Mechanism of the increase in cation permeability of human erythrocytes in low-chloride media. Involvement of the anion transport protein capnophorin

When human erythrocytes are suspended in low-Cl- media (with sucrose replacing Cl-), there is a large increase in both the net efflux and permeability of K+. A substantial portion (greater than 70% with Cl- less than 12.5 mM) of this K+ efflux is inhibited by the anion exchange inhibitor DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid). This inhibition cannot be explained as an effect of DIDS on net Cl- permeability (Pcl) and membrane potential, but rather represents a direct effect on the K+ permeability. When cells are reacted with DIDS for different times, the inhibition of K+ efflux parallels that of Cl- exchange, which strongly indicates that the band 3 anion exchange protein (capnophorin) mediates the net K+ flux. Since a noncompetitive inhibitor of anion exchange, niflumic acid, has no effect on net K+ efflux, the net K+ flow does not seem to involve the band 3 conformational change that mediates anion exchange. The data suggest that in low-Cl- media, the anion selectivity of capnophorin decreases so that it can act as a very low-conductivity channel for cations. Na+ and Rb+, as well as K+, can utilize this pathway.     

Identification of a defective cAMP-stimulated Cl- channel in cystic fibrosis fibroblasts

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.     

Chloride transport in human fibroblasts is activated by hypotonic shock

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.     

Functional carboxyl groups in the red cell anion exchange protein. Modification with an impermeant carbodiimide

Anion exchange in human red blood cell membranes was inactivated using the impermeant carbodiimide 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide (EAC). The inactivation time course was biphasic: at 30 mM EAC, approximately 50% of the exchange capacity was inactivated within approximately 15 min; this was followed by a phase in which irreversible exchange inactivation was approximately 100-fold slower. The rate and extent of inactivation was enhanced in the presence of the nucleophile tyrosine ethyl ester (TEE), suggesting that the inactivation is the result of carboxyl group modification. Inactivation (to a maximum of 10% residual exchange activity) was also enhanced by the reversible inhibitor of anion exchange 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) at concentrations that were 10(3)-10(4) times higher than those necessary for inhibition of anion exchange. The extracellular binding site for stilbenedisulfonates is essentially intact after carbodiimide modification: the irreversible inhibitor of anion exchange 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) eliminated (most of) the residual exchange activity: DNDS inhibited the residual (DIDS-sensitive) Cl- at concentrations similar to those that inhibit Cl- exchange of unmodified membranes: and Cl- efflux is activated by extracellular Cl-, with half-maximal activation at approximately 3 mM Cl-, which is similar to the value for unmodified membranes. But the residual anion exchange function after maximum inactivation is insensitive to changes of extra- and intracellular pH between pH 5 and 7. The titratable group with a pKa of approximately 5.4, which must be deprotonated for normal function of the native anion exchanger, thus appears to be lost after EAC modification.     

Hypotonic shock activated Cl- and K+ pathways in human fibroblasts.

The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress.     

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.     

The relationship between anion exchange and net anion flow across the human red blood cell membrane

The conductive (net) anion permeability of human red blood cells was determined from net KCl or K2SO4 effluxes into low K+ media at high valinomycin concentrations, conditions under which the salt efflux is limited primarily by the net anion permeability. Disulfonic stilbenes, inhibitors of anion exchange, also inhibited KCl or K2SO4 efflux under these conditions, but were less effective at lower valinomycin concentrations where K+ permeability is the primary limiting factor. Various concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) had similar inhibitory effects on net and exchange sulfate fluxes, both of which were almost completely DIDS sensitive. In the case of Cl-, a high correlation was also found between inhibition of net and exchange fluxes, but in this case about 35% of the net flux was insensitive to DIDS. The net and exchange transport processes differed strikingly in their anion selectivity. Net chloride permeability was only four times as high as net sulfate permeability, whereas chloride exchange is over 10,000 times faster than sulfate exchange. Net OH-permeability, determined by an analogous method, was over four orders of magnitude larger than that of Cl-, but was also sensitive to DIDS. These data and others are discussed in terms of the possibility that a common element may be involved in both net and exchange anion transport.     

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.     

Transport of pyruvate nad lactate into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride-independent carrier.

