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M Svelto

Publications and source records attributed to M Svelto.

At least 37 records · Page 2Linked to original sources
PubMed2001

Role of aquaporin-4 water channel in the development and integrity of the blood-brain barrier.

In this study, we have investigated the expression of aquaporin 4 during blood-brain barrier development in the optic tectum of chick embryos and newly hatched chicks, by means of western-blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and freeze-fracture and high-resolution immunogold electron microscopy. In the optic tecta of day-14 embryos, western blot analysis revealed an approx. 30 kDa band, immunoreactive for aquaporin-4, which was increased in day-20 embryos and in chicks. Semi-quantitative reverse transcriptase chain reaction experiments showed that there was already a high level of aquaporin-4 mRNA in day-9 embryos as well as in the subsequent stages and in newly hatched chicks. Immunohistochemically, reactivity for aquaporin-4 was detected in the optic tectum of day-14 embryos; similar results were obtained in telencephalon and cerebellum. Ultrastructurally, the microvessels of the tectum showed immunoreactivity for aquaporin-4 on the astroglial endfeet, which discontinuously surrounded endothelial cells joined by immature tight junctions. In the tectum, telencephalon and cerebellum of 20-day embryos and chicks, aquaporin-4 strongly labeled the ependymal cells and the subpial glial membranes, as well as the bodies and processes of astroglial cells. A continuous aquaporin-4 staining was found around the microvessel endothelial cells, which were sealed off from one another by extensive tight junctions. A complete astrocytic sheath, labeled by anti-aquaporin-4 gold particles, enveloped the endothelium-pericyte layer. Orthogonal arrays of particles were observed on fractured astrocytic membranes, starting from embryonic day 14 when the aquaporin-4 immunogold staining revealed clusters of gold particles, often forming square or rectangular clusters. The results showed that aquaporin-4 expression and organization of the intramembrane particles in orthogonal arrays followed the same temporal sequence. Finally, the lipopolysaccharide, a substance that induces blood-brain barrier disruption, determines a remarkable reduction in aquaporin-4 labeling, expressed by a few aquaporin-4 gold particles attached on swollen perivascular glial membranes. All these data show that aquaporin-4 expression occurs in the chick embryonic brain, in parallel with maturation and functioning of the blood-brain barrier and suggest that there is a close relationship between water transport regulation and brain development.

Animals↗
PubMed2000

Aquaporin-4-containing astrocytes sustain a temperature- and mercury-insensitive swelling in vitro.

In order to understand the molecular mechanism underlying astroglial swelling, we studied primary astrocyte cultures from newborn mouse and analyzed them for expression of functional water channels. Immunocytochemical analysis of mouse brain confirms the presence of AQP4 location in astrocytic endfeet with a polarized pattern, as found in rat. Using Southern blot PCR and Western blot analysis, we demonstrate that primary astrocyte cultures from mouse express the AQP4 water channel at both the RNA and protein levels. Two polypeptides, of 30 kDa and 32 kDa, were identified in the astrocytes. Densitometric analysis demonstrates that the 32-kDa form represents 25% of the total AQP4 protein. Moreover, immunofluorescence experiments show strong surface membrane expression of AQP4 protein in cultured cells, even though the polarity of the expression is not maintained. Furthermore, functional studies indicate that cultured astrocytes manifest rapid and temperature-independent volume changes in response to osmotic gradients, in agreement with a channel-mediated water transport. Water movement was found to be HgCl(2) insensitive, suggesting AQP4 and AQP7 as putative water channels. Using Western blot and PCR experiments, we exclude the presence of AQP7 in astrocytes, indicating that only AQP4 is responsible for the rapid water movement. Altogether, the results indicate that primary astrocyte cultures are a valid cell model for further investigation of the molecular mechanism of water movement in the brain and its physiological regulation.

Animals↗
PubMed2000

The cultured human gastric cells HGT-1 express the principal transporters involved in acid secretion.

HGT-1 is a human cell line sharing a number of physiological features with gastric parietal cells. HGT-1 cell monolayers were able to secrete H+ when stimulated with histamine (calculated external pH variation, deltapH(e) 0.46+/-0.05) as assessed using the impermeant, pH-sensitive fluorescence dye 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt (HPTS). Treatment with 100 microM omeprazole inhibited the histamine-induced apical acidification by about 60%. Intracellular pH (pH(i)) measurements using the fluorescent pH-sensitive dye 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) demonstrated the expression of a functional, omeprazole-sensitive H+/K+-pump. A monoclonal antibody directed against the alpha subunit of the H+/K+-ATPase immunoprecipitated a 95-kDa protein from HGT-1 cells and human stomach which corresponds to the expected molecular size of the native protein. HGT-1 cells were also positive for the anion exchanger AE2 that is expressed in gastric parietal cells. In addition, we identified a histamine- and pH(i)-sensitive Na+/H+ exchanger in HGT-1 cells, which might correspond to the functional expression of the NHE4 isoform that has been detected in gastric epithelial cells as well as in primary cultured parietal cells. HGT-1 cells therefore display the principal features of parietal cells and might represent an interesting cell culture model for studying the regulatory mechanisms involved in acid secretion.

