Presence Of DIDS Research Articles

Functional Role of Basolateral ClC-2 Channels in the Regulation of Duodenal Anion Secretion in Mice.

Although ClC-2 channels are important in colonic Cl- secretion, it is unclear about their roles in small intestinal anion secretion. Therefore, we sought to examine whether ClC-2 channels play important roles in anion secretion, particularly duodenal bicarbonate secretion (DBS). Duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers. Both duodenal short-circuit current (Isc) and HCO3- secretion in vitro were simultaneously recorded. DBS in vivo was measured by a CO2-sensitive electrode. Lubiprostone, a selective ClC-2 activator, concentration-dependently increased both duodenal Isc and DBS only when applied basolaterally, but not when applied apically. Removal of extracellular Cl- abolished lubiprostone-induced duodenal Isc, but did not alter HCO3- secretion even in the presence of DIDS, a Cl-/HCO3- exchanger inhibitor. However, further addition of glibenclamide, a CFTR channel blocker, abolished lubiprostone-evoked HCO3- secretion. Moreover, lubiprostone-induced HCO3- secretion was impaired in CFTR-/- mice compared to wild-type littermates. Luminal perfusion of duodenal lumen with lubiprostone did not alter basal DBS in vivo, but lubiprostone (i.p.) was able to induce DBS, which was also significantly inhibited by Cd2+, a ClC-2 channel blocker. [Ca2+]cyt level, Ca2+-activated K+ channel- and cAMP-mediated duodenal Isc, and HCO3- secretion were unchanged by lubiprostone. We have provided the first evidence for the novel functional role of basolateral ClC-2 channels in the regulation of duodenal anion secretion.

Study of the interaction between DNP and DIDS with human hemoglobin as binary and ternary systems: spectroscopic and molecular modeling investigation

The combination of several drugs is necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease in the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. Here, the interaction between 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 2,4-Dinitrophenol (DNP) with Hemoglobin (Hb) was investigated by different spectroscopic and molecular modeling techniques. Fluorescence analysis was used to estimate the effect of the DIDS and DNP on Hb as well as to define the binding properties of binary and ternary complexes. The distance r between donor and acceptor was obtained by the FRET and found to be 2.25 and 2.13 nm for DIDS and DNP in binary and 2.08 and 2.07 nm for (Hb–DNP) DIDS and (Hb–DIDS) DNP complexes in ternary systems, respectively. Time-resolved fluorescence spectroscopy confirmed static quenching for Hb in the presence of DIDS and DNP in both systems. Furthermore, an increase in ellipticity values of Hb upon interaction with DIDS and DNP showed secondary structural changes of protein that determine to disrupt of hydrogen bonds and electrostatic interactions. Our results showed that the Hb destabilize in the presence of DIDS and DNP. Molecular modeling of the possible binding sites of DIDS and DNP in binary and ternary systems in Hb confirmed the experimental results.

Inhibition of Rhbg‐mediated NH3 and NH4+ transport by DIDS (1098.2)

Rhbg is a non‐erythroid Rh glycoprotein expressed at the basolateral membrane of renal collecting duct. In previous studies, we demonstrated that Rhbg transported both NH4+ and NH3. As such, the Rh transporter is unique in being able to transport both the ionic and the gaseous components of ammonia. Other studies have shown that DIDS inhibited CO2 transport by AQP1. This suggested that DIDS might also inhibit transport of other gases such as NH3 by Rh proteins. This study was conducted to test this hypothesis. To do so we used ion‐selective microelectrodes and two‐electrode voltage clamp to measure changes in surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+. All experiments were conducted on Xenopus oocytes expressing Rhbg. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH4Cl caused a decrease in pHs by 0.85±0.12 and an inward current of ‐58±10 nA. The decrease in pHs is caused by NH3 influx whereas the inward current is due to electrogenic NH4+ influx. In the presence of DIDS, NH4Cl caused a significantly smaller decrease in pHs (0.57±0.07) and current (‐29±5 nA). Similarly, exposing Rhbg‐oocytes to 5mM MA/MA+ (a substitute to NH3/NH4+) caused a decrease in pHs of 0.27±0.04 and an inward current of ‐29±6 nA. In the presence of DIDS, the MA/MA+ induced changes in pHs (0.17±0.03) and ΔI (‐11±2 nA) were also inhibited. DIDS had no effect on NH3/NH4+ transport in H2O‐injected oocytes.Our data indicate that 1) DIDS inhibits transport of NH3 & MA by Rhbg without affecting endogenous NH3 & MA transport. 2) DIDS also inhibits the electrogenic transport of NH4+ & MA+ by Rhbg. 3) This is the first report to show inhibition of both gas (NH3) and ionic (NH4+) transport by Rhbg.Grant Funding Source: Supported by VA Merit grant

Efflux of glutathione and glutathione complexes from human erythrocytes in response to vanadate

The main objective of the present study was to investigate if vanadate is extruded from the cells in a glutathione dependent manner resulting in the appearance of extracellular glutathione and complexes of glutathione with vanadium. Vanadate significantly depleted intracellular non-protein sulfhydryl (NPSH) levels in a time- and concentration-dependent manner. The intracellular NPSH level was decreased to 0.0 ± 0.0 μmol/ml erythrocyte when exposed to 10 mM of vanadate for 4h. Extracellular NPSH level was increased concomitantly with the intracellular decrease and reached to 0.1410 ± 0.005 μmol/ml erythrocyte in 4h. Intracellular decrease and extracellular increase in NPSH levels were significantly inhibited in the presence of DIDS, a chloride-bicarbonate exchanger which also mediates phosphate and arsenate transport in erythrocytes. In parallel with the increase in extracellular NPSH levels, significant increases in extracellular glutathione levels were detected following exposure to vanadate. Extracellular glutathione levels reached to 0.0150 ± 0.0.001, 0.0330 ± 0.001, and 0.0576 ± 0.002 μmol/ml erythrocyte with 1, 5, and 10 mM of vanadate respectively. Dimercaptosuccinic acid treatment of supernatants significantly increased the glutathione levels measured in the extracellular media. Utilization of MK571 an MRP inhibitor decreased the rate of glutathione efflux from erythrocytes suggesting a role for this membrane transporter in the process. A known methylation inhibitor periodate oxidized adenosine decreased the rate of glutathione efflux from erythrocytes. This observed decrease in extracellular GSH levels suggests that GSH release partly requires a proper cellular methylation process and that part of GSH detected in the extracellular media may arise from GSH-vandium complexes. The results of the present study indicate that human erythrocyte efflux glutathione in reduced free form and in conjugated form/s that can be recovered with dimercaptosuccinic acid when exposed to vanadate.

DIDS exerts a cardioprotective effect on tunicamycin-induced apoptosis via reduction of endoplasmic reticulum stress

Aims4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a non-selective chloride channel blocker, has been shown to prevent apoptosis-induced by pathways of death receptor and mitochondria. However, the relationship between DIDS and apoptosis-induced by endoplasmic...

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

• To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. • Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). • Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). • The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.

Sodium–bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport

In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS; 0.5 mM). Replacing Cl(-) with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO(3) in 5% CO(2)) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.

Abstract 4454: Modulation of the early cytotoxic effects of Irvalec® by zinc and DIDS in lung tumor cells

Abstract Irvalec®, a marine-derived depsipeptide, has showed potent cytotoxic activity in vitro against a wide variety of human tumors, including breast, colon, pancreas, lung and prostate. Irvalec® is currently undergoing phase II clinical studies in non-small lung cancer. Exposure of tumor cells to Irvalec® induces necrotic cell death, a process associated with rapid loss of membrane integrity and subsequent cell permeabilization. In dose-response experiments using different tumor cell lines, very similar IC50 values were obtained after short (30 min) and long (72 h) exposure times to the drug, suggesting that the compound exerts its cytotoxic effect immediately after drug treatment. Tumor cells exposed to effective concentrations of Irvalec® underwent rapid and dramatic morphological changes, including cell blebbing, severe swelling, and plasma membrane permeabilization (as detected by propidium iodide (PI) uptake and LDH release) that irrevocably lead to cell lysis. Interestingly, membrane damage was observed only after reaching a threshold concentration of around 1-5 µM and only in the continuous presence of the compound in the culture medium. Compounds acting at the cell membrane have been reported to have similar cellular effects than those observed with Irvalec®. The activity of these compounds was inhibited by the use of different cell membrane protectors. To test if these protectors exert some effect against the cytotoxic activity of Irvalec® we exposed A549 cells to a panel of 20 different compounds and then treated with Irvalec®. The cytotoxic activity of the drug was evaluated by PI nuclear staining by fluorescence microscopy, time-lapse videomicroscopy and plate fluorimetry. Among the compounds tested, only DIDS (dihydro-4,4′ diisothiocyanostilbene-2,2′-disulphonic acid) and zinc salts resulted in a drastic reduction of the early effects of Irvalec® in A549 cells. In the presence of DIDS or zinc, a 70% and 90% decrease was observed, respectively, in the cytotoxicity of the drug. Altogether, these results suggest that Irvalec® induces a rapid hydroelectrolitic disbalance in tumor cells, causing severe alterations that lead to a necrotic cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4454.

