Plasma Membrane Water Permeability Research Articles

Intratumor mapping of intracellular water lifetime: metabolic images of breast cancer?

Shutter-speed pharmacokinetic analysis of dynamic-contrast-enhanced (DCE)-MRI data allows evaluation of equilibrium inter-compartmental water interchange kinetics. The process measured here – transcytolemmal water exchange – is characterized by the mean intracellular water molecule lifetime (τi). The τi biomarker is a true intensive property not accessible by any formulation of the tracer pharmacokinetic paradigm, which inherently assumes it is effectively zero when applied to DCE-MRI. We present population-averaged in vivo human breast whole tumor τi changes induced by therapy, along with those of other pharmacokinetic parameters. In responding patients, the DCE parameters change significantly after only one neoadjuvant chemotherapy cycle: while Ktrans (measuring mostly contrast agent (CA) extravasation) and kep (CA intravasation rate constant) decrease, τi increases. However, high-resolution, (1 mm)2, parametric maps exhibit significant intratumor heterogeneity, which is lost by averaging. A typical 400 ms τi value means a trans-membrane water cycling flux of 1013 H2O molecules s−1/cell for a 12 µm diameter cell. Analyses of intratumor variations (and therapy-induced changes) of τi in combination with concomitant changes of ve (extracellular volume fraction) indicate that the former are dominated by alterations of the equilibrium cell membrane water permeability coefficient, PW, not of cell size. These can be interpreted in light of literature results showing that τi changes are dominated by a PW(active) component that reciprocally reflects the membrane driving P-type ATPase ion pump turnover. For mammalian cells, this is the Na+,K+-ATPase pump. These results promise the potential to discriminate metabolic and microenvironmental states of regions within tumors in vivo, and their changes with therapy.

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4°C, 12°C, 21°C, and 37°C, the activation energy for water transport was determined to be Ea=51±3kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130°C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15°C.

Immunolocalization and translocation of aquaporin-5 water channel in sweat glands

BackgroundAquaporin-5 (AQP5) is a member of the water channel protein family. Although AQP5 was shown to be present in sweat glands, the presence or absence of regulated intracellular translocation of AQP5 in sweat glands remained to be determined. ObjectiveWe investigated whether AQP5 in sweat glands translocated during sweating, and also sought to determine the intracellular signal that triggers this translocation. MethodsImmunofluorescent analyses of AQP5 in mouse and human sweat glands were performed. Madin-Darby Canine kidney (MDCK) cell lines stably expressing human AQP5 were generated, and the regulated translocation of AQP5 in the polarized cells was assessed by immunofluorescent analysis and biotinylation assays. ResultsAQP5 showed rapid translocation to the apical membranes during sweating. In human eccrine sweat glands, immunoreactive AQP5 was detected in the apical membranes and the intercellular canaliculi of secretory coils, and in the basolateral membranes of the clear cells. Treatment of human AQP5-expressing MDCK cells with calcium ionophore A23187 resulted in a twofold increase of AQP5 in the apical membranes within 5min. ConclusionThe regulated AQP5 translocation may contribute to sweat secretion by increasing the water permeability of apical plasma membranes of sweat glands.

Lipid peroxidation, carbohydrate hydrolysis, and Amadori-Maillard reaction at early stages of dry seed aging

