H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells

Thwaites, David T.; Ford, Dianne; Glanville, Michael; Simmons, Nicholas L.

doi:10.1172/jci7192

Cited by 73 publications

(80 citation statements)

References 33 publications

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“…What has been less clear is why transport via endogenous H ϩ -oligopeptide symporters exhibits variable dependence upon extracellular sodium, whereas studies using recombinant transporters expressed exogenously indicate that sodium is not required for H ϩ gradient-driven dipeptide uptake (12,39). A recent series of elegant experiments has demonstrated that the apparent sodium dependence of the endogenous symporter activity in Caco-2 tissue culture cells is due to a functional coupling between Na ϩ /H ϩ exchange and oligopeptide uptake (40,41), and that this coupling occurs specifically with an exchanger located on the apical membrane (14). The importance of our work in relation to the previously established findings lies in the fact that we have shown that functional coupling likely occurs in live animals as well as in isolated cell systems with dramatic physiological consequences.…”

Section: Discussionmentioning

confidence: 99%

“…Although it has long been known that the rate of substrate absorption via the PepT1 and T2 oligopeptide-H ϩ symporters is greatly enhanced at a low extracellular pH (12,13), more recent data suggest that the resulting cytoplasmic acidification leads to the selective activation of apical Na ϩ /H ϩ exchangers (primarily NHE3) in intestinal Caco-2 cells (14). In addition, while this study was in progress, a report was published suggesting that the inhibition of dipeptide uptake caused by treatment with vasoactive intestinal peptide was indirect and manifest primarily through a modification of NHE3 activity (15).…”

Section: Namentioning

confidence: 99%

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A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

Nehrke

2003

Journal of Biological Chemistry

116

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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 (pH i ) 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 pH i , 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.

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Section: Discussionmentioning

confidence: 99%

Section: Namentioning

confidence: 99%

A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

Nehrke

2003

Journal of Biological Chemistry

116

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“…These effects of alterations of Ca 2ϩ in levels on pH in appear to be best explained by involvement of NHE isoform 3 (NHE-3). NHE-3 was identified functionally in the apical membrane of differentiated Caco-2 cells and was shown to be preferentially activated as a consequence of H ϩ influx across the apical membrane (32). Moreover, this antiporter has been shown to be inhibited by Ca 2ϩ -and PKC-dependent pathways (12,19), and therefore, the modulation of NHE-3 activity by Ca 2ϩ channel blockers or other compounds affecting Ca 2ϩ in concentrations in epithelial cells may thereby especially affect H ϩ gradient-dependent transport processes at the apical plasma membrane, such as H ϩ -coupled transport of ␤-lactams.…”

Section: Discussionmentioning

confidence: 99%

“…Since it has been shown that the influx of Ca 2ϩ across the plasma membrane into Caco-2 cells is associated with a reduction in pH in as a consequence of reduced Na ϩ and H ϩ exchange (NHE) activities (38), changes in Ca 2ϩ in levels caused by nifedipine could alter cefixime transport indirectly via modulation of NHE activities. It is known that the pH gradient across the apical membranes of Caco-2 cells limits the rate of transport of PEPT1 and that NHE activities play a crucial role in maintaining the driving force of the peptide transporter (30,32,34). To investigate whether Ca 2ϩ channel blockers or Ca 2ϩ ionophores affect pH in and thereby alter the driving force for cefixime uptake, pH in was measured when cells were superfused with nifedipine or ionomycin.…”

Section: Effects Of Camentioning

confidence: 99%

PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca ²⁺ Channel Blockers

Wenzel

Kuntz

Diestel

et al. 2002

Antimicrob Agents Chemother

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The ␤-lactam antibiotic group encompasses the penicillins, cephalosporins, carbapenems, and monobactams (13). The high intestinal absorption rates of orally active ␤-lactams are mainly explained by the fact that they serve as substrates for the intestinal peptide transporter PEPT1 (10). PEPT1 is located in the apical membranes of intestinal epithelial cells (23) and the Caco-2 human intestinal epithelial cell line (39). The peptide transporter translocates di-and tripeptides (17, 33) as well as a variety of peptidomimetic compounds, including selected immunostimulants (25, 28), angiotensin-converting-enzyme inhibitors (7, 31), and ␤-lactam antibiotics (36, 43), in a proton-coupled electrogenic mode (1).The affinities of ␤-lactams for PEPT1 show large variabilities, depending on the structure of the individual antibiotic (10, 42). Moreover, it was shown that different ␤-lactams can display completely different pH dependencies for transport into Caco-2 cells or into oocytes expressing heterologously cloned mammalian PEPT1. The optimum pH for transport of zwitterionic ␤-lactams such as cefadroxil is 6.5, whereas the dianionic cefixime is effectively transported only at pH values Ͻ6.0 (42). Since the intestinal epithelium has a fairly stable surface pH compartment (microclimate pH) with a pH that is Ͼ6.25 and Ͻ6.75 (22), interactions of ␤-lactams with PEPT1 in the normal gut and thus their in vivo absorption rates may be defined by the pH at the membrane surface. This might explain why cefixime, which is efficiently transported by PEPT1 in vitro at pH values below 6.0, shows a comparably low oral availability in vivo (3, 15). Other factors contributing to the absorption of ␤-lactams via PEPT1 may include regulation of transport capacity by protein kinase C (PKC)-and insulin-dependent pathways (9, 29). Understanding of those mechanisms responsible for alterations in the transport kinetics of PEPT1 could be important for specific enhancement of the absorption of drugs with low oral availabilities, such as cefixime, which is usually used for the treatment of respiratory tract infections (37). As it has been shown that rates of absorption of cefixime are increased by 30% when the Ca 2ϩ channel blocker nifedipine is coadministered with cefixime in humans (14), we were interested in studying the underlying mechanisms responsible for this increase. Although it had been suggested that neurohormonal regulation may be involved in mediating the effects of nifedipine on cefixime transport (18), we hypothesized that the alterations in intracellular free calcium ion (Ca 2ϩ in ) concentrations caused by nifedipine could enhance PEPT1 transport activity for cefixime.We therefore altered the Ca 2ϩ in concentration in Caco-2 cells, a human intestinal epithelial cell line that expresses PEPT1, with either Ca 2ϩ channel blockers or Ca 2ϩ ionophores and measured the effects on the apical influx of [ 14 -C]cefixime. Moreover, by use of intracellular pH (pH in ) measurements, we assessed whether alterations in Ca 2ϩ in levels secondarily ...

