Characterization of the anti-morphine action of CC12: An epoxygenase theory of morphine antinociception.

摘要

To study the significance of cytochrome P450-mediated arachidonic acid metabolism in opioid action, we generated a mouse (brain-Cpr -null) with brain neuron-specific deletion of the cytochrome P450 reductase (Cpr) gene. Deletion of Cpr was accomplished via crossing Cprlox/lox mice with CamKIIalpha-Cre expressing mice. CPR is necessary for all microsomal P450 function. Brain-Cpr -null mice showed loss of neuronal CPR, reduced brain P450 activity, and highly attenuated antinociception after morphine administration, as compared with wild-type controls.No differences in CPR expression were noted in the rostral ventromedial medulla (RVM) between wild-type and null mice. As expected, Cre expression in null mice paralleled the loss of CPR found in these mice. Expression of Cre was found in the cerebral cortex, hippocampus and PAG, but not in the RVM, of brain-Cpr-null mice. No Cre expression was found in wild-type control littermates. No genotype-associated differences were observed in brain morphine levels or brain opioid receptor properties. Additionally, no differences in baseline nociceptive responding, or in baseline responding to milder thermal or mechanical stimuli, were evident between null and WT mice. These data indicate that the deficit in morphine responding is not due to hyperalgesia in the null mice. Complementary studies in mice and rats confirmed that morphine antinociception was blocked by several epoxygenase and P450 inhibitors. Additionally, the selective epoxygenase inhibitor MS-PPOH demonstrated dose-dependent (3--300 nmol, icv) and long-lasting (up to 4 hours) inhibition of morphine antinociception. Furthermore, although the P450 isoform involved in morphine antinociceptive signaling is not currently known, the epoxygenase inhibitor MS-PPOH may be a useful tool in identifying the morphine-relevant P450 due to its long-lasting anti-morphine actions.Additional experiments tested other components of the epoxygenase theory. Several, but not all, EET regioisomers produced antinociception on both hot plate and mechanical tests of nociception in rats following intra-PAG or intra-RVM administration. However, this antinociception was not evident on the tail flick test, indicating that the EETs may not be the morphine analgesic mediator, or that other metabolites may be important for morphine antinociception on the tail flick test. Additional pilot studies tested the role for EETs in morphine antinociception by using an inhibitor of the main EET-metabolizing enzyme, soluble epoxide hydrolase (sEH). Icv administration with an inhibitor of sEH failed to enhance morphine antinociception.Systemic morphine produces antinociception by actions within the PAG, RVM, and dorsal horn of the spinal cord. To identify the CNS sites in which the morphine-P450 interaction occurs, 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. Additionally, intra-RVM CC12 pretreatment nearly completely blocked the effects of morphine following intracerebroventricular (icv) administration. These data show that P450 activity within the RVM is essential for supraspinal morphine antinociception. Although morphine is thought to act in both the PAG and RVM by presynaptic inhibition of inhibitory GABAergic transmission, the present findings indicate that the mechanism of morphine action differs between these two brainstem areas. Immunohistochemical analyses in brain-Cpr-null mice identified a select group of neurons within the PAG which lacked CPR. These neurons were proposed to be important for the morphine-resistant phenotype seen in these animals. However, pharmacological data in rats suggest that the RVM, and not the PAG, contains the morphine-relevant P450. A model showing a CPR-containing cell body located within the PAG with RVM-projecting, P450-containing terminals, is proposed and accounts for the disparate findings mentioned above. Further characterization of morphine-P450 interactions within the RVM circuits will enhance the understanding of the biochemistry of pain relief.Although the current thesis provides strong evidence in support of an epoxygenase theory of morphine analgesia, the epoxygenase (EETs and/or DHETs) or non-epoxygenase (HETEs, hepoxilins) AA metabolites which mediate morphine analgesia is unclear. While elucidation of the importance of a P450 epoxygenase enzyme in morphine analgesia creates a new avenue for the study of pain-relieving drugs, the scope and significance of this finding remains unknown. Further studies on the morphine-P450 antinociceptive interaction may help to discover new targets for analgesic drug development. (Abstract shortened by UMI.)