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.     

Localization of Erythrocyte Membrane Sulfhydryl Groups Essential for Glucose Transport

The reactions of three organic mercurial compounds, chlormerodrin, parachloromercuribenzoate (PCMB), and parachloromercuribenzenesulfonate (PCMBS) with intact red blood cells, hemolyzed red cells, hemoglobin solutions, and hemoglobin-free ghosts have been characterized. Both PCMB and PCMBS react with only 2 to 3 sulfhydryl groups per mole of hemoglobin in solution, whereas chlormerodrin reacts with 6 to 7. In hemoglobin-free ghosts, however, all three reagents react with a similar number of sulfhydryl groups, approximately 4 x 10^-17 moles per cell, or about 25 per cent of the total stromal sulfhydryl groups, which react with inorganic mercuric chloride. In the intact cell the membrane imposes a diffusion barrier; chlormerodrin and PCMB penetrate slowly, whereas PCMBS does not. Kinetic studies of chlormerodrin binding to intact cells reveal that the majority of stromal sulfhydryl groups is located inside the diffusion barrier, with only 1 to 1.5 per cent (or 1 to 1,400,000 sites per cell) located outside of this barrier. Reaction of PCMBS with intact cells is limited to this small fraction on the outer membrane surface. All three reagents are capable of inhibiting glucose transport in the red cell. With chlormerodrin and PCMBS it was demonstrated that the inhibition results from interactions with the sulfhydryl groups located on the outer surface of the membrane.     

Water permeability in human erythrocytes: identification of membrane proteins involved in water transport

The water permeability of human erythrocytes has been monitored by nuclear magnetic resonance (NMR) before and after treatment of the cells with various sulfhydryl reagents. Preincubation of the cells with N-ethylmaleimide (NEM), a non-inhibitory sulfhydryl reagent, results in a faster and more sensitive inhibition of water exchange by mercurials. The inhibition of water exchange by p-chloromercuribenzene sulfonate (PCMBS) was maximal at a binding of approximately 10 nmol PCMBS per mg protein when non-specific sulfhydryl groups are blocked by NEM. Inhibition by PCMBS has been correlated with the binding of 203Hg to erythrocyte membrane proteins. A significant binding of label to band 3 and the polypeptides in band 4.5 occurs, with approximately 1 mol of mercurial bound per mol of protein. Inhibition of water transport by sulfhydryl reagents does not induce major morphological changes in the cells as assessed by freeze-fracture and scanning electron microscopy.     

Chemical Modification of Membranes : II. Permeation paths for sulfhydryl agents

The amino-reactive reagent, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS),¹ considerably reduces the uptake of the sulfhydryl agent, parachloromercuriphenylsulfonic acid (PCMBS), but does not reduce its effects on cation permeability and on cation transport. These data indicate that PCMBS enters the membrane by at least two channels, one sensitive and the other insensitive to SITS, with only the latter leading to the cation-controlling sulfhydryl groups. Substitution of phosphate or sulfate for chloride results in an inhibition of PCMBS uptake via the SITS-insensitive pathway. These and other data lead to the conclusion that the SITS-sensitive pathway is the predominant one for anion permeation, and the insensitive one for cation permeation. Parachloromercuribenzoate (PCMB), an agent that is more lipid-soluble than PCMBS, penetrates faster but has a smaller effect on cation permeability. Its uptake is less sensitive to SITS. These and other observations suggest that the cation permeation path involves an aqueous channel in the membrane.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

Effects of sulfhydryl and other reagents on the diffusional permeability of dog erythrocytes to small solutes

The effects of p-chloromercuriphenylsulfonic acid (PCMBS), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), phloretin and thiourea on the diffusional permeability of dog erythrocytes to tritiated water and to small 14C-labeled lipophilic and hydrophilic solutes were measured at 37 degrees C by means of the linear diffusion technique. Permeability to 3HHO was significantly decreased by PCMBS but was not affected by the other reagents. The permeability to the small hydrophilic solutes acetamide and urea was decreased by phloretin and thiourea but only the permeability to acetamide was reduced to a statistically significant extent by PCMBS. The permeability to the lipophilic solutes methanol, ethanol and antipyrine was not affected by any of these agents. We interpret these results as an indication that the small lipophilic solutes probably move through lipid areas, that the small hydrophilic solutes probably move through protein associated areas in the erythrocyte membrane and that pathways for the small hydrophilic solutes are distinct from those for water. While the pathways for water may be associated with membrane protein they do not appear to be associated specifically with band 3 protein as has been suggested for human erythrocytes. Diffusional water movement through the dog erythrocyte occurs by two distinct pathways.     

