Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation

1 Department of Anesthesiology, 2 Department of Molecular Physiology and Biophysics, 3 Graduate Program in Cardiovascular Sciences, and 4 Department of Pediatrics, Section of Critical Care, Baylor College of Medicine, Houston, Texas Submitted 27 October 2008 ; accepted in final form 29 May 2009 Nitr...

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Published in:American journal of physiology. Heart and circulatory physiology 2009-07, Vol.297 (1), p.H417-H424
Main Authors: Chen, Jie, Crossland, Randy F, Noorani, Muzamil M. Z, Marrelli, Sean P
Format: Article
Language:English
Subjects:
Animals
Blotting, Western
Carotid Arteries - drug effects
Cyclic GMP-Dependent Protein Kinases - pharmacology
Gene expression
Immunoprecipitation
In Vitro Techniques
Inhibitor drugs
Isometric Contraction - drug effects
Male
Membrane Potentials - drug effects
Muscle, Smooth, Vascular - drug effects
Muscular system
Nitric oxide
Nitric Oxide - pharmacology
Patch-Clamp Techniques
Rats
Rats, Long-Evans
Reverse Transcriptase Polymerase Chain Reaction
Rodents
Signal Transduction - drug effects
TRPC Cation Channels - antagonists & inhibitors
Vasodilation - drug effects
Vasodilator Agents - pharmacology
Veins & arteries
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Summary:1 Department of Anesthesiology, 2 Department of Molecular Physiology and Biophysics, 3 Graduate Program in Cardiovascular Sciences, and 4 Department of Pediatrics, Section of Critical Care, Baylor College of Medicine, Houston, Texas Submitted 27 October 2008 ; accepted in final form 29 May 2009 Nitric oxide (NO) inhibits transient receptor potential channel 3 (TRPC3) channels via a PKG-dependent mechanism. We sought to determine 1 ) whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC); and 2 ) whether NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact CA by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and coimmunoprecipitated with TRPC3. Whole cell patch clamp demonstrated nonselective cation channel currents that were activated by UTP (60 µM) and completely inhibited by a TRPC channel inhibitor, La 3+ (100 µM). The UTP-stimulated current ( I UTP ) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody, but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I UTP . Exogenous NO [( Z )-1-{ N -methyl- N -[6( N -methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate)] or a cell-permeable cGMP analog (8-bromo-cGMP) significantly inhibited I UTP . Preapplication of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8-bromo-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 µM) in the presence or absence of La 3+ (100 µM) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 µM). Relaxation to sodium nitroprusside was significantly reduced in the La 3+ treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La 3+ -sensitive channels consistent with TRPC1/TRPC3. transient receptor potential channel; protein kinase G; transient receptor potential 3; transient receptor potential 1 Address for reprint requests and other correspondence: S. P. Marrelli, Dept. of Anesthesiology, Baylor College of Medicine, Suite 434-D, One Baylor Plaza, Houston, T
ISSN:0363-6135
1522-1539
DOI:10.1152/ajpheart.01130.2008