The kinetics and activation energy of entry of pyruvate and lactate into the erythrocyte were studied at concentrations below 4 and 15mM respectively. The Km and Vmax. values for both substrates are reported, and it is shown that pyruvate inhibits competitively with respect to lactate and vice versa. In both cases the Km for the carboxylate as a substrate was the same as its Ki as an inhibitor. Alpha-Cyano-4-hydroxycinnamate and its analogues inhibited the uptake of both lactate and pyruvate competitively. Inhibition was also produced by treatment of cells with fluorodinitrobenzene but not with the thiol reagents or Pronase. At high concentrations of pyruvate or lactate (20mM), uptake of the carboxylate was accompanied by an efflux of Cl-ions. This efflux of Cl- was inhibited by alpha-cyano-4-hydroxycinnamate and picrate and could be totally abolished by very low (less than 10 muM) concentrations of the inhibitor of Cl- transport, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid. This inhibitor titrated out the chlordie efflux induced by pyruvate, bicarbonate, formate and fluoride, in each case total inhibition becoming apparent when approximately 1.2x10(6) molecules of inhibitor were present per erythrocyte, that is, about one inhibitor molecule per molecule of the Cl- carrier. Evan when Cl- efflux was totally blocked pyruvate and lactate uptake occurred. Kinetic evidence is presented which suggests that the Cl- carrier can transport pyruvate and lactate with a high Km and high Vmax., but that an additional carrier with a low Km and a low Vmax. also exists. This carrier catalyses the exchange of small carboxylate anions with intracellular lactate, is competitively inhibited by alpha-cyano-4-hydroxycinnamate and non-competitively inhibited by picrate. The Cl- carrier shows a reverse pattern of inhibition. It is concluded that net efflux of lactic acid from the cell must occur on the Cl- carrier and involve exchange with HCO3 - followed by loss of CO2. The low Km carrier might be used in pyruvate/lactate or acetoacetate/beta-hydroxybutyrate exchanges involved in transferring reducing power across the cell membrane. The possibility that the Cl- carrier exists in cells other than the erythrocyte is discussed. It is concluded that its presence in other cell membranes together with a low intracellular Cl- concentration would explain why the pH in the cytoplasm is lower than that of the blood, and why permeable carboxylate anions do not accumulate within the cell when added from outside.     

Simultaneous recording of cell volume changes and intracellular pH or Ca2+ concentration in single osteosarcoma cells UMR-106-01.

We present a new technique for the simultaneous measurement of cell volume changes and intracellular ionic activities in single cells. The technique uses measurement of changes in the concentration of intracellularly trapped fluorescent dyes to report relative cell volume. By using pH- or Ca(2+)-sensitive dyes and recording at the ion-sensitive and -insensitive (isosbestic) wavelengths, the method can measure both cell volume changes and intracellular ionic activities. The technique was used to study the mechanisms of regulatory volume decrease (RVD) in the osteosarcoma cell line UMR-106-01 grown on cover slips. Swelling cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hypotonic medium was followed by stable cytosolic acidification and a decrease in cell volume back toward normal. The recovery of cell volume could be blocked by depolarization, treatment with ouabain, or depletion of cell Cl-. These suggest the conductive efflux of K+ and Cl- during RVD. The cytosolic acidification that accompanied cell swelling was not blocked by amiloride, bafilomycin A, or removal of Cl- and could not be reproduced by depletion of cellular ATP. These findings exclude Na+/H+ and Cl-/HCO-3 exchange, intracellularly generated acid, or increased metabolism, respectively, as the cause of the acidification. The cell swelling-induced acidification was inhibited by depolarization, suggesting the involvement of an electrogenic pathway. The acidification, as well as RVD, was inhibited by short incubation with deoxyglucose, and these effects could not be reversed by valinomycin. Thus, the anionic pathway(s) participating in RVD and the acidification are sensitive to the cellular level of ATP. Together, these studies indicate that RVD in UMR-106-01 cells in HEPES-buffered medium is mediated by the conductive efflux of K+, Cl-, and OH-.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.     

On the water and proton permeabilities across membranes from erythrocyte ghosts

The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton-permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport.     

Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes.

Cultured normal (N) cystic fibrosis (CF) keratinocytes were evaluated for their Cl(-)-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl- channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl- channel was found with a similar incidence in N and CF apical keratinocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successful in either N or CF keratinocytes. Forskolin was not able to activate Cl- channels in N and CF cell-attached patches. The Ca(2+)-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl- channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl- transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl- and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl(-)-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways.     

