Stanimirovic D B, Yamamoto T, Uematsu S, Spatz M
Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
J Neurochem. 1994 Feb;62(2):592-601. doi: 10.1046/j.1471-4159.1994.62020592.x.
The kinetic properties of endothelin-1 (ET-1) binding sites and the production of inositol phosphates (IPs; IP1, IP2, IP3), cyclic AMP, thromboxane B2, and prostaglandin F2 alpha induced by various endothelins (ET-1, ET-2, ET-3, and sarafotoxin S6b) were examined in endothelial cells derived from human brain microvessels (HBECs). The presence of both high- and low-affinity binding sites for ET-1 with KD1 = 122 pM and KD2 = 31 nM, and Bmax1 = 124 fmol/mg of protein and Bmax2 = 909 fmol/mg of protein, respectively, was demonstrated on intact HBECs. ET-1 dose-dependently stimulated IP accumulation with EC50 (IP3) = 0.79 nM, whereas ET-3 was ineffective. The order of potency for displacing ET-1 from high-affinity binding sites (ET-1 > ET-2 > sarafotoxin S6b > ET-3) correlated exponentially with the ability of respective ligands to induce IP3 formation. ET-1-induced IP3 formation by HBEC was inhibited by the ETA receptor antagonist, BQ123. The protein kinase C activator phorbol myristate ester dose-dependently inhibited the ET-1-stimulated production of IPs, whereas pertussis toxin was ineffective. Cyclic AMP production by HBECs was enhanced by both phorbol myristate ester and ET-1, and potentiated by combined treatment with ET-1 and phorbol myristate ester. Data indicate that protein kinase C plays a role in regulating the ET-1-induced activation of phospholipase C, whereas interaction of different messenger systems may regulate ET-1-induced accumulation of cyclic AMP. ET-1 also stimulated endothelial prostaglandin F2 alpha production, suggesting that activation of phospholipase A2 is most likely secondary to IP3-mediated intracellular calcium mobilization because both ET-1-induced IP3 and prostaglandin F2 alpha were inhibited by BQ123. These findings are the first demonstration of ET-1 (ETA-type) receptors linked to phospholipase C and phospholipase A2 activation in HBECs.
研究了人脑海微血管内皮细胞（HBECs）中内皮素-1（ET-1）结合位点的动力学特性以及各种内皮素（ET-1、ET-2、ET-3和沙雷肽素S6b）诱导的肌醇磷酸（IPs；IP1、IP2、IP3）、环磷酸腺苷、血栓素B2和前列腺素F2α的产生。在完整的HBECs上证实存在ET-1的高亲和力和低亲和力结合位点，KD1 = 122 pM，KD2 = 31 nM，Bmax1 = 124 fmol/mg蛋白质，Bmax2 = 909 fmol/mg蛋白质。ET-1以剂量依赖性方式刺激IP积累，EC50（IP3）= 0.79 nM，而ET-3无效。从高亲和力结合位点置换ET-1的效力顺序（ET-1 > ET-2 > 沙雷肽素S6b > ET-3）与各配体诱导IP3形成的能力呈指数相关。ETA受体拮抗剂BQ123抑制了HBEC中ET-1诱导的IP3形成。蛋白激酶C激活剂佛波醇肉豆蔻酸酯以剂量依赖性方式抑制ET-1刺激的IPs产生，而百日咳毒素无效。佛波醇肉豆蔻酸酯和ET-1均增强了HBEC中环磷酸腺苷的产生，并且ET-1与佛波醇肉豆蔻酸酯联合处理可使其增强。数据表明蛋白激酶C在调节ET-1诱导的磷脂酶C激活中起作用，而不同信使系统的相互作用可能调节ET-1诱导的环磷酸腺苷积累。ET-1还刺激了内皮细胞前列腺素F2α的产生，这表明磷脂酶A2的激活很可能继发于IP3介导的细胞内钙动员，因为ET-1诱导的IP3和前列腺素F2α均被BQ123抑制。这些发现首次证明了HBECs中与磷脂酶C和磷脂酶A2激活相关的ET-1（ETA型）受体。
