研究者詳細 - 安井　正人

論文 - 安井　正人

分割表示 >> ／全件表示 110 件中 1 - 110 件目

Adrenergic Receptor Antagonism Induces Neuroprotection and Facilitates Recovery from Acute Ischemic Stroke.

Monai H, Wang X, Yahagi K, Lou N, Mestre H, Xu Q, Abe Y, Yasui M, Iwai Y, Nedergaard M and Hirase H.

Proc. Natl. Acad. Sci. USA. (in press) 2019年

Characterization of Intra/Extracellular Water States Probed by Ultrabroadband Multiplex Coherent Anti-Stokes Raman Scattering (CARS) Spectroscopic Imaging.

Nuriya M, Yoneyama H, Takahashi K, Leproux P, Coudrc V, Yasui M, Kano H.

J. Phys. Chem. A. 123 （ 17 ） 3928 - 3934 2019年

Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis.

Watanabe-Matsumoto S, Moriwaki Y, Okuda T, Ohara S, Yamanaka K, Abe Y, Yasui M, and Misawa H.

Neurosci. Res. 133 48 - 57 2018年

Quantification of anomeric structural changes of glucose solution using near-infrared spectra.

Tanaka S, Kojic D, Tsenkova R and Yasui M.

Carbohydr. Res. 463 40 - 46 2018年

Comparison of diffusion-weighted MRI and anti-Stokes Raman scattering (CARS) measurements of the inter-compartmental exchange-time of water in expression-controlled aquaporin-4 cells.

Obata T, Kershaw J, Tachibana Y, Miyauchi T, Abe Y, Shibata S, Kawaguchi H, Ikoma Y, Takuwa H, Aoki I, Yasui M.

Sci Rep. 8 （ 1 ） 17954 2018年

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

Mestre H, Hablitz LM, Xavier AL, Feng W, Zou W, Pu T, Monai H, Murlidharan G, Castellanos Rivera RM, Simon MJ, Pike MM, Plá V, Du T, Kress BT, Wang X, Plog BA, Thrane AS, Lundgaard I, Abe Y, Yasui M, Thomas JH, Xiao M, Hirase H, Asokan A, Iliff JJ, Nedergaard M.

Elife. 7 2018年

Access to Document (DOI)

High-resolution plasma membrane-selective imaging by second harmonic generation.

Nuriya M, Mizuguchi T and Yasui M.

iScience 9 359 - 366 2018年

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes.

Glady A, Tanaka M, Moniaga CS, Yasui M and Hara-Chikuma M.

Biochem. Biophys. Rep. 14 7 - 15 2018年

Direct posttranslational modification of astrocytic connexin 43 proteins by the general anesthetic propofol in the cerebral cortex.

Nuriya M, Yasui D, Yamada T, Aoki T and Yasui M.

Biochem. Biophys. Res. Commun. 497 （ 2 ） 734 - 741 2018年

Effects of temperature, concentration, and isomer on the hydration structure in monosaccharide solutions.

Tomobe K, Yamamoto E, Yasui M and Yasuoka K.

Phys Chem Chem Phys. 2017年

Background norepinephrine primes astrocytic calcium responses to subsequent norepinephrine stimuli in the cerebral cortex.

Nuriya M, Takeuchi M and Yasui M.

Biochem. Bioph. Res. Commun. 483 （ 1 ） 732 - 738 2017年

査読有り

Improving Accuracy and Reproducibility of Vibrational Spectra for Diluted Solutions.

Kojic D, Tsenkova R and Yasui M.

Anal. Chim. Acta. 955 86 - 97 2017年

査読有り

Origin of the blueshift of water molecules at interfaces of hydrophilic cyclic compounds.

Tomobe K, Yamamoto E, Kojic D, Sato Y, Yasui M and Yasuoka K.

Science Adv. 3 2017年

Access to Document (DOI)

The Dual Effects of the Astrocyte-Specific Enhancer of the AQP4 M1 Promoter.

Abe Y, Goda W, Ikeshima-Kataoka H, and Yasui M.

FEBS Lett. 592 （ 23 ） 3906 - 3915 2017年

Evaluation of Urinary Aquaporin 2 and Plasma Copeptin as Biomarkers of Effectiveness of Desmopressin Acetate for the Treatment of Monosymptomatic Nocturnal Enuresis.

Hara T, Ohtomo Y, Endo A, Niijima S, Yasui M Shimizu T

J Urol. 198 （ 4 ） 921 - 927 2017年

Correlation between astrocyte activity and recovery from blood-brain barrier breakdown caused by brain injury.

Ikeshima-Kataoka H and Yasui M.

Neuroreport. 27 （ 12 ） 894 - 900 2016年

査読有り

High avidity chimeric monoclonal antibodies against the extracellular domains of human aquaporin-4 competing with NMO-IgG.

Miyazaki-Komine K, Takai Y, Huang P, Kusano-Arai O, Iwanari H, Misu T, Koda K, Mitomo K, Sakihama T, Toyama Y, Fujihara K, Hamakubo T, Yasui M and Abe Y.

Br J Pharmacol. 173 （ 1 ） 103 - 114 2016年

査読有り

Multimodal two-photon imaging using a second harmonic generation-specific dye.

Nuriya M, Fukushima S, Momotake A, Shinotsuka T, Yasui M and Arai T.

Nat Commun. 7 11557 2016年

査読有り

The binding property of a mAb against the extracellular domains of AQP4 directs AQP4 toward endocytosis.

Huang P, Takai Y, Kusano-Arai O, Ramadhanti J, Iwanari H, Miyauchi T,

Biochem. Biophys. Rep. 7 77 - 83 2016年

Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica.

Nishiyama S, Misu T, Nuriya M, Takano R, Takahashi T, Nakashima I, Yasui M, Itoyama Y, Aoki M, Fujihara K.

Biochem. Biophys. Rep. 7 45 - 51 2016年

研究論文（学術雑誌）, 共著, 査読有り

Roles of aquaporins in brain disorders

Yasui Masato

Brain and Nerve 67 （ 6 ） 733 - 738 2015年06月

ISSN 1881-6096

Aquaporin (AQP) is a water channel protein that is expressed in the cell membranes. AQPs are related to several kinds of human diseases such as cataract. In the mammalian central nervous system (CNS), AQP4 is specifically expressed in the astrocyte membranes lining the perivascular and periventricular structures. AQP4 plays a role in the development of brain edema associated with certain brain disorders. Neuromyelitis optica (NMO) is a demyelinating disorder, and patients with NMO develop autoimmune antibodies against AQP4 in their serum. Therefore, AQP4 is involved in NMO pathogenesis. A new concept referred to as "glymphatic pathway" has been recently proposed to explain the lymphatic system in the CNS. Dysfunction of the "glymphatic pathway" may cause several neurodegenerative diseases and mood disorders. Importantly, AQP4 may play a role in the "glymphatic pathway". Further investigation of AQP4 in CNS disorders is necessary, and a new drug against AQP4 is expected.

Origin of 1/f noise in hydration dynamics on lipid membrane surfaces

Yamamoto Eiji, Akimoto Takuma, Yasui Masato, Yasuoka Kenji

Scientific Reports 5 2015年03月

Water molecules on lipid membrane surfaces are known to contribute to membrane stability by connecting lipid molecules and acting as a water bridge. Although water structures and diffusivities near the membrane surfaces have been extensively studied, hydration dynamics on the surfaces has remained an open question. Here we investigate residence time statistics of water molecules on the surface of lipid membranes using all-atom molecular dynamics simulations. We show that hydration dynamics on the lipid membranes exhibits 1/f noise. Constructing a dichotomous process for the hydration dynamics, we find that residence times in each state follow a power-law with exponential cutoff and that the process can be regarded as a correlated renewal process where interoccurrence times are correlated. The results imply that the origin of the 1/f noise in hydration dynamics on the membrane surfaces is a combination of a power-law distribution with cutoff of interoccurrence times of switching events and a long-term correlation between the interoccurrence times. These results suggest that the 1/f noise attributed to the correlated renewal process may contribute to the stability of the hydration layers and lipid membranes.

Access to Document (DOI)

Dynamic subcellular localization of aquaporin-7 in white adipocytes

Miyauchi Takayuki, Yamamoto Hiroyuki, Abe Yoichiro, Yoshida Go J., Rojek Aleksandra, Sohara Eisei, Uchida Shinichi, Nielsen Søren, Yasui Masato

FEBS Letters 589 （ 5 ） 608 - 614 2015年02月

ISSN 0014-5793

Aquaporin-7 (AQP7) is expressed in adipose tissue, permeated by water and glycerol, and is involved in lipid metabolism. AQP7-null mice develop obesity, insulin resistance, and adipocyte hypertrophy. Here, we show that AQP7 is expressed in adipocyte plasma membranes, and is re-localized to intracellular membranes in response to catecholamine in mouse white adipose tissue. We found that internalization of AQP7 was induced by PKA activation and comparative gene identification 58 (CGI-58). This relocation was confirmed by functional studies in 3T3-L1 adipocytes. Collectively, these results suggest that AQP7 makes several contributions to adipocyte metabolism, in both cortical and intracellular membranes.

Access to Document (DOI)

Aquaporin 4-dependent expression of glial fibrillary acidic protein and tenascin-C in activated astrocytes in stab wound mouse brain and in primary culture

Ikeshima-Kataoka Hiroko, Abe Yoichiro, Yasui Masato

Journal of Neuroscience Research 93 （ 1 ） 121 - 129 2015年01月

ISSN 0360-4012

We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin-C (TN-C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4-deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN-C were lower in astrocytes from AQP4/KO mice than in those from wild-type (WT) mice. High levels of GFAP and TN-C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN-C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN-C expression in cells from WT mice, whereas the expressions of GFAP and TN-C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN-C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS-stimulated primary culture of astrocytes.

Access to Document (DOI)

Severely exacerbated neuromyelitis optica rat model with extensive astro cytopathy by high affinity anti-aquaporin-4 monoclonal antibody.

Kurosawa K, Misu T, Takai Y, Sato D K, Takahashi T, Abe Y, Iwanari H, Ogawa R, Nakashima I, Fujihara K, Hamakubo T, Yasui M and Aoki M.

Acta Neuropathol Com. 3 （ 1 ） 82 2015年

査読有り

The transmembrane transporter domain of glutamate transporters is a process tip localizer

Hayashi Mariko Kato, Yasui Masato

Scientific Reports 5 2015年

Glutamate transporters in the central nervous system remove glutamate released from neurons to terminate the signal. These transporters localize to astrocyte process tips approaching neuronal synapses. The mechanisms underlying the localization of glutamate transporters to these processes, however, are not known. In this study, we demonstrate that the trimeric transmembrane transporter domain fragment of glutamate transporters, lacking both N- and C-terminal cytoplasmic regions, localized to filopodia tips. This is a common property of trimeric transporters including a neutral amino acid transporter ASCT1. Astrocyte specific proteins are not required for the filopodia tip localization. An extracellular loop at the centre of the 4^th transmembrane helices, unique for metazoans, is required for the localization. Moreover, a C186S mutation at the 4^th transmembrane region of EAAT1, found in episodic ataxia patients, significantly decreased its process tip localization. The transmembrane transporter domain fragments of glutamate transporters also localized to astrocyte process tips in cultured hippocampal slice. These results indicate that the transmembrane transporter domain of glutamate transporters have an additional function as a sorting signal to process tips.

Access to Document (DOI)

Water confined in the local field of ions

Kojić Dušan, Tsenkova Roumiana, Tomobe Katsufumi, Yasuoka Kenji, Yasui Masato

ChemPhysChem 15 （ 18 ） 4077 - 4086 2014年12月

ISSN 1439-4235

Interionic distances are shorter in concentrated ionic solutions, thus instigating the interaction and overlap of hydration shells, as ions become separated by only one or two layers of water molecules. The simultaneous interaction of water with two oppositely charged ions has, so far, only been investigated by computer simulation studies, because the isolated vibrational spectroscopic signature of these molecules remains undetected. Our combined near-infrared spectroscopic and molecular dynamics simulation studies of alkali halide solutions present a distinct spectral feature, which is highly responsive to depletion of bulk water and merging of hydration shells. The analysis of this spectral feature demonstrates that absorption trends are in good agreement with the law of matching affinities, thus providing the first successful vibrational spectroscopic treatment of this topic. Combined with commonly observed near-infrared bands, this feature provides a spectral pattern that describes some relevant aspects of ionic hydration.

Access to Document (DOI)

Mucosal expression of aquaporin-4 in the stomach of histamine type 2 receptor knockout mice and Helicobacter pylori-infected mice

Fukuhara Seiichiro, Matsuzaki Juntaro, Tsugawa Hitoshi, Masaoka Tatsuhiro, Miyoshi Sawako, Mori Hideki, Fukushima Yasushi, Yasui Masato, Kanai Takanori, Suzuki Hidekazu

Journal of Gastroenterology and Hepatology (Australia) 29 （ S4 ） 53 - 59 2014年12月

ISSN 0815-9319

Background and Aim: Basolateral water channel, aquaporin-4 (AQP4), is known to be expressed in gastric parietal cells, especially in the basal side of gastric mucosa. However, the role of AQP4 in the stomach is still unknown. Histamine type 2 receptor (H₂R) knockout mice, which are characterized by suppressed gastric acid secretion, are known as formation of mucosal hyperplasia with cystic dilatation and spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach. The aim of the present study is to investigate whether the expression of AQP4 is changed by the condition of acid suppression and Helicobacter pylori infection. Methods: Male H₂R knockout mice and their controls (C57BL/6) were used. H.pylori was orally infected at the age of 5 weeks. The distributions of AQP4 and H⁺/K⁺-ATPase in the gastric mucosa were investigated by fluorescent immunohistochemistry. The mRNA expressions of AQP4, H⁺/K⁺- ATPase, sonic hedgehog (Shh), and trefoil factor-2 (TFF2) were investigated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Results: In the H₂R knockout mice, the distribution of AQP4-positive parietal cells was extended toward the surface of the fundic glands. Although the mRNA expression levels of AQP4 and H⁺/K⁺ ATPase were elevated in H₂R knockout mice at the age of 20 weeks, the elevations were not maintained by aging or H.pylori infection. In H₂R knockout mice with H.pylori infection, the expression level of TFF2mRNA was elevated while the ratio between AQP4 and H⁺/K⁺ ATPase mRNA expression was decreased compared with the H₂R knockout mice without H.pylori infection. Conclusions: In the H₂R knockout mice, massive SPEM was induced by H.pylori colonization and the ratio between AQP4 and H⁺/K⁺ ATPase mRNA expression was decreased.

