Diisothiocyanostilbene-2,2 ' - Disulphonate Research Articles

Crucial Residue Involved in L-Lactate Recognition by Human Monocarboxylate Transporter 4 (hMCT4)

BackgroundMonocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.Experimental approachTransport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.ResultsIn hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.ConclusionsOur results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

HCO3- potentiates the cAMP-dependent secretory response of the human distal colon through a DIDS-sensitive pathway.

We used the Ussing short-circuit technique to investigate the role of HCO3- in the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent secretory response of the human distal colon. In HCO3(-)-free 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-Ringer's, forskolin (10 mumol l-1 mucosal and serosal) evoked a sustained increase in short-circuit current (Isc) (delta Isc = 24 +/- 3 microA cm-2, n = 57). However, this was only 25% of the forskolin-stimulated Isc response in HCO3(-)-Ringer's (delta Isc = 84 +/- 8 microA cm-2, n = 57). The reduced response to forskolin in HCO3(-)-free HEPES-Ringer's was not due to inhibition of the secretory mechanism by HEPES, as replacing HCO3- with a different buffer, N-tris[hydroxymethyl)methyl-2-aminoethanesulphonic acid (TES), had a similar effect and inclusion of HEPES in the HCO3(-)-Ringer's did not reduce the secretory response. Similarly, it was not due to an indirect modulation of electrogenic Cl- secretion, as the forskolin-stimulated bumetanide-sensitive Isc was comparable in the two Ringer's. Rather it was due to the activation of a HCO3(-)-dependent Isc which was inhibited by serosal 4,4'-diisothiocyano-stilbene-2,2'-disulphonate (DIDS). This DIDS-sensitive Isc was not inhibited by acetazolamide, but it was inhibited by the replacement of bathing solution Cl- with gluconate, suggesting a role for a Na(+)-dependent Cl-/HCO3- exchanger in the cAMP-dependent secretory response of the human distal colon.

Mechanisms of ischemia-induced taurine release in mouse hippocampal slices

Taurine release in the hippocampus is markedly potentiated in various cell-damaging conditions, including ischemia and excitotoxic damage produced by glutamate. The increase in the levels of taurine may provide an important protective mechanism against excitotoxicity. The mechanisms of the enhanced release were now studied in mouse hippocampal slices using a superfusion system. The basal release of [ 3 H ]taurine was significantly increased in Na +-deficient media in normal conditions, whereas the ischemia-evoked release was decreased, indicating the participation of Na +-dependent transport processes. The involvement of taurine transport carriers in the release was confirmed with the structural analogs, hypotaurine and β-alanine. These amino acids potentiated the release by trans-stimulation in normoxia. In Na +-free conditions, this heteroexchange was not discernible, the carriers not being functional without Na +. In ischemia, the marked potentiation of taurine release by hypotaurine and β-alanine further indicates that the Na +-requiring transporters also operate in ischemia. The effects of membrane disruption on taurine release due to activation of phospholipases were estimated using phospholipase and protein kinase inhibitors, which had no marked effects on hippocampal taurine release. The chloride channel blockers, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonate (SITS) and diisothiocyanostilbene-2,2′-disulphonate (DIDS), reduced the ischemia-induced release, suggesting that taurine diffusion through an anion channel is partially responsible for the enhanced release in ischemia.

The characterization of butyrate transport across pig and human colonic luminal membrane.

1. Luminal membrane vesicles (LMV) were isolated from human and pig colonic tissues. They were characterized in terms of purity and ability to transport [14C]butyrate. 2. The activity of cysteine-sensitive alkaline phosphatase, and the abundance of villin, NHE2 and NHE3 proteins, markers of the colonic luminal membrane, were significantly enriched in the LMV compared with the original cellular homogenate. The LMV were free from contamination by other cellular organelles and basolateral membranes, as revealed by the negligible presence of either specific marker enzyme activity or characteristic immunogenic protein. 3. The transport of butyrate into the luminal membrane vesicles was enhanced 5-fold at pH 5.5 compared with pH 8.0. Butyrate transport was temperature dependent, and was stimulated in the presence of an outward-directed anion gradient in the order of butyrate > bicarbonate > propionate > chloride. Kinetic analysis of increasing substrate concentration showed saturation kinetics with an apparent Km value of 14.8 +/- 3.6 mM and a Vmax of 54 +/- 14 nmol min-1 (mg protein)-1. 4. Butyrate transport was significantly reduced in the presence of short chain fatty acids (SCFA), acetate, propionate and other monocarboxylates (pyruvate and L-lactate). Butyrate uptake was inhibited by several cysteine group modifying reagents such as p-chloromercuribenzosulphonic acid (pCMBS), p-chloromercuribenzoate (pCMB), mersalyl acid and HgCl2, but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) and 4,4'-dinitrostilbene-2,2'-disulphonate (SITS). 5. The described properties of butyrate transport across the luminal pole of the colon suggest the involvement of a carrier protein, in the form of a pH-activated anion exchange process. The transporter is distinct from the erythrocyte band-3 type anion exchanger and may belong to the monocarboxylate-type transport proteins (MCT1).

