Functional demonstration of connexin-protein binding using surface plasmon resonance.

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.

Use of antibodies in the analysis of connexin 43 turnover and phosphorylation.

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin.

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.

Deficient assembly and function of gap junctions in Trf1, a trafficking mutant of the human liver-derived cell line HuH-7.

The Trf1 cell line, selected from the human hepatoma cell line HuH-7, manifests altered trafficking of various plasma membrane proteins. In particular, there is a striking loss of State 2 asialoglycoprotein receptors. This cell line is shown here to also manifest defects in function and assembly of gap junctions comprising connexin43 (Cx43). No alteration of Cx43 expression or phosphorylation was apparent. Nevertheless, immunostaining of Cx43 revealed that fewer and smaller gap junctions were present at appositional membrane areas in Trf1 cells as compared with parental HuH-7. This correlated with a significant attenuation in gap junction-mediated communication between Trf1 cells as demonstrated by markedly decreased dye transfer and their reduced ability to propagate mechanically evoked Ca(2+) waves. Isoelectric focusing (IEF) of Cx43 in HuH-7 cells indicated that the pIs of this protein were significantly lower than that predicted from its amino acid sequence; no differences in pI were evident in Cx43 from Trf1 cells and the HuH-7 cell line. The effects of the Trf1 mutation on assembly and function of gap junctions indicate that this mutation influences trafficking of Cx43. Connexins differ in several respects from other membrane proteins thus far analyzed in Trf1 mutants: gap junctions localize exclusively to the lateral cell surface; they are not glycoproteins; and they do not play a role in endocytic pathways. The disruption of trafficking of Cx43 by this mutation suggests that the Trf1 phenotype is a defect at a common point along the trafficking pathway of cell-surface proteins, irrespective of their ultimate destination on the cell surface or their glycosylation profile.

TPA induced expression and function of human connexin 26 by post-translational mechanisms in stably transfected neuroblastoma cells.

Connexin 26 (Cx26) has been proposed to be a tumor suppressor gene and its expression may modulate development, cell growth and differentiation in various tissues, including the brain. 12-O-tetradecanoylphorbol-13-acetate (TPA) may serve as either tumor promoter (in mammary gland amd skin) or as a differentiating agent (in neuroblastoma and leukemic cells) and may also modulate expression, function and phosphorylation of gap junctions. In this study, to determine the effects of TPA on Cx26 expression and its function in neuroblastoma, we transfected N2A mouse neuroblastoma cells (which are gap junction deficient) with the coding region of human Cx26 gene (which lacks TPA response elements) and examined the changes of expression and function of Cx26 following 10 nM TPA treatment. Individual clones of transfectants stably expressed distinct levels of exogenous Cx26 as judged by Northern and Western blots, immunocytochemistry and electrophysiological recordings. Cx26 channels displayed unitary conductances of about 140-155 pS. Increase of Cx26 expression following TPA treatment was markedly observed using immunocytochemistry and Western blots of membrane fractions although it was not detected in Northern or Western blots of whole cells. This increase in Cx26 expression in the plasma membrane was accompanied by an increase of function as evidenced in measurements of junctional conductance. These results suggest that induction of exogenous Cx26 in neuroblastoma cells by TPA treatment is controlled by post-translational mechanisms.

Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia.

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence-specific anti-Cx43 antibodies (designated 13-8300, 18A, 16A and 71-0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13-8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71-0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13-8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13-8300, but elevated labelling of internalized Cx43 with 16A and 71-0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.

Gap junction disappearance in astrocytes and leptomeningeal cells as a consequence of protozoan infection.

Trypanosoma cruzi and Toxoplasma gondii are protozoan parasites capable of causing infections of the nervous system. In order to determine effects of infection by these organisms on intercellular communication in the brain, dye coupling and connexin abundance and distribution were examined in leptomeningeal cells and astrocytes infected with T. cruzi or T. gondii. For both cell types infected with either type of protozoan parasite, intercellular diffusion of intracellularly injected Lucifer Yellow was dramatically reduced. Immunocytochemistry with antibodies specific for connexin43 (in astrocytes) or both connexin43 and connexin26 (for leptomeningeal cells) demonstrated that punctate gap junctional staining was much reduced in infected cells, although uninfected neighbors could display normal connexin abundance and distribution. Western blot analyses revealed that connexin43 abundance in both cell types infected with either parasite was similar to that in uninfected cells. Phosphorylation state of connexin43 (inferred from electrophoretic mobility of connexin43 isoforms) was not significantly affected by the infection process. Immunocytochemistry of whole brains from animals acutely infected with either parasite also showed a marked reduction in connexin43 expression. We conclude that infection of both types of brain cells with either protozoan parasite results in a loss of intercellular communication and organized gap junction plaques without affecting expression levels or posttranslational processing of gap junction proteins. Presumably, these changes in gap junction distribution result from altered targeting of the junctional protein to the plasma membrane, and/or from changes in assembly of subunits into functional channels.

