Proteolytic Inactivation of Substance P and Neurokinin A in the Longitudinal Muscle Layer of Guinea Pig Small Intestine

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Abstract
Membrane vesicles, showing a 21 ± 2-fold enrichment in the activity of 5′-nucleotidase and a 11 ± 4-fold enrichment in the activity of angiotensin-converting enzyme relative to homogenate, were prepared from the myenteric plexus-containing longitudinal muscle layer of guinea pig ileum. Incubation of the vesicles with substance P and neurokinin A led to degradation of the peptides, and metabolites were isolated by reverse-phase HPLC and identified by amino acid composition. Cleavages of substance P between Glu6-Phe7, Phe7-Phe8, and Gly9-Leu10 and of neurokinin A between Gly8-Leu9 were observed and could be inhibited in a dose-dependent manner by phosphoramidon, an inhibitor of neutral endopeptidase 24.11. Formation of these metabolites was not completely inhibited by this agent, indicating that a phosphoramidon-insensitive form of endopeptidase 24.11 was present in the gut. Substance P was resistant to degradation by aminopeptidases, but neurokinin A was a substrate for bestatin-sensitive aminopeptidase(s), so that the neurokinin A (3–10) fragment represented the predominant metabolite in the chromatograms. The rate of formation of all the metabolites was not inhibited by ena-lapril and not enhanced by an increased Cl− concentration, indicating that angiotensin-converting enzyme was unimportant in the degradation process. Degradation of neurokinin A by the vesicles (Km 30 μM; Vmax 7.2 ±0.8 nmol min−1 mg of protein−1) was more rapid than degradation of substance P (Km 25 μM; Vmax 4.4 ± 0.4 nmol min−1 mg of protein−1).