Delayed separation and the plasma amino acids arginine and ornithine

Induction of gap junctional communication (GJC) is discussed as one possible mechanism underlying the cancer-preventive properties of carotenoids. Structurally different carotenoids have different effects on this GJC pathway. Beta-carotene, echinenone, canthaxanthin, cryptoxanthin and 4-hydroxy-beta-carotene efficiently induce GJC in murine fibroblasts. Decomposition products of canthaxanthin (e.g. 4-oxo-retinoic acid) enhance both GJC and expression of connexin 43 mRNA. Thus, the biological activity of canthaxanthin is due at least in part to formation of active decomposition products such as 4-oxo-retinoic acid. A number of other small molecules such as 1,25-dihydroxycholecalciferol or thyroid hormones serve as nuclear receptor ligands. Cholecalciferol, 3,3',5-triiodo-L-thyronine and L-thyroxine induce GJC to a similar extent as carotenoids or retinoic acid. Interactions between these signalling pathways may be involved in GJC regulation.     

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.     

Effects of race, sex, and socioeconomic status upon cardiovascular stress responsivity and recovery in youth

In rat brain, expression of the gap junction protein connexin30 increased during the first 3 weeks after birth and reached its maximum after 4 weeks, as shown by analysis with specific connexin30 antibodies. This contrasts with the prenatal onset of connexin43 expression. On cryosections of rat brain, connexin30 immunoreactivity was found near blood vessels and in ependymal as well as in leptomeningeal cells. Expression in the neuropil was first noticed 3 weeks after birth, showing the same spatial pattern of immunoreactivity as connexin43. This late onset of connexin30 expression in astrocytes was also seen in long-term glial cell cultures, where connexin30 was coexpressed with the astrocytic marker proteins S-100beta and glial fibrillary acid protein. In acute brain slices, connexin30 immunofluorescent signals were detected on processes of functionally identified astrocytes. Thus, our results show that connexin30 is expressed in three different cell types of the rodent brain. The late onset of connexin30 expression in astrocytes suggests that this gap junctional protein fulfills a role in intercellular communication among mature astrocytes.     

ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells

Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.     

A pump-prime aprotinin dose in cardiac surgery: appraisal of its effects on the hemostatic system

Neoplastic transformation is often associated with aberrant gap junctional intercellular communication. We assessed mutations and expression of the connexin43 (Cx43) gene in 49 intracranial meningiomas. SSCP analyses followed by direct DNA sequencing showed GCG-->GTG (Ala-->Val) transition mutation in codon 253 of the cytoplasmic carboxyl terminal of the Cx43 gene in 1 of 31 (3%) benign meningiomas and 1 of 14 (7%) anaplastic meningiomas. The same base change was present in normal tissue from these patients and also in 4 of 80 (5%) DNA samples extracted from lymphocytes of healthy Europeans, suggesting that this constitutes a newly identified Cx43 polymorphism. Western blot analyses showed expression of phosphorylated P1 (45 kD) and P2 (47 kD) Cx43 as well as the unphosphorylated form (42 kD) in 11 of 14 (79%) benign meningiomas. In contrast, the P2 form was not detectable in the majority (7 of 9; 78%) of atypical and anaplastic meningiomas. Since the presence of the P2 form is often associated with optimal function of the Cx43, these results suggest that loss or impaired gap junctional cell to cell communication may be associated with meningiomas displaying more rapid growth and a less favorable prognosis.     

