Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Kinetics of charge translocation in the passive downhill uptake mode of the Na/H antiporter NhaA of Escherichia coli

The Na⁺/H⁺ antiporter NhaA is the main Na⁺ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li⁺ and Na⁺ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na⁺ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent K_m values were 11 mM for Na⁺ and 7.3 mM for Li⁺ at basic pH and 180 mM for Na⁺ and 50 mM for Li⁺ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent K_m as well as turnover (V_max).     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

SALINITY ADAPTATION BY DUNALIELLA TERTIOLECTA. I. INCREASES IN CARBONIC ANHYDRASE ACTIVITY AND EVIDENCE FOR A LIGHT-DEPENDENT Na+/H+ EXCHANGE1,2

When Dunaliella tertiolecta, previously adapted to medium containing 0.5 M NaCl, is transferred to higher salinities, there is a lag in growth, suggesting an adaptation period. Since there is no significant difference in the Na⁺ content of cells grown between 0.5 and 3.5 M NaCl, a mechanism for Na⁺ extrusion or exclusion is indicated. Increasing the salinity of cell suspensions stimulates an incorporation of H⁺ by the cells, suggesting an H⁺/Na⁺ exchange. Cells adapted to higher salinities have, increased carbonic anhydrase activity, suggesting that increased CO₂ or HCO₃^? transport may be required at higher salinities. Growth, of D. tertiolecta at salinities above 2.5 M requires continuous illumination; therefore a light-driven H⁺/Na⁺ exchange accompanied by a HCO₃^? influx is proposed.     

The excretion of hydrogen ion by the isolated amphibian skin: Effects of antidiuretic hormone and amiloride

H⁺ extrusion by the isolated skins of two amphibia, Rana ridibunda and Bufo bufo was studied in order to test for the presence of exchange mechanisms of the type Na⁺/H⁺ and Cl^?/HCO₃^?, which have been described in several epithelial structures. The preparations were mounted in chambers of the Ussing type, so that the short-circuit current could be used as a function of Na⁺ transport and the pH-stat technique was utilized to determine the rates of H⁺ extrusion under different experimental conditions. These conditions were either the withdrawal of the ions intervening in the mentioned exchanges (Cl^- or Na⁺, or the addition of drugs with well-known effects on Na⁺ uptake and transport (antidiuretic hormone and amiloride).In the frog skin, H⁺ excretion was detected in solutions containing either Cl^? or SO₄^2?, with identical rates. Again, Na⁺ substitution by Mg²⁺ had no effect on H⁺ excretion rates, neither did the suppression of Na⁺ influx by amiloride or its stimulation by antidiuretic hormone. These experiments were repeated with similar results in gland-free preparations of the epidermis of frog skin separated from the corion by the action of collagenase.Experiments in toad skin showed that H⁺ excretion could not be detected when Cl^? was present in the outer medium, but became apparent if an impermeant anion, SO₄^2?, was used. This observation is compatible with the existence of an exchange mechanism of the type Cl^?/HCO₃^?. Secondly, in these preparations H⁺ extrusion increased after stimulation with antidiuretic hormone and decreased when amiloride was used or when Na⁺ was substituted by Mg²⁺, suggesting that at least a fraction of the total H⁺ efflux is linked to Na⁺ influx. In the isolated frog skin this mechanism does not seem to be operative.     

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of the na-requirement in cyanobacterial photosynthesis

The Na⁺ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li⁺ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O₂ evolution) as a function of Na⁺ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na⁺ (K_½[Na⁺]) were calculated. The value of both the low and the high K_½(Na⁺) was dependent upon extracellular DIC concentration. The low K_½(Na⁺) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K_½(Na⁺) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na⁺ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na⁺ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO₂ and HCO₃⁻ transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO₂ and HCO₃⁻ transport systems required Na⁺, directly or indirectly, for full activity. However, micromolar levels of Na⁺ were required for CO₂ transport while millimolar levels were required for HCO₃⁻ transport. These levels corresponded to those found for the low and high K_½(Na⁺) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO₂ transport increased with increased DIC concentration relative to HCO₃⁻ transport. This change was reflected in a decrease in the Na⁺ concentration required for maximum photosynthesis, in accord with the lower Na⁺-requirement for CO₂ transport. Lithium competitively inhibited Na⁺-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na⁺-dependent HCO₃⁻ transport. Lithium had little effect on CO₂ transport and only a small effect on the size of the pool it generated. Thus, CO₂ transport did not require a functional HCO₃⁻ transport system for full activity. Based on these observations and the differential requirement for Na⁺ in the CO₂ and HCO₃⁻ transport system, it was proposed that CO₂ and HCO₃⁻ were transported across the membrane by different transport systems.     

Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

Energy-dependent contraction of swollen heart mitochondria--activation by butacaine.

Butacaine and certain other local anesthetics markedly stimulate the rate, extent, and efficiency of respiration-dependent contraction of heart mitochondria in nitrate salts at alkaline pH. The local anesthetics also induce respiratory control associated with contraction (i.e., the elevated rate of respiration during contraction declines to a State 4-like controlled rate when contraction is complete) so that the reaction at alkaline pH closely resembles the rapid and highly efficient process seen at neutral pH. Respiration-dependent contraction appears to be an osmotic response to cation extrusion on an endogenous cation/H⁺ exchanger (G. P. Brierley, M. Jurkowitz, E. Chavez, and D. W. Jung, 1977, J. Biol. Chem.252, 7932–7939). At alkaline pH, net ion extrusion is slow and inefficient due to the elevated permeability of the membrane to monovalent cations through a putative uniport pathway. Butacaine and other local anesthetics seem to decrease influx-efflux cycling of cations at alkaline pH by restricting cation influx through this uniport. Passive swelling at pH 8.3 in nitrate salts indicates that the uniport reaction is sensitive to Ca²⁺ and has a cation-selectivity of Na⁺ > K⁺ > Li⁺. Butacaine does not inhibit passive swelling under these conditions but produces effects identical to those of classical uncouplers and consistent with increased H⁺ conductance and accelerated influx of cations by cation/H⁺ exchange in nonrespiring mitochondria. However, since contraction in respiring mitochondria is inhibited by uncouplers but stimulated by butacaine, it is apparent that butacaine is not an effective proton conductor in energized mitochondria.     

CO2-concentrating mechanisms: a direct role for thylakoid lumen acidification?

A testable mechanism of CO₂ accumulation in photolithotrophs, originally suggested by Pronina & Semenenko, is quantitatively analysed. The mechanism involves (as does the most widely accepted hypothesis) the delivery of HCO₃^? to the compartment containing Rubisco. It differs in proposing subsequent HCO₃^? entry (by passive uniport) to the thylakoid lumen, followed by carbonic anhydrase activity in the lumen; uncatalysed conversion of HCO₃^? to CO₂, even at the low pH of the lumen, is at least 300 times too slow to account for the rate of inorganic C acquisition. Carbonic anhydrase converts the HCO₃^? to CO₂ at the lower pH maintained in the illuminated thylakoid lumen by the light-driven H⁺ pump, generating CO₂ at 10 times or more the thylakoid HCO₃^? concentration. Efflux of this CO₂ can suppress Rubisco oxygenase activity and stimulate carboxylase activity in the stroma. This mechanism differs from the widely accepted hypotheses in the required location of carbonic anhydrase, i.e. in the thylakoid lumen rather than the stroma or pyrenoid, and in the need for HCO₃^? influx to thylakoids. The capacity for anion (assayed as Cl^?) entry by passive uniport reported for thylakoid membranes is adequate for the proposed mechanism; if the Cl^? channel does not transport HCO₃^?, HCO₃^? entry could be by combination of the Cl^? channel with a Cl^? HCO₃^? antiporter. This mechanism is particularly appropriate for organisms which lack overt accumulation of total inorganic C in cells, but which nevertheless have the gas exchange characteristics of an organism with a CO₂-concentrating mechanism.     

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production

The contribution of metabolic bicarbonate to cytosolic pH (pH_cyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO^?₃ decreased proton efflux from 18.6±5.0 to 1.64±0.29 μmol/min per g liver wet weight (w.w.) and pH_cyto from 7.17±0.06 to 6.87±0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69±0.26 μmol/min per g liver w.w. reflecting a reduction in metabolic CO₂ hydration, and hence a decrease of H⁺ and HCO^?₃ supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pH_cyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and amiloride since pH_cyto decreased by 0.16±0.05 and proton efflux by 0.60±0.14 μmol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pH_cyto regulation. The involvement of two mechanisms, most likely Na⁺/H⁺ antiport and Na⁺:HCO^?₃ symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO^?₃ in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pH_cyto regulation.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H⁺- and Na⁺-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (P_i) was accumulated by two kinetically discrete H⁺/P_i-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P_i concentrations. The high-affinity system, subjected to regulation by both extracellular P_i availability and intracellular polyphosphate stores, is mobilized during P_i-starvation. In cells grown at pH 9.5–10, P_i uptake is mediated by several kinetically discrete Na⁺-dependent systems that are specifically activated by Na⁺ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P_i concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P_i-starvation and appear to be controlled by extracellular P_i. They represent the first examples of high-capacity, Na⁺-driven P_i transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H⁺- and Na⁺-coupled P_i transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H⁺-coupled P_i transport systems are predominant. The contribution of the Na⁺/P_i cotransport systems to the total cellular P_i uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. Received: 15 December 2000/Revised: 14 May 2001     

