Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 +/- 0.06 versus pH 6.6 +/- 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton "leak", as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.     

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(+)-free medium and realkalinization occurred upon addition of Na+ to the medium in a concentration-dependent manner, with no apparent saturation. The rate of H(+)-efflux 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(+)-dependent H(+)-efflux was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2, [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H(+)-efflux were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H(+)-egress 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.     

Identification of a mitochondrial Na+/H+ exchanger

The electroneutral exchange of protons for Na+ and K+ across the mitochondrial inner membrane contributes to organellar volume and Ca2+ homeostasis. The molecular nature of these transporters remains unknown. In this report, we characterize a novel gene (YDR456w; renamed NHA2) in Saccharomyces cerevisiae whose deduced protein sequence is homologous to members of the mammalian Na+/H+ exchanger gene family. Fluorescence microscopy showed that a Nha2-green fluorescent protein chimera colocalizes with 4',6-diamidino-2-phenylindole staining of mitochondrial DNA. To assess the function of Nha2, we deleted the NHA2 gene by homologous disruption and found that benzamil-inhibitable, acid-activated 22Na+ uptake into mitochondria was abolished in the mutant strain. It also showed retarded growth on nonfermentable carbon sources and severely reduced survival during the stationary phase of the cell cycle compared with the parental strain, consistent with a defect in aerobic metabolism. Sequence comparisons revealed that Nha2 has highest identity to a putative Na+/H+ exchanger homologue (KIAA0267; renamed NHE6) in humans. Northern blot analysis demonstrated that NHE6 is ubiquitously expressed but is most abundant in mitochondrion-rich tissues such as brain, skeletal muscle, and heart. Fluorescence microscopy showed that a NHE6-green fluorescent protein chimera also accumulates in mitochondria of transfected HeLa cells. These data indicate that NHA2 and NHE6 encode homologous Na+/H+ exchangers and suggest they may be important for mitochondrial function in lower and higher eukaryotes, respectively.     

Pharmacological characterization of stably transfected Na+/H+ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties

A fibroblast mutant cell line devoid of Na+/H+ exchange was used to stably express cDNAs encoding the NHE1, NHE2, and NHE3 Na+/H+ antiporters. Pharmacological studies using amiloride and two of its 5-N-substituted derivatives, 5-N-dimethyl amiloride and 5-N-(methyl-propyl)amiloride (MPA), demonstrate that the NHE1 isoform is the ubiquitously expressed amiloride-sensitive Na+/H+ antiporter (Ki of 0.08 microM for MPA), whereas the NHE2 and NHE3 isoforms exhibit a lower affinity for these inhibitors (Ki of 0.5 microM and 10 microM, respectively, for MPA) and are therefore likely to be members of the epithelial Na+/H+ exchanger's family. In addition, we have used this system to test a new Na+/H+ exchanger inhibitor possessing anti-ischemic properties on myocardial cells [(3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. This compound inhibits competitively NHE1 (Ki of 0.16 microM) with a much greater affinity than NHE2 and NHE3 (Ki of 5 microM and 650 microM, respectively) and therefore appears to be much more discriminative between these two classes of antiporter isoforms than the amiloride-related molecules. These results suggest an explanation for the observed difference of physiological effects between amiloride and HOE694, and identify this new inhibitor as a useful tool for studies of Na+/H+ exchange.     

Mycobacterial phagosome maturation, rab proteins, and intracellular trafficking

One of the most prominent features of pathogenic mycobacteria, which include the potent human pathogens Mycobacterium tuberculosis and Mycobacterium leprae and their opportunistic relatives Mycobacterium avium and Mycobacterium marinum, is their ability to survive and multiply in phagosomes of mononuclear phagocytic cells. The phagocytosed mycobacteria reside in a vacuolar compartment which is exempted from maturation into the phagolysosome. Recently, the arrest of the maturation of phagosomes containing M. tuberculosis complex organisms (Mycobacterium bovis BCG) has been linked to the accumulation on the phagosomal membrane of the small GTP binding protein rab5, specific for the control of fusion within the early endosomal compartment. Furthermore, M. bovis BCG phagosome is devoid of rab7, a rab protein associated with the late endosome. The selective accumulation of rab5 and exclusion of rab7 defines the check point that has been compromised in mycobacterial phagosome maturation. Here we summarize these observations and relates them to other phenomena in the area of membrane and protein trafficking with the emphasis on phagosomes containing intracellular pathogens.     

