Apical Membrane Vesicles Research Articles

Relationship of the Donnan potential to the transmembrane pH gradient in tracheal apical membrane vesicles.

Experiments were performed to determine the factors which contribute to the transmembrane pH gradient (delta pH) and the potential gradient (delta psi) in apical plasma membrane vesicles isolated from bovine tracheal epithelium. As indicated by the accumulation of 14C-methylamine, the vesicles maintained a delta pH (inside acidic) which was dependent upon the external pH. The delta pH was also proportional to the ionic strength of the suspending medium, suggesting that the H+ distribution was dictated by a Donnan potential. Measurements of the distribution of 86Rb+ demonstrated an electrical potential gradient across the vesicle membrane, inside negative which was proportional to the medium ionic strength. delta pH changed in parallel with delta psi in response to a variety of imposed conditions. These results are compatible with the existence of a H+ conductance in the vesicle membrane. Thus the endogenous electrical and proton gradients may be manipulated and used as a general experimental tool to complement kinetic analysis in investigations of transport mechanisms using isolated vesicle preparations.

Detergent solubilization, functional reconstitution, and partial purification of epithelial amiloride-binding protein.

The amiloride-binding protein from cultured toad kidney cells (A6) was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), functionally reconstituted into liposomes, and partially purified. The specific binding of [3H]methylbromoamiloride ([3H]CH3BrA) was measured in intact A6 epithelia, A6 cell homogenate (H), apical plasma membrane vesicle (V1), and CHAPS-solubilized V1 and on material obtained after affinity chromatography of CHAPS-solubilized plasma membrane vesicles on agarose-immobilized wheat germ agglutinin (WGA). Specific [3H]CH3BrA binding to H, V1, and WGA material reached equilibrium after 10 min. Scatchard analysis of [3H]CH3BrA binding to V1 and WGA material revealed a homogeneous class of binding sites with KD's of 130 and 128 nM, respectively. These KD values were similar to the apparent inhibitory dissociation constant determined from amiloride inhibition of 22Na+ influx in both intact A6 epithelia and V1. The total number of specific binding sites was 4 pmol/mg of V1 protein, which represented a 10-fold enrichment compared to H, and 66.6 pmol/mg of WGA material (a 148-fold enrichment). From association/displacement kinetic studies of specific [3H]CH3BrA binding to V1, the rate constants of association (ka) and dissociation (kd) were calculated to be 3.6 X 10(5) M-1 s-1 and 49.5 X 10(-3) s-1, respectively. These values yield an equilibrium dissociation constant of 138 nM. In solubilized V1 protein, binding activity was enriched approximately 20-fold over H and was markedly dependent upon the relative concentrations of detergent and phospholipid. CHAPS solubilization of V1 resulted in an average 44% recovery of protein with 90% retention of the total number of specific [3H]CH3BrA binding sites. After WGA chromatography 2.7% of the applied protein and 46% of the specific binding sites were recovered.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells.

Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade.

Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium.

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.

Relationship between lipid fluidity and water permeability of bovine tracheal epithelial cell apical membranes.

Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.

Sodium-dependent transport of Pi by an established intestinal epithelial cell line (CaCo-2)

The uptake of inorganic phosphate (Pi) was analyzed in monolayers and in apical membrane vesicles (AMV) of the established intestinal cell line CaCo-2. AMV, prepared by a MgCl2 precipitation technique, were enriched approximately 10-fold in alkaline phosphatase activity. Pi uptake into intact cells as well as into AMV was specifically dependent on the presence of sodium. In the presence of high sodium concentrations, the apparent Km for Pi was 214 +/- 17 mumol/l in monolayers and 300 +/- 19.7 mumol/l in AMV. Increasing the sodium concentration increased the apparent affinity of the transport system for Pi but hardly affected the maximal velocity (Vmax). At 0.1 mmol/l Pi and pH 7.4, the apparent Km for sodium was approximately 70 mmol/l in intact cells as well as in AMV. The results obtained in both systems suggested the involvement of two sodium ions and one phosphate ion in the transport process. Advancing confluence--independently of the age of the monolayers--reduced sodium-dependent uptake of Pi significantly by a decrease in Vmax, whereas the apparent Km for Pi remained unchanged. It is concluded that the apical membrane of CaCo-2 cells contains a sodium-dependent transport system for Pi.

