Structure and permeability of ion-channels by integrated AFM and waveguide TIRF microscopy.

Membrane ion channels regulate key cellular functions and their activity is dependent on their 3D structure. Atomic force microscopy (AFM) images 3D structure of membrane channels placed on a solid substrate. Solid substrate prevents molecular transport through ion channels thus hindering any direct structure-function relationship analysis. Here we designed a ~70 nm nanopore to suspend a membrane, allowing fluidic access to both sides. We used these nanopores with AFM and total internal reflection fluorescence microscopy (TIRFM) for high resolution imaging and molecular transport measurement. Significantly, membranes over the nanopore were stable for repeated AFM imaging. We studied structure-activity relationship of gap junction hemichannels reconstituted in lipid bilayers. Individual hemichannels in the membrane overlying the nanopore were resolved and transport of hemichannel-permeant LY dye was visualized when the hemichannel was opened by lowering calcium in the medium. This integrated technique will allow direct structure-permeability relationship of many ion channels and receptors.

Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein.

We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI™ and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.

Isolation of extracellular recombinant fragment of rat connexin-43.

Analysis of membrane topology of connexin-43 (Cx-43) made it possible to determine one of its extracellular fragments (E2): Q173-1208. The nucleotide sequence of this fragment was cloned into pCBDQ and pHPML vectors containing the sequences of calmodulin-binding domain (CBD) and HPML-domain of Ca-ATPase of human hPMCA4b cells plasma membrane. This yields two chimeric proteins with N-terminal 6-histidine motif containing the extracellular fragment Cx43 E2 and one of hPMCA4b domains (Cx43-CBD and Cx43-HPML). The latter were inserted into the recombinant polypeptide to improve solubility and enhance immunogenicity of the product. Affinity-purified on Ni-NTA agarose recombinant Cx43-CBD was used for immunization of mice and obtaining of monoclonal antibodies. Primary selection of clones was carried out by solid-phase IEA with immobilized Cx43-HPML and by immunoblotting with Cx43-HPML. The positive clones were tested immunohistochemically on rat brain sections. This two-stage testing made it possible to select two hybridomas, which produced monoclonal antibodies to Cx43 in native conformation. The resultant antibodies can be used in vitro and in vivo for immunophenotyping of various Cx43-positive cells.

Purification and reconstitution of the connexin43 carboxyl terminus attached to the 4th transmembrane domain in detergent micelles.

In recent years, reports have identified that many eukaryotic proteins contain disordered regions spanning greater than 30 consecutive residues in length. In particular, a number of these intrinsically disordered regions occur in the cytoplasmic segments of plasma membrane proteins. These intrinsically disordered regions play important roles in cell signaling events, as they are sites for protein-protein interactions and phosphorylation. Unfortunately, in many crystallographic studies of membrane proteins, these domains are removed because they hinder the crystallization process. Therefore, a purification procedure was developed to enable the biophysical and structural characterization of these intrinsically disordered regions while still associated with the lipid environment. The carboxyl terminal domain from the gap junction protein connexin43 attached to the 4th transmembrane domain (TM4-Cx43CT) was used as a model system (residues G178-I382). The purification was optimized for structural analysis by nuclear magnetic resonance (NMR) because this method is well suited for small membrane proteins and proteins that lack a well-structured three-dimensional fold. The TM4-Cx43CT was purified to homogeneity with a yield of approximately 6 mg/L from C41(DE3) bacterial cells, reconstituted in the anionic detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], and analyzed by circular dichroism and NMR to demonstrate that the TM4-Cx43CT was properly folded into a functional conformation by its ability to form alpha-helical structure and associate with a known binding partner, the c-Src SH3 domain, respectively.

Up-regulation of connexin 43 and gap junctional intercellular communication by Coleusin Factor is associated with growth inhibition in rat osteosarcoma UMR106 cells.

