Isolating and characterizing three alternatively spliced mu opioid receptor variants: mMOR-1A, mMOR-1O, and mMOR-1P.

Extensive alternative pre-mRNA splicing of the mu opioid receptor gene, OPRM1, has demonstrated an array of splice variants in mice, rats and humans. Three classes of splice variants have been identified: full-length seven transmembrane (TM) domain variants with C-terminal splicing, truncated 6TM variants and single TM variants. The current studies isolates and characterizes an additional three full-length C-terminal splice variants generated from the mouse OPRM1 gene: mMOR-1A, mMOR-1O, and mMOR-1P. Using RT-qPCR, we demonstrated differential expression of these variants' mRNAs among selected brain regions, supporting region-specific alternative splicing. When expressed in Chinese Hamster Ovary cells, all the variants displayed high mu binding affinity and selectivity with subtle differences in the affinities toward some agonists. [³5S]γGTP binding assays revealed marked differences in agonist-induced G protein activation in both potency and efficacy among the variants. Together with the previous studies of mu agonist-induced phosphorylation and internalization in several carboxyl terminal splice variants, the current studies further suggest the existence of biased signaling of various agonists within each individual variant and/or among different variants.

The M1/M4 preferring agonist xanomeline is analgesic in rodent models of chronic inflammatory and neuropathic pain via central site of action.

The role of muscarinic receptor subtype-1 (M1) in chronic pain is unclear. In an attempt to gain an understanding of its role, we have tested xanomeline, an M1/M4-preferring agonist, together with nonselective (scopolamine and pirenzepine), and selective (MT-7 and MT-3) muscarinic receptor (M1 and M4, respectively) antagonists in a number of inflammatory and neuropathic pain models. Xanomeline potently and effectively reversed tactile allodynia and heat hyperalgesia associated with established neuropathic and inflammatory pain in both rat and mouse models. Scopolamine and pirenzepine completely blocked the analgesic response to xanomeline, confirming that the analgesic effect is mediated by the muscarinic system. The highly selective M1 receptor toxin, MT-7, almost completely abolished the analgesic response to xanomeline when administered supraspinally. However, the highly selective M4 receptor toxin, MT-3, only marginally reversed the analgesia when given supraspinally, and had no effect when given spinally. In conclusion, the data presented show that the nonselective muscarinic agonist xanomeline is analgesic in models of persistent pain and suggest that the activation of supraspinal M1 receptors, and to a lesser extent supraspinal M4 receptors, contributes to that analgesia.

The anti-apoptotic effect of leukotriene B4 in neutrophils: a role for phosphatidylinositol 3-kinase, extracellular signal-regulated kinase and Mcl-1.

The constitutive commitment of neutrophils to apoptosis is a key process for the control and resolution of inflammation and it can be delayed by various inflammatory mediators including leukotriene B4 (LTB4). The mechanisms by which LTB4 contributes to neutrophil survival are still unclear and the present work aims at identifying intracellular pathways underlying this effect. Inhibition of human neutrophil apoptosis by LTB4 was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and by the specific MEK inhibitor PD98059. In contrast, inhibitors of p38 MAPK, Jak2/3 and Src did not hinder the anti-apoptotic effect of LTB4. We also investigated the effects of members of the Bcl-2 family as they play a crucial role in the regulation of programmed cell death. When neutrophils were incubated with LTB4 for 1 to 6 h, the mRNA levels of the anti-apoptotic protein Mcl-1 were upregulated approximately 2-fold, while those of the pro-apoptotic protein Bax were downregulated 3- to 4-fold, as determined by real-time PCR. Accordingly, Western blot analysis revealed that the expression of Mcl-1 was upregulated in presence of LTB4, while flow cytometric analysis revealed that Bax protein was downregulated. Furthermore, the modulatory effects of LTB4 on Mcl-1 and Bax proteins were abolished in the presence of either wortmannin or PD98059. Taken together, these results demonstrate the participation of PI3-K and MEK/ERK kinases, as well as regulatory apoptotic proteins such as Mcl-1 and Bax, in the anti-apoptotic effects of LTB4 in human neutrophils.

Pharmacological characterization of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin analgesia and their differentiation from morphine.

The present study examined the pharmacology of dihydromorphine, 6-acetyldihydromorphine and dihydroheroin (3,6-diacetyldihydromorphine). Like morphine, dihydromorphine and its acetylated derivatives all were highly selective mu-opioids in receptor binding assays. All the compounds were potent mu-selective analgesics, as shown by their sensitivity towards the mu-selective opioid receptor antagonists naloxonazine and beta-funaltrexamine. However, the actions of dihydromorphine and its analogs were readily distinguished from those of morphine, differences that were surprising in view of the very limited structural differences among them that consisted of only the reduction of the 7,8-double bond. Like heroin and morphine-6beta-glucuronide, the analgesic actions of dihydromorphine and its two acetylated derivatives were antagonized by 3-O-methylnaltrexone at a dose that was inactive against morphine analgesia. Antisense mapping also distinguished between morphine and the dihydromorphine compounds. Antisense oligodeoxynucleotides targeting exon 2 of the cloned MOR-1 gene decreased dihydromorphine analgesia and that of its acetylated derivatives, but not morphine analgesia. Conversely, the exon 1 antisense that effectively lowered morphine analgesia was inactive against dihydromorphine and its analogs. Finally, dihydromorphine and its analogs retained their analgesic activity in a mouse model of morphine tolerance, consistent with incomplete cross-tolerance. Together, these findings imply that the mu-opioid receptor mechanisms mediating the analgesic actions of dihydromorphine and its acetylated analogs are distinct from morphine and more similar to those of heroin and morphine-6beta-glucuronide.

