RESUMEN
OBJECTIVES: Cryopreserved blood vessels are being increasingly employed in vascular reconstruction procedures but freezing/thawing is associated with significant cell death that may lead to graft failure. Vascular cells express connexin proteins that form gap junction channels and hemichannels. Gap junction channels directly connect the cytoplasm of adjacent cells and may facilitate the passage of cell death messengers leading to bystander cell death. Two hemichannels form a gap junction channel but these channels are also present as free non-connected hemichannels. Hemichannels are normally closed but may open under stressful conditions and thereby promote cell death. We here investigated whether blocking gap junctions and hemichannels could prevent cell death after cryopreservation. MATERIALS AND METHODS: Inclusion of Gap27, a connexin channel inhibitory peptide, during cryopreservation and thawing of human saphenous veins and femoral arteries was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assays and histological examination. RESULTS: We report that Gap27 significantly reduces cell death in human femoral arteries and saphenous veins when present during cryopreservation/thawing. In particular, smooth muscle cell death was reduced by 73% in arteries and 71% in veins, while endothelial cell death was reduced by 32% in arteries and 51% in veins. CONCLUSIONS: We conclude that inhibiting connexin channels during cryopreservation strongly promotes vascular cell viability.
Asunto(s)
Apoptosis/efectos de los fármacos , Conexinas/antagonistas & inhibidores , Criopreservación , Crioprotectores/farmacología , Arteria Femoral/efectos de los fármacos , Vena Safena/efectos de los fármacos , Adulto , Supervivencia Celular/efectos de los fármacos , Distribución de Chi-Cuadrado , Conexina 43/antagonistas & inhibidores , Conexina 43/metabolismo , Conexinas/metabolismo , Conexinas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Arteria Femoral/metabolismo , Arteria Femoral/patología , Arteria Femoral/trasplante , Humanos , Etiquetado Corte-Fin in Situ , Persona de Mediana Edad , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Oligopéptidos , Vena Safena/metabolismo , Vena Safena/patología , Vena Safena/trasplante , Proteína alfa-5 de Unión Comunicante , Proteína alfa-4 de Unión ComunicanteRESUMEN
Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca(2+) across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell-cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca(2+) solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations.
Asunto(s)
Conexina 43/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Péptidos/farmacología , Conexina 43/química , Conexina 43/genética , Conexinas/metabolismo , Fenómenos Electrofisiológicos , Células HeLa , Humanos , Potenciales de la Membrana/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.
Asunto(s)
Aleaciones de Cromo/efectos adversos , Citocinas/metabolismo , Daño del ADN , Nanopartículas del Metal/efectos adversos , Animales , Aleaciones de Cromo/metabolismo , Conexinas/metabolismo , Córnea/metabolismo , Radicales Libres/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Oligopéptidos , Transducción de Señal , Trofoblastos/metabolismoRESUMEN
Gap junctions (GJs) have been demonstrated to communicate cell death signals from apoptotic to healthy cells, thereby spatially extending apoptosis. Before being incorporated into GJs, hemichannels (hemi-GJs) are normally closed but recent evidence suggests that they can be opened by various messengers and conditions, thereby forming a pore through which molecules can enter or leave the cell potentially leading to cell death. The aim of this study was to determine the contribution of GJs and hemichannels in the communication of apoptosis toward surrounding cells. We induced apoptosis in C6 glioma cells stably transfected with connexin (Cx)43, with cytochrome C (cytC) using in situ electroporation and found that healthy surrounding cells underwent apoptotic transformation. Work with various cell death markers, wild-type (WT) and Cx43-expressing cells, inhibitors of GJs and/or hemichannels, and Cx43 gene silencing showed that GJs contribute to the spread of apoptosis in a zone next to where apoptosis was triggered whereas hemichannels also promoted cell death beyond this area. Buffering cytoplasmic Ca(2+) changes inhibited the spread of apoptosis in both cases. We conclude that Cx43 hemichannels, in concert with their GJ counterparts, play a role in communicating cytC-induced apoptotic cell death messages.