Potassium/proton exchange in brush-border membrane of rat ileum.

These experiments were designed to determine whether proton-driven 86Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of 86Rb was approximately 300 to 350% greater in the presence of a 100-fold H+ gradient than in its absence and was greater at 1, 2 and 5 minutes ("overshoot") than that at 90 minutes. Proton-driven 86Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3 mM amiloride did not significantly inhibit proton-driven 86Rb uptake; in contrast, proton-driven 22Na uptake was significantly inhibited by 0.3 mM amiloride by 34%. Similarly, 25 mM KCl inhibited proton-driven 86Rb uptake more than that of 22Na, while the inhibition of proton-driven 22Na uptake by 25 mM NaCl was greater than that of 86Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an outwardly directed K gradient. These studies demonstrate that a proton gradient stimulates 86Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.

The amiloride-sensitive sodium channel

Net Na+ movement across the apical membrane of high-electrical resistance epithelia is driven by the electrochemical potential energy gradient. This entry pathway is rate limiting for transepithelial transport, occurs via a channel-type mechanism, and is specifically inhibited by the diuretic drug amiloride. This channel is selective for Na+, Li+, and H+, saturates with increasing extracellular Na+ concentration, and is not affected, at least in frog skin epithelium, by changes in apical membrane surface potential. There also appears to be multiple inhibitory regions associated with each Na+ channel. We discuss the possible implications of a voltage-dependent block by amiloride in terms of macroscopic inhibitory phenomena. We describe the use of cultured epithelial systems, in particular, the toad kidney-derived A6 cell line, and the preparation of apical plasma membrane vesicles to study the Na+ entry process. We discuss experiments in which single, amiloride-sensitive channel activity has been detected and summarize current experimental approaches directed at the biochemical identification of this ubiquitous Na+ transport system.

Donnan equilibrium and pH gradient in isolated tracheal apical membrane vesicles.

Apical plasma membrane vesicles isolated from bovine tracheal epithelium were found to possess both an electrical potential gradient (delta psi) and a transmembrane pH gradient (delta pH). The delta psi was calculated from the distribution of 86Rb+ in the presence of the ionophore valinomycin, and delta pH was determined from the distribution of [14C]methylamine under conditions of apparent equilibrium. Maximal values for delta psi of -54.3 +/- 2.5 mV and for delta pH of 0.75 +/- 0.07 pH units were obtained under low ionic strength conditions. Increasing the ionic strength by the addition of 50 mM of either permeant or impermeant electrolytes reduced delta psi to near zero values. The delta pH varied in parallel with the delta psi. The results suggest that both 86Rb+ in the presence of valinomycin and H+ are in a Donnan equilibrium with impermeant negative charge in the vesicle interior.

Na-Pi cotransport in LLC-PK1 cells: fast adaptive response to Pi deprivation.

A short-term regulation of the Na-dependent transport of Pi in LLC-PK1 cells by the extracellular concentration of Pi is described. Pi deprivation of the cells for 10 min led to an increase (30%) of the Na-Pi cotransport activity, whereas the Na-dependent D-glucose transport system was not affected. This regulatory phenomenon was not affected by the presence of cycloheximide. The same adaptive response was observed in apical membrane vesicles isolated from rapidly adapted cells. In the isolated membranes, increased Na-Pi cotransport is characterized by an increase of the maximal rate of uptake (control: 193 +/- 15; adapted: 306 +/- 51 pmol X mg-1 X 15 s-1), whereas the apparent Km for Pi remained constant. The results suggest that LLC-PK1 cells possess a mechanism or mechanisms that allow a rapid "activation" and "inactivation" of Na-dependent Pi transport systems as a function of the concentration of the extracellular Pi.

Characterization of a Na∶K∶2Cl cotransport system in the apical membrane of a renal epithelial cell line (LLC-PK1)

86Rb uptake into LLC-PK1 cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na+ and Cl- for activity, and a "leak" component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of 86Rb, 22Na and 36Cl flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na+ : K+ : Cl- cotransport system. The kinetic parameters for Na+ and K+ interaction have been analyzed under initial linear zero trans conditions. The following values were obtained KmNa+ = 0.42 +/- 0.05 mmol/liter, Vmax = 303 +/- 24 pmol/mg/6 sec; KmK+ = 11.9 +/- 1.0 mmol/liter, VmaxK+ = 307 +/- 27 pmol/mg/6 sec. For Cl- interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na+ : 1K+ :2Cl- can be calculated. It is concluded that the apical membrane of LLC-PK1 cells contains a Na+ : K+ : 2Cl- co-transport system with properties similar to those described for the thick ascending limb of the loop of Henle.

