An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

A minireview on NHE1 inhibitors. A rediscovered hope in oncohematology.

BACKGROUND: Na(+)/H(+) exchanger-1 (NHE-1) is involved in pH regulation and is up-regulated in different malignancies. Activation of NHE-1 is one way for allowing cells to avoid intracellular acidification and protect them against apoptosis. Inhibitors of NHE-1 are able to decrease intracellular pH and induce apoptosis. Some statins can also act by partial inhibition of NHE-1. This review presents progress in understanding the mechanisms of action of these inhibitors, connections with certain genetic mutations and acquired treatment resistance, as well as new patents on them. METHODS: A MEDLINE search for original and review articles using key terms, Na(+)/H(+) exchanger, leukemia, cariporide, and amiloride. Recent patents with NHE-1 inhibitors published by United States Patent and Trademark Office are also presented. RESULTS AND CONCLUSIONS: Sorafenib is used for the treatment of acute myeloid leukemia patients carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation. 5-(N, N-hexamethylene)-amiloride can increase the suppression of FLT3 signaling by sorafenib. NHE-1 inhibitors are able to increase the sensitivity of chronic myeloid leukemia cells to tyrosine kinase inhibitors, including through the inhibition of P-glycoprotein. NHE-1 inhibitors are promising adjuvant drugs for overcoming acquired resistance to treatment in various malignant hemopathies.

A novel tescalcin-sodium/hydrogen exchange axis underlying sorafenib resistance in FLT3-ITD+ AML.

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is associated with inferior clinical prognosis. Sorafenib is effective in clearing leukemic blasts in chemorefractory FLT3-ITD(+) AML, but leukemia progression invariably occurs. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), known to be upregulated at leukemia progression during continuous sorafenib treatment and activate an Na(+)/H(+) exchanger type-1 (NHE1), may underlie tyrosine kinase inhibitor resistance. TESC was highly expressed in FLT3-ITD(+) AML lines MOLM-13 and MV4-11, and its knockdown by small-interfering RNA lowered intracellular pH (pHi) and induced apoptosis. The results were recapitulated by treatment with an NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA). Induction of sorafenib resistance in the MOLM-13 cell line (M13-RE) significantly increased its sensitivity to HMA. The later also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD(+) AML cells significantly reduced leukemia initiation in anti-CD122-primed NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of a TESC-NHE1-pHi axis in mediating sorafenib resistance in AML.

Overexpression of TaNHX3, a vacuolar Na⁺/H⁺ antiporter gene in wheat, enhances salt stress tolerance in tobacco by improving related physiological processes.

Salt stress is one of the major abiotic stresses affecting plant growth, development, and productivity. In this study, we functionally characterized a wheat vacuolar Na(+)/H(+) antiporter gene (TaNHX3). TaNHX3 is 78.9% identical with TaNHX2 in nucleic acid level, encoding a polypeptide of 522 amino acids (aa). TaNHX3 is targeted onto tonoplast after ER sorting and can complement the growth under salt stress in a yeast mutant with a defective vacuolar Na(+)/H(+) antiporter exchange. TaNHX3 transcripts were induced by applying salt stress in wheat cultivars. More TaNHX3 were detected in the salt-stress-resistant cultivar Ji 7369 compared with the salt-stress-sensitive cultivar Shimai 12 and Ji-Shi-3, an isogenic line derived from aforementioned cultivars with Shimai 12 genetic background. The ectopic TaNHX3 expression in tobacco significantly enhanced the plant tolerance to salt stress. Compared with control plants, the TaNHX3 overexpressing plants displayed no varied Na(+) contents and accumulated more Na(+) amount in plants. However, they exhibited higher fresh and dry weights, more accumulative nitrogen, phosphorus, and potassium, higher contents of chlorophyll, carotenoid, soluble protein, higher activities of the antioxidant enzymes including superoxide dismutase, catalase, and peroxidase, and lower malondialdehyde and H2O2 amount. Our results indicated that TaNHX3 plays an important role in regulating the cytosolic Na(+) transportation within vacuoles under high salinity, alleviating the Na(+) damage effects. The improved salt stress tolerance in TaNHX3 overexpressing tobacco plants is closely associated with the improvement of the aforementioned physiological processes. TaNHX3 can be used as a candidate gene for molecular breeding of salt-tolerant plants.

