Diisothiocyanostilbene-2,2'-disulfonate Research Articles

Effects of acetazolamide on Na+-HCO-3 cotransport in basolateral membrane vesicles isolated from rabbit renal cortex.

We evaluated the effects of acetazolamide on Na+-HCO3- cotransport in basolateral membrane vesicles isolated from the rabbit renal cortex. Na+ uptake stimulated by an imposed inward HCO3- gradient was not significantly reduced by 1.2 mM acetazolamide, indicating that acetazolamide does not directly inhibit Na+-HCO3- cotransport. 4,4'-Diisothiocyanostilbene-2,2'-disulfonate (DIDS)-sensitive Na+-base cotransport was found to be absolutely CO2/HCO3--dependent. We therefore tested whether acetazolamide-sensitive availability of HCO3- at the basolateral membrane could be rate-limiting for Na+-base cotransport under some conditions. In the presence of a CO2/HCO3- buffer system but absence of an initial HCO3- gradient, Na+ influx was stimulated fivefold by an outward NH4+ gradient. This stimulation of Na+ influx by an outward NH4+ gradient was inhibited greater than 75% by 0.6 mM acetazolamide, suggesting that acetazolamide blocked the ability of the NH4+ gradient to generate an inward HCO3- gradient. In the presence of an inward HCO3- gradient, Na+ influx was inhibited greater than 70% by an inward NH4+ gradient. This inhibition of Na+ influx was reduced to only 35% by 0.6 mM acetazolamide, suggesting that acetazolamide blocked the ability of NH4+ to collapse the inward HCO3- gradient. Similarly, Na+ influx in the presence of an inward HCO3- gradient was inhibited greater than 80% by an outward acetate gradient, and this inhibition was reduced to only 50% by acetazolamide. Thus, acetazolamide caused either inhibition or stimulation of Na+ uptake depending on the conditions with respect to pH and HCO3- gradients. The indirect interaction of acetazolamide with the basolateral membrane Na+-HCO3- cotransport system may be an important mechanism underlying inhibition of proximal tubule acid secretion by this agent.

Characterization of chloride channels in membrane vesicles from the kidney outer medulla

The basolateral membrane of the thick ascending loop of Henle (TALH) of the mammalian kidney is highly enriched in Na+/K+ ATPase and has been shown by electrophysiological methods to be highly conductive to Cl-. In order to study the Cl- conductive pathways, membrane vesicles were isolated from the TALH-containing region of the porcine kidney, the red outer medulla, and Cl- channel activity was determined by a 36Cl uptake assay where the uptake of the radioactive tracer is driven by the membrane potential (positive inside) generated by an outward Cl- gradient. The accumulation of 36Cl- inside the vesicles was found to be dependent on the intravesicular Cl- concentration and was abolished by clamping the membrane potential with valinomycin. The latter finding indicated the involvement of conductive pathways. Cl- channel activity was also observed using a fluorescent potential-sensitive carbocyanine dye, which detected a diffusion potential induced by an imposed inward Cl- gradient. The anion selectivity of the channels was Cl- greater than NO3- = I- much greater than gluconate. Among the Cl- transport inhibitors tested, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPAB), 4,4'-diisothiocyano-stilbene-2,2'-disulfonate (DIDS), and diphenylamine-2-carboxylate (DPC) showed IC50 of 110, 200 and 550 microM, respectively. Inhibition of 36Cl uptake by NPPAB and two other structural analogues was fully reversible, whereas that by DIDS was not. The nonreactive analogue of DIDS, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), was considerably less inhibitory than DIDS (25% inhibition at 200 microM). The irreversible inhibition by DIDS was prevented by NPPAB, whereas DPC was ineffective, consistent with its low inhibitory potency.(ABSTRACT TRUNCATED AT 250 WORDS)

PH homeostasis in promyelocytic leukemic HL60 cells.

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.

