Claude M. Gary-Bobo

Diffusion processes in lipid-water lamellar phases

Abstract The diffusion of 3HHO in lamellar lecithin-water and lecithin-cholesterol-water phases was measured as a function of water content and temperature. The diffusion coefficient was found to increase rapidly with the water content above 32 % in lecithin-water systems and above 20 % in lecithin-cholesterol-water systems. Below these values the diffusion coefficient was found to be essentially independent of water content. The evolution of the diffusion rate with water content can be accounted for in terms of the known structural parameters as well as the state of water in both systems. The diffusion of benzene in the lipid region of the phases was measured in both systems and found to increase with the water content. The evolution of the diffusion rate of benzene paralleled the variation in the fluidity of the hydrocarbon chains.

Effect of chemical modifiers of passive permeability on the conformation of spin-labeled erythrocyte membranes

Abstract Red blood cell membranes were labeled with stearic acid spin labels and with two protein spin labels (4-maleimido-2,2,6,6-tetramethylpiperidinooxyl and 4,2-iodoacetamido-2,2,6,6-tetramethylpiperidinooxyl) covalently bound to the membrane sulfhydryl group. Blocking the sulfhydryl groups with p- chloromercuribenzoate , p- chloromercuribenzosulfonate , N- ethylmaleimide and iodoacetamide provides information about the binding selectivity and the accessibility to their sites of the protein spin labels. The ESR spectra of labeled red blood cell membranes were studied in various experimental conditions. The ESR spectra of fatty acid spin-labeled intact erythrocytes are not modified by large variations of pH and osmolarity, indicating a stability of the phospholipid region. Alterations of the ESR spectra of the protein spin labels were produced upon exposure of the ghosts to chemical reagents that bind to amino groups (dinitrofluorobenzene, 2-methoxy-5-nitrotropone, trinitrobenzene sulfonic acid), indicating changes in the local environment surrounding the probes. The ESR spectra of the same protein spin labels are altered by pH variations of the external medium. These changes are different from those observed when the labeled ghosts are exposed to amino reagents. These data, observed by ESR, may be related to the well known effect of the NH2 reagents on the ionic permeability of the red blood cell.

Spin labelled sarcoplasmic reticulum vesicles: Ca2+-induced spectral changes.

A growing number of reports dealing with transport ATPases, either (Na + + K+)-activated or Ca2+-activated Mg2+-dependent ATPase, support the hypothesis that reactivity of their active center for phosphorylation is controlled by the bound cations [1--5]. To investigate this hypothesis we prepared rabbit skeletal muscle sarcoplasmic reticulum fragments [6 8], since these membranes are easily isolated as vesicles in which a CaZ+-dependent ATPase is the major protein. We labelled these vesicles with a paramagnetic iodoacetamide analog [9,10], and found that the bound label exhibits an e.s.r, spectrum which is insensitive to magnesium but sensitive to calcium concentration. The spectral change occurred between 1 and 3/~M at pH 8.0 and 4°C; when the same metal buffers were used to measure the K M of formation of the phosphorylated intermediate, the value of 1.3 #M was found. We discuss the possibility that this Ca 2÷ binding-induced spectral change indeed reveals a conformational change of the ATPase molecule upon Ca binding.

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.

Water diffusion in lecithin-water and lecithin-cholesterol-water lamellar phases at 22°

Abstract The diffusion of water (3HHO) was studied in lecithin (from egg yolk)-water lamellar phases, at 22°, as a function of their water content, Φ W . Depending upon Φ W , two diffusion rates can be distinguished, which are related to both sterical factors and hydration water in the phase. The results obtained in the lecithin-cholesterol-water systems support this interpretation.

Cation diffusion selectivity in a pore model. The phosphatidylcholine/water lamellar phase.

The diffusion coefficients D (cm2/s), of four monovalent cations K+, Na+, Rb+ and Cs+ and of Ca2+ have been measured in phosphatidylcholine/water lamellar phase as a function of phase hydration and temperature and in the presence of divalent cations. Diffusion rates vary strongly with phase hydration, between 10(-7) and 10(-6) cm2/s for monovalent and 10(-8) and 10(-7) for Ca2+. The activation energies obtained are relatively small (5--10 kcal/mol). As the phase water content increases, a series of diffusion sequences is obtained, corresponding to the sequences predicted by Eisenmans theory of alkali ion equilibrium selectivity. This diffusional selectivity, which depends exclusively upon non-equilibrium parameters (mobility) within the hydrophilic path is discussed in respect to current theories of pore selectivity.

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.