Browsing by Author "Saez, Juan C."

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    A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII
    (2021) Lopez, Ximena; Palacios-Prado, Nicolas; Guiza, Juan; Escamilla, Rosalba; Fernandez, Paola; Vega, Jose L.; Rojas, Maximiliano; Marquez-Miranda, Valeria; Chamorro, Eduardo; Cardenas, Ana M.; Maldifassi, Maria Constanza; Martinez, Agustin D.; Duarte, Yorley; Gonzalez-Nilo, Fernando D.; Saez, Juan C.
    Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation- measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1EGFP and rPanx1S394D-EGFP channels showed current at all voltages between +/- 100 mV, similar single channel currents with outward rectification, and unitary conductance (similar to 30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
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    Active acetylcholine receptors prevent the atrophy of skeletal muscles and favor reinnervation
    (2020) Cisterna, Bruno A.; Vargas, Anibal A.; Puebla, Carlos; Fernandez, Paola; Escamilla, Rosalba; Lagos, Carlos F.; Matus, Maria F.; Vilos, Cristian; Cea, Luis A.; Barnafi, Esteban; Gaete, Hugo; Escobar, Daniel F.; Cardozo, Christopher P.; Saez, Juan C.
    Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.
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    Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury
    (2023) Toro, Carlos A.; Johnson, Kaitlin; Hansen, Jens; Siddiq, Mustafa M.; Vasquez, Walter; Zhao, Wei; Graham, Zachary A.; Saez, Juan C.; Iyengar, Ravi; Cardozo, Christopher P.
    Membrane channels such as those formed by connexins (Cx) and P2X(7) receptors (P2X(7)R) are permeable to calcium ions and other small molecules such as adenosine triphosphate (ATP) and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx and Panx1 hemichannels (HCs). To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X(7)R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in spinal cord tissue just caudal from the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.
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    Boldine Prevents Renal Alterations in Diabetic Rats
    (2013) Hernandez-Salinas, Romina; Vielma, Alejandra Z.; Arismendi, Marlene N.; Boric, Mauricio P.; Saez, Juan C.; Velarde, Victoria
    Diabetic nephropathy alters both structure and function of the kidney. These alterations are associated with increased levels of reactive oxygen species, matrix proteins, and proinflammatory molecules. Inflammation decreases gap junctional communication and increases hemichannel activity leading to increased membrane permeability and altering tissue homeostasis. Since current treatments for diabetic nephropathy do not prevent renal damage, we postulated an alternative treatment with boldine, an alkaloid obtained from boldo with antioxidant, anti-inflammatory, and hypoglycemic effects. Streptozotocin-induced diabetic and control rats were treated or not treated with boldine (50 mg/Kg/day) for ten weeks. In addition, mesangial cells were cultured under control conditions or in high glucose concentration plus proinflammatory cytokines, with or without boldine (100 mu mol/L). Boldine treatment in diabetic animals prevented the increase in glycemia, blood pressure, renal thiobarbituric acid reactive substances and the urinary protein/creatinine ratio. Boldine also reduced alterations in matrix proteins and markers of renal damage. In mesangial cells, boldine prevented the increase in oxidative stress, the decrease in gap junctional communication, and the increase in cell permeability due to connexin hemichannel activity induced by high glucose and proinflammatory cytokines but did not block gap junction channels. Thus boldine prevented both renal and cellular alterations and could be useful for preventing tissue damage in diabetic subjects.
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    Confirmation of Connexin45 Underlying Weak Gap Junctional Intercellular Coupling in HeLa Cells
    (2020) Choi, Eun Ju; Palacios-Prado, Nicolas; Saez, Juan C.; Lee, Jinu
    Gap junctions (GJs) are intercellular channels that connect adjacent cells electrically and metabolically. The iodide-yellow fluorescent protein (I-YFP) gap junctional intercellular communication (GJIC) assay is a recently developed method with high sensitivity. HeLa cells have been widely used as GJ-deficient cells for GJ-related research. Herein, we present evidence showing that HeLa cells have functional GJs comprising connexin (Cx) 45 using the I-YFP GJ assay and CRISPR/Cas9 system. We conducted the I-YFP GJIC assay in HeLa cells, which revealed a weak level of GJIC that could not be detected by the Lucifer yellow scrape-loading assay. The mRNA expression of GJB5 (Cx31.1), GJA1 (Cx43), and GJC1 (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out GJC1 (Cx45) abolished GJIC, as analyzed by the I-YFP assay and dual whole-cell patch-clamp assay. These results suggest that HeLa cells express Cx45-based GJs and that the I-YFP GJIC assay can be used for cells with weak GJIC, such as Cx45-expressing HeLa cells. Further, GJC1 (Cx45)-knockout HeLa cells are more suitable as a GJ-null cell model for transfection experiments than wild-type HeLa cells. This experimental design was successfully applied to knock out Cx43 expression and GJIC in A549 lung cancer cells and can thus be used to identify major Cxs in other cell types and to establish GJ assay systems for different Cxs.