Access to Document (DOI)

Sustained down-regulation of β-dystroglycan and associated dysfunctions of astrocytic endfeet in epileptic cerebral cortex

Gondo Asako, Shinotsuka Takanori, Morita Ayaka, Abe Yoichiro, Yasui Masato, Nuriya Mutsuo

Journal of Biological Chemistry 289 （ 44 ） 30279 - 30288 2014年10月

ISSN 0021-9258

Epilepsy is characterized by the abnormal activation of neurons in the cerebral cortex, but the molecular and cellular mechanisms contributing to the development of recurrent seizures are largely unknown. Recently, the critical involvement of astrocytes in the pathophysiology of epilepsy has been proposed. However, the nature of plastic modulations of astrocytic proteins in the epileptic cortex remains poorly understood. In this study, we utilized the zero magnesium in vitro model of epilepsy and examined the potential molecular changes of cortical astrocytes, focusing specifically on endfeet, where specialized biochemical compartments exist. We find that the continuous epileptic activation of neurons for 1 h decreases the expression level of β-dystroglycan (βDG) in acute cortical brain slices prepared from mice. This change is completely abolished by the pharmacological blockade of NMDA-type glutamate receptors as well as by matrix metalloproteinase inhibitors. Consistent with the highly specialized localization of βDG at astrocytic endfeet, where it plays a pivotal role in anchoring endfeet-enriched proteins in astrocytes, the down-regulation of βDG is accompanied by a decrease in the expression of AQP4 but not laminin. Importantly, this down-regulation of βDG persists for at least 1 h, even after the apparent recovery of neuronal activation. Finally, we show that the down-regulation of βDG is associated with the dysfunction of the endfeet at the blood-brain interface as a diffusion barrier. These results suggest that the sustained downregulation of βDG leads to dysfunctions of astrocytic endfeet in the epileptic cerebral cortex and may contribute to the pathogenesis of epilepsy.

Access to Document (DOI)

Expression and localization of aquaporin-4 in sensory ganglia

Kato Jungo, Takai Yoshiki, Hayashi Mariko Kato, Kato Yasuhiro, Tanaka Manami, Sohma Yoshiro, Abe Yoichiro, Yasui Masato

Biochemical and Biophysical Research Communications 451 （ 4 ） 562 - 567 2014年09月

ISSN 0006-291X

Aquaporin-4 (AQP4) is a water channel protein that is predominantly expressed in astrocytes in the CNS. The rapid water flux through AQP4 may contribute to electrolyte/water homeostasis and may support neuronal activities in the CNS. On the other hand, little is known about the expression of AQP4 in the peripheral nervous system (PNS). Using AQP4^-/- mice as a negative control, we demonstrated that AQP4 is also expressed in sensory ganglia, such as trigeminal ganglia and dorsal root ganglia in the PNS. Immunohistochemistry revealed that AQP4 is exclusively localized to satellite glial cells (SGCs) surrounding the cell bodies of the primary afferent sensory neurons in the sensory ganglia. Biochemical analyses revealed that the expression levels of AQP4 in sensory ganglia were considerably lower than those in astrocytes in the CNS. Consistently, behavioral analyses did not show any significant difference in terms of mechanical and cold sensitivity between wild type and AQP4^-/- mice. Overall, although the pathophysiological relevance of AQP4 in somatosensory perception remains unclear, our findings provide new insight into the involvement of water homeostasis in the peripheral sensory system.

Access to Document (DOI)

Origin of subdiffusion of water molecules on cell membrane surfaces

Yamamoto Eiji, Akimoto Takuma, Yasui Masato, Yasuoka Kenji

Scientific Reports 4 2014年04月

Water molecules play an important role in providing unique environments for biological reactions on cell membranes. It is widely believed that water molecules form bridges that connect lipid molecules and stabilize cell membranes. Using all-atom molecular dynamics simulations, we show that translational and rotational diffusion of water molecules on lipid membrane surfaces exhibit subdiffusion and aging. Moreover, we provide evidence that both divergent mean trapping time (continuous-time random walk) and long-correlated noise (fractional Brownian motion) contribute to this subdiffusion. These results suggest that subdiffusion on cell membranes causes the water retardation, an enhancement of cell membrane stability, and a higher reaction efficiency.

Access to Document (DOI)

1 / f fluctuations of amino acids regulate water transportation in aquaporin 1

Yamamoto Eiji, Akimoto Takuma, Hirano Yoshinori, Yasui Masato, Yasuoka Kenji

Physical Review E - Statistical, Nonlinear, and Soft Matter Physics 89 （ 2 ） 2014年02月

ISSN 1539-3755

Aquaporins (AQPs), which transport water molecules across cell membranes, are involved in many physiological processes. Recently, it is reported that the water-water interactions within the channel are broken at the aromatic/arginine selectivity filter (ar/R region), which prevents proton transportation [U. K. Eriksson, Science 340, 1346 (2013)SCIEAS0036-807510.1126/science.1234306]. However, the effects of the conformational fluctuations of amino acids on water transportation remain unclear. Using all-atom molecular dynamics simulations, we analyze water transportation and fluctuations of amino acids within AQP1. The amino acids exhibit 1/f fluctuations, indicating possession of long-term memory. Moreover, we find that water molecules crossing the ar/R region obey a non-Poisson process. To investigate the effect of 1/f fluctuations on water transportation, we perform restrained molecular dynamics simulations of AQP1 and simple Langevin stochastic simulations. As a result, we confirm that 1/f fluctuations of amino acids contribute to water transportation in AQP1. These findings appreciably enhance our understanding of AQPs and suggest possibilities for developing biomimetic nanopores. © 2014 American Physical Society.

Access to Document (DOI)

Unprecedented cell-selection using ultra-quick freezing combined with aquaporin expression

Kato Yasuhiro, Miyauchi Takayuki, Abe Youichiro, Kojić Dušan, Tanaka Manami, Chikazawa Nana, Nakatake Yuhki, Ko Shigeru B H, Kobayashi Daisuke, Hazama Akihiro, Fujiwara Shoko, Uchida Tatsuya, Yasui Masato

PLoS ONE 9 （ 2 ） 2014年02月

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols. © 2014 Kato et al.

Access to Document (DOI)

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Shinotsuka Takanori, Yasui Masato, Nuriya Mutsuo

Biochemical and Biophysical Research Communications 444 （ 2 ） 171 - 176 2014年02月

ISSN 0006-291X

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca²⁺]_i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca²⁺]_i oscillations followed by larger and sustained [Ca²⁺]_i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca²⁺]_i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca²⁺]_i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca²⁺]_i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia. © 2014 Elsevier Inc. All rights reserved.

Access to Document (DOI)

Poisson property of the occurrence of flip-flops in a model membrane

Arai Noriyoshi, Akimoto Takuma, Yamamoto Eiji, Yasui Masato, Yasuoka Kenji

Journal of Chemical Physics 140 （ 6 ） 2014年

ISSN 0021-9606

How do lipid molecules in membranes perform a flip-flop? The flip-flops of lipid molecules play a crucial role in the formation and flexibility of membranes. However, little has been determined about the behavior of flip-flops, either experimentally, or in molecular dynamics simulations. Here, we provide numerical results of the flip-flops of model lipid molecules in a model membrane and investigate the statistical properties, using millisecond-order coarse-grained molecular simulations (dissipative particle dynamics). We find that there are three different ways of flip-flops, which can be clearly characterized by their paths on the free energy surface. Furthermore, we found that the probability of the number of the flip-flops is well fitted by the Poisson distribution, and the probability density function for the inter-occurrence times of flip-flops coincides with that of the forward recurrence times. These results indicate that the occurrence of flip-flops is a Poisson process, which will play an important role in the flexibilities of membranes. © 2014 AIP Publishing LLC.

Access to Document (DOI)

A general anaesthetic propofol inhibits aquaporin-4 in the presence of Zn²⁺

Kato Jungo, Hayashi Mariko Kato, Aizu Shinnosuke, Yukutake Yoshinori, Takeda Junzo, Yasui Masato

Biochemical Journal 454 （ 2 ） 275 - 282 2013年09月

ISSN 0264-6021

AQP4 (aquaporin-4), a water channel protein that is predominantly expressed in astrocyte end-feet, plays an important role in the brain oedema formation, and is thereby considered to be a potential therapeutic target. Using a stopped-flow analysis, we showed that propofol (2,6-diisopropylphenol), a general anaesthetic drug, profoundly inhibited the osmotic water permeability of AQP4 proteoliposomes in the presence of Zn2+ . This propofol inhibition was not observed in AQP1, suggesting the specificity for AQP4. In addition, the inhibitory effects of propofol could be reversed by the removal of Zn2+ . Other lipid membrane fluidizers also similarly inhibited AQP4, suggesting that themodulation of protein-lipid interactions plays an essential role in the propofol-induced inhibition of AQP4. Accordingly, we used Blue native PAGE and showed that the profound inhibition caused by propofol in the presence of Zn2+ is coupled with the reversible clustering of AQP4 tetramers. Site-directed mutagenesis identified that Cys253, located at the membrane interface connecting to the C-terminal tail, is responsible for Zn2+ -mediated propofol inhibition. Overall, we discovered that propofol specifically and reversibly inhibits AQP4 through the interaction between Zn2+ and Cys253. The findings provide new insight into the functional regulation of AQP4 and may facilitate the identification of novel AQP4-specific inhibitors. © 2013 Biochemical Society.

Access to Document (DOI)

Diffusion properties of molecules at the blood-brain interface

Nuriya Mutsuo, Shinotsuka Takanori, Yasui Masato

Cerebral Cortex 23 （ 9 ） 2118 - 2126 2013年09月

ISSN 1047-3211

Molecular diffusion in the extracellular space (ECS) plays a key role in determining tissue physiology and pharmacology. The blood-brain barrier regulates the exchange of substances between the brain and the blood, but the diffusion properties of molecules at this blood-brain interface, particularly around the astrocyte endfeet, are poorly characterized. In this study, we used 2-photon microscopy and acute brain slices of mouse neocortex and directly assessed the diffusion patterns of fluorescent molecules. By observing the diffusion of unconjugated and 10-kDa dextran-conjugated Alexa Fluor 488 from the ECS of the brain parenchyma to the blood vessels, we find various degrees of diffusion barriers at the endfeet: Some allow the invasion of dye inside the endfoot network while others completely block it. Detailed analyses of the time course for dye clearance support the existence of a tight endfoot network capable of acting as a diffusion barrier. Finally, we show that this diffusion pattern collapses under pathological conditions. These data demonstrate the heterogeneous nature of molecular diffusion dynamics around the endfeet and suggest that these structures can serve as the diffusion barrier. Therefore, astrocyte endfeet may add another layer of regulation to the exchange of molecules between blood vessels and brain parenchyma. © 2012 The Author.

Access to Document (DOI)

Immunological function of aquaporin-4 in stab-wounded mouse brain in concert with a pro-inflammatory cytokine inducer, osteopontin

Ikeshima-Kataoka Hiroko, Abe Yoichiro, Abe Takaya, Yasui Masato

Molecular and Cellular Neuroscience 56 65 - 75 2013年09月

ISSN 1044-7431

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3. days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS. © 2013 Elsevier Inc.

Access to Document (DOI)

A novel monoclonal antibody against the c-terminal region of aquaporin-4

Ramadhanti Julia, Huang Ping, Kusano-Arai Osamu, Iwanari Hiroko, Sakihama Toshiko, Misu Tasturo, Fujihara Kazuo, Hamakubo Takao, Yasui Masato, Abe Yoichiro

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 32 （ 4 ） 270 - 276 2013年08月

Aquaporin-4 (AQP4), the most abundant water channel in the brain, plays a central role in water homeostasis, neuronal activity, and migration of astrocytes in the central nervous system. Recent studies have demonstrated that AQP4 is a target of an autoantibody specifically detected in an autoimmune neurologic disease called neuromyelitis optica. Here we have generated a monoclonal antibody (MAb) against the C-terminal region of AQP4 using a baculovirus expressing mouse AQP4 as an immunogen. This antibody (clone E5206) recognized both human and mouse AQP4s in a denaturing condition and was able to precipitate AQP4 from cell lysates of CHO cells stably expressing AQP4. Western blot analysis using deletion mutants revealed that the epitope was located within a region between Asp³⁰³ and Leu³²⁰ in the C-terminal tail of AQP4. Although clone E5206 could not be used for immunostaining when cells or tissues were fixed with 4% paraformaldehyde or 10% formalin, it could be used when cells were fixed with 10% trichloroacetic acid and when a formalin-fixed tissue section was pretreated with antigen-retrieval reagents. This MAb can be a valuable tool for analysis of AQP4 in a variety of physiological and pathophysiological contexts, in human tissues and organs as well as in rodent models, both in vitro and in vivo. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

Access to Document (DOI)

Power-law trapping of water molecules on the lipid-membrane surface induces water retardation

Yamamoto Eiji, Akimoto Takuma, Hirano Yoshinori, Yasui Masato, Yasuoka Kenji

Physical Review E - Statistical, Nonlinear, and Soft Matter Physics 87 （ 5 ） 2013年05月

ISSN 1539-3755

Cell membranes provide unique local environments for biological reactions, where the diffusion of biomolecules as well as water molecules plays critical roles. Translational and rotational motions of water molecules near membranes are known to be slower than those in bulk. Using all-atom molecular dynamics simulations of a membrane, we show that the temperature dependence of the water molecular motions on the membrane surface is different from that in bulk. Decreasing temperature enhances the water retardation on the membrane surface, and the lateral motions of water molecules are correlated with the vertical motions. We find that trapping times of water molecules onto membrane surfaces are distributed according to a power-law distribution and that the power-law exponents depend linearly on temperature, suggesting a random energy landscape picture. Moreover, we find that water molecules on the membrane surfaces exhibit subdiffusions in translational motions. © 2013 American Physical Society.