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.

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.

N-terminal protein sequence analysis of the rabbit erythrocyte lactate transporter suggests identity with the cloned monocarboxylate transport protein MCT1

An improved purification for the rabbit erythrocyte lactate transporter, using aminoethyl-Sepharose chromatography, is described. The process of purification of the 40-50 kDa transporter, labelled with 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), was followed by Western blotting with anti-DIDS antibodies [Poole, R. C. and Halestrap, A. P. (1992) Biochem. J. 283, 855-862]. Fractions highly-enriched in transporter were further purified by SDS/PAGE and the 40-50 kDa DIDS-labelled polypeptide was subjected to N-terminal protein sequencing. This analysis identified the first 16 amino acids of the protein. With the exception of one conservative substitution, this protein sequence is identical to the N-terminal protein sequence predicted from a cDNA isolated from Chinese hamster ovary cells that encode a monocarboxylate transporter, MCT1 [Kim Garcia, C., Goldstein, J. L., Pathak, R. K., Anderson, R. G. W. and Brown, M. S. (1994) Cell 76, 865-873]. This observation, along with similarities in functional properties, leads us to conclude that lactate transport in rabbit erythrocytes is mediated by the MCT1 monocarboxylate transporter isoform.

Chloride and bicarbonate transport in chick embryonic red blood cells.

1. Unidirectional efflux of 36Cl- and H14CO3- from erythrocytes of 4- to 16-day-old chick embryos was measured under steady-state conditions at 37 degrees C and pH 7.7. The efflux rates were high, > 3 s-1, and were, therefore, measured by means of the continuous flow tube method. 2. At day 4 of development the range of permeability coefficients for bicarbonate and chloride (PHCO3 and PCl was 1-30 x 10(-4) cm s-1, with average values of respectively 10 x 10(-4) and 8 x 10(-4) cm s-1. However, the results can be divided into two groups, one with PHCO3 and PCl above 12 x 10(-4) cm s-1, and one with values below 5 x 10(-4) cm s-1. The same range of values was also obtained for day 6 erythrocytes, but the overlap is more conspicuous. At day 16, PHCO3 and PCl were respectively 9 x 10(-4) and 6 x 10(-4) cm s-1 (37 degrees C, pH 7.7). In adult chicken red blood cells PHCO3 and PCl were respectively 7 x 10(-4) and 4 x 10(-4) cm s-1, and in human red blood cells the respective values were 5.6 x 10(-4) and 4 x 10(-4) cm s-1. 3. Chloride self-exchange, measured at 0 degrees C, was almost completely inhibited by addition of 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) at both days 6 and 16 of embryonic development, supporting the finding that the embryonic chick erythrocytes also have a transmembrane anion exchanger similar to that of other red cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstitution of the lactate carrier from rat skeletal-muscle sarcolemma.

The lactate carrier was solubilized from purified rat skeletal-muscle sarcolemma with the detergent decanoyl-N-methyl-glucamide and the solubilized carrier was reconstituted into phospholipid vesicles. Reconstituted proteoliposomes showed a faster time course of L-lactate uptake than did protein-free liposomes. The rate of L-lactate uptake into the proteoliposomes was inhibited by the lactate-transport inhibitors p-chloromercuribenzenesulphonate, diethyl pyrocarbonate, alpha-cyano-4-hydroxycinnamate and quercetin. In contrast, the anion-exchange inhibitor DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulphonate) had almost no effect on the uptake. The extent of L-lactate uptake at equilibrium was not affected by the presence of the transport inhibitors, but was sensitive to osmotic strength. L-Lactate and pyruvate, but not D-lactate, inhibited L-lactate uptake when present at 10-fold excess. The properties of L-lactate transport in reconstituted proteoliposomes were similar to those observed in native sarcolemmal vesicles, i.e. the lactate carrier seems to retain its transport characteristics during the solubilization and reconstitution steps.

Identification and properties of pathways for K+ transport in guinea-pig and rat alveolar epithelial type II cells.