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    Endogenous pannexin1 channels form functional intercellular cell-cell channels with characteristic voltage-dependent properties
    (2022) Palacios-Prado, Nicolas; Soto, Paola A.; Lopez, Ximena; Choi, Eun Ju; Marquez-Miranda, Valeria; Rojas, Maximiliano; Duarte, Yorley; Lee, Jinu; Gonzalez-Nilo, Fernando D.; Saez, Juan C.
    The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exoge-nous human pannexin1 (hPanx1) cell-cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell-cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single -channel conductance of -175 pS, with a substate of -35 pS; the latter showed a pecu-liar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell-cell channels were also iden-tified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfer-ing RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell-cell cou-pling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibi-tor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the chan-nel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell-cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell-cell channels in different cell types might require special attention.
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    Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons
    (2022) Arredondo, Cristian; Cefaliello, Carolina; Dyrda, Agnieszka; Jury, Nur; Martinez, Pablo; Diaz, Ivan; Amaro, Armando; Tran, Helene; Morales, Danna; Pertusa, Maria; Stoica, Lorelei; Fritz, Elsa; Corvalan, Daniela; Abarzua, Sebastian; Mendez-Ruette, Maxs; Fernandez, Paola; Rojas, Fabiola; Kumar, Meenakshi Sundaram; Aguilar, Rodrigo; Almeida, Sandra; Weiss, Alexandra; Bustos, Fernando J.; Gonzalez-Nilo, Fernando; Otero, Carolina; Tevy, Maria Florencia; Bosco, Daryl A.; Saez, Juan C.; Kahne, Thilo; Gao, Fen-Biao; Berry, James D.; Nicholson, Katharine; Sena-Esteves, Miguel; Madrid, Rodolfo; Varela, Diego; Montecino, Martin; Brown, Robert H.; van Zundert, Brigitte
    Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/ FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.
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    Extracellular gentamicin reduces the activity of con nexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells
    (2014) Figueroa, Vania A.; Retamal, Mauricio A.; Cea, Luis A.; Salas, Jose D.; Vargas, Anibal A.; Verdugo, Christian A.; Jara, Oscar; Martinez, Agustin D.; Saez, Juan C.
    Gap 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.
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    Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro
    (2007) Eugenin, Eliseo A.; Gonzalez, Hernan E.; Sanchez, Helmuth A.; Branes, Maria C.; Saez, Juan C.
    Connexin43 (Cx43), a gap junction protein subunit, has been previously detected in Kupffer cells (KCs) during liver inflammation, however, KCs phagocytose cell debris that may include Cx43 protein, which could explain the detection of Cx43 in KCs. We determined that KCs express Cx43 and form gap junctions (GJs) both in vivo and in vitro. In liver sections of animals treated with LPS, Cx43 was detected at ED2+ cells interfaces, indicating formation of GJs between KCs in vivo. In vitro, unstimulated KCs cultures did not form functional GJs, and expressed low levels of Cx43 that showed a diffuse intracellular distribution. In contrast, KCs treated with LPS plus IFN-gamma, expressed a greater amount of Cx43 at both, protein and mRNA levels, and showed Cx43 at cell-cell contacts associated with higher dye coupling. In conclusion, activation of KCs in vivo or in vitro resulted in enhanced Cx43 expression levels and formation of GJ that might play relevant roles during liver inflammation. (c) 2007 Elsevier Inc. All rights reserved.
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    Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice
    (2014) Ardiles, Alvaro O.; Flores-Munoz, Carolina; Toro-Ayala, Gabriela; Cardenas, Ana M.; Palacios, Adrian G.; Munoz, Pablo; Fuenzalida, Marco; Saez, Juan C.; Martinez, Agustin D.
    The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca2+ concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LIP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LIP whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.
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    Remembrances of Dr. Michael VL Bennett by Iberoamerican Colleagues and Friends
    (2024) Abudara, Verónica; Araneda, Ricardo C.; Barrio, Luis; Berthoud, Viviana M.; Contreras, Jorge E.; Eugenin, Eliseo; Lerma, Juan; Orellana Roca, Juan Andrés; Palacios Prado, Nicolás; Perez-Armendariz, Elia Martha; Retamal, Mauricio A.; Saez, Juan C.
    On behalf of the Iberoamerican scientific community in the gap junction field, we offer a tribute to the late Dr. Michael V.L. Bennett, who passed away on November 16, 2023
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    Role of a RhoA/ROCK-Dependent Pathway on Renal Connexin43 Regulation in the Angiotensin II-Induced Renal Damage
    (2019) Gomez, Gonzalo I.; Velarde, Victoria; Saez, Juan C.
