Disulphonic Stilbene Research Articles

On the mechanism of transport of salicylate and p-hydroxybenzoic acid across human red cell membranes.

The pH-dependence of efflux of salicylate and p-hydroxybenzoic acid (PHB) from human red cells indicates that the un-ionized species penetrates the membrane. No effect of the anion channel blocker 4,4'-diisothiocyano-2,2'-disulphonic stilbene was observed. The temperature-dependence of efflux suggests that the energy barrier to transport of salicylate and PHB is the transfer of the acids from water into the membrane, rather than transport through the membrane interior. Intracellular binding of both acids was found to be pH-dependent.

Mechanisms of basolateral base transport in the renal proximal tubule.

Renal proximal tubules absorb HCO3- by secretion of H+ into the tubular lumen. This paper focuses on the mechanisms of HCO3- exit across the basolateral cell membrane. The major exit pathway is rheogenic sodium bicarbonate co-transport. This system transports Na+ and HCO3-, but not Cl-, in obligatory coupling at a fixed overall stoichiometry of three HCO3- to one Na+. The fact that HCO3- flux is reduced after inhibition of cytoplasmic and/or membrane-bound peritubular carbonic anhydrase indicates that HCO3- is not transported as such but is split during permeation into its buffer subspecies from which it is regenerated on the other side of the membrane. Since flow of OH- or of H+ (in opposite directions) can be excluded, it appears most likely that one HCO3- and one CO3(2-) move together with one Na+. Besides carbonic anhydrase inhibitors, disulphonic stilbenes and harmaline are known to block the co-transporter. In addition to rheogenic Na+ (HCO3-)3 co-transport, Na+-dependent and Na+-independent electroneutral Cl-/HCO-3 exchange have been identified. The latter mechanisms are particularly important in S3 segments of proximal tubule where Na+ (HCO3-)3 co-transport is missing. Further mechanisms which operate in parallel, but at lower rates, are electroneutral SO4(2-)/HCO3- exchange and, in some species, lactate/HCO3- exchange. Moreover, there may be some uncoupled OH- flux and it is reasonable to assume that OH- (or H+) flux is involved in the transport of dicarboxylic acids across the basolateral cell membrane.

The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates

A longevity gene called Indy (for 'I'm not dead yet'), with similarity to mammalian genes encoding sodium-dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane, but the specific transport mechanism mediated by INDY was not identified. To test whether INDY functions as an anion exchanger, we examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [14C]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [14C]citrate efflux was sensitive to inhibition by DIDS (4,4'-di-isothiocyano-2,2'-disulphonic stilbene), as demonstrated previously for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, efflux of [14C]succinate from INDY-expressing oocytes was stimulated by external citrate, alpha-oxoglutarate and fumarate, indicating succinate-citrate, succinate-alpha-oxoglutarate and succinate-fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pH(o)-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. We conclude that INDY functions as an exchanger of dicarboxylate and tricarboxylate Krebs-cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favours lifespan extension.

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit.

1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.

P2X7 receptors activate protein kinase D and p42/p44 mitogen-activated protein kinase (MAPK) downstream of protein kinase C.