1621 APICAL MEMBRANE LIPID FLUIDITY (LF) INFLUENCES PHOSPHATE (Pi) TRANSPORT IN FETAL KIDNEY

Plasma Pi in early life is high and renal Pi excretion low relative to that in adults. Renal tubular Pi transport kinetics and membrane LF was investigated in apical membrane vesicles (BBMV) in 6 fetal (F) lambs and pregnant ewes (E).Results:KmPi was similar in BBMV in the F and E but Vmax Pi was greater in F. Alkaline phosphatase enrichment, lipid saturation index and [cholesterol/phospholipid], [sphingomyelin/phosphatidylcholine] ratios were not different, however, protein/lipid ratios was higher in E. The results demonstrate greater renal tubular Pi transport capacity in F compared to E and increased VmaxPi in F is due, in part, to greater membrane LF. Relative increased VmaxPi in F may be due to difference in carrier function rather than to an increase in carrier concentration. This may involve more rapid cycling of Pi carrier from lumen to cytoplasmic surfaces in F.

Synthesis and characterization of methylbromoamiloride, a potential biochemical probe of epithelial Na+ channels

We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current of in vitro frog skin and 22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel.

Regulation of expression of the sodium-coupled hexose transporter in cultured LLC-PK1 epithelia.

A variety of techniques have been used to study the sodium-coupled hexose transporter in epithelia formed by LLC-PK1 cells. The expression of the transporter is affected by the density and age of the culture and by the concentration of glucose in the growth medium. Sodium-coupled hexose transport appears as the epithelium becomes confluent and increases further as the epithelium matures. The increased transport is associated with increased transport in apical plasma membrane vesicles. Epithelia grown in medium containing 5 mM glucose express more transporters than epithelia grown in medium containing 25 mM glucose. The increase in transport is not the result of an extracellular signal that is generated as a consequence of the concentration of glucose. The response to different hexoses that are or are not transported on the carrier indicates that it is the metabolism of glucose that acts as the signal for expression of more or fewer transporters. The results are compared to similar studies of the effects of substrate concentration on expression of transporters in cultured fibroblasts and the intestines in situ.

Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers

Single epithelial Na+ channels incorporated into planar lipid bilayers were studied to determine the effects of Na concentration on its own conductance. Amiloride-sensitive Na+ channels were obtained from apical membrane vesicles made from A6 cells, a continuous epithelial cell-line derived from amphibian kidney. Single-channel conductance was found to be a saturable function of Na+ concentration, with a Michaelis constant of approximately 17 or 47 mM, for a Gmax of approximately 4 or 44 pS, respectively.

Cl − transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

The Cl − transport properties of the luminal border of bovine tracheal epithelium have been investigated using a highly purified preparation of apical plasma membrane vesicles. Transport of Cl − into an intravesicular space was demonstrated by (1) a linear inverse correlation between Cl − uptake and medium osmolarity and (2) complete release of accumulated Cl − by treatment with detergent. The rate of Cl − uptake was highly temperature-sensitive and was enhanced by exchange diffusion, providing evidence for a carrier-mediated transport mechanism. Transport of Cl − was not affected by the ‘loop’ diuretic bumetanide or by the stilbene-derivative anion-exchange inhibitors SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid) and DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid). In the presence of the impermeant cation, tetramethylammonium (TMA +), uptake of Cl − was minimal; transport was stimulated equally by the substitution of either K + or Na + for TMA +. Valinomycin in the presence of K + enhanced further Cl − uptake, while amiloride reduced Na +-stimulated Cl − uptake towards the minimal level observed with TMA +. These results suggest the following conclusions: (1) the tracheal vesicle membrane has a finite permeability to both Na + and K +; (2) the membrane permeability to the medium counterion determines the rate of Cl − uptake; (3) Cl − transport is not specifically coupled with either Na + or K +; and, finally (4) Cl − crosses the tracheal luminal membrane via an electrogenic transport mechanism.

A two sodium ion/D-glucose symport mechanism: membrane potential effects on phlorizin binding.

