Characteristics of Patients Presenting at a University Outpatient Department for Complementary and Integrative Medicine.

BACKGROUND: Complementary and integrative medicine (CIM) is increasingly provided at university outpatient departments (OPDs) in Germany, but its scientific evaluation is sparse. Therefore, we aimed to investigate and evaluate feasibility, patients' characteristics and complaints at a university's CIM-OPD. METHODS: A prospective evaluation included new patients without age restriction. At baseline, and after 6 and 12 months, patients filled out paper questionnaires. Patients rated their mean subjectively perceived severity of the main complaint within the last 7 days on a numerical rating scale (NRS) from 0 = no complaints to 10 = maximum complaints, their perceived resilience capacity in everyday life within the last 7 days (0 = not resilient to 10 = very resilient), and their contentment with the treatment (0 = not content to 10 = very content). Diagnoses were provided by physicians and coded according to the International Statistical Classification of Diseases and Related Health Problems, 10th revision. All data were analyzed descriptively. RESULTS: During two years, 536 new patients {72.6% response, age (mean ± standard deviation [SD] and range) 49.6 ± 15.8 and 1-86 years, 75.7% female} chose to participate. The most frequent diagnosis groups were neoplasms (C00-C97, n = 143, 18.6%) and musculoskeletal diseases (M00-M99, n = 137, 17.9%). In n = 165 patients (30.8%), more than one diagnosis was provided. In a subgroup of 187 patients, who returned the questionnaire after 6 months, we compared baseline to 6-month values: severity of main complaint (mean ± SD) 5.2 ± 2.6 changed to 3.9 ± 2.6; resilience capacity 5.1 ± 2.6 to 5.6 ± 2.4. After 6 months, respondents rated their contentment with the treatment with (mean ± SD) 7.7 ± 2.6. Data after 12 months (n = 113) are comparable to data after 6 months. CONCLUSION: Patients of our CIM-OPD had a broad age range, were predominantly female, and suffered mostly from oncologic-related complaints and musculoskeletal diseases. In the responding subgroup after 6 months, patients were content with the treatment. These results should be verified by further prospective evaluations.Komplementäre und integrative Medizin (CIM) wird in Deutschland zunehmend in Hochschulambulanzen (OPDs) angeboten, deren wissenschaftliche Evaluation ist jedoch unzureichend. Deshalb war es unser Ziel, die Durchführbarkeit einer Evaluation, die Charakteristika und die Beschwerden der Patienten und Patientinnen an einer CIM-ODP zu untersuchen.Eine prospektive Evaluation schloss neue Patienten und Patientinnen ohne Altersbeschränkung ein. Zu Baseline sowie nach sechs und 12 Monaten füllten die Patienten und Patientinnen Papierfragebögen aus. Die Patienten und Patientinnen bewerteten ihre mittlere subjektiv empfundene Schwere der Hauptbeschwerden in den letzten sieben Tagen auf einer numerischen Ratingskala (NRS) von 0 = keine Beschwerden bis 10 = maximale Beschwerden, ihre mittlere subjektiv empfundene Belastbarkeit im Alltag in den letzten sieben Tagen (0 = nicht belastbar bis 10 = sehr belastbar) und ihre Zufriedenheit mit der Behandlung (0 = nicht zufrieden bis 10 = sehr zufrieden). Die Diagnosen wurden von den Ärzten und Ärztinnen gestellt und nach der International Statistical Classification of Diseases and Related Health Problems, 10. Revision, kodiert. Die Daten wurden deskriptiv ausgewertet.Im Laufe von zwei Jahren nahmen 536 neue Patienten und Patientinnen (72.6% Rücklauf, Alter (Mittelwert ± SD und Range) 49.6 ± 15.8 und 1­86 Jahre, 75.7% weiblich) teil. Die häufigsten Diagnosen waren Neoplasmen (C00-C97, n = 143, 18.6%) und Erkrankungen des Bewegungsapparates (M00-M99, n = 137, 17.9%). Bei n = 165 (30.8%) Patienten und Patientinnen wurde mehr als eine Diagnose vergeben. In einer Subgruppe von 187 Patienten und Patientinnen, die den Fragebogen nach 6 Monaten zurücksendeten, verglichen wir die Ausgangs-und 6-Monats-Werte: Schweregrad der Hauptbeschwerden (Mittelwert±SD) 5.2 ± 2.6 veränderte sich zu 3.9 ± 2.6; Belastbarkeit 5.1 ± 2.6 zu 5.6 ± 2.4. Nach sechs Monaten bewerteten die Befragten ihre Zufriedenheit mit der Behandlung mit (Mittelwert±SD) 7.7 ± 2.6. Die Daten nach 12 Monaten (n = 113) sind mit den Daten nach 6 Monaten vergleichbar.Die Patienten und Patientinnen unserer CIM-OPD hatten eine breite Altersspanne, überwiegend weiblich und litten zumeist unter onkologisch bedingten Beschwerden und Erkrankungen des Bewegungsapparates. Patienten und Patientinnen der nach sechs Monaten antwortenden Subgruppe waren mit der Behandlung zufrieden. Die Ergebnisse sollten durch weitere prospektive Evaluationen verifiziert werden.