Protein kinase D (PKD), also called protein kinase Cmu (PKCmu), is a serine/threonine kinase that has unique enzymic and structural properties distinct from members of the PKC family of proteins. In freshly isolated rat parotid acinar salivary cells, extracellular ATP rapidly increased the activity and phosphorylation of PKD. The stimulation by ATP required high concentrations, was mimicked by the P2X(7) receptor ligand BzATP [2'- and 3'-O-(4-benzoylbenzoyl)ATP], and was blocked by Mg(2+) and 4,4'-di-isothiocyano-2,2'-stilbene disulphonate (DIDS), suggesting that activation of PKD was mediated by P2X(7) receptors, which are ligand-gated non-selective cation channels. Phorbol ester (PMA) and the activation of muscarinic and substance P receptors also increased PKD activity. PKC inhibitors blocked ligand-dependent PKD activation and phosphorylation, determined by in vitro phosphorylation studies and by phospho-specific antibodies to two activation loop sites (Ser(744) and Ser(748)) and an autophosphorylation site (Ser(916)). ATP and BzATP also increased the tyrosine phosphorylation and activity of PKCdelta, and these stimuli also increased extracellular signal-regulated protein kinase (ERK) 1/2 activity in a PKC-dependent manner. PKD activation was not promoted by pervanadate (an inhibitor of tyrosine phosphatases) and was not blocked by PP1 (an inhibitor of Src family kinases) or genistein (a tyrosine kinase inhibitor), suggesting that tyrosine kinases and phosphatases did not play a major role in PKD activation. P2X(7) receptor-mediated signalling events were not dependent on Ca(2+) entry. These studies indicate that PKC is involved in cellular signalling initiated by P2X(7) receptors as well as by G-protein-coupled receptors, and demonstrate that PKD and ERK1/2 are activated in similar PKC-dependent signalling pathways initiated by these diverse receptor types.

Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells.

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

Inhibition of H,K-ATPase and Na,K-ATPase by DIDS, a disulphonic stilbene derivative.

Disulphonic stilbenes are effective inhibitors of an anion exchanger which is present in the plasma membranes of many cells (Cabantchik et al. 1978). In the present study, the effects of 4,4'-diisothiocyano-2,2'-disulphonic stilbene acid (DIDS) on the transport activity of the hydrochloric acid pump isolated from pig stomach (H,K-ATPase, EC 3.6.1.36) were tested. Half-maximal inhibition of proton transport carried out by the H,K-ATPase in the isolated vesicles was observed at micromolar concentrations of DIDS. The effects of DIDS on the adenosine-trisphosphatase and p-nitrophenyl-phosphatase activities of isolated H,K-ATPase were also studied and compared with those of the kinetically and structurally related Na,K-ATPase (EC 3.6.1.37). Half-maximal inhibition of the enzymatic activities of both enzymes were observed in the micromolar range of DIDS. The lipid bilayer of the gastric vesicle membrane is highly asymmetric and the original cytosolic side is facing the outside of the vesicle. Since DIDS does not readily cross the membrane, it is most likely that DIDS exerts its inhibitory effects by modifying the transport ATPases on their cytosolic sides.

Functional expression of basolateral Cl-/HCO3- exchange from rat jejunum in Xenopus laevis oocytes.

Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl-/HCO3- antiport was investigated by means of 36Cl- uptake. Two days after injection of 50 ng of poly(A)+ RNA, Cl- uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0.2 mM DIDS, whereas endogenous Cl- uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl- uptake was detected with mRNA size fraction of about 2-4 kb in length. The expressed Cl- uptake can occur against a Cl- concentration gradient and is unaffected by the known Cl- channel blocker anthracene-9-carboxylic acid. Cl- transport mechanism has properties similar to jejunal basolateral. Cl-/HCO3- exchange with regard to Na+ dependence.

New Findings Concerning Vertebrate Porin

Eukaryotic porin can be considered to be a good candidate for forming the channel component of the protein complex which, depending on the approach used, may realize its expression either as the outwardly-rectifying depolarization-induced chloride channel or as the volume-sensitive organic osmolyte-anion channel. As a basis for this proposition, we point to a series of correspondences in properties between mammalian porin and the ORDIC channel complex. Specifically, mammalian porin is expressed in the plasmalemma of different cells and chloride channels can be blocked by anti-human porin antibodies in astrocytes and endothelial cells. There is an indication of colocalisation of human porin and the cystic fibrosis (CF) gene product, CFTR, in the apical region of epithelial cells. The primary structure of porin from a CF patient was found to be normal. Cytosol and amniotic fluid fractions influence the channel characteristics of mammalian porin. Channel-active mammalian porin binds ATP and the stilbene disulphonate grouping of the chloride channel inhibitor DIDS. Human porin in black membranes is a pathway for taurine, and biogenic polyamines reduce the voltage dependence of human porin. Assuming the relationship between human porin and the ORDIC channel/VSOAC complex, studies on plasmalemma-integrated human porin have a relevance for CF research. In addition, we refer to a case study on a child with encephalomyopathy in which porin could not be detected using monoclonal anti-human porin antibodies. Our studies were based on purified and sequenced human porin from different cells and from different cell compartments. In addition, we raised antibodies against mature human porin or synthetic parts of the molecule. This provided a firm foundation for our topochemical work with which we were able to establish the multi-topological expression of eukaryotic porin channels. The data are summarized and discussed.

Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.

1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region.

An amino acid channel activated by hypotonically induced swelling of Leishmania major promastigotes.

Leishmania promastigotes accumulate amino acids (AAs) by an uphill transport mechanism that is dependent on membrane potential. The accumulated AAs provide the cell with an osmotic reservoir that can be utilized for osmoregulation. Exposure of Leishmania promastigotes to hypotonic media induced a rapid release of AAs that was proportional to the imposed osmotic gradients and independent of the ionic strength or the presence of Cl-, K+, Na+ or Ca2+ in the medium. The hypotonically activated AA release pathway was of relatively low chemical specificity. The solutes released included most of the zwitterionic and anionic AAs, predominantly alanine, hydroxyproline, glycine and glutamic acid, whereas cationic AAs were virtually excluded. AA release was markedly blocked by classical anion transport inhibitors such as the disulphonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) and its dihydro derivative H2DIDS and others, by restoration of isotonicity or by lowering the temperature (4 degrees C). The temperature profile of AA release showed a low energy of activation (Ea 46 +/- 1.3 (S.E.M.) kJ/mol) in the range 15-30 degrees C and a very high Ea (147 +/- 8 kJ/mol) in the range 4-15 degrees C. Parasites exposed to hypotonic media containing AAs also showed a hypotonically stimulated AA uptake under favourable solute concentration gradients. This uptake was analogous for L- and D-isomers of threonine. After hypotonic exposure, cells underwent a depolarization that was largely prevented by anion transport blockers. On the basis of all these results we propose that after hypotonic stress Leishmania promastigotes restore their internal volume by a regulated release of AAs, which involves activation of channels that allow the passage of both neutral and anionic AAs and possibly other anionic substances.

GABA-mediated inhibition of pacemaker neurons of rostral ventrolateral medulla by clonidine in vitro

In vitro, clonidine (1, 10, or 30 μM) dose-dependently and reversibly inhibited tonically active pacemaker neurons that correspond to the relatively fast-conducting reticulospinal vasomotor neurons of the rostral ventrolateral reticular nucleus of the medulla oblongata in rats. The clonidine-induced membrane-hyperpolarizing response of these neurons was abolished by either tetrodotoxin, bicuculline, a GABA A receptor antagonist, or 4,4′-diisothiocyano-1,2′-disulphonic stilbene acid, a Cl − channel blocker. We conclude that the clonidine-induced inhibition of the pacemaker neurons of the rostral ventrolateral reticular nucleus is indirect, mediated by synaptic release of γ-aminobutyric acid (GABA) or GABA-like substances, which activate Cl − channels of the pacemaker neurons of the rostral ventrolateral reticular nucleus.

Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein.

