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.

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.

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.

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.

Sleep quality and OPRM1 polymorphisms: a cross-sectional study among opioid-naive individuals

ABSTRACT O sistema opioidérgico envolve a regulação do sono e da vigília. É possível, portanto, que os polimorfismos genéticos no OPRM1 influenciem na qualidade do sono. Este estudo investigou a associação de polimorfismos do OPRM1 com a qualidade subjetiva do sono entre indivíduos sem tratamento prévio com opióides. Este estudo observacional de corte transversal envolveu 161 homens que nunca haviam se tornado opióides (média de idade = 27,74 anos; variação: 18 a 63 anos). A qualidade subjetiva do sono foi avaliada com a versão traduzida e validada em malaio do Índice de Qualidade do Sono de Pittsburgh (PSQI). O DNA foi extraído do sangue total e submetido à reação em cadeia da polimerase (PCR) para dois polimorfismos OPRM1 (118A> G e IVS2 + 691G> C). Sujeitos combinados com 118A e IVS2 + 691Galelos (haplótipo AC) apresentaram escores significativamente mais baixos do PSQI [média (DP) = 4,29 (1,76)] em comparação àqueles sem o haplótipo [4,99 (2,50)] (p = 0,004). Por outro lado, os indivíduos com genótipo heterozigótico combinado (GC / AG diplotipo) apresentaram escores significativamente mais altos do PSQI em comparação àqueles sem o diplótipo [6,04 (2,48) vs 4,54 (2,22), p = 0,004]. Em indivíduos sem tratamento prévio com opiáceos, o haplótipo AC e o diplótipo GC / AG para os polimorfismos 118A> G e IVS2 + 691G> C do OPRM1 estão associados a uma melhor e pior qualidade do sono, respectivamente.

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.

Uso y abuso de opioides en el área norte de la provincia de Cádiz / Use and abuse of opioids analgesics in the northern area of the Cádiz province

Introducción: La utilización de opioides en dolor crónico no oncológico (DCNO) es controvertida porque no está demostrada su utilidad a largo plazo. Hay pocos estudios sobre dependencia y adicción; del mismo modo se ha descrito un aumento de mortalidad en pacientes que reciben altas dosis de opioides en DCNO. Por esto, nos propusimos analizar a los pacientes que superaban la dosis diaria definida, DDD, en el área norte de la provincia de Cádiz. La DDD es la dosis diaria de mantenimiento media en adultos. Material y métodos: El área norte de la provincia de Cádiz tiene una población aproximada de 420.000 habitantes. Se detectaron a los pacientes que superaban la DDD de opiáceos en diciembre de 2014. Estudiamos la historia clínica de todos estos pacientes. Resultados: Se encontraron 23 pacientes que, por lo menos, duplicaban la DDD del opiáceo utilizado. Por tanto, la incidencia en nuestra área era de un paciente por cada 18.000 habitantes. Cuatro de ellos son pacientes oncológicos (17 %), el resto de dolor crónico no oncológico (83 %). Predomina claramente el dolor tipo osteo-articular (65 %), siendo el diagnóstico más frecuente el síndrome postlaminectomía con 8 casos (35 %). La edad media era de 58 años, oscilando entre la más joven, 34 años, que era la que más dosis consumía, y la de más edad con 84 años. La DDD/día mayor es la de la paciente más joven con 34 veces la DDD, 2 con ocho veces la DDD, 2 con siete veces la DDD, dos con cinco veces la DDD, 1 con cuatro veces, 4 con tres veces y, por último, 11 pacientes que consumían el doble de la DDD. Todos los pacientes tomaban un opiáceo para el dolor irruptivo, la gran mayoría (17 pacientes) tomaban comprimidos de fentanilo oral transmucoso (Effentora® o Actiq®). La mayoría de los pacientes tomaban oxicodona (7) o fentanilo transdérmico (8), como opioide de acción retardada. Dos pacientes no tomaban ningún opioide de acción retardada. Se observó que once pacientes (47 %) tenían algún antecedente psiquiátrico, dos de ellos tuvieron algún intento de autolisis. Conclusiones: El uso inadecuado de opioides se da sobre todo en pacientes no oncológicos. Había una relación clara con patología psiquiátrica. Hay relaciones de uso inadecuado y abuso de opiáceos con los de acción rápida, sobre todo fentanilos, aunque el abuso de opioides es poco frecuente, con lo que hay que ser cautos en iniciar tratamiento en DCNO. Hay que reevaluar de manera periódica la eficacia de los opioides, considerando la derivación a la Unidad de Dolor cuando la dosis de morfina supere los 180 mg de morfina o equivalente (AU)

