Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|R, Ca, Ch, H, M||WB, ELISA, ICC, IF, IHC, IP||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
References | 33 Available | See All References
|Reference overview||Pub Med ID|
|Inducible coexpression of connexin37 or connexin40 with connexin43 selectively affects intercellular molecular transfer. |
Joanna Gemel,Tasha K Nelson,Janis M Burt,Eric C Beyer
The Journal of membrane biology 245 2012
Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.
|Cell-to-cell coupling in engineered pairs of rat ventricular cardiomyocytes: relation between Cx43 immunofluorescence and intercellular electrical conductance. |
Megan L McCain,Thomas Desplantez,Nicholas A Geisse,Barbara Rothen-Rutishauser,Helene Oberer,Kevin Kit Parker,Andre G Kleber
American journal of physiology. Heart and circulatory physiology 302 2012
Gap junctions are composed of connexin (Cx) proteins, which mediate intercellular communication. Cx43 is the dominant Cx in ventricular myocardium, and Cx45 is present in trace amounts. Cx43 immunosignal has been associated with cell-to-cell coupling and electrical propagation, but no studies have directly correlated Cx43 immunosignal to electrical cell-to-cell conductance, g(j), in ventricular cardiomyocyte pairs. To assess the correlation between Cx43 immunosignal and g(j), we developed a method to determine both parameters from the same cell pair. Neonatal rat ventricular cardiomyocytes were seeded on micropatterned islands of fibronectin. This allowed formation of cell pairs with reproducible shapes and facilitated tracking of cell pair locations. Moreover, cell spreading was limited by the fibronectin pattern, which allowed us to increase cell height by reducing the surface area of the pattern. Whole cell dual voltage clamp was used to record g(j) of cell pairs after 3-5 days in culture. Fixation of cell pairs before removal of patch electrodes enabled preservation of cell morphology and offline identification of patched pairs. Subsequently, pairs were immunostained, and the volume of junctional Cx43 was quantified using confocal microscopy, image deconvolution, and three-dimensional reconstruction. Our results show a linear correlation between g(j) and Cx43 immunosignal within a range of 8-50 nS.
|Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits. |
Helga Reinshagen,Claudia Auw-Haedrich,Ruediger V Sorg,Daniel Boehringer,Philipp Eberwein,Johannes Schwartzkopff,Rainer Sundmacher,Thomas Reinhard
Acta ophthalmologica 89 2011
To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits.
|E-cadherin contributes to the bystander effect of TK/GCV suicide therapy and enhances its antitumoral activity in pancreatic cancer models. |
L Garcia-Rodríguez,D Abate-Daga,A Rojas,J R González,C Fillat
Gene therapy 18 2011
The thymidine kinase/ganciclovir (TK/GCV) cancer gene therapy approach is based on inducing GCV metabolite cytotoxicity in tumor cells expressing the herpes simplex virus TK gene and exposed to GCV. A bystander effect, mediated by gap junctions, accounts for the transfer of toxic metabolites from TK-expressing cells to neighboring cells. It has been proposed that E-cadherin participates in the formation and function of such gap junctions. In this study we investigate the influence of E-cadherin on TK/GCV suicide therapy with a panel of cellular and in vivo models of pancreatic ductal adenocarcinoma. We observed a strong correlation of E-cadherin expression and the TK/GCV bystander effect, associated with the modulation of gap junction communication and connexin expression or localization. Importantly, the co-expression of TK and E-cadherin genes in the adenoviral vector AdTat8TKIE improved TK/GCV cytotoxicity and triggered a potent antitumoral effect, superior to standard AdTat8TK/GCV in MIAPaCa-2 xenografts. The increased expression of E-cadherin resulted in the reduction of the bcl-2 content. Interestingly, the knockdown of bcl-2 sensitized cells to TK/GCV. Thus, we propose that by restoring E-cadherin in pancreatic tumor cells we will improve TK/GCV therapy, both by enhancing the bystander effect and by facilitating the induction of apoptosis.
|Connexin-26 is a key factor mediating gemcitabine bystander effect. |
Garcia-Rodríguez L, Pérez-Torras S, Carrió M, Cascante A, García-Ribas I, Mazo A, Fillat C
Mol Cancer Ther 10 505-17. 2011
Gemcitabine is a nucleoside analogue with anticancer activity. Inside the cell, it is sequentially phosphorylated to generate the active drug. Phosphorylated nucleoside analogues have been shown to traffic through gap junctions. We investigated the participation of gap junctional intercellular communication (GJIC) as a possible mechanism spreading gemcitabine cytotoxicity in pancreatic tumors. Immunohistochemical analysis of pancreatic cancer biopsies revealed increased connexin 26 (Cx26) content but loss of connexins 32 (Cx32) and 43 (Cx43) expression. Cx26 abundance in neoplastic areas was confirmed by Cx26 mRNA in situ hybridization. Heterogeneity on the expression levels and the localization of Cx26, Cx32, and Cx43 were identified in pancreatic cancer cells and found to be associated with the extent of GJIC, and correlated with gemcitabine bystander cytotoxic effect. The abundance of Cx26 at the contact points in tumoral regions prompted us to study the involvement of Cx26 in the GJIC of gemcitabine toxic metabolites and their influence on the antitumoral effects of gemcitabine. Knockdown of Cx26 led to decreased GJIC and reduced gemcitabine bystander killing whereas overexpression of Cx26 triggered increased GJIC and enhanced the gemcitabine cytotoxic bystander effect. Gemcitabine treatment of mice bearing tumors, with a high GJIC capacity, resulted in a significant delay in tumor progression. Interestingly, gemcitabine administration in mice bearing tumors that overexpress Cx26 triggered a dramatic tumor regression of 50% from the initial volume. This study shows that Cx26 participates in the gap junction-mediated bystander cytoxic effect of gemcitabine and provides evidence that upregulation of Cx26 improves gemcitabine anticancer efficacy. Mol Cancer Ther; 10(3); 505-17. ©2011 AACR.
|Influence of v5/6-his tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45. |
Desplantez T, Halliday D, Dupont E, Severs NJ, Weingart R
J Membr Biol 240 139-50 2011
HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V (j)-sensitive gating of I (j) (V (j), gap junction voltage; I (j), gap junction current), (2) contribution and (3) kinetics of I (j) deactivation and (4) single-channel conductance. The first three reflect alterations of fast V (j) gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.
|Hippocampal seizures alter the expression of the pannexin and connexin transcriptome. |
Mylvaganam S, Zhang L, Wu C, Zhang ZJ, Samoilova M, Eubanks J, Carlen PL, Poulter MO
J Neurochem 112 92-102. Epub 2009 Oct 13. 2010
Some forms of seizure activity can be stopped by gap junctional (GJ) blockade. Here, we found that GJ blockers attenuate hippocampal seizure activity induced by a novel seizuregenic protocol using Co(2+). We hypothesized that this activity may occur because of the altered expression of connexin (Cx) and/or pannexin (Panx) mRNAs and protein. We found a 1.5-, 1.4-, and 2-fold increase in Panx1, Panx2, and Cx43 mRNAs, respectively. Significant post-translational modifications of the proteins Cx43 and Panx1 were also observed after the Co(2+) treatment. No changes were observed in the presence of tetrodotoxin, indicating that seizure activity is required for these alterations in expression, rather than the Co(2+) treatment itself. Further analysis of the QPCR data showed that the Cx and Panx transcriptome becomes remarkably re-organized. Pannexin (Panxs 1 and 2) and glial connexin mRNA became highly correlated to one another; suggesting that these genes formed a transcriptomic network of coordinated gene expression, perhaps facilitating seizure induction. These data show that seizure activity up-regulates the expression of both glial and neuronal GJ mRNAs and protein while inducing a high degree of coordinate expression of the GJ transcriptome.
|Changes of connexin43 expression in non-pregnant porcine myometrium correlate with progesterone concentration during oestrous cycle. |
J Karasinski,J Galas,D Semik,A Fiertak,B Bilinska,W M Kilarski
Reproduction in domestic animals = Zuchthygiene 45 2010
Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non-pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre-pubertal pigs (n?=?7) and mature pigs at pre-ovulatory (n?=?7), luteal (n?=?5) and late luteal (n?=?3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1?mm(2) of the myometrial section was low in pre-pubertal pigs and significantly higher (p?0.022) in pre-ovulatory animals. In relation to pre-ovulatory animals the number of myometrial gap junctions was significantly lower (p?0.019) at the luteal phase and correlated with significantly higher (p?0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre-pubertal pigs and mature animals at pre-ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.
|Activator of G protein signaling 8 (AGS8) is required for hypoxia-induced apoptosis of cardiomyocytes: role of G betagamma and connexin 43 (CX43). |
M Sato, Q Jiao, T Honda, R Kurotani, E Toyota, S Okumura, T Takeya, S Minamisawa, SM Lanier, Y Ishikawa
The Journal of biological chemistry 284 31431-40 2009
Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G betagamma subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a G betagamma-dependent manner by providing a scaffold composed of G betagamma and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the G betagamma inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-G betagamma signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.Full Text Article
|ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43. |
Vladimir Akoyev,Dolores J Takemoto
Cellular signalling 19 2007
We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific plaquetosome arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.Full Text Article
|Effects of rotigaptide, a gap junction modifier, on defibrillation energy and resuscitation from cardiac arrest in rabbits. |
Jing-quan Zhong, Gabriel Laurent, Petsy Pui-Sze So, Xudong Hu, James K Hennan, Paul Dorian
Journal of cardiovascular pharmacology and therapeutics 12 69-77 2007
The gap junction modifier Rotigaptide (ZP123), which promotes cellular coupling, was hypothesized to decrease defibrillation thresholds during prolonged ventricular fibrillation (VF). Thirty-two New Zealand white rabbits were randomized to receive saline (control, n = 16) or Rotigaptide (n = 16). Following 4 min of untreated VF, biphasic defibrillation shocks were applied through chest wall patches, starting either at 300 volts (V) (n = 16) or 500 V (n = 16), with 200 V increasing steps to 900 V in case of shock failure. Rotigaptide significantly decreased defibrillation voltage requirements (average cumulative voltage of all shocks: 1206 +/- 709 V in control group vs. 844 +/- 546 V in treated group, P = .002). Rotigaptide had no effect on heart rate, QRS duration, QT interval, ventricular effective refractory period, monophasic action potential duration or on connexin 43 density using immunofluorescence. Rotigaptide improves the ability to defibrillate after untreated VF.
|Age-related changes in conducted vasodilation: effects of exercise training and role in functional hyperemia. |
Bearden, Shawn E, et al.
Am. J. Physiol. Regul. Integr. Comp. Physiol., 293: R1717-21 (2007) 2007
Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 +/- 0.1, adult = 3.9 +/- 0.1, old = 2.2 +/- 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond approximately 20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation.
|High-pressure freezing and freeze substitution of rat myocardium for immunogold labeling of connexin 43. |
Christian Mühlfeld, Joachim Richter
The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology 288 1059-67 2006
The value of high-pressure freezing (HPF) and freeze substitution (FS) for immunoelectron microscopy (immuno-EM) of the heart was investigated in bioptic specimens taken from isolated hearts of 0-, 5-, and 14-day-old rats at baseline and at 15, 30, 45, and 60 min after induction of ischemia. The target antigen chosen here was the gap junction protein connexin 43 (Cx43). After HPF and FS, immunogold labeling was applied for detection of Cx43. Gold particles associated with gap junction areas, free plasma membrane, and annular gap junctions (AGJs) were counted and distributions compared by contingency table analysis. HPF and FS resulted in excellent preservation of antigenicity for Cx43. The mostly good preservation of the ultrastructure was limited by mechanical damage at the border and by ice crystal formation in the center of the tissue blocks. In normal myocardium of newborns, gold particles associated with free plasma membrane were frequently observed, with AGJs only seldom. In older rats, the opposite relation was found. During ischemia, no distribution changes occurred in newborn or 14-day-old rats. In 5-day-old rats, however, ischemia induced a shift of Cx43 from gap junction plaques to AGJs. In conclusion, HPF and FS are an ideal alternative to chemical fixation for immuno-EM as the excellent preservation of antigenicity is combined with a well-preserved ultrastructure. The results indicate that the process of degradation of gap junctions via AGJs gradually increases during postnatal rat heart development, a process that may be accelerated by ischemia in an early developmental state.
|Expression of Kir2.1 and Kir6.2 transgenes under the control of the alpha-MHC promoter in the sinoatrial and atrioventricular nodes in transgenic mice. |
H Dobrzynski, R Billeter, I D Greener, J O Tellez, N J Chandler, T P Flagg, C G Nichols, A N Lopatin, M R Boyett
Journal of molecular and cellular cardiology 41 855-67 2006
Kir2.1 and Kir6.2 are ion channel subunits partly responsible for the background inward rectifier and ATP-sensitive K(+) currents (I(K1) and I(KATP)) in the heart. Very little is known about how the distribution of ion channel subunits is controlled. In this study, we have investigated the expression (at protein and mRNA levels) of GFP-tagged Kir2.1 and Kir6.2 transgenes under the control of the alpha-MHC promoter in the sinoatrial node (SAN), atrioventricular node (AVN), His bundle and working myocardium of transgenic mice. After dissection, serial 10-microm cryosections were cut. Histological staining was carried out to identify tissues, confocal microscopy was carried out to map the distribution of the GFP-tagged ion channel subunits and in situ hybridization was carried out to map the distribution of corresponding mRNAs. We demonstrate heterologous expression of the ion channel subunits in the working myocardium, but not necessarily in the SAN, AVN or His bundle; the distribution of the subunits does not correspond to the expected distribution of alpha-MHC. Both protein and mRNA expression does, however, correspond to the expected distributions of native Kir6.2 and Kir2.1 in the SAN, AVN, His bundle and working myocardium. The data demonstrate novel transcriptional and/or post-transcriptional control of ion channel subunit expression and raise important questions about the control of regional expression of ion channels.
|Postganglionic nerve stimulation induces temporal inhibition of excitability in rabbit sinoatrial node. |
Vadim V Fedorov, William J Hucker, Halina Dobrzynski, Leonid V Rosenshtraukh, Igor R Efimov, Vadim V Fedorov, William J Hucker, Halina Dobrzynski, Leonid V Rosenshtraukh, Igor R Efimov
American journal of physiology. Heart and circulatory physiology 291 H612-23 2006
Vagal stimulation results in complex changes of pacemaker excitability in the sinoatrial node (SAN). To investigate the vagal effects in the rabbit SAN, we used optical mapping, which is the only technology that allows resolving simultaneous changes in the activation pattern and action potentials morphologies. With the use of immunolabeling, we identified the SAN as a neurofilament 160-positive but connexin 43-negative region (n = 5). Normal excitation originated in the SAN center with a cycle length (CL) of 405 +/- 14 ms (n = 14), spread anisotropically along the crista terminalis (CT), and failed to conduct toward the septum. Postganglionic nerve stimulation (PNS, 400-800 ms) reduced CL by 74 +/- 7% transiently and shifted the leading pacemaker inferiorly (78%) or superiorly (22%) from the SAN center by 2-10 mm. In the intercaval region between the SAN center and the septal block zone, PNS produced an 8 +/- 1-mm(2) region of transient hyperpolarization and inexcitability. The first spontaneous or paced excitation following PNS could not enter this region for 500-1,500 ms. Immunolabeling revealed that the PNS-induced inexcitable region is located between the SAN center and the block zone and has a 2.5-fold higher density of choline acetyltransferase than CT but is threefold lower than the SAN center. The fact that the inexcitability region does not coincide with the most innervated area indicates that the properties of the myocytes themselves, as well as intercellular coupling, must play a role in the inexcitability induction. Optically mapping revealed that PNS resulted in transient loss of pacemaker cell excitability and unidirectional entrance conduction block in the periphery of SAN.
|Human atrial conduction and arrhythmogenesis correlates with conformational exposure of specific epitopes on the connexin40 carboxyl tail. |
Prapa Kanagaratnam, Emmanuel Dupont, Stephen Rothery, Steven Coppen, Nicholas J Severs, Nicholas S Peters
Journal of molecular and cellular cardiology 40 675-87 2006
BACKGROUND: Gap junction expression is considered to influence myocardial conduction and arrhythmogenesis, but studies in patients with atrial fibrillation (AF) have reported inconsistent results. We used human atrial conduction and arrhythmogenicity to provide clinical parameters with which to correlate quantification of connexin40 (Cx40) by different techniques to address the hypothesis that antibody-epitope binding properties may influence quantification methods. METHODS AND RESULTS: Atrial conduction properties were studied in patients undergoing coronary artery bypass grafting (N = 27) using multi-electrode array mapping. Patients were defined as having a vulnerable atrial substrate if pacing-induced AF lasted for more than 30 s. Using antibodies targeted at two different epitopes of the cytoplasmic carboxyl tail, Cx40 signal was quantified by immunoconfocal microscopy in biopsies taken from the mapped atria. Only patients with a vulnerable substrate subsequently developed post-operative AF (P 0.02). Immunoconfocal Cx40 signal measured by one antibody, designated S15C(R85), was lower in patients with sustained induced AF (0.013 +/- 0.009 microm(2)/microm(2) vs. 0.024 +/- 0.010 microm(2)/microm(2), P = 0.005) and was negatively correlated with atrial conduction velocity during sinus rhythm (P 0.05). However, these relationships did not exist when the Cx40 signal was measured using a different antibody (Y21Y(R968)). Further studies suggested that quantification technique was reproducible against the same epitope (P 0.001) but not against different epitopes indicating variable exposure of Cx40 epitopes. CONCLUSIONS: Quantification of Cx40 by immunoconfocal microscopy appears to be epitope dependent and evidence of epitope masking raises the possibility of more than one conformational form of the Cx40 carboxyl tail, suggesting functionally important conformations of the protein.
|Ultrastructural localization of connexins (Cx36, Cx43, Cx45), glutamate receptors and aquaporin-4 in rodent olfactory mucosa, olfactory nerve and olfactory bulb. |
John E Rash, Kimberly G V Davidson, Naomi Kamasawa, Thomas Yasumura, Masami Kamasawa, Chunbo Zhang, Robin Michaels, Diego Restrepo, Ole P Ottersen, Carl O Olson, James I Nagy
Journal of neurocytology 34 307-41 2005
Odorant/receptor binding and initial olfactory information processing occurs in olfactory receptor neurons (ORNs) within the olfactory epithelium. Subsequent information coding involves high-frequency spike synchronization of paired mitral/tufted cell dendrites within olfactory bulb (OB) glomeruli via positive feedback between glutamate receptors and closely-associated gap junctions. With mRNA for connexins Cx36, Cx43 and Cx45 detected within ORN somata and Cx36 and Cx43 proteins reported in ORN somata and axons, abundant gap junctions were proposed to couple ORNs. We used freeze-fracture replica immunogold labeling (FRIL) and confocal immunofluorescence microscopy to examine Cx36, Cx43 and Cx45 protein in gap junctions in olfactory mucosa, olfactory nerve and OB in adult rats and mice and early postnatal rats. In olfactory mucosa, Cx43 was detected in gap junctions between virtually all intrinsic cell types except ORNs and basal cells; whereas Cx45 was restricted to gap junctions in sustentacular cells. ORN axons contained neither gap junctions nor any of the three connexins. In OB, Cx43 was detected in homologous gap junctions between almost all cell types except neurons and oligodendrocytes. Cx36 and, less abundantly, Cx45 were present in neuronal gap junctions, primarily at mixed glutamatergic/electrical synapses between presumptive mitral/tufted cell dendrites. Genomic analysis revealed multiple miRNA (micro interfering RNA) binding sequences in 3'-untranslated regions of Cx36, Cx43 and Cx45 genes, consistent with cell-type-specific post-transcriptional regulation of connexin synthesis. Our data confirm absence of gap junctions between ORNs, and support Cx36- and Cx45-containing gap junctions at glutamatergic mixed synapses between mitral/tufted cells as contributing to higher-order information coding within OB glomeruli.Full Text Article
|Enalapril prevents perpetuation of atrial fibrillation by suppressing atrial fibrosis and over-expression of connexin43 in a canine model of atrial pacing-induced left ventricular dysfunction. |
Masao Sakabe, Akira Fujiki, Kunihiro Nishida, Masataka Sugao, Hidehiko Nagasawa, Takayuki Tsuneda, Koichi Mizumaki, Hiroshi Inoue
Journal of cardiovascular pharmacology 43 851-9 2004
Effects of enalapril on a canine model of atrial pacing-induced atrial fibrillation (AF) with rapid ventricular responses were determined. METHODS: Four weeks of atrial rapid pacing was performed on twenty-four beagles pretreated with placebo (Group I, n = 14) or enalapril 1 mg/kg (Group II, n = 10). Atrial effective refractory period (ERP), P-wave width, duration of AF, and left ventricular ejection fraction (LVEF) were evaluated every week. AF cycle length was determined by spectral analyses of fibrillation waves. Quantitative analysis of histology was added. RESULTS: After 4 weeks of pacing, P-wave width was longer in Group I than in Group II, and the duration of induced AF was significantly longer in Group I (59.6 +/- 66.3 seconds) than in Group II (3.6 +/- 3.4 seconds, P 0.05). AF cycle length was longer in Group I than in Group II despite similar shortening of atrial ERP. Mean ventricular rate during rapid atrial pacing was not different between the two groups. LVEF similarly decreased in both groups. Interstitial fibrosis and expression of connexin43 was greater in Group I than in Group II (interstitial fibrosis, 9.2 +/- 8.4 versus 1.9 +/- 2.1%, P 0.05; connexin43, 5.3 +/- 2.2 versus 1.1 +/- 1.1%, P 0.05). CONCLUSIONS: Enalapril suppressed atrial pacing-induced AF with tachycardia-mediated cardiomyopathy by suppressing interstitial fibrosis, connexin43 over-expression and conduction delay.
|Virtual electrode theory explains pacing threshold increase caused by cardiac tissue damage. |
Aleksandre T Sambelashvili, Vladimir P Nikolski, Igor R Efimov
American journal of physiology. Heart and circulatory physiology 286 H2183-94 2004
The virtual electrode polarization (VEP) effect is believed to play a key role in electrical stimulation of heart muscle. However, under certain conditions, including clinically, its existence and importance remain unknown. We investigated the influence of acute tissue damage produced by continuous pacing with strong current (40-mA, 4-ms biphasic pulses with 4-Hz frequency for 5 min) on stimulus-generated VEPs and pacing thresholds. A fluorescent optical mapping technique was used to obtain stimulus-induced transmembrane potential distribution around a pacing electrode applied to the ventricular surface of a Langendorff-perfused rabbit heart (n = 5). Maps and pacing thresholds were recorded before and after tissue damage. Spatial extents of electroporation and cell uncoupling were assessed by propidium iodide (n = 2) and connexin43 (n = 3) antibody staining, respectively. On the basis of these data, passive and active three-dimensional bidomain models were built to determine VEP patterns and thresholds for different-sized areas of the damaged region. Electrophysiological results showed that acute tissue damage led to disappearance of the VEP with an associated significant increase in pacing thresholds. Damage was expressed in electroporation and cell uncoupling within an approximately 1.0-mm-diameter area around the tip of the electrode. According to computer simulations, cell uncoupling, rather than electroporation, might be the direct cause of VEP elimination and threshold increase, which was nonlinearly dependent on the size of the damaged region. Fiber rotation with depth did not substantially affect the numerical results. The study explains failure to stimulate damaged tissue within the concepts of the VEP theory.
|Connexin35 mediates electrical transmission at mixed synapses on Mauthner cells. |
Pereda, A, et al.
J. Neurosci., 23: 7489-503 (2003) 2003
Auditory afferents terminating as "large myelinated club endings" on goldfish Mauthner cells are identifiable "mixed" (electrical and chemical) synaptic terminals that offer the unique opportunity to correlate physiological properties with biochemical composition and specific ultrastructural features of individual synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we demonstrate that gap junctions at these synapses contain connexin35 (Cx35). This connexin is the fish ortholog of the neuron-specific human and mouse connexin36 that is reported to be widely distributed in mammalian brain and to be responsible for electrical coupling between many types of neurons. Similarly, connexin35 was found at gap junctions between neurons in other brain regions, suggesting that connexin35-mediated electrical transmission is common in goldfish brain. Conductance of gap junction channels at large myelinated club endings is known to be dynamically modulated by the activity of their colocalized glutamatergic synapses. We show evidence by confocal microscopy for the presence of the NR1 subunit of the NMDA glutamate receptor subtype, proposed to be a key regulatory element, at these large endings. Furthermore, we also show evidence by FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor is present at postsynaptic densities closely associated with gap junction plaques containing Cx35 at mixed synapses across the goldfish hindbrain. Given the widespread distribution of electrical synapses and glutamate receptors, our results suggest that the plastic properties observed at these identifiable junctions may apply to other electrical synapses, including those in mammalian brain.
|Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells. |
Monica M Lurtz, Charles F Louis
American journal of physiology. Cell physiology 285 C1475-82 2003
The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+.
|Delivery of herpes simplex thymidine kinase bystander effect by engineered human mesothelial cells for the treatment of ovarian cancer. |
C Rancourt, C Bergeron, D Lane, G Garon, A Piché
Cytotherapy 5 509-22 2003
BACKGROUND: Resistance to conventional chemotherapy is a major clinical problem for ovarian cancer, and long-term survival for patients with advanced-stage disease is rare. Other therapeutic strategies, such as gene therapy, have been explored but several limitations exist, which include low viral vector transduction efficiency, host immune response to the vector, and vector toxicity. METHODS: We developed a cell-based therapy that exploits human mesothelial cells to deliver anticancer modalities for treatment of ovarian cancer. As a proof of concept, we genetically engineered mesothelial with the herpes simplex virus thymidine kinase/ganciclovir (HSVTK/GCV) system to deliver cytotoxicity to human ovarian cancer cells. This system is well characterized, and has been widely used in different gene-therapy based strategies. RESULTS: Our results demonstrate that HSVTK-modified mesothelial cells are sensitive to GCV killing in vitro and support the HSVTK bystander effect. Engineered mesothelial cells can deliver the HSVTK bystander effect to human ovarian cancer cell-lines, as well as to primary ovarian cancer cells. A significant reduction of tumor growth and prolongation of survival in s.c. and i.p. xenograft mouse models of ovarian cancer are obtained with as little as 1% of HSVTK-expressing mesothelial cells. Intraperitoneal administration of HSVTK-expressing mesothelial cells in an established mouse model of ovarian cancer results in a statistically significant prolonged survival of treated animals. Importantly, distribution studies showed that mesothelial cells localize preferentially to tumor sites. DISCUSSION: Our study demonstrates the feasibility of using a mesothelial cell-based therapy for treatment of ovarian cancer, and suggests that this strategy should be further explored.
|Cx43 and dual-pathway electrophysiology of the atrioventricular node and atrioventricular nodal reentry. |
Vladimir P Nikolski, Sandra A Jones, Matthew K Lancaster, Mark R Boyett, Igor R Efimov, Vladimir P Nikolski, Sandra A Jones, Matthew K Lancaster, Mark R Boyett, Igor R Efimov
Circulation research 92 469-75 2003
Fluorescent imaging has revealed that posterior nodal extensions provide the anatomical substrate for the dual-pathway electrophysiology of the atrioventricular (AV) node during normal conduction and reentry. The reentry can be intranodal, or as well as the posterior nodal extensions, it can involve an endocardial layer of atrial/atrial-nodal (A/AN) cells as part of the AV nodal reentry (AVNR) circuit. Using fluorescent imaging with a voltage-sensitive dye and immunolabeling of Cx43, we mapped the electrical activity and structural substrate in 3 types of AVNR induced by premature atrial stimulation in 8 rabbit hearts. In 6 cases, the AVNR pathway involved (1) a fast pathway (FP), (2) the A/AN layer, and (3) a slow pathway (SP). In 4 cases, reentry took the path (1) SP, (2) A/AN layer, and (3) FP. In 2 cases, reentry was intranodal, propagating between the 2 posterior nodal extensions. Immunolabeling revealed that the FP and SP are formed by Cx43-expressing bundles surrounded by tissue without Cx43. Cx43-expressing posterior nodal extensions are the substrate of AVNR during both intranodal and extranodal reentry.
|Skeletal muscle stem cells do not transdifferentiate into cardiomyocytes after cardiac grafting. |
Hans Reinecke, Veronica Poppa, Charles E Murry
Journal of molecular and cellular cardiology 34 241-9 2002
Skeletal muscle cell-derived grafts in the heart may benefit myocardial performance after infarction. Several studies have suggested that skeletal muscle stem cells (satellite cells) from adult muscle undergo transdifferentiation into cardiomyocytes after grafting into the heart, but expression of cardiac markers in graft cells has not been rigorously confirmed. To determine the fate of satellite cell-derived grafts in the heart, adult rat satellite cells were tagged in vitro with bromodeoxyuridine (BrdU) and grafted into normal hearts of syngeneic rats. At 4 and 12 weeks the graft cells formed multinucleated, cross-striated myofibers that expressed fast skeletal myosin heavy chain (MHC), thus indicating a mature skeletal muscle phenotype. Double staining for the BrdU tag and cardiac-specific markers was employed to identify transdifferentiation. Aside from four questionable cells, none of the 11 grafts examined expressed alpha-MHC, cardiac troponin I, or atrial natriuretic peptide. At 4 weeks, grafts expressed beta -MHC, a hallmark of slow twitch myofibers. By 12 weeks, however, the myofibers had atrophied and downregulated beta-MHC. Grafts never expressed the intercalated disk proteins N-cadherin or connexin43, hence electromechanical coupling did not occur. In conclusion, satellite cells differentiate into mature skeletal muscle and do not express cardiac-specific genes after grafting into the heart. Thus, transdifferentiation into cardiomyocytes did not occur.
|Expression of connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development. |
J F Bravo-Moreno, V Díaz-Sánchez, J G Montoya-Flores, E Lamoyi, J C Saéz, E M Pérez-Armendariz
The Anatomical record 264 13-24 2001
Connexin 43 (Cx43) is the most abundant and ubiquitously distributed gap junction protein in testicular cells. Lack of Cx43 expression results in male infertility. We investigated whether Cx43 is expressed and regulated in Leydig, Sertoli and germinal cells at different stages of postnatal development. Cx43 was detected using three different antibodies shown by immunoblotting to be highly specific. At different postnatal ages Cx43 localization was compared in serial or double labeled testicular cryosections with immunocytochemical distribution of steroidogenic enzyme, 3 betahydroxysteroid-dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GNCA1), which are specific markers of interstitial Leydig, Sertoli and germinal cells, respectively. In the interstitium, round cell clumps (RCC) with lipid droplets positive for 3betaHSD and Cx43 were frequently found at intertubular areas at birth and Cx43 was mainly localized at cell membrane appositions. From day 3, the number and size of 3betaHSD-positive RCC started to decrease, and reached a minimum at 7-14 dpp; Cx43 expressed by them is progressively downregulated. From day 21 an increase in the size and number of RCC positive for Cx43 and 3betaHSD was found that continued at 24, 26 and 28 days and reached a maximum at 35 and 60 dpp. Biphasic expression of interstitial Cx43 and 3betaHSD was also found to be positively and temporally correlated with fluctuations in intratesticular testosterone content at all ages studied. In the seminiferous cord (SC), Cx43 was expressed at birth between adjacent Sertoli cells (MIH positive) localized at the periphery, as well as in their cytoplasm projections that surround centrally localized gonocytes. From days 3 to 7, Cx43 labeling increased in Sertoli cells mainly at their apical border. At day 14, Cx43 distribution in Sertoli cells changed from apical to basal in parallel to migration of germinal (GNCA1-positive) cells from the periphery to the center of the SC. At all these ages, Cx43 was also localized at cell borders between Sertoli and germinal cells. In conclusion, this study demonstrates that Cx43 in Leydig cells is regulated during postnatal development in an age and functional dependent manner. In the tubule, it is demonstrated that Cx43 is modulated in Sertoli cells during the neonatal and prepubertal period. We also provide evidence for the first time that Cx43-gap junctions communicate between Sertoli and germinal cells before and during the first wave of spermatogenesis.
|Developmental regulation of connexin 43 expression in fetal mouse testicular cells. |
E M Pérez-Armendariz, E Lamoyi, J I Mason, D Cisneros-Armas, V Luu-The, J F Bravo Moreno
The Anatomical record 264 237-46 2001
Multiple connexins have been identified in testicular cells. Several lines of evidences indicate that, among them, connexin 43 (Cx43) may be unique for control of gonad development and spermatogenesis. To date, however, it is not known whether Cx43 is expressed in the fetal testis and what possible types of cellular interactions mediated by this connexin are critical to male fertility. In the present work, expression of Cx43 was investigated at various developmental ages in cryosections from mouse testis by using specific antibodies against Cx43. In serial or double-labeled sections, Cx43 localization was compared with immunocytochemical distribution of steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GCNA1), which are specific markers, respectively, of interstitial Leydig, Sertoli, and germinal cells. Sections were analyzed by fluorescence microscopy. We found that Cx43 immunofluorescence (IF) was uniformly distributed in the undifferentiated gonad at 11.5 days post coitus (dpc) and in cells of the mesonephric tubules. In the undifferentiated gonad, Cx43 was localized between primordial germ cells and somatic cells. At 12.5 dpc, when the gonad has undergone sexual differentiation, in the interstitium Cx43 was localized in Leydig cells and in the seminiferous cord it was localized between adjacent Sertoli cells. In Leydig and Sertoli cells, Cx43 labeling increased at 14.5, 16.5, and 18.5 dpc. From day 12.5 up to 18.5 dpc, Cx43 was also localized in cell borders between germinal and Sertoli cells. In conclusion, this study demonstrates that from the earliest stages of gonadal development, Cx43 is expressed in the principal cell types that participate in the control of male fertility. It also shows that Cx43 expression in Leydig and Sertoli cells increase during fetal life. Finally, it provides evidence that, throughout embryonic life, Cx43 forms gap junctions between Sertoli and germinal cells.
|Connexin43 in porcine myocardium and non-pregnant myometrium. |
J Karasiński, D Semik, W Kilarski, J Karasiński, D Semik, W Kilarski
Tissue cell 32 133-40 2000
It is generally accepted that connexin43 (Cx43) is a major constituent of heart and myometrial gap junctions. However, the presence of Cx43 gap junctions in non-pregnant myometrium is still poorly documented. Tissue sections of porcine heart and non-pregnant uterus and myometrial smooth muscle cell cultures were immunostained with monoclonal antibody against Cx43. In the heart, intensive immunostaining was confined to the intercalated discs as previously reported. In the non-pregnant uterus, punctuate immunostaining of Cx43 was seen throughout the myometrium along cell interfaces between myocytes. The expression of Cx43 was sustained in cultured smooth muscle cells isolated from non-pregnant myometrium. Western blotting has detected single isoform of Cx43 in both, cardiac and myometrial tissues. The electrophoretic mobility of porcine heart Cx43 was similar to that of myometrial isoform but different from the pattern of mobility of Cx43 of the rat heart. Hence, porcine myometrium may provide attractive model for studying cellular mechanisms triggering expression of gap junction protein in normal (non-pregnant) uterus.
|Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border. |
Coppen, S R, et al.
J. Histochem. Cytochem., 47: 907-18 (1999) 1999
The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.
|Requirement of a novel gene, Xin, in cardiac morphogenesis. |
D Z Wang, R S Reiter, J L Lin, Q Wang, H S Williams, S L Krob, T M Schultheiss, S Evans, J J Lin
Development (Cambridge, England) 126 1281-94 1999
A novel gene, Xin, from chick (cXin) and mouse (mXin) embryonic hearts, may be required for cardiac morphogenesis and looping. Both cloned cDNAs have a single open reading frame, encoding proteins with 2,562 and 1,677 amino acids for cXin and mXin, respectively. The derived amino acid sequences share 46% similarity. The overall domain structures of the predicted cXin and mXin proteins, including proline-rich regions, 16 amino acid repeats, DNA-binding domains, SH3-binding motifs and nuclear localization signals, are highly conserved. Northern blot analyses detect a single message of 8.9 and 5.8 kilo base (kb) from both cardiac and skeletal muscle of chick and mouse, respectively. In situ hybridization reveals that the cXin gene is specifically expressed in cardiac progenitor cells of chick embryos as early as stage 8, prior to heart tube formation. cXin continues to be expressed in the myocardium of developing hearts. By stage 15, cXin expression is also detected in the myotomes of developing somites. Immunofluorescence microscopy reveals that the mXin protein is colocalized with N-cadherin and connexin-43 in the intercalated discs of adult mouse hearts. Incubation of stage 6 chick embryos with cXin antisense oligonucleotides results in abnormal cardiac morphogenesis and an alteration of cardiac looping. The myocardium of the affected hearts becomes thickened and tends to form multiple invaginations into the heart cavity. This abnormal cellular process may account in part for the abnormal looping. cXin expression can be induced by bone morphogenetic protein (BMP) in explants of anterior medial mesoendoderm from stage 6 chick embryos, a tissue that is normally non-cardiogenic. This induction occurs following the BMP-mediated induction of two cardiac-restricted transcription factors, Nkx2.5 and MEF2C. Furthermore, either MEF2C or Nkx2.5 can transactivate a luciferase reporter driven by the mXin promoter in mouse fibroblasts. These results suggest that Xin may participate in a BMP-Nkx2.5-MEF2C pathway to control cardiac morphogenesis and looping.
|The differential expression of myometrial connexin-43, cyclooxygenase-1 and -2, and Gs alpha proteins in the upper and lower segments of the human uterus during pregnancy and labor. |
C Sparey, S C Robson, J Bailey, F Lyall, G N Europe-Finner, C Sparey, S C Robson, J Bailey, F Lyall, G N Europe-Finner
The Journal of clinical endocrinology and metabolism 84 1705-10 1999
There is evidence from many studies indicating that a number of specific quiescent and contractile associated proteins are temporally regulated in the myometrium during pregnancy. In this present investigation we provide data that strongly suggest that myometrial connexin-43, cyclooxygenase-1 and -2 (COX-1 and -2), and Gs alpha proteins are also spatially expressed within the human uterus during pregnancy and labor. Using paired lower and upper segment myometrial samples taken from individual women at term and during spontaneous labor, we have measured the expression of these proteins by immunoblotting with specific antibodies. We report that the myometrial gap junction connexin-43 protein is expressed at much greater levels in the upper uterine compared to the lower uterine segment and that this difference is even more pronounced during the course of labor. Conversely, myometrial COX-1 and -2 proteins appear to be expressed at much greater levels in the lower compared to the upper uterine segment. Moreover, the level of expression of both proteins is unaffected by the onset of parturition. In contrast, myometrial Gs alpha protein appears to be uniformly expressed in both lower and upper segments and is similarly down-regulated during parturition, as previously reported. The differential expression of COX-1 and -2 and connexin-43 in the uterus may allow cervical ripening before and dilatation during labor and facilitate effective propagation of contractions from fundus to cervix, which may be further facilitated by the down-regulation of Gs alpha at the onset of parturition.
|Role of endothelial nitric oxide synthase in endothelial cell migration. |
T Murohara, B Witzenbichler, I Spyridopoulos, T Asahara, B Ding, A Sullivan, D W Losordo, J M Isner, T Murohara, B Witzenbichler, I Spyridopoulos, T Asahara, B Ding, A Sullivan, D W Losordo, J M Isner
Arteriosclerosis, thrombosis, and vascular biology 19 1156-61 1999
Endothelium-derived nitric oxide (NO) and its precursor L-arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by Nomega-nitro-L-arginine methyl ester (L-NAME) (1 mmol/L) but not its inactive enantiomer D-NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by L-NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, L-NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas L-NAME did not affect endothelial DNA synthesis as assessed by analysis of [3H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by L-NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, L-NAME (1 mmol/L) inhibited surface expression of integrin alphavbeta3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, L-NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by L-NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin alphavbeta3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism.
|Connexin43 gene expression in the rabbit arterial wall: effects of hypercholesterolemia, balloon injury and their combination. |
Polacek, D, et al.
J. Vasc. Res., 34: 19-30 (1997) 1997
The specialized functions of endothelium require intercellular communication between endothelial cells within the monolayer, and between endothelium and other cells present in the vessel wall. This is accomplished by a combination of paracrine soluble mediators and direct gap-junctional intercellular communication (GJIC) mediated by a family of connexin proteins. A prominent connexin expressed by vascular cells in vivo and in vitro is connexin 43 (Cx43). We have investigated the in vivo gene regulation of Cx43 in the context of vascular pathology, as a result of mechanical injury, hypercholesterolemia or both. The aortoiliac bifurcation in the rabbit was examined following three types of insult: (1) diet-induced hypercholesterolemia resulting in macrophage-rich fatty streak lesions, (2) mechanical, stretch-denudation injury resulting in intimal smooth muscle cell (SMC) proliferation and (3) mechanical injury superimposed on hypercholesterolemia resulting in a complex vascular lesion having characteristics of both interventions. The normal rabbit iliac artery expressed approximately equal levels of Cx43 mRNA in the medial SMC layers and in the endothelium. In hypercholesterolemia-induced atherosclerosis, Cx43 expression was most prominent in macrophage foam cells even though normocholesterolemic precursor monocytes did not express Cx43 mRNA. Antibodies directed specifically to Cx43 protein confirmed the expression of macrophage gap junction protein in these cells. Medial SMC in hypercholesterolemia exhibited less Cx43 than their normal counterparts in control animals. Mechanical injury in the absence of hypercholesterolemia resulted in intimal thickening in which Cx43 expression in the intimal SMC was equivalent to that in the subjacent medial SMC, both being approximately equivalent to normal uninjured rabbit medial SMC expression. Cell-specific expression of Cx43 in combined mechanical injury/hypercholesterolemia was similar to that observed in hypercholesterolemia alone: Cx43 upregulation in macrophages, while medial SMC were downregulated. Normo- and hypercholesterolemic alveolar macrophages of the lung and Kupffer cells of the liver did not exhibit induction of Cx43 mRNA, nor did macrophages isolated from peritoneal or bronchial lavage fluid of the same animals. This work extends our previous finding of Cx43 upregulation in human atherectomy tissue and demonstrates that atherosclerotic lesions in situ, in a controlled animal model of atherosclerosis, exhibit cell-specific changes in Cx43 gene expression. Changes in medial SMC migration, proliferation and phenotype, as well as enhanced interactions between adherent/infiltrating monocytes and endothelium may be related to modified GJIC pathways in the vessel wall.
|Connexin43: a protein from rat heart homologous to a gap junction protein from liver. |
Beyer, E C, et al.
J. Cell Biol., 105: 2621-9 (1987) 1987
Northern blot analysis of rat heart mRNA probed with a cDNA coding for the principal polypeptide of rat liver gap junctions demonstrated a 3.0-kb band. This band was observed only after hybridization and washing using low stringency conditions; high stringency conditions abolished the hybridization. A rat heart cDNA library was screened with the same cDNA probe under the permissive hybridization conditions, and a single positive clone identified and purified. The clone contained a 220-bp insert, which showed 55% homology to the original cDNA probe near the 5' end. The 220-bp cDNA was used to rescreen a heart cDNA library under high stringency conditions, and three additional cDNAs that together spanned 2,768 bp were isolated. This composite cDNA contained a single 1,146-bp open reading frame coding for a predicted polypeptide of 382 amino acids with a molecular mass of 43,036 D. Northern analysis of various rat tissues using this heart cDNA as probe showed hybridization to 3.0-kb bands in RNA isolated from heart, ovary, uterus, kidney, and lens epithelium. Comparisons of the predicted amino acid sequences for the two gap junction proteins isolated from heart and liver showed two regions of high homology (58 and 42%), and other regions of little or no homology. A model is presented which indicates that the conserved sequences correspond to transmembrane and extracellular regions of the junctional molecules, while the nonconserved sequences correspond to cytoplasmic regions. Since it has been shown previously that the original cDNA isolated from liver recognizes mRNAs in stomach, kidney, and brain, and it is shown here that the cDNA isolated from heart recognizes mRNAs in ovary, uterus, lens epithelium, and kidney, a nomenclature is proposed which avoids categorization by organ of origin. In this nomenclature, the homologous proteins in gap junctions would be called connexins, each distinguished by its predicted molecular mass in kilodaltons. The gap junction protein isolated from liver would then be called connexin32; from heart, connexin43.
|Anti-Connexin 43, C-terminus - Data Sheet|