MicroRNAs and extracellular vesicles in cholangiopathies.

Cholangiopathies encompass a heterogeneous group of disorders affecting biliary epithelial cells (i.e. cholangiocytes). Early diagnosis, prognosis and treatment still remain clinically challenging for most of these diseases and are critical for adequate patient care. In the past decade, extensive research has emphasized microRNAs (miRs) as potential non-invasive biomarkers and tools to accurately identify, predict and treat cholangiopathies. MiRs can be released extracellularly conjugated with lipoproteins or encapsulated in extracellular vesicles (EVs). Research on EVs is also gaining attention since they are present in multiple biological fluids and may represent a relevant source of novel non-invasive biomarkers and be vehicles for new therapeutic approaches. This review highlights the most promising candidate miRs and EV-related biomarkers in cholangiopathies, as well as their relevant roles in biliary pathophysiology. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen. RESEARCH STRATEGY: PubMed search (April 2017) was done with the following terms: "microRNA", "miRNA", "miR", "extracellular vesicles", "EV", "exosomes", "primary biliary cholangitis", "primary biliary cholangitis", "PBC", "primary sclerosing cholangitis", "PSC", "cholangiocarcinoma", "CCA", "biliary atresia", "BA", "polycystic liver diseases", "PLD", "cholangiopathies", "cholestatic liver disease". Most significant articles in full-text English were selected. The reference lists of selected papers were also considered.

New Advances in Polycystic Liver Diseases.

Polycystic liver diseases (PLDs) include a heterogeneous group of congenital disorders inherited as dominant or recessive genetic traits; they are manifested alone or in association with polycystic kidney disease. Ductal plate malformation during embryogenesis and the loss of heterozygosity linked to second-hit mutations may promote the dilatation and/or development of a large number (> 20) of biliary cysts, which are the main cause of morbidity in these patients. Surgical procedures aimed to eliminate symptomatic cysts show short-term beneficial effects, but are not able to block the disease progression. Therefore, liver transplantation is the only curative option. Intense studies on the molecular mechanisms involved in the pathogenesis of PLDs have resulted in different clinical trials, some of them with promising outcomes. Here the authors summarize the key aspects of PLD etiology, pathogenesis, and therapy, highlighting the most recent advances and future research directions.

Dissemination of plasmid-mediated fosfomycin resistance fosA3 among multidrug-resistant Escherichia coli from livestock and other animals.

AIMS: To investigate plasmid-mediated fosfomycin resistance related to fosA3 in Escherichia coli isolates collected from different animals in Hong Kong, China, 2008-2010. METHODS AND RESULTS: In total, 2106 faecal specimens from 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents were cultured. The faecal colonization rates of fosfomycin-resistant E. coli were as follows: 11.2% in pigs, 8.6% in cattle, 7.3% in chickens, 2.4% in dogs, 0.8% in cats and 1.5% in rodents. The cultures yielded 1693 isolates of which 831 were extended-spectrum ß-lactamases (ESBL) producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be producers of ESBL and to have resistance to chloramphenicol, ciprofloxacin, cotrimoxazole, gentamicin and tetracycline. Of the 101 fosfomycin-resistant isolates, 97 (96.0%) isolates were fosA3 positive and 94 (93.1%) were bla(CTX) (-M) positive. PCR mapping showed that the fosA3-containing regions were flanked by IS26, both upstream and downstream in 81 (83.5%) isolates, and by an upstream bla(CTX-M-14) -containing transposon-like structure (ΔISEcp1-bla(CTX-M-14) -ΔIS903 or ISEcp1-IS10 -bla(CTX-M-14) -ΔIS903) and a downstream IS26 in 14 (14.4%) isolates. For the remaining two isolates, fosA3 was flanked by a downstream IS26 but the upstream part cannot be defined. In a random subset of 18 isolates, fosA3 was carried on transferable plasmids with sizes of 50-200 kb and the following replicons: F2:A-B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), N (n = 1), B/O (n = 1) and untypeable (n = 3). SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the emergence of fosA3-mediated fosfomycin resistance among multidrug-resistant E. coli isolates from various animals. IS26 transposon-like structures might be the main vehicles for dissemination of fosA3.

