Effect Of DIDS Research Articles

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.

PH-dependent fluoride transport in intestinal brush border membrane vesicles

Fluoride (F) absorption from the rat stomach and urinary bladder, hamster cheek pouch, and the renal tubules of several species are pH gradient-dependent. These observations led to the hypothesis that F crosses these epithelia in the form of the undissociated acid, HF. Several recent reports, however, have provided evidence that F absorption from the rat small intestine is insensitive to the lumenal pH. We report here our evidence that F uptake by rabbit intestinal brush border membrane vesicles (BBMV) occurred rapidly and with an overshoot only in the presence of an inward-directed proton gradient. In the absence of a proton gradient or in the presence of an outward-directed gradient, F uptake was slow and without an overshoot. In the presence of an inward-directed proton gradient, F uptake was partially inhibited by DIDS and DEP but not by diBAC. PCMBS inhibited F uptake by up to 83% in a dose-response manner. DiBAC appeared to reduce intravesicular pH slightly but the other reagents had no effect. When the uptake buffer contained chloride or nitrate, F uptake was partially inhibited compared to the mannitol or gluconate controls. It was concluded that F transport across the rabbit intestinal BBMV occurs via a carrier-mediated process which may involve cotransport of F with H + or exchange of F with OH −. The inhibitory effects of DIDS, DEP and PCMBS may occur by affecting this carrier-mediated transport.

A possible mechanism underlying corymine inhibition of glycine-induced Cl- current in Xenopus oocytes.

We previously reported that corymine, an alkaloid extracted from the leaves of Hunteria zeylanica native to Thailand, inhibited glycine-induced chloride current using a receptor expression model of Xenopus oocytes. In this study, we investigated the mechanism underlying the inhibitory action of this alkaloid on glycine current using the same model. Corymine inhibited glycine current in a noncompetitive fashion. Co-application with strychnine, a competitive glycine receptor antagonist, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, corymine decreased the ED50 value of strychnine, but did not change that of DIDS. Moreover, the inhibitory effects of corymine and either strychnine or DIDS were additive. The desensitization phase of glycine current showed two exponentials and corymine preferentially inhibited the fast component, whereas strychnine affected both of them to the same extent and DIDS preferentially inhibited the slow component. When these drugs were applied repeatedly, the inhibitory effects of corymine and strychnine were not use-dependent and reversible, while the effect of DIDS was use-dependent and irreversible. The inhibitory effect of corymine on gamma-aminobutyric acid (GABA) current was less potent than the effect on glycine current, while this alkaloid failed to affect acetylcholine and serotonin currents. These results demonstrate that corymine inhibits glycine-gated CI- channels by interacting with the site different from that of DIDS.

Modulation by extracellular Cl- of volume-activated organic osmolyte and halide permeabilities in HeLa cells.

Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.

Cross talk of bumetanide-sensitive and HCO3--dependent transporters activated by IBMX in renal epithelial A6 cells.

We studied cAMP-dependent regulation of ion transport in aldosterone-untreated renal epithelial A6 cells by measuring short-circuit current (Isc). Biphasic increases in Isc, a transient phase followed by a sustained one, were elicited in response to 1 mm 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase) which increased cytosolic cAMP concentration. IBMX increased the apical Cl- conductance. The sustained phase of Isc induced by IBMX was reduced by 50 microM bumetanide (Na+/K+/2 Cl- cotransporter inhibitor) or 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, an inhibitor of Cl-/HCO3- exchanger). Under the normal condition, the inhibitory effect of bumetanide was much larger than that of DIDS. On the other hand, under a low Cl- condition, the effect of DIDS was more effective than that of bumetanide. Further, under a Cl--free condition Na+/HCO3- symporter contributed to the IBMX-generated Isc. Taken together, our observations suggest that in A6 cells (i) IBMX stimulates Cl- secretion associated with an increase in apical Cl- conductances, (ii) the ionic components to generate the IBMX-induced Isc are mainly maintained by bumetanide-sensitive Na+/K+/2 Cl- cotransporter and DIDS-sensitive Cl-/HCO3- exchanger, (iii) Cl-/HCO3- exchanger coupled to Na+/HCO3- symporter under a low-Cl- condition or Na+/HCO3- symporter under a Cl--free condition contributes to the IBMX-induced Isc, compensating for diminishment of the Na+/K+/2Cl- cotransporter-mediated Cl- secretion, (iv) IBMX increases Cl- and HCO3- conductances in the apical membrane.

Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca 2+: correlation with mitochondrial permeability transition

The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca 2+ (0–500 μM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg 2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca 2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca 2+ plus t-butyl hydroperoxide ( t-bOOH) or Ca 2+ plus the thiol cross-linkers 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca 2+-induced alterations of thiol group reactivity, but dependent on Ca 2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca 2+ concentrations in range of 1–50 μM.

Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform.

Molecular cloning and expression have previously defined three members of the Na+/H+ exchanger (NHE) gene family NHE1 and NHE2 are sensitive to inhibition by amiloride and its 5'-amino alkyl-substituted analogues, whereas NHE3 is quite resistant to amiloride inhibition. Each of these exchangers has narrowly defined cation specificities for Na+ and Li+. Expression studies with NHE4 have not been as successful, with only a description of modest expression of activity (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994). We now report that NHE4 activity in stably transfected fibroblasts is activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), permitting functional characterization of this NHE isoform. The activating effect of DIDS was unique among the disulfonic stilbenes, and competition studies suggested a cross-linking mechanism. NHE4 is extremely resistant to amiloride and ethylisopropylamiloride inhibition and, unlike other NHE isoforms, affects K+/H+ exchange as well as Na+/H+ and Li+/H+ exchange. These findings demonstrate that NHE4 is a functionally distinct member of the NHE gene family and suggest a unique physiological role for this cation/H+ exchanger.

A Mathematical Model of the Pancreatic Ductal Epithelium

A mathematical model of the HCO-3-secreting pancreatic ductal epithelium was developed using network thermodynamics. With a minimal set of assumptions, the model accurately reproduced the experimentally measured membrane potentials, voltage divider ratio, transepithelial resistance and short-circuit current of nonstimulated ducts that were microperfused and bathed with a CO2/HCO-3-free, HEPES-buffered solution, and also the intracellular pH of duct cells bathed in a CO2/HCO-3-buffered solution. The model also accurately simulated: (i) the effect of step changes in basolateral K+ concentration, and the effect of K+ channel blockers on basolateral membrane potential; (ii) the intracellular acidification caused by a Na+-free extracellular solution and the effect of amiloride on this acidification; and (iii) the intracellular alkalinization caused by a Cl--free extracellular solution and the effect of DIDS on this alkalinization. In addition, the model predicted that the luminal Cl- conductance plays a key role in controlling both the HCO-3 secretory rate and intracellular pH during HCO-3 secretion. We believe that the model will be helpful in the analysis of experimental data and improve our understanding of HCO-3-transporting mechanisms in pancreatic duct cells.

Volume regulation and the efflux of amino acids from cells in incubated slices of rat cerebral cortex. I. Characteristics of transport mechanisms

The characteristics of amino acid efflux from pre-loaded cells in incubated slices of rat cerebral cortex have been investigated under basal conditions (isosmotic media, 315 mosmol/kg) and following mild hyposmotic shock (265 mOsmol/kg). Rates of efflux have been correlated with the extent of cell swelling in hyposmotic media. Hyposmolality accelerated the slow phase of cellular efflux of l-aspartate (+29%), γ-amino isobutyric acid (GABA) (+38%), l-glutamate (+28%) and glycine (+26%). The anion transport inhibitor 4,4′-di isothiocyanatostilbene-2,2′-sulfonic acid (DIDS, 25 or 100 μM) as well as trifluoperazine (TFP, 25 μM), an inhibitor of calmodulin activation, both retarded efflux in hyposmotic media, with associated cell swelling (increase in slice non-inulin space). The effects of DIDS and TFP were not additive. N-Ethylmaleimide (NEM, 100 μM) significantly retarded the efflux of neutral amino acids, with cell swelling: these effects were less pronounced in cells loaded with acidic amino acids. It is concluded that the hyposmotically-activated efflux of carboxylic amino acids, and associated cell swelling limitation, requires calmodulin activation and the presence of free sulfydryl groups.

Blockade of chloride channels by DIDS stimulates renin release and inhibits contraction of afferent arterioles.

