Nociceptin/orphanin FQ opioid peptide-receptor expression in pachyonychia congenita.

Nociceptin/orphanin FQ opioid peptide (NOP)-receptor (NOP-R) is a member of the opioid receptor family. NOP-R activation has demonstrated analgesic effects in preclinical pain models without the addiction risks associated with other opiate targets. Pachyonychia congenita (PC) is a palmoplantar keratoderma characterized by neuropathic pain in affected skin. A cohort of KRT6A gene mutation PC patients with no other explanation for their neuropathic pain offered a unique opportunity to assess potential of NOP-R as a therapeutic target. Plantar biopsies from 10 PC patients and 10 age/gender matched controls were performed at the ball (PC-affected) and the arch (PC-unaffected) of the foot. NOP-R expression was assessed by immunohistochemistry. Localization of NOP-R in subsets of epidermal nerve fibers was investigated using the pan-neuronal marker PGP9.5, markers for unmyelinated peptidergic fibers (calcitonin gene-related peptide [CGRP] and substance P [SP]), as well as for myelinated Aδ and Aß fibers (neurofilament H [NFH]). Robust NOP-R expression was detected in epidermal keratinocytes and in a subset of PGP9.5+ fibers in both epidermis and dermis, confirmed by western blot and absorption experiments with NOP-R peptide. NOP-R expression in keratinocytes was significantly reduced in PC-affected plantar skin compared with PC-unaffected skin. In addition, NOP-R expression occurred in dermal NFH+ myelinated fibers in all groups, although few CGRP+ fibers co-expressed NOP-R. Furthermore, most SP+ fibers also co-expressed NOP-R. These findings indicate that NOP-R is expressed on epidermal keratinocytes, as well as on epidermal and dermal nerve fibers and has potential as a promising target to treat neuropathic pain in PC.

Antinociceptive Effect of Ghrelin in a Rat Model of Irritable Bowel Syndrome Involves TRPV1/Opioid Systems.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS), defined as recurrent abdominal pain and changes in bowel habits, seriously affects quality of life and ability to work. Ghrelin is a brain-gut hormone, which has been reported to show antinociceptive effects in peripheral pain. We investigated the effect of ghrelin on visceral hypersensitivity and pain in a rat model of IBS. METHODS: Maternal deprivation (MD) was used to provide a stress-induced model of IBS in Wistar rats. Colorectal distension (CRD) was used to detect visceral sensitivity, which was evaluated by abdominal withdrawal reflex (AWR) scores. Rats that were confirmed to have visceral hypersensitivity after MD were injected with ghrelin (10 µg/kg) subcutaneously twice a week from weeks 7 to 8. [D-Lys3]-GHRP-6 (100 nmol/L) and naloxone (100 nmol/L) were administered subcutaneously to block growth hormone secretagogue receptor 1α (GHS-R1α) and opioid receptors, respectively. Expression of transient receptor potential vanilloid type 1 (TRPV1) and µ and κ opioid receptors (MOR and KOR) in colon, dorsal root ganglion (DRG) and cerebral cortex tissues were detected by western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemical analyses and immunofluorescence. RESULTS: Ghrelin treatment increased expression of opioid receptors and inhibited expression of TRPV1 in colon, dorsal root ganglion (DRG) and cerebral cortex. The antinociceptive effect of ghrelin in the rat model of IBS was partly blocked by both the ghrelin antagonist [D-Lys3]-GHRP-6 and the opioid receptor antagonist naloxone. CONCLUSION: The results indicate that ghrelin exerted an antinociceptive effect, which was mediated via TRPV1/opioid systems, in IBS-induced visceral hypersensitivity. Ghrelin might potentially be used as a new treatment for IBS.

Retest imaging of [11C]NOP-1A binding to nociceptin/orphanin FQ peptide (NOP) receptors in the brain of healthy humans.

[(11)C]NOP-1A is a novel high-affinity PET ligand for imaging nociceptin/orphanin FQ peptide (NOP) receptors. Here, we report reproducibility and reliability measures of binding parameter estimates for [(11)C]NOP-1A binding in the brain of healthy humans. After intravenous injection of [(11)C]NOP-1A, PET scans were conducted twice on eleven healthy volunteers on the same (10/11 subjects) or different (1/11 subjects) days. Subjects underwent serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (VT; a measure of receptor density) was determined by compartmental (one- and two-tissue) modeling in large regions and by simpler regression methods (graphical Logan and bilinear MA1) in both large regions and voxel data. Retest variability and intraclass correlation coefficient (ICC) of VT were determined as measures of reproducibility and reliability respectively. Regional [(11)C]NOP-1A uptake in the brain was high, with a peak radioactivity concentration of 4-7 SUV (standardized uptake value) and a rank order of putamen>cingulate cortex>cerebellum. Brain time-activity curves fitted well in 10 of 11 subjects by unconstrained two-tissue compartmental model. The retest variability of VT was moderately good across brain regions except cerebellum, and was similar across different modeling methods, averaging 12% for large regions and 14% for voxel-based methods. The retest reliability of VT was also moderately good in most brain regions, except thalamus and cerebellum, and was similar across different modeling methods averaging 0.46 for large regions and 0.48 for voxels having gray matter probability >20%. The lowest retest variability and highest retest reliability of VT were achieved by compartmental modeling for large regions, and by the parametric Logan method for voxel-based methods. Moderately good reproducibility and reliability measures of VT for [(11)C]NOP-1A make it a useful PET ligand for comparing NOP receptor binding between different subject groups or under different conditions in the same subject.

Targeting opioid receptors with pharmacological chaperones.

G protein-coupled receptors (GPCRs) are polytopic membrane proteins that have a pivotal role in cellular signaling. Like other membrane proteins, they fold in the endoplasmic reticulum (ER) before they are transported to the plasma membrane. The ER quality control monitors the folding process and misfolded proteins and slowly folding intermediates are targeted to degradation in the cytosol via the ubiquitin-proteasome pathway. The high efficiency of the quality control machinery may lead to the disposal of potentially functional receptors. This is the major underlying course for loss-of-function conformational diseases, such as retinitis pigmentosa, nephrogenic diabetes insipidus and early onset obesity, which involve mutant GPCRs. During the past decade, it has become increasingly evident that small-molecular lipophilic and pharmacologically selective receptor ligands, called pharmacological chaperones (PCs), can rescue these mutant receptors from degradation by stabilizing newly synthesized receptors in the ER and enhancing their transport to the cell surface. This has raised the interesting prospect that PCs might have therapeutic value for the treatment of conformational diseases. At the same time, accumulating evidence has indicated that wild-type receptors might also be targeted by PCs, widening their therapeutic potential. This review focuses on one GPCR subfamily, opioid receptors that have been useful models to unravel the mechanism of action of PCs. In contrast to most other GPCRs, compounds that act as PCs for opioid receptors, including widely used opioid drugs, target wild-type receptors and their common natural variants.

The opioid growth factor (OGF) and low dose naltrexone (LDN) suppress human ovarian cancer progression in mice.

OBJECTIVE: The opioid growth factor (OGF) and its receptor, OGFr, serve as a tonically active inhibitory axis regulating cell proliferation in normal cells and a variety of cancers, including human ovarian cancer. Blockade of OGF and OGFr with the nonselective opioid receptor antagonist naltrexone (NTX) upregulates expression of OGF and OGFr. Administration of a low dosage of NTX (LDN) blocks endogenous opioids from opioid receptors for a short period of time (4-6 h) each day, providing a window of 18-20 h for the upregulated opioids and receptors to interact. The present study investigated the repercussions of upregulating the OGF-OGFr axis by treatment with OGF or LDN on human ovarian tumorigenesis in vivo. METHODS: Female nude mice were transplanted intraperitoneally with SKOV-3 human ovarian cancer cells and treated on a daily basis with OGF (10 mg/kg), LDN (0.1 mg/kg), or an equivalent volume of vehicle (saline). Tumor burden, as well as DNA synthesis, apoptosis, and angiogenesis was assessed in tumor tissue following 40 days of treatment. RESULTS: OGF and LDN markedly reduced ovarian tumor burden (tumor nodule number and weight). The mechanism of action was targeted to an inhibition of tumor cell proliferation and angiogenesis; no changes in cell survival were noted. CONCLUSIONS: This study shows that a native opioid pathway can suppress human ovarian cancer in a xenograft model, and provides novel non-toxic therapies for the treatment of this lethal neoplasia.

Direct effect of morphine on breast cancer cell function in vitro: role of the NET1 gene.