Diseases of the musculoskeletal system and connective tissue in relation to temporomandibular disorders-A SWEREG-TMD nationwide case-control study.

INTRODUCTION: Temporomandibular disorders (TMD) are comprised by a heterogenous group of diagnoses with multifaceted and complex etiologies. Although diseases of the musculoskeletal system and connective tissue (MSD) have been reported as risk factors for developing TMD, no nationwide population-based registry studies have been conducted to investigate this possible link. The aim of this study was to investigate the association between MSD and TMD in a population-based sample using Swedish registry data, and to further investigate the difference in such association between patients diagnosed with TMD in a hospital setting and patients surgically treated for the condition. MATERIALS AND METHODS: Population based case-control study using Swedish nationwide registry data. Data was collected between 1998 and 2016 from 33 315 incident cases and 333 122 controls aged ≥18, matched for sex, age, and living area. Cases were stratified into non-surgical (NS), surgically treated once (ST1) and surgically treated twice or more (ST2). Information on MSD exposure (ICD-10 M00-M99) was collected between 1964 and 2016. Odds ratios were calculated using conditional logistic regression, adjusted for country of birth, educational level, living area, and mental health comorbidity. RESULTS: A significant association between MSD and the development of TMD was found for all diagnostic categories: arthropathies (OR 2.0, CI 1.9-2.0); systemic connective tissue disorders (OR 2.3, CI 2.1-2.4); dorsopathies (OR 2.2, CI 2.1-2.2); soft tissue disorders (OR 2.2, CI 2.2-2.3); osteopathies and chondropathies (OR 1.7, CI 1.6-1.8); and other disorders of the musculoskeletal system and connective tissue (OR 1.9, CI 1.8-2.1). The associations were generally much stronger for TMD requiring surgical treatment. The diagnostic group with the strongest association was inflammatory polyarthropathies, M05-M14 (OR 11.7, CI 8.6-15.9), which was seen in the ST2 group. CONCLUSIONS: Patients with MSD diagnoses have a higher probability of being diagnosed with TMD, in comparison to individuals without MSD. This association is even stronger for TMD that requires surgery. The results are in line with earlier findings, but present new population-based evidence of a possible causal relationship between MSD and TMD, even after adjusting for known confounders. Both dentists and physicians should be aware of this association and be wary of early signs of painful TMD among patients with MSD, to make early referral and timely conservative treatment possible.

EFFECTS OF A SUPPLEMENTAL ETORPHINE DOSE ON PULMONARY ARTERY PRESSURE AND CARDIAC OUTPUT IN IMMOBILIZED, BOMA-HABITUATED WHITE RHINOCEROS ( CERATOTHERIUM SIMUM): A PRELIMINARY STUDY.

