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Sample records for abcb1 atpase activity

  1. Membrane Assays to Characterize Interaction of Drugs with ABCB1.

    PubMed

    Fekete, Zsolt; Rajnai, Zsuzsanna; Nagy, Tünde; Jakab, Katalin Tauberné; Kurunczi, Anita; Gémes, Katalin; Herédi-Szabó, Krisztina; Fülöp, Ferenc; Tóth, Gábor K; Czerwinski, Maciej; Loewen, Greg; Krajcsi, Peter

    2015-12-01

    ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions. PMID:25926125

  2. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice.

    PubMed

    Xie, Jing-Dun; Huang, Yang; Chen, Dong-Tai; Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided.

  3. A novel screening strategy to identify ABCB1 substrates and inhibitors.

    PubMed

    von Richter, Oliver; Glavinas, Hristos; Krajcsi, Peter; Liehner, Stephanie; Siewert, Beate; Zech, Karl

    2009-01-01

    We tested the hypothesis whether data on ABCB1 ATPase activity and passive permeability can be used in combination to identify ABCB1 substrates and inhibitors. We determined passive permeability using an artificial membrane permeability assay (HDM-PAMPA) and ABCB1 function, i.e., vanadate-sensitive ATPase activity for a training set (40 INN drugs) and a validation set (26 development compounds). In parallel experiments, we determined ABCB1 function, i.e., vectorial transport in a Caco-2 cell monolayer, and ABCB1 inhibition, i.e., calcein AM extrusion out of K562-MDR cells, to cross-validate the results with cellular assays. We found that compounds that did not modulate ABCB1-ATPase did also not affect calcein AM extrusion and were not actively transported by ABCB1 in Caco-2 cell monolayers. The results corroborated the effect of passive permeability as an important covariate of active transport: active transport in Caco-2 monolayer was only apparent for compounds showing low passive permeability (<5.0 cmx10(-6)/s) in the HDM-PAMPA assay whereas compounds with high passive permeability (>50 cmx10(-6)/s) were shown to inhibit calcein AM efflux with IC50 values close to their respective Km value obtained for ABCB1-ATPase. The use of HDM-PAMPA in combination with ABCB1-ATPase offers a simple, inexpensive experimental approach capable of identifying ABCB1 inhibitors as well as transported substrates.

  4. Wallichinine reverses ABCB1-mediated cancer multidrug resistance.

    PubMed

    Lv, Min; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Huang, Jia-Rong; Wang, Kun; Wei, Meng-Ning; Cheng, Ke-Jun; Shi, Zhi

    2016-01-01

    Overexpression of ABCB1 in cancer cells is one of the main reasons of cancer multidrug resistance (MDR). Wallichinine is a compound isolated from piper wallichii and works as an antagonist of platelet activiating factor receptor to inhibit the gathering of blood platelet. In this study, we investigate the effect of wallichinine on cancer MDR mediated by ABCB1 transporter. Wallichinine significantly potentiates the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in ABCB1 overexpressing cancer cells. Furthermore, wallichinine do not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, wallichinine blocks the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. The predicted binding mode shows the hydrophobic interactions of wallichinine within the large drug binding cavity of ABCB1. At all, our study of the interaction of wallichinine with ABCB1 presented herein provides valuable clues for the development of novel MDR reversal reagents from natural products. PMID:27508017

  5. Wallichinine reverses ABCB1-mediated cancer multidrug resistance

    PubMed Central

    Lv, Min; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Huang, Jia-Rong; Wang, Kun; Wei, Meng-Ning; Cheng, Ke-Jun; Shi, Zhi

    2016-01-01

    Overexpression of ABCB1 in cancer cells is one of the main reasons of cancer multidrug resistance (MDR). Wallichinine is a compound isolated from piper wallichii and works as an antagonist of platelet activiating factor receptor to inhibit the gathering of blood platelet. In this study, we investigate the effect of wallichinine on cancer MDR mediated by ABCB1 transporter. Wallichinine significantly potentiates the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in ABCB1 overexpressing cancer cells. Furthermore, wallichinine do not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, wallichinine blocks the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. The predicted binding mode shows the hydrophobic interactions of wallichinine within the large drug binding cavity of ABCB1. At all, our study of the interaction of wallichinine with ABCB1 presented herein provides valuable clues for the development of novel MDR reversal reagents from natural products. PMID:27508017

  6. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice

    PubMed Central

    Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided. PMID:26633878

  7. Human ABCB1 confers cells resistance to cytotoxic guanidine alkaloids from Pterogyne nitens.

    PubMed

    Satake, Kazuhiro; Tsukamoto, Megumi; Mitani, Yuji; Regasini, Luis Octavio; da Silva Bolzani, Vanderlan; Efferth, Thomas; Nakagawa, Hiroshi

    2015-01-01

    Multidrug resistance (MDR) caused by human ABCB1 (P-glycoprotein/MDR1) is one of the major obstacles in chemotherapy. To understand the mechanism of MDR by ABCB1 and circumvent the MDR, in the present study, we established human ABCB1-expressing cells (Flp-In-293/ABCB1 cells) and examined the cytotoxic effects of four guanidine alkaloids from Pterogyne nitens (galegine, nitensidine A, pterogynidine and pterogynine) using Flp-In-293/Mock and Flp-In-293/ABCB1 cells. The activity of ABCB1 in Flp-In-293/ABCB1 cells were confirmed by typical substrates for ABCB1 (taxol and vinblastine) in MTT assay. Flp-In-293/ABCB1 cells were also resistant to the four guanidine alkaloids as well as taxol and vinblastine compared to Flp-In-293/Mock cells although the four guanidine alkaloids exhibited cytotoxicity against the two Flp-In-293 cells. Furthermore, the four guanidine alkaloids were also found to stimulate the ATPase activity of ABCB1 in ATPase assays. These results suggest that ABCB1 can confer the resistance to the cytotoxic guanidine alkaloids by transporting them.

  8. Trametinib modulates cancer multidrug resistance by targeting ABCB1 transporter

    PubMed Central

    Qiu, Jian-Ge; Zhang, Yao-Jun; Li, Yong; Zhao, Jin-Ming; Zhang, Wen-Ji; Jiang, Qi-Wei; Mei, Xiao-Long; Xue, You-Qiu; Qin, Wu-Ming; Yang, Yang; Zheng, Di-Wei; Chen, Yao; Wei, Meng-Ning; Shi, Zhi

    2015-01-01

    Overexpression of adenine triphosphate (ATP)-binding cassette (ABC) transporters is one of the main reasons of multidrug resistance (MDR) in cancer cells. Trametinib, a novel specific small-molecule mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor, is currently used for the treatment of melanoma in clinic. In this study, we investigated the effect of trametinib on MDR mediated by ABC transporters. Trametinib significantly potentiated the effects of two ABCB1 substrates vincristine and doxorubicin on inhibition of growth, arrest of cell cycle and induction of apoptosis in cancer cells overexpressed ABCB1, but not ABCC1 and ABCG2. Furthermore, trametinib did not alter the sensitivity of non-ABCB1 substrate cisplatin. Mechanistically, trametinib potently blocked the drug-efflux activity of ABCB1 to increase the intracellular accumulation of rhodamine 123 and doxorubicin and stimulates the ATPase of ABCB1 without alteration of the expression of ABCB1. Importantly, trametinib remarkably enhanced the effect of vincristine against the xenografts of ABCB1-overexpressing cancer cells in nude mice. The predicted binding mode showed the hydrophobic interactions of trametinib within the large drug binding cavity of ABCB1. Consequently, our findings may have important implications for use of trametinib in combination therapy for cancer treatment. PMID:25915534

  9. Semi-synthetic ocotillol analogues as selective ABCB1-mediated drug resistance reversal agents

    PubMed Central

    Zhang, Guan-Nan; Wang, Yi-Jun; Kathawala, Rishil J.; Si, Rui; Patel, Bhargav A.; Xu, Jinyi; Chen, Zhe-Sheng

    2015-01-01

    Overexpression of ATP-Binding Cassette transporters leads to multidrug resistance in cancer cells and results in the failure of chemotherapy. In this in-vitro study, we investigated whether or not (20S, 24R/S)-epoxy-12β, 25-dihydroxy-dommarane-3β-amine (ORA and OSA), a pair of semi-synthetic ocotillol analogue epimers, could inhibit the ABCB1 transporter. ORA (1 μM and 3 μM) significantly reversed the resistance to paclitaxel and vincristine in ABCB1-overexpressing SW620/Ad300 and HEK/ABCB1 cells, whereas OSA had no significant effects. In addition, ORA (3 μM) significantly increased the intracellular accumulation of [3H]-paclitaxel by suppressing the efflux function of ABCB1. Meanwhile, both ORA (3 μM) and OSA (3 μM) did not significantly alter the expression level or the subcellular location of ABCB1 protein. Moreover, the ABCB1 ATPase study suggested that ORA had a stronger stimulatory effect on the ATPase activity than OSA. ORA also exhibited a higher docking score as compared with OSA inside transmembrane domain of ABCB1. Overall, we concluded that ORA reverse ABCB1-mediated MDR by competitively inhibiting the ABCB1 drug efflux function. PMID:26296969

  10. Osimertinib (AZD9291), a Mutant-Selective EGFR Inhibitor, Reverses ABCB1-Mediated Drug Resistance in Cancer Cells.

    PubMed

    Zhang, Xiao-Yu; Zhang, Yun-Kai; Wang, Yi-Jun; Gupta, Pranav; Zeng, Leli; Xu, Megan; Wang, Xiu-Qi; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-01-01

    In recent years, tyrosine kinase inhibitors (TKIs) have been shown capable of inhibiting the ATP-binding cassette (ABC) transporter-mediated multidrug resistance (MDR). In this study, we determine whether osimertinib, a novel selective, irreversible EGFR (epidermal growth factor receptor) TKI, could reverse ABC transporter-mediated MDR. The results showed that, at non-toxic concentrations, osimertinib significantly sensitized both ABCB1-transfected and drug-selected cell lines to substrate anticancer drugs colchicine, paclitaxel, and vincristine. Osimertinib significantly increased the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant alteration in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 0.3 µM osimertinib for 72 h. In addition, ATPase assay showed osimertinib stimulated ABCB1 ATPase activity. Molecular docking and molecular dynamic simulations showed osimertinib has strong and stable interactions at the transmembrane domain of human homology ABCB1. Taken together, our findings suggest that osimertinib, a clinically-approved third-generation EGFR TKI, can reverse ABCB1-mediated MDR, which supports the combination therapy with osimertinib and ABCB1 substrates may potentially be a novel therapeutic stategy in ABCB1-positive drug resistant cancers. PMID:27649127

  11. Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function

    PubMed Central

    Atisha-Fregoso, Yemil; Lima, Guadalupe; Pascual-Ramos, Virginia; Baños-Peláez, Miguel; Fragoso-Loyo, Hilda; Jakez-Ocampo, Juan; Contreras-Yáñez, Irazú; Llorente, Luis

    2016-01-01

    Objective To compare drug efflux function of ABCB1 and ABCG2 transporters in rheumatoid arthritis (RA) patients with active disease and in remission. Methods Twenty two active RA patients (DAS28 ≥3.2) and 22 patients in remission (DAS28<2.6) were selected from an early RA clinic. All patients were evaluated at study inclusion and six months later. ABCB1 and ABCG2 functional activity was measured in peripheral lymphocytes by flow cytometry. The percentage of cells able to extrude substrates for ABCB1 and ABCG2 was recorded. Results Active patients had higher ABCB1 and ABCG2 activity compared with patients in remission (median [interquartile range]): 3.9% (1.4–22.2) vs (1.3% (0.6–3.2), p = 0.003 and 3.9% (1.1–13.3) vs 0.9% (0.5–1.9) p = 0.006 respectively. Both transporters correlated with disease activity assessed by DAS28, rho = 0.45, p = 0.002 and rho = 0.47, p = 0.001 respectively. Correlation was observed between the time from the beginning of treatment and transporter activity: rho = 0.34, p = 0.025 for ABCB1 and rho = 0.35, p = 0.018 for ABCG2. The linear regression model showed that DAS28 and the time from the onset of treatment are predictors of ABCB1 and ABCG2 functional activity, even after adjustment for treatment. After six months we calculated the correlation between change in DAS28 and change in the functional activity in both transporters and found a moderate and significant correlation for ABCG2 (rho = 0.28, p = 0.04) and a non-significant correlation for ABCB1 (rho = 0.22, p = 0.11). Conclusions Patients with active RA have an increased function of ABCB1 and ABCG2, and disease activity is the main determinant of this phenomena. PMID:27442114

  12. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.

    PubMed

    Shi, Zhi; Tiwari, Amit K; Shukla, Suneet; Robey, Robert W; Singh, Satyakam; Kim, In-Wha; Bates, Susan E; Peng, Xingxiang; Abraham, Ioana; Ambudkar, Suresh V; Talele, Tanaji T; Fu, Li-Wu; Chen, Zhe-Sheng

    2011-04-15

    Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions. PMID:21402712

  13. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells.

    PubMed

    Wang, Fang; Chen, Yifan; Huang, Lihua; Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; To, Kenneth Kin Wah; Gu, Yong; Fu, Liwu

    2015-12-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients.

  14. Cetuximab enhanced the efficacy of chemotherapeutic agent in ABCB1/P-glycoprotein-overexpressing cancer cells

    PubMed Central

    Liu, Tao; Huang, Yue; Zhao, Jianming; Wang, Xiaokun; Yang, Ke; Ma, Shaolin; Huang, Liyan; Wah To, Kenneth Kin; Gu, Yong; Fu, Liwu

    2015-01-01

    The overexpression of ATP-binding cassette (ABC) transporters is closely associated with the development of multidrug resistance (MDR) in certain types of cancer, which represents a formidable obstacle to the successful cancer chemotherapy. Here, we investigated that cetuximab, an EGFR monoclonal antibody, reversed the chemoresistance mediated by ABCB1, ABCG2 or ABCC1. Our results showed that cetuximab significantly enhanced the cytotoxicity of ABCB1 substrate agent in ABCB1-overexpressing MDR cells but had no effect in their parental drug sensitive cells and ABCC1, ABCG2 overexpressing cells. Furthermore, cetuximab markedly increased intracellular accumulation of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABCB1-overexpressing MDR cancer cells in a concentration-dependent manner. Cetuximab stimulated the ATPase activity but did not alter the expression level of ABCB1 or block phosphorylation of AKT and ERK. Interestingly, cetuximab decreased the cell membrane fluidity which was known to decrease the function of ABCB1. Our findings advocate further clinical investigation of combination chemotherapy of cetuximab and conventional chemotherapeutic drugs in ABCB1 overexpressing cancer patients. PMID:26506420

  15. P-glycoprotein (ABCB1) transports the primary active tamoxifen metabolites endoxifen and 4-hydroxytamoxifen and restricts their brain penetration.

    PubMed

    Iusuf, Dilek; Teunissen, Sebastiaan F; Wagenaar, Els; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2011-06-01

    P-glycoprotein (P-gp, ABCB1) is a highly efficient drug efflux pump expressed in brain, liver, and small intestine, but also in tumor cells, that affects pharmacokinetics and confers therapy resistance for many anticancer drugs. The aim of this study was to investigate the impact of P-gp on tamoxifen and its primary active metabolites, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen. We used in vitro transport assays and Abcb1a/1b(-/-) mice to investigate the impact of P-gp on the oral availability and brain penetration of tamoxifen and its metabolites. Systemic exposure of tamoxifen and its metabolites after oral administration of tamoxifen (50 mg/kg) was not changed in the absence of P-gp. However, brain accumulation of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were modestly, but significantly (1.5- to 2-fold), increased. Endoxifen, however, displayed a 9-fold higher brain penetration at 4 h after administration. Endoxifen was transported by P-gp in vitro. Upon direct oral administration of endoxifen (20 mg/kg), systemic exposure was slightly decreased in Abcb1a/1b(-/-) mice, but brain accumulation of endoxifen was dramatically increased (up to 23-fold at 4 h after administration). Shortly after high-dose intravenous administration (5 or 20 mg/kg), endoxifen brain accumulation was increased only 2-fold in Abcb1a/1b(-/-) mice compared with wild-type mice, suggesting a partial saturation of P-gp at the blood-brain barrier. Endoxifen, the clinically most relevant metabolite of tamoxifen, is a P-gp substrate in vitro and in vivo, where P-gp limits its brain penetration. P-gp might thus be relevant for tamoxifen/endoxifen resistance of P-gp-positive breast cancer and tumors positioned behind a functional blood-brain barrier. PMID:21378205

  16. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells.

    PubMed

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-12-22

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs.

  17. Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells

    PubMed Central

    Ma, Wenzhe; Feng, Senling; Yao, Xiaojun; Yuan, Zhongwen; Liu, Liang; Xie, Ying

    2015-01-01

    Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 μM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs. PMID:26689156

  18. Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters

    PubMed Central

    Zhang, Yun-Kai; Zhang, Guan-Nan; Wang, Yi-Jun; Patel, Bhargav A.; Talele, Tanaji T.; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-01-01

    ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [3H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [3H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 μM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment. PMID:27157787

  19. Impact of ABCB1 1236C > T-2677G > T-3435C > T polymorphisms on the anti-proliferative activity of imatinib, nilotinib, dasatinib and ponatinib

    PubMed Central

    Dessilly, Géraldine; Panin, Nadtha; Elens, Laure; Haufroid, Vincent; Demoulin, Jean-Baptiste

    2016-01-01

    Overexpression of ABCB1 (also called P-glycoprotein) confers resistance to multiple anticancer drugs, including tyrosine kinase inhibitors (TKIs). Several ABCB1 single nucleotide polymorphisms affect the transporter activity. The most common ABCB1 variants are 1236C > T, 2677G > T, 3435C > T and have been associated with clinical response to imatinib in chronic myelogenous leukaemia (CML) in some studies. We evaluated the impact of these polymorphisms on the anti-proliferative effect and the intracellular accumulation of TKIs (imatinib, nilotinib, dasatinib and ponatinib) in transfected HEK293 and K562 cells. ABCB1 overexpression increased the resistance of cells to doxorubicin, vinblastine and TKIs. Imatinib anti-proliferative effect and accumulation were decreased to a larger extent in cells expressing the ABCB1 wild-type protein compared with the 1236T-2677T-3435T variant relatively to control cells. By contrast, ABCB1 polymorphisms influenced the activity of nilotinib, dasatinib and ponatinib to a much lesser extent. In conclusion, our data suggest that wild-type ABCB1 exports imatinib more efficiently than the 1236T-2677T-3435T variant protein, providing a molecular basis for the reported association between ABCB1 polymorphisms and the response to imatinib in CML. Our results also point to a weaker impact of ABCB1 polymorphisms on the activity of nilotinib, dasatinib and ponatinib. PMID:27405085

  20. The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent mechanisms

    PubMed Central

    Natarajan, Karthika; Bhullar, Jasjeet; Shukla, Suneet; Burcu, Mehmet; Chen, Zhe-Sheng; Ambudkar, Suresh V.; Baer, Maria R.

    2013-01-01

    Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC50s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC50 of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization. PMID:23261525

  1. Wnt5A regulates ABCB1 expression in multidrug-resistant cancer cells through activation of the non-canonical PKA/β-catenin pathway

    PubMed Central

    Hung, Tsai-Hsien; Hsu, Sheng-Chi; Cheng, Ching-Yi; Choo, Kong-Bung; Tseng, Ching-Ping; Chen, Tse-Ching; Lan, Ying-Wei; Huang, Tsung-Teng; Lai, Hsin-Chih; Chen, Chuan-Mu; Chong, Kowit-Yu

    2014-01-01

    Multidrug resistance in cancer cells arises from altered drug permeability of the cell. We previously reported activation of the Wnt pathway in ABCB1-overexpressed human uterus sarcoma drug-resistant MES-SA/Dx5 cells through active β-catenin and associated transactivation activities, and upregulation of Wnt-targeting genes. In this study, Wnt5A was found to be significantly upregulated in MES-SA/Dx5 and MCF7/ADR2 cells, suggesting an important role for the Wnt5A signaling pathway in cancer drug resistance. Higher cAMP response elements and Tcf/Lef transcription activities were shown in the drug-resistant cancer cells. However, expression of Wnt target genes and CRE activities was downregulated in Wnt5A shRNA stably-transfected MES-SA/Dx5 cells. Cell viability of the drug-resistant cancer cells was also reduced by doxorubicin treatment and Wnt5A shRNA transfection, or by Wnt5A depletion. The in vitro data were supported by immunohistochemical analysis of 24 paired breast cancer biopsies obtained pre- and post-chemotherapeutic treatment. Wnt5A, VEGF and/or ABCB1 were significantly overexpressed after treatment, consistent with clinical chemoresistance. Taken together, the Wnt5A signaling pathway was shown to contribute to regulating the drug-resistance protein ABCB1 and β-catenin-related genes in antagonizing the toxic effects of doxorubicin in the MDR cell lines and in clinical breast cancer samples. PMID:25401518

  2. Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

    PubMed Central

    Chong, Kowit Yu; Hsu, Chih-Jung; Hung, Tsai-Hsien; Hu, Han-Shu; Huang, Tsung-Teng; Wang, Tzu-Hao; Wang, Chihuei; Chen, Chuan-Mu; Choo, Kong Bung; Tseng, Ching-Ping

    2015-01-01

    Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis. PMID:25590413

  3. Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

    PubMed Central

    Chufan, Eduardo E.; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T.; Durell, Stewart R.; Ambudkar, Suresh V.

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate

  4. The FLT3 and PDGFR inhibitor crenolanib is a substrate of the multidrug resistance protein ABCB1 but does not inhibit transport function at pharmacologically relevant concentrations.

    PubMed

    Mathias, Trevor J; Natarajan, Karthika; Shukla, Suneet; Doshi, Kshama A; Singh, Zeba N; Ambudkar, Suresh V; Baer, Maria R

    2015-04-01

    Background Crenolanib (crenolanib besylate, 4-piperidinamine, 1-[2-[5-[(3-methyl-3-oxetanyl)methoxy]-1H-benzimidazol-1-yl]-8-quinolinyl]-, monobenzenesulfonate) is a potent and specific type I inhibitor of fms-like tyrosine kinase 3 (FLT3) that targets the active kinase conformation and is effective against FLT3 with internal tandem duplication (ITD) with point mutations induced by, and conferring resistance to, type II FLT3 inhibitors in acute myeloid leukemia (AML) cells. Crenolanib is also an inhibitor of platelet-derived growth factor receptor alpha and beta and is in clinical trials in both gastrointestinal stromal tumors and gliomas. Methods We tested crenolanib interactions with the multidrug resistance-associated ATP-binding cassette proteins ABCB1 (P-glycoprotein), ABCG2 (breast cancer resistance protein) and ABCC1 (multidrug resistance-associated protein 1), which are expressed on AML cells and other cancer cells and are important components of the blood-brain barrier. Results We found that crenolanib is a substrate of ABCB1, as evidenced by approximate five-fold resistance of ABCB1-overexpressing cells to crenolanib, reversal of this resistance by the ABCB1-specific inhibitor PSC-833 and stimulation of ABCB1 ATPase activity by crenolanib. In contrast, crenolanib was not a substrate of ABCG2 or ABCC1. Additionally, it did not inhibit substrate transport by ABCB1, ABCG2 or ABCC1, at pharmacologically relevant concentrations. Finally, incubation of the FLT3-ITD AML cell lines MV4-11 and MOLM-14 with crenolanib at a pharmacologically relevant concentration of 500 nM did not induce upregulation of ABCB1 cell surface expression. Conclusions Thus ABCB1 expression confers resistance to crenolanib and likely limits crenolanib penetration of the central nervous system, but crenolanib at therapeutic concentrations should not alter cellular exposure to ABC protein substrate chemotherapy drugs.

  5. Use of a combined effect model approach for discriminating between ABCB1- and ABCC1-type efflux activities in native bivalve gill tissue.

    PubMed

    Faria, Melissa; Pavlichenko, Vasiliy; Burkhardt-Medicke, Kathleen; Soares, Amadeu M V M; Altenburger, Rolf; Barata, Carlos; Luckenbach, Till

    2016-04-15

    Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds. PMID:26929997

  6. Butorphanol, a synthetic opioid, sensitizes ABCB1-mediated multidrug resistance via inhibition of the efflux function of ABCB1 in leukemia cells.

    PubMed

    Wen, Jing; Zhang, Tao; Shan, Zhi-Ming; Qi, Min-Yue; Xiu, Huan-Huan; Liu, Lei; Wu, Shi-Zhe; Jia, Zhen; Xu, Kang-Qing

    2015-08-01

    Multidrug resistance (MDR) remains a formidable challenge in the use of chemotherapy and represents a powerful obstacle to the treatment of leukemia. ATP-binding cassette subfamily B member 1 (ABCB1) is a recognized factor which causes MDR and is closely related to poor outcome and relapse in leukemia. Ongoing research concerning the strategy for inhibiting the abnormally high activity of the ABCB1 transporter is critically needed. In the present study, we sought to elucidate the interaction between ABCB1 transporter and butorphanol. Our results showed that butorphanol significantly antagonized ABCB1-mediated drug efflux and increased the intracellular drug concentration by inhibiting the transport activity of ABCB1 in leukemia cells. Mechanistic investigations demonstrated that butorphanol did not alter the protein expression or localization of ABCB1 in HL60/VCR and K562/ADR cells. Furthermore, homology modeling indicated that butorphanol could fit into the large drug-binding cavity of ABCB1 and form a binding conformation. In conclusion, butorphanol reversed the ABCB1-mediated MDR in leukemia cells by directly suppressing the efflux activity of ABCB1. PMID:26062728

  7. Bis-cyclopropane analog of disorazole C1 is a microtubule-destabilizing agent active in abcb1-overexpressing human colon cancer cells

    PubMed Central

    Wu, Shaoyu; Guo, Zhijian; Hopkins, Chad D.; Wei, Ning; Chu, Edward; Wipf, Peter; Schmitz, John C.

    2015-01-01

    The novel, chemically stabilized disorazole analog, (−)-CP2-disorazole C1 (1) displayed potent anti-proliferative activity against a broad-spectrum of human colorectal cancer cells. HCT15 and H630R1 cell lines expressing high basal levels of the ABCB1 protein, known to cause multi-drug resistance, were also sensitive to growth inhibition by 1 but were resistant to both vincristine and docetaxel, two commonly used microtubule inhibitors. Compound 1 exhibited strong inhibition of tubulin polymerization at a level comparable to vincristine. In addition, treatment with 1 resulted in decreased protein levels of β-tubulin but not α-tubulin. An analysis of cellular proteins known to interact with microtubules showed that 1 caused decreased expression of c-Myc, APC, Rb, and additional key cellular signaling pathways in CRC cells. Treatment with compound 1 also resulted in G2/M cell cycle arrest and induction of apoptosis, but not senescence. Furthermore, endothelial spheroid sprouting assays demonstrated that 1 suppressed angiogenesis and can, therefore, potentially prevent cancer cells from spreading and metastasizing. Taken together, these findings suggest that the microtubule disruptor 1 may be a potential drug candidate for the treatment of mCRC. PMID:26506423

  8. Vatalanib sensitizes ABCB1 and ABCG2-overexpressing multidrug resistant colon cancer cells to chemotherapy under hypoxia.

    PubMed

    To, Kenneth K W; Poon, Daniel C; Wei, Yuming; Wang, Fang; Lin, Ge; Fu, Li-wu

    2015-09-01

    Cancer microenvironment is characterized by significantly lower oxygen concentration. This hypoxic condition is known to reduce drug responsiveness to cancer chemotherapy via multiple mechanisms, among which the upregulation of the ATP-binding cassette (ABC) efflux transporters confers resistance to a wide variety of structurally unrelated anticancer drugs. Vatalanib (PTK787/ZK22584) is a multitargeted tyrosine kinase inhibitor for all isoforms of VEGFR, PDGFR and c-Kit, which exhibit potent anticancer activity in vitro and in vivo. We investigated the potentiation effect of vatalanib on the anticancer activity of conventional cytotoxic drugs in colon cancer cell lines under both normoxic and hypoxic conditions. Mechanistically, vatalanib was found to inhibit ABCG2 and ABCB1 efflux activity, presumably by acting as a competitive inhibitor and interfering with their ATPase activity. Under hypoxic growth condition, ABCG2 and ABCB1-overexpressing cells sorted out by FACS technique as side population (SP) were found to be significantly more responsive to SN-38 (ABCG2 and ABCB1 substrate anticancer drug) in the presence of vatalanib. The anchorage independent soft agar colony formation capacity of the SP cells was remarkably reduced upon treatment with a combination of SN-38 and vatalanib, compared to SN-38 alone. However, vatalanib, at concentrations that produced the circumvention of the transporters-mediated resistance, did not appreciably alter ABCG2/ABCB1 mRNA or protein expression levels or the phosphorylation of Akt and extracellular signal-regulated kinase (ERK1/2). Our study thus advocates the further investigation of vatalanib for use in combination chemotherapy to eradicate drug-resistant cancer cells under hypoxia. PMID:26206183

  9. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters.

    PubMed

    Tiwari, Amit K; Sodani, Kamlesh; Wang, Si-Rong; Kuang, Ye-Hong; Ashby, Charles R; Chen, Xiang; Chen, Zhe-Sheng

    2009-07-15

    Nilotinib, a BCR-Abl tyrosine kinase inhibitor (TKI), was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia positive chronic myelogenous leukemia. Recently, it was shown that several human multidrug resistance (MDR) ATP-binding cassette (ABC) proteins could be modulated by specific TKIs. MDR can produce cancer chemotherapy failure, typically due to overexpression of ABC transporters, which are involved in the extrusion of therapeutic drugs. Here, we report for the first time that nilotinib potentiates the cytotoxicity of widely used therapeutic substrates of ABCG2, such as mitoxantrone, doxorubicin, and ABCB1 substrates including colchicine, vincristine, and paclitaxel. Nilotinib also significantly enhances the accumulation of paclitaxel in cell lines overexpressing ABCB1. Similarly, nilotinib significantly increases the intracellular accumulation of mitoxantrone in cells transfected with ABCG2. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of methotrexate (MTX), as well as E(2)17betaG a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2 similar to other established TKIs as well as fumitremorgin C. Nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a K(i) value of 0.69+/-0.083 microM as demonstrated by kinetic analysis of nilotinib. Overall, our results indicate that nilotinib could reverse ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be used to guide the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions. Also, these findings may be useful in clinical practice for cancer combination therapy with nilotinib.

  10. Paclitaxel sensitivity in relation to ABCB1 expression, efflux and single nucleotide polymorphisms in ovarian cancer

    PubMed Central

    Gao, Bo; Russell, Amanda; Beesley, Jonathan; Chen, Xiao Qing; Healey, Sue; Henderson, Michelle; Wong, Mark; Emmanuel, Catherine; Galletta, Laura; Johnatty, Sharon E.; Bowtell, David; Bowtell, David; Chenevix-Trench, Georgia; deFazio, Anna; Gertig, Dorota; Green, Adle; Webb, Penelope; Hung, Jillian; Moore, Sue; Traficante, Nadia; Fereday, Sian; Harrap, Karen; Sadkowsky, Troy; Pandeya, Nirmala; Stuart-Harris, Robin; Kirsten, Fred; Rutovitz, Josie; Clingan, Peter; Glasgow, Amanda; Proietto, Anthony; Braye, Stephen; Otton, Greg; Shannon, Jennifer; Bonaventura, Tony; Stewart, James; Begbie, Stephen; Friedlander, Michael; Bell, David; Baron-Hay, Sally; Ferrier, Alan; Gard, Greg; Nevell, David; Pavlakis, Nick; Valmadre, Sue; Young, Barbara; Camaris, Catherine; Crouch, Roger; Edwards, Lyndal; Hacker, Neville; Marsden, Donald; Robertson, Greg; Beale, Phillip; Beith, Jane; Carter, Jonothan; Dalrymple, Chris; Hamilton, Anne; Houghton, Roger; Russell, Peter; Links, Matthew; Grygiel, John; Hill, Jane; Brand, Alison; Byth, Karen; Jaworski, Richard; Harnett, Paul; Sharma, Raghwa; Achen, Anita; Wain, Gerard; Ward, Bruce; Papadimos, David; Crandon, Alex; Cummings, Margaret; Horwood, Ken; Obermair, Andreas; Perrin, Lew; Wyld, David; Nicklin, Jim; Davy, Margaret; Oehler, Martin K; Hall, Chris; Dodd, Tom; Healy, Tabitha; Pittman, Ken; Henderson, Doug; Miller, John; Pierdes, John; Blomfield, Penny; Challis, David; McIntosh, Robert; Parker, Andrew; Brown, Bob; Rome, Robert; Allen, David; Grant, Peter; Hyde, Simon; Laurie, Rohan; Robbie, Melissa; Healy, David; Jobling, Tom; Manolitsas, Tom; McNealage, Jane; Rogers, Peter; Susil, Beatrice; Sumithran, Eric; Simpson, Ian; Phillips, Kelly; Rischin, Danny; Fox, Stephen; Johnson, Daryl; Waring, Paul; Lade, Stephen; Loughrey, Maurice; O’Callaghan, Neil; Murray, William; Billson, Virginia; Pyman, Jan; Neesham, Debra; Quinn, Michael; Underhill, Craig; Bell, Richard; Ng, Leong-Fook; Blum, Robert; Ganju, Vinod; Hammond, Ian; Leung, Yee; McCartney, Anthony; Buck, Martin; Haviv, Izak; Purdie, David; Whiteman, David; Zeps, Nikolajs; Malt, Mary-Rose; Mellon, Anne; Robertson, Randall; Bergh, Trish Vanden; Jones, Marian; Mackenzie, Patricia; Maidens, Jane; Nattress, Kath; Chiew, Yoke-Eng; Stenlake, Annie; Sullivan, Helen; Alexander, Barbara; Ashover, Pat; Brown, Sue; Corrish, Tracy; Green, Lyn; Jackman, Leah; Ferguson, Kaltin; Martin, Karen; Martyn, Adam; Ranieri, Barbara; White, Jo; Jayde, Victoria; Bowes, Leanne; Mamers, Pamela; Galletta, Laura; Giles, Debra; Hendley, Joy; Alsop, Katherine; Schmidt, Trudy; Shirley, Helen; Ball, Colleen; Young, Cherry; Viduka, Suzanna; Tran, Hoa; Bilic, Sanela; Glavinas, Lydia; Brooks, Julia; Haber, Michelle; Norris, Murray; Harnett, Paul; Chenevix-Trench, Georgia; Balleine, Rosemary L.; deFazio, Anna

    2014-01-01

    ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells. PMID:24810093

  11. Regulation of expression of ABCB1 and LRP genes by mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and its role in generation of side population cells in canine lymphoma cell lines.

    PubMed

    Tomiyasu, Hirotaka; Watanabe, Manabu; Goto-Koshino, Yuko; Fujino, Yasuhito; Ohno, Koichi; Sugano, Sumio; Tsujimoto, Hajime

    2013-06-01

    The concept of the cancer stem cell (CSC) has been recognized as key for elucidation of the mechanisms that confer the multidrug resistance (MDR) phenotype to tumor cells, and the side population (SP) fraction has been shown to be enriched by cells with the CSC phenotype. The purpose of the present study was to identify the mechanism that induces a difference of phenotype between the SP and the remaining major population (MP) using two canine lymphoma cell lines. Expression levels of ABCB1 and LRP genes, which encode efflux pumps, were significantly higher in the SP than in the MP. Microarray analysis revealed up-regulation of the expression of transforming growth factor-β (TGF-β) type II receptor in SP compared with MP, and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was more up-regulated in the SP than in the MP. Stimulation of the MAPK/ERK pathway significantly increased the mRNA expression of both ABCB1 and LRP genes. These results indicate increased expression of the efflux pumps through the MAPK/ERK pathway in SP cells.

  12. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

    PubMed

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W

    2013-02-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

  13. ATPase Activity of Pea Cotyledon Submitochondrial Particles

    PubMed Central

    Grubmeyer, Charles; Spencer, Mary

    1980-01-01

    Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO3, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO3−, but inhibited by Cl−, indicating that Cl− stimulation is a distinguishing property of the pea mitochondrial ATPase complex. PMID:16661174

  14. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter

    PubMed Central

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-01-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  15. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

    PubMed

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-08-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance.

  16. ABCB1 regulation through LRPPRC is influenced by the methylation status of the GC -100 box in its promoter.

    PubMed

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Ferreira, Gerson; Cappelletti, Paola; Soares-Lima, Sheila; Pinto, Luis Felipe; Mencalha, André; Abdelhay, Eliana

    2014-08-01

    One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance. PMID:25089713

  17. Novel and functional ABCB1 gene variant in sporadic Parkinson's disease.

    PubMed

    Li, Yuequn; Li, Yonghua; Pang, Shuchao; Huang, Wenhui; Zhang, Aimei; Hawley, Robert G; Yan, Bo

    2014-04-30

    Parkinson's disease (PD) is a common progressive neurodegenerative disease. Most cases of PD are sporadic, which is caused by interaction of genetic and environmental factors. To date, genetic causes for sporadic PD remain largely unknown. ATP-binding cassette sub-family B member 1 (ABCB1) is a membrane-associated protein that acts as an efflux transporter for many substrates, including chemotherapeutic agents, anti-epilepsy medicine, antibiotics and drugs for PD. ABCB1 gene is widely expressed in human tissues, including endothelial cells of capillary blood vessels at blood-brain barrier sites. In PD patients, decreased ABCB1 levels have been reported. We speculated that misregulation of ABCB1 gene expression, caused by DNA sequence variants (DSVs) within its regulatory regions, may be involved in PD development. In this study, we genetically and functionally analyzed the proximal promoter of the human ABCB1 gene, which is required for constitutive expression, in sporadic PD patients and healthy controls. The results showed that a novel and heterozygous DSV g.117077G>A was identified in one PD patient, but in none of the controls. This DSV significantly altered the transcriptional activity of the ABCB1 gene promoter in transiently transfected HEK-293 cells. A heterozygous DSV g.116347T>C was only found in one control. Four single-nucleotide polymorphisms, g.116154T>C (rs28746504), g.117130A>G (rs2188524), g.117356C>G (rs34976462) and g.117372T>C (rs3213619), and one heterozygous deletion DSV g.116039del were found in PD patients and controls with similar frequencies. Therefore, our findings suggest that ABCB1 gene promoter DSVs may contribute to PD development as a rare risk factor. PMID:24572589

  18. ABCB1 in children's brain tumours.

    PubMed

    Coyle, Beth; Kessler, Maya; Sabnis, Durgagauri H; Kerr, Ian D

    2015-10-01

    Tumours of the central nervous system are the most common solid tumour, accounting for a quarter of the 1500 cases of childhood cancer diagnosed each year in the U.K. They are the most common cause of cancer-related death in children. Treatment consists of surgery followed by adjuvant chemotherapy and/or radiotherapy. Survival rates have generally increased, but many survivors suffer from radiotherapy-related neurocognitive and endocrine side effects as well as an increased risk of secondary cancer. Adjuvant chemotherapy is normally given in combination to circumvent chemoresistance, but several studies have demonstrated it to be ineffective in the absence of radiotherapy. The identification of children with drug-resistant disease at the outset could allow stratification of those that are potentially curable by chemotherapy alone. Ultimately, however, what is required is a means to overcome this drug resistance and restore the effectiveness of chemotherapy. Medulloblastomas and ependymomas account for over 30% of paediatric brain tumours. Advances in neurosurgery, adjuvant radiotherapy and chemotherapy have led to improvements in 5-year overall survival rates. There remain, however, significant numbers of medulloblastoma patients that have intrinsically drug-resistant tumours and/or present with disseminated disease. Local relapse in ependymoma is also common and has an extremely poor prognosis with only 25% of children surviving first relapse. Each of these is consistent with the acquisition of drug and radiotherapy resistance. Since the majority of chemotherapy drugs currently used to treat these patients are transport substrates for ATP-binding cassette sub-family B member 1 (ABCB1) we will address the hypothesis that ABCB1 expression underlies this drug resistance. PMID:26517917

  19. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae.

  20. [ATPase and phosphatase activity of drone brood].

    PubMed

    Bodnarchuk, L I; Stakhman, O S

    2004-01-01

    Most researches on insect enzymes concern carbohydrate and nitrogenous exchange. Data on ATPase activity for larval material of drone brood are absent in the available literature. The drone brood is one of the least investigated apiproducts. Allowing for the important role of ATPase in the vital functions of the insect cells our work was aimed at the study of ATPase of the drone blood activity and that of alkaline and acid phosphatases. When studying liophylised preparations of the drone brood homogenate we have found out high activity of Mg2+, Na+, K+-, Ca2+- and Mg2+-ATPase and of alkaline and acid phosphatase, that is the possible explanation of the high-intensity power and plastic processes proceeding during growth and development of larvae. PMID:16350755

  1. P-glycoprotein (ABCB1) activity decreases raltegravir disposition in primary CD4+P-gphigh cells and correlates with HIV-1 viral load

    PubMed Central

    Minuesa, Gerard; Arimany-Nardi, Cristina; Erkizia, Itziar; Cedeño, Samandhy; Moltó, José; Clotet, Bonaventura; Pastor-Anglada, Marçal; Martinez-Picado, Javier

    2016-01-01

    Objectives To evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets. Methods The cellular accumulation ratio of [3H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gphigh) and low P-gp activity (P-gplow); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects. Results [3H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gphigh cells accumulated less raltegravir (38.4% ± 9.6%) than P-gplow cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gphigh T cells sustained a higher HIV-1 replication than P-gplow cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001). Conclusions Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gphigh T cells eliminate intracellular raltegravir more readily than P-gplow cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gphigh T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance. PMID:27334660

  2. Na+/K+-ATPase: Activity and inhibition

    NASA Astrophysics Data System (ADS)

    Čolović, M.; Krstić, D.; Krinulović, K.; Momić, T.; Savić, J.; Vujačić, A.; Vasić, V.

    2009-09-01

    The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

  3. Sex differences in cyclosporine pharmacokinetics and ABCB1 gene expression in mononuclear blood cells in African American and Caucasian renal transplant recipients.

    PubMed

    Tornatore, Kathleen M; Brazeau, Daniel; Dole, Kiran; Danison, Ryan; Wilding, Gregory; Leca, Nicolae; Gundroo, Aijaz; Gillis, Kathryn; Zack, Julia; DiFrancesco, Robin; Venuto, Rocco C

    2013-10-01

    Cyclosporine exhibits pharmacokinetic and pharmacodynamic variability in renal transplant recipients (RTR) attributed to P-glycoprotein (P-gp), an ABCB1 efflux transporter that influences bioavailability and intracellular distribution. Data on race and sex influences on P-gp in RTR are lacking. We investigated sex and race influences on cyclosporine pharmacokinetics and ABCB1 gene expression in peripheral blood mononuclear cells (PBMC). Fifty-four female and male African American and Caucasian stable RTR receiving cyclosporine and mycophenolic acid completed a 12-hour study. ABCB1 gene expression was assessed in PBMCs pre-dose and 4 hours after cyclosporine. Statistical analysis used mixed effects models on transformed, normalized ABCB1 expression and cyclosporine pharmacokinetics. Sex and race differences were observed for the dose-normalized area under the concentration curve (AUC0-12 /Dose) [P = .0004], apparent clearance [P = .0004] and clearance/body mass index (CL/BMI) [P = .027] with slowest clearance and greatest drug exposure in females. Sex and race differences were found pre-dose and 4 hours for ABCB1 [P < .0001] with females having less expression than males. ABCB1 differences were observed between pre-dose and 4 hours [P = .0009]. Female RTR had slower cyclosporine clearance and lower ABCB1 gene expression in PBMC suggesting reduced efflux activity and greater intracellular drug exposure.

  4. Trypsin-induced ATPase Activity in Potato Mitochondria.

    PubMed

    Jung, D W; Laties, G G

    1976-04-01

    Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg(2+), 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg(2+)-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60 C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.

  5. Targeting ABCB1 and ABCC1 with their Specific Inhibitor CBT-1® can Overcome Drug Resistance in Osteosarcoma.

    PubMed

    Fanelli, Marilù; Hattinger, Claudia Maria; Vella, Serena; Tavanti, Elisa; Michelacci, Francesca; Gudeman, Beth; Barnett, Daryl; Picci, Piero; Serra, Massimo

    2016-01-01

    Clinical treatment response achievable with conventional chemotherapy in high-grade osteosarcoma (OS) is severely limited by the presence of intrinsic or acquired drug resistance, which in previous studies has been mainly addressed for overexpression of ABCB1 (MDR1/P-glycoprotein). This study was aimed to estimate the impact on OS drug resistance of a group of ATP binding cassette (ABC) transporters, which in other human tumors have been associated with unresponsiveness to the drugs that represent the backbone of multidrug treatment regimens for OS (doxorubicin, methotrexate, cisplatin). By using a group of 6 drug-sensitive and 20 drug-resistant human OS cell lines, the most relevant transporter which proved to be associated with the degree of drug resistance in OS cells, in addition to ABCB1, was ABCC1. We therefore evaluated the in vitro activity of the orally administrable ABCB1/ABCC1 inhibitor CBT-1(®) (Tetrandrine, NSC-77037). We found that in our OS cell lines this agent was able to revert the ABCB1/ABCC1-mediated resistance against doxorubicin, as well as against the drugs used in second-line OS treatments that are substrates of these transporters (taxotere, etoposide, vinorelbine). Our findings indicated that inhibiting ABCB1 and ABCC1 with CBT-1(®), used in association with conventional chemotherapeutic drugs, may become an interesting new therapeutic option for unresponsive or relapsed OS patients. PMID:26548759

  6. Nickel-induced cell death and survival pathways in cultured renal proximal tubule cells: roles of reactive oxygen species, ceramide and ABCB1.

    PubMed

    Dahdouh, Faouzi; Raane, Maximilian; Thévenod, Frank; Lee, Wing-Kee

    2014-04-01

    Nickel and nickel compounds are carcinogens that target the lungs and kidneys causing cell death or cell survival adaptation. The multidrug resistance P-glycoprotein ABCB1 protects cells against toxic metabolites and xenobiotics and is upregulated in many cancer cell types. Here, we investigated the role of ABCB1 in nickel-induced stress signaling mediated by reactive oxygen species (ROS) and ceramides. In renal proximal tubule cells, nickel chloride (0.1-0.25 mM) increased both ROS formation, detected by 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and cellular ceramides, which were determined by lipid dot blot and surface immunostaining, culminating in decreased cell viability, increased DNA fragmentation, augmented PARP-1 cleavage, and increased ABCB1 mRNA and protein. Inhibitors of the de novo ceramide synthesis pathway (fumonisin B1, L-cycloserine) and an antioxidant (α-tocopherol) attenuated nickel-induced toxicity as well as induction of ABCB1. ABCB1 protects against nickel toxicity as PSC833, an ABCB1 blocker, augmented the decrease in cell viability by nickel. Moreover, nickel toxicity was attenuated in renal MDCK cells stably overexpressing ABCB1. In agreement with previous data that demonstrated extrusion of (glucosyl)ceramides by ABCB1 (Lee et al. in Toxicol Sci 121:343, 2011), PSC833 increased total cellular ceramides by >2-fold after nickel treatment. Further, glucosylceramide synthase (GCS) mRNA is upregulated by nickel at 3 h by ~1.5-fold but declined with prolonged exposures (6-24 h). Inhibition of GCS with C9DGJ or knockdown of GCS with siRNA significantly attenuated nickel toxicity. In conclusion, nickel induces a ROS-ceramide pathway to cause apoptotic cell death as well as activate adaptive survival responses, including upregulation of ABCB1, which improves cell survival by extruding proapoptotic (glucosyl)ceramides. PMID:24448832

  7. Association of Extrarenal Adverse Effects of Posttransplant Immunosuppression With Sex and ABCB1 Haplotypes

    PubMed Central

    Venuto, Rocco C.; Meaney, Calvin J.; Chang, Shirley; Leca, Nicolae; Consiglio, Joseph D.; Wilding, Gregory E.; Brazeau, Daniel; Gundroo, Aijaz; Nainani, Neha; Morse, Sarah E.; Cooper, Louise M.; Tornatore, Kathleen M.

    2015-01-01

    Abstract Extrarenal adverse effects (AEs) associated with calcineurin inhibitor (CNI) and mycophenolic acid (MPA) occur frequently but are unpredictable posttransplant complications. AEs may result from intracellular CNI accumulation and low activity of P-glycoprotein, encoded by the ABCB1 gene. Since ABCB1 single nucleotide polymorphisms (SNPs) and sex influence P-glycoprotein, we investigated haplotypes and extrarenal AEs. A prospective, cross-sectional study evaluated 149 patients receiving tacrolimus and enteric coated mycophenolate sodium or cyclosporine and mycophenolate mofetil. Immunosuppressive AE assessment determined individual and composite gastrointestinal, neurologic, aesthetic, and cumulative AEs. Lipids were quantitated after 12-hour fast. ABCB1 SNPs: c.1236C>T (rs1128503), c.2677G>T/A (rs2032582), and c.3435C>T (rs1045642) were determined with haplotype associations computed using the THESIAS program, and evaluated by immunosuppression, sex and race using multivariate general linear models. Tacrolimus patients exhibited more frequent and higher gastrointestinal AE scores compared with cyclosporine with association to CTT (P = 0.018) and sex (P = 0.01). Aesthetic AE score was 3 times greater for cyclosporine with TTC haplotype (P = 0.005). Females had higher gastrointestinal (P = 0.022), aesthetic (P < 0.001), neurologic (P = 0.022), and cumulative AE ratios (P < 0.001). Total cholesterol (TCHOL), low-density lipoproteins (LDL), and triglycerides were higher with cyclosporine. The TTC haplotype had higher TCHOL (P < 0.001) and LDL (P = 0.005). Higher triglyceride (P = 0.034) and lower high-density lipoproteins (P = 0.057) were associated with TTT with sex-adjusted analysis. ABCB1 haplotypes and sex were associated with extrarenal AEs. Using haplotypes, certain female patients manifested more AEs regardless of CNI. Haplotype testing may identify patients with greater susceptibility to AEs and facilitate CNI

  8. Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization.

    PubMed

    Silva-Neta, Helena Lima; Torrezan, Elis; de Araújo Leite, Jocelmo Cássio; Santi-Gadelha, Tatiane; Marques-Santos, Luis Fernando

    2012-12-01

    Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

  9. Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases.

    PubMed

    Blum, J J; Hayes, A

    1984-01-01

    The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase

  10. P-glycoprotein (ABCB1) inhibited network of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum by systems-theoretical analysis.

    PubMed

    Lin, Hong; Wang, Lin; Jiang, Minghu; Huang, Juxiang; Qi, Lianxiu

    2012-10-01

    We constructed the significant low-expression P-glycoprotein (ABCB1) inhibited transport and signal network in chimpanzee compared with high-expression (fold change ≥2) the human left cerebrum in GEO data set, by using integration of gene regulatory activated and inhibited network inference method with gene ontology (GO) analysis. Our result showed that ABCB1 transport and signal upstream network RAB2A inhibited ABCB1, and downstream ABCB1-inhibited SMAD1_2, NCK2, SLC25A46, GDF10, RASGRP1, EGFR, LRPPRC, RASSF2, RASA4, CA2, CBLB, UBR5, SLC25A16, ITGB3BP, DDIT4, PDPN, RAB2A in chimpanzee left cerebrum. We obtained that the different biological processes of ABCB1 inhibited transport and signal network repressed carbon dioxide transport, ER to Golgi vesicle-mediated transport, folic acid transport, mitochondrion transport along microtubule, water transport, BMP signaling pathway, Ras protein signal transduction, transforming growth factor beta receptor signaling pathway in chimpanzee compared with the inhibited network of the human left cerebrum, as a result of inducing inhibition of mitochondrion transport along microtubule and BMP signal-induced cell shape in chimpanzee left cerebrum. Our hypothesis was verified by the same and different biological processes of ABCB1 inhibited transport and signal network of chimpanzee compared with the corresponding activated network of chimpanzee and the human left cerebrum, respectively.

  11. Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase.

    PubMed Central

    Arruda, Ana Paula; Da-Silva, Wagner S; Carvalho, Denise P; De Meis, Leopoldo

    2003-01-01

    The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3-4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism. PMID:12887329

  12. Aurora kinase A activates the vacuolar H+-ATPase (V-ATPase) in kidney carcinoma cells.

    PubMed

    Al-Bataineh, Mohammad M; Alzamora, Rodrigo; Ohmi, Kazuhiro; Ho, Pei-Yin; Marciszyn, Allison L; Gong, Fan; Li, Hui; Hallows, Kenneth R; Pastor-Soler, Núria M

    2016-06-01

    Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H(+)-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H(+) across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676-24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular

  13. Impact of Genetic Polymorphisms of ABCB1 (MDR1, P-Glycoprotein) on Drug Disposition and Potential Clinical Implications: Update of the Literature.

