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.     

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pH_i, pH_e) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na⁺/H⁺ exchanger NHE1 and Na⁺, HCO₃^? transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pH_e to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pH_i changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pH_i regulation by the two transporters.     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

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.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase

Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH_i), typically due to activation of Na⁺/H⁺ exchange (NHE). In the systemic vasculature, ET-1 increases pH_i, Na⁺/H⁺ exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH_i and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH_i and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3–5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH_i and NHE activity induced by increasing concentrations of ET-1 (10⁻¹⁰ to 10⁻⁸ M). We found that application of exogenous ET-1 increased pH_i and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH_i or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH_i.     

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.     

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)     

Interactions of external and internal H+ and Na+ with Na+/Na+ and Na+/H+ exchange of rabbit red cells: Evidence for a common pathway

Summary We have studied the kinetic properties of rabbit red cell (RRBC) Na⁺/Na⁺ and Na⁺/H⁺ exchanges (EXC) in order to define whether or not both transport functions are conducted by the same molecule. The strategy has been to determine the interactions of Na⁺ and H⁺ at the internal (i) and external (o) sites for both exchanges modes. RRBC containing varying Na _i and H _l were prepared by nystatin and DIDS treatment of acid-loaded cells. Na⁺/Na⁺ EXC was measured as Na _o -stimulated Na⁺ efflux and Na⁺/H⁺ EXC as Na _o -stimulated H⁺ efflux and pH _o -stimulated Na⁺ influx into acid-loaded cells.The activation of Na⁺/Na⁺ EXC by Na _o at pH _i 7.4 did not follow simple hyperbolic kinetics. Testing of different kinetic models to obtain the best fit for the experimental data indicated the presence of high (K _m 2.2 mM) and low affinity (K _m 108 mM) sites for a single- or two-carrier system. The activation of Na⁺/H⁺ EXC by Na _o (pH _i 6.6, Na _i <1 mM) also showed high (K _m 11 mM) and low (K _m 248 mM) affinity sites. External H⁺ competitively inhibited Na⁺/Na⁺ EXC at the low affinity Na _o site (K _H 52 nM) while internally H⁺ were competitive inhibitors (pK 6.7) at low Na _i and allosteric activators (pK 7.0) at high Na _i .Na⁺/H₊ EXC was also inhibited by acid pH _o and allosterically activated by H _i (pK 6.4). We also established the presence of a Na _i regulatory site which activates Na⁺/H⁺ and Na⁺/Na⁺ EXC modifying the affinity for Na _o of both pathways. At low Na _i , Na⁺/Na⁺ EXC was inhibited by acid pH _i and Na⁺/H⁺ stimulated but at high Na _i , Na⁺/Na⁺ EXC was stimulated and Na⁺/H⁺ inhibited being the sum of both pathways kept constant. Both exchange modes were activated by two classes of Na _o sites,cis-inhibited by external H _o , allosterically modified by the binding of H⁺ to a H _i regulatory site and regulated by Na _i . These findings are consistent with Na⁺/Na⁺ EXC being a mode of operation of the Na⁺/H⁺ exchanger.Na⁺/H⁺ EXC was partially inhibited (80–100%) by dimethyl-amiloride (DMA) but basal or pH _i -stimulated Na⁺/Na⁺ EXC (pH _i 6.5, Na _i 80 mM) was completely insensitive indicating that Na⁺/Na⁺ EXC is an amiloride-insensitive component of Na⁺/H⁺ EXC. However, Na⁺ and H⁺ efflux into Na-free media were stimulated by cell acidification and also partially (10 to 40%) inhibited by DMA: this also indicates that the Na⁺/H⁺ EXC might operate in reverse or uncoupled modes in the absence of Na⁺/Na⁺ EXC.In summary, the observed kinetic properties can be explained by a model of Na⁺/H⁺ EXC with several conformational states, H _i and Na _i regulatory sites and loaded/unloaded internal and external transport sites at which Na⁺ and H⁺ can compete. The occupancy of the H⁺ regulatory site induces a conformational change and the occupancy of the Na _i regulatory site modulates the flow through both pathways so that it will conduct Na⁺/H⁺ and/or Na⁺/Na⁺ EXC depending on the ratio of internal Na⁺:H⁺.     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Kinetic characterization of Na+/H+ antiport of Plasmodium falciparum membrane

Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H⁺ is not known. The possible involvement of a Na⁺/H⁺ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6-carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na⁺-medium and realkalinization occurred upon addition of Na⁺ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H⁺-at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na⁺-H⁺-was: 1) inhibited by the Na⁺/H⁺ inhibitors amiloride and 5-(N-ethyl-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH₆ (pH_i = 6.2 [Na⁺]_o = 30 mM); and 3) decreased with increasing pH_i (pH_o = 7.4; [Na⁺]_o = 30 mM). The pH_i and the pH_o dependencies of H⁺-were almost identical at all parasite stages. Only at pH_i > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H⁺-is mediated by a Na⁺/H⁺ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley-Liss, Inc.     

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.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Chaperone Stress 70 Protein (STCH) Binds and Regulates Two Acid/Base Transporters NBCe1-B and NHE1

Regulation of intracellular pH is critical for the maintenance of cell homeostasis in response to stress. We used yeast two-hybrid screening to identify novel interacting partners of the pH-regulating transporter NBCe1-B. We identified Hsp70-like stress 70 protein chaperone (STCH) as interacting with NBCe1-B at the N-terminal (amino acids 96–440) region. Co-injection of STCH and NBCe1-B cRNA into Xenopus oocytes significantly increased surface expression of NBCe1-B and enhanced bicarbonate conductance compared with NBCe1-B cRNA alone. STCH siRNA decreased the rate of Na⁺-dependent pH_i recovery from NH₄⁺ pulse-induced acidification in an HSG (human submandibular gland ductal) cell line. We observed that in addition to NBCe1-B, Na⁺/H⁺ exchanger (NHE)-dependent pH_i recovery was also impaired by STCH siRNA and further confirmed the interaction of STCH with NHE1 but not plasma membrane Ca²⁺ ATPase. Both NBCe1-B and NHE1 interactions were dependent on a specific 45-amino acid region of STCH. In conclusion, we identify a novel role of STCH in the regulation of pH_i through site-specific interactions with NBCe1-B and NHE1 and subsequent modulation of membrane transporter expression. We propose STCH may play a role in pH_i regulation at times of cellular stress by enhancing the recovery from intracellular acidification.     

Synthesis and Na+/H+ Exchanger‐1 Inhibitory Activity of Substituted (Quinolinecarbonyl)guanidine Derivatives

The Na⁺/H⁺ exchanger (NHE) is a protein expressed in many mammalian cell types. It is involved in intracellular pH (pH_i) homeostasis by exchanging extracellular Na⁺ for intracellular H⁺. To date, nine NHE isoforms (NHE1–NHE9) have been identified. NHE1 is the most predominant isoform expressed in mammalian cardiac muscle. A novel series of substituted (quinolinecarbonyl)guanidine derivatives were designed and synthesized as NHE inhibitors. Most compounds can inhibit NHE1‐mediated platelet swelling in a concentration‐dependent manner, among which compound 7f was the most active and more potent than cariporide. Furthermore, compound 7f has also been demonstrated to exhibit the in vivo cardioprotective effects against SD rat myocardial ischemic‐reperfusion injury superior to those of cariporide.     

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.     

Extracellular Mg regulates the intracellular Na concentration in rat sublingual acini

The intracellular free Na⁺ concentration ([Na⁺]_i) increases during muscarinic stimulation in salivary acinar cells. The present study examined in rat sublingual acini the role of extracellular Mg²⁺ in the regulation of the stimulated [Na⁺]_i increase using the fluorescent sodium indicator benzofuran isophthalate (SBFI). The muscarinic induced rise in [Na⁺]_i was approximately 4-fold greater in the absence of extracellular Mg²⁺. When Na⁺ efflux was blocked by the Na⁺,K⁺-ATPase inhibitor ouabain, the stimulated [Na⁺]_i increase was comparable to that seen in an Mg²⁺-free medium. Moreover, ouabain did not add further to the stimulated [Na⁺]_i increase in an Mg²⁺-free medium suggesting that removal of extracellular Mg²⁺ may inhibit the Na⁺ pump. In agreement with this assumption, ouabain-sensitive Na⁺ efflux and rubidium uptake were reduced by extracellular Mg²⁺ depletion. Our results suggest that extracellular Mg²⁺ may regulate [Na⁺]_i in sublingual salivary acinar cells by modulating Na⁺ pump activity.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

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.