Terapia asistida con animales

Treball presentat a l'assignatura de Deontologia i Veterinària Legal (21223

Experimental considerations for the assessment of in vivo and in vitro opioid pharmacology

Morphine and other mu-opioid receptor (MOR) agonists remain the mainstay treatment of acute and prolonged pain states worldwide. The major limiting factor for continued use of these current opioids is the high incidence of side effects that result in loss of life and loss of quality of life. The development of novel opioids bereft, or much less potent, at inducing these side effects remains an intensive area of research, with multiple pharmacological strategies being explored. However, as with many G protein-coupled receptors (GPCRs), translation of promising candidates from in vitro characterisation to successful clinical candidates still represents a major challenge and attrition point. This review summarises the preclinical animal models used to evaluate the key opioid-induced behaviours of antinociception, respiratory depression, constipation and opioid-induced hyperalgesia and tolerance. We highlight the influence of distinct variables in the experimental protocols, as well as the potential implications for differences in receptor reserve in each system. Finally, we discuss how methods to assess opioid action in vivo and in vitro relate to each other in the context of bridging the translational gap in opioid drug discovery

GRK Mediates μ-Opioid Receptor Plasma Membrane Reorganization

Differential regulation of the μ-opioid receptor (MOP) has been linked to the development of opioid tolerance and dependence which both limit the clinical use of opioid analgesics. At a cellular level, MOP regulation occurs via receptor phosphorylation, desensitization, plasma membrane redistribution, and internalization. Here, we used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to detect and quantify ligand-dependent changes in the plasma membrane organization of MOP expressed in human embryonic kidney (HEK293) cells. The low internalizing agonist morphine and the antagonist naloxone did not alter constitutive MOP plasma membrane organization. In contrast, the internalizing agonist DAMGO changed MOP plasma membrane organization in a pertussis toxin-insensitive manner and by two mechanisms. Firstly, it slowed MOP diffusion in a manner that was independent of internalization but dependent on GRK2/3. Secondly, DAMGO reduced the surface receptor number and the proportion of mobile receptors, and increased receptor clustering in a manner that was dependent on clathrin-mediated endocytosis. Overall, these results suggest the existence of distinct sequential MOP reorganization events at the plasma membrane and provide insights into the specific protein interactions that control MOP plasma membrane organization

The life cycle of the Mu-Opioid Receptor

Opioid receptors are undisputed targets for the treatment of pain. Unfortunately, targeting these receptors therapeutically poses significant challenges including addiction, dependence, tolerance and the appearance of side-effects such as respiratory depression and constipation. Moreover, misuse of prescription and illicit narcotics has resulted in the current opioid crisis. The mu-opioid receptor is the cellular mediator of the effects of most commonly used opioids and is a prototypical G protein-coupled receptor (GPCR) where new pharmacological, signalling and cell biology concepts have been coined. This review summarises our knowledge of the life cycle of this therapeutic target including its biogenesis, trafficking to and from the plasma membrane, and how the regulation of these processes impacts its function and is related to pathophysiological conditions

GRKs as Key Modulators of Opioid Receptor Function

Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl

G protein-coupled receptors are dynamic regulators of digestion and targets for digestive diseases

G protein-coupled receptors (GPCRs) are the largest family of transmembrane signaling proteins. Within the gastrointestinal tract, GPCRs expressed by epithelial cells sense contents of the lumen, and GPCRs expressed by epithelial cells, myocytes, neurons, and immune cells participate in communication amongst cells. GPCRs control digestion, mediate digestive diseases, and coordinate repair and growth. GPCRs are the target of over one third of therapeutic drugs, including many drugs used to treat digestive diseases. Recent advances in structural, chemical, and cell biology research have revealed that GPCRs are not static binary switches that operate from the plasma membrane to control a defined set of intracellular signals. Rather, GPCRs are dynamic signaling proteins that adopt distinct conformations and subcellular distributions when associated with different ligands and intracellular effectors. An understanding of the dynamic nature of GPCRs has provided insights into the mechanism of activation and signaling of GPCRs, and has revealed opportunities for drug discovery. We review the allosteric modulation, biased agonism, oligomerization, and compartmentalized signaling of GPCRs that control digestion and digestive diseases. We highlight the implications of these concepts for the development of selective and effective drugs to treat diseases of the gastrointestinal tract

