Vasoactive intestinal peptide in human nasal
mucosa.
J N Baraniuk, … , J H Shelhamer, M A Kaliner
J Clin Invest.
1990;
86(3)
:825-831.
https://doi.org/10.1172/JCI114780
.
Vasoactive intestinal peptide (VIP), which is present with acetylcholine in parasympathetic
nerve fibers, may have important regulatory functions in mucous membranes. The potential
roles for VIP in human nasal mucosa were studied using an integrated approach. The VIP
content of human nasal mucosa was determined to be 2.84 +/- 0.47 pmol/g wet weight (n =
8) by RIA. VIP-immunoreactive nerve fibers were found to be most concentrated in
submucosal glands adjacent to serous and mucous cells. 125I-VIP binding sites were
located on submucosal glands, epithelial cells, and arterioles. In short-term explant culture,
VIP stimulated lactoferrin release from serous cells but did not stimulate
[3H]glucosamine-labeled respiratory glycoconjugate secretion. Methacholine was more potent than VIP, and
methacholine stimulated both lactoferrin and respiratory glycoconjugate release. The
addition of VIP plus methacholine to explants resulted in additive increases in lactoferrin
release. Based upon the autoradiographic distribution of 125I-VIP binding sites and the
effects on explants, VIP derived from parasympathetic nerve fibers may function in the
regulation of serous cell secretion in human nasal mucosa. VIP may also participate in the
regulation of vasomotor tone.
Research Article
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Vasoactive Intestinal Peptide in Human Nasal Mucosa
James N. Baraniuk, Jens D. Lundgren,* Michiko Okayama, Joaquim Mullol, Marco
Merida,*
James H. Shelhamer,* and Micheal A. KalinerAllergicDiseases Section,LaboratoryofClinicalInvestigation, National Institute ofAllergy and Infectious Disease, Bethesda, Maryland 20892; *IntensiveCare Unit, ClinicalCenter, National Institutes ofHealth, Bethesda, Maryland 20892; and tDepartments ofOtolaryngologyandPediatrics,Georgetown University Hospital, Washington, DC
Abstract
Vasoactive intestinal peptide (VIP), which is present with
ace-tylcholine
inparasympathetic
nervefibers,
mayhave impor-tantregulatory functions
in mucous membranes. The potential roles for VIPin
human nasal mucosa were studied using an integrated approach. The VIP content of human nasal mucosa wasdetermined to be 2.84±0.47 pmol/g wet weight (n=8) by RIA. VIP-immunoreactive nervefibers
were found to be mostconcentrated
in submucosal glands adjacent to serous and mucouscells.125I-VIP
binding sites were located on submuco-salglands, epithelial
cells, andarterioles.
In short-term ex-plant culture, VIP stimulated lactoferrin release from serous cells butdid
notstimulate [3Hjglucosamine-labeled
respiratoryglycoconjugate
secretion. Methacholine was more potent thanVIP,
andmethacholine stimulated
bothlactoferrin
and respira-toryglycoconjugate
release. Theaddition of
VIPplusmetha-choline
toexplants resulted
inadditive increases
inlactoferrin release. Based upon the autoradiographic distribution of125I.
VIP binding sites and the effects on explants, VIP derived from
parasympathetic
nervefibers may function in theregula-tion of
serouscellsecretion
in human nasalmucosa.VIP may alsoparticipate
inthe regulation of
vasomotortone.(J.Clin.
Invest. 1990. 86:825-831.) Key
words:respiratory
glycoconju-gates *mucus*
lactoferrin
*parasympathetic
nervoussystemIntroduction
Vasoactive intestinal
peptide
(VIP;'
28 amino acid
residues;
1)
is
aneurotransmitter in
postganglionic
parasympathetic
neurons.
Preganglionic parasympathetic
nerves of thenasal
mucosa
originate in
thesuperior
salivatory
nucleusof
the sev-enthcranial
nerve, passthrough
theVidian nerve, and synapsewith
postganglionic cell bodies
inthesphenopalatine ganglion
(2-4).
Postganglionic cholinergic
neuronscontainVIP,
pep-tide with histidine
attheNH2
terminus and methionineattheCOOH terminus
(PHM),
and both cholineacetyl
transferaseand
acetylcholinesterase,
which indicate the presence oface-tylcholine (5-7).
Theneurons enterthemucosaviatheposte-Address reprint requeststo Dr. James N. Baraniuk, Laboratory of ClinicalInvestigation, Bldg. 10,Rm 1 -C-205, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Received forpublication 13 October 1989and in revisedform 7
May 1990.
1.Abbreviations usedinthis paper:P1,
period
1;P2,period
2; PHM, peptidewith histidine atthe NH2 terminus and methionineat theCOOHterminus;VIP, vasoactive intestinal
peptide.
The Journal of Clinical
Investigation,
Inc.Volume86,September
1990,
825-831rior nasal nerves and innervate submucosal glands, arterioles, and venules (3, 4, 7).
Stimulation
of parasympathetic neurons leads to the release of acetylcholine, which acts uponmusca-rinic
receptors on submucosal glands (8) and possibly vessels(9, 10).
In humans,
stimulation of
submucosal glands by exoge-nousmethacholine
(8) or central reflexes (1 1) leads tosecre-tion
of such
mucouscell products as mucousglycoconjugates,
and
serous cell products suchaslactoferrin,
lysozyme, secre-tory component, andsecretory
IgA(8, 11-13).Stimulation
of vessels by either cholinergic agonists or centralparasympa-thetic
reflexes
has been suggested to causevasodilation
(9, 10, 14, 15) and maycontribute
tothesecretion of
plasma productssuch
asalbumin and IgG. Parasympathetic efferent reflexes
may be
initiated
byafferent
sensory nervestimulation
(1 1, 13), orby "short circuit" stimulation of
postganglionic
parasympa-thetic
cellbodies
bynociceptive,
substanceP-containing
tri-geminal
neurons(16).
Inaddition,
electrical stimulation of
thesphenopalatine
ganglion
leadstoatropine inhibitable
nasalse-cretion
andatropine-resistant
vasodilation
(3).
