研究者詳細 - 安井　正人

論文 - 安井　正人

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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.

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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.

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