Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1

2008 ◽  
Vol 294 (2) ◽  
pp. G429-G440 ◽  
Author(s):  
Roger T. Worrell ◽  
Lisa Merk ◽  
Jeffrey B. Matthews
Keyword(s):  
Cell Line ◽  
Cell Model ◽  
Collecting Duct ◽  
Distal Colon ◽  
Crypt Cell ◽  
Ammonium Transport ◽  
Colonic Crypt ◽  
Protein Activity ◽  
T84 Cells ◽  
T84 Cell Line

Although colonic lumen NH4+levels are high, 15–44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH4+. More extensive studies have elucidated the transport of NH4+in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH4+secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH4+and maintain an apical-to-basolateral NH4+gradient. NH4+transport in the secretory direction was supported by basolateral NH4+loading on NKCC1, Na+-K+-ATPase, and the NH4+transporter, RhBG. NH4+was transported on NKCC1 in T84 cells nearly as well as K+as determined by bumetanide-sensitive86Rb-uptake.86Rb-uptake and ouabain-sensitive current measurement indicated that NH4+is transported by Na+-K+-ATPase in these cells to an equal extent as K+. T84 cells expressed mRNA for the basolateral NH4+transporter RhBG and the apical NH4+transporter RhCG. Net NH4+transport in the secretory direction determined by14C-methylammonium (MA) uptake and flux occurred in T84 cells suggesting functional RhG protein activity. The occurrence of NH4+transport in the secretory direction within a colonic crypt cell model likely serves to minimize net absorption of NH4+because of surface cell NH4+absorption. These findings suggest that we rethink the present limited understanding of NH4+handling by the distal colon as being due solely to passive absorption.

scholarly journals Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport

2004 ◽  
Vol 286 (3) ◽  
pp. F483-F489 ◽  
Author(s):  
Robin J. W. Diepens ◽  
Els den Dekker ◽  
Marcelle Bens ◽  
A. Freek Weidema ◽  
Alain Vandewalle ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Model ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Ruthenium Red ◽  
Distal Convoluted Tubule ◽  
Molecular Regulation ◽  
Hek 293 ◽  
Calbindin D

To unravel the molecular regulation of renal transcellular Ca2+ transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na+/Cl- cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca2+ channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca2+ transport, i.e., calbindin-D9k, calbindin-D28k, plasma membrane Ca2+-ATPase isoform 1b, and Na+/Ca2+ exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca2+ transport of 0.4 ± 0.1 nmol·h-1·cm-2, whereas net transcellular Ca2+ transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol·h-1·cm-2. Transcellular Ca2+ transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca2+ channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC50 of ruthenium red on Na+ currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D3, and 1-deamino-8-d-arginine vasopressin increased transcellular Ca2+ transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca2+ transport in the kidney in vitro.

Modulation of secretagogue-induced chloride secretion by intracellular bicarbonate

1994 ◽  
Vol 266 (5) ◽  
pp. G929-G934 ◽  
Author(s):  
P. C. Dagher ◽  
T. Z. Morton ◽  
C. S. Joo ◽  
A. Taglietta-Kohlbrecher ◽  
R. W. Egnor ◽  
...  
Keyword(s):  
Cell Line ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Tumor Cell Line ◽  
Distal Colon ◽  
Rat Colon ◽  
Sprague Dawley ◽  
T84 Cells ◽  
Cl Secretion ◽  
Rat Distal Colon

