John P. Mathis

Orphan opioid receptor antisense probes block orphanin FQ-induced hyperphagia

Orphanin FQ/nociceptin binds with high affinity to the orphan opioid receptor-like/K-3 (ORL1/KOR-3) clone, and stimulates feeding. The present study demonstrated that antisense oligodeoxynucleotides directed against either exons 1, 2 or 3 of the ORL1/KOR-3 clone reduced orphanin FQ/nociceptin-induced hyperphagia. A missense probe was ineffective. Naltrexone dose-dependently reduced orphanin FQ/nociceptin-induced hyperphagia. These data suggest that the receptor responsible for orphanin FQ/nociceptin-induced hyperphagia is encoded by the ORL1/KOR-3 clone.

Autoradiographic localization of 125I[Tyr14]orphanin FQ/nociceptin and 125I[Tyr10]orphanin FQ/nociceptin(1–11) binding sites in rat brain

The endogenous ligand for the orphan opioid receptor, orphanin FQ/nociceptin (OFQ), has recently been characterized. The OFQ peptide sequence contains paired basic amino acids, suggesting the possibility of posttranslational processing to a peptide containing the first 11 amino acids of the OFQ peptide. This peptide has been reported in the brain and it has a unique pharmacology. In the present study, we compared the autoradiographic distribution of 125I[Tyr14]OFQ and 125I[Tyr10]OFQ(1–11) in coronal rat brain sections. Nonspecific binding was defined with unlabeled OFQ or OFQ(1–11), respectively. Both radioligands demonstrated high levels of specific binding (>95% of total binding), with no appreciable binding in white matter areas with either ligand. 125I[Tyr14]OFQ binding was widely distributed throughout the rat brain. In contrast, 125I[Tyr10]OFQ(1–11) binding was more restricted. The highest 125I[Tyr14]OFQ binding levels measured in this study were found in the locus coeruleus, an area which contained very low 125I[Tyr10]OFQ(1–11) binding. Both ligands labeled the cortex, hippocampus and amygdala. In the thalamus, 125I[Tyr14]OFQ binding was prominent in most nuclei, whereas 125I[Tyr10]OFQ(1–11) binding was restricted to the midline thalamus. 125I[Tyr14]OFQ binding was heavy in the suprachiasmatic hypothalamus, and moderate in other hypothalamic nuclei. 125I[Tyr10]OFQ(1–11) binding in the hypothalamus, however, was present mainly in the ventromedial hypothalamic nucleus. Lower binding levels of both ligands were found in the caudate putamen. The distinct autoradiographic patterns of these two ligands are consistent with different binding sites, which might help explain their different functional activities. J. Comp. Neurol. 423:319–329, 2000.

DISSOCIATION OF AFFINITY AND EFFICACY IN KOR-3 CHIMERAS

KOR‐3 chimeras were constructed in which the first coding exon of KOR‐3 was exchanged for the corresponding first coding exon of either MOR‐1 (MOR‐1/KOR‐3) or DOR‐1 (DOR‐1/KOR‐3). All three clones were expressed in CHO cells and characterized with regards to their binding profiles for orphanin FQ/nociceptin (OFQ/N) and a variety of opioids as well as their functional activities in cyclase studies. 125I[Tyr14]OFQ/N labels both KOR‐3 (K D 37 pM) and the MOR‐1/KOR‐3 chimera (K D 39 pM) equally well. Although its affinity for the DOR‐1/KOR‐3 chimera is quite good (K D 135 pM), it is slightly lower than the other two. Competition studies confirm the high affinity of OFQ/N for all three clones. However, several competitors clearly distinguish the chimeras from KOR‐3. OFQ/N(1‐11) competes KOR‐3 (K i 55 nM) over 6‐fold more potently than either of the chimeras (K i values > 350 nM). Conversely, the modest affinity of naloxone benzoylhydrazone for KOR‐3 (310 nM) is greatly increased in both the MOR‐1/KOR‐3 (K i 69 nM) and DOR‐1/KOR‐3 (K i ) chimeras. The remainder of the opioids tested have no appreciable affinity against any of the clones. Functionally, OFQ/N inhibits forskolin‐stimulated cAMP accumulation in both the KOR‐3 and the MOR‐1/KOR‐3 chimera by almost 40%, with IC50 values in the low nanomolar range. Little activity is seen against the DOR‐1/KOR‐3 chimera. Naloxone benzoylhydrazone inhibits cAMP accumulation in the KOR‐3 and the DOR‐1/KOR‐3 chimera. Although naloxone benzoylhydrazone has higher affinity for the MOR‐1/KOR‐3 chimera in binding studies than KOR‐3 itself, it is inactive in cyclase studies using the MOR‐1/KOR‐3 chimera, implying that the replacement of the first coding exon increases affinity while decreasing intrinsic activity.