Apical membrane vesicles isolated from a continuous renal cell line, LLC-PK1, catalyze electrogenic Na+-stimulated hexose transport and Na+-dependent binding of 3H-labeled 1-[2-(beta-D-glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone [( 3H]phlorizin), a competitive ligand of this transport system. Phlorizin was not itself transported across the membrane and thus can serve as a probe of the binding step. The stoichiometry of Na+-dependent phlorizin binding in vesicles was 1:1, whereas Na+/hexose cotransport in vesicles exhibited a 2:1 stoichiometry. Na+ increased the affinity of phlorizin binding without affecting the total number of binding sites. An increased number of Na+-dependent phlorizin binding sites was observed under conditions of interior-negative membrane potential. These results are consistent with a model of the Na+/glucose cotransport cycle in which the unloaded transporter is negatively charged and its orientation influenced by membrane potential. Glucose and one sodium ion interact with the transporter, resulting in an uncharged complex. Binding of a second sodium ion triggers translocation of glucose and both sodium ions via formation of a loaded carrier complex bearing a single positive charge.

Aldosterone-stimulated sodium uptake by apical membrane vesicles from A6 cells.

Sodium fluxes in plasma membrane vesicles prepared from the cultured toad kidney epithelial cell line A6 are studied. The vesicles are enriched 7-10 times in apical membrane markers. Sodium uptake is osmotically sensitive and inhibited by low concentrations of amiloride (K0.5 = 7 X 10(-8) M at 1 mM NaCl). Vesicles prepared from aldosterone-treated cells (4.5 h at 10(-7) M aldosterone) show a 2-fold enhancement of amiloride-sensitive sodium flux relative to appropriate controls. The above observations are in good agreement with studies of sodium transport across the apical membrane of intact A6 epithelia. Thus, the amiloride-sensitive sodium transporter in the apical membrane of these cells is preserved in the vesicle preparation, making it possible to study the effects of aldosterone in the absence of nonmembrane-related phenomena.

Methylation increases sodium transport into A6 apical membrane vesicles: possible mode of aldosterone action.

When isolated apical membrane vesicles prepared from cultured A6 epithelia were incubated in vitro with the methyl donor S-adenosylmethionine, the control rate of amiloride-inhibitable sodium transport was doubled. The methylation inhibitors 3-deazaadenosine and S-adenosyl homocysteine returned the S-adenosyl-methionine-stimulated sodium transport to control levels. Neither these agents nor adenosine affected sodium transport into control vesicles. In vesicles incubated with S-adenosyl-[3H-methyl]methionine, both membrane phospholipids and proteins were labeled, and this labeling was inhibited by deazaadenosine. In vesicles prepared from A6 cells treated with aldosterone, sodium transport was twice the control value and S-adenosylmethionine did not cause any further stimulation of transport. In those vesicles, both lipid and protein methylation were increased. These results suggest that methylation, which increases the rate of amiloride-sensitive sodium transport is involved in the action of aldosterone at the apical membrane level in epithelia.

Neutral amino acid transport by plasma membrane vesicles of the rabbit choroid plexus

Uptakes of neutral l-amino acids into rabbit choroid plexus apical membrane vesicles were studied using l-[ 14C]amino acids. Apical membrane vesicles were prepared using Ca 2+ precipitation and uptakes of 14C-labeled substrates were measured by a rapid mixing and filtration procedure. Na-dependent, concentrative uptake was observed for proline, histidine and methylaminoisobutyric acid (MeAIB). Phenylalanine and d-glucose uptakes showed no significant Na dependence. Proline uptake was a saturable function of the proline concentration with J max 3.5 nmol/(mg min) and K t 0.3 mM. Competition experiments in the presence of Na indicated that proline and MeAIB are mutually competetive. Proline uptake was also inhibited 25% by phenylalanine, but MeAIB uptake was relatively unaffected. Neither proline nor MeAIB transport was significantly inhibited by glycine. We conclude that proline uptake across rabbit choroid plexus apical membrane vesicles is via an Na-dependent pathway which is shared by MeAIB and, to a minor extent, by phenylalanine, but from which glycine is excluded. Histidine uptake was inhibited by glycine, GABA, phenylalanine, proline and MeAIB. This suggests that histidine may utilize a different pathway in addition to that shared with proline and MeAIB. These transporters should play an active role in the regulation of amino acids in the CSF.