Na+₋sensing quantum dots for cell-based screening of intracellular Na+ concentrations ([Na+]i).

We have developed a Na-quantum dot (QD) nanosensor for [Na(+)]i measurements. Using this Na-QD, we determined the dynamic physiological responses of [Na(+)]i in nonexcitable human HEK-293F cells and excitable primary rat cardiac myocytes by pharmacologically manipulating the membrane permeability to Na(+), the Na-K-2Cl cotransporter, and the Na(+)/H(+) antiporter. These data suggest that the mechanisms of [Na(+)]i homeostasis can now be elucidated with this novel Na-QD nanosensor. This could have a broad impact on Na(+) channel drug discovery.

Roles of Na(+)/K(+)-dependent ATPase, Na(+)/H(+) antiporter and GLUT hexose transporters in the cryosurvival of dog spermatozoa: effects on viability, acrosome state and motile sperm subpopulation structure.

To assess the roles of Na(+)/K(+)-dependent ATPase, Na(+)/H(+) antiporter and GLUT hexose transporters in the cryosurvival of dog sperm, semen samples were frozen in a standard freezing medium supplemented with the specific inhibitors of these factors ouabain, amiloride or phloretin, respectively. The presence of ouabain did not counteract the effects of freeze-thawing on the percentages of motile sperm and altered acrosomes, although a small recovery effect was observed in motility parameter means. Amiloride had a similar effect, although motility was more intensely recovered. Phloretin significantly (P < 0.05) impaired viability when added at a maximal concentration of 10(-)4 M (57.3 ± 5.1% vs 76.5 ± 5.7% in cells frozen without inhibitors), although partial recovery of motility parameters was also observed. These effects were accompanied with specific changes in both motility parameters and the percentages of motile sperm in each of the 4 subpopulations comprising the motile sperm population of the ejaculate. Our findings indicate a role for Na(+)/K(+)-dependent ATPase and Na(+)/H(+) antiporter in the mechanisms involved in determining specific sperm motility patterns in response to freeze-thawing, although neither pump seems to be important for the resistance of cell membrane structures to freezing-thawing. In addition, a role for GLUTs in regulating water exchange in dog sperm during freeze-thawing seems unlikely. In contrast, the precise structure of dog sperm in terms of its motile subpopulations was found to condition both cryosurvival and sperm cell sensitivity to the inhibitors used.

Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach.

CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein-protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)-NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR-NHERF2-LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2-NHERF2 interaction. We show that this compound can disrupt the LPA2-NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl- channel function can be finely tuned by modulating PDZ domain-based protein-protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl- channels.

Rapid decrease of intracellular pH associated with inhibition of Na+/H+ exchanger precedes apoptotic events in the MNK45 and MNK74 gastric cancer cell lines treated with 2-aminophenoxazine-3-one.

The effects of Phx-3 on changes in intracellular pH (pHi) in the MKN45 and MKN74 human gastric cancer cell lines were evaluated in order to determine the mechanism for the proapoptotic effects of 2-aminophenoxazine-3-one (Phx-3) on these cells. Phx-3 (100 µM) reduced pHi in MKN45 from 7.45 to 5.8, and in MKN74 from 7.5 to 6.2 within 1 min of engagement with these cells. Such a decrease of pHi was closely correlated with the dose of this phenoxazine and continued for 4 h. The activity of Na+/H+ exchanger isoform l (NHE1), which is involved in H+ extrusion from the cells, was dose-dependently suppressed by Phx-3 in these cells, and was greatly suppressed in the presence of 100 µM Phx-3. This result indicates that the decrease of pHi in MKN45 and MKN74 cells is closely associated with the inhibition of NHE1 in these cells. The morphology of these cells at 24 h after treatment with Phx-3 indicated shrinkage of the cells and condensation of the nuclear chromatin structure, which are characteristic of the apoptotic events in these gastric cancer cells. Cytotoxicity of Phx-3 against MKN45 and MKN74 cells was extensive because almost all MKN45 cells lost viability at 24 h in the presence of 20 µM Phx-3, and nearly 50% of the MKN74 cells lost viability in the presence of 50 µM Phx-3. These results suggest that rapid and extensive decrease of pHi in human gastric cancer MKN45 and MKN74 cells caused by Phx-3 might disturb intracellular homeostasis, leading to apoptotic and cytotoxic events in these cells. Phx-3 is a good candidate for therapeutics of gastric cancer that is intractable to conventional chemopreventive therapies.

