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Cândida Lucas - One of the best experts on this subject based on the ideXlab platform.
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A member of the sugar Transporter family, Stl1p is the Glycerol/H+ symporter in Saccharomyces cerevisiae.
Molecular biology of the cell, 2005Co-Authors: Célia Ferreira, Cândida Lucas, Frank Van Voorst, António Martins, L. Neves, Rui Pedro Soares De Oliveira, Morten C. Kielland-brandt, Anders BrandtAbstract:Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up Glycerol by proton symport. To identify genes involved in active Glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on Glycerol medium. Deletion of STL1, which encodes a member of the sugar Transporter family, eliminates active Glycerol Transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where Glycerol symport is functional. Both the Stl1 protein and the active Glycerol Transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the Glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active Glycerol Transport system. In S. pombe, STL1 conferred the ability to take up Glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the Glycerol proton symporter in S. cerevisiae is encoded by STL1.
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Expression studies of GUP1 and GUP2, genes involved in Glycerol active Transport in Saccharomyces cerevisiae, using semi-quantitative RT-PCR
Current Genetics, 2004Co-Authors: Rui Oliveira, Cândida LucasAbstract:Glycerol active uptake in Saccharomyces cerevisiae , characterised physiologically as a proton symport, was previously described as repressed by glucose, induced by growth on non-fermentable carbon sources and unresponsive to growth under salt stress. GUP1 and GUP2 were identified and characterised as genes involved in Glycerol active uptake. Using semi-quantitative RT-PCR, GUP1 and GUP2 transcription was measured. Unlike active Transport activity determined previously, this was shown to be constitutive and not affected by either glucose repression or growth under salt stress. Furthermore, transcription of GUP1 and GUP2 was not affected in the gpd1gpd2 mutant strain grown under salt stress in the presence of small amounts of Glycerol, in which case a very high V _max of Glycerol uptake was reported. Intracellular compounds were determined. Glycerol, acetate and trehalose were found to be the major compounds accumulated. Surprisingly, the gpd1gpd2 mutant was found to produce significant amounts of Glycerol. Yet, the results provide no evidence for a correlation between the amount of each compound and the Glycerol Transport activity in any of the strains.
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New insights on Glycerol Transport in Saccharomyces cerevisiae.
FEBS letters, 2004Co-Authors: Luisa Neves, Fernanda Lages, Cândida LucasAbstract:Previous studies evidenced in Saccharomyces cerevisiae the activity of a H(+)/Glycerol symport, derepressed by growth on non-fermentable carbon sources, later associated with GUP1 and GUP2 genes. It was also demonstrated that only the combined deletion of GUP1, GUP2 together with GUT1 (Glycerol kinase) abolished active Transport in ethanol-induced cells. In this work, we show that a Glycerol H(+)/symport, with identical characteristics to the previously described, was found in gup1gup2gut1 grown under salt-stress, particularly high in cells collected during diauxic-shift. These results suggest different roles for Gup1/2p than Glycerol Transport. The gene encoding for Glycerol active uptake is thus yet unknown.
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Yeast orthologues associated with Glycerol Transport and metabolism
FEMS yeast research, 2004Co-Authors: L. Neves, Rui Pedro Soares De Oliveira, Cândida LucasAbstract:Glycerol is a key compound in the regulation of several metabolic pathways in Saccharomyces cerevisiae. From this yeast most of the genes involved in Glycerol consumption, production and Transport are now available. Some of the mechanisms involving Glycerol metabolism and Transport are common to other yeasts. This work presents a search for GPD1/2, GUT1, GUP1/2 and FPS1 orthologues in a series of hemiascomycetous yeasts. All the genes cloned were able to complement S. cerevisiae mutant phenotypes and presented a high degree of similarity to the corresponding genes in this yeast. A phylogenetic analysis is presented. The allocation of GUP genes in the membrane bound O-acyl transferases (MBOAT) family is suggested as more consistent than their inclusion in the TC-DB/Glycerol uptake family.
