PubMed Health⌕ Search

Biomedical subjects

B C Rossier

Publications and source records attributed to B C Rossier.

At least 91 records · Page 5Linked to original sources
PubMed1993

Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

Amino Acid Sequence↗
PubMed1993

Corticosterone induces 11 beta-HSD and mineralocorticoid specificity in an amphibian urinary bladder cell line.

We have examined the mineralocorticoid specificity in a TBM 18-23 cell line derived from the toad bladder epithelium. In cells grown on porous substrate, corticosterone was more potent than aldosterone in stimulating a sodium transport response, measured by the short-circuit current method 6 h after hormone addition [mean affinity constant (K0.5) for corticosterone = 1 nM vs. K0.5 for aldosterone = 8 nM]. The time course of effects and saturation kinetics were identical for both agonists, suggesting interaction with identical receptors. Whereas the dose-response relationship for aldosterone did not change with time of incubation (6 vs. 24 h), the dose-response curve for corticosterone became biphasic at 24-h incubation (apparent K0.5 as high as 40 nM), demonstrating that corticosterone became apparently less potent with time. Pretreatment with carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), restored full sensitivity at 24-h incubation to corticosterone. The 11 beta-HSD activity was low during the first 3 h of incubation in the presence of 3 nM corticosterone, and only a small fraction (approximately 7%) of corticosterone was metabolized. At 24-h incubation, 11 beta-HSD activity increased approximately 2.5-fold (P < 0.001, n = 8). We conclude that 11 beta-HSD activity is induced by its own substrate in TBM cells in parallel with the induction of the carbenoxolone-sensitive sodium transport response.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Ontogeny of alpha 1- and beta 1-isoforms of Na(+)-K(+)-ATPase in fetal distal rat lung epithelium.

Because immature, in contrast to mature, fetal lungs have ineffective Na transport, we wished to determine the ontogeny of Na(+)-K(+)-ATPase expression in fetal distal lung epithelium (FDLE). FDLE and fibroblasts (FLF) from 17- to 22-day gestational age fetal rats (term = 22 days) were grown in primary culture. Northern and slot-blot analyses utilizing isoform-specific cDNA probes determined that alpha 1- (3.7 kb) and beta 1- (2.7, 2.3, and 1.9 kb) transcripts were present in FDLE at levels approximately fivefold higher than in FLF. alpha 2-, alpha 3-, or beta 2-isoforms of Na(+)-K(+)-ATPase were not detected. In 17-day gestational age FDLE, only small amounts of alpha 1-mRNA levels were detectable, and there were approximately 10-fold less beta 1-isoform transcripts. By 20 days gestational age, the level of alpha 1-transcripts roughly doubled, whereas beta 1-levels increased approximately sixfold. Thus, during the transition from the canalicular to saccular stages of lung development, FDLE have a differentially regulated surge in mRNA levels of alpha 1- and beta 1-Na(+)-K(+)-ATPase isoforms and do not switch isoforms during lung development. Levels for both isoform transcripts then fell before birth, reaching values less than those seen for 17-day gestational age FDLE. FDLE vesicle Na(+)-K(+)-ATPase activity did not increase until 22 days gestational age.

Animals↗
PubMed1993

Expression of the epithelial Na+ channel in the developing rat lung.

The adult mature fetal, but not immature fetal, lung is capable of actively transporting Na+ from the alveolar space. The reason for the impaired Na+ transport in the immature lung is not known; however, the apical membrane Na+ channel is the rate-limiting step for epithelial Na+ transport. This study determined whether transcripts coding for the adult rat colonic epithelial Na+ channel (alpha rENaC) were present in the fetal and adult lung and whether they were developmentally regulated. Similarly sized alpha rENaC transcripts were identified in RNA isolated from fetal and adult whole rat lung, primary cultures of fetal and adult alveolar epithelium, and adult rat whole kidneys, suggesting that the lung alpha rENaC is a similar transcript to that found in the salt-deprived rat colonic epithelium. There were low mRNA levels in 17- to 18-day gestational age (GA) fetal lungs and epithelium (term GA = 22 days), but these levels increased markedly during the saccular stage of lung development (20 days GA) and remained high in adult lungs. The combined administration of thyroid-releasing hormone and dexamethasone to pregnant rats between 16 and 18 days GA induced the expression of lung alpha rENaC in their fetuses. We conclude that alpha rENaC is expressed in mature fetal and adult alveolar epithelium and that it is influenced by hormones known to alter maturation of the fetal lung.

Aging↗
PubMed1993

A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon.

Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.

Animals↗
PubMed1993

Mutation of a conserved proline residue in the beta-subunit ectodomain prevents Na(+)-K(+)-ATPase oligomerization.