Preámbulo: el último adiós

Treballs de l'alumnat del Grau de Comunicació Audiovisual, Facultat de Biblioteconomia i Documentació, Universitat de Barcelona, Projectes II - Grup5. Curs: 2014-2015, Tutor: Josep RoviraGuión: Anna Catalán y Laura Fuertes; Dirección: Laura Fuertes; Ayd. de Dirección: Sònia Canals; Aux. de Dirección: Anna Catalán; Coord. Producción: José Carlos Rodríguez i Kevin Roldán; Dirección de Arte y Vestuario: Anna Catalán; DOP (Dir. fotografía): Marina Miguel; Sonido Directo: Meritxell Figueras; Postproducción: Kevin Roldán (imagen) i Laura Fuertes y Sònia Canals (sonido); Etalonaje: José Carlos Rodríguez. Equip artístic: Personatge: Carlos, Actor/Actriu: David Aranda; Personatge: Álvaro, Actor/Actriu: Ferran Pericas; Personatge: Ana, Actor/Actriu: Loida Amorós; Personatge: Daniel, Actor/Actriu: Darius Macrea; Personatge: Pablo , Actor/Actriu: Miguel Herrera; Personatge: Lucia, Actor/Actriu: Itziar Sánchez; Personatge: Laura, Actor/Actriu: Sònia Canals; Personatge: Andrea, Actor/Actriu: Cristina Pérez.[Vídeo] Preámbulo es un cortometraje de unos quince minutos de duración aproximadamente que relata la historia de la necesidad de Carlos de superar la muerte de su mejor amigo Álvaro. La historia avanza de forma lineal a través de la explicación en primera persona del protagonista. Durante el cortometraje, alternamos escenas del presente y del pasado, así pues, observaremos mediante flashbacks el trágico accidente.[Memòria] El origen del proyecto se sitúa a mediados del mes de enero con la primera reunión del equipo técnico. La única premisa que existió fue la grabación de un cortometraje audiovisual que girase alrededor de la incomprensión y la desgracia de su personaje principal, hasta llevarlo al suicidio. Además de contar con las diferentes posibilidades que ofrece la temática, queríamos alejarnos de la típica historia amorosa. La propuesta final que ha acabado surgiendo nos permite jugar con el presente y el pasado de unas vidas que no volverán a ser las mismas; hablamos de la figura de unos amigos fieles, la diversidad de relaciones sentimentales de pareja y otros elementos, como por ejemplo, el embarazo sorpresa de la desaparecida pareja del personaje principal. A partir de estas premisas, nació el guión literario de ‘El último romántico’ (título inicial de nuestro proyecto), una historia con sentido para la reflexión. La obra quiere darle énfasis a una cita del dramaturgo Oscar Wilde: ‘A veces podemos pasarnos años sin vivir en absoluto, y de pronto toda nuestra vida se concentra en un solo instante’. Finalmente, y no menos importante, nos motiva la realización de la propuesta porque nos gusta la trama. Creemos que el proyecto puede tener recorrido después de su realización para la Universidad de Barcelona y, además, contamos con un trabajador equipo técnico, con experiencia y con una fantástica dinámica de trabajo para llevarlo a cabo

Synthesis, biological evaluation, and utility of fluorescent ligands targeting the μ-opioid receptor

Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pKi values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology

Phosphoproteomic characterization of the signaling network resulting from activation of chemokine receptor CCR2

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein-coupled receptors (GPCRs). Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition (DIA) MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function