4,4'-Diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS), a bifunctional inhibitor of anion exchange in erythrocytes, reacts with Lys-539 in band 3 at neutral pH and crosslinks to Lys-851 at alkaline pH. The accessibility of H2DIDS-labelled band 3 was determined using an anti-H2DIDS antibody and proteolysis. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that a polyclonal antibody raised against H2DIDS-labelled keyhole limpet hemocyanin bound a variety of stilbene disulphonates in the following order of affinities, H2DIDS having the highest affinity: H2DIDS > 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) > 4-acetamido-4'-isothiocyanostilbene-2,2'disulphonate (SITS) > 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) > 4,4'-diaminostilbene-2,2'-disulphonate (DADS). The antibody readily detected mono- or bifunctionally H2DIDS-labelled band 3 and proteolytic fragments on immunoblots. H2DIDS attached to Lys-539 is retained in a 7.5 kDa membrane-associated peptide after papain treatment of ghost membranes while the sequence around Lys-851 is more accessible. The band 3 proteolytic fragments protected by the membrane from proteolysis remained associated as a specific complex with a Stokes radius slightly smaller than the dimeric membrane domain after solubilization in detergent solution and retained 82% of the amino acid content of the membrane domain. Circular dichroism (CD) measurements of this H2DIDS-labelled complex showed that it had a very high helical content (86%). The loops connecting the transmembrane segments in H2DIDS-labelled band 3 are therefore not required to maintain transmembrane helix-helix interactions. Denatured band 3 prelabelled with H2DIDS was more readily immunoprecipitated with the anti-H2DIDS antibody than was native band 3 in detergent solution. Deglycosylation of band 3 or proteolytic cleavage of the extramembranous loops did not enhance immunoprecipitation of H2DIDS-labelled band 3. The stilbene disulphonate inhibitor site is therefore relatively inaccessible and is bound by a bundle of helices in the native band 3 protein.

Characterization of amino-acid transport in pig erythrocytes.

Conference Article| May 01 1993 Characterization of amino-acid transport in pig erythrocytes Joanne C. Dyke; Joanne C. Dyke 1School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WVI 4LY, U.K. Search for other works by this author on: This Site PubMed Google Scholar Daron A. Fincham Daron A. Fincham 1School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WVI 4LY, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (2): 307–309. https://doi.org/10.1042/bst0210307 Article history Received: December 16 1992 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Joanne C. Dyke, Daron A. Fincham; Characterization of amino-acid transport in pig erythrocytes. Biochem Soc Trans 1 May 1993; 21 (2): 307–309. doi: https://doi.org/10.1042/bst0210307 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: DIDS, 4,4′-isothiocyano-2,2′-stilbene disulphonate, S, extracellular amino acid concentration, v, initial rate of uptake This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells.

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

A red cell band 3 variant with altered stilbene disulphonate binding is associated with the Diego (Dia) blood group antigen.

1. We have shown that the Dia antigen of the Diego blood group system is associated with the presence of red cell band 3 Memphis, but not all band 3 Memphis samples carry the Dia antigen. 2. The band 3 Memphis associated with the Dia antigen was covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) more readily than was normal band 3 or band 3 Memphis not associated with the Dia antigen. This altered reactivity with H2DIDS has previously been noted for a band 3 Memphis sub-type designated variant 2. 3. This is the first example of a band 3 polymorphism associated with an antigenic change in the extracellular region of the band 3 polypeptide and with altered H2DIDS binding.

Pharmacological profile of inhibition of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein efflux in human HCT-8 intestinal epithelial cells

The efflux of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from human HCT-8 intestinal epithelial cultured cells was time-dependent, and after 5 hr 76% of the fluorochrome was extracellular. The pharmacological profile for inhibition of this efflux has been investigated, focusing on agents which modulate anion transport. BCECF efflux was sensitive to inhibition by 0.5 mM indomethacin (50% inhibition at 20 μM) which reduced efflux to values observed after depletion of ATP with azide and 2-deoxy-D-glucose. Indomethacin inhibition of BCECF efflux was not reversed with prostaglandin. The stilbene derivatives 4-acetamido-4′-isothiocyano-2-2′-disulphonic stilbene and 4,4′-diisothiocyano-2, 2′-disulphonic stilbene only resulted in partial inhibition of BCECF efflux, even at 1 mM. Furosemide, bumetamide, probenecid and 5-nitro-2-(3-phenylpropyl-amino)-benzoate only reduced BCECF efflux at 1 mM. The cationic agent vinblastine was as active as indomethacin as an inhibitor of BCECF efflux (50% inhibition with 10 μM) while actinomycin D was also a good inhibitor (50% inhibition with 100 μM). Several other cationic agents, including nifedipine, amiloride and reserpine, were ineffective as inhibitors of BCECF efflux in concentrations up to 1 mM. Thus, the pharmacological profile for inhibition of BCECF efflux does not fully equate with any recognised transport system. Agents such as cytochalasin B and chloroquine did not effect BCECF efflux suggesting accumulation and subsequent discharge from endosomes is not a pathway for secretion. BCECF may be a substrate for a cellular secretory detoxifying system in epithelial cells.

