Upregulation of Na/H exchanger by the AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O₂ deficiency glycolytic degradation of glucose leads to formation of lactate and H⁺, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H⁺ export. The present study explored whether AMPK influences Na⁺/H⁺ exchanger (NHE) activity and/or Na⁺-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH_i) was estimated utilizing BCECF fluorescence and Na⁺/H⁺ exchanger activity from the Na⁺-dependent re-alkalinization (ΔpH_i) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH_i and ΔpH_i in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pH_i and ΔpH_i was blunted in the presence of the Na⁺/H⁺ exchanger inhibitor cariporide (10 μM), but not by the H⁺ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.     

Hill Coefficient Analysis of Transmembrane Helix Dimerization

Sertoli cells are responsible for regulating a wide range of processes that lead to the differentiation of male germ cells into spermatozoa. Cytoplasmic pH (pH _i ) has been shown to be an important parameter in cell physiology, regulating namely cell metabolism and differentiation. However, membrane transport mechanisms involved in pH _i regulation mechanisms of Sertoli cells have not yet been elucidated. In this work, pH _i was determined using the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Addition of weak acids resulted in rapid acidification of the intracellular milieu. Sertoli cells then recovered pH _i by a mechanism that was shown to be sensitive to external Na⁺. pH _i recovery was also greatly reduced in the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and amiloride. These results point toward the action of an Na⁺-driven HCO₃⁻/Cl⁻ exchanger and/or an Na⁺/HCO₃⁻ cotransporter and the action of the Na⁺/H⁺ exchanger on pH _i regulation in the experimental conditions used. pH _i recovery was only slightly affected by ouabain, suggesting that the inhibition of Na⁺/K⁺-ATPase affects recovery indirectly, possibly via the shift on the Na⁺ gradient. On the other hand, recovery from the acid load was independent of the presence of concanamycin A, a specific inhibitor of the V-type ATPases, suggesting that these pumps do not have a relevant action on pH _i regulation in bovine Sertoli cells.     

Studies on the kinetics of Na+/H+ exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na⁺/H⁺ exchange at the level of an intact monolayer. Na⁺/ H⁺ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na⁺/H⁺ exchange activity was measured by monitoring changes in intracellular pH (pH _i ) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH _i , recovery mechanism, which is Na⁺ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH _i =7.68; pH _o =7.4) where Na⁺/H⁺ exchange is not detectable, transport rate increased as pH _i decreased. A positive cooperativity characterized the interaction of internal H⁺ with the exchanger, and suggests multiple H⁺ binding sites. In contrast, extracellular [Na⁺] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na⁺ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na⁺/H⁺ exchange activity by EIPA was competitive with respect to extracellular [Na⁺] and theK _i was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH _i and processes associated with their polarized expression and regulation.     

Calcium channels play a pivotal role in the sequence of ionic changes involved in initiation of mouse sperm acrosomal exocytosis

The sequence of ionic changes involved in initiation of acrosomal exocytosis in capacitated mouse spermatozoa was investigated. Earlier studies demonstrated that a large influx of Na⁺ is required for exocytosis, this Na⁺ apparently being associated with an increase in intracellular pH (pH_i) via an Na⁺-H⁺ exchanger. This rise in pH_i may in turn activate calcium channels and permit the influx of extracellular Ca²⁺ needed to trigger acrosomal exocytosis. In the present study, the dihydropyridine voltage-dependent calcium channel antagonist nifedipine was able to inhibit significantly exocytosis in sperm cells treated in various ways capable of stimulating acrosomal loss. The monovalent cation ionophore monensin can promote Na⁺ entry required for both capacitation and acrosomal exocytosis, as demonstrated by using chlortetracycline to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but was unable to trigger exocytosis. The requirement for internalization of a high concentration of Na⁺ can be bypassed by the addition of 25 mM NH₄CI to raise the pH_i of cells capacitated in 25NH₄CI to raise the pH_i of cells capacitated in 25 mM Na⁺ (insufficient Na⁺ to support exocytosis under usual conditions). Again, introduction of nifedipine was able to inhibit exocytosis. In a third experimental approach, amiloridestimulated exocytosis in capacitated cells was significantly inhibited by nifedipine. In contrast to these treatments directed at specific mechanisms, the ability of the Ca²⁺ inophore A23187 to promote more general entry of Ca²⁺ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine. Finally, monensin-treated cells exhibited a rise and then a fall in ⁴⁵Ca²⁺ uptake, the time course of which paralleled stimulation of acrosomal exocytosis in similarly treated cells. Nifedipine significantly reduced this uptake. The fact that nifedipine can block exocytosis induced by a variety treatments strongly suggests that voltage-dependent calcium channels play a pivotal role in the response. These results are consistent with the following sequence of ionic changes in capacitated cells leading to acrosomal exocytosis: [Na⁺]_i ↑ → [H]_i↓ → pH_i ↑ → activation of calcium channels → [Ca²⁺]_i ↑ → exocytosis. Given that zona-induced exocytosis is reportedly an indirect response, mediated by voltage-dependent calcium channels, and that the Na⁺-H⁺ exchanger in somatic cells can be activated by receptor-mediated mechanisms, we suggest that sperm-zona inter action promotes an influx of Na⁺ by activating an Na⁺-H⁺ exchanger and thereby initiating the above sequence of changes. © 1993 Wiley-Liss, Inc.     

