Grace C. Rossi

Antisense mapping the MOR-1 opioid receptor: evidence for alternative splicing and a novel morphine-6β-glucuronide receptor

Although MOR‐1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes results from alternative splicing of MOR‐1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine‐6β‐glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR‐1 while morphine‐6β‐glucuronide acts through a novel, previously unidentified receptor which is yet another MOR‐1 splice variant.

Novel receptor mechanisms for heroin and morphine-6β-glucuronide analgesia

Abstract The rapid metabolism of heroin to 6-acetylmorphine and its slower conversion to morphine has led many to believe that heroin and morphine act through the same receptors and that the differences between them are due to their pharmacokinetics. We now present evidence strongly implying that heroin and two potent mu drugs, fentanyl and etonitazine, act through a unique receptor mechanism similar to morphine-6β-glucuronide which is readily distinguished from morphine. Heroin, 6-acetylmorphine and morphine-6β-glucuronide show no analgesic cross tolerance to morphine in a daily administration paradigm, implying distinct receptors. Strains also reveal analgesic differences among the drugs. CXBK mice, which are insensitive to morphine, retain their analgesic sensitivity to heroin, 6-acetylmorphine, morphine-6β-glucuronide, fentanyl and etonitazine. Antisense mapping of the mu opioid receptor MOR-1 reveals that oligodeoxynucleotide probes against exon 2, which are inactive against morphine analgesia, block morphine-6β-glucuronide, heroin, fentanyl and etonitazine analgesia. Finally, an antisense probe targeting Giα1 blocks both heroin and morphine-6β-glucuronide, but not morphine, analgesia. These results indicate that heroin, 6-acetylmorphine, fentanyl and etonitazine all can produce analgesia through a novel mu analgesic system which is similar to that activated by morphine-6β-glucuronide.

Spinal analgesic activity of orphanin FQ/nociceptin and its fragments

Previous work reveals that orphanin FQ/nociceptin (OFQ/N) administered supraspinally produces an initial hyperalgesic response followed by analgesia. Spinally, OFQ/N elicits a rapidly appearing, naltrexone-reversible, dose-dependent analgesia in the tailflick assay without any indication of hyperalgesia. Two OFQ/N fragments, OFQ/N (1-7) and OFQ/N (1-11), are active, but far weaker. Blockade of sigma receptors with haloperidol enhances the analgesic potency of spinal OFQ/N, OFQ/N (1-7) and OFQ/N (1-11), but not as dramatically as supraspinal OFQ. Antisense probes targeting the second and third coding exons, but not the first exon, of the cloned mouse OFQ/N receptor (KOR-3) partially block OFQ/N analgesia.

Naloxone sensitive orphanin FQ-induced analgesia in mice.

Orphanin FQ, also known as nociceptin, is a heptadecapeptide with very high affinity for a novel member of the cloned opioid receptor family which produces hyperalgesia in mice. In addition to hyperalgesia, which is observed soon after administration of orphanin FQ, we now describe a delayed analgesic response. Unlike orphanin FQ-induced hyperalgesia, orphanin FQ-induced analgesia is readily reversed by the opioid antagonist naloxone, implying an opioid mechanism of action. In view of the very poor affinity of orphanin FQ for all the known traditional opioid receptors and the low affinity of opioids for the 125I[Tyr14]orphanin FQ binding site, orphanin FQ-induced analgesia is probably mediated through a novel orphanin FQ receptor subtype.

Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR-1F

The MOR‐1 gene is large, with a recent study reporting nine exons spanning 250 kb which combine to yield six different mu opioid receptor splice variants. We now report the isolation of exon 10, which is contained within yet another splice variant, MOR‐1F, which is composed of exons 1, 2, 3, 10, 6, 7, 8 and 9. Exon 10 comprises 186 bp which predict a unique 58 amino acid sequence extending beyond exon 3. It has been mapped between exons 4 and 6 and has flanking consensus splice sequences. On Northern blot analysis, the MOR‐1F mRNA is smaller than the other MOR‐1 mRNAs. When expressed in CHO cells, MOR‐1F binds the mu opioid radioligand [3H]DAMGO with high affinity (K D=1.04±0.03 nM). Competition studies demonstrated the selectivity of the variant for mu opioid ligands, supporting its classification within the mu opioid receptor family.

