Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.     

Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.     

Na+-independent Cl(-)-HCO-3- exchange in Madin-Darby canine kidney cells. Role in intracellular pH regulation

The role of plasma membrane Cl(-)-HCO-3-exchange in regulating intracellular pH (pHi) was examined in Madin-Darby canine kidney cell monolayers. In cells bathed in 25 mM HCO-3, pH 7.4, steady state pHi was 7.10 +/- 0.03 (n = 14) measured with the fluorescent pH probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Following acute alkaline loading, pHi recovered exponentially in approximately 4 min. The recovery rate was significantly decreased by Cl- or HCO-3 removal and in the presence of 50 microM 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS). Na+ removal or 10(-3) M amiloride did not inhibit the pHi recovery rate after an acute alkaline load. Following acute intracellular acidification, the pHi recovery rate was significantly inhibited by 10(-3) M amiloride but was not altered by Cl- removal or 50 microM DIDS. At an extracellular pH (pHo) of 7.4, pHi remained unchanged when the cells were bathed in either Cl- free media, HCO-3 free media, or in the presence of 50 microM DIDS. As pHo was increased to 8.0, steady state pHi was significantly greater than control in Cl(-)-free media and in the presence of 50 microM DIDS. It is concluded that Madin-Darby canine kidney cells possess a Na+-independent Cl(-)-HCO-3 exchanger with a Km for external Cl- of approximately 6 mM. The exchanger plays an important role in pHi regulation following an elevation of pHi above approximately 7.1. Recovery of pHi following intracellular acidification is mediated by the Na+/H+ antiporter and not the anion exchanger.     

Role of a Na+-dependent Cl-/HCO3- exchange in regulation of intracellular pH in fibroblasts

We previously reported that, in a HCO3(-)-free medium, cytoplasmic pH (pHi) of hamster fibroblasts (CCL39) is primarily regulated by an amiloride-sensitive Na+/H+ antiport (L'Allemain, G., Paris, S., and Pouysségur, J. (1984) J. Biol. Chem. 259, 5809-5815). Here we demonstrate the existence of an additional pHi-regulating mechanism in CCL39 cells, namely a Na+-dependent HCO3-/Cl- exchange. Evidence for this system is based on 36Cl- influx studies and on pHi measurements in PS120, a CCL39-derived mutant lacking the Na+/H+ antiport activity. 36Cl- influx rate is a saturable function of external [Cl-] (apparent Km approximately equal to 7 mM), is competitively inhibited by external HCO3- (KI approximately equal to 3 mM), and by stilbene derivatives (KI approximately equal to 20 microM for 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). Measurements of pHi recovery after an acute acid load indicate that PS120 cells possess an acid-extruding mechanism dependent on external HCO3-, which is inhibited by stilbene derivatives and requires external Na+. Since 22Na+ influx is stimulated upon addition of HCO3- to acid-loaded cells and this effect is completely abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, we conclude that Na+ is co-transported with HCO3-, in exchange for intracellular Cl-. In a HCO3(-)-containing medium, this pHi-regulating mechanism appears to have two essential physiological functions for the Na+/H+ antiport-deficient mutant: protection of the cells against excessive cytoplasmic acidification and establishment of a steady-state pHi permissive for growth, at neutral or slightly acidic pHo values (6.6-7.2).     

Chloride transport in embryonic cells: effect of ethanol and GABA

Ethanol and GABA (gamma-aminobutyric acid) and their interaction on 36Cl-influx were analyzed in cultured embryonic palate and limb mesenchymal cells in order to determine whether ethanol exerts its teratogenic action through a GABA receptor involved in embryogenesis. Cl- transport in secondary cultures of C57BL/6 palate mesenchymal cells was shown to consist of three systems including the electroneutral Cl-/HCO3- exchange (50%) and Na+/K+/Cl- cotransport (30%) pathways and the voltage-dependent Cl- channel (20%). Treatment with DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) or SITS (4-acetamido-4'-isocyano-stilbene-2,2' disulfonic acid) in SWV palate cells inhibited the Cl-/HCO3- exchange pathway, while treatment with DIDS and bumetanide inhibited both the exchange and cation cotransport pathways, the residual Cl- influx inferred to be the electrogenic pathway. Inhibition of Cl- transport by anthracene-9-carboxylic acid confirmed the presence of the electrogenic Cl- pathway. It was observed that the rate of Cl- transport was significantly greater in palate cells of C57BL/6 mice than those of SWV mice. Also the rate of Cl- transport was significantly greater in secondary cultures of palate cells from C57BL/6 mice than from primary cultures of limb cells from the same strain. No evidence could be obtained that ethanol (10 to 100 mM) or GABA (3 X 10(-5) M) or their combination stimulated total Cl- influx in either C57BL/6 or SWV palate mesenchymal cells, putative voltage-dependent Cl- influx in C57BL/6 palate cells, or total Cl- influx in primary cultures of C57BL/6 limb mesenchymal cells.     

