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DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) is a commonly used blocker of plasma membrane anion channels and transporters. We observed that DIDS undergoes decomposition while stored in DMSO (dimethyl sulfoxide) forming a biologically active compound. One decomposition product, called IADS, was identified and synthesized. Voltage-clamp and patch clamp experiments on Xenopus laevis oocytes and human erythrocytes revealed that IADS is able to activate a plasma membrane cation conductance in both cell types. Furthermore, we found that IADS induces hemolysis in red blood cells of healthy donors but fails to hemolyze erythrocytes of donors with cystic fibrosis. Thus, IADS stimulated activation of a cation conductance could form the basis for a novel diagnostic test of cystic fibrosis., (Copyright (c) 2006 S. Karger AG, Basel.)

Non-steroidal anti-inflammatory drugs (NSAID) elicit gastric damage through inhibition of the synthesis of prostaglandins that protect gastric cells and direct effect on mucous layer. As the latter effect is not well understood, we used acid hemolysis test in a model study on the cytotoxicity of nine NSAIDs. Human erythrocytes were used as model cells after their band 3 membrane protein was inhibited with DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonate) that strongly suppressed the entry of acid into cytosole and postponed acid-induced hemolysis. These drugs did not produce measurable hemolysis in media buffered at pH 7.2. However, in acidic media (pH 3.4) they markedly reduced to a variable extent the prelytic interval (time spent by acid to accumulate overcritically in cytosole) and time for 50% hemolysis (acid resistance). The cytotoxicity of NSAID to erythrocytes at acidic medium was expressed by the inverse of the concentration (C50%) that reduced twofold acid resistance. It was related to the hydrophobicity of drug as the log of C50% depended linearly on the log of its critical concentration for the formation of micelles. Hence, the cytotoxicity of NSAIDs to model cells in acidic media apparently involved the transfer of protonated forms and accumulation of the drug and acid into cytosole. We conclude, the protonophore mechanism could be involved in the direct damage of erythrocytes in acidic media. Based on this cytotoxicity the NSAIDs were ranked as aspirin < paracetamol < nimesulide < diclofenac < piroxicam < meloxicam < ibuprofen < naproxen < indomethacin. This is roughly the same row that expresses the relative in vivo gastropathogenicity of NSAIDs, hence, it is likely this mechanism might damage gastric epithelial cells by generation of influx of NSAID and back diffusion of acid and producing stress conditions and apoptosis.

The effect of postirradiation conditions on the haemolysis of y-irradiated (2.1 kGy) human erythrocytes was studied. Haemolysis was inhibited by incubation in mannitol and sucrose instead of saline, by hypertonicity of the medium and by calcium chelators. Dithiothreitol, butylated hydroxytoluene, deferoxamine, DIDS (in inhibitor of anion exchange) and furosemide (an inhibitor of K/Cl and K/Na/Cl cotransport) did not slow down the haemolysis. Apparently, the radiation-induced haemolysis is due to the formation of membrane pores leaky for electrolytes. From the inhibition of haemolysis by mannitol, apparent pore radius was estimated to be about 0.7 nm. The pores appear to be transient, the average pore number per cell being much less than unity.

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)