86Rb+ was used to study potassium uptake and efflux in type II pneumocytes freshly isolated from adult guinea-pig and rat lung. Both species exhibited a substantial ouabain-sensitive component of potassium influx. In rats, most of the ouabain-resistant influx was abolished by bumetanide and removal of extracellular chloride elicited no further effect. In contrast, only a proportion of the ouabain-insensitive uptake was inhibitable by bumetanide in guinea-pigs and this species showed an additional component of influx, which was chloride dependent and which was reduced by either the K(+)-H(+)-ATPase inhibitor, omeprazole, or by the stilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). The chloride-dependent component was also apparent in efflux experiments in guinea-pigs, but was absent in rats. Ouabain-insensitive ATPase activity was assayed in highly purified apical membranes from guinea-pig type II pneumocytes. This activity was inhibitable by omeprazole (apparent inhibition constant, Ki, was approximately 40 microM), was potassium dependent (apparent activation constant, Ka, was approximately 200 microM) and was doubled by the addition of nigericin. While potassium transport in rat type II cells is adequately accounted for by Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, our data suggest the additional presence of K(+)-Cl- cotransport and K(+)-H(+)-ATPase in guinea-pig type II pneumocytes. A model of how alveolar subphase acidification may occur is proposed.

PH control in rat skeletal muscle during exercise, recovery from exercise, and acute respiratory acidosis.

We used 31P magnetic resonance spectroscopy to compare the response of rat skeletal muscle to three kinds of proton load. During exercise (tetanic sciatic nerve stimulation), protons from lactic acid were buffered passively and consumed by net hydrolysis of phosphocreatine (PCr). During recovery from exercise, the pH-dependent efflux of protons produced by PCr resynthesis could be partially inhibited by amiloride or 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), implicating both sodium/proton and bicarbonate/chloride exchange, but was not inhibited by simultaneous respiratory acidosis. In early recovery, up to 30% of proton efflux was mediated by lactate/proton cotransport. During acute respiratory acidosis at rest, the eventual change in muscle pH was consistent with passive buffering and was unaffected by amiloride or DIDS, implying no significant contribution of proton fluxes.

Cell volume regulation in rat thymocytes.

1. DIOA (dihydroindenyl-oxy-alkanoic acid), a potent inhibitor of the K(+)-Cl- co-transport system, fully blocked regulatory volume decrease (RVD) in swelled rat thymocytes, with an IC50 of 2.2 +/- 0.5 x 10(-5) mol l-1 (mean +/- S.D., n = 4). Conversely, RVD was resistant to quinine, quinidine, apamin, cetiedil, amiloride, bumetanide and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonate). 2. DIOA-sensitive RVD followed mono-exponential kinetics, with t1/2 (half-lifetime) of 1-3 min and maximal capacity (Cmax) of about 55% of the initial cell swelling. Cmax and the initial rate of RVD (Vo) were both linear functions of the increase in cell volume. 3. RVD was: (i) slightly increased by replacing external Cl- by NO3-, (ii) reversed by replacing external Na+ by K+ (in the presence of external Cl-) and (iii) inhibited by cell K+ depletion. All these phenomena were blocked by DIOA (86 mumol l-1). 4. Increased membrane potassium permeability by valinomycin was unable to accelerate RVD or RVD reversal. 5. In the presence of DIOA, thymocytes responded like osmometers (the relative cell volume was a linear function of the reciprocal of the relative osmolality) in a large range of osmolalities. 6. The results strongly suggest that RVD in rat thymocytes is mediated by the K(+)-Cl- co-transport system.

Volume‐dependent K+ and Cl‐ fluxes in rat thymocytes.

1. Hypotonic stress unmasked inward and outward K+ and Cl- movements in rat thymocytes. This KCl flux stimulation was reduced by DIOA (dihydroindenyl-oxy-alkanoic acid), but not by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonate), quinidine, DPAC 144 (5-nitro-2-(2-phenylethyl-amino)-benzoic acid), bumetanide or ouabain. 2. In isotonic media (308 +/- 5 mosmol kg-1), the cells exhibited the following DIOA-sensitive fluxes: (i) a K+ efflux of 42.7 +/- 17.1 mmol (l cells.h)-1 (mean +/- S.D., n = 7), (ii) a Cl- efflux of 68 +/- 21 mmol (l cells.h)-1 (n = 3), (iii) a Rb+ influx of 9.7 +/- 3.9 mmol (l cells.h)-1 (n = 6) and (iv) a Cl- influx of 9.4 +/- 4.1 mmol (l cells.h)-1 (n = 6). 3. Hypotonic shock (183-200 mosmol kg-1) induced a sevenfold stimulation of DIOA-sensitive K+ and Cl- effluxes and a twofold stimulation of DIOA-sensitive Rb+ and Cl- influxes (with a Rb+ to Cl- stoichiometry of 1.04 +/- 0.31; mean +/- S.D., n = 6). 4. The DIOA-sensitive membrane carrier catalysed net outward KCl extrusion (the outward/inward flux ratio was 5-7 in isotonic media and 20 in hypotonic media at 189 mosmol kg-1). Inhibition of DIOA-sensitive 36Cl- efflux by cell K+ depletion suggested coupling of outward K+ and Cl- fluxes. Conversely, inward K+ and Cl- fluxes were found to be uncoupled in NO3- media and in K(+)-free media. 5. The results clearly show that rat thymocyte membranes possess a 1:1 K(+)-Cl- co-transport system which is strongly activated by hypotonic shock and catalyses net KCl extrusion.