Effect of stilbenedisulfonate binding on the state of association of the membrane-spanning domain of band 3 from bovine erythrocyte membrane

The membrane-spanning domain of bovine band 3, the anion transport protein of erythrocyte membrane, was purified in the presence of nonaethyleneglycol lauryl ether (C 12E 9) and the effect of a covalent attachment of 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS), a potent transport inhibitor, on the state of association of the domain isolated (the 58 kDa fragment) was studied via gel filtration, gel electrophoresis and sedimentation velocity experiments. It was indicated that the DIDS-unlabeled fragment in C 12E 9 solution forms heterogeneous aggregates which are larger in size than the dimer. This contrasted with the behavior that bovine band 3 is present as dimers or tetramers in the same medium (Nakashima and Makino (1980) J. Biochem. 88, 933–947). When DIDS was covalently attached, the fragment was present as a single molecular species which was indicated to be a dimer by molecular weight determination. The secondary structure of the fragment was not affected by DIDS. The change in the state of association caused by the DIDS-binding was also found in the presence of sucrose monolaurate (SE12), which was a more potent detergent for extraction of the 58 kDa fragment from membranes than C 12E 9. However, the complex with SE12 was extremely unstable.

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3.

A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682-1688, 1986) found that C1-/HCO-3 exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4'-diisothiocyano-2,2'-disulfonic stilbene), with a K1 similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4'-dibenzamido-2,2'-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (Ks1 = 93 +/- 24 nM) and a lower affinity site (Ks2 = 430 +/- 260 nM), which are closely similar to values for the red cell of 110 +/- 51 nM for the high-affinity site and 980 +/- 200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon, J. Gen. Physiol. 81:421-449, 1983). When Cl- replaces citrate in the buffer, the two sites collapse into a single one with Ks1 = 1500 +/- 400 nM, similar to the single Ks1 = 1200 +/- 200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon, J. Membrane Biol. 89:211-223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 microM-1 are characterized by a fast process, tau = 0.14 +/- 0.03 sec, similar to tau = 0.12 +/- 0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell 'band 3' closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.

Studies on the interaction of matrix-bound inhibitor with Band 3, the anion transport protein of human erythrocyte membranes

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4′-isothiocyanostilbene 2,2′-disulfonate (SITS; K i = 10 μ M)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4′-aminostilbene 2,2′-disulfonate (BADS; K i = 2 μ M ), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4°C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4°C, the weakly bound form was slowly converted to the strongly bound form. At 37°C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4′-diisothiocyanostilbene 2,2′-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37° C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37°C.

Affinity labeling of hemoglobin with 4,4'-diisothiocyanostilbene-2,2'-disulfonate: covalent cross-linking in the 2,3-diphosphoglycerate binding site.

The bifunctional reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) reacts with hemoglobin to give various products whose properties are dependent on the ligation state of the protein during the reaction. A major product obtained after reaction of (carbonmonoxy)hemoglobin with DIDS was a high oxygen affinity derivative [P50 = 1.4 mmHg, control P50 = 6 mmHg; 50 mM [bis(2-hydroxyethyl)-amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.4, 0.1 M Cl-, 25 degrees C] which contained two molecules of DIDS per tetramer resulting from adduct formation at each beta-chain amino terminus. In contrast, a major product of the reaction of deoxyhemoglobin with DIDS consisted of hemoglobin which had incorporated one molecule of DIDS per tetramer and was cross-linked between the beta-chain amino termini. This cross-linked hemoglobin was found to have a greatly decreased O2 affinity (P50 = 28 mmHg). Inhibition of the T to R transition due to the structural constraint produced by cross-linking the beta amino termini is likely to be a major factor in the decreased O2 affinity of this product. The structural and functional properties of this molecule make it a potential candidate for a cell-free blood substitute.

Identification, purification, and characterization of a stilbenedisulfonate binding glycoprotein from canine kidney brush border membranes. A candidate for a renal anion exchanger.

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.

