Biomedical subjects

E C Beyer

Publications and source records attributed to E C Beyer.

At least 73 records · Page 4Linked to original sources

PubMed1994

Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart.

Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofluorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution.

Animals↗

PubMed1994

Molecular cloning of two human cardiac gap junction proteins, connexin40 and connexin45.

Intercellular coupling of cardiac myocytes through gap junction channels facilitates normal cardiac impulse conduction. Multiple gap junction sequences (connexins) have been previously identified in mammalian and avian heart, but only one, connexin43 (Cx43), has been identified in the human heart. We used the polymerase chain reaction and genomic cloning to isolate DNA encoding the gap junction proteins human connexin40 (Cx40) and connexin45 (Cx45). Northern blots showed that specific probes for Cx43, Cx40 and Cx45 all hybridize to distinct mRNAs in human ventricular RNA. Immunohistochemistry with connexin-specific antibodies confirmed that Cx40, Cx43, and Cx45 all localized to intercalated disk regions in frozen sections of human left ventricle. The presence of multiple connexins in human ventricle may contribute to divergent mechanisms of regulation of cardiac conduction.

Amino Acid Sequence↗

PubMed1994

Characterization of the gap junction protein, connexin45.

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hep1, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285-298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [35S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [32P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.

Animals↗

PubMed1994

Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible.

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.

Amino Acid Sequence↗

PubMed1994

Distinct gap junction protein phenotypes in cardiac tissues with disparate conduction properties.

OBJECTIVES: We sought to characterize the connexin phenotypes of selected regions of the canine heart with different conduction properties to determine whether variations in connexin expression might contribute to the differences in intercellular resistance and conduction velocity that occur in different cardiac tissues. BACKGROUND: Gap junctions connect cardiac myocytes, allowing propagation of action potentials. Intercellular channels with different electrophysiologic properties are formed by different connexin proteins. METHODS: To determine which connexins were likely to be expressed in the sinus node, atrioventricular (AV) node and atrial and ventricular myocardium, messenger ribonucleic acids (RNAs) from each of these sites were hybridized with probes for connexin26, connexin31, connexin32, connexin37, connexin40, connexin43, connexin45, connexin46 and connexin50. Immunostaining with monospecific antibodies to connexin40, connexin43 and connexin45 was used to delineate the distribution of connexins in frozen sections of these different cardiac tissues. RESULTS: Only messenger RNAs coding for connexin40, connexin43 and connexin45 were detected by Northern blot analysis. By immunohistochemical staining, junctions in the sinus and AV nodes and proximal His bundle were virtually devoid of connexin43 but contained both connexin40 and connexin45. Gap junctions in the distal His bundle and the proximal bundle branches stained intensely for connexin40 and connexin43 and to a lesser extent for connexin45. Atrial gap junctions showed abundant staining of connexin43, connexin40 and connexin45. Ventricular gap junctions were characterized by abundant staining of connexin43 and connexin45 and much less staining of connexin40. CONCLUSIONS: Although most cardiac gap junctions contain connexin40, connexin43 and connexin45, the relative amounts of each of these connexins vary considerably in cardiac tissues with different conduction properties.

Animals↗

PubMed1994

Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities.

Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the open-closed gating of an aqueous pore permeable to ions and molecules of up to 1 kDa or 10-14 A in diameter. We symmetrically altered the ionic composition or asymmetrically added 6-carboxyfluorescein (6-CF, M(r) = 376), a fluorescent tracer, to pairs of connexin37-transfected mouse neuro2A cells to examine the ionic and dye permeability of human connexin37 channels. We demonstrate that the 300-pS channel formed by connexin37 has an effective relative anion/cation permeability ratio of 0.43, directly converts to at least one intermediate (63 pS) subconductance state, and that 6-CF dye transfer is accompanied by a 24% decrease in unitary channel conductance. These observations favor a new interpretation of the gap junction pore consistent with direct ion-channel interactions or electrostatic charge effects common to more conventional multistate ion channels. These results have distinct implications about the different forms of intercellular signaling (cationic, ionic, and/or biochemical) that can occur depending on the expression and conformation of the connexin channel proteins.

Animals↗

PubMed1994

Tissue-specific determinants of anisotropic conduction velocity in canine atrial and ventricular myocardium.