The effects of etorphine on the pulmonary vascular system of white rhinoceros ( Ceratotherium simum) have not been described and could play a role in the severe hypoxemia that develops after immobilization with etorphine-based drug combinations. Characterization of these effects requires measurement of pulmonary vascular pressures and cardiac output (CO). To refine a technique for pulmonary arterial catheterization, five boma-habituated white rhinoceros (three females and two males weighing 1,012-1,572 kg) were immobilized by remote injection with etorphine plus azaperone followed by butorphanol. This afforded the opportunity to perform a pilot study and acquire preliminary measurements of pulmonary arterial pressure (PAP) and CO before and after supplemental etorphine given intravenously. Ultrasonographic guidance was used to insert a sheath introducer into a linguofacial branch of a jugular vein. A 160-cm-long pulmonary artery catheter with a balloon and thermistor was then passed through the introducer and positioned with its tip in the pulmonary artery. It was not long enough to permit wedging for measurement of pulmonary artery occlusion pressure. Mean PAP was 35 mm Hg (minimum, maximum 32, 47 mm Hg) and increased ( P = 0.031) by 83% (28, 106%) after supplemental etorphine. Thermodilution CO was 120 L/min (92, 145 L/min) and increased 27% (3, 43%) ( P = 0.031). Heart rate was 100 (88, 112) beats/min and increased 20% (4, 45%) ( P = 0.031), whereas arterial partial pressure of oxygen was 35 mm Hg (30, 94 mm Hg) and decreased 47% (20, 72%) ( P = 0.031). The cardiovascular observations could result from etorphine-induced generalized sympathetic outflow, as has been reported in horses. Further studies of etorphine in isolation are needed to test this suggestion and to discern how the changes in pulmonary vascular pressures and blood flow might relate to hypoxemia in etorphine-immobilized white rhinoceros.

T394A Mutation at the µ Opioid Receptor Blocks Opioid Tolerance and Increases Vulnerability to Heroin Self-Administration in Mice.

The etiology and pathophysiology underlying opioid tolerance and dependence are still unknown. Because mu opioid receptor (MOR) plays an essential role in opioid action, many vulnerability-related studies have focused on single nucleotide polymorphisms of MOR, particularly on A118G. In this study, we found that a single-point mutation at the MOR T394 phosphorylation site could be another important susceptive factor in the development of opioid tolerance and dependence in mice. T394A mutation, in which a threonine at 394 was replaced by an alanine, did not alter agonist binding to MOR and opioid analgesia, but resulted in loss of etorphine-induced MOR internalization in spinal dorsal horn neurons and opioid analgesic tolerance induced by either morphine or etorphine. In addition, this mutation also caused an increase in intravenous heroin self-administration and in nucleus accumbens dopamine response to heroin. These findings suggest that T394 phosphorylation following MOR activation causes MOR internalization and desensitization, which subsequently contributes to the development of tolerance in both opioid analgesia and opioid reward. Accordingly, T394A mutation blocks opioid tolerance and leads to an increase in brain dopamine response to opioids and in opioid-taking behavior. Thus, the T394 may serve as a new drug target for modulating opioid tolerance and the development of opioid abuse and addiction. SIGNIFICANCE STATEMENT: The mechanisms underlying opioid tolerance and susceptibility to opioid addiction remain unclear. The present studies demonstrate that a single-point mutation at the T394 phosphorylation site in the C-terminal of mu opioid receptor (MOR) results in loss of opioid tolerance and enhanced vulnerability to heroin self-administration. These findings suggest that modulation of the MOR-T394 phosphorylation or dephosphorylation status may have therapeutic potential in management of pain, opioid tolerance, and opioid abuse and addiction. Accordingly, MOR-T394 mutation or polymorphisms could be a risk factor in developing opioid abuse and addiction and therefore be used as a new biomarker in prediction and prevention of opioid abuse and addiction.

Pulmonary gas exchange and acid-base status during immobilisation of black rhinoceroses (Diceros bicornis) in Zimbabwe.

When immobilising wildlife, adverse side effects can include hypoxaemia, acidosis and hypertension. Pulmonary gas exchange and acid-base status were evaluated during immobilisation of 25 free-ranging and one boma-held black rhinoceros (Diceros bicornis) in Zimbabwe. The effect of different body positions on arterial oxygenation was evaluated. A combination of the following drugs was used: an opioid (etorphine or thiafentanil), azaperone and an a2 -adrenoceptor agonist (detomidine or xylazine). Respiratory and heart rates, rectal temperature and pulse oximetry-derived haemoglobin oxygen saturation were recorded. Serial arterial blood samples were analysed immediately in the field. Marked hypoxaemia and hypercapnia were recorded in immobilised free-ranging black rhinoceroses. Arterial oxygenation was higher during sternal compared to lateral recumbency. Most rhinoceroses developed acidaemia of respiratory and metabolic origin. Initially high lactate concentrations in free-ranging rhinoceroses decreased during immobilisation. Pulse oximetry was unreliable in the detection of hypoxaemia. Positioning in sternal recumbency and routine use of oxygen supplementation are recommended in the management of immobilised rhinoceroses as measures to improve arterial oxygenation.

