Coupling modes and stoichiometry of Cl-/HCO3- exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3--buffered Cl--free media. These findings were used to develop a turnover cycle for Cl- and HCO3- transport by slc26a3. Cl- and HCO3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl--mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl- absorption and HCO3- secretion.     

Branchial and renal (Na+/K+) ATPase and carbonic anhydrase activities in a eurythermal freshwater teleost, Carassius auratus L

1. Plasma sodium and chloride levels were determined in goldfish, Carassius auratus L., following acclimation to 5, 15, 25 and 35 degrees C. Carbonic anhydrase and (Na+/K+)-stimulated ATPase activities of gill and kidney were also assayed at both acclimation temperature and 41 degrees C. 2. Consistent with earlier findings this eurythermal species exhibits more variation in plasma composition with temperature than do the more stenothermal salmonids. Seasonal changes were also observed. 3. Despite differences in detail the overall pattern of transport enzyme activity change with acclimation was comparable to that previously observed in trout. The goldfish is, however, notable for high levels of renal carbonic anhydrase activity, and presumably employs this system more than does the trout to drive urinary recovery of ions by H+:Na+ and HCO3-:Cl- exchanges.     

The Mechanism of Sodium and Chloride Uptake by the Gills of a Fresh-Water Fish, Carassius auratus : II. Evidence for NH4+ / NA+and HCO3- / Cl-exchanges

The addition of ammonium ions to the external medium results in an inhibition of the sodium influx and net uptake in Carassius auratus, while intraperitoneal injection of ammonium produces the opposite effect. The simultaneous chloride balance is not significantly affected by these treatments. The addition of bicarbonate ions to the external medium results in a reduction of the influx and net flux of chloride, while injection of bicarbonate produces the opposite effect. The simultaneous sodium balance is not significantly altered. The effects of the external additions are reversible after elimination of the excess ammonium or bicarbonate ions by rinsing. Inhibition of carbonic anhydrase in the gill by injection of acetazoleamide produces a simultaneous inhibition of both sodium and chloride exchanges. These results confirm the hypothesis of an exchange of sodium for ammonium, and of bicarbonate for chloride across the gill. A tentative schematic representation of the ionic absorption mechanisms in the branchial cell of the fresh-water teleosts is given. Similarities with other biological membranes and especially with the renal tubule are pointed out.     

Effects of lanthanum on sodium and chloride fluxes in the goldfishCarassius auratus

Summary The effect of lanthanum (La³⁺) on Na⁺ and Cl^– fluxes and on gill potentials was investigated in the goldfish,Carassius auratus. Initially La³⁺ caused large increases in both influx and efflux but after 15 min these decreased with influx returning to near normal levels while effluxes remained elevated, leading to a significant net loss of ions. Both La³⁺ and Ca²⁺ influenced gill potentials in an identical way. Possible physiological effects of La³⁺ on gill ion exchange mechanisms are discussed.     

Role of a Na+-dependent Cl-/HCO3- exchange in regulation of intracellular pH in fibroblasts

We previously reported that, in a HCO3(-)-free medium, cytoplasmic pH (pHi) of hamster fibroblasts (CCL39) is primarily regulated by an amiloride-sensitive Na+/H+ antiport (L'Allemain, G., Paris, S., and Pouysségur, J. (1984) J. Biol. Chem. 259, 5809-5815). Here we demonstrate the existence of an additional pHi-regulating mechanism in CCL39 cells, namely a Na+-dependent HCO3-/Cl- exchange. Evidence for this system is based on 36Cl- influx studies and on pHi measurements in PS120, a CCL39-derived mutant lacking the Na+/H+ antiport activity. 36Cl- influx rate is a saturable function of external [Cl-] (apparent Km approximately equal to 7 mM), is competitively inhibited by external HCO3- (KI approximately equal to 3 mM), and by stilbene derivatives (KI approximately equal to 20 microM for 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). Measurements of pHi recovery after an acute acid load indicate that PS120 cells possess an acid-extruding mechanism dependent on external HCO3-, which is inhibited by stilbene derivatives and requires external Na+. Since 22Na+ influx is stimulated upon addition of HCO3- to acid-loaded cells and this effect is completely abolished by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, we conclude that Na+ is co-transported with HCO3-, in exchange for intracellular Cl-. In a HCO3(-)-containing medium, this pHi-regulating mechanism appears to have two essential physiological functions for the Na+/H+ antiport-deficient mutant: protection of the cells against excessive cytoplasmic acidification and establishment of a steady-state pHi permissive for growth, at neutral or slightly acidic pHo values (6.6-7.2).     

Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl-/HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization.     

Cl-/HCO3- exchange at the apical membrane of Necturus gallbladder

The hypothesis of Cl-/HCO3- exchange across the apical membrane of the epithelial cells of Necturus gallbladder was tested by means of measurements of extracellular pH (pHo), intracellular pH (pHi), and Cl- activity (alpha Cli) with ion-sensitive microelectrodes. Luminal pH changes were measured after stopping mucosal superfusion with a solution of low buffering power. Under control conditions, the luminal solution acidifies when superfusion is stopped. Shortly after addition of the Na+/H+ exchange inhibitor amiloride (10(-3) M) to the superfusate, alkalinization was observed. During prolonged (10 min) exposure to amiloride, no significant pHo change occurred. Shortly after amiloride removal, luminal acidification increased, returning to control rates in 10 min. The absence of Na+ in the superfusate (TMA+ substitution) caused changes in the same direction, but they were larger than those observed with amiloride. Removal of Cl- (cyclamate or sulfate substitution) caused a short-lived increase in the rate of luminal acidification, followed by a return to control values (10-30 min). Upon re-exposure to Cl-, there was a transient reduction of luminal acidification. The initial increase in acidification produced by Cl- removal was partially inhibited by SITS (0.5 mM). The pHi increased rapidly and reversibly when the Cl- concentration of the mucosal bathing solution was reduced to nominally 0 mM. The pHi changes were larger in 10 mM HCO3-Ringer's than in 1 mM HEPES-Ringer's, which suggests that HCO3- is transported in exchange for Cl-. In both HEPES- and HCO3-Ringer's, SITS inhibited the pHi changes. Finally, intracellular acidification or alkalinization (partial replacement of NaCl with sodium propionate or ammonium chloride, respectively) caused a reversible decrease or increase of alpha Cli. These results support the hypothesis of apical membrane Cl-/HCO3- exchange, which can be dissociated from Na+/H+ exchange and operates under control conditions. The coexistence at the apical membrane of Na+/H+ and Cl-/HCO3- antiports suggests that NaCl entry can occur through these transporters.     

A Cl-/HCO-3-ATPase in the gills of Carassius auratus. Its inhibition by thiocyanate.

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl- and HCO-3, inhibited by SCN-. Biochemical characterization shows that HCO-3 stimulation (Km = 2.5 mequiv./l) is specifically inhibited in a competitive fashion by SCN- (Ki = 0.25 mequiv./l). This residual Mg2+-dependent activity is weakly affected by SCN-. In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO-3 (Km for chloride = 1 mequiv/l); no stimulation is observed in the absence of HCO-3. Thiocyanate exhibits a mixed type of inhibition (Ki = 0.06 mequiv./l) towards the Cl- stimulation of the enzyme. Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl-, but this enzyme has a relatively weak affinity for this substrate (Km = 14 mequiv./l).     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Mechanisms of ion and acid-base regulation at the gills of freshwater fish.

