Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Interactions of the chelating agent 2,3-dimercapto-propane-1-sulfonate with red blood cells in vitro. I. Evidence for carrier mediated transport

Uptake of the water soluble 1,2-dimercaptopropanol (BAL) derivative 2,3-dimercapto-1-sulfonate (DMPS) into human red blood cells was found in vitro and the mode of penetration studied in detail. The compound entered erythrocytes in a concentration dependent manner. In contrast to sealed ghosts where inside and outside concentrations reached the same value, DMPS accumulated in intact erythrocytes. Since no binding of DMPS could be detected, the reason for accumulation was assumed to be a conversion of DMPS into chelates or metabolites which penetrated the membrane in a slower rate. A facilitated transport of DMPS mediated by the anion carrier protein was concluded on the basis of the following similarities with the anion transport: inhibition of [¹⁴C]DMPS-uptake by N-ethylmaleimide (NEM), tetrathionate (90%), sulfate (50%), 5,5′-dithio bis(2-nitrobenzoic acid) (DTNB) (25%); inhibition of uptake and efflux by 4,4′-diisothiocyano-2,2′-stilbene disulfonate (DIDS) (80%), dipyridamole (55%); temperature dependency (activation energy 24 K_cal/mol); pH-dependency (pH optimum about 6.9); counter-transport; activation of uptake by preincubation with DMPS (transmembrane effect).     

Intracellular pH regulation in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.     

Anion movements across lamprey (Lampetra fluviatilis) red cell membrane

Chloride and bicarbonate movements across lamprey red cell membrane were investigated. The halftime for equilibration of radioactive chloride across the red cell membrane was 2.46 h, and apparent permeability for chloride-36 was approximately 10(-9) cm X s-1, a value similar to that observed for lipid bilayers. Chloride movements were not affected by the anion exchange inhibitor, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid (DIDS). Furthermore, intracellular buffering is effectively isolated from the extracellular compartment, as shown by the fact that practically no pH recovery occurred in the unbuffered extracellular medium after either acidification or alkalinization. These observations show that lamprey red cell membrane is quite impermeable to bicarbonate and other acid/base equivalents.     

The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.     

The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Reversible inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid of the plasma membrane (Ca(2+)+Mg2+)ATPase from kidney proximal tubules

Calcium accumulation by purified vesicles derived from basolateral membranes of kidney proximal tubules was reversibly inhibited by micromolar concentrations of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of anion transport. The inhibitory effect of this compound on Ca2+ uptake cannot be attributed solely to the inhibition of anion transport: (Ca(2+)+Mg2+)ATPase and ATP-dependent Ca2+ transport, respectively. The rate constant of EGTA-induced Ca2+ efflux from preloaded vesicles was not affected by DIDS, indicating that this compound does not increase the permeability of the membrane vesicles to Ca2+. In the presence of DIDS, the effects of the physiological ligands Ca2+, Mg2+, and ATP on (Ca(2+)+Mg2+)ATPase activity were modified. The Ca2+ concentration that inhibited (Ca(2+)+Mg2+)ATPase activity in the low-affinity range decreased from 91 to 40 microM, but DIDS had no effect on the Km for Ca2+ in the high-affinity, stimulatory range. Free Mg2+ activated (Ca(2+)+Mg2+)ATPase activity at a low Ca2+ concentration, and DIDS impaired this stimulation in a noncompetitive fashion. The inhibition by DIDS was eliminated when the free ATP concentration of the medium was raised from 0.3 to 8 mM, possibly due to an increase in the turnover of the enzyme caused by free ATP accelerating the E2----E1 transition, and leading to a decrease in the proportion of E2 forms under steady-state conditions. Alkaline pH totally abolished the inhibition of the (Ca(2+)+Mg2+)ATPase activity by DIDS, with a half-maximal effect at pH 8.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anion exchange and anion-cation co-transport systems in mammalian cells