Access to Document (DOI)

Dynamic interactions of cations, water and lipids and influence on membrane fluidity

Kagawa Rina, Hirano Yoshinori, Taiji Makoto, Yasuoka Kenji, Yasui Masato

Journal of Membrane Science 435 130 - 136 2013年05月

ISSN 0376-7388

Cell membranes, which consist of a lipid bilayer, play a critical role in cellular bioactivities. The lipid bilayer interacts dynamically with water as well as ions. Membrane fluidity is lesser in ionic solutions than in pure water, although how ions reduce membrane fluidity has not yet been clarified. Here, we performed 4×100-ns molecular dynamics simulations of a POPE (1-palmitoyl-2-oleoyl-phosphatidyl-ethanolamine [18:1]) lipid bilayer in the presence or absence of ions (NaCl, KCl, or CaCl₂) and compared the systems to evaluate the influence of ions on the structural dynamics of membranes. Our simulation revealed that membrane fluidity (no ion-system>Ca-system>K-system>Na-system) did not correspond to the distribution or the direct binding of cations to lipids (Ca²⁺>Na⁺>K⁺). Interestingly, we found that the presence of cations induces dehydration of lipids. The number of cations in the interface region corresponded to the degree of lipid dehydration and the reduction in membrane fluidity (Na⁺>K⁺>Ca²⁺). We therefore propose that membrane fluidity is not affected by direct interactions between cations and lipids, but by the dehydration of lipid molecules in the presence of cations located adjacent to the membrane. © 2013 Elsevier B.V.

Access to Document (DOI)

Endfeet serve as diffusion-limited subcellular compartments in astrocytes

Nuriya Mutsuo, Yasui Masato

Journal of Neuroscience 33 （ 8 ） 3692 - 3698 2013年02月

ISSN 0270-6474

Astrocytes extend their processes to make contact with neurons and blood vessels and regulate important processes associated with the physiology/pathophysiology of the brain. Their elaborate morphology, with numerous fine processes, could allow them to perform complex signal transductions with distinct compartments or to function as a spatial buffer depending on the diffusion properties of their intracellular molecules. Apart from calcium ions, however, the diffusion dynamics of molecules within astrocytes are poorly understood. In this study, we applied two-photon uncaging and fluorescence recovery after photobleaching of fluorescent molecules to acute cortical brain slices from mice to investigate the diffusion dynamics of molecules within astrocytes. We found that diffusion was significantly more restricted at the endfeet than at trunks and distal ends of other processes. Slow diffusion dynamics at the endfeet resulted in a large population of molecules being retained in a small region for tens of seconds, creating subcellular compartments that were isolated from other regions. In contrast, diffusion was fast and free at other processes. The same patterns were observed with the diffusions of a higher molecular weight (10 kDa) molecule and 2-NBDG, a fluorescent analog of glucose. These findings suggest that molecular diffusion is not uniform across the intracellular environment and that subcellular compartments are present in astrocytes. Therefore, similar to neurons, the elaborate and specialized structures of astrocytes may enable them to perform complex computations by providing distinct information storage/processing capacity among processes. © 2013 the authors.

Access to Document (DOI)

Characterization of the role of a mechanosensitive channel in osmotic down shock adaptation in Synechocystis sp PCC 6803

Nanatani Kei, Shijuku Toshiaki, Akai Masaro, Yukutake Yoshinori, Yasui Masato, Hamamoto Shin, Onai Kiyoshi, Morishita Megumi, Ishiura Masahiro, Uozumi Nobuyuki

Channels 7 （ 4 ） 238 - 242 2013年

ISSN 1933-6950

Synechocystis sp strain PCC 6803 contains one gene encoding a putative large conductance mechanosensitive channel homolog [named SyMscL (slr0875)]. However, it is unclear whether SyMscL contributes to the adaptation to hypoosmotic stress in Synechocystis. Here we report the in vivo characteristics of SyMscL. SyMscL was mainly expressed in the plasma membrane of Synechocystis. Cell volume monitoring using stopped-flow spectrophotometry showed that ΔsymscL cells swelled more rapidly than wild-type cells under hypoosmotic stress conditions. Expression of symscL was under circadian control, and its peak corresponded to the beginning of subjective night. These results indicate that SyMscL functioned as one component of the osmotic homeostatic regulatory system of the cell coordinating the response of Synechocystis to daily metabolic osmotic fluctuations and environmental changes. © 2013 Landes Bioscience.

Access to Document (DOI)

Establishment of monoclonal antibodies against the extracellular domain that block binding of NMO-IgG to AQP4

Miyazaki Kaori, Abe Yoichiro, Iwanari Hiroko, Suzuki Yota, Kikuchi Takahiro, Ito Takashi, Kato Jungo, Kusano-Arai Osamu, Takahashi Toshiyuki, Nishiyama Shuhei, Ikeshima-Kataoka Hiroko, Tsuji Shoji, Arimitsu Takeshi, Kato Yasuhiro, Sakihama Toshiko, Toyama Yoshiaki, Fujihara Kazuo, Hamakubo Takao, Yasui Masato

Journal of Neuroimmunology 260 （ 1-2 ） 107 - 116 2013年

ISSN 0165-5728

Neuromyelitis optica is a demyelinating disease characterized by a disease-specific autoantibody designated as NMO-IgG that specifically recognizes aquaporin-4, and the binding of NMO-IgG to AQP4 causes the progress of the disease. Prevention of the binding of NMO-IgG, therefore, may alleviate the disease. Here we have developed monoclonal antibodies against AQP4 with a baculovirus display system in order to obtain high affinity monoclonal antibodies against the extracellular domains of AQP4. Our monoclonal antibodies can block the binding of NMO-IgG in spite of their heterogeneity. Taken together, we propose that our monoclonal antibodies can be applied in clinical therapy for NMO patients. © 2013 Elsevier B.V.

Access to Document (DOI)

Common force field thermodynamics of cholesterol

Giangreco Francesco, Yamamoto Eiji, Hirano Yoshinori, Hodoscek Milan, Knecht Volker, Di Giosia Matteo, Calvaresi Matteo, Zerbetto Francesco, Yasuoka Kenji, Narumi Tetsu, Yasui Masato, Höfinger Siegfried

The Scientific World Journal 2013 2013年

Four different force fields are examined for dynamic characteristics using cholesterol as a case study. The extent to which various types of internal degrees of freedom become thermodynamically relevant is evaluated by means of principal component analysis. More complex degrees of freedom (angle bending, dihedral rotations) show a trend towards force field independence. Moreover, charge assignments for membrane-embedded compounds are revealed to be critical with significant impact on biological reasoning. © 2013 Francesco Giangreco et al.

Access to Document (DOI)

Direct visualization and quantitative analysis of water diffusion in complex biological tissues using CARS microscopy

Yu Ying Chun, Sohma Yoshiro, Takimoto Shinichi, Miyauchi Takayuki, Yasui Masato

Scientific Reports 3 2013年

To date, it has not been possible to measure microscopic diffusive water movements in epithelia and in the interstitial space of complex tissues and organs. Diffusive water movements are essential for life because they convey physiologically important small molecules, e.g. nutrients and signaling ligands throughout the extracellular space of complex tissues. Here we report the development of a novel method for the direct observation and quantitative analysis of water diffusion dynamics in a biologically organized tissue using Coherent Anti-Stokes Raman Scattering (CARS) microscopy. Using a computer simulation model to analyze the CARS O-H bond vibration data during H 2 O/D 2 O exchange in a 3D epithelial cyst, we succeeded in measuring the diffusive water permeability of the individual luminal and basolateral water pathways and also their response to hormonal stimulation. Our technique will be applicable to the measurement of diffusive water movements in other structurally complex and medically important tissues and organs.

Access to Document (DOI)

Aquaporin AqpZ is involved in cell volume regulation and sensitivity to osmotic stress in Synechocystis sp. strain PCC 6803

Akai Masaro, Onai Kiyoshi, Morishita Megumi, Mino Hiroyuki, Shijuku Toshiaki, Maruyama Hisataka, Arai Fumihito, Itoh Shigeru, Hazama Akihiro, Checchetto Vanessa, Szabò Ildikò, Yukutake Yoshinori, Suematsu Makoto, Yasui Masato, Ishiura Masahiro, Uozumia Nobuyuki

Journal of Bacteriology 194 （ 24 ） 6828 - 6836 2012年10月

ISSN 0021-9193

The moderately halotolerant cyanobacterium Synechocystis sp. strain PCC 6803 contains a plasma membrane aquaporin, AqpZ. We previously reported that AqpZ plays a role in glucose metabolism under photomixotrophic growth conditions, suggesting involvement of AqpZ in cytosolic osmolarity homeostasis. To further elucidate the physiological role of AqpZ, we have studied its gene expression profile and its function in Synechocystis. The expression level of aqpZ was regulated by the circadian clock. AqpZ activity was insensitive to mercury in Xenopus oocytes and in Synechocystis, indicating that the AqpZ can be categorized as a mercury-insensitive aquaporin. Stopped-flow light-scattering spectrophotometry showed that addition of sorbitol and NaCl led to a slower decrease in cell volume of the Synechocystis ΔaqpZ strain than the wild type. The ΔaqpZ cells were more tolerant to hyperosmotic shock by sorbitol than the wild type. Consistent with this, recovery of oxygen evolution after a hyperosmotic shock by sorbitol was faster in the ΔaqpZ strain than in the wild type. In contrast, NaCl stress had only a small effect on oxygen evolution. The amount of AqpZ protein remained unchanged by the addition of sorbitol but decreased after addition of NaCl. This decrease is likely to be a mechanism to alleviate the effects of high salinity on the cells. Our results indicate that Synechocystis AqpZ functions as a water transport system that responds to daily oscillations of intracellular osmolarity. © 2012, American Society for Microbiology.

Access to Document (DOI)

Structural features of aquaporin 4 supporting the formation of arrays and junctions in biomembranes

Höfinger Siegfried, Yamamoto Eiji, Hirano Yoshinori, Zerbetto Francesco, Narumi Tetsu, Yasuoka Kenji, Yasui Masato

Biochimica et Biophysica Acta - Biomembranes 1818 （ 9 ） 2234 - 2243 2012年09月

ISSN 0005-2736

A limited class of aquaporins has been described to form regular arrays and junctions in membranes. The biological significance of these structures, however, remains uncertain. Here we analyze the underlying physical principles with the help of a computational procedure that takes into account protein-protein as well as protein-membrane interactions. Experimentally observed array/junction structures are systematically (dis)assembled and major driving forces identified. Aquaporin 4 was found to be markedly different from the non-junction forming aquaporin 1. The environmental stabilization resulting from embedding into the biomembrane was identified as the main driving force. This highlights the role of protein-membrane interactions in aquaporin 4. Analysis of the type presented here can help to decipher the biological role of membrane arrays and junctions formed by aquaporin. © 2012 Elsevier B.V. All rights reserved.

Access to Document (DOI)

A phosphatidylinositol lipids system, lamellipodin, and Ena/VASP regulate dynamic morphology of multipolar migrating cells in the developing cerebral cortex

Yoshinaga Satoshi, Ohkubo Takahiro, Sasaki Shinji, Nuriya Mutsuo, Ogawa Yukino, Yasui Masato, Tabata Hidenori, Nakajima Kazunori

Journal of Neuroscience 32 （ 34 ） 11643 - 11656 2012年08月

ISSN 0270-6474

In the developing mammalian cerebral cortex, excitatory neurons are generated in the ventricular zone (VZ) and subventricular zone; these neurons migrate toward the pial surface. The neurons generated in theVZassume a multipolar morphology and remain in a narrow region called the multipolar cell accumulation zone (MAZ) for~24 h, in which they extend and retract multiple processes dynamically. They eventually extend an axon tangentially and begin radial migration using a migratory mode called locomotion. Despite the potential biological importance of the process movement of multipolar cells, the molecular mechanisms remain to be elucidated. Here, we observed that the processes of mouse multipolar cells were actin rich and morphologically resembled the filopodia and lamellipodia in growth cones; thus, we focused on the actin-remodeling proteins Lamellipodin (Lpd) and Ena/vasodilator-stimulated phosphoprotein (VASP). Lpd binds to phosphatidylinositol (3,4)-bisphosphate [PI(3,4)P₂] and recruits Ena/VASP, which promotes the assembly of actin filaments, to the plasma membranes. In situ hybridization and immunohistochemistry revealed that Lpd is expressed in multipolar cells in the MAZ. The functional silencing of either Lpd or Ena/VASP decreased the number of primary processes. Immunostaining and a Förster resonance energy transfer analysis revealed the subcellular localization of PI(3,4)P₂ at the tips of the processes. A knockdown experiment and treatment with an inhibitor for Src homology 2-containing inositol phosphatase-2, a 5-phosphatase that produces PI(3,4)P₂ from phosphatidylinositol (3,4,5)-triphosphate, decreased the number of primary processes. Our observations suggest that PI(3,4)P₂, Lpd, and Ena/VASP are involved in the process movement of multipolar migrating cells. ©2012 the authors.

