Research reportNociceptin receptors in the rat spinal cord during morphine 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 Gi/Go-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 [–Tyr14]nociceptin, a ligand which displays a high affinity for the ORL1 receptor [29]and is therefore a useful investigative tool.
Section snippets
Chemicals
Nociceptin (FGGFTGARKSARKYANQ) and [Tyr14]nociceptin were synthesized using solid phase techniques (H. Mazarguil, IPBS).
Bovine serum albumin (BSA) was from Euromedex, France; Lofentanil was a gift from Jansen Pharmaceuticals (Beerse, Belgium).
Iodination
[Tyr14]nociceptin was prepared using iodogen as oxidizing agent. Briefly, 10 μl of a 10−3 M solution of [Tyr14]nociceptin, 100 μl of 50 mM Tris–HCl pH 7.4, 25 μl CH3OH and 37 MBq (1 mCi) of carrier-free Na (Cis Bio International) were mixed. The
Binding parameters of [–Tyr14]nociceptin
Binding characteristics of [–Tyr14]nociceptin were analyzed on rat spinal cord sections at a single concentration (0.05 nM). After incubation of the sections with [–Tyr14]nociceptin followed by an ice-cold buffer wash, there was a rapid loss of total bound radioactivity, the amounts decreasing with the number of washes. Non-specific binding decreased rapidly, increasing the level of specific binding which remained constant after three washes. Labelling of the slices was routinely
Discussion
We report here the first detailed mapping of nociceptin receptors in the rat spinal cord and their biochemical and pharmacological properties in sections using quantitative autoradiography.
In slide-mounted rat spinal cord sections, [–Tyr14]nociceptin binds to a single class of binding sites in a time-dependent, reversible and saturable manner. [–Tyr14]nociceptin binding sites exhibit a high affinity for nociceptin and lofentanil that are high affinity ligands of the ORL1 receptor 3, 29.
Acknowledgements
This work was supported in part by funds from ARC (9924) and MENSR (97H0004), and MRC (Canada).
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