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O Chassande

Publications and source records attributed to O Chassande.

29 records · Page 2Linked to original sources
PubMed1991

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.

Animals↗
PubMed1990

[3H]phenamil binding protein of the renal epithelium Na+ channel. Purification, affinity labeling, and functional reconstitution.

This paper describes a large-scale purification procedure of the amiloride binding component of the epithelium Na+ channel. [3H]Phenamil was used as a labeled ligand to follow the purification. The first two steps are identical with those previously described [Barbry, P., Chassande, O., Vigne, P., Frelin, C., Ellory, C., Cragoe, E. J., Jr., & Lazdunski, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4836-4840]. A third step was a hydroxyapatite column. The purified material consisted of a homodimer of two 88-kDa proteins that migrated anomalously in SDS-PAGE to give an apparent Mr of 105,000. Deglycosylation by treatment with neuraminidase and endoglycosidase F or with neuraminidase and glycopeptidase F indicated that less than 5% of the mass of the native receptor was carbohydrate. Sedimentation analysis of the purified Na+ channel in H2O and D2O sucrose gradients and gel filtration experiments led to an estimated molecular weight of the [3H]phenamil receptor protein-detergent-phospholipid complex of 288,000 and of the native [3H]phenamil receptor protein of 158,000. [3H]Br-benzamil is another labeled derivative of amiloride that recognized binding sites that had the same pharmacological properties as [3H]phenamil binding sites and that copurified with them. Upon irradiation of kidney membranes, [3H]Br-benzamil incorporated specifically into a 185-kDa polypeptide chain under nonreducing electrophoretic conditions and a 105-kDa protein under reducing conditions. The same labeling pattern was observed at the different steps of the purification. Reconstitution of the purified phenamil receptor into large unilamellar vesicles was carried out. A low but significant phenamil- and amiloride-sensitive electrogenic Na+ transport was observed.

Affinity Labels↗
PubMed1990

Localization of the gene for amiloride binding protein on chromosome 7 and RFLP analysis in cystic fibrosis families.

The apical sodium channel is essential for sodium reabsorption by the kidney. Its activity is blocked by the diuretic amiloride. Using a human cDNA coding for the amiloride binding protein (ABP), the corresponding structural gene was mapped to human chromosome 7q34-q36 by in situ hybridization. This region flanks the region implicated in cystic fibrosis (7q32). Because an alteration of the amiloride sensitive sodium channel function has been suggested in cystic fibrosis, a possible link between the ABP gene and this disease was analyzed by restriction fragments length polymorphism (RFLP) analyses. From this study, it appears that the gene coding for ABP is not directly modified by mutations causing cystic fibrosis.

Amine Oxidase (Copper-Containing)↗
PubMed1990

Human kidney amiloride-binding protein: cDNA structure and functional expression.

Phenamil, an analog of amiloride, is a potent blocker of the epithelial Na+ channel. It has been used to purify the porcine kidney amiloride-binding protein. Synthetic oligonucleotides derived from partial sequences have been used to screen a human kidney cDNA library and to isolate the cDNA encoding the human amiloride-binding protein. The primary structure was deduced from the DNA sequence analysis. The protein is 713 residues long, with a 19-amino acid signal peptide. The mRNA was expressed in 293-S and NIH 3T3 cells, yielding a glycoprotein (i) that binds amiloride and amiloride analogs with affinities similar to the amiloride receptor associated with the apical Na+ channel in pig kidney membranes and (ii) that is immunoprecipitated with monoclonal antibodies raised against pig kidney amiloride-binding protein.

Amiloride↗
PubMed1989

Biochemical identification of two types of phenamil binding sites associated with amiloride-sensitive Na+ channels.

The existence of distinct forms of the epithelium Na+ channel that differ in their sensitivity to amiloride has been repeatedly suggested by physiological data. The biochemical basis for these differences was analyzed by using phenamil, the most potent inhibitor known so far for the epithelium Na+ channel. [3H]Phenamil of high radioactive specific activity (30 Ci/mmol) was prepared and used to titrate [3H]phenamil binding sites in pig kidney membranes. Kinetic experiments, equilibrium binding studies, and competition experiments indicated the presence in crude membrane preparations of two classes of independent binding sites. A first binding site was characterized by a high affinity for phenamil (Kd1 = 0.4 nM) and for amiloride (Kd1 = 0.1 microM). A second binding site recognized phenamil and amiloride with lower affinities [Kd2(phenamil) = 28 nM, Kd2(amiloride) = 4 microM]. The ratio of the respective amounts of low- and high-affinity binding sites was 14 +/- 2 in different membrane preparations (range: 6-22). The two types of binding sites for [3H]phenamil copurified and were still observed after purification of the epithelium Na+ channel to homogeneity. These results indicate that at least two types of pharmacologically distinguishable Na+ channels exist in the kidney. They correspond either to two isoforms of the apical Na+ channel or to one single type of channel under two different states of covalent regulation.

Amiloride↗
PubMed1988

The Na+/K+/Cl- cotransport in C6 glioma cells. Properties and role in volume regulation.

The role of the Na+/K+/Cl- cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by 'loop diuretics' with the following order of potency: benzmetanide greater than bumetanide greater than piretanide greater than furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide-sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl- cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl- cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso-osmotic medium deprived of either Na+, K+ or Cl- also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl- cotransport together with a cell swelling back to the normal volume were observed upon returning ion-deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl- cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.

Biological Transport↗
PubMed1988

Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+/H+ antiport and the Na+/Ca2+ exchanger.

Amiloride analogs inhibit a number of transmembrane Na+ transport systems: 1) the epithelium Na+ channel, 2) the Na+/H+ exchange system and 3) the Na+/Ca2+ exchange system. Structure--activity relationships using amiloride derivatives with selected modification of each of the functional groups of the molecule indicate that the 3 Na+ transporting systems have distinct pharmacological profiles. 5-N Disubstituted derivatives of amiloride, such as ethylisopropylamiloride are the most potent inhibitors of the Na+/H+ exchange system. Conversely, amiloride derivatives that are substituted on the guanidino moiety, such as phenamil, are potent inhibitors of the epithelium Na+ channel. It is thus possible, by using selected amiloride derivatives to inhibit selectively one or another of the Na+ transport systems.

Amiloride↗
PubMed1987

Purification and subunit structure of the [3H]phenamil receptor associated with the renal apical Na+ channel.

Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [3H]phenamil binding sites associated with the epithelial Na+ channel from pig kidney is 92 +/- 10 kDa. The [3H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na+ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na+ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO4/poly-acrylamide gel electrophoresis of the purified Na+ channel under nonreducing conditions showed the presence of a single major polypeptide chain of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na+ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na+ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges.

Amiloride↗
PubMed1986

Biochemical characterization of the Na+/K+/Cl- co-transport in chick cardiac cells.

Cultured chick cardiac cells possess a Na+K+Cl-co-transport system that is inhibited by the "loop diuretics" benzmetanide (IC50 = 0.3 microM), bumetanide (IC50 = 0.6 microM), piretanide (IC50 = 1.5 microM) and furosemide (IC50 = 5 microM). The K0.5 values for Cl- and Na+ activation of the bumetanide-sensitive 86Rb+ uptake are 59 mM and 40mM respectively. Bumetanide also inhibits a 22Na+ uptake component that is suppressed when external Cl- or K+ are substituted by impermeant ions. The ratio of bumetanide-sensitive 86Rb+ to 22Na+ uptake is close to 1. The cardiac Na+/K+/Cl- cotransport is a major uptake pathway for Na+ and K+. It accounts for 50% of the initial rate of 86Rb+ uptake and 17% of the initial rate of 22Na+ uptake by chick cardiac cells. It is activated two-fold by an hyperosmotic shock produced with 200 mM mannitol.

Animals↗