Takuto Fujii

Publications of Takuto Fujii

• 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.

• Bimodal effect of alkalization on the polycystin transient receptor potential channel, PKD2L1.

  Authors: Takahiro Shimizu, Taiga Higuchi, Takuto Fujii, Bernd Nilius, Hideki Sakai

  Pflügers Archiv : European journal of physiology. 02/2011; 461(5):507-13.

  Polycystic kidney disease 2-like 1(PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms constitutively active voltage-dependent nonselective cation channels in thePolycystic kidney disease 2-like 1(PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms constitutively active voltage-dependent nonselective cation channels in the plasma membrane. The mechanism of regulation of PKD2L1 channels, however, has been poorly understood. In the present study, we found a bell-shaped alkaline pH dependence of PKD2L1 channel activity at the single-channel and whole-cell levels in patch-clamp recordings in HEK293T cells overexpressing mouse PKD2L1: alkalization to pH 8.0-9.0 increased the PKD2L1 currents, but alkalization to pH 10.0 decreased them. Single-channel analysis revealed that alkalization changed the open probability of PKD2L1 channels, but not their single-channel conductance. In addition, the voltage dependence of PKD2L1 channels was negatively and positively shifted by treatment with solutions of pH 8.0-9.0 and pH 10.0, respectively. These results indicate that the voltage-dependent gating of PKD2L1 channels was modulated by alkalization through two different mechanisms. Interestingly, we observed rebound activation of the PKD2L1 channel on washout of the alkaline solution after PKD2L1 channel inhibition at pH 10.0, suggesting that alkalization to pH 10.0 decreased PKD2L1 currents by inactivating the channels. Consistently, the PKD2L1 tail currents were accelerated by alkalization. These results suggest that alkalization is a bimodal modulator of mouse PKD2L1 channels.

• 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.

• Increase in ouabain-sensitive K+-ATPase activity in hepatocellular carcinoma by overexpression of Na+, K+-ATPase alpha 3-isoform.

  Authors: Kazuto Shibuya, Junya Fukuoka, Takuto Fujii, Eri Shimoda, Takahiro Shimizu, Hideki Sakai, Kazuhiro Tsukada

  European journal of pharmacology. 07/2010; 638(1-3):42-6.

  Na(+),K(+)-ATPase is a housekeeping pump in virtually all animal cells. Recently, cardiac glycosides that inhibit Na(+),K(+)-ATPase have been reported to be candidate for novel anticancer drug. Here,Na(+),K(+)-ATPase is a housekeeping pump in virtually all animal cells. Recently, cardiac glycosides that inhibit Na(+),K(+)-ATPase have been reported to be candidate for novel anticancer drug. Here, we investigated clinical significance of Na(+),K(+)-ATPase alpha1-isoform (alpha 1NaK), alpha2-isoform (alpha 2NaK) and alpha 3-isoform (alpha 3NaK) in hepatocellular carcinoma (HCC). Interestingly, the expression levels of alpha 3NaK protein in HCC tissues were significantly higher than those in the accompanying non-tumor tissues, whereas no significant increases in expression of alpha 1NaK and alpha 2NaK proteins were observed in HCC compared to non-tumor tissues. The ouabain (10 microM)-sensitive K(+)-ATPase activities (Na(+),K(+)-ATPase activities) in the membrane fraction from HCC tissue were significantly higher than those from non-tumor tissues. The Na(+),K(+)-ATPase activity was positively and significantly correlated with the expression level of alpha 3NaK. Apparent affinity for Na(+) in the Na(+),K(+)-ATPase activity in HCC tissues was significantly lower than that in non-tumor tissues, consistent with an elevated expression of alpha 3NaK relative to alpha 1NaK. Our results suggest that overexpression of alpha 3NaK increases the Na(+),K(+)-ATPase activity of HCC cells.

• [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.

• 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.

  [(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K+–Cl− cotransporter (IC50 = 10 μM). Here we found that DIOA inhibited activities of P-type ATPases such as dog kidney[(Dihydroindenyl)oxy]acetic acid (DIOA) has been used as a potent inhibitor of K+–Cl− cotransporter (IC50 = 10 μM). Here we found that DIOA inhibited activities of P-type ATPases such as dog kidney Na+,K+-ATPase (IC50 = 53 μM), hog gastric H+,K+-ATPase (IC50 = 97 μM) and rabbit muscle Ca2+-ATPase (IC50 = 127 μM). 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 (IC50 = 95 μM) and the exogenous H+,K+-ATPase (IC50 = 75 μM). 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 μM) 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.