Gheorghe Benga

Evaluation of a nuclear magnetic resonance technique for the study of water exchange through erythrocyte membranes in normal and pathological subjects.

Abstract A critical evaluation has been performed of a NMR technique for the investigation of the water exchange time through erythrocyte membranes, based on doping with a paramagnetic salt. The conclusions are the following: 1. 1. The preparation of the samples for the NMR measurement plays an essential role in obtaining precise and reproducible values of the water exchange time through erythrocyte membranes. A method for optimum mixing of the whole blood with the doping solution is described. The values of the relaxation times of erythrocyte water protons obtained by using this method were reproducible to an accuracy ranging between 2%–6%. 2. 2. The theoretical basis of the method has been evaluated; the equations that should be employed for the calculation of the water exchange time are discussed. 3. 3. The water exchange time through normal erythrocyte membranes is 6.0 ± 0.5 ms . This value is not influenced by the presence of the doping paramagnetic solution; determinations by atomic absorption spectroscopy showed no significant penetration of the Mn2+ into red blood cells. 4. 4. The temperature dependence of the water exchange time through the erythrocyte membranes is characterised by values of the apparent activation energy ranging between 6 and 8 kcal/mol. These values are similar to the activation energies of the molecular rotational motion or of the diffusion of water in mono- or submonolayers of adsorbed water. 5. 5. It has been found that the exchange time of water through erythrocyte membranes increased by 13–55% in patients with certain diseases such as Gauchers disease, essential hyperlipemia, obstructive jaundice, chronic hepatitis, nephrotic syndrome. 6. 6. Our findings suggest that the NMR doping method is potentially useful for further studies of the physiology and pathology of erythrocyte water permeability.

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on the effects of inhibitors and of chemical modification of human membranes

SummaryThe changes in water diffusion across human erythrocyte membranes following exposure to various inhibitors and proteolytic enzymes have been studied on isolated erythrocytes suspended in isotonic buffered solutions. An important issue was to investigate whether the sulfhydryl reacting reagents that have been applied in osmotic experiments showed similar effects on diffusional permeability. It was found that mercurials, including mersalyl, were the only sulfhydryl reacting reagents that were efficient inhibitors. Under optimal conditions a similar degree of inhibition (around 45%) was found with all mercury-containing sulfhydryl reagents. Other reagents, including the sulfhydryl reagent DTNB, phloretin, or H2DIDS, the specific inhibitor of the anion transport system in erythrocyte membrane, did not appear to inhibit significantly the diffusional permeability. No changes in water diffusion were noticed after exposure of erythrocytes to trypsin and chymotrypsin. A new kind of experiment was that in which the effects of exposure of erythrocytes to two or more agents were studied. It was found that none of the chemical manipulations of membranes that did not affect water diffusion hampered the inhibitory action of mercurials. These findings show that the SH groups involved in water diffusion across erythrocyte membranes do not react with any of the other SH reagents aside from mercurials and that the molecular mechanism of water transport is not affected by chymotryptic cleavage of band 3 protein into the 60 and 35 kD fragments. The NMR method appears as a useful tool for studying changes in water diffusion in erythrocyte membranes following various chemical manipulations of the membranes with the aim of locating the water channel.

On measuring the diffusional water permeability of human red blood cells and ghosts by nuclear magnetic resonance

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes and ghosts by a doping nuclear magnetic resonance technique. In contrast to all previous investigations, systematic measurements were performed on blood samples obtained from a large group of donors. The mean values of P ranged from 2.2 X 10(-3) cm.s-1 at 5 degrees C to 8.1 X 10(-3) cm.s-1 at 42 degrees C. The reasons for some of the discrepancies in the permeability coefficients reported by various authors were found. In order to estimate the basal permeability, the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzenesulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 1.3 X 10(-3) cm.s-1 at 20 degrees C, 1.6 X 10(-3) cm.s-1 at 25 degrees C, 1.9 X 10(-3) cm.s-1 at 30 degrees C and 3.2 X 10(-3) cm.s-1 at 37 degrees C. The results reported here represent the largest series of determinations of water diffusional permeability of human red blood cells (without or with exposure to mercurials) available in the literature, and consequently the best estimates of the characteristics of this transport process. The values of P can be taken as references for the studies of water permeability in various cells or in pathological conditions.

