Further evidence for the interaction of μ- and δ-opioid receptors in the antinociceptive effects of the dual inhibitor of enkephalin catabolism, RB101(S)

Abstract

We have previously shown that RB101, a dual inhibitor of enkephalin-degrading enzymes, decreased carrageenin-evoked c-Fos protein expression at the spinal cord level in awake rats. Moreover, we have also shown that c-Fos expression is a useful marker of the possible direct or indirect interactions between neural pathways, such as opioid and cholecystokinin systems. We now investigated the respective roles of the three main types of opioid receptors (μ, δ, or κ) and their possible interactions, in the depressive effects of RB101 in inflammatory nociceptive conditions induced by intraplantar carrageenin (6 mg/150 μl of saline). We used β-funaltrexamine (β-FNA), naltrindole (NTI), and nor-binaltorphimine (BNI) as specific antagonists for μ-, δ- and κ-opioid receptors, respectively. c-Fos protein-immunoreactivity (c-Fos-IR) was evaluated as the number of c-Fos-IR nuclei in the lumbar spinal cord 90 min after carrageenin. c-Fos-IR nuclei were preferentially located in the superficial (I–II) and deep (V–VI) laminae of segments L4–L5 (areas containing numerous neurons responding exclusively, or not, to nociceptive stimuli). RB101(S) (30 mg/kg, i.v.) significantly reduced the total number of carrageenin-evoked c-Fos-IR nuclei (30% reduction, P<0.01). This effect was completely blocked by β-FNA (10 mg/kg, i.v.), or NTI (1 mg/kg, i.v.). In contrast, BNI (2.5 mg/kg, i.v.) did not reverse the reducing effects of RB101(S) on carrageenin-evoked c-Fos protein expression. These results suggest that functional interactions occur between μ- and δ-opioid receptors in enkephalin-induced antinociceptive effects.

Introduction

The opioid receptor family, a member of the superfamily of G-protein coupled receptors (GPCRs), consists of three receptor types: mu (μ), delta (δ), and kappa (κ). Opioid receptors have distinct pharmacological profiles and discrete, but overlapping distribution in the central nervous system [33], which may account for the possible existence of opioid receptor interaction, as has been suggested in the literature (see references in Refs. [19], [30]). Thus, several authors have shown that μ- and δ-opioid receptor agonists can interact in an additive or synergistic manner to produce antinociception (see references in Refs. [25], [47]). Moreover, observations in μ-receptor knock-out mice show that δ-opioid receptor-mediated analgesia and respiratory depression were reduced and abolished, respectively [34], [35]. The endogenous opioid peptides, enkephalins, interact with μ- and δ-opioid receptors. Thus, it was interesting to evaluate whether μ/δ interaction, previously suggested with exogenous agonists, may have a physiological relevance. To achieve this goal, we have used the dual inhibitor of enkephalin-degrading enzyme, RB101. This dual inhibitor of both enzymes involved in the degradation of the peptides, neutral endopeptidase (NEP) and aminopeptidase N (APN), increases their half-life, without modifying their release (see references in Ref. [41]). The endogenous opioid peptide-receptor systems mediate important physiological functions related to locomotion, motivation, reward, autonomic functions, immunomodulation, hormone secretion and pain perception. To evaluate whether enkephalins, protected from their enzymatic degradation, may modulate this latter function through μ/δ interaction, we have used an immunohistochemical approach. c-Fos protein expression, especially at the spinal level, is one of the long-term intracellular events, which could be used as an indirect marker of neuronal populations involved in nociceptive processes (see references in Ref. [20]). The ‘c-Fos technique’ has been shown to be very appropriate for analysis of the effects of various endogenous or exogenous substances involved in pain alleviation, especially at the spinal cord dorsal horn level (see references in Ref. [10]). In this study, we have investigated the effects of systemic administration of the diastereomer optically pure, RB101(S) (30 mg/kg, i.v.) on spinal c-Fos protein expression induced 1.5 h after an intraplantar (i.pl.) injection of carrageenin in awake rats pre-treated with β-funaltrexamine (β-FNA; 10 mg/kg, i.v.), naltrindole (NTI; 1 mg/kg, i.v.), or nor-binaltorphimine (BNI; 2.5 mg/kg, i.v.), which are selective μ-, δ-, and κ-opioid receptor antagonists, respectively. A preliminary report of this work appeared in abstract form at the annual meeting of the International Narcotics Research Conference in Seattle (USA, July 2000; [8]).