Calcium-activated chloride channels have been proposed to control renin release from juxtaglomerular cells and to be involved in the excitation-contraction coupling of the renal afferent arteriole. The hypothesis was tested on renin release from rat glomeruli and in microperfused rabbit afferent arterioles with the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Renin secretion was equally enhanced by omission of extracellular calcium and by addition of 0.5 mM DIDS. The inhibitory effect of calcium was blocked by DIDS. The stimulatory effects of low calcium [with or without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and DIDS were not additive. In the absence of chloride, basal renin release was suppressed and the stimulatory effect of DIDS was abolished. The DIDS-induced enhancement of renin release was not dependent on bicarbonate. Norepinephrine (5 x 10(-7)-1 x 10(-6) M) and angiotensin II (1 x 10(-8)-10(-6) M) evoked reversible and dose-dependent contractions of microperfused rabbit afferent arterioles. DIDS (0.5 mM) did not affect the basal diameter of the arterioles but strongly inhibited the response to angiotensin II and attenuated the duration of the contractile response to norepinephrine. The results support the hypothesis that DIDS-sensitive calcium-activated chloride channels are involved in regulation of renin release and in the afferent arteriolar contraction after angiotensin II but do not play a pivotal role in the response to norepinephrine.

Volume-sensitive chloride channels in the primary culture cells of human cervical carcinoma

Previous study shows volume-sensitive chloride currents are induced by hypotonicity in human cervical cancer cell lines, but not in normal cervical epithelium. To ascertain whether the preferential activation of these channels in cancer cell lines could be similarly and directly detected in cervical cancer tissues, we studied volume-sensitive chloride channels on the primary culture cells of invasive cervical carcinoma using the whole-cell patch-clamp technique. The process of regulatory volume decrease (RVD) was also studied using electronic cell sizing to measure cell volume. Results demonstrate that, in these cultured cells, RVD was mediated in part by chloride loss through the volume-sensitive Cl − channels. A small background current with a slope conductance of 0.32 ± 0.07 nS/pF at + 30 mV ( n = 60 cells from 10 different samples) was observed. Hypotonicity induced a fast activating and outward rectifying current which was reversed at about 0 mV, and the slope conductance at + 30 mV was increased by 10-fold to 3.62 ± 0.62 nS/pF. These effects were readily reversed by returning the cells to isotonic medium. Moreover, DIDS, NPPB, and 1,9-dideoxyforskolin, reversibly abolished the volume-sensitive Cl − currents. The EC 50 required for the inhibitory effect of DIDS, NPPB and 1,9-dideoxyforskolin was 150, 120, and 50 μM, respectively. Volume-sensitive Cl − channels were ubiquitously expressed in cultured cells from 10 samples of different cancer stages, histopathologic types, and state of HPV DNA positivity. Interestingly, similar outward rectifying chloride currents were activated by intracellular 300 μM GTPγS. It is proposed that this Cl − conductance may play an important role leading to RVD in human cervical cancer.

ION TRANSPORT IN THE INTESTINE OF ANGUILLA ANGUILLA: GRADIENTS AND TRANSLOCATORS

The transport of Na+, K+ and Cl- across the isolated epithelium of the eel intestine was studied using a combination of four experimental strategies: short-circuiting, measurements of intracellular potentials and ion concentrations, application of a variety of transport inhibitors and measurement of unidirectional fluxes with radioactive tracers. When short-circuited, the system performs a net transport of Cl- and Na+ towards the blood side, with a stoichiometry approaching 2, and a much smaller net transport of K+ towards the lumen. The system is totally driven by the sodium pump located in the basolateral barrier and the main coupling between the fluxes of the three ions is through the operation of a furosemide-sensitive transporter in the apical barrier, probably a 2Cl-/Na+/K+ symporter. The inhibitory effect of DIDS and picrylsulphonic acid on the short-circuit current, when added to the serosal side, suggests the presence of a sodium-dependent anionic shuttle located in the basolateral membrane. The short-circuit current is inhibited by H25, a non-specific inhibitor of the K+/Cl- symport, added to the serosal side. This effect occurs after a delay of at least 5 min and may result from the diffusion of the drug to the apical barrier, where it blocks the 2Cl-/Na+/K+ symport with much higher affinity.

Effects of Cl channel blockers on Ca-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.

