A new approach is proposed to investigate the metabolic perturbation induced by drugs in cells. The effects of various concentrations of amphotericin B on the aerobic [I-' 3C]glucose metabolism in glucose-grown repressed Succharomyces cerevisiue cells were studied as a function of time using I3C-, ' H-NMR and biochemical methods.The I3C enrichment of different compounds such as ethanol, glycerol and trehalose were determined by 'H-NMR spectroscopy. In the absence of amphotericin B, glycerol diffuses slowly from the internal to the external medium, whereas in its presence this diffusion is greatly facilitated by the formation of pores in the cell membrane. Amphotericin B has been found to exert a marked influence on the glucose consumption and the production of all metabolites; for example, at 1 FM, the glucose consumption and the production of ethanol decrease while the production of glycerol and trehalose increases. The I3C relative enrichments of ethanol, glycerol and trehalose are almost the same with and without the drug. Thus it can be concluded that amphotericin €3 induces a large effect on the production of these compounds in the cytosol but shows no significant influence on the mechanism of their formation. Upon addition of glucose, all the amino acid concentrations decrease continuously with time; this effect is more pronounced in the presence of the drug. The ratio of the integrated resonances of glutamate (C2 + C3)/C4 reflects the activity of pyruvate carboxylase relative to citrate synthase rather than to pyruvate dehydrogenase. Without amphotericin B, this ratio ( z 1.0) is pratically constant upon addition of glucose which suggests that the activities of pyruvate carboxylase and citrate synthase are equivalent. By contrast, upon coaddition of 25 mM glucose and 1 pM amphotericin B, the glutamate C4 resonance remains virtually unchanged while that of glutamate C2 is much smaller than in its absence and continuously decreases with time. It seems likely that amphotericin B induces a reduction in the activity of pyruvate carboxylase in the mitochondria.During the last 12 years, NMR spectroscopy has been used extensively to study the metabolic pathways in cells such as yeast [I -91, bacteria [lo-131, hepatocytes 114-161 and renal epithelial cells 117 -221. With 13C-labeled substrates, e. g. [l-'3C]glucose [I, 5, 6, 9, 12, 16, 191, [2-I3C] [14, 181, important information concerning the mechanism of formation of metabolites via different metabolic pathways has been obtained. However, most of these studies focussed on the fate of the substrates and, to our knowledge, few investigations have been devoted to the study of the influence of a drug on the cellular metabolism. The investigation of drug toxicity would be more interesting if one could determine, besides the lethal doses, how and where (i.e. in the cytosol, in the mitochondria or in the nucleus) the metabolic perturbations are induced by the drug in cells.Among the polyene antibiotics, amphotericin B has been widely used as an antifungal, antimicro...

We propose an experimental approach combining 'H-NMR and ' 3C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters: 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with [1-' 3C]glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2-'3C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent.The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1 .I. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques. Now that the investigation of cellular metabolism is in rapid progress, interest in understanding regulatory processes through measurement of the metabolite flux along the various pathways in cells is growing considerably. In recent years, thanks to the use of specifically labeled substrates, 13C-NMR was found to be a powerful tool for flux evaluation in isolated cells 11 -101, tissues [ll-151 and perfused organs [16-241 even though the I3C nucleus is much less sensitive than 'H and 31P. Many studies [I, 2, 9-11, 14, 16, 18-23] have been devoted to the flow of pyruvate and acetyl-CoA into the Krebs cycle frequently using the 13C fractional enrichment of some labeled compounds as experimental parameters. Chance et al.[I81 have developed a mathematical mode...

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The reciprocal effects of 2-deoxy-D-glucose (dGlc) and glucose (Glc) on the aerobic metabolism of Glc and dGlc in Glc-grown repressed Saccharomyces cerevisiae were studied at 30°C in a standard pyrophosphate medium containing about 4.5 X lo7 cells/ml. 'H-, 'T-NMR and biochemical techniques were used for quantitative evaluation of Glc and dGlc metabolites. The detection of intracellular dGlc and the determination of the intracellular dGlc6PldGlc ratio were realised using [l-'3C]dGlc and 'T-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 -t-0.03 mM/min (n = 3), 16 2 0.02 mM/min (n = 3), and 0.08 -t-0.01 mM/min (n = 3), respectively. This means that the Glc consumption is reduced about 41% and 70% in the presence of 5 mM and 20 mM dGlc, respectively. When dGlc is the unique carbon source, only a and p anomers of dGlc6P were formed. Their quantities are equivalent and reach the maximum values within 1 h of incubation and then decrease gradually. By contrast, Glc favours the consumption of dGlc and the synthesis of dGlc6P, dideoxy-trehalose (dGlc-dGlc), deoxy-trehalose (dGlc-Glc). In the presence of Glc, dGlc6P reaches a plateau after 1 h or 2 h of incubation while the quantities of trehalose (Glc-Glc), dGlc-dGlc, dGlc-Glc, which are small at 1 h, rapidly increase with time. The reasons why dGlc and Glc exert opposite effects on their metabolism are discussed.The production of Glc-Glc decreases with increasing the external dGlc concentration or the dGlc/ Glc ratio. The effect of dGlc on the biosynthesis of Glc-Glc can be explained by the competition of dGlc and Glc with respect to hexokinase. Although Glc favours the synthesis of dGlc6P, the maximum concentration of dGlc6P shows little dependence on the external dGlc concentration as long as glucose is not exhausted. The internal dGlc6PldGlc ratio at equilibrium, about 4.7 ? 0.7, is also found to be independent of the dGlc Concentration in the suspension. Only a small fraction of dGlc6P disappears to give rise to the formation of dGlc-dGlc and dGlc-Glc. At equilibrium the inverse reaction from dGlc6P to dGlc may be important to compensate for the fast reaction of dGlc phosphorylation by hexokinase. At least nine series of experiments were conducted and showed that, in pyrophosphate media and for incubation times less than 4 h, dGlc-dGlc was not observed in the absence of Glc. However, as for dGlc6P, the production of dGlc-dGlc does not depend very much on the external concentration of dGlc and Glc. The production of dGlc-Glc is proportional to the Glc concentration but insensitive to the dGlc concentration. Apart from dGlc6P, dGlc-Glc is the most abundant metabolite of dGlc and Glc. The synthesis of this compound results in an interaction between the precursors arising from two different carbon sources. Thus the effect of a drug strongly depends on the physiological state of cells. The presence of an energy source such as Glc was shown to be able to stimulate the drug consumption and the formation ...

The effect of mucosal La3+ on electrical parameters of isolated frog skins was studied on isolated frog skins with normally polarized or depolarized apical membrane. La3+ increases R8, the paracellular or shunt resistance and diminishes RNa, the resistance of the active sodium path, in both polarized and depolarized skins. The stimulatory effect of La3+ on short-circuit current (Is.c.) is correlated with this decrease in RNa. The characteristics of the stimulatory effect are: very rapid onset, ionic strength dependency, the possibility of being elicited by many other ions besides La3+. These features allow us to postulate that La3+ might affect the external interfacial potential which in turn affects the resistance of the sodium path.