Processes underlying aging of air-dry seeds were investigated. Naturally aged seeds of pea (Pisum sativum L.), cucumber (Cucumis sativus L.), and buckwheat (Fagopyrum esculentum Moench.) were separated into three fractions of different quality according to their room temperature phosphorescence (RTP): fraction I contained strong seeds; fraction II comprised weak seeds; and fraction III was composed of dead seeds. These seed fractions were used to identify the processes in air-dry seeds that account for the initial deterioration in seed quality at early stages of aging and for the possible transient improvement of seed quality at further aging. Experiments with seed powders showed that the increase in thermochemiluminescence (TCL) in the temperature range of 50–110°C was determined by the presence of lipid peroxidation products. No difference was observed between TCL levels in fraction I and fraction II seeds; hence, lipid peroxidation is not responsible for the transition of strong seeds to the fraction of weak seeds. The TCL intensity monitored upon heating in the range of 50–110°C increased only in the fraction of dead seeds. In fraction II seeds, a twofold increase in TCL upon heating at 150°C was observed in aged seeds; this indicates that the products of oligosaccharide hydrolysis (glucose) were more abundant in fraction II seeds than in fraction I seeds under similar conditions. This assumption was confirmed by direct determination of glucose content with a glucometer. Hence, the transition of air-dry seeds from fraction I (strong seeds) to fraction II (weak seeds) occurred due to the activation of carbohydrate hydrolysis in aging seeds. Since air-dry seeds contain no free water during aging, the seed moisture content decreases during hydrolysis, presumably, because the bound water participates in the hydrolysis reaction. The decrease in “improved” seeds of glucose content and the increase in moisture content suggest the activation of amino-carbonyl reaction. It is proposed that the amino-carbonyl (Amadori-Maillard) reaction is responsible for closing water channels in improved seeds, as well as for the decreased water permeability of cell membranes during seed imbibition.

Expression and localization of aquaporin‐1 in temporomandibular joint disc with internal derangement

Internal derangement is the most frequent arthropathy affecting the temporomandibular joint, where its commonest form is anterior disc displacement with or without reduction. Despite the frequency of the disorder, the biochemical features of displaced discs are still unclear. We investigated the expression pattern and localization of aquaporin-1, an important channel protein involved in plasma membrane water permeability, in patients with anterior disc displacement (both with and without reduction), with a view to assessing the characteristics of local tissue responses to the microenvironmental changes induced by abnormal mechanical loading of the displaced disc. Protein expression was studied by immunohistochemistry in different areas of discs from 18 patients with anterior disc displacement with or without reduction and in four normal controls. A greater proportion of cells immunopositive for aquaporin-1 were detected in diseased than in normal discs. Whereas protein expression was substantially similar in the different areas of normal discs, a significantly larger number of immunopositive cells were detected in the posterior band of displaced discs without reduction and in the anterior and intermediate bands of those with reduction. These findings suggest that aquaporin-1 is expressed and upregulated in temporomandibular joint with anterior disc displacement (both with and without reduction).

Increased Differentiation Capacity of Bone Marrow-Derived Mesenchymal Stem Cells in Aquaporin-5 Deficiency

Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5(-/-) mice showed significantly lower plasma membrane water permeability than those from AQP5(+/+) mice. In characterizing the cultured BMMSCs from AQP5(-/-) and AQP5(+/+) mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5(-/-) than AQP5(+/+) BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5(-/-) -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5(-/-) BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5(-/-) mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5(-/-) than AQP5(+/+) BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5(+/+) than AQP5(-/-) BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration.

Neuromyelitis Optica Immunoglobulin G present in sera from neuromyelitis optica patients affects aquaporin‐4 expression and water permeability of the astrocyte plasma membrane

NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process.

The effect of osmotic dehydrofreezing on the role of the cell membrane in carrot texture softening after freeze-thawing

To understand the protective mechanism of the osmotic dehydrofreezing technique on carrot texture after freeze-thawing, two mechanical texture parameters, fracture stress related to the cell wall and initial modulus related to the cell membrane, as well as cell membrane water permeability using PFG-NMR were evaluated. In particular, to understand the role of the cell membrane in texture alteration, tissue in which the cell membrane was exposed to chloroform vapor was used. Although dehydrofreezing protected texture from freezing damage, the effect was only observed with respect to fracture stress, with exhibited values close to those for raw tissue. However, there was no protective effect on initial modulus and water permeability, in which values did not differ from those of cell membrane-free tissue. More specifically, osmotic dehydrofreezing had no effect on the cell membrane induced by freeze-thawing.

