Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

Evidence for an anion exchanger in the mouse lacrimal gland acinar cell membrane

Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H⁺ (pH_i) and Cl^– (aCl_i) activities with double-barreled ion-selective microelectrodes. In a HCO ₃ ^– -free solution of pH 7.4 (HEPES/Tris buffered), pH_i was 7.25 andaCl_i was 33mm. By an exposure to a HCO ₃ ^– (25mm HCO ₃ ^– /5% CO₂, pH 7.4) solution for 15 min,aCl_i was decreased to 25mm and pH_i was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO ₃ ^– concentration [HCO ₃ ^– ]_i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO ₃ ^– -free solution reversed the changes inaCl_i and pH_i. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCl_i, pH_i, and [HCO ₃ ^– ]_i caused by admission/withdrawal of the HCO ₃ ^– , solution and decreased the buffer value. Replacement of all Cl^– with gluconate in the HCO ₃ ^– solution increased pH_i, and readmission of Cl^– decreased pH_i. The rates of these changes in pH_i were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO ₃ ^– solution, a stimulation of intracellular HCO ₃ ^– production by exposing the tissue to 25mm NH ₄ ⁺ increasedaCl_i significantly. While in the HCO ₃ ^– -free solution or in the HCO ₃ ^– , solution containing DIDS, exposure to NH ₄ ⁺ had little effect onaCl_i. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl^–/HCO ₃ ^– exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl^–/HCO ₃ ^– exchange is discussed.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H _i ) and external Na⁺ (Na _o ) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na _i ) and pH (pH _i ) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH _i (from 5.7 to 7.4), external pH (pH _o ), Na _i and Na _o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na _i <2.0 mmol/liter cell, Na _o = 150mm) increased sigmoidally (Hill coefficient 2.5) when pH _i fell below 7.0 and the external pH _o was 8.0, but increased linearly at pH _o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH _o levels (pH _o 8 and pH _o 6). The H⁺-driven Na⁺ influx reached saturation between pH _i 5.9 and 6.1. TheV _max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK _m for H _i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na _o was studied at pH _i 6.0, and Na _i lower than 2 mmol/liter cell at pH _o 6.0 and 8.0. The meanK _m for Na _o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na _i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV _max between individuals.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

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 NH₄Cl 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 HCO₃^–-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 A₁, 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 HCO₃^– transport is the dominant mechanism besides an NHE and V-ATPase to control pH_i in goldfish gill cells.Communicated by G. Heldmaier     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture

Summary Ehrlich ascites tumor cells contain a Na⁺ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC₅₀ of 25 m and by dimethylamiloride with an IC₅₀ of 0.6 m at 1mm external Na⁺. Decrease of external Na⁺ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na⁺/H⁺ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na⁺/H⁺ antiport for internal protons in quiescent cells is demonstrated by the following findings: 1. The internal pH, at which the half-maximal activation of the amiloride-sensitive Na⁺ uptake occurs, is shifted from 6.85 to 7.1 at 1mm external Na⁺. 2. The threshold value of external pH, below which a pronounced effect of amiloride on steadystate internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na⁺ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na⁺/H⁺ antiporter to internal protons. The Na⁺/H⁺ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5mm. The data are discussed in view of existing models of mitogenic activity of transitory pH changes.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH _i ) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO ₃ ^- and 5% CO₂, pH 7.25, pH _i was 7.19±0.02 (sem, n = 95). On removing HCO ₃ ^- and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO ₃ ^- and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH _i , but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO ₃ ^- led to a rise in pH _i , while substitution in the absence of HCO ₃ ^- led to a fall in pH _i . The above data indicate a significant role for a sodium-dependent Cl^–-HCO ₃ ^- exchange mechanism in the regulation of pH _i . Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO ₃ ^- led to a marked fall in pH _i , indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH _i . There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH _i . Larger changes in pH _i were observed in control compared to HCO ₃ ^- -free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH _i with either NH ₄ ⁺ or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO ₃ ^- was 5 mm/pH unit (procaine). However, in the presence of HCO ₃ ^- and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO ₃ ^- is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus : I. Role of HCO3?

