Rainbow Richard D, Norman Robert I, Everitt Diane E, Brignell Jennifer L, Davies Noel W, Standen Nicholas B
Department of Cardiovascular Sciences, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, UK.
Cardiovasc Res. 2009 Aug 1;83(3):493-500. doi: 10.1093/cvr/cvp143. Epub 2009 May 8.
Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents.
Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon.
ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.
动脉平滑肌（ASM）的电压门控钾离子（Kv）通道调节动脉张力，并通过蛋白激酶C（PKC）被血管收缩剂抑制。我们旨在确定内皮素-1（ET-1）和血管紧张素II（AngII），它们对Kv产生类似的抑制作用，是否使用相同的信号通路和PKC同工酶来发挥对Kv的作用，并比较PKC同工酶在对这些药物的收缩反应中的参与情况。
使用膜片钳技术记录新鲜分离的大鼠肠系膜ASM细胞的Kv电流，ET-1或AngII可抑制该电流。细胞内溶液中加入PKCε抑制肽可显著降低AngII的抑制作用，但不影响ET-1的抑制作用。传统的PKC抑制剂Gö 6976可降低ET-1对Kv的抑制作用，但PKCβ抑制剂LY333531则无此作用。PKCα和PKCε的选择性肽抑制剂与Tat载体肽相连，使其具有膜通透性，并用于表明抑制PKCα可阻止ET-1对Kv电流的抑制作用，但不影响AngII的抑制作用。相反，抑制PKCε可阻止AngII对Kv的抑制作用，但不影响ET-1的抑制作用。与Tat相连的抑制剂肽还用于研究PKCα和PKCε在肠系膜动脉环收缩反应中的参与情况，结果表明，抑制PKCα可显著降低ET-1引起的收缩，但对PKCε的抑制无影响。抑制PKCα对AngII引起的收缩无影响，但抑制PKCε可使其显著降低。
ET-1通过激活PKCα抑制肠系膜ASM的Kv通道，而AngII则通过PKCε来抑制。这意味着ET-1和AngII通过不同的PKC相互作用蛋白途径靶向ASM的Kv通道，因此每种血管收缩剂都能使其独特的PKC同工酶与Kv通道发生功能性相互作用。