    PubMed

    Wolking, Stefan; Schaeffeler, Elke; Lerche, Holger; Schwab, Matthias; Nies, Anne T

    2015-07-01

    ATP-binding cassette transporter B1 (ABCB1; P-glycoprotein; multidrug resistance protein 1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types. Numerous structurally unrelated compounds, including drugs and environmental toxins, have been identified as substrates. ABCB1 limits the absorption of xenobiotics from the gut lumen, protects sensitive tissues (e.g. the brain, fetus and testes) from xenobiotics and is involved in biliary and renal secretion of its substrates. In recent years, a large number of polymorphisms of the ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] gene have been described. The variants 1236C>T (rs1128503, p.G412G), 2677G>T/A (rs2032582, p.A893S/T) and 3435C>T (rs1045642, p.I1145I) occur at high allele frequencies and create a common haplotype; therefore, they have been most widely studied. This review provides an overview of clinical studies published between 2002 and March 2015. In summary, the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance. Future research activities are warranted, considering a deep-sequencing approach, as well as well-designed clinical studies with appropriate sample sizes to elucidate the impact of rare ABCB1 variants and their potential consequences for effect sizes.

  14. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    NASA Technical Reports Server (NTRS)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  15. Influence of a time-restricted feeding schedule on the daily rhythm of abcb1a gene expression and its function in rat intestine.

    PubMed

    Hayashi, Yohei; Ushijima, Kentarou; Ando, Hitoshi; Yanagihara, Hayato; Ishikawa, Eiko; Tsuruoka, Shu-Ichi; Sugimoto, Ko-Ichi; Fujimura, Akio

    2010-11-01

    P-glycoprotein (P-gp) is one of the ATP-binding cassette transporters and acts as an efflux pump for cytotoxic substances. P-gp mRNA expression and transporting activity show the daily rhythm and contribute to the chrono-pharmacokinetic profiles of many drugs. It is reported that the daily rhythm of abcb1a mRNA is regulated by a circadian clock-controlled output pathway. Time-restricted feeding is well known to shift the peripheral circadian phase of clock gene expression without changing the central clock function. This study was undertaken to examine the influence of a time-restricted feeding procedure during the light phase on the daily rhythms of abcb1a mRNA expression and P-gp activity. The abcb1a mRNA and P-gp activity showed a daily rhythm with a peak early in the dark phase in rat intestine under ad libitum feeding. Time-restricted feeding during the light phase shifted these rhythms to 12-h advance. The mRNA expression of clock genes (DBP and HLF, the transcript activators of abcb1a) also showed daily rhythms, and their phases were shifted by the time-restricted feeding procedure. The peak time of DBP mRNA expression was similar to that of abcb1a mRNA expression under ad libitum feeding and time-restricted feeding conditions. These results indicate that a time-restricted feeding procedure changes DBP mRNA expression, which in turn influences abcb1a mRNA expression and P-gp activity.

  16. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    PubMed

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  17. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain

    PubMed Central

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M.; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G.; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia

    2014-01-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family – Breast Cancer Resistance Protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) – play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested – even those able to enter the CNS in vivo – were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS. PMID:24993496

  18. High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters.

    PubMed

    Ivnitski-Steele, Irena; Larson, Richard S; Lovato, Debbie M; Khawaja, Hadya M; Winter, Stuart S; Oprea, Tudor I; Sklar, Larry A; Edwards, Bruce S

    2008-04-01

    Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers. PMID:18205550

  19. Measuring In Vitro ATPase Activity for Enzymatic Characterization.

    PubMed

    Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria

    2016-01-01

    Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary. PMID:27584824

  20. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    PubMed Central

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients’ survival. PMID:24380367

  1. A structural pathway for activation of the kinesin motor ATPase

    PubMed Central

    Yun, Mikyung; Zhang, Xiaohua; Park, Cheon-Gil; Park, Hee-Won; Endow, Sharyn A.

    2001-01-01

    Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPase. The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules. PMID:11387196

  2. Influence of activating hormones on human platelet membrane Ca/sup 2 +/-ATPase activity

    SciTech Connect

    Resink, T.J.; Dimitrov, D.; Stucki, S.; Buehler, F.R.

    1986-07-16

    Intact platelets were pretreated with hormones and thereafter membranes were prepared and Ca/sup 2 +/-ATPase activity determined. Thrombin decreased the V/sub max/ of Ca/sup 2 +/-ATPase after pretreatment of intact platelets. Platelet activating factor, vasopressin and ADP also decreased Ca/sup 2 +/-ATPase activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) or A23187 or ionomycin alone had no effect, while the simultaneous pretreatment with TPA and Ca/sup 2 +/-ionophore decreased Ca/sup 2 +/-ATPase activity. cAMP elevating agents prostaglandin E/sub 1/ (PGE/sub 1/) and forskolin had no influence per se on Ca/sup 2 +/-ATPase, but antagonized the inhibitory effect of thrombin. The data suggest a close connection between phosphoinositide metabolism and the Ca/sup 2 +/-ATPase system.

  3. Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport

    PubMed Central

    Michaelis, Martin; Rothweiler, Florian; Wurglics, Mario; Aniceto, Natália; Dittrich, Michaela; Zettl, Heiko; Wiese, Michael; Wass, Mark; Ghafourian, Taravat; Schubert-Zsilavecz, Manfred; Cinatl, Jindrich

    2016-01-01

    Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. PMID:26887049

  4. Cadmium inhibits motility, activities of plasma membrane Ca(2+)-ATPase and axonemal dynein-ATPase of human spermatozoa.

    PubMed

    Da Costa, R; Botana, D; Piñero, S; Proverbio, F; Marín, R

    2016-05-01

    Cd(2+) has been associated with decreased sperm motility in individuals exposed to this element, such as smokers. Among other factors, this lowered motility could be the result of inhibition exerted by Cd(2+) on the activity of the sperm ATPases associated with sperm motility. In this study, we evaluated the plasma membrane Ca(2+)-ATPase and the axonemal dynein-ATPase activities as well as sperm motility, in the presence of different free Cd(2+) concentrations in the assay media. It was found that spermatozoa incubated for 5 h in a medium containing 25 nm free Cd(2+) showed a significant inhibition of progressive motility, reaching values even lower at higher Cd(2+) concentrations. In addition, it was found that the activity of the plasma membrane Ca(2+)-ATPase reached maximal inhibition at 50 nm free Cd(2+), with a K50% inhibition of 18.3 nm free Cd(2+). The dynein-ATPase activity was maximally inhibited by 25 nm free Cd(2+) in the assay medium, with a K50% inhibition of 11.3 nm Cd(2+). Our results indicate that the decreased activity of the sperm ATPases might have a critical importance in the biochemical mechanisms underlying the decreased sperm motility of individuals exposed to Cd(2+). PMID:26259968

  5. ABCB1 C3435T and G2677T/A polymorphism decreased the risk for steroid-induced osteonecrosis of the femoral head after kidney transplantation.

    PubMed

    Asano, Takeshi; Takahashi, Kenji A; Fujioka, Mikihiro; Inoue, Shigehiro; Okamoto, Masahiko; Sugioka, Nobuyuki; Nishino, Hoyoku; Tanaka, Takashi; Hirota, Yoshio; Kubo, Toshikazu

    2003-11-01

    Advances in transplantation technology have brought about great benefits to patients suffering from organ failure, but the problem still remains of complications induced by steroids used for post-transplant immunosuppression. Among the side-effects caused by steroids, non-traumatic osteonecrosis of the femoral head (ONF) constitutes a serious problem. The same protocol for steroid administration induces ONF in some patients, but not in others, indicating the presence of individual difference in steroid sensitivity. We hypothesized that this difference might be mediated by the drug-transport protein, P-glycoprotein (P-gp), and investigated the relationship between single nucleotide polymorphisms in the multidrug resistance gene 1 (ABCB1, MDR1) encoding P-gp and ONF. Subjects comprised 136 patients receiving kidney transplantation. Thirty patients developed post-transplant ONF. Genomic DNA was extracted from peripheral blood, and genotypes of ABCB1 C3435T (exon 26) and G2677T/A (exon 21) were determined by direct sequencing. Multivariate analyses based on clinical information were performed to determine the relationship between ABCB1 genotypes and ONF. The dose/concentration (D/C) ratios of tacrolimus were also determined to estimate the activity of P-gp in patients with different genotypes of ABCB1 C3435T (CC, CT, TT), and in those who did and did not develop ONF. The ABCB1 3435TT genotype showed a significantly lower incidence of ONF (adjusted odds ratio = 0.10, P = 0.034). The D/C ratio in the 3435TT genotype was significantly higher than that in the 3435CC genotype. The D/C ratio in patients developing ONF was significantly higher than in those patients who did not develop ONF. The results suggest increased activity of P-gp in patients with the 3435TT genotype and in those who did not develop ONF. The ABCB1 2677 homozygous variant type also showed a lower incidence of ONF (adjusted odds ratio = 0.26, P = 0.056). The 3435T and 3435C alleles were in linkage

  6. Brain Na+, K+-ATPase Activity In Aging and Disease

    PubMed Central

    de Lores Arnaiz, Georgina Rodríguez; Ordieres, María Graciela López

    2014-01-01

    Na+/K+ pump or sodium- and potassium-activated adenosine 5’-triphosphatase (Na+, K+-ATPase), its enzymatic version, is a crucial protein responsible for the electrochemical gradient across the cell membranes. It is an ion transporter, which in addition to exchange cations, is the ligand for cardenolides. This enzyme regulates the entry of K+ with the exit of Na+ from cells, being the responsible for Na+/K+ equilibrium maintenance through neuronal membranes. This transport system couples the hydrolysis of one molecule of ATP to exchange three sodium ions for two potassium ions, thus maintaining the normal gradient of these cations in animal cells. Oxidative metabolism is very active in brain, where large amounts of chemical energy as ATP molecules are consumed, mostly required for the maintenance of the ionic gradients that underlie resting and action potentials which are involved in nerve impulse propagation, neurotransmitter release and cation homeostasis. Protein phosphorylation is a key process in biological regulation. At nervous system level, protein phosphorylation is the major molecular mechanism through which the function of neural proteins is modulted in response to extracellular signals, including the response to neurotransmitter stimuli. It is the major mechanism of neural plasticity, including memory processing. The phosphorylation of Na+, K+-ATPase catalytic subunit inhibits enzyme activity whereas the inhibition of protein kinase C restores the enzyme activity. The dephosphorylation of neuronal Na+, K+-ATPase is mediated by calcineurin, a serine / threonine phosphatase. The latter enzyme is involved in a wide range of cellular responses to Ca2+ mobilizing signals, in the regulation of neuronal excitability by controlling the activity of ion channels, in the release of neurotransmitters and hormones, as well as in synaptic plasticity and gene transcription. In the present article evidence showing Na+, K+-ATPase involvement in signaling pathways

  7. The effect of basketball training on the players' erythrocyte membrane acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities.

    PubMed

    Parthimos, T; Tsopanakis, C; Angelogianni, P; Schulpis, K H; Parthimos, N; Tsakiris, S

    2007-08-01

    The aim of this study was to investigate whether the activities of erythrocyte membrane acetylcholinesterase (AChE), (Na (+),K (+))-ATPase and Mg (2+)-ATPase are modulated by a basketball training. Blood was obtained from 10 basketball players pre- and postexercise. Total antioxidant status (TAS), lactate and pyruvate concentrations were determined with kits, while the enzyme activities were determined spectrophotometrically. Post-training blood lactate and pyruvate concentrations as well as AChE (2.90 +/- 0.05 vs. 3.98 +/- 0.09 Delta OD/min . mg protein, p < 0.01) and Na (+),K (+)-ATPase (0.58 +/- 0.04 vs. 1.27 +/- 0.12 micromol Pi/h . mg protein, p < 0.001) activities were remarkably increased, whereas TAS was significantly decreased. Mg (2+)-ATPase activity remained unaltered at the end of the training. In conclusion, the stimulation of AChE and Na (+),K (+)-ATPase by the training may be due to the rise of blood catecholamine oxidation contributing to TAS decrease and/or the increase of serotonin levels. This stress condition may modulate cholinergic and catecholaminergic/serotoninergic functions in players.

  8. The functional influences of common ABCB1 genetic variants on the inhibition of P-glycoprotein by Antrodia cinnamomea extracts.

    PubMed

    Sheu, Ming-Jyh; Teng, Yu-Ning; Chen, Ying-Yi; Hung, Chin-Chuan

    2014-01-01

    Antrodia cinnamomea is a traditional healthy food that has been demonstrated to possess anti-inflammatory, antioxidative, and anticacer effects. The purpose of this study was to evaluate whether the ethanolic extract of A. cinnamomea (EEAC) can affect the efflux function of P-glycoprotein (P-gp) and the effect of ABCB1 genetic variants on the interaction between EEAC and P-gp. To investigate the mechanism of this interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established and the expression of P-gp was confirmed by Western blot. The results of the rhodamine 123 efflux assay demonstrated that EEAC efficiently inhibited wild-type P-gp function at an IC50 concentration of 1.51 ± 0.08 µg/mL through non-competitive inhibition. The IC50 concentrations for variant-type 1236T-2677T-3435T P-gp and variant-type 1236T-2677A-3435T P-gp were 5.56 ± 0.49 µg/mL and 3.33±0.67 µg/mL, respectively. In addition, the inhibition kinetics of EEAC also changed to uncompetitive inhibition in variant-type 1236T-2677A-3435T P-gp. The ATPase assay revealed that EEAC was an ATPase stimulator and was capable of reducing verapamil-induced ATPase levels. These results indicate that EEAC may be a potent P-gp inhibitor and higher dosages may be required in subjects carrying variant-types P-gp. Further studies are required to translate this basic knowledge into clinical applications.

  9. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin.

    PubMed

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  10. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin

    PubMed Central

    Guo, Tingting; Huang, Jinhu; Zhang, Hongyu; Dong, Lingling; Guo, Dawei; Guo, Li; He, Fang; Bhutto, Zohaib Ahmed; Wang, Liping

    2016-01-01

    P-glycoprotein (P-gp) is one of the best-known ATP-dependent efflux transporters, contributing to differences in pharmacokinetics and drug-drug interactions. Until now, studies on pig P-gp have been scarce. In our studies, the full-length porcine P-gp cDNA was cloned and expressed in a Madin-Darby Canine Kidney (MDCK) cell line. P-gp expression was then determined in tissues and its role in the pharmacokinetics of oral enrofloxacin in pigs was studied. The coding region of pig Abcb1 gene was 3,861 bp, encoding 1,286 amino acid residues (Mw = 141,966). Phylogenetic analysis indicated a close evolutionary relationship between porcine P-gp and those of cow and sheep. Pig P-gp was successfully stably overexpressed in MDCK cells and had efflux activity for rhodamine 123, a substrate of P-gp. Tissue distribution analysis indicated that P-gp was highly expressed in brain capillaries, small intestine, and liver. In MDCK-pAbcb1 cells, enrofloxacin was transported by P-gp with net efflux ratio of 2.48 and the efflux function was blocked by P-gp inhibitor verapamil. High expression of P-gp in the small intestine could modify the pharmacokinetics of orally administrated enrofloxacin by increasing the Cmax, AUC and Ka, which was demonstrated using verapamil, an inhibitor of P-gp. PMID:27572343

  11. Association between ABCB1 genetic polymorphism and the effect on epilepsy following phenytoin treatment

    PubMed Central

    Sun, Fei; Cao, Bo-Qiang; Wang, Bo; Wu, Shi-Qiang; Jiang, De-Hua

    2016-01-01

    The aim of the study was to analyze the effect of ABCB1 genetic polymorphisms on the efficacy of phenytoin (PHT) treatment in epilepsy patients. In total, 200 epilepsy patients who were administered PHT were divided into the responsive and pharmaco-resistance groups depending on the clinical data of PHT treatment in epilepsy patients. The serum concentration of PHT was detected by high-performance liquid chromatography (HPLC). ABCB1 polymorphisms were analyzed by the polymerase chain reaction restriction-fragment length polymorphism method. The C1236T, C3435T and G2677T/A haplotypes were reconstructed for the ABCB1 gene using SHEsis programs. One-way analysis of variance was used for data analysis. In ABCB1 C1236T, the rate of the CC genotype in pharmaco-resistance (17.5%) was higher than that of the responsive group (2.1%), while the rate of the TT genotype in pharmaco-resistance (41.6%) was lower than that of the responsive group (55.4%) (P<0.05). In ABCB1 G2677T/A, the rate of the GG genotype in pharmaco-resistance (29.6%) was higher than that of the responsive group (9.7%), while the rate of the TT genotype in pharmaco-resistance (4.6%) was lower than that of the responsive group (30.4%) (P<0.05). The rate of the TTC haploid in pharmaco-resistance (24.1%) was higher than that of the responsive group (8.8%) (P<0.05). The PHT serum concentration had no statistical significance in the patients with different genotypes. In conclusion, there was no association between ABCB1 genetic polymorphism and PHT serum concentration, although the polymorphisms affected the efficacy of PHT treatment in patients with epilepsy. PMID:27602091

  12. Association between ABCB1 genetic polymorphism and the effect on epilepsy following phenytoin treatment

    PubMed Central

    Sun, Fei; Cao, Bo-Qiang; Wang, Bo; Wu, Shi-Qiang; Jiang, De-Hua

    2016-01-01

    The aim of the study was to analyze the effect of ABCB1 genetic polymorphisms on the efficacy of phenytoin (PHT) treatment in epilepsy patients. In total, 200 epilepsy patients who were administered PHT were divided into the responsive and pharmaco-resistance groups depending on the clinical data of PHT treatment in epilepsy patients. The serum concentration of PHT was detected by high-performance liquid chromatography (HPLC). ABCB1 polymorphisms were analyzed by the polymerase chain reaction restriction-fragment length polymorphism method. The C1236T, C3435T and G2677T/A haplotypes were reconstructed for the ABCB1 gene using SHEsis programs. One-way analysis of variance was used for data analysis. In ABCB1 C1236T, the rate of the CC genotype in pharmaco-resistance (17.5%) was higher than that of the responsive group (2.1%), while the rate of the TT genotype in pharmaco-resistance (41.6%) was lower than that of the responsive group (55.4%) (P<0.05). In ABCB1 G2677T/A, the rate of the GG genotype in pharmaco-resistance (29.6%) was higher than that of the responsive group (9.7%), while the rate of the TT genotype in pharmaco-resistance (4.6%) was lower than that of the responsive group (30.4%) (P<0.05). The rate of the TTC haploid in pharmaco-resistance (24.1%) was higher than that of the responsive group (8.8%) (P<0.05). The PHT serum concentration had no statistical significance in the patients with different genotypes. In conclusion, there was no association between ABCB1 genetic polymorphism and PHT serum concentration, although the polymorphisms affected the efficacy of PHT treatment in patients with epilepsy.

  13. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters.

    PubMed Central

    Conaway, R C; Conaway, J W

    1989-01-01

    A transcription factor required for synthesis of accurately initiated run-off transcripts by RNA polymerase II has been purified and shown to have an associated DNA-dependent ATPase (dATPase) activity that is strongly stimulated by the TATA region of promoters. This transcription factor, designated delta, was purified more than 3000-fold from extracts of crude rat liver nuclei and has a native molecular mass of approximately 230 kDa. DNA-dependent ATPase (dATPase) and transcription activities copurify when delta is analyzed by hydrophobic interaction and ion-exchange HPLC, arguing that transcription factor delta possesses an ATPase (dATPase) activity. ATPase (dATPase) is specific for adenine nucleotides; ATP and dATP, but not CTP, UTP, or GTP, are hydrolyzed. ATPase (dATPase) is stimulated by both double-stranded and single-stranded DNAs, including pUC18, ssM13, and poly(dT); however, DNA fragments containing the TATA region of either the adenovirus 2 major late or mouse interleukin 3 promoters stimulate ATPase as much as 10-fold more effectively than DNA fragments containing nonpromoter sequences. These data suggest the intriguing possibility that delta plays a critical role in the ATP (dATP)-dependent activation of run-off transcription through a direct interaction with the TATA region of promoters. Images PMID:2552440

  14. Dietary selenium increases the antioxidant levels and ATPase activity in the arteries and veins of poultry.

    PubMed

    Cao, Changyu; Zhao, Xia; Fan, Ruifeng; Zhao, Jinxin; Luan, Yilin; Zhang, Ziwei; Xu, Shiwen

    2016-07-01

    Selenium (Se) deficiency is associated with the pathogenesis of vascular diseases. It has been shown that oxidative levels and ATPase activity were involved in Se deficiency diseases in humans and mammals; however, the mechanism by how Se influences the oxidative levels and ATPase activity in the poultry vasculature is unclear. We assessed the effects of dietary Se deficiency on the oxidative stress parameters (superoxide dismutase, catalase, and hydroxyl radical) and ATPase (Na(+)K(+)-ATPase, Ca(++)-ATPase, Mg(++)-ATPase, and Ca(++)Mg(++)-ATPase) activity in broiler poultry. A total of 40 broilers (1-day old) were randomly divided into a Se-deficient group (L group, fed a Se-deficient diet containing 0.08 mg/kg Se) and a control group (C group, fed a diet containing sodium selenite at 0.20 mg/kg Se). Then, arteries and veins were collected following euthanasia when typical symptoms of Se deficiency appeared. Antioxidant indexes and ATPase activity were evaluated using standard assays in arteries and veins. The results indicated that superoxide dismutase activity in the artery according to dietary Se deficiency was significantly lower (p < 0.05) compared with the C group. The catalase activity in the veins and hydroxyl radical inhibition in the arteries and veins by dietary Se deficiency were significantly higher (p < 0.05) compared with the C group. The Se-deficient group showed a significantly lower (p < 0.05) tendency in Na(+)K(+)-ATPase activity, Ca(++)-ATPase activity, and Ca(++)Mg(++)-ATPase activity. There were strong correlations between antioxidant indexes and Ca(++)-ATPase activity. Thus, these results indicate that antioxidant indexes and ATPases may have special roles in broiler artery and vein injuries under Se deficiency. PMID:26637493

  15. Oscillations in glycolysis in Saccharomyces cerevisiae: the role of autocatalysis and intracellular ATPase activity.

    PubMed

    Kloster, Antonina; Olsen, Lars Folke

    2012-05-01

    We have investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The amplitude dependence on cell density shows the same behavior as that observed in cells in a CSTR. Furthermore, the amplitude decreases with increasing inhibition of the three ATPases (i) F(0)F(1) ATPase, (ii) plasma membrane ATPase (Pma1p) and (iii) vacuolar ATPase (V-ATPase). The amplitude of the oscillations also decreases by stimulating the ATPase activity, e.g. by FCCP or Amphotericin B. Thus, ATPase activity strongly affects the glycolytic oscillations. We discuss these data in relation to a simple autocatalytic model of glycolysis which can reproduce the experimental data and explain the role of membrane-bound ATPases . In addition we also studied a recent detailed model of glycolysis and found that, although this model faithfully reproduces the oscillations of glycolytic intermediates observed experimentally, it is not able to explain the role of ATPase activity on the oscillations.

  16. Substrate independent ATPase activity may complicate high throughput screening.

    PubMed

    Tuntland, Micheal L; Fung, L W-M

    2016-10-01

    Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening. PMID:27430931

  17. CYP2C9, CYP2C19, ABCB1 genetic polymorphisms and phenytoin plasma concentrations in Mexican-Mestizo patients with epilepsy.

    PubMed

    Ortega-Vázquez, A; Dorado, P; Fricke-Galindo, I; Jung-Cook, H; Monroy-Jaramillo, N; Martínez-Juárez, I E; Familiar-López, I; Peñas-Lledó, E; LLerena, A; López-López, M

    2016-06-01

    We aimed to explore the possible influence of CYP2C9 (*2, *3 and IVS8-109 A>T), CYP2C19 (*2, *3 and *17) and ABCB1 (1236C>T, 2677G>A/T and 3435C>T) on phenytoin (PHT) plasma concentrations in 64 Mexican Mestizo (MM) patients with epilepsy currently treated with PHT in mono- (n=25) and polytherapy (n=39). Genotype and allele frequencies of these variants were also estimated in 300 MM healthy volunteers. Linear regression models were used to assess associations between the dependent variables (PHT plasma concentration and dose-corrected PHT concentration) with independent variables (CYP2C9, CYP2C19 and ABCB1 genotypes, ABCB1 haplotypes, age, sex, weight, and polytherapy). In multivariate models, CYP2C9 IVS8-109 T was significantly associated with higher PHT plasma concentrations (t(64)=2.27; P=0.03). Moreover, this allele was more frequent in the supratherapeutic group as compared with the subtherapeutic group (0.13 versus 0.03, respectively; P=0.05, Fisher's exact test). Results suggest that CYP2C9 IVS8-109 T allele may decrease CYP2C9 enzymatic activity on PHT. More research is needed to confirm findings.

  18. The effect of ABCB1 polymorphisms on the outcome of breast cancer treatment

    PubMed Central

    Tulsyan, Sonam; Mittal, Rama Devi; Mittal, Balraj

    2016-01-01

    The ABCB1 gene encodes a permeability glycoprotein, which is one of the most extensively studied human adenosine-triphosphate (ATP)-dependent efflux transporters. Permeability glycoprotein is expressed in the apical membranes of tissues such as intestine, liver, blood–brain barrier, kidney, placenta, and testis and contributes to intracellular drug disposition. It is also highly expressed in tumor cells conferring drug resistance, which is one of the major problems in the efficacy of cancer chemotherapy treatment. ABCB1 is highly polymorphic, and three well-known single-nucleotide polymorphisms such as 1236C>T, 2677G>T/A, and 3435C>T have been found to be associated with altered messenger RNA levels, protein folding, and drug pharmacokinetics. Many association studies and meta-analyses have demonstrated the clinical impact of ABCB1 polymorphisms in breast cancer treatment outcomes with respect to therapeutic response, chemotoxicity, and overall survival. Therefore, the aim of this review was to evaluate the effects of ABCB1 polymorphisms on the outcome of breast cancer treatment which, in future, would be important for tailoring individualized anticancer therapy. PMID:27175090

  19. Targeting ABCB1-mediated tumor multidrug resistance by CRISPR/Cas9-based genome editing

    PubMed Central

    Yang, Yang; Qiu, Jian-Ge; Li, Yong; Di, Jin-Ming; Zhang, Wen-Ji; Jiang, Qi-Wei; Zheng, Di-Wei; Chen, Yao; Wei, Meng-Ning; Huang, Jia-Rong; Wang, Kun; Shi, Zhi

    2016-01-01

    The RNA-guided clustered regularly interspaced short palindromic (CRISPR) in combination with a CRISPR-associated nuclease 9 (Cas9) nuclease system is a new rapid and precise technology for genome editing. In the present study, we applied the CRISPR/Cas9 system to target ABCB1 (also named MDR1) gene which encodes a 170 kDa transmembrane glycoprotein (P-glycoprotein/P-gp) transporting multiple types of chemotherapeutic drugs including taxanes, epipodophyllotoxins, vinca alkaloids and anthracyclines out of cells to contribute multidrug resistance (MDR) in cancer cells. Our data showed that knockout of ABCB1 by CRISPR/Cas9 system was succesfully archieved with two target sgRNAs in two MDR cancer cells due to the alteration of genome sequences. Knockout of ABCB1 by CRISPR/Cas9 system significantly enhances the sensitivity of ABCB1 substrate chemotherapeutic agents and the intracellular accumulation of rhodamine 123 and doxorubicin in MDR cancer cells. Although now there are lots of limitations to the application of CRISPR/Cas9 for editing cancer genes in human patients, our study provides valuable clues for the use of the CRISPR/Cas9 technology in the investigation and conquest of cancer MDR. PMID:27725879

  20. Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines.

    PubMed

    Muthusamy, Ganesan; Balupillai, Agilan; Ramasamy, Karthikeyan; Shanmugam, Mohana; Gunaseelan, Srithar; Mary, Beaulah; Prasad, N Rajendra

    2016-09-01

    Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines. PMID:27262378

  1. Complete Knockout of Endogenous Mdr1 (Abcb1) in MDCK Cells by CRISPR-Cas9.

    PubMed

    Simoff, Ivailo; Karlgren, Maria; Backlund, Maria; Lindström, Anne-Christine; Gaugaz, Fabienne Z; Matsson, Pär; Artursson, Per

    2016-02-01

    Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

  2. The effect of ABCB1 polymorphisms on the outcome of breast cancer treatment.

    PubMed

    Tulsyan, Sonam; Mittal, Rama Devi; Mittal, Balraj

    2016-01-01

    The ABCB1 gene encodes a permeability glycoprotein, which is one of the most extensively studied human adenosine-triphosphate (ATP)-dependent efflux transporters. Permeability glycoprotein is expressed in the apical membranes of tissues such as intestine, liver, blood-brain barrier, kidney, placenta, and testis and contributes to intracellular drug disposition. It is also highly expressed in tumor cells conferring drug resistance, which is one of the major problems in the efficacy of cancer chemotherapy treatment. ABCB1 is highly polymorphic, and three well-known single-nucleotide polymorphisms such as 1236C>T, 2677G>T/A, and 3435C>T have been found to be associated with altered messenger RNA levels, protein folding, and drug pharmacokinetics. Many association studies and meta-analyses have demonstrated the clinical impact of ABCB1 polymorphisms in breast cancer treatment outcomes with respect to therapeutic response, chemotoxicity, and overall survival. Therefore, the aim of this review was to evaluate the effects of ABCB1 polymorphisms on the outcome of breast cancer treatment which, in future, would be important for tailoring individualized anticancer therapy. PMID:27175090

  3. Do drug transporter (ABCB1) SNPs and P-glycoprotein function influence cyclosporine and macrolides exposure in renal transplant patients? Results of the pharmacogenomic substudy within the symphony study.

    PubMed

    Llaudó, Inés; Colom, Helena; Giménez-Bonafé, Pepita; Torras, Joan; Caldés, Anna; Sarrias, Maria; Cruzado, Josep M; Oppenheimer, Federico; Sánchez-Plumed, Jaime; Gentil, Miguel Ángel; Ekberg, Henrik; Grinyó, Josep M; Lloberas, Núria

    2013-02-01

    The function of the efflux pump P-glycoprotein (Pgp) and ABCB1 single nucleotide polymorphisms (SNPs) should be considered as important tools to deepen knowledge of drug nephrotoxicity and disposition mechanisms. The aim of this study is to investigate the association of C3435T, G2677T, C1236T, and T129C ABCB1 SNPs with Pgp activity and exposure to different immunosuppressive drugs in renal transplant patients. Patients included in the Symphony Pharmacogenomic substudy were genotyped for ABCB1 SNPs. According to the design, patients were randomized into four immunosuppressive regimens: low and standard dose of cyclosporine (n = 30), tacrolimus (n = 13), and sirolimus (n = 23) concomitantly with mycophenolate and steroids. Pgp activity was evaluated in PBMC using the Rhodamine 123 efflux assay. TT carrier patients on C3435T, G2677T, and C1236T SNPs (Pgp-low pumpers) showed lower Pgp activity than noncarriers. Pgp-high pumpers treated with cyclosporine showed lower values of Pgp function than macrolides. There was a negative correlation between cyclosporine AUC and Pgp activity at 3 months. Results did not show any correlation between tacrolimus and sirolimus AUC and Pgp activity at 3 months. We found an important role of the ABCB1 SNPs Pgp function in CD3(+) peripheral blood lymphocytes from renal transplant recipients. Pgp activity was influenced by cyclosporine but not macrolides exposure.

  4. Effect of ABCB1 polymorphisms and atorvastatin on sitagliptin pharmacokinetics in healthy volunteers

    PubMed Central

    Aquilante, Christina L.; Wempe, Michael F.; Sidhom, Maha S.; Kosmiski, Lisa A.; Predhomme, Julie A.

    2013-01-01

    Objectives The objectives of this study were to determine if ABCB1 polymorphisms are associated with interindividual variability in sitagliptin pharmacokinetics, and if atorvastatin alters the pharmacokinetic disposition of sitagliptin in healthy volunteers. Methods In this open-label, randomized, two-phase crossover study, healthy volunteers were prospectively stratified according to ABCB1 1236/2677/3435 diplotype (n=9, CGC/CGC; n=10, CGC/TTT; and n=10, TTT/TTT). In one phase, participants received a single 100 mg dose of sitagliptin. In the other phase, participants received 40 mg of atorvastatin for five days, with a single 100 mg dose of sitagliptin administered on day 5. A 24 hour pharmacokinetic study followed each sitagliptin dose, and the study phases were separated by a 14-day washout period. Results Sitagliptin pharmacokinetic parameters did not differ significantly between ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotype groups during the monotherapy phase. Atorvastatin administration did not significantly affect sitagliptin pharmacokinetics, with GMRs (90% CIs) for sitagliptin Cmax, AUC0-∞, CLR, and fe of 0.93 (0.86, 1.01), 0.96 (0.91, 1.01), 1.02 (0.93, 1.12), and 0.98 (0.90, 1.06), respectively. Conclusions ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes did not influence sitagliptin pharmacokinetics in healthy volunteers. Furthermore, atorvastatin had no effect on the pharmacokinetics of sitagliptin in the setting of ABCB1 CGC/CGC, CGC/TTT, and TTT/TTT diplotypes. PMID:23407853

  5. Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.

    PubMed Central

    Ebus, J P; Stienen, G J

    1996-01-01

    1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin-skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/- 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)-independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/- 13 microM s-1 (mean +/- S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane-bound ATPase activity. The steady-state Ca(2+)-independent (basal) ATPase activity was 83 +/- 5 microM s-1 (n = 66). 3. Treatment of saponin-skinned preparations with Triton X-100 abolished 50 microM s-1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/- 5% of the basal activity, whereas 8 +/- 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s-1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)-activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/- 15 microM s-1 (n = 66). Experiments with CPA indicated that at least 9 +/- 6% of the maximal Ca(2+)-activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s-1. 5. Treatment of preparations with Triton X-100 abolished 15 +/- 3

  6. Rapid activation of gill Na+,K+-ATPase in the euryhaline teleost Fundulus heteroclitus

    USGS Publications Warehouse

    Mancera, J.M.; McCormick, S.D.

    2000-01-01

    The rapid activation of gill Na+,K+-ATPase was analyzed in the mummichog (Fundulus heteroclitus) and Atlantic salmon (Salmo salar) transferred from low salinity (0.1 ppt) to high salinity (25-35 ppt). In parr and presmolt, Salmo salar gill Na+,K+-ATPase activity started to increase 3 days after transfer. Exposure of Fundulus heteroclitus to 35 ppt seawater (SW) induced a rise in gill Na+,K+-ATPase activity 3 hr after transfer. After 12 hr, the values dropped to initial levels but showed a second significant increase 3 days after transfer. The absence of detergent in the enzyme assay resulted in lower values of gill Na+,K+-ATPase, and the rapid increase after transfer to SW was not observed. Na+,K+-ATPase activity of gill filaments in vitro for 3 hr increased proportionally to the osmolality of the culture medium (600 mosm/kg > 500 mosm/kg > 300 mosm/kg). Osmolality of 800 mosm/kg resulted in lower gill Na+,K+-ATPase activity relative to 600 mosm/kg. Increasing medium osmolality to 600 mosm/kg with mannitol also increased gill Na+,K+-ATPase. Cycloheximide inhibited the increase in gill Na+,K+-ATPase activity observed in hyperosmotic medium in a dose-dependent manner (10-4 M > 10-5 M > 10-6 M). Actinomycin D or bumetanide in the culture (doses of 10-4 M, 10-5 M, and 10-6 M) did not affect gill Na+,K+-ATPase. Injection of fish with actinomycin D prior to gill organ culture, however, prevented the increase in gill Na+,K+-ATPase activity in hyperosmotic media. The results show a very rapid and transitory increase in gill Na+,K+-ATPase activity in the first hours after the transfer of Fundulus heteroclitus to SW that is dependent on translational and transcriptional processes. (C) 2000 Wiley-Liss, Inc.

  7. Studies on lipids and the activity of Na,K-ATPase in lens fibre cells.

    PubMed Central

    Dean, W L; Delamere, N A; Borchman, D; Moseley, A E; Ahuja, R P

    1996-01-01

    Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes. PMID:8615795

  8. [ATPase activity of the guinea pig central nervous system tissue with experimental allergic encephalomyelitis].

    PubMed

    Metal'nikova, N P; Terlets'ka, Ia T; Belik, Ia V; Chepurko, V N

    1977-01-01

    It is established that the activity of Na+, K+ = ATPase and Mg2+, Ca2+ = ATPase lowers significantly at the paralytic stage of the disease. At earlier stages of the disease preceding the appearance of peculiar neurological symptoms (the seventh and fourteenth days of the incubation period) a decrease of the Mg2+, Ca2+ = ATPase activity was observed in the brain and spinal cord, whereas the Na+, K+ = ATPase activity in the brain remained at the control level up to the appearance of the disease clearly developed symptoms. The Na+, K+ = ATPase activity of the brain on the fourteenth day of the incubation period corresponded to the level of the activity at the paralytic stage of the disease.

  9. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood–Brain Barrier

    PubMed Central

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier‐Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M

    2016-01-01

    ABCB1 and ABCG2 work together at the blood–brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([11C]elacridar and [11C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single‐nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high‐dose tariquidar. In contrast to the ABCB1‐selective substrate (R)‐[11C]verapamil, [11C]elacridar and [11C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [11C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. PMID:26940368

  10. MDR1/ABCB1 gene polymorphisms in patients with chronic myeloid leukemia

    PubMed Central

    Lardo, Mabel; Castro, Marcelo; Moiraghi, Beatriz; Rojas, Francisca; Borda, Natalia; Rey, Jorge A

    2015-01-01

    Background Tyrosine kinase inhibitors (TKIs) are the recommended treatment for patients with chronic myeloid leukemia (CML). The MDR1/ABCB1 gene plays a role in resistance to a wide spectrum of drugs, including TKIs. However, the association of MDR1/ABCB1 gene polymorphisms (SNPs) such as C1236T, G2677T/A, and C3435T with the clinical therapeutic evolution of CML has been poorly studied. We investigated these gene polymorphisms in CML-patients treated with imatinib, nilotinib and/or dasatinib. Methods ABCB1-SNPs were studied in 22 CML-patients in the chronic phase (CP) and 2 CML-patients in blast crisis (BC), all of whom were treated with TKIs, and compared with 25 healthy controls using nested-PCR and sequencing techniques. Results Seventeen different haplotypes were identified: 7 only in controls, 6 only in CML-patients, and the remaining 4 in both groups. The distribution ratios of homozygous TT-variants present on each exon between controls and CML-patients were 2.9 for exon 12, and 0.32 for the other 2 exons. Heterozygous T-variants were observed in all controls (100%) and 75% of CML-patients. Wt-haplotype (CC-GG-CC) was observed in 6 CML-patients (25%). In this wt-group, two were treated with nilotinib and reached a major molecular response. The remaining 4 cases had either a minimal or null molecular response, or developed bone marrow aplasia. Conclusion Our results suggest that SNPs of the MDR1/ABCB1 gene could help to characterize the prognosis and the clinical-therapeutic evolution of CML-patients treated with TKIs. Wt-haplotype could be associated with a higher risk of developing CML, and a worse clinical-therapeutic evolution. PMID:26457282

  11. The Central Cavity of ABCB1 Undergoes Alternating Access During ATP Hydrolysis

    PubMed Central

    McDevitt, Christopher A.; Thomson, Andrew J.; Kerr, Ian D.; MacMillan, Fraser; Callaghan, Richard

    2016-01-01

    Understanding the process that underlies multi-drug recognition and efflux by P-glycoprotein (ABCB1) remains a key biological challenge. Structural data has recently become available for the murine and C. elegans homologues of ABCB1; however all structures were obtained in the absence of nucleotide. A feature of these structures was the presence of a central cavity that is inaccessible from the extracellular face of the protein. To determine the conformational dynamics of this region several residues in transmembrane helices TM6 (331, 343 and 354) and TM12 (980) were mutated to cysteine. Based upon structural predictions these residues are proposed to line, or reside proximal to, the central cavity. The mutant isoforms were labelled with a paramagnetic probe enabling the application of electron paramagnetic resonance (EPR) spectroscopic methods. Power saturation EPR spectra were recorded in the presence of hydrophobic (O2) or hydrophilic (NiEDDA) quenching agents to study the local environment of each residue. ABCB1 was trapped in both its nucleotide bound and post-hydrolytic conformations and EPR spectra were again recorded in the presence and absence of quenching agents. The EPR line shapes provide information on the movements of these residues within TM6 and TM12 during ATP hydrolysis. Rationalisation of the data with molecular dynamic simulations indicate that the cavity is converted to a configuration open to the aqueous phase following nucleotide binding, thereby suggesting alternating access to the cavity on opposite sides of the membrane during translocation. PMID:24597976

  12. Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity

    NASA Technical Reports Server (NTRS)

    Mcdonald, K. S.; Fitts, R. H.

    1993-01-01

    The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

  13. The Multidrug Resistance 1 Gene Abcb1 in Brain and Placenta: Comparative Analysis in Human and Guinea Pig

    PubMed Central

    Pappas, Jane J.; Petropoulos, Sophie; Suderman, Matthew; Iqbal, Majid; Moisiadis, Vasilis; Turecki, Gustavo; Matthews, Stephen G.; Szyf, Moshe

    2014-01-01

    The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain. PMID:25353162

  14. The multidrug resistance pump ABCB1 is a substrate for the ubiquitin ligase NEDD4-1

    PubMed Central

    Akkaya, Begum G.; Zolnerciks, Joseph K.; Ritchie, Tasha K.; Bauer, Bjoern; Hartz, Anika M.S.; Sullivan, James A.; Linton, Kenneth J.

    2016-01-01

    The ATP Binding Cassette transporter ABCB1 can export the neurotoxic peptide β-amyloid from endothelial cells that line the blood-brain barrier (BBB). This has the potential to lower cerebral levels of β-amyloid, but ABCB1 expression in the BBB appears to be progressively reduced in patients with Alzheimer's disease. The surface density of many membrane proteins is regulated by ubiquitination catalysed by ubiquitin E3 ligases. In brain capillaries of mice challenged with β-amyloid ex vivo, we show that the level of the ubiquitin ligase Nedd4 increases concomitant with reduction in Abcb1. In vitro we show that human ABCB1 is a substrate for human NEDD4-1 ligase. Recombinant ABCB1 was purified from Sf21 insect cells and incubated with recombinant NEDD4-1 purified from E. coli. The treated ABCB1 had reduced mobility on SDS-PAGE, and mass spectrometry identified eight lysine residues, K271, K272, K575, K685, K877, K885, K887 and K1062 that were ubiquitinated by NEDD4-1. Molecular modelling showed that all of the residues are exposed on the surface of the intracellular domains of ABCB1. K877, K885 and K887 in particular, are located in the intracellular loop of transmembrane helix 10 (TMH10) in close proximity, in the tertiary fold, to a putative NEDD4-1 binding site in the intracellular helix extending from TMH12 (PxY motif, residues 996-998). Transient expression of NEDD4-1 in HEK293 Flp-In cells stably expressing ABCB1 was shown to reduce the surface density of the transporter. Together, the data identify this ubiquitin ligase as a potential target for intervention in the pathophysiology of Alzheimer's disease. PMID:26006083

  15. Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis.

    PubMed

    Takahashi, Koji; Hayashi, Ken-ichiro; Kinoshita, Toshinori

    2012-06-01

    The phytohormone auxin is a major regulator of diverse aspects of plant growth and development. The ubiquitin-ligase complex SCF(TIR1/AFB) (for Skp1-Cul1-F-box protein), which includes the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) auxin receptor family, has recently been demonstrated to be critical for auxin-mediated transcriptional regulation. Early-phase auxin-induced hypocotyl elongation, on the other hand, has long been explained by the acid-growth theory, for which proton extrusion by the plasma membrane H(+)-ATPase is a functional prerequisite. However, the mechanism by which auxin mediates H(+)-ATPase activation has yet to be elucidated. Here, we present direct evidence for H(+)-ATPase activation in etiolated hypocotyls of Arabidopsis (Arabidopsis thaliana) by auxin through phosphorylation of the penultimate threonine during early-phase hypocotyl elongation. Application of the natural auxin indole-3-acetic acid (IAA) to endogenous auxin-depleted hypocotyl sections induced phosphorylation of the penultimate threonine of the H(+)-ATPase and increased H(+)-ATPase activity without altering the amount of the enzyme. Changes in both the phosphorylation level of H(+)-ATPase and IAA-induced elongation were similarly concentration dependent. Furthermore, IAA-induced H(+)-ATPase phosphorylation occurred in a tir1-1 afb2-3 double mutant, which is severely defective in auxin-mediated transcriptional regulation. In addition, α-(phenylethyl-2-one)-IAA, the auxin antagonist specific for the nuclear auxin receptor TIR1/AFBs, had no effect on IAA-induced H(+)-ATPase phosphorylation. These results suggest that the TIR1/AFB auxin receptor family is not involved in auxin-induced H(+)-ATPase phosphorylation. Our results define the activation mechanism of H(+)-ATPase by auxin during early-phase hypocotyl elongation; this is the long-sought-after mechanism that is central to the acid-growth theory.

  16. Na,K-ATPase activity in mouse muscle is regulated by AMPK and PGC-1α.

    PubMed

    Ingwersen, Maria S; Kristensen, Michael; Pilegaard, Henriette; Wojtaszewski, Jørgen F P; Richter, Erik A; Juel, Carsten

    2011-07-01

    Na,K-ATPase activity, which is crucial for skeletal muscle function, undergoes acute and long-term regulation in response to muscle activity. The aim of the present study was to test the hypothesis that AMP kinase (AMPK) and the transcriptional coactivator PGC-1α are underlying factors in long-term regulation of Na,K-ATPase isoform (α,β and PLM) abundance and Na(+) affinity. Repeated treatment of mice with the AMPK activator AICAR decreased total PLM protein content but increased PLM phosphorylation, whereas the number of α- and β-subunits remained unchanged. The K(m) for Na(+) stimulation of Na,K-ATPase was reduced (higher affinity) after AICAR treatment. PLM abundance was increased in AMPK kinase-dead mice compared with control mice, but PLM phosphorylation and Na,K-ATPase Na(+) affinity remained unchanged. Na,K-ATPase activity and subunit distribution were also measured in mice with different degrees of PGC-1α expression. Protein abundances of α1 and α2 were reduced in PGC-1α +/- and -/- mice, and the β(1)/β(2) ratio was increased with PGC-1α overexpression (TG mice). PLM protein abundance was decreased in TG mice, but phosphorylation status was unchanged. Na,K-ATPase V (max) was decreased in PCG-1α TG and KO mice. Experimentally in vitro induced phosphorylation of PLM increased Na,K-ATPase Na(+) affinity, confirming that PLM phosphorylation is important for Na,K-ATPase function. In conclusion, both AMPK and PGC-1α regulate PLM abundance, AMPK regulates PLM phosphorylation and PGC-1α expression influences Na,K-ATPase α(1) and α(2) content and β(1)/β(2) isoform ratio. Phosphorylation of the Na,K-ATPase subunit PLM is an important regulatory mechanism.

  17. Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity

    PubMed Central

    Bugreev, Dmitry V.; Mazin, Alexander V.

    2004-01-01

    Human Rad51 (hRad51) protein plays a key role in homologous recombination and DNA repair. hRad51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRad51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca2+ greatly stimulates DNA strand exchange activity of hRad51 protein. We found that Ca2+ exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. Our data demonstrate that, in the presence of Mg2+, the hRad51-ATP-ssDNA filament is quickly converted to an inactive hRad51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca2+ maintains the active hRad51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRad51 protein. This mechanism of Rad51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast Rad51 (yRad51) protein. PMID:15226506

  18. Comparison of developmental gradients for growth, ATPase, and fusicoccin-binding activity in mung bean hypocotyls

    NASA Technical Reports Server (NTRS)

    Basel, L. E.; Cleland, R. E.

    1992-01-01

    A comparison has been made of the developmental gradients along a mung bean (Vigna radiata L.) hypocotyl of the growth rate, plasma membrane ATPase, and fusicoccin-binding protein (FCBP) activity to determine whether they are interrelated. The hook and four sequential 7.5 millimeter segments of the hypocotyl below the hook were cut. A plasma membrane-enriched fraction was isolated from each section by aqueous two-phase partitioning and assayed for vanadate-sensitive ATPase and FCBP activity. Each gradient had a distinctive and different pattern. Endogenous growth rate was maximal in the second section and much lower in the others. Vanadate-sensitive ATPase activity was maximal in the third section, but remained high in the older sections. Amounts of ATPase protein, shown by specific antibody binding, did not correlate with the amount of vanadate-sensitive ATPase activity in the three youngest sections. FCBP activity was almost absent in the first section, then increased to a maximum in the oldest sections. These data show that the growth rate is not determined by the ATPase activity, and that there are no fixed ratios between the ATPase and FCBP.

  19. Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated1

    PubMed Central

    Barkla, Bronwyn J.; Vera-Estrella, Rosario; Maldonado-Gama, Minerva; Pantoja, Omar

    1999-01-01

    Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H+-translocating ATPase (V-ATPase) and Na+/H+ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na+ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H+ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na+/H+ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na+/H+ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C3 metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na+ sequestration, mediated by the V-ATPase and Na+/H+ antiport, is regulated through ABA-independent pathways. PMID:10398716

  20. New aspects of the glucose activation of the H(+)-ATPase in the yeast Saccharomyces cerevisiae.

    PubMed

    Souza, M A; Trópia, M J; Brandão, R L

    2001-10-01

    The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983. Many aspects of this signal transduction pathway are still obscure. In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process. It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase. The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated. Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed. Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H(+)-ATPase.

  1. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    PubMed

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  2. Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea.