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.     

Cyclic GMP Inhibits a Pharmacologically Distinct Na+/H+ Exchanger Variant in Cultured Rat Astrocytes via an Extracellular Site of Action

Abstract: There is growing evidence that cyclic GMP (cGMP) plays important roles in the brain. In cultured rat astrocytes, we observed that the cGMP-inducing C-type natriuretic peptide (CNP) and cGMP analogues caused a decrease in intracellular pH (pH_i). To examine whether this effect was due to inhibition of an Na⁺/H⁺ exchanger (NHE), we acidified cells by replacing extracellular Na⁺ by choline and examined the kinetics of the pH_i recovery that occurred on reintroduction of Na⁺ in the extracellular medium. Both CNP and amiloride analogues inhibited the Na⁺-dependent pH_i recovery, even in the nominal absence of CO₂/HCO₃^?. This indicated that CNP inhibited the activity of an exchanger that was Na⁺-dependent, HCO₃^?-independent, and sensitive to known inhibitors of NHE. However, comparison of the potencies of four distinct amiloride analogues revealed a pharmacological profile that was different from that of any other NHE characterized to date. cGMP mimicked the effect of CNP on sodium-dependent pH_i recovery, but the native nucleotide was as potent as membrane-permeant analogues. Intracellularly produced cGMP was very rapidly exported out of astrocytes. Probenecid and niflumic acid slowed down the rate of cGMP egression and inhibited the effect of CNP on Na⁺-dependent recovery, but not that of extracellular cGMP. Altogether, our data indicate that cGMP inhibits a novel type of NHE in astrocytes via an extracellular site of action. If these results with primary cultures transfer to brain, this phenomenon may constitute a mechanism by which natriuretic peptides exert some of their actions in the brain, as pH_i transients have been shown to modulate several important astrocytic functions.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

Comparison between drug-induced and K+-induced changes in molar acid extrusion fluxes (JH +) and in energy consumption rates in astrocytes

Neuronal excitation leads to an increase of the extracellular K⁺ concentration ([K⁺]_o) in brain. This increase has at least two energy-consuming consequences: (1) a depolarization-mediated change in intracellular pH (pH_i) in astrocytes due to depolarization-mediated increased activity of the acid-extruding Na⁺/bicarbonate transporter NBCe1 (driven by secondary active transport, supported by ion gradients established by the Na⁺, K⁺-ATPase); and (2) activation of cellular reuptake of K⁺ mediated by the Na⁺, K⁺-ATPase in both neurons and astrocytes. Astrocytic, but not neuronal increase in NBCe1 activity and pH_i is also seen after chronic treatment with either of the two anti-bipolar drugs carbamazepine or valproic acid. The third ‘classical’ anti-bipolar drug, ‘lithium’ increases astrocytic pH_i by a different mechanism (stimulation of the acid extruding Na⁺/H⁺ exchanger NHE1). The acid extruder fluxes, which depend upon the change in pH_i per time unit (ΔpH_i/Δt) and intracellular buffering power, have not been established in most of these situations. Therefore their stimulatory effects on energy metabolism has not been quantitated. This has been done in the present study in cultured mouse astrocytes. pH_i was determined using the fluorescent pH-sensitive indicator BCECF–AM and an Olympus IX71 live cell imaging fluorescence microscope. Molar acid extrusion fluxes (indicating transporter activity) were determined as pH_i changes/min during recovery after acid-loading with NH₃/NH₄ ⁺, NBCe1 mRNA and protein expression in the cultured cells by, respectively RT-PCR and Western blotting. Drug-induced up-regulation of acid extrusion flux was slow and less than physiologically seen after increase in K⁺ concentration. Energetically, K⁺ uptake is much costlier than NBCe1 activity.     

Intracellular pH Increase Driven by an Na+/H+ Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pH_i) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pH_iof 0.3 pH unit after activation by excess K⁺. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pH_iwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K⁺induced GVBD but the pH_iof K⁺-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pH_iincreases in K⁺-activated oocytes were otherwise proportional to the external Na⁺concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K⁺-induced increase of pH_ibut did not block GVBD. These two derivatives were able, however, to retard K⁺-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K⁺-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pH_iincrease, driven by a typical Na⁺/H⁺exchanger, follows activation of surf clam oocytes but that this pH_iincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.     

