title: TRPC3 channel contributes to nitric oxide release: significance during normoxia and hypoxia-reoxygenation.
abstract: OBJECTIVE
  Intracellular Ca(2+) ([Ca(2+)](i)) regulation in endothelial cells depends on transient receptor potential channels (TRPs), and the role of canonical TRPs (TRPCs) during hypoxia-reoxygenation (H-R) is unclear. We hypothesized that TRPC3 contributes to endothelial nitric oxide (NO) release and that H-R may reduce TRPC3 activity and the associated endothelial function, including NO release.
  
  
  RESULTS
  Measurements of [Ca(2+)](i) and patch-clamp study in primary cultured porcine coronary endothelial cells, measurements of NO and endothelium-dependent relaxation in porcine coronary arteries, and RT-PCR and western blot were conducted. Pre-treatment with SKF96365 (an inhibitor of TRPCs) or the selective TRPC3 inhibitor Pyr3 significantly decreased bradykinin-induced vasorelaxation. One hour of hypoxia followed by reoxygenation significantly reduced the vasorelaxation (70.3 ± 6.4 vs. 88.9 ± 3.5%) and NO concentration (24.0 ± 1.3 vs. 45.2 ± 2.8 nmol/L), and they were restored by pre-incubation with the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (96.4 ± 1.8% and 41.1 ± 4.7 nmol/L, respectively). In porcine coronary endothelial cells, H-R inhibited bradykinin-activated membrane current (8.6 ± 0.4 vs. 14.0 ± 1.5 pA/pF) and Pyr3-sensitive TRPC3 current (3.8 ± 0.3 vs. 6.3 ± 0.6 pA/pF; P< 0.01). H-R also inhibited bradykinin-induced Ca(2+) influx and the Ca(2+) influx via TRPC3. Cell surface expression of TRPC3 was decreased after H-R.
  
  
  CONCLUSIONS
  We have, for the first time, demonstrated that Ca(2+) entry via endothelial TRPC3 contributes to NO release and have revealed that H-R is associated with inhibition of TRPC3 activity. Inhibition of channel trafficking to the cell surface is involved in the underlying mechanism of the decrease of TRPC3 current and the reduction in Ca(2+) entry through TRPC3 during H-R. This study suggests that TRPC3 may have the potential to be a new target for endothelial protection during H-R.
authors.affiliation: Department of Surgery, The Chinese University of Hong Kong, Hong Kong and TEDA International Cardiovascular Hospital, Medical College, Nankai University, Tianjin, China.
authors.forename: Jun-Hao
authors.initials: JH
authors.lastname: Huang
authors.forename: Guo-Wei
authors.initials: GW
authors.lastname: He
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Xue
authors.forename: Xiao-Qiang
authors.initials: XQ
authors.lastname: Yao
authors.forename: Xiao-Cheng
authors.initials: XC
authors.lastname: Liu
authors.forename: Malcolm John
authors.initials: MJ
authors.lastname: Underwood
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D015687:Cell Hypoxia; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D007093:Imidazoles / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D009569:Nitric Oxide / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D011720:Pyrazoles / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D050052:TRPC Cation Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014664:Vasodilation / Q000187:drug effects
chemical_list: D007093:Imidazoles; D011720:Pyrazoles; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C559943:ethyl-1-(4-(2*3*3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate; D009569:Nitric Oxide; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D010100:Oxygen; D001920:Bradykinin