1. The effects of some chloride channel antagonists were studied on the calcium-activated chloride current (ICl(Ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium-activated chloride currents) in a concentration-dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), which is voltage-dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on tau was similar at holding potentials of -50 and +50 mV. 4. These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium-activated potassium currents). 5. DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 x 10(-4) M and 1.8 x 10(-3) M, respectively. 6. DIDS and SITS increased the calcium-activated potassium current (IK(Ca) evoked by noradrenaline and caffeine by 3-6 fold. 7. Anthracene-9-carboxylic acid (A-9-C) inhibited STICs in a voltage-dependent fashion and was about 3 fold more active at +50 mV than at -50 mV. A-9-C increased STIC tau and this effect was enhanced by depolarization. 8. A-9-C also inhibited caffeine-evoked IC1(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(Ca).9. The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.

The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.

1. In confluent primary cultures of rat hepatocytes, membrane effects of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) were recorded with conventional microelectrodes. In addition, cell pH and cell Ca2+ were monitored by use of the fluorescent dyes BCECF and fluo-3, respectively. Uptake of DIDS was determined by measuring intracellular DIDS fluorescence between 470 and 520 nm (excitation wavelength 390 nm). 2. In the presence of 0.2 mM DIDS, membrane voltages hyperpolarized from -44.0 +/- 1.8 to -73.1 +/- 1.9 mV (n = 16). This change was monophasic and occurred with a time constant of 170 +/- 25 s. The effect was only partly reversible. 3. Cable analysis revealed a concomitant decrease in the specific cell membrane resistance from 3.2 to 1.5 k omega cm2. 4. In ion substitution experiments, a 10-fold elevation of external K+ (from 2.5 to 25 mM) depolarized cell membranes by 6.2 +/- 1.5 mV (n = 5). In the presence of 0.2 mM DIDS, this membrane response was increased 5-fold to 32.2 +/- 4.1 mV. 5. Replacement of Cl- by 99% with gluconate depolarized the cells by 9.3 +/- 1.9 mV. In contrast, with 0.2 mM DIDS present, Cl- removal led to a membrane hyperpolarization of 5.9 +/- 0.9 mV (n = 4). 6. DIDS had no effect on cytosolic pH or Ca2+. 7. To determine the sidedness of the DIDS effect, i.e. to analyse if the increase in K+ conductance is mediated by uptake of the compound, DIDS was added in the presence of different substrates of hepatocellular anion transport. Taurocholate (50 microM) and frusemide (0.5 mM), which are both taken up via the sinusoidal multi-specific bile acid transporter, did not change DIDS-induced membrane hyperpolarization. 8. In contrast, 0.5 mM bromosulphthalein (BSP), a substrate of the bilirubin transporter, competitively inhibited the membrane hyperpolarization elicited by various concentrations of DIDS (0.1-1.0 mM). 9. Hepatocellular uptake of BSP is known to be, in part, Cl- dependent and to be competitively inhibited by Indocyanine Green. When 0.2 mM DIDS was added to a superfusate, in which 99% of Cl- had been exchanged by gluconate, the velocity of membrane hyperpolarization was decreased by 45%. In the presence of Indocyanine Green (0.1 mM) DIDS-induced membrane hyperpolarization was reduced to approximately 20%. 10. Addition of 0.2 mM DIDS to hepatocyte monolayers led to a time-dependent increase in cell fluorescence which was absent at 4 degrees C and which was completely blocked by 0.5 mM BSP.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a Proton-Translocating ATPase in a Tonoplast-Vesicle Fraction from Citrus