Gap junctions, formed by connexin (Cx) family proteins, permit direct exchange of regulatory ions and small signal molecules between neighbouring cells. Gap junctional intercellular communication (GJIC) plays an important role in maintaining the homeostasis and preventing cell transformation. Most of the tumour cells feature deficient or aberrant connexin expression and GJIC level, and restoration of connexin expression and GJIC is correlated with cell growth control. Numerous researches has suggested the possibility of connexins as potential anti-tumour targets for chemoprevention and chemotherapy. We investigated the ability of Coleusin Factor (CF, also named FSK88) to regulate the Cx43 expression and GJIC level in rat osteosarcoma UMR106 cells. The results have demonstrated that CF increased the mRNA and protein expression of Cx43 in both in a dose- and timedependent manner, and concomitant with up-regulation of Cx43, CF treatment up-regulated the diminished GJIC level in UMR106 cells as assayed by dye transfer experiments. In addition, Cx43 distribution at the plasma membrane was also enhanced dramatically by CF treatment. Furthermore, we discovered that CF was potent to inhibit the growth and proliferation of UMR106 cells. These results provide the first evidence that CF can regulate connexin and GJIC, indicating that Cx43 may be a target of CF to exert its anti-tumour effects.

Cloning, sequence analysis and phylogeny of connexin43 isolated from American black bear heart.

Conduction in the heart requires gap junctions. In mammalian ventricular myocytes these consist of connexin43 (Cx43). Hearts of non-hibernating species display conduction disturbances at reduced temperatures. These may exacerbate into lethal arrhythmias. Hibernating species are protected against these arrhythmias by a non-resolved mechanism. To analyze whether the amino acid composition of Cx43 from the hibernating American black bear displays specific features, we cloned the full coding sequence of Ursus americanus Cx43 and compared with that of other (non)hibernating species. UaCx43 displays 99.7% identity to rabbit Cx43 at the amino acid level. No specific features were observed in UaCx43 when compared to previously cloned Cx43 from hibernating and non-hibernating mammals. Phylogenetic tree reconstruction of this and other published full-length Cx43 sequences reveals a very high level of conservation from fish to men. Finally, one of the previously identified six mammalian characteristic amino acids, is not conserved in the black bear.

Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of Serine 368.

Indirect evidence suggests that the permeability of connexin 43 (Cx43) gap-junctional channels (connexons) to small organic molecules (M(r) < 1,000) is decreased by protein kinase C (PKC)-mediated phosphorylation of Ser-368. However, it is currently unknown whether this effect is produced directly by phosphorylation of this residue or whether cytoplasmic regulatory factors are required for the decrease in Cx43 gap-junctional channel permeability. Here we studied the effects of PKC-mediated phosphorylation on purified recombinant wild-type Cx43 and a PKC-unresponsive mutant (S368A). Our studies show that (a) PKC phosphorylates Ser-368, (b) the phosphorylation by PKC of purified and reconstituted connexons abolishes sucrose and Lucifer Yellow permeability, (c) the regulation of Cx43 by PKC is the direct result of phosphorylation of Ser-368 and does not involve intermediary regulatory factors, and (d) phosphorylation of Ser-368 produces a conformational change in purified Cx43 as demonstrated by changes in intrinsic Trp fluorescence and proteolytic digestion pattern. We conclude that phosphorylation of Ser-368 by PKC induces a conformational change of Cx43 that results in a decrease in connexon permeability.

Partial purification and characterization of proteins with growth promoting activities from ovine mammary gland secretions.

Developmental regulation of growth promoting activities in mammary secretions of pregnant Awassi ewes was defined, and growth factors contained in these secretions were partially purified and characterised. Mammary secretions from pregnant ewes enhanced fibroblast cell (AKR-2B) and mammary cell (CID-9 cell strain) proliferation to levels comparable to that induced by 10% Foetal calf serum. Major milk proteins in mammary secretions collected from pregnant ewes one month prior to lambing up to one week after lambing, were resolved by SDS-PAGE, while gelatinases were resolved by zymography. Gelatinase activity was noted prior to P134 and decreased thereafter to reach a minimum during lactation. This decrease was concomitant with the onset of casein production. It is during this critical developmental period that highest growth promoting activity in mammary secretions was detected. Secretions with highest growth promoting activity were fractionated by ion exchange and gel filtration chromatography. Two heat-resistant, trypsin/chymotrypsin sensitive, growth-promoting activities were characterised. The first, designated ovine mammary derived growth factor-1 (oMDGF-1), had around a 30 kDa molecular weight and eluted at 0.65 M NaCl gradient on cation ion exchange chromatography. The second, oMDGF-2, eluted under gel filtration conditions at a molecular weight of 50 kDa and 150 kDa. oMDGF-1 induced changes in Connexin 43, but not in beta-casein mRNA expression by CID-9 mammary cells. In conclusion, growth factor activities in ewe mammary secretions peak during gestation at a period that overlaps maximal gelatinase expression and precedes milk protein synthesis. The factors modulate mammary cell function and may play a role in mammary gland development.

