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IRS ID No . 44-058'5497
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SLEP
Philip Morris
REVISED 10/16/91
EXPENSES
(Toxicology)(IInternational)
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(Toxicology) (Ingredients)
08/29/'91 Southern Consulting Group, Inc .--Consultation
fees - Irving,H . LaValle (Risk Decision
Theorist) (General)
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.75
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210 .00
194 .0 .11
2015002959
367 .50 .
759 .03
('Toxicology)i (International)
2196 .15
2863 .35
90 .72
1159 .71
444 .15
(Toxicology) (International)
(Pathology) ('International)
(Toxicology) (International)
(Toxicology) (International)
Toxicology) (International)
(Toxicology) (International)l
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3 Ys
Continued Next Page
2015002'9G0
Confidential
Attorney Work Product
Attorney-Client Privileg e
Working Draft
August 11, 1998
(1190809.01
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t
Confidential
Attorney Work Produc t
Attorney-Client Privileg e
Holcomb, Larry (HES)(3-1065 )
Layard, Maxwell W. (Layard Assoc .)(3-1067)
Leber, A. Philip (Chem-Tox Consultanting)(3-1085)
Lee. Peter (PNL Stats . & Comp . Ltd.)(3-932, 3-1195)
Leslie, George B. (BIOASSAY)(3-1194)
Levy, Leonard S . (Univ. Birmingham)(3-967)
Lewis, Trent R. 0(3-537)
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PHILIP MORRIS
INTERNATIONAL CONSULTANTS
"EUROQUOTES"
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TABLE OF CONTENTS
Tab
As hf ord
. 1
Barnes
. 2
Bridges
. 3
. 4
. 5
: 6
Caldwell
Chanter
*Cline
. 7
de Wolff
*Idle
. 8
James
. 9
*Lee
. 10
. 11
Litchfield
Mannaioni
. 12
Nilsson
. 13
Palacek
. 14
. 15
. 16
. 17
. 18
Roberf roid
Samanek
* Springa 1l
Strubelt
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Chairman,
Department
of
Thoracic
Medicine
11012906
http://legacy.library.ucsf.edu/tid/kbc90c00/pdf
and
Honorary
London
11012806
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MINUTES
SRG Meeting
October 19`h-22" d 1998
Lainston House, Sparsholt, Winchester, UK
Attendees :
Richard Baker (Chairman)
Scott Appleton
Eva Sshumacher-Wittkopf
Stewart Massey
Adrian Payne
Graham Read
Derek Irwin
The TMA funded Covance study on nicotine retention is due to next month . The
study is expected to take approximately two months . If the study,is completed, the
results will be presented at the next SRG meeting .
(Action MD)
321494969
321494970
aQ1lcg
32149497 1
To :
cc :
From :
Date :
Subject :
Dear All,
Adrian has received this review of the Molecular Genetics of COPD by Professor Peter Barnes
(that is now in press (Thorax), on the " Molecular Genetics of COPD" and will probably be
published in Feb/March 1999) and suggested that I forward it FYI
Copdgefn .doc
This review should give you all an overview of what is known about this area, associated
factors and what/why further research may be important .
Professor Barnes is considering submitting a proposal to the SRG next year on genetic markers
of susceptibility to developing COPD .
Regards
. . .Anna-Lisa
322017516
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
http://legacy.library.ucsf.edu/tid/pfs60a99/pdf
24 September 199 9
Department of Thoracic Medicine
National Heart and Lung Institute
Dovehouse Street
32526181 5
&11c'. US DO', Philip Moms
...Anna-Lisa Fisher
32526181 6
PATfl, I11 DO,] v Philir Mnmc
Imperial Colleg e
6 October 1999
Anna-Lisa Fisher
R&D Centre
Regents Park Road
Millbrook
Southampton SO15 8TL
Dear Ms Fisher
Thank you for asking me to write a report on mechanisms of COPD with special reference to
smoking. I will try to let you have this within the next six months . I briefly discussed this
with Adrian Payne who suggested that an article of approximately 20 pages with references
would be ideal .
Best wishes.
Yours sincerely
e c"-,
Prvj . Q c~.~r-c S
t'l6se .-e .
http://legacy.library.ucsf.edu/tid/bzk23a99/pdf
32526190 6
FLAT.-' U% 0(U v Philip -
22 December 1999
Imperial Colleg e
OF SCIENCE, TECHNOLOGY AND MEDICIN E
Dr E 0 Gregg
Scientific and Regulatory Studies Manager
British American Tobacc o
R & D Centre
Regents Park Road
Millbroo k
Dear Euan
Re : Application of Dr R P Youn g
I apologise for the delay in sending you my report . This is an ambitious project that is
important and Dr Young has expertise in molecular genetics . This is an area where several
other larger labs are already actively involved . I am surprised about the concern about
confidentiality since these precise studies are already underway in at least two locations and
these are very obvious studies to do !
I think that the project is worthy of funding .
Best wishes and happy New Year .
Yours sincerely
Enc.
http://legacy.library.ucsf.edu/tid/znu71a99/pdf
32527993 6
BATCo US DOJ v Philip Myna
http://legacy.library.ucsf.edu/tid/znu71a99/pdf
325279937
&4TC, US COJ v Philip Mane
O. B. Cohan
cfe:- c7^t' '*-%*1 %* IVKUai.l *\st K \ ( l k m ) \ ) U | P i t i
* I * * * - . " * " f**U* l*r ti*a** raSnni- f f^jim-*.} i*M9>%ra>lz*mt*S<al lAaV.nwi**
ABSTRACT
Bata adrenoceptor subtypes m canine tracheal smooth muscle
have been investigated by radioligand binding and by physiological responses to beta agonists and sympathetic nerve stimulation in vim. Specie binding of | ffjdihydroaiprenotol to tracheal
smooth muscle membranes was of high affinity (K * 1.0 0.08
nM). as in peripheral lung membranes from the same animals.
but the concentratnn of binding sites (95.0 4.7 fmol/mg of
protein) was much lower than m lung (532 48 imoymg of
protein). Binding was stereoselective and agonists competed
with the rank order of potency isoproterenol > epinephrine >
norepinephrine, signifying a preponderance of beta-2 receptors.
Airway* an? relaxed both in vitnt and in avn hy beta adrenergic agunit*. indicating the presence of beta adrenoceptor!*
tin airway smooth muscle. Lands el at. (1967) originally proposed Ihm beta adrenoceptors could be aubdivided into two
classes based on the varying patencies of adrenergic agonists
in different organs. Heto-l receptors present in adipose tissue
mediated lipolysis. and in the heart mediated chr inotropic and
isniropic response*, whereas brtw2 receptor* medhted relaxation nl smooth mtiM-le in airway*, blood vessels and uterus.