Intrathecal delivery of a mutant micro-opioid receptor activated by naloxone as a possible antinociceptive paradigm.

Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.

dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.

We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.

Inhibition of akt/protein kinase B signaling by naltrindole in small cell lung cancer cells.

The phosphatidylinositol 3-kinase-Akt/protein kinase B (PKB) survival signaling is very important for cancer cell survival and growth. Constitutively active phosphatidylinositol 3-kinase-Akt/PKB signaling in small cell lung cancer (SCLC) is a major factor for the survival of SCLC cells. Inhibitors of this signaling pathway would be potential antitumor agents, particularly for SCLC. Here we report that naltrindole, which has been used as a classic delta opioid antagonist, inhibited growth and induced apoptosis in the three characteristic SCLC cell lines, NCI-H69, NCI-H345, and NCI-H510. Naltrindole treatment reduced constitutive phosphorylation of Akt/PKB on serine 473 and threonine 308 in cells. We found that the levels of constitutive phosphorylation of Akt/PKB on serine 473 correlate with the sensitivity of the three cell lines to naltrindole treatment. Furthermore, naltrindole treatment not only reduced the phosphorylation of the Akt/PKB upstream kinase phosphoinositide-dependent kinase-1, but also its downstream effectors glycogen synthase kinase-3beta and the Forkhead transcription factors AFX and FKHR. DNA array analysis of 205 apoptosis-related genes indicated that some Akt/PKB-dependent genes were either up- or down-regulated by naltrindole. Flow cytometric and microscopic analyses clearly showed that naltrindole induced apoptosis in SCLC cells. RNA interference experiments confirmed that naltrindole-induced cell death was associated with the Akt/PKB survival pathway. Together, these results show that naltrindole is a new inhibitor of the Akt/PKB signaling pathway, suggesting that naltrindole could be a potential lead for the development of a new type of inhibitors that target the constitutively active Akt/PKB signaling-dependent SCLC cells.

A Mechanistic Approach to the Development of Gene Therapy for Chronic Pain.

Treatment of chronic pain has created a "silent epidemic," a term that describes the serious public health problem of the abuse of opioid painkillers and other prescription drugs. Conventional pharmacotherapy is limited by the loss of effectiveness in the long-term and by potentially lethal side effects. Efforts need to be focused on the development of nonpharmacological approaches. As significant progress is made in the viral vector technology, gene therapy involving recombinant viruses as vehicles may become a viable alternative for treatment of severe pain. Virus-based gene therapy has several advantages: (1) the transfer of a therapeutic gene to produce/release bioactive therapeutic molecules in a specific location in the nervous system thus minimizing the risks of off-target side effects, and (2) sustained long-term production of the therapeutic agent. This review compiles recently developed strategies for gene therapy targeting specific mechanisms of specific chronic pain conditions. A few successful studies on animal models of chronic pain have been translated to human clinical trials.

The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor.

We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor.

Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence.

Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.

Effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells.

The molecular mechanism of opiate receptor down-regulation and desensitization was investigated by studying the effects of cycloheximide and tunicamycin on opiate receptor activities in neuroblastoma X glioma NG108-15 hybrid cells. Cycloheximide inhibited [35S]methionine and [3H]-glucosamine incorporation by hybrid cells, while tunicamycin inhibited [3H]glucosamine incorporation only. Exposing hybrid cells to these two agents did not alter the viability of the cell. Treatment of NG108-15 cells with cycloheximide or tunicamycin produced a decrease in [3H]diprenorphine binding dependent on both time and concentrations of inhibitors, with no measurable modification in the ability of etorphine to regulate intracellular cyclic AMP production. Cycloheximide attenuated [3H]-diprenorphine binding by decreasing both the number of sites, Bmax, and the affinity of the receptor, Kd. Tunicamycin treatment produced a decrease in Bmax with no apparent alteration in Kd values. Cycloheximide and tunicamycin did not potentiate the rate or magnitude of etorphine-induced down-regulation or desensitization of opiate receptor in NG108-15 cells. Furthermore, there was an apparent antagonism in cycloheximide action on receptor down-regulation. The reappearance of opiate binding sites after agonist removal was affected by these two inhibitors. Cycloheximide and tunicamycin eliminated the increase in [3H]diprenorphine binding in the chronic etorphine-treated cells after agonist removal. These two inhibitors did not alter the resensitization of hybrid cells to etorphine. Thus, the site of opiate agonist action to induce receptor down-regulation and desensitization is not at the site of protein synthesis or protein glycosylation. These data substantiate previously reported observations that receptor down-regulation and receptor desensitization are two different cellular adaptation processes.

