Biomedical subjects

P Barbry

Publications and source records attributed to P Barbry.

At least 37 records · Page 2Linked to original sources

PubMed1996

Molecular biology of the amiloride-sensitive epithelial Na+ channel.

The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits, alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5 pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha-, beta-, and gamma-proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-NH2 peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic and contain potential regulatory segments that are able to modulate the activity of the channel. Accordingly, in Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta- and gamma-chains of the epithelial Na+ channel lead to a hyperactivity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase in beta- and gamma-subunit transcription is observed, whereas the alpha-subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.

Amiloride↗

PubMed1996

[The amiloride sensitive sodium channel].

The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha, beta, and gamma proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-amide peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic, and contain potential regulatory segments that are able to modulate the activity of the channel. In Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta and gamma chains of the epithelial Na+ channel lead to a hyper-activity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting, by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase of the beta and gamma subunits transcription is observed, whereas the alpha subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not significantly altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are the positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.

Amiloride↗

PubMed1995

Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel.

The peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and structurally related peptides are present both in invertebrate and vertebrate nervous systems. Although they constitute a major class of invertebrate peptide neurotransmitters, the molecular structure of their receptors has not yet been identified. In neurons of the snail Helix aspersa, as well as in Aplysia bursting and motor neurons, FMRFamide induces a fast excitatory depolarizing response due to direct activation of an amiloride-sensitive Na+ channel. We have now isolated a complementary DNA from Helix nervous tissue; when expressed in Xenopus oocytes, it encodes an FMRFamide-activated Na+ channel (FaNaCh) that can be blocked by amiloride. The corresponding protein shares a very low sequence identity with the previously cloned epithelial Na+ channel subunits and Caenorhabditis elegans degenerins, but it displays the same overall structural organization. To our knowledge, this is the first characterization of a peptide-gated ionotropic receptor.

Amiloride↗

PubMed1995

Cloning, chromosomal localization, and physical linkage of the beta and gamma subunits (SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodium channel.

Three subunits of the amiloride-sensitive Na+ channel, named alpha, beta, and gamma, have previously been cloned in rat colon. The human lung alpha chain (SCNN1A) has also been cloned and its gene localized on chromosome 12p13. We now report the molecular cloning of the human lung beta (SCNN1B) and gamma (SCNN1G) chains. In situ hybridization and pulsed-field electrophoresis experiments demonstrate that both genes are located within a common 400-kb fragment on chromosome 16p12-p13. Screening of the cDNA library reveals two forms of the beta subunit that differ by the presence or absence of a 464-bp fragment in the 3' region. A frameshift in the short form modifies the COOH terminal sequence of the corresponding protein. Since several similar frameshifts mutations have recently been reported in patients affected by a rare form of hypertension, the existence of COOH truncated forms of the beta chain might be of physiological importance.

Amiloride↗

PubMed1995

A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease.

The effects of the mild cystic fibrosis (CF) mutation P574H were analysed and compared with those of three severe ones (delta I507, delta F508 and R560T). Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H >> delta F508 >> R560T approximately 0. Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that of the wild type CFTR-Cl- channel. This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. The P574H mutation modifies the gating mode of the channel with a large increase (approximately x 7) in the mean channel open time. Proline 574 might play an important role in the process connecting ATP hydrolysis at the nucleotide binding domain and opening and closing events of the CFTR-Cl- channel.

Animals↗

PubMed1995

Localization and regulation by steroids of the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney.

Polyclonal antibodies have been raised against the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel. The three subunits were detected by immunohistochemistry at the apical membrane of epithelial cells from the distal colon, the lung and the distal segments of the kidney tubules. No significant labelling was detected in lung alveoli, suggesting that it is not a major site of expression of the Na+ channel. Effects of a low Na+ diet or of dexamethasone treatment were measured at the mRNA level and at the protein level by immunohistochemistry. In the colon, steroids controlled Na+ channel activity via the stimulation of the transcription of beta and gamma subunits. The alpha mRNA was constitutively expressed. However, while neither alpha, beta nor gamma proteins were detected in the colon of control animals, they were all detected in the colon of steroid-treated animals. In the lung, Na+ channel expression was regulated by glucocorticoids the circulating level of which was sufficiently high to induce a maximal expression of the three subunits, even in control animals. Adrenalectomy drastically reduced expression of the three subunits. A surprising finding was the apparent absence of steroid effects on alpha, beta and gamma subunit expression in the kidney. Neither the expression of the mRNAs nor the expression of the proteins were significantly altered by aldosterone or by dexamethasone. These results could be due to mixed gluco- and mineralocorticoid regulations in different segments of the kidney tubule, but their interpretation also requires regulations that are apparently not found in the lung or colon.