Posttranslation modification of G protein-coupled receptor in relationship to biased agonism.

Biased signaling has been reported with a series of G protein-coupled receptors (GPCRs), including ß(2)-adrenergic receptor and µ-opioid receptor (OPRM1). The concept of biased signaling suggests that the agonists of one particular receptor may activate the downstream signaling pathways with different efficacies. Thus in an extreme case, agonists might activate different sets of signaling pathways, which provide a new route to develop drugs with increased efficacies and decreased side effects. Among the many factors, posttranslation modifications of receptor proteins have major roles in influencing the biased signaling. Take OPRM1, for example, the phosphorylation and palmitoylation of receptor can regulate the biased signaling induced by agonists. Thus, by modulating these posttranslation modifications, the biased signaling of GPCRs can be regulated. In addition, although it is not considered as posttranslation modification normally, the distribution of GPCRs on cell membrane, especially the distribution between lipid-raft and non-raft microdomains, also contributes to the biased signaling. Thus in this chapter, we described the methods used in our laboratory to study receptor phosphorylation, receptor palmitoylation, and membrane distribution of receptor by using OPRM1 as a model. A functional model was also provided on these posttranslational modifications at the last section of this chapter.

Membrane-delimited proteolytic regulation of opioid receptors.

Prolonged exposure of opioid receptors to agonists leads to their regulation by the classical process of clathrin-dependent internalization, followed by their intracellular degradation (down regulation). We have previously shown that the opioid agonist etorphine induced an additional process of down regulation of mu-opioid receptors (MOR) that occurred in intact MOR-transfected HEK-293 cells, as well as in isolated membranes. In the present study we show that etorphine similarly down regulated rat kappa-opioid receptors (KORs), which do not undergo the classical process of internalization and down regulation. This process was resistant to inhibitors of clathrin-coated pit formation (hypertonic sucrose, mono-dansyl-cadaverine) and was mainly mediated by membranous serine- and amino-peptidases. We further show that various opioid ligands, besides etorphine, induced down regulation of either KOR or MOR in isolated membranes. The ability of the various opioid ligands to induce membrane-delimited KOR or MOR down regulation did not correlate to their classical pharmacological profile, suggesting functional selectivity of the effect. Levorphanol, but not its stereoisomer dextrophan, induced membrane-delimited down regulation of both KOR and MOR, indicating that stereoselective binding to the receptor was necessary to initiate the process. Our findings that this proteolytic regulation of opioid receptors occurs not only in isolated membranes but also in intact cells and that it occurs even when the receptors are resistant to the conventional process of down regulation indicate its possible physiological role in the regulation of opioid activity.

Paradoxical relationship between RAVE (relative activity versus endocytosis) values of several opioid receptor agonists and their liability to cause dependence.

AIM: To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence. METHODS: The effects of (-)-cis-(3R,4S,2'R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2'S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [(35)S]GTP gamma S binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged mu-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested. RESULTS: All four agonists exhibited the same rank order of activity in stimulation of [(35)S]GTP gamma S binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202>or=morphine>F9204>or=DHE. CONCLUSION: Taken in combination with previous findings of these compounds' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence.

Down-regulation of c-Cbl by morphine accounts for persistent ERK1/2 signaling in delta-opioid receptor-expressing HEK293 cells.

Opioids display ligand-specific differences in the time course of ERK1/2 signaling. Whereas full agonists, like etorphine, induce only transient activation of ERK1/2, the partial agonist morphine mediates persistent stimulation of mitogenic signaling. Here we report that in stably delta-opioid receptor (DOR)-expressing HEK293 (HEK/DOR) cells, the transient nature of etorphine-induced ERK1/2 signaling is due to desensitization of epidermal growth factor (EGF) receptor-mediated activation of the Ras/Raf-1/ERK1/2 cascade. Desensitization of ERK1/2 activity by etorphine is associated with down-regulation of EGF receptors, an effect mediated by the ubiquitin ligase c-Cbl. In contrast, chronic morphine treatment failed to desensitize EGF receptors, resulting in unimpeded ERK1/2 signaling. The failure of morphine to desensitize ERK1/2 signaling is mediated by persistent activation of c-Src, which induces degradation of c-Cbl. The role of c-Src in opioid-specific ERK1/2 signaling is further demonstrated by pretreatment of the cells with PP2 and SKI-I as well as overexpression of a dominant negative c-Src mutant (c-Src(dn)) or a c-Src-resistant c-Cbl mutant (CblY3F), both of which facilitate desensitization of ERK1/2 signaling by morphine. Conversely, overexpression of c-Src as well as down-regulation of c-Cbl by small interfering RNA results in persistent etorphine-induced stimulation of ERK1/2 activity. Subcellular fractionation experiments finally attributed the ability of morphine to persistently activate c-Src to its redistribution from Triton X-100-insensitive membrane rafts to DOR and EGF receptor containing high density membrane compartments implicated in ERK1/2 signaling. These results demonstrate that agonist-specific differences in the temporal and spatial pattern of c-Src activation determine the kinetics of DOR-mediated regulation of ERK1/2 signaling.