Differential blockade of morphine and morphine-6β-glucuronide analgesia by antisense oligodeoxynucleotides directed against MOR-1 and G-protein α subunits in rats

Abstract An antisense oligodeoxynucleotide directed against the 5′-untranslated region of MOR-1 blocks the analgesic actions of the μ 1 analgesics morphine and [ d -Ala 2 , d -Leu 5 jenkephalin (DADL) when they are microinjected into the periaqueductal gray. In contrast, morphine-6β-glucuronide (M6G) analgesia is unaffected by this treatment. Antisense oligodeoxynucleotides directed against distinct G i α subunits also distinguish between morphine and M6G analgesia. A probe targeting G i α2 blocks morphine analgesia, as previously reported, but is inactive against M6G analgesia. Conversely, an antisense oligodeoxynucleotide against G i αI inhibits M6G analgesia without affecting morphine analgesia. The antisense oligodeoxynucleotide directed against G 0 α is ineffective against both compounds. These results confirm the prior association of G i α2 with morphine analgesia and strongly suggests that M6G acts through a different opioid receptor, as revealed by its insensitivity towards the MOR-1 antisense probe and differential sensitivity towards G-protein α subunit antisense oligodeoxynucleotides.

Medullary μ and δ opioid receptors modulate mesencephalic morphine analgesia in rats

Supraspinal opioid analgesia is mediated in part by connections between the midbrain periaqueductal gray (PAG) and rostral ventral medulla (RVM) which includes the nuclei raphe magnus and reticularis gigantocellularis. Serotonergic 5HT2 and 5HT3 receptor subtypes appear to participate in this pathway since general and selective serotonergic antagonists microinjected into the RVM significantly reduced morphine analgesia elicited from the PAG. Since both an enkephalinergic pathway between the PAG and RVM and intrinsic enkephalinergic cells in the RVM exist, the present study evaluated the abilities of general (naltrexone), μ-selective (β-funaltrexamine: B-FNA) andδ2-selective (naltrindole) opioid receptor subtype antagonists microinjected into the RVM to alter morphine (2.5 μg) analgesia elicited from the PAG as measured by the tail-flick and jump tests. Mesencephalic morphine analgesia was significantly reduced after pretreatment in the RVM with naltrexone (1–10 μg), B-FNA (0.5–5 μg) or naltrindole (0.5–5 μg). Naltrexone in the RVM failed to alter basal nociceptive thresholds and none of the opioid antagonists were effective in reducing mesencephalic morphine analgesia when they were microinjected into placements lateral or dorsal to the RVM. These data indicate that μ andδ2 opioid receptors in the RVM modulate the transmission of opioid pain-inhibitory signals from the PAG.

Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions

Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for μ-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6β-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors.

3-Methoxynaltrexone, a selective heroin/morphine-6β-glucuronide antagonist

Recent work has suggested that heroin and morphine‐6β‐glucuronide (M6G) both act through a novel mu opioid receptor subtype distinct from those mediating morphines actions. This very high affinity 3H‐M6G site is selectively competed by 3‐methoxynaltrexone. In vivo, 3‐methoxynaltrexone (2.5 ng, i.c.v.) selectively antagonizes the analgesic actions of heroin and M6G without interfering with mu (morphine and [d‐Ala2,MePhe4,Gly(ol)5]enkephalin), delta ([d‐Pen2,d‐Pen5]enkephalin), kappa1 (U50,488H) or kappa3 (naloxone benzoylhydrazone) analgesia. In dose–response studies, 3‐methoxynaltrexone (2.5 ng, i.c.v.) significantly shifted the ED50 values for heroin and its active metabolite, 6‐acetylmorphine, without affecting the morphine curve. These results indicate that 3‐methoxynaltrexone selectively blocks a novel 3H‐M6G binding site which is responsible for the analgesic actions of heroin and M6G. This ability to selectively antagonize heroin actions opens new possibilities in the development of therapeutics for the treatment of opioid abuse.

Synergistic brainstem interactions for morphine analgesia.

Morphine is a potent analgesic when microinjected into the periaqueductal gray (PAG), the rostral ventral medulla (RVM) which contains the nuclei raphe magnus and reticularis gigantocellularis and the dorsolateral pons (DLP) which includes the locus coeruleus. Coadministration of low morphine doses which are inactive alone into combinations of these three regions elicits dramatic analgesic responses, implying the existence of synergy. The most effective combination is the PAG/RVM, whereas the PAG/DLP and RVM/DLP combinations are much less efficacious. In addition to fixed combinations, inclusion of a low morphine dose in one region shifts the analgesic dose-response curves in the others. The marked synergy between the PAG and the RVM is sensitive to naloxonazine, implying a role for mu 1 receptors. Thus, these studies indicate the presence of intrinsic brainstem mu 1 receptor systems with synergistic interactions which can be pharmacologically distinguished from the brainstem mu 2 receptors mediating supraspinal/spinal synergy.