Study of the reaction of kidney collecting duct principal cells to hypotonic shock. Experiment and mathematical model

The reaction of rat kidney collecting duct principal cells to hypotonic shock was studied. The changes in cell relative volume were measured using fluorescent dye calcein, and a mathematical model based on our experimental results was developed. It was shown that regulatory volume decrease is mainly provided by significant release of osmolytes from the cell and decrease of the plasma membrane water permeability. Using our model, we calculated the membrane water permeability and found it to decrease from 2 · 10−1 to 2 · 10−2 cm/s. We conclude that for effective RVD to occur, a dramatic increase in the membrane permeability to K+, Cl− and organic anions is necessary.

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.

Endothelins decrease the expression of aquaporins and plasma membrane water permeability in cultured rat astrocytes

Some of the aquaporins (AQPs), a family of water channel proteins, are expressed in astrocytes. The expression of astrocytic AQPs is altered after brain insults, such as ischemia and head trauma. However, little is known about the regulation of AQP expressions. Endothelins (ETs), which are vasoconstrictor peptides, regulate several pathophysiological responses of astrocytes. In this study, the effects of ETs on AQP expressions and plasma membrane water permeability were examined in cultured rat astrocytes. Determination of AQP mRNA copy numbers revealed that AQP1 and AQP4 were expressed prominently in cultured astrocytes. ET-1 (100 nM) and Ala¹,³,¹¹,¹⁵-ET-1 (an ETB receptor agonist, 50 nM) decreased the AQP4 and AQP9 mRNA levels in cultured astrocytes, but the AQP1, -3, -5, and -8 mRNA levels remained unchanged. BQ788, an ETB receptor antagonist, reduced the effects of ET-1 on astrocytic AQP mRNAs, whereas FR139317, an ETA antagonist, had no effect. Immunoblot analyses revealed that treatment with ET-1 decreased the protein contents of AQP4 and AQP9 in both total cell lysates and plasma membrane fractions of cultured astrocytes. ET-1 and Ala¹,³,¹¹,¹⁵-ET-1 decreased hypoosmolarity-induced water influxes into cultured astrocytes. In the presence of 30 μM Hg²+, which inhibits water movement through AQP1 and AQP9, the hypoosmolarity-induced water influxes were reduced. Phloretin, an inhibitor of AQP9, did not greatly affect the water influxes. The ET-induced decreases in water influxes were obtained in the presence of Hg²+ and phloretin. These results suggest that ET is a factor that regulates AQP expressions and water permeability in astrocytes.

Intracellular pH sensing is altered by plasma membrane PIP aquaporin co-expression

The plant plasma membrane barrier can express aquaporins (PIP1 and PIP2) that show two intriguing aspects: (1) the potential of modulating whole membrane water permeability by co-expression of both types, which have recently been distinguished for showing a different capacity to reach the plasma membrane; and (2) the faculty to reduce water permeation through the pore after cytosolic acidification, as a consequence of a gating process. Our working hypothesis is that these two key features might enhance plasticity of the membrane water transport capacity if they jointly trigger any cooperative interaction. In previous work, we proved by biophysical approaches that the plasma membrane of the halophyte Beta vulgaris storage root presents highly permeable aquaporins that can be shut down by acidic pH. Root Beta vulgaris PIPs were therefore subcloned and expressed in Xenopus oocytes. Co-expression of BvPIP1;1 and BvPIP2;2 not only enhances oocyte plasma membrane water permeability synergistically but also reinforces pH inhibitory response from partial to complete shut down after cytosolic pH acidification. This pH dependent behavior shows that PIP1-PIP2 co-expression accounts for a different pH sensitivity in comparison with PIP2 expression. These results prove for the first time that PIP co-expression modulates the membrane water permeability through a pH regulatory response, enhancing in this way membrane versatility to adjust its water transfer capacity.