Adrenalectomy↗

PubMed1994

The human gene for diamine oxidase, an amiloride binding protein. Molecular cloning, sequencing, and characterization of the promoter.

The amiloride binding protein (ABP) is detected in many epithelium-rich and/or hematopoietic tissues (Lingueglia, E., Renard, S., Voilley, N., Waldmann, R., Chassande, O., Lazdunski, M., and Barbry, P. (1993) Eur. J. Biochem. 216, 679-687). The protein binds amiloride and some of its derivatives, such as phenamil, benzamil, and ethylpropylamiloride. These properties have previously suggested that ABP might be associated with an amiloride-sensitive Na+ channel. It corresponds in fact to an amiloride-sensitive diamine oxidase (DAO) that catalyzes the degradation of compounds such as putrescine or histamine. The analysis of the organization of the sequence of the human ABP/DAO gene reveals that the 2.4-kilobase messenger RNA is transcribed from two close origins identifying the proximal promoter. After sequencing, some corrections within the initial cDNA sequence have been made. Human ABP/DAO corresponds to a 751-residue polypeptide. The promoter activity of 1800 base pairs upstream of the transcription start sites of the long form has been analyzed. Two bulks of cis-activating sequences have been identified. One of them constitutes the proximal promoter. It contains a palindromic sequence previously described as E-PAL. This motif is essential for the full activity of the promoter and behaves like a composite element. This first molecular cloning of a human gene coding for a diamine oxidase will allow us to further understand its regulation during cell growth and/or embryonic development.

Amine Oxidase (Copper-Containing)↗

PubMed1994

Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone.

Long term regulation of the amiloride-sensitive Na+ channel activity by steroid hormones occurs via de novo protein synthesis. The messenger level of RCNaCh1, previously shown by expression cloning to be a component of this channel, was measured in colons from rats fed with a low sodium diet. After 1 week of this diet, the channel activity was increased in an all-or-none fashion, whereas the level of RCNaCh1 messenger remained constant. A cDNA coding for another subunit of the Na+ channel was obtained by polymerase chain reaction. The 650-amino acid protein, entitled RCNaCh2, is 58% homologous to RCNaCh1 and displays a similar structure. It had no intrinsic activity when expressed alone in Xenopus oocytes, but its co-expression with RCNaCh1 increased the channel activity 18 +/- 5-fold. The increase in messenger level for RCNaCh2 during the time course of the diet is likely to explain the positive regulation of the rat colon Na+ channel by steroids. Immunocytochemical localization of the RCNaCh1 subunit revealed an apical labeling in colon from sodium-depleted rats. No labeling was observed in colon from control animals. These results suggest that oligomerization is needed for the proper expression of RCNaCh1 at the cell surface.

Aldosterone↗

PubMed1994

Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones.

Molecular cloning of the amiloride-sensitive Na+ channel has permitted analysis of the mechanisms of its stimulation by steroids. In rat lung cells in primary culture, where its mRNA has been detected, the activity of an amiloride-sensitive channel, highly selective for Na+, is controlled by corticosteroids. Dexamethasone (0.1 microM) or aldosterone (1 microM) induced, after a minimum 10 h treatment, a large increase of the amiloride-induced hyperpolarization and of the amiloride-sensitive current. A parallel increase in the amount of the mRNA was observed. The corresponding gene is thus a target for steroid action. Using synthetic specific agonists and antagonists for mineralo- and glucocorticoid receptors, it has been shown that the steroid action on Na+ channel expression is mediated via glucocorticoid receptors. Triiodothyronine, known to modulate steroid action in several tissues, had no effect on both the amiloride-sensitive Na+ current and the level of the mRNA for the Na+ channel protein, but potentiates the stimulatory effect of dexamethasone. The increase in Na+ channel activity observed in the lung around birth can thus be explained by a direct increase in transcription of the Na+ channel gene.

