CC12, a P450/epoxygenase inhibitor, acts in the rat rostral, ventromedial medulla to attenuate morphine antinociception

doi:10.1016/j.brainres.2012.12.030

Brain Research

Volume 1499, 7 March 2013, Pages 1-11

https://doi.org/10.1016/j.brainres.2012.12.030 Get rights and content

Abstract

Brain cytochrome P450 epoxygenases were recently shown to play an essential role in mediating the pain-relieving properties of morphine. To identify the CNS sites containing the morphine-relevant P450s, the effects of intracerebral (ic) microinjections of the P450 inhibitor CC12 were determined on morphine antinociception in rats. CC12 inhibited morphine antinociception when both drugs were injected into the rostral ventromedial medulla (RVM), but not following co-injections into the periaqueductal gray (PAG) or into the spinal subarachnoid space. In addition, intra-RVM CC12 pretreatment nearly completely blocked the effects of morphine following intracerebroventricular (icv) administration. Although morphine is thought to act in both the PAG and RVM by pre-synaptic inhibition of inhibitory GABAergic transmission, the present findings show that 1) the mechanism of morphine action differs between these two brainstem areas, and 2) P450 activity within the RVM is important for supraspinal morphine antinociception. Characterization of morphine-P450 interactions within RVM circuits will further enhance the understanding of the biochemistry of pain relief.

Highlights

► Morphine activates brain μ opioid receptors to relieve pain through a cytochrome P450 epoxygenase mechanism. ► Morphine acts in several CNS areas, but the brain region(s) requiring cytochrome P450 activity are unknown. ► The P450 blocker CC12 inhibited morphine antinociception when injected into the rat rostral ventromedial medulla (RVM). ► Injections of CC12 into the periaqueductal gray or the spinal subarachnoid space did not affect morphine antinociception. ► These results show that cytochrome P450 activity within the RVM is essential for supraspinal morphine antinociception.

Introduction

Morphine, the prototypical μ opioid analgesic, remains one of the most commonly used treatments for pain. However, the clinical utility of this drug is limited by the side effects, which include respiratory depression, constipation, and tolerance. In addition, the rewarding effects of opioids can lead to addiction (Gutstein and Akil, 2006). It is well established that μ opioid agonists like morphine act at μ opioid receptors in the midbrain periaqueductal gray (PAG), the rostral ventromedial medulla (RVM; consisting of the nucleus raphe magnus and adjacent reticular formation), and the dorsal horn of the spinal cord to produce antinociception (Yaksh and Rudy, 1978, Yaksh et al., 1976). These three CNS sites comprise an interconnected pathway responsible for the descending modulation of pain processing (Heinricher and Ingram, 2008). Activation of this circuit, by electrical stimulation, stress, or analgesic drugs, results in the depression of incoming nociceptive signals at the level of the spinal cord (Heinricher and Ingram, 2008).

Previous work in our lab reported that CC12, a compound related to cimetidine, inhibits the antinociceptive activity of several types of pain-relieving drugs (Hough et al., 2007). Intracerebroventricular (icv) administration of CC12 blocked the antinociceptive effects of the μ opioid agonist morphine, the cannabinoid agonist WIN55–212, and the non-opioid analgesic improgan (Hough et al., 2007). It was shown that CC12 lacks affinity at a number of targets known to be involved in analgesic signaling (Hough et al., 2007). At the time, it was proposed that these three classes of analgesic drugs share a common, downstream, CC12-sensitive target, but the identity of this target was unknown. Subsequent studies found CC12 to be a potent inhibitor of a number of cytochrome P450 monooxygenase (P450) enzymes (Stadel et al., 2008), leading to the possibility that CC12 might block all three types of pain-relieving drugs via inhibition of P450 activity (Hough et al., 2007, Stadel et al., 2008). Several enzymes (including MAPK, phospholipase C, and phospholipase A₂) are known to participate in the supraspinal actions of μ opioid agonists, resulting in the release of a number of signaling molecules including inositol-1,4,5-triphosphate and arachidonic acid (Christie et al., 1999, Aoki et al., 2003, Law et al., 2000, Narita et al., 2003), but no evidence for P450-mediated opioid signal transduction existed.

