Current-voltage relationship of the basolateral membrane of a tight epithelium.

The polyene antibiotic nystatin is used to reduce selectively to zero the apical membrane resistance of the rabbit descending colon, allowing the measurement of the current-voltage curve of the basolateral membrane. The I--V relationship is described by the Goldman-Hodgkin-Katz equations allowing calculation of PNa/PK, PCl/PK and PK for the basolateral membrane. Cs+ is found to block inward current (serosa to mucosa) in a manner similar to that found in excitable membranes.     

Lithium absorption in tight and leaky segments of intestine

There is significant absorption of Li+ by human jejunum and ileum, but negligible absorption by human colon. Thus, a proximal-to-distal gradient of decreasing Li+ absorption and increasing junctional tightness exists in intestine as well as in renal tubule. For six leaky epithelia the relative permeabilities of K+, Na+, and Li+ by the junctional route are in the sequence PK greater than PNa greater than PLi and all fall within a factor of 2.5. In contrast, for tight epithelia PLi approximately PNa much greater than PK in the amiloride-sensitive channel of the apical membrane, but PK much greater than PLi approximately PNa in the basolateral membrane. The ability of several tight epithelia to sustain nonzero transepithelial Li+ absorption despite this basolateral barrier may be due to Na+/Li+ countertransport at the basolateral membrane, resulting in secondary active transport of Li+ across the epithelium.     

Ionic permeability of K, Na, and Cl in crayfish nerve. Regulation by membrane fixed charges and pH.

Teorell's fixed charge theory for membrane ion permeability was utilized to calculate specific ionic permeabilities from measurements of membrane potential, conductance, and specific ionic transference numbers. The results were compared with the passive ionic conductances calculated from the branched equivalent circuit membrane model of Hodgkin Huxley. Ionic permeabilities for potassium, sodium, and chloride of crayfish (Procambarus clarkii) medial giant axons were examined over an external pH range from 3.8 to 11.4. Action potentials were obtained over this pH range. Failures occurred below pH 3.8 during protonation of membrane phospholipid phosphate and carboxyl, and above pH 11.4 from calcium precipitation. In general, chloride permeability increases with membrane protonation, while cation permeability decreases. At pH 7.0, PK = 1.33 X 10(-5), PCl = 1.49 X 10(-6), PNa = 1.92 X 10(-8) cm/s. PK: PCl: PNa = 693:78:1. PCl is zero above pH 10.6 and is opened predominately by protonation of epsilon-amino, and partially by tyrosine and sulfhydryl groups from pH 10.6 to 9. PK is activated in part by ionization of phospholipid phosphate and carboxyl around pH 4, then further by imidazole from pH 5 to 7, and then predominately from pH 7 to 9 by most probably phosphatidic acid. PNa permeability parallels that of potassium from pH 5 to 9.4. Below pH 5 and above pH 9.4, PNa increases while PK decreases. Evidence was obtained that these ions possibly share common passive permeable channels. The data best support the theory of Teorell, that membrane fixed charges regulate permiability and that essentially every membrane ionizable group appears involved in various amounts in ionic permeability control.     

Electrical properties of normal and transformed mammalian cells.

The transmembrane potential difference, Em, and DC membrane resistance were measured in 3T3 and polyoma virus-transformed 3T3 cells. Em was a function of cell density and was -12 and -25 mV for the normal and transformed cells, respectively. The external concentrations of K+, Na+, and Cl were varied in order to study the nature of the differences between the two cell types. The relative permeability of ions was calculated to be: PNa/PK, 1.0; PCl/PK, 1.88; PNa/PCl, 0.53 for 3T3 cells, and 0.27, 1.75, and 0.15 for the transformed cells. In contrast to the normal cells, PNa/PK varied as a function of the external K+ concentration for the transformed cells. It was emphasized that the manipulation of variables directly affecting the electrical properties of cells also involves the indirect manipulation of a network of interconnected physiological determinants.     

Sodium channel selectivity. Dependence on internal permeant ion concentration

The selectivity of sodium channels in squid axon membranes was investigated with widely varying concentrations of internal ions. The selectivity ratio, PNa/PK, determined from reversal potentials decreases from 12.8 to 5.7 to 3.5 as the concentration of internal potassium is reduced from 530 to 180 to 50 mM, respectively. The internal KF perfusion medium can be diluted by tetramethylammonium (TMA), Tris, or sucrose solutions with the same decrease in PNa/PK. The changes in the selectivity ratio depend upon internal permeant ion concentration rather than ionic strength, membrane potential, or chloride permeability. Lowering the internal concentration of cesium, rubidium, guanidnium, or ammonium also reduces PNa/Pion. The selective sequence of the sodium channel is: Na greater than guanidinium greater than ammonium greater than K greater than Rb greater than Cs.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

The nature of the conductance increase induced by filipin in cholesterol-containing planar lipid bilayers

