Noriaki Takeguchi

Publications of Noriaki Takeguchi

• Inhibition of ecto-ATPase activity by curcumin in hepatocellular carcinoma HepG2 cells.

  Authors: Takuto Fujii, Takuma Minagawa, Takahiro Shimizu, Noriaki Takeguchi, Hideki Sakai

  The journal of physiological sciences : JPS. 09/2011; 62(1):53-8.

  Effects of curcumin, a major constituent of turmeric, on ecto-nucleotidases have not been clarified. Here, we investigated whether curcumin affects ecto-nucleotidase activities in humanEffects of curcumin, a major constituent of turmeric, on ecto-nucleotidases have not been clarified. Here, we investigated whether curcumin affects ecto-nucleotidase activities in human hepatocellular carcinoma HepG2 cells. In the cells, high levels of Mg(2+)-dependent activity of ecto-nucleotidases were observed in the presence of 1 mM adenosine triphosphate (ATP). The activity was inhibited by ecto-ATPase inhibitors such as suramin, ZnCl(2) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. On the other hand, the activity was significantly decreased at alkaline pH (pH 9) and was not inhibited by levamisole, an inhibitor of alkaline phosphatase. In the presence of ATP, curcumin inhibited the activity in a concentration-dependent manner (IC(50) = 6.2 μM). In contrast, curcumin had no effects on ecto-nucleotidase activity in the presence of ADP (1 mM) or AMP (1 mM). The K (m) value for ATP hydrolysis of curcumin-sensitive ecto-ATPase was similar to the value of NTPDase2, an isoform of ecto-nucleoside triphosphate diphosphohydrolase. These results suggest that curcumin is a potent inhibitor of ecto-ATPase and may affect extracellular ATP-dependent responses.

• [Molecular mechanisms of H(+) and Cl(-) secretion in gastric parietal cells].

  Authors: Takuto Fujii, Magotoshi Morii, Noriaki Takeguchi, Hideki Sakai

  Nihon yakurigaku zasshi. Folia pharmacologica Japonica. 08/2011; 138(2):51-5.

• Function of K⁺-Cl⁻ cotransporters in the acid secretory mechanism of gastric parietal cells.

  Authors: Takuto Fujii, Kyosuke Fujita, Noriaki Takeguchi, Hideki Sakai

  Biological & pharmaceutical bulletin. 01/2011; 34(6):810-2.

  Gastric proton pump (H⁺, K⁺-ATPase) secretes H⁺ of acid (HCl) via the luminal membrane of parietal cells. For the HCl secretion, Cl⁻- and K⁺-transporting proteins are required. Recent our studiesGastric proton pump (H⁺, K⁺-ATPase) secretes H⁺ of acid (HCl) via the luminal membrane of parietal cells. For the HCl secretion, Cl⁻- and K⁺-transporting proteins are required. Recent our studies have demonstrated that K⁺-Cl⁻ cotransporters (KCC3a and KCC4) are expressed in gastric parietal cells. KCC3a is associated with Na⁺, K⁺-ATPase in the basolateral membrane, and KCC4 is associated with H⁺, K⁺-ATPase in the apical canalicular membrane. This paper summarizes the functional association between KCCs and P-type ATPases and the contribution of these complexes to acid secretion in gastric parietal cells.

• The NH(2)-terminus of K(+)-Cl(-) cotransporter 3a is essential for up-regulation of Na(+),K(+)-ATPase activity.

  Authors: Takuto Fujii, Kyosuke Fujita, Takahiro Shimizu, Noriaki Takeguchi, Hideki Sakai

  Biochemical and biophysical research communications. 09/2010; 399(4):683-7.

  K(+)-Cl(-) cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na(+),K(+)-ATPase alpha1-subunitK(+)-Cl(-) cotransporter-3 has two major amino terminal variants, KCC3a and KCC3b. In LLC-PK1 cells, exogenously expressed KCC3a co-immunoprecipitated with endogenous Na(+),K(+)-ATPase alpha1-subunit (alpha1NaK), accompanying significant increases of the Na(+),K(+)-ATPase activity. Exogenously expressed KCC3b did not co-immunoprecipitate with endogenous alpha1NaK inducing no change of the Na(+),K(+)-ATPase activity. A KCC inhibitor attenuated the Na(+),K(+)-ATPase activity in rat gastric mucosa in which KCC3a is predominantly expressed, while it had no effects on the Na(+),K(+)-ATPase activity in rat kidney in which KCC3b is predominantly expressed. In these tissue samples, KCC3a co-immunoprecipitated with alpha1NaK, while KCC3b did not. Our results suggest that the NH(2)-terminus of KCC3a is a key region for association with alpha1NaK, and that KCC3a but not KCC3b can regulate the Na(+),K(+)-ATPase activity.

