Biomedical subjects

Juan C Sáez

Publications and source records attributed to Juan C Sáez.

18 recordsLinked to original sources

PubMed2007

Injury of skeletal muscle and specific cytokines induce the expression of gap junction channels in mouse dendritic cells.

Dendritic cells (DCs) in culture express at least connexin43, a protein subunit of gap junctions, and form gap junction channels, which could be important for T-cells activation. Here, we evaluated whether DCs express connexins in vivo and also to identify components of their microenvironment that regulate the functional expression of gap junctions. In vivo studies were performed in lymph nodes of mice under control conditions or after skeletal muscle damage. In double immunolabeling studies, connexin45 was frequently detected in DEC205(+) DCs in lymph nodes of control animals, whereas connexin43 was rarely found in DCs. However, connexin43 was upregulated in DCs after skeletal muscle damage. Upregulation of connexin43 gene expression by tissue damage was also confirmed in mice carrying a beta-galactosidase reporter gene in a connexin43 allele. The effect of several cytokines on the expression of functional gap junctions between cultured DCs was also tested. Under control conditions, cultured DCs did not communicate via gap junctions. However, after treatment with keratinocyte-conditioned medium or cytokine mixtures containing at least TNF-alpha and IL-1beta, they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by effective cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma, or IL-6) or other mixtures than the described above did not induce coupling via gap junctions. Increased levels of connexin43 and connexin45 protein and mRNA accompanied the appearance of cellular coupling. These studies provide demonstration of connexin expression and regulation by specific danger signals in DCs.

Animals↗

PubMed2006

S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents.

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.

Animals↗

PubMed2005

Chlorpromazine reduces the intercellular communication via gap junctions in mammalian cells.

In the work presented herein, we evaluated the effect of chlorpromazine (CPZ) on gap junctions expressed by two mammalian cell types; Gn-11 cells (cell line derived from mouse LHRH neurons) and rat cortical astrocytes maintained in culture. We also attempted to elucidate possible mechanisms of action of CPZ effects on gap junctions. CPZ, in concentrations comparable with doses used to treat human diseases, was found to reduce the intercellular communication via gap junctions as evaluated with measurements of dye coupling (Lucifer yellow). In both cell types, maximal inhibition of functional gap junctions was reached within about 1 h of treatment with CPZ, an recovery was almost complete at about 5 h after CPZ wash out. In both cell types, CPZ treatment increased the phosphorylation state of connexin43 (Cx43), a gap junction protein subunit. Moreover, CPZ reduced the reactivity of Cx43 (immunofluorescence) at cell interfaces and concomitantly increased its reactivity in intracellular vesicles, suggesting an increased retrieval from and/or reduced insertion into the plasma membrane. CPZ also caused cellular retraction reducing cell-cell contacts in a reversible manner. The reduction in contact area might destabilize existing gap junctions and abrogate formation of new ones. Moreover, the CPZ-induced reduction in gap junctional communication may depend on the connexins (Cxs) forming the junctions. If Cx43 were the only connexin expressed, MAPK-dependent phosphorylation of this connexin would induce closure of gap junction channels.

Animals↗

PubMed2005

Connexin-based gap junction hemichannels: gating mechanisms.

Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions.

Animals↗

PubMed2004

Expression of connexins during differentiation and regeneration of skeletal muscle: functional relevance of connexin43.

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.

Animals↗

PubMed2004

The formation of skeletal muscle myotubes requires functional membrane receptors activated by extracellular ATP.

Skeletal muscle differentiation follows an organized sequence of events including commitment, cell cycle withdrawal, and cell fusion to form multinucleated myotubes. The role of adenosine 5'-triphosphate (ATP)-mediated signaling in differentiation of skeletal muscle myoblasts was evaluated in C(2)C(12) cells, a myoblast cell line. Cell differentiation was inhibited by P2X receptor blockers or by degradation of endogenous ATP with apyrase. However, pertussis toxin, known to block only a group of P2Y receptors, did not alter the differentiation process. Cells were heterogeneous in their expression of functional P2X receptors, evaluated by the uptake of fluorescent permeability tracers (Lucifer yellow and ethidium bromide), and by immunofluorescence of P2X(7) receptors. Moreover, xestospongin C, a selective and membrane-permeable inhibitor of IP(3) receptors, inhibited both myotube formation and myogenin expression. Based on these results, we suggest that the known increase in intracellular Ca(2+) concentration required for differentiation is due at least in part to Ca(2+) influx through P2X receptors and Ca(2+) release from intracellular stores. The possible involvement of P2X receptors and other pathways that might set the intracellular Ca(2+) at the level required for myoblast differentiation as well as the possible involvement of gap junction channels in the intercellular transfer of second messengers involved in coordinating myogenesis is proposed.

