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

Abstract

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 M) and completely inhibited by a TRPC channel inhibitor, La 3ϩ (100 M). The UTP-stimulated current (IUTP) 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 cellpermeable 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 M) in the presence or absence of La 3ϩ (100 M) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 M). Relaxation to sodium nitroprusside was significantly reduced in the La 3ϩ 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 3ϩ -sensitive channels consistent with TRPC1/TRPC3. transient receptor potential channel; protein kinase G; transient receptor potential 3; transient receptor potential 1 THE CANONICAL TRANSIENT RECEPTOR potential channels (TRPCs) are nonselective cation channels that have been demonstrated to play a variety of roles in regulation of vascular tone (23). TRPC3 is one of the less well-characterized members of the TRPC family (TRPC1-7) within the vasculature. Nevertheless, TRPC3 has recently been shown to be involved in receptor-mediated constriction within the smooth muscle cells (SMCs) of cerebral arteries (18). Activation of the TRPC3 channel permits Ca 2ϩ and Na ϩ entry into the cell; however, the major mechanism of Ca 2ϩ entry in SMC is thought to be secondary to Na ϩ entry and smooth muscle depolarization (18). Depolarization from Na ϩ entry leads to activation of L-type Ca 2ϩ channels with subsequent Ca 2ϩ entry and smooth muscle contraction.Nitric oxide (NO) is well known as a vasodilator. However, the mechanism by which NO promotes smooth muscle relaxation is still incompletely resolved. At this time, it is believed that NO leads to smooth muscle relaxation by multiple pathways, including acti...

Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy

“…TRPC3 and TRPC6 are known to heterotetramerize based on immunoprecipitation and fluorescence resonance energy transfer studies (26). Genetic models involving expression of dominant negative proteins (8) may induce a promiscuous effect on the other channel and other TRPC channels; for instance, TRPC1 also multimerizes with TRPC3 (27). The present study supports this hypothesis, because neither single gene deletion model ameliorated hypertrophy/dysfunction with TAC, whereas the dKO model was more effective.…”

Nitro-Oleic Acid Inhibits Firing and Activates TRPV1- and TRPA1-Mediated Inward Currents in Dorsal Root Ganglion Neurons from Adult Male Rats

“…The effect of combined application of TRPV1 and TRPA1 antagonists on OA-NO 2 -evoked currents was comparable with that of the reducing agent DTT that competitively reacts with the electrophilic fatty acid (Salazar et al, 2008;Chen et al, 2009). Thus, the action of DTT on the OA-NO 2 -evoked currents is highly suggestive of an electrophilic interaction of OA-NO 2 with cysteine residues of TRP channels, such as that reported for various pungent compounds and AITC (Hinman et al, 2006;Macpherson et al, 2007).…”

TRPC expression in mesenchymal stem cells

“…1C). While this molecular weight does not correspond to the generally reported values of around 100 kDa [25], it is close to the isoform of TRPC1 detected in rat neurons [26]. Indeed, a short isoform has already been detected via RT-PCR in salivary gland cells [27] or in immature CD34 + cells [14] and shown to disappear and be progressively replaced by the unspliced TRPC1 form during that differentiation into megacaryocytes [14].…”