Mutation of calmodulin-binding site renders the Na+/H+ exchanger (NHE1) highly H(+)-sensitive and Ca2+ regulation-defective

The ubiquitous plasma membrane Na+/H+ exchanger (NHE1) is rapidly activated in response to various extracellular signals. To understand how the intracellular Ca2+ is involved in this activation process, we investigated the effect of Ca2+ ionophore ionomycin on activity of the wild-type or mutant NHE1 expressed in the exchanger-deficient fibroblasts (PS120). In wild-type transfectants, a short (up to 1 min) incubation with ionomycin induced a significant alkaline shift (approximately 0.2 pH unit) in the intracellular pH (pHi) dependence of the rate of 5-(N-ethyl-N-isopropyl) amiloride-sensitive 22Na+ uptake, without changes in the cell volume and phosphorylation state of NHE1. Mutations that prevented calmodulin (CaM) binding to a high affinity binding region (region A, amino acids 636-656) rendered NHE1 constitutively active by inducing a similar alkaline shift in pHi dependence of Na+/H+ exchange. These same mutations abolished the ionomycin-induced NHE1 activation. These data suggest that CaM-binding region A functions as an "autoinhibitory domain" and that Ca2+/CaM activates NHE1 by binding to region A and thus abolishing its inhibitory effect. Furthermore, we found that a short stimulation with thrombin and ionomycin had apparently no additive effects on the alkaline shift in the pHi dependence of Na+/H+ exchange and that deletion of region A also abolished such an alkaline shift induced by a short thrombin stimulation. The results strongly suggest that the early thrombin response and the ionomycin response share the same activation mechanism. Based on these data and the results shown in the accompanying paper (Bertrand, B., Wakabayashi, S., Ikeda, T., Pouysségur, J., and Shigekawa, M. (1994) J. Biol. Chem. 269, 13703-13709), we propose that CaM is one of the major "signal transducers" that mediate distinct extracellular signals to the "pHi sensor" of NHE1.     

Rapid computerized analysis of Na+/H+ exchange flux

The regulation of intracellular pH (pHi) is mediated by membrane transporters whose activity is directly controlled by pHi. Therefore, transport rates must be compared at identical pHi values in functional studies of these transporters. This is conventionally performed using scatter plots showing initial rates of proton flux versus intracellular pH. We present justification for determining proton flux over a wide range of pHi, by digitally smoothing a pHi trace and then directly taking the first derivative versus time of smoothed data. Compared to conventional least-squares analysis of initial rates, the derivative method generates much more information per experiment. Compared to other methods which fit pHi traces to a defined equation prior to rate calculation, the new method does not require that the pHi trace be well fit by any given mathematical function. The derivative technique is illustrated in an analysis of Na+/H+ exchange in Caco-2 cells. Intracellular pH is measured fluorometrically in cells loaded with BCECF (2',7'-bis[2-carboxyethyl]-5-(and-6)carboxyfluorescein). To validate the analysis of Na+/H+ exchange over an extended time range, we demonstrate that cellular acidification with NH4Cl does not change steady state Na+ content. We find that proton flux rates analyzed by the derivative method are equivalent to initial rates measured by least-squares analysis.     

Novel localization of a Na+/H+ exchanger in a late endosomal compartment of yeast. Implications for vacuole biogenesis