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Orthologous yeast genes associated with Glycerol Transport and metabolism
2003Co-Authors: Maria Luísa Vieira Das Neves, Rui Pedro Soares De Oliveira, Cândida LucasAbstract:Apresentacao efectuada nas "11as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", realizadas na Escola Superior Agraria de Braganca, em Maio de 2003.
Hiroaki Yuasa - One of the best experts on this subject based on the ideXlab platform.
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Competitive Inhibition of AQP7-mediated Glycerol Transport by Glycerol Derivatives
Drug metabolism and pharmacokinetics, 2014Co-Authors: Takahiro Katano, Katsuhisa Inoue, Yuko Ito, Kinya Ohta, Tomoya Yasujima, Hiroaki YuasaAbstract:Summary: Aquaporin 7 (AQP7) is an aquaglyceroporin that has recently been found to operate as a facilitative carrier rather than a channel for Glycerol, although its primary function is as a water channel. To probe into its substrate specificity, we examined the inhibitory effect of a series of acyl Glycerol derivatives on Glycerol Transport mediated by human AQP7 stably expressed in Madin-Darby canine kidney II cells. According to kinetic analyses, AQP7-mediated Glycerol Transport was found to be competitively inhibited by monoacetin, monobutyrin and diacetin. Therefore, it may be possible that they all could be recognized as substrates by AQP7. The inhibition constant ( Κ i ) of monoacetin (134 μΜ) was smaller than that of diacetin (420μΜ), but greater than the Michaelis constant for Glycerol (11.8 μΜ). Considering another finding that inhibition by triacetin was insignificant, it is likely that a decrease in the number of hydroxyl groups in the Glycerol molecule by acetyl derivatization leads to a decrease in affinity for AQP7. The K of monobutyrin (80 μΜ) was, on the other hand, comparable with that of monoacetin, suggesting that the extension of the acyl chain by two hydrocarbon units does not have an impact on affinity for AQP7.
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Functional Characteristics of Aquaporin 7 as a Facilitative Glycerol Carrier
Drug metabolism and pharmacokinetics, 2013Co-Authors: Takahiro Katano, Katsuhisa Inoue, Yuko Ito, Kinya Ohta, Tomoya Yasujima, Hiroaki YuasaAbstract:Summary: Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for Glycerol as well as for water. However, our recent studies have indicated that AQP9 and AQP10 operate in a carrier mode, which is of saturable nature, for Glycerol Transport. Assuming that such a functional characteristic could also be shared by AQP7, another aquaglyceroporin, we examined its Glycerol Transport function. The specific Transport of Glycerol by human AQP7, which was stably expressed in Madin-Darby canine kidney II cells, was indeed highly saturable, indicating the involvement of a carrier mode of operation mechanism. Kinetic analysis indicated that the specific Transport conformed to Michaelis-Menten kinetics with the Michaelis constant of 11.9 μΜ and was not associated with a nonsaturable Transport component as an indication of a simultaneous channel mode of operation, which was previously indicated for AQP10. AQP7-specific Glycerol Transport was furthermore found to be specifically inhibited by several compounds analogous to Glycerol and operate without requiring either Na + or H + . These characteristics of the carrier mode of AQP7 operation suggest that it is a facilitative carrier for Glycerol and, possibly, also for analogous compounds, providing a novel insight into its operation mechanism.