Since
VIPis
releasedalong with
acetylcholine
by
parasym-pathetic
nerves, VIP mayplay
animportant
rolein
regulating
nasal responses. However, there
is little understanding of
thefunctions of
VIPin
thenasal
mucosa.Therefore,
theconcen-tration of
VIP inhuman nasalmucosa wasmeasuredby
RIA,the locations of VIP-immunoreactive
nervefibers
weredeter-mined
byimmunohistochemistry,
thedistribution of
'251I-VIP
binding
sites
wasdetermined
by
autoradiography,
and theef-fects
onthe
secretion of
mucousandserouscell
products from
submucosal
glands
weredetermined by
exposing
humannasal
mucosal
fragments
toVIPin short
termexplant culture.
Based uponthis
integrated approach, inferences
weremade about therole of
VIP in theregulation of
vascular andglandular
re-sponsesof
human nasalmucosa.Methods
Tissuehandling.Human inferior turbinateswereobtainedatthetime ofsurgery from 35 patients with nasal obstructivesyndromes. No
patienthadhadarecentinfection.Atthetime ofsurgery,2%tetracaine HCl and 0.25%phenylephrine HClwereapplied topically onnasal
packs. The turbinateswereinjectedwith2-4 mlof1%lidocaine with
1:100,000
epinephrine.Anincisionwasmade from the lateral wall of the inferior turbinate at the level of the infundibulum through theinferior conchalbone, andtheninferiorlyalongthemedialaspectof
that bone. Themedialflapof turbinatethatremainedwaswrapped superiorlyandlaterallytoclosethe wound. Within 20minof
surgical
excision, the nasal mucosa wasdissected from the inferior conchal bone. Mucosal specimens for
autoradiography
were frozen in 2-methyl-butaneondry ice for 20s, then storedat-700C.Specimens
for explantculturewereplacedinL15 media(Biofluids, Rockville, MD)
supplementedwith100
,ug/ml
penicillin, 100;g/ml
streptomycin,
andTissuefixation. Fresh tissues for immunohistological examination were microwave fixed. Individual 5 X2 mm fragments of tissue were placed inplastic scintillation vials (Kimble, Toledo, Ohio) with 10 ml of 1.5% paraformaldhyde and 0.05% glutaraldehyde in pH 7.4, 50 mM sodium phosphate, 0.1 MNaCl (PBS) at room temperature and mi-crowaved (400W, Sharp Instruments, Mahwah, NJ) for 5 s (17). The temperatureof the solutionwasraisedto45±50C. After fixation, sam-ples were stored at40Cbefore being embedded in paraffin (American Histolabs,Gaithersburg, MD).
Turbinate extract preparationforRIA. Frozen turbinate tissue fromsingleindividualswasweighed (wet weight) and the tissue finely dissected withrazorblades.Three extract procedures wereconttasted. (a) Boiling 0.1Naceticacid in distilled water was added (20id/mg tissue) and boiled for 30 min (adapted from reference 18). During these 30 min, the preparation was homogenized in a ground glass tissue homogenizer, and sonicated atthreeintervals (Heat Systems-Ultra-sonics, Inc.,Plainview, NY) for 30seachat asetting of6. The suspen-sionwascentrifuged (1,700 g, 30
min,
40C)andthesupernatantwaslyophilized.
(b) Asolutionof cold 50% ethanol, 50% 0.1 N acetic acid, 0.02% sodium bisulfite in distilled water was added at 20Ml/mgtissue, and chilledat40Cfor 20 min (adapted from references 19 and 20). During this period, the mixture sonicated three times for 30 s each on ice. The tubeswerecentrifuged andthesupernatant waslyophilized.
(c) 2mlofacetone at40Cwereadded to 100-300 mg of turbinate tissue andsonicated as above (adapted from reference 21). After cen-trifugation, the supernatant was dried by Speed-Vac (Savant Indus-tries, Hicksville, NY).
Thepowdered extracts were resuspended in RIA buffer(0.1% BSA, pH 7.4, 0.1 Msodium phosphate, 0.05 MNaCl, 0.01% NaN3, 0.01% Tween-80) so that powdered extractequivalent to 10 mg of original tissuemass wassuspended in 100
Al
ofRIAbuffer.RIA. RIA reagents were purchased from Peninsula Laboratories (Belmont,CA)andincluded 3,326 g/mol VIP, 700 Ci/mmol
'25I-tyr-VIP, polyclonalrabbit antiserum to VIP, goat anti-rabbit gamma glob-ulin serum,and normalrabbitserum.Standardpeptide solutionswere
prepared for the range from 0.15to77.0 fmol per tube (0.5-256 pg per tube).As acontrol, samples of ethanol-acetic acidextractsof human nasalmucosaequivalentto5 mgof tissuewerealso addedtostandard
amountsofVIP todetermine if the addition of tissueextractsaffected the accuracy of theRIA.
Turbinateextracts werediluted and aliquottedto giveextracts
equalto 10, 3,and I mg of turbinate tissue per tube. Rabbit anti-VIP
serum wasadded. After overnight incubation at 4°C, 1251-VIP was added. Afterasecondovernight incubationat 4°C, polyclonal goat anti-rabbit gammaglobulinandnonimmune rabbitserumwereadded for2 h at roomtemperature.RIAbufferwasadded,thetubes centri-fugedat1,700 g for 40 min, and the supernatantsaspirated.Thepellets
werecounted inagamma scintillationcounter(Beckman Instruments, Irvine, CA)and thepercentage ofbound to total counts
(B/BO%)
wasdetermined.
The linearportion ofthestandard curve wasanalyzed by linear regression and theyield ofVIPpertube wasinterpolated.The femto-moles ofVIPpertube andpicomoles per gram turbinate tissuewere
calculated. Themean±SEMpicomolesper gram turbinate tissue for
eachextraction methodwasdetermined.
