ABSTRACT
It is widely accepted that a prolonged ouabain blockade of the Na(+),K(+)-ATPase makes cells detach from each other and from the substrate, leading to their death and that cellular resistance to ouabain is due to the presence of isoforms of Na(+),K(+)-ATPase with low affinity to this glycoside. In the present work the effect of reduced glutathione in the response of two types of renal cells to ouabain: MDCK, a ouabain-sensitive cell line and Ma104, a ouabain-resistant one, was studied. Glutathione protected MDCK cells from ouabain toxicity and inhibition of glutathione synthesis by L-buthionine-S,R-sulfoximine sensitized Ma104 cells to ouabain. As glutathione is involved with multidrug resistance (MDR) in cells expressing the multidrug resistance-related protein MRP1 and as Ma104 cells have a MDR phenotype, it was investigated whether Ma104 cells express this protein. The expression of the MRP1-mRNA in Ma104 cells was detected by reverse transcriptase-polymerase chain reaction and ribonuclease protection assay, and the protein was detected by Western blotting and immunofluorescence. Treatment of Ma104 cells with ouabain increased MRP1-mRNA expression and altered the localization of MRP1 in these cells. Our results suggest that some cells may have mechanisms to protect themselves from ouabain toxicity and that MRP1 may have a role in controlling the toxic effects of ouabain.
Subject(s)
ATP-Binding Cassette Transporters/analysis , Antioxidants/pharmacology , Gene Expression Regulation/drug effects , Glutathione/pharmacology , Ouabain/toxicity , ATP-Binding Cassette Transporters/genetics , Animals , Blotting, Western , Buthionine Sulfoximine/pharmacology , Catalase/pharmacology , Cell Line/drug effects , Drug Resistance, Multiple/genetics , Fluorescent Antibody Technique , Glutathione/antagonists & inhibitors , Multidrug Resistance-Associated Proteins , Ouabain/antagonists & inhibitors , Phenotype , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/pharmacologyABSTRACT
Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells, selected for resistance to one chemotherapeutic agent, simultaneously acquire resistance to several apparently unrelated drugs. The MDR phenotype is multifactorial. The best-studied mechanism involves the expression of a membrane protein that acts as an energy-dependent efflux pump, known as P-glycoprotein (Pgp), capable of extruding toxic materials from the cell. In this work, resistance to UVA radiation, but not to UVC nor UVB, was observed in an MDR leukemia cell line. This cell line overexpresses Pgp. To study the role of Pgp in the resistance to UVA radiation, two MDR modulators or reversing agents (verapamil and cyclosporin A) capable of blocking Pgp activity were used. Cell viability was assessed and the techniques of flow cytometry and fluorescence microscopy were employed to measure the extrusion of rhodamine 123 by the efflux pump. The results show that MDR modulators did not modify the resistance to UVA radiation. Furthermore, although cell viability was not significantly altered, Pgp function was impaired after UVA treatment, suggesting that this glycoprotein may be a physical target for oxidative damage, and that other factors may be responsible for the UVA resistance. In agreement with this, it was found that the resistant cell line presented a higher catalase activity than the parental (non-MDR) cell line.