Down-regulation of P-glycoprotein expression by sustained intracellular acidification in K562/Dox cells

We have investigated the involvement of intracellular pH (pH_i) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na⁺/H⁺ exchanger1 (NHE1) inhibitor cariporide and the “high K⁺” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH_i than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH_i and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH_i recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH_i and P-gp in K562/DOX cells.     

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.     

A Model of Na/H Exchanger and Its Central Role in Regulation of pH and Na in Cardiac Myocytes

A new kinetic model of the Na⁺/H⁺ exchanger (NHE) was developed by fitting a variety of major experimental findings, such as ion-dependencies, forward/reverse mode, and the turnover rate. The role of NHE in ion homeostasis was examined by implementing the NHE model in a minimum cell model including intracellular pH buffer, Na⁺/K⁺ pump, background H⁺, and Na⁺ fluxes. This minimum cell model was validated by reconstructing recovery of pH_i from acidification, accompanying transient increase in [Na⁺]_i due to NHE activity. Based on this cell model, steady-state relationships among pH_i, [Na⁺]_I, and [Ca²⁺]_i were quantitatively determined, and thereby the critical level of acidosis for cell survival was predicted. The acidification reported during partial blockade of the Na⁺/K⁺ pump was not attributed to a dissipation of the Na⁺ gradient across the membrane, but to an increase in indirect H⁺ production. This NHE model, though not adapted to the dimeric behavioral aspects of NHE, can provide a strong clue to quantitative prediction of degree of acidification and accompanying disturbance of ion homeostasis under various pathophysiological conditions.     

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.     

Ionic permeabilities induced by Bacillus thuringiensis in Sf9 cells

The effect of Bacillus thuringiensis insecticidal toxins on the monovalent cation content and intracellular pH (pH _i ) of individual Sf9 cells of the lepidopteran species Spodoptera frugiperda (fall armyworm) was monitored with the fluorescent indicators potassium-binding benzofuran isophthalate (PBFI) and 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The sequential removal of K⁺ and Na⁺ from the medium, in the presence of CryIC, a toxin which is highly active against Sf9 cells, caused sharp shifts in the fluorescence ratio of PBFI, demonstrating a rapid efflux of these ions. In Sf9 cells, pH _i depends strongly on the activity of a K⁺/H⁺ exchanger. In the absence of toxin, removal of K⁺ from the external medium resulted in a reversible acidification of the cells. In the presence of CryIC, pH _i equilibrated rapidly with that of the bathing solution. This effect was both time- and concentration-dependent. In contrast with CryIC, CryIIIA, a coleopteran-specific toxin, and CryIA(a), CryIA(b) and CryIA(c), toxins which are either inactive or poorly active against Sf9 cells, had no detectable effect on pH _i . B. thuringiensis endotoxins thus appear to act specifically by increasing the permeability of the cytoplasmic membrane of susceptible cells to at least H⁺, K⁺ and Na⁺.     

Na+/H+ exchange in crayfish neurons: dependence on extracellular sodium and pH

Summary Intracellular pH (pH_i) regulation was studied in crayfish neurons with pH-, and Na⁺-sensitive microelectrodes. It was confirmed to involve both a HCO ₃ ^– -dependent and a HCO ₃ ^– -independent mechanism. The latter was identified as the amiloride-sensitive Na⁺/H⁺ exchange described in vertebrate cells. Its dependence on extracellular pH (pH_e) and Na⁺ concentration ([Na⁺]_e) was studied in CO₂-free external solutions at 20°C. The steady state pH_i and the rate constant (k) of the exponential pH_i recovery following an acid load were determined. At pH_e=7.5 and [Na⁺]_e=200 mM, the average steady state pH_i was 7.09±0.12 (as compared to 7.30±0.10 in the presence of 5 mM bicarbonate). The dependence of the rate constant of recovery on [Na⁺]_e could be described by Michaelis-Menten kinetics; at pH_e=7.5 the apparentK _m andK _max were 39 mM and 1.4 mmol·l^–1·min^–1, respectively. Decreasing pH_e reduced the rate of recovery, the variations ofk with pH_e conforming to a simple titration curve with an apparent pK of 7.05±0.21. These kinetic properties of the Na⁺/H⁺ exchange in crayfish neurons are similar to those described in vertebrate cells.Preliminary results were presented at the First International Congress of Comparative Physiology and Biochemistry (Liège, Belgium, 1984)     

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.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

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