Abstract
In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co-transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange.
Highlights
Inthe presence of an outwardly directed hydroxyl gradient(pH 6.0 outside and pH 7.5 inside the vesicle), cholateuptake was markedly stimulatedand thebile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium (“overshoot”).In the absence of a pH gradient,uptake was relativeslylower and no overshoot was seen
Recent studies in our laboratory extended these observations by examining the structural determinants for Na+-dependent vesicle uptake of bile acids [4].While the uptake of both di- and trihydroxy conjugated bile acidswas markedly stimulated by an inwardly directed Na+ gradient, the uptake of unconjugated bile acids was not Na+-dependent
In theabsence of a pH gradient, an inside- examined the effects of pH gradients on the uptake of the positive valinomycin-induced K+ diffusion potential unconjugated bile acid cholate by rat basolateral liver plasma caused a slight increase in cholate uptake which was membrane vesicles. pH gradients have previouslybeen reinsensitive tofurosemide
Summary
As previously described [2]. Using this technique, uptakes could be measured reliably as early as 1.8s. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and 7.5 inside the vesicles), cholate (1p ~ ) uptake by basolateral liver plasma membrane vesicleswas rapid andthe bileacidwas transiently accumulated at a times with different membrane preparations. In the absence of a pH studies, uptake data were obtained for multiple membrane vesicle gradient (pH 6.0 or 7.5 both inside and outside the vesicle), preparations and pooled. A weighted least squaresfit of the individual the initial uptake rate (estimatedat 5 s) was relatively slower data points was performed on a computer as previously described [8]. 4-Acetamido-4‘-isothiocyanostiland no uptake 1.8 and overshoot wasobserved (Fig. 1, left panel). Cholate in the presence of a pH gradient was linear between 6.0 s (Fig. 1, right panel).
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