Characterization of Cl-HCO3 exchange in basolateral membrane of rat distal colon

2003 ◽  
Vol 285 (4) ◽  
pp. C912-C921 ◽  
Author(s):  
Mutsuhiro Ikuma ◽  
John Geibel ◽  
Henry J. Binder ◽  
Vazhaikkurichi M. Rajendran
Keyword(s):  
Anion Exchange ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Basolateral Membranes ◽  
Exchange Inhibitor ◽  
Rat Distal Colon ◽  
Different Types ◽  
Voltage Clamping

Sodium-independent Cl movement (i.e., Cl-anion exchange) has not previously been identified in the basolateral membranes of rat colonic epithelial cells. The present study demonstrates Cl-HCO3 exchange as the mechanism for 36Cl uptake in basolateral membrane vesicles (BLMV) prepared in the presence of a protease inhibitor cocktail from rat distal colon. Studies of 36Cl uptake performed with BLMV prepared with different types of protease inhibitors indicate that preventing the cleavage of the COOH-terminal end of AE2 protein by serine-type proteases was responsible for the demonstration of Cl-HCO3 exchange. In the absence of voltage clamping, both outward OH gradient (pHout/pHin: 7.5/5.5) and outward HCO3 gradient stimulated transient 36Cl uptake accumulation. However, voltage clamping with K-ionophore, valinomycin, almost completely (87%) inhibited the OH gradient-driven 36Cl uptake, whereas HCO3 gradient-driven 36Cl uptake was only partially inhibited (38%). Both electroneutral HCO3 and OH gradient-driven 36Cl uptake were 1) completely inhibited by DIDS, an anion exchange inhibitor, with a half-maximal inhibitory constant ( Ki) of ∼26.9 and 30.6 μM, respectively, 2) not inhibited by 5-nitro-2-(3-phenylpropylamino)benzoic acid(NPPB), a Cl channel blocker, 3) saturated by increasing extravesicular Cl concentration with a Km for Cl of ∼12.6 and 14.2 mM, respectively, and 4) present in both surface and crypt cells. Intracellular pH (pHi) was also determined with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetomethylester (BCECF-AM) in an isolated superfused crypt preparation. Removal of Cl resulted in a DIDS-inhibitable increase in pHi both in HCO3-buffered and in the nominally HCO3-free buffered solutions (0.28 ± 0.02 and 0.11 ± 0.02 pH units, respectively). We conclude that a carrier-mediated electroneutral Cl-HCO3 exchange is present in basolateral membranes and that, in the absence of HCO3, Cl-HCO3 exchange can function as a Cl-OH exchange and regulate pHi across basolateral membranes of rat distal colon.

scholarly journals Mechanism of n-butyrate uptake in the human proximal colonic basolateral membranes

2002 ◽  
Vol 282 (4) ◽  
pp. G676-G682 ◽  
Author(s):  
S. Tyagi ◽  
J. Venugopalakrishnan ◽  
K. Ramaswamy ◽  
P. K. Dudeja
Keyword(s):  
Fatty Acid ◽  
Anion Exchange ◽  
Short Chain Fatty Acid ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Organ Donor ◽  
Chain Fatty Acid ◽  
Ph Gradient ◽  
Basolateral Membrane Vesicles ◽  
Exchange Inhibitor

Current studies were undertaken to characterize the mechanism of short-chain fatty acid (SCFA) transport in isolated human proximal colonic basolateral membrane vesicles (BLMV) utilizing a rapid-filtration n-[14C]butyrate uptake technique. Human colonic tissues were obtained from mucosal scrapings from organ donor proximal colons. Our results, consistent with the existence of a HCO[Formula: see text]/SCFA exchanger in these membranes, are summarized as follows: 1) n-[14C]butyrate influx was significantly stimulated into the vesicles in the presence of an outwardly directed HCO[Formula: see text] and an inwardly directed pH gradient; 2) n-[14C]butyrate uptake was markedly inhibited (∼40%) by anion exchange inhibitor niflumic acid (1 mM), but SITS and DIDS (5 mM) had no effect; 3) structural analogs e.g., acetate and propionate, significantly inhibited uptake of HCO[Formula: see text] and pH-gradient-driven n-[14C]butyrate; 4) n-[14C]butyrate uptake was saturable with a Kmfor butyrate of 17.5 ± 4.5 mM and a Vmaxof 20.9 ± 1.2 nmol · mg protein−1· 5 s−1; 5) n-[14C]butyrate influx into the vesicles demonstrated a transstimulation phenomenon; and 6) intravesicular or extravesicular Cl−did not alter the anion-stimulated n-[14C]butyrate uptake. Our results indicate the presence of a carrier-mediated HCO[Formula: see text]/SCFA exchanger on the human colonic basolateral membrane, which appears to be distinct from the previously described anion exchangers in the membranes of colonic epithelia.

scholarly journals Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport

1993 ◽  
Vol 294 (2) ◽  
pp. 529-534 ◽  
Author(s):  
S A Pinches ◽  
S M Gribble ◽  
R B Beechey ◽  
A Ellis ◽  
J M Shaw ◽  
...  
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Independent Manner ◽  
Basolateral Membranes ◽  
Luminal Membrane ◽  
C Terminus ◽  
Normal Human

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

Bicarbonate-stimulated [14C]butyrate uptake in basolateral membrane vesicles of rat distal colon

1993 ◽  
Vol 105 (3) ◽  
pp. 725-732 ◽  
Author(s):  
Diedre A. Reynolds ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Basolateral Membrane Vesicles ◽  
Rat Distal Colon

scholarly journals A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine

1987 ◽  
Vol 247 (1) ◽  
pp. 141-146 ◽  
Author(s):  
H M Said ◽  
R Redha
Keyword(s):  
Folic Acid ◽  
Anion Exchange ◽  
Membrane Surface ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Basolateral Membrane Vesicles ◽  
Exchange Inhibitor ◽  
Carrier Mediated Transport ◽  
Structural Analogues ◽  
Disulphonic Acid

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.

