Nitric oxide and protein kinase G act on TRPC1 to inhibit 11,12-EET-induced vascular relaxation

Cardiovasc Res. 2014 Oct 1;104(1):138-46. doi: 10.1093/cvr/cvu190. Epub 2014 Aug 18.


Aims: 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.

Methods and 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.

Conclusion: 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.

Keywords: Epoxyeicosatrienoic acids; Nitric oxide; TRP channels; Vascular relaxation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 8,11,14-Eicosatrienoic Acid / analogs & derivatives*
  • 8,11,14-Eicosatrienoic Acid / pharmacology
  • Animals
  • Coronary Vessels / drug effects
  • Coronary Vessels / enzymology
  • Cyclic GMP / metabolism
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • Dose-Response Relationship, Drug
  • HEK293 Cells
  • Humans
  • Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / metabolism
  • Membrane Potentials
  • Muscle, Smooth, Vascular / drug effects*
  • Muscle, Smooth, Vascular / enzymology
  • Nitric Oxide / metabolism*
  • Nitric Oxide Donors / pharmacology
  • Phosphorylation
  • Protein Binding
  • Signal Transduction / drug effects
  • Swine
  • TRPC Cation Channels / genetics
  • TRPC Cation Channels / metabolism*
  • TRPV Cation Channels / metabolism
  • Transfection
  • Vasodilation / drug effects*
  • Vasodilator Agents / pharmacology*


  • Large-Conductance Calcium-Activated Potassium Channel alpha Subunits
  • Nitric Oxide Donors
  • TRPC Cation Channels
  • TRPV Cation Channels
  • Vasodilator Agents
  • transient receptor potential cation channel, subfamily C, member 1
  • Nitric Oxide
  • 11,12-epoxy-5,8,14-eicosatrienoic acid
  • Cyclic GMP-Dependent Protein Kinases
  • 8,11,14-Eicosatrienoic Acid
  • Cyclic GMP