Asunto(s)
Apoptosis , Conexina 43/biosíntesis , Uniones Comunicantes/metabolismo , Glioma/metabolismo , Transducción de Señal , Animales , Apoptosis/genética , Calcio/metabolismo , Línea Celular Tumoral , Conexina 43/genética , Citoplasma/genética , Citoplasma/metabolismo , Electroporación , Uniones Comunicantes/genética , Silenciador del Gen , Glioma/genética , Humanos , Ratas , Transducción de Señal/genéticaRESUMEN
The assembly of gap junctions was investigated in mammalian cells expressing connexin (Cx) 26, 32 and 43 fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Targeting of Cx32-CFP and 43-GFP to gap junctions and gap junctional communication was inhibited in cells treated with Brefeldin A, a drug that disassembles the Golgi. However gap junctions constructed of Cx26-GFP were only minimally affected by Brefeldin A. Nocodazole, a microtubule disruptor, had little effect on the assembly of Cx43-GFP gap junctions, but perturbed assembly of Cx26-GFP gap junctions. Co-expression of Cx26-YFP and Cx32-CFP in cells treated with Brefeldin A resulted in assembly of gap junctions constructed of Cx26-YFP. Two amino acids that distinguish Cx26 from Cx32 in transmembrane domains were mutated in Cx32 to investigate underlying mechanisms determining trafficking routes to gap junctions. One mutation, Cx32I28L, conferred on it partial Cx26-like trafficking properties as well the post-translational membrane insertion characteristics of Cx26, suggesting that a key determinant regulating trafficking was present in the first transmembrane domain. The results provide a protein trafficking basis for specifying and regulating connexin composition of gap junctions and thus selectivity of intercellular signaling, with Cx32 and 43 trafficking through the secretory pathway and Cx26 also following an alternative pathway.
Asunto(s)
Conexina 43/metabolismo , Conexinas/metabolismo , Retículo Endoplásmico/metabolismo , Uniones Comunicantes/metabolismo , Membranas Intracelulares/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Brefeldino A/farmacología , Células COS , Chlorocebus aethiops , Conexina 26 , Conexina 43/genética , Conexinas/genética , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Células HeLa , Humanos , Nocodazol/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína beta1 de Unión ComunicanteRESUMEN
Intercellular co-operation is a fundamental and widespread feature in tissues and organs. An important mechanism ensuring multicellular homoeostasis involves signalling between cells via gap junctions that directly connect the cytosolic contents of adjacent cells. Cell proliferation and intercellular communication across gap junctions are closely linked, and a number of pathologies in which communication is disrupted are known where connexins, the gap-junctional proteins, are modified. The proteins of gap junctions thus emerge as therapeutic targets inviting the development and exploitation of chemical tools and drugs that specifically influence intercellular communication. Connexin mimetic peptides that correspond to short specific sequences in the two extracellular loops of connexins are a class of benign, specific and reversible inhibitors of gap-junctional communication that have been studied recently in a broad range of cells, tissues and organs. This review summarizes the properties and uses of these short synthetic peptides, and compares their probable mechanism of action with those of a wide range of other less specific traditional gap-junction inhibitors.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Conexinas/fisiología , Uniones Comunicantes/fisiología , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Conexinas/química , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Uniones Comunicantes/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Estructura Secundaria de Proteína , Tráquea/fisiologíaRESUMEN
The assembly of connexins (Cxs) into gap junction intercellular communication channels was studied. An in vitro cell-free synthesis system showed that formation of the hexameric connexon hemichannels involved dimeric and tetrameric connexin intermediates. Cx32 contains two putative cytoplasmic calmodulin-binding sites, and their role in gap junction channel assembly was investigated. The oligomerization of Cx32 into connexons was reversibly inhibited by a calmodulin-binding synthetic peptide, and by W7, a naphthalene sulfonamide calmodulin antagonist. Removing the calmodulin-binding site located at the carboxyl tail of Cx32 limited connexon formation and resulted in an accumulation of intermediate connexin oligomers. This truncation mutant, Cx32Delta215, when transiently expressed in COS-7 cells, accumulated intracellularly and had failed to target to gap junctions. Immunoprecipitation studies suggested that a C-terminal sequence of Cx32 incorporating the calmodulin-binding site was required for the formation of hetero-oligomers of Cx26 and Cx32 but not for Cx32 homomeric association. A chimera, Cx32TM3CFTR, in which the third transmembrane and proposed channel lining sequence of Cx32 was substituted by a transmembrane sequence of the cystic fibrosis transmembrane conductance regulator, did not oligomerize in vitro and it accumulated intracellularly when expressed in COS-7 cells. The results indicate that amino-acid sequences in the third transmembrane domain and a calmodulin-binding domain in the cytoplasmic tail of Cx32 are likely candidates for regulating connexin oligomerization.