Association of cell and substrate adhesion molecules with connexin43 during intramembranous bone formation.

Prior studies in our laboratory have demonstrated an association of specific gap junction proteins with intramembranous bone formation in the avian mandible. The purpose of the present study was to extend these observations by determining if there was a relationship between the expression of one of the gap junction proteins examined previously (connexin43) and the expression of specific cell adhesion (CAM) and/or substrate adhesion (SAM) molecules [i.e. NCAM, A-CAM (N-cadherin) and tenascin (tenascin-C)] that have previously been shown to be associated with bone formation. Immunohistochemical localization of connexin43, tenascin, NCAM and N-cadherin was performed on serial sections of mandibles of chick embryos from 6 to 12 days of incubation. Analysis of adjacent serial sections revealed that the NCAM and tenascin immunostaining that appeared initially on the lateral aspect of Meckel's cartilage preceded the overt expression of trabecular bone. At subsequent stages, NCAM and tenascin staining gradually overlapped the region of connexin43 expression. In contrast, the expression of N-cadherin was found to colocalize with that of connexin43 from the first appearance of connexin43 expression. Most significantly, although the domains of NCAM and tenascin expression were initially separate from that of connexin43, bone formation originated only in the region where these domains intersected. These findings suggest that, of the CAMs and SAMs examined, N-cadherin appears to be associated with the establishment of cell contacts responsible for the presence and/or maintenance of connexin43-mediated gap junctional communication, while tenascin and NCAM appear to be associated, in a more specific manner, with processes that accompany the overt expression of the osteogenic phenotype.

Selective monoclonal antibody recognition and cellular localization of an unphosphorylated form of connexin43.

A sequence-specific monoclonal antibody directed against the gap junction protein connexin43 (Cx43) is shown here to be specific for the unphosphorylated form of this protein. In tissues and cultured cells containing different phosphorylated and unphosphorylated forms of Cx43, the antibody detected only the latter as shown by Western blotting of native and alkaline phosphatase-treated samples. Immunohistochemically, this monoclonal antibody did not recognize gap junctions in the vast majority of cultured cardiac myocytes, where nearly all detectable Cx43 is phosphorylated. In contrast, it was able to detect some intracellular Cx43 in tracheal smooth muscle cells and an epithelial cell line (Cl-9 cells), producing patterns of labeling consistent with those seen using a polyclonal antibody that recognizes both phosphorylated and unphosphorylated forms of Cx43. Immunostaining of gap junctions in the cultured cells indicates that both phosphorylated and unphosphorylated Cx43 are present in some assembled gap junctions, suggesting that assembled junctions do not contain exclusively the phosphorylated form of the protein. Annular gap junctions, believed to form as part of the pathway for internalization and degradation of gap junctions, were only occasionally and sparsely labeled by the monoclonal antibody, indicating that complete protein dephosphorylation is not required for uptake and degradation of gap junctions. Furthermore, the ability of this antibody to recognize only unphosphorylated Cx43, and not any of the phosphorylated forms present in the tissues and cell types examined, suggests that a unique phosphorylation site, perhaps present in the epitope recognized by this antibody, must be phosphorylated prior to phosphorylation of Cx43 at other sites.

Phosphorylation of connexin43 and the regulation of neonatal rat cardiac myocyte gap junctions.

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of 32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of 32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of 32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived from in vitro phosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243-382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.

Expression patterns of connexin43 protein during facial development in the chick embryo: associates with outgrowth, attachment, and closure of the midfacial primordia.

BACKGROUND: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. METHODS: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. RESULTS: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. CONCLUSIONS: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface.

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury.

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression.

Oligodendrocytes express gap junction proteins connexin32 and connexin45.

Oligodendrocytes, the myelin-forming glia of brain, are connected by gap junctions in situ and in culture. Cultured oligodendrocytes from adult bovine and porcine brains were studied using immunocytochemical, molecular, and electrophysiological techniques in order to characterize the gap junction types. The expression of connexin32 was substantiated by the detection of low, but significant, signals using connexin-specific probes in Northern and Western blot analyses. Connexin43, which comprises gap junctions in astrocytes, was not detectable in pure oligodendrocytic cultures; mRNAs of connexin40 and connexin37 and connexin26 were also not detected. By means of two specific antibodies directed to the recently cloned connexin45 and by RT-PCR we were able to identify this connexin as a second oligodendrocytic gap junction protein. Whole cell voltage clamp recording provided evidence for electrical coupling between pairs of cultured oligodendrocytes (mean junctional conductance 3.9 nS, n = 38 pairs) and intracellular Lucifer Yellow injection indicated that oligodendrocytes were usually only weakly dye coupled, with spread generally being restricted to nearest neighbors. Unitary conductances ranged from > 20 to < 150 pS with modes of distribution at about 100 to 120pS and 40 to 20 pS, respectively. These unitary conductances are consistent with the channel events expected for connexin32 and connexin45. The low degree of functional coupling between oligodendrocytes in vitro corresponds with the low levels of connexin32 and connexin45 messenger RNAs and protein expression.