Characterization of the adenosine triphosphatase of avian myeloblastosis virus.

The ATPase of avian myeloblastosis virus (AMV) is not a recognizable cellular enzyme. It hydrolyzes ATP, GTP, ITP, UTP, and dCTP at equal rates, is inhibited by high concentrations of dithiothreitol, and is partially inhibited by 1 × 10^?5mp-chloromercuribenzoic acid (PCMB) and p-chloromercuribenzene sulfonate acid (PCMBS). The inhibition by the mercurials is reversed by increasing the concentration of PCMB or PCMBS to 1 × 10^?3m. The enzyme requires phospholipid for activity. Incubation with phospholipase C inhibits activity and subsequent addition of lecithin-containing saturated fatty acids partially restores activity, whereas lecithin-containing unsaturated fatty acids further inhibit activity.     

Water exchange through erythrocyte membranes: Biochemical and nuclear magnetic resonance studies re-evaluating the effects of sulfhydryl reagents and of proteolytic enzymes on human membranes

Summary The water permeability of human red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on intact cells and resealed ghosts following exposure to various sulfydryl-reacting (SH) reagents and proteolytic enzymes. The main conclusions are the following: (i) When appropriate conditions for exposure of erythrocytes or ghosts to mercury-containing SH reagents (concentration, temperature and duration of incubation) were found, the maximal inhibition of water diffusion could be obtained with all mercurials (including HgCl₂ and mersalyl that failed to show their inhibitory action on RBC water permeability in some investigations). While previous studies claimed that long incubation times are required for the development of maximal inhibition of water diffusion by mercurials, the present results show that it can be induced in a much shorter time (5–15 min at 37°C) if relatively high concentrations of PCMBS (2–4mm) are used and no washings of the inhibitor are performed after incubation. Higher than optimal concentrations of mercurials and/or longer incubation times result in lower values of inhibition, sometimes a loss of inhibition, or can even lead to higher values of permeability compared to control RBCs. (ii) The conditions for inhibition by mercurials are drastically changed by preincubation of erythrocytes with noninhibitory SH reagents (such as NEM or IAM) or by exposure to proteolytic enzymes. If the cells are digested with papain, the duration of incubation with PCMBS should be decreased in order for inhibition to occur. This explains the lack of inhibition reported previously, when a relatively long duration of incubation with PCMBS was used subsequent to papain digestion. (iii) The degree of inhibition of water diffusion induced by mercurials appeared to be dependent upon the temperature of which the water permeability was measured. The values of maximal inhibition ranged from 45–50% at 37°C, increased 10–15% at 20°C and further increased at lower temperatures, reaching values above 75% below 10°C; these results clarify the conflicting reports of various authors. (iv) The inhibition of water diffusion, either reversible, or irreversible, was not accompanied by significant changes in the pattern of RBC membrane polypeptides fractionated by polyacrylamide gel electrophoresis. (v) The mean value of the activation energy of water diffusion (E _a,d) obtained on 42 donors was 25.6 kJ/mol. The values ofE _a,d increased in parallel with the values of the inhibition of water diffusion induced by PCMBS until the maximal inhibition was reached (whenE _a,d=41 kJ/mol) and then both sets of values decreased in parallel.     

Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter

Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.     

Re-examination of the localization of Mg-chelatase within the chloroplast

In a plastid-free assay, Mg-chelatase from pea ( Pisum sativum L. cv. Spring) and cucumber ( Cucumis sativus L. cv. Sumter) chloroplasts is inhibited to equal extents by the mercurial reagents. p -chloromercuribenzoate (PCMB) and p -chloromercuribenzene sulfonate (PCMBS). However, in intact chloroplasts PCMB inhibits Mg-chelatase fourfold more strongly than does PCMBS. Since PCMBS cannot penetrate membranes as readily as PCMB, Mg-chelatase may be localized interior to the inner chloroplast envelope. When Mg-chelatase is assayed with photosynthetically generated ATP, the presence of an external ATP trap does not inhibit activity, suggesting that the enzyme is not located in the interenvelope space. None of the components of Mg-chelatase are integral membrane proteins: Mg-chelatase activity is readily solubilized by washing the total chloroplast membranes in buffers of low MgCl₂ content. This precludes localization by purifying individual thylakoid and envelope membranes which requires low MgCl₂ concentrations.     