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“…The biogenesis and properties of fluid domes have been investigated in Madin-Darby canine kidney cell monolayers (49,(56)(57)(58)(59)(60)(61)(62) and in LLC-PK1 pig kidney-derived cells (63,64). For intestinal cells forming monolayers in culture (35), several studies have revealed that the fluid dome-forming cells are functionally equivalent to differentiated epithelial cells in the intestine involved in the transcellular transportation of solutes (45,46,48), since the functional properties of fluid domes are related to the differentiation-associated apical localization of functional sodium transporters, including sodium channels and Na ϩ /H ϩ exchangers (NHEs) controlling the transcellular passage of ions/water (65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76). In several of these reports, the combined measures of the electrical and functional transport properties and the activities of brush border-associated transporters have clearly established the relationship between the transcellular passage of fluid and the formation of fluid domes.…”

Section: Discussionmentioning

confidence: 99%

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells

Nicolas

Moal

2015

Infect Immun

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Diarrheal diseases affect millions of people, and 2.5 million children under the age of 5 years die from these diseases every year (1). Diarrheal disease develops through a multifactorial process that counteracts the net absorption of water as the result of an increase in the secretion or a decrease in the absorption of water. In the intestinal tract, the passage of water across the epithelial barrier is tightly regulated by both the transcellular and paracellular movements of fluid and electrolytes. Transcellular passage develops through an asymmetric intracellular distribution of membrane-associated pumps and channels, whereas paracellular permeability is regulated by structural and functional proteins located at the tight junctions (TJs) (2). Enteric bacterial pathogens have developed sophisticated strategies to manipulate the host's normal water balance by producing both structural and functional changes in the epithelial barrier (3). In particular, enterovirulent Escherichia coli strains alter the structural organization of polarized epithelial cells and/or deregulate the functional systems involved in the regulation of the transcellular or paracellular passage of fluids and electrolytes in the intestinal epithelial barrier by the production of deleterious cytotoxic or cytotonic toxins (3).The gastrointestinal system uses a variety of antisecretory or proabsorptive hormonal and protein agonists to balance the outflow of fluid and electrolytes. Those that have been more extensively studied are neuropeptide Y/peptide YY (NPY/PYY) (4) and antisecretory factor (AF) (5). AF is a 41-kDa endogenous protein which was originally purified from the pig pituitary gland by Lön-nroth and Lange (6). Its gene has been cloned and sequenced (7). AF is phylogenetically well preserved, since it appears to be a single protein with several conformational variants (8), and no AF-like proteins have been reported. AF is present in most tissues, including the nasal, respiratory, urinary, and gastrointestinal mucosae (9, 10), and is secreted into plasma and other tissue fluids in mammals (11-13). AF appears in rat tissues after a challenge with cholera toxin (CT) or Clostridium difficile toxin (CDT) (14-16). A region of AF that supports its antisecretory activity has been identified between residues 36 and 51, located in the N-terminal part of the full-length protein (14,(17)(18)(19)(20). Experimentally, AF inhibits the intestinal secretion of fluids induced by a variety of toxins, including CT (6,7,14,17,[21][22][23][24][25][26], Campylobacter toxin (24, 27), Escherichia coli heat-labile enterotoxin (LT) (18, 23) and heatstable enterotoxins (ST) (23,25,28), CDT (14,15,21), and Dinophysis toxin (24). Moreover, AF and the AF peptide containing the active peptide sequence from residues 36 to 51 have been shown to block the out-in permeation of 36 Cl in nerve cell membranes isolated from rabbit Dieter cells (20,29,30). Clinically, AF appears to be effective, since administration of a medicinal food containing AF-rich egg yolk powder (B2...

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H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells

Cited by 73 publications

References 33 publications

A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca ²⁺ Channel Blockers

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells

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H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells

Cited by 73 publications

References 33 publications

A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

A Reduction in Intestinal Cell pH Due to Loss of the Caenorhabditis elegans Na+/H+ Exchanger NHX-2 Increases Life Span

PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca 2+ Channel Blockers

Antisecretory Factor Peptide AF-16 Inhibits the Secreted Autotransporter Toxin-Stimulated Transcellular and Paracellular Passages of Fluid in Cultured Human Enterocyte-Like Cells

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PEPT1-Mediated Cefixime Uptake into Human Intestinal Epithelial Cells Is Increased by Ca ²⁺ Channel Blockers