Browsing by Author "Prieto Villalobos, Juan Carlos"

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    Astroglial Hemichannels and Pannexons: The Hidden Link between Maternal Inflammation and Neurological Disorders
    (2021) Prieto Villalobos, Juan Carlos; Alvear Soto, Tanhia Francheska; Liberona, Andres; Lucero, Claudia M.; Martinez Araya, Claudio J.; Balmazabal, Javiera; Inostroza, Carla A.; Ramírez Rojas, Gigliola; Gomez, Gonzalo I.; Orellana Roca, Juan Andrés
    Maternal inflammation during pregnancy causes later-in-life alterations of the offspring's brain structure and function. These abnormalities increase the risk of developing several psychiatric and neurological disorders, including schizophrenia, intellectual disability, bipolar disorder, autism spectrum disorder, microcephaly, and cerebral palsy. Here, we discuss how astrocytes might contribute to postnatal brain dysfunction following maternal inflammation, focusing on the signaling mediated by two families of plasma membrane channels: hemi-channels and pannexons. [Ca2+](i) imbalance linked to the opening of astrocytic hemichannels and pannexons could disturb essential functions that sustain astrocytic survival and astrocyte-to-neuron support, including energy and redox homeostasis, uptake of K+ and glutamate, and the delivery of neurotrophic factors and energy-rich metabolites. Both phenomena could make neurons more susceptible to the harmful effect of prenatal inflammation and the experience of a second immune challenge during adulthood. On the other hand, maternal inflammation could cause excitotoxicity by producing the release of high amounts of gliotransmitters via astrocytic hemichannels/pannexons, eliciting further neuronal damage. Understanding how hemichannels and pannexons participate in maternal inflammation-induced brain abnormalities could be critical for developing pharmacological therapies against neurological disorders observed in the offspring.
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    GABAergic Regulation of Astroglial Gliotransmission through Cx43 Hemichannels
    (2022) Jimenez-Dinamarca, Ivanka; Reyes-Lizana, Rachel; Lemunao-Inostroza, Yordan; Cardenas, Kevin; Castro-Lazo, Raimundo; Peña, Francisca; Lucero, Claudia M.; Prieto Villalobos, Juan Carlos; Retamal, Mauricio Antonio; Orellana Roca, Juan Andrés; Stehberg, Jimmy
    Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABA(A) receptor or efflux or K+ via the GABA(B) receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABA(A) and GABA(B) receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABA(A) receptor, as it was blunted by the GABA(A) receptor antagonist bicuculline but unaffected by GABA(B) receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.
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    Hypertensive Nephropathy: Unveiling the Possible Involvement of Hemichannels and Pannexons
    (2022) Lucero, Claudia M.; Prieto Villalobos, Juan Carlos; Marambio-Ruiz, Lucas; Balmazabal, Javiera; Alvear Soto, Tanhia Francheska; Vega, Matías; Barra, Paola; Retamal, Mauricio A.; Orellana Roca, Juan Andrés; Gómez, Gonzalo I.
    Hypertension is one of the most common risk factors for developing chronic cardiovascular diseases, including hypertensive nephropathy. Within the glomerulus, hypertension causes damage and activation of mesangial cells (MCs), eliciting the production of large amounts of vasoactive and proinflammatory agents. Accordingly, the activation of AT1 receptors by the vasoactive molecule angiotensin II (AngII) contributes to the pathogenesis of renal damage, which is mediated mostly by the dysfunction of intracellular Ca2+ ([Ca2+]i) signaling. Similarly, inflammation entails complex processes, where [Ca2+]i also play crucial roles. Deregulation of this second messenger increases cell damage and promotes fibrosis, reduces renal blood flow, and impairs the glomerular filtration barrier. In vertebrates, [Ca2+]i signaling depends, in part, on the activity of two families of large-pore channels: hemichannels and pannexons. Interestingly, the opening of these channels depends on [Ca2+]i signaling. In this review, we propose that the opening of channels formed by connexins and/or pannexins mediated by AngII induces the ATP release to the extracellular media, with the subsequent activation of purinergic receptors. This process could elicit Ca2+ overload and constitute a feed-forward mechanism, leading to kidney damage.
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    Role of the glycoprotein Spike S1 of the SARS-CoV-2 virus on the function of hemichannels formed by Cx43
    (2022) Prieto Villalobos, Juan Carlos; Orellana Roca, Juan Andrés; Pontificia Universidad Católica de Chile. Escuela de Medicina
    Introducción: El virus SARS-CoV-2 causante de la pandemia COVID-19 ha generado una crisis sanitaria mundial. A pesar del rápido desarrollo de vacunas contra el COVID-19, los mecanismos moleculares que subyacen a la infección por el SARS-CoV-2 y su impacto en la fisiología celular siguen siendo objeto de investigación. Un aspecto incierto es si el daño celular inducido por el COVID-19 se debe al SARS-CoV-2 per se, sus proteínas víricas (en particular la proteína de espiga S1 [spike S1]), y/o la inflamación sistémica inducida por la infección. Estudios anteriores indican que la activación de los hemicanales es crucial para la replicación viral y el daño celular evocado por diversas proteínas virales. Los hemicanales abarcan una familia de canales de la membrana plasmática que permiten el intercambio iónico y molecular entre el citosol y el espacio extracelular. De manera relevante, la apertura persistente de estos canales ha sido asociado a la patogénesis de diferentes enfermedades. Sin embargo, se desconoce si la proteína spike S1 afecta a la actividad de los hemicanales. Materiales y Métodos: Células HeLa transfectadas con la conexina-43 GFP (HeLa-Cx43GFP), o con la Cx43GFP más la enzima convertidora de la angiotensina 2 m Cherry (HeLa-Cx43GFP/ACE2mCherry) fueron tratadas durante 1-72 h con spike S1 (10-1000 pM). Luego se midió la actividad de los hemicanales mediante experimentos de captación de colorante y registros de fluorescencia en tiempo real por microscopia de epifluorescencia. Resultados: Spike S1 aumentó significativamente la captación de colorante en las células HeLa-Cx43GFP, fenómeno que se potenció en las células Cx43GFP/ACE2mCherry. Dos bloqueadores específicos de los hemicanales de Cx43 (Tat-L2 y gap19) suprimieron completamente estas respuestas y tampoco se observaron en las células HeLa parentales. Discusión: Se propone que la apertura persistente de los hemicanales de Cx43 podría contribuir al daño celular causado por el SARS-CoV-2. En este sentido, estos canales podrían ser un nuevo blanco terapéutico para hacer frente al cuadro clínico generado por el COVID prolongado o “Long-COVID”.
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    SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics
    (2023) Prieto Villalobos, Juan Carlos; Lucero, Claudia M.; Rovegno, Maximiliano; Gómez, Gonzalo I.; Retamal, Mauricio A.; Orellana Roca, Juan Andrés
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels.
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    SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics
    (2023) Prieto Villalobos, Juan Carlos; Lucero, Claudia M.; Rovegno, Maximiliano; Gómez, Gonzalo I.; Retamal, Mauricio A.; Orellana, Juan A.
    Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. Results: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. Conclusions: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.
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    TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway
    (2022) Lucero, Claudia M.; Marambio-Ruiz, Lucas; Balmazabal, Javiera; Prieto Villalobos, Juan Carlos; Leon, Marcelo; Fernandez, Paola; Orellana Roca, Juan Andrés; Velarde Aliaga, María Victoria; Saez, Juan C.; Gomez, Gonzalo, I
    Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-alpha plus IL-1 beta increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-alpha/IL-1 beta treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-alpha/IL-1 beta-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell-cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.