Redistribution of surface macromolecules in dissociated epithelial cells.

A number of ultrastructural and cytochemical techniques were used to study intact epithelial cells lining the frog urinary bladder: high resolution autoradiography after administration of [3H]glucosamine or [3H]fucose; 125I iodination of external protein; concanavalin A-peroxidase, periodic acid-chromic acid silver methenamine; and colloidal thorium. Results indicate that the material (probably glycoprotein) coating the apical surface differs from that which lines the lateral and basal surfaces. After dissociation and isolation of the epithelial cells, the material previously confined to the apical surface invaded progressively the opened "tight junctions" (about 5 min), then the lateral membranes (about 40 min), and finally the basal membrane (about 80 min): at that time, the whole cell surface was entirely enveloped by the apical material. Since, on the one hand, the reacting material was confined to the apical surface when the tight junctions were closed (in intact epithelial cells) and since, on the other hand, the apical material was sliding down the laterobasal membranes when the tight junctions were opened (in dissociated cells), it may be concluded that tight junctions contribute to maintain the cell surface specialization in epithelia.

Red cell membrane CO2 permeability in normal human blood and in blood deficient in various blood groups, and effect of DIDS.

The red cell membrane has an exceptionally high permeability for CO2, PCO2 approximately 0.15 cm/s, which is two to three orders of magnitude greater than that of some epithelial membranes and similarly greater than the permeability of the red cell membrane for HCO3-. As shown previously, this high PCO2 can be drastically inhibited by 10 microM 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate (DIDS), indicating that membrane proteins may be involved in this high gas permeability. Here, we have studied the possible contribution of several blood group proteins to CO2 permeation across the red cell membrane by comparing PCO2 of red cells deficient in specific blood group proteins with that of normal red cells. While PCO2 of normal red cells is approximately 0.15 cm/s and that of Fy(null) and Jk(null) red cells is similar, PCO2's of Colton null (deficient in aquaporin-1) and Rh(null) cells (deficient in Rh/RhAG) are both reduced to about 0.07 cm/s, i.e. to about one half. In addition, the inhibitory effect of DIDS is about half as great in Rh(null) and in Colton null red cells as it is in normal red cells. We conclude that aquaporin-1 and Rh/RhAG proteins contribute substantially to the high permeability of the human red cell membrane for CO2. Together these proteins are responsible for 50% or more of the CO2 permeability of red cell membranes. The CO2 pathways of both proteins can be partly inhibited by DIDS, which is why this compound very effectively reduces membrane CO2 permeability.

Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.

To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and Rhag-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag-/- and AQP1-/-. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions.

Hydrophobic cluster analysis and modeling of the human Rh protein three-dimensional structures.

Rh (Rhesus) is a major blood group system in man, which is clinically significant in transfusion medicine. Rh antigens are carried by an oligomer of two major erythroid specific polypeptides, the Rh (D and CcEe) proteins and the RhAG glycoprotein, that shared a common predicted structure with 12 transmembrane a-helices (M0 to M11). Non erythroid homologues of these proteins have been identified (RhBG and RhCG), notably in diverse organs specialized in ammonia production and excretion, such as kidney, liver and intestine. Phylogenetic studies and experimental evidence have shown that these proteins belong to the Amt/Mep/Rh protein superfamily of ammonium/methylammonium permease, but another view suggests that Rh proteins might function as CO2 gas channels. Until recently no information on the structure of these proteins were available. However, in the last two years, new insight has been gained into the structural features of Rh proteins (through the determination of the crystal structures of bacterial AmtB and archeaebacterial Amt-1. Here, models of the subunit and oligomeric architecture of human Rh proteins are proposed, based on a refined alignment with and crystal structure of the bacterial ammonia transporter AmtB, a member of the Amt/Mep/Rh superfamily. This alignment was performed considering invariant structural features, which were revealed through Hydrophobic Cluster Analysis, and led to propose alternative predictions for the less conserved regions, particularly in the N-terminal sequences. The Rh models, on which an additional Rh-specific, N-terminal helix M0 was tentatively positioned, were further assessed through the consideration of biochemical and immunochemical data, as well as of stereochemical and topological constraints. These models highlighted some Rh specific features that have not yet been reported. Among these, are the prediction of some critical residues, which may play a role in the channel function, but also in the stability of the subunit structure and oligomeric assembly. These results provide a basis to further understand the structure/function relationships of Rh proteins, and the alterations occurring in variant phenotypes.

Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis.

We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.

Cloning and functional characterization of a rat urea transporter: expression in the brain.

A cDNA coding for a rat urea transporter is described. The 1242 bp open reading frame codes for a 414 amino acids protein with 77.0% and 60.7% identity with the human UT11 and the rat UT2, respectively. When expressed in Xenopus oocytes, the protein induces a 10-fold rise in urea permeability, inhibited by mercurial reagents and phloretin. The rUT11 mRNA is massively expressed in the brain.

Interactions between Na+-dependent uptake of D-glucose, phosphate and L-alanine in rat renal brush border membrane vesicles.

D-Glucose decreases phosphate reabsorption in rat proximal tubule. It is also postulated that some amino acids interact with phosphate reabsorption. To investigate the mechanism of these interactions, phosphate, D-glucose and L-alanine transport kinetics were measured in brush border membrane vesicles isolated from superficial rat kidney cortex by the calcium precipitation technique. At pH 7.4, Na+-dependent phosphate transport was inhibited in the presence of either D-glucose (39 mM) or L-alanine (2.4 mM). In this model, with D-glucose or with L-alanine the V value of the phosphate uptake was decreased, whereas the apparent Km for the phosphate uptake was not affected. However, some inhibition of phosphate transport was observed in the presence of L-glucose, D-alanine or D-glucose after phlorizin preincubation. A 30% Na+-dependent L-alanine (0.1 mM) transport inhibition was observed in the presence of 5 mM phosphate. D-Glucose (1 mM) was also inhibited by 20% when 5 mM phosphate was added to incubation medium. According to several authors, in our model, D-glucose decreased the L-alanine transport and vice versa. Moreover, when the membrane potential was abolished, a clear inhibition of D-glucose by L-alanine persisted. These multiple interactions could be explained by the accelerated dissipation of the Na+ gradient insofar as the rate of the Na+ uptake was increased with D-glucose, L-alanine or phosphate and since the absence of variations in membrane potentials did not suppress these inhibitions.

Functional expression of urea channels in amphibian oocytes injected with frog urinary bladder mRNA.

In amphibian urinary bladder epithelium, vasopressin increases passive urea permeability, concomitant with the appearance of a facilitated urea transport. Amphibian oocytes from Xenopus laevis and Rana esculenta were microinjected with total or fractionated poly(A+) RNA isolated from frog urinary bladder epithelial cells. After several (3-5) days at 18 degrees C, the urea flux was assayed by measuring the uptake and efflux of [14C]urea in water-injected and mRNA-injected oocytes. A 2 to 3-fold increase of urea transport was detected in oocytes injected either with total mRNA or with a 6-10 kilobase mRNA fraction, when compared with water-injected oocytes. This expression of urea channels was inhibited by 0.1 mM phloretin (50% inhibition) and 0.1 mM nitrophenylthiourea (up to 70% inhibition). On the contrary, no expression was detected in brain mRNA-injected oocytes. These results show the specific functional expression of the phloretin- and NPTU-sensitive urea channel (or carrier) from frog urinary bladder epithelial cells, providing an approach for the expression cloning of these urea channels.

Evidence for proteic water pathways in the luminal membrane of kidney proximal tubule.

The osmotic permeability of the apical membrane of proximal tubule cells was studied on rat brush-border membrane vesicles by following their rate of shrinkage with a stopped-flow device coupled to light transmission recording. The mercuric sulfhydryl reagent para-chloromercuribenzenesulfonic acid (PCMBS) reduced the water permeability of the membrane, in a time- and dose-dependent manner, to 35% of the control value. Mercuric chloride was a more potent inhibitor and decreased the osmotic water permeability of the brush-border membrane to 15% of the control. This inhibition was reversed by an excess of cysteine, while cysteine per se did not modify the rate of vesicle shrinkage. These results suggest that most of the osmotic water movements across kidney brush-border membranes are through polar pathways which involve the integrity of the membrane proteins.

Osmotic water permeability and solute reflection coefficients of rat kidney brush-border membrane vesicles.

Solute reflection coefficients, sigma i, of rat kidney brush-border membrane vesicles were determined by the comparison of water flows induced by equiosmolal gradients of sucrose and NaCl, KCl or mannitol. The values of 0.53 for sigma NaCl and 0.56 for sigma KCl when compared with 0.92 for sigma mannitol suggested some interactions between salt and water pathways. Altering the membrane proteins with 0.4 mM HgCl2 decreased the osmotic water permeability of the vesicles by 70 to 80% and brought sigma NaCl and sigma KCl to a value not different from 1. This argued in favor of water protein pathways in the luminal membrane of kidney proximal cells which are partly accessible to NaCl and KCl.

Determination of solute reflection coefficients in kidney brush-border membrane vesicles by light scattering: influence of the refractive index.

Solute reflection coefficients sigma of cell membrane vesicles or liposomes are commonly determined by comparison of the water flow induced by a gradient of the studied solute and that of a reference molecule using light scattering techniques. However, variations in scattered light which are mainly related to change in vesicle volume are also influenced by the refractive index of the surrounding medium. Therefore comparing kinetics of vesicle shrinkage induced by hyperosmotic solutions which have different refractive indexes might lead to an under or over estimation of sigma. We determined sigma NaCl in rat kidney brush-border membrane vesicles by two different approaches using mannitol, a poorly permeant molecule, as reference. (1) The refractive index of the hyperosmotic NaCl solution was adjusted to that of mannitol by addition of polyvinyl pyrrolidone (Mr 40,000), without a significant increase in osmolality. Thereby the change in scattered light intensity induced by osmotic vesicle shrinkage due to gradients of NaCl and mannitol were comparable and led to a sigma NaCl value close to one instead of the previously published value of 0.53. (2) The reflection coefficient was calculated from the lifetime of vesicle shrinkage which is not refractive index-dependent. Again sigma NaCl was not different from one. These results suggest that the water proteic pathways found in the luminal membrane of proximal tubules are not shared by salts.

Simultaneous minute by minute determination of unidirectional and net water fluxes in frog urinary bladder. A reexamination of the two barriers in series hypothesis.

Unidirectional and net water fluxes were simultaneously estimated in frog urinary bladder. The minute by minute tritiated water (3HOH) transepithelial flux and the net volume of fluid traversing the tissue were employed. It was observed that: (1) the time course of the increase in the 3HOH flux induced by antidiuretic hormone had a very similar pattern to that reported for the increase in the net movement. (2) Unstirred layers strongly affected the magnitude of the antidiuretic hormone-induced increase in 3HOH fluxes while the time course of the response was almost non-affected. In non-stimulated bladders 3HOH fluxes were poorly modified by medium stirring. New steady-state conditions for 3HOH fluxes were established 1 min after stirring rate modifications. (3) The simultaneously determined net water flux was not affected by a modification in the unstirred layers, indicating that the variations in the measured net water fluxes are a good estimation of the changes in the mucosal border permeability. (4) The presence of an osmotic gradient during hormonal challenge (implying net water fluxes, cell swelling and dilation of the intracellular spaces) did not modify the time course of 3HOH movements. These results suggest that the time course of the increase in water permeability is an intrinsic characteristic of the experimental system that could result from the addition of permeability units that increase in number during the development of the hormonal action.

Zinc binding in intestinal brush-border membrane isolated from pig.

Zinc binding to brush-border membrane vesicles isolated from pig jejunum was investigated by a rapid filtration method, for long incubation periods (up to 180 min). Zn2+ influx revealed a large accumulation of the metal, reaching an apparent intravesicular volume of 160 microliters/mg protein at equilibrium, a volume 45-times that of an osmotically reactive sugar, sorbitol (3.6 microliters/mg protein). Changes in medium osmolarity had no effect on zinc accumulation. These results suggested a large degree of zinc binding to vesicular components (membrane or core). 65Zn efflux measurements led to the conclusion that two vesicular pools of zinc existed: a small external pool, accessible to different chelators (EGTA) or competitive cations, and a large intravesicular pool. Accumulated 65Zn was quickly removed from its internal sites only after the membrane had been permeabilized by the cation ionophore A23187 in association with an exchange molecule or a chelator. Scatchard plot analyses revealed, on one hand a first class of high-affinity extravesicular zinc binding sites (Ka = 8.6.10(3) M-1, n = 0.455 nmol Zn2+/mg protein) and a second class of extravesicular sites having a very low affinity (Ka = 22 M-1, n = 25.35 nmol Zn2+/mg protein) and, on the other hand one type of intravesicular sites (Ka = 3.3.10(4) M-1, n = 550 nmol Zn2+/mg protein). The intravesicular sites have a high affinity for zinc and are specific, since only nonlabelled zinc (or cadmium) but not calcium present in the bathing medium is exchanged with the internally accumulated labelled cation.

