Gu Z F, Menozzi D, Okamoto A, Maton P N, Bunnett N W
Digestive Diseases Branch, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Exp Physiol. 1993 Jan;78(1):35-48. doi: 10.1113/expphysiol.1993.sp003669.
The objectives of this investigation were to characterize neuropeptide-degrading enzymes on the surface of gastric muscle cells and to determine their physiological function. Neutral endopeptidase (NEP, EC 3.4.24.11) activity was measured using the fluorogenic substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. The NEP inhibitors phosphoramidon and DL-thiorphan (1 microM) inhibited degradation of the substrate by gastric muscle membranes by 100% and by freshly dispersed gastric muscle cells by 55-60%. The phosphoramidon or DL-thiorphan-inhibitable activity, attributed to NEP, of membranes was 112 +/- 4.0 pmol h-1 (micrograms protein)-1 and of cells was 4.2 +/- 0.8 nmol h-1 (10(6) cells)-1. This activity was associated with membranes prepared from cells and was not detected in the cytoplasm or in the cell incubation solution. Gastric muscle membranes were fractionated by electrophoresis and analysed by Western blotting using two NEP antisera. Both antisera recognized a protein in membranes with an electrophoretic mobility identical to that of recombinant human NEP and an apparent molecular mass of approximately 95 kDa. Neuropeptides were degraded by membranes with specific activities in the order of [Leu5]enkephalin > [Met5]enkephalin > gastrin-releasing peptide-10 (GRP-10) > [D-Ala2][Leu5]enkephalin > somatostatin-14. Phosphoramidon and DL-thiorphan similarly inhibited the degradation of GRP-10 (mean of 35% inhibition), somatostatin-14 (57%) and the aminopeptidase-resistant analogue, [D-Ala2][Leu5]enkephalin (75%). When aminopeptidases were inhibited with amastatin (10 microM) phosphoramidon inhibited degradation of [Leu5]enkephalin (54%) and [Met5]enkephalin (100%). Phosphoramidon increased the potency of the contractile effects of neuropeptides on muscle cells by > 280-fold for somatostatin-14, 17-fold for GRP-10, 18-fold for [Met5]enkephalin and 14-fold for [Leu5]enkephalin. The results show that an NEP-like enzyme on the surface of gastric muscle cells degrades and inactivates enkephalins, GRP-10 and somatostatin-14 and acts in a manner analogous to that of acetylcholinesterase in the neuromuscular junction of skeletal muscle.
本研究的目的是鉴定胃肌细胞表面的神经肽降解酶，并确定其生理功能。使用荧光底物戊二酰 - 丙氨酸 - 苯丙氨酸 - 4 - 甲氧基 - 2 - 萘胺来测定中性内肽酶（NEP，EC 3.4.24.11）的活性。NEP抑制剂磷酰胺素和DL - 硫醇苯丙氨酸（1 microM）可使胃肌细胞膜对底物的降解抑制100%，对新分离的胃肌细胞的降解抑制55 - 60%。归因于NEP的膜的磷酰胺素或DL - 硫醇苯丙氨酸可抑制的活性为112±4.0 pmol h-1（微克蛋白）-1，细胞的活性为4.2±0.8 nmol h-1（10^6个细胞）-1。该活性与从细胞制备的膜相关，在细胞质或细胞孵育溶液中未检测到。通过电泳对胃肌细胞膜进行分级分离，并用两种NEP抗血清进行蛋白质印迹分析。两种抗血清均识别膜中的一种蛋白质，其电泳迁移率与重组人NEP相同，表观分子量约为95 kDa。神经肽被膜降解，其比活性顺序为[亮氨酸5]脑啡肽>[甲硫氨酸5]脑啡肽>胃泌素释放肽 - 10（GRP - 10）>[D - 丙氨酸2][亮氨酸5]脑啡肽>生长抑素 - 14。磷酰胺素和DL - 硫醇苯丙氨酸同样抑制GRP - 10的降解（平均抑制率35%）、生长抑素 - 14的降解（57%）以及氨肽酶抗性类似物[D - 丙氨酸2][亮氨酸5]脑啡肽的降解（75%）。当用氨甲酰抑制剂（10 microM）抑制氨肽酶时，磷酰胺素抑制[亮氨酸5]脑啡肽的降解（54%）和[甲硫氨酸5]脑啡肽的降解（100%）。磷酰胺素使神经肽对肌细胞收缩作用的效力增加，对生长抑素 - 14增加>280倍，对GRP - 10增加17倍，对[甲硫氨酸5]脑啡肽增加18倍，对[亮氨酸5]脑啡肽增加14倍。结果表明，胃肌细胞表面的一种NEP样酶可降解并使脑啡肽、GRP - 10和生长抑素 - 14失活，其作用方式类似于骨骼肌神经肌肉接头中的乙酰胆碱酯酶。