Thix organ-specific subclaiwiljcation wa* challenged by later
work using selective beta adrenoceptor antagonist A which
shiwed a mixed frrta-1 and frrfa-2 receptor response i n isolated
heart preparations iCarlsson ft at.. 1972: Ablad et at., IB731.
Similar mixed responses were also found in airway smooth
muscle of dog iMoissier 7 a/.. 1971), guinea pig iFurrhgott vt
H n ^ l " r | > u M H o l i . m l ^ - n J - f .'V |SJU
*Tlii * ! * * MipiH-ntil in pun In NslH>nl I f t M i l i i W n f H r s l l h I ' m i r i m
1'itwni l.Mtii H l . - i t l m and rntann
M i l I rum i b r Ceonnl lr Toliirro
ftrwnb
* 1 ' i o r n i W - M I l r I V I M - I BrB. l>piimfni.4MfdK>>.Hini7wrHiih
H n - m l n l D u c a l * H<wl. | j * H - i U I . ' I K K K V H M I I il M r d i m l H o r M r h
Citonul l t.ftv* I I H L H I I f ratrHmr l > * " * h i H
' N J | I | ' M M I h i m u m Irrnn ihr SrtliO> N m r i r I A K I * > H I H A K I I I H M H
flnrtfhM
t>murr*Jinrtlnain/in1tnwn('i)i<l1uirfflmjlii.ili*ui|j>in\ iSummtt.
VI i
*Hrc>|wni l Satmnul lriiiu<r. til Mrslik K r v i K h l r i r l Ih-trltpntrni
4M
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then contracted with serotonin tOilaMl ami heft the contravtinn WHM
stable t.lD rain!, the retasation response to eectrical field stumiUuvn
Do tracbeatis t w h w i i f p n f i n i l n . We obtaiwd tracheae was determmed. This response was inhibited by I iiM propranolol. The
tiom Ayp- which were being used in other etpenrarm* which did net effect of seleciivr brio adrenergic agonists on the response tr> tlehl
imvhe (he useol drug* other than anesthetic*. Dogs were anesthf tired stimulation was determined by prior mcubatiun 130 nun) with either
nuh pewuhartnwl sodium t.10rng/kg t.v.t.and the trachea was rapidly prxctolul t:lp.M) or IPS :i&) 10.1 uMl.
Data analyale. Result* are espresied as mtin * S . Statistk-al
removed. The posipnor membrane portion, of ihe trachea containing
the trachealw muscle was dissected free of loou connective tissue and comparisons were made by unpaired Student** l test. Binding data
the epiihelium as stopped awry. The tracheal*! muscle was the* were analyzed by a nonlinear least.squire* cutve fitting computer
separated and finely mmred with *ct*or* in 10 vulume* of ice-cold program. For competition studies parameters were chosen to yield the
incubation buffer i50 mM Tri* HO. iH .!!. thru homogenized in a best fit ol data, as determined by the mmunal variance of experimental
Polytron tissue homogenizer (Bnhkmann Instruments. Inc, U'estbury. data about a curve generated by these parameters iMurlasrtaf.. I9B2J.
NVi at setting d tor A x IX tec period*. The resulting homogenate wa* Parameters Tor one and two affinity site interactions were tested. The
filtered through two layer* of cheesecloth 10 remove unbroken cell* and inhibitory dissociation constant l Ki I was determined from the tela! iunconnective tissue. The supernatant was centrifuged at aojOOD x for ship15 min aad the pellet worked and rccentrifuged in buffer at a concenIC
tration of OA to l.o ma of protein* per ml. Thi* particulate preparation
K.
I + |L]/K
wax either used directly in ihe binding away or stored at TOT for up
to .1 month* without change in binding characteristics. Protein wa* where U\. is the concentration of drug causing -Wi inhdiition of
determined by the method of Bradford 119781. using buviiir serum spenfir i'HIDHA bmdinK. IM is the concentration of I'HIDHA used
alrmmtn as the standard. Particulate* were aim prepired from periph- and Kit the dissociation constant of |'H|DHA determined in equiliberal lunjj of the u m r animals after dissecting sway majur atrwoy* and rium binding assay*. Saturation isotherm* were analyted arcoriiing l o
blond vessels, using the seme procedure.
a one siteor two site interaction and the best tit determined by triimmal
I'HIDHA binding assay. Membrane* lapproximately IdO vie of variance. The estimated dissociation constant I KM I for antagonist* tn
protein per aay> were incubated with | 'H]DHA in a final volume of the physiological studies wa* determined from the relationship:
025 ml. Nonspecific binding wait determined by inrubntion* in the
pretence or I *M /-propranolol. Each data point wis determined in
KM<
duplicate, and duplicate* did not vary by >ltici. Equilibrium incuba1LVL - I)
tiorjs were carried out at 2VC for IS min and termiaated by dilution
with & ml of ice-cold bull* r and rapid filtration through \Vbatman GF/ where | l | is the concentration of amagonsn. L' is the EDv.of agoniat
C filter*. toNowed by two further Aral washes. Fiher* were counted by in the presence of antagonist and L t>e EDv. of agonist alone."
Drugs and chemical*. |'H|DHA <>prcific activHy 101 ri/mmoll
Squid scintillation spectrometry- Specific binding, which was determined from the difference between total and nonspecific count* bound was obtained from New England Nuclear (Boston. MAI. Drug* ware
to the fitter*, comprised 60tnHo', ol total bindingat ligand concentra- obtained from the following sources: f-isoproterenril. /-eiwirphrihr hytion* ol leu* than 2 n\l. For equilibrium binding, concent rat HWK of drochloride, f-nurepmephnnrliydrochloride.atfopinesuluic. serinomn
I'HIDHA varying irom O.l 108 nM were used, and Inr competition and creatinine sulfate. acetylcholinrrhloride.oY.propranolol iSigmn Chemical Co.. iit. Umi*. MO): practolol. d- and f-proprahiilnl lAyerst Labokinetic studie*. a concentration hrtwern I and 2 nM wan u*ed.
J* vitro beta adrenergic responses. For physiological studies the ratories. New York. NYl: IPS.139 hydrochwrid* < AB Hassle, (ioteborx.
excited canine trachea wa* immediately immersed in Krehs-Henseleit Sweden): terbutaline sulfate (Astra Pharmaceutical Pmdurm. Inc..
solution with the billowing composition: NaCI. I ID mM: KCI. $.9 mM: Worcester. MAI: and phentolamine mesylate tCiba-leigy Corp..SumCaCI-. 3.* mM: MgSO.. 1.3 mM; NaH.PO.. 1.2 mM: NaHCO.. 2W mit. NJ). All drugs were made up freshly in distilled water immedf stely
mM: and glucose. 3.6 mM. which was gassed with 9l* O, and tf CO,. before use. Catecholammes ware made up in O.l mM ascorbic acid.
