Neuronal cytochrome P450 activity and opioid analgesia: relevant sites and mechanisms

Highlights

• Opioids act at spinal, brain stem and non-brain stem CNS sites to relieve pain.

• Critical areas are the periaqueductal gray (PAG) and rostral ventral medulla (RVM).

• Mu opioid analgesia requires neuronal cytochrome P450 activity.

• Nerve-terminal P450s in PAG-RVM projection neurons participate in opioid analgesia.

• Spinal opioid analgesia does not require neuronal cytochrome P450 activity.

Abstract

Recent studies suggest a functional role for neuronal cytochrome P450 monooxygenase (P450) activity in opioid analgesia. To characterize the relevant receptors, brain areas, and circuits, detailed in vitro and in vivo studies were performed with the highly selective μ opioid receptor agonist DAMGO in neuronal P450-deficient mutant (Null) and control mice. Homogenates of brain regions and spinal cord showed no differences in DAMGO-induced activation of [³⁵S]- GTPγS binding between Null and control mice, indicating no genotype differences in µ opioid receptor signaling, receptor affinities or receptor densities. Intracerebroventricular (icv) DAMGO produced robust, near-maximal, analgesic responses in control mice which were attenuated by 50% in Null mice, confirming a role for µ opioid receptors in activating P450-associated responses. Intra-periaqueductal gray (PAG) and intra-rostral ventromedial medulla (RVM) injections of DAMGO revealed deficits in Null (vs. control) analgesic responses, yet no such genotype differences were observed after intrathecal DAMGO administration. Taken with earlier published findings, the present results suggest that activation of µ opioid receptors in both the PAG and in the RVM relieves pain by mechanisms which include nerve-terminal P450 enzymes within inhibitory PAG-RVM projections. Spinal opioid analgesia, however, does not seem to require such P450 enzyme activity.

Introduction

Opioids remain an important category of medications for the treatment of many types of pain. Clinically useful opioids produce analgesia by activation of μ opioid receptors in the periaqueductal gray (PAG), the rostral ventromedial medulla (RVM), and the spinal dorsal horn (Heinricher and Ingram, 2008). In the PAG, μ stimulation inhibits pre-synaptic GABAergic activity, with the subsequent activation of descending, pain-relieving circuits, but details of these circuits remain unclear. At the neurochemical level, several cellular transduction mechanisms are used by μ opioid receptors (Williams et al., 2013, Law, 2011), but opening of voltage-gated potassium channels in pre-synaptic GABAergic terminals (thereby reducing GABA release) is a favored hypothesis to account for the activation of descending, pain-relieving circuits (Vaughan et al., 1997). As considered further below (see Discussion), both pre- and post-synaptic mechanisms are likely to be involved.

While exploring brain stem analgesic actions of opioids, our lab reported that knockout mice with deficiencies in brain neuronal cytochrome P450 monooxygenase (P450) lacked normal antinociceptive responses to morphine (Conroy et al., 2010). Although P450 enzymes are most commonly associated with drug metabolism, they also participate in numerous types of endogenous lipid oxidation (Spector, 2009, Morisseau and Hammock, 2013). Based on the defective responses in P450-deficient mice and other results, Conroy et al. (2010) suggested that μ opioids inhibit GABAergic activity by stimulating the release and P450- mediated epoxidation of arachidonic acid. The epoxide products of some fatty acids are reported to have analgesic or anti-allodynic activity (Terashvili et al., 2008, Wagner et al., 2013). Although the actual mechanism has not been established, μ opioid receptor activation was suggested to release arachidonic acid via sequential activation of phospholipase Cγ, inositol-1,4,5-triphosphate receptors and calcium-dependent PLA₂ (Conroy et al., 2010). More recent in vivo (Conroy et al., 2013, Hough et al., 2014b) and in vitro (Zhang and Pan, 2012) work supports this P450 epoxygenase theory of μ opioid receptor action.

Notwithstanding this recent support, many questions remain unanswered concerning the relationship between neuronal P450 activity and opioid analgesia. For example, based on results with morphine, μ opioid receptors have been proposed to utilize P450-related signal transduction, but a P450 requirement for highly selective μ agonists has not been studied. Similarly, brains from neuronal P450-deficient mice (which have defective morphine analgesia) have no deficits in whole brain μ opioid receptor number or affinity, but the possible relevance of brain P450 activity to μ opioid G protein-coupled signaling efficacy has not been studied. Finally, since opioids elicit analgesia from several CNS locations, the anatomical localization of the P450-relevant opioid receptors is of interest, but has not been determined. Presently, we describe detailed biochemical and CNS mapping studies with the highly selective μ agonist [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO) in control and neuronal P450-deficient knockout mice in order to answer these questions.