Biomedical subjects

J Bourguet

Publications and source records attributed to J Bourguet.

At least 19 recordsLinked to original sources

PubMed1995

Water pathways across a reconstituted epithelial barrier formed by Caco-2 cells: effects of medium hypertonicity.

Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (Jw), mannitol permeability (Ps), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mM polyethylene glycol-PEG), the spontaneously observed secretory Jw rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and Ps went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and "omega" figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Aquaporin-CHIP-related protein in frog urinary bladder: localization by confocal microscopy.

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.

Animals↗

PubMed1994

Antidiuretic response in the urinary bladder of Xenopus laevis: presence of typical aggrephores and apical aggregates.

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydro-osmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the antidiuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearance of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Activation of the vasopressin-sensitive water permeability pathway in the toad bladder by N-ethyl maleimide.

Vasopressin stimulates transepithelial water flow in the toad urinary bladder. We report here that N-ethyl maleimide (NEM) (0.1 mM) produces a similar increase in osmotic water flow when applied to the mucosal surface of the tissue. NEM-induced water flow is sensitive to inhibitors of hormone-induced water flow, including serosal acidification, or exposure to quinidine or cytoskeleton-disruptive drugs. NEM-induced water flow is additive with that induced by a submaximal, but not a maximal, dose of vasopressin. The response to mucosal NEM is not reversed on removal of the reagent, but established NEM-induced water flow can be inhibited by serosal acidification or quinidine. Like vasopressin, mucosal NEM induces the appearance of fusion profiles and intramembranous particle aggregates (putative water channels) in the apical plasma membrane of the granular cells, and the incidence of particle aggregates correlates with water flow. NEM does not cause an increase in intracellular cAMP. Our data suggest that NEM stimulates transepithelial water flow by irreversibly activating cellular mechanisms normally triggered by vasopressin, hence causing the insertion of water channels.

Animals↗

PubMed1993

Water handling in Caco-2 cells: effects of acidification of the medium.

Caco-2 cells were cultured on permeable supports. At confluence the minute-by-minute net water movement (Jw) was automatically recorded. Simultaneously, unidirectional [14C]mannitol, 22Na+, and/or 36Cl- fluxes and transepithelial resistances were measured. The water and mannitol permeabilities went progressively down between 9 and 16 days after seeding and then stabilized. In this last condition the hydrostatic permeability coefficient (Phydr) was 2.67 +/- 0.31 cm s-1 while the osmotic permeability coefficient (Posm) was 0.0017 +/- 0.0004 cm s-1. Phydr but not Posm was dependent on the temperature and on the presence of Na+ in the medium. A net secretory Jw was observed 16 days after seeding, in the absence of any osmotic, hydrostatic or chemical gradient. This secretory Jw was associated with net Cl- (1.43 +/- 0.43 muequiv h-1 cm-2) and Na+ (1.05 +/- 0.35 muequiv h-1 cm-2) secretions. Amiloride reduced, in open-circuit conditions, both Na+ and Cl- apical to basal fluxes, thus enhancing the net Na+ and Cl- exit. Acidification of the medium (pH 6.2) reversibly increased water and mannitol permeabilities in 10-day-old cultures. In 16-day-old cultures the same shift in medium pH did not change mannitol permeability, while stimulating water secretion. These results, obtained in the absence of supracellular structures (villae, crypts) and subepithelial components (muscular, vascular and conjunctive tissues) indicate that paracellular and transport-associated water pathways are sensitive to changes in the pH of the medium in Caco-2 cell layers.

Adenocarcinoma↗

PubMed1992

Effects of salt acclimation on water and urea permeabilities across the frog bladder: relationship with intramembrane particle aggregates.

1. In salt-acclimated frogs, water and urea bladder permeabilities are markedly higher than in tap water-acclimated animals. 2. Intra-membrane particle aggregates (IMPA) cover an unusually large surface area of the salt-acclimated frog bladder apical plasma membrane. 3. In saline-adapted animals, proteins extracted from the apical plasma membrane contain additional species of 19, 26, 31 and 53-61 kDa. These proteins might be related to the water channels contained by IMPA.

Adaptation, Physiological↗

PubMed1992

Selected polyclonal antibodies and ADH challenge in frog urinary bladder: a label-fracture study.

It is clearly established that the changes induced by antidiuretic hormone (ADH) in its target epithelial cells result from the insertion in the apical membrane of new components that contain channels for water. We have already undertaken an initial study of these channels by raising polyclonal antibodies against Triton X-100 apical extracts from ADH-treated bladders and approached their purification by different adsorption steps. In the present study, we used the label-fracture technique to investigate the localization of the binding sites of the obtained polyclonal antibodies on the apical membrane of ADH-stimulated frog urinary bladder. The results obtained clearly demonstrated a preferential labeling by the selected antibodies of morphological structures such as the groove arrays and the fusion images that are generally accepted as being involved in ADH-induced changes in water permeability of the apical membrane.

Adsorption↗

PubMed1991

Vesicles and particles of sodium bis(2-ethylhexyl) phosphate in binary and ternary systems.

Vesicles were identified in aqueous solution of pure sodium bis(2-ethylhexyl) phosphate, a short branched chain surfactant. Superficial tension measurements show that the vesicles appear above a molality of 0.02 (0.69 %w). These aggregates are equilibrium structures. The "packing parameter' theory established by Israelachvili et al. allows the prediction of the occurrence of such vesicles. If an organic solvent, such as xylene or ethylhexanoate, is added to the binary system, a different type of aggregate appears, the size of which is determined by several methods including electron microscopy and light scattering. Interfacial tension measurements show that these aggregates would be expected to form above a molality of 0.02. According to our experimental results, the microstructure of these aggregates can be described as micelles and/or vesicles, swollen or not.

Nephelometry and Turbidimetry↗

PubMed1991

ADH-induced water permeability and particle aggregates: alteration by a synthetic estrogen.

In the amphibian urinary bladder, the increase in water permeability induced by antidiuretic hormone (ADH) is accompanied by the appearance of apical intramembrane particle (IMP) aggregates that are believed to contain specific channels for water. In a previous work, we have shown that 3,3'-diallyldiethylstilbestrol (DADES), a synthetic estrogen which is a blocker of the glucose transporter, also inhibits the hydrosmotic response to ADH in the bladder. Our aim in the present study was to analyze the alterations of the membrane fine structure further and to correlate them with the water permeability changes. The results point to a selective inhibition of the ADH-induced net water flow, probably due to an interference with one of the last steps of the response to the hormone. This inhibition is associated with an increase in the density of the apical IMP aggregates, which are thus probably not operational. The resting net water flow is not inhibited and, surprisingly, typical IMP aggregates are frequently observed in the apical membrane after DADES treatment. The compound also induces the appearance of unusual loose IMP clusters that can only be seen on the apical membrane of the granular cells and that share several ultrastructural similarities with the ADH-induced aggregates. These results suggest that 1) apical DADES treatment stimulates the insertion of IMP aggregates in the apical membrane of the urinary bladder and 2) DADES inhibits the ADH-induced water flow by interfering with the aggregates and thus probably by blocking the specific water channels.

Animals↗

PubMed1991

Antidiuretic response: what markers for water channel components?

Antidiuretic hormone increases the water permeability of its target epithelial tissues by triggering the insertion into the apical cell membrane of aggregated intramembrane particles that contain channels specific for water. Little is known about the chemical composition of these membrane particles and of the water channel components. Present work describes a procedure for obtaining selected antibodies that specifically recognize ADH-induced components of the apical membrane in the amphibian urinary bladder epithelial cells.

Animals↗

PubMed1990

Effects of PCMBS on the water and small solute permeabilities in frog urinary bladder.

UNLABELLED: It has been reported that PCMBS (p-chloromercuribenzene sulfonate) blocks the water permeability of red cells and of the tubular kidney membranes. In this study we compare the effects of this mercurial compound on the permeability of water and other small solutes in the frog urinary bladder. We observed that: (i) 5 mM PCMBS applied at pH 5.0 to the mucosal side inhibited the net and unidirectional water fluxes induced by oxytocin without changing the delta Pf/delta Pd ratio. (ii) The oxytocin-induced urea and Na+ influxes were also inhibited by PCMBS. (iii) The unidirectional Cl- movement was first reduced and then increased during the course of PCMBS treatment. (iv) The short-circuit measured at low mucosal Na+ concentration (10 mM), diminished continuously, whereas the transepithelial resistance first increased and then diminished. (v) Mannitol, raffinose, alpha-methyl-glucose, antipyrine, caffeine and Rb+ movements were not changed significantly during the first 26 min of the water permeability inhibition. IN CONCLUSION: (i) The ADH-sensitive water, urea and Na+ transport systems were inhibited by PCMBS, (ii) PCMBS did not induce a nonspecific and general effect on the permeability of the membrane during the development of the water permeability inhibition, and (iii) in terms of water channels, the inhibition of water transport with the maintenance of a high Pf/Pd ratio suggests that PCMBS closes the water channels in an all or none manner, reducing their operative number in the apical border of frog bladder.

4-Chloromercuribenzenesulfonate↗

PubMed1990

[Exchanges of medical students from Rennes/Erlangen-Nuremberg: various considerations on these exchanges within the E.E.C. framework].

The current political trends of the European Economic Community (EEC) have made it more desirable than ever before for medical students to be able carry out part of their training in medical schools other than their Faculties of origin. The authors examine successively: 1) The results of 20 years of twinning-between the Medical Schools of Rennes (France) and Nürnberg-Erlangen (W. Germany). A considerable number of syllabuses and examinations are now recognized as equivalent. So far, nearly 500 students have taken advantage of the possibilities of exchanges lasting a few months or even a full University year. 2) The propositions submitted by the Medical Schools of Rennes and Strasbourg have been selected from the 800 presented by EEC Universities as port of the Erasmus programme. For the whole of the EEC, 12 medical projects have been started up since october 1989. Among the other projects accepted were those of Bonn and Hamburg, Ovideo and Valence, Naples and Barri, Dublin, Coïmbra, Brussels, Copenhagen, Leyden, etc. 3) In order to strengthen the North-South axis of the EEC and to widen further the horizons of European medical students (even beyond Europe), the Medical Facilities of Rennes and of Alcalà de Henares (University of Madrid) have just defined syllabus and examination equivalences as Rennes and Erlangen did some twenty years ago.

Education, Medical↗

PubMed1989

Effect of mercurial compounds on net water transport and intramembrane particle aggregates in ADH-treated frog urinary bladder.

It has been suggested that during the oxytocin-induced hydrosmotic response, water crosses the luminal membrane of urinary bladder epithelium cells through membrane-spanning proteins. Although specific inhibitors of osmotic water transport have not been found, certain sulfhydryl reagents such as mercurial compounds may help to identify the proteins involved in this permeation process. We tested the effects of p-chloromercuribenzene sulfonate (PCMBS) and of fluorescein-mercuric acetate (FMA) on the net water flux, the microtubule and microfilament structures of the frog urinary bladder, and the distribution of intramembrane particle aggregates in the luminal membrane. We observed that: (i) 5 mM PCMBS at pH 5 and 0.5 mM FMA at pH 8 added to the mucosal bath at the maximum of the response to oxytocin partially inhibited the net water flux. Inhibition then increased progressively when the preparation was repeatedly or continuously stimulated, until it reached a maximal inhibition at 120 min. This inhibition was not reversed even when cystein was added in the mucosal bath. PCMBS and FMA effects were also observed when cyclic AMP (3',5' cyclic adenosine monophosphate) was used to increase water permeability, (ii) PCMBS mucosal pretreatment did not modify the basal water flux but potentiated the inhibitory effect of PCMBS or FMA on the hydrosmotic response to oxytocin. (iii) Microtubule and microfilament network, visualized in target cells by immunofluorescence, was not affected by PCMBS. (iv) The maximal PCMBS or FMA inhibition was not associated with a reduction of aggregate surface area in the apical membrane. The persistence of the intramembrane particle aggregates associated with the oxytocin-induced hydrosmotic response during the net water flux inhibition by PCMBS, suggests that the PCMBS effect occurs possibly at the level of sulfhydryl groups of the water channel itself.

4-Chloromercuribenzenesulfonate↗

PubMed1989

Inhibition of the hydrosmotic response to antidiuretic hormone by 3,3'-diallyldiethylstilbestrol (DADES).

3,3'-diallyldiethylstilbestrol (DADES), a blocker of the facilitated diffusion of glucose, was found to interfere markedly with the hydrosmotic response to antidiuretic hormone and its related agonists. Frog urinary bladders were isolated and monitored for transmural net water flow. DADES was added either to the serosal or to the apical medium at concentrations ranging from 10(-4) M to 10(-6) M. Pretreatment for 30 min with apical 10(-4) M DADES drastically reduced the subsequent hydrosmotic response: (a) to oxytocin (4.4 x 10(-8) M) by 91.7 +/- 17.6% versus 6.2 +/- 7.8 in control; (b) to 8-bromo 3',5'-cyclic AMP by 93.5 +/- 19.4% versus 19.4 +/- 11.4%; (c) to serosal hyperosmolarity (mannitol 220 mOsm) by 99.3 +/- 0.5% versus 12.3 +/- 18.2%. This effect was dose-dependent. Inhibitory action of DADES was more effective on the apical side than on the serosal side (97.0 +/- 1.5 versus 45.8 +/- 10.8). Freeze-fracture studies revealed a modified distribution of the particles and unusual endocytotic pits and vesicles in the apical membrane of both granular and mitochondria-rich epithelial cells. These observations point to multiple and complex effects of the drug. Thus, it seems that DADES has numerous effects on urinary epithelium, which makes it a nonspecific inhibitor of water permeation. Conclusions on its use should therefore be drawn with suitable caution.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Wheat germ agglutinin (WGA) reduces ADH-induced water flow and induces cell surface changes in epithelial cells of frog urinary bladder.

The functional and structural changes induced by apical wheat germ agglutinin (WGA) 100 micrograms/ml exposure on frog urinary bladder have been investigated and the possible correlations between these effects discussed. Bladders, apically exposed to WGA for 30 min to 3 hr exhibit a marked reduction of their response to antidiuretic hormone (ADH) challenge and of their hydrosmotic reactivity. Structural changes triggered by WGA treatment are: 1. apical invaginations of the plasma membrane, interpreted as endocytotic in nature, taking into account the results of carbohydrate cytochemical detection and horseradish peroxidase (HRP) exposure: 2. cytoskeleton disorganization and microvilli collapse. These phenomena do not interfere with cortical granule traffic and are independent of ADH challenge: they occur in ADH-stimulated bladders as well as in bladders at rest. These findings could be interpreted as follows: binding of the divalent lectin WGA to its coat specific receptors would induce changes in the apical membrane structure which in turn could provoke disorganization and disruption of apical cytoskeletal elements associated with plasma membrane. Reduction of bladder response to ADH challenge could result from a reduced recycling of aggrephores, as they are associated with cytoskeletal elements in the subapical cytoplasm. Collapse of microvilli and endocytotic events also could result from apical cytoskeleton disruption, as microvilli are sustained by bundles of actin filaments interconnected with apical cytoskeletal filaments and as plasma membrane is associated with apical cytoskeleton. However, these two last events evidently occur in ADH-challenged or non-challenged bladders.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Microtubules and actin microfilaments in the amphibian bladder granular cells.

Microtubules and microfilaments were localized by an immunocytochemical method in the granular cells of the frog bladder after fixation and isolation. An extensive array of microtubules was observed in the granular cells with an orientation towards the luminal plasma membrane in the supranuclear zone. Actin filaments formed a continuous bundle that underlined the cellular membrane. After incubation in the presence of colchicine, nocodazole, or tubulozole, the microtubular network appeared fragmented but did not disappear completely. These observations are related to the role of the cytoskeleton in the permeability response of the frog bladder epithelium to vasopressin.

Actin Cytoskeleton↗

PubMed1989

Isolation of large sheets of apical material from frog urinary bladder epithelial cells by freeze-fracture.

In the amphibian urinary bladder, water permeability is correlated with the insertion of intramembrane particle aggregates (IMPAs) into the apical plasma membrane (AM) of the granular cells. These aggregates are believed to contain water channels. Characterization of the IMPAs by comparing AM fractions of antidiuretic hormone (ADH)-treated and resting epithelia requires isolation and purification of AM-rich material, free of other cytoplasmic aggregate-containing organelles, in both cases. A technique derived from freeze-fracture was chosen to isolate large sheets of apical membrane material from frog (Rana esculenta) urinary bladder epithelium. The apical side was plated on a polylysine-coated glass slide, frozen with liquid nitrogen, and fractured. A nylon mesh was inserted between the glass slide and the bladder, in order to bring the fracture plane back to the AM periodically. Fluorescent markers were used to characterize the material having fractured with the glass slide. Samples were observed by fluorescence and phase contrast microscopy. We obtained evidence that numerous patches of fractured AM remained on the glass surface without nuclei. A phase contrast picture was obtained only at a high magnification, indicating a low thickness of the recovered material. Further characterization was made with SDS-PAGE. Protein contents of samples were extracted under various experimental conditions and the patterns of ADH-treated, resting AM samples, or whole epithelial cell crude homogenates, were compared. Staining of some bands increased under certain conditions, whereas many others disappeared. Both morphological and biochemical approaches demonstrate that the recovered material was apical in origin.

Animals↗

PubMed1989

A method of isolation of apical membranous sheets from frog urinary bladder epithelium by stripping with gelatin.

We have developed a technique for recovering apical membranous sheets from amphibian urinary bladders by gelatin stripping. The tissue is mounted on a lucite support and the apical surface is first stuck onto a gelatin-coated glass slide at 30 degrees C. This sandwich is then chilled on ice and the bladder is pulled away from the slide. Preliminary results indicate that this simple technique could be used to remove membranous apical sheets of various sizes, almost devoid of cytoplasmic contamination and without significant damage to the underlying cell structures. The method could also be adapted to prepare perforated cells and to study the cohesive forces between the different layers of the tissue.

Animals↗