The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor.

Sodium/proton exchangers (NHX) are key players in the plant response to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space. The expression of most plant NHX genes is modulated by exposure of the organisms to salt stress or water stress. We explored the regulation of the vacuolar NHX1 gene from the salt-tolerant sugar beet plant (BvNHX1) using Arabidopsis plants transformed with an array of constructs of BvHNX1::GUS, and the expression patterns were characterized using histological and quantitative assays. The 5 UTR of BvNHX1, including its intron, does not modulate the activity of the promoter. Serial deletions show that a 337 bp promoter fragment sufficed for driving activity that indistinguishable from that of the full-length (2,464 bp) promoter. Mutating four putative cis-acting elements within the 337 bp promoter fragment revealed that MYB transcription factor(s) are involved in the activation of the expression of BvNHX1 upon exposure to salt and water stresses. Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

[Genetic transformation of buckwheat ( Fagopyrum esculentum Moench ) with AtNHX1 gene and regeneration of salt-tolerant transgenic plants].

The Arabidopsis thaliana tonoplast Na+ /H+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MS basal medium supplemented with 2.0mg/L 6-BA, 1.0mg/L KT, 0.lmg/L IAA, 50mg/L kanamycin and 500mg/L carbenicillin. 426 seedlings from 36 resistant calli originated from 864 explants (transformed about at 4.17 percentage) exhibited resistance to kanamycin. The transformants were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting analysis. After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived, while wild-type plants did not. After 3 days of stress treatment through different concentrations of NaCl, transgenic plants accumulated higher concentration of Na+ and proline than the control plants. However, the K+ concentration of transgenic plants declined in comparison with the control plants. Moreover, the rutin content of the roots, stems and leaves of transgenic buckwheat increased than those of the control plants. These results showed that it could be possible to improve the salt-tolerance of crops with genetic technology.

Intracellular pH signals in the induction of secondary pathways--the case of Eschscholzia californica.

Transient peaks of the cytoplasmic pH are essential elements in a number of signal cascades that activate environmental responses or developmental processes in plant cells but little is known about the mechanisms of their generation. In many plant cells, elicitation of the hypersensitive response is preceded by a perturbation of the ionic balance at the plasma membrane including the inhibition of the proton pump and the influx of H+ from the apoplast. A basically different mechanism of cytoplasmic acidification that is fed by vacuolar protons has been discovered in cell suspensions of the California Poppy (Eschscholzia californica). These cells react to a yeast glycoprotein elicitor with the overproduction of benzophenanthridine alkaloids. Low elicitor concentrations trigger the biosynthesis of these phytoalexins without invoking elements of the hypersensitive response. Accumulated data support the existence of a signal path that includes the following steps: Links between the above events that connect them within a distinct signal path are substantiated by the phenotypes of transformed cell lines that either display lowered Galpha levels due to antisense transformation or express Galpha-binding antibodies in the cytoplasm. All of these cell lines lack the elicitor-activation of PLA2 and of vacuolar proton fluxes and show an impaired phytoalexin response to low elicitor concentrations. High elicitor concentrations trigger alkaloid biosynthesis via an increase of jasmonate at a pH-independent signal path.

Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger.

The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

Blood cardioplegia supplementation with the sodium-hydrogen ion exchange inhibitor cariporide to attenuate infarct size and coronary artery endothelial dysfunction after severe regional ischemia in a canine model.