Birth of water channel proteins—the aquaporins

If we compare aquaporin (as a proteic pathway for water permeation across biological membranes) with a child we can say that he had a very long gestation period. His possible existence was predicted for a long time (Overton in 1985, Stein and Danielli in 1956), some of his features (transport of water and its reversible inhibition) were assigned by Macey and Farmer in 1970, however this child was first detected by Benga and coworkers in 1986. We clearly demonstrated for the first time the presence and location of a water channel at the human RBC membrane among the polypeptides migrating in the region having 35–60 kDa on the electrophoretogram of RBC membranes, labeled with 203Hg‐PCMBS in the conditions of specific inhibition of water diffusion; I suggested that a minor membrane protein that binds PCMBS is involved in water transport and also indicated the way in which the specific protein could be further characterized: by purification and reconstitution in liposomes. Our landmark papers in 1986 can be compared with the first detection of a child “in utero” by ultrasonography, since we discovered one of the essential components of the “aquaporin child” (a molecular weight of 35–60 kDa for the glycosylated component); we have also indicated the way to recognize him after birth (among other children of his group!): placing the isolated children in a certain environment and asking them to perform the same task (one should read: reconstitution studies in liposomes and measurement of water permeability), like aligning athletes for a running test. This was the only certain way to know that the child is really the fastest runner and not just one that is helping (by various means) another child to be fastest runner. A “new child” was observed in 1988 by Agre and coworkers, who identified a novel integral membrane protein in human RBCs having a non‐glycosylated component of 28 kDa and a glycosylated component migrating as a diffuse band of 35–60 kDa; they suggested that the new protein (nick‐named CHIP28 in 1991) may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 Agre and coworkers suggested that CHIP28 is a functional unit of membrane water channels; by reconstitution in liposomes it was demonstrated that CHIP28 is a water channel itself rather than a water channel regulator. In other words the child we first detected was recognized as having the predicted qualities only in 1992. In 1993 CHIP28 was renamed aquaporin 1. Looking in retrospect, asking the crucial question, when was the first water channel protein, aquaporin 1, discovered, a fair and clear cut answer would be: the first water channel protein, now called aquaporin 1, was identified or “seen” in situ in the human RBC membrane by Benga and coworkers in 1986. It was again “seen” when it was by chance purified by Agre and coworkers in 1988 and was again identified when its main feature, the water transport property was found by Agre and coworkers in 1992. If a comparison with the discovery of The New World of America is made, the first man who has “seen” a part, very small indeed, of The New Land was Columbus; later, others, including Amerigo Vespucci (from whom the name derived), have better “seen” a larger part of the new Continent and in the subsequent years many explorers discovered the complexity of the Americas!

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (Pd) and of the activation energy (Ea,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of Pd, these species could be divided into three groups. First, the RBCs from humans, cow, sheep and large kangaroos (Macropus giganteus and Macropus rufus) had Pd values approximately 5 x 10(-3) cm/s at 25 degrees and 7 x 10(-3) cm/s at 37 degrees C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had Pd values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of Ea,d was in most cases correlated with the values of Pd. A value of Ea,d approximately 26 kJ/mol was found for the RBCs from humans and the species having similar Pd values. Low values of Ea,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of Pd. The highest values of Ea,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of Ea,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that Pd in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.

Membrane defect affecting water permeability in human epilepsy.

