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Cardiac glycosides, known as inhibitors of Na + ,K + -ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na + ,K + -ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells., (© 2024. The Author(s).)

Otopetrins (Otop1-Otop3) belong to a newly identified family of proton (H + ) channels activated by extracellular acidification. Here, we found that Zn 2+ activates the mouse Otop3 (mOtop3) proton channels by using electrophysiological patch-clamp techniques. In mOtop3-expressing human embryonic kidney HEK293T cells, a biphasic inward mOtop3 H + current comprising a fast transient current followed by a sustained current was observed upon extracellular acidification at pH 5.0. No significant activation of the mOtop3 channel was observed at pH 6.5 and 7.4, but interestingly, Zn 2+ dose-dependently induced a sustained activation of mOtop3 under these pH conditions. Increasing the Zn 2+ concentration had no effect on the reversal potential of the channel currents, suggesting that Zn 2+ does not permeate through the mOtop3. The activation of the mOtop3 channel was specific to Zn 2+ among divalent metal cations. Our findings reveal a novel modulatory mechanism of mOtop3 proton channels by Zn 2+ ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson's disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H + ,K + -ATPase. The K + -dependent ATPase activity and the lysosomal K + -transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase, thapsigargin, and K + -competitive inhibitors of gastric H + ,K + -ATPase, such as vonoprazan and SCH28080. Interestingly, these H + ,K + -ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H + /K + -transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes., (© 2023. The Author(s).)

Bitterness is an important physiological function in the defense responses to avoid toxic foods. The taste receptor 2 family is well known to mediate bitter taste perception in Type II taste cells. Here, we report that the polycystic kidney disease 2-like 1 (PKD2L1) channel is a novel sensor for the bitter aftertaste in Type III taste cells. The PKD2L1 channel showed rebound activation after the washout of quinine, a bitter tastant, in electrophysiological whole-cell recordings of the PKD2L1-expressing HEK293T cells and Ca 2+ -imaging analysis of Type III taste cells isolated from wild-type PKD2L1 mice. In the short-term two-bottle preference and lick tests in vivo, the wild-type mice avoided normal water while the PKD2L1-knockout mice preferred normal water after they ingested the quinine-containing water. These results may explain the new mechanism of the quinine-triggered bitter aftertaste perception in Type III taste cells., (© 2023. The Author(s).)

Dirac fermion systems form a unique Landau level at the Fermi level-the so-called zero mode-whose observation itself will provide strong evidence of the presence of Dirac dispersions. Here, we report the study of semimetallic black phosphorus under pressure by ^{31}P-nuclear magnetic resonance measurements in a wide range of magnetic field up to 24.0 T. We have found a field-induced giant enhancement of 1/T_{1}T, where 1/T_{1} is the nuclear spin lattice relaxation rate: 1/T_{1}T at 24.0 T reaches more than 20 times larger than that at 2.0 T. The increase in 1/T_{1}T above 6.5 T is approximately proportional to the squared field, implying a linear relationship between the density of states and the field. We also found that, while 1/T_{1}T at a constant field is independent of temperature in the low-temperature region, it steeply increases with temperature above 100 K. All these phenomena are well explained by considering the effect of Landau quantization on three-dimensional Dirac fermions. The present study demonstrates that 1/T_{1} is an excellent quantity to probe the zero-mode Landau level and to identify the dimensionality of the Dirac fermion system.

Na + ,K + -ATPases are essential for maintaining the membrane potential in almost all cells, and their catalytic subunits have four isoforms (α1-α4). Volume-regulated anion channel (VRAC) plays an important role in the cell death signaling pathway in addition to its fundamental role in cell volume maintenance. First, we introduce that disruption of actin filaments cause the dysfunction of VRAC, which elicits resistance to cisplatin in the cancer cells. Next, we summarize the cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na + ,K + -ATPase α1-isoform (α1NaK) and VRAC in the membrane microdomain of the cancer cells. In this mechanism, sub-micromolar concentrations of cardiac glycosides bind to the receptor-type α1NaK, and generate VRAC activities concomitantly with a deceleration of cancer cell proliferation. Finally, we summarize the pathophysiological function of α3NaK, which is abnormally expressed in the intracellular vesicles of cancer cells. The cancer cell can survive even under loss of anchorage because they have the avoidance mechanism for anoikis. On cancer cell detachment, we found that intracellular α3NaK is translocated to the plasma membrane and this event contributes to the survival of the cells. Interestingly, cardiac glycosides inhibited the α3NaK translocation and cell survival. Our findings may open up new opportunities for the development of cancer medicines.

