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B C Rossier

Publications and source records attributed to B C Rossier.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.

Liddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

Animals↗
PubMed1998

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.

Amiloride↗
PubMed1998

Epithelial sodium channel regulatory proteins identified by functional expression cloning.

We describe here our current strategy for identifying and cloning proteins involved in the regulation of the epithelial sodium channel (ENaC). We have set up a complementation functional assay in the Xenopus laevis oocyte expression system. Using this assay, we have been able to identify a channel-activating protease (CAP-1) that can increase ENaC activity threefold. We propose a novel extracellular signal transduction pathway controlling ionic channels of the ENaC gene family that include genes involved in mechanotransduction (degenerins), in peptide-gated channels involved in neurotransmission (FaNaCh), in proton-gated channels involved in pH sensing (ASIC) or pain sensation (DRASIC).

Animals↗
PubMed1998

Unusual degradation of alpha-beta complexes in Xenopus oocytes by beta-subunits of Xenopus gastric H-K-ATPase.

The catalytic alpha-subunit of oligomeric P-type ATPases such as Na-K-ATPase and H-K-ATPase requires association with a beta-subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the beta-subunit of Xenopus gastric H-K-ATPase (betaHK) in Xenopus oocytes together with alpha-subunits of H-K-ATPase (alphaHK) or Na-K-ATPase (alphaNK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of Xenopus betaHK from metabolically labeled oocytes show that it is well expressed and, when synthesized without alpha-subunits, can leave the ER and become fully glycosylated. Xenopus betaHK can associate with both coexpressed alphaHK and alphaNK, but the alpha-beta complexes formed are degraded rapidly in or close to the ER and do not produce functional pumps at the cell surface as assessed by 86Rb uptake. A possible explanation of these results is that Xenopus betaHK may contain a tissue-specific signal that is important in the formation or correct targeting of functional alpha-beta complexes in the stomach but that cannot be recognized in Xenopus oocytes and in consequence leads to cellular degradation of the alpha-beta complexes in this experimental system.

Amino Acid Sequence↗
PubMed1998

[History of salt].

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History, 18th Century↗
PubMed1997

A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism.

Aldosterone-dependent epithelial sodium transport in the distal nephron is mediated by the absorption of sodium through the highly selective, amiloride-sensitive epithelial sodium channel (ENaC) made of three homologous subunits (alpha, beta, and gamma). In human, autosomal recessive mutations of alpha, beta, or gammaENaC subunits cause pseudohypoaldosteronism type 1 (PHA-1), a renal salt-wasting syndrome characterized by severe hypovolemia, high plasma aldosterone, hyponatremia, life-threatening hyperkaliemia, and metabolic acidosis. In the mouse, inactivation of alphaENaC results in failure to clear fetal lung liquid at birth and in early neonatal death, preventing the observation of a PHA-1 renal phenotype. Transgenic expression of alphaENaC driven by a cytomegalovirus promoter in alphaENaC(-/-) knockout mice [alphaENaC(-/-)Tg] rescued the perinatal lethal pulmonary phenotype and partially restored Na+ transport in renal, colonic, and pulmonary epithelia. At days 5-9, however, alphaENaC(-/-)Tg mice showed clinical features of severe PHA-1 with metabolic acidosis, urinary salt-wasting, growth retardation, and 50% mortality. Adult alphaENaC(-/-)Tg survivors exhibited a compensated PHA-1 with normal acid/base and electrolyte values but 6-fold elevation of plasma aldosterone compared with wild-type littermate controls. We conclude that partial restoration of ENaC-mediated Na+ absorption in this transgenic mouse results in a mouse model for PHA-1.

Amiloride↗
PubMed1997

An epithelial serine protease activates the amiloride-sensitive sodium channel.

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to the protease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodium channel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.

Amino Acid Sequence↗
PubMed1997

Lose salt and gain a friend! A tribute to Gerhard Giebisch.

In this little essay I describe recent advances in understanding the problem of salt sensitivity and salt resistance involved in the control of blood volume and blood pressure. Genetic evidence links the recently characterized epithelial sodium channel (ENaC) and the potassium channel (ROMK-1) to monogenic diseases in humans, characterized by a renal salt-losing syndrome. A loss of function mutations in ROMK-1 gene causes in some pedigrees the syndrome of Bartter, characterized by metabolic alkalosis and a severe salt-losing syndrome. A loss of function mutations in ENaC genes causes pseudohypoaldosteronism-type 1, characterized by hypovolaemia, hyperkaliaemia, metabolic acidosis and hypotension. ENaC and ROMK-1 are expressed in the apical membrane of principal cells of the cortical collecting duct and their role in Na/K balance is briefly reviewed.

Aldosterone↗
PubMed1997

Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics.

Abnormal regulation of ion channels by members of the ABC transport protein superfamily has been implicated in hyperinsulinemic hypoglycemia and in excessive Na+ absorption by airway epithelia in cystic fibrosis (CF). How ABC proteins regulate ion conductances is unknown, but must generally involve either the number or activity of specific ion channels. Here we report that the cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in CF, reverses the regulation of the activity of single epithelial sodium channels (ENaC) by cAMP. ENaC expressed alone in fibroblasts responded to activation of cAMP-dependent protein kinase with increased open probability (Po) and mean open time, whereas ENaC co-expressed with CFTR exhibited decreased Po and mean open time under conditions optimal for PKA-mediated protein phosphorylation. Thus, CFTR regulates ENaC at the level of single channel gating, by switching the response of single channel Po to cAMP from an increase to a decrease.

3T3 Cells↗
PubMed1997

Novel isoforms of the beta and gamma subunits of the Xenopus epithelial Na channel provide information about the amiloride binding site and extracellular sodium sensing.

We have previously identified three homologous subunits alpha, beta, and gamma of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed beta2 and gamma2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta2 and gamma2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after alphabeta2gamma XENaC cRNA injection in Xenopus oocyte did not differ from alphabetagamma XENaC. By contrast, the channel expressed after alphabetagamma2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (KI alphabetagamma2; 1.34 microM versus alphabetagamma 0.35 microM). These data indicate that the gamma (and/or gamma2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between gamma and gamma2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.

Amiloride↗
PubMed1997

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Amiloride↗
PubMed1997

Neonatal injection of native proton pump antigens induces autoimmune gastritis in mice.

BACKGROUND & AIMS: Autoimmune gastritis is associated with gastric H+, K(+)-adenosine triphosphatase (ATPase)-specific autoantibodies (HKAb). The (auto) antigen that triggers disease and the pathogenic role of the autoantibodies are unknown. The aim of this study was to analyze when these autoantibodies are produced during autoimmune gastritis in neonatally thymectomized mice and whether a native H+, K(+)-ATPase antigen preparation can induce disease in mice. METHODS: Autoantibodies were characterized by a novel assay based on immunoprecipitation of a functional H+, K(+)-ATPase expressed in Xenopus oocytes. Normal mice were injected intraperitoneally with H+, K(+)-ATPase-enriched gastric membranes in the absence of adjuvant. RESULTS: Conformational autoantibodies recognizing both H+, K(+)-ATPase subunits appeared simultaneously with the gastric lesions 1 month after thymectomy. Immunization of neonates, but not adults, induced a persistent autoimmune gastritis in the body mucosa, characterized by lymphocytic infiltrations, loss of parietal and chief cells, metaplasia, and H+, K(+)-ATPase-specific autoantibodies. The histopathological lesions of this new model are similar to those in humans and thymectomized mice. CONCLUSIONS: The onset of gastritis and autoantibody production parallels the expression of the H+, K(+)-ATPase during ontogeny. Exposure of the neonatal immune system to organ-specific antigens expressed late after birth induces autoimmune gastritis in adult mice.

Animals↗
PubMed1997

Polymorphisms in the carboxy-terminus of the epithelial sodium channel in rat models for hypertension.