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Dual Functional Characteristic of Human Aquaporin 10 for Solute Transport
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2011Co-Authors: Megumi Ishii, Katsuhisa Inoue, Jun Watanabe, Takahiro Katano, Kinya Ohta, Kimihiko Urano, Aki Miyamoto, Hiroaki YuasaAbstract:Background/Aims: Although aquaglyceroporins have been generally believed to operate in a channel mode, which is of nonsaturable nature, for Glycerol as well as for water, we recently found that human aquaporin 9 (hAQP9) operates in a carrier-mediated mode, which is of saturable nature, for Glycerol. Based on the finding, we assumed that such a characteristic might be shared by the other aquaglyceroporins and examined the functional characteristics of hAQP10, which is an intestine-specific aquaglyceroporin. Methods: Transport assays were conducted using Xenopus laevis oocytes expressing hAQP10 derived from the microinjected cRNA. Results: The Transport of Glycerol by hAQP10 was found to be highly saturable with a Michaelis constant of 10.4 µM and specifically inhibited by several Glycerol analogs such as monoacetin. Furthermore, when Glycerol was preloaded in hAQP10-expressing oocytes, its efflux was trans-stimulated by extracellular Glycerol. These results indicate the involvement of a carrier-mediated mechanism in Glycerol Transport by hAQP10. Interestingly, a channel mechanism was also found to be involved in part in hAQP10-mediated Glycerol Transport. Conclusion: The present study unveiled the uniquely dual functional characteristic of hAQP10 as a carrier/channel for solute Transport, providing a novel insight into its operation mechanism, which would help further elucidate its physiological role.
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Effect of Glycerol-related compounds on carrier-mediated Glycerol uptake in HCT-15 human colon cancer cell line.
Drug metabolism and pharmacokinetics, 2008Co-Authors: Nami Fujimoto, Yayoi Hayashi, Katsuhisa Inoue, Hiroaki YuasaAbstract:Summary: The effect of several compounds, which are structurally analogous to Glycerol, on carrier-mediated Glycerol uptake was examined in HCT-15 cells to help clarifying the functional characteristics of the Glycerol Transport system. The carrier-mediated uptake of Glycerol conformed to the Michaelis-Menten kinetics with a Michaelis constant of 21.1 μ M and the tested compounds were all suggested to inhibit it competitively with the values of the inhibition constant ( K i ) in the increasing order as follows: monobutyrin (41.0 μ M) μ M) μ M) μ M). Therefore, they all may possibly be substrates of the carrier-mediated Glycerol Transport system, for which the Glycerol esters (monoacetin and monobutyrin) have the highest affinities among them. It was also found that S-( + )-enantiomer of 1,2-propanediol ( K i = 484 μ M) has a higher affinity than its R-( – )-enantiomer ( K i = 19100 μ M), indicating enantioselective recognition. These results support the suggestion that a specific carrier protein is involved in Glycerol uptake in HCT-15 cells. It would be of interest to identify the carrier, which may be present also in some organs, and further investigate the possibility that Glycerol ester derivatives of drugs might be delivered via the carrier.
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Glycerol uptake in HCT-15 human colon cancer cell line by Na(+)-dependent carrier-mediated Transport.
Biological & pharmaceutical bulletin, 2006Co-Authors: Nami Fujimoto, Yayoi Hayashi, Katsuhisa Inoue, Hiroaki YuasaAbstract:It has recently been suggested that an Na+-dependent carrier-mediated Transport system is involved in intestinal Glycerol absorption. Such a Transport system is of general interest as a possible pathway of drug delivery and a target of drug development. However, the Na+-dependent mechanism of cellular Glycerol uptake has not been fully clarified in the small intestine or in any other organ. The purpose of the present study was to examine Glycerol uptake in the HCT-15 human colon cancer cell line, which was found to be able to perform Na+-dependent Glycerol uptake, to determine the Transport characteristics and help identify such Glycerol Transport systems. The uptake of Glycerol in HCT-15 cells was highly saturable with a Michaelis constant of 15.0 μM and a maximum uptake rate of 11.9 pmol/min/mg protein, accompanied by minimal unsaturable Transport; it was reduced markedly under Na+-free conditions, indicating Na+ requirement. Glycerol uptake was also reduced by 2,4-dinitrophenol, a metabolic inhibitor. These results suggest that a carrier-mediated Glycerol Transport system, which is Na+-dependent and secondarily active, is present in HCT-15 cells. The Transport system could be specific for Glycerol and some analogous compounds with hydroxyl groups, since Glycerol uptake was inhibited by some alcohols and compounds related to Glycerol, such as 1,2-propanediol and Glycerol 3-phosphpate. However, it may represent a high affinity Transport system, which is different from the one in the small intestine, because the Michaelis constant of 15.0 μM is about 50-fold lower than that observed in the rat small intestine. In conclusion, this is the first study to demonstrate an Na+-dependent carrier-mediated Glycerol Transport in an established cell line. This will help in identifying a group of Na+-dependent Glycerol Transport systems and elucidating their Transport mechanisms, although the one found in HCT-15 cells in this study seems to be different from one previously found in the rat small intestine.
