title: TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes.
abstract: 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca(2+) (0 [Ca(2+)](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 [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca(2+) 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.
authors.affiliation: Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK.
authors.forename: J
authors.initials: J
authors.lastname: Shi
authors.forename: M
authors.initials: M
authors.lastname: Ju
authors.forename: S N
authors.initials: SN
authors.lastname: Saleh
authors.forename: A P
authors.initials: AP
authors.lastname: Albert
authors.forename: W A
authors.initials: WA
authors.lastname: Large
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D053119:Benzophenanthridines / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D055724:Electrophysiological Phenomena; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011817:Rabbits; D013268:Stimulation, Chemical; D050052:TRPC Cation Channels / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D019138:Antibodies, Blocking; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D050052:TRPC Cation Channels; D014662:Vasoconstrictor Agents; C096351:transient receptor potential cation channel, subfamily C, member 1; D000804:Angiotensin II; D004533:Egtazic Acid; C016299:chelerythrine; D011493:Protein Kinase C; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium