K. Ohkubo et al., HUMAN NASAL MUCOSAL NEUTRAL ENDOPEPTIDASE (NEP) - LOCATION, QUANTITATION, AND SECRETION, American journal of respiratory cell and molecular biology, 9(5), 1993, pp. 557-567
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.