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).