title: [Construction of HEK293 cell lines expressing hCysLT₂ receptor and its application in screening of antagonists].
abstract: To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity.
  
  
  
  The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 μg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity.
  
  
  
  After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 μmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 μmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 μmol/L and 7.5 μmol/L.
  
  
  
  HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.
authors.affiliation: Department of Pharmacology, College of Medicine, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ka-Na
authors.initials: KN
authors.lastname: Lin
authors.forename: Xin-Xin
authors.initials: XX
authors.lastname: Wang
authors.forename: Xue-Qin
authors.initials: XQ
authors.lastname: Huang
authors.forename: Bei-Lei
authors.initials: BL
authors.lastname: Cai
authors.forename: San-Hua
authors.initials: SH
authors.lastname: Fang
authors.forename: Yun-Bi
authors.initials: YB
authors.lastname: Lu
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Zhang
authors.forename: Er-Qing
authors.initials: EQ
authors.lastname: Wei
mesh_terms: D004353:Drug Evaluation, Preclinical; D057809:HEK293 Cells; D006801:Humans; D020024:Leukotriene Antagonists; D018077:Receptors, Leukotriene / Q000235:genetics*; D014162:Transfection
chemical_list: D020024:Leukotriene Antagonists; D018077:Receptors, Leukotriene