• [Functional relationship between K(+)-Cl(-) cotransporters and P-type ATPases in gastric parietal cells]

  Authors: Hideki Sakai, Takuto Fujii, Noriaki Takeguchi

  Seikagaku. The Journal of Japanese Biochemical Society. 06/2009; 81(5):389-93.

• Involvement of aquaporin-5 in differentiation of human gastric cancer cells.

  Authors: Tomoko Watanabe, Takuto Fujii, Takeshi Oya, Naoki Horikawa, Yoshiaki Tabuchi, Yuji Takahashi, Magotoshi Morii, Noriaki Takeguchi, Kazuhiro Tsukada, Hideki Sakai

  The journal of physiological sciences : JPS. 04/2009; 59(2):113-22.

  Litttle is known about the function of aquaporin (AQP) water channels in human gastric cancer. In the upper or middle part of human stomach, we found that expression level of AQP5 protein inLitttle is known about the function of aquaporin (AQP) water channels in human gastric cancer. In the upper or middle part of human stomach, we found that expression level of AQP5 protein in intestinal type of adenocarcinoma was significantly higher than that in accompanying normal mucosa. AQP5 was localized in the apical membrane of the cancer cells. On the other hand, both AQP3 and AQP4 were not up-regulated in the adenocarcinoma. To elucidate the role of AQP5 in cancer cells, AQP5 was exogenously expressed in a cell line of poorly differentiated human gastric adenocarcinoma (MKN45). The AQP5 expression significantly increased the proportion of differentiated cells with a spindle shape, the activity of alkaline phosphatase, a marker for the intestinal epithelial cell type of cancer cells, and the expression level of laminin, an epithelial cell marker. Treatment of the MKN45 cells stably expressing AQP5 with HgCl(2), an inhibitor of aquaporins, significantly decreased the proportion of differentiated cells and the activity of alkaline phosphatase. Our results suggest that up-regulation of AQP5 may be involved in differentiation of human gastric cancer cells.

• Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells.

  Authors: Takuto Fujii, Yuji Takahashi, Akira Ikari, Magotoshi Morii, Yoshiaki Tabuchi, Kazuhiro Tsukada, Noriaki Takeguchi, Hideki Sakai

  The Journal of biological chemistry. 12/2008;

  We studied whether K(+)-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of theWe studied whether K(+)-Cl(-) cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles (TV), while H(+),K(+)-ATPase was expressed in both of them. In contrast, KCC1, KCC2 and KCC3 were not found in either SAV or TV. KCC4 coimmunoprecipitated with H(+),K(+)-ATPase in the lysate of SAV. Interestingly, the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H(+),K(+)-ATPase inhibitor (SCH28080) or the KCC inhibitor (DIOA). The KCC inhibitor suppressed the H(+) uptake into SAV and the H(+),K(+)-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K(+)-Cl(-) cotransport by KCC4 is tightly coupled with H(+)/K(+) antiport by H(+),K(+)-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H(+),K(+)-ATPase, KCC4 was coimmunoprecipitated with H(+),K(+)-ATPase. The rate of recovery of intracellular pH in the KCC4 expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4 non-expressing cells. Our results suggest that KCC4 and H(+),K(+)-ATPase are main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.

• Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H+,K+-ATPase.

  Authors: Magotoshi Morii, Masashi Yamauchi, Tomohiko Ichikawa, Takuto Fujii, Yuji Takahashi, Shinji Asano, Noriaki Takeguchi, Hideki Sakai

  The Journal of biological chemistry. 07/2008; 283(24):16876-84.

  Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/orGastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.

• K+-Cl- Cotransporter-3a Up-regulates Na+,K+-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells.

  Authors: Takuto Fujii, Yuji Takahashi, Yasuo Itomi, Kyosuke Fujita, Magotoshi Morii, Yoshiaki Tabuchi, Shinji Asano, Kazuhiro Tsukada, Noriaki Takeguchi, Hideki Sakai

  The Journal of biological chemistry. 04/2008; 283(11):6869-77.

  Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl(-) cotransporters (KCCs)Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K+-Cl(-) cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na+,K+-ATPase alpha1-subunit (alpha1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K+-dependent ATP-hydrolyzing activity (Na+,K+-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous alpha1NaK, and it recruited alpha1NaK in lipid rafts, accompanying increases of Na+,K+-ATPase activity and ouabain-sensitive Na+ transport activity that were suppressed by DIOA, whereas the total expression level of alpha1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with alpha1NaK. In conclusion, KCC3a forms a functional complex with alpha1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates alpha1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.

• Inhibition of P-type ATPases by [(dihydroindenyl)oxy]acetic acid (DIOA), a K+ -Cl- cotransporter inhibitor.

  Authors: Takuto Fujii, Yuta Ohira, Yasuo Itomi, Yuji Takahashi, Shinji Asano, Magotoshi Morii, Noriaki Takeguchi, Hideki Sakai

  European journal of pharmacology. 05/2007; 560(2-3):123-6.

  [(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K+ -Cl- cotransporter (IC(50)=10 microM). Here we found that DIOA inhibited activities of P-type ATPases such as dog[(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K+ -Cl- cotransporter (IC(50)=10 microM). Here we found that DIOA inhibited activities of P-type ATPases such as dog kidney Na+,K+-ATPase (IC(50)=53 microM), hog gastric H+,K+-ATPase (IC(50)=97 microM) and rabbit muscle Ca(2+)-ATPase (IC(50)=127 microM). In the membrane preparation of the LLC-PK1 cells stably expressing rabbit gastric H+,K+-ATPase, DIOA inhibited activities of the endogenous Na+,K+-ATPase (IC(50)=95 microM) and the exogenous H+,K+-ATPase (IC(50)=75 microM). 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl- channel blocker, had no effects on the DIOA-elicited inhibition of the P-type ATPases. These findings suggest that lower concentration of DIOA (< 20-30 microM) should be used for evaluation of the activity of K+ -Cl- cotransporter without affecting the activities of coexisting Na+,K+ -ATPase and/or H+,K+-ATPase in cells.

• Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2.

  Authors: Hideki Sakai, Tomoyuki Suzuki, Yuji Takahashi, Masashi Ukai, Katsunori Tauchi, Takuto Fujii, Naoki Horikawa, Tetsuji Minamimura, Yoshiaki Tabuchi, Magotoshi Morii, Kazuhiro Tsukada, Noriaki Takeguchi

  FEBS letters. 07/2006; 580(14):3368-74.

  Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared theTumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH(2) to thromboxane A(2) (TXA(2)), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA(2). The present results suggest that overexpression of TXS and subsequent excess production of TXA(2) in the cancer cells may be involved in the tumor growth of human colorectum.

• Overexpression of heat shock protein 70 restores the structural stability and functional defects of temperature-sensitive mutant of large T antigen at nonpermissive temperature.

  Authors: Yoshiaki Tabuchi, Ryosuke Kuribayashi, Ichiro Takasaki, Takeshi Doi, Hideki Sakai, Noriaki Takeguchi, Takashi Kondo, Kenzo Ohtsuka

  Cell stress & chaperones. 02/2006; 11(3):259-67.

  The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. InThe effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33 degrees C (permissive temperature) to 39 degrees C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21(waf1) pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33 degrees C, but it colocalized with T antigen in the nucleus at 39 degrees C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.

• Cyclic AMP-dependent Cl- secretion induced by thromboxane A2 in isolated human colon.

  Authors: Naoki Horikawa, Tomoyuki Suzuki, Takaoki Uchiumi, Tetsuji Minamimura, Kazuhiro Tsukada, Noriaki Takeguchi, Hideki Sakai

  The Journal of physiology. 03/2005; 562(Pt 3):885-97.

