Tesis doctorales: TESEO

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Ministerio de Educación, Cultura y Deporte - Gobierno de España

Título: ESTUDIO DE LA IMPLICACION DEL RECEPTOR P2X7 EN LOS EFECTOS PROINFLAMATORIOS DE ABACAVIR
Autor: COLLADO DÍAZ, VICTOR
Universidad: Universitat de València (Estudi General)
Departamento: Farmacología
Fecha de Lectura: 19/07/2018
Mención Europeo / Mención Internacional: Concedido
Programa de doctorado: Programa de Doctorado en Biomedicina y Farmacia por la Universitat de València (Estudi General)
Dirección:
- ALVAREZ RIBELLES, ANGELES (Director)
- ESPLUGUES MOTA, JUAN VICENTE (Codirector)
Tribunal:
- Morcillo Sánchez, Esteban Jesús (presidente)
- Barreiro del Río, Olga (secretario)
- ALCAIDE ALONSO, MARIA PILAR (vocal)
Descriptores:
- ANALISIS DE FARMACOS
- FARMACOLOGIA EXPERIMENTAL
Marcador: https://www.educacion.es/teseo/mostrarRef.do?ref=1699173Añadir a favoritos.
Resumen:
ABC, a nucleoside reverse transcriptase inhibitor (NRTI), is one of the most commonly used drugs in the treatment of the Human Immunodeficiency Virus (HIV) infection and has been related with an increase in cardiovascular risk. There is currently a controversy surrounding the use of ABC due to discrepancies between the results and conclusions of different studies and the lack of a clear mechanism of action that could justify its putative vascular toxicity. Different studies performed in our laboratory suggest that Abacavir is capable of inducing leukocyte accumulation, the first phase in the development of inflammatory cardiovascular diseases, and that this occurs through interaction between Mac-1 in leukocytes and endothelial ICAM-1. These effects were reproduced by didanosine - another NRTI purine analog - but not by other NRTI pyrimidine analogs, suggesting a structure-activity relationship in the proinflammatory action of ABC, further demonstrated by the implication of the P2X7 receptor in these effects. The present project aimed to corroborate the implication of the P2X7 receptor in the events in question, the importance of its location, and the mechanism of action by which ABC activates the receptor. The studies we have performed in “in vitro” and “in vivo” models confirm that the effect of ABC on leukocyte-endothelium interaction and the expression of Mac-1 is due to the P2X7 receptors located mainly in leukocytes. The activation of this membrane P2X7 purinergic receptor could be attributable to the direct action of ABC, the action of the endogenous ligand of the P2X7 receptor (ATP) or the action of CBV-TP, the active metabolite of ABC. Binding studies of the P2X7 receptor indicated that ABC is not capable of binding to the receptor at the binding site of the radioligand used, while ATP and CBV-TP do interact at that site. Both ATP and CBV-TP reproduced the effects of ABC on leukocyte-endothelium interaction and Mac-1 expression; however, the effects of ATP were not mediated specifically by the P2X7 receptor, whereas this receptor did mediate the effects of CBV-TP. The fact that we were not able to detect sufficient levels of CBV-TP to produce the evaluated effects led us to rule out this ligand as responsible for the effect of ABC. We could not exclude a role for ATP, because, although ABC did not produce changes in extracellular nucleotide levels (ATP, ADP, AMP, Adenosine), and affected neither the metabolism of these nucleotides nor the expression or enzymatic activity of the enzymes involved in this process, we did detect extracellular levels of ATP in our system (50-130 nM). Apirase (a compound that hydrolyzes ATP) reverted the effects of ABC without degrading it and was able to degrade ATP thus targeting ATP as the ligand responsible for the proinflammatory effects of ABC, raising the possibility that ABC was modulating the P2X7 receptor allosterically and sensitizing it to ATP. Thus, "docking" studies were performed and showed that ABC was not capable of interacting with binding site 1 (ATP’s binding site) but instead binds to binding site 2. Moreover, ABC and ATP, at concentrations that, by themselves, did not have effects, potentiated the proinflammatory effect of ABC, demonstrating that ABC sensitizes the receptor to the action of ATP. In addition, the proinflammatory effect of ABC was reverted by blocking connexin channels. This fact, together with the data of the intracellular reduction of ATP levels by ABC, suggests that this effect is dependent on connexin channels that release ATP into the extracellular medium. In conclusion, the proinflammatory effect of ABC is a result of the allosteric modulation of the ATP P2X7 receptor located mainly in leukocytes. At clinically relevant concentrations, this drug can activate the receptor, sensitizing it to the binding of endogenous ATP and promoting its release through the Cx43 channels present in leukocytes. This ATP acts extracellularly on the receptor itself, amplifying the process. Activation of the P2X7 receptor leads to overexpression of the β2 integrin Mac-1, which interacts with endothelial ICAM-1, causing adhesion of leukocytes to the endothelium and leading to an initial phase of the generation of an inflammatory process that can trigger adverse vascular effects compatible with the clinic associations described.

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