Mobilization of Ca2+ from intracellular stores in transfected neuro2a cells by activation of multiple opioid receptor subtypes.

In neuronal cell lines, activation of opioid receptors has been shown to mobilize intracellular Ca2+ stores. In this report, we describe the excitatory actions of opioid agonists on murine neuroblastoma neuro2a cells stably expressing either delta, mu, or kappa opioid receptors. Fura-2-based digital imaging was used to record opioid-induced increases in intracellular Ca2+ concentration ([Ca2+]i). Repeated challenges of delta, mu, or kappa opioid receptor expressing cells with 100 nM [D-Ala2,D-Leu5]-enkephalin (DADLE), [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO), or trans-(+/-)-3,4-dichloro N-methyl-N-(2-[1-pyrollidinyl] cyclohexyl) benzene acetamide (U-50488H), respectively, elicited reproducible Ca2+ responses. Non-transfected neuro2a cells did not respond to opioid agonists. Removal of extracellular Ca2+ from the bath prior to and during agonist challenge did not affect significantly the agonist-evoked increase in [Ca2+]i, indicating that the response resulted from the release of Ca2+ from intracellular stores. Naloxone reversibly inhibited responses in all three cell lines, confirming that they were mediated by opioid receptors. Expression of cloned opioid receptors in neuro2a cells, coupled with digital [Ca2+]i imaging, provides a model system for the study of opioid receptors and opioid-activated signaling processes. The fact that all three receptors coupled to the same intracellular signaling mechanism suggests that the primary functional difference between opioid responses in vivo results from their selective localization.

Neuroblastoma Neuro2A cells stably expressing a cloned mu-opioid receptor: a specific cellular model to study acute and chronic effects of morphine.

Several cellular systems display desensitization and downregulation of opioid receptors upon chronic treatment, suggesting that they could be used as a model system to understand opioid tolerance/dependence. However, a model system containing a homogeneous population of mu-opioid receptors, the receptors at which morphine and related opioids act, has been lacking. To approach this problem, the mu-opioid receptor (MOR-1) was stably expressed in murine neuroblastoma Neuro2A cells after transfection. The expressed receptor was negatively coupled to adenylyl cyclase through Gi/Go proteins, displayed high affinity ligand binding, and was expressed in high number (2.06 pmol/mg of [3H]diprenorphine binding sites). In addition, loss of ability of mu-opioids to acutely inhibit forskolin-stimulated cAMP formation was observed after 4-24 h of chronic exposure to these agonists with concentrations as low as 300-500 nM. The effects of chronic morphine or [D-Ala2,N-MePhe4,Gly-ol]enkephalin (DAMGO) administration were found to be time- and concentration-dependent. Cross 'tolerance' was also observed. Thus the IC50 value of DAMGO to inhibit adenylyl cyclase was increased by 27-fold from 4.3 nM in control cells to 117 nM in cells pretreated with 300 nM morphine; there was no effect on the inhibition of adenylyl cyclase mediated by muscarinic receptors. Further, receptor downregulation accompanied the desensitization process. However, different time-dependence for these two processes suggests, in line with other studies, that these are entirely different cellular adaptation processes. In addition, the opioid antagonist naloxone induced an acute increase in intracellular cAMP level (2-3 times above the control level) following chronic agonist exposure. This process was also concentration-dependent. Overall, these results suggest that the cell line utilized in this study has a homogeneous population of mu-opioid receptors, providing an ideal cellular model to study the molecular mechanisms underlying chronic morphine treatment.

mu-opioid receptor regulates CFTR coexpressed in Xenopus oocytes in a cAMP independent manner.

