vasoactive-intestinal-peptide and ubenimex

Neuromedin N (NN) induced a concentration-dependent contraction (ED50 = 2.3 +/- 0.2 microM) of the isolated longitudinal smooth muscle from guinea pig ileum. This effect was drastically enhanced (ED50 = 0.06 microM) by the aminopeptidases M and B inhibitor bestatin (10 microM), which elicited a 40-fold increase in NN potency. HPLC analysis indicated that the main NN catabolite generated by membranes from guinea pig longitudinal smooth muscle homogenate corresponded to des-Lys1-NN, which results from removal of the N-terminal lysyl residue of NN. The fact that the formation of des-Lys1-NN was fully prevented by bestatin (10 microM) further supports the involvement of aminopeptidases in NN degradation. We examined the catabolic fate of NN in vivo in the vascularly perfused dog ileum. Bolus administration or continuous infusion of the peptide led to rapid disappearance of NN. This was prevented by prior treatment of ileal segments with bestatin (10 microM) but not with arphamenine B (0.5 microM), which indicated that aminopeptidase M but not aminopeptidase B participated in NN proteolysis in vivo. We showed that 1 and 10 nmol NN trigger the release of 28 +/- 5 and 59 +/- 1 pmol, respectively, of endogenous vasoactive intestinal polypeptide-like immunoreactivity after infusion in the vascularly perfused dog ileum. This release was virtually doubled by prior treatment with 10 microM bestatin but not with 0.5 microM arphamenine B. Altogether, our data indicate that aminopeptidase M is largely responsible for NN degradation in vitro and in vivo in the gastrointestinal tract and could be considered the physiological inactivator of NN in the gut.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Dogs; Female; Guinea Pigs; Ileum; In Vitro Techniques; Leucine; Male; Methionyl Aminopeptidases; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neurotensin; Peptide Fragments; Protease Inhibitors; Vasoactive Intestinal Peptide

The effects of enzyme inhibitors on vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (PaO) and VIP-like immunoreactivity (VIP-LI) recovery were studied in isolated tracheal superfused guinea pig lungs. In the absence of inhibitors, VIP 0.38 (95% CI 0.33-0.54) nmol/kg animal, resulted in a 50% decrease in PaO and 33% of a 1 nmol/kg VIP dose was recovered as intact VIP. In the presence of two combinations of enzyme inhibitors, SCH 32615 (S, 10 microM) and aprotinin (A, 500 tyrpsin inhibitor units [TIU]/kg) or S and soybean trypsin inhibitor (T, 500 TIU/kg), VIP caused a significantly greater decrease in PaO and greater quantities of VIP were recovered from lung effluent (both P < 0.001). The addition of captopril, (3 microM), leupeptin (4 microM), or bestatin (1 microM) failed to further increase pulmonary relaxation or recovery of VIP-LI. When given singly, A, T, and S did not augment the effects or recovery of VIP. The efficacy of S (a specific inhibitor of neutral endopeptidase [NEP]) and A and T (serine protease inhibitors) thus implicated NEP and at least one serine protease as primary modulators of VIP activity in the guinea pig lung. We sought to corroborate this finding by characterizing the predominant amino acid sites at which VIP is hydrolized in the lung. When [mono(125I)iodo-Tyr10]VIP was offered to the lung, in the presence and absence of the active inhibitors, cleavage products consistent with activity by NEP and a tryptic enzyme were recovered. These data demonstrate that NEP and a peptidase with an inhibitor profile and cleavage pattern compatible with a tryptic enzyme inactivate VIP in a physiologically competitive manner.

Topics: Animals; Aprotinin; Captopril; Dose-Response Relationship, Drug; Endopeptidases; Guinea Pigs; Leucine; Leupeptins; Lung; Male; Muscle Relaxation; Muscle, Smooth; Perfusion; Protease Inhibitors; Time Factors; Trachea; Trypsin Inhibitors; Vasoactive Intestinal Peptide