Inhibition Of Gap Junctional Communication Research Articles

Reversible inhibition of gap junctional communication by tamoxifen in cultured cardiac myocytes.

Gap junction channels provide a cell-to-cell conduction pathway for direct exchange of ions and small molecules. The intercellular diffusion of a fluorescent dye, quantified in cardiac myocytes from neonatal rats by monitoring the fluorescence recovery after photobleaching, was found to be interrupted after short-term exposure (15 min) to tamoxifen, an anti-oestrogen drug often used in the treatment of human breast cancer. This diffusional uncoupling was dose dependent, occurred in the concentration range 3-25 microM and reversed after tamoxifen withdrawal. Some possible mechanisms of junctional channel closure have been examined. The cytosolic calcium concentration, examined using the fluorescent indicator Indo-1, did not vary during the short-term action of tamoxifen. A second anti-oestrogen agent (clomiphene) was able to impair gap junctional communication, whereas a third (nafoxidine) had no effect. Protein-kinase-C-inhibitor properties of tamoxifen did not seem to be involved in its uncoupling action. The characteristics of tamoxifen's action (i.e. channel inhibition delay, active concentration range, reversibility, etc.) were very similar to the previously observed effects of several other lipophilic compounds (e. g. 17beta-oestradiol, etc.) on junctional channels, and to recently reported effects of tamoxifen on voltage-gated calcium currents.

An inhibition of gap-junctional communication by cadherins.

The action of Ca(2+)-dependent cell-cell adhesion molecules (cadherins) on cell-to-cell channel-mediated intercellular communication was investigated in mouse L and rat Morris hepatoma cells. These cells fail to adhere to one another in aggregation assays and thus seem to lack cell adhesion molecules. Expression of exogenous cadherin induced strong cell-cell adhesion in both cell types, but had opposite effects of communication, causing inhibition in L cells and improvement in hepatoma cells. Both cells express the connexin43 cell-to-cell channel protein. By western blot we found no cadherin-specific changes in connexin43 protein in either cell type, but connexin43 gap junctional plaque staining, i.e. connexin43 localization to cell-cell junctions, was inhibited in L cells and facilitated in hepatoma cells. In addition we found that the inhibitory effect is largely abolished by blockers of glycosylation. Cadherin-cadherin interactions are known to trigger cell type-specific intracellular signal cascades resulting in diverse end effects, and gap junctional communication/plaque formation seems a further example of such cell type-specificity.

Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells.

Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione-sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.

Transforming growth factor β1 (TGFβ1) inhibits gap junctional communication during human trophoblast differentiation

Summary It was recently established that, during trophoblast differentiation, gap junctional communication (GJC) precedes the formation of the syncytiotrophoblast and is required for trophoblast cell fusion. Therefore, the end of cell-to-cell communication through gap junctions appears as a judicious criterion of cell fusion. Although GJC was seen to be stimulated by human Chorionic Gonadotropin (hCG), its regulation remains poorly understood. Transforming Growth Factor-β1 (TGF-β1), a multifunctional cytokine has been shown to modulate trophoblast differentiation. Therefore, the effects of TGF-β1 on intercellular dye diffusion have been investigated in cultured trophoblast of human term placenta, by means of the Fluorescence Recovery After Photobleaching (gap FRAP) technique. In parallel, trophoblast differnetiation, hCG production and human Chorionic Somatomammotropin (hCS, a specific syncytiotrophoblast hormonal product) expression were assessed. The presence of TGF-β1 (5 or 10 ng/ml) in the culture medium for two days partially inhibited syncytium formation. The percentage of coupled cells was significantly decreased (2.8 times) after two days in presence of 10 ng/ml of TGF β1. Simultaneously, hCG release in culture medium was reduced (at this concentration, to 0.65 and 0.79 after respectively two and three days). In these conditions, Western blot analysis of trophoblast cellular proteins revealed that, after two days, hCS expression was reduced by 40% compared to control. Furthermore, the stimulation of trophoblastic GJC by exogenous hCG (500 mIU/ml) was considerably reduced by simultaneous exposure to TGF-β1 (10 mg/ml). The addition of a polyclonal hCG antibody in excess decreased basal GJC. In the presence of hCG antibody, no significant additive inhibition by TGFβ1 was observed. In conclusion, TGF-β1 was found to inhibit intercellular communication and, subsequently, differentiation and concomitant placental hormone secretions.

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.

Involvement of nerve growth factor in the ovulatory cascade: trkA receptor activation inhibits gap junctional communication between thecal cells.

Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.