Na+/H+ exchangers catalyze the electrically silent countertransport of Na+ and H+, controlling the transmembrane movement of salt, water, and acid-base equivalents, and are therefore critical for Na+ tolerance, cell volume control, and pH regulation. In contrast to numerous well studied plasma membrane isoforms (NHE1-4), much less is known about intracellular Na+/H+ exchangers, and thus far no vertebrate isoform has been shown to have an exclusively endosomal distribution. In this context, we show that the yeast NHE homologue, Nhx1 (Nass, R., Cunningham, K. W., and Rao, R. (1997) J. Biol. Chem. 272, 26145-26152), localizes uniquely to prevacuolar compartments, equivalent to late endosomes of animal cells. In living yeast, we show that these compartments closely abut the vacuolar membrane in a striking bipolar distribution, suggesting that vacuole biogenesis occurs at distinct sites. Nhx1 is the founding member of a newly emergent cluster of exchanger homologues, from yeasts, worms, and humans that may share a common intracellular localization. By compartmentalizing Na+, intracellular exchangers play an important role in halotolerance; furthermore, we hypothesize that salt and water movement into vesicles may regulate vesicle volume and pH and thus contribute to vacuole biogenesis.     

Functional analysis of amino acid residues essential for activity in the Na+/H+ exchanger of fission yeast

We identified amino acid residues important for activity of sod2, the Na+/H+ antiporter of Schizosaccharomyces pombe. We mutated all eight His residues of sod2 into Arg. Only His367-->Arg affected function and resulted in complete inability of sod2 to allow growth of S. pombe in LiCl-containing medium. Mutant S. pombe (H367R) could not expel sodium in acidic (pH 4.0) medium and were defective in their ability to alkalinize external medium. When His367 was replaced by Asp, sodium export of S. pombe was suppressed at acidic pH while the sodium-dependent proton influx at pH 6.1 was increased compared to wild type. We also mutated three residues conserved in putative membrane regions of various eukaryotic and prokaryotic Na+/H+ exchangers. S. pombe containing Asp241-->Asn and Asp266, 267-->Asn mutations had greatly impaired growth in LiCl-containing medium. In addition, sodium-dependent proton influx at external pH 6. 1 was impaired. Sodium export from S. pombe cells at external pH 4.0 was also almost completely abolished by the D266,267N mutation; however, the D241N mutant protein retained almost normal Na+ export. The results demonstrate that His367, Asp241, and Asp266,267 are important in the function of the eukaryotic Na+/H+ exchanger sod2.     

Amiloride-insensitive Na+-H+ exchange: a candidate mediator of erythrocyte Na+-Li+ countertransport

Erythrocyte Na+-Li+ countertransport shows an increased activity in essential hypertension and diabetic nephropathy, but its nature remains unknown. This amiloride-insensitive membrane transport may not be a mode of operation of the amiloride-sensitive NHE1, the only Na+-H+ exchange isoform found in human erythrocytes. Whether an independent, although unknown, amiloride-insensitive isoform mediates Na+-Li+ countertransport is unclear. Na+-H+ exchange activity was measured in acid-loaded erythrocytes. Dimethylamiloride, a specific inhibitor of Na+-H+ exchange and phloretin, a known inhibitor of Na+-Li+ countertransport, gave a reduction in H+-driven Na+ influx (by 31 and 37%, respectively). This effect was additive, and a 66% reduction in H+-driven Na+ influx was found in the presence of both inhibitors. Internal acidification, a stimulus for Na+-H+ exchange, enhanced Na+-Li+ countertransport activity (from 287 +/- 55 to 1213 +/- 165 micromol x Lcell(-1) h(-1), mean +/- SEM, P = 0.003). This transport remained sensitive to phloretin under both conditions. Conversely, external acidification decreased Na+-Li+ countertransport activity (as expected for a Na+-H+ exchanger). Competition between internal H+ and Li+ or Na+ for a common binding site was present. Finally, similar kinetic parameters for external Na+ characterized Na+-Li+ countertransport and the phloretin-sensitive component of H+-driven Na+ influx. These findings suggest that both Na+-Li+ countertransport and the amiloride-insensitive, phloretin-sensitive component of H+-driven Na+ influx can be mediated by a previously unrecognized novel amiloride-insensitive Na+-H+ exchange isoform in human erythrocytes.     