1-Methyl-3-isobutylxanthine↗
PubMed2000

The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.

Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 microM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 microM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

Actins↗
PubMed1999

Visualization of AqpZ-mediated water permeability in Escherichia coli by cryoelectron microscopy.

Transport of water across the plasma membrane is a fundamental process occurring in all living organisms. In bacteria, osmotic movement of water across the cytoplasmic membrane is needed to maintain cellular turgor; however, the molecular mechanisms of this process are poorly defined. Involvement of aquaporin water channels in bacterial water permeability was suggested by the recent discovery of the aquaporin gene, aqpZ, in Escherichia coli. By employing cryoelectron microscopy to compare E. coli cells containing (AqpZ+) and lacking (AqpZ-) aquaporin, we show that the AqpZ water channel rapidly mediates large water fluxes in response to sudden changes in extracellular osmolarity. These findings (i) demonstrate for the first time functional expression of a prokaryotic water channel, (ii) evidence the bidirectional water channel feature of AqpZ, (iii) document a role for AqpZ in bacterial osmoregulation, and (iv) define a suitable model for studying the physiology of prokaryotic water transport.

Aquaporins↗
PubMed1999

Cloning, structural organization and chromosomal localization of the mouse aquaporin-8 water channel gene (Aqp8).

The gene encoding the mouse Aquaporin-8 water channel protein (Aqp8) was cloned and its genomic structure was defined. Aqp8 consists of six exons with boundaries at amino acids 1-4, 5-87, 88-129, 130-201, 202-246 and 247-261 which partially correspond to those of other known aquaporin genes. All splice sites conform to the GT-AG rule except the first one which is GT-GG. Primer extension and RNase protection analyses using mouse liver RNA demonstrated three initiation transcription sites located 385, 156 and 146 bp upstream from the translational start codon. No defined TATA box was found in the 5'-flanking region where numerous CAAT motifs and one GATA box were identified. Fluorescence in situ hybridization localized the Aqp8 locus to mouse chromosome 7F3. The 7F region is syntenic with human chromosomes 11, 16 and 10. These results (i) reveal marked structural distinction between the Aqp8 gene and the other known mammalian aquaporin genes, (ii) may now permit the molecular characterization of Aqp8 expression and (iii) represent a fundamental step for the construction of a target vector to generate transgenic Aqp8 knockout mice.

5' Untranslated Regions↗
PubMed1998

A heterotrimeric G protein of the Gi family is required for cAMP-triggered trafficking of aquaporin 2 in kidney epithelial cells.

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells.

Amino Acid Sequence↗
PubMed1998

Expression of aquaporin-4 in fast-twitch fibers of mammalian skeletal muscle.

In this study we analyzed the expression of aquaporin-4 (AQP4) in mammalian skeletal muscle. Immunohistochemical experiments revealed that affinity-purified AQP4 antibodies stained selectively the sarcolemma of fast-twitch fibers. By immunogold electron microscopy, little or no intracellular labeling was detected. Western blot analysis showed the presence of two immunopositive bands with apparent molecular masses of 30 and 32 kD specifically present in membrane fraction of a fast-twitch rat skeletal muscle (extensor digitorum longus, EDL) and not revealed in a slow-twitch muscle (soleus). PCR Southern blot experiments resulted in a selective amplification in EDL of a 960-bp cDNA fragment encoding for the full-length rat form of AQP4. Functional experiments carried out on isolated skeletal muscle bundle fibers demonstrated that the osmotic response is faster in EDL than in soleus fibers isolated from the same rat. These results provide for the first time evidence for the expression of an aquaporin in skeletal muscle correlated to a specific fiber-type metabolism. Furthermore, we have analyzed AQP4 expression in skeletal muscle of mdx mice in which a decreased density of orthogonal arrays of particles, a typical morphological feature of AQP4, has been reported. Immunofluorescence experiments showed a marked reduction of AQP4 expression suggesting a critical role in the membrane alteration of Duchenne muscular dystrophy.

Animals↗
PubMed1996

Expression and functional analysis of water channels in a stably AQP2-transfected human collecting duct cell line.

In this study, we describe the establishment of a stably transfected epithelial cell line with the cDNA for the rat aquaporin 2 (AQP2). To this end, we used a human cell line (HCD) derived from the cortical collecting duct and having characteristics of principal cells (Prié, D., Friedlander, G., Coureau, C., Vandewalle, A., Cassigena, R., and Ronco, P. M. (1995) Kidney Int. 47, 1310-1318). The HCD cells were first screened for the constitutive expression of AQPs. By Western blot analysis, we found a low expression of immunoreactive AQP2 and AQP4 proteins. In contrast, transfected cells (clone CD8) probed with AQP2 antiserum expressed an intense 29-kDa protein on immunoblot in addition to a broad band between 35-45 kDa corresponding to the glycosylated form of the protein, indicating that full maturity of the protein is attained in transfected cells. Immunofluorescence demonstrated that AQP2 was located in intracellular vesicles. After vasopressin stimulation, the staining redistributed from an intracellular site to the apical pole of the cells, an effect similar to that described on collecting duct principal cells in vivo (Sabolic, I., Katsura, T., Verbavatz, J. M., and Brown, D. (1995) J. Membr. Biol. 143, 165-175) and in perfused tubules (Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K., and Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1013-1017). The redistribution of AQP2 in CD8 cells was accompanied by an approximately 6-fold increase in osmotic water permeability coefficient (Pf), which was inhibited by 0.3 m HgCl2. These data indicate that functional vasopressin-sensitive water channels are expressed in transfected cells. The stably transfected cells represent a suitable model to unravel by direct experimental approach the intracellular signals involved in the translocation of AQP2 to the apical plasma membrane in the presence of vasopressin.

Amino Acid Sequence↗
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

Presence in frog urinary bladder of proteins immunologically related to the aquaporin-CHIP.

Aquaporin-CHIP, a 28 kDa channel forming protein already referred to as CHIP28, has been identified as the water channel in red blood cells as well as in mammalian renal tubule cells. Another member of the aquaporin family, WCH-CD, has been found in the apical membrane of collecting duct principal cells and may represent the ADH-sensitive water channel. The present study investigates the possible presence of CHIP28-like proteins in amphibian urinary bladder, where the presence of water channels has been postulated. For this purpose, we raised polyclonal antibodies against human erythrocyte CHIP28. Immune serum precipitated a protein of about 30 kDa from the whole homogenate of urinary epithelial cells. By Western blotting, in addition to the reaction with the 30 kDa component, the immune serum reacted with higher molecular weight components from the bladder homogenate. The 30 kDa band was detected by Western blot only in bladders having a high water permeability. Moreover, a 30 kDa protein was also recognized in frog red blood cell membranes by the anti-CHIP28 antibodies. In line with the immunoblotting studies, in immunohistofluorescence anti-CHIP28 antibodies stained frog red blood cells and urinary bladder epithelial cells. However, in whole tissue water permeability studies apical treatment with the anti-CHIP28 antibodies had no effect on either the hydrosmotic response to ADH or on the basal net water flow of the bladder. All together, these results indicate the presence in the frog red blood cells and urinary epithelium of proteins sharing immunological analogies with aquaporin-CHIP.

Animals↗
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

Polyclonal antibodies in study of ADH-induced water channels in frog urinary bladder.

It is now generally accepted that changes in water permeability in anti-diuretic hormone (ADH)-responsive target epithelial cells result from the insertion in the plasma apical membrane of new components that contain channels for water. The specificity of these channels suggests that they are formed by intrinsic proteins having access to both facies and spanning the whole membrane. We have previously shown that Triton X-100 apical extracts from ADH-stimulated frog urinary bladder contain some proteins inserted under hormonal stimulation. In the present study we have developed polyclonal antibodies using Triton X-100 extract as an immunogen. After considering the inhibitory effect exerted by the whole immune serum on the osmotic water flow, we used different adsorption steps to select, from the immune serum, antibodies to apical membrane proteins inserted in response to the hormone. Immunoblot analysis of these selected antibodies shows that they recognize seven to eight proteins, of which 55-, 35-, 26-, and 17-kDa proteins are always present. Antibodies to these four proteins, affinity purified on nitrocellulose sheets, inhibited ADH-induced osmotic water flow. Altogether these results strongly suggest that proteins of 55, 35, 26, and 17 kDa (or at least one of them) are likely to be involved in the mechanism of water transport.

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↗
PubMed1989

Fluorescence labeling of proteins related to ADH-induced change in frog bladder luminal membrane.

Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5'-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). We observed that: 1) EMA (1 mM) mucosal pretreatment did not modify either the basal water flux or the subsequent ADH-induced hydrosmotic response; 2) EMA added to the mucosal bath at the maximum response to ADH, significantly decreased net water flux by about 40%; similar results were obtained when 10(-5) M forskolin was used as a hydrosmotic agent. These results suggest that the inhibitory effect of EMA occurs at a post cAMP step, possibly at the level of the sulfhydryl groups of the water channels themselves. Fluorescence distribution in SDS-PAGE of Triton X-100 extracted proteins from bladder labeled with EMA in both control conditions and under ADH stimulation allowed us to identify apical membrane proteins, labeled during ADH stimulation and not labeled in water impermeable controls. Of particular importance are four proteins of 52, 32-35, 26, 17, kDa. These polypeptides are probably involved in ADH-stimulated water transport and may be components of the water channels.

Animals↗
PubMed1989

Isolation of frog urinary bladder plasma membranes with polycation coated beads.

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels. In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e. sucrose-MES buffer). By this procedure plasma membranes were obtained from both cAMP-stimulated cells and control cells. Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ ATPase; Na+, K+ ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low. Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP-treated and control samples.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

ADH-induced water permeability: what role for the microtubular network?

1. ADH-induced intramembrane particle aggregates in the apical membrane of the epithelial cells are specifically related to water permeability in the epithelium. 2. Colchicine and nocadozole (both of which bind to tubulin) inhibit ADH-induced osmotic water flow in the amphibian bladder. 3. Microtubules may be involved in the translocation of the aggrephores prior to their insertion into the plasma membrane.

Animals↗