We have previously demonstrated inhibition of basal Cl- secretion by intracellular bicarbonate concentration ([HCO3-]i) in rat distal colon. We now examined whether secretagogue-induced Cl- secretion is inhibited by [HCO3-]i as well. Stripped segments of distal colon from male Sprague-Dawley rats and the colon tumor cell line T84 were studied. Flux measurements were performed in the Ussing chamber under short-circuit conditions. [HCO3-]i was calculated from intracellular pH (pHi) values that were estimated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) and carbachol were used as secretagogues. In both distal colon and T84 cells, [HCO3-]i did not affect cAMP-induced Cl- secretion. However, carbachol-induced secretion was inhibited by [HCO3-]i; in rat colon, Cl- secretion decreased from 2.3 to 1.5 mueq.cm-2.h-1 when [HCO3-]i was increased from 15.0 to 28.4 mM (P < 0.05). In T84 cells, the change in short-circuit current decreased from 8.1 to 1.1 microA/cm2 over a range of [HCO3-]i from 0 to 15.6 mM (P < 0.001). We conclude that [HCO3-]i is an important modulator of carbachol-stimulated Cl- secretion in both rat distal colon and the T84 cell line. cAMP-mediated secretion is not affected by [HCO3-]i.

Positive and negative regulation of chloride secretion in T84 cells

1993 ◽  
Vol 265 (4) ◽  
pp. C859-C868 ◽  
Author(s):  
K. E. Barrett
Keyword(s):  
Cystic Fibrosis ◽  
Cell Line ◽  
Chloride Transport ◽  
Cell Types ◽  
Chloride Secretion ◽  
Cystic Fibrosis Gene ◽  
Secretory Process ◽  
T84 Cells ◽  
T84 Cell Line ◽  
Inflammatory Diarrhea

This review discusses recent findings regarding the mechanisms and regulation of chloride secretion in the T84 cell line, a widely used model for the study of transepithelial chloride transport, the properties of the cystic fibrosis gene product (cystic fibrosis transmembrane conductance regulator), and the interactions of inflammatory cell types with the intestinal epithelium. First, I review the features of the chloride secretory mechanism in this cell line, both as originally described by Dharmsathaphorn and co-workers and as modified by more recent findings. Second, I cover what is known of the intracellular regulation of the secretory process. Third, I review the ways in which the cell line has been utilized to dissect pathways of immune-epithelial interactions that may contribute to the pathogenesis of inflammatory diarrhea. Finally, I suggest areas of investigation with this cell line that may prove ripe for further study. The physiological and pathophysiological implications of findings obtained with the cell line are also discussed.

W1775 17 β-Estradiol Inhibits cAMP-Dependent Cl− Secretion in the Human Colonic Crypt Cell Line T84

2010 ◽  
Vol 138 (5) ◽  
pp. S-737
Author(s):  
Patrice Bouyer ◽  
Andreas Mykoniatis ◽  
Jun Tang ◽  
Xu Tang ◽  
Jeffrey B. Matthews
Keyword(s):  
Cell Line ◽  
Crypt Cell ◽  
Colonic Crypt ◽  
Cl Secretion

scholarly journals Effects of chemical chaperones on partially retarded NaCl cotransporter mutants associated with Gitelman's syndrome in a mouse cortical collecting duct cell line

2004 ◽  
Vol 19 (5) ◽  
pp. 1069-1076 ◽  
Author(s):  
J. C. de Jong ◽  
P. H. G. M. Willems ◽  
M. Goossens ◽  
A. Vandewalle ◽  
L. P. W. J. van den Heuvel ◽  
...  
Keyword(s):  
Cell Line ◽  
Collecting Duct ◽  
Duct Cell ◽  
Cortical Collecting Duct ◽  
Chemical Chaperones ◽  
Gitelman's Syndrome ◽  
Nacl Cotransporter

Polyunsaturated fatty acid metabolism in enterocyte models: T84 cell line vs. Caco-2 cell line

2013 ◽  
Vol 50 (2) ◽  
pp. 111-120 ◽  
Author(s):  
Pauline Beguin ◽  
Anne-Catherine Schneider ◽  
Eric Mignolet ◽  
Yves-Jacques Schneider ◽  
Yvan Larondelle
Keyword(s):  
Fatty Acid ◽  
Cell Line ◽  
Polyunsaturated Fatty Acid ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
T84 Cell Line