Role of gap junctions in lung neoplasia

Reduced gap junctional intercellular communication (GJIC) has been noted in many types of neoplastic cells and may contribute to the neoplastic phenotype. This study assessed GJIC (by fluorescent dye-coupling) and gap junction protein (connexin) expression in mouse and human lung carcinoma cell lines and investigated whether reduced GJIC was involved in their neoplastic phenotype. Dye-coupling and connexin43 (Cx43) expression were much lower in most of the carcinoma lines (16 of 22) compared to nontransformed lung epithelial cells. Other connexins were not detected. A poorly communicating mouse lung carcinoma cell line (E9) was transfected with Cx43 or transduced with Cx32 and several stable clones were isolated that had 2- to 4-fold increased dye coupling. When evaluated for growth in vitro, the population doubling times were increased and the saturation densities were decreased in the clones. When assessed for tumorigenicity, the parental E9 cells formed tumors with a 100% incidence (6/6 mice), whereas the clones varied in tumorigenic response (0-88% incidence). The best communicating clone (E9-2) was not tumorigenic. The highly communicating Cx32 clone, E9/32-9, gave a tumor incidence of 88%. These results suggest that restoration of GJIC by forced connexin expression can reduce the growth and tumorigenicity of lung carcinoma cells in a connexin-specific manner.     

Gap junctional intercellular communication contributes to hormonal responsiveness in osteoblastic networks

To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.     

Disruption of gap junctional intercellular communication in human renal cancer cell lines

OBJECTIVES: Gap junctional intercellular communication (GJIC) is believed to play an important role in the maintenance of cell homeostasis, and its disruption may be associated with carcinogenesis. However, GJIC has not been detected in many human cancers. We therefore studied the regulation of GJIC in human renal cancer cell lines. METHODS: We examined the human renal cancer cell lines, ACHN and NT, as well as Madin-Darby canine kidney (MDCK) cells as a positive control, using GJIC assays, Northern blotting to detect connexin 43 mRNA, immunofluorescent staining, and Western blotting of connexin 43 protein. RESULTS: GJIC of ACHN and NT was completely blocked. In ACHN cells, connexin 43 mRNA was not altered. However, connexin 43 protein was aberrantly localized and phosphorylated connexin 43 protein had disappeared. Both connexin 43 protein and its mRNA were undetectable in NT cells. CONCLUSIONS: GJIC in human renal cancer cell lines is impaired and various pathways may inhibit this mechanism in renal cancer. We believe that connexin plays an important role in renal carcinogenesis.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Gap junctional intercellular communication contributes to the contraction of rat osteoblast populated collagen lattices

The contraction of native collagen lattices by resident mesenchymal cells mimics the organization of collagen during development and repair. Lattice contraction is cell density dependent, suggesting that cell-to-cell communications may contribute to the process. This possibility was investigated by comparing lattice contraction by four rat osteoblastic cell lines: ROS 17/2.8 cells (ROS); ROS transfected with an antisense cDNA sequence of the gap junctional protein connexin 43 (RCx16); ROS transfected with connexin 45 cDNA, a connexin not normally expressed in ROS cells (ROS/Cx45); and ROS transfected with cDNA encoding carboxy-terminal truncated Cx45 (ROS/Cx45tr). The cell coupling indices, which reflect gap junctional communication, were quantitated by the fluorescent dye scrape loading. ROS cells were well coupled (index 3.0), ROS/Cx45tr were better coupled (index 4.2), ROS/Cx45 were poorly coupled (index 1.7), and RCx16 showed no coupling (index 1.1). As determined by immunoblotting, the level of connexin 43 protein was increased in both ROS/Cx45tr and ROS/Cx45 cell lines compared with ROS cells, while the level in RCx16 cells was reduced. ROS populated collagen lattices (PCLs) contracted significantly more at day 5 (177 mm2 to 67 mm2) than ROS/Cx45tr (84 mm2), ROS/Cx45 (108 mm2), or RCx16 (114 mm2). Myosin ATPase activity, which is required for lattice contraction, was equivalent in all four cell lines, indicating that it was not responsible for inhibiting PCL contraction. ROS cells in collagen appeared elongated compared with the other cell lines which were more rounded. These experiments suggest gap junctional communication contributes to PCL contraction by resident osteoblasts.     

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy

Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk- HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43(+)-tk+ and Cx43(+)-tk- while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, i.e. Cx43(+)-tk+/Cx43(-)-tk-. When GCV was given after the appearance of tumors, the size of the tumors from Cx43- cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.     

