Aminopeptidase activity in human nasal mucosa

Abstract

Background: Aminopeptidases activate bradykinin and degrade many inflammatory peptides.

Objective: The objective of this study was to identify the types of aminopeptidase activities in human nasal mucosa.

Methods: Human nasal mucosa was homogenized (n = 12), and cytoplasmic (S2) and membrane-rich (P2) fractions were obtained. Several aminopeptidase (Ap) activities were defined by (1) substrate specificity with leucine-enkephalin (leu-Ap) and alanine-nitroanilide (ala-Ap), (2) inhibitor studies with puromycin and bestatin, (3) enzyme activity histochemistry (zymography), (4) immunohistochemistry, and (5) gel electrophoresis. Human volunteers had methacholine, histamine, and allergen nasal provocations to determine the mechanisms controlling nasal aminopeptidase secretion in vivo.

Results: P2 was the largest reservoir of puromycin-resistant aminopeptidase activity (630 pmol leu-enk/min/mg protein). S2 contained 32 pmol leu-enk/min/mg activity, with 80% representing puromycin-resistant activity and 20% puromycin-sensitive aminopeptidase (PS-Ap). Ala-Ap was detected in both P2 and S2 fractions and was localized by zymography to epithelial and gland cells. Anti-rat brain-soluble PS-Ap IgG detected immunoreactive material in epithelium, glands, and endothelium. In nasal provocation studies, leu-AP correlated with glandular exocytosis but not vascular leak.

Conclusions: The predominant aminopeptidase in human nasal epithelial and submucosal gland cells was membrane-bound puromycin-resistant aminopeptidase. A novel soluble puromycin-resistant aminopeptidase and lower amounts of soluble PS-Ap were also detected.

Fig. 1

Inhibition of leucine-enkephalin–degrading aminopeptidase (leu-AP) by bestatin (open circles ) or puromycin (closed circles ) in human nasal mucosal S2 and P2 fractions.

Fig. 2

Substrate competition study in human nasal mucosal extracts. A , Leucine-enkephalin–degrading aminopeptidase (leu-Ap) activities in S2 fractions (open circles ) were inhibited and shifted right by addition of alanine-p -nitroanilide (ala-NA) (filled circles ). B, Leu-Ap activities in P2 fractions (open circles ) were inhibited and shifted right by adding ala-NA (filled circles ). C, Alanine aminopeptidase (ala-Ap) activity of S2 fractions (open circles ) was inhibited and shifted right by adding leucine enkephalin (leu-enk, filled circles) . D, Ala-Ap activities in P2 fractions (open circles ) were inhibited and shifted right by adding leu-enk (filled circles ).

Fig. 3

Native-PAGE and enzyme blot of human nasal mucosa extracts. Lane 1 , Concentrated S2 fraction; Lane 2 , concentrated P2 fraction; Lane 3 , S2 fraction; Lane 4 , P2 fraction. A , Native-PAGE gel was stained by Coomassie blue. B , Alanine-aminopeptidase activity zymogram.

Fig. 4

Puromycin-sensitive aminopeptidase (PS-Ap)-immunoreactive material in human nasal mucosa. A, PS-Ap–immunoreactive material (intensely stained black material) was present in glycocalyx and epithelial cells (e) . Goblet cells did not stain for PS-Ap. Endothelial surfaces of small veins (arrow heads *) stained slightly. B, Nonimmune serum negative control. C, PS-Ap immunoreactivity was noted in inner lumen of gland ducts and glandular cells (g) . D, Negative control. Nuclear fast red counterstain. Bars represent 200 μm.

Fig. 5

Puromycin-sensitive aminopeptidase (PS-Ap)-immunoreactive material in human nasal mucosa. A, PS-Ap–immunoreactive material (intensely stained black material) was present in glandular serous cells (s) but not in mucous cell vacuoles (m) . Endothelium of veins (v) was also stained. B, Nonimmune serum negative control. Nuclear fast red counterstain. Bar represents 50 μm.

Fig. 6

Zymogram of aminopeptidase M activity in human nasal mucosa. A,l- alanine-4-methoxy-2-naphthylamide–cleaving aminopeptidase was localized in epithelium (e) and submucosal glands (g) . B, Inhibition control. Adjacent sections preincubated with 1 mmol/L puromycin showed no aminopeptidase activity. C, Aminopeptidase activity was most concentrated in central region of glandular acini. D, Inhibition control for C . Bars represent 100 μm.

Fig. 7

Methacholine and histamine-induced nasal secretions. Correlations between nasal lavage fluid concentrations of leucine-aminopeptidase (leu-Ap) activity and total protein (TP) (A, D ), lactoferrin (LF) (B, E ), and albumin (ALB) (C, F ) are shown after methacholine (A, B, C ) and histamine (D, E, F ) provocations. Coefficients of variation (r ) were determined by linear regression.

Fig. 8

Bestatin-inhibitable leucine-enkephalin–degrading aminopeptidase (leu-Ap) activity in nasal lavage fluids after antigen challenge (mean ± SEM). Atopic subjects (n = 8) received serial saline nasal lavages for 50 minutes, then were challenged topically with allergen at 60 minutes. Lavages were then collected at 10-minute intervals between 70 and 120 minutes and at 20-minute intervals between 140 and 180 minutes.

Fig. 9

Allergen-induced nasal secretions. Data and coefficients of variation (r ) determined by linear regression are shown for secretion of leucine-enkephalin–degrading aminopeptidase (leu-Ap ) and A , total protein (TP ); B , lactoferrin (LF ); and C , IgG (IGG ) concentrations.