Addition Of DIDS Research Articles

Effects of VU0240551, a novel KCC2 antagonist, and DIDS on chloride homeostasis of neocortical neurons from rats and humans

The normal function of GABAA receptor-mediated inhibition is governed by several factors, including release of GABA, subunit composition and density of the receptors and in particular by the appropriate ionic gradient. In the human epileptogenic neocortex an impaired chloride (Cl−) gradient has been proposed, due to decreases of potassium-coupled chloride transport (KCC2) and voltage-gated Cl− channels (ClC). Regarding sodium- and potassium-coupled Cl− transport (NKCC1) both up- and downregulations have been proposed.We investigated changes of Cl− homeostasis of human and rat neocortical neurons (layer 2/3) with intracellular recordings and iontophoretic Cl− loading employing selective compounds. After cessation of iontophoresis, the IPSPA amplitudes of rat neurons recovered with a time constant (τrec) of 6.5s (n=21). In human neurons, τrec averaged 17.8s (n=36; 23 resections). Application of the novel KCC2 blocker VU0240551 (1μM) caused in rat neurons a reversible prolongation of τrec from 5.7 to 8.1s (n=11), corresponding to a VU0240551-sensitive Cl− transport rate (1/Δτrec) of 0.0504s−1. In human neurons, τrec increased on application of 1μM VU0240551, on average from 15.1 to 20.3s (n=17). The human neurons comprised two subgroups with different τrec when segregated according to a border given by the mean+2s.d. of rat neurons. In one group, τrec averaged 8.7s (n=6) and reversibly increased to 14.6s in the presence of 1μM VU0240551, corresponding to a Cl− transport rate of 0.0504s−1. The other group had an average τrec of 18.5s which increased in the presence of 1μM VU0240551 to 23.3s (n=11), indicating a much smaller rate (0.0151s−1). Addition of DIDS, a presumed blocker of anion exchanger (AE), increased the τrec of rat neurons from 7.5 to 8.8s (n=6) corresponding to a DIDS-sensitive rate of 0.0185s−1. In human neurons, DIDS increased τrec from 23.3 to 50.7s (n=7), corresponding to a DIDS-sensitive rate of 0.0200s−1.These data suggest a greatly reduced KCC2-mediated transport rate in most of the human neurons. The two subgroups observed in human tissue indicate a considerable variability of Cl− transport within a given tissue from almost normal to greatly impeded, predominated by a decline of KCC2 whereas AE is unaltered.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75μM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75μM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na+/H+ exchangers, Na+–K+ pump, Na+-driven and Na+-independent HCO3−/Cl− transporters and Na+/HCO3− co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4′-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3−/Cl− co-transporters present on the basolateral membrane.

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

Elucidation of the Mechanisms Underlying Hypo-osmotically Induced Turgor Pressure Regulation in the Marine Alga Valonia utricularis

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.

The complexity of intestinal absorption and exsorption of digoxin in rats

The potential multiple carrier-mediated mechanisms involved in the transport of digoxin in rat intestine were investigated by the rapid filtration method in rat intestinal brush-border vesicles (BBMV) and in vitro Ussing chambers. The uptake of digoxin showed a typical overshoot phenomenon in the presence of an inward proton gradient and an outward bicarbonate gradient, or an outward glutathione gradient in BBMV. Good fitting to an equation consisting of both saturable and linear terms was obtained using non-linear regression analysis. GF120918, a specific P-gp inhibitor, significantly increased the absorptive permeability of digoxin in rat ileum (7.02 × 10 −7 cm/s versus 2.11 × 10 −6 cm/s with GF120918) but the addition of DIDS (0.5 mM), an anionic transporter inhibitor, or bromosulfophthalein (0.1 mM), an Oatp inhibitor, in the presence of GF120918 decreased the absorptive permeability compared with GF120918 alone (2.11 × 10 −6 cm/s versus 1.46 × 10 −6 cm/s, p < 0.01 and 2.11 × 10 −6 cm/s versus 1.60 × 10 −6 cm/s, p < 0.05, respectively). The above results suggest the involvement of carrier-mediated uptake mechanism, possibly Oatp, in digoxin absorption. Interestingly, GF120918 (1 μM) did not abolish the polarized transport of digoxin in rat jejunum and ileum, and DIDS (0.5 mM), not a P-gp inhibitor, and MK571 (50 μM), an MRP-selective inhibitor, can also significantly decrease the exsorptive permeability of digoxin. This result indicates the involvement of non-P-gp efflux transporter in digoxin secretion and this transporter is DIDS and MK571-sensitive. Contrary to conventional concept, our studies show that intestinal absorption of digoxin may involve both active uptake and efflux transporters. Our study may have clinical implications in drug–drug or drug–food interactions involving transporters.

