Nociceptin receptors in the rat spinal cord during morphine tolerance

Abstract

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [${}^{125}\text{I}$–Tyr¹⁴]nociceptin. [${}^{125}\text{I}$–Tyr¹⁴]nociceptin appeared to interact with a single class of binding sites (K_D=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [${}^{125}\text{I}$–Tyr¹⁴]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

Introduction

Opioid receptors belong to the superfamily of G-protein coupled receptors characterized by seven membrane spanning helices. Molecular cloning of the μ, δ and κ opioid receptors led to the discovery of a new opioid receptor (ORL1) 2, 6, 23, 35. This receptor has approximately 65% amino acid sequence similarity to opioid receptors but does not bind opioids with high affinity. The endogenous ligand of the ORL1 receptor, nociceptin [21], also known as orphanin FQ [29], was recently isolated. Nociceptin is a heptadecapeptide (FGGFTGARKSARKLANQ) which exhibits a high affinity for the ORL1 receptor, and is likely to be derived from a larger precursor polypeptide, pro-nociceptin, whose cDNA and gene have been characterized 11, 21, 24, 27, 29.

Nociceptin and opioid receptors belong to the same structural family; however, they are functionally different and do not mediate the same pharmacological effects. Whereas opioids induce analgesia, nociceptin induces various responses, depending on the route of administration, including hyperalgesia [21], allodynia [26]and analgesia [38]. Furthermore, nociceptin has been described as being a functional anti-opioid [20], although it is unclear whether activation of the classical opioid receptors is involved in the antinociceptive effect of nociceptin.

Recent studies have examined the cellular signaling systems that are modulated by nociceptin activation of the ORL1 receptor. ORL1 receptor triggers the same G-protein signaling pathways, as do opioids. Activation of the ORL1 receptor by nociceptin causes inhibition of a pertussis toxin-sensitive forskolin-stimulated cyclic-AMP production 19, 21, 28, stimulation of K⁺ conductance in rat brain [34], and inhibition of pertussis-sensitive Ca²⁺ channels currents 4, 15. Thus, like opioid receptor subtypes, the ORL1 receptor can transduce intracellular signaling via G_i/G_o-mediated pathways 21, 28.

Both immunoreactive nociceptin-like and nociceptin receptor sites are present in high density in the dorsal horn of the spinal cord 2, 28, 37, 30, indicating a possible role for the peptide in nociceptive processing. In the spinal cord, nociceptin suppresses spinal reflex, C-fiber evoked wind-up and post-discharge of dorsal horn neurons 5, 32, 36, and produces thermal antinociception in the tail-flick [38]and paw-pressure [13]tests.

The present study was carried out to determine the precise location of nociceptin receptors in the spinal cord and its role in morphine tolerance. The binding sites were characterized using [${}^{125}\text{I}$–Tyr¹⁴]nociceptin, a ligand which displays a high affinity for the ORL1 receptor [29]and is therefore a useful investigative tool.