Effect of thiourea on PCMBS inhibition of osmotic water transport in human red cells

The organomercurial reagent p-chloromercuribenzene sulfonate (PCMBS) is an inhibitor of osmotic water permeability in the human red cell membrane. We have found that thiourea, when added along with PCMBS to a red cell suspension, interferes with this inhibition and at high enough concentrations prevents the inhibition from developing altogether. For a 2 mM PCMBS concentration K_i = ; 3 ± 1 mM. When thiourea is added at a later time, the PCMBS inhibition, which normally takes about 20 min to develop fully, is halted and remains fixed at the value attained by that time. Thiourea also inhibits the reversal of PCMBS inhibition by a 10 mM concentration of cysteine, the half-time for reversal increasing by more than an order of magnitude when [thiourea] = ; 50 mM. Possible implications for the nature of the water and urea transport pathways across the red cell membrane are discussed.     

Stoichiometry of hemolysis by the polyene antibiotic lucensomycin

The stoichiometry of hemolysis by the polyene antibiotic lucensomycin was investigated. It appears that hemolysis occurs only when a relatively high fraction (probably between 15 and 40%) of the cholesterol sites in the erythrocyte membrane have combined with the polyene. Also in phospholipid-cholesterol vesicles the increase of permeability requires occupancy of 40–50% of the existing cholesterol sites.As for the possible cooperative effect in the hemolytic process, it is probable that several (at least 9–10) lucensomycin-cholesterol adducts must interact on each side of the membrane to form an aqueous channel; the distribution of these adducts in the erythrocyte membrane occurs, however, apparently at random.     

Erythrocyte membrane sulfhydryl groups and cation permeability

Reaction of the slowly penetrating organic mercurial compound parachloromercuribenzene sulfonate (PCMBS) with intact erythrocytes has been characterized. Addition of concentrations of PCMBS which result in binding within the interior of the membrane of more than 1.9 × 10^?18 moles/cell produces alterations in Na⁺ and K⁺ permeability, but does not affect choline permeability. However, the increased cation permeability is observed only after a lag period of over two hours. After ten hours, a spontaneous slow “recovery” to normal rates of K⁺ leakage occurs at 25°C but not at 2°C. Subsequent to the effects on cation balance, increasing degrees of hemolysis occur, interpreted as colloid osmotic lysis. The relationships between the binding of the agent and its effects are as follows: a small, rapid initial uptake does not affect cation permeability; the subsequent slower uptake is associated with increased leakage of K⁺ and Na⁺; and the recovery at 25°C is associated with desorption of about half of the PCMBS due to competition by soluble thiol substances released into the medium from the cells. Desorption and “recovery” can be mimicked at any time by addition of small amounts of protein in the medium. The half of the PCMBS that cannot be desorbed is assumed to be bound by the hemoglobin inside the cell. The sulfhydryl groups involved in control of cation permeability constitute only a fraction of the total within the membrane (4–18%). They are located within the interior of the membrane separated from the medium and from the interior of the cell by diffusion barriers to PCMBS.     

Disulfiram may mediate erythrocyte hemolysis induced by diethyldithiocarbamate and 1,4-naphthoquinone-2-sulfonate.

The increase in 1,4-naphthoquinone-2-sulfonate (NQS)-induced hemolysis by the superoxide dismutase inhibitor diethyldithiocarbamate (DEDC) was formerly attributed to increased superoxide anion levels in the erythrocyte. Our results show that removal of DEDC after preincubation and prior to the addition of NQS did not produce a significant increase in hemolysis, which suggests that hemolysis is primarily caused by the reaction products of DEDC with NQS and not to the inactivation of superoxide dismutase. Disulfiram, the oxidized product of DEDC, was found to be the main product formed when excess DEDC was reacted with NQS. Oxygen uptake also occurred and hydrogen peroxide was formed. The latter caused the oxidation of DEDC to disulfiram as catalase prevented disulfiram formation. Disulfiram was found to readily hemolyze erythrocytes at low concentrations as well as to crosslink the proteins in the erythrocyte membrane. Furthermore, disulfiram-induced hemolysis was markedly enhanced in glutathione-depleted erythrocytes. Disulfiram was subsequently found to readily oxidize glutathione in red blood cells. When equimolar concentrations of DEDC and NQS were reacted, the major product formed was the diethyldithiocarbamate:1,4-naphthoquinone (DEDC:NQS) conjugate. However, the principal mediator of erythrocyte hemolysis when excess DEDC is reacted with 1,4-naphthoquinone-2-sulfonate is disulfiram, whose mode of action may be to modify membrane protein sulfhydryls.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