Asunto(s)
Calmodulina/antagonistas & inhibidores , Conexinas/química , Animales , Sitios de Unión , Células COS , Calmodulina/metabolismo , Cricetinae , Sulfonamidas/farmacologíaRESUMEN
Connexins (Cx), the protein subunits assembled into gap junction intercellular communication channels, are expressed in primary lymphoid organs and by circulating leukocytes. Human tonsil-derived T and B lymphocytes express Cx40 and 43; circulating human T, B, and NK lymphocytes express Cx43 and directly transfer between each other a low molecular dye indicative that functional gap junctions exist. We now identify specific properties in the immune system underwritten by gap junctions. Mixed lymphocytes cultured in the presence of two reagents with independent inhibitory action on gap junction communication, a connexin mimetic peptide and 18-alpha-glycyrrhetinic acid, markedly reduced the secretion of IgM, IgG, and IgA. The secretion of these immunoglobulins by purified B cells was also reduced by the two classes of gap junction inhibitors. Complex temporal inhibitory effects on the expression of mRNA encoding interleukins, especially IL-10, were also observed. The results indicate that intercellular signaling across gap junctions is an important component of the mechanisms underlying metabolic cooperation in the immune system.
Asunto(s)
Linfocitos B/inmunología , Comunicación Celular , Citocinas/biosíntesis , Uniones Comunicantes/fisiología , Inmunoglobulinas/biosíntesis , Linfocitos T/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Células Cultivadas , Niño , Conexinas/química , Conexinas/genética , Conexinas/fisiología , Ácido Glicirretínico/farmacología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-2/biosíntesis , Interleucina-2/genética , Lipopolisacáridos/farmacología , Péptidos/genética , Péptidos/farmacología , Fitohemaglutininas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/fisiologíaRESUMEN
Inter- and extracellular-mediated changes in intracellular Ca2+ concentration ([Ca2+]i) can ensure coordinated tissue function in the lung. Cultured rat alveolar epithelial cells (AECs) have been shown to respond to secretagogues with increases in [Ca2+]i and have been shown to be gap junctionally coupled. However, communication of [Ca2+]i changes in AECs is not well defined. Monolayers of AECs were mechanically perturbed and monitored for [Ca2+]i changes. Perturbation of AECs was administered by a glass probe to either mechanically stimulate or mechanically wound individual cells. Both approaches induced a change in [Ca2+]i in the stimulated cell that was propagated to neighboring cells (Ca2+ waves). A connexin mimetic peptide shown to uncouple gap junctions eliminated Ca2+ waves in mechanically stimulated cells but had no effect on mechanically wounded cells. In contrast, apyrase, an enzyme that effectively removes ATP from the extracellular milieu, had no effect on mechanically stimulated cells but severely restricted mechanically wounded Ca2+ wave propagation. We conclude that AECs have the ability to communicate coordinated Ca2+ changes using both gap junctions and extracellular ATP.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Células Epiteliales/metabolismo , Uniones Comunicantes/metabolismo , Alveolos Pulmonares/metabolismo , Adenosina Trifosfato/farmacología , Animales , Apirasa/farmacología , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Células Cultivadas , Conexinas/biosíntesis , Células Epiteliales/citología , Espacio Extracelular/metabolismo , Proteínas Activadoras de GTPasa/farmacología , Inmunohistoquímica , Masculino , Estimulación Física , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
The assembly of gap junction channels was studied using mammalian cells expressing connexin (Cx) 26, 32 and 43 in which the carboxyl terminus was fused to green, yellow or cyan fluorescent proteins (GFP, YFP, CFP). Intracellular targeting of Cx32-CFP and 43-GFP to gap junctions was disrupted by brefeldin A treatment and resulted in a severe loss of gap junctional intercellular communication reflected by low intercellular dye transfer. Cells expressing Cx43-GFP exposed to nocodazole showed normal targeting to gap junctions and dye transfer. Cx32 and 43 thus appear to be transported and assembled into gap junctions via the classical secretory pathway. In contrast, we found that assembly of Cx26-GFP into functional gap junctions was relatively unaffected by treatment of cells with brefeldin A, but was extremely sensitive to nocodazole