Functional characterization of the uncoupler-insensitive Na+ pump of the halotolerant bacterium,Ba1

Respiration initiates Na⁺ efflux from Na⁺-preloaded cells of the halotolerant bacterium, Ba₁. This efflux can take place against the concentration and electrochemical gradients. Since it is not inhibited by carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone or N'N'-dicyclohexylcarbodiimide, it seems unlikely that either Δp (electrochemical potential difference of H⁺ across the membrane) generated by the primary proton pump or ATP play a role in the transduction of the energy supplied by electron transport. The electrogenic extrusion of Na⁺ causes passive counterflow of protons and/or simultaneous flux of permeant anions. In the absence of permeant anions the charge compensation attained by influx of protons is not complete. The membrane potential which persists in this case is inside negative and insensitive to uncoupler. The influx of protons builds up a ΔpH of reversed sign (more acid inside), which is insensitive to uncoupler. The simultaneous efflux of Na⁺ and permeant anions diminishes the intracellular salt content and, as a corollary, causes volume contraction. Thus, the respiration-linked, uncoupler-insensitive Na⁺ pump may play a role in the regulation of the intracellular salt content.     

Phosphate transport in intestinal brush-border membrane

In the small intestine of the rabbit the process of Na⁺-dependent uptake of phosphate occurs only at the brush-border of duodenal enterocytes. Li⁺ can replace Na⁺. The process is activated when either K⁺, Cs⁺, Rb⁺, or choline is present in the intravesicular space. The presence of membrane-permeable anions is essential for maximum rates of phosphate transport. We conclude that the mechanism of the phosphate carrier is electrogenic at pH 6–8, probably two Na⁺ moving with each H₂PO ₄ ^– . This. will lead to the development of a positive charge within the vesicle. The variation of theK _m for H₂PO ₄ ^– with pH is thought to be the consequence of the affinity of the carrier protein for H₂PO ₄ ^– increasing as the pH increases. Polyclonal antibodies against membrane vesicles isolated from rabbit duodenum, jejunum, and ileum were prepared. The antibodies raised against the ileum and jejunum both activated the phosphate transport process, while the anti-duodenum antibody preparation inhibited phosphate transport.     

Sodium-Dependent Antiporters in Choroid Plexus Epithelial Cultures from Rabbit

Abstract: The mechanism of recovery from an acid load in primary cultures of rabbit choroid plexus epithelium (CPE) was examined, with emphasis on Na⁺-dependent antiports. Cells were incubated in saline solutions buffered to pH 7.38 with either HEPES or HCO₃^? plus 95% O₂/5% CO₂. Intracellular pH (pH_i) was determined from the steady-state distribution of [¹⁴C]benzoate. Recovery after acidification with NH₄Cl was rapid (t_1/2= 5 min) and was dependent on external Na⁺ (EC₅₀= 12 mM). Hexamethyleneamiloride and ethylisopropylamiloride, potent inhibitors of the Na⁺/H⁺ antiport, blocked 80% of recovery when [Na⁺] was 5 mM with IC₅₀ values of 100 nM. However, neither drug blocked recovery in normal [Na⁺]. 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of Cl^?/HCO₃^? antiports, blocked recovery of pH_i in a dose-related fashion in the presence of bicarbonate, but not in the presence of HEPES. No inhibition occurred with benzamil, an amiloride congener with high affinity for the Na⁺ channel, nor with dimethylbenzamil, an inhibitor of Na⁺/Ca²⁺ exchange. The carbonic anhydrase inhibitor acetazolamide also did not alter recovery from acidification. In CPE that had been pH-clamped with nigericin and KCl, the initial rate of ²²Na⁺ uptake was very rapid (227 pmol/μg of DNA/min at pH 6.2), was dependent on external [Na⁺] with an EC₅₀ value of 8 mM, and was inversely related to the pH of the medium. The maximal inhibition of ²²Na⁺ uptake by hexamethyleneamiloride was 60% with an IC₅₀ value of 76 nM. We conclude that both the Na⁺/H⁺ antiport and a DIDS-sensitive bicarbonate-dependent antiport are important mechanisms of regulation of the internal pH of rabbit CPE under acidifying conditions. Further, our data suggest that the rabbit choroid plexus Na⁺/H⁺ exchanger can be classified as amiloride insensitive, suggesting that this antiport may play a greater role in controlling transport mechanisms than does the pH of the CNS.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.