A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer

We describe a simple method for evaluating gap junctional communication (GJC) between cells in culture. The procedure involves pre-loading cells with two fluorescent dyes: calcein and DiI. Calcein is able to pass through gap junctions, while DiI is not. These pre-loaded cells are then plated with unlabeled cells. The number of cells receiving calcein from each pre-loaded cell can then be quantified after the cells settle on the plate. Potent and reversible inhibitors of GJC can be used in this system to evaluate dye transfer within a given period of time.     

Absorption and urinary excretion of the coffee diterpenes cafestol and kahweol in healthy ileostomy volunteers

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication

We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.     

Downregulation of immunodetectable connexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle

BACKGROUND: The regional wall motion impairment and predisposition to arrhythmias in human ventricular hibernation may plausibly result from abnormal intercellular propagation of the depolarizing wave front. This study investigated the hypothesis that altered patterns of expression of connexin43, the principal gap junctional protein responsible for passive conduction of the cardiac action potential, contribute to the pathogenesis of hibernation. METHODS AND RESULTS: Patients with poor ventricular function and severe coronary artery disease underwent thallium scanning and MRI to predict regions of normally perfused, reversibly ischemic, or hibernating myocardium. Twenty-one patients went on to coronary artery bypass graft surgery, during which biopsies representative of each of the above classes were taken. Hibernation was confirmed by improvement in segmental wall motion at reassessment 6 months after surgery. Connexin43 was studied by quantitative immunoconfocal laser scanning microscopy and PC image software. Analysis of en face projection views of intercalated disks revealed a significant reduction in relative connexin43 content per unit area in reversibly ischemic (76.7+/-34.6%, P<.001) and hibernating (67.4+/-24.3%, P<.001) tissue compared with normal (100+/-30.3%); ANOVA P<.001. The hibernating regions were further characterized by loss of the larger gap junctions normally seen at the disk periphery, reflected by a significant reduction in mean junctional plaque size in the hibernating tissues (69.5+/-20.8%) compared with reversibly ischemic (87.4+/-31.2%, P=.012) and normal (100+/-31.5%, P<.001) segments; ANOVA P<.001. CONCLUSIONS: These results indicate progressive reduction and disruption of connexin43 gap junctions in reversible ischemia and hibernation. Abnormal impulse propagation resulting from such changes may contribute to the electromechanical dysfunction associated with hibernation.     

A characterization of permolybdate and its effect on cellular tyrosine phosphorylation, gap junctional intercellular communication and phosphorylation status of the gap junction protein, connexin43

Biological and analytical characterizations of permolybdate (a mixture of H2O2 and molybdate) were done. Molybdate (10 mM) and molybdenum(V) chloride (3 mM) did not affect gap junctional intercellular communication (GJIC), phosphorylation status of connexin43 (Cx43) or cellular tyrosine phosphorylation in early passage hamster embryonic cells (mainly fibroblast-like). High concentrations of H2O2 (3-10 mM) affected some of the parameters. Acidified permolybdate was clearly more stable than the unadjusted permolybdate. The maximum biological potency of acidified permolybdate was found at a molar ratio of 2:1 (H2O2:molybdate). The mixtures of molybdenum(V) chloride and H2O2 gave a maximum effect at 4:1 molar ratio (H2O2:molybdenum(V)). This can be explained by decomposition of H2O2 and by the generation of less biologically active compounds. Spectrophotometric analyses of the mixtures corroborated the biological results. The Mo(V) electron spin resonance spectrum disappeared upon addition of H2O2 to Mo(V) solutions, and no spectrum appeared when H2O2 was mixed with Mo(VI). Thus, permolybdate is probably diperoxomolybdate, a Mo(VI) compound. Regardless of the parent metal salt, the H2O2/metal salt mixtures showed concentration-dependent biphasic responses with an initial decrease in GJIC followed by an increase. A dissociation between alteration in Cx43 phosphorylation status and GJIC was obtained under certain conditions. The biological activities of permolybdate were only partially mimicked by phenylarsine oxide, an alternative protein tyrosine phosphatase inhibitor.