treatment. Coexpression of Cx26-YFP and Cx32-CFP indicated a different intracellular distribution that was accentuated in the presence of brefeldin A, with the gap junctions in these cells constructed predominantly of Cx26-YFP. A site specific mutation in the first transmembrane domain that distinguished Cx32 from Cx26 (Cx32128L) resulted in the adoption of the trafficking properties of Cx26 as well as its unusual post-translational membrane integration characteristics. The results indicate that multiple intracellular connexin trafficking routes exist and provide a further mechanism for regulating the connexin composition of gap junctions and thus specificity in intercellular signalling.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Proteínas Luminiscentes/metabolismo , Transporte de Proteínas/fisiología , Animales , Brefeldino A/farmacología , Células COS , Conexina 26 , Conexinas/química , Conexinas/genética , Colorantes Fluorescentes/metabolismo , Uniones Comunicantes/química , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Mutación , Nocodazol/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.
Asunto(s)
Conexinas/fisiología , Uniones Comunicantes/fisiología , Péptidos/farmacología , Mucosa Respiratoria/fisiología , Adenosina Trifosfato/farmacología , Adenosina Trifosfato/fisiología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Señalización del Calcio , Comunicación Celular/efectos de los fármacos , Conexinas/química , Uniones Comunicantes/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Conejos , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Tráquea/fisiologíaRESUMEN
We have examined the effects of ouabain (1 mM), the gap junction inhibitors, 18 alpha-glycyrrhetinic acid (100 microM), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 microM) and palmitoleic acid (50 microM), and clotrimazole (10 microM) against endothelium-derived hyperpolarizing factor (EDHF)-mediated and K(+)-induced vasorelaxations in the rat mesentery. In the presence of indomethacin (10 microM) and 300-microM N(G)nitro-L-arginine methyl ester (L-NAME), carbachol caused EDHF-mediated relaxations (R(max)=85.3+/-4.0%). In the presence of ouabain, these responses were substantially reduced (R(max)=11.0+/-2.3%). 18 alpha-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these EDHF-mediated responses. K(+) caused vasorelaxation of preparations perfused with K(+)-free buffer (R(max)=73.7+/-2.4%), which were reduced by 10-microM indomethacin (R(max)=56.4+/-6.2%). K(+) vasorelaxation was essentially abolished by endothelial denudation. Both ouabain and 18 alpha-glycyrrhetinic acid opposed K(+) relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any effect. Direct cell-cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: (i) that gap junctional communication plays a major role in EDHF-mediated relaxations, (ii) that K(+)-vasorelaxation is endothelium-dependent (thus, K(+) is unlikely to represent an EDHF), and (iii) that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF.
Asunto(s)
Factores Biológicos/farmacología , Uniones Comunicantes/fisiología , Arteria Mesentérica Superior/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Potasio/farmacología , Animales , Bario/farmacología , Carbacol/farmacología , Cardiotónicos/farmacología , Colorantes , Endotelio Vascular/fisiología , Técnicas In Vitro , Masculino , Arteria Mesentérica Superior/citología , Agonistas Muscarínicos/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Ouabaína/farmacología , Ratas , Ratas WistarRESUMEN
To study the assembly of gap junctions, connexin--green-fluorescent-protein (Cx--GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26-- and Cx32--GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32--GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx--GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.
Asunto(s)
Conexinas/química , Uniones Comunicantes/química , Uniones Comunicantes/metabolismo , Secuencias de Aminoácidos , Animales , Western Blotting , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Conexina 26 , Citoplasma/química , Citoplasma/metabolismo , ADN Complementario/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil-derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil-derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and alpha-glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co-cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T- and B-lymphocyte co-cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes.
