RESUMO
Transient receptor potential (TRP) C1 and C3 (TRPC1 and TRPC3) are expressed in vascular smooth muscle cells and are thought to be involved in vascular contractility. In the present study, we determined the effect of systemic hypertension on TRPC1/TRPC3 channel expression and vascular contractility in rat carotid artery (CA). CA were studied from male spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY), and Long Evans (LE) rats. TRPC1/3 expression was determined by RT-PCR and Western blot. TRP channel function was evaluated by whole-cell patch clamp, using UTP (60 µM) to stimulate TRPC1/3 channels. Contractions of endothelium-denuded CA segments to UTP (1-300 µM) and phenylephrine (Phe; 0.1 nM-10 µM) were measured in an isometric tension bath. TRPC1 and TRPC3 mRNA was present in CA of both WKY and SHR. Western blot demonstrated 3.1 ± 1.2 times greater TRPC3 expression and 0.5 ± 0.2 times TRPC1 in SHR versus WKY CA. Isolated CA showed potentiated contraction to UTP in the SHR versus WKY. Activation of voltage-dependent Ca(2+) channels (VDCC) in UTP-mediated constriction only occurred in SHR CA. Contraction to Phe was unaltered between WKY and SHR CA and involved equal significant VDCC activation in both groups. Patch clamp demonstrated that the UTP-stimulated current (I(utp)) was greater in SHR compared to the normotensive WKY and LE rats with peak I(utp) (at -110 mV) of -63 ± 24 pA compared to -25 ± 4 pA, respectively. We demonstrate that UTP-mediated but not Phe-mediated constrictions are potentiated in the CA during hypertension. Expression of TRPC1 is decreased whereas TRPC3 is increased in SHR CA. Interestingly, VDCC activation only contributes to UTP-mediated contraction of SHR CAs whereas it contributes substantially and equally in Phe-mediated contraction. We speculate that the alteration of TRPC channel expression in hypertension leads to greater smooth muscle depolarization, VDCC activation, and vascular contractility in the UTP (but not Phe) signaling pathway.
RESUMO
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.