Yoshiki Shiba

Monoclonal antibody ECCD-1 inhibits intercellular communication in teratocarcinoma PCC3 cells

The monoclonal antibody ECCD-1 recognizing a certain class of cell surface proteins inhibits the Ca2+-dependent cell-to-cell adhesion in teratocarcinoma stem cells. In this paper, we studied the effect of ECCD-1 on cell-to-cell communication in PCC3 cells by measuring the transfer of lucifer yellow between cells. To this aim, PCC3 cells were cultured in the presence of ECCD-1 for various periods, and then the fluorescent dye was injected into a cell located in the center of cell colonies, followed by counting number of cells to which the dye was transferred. The results showed that ECCD-1 inhibits the dye transfer between cells, suggesting that the Ca2+-dependent cell-to-cell adhesion system (CDS) is essential for the functions of gap junction.

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling): Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [3H]phorbol-12,13-dibutyrate [( 3H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 X 10(5) PDBu molecules were bound to each cell. The binding of [3H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4 alpha-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [3H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.

Potentiation by Isoproterenol on Carbachol-induced K+ and Cl− Currents and Fluid Secretion in Rat Parotid

Abstract. Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid acinar cells, IPR and cpt-cAMP potentiated CCh-induced K+ and Cl− currents (IK and ICl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca2+ concentration ([Ca2+]i), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn2+ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca2+]i was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced by IPR through the potentiation of IK and ICl mainly by the increased cyclic AMP level and partially by the potentiated Ca2+ influx and [Ca2+]i increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca2+]i increase.

Protective effect of cAMP on tumor promoter-mediated inhibition of cell-cell communication☆

The formation and maintenance of electrical cell coupling is inhibited by 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). This inhibition could be prevented by db-cAMP and aminophylline when added together with TPA, though they did not restore the cell coupling once it had already been blocked by TPA. It is suggested that db-cAMP affects the early membrane effects of TPA in FL cells.

Gramicidin-perforated patch analysis on HCO3- secretion through a forskolin-activated anion channel in rat parotid intralobular duct cells.

Abstract. Forskolin-induced anion currents and depolarization were investigated to clarify the mechanism of HCO3− secretion in the intralobular duct cells of rat parotid glands. Anion currents of the cells were measured at the equilibrium potential of K+, using a gramicidin-perforated patch technique that negligibly affects intracellular anion concentration. The forskolin-induced anion current was sustained and significantly (54%) suppressed by glibenclamide (200 μm), a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. The anion current was markedly suppressed by addition of 1 mm methazolamide, a carbonic anhydrase inhibitor, and removal of external HCO3−. Forskolin depolarized the cells in the current-clamp mode. Addition of methazolamide and removal of external HCO3− significantly decreased the depolarizing level. These results suggest that activation of anion channels (mainly the CFTR Cl− channel located in luminal membranes) and production of cytosolic HCO3− induce the inward anion current and resulting depolarization. Inhibition of the Na+-K+-2Cl− cotransporter and the Cl−-HCO3− exchanger had no significant effect on the current or depolarization, indicating that the uptake of Cl− via the Na+-K+-2Cl− cotransporter or the Cl−-HCO3− exchanger is not involved in the responses. Taken together, we conclude that forskolin activates the outward movement (probably secretion) of HCO3− produced intracellularly, but not of Cl− due to lack of active Cl− transport in parotid duct cells, and that the gramicidin-perforated patch method is very useful to analyze anion transport.

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells.

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodamine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. The enhancement of binding of the beads by TPA was suppressed by addition of an adhesion-inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro). Treatment with TPA did not enhance nonspecific binding of beads coated with heat-denatured bovine serum albumin. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.

Carbachol-induced fluid movement through methazolamide-sensitive bicarbonate production in rat parotid intralobular ducts: Quantitative analysis of fluorescence images using fluorescent dye sulforhodamine under a confocal laser scanning microscope

