Characterization of opioid receptors on isolated canine gallbladder smooth muscle cells

Smooth muscle cells were isolated from the fundus of the canine gallbladder and examined for the presence of opioid receptors. The cells contracted in a concentration-dependent manner in response to three opioid peptides (Met-enkephalin, dynorphin1-13 and Leu-enkephalin), which are known derivatives of opioid precursors present in myenteric neurons of the gut. The order of potency was Met-enkephalin greater than dynorphin1-13 greater than Leu-enkephalin. The contractile response to opioid agonists was selectively inhibited by opioid antagonists (naloxone and Mr2266) but not by muscarinic, CCK/gastrin or tachykinin antagonists. Equivalent responses to the three opioid peptides exhibited differential sensitivity to preferential antagonists of mu (naloxone) and kappa (Mr2266) opioid receptors consistent with the presence of the three main types of opioid receptors (mu, delta and kappa) on canine gallbladder muscle cells.     

Anticonvulsant effects of mu (DAGO) and delta (DPDPE) enkephalins in rats

The effects of highly selective mu and delta opioid peptide agonists were determined in two rat models of experimentally-induced convulsions, the flurothyl threshold test and the maximal electroshock test. Intracerebroventricular injections of the mu selective enkephalin DAGO (0.3-2.2 nmol) resulted in a dose-related protection in both seizure models. Pretreatment with a low dose of naloxone (29 nmol) or the irreversible mu antagonist beta-FNA (21 nmol), but not the delta opioid antagonist ICI 154,129 (50 nmol), antagonized the anticonvulsant actions of DAGO. Intracerebroventricular injections of the delta selective enkephalin DPDPE (70-140 nmol) also resulted in seizure protection. These effects were selectively antagonized by the delta antagonist ICI 174,864 (2.8 nmol), but not by pretreatment with beta-FNA. Thus, using agonists and antagonists highly selective for mu and delta opioid receptors, anticonvulsant actions of enkephalin have been described against chemically- and electrically-induced convulsions in rats.     

Opioids regulate the release of human chorionic gonadotropin hormone from trophoblast tissue.

Opioid ligands were investigated for their effect on hCG release from trophoblast tissue obtained from term human placenta. Data obtained indicate that opiate agonists stimulate in vitro basal hCG release from trophoblast tissue. The potency of these opioid agonists correspond to their kappa receptor selectivity, i.e., the greater the selectivity the lower is the effective concentration causing maximum stimulation. Opioid antagonists inhibit the release of hCG due to their reversal of the stimulation caused by endogenous opioid peptides. Potency of the antagonists correspond also to their kappa receptor selectivity. Antagonists reverse the stimulation of hCG release caused by agonists indicating that the ligand's action is mediated by the placental kappa opioid receptors. The bell shaped response curves for agonists and antagonists suggest that opioids play a role in the regulation of hCG release from trophoblast tissue, but other mechanism(s) may also exist.     

Dimer-based model for heptaspanning membrane receptors

The existence of intramembrane receptor-receptor interactions for heptaspanning membrane receptors is now fully accepted, but a model considering dimers as the basic unit that binds to two ligand molecules is lacking. Here, we propose a two-state-dimer model in which the ligand-induced conformational changes from one component of the dimer are communicated to the other. Our model predicts cooperativity in binding, which is relevant because the other current models fail to address this phenomenon satisfactorily. Our two-state-dimer model also predicts the variety of responses elicited by full or partial agonists, neutral antagonists and inverse agonists. This model can aid our understanding of the operation of heptaspanning receptors and receptor channels, and, potentially, be important for improving the treatment of cardiovascular, neurological and neuropsychyatric diseases.     

Dimerization of morphine and orphanin FQ/nociceptin receptors: generation of a novel opioid receptor subtype

Although orphanin FQ/nociceptin (OFQ/N) receptors are a member of the opioid receptor family of receptors, they bind traditional opioids with very poor affinity. We now demonstrate that mu opioid receptors can physically associate with OFQ/N receptors, resulting in a complex with a unique binding selectivity profile. Immunoprecipitation of epitope-tagged OFQ/N receptors co-precipitates mu receptors. When the two receptors were co-expressed in CHO cells, [3H]OFQ/N retained its high binding affinity for its receptor. However, co-expression of the two receptors increased by up to 250-fold the affinity of a series of opioids in [3H]OFQ/N binding assays. This enhanced affinity was limited to agonists with high affinity for mu receptors. Selective kappa(1) and delta opioids did not lower binding. Despite the dramatic increase in affinity for the opioid agonists in co-expressing cells, the opioid antagonists naloxone and diprenorphine failed to compete [3H]OFQ/N binding.     

Stereospecific opioid drug receptors on excitable cell membranes

In the past decade evidence has accumulated showing that there are stereospecific opioid drug receptors located on the surface membranes of neurones and skeletal muscle fibres which can block action potentials when opioid drugs are applied to the cells. The nature of this evidence and the difficulties involved in this area of investigation are discussed in detail. Two different mechanisms have been described for this depression of excitability; one, a direct blocking action on sodium channels (or the calcium channels in the case of cells with calcium action potentials) and the other, a stimulation of membrane potassium conductance which results in hyperpolarization and increased membrane conductance both of which decrease excitability. For the most part these different mechanisms seem to prevail in different cell types as will be discussed. Some other points discussed are as follows: first, in higher doses (or concentrations) opioids produce a local anestheticlike effect which cannot be antagonized by opioid antagonists; next, the opioid antagonists which are easily easily available and commonly employed change from antagonists to agonists as their concentration is increased; and finally, the evidence required for the demonstration of stereospecific opioid receptors is considered in some detail.     

Modeling and simulation of the human delta opioid receptor

A model for the human delta opioid receptor has been generated via sequence alignment, structure building using the crystal structure of bovine rhodopsin as a template, and refinement by molecular dynamics simulation. The model building suggested that, in addition to the previously postulated interaction between D128 and Y308, an internal salt bridge also exists between residues D128 and R192, both of which are conserved in all the opioid receptors. The model and salt bridge were then shown to be stable during a 20-nsec simulation in a lipid bilayer. It is therefore proposed that both of these interactions play a role in stabilizing the inactive state of the receptor. The model is also used in an effort to rationalize many of the mutational studies performed on delta opioid receptors, and to suggest a plausible explanation for the differences between known delta opioid agonists and antagonists.     

Pyrrolo- and pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse.     

A single mutation in the 5-HT4 receptor (5-HT4-R D100(3.32)A) generates a Gs-coupled receptor activated exclusively by synthetic ligands (RASSL)

To better understand G-protein-coupled receptor (GPCRs) signaling, cellular and animal physiology, as well as gene therapy, a new tool has recently been proposed. It consists of GPCR mutants that are insensitive to endogenous ligands but sensitive to synthetic ligands. These GPCRs are called receptor activated solely by synthetic ligands (RASSL). Only two examples of such engineered receptors have been described so far: one G(i)-coupled (opioid receptors) and one G(s)-coupled (beta(2)-adrenergic receptors). Here, we describe the first RASSL related to serotonin receptors (D100(3.32)A G(s)-coupled 5-HT(4) receptor or 5-HT(4)-RASSL). 5-HT(4)-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still responds to synthetic ligands. These ligands have affinities in the range of nanomolar concentrations for the mutant receptor and exhibit full efficacy. More interestingly, two synthetic ligands behave as antagonists on the wild type but as agonists on the 5-HT(4)-RASSL.     

Central structures involved in opioid-induced feeding

This paper summarizes efforts to identify structures involved in the opioid regulation of feeding. Many opioid agonists and antagonists increase or decrease food intake when injected centrally, which suggests, but alone does not prove, that the opioid feeding system is located within the brain. Some conditions of hunger and feeding cause changes in opioid peptide levels in certain brain areas, notably the hypothalamus, which may indicate that the areas are components of this opioid system. Lesion studies have also identified some potentially important structures, inasmuch as lesions of these structures reduce the effectiveness of opioid agonists or antagonists to alter food intake. Finally, microinjection studies have mapped the brain in terms of the effects on feeding of opioid agonists and antagonists. Results of different types of studies are consistent in suggesting that parts of the hypothalamus, particularly the paraventricular and ventromedial nuclei and the lateral hypothalamic area, are important components of the opioid feeding system.     

Imaging mass spectrometry reveals elevated nigral levels of dynorphin neuropeptides in L-DOPA-induced dyskinesia in rat model of Parkinson's disease

L-DOPA-induced dyskinesia is a troublesome complication of L-DOPA pharmacotherapy of Parkinson's disease and has been associated with disturbed brain opioid transmission. However, so far the results of clinical and preclinical studies on the effects of opioids agonists and antagonists have been contradictory at best. Prodynorphin mRNA levels correlate well with the severity of dyskinesia in animal models of Parkinson's disease; however the identities of the actual neuroactive opioid effectors in their target basal ganglia output structures have not yet been determined. For the first time MALDI-TOF imaging mass spectrometry (IMS) was used for unbiased assessment and topographical elucidation of prodynorphin-derived peptides in the substantia nigra of a unilateral rat model of Parkinson's disease and L-DOPA induced dyskinesia. Nigral levels of dynorphin B and alpha-neoendorphin strongly correlated with the severity of dyskinesia. Even if dynorphin peptide levels were elevated in both the medial and lateral part of the substantia nigra, MALDI IMS analysis revealed that the most prominent changes were localized to the lateral part of the substantia nigra. MALDI IMS is advantageous compared with traditional molecular methods, such as radioimmunoassay, in that neither the molecular identity analyzed, nor the specific localization needs to be predetermined. Indeed, MALDI IMS revealed that the bioconverted metabolite leu-enkephalin-arg also correlated positively with severity of dyskinesia. Multiplexing DynB and leu-enkephalin-arg ion images revealed small (0.25 by 0.5 mm) nigral subregions with complementing ion intensities, indicating localized peptide release followed by bioconversion. The nigral dynorphins associated with L-DOPA-induced dyskinesia were not those with high affinity to kappa opioid receptors, but consisted of shorter peptides, mainly dynorphin B and alpha-neoendorphin that are known to bind and activate mu and delta opioid receptors. This suggests that mu and/or delta subtype-selective opioid receptor antagonists may be clinically relevant for reducing L-DOPA-induced dyskinesia in Parkinson's disease.     

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.     

Supraspinal circuitry mediating opioid antinociception: Antagonist and synergy studies in multiple sites

Supraspinal opioid antinociception is mediated by sensitive brain sites capable of supporting this response following microinjection of opioid agonists. These sites include the ventrolateral periaqueductal gray (vIPAG), the rostral ventromedial medulla (RVM), the locus coeruleus and the amygdala. Each of these sites comprise an interconnected anatomical and physiologically relevant system mediating antinociceptive responses through regional interactions. Such interactions have been identified using two pharmacological approaches: (1) the ability of selective antagonists delivered to one site to block antinociception elicited by opioid agonists in a second site, and (2) the presence of synergistic antinociceptive interactions following simultaneous administration of subthreshold doses of opioid agonists into pairs of sites. Thus, the RVM has essential serotonergic, opioid, cholinergic and NMDA synapses that are necessary for the full expression of morphine antinociception elicited from the vIPAG, and the vIPAG has essential opioid synapses that are necessary for the full expression of opioid antinociception elicited from the amygdala. Further, the vIPAG, RVM, locus coeruleus and amygdala interact with each other in synergistically supporting opioid antinociception.     

kappa-opioids induce a reversible inhibition of CFU-GM from CD133(+) cord blood cells

BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined.     

Differential activation and blockade of excitatory amino acid receptors in the mammalian and amphibian central nervous systems

1. Experiments were conducted in vitro on isolated spinal cords of frogs and immature rats and in vivo on cat spinal neurones. 2. The concept of two major types of excitatory amino acid receptors present in these preparations is summarized, one type (NMDA receptors) being activated specifically by N-methyl-D-aspartate (NMDA) and blocked by specific antagonists such as D(-)-2-amino-5-phosphonovalerate (APV), and a second type (non-NMDA receptors) characterized by insensitivity to specific NMDA antagonists. This second type may be comprised of two sub-types activated selectively by the agonists quisqualate and kainate. The putative transmitters L-glutamate and L-aspartate have mixed action on both NMDA and non-NMDA receptors. The major action of both transmitter candidates is considered to be on non-NMDA receptors, but the proportion of the composite responses mediated by NMDA receptors (at least for spinal neurones) appears to be greater for L-aspartate than for L-glutamate. 3. The preference of NMDA and non-NMDA receptors for a range of agonists is discussed. Some newer agonists are considered, in addition to several known agonists not previously discussed in terms of NMDA- and non-NMDA-receptor preference. Structure-activity relations of agonists are discussed. 4. The actions of some new amino acid antagonists are reported. Some of these have useful kainate and quisqualate blocking activity, in addition to their ability to block NMDA induced responses. 5. Evidence is presented suggesting that excitatory amino acid receptors are involved in both polysynaptic and monosynaptic excitation in the spinal cord, NMDA receptors mediating polysynaptic excitation and non-NMDA receptors monosynaptic excitation. 6. The unusual effect is reported of L-2-amino-4-phosphonobutyrate, which potently blocks spinal synaptic excitation in the absence of depressant action on excitatory amino acid-induced responses.     

Supportive or detrimental roles of P2Y receptors in brain pathology?—The two faces of P2Y receptors in stroke and neurodegeneration detected in neural cell and in animal model studies

This review describing the role of P2Y receptors in neuropathological conditions focuses on obvious differences between results demonstrating either a role in neuroprotection or in neurodegeneration, depending on in vitro and in vivo models. Such critical juxtaposition puts special emphasis on discussions of beneficial and detrimental effects of P2Y receptor agonists and antagonists in these models. The mechanisms reported to underlie the protection in vitro include increased expression of oxidoreductase genes, like carbonyl reductase and thioredoxin reductase; increased expression of inhibitor of apoptosis protein-2; extracellular signal-regulated kinase- and Akt-mediated antiapoptotic signaling; increased expression of Bcl-2 proteins, neurotrophins, neuropeptides, and growth factors; decreased Bax expression; non-amyloidogenic APP shedding; and increased neurite outgrowth in neuronal cells. Animal studies investigating the influence of P2Y receptors in middle cerebral artery occlusion (MCAO) models for stroke prove beneficial effects of P2Y receptor antagonists. In MCAO mice and rats, the application of broad-range P2 receptor antagonists decreased the infarct volume and improved neurological outcome. Moreover, antagonists of the P2Y₁ receptor, one of the most abundant P2Y receptor subtypes in brain tissue, decreased neuronal loss and improved spatial memory in rats after traumatic brain injury (TBI). Currently available data show a discrepancy between in vitro and in vivo models concerning the benefits of P2Y receptor activation in pathological conditions. In vitro models demonstrate protection by P2Y receptor agonists, but in vivo P2Y receptor activation deteriorates the outcome after MCAO and controlled cortical impact brain injury, a TBI model. To broaden the scope of the review, we additionally discuss publications that demonstrate detrimental effects of P2Y receptor agonists in vitro and publications showing protective effects of agonists in vivo. All these studies help to better understand the significant role of P2Y receptors especially in stroke models and to develop pharmacological strategies for the treatment of stroke.     

Pharmacologic methods for identification of receptors

Determinations of apparent equilibrium dissociation constants of drug-receptor interactions are made from both functional and radioligand binding studies. In each type of study, reversible reactions are assumed and the mass action law is applied. Functional studies are frequently used to determine the dissociation constant of a competitive antagonist but are less frequently used to obtain this constant for agonist compounds since the latter determination requires an experimental procedure that irreversibly inactivates a fraction of the receptors. In the present report, values of dissociation constant for prototype agonists and antagonists, determined from binding and from functional studies, are examined in two classical isolated preparations, rabbit aorta and guinea-pig ileum. In each preparation the dissociation constants from binding and functional experiments agree well for the antagonists but differ markedly for the agonists. Further, the dissociation constant values from binding are seen to be greater for the agonists than for the antagonists. When a chronic treatment regimen in the rabbit resulted in a pronounced change in the functional dissociation constant of subsequently administered norepinephrine, there was no significant change in either the binding constant of this agonist or in the pA2 value of the alpha antagonist, phentolamine. These, and the previously described results, are shown to be compatible with a simple two-state receptor model in which agonists bind with high and low affinity to each state while antagonists do not distinguish between the states. In this model, the ratio of low to high affinity states accounts for the failure of the binding procedure to detect changes in the agonists dissociation constant that are highly significant in the functional study. Whereas the model is based on data for these two classical preparations only, and may not be more generally applicable, the findings demonstrate the necessity for employing both functional and radioligand binding experiments when characterizing drug receptors.     

Opioid Modulation of Extracellular Signal-Regulated Protein Kinase Activity Is Ras-Dependent and Involves Gβγ Subunits

Abstract: Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([ d -Ala²,Me-Phe⁴,Gly-ol⁵]enkephalin)-, δ- ([ d -Pen², d -Pen⁵]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of G_i/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.