Alter separation ol the epithelium the iracheatit muscle was cut transvcrsel.v into strips - t o 3 mm wide which were mounted vertically in
Results
glass chambers filled with |A ml of Krebs-Henwleit solution, maint ^ i D H A biNdlng anturatlan atudlet. Specific binditiR or
tained at 3i*C and aerated with 94' O, and 6!7 CO,. l*oetric tension
was measured with strain gauges <0rass model FT 0.03 lorre-dlsplace* I'HIDHA t o don trachealiB membranes won saturable and o f
mem transducer) and recorded continuously (Grass model <D poly- high offinity. Saturation iaothemta were beat defined by inter*
graph*. The tissue baths were fitted with platinum elecirndes for action of | *H|DHA with a single populnlion of hindinit otic*
electrical field stimulation using bipharic pulses (supramaximal voltage and Scatchitrd analysis save an equilibrium dinsticiatiiin contt* msec duration. 12 Hr frequency! for 20 sec. Strips were allowed to Mant (Kul of 1.0 0.00 nM <n > 61. which was very simitar t o
eiaiilibrate lor 1 hr and retting tension was adjusted to 10 R. which was that determined in peripheral lung membranes from the same
optimal fur determining changes iti tension. Only strip* that developed
animals (0.98 0.0ft n M , n fil (fix. 1)., T h e maximum
a tension ol titeaier than 10 g to electrics! field stimulation at 12 H*
concentration of binding; sites ( B ^ , I to tracheal smooth muscle
far 20 sec srere wed in the sludv. ,
Muscle >tnri were ranirarted by 5 MM acetylrhutine. taeii. when membranen was 9A.6 4.7 fmol/mg of protein, whiih wan
cent radium, were stable, a cumulative dose-responne was performed tn considerably less than that determined in whole lunu memiMfproterenti) tiMii-lini Ml ut terbutaline il-.l M). using a 2-min branen {R32 4 8 fmol/mg of protein).
expmure tmte to each concentratibn. Theelfecl ol selective orio adre.
K i n e t i c s t u d i e a . Specific bindinx was rapid T , . '.S min),
nergic Wockade on these agonwt dW-Ksponse turn* was determined reachinx equilibrium al t n min. and was revemiUIrun aildition
h\ preincubation with erther thefti-o-lselective antagonist prartolnl orfpropranolol i T , u 3Jt mm). The kinetic Ki. calralatrd from
in sMl r the orfa-2 selrrthe amagoniM IPS X 0.1 M. These the ratKi o f the reverse rale curjHtnnt tn the forward rate
conrentratwn* ol antagani*! were chosen on the basis of cimpelitiDn constant wait calculated as 0.7(1 0.1.1 nM (n .*!). which was
studVk nh I "HIDHA hindmg. so that seleciivitv wiHild be retained.
in ttood ngreement with the Ki. deirrminrrl tn equilibrium
The ellert il selective Mit adrenergic antsgitaists on the beta adreneriiir telataiiim renim>e to electrical field Mimutoliim was also de- Murlir*.
C o m p e l l t i o n aludlea. Sprrinr liindinK t o f rm-hrnlis memtermined. The cholim-rgn re|HinewHlicl(edb\ 1 tM atrtipine. and
ihea^ihaaJreaergn riKmMh> lt)M pheniiilsmme The muwle wa branes was stereoselective, with / pmpMnoNI opiwoHiinntely
Methods
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Basnet at at.
W.225
8max - ' ' 8 fmoi/ina crorsm
R 9 . I . BKXJmsoM'HPHAtoacatncfiaaismooinrxrtda
membfarts. UHpanatspaoiCtxtxfngi*) art nonieaolie
bmomg mine presence ot 1 *Mpropranoioi{0)are shown.
Right panti: Seaward antfyas snowing a smgia daw ol
binding s>t* with equ*bhui aseooation constant () or
1.2 nM ami maximum receptor concentration (B*) of
1112 frnoymg ot prottn. Daja bom a nog* espenment
performed m duptcate are snown and art types! ol a
such experiments.
Mg.^. tnhibiHonotspaoli[aH)DHAbindinoioaog
tracheal smooth mueeie mtrnpranes by adrenergic
drugs. Left panel: inhibition by agonists, /-aoprottfnot ( ) . /-epintpnrine (O). Mwrnpncprmna (A) and
taroutaime (A). Right p a w inhibition by /propranolol ()) andtf-propranoioip ) . Each pant 11froma
angle experiment performed m duplicate and *
typical of three) auch expenmeme.
-9
(OB (AGONIST)
-S
CM)
-8
-7
-6
TABLE 1.
DiaaartaHon oonstaats WW ot adrenergic aganta lor mhibNon of
specJflcfolQHAbhxk^ 1 0 0 ^ t r a v e l emcothrnuecte
inai by the aelective brio antagonists IPS 339 and practolol waa
beat described by interactions with' two binding sites (fig. 31.
With IPS 3.19 most specific ' H binding 179.8 &??;. n - o>
waii to a high-affinity binding site which is presumably tht
betO'2 receptor, whereas the remainder of the binding waa to a
Agonats
site of lower affinity which U presumably the btto-\ receptor.
Msoprotarafwt
0.12 0.01
With practolol. the converse pattern waa seen with most spe/Epnephnne
1.2*01
cific J H I D H A binding <78.0 2.4rc*. n . - 4) to a low-affinity
ANwapnepnhna
153*0.3
aite (the 6t/o-2 receptor). As | 'HJDHA binding has equal affinTorbutakne
37.3*209
ity to 6rta*l and frefa-2 rereptora. the ratio of tota-l/bcta-i
Antagonist*
/Propranolol
0.0026x0.0007
aitea using either antagonist waa therefore approximately 1:4.