Alan S. Verkman - One of the best experts on this subject based on the ideXlab platform.
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Chemokine-dependent T cell migration requires aquaporin-3-mediated hydrogen peroxide uptake.
The Journal of experimental medicine, 2012Co-Authors: Mariko Hara-chikuma, Alan S. Verkman, Shunsuke Chikuma, Yoshinori Sugiyama, Kenji Kabashima, Shintaro Inoue, Yoshiki MiyachiAbstract:Chemokine-dependent trafficking is indispensable for the effector function of antigen-experienced T cells during immune responses. In this study, we report that the water/Glycerol channel aquaporin-3 (AQP3) is expressed on T cells and regulates their trafficking in cutaneous immune reactions. T cell migration toward chemokines is dependent on AQP3-mediated hydrogen peroxide (H2O2) uptake but not the canonical water/Glycerol Transport. AQP3-mediated H2O2 Transport is essential for the activation of the Rho family GTPase Cdc42 and the subsequent actin dynamics. Coincidentally, AQP3-deficient mice are defective in the development of hapten-induced contact hypersensitivity, which is attributed to the impaired trafficking of antigen-primed T cells to the hapten-challenged skin. We therefore suggest that AQP3-mediated H2O2 uptake is required for chemokine-dependent T cell migration in sufficient immune response.
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Aquaporins in Clinical Medicine
Annual Review of Medicine, 2012Co-Authors: Alan S. VerkmanAbstract:The aquaporins are a family of membrane water channels, some of which also Transport Glycerol. They are involved in a wide range of physiological functions (including water/salt homeostasis, exocrine fluid secretion, and epidermal hydration) and human diseases (including glaucoma, cancer, epilepsy, and obesity). At the cellular level, aquaporin-mediated osmotic water Transport across cell plasma membranes facilitates transepithelial fluid Transport, cell migration, and neuroexcitation; aquaporin-mediated Glycerol Transport regulates cell proliferation, adipocyte metabolism, and epidermal water retention. Genetic diseases caused by loss-of-function mutations in aquaporins include nephrogenic diabetes insipidus and congenital cataracts. The neuroinflammatory demyelinating disease neuromyelitis optica is marked by pathogenic autoantibodies against astrocyte water channel aquaporin-4. There remain broad opportunities for the development of aquaporin-based diagnostics and therapeutics. Disease-relevant aquapor...
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A cautionary note on cosmetics containing ingredients that increase aquaporin‐3 expression
Experimental dermatology, 2008Co-Authors: Alan S. VerkmanAbstract:Abstract: Aquaporin-3 (AQP3) is a membrane Transport protein that facilitates water and Glycerol Transport across cell plasma membranes in the basal layer of keratinocytes in normal skin. Motivated by a relation between AQP3 expression and skin water content, several companies have marketed cosmetics containing ingredients that increase AQP3 expression. However, caution seems warranted in targeting AQP3 to increase skin moisturization based on a recently discovered association in mice between epidermal AQP3 expression and skin tumor formation.
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Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption
Molecular and cellular biology, 2007Co-Authors: Mariko Hara-chikuma, Alan S. VerkmanAbstract:Aquaporin-3 (AQP3) is a water/Glycerol-Transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell Glycerol, its metabolite Glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular Glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated Glycerol Transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular Glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy.
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Defective water and Glycerol Transport in the proximal tubules of AQP7 knockout mice.
American journal of physiology. Renal physiology, 2005Co-Authors: Eisei Sohara, Alan S. Verkman, Tatemitsu Rai, Jun-ichi Miyazaki, Sei Sasaki, Shinichi UchidaAbstract:The aquaporin-7 (AQP7) water channel is known as a member of the aquaglyceroporins, which facilitate the Transport of Glycerol as well as water. Although AQP7 is abundantly expressed on the apical ...
A. S. Verkman - One of the best experts on this subject based on the ideXlab platform.
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The expression of differentiation markers in aquaporin-3 deficient epidermis
Archives of Dermatological Research, 2009Co-Authors: Mariko Hara-chikuma, A. S. Verkman, Shunsuke Chikuma, Kenzo Takahashi, Yoshiki MiyachiAbstract:Aquaporin-3 (AQP3) is a water/Glycerol Transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and Glycerol Transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca^2+ or 1α,25-dihydroxyvitamin D_3 (VD_3) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.
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roles of aquaporin 3 in the epidermis
Journal of Investigative Dermatology, 2008Co-Authors: Mariko Harachikuma, A. S. VerkmanAbstract:Aquaporin-3 (AQP3) is a membrane Transporter of water and Glycerol expressed in plasma membranes in the basal layer keratinocytes of epidermis in normal skin. AQP3 expression in human skin is increased in response to skin stress in diseases such as atopic eczema, to various agents such as retinoic acid, and in skin carcinomas. AQP3-knockout mice have reduced stratum corneum water content and elasticity compared with wild-type mice, as well as impaired wound healing and epidermal biosynthesis. Reduced AQP3-dependent Glycerol Transport in AQP3-deficient epidermis appears to be responsible for these phenotype findings, as evidenced by reduced Glycerol content in epidermis and stratum corneum in AQP3-knockout mice, and correction of the phenotype abnormalities by Glycerol replacement. Recent data implicate AQP3 as an important determinant in epidermal proliferation and skin tumorigenesis, in which AQP3-knockout mice are resistant to tumor formation by a mechanism that may involve reduced cell Glycerol content and ATP energy for biosynthesis. AQP3 is thus a key player in epidermal biology and a potential target for drug development.
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aquaporin 3 facilitates epidermal cell migration and proliferation during wound healing
Journal of Molecular Medicine, 2008Co-Authors: Mariko Harachikuma, A. S. VerkmanAbstract:Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/Glycerol-Transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was ~80% complete in wild-type mice at 5 days vs ~50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and Glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral Glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water Transport in epidermal cell migration and for AQP3-facilitated Glycerol Transport in epidermal cell proliferation.
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Physiological roles of Glycerol-Transporting aquaporins: the aquaglyceroporins
Cellular and Molecular Life Sciences CMLS, 2006Co-Authors: M. Hara-chikuma, A. S. VerkmanAbstract:A subclass of aquaporin (AQP) water channels, termed aquaglyceroporins, are also able to Transport Glycerol and perhaps urea and other small solutes. Although extensive data exist on the physiological roles of aquaporin-facilitated water Transport, until recently the biological significance of Glycerol Transport by the mammalian aquaglyceroporins has been unknown. There is now compelling evidence for involvement of aquaglyceroporin- facilitated Glycerol Transport in skin hydration and fat cell metabolism. Mice deficient in AQP3 have dry skin, reduced skin elasticity and impaired epidermal biosynthesis. Mice lacking AQP7 manifest progressive adipocyte fat accumulation and hypertrophy. These skin and fat phenotypes are attributable to impaired Glycerol Transport. A potential implication of these findings is the possibility of modulation of aquaglyceroporin expression or function in the therapy of skin diseases and obesity.
Katsuhisa Inoue - One of the best experts on this subject based on the ideXlab platform.