Aged garlic extract (AGE) has been shown to protect the skin against UV-induced damage, but effects of its volatile components remain unknown. We investigated the effects of the volatile fraction of AGE on the responses of cultured skin fibroblasts subjected to UV-B irradiation. UV-B irradiation (20 mJ/cm 2 ) reduced the cell viability to 55% of control. The nonvolatile and volatile fractions of AGE inhibited the UV-B-induced reduction of cell viability; the cell viabilities were 100% and 73%, respectively. The volatile fraction inhibited the UV-B-induced increase in apoptotic cell death by 28%. The volatile fraction also inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) induced by UV-B irradiation. GC-MS analysis revealed that a large number of volatile compounds were generated during aging of garlic. These results suggest that the volatile fraction of AGE has protective effects against the UV-B-induced death of skin fibroblasts, and that this effect may partly be due to an inhibition of apoptosis via the downregulation of MAPK signaling. The volatile compounds of AGE may have beneficial applications for skin health. PRACTICAL APPLICATIONS: In this study, we investigated the effects of AGE against cell damage of UV-B-irradiated human skin fibroblasts. The aging process of garlic produced characteristic volatile compounds that have significant protective effects against UV-induced cell damage. Our results demonstrated that the aging process is a suitable method to develop added value in garlic extracts to improve skin health., (© 2022 Wiley Periodicals LLC.)

Background: Performing dual-tasks is often required for completing activities of daily living. Limited research investigated the effects of dual-tasking on gait in people with Traumatic Brain Injury (TBI)., Purpose: To investigate the effects of cognitive tasks on gait in people with TBI Methods: Seven individuals with TBI and nine controls completed walking under three conditions; usual walking, walking with questions and answers, and walking with word generation while 3D motion analysis system captured gait., Results: Significant group x condition interactions were found in which TBI group showed greater changes in speed ( p < .01), cadence ( p = .07), and ankle kinematics ( p = .03) as cognitive task became more complex from usual walking to walking with word generation. TBI group decreased speed ( p = .02), stride length ( p = .01), and hip kinematics ( p = .03) as concurrent task became more complex. The control showed decreases in speed ( p = .01), stride length ( p = .01), and joint kinematics in the hip ( p = .03) and knee ( p = .01) as the complexity of concurrent cognitive task increased., Conclusion: People with TBI have greater difficulty walking with a cognitive task and show greater compromises in gait performance as the task complexity increases when compared to those without TBI. Clinicians should consider the use of progression in cognitive tasks for dual-task gait training.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na + ,K + -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na + ,K + -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [ 3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na + ,K + -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca 2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca 2+ chelator, a Ca 2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca 2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells., (© 2022 Wiley Periodicals LLC.)

Transient receptor potential vanilloid 1 (TRPV1) is a voltage-dependent non-selective cation channel activated by capsaicin, the main pungent ingredient of chili peppers, and noxious heat. Although TRPV1 channels produce outwardly rectifying currents even in the absence of capsaicin, little is known about the regulation mechanism of the TRPV1 currents. In the present study, we found that intracellular ATP regulates the basal activities of TRPV1 channels in a concentration-dependent manner. The ATP-dependent regulation of TRPV1 channels was mediated by phosphoinositides. Moreover, an increase in intracellular ATP concentration negatively shifted voltage-dependent activation of TRPV1 channels. These results suggest that the ATP-dependent production of phosphoinositides regulates the voltage-dependent gating of the basal TRPV1 channel activities in the absence of capsaicin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The gastric proton pump (H + ,K + -ATPase) responsible for the H + secretion of gastric acid is an essential therapeutic target for acid-related diseases. H + ,K + -ATPase belongs to a P 2 -type ATPase family. Here, we examined the effects of a newly synthesized dihydropyrazole derivative KYY-008 on the H + ,K + -ATPase. KYY-008 concentration-dependently inhibited the enzyme activity of the ATPase in the membrane fractions prepared from isolated hog gastric mucosa and from human kidney HEK293 cells in which gastric H + ,K + -ATPase is exogenously expressed. The IC 50 values in these samples were 3.4 μM and 3.7 μM, respectively. In addition, KYY-008 significantly inhibited the H + ,K + -ATPase-derived H + uptake into the tightly sealed vesicles prepared from the hog gastric mucosa. In contrast, KYY-008 has no effect on the activities of other P 2 -type ATPases such as Na + ,K + -ATPase and Ca 2+ -ATPase. KYY-008 did not change the ionic currents of voltage-dependent Ca 2+ channels, that were potential targets for some dihydropyrazole derivatives. Together, we discovered a new dihydropyrazole derivative which acts as a selective inhibitor of gastric H + ,K + -ATPase., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Beginning of metastasis, cancer cells detach from the primary tumor and they can survive even under loss of anchorage; however, the detachment-elicited mechanisms have remained unknown. Here, we found that Na + ,K + -ATPase α3-isoform (α3NaK) in human cancer cells is dynamically translocated from intracellular vesicles to the plasma membrane when the attached cells are detached and that this mechanism contributes to the survival of the detached (floating) cancer cells. α3NaK was detected in the plasma membrane of floating cancer cells in peritoneal fluids of patients, while it was in the cytoplasm of the cells in primary tumor tissues. On cancer cell detachment, we also found the focal-adhesion-kinase-dependent Ca 2+ response that induces the α3NaK translocation via nicotinic acid adenine dinucleotide phosphate pathway. Activation of AMP-activated protein kinase was associated with the translocated α3NaK in the plasma membrane. Collectively, our study identifies a unique mechanism for survival of detached cancer cells, opening up new opportunities for development of cancer medicines., Competing Interests: The authors declare that there are no conflicts of interest., (© 2021 The Author(s).)