o^Proptanoioi
019*0.03
ltt vitro response*; 6e/a aajoniatt. Isoproterenol caused a
relaxation of dog trachealis strips, which had been contracted
fmSttl
K
with acetylcholine, with an E D * , of 4.2 0.7 **M (n 8). Thtre
was no evidence for aignificant beta receptor deaensitiiation
79.8*27
0.0013 * 0.0002 0.29 0 04
during the cumulative dose-response to isoproterenol, aa in
IPS 339
0.30*0.11
16.3*2.7
22.0*2.5
Practolol
preliminary atudiea we found that the final cumulative doae
<10u * M 1 caused a similar relaxation in strips not exposed to
ICM
j j * W f M K # f C t t * thaVCOfVCeVttTV
K dHaanmo from tnt equation K ;
progressive increases in concentration. IPS 339 (O.t M> gave
t>anolsesfttceusffiQ9p%iflfttst*Qnof apaonc|*H)DHA twiOAQ fram a computer a marked parallel shift to the right in the isoproterenol dose*
cts^aring program.fc)iB^concentration ol j'H|0MAui<>d the away (1-2
response curve <ED- 78 1.5 p M ) with a K of 4.6 n M which
fiiMl and Ko it Mia oissoothon constantfromoojueonum among {l 0 oM)
for aw sweckve aniagonau K. * the Ossoeeten oj*a hish-aftrtiy ana and waa in good agreement with its K i for the high-affinity site 11.0
K, tftt lewarftnmi ite TM otta shown are meanslt $ t ot tnraa to vo n M ) determined from competition with | "HJDH/*. binding (fig.
asperats xperanantE.
4). Practolol (3 p M ) produced only a small hift in the doseMO timet more potent than 'propranolol (fig. 2: tabte 11. response curve to isoproterenol ( E D . , 9.7 2.S iM) with a KM
Among agonists the rank order of potency wae /-isoproterenol of 2.2 *iM which la intermediate between the high- and low.
> /-epinephrine > /-norepinephrine, indicating that t h t major- affinity binding sites determined by competition with J 'HIDHA
ity of beia receptor* were of the bcta-'2 subtype. The beta-2 binding. Thia may suggest interaction with both ftr/o-1 and
wleriheaapniatterrwtalinewaKapproxtrnaieiyequipoteniMiith rxfa-2 recrptors physiologicallv. With trrbutalinetED^ IrV)
rMrepinephrine which 1* similar In the finding of other* using 27 iM) IPS 339 gave a matked inhibition t E D . . :l.VH> lutu
lane tarmhranes which have predominantly brfa-2 receptor* nM) with a K H of 4 ^ n M . which was similar to that found with
isoproterenol. Practolol, however, had almoM no elfect <>n the
(Minntman el ol.. 19T91i. Inhibition ot specific ( HJDHA bind-
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Specific binding of I ' H J D H A t o canine tracheal smooth
. muscle membranes had t h e characteristics expected o f interactions w i t h bcla receptors, {finding wan o f high affinity and
similar to t h a t determined in peripheral lung membranes from
t h e same animals and also in hing membranen o f other specie*
fRugg *t at.. 1978; Barnes tt at.. 1979: Barnes c/ of., 1980).
Binding was stereoselective and the r a n k order of potency
among agonists suggested that the beta receptors in tracheal
smooth muscle were predominantly nf t h e beta-2 subtype. T h e
oero-2 selective agonist terbutaline Uad a low potency which
was simitar t o that reported in king membranes (Minnerann rt
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M'MMHAht. K. P.. HKOSTIUND. L. R. Akp M O U M W . P. B-: T h t aktwrtrmto*tea* saanflcky of M and o" * admwrf* trrrpuM* ia am (wan aai tana
I8M-IW). UU.
OMm. C SoNuniux, A. R. Fotro, C. C ROMDM. n AMI Fvuttf. UtflMracusa tetwrtn bii- and otUradmMrtptari in iha iatttattd I
trachea. Pharmacol. Rr*. Caataiaa. I I t ill-tU, l I T t .
u,iwa
RKMAMMOTt. J . B-: SUM of I W an. N r n t aa(y is Iha hull*. Aim. Rtv. Raipir.
JONANUOM. L". AN* WALMCK. B J BtMi-aaVmoNpton owdiaunt ratouttan of;
Ci*.lI:7A3-Mim
h point* W uarhn: EastrinwnU wrtb prrmkaraJ. a ariaratlrctivt adrany R u n . C. L.. B M W I T . D. B. ami N A K O M W . S. f_- Csniuanc* of W u , a i
actpiaraooaia.4.PhanB.PariaroL3S:3U-a*Ubi.
'
HataaVanoctotaraiaaNKalianIut^fNidmc*frMdiraetlM*diataiiidit.
LANIM. A. M . Awrout. A . M c A t U r r . 4 . P.. L C M C K A . P. P. AND BHOWX. T.
MaLPnirnacal.14tPM-IO0S.ltM.
6 , J*.- DiMrnmnatiea of ttctptor (vttnaa anivattrf by *ymptkaMitnttk
RttAnx, J. A.: Raaponn of iaolaitd ctniaa mm** w alteirinl attawiladoa
aainta. Narutt OaadlRldj M 7 - M * . INT.
ndaryJthoit.J.ApLPh-ol.48ia-*,iB7i.
LAKCKK. S. Zi Ptttymftte mndanon of ih* ttkaao f cattehoIaBHM*. FfcatSctirnt. R . MomrA. K. ANA KvmMatl. H^ Inamauan aad ptoiNltlt* of Iha
atacol. Rtv. a t : 337-ltt. JMO.
MKXh nuirla of <ba dot tMrhta. Jtfn. J. Phjmol. t i 393-310. ISM.
Lor&MtL. C..C AM> S t l u m i . Ji_- EfTnrt of nrtaakriol in atihrnauc patlanu.
TtttilNGCA. G. ANB SvKotavn. N. : tajtractie* of orallv adaHnftitrad mrtoareM.
Eur. 4. CKa. Phaimrol, M : 397-303.1M2.
LVUCH.K. M_ MncNStU H. W. AHD!>nuUB>M. M. P J Threat hint atrip a* ' mctslol and pwpianalol with ttepnnallttt M aMhmaiif*. Bur. J. Clla. Phar>
*<*). l i 1S3-IT0. I K S .
m in viitppupaiation 1 prnphtrit airway*: A <wnpaiion of otta-adftnoctpteraiamMi^aMocaidiaadaaaVhyianircaaUtiiitreaitlwIuaiiimpanduacfata.
Br. J. Pharmaml. M l 7t-T. 1S7&
MINNKMANV K. P.. H n t m u N n . L, JL AM> MOUNorr. P. B.: Simuliantoiu Sand rtprtnt raautata tat Dr. Prttr J. tatnii. Orpanattnl of Mtdkino.
dn tnntnaiioa of bia.| and btta-3 a*tnnrir m*pion> in timet coniumnjt HanNnrnunith Hatptttt. Dwrant Road. LaadoR W.|3. U.K.
both ttcrptorKihtypt*. Moi. Pharmacol. ! : M H C I9tfta.
