Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells

Epidermal growth factor receptor (EGFR) function is transregulated by a variety of stimuli, including agonists of certain G-protein-coupled receptors (GPCRs). One of the most ubiquitous GPCRs is the P2

P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells

T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.Fil: Barrera Avalos, Carlos. Universidad de Santiago de Chile; ChileFil: Briceño, Pedro. Universidad de Chile; ChileFil: Valdés, Daniel. Universidad de Santiago de Chile; ChileFil: Imarai, Mónica. Universidad de Santiago de Chile; ChileFil: Leiva Salcedo, Elías. Universidad de Santiago de Chile; ChileFil: Rojo, Leonel E.. Universidad de Santiago de Chile; ChileFil: Milla, Luis A.. Universidad de Santiago de Chile; ChileFil: Huidobro Toro, Juan Pablo. Universidad de Santiago de Chile; ChileFil: Robles Planells, Claudia. Universidad de Santiago de Chile; ChileFil: Escobar, Alejandro. Universidad de Chile; ChileFil: Di Virgilio, Francesco. Università di Ferrara; ItaliaFil: Moron, Victor Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Sauma, Daniela. Universidad de Chile; ChileFil: Acuña Castillo, Claudio. Universidad de Santiago de Chile; Chil

Potent Vasodilator and Cellular Antioxidant Activity of Endemic Patagonian Calafate Berries (Berberis microphylla) with Nutraceutical Potential

Hydroalcoholic extracts of Patagonian Calafate berry (Berberis microphylla) contain mono or disaccharide conjugated anthocyanins and flavonols. The Liquid Chromatography-Mass Spectrometry (LC-MS) chemical extract profile identified glycosylated anthocyanidins such as delphinidin-, petunidin- and malvidin-3-glucoside as the major constituents. The predominant flavonols were 3-O substituents quercetin-rutinoside or -rhamnoside. Anthocyanins doubled flavonols in mass (13.1 vs. 6 mg/g extract). Polyphenols vascular actions were examined in the rat arterial mesenteric bed bioassay; extract perfusion elicited concentration-dependent vasodilatation mimicked by conjugated anthocyanins standards. Vascular responses of main glycosylated anthocyanins were endothelium-dependent (p < 0.001) and mediated by NO production (p < 0.05). The anthocyanins antioxidant activity determined in isolated endothelial cells (CAA) showed a reduced redox potential as compared to the extract or quercetin. While in the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay, the anthocyanins showed an equivalent quercetin potency, the extract was 15-fold less active, proposing that the anthocyanin-induced vasodilation is not due to an antioxidant mechanism. The extract shows promising commercial nutraceutical potential

Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel’s ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators

ATP induces NO production in hippocampal neurons by P2X(7) receptor activation independent of glutamate signaling.

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2'(3')-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N(ω)-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity

NO production increased by ATP or Bz-ATP involves P2X₇R and nNOS activation in hippocampal slices.

<p>(<b>A</b>): BBG, a selective P2X₇ receptor antagonist, inhibits in a concentration-dependent manner the production of NO mediated by 100 µM ATP or 100 µM Bz-ATP. (<b>B</b>): N^ω-propyl-L-arginine, the selective nNOS inhibitor reduces the NO production elicited by ATP in a concentration-dependent manner. Results represent the mean ± S.E.M, of triplicate experiments, (n = 3–5 separate experiments). Student’s t-test *p<0.05 among nucleotides (A), or as compared to control (B); **p<0.01.</p

ATP or Bz-ATP induce NO production in hippocampal slices and cultured hippocampal neuron.

<p>(<b>A</b>): ATP and its analog Bz-ATP, a P2X₇R preferential agonist, induce NO production in a concentration-depending manner in hippocampal slices; (<b>B</b>): In Cultured rat hippocampal neurons (14 DIV), 100 µM ATP or 100 µM Bz-ATP induced a time-dependent surge of NO production, reaching maximal response at 15-min of continuous exposure (shaded bar). In (A) results are expressed as pmol of NO produced per slice. In (B), NO values elicited by nucleotides were normalized with respect to basal values; values represent the mean ± S.E.M, of triplicate experiments, Student’s t-test *p<0.05.</p

Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3

TRPM4 is a Ca2+-activated non-selective cationic channel that conducts monovalent cations. TRPM4 has been proposed to contribute to burst firing and sustained activity in several brain regions, however, the cellular and subcellular pattern of TRPM4 expression in medial prefrontal cortex (mPFC) during postnatal development has not been elucidated. Here, we use multiplex immunofluorescence labeling of brain sections to characterize the postnatal developmental expression of TRPM4 in the mouse mPFC. We also performed electrophysiological recordings to correlate the expression of TRPM4 immunoreactivity with the presence of TRPM4-like currents. We found that TRPM4 is expressed from the first postnatal day, with expression increasing up to postnatal day 35. Additionally, in perforated patch clamp experiments, we found that TRPM4like currents were active at resting membrane potentials at all postnatal ages studied. Moreover, TRPM4 is expressed in both pyramidal neurons and interneurons. TRPM4 expression is localized in the soma and proximal dendrites, but not in the axon initial segment of pyramidal neurons. This subcellular localization is consistent with a reduction in the basal current only when we locally perfused 9-Phenanthrol in the soma, but not upon perfusion in the medial or distal dendrites. Our results show a specific localization of TRPM4 expression in neurons in the mPFC and that a 9-Phenanthrol sensitive current is active at resting membrane potential, suggesting specific functional roles in mPFC neurons during postnatal development and in adulthood.Fondecyt Fondo Nacional de Desarrollo Cientifico y Tecnologico 11140731 1141132 1160518 Programa de Atraccion e Insercion de Capital Humano Avanzado (PAI) Comision Nacional de Investigacion Cientifica y Tecnologica 79140059 Comision Nacional de Investigacion Cientifica y Tecnologica Financiamiento Basal para Centros Cientificos y Tecnologicos de Excelencia FB 0807 National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) R01 NS042225 Iniciativa Cientifica Milenio of the Ministry of Economy, Development and Tourism (Chile