1.Na(+)/H(+) exchanger 1 expression and its effect on apoptosis in K562 and HL-60 cells with DNA damage.
Hua-Wen LI ; Li-Hong WANG ; Jian WANG ; Guo-Qiang CHANG ; Wei-Na JIN ; Ya-Ni LIN ; Wei GAO ; Ruo-Jun WANG ; Li MA ; Tian-Xiang PANG
Journal of Experimental Hematology 2011;19(4):857-860
This study was aimed to investigate the expression of Na(+)/H(+) exchanger 1 (NHE1) in K562 and HL-60 cells undergoing DNA damage induced by etoposide and to elucidate the regulating mechanism. Real-time quantitative PCR (RQ-PCR) and Western blot methods were used to determine the expression of NHE1 in K562 cells after the treating with etoposide. Meanwhile, the flow cytometry was used to detect the apoptosis of leukemic cells. The luciferase reporter vector containing NHE1 promoter was constructed to measure relative luciferase activity after treating with different etoposide concentrations. The results showed that the mRNA and protein of NHE1 increased in accordance with apoptosis ratio in HL-60 cells after treated with etoposide (p < 0.05), but no such obvious increase in K562 cells. Treatment with NHE1 specific inhibitor could block etoposide induced alkalization and reduce the apoptosis ratio of HL-60 cells. The expression pattern and apoptosis alteration was not similar in K562 cells. Relative luciferase activity of reporter vector containing NHE1 promoter however increased in K562 cells after treated with etoposide. It is concluded that the expression of NHE1 is up-regulated in the process of apoptosis of HL-60 cells induced by etoposide and depends on the pHi increasing caused by NHE1 up-regulation which is not found in K562 cells although the transcriptional activity increased.
Apoptosis
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Cation Transport Proteins
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metabolism
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DNA Damage
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Etoposide
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HL-60 Cells
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Humans
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K562 Cells
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Promoter Regions, Genetic
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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metabolism
2.Inhibition of NHE1 promotes hypoxia-induced differentiation of K562 leukemic cells.
Wei-Na JIN ; Jian WANG ; Guo-Qiang CHANG ; Ya-Ni LIN ; Li-Hong WANG ; Hua-Wen LI ; Wei GAO ; Qing-Hua LI ; Tian-Xiang PANG
Journal of Experimental Hematology 2011;19(3):661-665
This study was purposed to investigate the effect of hypoxia microenvironment on K562 leukemic cell differentiation, and characteristics of NHE1 involvement in this process. The K562 cells were treated with hypoxia-mimical agent CoCl₂ or under actual hypoxia culture, and the specific NHE1 inhibitor Cariporide was used to inhibit NHE1 activity. The fluorescent probe BCECF was used for pH(i) measurements. Gene expression was analyzed by RT-PCR. The morphological characteristics was determined by Wright's staining. Signaling pathways were detected by Western blot using phosphospecific antibodies. The results indicated that the hypoxia or mimetic hypoxia favored K562 cells differentiation with up-regulation of C/EBPα. Moreover, treatment with Cariporide under hypoxia synergistically enhanced leukemia cell differentiation. Treatment with Cariporide increased levels of phosphorylated ERK5 and P38 mitogen-activated protein kinase (MAPK). It is concluded that the hypoxia or mimetic hypoxia can induce the differentiation of K562 cells, the inhibition of NHE1 activity can promote the hypoxia-induced K562 cell differentiation. The enhancement of hypoxia-induced K562 differentiation by Cariporide via MAPK signal pathway suggests a possible therapeutic target of NHE1 under hypoxia microenvironment in the treatment of leukemias.
Cation Transport Proteins
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metabolism
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Cell Differentiation
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Cell Hypoxia
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Humans
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K562 Cells
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MAP Kinase Signaling System
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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metabolism
3.Role of Na(+)/H(+) exchanger 1 in apoptosis of HL-60 cells induced by etoposide and its mechanism.
Yan YAN ; Hua-Wen LI ; Qing-Hua LI ; Rong-Hua HU ; Tian-Xiang PANG
Journal of Experimental Hematology 2010;18(3):612-616
This study was aimed to investigate the role of Na(+)/H(+) exchanger 1 (NHE1) in apoptosis of HL-60 cells induced by etoposide. Real-time quantitative PCR (RQ-PCR) and Western blot methods were used to determine the expression of NHE1 in HL-60 cells after the treatment with etoposide. Meanwhile, laser scanning confocal microscopy was used to test the intracellular pH (pHi) of HL-60 cells. Cell apoptosis was measured by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The results showed that etoposide induced cell apoptosis after treatment for 24 hours. The expression level of NHE1 mRNA increased by 2.848 +/- 0.886 times after treatment with etoposide for 12 hours (p < 0.01), and the expression of NHE1 protein was also up-regulated (p < 0.01). The pHi of HL-60 cells increased from 7.11 to 7.46 after treatment with etoposide for 24 hours. Treatment with cariporide could block etoposide-induced alkalinisation and enhance the apoptosis HL-60 cells. It is concluded that the expression of NHE1 is up-regulated in process of apoptosis of HL-60 cells induced by etoposide and the apoptosis depends on the pH increase caused by NHE1 higher expression.