The effects of the polyene antibiotic filipin on the conductance and permeability of planar lipid bilayers were investigated under voltage-clamp conditions. The membrane conductance of lipid bilayers containing no cholesterol was not affected by filipin. In the presence of cholesterol containing lipid bilayers, filipin induced a 10(4)-10(5)-fold increase in transmembrane conductance. This conductance increase was dependent on the ionic species present in solution, decreasing in the following order: GCsCl greater than GNaAc greater than GKCl greater than GNaCl greater than CaCl2 greater than GNa2SO4 greater than GBaCl2 greater than GMgCl2. Reversal potential measurements in simple biionic conditions revealed the following relative permeability sequence: PK greater than PCl greater than PNa approximately Pac approximately PBa greater than PCs greater than PMg approximately PCa greater than Psulphate. The filipin-sterol mediated increase in membrane conductance was independent of the membrane potential. The increase in membrane current following a step alteration in membrane potential occurred instantaneously and had no dependence on the previous value of the holding membrane potential. We propose that the filipin-sterol complex forms ion channels in lipid membranes. These channels are found in a single configuration (open state) and select preferentially monovalent cations or anions over divalent ions. Our experimental results are discussed in relation to the effects of other polyene antibiotics on the membrane permeability, and also in relation to experimental problems previously reported with the use of filipin in planar lipid bilayers.     

Separation of hepatocyte plasma membrane domains by free flow electrophoresis

We have applied free flow electrophoresis to separate the canalicular and basolateral (sinusoidal and lateral) domains of rat hepatocyte plasma membranes. Hepatocyte plasma membranes were prepurified by rat zonal and discontinous sucrose gradient centrifugation. In electrophoretic separation, the canalicular membranes were more deflected toward the anode than the basolateral membranes. Na+-dependent taurocholate uptake could be measured in both membrane fractions, transport activity being highest in fractions containing the highest specific activity in the basolateral marker enzyme Na+-K+-ATPase. Thus, differences in electrophoretic mobility permit the separation of functional intact plasma membrane vesicles derived from basolateral and canalicular plasma membrane domains of rat hepatocyte.     

Cation and anion sequences in dark-adapted Balanus photoreceptor.

Anion and cation permeabilities in dark-adapted Balanus photoreceptors were determined by comparing changes in the membrane potential in response to replacement of the dominant anion (Cl-) or cation (Na+) by test anions or cations in the superfusing solution. The anion permeability sequence obtained was PI greater than PSO4 greater than PBr greater than PCl greater than Pisethionate greater than Pmethanesulfonate. Gluconate, glucuronate, and glutamate generally appeared more permeable and propionate less permeable than Cl-. The alkali-metal cation permeability sequence obtained was PK greater than PRb greater than PCx greater than PNa approximately PLi. This corresponds to Eisenman's IV which is the same sequencethat has been obtained for other classes of nerve cells in the resting state. The values obtained for the permeability ratios of the alkali-metal cations are considered to be minimal. The membrane conductance measured by passing inward current pulses in the different test cations followed the sequence, GK greater than GRb greater than GCs greater than GNa greater than GLi. The conductance ratios obtained for a full substitution of the test cation agreed quite well with permeability ratios for all the alkali-metal cations except K+ which was generally higher.     

Ion selectivity of aqueous leaks induced in the erythrocyte membrane by crosslinking of membrane proteins

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

A large conductance Cl- channel revealed by patch-recordings in human fibroblasts

A Cl- channel with large single-unit conductance and characteristic voltage-dependent inactivation was studied on cultured human fibroblasts. The channel was activated only after excision and lasting depolarization of the membrane patch. In inside-out configuration and in symmetrical 135 mM NaCl, the conductance was 300 pS. The channel was usually open at the membrane potentials between -20 to +20 mV, while more negative or positive voltages closed the channel. The time course of this apparent inactivation process was dependent on increasing potential. Recovery from inactivation was made possible by returning the membrane potential to 0 mV. The channel was selective to Cl- over Na+ with a PCl/PNa of 6. The order of permeability among anions was: I greater than Br = Cl greater than isethionate greater than F greater than glutamate. The channel was blocked by internal application of a derivative of the diphenylamine-2-carboxilate (Blocker 144) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.     

The permeability of the sodium channel in Myxicola to the alkali cations

Relative permeabilities to the alkali cations were determined, from the reversal potential (VRev), for the Na channel of internally perfused voltage-clamped Myxicola giant axons. PLi/PNa and PK/PNa are 0.94 and 0.076, respectively. Rb and Cs are not measurably permeant. VRev vs. the internal Na activity was well described by the constant field equation over a 300-fold range of internal Na concentrations. In agreement with findings on squid axons, the PK/PNa was found to increase when the K content of the internal perfusate was reduced (equivalent per equivalent substitution with TMA). Internal Rb and Cs also decreased the PK/PNa. The order of effectiveness of internal K, Rb, and Cs in increasing the Na selectivity of the Na channel was Cs greater than Rb greater than or equal to K. External Li increases the PK/PNa but this may be due to the formation of LiF internally. It may be that substances do not have to traverse the channel in order to affect the selectivity filter. Evidence is presented which suggests that the selectivity of the Na channel may be higher for Na in intact as compared to perfused giant axons. It was concluded that the channel selectivity properities do not reflect only some fixed structural features of the channel, but the selectivity filter has a labile organization.     

Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

Effect of oxytocin on the electrical potential and ion permeability of the apical membrane of frog gall bladder epithelial cells

The influence of oxytocin on the intracellular Na+ and K+ concentrations, the level of transmembrane potential differences, and on the relative ionic permeability (PNa/PK) of the apical zones of the superficial epithelium membrane was studied in experiments on the isolated frog gallbladder (GB). Oxytocine introduced into the outer incubation solution in a dose of 20 mulliunits/ml caused a reduction of transmembrane potential difference, and an increase of PNa/pk coefficient and an insignificant shift of the Na+ and K+ concentrations in the intracellular medium. Thirty minutes after the oxytocine action of the organ the membrane potential (MP) of the cells decreased from 52.7 mV to 38.7 mV (the cell is negatively charged inside), and PNa/PK increased from 0,083 (control) to 0,175 (test) with a simultaneous increase in the intracellular Na+ concentration by 18.3 milliequiv./kg of (H2O)i. Such a shift in the intracellular Na+ and K+ concentrations may cause a decrease of the MP by only--0.7 mV, but actually the membrane potential decreased by--14.0 mV. Thus, the reduction of the transmembrane potential difference results from increase of PNa/PK under the influence of oxytocine. No electrogenic ionic transport through the apical membrane of frog gallbladder epithelial cells was revealed.     

Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.     

Redistribution and fate of colchicine-induced alkaline phosphatase in rat hepatocytes: possible formation of autophagosomes whose membrane is derived from excess plasma membrane

The redistribution and fate of colchicine-induced alkaline phosphatase (ALPase) in rat hepatocytes were investigated by electron microscopic enzyme cytochemistry and biochemistry. ALPase activity markedly increased in rat hepatocytes after colchicine treatment (2.0 mg/kg body weight, intraperitoneal injection). At 20–24 h after colchicine treatment, the liver showed the highest activity of ALPase. Thereafter, ALPase activity decreased and returned to normal levels at 48 h. In normal hepatocytes from control rats, ALPase activity was seen only on the bile canalicular membrane. However, at 20–24 h after colchicine treatment, colchicine-induced ALPase was redistributed in the sinusoidal and lateral (basolateral) membranes as well as in the bile canalicular membrane. At 30–36 h after colchicine treatment, ALPase activity on the basolateral membrane gradually decreased. In contrast, ALPase in the bile canalicular membrane increased along with the enlargement of bile canaliculi, suggesting that ALPase in the basolateral membrane had been transported to the bile canalicular membrane. Furthermore, ALPase-positive vesicles, cisternae and autophagosome-like structures were frequently seen in the cytoplasm. ALPase was also positive in some lysosomal membranes. ALPase in hepatocytes at 48 h after colchicine treatment returned to almost the same location as in control hepatocytes. Altogether, it is suggested that excessively induced ALPase is at least partially retrieved by invagination of the bile canalicular membrane and then transported to lysosomes for degradation. In addition, this study indicates that excess plasma membrane might be a possible origin of autophagosomal membrane.     

Isolation and characterization of the putative canalicular bile salt transport system of rat liver

Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.     

Identification and localization of sodium-phosphate cotransporters in hepatocytes and cholangiocytes of rat liver

Hepatocytes and cholangiocytes release ATP into bile, where it is rapidly degraded into adenosine and P(i). In rat, biliary P(i) concentration (0.01 mM) is approximately 100-fold and 200-fold lower than in hepatocytes and plasma, respectively, indicating active reabsorption of biliary P(i). We aimed to functionally characterize canalicular P(i) reabsorption in rat liver and to identify the involved P(i) transport system(s). P(i) transport was determined in isolated rat canalicular liver plasma membrane (LPM) vesicles using a rapid membrane filtration technique. Identification of putative P(i) transporters was performed with RT-PCR from liver mRNA. Phosphate transporter protein expression was confirmed by Western blotting in basolateral and canalicular LPM and by immunofluorescence in intact liver. Transport studies in canalicular LPM vesicles demonstrated sodium-dependent P(i) uptake. Initial P(i) uptake rates were saturable with increasing P(i) concentrations, exhibiting an apparent K(m) value of approximately 11 muM. P(i) transport was stimulated by an acidic extravesicular pH and by an intravesicular negative membrane potential. These data are compatible with transport characteristics of sodium-phosphate cotransporters NaPi-IIb, PiT-1, and PiT-2, of which the mRNAs were detected in rat liver. On the protein level, NaPi-IIb was detected at the canalicular membrane of hepatocytes and at the brush-border membrane of cholangiocytes. In contrast, PiT-1 and PiT-2 were detected at the basolateral membrane of hepatocytes. We conclude that NaPi-IIb is most probably involved in the reabsorption of P(i) from primary hepatic bile and thus might play an important role in the regulation of biliary P(i) concentration.