This review examines the branchial mechanisms utilized by freshwater fish to regulate internal acid-base status and presents a model to explain the underlying basis of the compensatory processes. Rainbow trout, Oncorhynchus mykiss, and brown bullhead, Ictalurus nebulosus, were examined under a variety of experimental treatments which induced respiratory and metabolic acid-base disturbances. Acid-base regulation was achieved by appropriate adjustments of Na+ and Cl- net fluxes across the gills which, in turn, were accomplished by variable contributions of three different branchial mechanisms: 1) differential changes in Na+ and Cl- diffusive effluxes, 2) changes in internal substrate (H+, HCO3-) availability, and 3) morphological adjustments to the gill epithelium. Differential diffusive efflux of Na+ over Cl- was involved only during periods of metabolic alkalosis. The importance of internal substrate availability was demonstrated using a two-substrate model. According to the model, ionic flux rates (J(in)Cl-, J(in)Na+) are determined not only by the concentration of the external ion (Na+, Cl-) but also by the concentration of the internal counterion (H+, HCO3-). This system provides for an "automatic negative feedback" to aid in the compensation of metabolic acid-base disturbances. Morphological alteration of the gill epithelia and the associated regulation of chloride cell (CC) fractional area is an essential third mechanism which is especially important during respiratory acid-base disturbances. Specifically, fish vary the availability of the CC associated Cl-/HCO3- exchange mechanism by physical covering/uncovering of CCs by adjacent pavement cells.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

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.     

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108-116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na+/Cl-/glycine cotransport system is discussed.     

Biochemical demonstration of HCO3--und Cl--dependent ATPase activities in the rectum of larval dragonflies and inhibition of rectal chloride uptake by thiocyanate (author's transl)

Hydrogencarbonate and chloride activated, ouabain-insensitive ATPase activities are demonstrated in the salt-absorbing rectum of larval dragonflies. Maximal activation is achieved at approx. 30 mM HCO3- and 20 mM Cl-, respectively. The stimulation of each anion obeys Michaelis-Menten kinetics Km values are 4.65 mM for HCO3-- and 10.25 mM for Cl--activation. The activating anion of one type of ATPase simultaneously exerts an inhibitory effect on the other. Cl--activation is also reduced by Mg.ATP in concentrations above 0.5 mM and by Tris-Hepes buffer exceeding 2.5 mM. Both anion-dependent ATPase activities are found enriched in subcellular membraneous fractions of the rectum. Thiocyanate inhibits both activities and causes a significant decrease in rectal uptake of radioactive chloride from hypo-osmotic external solution. In the case of HCO3- dependent ATPase a competitive inhibition as SCN- was found with an inhibitor constant of Ki=0.5 mM.     

Evidence for a major route for zinc uptake in human red blood cells: [Zn(HCO3)2Cl]- influx through the [Cl-/HCO3-] anion exchanger

The initial rate of Zn2+ uptake in human red cells was measured by atomic absorption. A very important fraction of Zn2+ uptake was inhibited by DIDS with IC50 = 0.3 microM (and by furosemide and bumetanide with IC50 of 200 and 500 microM, respectively). DIDS-sensitive Zn2+ uptake exhibited the following properties: 1) It required the simultaneous presence of both external HCO3- and Cl-. 2) In Cl- containing media, it was strongly stimulated by external HCO3- following a sigmoidal (S-shaped) and saturable function, which was fitted by a Hanes equation, with n = 2 and an apparent dissociation constant (for external HCO3-) of 5.3 +/- 0.9 mM (mean +/- SD of four experiments). The maximal rate of Zn2+ uptake at saturating HCO3- concentrations was 50.7 +/- 4.8 mmol (liter cells x h)-1. 3) In HCO3- containing media, it was strongly stimulated by external Cl- following a Michaelis-like equation with an apparent dissociation constant (for external Cl-) of 88 +/- 11 mM (mean +/- SD of three experiments). 4) Bicarbonate-stimulated Zn2+ uptake was inhibited by physiological concentrations of phosphate (sulfate was a much less potent inhibitor than phosphate). A kinetic analysis of the data strongly suggested that zinc was transported by the anion carrier in the form of the monovalent anion complex: [Zn(HCO3)2Cl]-.     