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Competitive Inhibition of AQP7-mediated Glycerol Transport by Glycerol Derivatives
Drug metabolism and pharmacokinetics, 2014Co-Authors: Takahiro Katano, Katsuhisa Inoue, Yuko Ito, Kinya Ohta, Tomoya Yasujima, Hiroaki YuasaAbstract:Summary: Aquaporin 7 (AQP7) is an aquaglyceroporin that has recently been found to operate as a facilitative carrier rather than a channel for Glycerol, although its primary function is as a water channel. To probe into its substrate specificity, we examined the inhibitory effect of a series of acyl Glycerol derivatives on Glycerol Transport mediated by human AQP7 stably expressed in Madin-Darby canine kidney II cells. According to kinetic analyses, AQP7-mediated Glycerol Transport was found to be competitively inhibited by monoacetin, monobutyrin and diacetin. Therefore, it may be possible that they all could be recognized as substrates by AQP7. The inhibition constant ( Κ i ) of monoacetin (134 μΜ) was smaller than that of diacetin (420μΜ), but greater than the Michaelis constant for Glycerol (11.8 μΜ). Considering another finding that inhibition by triacetin was insignificant, it is likely that a decrease in the number of hydroxyl groups in the Glycerol molecule by acetyl derivatization leads to a decrease in affinity for AQP7. The K of monobutyrin (80 μΜ) was, on the other hand, comparable with that of monoacetin, suggesting that the extension of the acyl chain by two hydrocarbon units does not have an impact on affinity for AQP7.
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Functional Characteristics of Aquaporin 7 as a Facilitative Glycerol Carrier
Drug metabolism and pharmacokinetics, 2013Co-Authors: Takahiro Katano, Katsuhisa Inoue, Yuko Ito, Kinya Ohta, Tomoya Yasujima, Hiroaki YuasaAbstract:Summary: Aquaglyceroporins, which constitute a subgroup of aquaporin (AQP) water channels, had been believed to serve as channels for Glycerol as well as for water. However, our recent studies have indicated that AQP9 and AQP10 operate in a carrier mode, which is of saturable nature, for Glycerol Transport. Assuming that such a functional characteristic could also be shared by AQP7, another aquaglyceroporin, we examined its Glycerol Transport function. The specific Transport of Glycerol by human AQP7, which was stably expressed in Madin-Darby canine kidney II cells, was indeed highly saturable, indicating the involvement of a carrier mode of operation mechanism. Kinetic analysis indicated that the specific Transport conformed to Michaelis-Menten kinetics with the Michaelis constant of 11.9 μΜ and was not associated with a nonsaturable Transport component as an indication of a simultaneous channel mode of operation, which was previously indicated for AQP10. AQP7-specific Glycerol Transport was furthermore found to be specifically inhibited by several compounds analogous to Glycerol and operate without requiring either Na + or H + . These characteristics of the carrier mode of AQP7 operation suggest that it is a facilitative carrier for Glycerol and, possibly, also for analogous compounds, providing a novel insight into its operation mechanism.
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Dual Functional Characteristic of Human Aquaporin 10 for Solute Transport
Cellular physiology and biochemistry : international journal of experimental cellular physiology biochemistry and pharmacology, 2011Co-Authors: Megumi Ishii, Katsuhisa Inoue, Jun Watanabe, Takahiro Katano, Kinya Ohta, Kimihiko Urano, Aki Miyamoto, Hiroaki YuasaAbstract:Background/Aims: Although aquaglyceroporins have been generally believed to operate in a channel mode, which is of nonsaturable nature, for Glycerol as well as for water, we recently found that human aquaporin 9 (hAQP9) operates in a carrier-mediated mode, which is of saturable nature, for Glycerol. Based on the finding, we assumed that such a characteristic might be shared by the other aquaglyceroporins and examined the functional characteristics of hAQP10, which is an intestine-specific aquaglyceroporin. Methods: Transport assays were conducted using Xenopus laevis oocytes expressing hAQP10 derived from the microinjected cRNA. Results: The Transport of Glycerol by hAQP10 was found to be highly saturable with a Michaelis constant of 10.4 µM and specifically inhibited by several Glycerol analogs such as monoacetin. Furthermore, when Glycerol was preloaded in hAQP10-expressing oocytes, its efflux was trans-stimulated by extracellular Glycerol. These results indicate the involvement of a carrier-mediated mechanism in Glycerol Transport by hAQP10. Interestingly, a channel mechanism was also found to be involved in part in hAQP10-mediated Glycerol Transport. Conclusion: The present study unveiled the uniquely dual functional characteristic of hAQP10 as a carrier/channel for solute Transport, providing a novel insight into its operation mechanism, which would help further elucidate its physiological role.