HPLC. Nasaltissue wasextracted in ethanol-acetic acid, lyophi-lized, and reconstitutedineluentA(0.12% trifluoroaceticacid; Sigma Chemical Co., St.Louis,MO). After30
min,
nonsolublematerialwasremovedbycentrifugation.An 1
50-IA
aliquotwasappliedto ahigh performanceliquidchromatogram(114Mpumps, 421controller, and 164detector; Beckman Instruments, Inc.,Fullerton, CA)usingaC-18reverse-phasecolumn(ODS Ultrasphere,4.6X25 cm,5Jimporesize, BeckmanInstruments, Inc.).Sampleswereeluted5minafterinjection using45-min runsand alineargradient of 0-90%eluent B(0.12% trifluoroacetic acid in acetonitrile) in eluent A (22). Synthetic VIP
(Peninsula Laboratories)wasused tostandardize the column.
Frac-tionswerecollectedat
1-min
intervals,frozen, lyophilized,
andrecon-stituted in RIAbuffer. TheVIPcontentof each fraction was deter-minedbyRIA.
Indirect
immunohistochemistry.
6-Mtm-thickparaffin
sectionsweresequentially placed
inxylene,
graded
alcohols,
distilled water,PBS,
and PBS with 1% nonimmune goatserum
(11,
13).
Sections wereincubated with rabbitantisera(1:1,000)toVIP
(Peninsula
Laborato-ries)orwith nonimmune rabbitserumfor 18or44hat4VC.Theslideswerewashed inPBS, reblocked with 1% nonimmune goatserum in PBSfor 3 minat roomtemperature, and then 1:40 colloidal gold-la-beled goat anti-rabbit gamma
globulin (Auroprobe;
Janssen Pharma-ceuticalsPiscataway,
NJ)wasaddedfor 60 minatroomtemperature. Afterbeingwashed threetimes in PBS for5min each and three times indistilledwaterfor 3 min eachat roomtemperature, silverenhancing
solution(IntenSE;Janssen
Pharmaceuticals)
wasadded and the devel-opment of the stainedstructures wasmonitoredby
light microscopy.
Theslideswerewashed in distilled water,dehydratedingraded alco-hols,andcoverslips appliedwithPermount
(Sigma
ChemicalCo.).
Autoradiography.
Frozensections of tissuewerewarmedtoroomtemperature, incubated inpH 7.4,50 mM
Tris,
3 mMMgCl2
for 10minat
250C,
thenincubatedwith 1 nM251I-VIP
inpH 7.4,50 mMTris, 5 mM
MgCI2,
2%polypep
(Sigma
ChemicalCo.),
1MM thior-phan, 1MMphosphoramidon,
40mg/liter bacitracin,
5mg/liter
chy-mostatin, and4
mg/liter
leupeptin
for 3 h at37°C (adapted
from reference23).
Nonspecific
binding
wasdeterminedby adding
1JAM
VIP totheincubation mixture. After incubation the slideswerewashed twice inpH 7.4,50 mM Tris for5min eachat
4°C.
Theslidesweredried witha streamof cold
dry
air.Nuclear track emulsion
(NTB-2;
EastmanKodakCo., Rochester,
NY)wasmelted in thedark for4hat
45°C
and then mixed with 1% glycerolinwater.Inadarkroom,
slidesweredipped
inemulsion,
the backs of the slideswiped clean,
and theslidesplaced
vertically
for 2hto allow the emulsion to run off inan even fashion and
dry (24).
Coatedslideswere
placed
inplastic
boxeswithhygroscopic
calcium sulfate(W. A. Hammond DrieiteCo.,
Xenia, OH)
and storedat-200C.
Slideswereremovedatintervals andwarmedtoroomtemperature
in thedark.Theywere
developed
inD-19developer (Eastman
KodakCo.)
for 2 minat220C,
washed inwaterfor 30s,fixed in Kodak fixer for4min, andfinally
washed inwaterfor 15 min.Human nasal mucosal
explant
culture.Toquantify
the releaseofsecretory
cellproducts
in responsetoVIP,
freshhumannasalmucosawas cut into 3X 3mm
fragments (25,
26).Pairs offragments
wereplacedon
gelfoam
inpetri dishes,
and cultured in2 mlCMRL 1066containing
100Mg/ml
penicillin,
100Mg/ml
streptomycin,
0.5Ag/ml
amphotericin
(Gibco Laboratories,
GrandIsland,
NY),
and 1MCi/ml
[3Hlglucosamine
(NewEngland Nuclear, Boston,
MA).
The[3HJglu-cosamine becomes
incorporated
intonewly
synthesized respiratory
glycoconjugates (25,
26).
After24h,
the mediawaschanged
and 400KIU/ml
aprotinin
(Sigma
ChemicalCo.)
wasaddedtothe mixture. Afteranadditional 24h,
fresh media withaprotinin
wasaddedfor4h(period
I[Pl]).
The culturesupernatantfromthis baselineperiod
wascollected and then
replenished
forIhour(period
2[P2])
by aprotinin
containing
media with0.1, 1,
and5MMVIP(Peninsula
Laboratories),
1,
10,
and100MMmethacholine(Sigma
ChemicalCo.);
combinations ofVIPandmethacholine;
ormedia(control
plates). Supernatants
fromP1
and P2wereused forquantitation
ofrespiratory
glycoconjugates
and lactoferrin.
Respiratory
glycoconjugate
releasewasquantified by
precipitation
of
[3H]glycoconjugates
in 10% TCA and 1%phosphotungstic
acid (TCA/PTA)at5°Covernight.Theprecipitates
werepelleted by
cen-trifugation (1,200
g for 10min),
washed twice withTCA/PTA
and hydrolyzedin0.1 MNaOH.Aliquots
of theresuspended precipitates
wereusedforscintillation
counting.