Sulfate transport in human neutrophils

The mechanism by which SO4(2-) is transported across the plasma membrane of isolated human neutrophils was investigated. Unlike the situation in erythrocytes, SO4(2-) and other divalent anions are not substrates for the principal Cl-/HCO3- exchange system in these cells. At an extracellular concentration of 2 mM, total one-way 35SO4(2-) influx and efflux in steady-state cells amounted to approximately 17 mumol/liter of cell water per min. The intracellular SO4(2-) content was approximately 1 mM, approximately 25-fold higher than the passive distribution level. Internal Cl- trans stimulated 35SO4(2-) influx. Conversely, 35SO4(2-) efflux was trans stimulated by external Cl- (Km approximately 25 mM) and by external SO4(2-) (Km approximately 14 mM), implying the presence of a SO4(2-)/Cl- countertransport mechanism. The exchange is noncompetitively inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2' -disulfonate (SITS) (Ki approximately 50 microM) and competitively blocked by alpha-cyano-4-hydroxycinnamate (Ki approximately 230 microM) and by ethacrynate (Ki approximately 7 microM); furosemide and probenecid also suppressed activity. The carrier exhibits broad specificity for a variety of monovalent (NO3- approximately Cl- greater than Br- greater than formate- greater than I- approximately p-aminohippurate-) and divalent WO4(2-) greater than oxalate2- greater than SO4(2-) greater than MoO4(2-) greater than SeO4(2-) greater than AsO4(2-) anions. There was little, if any, affinity for HCO3-, phosphate, or glucuronate. The influx of SO4(2-) is accompanied by an equivalent cotransport of H+, the ion pair H+ + SO4(2-) being transported together in exchange for Cl-, thereby preserving electroneutrality. These findings indicate the existence of a separate SO4(2-)/Cl- exchange carrier that is distinct from the neutrophil's Cl-/HCO3- exchanger. The SO4(2-) carrier shares several properties in common with the classical inorganic anion exchange mechanism of erythrocytes and with other SO4(2-) transport systems in renal and intestinal epithelia, Ehrlich ascites tumor cells, and astroglia.     

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.     

Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion

Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.     

Cystic fibrosis transmembrane conductance regulator regulates luminal Cl-/HCO3- exchange in mouse submandibular and pancreatic ducts.

We have demonstrated previously the regulation of Cl-/HCO3- exchange activity by the cystic fibrosis transmembrane conductance regulator (CFTR) in model systems of cells stably or transiently transfected with CFTR (Lee, M. G., Wigley, W. C., Zeng, W., Noel, L. E., Marino, C. R., Thomas, P. J., and Muallem, S. (1999) J. Biol. Chem. 274, 3414-3421). In the present work we examine the significance of this regulation in cells naturally expressing CFTR. These include the human colonic T84 cell line and the mouse submandibular gland and pancreatic ducts, tissues that express high levels of CFTR in the luminal membrane. As in heterologous expression systems, stimulation of T84 cells with forskolin increased the Cl-/HCO3- exchange activity independently of CFTR Cl- channel activity. Freshly isolated submandibular gland ducts from wild type mice showed variable Cl-/HCO3- exchange activity. Measurement of [Cl-]i revealed that this was largely the result of variable steady-state [Cl-]i. Membrane depolarization with 5 mM Ba2+ or 100 mM K+ increased and stabilized [Cl-]i. Under depolarized conditions wild type and DeltaF/DeltaF mice had comparable basal Cl-/HCO3- exchange activity. Notably, stimulation with forskolin increased Cl-/HCO3- exchange activity in submandibular gland ducts from wild type but not DeltaF/DeltaF mice. Microperfusion of the main pancreatic duct showed Cl-/HCO3- exchange activity in both the basolateral and luminal membranes. Stimulation of ducts from wild type animals with forskolin had no effect on basolateral but markedly stimulated luminal Cl-/HCO3- exchange activity. By contrast, forskolin had no effect on either basolateral or luminal Cl-/HCO3- exchange activity of ducts from DeltaF/DeltaF animals. We conclude that CFTR regulates luminal Cl-/HCO3- exchange activity in CFTR-expressing cells, and we discuss the possible physiological significance of these findings regarding cystic fibrosis.