Morphine acts on mu opioid receptors in the brain and spinal cord to produce dramatic pain relief, but mu-associated side effects (including tolerance and substance abuse) limit its use. Despite substantial information on mu signaling, the mechanisms by which morphine activates brain stem analgesic circuits remain unknown. The goal of this proposal is to validate a new mechanism for morphine analgesia and to investigate new analgesic agents which may mimic morphine actions but lack side effects. Cytochrome P450s are a family of drug-metabolizing enzymes that also perform endogenous metabolic oxidations in the brain. Many P450s have arachidonic acid (AA) epoxygenase activity, i.e. they convert AA to epoxyeicosatrienoic acids (EETs). Based on three new findings and a literature review, an epoxygenase hypothesis is proposed for opioid analgesic action in the brain stem: Morphine -> receptor -> ? AA -> (P450) -> ? EET s -> ? K+ -> ? Analgesic Circuits Experiments to validate and exploit this hypothesis include: 1) Pilot studies show that newly-developed brain P450-deficient transgenic mice (BCPRN) have defective morphine antinociception. To validate this opioid analgesic phenotype, morphine experiments with BCPRN and control mice will investigate the importance of morphine dose, routes of administration, nociceptive tests and gender. 2) P450 epoxygenase inhibitors block morphine antinociception. Since morphine acts in the ventrolateral periaqueductal gray, the rostral ventromedial medulla, and the spinal dorsal horn, microinjections of P450 inhibitors and morphine into these regions of the rat CNS will identify the P450-relevant sites. Similar microinjections of morphine into BCPRN and control mice will confirm results from rats. 3) Morphine's mu-mediated side effects include modulation of respiration, locomotor activity, rotorod performance, and body temperature. Experiments with morphine and P450 inhibitors in rats and with morphine in BCPRN mice will discover which of these side effects utilize P450 mechanisms. 4) Additional experiments in rats with P450 inhibitors and with inhibitors of EET metabolism will further confirm and characterize the role of P450 expoxygenases in morphine analgesia. 5) Pilot results show that EETs, the products of AA epoxidation, have antinociceptive properties. Experiments to validate EET analgesia, to search for EET side effects, and to characterize EET mechanisms may reveal EETs to be a new class of experimental analgesic agents. The proposed studies will increase understanding of the mechanisms by which morphine relieves pain and produces unwanted side effects. Confirmation of the epoxygenase hypothesis may lead to new approaches for developing non-opioid, non-addicting pain relievers.