Saleh S N, Albert A P, Peppiatt C M, Large W A
Ion Channels and Cell Signalling, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 ORE, UK.
J Physiol. 2006 Dec 1;577(Pt 2):479-95. doi: 10.1113/jphysiol.2006.119305. Epub 2006 Sep 14.
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 (Icat1) with conductances states of about 15, 30 and 45 pS. At relatively high concentrations, Ang II (100 nM) inhibited Icat1 but evoked another cation channel (Icat2) with a conductance of approximately 2 pS. Ang II-evoked Icat1 and Icat2 were inhibited by the AT1 receptor antagonist losartan and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol (DAG) lipase inhibitor RHC80267 initially induced Icat1 which was subsequently inhibited to reveal Icat2. The DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (1 microM) activated Icat1 and Icat2 but inositol 1,4,5-trisphosphate did not evoke either conductance. The protein kinase C (PKC) inhibitor chelerythrine (3 microM) potentiated Ang II-evoked Icat1 and inhibited Icat2 whereas the PKC activator phorbol-12,13-dibutyrate (1 microM) reduced Ang II-induced Icat1 but activated Icat2. Moreover in cell-attached patches pretreated with chelerythrine, application of 100 nM Ang II activated Icat1. These data indicate that PKC inhibits Icat1 but stimulates Icat2. Agents that deplete intracellular Ca2+ stores also activated cation channel currents with similar properties to Icat2. Bath application of anti-TRPC6 and anti-TRPC1 antibodies to inside-out patches inhibited Icat1 and Icat2, respectively. Also flufenamic acid and zero external Ca2+ concentration, respectively, potentiated and reduced Ang II-evoked Icat1. 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 AT1 receptors linked to PLC. Icat1 is activated by DAG via a PKC-independent mechanism whereas Icat2 involves DAG acting via a PKC-dependent pathway. Higher concentrations of Ang II inhibit Icat1 by activating an inhibitory effect of PKC. It is proposed that TRPC6 and TRPC1 channel proteins are important components of Ang II-induced Icat1 and Icat2, respectively.
血管紧张素II（Ang II）是一种强效血管收缩剂，在控制血压方面发挥着重要作用；然而，关于Ang II诱发血管收缩反应的细胞机制的信息却很少。本研究的目的是使用膜片钳技术，在单通道水平上研究Ang II对新鲜分离的兔肠系膜动脉肌细胞中阳离子电导的影响。在细胞贴附式膜片中，浴槽施加低浓度的Ang II（1 nM）可激活阳离子通道电流（Icat1），其电导状态约为15、30和45 pS。在相对高浓度时，Ang II（100 nM）抑制Icat1，但诱发另一种电导约为2 pS的阳离子通道（Icat2）。Ang II诱发的Icat1和Icat2被AT1受体拮抗剂氯沙坦和磷脂酶C（PLC）抑制剂U73122抑制。二酰基甘油（DAG）脂肪酶抑制剂RHC80267最初诱导Icat1，随后被抑制以揭示Icat2。DAG类似物1-油酰基-2-乙酰基-sn-甘油（1 microM）激活Icat1和Icat2，但肌醇1,4,5-三磷酸不会诱发任何一种电导。蛋白激酶C（PKC）抑制剂白屈菜红碱（3 microM）增强Ang II诱发的Icat1并抑制Icat2，而PKC激活剂佛波醇-12,13-二丁酸酯（1 microM）降低Ang II诱导的Icat1但激活Icat2。此外，在预先用白屈菜红碱处理的细胞贴附式膜片中，施加100 nM Ang II可激活Icat1。这些数据表明PKC抑制Icat1但刺激Icat2。耗尽细胞内Ca2+储存的试剂也激活了与Icat2性质相似的阳离子通道电流。向外翻式膜片浴槽施加抗TRPC6和抗TRPC1抗体分别抑制Icat1和Icat2。同样，氟芬那酸和零细胞外Ca2+浓度分别增强和降低Ang II诱发的Icat1。免疫细胞化学研究显示TRPC6和TRPC1的表达，其中TRPC6优先分布在质膜中，TRPC1的表达遍布整个肌细胞。这些结果表明，Ang II通过刺激与PLC相连的AT1受体激活肠系膜动脉肌细胞中的两种不同阳离子电导。Icat1通过DAG经由非PKC依赖机制激活，而Icat2涉及DAG通过PKC依赖途径起作用。较高浓度的Ang II通过激活PKC的抑制作用来抑制Icat1。据推测，TRPC6和TRPC1通道蛋白分别是Ang II诱导的Icat1和Icat2的重要组成部分。
