Angiotensin II Inactivation Process in Cultured Mouse Spinal Cord Cells

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Abstract
The pattern of hydrolysis of [3H]angiotensin II ([3H]AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37°C. In the absence of peptidase inhibitors, 70% of [3H]AII was degraded, and the main labeled metabolite was [3H]tyrosine (40% of total radioactivity). Minor quantities of [3H]AII1–5 and [3H]AII4–8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the All-hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4,14.4) could play a critical role, as suggested by the formation of [3H]Val3-Tyr4 and [3H]-Tyr4-IIe5 in the presence of bestatin (2 ± 10−5M). This hypothesis was confirmed by the potency of dipeptidyl aminopeptidase III inhibitors to inhibit both [3H]AII hydrolysis and formation of these 3H-labeled dipeptides. An arylamidase-like activity could also be participating in [3H]AII hydrolysis, because higher concentrations of bestatin (10−4M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited [3H]tyrosine formation, increased protection of [3H]AII and [3H]AII1–7 formed, and provoked a slight accumulation of [3H]AII2–8. These results suggest that the formation of [3H]AII2–8 is due to the action of a bestatininsensitive acidic aminopeptidase and that the Pro7-Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase. Because care was taken to eliminate interference by enzymes leaking out into the incubation medium, the AII-hydrolyzing process described on these cultured mouse spinal cord cells presumably occurs under the action of membrane-bound peptidases.