Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Disulfonic stilbene derivatives open the Ca2+ release channel of sarcoplasmic reticulum

Ca2+ channels of isolated sarcoplasmic reticulum were incorporated into a planar lipid bilayer and their pharmacological properties were studied. The results show that the channel is a Ca2+-induced Ca2+ release channel like that observed in skinned muscle fibers and isolated vesicles. (i) The open channel probability was increased by the addition of micromolar amounts of Ca2+ to the cis (myoplasmic) side and further increased by millimolar ATP. (ii) The channel was closed by millimolar Mg2+ and micromolar ruthenium red. We found that two disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetoamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), when added to the cis side open the channel and lock it irreversibly at open without changing the single channel conductance. Ca2+ efflux from SR vesicles was also enhanced by SITS and DIDS, as monitored by a tracer assay. Further, Ag+ activated the channel transiently. These results suggest that certain amino and SH residues play important roles in gating the Ca2+ channel.     

Evidence that the erythrocyte calcium pump catalyzes a Ca2+:nH+ exchange

Treatment of whole erythrocytes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) results in inhibition of ATP and phosphate-dependent Ca2+ transport in subsequently prepared inside-out vesicles (IOV). Accumulation of phosphate into IOV in the presence of Ca2+ is virtually abolished by prior DIDS treatment, consistent with the presumed inhibition of the band III anion-exchange protein by this agent. No inhibition of Ca2+-activatable ATP hydrolysis is observed following DIDS treatment when open membranes are used to prevent development of ion gradients. This indicates that DIDS does not affect the inherent ATPase activity of the calcium pump (Waisman, D. M., Smallwood, J., Lafreniere, D., and Rasmussen, H. (1982) FEBS Lett. 145, 337-340). In IOV prepared from untreated cells, ATP-dependent Ca2+ uptake is stimulated by phosphate, sulfate, or chloride. Rates of Ca2+ uptake into DIDS-IOV are not increased by these anions. Lipid-permeable organic acids such as acetate, however, do promote Ca2+ transport in DIDS-IOV. Lipophilic anions incapable of transporting protons into the vesicle interior (nitrate and thiocyanate) support sustained uptake only when the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone is also added. These results support a model of the (Ca2+-Mg2+)-ATPase as a pump exchanging Ca2+ for protons, not transporting Ca2+ alone. Band III protein appears to promote Ca2+ transport in the presence of phosphate, sulfate, or chloride by exchanging external anion for the accumulating OH- (or HCO3-) produced by the calcium pump.     

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)     

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.     

Comparison of folic acid uptake characteristics by human placental choriocarcinoma cells at acidic and physiological pH

The aim of this work was to characterize the placental uptake of folic acid from the maternal circulation. Using 2 human trophoblast cell lines (BeWo and JAR), we verified that uptake of 3H-folic acid was pH-dependent, increasing significantly with decreasing extracellular pH. In BeWo cells, uptake of 3H-folic acid at pH 5.5 was (i) Na+-independent; (ii) inhibited by folic acid, 5-methyltetrahydrofolate (5-MTHF), and methotrexate (MTX); (iii) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS); (iv) inhibited by the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP); (v) not inhibited by blockers of receptor-mediated endocytosis (cytochalasin D and monensin); (vi) trans-inhibited by MTX and folic acid; and (vii) not affected by an anti-reduced folate transporter-1 (RFC) antibody. At pH 7.5, uptake of 3H-folic acid was (i) Na+-independent; (ii) inhibited by folic acid and MTX, but not by 5-MTHF; (iii) inhibited by SITS, but not by DIDS; (iv) not affected by FCCP; (v) inhibited by monensin (but not by cytochalasin D); (vi) trans-inhibited by folic acid (but not by MTX); and (vii) inhibited by an anti-RFC antibody. In conclusion, in BeWo cells, both RFC and receptor-mediated endocytosis seem to be involved in 3H-folic acid uptake at pH 7.5, whereas at pH 5.5, RFC and (or) a low pH-operating transporter distinct from RFC are involved.     

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.     

Regulation of the calcium release channel from skeletal muscle by suramin and the disulfonated stilbene derivatives DIDS,DBDS, and DNDS

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.     

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso- thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS- insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.     

4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid inhibits CD3-T cell antigen receptor-stimulated Ca2+ influx in human T lymphocytes

Stimulation of the CD3-T cell antigen receptor complex on T lymphocytes results in a rapid rise in intracellular calcium from both intra- and extracellular sources. The former is thought to be released from the endoplasmic reticulum in response to inositol trisphosphate, while the latter enters the cells through a membrane potential-sensitive transporter (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). In this report we show that the stilbene disulfonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), inhibited the ability of monoclonal anti-CD3 complex antibodies to stimulate an influx of calcium in the human T lymphocyte cell line, Jurkat. DIDS had no effect on either antibody binding to the receptor or receptor-stimulated phosphatidylinositol turnover. The Ki was approximately 25 microM in the presence of extracellular Cl- and 10 microM when labeling was performed in the absence of Cl-, suggesting that DIDS was competing with Cl- for binding to the cell membrane. The reduced form of DIDS, dihydroDIDS, was only 50% as effective as DIDS itself, and the monoisothiocyanate stilbene, 4-acetamido-4'-isothiocyantostilbene-2,2'-disulfonic acid, was totally ineffective, even to concentrations of 0.750 mM. Removal of extracellular Cl- also inhibited the antibody-stimulated influx of calcium. These data suggest that the function of the CD3-T cell receptor-activated calcium channel/transporter may be dependent on or regulated by extracellular Cl-.     

Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.     

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.     

Effect of disulphonic stilbenes on Ca2+ transport in smooth muscle plasma membranes

We studied the effects of two disulphonic stilbenes, 4',4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), on Ca2+ transport by plasma membrane vesicles from the circular muscle of the dog stomach. Both compounds inhibited ATP-dependent Ca2+ uptake and reduce the leak from loaded vesicles. The inhibition produced could not be significantly reduced by either permeant anions or by increasing the level of free Ca2+. The effects of DIDS could be rendered irreversible by incubating the membranes with this agent at 37 degrees C.     

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.     

An ATP transporter is required for protein translocation into the yeast endoplasmic reticulum.

The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.     

The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores

The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange.     

Inhibitor of anion transport, DIDS, releases Ca2+ from hepatic microsomes

Addition of 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) to Ca2+ loaded hepatic microsomal vesicles evoked a dose-dependent release of the accumulated Ca2+. Ca2+ uptake was also inhibited. The effects of DIDS do not seem to be due to the inhibitions of either Cl- or proton fluxes. The results indicate that DIDS inhibits Ca2+ uptake and releases Ca2+ by inhibiting the Ca2+-ATPase and the formation of the phosphorylated intermediate of the enzyme, and that it might interact with a specific site on the vesicle which is involved in the translocation of Ca2+ across the microsomal and mitochondrial membranes.     

Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids

The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.