BACKGROUND: Activation of the sodium-hydrogen ion exchange mechanism results in accumulation of intracellular calcium through the sodium-calcium ion antiport mechanism. Administration of a sodium-hydrogen ion exchange inhibitor before or during ischemia attenuates myocardial ischemia and reperfusion injury. However, the cardioprotection exerted by sodium-hydrogen ion exchange inhibitors as adjuncts to cardioplegia without perioperative administration has not been tested in a model of surgical reperfusion of acute coronary occlusion with cardiopulmonary bypass. This study tested the hypothesis that sodium-hydrogen ion exchange inhibitor-supplemented blood cardioplegia would reduce postcardioplegia injury after severe regional ischemia. METHODS: In anesthetized open-chest dogs, the left anterior descending coronary artery was occluded for 75 minutes, after which total cardiopulmonary bypass was initiated. After crossclamping, cold (4 degrees C) antegrade blood cardioplegia was delivered every 20 minutes for a total of 60 minutes of cardioplegic arrest. In 8 dogs, the blood cardioplegic solution was unsupplemented (vehicle group), whereas in 8 others the solution was supplemented with the sodium-hydrogen ion exchange inhibitor cariporide (10 micro mol/L, cariporide group). RESULTS: In the in vitro studies, the direct effects of cariporide on neutrophil function were determined. Isolated canine neutrophils were stimulated by platelet activating factor. Cariporide attenuated superoxide anion production in a concentration-dependent manner, with no appreciable effect at 10 micro mol/L (the concentration used in blood cardioplegia) and a peak effect at 100 micro mol/L. In the in vivo cardiopulmonary bypass model, infarct size was significantly (P <.05) smaller in the cariporide group than in the vehicle group (22.4% +/- 3.5% vs 40.1% +/- 5.1% of area at risk), although there were no group differences in postischemic regional wall motion after 2 hours of reperfusion (0.1% +/- 0.9% vs -0.2% +/- 0.3% systolic shortening). Transmural myocardial edema in the area at risk was significantly decreased in the cariporide group (80.6% +/- 0.5%) relative to the vehicle group (83.1% +/- 0.6%). Myeloperoxidase activity in the area at risk, an index of neutrophil accumulation, was significantly lower in the cariporide group than in the vehicle group (4.7 +/- 0.9 absorbence units/[min. g tissue] vs 10.3 +/- 2.3 absorbence units/[min. g tissue]). In isolated postischemic left anterior descending coronary artery rings, maximum relaxation in response to the endothelium-dependent vasodilator acetylcholine was significantly greater in the cariporide group than in the vehicle group (77.5% +/- 7.4% vs 51.4% +/- 8.0%), whereas smooth muscle relaxation in response to nitroprusside was comparable between groups. CONCLUSION: In this canine model, supplementation of blood cardioplegia with cariporide, a sodium-hydrogen ion exchange inhibitor, reduced infarct size, attenuated neutrophil accumulation in the area at risk, and reduced postischemic coronary artery endothelial dysfunction without directly inhibiting neutrophil activity. Cariporide as an adjunct to blood cardioplegia without perioperative administration attenuated surgical ischemia-reperfusion injury in jeopardized myocardium.

Targeting the myocardial sodium-hydrogen exchange for treatment of heart failure.

