Browsing by Author "Sáez, JC"

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    Expression of connexins during differentiation and regeneration of skeletal muscle
    (2005) Araya, R; Eckardt, D; Maxeiner, S; Krüger, O; Theis, M; Willecke, K; Sáez, JC
    The molecular mechanisms regulating skeletal muscle regeneration and differentiation are not well understood. We analyzed the expression of connexins (Cxs) 40, 43 and 45 in normal and regenerating tibialis anterior muscle and in primary cultures of differentiating myoblasts in adult and newborn mice, respectively. Cxs 45 and 43, but not 40, were strongly expressed in normal muscle and their expression was upregulated during regeneration. Furthermore, the functional role of Cx43 during differentiation and regeneration was examined after induced deletion of Cx43 in transgenic mice. In vivo, the inducible deletion of Cx43 delayed the formation of myofibers and prolonged the expression of myogenin during regeneration. In primary cultures of satellite cell-derived myoblasts, induced deletion of Cx43 led to decreased expression of myogenin and MyoD, dye coupling, creatine kinase activity and myoblast fusion. Thus, the expression of Cx45 and Cx43 is upregulated during skeletal muscle regeneration and Cx43 is required for normal myogenesis in vitro and adult muscle regeneration in vivo.
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    Gap junctional communication coordinates vasopressin-induced glycogenolysis in rat hepatocytes
    (1998) Eugenín, EA; González, H; Sáez, CG; Sáez, JC
    Because hepatocytes communicate via gap junctions, it has been proposed that Ca2+ waves propagate through this pathway and in the process activate Ca2+-dependent cellular responses. We tested this hypothesis by measuring vasopressin-induced glycogenolysis in shortterm cultures of rat hepatocytes. A 15-min vasopressin (10(-8) M) stimulation induced a reduction of glycogen content that reached a maximum 1-3 h later. Gap junction blockers, octanol or 18 alpha-glycyrrhetinic acid, reduced the effect by 70%. The glycogenolytic response induced by Ca2+ ionophore 8-bromo-A-21387, which acts on each hepatocyte, was not affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a synthetic peptide homologous to a domain of the extracellular loop I of the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced. Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP as a paracrine mediator. We propose that gap junctions allow for a coordinated vasopressin-induced glycogenolytic response despite the heterogeneity among hepatocytes.
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    Gating and regulation of connexin 43 (U43) hemichannels
    (2003) Contreras, JE; Sáez, JC; Bukauskas, FF; Bennett, MVL
    Connexin 43 (Cx43) nonjunctional or "unapposed" hemichannels can open under physiological or pathological conditions. We characterize hemichannels comprised of Cx43 or Cx43-EGFP (Cx43 with enhanced GFP fused to the C terminus) expressed in HeLa cells. Channel opening was induced at potentials greater than +60 mV. Open probability appeared to be very low. No comparable opening was detected in the parental, nontransfected HeLa cells. Conductance of fully open single hemichannels was approximate to220 pS, which is approximately double that of Cx43 cell-cell channels. Cx43 hemichannels exhibited two types of gating: fast transitions (<1 ms) between the fully open state and a substate of approximate to75 pS and slow transitions (>5 ms) between either open state and the fully closed state. Cx43-EGFP hemichannels exhibited only slow transitions (>5 ms) between closed and fully open states. These properties resemble those of the corresponding Cx43 and Cx43-EGFP cell-cell channels. Cx43 with EGFP on the N terminus (EGFP-Cx43) inserted into the surface and formed plaques but did not form hemichannels or cell-cell channels. Hemichannel blockers, 18beta-glycyrrhetinic acid or La3+, blocked depolarization-induced currents. Uptake of ethidium bromide (i) was faster in Cx43 and Cx43-EGFP than parental and EGFP-Cx43 cells, (it) was directly correlated with Cx43-EGFP expression, (M) was reduced by hemichannel blockers, and (iv) occurred at the same low rate in EGFP-Cx43 and parental cells. Although hemichannel opening was not detected electrophysiologically at the resting potential, infrequent or brief opening could account for ethidium bromide uptake. Opening of Cx43 hemichannels may mediate normal signaling or be deleterious.