P-type ion pumps (P-type ATPases) are involved in various fundamental biological processes. For example, the gastric proton pump (H + ,K + -ATPase) and sodium pump (Na + ,K + -ATPase) are responsible for secretion of gastric acid and maintenance of cell membrane potential, respectively. In this review, we summarize three topics of our studies. The first topic is gastric H + ,K + -ATPase associated with Cl - -transporting proteins (Cl - /H + exchanger ClC-5 and K + -Cl - cotransporter KCC4). In gastric parietal cells, we found that ClC-5 is predominantly expressed in intracellular tubulovesicles and that KCC4 is predominantly expressed in the apical membrane. Gastric acid (HCl) secretion may be accomplished by the two different complexes of H + ,K + -ATPase and Cl - -transporting protein. The second topic focuses on the Na + ,K + -ATPase α1-isoform (α1NaK) associated with the volume-regulated anion channel (VRAC). In the cholesterol-enriched membrane microdomains of human cancer cells, we found that α1NaK has a receptor-like (non-pumping) function and that binding of low concentrations (nM level) of cardiac glycosides to α1NaK activates VRAC and exerts anti-cancer effects without affecting the pumping function of α1NaK. The third topic is the Na + ,K + -ATPase α3-isoform (α3NaK) in human cancer cells. We found that α3NaK is abnormally expressed in the intracellular vesicles of attached cancer cells and that the plasma membrane translocation of α3NaK upon cell detachment contributes to the survival of metastatic cancer cells. Our results indicate that multiple functions of P-type ion pumps are generated by different membrane environments and their associated proteins.

Cisplatin is a widely used platinum-based anticancer drug in the chemotherapy of numerous human cancers. However, cancer cells acquire resistance to cisplatin. So far, functional loss of volume-sensitive outwardly rectifying (VSOR) Cl - channels has been reported to contribute to cisplatin resistance of cancer cells. Here, we analyzed protein expression patterns of human epidermoid carcinoma KB cells and its cisplatin-resistant KCP-4 cells. Intriguingly, KB cells exhibited higher β-actin expression and clearer actin filaments than KCP-4 cells. The β-actin knockdown in KB cells decreased VSOR Cl - currents and inhibited the regulatory volume decrease (RVD) process after cell swelling. Consistently, KB cells treated with cytochalasin D, which depolymerizes actin filaments, showed smaller VSOR Cl - currents and slower RVD. Cytochalasin D also inhibited cisplatin-triggered apoptosis in KB cells. These results suggest that the disruption of actin filaments cause the dysfunction of VSOR Cl - channels, which elicits resistance to cisplatin in human epidermoid carcinoma cells., (© 2020 Wiley Periodicals, Inc.)

Cisplatin [ cis -diamminedichloroplatinum (II)] is a platinum-based anticancer drug widely used for the treatment of various cancers. It forms interstrand and intrastrand cross-linking with DNA and block DNA replication, resulting in apoptosis. On the other hand, intrinsic and acquired cisplatin resistance restricts its therapeutic effects. Although some studies suggest that dramatic epigenetic alternations are involved in the resistance triggered by cisplatin, the mechanism is complicated and remains poorly understood. Recent studies reported that cytoskeletal structures regulate cisplatin sensitivity and that activities of membrane transporters contribute to the development of resistance to cisplatin. Therefore, we focus on the roles of actin filaments and membrane transporters in cisplatin-induced apoptosis. In this review, we summarize the relationship between actin cytoskeleton and membrane transporters in the cisplatin resistance of cancer cells., (Copyright © 2020 Shimizu, Fujii and Sakai.)

Pathophysiological functions of chloride intracellular channel protein 3 (CLIC3) in human gastric cancer have been unclear. In the tissue microarray analysis using 107 gastric cancer specimens, CLIC3 expression was negatively correlated with pathological tumor depth, and the patients with lower expression of CLIC3 exhibited poorer prognosis. CLIC3 was expressed in the plasma membrane of cancer cells in the tissue. CLIC3 expression was also found in a human gastric cancer cell line (MKN7). In whole-cell patch-clamp recordings of the cells expressing CLIC3, NPPB-sensitive outwardly rectifying Cl - currents were observed. Cell proliferation was significantly accelerated by knockdown of CLIC3 in MKN7 cells. On the other hand, the proliferation was attenuated by exogenous CLIC3 expression in human gastric cancer cells (KATOIII and NUGC-4) in which endogenous CLIC3 expression is negligible. Our results suggest that CLIC3 functions as a Cl - channel in the plasma membrane of gastric cancer cells and that decreased expression of CLIC3 results in unfavorable prognosis of gastric cancer patients.

Dynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H + in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

In the stomach, Sonic Hedgehog (Shh) is highly expressed in gastric parietal cells, and acts as a morphogen in early development of the organ. Here, we found that the cleaved N-terminal fragment of Shh (Shh-N) was abundantly expressed in hog gastric vesicles derived from the apical membrane of parietal cells. Interestingly, Shh-N recombinant significantly decreased K + -dependent ATP-hydrolyzing activity, which is sensitive to an inhibitor of H + ,K + -ATPase (SCH28080), in hog gastric tubulovesicles and membrane fractions of the H + ,K + -ATPase-expressing cells. In the living cells, Shh-N recombinant inhibited the SCH28080-sensitive 86 Rb + -uptake. Together, Shh-N may directly bind to extracellular side of H + ,K + -ATPase, and negatively regulates the pump activity. This is the first report to explore non-morphogenic property of Shh on ion transporters., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gastric proton pump (H + ,K + -ATPase) which is responsible for H + secretion of gastric acid (HCl) in gastric parietal cells is the major therapeutic target for treatment of acid-related diseases. H + ,K + -ATPase consists of two subunits, a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). N-glycosylation of βHK is essential for trafficking and stability of αHK in apical membrane of gastric parietal cells. Terminal sialic acid residues on sugar chains have an important role in various cellular functions. Recently, we succeeded in visualizing the sialylation and desialylation dynamics of βHK using a fluorescence bioimaging nanoprobe consisting of biocompatible polymers conjugated with lectins for detecting sialic acid. In H + ,K + -ATPase-expressing cell lines, rat gastric mucosa, and primary culture of rat gastric parietal cells, fluorescence imaging of sialic acid with the nanoprobe showed that sialylation of βHK is regulated by intragastric pH and that inhibition of gastric acid secretion induces desialylation of βHK. In biochemical and pharmacological studies, we revealed that enzyme activity of αHK is negatively regulated by desialylation of βHK. Our studies uncovered a novel negative-feedback mechanism of H + ,K + -ATPase in which sialic acids of βHK positively regulates H + ,K + -ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of βHK. In this topic, we introduce the overview of our research using the bioimaging nanoprobe.

Digitoxin and digoxin are plant-derived cardiac glycosides. They are Na + ,K + -ATPase (sodium pump) inhibitors, and have been used clinically for treatment and prevention of heart failure and various tachycardia. On the other hand, some epidemiological studies showed that digoxin users have a lower cancer risk compared to the non-users, and that cancer patients who had been treated with digoxin face on improvement of their survival. In various in vitro studies, cardiac glycosides at sub-μM concentrations, which have no significant effect on enzymatic and ion-transporting activities of Na + ,K + -ATPase, show anti-cancer effects. Na + ,K + -ATPase is ubiquitously expressed, so it remains unclear why low concentrations of cardiac glycosides have cancer-specific effects. Recently, we found that the receptor-type Na + ,K + -ATPase, which has no pumping activity, is associated with leucine-rich repeat-containing 8 family, member A(LRRC8A), one of the components of volume-regulated anion channel (VRAC), in the membrane microdomains of plasma membrane of cancer cells, and that this crosstalk contributes to the inhibition of the cancer cell growth by sub-μM cardiac glycosides. In this mechanism, cardiac glycosides bind to the receptor-type Na + ,K + -ATPase, and then stimulate the production of reactive oxygen species (ROS) via NADPH oxidase. The ROS activate VRAC within the membrane microdomains, thus eliciting anti-proliferative effects. VRAC is ubiquitously expressed, and it is normally activated by cell swelling. However, VRAC is activated by cardiac glycoside without cell swelling. On the other hand, the cardiac glycosides-induced effects were not observed in non-cancer cells. Our findings can partly explain why cardiac glycosides elicit selective effects in cancer cells.

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na + ,K + -ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na + ,K + -ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na + ,K + -ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na + ,K + -ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects., (Copyright © 2018. Published by Elsevier B.V.)

γ-Glutamyl- S-allylmercaptocysteine (GSAMC), a putative precursor compound of S-allylmercaptocysteine (SAMC), was isolated and identified from aged garlic extract (AGE). We analyzed the change of their contents in AGE during the aging process, chronologically from 1 to 22 months. The formation of these compounds occurred mostly during the early stage of the aging period: the SAMC content reached a maximum at approximately 4 months, whereas the GSAMC content reached a maximum at 1 month and then decreased during the subsequent aging period. To assess the possible relationship between the change of the two compounds during the aging process, we set up the model reactions with the hypothesis that GSAMC is produced from γ-glutamyl- S-allylcysteine (GSAC)/γ-gultamyl- S-1-propenylcysteine (GS1PC) and that SAMC is produced from GSAMC by endogenous γ-glutamyl transpeptidase (GGT) in garlic during the early stage of the aging process. We found that, in the model reactions, SAMC was produced from GSAMC by the garlic protein fraction having GGT activity and its production was suppressed by a GGT inhibitor. Furthermore, the production of GSAMC from allicin and GSAC/GS1PC was found in another model reaction. The reaction between allicin and GS1PC was faster than that between allicin and GSAC and, thus, may be involved in the production of GSAMC in the early stage of the aging process.