Introduction: The treatment of chronic noncancer pain (CNCP) with opioids is controversial because its effectiveness on the long-term outcomes has not been proven. The few trials focusing on dependence and addiction demonstrated increased mortality among CNCP patients receiving high doses of opioids. We therefore decided to analyze patients living in the northern area of the province of Cadiz who had exceeded the defined daily dose (DDD). The DDD is the assumed average maintenance dose per day for a drug used for its main indication in adults. Materials and methods: The northern area of Cadiz has a population of 360,000 inhabitants. Patients who exceeded the DDD of opioid in December 2014 were identified. The clinical history of each of these patients was studied retrospectively. Results: Twenty-three patients were identified who at least needed to double the DDD of their opioids. Consequently, the incidence of patients exceeding the DDD in the area under consideration was 1 per 15,000 inhabitants. Four of these were cancer patients (17 %) while the others suffered from CNCP (83 %). CNCP in these patients predominantly was of the osteo-articular type of pain (65 %), with the failed back syndrome, occurring in 8 cases (35 %), representing the most frequent diagnosis. Patient ages ranged from 34 to 84 Y with a median of 58 Y. The youngest of these (34 Y) requiring the largest amounts of drugs. The daily consumption of this youngest patient corresponded to 34 times the DDD, two other patients took 8 times the DDD, 1 four times, 4 three times and, finally, 11 patients needed to double the DDD. Todos los pacientes tomaban un opiáceo para el dolor irruptivo, la gran mayoría (17 pacientes) tomaban comprimidos de fentanilo oral transmucoso, effentora o actiq. Most of the patients were taking opioids for breaktrough pain, the vast majority of these (17 patients) using transmucosal fentanyl citrate formulations, i.e. Effentora® or Actiq®. Most of the patients took oxycodone (7) or transdermal fentanyl (8), as delayed action opioid. Two patients were not taking opioids with delayed action. It was noted that 11 patients (47 %) had a psychiatric history, 2 of them had a suicide attempt. Conclusions: The inappropriate use of opioids occurs mainly in non-cancer patients. There was a clear relationship with psychiatric disorders. There are relations of misuse and abuse of fast-acting drugs, especially fentanyls. Although the abuse of opioids is rare, so we must be cautious about starting treatment CNCP. It shoulf be bourne in mind that in these patients the effectiveness of opioids needs to be reassessed periodically, considering referral to the pain unit when opioid requirements exceeding 180 mg of morphine or its equivalent (AU)

Effect of prosthodontic planning on lateral occlusion scheme: a comparison between conventional and digital planning

Recently, digital wax-up is proposed as a tool to aid prosthodontic planning. However, there are no data about the effect of prosthodontic planning on lateral occlusion scheme. Objective : This study aims to evaluate the impact of conventional and digital prosthodontic planning on lateral occlusion scheme. Material and Methods : Dental models of 10 patients were collected. All models had Angle Class I occlusion and were undergoing prosthodontic treatment that would influence the lateral occlusion scheme. Each set of models had received both conventional wax-up and digital wax-up. In relation to the lateral occlusion scheme, the following variables were evaluated: the prevalence of the different lateral occlusion scheme, number of contacting teeth and percentage of each contacting tooth. Four excursive positions on the working side were included: 0.5, 1.0, 2.0 and 3.0 mm from the maximal intercuspation position. Results : The lateral occlusion scheme of the two wax-up models was subjected to alterations following excursion. There was a tendency for the prevalence of canine-guided occlusion to increase and for the prevalence of group function occlusion to decrease with increasing excursion. The number of contacting teeth was decreasing with the increasing magnitude of excursion. For the 0.5 mm and 1.0 mm positions, the two wax-ups had significantly greater contacts than the pre-treatment models, while at the 2.0 mm and 3.0 mm positions, all the models were similar. For all models, canines were the most commonly contacting teeth, followed by the teeth adjacent to them. No difference was observed between the two wax-ups in relation to the number of contacting teeth. Conclusion : Although the prosthodontic planning had influenced the pattern of the lateral occlusion scheme and contacts, there was no difference between the conventional and digital prosthodontic planning. .