Transgenic expression of AQP1 in the fiber cells of AQP0 knockout mouse: Effects on lens transparency

Mutations and knockout of aquaporin 0 (AQP0) result in dominant lens cataract. To date, several functions have been proposed for AQP0; however, two functions, water permeability and cell-to-cell adhesion have been supported by several investigators and only water channel function has been readily authenticated by in vitro and ex vivo studies. Lens shifts protein expression from the more efficient AQP1 in the equatorial epithelial cells to the less efficient water channel, AQP0, in the differentiating secondary fiber cells; perhaps, AQP0 performs a distinctive function. If AQP0 has only water permeability function, can the more efficient water channel AQP1 transgenically expressed in the fiber cells compensate and restore lens transparency in the AQP0 knockout (AQP0 −/−) mouse? To investigate, we generated a transgenic wild-type mouse line expressing AQP1 in the fiber cells using αA-crystallin promoter. These transgenic mice (TgAQP1 +/+) showed increase in fiber cell membrane water permeability without any morphological, anatomical or physiological defects compared to the wild type indicating that the main purpose of the shift in expression from AQP1 to AQP0 may not be to lessen the membrane water permeability. Further, we transgenically expressed AQP1 in the lens fiber cells of AQP0 knockout mouse (TgAQP1 +/+/AQP0 −/−) to determine whether AQP1 could restore AQP0 water channel function and regain lens transparency. Fiber cells of these mice showed 2.6 times more water permeability than the wild type. Transgene AQP1 reduced the severity of lens cataract and prevented dramatic acceleration of cataractogenesis. However, lens fiber cells showed deformities and lack of compact cellular architecture. Loss of lens transparency due to the absence of AQP0 was not completely restored indicating an additional function for AQP0. In vitro studies showed that AQP0 is capable of cell-to-cell adhesion while AQP1 is not. To our knowledge, this is the first report which uses an animal model to demonstrate that AQP0 may have an additional function, possibly cell-to-cell adhesion.

Effects of drugs on water permeability of erythrocyte membranes

The method of NMR-relaxation with the manganese doping has been applied to study changes of water permeability of red blood cell membranes affected by various concentrations of chlorhexidine digluconate and dimephosphone. It is shown that both investigated substances suppress the water permeability of the red blood cell membrane in a dose-dependent manner. Half-maximum inhibitory effect of studied substances was reached at the concentrations of 9 μM of chlorhexidine and 400 μM of dimephosphone.

Development of the Method to Measure the Water Permeability of Cell Membrane Using Two-laminar Flows in Microchannel

Development of the Method to Measure the Water Permeability of Cell Membrane Using Two-laminar Flows in Microchannel

Aquaporin 1-expressing MCF-7 mammary carcinoma cells show enhanced migration in vitro

Recent studies have demonstrated that aquaporin (AQP) expression facilitates cell migration and promotes angiogenesis and neutrophil motility. Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we investigated the expression of AQP in MCF-7 human mammary carcinoma cells and characterized its function in cell migration. Reverse Transcription–Polymerase Chain Reaction, Immunoblot and Immunofluorescence analysis demonstrated two populations of MCF-7 cell clones with low (AQP1L) and high (AQP1H) AQP1 expression and the AQP1 protein expression patterns in the plasma membrane of MCF-7 cells. MCF-7 cell clones (AQP1L and AQP1H) with low and about two-fold higher osmotic water permeability were identified by functional assays with corresponding low and high AQP1 expression. Cell migration rate was remarkably higher in AQP1H cells as compared to AQP1L cells, assessed by wound healing and transwell migration assays. Adenoviral-mediated mRNA and protein expression of AQP1 in AQP1L cells increased their water permeability and migration rate to the level similar to AQP1H cells. The results provided direct evidence that aquaporin-mediated plasma membrane water permeability played an important role in mammary carcinoma cell migration and may be associated with mammary carcinoma invasion and metastasis.

Curve fitting approach for measurement of cellular osmotic properties by the electrical sensing zone method. II. Membrane water permeability