McQUARRIE wrote that a disturbance in water balance perhaps affecting the central nervous system more specifically seems to be closely identified with the aetiology of epilepsy1. McQuarrie and Teglbjaerg2 studied water balance in relation to seizure frequency, establishing the ictogenic effect of excessive water ingestion, and the therapeutic value of dehydration. Schneider3 has reported that exacerbation of petit mal in children and adults is associated with reduction in urinary volume, and that clinical remission coincidences with an increase in water excretion. Reynolds4 has demonstrated disturbances in the whole body distribution of water in epilepsy, with a magnitude proportional to the frequency of attacks, concluding that the changes in body water and sodium are linked in some way to the aetiology of the disease. Wender and Strzyzewski5 have shown that water loading in epileptics produces seizures only in cases where there is also a marked increase in the water content of erythrocytes. There is thus abundant evidence linking epilepsy with water metabolism. Furthermore, diuretics, such as acetazolamid, are used in the therapy of epilepsies6. But the mechanism of the disturbance of water metabolism at the subcellular and molecular level has not been considered. We report here an investigation of water diffusion through erythrocyte membranes in epileptic children, made using a nuclear magnetic resonance (NMR) technique7. We found an abnormally low permeability in membranes from epileptics and suggest that a generalised membrane defect affecting water permeability is responsible for the disturbances of water metabolism in human epilepsy.

Effects of temperature on water diffusion in human erythrocytes and ghosts — nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.

NMR studies of diffusional water permeability of red blood cells from macropodid marsupials (kangaroos and wallabies)

1. The water permeabilities of the red blood cell (RBC) membranes of five species of macropodid marsupials were monitored by using a Mn(2+)-doping 1H nuclear magnetic resonance (NMR) technique. 2. Since this appears to be the first time that this type of measurement at 400 MHz for 1H has been reported, an analysis of instrumental parameters influencing the estimated value of the water exchange time (Te), and of the diffusional water permeability (Pd), was performed on samples of human RBC. 3. It was found that a short interpulse delay in the Carr-Purcell-Meiboom-Gill pulse sequence had to be used (around 100 microseconds) to avoid an underestimation of the relaxation times, that occurred as the result of molecular diffusion through non-uniform local magnetic fields in and around the cells. 4. There were no significant differences, in the water permeabilities of the RBC membranes, between the five species (Macropus rufogriseus, M. parma, M. eugenii, M. parryi and Wallabia bicolor). 5. There were also no significant differences between various colonies of M. eugenii living in different habitats. 6. The average values of Pd were 9.1 x 10(-3) cm/sec at 24.6 degrees C, 10.4 x 10(-3) cm/sec at 30 degrees C, 12.6 x 10(-3) cm/sec at 37 degrees C, and 14.7 x 10(-3) cm/sec at 42.1 degrees C; these were more than twice as high as those for human RBC. 7. In agreement with the high water permeability the RBC of macropodids displayed a relatively low activation energy of water diffusion across their membranes, approximately 21 kJ/mol, compared with 25 kJ/mol for human RBC.

Effects of temperature and pH on the water exchange through erythrocyte membranes: nuclear magnetic resonance studies.

SummaryThe temperature and pH dependence of water exchange has been studied on isolated erythrocytes suspended in isotonic buffered solutions. At pH 7.4 a break in the Arrhenius plot of water exchange time at around 26°C was found. The mean value of the apparent activation energy of the water exchange time at temperatures higher than that of the discontinuity was 5.7 kcal/mole (±0.4); at lower temperatures the values of the apparent activation energy were below 1.4 kcal/mole. The pH dependence of water exchange time of isolated erythrocytes revealed a marked increase of the water exchange time values in the acid range of pH; a much smaller variation of the same parameter occurs between pH 7.0 and 8.0. These finding could be correlated with other processes involving erythrocyte membranes that showed similar pH and temperature dependence and were considered to indicate state transitions in the membranes. It is suggested that the temperature and pH effects on water diffusion indicate that conformational changes and cooperative effects are implicated in the mechanism of this transport process.

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on resealed ghosts compared to human erythrocytes

SummaryThe water diffusion across human erythrocyte membrane 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 differences in their diffusional permeability were noticed for temperatures in the range 2–43°C. Contrary to what was previously noticed in erythrocytes, no significant increase in the water exchange time of ghosts in the acid range of pH occurred.