Fluid secretion is observed at the openings of ducts in the exocrine gland. It remains unclear whether the ducts are involved in fluid secretion in the salivary glands. In the present study, we investigated the exclusion of fluorescent dye from the duct lumen by carbachol (CCh) in isolated parotid intralobular duct segments to clarify the ability of the ducts for the fluid secretion. When the membrane-impermeable fluorescent dye, sulforhodamine, was added to the superfused extracellular solution, quantitative fluorescence images of the duct lumen were obtained under the optical sectioning at the level of the duct lumen using a confocal laser scanning microscope. CCh decreased the fluorescent intensity in the duct lumen during the superfusion of the fluorescent dye, and CCh flushed out small viscous substances stained with the fluorescent dye from isolated duct lumen, suggesting that CCh might induce fluid secretion in the duct, leading to the clearance of the dye and small stained clumps from the duct lumen. CCh-induced clearance of the fluorescent dye was divided into two phases by the sensitivity to external Ca2+ and methazolamide, an inhibitor for carbonic anhydrase. The initial phase was insensitive to these, and the subsequent late phase was sensitive to these. A major portion in the late phase was inhibited by removal of bicarbonate in the superfusion solution and DPC, but not low concentration of external Cl-, bumetanide or DIDS, suggesting that methazolamide-sensitive production of HCO3-, but not the Cl- uptake mechanism, might contribute to the CCh-induced clearance of the dye from the duct lumen. These results represent the first measurements of fluid movement in isolated duct segments, and suggest that carbachol might evoke fluid secretion possibly through Ca2+-activated, DPC-sensitive anion channels with HCO3- secretion in the rat parotid intralobular ducts.

Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory Cl− Movements in Salivary Acinar Cells

Elevations of cytoplasmic free calcium concentrations ([Ca2+]i) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl− through Ca2+-activated Cl− channels, while Cl− enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na+-K+-2Cl− cotransporter (NKCC) and/or parallel operations of Cl−-HCO3 − and Na+-H+ exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl− secretion, we analyzed carbachol (CCh)-activated Cl− currents in submandibular acinar cells using the “gramicidin-perforated patch recording configuration.” Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl− currents in the gramicidin-perforated patch recording were carried by Cl− efflux via Cl− channels, dependent upon Cl− entry through Cl− transporters expressed in the acinar cells. CCh-evoked oscillatory Cl− currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl− currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO3 − had significant effects, suggesting that the cotransporter rather than parallel operations of Cl−-HCO3 − and Na+-H+ exchangers is the primary Cl− uptake pathway. Pharmacological manipulation of the activities of the Ca2+-activated Cl− channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl− currents, while adjusting to the rate imposed by the Ca2+-activated Cl− channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl− movements may effectively provide a driving force for fluid secretion in intact acinar cells.

The relationship between membrane potential and ATP content in rat liver during ischemia

Both ischemia and ethionine decreased the membrane potential and ATP content in rat liver. In ethioninetreated rat liver, ischemia brought about a further decrease in membrane potential without significant decrease in ATP content.

Irsogladine maleate counters the interleukin‐1β‐induced suppression in gap‐junctional intercellular communication but does not affect the interleukin‐1β‐induced zonula occludens protein‐1 levels in human gingival epithelial cells

BACKGROUND AND OBJECTIVE Irsogladine maleate counters gap junctional intercellular communication reduction induced by interleukin-8 or Actinobacillus actinomycetemcomitans in cultured human gingival epithelial cells. Interleukin-1 beta is involved in periodontal disease. Little is known, however, about the effect of interleukin-1 beta on intercellular junctional complexes in human gingival epithelial cells. Furthermore, irsogladine maleate may affect the actions of interleukin-1 beta. In this study, we examined how interleukin-1 beta affected gap junctional intercellular communication, connexin 43 and zonula occludens protein-1, and how irsogladine maleate modulated the interleukin-1 beta-induced changes in the intercellular junctional complexes in human gingival epithelial cells. MATERIAL AND METHODS Human gingival epithelial cells were exposed to interleukin-1 beta, with or without irsogladine maleate. Connexin 43 and zonula occludens protein-1 were examined at mRNA and protein levels by real-time polymerase chain reaction and western blotting, respectively. Gap junctional intercellular communication was determined using the dye transfer method. The expression of zonula occludens protein-1 was also confirmed by immunofluorescence. RESULTS Interleukin-1 beta decreased connexin 43 mRNA levels, but increased zonula occludens protein-1 mRNA levels. Irsogladine maleate countered the interleukin-1 beta-induced reduction in gap junctional intercellular communication and connexin 43 levels. However, irsogladine maleate did not influence the increased zonula occludens protein-1 levels. CONCLUSION The effect of interleukin-1 beta on gap junctional intercellular communication and tight junctions of human gingival epithelial cells is different. The recovery of gap junctional intercellular communication by irsogladine maleate in the gingival epithelium may be a normal process in gingival epithelial homeostasis.