Anion-exchange blocker enhances cytoplasmic vacuole formation and cell death in serum-deprived mouse kidney epithelial cells in mice

An anion exchange blocker, DIDS, exhibits anti-apoptotic activity in response to several apoptotic stimuli, but has an opposite effect when apoptosis is induced by serum deprivation. After adding DIDS, serum-deprived MCT cells exhibited vacuole formation in their cytoplasm and underwent cell death. Caspase activity increased with the addition of DIDS to serum-deprived MCT cells, but Z-Asp-CH2-DCB, a caspase inhibitor, did not inhibit cell death in DIDS-treated, serum-deprived MCT cells. Cathepsin is considered to be important for vacuole formation and cell lysis, and pepstatin A, a cathepsin D inhibitor, partially inhibited vacuole formation in DIDS-treated, serum-deprived MCT cells, although caspase activation was not inhibited. 3-Methyladenin inhibited vacuole formation and cell death in DIDS-treated, serum-deprived MCT cells. These results suggest that DIDS-treated serum-deprived MCT cells undergo autophagy, not apoptosis.

Differential effects of Na-K-ATPase pump inhibition, chemical anoxia, and glycolytic blockade on membrane potential of rat optic nerve

Na +-K +-ATPase pump failure during either anoxia or ouabain perfusion induces rapid axonal depolarization by dissipating ionic gradients. In this study, we examined the interplay between cation and anion transporting pathways mediating axonal depolarization during anoxia or selective Na +-K +-ATPase inhibition. Compound resting membrane ( V m) potential of rat optic nerve was measured in a grease gap at 37 °C. Chemical anoxia (2 mM NaCN or NaN 3) or ouabain (1 mM) caused a loss of resting potential to 42 ± 11% and 47 ± 2% of control after 30 min, respectively. Voltage-gated Na +-channel blockade was partially effective in abolishing this depolarization. TTX (1 μM) reduced depolarization to 73 ± 10% (chemical anoxia) and 68 ± 4% (ouabain) of control. Quaternary amine Na + channel blockers QX-314 (1 mM) or prajmaline (100 μM) produced similar results. Residual ionic rundown largely representing co-efflux of K + and Cl − during chemical anoxia in the presence of Na +-channel blockade was further spared with DIDS (500 μM), a broad-spectrum anion transport inhibitor (95 ± 8% of control after 30 min in anoxia + TTX vs. 73 ± 10% in TTX alone). Addition of DIDS was slightly more effective than TTX alone in ouabain (74 ± 5% DIDS + TTX vs. 68 ± 4% in TTX alone, P < 0.05). Additional Na +-entry pathways such as the Na-K-Cl cotransporter were examined using bumetanide, which produced a modest albeit significant sparing of V m during ouabain-induced depolarization. Although cation-transporting pathways play the more important role in mediating pathological depolarization of central axons, anion-coupled transporters also contribute to a significant, albeit more minor, degree.

An apical K+-dependent HCO3− transport pathway opposes transepithelial HCO3− absorption in rat medullary thick ascending limb