We analyzed aged garlic extract (AGE) to understand its complex sulfur chemistry using post-column high-performance liquid chromatography with an iodoplatinate reagent and liquid chromatography high resolution mass spectrometry (LC-MS). We observed unidentified peaks of putative sulfur compounds. Three compounds were isolated and identified as γ-glutamyl-γ-glutamyl- S-methylcysteine, γ-glutamyl-γ-glutamyl- S-allylcysteine (GGSAC) and γ-glutamyl-γ-glutamyl- S-1-propenyl-cysteine (GGS1PC) by nuclear magnetic resonance and LC-MS analysis based on comparisons with chemically synthesized reference compounds. GGSAC and GGS1PC were novel compounds. Trace amounts of these compounds were detected in raw garlic, but the contents of these compounds increased during the aging process. Production of these compounds was inhibited using a γ-glutamyl transpeptidase (GGT) inhibitor in the model reaction mixtures. These findings suggest that γ-glutamyl tripeptides in AGE are produced by GGT during the aging process.

Objective: To compare cardiorespiratory responses between pool floor walking and overground walking (OW) in people poststroke., Design: Cross-sectional study., Setting: University-based therapeutic exercise facility., Participants: Participants (N=28) were comprised of 14 community-dwelling individuals poststroke (5.57±3.57y poststroke) and 14 age- and sex-matched healthy adults (mean age, 58.00±15.51y; male/female ratio, 9:5)., Interventions: Not applicable., Main Outcome Measures: A telemetric metabolic system was used to collect cardiorespiratory variables, including oxygen consumption (V˙o 2 ), energy expenditure (EE), and expired volume per unit time (V˙e), during 6-minute walking sessions in chest-depth water and on land at a matched speed, determined by average of maximum walking speed in water., Results: Individuals poststroke elicited no significant differences in cardiorespiratory responses between pool floor walking and OW. However, healthy controls showed significant increases in mean V˙o 2 values by 94%, EE values by 109%, and V˙e values by 94% (all P<.05) during pool floor walking compared with OW. A 2×2 mixed model analysis of variance revealed a significant group × condition interaction in V˙o 2 , in which the control group increased V˙o 2 from OW to pool floor walking, whereas the stroke group did not., Conclusions: Our results indicate that people poststroke, unlike healthy adults, do not increase EE while walking in water compared with on land. Unlike stationary walking on an aquatic treadmill, forward locomotion during pool floor walking at faster speeds may have increased drag force, which requires greater EE from healthy adults. Without demanding excessive EE, walking in water may offer a naturally supportive environment for gait training in the early stages of rehabilitation., (Copyright © 2017 American Congress of Rehabilitation Medicine. All rights reserved.)

Myricetin (3,3',4',5,5',7-hexahydroxyflavone), a major flavonoid in berries and red wine, has been recently used as a health food supplement based on its antioxidant and antitumor properties. We report here that myricetin preferentially exerts inhibitory effects on gastric H + , K + -ATPase. Myricetin inhibited H + , K + -ATPase with a sub-micromolar IC 50 value in an enzyme assay using freeze-dried tubulovesicles prepared from hog stomach. Na + , K + -ATPase and Ca 2+ -ATPase were also inhibited by myricetin in a dose-dependent manner, but the IC 50 values for these enzymes were approximately an order of magnitude higher compared to the H + , K + -ATPase. In structure-inhibitory functional analysis of sixteen myricetin derivatives, several phenolic hydroxy groups attached to the flavonoid backbone were highlighted as essential modifications for the inhibition of P 2 -type ATPases. Furthermore, oral administration of myricetin significantly attenuated histamine-induced gastric acid secretion in an in vivo mouse assessment. Therefore, myricetin derivatives seem to be useful seed compounds for developing new drugs and supplements to alleviate gastric acid secretion., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To investigate the inhibitory effect of a commercial proton pump inhibitor (lansoprazole) on the gastric proton pump H + ,K + -ATPase in vitro, we used orally disintegrating (OD) tablets including original brand-name and generic tablets. In the course of the development of generic products, dissolution and clinical tests are necessary to ensure their bioequivalence to the original brand-name products; by contrast, there is almost no opportunity to demonstrate their activity in vitro. This study initially compared the similarity of the dissolution of test generic tablets with that of the original brand-name tablets. The dissolution tests for 15 and 30-mg lansoprazole tablets found their dissolution properties were similar. Subsequently, the dissolution media were sampled and then their effects on the H + ,K + -ATPase activity were measured using tubulovesicles prepared from the gastric mucosa of hogs. We confirmed that the inhibitory effects of the generic tablets on H + ,K + -ATPase activity were consistent with those of the original brand-name tablets. Furthermore, lansoprazole contents in each tablet estimated from their inhibitory effects were in good agreement with their active pharmaceutical ingredient content. To our knowledge, this is the first technical report to compare the in vitro biochemical activity of lansoprazole OD tablets between the original brand-name and generic commercial products.