Summary Tonoplast vesicles were isolated from Valencia Late ( Citrus sinensis (L.) cv. Osbeck) leaves by sucrose cushion density gradient centrifugation. Measurements of marker enzymes for microsomal membranes like latent IDPase (Golgi apparatus), Antimycin-A-insensitive NADPH-cytochrome c reductase (endoplasmi reticulum) and cytochrome-c-oxidase (mitochondria), and measurements of the ATP-hydrolysis activity with inhibitors specific for ATPases of different microsom indicated a good purity of the tonoplast vesicle fraction. The rate of ATP-hydrolysis of the tonoplast H + -ATPase was optimal at pH 7.4. It was stimulated by Cl − with a maximum at 60 mM and was inhibited by 60 mM NO 3 − . The tonoplast H + -ATPase hydrolyzed [MgATP] 2− in preference to other Mg-nucleotides in the sequence ATP > GTP > UTP >- CTP > ADP. The apparent K m for [MgATP] 2− -hydrolysis was 0.38 ± 0.04 mM. The H + -ATPase activity of tonoplast vesicles was sensitive to the anion channel blocker DIDS (I 50 = 15 µM). Even in the absence of Cl − the rate of ATP hydrolysis was inhibited by 30% at 40 M DIDS, which indicates a direct effect of DIDS on the tonoplast H + -ATPase. Quinacrine-fluorescence quenching measurements of tonoplast vesicles showed that the tonoplast H + -ATPase was able to build up an MgATP-dependent electrochemical proton gradient across the vesicle membrane. This gradient broke down in the presence of the protonophore gramicidine A and by the inhibition of the tonoplast H + -ATPase by NO 3 − . Determination of latent H + -ATPase activity with 0.002 % Brij 58 showed that the tonoplast vesicle fraction contained approximately equal amounts of rightside-out vesicles and inside-out vesicles. In the tonoplast vesicle preparation five polypeptides (63, 52, 37, 30, and 16 kDa) cross-reacted with an antiserum against the tonoplast H + -ATPase from leaves of Kalanchoe daigremontiana . Our results indicate that the tonoplast vesicles of Citrus leaves contain an H + -ATPase with similar properties as the tonoplast H + -ATPase from other plant and fungal sources. This characterization of the enzyme is the basis for the investigation of the energetization and regulation of secondary active transport mechanisms of ions and metabolites at the tonoplast in response to environmental stress.

Characterization of the chloride carrier in the plasmalemma of the alga Valonia utricularis: the inhibition by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid

The effect of the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) on the Cl −-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235–248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, K AS , of the Cl −-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, K S , as well as the total surface concentration of the carrier, N o , were not affected. A Hill-plot of DIDS inhibition on K AS yielded an half-maximal inhibition concentration (IC 50) of 3.9 · 10 −5 M and a Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl −-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl −-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravacuolar DIDS or SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl −-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.

Blockade of ATP binding site of P 2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds

Extracellular ATP activates a P 2z-type purinergic receptor (purinoceptor) in rat parotid acinar cells that increases the intracellular free Ca 2+ concentration via the entry of extracellular Ca 2+ through an ATP-sensitive cation channel (Soltoff et al., Am J Physiol 262: C934–C940,1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [ 32PIATP to intact parotid cells (McMillian et al., Biochem J 255: 291–300,1988) and blocked the activation of the P 2z purinoceptor. The ATP-stimulated 45Ca 2+ uptake was blocked by DIDS, H 2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the ic 50 value of DIDS (∼35 μM), which has two SCN-groups, was much lower than that of SITS (∼ 125 μM), which has only one SCN- group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluoroscein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P 2z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked the covalent binding of DIDS to the plasma membrane. These results suggest that isothiocyanate compounds interact with the ATP binding site of this P 2 purinoceptor, and that isothiocyanate groups make an important contribution in determining the effectiveness of the stilbene disulfonate compounds in blocking the binding of nucleotide agonists to this purinoceptor.

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

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

O-121 - Effect of DIDS on adrenoceptor-mediated changes in intracellular pH in guinea-pig myocardium.

O-121 - Effect of DIDS on adrenoceptor-mediated changes in intracellular pH in guinea-pig myocardium.

The stilbene disulfonic acid DIDS stimulates the production of TNF-α in human lymphocytes

Exposure of human peripheral blood mononuclear cells (PBMC) to the stilbene derivative DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) (60 μM and above) significantly increased the release of tumor necrosis factor-α (TNF-α), as determined by TNF-α activity in the incubation media. When the TNF-α message was analyzed in PBMC by a reverse transcription/polymerase chain reaction (RT/PCR)-based procedure, it was found that incubation with DIDS (60μM) was followed by a time-dependent accumulation of TNF-α mRNA. Measurements of intracellular pH showed that the presence of increasing concentrations of DIDS resulted in a progressive intracellular alkalinization of PBMC. It is suggested that the known DIDS effect of inhibiting transmembrane anion exchange, i.e., chloride/bicarbonate exchange, might play a role in the stimulation of TNF-α production by PBMC exposed to DIDS.