In vivo tritium contamination effects on water exchange across the erythrocyte membranes: a nuclear magnetic resonance study

The effects produced by chronic in vivo tritium contamination on the process of water diffusion across the rat erythrocyte membranes were investigated using an NMR relaxation method. As the level of tritium intake increased, an inhibition of the water permeability was observed, reaching values of 40% for corresponding absorbed doses of about 100 mGy. The activation energy of the transport process was also significantly increased, suggesting that the mechanism of the water diffusion across the membrane had changed.     

Hypophosphite ion as a 31P nuclear magnetic resonance probe of membrane potential in erythrocyte suspensions.

Hypophosphorus acid has a single pKa of 1.1 and at physiological pH values it is therefore present almost entirely as the univalent hypophosphite ion. When added to a red cell suspension the ion crosses the cell membrane rapidly, via the anion exchange protein, and the intra- and extracellular populations of the ion give rise to separate 31P NMR resonances. From a single 31P NMR spectrum it was possible to determine the relative amounts of hypophosphite in the intra- and extracellular compartments and thereby estimate the corresponding concentrations. The ratio of intracellular to extracellular hypophosphite concentration was independent of the total hypophosphite concentration for cells suspended in NaCl solutions and was independent of hematocrit. The hypophosphite distribution ratio increased as extracellular NaCl was replaced iso-osmotically with citrate or sucrose, through it remained very similar to the corresponding hydrogen ion distribution ratio. Incorporation of the hypophosphite distribution ratio into the Nernst equation yielded an estimate of the membrane potential. For cells suspended in NaCl solutions the estimated potential was consistently around -10 mV.     

Water exchange through erythrocyte membranes V. Incubation with papain prevents the p-chloromercuri-benzensulfonate inhibition of water diffusion studied by a nuclear magnetic resonance technique

The changes in water diffusion across human erythrocyte membrane following exposure to proteolytic enzymes and to p-chloromercuribenzene sulfonate (PCMBS) have been studied on isolated erythrocytes suspended in isotonic solutions. Trypsin digested glycophorin without significantly changing the pattern of other polypeptides in erythrocyte membrane. On the contrary, with chymotrypsin or papain an extensive digestion of band 3 protein occured. No changes in water diffusion were noticed after exposure of erythrocytes to trypsin, chymotrypsin or papain. Neither trypsin nor chymotrypsin treatment prevented the inhibition of water diffusion induced by PCMBS. In contrast, exposure of erythrocytes to papain did hamper the inhibitory effect of subsequent incubation with PCMBS. Taking into account the degradation of band 3 protein by papain it appears that the binding site for PCMBS playing a role in the inhibition of water diffusion is located in this protein.     

Water exchange through erythrocyte membranes: p-choloromercuribenzene sulfonate inhibition of water diffusion in ghosts studied by a nuclear magnetic resonance technique

A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, A comparison of water diffusion in human erythrocytes and ghosts revealed a longer relaxation time in ghosts, corresponding to a decreased exchange rate. However, the diffusional permeability of ghosts was not significantly different from that of erythrocytes . The changes in water diffusion following exposure to p-chloromercuribenzene sulfonate (PCMBS) have been studied on ghosts suspended in isotonic solutions. It was found that a significant inhibitory effect of PCMBS on water diffusion occurred only after several minutes of incubation at 37°C. No inhibition was noticed after short incubation at 0°C as previously used in some labelling experiments. This indicates the location in the membrane interior of the SH groups involved in water diffusion across human erythrocyte membranes. The nuclear magnetic resonance ( n . m . r . ) method appears as a useful tool for studying changes in water diffusiofl in erythrocyte ghosts with the aim of locating the water channel.     

The determination of intracellular sodium concentration in human red blood cells: nuclear magnetic resonance measurements.

The intracellular sodium concentration and intracellular volume of human red blood cells were determined from 23Na and 1H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 +/- 3.1 mM. A comparison of different NMR methods used to determine sodium concentration is given.     

