The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments.

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Endocytosed ligands move through a series of progressively more acidic vesicles. These differences in pH (pHi) could reflect differences in ion transport mechanisms. Vesicles representing three stages of endocytosis, compartment for uncoupling of receptor and ligand (CURL), multivesicular bodies (MVB), and receptor recycling compartment (RRC), were studied, and all exhibited ATP-dependent electrogenic acidification that was a saturable function of medium chloride. Initial rates of acidification differed (RRC > CURL > MVB), and proton influx was similar for CURL and RRC but slower for MVB. Steady-state ATP-dependent pHi in the three vesicles was more similar. Vesicle membrane potential was substantial (+41 to +69 mV) in low-chloride medium and greatest for RRC but was low (-6 to +6 mV) in 140 mM KCl. These vesicles also exhibited -22 to -37 mV Donnan potentials. Steady-state pump-generated proton electrochemical gradients (delta mu H+) ranged from 114 to 175 mV and were greater for CURL and RRC than for MVB; however, delta mu H+ changed little over a 140-fold difference in chloride concentration. Proton leak rates were faster in CURL and RRC than in MVB, but proton efflux was similar. Finally, proton fluxes and permeabilities, calculated with regard to surface area, differed in the opposite direction (MVB > CURL > RRC). Thus, for the endocytic vesicles studied, intrinsic differences in proton flux and in vesicle geometry could be demonstrated that contributed to differences in pre-steady-state vesicle pHi.

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The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.

The mechanisms of bile acid uptake have been studied with primary monolayer cultures of rat hepatocytes. Hepatocytes were incubated with taurocholic acid (TC), glycocholic acid (GC), cholic acid (CA), glycochenodeoxycholic acid (GCDC), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), lithocholic acid (LCA), or cholylglycylhistamine (CCH), a neutral bile acid derivative for 10 s to 60 min in medium containing sodium chloride, sodium chloride with 1 mM ouabain, or choline chloride. Cells were washed free of radioactive tracer, cell-associated radioactivity was quantitated, and bile acid uptake rates, kinetic parameters of uptake, and steady-state bile acid content were calculated. Two mechanisms for bile acid uptake were identified. Uptake of TC, GC, CA, and GCDC occurred predominantly via a sodium-dependent, ouabain-suppressible saturable mechanism, presumably sodium-coupled transport. Estimates of apparent Km and Vmax for these bile acids were TC, 33 micro M and 0.36 nmol . min-1 . mg prot-1; GC, 18 micro M and 0.22 nmol . min-1 . mg prot-1; CA, 13 micro M and 0.10 nmol . min-1 . mg prot; and GCDC, 6 micro M and 0.21 nmol . min-1 . mg prot, respectively. Uptake via this sodium-coupled mechanism exhibited considerable substrate selectivity. It was enhanced by increased ring hydroxylation and amino acid conjugation and decreased by further conjugation with a neutral histamine group (CGH). In contrast, uptake of CDCA, DOCA, LCA, and CGH occurred primarily via a nonsaturable sodium-independent mechanism, possibly simple diffusion. This mechanism accounted for only a small portion of uptake of TC, GC, CA, and GCDC at low bile acid concentrations. Nonsaturable bile acid uptake rates appeared to correlate with decane-buffer partition coefficients and to be related to bile acid structure.

Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min. Inulin uptake and efflux in cultured hepatocytes and inulin uptake by perfused rat liver were kinetically compatible with the entry of inulin into a rapidly (t1/2, 1-2 min) turning-over (presumably endosomal) compartment that exchanged contents with the extracellular space and comprised -3% of hepatocyte volume, as well as entry into and concentration of inulin within slowly (t1/2, >1 hr) turning-over storage compartments. Based on inulin uptake, it is estimated that cultured hepatocytes endocytosed the equivalent of 20% or more of their volume and 5 or more times their plasma membrane surface area each hour. Neither chloroquine (1 mM) nor taurocholate (200 ,uM) affected inulin handling by cultured cells, whereas colchicine (10 ,uM) inhibited transfer to storage compartments by >50%. In conjunction with our previous observations, the present findings suggest that inulin endocytosed across the basolateral membrane is largely (Q80%) regurgitated back into plasma, with smaller amounts transported to intracellular storage compartments ("18%) or to bile (-2%). Transport of inulin via these pathways is unaffected by taurocholate and does not require vesicle acidification, whereas intact microtubular function is required for transfer to storage compartments or biliary secretion.The ability to internalize extracellular material appears to be a property shared by most, if not all, cells. Hepatocytes are actively endocytic and internalize a variety of ligands by receptor-mediated endocytosis (1, 2), yet relatively little is known regarding the overall rate at which hepatocytes internalize extracellular fluid or fluid phase markers (3, 4).We have recently reported evidence that the intact perfused rat liver transports a variety of fluid phase markers from perfusate to bile via a transcellular vesicular mechanism (5). In the present study, we have extended these observations in perfused liver and also characterized fluid phase endocytosis by rat hepatocytes in primary culture. In addition to measuring the rate at which hepatocytes endocytose extracellular fluid, our principal objectives were as follows. First, several recent reports indicate that fluid phase markers are internalized initially into a compartment that exchanges with extracellular fluid (6-8); however, the kinetics of this process, which presumably reflect membrane recycling, remain incompletely characterized. We therefore sought evidence of such a process in mammalian liver and characterized it kinetically. Our second objective was to integrate observations in perfused liver and culture...

In these studies, we have used several approaches to systematically explore the contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfused rat liver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), inulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran species in bile were the same as in perfusate. Taurocholate administration produced a 71% increase in bile flow but little or no (0-20%) increase in the output of horseradish peroxidase, microperoxidase, inulin, and dextran. Second, under nonsteady-state conditions in which the appearance in or disappearance from bile of selected markers was studied after their abrupt addition to or removal from perfusate, erythritol reached a B/P ratio of 1 within 2 min. Microperoxidase and dextran appeared in bile only after a lag period of 12 min and then slowly approached maximal values, whereas sucrose exhibited kinetically intermediate behavior. A similar pattern was observed after removal of >95% of the marker from the perfusate. Erythritol rapidly reapproached a B/P ratio of 1, whereas the B/P ratio for sucrose, dextran, and microperoxidase fell much more slowly and exceeded 1 for a full 30 min after perfusate washout. Finally, electron microscopy and fluorescence microscopy of cultured hepatocytes demonstrated the presence of horseradish peroxidase and fluoresceindextran, respectively, in intracellular vesicles, and fractionation of perfused liver homogenates revealed that at least 35-50% of sucrose, inulin, and dextran was associated with subcellular organelles.Collectively, these observations are most compatible with a transcytosis pathway that contributes minimally to the secretion of erythritol, but accounts for a substantial fraction of sucrose secretion and virtually all (>95%) of the blood-to-bile