Recycling and resensitization of delta opioid receptors

Exposure to opioids results in the activation of opioid receptors; this is followed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrin-coated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist's ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the resensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes.     

Lateralization of opioid receptors and their putative ligands in the visual cortex of the turtle

Opioid mu-agonist morphine, delta-agonist D-Ala2,D-Leu5-enkephalin (DADL) and kappa-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N1). The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Morphine and bremazocine predominantly inhibited the left cortex EP, whereas DADL was a potent inhibitor of the right cortex EP. Thus opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to mu- and kappa-type while delta-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Application of LVC- and RVC-extracts to the cortex surface led to EP inhibition, which was partially (60-80%) prevented by antagonist naloxone. LVC-extract proved to be a more potent inhibitor of the left cortex EP, whereas RVC-extract was found to be more effective when applied to the right cortex. It is suggested that not only opioid receptors, but also their endogenous ligands are lateralized in turtle visual cortex.     

Site-directed affinity-labeling of delta opioid receptors by

The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [-Ala², Leu⁵]-enkephalyl-Cys(Npys)⁶ and [-Ala²,Leu(CH₂SNpys)⁵]enkephalin, and dynorphin A analogues of [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide and [-Ala²,Cys(Npys)⁸]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [-Ala²,Cys(Npys)¹²]dynorphin A-(1-13) amide.     

Monoclonal antiidiotypic antibodies against delta opioid receptors as an electron microscopy probe.

Four different rat monoclonal antibodies were produced against delta opioid receptor using an antiidiotypic approach in which antibodies directed against the opioid agonist DADLE were used as immunogen. In the first step, seven hybridomas were selected on the basis of their ability to inhibit the DADLE-anti-DADLE antibody interaction. After purification from ascitic fluids, these monoclonal antibodies were characterized. Four antiidiotypic antibodies, named 5, 11, 16, and 51, directed toward different epitopes, recognized the delta opioid receptor: (i) they bound directly to the NG108-15 cells, (ii) they inhibited the [3H]DADLE binding on the NG108-15 cells, (iii) they immunoprecipitated a 52,500 dalton protein present on the surface of the NG108-15 cells. The four monoclonal antiidiotypic anti-opioid receptor antibodies were used to immunocytologically detect the opioid receptors under light and electron microscopy in the rat spinal cord. The regional distribution of the immunoreactivity corresponded to layers known to be rich delta opioid receptor subtype. Moreover, at the ultrastructural level, the labeling was located mainly on plasma membranes, especially on non-synaptic zones. Our results show that monoclonal antiidiotypic antibodies constitute a valuable tool for visualizing cell surface receptors.     

Electrophysiological changes in heart failure and their implications for arrhythmogenesis

Heart failure is the final common pathway of various cardiac pathologies and is associated with sudden cardiac death, mostly caused by ventricular arrhythmias. In this paper we briefly review the electrophysiological remodeling and the alterations in intracellular calcium handling, and the resulting arrhythmogenic mechanisms associated with heart failure. Intercellular uncoupling and fibrosis are identified as a major arrhythmogenic factors. Diet and ventricular wall stretch are discussed as modulating factors. Finally, emphasis is placed on the hitherto poorly studied aspects of right ventricular failure. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

Biaryl piperidines as potent and selective delta opioid receptor ligands

The design and synthesis of novel opiates are reported. Based on the message-address principle a novel class of 4,4- and 3,3-biaryl piperidines was designed and synthesized. Biological evaluation confirmed that these compounds exhibit high affinity and selectivity for the delta opioid receptor. Key structure–activity relationships that influence affinity, selectivity, functional activity and clearance are reported.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

Benzylideneoxymorphone: A new lead for development of bifunctional mu/delta opioid receptor ligands

Opioid analgesic tolerance remains a considerable drawback to chronic pain management. The finding that concomitant administration of delta opioid receptor (DOR) antagonists attenuates the development of tolerance to mu opioid receptor (MOR) agonists has led to interest in producing bifunctional MOR agonist/DOR antagonist ligands. Herein, we present 7-benzylideneoxymorphone (6, UMB 246) displaying MOR partial agonist/DOR antagonist activity, representing a new lead for designing bifunctional MOR/DOR ligands.     

Detection of kappa and delta opioid receptors in skin--outside the nervous system

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.     

Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.     

ErbB receptors and their ligands in the breast

ErbB receptor tyrosine kinases are membrane-bound receptors that possess intrinsic, ligand-activated, tyrosine kinase activity. Binding of growth factors to these receptors induces the formation of ErbB homo- and heterodimers and initiates a signalling cascade that traverses the cytoplasm to communicate with the nucleus and the cytoskeleton. The effect of this cascade is the regulation of cellular proliferation, differentiation, apoptosis, migration and adhesion. Although ErbB signalling is important for normal growth and development in the breast, a dysregulation of ErbB activity can lead to tumourigenesis. This review will focus on the role of ErbB signalling in both normal mammary gland development and breast cancer, with an emphasis on the mechanisms behind receptor activation and the therapeutic agents designed to inhibit ErbB activity.     

