RESUMEN
Cultures of neonatal rat heart cells contain predominantly myocytes and fibroblastic cells. Most abundant are groups of synchronously contracting myocytes, which are electrically well coupled through large gap junctions. Cardiac fibroblasts may be electrically coupled to each other and to adjacent myocytes, be it with low intercellular conductances. Nevertheless, synchronously beating myocytes interconnected via a fibroblast were present, demonstrating that nonexcitable cardiac cells are capable of passive impulse conduction. In fibroblast pairs as well as in myocyte-fibroblast cell pairs, no sensitivity to junctional voltage could be detected when transjunctional conductance was > 1-2 nS. However, in pairs coupled by a conductance of < 1 nS, complex voltage-dependent gating was evident; gap junction channel open probability decreased with increasing junctional voltage but a nongated residual conductance remained at all voltages tested. Single gap junction channel conductance between fibroblasts was approximately 21 pS, very similar to an approximately 18-pS channel conductance that was found between myocytes next to the major conductance of 43 pS. Single-channel conductance in heterologous myocyte-fibroblast gap junctions was approximately 32 pS, which matches the theoretical value of 29 pS for gap junction channels composed of a fibroblast connexon and the major myocyte connexon. A site-directed antibody against rat heart gap junction protein connexin43 recognized gap junctions between neonatal cardiomyocytes, as demonstrated by immunocytochemical labeling. In contrast, junctions between fibroblasts showed no labeling, while in myocyte-fibroblast junctions labeling occasionally was present. Our results suggest the existence of two gap junction proteins between neonatal rat cardiocytes, connexin43 and another yet unidentified connexin. An alternative explanation (cell-specific regulation of the conductance of connexin43 channels) is discussed.
Asunto(s)
Corazón/fisiología , Uniones Intercelulares/fisiología , Canales Iónicos/fisiología , Miocardio/citología , Animales , Animales Recién Nacidos , Células Cultivadas , Conexinas , Conductividad Eléctrica , Electrofisiología , Fibroblastos/fisiología , Técnica del Anticuerpo Fluorescente , Homeostasis , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Modelos Biológicos , Miocardio/metabolismo , Miocardio/ultraestructura , RatasRESUMEN
In the cardiac muscle, the electrical coupling of myocytes by means of gap (or communicating) junctions, allows the action potentials to be propagated. Connexin 43 (CX 43) is the major constitutive protein of the gap junctions in the mammalian myocardium. In this organ, the abundance of CX 43 and of its messenger, as well as the spatial expression of this protein, are developmentally regulated. These findings are complemented by the results presented in this article, which deals with the distribution of CX 43 in the ventricular myocytes of mouse heart during differentiation, between the 11 days post coitum embryo stage and adulthood. By immunoelectron microscopy experiments on ultrathin sections of cardiac ventricular tissue of one-week-old mouse, we have provided confirmation that the anti-CX 43 antibodies used here specifically recognized the gap junctions. Double labeling immunofluorescence experiments have been undertaken to localize, within the same cells, either CX 43 and desmin, or CX 43 and Con A or WGA receptor sites. From the earliest stage investigated (11 days post coitum) onwards, expression of CX 43 is always associated with desmin-positive cells, that is, with the myocytes. Up to birth, there is in the ventricular wall a gradient of expression of CX 43 which is superimposable on a gradient of expression of desmin. Immunoreactivity to anti-CX 43 and anti-desmin antibodies is high in the sub-endocardial trabeculae and low (or even undetectable for CX 43, in the early stages) in the sub-epicardial cell layers. In the embryonic stages, the expression sites of CX 43 are visible in the form of small dots, whose abundance increases as development proceeds. During these stages, the immunoreactive sites are distributed in a relatively homogeneous pattern throughout the membrane of the myocytes. One week after birth, the CX 43 expression is restricted to the two ends of the myocytes (where the intercalated discs develop), and the adjacent lateral regions. This polarization of CX 43 is more pronounced at the two and three weeks post natal stages and in the fully differentiated ventricular myocytes (adult stage) CX 43 is only present in the intercalated discs.