Asunto(s)
Comunicación Celular/fisiología , Conexinas/genética , Uniones Comunicantes/metabolismo , Sistema Inmunológico/fisiología , Subgrupos Linfocitarios/metabolismo , ARN Mensajero/análisis , Linfocitos B/metabolismo , Western Blotting , Células Cultivadas , Niño , Técnicas de Cocultivo , Conexina 26 , Conexina 43/análisis , Conexina 43/genética , Conexinas/análisis , Citometría de Flujo , Expresión Génica , Ácido Glicirretínico/farmacología , Humanos , Inmunoglobulina M/metabolismo , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Microscopía Fluorescente , Tonsila Palatina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Proteína alfa-5 de Unión ComunicanteRESUMEN
This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.
Asunto(s)
Señalización del Calcio , Conexina 43/metabolismo , Conexinas/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Calcio/metabolismo , Conexina 26 , Conexina 43/genética , Conexinas/genética , Retículo Endoplásmico/metabolismo , Matriz Extracelular/metabolismo , Uniones Comunicantes/metabolismo , Proteínas Fluorescentes Verdes , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Proteína beta1 de Unión ComunicanteRESUMEN
During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.
Asunto(s)
Conexinas/genética , Conexinas/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Animales , Conexina 26 , Conexinas/química , Femenino , Uniones Comunicantes/metabolismo , Regulación del Desarrollo de la Expresión Génica , Lactancia/genética , Lactancia/metabolismo , Ratones , Microscopía Inmunoelectrónica , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Proteína beta1 de Unión ComunicanteRESUMEN
The assembly of gap junction intercellular communication channels was studied by analysis of the molecular basis of the dysfunction of connexin 32 mutations associated with the X-linked form of Charcot-Marie-Tooth disease in which peripheral nervous transmission is impaired. A cell-free translation system showed that six recombinant connexin 32 mutated proteins-four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop-were inserted into microsomal membranes and oligomerised into connexon hemichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow. The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The results show a relationship between intracellular interruption of connexin trafficking, the efficiency of intercellular communication, and the severity of the disease phenotype. Intracellular retention was explained either by deficiencies in the ability of connexins to oligomerise or by mutational changes at two targeting motifs. The results point to dominance of two specific targeting motifs: one at the amino terminus and one at the membrane aspect of the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligomerisation and low intercellular dye transfer compared with wild-type connexin 32. The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot-Marie-Tooth X-linked disease.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Conexinas/genética , Uniones Comunicantes/fisiología , Ligamiento Genético , Mutación/fisiología , Cromosoma X , Animales , Células COS/metabolismo , Células COS/fisiología , Sistema Libre de Células , Conexinas/química , Conexinas/metabolismo , Humanos , Fenotipo , Procesamiento Proteico-Postraduccional , Proteína beta1 de Unión ComunicanteRESUMEN
Three point mutations of the connexin26 (GJB2) gene associated with hereditary deafness were studied using in vitro expression systems. Mutation M34T results in an amino acid substitution in the first transmembrane domain of the connexin protein, W77R is located in the second transmembrane domain and W44C is in the first extracellular loop. Wild-type and mutated connexin vectors were constructed and transfected into communication-deficient HeLa cells to obtain transient expression of the connexin proteins. Intercellular coupling was subsequently assessed by examining transfer of Lucifer yellow between cells. All three mutations resulted in impaired intercellular coupling. The mechanistic reasons for the functional inadequacies of the mutated proteins were investigated. First, intracellular trafficking and targeting of the expressed connexins were determined by immunohistochemistry. Mutation W77R was inefficiently targeted to the plasma membrane and retained in intracellular stores whereas the other two were targeted to the plasma membrane. Oligomerization assays showed that connexins M34T and W77R failed to assemble efficiently into hexameric gap junction hemichannels, but the W44C mutation did so. A cell-free translation system showed that the mutated proteins were inserted into microsomal membranes but the mutations have different effects on the post-translational properties of the expressed proteins. The results point to the conclusion that mutations in the transmembrane domains of connexin proteins influence gap junction assembly.