A highly conserved sequence motif (4 tyrosines and 1 proline: YYPYY) of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) beta 1-subunit ectodomain has been mutagenized to study its possible role in alpha/beta-assembly and sodium pump function. Single as well as double tyrosine mutants (tyrosine to phenylalanine: Y to F) of Xenopus laevis beta 1-subunits are able to associate with alpha 1-subunits and form functional Na-K pumps at the plasma membrane that are indistinguishable from wild-type alpha 1, beta 1-Na-K pumps (as assessed by measurements of ouabain binding, 86Rb flux, Na-K pump current, and activation by external potassium). In contrast, a single proline mutation (proline to glycine: P244G) reduced by > 90% the proper assembly and function of Na(+)-K(+)-ATPase, despite a normal rate of synthesis and core glycosylation. Our data indicate that proline-244 plays a critical role in the proper folding of the beta-subunit and its ability to associate efficiently with the alpha 1-subunit in the endoplasmic reticulum.

Animals↗
PubMed1993

Rat liver 11 beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid encodes oxoreductase activity in a mineralocorticoid-responsive toad bladder cell line.

The mineralocorticoid receptor displays equal affinity for aldosterone and corticosterone. It has been proposed that aldosterone selectivity in vivo is achieved by the conversion of corticosterone into its inactive metabolite 11-dehydrocorticosterone by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). To test this hypothesis, we transfected rat liver 11 beta HSD cDNA into TBM cells, a sodium-transporting cell line. These cells respond equally well to aldosterone and corticosterone, indicating that endogenous 11 beta HSD is expressed at low levels in TBM cells. Although exogenous rat liver 11 beta HSD was expressed at high levels in transfected cells, mineralocorticoid selectivity was not observed. By contrast, the biologically inactive 11-dehydrocorticosterone was readily converted into corticosterone, a potent agonist for sodium transport. Our results indicate that rat liver 11 beta HSD behaves predominantly as a reductase in TBM cells. Another 11 beta HSD isoform is likely to be responsible for the dehydrogenase reaction in aldosterone-responsive cells.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

The beta subunit modulates potassium activation of the Na-K pump.

We recently cloned the alpha 1 and the beta 1 and beta 3 subunits of the Na,K-ATPase of the toad Bufo marinus. To investigate possible functional differences between beta 1 and beta 3, we studied the potassium activation of Na-K pumps expressed in the oocyte of Xenopus laevis. Na-K pump activity was measured as K(+)-induced current in voltage-clamped oocytes. We could take advantage of the relative resistance to ouabain conferred by the Bufo alpha subunit to study specifically the exogenously expressed Na-K pumps after inhibition of the ouabain-sensitive endogenous Xenopus Na-K pumps. Coinjection of Bufo alpha 1 subunit cRNA with either beta 1 or beta 3 cRNAs results in the expression of functional Na-K pumps that share similar low ouabain sensitivity but differ in their K+ half activation constant (K1/2). Similar results were obtained with Xenopus alpha 1 and beta 1 or beta 3 subunits and with Bufo/Xenopus heterodimers. We conclude that some specific sequence of the beta subunit can influence the activation of the Na,K pump by extracellular K+ ions.

Animals↗
PubMed1992

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.

Amino Acid Sequence↗
PubMed1992

Processing, intracellular transport, and functional expression of endogenous and exogenous alpha-beta 3 Na,K-ATPase complexes in Xenopus oocytes.

The minimal functional Na,K-ATPase unit is composed of a catalytic alpha-subunit and a glycosylated beta-subunit. So far three putative beta-isoforms have been described, but only beta 1-isoforms have been identified clearly as part of a purified active enzyme complex. In this study we provide evidence that a putative beta 3-isoform might be the functional component of Xenopus oocyte Na,K-ATPase. beta 3-isoforms are expressed in the oocyte plasma membrane together with alpha-subunits, but beta 3-isoforms are synthesized to a lesser extent than alpha-subunits. The unassembled oocyte alpha-subunits accumulate in an immature trypsin-sensitive form most likely in the endoplasmic reticulum (ER). Injection of both beta 1- and beta 3-cRNA into oocytes abolishes the transport constraint of the oocyte alpha-subunit, renders it trypsin-resistant, and finally leads to an increased number of functional pumps at the plasma membrane. In addition, beta 3-isoforms as beta 1-isoforms depend on the concomitant synthesis of alpha-subunits to be able to leave the ER and to become fully glycosylated. Finally, alpha-beta 1 and alpha-beta 3 complexes expressed at the plasma membrane appear to have similar transport properties as assessed by ouabain binding, rubidium uptake, and electrophysiological measurements in oocytes coexpressing exogenous alpha 1- and beta 1- or beta 3-isoforms. Thus our data indicate that beta 3-isoforms have functional qualities similar to beta 1-isoforms. They can assemble and impose a structural reorganization to newly synthesized alpha-subunits which permits the exit from the ER and the expression of functional Na,K-pumps at the plasma membrane.

