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P Barbry

Publications and source records attributed to P Barbry.

53 records · Page 3Linked to original sources
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

Identification and properties of a novel type of Na+-permeable amiloride-sensitive channel in thyroid cells.

Amiloride-sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride-sensitive (K0.5 = 150 +/- 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+-flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5 = 47 +/- 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl-amiloride, hardly inhibited this 22Na+-influx component at a concentration of 21 microM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 +/- 10 nM and a maximal binding capacity of 5 +/- 1 pmol/mg protein. Patch-clamp experiments have directly demonstrated the existence of a phenamil- and amiloride-sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa+/PK+ = 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.

Amiloride↗
PubMed1989

A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels.

Endothelial cells from brain microvessels form the blood-brain barrier. Brain microvessels and endothelial cells isolated from rat brain microvessels express an amiloride-sensitive cationic channel that was characterized using [3H]phenamil binding and patch-clamp experiments. [3H]Phenamil, a labeled amiloride analog, recognizes a single family of binding sites with a dissociation constant of 20-30 nM and a maximum binding capacity of 8-15 pmol/mg protein. The pharmacological profile of the channel (phenamil greater than benzamil greater than amiloride) is very similar to that of the epithelium Na+ channel of mammalian kidney and of frog epithelia. Long-lasting currents were observed in patch-clamp experiments using excised outside-out patches. Application of amiloride or phenamil first produced a rapid flickering of channel activity and then its complete blockade. The mean unit channel conductance at 140 mM Na+ was 23 picosiemens. The selectivity of Na+ over K+ was estimated from reversal potentials to be 1.5:1. Properties of the channel in microvessels are clearly distinct from those of the Na+ channel of the kidney, suggesting the existence of several isoforms of cationic channels that are sensitive to amiloride and its derivatives. The low selectivity cationic channel of endothelial cells in brain microvessels might be important for controlling both Na+ and K+ movements across the blood-brain barrier.

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

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

[3H]phenamil, a radiolabelled diuretic for the analysis of the amiloride-sensitive Na+ channels in kidney membranes.

The interaction of amiloride and amiloride derivatives with the Na+ channels of pig kidney membranes was studied from 22Na+ uptake experiments. The order of potency of the different molecules tested is: phenamil greater than benzamil greater than amiloride, ethylisopropylamiloride. [3H]labelled phenamil was prepared and used to titrate Na+ channels in pig kidney membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]phenamil and unlabelled phenamil indicate that phenamil recognizes a single family of binding sites with a Kd value of 20 nM and a maximum binding capacity of 11.5 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]phenamil competition experiments is identical to that found for the inhibition of 22Na+ uptake by apical Na+ channels.

Amiloride↗
PubMed1986

Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system.

Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.

Amiloride↗
PubMed1986

The Na+/H+ antiport of eukaryotic cells: relationship between the kinetic properties of the system and its physiological function.

The Na+/H+ antiport is present in the plasma membrane of virtually all vertebrate cells and it plays a central role in cell homeostasis. The pharmacological properties and the characteristics of the interaction of extracellular Na+, Li+, H+ and of intracellular H+ with the Na+/H+ antiport are reviewed herein. The kinetic properties of the system are shown to be essential for defining its four main physiological functions: transepithelial ion transport, control of the pHi, control of the intracellular Na+ concentration, and control of the cell volume. The activity of the Na+/H+ antiport can be modulated by a large number of effectors which are thought to act via protein kinases. At least three mechanisms of activation of the Na+/H+ exchanger are defined from the analysis of the kinetic properties of the system. Activation of the Na+/H+ antiport leads to very different consequences, depending upon the activity of other ion transporting systems in the membrane.

Animals↗
PubMed1985

[3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys.

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.

Amiloride↗
PubMed1985

Biochemical properties of the Na+/H+ exchange system in rat brain synaptosomes. Interdependence of internal and external pH control of the exchange activity.

Properties of the Na+/H+ exchange system in synaptosomes have been studied primarily by using acridine orange fluorescence to follow H+ efflux. Results obtained from 22Na+ uptake experiments and [3H]ethylpropylamiloride binding experiments are also presented for comparison. The basal properties of the Na+/H+ antiport in synaptosomes are similar to those found in other systems; (i) the stoichiometry of Na+/H+ exchange is 1:1; (ii) Li+ can be successfully substituted for Na+; its affinity for the exchanger (KLi+ = 3 mM) is higher than that of Na+ (KNa+ = 12 mM), but the maximal rate of H+ efflux in the presence of Li+ is about 3 times lower than the maximal rate of H+ efflux in the presence of Na+; and (iii) the Na+/H+ antiport is inhibited by amiloride derivatives with the rank order:ethylisopropylamiloride greater than ethylpropylamiloride greater than amiloride greater than benzamil. The most important finding of this paper is that the external pH dependence of the synaptosomal Na+/H+ antiport is controlled by the value of internal pH and vice versa. For example apparent pHo values for half-maximum activation of the Na+/H+ exchanger are pHo = 7.12 when pHi = 6.4 and pHo = 7.95 when pHi = 7.3. Therefore, a 0.9 pH unit increase in internal pH produces a shift of at least a 0.83 pH unit in the external pH dependence. In addition, changing pHo from 7.75 to 8.50 also shifts the half-maximum pHi value for activation of the Na+/H+ antiport from 6.67 to 7.54.

Acridine Orange↗
PubMed1984

Cyclic AMP as a transcriptional inhibitor of upper eukaryotic gene transcription.

Recently, glucagon and its second messenger, cyclic AMP, have been shown to stimulate the transcription rate of several upper eukaryotic genes (1-5). We show here that glucagon can also block gene transcription. Both glucagon and cyclic AMP were found to inhibit the transcription of the genes encoding three liver glycolytic enzymes, including L-type pyruvate kinase and aldolase B. Thus, cyclic AMP proves to be not only an activator but also an inhibitor of gene transcription in eukaryotes.

Animals↗
PubMedDate not supplied

Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation.

Cystic fibrosis is associated with a defect in epithelial chloride ion transport which is caused by mutations in a membrane protein called CFTR (cystic fibrosis transmembrane conductance regulator). Heterologous expression of CFTR produces cyclicAMP-sensitive Cl(-)-channel activity. Deletion of phenylalanine at amino-acid position 508 in CFTR (delta F508 CFTR) is the most common mutation in cystic fibrosis. It has been proposed that this mutation prevents glycoprotein maturation and its transport to its normal cellular location. We have expressed both CFTR and delta F508 CFTR in Vero cells using recombinant vaccinia virus. Although far less delta F508 CFTR reached the plasma membrane than normal CFTR, sufficient delta F508 CFTR was expressed at the plasma membrane to permit functional analysis. delta F508 CFTR expression induced a reduced activity of the cAMP-activated Cl- channel, with conductance, anion selectivity and open-time kinetics similar to those of CFTR, but with much greater closed times, resulting in a large decrease of open probability. The delta F508 mutation thus seems to have two major consequences, an abnormal translocation of the CFTR protein which limits membrane insertion, and an abnormal function in mediating Cl- transport.

Cells, Cultured↗