OBJECTIVE: To investigate whether mutations in the C-terminus of the three subunits of the rat epithelial sodium channel (alphabetagamma-rENaC) contribute to the hypertensive phenotype in five rat models for essential hypertension. DESIGN: We sequenced the C-terminal regions of alpha-, beta- and gamma-rENaC genes in five different hypertensive rat strains [spontaneously hypertensive rats (SHR), Dahl salt-sensitive (SS/Jr) rats, Milan hypertensive (MHS) rats, Sabra hypertensive (SBH) rats and Lyon hypertensive rats (LHR)] and their normotensive controls [Wistar-Kyoto (WKY) rats, Dahl salt-resistant (SR/Jr) rats, Milan normotensive (MNS) rats, Sabra normotensive (SBN) rats and Lyon normotensive rats (LNR)]. Identified polymorphisms were tested for cosegregation with blood pressure as well as for increased epithelial sodium channel (ENaC) activity. METHODS: Genomic DNA extracted from hypertensive and normotensive rat strains was amplified by the polymerase chain reaction and polymerase chain reaction fragments were sequenced. Cosegregation analysis was performed to test for correlations between blood pressure and different genotypes. The effects of a polymorphism on ENaC activity were assessed by functional expression in Xenopus laevis oocytes. The chromosomal location of the gene for gamma-ENaC was determined by linkage analysis in an F2 (MHS x MNS) population. RESULTS: We found no polymorphisms at the C-terminus of alpha- and beta-rENaC in the five rat models tested. We identified two polymorphisms at the C-terminus of the gamma-subunit, one leading to an amino acid change. Milan strains (MNS and MHS) were polymorphic for this mutation. By cosegregation analysis we could exclude the possibility that there was a correlation between blood pressure and this polymorphism. Functional expression of the polymorphism caused no increase in ENaC activity assessed by measurement of the amiloride-sensitive sodium current in Xenopus oocytes. The gene for the gamma-ENAC was located on rat chromosome 1. CONCLUSIONS: No polymorphisms at the C-terminus of the three subunits of the epithelial sodium channel cosegregating with blood pressure were detected in five different genetic rat models for hypertension. If an altered ENaC activity contributes to the pathogenesis of hypertension in these rats, it must thus arise from mutations in other parts of the protein, from mutations outside the coding region impairing the proper regulation of one of the subunits or from mutations in an ENaC-associated protein.

Animals↗
PubMed1997

A reappraisal of aldosterone effects on the kidney: new insights provided by epithelial sodium channel cloning.

Sodium homeostasis is crucial for the control of extra-cellular volume and blood pressure. Regulation of sodium reabsorption is mainly achieved in the distal nephron by the mineralocorticoid aldosterone, but the molecular pathway of aldosterone action has largely remained unclear. Molecular genetic analysis of inherited diseases disturbing sodium homeostasis has now demonstrated that the amiloride-sensitive epithelial sodium channel is a major effector of aldosterone action. Mechanisms by which aldosterone regulates the epithelial sodium channel activity are beginning to emerge and will be of great importance for a better understanding of salt-sensitive hypertension.

Aldosterone↗
PubMed1997

Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.

Transepithelial Na+ reabsorption across tight epithelia is regulated by aldosterone. The amiloride-sensitive epithelial sodium channel (ENaC) is a major target for the natriferic action of aldosterone. In this study, the effect of aldosterone on ENaC mRNA abundance and the rate of protein synthesis for each of the three ENaC subunits (alpha, beta and gamma) in the A6 kidney cell line were examined. In cells grown on plastic, aldosterone induced a large and rapid increase in epithelial sodium channel (ENaC) beta and gamma subunit mRNA abundance, but this effect is not translated into the synthesis of the corresponding proteins. In cells grown on a porous substrate, amiloride-sensitive electrogenic sodium transport was expressed and was upregulated by aldosterone (300 nM) as early as 1 h after the addition of the hormone. The alpha, beta, and gamma mRNA abundance was not changed by aldosterone during the first 3 h of stimulation, whereas a fourfold increase over control was observed after 24 h. The rate of synthesis of alpha subunit was significantly increased above control already 60 min after aldosterone addition, whereas beta subunit synthesis increased only 6 h after hormone addition, with no significant change for the gamma subunit. The half-lives of each subunit as assessed by 35S methionine pulse-chase experiments were short (between 40 and 50 min) and were not modified by aldosterone. Taking into account the short half-life of ENaC protein and assuming that the synthesis of the alpha subunit is a limiting factor in the assembly and expression of new channels at the cell surface, it is proposed that the aldosterone regulation of sodium transport might be, in part, mediated by de novo synthesis of the channel protein.

Aldosterone↗