Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells

1994 ◽  
Vol 267 (1) ◽  
pp. C84-C93 ◽  
Author(s):  
E. J. McMurchie ◽  
S. L. Burnard ◽  
G. S. Patten ◽  
E. J. Lee ◽  
R. A. King ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Subjects ◽  
Maximum Velocity ◽  
Hill Coefficient ◽  
Transport Activity ◽  
Uptake Activity ◽  
Dimethyl Amiloride ◽  
Mouth Wash ◽  
Na Uptake

Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.

Characterization of Na(+)-H+ exchange in segments of rat mesenteric artery

1992 ◽  
Vol 262 (6) ◽  
pp. H1651-H1656
Author(s):  
C. D. Foster ◽  
W. A. Hill ◽  
T. W. Honeyman ◽  
C. R. Scheid
Keyword(s):  
Fluorescence Emission ◽  
Mesenteric Arteries ◽  
Hill Coefficient ◽  
Excitation Wavelength ◽  
Maximal Velocity ◽  
Resistance Vessels ◽  
Rat Mesenteric Artery ◽  
On Line ◽  
Dimethyl Amiloride

To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) “on line” in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7”-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.

Lysine uptake by human placental microvillous membrane: comparison of system y+ with basal membrane

1995 ◽  
Vol 268 (3) ◽  
pp. C755-C761 ◽  
Author(s):  
T. C. Furesz ◽  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Basal Membrane ◽  
System Model ◽  
Independent System ◽  
Lysine Concentration ◽  
Menten Constant ◽  
Na Uptake

Transport of lysine by microvillous membranes was investigated by characterization of L-[3H]lysine uptake in membrane vesicles isolated from human placentas. At least one Na(+)-independent system was observed at 22 degrees C and two systems at 37 degrees C. Lysine concentration dependence data were fit by a one- or two-system model with a Michaelis-Menten constant (Km) of 124 +/- 28 microM and a maximum velocity (Vmax) of 33.1 +/- 7.7 pmol.mg protein-1.min-1 at 22 degrees C and with Km values of 1 +/- 0.6 and 245 +/- 51 microM and Vmax values of 0.14 +/- 0.07 and 45.8 +/- 8.7 pmol.mg protein-1.30 s-1 at 37 degrees C. In the presence of N-ethylmaleimide, the uptake (37 degrees C) data were fit by a one-system model with kinetic parameters similar to the lower Km system. Uptake of L-lysine in the absence of Na+ was inhibited completely by L-arginine, L-histidine, and L-homoarginine. In the presence of Na+, uptake was inhibited completely by these same three amino acids and L-leucine but only partially by other neutral amino acids. To compare directly microvillous and basal membrane from the same placenta, we examined the inhibition of 20 microM lysine uptake in the presence of Na+. Inhibition by L-leucine was similar in the two membranes. However, L-homoserine, L-alanine, and L-phenylalanine over a wide concentration range inhibited substantially less in microvillous (at both temperatures) than in basal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells

2000 ◽  
Vol 278 (4) ◽  
pp. L640-L648 ◽  
Author(s):  
Wolf Heberlein ◽  
Ralf Wodopia ◽  
Peter Bärtsch ◽  
Heimo Mairbäurl
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
A549 Cells ◽  
Alveolar Epithelial Cells ◽  
Transport Activity ◽  
Excitable Cells ◽  
Alveolar Epithelial ◽  
Oxygen Sensitive ◽  
Na Uptake

In oxygen-sensitive excitable cells, responses to hypoxia are initiated by membrane depolarization due to closing of the K channels that is thought to be mediated by a decrease in reactive oxygen species (ROS). Because the mechanisms of hypoxic inhibition of ion transport of alveolar epithelial cells (Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Am J Physiol Lung Cell Mol Physiol 271: L70–L78, 1996; Mairbäurl H, Wodopia R, Eckes S, Schulz S, and Bärtsch P. Am J Physiol Lung Cell Mol Physiol 273: L797–L806, 1997) are not yet understood, we tested the possible involvement of a hypoxia-induced change in ROS that might control transport activity. Transport was measured as86Rb and22Na uptake in A549 cells exposed to normoxia, hyperoxia, or hypoxia together with ROS donors and scavengers. H2O2< 1 mM did not affect transport, whereas 1 mM H2O2activated22Na uptake (+200%) but inhibited86Rb uptake (−30%). Also hyperoxia, aminotriazole plus menadione, and diethyldithiocarbamate inhibited86Rb uptake. N-acetyl-l-cysteine, diphenyleneiodonium, and tetramethylpiperidine- N-oxyl, used to reduce ROS, inhibited86Rb uptake, thus mimicking the hypoxic effects, whereas deferoxamine, superoxide dismutase, and catalase were ineffective. Also, hypoxic effects on ion transport were not prevented in the presence of H2O2, diethyldithiocarbamate, and N-acetyl-l-cysteine. These results indicate that ion transport of A549 cells is significantly affected by decreasing or increasing cellular ROS levels and that it is possible that certain species of ROS might mediate the hypoxic effects on ion transport of alveolar epithelial cells.

scholarly journals Shiga Toxin (Stx)-Binding Glycosphingolipids of Primary Human Renal Cortical Epithelial Cells (pHRCEpiCs) and Stx-Mediated Cytotoxicity

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 139
Author(s):  
Johanna Detzner ◽  
Elisabeth Krojnewski ◽  
Gottfried Pohlentz ◽  
Daniel Steil ◽  
Hans-Ulrich Humpf ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Shiga Toxin ◽  
Saturated Fatty Acids ◽  
Human Kidney ◽  
Enterohemorrhagic Escherichia Coli ◽  
Highly Sensitive ◽  
Liquid Ordered ◽  
Uremic Syndrome

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic–uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.

scholarly journals Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner

2021 ◽  
Author(s):  
Hui-Yu Chen ◽  
Shan-Hua Lin ◽  
Ling-Hsin Cheng ◽  
Jeng-Jong Wu ◽  
Yi-Chen Lin ◽  
...  
Keyword(s):  
Flowering Time ◽  
Nitrate Transport ◽  
Dependent Manner ◽  
Transport Activity ◽  
Node Number ◽  
Shoot Architecture ◽  
Glucuronidase Reporter ◽  
Uptake Activity ◽  
Delayed Flowering ◽  
Severe Growth

Abstract Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and β-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

scholarly journals Hydrogen Production by Termite Gut Protists: Characterization of Iron Hydrogenases of Parabasalian Symbionts of the Termite Coptotermes formosanus

2007 ◽  
Vol 6 (10) ◽  
pp. 1925-1932 ◽  
Author(s):  
Jun-Ichi Inoue ◽  
Kanako Saita ◽  
Toshiaki Kudo ◽  
Sadaharu Ui ◽  
Moriya Ohkuma
Keyword(s):  
Specific Activity ◽  
Short Form ◽  
Coptotermes Formosanus ◽  
High Hydrogen ◽  
Physiology And Biochemistry ◽  
Termite Gut ◽  
Genes Encoding ◽  
Gut Symbionts ◽  
Uptake Activity

ABSTRACT Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H2) that is emitted by the termites; however, little is known about the physiology and biochemistry of H2 production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H2 production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H2 evolution rather than H2 uptake. H2 evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H2 evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H2 uptake activity of the endosymbiotic bacteria compensated for the strong H2 evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H2 production in termite guts has been largely underestimated.

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