The amiloride-sensitive Na+/H+ antiport in 3T3 fibroblasts

BALB/c 3T3 fibroblasts have an amiloride-sensitive Na+ uptake mechanism which is hardly detectable under normal physiological conditions. The activity of this Na+ transport system can be increased to a large extent by treatments that decrease the internal pH such as loss of intracellular NH4+ as NH3 or incubation with nigericin in the presence of a low external K+ concentration. These treatments have made possible an analysis of the interaction of the Na+/H+ antiport with amiloride and of the external pH dependence of the system. The addition of fetal bovine serum to quiescent 3T3 cells stimulates the initial rate of the amiloride-sensitive 22Na+ uptake by only 50%. However, after treatment of the cells with ammonia or nigericin, serum produces a 40-fold stimulation of the rate of the amiloride-sensitive 22Na+ uptake. Control experiments show that serum does not stimulate the activity of the Na+/H+ antiport by an indirect mechanism involving a depolarization of the membrane or a modification of the internal Ca2+ concentration. It is suggested that some serum component directly interacts with the Na+/H+ exchanger to modify its catalytic properties.     

On the mechanism of amiloride-sensitive nonelectrogenic Na+-H+ exchange in cell membranes: Na+/H+ antiport or Na+/OH- symport?

The amiloride-sensitive and nonelectrogenic Na+-H+ exchange system of eucaryotic cells is currently a topic of great interest. The results of membrane transport in the presence of protons are shown to be similar in two cases: when H+ is transferred in one direction or OH- -in the opposite direction. Therefore, in principle Na+-H+ exchange can be performed by two different mechanisms: Na+/H+ antiport or Na+/OH- symport. However, the kinetic properties of these mechanisms turn out to be quite different. The present study analyses the simplest models of antiport and symport and delineates their important differences. For this purpose the Lineweaver-Burk plot presented as Na+ reverse flow entering a cell 1/JNa (or H+ leaving a cell) versus the reverse concentration of Na+ outside 1/[Na+]0 is most useful. If a series of lines with external pH as a parameter have a common point of intersection placed on the ordinate, it indicates the availability of Na+/H+ antiport. In case of Na+/OH- symport a point of intersection is shifted to the left of the ordinate axis. According to data available in the literature, Na+/H+ antiport manifests itself in dog kidney cells and in hamster lung fibroblasts. In the skeletal muscles of chicken and in rat thymus lymphocytes however, a Na+/OH- symport is apparently present.     

Effect of mutations affecting Na+: H+ antiport activity on tumorigenic potential of hamster lung fibroblasts

Mutants unable to regulate intracellular pH through the Na+: H+ antiport system were found to evolve tumors less frequently than wild-type CCL39 hamster lung fibroblasts, after transplantation in athymic nude mice. When rare tumors arose, they comprised cells which were transformed in vitro, but which upon retransplantation grew at a lower rate than tumor cells originating from CCL39 cells. Both parental and mutant cells became transformed after transfection of the activated Harvey ras oncogene, but transfectants derived from the mutants had a weaker tumorigenic potential. These results suggest that transformed characteristics can be acquired independently from the Na+: H+ antiporter. However, the presence of this system provides a selective growth advantage when cells are confronted with natural environments, as it occurs during the expansion of tumors in a host.     

The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Na+/H+ antiport

ATP-dependent trapping of [14C]methylamine was demonstrated in vesicles selectively derived from the sinusoidal plasma membrane of rat hepatocytes; activity was lacking in vesicles prepared from the canalicular domain of the plasma membrane of rat hepatocytes. The proton movement was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, strophanthidin, vanadate, amiloride, and absence of sodium. 22Na efflux from sinusoidal membrane vesicles increased inversely to extravesicular pH. The results indicate that the sinusoidal plasma membrane of rat hepatocytes contains a Na+/H+ antiport.     

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.     

Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport

Mg2+ efflux from Mg2+-preloaded chicken erythrocytes is caused by an electroneutral Na+/Mg2+ antiport. It depends specifically on extracellular Na+, according to Michaelis-Menten kinetics (Km = 25 mM), and is reversibly noncompetitively inhibited by amiloride (Ki = 0.59 mM). In contrast to Na+/H+ antiport, Li+, Ca2+ and N-ethylmaleimide do not interfere with Na+/Mg2+ antiport. The Na+/Mg2+ antiport is driven by the intracellular/extracellular Mg2+ gradient.     

Evidence for Na+/H+ antiport inMethanospirillum Hungatei

The growth and methane formation ofMethanospirillum hungatei were inhibited by an inhibitor of Na⁺/H⁺ antiport amiloride. After addition of NaCl or LiCl, when the cells had a lower intracellular pH and were deenergized, they extruded protons into the external medium. The acidification of the external medium was stimulated by protonophores and inhibited by amiloride. These findings suggest the existence of an Na⁺/H⁺ antiport in the cytoplasmic membrane ofM. hungatei and its role in the energetics of methanogenic bacteria.     

Calcium mobilization and Na+/H+ antiport activation by endothelin in human skin fibroblasts

Endothelin (ET-1) has been shown to exert vasoconstrictor activity in vivo and mobilize Ca2+ in vascular smooth muscle cells in culture. In this paper we show that the human skin fibroblast exhibits specific receptors to ET-1 and that activation of these receptors results in increased intracellular Ca2+ (Ca2+i) and accelerated Na+/H+ antiport activity. ET-1 raised Ca2+i in a dose-response manner; the peak Ca2+i rise was from basal levels of 112.2 +/- 21.9 to 299.2 +/- 49.7 nM at 300 nM ET-1. This rise was attenuated by removal of extracellular Ca2+i0. Although ET-1 did not alter basal intracellular pH, it enhanced Na+/H+ antiport activity of acidified cells. Fibroblasts demonstrated 156 +/- 18 (mean +/- SE) ET-1 receptors per unit cell and an equilibrium dissociation constant of 203.4 +/- 35.6 pM. Inasmuch as ET-1 plays a role in the metabolism of cells such as the undifferentiated fibroblast, an important action of this peptide may be to act as a growth factor.     