Theratiosofdisintegrations
per minutefor P2to P1(secretory
index)
werecalculated foreach treat-ment.Thesecretory
indiceswerecompared
with controlcultures,
and the percentchange
from control(mean±SEM)
wascalculated. Resultswere
statistically
compared
tocontrolsusing
thepaired
ttest.Lactoferrin ELISA. Quantitation of lactoferrin release into the culturemedia from P1 and P2 foreachtreatmentwasperformed by usinganoncompetitive ELISA (1 1). Microtiter plates were coated with 50
gl
of rabbit anti-human lactoferrin (Dako Corp., Santa Barbara, CA) diluted 1:1,000 in 0.1 Mcarbonatebuffer, pH 9.6,andincubated at40C overnight. The wellswerewashedwith 4 vol ofabuffer (PT) consisting of PBS, pH 7.4, with 0.05% Tween 80 (FisherScientific,Fair Lawn,NJ).Afterblocking nonspecific binding sites with 200yd
of1%goatserum(Gibco Laboratories) inPTfor 30 minat room tempera-ture,50
,d
of mediaorstandard(diluted in PT)wasadded to each well andincubated at370C for 90 min. Then 50,d
of goat anti-human lactoferrin conjugated tohorseradish peroxidase (Organon Teknika-Cappel, WestChester, PA)wasaddedandincubatedat370Cfor 90 min.The colorreactionwasdeveloped withano-phenylenediamine dihydrochloride substrate(SigmaChemical Co.), and then the optical densities of the plateswerereadat490nm on anELISAreader (Dy-natechLaboratories,Alexandria, VA). The ratios of optical densities from the supernatants collected during P2to thatfor P1 (secretory index) were calculated for each treatment as above, and the mean(±SEM) percent change insecretoryindices fromcontrolvalueswas
calculated. The secretory indices for eachtreatment werecompared with the control values by pairedttest.
Results
RIA. The sigmoid standard curve was linear between 1.2 and 38.5 fmol (4 and 128 pg) per
tube.
Using linear regression,
the squares of thecorrelation
coefficients for
the standard curvesa
b
f
W
I
4.
c
werebetween
0.96
and0.99.
Thesensitivity (concentrationatB/Bo
=50%)
of
theassay was5.83±0.62fmolper tube (n = 5). Theaddition
ofethanol-acetic acid
extractequivalent
to5 mg of nasalmucosadid
notaffect
theshape of
the standardcurveorthe
slope
of thelinear portion,
eventhough
the additiondid
shift thecurve
slightly
totheright. The
VIPcontentofhuman
turbinate nasal mucosa was
estimated by interpolation of
B/Bo%
values from thelinear portion of
the standardcurve.Samples
extracted withboiling acetic acid yielded
2.84±0.47
pmol
per g wet weighttissue
(n = 8). Contentranged from 1.07
to4.61.Samples extracted with ethanol-ace-tic acidyielded
2.84±0.38 pmol
perg(n
= 17patients) with
arange
from 0.70
to 5.80. Samples extracted with acetoneyielded 0.80±0.10
pmolperg(n = 4), with a rangebetween 0.23 and1.03.
HPLC. Synthetic VIP (25
,ug)
elutedfrom the HPLC col-umnwithasingle,narrowpeakat25.96min. Usingthe RIA, VIPelutedonly
infractions
collectedat25 and 26min.
When turbinate tissuewaseluted from the HPLC column, VIPim-munoreactive materialwascollected only at25 and 26 min. All other fractions for both the standards and tissue contained no
immunoreactive
VIP.Indirect
immunohistochemistry.
Humaninferior turbinate
nasalmucosais covered by
ciliated
pseudostratified columnarrespiratory epithelium
(Fig. 1). Beneath the basement mem-brane arefenestrated capillaries (27).
Beneath this vascular..)...@..
A~~~~~~~~~~~~~.q.~~~~~~~~~~~~~~~~
M
q
--S
M
r -_
I.. _
,,
:
...S
...:..
.R#
e &"
.K. >.. s
S
M
V.
Figure1. VIPimmunoreactivenervefibers in human nasalmucosa.
(a)
VIPnervefibers(arrowheads)
are seenina nervebundle(NB)
and in thewallofasmall venule(V).
Hematoxylin
counterstain.(b)
AVIPnervefiber(arrowheads)
isseeninnervating
mucouscells(M)
ofa submu-cosalglandacinus.Nuclearfast red counterstain.(c)
VIPnervefibersareseeninnervating
serouscells(S)
andmucouscells(M)
ofa submuco-salgland. Nuclear fastredcounterstain.(d)
Negative
control.VIPantiserum adsorbedwithexcessVIPdidnotidentify
VIPimmunoreactivestructuresin theseserous
(S)
andmucous(M)
aciniiorvenule. Nuclearfast red counterstain.Bars,
25Am.I
zone are
interspersed
tubuloacinar seromucous submucosal glands and their ducts. In the connective tissue between sub-mucosal glands are large venous sinusoids. Coiled arterioles and nerve bundles are locateddeeper in the mucosa.VIP-immunoreactivematerial was identified readily in tis-sue fixed by microwave irradiation. Microwave fixation al-lowed
rapid tissue
processing and excellent preservation oftissue histology,
andimproved
the identification of immuno-reactive nerve fibers whencompared with other fixationtech-niques
( 17).This
mayreflect
the rapidity of tissue fixation by microwave irradiation.Staining sections with
nuclear fast red (Sigma Chemical Co.) allowedtissue definition
which did not obscure nervefibers.
Otherstains
obscured detection of immunoreactive VIP nerves which could otherwise be seen in unstained sections.VIP immunoreactive nerve fibers were most frequently encountered
in
submucosal glands (Fig. 1). The fibers wereoften in direct
contactwith acinar cells. There was apparent contactwith
both serous and mucous cells, and with themyo-epithelial cells that
surround eachacinus.
VIPfibers
were seenin
nerve bundles. Some VIPnerve fibers were also detected in the wallsof arterial
and venous vessels. More fibers were seenin
glands than vessels. Nofibers
werefound in the epithelium.Autoradiography.
'25I-VIP binding
sites on the epithelium and submucosalglands of
human nasal mucosa were detected asdeposits of silver grains using darkfield
microscopy (Fig. 2). It was notpossible
to determine if1251I-VIP
boundpreferen-tially
tospecific
subsetsof
cells such as goblet cells, serous cells, or mucous cellssince the
useof
mucous cell-specific stains such asalcian
blue damaged the emulsion and dispersed theI:
s
..-* \
g
4
a.
I_.
._ e
i, .
I
**? J
"-iv *
silver grains.
There alsoappeared
tobebinding of
'251-VIP
to the vascular smooth muscleof
arterioles(Fig. 3).
