Elsevier

Neuropeptides

Volume 37, Issue 4, August 2003, Pages 211-219
Neuropeptides

Expression and G-protein coupling of μ-opioid receptors in the spinal cord and dorsal root ganglia of polyarthritic rats

https://doi.org/10.1016/S0143-4179(03)00045-3Get rights and content

Abstract

Although chronic inflammatory pain is known to be associated with hypersensitivity to μ opioid receptor agonists, no evidence for changes in the expression and/or characteristics of central μ opioid receptors has yet been reported in relevant models of this type of pain. In the present study, both immunohistochemical and autoradiographic approaches were used to address this question in polyarthritic rats, on the 4th week after intradermal injection of complete Freund’s adjuvant, when inflammatory pain was at its maximum. Immunohistochemical labeling with specific anti-μ opioid receptor antibodies and autoradiographic labeling with [3H]DAMGO showed an upregulation of μ opioid receptors in the dorsal root ganglia but no changes in the density of these receptors in the dorsal horn at the level of L4–L6 segments in polyarthritic compared to age-paired control rats. On the other hand, autoradiographic quantification of the concentration-dependent increase in [35S]GTP-γ-S binding by the μ-opioid receptor agonist DAMGO did not show any significant differences within the lumbar dorsal horn between polyarthritic and control rats. These data indicate that chronic inflammatory pain caused by polyarthritis was associated with an increased expression of μ-opioid receptors in dorsal root ganglion sensory neurones that did not result in an increased spinal density of these receptors, in spite of their well established axonal transport in the central portion of primary afferent fibres to the dorsal horn. In contrast, axonal transport of μ-opioid receptors in the peripheral portion of these fibres probably accounts for the increased receptor density in inflamed tissues already reported in the literature.

Introduction

Behavioural data clearly showed that inflammatory pain is associated with central supersensitivity to opioids, notably when these compounds are administered directly at the spinal level where is located the first synaptic relay in the transmission of nociceptive messages (see Cesselin et al., 1999, for a review). However, only functional alterations in spinal opioid receptors, especially those of the μ type (MOR), have been reported to be associated with peripheral inflammation, notably in a relevant model for chronic inflammatory pain, the polyarthritic rat (Kayser and Guilbaud, 1983; Besson and Guilbaud, 1988; Ballet et al., 1998). Indeed, to date, all investigations failed to reveal any modifications in the density and/or affinity of opioid receptor binding sites in the spinal cord that would explain these functional changes (Cesselin et al., 1980; Millan et al., 1986; Delay-Goyet et al., 1989; Kar et al., 1994).

Peripheral antinociceptive effects of exogenous opioids have also been shown to be enhanced under inflammatory conditions (see Stein et al., 1999, for a review). In line with these behavioural observations, an increased density of opioid receptors was found on cutaneous nerve fibres in inflamed tissues (Hassan et al., 1993). Opioid receptors on these fibres are synthesised in neuronal cell bodies located in dorsal root ganglia (DRG). Interestingly, one as well as three days after carrageenan induced-inflammation of a hindpaw or four days following intraplantar injection of complete Freund’s adjuvant (Ji et al., 1995; Zhang et al., 1998a), an elevated expression of opioid receptors has been reported to occur in DRG. Whether or not such a change also persists under chronic inflammatory pain conditions, i.e. in polyarthritic rats, is presently unknown.

With the cloning of opioid receptors, including MOR (Chen et al., 1993; Thompson et al., 1993), it has become possible to study opioid receptor expression using in situ hybridisation (Mansour et al., 1994) and immunohistochemistry (Ji et al., 1995) at the cellular level. In the spinal cord, MOR-immunoreactivity has been detected in the plasmalemma of somas and dendrites and on unmyelinated axon terminals located principally in dorsal horn (Arvidsson et al., 1995; Cheng et al., 1996). In DRG, MOR-immunostaining mainly concerned small-size neurones although some medium to large-size neurones were also immunopositive (Ji et al., 1995; Zhang et al., 1998a).

MOR belongs to the superfamily of guanine nucleotide-binding regulatory protein (G-protein)-coupled receptors (Dhawan et al., 1996). Agonist occupancy of MOR has several cellular consequences including: (1) inhibition of adenylyl cyclase activity; (2) activation of an inwardly rectifying potassium conductance; and (3) inhibition of N- and T-types of Ca2+ currents. All of these actions are mediated by pertussis toxin-sensitive G-proteins of the Gi/Go family (Dhawan et al., 1996). Evaluation of the coupling efficiency between MOR and Gi/Go is now possible thanks to the measurement of the binding of [35S]guanylyl-5-O-(thio)-triphosphate ([35S]GTP-γ-S) to brain membranes and sections exposed to selective agonists (Sim et al., 1995; Rodriguez-Puertas et al., 2000).

In the present study, these recently developed techniques derived from the knowledge of MOR at the molecular level, i.e. MOR immunohistochemistry and autoradiography of DAMGO-stimulated [35S]GTP-γ-S binding, together with autoradiographic analysis of specific [3H]DAMGO binding, were used to re-address the question of possible changes in MOR within the spinal cord and/or DRG, which would account for the supersensitivity to opioids of rats suffering from chronic inflammatory pain. Experiments were performed in polyarthritic rats, at the 4th week after intradermal injection of complete Freund’s adjuvant, when inflammatory pain was at its maximum (Calvino et al., 1987; Besson and Guilbaud, 1988).

Section snippets

Animals

Polyarthritis was induced at the Centre d’Elevage Charles River (Saint-Aubin-lès-Elbeuf, France) by an intradermal injection of 0.05 ml of killed Mycobacterium butyricum suspended (10 mg/ml) in mineral oil (complete Freund’s adjuvant) (Gouret et al., 1976). The injection was made at approximately 3 cm from the base of the tail of 6-week-old male Sprague–Dawley rats weighing 150–175 g. Animals were used for experiments during the fourth week following the injection, when the paws and tail were

[3H]DAMGO autoradiographic labeling of spinal cord and DRG sections

In the spinal cord, specific [3H]DAMGO binding was detected only in the superficial layers of the dorsal horn (not shown), where the quantification of binding was made at the lumbar level (L4–L6). No significant change in the specific binding of the radioligand was observed in the spinal cord of polyarthritic animals (Table 1) compared to controls. By contrast, specific [3H]DAMGO binding was markedly higher (∼+300%) in lumbar DRG sections of polyarthritic rats than in those of control animals (

Discussion

Since behavioural, electrophysiological and biochemical data (Kayser and Guilbaud, 1983; Hylden et al., 1991; Stanfa et al., 1992; Ballet et al., 1998) demonstrated a functional supersensitivity of MOR in rats suffering from chronic inflammatory pain, relevant immunohistochemical and autoradiographic approaches have been used in the present study in an attempt to detect possible changes in spinal and DRG MOR characteristics that would underlie these functional modifications.

We first examined

Acknowledgements

This research was supported by grants from INSERM and Bristol-Myers Squibb Foundation (Unrestricted Biomedical Research Grant Program). S. Ballet was a recipient of a MENESR (Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche) fellowship during the performance of these studies.

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    Abbreviations: Avidin–biotin-peroxidase complex, ABC; d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2, CTOP; [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, DAMGO; dorsalrootganglion, DRG; fluorescein isothiocyanate, FITC; [35S] guanylyl-5-O-(thio)-triphosphate, [35S]GTP-γ-S; IR, immunoreactive; μ-opioid receptor, MOR; sodium phosphate buffer, PB; phosphate buffered saline–normal goat serum, PBS–NGS.

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