RESUMO

This work studies water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, applying a technique where cellular content is replaced with a known medium, with the possibility of measuring intracellular pressure. Consequences on water transport-produced by well-known anisotonic gradients and by the intracellular effect of probable aquaporin inhibitors-were tested. In this way, the specific intracellular inhibition of hAQP1 by the diuretic drug furosemide was demonstrated. In addition, experiments imposing anisotonic mannitol gradients with a constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response when the gradient is higher than 150 mosmol kg (W) (-1) . These results would indicate that the passage of water molecules through hAQP1 may have a maximum rate. As a whole, this work demonstrates the technical advantage of controlling both intracellular pressure and medium composition in order to study biophysical properties of hAQP1, and contributes information on water channel behavior under osmotic challenges and the discovery of new inhibitors.

Assuntos

RESUMO

Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

Assuntos

RESUMO

This review focuses on studies of water movement across biological membranes performed over the last 50 years. Different scientific approaches had tried to elucidate such intriguing mechanism, from hypotheses emphasizing the role of the lipid bilayer to the cloning of aquaporins, the ubiquitous proteins described as specific water channels. Pioneering and clarifying biophysical work are reviewed beside results obtained with the help of recent sophisticated techniques, to conclude that great advances in the subject live together with old questions without definitive answers.

RESUMO

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

Assuntos

RESUMO

The Cl(-)/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pH(i) was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pH(i) to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pH(i) became alkaline and was not regulated, after 48 h adaptation; steady-state pH(i) reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl(-)/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pH(i) and that AE2 would play a key role in cell homeostasis.

Assuntos

RESUMO

BACKGROUND INFORMATION: The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS: In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS: These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.

Assuntos

RESUMO

BACKGROUND INFORMATION: Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. RESULTS: An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 microm x s(-1)), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl(2) (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. CONCLUSIONS: Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl(2), not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.

Assuntos

RESUMO

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (deltaOsm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 ñ 0.049 cm/s vs. 0.156 ñ 0.022 cm/s and 0.0012 ñ 0.0001 cm/s vs. 0.0019 ñ O.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 mug/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48 per cent decrease in the absorptive Jwt values. (AU)

Assuntos

RESUMO

The net absorptive water flux (Jw), the transepithelial potential difference (PD) and the short-circuit current (Isc) were simultaneously measured in the human small intestine in vitro with the following results: 1) An absorptive Jw was observed when the jejunum or the ileum were mounted between two identical standard solutions in the presence of an hydrostatic pressure gradient (delta P) of 13 cm of water (mucosal side positive). 2) The absorptive Jw was a linear function of the applied delta P or the imposed osmotic transepithelial gradient (deltaOsm) in both intestinal segments. The hydrostatic (Phydr) and osmotic (Posm) permeabilities to water for jejunum and ileum were: 0.349 ñ 0.049 cm/s vs. 0.156 ñ 0.022 cm/s and 0.0012 ñ 0.0001 cm/s vs. 0.0019 ñ O.0003, respectively. 3) A fraction of this absorptive Jw was independent of the presence of any hydrostatic, osmotic or chemical gradient and represented the transport associated to movement of water (Jwt). 4) PD and Isc values were similar in the jejunum and in the ileum but the transepithelial resistance (Rt) was significantly greater in ileum than in jejunum. 5) 2 mug/ml of E. coli heat-stable enterotoxin (STa) caused a significant inhibition of the absorptive Jw without modification of Phydr, Posm or Isc. 6) After STa treatment, the absorptive Jwt reverted to a secretory one in the jejunum. In the ileum, STa action caused a 48 per cent decrease in the absorptive Jwt values.

Assuntos

RESUMO

El trabajo describe la metodología necesaria para obtener, a partir de la vejiga urinaria de la rana, capas de células aisladas apropiadas para estudios por "patch-clamp". Este resultado se logró combinando presión negativa, digestión enzimática y microdisección. Se obtuvieron registros de canales únicos en las membranas basolaterales, en una situación en la que el epitelio mantenía su polaridad y estructura general

Assuntos

RESUMO

El trabajo describe la metodología necesaria para obtener, a partir de la vejiga urinaria de la rana, capas de células aisladas apropiadas para estudios por "patch-clamp". Este resultado se logró combinando presión negativa, digestión enzimática y microdisección. Se obtuvieron registros de canales únicos en las membranas basolaterales, en una situación en la que el epitelio mantenía su polaridad y estructura general (AU)

Assuntos

Assuntos

Assuntos