J. Membrane Biol. 89, 127-130 (1986) The Jouma[ of Membrane Biology 9 Springer-Verlag 1986 Water Exchange through Erythrocyte Membranes: Nuclear Magnetic Resonance Studies on Resealed Ghosts Compared to Human Erythrocytes Gheorghe Benga, Victoria Borza, Octavian Popescu, Victor I. Pop, and Aria Mure~an Department of Cell Biology, Faculty of Medicine, Medical and Pharmaceutical Institute, Cluj-Napoca, Roumania Summary. The water diffusion across human erythrocyte mem- brane has been studied on intact cells and resealed ghosts by a doping NMR technique. Although the water exchange time of ghosts was longer than that of erythrocytes, no significant differ- ences in their diffusional permeability were noticed for tempera- tures in the range 2-43~ Contrary to what was previously no- ticed in erythrocytes, no significant increase in the water exchange time of ghosts in the acid range of pH occurred. Key Words erythrocyte membranes - water permeability 9 resealed ghosts. NMR 9 pH effects Introduction Because of its relatively simple structure the red blood cell has been a favorite object for studying water permeability. A great deal of work has been done on osmotic and diffusional water permeability in human red blood cells. The information on os- motic permeability of erythrocyte membranes has been recently reviewed by Sha'afi (1981) and that on diffusional water permeability by Morariu and Benga (1984). During recent years methods of preparing red blood cell ghosts by hypotonic hemolysis have very much improved. Since such ghosts are devoid of intracellular structure and consist primarily of the cell membrane, they are widely used in studies of composition, structure and function of the red blood cell membrane. It is assumed that hypotonic ghosts have a membrane composition very similar to that of the intact cell (Wood & Passow, 1981). Moreover, two types of hypotonic ghosts have been prepared having a membrane permeability very close to that of the intact cell: (a) the resealed (pink) ghosts (Schwoch & Passow, 1973), which retain a small amount of the original hemoglobin, and (b) white ghosts (Bjerrum, 1979), which are free from visible contamination with intracellular components including hemoglobin. In spite of potential advantages offered by hy- potonic ghosts as an object for investigating various processes, there are few studies, performed by the radio-tracer method, on the water permeability of ghosts (Bjerrum, 1979; Brahm, 1982). Comparative studies of water diffusion in erythrocytes and ghosts would give us a better understanding of the molecular mechanisms of this transport process. Moreover, such studies could help in interpreting the effect of pH on water diffusion. The aims of our paper were: (a) to compare the water diffusion in erythrocytes and ghosts by the doping NMR method; (b) to investigate the effect of pH in ghosts; and (c) to establish the characteristics of water permeability of human red blood cell mem- brane on a relatively large number of subjects at various temperatures. Materials and Methods BLOOD SAMPLE PREPARATIONS Human blood was obtained by venipuncture in heparinized tubes and used within 4 hr. The donors were healthy male or female subjects, 20 to 40 years old. The erythrocytes were isolated by eentrifugation, and washed three times in 166 mM NaCI. For the preparation of resealed (pink) ghosts the procedure of Bodemann and Passow, as described by Wood and Passow (1981) has been used. In some experiments white ghosts were prepared as de- scribed by Bjerrum (1979). Finally the erythrocytes or the ghosts were suspended in 150 mM NaC1, 5.5 mM glucose, 5 mM HEPES (pH 7.4) and 0.5% bovine serum albumin at a cytocrit of 50%. NMR MEASUREMENTS Samples for NMR 1 measurements were prepared by carefully mixing 0.2 ml erythrocyte or ghost suspensions and 0.1 ml dop- Abbreviations: NMR = nuclear magnetic resonance;/'1 = longitudinal (spin-lattice) relaxation time; T2 = transversal (spin- spin) relaxation time.