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Ctr Acknowledged
D. B . Cohert + 4s-
t` -
C7 ti
.uaMtodaumnnrlrMdlatribulionolaulon0mlonle pptnA in airway fmooth muscle ol tanN rrnrn traehea 10 IamMMI brplcMain : PWldlhytlrnrF4
penaol . fuguaraala and PN/quinudidlnyl iumltata w .n used to nbal Ortwmannak . alpna
adraMple, and murerNnlc ncaptnrs, ra.paenraty- using aeperimental cprdllluns that aara
ma.rmal specific eaerptor hindlnp wsraad dlner .ncaa wnrr rnund In Iha longitudinal disldGdion
el .aCh qe.plor and en dirrelbutlun of the rarlous neaplan In .eeh pan0er alnray . aalaROaP
tots rran present in Mah density th .ouanaullM alrwaya . r.ah Iha hlpMat Wrn11y In MnneNnVar.
Alplur .e.ptnrs wan eprrau In large sinraya, but numereur In small bronehlo4s, whareat eho
(radioligands) (9-12) . Because lungs nized glus microscope sections . Thesv slidec
are made up of over 40 different cell were eilher used directly in the binding aslypes, assays alone are inadequate to say or stored al -70 C .
censly shown that autonomic receptors speciflc receptor binding ror each radiocan be labeled specifically in frozen )igmd (13-1!) ; I'Hldihydroalprenolol
sections of h :ng and localized by auto- (qIH)DHA)(speci/icaclivily, 101 Ci/mmol;
radiography (13-15). Using experimen- New England Nuclear Corp ., Boston. MA)
lal conditions, which proved to be opti- was used to label bel .-adrenocepsors,
mal for specific receptor binding, we 1'Hiprarosin qrHIPZllspecUic activity, 20
have now studied the distribution of Cf/mmol ; New England Nuclear) was used
alpha-adrenergic, beta-adrenergic, and to label alpha-recepton, and Irrt)qulnuclimuscarinic receptors in smooth muscle dinylbenrilale(IIH)QNR)(speciRcanivily,
of airways from trachea to terminal 33 Ci/mmop wa~ used to labe)muscarinic
.
receptors . With ) H)DHA d 11HIPZ, seebronchides
tions wele incubated at 256 C for 20 min,
Methoda
IFerrets (Musrelo parorius) were anesthetized From the Cardlovascular Research Institute
Avilh pentobarbilal sodium (35 mg/kg by In- and the Depanmmis of Analomy and Medicine .
craperiloneal inJeclionh and the trachea Univershy of Californla- San Francisco, San
and thoracic contents werequickly removed . Francisco . Califomia .
Endividual lobes of Ihe lung werr separated, e Supponed /n pan by Propram Project Grant
eannulated- and inOMed wlth tissueambed- No . HL .24136 from Ihe National Inunule, of
ding fluid (OCT : Lab-Tek Produqs, Naper- Heabh and by Contran No . 1111 from rhe
vllle, IL) diluted 1 :4 with phosphale-buf- Council for Tobaao ResearcRUSA . Ine .
reprinn should be addrcaed to
fered saline . The infiated lobe and tracheal ' Requeus tor
. Barnn, Depanmenl of Medicine .
rinas 5 mm in length w~ere rapidly froten inHammerrmith
~' R J
Hmpilal, Ducane Road-Londun,
freon 22 cooled by tiquid nilrogen. Frozen w .12. UK .
secdons 61o 8 pm thick were em on a eryo- Recipient of a Tra .elElna Fello-ship bom
stat at 1S C and thaw-moumed onto aelati- the Medical Research Counca of Great Bnlnn .
Taf
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PUBLICRTrONS
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d and with l'H/QNB, for 60 min . Concennalionsof radloligand were used that were ap
- prorimately equal to the dissociation constanl (Ka) for binding to ferret lung sections .
as previously delermined (13-15). Nonspecific binding was,dnumined by incubation
of adjacenl secrions from the same animal
in ihe same eoncentration of radioligand
but with an exceas of unlabeled anlagonin,
so that ccptrtr binding was competitively
inhibited . For /tiHIDHA we used I PM
(-)propranolol, for pH)PZ . IOvM Dhentolamine, and for l'H/QNB, I pM atropine .
After washing for 10 min in ice-cold buffer
(S0 mht Tris Hdi pH . 7.4), sections were
rapidly dried in a stream of cold air to prevenl diffusion of tadioligand, and stored In
a dessicalor overrbight .
Arrrorodrogmphy
Autoradiogmphy was performed using the
method of Young and Kuhar (16) to retain
reversibly bound (i .e ., diffusible) mdioligands at reeeploo sites. Glass coverslips .
which had previously been eoaled in pisoto
graphic emulsion, were pieced over the sections and 6xed to one end of the slide with
cyanoacrylme adhesive . The emulsion was
held in contact whh the section by binder
clips, then stored in light-proof boxes at
4' C . Optimal exposure times were found
io be 3 months for pHJDHA, 4 months for
lrHIPZ, and S months for pHJQNB. After
exposure, the cov'erslip was partially sepa
raled from the slide so that the emulsion
could be developed and the section stained
with 20a cresyl vuolet . The sections were
mounted and viewed under brightfield and
dartfield illuminsition . Autoradiographic
grain counts were performed using a cali
brated eyepiece and a x 100 objective lens .
Grains were counted over areas of smooth
muscle in several airways from each section,
and the airway dimensions were recorded .
There is some eonfusion over the definition
or "small airways," particularly when differenl species are compared . In our ssudy,
imrapulmonary airways were categorized as
cither O) cartilaginous airways (bronchi)
measuring I to 2 min in diameter and corra
+ponding to subsegmental bronchi of
sourth or fs(Ih gene,ration, or (?) noncanila
ginous airways (bkanehiotes) . Bronchioles
aere arbitrarily defined as proximal (> 0 .3
mm) or dislal (< 0.3 mm). Distal bronehi
ales Included terminal bronchioles and re
spiratory bronchioles.
Dald Ana)ys&
Resuhs are expressed as means m SE . Speafrc grain counts were determined by
c,+unming grains/unit area . then subtracting
background counts and nonspecific counts
flom the same area in an adjacent section.