Interactions of bromide, iodide, and fluoride with the pathways of chloride transport and diffusion in human neutrophils

Isolated human neutrophils possess three distinct pathways by which Cl- crosses the plasma membrane of steady state cells: anion exchange, active transport, and electrodiffusion. The purpose of the present work was to investigate the selectivity of each of these separate processes with respect to other external halide ions. (a) The bulk of total anion movements represents transport through an electrically silent anion-exchange mechanism that is insensitive to disulfonic stilbenes, but which can be competitively inhibited by alpha-cyano-4-hydroxycinnamate (CHC; Ki approximately 0.3 mM). The affinity of the external translocation site of the carrier for each of the different anions was determined (i) from substrate competition between Cl- and either Br-, F-, or I-, (ii) from trans stimulation of 36Cl- efflux as a function of the external concentrations of these anions, (iii) from changes in the apparent Ki for CHC depending on the nature of the replacement anion in the bathing medium, and (iv) from activation of 82Br- and 125I- influxes by their respective ions. Each was bound and transported at roughly similar rates (Vmax values all 1.0-1.4 meq/liter cell water.min); the order of decreasing affinities is Cl- greater than Br- greater than F- greater than I- (true Km values of 5, 9, 23, and 44 mM, respectively). These anions undergo 1:1 countertransport for internal Cl-. (b) There is a minor component of total Cl- influx that constitutes an active inward transport system for the intracellular accumulation of Cl- [( Cl-]i approximately 80 meq/liter cell water), fourfold higher than expected for passive distribution. This uptake is sensitive to intracellular ATP depletion by 2-deoxy-D-glucose and can be inhibited by furosemide, ethacrynic acid, and CHC, which also blocks anion exchange. This active Cl- uptake process binds and transports other members of the halide series in the sequence Cl- greater than Br- greater than I- greater than F- (Km values of 5, 8, 15, and 41 mM, respectively). (c) Electrodiffusive fluxes are small. CHC-resistant 82Br- and 125I- influxes behave as passive leak fluxes through low-conductance ion channels: they are nonsaturable and strongly voltage dependent. These anions permeate the putative Cl- channel in the sequence I- greater than Br- greater than Cl- with relative permeability ratios of 2.2:1.4:1, respectively, where PCl approximately 5 X 10(-9) cm/s.     

Cyclic AMP inhibits Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder epithelium

Intracellular microelectrode techniques were employed to study the effect of cyclic AMP on apical membrane Cl-/HCO3- exchange and electrodiffusive HCO3- transport in Necturus gallbladder epithelium. Intracellular cAMP levels were raised by addition of either the phosphodiesterase inhibitor theophylline (3 X 10(-3) M) or the adenylate cyclase activator forskolin (10(-5) M) to the serosal bathing solution. Measurements of pH in a poorly buffered control mucosal solution upon stopping superfusion show acidification, owing to secretion of both H+ and HCO3-. When the same experiment is performed after addition of amiloride or removal of Na+ from the mucosal bathing medium, alkalinization is observed since H+ transport is either inhibited or reversed, whereas HCO3- secretion persists. The changes in pH in both amiloride or Na-free medium were significantly decreased in theophylline-treated tissues. Theophylline had no effect on the initial rates of fall of intracellular Cl- activity (aCli) upon reducing mucosal solution [Cl-] to either 10 or 0 mM, although membrane voltage and resistance measurements were consistent with stimulation of apical membrane electrodiffusive Cl- permeability. Estimates of the conductive flux, obtained by either reducing simultaneously mucosal [Cl-] and [HCO3-] or lowering [Cl-] alone in the presence of a blocker of anion exchange (diphenylamine-2-carboxylate), indicate that elevation of intracellular cAMP inhibited the anion exchanger by approximately 50%. Measurements of net Cl- uptake upon increasing mucosal Cl- from nominally zero to levels ranging from 2.5 to 100 mM suggest that the mechanism of inhibition is a decrease in Vmax. Consistent with these results, the rate of intracellular alkalinization upon reducing external Cl- was also inhibited significantly by theophylline. Reducing mucosal solution [HCO3-] from 10 to 1 mM under control conditions caused intracellular acidification and an increase in aCli. Theophylline inhibited both changes, by 62 and 32%, respectively. These data indicate that elevation of intracellular cAMP inhibits apical membrane anion (Cl-/HCO3-) exchange. Studies of the effects of rapid changes in mucosal [HCO3-] on membrane voltages and the apparent ratio of membrane resistances, both in the presence and in the absence of theophylline, with or without Cl- in the mucosal solution, do not support the hypothesis that cAMP produces a sizable increase in apical membrane electrodiffusive HCO3- permeability.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.     

