocks G beta gamma-mediated signaling. E xpression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21(ras), also attenuated nociceptin (OFQ)-stimul ated MAP kinase activation. he phosphatidylinositol 3-kinase (PI-3K) i nhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) ac tivity by bisindolylmaleimide I or cellular depletion of PKC had no ef fect. In a similar manner, in cells expressing mu-opioid receptor, [D- Ala(2),N-Me-Phe(4),Gly-ol]-enkephalin (DAMGO; a mu-opioid receptor-sel ective agonist) stimulated PTX-sensitive MAP kinase activation that wa s inhibited by wortmannin, LY294002, beta ARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These resu lts indicate that both ORL-1 (OFQR) and ii-opioid receptors mediate MA P kinase activation via a signaling pathway using the py-subunit of Gi , a PI-3K, and SOS, independent of PKC activity. In cells expressing b oth ORL-1 (OFQR) and mu-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased s ubsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demon strate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate m u-opioid receptor signaling in a cellular system.