Browsing by Author "Sáez, Juan C."

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    Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes
    (2024) Gómez, Gonzalo I.; García-Rodríguez, Claudia; Marillán, Jesus E.; Vergara, Sergio A.; Alvear, Tanhia F.; Farias-Pasten, Arantza; Sáez, Juan C.; Retamal, Mauricio A.; Rovegno, Maximiliano; Ortiz, Fernando C.; Orellana Roca, Juan Andrés
    Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+](i) linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+](i). The [Ca2+](i)-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.
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    Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage
    (2024) Gómez, Gonzalo I.; Alvear, Tanhia F.; Roa, Daniela A.; Farias-Pasten, Arantza; Vergara, Sergio A.; Mellado, Luis A.; Martinez-Araya, Claudio J.; Prieto Villalobos, Juan; García-Rodríguez, Claudia; Sánchez, Natalia; Sáez, Juan C.; Ortíz, Fernando C.; Orellana, Juan A.
    Background: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. Results: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1β and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. Conclusion: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.
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    Lack of canonical activities of connexins in highly aggressive human prostate cancer cells
    (2024) Asencio Barría, Catalina Andrea; Véliz García, Loreto Pamela; Flores-Faúndez, Emilia; Azócar, Lorena; Echeverría, Carolina E.; Torres Estay, Verónica; Orellana, Viviana; Ramírez Santelices, Catalina; Sotomayor Fahrenkrog, Paula Camila Stefanía; Cancino, Jorge; Kerr, Bredford; Fernandez-Olivares, Ainoa; Retamal, Mauricio A.; Sáez, Juan C.; Godoy, Alejandro S.
    Abstract Connexins (Cxs) have the ability to form channels that allow the exchange of ions/metabolites between adjacent cells (gap junction channels, GJC) or between the intra- and extra-cellular compartments (hemichannels, HC). Cxs were initially classified as tumor suppressors. However, more recently, it has been shown that Cxs exert anti- and pro-tumorigenic effects depending on the cell and tissue context. In prostate cancer (PCa), the expression and functionality of Cxs remain highly controversial. Here, we analyzed the expression pattern of Cx26, Cx32, Cx37, Cx40, Cx43 and Cx45 in PCa cell lines with increasing levels of tumor aggressiveness (LNCaP < LNCaP-C4-2 < Du-145 < PC-3). In addition, GJ and HC activities were evaluated in the PCa cell lines using dye coupling and dye uptake assays, respectively. Lastly, the cellular localization of Cx26, Cx32, and Cx43 was analyzed in LNCaP and PC-3 cell lines using immunofluorescence analyses. Our results showed a positive association between the mRNA levels of Cx26, Cx37 and Cx45 and the degree of aggressiveness of PCa cells, a negative association in the case of Cx32 and Cx43, and no clear pattern for Cx40. At the protein level, a negative relationship between the expression of Cx26, Cx32 and Cx43 and the degree of aggressiveness of PCa cell lines was observed. No significant differences were observed for the expression of Cx37, Cx40, and Cx45 in PCa cell lines. At the functional level, only LNCaP cells showed moderate GJ activity and LNCaP and LNCaP-C4-2 cells showed HC activity. Immunofluorescence analyses confirmed that the majority of Cx26, Cx32, and Cx43 expression was localized in the cytoplasm of both LNCaP and PC3 cell lines. This data indicated that GJ and HC activities were moderately detected only in the less aggressive PCa cells, which suggest that Cxs expression in highly aggressive PCa cells could be associated to channel-independent roles.