Inhibition of l-lactate transport and band 3-mediated anion transport in erythrocytes by the novel stilbenedisulphonate N,N,N′,N′-tetrabenzyl-4,4′-diaminostilbene-2,2′-disulphonate (TBenzDS)

(1) The synthesis of the novel stilbenedisulphonate N,N,N′,N′-tetrabenzyl-4,4′-diaminostilbene-2,2′-disulphonate (TBenzDS) is described, and its interaction with the lactate transporter and band 3 protein of erythrocytes investigated. At 10% haematocrit the IC 50 (concn. required for 50% inhibition) for inhibition of transport of 0.5 mM l-lactate into rat erythrocytes at 7°C was approx. 1.6 μM, as low as any other inhibitor of the transporter. In human erythrocytes at 10% haematocrit the IC 50 value was increased from approx. 3 μM to 9 μM upon raising the temperature from 7°C to 25°C. (2) TBenzDS inhibited transport of l-lactate into rat erythrocytes in a manner that was competitive with the substrate, as is the case for some other stilbene disulphonate derivatives (Poole, R.C. and Halestrap, A.P. (1991) Biochem. J. 275, 307–312). (3) Increasing the haematocrit from 5 to 20% caused a 3-fold increase in the IC 50 value for inhibition of l-lactate transport in rat erythrocytes. (4) TBenzDS was found to bind to erythrocyte membranes, with a partition coefficient ( P m) of 6000–7000 under all conditions tested. (5) TBenzDS also inhibited band 3-mediated sulphate transport in rat erythrocytes; 50% inhibition required approx. 2.5 μM TBenzDS for cells at 10% haematocrit. (6) TBenzDS is fluorescent, and an enhancement of this fluorescence occurs upon addition of BSA or erythrocyte membranes. The fluorescence enhancement caused by erythrocyte membranes is due to binding of the inhibitor to the band 3 protein at the same site as the stilbenedisulphonate 4,4′-diisothiocyanodihydrostilbene-2,2′-disulphonate (H 2DIDS).

Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of Cl channels by organic and inorganic blockers in vascular smooth muscle cells.

The effects of Cl channel blockers on large-conductance (LC-type) Cl channels of cultured vascular smooth muscle cells (VSMC) were studied in order to characterize the channel pharmacologically. Intracellular disulphonic stilbene derivatives, such as 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2'-disulphonic acid (SITS) inhibited Cl channel activity in a dose-dependent manner. An obvious inhibitory effect of DIDS in this condition was obtained at concentrations higher than 5 microM, and the complete inhibition was obtained at around 100 microM, which was almost 10 times less than the effective dose of SITS. The inhibitory effect of DIDS was reversible at a drug concentration of lower than 50 microM. Single-channel conductance decreased as the concentration of DIDS increased. This decrease in the conductance was a consequence of unresolved openings of the channel due to fast blocking and unblocking rates of the drug. The Cl channel was also obviously inhibited by extracellular DIDS at a concentration of 1 mM. In addition, in cell-attached patches, 500 microM DIDS applied extracellularly inhibited Cl channel activated by the application of polymyxin B. We also investigated the effect of Zn on Cl channels in VSMC. Intracellular Zn dose dependently and reversibly blocked the channel at the positive intracellular potential range, whereas at the negative intracellular potential range it did not block the channel activity. Results in this study suggest the diversity of Cl channels among various tissues.