Animals↗
PubMed1992

Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase alpha subunit confers ouabain resistance.

The cardiac glycoside ouabain inhibits Na,K-ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild-type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K-pump in Xenopus oocytes injected with wild-type and mutated alpha 1 and wild-type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K-ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000-fold compared with wild-type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.

Amino Acid Sequence↗
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↗
PubMed1992

Sodium-independent effect of aldosterone on initial rate of ouabain binding in A6 cells.

To estimate the early effect of aldosterone on the number of active Na(+)-K+ pumps at the basolateral membrane of amphibian tight epithelia, we have measured the initial rate (at 4 min) of [3H]ouabain binding to the basolateral membrane of intact monolayers of A6 cells grown on permeable supports. Within 3 h, aldosterone induced a threefold increase of the Na transport and, simultaneously, a twofold increase of the binding rate of ouabain. Because the affinity of ouabain, estimated either by equilibrium binding studies or inhibition kinetics, was not modified by aldosterone, the effect on the initial rate of ouabain binding was due to an increase in the number of binding sites. This effect on ouabain binding was not prevented by 10 microM amiloride, which reduced the transepithelial sodium transport below control level. By contrast, the effect of aldosterone on ouabain binding was abolished by cycloheximide (5 micrograms/ml) or actinomycin D (2 micrograms/ml), doses which inhibited the aldosterone-dependent sodium transport response. These data suggest that aldosterone elicits an early, sodium-independent, protein synthesis-dependent increase in the expression of active Na(+)-K(+)-ATPase molecules.

Aldosterone↗
PubMed1992

Aldosterone regulation of gene transcription leading to control of ion transport.

Aldosterone, like other steroid hormones, initiates its effects by binding to intracellular receptors; these receptors are then able to control the transcription of several genes. The products of these genes eventually modulate the activity of ionic transport systems located in the apical and the basolateral membrane of specialized epithelial cells, thereby modulating the excretion of Na+ and K+ ions. Considerable progress has been made recently in understanding these mechanisms and the structure of the proteins involved in these processes. A novel principle has been discovered to explain the selective effect of aldosterone on its target epithelia. These tissues exclude competing glucocorticoid hormones by the activity of the 11 beta-hydroxysteroid dehydrogenase to allow aldosterone, an enzyme-resistant steroid, to bind to its receptors. Aldosterone induces numerous changes in the activity of membrane ion transport systems and enzymes and cell morphology. Although the enhancement of Na,K-ATPase synthesis and the increase of the number of active Na+ channels in the apical membrane appear as both direct and primary effects, the mechanisms of the other effects remain to be determined. The knowledge of the primary structure of several elements of the aldosterone response system (e.g., mineralocorticoid receptor and Na,K-ATPase) allows us to understand abnormal regulation of Na+ balance at the molecular level and, potentially, to identify genetic alterations responsible for these defects.

Aldosterone↗
PubMed1991

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.

Adenosine Triphosphatases↗
PubMed1991

Coexpression of alpha 1 with putative beta 3 subunits results in functional Na+/K+ pumps in Xenopus oocytes.

The active Na+/K+ pump is composed of an alpha and a beta subunit. Until now, three putative isoforms of the beta subunit have been identified that share sequence similarity. We have expressed the beta 1 and beta 3 isoforms of Xenopus laevis Na+/K(+)-ATPase in Xenopus oocytes to compare functional properties of the Na+/K+ pump, including either of these two isoforms. Na+/K+ pump current, estimated as K(+)-induced outward current in voltage-clamped oocytes, was doubled by coexpression of alpha 1 subunits with either isoform of the beta subunit compared to expression of alpha 1 subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K+ pump were similar with both beta isoforms, indicating that both beta 1 and beta 3 isoforms can support expression at the oocyte surface of an active Na+/K+ pump with similar functional properties.

Animals↗
PubMed1991

Functional expression of N-terminal truncated alpha-subunits of Na,K-ATPase in Xenopus laevis oocytes.

N-terminal deletion mutants of Na,K-ATPase alpha 1 isoforms initiating translation at Met34 (alpha 1T1) or at Met43 (alpha 1T2) were expressed in X. laevis oocytes. Compared to beta 3 cRNA injected controls, the co-expression of alpha 1wt, alpha 1T1, alpha 1T2 with beta 3 subunits results in a 2- to 3-fold increase of ouabain binding sites, parallelled by a concomitant increase in Na,K-pump current. The apparent K1/2 for potassium activation of the alpha 1T2/beta 3 Na,K-pumps is significantly higher than that of the alpha 1wt/beta 3 or alpha 1T1/beta 3 Na,K-pumps expressed at the cell surface. Total deletion of the lysine-rich N-terminal domain thus allows the expression of active Na,K-pump but with distinct cation transport properties.

Amino Acid Sequence↗