It was notpossible
todifferentiate between
smooth muscle orendothelial
binding
of1251I-VIP
due to the scatter ofradiation from 1251
decay. However, venous sinusoids bound very
little '25I-VIP,
suggesting that the endothelium of
thesethin
walled vessels had veryfew
or no VIPbinding sites.
Theaddition of
excess VIP ablated binding to epithelium, glands, and arterioles (Figs.2fand
3
d).
Human
nasal
mucosal
explant culture. Methacholine
(MC) between 1 and 100 ,uMinduced
dose dependantsecretion of
lactoferrin
and[3H]glucosamine
labeledrespiratory
glycocon-jugates
(TableI).
Releaseof lactoferrin
wassignificantly
stimu-lated at 1gM
MC whereasglycoconjugate
release wasun-changed from control,
indicating that either lactoferrin
con-taining
serous cells responded to lower concentrations ofmethacholine
than glycoconjugate containing cells, or that changesin
lactoferrin secretion
were morereadily
detected than changes inglycoconjugate secretion.
VIPin
dosesof
0.1-5
IgM
induced
significant lactoferrin secretion,
butdid
notaffect glycoconjugate secretion.
Atconcentrations of
1 uM,methacholine
was more potent than VIP atinducing
lactofer-rin secretion.
Since
VIP andacetylcholine
may besimultaneously
re-leasedfrom
parasympathetic
nerves,the effects of
combina-tions
of methacholine
and VIPwerestudied.
Theaddition of
VIPto 1 uM
methacholine did
notstimulate
glycoconjugate
release or augment
the
releaseof lactoferrin. The addition of
VIPto 10
1AM
methacholine did
notaffect
glycoconjugate
re-lease.Lactoferrin release
wasincreased
inadditive
but notFigure 2.
'25I-VIP
bindingsites inhumannasalmucosa.(A)Brightfield imageofepithelium (e) and laminapropria.Submucosalglands(g)and sinusoids(s)are seen.(B)Darkfieldimageof the
samefieldasA,indicating'25I-VIP
binding
to theepithelium(e)andsubmucosalglands(g). Sinusoids(s)didnotdemonstratebinding.
(C) Brightfield imageofadensecollection of sub-mucosalglands (g)andadjacentvenous sinu-soids(s). (D)Darkfieldimageof thesamefieldas(C)showing 1251-VIP bindingtosubmucosal glands (g)butnotsinusoids (s).(E)
Brightfield
image fromaconsecutivelycuttissue section showingvessels(v)andsubmucosal
glands (g)
on aslide treated with1
AM
VIP.(F)
Darkfield imageof thesamefieldasE,showing
thatthe addition ofexcessVIPablated thebinding
of'251-VIP.
Toluidine bluecounterstaining.
Bars, 50 Am.Baraniuk,
Lundgren,
Okayama, Mullol, Merida, Shelhamer,
andKaliner.-. *fi~ a :.
a ..<.-...- A... 1
< f;'' l/
tP _ b I ,,
_ A
~
.q -0,
vle
J.
ill.*.4
-..".0,
AkA' -~,
'd n.tr~
,.,
i.
.. 'U."
Figure3. '25I-VIP bindingsites in human nasalmucosa.(A)Brightfield imageofanarteriole(a),submucosalglands (g),andsinusoids(s). (B) Darkfieldimageof thesamefieldasA, showing '25I-VIP bindingtothe arteriole(a)andglands (g),butnobindingtothevenousstructures(s). (C) Brightfield image showingseveral vessels with vascular smooth muscle in their walls(arrowheadsindicaterepresentative areas).Several
ar-terioles(a),thin muscularveins,andvenoussinusoids(s)areshown.(D)DarkfieldimageofthesamefieldasC showssilvergrainsoverthe
areasofvascular smooth muscle(arrowheads).Areaswithoutsmooth muscle did not appear to demonstratebinding.Toluidine blue counter-staining. Bars,50,gm.
synergistic fashion by
5AM
VIPplus
10AM
methacholine. These resultsindicate
that VIPstimulated
serous cellsecre-tion,
but that VIPwasless potent thanmethacholine.
VIPdid notpotentiate
theeffects of methacholine
onsecretion from
either
serouscells or[3H]glucosamine-labeled
respiratory
gly-coconjugate-containing
cells.TableI.
Effects
of
VIPand Methacholineonthe ReleaseofLactoferrin
and[3H]Glucosamine-labeled
Respiratory
Glycoconjugates
from
HumanNasalMucosa inExplant
CultureVIP MC n Lactoferrin Glycoconjugates AM MM % Change P %Change P
0 100 16 460±96 <0.001 31.8±7.4 <0.001
0 10 7 563±177 <0.02 17.0±12.0 NS 0 1 6 124±24 <0.005 -0.6±2.6 NS
5 0 14 132±37 <0.01 3.9±5.1 NS
1 0 9 114±29 <0.01 -2.2±3.8 NS
0.1 0 9 90±35 <0.05 8.9±12.1 NS
5 10 9 759±320 <0.05 16.6±9.9 NS
1 10 7 574±203 <0.02 13.0±5.1 <0.02 0.1 10 6 411±125 <0.01 16.6±6.9 <0.01
Mean±SEM;unpairedt test.
Discussion
VIPwaspresentinhuman nasalmucosa.The concentrationof
acid ethanol extractable VIP(2.84±0.38 pmolper gtissue)was
slightlyless thanthe concentration ofneuropeptideswhichwe
have measured previously. Neuropeptide Y(NPY), a
neuro-transmitter ofsympathetic neurons in human nasal mucosa
(28, 29), hasaconcentration of 3.13±0.79 pmolper gtissue.
Theconcentration of VIPwasgreaterthan the concentration of calcitonin gene-related peptide (CGRP; 0.54±0.08 pmol
per g), a neurotransmitter oftrigeminal, nociceptive, type C
sensory neurons(16, 30-32). Therelative enrichment of VIP
reflects theabundant supplyofparasympathetic, cholinergic
nervestothenasalmucosa.
