Ribonucleic Acid Synthesis Dependent on Exogenous Triphosphates in Nystatin-Treated Cells of
            <i>Kluyveromyces lactis</i>

Amphotericin B (AmB) and filipin effects on L and HeLa cells were compared by monitoring drug-induced potassium leakage from cells, changes in radioactive uridine incorporation into cellular ribonucleic acid, protein leakage from cells, and cell viability. L cells were much more susceptible to both AmB and filipin than were HeLa cells, but the overall dose response was similar. For AmB, the various effects were easily separable. Potassium leakage occurred at the lowest concentrations of AmB and was reversible. Inhibition of uridine incorporation and loss of viability occurred at intermediate levels, and protein loss occurred at higher levels. In contrast, filipin was much more potent; its effects on potassium leakage were only minimally reversible, and the separation of the permeabilizing effects from complete cell lysis was possible only over a limited concentration range and for a short time.Amphotericin B (AmB) belongs to the class of polyene antibiotics, which bind to the sterol component of the membranes of all eucaryotic cells (6,13). At sufficiently high concentrations, the polyene antibiotics cause an increase in membrane permeability, with leakage of metabolites and macromolecules from the cell and resultant cell death. Previous studies with fungi and animal cells in culture have shown that at low concentrations AmB can induce nonlethal permneabilizing changes in cell membranes which enlhance uptake of certain small molecules (16) and even macromolecules (9). These properties of AmB have been exploited to achieve the penetration into cells of pharmacologically active compounds (9, 12, 17) that do not penetrate the untreated cell membranes.We have undertaken the present study to compare the nonlethal and lethal effects of AmB on cultured L and HeLa cells, and to determine under which conditions they can be separated and whether the cell damages can be repaired. Similar studies have also been performed with filipin to determine whether permeability changes induced by this polyene antibiotic are separable from lethality. MATERIALS AND METHODSChemicals. AmB in the form of Fungizone was purchased from E. R. Squibb and Sons, Inc., Princeton, N.J. It was dissolved in sterile water before use.The concentrations of the AmB solutions were calculated on the basis of the amounts of AmB in the Fungizone, as stated by the manufacturer. Filipin, in the form of the unfractionated antibiotic complex, which was 86% pure, was donated by The Upjohn Co., Kalamazoo, Mich. The amounts noted refer to the crude product. Filipin was dissolved in methanol before use.[5-3H]uridine (specific activity, 8 Ci/mmol) was purchased from Schwarz/Mann, Orangeburg, N.Y. L-[methyl-3H]methionine (specific activity, 5.0 mCi/ 0.071 mg) was purchased from the New England Nuclear Corp., Boston, Mass. Triton X-100 (Triton) was purchased from Packard Instrumental Co., Downers Grove, Ill. Lithium diluent was purchased from Instrumental Laboratory, Inc., Lexington, Mass.Cell culture. HeLa and L 929 cells were maintained and assayed in monolayer in Ea...

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“…The inhibition of uridine incorporation probably reflects more serious membrane damage. This assumption is in line with work by others (1).…”

Section: Discussionsupporting

confidence: 79%