Chamber formation and morphogenesis in the developing mammalian heart.

In this study we challenge the generally accepted view that cardiac chambers form from an array of segmental primordia arranged along the anteroposterior axis of the linear and looping heart tube. We traced the spatial pattern of expression of genes encoding atrial natriuretic factor, sarcoplasmic reticulum calcium ATPase, Chisel, Irx5, Irx4, myosin light chain 2v, and beta-myosin heavy chain and related these to morphogenesis. Based on the patterns we propose a two-step model for chamber formation in the embryonic heart. First, a linear heart forms, which is composed of "primary" myocardium that nonetheless shows polarity in phenotype and gene expression along its anteroposterior and dorsoventral axes. Second, specialized ventricular chamber myocardium is specified at the ventral surface of the linear heart tube, while distinct left and right atrial myocardium forms more caudally on laterodorsal surfaces. The process of looping aligns these primordial chambers such that they face the outer curvature. Myocardium of the inner curvature, as well as that of inflow tract, atrioventricular canal, and outflow tract, retains the molecular signature originally found in linear heart tube myocardium. Evidence for distinct transcriptional programs which govern compartmentalization in the forming heart is seen in the patterns of expression of Hand1 for the dorsoventral axis, Irx4 and Tbx5 for the anteroposterior axis, and Irx5 for the distinction between primary and chamber myocardium.

Stimulated phosphorylation of intracellular connexin43.

A monoclonal antibody, Zymed 13-8300, was previously reported to only detect nonphosphorylated connexin43 (Nagy et al., Exp. Cell Res. 236, 127-136, 1997). We show that 13-8300 can detect several phosphorylated species of connexin43 in Western blots after stimulation of two fibroblast cell systems with fresh growth medium, 12-O-tetradecanoyl phorbol-13-acetate, pervanadate, or permolybdate. In one of the cell systems, at least three forms of phosphorylated connexin43 could migrate at the same position during electrophoresis. The comigration of differentially phosphorylated species may complicate the molecular and functional analysis of phosphorylation sites in Cx43. Immunofluorescence experiments indicated that the newly generated phosphorylated Cx43 forms mainly had a perinuclear location. Also, in cells treated with brefeldin A for 8 h, in which the majority of connexin43 was intracellular, phosphorylation was induced by the agents. Phosphorylation of intracellular connexin43 can therefore be induced by several stimuli.

Differential expression of gap-junction gene connexin 31 in seminiferous epithelium of rat testes.

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of certain gap-junction connexins. Our findings by RT-PCR indicate that the Cx31 gene is expressed in testis tissue of adult and postnatal rats. During the postnatal spermatogenic process, the Cx31-specific signal became detectable at 15 dpp and onward by in situ hybridization, and apparently localized in the basal compartment of seminiferous epithelium where active spermatogonia and early primary spermatocytes reside. No signal was found in the luminal region. In adult testes, spermatids of elongation phase were also Cx31 positive. Immunohistochemical analysis with mouse anti-Cx31 antibody gave a similar staining pattern, providing further evidence that the gap-junction protein is abundant in the basal seminiferous epithelium, in accordance with the cellular distribution of Cx31 mRNA. These results represent the first demonstration of Cx31 expression at both transcriptional and protein levels in the seminiferous epithelium of rat testes. Thus, Cx31 may play a role in cell-cell communication during spermatogenesis.

Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation.

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.

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

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

The gap junction protein connexin43 is degraded via the ubiquitin proteasome pathway.