Access to Document (DOI)

Diffusive nature of xenon anesthetic changes properties of a lipid bilayer

Yamamoto Eiji, Akimoto Takuma, Shimizu Hiroyuki, Hirano Yoshinori, Yasui Masato, Yasuoka Kenji

Journal of Physical Chemistry B 116 （ 30 ） 8989 - 8995 2012年08月

ISSN 1520-6106

Effects of general anesthesia can be controllable by the ambient pressure. We perform molecular dynamics simulations for a 1-palmitoyl-2-oleoyl phosphatidylethanolamine lipid bilayer with or without xenon molecules by changing the pressure to elucidate the mechanism of the pressure reversal of general anesthesia. According to the diffusive nature of xenon molecules in the lipid bilayer, a decrease in the orientational order of the lipid tails, an increase in the area and volume per lipid molecule, and an increase in the diffusivity of lipid molecules are observed. We show that the properties of the lipid bilayer with xenon molecules at high pressure come close to those without xenon molecules at 0.1 MPa. Furthermore, we find that xenon molecules are concentrated in the middle of the lipid bilayer at high pressures by the pushing effect and that the diffusivity of xenon molecules is suppressed. These results suggest that the pressure reversal originates from a jamming and suppression of the diffusivity of xenon molecules in lipid bilayers. © 2012 American Chemical Society.

Access to Document (DOI)

Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

Hiyoshi H., Abdelhady S., Segerström L., Sveinbjörnsson B., Nuriya M., Lundgren T. K., Desfrere L., Miyakawa A., Yasui M., Kogner P., Johnsen J. I., Andäng M., Uhlén P.

British Journal of Cancer 106 （ 11 ） 1807 - 1815 2012年05月

ISSN 0007-0920

Background: Cellular quiescence is a state of reversible proliferation arrest that is induced by anti-mitogenic signals. The endogenous cardiac glycoside ouabain is a specific ligand of the ubiquitous sodium pump, Na,K-ATPase, also known to regulate cell growth through unknown signalling pathways. Methods: To investigate the role of ouabain/Na,K-ATPase in uncontrolled neuroblastoma growth we used xenografts, flow cytometry, immunostaining, comet assay, real-time PCR, and electrophysiology after various treatment strategies. Results: The ouabain/Na,K-ATPase complex induced quiescence in malignant neuroblastoma. Tumour growth was reduced by >50% when neuroblastoma cells were xenografted into immune-deficient mice that were fed with ouabain. Ouabain-induced S-G2 phase arrest, activated the DNA-damage response (DDR) pathway marker γH2AX, increased the cell cycle regulator p21 ^Waf1/Cip1 and upregulated the quiescence-specific transcription factor hairy and enhancer of split1 (HES1), causing neuroblastoma cells to ultimately enter G0. Cells re-entered the cell cycle and resumed proliferation, without showing DNA damage, when ouabain was removed. Conclusion: These findings demonstrate a novel action of ouabain/Na,K-ATPase as a regulator of quiescence in neuroblastoma, suggesting that ouabain can be used in chemotherapies to suppress tumour growth and/or arrest cells to increase the therapeutic index in combination therapies. © 2012 Cancer Research UK.

Access to Document (DOI)

[Role of aquaporin, water channel in the brain].

Yasui Masato

Nihon yakurigaku zasshi. Folia pharmacologica Japonica 139 （ 5 ） 230 2012年05月

ISSN 0015-5691

An astrocyte-specific enhacer of the aquaporin-4 gene functions through a consensus sequence of POU transcription factors in concert with multiple upstream elements

Abe Yoichiro, Ikeshima-Kataoka Hiroko, Goda Wakami, Niikura Takako, Yasui Masato

Journal of Neurochemistry 120 （ 6 ） 899 - 912 2012年03月

ISSN 0022-3042

Aquaporin-4, a predominant water channel in the brain, is specifically expressed in astrocyte endfeet and plays a central role in water homeostasis, neuronal activity, and cell migration in the brain. It has two dominant isoforms called M1 and M23, whose mRNA is driven by distinct promoters located upstream of exons 0 and 1 of the aquaporin-4 gene, respectively. To identify cis-acting elements responsible for the astrocyte-specific transcription of M1 mRNA, the promoter activity of the 5′-flanking region upstream of exon 0 in primary cultured mouse astrocytes was examined by luciferase assay, and sequences, where nuclear factors bind, were identified by electrophoretic mobility shift assay. An astrocyte-specific activity enhancing transcription from the M1 promoter was observed within ∼2 kb from the transcriptional start sites of M1 mRNA. At least five elements clustered within the 286-bp region were found to function as a novel astrocyte-specific enhancer. Among the five elements, a consensus sequence of Pit-1/Oct/Unc-86 (POU) transcription factors was indispensable to the astrocyte-specific enhancer since disruption of the POU motif completely abolished the enhancer activity in astrocytes. However, the POU motif alone had little activity, indicating the requirement for cooperation with other upstream elements to exert full enhancer activity. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Access to Document (DOI)

Pharmaceutical implications of curcumin in the development of drugs for cf pharmacotherapy

Sohma Yoshiro, Yu Ying chun, Miki Haruna, Nakamura Yumi, Hanyuda Akiko, Lin Hui Yi, Yasui Masato, Bompadre Silvia, Hwang Tzyh Chang

223 - 234 2012年02月

The Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel plays an essential role in salt and water transport across epithelia and mutations of CFTR causing its dysfunction result in the genetic disease cystic fibrosis (CF). Various mutations induce CFTR channel dysfunction and various pharmacological approaches are currently being explored. The G551D mutation in CFTR is a common cause of CF, characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Recently we investigated the potentiation effect of curcumin, especially its combined effect with a flavonoid genistein, on G551D-CFTR activity using the patch-clamp technique. Curcumin potentiated G551D-CFTR less than genistein at their maximally effective concentrations. However, curcumin showed an additive effect with genistein and, more importantly, a significant synergistic effect with genistein in a low concentration range. This does not only suggest that multiple mechanisms are involved in action of the CFTR potentiators, but also pose pharmaceutical implications of curcumin in the development of drugs for CF pharmacotherpy. © 2012 Nova Science Publishers, Inc. All rights reserved.

Increased expression of aquaporin-4 with methylmercury exposure in the brain of the common marmoset

Yamamoto Megumi, Takeya Motohiro, Ikeshima-Kataoka Hiroko, Yasui Masato, Kawasaki Yasuaki, Shiraishi Mitsuya, Majima Eiji, Shiraishi Seiji, Uezono Yasuhito, Sasaki Masanori, Eto Komyo

Journal of Toxicological Sciences 37 （ 4 ） 749 - 763 2012年

ISSN 1880-3989

The relationship between methylmercury (MeHg) exposure and aquaporin (AQP) expression in the brain is currently unknown. To investigate this, we used a common marmoset model of acute MeHg exposure to examine AQP1, AQP4 and AQP11 gene expression. MeHg (1.5 mg Hg/kg/day p.o.) was given to three marmosets for 14 days, followed by 14 days without. All treated marmosets showed slight akinesia before sacrifice. In the frontal lobe, occipital lobe and cerebellum, total mercury concentrations following MeHg administration were 26.7, 31.4, and 22.6 μg/g, respectively. Slight apoptosis was observed in the occipital lobe. Immunohistochemistry showed increased expression of glial fibrillary acidic protein, its mRNA and Iba1 with MeHg, indicating that neuronal injury activated astrocytes and microglia. There was no significant difference between control and MeHg-administered groups in AQP1 protein or AQP11 mRNA in the frontal lobe, occipital lobe or cerebellum. The ratio of AQP4 mRNA expression in MeHg-administered marmosets to the mean AQR4 expression in the controls (n = 3) were 1.3, 1.5 and 1.2, 1.7, 1.9 and 1.5, and 1.5, 1.6 and 1.2 for the frontal lobe, occipital lobe and cerebellum, respectively. Western blotting showed significantly increased AQP4 protein in the occipital lobe and cerebellum with MeHg administration, but no obvious up-regulation in the frontal lobe. Immunofluorescence analysis with double staining revealed low AQP4 expression in the cell body of reactive astrocytes in the MeHg-administered group. These results indicate that AQP4 expression might be stimulated by MeHg exposure in astrocytes in the occipital lobe and cerebellum, suggesting a role for AQP4 in MeHg neurotoxicity via astrocyte dysfunction.

Access to Document (DOI)

Non-Gaussian fluctuations resulting from power-law trapping in a lipid bilayer

Akimoto Takuma, Yamamoto Eiji, Yasuoka Kenji, Hirano Yoshinori, Yasui Masato

Physical Review Letters 107 （ 17 ） 2011年10月

ISSN 0031-9007

Anomalous diffusion in lipid bilayers is usually attributed to viscoelastic behavior. We compute the scaling exponent of relative fluctuations of the time-averaged mean square displacement in a lipid bilayer, by using a molecular dynamics simulation. According to the continuous time random walk theory, this exponent indicates non-Gaussian behavior caused by a power-law trapping time. Our results provide the first evidence that a lipid bilayer has not only viscoelastic properties but also trapping times distributed according to a power law. © 2011 American Physical Society.

Access to Document (DOI)

Plasma membrane aquaporin AqpZ protein is essential for glucose metabolism during photomixotrophic growth of Synechocystis sp. PCC 6803

Akai Masaro, Onai Kiyoshi, Kusano Miyako, Sato Mayuko, Redestig Henning, Toyooka Kiminori, Morishita Megumi, Miyake Hiroshi, Hazama Akihiro, Checchetto Vanessa, Szabò Ildikò, Matsuoka Ken, Saito Kazuki, Yasui Masato, Ishiura Masahiro, Uozumi Nobuyuki

Journal of Biological Chemistry 286 （ 28 ） 25224 - 25235 2011年07月

ISSN 0021-9258

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity atpHvalues ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Access to Document (DOI)

Curcumin and genistein additively potentiate G551D-CFTR

Yu Ying Chun, Miki Haruna, Nakamura Yumi, Hanyuda Akiko, Matsuzaki Yohei, Abe Yoichiro, Yasui Masato, Tanaka Kazuhiko, Hwang Tzyh Chang, Bompadre Silvia G., Sohma Yoshiro

Journal of Cystic Fibrosis 10 （ 4 ） 243 - 252 2011年07月

ISSN 1569-1993

Background: The G551D mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is a common cause of cystic fibrosis (CF). G551D-CFTR is characterized by an extremely low open probability despite its normal trafficking to the plasma membrane. Numerous small molecules have been shown to increase the activity of G551D-CFTR presumably by binding to the CFTR protein. Methods: We investigated the effect of curcumin, genistein and their combined application on G551D-CFTR activity using the patch clamp technique. Results: Curcumin increased G551D-CFTR whole-cell and single-channel currents less than genistein did at their maximally effective concentrations. However, curcumin further increased the channel activity of G551D-CFTR that had been already maximally potentiated by genistein, up to ~. 50% of the WT-CFTR level. In addition, the combined application of genistein and curcumin over a lower concentration range synergistically rescued the gating defect of G551D-CFTR. Conclusions: The additive effects between curcumin and genistein not only support the hypothesis that multiple mechanisms are involved in the action of CFTR potentiators, but also pose pharmaceutical implications in the development of drugs for CF pharmacotherapy. © 2011 European Cystic Fibrosis Society.

Access to Document (DOI)

Progesterone maintains amniotic tight junctions during midpregnancy in mice

Kobayashi Ken, Miwa Hideki, Yasui Masato

Molecular and Cellular Endocrinology 337 （ 1-2 ） 36 - 42 2011年04月

ISSN 0303-7207

The amniotic epithelium is in direct contact with the amniotic fluid and restricts fluid flux via the paracellular pathway by means of tight junctions (TJs). Several factors affect TJs to modulate the paracellular flux. Progesterone contributes to the antenatal formation and disappearance of TJs in uterine and mammary epithelial tissues. In this study, we investigated whether progesterone positively or negatively influences amniotic TJs. The administration of RU-486, a progesterone receptor (PR) antagonist, into pregnant mice adversely affects the localization and expression of claudin-3 and claudin-4 in the amniotic epithelium. RU-486 administration also increased the permeability of the amniotic membrane. In organ-cultured amniotic membranes, progesterone induced increases in claudin-3 and claudin-4 expression in a dose-dependent manner but did not influence their localization. PRs were also present in the amniotic epithelium during midpregnancy but they disappeared during late pregnancy. These results indicate that the progesterone/PR pathway maintains TJs in the amniotic epithelium during midpregnancy. © 2011 Elsevier Ireland Ltd.

Access to Document (DOI)

Apoptosis in mouse amniotic epithelium is induced by activated macrophages through the TNF receptor type 1/TNF pathway

Kobayashi Ken, Umezawa Kazuo, Yasui Masato

Biology of Reproduction 84 （ 2 ） 248 - 254 2011年02月

ISSN 0006-3363

The amniotic epithelium is in direct contact with the amniotic fluid and has tight junctions. The amniotic tight junctions function as a barrier to restrict fluid flux via the amniotic membrane during midpregnancy in the mouse. However, during late pregnancy, amniotic fluid volume significantly decreases in association with the disruption of amniotic tight junctions. The disruption of amniotic tight junctions is caused by apoptosis in the amniotic epithelium on Embryonic Day 17 (E17). In this study, we examine the molecular mechanisms underlying apoptosis of the amniotic epithelium of the mouse. We found that from E16, the number of activated macrophages that express high levels of NOS2 and tumor necrosis factor (TNF) increase in amniotic fluid. TNF receptor type 1 (TNFR1) was detectable from E16 onward. On E17, amniotic epithelial cells expressing TNFR1 became TUNEL positive, suggesting that TNF/TNFR1 signaling may initiate apoptosis. To further confirm the role of TNF/TNFR1 signaling, WP9QY, a TNFR1 antagonist, was injected into the amniotic cavity and was found to significantly reduce the numbers of apoptotic cells in the E17 amniotic epithelium. Furthermore, dehydroxymethylepoxyquinomicin, a specific nuclear factor-kappa B inhibitor, was found to inhibit TNF production in macrophages and amniotic apoptosis in vivo. Finally, we showed that injection of TNF into the amniotic cavity induces early onset of apoptosis. These results indicate that amniotic apoptosis is induced by the TNF pathway via TNFR1 expressed in the amniotic epithelial cells and that activation of macrophages may trigger amniotic apoptosis. © 2011 by the Society for the Study of Reproduction, Inc.