R:ceptor density was then determined by
a+rreclion for specific activity of the radiohgand and the time of exposure using the
fprmula :
It = (gd/1) x (A/5.C) x 2
7>9
TABLE I
DISTRIBUTION OF 6ETA-ADRENEROIC . ALPHA#DRENERGIC . AND CHOLINERGIC RECEPTORS
IN AIRWAY SMOOTH MUSCLE OF FERRET
Reeeptor Dansay
IOIndlny s/rerr r m')
Oumeler A'M'ar tmm) BetaAdrenerpm AMpha-Adrenerprc Chormeroc
Tbacnea
6
1D7
s4
-1
T9 :0.0 - 263 x
BronohN 1-2 BO-0 ar 6 .1
9.1 29-9 7852
Pioxlmat aronchiolas O.y-09 122 a 20 99 s 7
.t 139 a
D/slar erpnemotes e 0 .3 160 z 15 163 : 6.9 102 o
Ba
96
77
tl
- RKrptp dM41Y eNlrieiryn hym .n .el,nn eefCxMn ,n MR"npf Rtlyln e,e nyyn r eF 110m e, Nen ] 4re,e
Ratulls
There was a striking difference in the
pattern of labeling of airway smooth
muscle with the different radioligands
~~
'a1aYe'
'
F/g. 1 . 6oCa11raUpi o1lMtaidranoraesplON in snwar amootll muscle 01 (aMet (al Darallaia llluminer
tioqatqna the distribution or auloraalo9raphie e1rMt qralna 66 br/pht datla in an inlralnrtmpnary branehu9: PHIDNA deneely labela sm qth muaete ISM). (b) . B/lahlliate view 61 the aba shown InteL (e) OarMnatQ waw ot 7ha sune araa In an adlarant section incubated wnh PM)DNA In the preunea ar an areesa
or pFopranol4l, showinp rq speeilie IaIN1rnB . (ol Dsr4hald view Ol a ametl nranehlo4 ahowinp danN
Iabeling of sn1oolh musete . Note the rarlehn0 ot aunoundinB aN/plir wana . (ar Br/qhthete Yiew or Ina
atea ahown ln (d1-(1) Deraaatd view ot the aame anta trCm an adjacent aaction Incu Wletl wnn l'MJOMA
and Dropranolol. Sule bat . 100 rrn.
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026945
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10
Fig. a Lorallranon ot aipnaatlunocePlon .. arnsy rmootn muecN 01 ferral Is) Duldbrd vrww o1 an
/myprununuy broncttus . arowina /abs4na by plriprarosrn of eonlrobum (EP)oul arewsf no labeuna of
ampoth muscro taM). Baeeprouns rs conautlerably hlaner than,n Npura I ID) . ananerom wew of the er.a
Shown In (s) (e) . Darkllettl view of tM same araa nom an adlaceni seenon incubaleP with PMloraroNn
antl an etcess 01 Dflente4Mns . (e/ Dark6ela wew o/ a amao bronelnoM sMwma tlenR labeling o1 aU
watl smDDM muubtaMl n) BrrpMnvta wsw of the area snown m(tl) lD Darkl,Ntl view o11M esme aroa
/rorinanadpCeresaetlon,ncuwtetlwunpNlprarosmantlane .eeasofpnemmaro,ne.aca+eWr . r0a,.n .
to studyitdg autonomic rei-eptors in airway smooth muscle using autoradiographic localization of specific radioligands . Wc have previously established
that specific binding of each radioligand had the characteristics expected
of interaction with its receptor in lung
sections, and were able to determine
the experimental conditions ner :essary
to produce maximal specific binding . It
may be difficult to precisely determine
receptor densities from autoradiographic grain counts, but it is possible
to obtain estimates of relative reoeptor
density (17). Such estimates involve
several as'sumptions, but berarrse these
apply equally to each radioligand, it Is
valid to compare both the relative distribution of different retxptors in the
same structure as well as the relative
density of each reecptor in differant
structures .
Beta-adrenoceptors were numerous
in smooth muscle of all airways, but
the density increased al airways became smaller, with the smallest bronchioles having a very high density of
beta-receptors. This is in agreement
with the finding that beta-agonists produce relaxation of lung parenchymal
strips as well as isolated tracheal and
bronchial smooth muscle (3-B) . Alphaadrenergic contractile responses have
been described in smooth muscle strips
in many species (23-23), but they may
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PUBLICATIONS
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re+
eated, but ahey may be difficult ro interpret in the face of coc%ictenl abnormalities in large airway function .
Studies on peripheral lung strips snffer
.*om the problem of helerogencity in
contractile efements, as di.cussed
above . The autoradiographic method .
however, has the advantage that
smooth mttscle of Ihe smallest airwac+
may be studied . This method may be
useful in delermining the c%istencc of
abnormalities in airway autonomic receptors in disease. /n a guinea pip
model of aslhma, a decrease in pulmonary beta-receptors was accompanied
by an increase in alpha-receptors (35) .
and in chronic airway obstruction there
was an apparent increase in alpha-receptors (11).
Because these studies involved radioligand binding to homogenates of
whole lung, it was uncertain whether
the changes occurred in airway smooth
muscle, or In other lung components.
The autoradiographic method we have
described should provide answers to
these important questions . Although
we have studied adrenergic and cholinergic receptors in the airways, the technique is equally applicable to other rtceptors (e .g ., for histamine, serotonin,
prostaglandins. and regulatory peptides) . This technique offers a new strategy for the study of regulation of
small airways and other structures
within the lung . The demonstration of
specific binding sites does not necessarily indicate that tbey are functionally
signifieant, and in the future it will be
important to correlate the presence or
changes in binding sites in a tissue.
with some measurement of receptor
function in that tissue.
Fig. 3. LOCatiranbn of muiear/nie rKeptora in aineay smooth muscle ol tana,t . ia) DVkiield view pl an
mrmpidmanary siOnenus showing dense labeling by rNioNa of arnootn muaclo {arA) Di- erialltaeid
vl.o of ineatN rhown in (a) .OCI. DarMiald vifw o11M aanNarra fromanad/acpnt .action incutNUdwitn
rlqnNa and an auce9{ of atropille. (d). Damturld vlew o/ a pro}linai broncniole Showing aparsN labeling
of 691001111 musnr Bldl . (e) ariamludd view ot 1M area shown in (dl dt Darknald vUnr ot iM same area
Irom an tidReenl paction inoubalUd wtth pH1DNa and an a .oe.as of atroplna . (a). Darkfleid view of d4,ai
6ronciuete srunrinp no upeeifn : labeling by rlI2ON8 terta111 . (n) . anpntliao view of aL. snown in (at %
DarYhald viaw at {ama arE NPm adiacent sectWn inoubanld with PNiONa and an .npsa of atMqna .