We studied the regulation of intracellular pH (pH_i) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2′,7′-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH_i. Intrinsic buffering power (β_I) was 10.5 mM (pH unit)⁻¹ at pH_i 7.0, and decreased linearly with pH_i; the best-fit line to the data had a slope of −10.0 mM (pH unit)⁻². In the absence of HCO₃ ⁻, pH_i recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH_i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH_i. Acid extrusion was inhibited 68% (pH_i 6.23) by substituting Li⁺ for Na⁺ in the bath solution. Switching from a CO₂/HCO₃ ⁻-free to a CO₂/HCO₃ ⁻-containing bath solution caused mean steady state pH_i to increase from 6.82 to 6.90, due to a Na⁺-driven HCO₃ ⁻ transporter. The HCO₃ ⁻-induced pH_i increase was unaffected by amiloride, but was inhibited 75% (pH_i 6.85) by 400 μM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and 65% (pH_i 6.55–6.75) by pretreating astrocytes for up to ∼6.3 h with 400 μM 4-acetamide-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). The CO₂/HCO₃ ⁻-induced pH_i increase was blocked when external Na⁺ was replaced with N-methyl-d-glucammonium (NMDG⁺). In the presence of HCO₃ ⁻, the Na⁺-driven HCO₃ ⁻ transporter contributed to the pH_i recovery from an acid load. For example, HCO₃ ⁻ shifted the plot of acid-extrusion rate vs. pH_i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO₃ ⁻ increased acid extrusion 3.8-fold (pH_i 6.20). When astrocytes were acid loaded in amiloride, with Li⁺ as the major cation, HCO₃ ⁻ failed to elicit a substantial increase in pH_i. Thus, Li⁺ does not appear to substitute well for Na⁺ on the HCO₃ ⁻ transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na⁺-driven HCO₃ ⁻ transporter are the predominant acid extruders in astrocytes.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

Summary The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na⁺-enriched cells. Subsequent incubation in K⁺-containing NaCl medium results in the reaccumulation of K⁺, Cl^–, water and the extrusion of Na⁺. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (K _M for K ₀ ⁺ =3.5mm;J _max=30.1 mEq/kg dry min), which in these experiments was coupled 1K⁺/1 Na⁺. The second is the Cl^–-dependent (Na⁺+K⁺) cotransport system (K _M for K ₀ ⁺ =6.8mm;J _max=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K⁺1Na⁺2Cl^–, the exchange of K _i ⁺ for K ₀ ⁺ . The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Functional roles of Na+ and H+ in SO 4 2− transport by rabbit ileal brush border membrane vesicles

Summary Sulphate uptake by rabbit ileal brush border membrane vesicles was stimulated by a transmembrane sodium gradient ([Na⁺] _o >[Na⁺] _i ), but not by a similar potassium gradient.³⁵SO ₄ ^2– influx (J _oi ^SO4 ) from outside (o) to inside (i) these vesicles was a hyperbolic function of [SO ₄ ^2– ] _o and the affinity constant for anion transport was strongly influenced by [Na⁺] _o (100mm Na⁺,K _t ^SO4 =0.52mm SO ₄ ^2– ; 10mm Na⁺,K _t ^SO4 =4.32mm SO ₄ ^2– ).J _t ^SO4 was a sigmoidal function of [Na⁺] _o at pH 7.4 for both low (0.2m) and high (4.0mm) [SO ₄ ^2– ] _o . The Na⁺-dependency ofJ _t ^SO4 was examined at pH 6.0, 7.4, and 8.0 (same pH inside and outside). At pH 6.0 and 7.4 sigmoidal Na⁺-dependentJ _t ^SO4 exhibited nonlinear Eadie-Hofstee plots indicative of a transport mechanism capable of binding a variable number of sodium ions over the [Na⁺] _o range used. Hill plots of anion transport under these conditions displayed slopes near unity at low [Na⁺] _o and slopes approximating 2.0 at higher cation concentrations. At pH 8.0, Na⁺-dependentJ _t ^SO4 was hyperbolic and showed linear Eadie-Hofstee and Hill plots, the latter with a single slope near 1.0. When a H⁺ gradient was imposed across the vesicle wall (pH _i =8.0, pH _o =6.0), Na⁺-dependentJ _t ^SO4 was hyperbolic and significantly increased at each [Na⁺] _o over values observed using bilateral pH 8.0. In contrast, a H⁺ gradient oriented in the opposite direction (pH _i =6.0, pH _o =8.0) led to Na⁺-dependentJ _t ^SO4 that was sigmoidal and significantly lower at each [Na⁺] _o than values found using bilateral pH 6.0. Electrogenicity ofJ _t ^SO4 at pH 8.0 for both high and low [Na⁺] _o was demonstrated by using a valinomycin-induced transmembrane electrical potential difference. At pH 6.0, electrogenicJ _t ^SO4 occurred only at low [Na⁺] _o (5mm); anion transfer was electroneutral at 50mm Na⁺. A model is proposed for proton regulation of sodium sulphate cotransport where flux stoichiometry is controlled by [H⁺] _i and sodium binding affinity is modified by [H⁺] _o . Preliminary experiments with rabbit proximal tubular brush border membrane vesicles disclosed similarJ _t ^SO4 kinetic properties and a common transport mechanism may occur in both tissues.     

Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick

Summary The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H⁺ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K⁺ plus valinomycin voltage clamp in order to prevent electrical coupling of ion fluxes. Net H⁺ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na⁺/H⁺ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na⁺ gradient. The kinetics of the Na⁺/H⁺ exchange reaction at pH 6.0 [K _t for Na⁺=57mm,V _max=42 mmol H⁺/liter 3OMG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (K _t for Na⁺=15mm,V _max=1.7 mmol H⁺/liter 3OMG space/min). Experiments involving imposed K⁺ gradients suggest that these cells have negligible K⁺/H⁺ exchange capability. They exhibit limited but measurable H⁺ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.