Voltage-dependent chloride conductance of the squid axon membrane and its blockade by some disulfonic stilbene derivatives

When giant axons of squid, Sepioteuthis, were bathed in a 100 mM Ca-salt solution containing tetrodotoxin (TTX) and internally perfused with a solution of 100 mM tetraethylammonium-salt (TEA-salt) or tetramethylammonium-salt (TMA-salt), the membrane potential was found to become sensitive to anions, especially Cl-. Membrane currents recorded from those axons showed practically no time-dependent properties, but they had a strong voltage-dependent characteristic, i.e., outward rectification. Cl- had a strong effect upon the voltage-dependent membrane currents. The nonlinear property of the currents was almost completely suppressed by some disulfonic stilbene derivatives applied intracellularly, such as 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which are blockers of chloride transport. On the basis of these experimental results, it is concluded that a voltage-dependent chloride-permeable channel exists in the squid axon membrane. The chloride permeability (PCl) is a function of voltage, and its value at the resting membrane (Em = -60 mV) is calculated, using the Goldman-Hodgkin-Katz equation, to be 3.0 X 10(-7) cm/s.     

Mechanism for electrosilent Ca2+ transport to cause calcification of spicules in sea urchin embryos

Embryos of the sea urchin, Hemicentrotus pulcherrimus, kept in sea water containing the calcium antagonists, diltiazem and verapamil, or an anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), during a developmental period between the mesenchyme blastula and the pluteus corresponding stage, became abnormal plutei with poorly developed arms and quite small spicules. Treatment with ethacrynic acid and furosemide, inhibitors of chloride transport, during the same period of development yielded quasi-normal plutei with poor spicules and somewhat developed arms. In late gastrulae, the inhibitory effects of these calcium antagonists and DIDS on the uptake of 45Ca2+ in whole embryos were as strong as those on 45Ca deposition in spicules, whereas the effects of chloride transport inhibitors on calcium deposition in the spicules were markedly stronger than on its uptake in whole embryos. Electrosilent uptake of Ca2+ seems to be established mainly by coupled influx of chloride in the cells which mediate spicule calcification, and by concomitant influx of anions in the other cells. In swimming blastulae, 45Ca2+ uptake was inhibited by calcium antagonists and DIDS, but not by chloride transport inhibitors. Ca2+ uptake probably becomes coupled with chloride influx only in embryos in which spicule calcification occurs.     

Arsenite, arsenate and vanadate affect human erythrocyte membrane

Effects of arsenite, arsenate and vanadate on human erythrocyte membrane have been assessed according to their routes passing through the membrane, their binding modes to the membrane and their influences on membrane proteins and lipids. The uptake of arsenate (1.0 mM) by cells approached a limit with intracellular arsenic of about 0.2 mM in 5 h, and was strongly inhibited (approximately 95%) by 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS), indicating that arsenate, similar to vanadate, passed across the membrane through the anion exchange protein, band 3. Arsenite (1.0 mM) influx reached a maximum of about 0.4 mM in 30 min, and was not inhibited by DIDS. The transformed species of arsenite bound to the membrane from cytosol. In contrast, arsenate bound rapidly from the outside, followed by releasing and re-binding. The binding to the membrane via sulfhydryl was indicated by the decrease of the sulfhydryl level of membrane proteins. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) analysis revealed that the proteins, bands 1-3, were among the targets of arsenite, arsenate and vanadate. Their binding to the membrane also induced changes in the fluidity of membrane lipids and in the negative charge density in the outer surface of the membrane.     

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.     