Erythrocyte-plasma water exchange studied by nuclear magnetic resonance spin-spin (T2) relaxation. Relation between the time the water molecule stays in the erythrocyte and its volume

A simple, precise and rapid pulse nuclear magnetic resonance technique for measuring the rate of water exchange across the erythrocyte membrane is presented. The effects of cell volume on cell water lifetime may be eliminated by the introduction of a cell water diffusion coefficient. This parameter is applied to the study of eleven blood samples.     

The kinetics of water exchange across the chloroplast membrane

The kinetics of water exchange across the membrane of class II chloroplasts has been studied by two NMR methods. Both methods utilize Dy(en)3+ (en = ethylenediamine) to induce a transmembranal chemical shift the order of 40 Hz in the water proton resonance. The shift reagent is impermeant to the chloroplast membrane, inert as a redox reagent, soluble at millimolar concentrations at neutral pH, and associated with a large, virtually temperature independent molar shift (0.10-0.12 ppm/mM). Water exchange across the membrane is monitored by two independent experiments. In the first, chemical exchange causes line broadening in the water proton resonance in the high-resolution spectrum. Measurement of the incremental linewidth as a function of transmembranal chemical shift determines the exchange kinetics as well as the fractions of water protons in internal and external media. In the second experiment, chemical exchange causes the transverse relaxation time, as measured by the Carr-Purcell-Gill-Meiboom technique, to be dependent on the 180 degree pulse spacing. The two experiments, while independent of each other, depend on the same set of theoretical parameters. These parameters are overdetermined by simultaneous analysis of both experiments. The mean lifetime of a water proton in the inner thylakoid space is found to be 1.1 +/- 0.8 ms at 25 degrees C and 2.75 +/- 0.4 ms at 3 degrees C in NH2OH/EDTA-treated chloroplasts. Values derived from dark-adapted chloroplasts that are active with respect to oxygen evolution are 1.1 +/- 0.3 ms (25 degrees C) and 1.75 +/- 0.4 ms (3 degrees C). The internal thylakoid volume is also determined in principle by the data, but uncertainties in the membrane volume and the transmembranal chemical shift severely limits the accuracy of this measurement.     

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

Summary The 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.Institute for Isotopic and Molecular Technology.     

1H nuclear magnetic resonance assay of erythrocyte triosephosphate isomerase

A direct method for measuring the activity of erythrocyte triosephosphate isomerase using 1H NMR spectroscopy was developed. NMR spectroscopy allows the simultaneous monitoring of the substrate and the product of the reaction by virtue of the differences in the NMR spectrum of each chemical species. The assay conditions were based on a modification of a conventional spectrophotometric method. The enzymatic activity measured using NMR gave results comparable to those obtained in a standard assay. The results were used in the kinetic characterization of triosephosphate isomerase in hemolysates from subjects with homozygous or heterozygous deficiency of the enzyme. In general, NMR spectroscopy has the potential for wide application in the rapid development of new enzyme assays.     

Time dependence of the effect ofp-chloromercuribenzoate on erythrocyte water permeability: A pulsed nuclear magnetic resonance study

Summary Pulsed nuclear magnetic resonance spectroscopy is employed to determine the time dependence of the change in erythrocyte water permeability following exposure top-chloromercuribenzoate (PCMB) orp-chloromercuribenzene sulfonic acid (PCMBS). pH variation was used to examine the environment of the sulfhydryl groups reactive to these drugs. PCMB reacted with at least two sulfhydryl groups which affect water permeability. This was shown by the double exponential character of the change in erythrocyte diffusional permeability with time after PCMB addition. However, only one inhibition rate process could be distinguished following PCMBS exposure, suggesting that one site bound by PCMB is not accessible to PCMBS. This site is postulated to be located in a hydrophobic region of the membrane, whereas the site reached by both drugs is located in the normal anion permeation channel. The effect of pH on the degree of inhibition due to each component and the inhibition rates is explained in terms of its effect on solubility of the reagents in the membrane and variation of the dissociated-to-undissociated ratio of PCMB.     