The objective of this study was to characterize the signaling mechanisms of the mu-opioid receptor in its coupling to the cystic fibrosis transmembrane conductance regulator (CFTR) when coexpressed in Xenopus oocytes. Because oocytes do not contain endogenous cAMP-regulated ion channels, the cAMP-modulated CFTR was coexpressed with receptors as a 'reporter' channel. Agonist treatment of oocytes coexpressing mu-opioid receptors, beta2-adrenergic receptors and CFTR produced Cl- currents in a dose-related manner and immunocytochemical analysis confirmed receptor expression. These data suggest that opioid agonists could activate adenylyl cyclase in this system to elevate cAMP levels. Heterotrimeric G protein betagamma-subunits acting on adenylyl cyclase type II would increase cAMP levels. The probable presence of adenylyl cyclase type II and other components of opioid signal transduction such as G(i alpha2), were demonstrated by RT-PCR. However, measurement of cAMP levels in individual oocytes by radioimmunoassay showed that opioid agonist application to oocytes expressing mu-opioid receptors, beta2-adrenergic receptors and CFTR did not increase cAMP levels, whereas application of the beta2-adrenergic agonist, isoproterenol, or IBMX alone did increase cAMP levels. Opioid-induced CFTR activation was not affected by either application of the broad spectrum kinase inhibitor, H7, nor by application of the specific PKA inhibitor, KT5720. Injection of free betagamma-subunits, which could activate the endogenous type II cyclase, was unable to produce measurable currents in oocytes expressing the CFTR. These studies indicate that opioid activation of the CFTR is not mediated through a cAMP/PKA pathway, by either betagamma-subunit activation of an adenylyl cyclase type II or promiscuous coupling to G(s alpha).

Functional expression of adrenergic and opioid receptors in Xenopus oocytes: interaction between alpha 2- and beta 2-adrenergic receptors.

We functionally expressed alpha 2-adrenergic, beta 2-adrenergic, and delta-opioid receptors in Xenopus laevis oocytes. We detected receptor function as changes in currents carried by adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride channels provided by the cystic fibrosis transmembrane conductance regulator (CFTR) and recorded by two-electrode voltage clamp. Co-application of forskolin and isobutylmethylxanthine (IBMX) or IBMX alone produced currents with a reversal potential indicative of chloride ions only in oocytes previously injected with mRNA encoding CFTR. Isoproterenol produced concentration-dependent responses in oocytes injected with mRNA encoding beta 2-adrenergic receptors and CFTR, and co-administration of propranolol antagonized these responses. Similarly, the alpha 2-adrenergic agonist UK14304 increased IBMX-induced currents only in oocytes injected with mRNA encoding alpha 2-adrenergic receptors and CFTR, and idazoxan antagonized these enhancements. The delta-opioid agonist DADLE produced concentration-related, naloxone-reversible increases in IBMX- and forskolin-induced currents only in oocytes injected with mRNA encoding delta-opioid receptors and CFTR. In oocytes co-injected with alpha 2, beta 2, and CFTR mRNAs, isobolographic analysis revealed an additive interaction between alpha 2- and beta 2-adrenergic receptors. These studies establish the oocyte as a cell system for studying the interactions among cAMP-modulating G protein-coupled receptors and provide another example of alternative coupling of alpha 2-adrenergic and delta-opioid receptors to G proteins, possibly Gs proteins, other than Gi proteins.

Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned mu, delta, and kappa opioid receptors.

A rat brain cDNA library was screened for clones homologous to the recently cloned mouse delta-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57-58% identical to the amino acid sequences of the cloned delta, mu and kappa opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.

Role of carboxyl terminus of mu-and delta-opioid receptor in agonist-induced down-regulation.