  Increased release of thromboxane A(2) (TXA(2)) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA(2) analogue (STA(2))Increased release of thromboxane A(2) (TXA(2)) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA(2) analogue (STA(2)) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA(2) stimulated Cl- secretion in a concentration-dependent manner with an EC(50) of 0.06 microm. The STA(2)-induced Cl- secretion was significantly inhibited by ONO-3708 (10 microm), a specific TXA(2) receptor antagonist. The effect of STA(2) (0.3 microm) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA(2)-induced Cl- secretion in the human colonic mucosa (IC(50) value 1.18 microm). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA(2)-induced Cl- secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 microm), a membrane-permeant cAMP antagonist. STA(2) (0.3 microm) significantly increased the intracellular cAMP levels and the short-circuit current via TXA(2) receptor in a human colonic cell line. These results suggest that the TXA(2)-induced Cl- secretion in the colon is mediated via the cAMP pathway in addition to the Ca(2+)-calmodulin pathway which was previously reported.

• Is the ClC-2 chloride channel involved in the Cl- secretory mechanism of gastric parietal cells?

  Authors: Katsuhito Hori, Yuji Takahashi, Naoki Horikawa, Tetsushi Furukawa, Kazuhiro Tsukada, Noriaki Takeguchi, Hideki Sakai

  FEBS letters. 10/2004; 575(1-3):105-8.

  It has been controversial whether the ClC-2 chloride channel is involved in hydrochloric acid secretion of gastric parietal cells. Here, we investigated whether ClC-2 is the apical Cl- channelIt has been controversial whether the ClC-2 chloride channel is involved in hydrochloric acid secretion of gastric parietal cells. Here, we investigated whether ClC-2 is the apical Cl- channel associated with gastric acid secretion. Two anti-ClC-2 antibodies used in this study reacted with cloned ClC-2 protein expressed in HEK293 cells. In isolated rabbit gastric glands, significant expression of ClC-2 mRNA was observed, but the presence of ClC-2 protein was not clear. Furthermore, no expression of ClC-2 protein was observed in isolated rat and human gastric mucosa. Immunohistochemistry on the rat gastric mucosa showed no significant expression of ClC-2 protein in the parietal cells which showed abundant expression of H+,K+-ATPase. These results indicate that ClC-2 may not be a Cl- -transporting protein for gastric acid secretion in parietal cells.

• Up-regulation of Na(+),K(+)-ATPase alpha 3-isoform and down-regulation of the alpha1-isoform in human colorectal cancer.

  Authors: Hideki Sakai, Tomoyuki Suzuki, Mizuki Maeda, Yuji Takahashi, Naoki Horikawa, Tetsuji Minamimura, Kazuhiro Tsukada, Noriaki Takeguchi

  FEBS letters. 05/2004; 563(1-3):151-4.

  We investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of theWe investigated expression levels of Na(+),K(+)-ATPase alpha-isoforms and their ATPase activities in human colorectal cancer tissue and the accompanying normal mucosa. A decrease in expression of the alpha1-isoform protein was observed in all sampled cancer tissues compared with the normal mucosae. The level of ouabain (5 microM)-sensitive Na(+),K(+)-ATPase activity in carcinomas was 81+/-5% that of in the normal mucosae. The mRNA expression of alpha2- and alpha 4-isoforms was decreased in almost all the carcinoma samples. Interestingly, the expression level of the alpha 3-isoform protein in the cancer tissue was higher than that of the normal mucosa. These results indicate that a decrease in the alpha1-isoform expression and an increase in the alpha 3-isoform expression may be associated with colorectal cancer.

• The cavity structure for docking the K(+)-competitive inhibitors in the gastric proton pump.

  Authors: Shinji Asano, Ayumi Yoshida, Hiroaki Yashiro, Yusuke Kobayashi, Anna Morisato, Haruo Ogawa, Noriaki Takeguchi, Magotoshi Morii

  The Journal of biological chemistry. 05/2004; 279(14):13968-75.

