Permeability Of Human Erythrocytes Research Articles

Identification and characterization of potent and selective aquaporin-3 and aquaporin-7 inhibitors

The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H2O2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC50 of ∼0.1-0.4 μm but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC50, ∼0.7-0.9 μm), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, ∼0.2 μm). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.

The Immobilization of Gossypol Derivative on N-Polyvinylpyrrolidone Increases its Water Solubility and Modifies Membrane-Active Properties

The conjugate of the gossypol derivative megosin (1) with N-polyvinylpyrrolidone named rometin (2) was synthesized. The effects of 1 and 2 on the structure and permeability of human erythrocytes and rat liver mitochondria were compared. Compound 1 induced dose-dependent erythrocyte hemolysis and increased mitochondrial permeability, with concomitant changes in membrane structure as determined by ESR and fluorescence anisotropy methods. Immobilization of 1 on N-polyvinylpyrrolidone (compound 2) increased its water solubility and reduced the intensity of its effects on erythrocyte membrane integrity and mitochondrial permeability, which correlated with a decrease in the membranes structural changes induced by the compound. Although the same concentrations of free and N-polyvinylpyrrolidone bound 1 were used, far less (14)C-labeled 1 was incorporated into the membranes from complex than free 1. The increase in water solubility and the reduction of membrane-active properties of 1 after immobilization on N-polyvinylpyrrolidone could explain our previous observation of the decreased toxicity of 1.

Characterization of a phenylglyoxal-sensitive passive Ca2+ permeability in human erythrocytes.

In viable human erythrocytes, passive downhill entry of Ca2+ through the plasma membrane is opposed by an active extrusion Ca2+-pump mechanism which maintains a free [Ca2+]i approximately 10(-7) M. This passive Ca2+ permeability can be determined reliably in cells with a completely arrested Ca2+ pump, as can be achieved in cells which are both ATP-depleted and vanadate-treated. With this approach, whole-cell preparations from banked human erythrocytes exhibited passive net uptake of Ca2+ that has characteristics of a carrier-mediated process which could be shunted by the addition of the Ca2+ ionophore A23187. Passive Ca2+ uptake was apparently independent of extracellular Na+, K+ or Cl- and was not responsive to a variety of Ca2+ channel entry blockers. However, in the presence of phenylglyoxal, a specific, covalent modifier of arginyl residues, the initial (0 to 60 min) rate of passive Ca2+ uptake was inhibited in a dose-dependent manner. This can be taken in support of a proteinaceous carrier-mediated passive Ca2+ entry mechanism that involves one or several functional arginyl residue(s) and may represent Ca2+ back-diffusion through the arrested Ca2+ translocation pump.

Alteration of Na + permeability in human erythrocytes as studied by 23Na-NMR and inhibition of the kidney Na +,K +-ATPase activities with saponins: Interaction of Gleditsia saponins with human erythrocyte membranes

Interaction of Gleditsia saponin C 1, bis-monoterpenyl echinocystic acid 3,28-O-bisglycoside, with human erythrocyte membranes and artificial membranes was investigated by means of the hemolytic activities, alteration of Na + permeability by 23Na-NMR spectroscopy and inhibition of Na +,K +-ATPase activities. Interaction of Gleditsia saponin C ( 1), bis-monoterpenyl echinocystic acid 3,28-O-bisglycoside, with human erythrocyte membranes and artificial membranes was investigated by means of the hemolytic activities, alteration of Na + permeability by 23Na-NMR spectroscopy and inhibition of Na +,K +-ATPase activities.

Studies on the chemical modification of monensin. IV. Synthesis, sodium ion permeability, and biological activity of 7-O-acyl- and 7-O-alkylmonensins.

7-O-Acyl-(4a-e) and 7-O-alkylmonensins (5a-d) were prepared from monensin (1). Their lipophilicity, sodium ion permeability in human erythrocytes, antibacterial activity and effect on rat tail artery were examined. There was a correlation between lipophilicity and sodium ion permeability as well as between lipophilicity and antibacterial activity. We also found that the compound having larger sodium ion permeability, showed stronger contraction of rat tail artery. 7-O-Benzylmonensin (5c) exhibited higher lipophilicity and larger sodium ion permeability than monensin (1) among the tested monensin derivatives. In addition, antibacterial activity and contractile effect on rat tail artery of 5c were comparable to those of 1.

Mode of action of the gramicidin S analogs lacking hydrophilic amino acid residues on biomembranes.

The gramicidin S analog lacking basic ornithine residues, cyclo(-Val-Ala-Leu-delta Phe-Pro-)2 (where delta Phe represents alpha, beta-dehydrophenylalanine), increased the K+ permeability of human erythrocytes and Staphylococcus aureus similarly to the parent gramicidin S. This analog altered the normal discoid shape of human erythrocytes to an invaginated form. The direction of the shape change was opposite to the case of gramicidin S causing crenated cells. We suppose that the analog accumulated predominantly into the inner half monolayer of membrane and destabilized the membrane structure, resulting in a break in the membrane.