Chloride-stimulated sulfate efflux in Ehrlich ascites tumor cells: evidence for 1:1 coupling

The kinetics of Cl-SO4-(2) exchange in Ehrlich ascites tumor cells was investigated in an attempt to determine the stoichiometry of this process. When tumor cells, equilibrated in Cl--free, 25 mM SO4-(2) medium are placed in SO4-(2)-free, 25 mm Cl-medium, both the net amount and rate of Cl-uptake far exceeds SO4-(2) loss.. Addition of the anion transport inhibitor SITS (4-acetamido-4,-isothiocyano-stilbene-2,2'-disulfonic acid) greatly reduces sulfate efflux (97%), but has no measurable effect on chloride uptake. Addition of furosemide, a Cl-transport inhibitor, reduces chloride uptake 94% but is without effect on sulfate efflux. These findings suggest that a chloride permeability pathway exists distinct from that utilized by SO4-(2). SITS, when added to furosemide treated cells, further reduces chloride uptake as well as inhibiting sulfate efflux, and under these experimental conditions, a linear relationship exists between SITS-sensitive, net chloride uptake and sulfate loss. The slope of this line is 1.05 (correlation coefficient = 0.996) which suggests the stoichiometry of Cl-SO4-(2) exchange is 1:1. Assuming a 1:1 stoichiometry, measurement of the initial chloride influx and initial sulfate efflux indicate that 92% of net chloride uptake is independent of sulfate efflux. Taken altogether, these results support the contention that the tumor cell possesses a permeability pathway which facilitates the exchange of one sulfate for one chloride. Under conditions where anion transport is not inhibited, this coupling is obscured by a second and quantitatively more important pathway for chloride uptake. This pathway is SITS-insensitive, although partially inhibited by furosemide.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

A sensitive technique for the determination of anion exchange activities in brush-border membrane vesicles. Evidence for two exchangers with different affinities for HCO3- and SITS in rat intestinal epithelium

A large percentage (up to 70%) of 36Cl- influx in brush-border membrane vesicles from rat small intestine under equilibrium exchange conditions was found to be mediated by SITS-inhibitable anion exchange. This Cl-/anion exchange could be measured 10-15-times more sensitive by determining the uptake of trace amounts of 125I- driven by a large Cl- gradient (in greater than out) generated by passing the vesicles through an anion-exchange column. Voltage clamping of the vesicle membrane with K+ and valinomycin did not effect the chloride driven 125I- uptake, showing that the 'overshooting' I- uptake was not mediated by an electrical diffusion potential, as might be generated by the Cl- gradient in the presence of a chloride channel. The Cl-/anion exchange was further characterized in brush-border membrane vesicles from both rat ileum and jejunum by studying the inhibitory action of various anions on the Cl- driven I- uptake. NO3-, Cl-, SCN- and formate at 2 mM could inhibit Cl-/I- exchange for more than 80%. The ileal brush-border membrane vesicles displayed a clear heterogeneity with respect to the inhibitory action of SO2-(4), SITS and HCO-3 on Cl-/I- exchange. Approximately 30% of the Cl-/I- exchange was insensitive to SO2-(4) and showed a relatively low sensitivity to SITS (IC50 = 1 mM) but could be inhibited for 80% by 2 mM HCO-3. Presumably this component represents Cl-/OH- or Cl-/HCO-3 exchange. The residual 70% showed a high sensitivity to SO2-(4) (IC50 = 0.5 mM) and SITS (IC50 = 2.5 microM) but was less sensitive to HCO-3. This part of the exchange activity showed inhibition characteristics very similar to the Cl-/I- exchange in the jejunal vesicles. The latter process was also inhibited for 80% by 2 mM oxalate. As discussed in this paper both exchangers may be involved in the electroneutral transport of NaCl across the apical membrane of the small intestinal villus cell.     

Chloride permeability of membrane vesicles isolated from Torpedo californica electroplax.

The Cl- permeability of membrane vesicles prepared from the electric organ of the marine ray Torpedo californica was studied by means of radioactive tracer exchange and by measuring the changes in the scattered-light intensity caused by osmotically induced volume changes. Both types of experiments indicate that a substantial fraction of the vesicles is extremely permeable of Cl-. Furthermore, this permeability pathway is inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, a well-known inhibitory of anion transport in a variety of systems. The properties of this permeability pathway are consistent with its identification as the voltage-aged Cl- channel studied in planar bilayers.