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Effect of Glycerol-related compounds on carrier-mediated Glycerol uptake in HCT-15 human colon cancer cell line.
Drug metabolism and pharmacokinetics, 2008Co-Authors: Nami Fujimoto, Yayoi Hayashi, Katsuhisa Inoue, Hiroaki YuasaAbstract:Summary: The effect of several compounds, which are structurally analogous to Glycerol, on carrier-mediated Glycerol uptake was examined in HCT-15 cells to help clarifying the functional characteristics of the Glycerol Transport system. The carrier-mediated uptake of Glycerol conformed to the Michaelis-Menten kinetics with a Michaelis constant of 21.1 μ M and the tested compounds were all suggested to inhibit it competitively with the values of the inhibition constant ( K i ) in the increasing order as follows: monobutyrin (41.0 μ M) μ M) μ M) μ M). Therefore, they all may possibly be substrates of the carrier-mediated Glycerol Transport system, for which the Glycerol esters (monoacetin and monobutyrin) have the highest affinities among them. It was also found that S-( + )-enantiomer of 1,2-propanediol ( K i = 484 μ M) has a higher affinity than its R-( – )-enantiomer ( K i = 19100 μ M), indicating enantioselective recognition. These results support the suggestion that a specific carrier protein is involved in Glycerol uptake in HCT-15 cells. It would be of interest to identify the carrier, which may be present also in some organs, and further investigate the possibility that Glycerol ester derivatives of drugs might be delivered via the carrier.
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Glycerol uptake in HCT-15 human colon cancer cell line by Na(+)-dependent carrier-mediated Transport.
Biological & pharmaceutical bulletin, 2006Co-Authors: Nami Fujimoto, Yayoi Hayashi, Katsuhisa Inoue, Hiroaki YuasaAbstract:It has recently been suggested that an Na+-dependent carrier-mediated Transport system is involved in intestinal Glycerol absorption. Such a Transport system is of general interest as a possible pathway of drug delivery and a target of drug development. However, the Na+-dependent mechanism of cellular Glycerol uptake has not been fully clarified in the small intestine or in any other organ. The purpose of the present study was to examine Glycerol uptake in the HCT-15 human colon cancer cell line, which was found to be able to perform Na+-dependent Glycerol uptake, to determine the Transport characteristics and help identify such Glycerol Transport systems. The uptake of Glycerol in HCT-15 cells was highly saturable with a Michaelis constant of 15.0 μM and a maximum uptake rate of 11.9 pmol/min/mg protein, accompanied by minimal unsaturable Transport; it was reduced markedly under Na+-free conditions, indicating Na+ requirement. Glycerol uptake was also reduced by 2,4-dinitrophenol, a metabolic inhibitor. These results suggest that a carrier-mediated Glycerol Transport system, which is Na+-dependent and secondarily active, is present in HCT-15 cells. The Transport system could be specific for Glycerol and some analogous compounds with hydroxyl groups, since Glycerol uptake was inhibited by some alcohols and compounds related to Glycerol, such as 1,2-propanediol and Glycerol 3-phosphpate. However, it may represent a high affinity Transport system, which is different from the one in the small intestine, because the Michaelis constant of 15.0 μM is about 50-fold lower than that observed in the rat small intestine. In conclusion, this is the first study to demonstrate an Na+-dependent carrier-mediated Glycerol Transport in an established cell line. This will help in identifying a group of Na+-dependent Glycerol Transport systems and elucidating their Transport mechanisms, although the one found in HCT-15 cells in this study seems to be different from one previously found in the rat small intestine.