Action of membrane-active compounds on mammalian cells. Permeabilization of human cells by ionophores to inhibitors of translation and transcription.

Abstract

1. Hygromycin B, an inhibitor of protein synthesis in eukaryotic cell-free systems, does not block translation in intact HeLa cells, as mammalian cells are normally impermeable to this antibiotic. However, the presence in the culture medium of the ionophore nigericin at concentrations that did not interphere with protein synthesis rendered human HeLa cells permeable to hygromycin B. A similar effect was achieved with a number of ionophores, such as valinomycin, monensin, gramicidin D, bromolasalocid, A23187, amphotericin B and nystatin, as well as with the cardioglycoside antibiotic ouabain, which blocks (Na+, K+)-ATPase activity. The permeabilization of HeLa cells by nigericin to several inhibitors of translation was also studied. The inhibition of protein synthesis by destomycin A, anthelmycin, gougerotin, edeine, tetracycline and several nucleotide derivatives in cultured HeLa cells was greatly enhanced by the presence of 0.8 μM nigericin. 2. Concentrations of α-amanitin above 200 μ/ml were required to inhibit RNA synthesis completely in intact cells. Once more, the presence of nigericin reduced 20–50-fold the concentration of α-amanitin necessary to inhibit transcription. Interestingly enough, nigericin alone proved to be a very effective inhibitor of protein synthesis in HeLa cells: 2 μM nigericin almost completely abolished the translation capacity of the cells, whereas transcription was much more resistant to inhibition by nigericin. 3. The concentration of monovalent cations in the culture medium drastically affected: (a) the permeability of HeLa cells to hygromycin B, (b) the inhibition of protein synthesis by nigericin and (c) the permeability of HeLa cells to hygromycin B induced by nigericin. A very simple method of permeabilizing cultured cells to low-molecular-weight compounds has been devised. This method, which permits translation in HeLa cells to continue at control levels under the permeabilization conditions, is very mild and is based on the manipulation of the monovalent ion concentrations of the culture medium.