We investigated the degradation of the gap junction protein connexin43 in E36 Chinese hamster ovary cells and rat cardiomyocyte-derived BWEM cells. Treatment of E36 cells with the lysosomotropic amine, primaquine, for 16 h doubled the amount of connexin43 detected by immunoblotting and modestly increased the half-life of connexin43 in pulse-chase studies, suggesting that the lysosome played a minor role in connexin43 proteolysis. In contrast, treatment with the proteasomal inhibitor N-acetyl-L-leucyl-L-leucinyl-norleucinal led to a 6-fold accumulation of connexin43 and increased the half-life of connexin43 to approximately 9 h. The role of ubiquitin in connexin43 degradation was examined in an E36-derived mutant, ts20, which contains a thermolabile ubiquitin-activating enzyme, E1. E36 and ts20 cells grown at the permissive temperature contained similar amounts of connexin43 detectable by immunoblotting. Heat treatment dramatically reduced the amount of connexin43 detected in E36 cells, while connexin43 levels in heat-treated ts20 cells did not change. E36 cells that were heat-treated in the presence of N-acetyl-L-leucyl-L-leucinyl-norleucinal did not lose their connexin43. Pulse-chase experiments showed the reversibility of the block to connexin43 degradation in ts20 cells that were returned to the permissive temperature. Finally, sequential immunoprecipitation using anti-connexin43 and anti-ubiquitin antibodies demonstrated polyubiquitination of connexin43. These results indicate that ubiquitin-mediated proteasomal proteolysis may be the major mechanism of degradation of connexin43.

Inhibition of glycosylation induces formation of open connexin-43 cell-to-cell channels and phosphorylation and triton X-100 insolubility of connexin-43.

We transfected the cDNA for the cell-to-cell channel protein connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express little Cx43 and lack gap junctional communication (open cell-to-cell channels). We found that cells overexpressing Cx43 nonetheless lacked open cell-to-cell channels, but that inhibition of glycosylation by tunicamycin induced open channels in these cells. Tunicamycin also induced biochemical changes in Cx43 protein; the level increased, and a considerable fraction became phosphorylated and Triton X-100 insoluble, in contrast to untreated cells where Cx43 was non-phosphorylated and Triton X-100 soluble. Although tunicamycin caused the formation of open channels, channels were not found aggregated into gap junctional plaques, as they are when they have been induced by elevation of intracellular cAMP. The results suggest that although Cx43 itself is not glycosylated, other glycosylated proteins influence Cx43 posttranslational modification and the formation of Cx43 cell-to-cell channels.

Down-regulation by butylated hydroxytoluene of the number and function of gap junctions in epithelial cell lines derived from mouse lung and rat liver.

The mouse pneumotoxicant and lung and liver tumor promoter butylated hydroxytoluene (BHT) was examined for its effects on gap junctional intercellular communication (GJIC) in mouse lung epithelial (C10) and rat liver epithelial (WB-F344) cell lines. GJIC, as measured by fluorescent dye microinjection, was inhibited in both types of cells by BHT in dose- and time-dependent fashions. Inhibition was detected in WB-F344 cells at BHT concentrations > or = 62.5 microM and in C10 cells at concentrations > or = 150 microM after 4 h treatment. Inhibition occurred within 15-30 min and was reversed by removing BHT from the culture medium. The highly toxic BHT metabolite 6-t-butyl-2-(hydroxy-t-butyl)-4-methylphenol (BHTOH) and the non-toxic BHT metabolite, 2,6-di-t-butyl-4-hydroxymethylphenol (BHTBzOH) were also tested. In both cell lines BHTOH was a more potent inhibitor of GJIC than BHT, whereas BHTBzOH was ineffective. The mechanisms of inhibition of GJIC by BHT were also examined. The initial rapid inhibition detected within 15-30 min may have been due to gap junction channel closure or blockage, since no changes in gap junction number, connexin (Cx) 43 levels or Cx43 phosphorylation were observed. By 2-4 h, however, gap junctions were internalized into the cytoplasm, the number of immunodetectable plasma membrane gap junctions was reduced and phosphorylated Cx43-P2 was decreased. Treatment of the cells for 24 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) prevented inhibition of GJIC by TPA, but not by BHT. Western blot analyses of TPA-treated WB-F344 or C10 cells revealed the presence of a hyperphosphorylated form of Cx43 (Cx43-P3) and no reduction in Cx43-P2, in contrast to BHT-treated cells. These data suggest that BHT and TPA inhibit lung and liver epithelial cell GJIC through distinct mechanisms.

Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone.