Bicarbonate exchange kinetics at equilibrium across the erythrocyte membrane by 13C NMR

The rate of exchange of 13C-labelled bicarbonate across the membranes of human erythrocytes in suspension, at thermal and chemical equilibrium, was measured using 13C NMR spectroscopy: the permeability coefficient (3.34 X 10(-4) cm s-1) agrees well with previous values obtained with other methods. Data analysis was complicated by the need to consider the Donnan ratio of the charged species inside and outside the cells. This work appears to be the first, involving the present NMR procedure, for studying fast membrane transport of a molecule other than water.     

Effective bilayer expansion and erythrocyte shape change induced by monopalmitoyl phosphatidylcholine. Quantitative light microscopy and nuclear magnetic resonance spectroscopy measurements.

When human erythrocytes are treated with exogenous monopalmitoyl phosphatidylcholine (MPPC), the normal biconcave disk shape red blood cells (RBC) become spiculate echinocytes. The present study examines the quantitative aspect of the relationship between effective bilayer expansion and erythrocyte shape change by a newly developed method. This method is based on the combination of direct surface area measurement of micropipette and relative bilayer expansion measurement of 13C crosspolarization/magic angle spinning nuclear magnetic resonance (NMR). Assuming that 13C NMR chemical shift of fatty acyl chain can be used as an indicator of lateral packing of membrane bilayers, it is possible for us to estimate the surface area expansion of red cell membrane induced by MPPC from that induced by ethanol. Partitions of lipid molecules into cell membrane were determined by studies of shape change potency as a function of MPPC and red cell concentration. It is found that 8(+/- 0.5) x 10(6) molecules of MPPC per cell will effectively induce stage three echinocytes and yield 3.2(+/- 0.2)% expansion of outer monolayer surface area. Surface area of normal cells determined by direct measurements from fixed geometry of red cells aspirated by micropipette was 118.7 +/- 8.5 microns2. The effective cross-sectional area of MPPC molecules in the cell membrane therefore was determined to be 48(+/- 4) A2, which is in agreement with those determined by x-ray from model membranes and crystals of lysophospholipids. We concluded that surface area expansion of RBC can be explained by a simple consideration of cross-sectional area of added molecules and that erythrocyte shape changes correspond quantitatively to the incorporated lipid molecules.     

Effects of p-chloromercuribenzene sulfonate on water transport across the marsupial erythrocyte membrane

The effects of exposure of red blood cells (RBC) of three species of marsupial to a mercury-containing sulfhydryl-modifying reagent, p-chloromercuribenzene sulfonate (PCMBS), on the water diffusional permeability ( P (d)) of their membranes were monitored by using an Mn(2+)-doping (1)H nuclear magnetic resonance (NMR) technique at 400 MHz. For koala ( Phascolarctos cinereus), RBC the maximal inhibition was reached at 37 degrees C in 60 min with 1 mmol.l(-1) PCMBS or in 15-30 min with 2 mmol. l(-1) PCMBS. In contrast, in the case of red kangaroo ( Macropus rufus) or swamp wallaby ( Wallabia bicolor) RBC, maximal inhibition required an incubation of 90 min at 37 degrees C with 2 mmol.l(-1) PCMBS. For the RBC of all three species the value of maximal inhibition was very high, being 50-70% when measured at 25 degrees C, 60-80% at 30 degrees C and 60-70% at 37 degrees C. The lowest values of P (d) appeared to be around 2 x 10(-3)-3 x 10(-3) cm.s(-1) in the temperature range of 25-37 degrees C. The mean value of the activation energy of water diffusion ( E (a,d)) was approximately 20-25 kJ.mol(-1) for control and approximately 40 kJ.mol(-1) for PCMBS-inhibited RBCs. These results show that marsupial RBC have a basal permeability to water similar to that previously reported for human RBC, but a higher value of the PCMBS-inhibitable water permeability. This indicates that the higher water permeability of marsupial RBC compared with human RBC is associated with a higher fraction of protein-mediated water permeability.