Isolation and characterization of apical membrane vesicles of the rat distal colon

1995 ◽  
Vol 195 (1) ◽  
pp. 333-342 ◽  
Author(s):  
Oliver Schröder ◽  
Ralf Gerhard ◽  
Wolfgang F. Caspary ◽  
Jürgen Stein
Keyword(s):  
Apical Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Isolation And Characterization ◽  
Rat Distal Colon ◽  
Apical Membrane Vesicles

scholarly journals Parallel intermediate conductance K+ and Cl− channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon

2020 ◽  
Vol 319 (2) ◽  
pp. G142-G150
Author(s):  
Shabina Rehman ◽  
Karthikeyan Narayanan ◽  
Andrew J. Nickerson ◽  
Steven D. Coon ◽  
Kazi Mirajul Hoque ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Significant Contribution ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Channel Activity ◽  
Basolateral Membranes ◽  
Cl Channel ◽  
Rat Distal Colon

This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller but significant contribution from K+-Cl− cotransporter-1 activity.

Distribution and regulation of apical Cl/anion exchanges in surface and crypt cells of rat distal colon

1999 ◽  
Vol 276 (1) ◽  
pp. G132-G137 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Spatial Distribution ◽  
Concentration Gradient ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Na Depletion ◽  
Crypt Cells ◽  
Apical Membrane Vesicles

Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3 and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3 and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent 36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3 concentration gradient-driven36Cl uptake (i.e., Cl/HCO3 exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3 also stimulates36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3 exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.

Cl-HCO3 and Cl-OH exchanges mediate Cl uptake in apical membrane vesicles of rat distal colon

1993 ◽  
Vol 264 (5) ◽  
pp. G874-G879 ◽  
Author(s):  
V. M. Rajendran ◽  
H. J. Binder
Keyword(s):  
Apical Membrane ◽  
Kinetic Constant ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Hyperbolic Curve ◽  
Apical Membrane Vesicles

This study describes Cl-HCO3 and Cl-OH exchanges as the mechanism for Cl uptake by apical membrane vesicles (AMV) of rat distal colon. Although HCO3 gradient-stimulated 36Cl uptake was additionally stimulated by the additional presence of a pH gradient, pH gradient-stimulated 36Cl uptake was not further enhanced by a HCO3 gradient. HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake was not inhibited by voltage clamping, with K and its ionophore valinomycin, but was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor, with an apparent inhibitory constant of 7.8 and 106.0 microM, respectively. Increasing intravesicular OH concentration in the absence of HCO3 (with fixed extravesicular Cl concentration) yielded a sigmoidal curve for 36Cl uptake. In contrast, increasing intravesicular OH concentration in the presence of equimolar intra- and extravesicular HCO3 (25 mM) yielded a saturable hyperbolic curve. Increasing extravesicular Cl concentration saturated both HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake, with a kinetic constant for Cl of approximately 11.9 and 22.6 mM, respectively. We conclude that Cl uptake in AMV of rat distal colon occurs via two separate anion (Cl-HCO3 and Cl-OH) exchange processes. We speculate that one of these two anion exchanges may be responsible for transcellular Cl movement, while the other may be important in the regulation of intracellular pH homeostasis.

Calcium transport by rat duodenal villus and crypt basolateral membranes

1987 ◽  
Vol 252 (2) ◽  
pp. G170-G177 ◽  
Author(s):  
J. R. Walters ◽  
M. M. Weiser
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Cell Fraction ◽  
Basolateral Membranes ◽  
Pump Activity ◽  
Crypt Cells

Rat duodenal cells were isolated sequentially to give fractions enriched for villus and crypt cells. From each of these fractions, basolateral-enriched membrane vesicles were prepared and ATP-dependent calcium uptake was studied. Calcium uptake was sensitive to temperature, was inhibited by vanadate and by A23187, and was lower in vitamin D-deficient animals. In normal animals, calcium transport was approximately twofold greater in villus-tip than in crypt cell-fraction basolateral membranes though the affinity of the uptake for calcium was similar (Km = 0.3 microM). In vitamin D-deficient animals, the crypt-to-villus gradient was reduced, and in all fractions, calcium transport was similar to or lower than that in the crypts of normal animals. Six hours after vitamin D-deficient animals were repleted with 1,25-dihydroxycholecalciferol, a significant increase in calcium transport by everted gut sacs was present; however, basolateral calcium transport was significantly increased in only the mid-villus fractions, and no change was seen in the villus-tip fractions. Thus vitamin D appears necessary for the development of increased basolateral membrane calcium pump activity in duodenal villus cells, but not all cells in vitamin D-deficient rats are able to respond to 1,25-dihydroxycholecalciferol.

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