scholarly journals Biochemical evidence for ATPase activity in CFTR-enriched apical membrane vesicles from tracheal epithelium

2000 ◽  
Vol 1467 (1) ◽  
pp. 7-17
Author(s):  
Hélène Villette ◽  
Lionel G Lelièvre
1987 ◽  
Vol 253 (3) ◽  
pp. C416-C425 ◽  
Author(s):  
A. Elgavish ◽  
D. R. DiBona ◽  
P. Norton ◽  
E. Meezan

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.


1999 ◽  
Vol 276 (1) ◽  
pp. G132-G137 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder

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.


1993 ◽  
Vol 264 (5) ◽  
pp. G874-G879 ◽  
Author(s):  
V. M. Rajendran ◽  
H. J. Binder

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.


1994 ◽  
Vol 106 (1) ◽  
pp. 125-133 ◽  
Author(s):  
Edward J. Cragoe ◽  
Pradeep K. Dudeja ◽  
James M. Harig ◽  
Melissa L. Baldwin ◽  
Krishnamurthy Ramaswamy ◽  
...  

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