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    Histamine reduces gap junctional communication of human tonsil high endothelial cells in culture
    (2004) Figueroa, XF; Alviña, K; Martínez, AD; Garcés, G; Rosemblatt, M; Boric, MP; Sáez, JC
    The regulation of gap junctional communication by histamine was studied in primary cultures of human tonsil high endothelial cells (HUTECs). We evaluated intercellular communication, levels, state of phosphorylation, and cellular distribution of gap junction protein subunits, mainly connexin (Cx)43. Histamine induced a time-dependent reduction in dye coupling (Lucifer yellow) associated with reduction in connexin43 localized at cell-cell appositions (immunofluorescence), without changes in levels and phosphorylation state of connexin43 (immunoblots). These effects were prevented with chlorpheniramine, an H-1 receptor blocker; indomethacin, a cyclooxygenase blocker; or GF109203X, a protein kinase C inhibitor. Treatment with phorbol myristate acetate, a protein kinase C activator, and 4bromo (4Br)-A23187, a calcium ionophore, mimicked the histamine-induced effects on dye coupling. 8Bromo-cAMP doubled the dye coupling extent and prevented the histamine-induced reduction in incidence of dye coupling. After 24-h histamine treatment, known to desensitize H, receptors, reapplication of histamine increased cell coupling in a way prevented by ranitidine, an H-2 receptor blocker. Thus, activation of H-1 and H-2 receptors, which increase intracellular levels of free Ca2+ and cAMP, respectively, may affect gap junctional communication in opposite ways. Stabilization of actin filaments with phalloidine diminished but did not totally prevent histamine-induced cell shape changes and reduction in dye coupling. Hence, the histamine-induced reduction in gap junctional communication between HUTEC is mediated by cytoskeleton-dependent and -independent mechanisms and might contribute to modulate endothelial function in lymphoid tissue. (C) 2004 Elsevier Inc. All fights reserved.
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    Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α
    (2001) Eugenín, EA; Eckardt, D; Theis, M; Willecke, K; Bennett, MVL; Sáez, JC
    Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha -glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.
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    Regulation of hepatic connexins in cholestasis
    (2002) González, HE; Eugenín, EA; Garcés, G; Solís, N; Pizarro, M; Accatino, L; Sáez, JC
    Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-alpha and interleukins-1beta and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.
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    S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes
    (2006) Retamal, MA; Cortés, CJ; Reuss, L; Bennett, MVL; Sáez, JC
    Marked increase in cell permeability ascribed to open connexin (Cx)43 hemichannels is induced by metabolic inhibition (MI) of cortical astrocytes in culture, but the molecular mechanisms are not established. Dephosphorylation and/or oxidation of Cx43 hemichannels was proposed as a potential mechanism to increase their open probability. We now demonstrate that MI increases the number of hemichannels on the cell surface assayed by biotinylation and Western blot, and that this change is followed by increased dephosphorylation and S-nitrosylation. The increase in rate of dye uptake caused by MI is comparable to the increase in surface expression; thus, open probability and permeation per hemichannel may be unchanged. Reducing agents did not affect dephosphorylation of Cx43 hemichannels but reduced dye uptake and S-nitrosylation. Uptake was also reduced by elevated intracellular but not extracellular levels of reduced glutathione. Moreover, nitric oxide donors induced dye uptake and nitrosylation of surface Cx43 but did not affect its abundance or phosphorylation. Thus, permeability per channel is increased, presumably because of increase in open probability. We propose that increased dye uptake induced by MI is mediated by an increased number of Cx43 hemichannels in the surface and is associated with multiple molecular changes, among which nitrosylation of intracellular Cx43 cysteine residues may be a critical factor.
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    TNF-α plus IFN-γ induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses
    (2003) Eugenín, EA; Brañes, MC; Berman, JW; Sáez, JC
    In this work, the effects of bacterial LPS, TNF-alpha, and IFN-gamma on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-alpha, or IFN-gamma alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 alpha-glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF-alpha significantly reduced the LPS plus IFN-gamma-induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18-alpha-glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF-alpha plus IFN-gamma. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates.