AB1745 Sigma-AldrichAnti-Connexin 45 Antibody, near CT, cytoplasmic

Gap junction (GJ) channels have been recognized as an important mechanism for synchronizing neuronal networks. Herein, we investigated the participation of GJ channels in the pilocarpine-induced status epilepticus (SE) by analyzing electrophysiological activity following the blockade of connexins (Cx)-mediated communication. In addition, we examined the regulation of gene expression, protein levels, phosphorylation profile and distribution of neuronal Cx36, Cx45 and glial Cx43 in the rat hippocampus during the acute and latent periods. Electrophysiological recordings revealed that the GJ blockade anticipates the occurrence of low voltage oscillations and promotes a marked reduction of power in all analyzed frequencies.Cx36 gene expression and protein levels remained stable in acute and latent periods, whereas upregulation of Cx45 gene expression and protein redistribution were detected in the latent period. We also observed upregulation of Cx43 mRNA levels followed by changes in the phosphorylation profile and protein accumulation. Taken together, our results indisputably revealed that GJ communication participates in the epileptiform activity induced by pilocarpine. Moreover, considering that specific Cxs undergo alterations through acute and latent periods, this study indicates that the control of GJ communication may represent a focus in reliable anti-epileptogenic strategies.

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.

During mammalian oogenesis, intercellular communication between oocytes and the surrounding follicle cells through gap junction channels is crucial for oocyte development and maturation. The channel properties of gap junctions may be affected by the composition or combination of connexins, the expression of which is regulated by gonadotropins and other factors. Thus, identification and expression analysis of connexin genes in oocytes and follicle cells will help us to better understand how oogenesis and folliculogenesis are regulated in a species-specific manner in mammals. We previously reported the spatiotemporal expression of multiple connexin genes in porcine follicle cells. Here, we searched for connexin genes specifically expressed in porcine oocytes that may be involved in the formation of gap junctions between oocytes and follicle cells. To achieve this, we constructed an oocyte-specific cDNA library to identify which connexin genes are expressed in these cells and found that gap junction protein, alpha 10, which encodes connexin-60, and a porcine ortholog of mouse gap junction protein, gamma 1 encoding connexin-45, are the major connexins expressed in porcine oocytes during folliculogenesis. Immunostaining and in situ hybridization of sectioned porcine ovaries confirmed oocyte expression of these genes at 3 different stages of ovary development. Furthermore, their gap junction channel activity was assessed using a heterologous cell system. However, gap junction protein, alpha 4, which encodes connexin-37 and is expressed in the oocytes of several other mammals, was undetectable. We demonstrate that there is diversity in the connexin genes expressed in mammalian oocytes, and hence in the gap junctions connecting oocytes and cumulus cells.

Haplodeficient mice expressing carboxyl-terminally truncated Cx43 (K258stop/KO), instead of the wild-type Cx43 isoform, reach adulthood and reveal no abnormalities in heart morphology. Here, we have analyzed the expression of K258stop protein and the morphology of gap junctions in adult hearts of these mice. Coimmunofluorescence analysis revealed reduced juxtaposition of K258stop with other junctional proteins at the intercalated disc. Immunoprecipitation studies documented changes in the interaction with previously described Cx43 binding proteins. Quantitative transmission electron and confocal microscopy confirmed the localization of K258stop gap junctions to the periphery of the intercalated disc and further revealed an increase in the size of K258stop gap junction plaques and a reduction in their number. Dual whole cell patch clamp analysis confirmed that K258stop gap junctions were functional, with single channel properties similar to those described in exogenous systems. We conclude that the carboxyl-terminal domain of Cx43 (Cx43CT) is involved in regulating the localization, number and size of Cx43 plaques in vivo. Conversely, protein interactions or posttranslational modifications taking place within the Cx43CT are not required for the assembly of functional gap junctions in the intercalated disc.

Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.

Myocardial infarction leads to extensive changes in the organization of cardiac myocytes and fibroblasts, and changes in gap junction protein expression. In the immediate period following ischemia, reperfusion causes hypercontraction, spreading the necrotic lesion. Further progressive infarction continues over several weeks. In reperfusion injury, the nonspecific gap junction channel uncoupler heptanol limits necrosis. We hypothesize that gap junction coupling and fibroblast invasion provide a substrate for progressive infarction via a gap junction mediated bystander effect.A sheep coronary occlusion infarct model was used with samples collected at 12, 24 and 48 h, and 6, 12 and 30 d (days) post-infarction. Immunohistochemical labelling of gap junction connexins Cx40, Cx43, and Cx45 was combined with cell-specific markers for fibroblasts (anti-vimentin) and myocytes (anti-myomesin). Double and triple immunolabelling and confocal microscopy were used to follow changes in cardiac myocyte morphology, fibroblast content and gap junction expression after myocardial infarction. Gap junction protein levels and fibroblast numbers were quantified.Within 12 h of ischemia, myocyte viability is impaired within small islands in the ischemic region. These islands spread and fuse into larger infarct zones until 12 d post-infarction. Thereafter, surviving myocytes within the infarct and in the border-zone appear to become stabilized. Distant from the infarct, continuing myocyte disruption is regularly observed, even after 30 d. Cx43 becomes redistributed from intercalated discs to the lateral surface of structurally compromised myocytes within 12 d. Cx45 expressing fibroblasts infiltrate the damaged region within 24 h, becoming most numerous at 6-12 d post-infarction, with peak Cx45 levels at 6 d. Later, Cx43 expressing fibroblasts are observed, and the related Cx43 label increases over the 30 d observation period, even though fibroblast numbers decline after 12 d. Cx40 was only seen in vascular endothelium.Progressive infarction, identified by myocyte sarcomere disruption and subsequent cell loss, occurs in parallel with fibroblast invasion and gap junction remodeling. Two fibroblast phenotypes occur within infarcts, expressing either Cx43 or Cx45. Coupled fibroblasts may play a number of roles in tissue remodeling following myocardial infarction, including provision of a possible substrate for progressive infarction via a gap junction mediated bystander effect.

Cells in blood vessel walls express connexin (Cx)43, Cx40, and Cx37. We recently characterized gap junction channels in rat basilar artery smooth muscle cells and found features attributable not only to these three connexins but also to an unidentified connexin, including strong voltage dependence and single channel conductance of 30-40 pS. Here, we report data consistent with identification of Cx45. Immunofluorescence using anti-human Cx45 and anti-mouse Cx45 antibodies revealed labeling between alpha-actin-positive cells, and RT-PCR of mRNA from arteries after endothelial destruction yielded amplicons exhibiting 90-98% identity with mouse Cx45 and human Cx45. Dual-perforated patch clamping was performed after exposure to oligopeptides that interfere with docking of Cx43, Cx40, or Cx45. Cell pairs pretreated with blocking peptides for Cx43 and Cx40 exhibited strongly voltage-dependent transjunctional conductances [voltage at which voltage-dependent conductance declines by one-half (V1/2) = +/-18.9 mV] and small single channel conductances (31 pS), consistent with the presence of Cx45, whereas cell pairs pretreated with blocking peptide for Cx45 exhibit weaker voltage-dependent conductances (V1/2 = +/-37.9 mV), consistent with block of Cx45. Our data suggest that Cx45 is transcribed, expressed, and forms functional gap junction channels in rat cerebral arterial smooth muscle.

Gap junctions (GJs) provide for a unique system of intercellular communication (IC) allowing rapid transport of small molecules from cell to cell. GJs are formed by a large family of proteins named connexins (Cxs). Cx43 has been considered as the predominantly expressed Cx by hematopoietic-supporting stroma. To investigate the role of the Cx family in hemopoiesis, we analyzed the expression of 11 different Cx species in different stromal cell lines derived from murine bone marrow (BM) or fetal liver (FL). We found that up to 5 Cxs are expressed in FL stromal cells (Cx43, Cx45, Cx30.3, Cx31, and Cx31.1), whereas only Cx43, Cx45, and Cx31 were clearly detectable in BM stromal cells. In vivo, the Cx43-deficient 14.5- to 15-day FL cobblestone area-forming cells (CAFC)-week 1-4 and colony-forming unit contents were 26%-38% and 39%-47% lower than in their wild-type counterparts, respectively. The reintroduction of the Cx43 gene into Cx43-deficient FL stromal cells was able to restore their diminished IC to the level of the wild-type FL stromal cells. In addition, these Cx43-reintroduced stromal cells showed an increased support ability (3.7-fold) for CAFC-week 1 in normal mouse BM and 5-fold higher supportive ability for CAFC-week 4 in 5-fluorouracil-treated BM cells as compared with Cx43-deficient FL stromal cells. These findings suggest that stromal Cx43-mediated IC, although not responsible for all GJ-mediated IC of stromal cells, plays a role in the supportive ability for hemopoietic progenitors and stem cells. (Blood. 2000;96:498-505)