Biomedical subjects

E C Beyer

Publications and source records attributed to E C Beyer.

At least 37 records · Page 2Linked to original sources

PubMed2000

PKC isoenzymes in the chicken lens and TPA-induced effects on intercellular communication.

PURPOSE: Because lens connexins are phosphoproteins and intercellular communication between lens cells may be modulated by connexin phosphorylation, experiments were designed to characterize the expression of protein kinase C (PKC) isoenzymes in the chicken lens and in lentoid-containing cultures and to study the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment on the distribution of PKC isoenzymes and intercellular communication. METHODS: The presence and distribution of PKC isoenzymes were studied by immunoblot analysis and immunofluorescence in chicken lens sections and in cell cultures under control conditions and after treatment with TPA. Intercellular communication was assessed by transfer of microinjected Lucifer yellow. RESULTS: PKC alpha, gamma, iota, epsilon, and mu were detected in lens homogenates by immunoblot analysis. The levels of PKC alpha, gamma, iota, and mu decreased between the 7th and the 18th embryonic days. Levels of PKC epsilon remained relatively constant during the period of study. Similarly, lens cells in culture expressed isoenzymes alpha, gamma, epsilon, iota, and mu. PKC beta was not detected in lens or culture homogenates. In lens sections, all PKC isoenzymes analyzed were present in epithelial cells, in the annular pad region, and in the posterior aspect of fiber cells. The anti-PKC gamma antibody also stained fiber cell membranes. Analysis of lentoid cultures by immunofluorescence revealed that PKC gamma, epsilon, and iota and minimal amounts of PKC alpha were present in lentoid cells. Treatment with 200 nM TPA for 15 to 30 minutes induced translocation of PKC gamma to the plasma membrane of lentoid cells and significantly reduced the transfer of microinjected Lucifer yellow. CONCLUSIONS: Several PKC isoenzymes are expressed by lens cells in situ and in culture. The gamma isoenzyme, present in lens fibers, was activated in lentoid cells by TPA, a known activator of PKC. We have previously demonstrated TPA-induced phosphorylation of the gap junction protein connexin56 (Cx56). The new data presented in the current study demonstrate that TPA treatment also decreased intercellular communication. Taken together, the results suggest that differential phosphorylation of Cx56 by PKCgamma may induce a conformational change in the protein which, in turn, might lead to channel closure.

Animals↗

PubMed1999

Cultured chicken embryo lens cells resemble differentiating fiber cells in vivo and contain two kinetic pools of connexin56.

The lens is an avascular organ in which gap junctions play a pivotal role for cell physiology and transparency. Here we evaluate a lens culture system as a model for studies of lens gap junction dynamics. In culture, chicken embryo lens cells initially form a monolayer of epithelial cells. Subsequently, the epithelial cells differentiate into lentoids, birefringent multicellular structures composed of fiber-like cells. We examined the cultures for the expression of cellular markers and lens fiber specific proteins using immunofluorescence and immunoblot analysis. We also determined the half-life of connexin56 (Cx56), a fiber-specific gap junction protein. All lens cells in culture expressed actin, endoplasmic reticulum proteins and N-cadherin. Only lentoid cells expressed the lens fiber connexins, Cx45.6 and Cx56. Cx56 localized at appositional membranes and did not co-localize with endoplasmic reticulum proteins or N-cadherin. Two pools of Cx56 were detected in these cultures, one with a half-life of a few hours and the other with a half-life of days. The two pools contained phosphorylated forms of Cx56 of different apparent molecular weights. These results suggest that lens cells in culture can be used as a model for the study of lens biology. They also suggest that phosphorylation of Cx56 might be regulating the stability of the protein.

Animals↗

PubMed1999

Co-expression of lens fiber connexins modifies hemi-gap-junctional channel behavior.

Lens fiber cells contain two gap junction proteins (Cx56 and Cx45.6 in the chicken). Biochemical studies have suggested that these two proteins can form heteromeric connexons. To investigate the biophysical properties of heteromeric lens connexons, Cx56 was co-expressed with Cx45.6 (or its mouse counterpart, Cx50) in Xenopus oocytes. Whole-cell and single-channel currents were measured in single oocytes by conventional two-microelectrode voltage-clamp and patch clamp techniques, respectively. Injection of Cx56 cRNA induced a slowly activating, nonselective cation current that activated on depolarization to potentials higher than -10 mV. In contrast, little or no hemichannel current was induced by injection of Cx50 or Cx45.6 cRNA. Co-expression of Cx56 with Cx45.6 or Cx50 led to a shift in the threshold for activation to -40 or -70 mV, respectively. It also slowed the rate of deactivation of the hemichannel currents. Moreover, an increase in the unitary conductance, steady state probability of hemichannel opening and mean open times at negative potentials, was observed in (Cx56 + Cx45.6) cRNA-injected oocytes compared with Cx56 cRNA-injected oocytes. These results indicate that co-expression of lens fiber connexins gives rise to novel channels that may be explained by the formation of heteromeric hemichannels that contain both connexins.

Animals↗

PubMed1999

Molecular mechanism underlying a Cx50-linked congenital cataract.

Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526-532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of approximately 5 microS 4-7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of approximately 8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.

Animals↗

PubMed1998

Rapid turnover of connexin43 in the adult rat heart.

Remodeling of the distribution of gap junctions is an important feature of anatomic substrates of arrhythmias in patients with healed myocardial infarcts. Mechanisms underlying this process are poorly understood but probably involve changes in gap junction protein (connexin) synthesis, assembly into channels, and degradation. The half-life of the principal cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours in primary cultures of neonatal myocytes, but it is unknown whether rapid turnover of Cx43 occurs in the adult heart or is unique to disaggregated neonatal myocytes that are actively reestablishing connections in vitro. To characterize connexin turnover dynamics in the adult heart and to elucidate its potential role in remodeling of gap junctions, we measured Cx43 turnover kinetics and characterized the proteolytic pathways involved in Cx43 degradation in isolated perfused adult rat hearts. Hearts were labeled for 40 minutes with Krebs-Henseleit buffer containing [35S]methionine, and then chase perfusions were performed with nonradioactive buffer for 0, 60, 120, and 240 minutes. Quantitative immunoprecipitation assays of Cx43 radioactivity in 4 hearts at each time point yielded a monoexponential decay curve indicating a Cx43 half-life of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation were elucidated by perfusing isolated rat hearts for 4 hours with specific inhibitors of either lysosomal or proteasomal proteolysis. Immunoblot analysis demonstrated significant increases ( approximately 30%) in Cx43 content in hearts perfused with either lysosomal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perfused with lysosomal inhibitors consisted of phosphorylated isoforms, whereas nonphosphorylated Cx43 accumulated selectively in hearts perfused with a specific proteasomal inhibitor. These results indicate that Cx43 turns over rapidly in the adult heart and is degraded by multiple proteolytic pathways. Regulation of Cx43 degradation could play an important role in gap junction remodeling in response to cardiac injury.

Animals↗

PubMed1998

Differential expression of gap junction proteins in the canine sinus node.

Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: approximately 55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.

Animals↗

PubMed1998

Disparate effects of deficient expression of connexin43 on atrial and ventricular conduction: evidence for chamber-specific molecular determinants of conduction.

BACKGROUND: Myocardial conduction depends on intercellular transfer of current at gap junctions. Atrial myocytes express three different gap junction channel proteins-connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40)-- whereas ventricular myocytes express only Cx43 and Cx45. However, the physiological roles of individual connexins are unknown. We have previously shown that mice heterozygous for a null mutation in the gene encoding Cx43 (Cx43(+/-) mice) express 50% of the normal amount of Cx43 in ventricular myocardium and exhibit marked slowing of ventricular conduction. METHODS AND RESULTS: To determine whether atrial conduction is affected in Cx43(+/-) mice, we measured atrial conduction velocity in isolated hearts, performed detailed ECG and electrophysiological studies in intact animals, and determined the amount of cardiac connexins in atrial and ventricular tissue. Ventricular conduction velocity was reduced by 38% in Cx43(+/-) mice compared with wild-types, but atrial conduction velocity in the same hearts was normal. QRS duration was significantly greater in Cx43(+/-) mice than in wild-types, but P-wave duration and amplitude did not differ. Atrial expression of Cx43 was reduced by 50%. CONCLUSIONS: These results indicate that Cx43 is a principal conductor of intercellular current in the ventricle because ventricular conduction is significantly slowed when Cx43 content is reduced by only 50%. In contrast, a similar reduction in Cx43 content in atrial muscle has no effect on atrial conduction, suggesting that Cx40 (which is expressed in atrial but not ventricular myocytes) is a major electrical coupling protein in atrial muscle. Thus, Cx43 and Cx40 may be chamber-specific determinants of myocardial conduction.

Animals↗

PubMed1998

Regulation of connexin43 expression and function by prostaglandin E2 (PGE2) and parathyroid hormone (PTH) in osteoblastic cells.

Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of negatively charged dyes between UMR 106-01 cells. This stimulatory effect lasted for at least 4 h. Both PGE2 and PTH increased steady-state levels of Cx43 mRNA, but only after 2-4 h of incubation. Transfection with a Cx43 gene construct linked to luciferase showed that this effect of PTH was the result of transcriptional upregulation of Cx43 promoter. Stimulation of dye coupling and Cx43 gene transcription were reproduced by forskolin and 8Br-cAMP. Exposure to PGE2 for 30 min increased Cx43 abundance at appositional membranes in UMR 106-01, whereas total Cx43 protein levels increased only after 4-6 h of incubation with either PGE2 or PTH. Inhibition of protein synthesis by cycloheximide did not affect this early stimulation of dye coupling, but it significantly inhibited the sustained effect of PTH and forskolin on cell coupling. In summary, both PTH and PGE2, presumably through cAMP production, enhance gap junctional communication in osteoblastic cell cultures via two mechanisms: initial rapid redistribution of Cx43 to the cell membrane, and later stimulation of Cx43 gene expression. Modulation of intercellular communication represents a novel mechanism by which osteotropic factors regulate the activity of bone forming cells.

Animals↗

PubMed1998

Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes.

OBJECTIVE: The present studies were performed to examine the degradation of connexin43-containing gap junctions by the lysosome or the proteasome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. METHODS: Primary cultures were prepared from neonatal rat ventricular myocytes. Connexin43 was detected by immunoblotting, immunofluorescence, or immunoprecipitation. Gap junction profiles were detected by transmission electron microscopy. RESULTS: Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures treated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or ammonium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse-chase experiments showed that the half-life of connexin43 was 1.4 h in control cultures, but was prolonged to 2.0 or 2.8 h in cultures treated with chloroquine or lactacystin, respectively. Immunofluorescence and electron microscopy showed a significant increase in the number of gap junction profiles in myocytes treated with either chloroquine or lactacystin. Heat treatment of cultures (43.5 degrees C for 30 min) produced a rapid loss of connexin43 as detected by immunoblotting or immunofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexin43 levels and distribution returned to normal by 3 h following a heat shock and were resistant to a subsequent repeat heat stress. The heat shock also led to production of HSP70 as detected by immunoblotting. CONCLUSIONS: These data suggest that Cx43 gap junctions in myocytes are degraded by the proteasome and the lysosome, that this proteolysis can be augmented by heat stress, and that inducible factors such as HSP70 may protect against Cx43 degradation.

Acetylcysteine↗

PubMed1998

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes.

OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.

Angiotensin II↗

PubMed1997

Mouse connexin40: gene structure and promoter analysis.

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3'-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5' of the transcriptional start site and 400 bp 3' of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297.

Animals↗

PubMed1997

Degradation of connexin43 gap junctions involves both the proteasome and the lysosome.

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1-3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of approximately 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.

Animals↗

PubMed1997

Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.

To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.

Action Potentials↗

PubMed1997

The gap-junction protein connexin 56 is phosphorylated in the intracellular loop and the carboxy-terminal region.

The lens gap-junction protein, connexin 56, is modified by phosphorylation. Two-dimensional mapping of tryptic phosphopeptides of 32P-labeled connexin 56 from primary chicken-lens cultures showed that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) induced an increase in phosphorylation of connexin 56 at specific constitutively phosphorylated sites. Treatment with 8-Br-cAMP or forskolin did not induce substantial changes in connexin 56 phosphorylation. Two phosphorylation sites within connexin 56, S493 and S118, were identified after HPLC purification and peptide sequencing of tryptic phosphopeptides from bacterially expressed connexin 56 fusion proteins phosphorylated by protein kinase C or protein kinase A in vitro. Comparisons of the two-dimensional maps of tryptic phosphopeptides from in vitro phosphorylated connexin 56 fusion proteins and in vivo phosphorylated connexin 56 showed that S493 and S118 were constitutively phosphorylated in lentoid-containing cultures, and that treatment with TPA induced an increase in phosphorylation of the peptides containing S118. It is suggested that phosphorylation of connexin 56 at S118 is involved in the TPA-induced decrease in intercellular communication and acceleration of connexin 56 degradation.

Amino Acid Sequence↗

PubMed1997

Differential regulation of connexin43 and connexin37 in endothelial cells by cell density, growth, and TGF-beta1.