Studies of zinc transport into brush-border membrane vesicles isolated from pig small intestine.

Zinc transport into brush-border membrane vesicles was investigated by measuring uptake rates at a very short incubation time (2 seconds), during the initial linear uptake. A divalent cation chelator (EGTA) was added to the stop and washout solutions in order to remove the zinc bound to the external surface of the vesicles. Under these conditions, we showed that zinc enters the vesicles by (1) a saturable carrier-mediated process, and (2) an unsaturable pathway. The kinetic parameters we calculated were an affinity of 0.215 +/- 0.039 mM, a Jmax of 17.2 +/- 1.7 nmol.min-1.(mg protein)-1 and an unsaturable constant of 0.025 +/- 0.006 (n = 6). The imposition of an outwardly directed K+ gradient (negative inside) did not affect the Jmax value of the zinc uptake but increased the Km value significantly. This suggests that, at least a portion of zinc which crosses the membrane does not do so in a cationic form. Zinc uptake was decreased or increased according to the nature of accompanying anions (Cl-, SO4(2)-, SCN-) in the absence of any membrane potential. With highly permeant anions such as thiocyanates, zinc uptake was considerably augmented, suggesting a movement of zinc in a complexed form involving the presence of negative species. We also showed that cadmium competitively inhibited the zinc uptake; we measured a Ki value of 0.21 mM, indicating a similar affinity of cadmium for the carrier as zinc itself. By contrast, the presence of calcium had little effect on zinc entry into vesicles. The calcium ionophore A23187 had only a slight stimulating effect on zinc uptake. These results indicate that zinc and calcium transports are probably independent of each other.

Detergent extraction of membrane proteins related to the action of antidiuretic hormone.

Antidiuretic hormone (ADH) induces, in the apical plasma membrane of target cells, the insertion of intramembranous particle aggregates that probably contain water channels. A mild attack of this membrane by a polyoxyethylene nonylphenyl detergent, which reversibly depressed ADH-induced water permeability, has been found to modify aggregate structure while extracting additional proteins. This simple procedure could be a valuable approach to the problem of aggregate isolation and characterization.

Sequence and functional expression of an amphibian water channel, FA-CHIP: a new member of the MIP family.

A new member of the family of water channel proteins (aquaporin-CHIP) related to the major intrinsic protein (MIP) family is described. The cDNA coding for this amphibian CHIP was cloned from frog (Rana esculenta) urinary bladder, a model for the kidney collecting duct, using a RT-PCR cloning strategy. The encoded protein, designated FA-CHIP (frog aquaporin-CHIP), shows 77.4%, 42.4% and 35.6% identity with the three proteins now referred to as the aquaporins of the MIP family, i.e., human CHIP28, WCH-CD and gamma-TIP, respectively. Xenopus leavis injected with FA-CHIP cRNA exhibited a marked increase of the osmotic water permeability.

Molecular and functional characterization of an amphibian urea transporter.

We report the characterization of a frog (Rana esculenta) urea transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box urea transporter signature sequence were found in this amphibian urea transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thiourea, but the fUT-mediated urea transport was strongly inhibited by this compound. Thus, this amphibian urea transporter displays transport characteristics in between those of UT-A2 and UT-B.

The effect of hypertonic media on water permeability of frog urinary bladder. Inhibition by catecholamines and prostaglandin E 1 .