BACKGROUND: Experimental data suggest that postoperative analgesia in general and opioids in particular may affect the risk of metastases after primary cancer surgery. Perioperative single-gene activation may also spark metastatic disease. The NET1 gene promotes migration in adenocarcinoma cells. We investigated opioid receptor expression in both breast cancer cell lines and the direct effect of morphine and NET-1 on breast cancer cell migration in vitro. METHODS: Proliferation and migration of oestrogen receptor-negative MDA-MB-231 and oestrogen receptor-positive MCF7 breast cancer cells were studied after incubation with morphine 10-100 ng ml(-1) and control. NET1 gene expression was determined by polymerase chain reaction. The effect of NET1 on cell migration was determined using gene silencing with siRNA and stimulation with lysophosphatidic acid (LPA). The effect of morphine on NET1 expression and migration of cells with silenced NET1 was investigated. RESULTS: The NET1 gene was expressed in both cell lines and stimulated by LPA (2.9-fold in MCF7 and 78-fold in MDA-MB-231). NET1 expression was decreased by 96% after gene silencing in both cell lines with corresponding changes in migration. Despite the lack of opioid receptor expression, morphine increased the expression of NET1 (by 94% in MCF7 and by 263% in MDA-MB-231 cells). Morphine also increased migration by 17-27% and 7-53% in MCF7 and MDA-MB-231, respectively. Silencing the NET1 gene reversed the effect of morphine on migration. CONCLUSIONS: The NET1 gene, but not opioid receptors, is expressed in breast adenocarcinoma cells and may facilitate their migration. Morphine increased both expression of NET1 and cell migration but not when NET1 was silenced, implying that NET1 contributes to mediating the direct effect of morphine on breast cancer cell migration.

Immunohistological detection of mu, delta and kappa opioid-like receptors in the gill, gonad, and hemocytes of the scallop Chlamys farreri.

Endogenous opioid peptides and opioid receptors form a neuromodulatory system, which plays an important part in the control of physiological pathways. In addition, some opioid peptides can function as endogenous messengers of the immune system and participate in the regulation of the immune response. The present studies indicated that mu, delta, and kappa opioid-like receptors were present in the gill and gonad of the scallop Chlamys farreri. Furthermore, the significance of opioid peptides involvement with the immune system is ascertained from the presence of mu, delta, and kappa opioid-like receptors on hemocytes of the scallop. Our report constitutes the first characterization of mu, delta, and kappa opioid-like receptors in the gill and gonad of the scallop Chlamys farreri.

A quantum dot-labeled ligand-receptor binding assay for G protein-coupled receptors contained in minimally purified membrane nanopatches.

A robust method to directly measure ligand-receptor binding interactions using fluorescence cross-correlation spectroscopy (FCCS) is described. The example receptor systems demonstrated here are the human micro-opioid receptor, a representative G protein-coupled receptor (GPCR), and Streptavidin, but these general protocols can be extended for the analysis of many membrane receptors. We present methods for the preparation of GPCR-containing membrane nanopatches that appear to have the shapes of nanovesicles, labeling of proteins in membrane vesicles, in addition to the coupling of quantum dots (QDs) to peptide ligands. Further, we demonstrate that reliable binding information can be obtained from these partially purified receptors.

[Distribution of met-enkephalin expression in the visual pathway. Experimental study by inmunocytochemistry]. / Distribución de la metencefalina en la vía óptica. Estudio experimental inmunocitoquímico.

PURPOSE: The localization and distribution of neuropeptide expression in the cat visual pathway can provide information about the function of that pathway. METHOD: Study of optic pathway in eight cats. Following extraction of the brain, slices were prepared using a microkeratome. The slices were examined by indirect immunocytochemistry using anti-metenkephalin as antibody to determine the presence or absence of this pentapeptide in the visual pathway. RESULTS: Met-enkephalin receptors in both cortical and subcortical regions of the brain were detected. This suggests that met-enkephalin could be involved in the visual mechanism. CONCLUSIONS: The presence of met-enkephalin receptors in both cortical and subcortical regions of the brain suggests that this pentapeptide could be involved in the visual mechanism.

Visualizing endogenous opioid receptors in living neurons using ligand-directed chemistry.

Identifying neurons that have functional opioid receptors is fundamental for the understanding of the cellular, synaptic and systems actions of opioids. Current techniques are limited to post hoc analyses of fixed tissues. Here we developed a fluorescent probe, naltrexamine-acylimidazole (NAI), to label opioid receptors based on a chemical approach termed 'traceless affinity labeling'. In this approach, a high affinity antagonist naltrexamine is used as the guide molecule for a transferring reaction of acylimidazole at the receptor. This reaction generates a fluorescent dye covalently linked to the receptor while naltrexamine is liberated and leaves the binding site. The labeling induced by this reagent allowed visualization of opioid-sensitive neurons in rat and mouse brains without loss of function of the fluorescently labeled receptors. The ability to locate endogenous receptors in living tissues will aid considerably in establishing the distribution and physiological role of opioid receptors in the CNS of wild type animals.

