Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation.
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 microM) and completely inhibited by a TRPC channel inhibitor, La(3+) (100 microM). 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 microM) in the presence or absence of La(3+) (100 microM) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 microM). 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.
Chen
Jie
J
Department of Anesthesiology, Section of Critical Care, Baylor College of Medicine, Houston, TX 77030, USA.
Crossland
Randy F
RF
Noorani
Muzamil M Z
MM
Marrelli
Sean P
SP
D000818:Animals; D015153:Blotting, Western; D002339:Carotid Arteries / Q000187:drug effects; D017869:Cyclic GMP-Dependent Protein Kinases / Q000494:pharmacology*; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D009569:Nitric Oxide / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D051381:Rats; D020318:Rats, Long-Evans; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors*; D014664:Vasodilation / Q000187:drug effects*; D014665:Vasodilator Agents / Q000494:pharmacology*
D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D014665:Vasodilator Agents; C096351:transient receptor potential cation channel, subfamily C, member 1; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases