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F Verrey

Publications and source records attributed to F Verrey.

At least 55 records · Page 3Linked to original sources
PubMed1994

Antidiuretic hormone action in A6 cells: effect on apical Cl and Na conductances and synergism with aldosterone for NaCl reabsorption.

The effect of antidiuretic hormone on transepithelial Na+ and Cl- transport and its modulation by aldosterone (10(-6) M) was studied in the Xenopus laevis distal nephron cell line A6-C1 by measuring transepithelial electrophysiological parameters and bidirectional anion fluxes. Vasotocin (or vasopressin) induced a biphasic increase in transepithelial short-circuit current (Isc). Early and late effects were potentiated by aldosterone and could be mimicked by forskolin and BrcAMP, implicating cAMP as a mediator. The early increase in Isc (maximum 1-2 min after hormone addition) was resistant to 50 microM amiloride. Electrophysiological experiments with apical ion substitutions or basolateral bumetanide (0.5 mM), as well as flux studies with 125I- or 36Cl-, indicated that this current represented Cl- secretion. The late increase in Isc appeared with a lag of 2-5 min and was maximal after 15-25 min. It corresponded to an increase in Na+ reabsorption, since it was amiloride sensitive. Bidirectional 36Cl- flux measurements in aldosterone-treated monolayers maintained under open-circuit conditions showed that the large vasotocin-induced increase in Cl- permeability led, in these conditions, to a threefold increase of a baseline Cl- reabsorption. This study shows that vasotocin induces in A6-Cl cells both a rapid increase in Cl- permeability and a slower increase in Na+ transport. The Cl- permeability, which leads to Cl- secretion under short-circuit conditions, contributes, under the more physiological open-circuit conditions, to the transport of Na+ by allowing its co-reabsorption with Cl-.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Aldosterone induces early activation and late accumulation of Na-K-ATPase at surface of A6 cells.

In A6 cells cultured on filters, aldosterone (10(-6) M) induces an early increase in the initial rate of ouabain binding to intact monolayers (in K-free buffer) that parallels an early increase in Na transport. This effect is independent of apical Na influx and precedes an increase in the synthesis of Na-K-adenosinetriphosphatase (ATPase) subunits. In the present study we observed in addition a late aldosterone-induced increase in the rate of ouabain binding (2.5 times from 3 to 120 h aldosterone). The role of Na-K-ATPase accumulation and/or translocation to the cell surface was tested by Western blotting, saturation ouabain binding, and cell-surface labeling using sulfosuccinimidobiotin or enzyme-mediated radioiodination. Only cell-surface beta 1-subunit was detected by blotting with streptavidin or autoradiography, because the alpha 1-subunit was not efficiently labeled. Three hours after hormone addition, none of the three parameters had increased significantly, whereas after 20 and 120 h similar increases (approximately 1.6 and 2.3 times, respectively) were detected by all three methods. In addition, it was shown that increasing intracellular Na with amphotericin B or a K-free preincubation also stimulated the rate of ouabain binding without increasing the surface labeling of beta 1-subunits. Taken together, these results suggest that a short aldosterone treatment or an increase in intracellular Na leads to an increase in the rate of ouabain binding that is due to an in situ activation of cell-surface Na-K-ATPase molecules. In contrast, the late increase in the rate of ouabain binding parallels an increase in the number of pumps.

Aldosterone↗
PubMed1993

Determinants of oligomeric structure in the chicken liver glycoprotein receptor.

The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation.

Amino Acid Sequence↗
PubMed1993

Vasopressin-dependent control of basolateral Na/H-exchange in highly differentiated A6-cell monolayers.

We have used a well-differentiated A6-cell preparation (A6-C1) to study cellular location and vasopressin control of Na/H-exchange activity. After cell acidification, cell pHi (measured by BCECF-fluorescence) only recovered by the addition of Na medium to the basolateral cell surface; this pHi recovery was inhibited by dimethylamiloride (2 microM) consistent with basolateral location of Na/H-exchange activity. Addition of vasopressin produced stimulation of Na/H-exchange activity and increased the affinity of the exchanger for Na+. Stimulation of Na/H exchange was mimicked by pharmacological activation of protein kinase A (forskolin, 8-Br-cAMP) and not by pharmacological activation of protein kinase C (TPA). It is concluded that basolaterally located Na/H-exchange in A6-C1 cells is activated by vasopressin.

Animals↗
PubMed1993

Polarized membrane movements in A6 kidney cells are regulated by aldosterone and vasopressin/vasotocin.

The polarity of cell-surface membrane movements and their regulation by adrenal steroid hormones (10(-6) M aldosterone) and vasopressin or vasotocin were studied in A6 cells. This cell line is derived from the Xenopus laevis distal nephron and displays regulated Na+ reabsorption but is devoid of regulated water transport. Apical and basolateral membrane movements and their hormonal regulation were characterized by measuring the uptake of the fluid phase marker horseradish peroxidase (HRP) and the secretion of proteins on both sides of cell monolayers cultured on filters. The intracellular accumulation of HRP was visualized by electron microscopy and quantified by the measure of cell-associated peroxidase activity. The rate of intracellular HRP accumulation corresponded to 0.01 nl/minute/filter (4.7 cm2) from the apical side and was 20-32 times faster from the basolateral side. In contrast, the level of protein secretion was 3.5 times higher apically than basolaterally. Among the secreted proteins some were found to be secreted essentially apically, and others basolaterally. Vasotocin increased apical endocytosis (1.88-fold) and apical protein secretion (1.49-fold) in cells pretreated with aldosterone. Basolaterally, only the endocytosis was increased, and to a smaller extent (1.36-fold). These effects of vasotocin depended on aldosterone pretreatment and could be mimicked with forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP). Measurements of intracellular cAMP levels showed that there was a rankorder correlation between the induced level of intracellular cAMP and that of apical endocytosis. This study shows that vasotocin has a polarized stimulatory action on apical endocytosis and protein secretion in A6 cells, and that the mediation of this action by cAMP is aldosterone dependent.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity.

High resistance epithelia express on their apical side an amiloride-sensitive sodium channel that controls sodium reabsorption. A cDNA was found to encode a 1,420-amino acid long polypeptide with no signal sequence, a putative transmembrane segment, and three predicted amphipathic alpha helices. A corresponding 5.2-kb mRNA was detected in Xenopus laevis kidney, intestine, and oocytes, with weak expression in stomach and eyes. An antibody directed against a fusion protein containing a COOH-terminus segment of the protein and an antiidiotypic antibody known to recognize the amiloride binding site of the epithelial sodium channel (Kleyman, T. R., J.-P. Kraehenbuhl, and S. A. Ernst. 1991. J. Biol. Chem. 266:3907-3915) immunoprecipitated a similar protein complex from [35S]methionine-labeled and from apically radioiodinated Xenopus laevis kidney-derived A6 cells. A single integral of 130-kD protein was recovered from samples reduced with DTT. The antibody also cross-reacted by ELISA with the putative amiloride-sensitive sodium channel isolated from A6 cells (Benos, D. J., G. Saccomani, and S. Sariban-Sohraby. 1987. J. Biol. Chem. 262:10613-10618). Although the protein is translated, cRNA injected into oocytes did not reconstitute amiloride-sensitive sodium transport, while antisense RNA or antisense oligodeoxynucleotides specific for two distinct sequences of the cloned cDNA inhibited amiloride-sensitive sodium current induced by injection of A6 cell mRNA. We propose that the cDNA encodes an apical plasma membrane protein that plays a role in the functional expression of the amiloride-sensitive epithelial sodium channel. It may represent a subunit of the Xenopus laevis sodium channel or a regulatory protein essential for sodium channel function.

Amiloride↗
PubMed1990

Polarized transport of the polymeric immunoglobulin receptor in transfected rabbit mammary epithelial cells.

A cDNA for the rabbit low Mr polymeric immunoglobulin (poly-Ig) receptor was expressed in an immortalized rabbit mammary cell line. The intracellular routing of the receptor and its cell surface expression was analyzed in stably transfected cells grown on permeable supports. Initially the cells formed a monolayer with no transmural electrical resistance. All monolayer cells expressed the poly-Ig receptor and cytokeratin 7 filaments characteristic of luminal mammary cells but absent in myoepithelial cells. Within 7 d in culture, the cells underwent cytodifferentiation and formed a bilayer with a transepithelial electrical resistance of approximately 500 omega x cm2. Upper layer cells formed tight junctions with adjacent cells and gap junctions with basal cells. Expression of the poly-Ig receptor and cytokeratin 7 was restricted to the cells from the upper layer. The kinetics of receptor biosynthesis and processing was similar to that reported for rabbit mammary gland and rat liver. The receptor was cleaved at the apical cell surface and release of secretory component into the apical medium occurred with a half-time of approximately 2 h. Selective cell surface trypsinization combined with pulse-chase experiments served to determine at which cell surface domain newly synthesized receptor appeared first. The receptor was digested with a half-time of approximately 60 min with trypsin present in the basolateral medium and 90 min with apical trypsin. These data are consistent with selective targeting of newly synthesized receptor to the basolateral surface. The results indicate that transcytosis of the receptor from basolateral to apical membrane in the presence or the absence of its ligand requires approximately 30 min. Cleavage of the receptor by endogenous protease is not concomitant with its appearance at the apical surface, but requires additional time, thus explaining the presence of intact receptor on the apical membrane.

Animals↗
PubMed1990

Endocytosis via coated pits mediated by glycoprotein receptor in which the cytoplasmic tail is replaced by unrelated sequences.

Rat 6 fibroblast cell lines expressing wild-type chicken liver glycoprotein receptor (CHL) or chimeric receptors with alternate cytoplasmic tails were produced to study the role of the cytoplasmic tail in mediating receptor localization in coated pits and endocytosis of ligand. Cells expressing CHL or cells expressing a hybrid receptor that contains the cytoplasmic tail of the asialoglycoprotein receptor display high-efficiency endocytosis of N-acetylglucosamine-conjugated bovine serum albumin in experiments designed to measure an initial internalization step, as well as in studies of continuous uptake and degradation. Substitution of the cytoplasmic tail by the equivalent domain of rat Na,K-ATPase beta subunit or by a stretch of Xenopus laevis globin beta chain does not abolish endocytosis but decreases the endocytosis rate constant from 15%-16%/min to 2.4% and 6.5%/min, respectively. Electron microscopy was used to visualize the glycoprotein binding sites at the surface of Rat 6 cells transfected with the various receptors. The percentage of receptors found in coated areas ranged from 32% for CHL to 9% for the Na,K-ATPase hybrid, indicating that clustering in coated pits correlates with efficiency of endocytosis. We concluded that replacement of the CHL cytoplasmic tail with unrelated sequences does not prevent, but decreases to varying extents, coated-pit localization and endocytosis efficiency. The construct with NH2-terminal globin tail lacks a signal for high-efficiency localization in coated pits but nevertheless is directed to the pits by an alternative mechanism.

Amino Acid Sequence↗
PubMed1989

A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.

In all cellular systems studied so far, the catalytic alpha- and the glycosylated beta-subunit of Na+-K+-ATPase are coordinately synthesized and are assembled into stoichiometric alpha, beta-complexes. In contrast to these data, in this study we show that the fully grown oocyte of Xenopus laevis synthesizes much less beta-subunit than alpha-subunit. The alpha-subunit produced in excess over the beta-subunit is membrane associated but highly trypsin sensitive and can be compared with the immature alpha-subunit population identified in epithelial cells immediately after synthesis (K. Geering, J. P. Kraehenbuhl, and B.C. Rossier, J. Cell Biol. 105: 2613-2619, 1987). The Xenopus oocyte thus turns out to be a unique system to study the functional role of the beta-subunit. Injection of beta-subunit-specific mRNA transcribed in vitro from a beta-cDNA clone (derived from Xenopus kidney, A6 cells) into oocytes results in translation of a glycosylated beta-subunit. The synthesis of this exogenous beta-subunit increases significantly the proportion of trypsin-resistant oocyte alpha-subunits able to perform cation-dependent conformational changes. In addition, 25-65% more ouabian binding sites are expressed at the plasma membrane in beta-mRNA-injected oocytes. In contrast, newly synthesized alpha-subunit translated after injection of size-fractionated mRNA enriched in alpha-mRNA remains trypsin sensitive as the oocyte alpha-subunit. These data suggest that association of the beta-subunit to the alpha-subunit provokes a structural rearrangement of the alpha-subunit that might be a first step toward the functional maturation of the Na+-K+-ATPase and its expression at the plasma membrane.

Animals↗
PubMed1989

Primary sequence of Xenopus laevis Na+-K+-ATPase and its localization in A6 kidney cells.

Polyclonal antibodies raised against the alpha- and beta-subunits of amphibian kidney Na+-K+-ATPase were used to screen an expression library from Xenopus laevis kidney epithelial cells (A6 cell line). cDNAs coding for each Na+-K+-ATPase subunit were identified and used to isolate near full-length cDNAs. The complete nucleotide sequence and the deduced amino acid sequence were determined. The alpha-subunit is an alpha (alpha I)-isoform. The alpha- and beta-subunits are more closely related to the mammalian and avian than the fish sequences. Antibodies raised against the fusion proteins produced by the two clones served to immunoprecipitate proteins from biosynthetically labeled or selectively surface-radioiodinated A6 cells grown on a porous substrate. The alpha- and the beta-subunits of Na+-K+-ATPase were found associated early in the course of biosynthesis and were restricted to the basolateral plasma membrane.

Amino Acid Sequence↗
PubMed1989

Aldosterone induces a rapid increase in the rate of Na,K-ATPase gene transcription in cultured kidney cells.

Aldosterone (300 nM) induces a 4-fold increase over a 6-hr stimulation in A6 kidney cells from Xenopus laevis in the abundance of mRNA beta 1 and a 2-fold in that of mRNA alpha 1 coding for each Na,K-ATPase subunit, which is in agreement with a previous report. After a 3-hr stimulation already, aldosterone elicited a significant increase of mRNA beta 1 (2.51-fold +/- 0.47, n = 3, P less than 0.05) and a nonsignificant increase of mRNA alpha 1 (1.26-fold +/- 0.30, n = 3, NS). Increasing doses of cycloheximide up to 3 micrograms ml-1 led to 90% inhibition of protein synthesis, but failed to block the differential effect of aldosterone on mRNA abundance over a 6-h incubation period. The rate of transcription was measured by a nuclear run-on assay. The basal rate of mRNA alpha 1 transcription exceeded that of mRNA beta 1 by 2.8-fold. Aldosterone (300 nM) stimulated the transcription of the two subunit genes. Fifteen minutes after aldosterone addition there was a significant and parallel increase in the rate of transcription of the alpha 1 subunit (1.98-fold +/- 0.20, n = 3, P less than 0.02) and that of the beta 1 subunit (2.13 +/- 0.32, n = 3, P less than 0.04). After a 45-min stimulation period the transcription rate of the alpha 1 subunit remained at the level observed at 15 min (1.84-fold +/- 0.14, n = 4, P less than 0.01), while the transcription rate of the beta 1 subunit increased further (2.89-fold +/- 0.38, n = 4, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1987

Regulation by aldosterone of Na+,K+-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. Mineralocorticoids modulate the expression of a number of proteins. Na+,K+-ATPase has been identified as an aldosterone-induced protein (Geering, K., M. Girardet, C. Bron, J. P. Kraehenbuhl, and B. C. Rossier, 1982, J. Biol. Chem., 257:10338-10343). Using A6 cells (kidney of Xenopus laevis) grown on filters we demonstrated by Northern blot analysis that the induction of Na+,K+-ATPase was mainly mediated by a two- to fourfold accumulation of both alpha- and beta-subunit mRNAs. The specific competitor spironolactone decreased basal Na+ transport, Na+,K+-ATPase mRNA, and the relative rate of protein biosynthesis, and it blocked the response to aldosterone. Cycloheximide inhibited the aldosterone-dependent sodium transport but did not significantly affect the cytoplasmic accumulation of Na+,K+-ATPase mRNA induced by aldosterone.

Aldosterone↗