Gap-junction disassembly and connexin 43 dephosphorylation induced by 18β-glycyrrhetinic acid

Gap-junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor-promoting chemicals have been identified that inhibit cell-cell gap-junctional coupling. Here, we provide morphological evidence that 18 beta-glycyrrhetinic acid (18 beta-GA), a saponin isolated from licorice root that is an inhibitor of gap-junctional communication, caused the disassembly of gap-junction plaques in WB-F344 rat liver epithelial cells. This effect was dose (5-40 microM) and time dependent (1-4 h treatment). Gap-junction channels in WB-F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43-P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18 beta-GA was correlated with decreases in Cx43-P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18 beta-GA-induced dephosphorylation of Cx43-P2 and not its degradation or the inhibition of Cx43-NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18 beta-GA causes type 1 or type 2A protein phosphatase-mediated Cx43 dephosphorylation coincident with the disassembly of gap-junction plaques.

Induction of nitric oxide synthase inhibits gap junction permeability in cultured rat astrocytes.

Nitric oxide (.NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the NG-methyl-L-arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of L-[3H]arginine to L-[3H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 microgram/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 microgram/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O2.-) scavenger superoxide dismutase. Incubation of the cells with both the .NO donor S-nitroso-N-acetylpenicillamine and the O2.(-)-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between .NO and O2.-, to form the peroxynitrite anion (ONOO-), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO- derivative hydroxyl radical (.OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO- caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO-). The pathophysiological relevance of ONOO(-)-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.

Evidence That Disruption of Connexon Particle Arrangements in Gap Junction Plaques Is Associated with Inhibition of Gap Junctional Communication by a Glycyrrhetinic Acid Derivative

Glycyrrhetinic acid exhibits many pharmacological activities, including the ability to block gap junctional communication. However, the mechanism of glycyrrhetinic action is not clear. Others have shown that glycyrrhetinic acid apparently binds to a single proteinatious binding site in the plasma membrane. We present evidence that while exposure to glycyrrhetinic acid derivatives may not affect protein synthesis or location, it does seem to alter connexon particle packing in gap junction plaques.

799 Chronic inhibition of phosphatases 1 and 2A in rat brain — A non-transgenic model of Alzheimer's disease

799 Chronic inhibition of phosphatases 1 and 2A in rat brain — A non-transgenic model of Alzheimer's disease

Lindane-Induced Elimination of Gap Junctional Communication in Rat Uterine Myocytes Is Mediated by an Arachidonic Acid-Sensitive cAMP-Independent Mechanism

Previous studies by this laboratory showed that the pesticide lindane rapidly and potently inhibits gap junctional communication in myometrial smooth muscle cells. This study examined the possible role of cAMP or arachidonic acid in lindane′s elimination of myometrial gap junctional communication. Lindane produced concentration-dependent increases in cAMP of 1.21, 2.94, 6.06, and 8.69 pmol/mg protein with 0.1, 1, 30, and 100 μM lindane, respectively, compared to solvent-treated controls (1.27 pmol/mg protein). Lindane also increased release of tritiated arachidonic acid to 342, 509, 852, 1236, 1639, and 4454 dpm/μg protein with 0.01, 0.1, 1, 10, and 100 μM lindane, respectively, compared to solvent controls (342 dpm/mu g protein). Transfer of Lucifer Yellow dye was used as a measure of gap junctional communication. Both 8-br-cAMP (98, 97, 54, and 4% transfer seen with 0, 1, 10, and 100 μM cAMP) and arachidonic acid (98, 73, 54, 31, and 0% dye transfer for 0.1, 1, 10, 100, and 1000 nM arachidonic acid) depressed dye transfer in cultured myocytes. Although the adenylate cyclase inhibitor 2′,3′-dideoxyadenosine completely reversed forskolin-induced depression of dye transfer (1 μM forskolin, 22% transfer), it had no effect with lindane, indicating that lindane′s depression of dye transfer was independent of adenylate cyclase activation. Lindane′s inhibition of dye transfer was effectively reversed by growing myometrial cells under arachidonic acid-free conditions in the presence of eicosapentaenoic acid, a fatty acid that competes with arachidonic acid for the sn-1,2 position of membrane phospholipids: 0, 15, 40, and 88% dye transfer occurred in the presence of 0.01, 0.1, 1, and 10 μM eicosapentaenoic acid with 30 μM lindane. This implies that arachidonic acid release may be a critical event associated with lindane′s inhibition of gap junctional communication in uterine myocytes.