Absorption of HCO(3)(-) in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na(+)/H(+) exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO(3)(-)/OH(-)/H(+) transport pathways in the rat MTAL and to determine their role in transepithelial HCO(3)(-) absorption. MTALs were perfused in vitro in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2). Lumen addition of either 120 mM Cl(-) or 50 mM Na(+) (50 microM EIPA present) had no effect on intracellular pH (pH(i)). Lumen Cl(-) addition also had no effect on pH(i) in the presence of 145 mM Na(+) or in the nominal absence of HCO(3)(-)/CO(2). Thus there was no evidence for apical Cl(-)/HCO(3)(-) (OH(-)) exchange, Na(+)-dependent Cl(-)/HCO(3)(-) exchange, or Na(+)-HCO(3)(-) cotransport. In contrast, in tubules studied in Na(+)- and Cl(-)-free solutions containing 25 mM HCO(3)(-), 5% CO(2) and 120 mM K(+), removal of luminal K(+) induced a rapid and pronounced decrease in pH(i) (DeltapH(i) = 0.56 +/- 0.06 pH U). pH(i) recovered following lumen K(+) readdition. The initial rate of net base efflux induced by lumen K(+) removal was decreased 85% at the same pH(i) in the nominal absence of HCO(3)(-)/CO(2), indicating a dependence on HCO(3)(-)/CO(2) and arguing against apical K(+)/H(+) exchange. A combination of the apical K(+) channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K(+)-induced pH(i) changes, arguing against electrically coupled K(+) and HCO(3)(-) conductances. The effect of lumen K(+) on pH(i) was inhibited by 1 mM H(2)DIDS. In addition, lumen addition of DIDS increased transepithelial HCO(3)(-) absorption from 10.7 +/- 0.7 to 14.9 +/- 0.7 pmol x min(-1) x mm(-1) (P < 0.001) and increased pH(i) slightly in MTAL studied in physiological solutions (25 mM HCO(3)(-) and 4 mM K(+)). Lumen DIDS stimulated HCO(3)(-) absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K(+)-dependent HCO(3)(-) transport pathway that mediates coupled transfer of K(+) and HCO(3)(-) from cell to lumen in the MTAL. This mechanism, possibly an apical K(+)-HCO(3)(-) cotransporter, functions in parallel with apical Na(+)/H(+) exchange and opposes transepithelial HCO(3)(-) absorption.

Mechanisms of chloride secretion induced by thermostable direct haemolysin of Vibrio parahaemolyticus in human colonic tissue and a human intestinal epithelial cell line.

Thermostable direct haemolysin (TDH) produced by Vibrio parahaemolyticus is thought to play an important role in the severe diarrhoea caused by this organism. This study investigated the enterotoxicity of TDH for human intestinal cells. Addition of TDH to the mucosal side of human colonic tissue in Ussing chambers caused increased short circuit currents (Isc), a process that was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca2+ -activated chloride (Cl-) channels. With human colonic epithelial (Caco-2) cells, high Isc and intracellular Ca2+ concentrations ([Ca2+]in) were detected after the addition of TDH to the apical side of the cell monolayer. The Isc decreased with the addition of DIDS, but not with glybenclamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid, or gadolinium chloride. No Isc increase with TDH was observed when the Cl- in the medium was replaced by gluconate or when Ca2+ was depleted. Similarly, TDH did not raise [Ca2+]in after depletion of extracellular Ca2+. R7, a mutant form of TDH, reduced the effects of TDH on Isc and [Ca2+]in, as did protein kinase C (PKC) inhibitors. Thus, TDH increases Cl- secretion in human colonic epithelial cells, apparently through mechanisms involving cell binding and Ca2+ influx, followed by elevation of [Ca2+]in associated with PKC phosphorylation.

Na(+) and Cl(-) transport by the urinary bladder of the freshwater rainbow trout (Oncorhynchus mykiss).

Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (¿Na(+) = 2.12 mM, ¿Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.

Activation of Ionic Channels by Deoxycholate in Frog and Human Cell Lines

Humans, after extensive ileal resection, frequently suffer from diarrhoea, which may be due to an increased delivery of deoxycholate (DOC) to the large intestine. In the frog skin the addition of DOC (0.5 mM) to the apical side induced the activation of amiloride-sensitive Na+ channels and an increase in the unidirectional Cl- fluxes. Here we used two established cell lines (A6 and Caco2) to study the effect of DOC on ion channels at cell and membrane level using the patch-clamp technique. In A6 cells subcultured directly on Petri dishes and studied in the whole-cell configuration, DOC induced an increase in cell conductance of 110.3 +/- 4 pS pF-1 (N = 8) which was reduced to 89 +/- 14 pS pF-1 (N = 8) by the addition of DIDS (0.5 mM), The absolute values of these two effects were not statistically different (P < 0.2). In Caco2 cells, the addition of DOC (0.5 mM) induced, after 1 min, an increase in cell conductance of 583 +/- 16 pS pF-1 (N = 8) which was reduced to 560.4 +/- 16 pS pF-1 (N = 8) by DIDS (0.5 mM) and N-phenylanthranilic acid (DPC; 0.5 mM). The two values were not statistically different (P < 0.4). In Caco2 cells subcultured under the same conditions, DOC induced an increase in cell conductance of 1710 +/- 64 pS pF-1 (N = 6). Subsequent addition of amiloride (0.1 mM) reduced the cell conductance to 1558 +/- 33 pS pF-1 (N = 6). These two mean values were statistically different allowing for an error of the second kind < 0. 05. In cells in which DOC produced a conductance increase of 1010 +/- 10 pS pF-1, gadolinium (0.5 mM) induced a fall in cell conductance of 1800 +/- 10 pS pF-1. In Caco2 cells, addition of DOC (0.5 mM) to the bath reversibly induced the appearance of or an increase in channel activity in patches studied in cell-attached and excised inside-out configuration. In inside-out experiments (N = 13) DOC (0. 5 mM) induced the appearance of channel activity with conductances and reversal potentials (Er) of 27.7 +/- 1.9 pS and 0.8 +/- 5.7 mV, respectively. In cell-attached patches (N = 13) these values were 24.9 +/- 4.4 pS and -18.1 +/- 6.4 mV. In excised inside-out patches from Caco2 cells, subjected to electrochemical gradients for Na+, K+ and Cl-, (+85, -85 and 0 mV, respectively), addition of DOC also induced an increase in the baseline conductance and a shift in the reversal potential from values around +25 mV to values around 0 mV. Bile salts activated both anionic and cationic channels and did not require the presence of intracellular factors for these effects. We suggest that they act at the membrane level.

DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) increases iodide trapping, inhibits thyroperoxidase and antagonizes the TSH-induced apical iodide efflux in porcine thyroid cells

4,4′-Di-isothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of several anionic channels and transporters including the band 3 protein of the red blood cell membrane was tested on iodide metabolism in cultured porcine thyroid cells. We used three experimental cell culture models: (i) forskolin-stimulated correctly inside-in polarized follicle-associated thyroid cells cultured onto plastic support (ii) suspensions of isolated cells derived from such cultures (iii) polarized monolayers in bicameral chambers. DIDS was observed to increase free-iodide trapping in all conditions. Organification of iodide by follicle-associated cell cultures incubated for 6 h decreased as a function of DIDS concentration with an IC 50 of 5×10 −5 M. This block in organification is accounted for a block in thyroperoxidase activity as in vitro both purified lactoperoxidase and purified porcine thyroperoxidase were inhibited by DIDS with a similar dose-dependency the IC 50 being also of 5×10 −5 M. Both control and DIDS-treated cells in suspension, actively trapped iodide and reached a steady concentration in about 50 min; however the plateau was 4.4-fold higher in (10 −3 M) DIDS-treated cells. Acute TSH-stimulation at this plateau of 125I-preloaded cells in suspension in the presence of 2 mM methimazole (MMI) induced a fast release of iodide from these cells as expected (first step of the TSH-biphasic effect). This TSH-induced iodide efflux was however completely inhibited by DIDS (10 −3 M). Furthermore, addition of DIDS to the apical compartment of TSH-prestimulated cell monolayers in bicameral chambers resulted in an increase in intracellular-iodide concentration and in an inhibition of iodide efflux into the apical medium. Taken together, the present results demonstrate that DIDS mainly interacts with two main components of the thyroid apical cell membrane: thyroperoxidase and a cAMP-sensitive iodide channel.

Inhibition of Bicarbonate Transport Protects Embryonic Heart Against Reoxygenation-induced Dysfunction

