Subcellular Redistribution of Root Aquaporins Induced by Hydrogen Peroxide.

Aquaporins are water channel proteins that mediate the fine-tuning of cell membrane water permeability during development or in response to environmental stresses. The present work focuses on the oxidative stress-induced redistribution of plasma membrane intrinsic protein (PIP) aquaporins from the plasma membrane (PM) to intracellular membranes. This process was investigated in the Arabidopsis root. Sucrose density gradient centrifugation showed that exposure of roots to 0.5 mM H2O2 induces significant depletion in PM fractions of several abundant PIP homologs after 15 min. Analyses by single-particle tracking and fluorescence correlative spectroscopy showed that, in the PM of epidermal cells, H2O2 treatment induces an increase in lateral motion and a reduction in the density of a fluorescently tagged form of the prototypal AtPIP2;1 isoform, respectively. Co-expression analyses of AtPIP2;1 with endomembrane markers revealed that H2O2 triggers AtPIP2;1 accumulation in the late endosomal compartments. Life-time analyses established that the high stability of PIPs was maintained under oxidative stress conditions, suggesting that H2O2 triggers a mechanism for intracellular sequestration of PM aquaporins without further degradation. In addition to information on cellular regulation of aquaporins, this study provides novel and complementary insights into the dynamic remodeling of plant internal membranes during oxidative stress responses.

Immunolocalization of aquaporins 1, 2 and 7 in rete testis, efferent ducts, epididymis and vas deferens of adult dog

The transepithelial movement of water into the male reproductive tract is an essential process for normal male fertility. Protein water channels, referred to as aquaporins (AQPs), are involved in increasing the osmotic permeability of membranes. This study has examined the expression of AQP1, AQP2, and AQP7 in epithelial cells in adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis, initial segment, caput, corpus and cauda epididymidis, and vas deferens were investigated by immunohistochemistry and Western blotting procedures to show the localization and distribution of the AQPs. AQP1 was noted in rete testis, in efferent ducts, and in vessels in the intertubular space, suggesting that AQP1 participated in the absorption of the large amount of testicular fluid occurring characteristically in the efferent ducts. AQP2 expression was found in the rete testis, efferent ducts and epididymis, whereas AQP7 was expressed in the epithelium of the proximal regions of the epididymis and in the vas deferens. This is the first time that AQP2 and AQP7 have been observed in these regions of mammalian excurrent ducts, but their functional role in the dog male reproductive tract remains unknown. Investigations of AQP biology could be relevant for clinical studies of the male reproductive tract and to technologies for assisted procreation.

The expression of aquaporins 1 and 9 in adult rat epididymis is perturbed by chronic exposure to ethanol

Aquaporins (AQPs), notably AQP-1 and AQP-9, may contribute to reabsorption of fluid and solute across the epididymis. Ethanol is related to be a toxicant affecting directly or indirectly the epididymis and the sperm motility. This study examined the expression of AQP-1 and AQP-9 in adult epididymis of the UChA and UChB 10% (v/v) ethanol-preferring rats, focusing the ethanol-induced hormonal disturbances upon the regulation of these AQPs. Chronic ethanol intake significantly decreased body weight, while UChA and UChB rats displayed a marked loss of epididymal weights. Both ethanol-consuming animals had a severe reduction of testosterone levels, whereas LH and 17β-estradiol were unchanged. Throughout the epididymis, a strong reaction to AQP-1 was observed in myoid and endothelial cells of the UChB ethanol-preferring rats, differently from a moderate intensity in the initial segment of the UChA rats. In addition, AQP-9 showed a strong immunoreaction in the apical membrane of principal cells at initial segment. In cauda epididymis, the level of AQP-9 was reduced along the microvillus projections in both UChA and UChB rats compared to controls. We conclude that chronic ethanol consumption modulates the androgen levels, thereby modifying the expression pattern of AQP-1 and 9 in the epididymis. © 2011 Elsevier Ltd.