Histogranin-like antinociceptive and anti-inflammatory derivatives of o-phenylenediamine and benzimidazole.

Histogranin (HN)-like nonpeptides were designed and synthesized using benzimidazole (compound 1) and o-phenylenediamine (compounds 2-7) as scaffolds for the attachment of phenolic hydroxyl and basic guanidino pharmacophoric elements present in HN. The benzimidazole derivative N-5-guanidinopentanamide-(2R)-yl-2-(p-hydroxybenzyl)-5-carboxybenzimidazole (1) and the o-phenylenediamine derivative N-5-guanidinopentanamide-(2S)-yl-2-N-(p-hydroxyphenylacetyl) phenylenediamine (2) were more potent analgesics than HN in both the mouse writhing (5.5 and 3.5 as potent as HN, respectively) and tail-flick (11.8 and 8.0 as potent as HN, respectively) pain assays. Improvements in the potencies and times of action of compound 2 in the mouse writhing test were obtained by attaching carboxyl (6)or p-Cl-benzoyl (7) groups at position 4 of the (2R) o-phenylenediamine derivative (5). In rats, compounds 2 (80 nmol i.t.), 6 (36 nmol i.t.), and 7 (18 nmol i.t.) were effective in blocking both persistent inflammatory pain in the formalin test and hyperalgesia in the complete Freund adjuvant assay. Compounds 2, 6, and 7, but not compound 1 at 10 nmol (i.c.v.) also mimicked the HN (60 nmol i.c.v.) blockade of N-methyl-D-aspartate (NMDA)-induced convulsions in mice. Finally, in primary cultures of rat alveolar macrophages, HN and compounds 1, 2, 6, and 7 (10(-8) M) significantly blocked lipopolysaccharide-induced cyclooxygenase-2 induction and prostaglandin E(2) secretion. These studies indicate that both derivatives of benzimidazole and o-phenylenediamine mimic the in vivo antinociceptive and in vitro anti-inflammatory effects of HN, but the HN protection of mice against NMDA-induced convulsions is mimicked only by the o-phenylenediamine derivatives.

Bioactive peptidic analogues and cyclostereoisomers of the minimal antinociceptive histogranin fragment-(7-10).

Novel analogues of the minimal antinociceptive histogranin (HN) fragment Gly(7)-Gln-Gly-Arg(10), in which amino acids in positions 8, 9, and 10 were replaced by lipophilic amino acids and corresponding d-amino acid residues in combination with N- to C-terminal cyclization, were synthesized and tested in various animal models of pain. All synthetic compounds were potent and efficacious analgesics in the mouse writhing test. Cyclic [-Gly-Ala-Tyr-d-Arg-] (9) and cyclic [-Gly p-Cl-Phe-Tyr-d-Arg-] (10) were the most potent analgesics, being 17 and 135 times as potent as HN, respectively (AD(50) of 1.37 and 0.17 nmol/mouse icv, as compared with 23 nmol/mouse for HN). The times of action of compounds 9 and 10 were also much improved with half-maximal effects still being observed 60 min and >90 min after their administration, respectively, as compared with 8.1 min for the parent peptide HN-(7-10) and 22.1 min for HN. At analgesic doses, compounds 9 and 10 were devoid of motor effect as assessed by the mouse rotarod assay. As already observed with HN, compounds 9 (10 nmol/rat; i.t.) and 10 (0.5 nmol/rat; i.t.) were effective in blocking persistent inflammatory pain in the formalin test and hyperalgesia induced by intraplantar administration of complete Freund adjuvant. In addition, the analgesic effects evoked by compounds 9 (10 nmol/mouse; icv) and 10 (1 micromol/kg; i.v.) in the mouse writhing test and compound 9 (10 nmol/mouse; icv) in the mouse tail flick assay were similarly antagonized by the dopamine D(2) receptor antagonist raclopride (1 nmol/mouse; icv) but not the opiate antagonist naloxone (1 nmol/mouse; i.c.v). Finally, the various cyclic compounds competed with the binding of [(3)H]raclopride in rat brain membrane preparations. Their ability to compete with the binding of the D(2) ligand correlated well with their potency in alleviating pain in the mouse writhing test (r = 0.95). These results indicate that the analgesic activity of the minimal active core in HN can be improved by changes that favor its interaction with the dopamine D(2) receptor.

Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1O and hMOR-1X.

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.

The role of descending fibers from the rostral ventromedial medulla in opioid analgesia in rats.

There has been controversy as to whether the contribution of descending fibers from the rostral ventromedial medulla to opioid analgesia depends on the nature of the noxious stimulus eliciting pain. In the present study, inactivation of descending fibers by microinjection of muscimol (50 ng) in the rostral ventromedial medulla abolished morphine analgesia in the tail immersion and hot plate tests but decreased morphine analgesia by 60% in the formalin test. Analysis of the dose-response relation for morphine after inactivation of descending fibers revealed that, except for the tail immersion test, high doses of morphine could not overcome the block induced by muscimol. Also, morphine analgesia elicited supraspinally was not detectable when descending fibers were inactivated, suggesting that the analgesic effect of morphine in the brain requires a relay via the rostral ventromedial medulla. The analgesic effect of buprenorphine also depends on the integrity of descending fibers from the rostral ventromedial medulla. The results indicate that descending fibers from the rostral ventromedial medulla are critically important to the analgesic effect of opioids, regardless of the type of noxious stimulation eliciting pain. Residual analgesic effects of opioids after inactivation of descending fibers may be due to peripheral effects in the presence of inflammation.