The predictive power of brain mRNA mappings for in vivo protein density: a positron emission tomography correlation study.

Substantial efforts are being spent on postmortem mRNA transcription mapping on the assumption that in vivo protein distribution can be predicted from such data. We tested this assumption by comparing mRNA transcription maps from the Allen Human Brain Atlas with reference protein concentration maps acquired with positron emission tomography (PET) in two representative systems of neurotransmission (opioid and serotoninergic). We found a tight correlation between mRNA expression and specific binding with 5-HT1A receptors measured with PET, but for opioid receptors, the correlation was weak. The discrepancy can be explained by differences in expression regulation between the two systems: transcriptional mechanisms dominate the regulation in the serotoninergic system, whereas in the opioid system proteins are further modulated after transcription. We conclude that mRNA information can be exploited for systems where translational mechanisms predominantly regulate expression. Where posttranscriptional mechanisms are important, mRNA data have to be interpreted with caution. The methodology developed here can be used for probing assumptions about the relationship of mRNA and protein in multiple neurotransmission systems.

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.

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.

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.

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.

Morfina endógena a nivel central y periférico / Central and peripheral endogenous morphine

Morphine was first identified in opium from Papaver somniferum, and is still one of the strongest known analgesic compounds used in hospital. Since the beginning of the 80s, endogenous morphine, with an identical structure to that of morphine isolated from poppies, has been characterised in numerous mammalian cells and tissues. In mammals, the biosynthesis of endogenous morphine is associated with dopamine, as demonstrated in the SH-SY5Y human neuronal catecholamine-producing cell line. More recently, morphine andmorphine-6-glucuronide has been shown to be present in the humanneuro blastoma SH-SY5Y cell line and that morphine is secreted from the large dense core vesicles in response to nicotine stimulation via a Ca2+-dependent mechanism suggesting its implication in neurotransmission. An increasing number of publications have demonstrated its presence and implication in different biological processes at the central and peripheral levels. The present review reports the major data concerning endogenous morphine presence and implication in physiological processes (AU)

Morfina endógena a nivel central y periférico. La morfina se identificó por primera vez en el opio procedentede Papaver somniferum, y sigue siendo uno de los analgésicos más potentes conocidos empleados en los hospitales. Desde comienzos dela década de los 80s, la morfina endógena, con una estructura idéntica a la morfina aislada de las amapolas, se caracterizado en numerosas células y tejidos de mamíferos. En mamíferos, la biosíntesis dela morfina endógena está asociada a la dopamina, como se ha demostrado en la línea celular neuronal humana productora de catecolaminasSH-SY5Y. Más recientemente, se ha demostrado la presencia de morfina y mofina-6-glucorónido en la línea celular de neuroblastoma humano SH-SY5Y y que esta morfina es secretada desde vesículas densas en respuesta a estimulación con nicotina vía un mecanismo dependiente de Ca2+ sugiriendo su implicación en l la neurotransmisión. Un número cada vez mayor de publicaciones han demostrado su presencia e implicación en diferentes procesos biológicos a niveles central y periférico. La presente revisión recoge los datos más importantes sobre la presencia e implicación en procesos fisiológicos de la morfina endógena (AU)

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.

ORL-1 receptor mediates the action of nociceptin on ascending myenteric reflex pathways in rats.

