title: Nitric oxide and protein kinase G act on TRPC1 to inhibit 11,12-EET-induced vascular relaxation.
abstract: OBJECTIVE
  Vascular endothelial cells synthesize and release vasodilators such as nitric oxide (NO) and epoxyeicosatrienoic acids (EETs). NO is known to inhibit EET-induced smooth muscle hyperpolarization and relaxation. This study investigates the underlying mechanism of this inhibition.
  
  
  RESULTS
  Through measurements of membrane potential and arterial tension, we show that 11,12-EET induced membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. These responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, an NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, an NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-bromo cyclic GMP (8-Br-cGMP) on the EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with an excessive supply of exogenous PKG substrates, TAT-TRPC1(S172) and TAT-TRPC1(T313). Furthermore, a phosphorylation assay demonstrated that PKG could directly phosphorylate TRPC1 at Ser(172) and Thr(313). In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically associated with each other in smooth muscle cells.
  
  
  CONCLUSIONS
  Our findings demonstrate a novel role of the NO-cGMP-PKG pathway in the inhibition of 11,12-EET-induced smooth muscle hyperpolarization and relaxation via PKG-mediated phosphorylation of TRPC1.
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Ma
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Stem Cell and Regenerative Medicine Consortium, The University of Hong Kong, Hong Kong, China.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
authors.affiliation: Department of Surgery, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Song
authors.initials: S
authors.lastname: Wan
authors.affiliation: School of Biomedical Sciences and Li Ka Shing institute of Health Science, The Chinese University of Hong Kong, Hong Kong, China Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China yao2068@cuhk.edu.hk.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Yao
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D003331:Coronary Vessels / Q000187:drug effects / Q000201:enzymology; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D013552:Swine; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000378:metabolism; D014162:Transfection; D014664:Vasodilation / Q000187:drug effects*; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D020030:Nitric Oxide Donors; D050052:TRPC Cation Channels; D050916:TRPV Cation Channels; D014665:Vasodilator Agents; C096351:transient receptor potential cation channel, subfamily C, member 1; D009569:Nitric Oxide; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D017869:Cyclic GMP-Dependent Protein Kinases; D015126:8,11,14-Eicosatrienoic Acid; D006152:Cyclic GMP
keywords: Epoxyeicosatrienoic acids; Nitric oxide; TRP channels; Vascular relaxation