Access to Document (DOI)

Analysis of aquaporin-mediated diffusional water permeability by coherent anti-Stokes Raman scattering microscopy

Ibata Keiji, Takimoto Shinichi, Morisaku Toshinori, Miyawaki Atsushi, Yasui Masato

Biophysical Journal 101 （ 9 ） 2277 - 2283 2011年

ISSN 0006-3495

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P_f), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P_d). Here, we report an accurate and instantaneous method for measuring the P_d of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H₂O/D₂O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ_CARS) for the external solution H₂O/D₂O exchange was 16.1 ms, whereas the intracellular H₂O/D₂O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ_CARS for the intracellular H ₂O/D₂O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P_d. The average P_d values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10^-3 and 8.3 ± 2.6 × 10^-4 cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs. © 2011 Biophysical Society.

Access to Document (DOI)

Acid suppression by proton pump inhibitors enhances aquaporin-4 and KCNQ1 expression in gastric fundic parietal cells in mouse

Matsuzaki Juntaro, Suzuki Hidekazu, Minegishi Yuriko, Sugai Etsuko, Tsugawa Hitoshi, Yasui Masato, Hibi Toshifumi

Digestive Diseases and Sciences 55 （ 12 ） 3339 - 3348 2010年12月

ISSN 0163-2116

Background: The widespread use of proton pump inhibitors (PPIs) is known to cause sporadic gastric fundic gland polyps (FGPs). Altered expression and localization of the water or ion transport proteins might contribute to the excess fluid secretion into the cystic lumen for the development of FGPs. Aims: We investigated the alteration of the murine gastric fundic mucosa after PPI treatment, and examined the expression of water channel aquaporin-4 (AQP4) and potassium channel KCNQ1, which are expressed only in the parietal cells in the gastric mucosa. Methods: Male 5-week-old C57BL/6J mice were administered lansoprazole (LPZ) by subcutaneous injection for 8 weeks. The expression of AQP4 and KCNQ1 were investigated by Western blotting, quantitative RT-PCR, and immunohistochemistry. The expression of mucin-6 (Muc6), pepsinogen, and sonic hedgehog (Shh) were also investigated as mucosal cell lineage markers. Results: Gastric mucosal hyperplasia with multiple cystic dilatations, exhibiting similar histological findings to the FGPs, was observed in the LPZ-treated mice. An increase in the number of AQP4-positive parietal cells and KCNQ1-positive parietal cells was observed. The extension of the distribution of AQP4-positive cells toward the surface of the fundic glands was also observed. The expression levels of AQP4 mRNA and protein were significantly enhanced. The expression of KCNQ1 mRNA was correlated with that of AQP4 mRNA in the LPZ-treated mice. Mucous neck-to-zymogenic cell lineage differentiation was delayed in association with decreased expression of Shh in the LPZ-treated mice. Conclusions: PPI administration increased the number of parietal cells with enhanced expression of AQP4 and KCNQ1. © 2010 Springer Science+Business Media, LLC.

Access to Document (DOI)

Inflammatory mediators weaken the amniotic membrane barrier through disruption of tight junctions

Kobayashi Ken, Miwa Hideki, Yasui Masato

Journal of Physiology 588 （ 24 ） 4859 - 4869 2010年12月

ISSN 0022-3751

In chorioamnionitis, intra-amniotic infections render the amniotic fluid an adverse environment for the fetus and increase the risk of fetal mortality and morbidity. It remains unclear how infection crosses the amniotic barrier, which is made up of tight junctions (TJs). In this study, we investigated whether amniotic TJs are disrupted in inflammatory conditions such as chorioamnionitis. Amniotic TJs were disrupted by single applications of interleukin (IL)-1β, IL-6, tumour necrosis factor-α(TNF-α), and prostaglandin E2. In organ-cultured amniotic membranes, these inflammatory mediators decreased the claudin-3 and claudin-4 levels at the apical junction at different times. Injecting IL-6 into the amniotic cavity concurrently induced the disruption of amniotic TJs by decreasing the claudin-3 and claudin-4 levels at the apical junction, and the dysfunction of the amniotic barrier; in contrast, injecting TNF-α weakened the amniotic barrier by inducing apoptosis of the amniotic epithelial cells, with no decrease in claudin-3 and claudin-4 at the apical junction. Furthermore, inflammation in the amniotic membrane, which was induced by the administration of lipopolysaccharide to pregnant mice, concurrently caused dysfunction of the amniotic barrier and disruption of TJs, involving the decrease of claudin-3 and claudin-4 levels at the apical junction and apoptosis in the amniotic epithelium. These results indicate that the adverse effects of the inflammatory mediators on amniotic TJs cause severe dysfunction of the amniotic barrier.Chorioamnionitis increases the risk of mortality and morbidity in newborn infants because of amniotic fluid infection. However, the mechanism by which this infection crosses the amniotic barrier and spreads into the amniotic fluid remains unclear. In this study, we have shown that inflammation weakens the amniotic barrier and that this effect is induced by inflammatory mediators, such as interleukin-1β, interleukin-6, tumour necrosis factor-α, and prostaglandin E2, in distinctly different ways. Knowledge of the mechanism underlying the weakening of the amniotic barrier aids in increasing our understanding of the mechanism underlying antenatal infection. © 2010 The Authors. Journal compilation © 2010 The Physiological Society.

Access to Document (DOI)

Cellular and subcellular localization of aquaporins 1, 3, 8, and 9 in amniotic membranes during pregnancy in mice

Kobayashi Ken, Yasui Masato

Cell and Tissue Research 342 （ 2 ） 307 - 316 2010年11月

ISSN 0302-766X

The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (E12), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between E10 and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at E14 in the lateral membrane and apical junction; at E16 in the lateral membrane alone; at E17 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes. © 2010 Springer-Verlag.

Access to Document (DOI)

A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR

Shimizu Hiroyasu, Yu Ying Chun, Kono Koichi, Kubota Takahiro, Yasui Masato, Li Min, Hwang Tzyh Chang, Sohma Yoshiro

Journal of Physiological Sciences 60 （ 5 ） 353 - 362 2010年09月

ISSN 1880-6546

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. © 2010 The Physiological Society of Japan and Springer.

Access to Document (DOI)

Regulation of water permeability through aquaporin-4

Yukutake Y., Yasui M.

Neuroscience 168 （ 4 ） 885 - 891 2010年07月

ISSN 0306-4522

Aquaporin-4 (AQP4) is a predominant water channel protein in mammalian brains that is distributed with the highest density in the perivascular and subpial astrocyte end-feet. AQP4 is a critical component of an integrated water and potassium homeostasis. Expression and regulation of AQP4 have been studied to understand the roles of AQP4 in physiology and several pathological conditions. Indeed, AQP4 has been implicated in several neurological conditions, such as brain edema and seizure. AQP4 is dynamically regulated at different levels: channel gating, subcellular distribution, phosphorylation, protein-protein interactions and orthogonal array formation. In this review, we focus on the short-term regulation of AQP4. Phosphorylation of AQP4 is important; AQP4 is inhibited when Ser180 is phosphorylated and activated when Ser111 is phosphorylated. AQP4 is also regulated by several metal ions. These metal ions inhibit AQP4 by interacting with the Cys178 residue located in the cytoplasmic loop D, suggesting that AQP4 is regulated by intracellular signaling pathways in response to extracellular stimuli. Recently, it was demonstrated that AQP4 may be inhibited by arylsulfonamides, antiepileptic drugs and other related chemical compounds. Structural analysis of AQP4 may guide a drug design for AQP4. © 2010 IBRO.

Access to Document (DOI)

Molecular mechanisms of how mercury inhibits water permeation through aquaporin-1

Hirano Yoshinori, Okimoto Noriaki, Kadohira Ikuko, Suematsu Makoto, Yasuoka Kenji, Yasui Masato

Biophysical Journal 98 （ 8 ） 1512 - 1519 2010年04月

ISSN 0006-3495

Aquaporin (AQP) functions as a water-conducting pore. Mercury inhibits the water permeation through AQP. Although site-directed mutagenesis has shown that mercury binds to Cys¹⁸⁹ during the inhibition process, it is not fully understood how this inhibits the water permeation through AQP1. We carried out 40 ns molecular dynamics simulations of bovine AQP1 tetramer with mercury (Hg-AQPI) or without mercury (Free AQP1). In Hg-AQPI, Cys¹⁹¹ (Cys¹⁸⁹ In human AQP1) is converted to CyS-SHg⁺ in each monomer. During each last 10 ns, we observed water permeation events occurred 23 times in Free AQP1 and never In Hg-AQPL Mercury binding did not influence the whole structure, but did induce a collapse in the orientation of several residues at the ar/R region. In Free AQP1, backbone oxygen atoms of Gly ¹⁹⁰, Cys¹⁹¹, and Gly¹⁹² lined, and were oriented to, the surface of the water pore channel. In Hg-AQP1, however, the SHg⁺ of Cys191-SHg⁺ was oriented toward the outside of the water pore. As a result, the backbone oxygen atoms of Gly¹⁹⁰, Cys¹⁹¹, and Gly¹⁹² became disorganized and the ar/R region collapsed, thereby obstructing the permeation of water. We suggest that mercury disrupts the water pore of AQP1 through local conformational changes in the ar/R region. © 2010 by the Biophysical Society.

Access to Document (DOI)

Regulation of Kv2.1 phosphorylation in an animal model of anoxia

Ito Takashi, Nuriya Mutsuo, Yasui Masato

Neurobiology of Disease 38 （ 1 ） 85 - 91 2010年04月

ISSN 0969-9961

Conditions such as hypoxia and anoxia inflict serious damage to the brain and continue to be major medical problems. However, the molecular mechanisms that give rise to such damage are not well understood. To elucidate these mechanisms, we established a clinically relevant rodent model of anoxia/recovery by monitoring blood gas levels after oxygen deprivation. Using this animal model, we examined the regulation of Kv2.1, a voltage-gated potassium channel that plays pivotal roles in the homeostasis and survival of neurons. We found that exposure to anoxia induces rapid dephosphorylation of Kv2.1 in the brain, which can be blocked by pre-administration of a NMDA-type glutamate receptor antagonist, memantine. Furthermore, this change is rapidly reversed as the animal recovers from anoxic stress. These results suggest that Kv2.1 is tightly regulated in a clinically relevant animal model of anoxia and further implicate its role in the homeostasis of neurons during anoxic stress. © 2010 Elsevier Inc. All rights reserved.

Access to Document (DOI)

Expression and Distribution of Tight Junction Proteins in Human Amnion During Late Pregnancy

Kobayashi K., Kadohira I., Tanaka M., Yoshimura Y., Ikeda K., Yasui M.

Placenta 31 （ 2 ） 158 - 162 2010年02月

ISSN 0143-4004

Amnion is the innermost layer of the fetal membrane and has been suggested to regulate the volume of amniotic fluid via the amniotic epithelium. The transepithelial pathway is generally restricted by tight junctions (TJs). Thus far, human amniotic TJs have not been identified. In this study, we determined whether the human amniotic epithelium contains TJs. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting analyses showed that the human amniotic epithelium has TJ components, such as occludin, ZO-1, and at least 2 types of claudins, i.e., claudin-4 and claudin-7. The TJ components were found to localize in the lateral membranes and cytoplasm at 35 weeks of gestation; these components disappeared from the lateral membrane at 37 weeks of gestation. Organ culturing of the amnion at 37 weeks gestation induced the relocalization of the TJ proteins from the cytoplasm to the lateral membranes. Furthermore, in cultured amniotic epithelial cells, dexamethasone induced the downregulation of the protein expression of TJs. These findings suggest that the human amniotic epithelium has TJs that disrupt during late pregnancy. The disruption may be induced by several factors such as glucocorticoids present in the amniotic fluid during late pregnancy. © 2009 Elsevier Ltd. All rights reserved.

Access to Document (DOI)

Membrane potential dynamics of axons in cultured hippocampal neurons probed by second-harmonic-generation imaging

Nuriya Mutsuo, Yasui Masato

Journal of Biomedical Optics 15 （ 2 ） 2010年

ISSN 1083-3668

The electrical properties of axons critically influence the nature of communication between neurons. However, due to their small size, direct measurement of membrane potential dynamics in intact and complex mammalian axons has been a challenge. Furthermore, quantitative optical measurements of axonal membrane potential dynamics have not been available. To characterize the basic principles of somatic voltage signal propagation in intact axonal arbors, second-harmonic-generation (SHG) imaging is applied to cultured mouse hippocampal neurons. When FM4-64 is applied extracellularly to dissociated neurons, whole axonal arbors are visualized by SHG imaging. Upon action potential generation by somatic current injection, nonattenuating action potentials are recorded in intact axonal arbors. Interestingly, however, both current-and voltage-clamp recordings suggest that nonregenerative subthreshold somatic voltage changes at the soma are poorly conveyed to these axonal sites. These results reveal the nature of membrane potential dynamics of cultured hippocampal neurons, and further show the possibility of SHG imaging in physiological investigations of axons. © 2010 Society of Photo-Optical Instrumentation Engineers.

Access to Document (DOI)

Rapid and reversible inhibition of aquaporin-4 by zinc

Yukutake Yoshinori, Hirano Yoshinori, Suematsu Makoto, Yasui Masato

Biochemistry 48 （ 51 ） 12059 - 12061 2009年12月

ISSN 0006-2960

Aquaporin-4 (AQP4) is the predominant water channel in the brain. Although AQP4 plays an important role in brain water homeostasis, the molecular mechanisms of AQP4 regulation are not fully understood. In this report, we show how Zn²⁺ rapidly and reversibly decreases the water permeability of AQP4 when it is reconstituted into proteoliposomes. Mutagenesis analysis identified Cys178, located in cytoplasmic loop D, as a target residue of ZnCl₂ inhibition. Moreover, treatment with diamide enhanced the inhibitory effects of ZnCl₂. These results suggest that the water permeability of AQP4 may be regulated by dynamic changes in intracellular Zn²⁺ concentration linked to the cellular redox state. © 2009 American Chemical Society.

Access to Document (DOI)

教育講演5　中枢神経系におけるアクアポリン-4 (AQP4) の役割

安井正人

臨床神経学 49 （ 11 ） 786 - 788 2009年11月

Dynamic Changes in Amniotic Tight Junctions during Pregnancy

Kobayashi K., Inai T., Shibata Y., Yasui M.

Placenta 30 （ 10 ） 840 - 847 2009年10月

ISSN 0143-4004

The amniotic membrane encloses and retains amniotic fluid during pregnancy. In general, fluid flux is regulated by epithelial tissues, which have tight junctions (TJs). However, TJs have not yet been identified in the amniotic epithelium. In this study, we have determined whether the mouse amniotic epithelium contains TJs. Freeze-fracture electron microscopy revealed the presence of strand-like TJs in the amniotic epithelium. Amniotic TJs were composed of occludin; zona occludens (ZO)-1; and claudins 1, 3, 4, and 7. These claudins underwent developmental changes during pregnancy. The localization patterns of the claudins and their detergent solubility drastically changed between embryonic day (E) 16 and E17; the volume of the amniotic fluid also decreased sharply. Furthermore, in vitro assessment of amniotic membrane permeability showed that the amniotic membrane was more permeable on E17 than on E16. On E17, TJ components were sparsely distributed in parts of the amniotic epithelium. The results of Annexin V-fluorescein staining and Terminal dUTP nick-end labeling (TUNEL) assay revealed ongoing apoptosis in all the cells in such regions. The above findings suggest that TJs in the amniotic epithelium maintain amniotic fluid volume during pregnancy, while apoptosis of amniotic epithelial cells between E16 and E17 causes disruption of the TJs. © 2009 Elsevier Ltd. All rights reserved.

Access to Document (DOI)

Activity-dependent regulation of HCN1 protein in cortical neurons

Arimitsu Takeshi, Nuriya Mutsuo, Ikeda Kazushige, Takahashi Takao, Yasui Masato

Biochemical and Biophysical Research Communications 387 （ 1 ） 87 - 91 2009年09月

ISSN 0006-291X

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I_h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons. © 2009 Elsevier Inc. All rights reserved.

Access to Document (DOI)

Drosophila big brain does not act as a water channel, but mediates cell adhesion

Tatsumi Kimiko, Tsuji Shoji, Miwa Hideki, Morisaku Toshinori, Nuriya Mutsuo, Orihara Minako, Kaneko Kazunari, Okano Hideyuki, Yasui Masato

FEBS Letters 583 （ 12 ） 2077 - 2082 2009年06月

ISSN 0014-5793

The neurogenic gene Drosophila big brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis. © 2009 Federation of European Biochemical Societies.

Access to Document (DOI)

Aquaporin 6 binds calmodulin in a calcium-dependent manner

Rabaud Nicole E., Song Linhua, Wang Yiding, Agre Peter, Yasui Masato, Carbrey Jennifer M.

Biochemical and Biophysical Research Communications 383 （ 1 ） 54 - 57 2009年05月

ISSN 0006-291X

Aquaporin 6 (AQP6) is an anion channel that is expressed primarily in acid secreting α-intercalated cells of the kidney collecting duct. In addition, AQP6 anion channel permeability is gated by low pH. Inspection of the N-terminus of AQP6 revealed a putative calmodulin binding site. AQP6-expressing CHO-K1 cell lysates were mixed with calmodulin beads and AQP6 was pulled down in the presence of calcium. Mutagenesis of the N-terminal calmodulin binding site in full length mouse AQP6 resulted in a loss of calmodulin binding activity. Mouse and human AQP6 calmodulin binding site peptides bound dansyl-calmodulin with a dissociation constant of approximately 1 μM. The binding of AQP6 to calmodulin may be an important key to determining the physiological role of AQP6 in the kidney. © 2009 Elsevier Inc. All rights reserved.

Access to Document (DOI)

TSHR mutations as a cause of congenital hypothyroidism in Japan

Narumi Satoshi, Muroya Koji, Abe Yoichiro, Yasui Masato, Asakura Yumi, Adachi Masanori, Hasegawa Tomonobu

Journal of Clinical Endocrinology and Metabolism 94 （ 4 ） 1317 - 1323 2009年04月

ISSN 0021-972X

Context: The prevalence of congenital hypothyroidism (CH) associated with mutations in the TSH receptor gene (TSHR) has not been established. Objective: We examined the frequency of TSHR mutations among patients with permanent primary CH and in the general population in Japan. Subjects and Methods: We enrolled 102 patients with permanent primary CH [70 with "moderate to severe CH" (TSH, >10 mU/liter) and 32 with "mild CH" (TSH, 5-10 mU/liter)], who were identified through newborn screening among 353,000 newborns born in Kanagawa prefecture from October 1979 to June 2006. These subjects were tested for TSHR mutations by PCR-based direct sequencing. We further characterized molecular functions of identified mutant TSHRs in vitro. Results: We found three patients with moderate to severe CH who had biallelic mutations in TSHR and three patients with mild CH who had monoallelic mutations. Observed mutations included one previously characterized mutation (p.R450H) and three uncharacterized mutations (p.G132R, p.A204V, and p.D403N). In vitro experiments confirmed loss of functions of these four mutants. Among four mutations, p.R450H was particularly frequent: six of nine mutant alleles harbored p.R450H. All six alleles with p.R450H commonly carried a minor single nucleotide polymorphism, suggesting a founder effect. We estimated the prevalence of biallelic TSHR mutations to be 4.3% (three in 70) in Japanese patients with moderate to severe CH, and 1 in 118,000 (three in 353,000) in the general Japanese population. Conclusions: In Japan, TSHR mutations are relatively common among patients with CH, and a founder mutation (p.R450H) accounts for about 70% of mutants. Copyright © 2009 by The Endocrine Society.

Access to Document (DOI)

PH regulated anion permeability of aquaporin-6

Yasui Masato

Handbook of Experimental Pharmacology 190 299 - 308 2009年

ISSN 0171-2004

The kidney is a model organ for transport physiology (Nielsen 1996). AQPs are well-characterized in mammalian kidneys, where they facilitate transep-ithelial water reabsorption. Most renal AQPs are expressed either in proximal tubule cells or in collecting duct principal cells, which are known as sites for water reab-sorption. AQP1 is present in both apical and basolateral membranes of proximal tubules, and in descending limbs of Henle's loop where 70% of filtrated water is isoosmotically reabsorbed (King and Agre 1996). AQP2 is expressed in principal cells of the collecting duct; in response to vasopressin, AQP2 translocates from in-tracellular vesicles to the apical plasma membranes, thereby increasing water permeability to concentrate urine (Nielsen et al. 1993, 1995; Knepper 1997; Schrier 2006). AQP3 and AQP4 reside in the basolateral membranes of collecting duct principal cells, where they may provide the exit pathways for urine. AQP7, AQP8, and AQP11 are also present in the proximal tubules (Nielsen et al. 1998). A rat cDNA clone encoding AQP6 was isolated by PCR-based homologous cloning from a rat kidney cDNA library (Ma et al. 1993; Yasui et al. 1999). AQP6 has high sequence homology to AQP0, AQP2, and AQP5. A human AQP6 was also cloned (Ma et al. 1996). Interestingly, the genes encoding AQP2, AQP5, and AQP6 are mapped to chromosome band 12q13 as a family gene cluster at this locus (Ma et al. 1997). Nevertheless, AQP6 is distinct from AQP0, AQP2, and AQP5 in terms of function. Among the renal aquaporins mentioned above, AQP6 has a unique distribution and a distinct function. © 2009 Springer Berlin Heidelberg.

Access to Document (DOI)

Regulation, structure and function of brain aquaporin

Yasui Masato

Clinical Neurology 49 （ 11 ） 786 - 788 2009年

ISSN 0009-918X

Discovery of aquaporin water channel proteins has provided insight into the molecular mechanism of membrane water permeability. In mammalian brain, Aquaporin-4 (AQP4) is the main water channel and is distributed with highest density in the perivascular and subpial astrocyte end-feet. AQP4 is a critical component of an integrated water and potassium homeostasis. Indeed, AQP4 has been implicated in several neurologic conditions, such as brain edema, seizure and even mood disorders. Expression and regulation of AQP4 have been studied to understand the roles of AQP4 in physiological and pathological conditions. Here we discuss about the mechanisms how AQP4 is dynamically regulated at different levels; channel gating, subcellular distribution, phosphorylation, protein-protein interactions and orthogonal array formation. Interestingly, AQP4 has been identified as a target antigen of autoimmune attack in neuromyelitis optica (NMO). We have evaluated putative epitopes on AQP4 for NMO-IgG binding. We have also studied Drosophila Big Brain (Bib), since Bib has high sequence homology to AQP-4, and play an important role for Drosophila neurogenesis. AQP4 may be a potential therapeutic target in several neurologic conditions. Further studies from different aspects are required to develop new drugs against AQP4.

Access to Document (DOI)

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

Laforenza Umberto, Gastaldi Giulia, Polimeni Mariarosa, Tritto Simona, Tosco Marisa, Ventura Ulderico, Scaffino Manuela F., Yasui Masato

BMC Physiology 9 （ 1 ） 2009年

Background. Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel. Results. RT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding. Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein. The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells. Conclusion. The results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi. © 2009 Laforenza et al.

Access to Document (DOI)

Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

Kadohira Ikuko, Abe Yoichiro, Nuriya Mutsuo, Sano Kazumi, Tsuji Shoji, Arimitsu Takeshi, Yoshimura Yasunori, Yasui Masato

Biochemical and Biophysical Research Communications 377 （ 2 ） 463 - 468 2008年12月

ISSN 0006-291X

Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [³²P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition. © 2008 Elsevier Inc. All rights reserved.

Access to Document (DOI)

[Roles of aquaporins in the central nervous system].

Yasui Masato

Masui. The Japanese journal of anesthesiology 57 Suppl 2008年11月

ISSN 0021-4892

Erratum

Yukutake Yoshinori, Tsuji Shoji, Hirano Yoshinori, Adachi Takeshi, Takahashi Toshiyuki, Fujihara Kazuo, Agre Peter, Yasui Masato, Suematsu Makoto

Biology of the Cell 100 （ 9 ） 561 2008年09月

ISSN 0248-4900

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes

Yukutake Yoshinori, Tsuji Shoji, Hirano Yoshinori, Adachi Takeshi, Takahashi Toshiyuki, Fujihara Kazuo, Agre Peter, Yasui Masato, Suematsu Makoto

Biology of the Cell 100 （ 6 ） 355 - 363 2008年06月

ISSN 0248-4900

Background information. Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys¹⁸⁹ as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury-insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys¹⁸⁹ of AQP1. Indeed, the osmotic water permeability (P_f) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl_2-. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni²⁺-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results. The water permeability of AQP4 proteoliposomes with or without HgCl₂ was measured with a stopped-flow apparatus. Surprisingly, the P_f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl₂ within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg²⁺ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site-directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys¹⁷⁸ which is located at loop D facing the intracellular side, is a target responding to Hg²6+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the P_f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.

Access to Document (DOI)

The difference of aquaporin 5 distribution in acinar and ductal cells in lacrimal and parotid glands

Sasaki Yasumasa, Tsubota Kazuo, Kawedia Jitesh D., Menon Anil G., Yasui Masato

Current Eye Research 32 （ 11 ） 923 - 929 2007年11月

ISSN 0271-3683

Purpose: This study was designed to clarify the physiological function and tissue distribution of aquaporin 5 (AQP5) in the lacrimal and parotid glands. Methods: Saliva and tear volumes were compared in AQP5 knockout (AQP5-/-) mice and wild-type mice. Immunohistochemistry and immunoblot analysis were performed in wild-type and AQP5-/- mice. Results: Immunofluorescence of AQP5 staining showed that AQP5 was localized mainly in the ductal cells rather than in the acinar cells of the lacrimal gland. In contrast, in the parotid gland, AQP5 was observed abundantly in acinar cells with undetectable staining in ductal cells. Tear secretion was not changed in AQP5-/- mouse, although saliva secretion was significantly reduced. Conclusions: AQP5 distribution in acinar cells and ductal cells was completely opposite in the lacrimal and parotid glands. The physiological role of AQP5 might be dependent on the characteristic tissue distribution of the protein in the lacrimal and parotid glands. Copyright © Informa Healthcare USA, Inc.

Access to Document (DOI)

Molecular cloning and characterization of mouse aquaporin 6

Nagase Hiroaki, Ågren Johan, Saito Akiko, Liu Kun, Agre Peter, Hazama Akihiro, Yasui Masato

Biochemical and Biophysical Research Communications 352 （ 1 ） 12 - 16 2007年01月

ISSN 0006-291X

In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions. © 2006 Elsevier Inc. All rights reserved.

