Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

Macropinocytosis is differentiated from other types of endocytosis by its unique susceptibility to inhibitors of Na⁺/H⁺ exchange. Yet, the functional relationship between Na⁺/H⁺ exchange and macropinosome formation remains obscure. In A431 cells, stimulation by EGF simultaneously activated macropinocytosis and Na⁺/H⁺ exchange, elevating cytosolic pH and stimulating Na⁺ influx. Remarkably, although inhibition of Na⁺/H⁺ exchange by amiloride or HOE-694 obliterated macropinocytosis, neither cytosolic alkalinization nor Na⁺ influx were required. Instead, using novel probes of submembranous pH, we detected the accumulation of metabolically generated acid at sites of macropinocytosis, an effect counteracted by Na⁺/H⁺ exchange and greatly magnified when amiloride or HOE-694 were present. The acidification observed in the presence of the inhibitors did not alter receptor engagement or phosphorylation, nor did it significantly depress phosphatidylinositol-3-kinase stimulation. However, activation of the GTPases that promote actin remodelling was found to be exquisitely sensitive to the submembranous pH. This sensitivity confers to macropinocytosis its unique susceptibility to inhibitors of Na⁺/H⁺ exchange.     

Inverse Relationship Between Membrane Lipid Fluidity and Activity of Na+/H+ Exchangers, NHE1 and NHE3, in Transfected Fibroblasts

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of ≈50% in the specific activities of both NHE1 and NHE3. Only V _max was affected; K _Na was unchanged. This effect was specific, as Na⁺, K⁺, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4°C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na⁺/H⁺ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity. Received: 23 January 1997/Revised: 1 August 1997     

Identification of the renal Na+/H+ exchanger with N,N′-dicyclohexylcarbodiimide (DCCD) and amiloride analogues

Summary Dicyclohexylcarbodiimide (DCCD) and the 5-ethylisopropyl-6-bromo-derivative of amiloride (Br-EIPA) have been used as affinity and photoaffinity labels of the Na⁺/H⁺ exchanger in rat renal brush-border membranes. Intravesicular acidification by the Na^–/H⁺ exchanger was irreversibly inhibited after incubation of vesicles for 30 min with DCCD. The substrate of the antiporter, Na⁺, and the competitive inhibitor, amiloride, protected from irreversible inhibition. The Na⁺-dependent transport systems for sulfate, dicarboxylates, and neutral, acidic, and basic amino acids were inhibited by DCCD, but not protected by amiloride. An irreversible inhibition of Na⁺/H⁺ exchange was also observed when brush-border membrane vesicles were irradiated in the presence of Br-EIPA. Na⁺ and Li⁺ protected. [¹⁴C]-DCCD was mostly incorporated into three brush-border membrane polypeptides with apparent molecular weights of 88,000, 65,000 and 51,000. Na⁺ did not protect but rather enhanced labeling. In contrast, amiloride effectively decreased the labeling of the 65,000 molecular weight polypeptide. In basolateral membrane vesicles one band was highly labeled by [¹⁴C]-DCCD that was identified as the -subunit of the Na⁺, K⁺-ATPase. [¹⁴C]-Br-EIPA was mainly incorporated into a brushborder membrane polypeptide with apparent molecular weight of 65,000. Na⁺ decreased the labeling of this protein. Similar to the Na⁺/H⁺ exchanger this Na⁺-protectable band was absent in basolateral membrane vesicles. We conclude that a membrane protein with an apparent molecular weight of 65,000 is involved in rat renal Na⁺/H⁺ exchange.     

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na⁺/H⁺ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na⁺/H⁺ exchange in a competitive manner with a K_i of 2.5 and 5.9 micromolar for ΔpH-dependent ²²Na⁺ influx in tonoplast vesicles and Na⁺-dependent H⁺ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [³H]MIA to tonoplast membranes revealed a high affinity binding component with a K_d of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na⁺/H⁺ antiport. Photolabeling of the tonoplast with [³H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.     

Activators of Epithelial Na+ Channels Inhibit Cytosolic Feedback Control. Evidence for the Existence of a G Protein-Coupled Receptor for Cytosolic Na+

We have previously shown that epithelial Na⁺ channels in mouse mandibular gland duct cells are controlled by cytosolic Na⁺ and Cl⁻, acting, respectively, via G _o and G _i proteins. Since we found no evidence for control of epithelial Na⁺ channels by extracellular Na⁺ ([Na⁺] _o ), our findings conflicted with the long-held belief that Na⁺ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na⁺] _o . Instead, we now show that PCMPS acts by rendering epithelial Na⁺ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na⁺ and Cl⁻ on Na⁺ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na⁺ channels by cytosolic Na⁺, while leaving inhibition by cytosolic Cl⁻ unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of G _o in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na⁺ receptor. Received: 1 October 1997/Revised: 24 December 1997     

