P-31 AND C-13 NMR SPECTROSCOPIC STUDY OF WILD-TYPE AND MULTIDRUG-RESISTANT EHRLICH ASCITES TUMOR-CELLS

Steady-state P-31 NMR spectra of wild type EHR2 Ehrlich ascites tumor cells and multidrug resistant EHR2/DNR+ cells, immobilized in agarose threads, and continuously perfused with medium, showed temperature-dep endent differences in the levels of intracellular phosphate metabolite s. At 37-degrees-C, the EHR2/DNR+ cells contained four times more phos phocreatine (PCr) than the EHR2 cells. At 20-degrees-C, the EHR2 cells contained 80% more of phosphodiesters (PDE), the levels of PCr being equal. The quantitative metabolite level data are based on T1 relaxati on times data and are normalized for the protein content of the cells. Perfusion of the cells with azide, an inhibitor of mitochondrial resp iration, had no effect on the ATP level, and caused no changes in gluc ose consumption and lactate production. Azide perfusion, combined with glucose depletion, caused rapid drop in the ATP content, which was re established after renewed perfusion with glucose. Similarly, perfusion with 2,4-dinitrophenol, an uncoupler of the respiration chain, had no effect on the phosphate metabolites. These results demonstrate that a erobic glycolysis is the main route by which glucose is metabolised un der the conditions used (glucose concentration in medium 2 g/L). Rates of uptake and phosphorylation of 2-deoxy-D-glucose were measured by f ollowing the formation of intracellular [6-C-13]2-deoxy-D-glucose-6-ph osphate by C-13 NMR; at 37-degrees-C the observed rates for EHR2 and E HR2/DNR+ cells were equal, about 10 nmol/(min x mg protein), whereas a t 20-degrees-C the wild type cells produced the 6-phosphate at an appr oximately twice the rate found for the resistant cells [about 4 and 2 nmol/(min x mg protein), respectively]. The latter difference was also manifested during perfusion of the cells with unlabelled 2-deoxy-D-gl ucose at 20-degrees-C, following the rate of ATP depletion and formati on of 2-deoxy-D-glucose-6-phosphate by P-31 NMR. The multidrug resista nt Ehrlich ascites cells differ from most of previously investigated m ultidrug resistant cells as they do not show enhanced rates of glycoly sis relative to the wild type cells; since the EHR2/DNR+ cells exhibit a low level of multidrug resistance, and their resistance has been in duced in vivo, the results presented may be regarded as more relevant to clinical resistance cases.