Scale bar w 1os :.n
than did tracheal smooth muscle . Thus, muscarinic cholinergic recepSmooth muscle of proximal bronchi- tors should be closely related to cholinoles had a much lower density, and the ergic axoas . This is in marked contrast
distal bronchioles had almost no cho- to the distribution of adrenergic receplinergic receptors . This distribution is tors . In most species, direct sympatheconsistent with physiolegic studies in tic Bsnervation of airway smooth musanimals showing that the greatest bron- cle is rather sparse in large airways and
choconstriction after va$al stimulation absent from bronchioles (33) . Adreneror cholinergic agonists Is in large rather gic axons in ferret lung show a similar
than in small airways, both in vivo (29, distribution (determined by fluores3D) and (n vitro (31) . Similarty, in asth- cence histochemistry) (13) . Adrenergic
matie subjects, antichtilinergie drugs receptors in small airway smooth musappear to cause dilation mainly in cle would, therefore, be regulated by
bronchi, whereas beta-qgonisas dilate circulating calecholamines .
all airways (32). The pattern of distri- Although there may be marked spebution of cholinergic reorpton Is prob- des differences in the response of airably related to parasympathetic inner- way smooth muscle to autacolds and
vation, which Is dense in bronchial prostaglandins, differences in responses
smooth muscle but sparser in bronchi- to autonomic drugs are much less apolar smooth muscle and vinually ab- parrnt (34) . Our findings in ferret airsent from terminal bronchioles (33) . ways can, therefore, probably be cxThis Is not surprising because acetyl- trapolated to other species . Including
choline, when released from choliner- humans . Littte Is known about the
gic a\ons, is rapidly Inactivated by cho- function of small airways in human
lineslerase and does not function as a disease . Several physiologic tests of
circulating hormone. small airway function have been advo-
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AeknowkdamrNr
The writers wish lo Ihank Leona Lauricelta
for expen photographic assistance, and
Beth Cost and Palty Snell for their assittanee in preparina the manuuript .
Rafarane.a
1 . Hahn HL, Nadel JA . MetDod& of uudy of
airway smooth muscle and Its phynoloay In:
Widdhombe JO. ed . Internalional enryclopedla
of pharmacoloay, tand IheraPeuiicn : ra.piraiory
phnmacotoay. London : i'tryawn Prnt, 19a1 :
sor-at .
~ rcArraus
10346183
026547
. 762
nous adminiuration of slow aainq substance
of anaphSla%dw histamine. bradykinin and pmsglandin F1 .*r,r on pulmqnar5 mechanics in the
,aurcaa pig . 1 Clin Inveu 1974; 3~:1679-83 .
S . Lulich V :\1 . Mitchell, HW, Spqrrou MP .
The car lung strip as an in sitm preparaion of
. peripheral ain.a>s : a compariwn of bcraodrenoaplor agoriists. amacoids and anaphyiactic
chaTlenge o1,the luns strip and trachea . Br 1
Pharmarol 1976; 3g :11-9.
6. Seigl PKS. Rossi V. L7ruchorski RR . 1so
Ined lung strips of guinea pigs : respunses of
bera.adrenergic agonins and amalntiists . Eur J
Pharmacol 1979 ; 34:1-7.
7. Kldnniver PM'. Eyre P. The lunl Wrenchyma strip preparation of nd and dog. Responses
to anaphylaaic mediason and sympathetic bron, cAudilators . Ra Comun Chem Pathol Phvmaco! 1980: 27 :431-67 .
B . Goldle RG. Paterson JW. Wate JL A comparatise study of betaadrr,enoceprors in human
and porcine lung parenchyma strip. Br J Pharmacol 1982 : 76:323-6.
9. Rugf EL. Bamen DR N4honti SR . Coenisrence of P,- and A:-adrenocepton in mammalian
lung : evidence from dired binding studies . Mol
Ph .rmml 1978; 14:996-1003.
10. Bames P. Rarliner J ., Hamilton CA . Dollery C- Demonsuation of plpha,-adrenoceplors
in guina pi8 lung using I'Hlpraeosin . Life Sci
1979: 25 :f207-14 .
II. Barnes PJ . Karliner 1S. Dollery CT . Human lung-adrenocepfoas sthdied by radioligand
binding . Clin Sd /980: 51:437-61 .
12. Chens JB. Tornley RG . Comparimn of
muscarinic and bera-adrenergic receptors bewxn bovine peripheral fung and tracheal
smooth muu4es: a slNking differcnee . Life Sei
1982 ; 50:2079-86 .
13 . Bames Pl, Bnbaum CB, Nadel lA. Robens
:M . Localisarion of beraadrenorepton in mammaihrn lung by li;hl microscopic autoradlo8raphy. Nmure 1982 : 299:444-7 .
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PUBLICATIONS
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gi
/
H.PPIMG 0F VASOACTIVE INTESTINAL PEPTIDE (qIP) RECEPTORS
USING. CYCLIC A38 (cAfff) O9NHOGYIOGNGMISTRY . S-U . tura_y
C . . baabam, P .J . Harnes and u .M- culd . Grdlovasc .IIar
of
Public
Health . Boston . nA
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ly :
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PUBLICATIONS Q1
10333448
-45
D. B . Cohea `'
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maal GYRk . Maalorytoma nYCUIp rNm a YYp .wn iIaM anE aLayysQ+rwd wM . cellaypvaa to
Pnvlos a an fuapenawnot maatorytwna Ma er ~ 93M wntr . TM pw.~ ef aurunomk waP
lo/a nI/ aiN1JM ay nUnl rOleOllpp~tlbin0l.q aiYr . ..a 4r aMUimg pnYm .eObyk .nDU.NLon
d mMlator rlNau In eM rbbllparM Elndlny asrya MFamN,Mnle nleePton aMO aaMnMtad
.U ehou,.w,
p'r 1'Mp~nF'oa .penewl mnouq, nlr .e . .r..qia rseepew. W PHlwaamin a1nOn'p v
yk r . .>pmn by hH14ulnuGltllnrl Dantliab OinClnq . Nen.yepnc OuWmp wn oaurm/natl m seoa
eae' nr ineuen~enln ur pe .u .e.oltM .p .[mc anaqo .YSn Moypqlw.On.^fMamus, anC ans
plnq sapadMlr. TnaNnel of aNanemk apoMab on Imrnunebpk .ne non/mmunaioylc Mals
R11M
raNaM
wq
alpnYWrlnarple apornet pMrqlpbnna wrthantl wnlrout p10prYW WI . arW tM enelhw .9ta aFn41
:
rw curmdanl . rMw Yey ma.foryton~ eell. MO a nqn e.elry of pey..ac.pb.a lZf .5nn
SE ML-nppMNpll . n-31 et+tneh IM p .aGOmIMm aubrrpa .pparaU m tw Eau,.
3,aGG: mean .
PUBL ICflTIONS
10333399
014410
1020
owrwn .
