Permeability of phospholipid membrane for small polar molecules determined from osmotic swelling of giant phospholipid vesicles

A method for determining permeability of phospholipid bilayer based on the osmotic swelling of micrometer-sized giant unilamellar vesicles (GUVs) is presented as an alternative to the two established techniques, dynamic light scattering on liposome suspension, and electrical measurements on planar lipid bilayers. In the described technique, an individual GUV is transferred using a micropipette from a sucrose/glucose solution into an isomolar solution containing the solute under investigation. Throughout the experiment, vesicle cross-section is monitored and recorded using a digital camera mounted on a phase-contrast microscope. Using a least-squares procedure for circle fitting, vesicle radius R is computed from the recorded images of vesicle cross-section. Two methods for determining membrane permeability from the obtained R(t) dependence are described: the first one uses the slope of R(t) for a spherical GUV, and the second one the R(t) dependence around the transition point at which a flaccid vesicle transforms into a spherical one. We demonstrate that both methods give consistent estimates for membrane permeability.Comment: 40 pages, 8 figures, to appear in Advances in Planar Lipid Membranes and Liposomes vol. 1

Sodium Movement in High Sodium Feline Red Cells

The transport of Na in the cat red cells has been studied under various experimental conditions. The unidirectional radioactive Na influx increased with increasing temperature until it reached a maximum value at 37°C ± 2°C and then decreased with a further increase in temperature. Errors stated in this paper represent 1.0 standard errors of the mean. The apparent activation energy was calculated in the region between 25 and 37°C and was found to be 4.9 ± 0.5 kcal/mole. Copper at a concentration of 0.04 mM inhibited this influx by 65%. When cells were suspended in isosmotic KCl buffer, cell volume was found to decrease initially with time. This unusual behavior is discussed in terms of Na to K preference of the cell membrane. In cat red cells, Na influx was found to increase about 13-fold when cell volume was decreased from 1.16 normal to 0.87. This effect could not be reproduced when the medium osmolarity was changed only by the addition of urea, a permeating molecule. On the other hand, K influx was found to decrease from 0.24 ± 0.03 mEq/liters RBC, hr at a relative cellular volume equal to 1.0 to 0.11 ± 0.01 mEq/liters RBC, hr at a cell volume of 0.75. Na influx in human red cells did not show any significant dependence on cell volume. The properties of Na movement in the cat red cells are compared to those of human red cells

Cation Transport in Dog Red Cells

Studies have been made on the cation transport system of the dog red cell, a system of particular interest because it has been shown that there is a marked dependence of cation fluxes on the cell volume. We have found that a 10% decrease in cell volume causes a large increase in 1 hr uptake of 24Na as well as a considerable inhibition of 42K uptake. This effect cannot be produced by a difference in medium osmolality but rather requires the cell volume to change. Dog red cell uptake of 24Na is not inhibited by iodoacetate. Phloretin inhibits 24Na uptake and lactate production, and virtually abolishes the volume effect on Na uptake. These several observations may be accounted for in terms of a working hypothesis which presupposes a cation carrier complex which pumps K into and Na out of cells of normal volume. When the cells are shrunken the carrier specificity shifts to an external Na-specific mode and there is a large increase in 24Na uptake, driven by the inwardly directed Na electrochemical potential gradient

Effect of Osmolality on the Hydraulic Permeability Coefficient of Red Cells

The osmotic water permeability coefficient, Lp, for human and dog red cells has been measured as a function of medium osmolality, and found to depend on the osmolality of the bathing medium. In the case of human red cells Lp falls from 1.87 x 10-11 cm3/dyne sec at 199 mOSM to 0.76 x 10-11 cm3/dyne sec at 516 mOSM. A similar decrease was observed for dog red cells. Moreover, Lp was independent of the direction of water movement and the nature of the solute used to provide the osmotic pressure gradient; it depended only on the final osmolality of the medium. Furthermore, Lp was not affected by pH in the range of 6 to 8 nor by the presence of drugs such as valinomycin (1 x 10-6 M) and tetrodotoxin (3.2 x 10-6 M). The instantaneous nature of the response to changes in external osmolality suggests that the hydraulic conductivity of the membrane is controlled by a thin layer at the outer face of the membrane