It has not been well established whether the mechanisms participating in pH regulation in the anoxic–reoxygenated developing myocardium resemble those operating in the adult. We have specially examined the importance of Na+/H+exchange (NHE) and HCO3-dependent transports in cardiac activity after changes in extracellular pH (pHo). Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to single or repeated anoxia (1 min) followed by reoxygenation (10 min). The chronotropic, dromotropic and inotropic responses of the hearts were determined in standard HCO3−buffer at pHo7.4 and at pHo6.5 (hypercapnic acidosis). In distinct experiments, acidotic anoxia preceded reoxygenation at pHo7.4. NHE was blocked with amiloride derivative HMA (1μmol/l) and HCO3-dependent transports were inactivated by replacement of HCO3or blockade with stilbene derivative DIDS (100μmol/l). Anoxia caused transient tachycardia, depressed mechanical function and induced contracture. Reoxygenation temporarily provoked cardiac arrest, atrio-ventricular (AV) block, arrhythmias and depression of contractility. Addition of DIDS or substitution of HCO3at pHo7.4 had the same effects as acidosisper se, i.e. shortened contractile activity and increased incidence of arrhythmias during anoxia, prolonged cardioplegia and provoked arrhythmias at reoxygenation. Under anoxia at pHo6.5/reoxygenation at pHo7.4, cardioplegia, AV block and arrhythmias were all markedly prolonged. Interestingly, in the latter protocol, DIDS suppressed AV block and arrhythmias during reoxygenation, whereas HMA had no effect. Thus, intracellular pH regulation in the anoxic–reoxygenated embryonic heart appears to depend predominantly on HCO3availability and transport. Furthermore, pharmacological inhibition of anion transport can protect against reoxygenation-induced dysfunction.

Sensitization to hyperthermia by intracellular acidification of C6 glioma cells.

Hyperthermia has been introduced as a new modality of treatment for glioma. In these experiments, the cytotoxicity of hyperthermia in C6 glioma cells was enhanced by increasing the intracellular acidity with amiloride and/or 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS). Intracellular pH (pHi) is regulated mainly by Na+/H+ and HCO3-/Cl- antiports through the cell membrane, and amiloride acts on the former, DIDS on the latter to lower pHi. The cellular thermosensitivity to clinically achievable brain hyperthermia at 42 degrees C was enhanced by 0.5 mM amiloride (Na+/H+ antiport inhibitor). T0 values (T0 = the heating period required to reduce experimental survival rate by 1/e) at 42 degrees C without and with amiloride was 192 and 81 min, respectively. The addition of DIDS (HCO3-/Cl- antiport inhibitor) further enhanced. T0 value was 25 min. Fluorophotometric measurement of pHi was employed using the pH sensitive dye, bis(carboxyethyl)carboxyfluorescein, which is trapped in viable cells. The average pHi in control C6 glioma cells in pH 7.2 media was 7.21. In the untreated cells heated at 42 degrees C for 1 hour, the pHi was 7.12. The pHi of the cells heated in the presence of amiloride was decreased to 6.83. The pHi was further lowered to 6.67 by the treatment with amiloride in combination with DIDS for 2 hours. Hyperthermia with amiloride and DIDS may be a more effective treatment for malignant gliomas.

Influence of heat on Na(+)-dependent HCO3-/Cl- exchanger function in a thermoresistant cell line.

During previous studies on the relationship between thermosensitivity and intracellular pH (pHi) regulation in mammalian cells, we observed that the thermoresistant TR-4 cells appeared to be resistant to manipulations of the Na+/H+ antiport, one of the two primary membrane regulators of pHi. We hypothesized that this might be due to up-regulation of the alternate pHi regulator, the Na(+)-dependent HCO3-/Cl- exchanger, in the TR-4 cells. We have now evaluated the effect of heat exposure on the function of the Na(+)-dependent HCO3-/Cl- exchanger in both the parent RIF-1 and the thermoresistant variant TR-4 cells. We also assessed thermosensitivity of the cell lines under conditions of either pHe 7.2 or 6.8, with NaHCO3, with or without the addition of DIDS, an inhibitor of HCO3-/Cl- exchanger function. After 2h of heating at 43 degrees C, relative exchanger function declined to around 50% for the TR-4 cells and 10% for the RIF-1 cells. DIDS (0.2 mM) enhanced thermal cytotoxicity in both cells lines, by around 1 log when the cells were heated at neutral conditions, and by around 1.5 log when the condition became acidic. Results from pHi measurements during heating reflected the clonogenic survival data in that lower pHi levels were associated with the conditions when DIDS was present. We conclude that in the TR-4 cells, the Na(+)-dependent HCO3-/Cl- exchanger appears to be a more important regulator of pHi than the Na+/H+ antiport.

Purinergic regulation of anion secretion by cystic fibrosis pancreatic duct cells.

