Browsing by Author "Sáez, Juan"
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Item Extracellular gentamicin reduces the activity of con nexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells(Frontiers Research Foundation, 2014) Figueroa, Vania; Retamal, Mauricio; Cea, Luis; Salas, José; Vargas, Aníbal; Verdugo, Christián; Jara, Oscar; Martínez, Agustín; Sáez, JuanGap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.Item Opening of pannexin- and con nexin-based channels increases the excitability of nodose ganglion sensory neurons(Frontiers Research Foundation, 2014) Retamal, Mauricio; Alcayaga, Julio; Verdugo, Christian; Bultynck, Geert; Leybaert, Luc; Sáez, Pablo; Fernández, Ricardo; León, Luis; Sáez, JuanSatellite glial cells (SGCs) are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system (CNS), astrocytes present connexin43 (Cx43) hemichannels and pannexin1 (Panx1) channels, and the opening of these channels allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complexes (NPJcs) using confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and in sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to a Ca2+/Mg2+-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT). Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because in those from Panx1 knockout mice showed a reduced increase of neuronal activity induced by Ca2+/Mg2+-free extracellular conditions. The data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons.Item Regulation of Intercellular Calcium Signaling Through Calcium Interactions with Connexin-Based Channels(2012) Orellana, Juan; Sánchez, Helmuth; Schalper, Kurt; Figueroa, Vania; Sáez, JuanThe synchronization of numerous cellular events requires complex electric and metabolic cell-cell interactions. Connexins are a family of membrane proteins that constitute the molecular basis of two kinds of channels: gap junction channels (GJCs), which allow direct cytoplasm-cytoplasm communication, and hemichannels (HCs) that provide a pathway for exchanges between the intra and extra-cellular milieu. Both kind of connexin-based channels support intercellular communication via intercellular propagation of calcium waves. Here, we review evidence supporting the role of Ca 2+ in the regulation of GJCs and HCs formed by connexins. Also it is speculated how these connexin-based channels could contribute to the propagation of intercellular Ca 2+ signals.Item Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala(Federation of American Societies for Experimental Biology, 2012) Stehberg, Jimmy; Moraga-Amaro, Rodrigo; Salazar, Christian; Becerra, Alvaro; Echeverría, Cesar; Orellana, Juan; Bultynck, Geert; Ponsaerts, Raf; Leybaert, Luc; Simon, Felipe; Sáez, Juan; Retamal, MauricioRecent in vitro evidence indicates that astrocytes can modulate synaptic plasticity by releasing neuroactive substances (gliotransmitters). However, whether gliotransmitter release from astrocytes is necessary for higher brain function in vivo, particularly for memory, as well as the contribution of connexin (Cx) hemichannels to gliotransmitter release, remain elusive. Here, we microinfused into the rat basolateral amygdala (BLA) TAT-Cx43L2, a peptide that selectively inhibits Cx43-hemichannel opening while maintaining synaptic transmission or interastrocyte gap junctional communication. In vivo blockade of Cx43 hemichannels during memory consolidation induced amnesia for auditory fear conditioning, as assessed 24 h after training, without affecting short-term memory, locomotion, or shock reactivity. The amnesic effect was transitory, specific for memory consolidation, and was confirmed after microinfusion of Gap27, another Cx43-hemichannel blocker. Learning capacity was recovered after coinfusion of TAT-Cx43L2 and a mixture of putative gliotransmitters (glutamate, glutamine, lactate, d-serine, glycine, and ATP). We propose that gliotransmitter release from astrocytes through Cx43 hemichannels is necessary for fear memory consolidation at the BLA. Thus, the present study is the first to demonstrate a physiological role for astroglial Cx43 hemichannels in brain function, making these channels a novel pharmacological target for the treatment of psychiatric disorders, including post-traumatic stress disorder.