Apoptosis
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drug effects
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Cation Transport Proteins
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genetics
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metabolism
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DNA Fragmentation
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Etoposide
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pharmacology
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Gene Expression Regulation, Leukemic
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HL-60 Cells
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Humans
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RNA, Messenger
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genetics
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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genetics
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metabolism
4.Expression of Na+-H+ exchanger 1 in human gastric carcinoma tissue and its clinical significance.
Yan-yang NIU ; Pei-wu YU ; Bo TANG ; Yan SHI ; Ying-xue HAO
Chinese Journal of Gastrointestinal Surgery 2010;13(8):604-607
OBJECTIVETo determine the expression of Na+/H+ exchanger 1(NHE1) in human gastric carcinoma tissue and to investigate the association between NHE1 expression and clinicopathological characteristics.
METHODSThe expressions of NHE1 mRNA and protein were detected in both gastric carcinoma tissue (n=60) and adjacent gastric mucosa tissue (n=30) by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The association between the expression and the clinicopathological characteristics was analyzed.
RESULTSThe relative expression levels of NHE1 mRNA and protein in gastric carcinoma tissue were 0.786+/-0.291 and 1.442+/-0.175, which were significantly higher than those in adjacent gastric mucosa tissue (0.369+/-0.052 and 0.348+/-0.029) (P<0.01). The expression of NHE1 mRNA was positively correlated with NHE1 protein in the gastric carcinoma tissue (r=0.264, P<0.05). The expressions of NHE1 mRNA and protein were associated with the depth of invasion, lymph node metastasis, and TNM staging (P<0.05). However, no statistical difference was found in age, gender, and tumor differentiation (P>0.05).
CONCLUSIONThe expression levels of NHE1 mRNA and protein are significantly up-regulated in gastric carcinoma tissue, which may be involved in the development of gastric carcinoma.
Adult ; Aged ; Carcinoma ; metabolism ; pathology ; Cation Transport Proteins ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; RNA, Messenger ; metabolism ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; metabolism ; Stomach ; metabolism ; pathology ; Stomach Neoplasms ; metabolism ; pathology
5.Effect of NHE1 on stem cell differentiation into cardiomyocytes.
Lei LEI ; Lin DOU ; Long YAN ; Zhongying DOU ; Huayan WANG
Chinese Journal of Biotechnology 2008;24(10):1790-1795
Sodium/proton exchanger 1 (NHE1) plays an important role in the cardiomyocyte development. To study the effect of NHE1 activity in stem cells differentiation into cardiomyocytes, we treated P19 stem cells with dimethyl sulfoxide (DMSO) to initiate cardiomyocyte differentiation. In separate experiments, P19 cells were incubated with NHE1 specific inhibitor EMD87580 during the DMSO induction. The formed embryoid bodies (EBs) were detected with cell morphology detection, immunohistochemisty staining and RT-PCR analysis of expression of cardio-specific gene markers. Results showed that P19 cells were able to differentiate into cardiomyocytes and form the beating cell clusters. However, when cells treated with NHE1 inhibitor EMD87580, they could still form the EBs and proliferate when cell clusters adhered on the culture plate, but cells were unable to differentiate. This observation indicates that inhibition of NHE1 activity affected P19 stem cells differentiating into cardiomyocytes.
Animals
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Cation Transport Proteins
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antagonists & inhibitors
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Cell Differentiation
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drug effects
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Cell Line
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Culture Media
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Dimethyl Sulfoxide
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pharmacology
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Guanidines
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pharmacology
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Mice
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Myocytes, Cardiac
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cytology
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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antagonists & inhibitors
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Stem Cells
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cytology
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drug effects
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Sulfones
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pharmacology
6.Effects of sodium hydrogen exchanger-1 hammerhead ribozyme on proliferation of pulmonary artery smooth muscle cells in rats in vitro.
Wei YAO ; Jun-Yu LU ; Gui-Sheng QIAN ; Xiao-Jing YANG
Chinese Journal of Applied Physiology 2003;19(1):39-42
AIMTo explore the effects of sodium hydrogen exchanger (NHE-1) specific hammerhead ribozyme on the expression and activity of NHE-1 and pHi in pulmonary artery smooth muscle cells (PASMCs) in rats, and its role in PASMCs proliferation.
METHODSAccording to the secondary structure of NHE-1 mRNA in rats, NHE-1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and NHE-1 hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 60 microg/ml G418. Then, the expression of NHE-1 mRNA was detected by RT-PCR, intracellular pH was measured with fluorescent probe-BCECF, 22Na and 3H-TdR incorporation were determined respectively.
RESULTSCompared with the cells transfected with pLXSN and non-transfected cells, NHE-1 mRNA, pHi value, 3H-TdR and 22Na incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non-transfected group.
CONCLUSIONNHE-1 hammerhead ribozyme can cleave the target RNA specifically, reduce the expression of NHE-1 mRNA, induce intracellular acidosis and consequently prohibit the proliferation of PASMCs.
Animals ; Cell Proliferation ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; cytology ; Pulmonary Artery ; cytology ; pathology ; RNA, Catalytic ; genetics ; RNA, Messenger ; genetics ; Rats ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; metabolism ; Transfection
7.Inhibition of NHE1 down-regulates IL-8 expression and enhances p38 phosphorylation.
Wei GAO ; Yu-Juan ZHANG ; Hai-Rui ZHANG ; Wei-Na JIN ; Guo-Qiang CHANG ; Hong-Ju ZHANG ; Li MA ; Ya-Ni LIN ; Qing-Hua LI ; Rong-Xin RU ; Tian-Xiang PANG
Journal of Experimental Hematology 2013;21(1):45-48
This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins
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antagonists & inhibitors
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Down-Regulation
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Guanidines
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pharmacology
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Humans
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Imidazoles
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pharmacology
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Interleukin-8
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metabolism
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K562 Cells
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Phosphorylation
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drug effects
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Pyridines
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pharmacology
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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antagonists & inhibitors
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Sulfones
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pharmacology
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p38 Mitogen-Activated Protein Kinases
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metabolism
8.Increasing sensitivity of leukemia cells to imatinib by inhibiting NHE1 and p38MAPK signaling pathway.
Rong-Hua HU ; Wei-Na JIN ; Guo-Qiang CHANG ; Ya-Ni LIN ; Jian WANG ; Yong-Xin RU ; Qing-Hua LI ; Tian-Xiang PANG
Journal of Experimental Hematology 2012;20(6):1341-1345
This study was aimed to investigate whether the inhibition of NHE1 activity and intracellular acidification can reverse resistance of leukemia cells to the imatinib and to explore downstream signal molecule networks of BCR/ABL in the cells of chronic myelocytic leukemia (CML) patients. The mRNA and protein expression of P-glycoprotein (Pgp) and the drug accumulation were assayed after acidifying the primary leukemia cells of patients or K562/DOX and K562/G01 cells. The effects of intracellular acidification of primary leukemia cells on the phosphorylation level changes of ERK1/2 and p38 MAPK were analyzed by Western blot. The results showed that the intracellular concentration of drugs in the advanced patients increased and the sensitivity of K562/DOX and K562/G01 cells to imatinib was enhanced after intracellular acidification or treatment with NHE1 inhibitor cariporide. With downregulation of intracellular pH, the phosphorylation of p38 MAPK decreased in advanced patients and the phosphorylation of ERK1/2 increased within 3 min and then decreased after 30 min. SB203580, the specific inhibitor of p38 MAPK, displayed a synergistic effect with the inhibitor of NHE1 to downregulate the mRNA and protein expression of Pgp. It is concluded that the inhibiton of NHE1 can significantly decrease the protein expression of Pgp in K562/DOX and K562/G01 cells, increase the accumulation of Rhodamine123 and doxorubicin in the cells of advanced patients and enhance the sensitivity of cells to imatinib in which the p38 MAPK signal transduction pathways involves.
ATP-Binding Cassette, Sub-Family B, Member 1
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metabolism
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Benzamides
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pharmacology
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Cation Transport Proteins
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antagonists & inhibitors
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metabolism
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Drug Resistance, Neoplasm
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drug effects
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Enzyme Inhibitors
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pharmacology
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Gene Expression Regulation, Leukemic
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Humans
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Imatinib Mesylate
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Imidazoles
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pharmacology
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K562 Cells
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MAP Kinase Signaling System
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Piperazines
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pharmacology
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Pyridines
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pharmacology
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Pyrimidines
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pharmacology
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Sodium-Hydrogen Exchanger 1
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Sodium-Hydrogen Exchangers
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antagonists & inhibitors
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metabolism
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p38 Mitogen-Activated Protein Kinases
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antagonists & inhibitors
;
metabolism