Angiotensin II activates two cation conductances with distinct TRPC1 and TRPC6 channel properties in rabbit mesenteric artery myocytes

Angiotensin II (Ang II) is a potent vasoconstrictor with an important role in controlling blood pressure; however, there is little information on cellular mechanisms underlying Ang II-evoked vasoconstrictor responses. The aim of the present study is to investigate the effect of Ang II on cation conductances in freshly dispersed rabbit mesenteric artery myocytes at the single-channel level using patch-clamp techniques. In cell-attached patches, bath application of low concentrations of Ang II (1 nM) activated cation channel currents (I-cat1) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited I-cat1 but evoked another cation channel (I-cat2) with a conductance of approximately 2 pS. Ang II-evoked I-cat1 and I-cat2 were inhibited by the AT(1) receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced I-cat1 which was subsequently inhibited to reveal I-cat2. The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 mu M) activated I-cat1 and I-cat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 mu M) potentiated Ang II-evoked I-cat1 and inhibited I-cat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 mu M) reduced Ang II-induced I-cat1 but activated I-cat2. Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated I-cat1. These data indicate that PKC inhibits I-cat1 but stimulates I-cat2. Agents that deplete intracellular Ca2+ stores also activated cation channel currents with similar properties to I-cat2. Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited I-cat1 and I-cat2, respectively. Also flufenamic acid and zero external Ca2+ concentration, respectively, potentiated and reduced Ang II-evoked I-cat1. Immunocytochemical studies showed TRPC6 and TRPC1 expression with TRPC6 preferentially distributed in the plasma membrane and TRPC1 expression located throughout the myocyte. These results indicate that Ang II activates two distinct cation conductances in mesenteric artery myocytes by stimulation of AT(1) receptors linked to PLC. I-cat1 is activated by DAG via a PKC-independent mechanism whereas I-cat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit I-cat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced I-cat1 and I-cat2, respectively.