Chemical Modification of Hemoglobin with 4,4′-Diisothiocyanostilbene-2,2′-Disulfonate (DIDS): Initial Studies of a Potential Blood Substitute

Several bifunctional 2,3-diphosphoglycerate (DPG) analogs will cross-link residues in the DPG binding site and prevent subunit dissociation of undenatured hemoglobin. By selecting reagents of proper structure and appropriate reaction conditions, it is possible to produce modified hemoglobins with oxygen equilibria shifted towards either the low affinity or the nigh affinity state. One such reagent is 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS). Depending upon the specific reaction conditions, different DIDS products of hemoglobin result. The major deoxyhemoglobin reaction product is a molecule in which one DIDS per tetramer is incorporated, cross-linking between the two β chain amino terminal valyl residues. This derivative, Hb(DIDS)-T, has greatly decreased O2 affinity with a P50 of 24 mmHg compared to P50 of 5 mmHg for normal hemoglobin (50 mM bis-Tris, pH 7.4, 0.1 M Cl-, and 25°C). Its cooperativity is reduced moderately (nmax = 2.1) but the addition of 2,3-DPG has no effect on its oxygen affin...

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.

Further evidence for coupling of sodium and proton movements in dog red blood cells

Using 4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS) and tributyltin the sodium transport pathway activated by shrinkage in dog red blood cells is shown to behave as expected for an electroneutral Na +/H + exchanger. When the driving forces for sodium and protons are equal, flow through the pathway stops. Amiloride inhibits the shrinkage-induced Na +/H + exchange.

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 35SO2−4 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.

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.

Labeling of human erythrocyte band 3 with maltosylisothiocyanate. Interaction with the anion transporter.

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.

Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors.

The band 3 protein of red cells is a transmembrane ion transport protein that catalyzes the one-for-one exchange of anions across the cell membrane. 35Cl NMR studies of Cl- binding to the transport sites of band 3 show that inhibitors of anion transport can be grouped into three classes: (1) transport site inhibitors (examined in this paper), (2) channel-blocking inhibitors (examined in the second of three papers in this issue), and (3) translocation inhibitors (examined in the third of three papers in this issue). Transport site inhibitors fully or partially reduce the affinity of Cl- for the transport site. The dianion 4,4'-di-nitrostilbene-2,2'-disulfonate (DNDS) and the arginine-specific reagent phenylglyoxal (PG) each completely eliminate the transport site 35Cl NMR line broadening, and each compete with Cl- for binding. These results indicate that DNDS and PG share a common inhibitory mechanism involving occupation of the transport site: one of the DNDS negative charges occupies the site, while PG covalently modifies one or more essential positive charges in the site. In contrast, 35Cl NMR line broadening experiments suggest that 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) leaves the transport site partially intact so that the affinity of Cl- for the site is reduced but not destroyed. This result is consistent with a picture in which DIDS binds near the transport site and partially occupies the site.

Mechanism of coupling between Cl − and OH − transport in renal brush-border membranes

The coupling mechanism for Cl − and H +/OH − transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H + and Cl − gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H + gradients and Cl −, gluconate, or sulfate gradients, each with and without a K +/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO 3 − or 5 mM formate. Rate of H +/OH − transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl − caused enhanced H +/OH − transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO 4 2−, or HCO 3 −, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl −/OH − transport was not inhibited by 100 μM DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) or 100 μM DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate). SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) was found to be a protonphore at concentrations > 500 μM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na +-H + exchange (H + flux = 107 ± 9 nmol/s per mg, 100 mM Na +) and electroneutral, DBDS inhibitable Cl −-HCO 3 − exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl −-OH − or HCO 3 − exchange does not measurably contribute to Cl − transport in the proximal tubule brush border. Cl −-formate exchange with formic acid recyling appears to be the only electroneutral coupling mechanism between Cl − and OH − transport demonstrable in renal brush-border membrane vesicles.

Relationship of the decreases in protein synthesis and intracellular Na+ during friend murine erythroleukemic cell differentiation.