A role for the volume regulated anion channel in volume regulation in the murine CNS cell line, CAD

The role of the volume regulated anion channel (VRAC) in a model CNS neuronal cell line, CAD, was investigated. Changes in cell volume following hypotonic challenges were measured using a video-imaging technique. The effect of the Cl(-) channel antagonists tamoxifen (10 microm) and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 microm) on regulatory volume decrease (RVD) were measured. The whole-cell voltage-clamp technique was used to characterize ICl(swell), the current underlying the VRAC. Using the video-imaging technique, CAD cells were found to swell and subsequently exhibit RVD when subjected to a sustained hypotonic challenge from 300 mOsmol kg(-1) H(2)O to 210 mOsmol kg(-1) H(2)O. In the presence of tamoxifen (10 microm) or DIDS (100 microm) RVD was abolished, suggesting a role for the VRAC. A hypotonic solution (230 mOsmol kg(-1) H(2)O) evoked ICl(swell), an outwardly rectifying current displaying time-independent activation, which reversed upon return to isotonic conditions. The reversal potential (E(rev)) for ICl(swell) was -14.7 + or - 1.4 mV, similar to the theoretical E(rev) for a selective Cl(-) conductance. ICl(swell) was inhibited in the presence of DIDS (100 microm) and tamoxifen (10 microm), the DIDS inhibition being voltage dependent. Osmotic swelling elicits an outwardly rectifying Cl(-) conductance in CAD cells. The ICl(swell) observed in these cells is similar to that observed in other cells, and is likely to provide a pathway for the loss of Cl(-) which leads to water loss and RVD. As ischaemia, brain trauma, hypoxia and other brain pathologies can cause cell swelling, CAD cells represent a model cell line for the study of neuronal cell volume regulation.

Vasomotion has chloride-dependency in rat mesenteric small arteries

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(Ca,cGMP))) chloride conductances are important. The effect of anion substitution and Cl(-) channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl(-) (o) replacement with aspartate blocked vasomotion while 36mM SCN(-) (o) (substituted for Cl(-)) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca(2+)](i) disappeared with 36mM SCN(-). DIDS and Zn(2+) blocked I (Cl(Ca,cGMP)) but not I (Cl(Ca)). Vasomotion was not sensitive to DIDS and Zn(2+), and DIDS and Zn(2+) induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn(2+) was sensitive to 36mM SCN(-) (o). The anion substitution data indicate that Cl(-) is crucial for the V (m) and [Ca(2+)](i) oscillations underlying vasomotion. The Cl(-) channel blocker data are consistent with both CaCCs being important.

Effects of Na + and Cl − Channel Blockers on Guinea‐pig (GP) Isolated Tracheal epithelial cell (EC) shrinkage in response to hyperosmolar challenge

In GP trachea that are precontracted with methacholine (MCh; 3H10−7 M), hyperosmolar D-mannitol (D-M) induces changes in epithelial Na+ and ClΓ transport and smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). We examined the effects of sequentially-added ion channel blockers, MCh and D-M (30 mosM) on EC cell volume (CV) changes in ECs isolated enzymatically (2% protease, 1 h) from GP-trachea and suspended in Krebs-Henseleit solution, under conditions in which these agents are used in the isolated, perfused trachea preparation to examine D-M-induced EpDRF release. CV was measured with a cell sizer. D-M elicited shrinkage of EC (~15 %) under conditions in which it caused EpDRF-mediated relaxation. The Na+ channel blocker, amiloride (A; 10−4 M), did not affect EC CV. MCh had no effect on CV (±A). Shrinkage stimulated by D-M was unaffected by A. On the other hand, incubation with the ClΓ blocker, DIDS (10−4 M), resulted in an immediate and prolonged decrease in CV (~9%). Again, MCh had no effect on CV (±DIDS). However, in the presence of DIDS the CV decrease in response to D-M was diminished. Thus, the inhibitory effect of A on EpDRF release is not due to an effect on EC shrinkage, whereas the inhibitory effect of DIDS on EpDRF release may be linked to the decrease in the CV response. The findings and conclusions in this abstract have not been formally disseminated by the National Institute for Occupational Safety and Health and should not be construed to represent any agency determination of policy. Funded by NIOSH.

