Elsevier

Molecular Brain Research

Volume 43, Issues 1–2, 31 December 1996, Pages 96-104
Molecular Brain Research

Research report
Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus

https://doi.org/10.1016/S0169-328X(96)00165-9Get rights and content

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 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.

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. Pharmacological studies have defined at least three classes of opioid receptors, termed δ, κ, and μ, that differ in their ligand selectivity and distribution in the nervous system 11, 24. Recent cDNA cloning studies have revealed primary structures of δ-, κ-, and μ-opioid receptors, and have also shown that these receptors are members of the 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 cyclase, increase K+ conductance, and inactivate Ca2+ channels through a pertussis toxin-sensitive G-protein 11, 12, 24. Recently, opioid receptor homologues designated as ROR-C [7]and LC132 [1]have been cloned from rat cerebrum cDNA library by cross-hybridization or by polymerase chain reaction (PCR). The deduced amino acid sequences of ROR-C and LC132, its human counterpart ORL1 [18]and mouse counterpart KOR-3 [22]share high 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 expressed 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 ORL1 and LC132 was reported independently by two groups 14, 23. The amino acid sequence of the ROR-C ligand purified from bovine brain was identical to those of the peptides designated as nociceptin in rat brain and orphanin FQ in porcine brain. Therefore we used the endogenous ROR-C ligand and nociceptin equivalently in the following description.

Hyperalgesia is defined as pain due to a stimulus that is normally painful, whereas the closely related term allodynia defined as pain due to a stimulus that does not normally provoke pain [10]. Although hyperalgesia and allodynia are often associated consequences of damage to peripheral nerves or the central nervous system, it has recently been suggested that different receptor systems in the spinal cord may be involved in these two sensory disorders [34]. It was previously reported that intrathecal (i.t.) administration of a high dose of morphine into rats induced hyperesthesia or allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli [35]. Clinical reports have also suggested that the allodynia may also occur in humans after high doses of i.t. morphine [27]. In this paper, we report the purification of nociceptin from bovine brain, expression patterns of nociceptin and mRNA for the precursor protein in the brain and spinal cord, and involvement of nociceptin in allodynia.

Section snippets

Materials

[α-32P]dCTP (3000 Ci/mmol) was obtained from Du Pont-New England Nuclear (Wilmington, DE, USA). Naloxone benzoylhydrazone was obtained from Research Biochemicals (Natik, MA, USA). All other chemicals were of reagent grade. CHO cells in culture were transfected with the mammalian expression plasmid pRORS13-3, carrying the entire sequence of ROR-C [7]. Clones expressing ROR-C (CHO-ROR-C) were isolated by screening G418-resistant clones by RNA blot hybridization analysis using ROR-C cDNA as probe.

Purification of an endogenous ligand for ROR-C from bovine brain

Purification of an endogenous ligand for ROR-C from bovine brain

In order to purify an endogenous ligand for ROR-C, we chose bovine brain as starting material because bovine brain can be obtained in large amounts. In preliminary experiments, we found that concentrates of crude extracts from some regions of bovine brain such as hypothalamus and entorhinal cortex, but not cerebellum, inhibited forskolin-induced cAMP formation in CHO-ROR-C cells and that this inhibitory activity disappeared with treatment of crude extracts with chymotrypsin and trypsin,

Discussion

In the present study, we purified a heptadecapeptide as an endogenous ligand for ROR-C from bovine brain on the basis of inhibition of forskolin-induced cAMP formation in CHO-ROR-C cells. The amino acid sequence of the peptide was identical to that purified from rat and porcine brain, suggesting the importance of this peptide in brain functions beyond species. In fact, it was reported that the synthetic peptide induced hyperalgesia and decreased locomotor activity when administered

Acknowledgements

We are grateful to Dr. Terutoshi Kimura of the Peptide Institute (Osaka) for kind supply of synthetic nociceptin. This work was supported in part by Grants-in-Aids for Scientific Research on Priority Areas, Scientific Research (B) (06454171), Scientific Research (C) (08672042) and for Encouragement of Young Scientists (06671243) from the Ministry of Education, Science, and Culture of Japan, by a research grant for intractable diseases from the Ministry of Health and Welfare of Japan and by

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