Phosphoramidon和Thiorphan对豚鼠气管上皮细胞内皮素-1代谢的影响

The effects of phosphoramidon and thiorphan on production and metabolism of endothelin in guinea pig tracheal epithelial cell culture

目的 观察内皮素转换酶和主要的内皮素降解酶ＮＥＰ 对豚鼠气管上皮细胞培养上清液内皮素１（ＥＴ１） 浓度的影响。方法 无菌条件下取豚鼠气管，并用Ｓｃｒａｐｅｒ 机械轻刮下上皮细胞，在５ ％ ＣＯ２ ３７°ＣＤＭＥＭ／Ｆ１２ 培养液中培养，不加或分别按０ ．１ｍ ｍｏｌ／Ｌ 浓度加入中性肽链内切酶内皮素转换酶双重抑制剂Ｐｈｏｓｐｈｏｒａｍｉｄｏｎ 和中性肽链内切酶抑制剂Ｔｈｉｏｒｐｈａｎ 继续培养１０ 小时。收集上清液，放免法测定ＥＴ１ 。结果 与对照组［（５８ ．５１ ±１４ ．２９）ｐｇ／ｍｇ 蛋白］ 比较，Ｐｈｏｓｐｈｏｒａｍｉｄｏｎ 组［（４０ ．４３±１５ ．３７）ｐｇ／ｍｇ 蛋白］ＥＴ１ 降低有统计意义（ Ｐ＝ ０ ．０２８８） ，而Ｔｈｉｏｒｐｈａｎ 组［（８８ ．５１ ±１９ ．９８）ｐｇ／ｍｇ 蛋白）］ＥＴ１ 浓度明显增高（ Ｐ＝ ０ ．００３９）。结论 中性肽链内切酶抑制剂Ｔｈｉｏｒｐｈａｎ 因抑制中性肽链内切酶而减少内皮素降解，使豚鼠气管上皮细胞培养上清液中内皮素浓度增加。中性肽链内切酶内皮素转换酶双重抑制剂ｐｈｏｓｐｈｏｒａｍｉｄｏｎ 因抑制内皮素转换酶，使内皮素形成减少。

Objective To observe the effects of endothelin converting enzyme (ECE) and neutral endopeptidase (NEP) on the conversion and degradation of endothelins in guinea pig tracheal epithelial cell in cultrue with a dual inhibitor of NEP and ECE, the phosphoramidon, and a selective inhibitor of NEP, the thiorphan. Methods The trachea of Hartley guinea pig was harvested under sterile condition. The epithelial cells were removed from the mucosal surface with cell scraper and seeded at a concentration of 5×10 5 cells/well in 24 well culture plate with DMEM/F12 and cultured at 37 °C in an atmosphere containing 5% CO 2. The cells reached confluency after 7 days. Then the cells were incubated for 10h with phosphoramidon or thiorphan respectively at concentration 0.1mmol/L. The supernatants were then collected and the concentrations of immunoreactive ET(ir ET) were measured by RIA.Results Comparison of the concentrations of ir ET between the control group (58.51±14.29)pg/mg protein and phosphoramidon group (40.43 ±15.37)pg/mg protein showed that phosphoramidon inhibited the release of ir ET( P =0.0288) by decreasing the formation of ET through inhibiting ECE. In contrast, thiorphan induced the increase of concentration of ir ET significantly (88.51±19.98)pg/mg protein, ( P =0.00387) by blocking the degradation of ET. Conclusion It is suggested that the ET is released and degraded by tracheal epithelial cell via ECE and NEP pathways. In prospective, ECE inhibitor may become a new path way in treat ment of bronchial asthma. (Shanghai Med J, 1999,22∶677 680)