Asunto(s)
Envejecimiento/fisiología , Corazón/embriología , Corazón/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Miocardio/citología , Animales , Animales Recién Nacidos , Diferenciación Celular , Conexinas , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Edad Gestacional , Ventrículos Cardíacos , Proteínas de la Membrana/análisis , Ratones , Microscopía Inmunoelectrónica , Miocardio/ultraestructuraRESUMEN
Endothelial cells of the microvasculature differ both structurally and functionally from endothelial cells of larger vessels. To assess whether these cells also differ in terms of direct cell-to-cell communication, we compared gap junction-mediated intercellular coupling and connexin (Cx) expression in monolayer cultures of bovine microvascular and large vessel (aortic and pulmonary artery) endothelial cells. In confluent monolayers, junctional communication (as assessed by transfer of Lucifer Yellow) was greater between large vessel than between microvascular endothelial cells. Basal levels of connexin 43 (Cx43) and Cx43 mRNA were also greater in large vessel than in microvascular endothelial cells. When monolayers of microvascular endothelial cells were mechanically wounded, junctional communication was increased between migrating cells at the wound edge. In contrast, coupling between large vessel endothelial cells was not increased after wounding. The wound-induced increase in coupling between microvascular endothelial cells was accompanied by an increase in Cx43 and Cx43 mRNA. In contrast, Cx43 expression was unaltered after wounding monolayers of large vessel endothelial cells. These studies revealed differences in basal and wound-induced levels of coupling and Cx43 expression in microvascular and large vessel endothelial cells in vitro, raising the possibility that the role of coupling in endothelial cell function may be different in these different cell types.
Asunto(s)
Comunicación Celular , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Aorta/lesiones , Aorta/metabolismo , Células Cultivadas , Conexinas , Endotelio Vascular/citología , Técnica de Fractura por Congelación , Uniones Intercelulares/fisiología , Proteínas de la Membrana/genética , Microcirculación , Arteria Pulmonar/lesiones , Arteria Pulmonar/metabolismo , ARN Mensajero/metabolismo , Heridas y Lesiones/metabolismoRESUMEN
Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.
Asunto(s)
Epítopos/análisis , Uniones Intercelulares/química , Proteínas de la Membrana/análisis , Miocardio/química , Secuencia de Aminoácidos , Animales , Conexinas , Disulfuros , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/inmunología , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Miocardio/ultraestructura , Oligopéptidos/inmunología , Conformación Proteica , RatasRESUMEN
Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.
Asunto(s)
Química Encefálica , Proteínas de la Membrana/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Northern Blotting , Cromatografía de Afinidad , Conexinas , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Miocardio/química , Oligopéptidos/síntesis química , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
We have explored the expression of gap junctional proteins and corresponding mRNAs by insulin-producing B-cells of native rat pancreas and of a transplantable rat insulinoma. By immunostaining cryostat sections (indirect immunofluorescence) and crude membrane preparations (Western blots) with antibodies against connexins 26, 32, and 43 and by hybridizing total islet and insulinoma RNA (Northern blot) with cRNAs for the latter two proteins, we have found that normal and tumoral B-cells express connexin 43 but do not show detectable levels of either connexin 32 or 26. By evaluating the conductance (dual patch-clamp whole-cell recording) and permeability of junctional channels (microinjection of Lucifer yellow), we have found that control B-cells show low levels of electrical and dye coupling in only a portion of the pairs studied. By studying B-cells of glibenclamide-treated rats, we have found that sustained stimulation of insulin release in vivo is associated with a two-fold increase in the level of connexin 43 gene transcripts and in the incidence of both ionic and dye coupling. These observations indicate that (1) connexin 43 is a major component of communicating channels between insulin-producing cells; (2) some but not all B-cells are electrically coupled by low conductance junctional channels; and (3) connexin 43 gene transcripts and incidence of junctional coupling are modulated in parallel during sustained stimulation of B-cell functioning in vivo.
Asunto(s)
Islotes Pancreáticos/citología , Proteínas de la Membrana/biosíntesis , Animales , Comunicación Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Conexinas , Conductividad Eléctrica/efectos de los fármacos , Colorantes Fluorescentes , Regulación de la Expresión Génica/efectos de los fármacos , Gliburida/farmacología , Técnicas In Vitro , Insulina/biosíntesis , Uniones Intercelulares/fisiología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Isoquinolinas , Masculino , Microinyecciones , Sondas ARN , Ratas , Ratas EndogámicasRESUMEN
We found that a rat liver epithelial cell line (IAR 20) expresses connexin 43, the major cardiac gap-junction protein, but not connexin 26 or connexin 32, major liver gap-junction proteins. The effects of TPA on connexin 43 expression in IAR 20 were investigated using northern blot analysis, western blot analysis, and an immunofluorescence technique. Gap-junctional intercellular communication (GJIC) in this cell line decreased within 60 min of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment and recovered after 24 h. The number of immunofluorescence spots of connexin 43 on IAR 20 was closely related to the change in GJIC induced by TPA. However, TPA did not change the level of mRNA measured by northern blot analysis. Moreover, connexin 43 protein expression analyzed by western blotting suggests that connexin 43 proteins were still present in TPA-treated cells at a similar level. These results suggest that GJIC of these rat liver epithelial cells was mediated by connexin 43 protein and that TPA inhibited GJIC by inhibiting posttranslational processing of connexin 43 proteins, e.g., localization or assembly.