The molecular basis of opioid receptor function.

An extensive body of pharmacological data demonstrates the existence of at least three opioid receptor subtypes mediating the diverse effects of opioids. Distinct binding and activity profiles of highly selective ligands, variable sensitivity to naloxone antagonisms, and selective protection and inactivation experiments strongly suggest that mu-, delta-, and kappa-opioid receptors represent recent discrete molecular entities. Purification and affinity labeling of receptor subunits are beginning to provide confirmation of this concept. The delta-opioid receptor affinity labeled and purified to homogeneity from NG108-15 cells comprises a glycoprotein subunit of Mr58,000 with one mol ligand bound/mol protein. Antibodies to this protein recognize native receptor in detergent solution and selectively bind to the Mr58,000 protein on immunoblots of partially purified preparations. Purification of the mu-opioid receptor from bovine striatum reveals a glycoprotein of Mr 65,000 which demonstrates opioid binding activity. Purification and affinity-labeling studies from other laboratories suggest a smaller size of Mr 58,000 for the mu-receptor however. The kappa-opioid receptor from guinea pig brain exhibits a unique mobility on sucrose density gradient centrifugation but has not been characterized in purified form. The primary structure of the opioid receptors, although unknown at present, will most likely reflect structural features of other inhibitory receptors coupled to G-proteins, with seven transmembrane helices and a large third cytoplasmic loop. Biochemical evidence clearly demonstrates the coupling of opioid receptors to Gi, accounting for opioid inhibition of adenylyl cyclase in neuronal cell culture and brain. Opioid inhibition of adenylyl cyclase has been reconstituted in IAP-treated NG108-15 cell membranes with a Gi preparation from brain. Electrophysiological evidence suggests that mu- and delta-opioid receptors can couple to a G-protein which mediates activation of inwardly rectifying potassium channels, perhaps to the same Gk mediating muscarinic potassium channel activation in heart. kappa-Opioid receptors are coupled to inhibition of voltage-dependent calcium channels in several neuronal systems. In NG108-15 cells opioid inhibition of calcium conductance is IAP sensitive and can be reconstituted with G-proteins purified from brain. Differences in the primary structure of mu-, delta-, and kappa-opioid receptors, as well as possible novel opioid receptor subtypes, will be defined by molecular cloning of recombinant DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594 ) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. EXPERIMENTAL APPROACH: We assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). KEY RESULTS: N/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594 , movement of ligand and receptor from the cell surface to the cytosol can be measured. CONCLUSIONS AND IMPLICATIONS: In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ-driven cellular responses.

Analysis of the distribution of spinal NOP receptors in a chronic pain model using NOP-eGFP knock-in mice.

BACKGROUND AND PURPOSE: The nociceptin/orphanin FQ opioid peptide (NOP) receptor system plays a significant role in the regulation of pain. This system functions differently in the spinal cord and brain. The mechanism by which the NOP receptor agonists regulate pain transmission in these regions is not clearly understood. Here, we investigate the peripheral and spinal NOP receptor distribution and antinociceptive effects of intrathecal nociceptin/orphanin FQ (N/OFQ) in chronic neuropathic pain. EXPERIMENTAL APPROACH: We used immunohistochemistry to determine changes in NOP receptor distribution triggered by spinal nerve ligation (SNL) using NOP-eGFP knock-in mice. Antinociceptive effects of intrathecal N/OFQ on SNL-mediated allodynia and heat/cold hyperalgesia were assessed in wild-type mice. KEY RESULTS: NOP-eGFP immunoreactivity was decreased by SNL in the spinal laminae I and II outer, regions that mediate noxious heat stimuli. In contrast, immunoreactivity of NOP-eGFP was unchanged in the ventral border of lamina II inner, which is an important region for the development of allodynia. NOP-eGFP expression was also decreased in a large number of primary afferents in the L4 dorsal root ganglion (DRG) of SNL mice. However, SNL mice showed increased sensitivity, compared to sham animals to the effects of i.t administered N/OFQ with respect to mechanical as well as thermal stimuli. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that the spinal NOP receptor system attenuates injury-induced hyperalgesia by direct inhibition of the projection neurons in the spinal cord that send nociceptive signals to the brain and not by inhibiting presynaptic terminals of DRG neurons in the superficial lamina.

