| Autor: |
Gu, ZF, Menozzi, D, Okamoto, A, Maton, PN, Bunnett, NW |
| Zdroj: |
Experimental Physiology; January 1993, Vol. 78 Issue: 1 p35-48, 14p |
| Abstrakt: |
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. |
| Databáze: |
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