Electrical conduction is very rapid and highly anisotropic in atrial fiber bundles, such as the crista terminalis. In contrast to left ventricular myocardium in which the ratio of longitudinal to transverse conduction velocities is approximately 3, propagation velocity in the crista terminalis is approximately 10 times greater in the longitudinal than in the transverse direction. To elucidate potential determinants of these distinct conduction properties, we characterized structural and molecular features of intercellular coupling in the crista terminalis and left ventricular myocardium of the canine heart. Analysis of the number and spatial orientation of myocyte interconnections at gap junctions revealed that a typical left ventricular myocyte was connected to 11.3 +/- 2.2 other myocytes. Approximately equal numbers of connections occurred between ventricular myocytes juxtaposed in side-to-side and end-to-end orientation. In contrast, a typical myocyte of the crista terminalis was connected to only 6.4 +/- 1.7 other cells (P < .05), but nearly 80% of these connections occurred between cells oriented in an end-to-end configuration. In comparison with the ventricular pattern, this spatial distribution of connections would limit intercellular current transfer between laterally apposed cells and thereby enhance anisotropy of conduction velocity in the longitudinal and transverse directions. Ultrastructural analysis showed that crista terminalis myocytes were connected by numerous small gap junctions that occurred in relatively simple, straight intercalated disks. Northern blot analysis showed approximately equivalent amounts of mRNAs encoding the gap junction channel proteins connexin43 and connexin45 but approximately four times more connexin40 mRNA in crista terminalis than in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Selective dye and ionic permeability of gap junction channels formed by connexin45.

Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the gating of an aqueous pore permeable to ions and molecules of up to 1 kD or 8 to 14 A in diameter. We performed ion-substitution and dye-transfer experiments to determine the relative Cl-/K+ conductance and dye permeability of anionic fluorescein derivatives in chick connexin45 (Cx45) channels. We demonstrate that Cx45 forms a 26 +/- 6-picosiemen (pS) channel with a maximum detectable Cl- permeability of 0.2 relative to K+ or Cs+. Although homogeneous channel conductances were observed in multichannel recordings, the open probability estimates were indicative of nonhomogeneous gating behavior and occasional cooperativity. A second conductance state of 19 +/- 4 pS begins to predominate at higher voltages. Cx45 gap junctions are permeable to 2',7'-dichlorofluorescein but are not permeable to the more polar 6-carboxyfluorescein dye. These observations suggest that the Cx45 pore diameter is approximately 10 A and is associated with a fixed negative charge within the junctional channel.

Animals↗

PubMed1994

Expression of multiple gap junction proteins in human fetal and infant hearts.

Mammalian cardiac myocytes express multiple gap junction channel proteins or connexins. Expression patterns of the avian homologues of the mammalian cardiac connexins change during cardiac morphogenesis in association with changes in the electrophysiologic properties of intercellular junctions in chick cardiac myocytes. To determine whether expression of cardiac connexins is developmentally regulated in humans, we characterized connexin mRNA and protein content and distribution in hearts of 11 human fetuses (74 to 122 d gestational age), seven children (0.5 mo to 3 y of age), and two adults. Northern blot analysis identified transcripts of connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45) genes in all hearts analyzed. Cx40 mRNA was approximately 5-fold more abundant in samples from fetal hearts than in hearts of children or adults. However, fetal samples used for RNA extraction included atrial as well as ventricular myocardium, whereas samples from children and adults were exclusively ventricular. Northern analysis of adult human right atrial appendages revealed abundant Cx40 mRNA, thus suggesting that the greater amount of Cx40 signal seen on Northern blots from fetal hearts could have been attributable to atrial contributions. Neither Cx43 nor Cx45 mRNA varied significantly in amount in samples from the different developmental stages analyzed. Immunofluorescence identified abundant Cx43 in the known distribution of gap junctions in myocytes in sections of all hearts. Cx45 staining was inconspicuous in fetal hearts but was readily apparent in cardiac myocytes in hearts of older subjects. In contrast, Cx40 staining in the ventricle was confined to mural coronary arteries, apparently in endothelial cells, whereas in the atrium Cx40 staining at myocyte junctions was abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗

PubMed1994

Characterization of the gap junction protein connexin56 in the chicken lens by immunofluorescence and immunoblotting.

PURPOSE: To characterize the chicken lens gap junction protein, connexin56 (Cx56). METHODS: The methods used were immunoblotting, immunofluorescence, alkaline phosphatase treatment, in vitro translation, and primary tissue culture. RESULTS: Connexin56 translated in vitro showed a single band with an electrophoretic mobility of approximately 66 kd. Multiple bands of 67 to 90 kd were detected in immunoblots of chicken lens homogenates using antibodies raised against a peptide from Cx56. Most, if not all, of these bands represented different phosphorylated forms of Cx56, because immunoreactive Cx56 was detected as a doublet of 65 to 67 kd after treatment of lens homogenates with alkaline phosphatase. Indirect immunofluorescence demonstrated that Cx56 was localized at membrane appositions between lens fibers and bow region cells. Levels of Cx56 increased from embryonic days 4 to 15; thereafter, levels remained fairly constant until hatching, after which they declined. Before embryonic day 9, the slowest migrating bands were not as abundant as they were at later ages. After embryonic day 20, less Cx56 was observed by immunofluorescence in the nucleus than in the cortex; however, both regions had similar levels of Cx56 as measured by immunoblotting. The pattern of bands differed between the two lens regions, suggesting differential protein modification. Immunoreactive Cx56 bands of 35 to 38 kd were detected unless homogenates were prepared in the presence of ethylenediaminetetraacetic acid (EDTA). Cx56 was also detected in lentoid-containing primary cultures derived from chicken lens. CONCLUSIONS: Cx56 is a phosphoprotein. Its appearance and modification by phosphorylation, as detected by immunoblotting, correlate with lens fiber differentiation.

Amino Acid Sequence↗

PubMed1994

Bovine connexin44, a lens gap junction protein: molecular cloning, immunologic characterization, and functional expression.

PURPOSE: To identify, clone molecularly, characterize immunochemically, and express functionally a bovine lens gap junction protein (connexin). METHODS: The methods used were polymerase chain reaction, genomic cloning, RNA and DNA blotting, bacterial expression of a fusion protein, immunoblotting, alkaline phosphatase treatment, Xenopus oocyte expression, and voltage clamp technique. RESULTS: A bovine genomic clone encoding a polypeptide of 44,424 d, termed connexin44 (Cx44), was isolated. Cx44 was most closely related to the lens connexins rat Cx46 and chicken Cx56. The Cx44 DNA hybridized to a 2.5 kb mRNA detected only in lens RNA. The carboxyl terminal 161 amino acids from Cx44 were expressed in bacteria fused to maltose binding protein (MBP). The Cx44/MBP fusion protein reacted in immunoblots with anti-rat Cx46(411 to 416) antibodies and with the monoclonal antibody 5H1, but not with a monoclonal antibody to MP70 nor with antibodies to other connexins. Cx44 translated in vitro from the cloned DNA showed a single band with an apparent electrophoretic mobility of approximately 50 kd on polyacrylamide gels containing sodium dodecyl sulfate. Multiple bands of 53 to 57 kd were detected by immunoblotting in homogenates of bovine lens; these bands were reduced to a broad band of approximately 50 kd by alkaline phosphatase treatment, suggesting that they represented phosphorylated forms of Cx44. Cx44 RNA injected in single oocytes induced a large and characteristic time- and voltage-dependent current. Overexpression of Cx44 produced depolarization and cell lysis. Junctional currents that could be regulated by transjunctional voltage were induced between paired oocytes injected with Cx44 RNA. Observations in paired oocytes suggested the assembly of hemichannels into junctional channels. CONCLUSIONS: Cx44 is a phosphoprotein component of bovine lens fiber gap junctions. Although it has a relatively distinct sequence, it shares sequence similarity, immunologic cross-reactivity, and electrophysiological properties with rat Cx46. These data suggest that Cx44 is the protein previously identified in several immunohistochemical studies of bovine lens gap junctions that used anti-rat Cx46 antibodies. They also suggest that the formation of intercellular channels by pairing of hemichannels might prevent the cell lysis induced by the opening of unpaired hemichannels.

Amino Acid Sequence↗

PubMed1993

Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression.

We used primers corresponding to the amino-terminal sequence shared by rat connexin-46 and ovine MP70 and a consensus sequence of the second extracellular loop conserved in all connexins to amplify and subsequently clone from chick genomic DNA a new member of the connexin family of gap junction proteins, chick connexin-56. The derived chick connexin-56 polypeptide contains 510 amino acids with a predicted molecular mass of 55,857 daltons. Although identical in the first 70 amino acids to rat connexin-46, chick connexin-56 diverges significantly in length and composition in predicted cytoplasmic regions, which have previously been inferred to determine functional and regulatory specificity. We were able to detect hybridization of connexin-56 probes only to RNA derived from lens. Connexin-56 was functionally expressed by the stable transfection of communication-deficient Neuro2A cells. The connexin-56-transfected cells demonstrated intercellular coupling by transfer of microinjected 6-carboxyfluorescein. Double whole-cell patch clamp recordings demonstrated electrical coupling. The induced intercellular conductances were insensitive to uncoupling by heptanol, octanol, or acidification. This behavior of chick connexin-56 may explain previous observations of the unusual physiology of lens fiber gap junctions.

Amino Acid Sequence↗

PubMed1993

Distinct patterns of connexin expression in canine Purkinje fibers and ventricular muscle.

Electrical conduction is more rapid in Purkinje fibers than in ventricular muscle, which are distinct cardiac tissues that have different active and passive electrophysiological properties. We have recently demonstrated that canine myocardium contains multiple gap junction proteins or connexins that form channels with unique electrophysiological properties. To determine whether differences in connexin expression may account, in part, for the characteristic conduction properties of Purkinje fibers and ventricular muscle, we assessed the amounts of mRNA for two connexins, Cx40 and Cx43, in these tissues obtained from canine hearts by Northern blot analysis and in situ hybridization. We also characterized the distribution and relative abundance of these two connexins in gap junctions with immunocytochemistry. A significantly greater amount of Cx40 mRNA was observed in Purkinje fibers compared with ventricular muscle, a difference that was at least threefold according to quantitative in situ hybridization (p < 0.001) and densitometric analysis of Northern blots. Purkinje fibers also demonstrated greater immunostaining intensity when incubated with anti-Cx40 antibodies than did ventricular muscle. In contrast, Cx43 mRNA and protein appeared to be abundant in both tissues. Quantitative in situ hybridization demonstrated a modest but not statistically significant increase in Cx43 mRNA in Purkinje fibers compared with ventricular myocardium. These results indicate that Purkinje fibers and ventricular muscle express distinct patterns of connexins. This tissue-specific pattern of connexin expression could contribute to differences in the conduction properties of Purkinje fibers and ventricular muscle.

Animals↗

PubMed1993

Connexin expression in the developing avian cardiovascular system.

Recent observations have suggested that the patterns of expression of the gap junction protein connexin43 in the developing cardiovascular system of the avian embryo diverge significantly from the patterns previously seen in mammalian species. Therefore, a detailed analysis of connexin43 expression in the chicken embryo was performed by use of immunofluorescent localization with two different connexin43-specific antipeptide antibodies as well as Western and Northern blot analysis. Connexin43 protein was not detected in the avian myocardium, the venous system, or the smaller vessels of the arterial system. Rather, it was limited exclusively to the vessels of the arterial outflow tract in a concentric pattern that became evident by embryonic day 8. Double staining with anti-alpha-smooth muscle actin and connexin43 demonstrated colocalization in the media of outflow tract vessel walls. The developmental expression of connexin43 was found to mirror the spatial patterning of secondary actin; connexin43, however, preceded the expression of secondary actin by a period of 1-2 days. In contrast, antibodies to a related gap junction protein (connexin42) revealed an absence of immunostaining in the avian outflow tract. Double staining with anti-connexin42 and anti-A-cell adhesion molecule (specific for avian intercalated discs) demonstrated colocalization between cardiac myocytes, indicating that connexin42 is a constituent of avian myocardial gap junctions. In light of these findings, developmental expression of differing myocardial connexins may reconcile previous studies showing different physiological properties of avian and mammalian cardiac gap junctions.

Animals↗

PubMed1993

Multiple connexins colocalize in canine ventricular myocyte gap junctions.

We have recently shown that adult canine ventricular myocytes express three distinct gap junction channel proteins, connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). These proteins have unique cytoplasmic domains that likely confer connexin-specific physiological properties. To determine whether the three distinct channel proteins are distributed in identical or different populations of gap junctions, we performed double-label immunofluorescence on disaggregated canine ventricular myocytes incubated simultaneously with a mouse monoclonal anti-Cx43 and affinity-purified polyclonal rabbit antibodies against Cx40 or Cx45. Analysis of double-labeled cardiac myocytes using laser scanning confocal microscopy revealed virtually identical patterns of immunoreactivity for both the Cx43/Cx40 and Cx43/Cx45 pairs. Double-label immunoelectron microscopy confirmed that ultrastructurally identified cardiac myocyte gap junctions contain multiple channel proteins. Thus, three channel proteins colocalize in canine cardiac myocyte gap junctions. The presence of multiple functionally distinct connexins suggests complex possibilities regarding the composition of individual channels and the regulation of intercellular coupling.

Animals↗

PubMed1993

Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins.

Alcohols↗

PubMed1993

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

Alkaline Phosphatase↗

PubMed1993

Gap junctions.

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Animals↗