Chronic exposure of mu-and delta-opioid receptors to their agonists leads to different rates in receptor down-regulation. In order to analyze the role of the carboxyl terminus of mu-and delta-opioid receptors in the difference in the rate of down-regulation, two chimeras of these receptors were generated by swapping the carboxyl termini; MORTAGDT and DORTAGMT. These chimeras were tagged at the N-terminus with hemagglutinin (HA) epitope (YPYDVPDYA), which can be recognized by the monoclonal antibody 12CA5, and then stably expressed in Neuro 2A (N2A) cells. The swapping of the carboxyl termini did not alter the ligand selectivity of these receptor chimeras. However, they did exhibit a reduction in agonist potency to inhibit forskolin-stimulated adenylyl cyclase activity for all agonists tested except etorphine which had a potency comparable to that of wild type receptors. Treatment of the N2A cells expressing MORTAGDT with 50 nM etorphine produced a faster rate of receptor down-regulation when compared to the wild type mu-opioid receptor. Immunofluorescence microscopy of the MORTAGDT chimera using a monoclonal antibody against HA confirmed internalization of the receptors after treatment with etorphine for 1 and 6h. There was a reduction in the HA-immunoreactivity at the cell surface of the MORTAGDT chimera concurrent with more noticeable HA-immunoreactivity inside the cell compared to the wild type receptor. On the other hand, the rate of down-regulation of DORTAGMT receptors was seen to be the same as the wild type delta-opioid receptor after etorphine treatment. Immunofluorescence studies showed more reduction in cell surface staining of the DORTAGMT chimera compared to the wild type receptor. These data suggest the involvement of the carboxyl terminus in agonist-induced down-regulation and internalization of the nu-opioid receptor. However, different mechanisms that are unrelated to the carboxyl terminus may operate in the down-regulation of delta-opioid receptor.

Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure.

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual G alpha subunit and is similar to that of other opioid receptors.

The purpose of the present study was to examine the coupling pattern of a recently cloned kappa-opioid receptor stably transfected in CHO cells to individual G alpha subunits with subsequent comparison to that observed previously for delta- and mu-opioid receptors. Data presented in the current study indicate the successful stable expression of a kappa-opioid receptor in CHO cells. This is supported by experiments in which ligands with selectivity for kappa-, but not delta- or mu-opioid receptors demonstrated high affinity for the expressed receptor and were able to potently and efficaciously produce inhibition of adenylyl cyclase activity. In addition, only kappa-opioid agonists were able to induce dose-dependent increases in the incorporation of [32P]azidoanilido-GTP into four G alpha subunits, three of which were identified as Gi3 alpha, Gi2 alpha and Go2 alpha. Further, the amount of kappa-opioid agonists required to induce 50% maximal labeling of any individual G alpha subunit was similar. Although kappa-opioid agonists produced equivalent maximal labelling of Gi3 alpha, Gi2 alpha and Go2 alpha, significantly less agonist-induced labeling was observed for an unknown G-protein designated as G? alpha. Although these results are slightly different than those observed previously for both delta- and mu-opioid receptors, it appears that all opioid receptors stably transfected in CHO cells interact with multiple G-proteins and that this coupling is not selective for any individual G alpha subunit.

Immunofluorescent identification of a delta (delta)-opioid receptor on primary afferent nerve terminals.

Antisera were produced against synthetic peptides predicted from the recent cloning of a delta opioid receptor, DOR-1. Confocal microscopic examination of immunostained spinal cord sections revealed that DOR-1 immunoreactive (-ir) nerve fibers and terminals form a moderately dense plexus within the superficial dorsal horn of rats and mice. These fibers decreased dramatically following dorsal rhizotomy and consistent with these observations a population of small diameter neurons in ganglia exhibited DOR-1-ir. DOR-1-ir ganglion neurons were also immunoreactive for calcitonin gene-related peptide (CGRP), and their terminals in the spinal cord contained both CGRP- and DOR-1-ir, the latter presumably located as a 'presynaptic' receptor. Interestingly, terminals containing DOR-1-ir appeared to be closely apposed by fibers and terminals containing enkephalin (ENK)-ir, which suggests that ENK may be a physiologically relevant ligand for the receptor encoded by DOR-1, and that DOR-1 may act to regulate the release of transmitters from small diameter primary afferent neurons.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity.

Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions.