Jaime Wietzerbin

Lanthanum inhibition of the action of oxytocin on the water permeability of the frog urinary bladder: Effect on the serosal and the apical membrane

SummaryThe effect of La3+ ion on the hydrosmotic response of the frog urinary bladder to oxytocin has been studied. When added to the serosal medium, in the concentration range 10−4M to 10−3M, La3+ inhibits the response to oxytocin, theophylline and cyclic-AMP, but not to hypertonicity. When added to the apical medium at a concentration greater than 5×10−3M, La3+ exhibits an inhibitory effect only during respective stimulation, either by hormone or by hypertonicity, but has no significant effect on the developed response. The significance of this particular mode of action is discussed in terms of possible electrical potential variations of the apical membrane during stimulation.

Interaction of 14C-labelled amphotericin B derivatives with human erythrocytes: relationship between binding and induced K+ leak

Four 14C-labelled amphotericin B (Am B) derivatives with different net electric charges were examined: zwitterionic N-fructosyl Am B, positively charged N-fructosyl Am B methyl ester, negatively charged N-acetyl Am B and neutral N-acetyl Am B methyl ester. The binding of these four derivatives to human red cells and their octanol-water partition coefficients were measured. Simple partitioning between red cells and buffer was found for the four compounds, regardless of concentration, within a range of 10(-8) and 10(-4) M. This indicates the absence of cooperativity and saturability of binding at least in this concentration range. The constant partition coefficients were found to be three to five times higher for the two methyl ester derivatives than for the two non-esterified compounds. All partition coefficients were proportional to those found for the octanol-water system. Efficiency in inducing K+ leak from red cells was measured during the binding experiments. Despite the higher partition coefficients of the two methyl ester derivatives, they were found to have much lower ionophoric efficiency than the two non-esterified compounds. These results are discussed in terms of the mechanism of permeability pathway formation by polyene antibiotics.

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.

Cellular pH and the ADH-induced hydrosmotic response in different ADH target epithelia

The hydrosmotic response elicited by oxytocin in the frog skin epithelium (Rana esculenta) was reversibly inhibited by 70% when the medium pH was reduced to 6.2 by CO2 bubbling on the serosal side. On the contrary, the response to 8-bromo cyclic AMP (8 Br-CAMP) was not affected by medium acidification, even after corion removal. In other experiments intracellular pH was measured, employing the dimetyl-oxazolidine-dione distribution technique, in frog urinary bladder and the isolated frog skin epithelium. As previously observed in the case of oxytocin, 8 Br-CAMP increased intracellular pH in frog urinary bladder. Incubation with oxytocin also augmented the intracellular pH in the isolated frog skin epithelium but 8 Br-CAMP did not modify cell proton concentration in this tissue. From previous and present results it can be summarized that: 1) The intracellular alkalinization effect clicited by oxytocin addition and the inhibition in the hydrosmotic response induced by medium acidification were qualitatively similar in both tested target epithelia. 2) On the contrary, a post cyclic AMP step sensitive to changes in intracellular pH was not observed in frog skin, as is the case in frog urinary bladder. 3) The 8 Br-CAMP induced intracellular alkalinization effect was only observed in frog urinary bladder.

Effects of mucosal lanthanum on electrical parameters of isolated frog skin

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 Rs, 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.

Influence of membrane polarization and hormonal stimulation on the action of lanthanum on frog skin sodium permeability

SummaryThe effect of mucosal La3+ on shortcircuit current (s.c.c.) has been studied on isolated frog skins with normaly polarized or depolarized apical membrane. With skins in “polarized condition”. La3+ stimulates transiently the s.c.c. and after a long-lasting presence on the mucosal side (1 h) attenuates significantly the stimulation promoted by oxytocin. With skins in “depolarized conditions”, lanthanum stimulates permanently the s.c.c. and does not modify the oxytocin effect, even over long period of continuous stimulation. In contrast, an inhibitory action of mucosal La3+ develops, when skins submitted to repetitive hormonal stimulation are forced to oscillate from the stimulated to the normal resting state. The significance of this particular mode of action is discussed in terms of a possible electrical potential variation of apical membrane during oxytocin stimulation.

Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K+- and Na+-rich media

In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.

Reciprocal Effects of Glucose and Drugs on Their Metabolism in S. Cerevisiae Studied by Multi-Nuclear NMR Spectroscopy

The metabolism in cells was extensively investigated by NMR spectroscopy during the last 15 years. However little information concerning the influence of drugs on cellular metabolism is available. The effect of an energy source such as glucose, lipid, amino-acids.. on drug metabolism is practically unknown. Since the metabolism of a drug in patients always takes place in the presence of glucose, lipid, amino-acids or other substrates, knowledge of the effect of an energy source as glucose would be highly important for medical treatment. Therefore it is of considerable interest to study the reciprocal effects of drugs and glucose on their own metabolism.