title: [Roles of STIM2 and TRPC3 in the CaR mediated Ca2+ entry and NO generation in human umbilical vein endothelial cells].
abstract: OBJECTIVE
  To study the roles of stromal interaction molecule 2 (STIM2) and transient receptor potential canonical 3 (TRPC3) in extracellular Ca(2+)-sensing receptor (CaR)-induced extracellular Ca2+ influx and the production of nitric oxide (NO) in human umbilical vein endothelial cells (HUVEC).
  
  
  METHODS
  (1) The interaction of STIM2 and TRPC3 was determined using the immunofluorescence technique. (2) The expressions of STIM2 and TRPC3 genes were silenced in HUVEC by transfection constructed STIM2 and TRPC3 RNA interference plasmids. The interference efficiency of STIM2, TRPC3 protein and mRNA levels were determined by Western blot and real time RT-PCR, respectively. (3) The second to fifth passage of HUVEC were divided into: STIM2-002 short hairpin RNA (STIM2-002 shRNA ) + spermine + Ca2+ group and TRPC3-004 short hairpin RNA (TRPC3-004 shRNA ) + spermine + Ca2+ group; control group (spermine + Ca2+ group) and vehicle+ spermine + Ca2+ group. The four groups of cells were incubated with CaR agonist spermine, the intracellular Ca2+ concentration ([Ca2+]i) was detected using the fluorescence Ca2+ indicator Fura-2/AM, and the production of NO was determined by DAF-FM (NO fluorescent probe) of each group in HUVEC.
  
  
  RESULTS
  (1) Immunofluorescence technique results showed that STIM2 and TRPC3 proteinswere present in the cytoplasm of HUVEC. (2) The results of transfection constructed STIM2 and TRPC3 RNA interference plasmids demonstrated that shRNA targeted to the STIM2 and TRPC3 genes decreased STIM2 and TRPC3 mRNA levels by 88.2% and 74.0%, respectively (P < 0.05), simultaneously, the STIM2 and TRPC3 protein levels were decreased by 79.9% and 71.8%, respectively (P < 0.05). (3) Compared with spermine + Ca2+ group, the [Ca2+]i and the net NO fluorescence intensity of spermine + Ca(2+) + ShSTIM2-002 group, spermine + Ca(2+) + ShTRPC3-004 group and spermine + Ca2+ Vehicle group were not changed (P > 0.05).
  
  
  CONCLUSIONS
  STIM2 and TRPC3 do not participate in CaR-mediated Ca2+ influx and NO production individually.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Zhong
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: La-Mei
authors.initials: LM
authors.lastname: Wang
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Pang
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Sun
authors.forename: Fang
authors.initials: F
authors.lastname: He
mesh_terms: D002118:Calcium / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells / Q000502:physiology*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D000071738:Stromal Interaction Molecule 2; D050052:TRPC Cation Channels / Q000502:physiology*
chemical_list: D015815:Cell Adhesion Molecules; C513279:STIM2 protein, human; D000071738:Stromal Interaction Molecule 2; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D009569:Nitric Oxide; D002118:Calcium