Yoshinobu Kanno

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus): I. Interaction between cell membranes and virus in the early stage☆

Abstract A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.

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.

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.

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.

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.

Close relationship between modulation of serum-induced stimulation of DNA synthesis and changes in gap-junctional intercellular communication in quiescent 3T3-L1 cells caused by cyclic AMP and the tumor-promoting phorbol ester TPA☆

Involvement of gap-junctional intercellular communication in the stimulation of growth was investigated in quiescent 3T3-L1 cells. When the cells in monolayer were growth-arrested by culture in a low concentration of calf serum, addition of dibutyryl cyclic AMP enhanced dye-coupling and suppressed the enhancement of DNA synthesis, induced by calf serum, in quiescent cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed dye-coupling in quiescent cells and enhanced DNA synthesis in both quiescent and serum-treated cells. When about 5000 cells were cultured in contact to form a colony, growth arrest of the cells was observed in the central region of such colonies rather than in the peripheral region, but addition of calf serum induced DNA synthesis in the cells in both the peripheral and central regions of the colonies. Addition of TPA enhanced serum-induced DNA synthesis in the cells in the central region of colonies rather than in the peripheral region. These results suggest that the ability of quiescent cells to escape from growth arrest is inversely correlated to the extent of gap-junctional intercellular communication.

Visualization of the secretory process involved in Ca2+-activated fluid secretion from rat submandibular glands using the fluorescent dye, calcein.

The central feature of fluid and electrolyte secretion by salivary acinar cells is transepithelial Cl- movement as a driving force for the secretion. However, little is known about the membrane localization and regulation by agonists of various anion channels. To characterize the anion transport and fluid secretion, we visualized the secretory process induced by the cholinergic agonist, carbachol (CCh), using the anionic fluorescent dye, calcein, under a confocal laser scanning microscope. The fluorescence of calcein loaded into the isolated acini was spread diffusely throughout the cytoplasm and was less intense in the secretory vesicles which occupied the apical pole. Cytoplasmic calcein was released into intercellular canaliculi just after the addition of CCh, depending upon a rise in [Ca2+]i by Ca2+ release from intracellular stores. Thereafter, the formation of watery vacuoles connected with intercellular canaliculi was visualized in the calcein-loaded acini, depending upon external Ca2+. Both the calcein release and vacuole formation were inhibited by suppressing the Ca(2+)-activated K+ efflux. The calcein release was also affected by the external anion substitution, suggesting that calcein is released through an anion channel. In the isolated, perfused glands, CCh-induced fluid secretion was sustained in two phases, whereas the loaded calcein was initially and transiently released into the saliva. By revealing the [Ca2+]i dependence and sensitivities to channel blockers, our results suggest that the initial phase of CCh-induced fluid secretion was evoked in association with the release of the organic anion, calcein, and the late phase of fluid secretion, during which calcein is less permeable, was associated with the formation of watery vacuoles. Thus, the anion channels possessing the distinct property of anion permeation may be activated in the initial phase and late phase. These results indicate that the anionic fluorescent dye, calcein, is useful for visualizing the process of Ca(2+)-dependent fluid secretion, and for clarifying the relation between fluid secretion and anion transport.

Different localizations of 21 and 27 kDa gap-junction proteins in rat salivary glands

Antibodies against 21 and 27 kDa gap-junction proteins from rat liver were used to examine the identification and localization of gap-junction proteins in rat salivary glands. Acinar cells of the submandibular glands and parotid glands stained well for the 27 kDa gap junction protein and less intensely for the 21 kDa protein. Acinar cells of the sublingual glands were stained heavily for the 27 kDa gap junction protein and stained well for 21 kDa gap junction protein. No 27 kDa protein was observed in the ducts of the salivary glands. The 21 kDa gap-junction protein was distributed in some of the intercalated ducts in the parotid and submandibular glands. Immunoblotting of an extract of parotid glands with antibodies against 21 and 27 kDa gap-junction proteins revealed the presence of 21 and 27 kDa proteins in the parotid glands. It is concluded that the 27 kDa gap-junction protein in tistributed as a major component of the gap junctions in the acinar cells of all the salivary glands; the 21 kDa protein is localized as a minor component in the acinar cells and some portions of the intercalated ducts in the salivary glands. It is possible that these gap-junction proteins might contribute to the regulation of function of the salivary glands.

Involvement of intracellular calcium ions in the release of the fluorescent dye calcein by cholinergic and α-adrenergic agonists from rat parotid acinar cells

Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10−8 M to 10−6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca2+]i). Addition of NA at 10−6 M increased [Ca2+]i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10−6 M CCh, but it abolished the sustained increase in [Ca2+]i. The transient increase in [Ca2+]i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10−6 M), but not 10−3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca2+]i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca2+]i by muscarinic and α-adrenergic agonists and may represent the initial process of salivary secretion.