GRIN1 regulates micro-opioid receptor activities by tethering the receptor and G protein in the lipid raft.

The lipid raft location of mu-opioid receptor (MOR) determines the receptor activities. However, the manner in which MOR is anchored within the lipid rafts is undetermined. Using the targeted proteomic approach and mass spectrometry analyses, we have identified GRIN1 (G protein-regulated inducer of neurite outgrowth 1) can tether MOR with the G protein alpha-subunit and subsequently regulate the receptor distribution within the lipid rafts. Glutathione S-transferase fusion pulldown and receptor mutational analyses indicate that GRIN1-MOR interaction involves a receptor sequence (267)GSKEK(271) within the MOR third intracellular loop that is not involved in Galpha interaction. The GRIN1 domains involved in MOR interaction are also distinct from those involved in Galpha interaction. Pertussis toxin pretreatment reduced the amount of GRIN1 co-immunoprecipitated with MOR but not the amount with Galpha. Furthermore, overexpression of GRIN1 significantly enhanced the amount of MOR in lipid raft and the receptor signaling magnitude as measured by Src kinase activation. Such increase in MOR signaling was demonstrated further by determining the GRIN1-dependent pertussis toxin-sensitive neurite outgrowth. In contrast to minimal neurite outgrowth induced by etorphine in control neuroblastoma N2A cells, overexpression of GRIN1 resulted in the increase in etorphine- and non-morphine-induced neurite outgrowth in these cells. Knocking down endogenous GRIN1 by small interfering RNA attenuated the agonist-induced neurite outgrowth. Disrupting lipid raft by methyl-beta-cyclodextrin also blocked neurite outgrowth. Hence, by tethering Galpha with MOR, GRIN1 stabilizes the receptor within the lipid rafts and potentiates the receptor signaling in the neurite outgrowth processes.

delta-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation.

delta-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the "pro-survival" kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastomaxglioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen(2,5)]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G(i/o) proteins, because pre-treatment with pertussis toxin, but not over-expression of the G(q/11) scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gbetagamma scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

delta-Opioid receptors stimulate ERK1/2 activity in NG108-15 hybrid cells by integrin-mediated transactivation of TrkA receptors.

This study demonstrates that activation of delta-opioid receptors (DORs) in neuroblastomaxglioma (NG108-15) hybrid cells by [D-Ala2, D-Leu5]enkephalin (DADLE) and etorphine significantly enhances cell adhesion to fibronectin-coated wells. This effect is blocked by both naloxone and integrin binding RGDT peptides. In addition, cell adhesion turned out to be a prerequisite for DOR-stimulated transactivation of Tropomyosin-related kinase A (TrkA) and extracellular signal-regulated kinases 1/2 (ERK1/2). Because inhibition of TrkA activation by AG879 completely blocked DOR- and integrin-mediated ERK1/2 signaling, the present results indicate that in NG108-15 cells DOR-stimulated ERK1/2 activation is mediated by integrin-induced transactivation of TrkA.

Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases.

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the delta-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala(2), D-Leu(5)]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH(3) revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-delta, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.

Different kinases desensitize the human delta-opioid receptor (hDOP-R) in the neuroblastoma cell line SK-N-BE upon peptidic and alkaloid agonists.