    PubMed

    Ramnanan, Christopher J; Storey, Kenneth B

    2006-02-01

    Entry into the hypometabolic state of estivation requires a coordinated suppression of the rate of cellular ATP turnover, including both ATP-generating and ATP-consuming reactions. As one of the largest consumers of cellular ATP, the plasma membrane Na+/K+-ATPase is a potentially key target for regulation during estivation. Na+/K+-ATPase was investigated in foot muscle and hepatopancreas of the land snail Otala lactea, comparing active and estivating states. In both tissues enzyme properties changed significantly during estivation: maximal activity was reduced by about one-third, affinity for Mg.ATP was reduced (Km was 40% higher), and activation energy (derived from Arrhenius plots) was increased by approximately 45%. Foot muscle Na+/K+-ATPase from estivated snails also showed an 80% increase in Km Na+ and a 60% increase in Ka Mg2+ as compared with active snails, whereas hepatopancreas Na+/K+-ATPase showed a 70% increase in I50 K+ during estivation. Western blotting with antibodies recognizing the alpha subunit of Na+/K+-ATPase showed no change in the amount of enzyme protein during estivation. Instead, the estivation-responsive change in Na+/K+-ATPase activity was linked to posttranslational modification. In vitro incubations manipulating endogenous kinase and phosphatase activities indicated that Na+/K+-ATPase from estivating snails was a high phosphate, low activity form, whereas dephosphorylation returned the enzyme to a high activity state characteristic of active snails. Treatment with protein kinases A, C or G could all mediate changes in enzyme properties in vitro that mimicked the effect of estivation, whereas treatments with protein phosphatase 1 or 2A had the opposite effect. Reversible phosphorylation control of Na+/K+-ATPase can provide the means of coordinating ATP use by this ion pump with the rates of ATP generation by catabolic pathways in estivating snails. PMID:16449562

  3. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation1[OPEN

    PubMed Central

    Okumura, Masaki; Inoue, Shin-ichiro; Kuwata, Keiko

    2016-01-01

    Plant plasma membrane H+-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H+-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha. However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H+-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H+-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H+-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H+-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H+-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H+-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H+-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. PMID:27016447

  4. Leishmania amazonensis: effects of heat shock on ecto-ATPase activity.

    PubMed

    Peres-Sampaio, Carlos Eduardo; de Almeida-Amaral, Elmo Eduardo; Giarola, Naira Ligia Lima; Meyer-Fernandes, José Roberto

    2008-05-01

    In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress. PMID:18295760

  5. The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase.

    PubMed

    Bredeston, Luis M; González Flecha, F Luis

    2016-07-01

    Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP. PMID:27086711

  6. Association between ABCB1 polymorphisms and haplotypes and Alzheimer's disease: a meta-analysis.

    PubMed

    Zhong, Xin; Liu, Ming-Yan; Sun, Xiao-Hong; Wei, Min-Jie

    2016-01-01

    Although several epidemiological studies have investigated the association between ATP-binding cassette subfamily B member 1 (ABCB1) gene polymorphisms and Alzheimer's disease (AD) susceptibility, controversial results exist. Here, we performed a meta-analysis to assess whether ABCB1 polymorphisms 3435C > T (rs1045642), 2677G > T/A (rs2032582), 1236C > T (rs1128503) and haplotypes were associated with AD risk. Nine independent publications were included and analyzed. Crude odds ratio (OR) and 95% confidence interval (CI) were applied to investigate the strength of the association. Sensitivity analysis was conducted to measure the robustness of our analysis. A funnel plot and trim and fill method were used to test and adjust for publication bias. The results showed a significant association between the 3435C > T single nucleotide polymorphism (SNP) and AD susceptibility (CT vs. CC: OR = 1.24, 95% CI = 1.06-1.45, P = 0.01; CT + TT vs. CC: OR = 1.21, 95% CI = 1.04-1.41, P = 0.01) in the total population, as well as in Caucasian subgroup. The 2677G > T/A SNP was related to a decreased AD risk in Caucasian subgroup (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47-0.98, P = 0.04). Moreover, the ABCB1 haplotype analysis showed that the 1236T/2677T/3435C haplotype was associated with a higher risk of AD (OR = 1.99, 95% CI = 1.24-3.18, P = 0.00). Our results suggest that the ABCB1 3435C > T SNP, the 2677G > T/A SNP and 1236T/2677T/3435C haplotype are significantly associated with AD susceptibility. PMID:27600024

  7. Association between ABCB1 polymorphisms and haplotypes and Alzheimer’s disease: a meta-analysis

    PubMed Central

    Zhong, Xin; Liu, Ming-Yan; Sun, Xiao-Hong; Wei, Min-Jie

    2016-01-01

    Although several epidemiological studies have investigated the association between ATP-binding cassette subfamily B member 1 (ABCB1) gene polymorphisms and Alzheimer’s disease (AD) susceptibility, controversial results exist. Here, we performed a meta-analysis to assess whether ABCB1 polymorphisms 3435C > T (rs1045642), 2677G > T/A (rs2032582), 1236C > T (rs1128503) and haplotypes were associated with AD risk. Nine independent publications were included and analyzed. Crude odds ratio (OR) and 95% confidence interval (CI) were applied to investigate the strength of the association. Sensitivity analysis was conducted to measure the robustness of our analysis. A funnel plot and trim and fill method were used to test and adjust for publication bias. The results showed a significant association between the 3435C > T single nucleotide polymorphism (SNP) and AD susceptibility (CT vs. CC: OR = 1.24, 95% CI = 1.06–1.45, P = 0.01; CT + TT vs. CC: OR = 1.21, 95% CI = 1.04–1.41, P = 0.01) in the total population, as well as in Caucasian subgroup. The 2677G > T/A SNP was related to a decreased AD risk in Caucasian subgroup (TT + TA + AA vs. GT + GA + GG: OR = 0.68, 95% CI = 0.47–0.98, P = 0.04). Moreover, the ABCB1 haplotype analysis showed that the 1236T/2677T/3435C haplotype was associated with a higher risk of AD (OR = 1.99, 95% CI = 1.24–3.18, P = 0.00). Our results suggest that the ABCB1 3435C > T SNP, the 2677G > T/A SNP and 1236T/2677T/3435C haplotype are significantly associated with AD susceptibility. PMID:27600024

  8. Protein Kinase C-α Interaction with F0F1-ATPase Promotes F0F1-ATPase Activity and Reduces Energy Deficits in Injured Renal Cells*

    PubMed Central

    Nowak, Grażyna; Bakajsova, Diana

    2015-01-01

    We showed previously that active PKC-α maintains F0F1-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F0F1-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F0F1-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and β-subunits, were decreased by injury, an event associated with 54% inhibition of F0F1-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F0F1-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, β-, and γ-subunits, decreased F0F1-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, β-, and γ-subunits of F0F1-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F0F1-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or β- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, β-, and γ-subunits of F0F1-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser146 on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F0F1-ATPase, 2) interacts with α-, β-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F0F1-ATPase activity and ATP content after injury in RPTC. PMID:25627689

  9. An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics.

    PubMed

    Wolf, S J; Bachtiar, M; Wang, J; Sim, T S; Chong, S S; Lee, C G L

    2011-10-01

    The human ABCB1 protein, (P-glycoprotein or MDR1) is a membrane-bound glycoprotein that harnesses the energy of ATP hydrolysis to drive the unidirectional transport of substrates from the cytoplasm to the extracellular space. As a large range of therapeutic agents are known substrates of ABCB1 protein, its role in the onset of multidrug resistance has been the focus of much research. This role has been of particular interest in the field of pharmacogenomics where genetic variation within the ABCB1 gene, particularly in the form of single nucleotide polymorphisms (SNPs), is believed to contribute to inter-individual variation in ABCB1 function and drug response. In this review we provide an update on the influence of coding region SNPs within the ABCB1 gene on drug pharmacokinetics. By utilizing the crystal structure of the mouse ABCB1 homolog (Abcb1a), which is 87% homologous to the human sequence, we accompany this discussion with a graphical representation of residue location for amino acids corresponding to human ABCB1 coding region SNPs. Also, an assessment of residue conservation, which is calculated following multiple sequence alignment of 11 confirmed sequences of ABCB1 homologs, is presented and discussed. Superimposing a 'heat map' of residue homology to the Abcb1a crystal structure has permitted additional insights into both the conservation of individual residues and the conservation of their immediate surroundings. Such graphical representation of residue location and conservation supplements this update of ABCB1 pharmacogenetics to help clarify the often confounding reports on the influence of ABCB1 polymorphisms on drug pharmacokinetics and response.

  10. L-Cysteine and glutathione restore the reduction of rat hippocampal Na+, K+-ATPase activity induced by aspartame metabolites.

    PubMed

    Simintzi, Irene; Schulpis, Kleopatra H; Angelogianni, Panagoula; Liapi, Charis; Tsakiris, Stylianos

    2007-07-31

    Studies have implicated aspartame (ASP) ingestion in neurological problems. The aim of this study was to evaluate hippocampal Na(+),K(+)-ATPase and Mg(2+)-ATPase activities after incubation with ASP or each of ASP metabolites, phenylalanine (Phe), methanol (MeOH) and aspartic acid (asp) separately. Suckling rat hippocampal homogenates or pure Na(+),K(+)-ATPase were incubated with ASP metabolites. Na(+),K(+)-ATPase and Mg(2+)-ATPase activities were measured spectrophotometrically. Incubation of hippocampal or pure Na(+),K(+)-ATPase with ASP concentrations (expected in the cerebrospinal fluid (CSF)) after ASP consumption of 34, 150 or 200mg/kg resulted in hippocampal enzyme activity reduction of 26%, 50% or 59%, respectively, whereas pure enzyme was remarkably stimulated. Moreover, incubation with hippocampal homogenate of each one of the corresponding in the CSF ASP metabolites related to the intake of common, high/abuse doses of the sweetener, inhibited Na(+),K(+)-ATPase, while pure enzyme was activated. Hippocampal Mg(2+)-ATPase remained unaltered. Addition of l-cysteine (cys) or reduced glutathione (GSH) in ASP mixtures, related with high/toxic doses of the sweetener, completely or partially restored the inactivated membrane Na(+),K(+)-ATPase, whereas the activated pure enzyme activity returned to normal. CSF concentrations of ASP metabolites related to common, abuse/toxic doses of the additive significantly reduced rat hippocampal Na(+),K(+)-ATPase activity, whereas pure enzyme was activated. Cys or GSH completely or partially restored both enzyme activities.

  11. 1236 C/T and 3435 C/T polymorphisms of the ABCB1 gene in Mexican breast cancer patients.

    PubMed

    Gutierrez-Rubio, S A; Quintero-Ramos, A; Durán-Cárdenas, A; Franco-Topete, R A; Castro-Cervantes, J M; Oceguera-Villanueva, A; Jiménez-Pérez, L M; Balderas-Peña, L M A; Morgan-Villela, G; Del-Toro-Arreola, A; Daneri-Navarro, A

    2015-02-13

    MDR1, which is encoded by the ABCB1 gene, is involved in multidrug resistance (hydrophobic), as well as the elimination of xenotoxic agents. The association between ABCB1 gene polymorphisms and breast cancer risk in different populations has been described previously; however, the results have been inconclusive. In this study, we examined the association between polymorphisms 3435 C/T and 1236 C/T in the ABCB1 gene and breast cancer development in Mexican women according to their menopausal status and molecular classification. Molecular subtypes as well as allele and genotype frequencies were analyzed. A total of 248 women with initial breast cancer diagnosis and 180 ethnically matched, healthy, unrelated individuals were enrolled. Polymerase chain reaction-restriction fragment length polymorphism was performed to detect polymorphisms 3435 C/T and 1236 C/T in the ABCB1 gene. Premenopausal T allele carriers of the 3435 C/T polymorphism showed a 2-fold increased risk of breast cancer with respect to the reference and postmenopausal groups, as well as triple-negative expression regarding the luminal A/B molecular subrogated subtypes. In contrast, the CT genotype of the 1236 polymorphism was a protective factor against breast cancer. We conclude that the T allele carrier of the 3435 C/T polymorphism in the ABCB1 gene in combination with an estrogen receptor-negative status may be an important risk factor for breast cancer development in premenopausal women.

  12. ABCB1 Overexpression Is a Key Initiator of Resistance to Tyrosine Kinase Inhibitors in CML Cell Lines.

    PubMed

    Eadie, Laura N; Hughes, Timothy P; White, Deborah L

    2016-01-01

    The tyrosine kinase inhibitor (TKI) imatinib has resulted in excellent responses in the majority of Chronic Myeloid Leukaemia (CML) patients; however, resistance is observed in 20-30% of patients. More recently, resistance to the second generation TKIs, nilotinib and dasatinib, has also been observed albeit at a lower incidence. ABCB1 has previously been implicated in TKI export and its overexpression linked to TKI resistance. In this study the dynamics of nilotinib resistance was studied in CML cell lines with particular focus on ABCB1 expression levels during development of resistance. Results revealed ABCB1 overexpression is likely an important initiator of nilotinib resistance in vitro. ABCB1 overexpression was also observed in cell lines as an intermediate step during development of resistance to imatinib and dasatinib in vitro. We conclude that ABCB1 overexpression may provide an initial platform to facilitate development of additional mechanisms for resistance to TKIs. This provides a rationale for investigating this phenomenon in patients undergoing TKI therapy. PMID:27536777

  13. ABCB1 Overexpression Is a Key Initiator of Resistance to Tyrosine Kinase Inhibitors in CML Cell Lines

    PubMed Central

    Hughes, Timothy P.; White, Deborah L.

    2016-01-01

    The tyrosine kinase inhibitor (TKI) imatinib has resulted in excellent responses in the majority of Chronic Myeloid Leukaemia (CML) patients; however, resistance is observed in 20–30% of patients. More recently, resistance to the second generation TKIs, nilotinib and dasatinib, has also been observed albeit at a lower incidence. ABCB1 has previously been implicated in TKI export and its overexpression linked to TKI resistance. In this study the dynamics of nilotinib resistance was studied in CML cell lines with particular focus on ABCB1 expression levels during development of resistance. Results revealed ABCB1 overexpression is likely an important initiator of nilotinib resistance in vitro. ABCB1 overexpression was also observed in cell lines as an intermediate step during development of resistance to imatinib and dasatinib in vitro. We conclude that ABCB1 overexpression may provide an initial platform to facilitate development of additional mechanisms for resistance to TKIs. This provides a rationale for investigating this phenomenon in patients undergoing TKI therapy. PMID:27536777

  14. Effect of alpha-actinin on actin structure. Actin ATPase activity.

    PubMed

    Singh, I; Goll, D E; Robson, R M

    1981-08-28

    Alpha-Actinin increases the ATPase activity of actin by up to 84%, depending un pH, divalent cations present and the added Mg2+: ATP ratio. Dithiothreitol decreases actin ATPase activity approx. 20% but does not reduce the ability of alpha-actinin to increase actin ATP activity. Increasing amounts of added alpha-actinin up to 1 mos alpha-actinin to 49 mol actin cause in increasing increment in actin ATPase activity, but adding alpha-actinin beyond 1 mol alpha-actinin to 49 mol actin elicits only small additional increments in activity. Actin ATPase activity ranges from approx 100 nmol Pi/mg actin per h (4.3 mol Pi/mol actin per h) at high levels (10 mM) of ATP in the presence of lower amounts (1 mM) of added mg2+ to approx. 12.5 nmol Pi/mg actin per h (0.52 mol Pi/mol actin per h) at high pH (8.5) or at low levels (0.5-1.0 mM) of ATP in the presence of higher amounts (10 mM) of added Mg2+ ATp uncomplexed with Mg2+ inhibits the ability of alpha-actinin to increase F-actin ATPase activity. Activities with different divalent cations showed that the actin ATPase in these studies, which was 1/100 as great as Mg2+-modified actomyosin ATPase activity, was not due to trace amounts of myosin contaminating the actin preparations. The results are consistent with the concept that alpha-actinin can alter the structure of actin monomers. PMID:6456018

  15. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats.

    PubMed

    Belcastro, A N; Turcotte, R; Rossiter, M; Secord, D; Maybank, P E

    1984-01-01

    The activation characteristics of Mg-ATP and Ca2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min-1 with an 8% incline. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 microM Ca2+ at pH 7.0 and 30 degrees C. For control skeletal muscle, at 1 and 10 microM Ca2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP (P less than 0.05). For E animals the myofibril ATPase activities at 10 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ and all Mg-ATP concentrations were similar to C (P greater than 0.05). At 1.0 microM Ca2+ the activities at 3 and 5 mM Mg-ATP were greater for the E animals (P less than 0.05). Increasing KCl concentrations resulted in greater inhibition for E animals. With cardiac muscle, the myofibril ATPase activities at 1.0 microM free Ca2+ were lower for E at all Mg-ATP levels (P less than 0.05). In contrast, at 10 microM Ca2+, the E group exhibited an elevated myofibril ATPase activity. The results indicate that Mg-ATP and Ca2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise. PMID:6230276

  16. Cholesterol-mediated activation of P-glycoprotein: distinct effects on basal and drug-induced ATPase activities.

    PubMed

    Belli, Sara; Elsener, Priska M; Wunderli-Allenspach, Heidi; Krämer, Stefanie D

    2009-05-01

    Cholesterol promotes basal and verapamil-induced ATPase activity of P-glycoprotein (P-gp). We investigated whether these effects are related to each other and to the impact of the sterol on bilayer fluidity and verapamil membrane affinity. P-gp was reconstituted in egg-phosphatidylcholine (PhC) liposomes with or without cholesterol, 1,2-dipalmitoyl-phosphatidylcholine (DPPC), alpha-tocopherol (alpha-Toc) or 2,2,5,7,8-pentamethyl-6-chromanol (PMC). Basal and verapamil-induced ATPase activities were studied with an enzymatic assay. Membrane fluidity was characterized with diphenyl-hexatriene anisotropy measurements and membrane affinity by equilibrium dialysis. DPPC (70% mol/mol) decreased the fluidity of PhC bilayers to the same level as 20% cholesterol. PMC (20%) and alpha-Toc (20%) decreased the fluidity to lesser extents. alpha-Toc and PMC, but not DPPC increased the verapamil membrane affinity. While 20% cholesterol strikingly enhanced the basal ATPase activity, none of the other constituents had a similar effect. In contrast, verapamil stimulation of P-gp ATPase activity was not only enabled by cholesterol but also by alpha-Toc and DPPC. PMC had no effect. In conclusion, cholesterol exerts distinct effects on basal and verapamil-induced ATPase activity. The influence on basal ATPase activity is sterol-specific while its effect on verapamil-induced ATPase activity is unspecific and not related to its influence on membrane fluidity and on verapamil membrane affinity.

  17. Genomewide analysis of ABCBs with a focus on ABCB1 and ABCB19 in Malus domestica.

    PubMed

    Ma, Juan Juan; Han, Mingyu

    2016-03-01

    The B subfamily of ATP-binding cassette (ABC) proteins (ABCB) plays a vital role in auxin efflux. However, no systematic study has been done in apple. In this study, we performed genomewide identification and expression analyses of the ABCB family in Malus domestica for the first time. We identified a total of 25 apple ABCBs that were divided into three clusters based on the phylogenetic analysis. Most ABCBs within the same cluster demonstrated a similar exon-intron organization. Additionally, the digital expression profiles of ABCB genes shed light on their functional divergence. ABCB1 and ABCB19 are two well-studied auxin efflux carrier genes, and we found that their expression levels are higher in young shoots of M106 than in young shoots ofM9. Since young shoots are the main source of auxin synthesis and auxin efflux involves in tree height control. This suggests that ABCB1 and ABCB19 may also take a part in the auxin efflux and tree height control in apple. PMID:27019441

  18. Agrin regulation of alpha3 sodium-potassium ATPase activity modulates cardiac myocyte contraction.

    PubMed

    Hilgenberg, Lutz G W; Pham, Bryan; Ortega, Maria; Walid, Saif; Kemmerly, Thomas; O'Dowd, Diane K; Smith, Martin A

    2009-06-19

    Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of alpha3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the alpha3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the alpha3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na(+) in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-alpha3 Na,K-ATPase interaction and suggest that agrin modulation of the alpha3 Na,K-ATPase is important in regulating heart function.

  19. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    SciTech Connect

    Kohio, Hinissan P.; Adamson, Amy L.

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  20. Luminal flow modulates H+-ATPase activity in the cortical collecting duct (CCD).

    PubMed

    Liu, Wen; Pastor-Soler, Núria M; Schreck, Carlos; Zavilowitz, Beth; Kleyman, Thomas R; Satlin, Lisa M

    2012-01-01

    Epithelial Na(+) channel (ENaC)-mediated Na(+) absorption and BK channel-mediated K(+) secretion in the cortical collecting duct (CCD) are modulated by flow, the latter requiring an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), microtubule integrity, and exocytic insertion of preformed channels into the apical membrane. As axial flow modulates HCO(3)(-) reabsorption in the proximal tubule due to changes in both luminal Na(+)/H(+) exchanger 3 and H(+)-ATPase activity (Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, Wang T. Am J Physiol Renal Physiol 290: F289-F296, 2006), we sought to test the hypothesis that flow also regulates H(+)-ATPase activity in the CCD. H(+)-ATPase activity was assayed in individually identified cells in microperfused CCDs isolated from New Zealand White rabbits, loaded with the pH-sensitive dye BCECF, and then subjected to an acute intracellular acid load (NH(4)Cl prepulse technique). H(+)-ATPase activity was defined as the initial rate of bafilomycin-inhibitable cell pH (pH(i)) recovery in the absence of luminal K(+), bilateral Na(+), and CO(2)/HCO(3)(-), from a nadir pH of ∼6.2. We found that 1) an increase in luminal flow rate from ∼1 to 5 nl·min(-1)·mm(-1) stimulated H(+)-ATPase activity, 2) flow-stimulated H(+) pumping was Ca(2+) dependent and required microtubule integrity, and 3) basal and flow-stimulated pH(i) recovery was detected in cells that labeled with the apical principal cell marker rhodamine Dolichos biflorus agglutinin as well as cells that did not. We conclude that luminal flow modulates H(+)-ATPase activity in the rabbit CCD and that H(+)-ATPases therein are present in both principal and intercalated cells. PMID:21957178

  1. Association of single nucleotide polymorphisms of ABCB1, OPRM1 and COMT with pain perception in cancer patients.

    PubMed

    Wang, Xu-shi; Song, Hai-bin; Chen, Si; Zhang, Wei; Liu, Jia-qi; Huang, Chao; Wang, Hao-ran; Chen, Yuan; Chu, Qian

    2015-10-01

    Pain perception is influenced by multiple factors. The single nucleotide polymorphisms (SNPs) of some genes were found associated with pain perception. This study aimed to examine the association of the genotypes of ABCB1 C3435T, OPRM1 A118G and COMT V108/158M (valine 108/158 methionine) with pain perception in cancer patients. We genotyped 146 cancer pain patients and 139 cancer patients without pain for ABCB1 C3435T (rs1045642), OPRM1 A118G (rs1799971) and COMT V108/158M (rs4680) by the fluorescent dye-terminator cycle sequencing method, and compared the genotype distribution between groups with different pain intensities by chi-square test and pain scores between groups with different genotypes by non-parametric test. The results showed that in these cancer patients, the frequency of variant T allele of ABCB1 C3435T was 40.5%; that of G allele of OPRM1 A118G was 38.5% and that of A allele of COMT V108/158M was 23.3%. No significant difference in the genotype distribution of ABCB1 C3435T (rs1045642) and OPRM1 A118G (rs1799971) was observed between cancer pain group and control group (P=0.364 and 0.578); however, significant difference occurred in the genotype distribution of COMT V108/158M (rs4680) between the two groups (P=0.001). And the difference could not be explained by any other confounding factors. Moreover, we found that the genotypes of COMT V108/158M and ABCB1 C3435T were associated with the intensities of pain in cancer patients. In conclusion, our results indicate that the SNPs of COMT V108/158M and ABCB1 C3435T significantly influence the pain perception in Chinese cancer patients.

  2. Incorporation of ABCB1-mediated transport into a physiologically-based pharmacokinetic model of docetaxel in mice

    PubMed Central

    Hudachek, Susan F.

    2015-01-01

    Docetaxel is one of the most widely used anticancer agents. While this taxane has proven to be an effective chemotherapeutic drug, noteworthy challenges exist in relation to docetaxel administration due to the considerable interindividual variability in efficacy and toxicity associated with the use of this compound, largely attributable to differences between individuals in their ability to metabolize and eliminate docetaxel. Regarding the latter, the ATP-binding cassette transporter B1 (ABCB1, PGP, MDR1) is primarily responsible for docetaxel elimination. To further understand the role of ABCB1 in the biodistribution of docetaxel in mice, we utilized physiologically-based pharmacokinetic (PBPK) modeling that included ABCB1-mediated transport in relevant tissues. Transporter function was evaluated by studying docetaxel pharmacokinetics in wild-type FVB and Mdr1a/b constitutive knockout (KO) mice and incorporating this concentration–time data into a PBPK model comprised of eight tissue compartments (plasma, brain, heart, lung, kidney, intestine, liver and slowly perfused tissues) and, in addition to ABCB1-mediated transport, included intravenous drug administration, specific binding to intracellular tubulin, intestinal and hepatic metabolism, glomerular filtration and tubular reabsorption. For all tissues in both the FVB and KO cohorts, the PBPK model simulations closely mirrored the observed data. Furthermore, both models predicted AUC values that were with 15 % of the observed AUC values, indicating that our model-simulated drug exposures accurately reflected the observed tissue exposures. Overall, our PBPK model furthers the understanding of the role of ABCB1 in the biodistribution of docetaxel. Additionally, this exemplary model structure can be applied to investigate the pharmacokinetics of other ABCB1 transporter substrates. PMID:23616082

  3. Methylphenidate treatment increases Na(+), K (+)-ATPase activity in the cerebrum of young and adult rats.

    PubMed

    Scherer, Emilene B S; Matté, Cristiane; Ferreira, Andréa G K; Gomes, Karin M; Comim, Clarissa M; Mattos, Cristiane; Quevedo, João; Streck, Emilio L; Wyse, Angela T S

    2009-12-01

    Methylphenidate is a central nervous system stimulant used for the treatment of attention-deficit hyperactivity disorder. Na(+), K(+)-ATPase is a membrane-bound enzyme necessary to maintain neuronal excitability. Considering that methylphenidate effects on central nervous system metabolism are poorly known and that Na(+), K(+)-ATPase is essential to normal brain function, the purpose of this study was to evaluate the effect of this drug on Na(+), K(+)-ATPase activity in the cerebrum of young and adult rats. For acute administration, a single injection of methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline was given to rats on postnatal day 25 or postnatal day 60, in the young and adult groups, respectively. For chronic administration, methylphenidate (1.0, 2.0, or 10.0 mg/Kg) or saline injections were given to young rats starting at postnatal day 25 once daily for 28 days. In adult rats, the same regimen was performed starting at postnatal day 60. Our results showed that acute methylphenidate administration increased Na(+), K(+)-ATPase activity in hippocampus, prefrontal cortex, and striatum of young and adult rats. In young rats, chronic administration of methylphenidate also enhanced Na(+), K(+)-ATPase activity in hippocampus and prefrontal cortex, but not in striatum. When tested in adult rats, Na(+), K(+)-ATPase activity was increased in all cerebral structures studied. The present findings suggest that increased Na(+), K(+)-ATPase activity may be associated with neuronal excitability caused by methylphenidate.

  4. Effects of rifampicin, dexamethasone, St. John's Wort and Thyroxine on maternal and foetal expression of Abcb1 and organ distribution of talinolol in pregnant rats.

    PubMed

    Saljé, Karen; Lederer, Kirstin; Oswald, Stefan; Dazert, Eike; Warzok, Rolf; Siegmund, Werner

    2012-08-01

    It is well accepted that ABCB1 plays a critical role in absorption, distribution and elimination of many xenobiotics and drugs. Only little is known about the regulation and function of ABCB1 during pregnancy. Thus, the aim of this study is to investigate maternal, placental and foetal Abcb1 expression and function in pregnant rats after induction with rifampicin, dexamethasone, St. John's wort (SJW) or thyroxine. Wistar rats were orally treated with rifampicin (250 mg/kg), SJW (1.0 g/kg), thyroxine (9 μg/kg), dexamethasone (1 mg/kg) or 0.5% methylcellulose suspension (control) for 9 days during late pregnancy (each N = 5). Afterwards, organ mRNA expression and protein content of Abcb1a were determined. Tissue concentrations of the ABCB1 probe drug talinolol were measured after repeated administration of the drug (100 mg/kg, 9 days) and after induction with oral rifampicin (250 mg/kg, 9 days, N = 5). Abcb1 expression was substantially lower in foetal than in maternal organs. Abcb1 was significantly induced by SJW in the maternal jejunum and placenta, by dexamethasone in foetal brain and liver and by thyroxine in the placenta and maternal and foetal brain. Rifampicin induced Abcb1 in all maternal and foetal organs. However, organ distribution of talinolol was not influenced by comedication of rifampicin. In conclusion, maternal and foetal Abcb1 organ expression in pregnant rats is inducible by nuclear receptor agonists. Although rifampicin regulates maternal and foetal Abcb1 expression, organ distribution of talinolol remains unchanged most likely caused by the known inhibitory effect of rifampicin on Abcb1 function.

  5. Ca2+-dependent phosphatase and Ca2+-dependent ATPase activities in plasma membranes of eel gill epithelium--III. Stimulation of branchial high-affinity Ca2+-ATPase activity during prolactin-induced hypercalcemia in American eels.

    PubMed

    Flik, G; Wendelaar Bonga, S E; Fenwick, J C

    1984-01-01

    Infusions of ovine prolactin for 10 days induced hypercalcemia in unfed American eels, Anguilla rostrata LeSueur, that tentatively was related to stimulation of branchial Ca2+-uptake mechanisms. Analysis of ATPase activities in the plasma membranes of the branchial epithelium in prolactin treated eels showed a specific stimulation of high-affinity Ca2+-ATPase. The results of this study form further evidence that the high-affinity Ca2+-ATPase activity represents the Ca2+-pump of the branchial epithelium.

  6. Ca/sup 2 +/-CaM-ATPase activity after corticosterone binding to synaptosomal plasma membrane

    SciTech Connect

    Iqbal, Z.; Sze, P.Y.

    1986-03-01

    Studies conducted in the laboratory have demonstrated that corticosterone (CS) binds specifically to synaptosomal plasma membrane (SPM) and modify the cellular events in the synaptosomes. On an exposure of a rat brain synaptosomes to physiological concentrations (<1..mu..M) of CS, the uptake of /sup 45/Ca/sup 2 +/ into synaptosomes was increased by 40-50%. Similarly the binding of /sup 45/Ca/sup 2 +/ to isolated SPM was also enhanced by 50% in the presence of CS. However when intact synaptosomes were pre-incubated with CS, the capacity of membranes to bind /sup 45/Ca/sup 2 +/ was increased by 2-fold. After the incubation of intact synaptosomes with < ..mu..M CS, the activity of trifluoperazine sensitive and CaM dependent Ca/sup 2 +/-ATPase at 1..mu..M Ca/sup 2 +/ was found to be stimulated by 20-30% whereas the activity of Ca/sup 2 +/-ATPase at 1mM Ca/sup 2 +/ remained unaffected. Exposure of synaptosomes to CS was also effective in protecting the reduction of ouabain-sensitive ATPase (Na/sup +/-K/sup +/-ATPase) activity caused by 0.5% ethanol used as a solvent medium for CS. These findings suggest that one of the physiological actions of CS in synaptosomes, after binding to synaptosomal plasma membranes, is an enhancement of Ca/sup 2 +/ transport and an increase of Ca/sup 2 +/-CaM-ATPase activity.

  7. The Apparent Rates of Crossbridge Attachment and Detachment Estimated from Atpase Activity in Insect Flight Muscle

    PubMed Central

    Güth, K.; Poole, K. J. V.; Maughan, D.; Kuhn, H. J.

    1987-01-01

    The ATPase activity of single fibers of small fiber bundles (one to three fibers) of insect flight muscle was measured when fibers were repetitively released and restretched by 1.5% of their initial length. The ATPase activity increased with increasing duration of release-restretch pulses applied at a constant repetition frequency, reaching a maximum at a duration of ∼20 ms. For a given duration, the average ATPase activity also increased with increasing frequency of applied length changes and reached a maximum (200% of the isometric ATPase) at a frequency of ∼50 Hz. The data could be fitted to a two-state model in which the apparent rate of crossbridge detachment is enhanced when the crossbridges are mechanically released. Estimates of the apparent rates of attachment and detachment in the isometrically contracting state and of the enhanced detachment rate of unloaded crossbridges were derived from fits to the two-state model. After short pulses of releasing and restretching the fiber the force was low and increased after the restretch in a roughly exponential manner to the initial level. The rate at which force increased after a release-restretch pulse was similar to the sum of the apparent attachment and detachment rates for the isometrically contracting muscle derived from the ATPase activity measurements. PMID:19431712

  8. Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages.

    PubMed

    Schachter, Julieta; Delgado, Kelly Valcárcel; Barreto-de-Souza, Victor; Bou-Habib, Dumith Chequer; Persechini, Pedro Muanis; Meyer-Fernandes, José Roberto

    2015-05-01

    Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages.

  9. Organophosphate inhibition of avian salt gland Na, K-ATPase activity

    USGS Publications Warehouse

    Eastin, W.C.; Fleming, W.J.; Murray, H.C.

    1982-01-01

    1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6-11. 2. After 11 days, ducks drinking saltwater had lost more weight and had higher plasma Na and uric acid concentrations and osmolalities than birds drinking freshwater. 3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity. 4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.

  10. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+-ATPase inhibitors

    PubMed Central

    Chen, Ronald JY; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason TC

    2011-01-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na+/K+-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na+/K+-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na+/K+-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na+/K+-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na+/K+-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na+/K+-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na+/K+-ATPase in the brain could be potential drugs for the treatment of ischemic stroke. PMID:21293466

  11. Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

    PubMed

    Chen, Ronald J Y; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason T C

    2011-02-01

    The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

  12. [Na,K-ATPase activity of erythrocytes of rats during prolonged starvation].

    PubMed

    Skverchinskaia, E A; Tavrovskaia, T V; Novozhilov, A V

    2013-01-01

    Activity of Na,K-ATPase (Na+,K+-adenosine triphosphatase, EC 3.6.3.9) in the whole erythrocytes was studied in dynamics of the complete rat alimentary starvation for 1, 3, 5, 7-8, and 10-12 days with water drinking ad libitum. There has been established a change of the erythrocyte Na,K-ATPase activity depending on the phase of starvation (the period connected with a certain level of metabolism). After the state on an empty stomach and adaptation to endogenous nutrition (the 0-I phase), from the 3rd to the 7-8th starvation day, the II phase, the period of compensated adaptation occurs (the euglycemia is preserved, the plateau level is preserved, the plateau level is achieved for protein loss and hormonal stimulation). Changes of the Na,K-ATPase activity level within the limits of the II phase were insignificant (p < 0.05), but loses of potassium content in plasma and erythrocytes have been from the 5th starvation day. The III phase (the 12-13th day) is the beginning of the terminal period and is characterized by a decrease of the Na,K-ATPase activity (the oubain-sensitive activity) and of Mg2+-ATPase (the oubain-independent activity), by a decrease of the plasma sodium level (prior to that, this level remained practically unchanged). Ad causes of the revealed decrease of the ATPase activities at the long-term starvation, there are considered aging of population of circulating erythrocytes (the absence of reticulocytes and young erythrocytes), depletion of cell energetic resources (hypoglycemia and glycopenia), effect of endogenous oubain, and endotoxemia.

  13. Selective decrease of Na+/k+ -ATPase activity in the brain of hypothyroid rats.

    PubMed

    Pacheco-Rosado, Jorge; Arias-Citalán, G; Ortiz-Butrón, R; Rodríquez-Páez, L

    2005-01-01

    The present work was performed in order to know if mild hypothyroidism in rats modifies the activity of the Na+/K+ -ATPase in different regions of the brain. Male Wistar rats (300-350 g) were randomly divided into three groups: (1) control group (n=8) drank tap water. (2) hypothyroid group (n=8) treated with 60 mg/kg of methimazole in drinking water; and (3) replaced group (n=8) treated with 60 mg/kg of methimazole plus 35 microg/kg of thyroid hormone (T3) in drinking water. After four weeks of treatment, the rats of all groups were sacrificed by decapitation. The cortex, amygdala, hippocampus and cerebellum were dissected and frozen at -70 degrees C until assay. For enzymatic assay, the tissues were homogenized. The Na+/K+ -ATPase activity was determined by quantifying inorganic phosphate after the samples were incubated with ATP in the presence and absence of 1 mM ouabain. The Na+/K+ -ATPase activity is expressed as pmoles Pi/hr/mg protein. The results showed that the Na+/K+ -ATPase activity in the cortex, amygdala and hippocampus, but not in cerebellum, was lower in hypothyroid group than in control group (p<0.05). The co-administration of methimazole and T3 avoided the decrease of Na+/K+ -ATPase activity, except in amygdala. According to the results obtained we concluded that methimazole treatment decreased the Na+/K+- ATPase activity in the brain's regions which are related to seizures onset. That decrement in enzyme activity was avoided with the coadministration of thyroid hormone.

  14. VEGF, eNOS, and ABCB1 genetic polymorphisms may increase the risk of osteonecrosis of the femoral head.

    PubMed

    Zhou, Z C; Gu, S Z; Wu, J; Liang, Q W

    2015-01-01

    We investigated the associations between vascular endothelial growth factors (VEGF), endothelial nitric oxide synthase (eNOS), and ATP-binding cassette subfamily B member 1 transporter (ABCB1) polymorphisms and the risk of osteonecrosis of the femoral head (ONFH). Published studies were reviewed and analyzed based on predefined selection criteria. The strength of the association between VEGF, eNOS, and ABCB1 polymorphisms and ONFH risk was evaluated based on the odds ratio with corresponding 95%CIs. Meta-analysis was performed using the Comprehensive Meta-analysis 2.0 software. A total of 135 relevant articles were retrieved, of which 10 studies met the selection criteria, and included a total of 1025 patients with ONFH and 1730 healthy controls. The meta-analysis study results revealed that the VEGF rs2010963 G>C polymorphism increased the risk of ONFH, while the VEGF rs2010963 G>C and ABCB1 rs1045642 C>T polymorphisms increased the risk of ONFH under the allele model. In conclusion, the VEGF, eNOS, and ABCB1 polymorphisms may contribute to ONFH, but further studies including larger sample sizes are needed to confirm the results. PMID:26535684

  15. Variants in CDA and ABCB1 are predictors of capecitabine-related adverse reactions in colorectal cancer

    PubMed Central

    García, María I.; García-Alfonso, Pilar; Robles, Luis; Grávalos, Cristina; González-Haba, Eva; Marta, Pellicer; Sanjurjo, María; López-Fernández, Luis A.

    2015-01-01

    Adverse reactions to capecitabine-based chemotherapy limit full administration of cytotoxic agents. Likewise, genetic variations associated with capecitabine-related adverse reactions are associated with controversial results and a low predictive value. Thus, more evidence on the role of these variations is needed. We evaluated the association between nine polymorphisms in MTHFR, CDA, TYMS, ABCB1, and ENOSF1 and adverse reactions, dose reductions, treatment delays, and overall toxicity in 239 colorectal cancer patients treated with capecitabine-based regimens. The ABCB1*1 haplotype was associated with a high risk of delay in administration or reduction in the dose of capecitabine, diarrhea, and overall toxicity. CDA rs2072671 A was associated with a high risk of overall toxicity. TYMS rs45445694 was associated with a high risk of delay in administration or reduction in the dose of capecitabine, HFS >1 and HFS >2. Finally, ENOSF1 rs2612091 was associated with HFS >1, but was a poorer predictor than TYMS rs45445694. A score based on ABCB1-CDA polymorphisms efficiently predicts patients at high risk of severe overall toxicity (PPV, 54%; sensitivity, 43%) in colorectal cancer patients treated with regimens containing capecitabine. Polymorphisms in ABCB1, CDA, ENOSF1,and TYMS could help to predict specific and overall severe adverse reactions to capecitabine. PMID:25691056

  16. Ensemble Rule-Based Classification of Substrates of the Human ABC-Transporter ABCB1 Using Simple Physicochemical Descriptors.

    PubMed

    Demel, Michael A; Kraemer, Oliver; Ettmayer, Peter; Haaksma, Eric; Ecker, Gerhard F

    2010-03-15

    Within the last decades, the detailed knowledge on the impact of membrane bound drug efflux transporters of the ATP binding cassette (ABC) protein family on the pharmacological profile of drugs has enormously increased. Especially, ABCB1 (P-glycoprotein, P-gp, MDR1) has attracted particular interest in medicinal chemistry, since it determines the clinical efficacy, side effects and toxicity risks of drug candidates. Based on this, the development of in silico models that provide rapid and cost-effective screening tools for the classification of substrates and nonsubstrates of ABCB1 is an urgent need in contemporary ADMET profiling. A characteristic hallmark feature of this transporter is its polyspecific ligand recognition pattern. In this study we describe a method for classifying ABCB1 ligands in terms of simple, conjunctive rules (RuleFit) based on interpretable ADMET features. The retrieved results showed that models based on large, very diverse data sets gave better classification performance than models based on smaller, more homogenous training sets. The best model achieved gave a correct classification rate of 0.90 for an external validation set. Furthermore, from the interpretation of the best performing model it could be concluded that in comparison to nonsubstrates ABCB1 substrates generally show a higher number of hydrogen-bond acceptors, are more flexible and exhibit higher logP values.

  17. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    PubMed

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors.

  18. ATPase activity measurement of DNA replicative helicase from Bacillus stearothermophilus by malachite green method.

    PubMed

    Yang, Mu; Wang, Ganggang

    2016-09-15

    The DnaB helicase from Bacillus stearothermophilus (DnaBBst) was a model protein for studying the bacterial DNA replication. In this work, a non-radioactive method for measuring ATPase activity of DnaBBst helicase was described. The working parameters and conditions were optimized. Furthermore, this method was applied to investigate effects of DnaG primase, ssDNA and helicase loader protein (DnaI) on ATPase activity of DnaBBst. Our results showed this method was sensitive and efficient. Moreover, it is suitable for the investigation of functional interaction between DnaB and related factors. PMID:27372608

  19. Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity.

    PubMed

    Hirata, Y; Okada, K

    1990-06-01

    The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.

  20. Glucose-induced activation of the plasma membrane H(+)-ATPase in Fusarium oxysporum.

    PubMed

    Brandão, R L; Castro, I M; Passos, J B; Nicoli, J R; Thevelein, J M

    1992-08-01

    Addition of glucose and other sugars to derepressed cells of the fungus Fusarium oxysporum var. lini triggered activation of the plasma membrane H(+)-ATPase within 5 min. Glucose was the best activator while galactose and lactose had a lesser effect. The activation was not prevented by previous addition of cycloheximide and it was fully reversible when the glucose was removed. The activation process in vivo also caused changes in the kinetic properties of the enzyme. The non-activated enzyme had an apparent Km of about 3.2 mM for ATP whereas the activated enzyme showed an apparent Km of 0.26 mM. In addition, the pH optimum of the H(+)-ATPase changed from 6.0 to 7.5 upon activation. The activated enzyme was more sensitive to inhibition by vanadate. When F. oxysporum was cultivated in media containing glucose as the major carbon source, enhanced H(+)-ATPase activity was largely confined to the period corresponding to the lag phase, i.e. just before the start of acidification of the medium. This suggests that the activation process might play a role in the onset of extracellular acidification. Addition of glucose to F. oxysporum var. lini cells also caused an increase in the cAMP level. No reliable increase could be demonstrated for the other sugars. Addition of proton ionophores such as DNP and CCCP at pH 5.0 caused both a large increase in the intracellular level of cAMP and in the activity of the plasma membrane H(+)-ATPase. Inhibition of the DNP-induced increase in the cAMP level by acridine orange also resulted in inhibition of the activation of plasma membrane H(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. PMID:26944019

  2. P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

    PubMed

    Loo, Tip W; Clarke, David M

    2016-04-01

    P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids.

  3. Knockdown of c-MET induced apoptosis in ABCB1-overexpressed multidrug-resistance cancer cell lines.

    PubMed

    Hung, T-H; Li, Y-H; Tseng, C-P; Lan, Y-W; Hsu, S-C; Chen, Y-H; Huang, T-T; Lai, H-C; Chen, C-M; Choo, K-B; Chong, K-Y

    2015-05-01

    Inappropriate c-MET signaling in cancer can enhance tumor cell proliferation, survival, motility, and invasion. Inhibition of c-MET signaling induces apoptosis in a variety of cancers. It has also been recognized as a novel anticancer therapy approach. Furthermore, reports have also indicated that constitutive expression of P-glycoprotein (ABCB1) is involved in the HGF/c-MET-related pathway of multidrug resistance ABCB1-positive human hepatocellular carcinoma cell lines. We previously reported that elevated expression levels of PKCδ and AP-1 downstream genes, and HGF receptor (c-MET) and ABCB1, in the drug-resistant MES-SA/Dx5 cells. Moreover, leukemia cell lines overexpressing ABCB1 have also been shown to be more resistant to the tyrosine kinase inhibitor imatinib mesylate. These findings suggest that chemoresistant cancer cells may also develop a similar mechanism against chemotherapy agents. To circumvent clinical complications arising from drug resistance during cancer therapy, the present study was designed to investigate apoptosis induction in ABCB1-overexpressed cancer cells using c-MET-targeted RNA interference technology in vitro and in vivo. The results showed that cell viability decreased and apoptosis rate increased in c-MET shRNA-transfected HGF/c-MET pathway-positive MES-SA/Dx5 and MCF-7/ADR2 cell lines in a dose-dependent manner. In vivo reduction of tumor volume in mice harboring c-MET shRNA-knockdown MES-SA/Dx5 cells was clearly demonstrated. Our study demonstrated that downregulation of c-MET by shRNA-induced apoptosis in a multidrug resistance cell line.

  4. [Changes in acetylcholinesterase and ATPase activity and certain structural features of the erythrocyte membrane in experimental myocardial ischemia].

    PubMed

    Chernukh, A M; Kopteva, L A; Shevchenko, A S

    1980-09-01

    Acute heart ischemia induced by ligation of the left coronary artery is associated with variation in the activity of acetylcholinesterase, Na, K-ATPase and Ca, Mg-ATPase in rat erythrocytes. The maximum pronounced variations in the enzymatic activity and in the membrane capacity of erythroblasts for binding direct turquoise are recorded on the 7th day of the experimental myocardial infarction.

  5. Characterization of ATPase activity of the AAA ARC from Bifidobacterium longum subsp. infantis.

    PubMed

    Guzmán-Rodríguez, Mabel; de la Rosa, Ana Paulina Barba; Santos, Leticia

    2015-01-01

    Bifidobacteria are considered to be probiotics that exist in the large intestine and are helpful to maintain human health. Oral administration of bifidobacteria may be effective in improving the intestinal flora and environment, stimulating the immune response and possibly preventing cancer. However, for consistent and positive results, further well-controlled studies are urgently needed to describe the basic mechanisms of this microorganism. Analysis of the proteasome-lacking Bifidobacterium longum genome reveals that it possesses a gene, IPR003593 AAA ATPase core, which codes a 56 kDa protein containing one AAA ATPase domain. Phylogenetic classification made by CLANS, positioned this sequence into the ARC divergent branch of the AAA ATPase family of proteins. N-terminal analysis of the sequence indicates this protein is closely related to other ATPases such as the Rhodococcus erythropolis ARC, Archaeoglobus fulgidus PAN, Mycobacterium tuberculosis Mpa and the human proteasomal Rpt1 subunit. This gene was cloned, the full-length recombinant protein was overexpressed in Escherichia coli, purified as a high-molecular size complex and named Bl-ARC. Enzymatic characterization showed that Bl-ARC ATPase is active, Mg(+2)-dependent and sensitive to N-ethylmaleimide. Gene organization positions bl-arc in a region flanked by a cluster of genes that includes pup, dop and pafA genes. These findings point to a possible function as a chaperone in the degradation pathway via pupylation.

  6. Multiplicity of acquired cross-resistance in paclitaxel-resistant cancer cells is associated with feedback control of TUBB3 via FOXO3a-mediated ABCB1 regulation

    PubMed Central

    Aldonza, Mark Borris D.; Hong, Ji-Young; Alinsug, Malona V.; Song, Jayoung; Lee, Sang Kook

    2016-01-01

    Acquired drug resistance is a primary obstacle for effective cancer therapy. The correlation of point mutations in class III β-tubulin (TUBB3) and the prominent overexpression of ATP-binding cassette P-glycoprotein (ABCB1), a multidrug resistance gene, have been protruding mechanisms of resistance to microtubule disruptors such as paclitaxel (PTX) for many cancers. However, the precise underlying mechanism of the rapid onset of cross-resistance to an array of structurally and functionally unrelated drugs in PTX-resistant cancers has been poorly understood. We determined that our established PTX-resistant cancer cells display ABCB1/ABCC1-associated cross-resistance to chemically different drugs such as 5-fluorouracil, docetaxel, and cisplatin. We found that feedback activation of TUBB3 can be triggered through the FOXO3a-dependent regulation of ABCB1, which resulted in the accentuation of induced PTX resistance and encouraged multiplicity in acquired cross-resistance. FOXO3a-directed regulation of P-glycoprotein (P-gp) function suggests that control of ABCB1 involves methylation-dependent activation. Consistently, transcriptional overexpression or downregulation of FOXO3a directs inhibitor-controlled protease-degradation of TUBB3. The functional PI3K/Akt signaling is tightly responsive to FOXO3a activation alongside doxorubicin treatment, which directs FOXO3a arginine hypermethylation. In addition, we found that secretome factors from PTX-resistant cancer cells with acquired cross-resistance support a P-gp-dependent association in multidrug resistance (MDR) development, which assisted the FOXO3a-mediated control of TUBB3 feedback. The direct silencing of TUBB3 reverses induced multiple cross-resistance, reduces drug-resistant tumor mass, and suppresses the impaired microtubule stability status of PTX-resistant cells with transient cross-resistance. These findings highlight the control of the TUBB3 response to ABCB1 genetic suppressors as a mechanism to reverse the

  7. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

    SciTech Connect

    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina; Laufer, Stefan; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  8. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    PubMed

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-01

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators. PMID:27226582

  9. Changes in the level and activation state of the plasma membrane H(+)-ATPase during aging of red beet slices.

    PubMed Central

    Papini, R; De Michelis, M I

    1997-01-01

    The effect of aging on the plasma membrane (PM) H(+)-ATPase of red beet (Beta vulgaris L.) parenchyma discs was analyzed in PM purified by aqueous two-phase partitioning. Aging increased both the activity in the amount of immunodetectable H(+)-ATPase in the PM. The activity assayed at slightly alkaline pH values increased earlier and more strongly than that assayed at acidic pH values, so that the pH curve of the enzyme from aged beet discs was shifted toward more alkaline values. Aging decreased the stimulation of the PM H(+)-ATPase activity by controlled trypsin treatments or by lysophosphatidylcholine. After trypsin treatment the pH dependence of H(+)-ATPase from dormant or aged beet discs became equal. These results indicate that aging not only increases the level of H(+)-ATPase in the PM, but also determines its activation, most likely by modifying the interaction between the autoinhibitory carboxyl-terminal domain and the catalytic site. When the PM H(+)-ATPase activity was assayed at a slightly alkaline pH, the tyrosine modifier N-acetylimidazole inhibited the H(+)-ATPase in the PM from dormant beet discs much less than in the PM from aged discs, suggesting that modification of a tyrosine residue may be involved in the activation of the PM H(+)-ATPase induced by aging. The results are discussed with regard to aging-induced development of transmembrane transport activities. PMID:9232872

  10. [Effect of electromagnetic field of extremely low frequency on ATPase activity of actomyosin].

    PubMed

    Tseĭslier, Iu V; Sheliuk, O V; Martyniuk, V S; Nuryshchenko, N Ie

    2012-01-01

    The Mg2+/Ca2+ and K(+)-ATPase actomyosin activity of rabbit skeletal muscle was evaluated by the Fiske-Subbarow method during a five-hour exposition of protein solutions in electromagnetic field of extremely low frequency of 8 Hz and 25 microT induction. The results of the study of the ATPase activity of actomyosin upon electromagnetic exposure have shown statistically significant changes that are characterized by a rather complex time dynamics. After 1, 2 and 4 hours of exposure of protein solutions the effect of ELF EMF exposure inhibits the ATPase activity compared to control samples, which are not exposed to the magnetic field. By the third and fifth hours of exposure to the electromagnetic field, there is a significant increase in the ATPase activity of actomyosin. It should be noted that a similar pattern of change in enzyme activity was universal, both for the environment by Mg2+ and Ca2+, and in the absence of these ions in the buffer. This can evidence for Ca(2+)-independent ways of the infuence of electromagnetic field (EMP) on biologic objects. In our opinion, the above effects are explained by EMP influence on the dynamic properties of actomyosin solutions, which are based on the processes of spontaneous dynamic formation of structure.

  11. Acute delta 9-tetrahydrocannabinol exposure alters Ca2+ ATPase activity in neuroendocrine and gonadal tissues in mice.

    PubMed

    Dalterio, S L; Bernard, S A; Esquivel, C R

    1987-05-01

    Acute administration of delta 9-tetrahydrocannabinol (THC) (50 mg/kg) at puberty (35-40 days) significantly (P less than 0.05) reduced Ca2+ ATPase activity in hypothalamic plasma membranes but increased, although not significantly, enzyme activity in hypothalamic tissue obtained from adult mice. In contrast, testicular Ca2+ ATPase activity was increased in pubertal THC-treated males, and significantly reduced in adults. Pituitary Ca2+ ATPase activity exhibited a dose-related decrease after acute THC administration at 0.5, 5 or 50 mg/kg, but there were no differential effects of age. Pituitary plasma membranes obtained from THC-treated males did not respond to in vitro exposure to luteinizing hormone releasing hormone (LHRH, 10(-7) M) with the marked reduction (approximately 40%) in Ca2+ ATPase activity observed in pituitaries from oil-treated controls. In addition, effects of THC appear specific for Ca2+ ATPase activity, since Mg2+ ATPase and Na+/K+ ATPase activities were not affected. These findings indicate that acute in vivo administration of THC influences Ca2+ membrane transport, in particular Ca2+ ATPase activity. These effects occur at each level of the hypothalamic-pituitary-gonadal (HPG) axis, are related to dose and developmental age at exposure, and also appear specific for Ca2+-dependent ATPase activity. Furthermore, THC exposure modulates pituitary sensitivity to LHRH receptor-mediated effects on Ca2+ ATPase activity. Therefore, effects on Ca2+ membrane transport may be involved in acute THC actions on hormonal activity at these HPG sites.

  12. Increased oxidative stress and decreased activities of Ca2+/Mg2+-ATPase and Na+/K+-ATPase in the red blood cells of the hibernating black bear

    USGS Publications Warehouse

    Chauhan, V.P.S.; Tsiouris, J.A.; Chauhan, A.; Sheikh, A.M.; Brown, W. Ted; Vaughan, M.

    2002-01-01

    During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca2+/Mg2+-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na+/K+-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca2+/Mg2+-ATPase and Na+/K+-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression. ?? 2002 Elsevier Science Inc. All rights reserved.

  13. [Effect of glycine and strychnine on Cl(-)-activated Mg2+-ATPase from bream brain microsomes (Abamis brama L.)].

    PubMed

    Menzikov, S A; Menzikova, O V

    2001-01-01

    The effect of glycine and strychnine on Mg2+-ATPase from the microsomal fraction of the bream (Abramis brama L.) brain was studied. The glycine in the concentration range 10(-7)-10(-4) M activates the enzyme. The effect of glycine on Mg2+-ATPase is obviated by 100 microM strychnine. The strychnine in the concentration range 5-90 microM activates the basal Mg2+-ATPase but decreases the effect of the enzyme activation by 10(-4) M glycine. The effect of Cl- on Mg2+-ATPase depends on the substrate concentration (Mg2+-ATP) and is not observed in the presence of 100 microM strychnine. A receptor-dependent pathway of glycine and strychnine action on Cl(-)-activated Mg2+-ATPase from bream brain microsomes is proposed.