Functional characterization of Cl-/HCO3- exchange in villous cells of the mouse ileum

At least three kinds of Cl(-)/HCO(3)(-) exchangers, SLC26A3, SLC26A6 and AE2, have been demonstrated to be expressed in the intestinal epithelial cell. To examine the functional expression of these exchangers in the native enterocyte, we studied the Cl(-)/HCO(3)(-)- exchange activity in isolated villi from the mouse ileum by microfluorometric intracellular pH (pH(i)) measurement. The pH(i) value increased upon Cl(-) removal when the villus was superfused with an HCO(3)(-)/CO(2)-buffered solution, while the response was blunted when superfused with an HCO(3)(-)/CO(2)-free, Hepes-buffered solution. The recovery of pH(i) value induced by Cl(-) re-addition (after initial Cl(-) removal) was totally or partially mimicked by the addition of Br(-), I(-), F(-), NO(3)(-), or SO(4)(2-) (in the absence of Cl(-)). The increase in pH(i) value induced by Cl(-) removal was partially inhibited in the presence of DIDS (30 muM), tenidap (10 muM), niflumic acid (30 muM) or NPPB (30 muM). Increasing the K(+) concentration from 5 mM to 60 mM in the superfusion solution induced a reversible increase in pH(i) value under the HCO(3)(-)/CO(2)-buffered condition, while it had hardly any effect on pH(i) under the Hepesbuffered condition. The K(+)-induced pH(i) changes were partially suppressed by removing Cl(-) from the superfusion solution. These results, together with the reported findings of mouse slc26a3, slc26a6 and AE2 in heterologously expressed systems, suggest the possibility that these three exchangers may all be functionally expressed in mouse ileal villous cells.

Acid?base regulation in isolated gill cells of the goldfish (Carassius auratus)

Mechanisms of acid release and intracellular pH (pH(i)) homeostasis were analysed in goldfish (Carassius auratus) gill cells in primary culture. The rate of acid secretion was measured using a cytosensor microphysiometer, and pH(i) was determined using the fluorescent probe 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF). Amiloride, a Na(+) channel and Na(+)/H(+) exchanger (NHE) inhibitor, had no effect on pH(i), but acid secretion of the gill cells was significantly impaired. In the presence of amiloride, the intracellular acidification (achieved using the NH(4)Cl pulse technique) was more severe than in the absence of amiloride, and recovery from the acidosis was slowed down. Accordingly, acid secretion of gill cells was severely reduced in the absence of extracellular Na(+). Under steady-state conditions, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid (DIDS), a HCO(3)(-)-transport inhibitor, caused a slow acidification of pH(i), and acid secretion was significantly reduced. No recovery from intracellular acidification was observed in the presence of DIDS. Bafilomycin A(1), an inhibitor of V-ATPase, had no effect on steady-state pH(i) and recovery from an intracellular acidification, whereas the rate of acid secretion under steady-state conditions was slightly reduced. Immunohistochemistry clearly revealed the presence of the V-ATPase B-subunit in goldfish gill lamellae. Taken together, these results suggest that a Na(+)-dependent HCO(3)(-) transport is the dominant mechanism besides an NHE and V-ATPase to control pH(i) in goldfish gill cells.

Functional and Molecular Characterization of a Volume-activated Chloride Channel in Rabbit Corneal Epithelial Cells