Apparent inhibition of Na+/H+ exchange by amiloride and harmaline in acridine orange studies

Amiloride and harmaline were tested as inhibitors of proton movements in brush-border membrane vesicles from rat kidney cortex. Transmembrane pH differences were visualized using acridine orange. Fluorescence quenching due to Na⁺ gradient-driven intravesicular acidification was inhibited by amiloride and harmaline. However, a similar inhibition was observed for the Na⁺ gradient-driven electrogenic proton movements in the presence of gramicidin. Moreover, amiloride and harmaline decreased the fluorescence signal of electrogenic proton movements driven by a K⁺ gradient in the presence of valinomycin. The degree of inhibition of intravesicular acidification by both drugs was concentration dependent. Half-maximal inhibition (I₅₀) of Na⁺/H⁺ exchange and K⁺ gradient-driven proton movements occurred at 0.21 and 0.6 amiloride, respectively. The I₅₀ for harmaline was 0.21 mM in both cases. Amiloride also decreased the initial quenching of acridine orange fluorescence due to a preset pH gradient without affecting the rate of dissipation of the pH gradient. This effect was independent of the buffer capacity. In contrast, harmaline seemed to dissipate pH gradient in the same way as a permeant buffer. Amiloride and harmaline led to a concentration-dependent fluorescence decrease even in aqueous solution. The results suggest an interaction of amiloride and harmaline with acridine orange which overlaps a possible specific inhibition of Na⁺/H⁺ exchange by these drugs.     

Na+/H+ exchange in ehrlich ascites tumor cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate

Summary Exposure of Ehrlich cells to isotonic Na⁺-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K⁺-propionate medium but may be induced by the ionophore nigericin, which mediates K⁺/H⁺ exchange. Cell swelling in Na⁺-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na⁺/H⁺ exchange. Consequently, swelling of Ehrlich cells in Na⁺-propionate medium is due to the operation of an amiloride-sensitive, Na⁺-specific mechanism. It is concluded that this mechanism is a Na⁺/H⁺ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO₄ in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO₄ inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO₄ causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO₄ causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na⁺/H⁺ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO₄ activates this Na⁺/H⁺ exchange system.     

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.     

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.     

The H+ Pump in Frog Skin (Rana esculenta): Identification and Localization of a V-ATPase

We here report on studies on the frog skin epithelium to identify the nature of its excretory H⁺ pump by comparing transport studies, using inhibitors highly specific for V-ATPases, with results from immunocytochemistry using V-ATPase-directed antibodies. Bafilomycin A₁ (10 μm) blocked H⁺ excretion (69 ± 8% inhibition) and therefore Na⁺ absorption (61 ± 17% inhibition after 60 min application, n= 6) in open-circuited skins bathed on their apical side with a 1 mm Na₂SO₄ solution, ``low-Na<sup>+</sup> conditions' under which H<sup>+</sup> and Na<sup>+</sup> fluxes are coupled 1:1. The electrogenic outward H<sup>+</sup> current measured in absence of Na<sup>+</sup> transport (in the presence of 50 μm amiloride) was also blocked by 10 μm bafilomycin A<sub>1</sub> or 5 μm concanamycin A. In contrast, no effects were found on the large and dominant Na<sup>+</sup> transport (short-circuit current), which develops with apical solutions containing 115 mm Na<sup>+</sup> (``high-Na⁺ conditions'), demonstrating a specific action on H⁺ transport. In immunocytochemistry, V-ATPase-like immunoreactivity to the monoclonal antibody E11 directed to the 31-kDa subunit E of the bovine renal V-ATPase was localized only in mitochondria-rich cells (i) in their apical region which corresponds to apical plasma membrane infoldings, and (ii) intracellularly in their neck region and apically around the nucleus. In membrane extracts of the isolated frog skin epithelium, the selectivity of the antibody binding was tested with immunoblots. The antibody labeled exclusively a band of about 31 kDa, very likely the corresponding subunit E of the frog V-ATPase. Our investigations now deliver conclusive evidence that H⁺ excretion is mediated by a V-ATPase being the electrogenic H⁺ pump in frog skin. Received: 21 May 1996/Revised: 24 December 1996     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