Effect of PCMBS on water transfer across biological membranes

P-chloromercuriphenylsulfonate, PCMBS, and 5, 5′ dithiobis-(2-nitrobenzoic acid), DTNB at a concentration of 1 mM are found to inhibit the rate of water transport across human red cell membrane. In addition PCMBS inhibits the rates of transport of small hydrophilic but not hydrophobic nonelectrolytes. Other sulfhydryl reagents such as N-ethylmaleimide and iodoacetamide have no significant effect on the rate of water transfer in these cells. The results suggest that there are at least two populations of membrane bound SH-groups which differ in their topical location which participate in the control of water transfer. One is located closer to the outer surface of the membrane, and thus is readily accessible to PCMBS while the other component is probably located in the membrane interior. These two populations can be dissociated by pH. The effect of PCMBS on water transfer can be greatly influenced by pH and temperature. The main effect of temperature and pH is on the permeability of the membrane to the drug. The same concentration of PCMBS is also found to inhibit to a lesser degree water transfer across other biological membranes.     

Effect of mercurial compounds on net water transport and intramembrane particle aggregates in ADH-treated frog urinary bladder

Summary It has been suggested that during the oxytocin-induced hydrosmotic response, water crosses the luminal membrane of urinary bladder epithelium cells through membranespanning proteins. Although specific inhibitors of osmotic water transport have not been found, certain sulfhydryl reagents such as mercurial compounds may help to identify the proteins involved in this permeation process. We tested the effects ofp-chloromercuribenzene sulfonate (PCMBS) and of fluoresceinmercuric acetate (FMA) on the net water flux, the microtubule and microfilament structures of the frog urinary bladder, and the distribution of intramembrane particle aggregates in the luminal membrane.We observed that: (i) 5mm PCMBS at pH 5 and 0.5mm FMA at pH 8 added to the mucosal bath at the maximum of the response to oxytocin partially inhibited the net water flux. Inhibition then increased progressively when the preparation was repeatedly or continuously stimulated, until it reached a maximal inhibition at 120 min. This inhibition was not reversed even when cystein was added in the mucosal bath. PCMBS and FMA effects were also observed when cyclic AMP (3,5 cyclic adenosine monophosphate) was used to increase water permeability. (ii) PCMBS mucosal pretreatment did not modify the basal water flux but potentiated the inhibitory effect of PCMBS or FMA on the hydrosmotic response to oxytocin. (iii) Microtubule and microfilament network, visualized in target cells by immunofluorescence, was not affected by PCMBS. (iv) The maximal PCMBS or FMA inhibition was not associated with a reduction of aggregate surface area in the apical membrane.The persistence of the intramembrane particle aggregates associated with the oxytocin-induced hydrosmotic response during the net water flux inhibition by PCMBS, suggests that the PCMBS effect occurs possibly at the level of sulfhydryl groups of the water channel itself.     

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

The 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 to erythrocytes to trypsin and chymotrypsin. A new kind of experiments 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 membrane 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.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

NMR study of the selective inhibition of water permeability of rat erythrocyte membrane

The inhibition of water diffusion across the rat erythrocyte membrane was studied by NMR using two basically different types of inhibitory agents: PCMB andin vivo irradiation. The contribution of lipid and protein to water permeability revealed the inhibitory effect of each pathway. Internal contamination with tritium (25–115 mGy) reduces water permeability due to protein modifications; for doses higher than 100 mGy the lipid mediated mechanism seems also to be impaired. The same procedure enables one to assess the extent to which the higher water permeability of rat, compared to human, erythrocyte is due to one of the two pathways.