VIP-immunoreactive nerve fibers were located predomi-nantlyaround submucosal glands. Individual fiberswerealso
seen inthewallsofvenules and arterioles. The distribution of
VIPcontrasts with that of CGRP(32)from sensoryneurons,
andNPY(29)ofsympatheticneurons.CGRP fibersand NPY
fibers densely innervate arterial vessels, whereas individual
CGRP fibers and NPY fibers were present in the walls of
venous vessels and occasionally in submucosal gland acini.
CGRP fibers were also found in the nasal epithelium. VIP
fibers in the trachea and bronchi have been found around
bloodvessels, insmooth musclebundles, submucosalglands, andneartheepithelium (33, 34).
A
.I
I
O
,I .0
'25l-VIP
binding
sites
werefound
onsubmucosal
glands,
epithelium,
and in the wallsof
arteriolesand veins in human nasal mucosa. This was similar to the distribution of VIPfound
in the lowerrespiratory tract, where VIP receptors have beendemonstrated
on tracheobronchialepithelium,
submu-cosal glands,
andalveolar walls(23).
The highest density of VIPreceptors
were found on pulmonaryvascular
smooth muscle,and
bronchial smooth
muscleof trachea and bronchi(35, 36).
VIP-containing
neurons also contain aclosely
related pep-tide: PHM(37, 38).
Although VIP, PHM, and acetylcholine areall
present inthe
peripheral
neurosecretory varicosities ofpostganglionic parasympathetic
neurons(39),
the amounts ofeach
released during
neural transmission appears todepend
upon the nerve
impulse
frequency (39). At low rates,acetyl-choline is
selectively
released. Athigh
rates,acetylcholine
to-gether with
VIPand PHMarereleased. VIP may augment thepostsynaptic acetylcholine-induced
secretory response in glands (e.g., catsalivary glands, reference 40),
but may also havepresynaptic inhibitory effects which could
act tolimit
neuropeptide release.
This mechanism would conserve the amountof stored
peptides
since thereare nore-uptake
mecha-nisms, and VIPand
PHM canonly
beresupplied by
axonal transportfrom
the cellbody (37, 39).
Thisprinciple
may be veryimportant in understanding the physiology of
parasympa-thetic
neurons.VIP
stimulates
many aspectsof exocrine function and in-creases the contentof chloride in
intestinal and bronchialse-cretions,
andpancreatic
secretion of bicarbonate, water, andmacromolecules
(33). Inrespiratory
tissues, however, theef-fects of
VIPon glands have beeninconsistent.
Tracheal tissuesfrom ferret
(41, 42)and dog (43) release modest amounts ofrespiratory
glycoconjugates ("mucus") in response to VIP. VIPinduces
serous cellexocytosis from
ferret tracheal explants(42). Feline
trachealexplants
do not respond to VIP (44).However, isolated feline
submucosal glands do respond(44),
and VIP augments the
secretory
responsesof methacholine
(44)
and other secretagogues(45).
Inhumans, incubation of
tracheal explants with 0.2-2,000
nM VIPinhibited
[3H]glyco-conjugate
releasefrom normal
subjects,
but had noeffect
uponexplants from
subjects
with
chronic bronchitis (46).
The present
explant
resultsindicate
that VIPstimulated
lactoferrin secretion from
serous cells. VIP was less potent thanmethacholine
anddid
notsignificantly
augmentmusca-rinic
receptor-mediated stimulation of lactoferrin secretion.
VIP
did
notstimulate
[3H]glucosamine-labeled
respiratory
glycoconjugate
release anddid
notaffect
methacholine-in-duced
secretion.
The releaseof lactoferrin
was much moreresponsive
than[3H]glycoconjugate
release.
Either
serous cells were moresensitive
tomethacholine
thanglycoconjugate-containing cells,
orlactoferrin
wasamoresensitive
markerof
submucosal gland secretion
thanglycoconjugates.
Therefore,
VIPaugments the
effects of cholinergic stimulation
bycausing
selective enrichment of
serous cellproducts in
nasalsecretions.
The
activation
of
VIPreceptors on serous cellsof
submucosal glands represents anadditional
component which must beconsidered
whenstudying
parasympathetic reflexes
in human upperrespiratory
tract(1
1).
'25I-VIP
binding sites
werealsofound
on vessels. VIPin-duces
dilation of
humansubmandibular arteries
invitro
(15)
and
feline
nasal vessels invivo (47, 48).
VIP augments the cutaneousplasma extravasation
that canbeinduced
by sub-stance P(49).
Alterations of
VIP metabolism may contribute to severalclinical
syndromes. Increased VIP immunohistochemicalstaining in nasal
mucosahas beenreported in patients with anallergic diathesis
andsymptomsof
paroxysmal nasalobstruc-tion (50).
Increasedreleaseof
VIPand other neurotransmitters bythe
parasympathetic
route couldcontribute
tothe chronic vascularcongestion
and hypersecretion characteristic of someobstructive nasal syndromes.
In contrast,VIP-immunoreac-tive
nervefibers
may be absentfrom bronchial
walls inpa-tients
whodie of
status asthmaticus (5 1). These asthmaticpa-tients
werealsoreported
tohaveincreased relative
densitiesof
substance
P-immunoreactive
nervefibers in their
bronchial mucosa (52). Thesefindings
suggest that abnormalities ofneuropeptide containing
nerves could contribute to thepatho-genesis of respiratory diseases. Prospective studies
withcare-fully defined patient
groups andappropriate
controls arere-quired
toimplicate neuropeptides
inspecific
disease states. Theseobservations indicate
that VIP is present in human nasalmucosa
and thatVIP-immunoreactive neurons
inner-vatesubmucosal glands and vessels. VIP binding sites are pres-ent onthe
epithelium, submucosal
glands, and the wallsof
arterioles.
VIPstimulated lactoferrin
releasefrom
human nasal mucosalfragments. Since
serous cell products includespecific
andnonspecific antimicrobial factors, this selective
response
could
be animportant regulator of
mucosal hostde-fense mechanisms.
Acknowledaments
This work wassupported inpart by agrantfrom TheProcterand GambleCompany,Inc.(J.N.Baraniuk), bythe Danish Medical
Re-search Council(J. D.Lungren) andbyagrant from Glaxo,Inc.(J. Mullol).