Modulation of the Ca 2+- or Pb 2+-activated K +-selective channels in human red cells. I. Effects of propranolol

To study the effect of propranolol on the Ca 2+- or Pb 2+-activated K + permeability in human erythrocytes K + effluxes were compared with single-channel currents. The results demonstrate that propranolol has a twofold effect: (1) it renders the channel protein more sensitive to Ca 2+ or Pb 2+; and (2) it simultaneously inhibits channel activity and slightly reduces single-channel conductance. The number of active channels is not affected.

Effects of gossypol on the activity of kidney ( Na+ + K+)-ATPase and the functions of erythrocyte membrane

Gossypol extracted from cottonseed oil, reputed to exert contraceptive action in males, may induce a side effect of hypokalemia. The cellular and molecular mechanisms by which gossypol produces hypokalemia are not quite understood. We have examined the inhibitory effect of gossypol on the activity of ( Na + + K +)-ATPase purified from the outer medulla of rabbit kidney, the half maximal inhibitory concentration (IC 50) is 6.5 μm. The kinetic studies using this enzyme preparation show that gossypol is noncompetitive with ATP, Mg 2+, Na + and K + with apparent Ki of 15.0, 13.0, 14.0 and 4.4 μm, respectively. On the other hand, in order to estimate the effects of gossypol on membrane transport of intact cells, we have investigated the effects of gossypol on the activity of ( Na + + K +)-ATPase, membrane integrity and permeability of human erythrocytes. It shows that gossypol inhibited the enzyme activity (<5 μm) and expressed the hemolysis (<50 μm) in vitro in a concentration-dependent manner, and increased the K +-efflux of the cells (10–40 μm). The above effects are antagonized by 1–2% bovine serum albumin. The data demonstrate that gossypol is a specific and potent membrane active agent. These results may be relevant to the in vivo actions of gossypol.

Natural variation in passive sodium permeability in human erythrocytes.

The rate of influx of 22Na+ into human erythrocytes (RBC) varies greatly depending upon the donor. A high rate of influx may be related to a congenital predisposition to essential hypertension. In Northern Europeans, we find a threefold difference in the rate of 22Na+ influx between those with the least (LP) and most highly permeable (HP) RBC (from less than 0.15 to greater than 0.60 mmol Na+/liter RBC/hr). In order to further define determinants of these apparently hereditary differences in passive membrane Na+ transport, we identified two groups of normal laboratory and hospital personnel differing markedly (greater than twofold) in RBC 22Na+ influx rate. We find that the loop diuretics furosemide and bumetanide decrease by about 50% the influx of 22Na+ into HP RBC, but have a lesser influence on LP RBC. Impermeant polyanions such as citrate and pyrophosphate also specifically diminish 22Na+ influx into HP, but not LP, RBC. Therefore, the exaggerated 22Na+ influx into HP RBC probably occurs through a discrete pathway (perhaps via "Na/K/Cl cotransport"), which appears to be almost absent in LP RBC. The differences between HP and LP RBC most likely do not involve polymorphisms of RBC anion transport per se. The rate of RBC anion (35SO4(2-)) transport is the same in HP and LP RBC and is equally inhibited by furosemide and (to a lesser extent) bumetanide. Furthermore, the potent inhibitor of RBC anion transport, DIDS (diisothiocyanostilbene disulfonate) does not affect RBC Na+ permeability in either group. Nonetheless, the preferential reduction of Na+ permeation of HP RBC by loop diuretics may be of help in experimentally distinguishing HP from LP phenotypes. This information may be crucial in unraveling the structural basis of intrinsic differences in cell membrane Na+ permeability and their possible relationship to essential hypertension.

Effects of vanadate, menadione and menadione analogs on the Ca 2+-activated K + channels in human red cells. Possible relations to membrane-bound oxidoreductase activity

The modulation of the Ca 2+- (or Pb 2+-)activated K + permeability in human erythrocytes by vanadate, menadione and chloro-substituted menadione analogs was investigated by measurements of K + fluxes and single-channel currents. Vanadate and menadione stimulate the K + permeability by increasing the probability of channel openings; the menadione analogs, on the other hand, inhibit the K + permeability by increasing the probability of channel closings. The compounds used in these experiments also interact with oxidoreductases; it is demonstrated that menadione analogs in contrast to menadione strongly inhibit the membrane-bound dehydrogenase in the erythrocytes. Concentrations of Pb 2+ above 10 μmol/l, but not of Ca 2+, inhibit the enzyme activity as well as the K + permeability. The parallel effects on dehydrogenase activity and the K + channels suggest a direct relationship between these two systems in the membrane of erythrocytes.

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca2+ activate a K+-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K+ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs+, tetraethylammonium and Ba2+ and of single-channel activity by quinine and Ba2+ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na+ and Rb+) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.