  2-Methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) is a reversible inhibitor specific for the gastric proton pump. The inhibition pattern is competitive with K(+). Here we2-Methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) is a reversible inhibitor specific for the gastric proton pump. The inhibition pattern is competitive with K(+). Here we studied the binding sites of this inhibitor on the putative three-dimensional structure of the gastric proton pump alpha-subunit that was constructed by homology modeling based on the structure of sarcoplasmic reticulum Ca(2+) pump. Alanine and serine mutants of Tyr(801) located in the fifth transmembrane segment of the gastric proton pump alpha-subunit retained the (86)Rb transport and K(+)-dependent ATPase (K(+)-ATPase) activities. These mutants showed 60-80-times lower sensitivity to SCH 28080 than the wild type in the (86)Rb transport activity. The K(+)-ATPase activities of these mutants were not completely inhibited by SCH 28080. The sensitivity to SCH 28080 was dependent on the bulkiness of the side chain at this position. Therefore, the side chain of Tyr(801) is important for the interaction with this inhibitor. In the three-dimensional structure of the E(2) form (conformation with high affinity for K(+)) of the gastric proton pump, Tyr(801) faces a cavity surrounded by the first, fourth, fifth, sixth, and eighth transmembrane segments and fifth/sixth, seventh/eighth, and ninth/tenth loops. SCH 28080 can dock in this cavity. However, SCH 28080 cannot dock in the same location in the E(1) form (conformation with high affinity for proton) of the gastric proton pump due to the drastic rearrangement of the transmembrane helices between the E(1) and E(2) forms. These results support the idea that this cavity is the binding pocket of SCH 28080.

• Leukotrienes-mediated effects of water extracts from Sargassum horneri, a marine brown alga, on Cl- absorption in isolated rat colon.

  Authors: Hideki Sakai, Takaoki Uchiumi, Jung-Bum Lee, Yuta Ohira, Jun-ichi Ohkura, Tomoyuki Suzuki, Toshimitsu Hayashi, Noriaki Takeguchi

  The Japanese journal of physiology. 03/2004; 54(1):71-7.

  Sargassum horneri is an edible marine brown alga distributed along the seacoast of Japan. Here we examined effects on the water-soluble (ethanol-insoluble) extracts (EIS) from Sargassum horneri onSargassum horneri is an edible marine brown alga distributed along the seacoast of Japan. Here we examined effects on the water-soluble (ethanol-insoluble) extracts (EIS) from Sargassum horneri on ion transports across the isolated rat colonic mucosa set in Ussing chambers. The nonpolysaccharide fraction of EIS (EIS-2) significantly decreased short-circuit current (Isc) across the mucosa, and increased the tissue conductance (Gt). The half-maximal effect of EIS-2 was obtained at 20 microg/ml. In contrast, the polysaccharide fraction of EIS (EIS-1; 100 microg/ml) had little effect on Isc and Gt. The effect of EIS-2 depended on the presence of Cl- and HCO3- but not K+ in the bathing solution. These results suggest that EIS-2 stimulates Cl)absorption in the colonic mucosa. The EIS-2-induced changes in Isc and Gt were inhibited by 3-(1-[p-chlorobenzyl]-5-[isopropyl]-3-t-butylthioindol-2-yl)-2,2-dimethyl-propanoic acid sodium (MK-886; 10 microM), a 5-lipoxygenase-activating protein inhibitor, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 100 microM), a Cl- channel blocker. EIS-2 attenuated the prostaglandin E2 (0.5 microM)-increased Isc, and the half-maximal effect of EIS-2 was obtained at 50 microg/ml. The present study suggests that the EIS-2 stimulates Cl- absorption mediated by basolateral leukotriene-sensitive Cl- channels and apical Cl-/HCO3- exchanger in the rat colonic mucosa.

• Molecular and cellular regulation of the gastric proton pump.

  Authors: Shinji Asano, Magotoshi Morii, Noriaki Takeguchi

  Biological & pharmaceutical bulletin. 02/2004; 27(1):1-12.

  The gastric H+, K+-ATPase is a proton pump that is responsible for gastric acid secretion and that actively transports protons and K+ ions in opposite directions to generate in excess of aThe gastric H+, K+-ATPase is a proton pump that is responsible for gastric acid secretion and that actively transports protons and K+ ions in opposite directions to generate in excess of a million-fold gradient across the membrane under physiological conditions. This pump is also a target molecule of proton pump inhibitors which are used for the clinical treatment of hyperacidity. In this review, we wish to summarize the molecular regulation of this pump based on mutational studies, particularly those used for the identification of binding sites for cations and specific inhibitors. Recent reports by Toyoshima et al (2000, 2002) presented precise three-dimensional (3-D) structures of the sarcoplasmic reticulum (SR) Ca2+-ATPase, which belongs to the same family as the gastric H+, K+-ATPase. We have studied the structure-function relationships for the gastric H+, K+-ATPase using 3-D structures constructed by homology modeling of the related SR Ca2+-ATPase, which was used as a template molecule. We also discuss in this review, the regulation of cell surface expression and synthesis control of the gastric proton pump.