References
1. Said, S. I., and V. Mutt, editors. 1988. Vasoactive intestinal peptideandrelatedpeptides.Ann.NYAcad.Sci. 527:1-689.
2.Konno, A.,andK.Togawa. 1979.Roleofthevidian nervein nasalallergy.Ann.Otol. Rhinol. Laryngol. 88:258-266.
3. Uddman, R., and F. Sundler. 1986.Innervation of the upper
airways. Clin. Chest Med. 7:210-219.
4.Uddman, R.,and F. Sundler. 1987.Neuropeptides inthe
air-ways: areview.Am.Rev. Respir. Dis. 136(Suppl.):S3-S8.
5. Costa, M., J. B. Furness, I. L. Gibbons, J. L. Morris, J. C.
Bornstein, I. L.Llewellyn-Smith, and R. Murphy. 1988. Colocaliza-tion ofVIPwith other neuropeptides and neurotransmitters in the autonomicnervoussystem. Ann.NYAcad.Sci.527:103-109.
6.Sundler, F.,E.Ekblad,T.Grunditz,R.Hakanson,and R.
Udd-man. 1988.VIP intheperipheralnervoussystem. Ann.NYAcad. Sci. 527:143-147.
7.Klaassen,A.B.M.,Y. J. B.vanMegen,W.Kuihpers,and P.van
den Brock. 1988. Utonomic innervation ofthe nasalmucosa. ORL
(Oto-Rhino-Laryngol.).
50:32-41.8.Raphael, G. D.,H. M.Druce,J. N.Baraniuk,and M. Kaliner.
1988.Pathophysiology of rhinitis. I.Assessmentofthesourcesof
pro-teininmethacholine-induced nasalsecretions.Am.Rev. Respir.Dis.
138:413-420.
9.Malm, L.,F.Sundler, andR.Uddman. 1980. Effects of
vasoac-tive intestinalpeptide(VIP)onresistance andcapacitancevessels in nasal mucosa. ActaOtolaryngol. (Stockh.).90:304-308.
10. Larsson, O., M.Duner-Engstrom,J. M.Lundberg,B. B.
Fre-holm,and A.Anggard. 1986. Effects of VIP,PHMandsubstancePon
blood vessels and secretory elements ofthe human submandibular gland.Regul. Pept. 13:319-326.
11. Raphael, G. D., M. Huptschein-Raphael, and M. A. Kaliner. 1989. Gustatory rhinitis: a syndrome of food induced rhinorrhea. J.
Allergy Clin. Immunol.83:110-115.
12. Baraniuk, J. N., G. D. Raphael, M. Merida, and M. Kaliner. 1988.Histochemical localization of macromolecules secreted by nasal mucosa.Am. Rev. Respir. Dis. 137:23 1.
13. Raphael,G.D.,S.D.Meredith, J. N. Baraniuk, H. M. Druce,
S.M.Banks, andM. A.Kaliner. 1989. The pathophysiologyof rhinitis.
II. Assessment ofthesourcesof protein inhistamine-induced nasal
secretions.Am.Rev.Respir.Dis. 139:791-800.
14.Hakanson, R., F.Sundler,and R. Uddman. 1982. Distribution and topography ofperipheral VIP nerve fibres: functional
implica-tions.InVasoactiveIntestinal Peptide.S.I. Said, editor.Raven Press, NY. 121-144.
15.Lundberg,J.M., A.Anggard,J.Fahrenkrug, 0.Johansson, and T.Hokfelt. 1982.Vasoactiveintestinalpeptidein cholinergic neurons
of exocrine glands.InVasoactive Intestinal Peptide. S.I. Said, editor. RavenPress, NY.373-389.
16. Lundblad, L. 1984. Protectivereflexesandvasculareffects in thenasal mucosaelicited by activation of capsaicin-sensitivesubstance
P-immunoreactive trigeminal neurons. Acta Physiol. Scand. Suppl. 529:1-42.
17.Login, G. R.,S. J.Schnitt,and A. M. Dvorak.1987.Methods in
laboratory investigation: rapid microwave fixation ofhuman tissues for light microscopic immunoperoxidase identification of diagnosti-cally useful antigens. LabInvest.57:585-591.
18. Skofitsch, G., andD.M.Jacobowitz. 1985. Quantificationof
calcitoningenerelatedpeptide inratcentralnervoussystem.Peptides (NY). 6:1069-1073.
19. Theodorsson-Norheim, E., E. Hensen, and J. M. Lundberg.
1985.Radioimmunoassay for neuropeptideY(NPY):characterization ofimmunoreactivityinplasma andtissue extracts.Scand.J.Clin. Lab.
Invest. 45:355-365.
20.Cooper, C. L., C.A.Marsden,andG.W.Bennett. 1987. Mea-surementofcatecholamines, indoleamines, thyrotropin releasing hor-mone, and substance P in rat and humanspinalcordusingacommon
extractionmethod.J. Neurosci. Methods. 22:31-39.
21.Pandian,M.R., A.Horvat,and S. I.Said. 1982.VIP. In
Va-soactive IntestinalPeptide.S. I.Said, editor.RavenPress,NY.35-50. 22.McDonald,T.J., F. L.Christofi,B. D.Brooks,W.Barnett,and M. A.Cook. 1988. Characterization ofcontentandchromatographic forms ofneuropeptides inpurified nerve varicosities prepared from guinea pigmyentericplexus.Regul.Pept. 21:69-83.
23. Carstairs,J. R.,and P. J. Barnes. 1986. Visualization of
va-soactive intestinalpeptidereceptors in human andguinea pig lung.J.
Pharmacol. Exp. Ther. 239:249-55.
24. Rogers, A. W. 1984. Practical Autoradiography. Review 20.
Amersham Corp.,ArlingtonHeights,IL.
25. Patow, C. A.,J.Shelhamer,Z. Marom, C.Logun,and M. A.
Kaliner. 1984.Analysis ofhuman nasalmucousglycoproteins.Am.J.
Otolaryngol.5:334-343.
26.Lundgren,J.D.,C. J.Wiedermann, C. Logun,J.Plutchok,M.
Kaliner,and J. H. Shelhamer. 1989. Substance P receptormediated secretion ofrespiratory glycoconjugate fromfelineairways in vitro.