Access to Document (DOI)

Determinants of AQP6 trafficking to intracellular sites versus the plasma membrane in transfected mammalian cells

Beitz Eric, Liu Kun, Ikeda Masahiro, Guggino William B., Agre Peter, Yasui Masato

Biology of the Cell 98 （ 2 ） 101 - 109 2006年02月

ISSN 0248-4900

Background information. Most AQPs (aquaporins) function at the plasma membrane, however AQP6 is exclusively localized to membranes of intracellular vesicles in acid-secreting type-A intercalated cells of renal collecting ducts. The intracellular distribution indicates that AQP6 has a function distinct from trans-epithelial water movement. Results. We show by mutational analyses and immunofluorescence that the N-terminus of AQP6 is a determinant for its intracellular localization. Presence or absence at the plasma membrane of AQP6 constructs was confirmed by electrophysiological methods. Addition of a GFP (green fluorescent protein) or a HA (haemagglutinin) epitope tag (GFP-AQP6 or HA-AQP6) to the N-terminus of AQP6, directed AQP6 to the plasma membranes of transfected Madin-Darby canine Kidney cells. In contrast, addition of a GFP tag to the C-terminus (AQP6-GFP) caused the protein to remain intracellular, similar to untagged wild-type AQP6. Replacement of the N-terminus of AQP6 by that of AQP1 also directed AQP6 to the plasma membranes, whereas the N-terminus of AQP6 retained AQP1 in cytosolic sites. Conclusion. Our results suggest that the N-terminus of AQP6 is critical for trafficking of the protein to the intracellular sites. Moreover, our studies provide an approach for future identification of proteins involved in vesicle sorting in the acid-secreting type-A intercalated cells.

Access to Document (DOI)

Identification of AQP5 in lipid rafts and its translocation to apical membranes by activation of M₃ mAChRs in interlobular ducts of rat parotid gland

Ishikawa Yasuko, Yuan Zhenfang, Inoue Noriko, Skowronski Mariusz T., Nakae Yoshiko, Shono Masayuki, Cho Gota, Yasui Masato, Agre Peter, Nielsen Søren

American Journal of Physiology - Cell Physiology 289 （ 5 58-5 ） 2005年11月

ISSN 0363-6143

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M₃ muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M₃ mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca²⁺ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. Copyright © 2005 the American Physiological Society.

Access to Document (DOI)

Conversion of aquaporin 6 from an anion channel to a water-selective channel by a single amino acid substitution

Liu Kun, Kozono David, Kato Yasuhiro, Agre Peter, Hazama Akihiro, Yasui Masato

Proceedings of the National Academy of Sciences of the United States of America 102 （ 6 ） 2192 - 2197 2005年02月

ISSN 0027-8424

Aquaporin (AQP) 6 belongs to the aquaporin water channel family. Unlike other aquaporins, AQP6 functions not as a water channel but as an anion-selective channel. Single-channel analyses have shown AQP6 to flicker rapidly between closed and open status. The atomic structure of AQP1 and amino acid sequence alignments of the mammalian aquaporins reveal two well conserved glycine residues: Gly-57 in transmembrane helix (TM) 2 and Gly-173 in TM5 reside at the contact point where the two helices cross in human AQP1. Uniquely, all known mammalian orthologs of AQP6 have an asparagine residue (Asn-60) at the position corresponding to Gly-57. Here we show that a single residue substitution (N60G in rat AQP6) totally eliminates the anion permeability of AQP6 when expressed in Xenopus oocytes, but the N60G oocytes exhibit significantly higher osmotic water permeability under basal conditions. Replacement of the glycine at this site in AQP0, AQP1, and AQP2 blocked expression of the mutants at the oocyte plasma membrane. We propose that the asparagine residue at the contact point between TM2 and TM5 in AQP6 may function as a teeter board needed for rapid structural oscillations during anion permeation.

Access to Document (DOI)

Molecular mechanisms and drug development in aquaporin water channel diseases

Yasui Masato

Journal of Pharmacological Sciences 96 （ 3 ） 260 - 263 2004年11月

ISSN 1347-8613

The discovery of the water channel aquaporin has greatly expanded our understanding of the regulation of the water permeability of biological membranes. The atomic structure of aquaporin-1 (AQP1) demonstrated how aquaporin is freely permeated by water but not protons and provided marked insight into several human disorders. Eleven mammalian aquaporins have been identified, each with a distinct distribution, and these are selectively permeated by water or water plus glycerol. Aquaporins are suspected in numerous pathological conditions involving fluid transport such as brain edema. Knowledge of aquaporin structure may provide insight into the development of new therapeutics through appropriate drug design.

Access to Document (DOI)

Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis

Beitz Eric, Pavlovic-Djuranovic Slavica, Yasui Masato, Agre Peter, Schultz Joachim E.

Proceedings of the National Academy of Sciences of the United States of America 101 （ 5 ） 1153 - 1158 2004年02月

ISSN 0027-8424

The selectivity of aquaporins for water and solutes is determined by pore diameter. Paradoxically, the wider pores of glycerol facilitators restrict water passage by an unknown mechanism. Earlier we characterized an aquaglyceroporin from Plasmodium falciparum with high permeability for both glycerol and water. We use point mutations to demonstrate that amino acids directly lining the pore are not responsible for the excellent water permeability of the Plasmodium aquaglyceroporin but affect permeability of pentitols. Within a conserved WET triad in the extracellular C-loop we identified a Plasmodium aquaglyceroporin-specific glutamate (E125) located in proximity to a conserved arginine (R196) at the pore mouth. Mutation of E125 to serine largely abolished water permeability. Concomitantly, the activation energy for water permeation was increased by 4 kcal/mol. Mutation of the adjacent tryptophan to cysteine led to irreversible inhibition of water passage by Hg²⁺. This unequivocally proves the proximity of the couple WI24/E125 close to the pore mouth. We conclude that in the Plasmodium aquaglyceroporin the electrostatic environment at the extracellular pore entry regulates water permeability.

Access to Document (DOI)

Characterization of aquaporin-6 as a nitrate channel in mammalian cells. Requirement of pore-lining residue threonine 63

Ikeda Masahiro, Beitz Eric, Kozono David, Guggino William B., Agre Peter, Yasui Masato

Journal of Biological Chemistry 277 （ 42 ） 39873 - 39879 2002年10月

ISSN 0021-9258

Aquaporins (AQP) were originally regarded as plasma membrane channels that are freely permeated by water or small uncharged solutes but not by ions. Unlike other aquaporins, AQP6 overexpressed in Xenopus laevis oocytes was previously found to exhibit Hg²⁺ or pH-activated ion conductance. AQP6 could not be analyzed electrophysiologically in mammalian cells, however, because the protein is restricted to intracellular vesicles. Here we report that addition of a green fluorescence protein (GFP) tag to the N terminus of rat AQP6 (GFP-AQP6) redirects the protein to the plasma membranes of transfected mammalian cells. This permitted measurement of rapid, reversible, pH.induced anion currents by GFP-AQP6 in human embryonic kidney 293 cells. Surprisingly, anion selectivity relative to Cl^- revealed high nitrate permeability even at pH 7.4; P_NO3/P_Cl >9.8. Site-directed mutation of a pore-lining threonine to isoleucine at position 63 at the midpoint of the channel reduced NO₃
^-/Cl^- selectivity. Moreover, no anomalous mole-fraction behavior was observed with NO₃
^-/Cl^- mixtures, suggesting a single ion-binding pore in each subunit. Our studies indicate that AQP6 exhibits a new form of anion permeation with marked specificity for nitrate conferred by a specific pore-lining residue, observations that imply that the primary role of AQP6 may be in cellular regulation rather than simple fluid transport.

Access to Document (DOI)

Aquaporins and disease

King Landon S., Yasui Masato

Trends in Endocrinology and Metabolism 13 （ 8 ） 355 - 360 2002年10月

ISSN 1043-2760

Recent discovery of a family of water-specific membrane channel proteins, the aquaporins, has provided new insights into the molecular basis of membrane water permeability. Eleven mammalian aquaporins have been identified to date, with homolog present across the spectrum of life, including bacteria, yeast and plants. The distribution of the mammalian aquaporins predicts their participation in a range of pathophysiological events. Empirical evidence of a physiological role for aquaporins is emerging from studies in both mice and humans, and suggests that aquaporins are likely to play significant roles in human pathophysiology.

Access to Document (DOI)

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation.

Hazama Akihiro, Kozono David, Guggino William B., Agre Peter, Yasui Masato

Journal of Biological Chemistry 277 （ 32 ） 29224 - 29230 2002年08月

ISSN 0021-9258

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.

Aquaporin water channels - From atomic structure to clinical medicine

Agre Peter, King Landon S., Yasui Masato, Guggino Wm B., Ottersen Ole Petter, Fujiyoshi Yoshinori, Engel Andreas, Nielsen Søren

Journal of Physiology 542 （ 1 ） 3 - 16 2002年07月

ISSN 0022-3751

The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single-file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes - ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

Access to Document (DOI)

Aquaporin water channels

Kozono David, Yasui Masato, King Landon S., Agre Peter

Journal of Clinical Investigation 109 （ 11 ） 1395 - 1399 2002年

ISSN 0021-9738

Access to Document (DOI)

Chapter 1 discovery of the aquaporins and their impact on basic and clinical physiology

Agre Peter, Borgnia Mario J., Yasui Masato, Neely John D., Carbrey Jennifer, Kozono David, Beitz Eric, Hoffert Jason, Leitch Virginia, King Landon S.

Current Topics in Membranes 51 1 - 38 2001年

ISSN 1063-5823

Functional impairment of lens aquaporin in two families with dominantly inherited cataracts

Francis Peter, Chung Jean Ju, Yasui Masato, Berry Vanita, Moore Anthony, Wyatt M. Keith, Wistow Graeme, Bhattacharya Shomi S., Agre Peter

Human Molecular Genetics 9 （ 15 ） 2329 - 2334 2000年09月

ISSN 0964-6906

Opacities in the crystalline lens of eye appear with high frequency in the general population. Dominantly inherited cataracts with differing clinical features were found in two families carrying different point mutations in the gene encoding lens water channel protein AQP0 (major intrinsic protein, MIP). Families with E134G have a uni-lamellar cataract which is stable after birth, whereas families with T138R have multi-focal opacities which increase throughout life. To establish pathophysiological relevance of cataract formation, the Xenopus laevis oocyte expression system was employed to evaluate functional defects in the mutant proteins, E134G and T138R. Both substitutions cause loss of membrane water channel activity due to impaired trafficking of the mutant proteins to the oocyte plasma membrane. Although missense mutations in AQP1 and AQP2 proteins are known to result in recessive traits in vivo and in vitro, when E134G or T138R are co-expressed with wild-type AQP0 protein, the mutant proteins exhibit dominant negative behaviour. To our knowledge, these studies represent the first in vitro demonstration of functionally defective AQP0 protein from humans with congenital cataracts. Moreover, these observations predict that less severe defects in the AQP0 protein may contribute to lens opacity in patients with common, less fulminant forms of cataracts.

Aquaporins in health and disease

King Landon S., Yasui Masato, Agre Peter

Molecular Medicine Today 6 （ 2 ） 60 - 65 2000年

ISSN 1357-4310

The molecular basis of membrane water-permeability remained elusive until the recent discovery of the aquaporin water-channel proteins. The fundamental importance of these proteins is suggested by their conservation from bacteria through plants to mammals. Ten mammalian aquaporins have thus far been identified, each with a distinct distribution. In the kidney, lung, eye and brain, multiple water-channel homologs are expressed, providing a network for water transport in those locations. It is increasingly clear that alterations in aquaporin expression or function can be rate-limiting for water transport across certain membranes. Aquaporins are likely to prove central to the pathophysiology of a variety of clinical conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for therapy in diseases of altered water homeostasis.

Access to Document (DOI)

Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance

Promeneur Dominique, Kwon Tae Hwan, Yasui Masato, Kim Gheun Ho, Frøkiæer Jørgen, Knepper Mark A., Agre Peter, Nielsen Søren

American Journal of Physiology - Renal Physiology 279 （ 6 48-6 ） 2000年

ISSN 0363-6127

In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 ± 18 and 151 ± 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH₄Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 ± 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.

Aquaporin-6

Yasui Masato, Kwon Tae Hwan, Knepper Mark A., Nielsen Søren, Agre Peter

Proceedings of the National Academy of Sciences of the United States of America 96 （ 10 ） 5808 - 5813 1999年05月

ISSN 0027-8424

All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism.

Access to Document (DOI)

Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue

Nishimoto Goro, Zelenina Marina, Li Dailin, Yasui Masato, Aperia Anita, Nielsen Søren, Nairn Angus C.

American Journal of Physiology - Renal Physiology 276 （ 2 45-2 ） 1999年02月

ISSN 0363-6127

Aquaporin-2 (AQP2), the protein that mediates arginine vasopressin (AVP)-regulated apical water transport in the renal collecting duct, possesses a single consensus phosphorylation site for cAMP-dependent protein kinase A (PKA) at Ser²⁵⁶. The aim of this study was to examine whether AVP, and other agents that increase cAMP levels, could stimulate the phosphorylation of AQP2 in intact rat renal tissue. Rat renal papillae were prelabeled with ³²P and incubated with vehicle or drugs, and then AQP2 was immunoprecipitated. Two polypeptides corresponding to nonglycosylated (29 kDa) and glycosylated (35-48 kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and dose-dependent increase in phosphorylation of both glycosylated and nonglycosylated AQP2. The threshold dose for a significant increase in phosphorylation was 10 pM, which corresponds to a physiological serum concentration of AVP. Maximal phosphorylation was reached within 1 min of AVP incubation. This effect on AQP2 phosphorylation was mimicked by the vasopressin (V₂) agonist, 1-desamino-[8-D-arginine]vasopressin (DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of Ser²⁵⁶ after incubation of papillae with AVP. The results indicate that AVP stimulates phosphorylation of AQP2 at Ser²⁵⁶ via activation of PKA, supporting the idea that this is one of the first steps leading to increased water permeability in collecting duct cells.

Perinatal changes in expression of aquaporin-4 and other water and ion transporters in rat lung

Yasui Masato, Serlachius Eva, Löfgren Malin, Belusa Roger, Nielsen Søren, Aperia Anita

Journal of Physiology 505 （ 1 ） 3 - 11 1997年11月

ISSN 0022-3751

1. At birth, rapid removal of lung liquid from potential airspaces is required to establish pulmonary gas exchange. To investigate the role for water channels, aquaporins (AQP) and ion transporters in this process, the mRNA expression of AQP, Na⁺,K⁺-ATPase and the amiloride-sensitive Na⁺ channel (ENaC) were studied in the fetal and postnatal rat lung. 2. The mRNA expression of all transporters studied increased postnatally. 3. The following water channels were expressed in the lung, AQP1, 4 and 5. The most specific perinatal induction pattern was observed for AQP4. A sharp and transient increase of AQP4 mRNA occurred just after birth coinciding with the time course for clearance of lung liquid. This transient induction of AQP4 mRNA at birth was lung-tissue specific. Around birth there was a moderate increase in AQP1 mRNA, which was not transient. AQP5 increased continuously until adulthood. 4. Fetal lung AQP4 mRNA was induced by both β-adrenergic agonists and glucocorticoid hormone, which are factors that have been suggested to accelerate the clearance of lung liquid. 5. Immunocytochemistry revealed that AQP4 was located in the basolateral membranes of bronchial epithelia in newborn rats, consistent with the view that this is the major site for perinatal lung liquid absorption. 6. The Na⁺,K⁺-ATPase α1 subunit and ENaC α-subunit mRNA also increased around birth, suggesting that they co-operatively facilitate lung liquid clearance at birth. 7. These data indicate that removal of lung liquid at birth is associated with pronounced and well-synchronized changes in the expression of AQP and the ion transporters studied. The transient perinatal induction of AQP4, which could be prenatally induced by β-adrenergic agonists, and the localization of this water channel strongly suggest that it plays a critical role for removal of lung liquid at the time of birth.

Mouse water channel aquaporin 4

Zelenin S. M., Yasui M., Serlachius E., Lofgren M., Aperia A.

FASEB Journal 11 （ 9 ） 1997年

ISSN 0892-6638

The goal of this study was to examine the regulation and gene structure of AQP4, the water channel that is expressed in brain, lung and kidney. We found that AQP4 expression is developmentally regulated in all tissues. In the lung AQP4 mRNA is transiently increased around birth, indicating its role for lung water clearance, and is inducible by glucocorticoids and 3 -adrenergic agonists. In the brain AQP4 may play a role as an osmoreceptor. We have screened a eDNA library from mouse embryonic brain and mouse genomic library, cloned eDNA and AQP4 gene and found alternative splicing and different starts of transcription. The shorter form of mRNA has another reading frame (from Leu113), which codes for a protein with unknown function. AQP1 gene restriction mapping and partial sequencing of promoters and coding regions demonstrate alternative start of transcription at +3 kb position before beginning of AQP4 translation and the presence of the putative reglJlatory elements AP1, c-Jun and CTF/NF-I. The exon-intron boundaries of the mouse AQP4 gene were identified and target vectors and clones of embryonic stem cells as first step of knockout model generation have been prepared. This study was supported by grants from Swedish Medical Council, the Swedish ttear and Lung Foundation, the Nov() Nordisk Foundation and Karen Elise Jensen foundation. S.Zelenin was supported by Wenner Gren center Foundation.

Adenylate cyclase-coupled vasopressin receptor activates aqp2 promoter via a dual effect on cre and api elements

Yasui Masato

American Journal of Physiology - Heart and Circulatory Physiology 41 （ 4 ） 1997年

ISSN 0363-6135

-Vasopressin plavs an essential role for the regulation of water balance by activating the collecting duct-specific water channel, aquaporin-2 (AQP2). Here we present evidence that vasopressin may also act as a longterm, transcriptional regulator of AQP2. The studies were performed on LLC-PKi cells, which normally express V2 receptor (V2R) and which were transfected with a fragment of the human AQP2 promoter. Activation of the adenylate cyclase-coupled V2R in LLC-PK! cells induced phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP) responsive element binding protein (CREB) and expression of c-Fos. Binding of these factors to the CRE and API site did, in combination, lead to AQP2 promoter activation. These results establish the role of vasopressin as a regulator of transcription and are the first example of how a message from a highly specific receptor is, via a dual effect of the cAMP signal on CREB and immediate early gene expression, transduced to the transcription of a final target protein with known biological effects. gene regulation; phosphorylation; adenosine 3',5'-cyclic monophosphate; a.quaporin-2 I Copyright ©1997 the American Physiological Society.

Glucocorticoids stimulate the maturation of H,K-ATPase in the infant rat stomach

Wang Zheng Ming, Aizman Roman, Grahnquist Lena, Yasui Masato, Hemphälä Åsa, Celsi Gianni

Pediatric Research 40 （ 5 ） 658 - 663 1996年11月

ISSN 0031-3998

The development of gastric H,K-ATPase from fetal to adult life was studied in the rat. The α and β H,K-ATPase mRNA abundance, the protein abundance, and the enzyme activity increased postnatally. The sharpest increase in mRNA and enzyme activity was observed in the weaning period. Several intestinal enzymes are known to be stimulated by glucocorticoids at the time of weaning. To study the role of glucocorticoids in the maturation of gastric H,K-ATPase, we treated 10-d-old rats with a single injection of betamethasone. Twenty-four hours after betamethasone injection, the enzyme activity was significantly higher than in the control animals (2.6-fold, p < 0.05). The abundance of catalytic α H,K-ATPase protein was also increased (2.5-fold, p < 0.01). The time-dependent effect of betamethasone on α H,K- ATPase mRNA was determined from 6 to 24 h after treatment. Glucocorticoids did not significantly alter the mRNA abundance within 18 h. Twenty four hours after injection, the gastric H,K-ATPase mRNA was significantly increased compared with controls (2.8- and 2.2-fold increase for α and β subunits, respectively, p < 0.01 for both). In conclusion this study indicates that glucocorticoids may regulate the long term maturation of gastric H,K-ATPase by indirectly stimulating enzyme synthesis.

Development of urinary concentrating capacity

Yasui M., Makples D., Belusa R., Eklöf A. C., Celsi G., Nielsen S., Aperia A.

American Journal of Physiology 271 （ 2 PART 2 ） 1996年

ISSN 0002-9513

The capacity to concentrate urine develops progressively during postnatal life in most mammalian species. Here we have examined whether low expression of the arginine vasopressin (AVP)-activated water channel aquaporin-2 (AQP-2) may be a limiting factor for the concentrating capacity in the infant rats. Urine osmolality in response to 24-h dehydration increased significantly from 10 to 40 days of age. The most rapid increase occurred during the weaning period, i.e., days 15-20. A similar developmental pattern was observed for AQP-2 mRNA levels in the renal medulla. AQP-2 protein levels also increased markedly from day 10 to 40. Immunohistochemistry revealed that AQP-2 was exclusively located in collecting duct principal cells both in infant and adult rats but that the signal was much weaker in infants. To further examine the relationship between urinary concentrating capacity and AQP-2 expression, we treated rats with a single injection of betamethasone, which is known to accelerate maturation in several organs. Twenty-four hours after treatment, there was an increase in urine osmolality, renal medullary AQP-2 mRNA, and AQP-2 protein levels in infant but not in adult rats. A single injection of a specific V2 agonist caused within 6 h significant increase of AQP-2 mRNA in both infant arid adult. The expression of the mRNA of three other transporters involved in the concentrating process, medullary Na+-K+ATPase a-subunit, Na-K-2Cl cotransporter, and epithelial chloride channel also increased during the weaning period and were upregulated by glucocorticoids. We conclude that there is a well-synchronized development of the many of the components that determine the concentrating capacity and that the low expression of AQP-2 is one of the limiting factors for low concentrating capacity in infants. Copyright ©1996 the American Physiological Society.

Differential effects of glucocorticoids and mineralocorticoids on the mRNA expression of colon ion transporters in infant rats

Wang Z. M., Yasui M., Celsi G.

Pediatric Research 38 （ 2 ） 164 - 168 1995年

ISSN 0031-3998

Several epithelial ion transporters are developmentally regulated in the preweaning period, at the time when the circulating levels of glucocorticoid and mineralocorticoid hormones increase. The specific role of glucocorticoids and mineralocorticoids in the maturation of epithelial ion transport is still disputed. In this study, we investigated the effect of corticosteroids on the mRNA expression of ion transporters in the infant rat colon, a glucocorticoid- and mineralocorticoid-sensitive organ. The expression of the Na,K-ATPase, the H,K-ATPase and the amiloride-sensitive Na⁺ channel mRNA was investigated in control rats from fetal to adult life. We found that the mRNA of the three transporters is temporally up-regulated in the preweaning period. Rats were then injected with a single dose of betamethasone or aldosterone at 10 d of age. The main effect was the glucocorticoid stimulation of the Na,K-ATPase mRNA within 6 h (4-fold). Glucocorticoids did not alter H,K-ATPase nor Na⁺ channel mRNA within 6 h. Aldosterone moderately (1.7-fold) stimulated Na⁺ channel within 6 h, but did not alter Na,K-ATPase nor H,K-ATPase mRNA. Twenty-four hours after injection, both glucocorticoids and mineralocorticoids had less pronounced and distinct effects. In tissue with lower aldosterone receptor abundance (renal cortex) or with no aldosterone receptor (stomach), glucocorticoids induce a similarly rapid increases in Na,K-ATPase mRNA (4-fold within 6 h), whereas aldosterone had no effect within 6 h. However, glucocorticoids did not stimulate Na,K-ATPase mRNA in the brain, a tissue rich in glucocorticoid receptors. This study indicates that Na,K-ATPase is a primary target for glucocorticoids in the preweaning period, and suggests that glucocorticoid induction of Na,K-ATPase mRNA may play an important role in the maturation of epithelial ion transport capacity. The effect is probably mediated by glucocorticoids and not by mineralocorticoid receptors. However, it seems that an auxiliary factor is required for glucocorticoid-dependent stimulation of Na,K-ATPase mRNA.

Glucocorticoids regulate the transcription of Na⁺-K⁺-ATPase genes in the infant rat kidney

Wang Z. M., Yasui M., Celsi G.

American Journal of Physiology - Cell Physiology 267 （ 2 36-2 ） 1994年

ISSN 0363-6143

Glucocorticoids modulate the maturation of Na⁺-K⁺-ATPase mRNA in a tissue- and age-dependent manner. In this study, we report the effect of glucocorticoids on Na⁺-K⁺-ATPase gene transcription in the infant rat kidney. Ten-day-old rats were treated with one intraperitoneal injection of betamethasone. In glucocorticoid-treated rats, there was a significant increase in renal cortical α₁- and β₁-mRNAs (3.08 ± 0.34- and 4.06 ± 0.10-fold). Pretreatment with cycloheximide, an inhibitor of protein synthesis, did not abolish the increase in α₁- and β₁-mRNA after glucocorticoids. The α₁- and β₁-gene transcription rates were significantly increased in nuclei isolated from kidneys of glucocorticoid- treated rat (2.16 ± 0.05- and 3.12 ± 0.50-fold). Interaction between nuclear proteins and Na⁺-K⁺-ATPase α₁-promoter was studied by gel retardation assay. Nuclear protein from glucocorticoid-treated rats retarded a fragment of α₁-promoter that includes a half-consensus glucocorticoid response element (GRE) at position -750 bp but did not retard a fragment including a half-consensus GRE at position -481. Retardation of α₁- promoter was inhibited by incubation with molar excess of GRE or with a monoclonal antibody against glucocorticoid receptor. We conclude that in the infant kidney, glucocorticoids directly stimulate the transcription of α₁- and β₁-Na⁺-K⁺-ATPase subunits. It is likely that the binding of glucocorticoid receptor to α₁-Na⁺-K⁺-ATPase promoter requires the presence of an auxiliary factor.

[¹³C] breath test of medium-chain triglycerides and oligosaccharides in neonates

Hoshi J., Nishida H., Yasui M., Ohishi M., Takahashi M.

Acta Paediatrica Japonica (Overseas Edition) 34 （ 6 ） 674 - 677 1992年

ISSN 0374-5600

It is suitable to examine the utilization of carbohydrates and fats using stable isotope-labelled substrates in neonates because of their non-radioactivity. Administering medium chain triglycerides (MCT) and oligosaccharides is of use in enteral nutrition for a patient with a limited water intake such as a neonate. In this study, the oxidation of MCT and maltose administered orally as an energy supplement in neonates has been examined using a stable isotope-labelled breath test. Five normal term neonates and five growing preterm infants were given [¹³C]-trioctanoin orally and three growing preterm infants were given [¹³C]-glucose and [¹³C]-maltose orally. The [¹³C] enrichment in carbon dioxide was analyzed by isotope ratio mass spectrometry, and oxidation rates over 6 hr and 12 hr respectively, were calculated. The oxidation rates for [¹³C]-octanoin after 6 hr were 46.2 ± 3.6% in preterm infants and 53.5 ± 13.8% in normal neonates, respectively (no significant difference), and 58.4 ± 9.4% and 52.8 ± 6.0% for [¹³C]-glucose and [¹³C]-maltose, respectively (not significant). The results demonstrate that orally administered MCT and maltose are oxidized sufficiently in preterm infants.

　前のページ - 次のページ　

安井 正人

論文 - 安井 正人

安井　正人

論文 - 安井　正人