Electroneutral anion transfer in the Ehrlich ascites tumour cell has been found to occur by two separate mechanisms. One is an exchange diffusion system with many similarities to that found in erythrocytes, e.g. saturation kinetics with 'self-inhibition', a relatively pronounced temperature dependence, competitive interactions of Br-, NO3- and SCN-, and a low conductive PCl- of 4 x 10(-8) cm s-1. The main differences are that the Cl- flux in Ehrlich cells at 38 degrees C is one thousandth of the flux in red cells, and that the specificity of the system is less pronounced. It is suggested that the density of anion exchange sites in Ehrlich cells could be the same as in red blood cells, but with a lower turnover rate. The other system is an anion-cation co-transport system capable of mediating a secondary active Cl- influx. This system has a volume-regulatory function and is activated by a reduction in cell volume and intracellular [Cl-]. The two transport systems can be separated by using DIDS as an inhibitor of anion exchange and bumetanide as an inhibitor of co-transport. Under normal steady-state conditions Cl- flux is dominated by the exchange system. It is suggested that intracellular pH regulation can be achieved by the two systems operating in parallel, because the chloride disequilibrium maintained by the co-transport system can drive an influx of bicarbonate through the exchange mechanism.     

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Sulphate and phosphate transport in the renal proximal tubule

Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane.     

A novel technique for identification of sites of anion transport in intact cells and tissues using a fluorescent probe

Abstract The purported blocker of anion transport 4, 4′ di-isothiocyano-2-2′ stilbene disulfonate (DIDS) has been shown to partially inhibit ³⁶Cl^? influx, ³⁶CIO^?₃ influx and ³⁵SO^2?₄ influx into Pisum salivum L. cv. Feltham First seedlings. This inhibitory effect could be prevented by pretreatment with the respective unlabelled medium. There was no effect of DIDS on ¹⁴C methylamine influx. The results are consistent with the hypothesis that the binding of DIDS to the site of anion-carrier interaction is responsible for its observed inhibitory effects on anion fluxes. The fluorescent properties of DIDS upon binding to membrane proteins was exploited in an attempt to examine the major sites of anion pumping in whole roots. The results show clearly that in the presence of DIDS the epidermal layers became brightly fluorescent, while cortical layers did not fiuoresce. Lycopersicum esculentum cells taken from locular fluid were plasmolysed using sucrose solution, and the patterns of fluorescence in the presence of DIDS showed in an unambiguous way that the fluorescence is associated with cell membranes. The potential usefulness of this technique to probe sites of anion transport in whole plants and tissues is discussed.     

Sat1 is dispensable for active oxalate secretion in mouse duodenum

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.     

Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.     

Sulphydryl groups involved in Na+-Li+ exchange in human erythrocytes

Oxidative stress causes cellular injury that is thought to be due to increased cytosolic cation levels. Disturbances of a variety of mechanisms which normally maintain intracellular anion/cation homeostasis, occur during oxidative stress. Reactivity of the SH- groups essential for oubain-resistant Na(+)-Li(+) exchange by N-ethylmaleimide (NEM) and selenite was studied in human erythrocytes. In addition, the reactivity of the substances on SH- groups and Li(+) influx have been studied as a function of pH of the medium. The results show that NEM induces an irreversible inhibition of Li(+) influx. It diminishes progressively with the increasing pH of the medium. Whereas we obtain increasing intracellular Li(+) concentration with the rising selenite concentration in the medium. The maximum effect with this substance is reached at about pH 8.0. We can state that the -SH reagents (NEM and selenite) studied behave differently: NEM inhibits Li(+) influx by modifying the essential SH-groups of the membrane proteins in such a way that the exchange is reduced, whereas it maintains the Na(+) permeability almost unaltered. The slight increase in intracellular Na(+) induced by selenite suggests that the oxidative changes in the intracellular sulphydryl groups may constitute an important mechanism for the regulation of the intracellular cations.     

Changes in the structural-functional state of erythrocyte membranes treated with anion transport inhibitors

Effect of 4,4-dyisotiocyanostilben-2,2-disulfonate (DIDS) and 1-ftor-2,4-dinitrobenzol (NDFB) on the rate of phosphate ion transport in erythrocytes, filtrability and thermal stability of erythrocytes and on the structural state of the erythrocyte membrane estimated by UV-fluorescence, PAAG--electrophoresis and measuring of the activity of membrane-bound acetylcholinesterase (AChE) has been studied. Unpenetrating anion transport inhibitor DIDS is shown to induce structural modifications of bands 3 of protein and AChE, while DNFB penetrating the membrane causes a significant reorganization of many membrane proteins (including spectrin) resulting in changes of transport and mechanical properties of erythrocytes.