Exp. Lung Res. 15:17-29.
27.Cauna, N.,and K. H.Hinderer. 1969. Finestructureofblood
vessels ofthe human nasal respiratory mucosa. Ann. Otol. Rhinol.
Laryngol.
78:865-879.28. Potter,E. K. 1988. NeuropeptideYas anautonomic
neuro-transmitter. Pharmacol. Ther. 37:251-273.
29.Baraniuk,J.N., S.Castellino,M.Merida,and M. A.Kaliner.
1989.NeuropeptideY(NPY)inhuman nasalmucosa. Am.Rev.
Re-spir.Dis. 139:A238.
30. Hua, X. Y. 1986. Tachykinins and calcitonin gene related
peptideinrelationtoperipheral functions ofcapsaicin-sensitive
sen-sorynerves. ActaPhysiol. Scand. 127(Suppl. 551):1-45.
31.Holtzer,P. 1988. Local effector functions of
capsaicin-sensitive
sensory nerve endings: involvement of tachykinins, calcitonin gene-relatedpeptideand otherneuropeptides. Neuroscience. 24:739-768.
32. Baraniuk, J. N., M. Merida, I. Linnoila, J. Shelhamer, and M. A. Kaliner. 1989. Calcitonin gene related peptide (CGRP) in humannasal mucosa. J. Allergy Clin. Immunol. 83:304.
33. Lundberg, J. M., L. Lundblad, C. R. Martling, A. Saria, J.
Stjarne,A. Anggard. 1987. Coexistence of multiple peptides and classic
transmitters in airway neurons: functional andpathophysiologic aspects.Am.Rev.Respir. Dis. 136(Suppl.):S16-S22.
34. Lundberg,J. M., and A. Saria. 1987. Polypeptide-containing neurons inairwaysmoothmuscle. Am. Rev.Physiol.49:557-572.
35.Said, S. I. 1982. Vasoactive peptidesin the lung, with special
referencetovasoactive intestinalpeptide. Exp. Lung Res. 3:343-348.
36. Lazarus, S.C.,C. B. Basbaum, P. J. Barnes, and W. M. Gold. 1986. cAMPimmunocytochemistryprovides evidence for functional VIP receptors in trachea. Am.J.Physiol. 251:C115-Cl19.
37. Fahrenkrug,J., T. Bek, and T.Hokfelt. 1985. VIP and PHI in cat neurons:colocalizationbutvariabletissuecontentpossibly due to
differentialprocessing. Regul. Pept. 12:21-34.
38. Itoh, N., K. Obata, N. Yanaihara, and H. Okamoto. 1983. Human preprovasoactive intestinal polypeptide contains a novel
PHI-27-likepeptide: PHM-27.Nature(Lond.).304:547-549. 39. Hokfelt, T., K. Fuxe, and B. Pernow. 1986. Coexistence of neuronal messengers: a newprincipleinchemicaltransmission. Prog. Brain Res.68:1-343.
40. Lundberg,J. M., A. Angaard,andJ. Fahrenkrug. 1981. Com-plementary roleof vasoactive intestinal peptide(VIP) and
acetylcho-line forcatsubmandibulargland bloodflowandsecretion. Acta Phys-iol. Scand. 113:329-336.
41.Peatfield,A.C., P. J. Barnes, and C. Bratcher. 1983. Vasoactive
intestinal peptide stimulated tracheal submucosalgland secretion in
ferret.Am.Rev.Respir. Dis. 128:89-93.
42.Gashi,A.A., D. B. Borson, W. E.Finkbeiner,J. A.Nadel,and C. B. Basbaum. 1986.Neuropeptides degranulateserouscellsof ferret
trachealglands.Am.J.Physiol.251:C223-C229.
43.Coles, S. J.,K.R.Bhaskar,D. D.O'Sullivan,K. H. Neill,and L. M. Reid. 1984. Airwaymucus compositionandregulation of its
secretion by neuropeptides in vitro. In: Mucus andmucosae. CIBA
Found. Symp. 109:40-60.
44.Shimura, S., S.Sasaki,H.Sasaki,and T.Takishima. 1988. VIP augments cholinergic-induced glycoconjugate secretion in tracheal
submucosalglands.J.Appl.Physiol. 65:2537-2544.
45. Richardson, P. S., and S. E. Webber. 1987. The control of
mucoussecretionin theairways bypeptidergic mechanisms.Am.Rev. Respir. Dis. 136(Suppl.):S72-S76.
46. Coles, S.J., S. I. Said, and L. M. Reid. 1981. Inhibition by vasoactiveintestinalpeptide of glycoconjugateandlysozyme secretion byhumanairwaysinvitro.Am.Rev.Respir. Dis. 124:531-536.
47.Hakanson, R.,F.Sundler,and R.Uddman. 1982. Distribution
and topography ofperipheral VIP nerve fibres: functional implica-tions.InVasoactive IntestinalPeptide.S. I.Said,editor.RavenPress,
NY 121-144.
48. Malm, L., F. Sundler, and R. Uddman. 1980. Effectsof
va-soactive intestinalpeptide (VIP)onresistanceandcapacitancevessels
innasalmucosa. ActaOtolaryngol. (Stockh.)90:304-308.
49.Khalil,Z., P. V.Andrew,and R. D. Helme. 1988.VIP modu-latessubstance P inducedplasma extravasationinvivo. Eur. J. Phar-macol. 151:281-287.
50. Kurian, S. S., M. A. Blank, and M. N. Sheppard. 1983.
Va-soactive intestinalpolypeptide (VIP)invasomotorrhinitis. Clin. Bio-chem. 11:425-426.
51.Ollerenshaw,S.L.,D. L.Jarvis,A.J.Woolcock,C. E.Sullivan,
and T.Scheibner. 1989. Absenceofimmunoreactive vasoactive intes-tinalpolypeptidefrom thelungsofpatientswithasthma. N.
Engl.
J. Med. 320:1244-1248.52.Ollerenshaw,S.L.,D. L.Jarvis,A.J.Woolcock,T.Scheibner,
and C. E.Sullivan. 1989.Substance Pimmunoreactivenervefibresin