The earliest known ionic event during Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway is a 45% drop in intracellular sodium concentration ([Na+]i) due to a decrease in Na+ influx (Lannigan, D. A., and Knauf, P. A. (1985) J. Biol. Chem. 260, 7322-7324). We have analyzed the mechanism of the decreased Na+ influx. The Na+ influx in uninduced cells was insensitive to dimethylamiloride, bumetanide, and diisothiocyanostilbene disulfonate. The intracellular pH (pHi) did not change up to 15 h after dimethyl sulfoxide induction, at which time Na+ influx has decreased by approximately 40%; thus, the decrease in Na+ influx is not coupled to a change in pHi. A substantial amount of the decrease in Na+ influx seems to result from a drop in amino acid-dependent Na+ transport. This reduction in amino acid-dependent Na+ influx reflects a decrease in net Na+ influx rather than solely in Na+/Na+ exchange and can account for an appreciable portion of the reduction in [Na+]i seen during differentiation. The drop in amino acid-dependent Na+ influx could not be explained by membrane depolarization but was correlated with a decrease in protein synthesis. Inhibition of protein synthesis in uninduced cells by cycloheximide also caused a decrease in Na+ influx. We conclude that during differentiation the reduction in protein synthesis decreases amino acid-dependent Na+ influx which in turn causes a drop in [Na+]i leading to a reduction in the Na+/K+ pump rate.

Characterization of the anion transport channel protein in human erythrocytes. Induced circular dichroism of inhibitors bound to the anion transport channel

The induced circular dichroism (CD) of erythrocyte ghosts with anion-transport inhibitors has been studied. A ghost-EITC (eosin 5-isothiocyanate) system shows an induced CD spectrum at the wavelength region corresponding to the absorption bands of EITC. Also a ghost-EMI (eosin 5-maleimide) system shows induced CD, but has bands of opposite sign to the EITC system. From the change of the CD intensity, the number of EITC molecules bound to one erythrocyte was estimated to be about 1.4·10 6, being close to the number of band 3 copies per ghost. The CD spectra of EITC and EMI systems show that a configurational structure of the moiety anchoring the EMI molecule is the reverse to that of EITC. The preferred conformation of bound EITC may be twisted in a right-handed sense. From the signs of the induced CD bands in ghost-stilbene disulfonate systems, the chirality of twisted stilbene derivatives seems to be a left-handed sense, as is the case for the EMI derivative. The CD spectra of EITC in the presence of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) shows that the binding site of EITC may not be identical with that of DIDS. The results observed in this study reflect the ternary arrangement of the functional amino groups in anion recognition sites.

Concentration-dependent effects of disulfonic stilbenes on colonic chloride transport.

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to determine effects of the disulfonic stilbenes 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and the diuretic furosemide on unidirectional and net Cl fluxes. Results from these studies reveal that SITS (1 mM) added to either the serosal or mucosal bathing solution reduced both unidirectional Cl fluxes with no significant change in net Cl flux. The effects of SITS do not appear to be mediated by an effect on the shunt permeability since SITS (1 mM) did not alter either the intercept or slope of the Na concentration dependence of the serosal-to-mucosal Na flux. Furosemide (1 mM) decreased the serosal-to-mucosal Cl flux without altering short-circuit current (Isc) when added to the luminal bathing solution and reduced both unidirectional fluxes and increased Isc when added to the serosal bathing solution. DIDS (0.5 mM) added to the luminal bathing solution did not alter unidirectional Cl fluxes or Isc. However, serosal addition of DIDS produced dose-dependent changes in Cl transport. At 5 microM DIDS reduced the mucosal-to-serosal Cl flux without altering the serosal-to-mucosal flux or Isc. At 50 microM DIDS reduced the mucosal-to-serosal Cl flux and increased Isc, and at 0.5 mM DIDS increased the serosal-to-mucosal Cl flux, reduced the mucosal-to-serosal Cl flux, and increased Isc and transepithelial conductance. The effect of 0.5 mM DIDS on Isc was reduced by Ca removal from the serosal bathing solution and by the loop diuretics furosemide and bumetanide.(ABSTRACT TRUNCATED AT 250 WORDS)

Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl-HCO3 exchange.

An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4',5'-dimethyl-5(and -6)-carboxyfluorescein by exposing them to the dye's permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3(-)-free Ringer's solution, pHi recovered exponentially from acid loads applied by NH+4 prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer's solution, external Cl- removal caused pHi to reversibly increase by approximately equal to 0.3. This pHi increase was substantially reduced by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by approximately equal to 0.2. This pHi change was greatly diminished by DIDS, by removal of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi.