A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span

Na+/H+ exchangers are involved in cell volume regulation, fluid secretion and absorption, and pH homeostasis. NHX-2 is a Caenorhabditis elegans Na+/H+ exchanger expressed exclusively at the apical membrane of intestinal epithelial cells. The inactivation of various intestinal nutrient transport proteins has been shown previously to influence aging via metabolic potential and a mechanism resembling caloric restriction. We report here a functional coupling of NHX-2 activity with nutrient uptake that results in long lived worms. Gene inactivation of nhx-2 by RNAi led to a loss of fat stores in the intestine and a 40% increase in longevity. The NHX-2 protein was coincidentally expressed with OPT-2, an oligopeptide transporter that is driven by a transmembrane proton gradient and that is also known to be involved in fat accumulation. Gene inactivation of opt-2 led to a phenotype resembling that of nhx-2, although not as severe. In order to explore this potential functional interaction, we combined RNA interference with a genetically encoded, fluorescence-based reagent to measure intestinal intracellular pH (pHi) in live worms under physiological conditions. Our results suggest first that OPT-2 is the main dipeptide uptake pathway in the nematode intestine, and second that dipeptide uptake results in intestinal cell acidification, and finally that recovery following dipeptide-induced acidification is normally a function of NHX-2. The loss of NHX-2 protein results in decreased steady-state intestinal cell pHi, and we hypothesize that this change perturbs proton-coupled nutrient uptake processes such as performed by OPT-2. Our data demonstrate a functional role for a Na+/H+ exchanger in nutrient absorption in vivo and lays the groundwork for examining integrated acid-base physiology in a non-mammalian model organism.     

Uroguanylin regulates net fluid secretion via the NHE2 isoform of the Na/H+ exchanger in an intestinal cellular model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na(+)/H(+) exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na(+)/H(+) exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (J(v)) was studied by optic techniques and intracellular pH (pH(i)) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na(+) absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pH(i). In the assayed conditions, cell exposure to apical UGN increases net secretory J(v), without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net J(v) was produced mainly by a reduction in normal Na(+) absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pH(i) is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pH(i) regulation.     

Analysis of Ca2+ Signaling Motifs That Regulate Proton Signaling through the Na+/H+ Exchanger NHX-7 during a Rhythmic Behavior in Caenorhabditis elegans

Membrane proton transporters contribute to pH homeostasis but have also been shown to transmit information between cells in close proximity through regulated proton secretion. For example, the nematode intestinal Na⁺/H⁺ exchanger NHX-7 causes adjacent muscle cells to contract by transiently acidifying the extracellular space between the intestine and muscle. NHX-7 operates during a Ca²⁺-dependent rhythmic behavior and contains several conserved motifs for regulation by Ca²⁺ input, including motifs for calmodulin and phosphatidylinositol 4,5-bisphosphate binding, protein kinase C- and calmodulin-dependent protein kinase type II phosphorylation, and a binding site for calcineurin homologous protein. Here, we tested the idea that Ca²⁺ input differentiates proton signaling from pH housekeeping activity. Each of these motifs was mutated, and their contribution to NHX-7 function was assessed. These functions included pH recovery from acidification in cells in culture expressing recombinant NHX-7, extracellular acidification measured during behavior in live moving worms, and muscle contraction strength as a result of this acidification. Our data suggest that multiple levels of Ca²⁺ input regulate NHX-7, whose transport capacity normally exceeds the minimum necessary to cause muscle contraction. Furthermore, extracellular acidification limits NHX-7 proton transport through feedback inhibition, likely to prevent metabolic acidosis from occurring. Our findings are consistent with an integrated network whereby both Ca²⁺ and pH contribute to proton signaling. Finally, our results obtained by expressing rat NHE1 in Caenorhabditis elegans suggest that a conserved mechanism of regulation may contribute to cell-cell communication or proton signaling by Na⁺/H⁺ exchangers in mammals.     

Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5, 6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H(+)-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H(+)-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H(+)-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A(1), specific inhibitors of vacuolar H(+)-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mm, an inhibitor of P-type H(+)-ATPase, nor ethylisopropylamiloride at 0.2 mm, an inhibitor of Na(+)/H(+)-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H(+)-ATPase is responsible for active extrusion of protons from the parasite cells.     

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.     

Functional characterization of a NapA Na(+)/H(+) antiporter from Thermus thermophilus

Na(+)/H(+) antiporters are ubiquitous membrane proteins and play an important role in cell homeostasis. We amplified a gene encoding a member of the monovalent cation:proton antiporter-2 (CPA2) family (TC 2.A.37) from the Thermus thermophilus genome and expressed it in Escherichia coli. The gene product was identified as a member of the NapA subfamily and was found to be an active Na(+)(Li(+))/H(+) antiporter as it conferred resistance to the Na(+) and Li(+) sensitive strain E. coli EP432 (DeltanhaA, DeltanhaB) upon exposure to high concentration of these salts in the growth medium. Fluorescence measurements using the pH sensitive dye 9-amino-6-chloro-2-methoxyacridine in everted membrane vesicles of complemented E. coli EP432 showed high Li(+)/H(+) exchange activity at pH 6, but marginal Na(+)/H(+) antiport activity. Towards more alkaline conditions, Na(+)/H(+) exchange activity increased to a relative maximum at pH 8, where by contrast the Li(+)/H(+) exchange activity reached its relative minimum. Substitution of conserved residues D156 and D157 (located in the putative transmembrane helix 6) with Ala resulted in the complete loss of Na(+)/H(+) activity. Mutation of K305 (putative transmembrane helix 10) to Ala resulted in a compromised phenotype characterized by an increase in apparent K(m) for Na(+) (36 vs. 7.6 mM for the wildtype) and Li(+) (17 vs. 0.22 mM), In summary, the Na(+)/H(+) antiport activity profile of the NapA type transporter of T. thermophilus resembles that of NhaA from E. coli, whereas in contrast to NhaA the T. thermophilus NapA antiporter is characterized by high Li(+)/H(+) antiport activity at acidic pH.     

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.     

Polarity of alveolar epithelial cell acid-base permeability

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO-free Na(+) buffer (140 mM Na(+), 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked > or =80% by 100 microM dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na(+)/H(+) exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.     

Hormonal regulation of chicken intestinal NHE and SGLT-1 activities

The effects of aldosterone and arginine vasotocin (AVT) on intestinal Na(+)/H(+) exchange (NHE) and Na(+)-sugar cotransport (SGLT-1) activities have been investigated using brush-border membrane vesicles isolated from Hubbard chicken small and large intestines, and they were compared with those induced by either Na(+) depletion or dehydration. Na(+) depletion was induced by feeding the chickens with either a low- or a high-Na(+) diet for either 0.5, 1, 2, 4, or 8 days. Ileal and colonic NHE2 activity increased with the duration of the Na(+) depletion, whereas that of intestinal SGLT-1 decreased, reaching a plateau after 2 days of treatment. Three-hour incubation of the intestine with aldosterone produced the same effects on NHE activity as does Na(+) depletion, without altering SGLT-1 activity. However, 3-h incubation of the intestine with AVT increased intestinal SGLT-1 activity, without affecting intestinal NHE activity. It is concluded that aldosterone regulates apical ileal and colonic NHE2 activity, whereas that of SGLT-1 is regulated by AVT.     

Proton transfer reactions associated with the reaction of the fully reduced,purified cytochrome C oxidase with molecular oxygen and ferricyanide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.     

Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. The nature of the link between K+ and Na+ transport

We have studied the links between the mechanisms of Na(+), K(+) and H(+) movements in glycolysing Mycoplasma mycoides var. Capri cells. In the light of the results reported in the preceding paper [Benyoucef, Rigaud & Leblanc (1982) Biochem. J.208, 529-538], we investigated certain properties of the membrane-bound ATPase of Mycoplasma cells, with special reference to its ionic requirements and sensitivity to specific inhibitors. Our findings show, first, that, although Na(+) stimulated ATPase activity, K(+) did not affect it, and, secondly, that NN'-dicyclocarboidi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD) were potent inhibitors of the basal ATPase activity, which was unaffected by vanadate and ouabain. We also investigated the movements of Na(+) and H(+) under the experimental conditions applied to the study of the K(+) uptake reported in the preceding paper, and found that when ;Na(+)-loaded cells' previously equilibrated with (22)Na(+) were diluted in a sodium-free medium, addition of glucose induced a rapid efflux of (22)Na(+). This energy-dependent efflux was independent of the presence of KCl in the medium. Studies of the changes in internal pH by 9-aminoacridine fluorescence or [(14)C]methylamine distribution indicated that the movement of Na(+) was coupled to that of protons moving in the opposite direction, a finding that supports the presence of an Na(+)/H(+) antiport. When Na(+)-loaded cells are diluted in an Na(+)-rich medium the Na(+)/H(+) antiport is still active, but cannot decrease the intracellular Na(+) concentration. Under such conditions, net (22)Na(+) extrusion is specifically dependent on the presence of K(+) in the medium. The present results and those derived from the study of K(+) accumulation (the preceding paper) can be rationalized by assuming that Mycoplasma mycoides var. Capri cells contain two transport systems for Na(+) extrusion: an Na(+)/H(+) antiport and an ATP-consuming Na(+)/K(+)-exchange system.     

Altered cardiac Na(+)/H(+) exchange in phospholipase D-treated sarcolemmal vesicles

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.     

Sodium channels are required during in vivo sodium chloride hyperosmolarity to stimulate increase in intestinal endothelial nitric oxide production

NaCl hyperosmolarity increases intestinal blood flow during food absorption due in large part to increased NO production. We hypothesized that in vivo, sodium ions enter endothelial cells during NaCl hyperosmolarity as the first step to stimulate an increase in intestinal endothelial NO production. Perivascular NO concentration ([NO]) and blood flow were determined in the in vivo rat intestinal microvasculature at rest and under hyperosmotic conditions, 330 and 380 mosM, respectively, before and after application of bumetanide (Na(+)-K(+)-2Cl(-) cotransporter inhibitor) or amiloride (Na(+)/H(+) exchange channel inhibitor). Suppressing amiloride-sensitive Na(+)/H(+) exchange channels diminished hypertonicity-linked increases in vascular [NO], whereas blockade of Na(+)-K(+)-2Cl(-) channels greatly suppressed increases in vascular [NO] and intestinal blood flow. In additional experiments we examined the effect of sodium ion entry into endothelial cells. We proposed that the Na(+)/Ca(2+) exchanger extrudes Na(+) in exchange for Ca(2+), thereby leading to the calcium-dependent activation of endothelial nitric oxide synthase (eNOS). We blocked the activity of the Na(+)/Ca(2+) exchanger during 360 mosM NaCl hyperosmolarity with KB-R7943; complete blockade of increased vascular [NO] and intestinal blood flow to hyperosmolarity occurred. These results indicate that during NaCl hyperosmolarity, sodium ions enter endothelial cells predominantly through Na(+)-K(+)-2Cl(-) channels. The Na(+)/Ca(2+) exchanger then extrudes Na(+) and increases endothelial Ca(2+). The increase in endothelial Ca(2+) causes an increase in eNOS activity, and the resultant increase in NO increases intestinal arteriolar diameter and blood flow during NaCl hyperosmolarity. This appears to be the major mechanism by which intestinal nutrient absorption is coupled to increased blood flow.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

Na(+)/H(+)-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na(+)-dependent processes to acid extrusion, 2) sensitivity to Na(+)/H(+) exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pH(i)) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na(+) concentration ([Na(+)](o)) during pH(i) recovery decreased H(+) efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na(+). The Na(+)/H(+) exchange inhibitors ethylisopropylamiloride and amiloride inhibited H(+) efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na(+)](o) and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na(+)/H(+) exchange by isoforms NHE1, NHE2, and NHE3.     

