Novel perspectives in the physio-pharmacological regulation of opioid receptors

The opioid peptide receptors encompass three major “classical” subclasses, namely, μ, δ and κ (MOP, DOP and KOP receptors respectively) and a more recently identified subclass, the NOP receptor. The endogenous opioid and dopaminergic systems are known to interact in the modulation of motor functions under physio-pathological conditions. In particular, degeneration of dopamine (DA) neurons and loss of DA innervation in Parkinson’s disease (PD) is known to trigger adaptive changes in opioid systems in the basal ganglia (BG), which may be viewed either as pathogenic or compensatory. In the present thesis we investigated the role of DA, and specifically the role of D2 receptor-mediated transmission, in the motor effects exerted by DOP and NOP receptors ligands. Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a dual control over motor function, low doses being facilitating and high ones being inhibitory. Since the same dual response has been observed after administration of NOP receptor antagonists, we hypothesized that the motor effects exerted by NOP receptors ligands might involve different DA receptor subtypes. To test our hypothesis we used both pharmacological (i.e. DA receptor selective ligands) and genetic (i.e. mice lacking the D2 receptor (D2R-/-) or its long isoform (D2L-/-)) tools. The D2/D3 antagonist raclopride prevented the motor facilitation induced by low doses of N/OFQ or the NOP antagonist J-113397, but was ineffective in preventing the motor impairment induced by high doses of J-113397. These effects suggest that motor facilitation and motor inhibition might rely on different D2 receptor subpopulations. Therefore to dissect out the contribution of pre- and post-synaptic D2 receptors, D2 knockout mice were used. We observed that the motor facilitation induced by low doses of N/OFQ or J-113397 was lost in D2R-/- and D2L-/- mice. The inhibitory effect of high doses of J-113397 was retained in D2L-/- mice, but disappeared in D2R-/- mice, whereas motor inhibition induced by high doses of N/OFQ occurs even in the absence of D2 receptors. Our results demonstrate that motor stimulation induced by low doses of N/OFQ or J-113397 is mediated by D2L receptors whereas motor inhibition induced by J-113397 involves D2S, possibly pre-synaptic, receptors. N/OFQ-induced hypolocomotion is independent from D2 receptors, and possibly due to direct inhibition of nigral DA cells. The same combined approach was followed to study the role of D2 transmission in DOP receptor-mediated motor effects. Evidence that DOP receptor is an attractive target for the treatment of neuropsychiatric disorders, among which PD, has been growing in recent years. The DOP receptor agonist SNC-80 did not increase motor activity in C57BL naïve mice. However, it restored motor activity in mice rendered hypokinetic with raclopride, or emiparkinsonian 6-OHDA lesioning. This finding corroborates the hypothesis that the DOP receptor system plays a compensatory role under conditions where DA transmission and, consequently, motor function is impaired. To further confirm the involvement of D2 receptors in the motor action exerted by DOP receptor agonist, SNC-80 was administered to D2R-/- and D2L-/- mice, and wild-type controls. SNC-80 was ineffective in D2R+/+ and D2L+/+ mice but improved motor function in D2R-/- and D2L-/- mice. These results show that the absence of post-synaptic D2 receptors discloses a motor promoting action of DOP receptor ligands, suggesting the existence of a D2/DOP receptor interaction both at the membrane and network level. Receptor signalling is typically accompanied by desensitization, a complex feedback regulatory process whereby receptor responsiveness decreases on continued agonist stimulation. Signaling of opioid receptors is regulated negatively by regulators of G protein signalling (RGS) proteins and in vitro studies provided evidence for a DOP receptor-specific effect of RGS4. Small molecule inhibitors of RGS-box-Gα interactions should serve to enhance G-protein signals and act as adjuvants or “potentiators” of GPCR agonists. Therefore, we tested the hypothesis that the coapplication of SNC-80 and CCG-203769, a small molecule inhibitor of RGS4, developed by R. Neubig at the University of Michigan, could potentiate the effects of SNC-80 on motor activity, both under physiological and parkinsonian conditions. We showed that the coapplication of sub-threshold doses of CCG-203769 disclosed a mild, but significant, effect of SNC-80 on motor activity in naïve mice. In addition, it synergized with SNC-80 in restoring motor activity in raclopride-treated, or 6-OHDA hemilesioned rats. These data show that RGS4 contribute to extinguish DOP-triggered motor responses, opening the way to the use of RGS4 inhibitors as adjuncts to DOP agonists in PD. Overall, the data provide evidence for an opposite role of DA transmission in the modulation of N/OFQergic and enkephalinergic opioid systems. Moreover, these results demonstrate that endogenous RGS4 exerts a regulatory role on DOP receptor signalling and that the inhibition of RGS4 might be beneficial in modulating DOP transmission in pathological conditions. Finally, this study proposes new possible targets to regulate motor activity, which may translate into new drugs useful in the treatment of PD and other brain disorders.