We studied the growth-related expression of the gap junction proteins connexin43 (Cx43) and connexin37 (Cx37) to characterize mechanisms of their differential regulation in cultured bovine aortic endothelial cells. During growth to confluency, Cx43 mRNA levels were high in subconfluent cells and decreased at confluency; Cx37 mRNA was weakly detectable until the cultures became confluent, when Cx37 levels became similar to those of Cx43. Immunoprecipitation, immunoblots, and immunostaining demonstrated that Cx43 synthesis and content paralleled the changes in mRNA levels. These data suggested regulation of connexin expression related to growth status or cell density. We tested this hypothesis by inhibiting growth with transforming growth factor-beta1 (TGF-beta1). TGF-beta1 treatment caused an upregulation of Cx43 synthesis, content, and apparent half-life and an upregulation of mRNA, independent of changes in cell density. Increases in Cx43 synthesis preceded increases in mRNA, suggesting both translational and transcriptional regulation, whereas the increased half-life suggested post-translational regulation, as well. Immunostaining revealed the development of intense vesicular staining in the treated cells, which may explain the increased half-life. TGF-beta1 treatment also suppressed the upregulation of Cx37 expression. These alterations in connexin expression may have implications for endothelial communication under conditions of elevated vascular TGF-beta1 concentrations such as in wound healing.

Animals↗

PubMed1997

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37.

Homomeric gap junction channels are composed solely of one connexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different from each other. A heteromeric gap junction channel is one that contains different connexins within either or both hemichannels. The existence of heteromeric forms has been suggested, and many cell types are known to coexpress connexins. To determine if coexpressed connexins would form heteromers, we cotransfected rat connexin43 (rCx43) and human connexin37 (hCx37) into a cell line normally devoid of any connexin expression and used dual whole cell patch clamp to compare the observed gap junction channel activity with that seen in cells transfected only with rCx43 or hCx37. We also cocultured cells transfected with hCx37 or rCx43, in which one population was tagged with a fluorescent marker to monitor heterotypic channel activity. The cotransfected cells possessed channel types unlike the homotypic forms of rCx43 or hCx37 or the heterotypic forms. In addition, the noninstantaneous transjunctional conductance-transjunctional voltage (Gj/Vj) relationship for cotransfected cell pairs showed a large range of variability that was unlike that of the homotypic or heterotypic form. The heterotypic cell pairs displayed asymmetric voltage dependence. The results from the heteromeric cell pairs are inconsistent with summed behavior of two independent homotypic populations or mixed populations of homotypic and heterotypic channels types. The Gj/Vj data imply that the connexin-to-connexin interactions are significantly altered in cotransfected cell pairs relative to the homotypic and heterotypic forms. Heteromeric channels are a population of channels whose characteristics could well impact differently from their homotypic counterparts with regard to multicellular coordinated responses.

Animals↗

PubMed1997

Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells.

Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.

Animals↗

PubMed1996

Expression of zebrafish connexin43.4 in the notochord and tail bud of wild-type and mutant no tail embryos.

Communication via gap junctions provides a mechanism for the cell-cell transfer and coordination of developmental signals. The spatial restriction of gap junctions may also serve to organize cells into domains of coordinated behavior. To investigate the role of gap junctions during embryogenesis, we have characterized the expression of a member of the gap junction gene family, zebrafish connexin43.4, a homolog of connexin45 in chicken and mammals. Expression of connexin43.4 was induced in the early gastrula, coincident with the first definitive assignments of axial cell fate and the onset of the cell movements comprising convergence and extension in zebrafish. In situ hybridization and immunohistochemistry revealed that during gastrulation connexin43.4 mRNA and protein were progressively enriched in the germ ring and in the notochord primordia on the dorsal side of the embryo. Later in development connexin43.4 expression was detected in the notochord, the paraxial mesoderm, and the tail bud but was not observed after the differentiation of these tissues. In no tail mutant embryos which are defective in tail formation and proper morphogenesis of the notochord, connexin43.4 expression was absent during gastrulation from the caudal embryonic shield and notochord primordia. During somite stages in no tail embryos, connexin43.4 expression remained absent in the notochordal precursor cells and was lost in the tail bud. Thus, the no tail gene product, a transcription factor, was required for the expression of connexin43.4 in both the notochord and tail bud during morphogenesis. By microinjection of mRNA coding for a connexin43.4/green fluorescent protein fusion in the 1-cell zebrafish embryo, we showed that connexin43.4 is capable of assembling into structures reminiscent of gap junctions. The progressively restricted, developmental expression of the zebrafish connexin43.4 gene suggests that this gap junctional protein participates in the coordination of gastrulation and the formation of the notochord and tail.

Animals↗