  14. The ABCB1, rs9282564, AG and TT Genotypes and the COMT, rs4680, AA Genotype are Less Frequent in Deceased Patients with Opioid Addiction than in Living Patients with Opioid Addiction.

    PubMed

    Christoffersen, Dorte J; Damkier, Per; Feddersen, Søren; Möller, Sören; Thomsen, Jørgen L; Brasch-Andersen, Charlotte; Brøsen, Kim

    2016-10-01

    Sudden death due to acute intoxication occurs frequently in patients with opioid addiction (OA). To examine whether certain genotypes were associated with this, we examined the frequencies of 29 SNPs located in candidate genes related to opioid pharmacology: ABCB1, OPRM1, UGT2B7, CYP3A5, CYP2B6, CYP2C19, CYP2D6, COMT, KCNJ6 and SCN9A in 274 deceased patients with OA (DOA), 309 living patients with OA (LOA) and in 394 healthy volunteers (HV). The main hypothesis of the study was that subjects homozygous for the variant 3435T in ABCB1 (rs1045642) occur more frequently in DOA than in LOA and HV because morphine and methadone more readily cross the blood barrier in these subjects due to a lower efflux transporter activity of the ABCB1 (p-glycoprotein) transporter. Our results did not support this hypothesis, because no statistically significant difference (p = 0.506) in the frequency of the TT genotype of rs1045642 was observed between the DOA, LOA and HV cohorts. However, for another ABCB1 variant, rs9282564, we found that the frequencies of the AG and TT genotypes were 13, 21 and 25% in DOA, LOA and HV, respectively, and after correcting for age, sex and multiple testing, the differences between DOA and LOA were statistically significantly different (p = 0.027). The COMT rs4680 AA genotype frequencies were 25%, 35% and 31% in DOA, LOA and HV, respectively, and the difference between DOA and LOA was also statistically significant (p = 0.0028). In conclusion, this study generated two hypotheses suggesting possible associations of a reduced risk of death and carrying, respectively, the ABCB1 rs9282564 AG and TT genotypes and the COMT rs4680 AA genotype among patients with OA. These findings should be confirmed in independent cohorts, and if a causal relationship between these variants and fatal poisoning in OA is confirmed, then it may be possible at least in theory to personalize prevention of sudden death in this patient group.

  15. Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

    DOE PAGES

    Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; Connolly, Lynn; Bendebury, Anastasia; Finer-Moore, Janet; Holton, James; Cheng, Yifan; Stroud, Robert M.; Cox, Jeffery S.

    2015-04-09

    We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less

  16. Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

    SciTech Connect

    Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; Connolly, Lynn; Bendebury, Anastasia; Finer-Moore, Janet; Holton, James; Cheng, Yifan; Stroud, Robert M.; Cox, Jeffery S.

    2015-04-09

    We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

  17. The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

    PubMed Central

    Austin, S; Dixon, R

    1992-01-01

    The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Images PMID:1534752

  18. Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

    PubMed Central

    Lu, Connie; Turley, Stewart; Marionni, Samuel T.; Park, Young-Jun; Lee, Kelly K.; Patrick, Marcella; Shah, Ripal; Sandkvist, Maria; Bush, Matthew F.; Hol, Wim G. J.

    2013-01-01

    SUMMARY The Type II Secretion System (T2SS), a multi-protein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multi-domain T2SS assembly ATPase GspEEpsE had so far not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspEEpsE are obtained when fused to Hcp1 as an assistant hexamer, as shown by native mass spectrometry. The enzymatic activity of the GspEEpsE-Hcp1 fusions is ~20 times higher than that of a GspEEpsE monomer indicating that increasing the local concentration of GspEEpsE by the fusion strategy was successful. Crystal structures of GspEEpsE-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspEEpsE with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspEEpsE-Hcp1 fusions suggest that even further variability in GspEEpsE hexamer architecture is likely. PMID:23954505

  19. Impact of membrane-associated hydrogenases on the F₀F₁-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N'-dicyclohexylcarbodiimide in the mutants lacking hydrogenases.

    PubMed

    Blbulyan, Syuzanna; Trchounian, Armen

    2015-08-01

    Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH.

  20. Nilotinib reverses ABCB1/P-glycoprotein-mediated multidrug resistance but increases cardiotoxicity of doxorubicin in a MDR xenograft model.

    PubMed

    Zhou, Zhi-Yong; Wan, Li-Li; Yang, Quan-Jun; Han, Yong-Long; Li, Dan; Lu, Jin; Guo, Cheng

    2016-09-30

    The BCR-Abl tyrosine kinase inhibitor (TKI), nilotinib, was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia-positive chronic myelogenous leukemia. Recent studies have shown that nilotinib induces potent sensitization to anticancer agents by blocking the functions of ABCB1/P-glycoprotein (P-gp) in multidrug resistance (MDR). However, changes in P-gp expression or function affect the cardiac disposition and prolong the presence of both doxorubicin (DOX) and doxorubicinol (DOXol) in cardiac tissue, thus, enhancing the risk of cardiotoxicity. In this study, we used a MDR xenograft model to evaluate the antitumor activity, tissue distribution and cardiotoxicity of DOX when co-administered with nilotinib. This information will provide more insight into the pharmacological role of nilotinib in MDR reversal and the risk of DOX cardiotoxicity. Our results showed that nilotinib significantly enhanced DOX cytotoxicity and increased intracellular rhodamine 123 accumulation in MG63/DOX cells in vitro and strongly enhanced DOX inhibition of growth of P-gp-overexpressing MG63/DOX cell xenografts in nude mice. Additionally, nilotinib significantly increased DOX and DOXol accumulation in serum, heart, liver and tumor tissues. Importantly, nilotinib induced a disproportionate increase in DOXol in cardiac tissue. In the co-administration group, CBR1 and AKR1A1 protein levels were significantly increased in cardiac tissue, with more severe necrosis and vacuole formation. These results indicate that nilotinib reverses P-gp- mediated MDR by blocking the efflux function and potentiates DOX-induced cardiotoxicity. These findings represent a guide for the design of future clinical trials and studies of pharmacokinetic interactions and may be useful in guiding the use of nilotinib in combination therapy of cancer in clinical practice.

  1. V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

    PubMed Central

    Monteiro, Joana; Aires, Rita; Becker, Jörg D.; Jacinto, António; Certal, Ana C.; Rodríguez-León, Joaquín

    2014-01-01

    The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

  2. V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

    PubMed

    Monteiro, Joana; Aires, Rita; Becker, Jörg D; Jacinto, António; Certal, Ana C; Rodríguez-León, Joaquín

    2014-01-01

    The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

  3. Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity.

    PubMed

    de Almeida-Amaral, Elmo Eduardo; Caruso-Neves, Celso; Pires, Vanessa Maria Pereira; Meyer-Fernandes, José Roberto

    2008-02-01

    We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite. PMID:17825292

  4. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    NASA Technical Reports Server (NTRS)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  5. Molecular model of the outward facing state of the human P-glycoprotein (ABCB1), and comparison to a model of the human MRP5 (ABCC5)

    PubMed Central

    Ravna, Aina W; Sylte, Ingebrigt; Sager, Georg

    2007-01-01

    Background Multidrug resistance is a particular limitation to cancer chemotherapy, antibiotic treatment and HIV medication. The ABC (ATP binding cassette) transporters human P-glycoprotein (ABCB1) and the human MRP5 (ABCC5) are involved in multidrug resistance. Results In order to elucidate structural and molecular concepts of multidrug resistance, we have constructed a molecular model of the ATP-bound outward facing conformation of the human multidrug resistance protein ABCB1 using the Sav1866 crystal structure as a template, and compared the ABCB1 model with a previous ABCC5 model. The electrostatic potential surface (EPS) of the ABCB1 substrate translocation chamber, which transports cationic amphiphilic and lipophilic substrates, was neutral with negative and weakly positive areas. In contrast, EPS of the ABCC5 substrate translocation chamber, which transports organic anions, was generally positive. Positive-negative ratios of amino acids in the TMDs of ABCB1 and ABCC5 were also analyzed, and the positive-negative ratio of charged amino acids was higher in the ABCC5 TMDs than in the ABCB1 TMDs. In the ABCB1 model residues Leu65 (transmembrane helix 1 (TMH1)), Ile306 (TMH5), Ile340 (TMH6) and Phe343 (TMH6) may form a binding site, and this is in accordance with previous site directed mutagenesis studies. Conclusion The Sav1866 X-ray structure may serve as a suitable template for the ABCB1 model, as it did with ABCC5. The EPS in the substrate translocation chambers and the positive-negative ratio of charged amino acids were in accordance with the transport of cationic amphiphilic and lipophilic substrates by ABCB1, and the transport of organic anions by ABCC5. PMID:17803828

  6. A unique DNase activity shares the active site with ATPase activity of the RecA/Rad51 homologue (Pk-REC) from a hyperthermophilic archaeon.

    PubMed

    Rashid, N; Morikawa, M; Kanaya, S; Atomi, H; Imanaka, T

    1999-02-19

    A RecA/Rad51 homologue from Pyrococcus kodakaraensis KOD1 (Pk-REC) is the smallest protein among various RecA/Rad51 homologues. Nevertheless, Pk-Rec is a super multifunctional protein and shows a deoxyribonuclease activity. This deoxyribonuclease activity was inhibited by 3 mM or more ATP, suggesting that the catalytic centers of the ATPase and deoxyribonuclease activities are overlapped. To examine whether these two enzymatic activities share the same active site, a number of site-directed mutations were introduced into Pk-REC and the ATPase and deoxyribonuclease activities of the mutant proteins were determined. The mutant enzyme in which double mutations Lys-33 to Ala and Thr-34 to Ala were introduced, fully lost both of these activities, indicating that Lys-33 and/or Thr-34 are important for both ATPase and deoxyribonuclease activities. The mutation of Asp-112 to Ala slightly and almost equally reduced both ATPase and deoxyribonuclease activities. In addition, the mutation of Glu-54 to Gln did not seriously affect the ATPase, deoxyribonuclease, and UV tolerant activities. These results strongly suggest that the active sites of the ATPase and deoxyribonuclease activities of Pk-REC are common. It is noted that unlike Glu-96 in Escherichia coli RecA, which has been proposed to be a catalytic residue for the ATPase activity, the corresponding residual Glu-54 in Pk-REC is not involved in the catalytic function of the protein.

  7. Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant

    PubMed Central

    Hawwa, Ahmed F; McKiernan, Patrick J; Shields, Michael; Millership, Jeff S; Collier, Paul S; McElnay, James C

    2009-01-01

    AIMS The aim of this study was to investigate the influence of genetic polymorphisms in ABCB1 on the incidence of nephrotoxicity and tacrolimus dosage-requirements in paediatric patients following liver transplantation. METHODS Fifty-one paediatric liver transplant recipients receiving tacrolimus were genotyped for ABCB1 C1236>T, G2677>T and C3435>T polymorphisms. Dose-adjusted tacrolimus trough concentrations and estimated glomerular filtration rates (EGFR) indicative of renal toxicity were determined and correlated with the corresponding genotypes. RESULTS The present study revealed a higher incidence of the ABCB1 variant-alleles examined among patients with renal dysfunction (≥30% reduction in EGFR) at 6 months post-transplantation (1236T allele: 63.3% vs 37.5% in controls, P= 0.019; 2677T allele: 63.3% vs. 35.9%, p = 0.012; 3435T allele: 60% vs. 39.1%, P= 0.057). Carriers of the G2677->T variant allele also had a significant reduction (%) in EGFR at 12 months post-transplant (mean difference = 22.6%; P= 0.031). Haplotype analysis showed a significant association between T-T-T haplotypes and an increased incidence of nephrotoxicity at 6 months post-transplantation (haplotype-frequency = 52.9% in nephrotoxic patients vs 29.4% in controls; P= 0.029). Furthermore, G2677->T and C3435->T polymorphisms and T-T-T haplotypes were significantly correlated with higher tacrolimus dose-adjusted pre-dose concentrations at various time points examined long after drug initiation. CONCLUSIONS These findings suggest that ABCB1 polymorphisms in the native intestine significantly influence tacrolimus dosage-requirement in the stable phase after transplantation. In addition, ABCB1 polymorphisms in paediatric liver transplant recipients may predispose them to nephrotoxicity over the first year post-transplantation. Genotyping future transplant recipients for ABCB1 polymorphisms, therefore, could have the potential to individualize better tacrolimus immunosuppressive therapy and

  8. Identification of a putatively multixenobiotic resistance related Abcb1 transporter in amphipod species endemic to the highly pristine Lake Baikal.

    PubMed

    Pavlichenko, Vasiliy V; Protopopova, Marina V; Timofeyev, Maxim; Luckenbach, Till

    2015-04-01

    The fauna of Lake Baikal in Eastern Siberia, the largest freshwater body on Earth, is characterized by high degrees of biodiversity and endemism. Amphipods, a prominent taxon within the indigenous fauna, occur in an exceptionally high number of endemic species. Considering the specific water chemistry of Lake Baikal with extremely low levels of potentially toxic natural organic compounds, it seems conceivable that certain adaptions to adverse environmental factors are missing in endemic species, such as cellular defense mechanisms mitigating toxic effects of chemicals. The degree to which the endemic fauna is affected by the recently occurring anthropogenic water pollution of Lake Baikal may depend on the existence of such cellular defense mechanisms in those species. We here show that endemic amphipods express transcripts for Abcb1, a major component of the cellular multixenobiotic resistance (MXR) defense against toxic chemicals. Based on a partial abcb1 cDNA sequence from Gammarus lacustris, an amphipod species common across Northern Eurasia but only rarely found in Lake Baikal, respective homologous sequences were cloned from five amphipods endemic to Lake Baikal, Eulimnogammarus verrucosus, E. vittatus, E. cyaneus, E. marituji, and Gmelinoides fasciatus, confirming that abcb1 is transcribed in those species. The effects of thermal (25 °C) and chemical stress (1-2 mg L(-1) phenanthrene) in short-term exposures (up to 24 h) on transcript levels of abcb1 and heat shock protein 70 (hsp70), used as a proxy for cellular stress in the experiments, were exemplarily examined in E. verrucosus, E. cyaneus, and Gammarus lacustris. Whereas increases of abcb1 transcripts upon treatments occurred only in the Baikalian species E. verrucosus and E. cyaneus but not in Gammarus lacustris, changes of hsp70 transcript levels were seen in all three species. At least for species endemic to Lake Baikal, the data thus indicate that regulation of the identified amphipod abcb1 is

  9. Modulation of Na+/K+ ATPase Activity by Hydrogen Peroxide Generated through Heme in L. amazonensis.

    PubMed

    Rocco-Machado, Nathália; Cosentino-Gomes, Daniela; Meyer-Fernandes, José Roberto

    2015-01-01

    Leishmania amazonensis is a protozoan parasite that occurs in many areas of Brazil and causes skin lesions. Using this parasite, our group showed the activation of Na+/K+ ATPase through a signaling cascade that involves the presence of heme and protein kinase C (PKC) activity. Heme is an important biomolecule that has pro-oxidant activity and signaling capacity. Reactive oxygen species (ROS) can act as second messengers, which are required in various signaling cascades. Our goal in this work is to investigate the role of hydrogen peroxide (H2O2) generated in the presence of heme in the Na+/K+ ATPase activity of L. amazonensis. Our results show that increasing concentrations of heme stimulates the production of H2O2 in a dose-dependent manner until a concentration of 2.5 μM heme. To confirm that the effect of heme on the Na+/K+ ATPase is through the generation of H2O2, we measured enzyme activity using increasing concentrations of H2O2 and, as expected, the activity increased in a dose-dependent manner until a concentration of 0.1 μM H2O2. To investigate the role of PKC in this signaling pathway, we observed the production of H2O2 in the presence of its activator phorbol 12-myristate 13-acetate (PMA) and its inhibitor calphostin C. Both showed no effect on the generation of H2O2. Furthermore, we found that PKC activity is increased in the presence of H2O2, and that in the presence of calphostin C, H2O2 is unable to activate the Na+/K+ ATPase. 100 μM of Mito-TEMPO was capable of abolishing the stimulatory effect of heme on Na+/K+ ATPase activity, indicating that mitochondria might be the source of the hydrogen peroxide production induced by heme. The modulation of L. amazonensis Na+/K+ ATPase by H2O2 opens new possibilities for understanding the signaling pathways of this parasite. PMID:26070143

  10. A comprehensive study of polymorphisms in the ABCB1, ABCC2, ABCG2, NR1I2 genes and lymphoma risk

    PubMed Central

    Campa, Daniele; Butterbach, Katja; Slager, Susan L; Skibola, Christine F; de Sanjosé, Silvia; Benavente, Yolanda; Becker, Nikolaus; Foretova, Lenka; Maynadie, Marc; Cocco, Pierluigi; Staines, Anthony; Kaaks, Rudolf; Boffetta, Paolo; Brennan, Paul; Conde, Lucia; Bracci, Paige M; Caporaso, Neil E; Strom, Sara S; Camp, Nicola J; Cerhan, James R; Canzian, Federico; Nieters, Alexandra

    2012-01-01

    Owing to their role in controlling the efflux of toxic compounds, transporters are central players in the process of detoxification and elimination of xenobiotics, which in turn is related to cancer risk. Among these transporters, ATP-binding cassette B1/multidrug resistance 1 (ABCB1/MDR1), ABCC2/multidrug resistance protein 2 (MRP2), and ABCG2/breast cancer resistance protein (BCRP) affect susceptibility to many hematopoietic malignancies. The maintenance of regulated expression of these transporters is governed through the activation of intracellular “xenosensors” like the nuclear receptor 1I2/pregnane X receptor (NR1I2/PXR). SNPs in genes encoding these regulators have also been implicated in the risk of several cancers. Using a tagging approach, we tested the hypothesis that common polymorphisms in the transporter genes ABCB1, ABCC2, ABCG2, and the regulator gene NR1I2 could be implicated in lymphoma risk. We selected 68 SNPs in the 4 genes, and we genotyped them in 1,481 lymphoma cases and 1,491 controls of the European cases-control study (EpiLymph) using the Illumina™ GoldenGate assay technology.Carriers of the SNP rs6857600 minor allele in ABCG2, was associated with a decrease in risk of B-cell lymphoma (B-NHL) overall (p<0.001). Furthermore, a decreased risk of chronic lymphocytic leukemia (CLL) was associated with the ABCG2 rs2231142 variant (p=0.0004), which could be replicated in an independent population. These results suggest a role for this gene in B-NHL susceptibility, especially for CLL. PMID:21918980

  11. Activation and Deactivation of H+-ATPase in Intact Chloroplasts 1

    PubMed Central

    Shahak, Yosepha

    1982-01-01

    The light activation mechanism of the latent H+-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H2O2 or ferricyanide) and slowed down by dithiothreitol. It is suggested that the physiological mechanism for regulation of the H+-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes. PMID:16662486

  12. Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.

    PubMed

    Giarola, Naira Lígia Lima; de Almeida-Amaral, Elmo Eduardo; Collopy-Júnior, Itallo; Fonseca-de-Souza, André Luiz; Majerowicz, David; Paes, Lisvane Silva; Gondim, Katia C; Meyer-Fernandes, José Roberto

    2013-04-01

    In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction

  13. Effect of Hindlimb Unweighting on Single Soleus Fiber Maximal Shortening Velocity and ATPase Activity

    NASA Technical Reports Server (NTRS)

    McDonald, K. S.; Fitts, R. H.

    1993-01-01

    This study characterizes the time course of change in single soleus muscle fiber size and function elicited by hindlimb un weighting (HU) and analyzes the extent to which varying durations of HU altered maximal velocity of shortening (V(sub o)), myofibrillar adenosinetriphosphatase (ATPase), and relative content of slow and fast myosin in individual soleus fibers. After 1, 2, or 3 weeks of HU, soleus muscle bundles were prepared and stored in skinning solution at -20 C. Single fibers were isolated and mounted between a motor arm and a transducer, and fiber force, V(sub o), and ATPase activity were measured. Fiber myosin content was determined by one-dimensional sodium dodecyl sulfate- (SDS) polyacrylamide gel electrophoresis. After 1, 2, and 3 weeks of HU, soleus fibers exhibited a progressive reduction in fiber diameter (16, 22, and 42%, respectively) and peak force (42, 48, and 7%, respectively). Peak specific tension was significantly reduced after 1 week of HU (18%) and showed no further change in 2-3 weeks of HU. During 1 and 3 wk of HU, fiber V(sub o) and ATPase showed a significant increase. By 3 week, V(sub o) had increased from 1.32 +/- 0.04 to 2.94 +/- 0.17 fiber lengths/s and fiber ATPase from 291 +/- 16 to 1064 +/- 128 micro-M min(sub -1) mm(sub -3). The percent fibers expressing fast myosin heavy chain increased from 4% to 29% by 3 week of HU, and V(sub o) and ATPase activity within a fiber were highly correlated. However, a large population of fibers after 1, 2, and 3 weeks of HU showed increases in V(sub o) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers. The mechanism eliciting increased fiber V(sub o) and ATPase activity was not obvious but may have been due to increases in fast myosin that went undetected on SDS gels and/or other factors unrelated to the myosin filament.

  14. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    SciTech Connect

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  15. In vitro antioxidant and H+, K+-ATPase inhibition activities of Acalypha wilkesiana foliage extract

    PubMed Central

    Prakash Gupta, Rajesh Kashi; Pradeepa; Hanumanthappa, Manjunatha

    2013-01-01

    Aims: The aim of this study was to evaluate the antioxidant activty and anti-acid property of Acalypha wilkesiana foliage extract. Materials and Methods: Hot and cold aqueous extracts were prepared from healthy leaves of A. wilkesiana. Free radical scavenging activity and H+, K+-ATPase inhibition activities of aqueous foliage extracts was screened by in vitro models. Statistical Analysis Used: All experiments were performed in triplicate and results are expressed as mean ± SEM. Results: A. wilkesiana hot aqueous extract (AWHE) showed significant antioxidants and free radical scavenging activity. Further, AWHE has shown a potent H+, K+-ATPase inhibitory activity (IC50: 51.5 ± 0.28 μg/ml) when compare to standard proton pump inhibitor omeprazole (56.2 ± 0.64 μg/ml); however, latter activity is equal to A. wilkesiana cold aqueous extract (AWCE). Quantitative analysis of AWHE has revealed more content of phenols and flavonoids; this is found to be the reason for good antioxidant activity over AWCE. Molecular docking was carried out against H+, K+-ATPase enzyme crystal structure to validate the anti-acid activity of A. wilkesiana major phytochemicals. Conclusions: The present study indicates that the constituents of AWHE and AWCE have good antacid and free radical scavenging activity. PMID:24082698

  16. Gill ATPase activity in Procambarus clarkii as an indicator of heavy metal pollution

    SciTech Connect

    Torreblanca, A.; Del Ramo, J.; Diaz-Mayans, J. )

    1989-06-01

    Lake Albufera and the surrounding rice field waters are subjected to very heavy loads of sewage and toxic industrial residues, including heavy metals, from the many urban and waste waters of this area. The American red crayfish, Procambarus clarkii have a high resistance to toxic effects of heavy metals. The sublethal effects of heavy metals on gills of fish and aquatic invertebrates have been extensively studied. Some metabolic disturbances and histologic damages have been reported, as well as osmoregulation alterations. However, little work has been done about the effect of heavy metals on Na,K and Mg-ATPases of freshwater invertebrate gills. Na,K-ATPase is the prime mediator of ion transport across cellular membranes and plays a central role in whole body ion regulation in marine and estuarine animals. Na,K-ATPase has been reviewed and assessed as a potentially useful indicator of pollution stress in aquatic animals. The purpose of this study is look for the relation, if any, between crayfish gill ATP-ase activity changes and metal exposure in laboratory. This find would allow the authors to assay this potential indicator in the field.

  17. Genetic association of NOS1 exon18, NOS1 exon29, ABCB1 1236C/T, and ABCB1 3435C/T polymorphisms with the risk of Parkinson's disease

    PubMed Central

    Huang, Hongbin; Peng, Cong; Liu, Yong; Liu, Xu; Chen, Qicong; Huang, Zunnan

    2016-01-01

    Abstract Background: Parkinson's disease (PD) is the second most frequent neurodegenerative disorder. Previous publications have investigated the association of NOS1 and ABCB1 polymorphisms with PD risk. However, those studies have provided some contradictory results. Methods: Literature searches were performed using PubMed, Embase, PDgene, China National Knowledge Infrastructure database, and Google Scholar. Odds ratios (ORs) with 95% confidence intervals (CIs) were applied to evaluate the strength of association. Results: The analysis results indicated that NOS1 exon18 polymorphism was associated with developing PD in 4 genetic models (allelic: OR = 1.25, 95%CI 1.09–1.44, P = 0.001; homozygous: OR = 1.79, 95%CI 1.32–2.45, P < 0.001; recessive: OR = 1.70, 95%CI 1.26–2.28, P < 0.001; dominant: OR = 1.22, 95%CI 1.02–1.46, P = 0.03), whereas exon29 polymorphism was not correlated to PD susceptibility. In addition, ABCB1 1236C/T polymorphism was related to PD in the recessive (OR = 0.80, 95%CI 0.66–0.97, P = 0.025) and overdominant (OR = 1.21, 95%CI 1.03–1.43, P = 0.02) models, which might indicate the opposite effects of 2 minor variants of this locus on Parkinson's disease. However, this associated result was not robust enough to withstand statistically significant correction. On the other hand, no association was found between ABCB1 3435C/T polymorphism and the predisposition to PD in 5 genetic models, and such an absence of relationship was further confirmed by subgroup analysis in Caucasians and Asians. Whether the polymorphisms of these 4 loci were linked to PD or not, our study provided some interesting findings that differ from the previous results with regard to their genetic susceptibility. Conclusion: The NOS1 exon18 and ABCB1 1236C/T variants might play a role in the risk of Parkinson's disease, whereas NOS1 exon29 and ABCB1 3435C/T polymorphisms might not contribute to PD susceptibility. PMID

  18. Influence of ABCB1 polymorphisms and docetaxel pharmacokinetics on pathological response to neoadjuvant chemotherapy in breast cancer patients.

    PubMed

    Lévy, Pierre; Gligorov, Joseph; Antoine, Martine; Rezai, Keyvan; Lévy, Eric; Selle, Frédéric; Saintigny, Pierre; Lokiec, François; Avenin, Danielle; Beerblock, Karine; Lotz, Jean-Pierre; Bernaudin, Jean-François; Fajac, Anne

    2013-06-01

    We have previously reported an association between ABCB1 C3435T polymorphism and docetaxel pharmacokinetics in breast cancer patients. We therefore investigated whether these parameters could account for variations in pathological response. Five ABCB1 polymorphisms including C3435T polymorphism were analyzed in breast cancer patients receiving neoadjuvant chemotherapy with doxorubicin and docetaxel (n = 101). Pathological response was assessed using the Sataloff classification. Pharmacokinetic analysis was performed for the first course of docetaxel (n = 84). No significant association was found between ABCB1 polymorphisms or docetaxel pharmacokinetics and pathological complete response. C3435T genotype was an independent predictive factor of good response in breast (response >50 %, i.e., Sataloff T-A and T-B): OR: 4.6 (95 % CI: 1.3-16.1), p = 0.015, for TT patients versus CT and CC patients. Area under the plasma concentration-time curve (AUC) of docetaxel was the only independent predictive factor of the total absence of response in breast (Sataloff T-D): OR: 14.3, (95 % CI: 1.7-118), p = 0.015, for AUC of docetaxel <3,500 μg h/L versus ≥3,500 μg h/L. These results suggest that C3435T polymorphism and docetaxel exposure are involved in the response to neoadjuvant chemotherapy in breast cancer patients and may be useful to optimize individualized therapy.

  19. Host genetic variants of ABCB1 and IL15 influence treatment outcome in paediatric acute lymphoblastic leukaemia

    PubMed Central

    Lu, Y; Kham, S K Y; Ariffin, H; Oei, A M I; Lin, H P; Tan, A M; Quah, T C; Yeoh, A E J

    2014-01-01

    Background: Host germline variations and their potential prognostic importance is an emerging area of interest in paediatric ALL. Methods: We investigated the associations between 20 germline variations and various clinical end points in 463 children with ALL. Results: After adjusting for known prognostic factors, variants in two genes were found to be independently associated with poorer EFS: ABCB1 T/T at either 2677 (rs2032582) or 3435 (rs1045642) position (P=0.003) and IL15 67276493G/G (rs17015014; P=0.022). These variants showed a strong additive effect affecting outcome (P<0.001), whereby patients with both risk genotypes had the worst EFS (P=0.001), even after adjusting for MRD levels at the end of remission induction. The adverse effect of ABCB1 T/T genotypes was most pronounced in patients with favourable cytogenetics (P=0.011) while the IL15 67276493G/G genotype mainly affected patients without common chromosomal abnormalities (P=0.022). In both cytogenetic subgroups, increasing number of such risk genotypes still predicted worsening outcome (P<0.001 and=0.009, respectively). Conclusion: These results point to the prognostic importance of host genetic variants, although the specific mechanisms remain unclarified. Inclusion of ABCB1 and IL15 variants may help improve risk assignment strategies in paediatric ALL. PMID:24434428

  20. P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

    PubMed

    Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

    2010-03-01

    We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. PMID:20004641

  1. Effect of the Electrochemical Proton Gradient and Anions on the ATPase Activity of Soybean Submitochondrial Particles 1

    PubMed Central

    Martins, Ione S.; Martins, Orlando B.; de Gómez-Puyou, Marietta Tuena; Gómez-Puyou, Armando

    1988-01-01

    Submitochondrial particles from soybean (Glycine max L. cv Jupiter) hypocotyls with an ATPase activity of 0.3 to 1.0 micromole per minute per milligram were prepared by sonication with Mg-ATP. The particles catalyzed ATP synthesis with NADH and succinate; the ratios of ATP/O with these substrates were 1.0 and 0.1, respectively. As monitored by oxonol-VI, the particles built up and maintained a membrane potential that was higher with NADH than with succinate or Mg-ATP. The ATPase activity of the particles increased two to threefold by preincubation with 50 millimolar phosphate at a temperature of 38°C. The increase in ATPase activity became higher (five to sixfold) when particles were preincubated with Mg-ATP plus phosphate. Under the latter conditions, collapse of Δ̄μH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone prevented the activation. An increase in ATPase activity of the particles was also observed with NADH and succinate, although activation was lower with succinate. With these substrates, phosphate did not increase ATPase activation. When particles were preincubated with Mg-ATP, anions that stimulate ATP hydrolysis (malate, malonate, and bicarbonate) had an activating effect similar to that of phosphate. The data suggest that the soybean mitochondrial ATPase can be activated by Δ̄μH but that this activation is increased by the binding of certain anions to a conformation of the enzyme that appears during hydrolytic cycles. PMID:16666151

  2. Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

    PubMed

    Ledoux, Sarah; Guthrie, Christine

    2016-06-01

    Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. PMID:27072132

  3. Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants.

    PubMed Central

    Schaller, A; Oecking, C

    1999-01-01

    Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed. PMID:9927643

  4. Trifluoperazine inhibits the MgATPase activity and in vitro motility of conventional and unconventional myosins.

    PubMed

    Sellers, James R; Wang, Fei; Chantler, Peter D

    2003-01-01

    Trifluoperazine, a calmodulin antagonist, has recently been shown to inhibit the MgATPase activity of scallop myosin in the absence of light chain dissociation (Patel et al. (2000) J Biol Chem 275: 4880-4888). To investigate the generality of this observation and the mechanism by which it occurs, we have examined the ability of trifluoperazine to inhibit the enzymatic properties of other conventional and unconventional myosins. We show that trifluoperazine can inhibit the actin-activated MgATPase activity of rabbit skeletal muscle myosin II heavy meromyosin (HMM), phosphorylated turkey gizzard smooth muscle myosin II HMM, phosphorylated human nonmuscle myosin IIA HMM and myosin V subfragment-1 (S1). In all cases half maximal inhibition occurred at 50-75 microM trifluoperazine while light chains (myosin II) or calmodulin (myosin V) remained associated with the heavy chains. In vitro motility of all myosins tested was completely inhibited by trifluoperazine. Chymotryptic digestion of baculovirus-expressed myosin V HMM possessing only two calmodulin binding sites yielded a minimal motor fragment with no bound calmodulin. The MgATPase of this fragment was inhibited by trifluoperazine over the same range of concentrations as the S1 fragment of myosin.

  5. Inhibitory Effect of Fluoride on Na+,K+ ATPase Activity in Human Erythrocyte Membrane.

    PubMed

    A, Shashi; G, Meenakshi

    2015-12-01

    The present study was performed to evaluate the role of long-term consumption of excessive fluoride on electrolyte homeostasis and their transporting mechanisms in erythrocytes of subjects afflicted with dental and skeletal fluorosis. A total of 620 adult (20-50 years) Indian residents participated in this study: 258 men and 242 women exposed to high concentrations of fluoride and 120 age and gender-matched control subjects. Erythrocytes were isolated from blood samples, washed, and used for the estimation of intraerythrocyte sodium and potassium concentrations. Na+,K+ ATPase activity was determined spectrophotometrically from a ghost erythrocyte membrane prepared by osmotic lysis. Erythrocyte analytes were correlated with the water and serum fluoride concentrations by Pearson's bivariate correlation and regression analysis. Results indicated a significant increase in intraerythrocyte sodium (F=14306.265, P<0.0001) in subjects from endemic fluorosis study groups as compared to controls. A significant (P<0.05) positive correlation of intracellular sodium was found with water and serum fluoride concentrations. Mean concentration of intraerythrocytic potassium ions showed significant reduction (F=9136.318, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) negative correlation of potassium ions was noted with water and serum fluoride concentrations. Na+,K+ ATPase activity was significantly declined (F=1572.763, P<0.0001) in subjects exposed to fluoride. A significant (P<0.05) inverse relationship of Na+,K+ ATPase activity was revealed with water and serum fluoride concentrations.

  6. Different effect of cadmium and copper on H+-ATPase activity in plasma membrane vesicles from Cucumis sativus roots.

    PubMed

    Janicka-Russak, Małgorzata; Kabała, Katarzyna; Burzynski, Marek

    2012-06-01

    The effect of heavy metals on plasma membrane (PM) H(+)-ATPase (EC 3.6.3.14) activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H(+)-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 μM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H(+)-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H(+)-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H(+)-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H(+)-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H(2)O(2) in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H(+)-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H(+)-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants.

  7. Different effect of cadmium and copper on H+-ATPase activity in plasma membrane vesicles from Cucumis sativus roots

    PubMed Central

    Janicka-Russak, Małgorzata; Kabała, Katarzyna; Burzyński, Marek

    2012-01-01

    The effect of heavy metals on plasma membrane (PM) H+-ATPase (EC 3.6.3.14) activity in cucumber (Cucumis sativus) roots was studied. The aim of this work was to explain the mechanism of modification of the PM H+-ATPase activity in plants subjected to heavy metals. Plants were treated with 10 μM Cd or Cu for 6 d. After 3 d exposure to the heavy metals, some of the plants were transferred to control conditions for a further 3 d (3/3 plants). The activity of PM H+-ATPase was found to be increased in plants treated with heavy metals. The highest activity measured as proton transport was observed in 3/3 plants. Estimation of transcript levels of C. sativus PM H+-ATPase in roots indicated that the action of Cd, but not Cu, affected the gene expression level. Transcript levels of C. sativus PM H+-ATPase (CsHA2, CsHA3, CsHA4, CsHA8, and CsHA9) genes increased in roots treated with Cd. Moreover, Western blot analysis with antibody against phosphothreonine and 14-3-3 protein indicated that increased activity of PM H+-ATPase under heavy-metal stress resulted from phosphorylation of the enzyme. It was found that Cu markedly increased the activity of catalase and ascorbate peroxidase and reduced the level of H2O2 in cucumber roots. In contrast, Cd did not affect these parameters. These results indicate that Cd and Cu can, in different ways, lead to modification of PM H+-ATPase activity. Additionally, it was observed that treatment of plants with heavy metals led to an increased level of heat-shock proteins in the tissues. This suggests that the plants had started adaptive processes to survive adverse conditions, and increased PM H+-ATPase activity could further enhance the repair processes in heavy-metal-stressed plants. PMID:22451724

  8. Oxysterols decrease apical-to-basolateral transport of Aß peptides via an ABCB1-mediated process in an in vitro Blood-brain barrier model constituted of bovine brain capillary endothelial cells.

    PubMed

    Saint-Pol, Julien; Candela, Pietra; Boucau, Marie-Christine; Fenart, Laurence; Gosselet, Fabien

    2013-06-23

    It is known that activation of the liver X receptors (LXRs) by natural or synthetic agonists decreases the amyloid burden and enhances cognitive function in transgenic murine models of Alzheimer's disease (AD). Recent evidence suggests that LXR activation may affect the transport of amyloid ß (Aß) peptides across the blood-brain barrier (the BBB, which isolates the brain from the peripheral circulation). By using a well-characterized in vitro BBB model, we demonstrated that LXR agonists (24S-hydroxycholesterol, 27-hydroxycholesterol and T0901317) modulated the expression of target genes involved in cholesterol homeostasis (such as ATP-binding cassette sub-family A member 1 (ABCA1)) and promoted cellular cholesterol efflux to apolipoprotein A-I and high density lipoproteins. Interestingly, we also observed a decrease in Aß peptide influx across brain capillary endothelial cells, although ABCA1 did not appear to be directly involved in this process. By focusing on others receptors and transporters that are thought to have major roles in Aß peptide entry into the brain, we then demonstrated that LXR stimulation provoked an increase in expression of the ABCB1 transporter (also named P-glycoprotein (P-gp)). Further investigations confirmed ABCB1's involvement in the restriction of Aß peptide influx. Taken as a whole, our results not only reinforce the BBB's key role in cerebral cholesterol homeostasis but also demonstrate the importance of the LXR/ABCB1 axis in Aß peptide influx-highlighting an attractive new therapeutic approach whereby the brain could be protected from peripheral Aß peptide entry.

  9. Loss of Vacuolar H+-ATPase (V-ATPase) Activity in Yeast Generates an Iron Deprivation Signal That Is Moderated by Induction of the Peroxiredoxin TSA2 *

    PubMed Central

    Diab, Heba I.; Kane, Patricia M.

    2013-01-01

    Vacuolar H+-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125–7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (PFIT2-GFP) or the TSA2 promoter (PTSA2-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated PFIT2-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased PFIT2-GFP expression and, surprisingly, restored PTSA2-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and PFIT2-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis. PMID:23457300

  10. Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis

    SciTech Connect

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy

    2013-12-10

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

  11. Cohesin’s ATPase Activity Couples Cohesin Loading onto DNA with Smc3 Acetylation

    PubMed Central

    Ladurner, Rene; Bhaskara, Venugopal; Huis in ’t Veld, Pim J.; Davidson, Iain F.; Kreidl, Emanuel; Petzold, Georg; Peters, Jan-Michael

    2014-01-01

    Summary Background Cohesin mediates sister chromatid cohesion by topologically entrapping sister DNA molecules inside its ring structure. Cohesin is loaded onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on the adenosine triphosphatase (ATPase) activity of cohesin’s Smc1 and Smc3 subunits. Subsequent cohesion establishment during DNA replication depends on Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which “locks” cohesin on DNA by inactivating the cohesin release factor Wapl. Results Human cohesin ATPase mutants associate transiently with DNA in a manner that depends on the loading complex but cannot be stabilized on chromatin by depletion of Wapl. These mutants cannot be acetylated, fail to interact with sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the ATPase mutants is not a consequence of their transient association with DNA but is directly caused by their inability to hydrolyze ATP because acetylation of wild-type cohesin also depends on ATP hydrolysis. Conclusions Our data indicate that cohesion establishment involves the following steps. First, cohesin transiently associates with DNA in a manner that depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to entrapment of DNA and converts Smc3 into a state that can be acetylated. Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl, and stabilization of cohesin on DNA. Our finding that cohesin’s ATPase activity is required for both cohesin loading and Smc3 acetylation raises the possibility that cohesion establishment is directly coupled to the reaction in which cohesin entraps DNA. PMID:25220052

  12. Redox Activation of the Universally Conserved ATPase YchF by Thioredoxin 1

    PubMed Central

    Hannemann, Liya; Suppanz, Ida; Ba, Qiaorui; MacInnes, Katherine; Drepper, Friedel; Warscheid, Bettina

    2016-01-01

    Abstract Aims: YchF/Ola1 are unconventional members of the universally conserved GTPase family because they preferentially hydrolyze ATP rather than GTP. These ATPases have been associated with various cellular processes and pathologies, including DNA repair, tumorigenesis, and apoptosis. In particular, a possible role in regulating the oxidative stress response has been suggested for both bacterial and human YchF/Ola1. In this study, we analyzed how YchF responds to oxidative stress and how it potentially regulates the antioxidant response. Results: Our data identify a redox-regulated monomer–dimer equilibrium of YchF as a key event in the functional cycle of YchF. Upon oxidative stress, the oxidation of a conserved and surface-exposed cysteine residue promotes YchF dimerization, which is accompanied by inhibition of the ATPase activity. No dimers were observed in a YchF mutant lacking this cysteine. In vitro, the YchF dimer is dissociated by thioredoxin 1 (TrxA) and this stimulates the ATPase activity. The physiological significance of the YchF-thioredoxin 1 interaction was demonstrated by in vivo cross-linking, which validated this interaction in living cells. This approach also revealed that both the ATPase domain and the helical domain of YchF are in contact with TrxA. Innovation: YchF/Ola1 are the first redox-regulated members of the universally conserved GTPase family and are inactivated by oxidation of a conserved cysteine residue within the nucleotide-binding motif. Conclusion: Our data provide novel insights into the regulation of the so far ill-defined YchF/Ola1 family of proteins and stipulate their role as negative regulators of the oxidative stress response. Antioxid. Redox Signal. 24, 141–156. PMID:26160547

  13. Leishmania amazonensis: Increase in ecto-ATPase activity and parasite burden of vinblastine-resistant protozoa.

    PubMed

    Giarola, Naira Lígia Lima; Silveira, Thaís Souza; Inacio, Job Domingos Filho; Vieira, Lisvane Paes; Almeida-Amaral, Elmo Eduardo; Meyer-Fernandes, José Roberto

    2014-11-01

    Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of L. amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and

  14. A new steroidal saponin from AGAVE brittoniana and its biphasic effect on the Na+-ATPase activity.

    PubMed

    Silva, Graziela M; De Souza, Aloa M; Lara, Luciene S; Mendes, Tatiana P; da Silva, Bernadete P; Lopes, Anibal G

    2005-01-01

    A new steroidal saponin, 3-{(O-6-deoxy-a-L-mannopyranosyl-(1 --> 4)-O-beta-D-glucopyranosyl-(1 --> 3)-O-[O-beta-D-glucopyranosyl-(1 --> 3)-beta-D-glucopyranosyl-(1 --> 2)]-O-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranosyl)oxy}-6-hydroxy-(3beta,5alpha,6alpha,25R)-spirostan-12-one, was isolated from Agave brittoniana Trel. The structure was determined by extensive NMR spectroscopy studies and chemical conversions. Its effects on the Na+-ATPase and (Na+ + K+)-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that this steroidal saponin exerts a biphasic effect on the Na+-ATPase activity. It is concluded that the effect of the aqueous extract as a diuretic is due, at least in part, to the action of saponin on the ouabain-insensitive Na+-ATPase.

  15. Genome-wide association data suggest ABCB1 and immune-related gene sets may be involved in adult antisocial behavior.

    PubMed

    Salvatore, J E; Edwards, A C; McClintick, J N; Bigdeli, T B; Adkins, A; Aliev, F; Edenberg, H J; Foroud, T; Hesselbrock, V; Kramer, J; Nurnberger, J I; Schuckit, M; Tischfield, J A; Xuei, X; Dick, D M

    2015-04-28

    Adult antisocial behavior (AAB) is moderately heritable, relatively common and has adverse consequences for individuals and society. We examined the molecular genetic basis of AAB in 1379 participants from a case-control study in which the cases met criteria for alcohol dependence. We also examined whether genes of interest were expressed in human brain. AAB was measured using a count of the number of Antisocial Personality Disorder criteria endorsed under criterion A from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Participants were genotyped on the Illumina Human 1M BeadChip. In total, all single-nucleotide polymorphisms (SNPs) accounted for 25% of the variance in AAB, although this estimate was not significant (P=0.09). Enrichment tests indicated that more significantly associated genes were over-represented in seven gene sets, and most were immune related. Our most highly associated SNP (rs4728702, P=5.77 × 10(-7)) was located in the protein-coding adenosine triphosphate-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1). In a gene-based test, ABCB1 was genome-wide significant (q=0.03). Expression analyses indicated that ABCB1 was robustly expressed in the brain. ABCB1 has been implicated in substance use, and in post hoc tests we found that variation in ABCB1 was associated with DSM-IV alcohol and cocaine dependence criterion counts. These results suggest that ABCB1 may confer risk across externalizing behaviors, and are consistent with previous suggestions that immune pathways are associated with externalizing behaviors. The results should be tempered by the fact that we did not replicate the associations for ABCB1 or the gene sets in a less-affected independent sample.

  16. Genome-wide association data suggest ABCB1 and immune-related gene sets may be involved in adult antisocial behavior

    PubMed Central

    Salvatore, J E; Edwards, A C; McClintick, J N; Bigdeli, T B; Adkins, A; Aliev, F; Edenberg, H J; Foroud, T; Hesselbrock, V; Kramer, J; Nurnberger, J I; Schuckit, M; Tischfield, J A; Xuei, X; Dick, D M

    2015-01-01

    Adult antisocial behavior (AAB) is moderately heritable, relatively common and has adverse consequences for individuals and society. We examined the molecular genetic basis of AAB in 1379 participants from a case–control study in which the cases met criteria for alcohol dependence. We also examined whether genes of interest were expressed in human brain. AAB was measured using a count of the number of Antisocial Personality Disorder criteria endorsed under criterion A from the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Participants were genotyped on the Illumina Human 1M BeadChip. In total, all single-nucleotide polymorphisms (SNPs) accounted for 25% of the variance in AAB, although this estimate was not significant (P=0.09). Enrichment tests indicated that more significantly associated genes were over-represented in seven gene sets, and most were immune related. Our most highly associated SNP (rs4728702, P=5.77 × 10−7) was located in the protein-coding adenosine triphosphate-binding cassette, sub-family B (MDR/TAP), member 1 (ABCB1). In a gene-based test, ABCB1 was genome-wide significant (q=0.03). Expression analyses indicated that ABCB1 was robustly expressed in the brain. ABCB1 has been implicated in substance use, and in post hoc tests we found that variation in ABCB1 was associated with DSM-IV alcohol and cocaine dependence criterion counts. These results suggest that ABCB1 may confer risk across externalizing behaviors, and are consistent with previous suggestions that immune pathways are associated with externalizing behaviors. The results should be tempered by the fact that we did not replicate the associations for ABCB1 or the gene sets in a less-affected independent sample. PMID:25918995

  17. γ-Benzylidene digoxin derivatives synthesis and molecular modeling: Evaluation of anticancer and the Na,K-ATPase activity effect.

    PubMed

    Alves, Silmara L G; Paixão, Natasha; Ferreira, Letícia G R; Santos, Felipe R S; Neves, Luiza D R; Oliveira, Gisele C; Cortes, Vanessa F; Salomé, Kahlil S; Barison, Andersson; Santos, Fabio V; Cenzi, Gisele; Varotti, Fernando P; Oliveira, Soraya M F; Taranto, Alex G; Comar, Moacyr; Silva, Luciana M; Noël, François; Quintas, Luis Eduardo M; Barbosa, Leandro A; Villar, José A F P

    2015-08-01

    Cardiotonic steroids (CS), natural compounds with traditional use in cardiology, have been recently suggested to exert potent anticancer effects. However, the repertoire of molecules with Na,K-ATPase activity and anticancer properties is limited. This paper describes the synthesis of 6 new digoxin derivatives substituted (on the C17-butenolide) with γ-benzylidene group and their cytotoxic effect on human fibroblast (WI-26 VA4) and cancer (HeLa and RKO) cell lines as well as their effect on Na,K-ATPase activity and expression. As digoxin, compound BD-4 was almost 100-fold more potent than the other derivatives for cytotoxicity with the three types of cells used and was also the only one able to fully inhibit the Na,K-ATPase of HeLa cells after 24h treatment. No change in the Na,K-ATPase α1 isoform protein expression was detected. On the other hand it was 30-40 fold less potent for direct Na,K-ATPase inhibition, when compared to the most potent derivatives, BD-1 and BD-3, and digoxin. The data presented here demonstrated that the anticancer effect of digoxin derivatives substituted with γ-benzylidene were not related with their inhibition of Na,K-ATPase activity or alteration of its expression, suggesting that this classical molecular mechanism of CS is not involved in the cytotoxic effect of our derivatives.

  18. Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains

    SciTech Connect

    Chen, Baowei; Squier, Thomas C.; Bigelow, Diana J.

    2004-04-13

    High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 involving the nucleotide-binding (N) domain is required to permit the transfer of the -phosphoryl group of ATP to Asp351 in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientation of the N-domain relative to the P-domain in both different crystal structures of the Ca-ATPase following calcium activation, and structures of other P-type ATPases, suggests the presence of conformational heterogeneity in solution which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer (FRET) to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino] naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P- and N-domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains is unaffected by enzyme phosphorylation. However, calcium-activation results in a 3.4 increase in the average spatial separation, which increases from 29.4 to 32.8 , in good agreement with the high-resolution structures where these sites are respectively separated by 31.6 (1 IWO.pdb) and 35.9 (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. However, there is substantial conformational heterogeneity for all enzyme states measured, indicating that formation of catalytically important transition states involves a subpopulation of enzyme intermediates. These results suggest that the

  19. Polymorphisms in the BRCA1 and ABCB1 genes modulate menopausal hormone therapy associated breast cancer risk in postmenopausal women.

    PubMed

    2010-04-01

    Menopausal hormone therapy (HT) is associated with an increased breast cancer risk among postmenopausal women. In this study, we investigated genetic effect modification of HT associated breast cancer risk in 3,149 postmenopausal breast cancer patients and 5,489 controls from the two German population-based case-control studies MARIE and GENICA. Twenty-eight polymorphisms of 14 candidate genes including two drug and hormone transporter genes (ABCB1/MDR1 and SHBG), four genes involved in cell cycle regulation (BRCA1, P21/CDKN1A, STK15/AURKA and TP53), six cytokine genes (IGFBP3, IL6, TGFB1, TNF, LTA and IGF1), and two cytokine receptor genes (EGFR and ERBB2) were genotyped using validated methods. Conditional logistic regression was used to assess multiplicative statistical interaction between polymorphisms and duration of estrogen-progestagen therapy and estrogen monotherapy use with regard to breast cancer risk assuming log-additive and co-dominant modes of inheritance. Women homozygous for the major ABCB1_rs2214102_G allele were found to be at a significantly increased breast cancer risk associated with combined estrogen-progestagen therapy [odds ratio (OR) = 1.17, 95% confidence interval (CI) = 1.12-1.23, P (interaction) = 0.022]. Additionally, risk associated with estrogen monotherapy was modified by BRCA1_rs799917. We observed a trend with increasing minor T alleles leading to the highest risk in homozygous carriers of the minor allele [OR (95% CI) = 1.17 (0.98-1.39), 1.06 (0.98-1.14), and 1.02 (0.94-1.11) for homozygous minor, heterozygous, and homozygous major allele carriers, respectively; P (interaction) = 0.032]. Our results suggest that genetic variants in ABCB1 and BRCA1 may modify the effect of HT on postmenopausal breast cancer risk.

  20. Calcium Release-Activated Calcium (CRAC) Channels Mediate the β2-Adrenergic Regulation of Na,K-ATPase

    PubMed Central

    Keller, Michael J.; Lecuona, Emilia; Prakriya, Murali; Cheng, Yuan; Soberanes, Saul; Scott Budinger, G.R.; Sznajder, Jacob I.