In a companion paper, we demonstrated that dynamic range limitations can confound measurement of the osmotically inactive volume using electrical sensing zone instruments (e.g., Coulter counters), and presented an improved parameter estimation method in which a lognormal function was fit to the cell volume distribution to allow extrapolation beyond the bounds of the data. Presently, we have investigated the effect of dynamic range limitations on measurement of the cell membrane water permeability ( L p ), and adapted the lognormal extrapolation method for estimation of L p from transient volume data. An alternative strategy (the volume limit adjustment method, in which the measured isotonic volume distribution is used to generate model predictions for curve fitting, and the bounds of the dynamic range are adjusted such that extrapolation is not required) was also developed. The performance of these new algorithms was compared to that of a conventional parameter estimation method. The best-fit L p values from in vitro experiments with mouse insulinoma (MIN6) cells differed significantly for the different parameter estimation techniques ( p < 0.001). Using in silico experiments, the volume limit adjustment method was shown to be the most accurate (relative error 0.4 ± 3.2%), whereas the conventional method underestimated L p by 19 ± 2% for MIN6 cells. Parametric analysis revealed that the error associated with the conventional method was sensitive to the dynamic range and the width of the volume distribution. Our initial implementation of the lognormal extrapolation method also yielded significant errors, whereas accuracy of this algorithm improved after including a normalization scheme.

Aquaporin‐1 activity of plasma membrane affects HT20 colon cancer cell migration

Recent studies revealed an important role of aquaporins (AQPs) in cell migration and migration-associated cell function such as angiogenesis, wound healing, and neutrophil motility. Migration of tumor cells is a crucial step in tumor invasion and metastasis. In the present study, we investigated the expression of AQP1 in human HT20 colon cancer cells and characterized its function in cell migration. By reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis, expression of AQP1 was identified in HT20 cell lines. Immunofluorescence analysis indicated expression of AQP1 protein in the plasma membrane of HT20 cells. The recombinant adenovirus expressing human AQP1 increased the mRNA and protein expression of AQP1 in HT20 cells. In contrary, the RNA interference vector of AQP1 effectively inhibited the mRNA and protein expression of AQP1 in HT20 cells. Adenovirus-mediated high expression of AQP1 in HT20 cells increased relative plasma membrane water permeability and migration rate in both wound healing and invasive transwell migration assays. In contrary, RNA interference vector-mediated low expression of AQP1 in HT20 cells reduced relative plasma membrane water permeability and migration rate. AQP1 expression induced relocalization of actin protein and activation of RhoA and Rac. In nude mice, AQP1 increased extravasation of HT20 Cells in lung after tail vein injection. The results provided the direct evidence that aquaporin-mediated plasma membrane water permeability plays an important role in colon cancer cell migration and may be associated with colon cancer invasion and metastasis.

Tumor necrosis factor-α decreases aquaporin-3 expression in DJM-1 keratinocytes

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein that is strongly expressed at the plasma membranes of keratinocytes in skin. There is evidence for involvement of AQP3-facilitated water and glycerol transport in skin hydration and wound repair, respectively. In this study, we show that tumor necrosis factor-α (TNF-α) and TNF receptor-1 signaling decreased AQP3 protein expression and plasma membrane water permeability in DJM-1 keratinocytes. TNF-α also decreased AQP3 mRNA expression and promoter activity, indicating that TNF-α suppresses AQP3 gene transcription. In addition, inhibitors of p38 and extracellular signal-regulated kinase (ERK) abolished the effect of TNF-α on AQP3 expression level, whereas inhibitors for NF-κB did not. These data indicate that TNF-α decreases AQP3 gene expression through p38 and ERK activation, and suggest that the decrease in AQP3 expression caused by TNF-α might be related to the phenotypes of skin inflammation, such as dry skin.

Role of aquaporins in leaf physiology

Playing a key role in plant growth and development, leaves need to be continuously supplied with water and carbon dioxide to fulfil their photosynthetic function. On its way through the leaf from the xylem to the stomata, water can either move through cell walls or pass from cell to cell to cross the different tissues. Although both pathways are probably used to some degree, evidence is accumulating that living cells contribute substantially to the overall leaf hydraulic conductance (K(leaf)). Transcellular water flow is facilitated and regulated by water channels in the membranes, named aquaporins (AQPs). This review addresses how AQP expression and activity effectively regulate the leaf water balance in normal conditions and modify the cell membrane water permeability in response to different environmental factors, such as irradiance, temperature, and water supply. The role of AQPs in leaf growth and movement, and in CO(2) transport is also discussed.