Rol de la homeostasis de calcio (Ca2+) del retículo endoplasmático en la respuesta a proteínas mal plegadas (UPR) durante la respuesta sistémica adquirida (SAR) en arabidopsis thaliana

Tesis (Magíster en Biotecnología)Esta tesis fue realizada en el Centro de Biotecnología Vegetal de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por el proyecto Núcleo DI-590-14/NEl ion calcio (Ca2+) juega un rol fundamental dentro de la célula como segundo mensajero, y se ha demostrado que la homeostasis de este ion está involucrada en diversos procesos celulares, entre ellos, el establecimiento de SAR, por lo que, en este trabajo, se analizará cómo afecta al establecimiento de SAR la alteración de la dinámica de Ca2+, dada entre el citoplasma y el RE, y su relación con la respuesta de UPR. Para esto, se analizó la bomba Ca2+/ATPasa presente en el RE, ACA2, y el efecto de su ausencia en el establecimiento de SAR. Se utilizaron plantas silvestres y mutantes en esta bomba de Ca2+ para analizar el establecimiento de SAR en respuesta a la infección por Pseudomonas syringae, la activación y secreción de proteínas de defensa y la participación del RE y de genes de UPR, en la respuesta de defensa. Además, se compararon los resultados con plantas mutantes para los factores de transcripción activados en respuesta a estrés biótico. Estos resultados demuestran el rol central de ACA2, en la dinámica de Ca2+ existente entre citoplasma y RE, durante la respuesta a proteínas mal plegadas y el establecimiento de SAR en Arabidopsis thaliana.Calcium ion (Ca+2) plays key roles in cells as a second messenger, and it has been demonstrated that its homeostasis is involved in several processes, among them, SAR establishment. Therefore, in this work, the way in which SAR establishment is affected by Ca+2 dynamics, given between the cytoplasm and RE, and its relation with UPR response, will be analyzed. For this purpose, the absence of a Ca+2/ATPase pump from RE, ACA2, was analyzed during SAR establishment. Wild-type plants and mutant plants for this Calcium pump were used to analyze SAR establishment as a response to infection by Pseudomonas syringae, the activation and secretion of defense proteins, and the participation of RE and UPR genes in defense response. In addition, obtained results were compared with those of mutant plants for the transcription factors that are activated as a response to biotic stress. This results demonstrate the central role of ACA2 in the Ca+2 dynamics between cytoplasm and RE, during the Unfolded Protein Response and SAR establishment in Arabidopsis thaliana

UFR2709, a nicotinic acetylcholine receptor antagonist, decreases ethanol intake in alcohol-preferring rats

Brain nicotinic acetylcholine receptors (nAChRs), a heterogeneous family of pentameric acetylcholine-gated cation channels, have been suggested as molecular targets for the treatment of alcohol abuse and dependence. Here, we examined the effect of the competitive nAChR antagonist UFR2709 on the alcohol consumption of high-alcohol-drinking UChB rats. UChB rats were given free access to ethanol for 24-h periods in a two-bottle free-choice paradigm and their ethanol and water intake were measured. The animals were i.p injected daily for 17 days with a 10, 5, 2.5 or 1 mg/kg dose of UFR2709. Potential confounding motor effects of UFR2709 were assessed by examining the locomotor activity of animals administered the highest dose of UR2709 tested (10 mg/kg i.p.). UFR2709 reduced ethanol consumption and ethanol preference and increased water consumption in a dose-dependent manner. The most effective dose of UFR2709 was 2.5 mg/kg, which induced a 56% reduction in alcohol consumption. Administration of UFR2709 did not affect the weight or locomotor activity of the rats, suggesting that its effects on alcohol consumption and preference were mediated by specific nAChRs

Prolonged AT1R activation induces CaV1.2 channel internalization in rat cardiomyocytes

Abstract The cardiac L-type calcium channel is a multi-subunit complex that requires co-assembling of the pore-forming subunit CaV1.2 with auxiliary subunits CaVα2δ and CaVβ. Its traffic has been shown to be controlled by these subunits and by the activation of various G-protein coupled receptors (GPCR). Here, we explore the consequences of the prolonged activation of angiotensin receptor type 1 (AT1R) over CaV1.2 channel trafficking. Bioluminescence Resonance Energy Transfer (BRET) assay between β-arrestin and L-type channels in angiotensin II-stimulated cells was used to assess the functional consequence of AT1R activation, while immunofluorescence of adult rat cardiomyocytes revealed the effects of GPCR activation on CaV1.2 trafficking. Angiotensin II exposure results in β-arrestin1 recruitment to the channel complex and an apparent loss of CaV1.2 immunostaining at the T-tubules. Accordingly, angiotensin II stimulation causes a decrease in L-type current, Ca2+ transients and myocyte contractility, together with a faster repolarization phase of action potentials. Our results demonstrate that prolonged AT1R activation induces β-arrestin1 recruitment and the subsequent internalization of CaV1.2 channels with a half-dose of AngII on the order of 100 nM, suggesting that this effect depends on local renin-angiotensin system. This novel AT1R-dependent CaV1.2-trafficking modulation likely contributes to angiotensin II-mediated cardiac remodeling

TRPM7 mediates kidney injury, endothelial hyperpermeability and mortality during endotoxemia

Acute kidney injury and endothelial hyperpermeability are main features observed during severe sepsis with low survival rate. Transient receptor potential melastatin 7 (TRPM7) calcium channel inhibition protects against endotoxemia-induced kidney injury potentially by means of endothelial hyperpermeability decreasing. Remarkably, TRPM7 inhibition improves survival in endotoxemic animals. Sepsis is the main cause of mortality in patients admitted to intensive care units. During sepsis, endothelial permeability is severely augmented, contributing to renal dysfunction and patient mortality. Ca2+ influx and the subsequent increase in intracellular [Ca2+](i) in endothelial cells (ECs) are key steps in the establishment of endothelial hyperpermeability. Transient receptor potential melastatin 7 (TRPM7) ion channels are permeable to Ca2+ and are expressed in a broad range of cell types and tissues, including ECs and kidneys. However, the role of TRPM7 on endothelial hyperpermeability during sepsis has remained elusive. Therefore, we investigated the participation of TRPM7 in renal vascular hyperpermeability, renal dysfunction, and enhanced mortality induced by endotoxemia. Our results showed that endotoxin increases endothelial hyperpermeability and Ca2+ overload through the TLR4/NOX-2/ROS/NF-kappa B pathway. Moreover, endotoxin exposure was shown to downregulate the expression of VE-cadherin, compromising monolayer integrity and enhancing vascular hyperpermeability. Notably, endotoxin-induced endothelial hyperpermeability was substantially inhibited by pharmacological inhibition and specific suppression of TRPM7 expression. The endotoxin was shown to upregulate the expression of TRPM7 via the TLR4/NOX-2/ROS/NF-kappa B pathway and induce a TRPM7-dependent EC Ca2+ overload. Remarkably, in vivo experiments performed in endotoxemic animals showed that pharmacological inhibition and specific suppression of TRPM7 expression inhibits renal vascular hyperpermeability, prevents kidney dysfunction, and improves survival in endotoxemic animals. Therefore, our results showed that TRPM7 mediates endotoxemia-induced endothelial hyperpermeability, renal dysfunction, and enhanced mortality, revealing a novel molecular target for treating renal vascular hyperpermeability and kidney dysfunction during endotoxemia, sepsis, and other inflammatory diseases.Fondo Nacional de Desarrollo CientÍfico y Tecnológico (National Fund for Scientific and Technological Development) 1161646 1161288 116090