Voltage-dependent inactivation of ion channels contributes to the regulation of the membrane potential of excitable cells. Mouse polycystic kidney disease 2-like 1 (PKD2L1) forms voltage-dependent nonselective cation channels, which are activated but subsequently inactivated in response to membrane depolarization. Here, we found that the mutation of an asparagine 533 residue (N533Q) in the outer pore loop region of PKD2L1 caused a marked increase in outward currents induced by depolarization. In addition, the tail current analysis demonstrated that the N533Q mutants are activated during depolarization but the subsequent inactivation does not occur. Interestingly, the N533Q mutants lacked the channel activation triggered by the removal of stimuli such as extracellular alkalization and heating. Our findings suggest that the N533 residue in the outer pore loop region of PKD2L1 has a key role in the voltage-dependent channel inactivation.

The purpose of this study was to investigate the effects of an 8-week Taekwondo (TKD) intervention on balance in children with autism spec-trum disorder (ASD). A total of 14 children with ASD participated in this study. Eight children (eight males; mean age, 10.25±2.38 yr) completed TKD intervention (50 min/2 times/8 week), and six children received no intervention serving as controls (five males, one female; mean age, 10.00±2.83 yr). A computed posturography system with a long forceplate (NeuroCom Balance Master) was used to evaluate static (double and single leg stance with various test conditions) and functional balance (step-quick-turn). Balance was measured before and after the intervention. A mixed-model analysis of variance showed a significant group by time interaction in single leg stance balance. After the intervention, the TKD group displayed a greater improvement in single leg stance balance with eyes closed condition than the control group (P=0.046). Within-group analysis showed that the TKD group significantly improved single leg stance balance with eyes open condition (P=0.014). In addition, TKD group displayed trends of improvements in double leg stance balance with unstable surface under eyes closed condition (ES=0.83) and step-quick-turn (Cohen d [ES]=0.70). The control group did not show any significant changes in balance outcomes. In conclusion, TKD training can help children with ASD improve their balance. Children with ASD also showed a high rate of adherence (92%) to the TKD training. Our findings suggest that TKD can be a fun, feasible, and effective therapeutic option for balance improvement of children with ASD.

Proton(H+) of gastric acid(HCl) is actively secreted by gastric proton pump (H+, K+- ATPase) in the parietal cells. The proton pump is expressed in both tubulovesicles and apical membrane of the cells. In resting parietal cells, tubulovesicles are present in intracellular compartments underlying the apical membrane and forming a reticulated meshwork. Upon stimulation, tubulovesicles fuse each other and connect with the apical membrane, resulting in massive acid secretion. On the other hand, the mechanism of apical Cl- transport for HCl secretion is not fully understood, although several Cl- transporters and Cl- channels have been reported to be the candidate. Here, we summarized the function of Cl- transporters such as KCC4, a K+-Cl- cotransporter, and ClC-5, a Cl-/H+ exchanger, in gastric acid secretion.

The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK., (Copyright © 2016 Elsevier B.V. All rights reserved.)

As a physiological phenomenon, acid secretion from the stomach was known already at least in the 17th century. But its mechanism was elucidated in more recent times only. At the end of the 20th century, gastric H(+)/K(+)-ATPase in the parietal cells was found to be responsible for a final step of H(+) secretion in these cells. In this century, several Cl(-)-transporting proteins for gastric acid (hydrochloric acid; HCl) secretion have been found. As inhibitors of gastric acid secretion, histamine H2 receptor antagonists (H2 blockers) were developed in the 1970's. This discovery brought a great benefit; that is, peptic ulcers became treatable by administration of a drug. In 1980's, proton pump inhibitors (PPIs) were developed. The target of PPIs is gastric H(+)/K(+)-ATPase and the PPIs exert generally more potent effects compared with H2 blockers. Most recently, several K(+)-competitive inhibitors of the ATPase are being developed. Here, we introduce gastric H(+)/K(+)-ATPase and its related proteins for gastric acid secretion, and several gastric diseases and their treatment by medicines.