Evidence for the co-localization of another connexin with connexin-43 at astrocytic gap junctions in rat brain.

Gap junctions between astrocytes as well as between astrocytes and oligodendrocytes in rat brain were immunohistochemically labelled with a monoclonal and an affinity-purified polyclonal antibody generated against connexin-26. By light microscopy, the immunolabelling patterns obtained were, with a few exceptions, remarkably similar to previously described distribution patterns of the gap junctional protein connexin-43, which is expressed by astrocytes and is localized at astrocytic gap junctions. By electron microscopy, immunoreactivity with these two anti-connexin-26 antibodies was restricted to astrocytes; inter-astrocytic gap junctional membranes were symmetrically labelled, heterologous oligo-astrocytic junctional membranes were asymmetrically labelled only on the astrocyte side and oligo-oligodendrocyte junctions were unlabelled. Two additional anti-connexin-26 antibodies that were found to produce punctate labelling in leptomeninges and liver failed to do so in brain parenchyma, consistent with reports indicating the absence of authentic connexin-26 in this tissue. Antibodies that labelled astrocytic gap junctions exhibited no cross-reaction with connexin-43 or connexin-32, as demonstrated by western blotting, but recognized liver connexin-26 as well as several brain proteins, including an approximately 32000 mol. wt protein that did not correspond to connexin-32 and a 26000 mol. wt protein that co-migrated with liver connexin-26. These results suggest that connexin-26, or more likely a protein having sequence homology with connexin-26, is targeted to astrocytic gap junctions and raise the possibility of the existence of connexins that may be co-expressed with connexin-43 in most, but perhaps not all, astrocytes.

Connexin32 in oligodendrocytes and association with myelinated fibers in mouse and rat brain.

The distribution and cellular localization of connexin32 (Cx32) in the brain and spinal cord of the mouse and rat was investigated by light microscope (LM) and electron microscope (EM) immunohistochemistry by using several different antibodies against Cx32. By double immunofluorescence staining for Cx32 and either the oligodendrocyte markers cyclic nucleotide phosphodiesterase (CNPase) or Rip, Cx32 was consistently found in oligodendrocyte cell bodies and proximal processes. Cx32 immunoreactivity was also clearly visualized along CNPase- and Rip-positive myelinated fibers. Both immunopositive cells and fibers were heterogeneously distributed and were often more intensely labeled when dispersed in or associated with regions of gray matter than when concentrated in major white matter tracts. Labeling of myelin sheaths along fibers was restricted to subpopulations of myelinated axons. In the cerebellar cortex, for example, it was selectively localized to sheaths around Purkinje cell axons. Punctate staining, distinct from that corresponding to cells or fibers, was evident in the olfactory bulb and hippocampus. By EM, oligodendrocytes exhibited cytoplasmic labeling associated with rough endoplasmic reticulum and Golgi apparatus. Their processes were intermittently stained, most intensely when surrounding myelinated fibers and occasionally in paranodal loops. Cx32-immunoreactive gap junctions with symmetric labeling (staining on both junctional membranes) were observed between oligodendrocytic somata and processes as well as between presumptive oligodendrocytic processes. Unidentifiable elements forming asymmetrically labeled gap junctions (staining only one side of junctional membranes) were less frequently encountered. Western blot analysis confirmed anti-Cx32 antibody detection of Cx32 in whole brain homogenates and an enrichment of the protein in isolated myelin fractions. These results are consistent with earlier ultrastructural studies showing the occurrence of inter-oligodendrocytic gap junctions, but indicate that these may be more prevalent than previously thought. Furthermore, the results suggest a specialized role of gap junctions composed of Cx32 along myelinated fibers belonging to subpopulations of neurons.

Connexin-43 in rat spinal cord: localization in astrocytes and identification of heterotypic astro-oligodendrocytic gap junctions.

