Aquaporins in adipocytes:insights into the pathophysiology of human obesity

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014

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.

Ethephon increases rubber tree latex yield by regulating aquaporins and alleviating the tapping-induced local increase in latex total solid content

The concentration of phloem solute generally falls from leaves to roots. However, a local increase in latex total solid content (LILTSC) was identified near the tapping cut of rubber trees. To understand the mechanism of ethephon-stimulated latex yield, the formation and ethephon (an ethylene releaser) alleviation of the LILTSC near the tapping cut were examined. It was found that the LILTSC near the tapping cut of a tapped rubber tree was caused by the tapping-accelerated rubber biosynthesis which began following the first tapping and became significant after the fourth tapping. Ethephon stimulation markedly reduced the LILTSC. The latex yield change pattern upon ethephon stimulation was associated with the kinetic change of LILTSC and the decomposition dynamic of ethephon into ethylene. Once the LILTSC was reduced by ethylene release upon ethephon stimulation, the latex yield increased; however, when the ethylene release upon ethephon stimulation receded, the LILTSC was restored and the effect of ethephon stimulation dissipated. The reduction of LILTSC by ethephon stimulation could be ascribed to the translocation property of ethylene in plants and its regulation of aquaporins. Because maximum ethylene release upon tapping-cut-ethephon-application occured close to the tapping cut, the aquaporins were more up-regulated in this region, leading to a reduction of the LILTSC and an increase in latex yield. All these results suggest that the LILTSC near the tapping cut was caused by tapping; the ethephon-induced aquaporin up-regulation and LILTSC reduction are involved in the mechanism of ethephon-promoted latex yield.

Hydrophobic Peptide Channels and Encapsulated Water Wires

Peptide nanotubes with filled and empty pores and close-packed structures are formed in closely related pentapeptides. Enantiomorphic sequences, Boc-(D)Pro-Aib-Xxx-Aib-Val-OMe (Xxx = Leu, 1; Val, 2; Ala, 3; Phe, 4) and Boc-Pro-Aib-(D)Xxx-Aib-(D)Val-OMe ((XXX)-X-D = (D)Leu, 5; (D)Val, 6; (D)Ala, 7; (D)Phe, 8), yield molecular structures with a very similar backbone conformation but varied packing patterns in crystals. Peptides 1, 2, 5, and 6 show tubular structures with the molecules self-assembling along the crystallographic six-fold axis (c-axis) and revealing a honeycomb arrangement laterally (ab plane). Two forms of entrapped water wires have been characterized in 2: 2a with d(O center dot center dot center dot O) = 2.6 angstrom and 2b with d(O center dot center dot center dot O) = 3.5 angstrom. The latter is observed in 6 (6a) also. A polymorphic form of 6 (6b), grown from a solution of methanol-water, was observed to crystallize in a monoclinic system as a close-packed structure. Single-file water wire arrangements encapsulated inside hydrophobic channels formed by peptide nanotubes could be established by modeling the published structures in the cases of a cyclic peptide and a dipeptide. In all the entrapped water wires, each water molecule is involved in a hydrogen bond with a previous and succeeding water molecule. The O-H group of the water not involved in any hydrogen bond does not seem to be involved in an energetically significant interaction with the nanotube interior, a general feature of the one-dimensional water wires encapsulated in hydrophobic environements. Water wires in hydrophobic channels are contrasted with the single-file arrangements in amphipathic channels formed by aquaporins.

根系吸水机理研究进展

植物根系除支撑和固定其地上部这一力学功能外的一个重要功能就是从土壤中吸收水分和养分以满足植物地上部生长所需.表征植物根吸水能力的一个重要的水力学参数是水力导度(用单位时间单位面积的水流速率来表示),可在细胞(细胞水力导度)、单根和整株根系水平上来表达,其中单根导度可分为径向导度和轴向导度,仅径向导度反映了单根吸收水分的能力,而轴向导度则反映了植物根系输导水分的能力,但在整株根系水平上则以通过整个根系的水流通量与根木质部和根表土壤间的水势差之比来表示,既包括径向导度也包括轴向导度.近年来,对植物根系吸水或根水力导度研究已取得了许多重要进展,这对阐明根系吸水机理和地上地下部关系起到了重要作用.下面做一简要综述.1　根系吸收水分的主要部位根系吸水的部位一般认为在距根尖10～100ｍｍ的区域内,这种看法是基于解剖上的证据.从根表面到根中心,依次为根表皮、下表皮、皮层、内皮层、中柱,其中根表皮是有最高吸收活性的根区,但一般仅可存活几天,而内皮层将皮层和中柱分开形成了根内侧的一个界面,老根一般有周皮或栓质化的内皮层,有很强的不透水性(即阻力很大).但Ｓａｎｄｅｒｓｏｎ[1]对大麦的研究发现,虽然老根区内重度栓化的内皮层已...

Arsenite transport into paddy rice (Oryza sativa) roots

Here the mechanism of arsenite transport into paddy rice (Oryza sativa) roots, uptake of which is described by Michaelis-Menten kinetics, is reported. A recent study on yeast (Saccharomyces cerevisiae) showed that undissociated arsenite (its pK_a is 9.2) was transported across the plasma membrane via a glycerol transporting channel. To investigate whether the same mechanism of transport was involved for rice, competitive studies with glycerol, which is transported into cells via aquaporins, were performed. Glycerol competed with arsenite for transport in a dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were the same. Arsenate transport was unaffected by glycerol, confirming that arsenate and arsenite are taken up into cells by different mechanisms. Antimonite, an arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also competed with arsenite transport in a dose-dependent manner, providing further evidence that arsenite is transported into rice roots via glycerol transporting channels. Mercury (Hg²⁺) inhibited both arsenite and arsenate uptake, suggesting that inhibition of influx was due to general cellular stress rather than the specific action of Hg²⁺ on aquaporins. Arsenite uptake by pea (Pisum sativum) and wheat (Triticum aestivum) was also described by Michaelis-Menten kinetics.

Biological membranes : biophysical properties and aquaporin function

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2014