Identification of a high-affinity orphanin FQ/nociceptin(1-11) binding site in mouse brain.

The presence of pairs of basic amino acids within the orphanin FQ/Nociceptin (OFQ/N) sequence has raised the possibility that truncated versions of the peptide might be physiologically important. OFQ/N(1–11) is pharmacologically active in mice, despite its poor affinity in binding assays (Ki > 250 nM) for the OFQ/N receptor. Using an analog of OFQ/N(1–11), [125I][Tyr10]OFQ/N(1–11), we identified a high‐affinity binding site (KD 234 pM; Bmax 43 fmol/mg protein) with a selectivity profile distinct from the OFQ/N receptor and all the traditional opioid receptors. This site had very high affinity for OFQ/N and its related peptides. The most striking differences between the new site and the OFQ/N receptor previously observed in brain were seen with traditional opioids. Dynorphin A analogs and α‐neoendorphin competed with [125I][Tyr10]OFQ/N(1–11) binding in mouse brain with Ki values below 10 nM, while naloxone benzoylhydrazone (Ki 3.9 nM) labeled the [125I][Tyr10]OFQ/N(1–11) binding site as potently as many traditional opioid receptors. Several other opioids, including fentanyl, (−)cyclazocine, levallorphan, naltrindole, and diprenorphine, also displayed moderate affinities for this site. Finally, the [125I][Tyr10]OFQ/N(1–11) site had a unique regional distribution consistent with a distinct receptor. Thus, [125I][Tyr10]OFQ/N(1–11) labels a novel site in brain with a selectivity profile intermediate between that of either opioid or OFQ/N receptors. Synapse 34:181–186, 1999.

Morphine and morphine-6β-glucuronide-induced feeding are differentially reduced by G-protein α-subunit antisense probes in rats

Although morphine and its active metabolite, morphine-6beta-glucuronide (M6G), each induce mu-opioid receptor-sensitive feeding, different antisense oligodeoxynucleotide (AS ODN) probes directed against the MOR-1 clone produce distinct effects. Thus, MOR-1 AS ODN probes directed against exons 1 or 4 reduce morphine-, but not M6G-induced feeding, whereas probes directed against exons 2 or 3 reduce M6G-, but not morphine-induced feeding. AS ODN probes directed against different G-protein alpha-subunits differentially reduced morphine (G(ialpha2)) and M6G (G(ialpha1))-induced analgesia. The present study evaluated the ability of AS ODN probes directed against G-protein alpha-subunits to reduce feeding induced by morphine and M6G in rats. The AS ODN probes (25 microg, i.c.v.) were administered once 24 h prior to morphine (5 microg, i.c.v.) or M6G (250 ng) and spontaneous free feeding was assessed 1, 2 and 4 h thereafter. In agreement with analgesic studies, morphine-induced feeding was significantly reduced by the G(ialpha2) AS ODN probe. Morphine-induced feeding was unaffected by AS ODN probes directed against either G(ialpha1), G(ialpha3), G(oalpha), G(x/zalpha), G(qalpha) or a nonsense control probe, and was significantly enhanced by pretreatment with the G(salpha) probe. In contrast, M6G-induced feeding was significantly reduced by AS ODN probes directed against either G(ialpha1), G(ialpha3) or G(x/zalpha), whereas AS ODN probes targeting G(ialpha2), G(oalpha), G(salpha), G(qalpha) or a nonsense control probe were ineffective. When M6G-induced feeding was assessed at a dose (500 ng) which was sensitive to MOR-1 AS ODN effects, none of the G-protein alpha-subunit AS ODN probes were effective. These data indicate that morphine and M6G-induced feeding are mediated through different G-protein alpha-subunits, and provide further evidence for separate and distinct molecular mechanisms mediating these functional responses through different opioid receptors. This strongly suggests that M6G may act through a novel opioid receptor displaying a distinct pharmacological mechanism.

Antinociceptive analogs of orphanin FQ/nociceptin(1–11)

The presence of pairs of basic amino acids within the sequence of orphanin FQ/nociceptin (OFQ/N) peptide, the endogenous ligand for the ORL1/KOR-3 receptor, has raised the possibility that processing might generate pharmacologically important truncated peptides, including OFQ/N(1-11). OFQ/N(1-11) is pharmacologically active in vivo with a potency comparable to OFQ/N. Several tyrosine-containing analogs of OFQ/N(1-11) have been synthesized and examined for antinociceptive activity. Like OFQ/N(1-11), [Tyr1]OFQ/N(1-11), [Tyr10]OFQ/N(1-11) and [IodoTyr10]OFQ/N(1-11) given supraspinally in mice were antinociceptive in the tailflick assay in mice. The tyrosine analogs showed similar potencies as OFQ/N(1-11) but longer durations of action. This response was readily reversed by the opioid antagonist naloxone despite poor affinities for these analogs at opioid receptors. Another compound, [Tyr11]OFQ/N(1-11) was highly epileptogenic, inducing naloxone-sensitive seizures in greater than 50% of the mice tested at doses comparable to those examined with the other analogs. These results indicate that it is possible to make analgesic OFQ/N(1-11) analogs. The activity of [IodoTyr10]OFQ/N(1-11) suggests that it may prove useful as a radioligand in exploring potential OFQ/N(1-11) binding sites.