No tolerance to peripheral morphine analgesia in presence of opioid expression in inflamed synovia.

Pain treatment with centrally acting opiates is limited by tolerance. Tolerance is a decreasing effect of a drug with prolonged administration of that drug or of a related (e.g., endogenous) compound acting at the same receptor. This is often associated with a downregulation of receptors. In peripheral inflamed tissue, both locally expressed opioid peptides and morphine can produce powerful analgesia mediated by similar populations of opioid receptors. We hypothesized that the chronic presence of endogenous opioids in inflamed joints might convey downregulation of peripheral opioid receptors and tolerance to the analgesic effects of intraarticular morphine. We assessed these effects after arthroscopic surgery in patients with and without histologically verified synovial cellular infiltration, and we examined synovial opioid peptides and opioid receptors by immunocytochemistry and autoradiography, respectively. We found that, despite an abundance of opioid-containing cells in pronounced synovitis, morphine is at least as effective as in patients without such cellular infiltrations, and there is no major downregulation of peripheral opioid receptors. Thus, opioids expressed in inflamed tissue do not produce tolerance to peripheral morphine analgesia. Tolerance may be less pronounced for peripherally than for centrally acting opioids, which provides a promising perspective for the treatment of chronic pain in arthritis and other inflammatory conditions.

Overexpression of the opioid growth factor receptor potentiates growth inhibition in human pancreatic cancer cells.

The opioid growth factor (OGF), [Met5]-enkephalin, and OGF receptor (OGFr) form an inhibitory axis regulating the growth of human pancreatic cancer. This study examined whether overexpression of OGFr decreases the growth of pancreatic cells in vitro. MIA PaCa-2 cells were transfected with OGFr cDNA, and six clonal lines were examined for protein expression and function. OGFr binding assays revealed a 2.3- to 5.6-fold increase in binding capacity from wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by immunohistochemistry and Western blotting, was enhanced in clonal cell lines compared to controls. Doubling times of OGFr clonal lines were 47-91% longer than in the WT/EV groups for all but one clonal line. DNA synthesis of cells overexpressing OGFr was diminished from the WT/EV groups by 28-52%. Addition of exogenous OGF further reduced (14-31%) the cell growth of clonal lines, and the effects of exogenous OGF were receptor-mediated. Exposure of cells overexpressing OGFr to naltrexone increased the cell number by up to 9.4-fold. OGF was identified as the only opioid peptide to depress cell replication in the transfected cell lines. Neutralization of endogenous OGF with antibodies to this peptide elevated the cell number in clonal cell lines. These data identify OGFr at the molecular level as integral to regulating the cell replication of human pancreatic cancer, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

Comparative neurochemical study of the thoracic and sacral intermediolateral nuclei in the rat.

BACKGROUND AND PURPOSE: In view of the known functional differences, the neurochemical character of the thoracic and sacral intermediolateral nuclei were compared. METHODS: Neurons and the afferent fiber components were labeled using antibodies raised against neurofilament, neural nuclear protein, cholinacetyltransferase, nitric oxide synthase, neurokinin receptor-1, substance P, calcitonin gene-related peptide, micro-opiate receptor-1 and vesicular glutamate transporter type 1 in rats. Biontinylated isolectin IB4 was used to label unmyelinated primary afferent fibers. Specimens were analyzed with confocal laser microscope. RESULTS AND CONCLUSIONS: The thoracic and sacral intermediolateral nuclei are similar in the chemical character of the neurons. In the thoracic segments the dendrites of the labeled neurons followed a transverse path towards the neurons located at both sides of the midline above the central canal. The transverse orientation of the dendrites in the sacral segment was less evident. Calcitonin gene-related peptide, isolectin IB4 and micro-opiate receptor-1 immunopositive afferent fibers arborize only in the sacral intermediolateral nucleus. We conclude that fine caliber primary afferent fibers, departing from the adjacent superficial dorsal horn, terminate in the sacral intermediolateral nucleus. It is probable that the preganglionic parasympathetic neurons in the nucleus receive synapses from the primary afferent fibers.

Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications.

The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization.

Non-cannabinoid CB1, non-cannabinoid CB2 antinociceptive effects of several novel compounds in the PPQ stretch test in mice.

