内皮素-1/内皮素A受体介导花生四烯酸释放过程中涉及的钙离子通道和G蛋白的特性
Characterization of Ca2+ channels and G proteins involved in arachidonic acid release by endothelin-1/endothelinA receptor.
作者信息
Kawanabe Yoshifumi, Nozaki Kazuhiko, Hashimoto Nobuo, Masaki Tomoh
机构信息
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Institutes of Medicine, Room 520, 77 Avenue Louis Pasteur, Boston, MA 02115.
出版信息
Mol Pharmacol. 2003 Sep;64(3):689-95. doi: 10.1124/mol.64.3.689.
Endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in Chinese hamster ovary cells expressing endothelinA receptors (CHO-ETAR). These channels can be distinguished by their sensitivity to Ca2+ channel blockers 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365; NSCC-2 is sensitive to both blockers, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. In this study, we examined the mechanism of ET-1-induced arachidonic acid (AA) release. Both SK&F 96365 and LOE 908 inhibited ET-1-induced AA release with the IC50 values correlated to those of ET-1-induced Ca2+ influx. Moreover, combined treatment with these blockers abolished ET-1-induced AA release. Wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase (PI3K), partially inhibited ET-1-induced AA release. LOE 908, but not SK&F 96365, inhibited ET-1-induced AA release in wortmannin-treated CHO-ETAR. ET-1 also induced AA release in CHO cells expressing ETAR truncated at the carboxyl terminal downstream of Cys385 (CHO-ETARDelta385) or an unpalmitoylated (Cys383 Cys385-388--> Ser383Ser385-388) ETAR (CHO-SerETAR), each of which is coupled with Gq or Gs/G12, respectively. In CHO-SerETAR, a dominant-negative mutant of G12 inhibited AA release. SK&F 96365 inhibited ET-1-induced AA release in CHO-ETARDelta385, whereas LOE 908 inhibited it in CHO-SerETAR. These results indicate the following: 1) ET-1-induced AA release depends on Ca2+ influx through NSCC-1, NSCC-2, and SOCC in CHO-ETAR; 2) Gq and G12 mediate AA release through ETAR in CHO cells; and 3) PI3K is involved in ET-1-induced AA release, which depends on NSCC-2 and SOCC.
内皮素 -1（ET -1）可激活表达内皮素A受体的中国仓鼠卵巢细胞（CHO - ETAR）中的两种钙离子通透型非选择性阳离子通道（分别命名为NSCC -1和NSCC -2）以及一种储存式钙离子通道（SOCC）。这些通道可通过它们对钙离子通道阻滞剂1 -（β - [3 -（4 - 甲氧基苯基）丙氧基]-4 - 甲氧基苯乙基）-1H - 咪唑盐酸盐（SK&F 96365）和（R,S）-（3,4 - 二氢 -6,7 - 二甲氧基 - 异喹啉 -1 - 基）-2 - 苯基 -N,N - 二[2 -（2,3,4 - 三甲氧基苯基）乙基]乙酰胺甲磺酸盐（LOE 908）的敏感性来区分。NSCC -1对LOE 908敏感而对SK&F 96365有抗性；NSCC -2对两种阻滞剂均敏感，而SOCC对LOE 908有抗性且对SK&F 96365敏感。在本研究中，我们检测了ET -1诱导花生四烯酸（AA）释放的机制。SK&F 96365和LOE 908均抑制ET -1诱导的AA释放，其半数抑制浓度（IC50）值与ET -1诱导的钙离子内流相关。此外，联合使用这些阻滞剂可消除ET -1诱导的AA释放。渥曼青霉素和LY294002，即磷脂酰肌醇3 -激酶（PI3K）的抑制剂，可部分抑制ET -1诱导的AA释放。在经渥曼青霉素处理的CHO - ETAR中，LOE 908可抑制ET -1诱导的AA释放，而SK&F 96365则不能。ET -1还可诱导表达在Cys385下游羧基末端截短的ETAR（CHO - ETARDelta385）或未棕榈酰化的（Cys383 Cys385 - 388→Ser383Ser385 - 388）ETAR（CHO - SerETAR）的CHO细胞释放AA，其中每种细胞分别与Gq或Gs/G12偶联。在CHO - SerETAR中，G12的显性负性突变体可抑制AA释放。SK&F 96365可抑制CHO - ETARDelta385中ET -1诱导的AA释放，而LOE 908可抑制CHO - SerETAR中的该释放。这些结果表明：1）ET -1诱导的AA释放在CHO - ETAR中依赖于通过NSCC -1、NSCC -2和SOCC的钙离子内流；2）Gq和G12通过CHO细胞中的ETAR介导AA释放；3）PI3K参与ET -1诱导的AA释放，这依赖于NSCC -2和SOCC。
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