Browsing by Author "Willecke, Klaus"

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Open Access
Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes
(1989) Traub, Otto; Look, Jutta; Dermietzel, Rolf; Brümmer, Franz; Hülser, Dieter F.; Willecke, Klaus
Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.
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
Immunochemical and electrophysiological characterization of murine connexin40 and -43 in mouse tissues and transfected human cells
(1994) Traub, Otto; Eckert, Reiner; Lichtenberg-Fraté, Hella; Elfgang, Claudia; Bastide, Bruno; Scheidtmann, Karl Heinz; Hülser, Dieter F.; Willecke, Klaus
Human HeLa or SkHep1 cells, defective in intercellular communication through gap junctions, were transfected with coding sequences of murine connexin40 (Cx40) and -43. The transfected cells were restored in gap junctional coupling as shown by 100-fold increased electrical conductance. When studied by the double whole-cell patch-clamp technique, Cx40 HeLa transfectans exhibited single channel conductances of γ=121 ± 7 pS and γ=153 ± 5 pS. They were voltage gated with an equivalent gating charge of z=4.0 ± 0.5 for a voltage of half-maximal inactivation U 9= 44 ± 7 mV. The corresponding values or connexin43 (Cx43) HeLa transfectants are: γ=60 ± 4 pS and γ=40 ± 2 pS as well as z=3.7 ± 0.8 and U 0 = 73 ± 7 mV. Transfer of the dye Lucifer Yellow was always considerably lower in Cx4- than in Cx43-transfectants though their total junctional conductance was similar or even higher than for Cx43-transfectants.
Open Access
Isolation and characterization of Chinese hamster cells defective in cell-cell coupling via gap junctions
(1983) Willecke, Klaus; Müller, Dagmar; Drüge, Petra Maria; Frixen, Uwe; Schäfer, Reinhold; Dermietzel, Rolf; Hülser, Dieter F.
Chinese hamster Wg3-h-o cells which were descended from DON cells have been mutagenized and selected for derivatives defective in metabolic cooperation via gap junctions (i.e., mec−). The selection protocol included four consecutive cycles of cocultivating mutagenized cells, deficient in hypoxanthine phosphoribosyltransferase (HPRT) and wild-type cells in the presence of thioguanine (cf Slack, C, Morgan, R H M & Hooper, M L, Exp cell res 117 (1978) 195-205) [8]. We carried out the last two selection cycles in the presence of 1 mM dibutyryl cyclic adenosine monophosphate (db-cAMP). The isolated Chinese hamster CI-4 cells which expressed the mec− phenotype most stringently showed the following characteristics: 1. 1. In standard culture medium no cell-cell coupling was detected among CI-4 cells when assayed by injections of the fluorescent dye Lucifer yellow or by electrical measurements. Between 73 and 100% of the mec+ parental cells were coupled under these conditions. Up to 14% positive contacts were found between CI-4 cells and Chinese hamster Don cells (mec+). Confluent CI-4 cells grown in the presence of 1 mM db-cAMP showed 9% coupled cells. 2. 2. No gap junction plaques were found on electron micrographs of freeze-fractured, confluent CI-4 cells. The mec+ parental cells showed small gap junction plaques (0.013% of the total cell surface analyzed). 3. 3. CI-4 cells exhibited 16% positive contacts and the parental Wg3-h-o cells showed 92% positive contacts in autoradiographic measurements of metabolic cooperation with DON cells. On an extracellular matrix, prepared from normal embryonic fibroblasts, metabolic cooperation between CI-4 and DON cells was autoradiographically measured to be 68%. Other cells of spontaneous mec− phenotype (for example mouse L cells or human fibrosarcoma HT1080 cells) also appeared to exhibit increased metabolic cooperation when grown on an extracellular matrix and assayed by autoradiographic measurements. When tested by Lucifer yellow injections, however, only very few positive contacts were found for CI-4/DON cell pairs and no positive contacts were found among mouse L cells grown on an extracellular matrix. 4. 4. The mec− defect in the genome of CI-4 cells was cured in somatic cell hybrids with mouse embryonic fibroblasts or with mouse embryonal carcinoma cells. The results of isozyme and karyotype studies of mec−, as well as mec+ somatic cell hybrids suggest that mouse chromosome 16 may be involved in complementation of the mec− defect.