Immunolocalization of aquaporins 1 and 9 in the ram efferent ducts and epididymis

Aquaporins (AQPs) are essential membrane protein channels for the transport of water across membranes. Fluid movement in the epididymis is important for modulation of the luminal environment, in which sperm mature and reside. This study was designed to understand the morphology and localization of AQPs in ram efferent ducts (ED) and epididymis. For this purpose, the epididymis of seven animals were removed for histologic and immunohistochemical analyses. AQP1 immunoreactivity was observed in the apex of the ED, and AQP9 was found adjacent to the nuclei of the epithelial cells of the ED. The epithelial lining of ram epididymis is pseudostratified columnar and presents principal, basal, apical and narrow cells. In the initial segment (IS), a moderate reaction for AQP1 was observed in the apical cytoplasm of epithelial cells. An intense reactivity for AQP1 was noted over the microvilli of principal cells and in spermatozoa in the caput. In the corpus and cauda, AQP1 was noted only over the endothelial cells of vascular channels located in intertubular spaces. A weak-to-moderate reaction for AQP9 was observed in the nuclei of epithelial cells in the IS, caput and corpus of the epididymis. In the cauda, an intense reaction to AQP9 was observed in the epithelial border. In the IS, caput and corpus, the reactivity for AQP9 differed from those observed in domestic animals. The cauda showed a pattern similar to that previously described. These results indicate that AQPs 1 and 9 have reversed locations and roles in rams, suggesting activity variations related with fluid and solute absorption throughout the epididymis.

Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9

Previous work showed that aquaporin 1 (AQP1), AQP4-M23, and AQP5 each has a characteristic CO(2)/NH(3) and CO(2)/H(2)O permeability ratio. The goal of the present study is to characterize AQPs 0-9, which traffic to the plasma membrane when heterologously expressed in Xenopus oocytes. We use video microscopy to compute osmotic water permeability (P(f)) and microelectrodes to record transient changes in surface pH (ΔpH(S)) caused by CO(2) or NH(3) influx. Subtracting respective values for day-matched, H(2)O-injected control oocytes yields the channel-specific values P(f)* and ΔpH(S)*. We find that P(f)* is significantly >0 for all AQPs tested except AQP6. (ΔpH(S)*)(CO(2)) is significantly >0 for AQP0, AQP1, AQP4-M23, AQP5, AQP6, and AQP9. (ΔpH(S)*)(NH(3)) is >0 for AQP1, AQP3, AQP6, AQP7, AQP8, and AQP9. The ratio (ΔpH(S)*)(CO(2))/P(f)* falls in the sequence AQP6 (∞) > AQP5 > AQP4-M23 > AQP0 ≅ AQP1 ≅ AQP9 > others (0). The ratio (ΔpH(S)*)(NH(3))/P(f)* falls in the sequence AQP6 (∞) > AQP3 ≅ AQP7 ≅ AQP8 ≅ AQP9 > AQP1 > others (0). Finally, the ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) falls in the sequence AQP0 (∞) ≅ AQP4-M23 ≅ AQP5 > AQP6 > AQP1 > AQP9 > AQP3 (0) ≅ AQP7 ≅ AQP8. The ratio (ΔpH(S)*)(CO(2))/(-ΔpH(S)*)(NH(3)) is indeterminate for both AQP2 and AQP4-M1. In summary, we find that mammalian AQPs exhibit a diverse range of selectivities for CO(2) vs. NH(3) vs. H(2)O. As a consequence, by expressing specific combinations of AQPs, cells could exert considerable control over the movements of each of these three substances

Expression of heterologous aquaporins for functional analysis in Saccharomyces cerevisiae