The present study explored regulation of anion secretion across cystic fibrosis pancreatic ductal epithelium by extracellular ATP with the short-circuit current (Isc) technique. CFPAC-1 cells grown on Millipore filters formed polarized monolayers with junctional complexes as revealed by light and electron microscopy. The cultured monolayers exhibited an increase in Isc in response to apical application of ATP in a concentration-dependent manner (concentration eliciting 50% of maximal response = 3 microM). Replacement of Cl- in the bathing solution or treatment of the cells with a Cl- channel blocker, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), markedly reduced Isc, indicating that a substantial portion of ATP-activated Isc was Cl- dependent. The effects of different adenosine nucleosides and/or nucleotides on Isc were also studied to identify the type of purinoceptors involved. The order of potency, ATP = UTP > ADP > adenosine, was consistent with that for P2 purinoceptors. Reactive blue 2 (100 microM), a P2 antagonist, was found to inhibit 86% of ATP-induced Isc. ATP-induced Isc was also inhibited by pretreatment of the cells with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (50 microM). Confocal microscopic study also demonstrated a rise in intracellular Ca2+ with stimulation by extracellular ATP, indicating a role of intracellular Ca2+ in mediating the ATP response. ATP-induced Isc was observed in monolayers whose basolateral membranes had been permeabilized by nystatin, which was also sensitive to apical addition of DIDS, suggesting that Isc was mediated by apical Cl- channels. The results of the present study demonstrate the presence of a purinergic regulatory mechanism involving P2U receptor and Ca2+ mobilization in pancreatic duct anion secretion.

The chronic administration of drugs that inhibit the regulation of intracellular pH: in vitro and anti-tumour effects

Mean values of extracellular pH (pHe) in tumours tend to be about 0.5 pH units lower than in normal tissues, whereas values of intracellular pH (pHi) in tumours and normal tissues are similar. Previous studies have shown that drugs that acidify cells at lower pHe such as nigericin, used alone or with agents that inhibit the regulation of pHi, have toxicity to cultured cells at pHe < 6.5 in short-term exposure; these agents also lead to modest anti-tumour effects in mice when given acutely. To evaluate the long-term effects of these drugs at levels of pHe that might occur commonly in tumours, we exposed cells for up to 72h at pHe 6.8 or 7.2 in vitro. Nigericin (0.033 microM) caused time-dependent cell killing of murine KHT and EMT-6 cells at pHe 6.8 (but not at pHe 7.2) with a surviving fraction approximately 5 x 10(-3) after 72 h exposure. Cell killing was increased in the presence of 4,4-diisothiocyanstilbene 2,2-disulphonic acid (DIDS), an inhibitor of Na+-dependent HCO3-/CI- exchange, and to a lesser extent in the presence of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an inhibitor of Na+/H+ exchange. Cell killing was exquisitely sensitive to the level of pHe. Osmotic pumps were used to obtain a 72 h continuous infusion of nigericin in mice; this led to dose-dependent killing of cells in KHT tumours with surviving fraction of approximately 0.1 at maximum tolerated doses. Hydralazine, which may cause tumour hypoxia and lower pHi as well as pHe, caused cytotoxity when given alone by chronic infusion, and enhanced the cytotoxicity due to nigericin. The addition of DIDS and/or EIPA (using two pumps) further enhanced anti-tumour toxicity, with a surviving fraction of approximately 0.002 at tolerated doses of the four drugs used to treat KHT tumours. The experiments demonstrate the activity of drugs that inhibit the regulation of pHi against murine tumours when delivered by chronic infusion.

Both CFTR and outwardly rectifying chloride channels contribute to cAMP-stimulated whole cell chloride currents.

From whole cell patch-clamp recordings at 35 degrees C utilizing either nystatin perforation or conventional methods with 5 mM MgATP in the pipette solution, it was demonstrated that both cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels and outwardly rectifying Cl- channels (ORCC) contribute to adenosine 3',5'-cyclic monophosphate (cAMP)-activated whole cell Cl- currents in cultured human airway epithelial cells. These results were similar whether recordings were performed on two normal human cell lines or on two cystic fibrosis (CF) cell lines stably complemented with wild-type CF gene. These results were obtained by exploiting dissimilar biophysical properties of CFTR and ORCC currents such as the degree of rectification of the current-voltage relationship, the difference in sensitivity to Cl- channel-blocking drugs such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), calixarenes, and diphenylamine carboxylic acid (DPC), and the opposing Cl- relative to I- permeabilities of the two channels. In normal cells or complemented CF cells, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate stimulated outwardly rectifying whole cell Cl- currents. Addition of DIDS in the presence of cAMP inhibited the outwardly rectifying portion of the cAMP-activated Cl- current. The remaining cAMP-activated, DIDS-insensitive, linear CFTR Cl- current was inhibited completely by DPC. Additional results showed that not only do ORCC and CFTR Cl- channels contribute to cAMP-activated Cl- currents in airway epithelial cells where wild-type CFTR is expressed but that both channels fail to respond to cAMP in delta F508-CFTR-containing CF airway cells. We conclude that CFTR not only functions as a cAMP-regulated Cl- channel in airway epithelial cells but also controls the regulation of ORCC.