Although the past number of years have seen a substantial improvement in the therapeutic approaches for the treatment of heart failure, mortality rates continue to be high. Moreover, the incidence of heart failure is expanding rapidly. Sodium-hydrogen exchange (NHE) is a key target for the treatment of heart failure. NHE is a major mechanism for intracellular pH regulation in most cell types, including the cardiac cell. Seven isoforms of NHE have been identified so far although cardiac cells possess primarily the ubiquitous NHE-1 subtype. NHE-1 is a major contributor to ischaemic and reperfusion injury and NHE-1 inhibitors exert marked cardioprotective effects, particularly when administered before ischaemia, findings which have now been extended to clinical trials. It is emerging that NHE-1 also contributes to chronic maladaptive myocardial responses to injury (myocardial remodelling) and may contribute to the development of heart failure. Experimental studies using both in vitro approaches as well as animal models of heart failure have consistently demonstrated a beneficial effect of NHE-1 inhibitors in terms of inhibition of hypertrophy in response to various stimuli as well as inhibiting heart failure after coronary artery ligation. These effects occurred independently of any infarct size reducing effects of NHE-1 inhibitors or on any direct effects on afterload thus indicating a direct effect on the myocardial remodelling process. In fact, it appears that NHE-1 may represent a common downstream mediator for various hypertropic factors such as angiotensin II, endothelin-1 and beta(1) adrenergic receptor activation. NHE-1 inhibition, therefore, represents a potentially effective new therapeutic approach for the treatment of heart failure.

Profiling of drugs for membrane activity using liposomes as an in vitro model system.

The increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potentialfor these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K+ and protonophoric uncouplers for H+; (ii) ion antiporters which exchange H+ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.

The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux.

The NHA1 gene of Saccharomyces cerevisiae, transcribed into a 3.5 kb mRNA, encodes a protein mediating Na+ and K+ efflux through the plasma membrane that is required for alkali cation tolerance at acidic pH. Deletion of the gene in a wild-type strain resulted in higher sensitivity to both K+ and Na+ at acidic pH. Measurements of cation loss in strains carrying deleted or overexpressed alleles of NHA1 demonstrated its role in K+ and Na+ efflux. In addition, high K+ and Na+ efflux observed upon alkalinization of the cytoplasm implies a role of Nha1p in the regulation of intracellular pH. Moreover, the overexpression of ENA1 and NHA1 genes in an ena1-4 delta-nha1 delta strain showed that the Nha1 alkalication antiporter is responsible for growth on high concentrations of KCl and NaCl at acidic pH, and Ena alkali-cation ATPases are necessary at higher pH values. Both systems have a complementary action to maintain the intracellular steady-state concentration of K+ and Na+.

The relationship between thermosensitivity and intracellular pH in cells deficient in Na+/H+ antiport function.

BACKGROUND AND PURPOSE: We have demonstrated previously the relationship between intracellular pH (pHi) level and heat survival in mammalian cells. To explore this in further detail, we studied thermosensitivity in CCL 39 and their variant PS120 cells, which lack Na+/H+ antiport function. MATERIALS AND METHODS: CCL39 and PS120 cells were heated with or without amiloride, or 5-(N-ethyl(-N-isopropyl) amiloride (EIPA), inhibitors of Na+/H+ antiport function. Antiport activity and pHi measurements were made using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). A clonogenic assay was used to assess survival after heating. RESULTS: Enhanced cytotoxicity was observed when CCL39 cells were heated with either EIPA (15 microM) or amiloride (2.5 mM) at pHe7.3 in the presence of NaHCO3. Under the same conditions, thermal enhancement of PS120 cells was observed only with amiloride at 2.5 mM. When the cells were heated at pHe 6.5 in bicarbonate-free medium, both EIPA and amiloride enhanced thermal cytotoxicity in CCL39 cells, but only the higher dose of amiloride sensitized the variant PS120 cells. Surviving fraction was related to pHi, but the data fell into two clusters, depending on whether or not both Na+/H+ antiport and the Na(+)-dependent HCO3-/Cl- exchangers were functioning. CONCLUSIONS: We confirm that Na+/H+ antiport function can mediate thermosensitivity, and corroborate a linear correlation between pHi level and log survival after heating, but suggest that this relationship is complicated by other factors such as membrane exchanger function, and extracellular pH levels during heating.

Transient expression of Na+/H+ exchanger isoform NHE-2 in LLC-PK1 cells: inhibition of endogenous NHE-3 and regulation by hypertonicity.