One important role of the junctional communication in the ovarian follicle is to mediate transmission of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumption of meiosis. Our experiments were directed at exploration of mechanisms involved in the LH-induced communication breakdown in the preovulatory ovarian follicle. Immunofluorescence and Western blot analysis, using highly specific antibodies, showed that connexin-43 (Cx43), the ovarian gap junction protein, is present in the cytoplasmic membranes of the follicular cells in multiple phosphorylated forms. The relative amounts of the different forms of Cx43 vary in response to LH: short time exposure (10 min) stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, while longer incubations (8 and 24 h) with this hormone resulted in elimination of the protein. Forskolin mimicked the LH-induced phosphorylation/dephosphorylation, as well as the decrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation of Cx43 and a later reduction of the amount of Cx43. The direct PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphorylation of Cx43 that was completely blocked by the protein kinase C inhibitor, staurosporine. This kinase inhibitor partially interfered with LH, but not forskolin-induced phosphorylation of Cx43. Analysis of the effect of LH on Cx43 gene expression revealed a significant decrease (45%) in Cx43 mRNA level at 24 h of incubation. A drop of Cx43 mRNA was also induced by GnRHa. Our results suggest that the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation of its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.

Expression of gap junction connexin 43 and connexin 43 mRNA in different regional tissues of intestine in dog.

To determine the distribution of gap junction protein connexin 43 (Cx43) and the expression of Cx43 mRNA in different regions of canine small intestine and colon, modified Western blot and Northern blot techniques were used with a site-directed antibody raised against a synthetic peptide of Cx43 Anti-Cx43 (252-271) and a probe of 1.3-kb Cx43 cDNA. Equal amounts of plasma membrane enriched fraction (10 micrograms protein) from small intestinal and colonic circular muscle, longitudinal muscle, and colonic submucous plexus border of circular muscle (interstitial cells of Cajal rich layer, ICC) of the dog were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and blotted by chemiluminescent assay. Different yet characteristic ratios of the immunoreactive gap junction proteins located at 43 and 40 kDa were found in membranes from small intestinal and colonic circular smooth muscle, and ICC. These results suggest that gap junction Cx43 in dog heart, dog small intestinal and colonic circular muscle, and ICC is homologous to rat heart. Furthermore, with the use of Northern blot hybridization with a 1.3-kb Cx43 cDNA, a 3.0-kb message was observed in small intestinal and colonic circular muscles, longitudinal muscles, and ICC. However, the mRNA signal of small intestinal circular muscle was the strongest and that of longitudinal muscle was weakest, especially from colon longitudinal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatase inhibitors, gap junctional intercellular communication and [125I]-EGF binding in hamster fibroblasts.

A number of phosphatase inhibitors (okadaic acid, calyculin A, aluminium fluoride, sodium molybdate, sodium orthovanadate, pervanadate and vanadyl sulphate) were investigated for their effects on gap junctional intercellular communication (GJIC) and [125I]-epidermal growth factor (EGF) binding in early passage Syrian hamster embryo cells (mainly fibroblast-like cells) and in V79 Chinese hamster lung fibroblasts. Only pervanadate decreased GJIC significantly. After the initial pervanadate-induced decrease the GJIC recovered rapidly. Only pervanadate was able to change the band pattern of the gap junction protein connexin43 (cx43) in Western blots. Together this may indicate either that there is a low turnover of phosphate groups in cx43 under basal conditions or that the putative phosphatases are not sensitive to most of the phosphatase inhibitors applied. In contrast, pervanadate, orthovanadate and molybdate decreased [125I]-EGF binding. 12-O-Tetradecanoylphorbol-13-acetate (TPA) is able to induce the phosphorylation of both cx43 and the EGF receptor, concomitantly with a decrease in GJIC and [125I]-EGF binding. These effects are reversible after removal of TPA. It could be imagined that other phosphatases would act on cx43 and the EGF receptor after the forced phosphorylation of the two molecules. Thus TPA was used to downregulate GJIC and [125I]-EGF binding and phosphatase inhibitors were applied in the upregulation phase. Only pervanadate affected the upregulation of GJIC, and pervanadate, orthovanadate and molybdate affected the upregulation of [125I]-EGF binding. Thus it is not an identical complement of phosphatases that act on cx43 and the EGF receptor. All the downregulating agents are assumed to be phosphotyrosine phosphatase inhibitors.