Propagation of intercellular calcium waves in PC12 cells overexpressing a carboxy-terminal fragment of amyloid precursor protein

We have recently found that PC 12 cells overexpressing a C-terminal 97 amino acid fragment containing the β/A4 amyloid peptide of amyloid precursor protein (APP) exhibit an induced production of the gap junctional protein connexin43 (Cx43) and an induction of gap junctional communication as assessed by dye-transfer. In studies of two β/A4-transfected PC12 clones, we show here that these cells, unlike normal PC12 cells or those transfected with vector alone, have the capacity for intercellular transmission of calcium waves as determined by imaging of calcium with fura-2. Intercellular wave propagation occurred in the absence of extracellular calcium and was blocked by known inhibitors of gap junctional communication (octanol and 12- O-tetradecanoyl-phorbol-l3-acetate), suggesting mediation by gap junctions. The results indicate a disruptive influence of the C-terminal region of APP or of β/A4 amyloid peptide on intercellular signaling via gap junctions, which may be relevant to the normal functions of APP or to pathology in Alzheimer's disease.

Transforming growth factor β1 (TGF-β1) inhibits GAP junctional communication and peptide hormone production during human trophoblast differentiation

Transforming growth factor β1 (TGF-β1) inhibits GAP junctional communication and peptide hormone production during human trophoblast differentiation

Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells.

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.

Glutathione depletion potentiates 12- O-tetradecanoyl phorbol-13-acetate(TPA)-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells: relationship to intracellular oxidative stress

Treatment of WB-F344 liver epithelial cells with buthionine sulfoximine (BSO, 100 /∗M) for 24 h caused a greater than 95% depletion in cellular glutathione (GSH) and potentiated the ability of 12- O-tetradecanoyl phorbol-13-acetate (TPA) to inhibit gap junctional intercellular communication (GJIC) between the cells (IC 50 shifted from 5 μM to 2 μM). Similarly, acute depletion of GSH by up to 30%, either with the thiol oxidant diamide or with BSO, also potentiated the inhibitory effect of the phorbol ester on GJIC. The treatment of the control cells with TPA caused a concomitant increase in the accumulation of oxidation products of 2′,7′-dichlorofluorescein (DCF), indicating elevated production of oxidants in the cells during the blockade of GJIC. The depletion of GSH over a 24 h period with BSO itself increased the flux of oxidants in the cells but did not inhibit GJIC. Treatment of these GSH-depleted cells with TPA caused an additive elevation in the accumulation of oxidised DCF metabolites. Direct application of H 2O 2 (25–200 μM) or benzoyl peroxide (25–150 μM) to the control cells for 60 min caused weak, dose-dependent inhibitions of gap junctional communication in these cells but these responses were accompanied by the induction of acute, sub-lethal cytotoxicity. The depletion of GSH from the cells did not potentiate these responses to the peroxides but did facilitate synergistic inhibition of gap junctional communication in response to both TPA and sub-toxic doses of either peroxide. The results of the above studies indicate that oxidants are produced in WB-F344 cells in response to TPA and that these function in a co-operative manner with other cellular responses to the phorbol ester in the inhibition of gap junctional communication. This may explain why priming the cells for the induction of oxidative stress by the depletion of GSH potentiates the inhibitory activity of TPA on gap junctional communication.

Lindane Inhibition of Gap Junctional Communication in Myometrial Myocytes Is Partially Dependent on Phosphoinositide-Generated Second Messengers

The ability of environmental contaminants to modulate gap junctional communication between uterine smooth muscle cells is generally unknown, despite recognition that myometrial gap junctions may play a role in synchronizing uterine contractions during parturition. The present study tested the hypothesis that the organochlorine pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junctional communication in myometrial myocytes due to the release of phosphoinositide-dependent second messengers. The effect on gap junctional communication by lindane was tested in cultured rat myometrial smooth muscle cells by monitoring transfer of the fluorescent dye Lucifer yellow. A rapid, concentration-dependent, but reversible inhibition of dye transfer was noted with 4-min exposures, and inhibition was complete with 10 microM lindane. Lindane also stimulated the production of the Ca(2+)-releasing species inositol 1,4,5-trisphosphate which peaked at 5 min (100 pmol/mg protein) and remained elevated after a 15-min exposure. To examine the possible inhibitory role of Ca2+ on gap junctions, the Ca2+ ionophore 4-br-A23187 was used. Although A23187 also inhibited gap junctional communication, inhibition was not complete even at concentrations that appeared cytotoxic (70% inhibition at 2 microM A23187). Cells were then loaded with the Ca2+ chelator BAPTA-AM, which blocked the lindane-induced rise in calcium, and dye transfer experiments with lindane were repeated in Ca(2+)-free medium. Inhibition of dye transfer was still complete under these conditions, showing that increased intracellular calcium was not required for lindane-induced inhibition of gap junctional communication. Subsequently, 10 microM lindane was shown to produce a sustained increase in protein kinase C (PKC) activity (31, 17, and 15 pmol of PKC peptide phosphorylated/min/mg protein for 2-, 5-, and 10-min exposures, respectively). Known activators of PKC, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol, abolished gap junctional communication at nanomolar concentrations. Although use of the PKC inhibitor staurosporine failed to reverse lindane's inhibitory action, depletion of PKC activity through prolonged exposure to TPA partially reversed lindane's effect. This suggests that PKC activation potentiates but does not solely mediate lindane's inhibitory action on gap junctional communication.

Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated.

Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n-6 and n-3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 microM arachidonic acid (20:4 n-6) resulted in a rapid and transient loss of communication competence. The response to 20 microM 20:4 (n-6) was prolonged (> 210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20:4 (n-6). The fatty acids, 18:3 (n-6), 20:5 (n-3), 22:6 (n-3) and the 15-hydroxy- and the 15-hydroperoxy-derivatives of 20:4 (n-6) were also powerful inhibitors of GJC, while 23:4 (n-6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20:0, and the methyl ester of 20:4 (n-6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20:4 (n-6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n-6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20:4 (n-6) was antagonized by preincubating WB cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethyl-xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20:4 (n-6). As expected, other inhibitors of gap junctional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancement in the phosphorylation of Cx43 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of gap junctional communication, trophoblast differentiation and hCS expression by heptanol in human trophoblast in culture

Inhibition of gap junctional communication, trophoblast differentiation and hCS expression by heptanol in human trophoblast in culture

Effects of Selected Anti-Tumor-Promoting Chemicals on Metabolic Cooperation between Chinese Hamster V79 Cells

Many tumor-promoting chemicals inhibit gap junctional communication between cells. We investigated the possibility that antipromoting chemicals may act inversely and enhance gap junctional communication. The V79/metabolic cooperation assay is an in vitro test that measures gap junctional communication indirectly by determining the extent of metabolic cooperation between mutant and wild-type V79 Chinese hamster lung fibroblasts in culture. Six in vivo antipromoters (caffeine, 3-isobutyl-1-methylxanthine (IBMX), phenidone, dibromoacetophenone, tosylphenylalanine chloromethyl ketone (TPCK), and acetic acid) were tested in this assay to assess their effects on metabolic cooperation. Caffeine, IBMX, phenidone, and dibremoacetophenone had no effect on metabolic cooperation, while TPCK slightly inhibited metabolic cooperation in one V79 assay. Acetic acid appeared to facilitate metabolic cooperation. In tests where an antipromoter was combined with the established tumor promoter phorbol 12-myristate 13-acetate (PMA), acetic acid, caffeine, and IBMX counteracted PMA-induced inhibition of metabolic cooperation, while phenidone, dibromoacetophenone, and TPCK had little effect. These results indicate that some antipromoters interfere with the ability of a tumor-promoting chemical to inhibit metabolic cooperation and suggest that alteration of gap junctional communication can be a mechanism of antipromoter action.

Dissociation of PDGF receptor tyrosine kinase activity from PDGF-mediated inhibition of gap junctional communication.

Gap junction-mediated intercellular communication (GJC) may play an important role in cell proliferation and transformation since GJC is inhibited by growth factors, oncogenes, tumor promoters, and carcinogens. We have studied inhibition of GJC by platelet-derived growth factor-BB (PDGF) in the mouse fibroblast cell line C3H/10T1/2 and have sought to determine whether PDGF-induced inhibition of GJC is mediated by the PDGF receptor tyrosine kinase (RTK). PDGF-mediated inhibition of GJC was rapid and transient, with maximal inhibition occurring 40 min after PDGF addition and GJC returning to control levels after 70 min. The effect of PDGF on GJC was concentration-dependent, with maximal inhibition of 90% or greater occurring at 10 ng/ml PDGF. Stimulation of RTK activity, as determined by antiphosphotyrosine immunoblot analysis of PDGF receptor and the receptor substrates phospholipase C-gamma I (PLC-gamma I) and guanosine triphosphatase activating protein (GAP), was also concentration-dependent. Inhibition of GJC required a greater concentration of PDGF than did stimulation of RTK activity. The tyrosine kinase inhibitor genistein blocked PDGF-induced RTK activity, as measured by PDGF receptor, PLC-gamma I, and GAP tyrosine phosphorylation, in a concentration-dependent manner but did not affect PDGF-mediated inhibition of GJC. Genistein alone had no effect on GJC or PDGF receptor expression. PDGF treatment in the presence or absence of genistein resulted in phosphorylation of the connexin 43 protein on nontyrosine residues. These results suggest that inhibition of GJC by ligand-activated PDGF receptor is dissociable from the RTK activity responsible for PDGF, PLC-gamma I, and GAP phosphorylation.