Identification and partial purification of the erythrocyte L-lactate transporter.

1. Intact erythrocytes were incubated with 100 microM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig erythrocytes, were detected by this technique. In human erythrocytes, which have 10-fold less transport activity, no labelled polypeptide in this molecular mass range was detected. 2. Labelling of these 35-50 kDa polypeptides was decreased markedly in the presence of the specific inhibitors of lactate transport alpha-cyano-4-hydroxycinnamate and 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), which compete with DIDS for binding to the transporter. However, the weakly bound inhibitor 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) afforded little protection against labelling by DIDS. 3. The lactate transporter from rat erythrocytes was solubilized with decanoyl-N-methyl glucamide (MEGA-10) and partially purified by Mono-Q anion-exchange chromatography, with transport activity eluting at 0.1-0.15 M-NaCl. The 35-45 kDa DIDS-labelled polypeptide from rat erythrocytes was eluted in the same peak of protein as lactate transporter activity during Mono-Q chromatography. 4. These observations provide strong evidence that the lactate transporter is a polypeptide of 35-45 kDa in rat erythrocytes and of 40-50 kDa in rabbit and guinea pig erythrocytes.

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles

The exothermic thermal denaturation transition of band 3, the anion transporter of the human crythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190–300 kcal mol −1) with an accompanying decrease in thermal denaturation temperatures (48–60°C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4′-diisothiocyanostilbene-2,2′-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75°C) and DIDS-insensitive (62°C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant diffence was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C 12E 8) are required for optimal thermal stability of band 3.

Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes. Identification of some new high-affinity inhibitors

1. Inhibition of L-lactate transport into rat erythrocytes by stilbenedisulphonates was studied under conditions which allowed the contribution of reversible and irreversible inhibition to be assessed. 2. At low temperatures (7 degrees C), 4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) and other stilbenedisulphonates were found to inhibit lactate transport instantaneously, in a manner which was fully reversible. The most potent reversible inhibitors were 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), DIDS and 4-acetamido-4'isothiocyanostilbene-2,2'-disulphonate (SITS), for which apparent Ki values at 0.5 mM-L-lactate were approx. 36, 53 and 130 microM respectively. 3. DIDS and DBDS were competitive inhibitors with respect to L-lactate, with Ki values of approx 40 microM and 22 microM respectively. 4. After incubation for 1 h at 37 degrees C with DIDS or its dihydro derivative (H2DIDS), which contain the amino-reactive isothiocyanate group, most of the inhibition observed was irreversible. Under these conditions the IC50 value (concn. causing 50% inhibition) for irreversible inhibition by both compounds was approx 100 microM. SITS was much less potent as an irreversible inhibitor of L-lactate transport, approx. 20% inhibition being obtained at 100 microM. 5. The reversible inhibitor DBDS (1 mM) afforded protection against irreversible inhibition by DIDS and H2DIDS (100 microM); protection was 60 and 65% respectively after a 60 min incubation. This indicates that specific binding of the irreversible inhibitors is required before covalent modification can take place. 6. These compounds may be useful high-affinity probes for lactate transport in other tissues and might act as affinity labels for the transport protein(s).

Chloride and bicarbonate transport in fetal red cells.