Rab11 is an apically located small GTP-binding protein in epithelial tissues

1996 ◽  
Vol 270 (3) ◽  
pp. G515-G525 ◽  
Author(s):  
J. R. Goldenring ◽  
J. Smith ◽  
H. D. Vaughan ◽  
P. Cameron ◽  
W. Hawkins ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Distal Colon ◽  
Gastric Fundus ◽  
Pancreatic Acinar Cells ◽  
Rab Proteins ◽  
Bile Canaliculi ◽  
Gastric Parietal Cell ◽  
Brush Borders ◽  
Glandular Cells ◽  
Collecting Duct Cells

Rab proteins are involved in many aspects of dynamic vesicle processing within eukaryotic cells. We have previously identified Rab11 in gastric parietal cell tubulovesicle membranes. We have produced a monoclonal antibody that is specific for Rab11. In all rabbit tissues examined, Rab11 immunoreactivity was highly enriched in epithelial cells. In the gastric fundus, parietal cells were stained in a pattern consistent with localization on tubulovesicles. Surface mucous cells of both the fundus and antrum demonstrated punctate subapical staining. Ileal and proximal colonic enterocyte labeling was observed deep to the brush borders. In the distal colon, staining was observed in the apical regions of mid-crypt cells. In skin and esophagus, punctate immunoreactivity was present in the medial layers of the squamous epithelia. Prominent Rab11 immunoreactivity was present in hepatocytes deep to the bile canaliculi. Punctate subapical staining was observed in pancreatic acinar cells. Apical staining was also observed in collecting duct cells and in the glandular cells of the prostate. These results indicate the Rab11 is expressed in apical vesicular populations in discrete epithelial cell populations.

Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD-3)

2004 ◽  
Vol 287 (4) ◽  
pp. F628-F638 ◽  
Author(s):  
Mary E. Handlogten ◽  
Seong-Pyo Hong ◽  
Connie M. Westhoff ◽  
I. David Weiner
Keyword(s):  
Collecting Duct ◽  
Duct Cell ◽  
Ammonium Transport ◽  
Transport Activity ◽  
Acid Base ◽  
Ammonia Transport ◽  
Medullary Collecting Duct ◽  
Extracellular Sodium ◽  
Permeable Support ◽  
Exchange Activity

The renal collecting duct is the primary site for the ammonia secretion necessary for acid-base homeostasis. Recent studies have identified the presence of putative ammonia transporters in the collecting duct, but whether the collecting duct has transporter-mediated ammonia transport is unknown. The purpose of this study was to examine basolateral ammonia transport in the mouse collecting duct cell (mIMCD-3). To examine mIMCD-3 basolateral ammonia transport, we used cells grown to confluence on permeable support membranes and quantified basolateral uptake of the radiolabeled ammonia analog [14C]methylammonia ([14C]MA). mIMCD-3 cell basolateral MA transport exhibited both diffusive and transporter-mediated components. Transporter-mediated uptake exhibited a Kmfor MA of 4.6 ± 0.2 mM, exceeded diffusive uptake at MA concentrations below 7.0 ± 1.8 mM, and was competitively inhibited by ammonia with a Kiof 2.1 ± 0.6 mM. Transporter-mediated uptake was not altered by inhibitors of Na+-K+-ATPase, Na+-K+-2Cl−cotransporter, K+channels or KCC proteins, by excess potassium, by extracellular sodium or potassium removal or by varying membrane potential, suggesting the presence of a novel, electroneutral ammonia-MA transport mechanism. Increasing the outwardly directed transmembrane H+gradient increased transport activity by increasing Vmax. Finally, mIMCD-3 cells express mRNA and protein for the putative ammonia transporter Rh B-glycoprotein (RhBG), and they exhibit basolateral RhBG immunoreactivity. We conclude that mIMCD-3 cells express a basolateral electroneutral NH4+/H+exchange activity that may be mediated by RhBG.

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