ATP dependence is not an intrinsic property of Na<sup>+</sup>/H<sup>+</sup>exchanger NHE1: requirement for an ancillary factor

Aharonovitz, Orit; Demaurex, Nicolas; Woodside, Michael; Grinstein, Sergio

doi:10.1152/ajpcell.1999.276.6.c1303

Cited by 37 publications

(20 citation statements)

References 42 publications

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“…Consistent with previous studies (21)(22)(23)(24)(25)(26)(27)(28), we find that pretreatment of CHO cells for 30 min with 5 mM 2-deoxy-D-glucose and 5 g͞ml oligomycin resulted in complete loss of NHE1 activity in subsequent recordings without addition of ATP to the pipette solution (data not shown). The omission of MgATP from the pipette solution alone, however, does not affect NHE1 activity.…”

Section: Resultssupporting

confidence: 93%

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Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Fuster

Moe

Hilgemann

2004

Proc. Natl. Acad. Sci. U.S.A.

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Sodium͞hydrogen exchangers (NHEs) are ubiquitous ion transporters that serve multiple cell functions. We have studied two mammalian isoforms, NHE1 (ubiquitous) and NHE3 (epithelial-specific), by measuring extracellular proton (H ؉ ) gradients during whole-cell patch clamp with perfusion of the cell interior. Maximal Na ؉ -dependent H ؉ fluxes (JH؉) are equivalent to currents >20 pA for NHE1 in Chinese hamster ovary fibroblasts, >200 pA for NHE1 in guinea pig ventricular myocytes, and 5-10 pA for NHE3 in opossum kidney cells. Sodium͞hydrogen exchangers (NHEs) are present in simple prokaryotes, lower eukaryotes, and higher eukaryotes including plants, fungi, and animals (1). In prokaryotes, yeast, and plants, the driving force of ⌬H ϩ energizes NHEs to export Na ϩ from the cytosol, whereas in animal cells, ⌬Na ϩ drives NHEs to extrude H ϩ . In mammals, eight NHE isoforms (NHE1-NHE8) are cloned (1). NHE1 is a ubiquitous plasma membrane transporter that regulates pH and cell volume. It is implicated in regulating diverse cellular functions including proliferation, adhesion, and migration (2-4). NHE3 is present on endosomal and plasma membranes of epithelial cells, in which it plays a pivotal role in transepithelial Na ϩ transport in multiple organs (5, 6). The functions of other isoforms, present on both cell-surface (NHE2, NHE4, and NHE8) and organellar (NHE6 and NHE7) membranes, remain largely undefined (7,8).NHE activities are typically determined by changes in bulk cytoplasmic pH by using fluorescent pH-sensitive dyes, Na 22 isotopic flux, or radioactive͞fluorescent membrane-permeant buffer trapping. A common protocol is to acidify the cytoplasm and then quantify acid extrusion by NHEs under near V max conditions during Na ϩ -induced pH recovery. Pervasive problems of this approach include low sensitivity, poor time resolution, changes of ion concentrations, and very limited control of the cytoplasmic milieu. Here we describe an approach using pH microelectrodes during whole-cell patch clamp recording. Extracellular H ϩ gradients caused by H ϩ transport are detected by moving cells close to and away from a pH microelectrode in the presence of low buffer concentrations and measuring the H ϩ gradient with cancellation of electrode drift (9-11). Extensive manipulation of the cytoplasmic medium is possible by pipette perfusion, and H ϩ fluxes can be quantified by simulation of buffer diffusion. Using this ''self-referencing'' pH microelectrode technique, we provide different perspectives on the regulatory properties of NHE1 and NHE3 isoforms, in particular their lipid-and mechanosensitive properties. Specifically, we demonstrate that individual phosphatidylinositides have rapid, specific effects on NHE1 and NHE3 and that NHE1 can be very sensitive to rearrangements of its surrounding lipid domain. However, hydrophobic mismatch does not seem to be the primary molecular signal that mediates NHE1 volume sensing. MethodsCell Culture. Chinese hamster ovary (CHO) fibroblasts were maintained in F-12K nutrient mixture, Kai...

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Section: Resultssupporting

confidence: 93%

“…Grinstein and coworkers (20,(22)(23)(24)29) reported that NHE1 is sensitive to ATP depletion. ATP depletion must be quite extensive to inhibit NHE1, because the simple removal of ATP from the cytoplasmic solutions does not cause NHE1 rundown.…”

Section: Discussionmentioning

confidence: 99%

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Fuster

Moe

Hilgemann

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…41 Because alanine, a noncarbohydrate source of energy, was able to abolish the inhibitory effect produced by the perfusion of 2-DG only in the absence of DL-cycloserine (an alanine aminotransferase inhibitor), we can assume that (1) 2-DG perfusion produced ATP depletion, which has been observed previously, 41 and (2) alanine is converted into pyruvate and can be used by the PT cells as an energy source. In addition, these data suggest that decreased glycolytic metabolism as well as ATP depletion might inhibit NHE3 in the PT.…”