Aldosterone↗

PubMed1994

Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel.

A key protein component of the amiloride-sensitive sodium channel has been cloned from rat colon and human lung. It may represent the first member of a new family of ionic channels expressed from nematode to human. The biochemical properties of the rat protein, a 699 amino acids long polypeptide, have been analyzed. Four polyclonal antibodies raised against distinct parts of the channel immunoprecipitated a glycosylated protein of 96 kDa after cRNA expression in oocytes as well as after in vitro translation. When expressed alone into oocytes, the protein was not stable; most of it remains stacked into the endoplasmic reticulum. This results in a very low yield of complete maturation of the protein at the cell surface after expression from the pure cRNA. To determine the membrane topology of the protein, in vitro translation by a rabbit reticulocyte lysate was performed followed by insertion into canine pancreatic microsomes and protease digestion. Analysis revealed a model with only two transmembrane alpha helices and a large extracellular domain of about 500 amino acids. The NH2 and COOH termini are cytoplasmic. Protease digestion results suggest the possible presence of a structural element that could have a function similar to that of the H5 segment in K+ channels. The model indicates that there is no cytoplasmic site for protein kinase A phosphorylation. The well known regulation of the channel activity by hormones that activate this kinase such as vasopressin might thus be situated on another channel component.

Amiloride↗

PubMed1994

The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel.

Cystic fibrosis is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs. Epithelial cells of cystic fibrosis patients have a decreased capacity to secrete chloride in response to cAMP-mobilizing agents because of the mutation of a single gene. The gene product, the cystic fibrosis transmembrane conductance regulator or CFTR, is a chloride channel. The most frequent mutation is a deletion of phenylalanine in position 508 (delta F508-CFTR) that reduces both the expression of the CFTR protein at the cell surface, and the activity of the Cl- channel. This work presents the properties of NS004, a substituted benzimidazolone, which is the first activator of normal and mutant CFTR-associated chloride channels to be described. NS004 activated CFTR and delta F508-CFTR Cl- channels expressed in Xenopus oocytes, and increased 125I efflux (via the Cl- channel) from Vero cells expressing CFTR and delta F508-CFTR. Application of NS004 to the external side of outside-out patches excised from these CFTR- and delta F508-CFTR-expressing cells induced a marked and reversible increase in channel activity.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Diamine oxidase is the amiloride-binding protein and is inhibited by amiloride analogues.

Diamine oxidase (histaminase), an enzyme that oxidatively deaminates putrescine and histamine, was purified from human placenta and from pig kidney. Both NH2-terminal sequences are highly homologous to the human kidney amiloride-binding protein, previously thought to be a component of the amiloride-sensitive Na+ channel. Monoclonal antibodies raised against the pig kidney amiloride-binding protein immunoprecipitate a polypeptide with the same M(r) (105,000) as that of pig kidney diamine oxidase. That polypeptide has both diamine oxidase activity and the capacity to bind [3H]phenamil, a tritiated amiloride derivative. Cells stably transfected with human kidney amiloride-binding protein cDNA express a high diamine oxidase activity. In transfected cells as well as with the purified enzyme, this activity was inhibited by amiloride and by some of its derivatives, such as phenamil and ethylpropylamiloride. Amiloride inhibition seems to be due to drug binding at the active site of the enzyme. These data indicate that human placental diamine oxidase is identical to the human kidney amiloride-binding protein and that amiloride analogues may have wider physiological effects besides those on epithelial ion transport.

Amiloride↗

PubMed1994

The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning.

Water balance in the lung is controlled via active Na+ and Cl- transport. Electrophysiological measurements on lung epithelial cells demonstrated the presence of a Na+ channel that is inhibited by amiloride (K0.5 = 90 nM) and some of its derivatives such as phenamil (K0.5 = 19 nM) and benzamil (K0.5 = 14 nM) but not by ethylisopropylamiloride. An amiloride-sensitive Na+ channel of 4 pS was recorded from outside-out patches excised from the apical membrane. This channel is highly selective for Na+ (PNa+/PK+ > or = to 10). Isolation of a human lung cDNA led to the primary structure of the lung Na+ channel. The corresponding protein is 669 residues long and has two large hydrophobic domains. An amiloride-sensitive Na(+)-selective current apparently identical to the one observed in lung epithelial cells was recorded after expression of the cloned channel in oocytes. The level of the mRNA for the Na+ channel was highly increased from fetal to newborn and adult stages. This observation indicates that the increased Na+ reabsorption that occurs at birth as a necessary event to pass to an air-breathing environment is probably associated with control of transcription of this Na+ channel. The human gene for the lung Na+ channel was mapped on chromosome 12p13.

Amiloride↗

PubMed1993

Molecular cloning and functional expression of different molecular forms of rat amiloride-binding proteins.

The colon and lung amiloride-binding proteins were cloned from rat tissues. Two sizes of transcripts were identified. The 2.7-kb transcript codes for an 85-kDa protein, whereas the 1.2-kb transcript codes for a 25-kDa polypeptide. The 2.7-kb transcript was detected in the proximal and distal colon and in duodenum, liver, placenta and thymus. The 1.2-kb transcript was the only form present in lung and spleen, and it was also detected in placenta and colon. The short form corresponds to the 3' terminus of the longer one. It is formed by alternative transcription under the control of an internal promoter. Cells stably transfected with cDNAs encoding these two proteins were used for binding studies using [3H]phenamil, a potent blocker of the epithelial Na+ channel, derived from amiloride. Both the long and short forms of the protein bind amiloride and some of its derivatives, but they have distinct pharmacologies. The order of potency of the different amiloride derivatives to inhibit [3H]phenamil binding was phenamil (K0.5 = 10 nM) > benzamil (K0.5 = 43 nM) > amiloride (K0.5 = 1.4 microM) approximately ethylisopropylamiloride (K0.5 = 1.6 microM) for the long form, whereas it was phenamil (K0.5 = 68 nM) > amiloride (K0.5 = 3.2 microM) approximately ethylisopropylamiloride (K0.5 = 4 microM) approximately benzamil (K0.5 = 6.3 microM) for the short form. Although the binding proteins described here are distinct from the pore-forming protein of the epithelial Na+ channel, the pharmacological profile of the long form of the ABP is identical to that described previously in pig and human kidney, and similar to that expected for an epithelial Na+ channel. The pharmacological profile of the short form resembles that previously described for an amiloride-binding protein in pneumocytes. Results presented in this paper suggest that previously purified preparations showing Na+ channel activity contain different forms of the amiloride-binding protein, possibly associated with other proteins. The similarity between amiloride-binding proteins and a protein identified in seminal vesicles suggests that amiloride-binding proteins are the first members of a new family of epithelia-specific proteins.

Amine Oxidase (Copper-Containing)↗

PubMed1993

An epithelial high-affinity amiloride-binding site, different from the Na+ channel.

Specific binding of the radioactive amiloride analogues [3H]phenamil and [3H]benzamil was studied in plasma membrane from chicken lower intestine. A single population of sites whose affinities and specificities towards pyrazinecarboxamides roughly resemble those of the epithelial Na+ channel, was identified. However, a matched comparison of pyrazinecarboxamide binding and Na+ transport inhibition revealed substantial differences between the high-affinity [3H]phenamil-binding site detected, and the site whose occupancy by phenamil blocks Na+ transport. First, 5-(N-ethyl-N-isopropyl)-amiloride was found to displace bound [3H]phenamil at concentrations that are at least 10-fold lower than those needed to block the channel. Second, the rates at which [3H]phenamil associates and dissociates from this site are lower than the rates at which Na+ channels are inhibited and reactivated, under similar conditions. A site with high affinity to both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride was detected also in membranes from other epithelia. We conclude that tight epithelia contain a major high-affinity amiloride receptor other than the Na(+)-conducting channel, the Na+/H+ antiport or the Na+/Ca2+ exchanger. This site could be associated with a pool of nonconducting channels, another (but structurally related) channel, or a totally unrelated protein.

Aldosterone↗

PubMed1993

Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins.

A complementary DNA encoding an amiloride-sensitive Na+ channel has been cloned and characterized from rat colon. The protein encoded by the cDNA has a sequence of 699 amino acids (79 kDa) containing several putative membrane spanning domains and potential phosphorylation sites. It forms a channel that has the electrophysiological and pharmacological properties characteristic of the epithelial Na+ channel. Homologies (including in transmembrane domains) have been found between a part of the channel sequence and the Mec4 gene product of Caenorhabditis elegans, a protein associated with mutation-induced neuronal degeneration.

Amiloride↗

PubMed1993

Oncogene-mediated propagation of tracheal epithelial cells from two cystic fibrosis fetuses with different mutations. Characterization of CFT-1 and CFT-2 cells in culture.

Primary tracheal epithelial cells obtained from two fetuses with cystic fibrosis (CF) were successfully transfected with a plasmid vector recombined with the large T oncogene of SV40. The resulting tracheal cells were propagated in culture for up to 25 passages and retained the mutations of the CF genes carried by the two fetuses, one heterozygous for the S549N and N1303K substitutions (CFT-1 cells), and the other homozygous for the most common deletion delta F508 (CFT-2 cells). The transfected cells: (a) expressed the SV40 large T oncogene, as determined by immunofluorescence and Northern blot analysis; (b) retained typical epithelial morphology, as assessed by the presence of microvilli, desmosomes, gap junctions, and cytokeratin expression; (c) were fully responsive to the cAMP-stimulating agents isoproterenol, forskolin and vasoactive intestinal peptide for cAMP production and PKA activation; (d) do not produce any tumour in the athymic nude mice; (e) were diploid and tetraploid with a normal chromosomal complement at early passages, and (f) exhibited the abnormal regulation of chloride conductance characteristic of CF. These results indicate that CFT-1 and CFT-2 cells constitute a suitable model for: (a) comparison of the maturation and function of the CFTR protein mutated in the two nucleotide-binding domains; (2) analysis of the biochemical defect in CF epithelial airway cells, (c) development of new therapeutic agents, and correction of the CF defect by gene replacement therapy in vitro.

Animals↗

PubMed1991

Functional insertion of the SV40 large T oncogene in cystic fibrosis intestinal epithelium. Characterization of CFI-3 cells.

Intestinal epithelial cells were isolated from a fetus with cystic fibrosis (CF) and transfected with a plasmid vector recombined with the ori- mutant of SV40. A population of proliferative cells was then subcloned and designated as CFI-3. These cells had a doubling time of 24 h and were maintained in culture for up to 25 passages. At passage 8, CFI-3 cells did not produce any tumors in nude mice. Northern blot and immunofluorescence studies indicated that the extended lifespan of CFI-3 cells results in genomic insertion of SV40 LT. Intestinal CFI-3 cells are epithelial, according to the expression of the human cytokeratin 18 gene and poorly differentiated by phase-contrast and electron microscopy. Functional membrane receptors activated by vasoactive intestinal peptide (VIP), its natural analogue pituitary adenylate cyclase activating peptide (PACAP-38), and isoproterenol were observed in CFI-3 cells. Restriction fragment length polymorphism analysis of the PstI KM19 site revealed that the cftr locus was identical in the chorionic villi and in CFI-3 cells. The manifestation of CF in this family was not related to the common mutation delta F508, since this fetus was heterozygous for the substitutions S549N and N1303K. Chloride transport, assessed by the 125I efflux, was induced in CFI-3 cells by the calcium inophore ionomycin, but not by the adenylate cyclase activator forskolin, and was inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid. These results were confirmed in patch clamp studies in which the cpt cAMP analogue failed to stimulate membrane currents, while the calcium ionophore ionomycin stimulated inward currents. We conclude that intestinal CFI-3 cells retain the CF phenotype relating to defective regulation of Cl- channels, and therefore constitute a suitable model, 1) for elucidating the function of CFTR protein, 2) developing new therapeutic agents, and 3) correcting the CF defect by gene replacement therapy in vitro.

Antigens, Polyomavirus Transforming↗