Rapid activation of Na+/H+ exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance

There is increasing evidence for an additional acute, nongenomic action of the mineralocorticoid hormone aldosterone on renal epithelial cells, leading to a two-step model of mineralocorticoid action on electrolyte excretion. We investigated the acute effect of aldosterone on intracellular free Ca2+ and on intracellular pH in an aldosterone-sensitive Madin-Darby canine kidney cell clone. Within seconds of application of aldosterone, but not of the glucocorticoid hydrocortisone, there was a 3-fold sustained increase of intracellular Ca2+ at a half-maximal concentration of 10(-10) mol/liter. Omission of extracellular Ca2+ prevented this hormone response. In the presence of extracellular Ca2+ aldosterone led to intracellular alkalinization. The Na+/H+ exchange inhibitor ethyl-isopropanol-amiloride (EIPA) prevented the aldosterone-induced alkalinization but not the aldosterone-induced increase of intracellular Ca2+. Omission of extracellular Ca2+ also prevented aldosterone-induced alkalinization. Instead, aldosterone led to a Zn(2+)-dependent intracellular acidification in the presence of EIPA, indicative of an increase of plasma membrane proton conductance. Under control conditions, Zn2+ prevented the aldosterone-induced alkalinization completely. We conclude that aldosterone stimulated net-entry of Ca2+ from the extracellular compartment and a plasma membrane H+ conductance as prerequisites for the stimulation of plasma membrane Na+/H+ exchange which in turn modulates K+ channel acitivity. It is probable that the aldosterone-sensitive H+ conductance maintains Na+/H+ exchange activity by providing an acidic environment in the vicinity of the exchanger. Thus, genomic action of aldosterone determines cellular transport equipment, whereas the nongenomic action regulates transporter activity that requires responses within seconds or minutes, which explains the rapid effects on electrolyte excretion.     

Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

1. Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. 2. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. 3. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling. 4. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. 5. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV. 6. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. 7. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. 8. Western analysis with a monoclonal anti gastric H+/K+ ATPase beta-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. 9. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.     

Enhanced Na+/H+ exchange in Cushing's syndrome reflects functional hypermineralocorticoidism

BACKGROUND: Patients with Cushing's syndrome exhibit a bimodal distribution of maximal rates of the erythrocyte amiloride-sensitive Na+/H+ exchange (NHE). Enhanced erythrocyte NHE has recently been found in patients with primary aldosteronism. OBJECTIVE: To test the hypothesis that occult hypermineralocorticoidism in a subset of patients with Cushing's syndrome is responsible for the greater than normal NHE. METHODS: NHE was measured as maximal initial rate (Vmax) of amiloride-inhibited efflux of H+ into an alkaline Na+-containing medium, for 47 patients with hypercortisolism (20 with pituitary adenomas, 18 with adrenal adenomas, and nine with ectopic production of adrenocorticotropin). Clinical appearance, blood pressure levels, plasma aldosterone and deoxycorticosterone levels, serum electrolytes, and urine (tetrahydrocortisol plus 5-alpha-tetrahydrocortisol) : tetrahydrocortisone ratios were assessed for all patients. Twenty patients (10 with greater than normal NHE and 10 with low-to-normal NHE) were randomly selected from 47 patients with hypercortisolism, and treated with 200 mg/day spironolactone for 7 days. NHE in these patients was assessed before starting the treatment and 2 days after its cessation. RESULTS: Greater than normal NHE (Vmax) was associated with peripheral edema, high diastolic blood pressure, hypokalemia, and high urine (tetrahydrocortisol plus 5-alpha-tetrahydrocortisol) : tetrahydrocortisone ratios. The enhanced NHE was rapidly normalized by treatment with spironolactone. CONCLUSION: Erythrocyte NHE in patients with hypercortisolism and functional hypermineralocorticoidism is greater than normal due to incomplete peripheral conversion of cortisol (which binds to mineralocorticoid receptors) into metabolically inactive cortisone.     

Activation of Na+/H+ exchange is required for regulatory volume decrease after modest "physiological" volume increases in jejunal villus epithelial cells

Epithelial cell volume increases that occur because of the uptake of Na+-cotransported solutes or hypotonic dilution are followed by a regulatory volume decrease (RVD) due to the activation of K+ and Cl- channels. We studied the relationship of Na+/H+ exchange (NHE) to this RVD in suspended guinea pig jejunal villus cells, using electronic sizing to measure cell volume changes and fluorescent spectroscopy of cells loaded with 2', 7'-bis(carboxyethyl)-5()-carboxyfluorescein to monitor intracellular pH (pHi). When the volume increase achieved by these cells during Na+ solute absorption was duplicated by a modest 5-7% hypotonic dilution, their pHi first acidified and then alkalinized. This alkalinization was blocked by 5-(N-methyl-N-isobutyl) amiloride (MIA; 1 microM), an inhibitor of NHE. The RVD subsequent to 5-7% hypotonic dilution was prevented by Na+-free medium and by amiloride and non-amiloride derivatives. The order of potency of these inhibitors was as follows: MIA > 5-(N,N-dimethyl) amiloride > cimetidine > clonidine, in keeping with the pattern attributable to NHE-1 as the isoform of NHE responsible for increase in pHi after modest volume increases. A substantial 30% hypotonic dilution caused acidification, and RVD following this larger volume increase was not affected by MIA. To assess the effect of hypotonicity on the activity of NHE, we measured the rate of MIA-sensitive pHi recovery from an acid load (dpHi/dt) in 5 and 30% hypotonic media. pHi recovery was faster in 5% hypotonic medium compared with isotonic medium and slowest in 30% hypotonic medium, which suggested that the activity of NHE was stimulated in the slightly hypotonic medium, but inhibited in the 30% hypotonic medium. To determine the role of activated NHE in RVD after a modest volume increase, cells were hypotonically diluted 7% in MIA to prevent RVD and then alkalinized by NH4Cl or acidified by propionic acid addition. Only after alkalinization was there complete volume regulation. We conclude that in Na+-absorbing enterocytes, the NHE-1 isoform of Na+/H+ exchange is stimulated by volume increases that duplicate the "physiological" volume increase occurring when these cells absorb Na+-cotransported solutes. The subsequent alkalinization of pHi is a required determinant of the osmolyte loss that underlies this distinct volume regulatory mechanism.     

Bicyclic acylguanidine Na+/H+ antiporter inhibitors

Blockade of the Na+/H+ exchange has been shown to diminish the serious consequences of myocardial ischemia. The aim of this investigation was to alter the structure of the common benzoylguanidine NHE inhibitors in such a way that the 3-methylsulfonyl and 4-alkyl group form a ring. The new benz-fused five-, six-, and seven-membered ring sulfones were prepared by internal Heck reaction. Benz-fused five-membered ring sulfones could also be prepared by internal aldol-type condensation using ketones or nitriles as acceptor groups. In the final step, the carboxyl groups were converted to acylguanidines preferentially by guanidine treatment of the esters or acid chlorides. The compounds were tested as their methanesulfonate salts. The inhibition of the Na+/H+ antiport activity was determined by observing the uptake of 22Na+ into acidified rabbit erythrocytes. Additionally, the inhibition of the antiport activity was assessed also by the platelet swelling assay (PSA), in which the swelling of human platelets was induced by the incubation in the presence of a weak organic acid. On average, the IC50 values in the PSA turned out to be about 10-fold higher than in the erythrocyte assay primarily due to a higher Na+ concentration in the PSA; however, the order of the compounds' potency was not substantially altered. The new compounds were found to be highly active with peak values ranging within the cariporide and EMD 96785 standards.     

Intracellular pH regulation in bovine aortic endothelial cells: evidence of both Na+/H+ exchange and Na+-dependent Cl-/HCO3- exchange

Regulation of intracellular pH (pHi) was studied in cultured bovine aortic endothelial cells, an important cell system for cardiovascular research. Suspended cells were acidified by the NH4Cl prepulse technique as well as by exposure to CO2/HCO3-. Subsequent rates of pHi recovery were monitored using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF). In HCO3(-)-free solutions, an EIPA-sensitive, Na+-dependent mechanism fully accounted for realkalinization, namely the Na+/H+ exchanger (NHE). In the presence of HCO3-, an additional acid efflux mechanism was found. This one was dependent on Na+ and intracellular Cl-, EIPA-insensitive but DIDS-sensitive, and therefore represented a Na+-dependent Cl-/HCO3- exchanger (NCBE). In summary, two acid-extruding mechanisms were identified in bovine aortic endothelial cells: NHE and NCBE.     

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4

Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.