Effects of bicarbonate on lithium transport in human red cells

Lithium influx into human erythrocytes increased 12-fold, when chloride was replaced with bicarbonate in a 150 mM lithium medium (38 degrees C. pH 7.4). The increase was linearly related to both lithium- and bicarbonate concentration, and was completely eliminated by the amino reagent 4, 4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). DIDS binds to an integral membrane protein (mol wt approximately 10(5) dalton) involved in anion exchange. Inhibition of both anion exchange and of bicarbonate-stimulated lithium influx was linearly related to DIDS binding. 1.1 X 10(6) DIDS molecules per cell caused complete inhibition of both processes. Both Cl- and Li+ can apparently be transported by the anion transport mechanism. The results support our previous proposal that bicarbonate-induced lithium permeability is due to transport of lithium-carbonate ion pairs (LiCO-3). DIDS-sensitive lithium influx had a high activation energy (24 kcal/mol), compatible with transport by the anion exchange mechanism. We have examined how variations of passive lithium permeability, induced by bicarbonate, affect the sodium-driven lithium counter-transport in human erythrocytes. The ability of the counter-transport system to establish a lithium gradient across the membrane decrease linearly with bicarbonate concentration in the medium. The counter-transport system was unaffected by DIDS treatement. At a plasma bicarbonate concentration of 24 mM, two-thirds of the lithium influx is mediated by the bicarbonate-stimulated pathway, and the fraction will increase significantly in metabolic alkalosis.     

Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO4(2-) as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1-3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 microM for DBDS and 3.4 and 55 microM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14-24 A and that the pore constriction is approximately 10 A in diameter.     

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.     

Conformational change of band 3 protein induced by diethyl pyrocarbonate modification in human erythrocyte ghosts

Diethyl pyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethyl pyrocarbonate inhibited the extracellular binding of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to band 3 protein. Furthermore, the extracellular 4,4'-dinitrostilbene-2,2'-disulfonic acid protected the membranes from the intracellular modification by diethyl pyrocarbonate. These results suggest that the extracellular binding of 4,4'-dinitrostilbene-2,2'-disulfonic acid to band 3 protein induces the conformational change of the intracellular counterpart of band 3 protein and the diethyl pyrocarbonate susceptible residue(s) is (are) hidden from the cytosolic surface of the cell membrane in connection with the conformational change. Conversely, under the conditions where the diethyl pyrocarbonate modification is confined to the intracellular side of the membrane, the extracellular binding site of [3H]dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid is hidden from the cell surface.     

Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'- dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.     

The role of anion channels in the mechanism of acrosome reaction in bull spermatozoa.

The involvement of anion channels in the mechanism of the acrosome reaction (AR) was investigated. The AR was induced by Ca2+ or by addition of the Ca2+ ionophore A23187. The occurrence of AR was determined by following the release of acrosin from the cells. In order to investigate the role of anion channels in the AR, several anion-channel inhibitors were tested, mainly DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). Other blockers, like SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid), furosemide, probenecid and pyridoxal 5-phosphate, were also tested. We found that DIDS binds covalently to sperm plasma membrane in a time- and concentration-dependent manner. Maximal binding occurs after 2 h with 0.3 mM DIDS. DIDS and SITS inhibit AR in a concentration-dependent manner. The IC50 of DIDS and SITS in the presence of A23187 is 0.15 and 0.22 mM, respectively. Tributyltin chloride (TBTC), an Cl-/OH- exchanger, partially overcomes DIDS inhibition of the AR. HCO3- is required for a maximal acrosin release and Ca(2+)-uptake, in the presence or absence of A23187. It is known that HCO3- activates adenylate cyclase and therefore, increases the intracellular level of cAMP. The inhibition of the AR by DIDS decreases from 95 to 50% when (dibutyryl cyclic AMP (dbcAMP) was added, i.e., HCO3- is no longer required while elevating the level of cAMP in an alternative way. Moreover, we show that the stimulatory effect of HCO3- on Ca(2+)-uptake is completely inhibited by DIDS. We conclude that DIDS inhibits AR by blocking anion channels, including those that transport HCO3- into the cell.     

Hypo-osmolar stimulation of transepithelial Cl- secretion in cultured human T84 intestinal epithelial layers.