We characterized the functional and molecular properties of a volume-regulated anion channel (VRAC) in SV40-immortalized rabbit corneal epithelial cells (tRCE), since they mediate a robust regulatory volume decrease (RVD) response during exposure to a hypotonic challenge. Whole-cell patch clamp-monitored chloride currents and light-scattering measurements evaluated temporal cell-volume responsiveness to hypoosmotic challenges. Exposure to 200 mOsm medium elicited an outwardly-rectifying current (VACC), which was reversible upon reperfusion with isotonic (300 mOsm) medium. VACC and RVD were chloride-dependent because either chloride removal or application of NPPB (100 microM) suppressed these responses. VACC behavior exhibited voltage-dependent inhibition in the presence of DIDS (500 microM), whereas inhibition by both NPPB (100 microM) and niflumic acid (500 microM) was voltage-independent. VACC was insensitive to glibenclamide (250 microM), verapamil (500 microM) or removal of extracellular calcium. Phorbol dibutyrate, PDBu, (100 nM) had no effect on activated VACC. However, preincubation with PDBu prior to hypotonic challenge prevented VACC and RVD responses as well as prolonged characteristic time. An inactive phorbol ester analogue had no effect on RVD behavior. Moreover, Northern blot analysis verified expression of ClC-3 gene transcripts. The presence of ClC-3 transcripts along with the correspondence between the effects of known ClC-3 inhibitors on VACC and RVD suggest that ClC-3 activation underlies these responses to hypotonic-induced cell swelling.

Different mechanisms mediate uptake of lead in a rat astroglial cell line.

The mechanism by which lead (Pb) enters astrocytes was examined in a rat astroglial cell line in order to characterize specific pathways for transport. Pb uptake was saturable at pH 5.5 and 7.4, although quantitative differences existed in the Michaelis-Menten constants. At pH 7.4, the Vmax and Km were 2700 fmoles/mg protein/min and 13.4 microM, respectively, whereas the Vmax and Km were 329 fmoles/mg and 8.2 microM in the buffer at pH 5.5, respectively. The presence of extracellular iron inhibited uptake in a buffer at pH 5.5 but not at pH 7.4. Cells treated with the iron chelator deferoxamine displayed higher levels of the iron transporter divalent metal transporter 1 (DMT1) mRNA and protein, and consistent with increased DMT1 expression, the treated cells displayed greater uptake of Pb in the buffer at pH 5.5 but not at pH 7.4. Alternatively, at pH 7.4, the transport of Pb was blocked by the anion transporter inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), which bound to cell surface proteins at concentrations that were similar to those that blocked Pb uptake. DIDS did not inhibit uptake of Pb in the buffer at pH 5.5. Greater uptake of Pb was observed in a buffer containing sodium bicarbonate, which was abrogated in the presence of DIDS. In summary, the astroglial cell line displays two distinct pH-sensitive transport mechanisms for Pb.

The interaction of haemoglobin, magnesium, organic phosphates and band 3 protein in nucleated and anucleated erythrocytes.

The anion influx was measured in order to study the interaction among organic phosphates, magnesium, haemoglobin and the N-terminal of the cytoplasmic domain of band 3 protein in human, chicken and trout erythrocytes. The rate constant for SO(4)(2-) influx in human and trout erythrocytes increased significantly when it was measured with an increased concentration of intracellular Mg(2+). The SO(4)(2-) influx was also measured in human erythrocyte ghosts in the presence and absence of Mg(2+). The smaller activation provoked by Mg(2+) in ghosts could be caused by the presence of a small quantity of haemoglobin which remained inside. The SO(4)(2-) uptake in chicken erythrocytes in the presence and in absence of Mg(2+) was characterized by very similar rate constants. The results suggest that the small increase in intracellular Mg(2+) in the erythrocytes involves an increase in the formation of Mg(2+)-ATP and Mg(2+)-2,3 BPG complexes reducing the affinity of the organic phosphates for Hb. This new situation may influence the functions of the anion transporter with consequent variations of SO(4)(2-) influx throughout the erythrocyte membrane in human and in trout erythrocytes, whereas in chicken RBCs this function cannot occur and, in fact, no increase in sulphate influx was noticeable. The measurement of Hb/O(2) affinity by the use of alternating fixed and variable concentrations of organic phosphates and Mg(2+), confirms the interactions between these elements and their effect on the mechanism of the affinity. When we measured the sulphate influx in the presence of DIDS we found some differences in the three types of cells.

Okadaic acid, a protein phosphatase inhibitor, stimulates the activity of Na+/H+ and Na+-independent Cl-/HCO3- exchangers in human lymphocytes.

The cytosolic pH (pHi) regulatory mechanisms of peripheral blood human lymphocytes and the effect of okadaic acid on the activity of these mechanisms were studied by means of fluorescence imaging microscopy of 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (BCECF)-loaded individual cells. Lymphocytes recover from a CO(2)-induced acid load in an extracellular Na+-dependent, intracellular Cl- -independent fashion. This pHi recovery is highly sensitive to the anion exchange inhibitor 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and minimally sensitive to the Na+/H+ exchange inhibitor amiloride, suggesting that it is mostly due to the action of a Na+-dependent HCO3- transporter. Extracellular Cl- and Na+ removal experiments point to the existence of a DIDS-sensitive Na+-independent Cl-/HCO3- exchanger. Preincubation with okadaic acid stimulates the pHi recovery rate from a CO2-induced acid load in the presence of DIDS (0.002 pHu/min vs. 0.065 pHu/min), but not in the presence of amiloride. Okadaic acid also accelerates the pHi elevation induced by Cl- removal (0.039 pHu/min vs. 0.067 pHu/min). In summary, these results indicate that okadaic acid stimulates the activity of Na+/H+ and Na+-independent Cl-/HCO3- exchangers, but has no effect on the activity of the Na+-dependent HCO3- transporter of human lymphocytes.

Cyclic AMP and anionic currents in porcine ciliary epithelium.

To investigate whether in the ciliary epithelium of isolated porcine ciliary body cyclic 3',5' adenosine monophosphate (cAMP) activates transmembrane anionic currents. Changes in membrane potential induced either by the adenylcyclase activator forskolin (10 microM; n = 4) or the stable membrane permeable cAMP analog 8-bromo-adenosine 3',5'-cyclic monophosphothioate (8-br-cAMP; 30 microM; n = 4) were measured with intracellular microelectrodes. The effect of the drugs were assessed in the absence or in the presence of the non-selective anionic channel/transporter inhibitor diisothiocyanatostilbene-2,2' disulfonic acid (DIDS; 1 mM; n = 4). Significant (p < 0.001) membrane potential depolarization were induced by both forskolin (11.8 +/- 0.3 mV) or 8-br-cAMP (9.3 +/- 0.4 mV). In the presence of DIDS, a significant (p < 0.001) inhibition of the depolarization evoked by forskolin (0.9 +/- 1.1 mV) and 8-bromo-cAMP (0.7 +/- 0.2 mV) was observed. In the ciliary epithelium of isolated porcine ciliary body cAMP induces membrane potential depolarization through a process that could involve anionic channels.

DRA expression and brush border membrane anion exchange characteristics in rat and rabbit duodenum

Backround and Aims: Duodenal bicarbonate secretion in the resting state is in part mediated by an apical anion exchanger whose molecular nature is unknown. Recent data suggest that the dra gene, which is defective in congenital chlorid diarrhea, encodes an anion exchanger which, in oocytes, mediates sulfate as well as Cl uptake. We now investigated if the DRA gene product may be the apical anion exchanger in the duodenum. Methods: To test this hypothesis, we cloned rat and rabbit DRA cDNA fragments and studied the relative expression levels of DRA and NHE3 (Na+/H+ exchanger isoform 3) in the different segments of the rat and rabbit intestine by Northern analysis and semiquantitative PCR. We then isolated rat and rabbit duodenal and jejunal brush border membrane vesicles and studied 36Clund 35S0~uptake into Cl, HCOj, SO~and gluconate-loaded vesicles in the absence and presence of a pH-gradient (to distinguish SO~from HS04 transport) and the absence and presence of DIDS. We also examined HC03secretion in stripped rat and rabbit intestinal mucosa in the Ussing-chamber. Results: DRA was highly expressed in rat and rabbit intestinal tract, with the expression pattern colon>duodenum>ileum>jejunum in the rat and ileumc-colon> duodenum in the rabbit, and clearly differed from the expression pattern for NHE3 which was colon>ileum> jejunum>duodenum in both species. This excess DRA over NHE3 expression in the small intestine but not the proximal colon may explain why the resting state (absorbing) small intestine in the Ussing-chamber displays electroneutral HC03secretion whereas the proximal colon acidifies the luminal perfusate under resting conditions. Both rat and rabbit BBMvs displayed DIDS-sensitive CIICI-, HC03-/Ct, SO~-/Cl, and CIISO~exchange, findings compatible with DRA characteristics. Anion affinity for the internal and external anion binding sites differed somewhat between rat and rabbit but were Cl >HCOj in both species. Summary and Conclusions: The high duodenal DRA expression levels and the fact that the transport characteristics of rat and rabbit BBM anion exchange corresponds with those found in oocytes and Hek293 cells suggests that DRA is the likely transport protein which mediates electroneutral HCOj secretion in the duodenum.

A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase.

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.