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

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 (K_m 30 μM; V_max 7.2 ±0.8 nmol min⁻¹ mg of protein⁻¹) was more rapid than degradation of substance P (K_m 25 μM; V_max 4.4 ± 0.4 nmol min⁻¹ mg of protein⁻¹).