Intact epithelial monolayers of T84 human colonic adenocarcinoma cells were exposed from the basolateral surfaces to hypo-osmotic media; in responsive tissues this resulted in a transient stimulation of inward short-circuit current (SCC) to a peak of 12.9 +/- 1.5 (S.E., n = 10) microA/cm2 which declined to prestimulation values of SCC (2.1 microA/cm2) within 5 min. Exposure of T84 cells to hypo-osmotic media results in an increase in cytosolic [Ca2+]i, dependent on extracellular Ca2+ influx. The cell-swelling activated SCC is abolished upon medium Cl- replacement and by 100 microM bumetanide applied to the basal-surfaces, consistent with the inward SCC resulting from transepithelial Cl- secretion. 100 microM DIDS (4,4'-diisothiocyanantostilbene-2,2'-disulphonic acid) also abolished the cell-swelling activated increase in SCC; DIDS is without effect upon the VIP-stimulated SCC, suggesting distinct Cl- channels are involved in the two responses.     

Involvement of anion channel in signal transduction of early defense responses elicited by harpinPss in tobacco]

No matter when anion channel inhibitors, DIDS (4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid) and A9C (anthracene-9-carboxylic acid) added (before, at the same time of or after harpinPss treatment), they can inhibit harpinPss-induced hypersensitive response in tobacco seedlings and release of active oxygen and extracellular alkalinization in tobacco suspension cells. DIDS and A9C also inhibit harpinPss-induced Ca2+ influx. In all these cases, DIDS is more efficient than A9C. It is postulated that anion channel positively regulates calcium channel in plasma membrane, and harpinPss may function through signal transduction mediated by anion channel and calcium channel to regulate cellular Ca2+ concentration and defense responses.     

阴离子通道参与了harpin_(Pss)诱导的烟草细胞早期防卫反应的信号传导

无论在harpin_(Pss)之前、同时、还是之后向烟草植株或悬浮培养系加阴离子通道的抑制剂DIDS(4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid)或AgC(anthracene-9-carboxylic acid),都可以抑制harpin_(Pss)诱导的烟草植株过敏反应和悬浮细胞的活性氧的释放及胞外碱性化。DIDS和A9C还可以抑制harpin_(Pss)诱导的Ca~(2 )内流。而且DIDS的抑制效率比A9C高。推测质膜上的阴离子通道对钙离子通道有着正调节作用,harpin_(Pss)通过阴离子通道和钙离子通道介导的信号传导途径,调节胞内Ca~(2 )浓度,从而启动这些防卫反应。     

Role of ABH blood group antigens in the stimulation of a DIDS-sensitive Ca2+ influx pathway in human erythrocytes by Ulex europaeus agglutinin I and a monoclonal anti A1 antibody

Of eleven agglutinating lectins tested, only one, Ulex europaeus agglutinin I (UEA1), stimulated Ca2+ uptake in quin2-loaded erythrocytes by about 2-fold. UEA1 is known to be an alpha-L-fucose and ABH blood group specific lectin. The 45Ca2+ influx induced by UEA1 was absent in the presence of extracellular fucose (5 and 15 mM) and depended on the ABH blood group of the donor, the stimulatory potency of the lectin decreasing in the order H greater than A2 greater than A1. Ca2+ entry blockers, such as cobalt and verapamil, did not affect the 45Ca2+ influx induced by UEA1. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited dose-dependently with a Ki of 1-2 microM. 10 microM DIDS, 10 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 20 microM dipyridamole fully blocked the 45Ca2+ influx induced by UEA1. The effect of UEA1 on 45Ca2+ influx was absent in K+ and Mg2+ media and was less pronounced in choline than in Na+ media. The 45Ca2+ influx induced by the lectin was abolished by preincubation with 12-O-tetradecanoylphorbol 13-acetate (TPA, 60 ng/ml). A monoclonal antibody raised against A1 erythrocytes (Bric 54) accelerated 45Ca2+ influx in quin2 loaded A1 erythrocytes by about 2-fold. No effect was seen in A2 and H erythrocytes. The 45Ca2+ influx elicited by Bric 54 exhibited a sensitivity towards inhibition by DIDS and TPA, as well as a dependence on the cation composition of the incubation medium similar to that observed with UEA1. The effects of UEA1 and Bric 54 were not additive. These observations suggest that the Ca2+ influx induced by UEA1 and Bric 54 is mediated by the same transport pathway. Since both the lectin and the antibody exhibit ABH blood group specificity, it appears reasonable to conclude that ABH antigens can serve as recognition sites for activation of a Ca2+ influx pathway in human erythrocytes, which is sensitive to inhibitors of the band 3 anion-exchanger.