In a previous work, we described a differential desensitization of the human delta-opioid receptor (hDOP-R) by etorphine (a non-selective and alkaloid agonist) and delta-selective and peptidic agonists (DPDPE ([D-Pen(2,5)]enkephalin) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2))) in the neuroblastoma cell line SK-N-BE (Allouche et al., Eur. J. Pharmacol., 371, 235, 1999). In the present study, we explored the putative role of different kinases in this differential regulation. First, selective chemical inhibitors of PKA, PKC and tyrosine kinases were used and we showed a significant reduction of etorphine-induced opioid receptor desensitization by the bisindolylmaleimide I (PKC inhibitor) while genistein (tyrosine kinase inhibitor) was potent to impair desensitization induced by the different agonists. When the PKA was inhibited by H89 pretreatment, no modification of opioid receptor desensitization was observed whatever the agonist used. Second, we further studied the role of G protein-coupled receptor kinases (GRKs) and by using western-blot experiments we observed that only the GRK2 isoform was expressed in the SK-N-BE cells. Next, the neuroblastoma cells were transfected with the wild type GRK2 or its dominant negative mutant GRK2-K220R and the inhibition on cAMP level was determined in naïve and agonist-pretreated cells. We showed that over-expression of GRK2-K220R totally abolished etorphine-induced receptor desensitization while no effect was observed with peptidic agonists and over-expression of GRK2 selectively impaired cAMP inhibition promoted by etorphine suggesting that this kinase was involved in the regulation of hDOP-R activated only by etorphine. Third, correlation between functional experiments and phosphorylation of the hDOP-R after agonist activation was assessed by western-blot using the specific anti-phospho-DOP-R Ser(363) antibody. While all agonists were potent to increase phosphorylation of opioid receptor, we showed no impairment of receptor phosphorylation level after PKC inhibitor pretreatment. Upon agonist activation, no enhancement of receptor phosphorylation was observed when the GRK2 was over-expressed while the GRK2-K220R partially reduced the hDOP-R Ser(363) phosphorylation only after peptidic agonists pretreatment. In conclusion, hDOP-R desensitization upon etorphine exposure relies on the GRK2, PKC and tyrosine kinases while DPDPE and deltorphin I mediate desensitization at least via tyrosine kinases. Although the Ser(363) was described as the primary phosphorylation site of the mouse DOP-R, we observed no correlation between desensitization and phosphorylation of this amino acid.

Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell.

In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.

Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells.

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.

High-purity selection and maintenance of gene expression in human neuroblastoma cells stably over-expressing GFP fusion protein. Application for opioid receptors desensitization studies.

Chronic use of opiates such as morphine is associated with drug tolerance, which is correlated with the desensitization of opioid receptors. This latter process involves phosphorylation of opioid receptors by G protein-coupled receptors kinases (GRKs) and subsequent uncoupling by beta-arrestins. To explore these molecular mechanisms, neuronal cell lines, endogenously expressing the opioid receptors, provide an ideal cellular model. Unfortunately, there are two major drawbacks: (1) these cells are refractory to cDNA introduction, resulting in low transfection efficiency; (2) continuous culturing of transfected cells invariably leads to phenotypic drift of the cultures even after an antibiotic selection. So, these cells were dropped in favor of heterologous expression systems, which are easier to transfect but whose relevance as adequate cellular model for studying opioid receptor regulation should be questioned, as recently demonstrated by [Haberstock-Debic, H., Kim, K.A.,Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J. Neurosci. 25, 7847-7857]. In this work, we describe a method, based on fluorescence-activated cell sorting (FACS), to select and maintain a high proportion of transfected SK-N-BE cells (a neuronal cell line endogenously expressing human Delta-Opioid Receptor (hDOR)), expressing the beta-arrestin1 fused to green fluorescent protein (GFP). While in functional experiments, we were not able to observe a major effect in non-sorted SK-N-BE cells expressing beta-arrestin1-GFP, the enrichment by 18-fold with FACS resulted in a robust increase of beta-arrestin1-GFP expression associated with strong hDOR desensitization. Moreover, this method also allows to counteract the phenotypic drift and to maintain a high-purity selection of SK-N-BE cells expressing beta-arrestin1-GFP. Thus, this approach provides a valuable tool for exploring opioid receptors desensitization in neuronal cells.

Overdose during chemical restraint in a black rhinoceros (Diceros bicornis).