In this study the yeast Saccharomyces cerevisiae, which is a genetically tractable model for analysis of osmoregulation, has been used for analysis of heterologous aquaporins. Aquaporin water channels play important roles in the control of water homeostasis in individual cells and multicellular organisms. We have investigated the effects of functional expression of the mammalian aquaporins AQP1 and AQP5 and the aquaglyceroporins AQP3 and AQP9. Expression of aquaporins caused moderate growth inhibition under hyperosmotic stress, while expression of aquaglyceroporins mediated strong growth inhibition due to glycerol loss. Water transport was monitored in protoplasts, where the kinetics of bursting was influenced by presence of aquaporins but not aquaglyceroporins. We observed glycerol transport through aquaglyceroporins, but not aquaporins, in a yeast strain deficient in glycerol production, whose growth depends on glycerol inflow. In addition, a gene reporter assay allowed to indirectly monitor the effect of AQP9-mediated enhanced glycerol loss on osmoadaptation. Transport activity of certain aqua(glycero)porins was diminished by low pH or CuSO 4, suggesting that yeast can potentially be used for screening of putative aquaporin inhibitors. We conclude that yeast is a versatile system for functional studies of aquaporins, and it can be developed to screen for compounds of potential pharmacological use. © Springer-Verlag 2006.

Human aquaporins:regulators of transcellular water flow

Background - Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistent with their expression in most tissues, AQPs are associated with diverse physiological and pathophysiological processes. Scope of review - AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volume regulation (CVR) is particularly notable. This review examines the regulatory role of AQPs in transcellular water flow, especially in CVR. We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema. Major conclusions - AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapid movement of water across diverse cell membranes and playing regulatory roles in CVR. Gating mechanisms have been proposed for human AQPs, but have only been reported for plant and microbial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow. General significance - Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.

Beyond water homeostasis:diverse functional roles of mammalian aquaporins

Background - Aquaporin (AQP) water channels are best known as passive transporters of water that are vital for water homeostasis. Scope of review - AQP knockout studies in whole animals and cultured cells, along with naturally occurring human mutations suggest that the transport of neutral solutes through AQPs has important physiological roles. Emerging biophysical evidence suggests that AQPs may also facilitate gas (CO2) and cation transport. AQPs may be involved in cell signalling for volume regulation and controlling the subcellular localization of other proteins by forming macromolecular complexes. This review examines the evidence for these diverse functions of AQPs as well their physiological relevance. Major conclusions - As well as being crucial for water homeostasis, AQPs are involved in physiologically important transport of molecules other than water, regulation of surface expression of other membrane proteins, cell adhesion, and signalling in cell volume regulation. General significance - Elucidating the full range of functional roles of AQPs beyond the passive conduction of water will improve our understanding of mammalian physiology in health and disease. The functional variety of AQPs makes them an exciting drug target and could provide routes to a range of novel therapies.

Sugarcane genes associated with sucrose content

Background -: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. Results -: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. Conclusion -: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference.

Neuroimaging with diffusion-weighted imaging is routinely used for clinical diagnosis/prognosis. Its quantitative parameter, the apparent diffusion coefficient (ADC), is thought to reflect water mobility in brain tissues. After injury, reduced ADC values are thought to be secondary to decreases in the extracellular space caused by cell swelling. However, the physiological mechanisms associated with such changes remain uncertain. Aquaporins (AQPs) facilitate water diffusion through the plasma membrane and provide a unique opportunity to examine the molecular mechanisms underlying water mobility. Because of this critical role and the recognition that brain AQP4 is distributed within astrocytic cell membranes, we hypothesized that AQP4 contributes to the regulation of water diffusion and variations in its expression would alter ADC values in normal brain. Using RNA interference in the rodent brain, we acutely knocked down AQP4 expression and observed that a 27% AQP4-specific silencing induced a 50% decrease in ADC values, without modification of tissue histology. Our results demonstrate that ADC values in normal brain are modulated by astrocytic AQP4. These findings have major clinical relevance as they suggest that imaging changes seen in acute neurologic disorders such as stroke and trauma are in part due to changes in tissue AQP4 levels.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Background: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.Results: The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.Conclusion: Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.