The mechanism of vanadium action on selective K +-permeability in human erythrocytes

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO 2+, +4 oxidation state) to vanadate (VO 3 −, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K + permeability, which is very similar to the Gardos Ca 2+-induced K + permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K +. In ultrapure media with very low Ca 2+, however, vanadyl has no effect on K + permeability. These experiments suggest that Ca 2+ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K + permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca 2+ from EDTA. Vanadate, an inhibitor of Ca 2+-ATPase, causes a selective increase in K + permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca 2+. Earlier observations of effects of vanadyl and vanadate on erythrocyte K + permeability can thus be explained on the basis of inhibition of the Ca 2+ pump by vanadium, leading to an increase in intracellular Ca 2+ concentration.

Physiological shear stresses enhance the Ca2+ permeability of human erythrocytes.

A membrane-bound Ca2+ pump, capable of extruding Ca2+ at a rate of 4−30 × 10−3 mol Ca2+ per l of cells per h in optimal conditions1, enables mammalian erythrocytes to maintain a large transmembrane Ca2+ concentration gradient (10−3M outside compared with 10−6M inside)2. This rate is much greater than the passive Ca2+ influx, estimated3 to be 6−10 × 10−6 mol per l of cells per h. It has been suggested that the apparently large excess capacity of the Ca2+ pump both gives the required Ca2+ sensitivity to a pump incapable of regulating its Ca2+ affinity4 and ensures against Ca2+ toxicity in certain red cell activities5. The results presented here, however, show that circulatory shear stresses enhance the Ca2+ permeability of the membrane, and suggest that greater demands are imposed on the Ca2+ pump in vivo than had been previously thought. We find that the passive permeability to Ca2+ is increased by up to an order of magnitude and the Ca2+-stimulated ATPase activity is strongly enhanced when red cells are sheared at physiological rates in a rotational viscometer.

Vanadium increases selective K +-permeability in human erythrocytes

In human erythrocytes that had been depleted of ATP by incubation with iodoacetate and adenosine, vanadate induces a 10–15-fold increases of K +-permeability. The effect is similiar to that produced by calcium ions. Like the calcium-induced permeability change, the vanadate-induced effect is preceded by a lag period. Preincubation without substrates for ATP synthesis reduces the length of the lag period following the addition of either vanadate or calcium. The selective change of K +-permeability was brought about by vanadate anions (+5 oxidation state) as well as by vanadyl cations (+4 oxidation state). In both cases, the presence of EDTA prevented the permeability change. Blocking of the anion-transport system of the human erythrocytes by H 2DIDS was used to discriminate between the unstable forms of vanadate anion and vanadyl cation in producing the potassium loss. The observation that H 2DIDS had little if any effect on the efficiency and the previous reported fact by Cantley, L.C. and Aisen, Ph. (J. Biol. Chem., 254 (1979) 1781) that vanadate appears mostly as vanadyl in the cell interior suggests that, similar to Ca 2+, Mg 2+ or Pb 2=, vanadyl (VO 2+) can open the “potassium channel” in the erythrocyte membrane.

A simple technique of measuring membrane permeability of human erythrocytes to water and other highly permeable solutes

A new way of measuring high diffusional permeabilities Pd of erythrocyte membranes is presented using THO and 14C-glycol as test solutes. The method is essentially based on the work of Redwood et al. (I). The underlying principle is to measure tracer diffusion through a column of tightly packed erythrocytes in phosphate-buffered isotonic saline (hematocrit 98%). A bulk diffusion coefficient, Def , is thus obtained. Based on an appropriate physical model, diffusional permeabilities of the membrane may be calulated from Def , if three other quantities are known:

The effects of the anticoagulants ACD and CPD on the glycerol permeability of human erythrocytes

The effects of the anticoagulants ACD and CPD on the glycerol permeability of human erythrocytes

Human red cell sodium and potassium in metabolic alkalosis.

Erythrocyte sodium and potassium was studied in 26 patients with metabolic alkalosis. Sodium accumulated in red cells during alkalosis. Red cell potassium increased when the plasma concentration of potassium was normal but decreased when the alkalosis was complicated by low extracellular potassium. Seven of the patients were uremic, but azotemia could not explain the accumulation of sodium in the red cells. The changes of sodium and potassium content in erythrocytes from alkalotic patients are interpreted in the light of in vitro examinations of the effect of bicarbonate ions on the cation permeability of human erythrocytes.

Inhalation anesthetics and permeability of human erythrocytes to monosaccharides.

Inhalation anesthetics and permeability of human erythrocytes to monosaccharides.

Calcium ions and the permeability of human erythrocytes.

Calcium ions and the permeability of human erythrocytes.

Cholinesterase activity and potassium permeability in human erythrocytes.

SummaryInhibition of human erythrocyte cholinesterase with or without exogenous AcCh had no effect on loss of potassium from cells during storage at 4°C or during incubation at 37.5°C. The re-entry at 37.5°G of potassium into cells previously stored for 24 hours at 4°C to permit its leakage into the serum was also unaffected.