Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

Using 5' RACE, we have isolated four additional exons of the mu opioid receptor gene (Oprm), resulting in a gene spanning over 250 kb. The four new exons are contained within eight additional splice variants containing exon 11 at the 5' terminus. Exon 11, which is under the control of a previously unknown upstream promoter, and exon 12 are located approximately 10 kb and approximately 8 kb upstream from exon 1, respectively. Exon 13 and 14 are located between exons 1 and 2. The regional distributions of the variants, as determined by reverse transcription-PCR, varied among themselves and were distinct from that of MOR-1, implying region-specific RNA processing. Three variants (MOR-1H, MOR-1I, and MOR-1J) contained two potential translational start points, with the translational start point in exon 1 producing proteins identical to the original MOR-1 protein. When expressed, the receptor binding of these three variants was indistinguishable from that of MOR-1. The remaining eight proteins using the translation start point in exon 11 were all truncated, with three (MOR-1G, MOR-1M, and MOR-1N) predicting proteins of only six transmembrane domains and the rest giving proteins under 10 kDa. Western blots with an exon 11-specific antiserum revealed bands consistent with the six transmembrane domain proteins within the brain, but the shorter proteins were not detected. Thus, the MOR-1 protein can be generated by four different splice variants of the Oprm gene under the control of two physically distinct promoters. Although the truncated proteins are expressed in brain with a unique regional distribution, their functional significance remains unknown.

Ultrastructural localization of nitrotyrosine within the caudate-putamen nucleus and the globus pallidus of normal rat brain.

Nitration of protein tyrosine residues by nitric oxide (NO)-derived reactive species results in the production of stable nitrotyrosine (NT) moieties that are immunochemically detectable in many regions of normal brain and enriched in those areas containing constitutive nitric oxide synthase (cNOS). These include the caudate-putamen nucleus (CPN) and the globus pallidus, which receives major inhibitory input from the CPN. To determine the functional sites for NT production in these critical motor nuclei, we examined the electron microscopic immunocytochemical localization of NT and cNOS in rat brain. In the CPN, NT was localized to the somata and dendrites of cNOS-containing interneurons and spiny neurons, some of which received input from cNOS-labeled terminals. The NT immunoreactivity was most prevalent on outer mitochondrial membranes and nearby segments of the plasma membranes in dendrites and within asymmetric synapses on dendritic spines. In the CPN and globus pallidus, there was also a prominent labeling of NT in astrocytic processes, small axons, and tubulovesicles and/or synaptic vesicles in axon terminals. These terminals formed mainly asymmetric synapses in the CPN and inhibitory-type synapses in the globus pallidus where they often apposed cNOS-containing terminals that also formed asymmetric, excitatory-type synapses. Our results suggest that NT is generated by mechanisms requiring the dual actions of excitatory transmitters and NO derived either from interneurons in the CPN or from excitatory afferents in the globus pallidus. The findings also implicate NT in the physiological actions of NO within the striatal circuitry and, particularly, in striatopallidal neurons severely affected in Huntington's disease.

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.

Pharmacological characterization of morphine-6-sulfate and codeine-6-sulfate.

Morphine-6-sulfate (M6S) and codeine-6-sulfate (C6S) are mu-selective opiates which have been isolated from brain. M6S is an effective analgesic, with a 30-fold greater potency than morphine in the mouse radiant heat tailflick assay and similar to the active morphine metabolite morphine-6beta-glucuronide (M6G). M6S analgesia is reversed by 3-methoxynaltrexone at low antagonist doses which are inactive against morphine, suggesting that M6S may be acting through the same mechanisms as M6G. Consistent with this possibility, antisense mapping of the MOR-1 clone revealed that M6S analgesia was lowered by probes targeting exon 2 and not by targeting exon 1, a sensitivity profile similar to that of M6G and not morphine. C6S also has analgesic activity at doses approximately 10-fold greater than M6S. However, its characterization was impeded by the appearance of seizures at doses below full analgesic activity. Thus, M6S is a potent analgesic with pharmacological properties similar to M6G. C6S has limited utility due to its high level of toxicity.

Identification and characterization of three new alternatively spliced mu-opioid receptor isoforms.

We have identified four new mu-opiod receptor (MOR)-1 exons, indicating that the gene now contains at least nine exons spanning more than 200 kilobases. Replacement of exon 4 by combinations of the new exons yields three new receptors. When expressed in Chinese hamster ovary cells, all three variants displayed high affinity for mu-opioid ligands, but kappa and delta drugs were inactive. However, there were subtle, but significant, differences in the binding profiles of the three variants among themselves and from MOR-1. Immunohistochemically, the major variant, MOR-1C, displayed a regional distribution quite distinct from that of MOR-1. Region-specific processing also was seen at the mRNA level. Antisense mapping revealed that the four new exons were all involved in morphine analgesia. Together with two other variants generated from alternative splicing of exon 4, there are now six distinct MOR-1 receptors.

Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1.