The frog urinary bladder undergoes, in some conditions, a marked increase of its water permeability when incubated in hypertonic media. This increase was observed with various nonpermeant solutes. It seems to result from the shrinkage of an osmo-sensitive compartment of the tissue, probably the epithelial cells. Many similarities were found between this effect and the physiological increase in water permeability (hydrosmotic response) elicited by antidiuretic hormone (ADH): both were dependent on the physiological state of the animals, and although the response was slower after hyperosmolar than after hormonal challenge, the patterns of response were similar, and in both cases markedly dependent on bathing solution temperature. Norepinephrine and prostaglandin E(1), which in this tissue reduce the hydrosmotic action of ADH, presumably by inhibiting the adenyl cylase also reduced the effect of hyperosmolarity. Conversely this effect was potentiated by incubation in the presence of oxytocin, exogenous cyclic AMP, and theophylline, conditions in which the intracellular concentration of cyclic AMP is increased. These data demonstrate that the response to hyperosmolarity is elicited, at least partly, by mechanisms also involved in the physiological hydrosmotic response to ADH.

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Purification and characterization.

Exocytotic processes play a major role in the hormonal control of water permeability in the amphibian urinary bladder. Different treatments such as antidiuretic hormone (ADH) stimulation, incubation with phorbol ester or mild detergent and mechanical stretch of the bladder, consistently induce a liberation of two major polypeptides of 76 and 14 kDa molecular mass into the luminal medium. Each of these polypeptides represents 3 to 5% of the total protein of epithelial cell homogenates and 20 to 50% of the released material. Proportions of released 76 kDa polypeptide in urinary bladders of toads (Bufo marinus) and frogs (Rana esculenta) were similar but, in the frog extracts, two bands ("doublet") were resolved at the level of 76 kDa. In high performance liquid chromatography (HPLC), using gel filtration and ion exchange chromatography, the frog 76 kDa protein was resolved into two polypeptides of 80,000 to 100,000 and 60,000 to 80,000 daltons while the 14 kDa protein included two polypeptides, each with a molecular mass of approximately 14,000 daltons. Isoelectric focusing of the material released during a mechanical stretch of the tissue ("stretch extract") or of isolated purified proteins from the frog urinary bladder showed that the 14 kDa polypeptides were resolved in two major groups of polypeptides, one in the range of pH 7.4 to 7.8, the other at pH 5.6. The lower band of the 76 kDa doublet also comprised some diffuse bands (5.0 less than pI less than 5.2) while the other polypeptide of the doublet presented a sharp band at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the FA-CHIP water channel in frog urinary bladder.

Like mammalian kidney collecting duct, the water permeability of frog urinary bladder epithelial cells is antidiuretic hormone (ADH)-sensitive. In kidney, this permeability is mediated by water channels named aquaporins. We recently reported the cloning of the frog aquaporin CHIP (FA-CHIP), a water channel from frog urinary bladder. FA-CHIP has 79% identity with rat Aquaporin 1 (AQP1) and only 42% identity with the kidney collecting duct Aquaporin 2 (AQP2). The purpose of this study was to examine the localization of FA-CHIP in frog urinary bladder. We raised antibodies against peptides of 15 to 17 residues, encompassing the N-ter and C-ter regions of FA-CHIP. Anti-FA-CHIP antibodies were used for Western blotting, indirect immunofluorescence microscopy and gold labeling electron microscopy in urinary bladder and other frog tissues. By Western blotting of frog urinary bladder total homogenate, the antibodies recognized a band of 29 kDa and glycosylated forms of the protein between 40 and 70 kDa. No signal was found on membrane preparations from epithelial cell homogenate. FA-CHIP was also found in frog skin, brain, gall bladder, and lung. In immunofluorescence microscopy on urinary bladder sections, FA-CHIP was localized to endothelial cells of blood capillaries and on mesothelial cells of the serosal face. Red blood cells, epithelial and basal cells were unstained. The localization of FA-CHIP in cell plasma membranes was confirmed by gold labeling electron microscopy. In other positive tissues, FA-CHIP was also localized to capillaries. In brain, plasma membranes of epithelial cells were also stained. In conclusion, like its mammalian homologue AQP1, FA-CHIP appears to be localized to constitutively water permeable cells of frog. Therefore, it belongs to the AQP1 family of proteins although unlike AQP1, FA-CHIP is absent from red blood cells and kidney. In frog urinary bladder and skin, FA-CHIP probably plays an important role in water transport across the barriers in series with the ADH-sensitive epithelial cells.