1. Chloride (JCl) and bicarbonate (JHCO3) self-exchange flux in fetal human red cells was studied at 0-38 degrees C as 36Cl- and [14C]HCO3- efflux. 2. Both at 0 and 38 degrees C JCl showed a bell-shaped pH dependence with a broad maximum at pH 7-8. JCl was 99.7% inhibited by the binding of 1.1 x 10(6) 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) molecules per cell membrane. 3. By raising the temperature from 0 to 38 degrees C JCl, at external concentration (C0) = 150 mM, increased about 200 times to 5 x 10(-8) mol/(cm2 s), the half-time of the tracer efflux being 80 ms at 38 degrees C and pH 7.2. Under conditions where Co = (110 mM-Cl- + 25 mM-HCO3-), JCl also increased by about 200 times, while JHCO3 increased only about 100 times, as temperature was raised from 0 to 38 degrees C. 4. The apparent activation energy (EA) of Janion was not constant, but increased gradually with decreasing temperature. Assuming that the change of EA with temperature consists of two components JCl, under both experimental conditions, had average values of EA = 117-120 under and EA = 73-78 kJ/mol above a 'breaking' point at 15-20 degrees C, while JHCO3 showed an EA = 100 below and EA = 48 kJ/mol above 25 degrees C. 5. We conclude that the anion transport protein becomes built into the membrane at a very early stage of life, and that the kinetics of Janion in fetal red cells show characteristics that are similar to those of adult red cells. 6. Our results suggest that the CO2 transport capacity of the fetal blood can be exploited during rest nearly as efficiently as that of adult blood.

A study of sulphate transport by lactating rat mammary tissue slices: Evidence for anion exchange

1. 1. The efflux of radiolabelled sulphate from lactating rat mammary tissue slices has been studied. Sulphate efflux was found to be time- and temperature-dependent. 2. 2.4,4'-Diisothiocyanostilbene-2,2'-disulphonate (DIDS) inhibited a portion of sulphate release, whereas bumetanide was without effect. 3. 3. The anions chloride, iodide and sulphate trans-stimulated sulphate efflux when added to the incubation medium. The increase in the efflux rate of sulphate found with chloride could be markedly inhibited by DIDS. Thiocyanate, unlike the other anions tested, only had a small effect. 4. 4. The results strongly suggest that there is an anion exchange mechanism in the mammary gland which can mediate the transport of sulphate. This transporter may be important for the metabolism of sulphate by the mammary gland and may also help determine milk anion concentrations.

The effect of DIDS on fluid reabsorption from the proximal convoluted tubule of the rat: dependence on the presence of bicarbonate.

The effects of the anion transport inhibitor DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulphonate) on fluid reabsorption by the rat proximal convoluted tubule were investigated by the stationary microperfusion (split-drop) method. Addition of 10(-5) mol 1-1 DIDS to luminal and capillary perfusates in the presence of peritubular bicarbonate (25 mmol 1-1) increased the reabsorptive half-time (t 1/2) from 35.1 +/- 4.5 to 57.0 +/- 6.2 s, corresponding to a decrease in fluid reabsorption (Jv) from 1.33 +/- 0.06 to 0.89 +/- 0.09 nl min-1 mm-1. Omission of bicarbonate per se from the peritubular capillaries reduced Jv to 0.41 +/- 0.03 nl min-1 mm-1 and this reabsorptive flux was completely inhibited by the addition of 2 mmol 1-1 NaCN to both the luminal and peritubular perfusates. In the absence of bicarbonate, the addition of DIDS at either 10(-4) or 10(-5) mol 1-1 to both lumen and capillary perfusates had no significant effect on either t 1/2 or Jv. These results show that the inhibitory action of DIDS on fluid reabsorption is dependent on the presence of bicarbonate. However, the proximal tubule also reabsorbs water in the absence of bicarbonate by a mechanism that is dependent on cellular metabolism, but is insensitive to DIDS at the concentrations used in this study.

Transport of selenate and sulphate across the intestinal brush-border membrane of pig jejunum by two common mechanism.

The objectives of the present study were to find out whether selenate and sulphate are transported by a common Na+-dependent transport mechanism across the intestinal brush-border membrane and whether selenate is also transported by the anion-exchange system transporting sulphate across the brush-border membrane. Results from cis-inhibition as well as from trans-stimulation experiments show that selenate and sulphate are transported across the brush-border membrane by both Na+ co-transport and anion exchange. The influence of various dicarboxylates (oxalate, oxaloacetate, glutamate) and of the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) on selenate and sulphate transport across the brush-border membrane was also investigated. Under Na+-gradient conditions selenate uptake across the brush-border membrane was reduced by oxalate and oxaloacetate but not by L-glutamate or L-leucine. Additionally sulphate uptake under this condition was significantly inhibited by oxalate (about 50%) and DIDS (25%). Sulphate transport by anion exchange was inhibited about 80% by both oxalate and DIDS. These results suggest that under Na+-gradient conditions sulphate (selenate) uptake in part occurs via anion exchange. Furthermore, oxalate seems to inhibit both Na+-sulphate (selenate) co-transport and sulphate (selenate) transport by anion exchange.