    2014-01-01

    β2-adrenergic agonists have been shown to regulate Na,K-ATPase in the alveolar epithelium by recruiting Na,K-ATPase-containing vesicles to the plasma membrane of alveolar epithelial cells (AEC). Here, we provide evidence that β2-agonists induce store-operated calcium entry (SOCE) in AECs. This calcium entry is necessary for β2-agonist-induced recruitment of Na,K-ATPase to the plasma membrane of AECs. Specifically, we show that β2-agonists induce SOCE via stromal interaction molecule 1 (STIM1)-associated calcium release-activated calcium (CRAC) channels. We also demonstrate that the magnitude of SOCE affects the abundance of Na,K-ATPase at the plasma membrane of AECs. PMID:25447523

  1. ABCB1 and ABCC2 and the risk of distant metastasis in Thai breast cancer patients treated with tamoxifen

    PubMed Central

    Sensorn, Insee; Sukasem, Chonlaphat; Sirachainan, Ekaphop; Chamnanphon, Montri; Pasomsub, Ekawat; Trachu, Narumol; Supavilai, Porntip; Pinthong, Darawan; Wongwaisayawan, Sansanee

    2016-01-01

    Background Genetic polymorphisms of drug-metabolizing enzymes and transporters have been extensively studied with regard to tamoxifen treatment outcomes. However, the results are inconclusive. Analysis of organ-specific metastasis may reveal the association of these pharmacogenetic factors. The aim of this study is to investigate the impact of CYP3A5, CYP2D6, ABCB1, and ABCC2 polymorphisms on the risk of all distant and organ-specific metastases in Thai patients who received tamoxifen adjuvant therapy. Methods Genomic DNA was extracted from blood samples of 73 patients with breast cancer who received tamoxifen adjuvant therapy. CYP3A5 (6986A>G), CYP2D6 (100C>T), ABCB1 (3435C>T), and ABCC2 (−24C>T) were genotyped using allelic discrimination real-time polymerase chain reaction assays. The impacts of prognostic clinical factors and genetic variants on disease-free survival were analyzed using the Kaplan–Meier method and Cox regression analysis. Results In the univariate analysis, primary tumor size >5 cm was significantly associated with increased risk of distant metastasis (P=0.004; hazard ratio [HR] =3.05; 95% confidence interval [CI], 1.44–6.47). In the multivariate analysis, tumor size >5 cm remained predictive of distant metastasis (P<0.001; HR=5.49; 95% CI, 2.30–13.10). ABCC2 −24CC were shown to be associated with increased risk of distant metastasis (P=0.040; adjusted HR=2.34; 95% CI, 1.04–5.27). The combined genotype of ABCC2 −24CC − ABCB1 3435 CT+TT was associated with increased risk of distant and bone metastasis (P=0.020; adjusted HR=2.46; 95% CI, 1.15–5.26 and P=0.040; adjusted HR=3.70; 95% CI, 1.06–12.89, respectively). Conclusion This study indicates that polymorphisms of ABCC2 and ABCB1 are independently associated with bone metastasis. Further prospective studies with larger sample sizes are needed to verify this finding. PMID:27110128

  2. Effects of diene valepotriates from Valeriana glechomifolia on Na+/K+-ATPase activity in the cortex and hippocampus of mice.

    PubMed

    Müller, Liz G; Salles, Luisa; Lins, Helena A; Feijó, Priscilla R O; Cassel, Eduardo; Vargas, Rubem; von Poser, Gilsane L; Noël, François; Quintas, Luis E M; Rates, Stela M K

    2015-02-01

    Diene valepotriates obtained from Valeriana glechomifolia present antidepressant-like activity, mediated by dopaminergic and noradrenergic neurotransmissions. Also, previous studies have shown inhibitory activity of diene valepotriates towards Na(+)/K(+)-ATPase from the rat brain in vitro. Nevertheless, in vivo studies regarding the action of diene valepotriates on this enzyme are still lacking. Considering that Na(+)/K(+)-ATPase cerebral activity is involved in depressive disorders, the aim of this study was to investigate the effects of acute (5 mg/kg, p. o.) and repeated (5 mg/kg, p. o., once a day for three days) diene valepotriate administration on Na(+)/K(+)-ATPase activity in the cortex and hippocampus of mice submitted or not submitted to the forced swimming test. In addition, the protein expression of Na(+)/K(+)-ATPase α1, α2, and α3 isoforms in the cortex of mice repeatedly treated with diene valepotriates (and submitted or not submitted to the forced swimming test) was investigated. Diene valepotriates significantly decreased mice immobility time in the forced swimming test when compared to the control group. Only the animals repeatedly treated with diene valepotriates presented increased Na(+)/K(+)-ATPase activity in the cerebral cortex, and the exposure to the forced swimming test counteracted the effects of the diene valepotriates. No alterations in the hippocampal Na(+)/K(+)-ATPase activity were observed. Repeated diene valepotriate administration increased the cortical content of the α2 isoform, but the α3 isoform protein expression was augmented only in mice repeatedly treated with diene valepotriates and forced to swim. Mice treated with the vehicle and submitted to the forced swimming test also presented an increase in the content of the α2 isoform, but no alterations in Na(+)/K(+)-ATPase activity. These results suggest that cortical Na(+)/K(+)-ATPase may represent a molecular target of the diene valepotriates in vivo and long

  3. Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.

    PubMed Central

    Ishii, T; Lemas, M V; Takeyasu, K

    1994-01-01

    Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase

  4. Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

    PubMed

    Tang, Seng Chuan; Nguyen, Luan N; Sparidans, Rolf W; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

    2014-03-15

    Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (~40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ∼25-fold, and brain-to-plasma ratios ~14-fold higher in Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b(-/-) mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ~70-fold higher in Abcb1a/1b(-/-) than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-to-plasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2(-/-) mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.

  5. Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development.

    PubMed

    Zhou, Yuchan; Pan, Xiaoping; Qu, Hongxia; Underhill, Steven J R

    2014-02-01

    Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.

  6. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that

  7. Relationship between body size, Na+-K+-ATPase activity, and membrane lipid composition in mammal and bird kidney.

    PubMed

    Turner, Nigel; Haga, Kurt L; Hulbert, A J; Else, Paul L

    2005-01-01

    We investigated the relationship between body size, Na(+)-K(+)-ATPase molecular activity, and membrane lipid composition in the kidney of five mammalian and eight avian species ranging from 30-g mice to 280-kg cattle and 13-g zebra finches to 35-kg emus, respectively. Na(+)-K(+)-ATPase activity was found to be higher in the smaller species of both groups. In small mammals, the higher Na(+)-K(+)-ATPase activity was primarily the result of an increase in the molecular activity (turnover rate) of individual enzymes, whereas in small birds the higher Na(+)-K(+)-ATPase activity was the result of an increased enzyme concentration. Phospholipids from both mammals and birds contained a relatively constant percentage of unsaturated fatty acids; however, phospholipids from the smaller species were generally more polyunsaturated, and a complementary significant allometric increase in monounsaturate content was observed in the larger species. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid [22:6(n-3)] displayed the greatest variation with body mass, scaling with allometric exponents of -0.21 and -0.26 in the mammals and birds, respectively. This allometric variation in fatty acid composition was correlated with Na(+)-K(+)-ATPase molecular activity in mammals, whereas in birds molecular activity only correlated with membrane cholesterol content. These relationships are discussed with respect to the metabolic intensity of different-sized animals.

  8. Mutant LV(476-7)AA of A-subunit of Enterococcus hirae V1-ATPase: High affinity of A3B3 complex to DF axis and low ATPase activity.

    PubMed

    Alam, Jahangir; Yamato, Ichiro; Arai, Satoshi; Saijo, Shinya; Mizutani, Kenji; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki; Iwata, So; Kakinuma, Yoshimi; Murata, Takeshi

    2013-01-01

    Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble functional domain V1 (A3B3DF) and an integral membrane domain Vo (ac), where V1 and Vo domains are connected by a central stalk, composed of D-, F-, and d-subunits; and two peripheral stalks (E- and G-subunits). We identified 120 interacting residues of A3B3 heterohexamer with D-subunit in DF heterodimer in the crystal structures of A3B3 and A3B3DF. In our previous study, we reported 10 mutants of E. hirae V1-ATPase, which showed lower binding affinities of DF with A3B3 complex leading to higher initial specific ATPase activities compared to the wild-type. In this study, we identified a mutation of A-subunit (LV(476-7)AA) at its C-terminal domain resulting in the A3B3 complex with higher binding affinities for wild-type or mutant DF heterodimers and lower initial ATPase activities compared to the wild-type A3B3 complex, consistent with our previous proposal of reciprocal relationship between the ATPase activity and the protein-protein binding affinity of DF axis to the A3B3 catalytic domain of E. hirae V-ATPase. These observations suggest that the binding of DF axis at the contact region of A3B3 rotary ring is relevant to its rotation activity. PMID:24404436

  9. Effect of ceritinib (LDK378) on enhancement of chemotherapeutic agents in ABCB1 and ABCG2 overexpressing cells in vitro and in vivo

    PubMed Central

    Hu, Jing; Zhang, Xu; Wang, Fang; Wang, Xiaokun; Yang, Ke; Xu, Meng; To, Kenneth K.W.

    2015-01-01

    Multidrug resistance (MDR) is the leading cause of treatment failure in cancer chemotherapy. The overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1, ABCC1 and ABCG2, play a key role in mediating MDR by pumping anticancer drugs out from cancer cells. Ceritinib (LDK378) is a second-generation tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) currently in phase III clinical trial for the treatment of non-small cell lung cancer. Here, we found that ceritinib remarkably enhanced the efficacy of chemotherapeutic drugs in ABCB1 or ABCG2 over-expressing cancer cells in vitro and in vivo. Ceritinib significantly increased the intracellular accumulation of chemotherapeutic agents such as doxorubicin (DOX) by inhibiting ABCB1 or ABCG2-mediated drug efflux in the transporters-overexpressing cells. Mechanistically, ceritinib is likely a competitive inhibitor of ABCB1 and ABCG2 because it competed with [125I]-iodoarylazidoprazosin for photo affinity labeling of the transporters. On the other hand, at the transporters-inhibiting concentrations, ceritinib did not alter the expression level of ABCB1 and ABCG2, and phosphorylation status of AKT and ERK1/2. Thus the findings advocate further clinical investigation of combination chemotherapy of ceritinib and other conventional chemotherapeutic drugs in chemo-refractory cancer patients. PMID:26556876

  10. [Effect of picrotoxin on the Cl-activated ATPase microsomal fraction of bream brain (Abramis brama L.)].

    PubMed

    Menzikov, S A; Menzikova, O V

    2001-01-01

    It is found that picrotoxine in range concentrations 0.1-10 microM stimulates the basal Mg(2+)-ATPase from microsomal fraction of fish bream (Abramis brama L.), however decreases activating effect of 10(-5) M GABA on the enzyme. The stimulative effect of picrotoxine dependants on duration of preincubation with microsomes. It was established that basal Mg(2+)-ATP-ase activity was activated by anions (Cl- > Br- > I-). The activated effect of anions on the Mg(2+)-ATP-ase is decreased in the presence 1 microM picrotoxine. It was shown that in the dependence on concentration of the Mg(2+)-ATP (0.2 or 1 mM) in the incubation medium the picrotoxine serves as on activator or inhibitor of the enzyme activity. It is supposed that picrotoxine allosterially influences on the enzyme by the receptor-dependent way.

  11. Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts

    PubMed Central

    Pierre, Sandrine V.; Yang, Changjun; Yuan, Zhaokan; Seminerio, Jennifer; Mouas, Christian; Garlid, Keith D.; Dos-Santos, Pierre; Xie, Zijian

    2007-01-01

    Objective Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. Methods and Results In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 μM followed by an 8-minute washout before 30 minutes of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cγ1/protein kinase Cε (PLC-γ1/PKCε) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-γ1/PKCε pathway, but also cardiac protection. This protection was also blocked by a PKCε translocation inhibitor peptide (PKCε TIP). Conclusion Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-γ1 and PKCε. PMID:17157283

  12. Alterations in erythrocyte membrane fluidity and Na+/K+ -ATPase activity in chronic alcoholics.

    PubMed

    Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

    2010-06-01

    Ethanol disorders biological membranes causing perturbations in the bilayer and also by altering the physicochemical properties of membrane lipids. But, chronic alcohol consumption also increases nitric oxide (NO) production. There was no systemic study was done related to alcohol-induced production of NO and consequent formation of peroxynitrite mediated changes in biophysical and biochemical properties, structure, composition, integrity and function of erythrocyte membranes in chronic alcoholics. Hence, keeping all these conditions in mind the present study was undertaken to investigate the role of over produced nitric oxide on red cell membrane physicochemical properties in chronic alcoholics. Human male volunteers aged 44 +/- 6 years with similar dietary habits were divided into two groups, namely nonalcoholic controls and chronic alcoholics (~125 g of alcohol at least five times per week for the past 10-12 years). Elevated nitrite and nitrate levels in plasma and lysate, changes in erythrocyte membrane individual phospholipid composition, increased lipid peroxidation, protein carbonyls, cholesterol and phospholipids ratio (C/P ratio) and anisotropic value (gamma) with decreased sulfhydryl groups and Na(+)/K(+)-ATPase activity in alcoholics was evident from this study. RBC lysate NO was positively correlated with C/P ratio (r = 0.547) and anisotropic (gamma) value (r = 0.428), Na(+)/K(+)-ATPase activity was negatively correlated with RBC lysate NO (r = -0.372) and anisotropic (gamma) value (r = -0.624) in alcoholics. Alcohol-induced overproduction of nitric oxide reacts with superoxide radicals to produce peroxynitrite, which appears to be responsible for changes in erythrocyte membrane lipids and the activity of Na(+)/K(+)-ATPase.

  13. D-Methionine attenuated cisplatin-induced vestibulotoxicity through altering ATPase activities and oxidative stress in guinea pigs

    SciTech Connect

    Cheng, P.-W.; Liu, S.-H.; Young, Y.-H.; Lin-Shiau, Shoei-Yn . E-mail: syl@ha.mc.ntu.edu.tw

    2006-09-01

    Cisplatin has been used as a chemotherapeutic agent to treat many kinds of malignancies. Its damage to the vestibulo-ocular reflex (VOR) system has been reported. However, the underlying biochemical change in the inner ear or central vestibular nervous system is not fully understood. In this study, we attempted to examine whether cisplatin-induced vestibulotoxicity and D-methionine protection were correlated with the changes of ATPase activities and oxidative stress of ampullary tissue of vestibules as well as cerebellar cortex (the inhibitory center of VOR system) of guinea pigs. By means of a caloric test coupled with electronystagmographic recordings, we found that cisplatin exposure caused a dose-dependent (1, 3, or 5 mg/kg) vestibular dysfunction as revealed by a decrease of slow phase velocity (SPV). In addition, cisplatin significantly inhibited the Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities in the ampullary tissue with a good dose-response relationship but not those of cerebellar cortex. Regression analysis indicated that a decrease of SPV was well correlated with the reduction of Na{sup +}, K{sup +}-ATPase and Ca{sup 2+}-ATPase activities of the ampullary tissue. D-Methionine (300 mg/kg) reduced both abnormalities of SPV and ATPase activities in a correlated manner. Moreover, cisplatin exposure led to a significant dose-dependent increase of lipid peroxidation and nitric oxide concentrations of the vestibules, which could be significantly suppressed by D-methionine. However, cisplatin did not alter the levels of lipid peroxidation and nitric oxide of the cerebellum. In conclusion, cisplatin inhibited ATPase activities and increased oxidative stress in guinea pig vestibular labyrinths. D-Methionine attenuated cisplatin-induced vestibulotoxicity associated with ionic disturbance through its antioxidative property.

  14. A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast.

    PubMed

    Pérez-Castiñeira, José R; Hernández, Agustín; Drake, Rocío; Serrano, Aurelio

    2011-07-15

    V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

  15. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line

    SciTech Connect

    Zaja, Roko; Caminada, Daniel; Loncar, Jovica; Fent, Karl; Smital, Tvrtko

    2008-03-01

    The development of the multidrug resistance (MDR) phenotype in mammals is often mediated by the overexpression of the P-glycoprotein1 (Pgp, ABCB1) or multidrug resistance-associated protein (MRP)-like ABC transport proteins. A similar phenomenon has also been observed and considered as an important part of the multixenobiotic resistance (MXR) defence system in aquatic organisms. We have recently demonstrated the presence of ABC transporters in the widely used in vitro fish model, the PLHC-1 hepatoma cell line. In the present study we were able to select a highly resistant PLHC-1 sub-clone (PLHC-1/dox) by culturing the wild-type cells in the presence of 1 {mu}M doxorubicin. Using quantitative PCR a 42-fold higher expression of ABCB1 gene was determined in the PLHC-1/dox cells compared to non-selected wild-type cells (PLHC-1/wt). The efflux rates of model fluorescent Pgp1 substrates rhodamine 123 and calcein-AM were 3- to 4-fold higher in the PLHC-1/dox in comparison to the PLHC-1/wt cells. PLHC-1/dox were 45-fold more resistant to doxorubicin cytotoxicity than PLHC-1/wt. Similarly to mammalian cell lines, typical cross-resistance to cytotoxicity of other chemotherapeutics such as daunorubicin, vincristine, vinblastine, etoposide and colchicine, occurred. Furthermore, cyclosporine A, verapamil and PSC833, specific inhibitors of Pgp1 transport activity, completely reversed resistance of PLHC-1/dox cells to all tested drugs, resulting in EC50 values similar to the EC50 values found for PLHC-1/wt. In contrast, MK571, a specific inhibitor of MRP type of efflux transporters, sensitized PLHC-1/dox cells, neither to doxorubicin, nor to any other of the chemotherapeutics used in the study. These data demonstrate for the first time that a specific Pgp1-mediated doxorubicin resistance mechanism is present in the PLHC-1 fish hepatoma cell line. In addition, the fact that low micromolar concentrations of specific inhibitors may completely reverse a highly expressed doxorubicin

  16. Fluidity-dependent Mg2(+)-ATPase activity in membranes from Leishmania donovani promastigotes.

    PubMed Central

    Dutta, M; Bandyopadhyay, R; Ghosh, C; Basu, M K

    1990-01-01

    The state of the lipid phase of the membrane plays a key role in the exposure of various receptors, antigens and enzymes on the membrane surface. The fluidity of membranes of Leishmania donovani promastigotes was monitored by two independent methods, i.e. influx of sterol from liposomes and removal of phospholipids by treatment with phospholipase C. The altered sterol/phospholipid ratio, in both cases, provided evidence that the activity of the functionally important membrane-bound enzyme Mg2(+)-ATPase is modulated by the state of the lipid phase of the membrane. PMID:2137691

  17. Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Croset, M; Crawford, N

    1986-02-01

    Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar

  18. Small terminase couples viral DNA-binding to genome-packaging ATPase activity

    PubMed Central

    Roy, Ankoor; Bhardwaj, Anshul; Datta, Pinaki; Lander, Gabriel C.; Cingolani, Gino

    2012-01-01

    SUMMARY Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here, we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology. PMID:22771211

  19. Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

    PubMed Central

    Daryadel, Arezoo; Bourgeois, Soline; Figueiredo, Marta F. L.; Gomes Moreira, Ana; Kampik, Nicole B.; Oberli, Lisa; Mohebbi, Nilufar; Lu, Xifeng; Meima, Marcel E.; Danser, A. H. Jan; Wagner, Carsten A.

    2016-01-01

    The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H+-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H+-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H+-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H+-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H+-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H+-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H+-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex

  20. Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

    PubMed

    Kriegel, Anne; Andrés, Zaida; Medzihradszky, Anna; Krüger, Falco; Scholl, Stefan; Delang, Simon; Patir-Nebioglu, M Görkem; Gute, Gezahegn; Yang, Haibing; Murphy, Angus S; Peer, Wendy Ann; Pfeiffer, Anne; Krebs, Melanie; Lohmann, Jan U; Schumacher, Karin

    2015-12-01

    The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH. PMID:26589552

  1. V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

    PubMed

    Florey, Oliver; Gammoh, Noor; Kim, Sung Eun; Jiang, Xuejun; Overholtzer, Michael

    2015-01-01

    Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation. PMID:25484071

  2. Characterization of ATPase Activity of P2RX2 Cation Channel.

    PubMed

    Mittal, Rahul; Grati, M'hamed; Sedlacek, Miloslav; Yuan, Fenghua; Chang, Qing; Yan, Denise; Lin, Xi; Kachar, Bechara; Farooq, Amjad; Chapagain, Prem; Zhang, Yanbin; Liu, Xue Z

    2016-01-01

    P2X purinergic receptors are plasma membrane ATP-dependent cation channels that are broadly distributed in the mammalian tissues. P2RX2 is a modulator of auditory sensory hair cell mechanotransduction and plays an important role in hair cell tolerance to noise. In this study, we demonstrate for the first time in vitro and in cochlear neuroepithelium, that P2RX2 possesses the ATPase activity. We observed that the P2RX2 V60L human deafness mutation alters its ability to bind ATP, while the G353R has no effect on ATP binding or hydrolysis. A non-hydrolysable ATP assay using HEK293 cells suggests that ATP hydrolysis plays a significant role in the opening and gating of the P2RX2 ion channel. Moreover, the results of structural modeling of the molecule was in agreement with our experimental observations. These novel findings suggest the intrinsic ATPase activity of P2RX2 and provide molecular insights into the channel opening. PMID:27252659

  3. Characterization of ATPase Activity of P2RX2 Cation Channel

    PubMed Central

    Mittal, Rahul; Grati, M'hamed; Sedlacek, Miloslav; Yuan, Fenghua; Chang, Qing; Yan, Denise; Lin, Xi; Kachar, Bechara; Farooq, Amjad; Chapagain, Prem; Zhang, Yanbin; Liu, Xue Z.

    2016-01-01

    P2X purinergic receptors are plasma membrane ATP-dependent cation channels that are broadly distributed in the mammalian tissues. P2RX2 is a modulator of auditory sensory hair cell mechanotransduction and plays an important role in hair cell tolerance to noise. In this study, we demonstrate for the first time in vitro and in cochlear neuroepithelium, that P2RX2 possesses the ATPase activity. We observed that the P2RX2 V60L human deafness mutation alters its ability to bind ATP, while the G353R has no effect on ATP binding or hydrolysis. A non-hydrolysable ATP assay using HEK293 cells suggests that ATP hydrolysis plays a significant role in the opening and gating of the P2RX2 ion channel. Moreover, the results of structural modeling of the molecule was in agreement with our experimental observations. These novel findings suggest the intrinsic ATPase activity of P2RX2 and provide molecular insights into the channel opening. PMID:27252659

  4. Population pharmacokinetic approach to evaluate the effect of CYP2D6, CYP3A, ABCB1, POR and NR1I2 genotypes on donepezil clearance

    PubMed Central

    Noetzli, Muriel; Guidi, Monia; Ebbing, Karsten; Eyer, Stephan; Wilhelm, Laurence; Michon, Agnès; Thomazic, Valérie; Stancu, Ioana; Alnawaqil, Abdel-Messieh; Bula, Christophe; Zumbach, Serge; Gaillard, Michel; Giannakopoulos, Panteleimon; von Gunten, Armin; Csajka, Chantal; Eap, Chin B

    2014-01-01

    Aims A large interindividual variability in plasma concentrations has been reported in patients treated with donepezil, the most frequently prescribed antidementia drug. We aimed to evaluate clinical and genetic factors influencing donepezil disposition in a patient population recruited from a naturalistic setting. Methods A population pharmacokinetic study was performed including data from 129 older patients treated with donepezil. The patients were genotyped for common polymorphisms in the metabolic enzymes CYP2D6 and CYP3A, in the electron transferring protein POR and the nuclear factor NR1I2 involved in CYP activity and expression, and in the drug transporter ABCB1. Results The average donepezil clearance was 7.3 l h−1 with a 30% interindividual variability. Gender markedly influenced donepezil clearance (P < 0.01). Functional alleles of CYP2D6 were identified as unique significant genetic covariate for donepezil clearance (P < 0.01), with poor metabolizers and ultrarapid metabolizers demonstrating, respectively, a 32% slower and a 67% faster donepezil elimination compared with extensive metabolizers. Conclusion The pharmacokinetic parameters of donepezil were well described by the developed population model. Functional alleles of CYP2D6 significantly contributed to the variability in donepezil disposition in the patient population and should be further investigated in the context of individual dose optimization to improve clinical outcome and tolerability of the treatment. PMID:24433464

  5. [Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae].

    PubMed

    Dias, Decivaldo S; Coelho, Milton V

    2007-01-01

    ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90% of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity. PMID:17420863

  6. Down-regulated Na+/K+-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats

    PubMed Central

    Huang, Hao; Chen, Yang-Mei; Zhu, Fei; Tang, Shi-Ting; Xiao, Ji-Dong; Li, Lv-Li; Lin, Xin-Jing

    2015-01-01

    This study was aimed to examine whether the Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na+/K+-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na+/K+-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na+/K+-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na+/K+-ATPase activity. Our results suggest that down-regulated Na+/K+-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease. PMID:26722460

  7. Effect of Rejection on Electrophysiologic Function of Canine Intestinal Grafts: Correlation with Histopathology and Na–K-ATPase Activity

    PubMed Central

    TAKEYOSHI, IZUMI; ZHANG, SHIMIN; KOKUDO, YASUTAKA; NAKAMURA, KENJIRO; IKOMA, AKIRA; ZHU, YUE; STARZL, THOMAS E.; TODO, SATORU

    2010-01-01

    To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na–K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18–24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were switched between a pair of dogs (allograft). Animals receiving intestinal autotransplantation were used as controls. Allograft recipients were sacrificed 3, 4, 5, 7, or 9 days after transplantation, and autograft recipients were sacrificed 3, 7, or 14 days after transplantation. Immunosuppression was not used. Electrophysiologic measurements were done with an Ussing chamber. Histological analysis was performed blindly using whole thickness sections. Na–K–ATPase activity in the mucosal tissue, which is said to regulate the potential difference, was also measured. Potential difference, resistance, and Na–K-ATPase activity of the allograft intestine decreased with time and were significantly lower 7 and 9 days after transplantation compared to host intestine, normal intestine, and graft intestine of controls (autograft). Potential difference, resistance, and Na–K-ATPase activity of the native intestinal tissue and the autografts did not decrease with time. Detection of histologically mild rejection of the intestine, which is important for appropriate immunosuppressive treatment in clinical cases, could not be achieved based on electrophysiology or Na–K-ATPase activity. Deterioration of electrophysiologic function during rejection correlated with the histological rejection process and Na–K-ATPase activity; however, electrophysiology may not be a reliable tool for monitoring grafts, since it cannot detect early intestinal rejection. PMID:8519738

  8. V-ATPase-activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis

    PubMed Central

    Zhang, Yi; Irani, Niloufer G.; Rubbo, Simone Di; Neumetzler, Lutz; Krishnamoorthy, Praveen; Van Houtte, Isabelle; Mylle, Evelien; Bischoff, Volker; Vernhettes, Samantha; Winne, Johan; Friml, Jiří; Stierhof, York-Dieter

    2016-01-01

    In plants, vacuolar H+-ATPase (V-ATPase) activity acidifies both the trans-Golgi network/early endosome (TGN/EE) and the vacuole. This dual V-ATPase function has impeded our understanding in how the pH homeostasis within the plant TGN/EE controls exo- and endocytosis. Here, we show that the weak V-ATPase mutant deetiolated3 (det3) displayed a pH increase in the TGN/EE, but not in the vacuole, strongly impairing secretion and recycling of the brassinosteroid receptor and the cellulose synthase complexes to the plasma membrane, in contrast to mutants lacking tonoplast-localized V-ATPase activity only. The brassinosteroid insensitivity and the cellulose deficiency defects in det3 were tightly correlated with reduced Golgi and TGN/EE motility. Thus, our results provide strong evidence that acidification of the TGN/EE, but not of the vacuole, is indispensable for functional secretion and recycling in plants. PMID:27250258

  9. Alterations in erythrocyte plasma membrane ATPase activity and adenine nucleotide content in a spontaneously diabetic subline of the Chinese hamster.

    PubMed

    Bettin, D; Klöting, I; Kohnert, K D

    1996-01-01

    The CHIG/Han subline of the Chinese hamster develops noninsulin-dependent diabetes mellitus characterized by hyperinsulinemia and different degrees of glucose intolerance. To study whether these abnormalities could affect transmembrane cation transport activity, we determined membrane ATPase activity and ATP concentrations in red blood cells of diabetes-resistant CHIA and diabetes-susceptible CHIG sublines of the Chinese hamster. Mg(2+)-ATPase activity was increased in red blood cell membranes of diabetic hamsters compared with that of nondiabetic CHIG and the diabetes-resistant CHIA animals and correlated with plasma triglyceride and cholesterol levels. Ca(2+)-ATPase and Na+/K+ATPase activity were not significantly different between diabetic and nondiabetic hamsters, but for the Na+/K(+)-ATPase, Km was decreased and the Vmax value increased in membrane preparations from severely diabetic hamsters. Both ATP and ADP content were lower in erythrocytes from diabetic than nondiabetic hamsters. Independently of the levels of glycemia, AMP concentrations were higher in CHIG than in CHIA hamsters. While ATP/AMP ratios were found to be decreased in erythrocytes from diabetes-susceptible CHIG hamsters compared to the diabetes-resistant CHIA animals, they were significantly correlated with the levels of glycemia. Furthermore, the relationship between blood glucose levels and kidney weight in hamsters of the diabetes-susceptible CHIG subline was such, that severely hyperglycemic animals displayed the greatest increase in kidney wet weight. These results indicate that the progressive metabolic deterioration in the development of noninsulin-dependent diabetes is associated with significant changes in the activity and kinetic parameters of cellular ATPases which could probably indicate early membrane alterations which may eventually result in the late microangiopathic complications of diabetes. PMID:8820985

  10. [GABA(A)-Coupled Cl-/HCO3(-)-ATPase: Candidate for an Novel Primary Active Transporter in Neuronal Membranes].

    PubMed

    Menzikov, S A

    2015-01-01

    Cl(-)-transport systems in cell membranes from various origins (including neurons) play an important role in different processes of their vital functions. Various transport mechanisms involved in the maintenance of intracellular concentration of Cl- that differs from concentration equilibrium have been considered. This review provides the biochemical properties of the GABA(A)-coupled Cl-/HCO3(-)-ATPase which is a candidate for an novel primary active system in neuronal membranes. Special emphasis has been placed on a review of the prerequisites for the existence of the GABA(A)-coupled ATPase. This work provides data for the benefit not only functional but also the alleged structural coupling of the enzyme with GABA(A)-receptors. It is concluded on the importance of the found ATPase in primary active transport processes across the plasma membrane of neuronal cells with different level of the organization.

  11. Structure of a topoisomerase II-DNA-nucleotide complex reveals a new control mechanism for ATPase activity

    PubMed Central

    Schmidt, Bryan H.; Osheroff, Neil; Berger, James M.

    2012-01-01

    Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes by a complex, ATP-dependent strand passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the first structure of a fully-catalytic Saccharomyces cerevisiae topoisomerase II homodimer, complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between the bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. PMID:23022727

  12. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    PubMed

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp.

  13. Design and synthesis of human ABCB1 (P-glycoprotein) inhibitors by peptide coupling of diverse chemical scaffolds on carboxyl and amino termini of (S)-valine-derived thiazole amino acid.

    PubMed

    Singh, Satyakam; Prasad, Nagarajan Rajendra; Chufan, Eduardo E; Patel, Bhargav A; Wang, Yi-Jun; Chen, Zhe-Sheng; Ambudkar, Suresh V; Talele, Tanaji T

    2014-05-22

    P-glycoprotein (P-gp) serves as a therapeutic target for the development of multidrug resistance reversal agents. In this study, we synthesized 21 novel compounds by peptide coupling at corresponding carboxyl and amino termini of (S)-valine-based bis-thiazole and monothiazole derivatives with diverse chemical scaffolds. Using calcein-AM efflux assay, we identified compound 28 (IC50 = 1.0 μM) carrying 3,4,5-trimethoxybenzoyl and 2-aminobenzophenone groups, respectively, at the amino and carboxyl termini of the monothiazole zwitter-ion. Compound 28 inhibited the photolabeling of P-gp with [(125)I]-iodoarylazidoprazosin with IC50 = 0.75 μM and stimulated the basal ATP hydrolysis of P-gp in a concentration-dependent manner (EC50 ATPase = 0.027 μM). Compound 28 at 3 μM reduced resistance in cytotoxicity assay to paclitaxel in P-gp-expressing SW620/Ad300 and HEK/ABCB1 cell lines. Biochemical and docking studies showed site-1 to be the preferable binding site for 28 within the drug-binding pocket of human P-gp. PMID:24773054

  14. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

    PubMed

    Teng, Yu-Ning; Hsieh, Yow-Wen; Hung, Chin-Chuan; Lin, Hui-Yi

    2015-01-28

    Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 μM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of <1 μM and efficiently inhibited 200 μM verapamil-stimulated ATPase activity, indicating a high affinity of demethoxycurcumin for P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

  15. Sphingosine inhibits the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity.

    PubMed

    Benaim, Gustavo; Pimentel, Adriana A; Felibertt, Pimali; Mayora, Adriana; Colman, Laura; Sojo, Felipe; Rojas, Héctor; De Sanctis, Juan B

    2016-04-29

    The increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) is the key variable for many different processes, ranging from regulation of cell proliferation to apoptosis. In this work we demonstrated that the sphingolipid sphingosine (Sph) increases the [Ca(2+)]i by inhibiting the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), in a similar manner to thapsigargin (Tg), a specific inhibitor of this Ca(2+) pump. The results showed that addition of sphingosine produced a release of Ca(2+) from the endoplasmic reticulum followed by a Ca(2+) entrance from the outside mileu. The results presented in this work support that this sphingolipid could control the activity of the SERCA, and hence sphingosine may participate in the regulation of [Ca(2+)]I in mammalian cells.

  16. Influence of phosphate and pH on myofibrillar ATPase activity and force in skinned cardiac trabeculae from rat.

    PubMed Central

    Ebus, J P; Stienen, G J; Elzinga, G

    1994-01-01

    1. The effects of inorganic phosphate (Pi) and pH on maximal calcium-activated isometric force and MgATPase activity were studied in chemically skinned cardiac trabeculae from rat. ATP hydrolysis was coupled enzymatically to the breakdown of NADH, and its concentration was determined photometrically. Measurements were performed at 2.1 microns sarcomere length and 20 degrees C. ATPase activity and force were also determined when square-wave-shaped length changes were applied, with a frequency of 23 Hz and an amplitude of 2.5%. 2. At pH 7.0 without added Pi, the average isometric force (+/- S.E.M.) was 51 +/- 3 kN m-2 (n = 23). The average isometric ATPase activity was 0.43 +/- 0.02 mM s-1 (n = 23). During the changes in length ATPase activity increased to 152 +/- 3% of the isometric value, while the average force level decreased to 48 +/- 2%. 3. Isometric force gradually decreased to 31 +/- 2% of the control value when the Pi concentration was increased to 30 mM. Isometric ATPase activity, however, remained constant for Pi concentrations up to 5 mM and decreased to 87 +/- 3% at 30 mM Pi. When Pi accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [Pi]. The decrease in relative force was found to be 44 +/- 4% per decade. 4. During the length changes, ATPase activity and average force showed, apart from the increase in ATPase activity and decrease in average force, the same dependence on Pi as the isometric values. Stiffness, estimated from the amplitude of the force responses during the length changes, decreased in proportion to isometric force when the Pi concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the Pi concentration were relatively small. These results suggest that the effect of Pi on the transitions which influence ATP turnover is rather insensitive to changes in cross

  17. A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.

    PubMed

    Uchimura, Seiichi; Fujii, Takashi; Takazaki, Hiroko; Ayukawa, Rie; Nishikawa, Yosuke; Minoura, Itsushi; Hachikubo, You; Kurisu, Genji; Sutoh, Kazuo; Kon, Takahide; Namba, Keiichi; Muto, Etsuko

    2015-01-19

    Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein-MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.

  18. Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane

    PubMed Central

    Sousa, Leilismara; Garcia, Israel J. P.; Costa, Tamara G. F.; Silva, Lilian N. D.; Renó, Cristiane O.; Oliveira, Eneida S.; Tilelli, Cristiane Q.; Santos, Luciana L.; Cortes, Vanessa F.; Santos, Herica L.; Barbosa, Leandro A.

    2015-01-01

    Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels. PMID:26197432

  19. Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane.

    PubMed

    Sousa, Leilismara; Garcia, Israel J P; Costa, Tamara G F; Silva, Lilian N D; Renó, Cristiane O; Oliveira, Eneida S; Tilelli, Cristiane Q; Santos, Luciana L; Cortes, Vanessa F; Santos, Herica L; Barbosa, Leandro A

    2015-01-01

    Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels.

  20. Seasonal variation in nitrogen net uptake and root plasma membrane H+-ATPase activity of Scots pine seedlings as affected by nutrient availability.

    PubMed

    Iivonen, Sari; Vapaavuori, Elina

    2002-01-01

    We examined changes in nitrogen (N) net uptake and activity and amount of plasma membrane H+-ATPase (PM-ATPase) in roots of hydroponically cultured Scots pine (Pinus sylvestris L.) seedlings throughout a simulated second growing season. Seedlings were grown with low (0.25 mM N) or high (2.5 mM N) nutrient availability to determine whether root PM-ATPase is dependent on an external nutrient supply. Climatic conditions in the growth chamber simulated the mean growing season from May to mid-October in southern Finland. Root PM-ATPase activity varied considerably during the growing season and was higher in current-year roots than in previous-year roots. Total PM-ATPase activity of current-year roots was highest at the end of the growing season, whereas PM-ATPase activity per unit fresh mass of current-year roots and specific absorption rate of N were highest in mid-July and decreased at the end of the growing season. This indicates that the decrease in PM-ATPase activity per unit fresh mass of the roots at the end of the growing season was compensated by the increased size of the root system. Seasonal variation in PM-ATPase activity had no clear dependence on root zone temperature. The response of PM-ATPase to root zone temperature was dependent on the developmental stage of the seedling. High nutrient availability resulted in increased root PM-ATPase activity and an extended period of root growth in autumn. PMID:11772550

  1. Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

    PubMed Central

    Krebs, Melanie; Beyhl, Diana; Görlich, Esther; Al-Rasheid, Khaled A. S.; Marten, Irene; Stierhof, York-Dieter; Hedrich, Rainer; Schumacher, Karin

    2010-01-01

    The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H+-ATPase (V-ATPase) and the vacuolar H+-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn2+/H+-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na+/H+-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth. PMID:20133698

  2. Effects of sex hormones on genioglossal muscle contractility and SR Ca2+-ATPase activity in aged rat.

    PubMed

    Liu, Yue-Hua; Qi, Juan; Hou, Yu-Xia; Wang, Fei

    2008-04-01

    This investigation was designed to examine whether short-term administration of sex hormones could produce changes in contractile properties and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function in genioglossal muscle (GG) of aged male rats. Twenty-four aged male rats were randomly divided into three groups to receive an intramuscular injection of either 0.1mg/kg oestrogen (group A), 2.5 mg/kg testosterone (group B), or 0.2 ml sterile peanut oil (group C, control), twice a week, during 4 weeks. After hormone treatment, in vivo isometric contractile properties were determined using surgically prepared GG muscles with platinum electrodes for stimulation of the medial branch of the hypoglossus nerve. Sarcoplasmic reticulum Ca(2+)-ATPase activity was measured in muscle homogenates by detecting the amount of inorganic phosphorus ion released in a standard coupled enzyme assay. SERCA1 mRNA level was observed using a real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). It was found that, compared with group C, testosterone treatment decreased the fatigue resistance in GG muscles (p<.05), whereas no change was observed in the isometric twitch (P(t)) and tetanic tension (P(0)) (p>.05). By contrast, in oestrogen treated GG muscles, no significant modification was found either in the contractile or in endurance properties (p>.05). The change in GG fatigue resistance of group B was associated with a marked decrease in SR Ca(2+)-ATPase activity when compared with that of the control group (p<.01). Furthermore, SERCA1 mRNA level was also down regulated in group B (p<.05). No prominent differences in SR Ca(2+)-ATPase activity and SERCA1 mRNA expression existed between group A and group C (p>.05). The present results show that exogenous testosterone produces more pronounced changes in GG muscle fatigue resistance than oestrogen does by acting at SR Ca(2+)-ATPase activity and SERCA gene expression.

  3. Managing Brain Extracellular K+ during Neuronal Activity: The Physiological Role of the Na+/K+-ATPase Subunit Isoforms

    PubMed Central

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    During neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease. PMID:27148079

  4. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    PubMed

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  5. The LEF-4 subunit of baculovirus RNA polymerase has RNA 5'-triphosphatase and ATPase activities.

    PubMed

    Jin, J; Dong, W; Guarino, L A

    1998-12-01

    The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that is required for transcription of viral late genes. This polymerase is composed of four equimolar subunits, LEF-8, LEF-4, LEF-9, and p47. The LEF-4 subunit has guanylyltransferase activity, suggesting that baculoviruses may encode a full complement of capping enzymes. Here we show that LEF-4 is a bifunctional enzyme that hydrolyzes the gamma phosphates of triphosphate-terminated RNA and also hydrolyzes ATP and GTP to the respective diphosphate forms. Alanine substitution of five residues previously shown to be essential for vaccinia virus RNA triphosphatase activity inactivated the triphosphatase component of LEF-4 but not the guanylyltransferase domain. Conversely, mutation of the invariant lysine in the guanylyltransferase domain abolished the guanylyltransferase activity without affecting triphosphatase function. We also investigated the effects of substituting phenylalanine for leucine at position 105, a mutation that results in a virus that is temperature sensitive for late gene expression. We found that this mutation had no significant effect on the ATPase or guanylyltransferase activity of LEF-4 but resulted in a modest decrease in RNA triphosphatase activity. PMID:9811739

  6. A novel inhibitor of vacuolar ATPase, FR167356, which can discriminate between osteoclast vacuolar ATPase and lysosomal vacuolar ATPase

    PubMed Central

    Niikura, Kazuaki; Takano, Mikiko; Sawada, Masae

    2004-01-01

    Vacuolar ATPase (V-ATPase) has been proposed as a drug target in lytic bone diseases. Studies of bafilomycin derivatives suggest that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is selective inhibition of osteoclast V-ATPase. Previous efforts to develop therapeutic inhibitors of osteoclast V-ATPase have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, we tried to find novel potent and specific V-ATPase inhibitors, which have new structural features and inhibition selectivity, from random screening using osteoclast microsomes. Finally, a novel V-ATPase inhibitor, FR167356, was obtained through chemical modification of a parental hit compound. FR167356 inhibited not only H+ transport activity of osteoclast V-ATPase but also H+ extrusion from cytoplasm of osteoclasts, which depends on the V-ATPase activity. As expected, FR167356 remarkably inhibited bone resorption in vitro. FR167356 also showed inhibitory effects on other V-ATPases, renal brush border V-ATPase, macrophage microsome V-ATPase and lysosomal V-ATPase. However, FR167356 was approximately seven-fold less potent in inhibiting lysosomal V-ATPase compared to osteoclast V-ATPase. Moreover, LDL metabolism in cells, which depends on acidification of lysosome, was blocked merely at higher concentration than bone resorption, suggesting that FR167356 inhibits V-ATPase of osteoclast ruffled border membrane still more selectively than lysosome at the cellular level. These results from the experiments seem to indicate that osteoclast V-ATPase may be different from lysosomal V-ATPase in respect of their structure. FR167356 had a novel chemical structural feature as well as inhibitory characteristics distinctly different from any previously known V-ATPase inhibitor family. Therefore, FR167356 is thought to be a useful tool for estimating the essential characteristics of V-ATPase inhibitors for drug development. PMID:15148249

  7. Gill Na{sup +}, K{sup +}-ATPase activity in largemouth bass (Micropterus salmoides) inhabiting reservoirs contaminated with mercury

    SciTech Connect

    Brundage, S.; Jagoe, C.H.; Shaw-Allen, P.

    1995-12-31

    Active transport of Na{sup +} and K{sup +} for osmoregulation in fish involves gill Na{sup +}, K{sup +}-ATPase, a membrane-bound enzyme powered by hydrolysis of ATP. Na{sup +}, K{sup +}-ATPase is inhibited by many dissolved metals including Al, Cd, Cu and Hg, resulting in ionoregulatory dysfunction. However, dissolved Hg concentrations are quite low in most aquatic systems, and dietary sources are the most important contributors to Hg burdens in fish. One recent study demonstrated relationships between muscle Hg concentration and gill Na{sup +}, K{sup +}-ATPase in a marine fish, suggesting that Hg accumulated via diet can affect osmoregulation. The authors tested for such a relationship in several age-classes of a freshwater fish (Micropterus salmoides) collected from three reservoirs. Fish from Par Pond and L Lake, on the USDOE Savannah River Site in South Carolina had relatively high Hg content: for Par Pond, muscle and liver ranged from 1.58--12.01 and 1.46--23.22 {micro}g Hg/g dry mass, respectively, and for L Lake muscle and liver ranged from 3.11--5.16 and 1.28--12.59 {micro}g Hg/g dry mass, respectively. Bass from an offsite location, Thurmond Lake, had significantly (P <0.05 by Kruskal-Wallis test) less Hg (muscle and liver range 0.61--2.39 and 0.28--2.32 {micro}g Hg/g dry mass, respectively). In all reservoirs, liver Hg varied more among individuals than muscle Hg. Water chemistry was similar in all reservoirs. Fish from the three reservoirs did not differ significantly in gill ATPase activity, and a correlation between tissue Hg and Na{sup +}, K{sup +}-ATPase activity was not evident.

  8. Walker-A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

    PubMed Central

    Nagy, Maria; Wu, Hui-Chuan; Liu, Zhonghua; Kedzierska-Mieszkowska, Sabina; Zolkiewski, Michal

    2009-01-01

    Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide-binding P-loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker-A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T-to-N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP-bound conformation and the high-affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker-A Thr in sensing the nucleotide's γ-phosphate and in maintaining an allosteric linkage between the P-loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate-remodeling activity. PMID:19177562

  9. Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress.

    PubMed

    Navarro, Anna; Weißbach, Susann; Faria, Melissa; Barata, Carlos; Piña, Benjamin; Luckenbach, Till

    2012-10-15

    Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages. PMID:22819804

  10. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity

    PubMed Central

    Lipps, Georg; Röther, Susanne; Hart, Christina; Krauss, Gerhard

    2003-01-01

    Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers ∼8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved indepen dently from the eubacterial and archaeal/eukaryal proteins of DNA replication. PMID:12743045

  11. Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.

    PubMed

    Sherman, David J; Lazarus, Michael B; Murphy, Lea; Liu, Charles; Walker, Suzanne; Ruiz, Natividad; Kahne, Daniel

    2014-04-01

    The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.

  12. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    PubMed

    Balan, Shabeesh; Bharathan, Sumitha Prameela; Vellichiramal, Neetha Nanoth; Sathyan, Sanish; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE

  13. The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity.

    PubMed

    Martuseviciene, Giedre; Hofman-Bang, Jacob; Clausen, Torben; Olgaard, Klaus; Lewin, Ewa

    2011-04-01

    The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be linked to increased hormone secretion. In this study, we used an in vivo rat model to determine the importance of sodium/potassium-ATPase in PTH secretion. Glands were exposed and treated in situ with vehicle or ouabain, a specific inhibitor of sodium/potassium-ATPase. PTH secretion was significantly increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by (86)rubidium uptake, which was found to be significantly decreased in ouabain-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model. PMID:21209610

  14. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy

    PubMed Central

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C.; Thomas, George; Tauler, Albert

    2015-01-01

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1. PMID:26356814

  15. V-ATPase: a master effector of E2F1-mediated lysosomal trafficking, mTORC1 activation and autophagy.

    PubMed

    Meo-Evoli, Nathalie; Almacellas, Eugènia; Massucci, Francesco Alessandro; Gentilella, Antonio; Ambrosio, Santiago; Kozma, Sara C; Thomas, George; Tauler, Albert

    2015-09-29

    In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

  16. Proteomic analysis of V-ATPase-rich cells harvested from the kidney and epididymis by fluorescence-activated cell sorting

    PubMed Central

    Da Silva, Nicolas; Pisitkun, Trairak; Belleannée, Clémence; Miller, Lance R.; Nelson, Raoul; Knepper, Mark A.; Brown, Dennis

    2010-01-01

    Proton-transporting cells are located in several tissues where they acidify the extracellular environment. These cells express the vacuolar H+-ATPase (V-ATPase) B1 subunit (ATP6V1B1) in their plasma membrane. We provide here a comprehensive catalog of the proteins that are expressed in these cells, after their isolation by enzymatic digestion and fluorescence-activated cell sorting (FACS) from transgenic B1-enhanced green fluorescent protein (EGFP) mice. In these mice, type A and B intercalated cells and connecting segment cells of the kidney, and narrow and clear cells of the epididymis, which all express ATP6V1B1, also express EGFP, while all other cell types are negative. The proteome of renal and epididymal EGFP-positive (EGFP+) cells was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and compared with their respective EGFP-negative (EGFP−) cell populations. A total of 2,297 and 1,564 proteins were detected in EGFP+ cells from the kidney and epididymis, respectively. Out of these proteins, 202 and 178 were enriched by a factor greater than 1.5 in EGFP+ cells compared with EGFP− cells, in the kidney and epididymis respectively, and included subunits of the V-ATPase (B1, a4, and A). In addition, several proteins involved in intracellular trafficking, signaling, and cytoskeletal dynamics were identified. A novel common protein that was enriched in renal and epididymal EGFP+ cells is the progesterone receptor, which might be a potential candidate for the regulation of V-ATPase-dependent proton transport. These proteomic databases provide a framework for comprehensive future analysis of the common and distinct functions of V-ATPase-B1-expressing cells in the kidney and epididymis. PMID:20181927

  17. Combined analysis of circulating β-endorphin with gene polymorphisms in OPRM1, CACNAD2 and ABCB1 reveals correlation with pain, opioid sensitivity and opioid-related side effects

    PubMed Central

    2013-01-01

    Background Opioids are associated with wide inter-individual variability in the analgesic response and a narrow therapeutic index. This may be partly explained by the presence of single nucleotide polymorphisms (SNPs) in genes encoding molecular entities involved in opioid metabolism and receptor activation. This paper describes the investigation of SNPs in three genes that have a functional impact on the opioid response: OPRM1, which codes for the μ-opioid receptor; ABCB1 for the ATP-binding cassette B1 transporter enzyme; and the calcium channel complex subunit CACNA2D2. The genotyping was combined with an analysis of plasma levels of the opioid peptide β-endorphin in 80 well-defined patients with chronic low back pain scheduled for spinal fusion surgery, and with differential sensitivity to the opioid analgesic remifentanil. This patient group was compared with 56 healthy controls. Results The plasma β-endorphin levels were significantly higher in controls than in pain patients. A higher incidence of opioid-related side effects and sex differences was found in patients with the minor allele of the ABCB1 gene. Further, a correlation between increased opioid sensitivity and the major CACNA2D2 allele was confirmed. A tendency of a relationship between opioid sensitivity and the minor allele of OPRM1 was also found. Conclusions Although the sample cohort in this study was limited to 80 patients it appears that it was possible to observe significant correlations between polymorphism in relevant genes and various items related to pain sensitivity and opioid response. Of particular interest is the new finding of a correlation between increased opioid sensitivity and the major CACNA2D2 allele. These observations may open for improved strategies in the clinical treatment of chronic pain with opioids. PMID:23402298

  18. H(+)/K(+) ATPase activity is required for biomineralization in sea urchin embryos.

    PubMed

    Schatzberg, Daphne; Lawton, Matthew; Hadyniak, Sarah E; Ross, Erik J; Carney, Tamara; Beane, Wendy S; Levin, Michael; Bradham, Cynthia A

    2015-10-15

    The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles. PMID:26282894

  19. Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity.

    PubMed

    Ito, Kohji; Kashiyama, Taku; Shimada, Kiyo; Yamaguchi, Akira; Awata, Jun ya; Hachikubo, You; Manstein, Dietmar J; Yamamoto, Keiichi

    2003-12-26

    The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.

  20. H(+)/K(+) ATPase activity is required for biomineralization in sea urchin embryos.

    PubMed

    Schatzberg, Daphne; Lawton, Matthew; Hadyniak, Sarah E; Ross, Erik J; Carney, Tamara; Beane, Wendy S; Levin, Michael; Bradham, Cynthia A

    2015-10-15

    The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles.

  1. Osmoregulation and salt gland Na, K-ATPase activity following exposure to the anticholinesterase fenthion

    USGS Publications Warehouse

    Rattner, B.A.; Fleming, W.J.; Murray, H.C.

    1982-01-01

    Salt gland function and osmoregulation in aquatic birds drinking hyperosmotic water has been suggested to be impaired by organophosphorus insecticides. To test this hypothesis, adult ducks (Anas rubripes) were provided various regimens of fresh or salt (1.5% NaCl) water (FW, SW) and mash containing vehicle or 21 ppm fenthion (Fn) on days 1-7 and 7-12 of this study. The 8 treatments (day 1-7:day 7-12) included :FW:FW, FW:FW+Fn, FW:SW, FW+Fn:SW, FW:SW+Fn, FW+Fn:SW+FN, SW;SW, and SW:5W+Fn. Ducks were bled by jugular venipuncture on days 1,7 and 12, and then sacrificed. Brain and salt gland acetylcholinesterase activities were substantially inhibited (44-52% and 14-26%) by Fn. However, plasma Na, Cl and osmolality, as indirect but cumulative indices of salt gland function, were uniformly elevated in all SW groups including those receiving Fn. In a second experiment, salt gland Na,K-ATPase activity was reduced after in vitro incubation with DDE (40 and 400 ?M; positive control), but was unaffected by Fn and its oxygen analog (0.04-400 ?M). The present findings suggest that environmentally realistic concentrations of organophosphorus insecticides do not affect osmoregulatory function in adult ducks.