Self-Inhibition in Amiloride-sensitive Sodium Channels in Taste Receptor Cells

Electrophysiological recording techniques were used to study the Na⁺ dependence of currents through amiloride-sensitive sodium channels (ASSCs) in rat taste cells from the fungiform and vallate papillae. Perforated patch voltage clamp recordings were made from isolated fungiform and vallate taste receptor cells (TRCs) and Na⁺ transport was measured across lingual epithelia containing fungiform or vallate taste buds in a modified Ussing chamber. In isolated fungiform TRCs that contain Na⁺ currents sensitive to the diuretic amiloride, Na⁺ ions inhibit their own influx through ASSCs, a process known as sodium self-inhibition. Due to the interaction between self-inhibition and the driving force for Na⁺ entry, self-inhibition is most evident in whole-cell recordings at Na⁺ concentrations from 50 to 75 mM. In amiloride-sensitive cells, the Na permeability is significantly higher in extracellular solutions containing 35 mM Na⁺ than in 70 or 140 mM Na⁺. Compared with the block by amiloride, the development of self-inhibition is slow, taking up to 15 s to become maximally inhibited. Approximately one third of fungiform TRCs and all vallate TRCs lack functional ASSCs. These amiloride-insensitive TRCs show no signs of self-inhibition, tying this phenomenon to the presence of ASSCs. The sulfhydryl reagent, p-hydroxymercuribenzoate (p-HMB; 200 μM), reversibly removed self-inhibition from amiloride-sensitive Na⁺ currents, apparently by modifying cysteine residues in the ASSC. Na⁺ currents in amiloride-insensitive TRCs were unaffected by p-HMB. In sodium transport studies in fungiform taste bud–containing lingual epithelia, ∼40% of the change in short-circuit current (I_sc) after addition of 500 mM NaCl to the mucosal chamber is amiloride sensitive (0.5 mM). p-HMB significantly enhanced mucosal NaCl-induced changes in these epithelia at mucosal Na⁺ concentrations of 50 mM and above. In contrast, the vallate-containing epithelia, which are insensitive to amiloride, showed no enhancement of I_sc during p-HMB treatment. These findings suggest that sodium self-inhibition is present in ASSCs in taste receptor cells where it may play a crucial role in performance of salt-sensitive pathways in taste tissue during sodium stimulation. This phenomenon may be important in the process of TRC adaptation, in the conservation of cellular resources during chronic sodium exposure, or in the gustatory response to water.     

Inhibition by amiloride of sodium transport into rabbit kidney medulla microsomes

Sodium transport into rabbit kidney medulla microsomes was 50% inhibited by amiloride. This Na⁺ uptake was shown to represent transport when the uptake process was reversed by the ionophore nigericin. The transport was complete within 60 min and proportional to the microsomal protein concentration. The effect of amiloride on transport was specific since the similar compound sulfaguanidine failed to affect microsomal Na⁺ transport. Amiloride-sensitive Na⁺ transport into microsomes was inhibited 70% by decreasing the pH (from 7.0 to 5.9), but was unaffected by the presence of a pH gradient. The kinetics of Na⁺ transport could be explained by a simple model, assuming that amiloride lowered the rate of Na⁺ entrance into the vesicles but had no effect on the rate of efflux. The failure of amiloride to effect efflux from the vesicles was also demonstrated directly.     

Plasma membrane K+/H+-ATPase From leishmania donovani

Leishmania donovani has an active ^K+/H⁺ exchange system on the surface membrane. Modulation of external K⁺ concentration resulted in a corresponding change in internal pH (pH_i) suggesting a link between proton and potassium transport. Although a Na⁺/H⁺ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K⁺/H⁺ exchange. Reduction of cellular ATP with NaN₃ and KCN inhibits K⁺/H⁺ exchange showing thereby that the process is energy dependent. The K⁺/H⁺ exchange is sensitive to inhibitors of the gastric K⁺/H⁺-ATPase. It is concluded that the H⁺-ATPase previously reported on the plasma membrane of L. donovani is in fact a K⁺/H⁺-ATPase. © 1994 wiley-Liss, Inc.     

Recognition of Adenosine Receptors by Amiloride and Its Analogues

Abstract

Amiloride and its analogues displace the adenosine A, receptor ligands [³H]CPDPX and [³H]PIA from their binding sites in calf brain membranes in a GTP-insensitive manner. High [NaCl] or low pH reduces the affinity of amiloride for A, receptors, whereas the affinity of [³H]CPDPX is not affected. Notwithstanding this difference in modulation, the interaction between amiloride and A, receptors appears competitive in nature. The structure-affinity relationships differ from those for classic amiloride-sensitive Na' transport systems, indicating that a coupling between the A, receptor and one of these systems is very unlikely. Amiloride and its analogues may reprcsent a novel class of A, receptor antagonists.     

Homeostasis of Sodium and Potassium Ions in Erythrocytes of the Lamprey Lampetra fluviatilis: Effect of Ion Transport and Metabolic Blockers, and Ionophores

After incubation of lamprey Lampetra fluviatilis erythrocytes in the standard medium for 90–120 min, intracellular Na⁺ and K⁺ content remained unchanged (28.7 ± 1.1 and 66.3 ± 1.5 mmol/l cells, respectively, n = 33). The erythrocyte ion content also did not change after treatment of the cells with ion transport inhibitors, Ba^{2 +} and amiloride. Addition of 0.1 mM ouabain to the incubation medium led to a decrease of K⁺ content by 8.4 ± 1.2 and to an increase of Na⁺ content by 2.4 ± 0.8 mmol/l/2 h. Similar reciprocal changes in the cellular ion composition were observed after treatment of the erythrocytes by oxidative metabolism inhibitors (rotenone and CCCP—carbonyl cyanide m-chlorophenyl-hydrazone). The metabolic blockers produced more significant ion composition changes in comparison with ouabain. An increase of intracellular Na⁺ content under effect of CCCP was completely inhibited by amiloride. It can be suggested that inhibition of oxidative metabolism is accompanied by a cell acidification and Na⁺/H⁺ exchange activation. Erythrocyte acidification by a K⁺/H⁺ ionophore led to a rapid cellular Na⁺ accumulation, which indicates the presence of a Na⁺/H⁺ exchanger with high activity. The K⁺ ionophore valinomycin produced a relatively small K⁺ loss from the lamprey erythrocytes to indicate a low anion conductance of the cells. The data obtained indicate an important role of oxidative metabolism in the monovalent ion homeostasis in the lamprey red blood cells.     

Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na⁺ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na⁺ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na⁺ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na⁺ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na⁺ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na⁺ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na⁺ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na⁺ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na⁺. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na⁺ uptake, whereas benzamil, the Na⁺ channel blocker, had no effect, it was concluded that the amiloride sensitive Na⁺ uptake was predominantly or exclusively due to a Na⁺-H⁺ exchanger activity. K⁺ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na⁺ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10⁻⁷ M insulin, 10⁻¹⁰ M angiotensin II, or 10⁻⁸ M human parathyroid hormone (PTH) did not influence Na⁺ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na⁺-H⁺ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na⁺-H⁺ exchange is probably the unique mechanism of Na⁺ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc.     

Ion pathways in renal brush border membranes

The absorbance change of the weak base dye probe, Acridine orange, was used to monitor alterations of pH gradients across renal brush border membrane vesicles. The presence of Na⁺/H⁺ or Li⁺/H⁺ exchange was demonstrated by diluting Na₂SO₄ or Li₂SO₄ loaded vesicles into Na⁺- or Li⁺-free solutions, which caused dye uptake. About 20% of the uptake was abolished by lipid permeable cations such as valinomycin-K⁺ or tetraphenylphosphonium, indicating perhaps the presence of a finite Na⁺ conductance smaller than electroneutral Na⁺/H⁺ exchange. The protonophore tetrachlorosalicylanilide raised the rate of dye uptake under these conditions, hence the presence of an Na⁺ conductance greater than the H⁺ conductance was suggested. K⁺ gradients also induced changes of pH, at about 10% of the Na⁺ or Li⁺ rate. Partial inhibition (21%) was seen with 0.1 mM amiloride indicating that K⁺ was a low affinity substrate for the Na⁺/H⁺ exchange. Acceleration both by tetrachlorosalicylanilide (2-fold) and valinomycin (4-fold) suggested the presence of 2 classes of vesicles, those with high and those with low K⁺ conductance. The larger magnitude of the valinomycin dependent signal suggested that 75% of the vesicles had a low K⁺ conductance. Inward Cl^? gradients also induced acidification, partially inhibited by the presence of tetraphenylphosphonium, and accelerated by tetrachlorosalicylanilide. Thus both a Cl^? conductance greater than the H⁺ conductance and a Cl^?/OH^? exchange were present. The rate of Na⁺/H⁺ exchange was amiloride sensitive with a pH optimum of 6.5 and an apparent K_m for Na⁺ or Li⁺ of about 10 mM and an E_A of 14.3 kcal per mol. A 61-fold Na₂SO₄ gradient resulted in a pH gradient of 1.64 units which increased to 1.8 with gramicidin. An equivalent NaCl gradient gave a much lower ΔpH even in the presence of gramicidin showing that the H⁺ and Cl^? pathways could alter the effects of the Na⁺/H⁺ exchange.     

Scanning ion‐selective electrode technique and X‐ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt‐sensitive cucumber and salt‐tolerant pumpkin under NaCl stress

Grafting onto salt‐tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na⁺ in salt‐tolerant pumpkin and salt‐sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion‐selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na⁺, and a correspondingly increased H⁺ influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na⁺/H⁺ exchange in the root was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or vanadate [a plasma membrane (PM) H⁺‐ATPase inhibitor], indicating that Na⁺ exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na⁺/H⁺ antiporter across the PM, and the Na⁺/H⁺ antiporter system in salt stressed pumpkin roots was sufficient to exclude Na⁺. X‐ray microanalysis showed higher Na⁺ in the cortex, but lower Na⁺ in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na⁺, limit the radial transport of Na⁺ to the stele and thus restrict the transport of Na⁺ to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na⁺ to the shoot than cucumber roots.