Browsing by Author "Zempel, Günther"

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Open Access
Arachidonovaja kislota obratimo blokiruet vysokopronicaemye mežkletočnye kontakty
(1994) Hülser, Dieter F.; Zempel, Günther; Reuss, Bernhard; Suhr, Dierk; Sarovskaja, Julija J.; Murav'eva, O. V.; Dunina-Barkovskaja, Antonina; Margolis, Leonid B.
The effect of arachidonic on intercellular coupling via gap junctions has been studied in BICR/M1R k cells - a mammary tumor cell line of the Marshall ratt. Arachidonic acid is shown to reversibly block both ionic and dye coupling in a dose-dependent manner. The cells recoupled after the washout with either serum- or albumin (essentially fatty acid-free)-containing solution. The intercellular pH decreased from 7,2 to 7,0 after arachidonic acid treatment; the same pH shift in the absence of arachidonic acid, however, had no effect on the junctional permeability. Flow cytometric measurments revealed an arachidonic acid-induced increase of the cytoplasmic free Ca 2+ concentration which was also reversible upon albumin treatment. Intracellular Ca 2+ or H+ are unlikely to be involved in the mechanism of the arachidonic acid effect on intercellular coupling: high resolution measurments using double whole-cell technique also show reversible blockage of the junctional conductance in the presence of arachidonic acid while the pipette solution was buffered with 10 mM HEPES and 10 mM EGTA to clamp intracellular calcium and proton concentrations. We suggest that arachiconic acid directly affects the gap junction channels, probably interfering with the lipid-protein interactions.
Open Access
Growth inhibition of oncogene transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions
(1991) Martin, Wolfgang; Zempel, Günther; Hülser, Dieter F.; Willecke, Klaus
We have studied the proliferation of rat 208F cells (a derivative of Rat-1 cells) transformed by activated c-Ha-ras, v-fgr, v-raf, v-fms, or v-src oncogenes during cocultivation with an excess of early passage rat embryonic fibroblasts or immortal 208F cells. The total number and size of foci formed by oncogene-transformed 208F cells were strongly reduced by cocultured normal fibroblasts. The extent of growth suppression of transformed foci appears to be dependent on the type of transforming oncogene and on the type of normal fibroblasts rather than on the extent of gap-junctional communication between transformed and normal cells. Total inhibition of fluorescent dye transfer between normal and transformed cells by the 3β-O-hemisuccinate of 18α-glycyrrhetinic acid (18α-carbenoxolone), an inhibitor of gap-junctional communication in human fibroblasts, did not prevent growth inhibition of transformed cells in the cocultivation assay. Since adjacent cells remained electrically coupled in the presence of this inhibitor it is possible that the strongly reduced metabolic cooperation, as indicated by the lack of fluorescent dye transfer, is sufficient for mediating the growth-inhibitory effect of normal fibroblasts. 208F cell-conditioned medium, however, also caused strong growth inhibition of transformed derivatives, suggesting that the effect is in part mediated by release of stable growth inhibitor(s) from 208F cells.
Open Access
Patch-clamp measurements of gap-junction channels in cultured cells
(1992) Hülser, Dieter F.; Eckert, Reiner; Zempel, Günther; Paschke, Dietmar; Dunina-Barkovskaja, Antonina
Direct intercellular communication in most tissues is made possible by proteinaceous pores called gap-junction channels. These channels bridge the extracellular gap between apposed cells and connect their intracellular compartments both electrically and metabolically. The extracellular parts of two hemichannels - the connexons - are linked thus forming a communicating gap-junction channel. A connexon is a hexamer of protein subunits which are members of the connexin family. Since connexin 32 (Cx32) was the first gap-junction channel protein to be sequenced from hepatocytes, it serves as a reference to which all other gap-junction proteins are compared. The individual channel conductance may vary between 25 and 150 pS. Gap-junction channels of some tissues are more voltage sensitive (e.g. liver) than others (e.g. heart). The question whether these differences in electrical properties may be attributed to the different connexins being expressed in these tissues is still unanswered. Several approaches to resolve this problem will be discussed in this contribution, all are based on double whole-cell patch-clamp measurements using isolated cell pairs, as follows: (1) Cells with two different channel conductances perfused with anti connexin antibodies to specifically block one channel species; (2) Cells with only one connexin species selected by immunological characterization; (3) Weakly coupled HeLa cells transfected with specific connexin genes, a method which resulted in better correlations between connexin type and single channel properties.
Open Access
Production of tissue plasminogen activator (t-PA) with differentiated F9-embryonic carcinoma cells grown as multicell spheroids
(1991) Brenner, Joachim; Zempel, Günther; Hülser, Dieter F.
F9 cells can only temporarily be cultivated as single cell suspension. The growth of multicell spheroids from a single cell suspension is shown. Within 24 hours the cells aggregated to multicell spheroids in spinner flasks. The aggregation was almost finished within 24 h and was independent of the initial cell concentration. but could be influenced by the geometry of the cultu re flask and the stirring velocity. A significant cell proliferation of the anchorage-dependent F9 cells was only detectable in the aggregated state. A narrow size distribution of multicell spheroids in a 3 days old culture revealed identical spheroid sizes. Under our conditions. with an inital cell density of 10 5 cells/ml a cell concentration of 6x10 6 cells/ml was reached within 4 days of spheroid culture. Cells in monolayers and in multicell spheroids were well coupled by gap junctions. Differentiated F9 cells showed a fibroblastoid morphology in contrast to the epitheloid morphology of undifferentiated F9 stem cells. This was not the case for multicell spheroids where the cells were also well coupled.
Open Access
Retinoic acid modulates gap junctional permeability: a comparative study of dye spreading and ionic coupling in cultured cells
(1991) Brümmer, Franz; Zempel, Günther; Bühle, Peter; Stein, Johannes-Christoph; Hülser, Dieter F.
All-trans retinoic acid (RA), which was recently identified as a morphogen, affects gap junctional permeability in a dose- and time-dependent manner. In five different established mammalian cell lines (FL, BRL, BICR/M1Rk, HEL37, BT5C1) 100 μmol/liter RA reduced Lucifer yellow spreading within 30 min to 20-50% of the control. Ionic coupling, however, remained almost unaffected under the same conditions. Freezefractured membranes of untreated and RA-treated cells were similar with regard to frequency and sizes of gap junction plaques. With concentrations of less than 10 μmol/liter RA the dye spreading increased significantly in the human amniotic cell line FL, pointing to a possible modulatory effect of RA on junctional communication.