Increased vacuolar Na(+)/H(+) exchange activity in Salicornia bigelovii Torr. in response to NaCl

Shoots of the halophyte Salicornia bigelovii are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. In glycophytes, sensitivity to salt has been associated with an inability to remove sodium ions effectively from the cytoplasm in order to protect salt-sensitive metabolic processes. Therefore, in Salicornia bigelovii efficient vacuolar sequestration of sodium may be part of the mechanism underlying salt tolerance. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole via a Na(+)/H(+) exchanger. In current studies, increased vacuolar pyrophosphatase activity (hydrolysis of inorganic pyrophosphate and proton translocation) and protein accumulation were observed in Salicornia bigelovii grown in high concentrations of NaCl. Based on sodium-induced dissipation of a pyrophosphate-dependent pH gradient in vacuolar membrane vesicles, a Na(+)/H(+) exchange activity was identified and characterized. This activity is sodium concentration-dependent, specific for sodium and lithium, sensitive to methyl-isobutyl amiloride, and independent of an electrical potential. Vacuolar Na(+)/H(+) exchange activity varied as a function of plant growth in salt. The affinity of the transporter for Na(+) is almost three times higher in plants grown in high levels of salt (K(m)=3.8 and 11.5 mM for plants grown in high and low salt, respectively) suggesting a role for exchange activity in the salt adaptation of Salicornia bigelovii.     

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.     

Na+/H+ exchange and pH regulation in the control of neutrophil chemokinesis and chemotaxis

Large proton fluxes accompany cell migration, but their precise role remains unclear. We studied pH regulation during the course of chemokinesis and chemotaxis in human neutrophils stimulated by attractant peptides. Activation of cell motility by chemoattractants was accompanied by a marked increase in metabolic acid generation, attributable to energy consumption by the contractile machinery and to stimulation of the NADPH oxidase and the ancillary hexose monophosphate shunt. Despite the increase in acid production, the cytosol underwent a sizable alkalinization, caused by acceleration of Na(+)/H(+) exchange. The development of the alkalinization mirrored the increase in the rate of cell migration, suggesting a causal relationship. However, elimination of Na(+)/H(+) exchange by omission of external Na(+) or by addition of potent inhibitors was without effect on either chemokinesis or chemotaxis, provided the cytosolic pH remained near neutrality. At more acidic levels, cell motility was progressively inhibited. These observations suggest that Na(+)/H(+) exchange plays a permissive role in cell motility but is not required for the initiation or development of the migratory response. Chemokinesis also was found to be exquisitely sensitive to extracellular acidification. This property may account for the inability of neutrophils to access abscesses and solid tumors that have been reported to have inordinately low pH.     

Proton diet for the sodium pump

In the absence of Na(+) and K(+) ions the Na,K-ATPase shows a pH-dependent ATP hydrolysis that can be inhibited by ouabain. At pH 7.2 this activity is 5% of the maximal under physiological conditions. It could be inferred that this activity is associated with H(+) transport in both directions across the membrane and facilitates an H-only mode of the sodium pump under such unphysiological conditions. By the analysis of experiments with reconstituted proteoliposomes an overall electroneutral transport mode has been proven. The stoichiometry was determined to be 2 H(+)/2 H(+)/1 ATP and is comparable to what is known from the closely related H,K-ATPase. By time-resolved ATP-concentration jump experiments it was found that at no time was the third, Na(+)-specific binding site of the pump occupied by protons. A modified Post-Albers pump cycle is proposed, with H(+) ions as congeners for Na(+) and K(+), by which all experiments performed can be explained.