4,4′-Di-isothiocyanatostilbene-2,2′-disulphonic acid (‘DIDS’) activates protein kinase C and Na+/H+ exchange in human platelets via α2A-adrenergic receptors

Most agonists stimulate platelets by inducing Ca2+ mobilization, Ca2+ influx and protein kinase C (PKC) activation leading to Na+/H+ exchange, exposure of fibrinogen-binding sites and aggregation. In contrast, previous studies showed that adrenaline induces exposure of fibrinogen-binding sites and aggregation without appreciable changes in cystolic Ca2+ content or PKC activity. In the present study we investigated platelet responses mediated via alpha 2A-adrenergic receptors, using 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), which is known to bind to this type of receptor. Addition of DIDS (2-20 microM) induced (i) a rise in cytosolic pH of 0.23 +/- 0.05 pH unit (n = 5) as detected by BCECF fluorescence, due to activation of the Na+/H+ exchanger, (ii) a 3.5-4-fold increase in the phosphorylation of the 47 kDa protein, a major substrate of PKC, (iii) exposure of 81,072 +/- 7293 (n = 3) binding sites for 125I-fibrinogen per platelet, and (iv) irreversible aggregation. These responses occurred without changes in cytosolic [Ca2+], secretion of dense-granule contents and enhanced phosphoinositide metabolism, and were not affected by inhibition of thromboxane A2 generation (30 microM indomethacin). The alpha 2A-adrenergic-receptor antagonists oxymetazoline (500 microM) and yohimbine (1 mM) completely abolished DIDS-induced responses. Inhibition of PKC (1 microM staurosporine) prevented phosphorylation of the 47 kDa protein, the increase in Na+/H+ exchange and exposure of fibrinogen-binding sites. Thus our present data suggest that activation of PKC is an early event in DIDS-induced platelet activation via the alpha 2A-adrenergic receptor, which precedes any of the other known signal-transducing sequences.

Anion-dependent transport of thallous ions through human erythrocyte membrane.

Undirectional fluxes of 204Tl+ through the human red blood cell membrane were measured. The inward rate coefficient measured in a K(+)-free saline was 15.6 +/- 0.6 hr-1. The influx of Tl+ could be partially inhibited with 0.1 mM ouabain (by 28%), 0.1 mM DIDS (by 50%) or 1 mM furosemide (by 51%). The inhibitory effects of ouabain and DIDS or furosemide were additive. Half-maximal responses were seen at 0.72 microM and 0.22 mM concentrations of DIDS and furosemide, respectively. A similar action of these blockers on Tl+ influx was observed in the erythrocytes incubated in MgCl2-sucrose media. The outward rate coefficient of 204Tl was also inhibited by DIDS and furosemide (by 65 and 52%, respectively). Rate coefficients of 204Tl influx and efflux decreased significantly in the red cells exposed to Cl(-)-free media (NaNO3 or Mg(NO3)2-sucrose). Under these conditions addition of DIDS and furosemide led to only a small inhibition of Tl+ fluxes. There was a linear increase in Tl+ influx with rising of external Cl- concentration within 80-155 mM or HCO3- concentration from 20 to 40 mM when the sum of anions was kept constant (155 mM) with NO3-. The HCO3(-)-stimulated Tl+ influx was completely blocked by 0.05 mM DIDS but only 67% by 1 mM furosemide. The present study provides direct evidence for the occurrence of Cl- (HCO3-)-dependent, DIDS-sensitive movement of Tl+ across the human erythrocyte membrane in both directions. Under physiological conditions, about half of net Tl+ fluxes occurs due to an anion exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)