Role for C-tail residues in delta opioid receptor downregulation

The delta opioid receptor, a member of the G-protein-coupled receptor superfamily, was used as a model system to characterize opioid receptor downregulation. Metabolic labeling followed by immunoprecipitation resulted in the isolation of the epitope-tagged mouse delta opioid receptor as a approximately 60-kDa protein. Prolonged agonist treatment with 100 nM d-Ala2, d-Leu5-enkephalin (DADLE) caused significant (approximately 60%) reduction in the level of receptor. The delta opioid receptor contains a number of phosphorylatable residues in the C tail. Point mutations of the majority of Ser/Thr sequences did not affect the level of downregulation, whereas mutation of Thr353 to Ala did. In order to test if phosphorylation at this site is involved in receptor downregulation, we generated a Thr353Glu mutant that would mimic the phosphorylated Thr at this site. This mutant exhibited a significantly higher extent of downregulation than the Thr353Ala mutant. In order to critically evaluate the requirement of Thr353 in receptor downregulation, we examined the downregulation of wildtype rat delta receptor (which does not contain Ala353) and an Ala353Thr point-mutant rat delta receptor. The wild-type receptor exhibited poor agonist-mediated downregulation, whereas Ala353Thr mutant exhibited increased downregulation. These results and results from additional studies with rat/mouse chimeric receptors support a role for phosphorylation of sites within the C tail in efficient downregulation of delta opioid receptors.     

Physiological analysis of delta opioid receptors in the guinea pig myenteric plexus

Ilea taken from guinea pigs that had been chronically exposed to morphine exhibit a greater tolerance to morphine and normorphine than to the opioid peptides D-ala2-D-leu5-enkephalin (DADLE) or D-met2-pro5-enkephalinamide (DMPE). This differential tolerance strongly implies the existence of at least two different types of opioid receptor in the guinea pig myenteric plexus or two different mechanisms of interaction between opioids and their receptor complex. Since DADLE is considered to be the prototypic ligand for the delta receptor, the above results imply the presence of delta receptors in the guinea pig myenteric plexus and furthermore, that this subtype of opioid receptor is associated with the modulation of release of enteric acetylcholine.     

Syntheses of novel high affinity ligands for opioid receptors

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring ‘open’ derivatives display very high affinity for μ and κ receptors and much less affinity for δ. The observation that these target compounds have much higher receptor affinity than the corresponding ring ‘closed’ carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.     

Opioid receptors and their selective ligands

Opioid receptors (micro, delta, and kappa) belong to a large family of G protein-coupled receptors and play an important physiological role. Stimulation of these receptors triggers analgesic effects and affects the function of gastrointestinal tract. The discovery of opioid peptides, which are endogenous ligands of opioid receptors, including delta-selective enkephalins, kappa-selective dynorphins, and micro-selective endomorphins, initiated their structure-activity relationship studies. For the last 30 years, hundreds of analogs of opioid peptides have been synthesized in an effort to obtain the compounds more active, selective, and resistant to biodegradation than the endogenous ligands. Different unnatural amino acids, as well as cyclisation procedures, leading to conformationaly restricted analogs, were employed. All these modifications resulted in obtaining very selective agonists and antagonists with high affinity at micro-, dlta-, and kappa-opioid receptors, which are extremely useful tools in further studies on the pharmacology of opioid receptors in a mammalian organism.     

Activation of delta opioid receptors induces receptor insertion and neuropeptide secretion

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca(2+) release from IP(3)-sensitive stores and Ca(2+) entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y(1) receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca(2+)-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.     

Cannabinoid receptors and their endogenous ligands

Delta9-Tetrahydrocannabinol, a major psychoactive component of marijuana, has been shown to interact with specific cannabinoid receptors, thereby eliciting a variety of pharmacological responses in experimental animals and human. In 1990, the gene encoding a cannabinoid receptor (CB1) was cloned. This prompted the search for endogenous ligands. In 1992, N-arachidonoylethanolamine (anandamide) was isolated from pig brain as an endogenous ligand, and in 1995, 2-arachidonoylglycerol was isolated from rat brain and canine gut as another endogenous ligand. Both anandamide and 2-arachidonoylglycerol exhibit various cannabimimetic activities. The results of structure-activity relationship experiments, however, revealed that 2-arachidonoylglycerol, but not anandamide, is the intrinsic natural ligand for the cannabinoid receptor. 2-Arachidonoylglycerol is a degradation product of inositol phospholipids that links the function of cannabinoid receptors with the enhanced inositol phospholipid turnover in stimulated tissues and cells. The possible physiological roles of cannabinoid receptors and 2-arachidonoylglycerol in various mammalian tissues such as those of the nervous system are discussed.