Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Analysis of Potassium and Calcium Imaging to Assay the Function of Opioid Receptors
As the activation of opioid receptors leads to the modulation of potassium and calcium channels, the ion imaging represents an attractive method to analyze the function of the receptors. Here, we describe the imaging of potassium using the FluxOR™ potassium ion channel assay, and of calcium using Fura-2 acetoxymethyl ester. Specifically, we (1) characterize the activation of the G-protein-coupled inwardly rectifying potassium 2 channel by agonists of μ- and δ-opioid receptors with the aid of the FluxOR™ assay in cultured mouse dorsal root ganglion neurons, and (2) describe calcium imaging protocols to ...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Fluorescence-Based, High-Throughput Assays for μ-Opioid Receptor Activation Using a Membrane Potential-Sensitive Dye
The development of new and improved opioid analgesics requires high-throughput screening (HTS) methods to identify potential therapeutics from large libraries of lead compounds. Here we describe two simple, real-time fluorescence-based assays of μ-opioid receptor activation that may be scaled up for HTS. In AtT-20 cells expressing the μ-opioid receptor (MOPr), opioids activate endogenous G protein gated inwardly rectifying K channels (GIRK channels), leading to membrane hyperpolarization. In Chinese hamster ovary cells expressing MOPr, adenylyl cyclase activation via forskolin results in membrane hyperpolarization, w...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
[35S]GTPγS Autoradiography for Studies of Opioid Receptor Functionality
The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR) that are Gi-coupled which make them suitable for studying the receptor functionality. The [35S]G...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Bioluminescence Resonance Energy Transfer (BRET) to Detect the Interactions Between Kappa Opioid Receptor and Non visual Arrestins
Bioluminescence resonance energy transfer (BRET) is a very sensitive technique employed to study protein–protein interactions, including G-protein-coupled receptors (GPCRs) hetero- and homo-dimerization. Recently, BRET has also been used to investigate the interaction between GPCRs (e.g., β2 adrenergic receptor, muscarinic M2 receptor, dopaminergic D2 receptor) and non-visual arrestins. Here a BRET protocol is described to investigate interactions between the kappa opioid receptor (KOR) and non visual arrestins (arrestin-2 and arrestin-3) in HEK-293 cells, both under basal conditions and after exposure to KOR li...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Monitoring Opioid Receptor Dimerization in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)
Bioluminescence resonance energy transfer (BRET) is a natural phenomenon that has been successfully applied for the study of protein–protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the finding of new functions and new way of signaling and trafficking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fluorescent protein (such as EYFP), it is possible to study opioid receptor dimerization using BRET. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
In Vivo Techniques to Investigate the Internalization Profile of Opioid Receptors
G-protein-coupled receptors (GPCRs) regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Receptor internalization is commonly observed following agonist binding and activation. Receptor trafficking events have been well characterized in cell systems, but the in vivo significance of GPCR internalization is still poorly understood. To address this issue, we have developed an innovative knock-in mouse model, where an opioid receptor is directly visible in vivo. These knockin mice express functional fluorescent delta opioid receptors (DOR-eGFP) in place of the endogenous rece...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Real-Time Imaging of Mu Opioid Receptors by Total Internal Reflection Fluorescence Microscopy
Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand-dependent endocytosis and recycling have been associated with opioid tolerance and dependence. Ligands of MOR can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Immunohistochemical Analysis of Opioid Receptors in Peripheral Tissues
Immunohistochemical staining is widely used to identify opioid receptors in specific cell types or anatomical structures throughout the nervous system. Opioid receptors are not restricted to the central nervous system, but are also present in peripheral sensory neurons, where their activation exerts analgesic effects without inducing centrally mediated side effects. Here, we describe immunohistochemical analysis of opioid receptors in the peripheral sensory neuron cell bodies, along the axons and their peripheral endings in the hind paw skin, as well as in the spinal cord, under naïve and sciatic nerve damage conditio...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Detection of Mu Opioid Receptor (MOPR) and Its Glycosylation in Rat and Mouse Brains by Western Blot with Anti-μC, an Affinity-Purified Polyclonal Anti-MOPR Antibody
Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Identification and Verification of Proteins Interacting with the Kappa Opioid Receptor (KOPR)
Proteins that interact with the human kappa opioid receptor (hKOPR) may contribute to regulation and signaling of the receptor. In this paper, we focus on the protein 14-3-3zeta that regulates anterograde transport of the hKOPR from the endoplasmic reticulum (ER) to the Golgi apparatus. 14-3-3zeta interacts with the C-terminal domain of the receptor and promotes cell surface expression of the hKOPR by inhibiting coatomer protein I (COPI) and RVR motif-mediated ER retension of the hKOPR. Here we describe three experimental procedures we used to evaluate the interaction between hKOPR and 14-3-3zeta: co-immunoprecipitation, p...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
High-Throughput Gene Expression Profiling of Opioid-Induced Alterations in Discrete Brain Areas
Whole-genome screening methods are unique approach to search for novel genes and molecular pathways involved in drug action. High-throughput profiling allows the gene expression levels of tens of thousands of transcripts to be measured simultaneously. Here, we describe transcriptional profiling in a specific area of the brain using DNA microarrays and next-generation sequencing. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Renilla Luciferase Reporter Assay to Study 3′UTR-Driven Posttranscriptional Regulations of OPRM1
The regulation of MOR expression at the level of mRNA is relevant for its role in pain transmission and in other functions involving opioid receptors. Recently, the role of the 3′UTR in the posttranscriptional regulation of MOR expression has been highlighted. Here we describe a Renilla luciferase reporter assay for the study of the effect of any selective treatment on the 3′UTR-dependent regulation of OPRM1 in a model of glial cells. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Analysis of Epigenetic Mechanisms Regulating Opioid Receptor Gene Transcription
Opioid drugs are generally used for moderate and severe pain reductions which act through opioid receptors. Studies on transcriptional regulation of opioid receptors are still invaluable because not only transcription is the first step to produce protein products in cells, but the receptor transcription levels also affect the pain reduction by opioids, as observed in studies of heterozygous opioid receptor knockout mice. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Computational Structural Biology of Opioid Receptors
The publication of high-resolution structures for all of the opioid receptor subfamilies has unveiled exciting opportunities for mechanistic insight into the molecular mechanisms underlying the biology of nociception, reward, and higher cognitive functions, as well as promises for progress in several clinical areas such as pain management, physiological dependence, addiction, and mood disorders. To turn this promise into novel and improved therapeutic entities, however, this information needs to be supplemented with research strategies that explore the dynamic behavior of the proteins and their interactions with other rece...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
Overview of Genetic Analysis of Human Opioid Receptors
The human μ-opioid receptor gene (OPRM1), due to its genetic and structural variation, has been a target of interest in several pharmacogenetic studies. The μ-opioid receptor (MOR), encoded by OPRM1, contributes to regulate the analgesic response to pain and also controls the rewarding effects of many drugs of abuse, including opioids, nicotine, and alcohol. Genetic polymorphisms of opioid receptors are candidates for the variability of clinical opioid effects. The non-synonymous polymorphism A118G of the OPRM1 has been repeatedly associated with the efficacy of opioid treatments for pain and various types of depende...
Source: Springer protocols feed by Pharmacology/Toxicology - October 9, 2014 Category: Drugs & Pharmacology Source Type: news