Connexin-43 in relation to gap junctions between astrocytes and between other cell types in rat spinal cord was investigated immunohistochemically. In gray matter, connexin-43 was distributed thoughout all laminae, but was more concentrated in the substantia gelatinosa and around the central canal. Ultrastructurally, immunostaining was present in the cytoplasm of, and at gap junctions between, fine astrocytic processes, most of which ensheathed neuronal elements. In white matter, connexin-43 was localized to somata of fibrous astrocytes, their glial fibrillary acidic protein-positive processes running parallel to myelinated axons, and at gap junctions between these processes. Labelling was also evident in thick radially-directed astrocytic processes displaying pockets of staining near immunopositive gap junctions. Near the cord surface, staining was present in cell bodies of subpial astrocytes and at gap junctions between their tangential processes which formed most of the glia limitans. Radially-directed processes of subpial astrocytes formed symmetrically- and asymmetrically-labelled gap junctions with each other and extended fine branches into surrounding white matter where they made contact and often formed gap junctions with oligodendrocytic processes at the outer surface of myelinated fibres. Immunopositive astrocyte processes also made heterologous gap junctions with unstained oligodendrocyte cell bodies. Ependymal cells lining the central canal exhibited apical cytoplasmic labelling, as well as symmetrically-labelled gap junctions at their apices. Ependymal cells also formed asymmetrically-labelled gap junctions at which the junctional membranes of unlabelled cells, presumed to be tanycytes, were unstained. The results indicate the expression of connexins in addition to connexin-43 at asymmetrically-labelled gap junctions between some astrocytic processes, between astrocytes and oligodendrocytes and between some ependymal cells. The presence of gap junctions between astrocyte and oligodendrocyte processes at the outer surface of myelin suggests incorporation of the latter into the extensive gap junctionally-coupled astrocytic syncytium.

Elevated connexin43 immunoreactivity at sites of amyloid plaques in Alzheimer's disease.

The distribution of the astrocytic gap junctional protein, connexin43 (Cx43) was compared immunohistochemically with that of amyloid plaques in Alzheimer's Disease (AD) brain. By light microscopy, cortical areas containing numerous beta/A4 amyloid plaques exhibited increased immunostaining density for Cx43 and some plaques corresponded exactly to sites of intensified Cx43 immunoreactivity. By electron microscopy, Cx43 was localized to astrocytic gap junctions in AD brain. Increased Cx43 expression in AD may represent an attempt to maintain tissue homeostasis by augmented intercellular communication via gap junction formation between astrocytic processes that invest senile plaques, or alternatively, an aberrant induction of astrocytic Cx43 expression which may further compromise homeostasis and exacerbate pathological conditions in the microenvironment of amyloid plaques.

Induction of connexin43 and gap junctional communication in PC12 cells overexpressing the carboxy terminal region of amyloid precursor protein.

Previous studies have shown that PC12 cells overexpressing beta/A4 amyloid peptide display altered morphology characterized by pronounced membrane ruffling and extensive intercellular appositions. Having observed other cell types in which these features accompany increased connexin43 (Cx43) production and gap junctional communication, we examined Cx43 in normal and beta/A4-transfected PC12 cells. Studies of two beta/A4-transfected PC12 clones revealed an induction of Cx43 expression by Western blotting, intracellular and plasma membrane-associated Cx43 in some cells of cultures processed by immunofluorescence, dye-transfer between some cells microinjected with Lucifer Yellow, and gap junctions between cells examined by EM. Normal and vector-transfected PC12 cells exhibited none of these properties. Increased immunofluorescence in some clusters of beta/A4-transfected cells was also observed with a monoclonal antibody against connexin32. The results suggest that beta/A4 amyloid peptide may cause aberrant intercellular communication and gap junction formation through induction or increased expression of connexins in cells that are not normally coupled or only poorly coupled by gap junctions.

Reversible intercellular coupling by regulated expression of a gap junction channel gene.

Direct intercellular coupling through gap junction channels has been implicated in diverse processes including cellular differentiation, growth control, metabolic cooperativity and electronic coupling and natural and induced mutations in connexin genes have been described in human and experimental disease states. Genetic systems in which the extent of coupling could be reversibly regulated would provide an important approach for examining these potential functional roles, both in vitro and in vivo. Here we describe the generation and characterization of cell lines in which the extent of coupling is reversibly controlled at the transcriptional level. Plasmids encoding a tetracycline-controlled transactivator and a tetracycline-responsive connexin32 target gene were introduced in the communication-deficient SKHep1 cell line. Quantitative immunoblotting and confocal immunofluorescence microscopy with connexin32-specific antibodies demonstrated that expression of connexin32 in stable transfectants was tightly regulated by tetracycline treatment. Moreover, transfectants exhibited a highly coupled phenotype which was rapidly and reversibly converted to the communication deficient parental state after tetracycline treatment. Time constants for decay of the messenger RNA, protein and functional coupling were similar (approximately 4 hrs), implying that transcription was rate-limiting and that separate long-lived pools of connexin32 protein were absent. In contrast to other approaches in which the extent of coupling is pharmacologically regulated by altering channel gating characteristics or by generalized blockade of transcription or translation, in this system intercellular communication is regulated by directly controlling connexin gene expression.

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels.

The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.