• Inhibition of small-conductance Cl- channels by the interleukin-1beta-stimulated production of superoxide in rabbit gastric parietal cells.

  Authors: Hideki Sakai, Yuta Ohira, Akiko Tanaka, Tomoyuki Suzuki, Akira Ikari, Magotoshi Morii, Noriaki Takeguchi

  The Journal of physiology. 09/2003; 551(Pt 1):207-17.

  We have shown previously that the G protein-coupled production of superoxide anion (O2-) leads to closure of small-conductance Cl- channels (0.3-0.4 pS) in the basolateral membrane of rabbit parietalWe have shown previously that the G protein-coupled production of superoxide anion (O2-) leads to closure of small-conductance Cl- channels (0.3-0.4 pS) in the basolateral membrane of rabbit parietal cells. In the present study, effects of interleukin-1beta (IL-1beta) on the Cl- channel were investigated. In the whole-cell patch-clamp recording, IL-1beta (0.3-10 ng ml-1) inhibited the whole-cell Cl- current recorded from a parietal cell within isolated rabbit gastric glands. Variance noise analysis of the whole-cell Cl- current showed that the single channel conductance of the Cl- channel that is sensitive to IL-1beta is 0.37 pS. The IL-1beta (1 ng ml-1)-induced decrease of the Cl- current was abolished by anti-IL-1beta antibody (2 microg ml-1), recombinant IL-1 receptor antagonist (500 ng ml-1), GDPbetaS (500 microM) and superoxide dismutase (100 units ml-1), a scavenger of O2-. Northern blot analysis showed that the mRNA of the IL-1 receptor was selectively expressed in rabbit gastric parietal cells. In the dihydrofluorescein diacetate-loaded single parietal cells in gastric glands, IL-1beta (0.3-10 ng ml-1) stimulated the production of oxygen radicals. Y-27632 (1-10 microM), a specific Rho-kinase inhibitor, and fluvastatin (10 microM), an indirect inhibitor for Rho proteins, significantly inhibited the IL-1beta-induced effects on the channel activity and production of oxygen radicals. IL-1beta (0.3-10 ng ml-1) activated Rho in the parietal cells. These results indicate that IL-1beta binds to the IL-1 receptor of gastric parietal cells and inhibits the small-conductance Cl- channel via the G protein-mediated Rho/Rho-kinase-dependent production of O2-.

• Quantity and quality control of gastric proton pump in the endoplasmic reticulum by ubiquitin/proteasome system.

  Authors: Tohru Kimura, Hokara Ishizuka, Ayumi Yoshida, Magotoshi Morii, Noriaki Takeguchi, Shinji Asano

  Biochemistry. 06/2003; 42(17):4771-9.

  The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ERThe gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the noncatalytic beta-subunit. These subunits are assembled in the endoplasmic reticulum (ER) and leave the ER to reach to the cell surface as a functional holoenzyme. We studied the quantity control mechanism of the H(+),K(+)-ATPase in the ER by using a heterologous expression system in human embryonic kidney 293 cells. The alpha-subunit in the alpha-expressing cells was degraded more rapidly than in the alpha+beta-expressing cells. It was stabilized, however, in the presence of a proteasome inhibitor, lactacystin. Polyubiquitination of the alpha-subunit was observed in the alpha-expressing cells as well as in the alpha+beta-expressing cells. The extent of polyubiquitination was higher in the former alpha-expressing cells especially in the presence of lactacystin. On the other hand, polyubiquitination of the beta-subunit was not observed in the absence and presence of lactacystin. When the alpha-subunit was coexpressed with a mutant beta-subunit that lacks alpha/beta assembly capacity, degradation of the alpha-subunit was accelerated in parallel with increased polyubiquitination of the alpha-subunit. These results indicate that the ubiquitin/proteasome system is involved in degradation of the unassembled alpha-subunits in the ER to control the cell surface expression of the functional alpha/beta holoenzymes.