Ž . Molecular Brain Research 43 1996 96–104 Research report Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus Emiko Okuda-Ashitaka a , Shinro Tachibana b , Takeshi Houtani c , Toshiaki Minami d , Yasuo Masu a , Miyuki Nishi e , Hiroshi Takeshima e , Tetsuo Sugimoto c , Seiji Ito a, ) a Department of Medical Chemistry, Kansai Medical UniÕersity, 10–15 Fumizono, Moriguchi 570, Japan b Tsukuba Research Laboratories, Eisai Co., Tsukuba Science City 300-26, Japan c Department of Anatomy, Kansai Medical UniÕersity, Moriguchi 570, Japan d Department of Anesthesiology, Osaka Medical College, Takatsuki 569, Japan e Department of Neurochemistry, Tokyo Institute of Psychiatry, Setagaya, Tokyo 156, Japan Accepted 11 June 1996 Abstract We reported here purification and characterization of a novel heptadecapeptide in bovine brain as an endogenous ligand for ROR-C, an opioid receptor homologue cloned from rat cerebrum. The amino acid sequence of the peptide that we purified is identical to those recently identified as nociceptin in rat brain and orphanin FQ in porcine brain. The peptide inhibited the forskolin-induced cyclic AMP accumulation in ROR-C expressing Chinese hamster ovary cells. Studies on inhibitory activity of cyclic AMP accumulation and Northern blot analysis showed that the peptide and its precursor mRNA are present in a number of brain regions, less abundant in the spinal cord, and negligible in the cerebellum. In situ hybridization analysis revealed that hybridization-positive neurons were distributed in the Ž . superficial layer lamina I of the dorsal horn and were also interspersed between the tract of Lissauer in the spinal cord. Intrathecal administration of the peptide into conscious mice induced allodynia, a pain response to innocuous tactile stimuli, in a bell-shaped manner. These results demonstrate that the peptide exists in the brain and spinal cord and plays an important role in pain transmission. Keywords: ROR-C; Opioid receptor; Endogenous ligand; Allodynia; Spinal cord 1. Introduction Morphine, a major component of opium, is a powerful analgesic that has been used for centuries for the treatment of pain. Attempts to determine its mechanism of action led to the discovery of the endogenous opioid peptides: the enkephalins, the endorphins and the dynorphins. Pharma- cological studies have defined at least three classes of opioid receptors, termed d , k , and m, that differ in their ligand selectivity and distribution in the nervous system w x 11,24 . Recent cDNA cloning studies have revealed pri- mary structures of d-, k-, and m-opioid receptors, and have also shown that these receptors are members of the w x G-protein-coupled superfamily 5,6,9,12,13,15,19,31,38 . Despite pharmacological and physiological heterogeneities, the three types of opioid receptors inhibit adenylate cy- ) Ž. Corresponding author. Fax: q81 6 992-1781. clase, increase K q conductance, and inactivate Ca 2q chan- nels through a pertussis toxin-sensitive G-protein w x 11,12,24 . Recently, opioid receptor homologues desig- wx wx nated as ROR-C 7 and LC132 1 have been cloned from rat cerebrum cDNA library by cross-hybridization or by Ž . polymerase chain reaction PCR . The deduced amino acid sequences of ROR-C and LC132, its human counterpart w x w x ORL 18 and mouse counterpart KOR-3 22 share high 1 homologies with those of the previously cloned opioid receptors, but the molecules expressed from the cDNAs did not show high affinities for opioid ligands. In order to isolate an endogenous ligand for ROR-C, we stably ex- Ž . pressed ROR-C in Chinese hamster ovary CHO-ROR-C cells and purified the ligand from bovine brain on the basis of inhibition of forskolin-induced cAMP accumulation in CHO-ROR-C cells. In the course of characterization of the ROR-C ligand, an endogenous ligand for ORL and LC132 1 w x was reported independently by two groups 14,23 . The 0169-328Xr96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 96 00165-9