A juvenile female black rhinoceros (Diceros bicornis) was successfully treated after overdose of drugs used for chemical restraint. Subsequent general anaesthesia for surgical reduction of a recurrent rectal prolapse was uneventful. Over a 25-minute period before transportation to the veterinary hospital, the animal received a total dose of 1.225 mg etorphine, 30 mg acepromazine and 30 mg detomidine. Based on an estimated mass of 200 kg, these corresponded to doses of 6.1 microg kg(-1) etorphine, 150 microg kg(-1) acepromazine, and 150 microg kg(-1) detomidine which constitutes considerable overdose for each drug given separately, notwithstanding the synergy that probably resulted when the three drugs were present concurrently. The estimated body mass may have substantially overestimated the actual body mass and exacerbated overdosage. The animal was recumbent and apnoeic on arrival at the hospital. Heart sounds were auscultated and a weak peripheral pulse was palpated; no pulse deficits were detected, although the heart rate was low. The trachea was intubated, inspired breath was enriched with oxygen and the lungs ventilated manually. Diprenorphine (1.5 mg) was given intravenously and spontaneous breathing resumed 11 minutes later. After induction of general anaesthesia using isoflurane, emergency surgery for correction of rectal prolapse was performed, from which the animal recovered uneventfully. The case highlights some of the practical problems that may be encountered in dealing with dangerous and unfamiliar species.

Opioid control of MAP kinase cascade.

Activation of G protein-coupled receptors (GPCRs) may result in phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2). The signaling pathway involves ectodomain shedding, generating epidermal growth factor (EGF)-like ligands, which in turn stimulate the mitogen-activated protein kinase (MAPK) via EGF receptors. The present study investigates into the control of MAPKs by opioidergic GPCRs in human embryonic kidney cells (HEK 293). Experiments were conducted with cells expressing opioid receptors, G protein-coupled receptor kinases, and ERKs. The outcome of our studies let us suggest that EGF-like ligands released by opioid receptor stimulation utilize different EGF receptors to phosphorylate ERKs, while EGF utilizes type 1 receptors. Differences between multiple opioid receptors are apparent with respect to the activation of ERKs. EGF rapidly triggers internalization of the fluorescent EGF receptor type 1, but we failed to observe any sequestration of this receptor type upon exposure of cells to an opioid, since opioids most likely trigger stimulation of a different EGF receptor type. In conclusion, G protein-coupled opioid receptors control the MAPK cascade in a similar fashion as described for non-opioid GPCRs, although distinct differences exist between mu-, delta- and kappa-receptors. EGF-induced ERK activation is mediated by EGF receptor type 1 while opioid receptor activation seems to brings about stimulation via EGF receptor type.

Binding affinity to and dependence on some opioids in Sf9 insect cells expressing human mu-opioid receptor.

AIM: To investigate the receptor binding affinity and naloxone-precipitated cAMP overshoot of dihydroetorphine, fentanyl, heroin, and pethidine in Sf9 insect cells expressing human mu-opioid receptor (Sf9-mu cells). METHODS: Competitive binding assay of [3H]ohmefentanyl was used to reveal the affinity for mu-opioid receptor in Sf9-mu cells. [3H]cAMP RIA was used to determine cAMP level. Antinociceptive activity was evaluated using degree 55 mouse hot plate test. Naloxone-precipitated withdrawal jumping was used to reflect physical dependence in mice. RESULTS: All drugs displayed antinociceptive activity and produced physical dependence in mice. The K(i) values of dihydroetorphine, fentanyl, heroin, and pethidine in competitive binding assay were (0.85+/-0.20) nmol, (59.1+/-11.7) nmol, (0.36+/-0.13) micromol, and (12.2+/-3.8) micromol respectively. The binding affinities of these drugs for mu-opioid receptor in Sf9-mu cells were paralleled to their antinociceptive activities in mice. After chronic pretreatment with these drugs, naloxone induced cAMP withdrawal overshoot in Sf9-mu cells. The dependence index in Sf9-mu cells was calculated as K(i) value in competitive binding assay over EC(50) value in naloxone-precipitated cAMP assay. The physical dependence index in mice was calculated as antinociceptive ED(50)/withdrawal jumping cumulative ED(50). There was a good linear correlation between dependence index in Sf9-mu cells and physical dependence index in mice. CONCLUSION: The Sf9-mu cells could be used as a cell model to evaluate the receptor binding affinity and physical dependent liability of analgesic agents.