    In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: "In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43". In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1 beta), 3) fibrosis (immune detection of type III collagen; Col III) and 4) activity of RhoA/ROCK (amount of phosphorylated MYPT1; p-MYPT1). The ratio Uprot/UCrea, SBP, oxidative stress, inflammation, amount of Cx43 and p-MYPT1 remained high 2 weeks after suspending AngII treatment in rats treated for 4 weeks with AngII. These responses were not observed in rats treated with AngII for less than 4 weeks, in which all measurements returned spontaneously close to the control values after suspending AngII treatment. Rats treated with AngII for 6 weeks and co-treated for the last 4 weeks with Fasudil, an inhibitor of ROCK, showed high SBP but did not present renal damage or increased amount of renal Cx43. Therefore, renal damage induced by AngII correlates with the activation of RhoA/ROCK and the increase in Cx43 amounts and can be prevented by inhibitors of this pathway.
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    Role of Akt and Ca2+ on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition
    (2015) Salas, Daniela; Puebla, Carlos; Lampe, Paul D.; Lavandero, Sergio; Saez, Juan C.
    Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphotylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion. (C) 2015 Elsevier B.V. All rights reserved.
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    Skeletal Muscle Atrophy Induced by Diabetes Is Mediated by Non-Selective Channels and Prevented by Boldine
    (2023) Cea, Luis A.; Vasquez, Walter; Hernandez-Salinas, Romina; Vielma, Alejandra Z.; Castillo-Ruiz, Mario; Velarde, Victoria; Salgado, Magdiel; Saez, Juan C.
    Individuals with diabetes mellitus present a skeletal muscle myopathy characterized by atrophy. However, the mechanism underlying this muscular alteration remains elusive, which makes it difficult to design a rational treatment that could avoid the negative consequences in muscles due to diabetes. In the present work, the atrophy of skeletal myofibers from streptozotocin-induced diabetic rats was prevented with boldine, suggesting that non-selective channels inhibited by this alkaloid are involved in this process, as has previously shown for other muscular pathologies. Accordingly, we found a relevant increase in sarcolemma permeability of skeletal myofibers of diabetic animals in vivo and in vitro due to de novo expression of functional connexin hemichannels (Cx HCs) containing connexins (Cxs) 39, 43, and 45. These cells also expressed P2X(7) receptors, and their inhibition in vitro drastically reduced sarcolemma permeability, suggesting their participation in the activation of Cx HCs. Notably, sarcolemma permeability of skeletal myofibers was prevented by boldine treatment that blocks Cx43 and Cx45 HCs, and now we demonstrated that it also blocks P2X(7) receptors. In addition, the skeletal muscle alterations described above were not observed in diabetic mice with myofibers deficient in Cx43/Cx45 expression. Moreover, murine myofibers cultured for 24 h in high glucose presented a drastic increase in sarcolemma permeability and levels of NLRP3, a molecular member of the inflammasome, a response that was also prevented by boldine, suggesting that, in addition to the systemic inflammatory response found in diabetes, high glucose can promote the expression of functional Cx HCs and activation of the inflammasome in skeletal myofibers. Therefore, Cx43 and Cx45 HCs play a critical role in myofiber degeneration, and boldine could be considered a potential therapeutic agent to treat muscular complications due to diabetes.
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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.
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    Vitamin E Blocks Connexin Hemichannels and Prevents Deleterious Effects of Glucocorticoid Treatment on Skeletal Muscles
    (2020) Balboa, Elisa; Saavedra, Fujiko; Cea, Luis A.; Ramirez, Valeria; Escamilla, Rosalba; Vargas, Anibal A.; Regueira, Tomas; Saez, Juan C.
    Glucocorticoids are frequently used as anti-inflammatory and immunosuppressive agents. However, high doses and/or prolonged use induce undesired secondary effects such as muscular atrophy. Recently, de novo expression of connexin43 and connexin45 hemichannels (Cx43 HCs and Cx45 HCs, respectively) has been proposed to play a critical role in the mechanism underlying myofiber atrophy induced by dexamethasone (Dex: a synthetic glucocorticoid), but their involvement in specific muscle changes promoted by Dex remains poorly understood. Moreover, treatments that could prevent the undesired effects of glucocorticoids on skeletal muscles remain unknown. In the present work, a 7-day Dex treatment in adult mice was found to induce weight loss and skeletal muscle changes including expression of functional Cx43/Cx45 HCs, elevated atrogin immunoreactivity, atrophy, oxidative stress and mitochondrial dysfunction. All these undesired effects were absent in muscles of mice simultaneously treated with Dex and vitamin E (VitE). Moreover, VitE was found to rapidly inhibit the activity of Cx HCs in freshly isolated myofibers of Dex treated mice. Exposure to alkaline pH induced free radical generation only in HeLa cells expressing Cx43 or Cx45 where Ca2+ was present in the extracellular milieu, response that was prevented by VitE. Besides, VitE and two other anti-oxidant compounds, Tempol and Resveratrol, were found to inhibit Cx43 HCs in HeLa cells transfectants. Thus, we propose that in addition to their intrinsic anti-oxidant potency, some antioxidants could be used to reduce expression and/or opening of Cx HCs and consequently reduce the undesired effect of glucocorticoids on skeletal muscles.