4-(2-Butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB) has been used as an inhibitor of volume-regulated anion channel (VRAC), which is expressed in almost all cells (IC50 is around 4 µM). Here, we found that DCPIB significantly inhibited the activities of gastric proton pump (H+,K+-ATPase) in isolated gastric tubulovesicles and the membrane sample of the H+,K+-ATPase-expressing cells, and their IC50 values were around 9 µM. In the tubulovesicles, no significant expression of leucine rich repeat containing 8 family member A (LRRC8A), an essential component of VRAC, was observed. The inhibitory effect of DCPIB was also found in the membrane sample obtained from the cells in which LRRC8A had been knocked down. On the other hand, DCPIB had no significant effect on the activity of Na+,K+-ATPase or Ca2+-ATPase. In the H+,K+-ATPase-expressing cells, DCPIB inhibited the 86Rb+ transport activity of H+,K+-ATPase but not that of Na+,K+-ATPase. DCPIB had no effect on the activity of Cl- channels other than VRAC in the cells. These results suggest that DCPIB directly inhibits H+,K+-ATPase activity. DCPIB may be a beneficial tool for studying the H+,K+-ATPase function in vitro., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Butyrate is a fatty acid with an important role in the maintenance of colonic homeostasis. It induces apoptosis in colonic epithelial cells and contributes to the pathogenesis of ulcerative colitis. However, just how how butyrate triggers apoptosis is poorly understood. In our model system of mouse colonic epithelial MCE301 cells, whole-cell patch-clamp recordings revealed the presence of swelling-activated outwardly rectifying chloride ion (Cl(-)) currents. These currents exhibited time-dependent inactivation upon strong depolarization, a low field strength anion selectivity (I(-) > Br(-) > Cl(-) > F(-)), and a sensitivity to 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), a specific blocker of volume-sensitive outwardly rectifying (VSOR) Cl(-) channels. The results of flow cytometric analysis and caspase 3/7 assays demonstrated that exposure of MCE301 cells to sodium butyrate for 16 h triggered apoptotic cell shrinkage, phosphatidylserine exposure, and caspase 3/7 activation. Importantly, the VSOR Cl(-) channel blocker was able to inhibit sodium butyrate-induced apoptotic processes. These results suggest that activation of the VSOR Cl(-) channel is essential for sodium butyrate-triggered apoptosis in MCE301 cells.

Polycystic kidney disease 2-like 1 (PKD2L1), previously called transient receptor potential polycystin 3 (TRPP3), forms a voltage-dependent nonselective cation channel that exhibits large tail currents triggered by repolarization after depolarization. Since it has previously been proposed that temperature sensitivity of some TRP channels is linked to the voltage-dependent gating, we here investigated heating effects on PKD2L1 currents in human embryonic kidney HEK293T cells overexpressing mouse PKD2L1. Tail PKD2L1 currents were increased by heating to 32 °C, but decreased at more than 36 °C. Voltage dependency of the PKD2L1 channel was shifted by heating in a bimodal fashion: an increase in temperature to 32 °C and to 36 °C shifted the activation curves toward the left and the right, respectively. In addition, heating accelerated deactivation of tail PKD2L1 currents. To analyze the channel gating kinetics, single-channel events of the PKD2L1 channel were recorded at hyperpolarized potentials under whole-cell configurations. A rise in temperature decreased the open probability of the channel. Dwell-time analysis showed that both open and closed dwell times during heating were shorter than those at room temperature. Interestingly, a rapid temperature drop after heating markedly enhanced the PKD2L1 currents at both single-channel and whole-cell levels. The rebound activation of the PKD2L1 channel was due to an increase in the open probability but not in the single-channel conductance. These results suggest that heating opens but subsequently inactivates PKD2L1 channels, which is essential for the rebound activation of the channel after heating.

Thromboxane A2 (TXA2) is known to stimulate colonic cancer cell proliferation, although the mechanism has not been clarified. In this study, we compared the expression levels of Kv7.1 K(+) channels between human colorectal cancer tissue and the accompanying non-tumor mucosa. Kv7.1 proteins were found to be consistently up-regulated in the cancer tissues from different patients. Kv7.1 was also expressed in human colonic cancer cell lines. Treatment of colonic cancer cells with 9,11-epithio-11,12-methano-thromboxane A2 (STA2), a stable analogue of TXA2, significantly increased whole-cell K(+) currents sensitive to chromanol 293B, an inhibitor of Kv7.1 channels, in parallel with an increased expression of Kv7.1 proteins. In contrast, TXB2, an inactive metabolite of TXA2, had no effects on expression level and function of Kv7.1. A TXA2 receptor antagonist (SQ29548) and an inhibitor of cAMP-dependent protein kinase (Rp-8-Br-MB-cAMPS) inhibited STA2-induced increases in both Kv7.1 expression and chromanol 293B-sensitive K(+) currents. Interestingly, STA2-stimulated proliferation of colonic cancer cells was inhibited by chromanol 293B. These results suggest that Kv7.1 channels are involved in the TXA2-induced cancer cell proliferation and that they are up-regulated by the TXA2 receptor-mediated cAMP pathway.