Adenosine Triphosphate↗

PubMed2004

Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue.

Gap junction channels and hemichannels formed of connexin subunits are found in most cell types in vertebrates. Gap junctions connect cells via channels not open to the extracellular space and permit the passage of ions and molecules of approximately 1 kDa. Single connexin hemichannels, which are connexin hexamers, are present in the surface membrane before docking with a hemichannel in an apposed membrane. Because of their high conductance and permeability in cell-cell channels, it had been thought that connexin hemichannels remained closed until docking to form a cell-cell channel. Now it is clear that at least some hemichannels can open to allow passage of molecules between the cytoplasm and extracellular space. Here we review evidence that gap junction channels may allow intercellular diffusion of necrotic or apoptotic signals, but may also allow diffusion of ions and substances from healthy to injured cells, thereby contributing to cell survival. Moreover, opening of gap junction hemichannels may exacerbate cell injury or mediate paracrine or autocrine signaling. In addition to the cell specific features of an ischemic insult, propagation of cell damage and death within affected tissues may be affected by expression and regulation of gap junction channels and hemichannels formed by connexins.

Animals↗

PubMed2004

Histamine reduces gap junctional communication of human tonsil high endothelial cells in culture.

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 H1 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 H1 receptors, reapplication of histamine increased cell coupling in a way prevented by ranitidine, an H2 receptor blocker. Thus, activation of H1 and H2 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.

Cells, Cultured↗

PubMed2003

Gating and regulation of connexin 43 (Cx43) hemichannels.

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 approximately 220 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 approximately 75 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, (ii) was directly correlated with Cx43-EGFP expression, (iii) 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.

Connexin 43↗

PubMed2003

TNF-alpha plus IFN-gamma induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses.

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.

Adjuvants, Immunologic↗

PubMed2003

Connexin43 is expressed in mouse fetal ovary.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3betaHSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc. In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

New roles for astrocytes: gap junction hemichannels have something to communicate.

Gap junctions are clusters of aqueous channels that connect the cytoplasm of adjoining cells. Each cell contributes a hemichannel, or connexon, to each cell-cell channel. The cell-cell channels are permeable to relatively large molecules, and it was thought that opening of hemichannels to the extracellular space would kill cells through loss of metabolites, collapse of ionic gradients and influx of Ca(2+). Recent findings indicate that specific non-junctional hemichannels do open under both physiological and pathological conditions, and that opening is functional or deleterious depending on the situation. Most of these studies utilized cells in tissue culture that expressed a specific gap junction protein, connexin 43. Several such examples are reviewed here, with a particular focus on astrocytes.

Animals↗

PubMed2002

Identification of second messengers that induce expression of functional gap junctions in microglia cultured from newborn rats.

The effect of several second messengers on the functional expression of gap junctions was investigated in primary cultures of newborn rat microglia. As previously reported, microglia cultured under resting conditions expressed low levels of the gap junction protein connexin 43, and exhibited little dye coupling. After treatment with 4bromo-A23187, a Ca(2+) ionophore, the incidence of dye coupling between microglia increased progressively over a 12-h period. Dye coupling was markedly reduced by gap junction blockers. Induction of dye coupling by 4bromo-A23187 was prevented by the addition of a synthetic peptide with the same sequence as a region of the extracellular loop 1 of connexin 43 (residues 53-66). The increase in dye coupling induced by 4bromo-A23187 was associated with increased connexin 43 mRNA and protein levels. Treatment of microglia with phorbol 12-myristate 13-acetate, an activator of protein kinase C, did not promote gap junctional communication in untreated microglia and reversed 4bromo-A23187-induced dye coupling. Thus, gap junctional communication between microglia can be regulated oppositely by calcium- and protein kinase C-dependent pathways. Activators of cGMP-dependent protein kinase (8bromo-cGMP) or protein kinase A (8bromo-cAMP) had no effect on untreated microglia or on 4bromo-A23187-induced dye coupling. Differential regulation of gap junctions by intracellular calcium concentration and protein kinase C activity may help to explain how various stimuli evoke differences in microglia responses, such as synthesis and secretion of cytokines and proteases.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.