Section: Discussionmentioning

confidence: 63%

“…38 In agreement with our findings, some studies have shown that glucose metabolism modulates other members of the NHE family. 9,31,[39][40][41][42][43][44] These transporters have been shown to be inhibited by glycolytic inhibitors, such as 2-DG, 9,45-47 and have also been observed to be more sensitive to glycolytic inhibitors than oxidative phosphorylation inhibitors. [48][49][50] Additionally, our study suggests, for the first time, that the apical uptake of glucose is important for maintaining NHE3 activity, because glucose was perfused only apically in the tubules, and that glucose metabolism occurs in the PT and is important for maintaining NHE3 activity.…”

Section: Discussionmentioning

confidence: 99%

Functional Role of Glucose Metabolism, Osmotic Stress, and Sodium-Glucose Cotransporter Isoform-Mediated Transport on Na+/H+ Exchanger Isoform 3 Activity in the Renal Proximal Tubule

Pessoa

Campos

Carraro‐Lacroix

et al. 2014

Journal of the American Society of Nephrology

161

114

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Na + -glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na + -H + exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLTmediated glucose uptake regulates NHE3-mediated NaHCO 3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLTmediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule. 25: 202825: -203925: , 201425: . doi: 10.1681 The kidney proximal tubule (PT) is the site where the reabsorption of approximately 70% of filtered sodium bicarbonate occurs. It is mainly performed by the Na + /H + exchanger isoform 3 (NHE3). 1 The physiologic importance of NHE3 became evident after the development of NHE3 knockout mice, which presented mild metabolic acidosis and volume depletion with reduced BP, underscoring the role of NHE3 in volume homeostasis. 2 It has been shown that NHE3 physically and functionally interacts with dipeptidyl-peptidase IV, an enzyme that degrades and inactivates the incretin hormone glucagon like peptide-1. 3 The inhibition of dipeptidyl-peptidase IV and the action of glucagon like peptide-1 were shown to inhibit NHE3 and promote natriuresis. 3-8 Additionally, various conditions and substances related to glucose metabolism, including diabetes, insulin, ATP, and glucose, modulate NHE3 in different tissues, J Am Soc Nephrol

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“…The subsequent intracellular acidification was expected to stimulate the Na + /H + exchanger NHE1, which is expressed in erythrocytes [85,86]. Activation of the carrier leads to entry of Na + and thus to cell swelling [87].…”

Section: Discussionmentioning

confidence: 99%

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

Bissinger

Malik

Bouguerra

et al. 2015

Basic Clin Pharma Tox

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The K + ,H + ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca 2+ -permeable unselective cation channels leading to Ca 2+ entry, increase in cytosolic Ca 2+ activity ([Ca 2+ ] i ) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca 2+ ] i from Fluo3 fluorescence, pH i from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. Materials and MethodsErythrocytes, solutions and chemicals. Li-heparin-anticoagulated fresh blood samples were kindly provided by the blood bank of the University of T€ ubingen. The study was approved by the ethics committee of the University of T€ ubingen (184/2003 V). The blood was centrifuged at 120 9 g for 20 min. at 21°C and the platelet-and leucocyte-containing supernatant were disposed. Erythrocytes were incubated in vitro at a haematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO 4 , 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 5 glucose and 1 CaCl 2 , at 37°C for 48 hr. Where indicated, erythrocytes were exposed to nigericin (Enzo Life Sciences, L€ orrach, Germany) at the indicated concentrations.Annexin V binding and forward scatter. After incubation under the respective experimental condition, a 150-ll cell suspension was centrifuged at 350 rcf for 3 min. and, after trashing the supernatant,

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ATP dependence is not an intrinsic property of Na⁺/H⁺exchanger NHE1: requirement for an ancillary factor

Cited by 37 publications

References 42 publications

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Functional Role of Glucose Metabolism, Osmotic Stress, and Sodium-Glucose Cotransporter Isoform-Mediated Transport on Na+/H+ Exchanger Isoform 3 Activity in the Renal Proximal Tubule

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

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ATP dependence is not an intrinsic property of Na+/H+exchanger NHE1: requirement for an ancillary factor

Cited by 37 publications

References 42 publications

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Lipid- and mechanosensitivities of sodium/hydrogen exchangers analyzed by electrical methods

Functional Role of Glucose Metabolism, Osmotic Stress, and Sodium-Glucose Cotransporter Isoform-Mediated Transport on Na+/H+ Exchanger Isoform 3 Activity in the Renal Proximal Tubule

Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin

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ATP dependence is not an intrinsic property of Na⁺/H⁺exchanger NHE1: requirement for an ancillary factor