Asunto(s)
Comunicación Celular/efectos de los fármacos , Uniones Intercelulares/fisiología , Proteínas de la Membrana/fisiología , Acetato de Tetradecanoilforbol/farmacología , Animales , Northern Blotting , Línea Celular , Conexinas , Epitelio/efectos de los fármacos , Epitelio/fisiología , Expresión Génica/efectos de los fármacos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/ultraestructura , Cinética , Hígado , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , ARN Mensajero/análisis , ARN Mensajero/genética , RatasRESUMEN
Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.
Asunto(s)
Astrocitos/ultraestructura , Uniones Intercelulares/fisiología , Canales Iónicos/fisiología , Proteínas de la Membrana/fisiología , Animales , Astrocitos/química , Células Cultivadas , Conexinas , Cuerpo Estriado/embriología , Cuerpo Estriado/ultraestructura , ADN/genética , Electrofisiología , Técnica del Anticuerpo Fluorescente , Immunoblotting , Inmunohistoquímica , Activación del Canal Iónico/fisiología , Potenciales de la Membrana , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Hibridación de Ácido Nucleico , RatasRESUMEN
A cDNA probe coding for rat connexin 43 (Beyer et al., 1987), a gap junctional protein, was used to detect specific mRNA and estimate its relative abundance in mouse heart at different developmental stages: 11, 14 and 19 days post-coïtum (dpc); 1, 2 and 3 weeks post-partum (wpp), and at the adult stage. On Northern blots of total cellular RNA, a single 3.0 kb message was detected at all stages of development, and the differential intensities of labeling indicated developmental changes in mRNA abundance. mRNA levels were further investigated by dot-blotting. Densitometric analyses of dot-blot autoradiograms showed a five-fold increase of the mRNA level between 11 dpc and 1 wpp, then a gradual decrease until the adult stage where it reached a value close to that detected at 11 dpc. By comparison, myosin heavy chains and glycerldehyde-3-phosphate dehydrogenase mRNAs were found to peak at 3 wpp and 14 dpc, respectively. The presence and the relative abundance of connexin 43 were investigated at the same developmental stages as previously by immunoblotting of whole-ventricle fractions using antipeptide antibodies specific for this junctional protein. Quantitative data obtained from densitometric analyses of immunoblots showed that from 14 dpc to 1 wpp intensity of labeling of connexin 43 was roughly multiplied by a factor of 10. It peaked at 3 wpp before dropping to about 20% at the adult stage. The data obtained with both the cDNA probe and the antibodies were significant as shown by variance analyses. They suggest that expression of cardiac connexin 43 is developmentally-regulated: at the early stages of heart development the expression levels of the protein would seem to be mainly regulated by mRNA abundance; beyond 2 weeks after birth, the levels of connexin 43 would seem rather to depend upon its stability and/or the efficiency of the translation.
Asunto(s)
Corazón/crecimiento & desarrollo , Proteínas de la Membrana/biosíntesis , Miocardio/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Conexinas , Regulación de la Expresión Génica , Corazón/embriología , Uniones Intercelulares/química , Proteínas de la Membrana/genética , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , ARN Mensajero/metabolismoRESUMEN
According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314-322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrahin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions. The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43. Proteins of Mr 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.
Asunto(s)
Química Encefálica/fisiología , Citoplasma/análisis , Mamíferos/metabolismo , Proteínas de la Membrana , Miocardio/análisis , Secuencia de Aminoácidos , Animales , Anticuerpos/aislamiento & purificación , Northern Blotting , Pollos , Conexinas , Electroforesis en Gel de Poliacrilamida , Cobayas , Humanos , Immunoblotting , Técnicas In Vitro , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Persona de Mediana Edad , Datos de Secuencia Molecular , Ratas , Trucha , XenopusRESUMEN
SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100 mM) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.
Asunto(s)
Disulfuros/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de la Membrana/metabolismo , Miocardio/ultraestructura , Compuestos de Sulfhidrilo/metabolismo , Animales , Conexinas , Etilmaleimida/farmacología , Uniones Intercelulares/análisis , Uniones Intercelulares/ultraestructura , Yodoacetamida/farmacología , Proteínas de la Membrana/análisis , Microscopía Electrónica , Miocardio/metabolismo , RatasRESUMEN
Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constituent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins of Mr 32 and 22 kD in rat and mouse liver, of Mr 41 kD in rat cerebellum, of Mr 43 kD in uterus, stomach and kidney of rat, of Mr 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.