It has been reported that chloride-proton exchanger ClC-5 and vacuolar-type H(+)-ATPase are essential for endosomal acidification in the renal proximal cells. Here, we found that ClC-5 is expressed in the gastric parietal cells which secrete actively hydrochloric acid at the luminal region of the gland, and that it is partially localized in the intracellular tubulovesicles in which gastric H(+),K(+)-ATPase is abundantly expressed. ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase in the lysate of tubulovesicles. The ATP-dependent uptake of (36)Cl(-) into the vesicles was abolished by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH28080), an inhibitor of H(+),K(+)-ATPase, suggesting functional expression of ClC-5. In the tetracycline-regulated expression system of ClC-5 in the HEK293 cells stably expressing gastric H(+),K(+)-ATPase, ClC-5 was co-immunoprecipitated with H(+),K(+)-ATPase, but not with endogenous Na(+),K(+)-ATPase. The SCH28080-sensitive (36)Cl(-) transporting activity was observed in the ClC-5-expressing cells, but not in the ClC-5-non-expressing cells. The mutant (E211A-ClC-5), which has no H(+) transport activity, did not show the SCH28080-sensitive (36)Cl(-) transport. On the other hand, both ClC-5 and its mutant (E211A) significantly increased the activity of H(+),K(+)-ATPase. Our results suggest that ClC-5 and H(+),K(+)-ATPase are functionally associated and that they may contribute to gastric acid secretion.

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H(+),K(+)-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H(+),K(+)-ATPase significantly increased the ATPase activity without changing the expression level of H(+),K(+)-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H(+),K(+)-ATPase activity. In contrast, knockdown of endogenous ERp57 in H(+),K(+)-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H(+),K(+)-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na(+),K(+)-ATPase. These results suggest that ERp57 positively regulates H(+),K(+)-ATPase activity apart from its chaperoning function., (© 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

TMEM16 (transmembrane protein 16) proteins, which possess eight putative transmembrane domains with intracellular NH2- and COOH-terminal tails, are thought to comprise a Cl(-) channel family. The function of TMEM16F, a member of the TMEM16 family, has been greatly controversial. In the present study, we performed whole cell patch-clamp recordings to investigate the function of human TMEM16F. In TMEM16F-transfected HEK293T cells but not TMEM16K- and mock-transfected cells, activation of membrane currents with strong outward rectification was found to be induced by application of a Ca(2+) ionophore, ionomycin, or by an increase in the intracellular free Ca(2+) concentration. The free Ca(2+) concentration for half-maximal activation of TMEM16F currents was 9.6 μM, which is distinctly higher than that for TMEM16A/B currents. The outwardly rectifying current-voltage relationship for TMEM16F currents was not changed by an increase in the intracellular Ca(2+) level, in contrast to TMEM16A/B currents. The Ca(2+)-activated TMEM16F currents were anion selective, because replacing Cl(-) with aspartate(-) in the bathing solution without changing cation concentrations caused a positive shift of the reversal potential. The anion selectivity sequence of the TMEM16F channel was I(-) > Br(-) > Cl(-) > F(-) > aspartate(-). Niflumic acid, a Ca(2+)-activated Cl(-) channel blocker, inhibited the TMEM16F-dependent Cl(-) currents. Neither overexpression nor knockdown of TMEM16F affected volume-sensitive outwardly rectifying Cl(-) channel (VSOR) currents activated by osmotic swelling or apoptotic stimulation. These results demonstrate that human TMEM16F is an essential component of a Ca(2+)-activated Cl(-) channel with a Ca(2+) sensitivity that is distinct from that of TMEM16A/B and that it is not related to VSOR activity.

K(+)-Cl(-) cotransporter-3a (KCC3a) is associated with Na(+),K(+)-ATPase α1-subunit (α1NaK) in lipid rafts of gastric acid-secreting cells and positively regulates Na(+),K(+)-ATPase activity. Here, effects of cholesterol on association of KCC3a with α1NaK in lipid rafts were studied in LLC-PK1 cells stably expressing KCC3a. In the cells, lipid rafts destructed by methyl-β-cyclodextrin (MβCD) could be reconstructed by exogenous addition of cholesterol accompanying a shift of both KCC3a and α1NaK from non-rafts to rafts. The KCC3a-increased Na(+),K(+)-ATPase activity was abolished by MβCD, and recovered by repletion of cholesterol without changing expression levels of KCC3a and α1NaK in the cells. KCC3a was co-immunoprecipitated with α1NaK even after destruction of lipid rafts by MβCD, indicating that molecular association of KCC3a with α1NaK still retains in the non-raft environment. Our results suggest that cholesterol is essential for eliciting up-regulation of Na(+),K(+)-ATPase activity by KCC3a in the KCC3a-α1NaK complex., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 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.

Polycystic 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.

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 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.

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-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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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., ((c) 2010 Elsevier B.V. All rights reserved.)

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 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.

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 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, whereas H+,K+-ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles. 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 ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). 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 the 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.

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/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.

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.