Refined estimation of kinetic parameters of the Na+/H+ antiport in human fibroblasts and platelets

A technique is presented to estimate the initial rates of Na(+)-dependent alkalinization of acidified human fibroblasts and platelets and assess the kinetics of the Na+/H+ antiport in these cells. Cytosolic pH (pHi) exhibits an exponential recovery following cellular acidification. Thus, the length of the time interval selected to monitor changes in pHi (delta pHi) is critical to estimating the kinetics of the Na+/H+ antiport. We compared kinetic parameters of the Na+/H+ antiport, using computed and observed changes in delta pHi, for arbitrarily selected time intervals following Na(+)-dependent activation. In both cells, significant increases in both the [Na+] for half-maximal activation (K0.5) and maximal velocities (Vmax) were observed as delta pHi was decreased. We conclude that kinetic parameters derived from initial rate determinations enable a more accurate characterization of the Na+/H+ antiport.     

Two G protein-coupled receptors activate Na+/H+ exchanger isoform 1 in Chinese hamster lung fibroblasts through an ERK-dependent pathway

The sodium hydrogen exchanger isoform 1 (NHE1) is present in nearly all cells. Regulation of proton flux via the exchanger is a permissive step in cell growth and tumorgenesis and is vital in control of cell volume. The regulation of NHE1 by growth factors involves the Ras-extracellular signal regulated kinase (ERK) pathway, however, the mechanism for G protein-coupled receptor (GPCR) activation of NHE1 is not well established. In this report, the relationship between GPCRs, ERK, and NHE1 in CCL39 cells is investigated. We give evidence that two agonists, the specific alpha(1)-adrenergic agonist, phenylephrine and the water-soluble lipid mitogen, lysophosphatidic acid (LPA) activate NHE1 in CCL39 cells. Activation of ERK by phenylephrine and LPA occurs in a dose- and time-dependent manner. Optimal ERK activation was observed at 10 min and displayed a maximum stimulation at 100 microM phenylephrine and 10 microM LPA. alpha(1)-Adrenergic stimulation also led to a rise in steady-state pH(i) of 0.16+/-0.02 pH units, and incubation with LPA induced a 0.43+/-0.06 pH unit increase in pH(i). Phenylephrine-induced activation of NHE1 transport and ERK activity was inhibited by pretreating the cells with the MEK inhibitor PD98059. While only half of the LPA activatable exchange activity was abolished by PD98059 and U0126. To further demonstrate the specificity of the phenylephrine and LPA regulation of NHE1 and ERK, CCL39 cells were transfected with a kinase inactive MEK. The data indicate that ERK activation is essential for phenylephrine stimulation of NHE1, and that ERK and RhoA are involved in LPA stimulation of NHE1 by more than one mechanism. In addition, evidence of the convergence of these two pathways is shown by the loss of NHE1 activity when both pathways are inhibited and by the partial additivity of the two agonists on ERK and NHE1 activity. These studies indicate a direct involvement of ERK in the alpha(1)-adrenergic activation of NHE1 and a significant role for both ERK and RhoA in LPA stimulation of NHE1 in CCL39 fibroblasts.     

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.     

Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diferric transferrin reduction stimulates the Na+/H+ antiport of HeLa cells

Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.     

Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts

Chinese hamster embryo fibroblast cells (CHEF/18) possess a plasma membrane-associated, amiloride-sensitive Na+/H+ antiporter that affects intracellular pH (pHi) and is activated by growth factor addition. Our results using 14C-benzoic acid distribution indicate that both epidermal growth factor (EGF) and thrombin are capable of causing rapid rises in the pHi of CHEF/18 cells. The maximal shift induced by these factors is 0.20 to 0.25 pH units above the basal unstimulated level. Distinctive differences were observed between the modes of action of these two growth factors. Sequential additions revealed that the rise in pHi due to EGF was additive with that caused by diacylglycerols (DAG), while that of thrombin was not. Furthermore, exposure of cells to the phorbol ester PMA for a prolonged period of time in order to down-regulate protein kinase C (pkC), or treatment with the pkC inhibitor H-7, abolished the pHi response to thrombin but not to EGF. In contrast, incubation of cells in nominally calcium-free medium or with the calmodulin antagonists W-7 or trifluoperazine (TFP) decreased only the ability of EGF to cause changes in pHi. These data suggest that there are two distinct mechanisms for activation of the Na+/H+ antiporter in CHEF/18 fibroblast cells and thus provide an example of the use of alternative modes for the modulation of intracellular processes.     

Apparent absence of Na+/H+ antiport activity in the two-cell mouse embryo

We have used the pH-sensitive dye BCECF to investigate the regulation of intracellular pH (pHi) by two-cell stage mouse embryos in bicarbonate-free medium. There is no indication of a Na+/H+ antiport active in regulating pHi, as recovery from acid-loading was insensitive to amiloride, ethylisopropylamiloride, or the absence of extracellular Na+. Instead, protons appear to be in equilibrium across the plasma membrane, as indicated by the response of pHi to changes in external K+. The embryos have an intracellular buffering power in the normal range (25.3 mM/pH); their apparent permeability to protons is, however, very high (0.22 cm/sec).     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.