The analgesic and anti-hyperalgesic effects of cannabinoid- and vanilloid-like compounds, plus the fatty acid amide hydrolase (FAAH) inhibitor Cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597), and acetaminophen, were evaluated in the phenyl-p-quinone (PPQ) pain model, using different routes of administration in combination with opioid and cannabinoid receptor antagonists. All the compounds tested produced analgesic effects. Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide ((R)-methanandamide) were active by three routes of administration: i.p., s.c. and, p.o. Delta(9)-THC produced ED(50)s of 2.2 mg/kg (0.3-15.6) i.p., 9 mg/kg (4.3-18.9) s.c., and 6.4 mg/kg (5.5-7.6) p.o. Similarly, (R)-methanandamide yielded ED(50)s of 2.9 mg/kg (1-8) i.p., 11 mg/kg (7-17) s.c., and 11 mg/kg (0.9-134) p.o. N-vanillyl-arachidonyl-amide (arvanil) was active by two routes, producing ED(50)s of 4.7 mg/kg (3.0-7.4) s.c. and 0.06 mg/kg (0.02-0.2) i.p. Palmitoylethanolamide, URB597, and acetaminophen were active i.p., resulting in ED(50)s of 3.7 mg/kg (3.2-4.2), 22.9 mg/kg (11.1-47.2), and 160 mg/kg (63-405), respectively. None of the cannabinoid or opioid receptor antagonists tested blocked the compounds evaluated, with two exceptions: the antinociceptive effects of Delta(9)-THC and URB597 were completely blocked by SR141716A, a cannabinoid CB(1) receptor antagonist. Western immunoassays performed using three opioid receptor antibodies, a cannabinoid CB(1) receptor antibody and a transient receptor potential vanilloid type 1(TRPV(1)) receptor antibody, yielded no change in receptor protein levels after short-term arvanil, (R)-methanandamide or Delta(9)-THC administration. These data suggest that all the compounds tested, except Delta(9)-THC and URB597, produced analgesia via a non-cannabinoid CB(1), non-cannabinoid CB(2) pain pathway not yet identified.

Comparison of opioid receptor distributions in the rat ileum.

The cellular expression patterns of mu-, delta- and kappa-opioid receptors in the rat ileum were examined using fluorescence immunohistochemistry. Double-labelling was used to examine cellular receptor co-localisation as a pre-requisite for intracellular molecular interactions, such as heterodimerisation. Tissues were stained as whole-mount preparations. Strong, broadly distributed immunoreactivity (ir) was observed for each receptor in the myenteric and submucous plexuses. Although intracellular mu- and delta-ir patterns differed in ganglion neurons, mu/delta co-expression was extensive in these cells. mu/delta co-expression was also observed in interstitial cells, which were diffusely distributed in submucous plexus preparations but generally located adjacent to myenteric plexus structures. Punctate kappa-ir was seen broadly in nerve fibres in both plexuses, suggesting localisation in varicosities. Neuronal mu/kappa co-localisation was not apparent, although kappa-ir fibres were often apposed against mu-ir cells. mu/kappa co-localisation was detected in interstitial cells in submucous plexus preparations. Similarities in mu and delta expression patterns might reflect similar functional properties previously detected for these receptors. This study indicates that the rat gastrointestinal tract might provide a useful tool for the future study of molecular interactions between opioid receptor types.

Low efficacy opioids: implications for sex differences in opioid antinociception.

It is becoming increasingly evident that the sex of an organism is a critical determinant of responsiveness to opioid analgesics. However, the factors that determine the magnitude and direction of sex differences in opioid antinociception have not been fully elucidated. One factor that has received attention is the relative efficacy of the opioid. This review summarizes recent findings in which opioid efficacy was systematically manipulated as an independent variable to probe underlying sex differences in opioid system function. Overall, in rodents and nonhuman primates, mu and kappa opioids are generally more potent and effective in males than in females. The data indicate that although sex differences in the potency of high efficacy opioids such as morphine are generally less than 3.0-fold, sex differences with lower efficacy opioids can be greater than 90-fold. Moreover, that these drugs can function as full agonists in males while functioning as antagonists in females under identical conditions suggests some fundamental sex difference in opioid system function. In addition to efficacy, a number of other variables can affect the outcomes of these studies, including the drug history, genotype, and nociceptive stimulus modality, duration, and intensity. These factors may interact with opioid efficacy to determine the specific conditions under which sex differences are observed. The testing of low efficacy opioids by other laboratories and under other experimental conditions will determine the extent to which this variable affords a strategic research tool. The potential utility of low efficacy opioids in other domains of behavioral pharmacology is also discussed.