TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes

摘要

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca²⁺ buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15–45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1–100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca²⁺ buffering conditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca²⁺ buffering. In addition, increasing intracellular Ca²⁺ levels ([Ca²⁺]i) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca²⁺ (0 [Ca²⁺]o) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1–100 nm Ang II and blocked the inhibitory action of [Ca²⁺]i on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca²⁺]o and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca²⁺ influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.