BACKGROUND AND AIMS: Nociceptin is the endogenous agonist of the "orphan" opioid receptor-1 (ORL-1). We investigated whether activation of the ORL-1 receptor influences smooth muscle contractility and enteric neurotransmission within ascending myenteric reflex pathways of rats. METHODS: Reverse transcriptase polymerase chain reaction was performed to evaluate the presence of ORL-1 receptors. The ascending part of the ascending myenteric reflex in rats was studied in ileal segments using a 3-chambered organ bath. Intracellular recordings were performed to evaluate pharmacologic effects on excitatory and inhibitory junction potentials (EJP; IJP). Single- and double-labeling immunohistochemistry was used to examine the distribution of ORL-1 within the intestinal wall. RESULTS: ORL-1 expression and immunoreactivity was found in the large majority of myenteric neurons. In addition to the cholinergic myenteric neurons, all nitrergic myenteric neurons expressed the ORL-1 receptor. Nociceptin significantly reduced cholinergic twitch contractions, an effect that was reversed by the ORL-1 receptor antagonist [Nphe(1)]nociceptin(1-13)NH(2). Neither nociceptin nor [Nphe(1)]nociceptin(1-13)NH(2) had a direct influence on smooth muscle contractility. Nociceptin significantly reduced ascending myenteric reflex contractions and prolonged the latency from stimulation to contraction. Both effects were antagonized by [Nphe(1)]nociceptin(1-13)NH(2). Intracellular recordings demonstrated that nociceptin reduces the cholinergically mediated EJP and the nitrergic phase of IJP in a concentration-dependent manner, effects that were reversible in presence of [Nphe(1)]nociceptin(1-13)NH(2). CONCLUSIONS: We conclude that activation of ORL-1 receptors on myenteric neurons reduce excitatory and inhibitory neurotransmission within the gastrointestinal tract. This is accompanied by a reduction of the small intestinal peristaltic reflex response. These effects might be used pharmacologically.

Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor.

Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, Kd, without purification of the receptor protein. Accurately measured Kd's are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human micro opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches ( approximately 100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides Kd and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.

Role of hypothalamus nociceptin/orphanin FQ in pre-ovulatory luteinizing hormone surge of estrogen and progesterone-primed, ovariectomized rats.

AIM: To investigate the role of hypothalamus nociceptin/orphanin FQ (OFQ) and its endogenous receptor, the opioid receptor-like1 receptor (ORL1 receptor) in the estrus cycle of female rats. METHOD: Radioimmunoassay was used to detect the effect of the intracerebroventricular (icv) administration of OFQ and/or the ORL1 receptor antagonist [Nphe1]Nociceptin(1-13)NH2, that is, NC13 on luteinizing hormone (LH) levels of estrogen- and progesterone (EBP)-primed, ovariectomized (OVX) rats (EBP-primed OVX rats). RT-PCR, Western blotting, and immunohistochemistry techniques were adopted to observe the changes of OFQ and the ORL1 receptor in the pre-optic area (POA) and the medial basal hypothalamus (MBH) of the estrus cycle of female rat. RESULTS: Pre-ovulatory LH surges in EBP-primed, OVX rats were significantly reduced by icv administration of 20 and 200 nmol OFQ (P<0.05), and the effect of 20 nmol OFQ could be abolished by pretreatment with 20 nmol NC13. The OFQ mRNA level in the POA on pro-estrus was lowered markedly compared to diestrus and estrus (P<0.05), while the mRNA and protein levels of the ORL1 receptor showed no significant changes in the POA and MBH across the estrus cycle. Meanwhile, the number of OFQ-immunoreactive neurons in the medial POA, ventromedial hypothalamus, and the arcuate nucleus on pro-estrus was significantly decreased compared to diestrus and estrus (P<0.05). CONCLUSION: The inhibitory effect of OFQ on the LH surge of EBP-primed, OVX rats and its downregulation in POA and MBH on pro-estrus suggests that it might play a negative modulatory role in the estrus cycle.