Recently, experiments with several different kinds of P450 inhibitors and a new line of transgenic mice lacking functional neuronal P450 activity provided strong support for the hypothesis that μ opioids activate brain analgesic circuits through cytochrome P450 epoxygenase signaling (Conroy et al., 2010). Consistent with earlier results, it was proposed that μ opioid receptors signal through activation of phospholipase A₂, arachidonate release, P450-catalyzed epoxidation of arachidonate, and formation of pain-relieving epoxyeicosatrienoic acids (see Supplemental Fig. 1 of Conroy et al., 2010). Since CC12 is a P450 inhibitor (Stadel et al., 2008), the results of Conroy et al. make it likely that CC12 blocks morphine antinociception (Hough et al., 2007) by inhibiting brain P450 enzymes. Presently, intracerebral (ic) and intrathecal microinjections were employed to localize the CNS sites in which the P450 epoxygenase inhibitor CC12 acts to attenuate morphine antinociception.

Section snippets

Results

The effects of the P450 inhibitor CC12 microinjected into the PAG, RVM and caudal medulla were studied on the antinociception produced by morphine administration into these brain areas. Microinjections of morphine sulfate (5 μg) into the PAG in saline- pretreated animals increased nociceptive latencies in both the tail flick (Fig. 1A) and hot plate (Fig. 1B) tests. ANOVA of the data in Fig. 1A (between groups: CC12, morphine; within groups: time) found significant main effects of morphine (F_1,19

Discussion

Mu opioid receptors are densely expressed in the PAG, RVM, and dorsal horn of the spinal cord (Gutstein et al., 1998, Mansour et al., 1995, Thompson et al., 1993), and morphine injected into any of these sites produces behavioral antinociception. When administered systemically, morphine causes antinociception by activating μ opioid receptors in all three sites (Yaksh et al., 1976, Yaksh et al., 1988, Yaksh and Rudy, 1978, Yeung and Rudy, 1980). The action of morphine in the PAG stimulates the

Drugs and solutions

Naltrexone hydrochloride and morphine sulfate were purchased from Sigma-RBI (St. Louis, MO). CC12 was synthesized as previously described (Hough et al., 2007). Morphine sulfate was dissolved in saline for all studies. For ic and icv testing, CC12 were dissolved in saline. For intrathecal testing, CC12 and naltrexone were dissolved in 50% DMSO.

Animals

Male Sprague-Dawley rats (250–350 g at time of testing, Taconic Farms, Germantown, NY) were maintained on a 12-h light/dark cycle (lights on from 0700 to

Acknowledgment

This work was supported by grants (DA03816, DA027835) from the National Institute on Drug Abuse.

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¹: present address: NINDS/NIH, 5625 Fishers Lane Rm 4S-04, Rockville, MD 20852, USA

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Research ReportCC12, a P450/epoxygenase inhibitor, acts in the rat rostral, ventromedial medulla to attenuate morphine antinociception

Abstract

Highlights

Introduction

Section snippets

Results

Discussion

Drugs and solutions

Animals

Acknowledgment

Neurosci. Lett.

Neuropharmacol

Pharmacol. Biochem. Behav.

Pharmacol. Biochem. Behav.

Brain Res.

Neurosci. Lett.

Eur. J. Pharmacol.

Prog. Brain Res.

Pain

Pain

Neuroscience

Neuropharmacology

Brain Res.

Brain Res.

Brain Res.

J. Chem. Neuroanat.

Prog. Neurobiol.

Neuroscience

Brain Res.

Pain

Brain Res.

Brain Res.

Neuroscience

Brain Res.

Behav. Biol.

Neuron

Brain Res.

Research Report
CC12, a P450/epoxygenase inhibitor, acts in the rat rostral, ventromedial medulla to attenuate morphine antinociception