Morphine produces analgesia by activating mu opioid receptors encoded by the MOR-1 gene. Although morphine-6 beta-glucuronide (M6G), heroin and 6-acetylmorphine also are considered mu opioids, recent evidence suggests that they act through a distinct receptor mechanism. We examined this question in knockout mice containing disruptions of either the first or second coding exon of MOR-1. Mice homozygous for either MOR-1 mutation were insensitive to morphine. Heroin, 6-acetylmorphine and M6G still elicited analgesia in the exon-1 MOR-1 mutant, which also showed specific M6G binding, whereas M6G and 6-acetylmorphine were inactive in the exon-2 MOR-1 mutant. These results provide genetic evidence for a unique receptor site for M6G and heroin analgesia.

Pharmacological characterization of endomorphin-1 and endomorphin-2 in mouse brain.

The recently isolated peptides endomorphin-1 and endomorphin-2 have been suggested to be the endogenous ligands for the mu receptor. In traditional opioid receptor binding assays in mouse brain homogenates, both endomorphin-1 and endomorphin-2 competed both mu1 and mu2 receptor sites quite potently. Neither compound had appreciable affinity for either delta or kappa1 receptors, confirming an earlier report. However, the two endomorphins displayed reasonable affinities for kappa3 binding sites, with Ki values between 20 and 30 nM. Both endomorphins competed 3H-[D-Ala2, MePhe4,Gly(ol)5] enkephalin binding to MOR-1 receptors expressed in CHO cells with high affinity. In mouse brain homogenates 125I-endomorphin-1 and 125I-endomorphin-2 binding was selectively competed by mu ligands. 125I-Endomorphin-1 and 125I-endomorphin-2 also labeled MOR-1 receptors expressed in CHO cells with high affinity. Autoradiography of the two 125I-labeled endomorphins demonstrated regional patterns in the brain similar to those previously observed for mu drugs. Pharmacologically, the endomorphins were potent analgesics. Although they were equipotent supraspinally, endomorphin-1 was more potent spinally. Endomorphin analgesia was effectively blocked by naloxone, as well as the mu-selective antagonists beta-funaltrexamine and naloxonazine. In CXBK mice, which are insensitive to supraspinal morphine, neither endomorphin was active, consistent with a mu mechanism of action. Finally, the endomorphins inhibited gastrointestinal transit. In conclusion, these results support the mu selectivity of these agents.

Pharmacological characterization of orphanin FQ/nociceptin and its fragments.

The cloning of a fourth member of the opioid receptor family has led to the discovery of a new neuropeptide termed orphanin FQ or nociceptin (OFQ/N). Studies in CD-1 mice confirm the ability of OFQ/N to rapidly induce hyperalgesia within 15 min which is insensitive to opioid antagonists. This is followed in the next 30 min by loss of hyperalgesia and the appearance of analgesia in the tailflick assay which is readily reversed by opioid antagonists. However, the very poor affinity of OFQ/N for all the traditional opioid receptors and the insensitivity of OFQ/N analgesia to antisense oligodeoxynucleotides active against MOR-1, DOR-1 or KOR-1 sequences that selectively block mu, delta or kappa1 analgesia, respectively, make it unlikely that OFQ/N analgesia is mediated through typical opioid receptors. Blockade of the antiopioid sigma system by haloperidol enhances the analgesic potency of OFQ/N of more than 100-fold. This effect is pronounced in BALB-C and Swiss-Webster mice. Although OFQ/N alone has little analgesic activity in these mice, the blockade of sigma systems with haloperidol uncovers a robust analgesic response in both strains. Two shorter OFQ/N fragments, OFQ/N(1-7) and OFQ/N(1-11), also are analgesic in CD-1 mice and their actions are reversed by the opioid antagonist diprenorphine despite very poor affinities of both peptides against [125I]OFQ/N binding and all the opioid receptors. In antisense studies, a probe targeting the first coding exon of KOR-3 eliminates OFQ/N hyperalgesia, but not OFQ/N analgesia. Conversely, antisense probes based on the second and third coding exons are inactive against OFQ/N hyperalgesia but readily reverse kappa3 opioid analgesia. These results suggest that OFQ/N elicits both analgesia and hyperalgesia through pharmacologically distinct receptors that do not correspond to traditional opioid receptors.

Intrafamilial phenotypic heterogeneity of the Poland complex: a case report.

Three cases of familial unilateral gluteal hypoplasia are reported. The index case in addition to having gluteal hypoplasia also has unilateral pectoral muscle hypoplasia. Another relative has unilateral symbrachydactyly of the distal phalanges of one foot. All four affected individuals in our pedigree were female. We propose that our cases are best classified as part of the Poland complex of anomalies. Our cases emphasize that intrafamilial phenotypic heterogeneity is possible within the Poland complex.

Aicardi syndrome with multiple tumors: a case report with literature review.

A 5-year-old girl with Aicardi syndrome, choroid plexus papilloma and multiple gastric hyperplastic polyps is reported. Gastric polyposis is unusual in the pediatric age group and has not previously been reported in a patient with Aicardi syndrome. A variety of uncommon benign and malignant tumors have been associated with Aicardi syndrome; this literature is briefly reviewed. The increased frequency of tumors in Aicardi syndrome should be kept in mind when evaluating these patients.