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.

Animals↗

PubMed2002

Activation of human polymorphonuclear cells induces formation of functional gap junctions and expression of connexins.

BACKGROUND: During inflammatory responses activated polymorphonuclear cells (PMNs) adhere to each other and form clusters within the vasculature or injured tissues. We hypothesized that conditions that partially mimic the chemical environment of inflammatory foci induce the expression of functional gap junctions (GJs) between cultured PMNs. MATERIAL/METHODS: Human PMNs were treated with bacterial lipopolysaccharide (LPS), TNF-a, LPS plus medium conditioned by LPS-treated endothelial cells (ECs) or TNF-a plus ECs conditioned medium. Gap junctional communication was evaluated with the dye coupling technique using a permeant and an impermeant GJ fluorescent dye and GJ blockers. The expression of connexins, GJ protein subunits, was evaluated by immunocytochemistry and immunoblotting. Cytochalasin-D and nocodazole were used to evaluate the involvement of cytoskeleton in the induction of dye coupling. RESULTS: Treatment with LPS or TNF-a induced the formation of PMN aggregates, but cells were not dye coupled. If the latter protocols occurred in medium conditioned by LPS-treated ECs or resting ECs, respectively, intercellular transfer only of the GJ permeant molecule was observed in most clustered cells. Dye coupling was reversibly inhibited by GJ blockers and prevented by cytochalasin-D, a microfilament disrupter, but not by nocodazole, a microtubule disrupter. Treatments that induced dye coupling also induced connexin43 and connexin40, but not connexin32 immunoreactivity. None of these connexins was detected in circulating cells. CONCLUSIONS: EC-derived factor(s) and microfilament integrity are required for dye coupling between LPS- and TNF-a-treated PMNs. GJ formation between PMNs is correlated with the presence of connexins 43 and 40, but not 32 and requires intact microfilaments.

Animals↗

PubMed2001

Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture.

Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to "chemical ischemia" by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18alpha-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La(3+) also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.

Adenosine Triphosphate↗

PubMedDate not supplied

Functioning of cx43 hemichannels demonstrated by single channel properties.

It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.

Cell Membrane Permeability↗

PubMedDate not supplied

Presence and importance of connexin43 during myogenesis.

We analyzed the expression of connexin(Cx)43 in proliferating and differentiating C(2)C(12) cells and in myoblasts obtained from newborn mice. Cx43 was present in both cell types and under both conditions. The functional role of gap junctional communication (GJC) during terminal differentiation was evaluated in C(2)C(12) myoblasts in the presence or absence of the gap junction blocker 18beta-glycyrrhetinic acid (beta-GA). Differentiation was temporally analyzed through myogenin expression, activity of creatine kinase (CK), and yield of multinucleated cells. In cells treated with beta-GA, the CK activity and myotube formation were reversibly blocked. While in control cultures positive myogenin expression was seen in cell clusters, in beta-GA treated cultures the myogenin immunoreactivity was detected in few, preferentially sparse cells. The role of Cx43 during terminal differentiation was evaluated in cultures of myoblasts obtained from Cx43(Cre-ER(T)/fl) transgenic mice. Inducible deletion of Cx43 was obtained upon activation of Cre-ER(T) via 4-OH-tamoxifen applications. Cx43 deletion led to a drastic decrease in myogenin expression at 24 h of differentiation as compared to myoblasts from control mice. Our results indicate that Cx43-containing gap junctions are required for normal skeletal muscle terminal differentiation. These channels might provide a pathway for the intercellular transfer of signals involved in myogenesis.

Animals↗