HUMAN NASAL MUCOSAL NEUTRAL ENDOPEPTIDASE (NEP) - LOCATION, QUANTITATION, AND SECRETION

Neutral endopeptidase (E.C.3.4.24.11, enkephalinase, NEP) is a potenti ally important enzyme capable of regulating the activity of neuropepti des released in the respiratory mucosa. In order to confirm the existe nce of NEP in the human respiratory mucosa, inferior nasal turbinate m ucosae obtained at surgery and nasal secretions induced by topical pro vocations with methacholine, histamine, and allergen were analyzed for : (1) NEP activity (pmol product/min/ml) by enzymatic degradation of [ H-3]leu-enkephalin, (2) the presence of NEP-immunoreactive material by Western blot analysis, and (3) cellular localization of NEP distribut ion by immunohistochemistry. NEP activity in human nasal secretions ob tained after normal saline challenge was 0.15 +/- 0.06 pmol/min/ml. Se cretion increased to 0.86 +/- 0.26 pmol/min/ml after methacholine prov ocation and 1.69 +/- 0.74 pmol/min/ml after histamine provocation. The increase in NEP activity in methacholine-induced secretions was preve nted by atropine (0.13 +/- 0.06 pmol/min/nil). After methacholine, his tamine, and antigen nasal provocation, the kinetics of NEP appearance correlated more closely to the glandular marker, lactoferrin, than wit h the vascular markers albumin and IgG. In homogenates of nasal mucosa , the membrane fraction contained significantly more NEP on a per mg p rotein basis than did the soluble fraction (227.6 +/- 50.52 versus 9.6 1 +/- 3.18 pmol/min/mg protein, respectively, P < 0.01, n = 6). NEP in the membrane fraction was detected as a single band migrating at 97 k D on Western blots using antibodies specific for NEP and the common ac ute lymphoblastic leukemia antigen (CALLA). Immunoreactive NEP was loc alized to serous cells of the submucosal glands, epithelial cells, and endothelial and myoepithelial cells of small vessels. Staining for NE P in the serous cells was of the same intensity as that in epithelial cells. These results indicate that 97 kD NEP-immunoreactive material e xists in discrete locations in the nasal mucosa, including the epithel ium, serous cells of the submucosal glands, and vessel walls, and that NEP activity is detected as a minor component in nasal secretions enr iched by glandular products. In addition to the modulating functions o f NEP on neuropeptide-mediated activities on vessels and glands, it is possible that NEP in secretions plays a role in regulating mucosal re sponses to luminal neuropeptides or other as yet uncharacterized NEP s ubstrates.