Na+/H+ exchanger isoforms NHE-2 and NHE-3 demonstrate distinct tissue expression patterns in renal epithelial cells. NHE-2 is predominantly expressed in the inner medulla whereas NHE-3 is highly expressed in the proximal tubule cells. The purpose of the current experiments was to study the characteristics of NHE-2 upon its own expression in cultured proximal tubule cells, LLC-PK1. Toward this end, LLC-PK1 cells were subjected to six cycles of proton suicide. The mutant cells, when grown to confluence and assayed for Na+/H+ exchanger by 22Na+ influx, showed significant reduction in NHE activity as compared to the parent cells (10.4 nmole/mg prot/4 min in parent cells vs. 1.8 in mutant cells, P < 0.001, n = 4). This remaining exchanger activity was mostly mediated via NHE-3 as shown by inhibition of the Na influx following PKC stimulation (65% with PMA vs. 100% without PMA. P < 0.05, n = 4). The mutant cells were transiently transfected with a pCMV/NHE-2 expression vector using calcium phosphate precipitation method. Northern blot analysis showed the expression of a 3.4 kb transcript only in the transfected cells. The expression peaked at 48 hr and diminished by 96 hr. The exchanger activity at 48 hr after transfection was mostly due to NHE-3 (as shown by inhibition in the presence of PMA) but was significantly lower than in sham transfected cells (1.2 nmoles/mg prot. in NHE-2-transfected and 2.1 in sham-transfected, P < 0.05, n = 4). At 60 hr after transfection, the cells exhibited PMA-stimulated Na influx (>28%) indicating functional expression of NHE-2. Increasing the osmolality of the media to 510 mOsm/l stimulated the Na+/H+ exchanger in NHE-2 transfected cells but inhibited the exchanger activity in sham transfected cells. In conclusion, NHE-2 appears as a 3.4 kb transcript in transfected LLC-PK1 cells and functional expression of NHE-2 is preceded by inhibition of endogenous NHE-3 activity. The NHE-2 is stimulated by hypertonicity, indicating a likely role for this isoform in cell volume regulation.

The effect of heat on Na+/H+ antiport function and survival in mammalian cells.

PURPOSE: Because intracellular pH (pHi) is a determinate of thermosensitivity, it is important to understand the relationship between heat cytotoxicity and the mechanisms responsible for pHi regulation, such as the Na+/H+ antiport. The objective of this study is to elucidate the relationship between heat damage and Na+/H+ antiport activity. METHODS AND MATERIALS: Various cell lines, EMT6, RIF-1, and its thermoresistant variant TR-4, and CCL39, and its variant that lacks the Na+/H+ antiport (PS120), were all heated using a water bath. Parallel assessments of antiport function and pHi were made using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). RESULTS: Exposure of EMT6 cells to 43-46 degrees C for 30-60 min caused progressive decline in antiport activity, in parallel with cytotoxicity. When the same degree of cytotoxicity was induced by ionizing radiation, no alteration in Na+/H+ antiport function was observed. Despite a 10-fold lower survival in RIF-1 compared to TR-4 cells after heating, there was no difference in the thermosensitivity of their antiports. Antiport activity in the TR-4 cells, however, was higher than that of RIF-1 cells both before and during heating. Intracellular pH for TR-4 cells decreased minimally during heating, in contrast to a decline of 1 pH unit in RIF-1 cells despite similar relative levels of antiport activity, suggesting that in this pair of cell lines, antiport activity does not play a major pHi regulatory role. PS120 and CCL39 cells and similar survival levels when heated at pHe 7.2 in the presence of NaHCO3, which allows function of the other major regulator of pHi, the Na+ -dependent HCO3-/Cl- exchanger. This occurred despite a drop in pHi in the PS120 cells during heating. A reduced survival was observed, however, in PS120 cells after 43 degrees C for 30-60 min at either pHe 6.5 or pHe 7.2 in the absence of NaHCO3. Intracellular pH was consistently greater for PS120 than CCL39 cells. CONCLUSION: We demonstrated that damage to the Na+/H+ antiport likely reflects early heat-induced change in membrane function, but is not a primary target for heat cytotoxicity. Although there is an association between survival, antiport function, and pHi level under most treatment conditions, the precise role of the Na+/H+ antiport in mediating thermal cytotoxicity remains uncertain.