  2. Effects of Celangulin IV and V From Celastrus angulatus Maxim on Na+/K+-ATPase Activities of the Oriental Armyworm (Lepidoptera: Noctuidae).

    PubMed

    Cheng, Dan; Feng, Mingxing; Ji, Yufei; Wu, Wenjun; Hu, Zhaonong

    2016-01-01

    Na(+)/K(+)-ATPase (sodium pump) is an important target for the development of botanical pesticide as it is responsible for transforming chemical energy in ATP to osmotic work and maintaining electrochemical Na(+ )and K(+ )gradients across the cell membrane of most animal cells. Celangulin IV (C-IV) and V (C-V), which are isolated from the root bark of Celastrus angulatus, are the major active ingredients of this insecticidal plant. The activities of C-IV and C-V on Na(+)/K(+)-ATPase were investigated by ultramicro measuring method to evaluate the effects of C-IV and C-V on Na(+)/K(+)-ATPase activities of the brain from the fifth Mythimna separata larvae and to discuss the insecticidal mechanism of C-IV and C-V. Results indicate that inhibitory activities of Na(+)/K(+)-ATPase by C-IV and C-V possess an obvious concentration-dependent in vitro. Compared with C-IV, the inhibition of C-V on Na(+)/K(+)-ATPase was not striking. In vivo, at a concentration of 25 mg/liter, the inhibition ratio of C-IV on Na(+)/K(+)-ATPase activity from the brain in narcosis and recovery period was more remarkable than that of C-V. Furthermore, the insects were fed with different mixture ratios of C-IV and C-V. The inhibition extent of Na(+)/K(+)-ATPase activity was corresponded with the dose of C-IV. However, C-V had no notable effects. This finding may mean that the mechanism of action of C-IV and C-V on Na(+)/K(+)-ATPase were different. Na(+)/K -ATPase may be an action target of C-IV and C-V.

  3. Effects of Celangulin IV and V From Celastrus angulatus Maxim on Na+/K+-ATPase Activities of the Oriental Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Cheng, Dan; Feng, Mingxing; Ji, Yufei; Wu, Wenjun; Hu, Zhaonong

    2016-01-01

    Na+/K+-ATPase (sodium pump) is an important target for the development of botanical pesticide as it is responsible for transforming chemical energy in ATP to osmotic work and maintaining electrochemical Na+ and K+ gradients across the cell membrane of most animal cells. Celangulin IV (C-IV) and V (C-V), which are isolated from the root bark of Celastrus angulatus, are the major active ingredients of this insecticidal plant. The activities of C-IV and C-V on Na+/K+-ATPase were investigated by ultramicro measuring method to evaluate the effects of C-IV and C-V on Na+/K+-ATPase activities of the brain from the fifth Mythimna separata larvae and to discuss the insecticidal mechanism of C-IV and C-V. Results indicate that inhibitory activities of Na+/K+-ATPase by C-IV and C-V possess an obvious concentration-dependent in vitro. Compared with C-IV, the inhibition of C-V on Na+/K+-ATPase was not striking. In vivo, at a concentration of 25 mg/liter, the inhibition ratio of C-IV on Na+/K+-ATPase activity from the brain in narcosis and recovery period was more remarkable than that of C-V. Furthermore, the insects were fed with different mixture ratios of C-IV and C-V. The inhibition extent of Na+/K+-ATPase activity was corresponded with the dose of C-IV. However, C-V had no notable effects. This finding may mean that the mechanism of action of C-IV and C-V on Na+/K+-ATPase were different. Na+/K -ATPase may be an action target of C-IV and C-V. PMID:27324586

  4. Effects of Celangulin IV and V From Celastrus angulatus Maxim on Na+/K+-ATPase Activities of the Oriental Armyworm (Lepidoptera: Noctuidae).

    PubMed

    Cheng, Dan; Feng, Mingxing; Ji, Yufei; Wu, Wenjun; Hu, Zhaonong

    2016-01-01

    Na(+)/K(+)-ATPase (sodium pump) is an important target for the development of botanical pesticide as it is responsible for transforming chemical energy in ATP to osmotic work and maintaining electrochemical Na(+ )and K(+ )gradients across the cell membrane of most animal cells. Celangulin IV (C-IV) and V (C-V), which are isolated from the root bark of Celastrus angulatus, are the major active ingredients of this insecticidal plant. The activities of C-IV and C-V on Na(+)/K(+)-ATPase were investigated by ultramicro measuring method to evaluate the effects of C-IV and C-V on Na(+)/K(+)-ATPase activities of the brain from the fifth Mythimna separata larvae and to discuss the insecticidal mechanism of C-IV and C-V. Results indicate that inhibitory activities of Na(+)/K(+)-ATPase by C-IV and C-V possess an obvious concentration-dependent in vitro. Compared with C-IV, the inhibition of C-V on Na(+)/K(+)-ATPase was not striking. In vivo, at a concentration of 25 mg/liter, the inhibition ratio of C-IV on Na(+)/K(+)-ATPase activity from the brain in narcosis and recovery period was more remarkable than that of C-V. Furthermore, the insects were fed with different mixture ratios of C-IV and C-V. The inhibition extent of Na(+)/K(+)-ATPase activity was corresponded with the dose of C-IV. However, C-V had no notable effects. This finding may mean that the mechanism of action of C-IV and C-V on Na(+)/K(+)-ATPase were different. Na(+)/K -ATPase may be an action target of C-IV and C-V. PMID:27324586

  5. Plasma membrane H(+) -ATPase gene expression, protein level and activity in growing and non-growing regions of barley (Hordeum vulgare) leaves.

    PubMed

    Visnovitz, Tamás; Solti, Adám; Csikós, György; Fricke, Wieland

    2012-04-01

    Plasma membrane proton ATPase (PM-H⁺-ATPase) is the key means through which plant cells energize nutrient uptake and acidify the apoplast. Both of these processes aid cell elongation; yet, it is not known how such a suspected role of the PM-H⁺-ATPase in growth is reflected through changes in its transcript level and activity in grass leaves. In the present study on leaf three of barley, the elongation zone and the emerged blade, which contained fully expanded cells were analyzed. Plasma membranes were isolated and used to assay the activity (ATPase assay) and abundance (western blotting) of PM-H⁺-ATPase protein. Expression of mRNA was quantified using real-time polymerase chain reaction (qPCR). PM-H⁺-ATPase transcript and protein level and activity differed little between growing and non-growing leaf regions when values were related to unit extracted total RNA and cell number, respectively. However, when values were related to unit surface area of plasma membrane, they were more than twice as high in growing compared with non-growing leaf tissue. It is concluded that this higher surface density of PM-H⁺-ATPase activity in growing barley leaf tissue aids apoplast acidification and cell expansion. PMID:22257033

  6. Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle

    PubMed Central

    Frank, Gabriel A.; Shukla, Suneet; Rao, Prashant; Borgnia, Mario J.; Bartesaghi, Alberto; Merk, Alan; Mobin, Aerfa; Esser, Lothar; Earl, Lesley A.; Gottesman, Michael M.; Xia, Di

    2016-01-01

    The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP. PMID:27190212

  7. Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle.

    PubMed

    Frank, Gabriel A; Shukla, Suneet; Rao, Prashant; Borgnia, Mario J; Bartesaghi, Alberto; Merk, Alan; Mobin, Aerfa; Esser, Lothar; Earl, Lesley A; Gottesman, Michael M; Xia, Di; Ambudkar, Suresh V; Subramaniam, Sriram

    2016-07-01

    The multidrug transporter P-glycoprotein (P-gp, ABCB1) is an ATP-dependent pump that mediates the efflux of structurally diverse drugs and xenobiotics across cell membranes, affecting drug pharmacokinetics and contributing to the development of multidrug resistance. Structural information about the conformational changes in human P-gp during the ATP hydrolysis cycle has not been directly demonstrated, although mechanistic information has been inferred from biochemical and biophysical studies conducted with P-gp and its orthologs, or from structures of other ATP-binding cassette transporters. Using single-particle cryo-electron microscopy, we report the surprising discovery that, in the absence of the transport substrate and nucleotides, human P-gp can exist in both open [nucleotide binding domains (NBDs) apart; inward-facing] and closed (NBDs close; outward-facing) conformations. We also probe conformational states of human P-gp during the catalytic cycle, and demonstrate that, following ATP hydrolysis, P-gp transitions through a complete closed conformation to a complete open conformation in the presence of ADP.

  8. 3-Methylcrotonylglycine disrupts mitochondrial energy homeostasis and inhibits synaptic Na(+),K (+)-ATPase activity in brain of young rats.

    PubMed

    Moura, Alana Pimentel; Ribeiro, César Augusto João; Zanatta, Ângela; Busanello, Estela Natacha Brandt; Tonin, Anelise Miotti; Wajner, Moacir

    2012-03-01

    Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO(2) production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I-IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na(+),K(+)-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO(2) production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na(+),K(+)-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na(+),K(+)-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na(+),K(+)-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.

  9. CALIX[4]ARENE C-99 INHIBITS MYOSIN ATPase ACTIVITY AND CHANGES THE ORGANIZATION OF CONTRACTILE FILAMENTS OF MYOMETRIUM.

    PubMed

    Labyntseva, R D; Bevza, A A; Lul'ko, A O; Cherenok, S O; Kalchenko, V I; Kosterin, S O

    2015-01-01

    Calix[4]arenes are cup-like macrocyclic (polyphenolic) compounds, they are regarded as promising molecular "platforms" for the design of new physiologically active compounds. We have earlier found that calix[4]arene C-99 inhibits the ATPase activity of actomyosin and myosin subfragment-1 of pig uterus in vitro. The aim of this study was to investigate the interaction of calix[4]arene C-99 with myosin from rat uterine myocytes. It was found that the ATPase activity of myosin prepared from pre-incubated with 100 mM of calix[4]arene C-99 myocytes was almost 50% lower than in control. Additionally, we have revealed the effect of calix[4]arene C-99 on the subcellular distribution of actin and myosin in uterus myocytes by the method of confocal microscopy. This effect can be caused by reorganization of the structure of the contractile smooth muscle cell proteins due to their interaction with calix[4]arene. The obtained results demonstrate the ability of calix[4]arene C-99 to penetrate into the uterus muscle cells and affect not only the myosin ATPase activity, but also the structure of the actin and myosin filaments in the myometrial cells. Demonstrated ability of calix[4]arene C-99 can be used for development of new pharmacological agents for efficient normalization of myometrial contractile hyperfunction.

  10. Effect of cinnamon, clove and some of their constituents on the Na(+)-K(+)-ATPase activity and alanine absorption in the rat jejunum.

    PubMed

    Kreydiyyeh, S I; Usta, J; Copti, R

    2000-09-01

    The effect of a water extract of some spices on the in vitro activity of the rat jejunal Na(+)-K(+)-ATPase was investigated. Extracts of nutmeg, cinnamon, clove, cumin, coriander, turmeric and caraway all inhibited the ATPase, while anise seed and white pepper exerted no significant effects. The extracts of clove and cinnamon had the most potent inhibitory effect on the intestinal ATPase as compared to extracts of other spices. They also inhibited the in vitro Na(+)-K(+)-ATPase activity in a crude kidney homogenate and the activity of an isolated dog kidney Na(+)-K(+)-ATPase. The alcoholic extract of cinnamon, compared to the aqueous extract, had a stronger inhibitory action on the jejunal enzyme and a lower IC(50) value, which was not significantly different from the one observed with cinnamaldehyde, the major volatile oil present cinnamon, suggesting that in alcoholic extracts cinnamaldehyde is the major inhibitory component. The IC(50) values of eugenol, aqueous clove extract and ethanolic clove extract all fell within the same range and were not significantly different from each other, suggesting that eugenol is the major inhibitory component in both alcoholic and aqueous extracts. Based on the IC(50) values, the order of sensitivity of the enzyme to the spices extracts is as follows: isolated dog kidney ATPase>rat kidney ATPase>rat intestine ATPase. The aqueous extracts of clove and cinnamon also significantly lowered the absorption of alanine from the rat intestine. It was concluded that the active principle(s) in clove and cinnamon can permeate the membrane of the enterocytes and inhibit the Na(+)-K(+)-ATPase that provides the driving force for many transport processes. PMID:10930696

  11. Nutrient transport in the small intestine: Na+,K+-ATPase expression and activity in the small intestine of the chicken as influenced by dietary sodium.

    PubMed

    Gal-Garber, O; Mabjeesh, S J; Sklan, D; Uni, Z

    2003-07-01

    The Na+-K+-ATPase, localized in the basolateral membrane of enterocytes plays a major role in nutrient transport in the small intestine by transferring K+ ions into and Na+ out of the cell. Within the enterocyte, homeostasis is maintained by active exclusion of Na from the cell by the Na+,K+-adenosine triphosphatase (ATPase) or sodium pump. Because much of the intestinal nutrient transport is by Na cotransporters, Na+,K+-ATPase may be used to evaluate nutrient uptake. In this study, nutrient transport was evaluated by determining expression and activity of Na+-K+-ATPase in the jejunum of chicks fed diets with different concentrations of Na. Expression of the chicken Na+-K+-ATPase gene was examined following isolation of an 1,140 bp cDNA fragment of the alpha-subunit using a reverse transcription (RT)-PCR reaction with specific primers. This fragment was sequenced and showed 95 to 98% homology with the mammalian alpha-subunit of the Na+-K+-ATPase genes. This cDNA fragment was used as a specific probe in Northern blot hybridization for determination of expression in the chicken jejunum. Expression of mRNA of Na+-K+-ATPase was enhanced at low dietary Na but was unchanged at high dietary Na concentrations. In contrast, activity of the enzyme was low with low dietary Na and unchanged at high dietary Na. The Vmax of the Na+-K+-ATPase was unchanged, but affinity was altered by dietary Na concentrations. Thus, determination of expression and activity of intestinal Na+-K+-ATPase allows clearer understanding of changes in intestinal uptake due to dietary Na.

  12. The structural coupling between ATPase activation and recovery stroke in the myosin II motor

    SciTech Connect

    Koppole, Sampath; Smith, Jeremy C; Fischer, S.

    2007-07-01

    Before the myosin motor head can perform the next power stroke, it undergoes a large conformational transition in which the converter domain, bearing the lever arm, rotates {approx} 65{sup o}. Simultaneous with this 'recovery stroke', myosin activates its ATPase function by closing the Switch-2 loop over the bound ATP. This coupling between the motions of the converter domain and of the 40 {angstrom}-distant Switch-2 loop is essential to avoid unproductive ATP hydrolysis. The coupling mechanism is determined here by finding a series of optimized intermediates between crystallographic end structures of the recovery stroke (Dictyostelium discoideum), yielding movies of the transition at atomic detail. The successive formation of two hydrogen bonds by the Switch-2 loop is correlated with the successive see-saw motions of the relay and SH1 helices that hold the converter domain. SH1 helix and Switch-2 loop communicate via a highly conserved loop that wedges against the SH1-helix upon Switch-2 closing.

  13. Optimisation of Recombinant Production of Active Human Cardiac SERCA2a ATPase

    PubMed Central

    Antaloae, Ana V.; Montigny, Cédric; le Maire, Marc; Watson, Kimberly A.; Sørensen, Thomas L.-M.

    2013-01-01

    Methods for recombinant production of eukaryotic membrane proteins, yielding sufficient quantity and quality of protein for structural biology, remain a challenge. We describe here, expression and purification optimisation of the human SERCA2a cardiac isoform of Ca2+ translocating ATPase, using Saccharomyces cerevisiae as the heterologous expression system of choice. Two different expression vectors were utilised, allowing expression of C-terminal fusion proteins with a biotinylation domain or a GFP- His8 tag. Solubilised membrane fractions containing the protein of interest were purified onto Streptavidin-Sepharose, Ni-NTA or Talon resin, depending on the fusion tag present. Biotinylated protein was detected using specific antibody directed against SERCA2 and, advantageously, GFP-His8 fusion protein was easily traced during the purification steps using in-gel fluorescence. Importantly, talon resin affinity purification proved more specific than Ni-NTA resin for the GFP-His8 tagged protein, providing better separation of oligomers present, during size exclusion chromatography. The optimised method for expression and purification of human cardiac SERCA2a reported herein, yields purified protein (> 90%) that displays a calcium-dependent thapsigargin-sensitive activity and is suitable for further biophysical, structural and physiological studies. This work provides support for the use of Saccharomyces cerevisiae as a suitable expression system for recombinant production of multi-domain eukaryotic membrane proteins. PMID:23951256

  14. Association of ABCB1 and ABCG2 single nucleotide polymorphisms with clinical findings and response to chemotherapy treatments in Kurdish patients with breast cancer.

    PubMed

    Ghafouri, Houshiyar; Ghaderi, Bayazid; Amini, Sabrieh; Nikkhoo, Bahram; Abdi, Mohammad; Hoseini, Abdolhakim

    2016-06-01

    The possible interaction between gene polymorphisms and risk of cancer progression is very interesting. Polymorphisms in multi-drug resistance genes have an important role in response to anti-cancer drugs. The present study was aimed to evaluate the possible effects of ABCB1 C3435T and ABCG2 C421A single nucleotide polymorphisms on clinical and pathological outcomes of Kurdish patients with breast cancer. One hundred breast cancer patients and 200 healthy controls were enrolled in this case-control study. Clinical and pathological findings of all individuals were reported, and immunohistochemistry staining was used to assess the tissue expression of specific breast cancer proteins. The ABCB1 C3435T and ABCG2 C421 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The distribution of different genotypes between patient and control groups was only significant for ABCG2 C421A. A allele of ABCG2 C421A polymorphisms were significantly higher in patients than in controls. Patients with AA genotype of ABCG2 C421A were at higher risk of progressing breast cancer. Patients with A allele of ABCG2 had complete response to chemotherapeutic agents. There was no statistically significant association between ABCB1 C3435T and ABCG2 C421A polymorphisms and tissue expression of ER, PR, Her2/neu, and Ki67. The ABCB1 C3435T has no correlation with clinical findings and treatment with chemotherapy drugs. The A allele of ABCG2 C421A may be a risk factor for progression of breast cancer in Kurdish patients. In addition, breast cancer patients with C allele of this polymorphism have weaker response to treatments with anthracyclines and Paclitaxol.

  15. ATPase active-site electrostatic interactions control the global conformation of the 100 kDa SecA translocase.

    PubMed

    Kim, Dorothy M; Zheng, Haiyan; Huang, Yuanpeng J; Montelione, Gaetano T; Hunt, John F

    2013-02-27

    SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes. PMID:23167435

  16. Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit.

    PubMed

    Fujii, Takuto; Watanabe, Midori; Shimizu, Takahiro; Takeshima, Hiroshi; Kushiro, Keiichiro; Takai, Madoka; Sakai, Hideki

    2016-06-01

    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.

  17. Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli.

    PubMed

    Reimann, Sven; Poschmann, Gereon; Kanonenberg, Kerstin; Stühler, Kai; Smits, Sander H J; Schmitt, Lutz

    2016-08-15

    Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB. PMID:27279651

  18. Iron nanoparticle-induced activation of plasma membrane H(+)-ATPase promotes stomatal opening in Arabidopsis thaliana.

    PubMed

    Kim, Jae-Hwan; Oh, Youngjun; Yoon, Hakwon; Hwang, Inhwan; Chang, Yoon-Seok

    2015-01-20

    Engineered nanomaterials (ENMs) enable the control and exploration of intermolecular interactions inside microscopic systems, but the potential environmental impacts of their inevitable release remain largely unknown. Plants exposed to ENMs display effects, such as increase in biomass and chlorophyll, distinct from those induced by exposure to their bulk counterparts, but few studies have addressed the mechanisms underlying such physiological results. The current investigation found that exposure of Arabidopsis thaliana to nano zerovalent iron (nZVI) triggered high plasma membrane H(+)-ATPase activity. The increase in activity caused a decrease in apoplastic pH, an increase in leaf area, and also wider stomatal aperture. Analysis of gene expression indicated that the levels of the H(+)-ATPase isoform responsible for stomatal opening, AHA2, were 5-fold higher in plants exposed to nZVI than in unexposed control plants. This is the first study to show that nZVI enhances stomatal opening by inducing the activation of plasma membrane H(+)-ATPase, leading to the possibility of increased CO2 uptake. PMID:25496563

  19. Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae).

    PubMed

    Belli, N M; Faleiros, R O; Firmino, K C S; Masui, D C; Leone, F A; McNamara, J C; Furriel, R P M

    2009-03-01

    Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 per thousand salinity), as well as potential routes of Na+ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na+ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.

  20. Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

    PubMed Central

    Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

    1989-01-01

    The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252

  1. Effects of vanadium on population growth and Na-K-ATPase activity of the brackish water hydroid Cordylophora caspia

    SciTech Connect

    Ringelband, U.; Karbe, L.

    1996-07-01

    Vanadium, a relatively abundant heavy metal, enters the environment naturally through rock weathering. A large fraction of vanadium input is of human origin. The combustion of petroleum- and coal-products, which contain relatively high concentrations of vanadium, is one of the most important sources of the enrichment of vanadium in the environment. As it is used as an alloy, and vanadium rich iron-ores of various origin are used in steel production, the residual slag-stones of the steel industry can contain considerable vanadium concentrations. Wherever slag-stones serve as a cheap and convenient material in riverbank reinforcement, vanadium can leach into the aquatic environment. Vanadium is regarded as an essential trace element for higher animals. Cantley et al. indicated a regulatory function of vanadate in vivo. Although considerable information is available on the toxic effects of vanadium on humans, very little is known about the toxicity of vanadium towards aquatic organisms, especially invertebrates. Bell and Sargent have shown an inhibition of Na-K-ATPase activity in gills of the eel Anguilla anguilla. Holleland and Towle have demonstrated the inhibition of Na-K-ATPase activity in the gills of the shore crab Carcinus maenas. The aim of this study was to determine the toxicity of vanadium towards the brackish water hydroid Cordylophora caspia. Hydroids are known to be particularly sensitive to heavy metals and their asexual reproduction can be used in a well-established population growth test. Furthermore, the effects of vanadium on Na-K-ATPase activity in hydroids were studied in in vivo experiments, wherein the animals were exposed to sublethal concentrations of vanadium. In addition, the inhibition of Na-K-ATPase was measured in vitro, by adding vanadium to a microsomal preparation. 16 refs., 4 figs.

  2. Preliminary study of gill NA+,K+-ATPase activity in juvenile spring chinook salmon following electroshock or handling stress

    USGS Publications Warehouse

    VanderKooi, S.P.; Gale, William L.; Maule, A.G.

    2000-01-01

    We compared gill Na+,K+-ATPase in subyearling and yearling spring chinook salmon Oncorhynchus tshawytscha 3 h, 24 h, and 7 d after exposure to either a short pulsed DC electroshock (300 V, 50 Hz, 8-ms pulse duration) or an acute handling stress. Mean gill Na+,K+-ATPase values ranged from 7.5 to 11.8 ??mol inorganic phosphate (Pi) ?? (mg protein)-1 ?? h-1. No significant differences were detected, with the exception of electroshocked subyearlings 7 d after treatment. Increased activity was attributed to the presence of two influential values. No significant differences were detected after removal of these observations, so the increase was not considered biologically significant. Inclusion of the outliers did not alter our interpretation of the results given that the observed increase was slight compared with the magnitude of changes reported under experimental conditions and in migrating juvenile salmonids. The treatment groups underwent a typical stress response and had significantly elevated cortisol and glucose levels 3 h after treatment. Recovery to control levels occurred within 24 h for cortisol and from 24 h to 7 d for glucose. Our results lead to the conclusion that neither acute electroshock nor acute handling stress alters Na+,K+-ATPase activity in juvenile spring chinook salmon.

  3. Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

    PubMed Central

    Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B. Tracy

    2013-01-01

    It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54–RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules. PMID:24240239

  4. Bufadienolides from parotoid gland secretions of Cuban toad Peltophryne fustiger (Bufonidae): Inhibition of human kidney Na(+)/K(+)-ATPase activity.

    PubMed

    Perera Córdova, Wilmer H; Leitão, Suzana Guimarães; Cunha-Filho, Geraldino; Bosch, Roberto Alonso; Alonso, Isel Pascual; Pereda-Miranda, Rogelio; Gervou, Rodrigo; Touza, Natália Araújo; Quintas, Luis Eduardo M; Noël, François

    2016-02-01

    Parotoid gland secretions of toad species are a vast reservoir of bioactive molecules with a wide range of biological properties. Herein, for the first time, it is described the isolation by preparative reversed-phase HPLC and the structure elucidation by NMR spectroscopy and/or mass spectrometry of nine major bufadienolides from parotoid gland secretions of the Cuban endemic toad Peltophryne fustiger: ψ-bufarenogin, gamabufotalin, bufarenogin, arenobufagin, 3-(N-suberoylargininyl) marinobufagin, bufotalinin, telocinobufagin, marinobufagin and bufalin. In addition, the secretion was analyzed by UPLC-MS/MS which also allowed the identification of azelayl arginine. The effect of arenobufagin, bufalin and ψ-bufarenogin on Na(+)/K(+)-ATPase activity in a human kidney preparation was evaluated. These bufadienolides fully inhibited the Na(+)/K(+)-ATPase in a concentration-dependent manner, although arenobufagin (IC50 = 28.3 nM) and bufalin (IC50 = 28.7 nM) were 100 times more potent than ψ-bufarenogin (IC50 = 3020 nM). These results provided evidence about the importance of the hydroxylation at position C-14 in the bufadienolide skeleton for the inhibitory activity on the Na(+)/K(+)-ATPase.

  5. Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly.

    PubMed

    Wu, Guowei; Adachi, Hironori; Ge, Junhui; Stephenson, David; Query, Charles C; Yu, Yi-Tao

    2016-03-15

    Pseudouridine (Ψ) is the most abundant internal modification identified in RNA, and yet little is understood of its effects on downstream reactions. Yeast U2 snRNA contains three conserved Ψs (Ψ35, Ψ42, and Ψ44) in the branch site recognition region (BSRR), which base pairs with the pre-mRNA branch site during splicing. Here, we show that blocks to pseudouridylation at these positions reduce the efficiency of pre-mRNA splicing, leading to growth-deficient phenotypes. Restoration of pseudouridylation at these positions using designer snoRNAs results in near complete rescue of splicing and cell growth. These Ψs interact genetically with Prp5, an RNA-dependent ATPase involved in monitoring the U2 BSRR-branch site base-pairing interaction. Biochemical analysis indicates that Prp5 has reduced affinity for U2 snRNA that lacks Ψ42 and Ψ44 and that Prp5 ATPase activity is reduced when stimulated by U2 lacking Ψ42 or Ψ44 relative to wild type, resulting in inefficient spliceosome assembly. Furthermore, in vivo DMS probing analysis reveals that pseudouridylated U2, compared to U2 lacking Ψ42 and Ψ44, adopts a slightly different structure in the branch site recognition region. Taken together, our results indicate that the Ψs in U2 snRNA contribute to pre-mRNA splicing by directly altering the binding/ATPase activity of Prp5. PMID:26873591

  6. Deficiency in Na,K-ATPase alpha isoform genes alters spatial learning, motor activity, and anxiety in mice.

    PubMed

    Moseley, Amy E; Williams, Michael T; Schaefer, Tori L; Bohanan, Cynthia S; Neumann, Jon C; Behbehani, Michael M; Vorhees, Charles V; Lingrel, Jerry B

    2007-01-17

    Several disorders have been associated with mutations in Na,K-ATPase alpha isoforms (rapid-onset dystonia parkinsonism, familial hemiplegic migraine type-2), as well as reduction in Na,K-ATPase content (depression and Alzheimer's disease), thereby raising the issue of whether haploinsufficiency or altered enzymatic function contribute to disease etiology. Three isoforms are expressed in the brain: the alpha1 isoform is found in many cell types, the alpha2 isoform is predominantly expressed in astrocytes, and the alpha3 isoform is exclusively expressed in neurons. Here we show that mice heterozygous for the alpha2 isoform display increased anxiety-related behavior, reduced locomotor activity, and impaired spatial learning in the Morris water maze. Mice heterozygous for the alpha3 isoform displayed spatial learning and memory deficits unrelated to differences in cued learning in the Morris maze, increased locomotor activity, an increased locomotor response to methamphetamine, and a 40% reduction in hippocampal NMDA receptor expression. In contrast, heterozygous alpha1 isoform mice showed increased locomotor response to methamphetamine and increased basal and stimulated corticosterone in plasma. The learning and memory deficits observed in the alpha2 and alpha3 heterozygous mice reveal the Na,K-ATPase to be an important factor in the functioning of pathways associated with spatial learning. The neurobehavioral changes seen in heterozygous mice suggest that these mouse models may be useful in future investigations of the associated human CNS disorders.

  7. A pure chloride channel mutant of CLC-5 causes Dent's disease via insufficient V-ATPase activation.

    PubMed

    Satoh, Nobuhiko; Yamada, Hideomi; Yamazaki, Osamu; Suzuki, Masashi; Nakamura, Motonobu; Suzuki, Atsushi; Ashida, Akira; Yamamoto, Daisuke; Kaku, Yoshitsugu; Sekine, Takashi; Seki, George; Horita, Shoko

    2016-07-01

    Dent's disease is characterized by defective endocytosis in renal proximal tubules (PTs) and caused by mutations in the 2Cl(-)/H(+) exchanger, CLC-5. However, the pathological role of endosomal acidification in endocytosis has recently come into question. To clarify the mechanism of pathogenesis for Dent's disease, we examined the effects of a novel gating glutamate mutation, E211Q, on CLC-5 functions and endosomal acidification. In Xenopus oocytes, wild-type (WT) CLC-5 showed outward-rectifying currents that were inhibited by extracellular acidosis, but E211Q and an artificial pure Cl(-) channel mutant, E211A, showed linear currents that were insensitive to extracellular acidosis. Moreover, depolarizing pulse trains induced a robust reduction in the surface pH of oocytes expressing WT CLC-5 but not E211Q or E211A, indicating that the E211Q mutant functions as a pure Cl(-) channel similar to E211A. In HEK293 cells, E211A and E211Q stimulated endosomal acidification and hypotonicity-inducible vacuolar-type H(+)-ATPase (V-ATPase) activation at the plasma membrane. However, the stimulatory effects of these mutants were reduced compared with WT CLC-5. Furthermore, gene silencing experiments confirmed the functional coupling between V-ATPase and CLC-5 at the plasma membrane of isolated mouse PTs. These results reveal for the first time that the conversion of CLC-5 from a 2Cl(-)/H(+) exchanger into a Cl(-) channel induces Dent's disease in humans. In addition, defective endosomal acidification as a result of insufficient V-ATPase activation may still be important in the pathogenesis of Dent's disease.

  8. Effect of vanadium on renal Na+,K+-ATPase activity in diabetic rats: a possible role of leptin.

    PubMed

    Morsy, Mohamed D; Abdel-Razek, Hesham A; Osman, Osama M

    2011-03-01

    Several researches attempt to protect diabetic patients from the development of nephropathy. Involvement of leptin and renal Na+,K+-ATPase enzyme in diabetic nephropathy (DN) development is a recent field for researches. Vanadium, as a trace element with insulin mimetic effect, may act synergistically with insulin to protect against the development of DN. Sixty male Sprague Dawley rats were divided into six groups: control group (C), vanadium control group (CV), streptozotocin-induced diabetic group (D), insulin-treated diabetic group (DI), vanadium-treated diabetic group (DV), and combined insulin and vanadium-treated diabetic group. Six weeks later, systolic blood pressure (SBP) was measured and retro-orbital blood samples were collected to estimate glycosylated hemoglobin (HbA(₁c)), serum sodium (Na+) and creatinine, blood urea nitrogen (BUN) and plasma leptin levels. Preparation of microsomal fraction of renal tissue homogenate for estimation of Na+,K+-ATPase activity was done. The D group showed a significant increase in SBP, HbA(₁c), serum Na+, creatinine, and BUN levels and Na+,K+-ATPase activity in microsomal fraction of renal tissue homogenate while plasma leptin level decreased significantly compared with C and CV groups. Both DI and DV groups showed a significant improvement in all the above measured parameters compared with D group while there were no significant changes between the DI and DV groups. Concomitant treatment with insulin and vanadium resulted in a significant improvement in all the measured parameters compared to each alone. Vanadium in combination with insulin ameliorates DN markers and reduces renal Na+,K+-ATPase overactivity in diabetic rats. An effect that may be partially mediated through correction of hypoleptinemia observed in these animals.

  9. Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

    PubMed Central

    Hakimjavadi, Hesamedin; Lingrel, Jerry B.

    2015-01-01

    The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K+ concentration in the T-tubules, through its K+ substrate affinity. Apparent K+ affinity was determined from measurements of the K1/2 for K+ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K+ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q10 was 2.1 over the range of 22–37°C. The K1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K+ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K+ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K+ increases in proportion to the frequency and duration of action potential firing. This K1/2,K predicts a low fractional occupancy of K+ substrate sites at the resting extracellular K+ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K+ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles. PMID:26371210

  10. The 70S ribosome modulates the ATPase activity of Escherichia coli YchF

    PubMed Central

    Becker, Marion; Gzyl, Katherine E.; Altamirano, Alvin M.; Vuong, Anthony; Urbahn, Kirstin; Wieden, Hans-Joachim

    2012-01-01

    YchF is one of two universally conserved GTPases with unknown cellular function. As a first step toward elucidating YchF’s cellular role, we performed a detailed biochemical characterization of the protein from Escherichia coli. Our data from fluorescence titrations not only confirmed the surprising finding that YchFE.coli binds adenine nucleotides more efficiently than guanine nucleotides, but also provides the first evidence suggesting that YchF assumes two distinct conformational states (ATP- and ADP-bound) consistent with the functional cycle of a typical GTPase. Based on an in vivo pull-down experiment using a His-tagged variant of YchF from E. coli (YchFE.coli), we were able to isolate a megadalton complex containing the 70S ribosome. Based on this finding, we report the successful reconstitution of a YchF•70S complex in vitro, revealing an affinity (KD) of the YchFE.coli•ADPNP complex for 70S ribosomes of 3 μM. The in vitro reconstitution data also suggests that the identity of the nucleotide-bound state of YchF (ADP or ATP) modulates its affinity for 70S ribosomes. A detailed Michaelis-Menten analysis of YchF’s catalytic activity in the presence and the absence of the 70S ribosome and its subunits revealed for the first time that the 70S ribosome is able to stimulate YchF’s ATPase activity (~10-fold), confirming the ribosome as part of the functional cycle of YchF. Our findings taken together with previously reported data for the human homolog of YchF (hOLA1) indicate a high level of evolutionary conservation in the enzymatic properties of YchF and suggest that the ribosome is the main functional partner of YchF not only in bacteria. PMID:22995830

  11. Association of genotypes and haplotypes of multi-drug transporter genes ABCB1 and ABCG2 with clinical response to imatinib mesylate in chronic myeloid leukemia patients.

    PubMed

    Au, Anthony; Aziz Baba, Abdul; Goh, Ai Sim; Wahid Fadilah, S Abdul; Teh, Alan; Rosline, Hassan; Ankathil, Ravindran

    2014-04-01

    The introduction and success of imatinib mesylate (IM) has become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, the high efficacy of IM has been hampered by the issue of clinical resistance that might due to pharmacogenetic variability. In the current study, the contribution of three common single nucleotide polymorphisms (SNPs) of ABCB1 (T1236C, G2677T/A and C3435T) and two SNPs of ABCG2 (G34A and C421A) genes in mediating resistance and/or good response among 215 CML patients on IM therapy were investigated. Among these patients, the frequency distribution of ABCG2 421 CC, CA and AA genotypes were significantly different between IM good response and resistant groups (P=0.01). Resistance was significantly associated with patients who had homozygous ABCB1 1236 CC genotype with OR 2.79 (95%CI: 1.217-6.374, P=0.01). For ABCB1 G2677T/A polymorphism, a better complete cytogenetic remission was observed for patients with variant TT/AT/AA genotype, compared to other genotype groups (OR=0.48, 95%CI: 0.239-0.957, P=0.03). Haplotype analysis revealed that ABCB1 haplotypes (C1236G2677C3435) was statistically linked to higher risk to IM resistance (25.8% vs. 17.4%, P=0.04), while ABCG2 diplotype A34A421 was significantly correlated with IM good response (9.1% vs. 3.9%, P=0.03). In addition, genotypic variant in ABCG2 421C>A was associated with a major molecular response (MMR) (OR=2.20, 95%CI: 1.273-3.811, P=0.004), whereas ABCB1 2677G>T/A variant was associated with a significantly lower molecular response (OR=0.49, 95%CI: 0.248-0.974, P=0.04). However, there was no significant correlation of these SNPs with IM intolerance and IM induced hepatotoxicity. Our results suggest the usefulness of genotyping of these single nucleotide polymorphisms in predicting IM response among CML patients. PMID:24581936

  12. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  13. Effects of type 1 diabetes, sprint training and sex on skeletal muscle sarcoplasmic reticulum Ca2+ uptake and Ca2+-ATPase activity.

    PubMed

    Harmer, A R; Ruell, P A; Hunter, S K; McKenna, M J; Thom, J M; Chisholm, D J; Flack, J R

    2014-02-01

    Calcium cycling is integral to muscle performance during the rapid muscle contraction and relaxation of high-intensity exercise. Ca(2+) handling is altered by diabetes mellitus, but has not previously been investigated in human skeletal muscle. We investigated effects of high-intensity exercise and sprint training on skeletal muscle Ca(2+) regulation among men and women with type 1 diabetes (T1D, n = 8, 3F, 5M) and matched non-diabetic controls (CON, n = 8, 3F, 5M). Secondarily, we examined sex differences in Ca(2+) regulation. Subjects undertook 7 weeks of three times-weekly cycle sprint training. Before and after training, performance was measured, and blood and muscle were sampled at rest and after high-intensity exercise. In T1D, higher Ca(2+)-ATPase activity (+28%) and Ca(2+) uptake (+21%) than in CON were evident across both times and days (P < 0.05), but performance was similar. In T1D, resting Ca(2+)-ATPase activity correlated with work performed until exhaustion (r = 0.7, P < 0.01). Ca(2+)-ATPase activity, but not Ca(2+) uptake, was lower (-24%, P < 0.05) among the women across both times and days. Intense exercise did not alter Ca(2+)-ATPase activity in T1D or CON. However, sex differences were evident: Ca(2+)-ATPase was reduced with exercise among men but increased among women across both days (time × sex interaction, P < 0.05). Sprint training reduced Ca(2+)-ATPase (-8%, P < 0.05), but not Ca(2+) uptake, in T1D and CON. In summary, skeletal muscle Ca(2+) resequestration capacity was increased in T1D, but performance was not greater than CON. Sprint training reduced Ca(2+)-ATPase in T1D and CON. Sex differences in Ca(2+)-ATPase activity were evident and may be linked with fibre type proportion differences.

  14. Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.

    PubMed

    Pollock, Naomi L; McDevitt, Christopher A; Collins, Richard; Niesten, Petronella H M; Prince, Stephen; Kerr, Ian D; Ford, Robert C; Callaghan, Richard

    2014-01-01

    ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein. PMID:24036079

  15. Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).

    PubMed

    Maki, Nazli; Dey, Saibal

    2006-07-14

    The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

  16. An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors.

    PubMed

    Chen, Hong-Lin; Zhao, Jing; Zhang, Guan-Jun; Kou, Jun-Ping; Yu, Bo-Yang

    2016-06-01

    Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro. PMID:27473959

  17. A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity

    PubMed Central

    Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M.; Tauchert, Marcel J.; Agafonov, Dmitry E.; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L.; Lührmann, Reinhard

    2016-01-01

    The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation. PMID:27377154

  18. Two ATPases

    PubMed Central

    Senior, Alan E.

    2012-01-01

    In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. PMID:22822068

  19. Oral and inhaled corticosteroids: Differences in P-glycoprotein (ABCB1) mediated efflux

    SciTech Connect

    Crowe, Andrew Tan, Ai May

    2012-05-01

    There is concern that P-glycoprotein mediated efflux contributes to steroid resistance. Therefore, this study examined bidirectional corticosteroid transport and induction capabilities for P-glycoprotein (P-gp) to understand which of the systemic and inhaled corticosteroids interacted with P-gp to the greatest extent. Hydrocortisone, prednisolone, prednisone, methylprednisolone, and dexamethasone represented systemically active drugs, while fluticasone propionate, beclomethasone dipropionate, ciclesonide and budesonide represented inhaled corticosteroids. Aldosterone and fludrocortisone represented mineralocorticoids. All drugs were detected using individually optimised HPLC protocols. Transport studies were conducted through Caco-2 monolayers. Hydrocortisone and aldosterone had efflux ratios below 1.5, while prednisone showed a P-gp mediated efflux ratio of only 1.8 compared to its active drug, prednisolone, with an efflux ratio of 4.5. Dexamethasone and beclomethasone had efflux ratios of 2.1 and 3.3 respectively, while this increased to 5.1 for methylprednisolone. Fluticasone showed an efflux ratio of 2.3. Protein expression studies suggested that all of the inhaled corticosteroids were able to induce P-gp expression, from 1.6 to 2 times control levels. Most of the systemic corticosteroids had higher passive permeability (> 20 × 10{sup −6} cm/s) compared to the inhaled corticosteroids (> 5 × 10{sup −6} cm/s), except for budesonide, with permeability similar to the systemic corticosteroids. Inhaled corticosteroids are not transported by P-gp to the same extent as systemic corticosteroids. However, they are able to induce P-gp production. Thus, inhaled corticosteroids may have greater interactions with other P-gp substrates, but P-gp itself is less likely to influence resistance to the drugs. -- Highlights: ► Inhaled corticosteroids are only weak substrates for P-gp, including budesonide. ► Inhaled corticosteroid potent P-gp inducers especially

  20. Effect of diet of varying protein concentrations on the activity of erythrocyte membrane Ca2+Mg2+ ATPase in dogs.

    PubMed

    Adewoye, E O; Ige, A O; Adeyanju, O; Bekibele, E B

    2010-11-25

    Alterations in protein diet have been reported to result in alterations in calcium homeostasis in the body. Ca2+Mg2+ATPase is an ubiquitous enzyme important in calcium homeostasis in the body. The effect of varying protein diet on the activities of Ca2+ pump across cell membranes is however yet to be fully elucidated. In this study, the activity of erythrocyte membrane calcium pump in response to varying protein concentration in diet was therefore studied in the dog. The study was carried out in 24 dogs, randomly divided into 4 groups. The groups were fed with diets containing 30%, 26%, 16% and 0% proteins (high, medium, low and zero) for six weeks respectively. Blood samples were collected from each animal to determine packed cell volumes, hematocrit, blood urea, electrolyte studies and erythrocyte ghost membrane studies. The effects of Ca2+ and ATP on the activity of Ca2+Mg2+ ATPase were determined in the isolated ghost membrane. The result of the study shows that there was a protein diet dependent increase in the activity of Ca2+Mg2+ ATPase in the presence and absence of ATP in all the groups with the highest activity recorded in the high protein diet group and the lowest activity observed in the zero protein group. There was also a protein diet dependent increase in the protein concentration of the membranes in all groups observed with the highest protein concentration recorded in the high protein diet group and the lowest activity observed in the zero protein group. There was a significant decrease in K+ concentration (P <0.05) and a significant increase in urea concentration of animals fed with high protein diet (P <0.05). There was also a significant increase (P <0.05) in HCO3- concentration in the animals fed with medium protein diet and no significant difference in the PCV and heamatocrit values in all groups. This study has shown that high protein diets increase the activity of the Ca2+Mg2+ ATPase in the presence and absence of ATP.

  1. Amyloid beta-peptide impairs ion-motive ATPase activities: evidence for a role in loss of neuronal Ca2+ homeostasis and cell death.

    PubMed

    Mark, R J; Hensley, K; Butterfield, D A; Mattson, M P

    1995-09-01

    The amyloid beta-peptide (A beta) that accumulates as insoluble plaques in the brain in Alzheimer's disease can be directly neurotoxic and can increase neuronal vulnerability to excitotoxic insults. The mechanism of A beta toxicity is unclear but is believed to involve generation of reactive oxygen species (ROS) and loss of calcium homeostasis. We now report that exposure of cultured rat hippocampal neurons to A beta 1-40 or A beta 25-35 causes a selective reduction in Na+/K(+)-ATPase activity which precedes loss of calcium homeostasis and cell degeneration. Na+/K(+)-ATPase activity was reduced within 30 min of exposure to A beta 25-35 and declined to less than 40% of basal level by 3 hr. A beta did not impair other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. Experiments with ouabain, a specific inhibitor of the Na+/K(+)-ATPase, demonstrated that impairment of this enzyme was sufficient to induce an elevation of [Ca2+]i and neuronal injury. Impairment of Na+/K(+)-ATPase activity appeared to be causally involved in the elevation of [Ca2+]i and neurotoxicity since suppression of Na+ influx significantly reduced A beta- and ouabain-induced [Ca2+]i elevation and neuronal death. Neuronal degeneration induced by ouabain appeared to be of an apoptotic form as indicated by nuclear condensation and DNA fragmentation. The antioxidant free radical scavengers vitamin E and propylgallate significantly attenuated A beta-induced impairment of Na+/K(+)-ATPase activity, elevation of [Ca2+]i and neurotoxicity, suggesting a role for ROS. Finally, exposure of synaptosomes from postmortem human hippocampus to A beta resulted in a significant and specific reduction in Na+/K(+)-ATPase and Ca(2+)-ATPase activities, without affecting other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. These data suggest that impairment of ion-motive ATPases may play a role in the pathogenesis of neuronal injury in Alzheimer's disease. PMID:7666206

  2. A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

    PubMed Central

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-01-01

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

  3. [Combined effect of diazepam and GABAA-ergic ligands on the activity of Cl(-)-ATPase from plasma membrane of bream brain (Abramis brama L.)].

    PubMed

    Menzikov, S A; Menzikova, O V

    2004-01-01

    We studied the combined effect of diazepam and GABAA-ergic ligands on the activity of Cl- -ATPase from plasma membrane of bream brain. of The membranes were preincubated and incubated with diazepam as well as with other GABAA-ergic ligands at physiological pH (7.4), i.e. under the conditions when Cl- -ATPase activity is undetectable. GABA (0.1-100 microM) induced Cl- -ATPase activity with the maximum effect at 10 microM. Diazepam (0.1 microM) enhanced the effect of low GABA concentrations (0.1-1 microM) on Cl- -ATPase activity but had no effect on the enzyme in the presence of high GABA concentrations (10-100 microM). At the same time, GABA (1 microM) enhanced the effect of low diazepam concentrations (0.1-1 microM) on the enzyme activity but had no effect on it in the presence of high concentrations of the ligand. Blockers of GABAA-ergic receptors, picrotoxin (50 microM) and bicuculline (5 microM), canceled the combined effect of diazepam and GABA on the enzyme activity. The obtained data demonstrate that the combined effect of diazepam and GABAA-ergic ligands on Cl- -ATPase activity at physiological pH is similar to the effect of these ligands on GABAA/benzodiazepine/Cl- channel.

  4. Alterations in activities of acid phosphatase, alkaline phosphatase, ATPase and ATP content in response to seasonally varying Pi status in okra (Abelmoschus esculentus).

    PubMed

    Sen, Supatra; Mukherji, S

    2004-04-01

    Phosphorus (P) is the second most important macronutrient for plant growth. Plants exhibit numerous physiological and metabolic adaptations in response to seasonal variations in phosphorus content. Activities of acid and alkaline phosphatases, ATPase and ATP content were studied in summer, rainy and winter seasons at two different developmental stages (28 and 58 days after sowing) in Okra. Activities of both acid and alkaline phosphatases increased manifold in winter to cope up with low phosphorus content. ATP content and ATPase activity were high in summer signifying an active metabolic period. Phosphorus deficiency is characterized by low ATP content and ATPase activity (which are in turn partly responsible for a drastic reduction in growth and yield) and enhanced activities of acid and alkaline phosphatases which increase the availability of P in P-deficient seasons.

  5. Effect of activators and blockers of ligand-regulated ion channels on the activity of the Cl-stimulated Mg2+-ATPase of the plasma membrane fraction from bream (Abramis brama L.) brain.

    PubMed

    Menzikov, S A; Menzikova, O V

    2002-02-01

    Effects of GABA, glycine, acetylcholine, and glutamate (agonists of the GABAa/benzodiazepine, glycine, choline, and glutamate receptors, respectively) at concentrations in the range 10(-8)-10(-4) M on the activity of "basal" Mg(2+)-ATPase of the plasma membrane fraction from bream brain and on its activation by Cl(-) were investigated. GABA and glycine activated "basal" Mg(2+)-ATPase activity and suppressed its activation by Cl(-). Acetylcholine and glutamate activated "basal" Mg(2+)-ATPase to a lesser extent and did not suppress the activation of the enzyme by Cl(-). The activation of "basal" Mg(2+)-ATPase by neuromediators was decreased by blockers of the corresponding receptors (picrotoxin, strychnine, benztropine mesylate, and D-2-amino-5-phosphonovaleric acid). In addition, picrotoxin and strychnine eliminated the inhibiting effect of GABA and glycine, respectively, on the Cl(-)-stimulated Mg(2+)-ATPase activity. Agonists of the GABAa/benzodiazepine receptor--phenazepam (10(-8)-10(-4) M) and pentobarbital (10(-6)-10(-3) M)--activated the "basal" Mg(2+)-ATPase activity and decreased the Cl(-)-stimulated Mg(2+)-ATPase activity. The dependence of both enzyme activities on ligand concentration is bell-shaped. Moreover, phenazepam and pentobarbital increased the "basal" Mg(2+)-ATPase activity in the presence of 10(-7) M GABA and did not influence it in the presence of 10(-4) M GABA and 10(-6) M glycine. The data suggest that in the fish brain membranes the Cl(-)-stimulated Mg(2+)-ATPase interacts with GABAa/benzodiazepine and glycine receptors but not with m-choline and glutamate receptors.

  6. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

    PubMed

    Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

    2014-09-15

    Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis.

  7. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus

    PubMed Central

    Arnaldo, Francis B.; Villar, Van Anthony M.; Konkalmatt, Prasad R.; Owens, Shaun A.; Asico, Laureano D.; Jones, John E.; Yang, Jian; Lovett, Donald L.; Armando, Ines; Concepcion, Gisela P.

    2014-01-01

    Dopamine-mediated regulation of Na+-K+-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na+-K+-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na+-K+-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na+-K+-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na+-K+-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na+-K+-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na+-K+-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. PMID:25080496

  8. Interaction of pentobarbital with gabaergic drugs acting on the Cl(-)-ATPase activity of the plasma membranes from bream brain (Abramis brama L.).

    PubMed

    Menzikov, Sergey; Menzikova, Olga

    2002-12-16

    The present study was designed to investigate the role of the interaction of pentobarbital with gamma-aminobutyric acid (GABA)ergic drugs acting on the Cl(-)-adenosine triphosphate (ATP)ase activity of the plasma membranes fraction of bream brain. The preincubation and then incubation of the membranes with pentobarbital as well as with other GABAergic ligands was conducted at physiologic pH (7.4), i.e. at the condition where the Cl(-)-ATPase activity is not detected. Pentobarbital (1-100 microM) induces Cl(-)-ATPase activity, however at high concentration (1,000 microM) no effect of the ligand was found. In addition pentobarbital (50 microM) enhances the effect of low concentration of GABA (1 microM) on the Cl(-)-ATPase activity, but inhibits the action of high concentration of GABA (100 microM) on the enzyme. Whereas no activating effect of pentobarbital in the presence of baclofen (1 microM) was found. The blocker of GABA(A)-receptors, picrotoxin (50 microM) and bicuculline (5 microM) eliminated the action of pentobarbital on the enzyme. The present results provide evidence for the first time that at physiologic pH in incubation medium the interaction of pentobarbital with GABAergic drugs on the Cl(-)-ATPase activity is similar to the effects of these ligands on the GABA(A)-receptor.

  9. Hypoxia Leads to Na,K-ATPase Downregulation via Ca2+ Release-Activated Ca2+ Channels and AMPK Activation

    PubMed Central

    Gusarova, Galina A.; Trejo, Humberto E.; Dada, Laura A.; Briva, Arturo; Welch, Lynn C.; Hamanaka, Robert B.; Mutlu, Gökhan M.; Chandel, Navdeep S.; Prakriya, Murali; Sznajder, Jacob I.

    2011-01-01

    To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca2+ from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca2+ release-activated Ca2+ (CRAC) channels. This increase in intracellular Ca2+ induces Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca2+ concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca2+ signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction. PMID:21730292

  10. Effect of hypoxia on the calcium and magnesium content, lipid peroxidation level, and Ca²⁺-ATPase activity of syncytiotrophoblast plasma membranes from placental explants.

    PubMed

    Chiarello, Delia I; Marín, Reinaldo; Proverbio, Fulgencio; Benzo, Zully; Piñero, Sandy; Botana, Desirée; Abad, Cilia

    2014-01-01

    In the current study the possible relationship between the Ca(2+)/Mg(2+) ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca(2+)-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca(2+) content, diminished their Ca(2+)-ATPase activity, and kept their Mg(2+) content unchanged. Membranes preincubated with different concentrations of Ca(2+) increased their Ca(2+) content without changes in their Mg(2+) content. There is a direct relationship between Ca(2+) content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca(2+) content and Ca(2+)-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg(2+) showed a higher Mg(2+) content without changing their lipid peroxidation and Ca(2+)-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca(2+)-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca(2+) content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg(2+) might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals. PMID:25180187

  11. Influence of cadmium concentration and length of exposure on metabolic rate and gill Na+/K+ ATPase activity of golden shiners (Notemigonus crysoleucas).

    PubMed

    Peles, John D; Pistole, David H; Moffe, Mickey

    2012-06-01

    Although metabolic rate is considered to be useful as a general indicator of the biological effects of exposure to metals, it is seldom measured in conjunction with specific physiological, biochemical or cellular parameters. The purpose of this investigation was to examine the influence of cadmium (Cd) exposure on metabolic rate and gill Na(+)/K(+) ATPase activity in golden shiners (Notemigonus crysoleucas). Shiners were exposed to six levels of Cd (ranging from control to the maximum sublethal concentration) for 24- and 96-h periods. After 24-h, metabolic rate and Na(+)/K(+) ATPase activity of individual fish were strongly correlated. Shiners exposed to the four highest Cd concentrations (500, 800, 1100, and 1400 μg L(-1)) for 24-h exhibited a shock response that was characterized by mean values for metabolic rate and Na(+)/K(+) ATPase activity that were significantly lower compared to the control. Although results for 96-h exposures reflect a repair/recovery phase, there was no significant correlation between metabolic rate and Na(+)/K(+) ATPase activity. Metabolic rate of shiners was significantly elevated (65-100%) at all concentrations compared to the control after 96-h, whereas Na(+)/K(+) ATPase activity did not differ from the control. Elevated metabolic rate after 96-h likely reflects the influence of a variety of energetically demanding processes associated with repair and recovery.

  12. Modulation By K+ Plus NH4+ of Microsomal (Na+, K+)-ATPase Activity in Selected Ontogenetic Stages of the Diadromous River Shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

    PubMed Central

    Leone, Francisco A.; Bezerra, Thais M. S.; Garçon, Daniela P.; Lucena, Malson N.; Pinto, Marcelo R.; Fontes, Carlos F. L.; McNamara, John C.

    2014-01-01

    We investigate the synergistic stimulation by K+ plus NH4+ of (Na+, K+)-ATPase activity in microsomal preparations of whole zoea I and decapodid III, and in juvenile and adult river shrimp gills. Modulation of (Na+, K+)-ATPase activity is ontogenetic stage-specific, and particularly distinct between juveniles and adults. Although both gill enzymes exhibit two different sites for K+ and NH4+ binding, in the juvenile enzyme, these two sites are equivalent: binding by both ions results in slightly stimulated activity compared to that of a single ionic species. In the adult enzyme, the sites are not equivalent: when one ion occupies its specific binding site, (Na+, K+)-ATPase activity is stimulated synergistically by ≈50% on binding of the complementary ion. Immunolocalization reveals the enzyme to be distributed predominantly throughout the intralamellar septum in the gill lamellae of juveniles and adults. Western blot analyses demonstrate a single immunoreactive band, suggesting a single (Na+, K+)-ATPase α-subunit isoform that is distributed into different density membrane fractions, independently of ontogenetic stage. We propose a model for the modulation by K+ and NH4+ of gill (Na+, K+)-ATPase activity. These findings suggest that the gill enzyme may be regulated by NH4+ during ontogenetic development in M. amazonicum. PMID:24586919

  13. Effect of Hypoxia on the Calcium and Magnesium Content, Lipid Peroxidation Level, and Ca2+-ATPase Activity of Syncytiotrophoblast Plasma Membranes from Placental Explants

    PubMed Central

    Chiarello, Delia I.; Benzo, Zully; Piñero, Sandy; Botana, Desirée; Abad, Cilia

    2014-01-01

    In the current study the possible relationship between the Ca2+/Mg2+ ratio of human syncytiotrophoblast plasma membranes and their lipid peroxidation and Ca2+-ATPase activity was determined. Syncytiotrophoblast plasma membranes of placental explants cultured under hypoxia increased their lipid peroxidation and Ca2+ content, diminished their Ca2+-ATPase activity, and kept their Mg2+ content unchanged. Membranes preincubated with different concentrations of Ca2+ increased their Ca2+ content without changes in their Mg2+ content. There is a direct relationship between Ca2+ content and lipid peroxidation of the membranes, as well as an inverse relationship between their Ca2+ content and Ca2+-ATPase activity. On the contrary, preincubation of membranes with different concentrations of Mg2+ showed a higher Mg2+ content without changing their lipid peroxidation and Ca2+-ATPase activity. Explants cultured under hypoxia in the presence of 4 mM MgSO4 showed similar values of lipid peroxidation and Ca2+-ATPase activity of their membranes compared to those of explants cultured under normoxia. Increased Ca2+ content of the membranes by interacting with negatively charged phospholipids could result in destabilizing effects of the membrane structure, exposing hydrocarbon chains of fatty acids to the action of free radicals. Mg2+ might exert a stabilizing effect of the membranes, avoiding their exposure to free radicals. PMID:25180187

  14. Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein*

    PubMed Central

    Peng, Wentao; Lin, Zhijie; Li, Weirong; Lu, Jing; Shen, Yuequan; Wang, Chunguang

    2013-01-01

    The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function. PMID:23979136

  15. Cadmium induced inhibition of Na sup + /K sup + ATPase activity in tissues of crab Scylla serrata (Forskal)

    SciTech Connect

    Dhavale, D.M. ); Masurekar, V.B. ); Giridhar, B.A. )

    1988-06-01

    Heavy metals discharged from industries these days are a major source of pollution which has become threat to all forms of life. A measure of metabolism may be a most sensitive parameter since it integrates many factors such as enzyme activity, biochemical contents and physiological response. The ability to accurately characterize enzymes with respect to their distribution and kinetics makes them attractive indices of stress. The concept of Na{sup +}/K{sup +}-ATPase being intimately involved in active transport of ions across biological membranes has gained wide acceptance in recent years. Interference with osmoregulation may restrict the animal's ability to adapt to salinity changes. Several studies have shown that crustacenas are highly sensitive to metal pollutants, particularly when acclimated to low salinities. The present study was initiated to compare the characteristics of Na{sup +}/K{sup +}-ATPase activity in hepatopancreas and gills of crabs Scylla serrata (Forskal) exposed to acute and sublethal concentrations of cadmium chloride for defined periods with those from undosed animals.

  16. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus.

    PubMed

    Mathur, Chhavi; Savithri, Handanahal S

    2012-10-12

    Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  17. Carbofuran induced oxidative stress mediated alterations in Na⁺-K⁺-ATPase activity in rat brain: amelioration by vitamin E.

    PubMed

    Jaiswal, Sunil Kumar; Siddiqi, Nikhat Jamal; Sharma, Bechan

    2014-07-01

    Pesticides cause oxidative stress and adversely influence Na(+)-K(+)-ATPase activity in animals. Since impact of carbofuran has not been properly studied in the mammalian brain, the ability of carbofuran to induce oxidative stress and modulation in Na(+)-K(+)-ATPase activity and its amelioration by vitamin E was performed. The rats divided into six groups received two different doses of carbofuran (15% and 30% LD50) for 15 days. The results suggested that the carbofuran treatment caused a significant elevation in levels of malonaldehyde and reduced glutathione and sharp inhibition in the activities of super oxide dismutase, catalase, and glutathione-S-transferase; the effect being dose dependent. Carbofuran at different doses also caused sharp reduction in the activity of Na(+)-K(+)-ATPase. The pretreatment of vitamin E, however, showed a significant recovery in these indices. The pretreatment of rats with vitamin E offered protection from carbofuran-induced oxidative stress.

  18. Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity.

    PubMed

    Cheng, Iteen; Mikita, Natalie; Fishovitz, Jennifer; Frase, Hilary; Wintrode, Patrick; Lee, Irene

    2012-05-01

    Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen-deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232-252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (K(λN)) needed to achieve 50% maximal ATPase stimulation. Comparing the K(λN) values reveals that the region encompassing 232-252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240-252 of ELon is important for ATPase activity, substrate translocation and protein degradation.

  19. Rotary ATPases

    PubMed Central

    Stewart, Alastair G.; Sobti, Meghna; Harvey, Richard P.; Stock, Daniela

    2013-01-01

    Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors. PMID:23369889

  20. Detailed characterization of the cooperative mechanism of Ca(2+) binding and catalytic activation in the Ca(2+) transport (SERCA) ATPase.

    PubMed

    Zhang, Z; Lewis, D; Strock, C; Inesi, G; Nakasako, M; Nomura, H; Toyoshima, C

    2000-08-01

    Expression of heterologous SERCA1a ATPase in Cos-1 cells was optimized to yield levels that account for 10-15% of the microsomal protein, as revealed by protein staining on electrophoretic gels. This high level of expression significantly improved our characterization of mutants, including direct measurements of Ca(2+) binding by the ATPase in the absence of ATP, and measurements of various enzyme functions in the presence of ATP or P(i). Mutational analysis distinguished two groups of amino acids within the transmembrane domain: The first group includes Glu771 (M5), Thr799 (M6), Asp800 (M6), and Glu908 (M8), whose individual mutations totally inhibit binding of the two Ca(2+) required for activation of one ATPase molecule. The second group includes Glu309 (M4) and Asn796 (M6), whose individual or combined mutations inhibit binding of only one and the same Ca(2+). The effects of mutations of these amino acids were interpreted in the light of recent information on the ATPase high-resolution structure, explaining the mechanism of Ca(2+) binding and catalytic activation in terms of two cooperative sites. The Glu771, Thr799, and Asp800 side chains contribute prominently to site 1, together with less prominent contributions by Asn768 and Glu908. The Glu309, Asn796, and Asp800 side chains, as well as the Ala305 (and possibly Val304 and Ile307) carbonyl oxygen, contribute to site 2. Sequential binding begins with Ca(2+) occupancy of site 1, followed by transition to a conformation (E') sensitive to Ca(2+) inhibition of enzyme phosphorylation by P(i), but still unable to utilize ATP. The E' conformation accepts the second Ca(2+) on site 2, producing then a conformation (E' ') which is able to utilize ATP. Mutations of residues (Asp813 and Asp818) in the M6/M7 loop reduce Ca(2+) affinity and catalytic turnover, suggesting a strong influence of this loop on the correct positioning of the M6 helix. Mutation of Asp351 (at the catalytic site within the cytosolic domain

  1. The effect of juvenile hormone III, methyl farnesoate, and methoprene on Na/K-ATPase activity in larvae of the brine shrimp, Artemia.

    PubMed

    Ahl, J S; Brown, J J

    1991-01-01

    1. Ion transport enzyme (Na/K-ATPase) activity in stage III larvae of the brine shrimp, Artemia, remains elevated throughout the stadium when populations are exposed to methoprene in artificial seawater. 2. Infusion of methoprene, juvenile hormone, or methyl farnesoate causes increased Na/K-ATPase activity in homogenates of mid-stadium larvae that would otherwise exhibit low activity. 3. The sensitivity of the enzyme system to extremely low concentrations of the juvenoids suggests that this may be a common mode of action of these compounds. Additionally it suggests that the enzyme may be under the influence of a similar compound present in the larvae. PMID:1682091

  2. Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

    PubMed Central

    Xu, Ming; Xia, Min; Li, Xiao-Xue; Han, Wei-Qing; Boini, Krishna M.; Zhang, Fan; Zhang, Yang; Ritter, Joseph K; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H+-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase–mediated production of O2−. in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H+-ATPase apparently attenuated FasL-caused pH decrease. V1 H+-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H+-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H+-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs. PMID:22357614

  3. [Kinetics of inhibitory effect of calix[4]arene C-90 on activity of transporting plasma membrane Ca2+, Mg2+-ATPase of smooth muscle cells].

    PubMed

    Veklich, T O; Shkrabak, O A; Mazur, Iu Iu; Rodik, R V; Kal'chenko, V I; Kosterin, S O

    2014-01-01

    In experiments on the suspension of myometrium cell plasma membrane, processed by 0.1% digitonin, the inhibitory action of calix[4]arene C-90 (5,11,17,23-tetra(threeftor)methyl(phenilsulphonilimino)-methylamino-25,26,27,28-tetrapropoxy-calix[4]arene) on the activity of Ca2+,Mg2+-ATPase was investigated. The authors also examined the influence of calix[4]arene in different concentration on affinity of enzyme (Ca2,Mg2+-ATPase) for the ATP and ions of Mg and Ca, and its influence on cooperative effect and maximum velocity of ATP hydrolysis. It is shown that calix[4]arene does not influence the affinity of Ca2+,Mg2+-ATPase for the ATP, which means that these two compounds have different binding centers. Also calix[4]arene has no influence on affinity and cooperative effect of Ca ions, if it is used in concentration lower than 50 μM. Calix[4]arene slightly increases coefficient of Ca2+,Mg2+-ATPase activation by magnesium chloride. In all three cases, where ATP, Mg and Ca ions are used to test the impact of calix[4]arene, maximum velocity of ATP hydrolysis significantly decreases. All these results clarify that calix[4]arene implements its inhibitory action through mechanism of uncompetitive inhibition of Ca2+,Mg2+-ATPase activity.

  4. Extracellular Na+ levels regulate formation and activity of the NaX/alpha1-Na+/K+-ATPase complex in neuronal cells

    PubMed Central

    Berret, Emmanuelle; Smith, Pascal Y.; Henry, Mélaine; Soulet, Denis; Hébert, Sébastien S.; Toth, Katalin; Mouginot, Didier; Drolet, Guy

    2014-01-01

    MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na+]out). The mechanism underlying Na+-sensing involves Na+-flow through the NaX channel, directly regulated by the Na+/K+-ATPase α1-isoform which controls Na+-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na+]in). Here we aim to determine whether environmental changes in Na+ could actively modulate the NaX/Na+/K+-ATPase complex activity. We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na+] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na+] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na+/K+-ATPase remained unaltered. This unbalance between NaX and Na+/K+-ATPase pump proportion would induce a bigger portion of Na+/K+-ATPase-control-free NaX channel. Thus, we suggest that hypernatremic environment increases NaX/Na+/K+-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump. PMID:25538563

  5. The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

    PubMed

    Silva, Lilian N D; Ribeiro-Neto, José A; Valadares, Jéssica M M; Costa, Mariana M; Lima, Luciana A R S; Grillo, Luciano A M; Cortes, Vanessa F; Santos, Herica L; Alves, Stênio N; Barbosa, Leandro A

    2016-08-01

    Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.

  6. Hydrolytic and pumping activity of H+-ATPase from leaves of sugar beet (Beta vulgaris L.) as affected by salt stress.

    PubMed

    Wakeel, Abdul; Hanstein, Stefan; Pitann, Britta; Schubert, Sven

    2010-06-15

    Cell wall extensibility plays an important role in plant growth. According to the acid-growth theory, lower apoplastic pH allows extension growth by affecting cell wall extensibility. A lowered apoplastic pH is presumed to activate wall-loosening enzymes that control plant growth. Plasma membrane (PM) H(+)-ATPases play a major role in the apoplastic acidification by H(+) transport from cytosol to the apoplast. A salt-induced decrease in H(+)-pumping activity of plasma membrane H(+)-ATPases in salt-sensitive maize plants has previously been found. This led us to formulate the hypothesis that salt-resistant plant species such as sugar beet (Beta vulgaris L.) may have a mechanism to eliminate the effect of higher salt concentrations on plasma membrane H(+)-ATPase activity. In the present study, sugar beet plants were grown in 1mM NaCl (control) or 150 mM NaCl in hydroponics. H(+)-ATPase hydrolytic and pumping activities were measured in plasma membrane vesicles isolated from sugar beet shoots. We found that plasma membrane H(+)-ATPase hydrolytic and pumping activities were not affected by application of 150 mM NaCl. Moreover, apoplastic pH was also not affected under salt stress. However, a decrease in plant growth was observed. We assume that growth reduction was not due to a decrease in PM-H(+)-ATPase activity, but that other factors may be responsible for growth inhibition of sugar beet plants under salt stress.

  7. Δ²,³-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases.

    PubMed

    Noël, François; Pimenta, Paulo Henrique Cotrim; Dos Santos, Anderson Rouge; Tomaz, Erick Carlos Loureiro; Quintas, Luis Eduardo Menezes; Kaiser, Carlos Roland; Silva, Claudia Lucia Martins; Férézou, Jean-Pierre

    2011-01-01

    Looking at a new putative target for the large spectrum antiparasitic drug ivermectin, we recently showed that avermectin-derived drugs are active against promastigote and amastigote forms of Leishmania amazonensis at low micromolar concentrations. However, we then reported that at this concentration range ivermectin is also able to inhibit three important mammalian P-type ATPases so that unacceptable adverse effects could occur if this drug were used at such high doses therapeutically. The present work aimed to test the activity of ten ivermectin analogs on these rat ATPases in search of a compound with similar leishmanicidal activity but with no effect on the mammalian (host) ATPases at effective concentrations. We synthesized three new ivermectin analogs for testing on rat SERCA (1a and 1b), Na+, K+-ATPase (α₁ and α₂/α₃ isoforms) and H+/K+-ATPase activity, along with seven analogs already characterized for their leishmanicidal activity. Our main finding is that one of the prepared derivatives, Δ²,³-ivermectin ethyl secoester 8, is equipotent to ivermectin 1 for the in vitro leishmanicidal effects but is nearly without effect on the rat ATPases, indicating that it could have a better therapeutic index in vivo and could serve as a candidate for hit-to-lead progression. This conclusion is further supported by the fact that compound 8 produced only 6% (vs 77% for ivermectin) inhibition of the human kidney enzyme at 5 μM, a concentration corresponding to the IC₅₀ for the activity against L. amazonensis amastigotes. PMID:21088826

  8. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

    PubMed Central

    Chang, FuJung; Riera, Alberto; Evrin, Cecile; Sun, Jingchuan; Li, Huilin; Speck, Christian; Weinreich, Michael

    2015-01-01

    To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo. DOI: http://dx.doi.org/10.7554/eLife.05795.001 PMID:26305410

  9. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication.

    PubMed

    Chang, FuJung; Riera, Alberto; Evrin, Cecile; Sun, Jingchuan; Li, Huilin; Speck, Christian; Weinreich, Michael

    2015-08-25

    To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant 'Cdc6-E224Q' promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

  10. Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+ exchange

    PubMed Central

    Silva, Paulo

    2009-01-01

    Over the last decades several efforts have been carried out to determine the mechanisms of salt homeostasis in plants and, more recently, to identify genes implicated in salt tolerance, with some plants being successfully genetically engineered to improve resistance to salt. It is well established that the efficient exclusion of Na+ excess from the cytoplasm and vacuolar Na+ accumulation are the most important steps towards the maintenance of ion homeostasis inside the cell. Therefore, the vacuole of plant cells plays a pivotal role in the storage of salt. After the identification of the vacuolar Na+/H+ antiporter Nhx1 in Saccharomyces cerevisiae, the first plant Na+/H+ antiporter, AtNHX1, was isolated from Arabidopsis and its overexpression resulted in plants exhibiting increased salt tolerance. Also, the identification of the plasma membrane Na+/H+ exchanger SOS1 and how it is regulated by a protein kinase SOS2 and a calcium binding protein SOS3 were great achievements in the understanding of plant salt resistance. Both tonoplast and plasma membrane antiporters exclude Na+ from the cytosol driven by the proton-motive force generated by the plasma membrane H+-ATPase and by the vacuolar membrane H+-ATPase and H+-pyrophosphatase and it has been shown that the activity of these proteins responds to salinity. In this review we focus on the transcriptional and post-transcriptional regulation by salt of tonoplast proton pumps and Na+/H+ exchangers and on the signalling pathways involved in salt sensing. PMID:19820346

  11. Structures, chemotaxonomic significance, cytotoxic and Na(+),K(+)-ATPase inhibitory activities of new cardenolides from Asclepias curassavica.

    PubMed

    Zhang, Rong-Rong; Tian, Hai-Yan; Tan, Ya-Fang; Chung, Tse-Yu; Sun, Xiao-Hui; Xia, Xue; Ye, Wen-Cai; Middleton, David A; Fedosova, Natalya; Esmann, Mikael; Tzen, Jason T C; Jiang, Ren-Wang

    2014-11-28

    Five new cardenolide lactates (1–5) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (6–16 and 18–21) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 1–3 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (17–21) and cardenolide lactates (1–5) provided unique chemotaxonomic markers for this genus. Compounds 1–21 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate. PMID:25270760

  12. Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH

    PubMed Central

    Senouci-Rezkallah, Khadidja; Jobin, Michel P; Schmitt, Philippe

    2015-01-01

    This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells. PMID:25740257

  13. pH-Dependent Conformational Changes in the HCV NS3 Protein Modulate Its ATPase and Helicase Activities

    PubMed Central

    Ventura, Gustavo Tavares; da Costa, Emmerson Corrêa Brasil; Capaccia, Anne Miranda; Mohana-Borges, Ronaldo

    2014-01-01

    The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication. PMID:25551442

  14. F{sub o}F{sub 1}-ATPase activity regulated by external links on {beta} subunits

    SciTech Connect

    Cheng, Jie; Zhang, Xiao-ai; Shu, Yao-Gen; Yue, Jia-Chang

    2010-01-01

    F{sub o}F{sub 1}-ATPase activity is regulated by external links on {beta} subunits with different molecular weight. It is inhibited when anti-{beta} subunit antibody, streptavidin and H9 antibody link on the {beta} subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-{beta} antibody target was on the non-catalytic site of the {beta} subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the {beta} subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.

  15. Short-Term Treatment with Cell Wall Degrading Enzymes Increases the Activity of the Inositol Phospholipid Kinases and the Vanadate-Sensitive ATPase of Carrot Cells 1

    PubMed Central

    Chen, Qiuyun; Boss, Wendy F.

    1990-01-01

    Treating carrot (Daucus carota L.) suspension culture cells with a mixture of cell wall degrading enzymes, Driselase, resulted in an increase in the percentage of [3H]phosphatidylinositol bisphosphate. Analysis of the lipid kinase activities in the isolated plasma membranes after whole cell treatment indicated that treatment with Driselase (2% weight/volume; the equivalent of 340 units per milliliter of hemicellulase and 400 units per milliliter of cellulase activity) or treatment with hemicellulase (31.7% weight/volume, 20.7 units per milliliter) resulted in an increase in the inositol phospholipid kinase activity. However, treatment with cellulase alone had no effect at 0.5% (weight/volume, 17.2 units per milliliter) or inhibited the kinase activity at 1% (weight/volume, 34.4 units per milliliter). The active stimulus in Driselase was heat sensitive. The plasma membrane vanadate-sensitive ATPase activity also increased when the cells were treated with Driselase. A time course study indicated that both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase responded to as little as 5 seconds of treatment with 2% Driselase. However, at the lowest concentration of Driselase (0.04%, weight/volume) that resulted in an increase in inositol phospholipid kinase activity, the ATPase activity was not affected. Because inositol phospholipids have been shown to activate the vanadate-sensitive ATPase from plants (AR Memon, Q Chen, WF Boss [1989] Biochem Biophys Res Commun 162: 1295-1301), a stimulus-response pathway involving both the inositol phospholipid kinases and the plasma membrane vanadate-sensitive ATPase activity is discussed. Images Figure 2 Figure 6 PMID:16667922

  16. Modulation of Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle. Effects of mineralocorticoids and sodium.

    PubMed Central

    Grossman, E B; Hebert, S C

    1988-01-01

    This study investigates the effect of variations in mineralocorticoid as well as cell sodium delivery and uptake on Na-K-ATPase activity in the mouse medullary thick ascending limb of Henle (mTALH). Pharmacologic doses of the mineralocorticoid deoxycorticosterone acetate (DOCA) resulted in a 28% increase of Na-K-ATPase activity. Furosemide-induced inhibition of sodium uptake by the mTALH cell also resulted in Na-K-ATPase activity reduction (45%). Sodium deprivation did not cause a clear change in enzyme activity, either at 3 d or 2 wk, likely reflecting the result of the opposing influences of decreased sodium delivery and increased endogenous aldosterone. Finally, the behavior of Na-K-ATPase activity at 3 d of sodium deprivation in the mTALH contrasted with a 60% increase in activity observed in the cortical collecting tubule, a nephron segment known to be responsive to mineralocorticoid, and this heterogeneity of response may suggest an important role for the mTALH in maintaining salt homeostasis. PMID:2830316

  17. Role of the D1-D2 Linker of Human VCP/p97 in the Asymmetry and ATPase Activity of the D1-domain

    PubMed Central

    Tang, Wai Kwan; Xia, Di

    2016-01-01

    Human AAA+ protein p97 consists of an N-domain and two tandem ATPase domains D1 and D2, which are connected by the N-D1 and the D1-D2 linkers. Inclusion of the D1-D2 linker, a 22-amino acid peptide, at the end of p97 N-D1 truncate has been shown to activate ATP hydrolysis of its D1-domain, although the mechanism of activation remains unclear. Here, we identify the N-terminal half of this linker, highly conserved from human to fungi, is essential for the ATPase activation. By analyzing available crystal structures, we observed that the D1-D2 linker is capable of inducing asymmetry in subunit association into a p97 hexamer. This observation is reinforced by two new crystal structures, determined in the present work. The effect of D1-D2 linker on the ATPase activity of the D1-domain is correlated to the side-chain conformation of residue R359, a trans-acting arginine-finger residue essential for ATP hydrolysis of the D1-domain. The activation in D1-domain ATPase activity by breaking perfect six-fold symmetry implies functional importance of asymmetric association of p97 subunits, the extent of which can be determined quantitatively by the metric Asymmetric Index. PMID:26818443

  18. Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

    PubMed

    Hsu, Jui-Ling; Liu, Fan-Lun; Hsu, Lih-Ching; Chang, Hsun-Shuo; Leu, Wohn-Jenn; Yu, Chia-Chun; Chang, Wei-Ling; Chen, Ih-Sheng; Kung, Fan-Lu; Guh, Jih-Hwa

    2015-09-15

    Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

  19. Effects of different kinds of fluorides on enolase and ATPase activity of a fluoride-sensitive and fluoride-resistant Streptococcus mutans strain.

    PubMed

    van Loveren, C; Hoogenkamp, M A; Deng, D M; ten Cate, J M

    2008-01-01

    Enolase and ATPase are sensitive to fluoride. It is unclear whether this sensitivity differs for F-sensitive and F-resistant cells or for different types of fluoride. Permeabilized cells of the fluoride-sensitive strain Streptococcus mutans C180-2 and its fluoride-resistant mutant strain C180-2 FR were preincubated at pH 7 or 4 with NaF, the amine fluorides Olaflur and Dectaflur and amine chloride controls. After preincubations, enolase and ATPase activities of the cells were assessed. Enolase activity was more inhibited after preincubation at pH 7 with NaF than with Olaflur. Amine chloride stimulated, although not with statistical significance, the enolase activity of both strains. After preincubation at pH 4 the enolases were strongly inactivated, but the fluoride-resistant strain's enolase to a lesser extent. The results suggested that amine acts to protect enolase activity against the detrimental low pH effect. Gene sequencing showed that the enolase genes of the fluoride-resistant and fluoride-sensitive strain were identical. ATPase activity was not reduced after NaF preincubation at either pH 7 or pH 4. The amine fluorides and their chloride controls in the preincubation mixture reduced the ATPase activity significantly at both pH values. In conclusion, our results showed that preincubation with amine fluoride did not inhibit enolase activity more effectively than NaF. The amine part of the molecule may protect enolase activity against preincubations at low pH. ATPase activity was not inhibited by NaF preincubation but was significantly inhibited after preincubation with amine fluorides and amine chlorides.

  20. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    SciTech Connect

    Mathur, Chhavi; Savithri, Handanahal S.

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Pepper vein banding potyvirus VPg harbors Walker motifs. Black-Right-Pointing-Pointer VPg exhibits ATPase activity in the presence of NIa-Pro. Black-Right-Pointing-Pointer Plausible structural and functional interplay between VPg and NIa-Pro. Black-Right-Pointing-Pointer Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  1. Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings

    SciTech Connect

    Uemura, Matsuo; Steponkus, P.L. )

    1989-11-01

    The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H{sup +}-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments.

  2. Leishmania amazonensis: heme stimulates (Na(+)+K(+))ATPase activity via phosphatidylinositol-specific phospholipase C/protein kinase C-like (PI-PLC/PKC) signaling pathways.

    PubMed

    Almeida-Amaral, Elmo Eduardo; Cardoso, Viviane Carrozino; Francioli, Fernanda Gomes; Meyer-Fernandes, José Roberto

    2010-04-01

    In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na(+)+K(+))ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH(3) and U73122, specific inhibitors of PI-PLC. (Na(+)+K(+))ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50nM. This effect was completely reversed by 10nM calphostin C, an inhibitor of PKC. Thus, the effect of 50nM heme on (Na(+)+K(+))ATPase activity is completely abolished by ET-18-OCH(3) and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na(+)+K(+))ATPase activity through a PI-PLC/PKC signaling pathway. PMID:20045694

  3. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB.

    PubMed

    Tikhonova, Elena B; Devroy, Vishakha K; Lau, Sze Yi; Zgurskaya, Helen I

    2007-02-01

    Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter. PMID:17214741

  4. Electric field strength of membrane lipids from vertebrate species: membrane lipid composition and Na+-K+-ATPase molecular activity.

    PubMed

    Starke-Peterkovic, Thomas; Turner, Nigel; Else, Paul L; Clarke, Ronald J

    2005-03-01

    Intramembrane electric field strength is a very likely determinant of the activity of ion-transporting membrane proteins in living cells. In the absence of any transmembrane electrical potential or surface potential, its magnitude is determined by the dipole potential of the membrane's lipid components and their associated water of hydration. Here we have used a fluorometric method to quantify the dipole potential of vesicles formed from lipids extracted from kidney and brain of 11 different animal species from four different vertebrate classes. The dipole potential was compared with the fatty acid composition and with the Na(+)-K(+)-ATPase molecular activity of each preparation. The magnitude of the dipole potential was found to be relatively constant across all animal species, i.e., 236-334 mV for vesicles prepared from the total membrane lipids and 223-256 mV for phospholipids alone. The significantly lower value for phospholipids alone is potentially related to the removal of cholesterol and/or other common soluble lipid molecules from the membrane. Surprisingly, no significant dependence of the dipole potential on fatty acid composition was found. This may, however, be due to concomitant compensatory variations in lipid head group composition. The molecular activity of the Na(+)-K(+)-ATPase was found to increase with increasing dipole potential. The fact that the dipole potential is maintained at a relatively constant value over a wide range of animal species suggests that it may play a fundamental role in ensuring correct ion pump conformation and function within the membrane.

  5. Fusicoccin-induced catalase inhibitor is produced independently of H+-ATPase activation and behaves as an organic acid.

    PubMed

    Beffagna, Nicoletta; Riva, Marzia Alessandra

    2011-06-01

    The phytotoxin fusicoccin (FC) was found to induce an increase in apoplastic H₂O₂ content in Arabidopsis thaliana cells, apparently linked to the presence of an as yet unidentified catalase inhibitor detectable even in the external medium of FC-treated cells. This study, aimed to further characterize the inhibitor's features, shows that (1) FC-induced H₂O₂ accumulation increases as a function of FC concentration and correlates to the amount of inhibitor released at apoplastic level. The pattern of H+ efflux, conversely, does not fit with that of these two parameters, suggesting that neither the production nor the release of the catalase inhibitor is linked to the main role of FC in activating the plasma membrane (PM) H+-ATPase; (2) treatment with 10 µM erythrosine B (EB) early and totally inhibits net H+ and K+ fluxes across the PM, indicative of the H+ pump activity; nevertheless, also in these conditions a huge FC-induced H₂O₂ accumulation occurs, confirming that this effect is not related to the FC-induced PM H+-ATPase activation; (3) the inhibitor's release increases with time in all conditions tested and is markedly affected by extracellular pH (a higher pH value being associated to a larger efflux), in agreement with a weak acid release; and (4) the inhibitor can be almost completely recovered in a CH₂C₂-soluble fraction extracted from the incubation medium by sequential acid-base partitioning which contains nearly all of the organic acids released. These final results strongly suggest that the metabolite responsible for the FC-induced catalase inhibition belongs to the organic acid class.

  6. XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

    SciTech Connect

    Yamada, N; Hinz, J; Kopf, V L; Segalle, K; Thompson, L

    2004-02-25

    Homologous recombinational repair is a major DNA repair pathway that preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 appears to exist in a single complex with Rad51C. To begin to examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of the mutants' dysfunction, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon coexpression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in HRR.

  7. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    PubMed Central

    Kus, Tulay; Aktas, Gokmen; Kalender, Mehmet Emin; Demiryurek, Abdullah Tuncay; Ulasli, Mustafa; Oztuzcu, Serdar; Sevinc, Alper; Kul, Seval; Camci, Celaletdin

    2016-01-01

    Background Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001–3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. Conclusion ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy. PMID:27574448

  8. Fine structure and Ca-ATPase activity of the stratum intermedium cells during odontogenesis in gars, Lepisosteus, Actinopterygii.

    PubMed

    Sasagawa, Ichiro; Ishiyama, Mikio

    2002-01-01

    This is the first report on the stratum intermedium in vertebrates other than mammals. The aim of this study is to elucidate the fine structure and cytochemical features of the stratum intermedium during the stages of enameloid formation in Lepisosteus. Inner dental epithelium, stratum intermedium, stellate reticulum, and outer dental epithelium are consistently present in the tooth germs of Lepisosteus. The stratum intermedium cells are oval in shape, contain elliptical nuclei, and extend many small processes. It is implied that the structure of the enamel organ is different among actinopterygians, and that constitution of the enamel organ in Lepisosteus resembles that in higher vertebrates. Marked Ca-ATPase activity is observed at the cell membrane of the stratum intermedium cells, suggesting that the cells are involved in calcium transport during the stages of enameloid formation.

  9. AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis

    PubMed Central

    Vadász, István; Dada, Laura A.; Briva, Arturo; Trejo, Humberto E.; Welch, Lynn C.; Chen, Jiwang; Tóth, Péter T.; Lecuona, Emilia; Witters, Lee A.; Schumacker, Paul T.; Chandel, Navdeep S.; Seeger, Werner; Sznajder, Jacob I.

    2008-01-01

    Hypercapnia (elevated CO2 levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO2-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO2 levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO2-triggered increase in intracellular Ca2+ concentration and Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). Chelating intracellular Ca2+ or abrogating CaMKK-β function by gene silencing or chemical inhibition prevented the CO2-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-ζ and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-α1 prevented CO2-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a β-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO2 levels are sensed by AECs and that AMPK mediates CO2-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with β-adrenergic agonists and cAMP. PMID:18188452

  10. Paradoxical activation of the sodium chloride cotransporter (NCC) without hypertension in kidney deficient in a regulatory subunit of Na,K-ATPase, FXYD2.

    PubMed

    Arystarkhova, Elena; Ralph, Donna L; Liu, Yi Bessie; Bouley, Richard; McDonough, Alicia A; Sweadner, Kathleen J

    2014-12-01

    Na,K-ATPase generates the driving force for sodium reabsorption in the kidney. Na,K-ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na(+) retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K-ATPase activity was documented in the Fxyd2(-/-) mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K-ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide-sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na(+) reabsorption in this segment. Specifically, NCC total increased 30% and NCC phosphorylated at T53 and S71, associated with activation, increased 4-6 fold. The phosphorylation of the closely related thick ascending limb (TAL) apical NKCC2 (Slc12a1) increased at least twofold. Abundance of the total and cleaved (activated) forms of ENaC α-subunit was not different between genotypes. Nonetheless, no elevation of blood pressure was evident despite the fact that NCC and NKCC2 are in states permissive for Na(+) retention. Activation of NCC and NKCC2 may reflect an intracellular linkage to elevated Na,K-ATPase activity or a compensatory response to Na(+) loss proximal to the TAL and DCT.

  11. Paradoxical activation of the sodium chloride cotransporter (NCC) without hypertension in kidney deficient in a regulatory subunit of Na,K-ATPase, FXYD2.

    PubMed

    Arystarkhova, Elena; Ralph, Donna L; Liu, Yi Bessie; Bouley, Richard; McDonough, Alicia A; Sweadner, Kathleen J

    2014-12-01

    Na,K-ATPase generates the driving force for sodium reabsorption in the kidney. Na,K-ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na(+) retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K-ATPase activity was documented in the Fxyd2(-/-) mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K-ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide-sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na(+) reabsorption in this segment. Specifically, NCC total increased 30% and NCC phosphorylated at T53 and S71, associated with activation, increased 4-6 fold. The phosphorylation of the closely related thick ascending limb (TAL) apical NKCC2 (Slc12a1) increased at least twofold. Abundance of the total and cleaved (activated) forms of ENaC α-subunit was not different between genotypes. Nonetheless, no elevation of blood pressure was evident despite the fact that NCC and NKCC2 are in states permissive for Na(+) retention. Activation of NCC and NKCC2 may reflect an intracellular linkage to elevated Na,K-ATPase activity or a compensatory response to Na(+) loss proximal to the TAL and DCT. PMID:25472608

  12. Localisation of calcium ions and calcium-ATPase activity within myelinated nerve fibres of the adult guinea-pig optic nerve.

    PubMed Central

    Maxwell, W L; Watt, C; Pediani, J D; Graham, D I; Adams, J H; Gennarelli, T A

    1991-01-01

    There is no published description of the distribution of free Ca2+, nor of the distribution of Ca(2+)-ATPase activity associated with the maintenance of low axoplasmic Ca2+ concentrations, in normal central myelinated nerve fibres. We have used the oxalate-pyroantimonate technique to localise free Ca2+, together with the lead-citrate technique to localise Ca(2+)-ATPase activity within myelinated fibres from the adult guinea-pig optic nerve. Pyroantimonate precipitate occurred within the axoplasm at nodes of Ranvier and the internode, at areas of myelin disruption, within Schmidt-Lanterman incisures (SLI) and glial paranodal loops. But precipitate was absent from the axoplasm beneath SLI and at the paranode. Ca(2+)-ATPase activity was localised in axonal smooth endoplasmic reticulum (SER), the outer membrane of mitochondria, the nodal axolemma, the glial membranes of the paranodal loops, the SLI and the external aspect of the myelin sheath. We have demonstrated large domains within the axons of CNS fibres where calcium is present or absent. Moreover, we have shown that, where calcium is absent, there is localisation of Ca(2+)-ATPase activity, which would serve to remove calcium from the adjacent axoplasm. Our results are compared with information obtained from PNS fibres and some differences of distribution discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:1833365

  13. Control of Protein and Sterol Trafficking by Antagonistic Activities of a Type IV P-type ATPase and Oxysterol Binding Protein Homologue

    PubMed Central

    Muthusamy, Baby-Periyanayaki; Raychaudhuri, Sumana; Natarajan, Paramasivam; Abe, Fumiyoshi; Liu, Ke; Prinz, William A.

    2009-01-01

    The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Δ, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Δ cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein. PMID:19403696

  14. Effect of salinity on hemolymph osmotic pressure, sodium concentration and Na+-K+-ATPase activity of gill of Chinese crab, Eriocheir sinensis

    NASA Astrophysics Data System (ADS)

    Liu, Hongyu; Pan, Luqing; Fu, Lü

    2008-02-01

    The effects of salinity on hemolymph osmotic pressure, Na+ concentration and Na+-K+-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na+ concentration increased significantly ( P<0.05), and the Na+-K+-ATPase activity of gills decreased significantly ( P<0.05) when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na+ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na+-K+-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na+ concentration and lower Na+-K+-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct ( P>0.05); However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1 d, and then tended to be stable from day 1 to day 15.

  15. Control of protein and sterol trafficking by antagonistic activities of a type IV P-type ATPase and oxysterol binding protein homologue.

    PubMed

    Muthusamy, Baby-Periyanayaki; Raychaudhuri, Sumana; Natarajan, Paramasivam; Abe, Fumiyoshi; Liu, Ke; Prinz, William A; Graham, Todd R

    2009-06-01

    The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Delta, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Delta cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

  16. Effects of intermittent fasting on age-related changes on Na,K-ATPase activity and oxidative status induced by lipopolysaccharide in rat hippocampus.

    PubMed

    Vasconcelos, Andrea Rodrigues; Kinoshita, Paula Fernanda; Yshii, Lidia Mitiko; Marques Orellana, Ana Maria; Böhmer, Ana Elisa; de Sá Lima, Larissa; Alves, Rosana; Andreotti, Diana Zukas; Marcourakis, Tania; Scavone, Cristoforo; Kawamoto, Elisa Mitiko

    2015-05-01

    Chronic neuroinflammation is a common characteristic of neurodegenerative diseases, and lipopolysaccharide (LPS) signaling is linked to glutamate-nitric oxide-Na,K-ATPase isoforms pathway in central nervous system (CNS) and also causes neuroinflammation. Intermittent fasting (IF) induces adaptive responses in the brain that can suppress inflammation, but the age-related effect of IF on LPS modulatory influence on nitric oxide-Na,K-ATPase isoforms is unknown. This work compared the effects of LPS on the activity of α1,α2,3 Na,K-ATPase, nitric oxide synthase gene expression and/or activity, cyclic guanosine monophosphate, 3-nitrotyrosine-containing proteins, and levels of thiobarbituric acid-reactive substances in CNS of young and older rats submitted to the IF protocol for 30 days. LPS induced an age-related effect in neuronal nitric oxide synthase activity, cyclic guanosine monophosphate, and levels of thiobarbituric acid-reactive substances in rat hippocampus that was linked to changes in α2,3-Na,K-ATPase activity, 3-nitrotyrosine proteins, and inducible nitric oxide synthase gene expression. IF induced adaptative cellular stress-response signaling pathways reverting LPS effects in rat hippocampus of young and older rats. The results suggest that IF in both ages would reduce the risk for deficits on brain function and neurodegenerative disorders linked to inflammatory response in the CNS. PMID:25818175

  17. Regulation by the exogenous polyamine spermidine of Na,K-ATPase activity from the gills of the euryhaline swimming crab Callinectes danae (Brachyura, Portunidae).

    PubMed

    Silva, E C C; Masui, D C; Furriel, R P M; Mantelatto, F L M; McNamara, J C; Barrabin, H; Leone, F A; Scofano, H M; Fontes, C F L

    2008-04-01

    Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine>spermidine>putrescine. Spermidine affected K(0.5) values for Na(+) with minor alterations in K(0.5) values for K(+) and NH(4)(+), causing a decrease in maximal velocities under saturating Na(+), K(+) and NH(4)(+) concentrations. Phosphorylation measurements in the presence of 20 microM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na(+), both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na(+), the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na(+) at the Na(+)-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments.

  18. Changes of plasma membrane AtPase activity, membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities.

    PubMed

    Zhao, Zhong-Qiu; Zheng, Hai-Lei; Zhu, Yong-Guan

    2004-01-01

    The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0 per thousand, 10 per thousand, 20 per thousand, 30 per thousand, 40 per thousand) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A. marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity (A. marina and roots of K. candel: 0-30 per thousand; leaves of K. candel: 0-20 per thousand), the activity of ATPase increased with increasing salinity, while high salinity (above 30 per thousand or 20 per thousand) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0-10 per thousand (K. candel) or 0-20 per thousand (A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

  19. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    PubMed

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  20. Evaluation of CYP2C19, P2Y12, and ABCB1 polymorphisms and phenotypic response to clopidogrel in healthy Indian adults

    PubMed Central

    Sridharan, Kannan; Kataria, Rachna; Tolani, Drishti; Bendkhale, Shital; Gogtay, Nithya J.; Thatte, Urmila M.

    2016-01-01

    Introduction: CYP2C19 and P2Y12 polymorphisms have been claimed to alter the pharmacodynamic response to clopidogrel. ABCB1 polymorphism has been associated with the efflux of clopidogrel resulting in decreased bioavailability. Due to paucity of data from Indian population, the present study was undertaken to evaluate the association of genetic polymorphisms of CYP2C19, P2Y12, and ABCB1 with inhibition of platelet aggregation (IPA) by clopidogrel. Methods: Healthy adults (n = 90) of either gender were administered single dose of 300 mg clopidogrel. Baseline, 4 h postdose, and day 7 assessment of platelet aggregation and genotype of CYP2C19, P2Y12, and ABCB1 were carried out using standardized laboratory methods. The difference in the maximum platelet aggregation (MPA) between baseline and 4 h postdose was considered as delta-MPA (DMPA), and percentage change of MPA at 4 h from baseline was considered as IPA. Those with an IPA of <30% were considered as poor responders. Inferential statistics was applied to find out significant difference of these parameters between various groups of genetic polymorphisms. Results: Mean (standard deviation [SD]) of MPA (%) at baseline, 4 h postdose, and day 7 were 78 (5), 56 (16), and 71 (8), respectively. Similarly, mean (SD) of DMPA (%) and IPA (%) were 23 (17) and 29 (21), respectively. A total of 54/90 (60%) cases were found to be poor responders to clopidogrel. A wild genotype (*1/*1) of CYP2C19 was observed in 35 (40.2%), 42 (48.3%) had *1/*2, 2 (2.3%) individuals had *1/*3, and 8 (9.2%) had *2/*2 mutant genotypes. Although statistically not significant (P = 0.09), a trend was observed in having decreased inhibition values (both MPA and IPA) as we proceed from wild genotype (*1/*1) to mutant genotypes in the order of *1/*2, *1/*3, and *2/*2. Similarly, in P2Y12, a wild haplotype (H1/H1) was present in 77 (89.5%) and 9 (10.5%) individuals had H1/H2 type. A statistically significant difference in DMPA and IPA was observed with

  1. Revisiting the mechanisms of copper toxicity to rainbow trout: Time course, influence of calcium, unidirectional Na(+) fluxes, and branchial Na(+), K(+) ATPase and V-type H(+) ATPase activities.

    PubMed

    Chowdhury, M Jasim; Girgis, Mina; Wood, Chris M

    2016-08-01

    In order to resolve uncertainties as to the mechanisms of toxic action of Cu and the protective effects of water [Ca], juvenile rainbow trout were acclimated to baseline soft water (SW, [Na(+)]=0.07, [Ca(2+)]=0.15, [Mg(2+)]=0.05mmolL(-1)) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27mmolL(-1)). The 96-h LC50 was 7-fold higher (63.8 versus 9.2μgCuL(-1); 1.00 versus 0.14μmolCuL(-1)) at [Ca]=3.0 versus 0.15mmolL(-1). Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24h, the gill Cu load (LA50≈0.58μgCug(-1); 9.13nmolCug(-1)) predictive of 50% mortality by 96h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na(+) losses (J(Na)net) by increasing unidirectional Na(+) efflux rates (J(Na)out) and inhibiting unidirectional Na(+) uptake rates (J(Na)in). The effect on J(Na)out was virtually immediate, whereas the effect on J(Na)in developed progressively over 24h and was associated with an inhibition of branchial Na(+), K(+) ATPase activity. The J(Na)in inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL(-1)) than the J(Na)out stimulation (100μg Cu L(-1)). Elevated Ca protected against both effects, as well as against the inhibition of Na(+), K(+) ATPase activity. Branchial V-type H(+) ATPase activity was also inhibited by Cu exposure (100μgCuL(-1)), but only after 24h at high [Ca] (3.0mmolL(-1)). These novel results therefore reinforce the applicability of BLM theory to Cu, clarify that whether Na(+) influx or efflux is more sensitive depends on the duration of Cu exposure, show that elevated water [Ca], independent of alkalinity, is protective against both mechanisms of Cu toxicity, and identify V-type H(+)ATPase as a new Cu target for future investigation.

  2. Revisiting the mechanisms of copper toxicity to rainbow trout: Time course, influence of calcium, unidirectional Na(+) fluxes, and branchial Na(+), K(+) ATPase and V-type H(+) ATPase activities.

    PubMed

    Chowdhury, M Jasim; Girgis, Mina; Wood, Chris M

    2016-08-01

    In order to resolve uncertainties as to the mechanisms of toxic action of Cu and the protective effects of water [Ca], juvenile rainbow trout were acclimated to baseline soft water (SW, [Na(+)]=0.07, [Ca(2+)]=0.15, [Mg(2+)]=0.05mmolL(-1)) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27mmolL(-1)). The 96-h LC50 was 7-fold higher (63.8 versus 9.2μgCuL(-1); 1.00 versus 0.14μmolCuL(-1)) at [Ca]=3.0 versus 0.15mmolL(-1). Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24h, the gill Cu load (LA50≈0.58μgCug(-1); 9.13nmolCug(-1)) predictive of 50% mortality by 96h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na(+) losses (J(Na)net) by increasing unidirectional Na(+) efflux rates (J(Na)out) and inhibiting unidirectional Na(+) uptake rates (J(Na)in). The effect on J(Na)out was virtually immediate, whereas the effect on J(Na)in developed progressively over 24h and was associated with an inhibition of branchial Na(+), K(+) ATPase activity. The J(Na)in inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL(-1)) than the J(Na)out stimulation (100μg Cu L(-1)). Elevated Ca protected against both effects, as well as against the inhibition of Na(+), K(+) ATPase activity. Branchial V-type H(+) ATPase activity was also inhibited by Cu exposure (100μgCuL(-1)), but only after 24h at high [Ca] (3.0mmolL(-1)). These novel results therefore reinforce the applicability of BLM theory to Cu, clarify that whether Na(+) influx or efflux is more sensitive depends on the duration of Cu exposure, show that elevated water [Ca], independent of alkalinity, is protective against both mechanisms of Cu toxicity, and identify V-type H(+)ATPase as a new Cu target for future investigation. PMID:27262060

  3. Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

    PubMed Central

    Nai, Changsheng; Xuan, Haochen; Zhang, Yingying; Shen, Mengxiao; Xu, Tongda; Pan, Defeng; Zhang, Congwei; Zhang, Yanbin; Li, Dongye

    2015-01-01

    The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R) injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications. PMID:26681967

  4. The Influence of C3435T Polymorphism of the ABCB1 Gene on Genetic Susceptibility to Depression and Treatment Response in Polish Population - Preliminary Report

    PubMed Central

    Jeleń, Agnieszka Maria; Sałagacka, Aleksandra; Żebrowska, Marta Karolina; Mirowski, Marek; Talarowska, Monika; Gałecki, Piotr; Balcerczak, Ewa Izabela

    2015-01-01

    Background: Despite the high prevalence of depression, the mechanism of the origin of this disease as well as the causes of resistance to therapy in some patients are still not fully understood. Increasingly, the possible role of genetic factors is considered. One of them is polymorphisms in the ABCB1 (MDR1) gene which encodes P-glycoprotein, responsible for the transport of xenobiotics, including antidepressant drugs, through the blood-brain barrier. Methods: C3435T was evaluated in 90 patients with recurrent depressive disorders (rDD). Genotyping was performed using a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Results: The obtained results indicate that the TT genotype occurred more frequently among patients with rDD than in healthy volunteers (p=0.0441). Also, at least one C allele was present significantly less frequent in the study group than in healthy individuals (p=0.0300). The severity of depressive symptoms was higher among patient with the CC genotype in comparison with the other genotypes (p=0.0106) but treatment response to antidepressants was better in this group than among patients with CT or TT genotypes (p=0.0301). Likewise, patients with the T allele have a significantly lower severity of symptoms (p=0.0026) and decreased therapy effectiveness (p=0.0142) than C allele carriers. Conclusions: This study suggests that C3435T polymorphisms in the ABCB1 gene are strongly associated with a predisposition to depression development, the severity of depressive symptoms and the effectiveness of therapy with using different groups of antidepressant agents. PMID:26664259

  5. Pharmacokinetics of a Once-Daily Dose of Tacrolimus Early After Liver Transplantation: With Special Reference to CYP3A5 and ABCB1 Single Nucleotide Polymorphisms.

    PubMed

    Miyata, Yoichi; Akamatsu, Nobuhisa; Sugawara, Yasuhiko; Kaneko, Junichi; Yamamoto, Takehito; Suzuki, Hiroshi; Arita, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Tamura, Sumihito; Kokudo, Norihiro

    2016-01-01

    BACKGROUND The aim of the present study was to investigate the pharmacokinetics of the once-daily tacrolimus formulation (QD form) in relation to polymorphisms of the donor cytochrome P450 family 3 sub-family A polypeptide 5 (CYP3A5) gene and recipient adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1) gene. MATERIAL AND METHODS A total of 80 consecutive living-donor liver transplant (LDLT) recipients were started on the QD form of tacrolimus (day 1), and 60 patients were completely followed for 7 days early after liver transplantation in order to evaluate the pharmacokinetics. RESULTS The concentration/dose (C/D) ratio in recipients with the donor CYP3A5 *1 allele was significantly lower throughout the observation period compared with those with the CYP3A5 genotype *3/*3 (p<0.001), while no effect of single-nucleotide polymorphisms (SNPs) of ABCB1 was observed. The administered doses required to achieve the target trough level were significantly higher on day 7 than on day 1 among all groups, regardless of the differences in the SNPs, especially among those with donor CYP3A5 *1 allele. The tacrolimus concentration was kept within the targeted level all through the study regardless of SNPs. CONCLUSIONS The donor CYP3A5 *1 allele correlated with the lower C/D ratio after administration of the QD form, and higher doses of QD-form tacrolimus and careful monitoring for the trough level should be considered, especially in recipients with the donor CYP3A5 *1 allele. PMID:27503662

  6. Concerted but Noncooperative Activation of Nucleotide and Actuator Domains of the Ca-ATPase Upon Calcium Binding

    SciTech Connect

    Chen, Baowei; Mahaney, James E.; Mayer, M. Uljana; Bigelow, Diana J.; Squier, Thomas C.

    2008-11-25

    Calcium-dependent domain movements of the nucleotide (N) and actuator (A) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FlAsH-EDT2). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT2. Distance measurements using the nucleotide analog TNP-ATP, which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 Å increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound respectively to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both N- and A-domains domains display virtually identical calcium dependencies (i.e., Kd = 4.8 ± 0.4 μM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates ATP utilization upon occupancy of the second high-affinity calcium site to enhance transport efficiency.

  7. Genome segment 6 of Antheraea mylitta cypovirus encodes a structural protein with ATPase activity

    SciTech Connect

    Chavali, Venkata R.M.; Madhurantakam, Chaithanya; Ghorai, Suvankar; Roy, Sobhan; Das, Amit K.; Ghosh, Ananta K.

    2008-07-20

    The genome segment 6 (S6) of the 11 double stranded RNA genomes from Antheraea mylitta cypovirus was converted into cDNA, cloned and sequenced. S6 consisted of 1944 nucleotides with an ORF of 607 amino acids and could encode a protein of 68 kDa, termed P68. Motif scan and molecular docking analysis of P68 showed the presence of two cystathionine beta synthase (CBS) domains and ATP binding sites. The ORF of AmCPV S6 was expressed in E. coli as His-tag fusion protein and polyclonal antibody was raised. Immunoblot analysis of virus infected gut cells and purified polyhedra using raised anti-p68 polyclonal antibody showed that S6 encodes a viral structural protein. Fluorescence and ATPase assay of soluble P68 produced in Sf-9 cells via baculovirus expression system showed its ability to bind and cleave ATP. These results suggest that P68 may bind viral RNA through CBS domains and help in replication and transcription through ATP binding and hydrolysis.

  8. Light induces changes in activities of Na+/K+-ATPase, H+/K+-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam, Tridacna squamosa

    PubMed Central

    Ip, Yuen K.; Ching, Biyun; Hiong, Kum C.; Choo, Celine Y. L.; Boo, Mel V.; Wong, Wai P.; Chew, Shit F.

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH+4 or H+, and activities of enzymes involved in converting NH+4 to glutamate/glutamine in inner mantle, outer mantle, and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH+4 in substitution for K+ to activate Na+/K+-ATPase (NKA), manifested as a significant increase in the Na+/NH+4-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H+ released from CaCO3 deposition could react with NH3 to form NH+4 in the extrapallial fluid, and NH+4 could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH+4 and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM)-sensitive-V-H+-ATPase (VATPase) in the inner mantle, and significant increases in the Na+/K+-activated-NKA, H+/NH+4-activated-H+/K+-ATPase, and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na+ homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH+4 from the environment. This is the first report on light having direct enhancing effects on activities of certain transporters

  9. Light induces changes in activities of Na(+)/K(+)-ATPase, H(+)/K(+)-ATPase and glutamine synthetase in tissues involved directly or indirectly in light-enhanced calcification in the giant clam, Tridacna squamosa.

    PubMed

    Ip, Yuen K; Ching, Biyun; Hiong, Kum C; Choo, Celine Y L; Boo, Mel V; Wong, Wai P; Chew, Shit F

    2015-01-01

    The objective of this study was to determine the effects of 12 h of exposure to light, as compared with 12 h of exposure to darkness (control), on enzymatic activities of transporters involved in the transport of NH(+) 4 or H(+), and activities of enzymes involved in converting NH(+) 4 to glutamate/glutamine in inner mantle, outer mantle, and ctenidia of the giant clam, Tridacna squamosa. Exposure to light resulted in a significant increase in the effectiveness of NH(+) 4 in substitution for K(+) to activate Na(+)/K(+)-ATPase (NKA), manifested as a significant increase in the Na(+)/NH(+) 4-activated-NKA activity in the inner mantle. However, similar phenomena were not observed in the extensible outer mantle, which contained abundant symbiotic zooxanthellae. Hence, during light-enhanced calcification, H(+) released from CaCO3 deposition could react with NH3 to form NH(+) 4 in the extrapallial fluid, and NH(+) 4 could probably be transported into the shell-facing inner mantle epithelium through NKA. Light also induced an increase in the activity of glutamine synthetase, which converts NH(+) 4 and glutamate to glutamine, in the inner mantle. Taken together, these results explained observations reported elsewhere that light induced a significant increase in pH and a significant decrease in ammonia concentration in the extrapallial fluid, as well as a significant increase in the glutamine concentration in the inner mantle, of T. squamosa. Exposure of T. squamosa to light also led to a significant decrease in the N-ethylmaleimide (NEM)-sensitive-V-H(+)-ATPase (VATPase) in the inner mantle, and significant increases in the Na(+)/K(+)-activated-NKA, H(+)/NH(+) 4-activated-H(+)/K(+)-ATPase, and NEM-sensitive-VATPase activities in ctenidia, indicating that light-enhanced calcification might perturb Na(+) homeostasis and acid/base balance in the hemolymph, and might involve the active uptake of NH(+) 4 from the environment. This is the first report on light having direct

  10. ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB.

    PubMed

    Koronakis, V; Hughes, C; Koronakis, E

    1993-06-01

    The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol. PMID:8361361

  11. Effects of Ouabain on Proliferation of Human Endothelial Cells Correlate with Na+,K+-ATPase Activity and Intracellular Ratio of Na+ and K.

    PubMed

    Tverskoi, A M; Sidorenko, S V; Klimanova, E A; Akimova, O A; Smolyaninova, L V; Lopina, O D; Orlov, S N

    2016-08-01

    Side-by-side with inhibition of the Na+,K+-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na+ and K+ ratio ([Na+]i/[K+]i). Thus, we compared the dose- and time-dependences of the effect of ouabain on intracellular [Na+]i/[K+]i ratio, Na+,K+-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na+]i/[K+]i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na+,K+-ATPase activity by 25-30%, as measured by the rate of (86)Rb(+) influx. Higher ouabain concentrations inhibited Na+,K+-ATPase, increased [Na+]i/[K+]i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na+]i/[K+]i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na+]i/[K+]i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na+ and K+ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na+,K+-ATPase and decrease in the [Na+]i/[K+]i ratio. PMID:27677555

  12. Structure–Activity Relationship Study Reveals ML240 and ML241 as Potent and Selective Inhibitors of p97 ATPase

    PubMed Central

    Chou, Tsui-Fen; Li, Kelin; Frankowski, Kevin J; Schoenen, Frank J; Deshaies, Raymond J

    2013-01-01

    To discover more potent p97 inhibitors, we carried out a structure–activity relationship study of the quinazoline scaffold previously identified from our HTS campaigns. Two improved inhibitors, ML240 and ML241, inhibit p97 ATPase with IC50 values of 100 nm. Both compounds inhibited degradation of a p97-dependent but not a p97-independent proteasome substrate in a dual-reporter cell line. They also impaired the endoplasmic-reticulum-associated degradation (ERAD) pathway. Unexpectedly, ML240 potently stimulated accumulation of LC3-II within minutes, inhibited cancer cell growth, and rapidly mobilized the executioner caspases 3 and 7, whereas ML241 did not. The behavior of ML240 suggests that disruption of the protein homeostasis function of p97 leads to more rapid activation of apoptosis than is observed with a proteasome inhibitor. Further characterization revealed that ML240 has broad antiproliferative activity toward the NCI-60 panel of cancer cell lines, but slightly lower activity toward normal cells. ML240 also synergizes with the proteasome inhibitor MG132 to kill multiple colon cancer cell lines. Meanwhile, both probes have low off-target activity toward a panel of protein kinases and central nervous system targets. Our results nominate ML240 as a promising starting point for the development of a novel agent for the chemotherapy of cancer, and provide a rationale for developing pathway-specific p97 inhibitors. PMID:23316025

  13. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

    PubMed Central

    Livnat-Levanon, Nurit; I. Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

  14. Diphenyl diselenide elicits antidepressant-like activity in rats exposed to monosodium glutamate: A contribution of serotonin uptake and Na(+), K(+)-ATPase activity.

    PubMed

    Quines, Caroline B; Rosa, Suzan G; Velasquez, Daniela; Da Rocha, Juliana T; Neto, José S S; Nogueira, Cristina W

    2016-03-15

    Depression is a disorder with symptoms manifested at the psychological, behavioral and physiological levels. Monosodium glutamate (MSG) is the most widely used additive in the food industry; however, some adverse effects induced by this additive have been demonstrated in experimental animals and humans, including functional and behavioral alterations. The aim of this study was to investigate the possible antidepressant-like effect of diphenyl diselenide (PhSe)2, an organoselenium compound with pharmacological properties already documented, in the depressive-like behavior induced by MSG in rats. Male and female newborn Wistar rats were divided in control and MSG groups, which received, respectively, a daily subcutaneous injection of saline (0.9%) or MSG (4g/kg/day) from the 1st to 5th postnatal day. At 60th day of life, animals received (PhSe)2 (10mg/kg, intragastrically) 25min before spontaneous locomotor and forced swimming tests (FST). The cerebral cortices of rats were removed to determine [(3)H] serotonin (5-HT) uptake and Na(+), K(+)-ATPase activity. A single administration of (PhSe)2 was effective against locomotor hyperactivity caused by MSG in rats. (PhSe)2 treatment protected against the increase in the immobility time and a decrease in the latency for the first episode of immobility in the FST induced by MSG. Furthermore, (PhSe)2 reduced the [(3)H] 5-HT uptake and restored Na(+), K(+)-ATPase activity altered by MSG. In the present study a single administration of (PhSe)2 elicited an antidepressant-like effect and decrease the synaptosomal [(3)H] 5-HT uptake and an increase in the Na(+), K(+)-ATPase activity in MSG-treated rats.

  15. Effect of plictran on beef heart mitochondrial ATPases.

    PubMed

    Mehrotra, B D; Prasada Rao, K S; Desaiah, D

    1985-07-01

    The in vitro effects of plictran on oligomycin-sensitive Mg2+-ATPase and Ca2+-ATPase activities in beef heart mitochondria were studied. Beef heart mitochondrial fractions were prepared by the conventional centrifugation method. ATPase activities were measured by determining the inorganic phosphate released by the hydrolysis of ATP. Plictran inhibited both oligomycin-sensitive (o.s.) Mg2+-ATPase and Ca2+ ATPase activities at nanomolar concentrations. However, plictran did not affect the oligomycin-insensitive (o.i.) Mg2+-ATPase activity at any concentration studied. Substrate activation kinetics revealed that plictran inhibited o.s. Mg2+-ATPase uncompetitively and Ca2+-ATPase non-competitively. These results clearly indicate that plictran affects ATP synthesis and calcium ion transport in beef heart mitochondria.

  16. Discovery of Tricyclic Clerodane Diterpenes as Sarco/Endoplasmic Reticulum Ca(2+)-ATPase Inhibitors and Structure-Activity Relationships.

    PubMed

    De Ford, Christian; Calderón, Carlos; Sehgal, Pankaj; Fedosova, Natalya U; Murillo, Renato; Olesen, Claus; Nissen, Poul; Møller, Jesper V; Merfort, Irmgard

    2015-06-26

    Tricyclic clerodane diterpenes (TCDs) are natural compounds that often show potent cytotoxicity for cancer cells, but their mode of action remains elusive. A computationally based similarity search (CDRUG), combined with principal component analysis (ChemGPS-NP) and docking calculations (GOLD 5.2), suggested TCDs to be inhibitors of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, which is also the target of the sesquiterpene lactone thapsigargin. Biochemical studies were performed with 11 TCDs on purified rabbit skeletal muscle sarcoplasmic reticulum membranes, which are highly enriched with the SERCA1a isoform. Casearborin D (2) exhibited the highest affinity, with a KD value of 2 μM and giving rise to complete inhibition of SERCA1a activity. Structure-activity relationships revealed that functionalization of two acyl side chains (R1 and R4) and the hydrophobicity imparted by the aliphatic chain at C-9, as well as a C-3,C-4 double bond, play crucial roles for inhibitory activity. Docking studies also suggested that hydrophobic interactions in the binding site, especially with Phe256 and Phe834, may be important for a strong inhibitory activity of the TCDs. In conclusion, a novel class of SERCA inhibitory compounds is presented. PMID:25993619

  17. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    PubMed

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells.

  18. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    PubMed

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression. PMID:21531199

  19. Modulation of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase activity and oxidative modification during the development of adjuvant arthritis.

    PubMed

    Strosova, Miriam K; Karlovska, Janka; Zizkova, Petronela; Kwolek-Mirek, Magdalena; Ponist, Silvester; Spickett, Corinne M; Horakova, Lubica

    2011-07-01

    Adjuvant arthritis (AA) was induced by intradermal administration of Mycobacterium butyricum to the tail of Lewis rats. In sarcoplasmic reticulum (SR) of skeletal muscles, we investigated the development of AA. SR Ca(2+)-ATPase (SERCA) activity decreased on day 21, suggesting possible conformational changes in the transmembrane part of the enzyme, especially at the site of the calcium binding transmembrane part. These events were associated with an increased level of protein carbonyls, a decrease in cysteine SH groups, and alterations in SR membrane fluidity. There was no alteration in the nucleotide binding site at any time point of AA, as detected by a FITC fluorescence marker. Some changes observed on day 21 appeared to be reversible, as indicated by SERCA activity, cysteine SH groups, SR membrane fluidity, protein carbonyl content and fluorescence of an NCD-4 marker specific for the calcium binding site. The reversibility may represent adaptive mechanisms of AA, induced by higher relative expression of SERCA, oxidation of cysteine, nitration of tyrosine and presence of acidic phospholipids such as phosphatidic acid. Nitric oxide may regulate cytoplasmic Ca(2+) level through conformational alterations of SERCA, and decreasing levels of calsequestrin in SR may also play regulatory role in SERCA activity and expression.

  20. A small molecule inhibitor for ATPase activity of Hsp70 and Hsc70 enhances the immune response to protein antigens

    NASA Astrophysics Data System (ADS)

    Baek, Kyung-Hwa; Zhang, Haiying; Lee, Bo Ryeong; Kwon, Young-Guen; Ha, Sang-Jun; Shin, Injae

    2015-12-01

    The ATPase activities of Hsp70 and Hsc70 are known to be responsible for regulation of various biological processes. However, little is known about the roles of Hsp70 and Hsc70 in modulation of immune responses to antigens. In the present study, we investigated the effect of apoptozole (Az), a small molecule inhibitor of Hsp70 and Hsc70, on immune responses to protein antigens. The results show that mice administered with both protein antigen and Az produce more antibodies than those treated with antigen alone, showing that Az enhances immune responses to administered antigens. Treatment of mice with Az elicits production of antibodies with a high IgG2c/IgG1 ratio and stimulates the release of Th1 and Th2-type cytokines, suggesting that Az activates the Th1 and Th2 immune responses. The observations made in the present study suggest that inhibition of Hsp70 and Hsc70 activities could be a novel strategy designing small molecule-based adjuvants in protein vaccines.

  1. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells.

    PubMed

    Seo, Jeong-Ah; Kim, Boyun; Dhanasekaran, Danny N; Tsang, Benjamin K; Song, Yong Sang

    2016-02-01

    Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca(2+) homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca(2+) concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca(2+) flux was evident after the curcumin treatment (15 µM). Treatment with Ca(2+) chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca(2+) concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca(2+) elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca(2+) homeostasis and thereby promotes apoptosis in ovarian cancer cells. PMID:26607901

  2. Structural requirements for inhibitory effects of bisphenols on the activity of the sarco/endoplasmic reticulum calcium ATPase

    PubMed Central

    Woeste, Matthew; Steller, Jeffrey; Hofmann, Emily; Kidd, Taylor; Patel, Rahul; Connolly, Kevin; Jayasinghe, Manori; Paula, Stefan

    2013-01-01

    Bisphenols (BPs) are a class of small organic compounds with widespread industrial applications. Previous studies have identified several BPs that interfere with the activity of the ion-translocating enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). In order to define the molecular determinants of BP-mediated SERCA inhibition, we conducted enzyme activity assays with rabbit SERCA to determine the inhibitory potencies of 27 commercially available BPs, which were the basis for structure-activity relationships. The most potent BPs inhibited SERCA at low micromolar concentrations and carried at their two phenyl rings multiple non-polar substituents, such as small alkyl groups or halides. Furthermore, the presence of methyl groups or a cyclohexyl group at the central carbon atom connecting the two phenyl moieties correlated with good potencies. For a characterization and visualization of inhibitor/enzyme interactions, molecular docking was performed, which suggested that hydrogen bonding with Asp254 and hydrophobic interactions were the major driving forces for BP binding to SERCA. Calcium imaging studies with a selection of BPs showed that these inhibitors were able to increase intracellular calcium levels in living human cells, a behavior consistent with that of a SERCA inhibitor. PMID:23643898

  3. Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.

    1996-01-01

    Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

  4. DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

    PubMed

    Zheng, Tao; Jiang, Pan; Cao, Bo; Cheng, Qiuxiang; Kong, Lingxin; Zheng, Xiaoqing; Hu, Qinghai; You, Delin

    2016-01-15

    Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.

  5. Leaf ion homeostasis and plasma membrane H(+)-ATPase activity in Vicia faba change after extra calcium and potassium supply under salinity.

    PubMed

    Morgan, Sherif H; Maity, Pooja Jha; Geilfus, Christoph-Martin; Lindberg, Sylvia; Mühling, Karl Hermann

    2014-09-01

    Salt stress in plants impacts apoplastic ion activities and cytosolic ionic homeostasis. The ameliorating effects exerted by calcium or potassium on compartmentation of ions in leaves under salinity are not fully understood. To clarify how calcium or potassium supply could ameliorate ion homeostasis and ATPase activities under salinity, 5 mM CaSO4 or 10 mM K2SO4 were added with, or without, 100 mM NaCl for 7 d and 21 d to Vicia faba grown in hydroponics. The apoplastic pH was detected with Oregon Green dextran dye in intact second-uppermost leaves by microscopy-based ratio imaging. The cytosolic Ca(2+), Na(+), K(+) activities and pH were detected in protoplasts loaded with the acetoxy methyl-esters of Fura-2, SBFI, PBFI and BCECF, respectively, using epi-fluorescence microscopy. Furthermore, total Ca(2+), Na(+), K(+) concentrations and growth parameters were investigated. The ATPase hydrolyzing activity increased with time, but decreased after long salinity treatment. The activity largely increased in calcium-treated plants, but was depressed in potassium-treated plants after 7 d. The calcium supply increased Vmax, and the ATPase activity increased with salinity in a non-competitive way for 7 d and 21 d. The potassium supply instead decreased activity competitively with Na(+), after 21 d of salinity, with different effects on Km and Vmax. The confirmed higher ATPase activity was related with apoplast acidification, cytosol alkalinization and low cytosolic [Na(+)], and thus, might be an explanation why extra calcium improved shoot and leaf growth.

  6. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    PubMed

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  7. Active compounds in Chinese herbs and medicinal animal products which promote blood circulation via inhibition of Na+, K+-ATPase.

    PubMed

    Tzen, Jason Tc; Chen, Ronald Jy; Chung, Tse-Yu; Chen, Yi-Ching; Lin, Nan-Hei

    2010-01-01

    The therapeutic effect of cardiac glycosides for congestive heart failure lies in their reversible inhibition on Na+, K+-ATPase located in human myocardium. Several steroid-like compounds containing a core structure similar to cardiac glycosides have been found in many Chinese herbs and medicinal animal products conventionally used to promote blood circulation. They are putatively responsible for the therapeutic effect of those medicinal products via the same mechanism of inhibiting Na+, K+-ATPase. Inhibitory potency on Na+, K+-ATPase by ginsenosides, one of the identified steroid-like compounds, is significantly affected by sugar attachment that might cause steric hindrance of their binding to Na+, K+-ATPase. Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, substantially inhibit Na+, K+-ATPase. However, their inhibitory potency is abolished when sugar moieties are linked to the C-6 or C-20 position of the steroid-like structure. In contrast, no appreciable contents of steroid-like compounds are found in danshen, a well-known Chinese herb traditionally regarded as an effective medicine promoting blood circulation. Instead, magnesium lithospermate B (MLB), the major soluble ingredient in danshen, is assumed to be responsible for the therapeutic effect by inhibiting Na+, K+-ATPase in a manner comparable to cardiac glycosides. Neuroprotective effects of cardiac glycosides, ginsenosides and MLB against ischemic stroke were accordingly observed in a cortical brain slice-based assay model. Whether the neuroprotection is also triggered by inhibition of Na+, K+-ATPase remains to be investigated. Molecular modeling suggests that cardiac glycosides, ginsenosides and MLB presumably bind to the same extracellular pocket of the Na+, K+-ATPase alpha subunit. PMID:20438664

  8. Active compounds in Chinese herbs and medicinal animal products which promote blood circulation via inhibition of Na+, K+-ATPase.

    PubMed

    Tzen, Jason Tc; Chen, Ronald Jy; Chung, Tse-Yu; Chen, Yi-Ching; Lin, Nan-Hei

    2010-01-01

    The therapeutic effect of cardiac glycosides for congestive heart failure lies in their reversible inhibition on Na+, K+-ATPase located in human myocardium. Several steroid-like compounds containing a core structure similar to cardiac glycosides have been found in many Chinese herbs and medicinal animal products conventionally used to promote blood circulation. They are putatively responsible for the therapeutic effect of those medicinal products via the same mechanism of inhibiting Na+, K+-ATPase. Inhibitory potency on Na+, K+-ATPase by ginsenosides, one of the identified steroid-like compounds, is significantly affected by sugar attachment that might cause steric hindrance of their binding to Na+, K+-ATPase. Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, substantially inhibit Na+, K+-ATPase. However, their inhibitory potency is abolished when sugar moieties are linked to the C-6 or C-20 position of the steroid-like structure. In contrast, no appreciable contents of steroid-like compounds are found in danshen, a well-known Chinese herb traditionally regarded as an effective medicine promoting blood circulation. Instead, magnesium lithospermate B (MLB), the major soluble ingredient in danshen, is assumed to be responsible for the therapeutic effect by inhibiting Na+, K+-ATPase in a manner comparable to cardiac glycosides. Neuroprotective effects of cardiac glycosides, ginsenosides and MLB against ischemic stroke were accordingly observed in a cortical brain slice-based assay model. Whether the neuroprotection is also triggered by inhibition of Na+, K+-ATPase remains to be investigated. Molecular modeling suggests that cardiac glycosides, ginsenosides and MLB presumably bind to the same extracellular pocket of the Na+, K+-ATPase alpha subunit.

  9. In vitro evidence that phytanic acid compromises Na(+),K(+)-ATPase activity and the electron flow through the respiratory chain in brain cortex from young rats.

    PubMed

    Busanello, Estela Natacha Brandt; Viegas, Carolina Maso; Moura, Alana Pimentel; Tonin, Anelise Miotti; Grings, Mateus; Vargas, Carmen R; Wajner, Moacir

    2010-09-17

    Phytanic acid (Phyt) tissue concentrations are increased in Refsum disease and other peroxisomal disorders characterized by neurologic damage and brain abnormalities. The present work investigated the in vitro effects of Phyt, at concentrations found in these peroxisomal disorders, on important parameters of energy metabolism in brain cortex of young rats. The parameters analyzed were CO(2) production from labeled acetate and glucose, the activities of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, as well as of the respiratory chain complexes I-IV, creatine kinase and Na(+),K(+)-ATPase. Our results show that Phyt did not alter citric acid cycle enzyme activities, or CO(2) production from acetate, reflecting no impairment of the functionality of the citric acid cycle. In contrast, respiratory chain activities were reduced at complexes I, II, I-III, II-III and IV. Membrane synaptical Na(+),K(+)-ATPase activity was also reduced by Phyt, with no alteration of creatine kinase activity. Considering the importance of the electron flow through the respiratory chain for brain energy metabolism (oxidative phosphorylation) and of Na(+),K(+)-ATPase activity for maintaining membrane potential necessary for neurotransmission, the data indicate that Phyt impairs brain bioenergetics at the level of energy formation, as well as neurotransmission. It is presumed that Phyt-induced impairment of these important systems may be involved at least in part in the neurological damage found in patients affected by disorders in which brain Phyt concentrations are increased.

  10. Resveratrol, an activator of SIRT1, upregulates sarcoplasmic calcium ATPase and improves cardiac function in diabetic cardiomyopathy

    PubMed Central

    Sulaiman, M.; Matta, M. J.; Sunderesan, N. R.; Periasamy, M.; Gupta, M.

    2010-01-01

    Reduced sarcoplasmic calcium ATPase (SERCA2a) expression has been shown to play a significant role in the cardiac dysfunction in diabetic cardiomyopathy. The mechanism of SERCA2a repression is, however, not known. This study was designed to examine the effect of resveratrol (RSV), a potent activator of SIRT1, on cardiac function and SERCA2a expression in chronic type 1 diabetes. Adult male mice were injected with streptozotocin (STZ) and fed with either a regular diet or a diet enriched with RSV. STZ administration produced progressive decline in cardiac function, associated with markedly reduced SERCA2a and SIRT1 protein levels and increased collagen deposition; RSV treatment to these mice had a tremendous beneficial effect both in terms of improving SERCA2a expression and on cardiac function. In cultured cardiomyocytes, RSV restored SERCA2 promoter activity, which was otherwise highly repressed in high-glucose media. Protective effects of RSV were found to be dependent on its ability to activate Silent information regulator (SIRT) 1. In cardiomyocytes, overexpression of SIRT1 was found sufficient to activate SERCA2 promoter in a dose-dependent manner. In contrast, pretreatment of cardiomyocytes with SIRT1 antagonist, splitomycin, blocked these beneficial effects of RSV. In addition, SIRT1 knockout (+/−) mice were also found to be more sensitive to STZ-induced decline in SERCA2a mRNA. The data demonstrate that, in chronic diabetes, 1) the enzymatic activity of cardiac SIRT1 is reduced, which contributes to reduced expression of SERCA2a and 2) through activation of SIRT1, RSV enhances expression of SERCA2a and improves cardiac function. PMID:20008278

  11. Angiotensin II AT1 receptor stimulates Na+–K+ ATPase activity through a pathway involving PKC-ζ in rat thyroid cells

    PubMed Central

    Marsigliante, S; Muscella, A; Elia, M G; Greco, S; Storelli, C

    2003-01-01

    Angiotensin II (Ang II) receptor subtype 1, AT1, is expressed by the rat thyroid. A relationship between thyroid function and several components of the renin-angiotensin system has also been established, but the Ang II cellular effects in thyrocytes and its transduction signalling remain undefined. The aim of the present paper was to investigate the modulation of the activity of the Na+-K+ ATPase by Ang II and its intracellular transduction pathway in PC-Cl3 cells, an established epithelial cell line derived from rat thyroid. Here we have demonstrated, by RT-PCR analysis, the expression of mRNA for the Ang II AT1 receptor in PC-Cl3 cells; mRNA for the Ang II AT2 receptor was not detected. Ang II was not able to affect the intracellular Ca2+ concentration in fura-2-loaded cells, but it stimulated the translocation from the cytosol to the plasma membrane of atypical protein kinase C-zeta (PKC-ζ) and -iota (PKC-ι) isoforms with subsequent phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1 and 2). Translocated atypical PKCs displayed temporally different activations, the activation of PKC-ζ being the fastest. PC-Cl3 cells stimulated with increasing Ang II concentrations showed dose- and time-dependent activation of the Na+-K+ ATPase activity, which paralleled the PKC-ζ translocation time course. Na+-K+ ATPase activity modulation was dependent on PKC activation since the PKC antagonist staurosporine abolished the stimulatory effect of Ang II. The inhibition of the ERK kinases 1 and 2 (MEK1 and 2) by PD098059 (2′-amino-3′-methoxyflavone) failed to block the effect of Ang II on the Na+-K+ ATPase activity. In conclusion, our results suggest that Ang II modulates Na+-K+ ATPase activity in PC-Cl3 cells through the AT1 receptor via activation of atypical PKC-ζ while the Ang II-activated PKC-ζ appears to have other as yet unknown functions. PMID:12527732

  12. Astrocytes drive upregulation of the multidrug resistance transporter ABCB1 (P-Glycoprotein) in endothelial cells of the blood-brain barrier in mutant superoxide dismutase 1-linked amyotrophic lateral sclerosis.

    PubMed

    Qosa, Hisham; Lichter, Jessica; Sarlo, Mark; Markandaiah, Shashirekha S; McAvoy, Kevin; Richard, Jean-Philippe; Jablonski, Michael R; Maragakis, Nicholas J; Pasinelli, Piera; Trotti, Davide

    2016-08-01

    The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313.

  13. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase

    PubMed Central

    1987-01-01

    The microvillus 110-kD protein-calmodulin complex (designated 110K-CM) shares several properties with all myosins. In addition to its well- defined ATP-dependent binding interaction with F-actin, 110K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K+ and EDTA (0.24 mumol P1/mg per min) or in the presence of Ca++ (0.40 mumol P1/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 mumol P1/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of 110K-CM is also enhanced by the addition of 5-10 microM Ca++, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca++. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of 110K- CM's enzymatic activity in the presence of 5-10 microM Ca++ and actin. 110K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified 110K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the 110K-CM complex is functionally analogous to the mechanoenzyme myosin. PMID:2956266

  14. Photosynthetic control of the plasma membrane H+-ATPase in Vallisneria leaves. I. Regulation of activity during light-induced membrane hyperpolarization.

    PubMed

    Harada, Akiko; Okazaki, Yoshiji; Takagi, Shingo

    2002-04-01

    In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, red, blue, or blue plus far-red light induced a typical membrane hyperpolarization, whereas far-red light alone had little effect. Both N,N'-dicyclohexylcarbodiimide, a potent inhibitor of H+-ATPase, and carbonylcyanide m-chlorophenylhydrazone, an uncoupler, produced a considerable membrane depolarization in the dark-adapted cells and a complete suppression of the light-induced hyperpolarization. Although 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport, did not affect the membrane potential in darkness, it completely inhibited the light-induced membrane hyperpolarization. In vivo illumination of the leaves with red light caused a substantial decrease in the Km for ATP, not only of the vanadate-sensitive ATP-hydrolyzing activity in leaf homogenate, but also of the ATP-dependent H+-transporting activity in plasma membrane (PM) vesicles isolated from the leaves by aqueous polymer two-phase partitioning methods. The effects of red light were negated by the presence of DCMU during illumination. In vivo illumination with far-red light had no effect on the Km for ATP of H+-transporting activity. These results strongly suggest that an electrogenic component in the membrane potential of the mesophyll cell is generated by the PM H+-ATPase, and that photosynthesis-dependent modulation of the enzymatic activity of the PM H+-ATPase is involved in the light-induced membrane hyperpolarization. PMID:11941462

  15. Short- and long-term, salinity-induced modulation of V-ATPase activity in the posterior gills of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae).

    PubMed

    Firmino, Kelly Cristina Silva; Faleiros, Rogério Oliveira; Masui, Douglas Chodi; McNamara, John Campbell; Furriel, Rosa Prazeres Melo

    2011-09-01

    To better understand the biochemical mechanisms underlying anisosmotic extracellular regulation in the freshwater Brachyura, we kinetically characterized the V-ATPase from the posterior gills of Dilocarcinus pagei, acclimated for 10days to salinities up to 21‰. Specific activity was highest in fresh water (26.5±2.1U mg(-1)), decreasing in 5‰ to 21‰, attaining 3-fold less at 15‰. Apparent affinities for ATP and Mg(2+) respectively increased 3.2- and 2-fold at 10‰, suggesting expression of different isoenzymes. In a 240-h time-course study of exposure to 21‰, maximum specific activity decreased 2.5- to 4-fold within 1 to 24h while apparent affinities for ATP and Mg(2+) respectively increased by 12-fold within 24h and 2.4-fold after 1h, unchanged thereafter. K(I) for bafilomycin A(1) decreased 150-fold after 1h, remaining constant up to 120h. This is the first kinetic analysis of V-ATPase specific activity in crustacean gills during salinity acclimation. Our findings indicate active gill Cl(-) uptake by D. pagei in fresh water, and short- and long-term down-regulation of V-ATPase-driven ion uptake processes during salinity exposure, aiding in comprehension of the biochemical adaptations underpinning the establishment of the Brachyura in fresh water.

  16. Effects of exposure to sublethal propiconazole on the antioxidant defense system and Na+-K+-ATPase activity in brain of rainbow trout, Oncorhynchus mykiss.

    PubMed

    Li, Zhi-Hua; Zlabek, Vladimir; Grabic, Roman; Li, Ping; Machova, Jana; Velisek, Josef; Randak, Tomas

    2010-07-01

    Propiconazole (PCZ), a triazole fungicide, is widely present in the aquatic environment, but little is known regarding its chronic toxicity in the fish brain. This study assessed the effects of long-term exposure to PCZ on the antioxidant defense system and Na(+)-K(+)-ATPase activity of rainbow trout brain. Fish were exposed to sublethal concentrations of PCZ (0.2, 50, and 500 microg/l) for 7, 20, and 30 days, respectively. Oxidative stress indices (reactive oxygen species, lipid peroxidation, and carbonyl protein) and antioxidant parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and reduced glutathione) were measured, as well as Na(+)-K(+)-ATPase activity. Adaptive responses to PCZ-induced stress were observed at 7 days. With prolonged exposure, significantly higher levels of oxidative indices were indicative of oxidative stress, as also were the significant inhibition of antioxidant enzyme activity and reduced glutathione content. Na(+)-K(+)-ATPase activity was significantly inhibited after prolonged exposure. Chemometrics of all parameters by principal component analysis, enabled the separation of sampled individuals into four groups with 93.39% of total accumulated variance. A low level of oxidative stress can induce the adaptive responses of the antioxidant defense system, while prolonged exposure to PCZ may lead to serious oxidative damage in fish brain. We suggest that selected biochemical markers in fish brain could be used as potential biomarkers for monitoring residual fungicides present in the aquatic environments.

  17. Low frequency and low intensity pulsed electromagnetic field exerts its antiinflammatory effect through restoration of plasma membrane calcium ATPase activity.

    PubMed

    Selvam, Ramasamy; Ganesan, Kalaivani; Narayana Raju, K V S; Gangadharan, Akkalayi Chandrapuram; Manohar, Bhakthavatchalam Murali; Puvanakrishnan, Rengarajulu

    2007-06-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting 1% of the population worldwide. Pulsed electromagnetic field (PEMF) has a number of well-documented physiological effects on cells and tissues including antiinflammatory effect. This study aims to explore the antiinflammatory effect of PEMF and its possible mechanism of action in amelioration of adjuvant induced arthritis (AIA). Arthritis was induced by a single intradermal injection of heat killed Mycobacterium tuberculosis at a concentration of 500 microg in 0.1 ml of paraffin oil into the right hind paw of rats. The arthritic animals showed a biphasic response regarding changes in the paw edema volume. During the chronic phase of the disease, arthritic animals showed an elevated level of lipid peroxides and depletion of antioxidant enzymes with significant radiological and histological changes. Besides, plasma membrane Ca(2+) ATPase (PMCA) activity was inhibited while intracellular Ca(2+) level as well as prostaglandin E(2) levels was noticed to be elevated in blood lymphocytes of arthritic rats. Exposure of arthritic rats to PEMF at 5 Hzx4 microT x 90 min, produced significant antiexudative effect resulting in the restoration of the altered parameters. The antiinflammatory effect could be partially mediated through the stabilizing action of PEMF on membranes as reflected by the restoration of PMCA and intracellular Ca(2+) levels in blood lymphocytes subsequently inhibiting PGE(2) biosynthesis. The results of this study indicated that PEMF could be developed as a potential therapy for RA in human beings.

  18. Direct measurement of metal-ion chelation in the active site of the AAA+ ATPase magnesium chelatase.

    PubMed

    Viney, Joanne; Davison, Paul A; Hunter, C Neil; Reid, James D

    2007-11-01

    Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis. This complex enzyme has at least three substrates and couples ATP hydrolysis to the insertion of Mg2+ into protoporphyrin IX. We directly observed metal-ion chelation fluorometrically, providing the first data describing the on-enzyme reaction. We describe the transient-state kinetics of magnesium chelatase with direct observation of the evolution of an enzyme-product complex EMgDIX. We demonstrate that MgATP2- binding occurs after the rate-determining step. As nucleotide hydrolysis is essential for the overall reaction this must also occur after the rate-determining step. This provides the first evidence for the synchronization of the ATPase and chelatase pathways and suggests a mechanism where nucleotide binding acts to clamp the chelatase in a product complex. Comparison of rate constants for the slow step in the reaction with further transient kinetics under conditions where multiple turnovers can occur reveals that an additional activation step is required to explain the behavior of magnesium chelatase. These data provide a new view of the sequence of events occurring in the reaction catalyzed by magnesium chelatase.

  19. The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity

    PubMed Central

    Tameling, Wladimir I. L.; Elzinga, Sandra D. J.; Darmin, Patricia S.; Vossen, Jack H.; Takken, Frank L. W.; Haring, Michel A.; Cornelissen, Ben J. C.

    2002-01-01

    Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP. PMID:12417711

  20. Mg2+-dependent ATPase activity in cardiac myofibrils from the insulin-resistant JCR:LA-cp rat.

    PubMed

    Misra, T; Russell, J C; Clark, T A; Pierce, G N

    2001-01-01

    There is a great deal of information presently available documenting a cardiomyopathic condition in insulin-deficient models of diabetes. Less information is available documenting a similar status in non insulin-dependent models of diabetes. We have studied the functional integrity of the myofibrils isolated from hearts of JCR:LA rats. The JCR:LA rat is hyperinsulinemic, hyperlipidemic, glucose intolerant and obese. As such, it carries many of the characteristics found in humans with non insulin-dependent diabetes mellitus. These animals also have many indications of heart disease. However, it is not clear if the hearts suffer from vascular complications or are cardiomyopathic in nature. We examined Mg2+-dependent myofibrillar ATPase in hearts of JCR:LA-cp/cp rats and their corresponding control animals (+/?) and found no significant differences (P> 0.05). This is in striking contrast to the depression in this activity exhibited by cardiac myofibrils isolated from insulin-deficient models of diabetes. Our data demonstrate that myofibrillar functional integrity is normal in JCR:LA-cp rats and suggest that these hearts are not in a cardiomyopathic state. Insulin status may be critical in generating a cardiomyopathic condition in diabetes.

  1. Uncoupling of attenuated myo-(3H)inositol uptake and dysfunction in Na(+)-K(+)-ATPase pumping activity in hypergalactosemic cultured bovine lens epithelial cells

    SciTech Connect

    Cammarata, P.R.; Tse, D.; Yorio, T. )

    1991-06-01

    Attenuation of both the active transport of myo-inositol and Na(+)-K(+)-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagle's minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na(+)-K(+)-ATPase and myo-inositol uptake. The Na(+)-K(+)-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-(3H)inositol was lowered after 20 h of incubation in galactose and remained below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-(3H)inositol uptake. No significant difference in the rates of passive efflux of myo-(3H)inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil.2+ reversible and occurred independently of changes in Na(+)-K(+)-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

  2. afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2).

    PubMed

    Lee, Ping-Chin; Umeyama, Takashi; Horinouchi, Sueharu

    2002-03-01

    AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3' to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the DeltaafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation of afsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues.

  3. Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids.

    PubMed

    Hamaguchi, Ryohei; Haginaka, Jun; Tanimoto, Toshiko; Kuroda, Yukihiro

    2014-02-01

    Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green-dextran-tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.

  4. Marked inhibition of Na+, K(+)- ATPase activity and the respiratory chain by phytanic acid in cerebellum from young rats: possible underlying mechanisms of cerebellar ataxia in Refsum disease.

    PubMed

    Busanello, Estela Natacha Brandt; Zanatta, Ângela; Tonin, Anelise Miotti; Viegas, Carolina Maso; Vargas, Carmen Regla; Leipnitz, Guilhian; Ribeiro, César Augusto João; Wajner, Moacir

    2013-02-01

    Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.

  5. A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica.

    PubMed

    Singh, Meetali; Shah, Varun; Tatu, Utpal

    2014-04-17

    Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with Kd values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12×10(-4) min(-1) μM(-1). Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.

  6. Basic FGF attenuates amyloid beta-peptide-induced oxidative stress, mitochondrial dysfunction, and impairment of Na+/K+-ATPase activity in hippocampal neurons.

    PubMed

    Mark, R J; Keller, J N; Kruman, I; Mattson, M P

    1997-05-01

    Basic fibroblast growth factor (bFGF) exhibits trophic activity for many populations of neurons in the brain, and can protect those neurons against excitotoxic, metabolic and oxidative insults. In Alzheimer's disease (AD), amyloid beta-peptide (A beta) fibrils accumulate in plaques which are associated with degenerating neurons. A beta can be neurotoxic by a mechanism that appears to involve induction of oxidative stress and disruption of calcium homeostasis. Plaques in AD brain contain high levels of bFGF suggesting a possible modulatory role for bFGF in the neurodegenerative process. We now report that bFGF can protect cultured hippocampal neurons against A beta25-35 toxicity by a mechanism that involves suppression of reactive oxygen species (ROS) accumulation and maintenance of Na+/K+-ATPase activity. A beta25-35 induced lipid peroxidation, accumulation of H2O2, mitochondrial ROS accumulation, and a decrease in mitochondrial transmembrane potential; each of these effects of A beta25-35 was abrogated in cultures pre-treated with bFGF. Na+/K+-ATPase activity was significantly reduced following exposure to A beta25-35 in control cultures, but not in cultures pre-treated with bFGF. bFGF did not protect neurons from death induced by ouabain (a specific inhibitor of the Na+/K+-ATPase) or 4-hydroxynonenal (an aldehydic product of lipid peroxidation) consistent with a site of action of bFGF prior to induction of oxidative stress and impairment of ion-motive ATPases. By suppressing accumulation of oxyradicals, bFGF may slow A beta-induced neurodegenerative cascades. PMID:9187334

  7. The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

    PubMed Central

    Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K.; Bindics, János; Ślusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L.; Tamaru, Hisashi

    2014-01-01

    Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. PMID:25344531

  8. Mechanism of noradrenaline-induced stimulation of Na-K ATPase activity in the rat brain: implications on REM sleep deprivation-induced increase in brain excitability.

    PubMed

    Mallick, Birendra Nath; Singh, Sudhuman; Singh, Abhishek

    2010-03-01

    Rapid eye movement (REM) sleep is a unique phenomenon expressed in all higher forms of animals. Its quantity varies in different species and with ageing; it is also affected in several psycho-somatic disorders. Several lines of studies showed that after REM sleep loss, the levels of noradrenaline (NA) increase in the brain. The NA in the brain modulates neuronal Na-K ATPase activity, which helps maintaining the brain excitability status. The detailed mechanism of increase in NA level after REM sleep loss and the effect of NA on stimulation of Na-K ATPase in the neurons have been discussed. The findings have been reviewed and discussed with an aim to understand the role of REM sleep in maintaining brain excitability status.

  9. Glucostatic regulation of (+)-(/sup 3/H)amphetamine binding in the hypothalamus: correlation with Na/sup +/, K/sup +/-ATPase activity

    SciTech Connect

    Angel, I.; Hauger, R.L.; Luu, M.D.; Giblin, B.; Skolnick, P.; Paul, S.M.

    1985-09-01

    Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37/sup 0/C) resulted in a time-dependent decrease in specific (+)-(/sup 3/H)amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-(/sup 3/H)amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-(/sup 3/H)amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-(/sup 3/H)amphetamine binding, suggesting the involvement of Na/sup +/, K/sup +/-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na/sup +/,K/sup +/-ATPase activity and the number of specific high-affinity binding sites for (/sup 3/H)ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-(/sup 3/H)amphetamine and (/sup 3/H)ouabain binding. These data suggest that the (+)-(/sup 3/H)amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na/sup +/,K/sup +/-ATPase activity, and the latter may be involved in the glucostatic regulation of appetite.

  10. BAP, a mammalian BiP-associated protein, is a nucleotide exchange factor that regulates the ATPase activity of BiP.

    PubMed

    Chung, Kyung Tae; Shen, Ying; Hendershot, Linda M

    2002-12-01

    We identified a mammalian BiP-associated protein, BAP, using a yeast two-hybrid screen that shared low homology with yeast Sls1p/Sil1p and mammalian HspBP1, both of which regulate the ATPase activity of their Hsp70 partner. BAP encoded an approximately 54-kDa protein with an N-terminal endoplasmic reticulum (ER) targeting sequence, two sites of N-linked glycosylation, and a C-terminal ER retention sequence. Immunofluorescence staining demonstrated that BAP co-localized with GRP94 in the endoplasmic reticulum. BAP was ubiquitously expressed but showed the highest levels of expression in secretory organ tissues, a pattern similar to that observed with BiP. BAP binding was affected by the conformation of the ATPase domain of BiP based on in vivo binding studies with BiP mutants. BAP stimulated the ATPase activity of BiP when added alone or together with the ER DnaJ protein, ERdj4, by promoting the release of ADP from BiP. Together, these data demonstrate that BAP serves as a nucleotide exchange factor for BiP and provide insights into the mechanisms that control protein folding in the mammalian ER.

  11. ABCB1, ABCC2, SCN1A, SCN2A, GABRA1 gene polymorphisms and drug resistant epilepsy in the Chinese Han population.

    PubMed

    Zhou, Luo; Cao, Yuze; Long, Hongyu; Long, Lili; Xu, Lin; Liu, Zhaoqian; Zhang, Ying; Xiao, Bo

    2015-06-01

    Drug resistance is common in epilepsy despite multiple available medications. Single nucleotide polymorphisms (SNP) may influence drug efficacy in epilepsy. We therefore aimed to clarify the association between polymorphisms of several controversial SNP loci and drug resistance in Chinese Han epilepsy patients from central China. Among all the 391 recruited subjects, 235 and 156 patients were classified into a drug responsive and resistant group, respectively, according to the definition of drug resistance proposed by the International League Against Epilepsy. The candidate SNP loci, including ATP-binding cassette (ABC) subfamily gene ABCB1 rs2032582 and rs1045642; ABC subfamily gene ABCC2 rs717620 and rs2273697; sodium channel subunit gene SCN1A rs3812718, SCN2A rs2304016; γ-amino butyric acid type A (GABAA) receptor subunit subtype gene GABRA1 rs2279020 were genotyped following the Illumina protocols. There were no significant differences in allelic or genotypic frequencies between the drug responsive and resistant patients. The polymorphisms of the above SNP loci may not be associated with drug resistance of epilepsy in the Chinese Han population.

  12. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  13. Na⁺,K⁺-ATPase activity in the posterior gills of the blue crab, Callinectes ornatus (Decapoda, Brachyura): modulation of ATP hydrolysis by the biogenic amines spermidine and spermine.

    PubMed

    Garçon, Daniela P; Lucena, Malson N; França, Juliana L; McNamara, John C; Fontes, Carlos F L; Leone, Francisco A

    2011-11-01

    We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis-Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.

  14. Gill-specific (Na(+), K(+))-ATPase activity and α-subunit mRNA expression during low-salinity acclimation of the ornate blue crab Callinectes ornatus (Decapoda, Brachyura).

    PubMed

    Leone, Francisco A; Garçon, Daniela P; Lucena, Malson N; Faleiros, Rogério O; Azevedo, Sergio V; Pinto, Marcelo R; McNamara, John C

    2015-08-01

    We evaluate (Na(+), K(+))-ATPase activity, and protein and gene expression of the α-subunit in posterior gills 6 and 7 of Callinectes ornatus, a euryhaline crab, during a 10-day acclimation period from seawater (33‰ S) to low salinity (21‰ S). (Na(+), K(+))-ATPase activity decreased within 1h after transfer to 21‰ S, values recovering by 24h and attaining a maximum of ≈180 nmol Pi min(-1) mg(-1) after 10 days (≈2.5-fold increase). (Na(+), K(+))-ATPase activity is ≈1.5-fold greater in gill 6 than in gill 7, independently of salinity. Relative expression of (Na(+), K(+))-ATPase α-subunit mRNA increased in both gills within 1- to 2-h exposure to low salinity, reaching an ≈8-fold maximum after 24-h exposure, decreasing slightly by 10 days acclimation to low salinity. This increase in α-subunit mRNA expression may underpin the increased (Na(+), K(+))-ATPase activity seen after 10 days acclimation to low salinity. Enzyme affinity for ATP was greater in gill 6 than in gill 7, in contrast to ouabain affinity that was greater in gill 7. Western blotting analysis identified a single immunoreactive band against the (Na(+), K(+))-ATPase α-subunit with an Mr of ≈105 kDa, independently of gill number and low salinity acclimation. Despite these differences, gills 6 and 7 appear to perform similar functions in salt uptake from the dilute medium. The partial cDNA sequence obtained for the gill (Na(+), K(+))-ATPase of C. ornatus (GenBank deposit KF056804) showed 97 to 91% identities with similar sequences from other portunid crab gills. The regulation of gill (Na(+), K(+))-ATPase activity during acclimation to low salinity is discussed.

  15. Kinetic analysis of gill (Na⁺,K⁺)-ATPase activity in selected ontogenetic stages of the Amazon River shrimp, Macrobrachium amazonicum (Decapoda, Palaemonidae): interactions at ATP- and cation-binding sites.

    PubMed

    Leone, Francisco Assis; Masui, Douglas Chodi; de Souza Bezerra, Thais Milena; Garçon, Daniela Pereira; Valenti, Wagner Cotroni; Augusto, Alessandra Silva; McNamara, John Campbell

    2012-04-01

    We investigated modulation by ATP, Mg²⁺, Na⁺, K⁺ and NH₄⁺ and inhibition by ouabain of (Na⁺,K⁺)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, Macrobrachium amazonicum. (Na⁺,K⁺)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP (K(M) = 0.09 ± 0.01 mmol L⁻¹) of the decapodid III (Na⁺,K⁺)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na⁺,K⁺-ATPase activity by K⁺ also revealed a threefold greater affinity for K⁺ (K₀.₅ = 0.91 ± 0.04 mmol L⁻¹) in decapodid III than in other stages; NH₄⁺ had no modulatory effect. The affinity for Na⁺ (K₀.₅ = 13.2 ± 0.6 mmol L⁻¹) of zoea I (Na⁺,K⁺)-ATPase was fourfold less than other stages. Modulation by Na⁺, Mg²⁺ and NH₄⁺ obeyed cooperative kinetics, while K⁺ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg²⁺ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg²⁺-stimulated ATPases other than (Na⁺,K⁺)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na⁺-ATPase may be involved in the ontogeny of osmoregulation in larval M. amazonicum. The NH₄⁺-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.

  16. Physiological Studies on Pea Tendrils. III. ATPase Activity and Contractility Associated with Coiling

    PubMed Central

    Jaffe, M. J.; Galston, A. W.

    1967-01-01

    Extracts of the tendrils of Pisum sativum, Var. Alaska, exhibit adenosine triphosphatase activity which is inversely proportional to the amount the tendrils have coiled. The specific viscosity of the extract decreases when ATP is added. This evidence indicates a possible role of a contractile adenosine triphosphatase in coiling. PMID:16656580

  17. Structural lipid changes and Na(+)/K(+)-ATPase activity of gill cells' basolateral membranes during saltwater acclimation in sea lamprey (Petromyzon marinus, L.) juveniles.

    PubMed

    Lança, Maria João; Machado, Maria; Ferreira, Ana Filipa; Quintella, Bernardo Ruivo; de Almeida, Pedro Raposo

    2015-11-01

    Seawater acclimation is a critical period for anadromous species and a process yet to be understood in lampreys. Considering that changes in lipid composition of the gill cells' basolateral membranes may disrupt the major transporter Na(+)K(+)-ATPase, the goal of this study was to detect changes at this level during juvenile sea lamprey seawater acclimation. The results showed that saltwater acclimation has a direct effect on the fatty acid composition of gill cells basolateral membrane's phospholipids. When h