Orphanin FQ/nociceptin and naloxone benzoylhydrazone activate distinct receptors in BE(2)-C human neuroblastoma cells

kappa(3) opioid receptors have a unique binding and analgesic profile, as originally defined by naloxone benzoylhydrazone (NalBzoH). Although antisense studies demonstrated the close relationship between kappa(3) opioid and Orphan opioid receptor-like receptor (ORL1) and implied they were generated from the same gene, these studies also revealed differences in the sensitivity profiles of NalBzoH and orphanin FQ/nociceptin (OFQ/N), indicating that they were not identical. To help define the relationship between kappa(3) and ORL1 receptors, we utilized BE(2)-C human neuroblastoma cells that natively express functional ORL1 and kappa(3) opioid receptors. (125)I-[Tyr(14)]OFQ/N binds to a single population of receptors in BE(2)-C cells. Competition binding and adenylyl cyclase studies clearly illustrated marked selectivity differences between the ORL1 and the kappa(3) sites. Furthermore, antisense DNA targeting ORL1 blocked the inhibition of cAMP by OFQ/N, but not by NalBzoH. Thus, the receptor mechanisms mediating the activity of OFQ/N and NalBzoH in BE(2)-C cells are distinct.

[125I]orphanin FQ/nociceptin binding in Raji cells

Western blots using an antibody which recognizes the orphanin FQ/nociceptin (OFQ/N) receptor reveals a band at approximately 69 kD in several cell lines, including the Raji human B cell lymphoma cell line. RT‐PCR confirms the presence of this receptor in the Raji cells. Binding studies revealed a high affinity [125I][Tyr14]OFQ/N site in the Raji cells. The affinity of [125I][Tyr14]OFQ/N in the Raji cells (KD 68.4 pM) was similar to that in the transfected receptor (KD 36.7 pM). Its selectivity profile also was quite similar. OFQ/N competed binding quite potently (Ki 65 pM), as did [Tyr14]OFQ/N (Ki 33 pM). Traditional opioids displayed no appreciable affinity for the binding at any concentration examined, with the exception of naloxone benzoylhydrazone, which had only a very modest affinity. The receptors in the Raji cells were functionally active. OFQ/N inhibited forskolin‐stimulated cyclase by 72% with an IC50 value of approximately 1 nM. Synapse 34:187–191, 1999.

Characterizing κ3 opioid receptors with a selective monoclonal antibody

To help characterize κ3 receptors and establish their relationship to traditional μ and δ receptors, we have generated a κ3‐selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)‐C cells, a human neuroblastoma cell line containing μ, κ3, and δ opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)‐C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of 3H‐NalBzoH (κ3) binding without affecting 3H‐DAMGO (μ) or 3H‐naltrindole (δ) binding in BE(2)‐C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)‐C cells by the κ3 agonist NalBzoH but not the μ agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes κ3 receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked κ3 but not morphine (μ) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)‐C. These studies demonstrate the selectivity of mAb8D8 for κ3 receptors and provide additional support for the existence of this unique opioid receptor subtype.

Carboxyl terminal peptides derived from prepro-orphanin FQ/nociceptin (ppOFQ/N) are produced in the hypothalamus and possess analgesic bioactivities

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand for the ORL-1/KOR-3 receptor, produces a wide variety of behavioral responses. Its precursor protein, prepro-OFQ/N (ppOFQ/N) contains several series of amino acids bounded by pairs of basic amino acids, raising the possibility that additional functional neuropeptides could be generated by proteolytic posttranslational processing. Several of these processing products have been shown to have pharmacological activity, including the 17 amino acid peptide OFQ/N (mppOFQ/N(140-157)) which is a major product of this precursor in the hypothalamus. Here we have used a newly developed radioimmunoassay and RP-HPLC to detect mppOFQ/N(160-187) in mouse hypothalamic extracts. Murine ppOFQ/N(160-187) has potent analgesic activity supraspinally (3.4 nmol, i.c.v.) and spinally (4.3 nmol, i.t.). This analgesic activity was reversed by the opioid antagonist naloxone (5 mg/kg, s.c.) and kappa(1)-selective opioid antagonist nor-BNI (60 microg, i.c.v.), despite the inability of ppOFQ/N(160-187) to compete binding in mu, delta, kappa(1), kappa(3), or OFQ/N binding assays. These findings suggest that murine ppOFQ/N(160-187) may be a physiologically relevant neuropeptide with a novel mechanism of action.