Corp., Boston, MA .
The drugs and chemicals used were : acetylcholine chloride (ACh) ; arropine sulfate,
bovine serum albumimfracuon V (BSA),
compound 48/B0, ( x )epinephrine hydrochloride, histamine diphosphate. ( m hsoproterenol, ( x )arcerenol (norepinephrine hydrochloride), (x )propnnotol hydrbchloride, penicillin O, streptomycin (Sigma Chemical Co ., St .
Louis, MO), phentolaminr mesylale (CibaGiegy Co., Summit, NJ), terbutaline sulfate
(Astra Pharmaceutical Products, Inc . . Worcester, MA) . (-)pherlylephnne hydrochloride
(Winthrop Laboratories, New York, NY),
shon ragweed (ombrosia arremnifolra) pollen extract (Hollister-Stier Laboratories, Spokane, WA), acepromazine maleate (Averst
Izboratories, Ina, New York, NY), sodium
thiamylal (Bio-Ceutic Laborarories, Inc . St .
Joseph, MO), Dulbecco's modified Eagle
medium / DME), mrnunum essent tal medium
(Eaglel-JOKLIK-modified (MEM-IOKLIK),
4-( 2-hydroxyelhyl)-1-piperazine<I han esulfonic acid (HEPES) (Boehfmger Mannheim
Biochemicals, Indianapolis ; IN) . felal calf serum (Hyclone Sterile Systems, Logan, UT),
amphotericin B with deoxycholate (Fungazone; E. R- Squibb Co., Mirad a, CA ), trypan
blue (GIBCQ. Grand Island, NY), toluidine
blue (Fisher ScientificCa . Pittsburgh, PA),
protease-free cotlagenase (CPLSA, Worthrngton Chemical Co., Freehold . NJ), and deoxyribonuclease (DNAase) (Millipore Corporation, Bedford, MA).
The following reagents were used for
fluorometric histamine analysis ; perch7oric
acid, hydrochtoric acid . mctbanol IJ . 7: Baker
Chemical Ca . Phillipsburg; NJ), n-heptane,
I-butanol alcohol (MCB Manufacturing
Chemists, Inc, Cincinnati, OH) . o-phthalaldehyde (Dionex Co ., Sunnyvale, CA), sodium hydroxide (Maliinckrudr Inc, Paris, KY),
and phosphoric acid (Fisher Scientific Co .,
Pittsburgh, PA) .
The experimenlal procedure followed adhered to Ihe published "Gaiding Principles
in the Care and Use of Animals" approved
by the Council of The American Physiological Society and specific protocols approved
by the Committee on Animal Care of Ihe
University of Califorma, San Francisco
a~wrca
un
pqn
Masmcyrorna Biopsy and Disaggregarion for 20 min ; for saturation binding studies,
Ma-stocytonia nodules were excised from a cnncentrationsrapgedfrom0 .1 to2OnMand
4-yr-oldGerdnanShepherddogthaAhadnrul- forcompcthionsAudiesfrorrilto2nM .No
tiplesutrcutaneouslesionslocatedneainlyover specificbinding .~asmeasuredbyincubatije
the abdomen, chorax, and hindquarters . irstheprosenceoflM(-)propranoloLWhen
These tumors gretv slowly, doubling in size catecholamines ivere used in competition
over the course of I yr . The nodules ranged studies . 100 M ascorbic acid was added to
from 5 to 25 mm in diameter at the time of tbeincubation tubes topreve,nt oxldattorr . AB
biopry. A total of 22 nodules were excised over samples were run in duplicate or triplicate .
the modulation
of
suspensions obtained from disaggregation h-stamme release by beta-adtenergiq alphawere centrifuged (200 x g for 10 rd .in), and adrenergic, and chblinergic agonisu and anthe resultant cell pellet was washed tvSice with
tagonists. Histamiote release .vas induced eicaldum-free 7yrode's buffer before resuspen- ther nommmunologically by challenge with
sioninanappropriatemedium .Mastocytoma
compound 48/80, or immunologieaUy by
cells were identified by their characteristic challenging passively sensit'urtd cells with angranules. whicl, stained merachromatically tigea For pharmacologic studies requiring
Cells were resuspended in 7yrode s buffer con- For studies involving immtdnologic hismtainrng 25 mM HEPES at a concentration of mine release, cells were first sensitized pasapproximately 0 .5 x]0 cells/ml (range, 0.3 sively to ragweed antigen byy resuspending
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
PuBLrC~iTrONS 014411~
10333400
1021
masched with 41p bes to which only the releasing agent was added . In addition, spontaneous histamine release was also examined in
quadruplicate iri tubes containing eells, but
neither releasing agent nor autonomrcagent .
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
I !.~
~
rf
G(ara Analysrs
agent had also been added . In order to e>:amme statistically all the experiments with a par-
licular autonomic ageni as a group, the histamine release values were normalized by disignatmg the maximal release as 100% and
expressing all other values as a percent of the
wherecontrollsthepercent hrstaminereleased
by the releasing agenl and experimental is the
percent histamine released by the releasing
agent in the presenceof the autonomic agent .
The values to be inserted in this formula
were obtained as follows : t he percent release
wit h t he releasing agent cor responding t o the
E Ds was calculated by subtracting rhe spontaneous release from the maximal release obtained and dividing by 2 . In order to dctermtne the corresponding percent release in the
presence of the autonomic agent . dose-response curves were drawn graphically . The
point on rhe dose-response curve fa t he releasing agent corresponding to the ED,o,'alue
was found by adding t he spontaneous release
to the F Dso value. From this poi nl, a perpendicular line was dropped to the abscissa . The
percent telease was noted at the pointat which
this line cut the dose-response curve constructed for the autonuntic agent . The required value for the formula was then calculated b} subtracting the spontaneous release
in the presence of the autonomic agent from
the value obtained from the graph, ai described prevtously. Experiments with different concentrations of the autonomic agents
wcre analyvrd individually by Student'ss test
and s a group by 1woway analysis of varlance using rrplf :ate values .
PUBLICATIQNS
10333401
~.
1 (r
e .e snswn
Histamine Assay
Histamfne was determined by the o-phthalaldehyde spectrofluorometric procedure of
Shore and coworkers (13) . but modified for
autoanalysis (14) with an automated spectrofluorometric analyzer (Alpkem Corp .,
Clakamas, OR). The autonomic agents, compound 48/80 and ragweed antigen, wereeach
tested in the concentrations used in these experiments for auto0uorescenceand for their
possible effects on the determ-inauon of
known histamine standardsReaulte
Radro(igand Binding
014412
<
1022
~
~
-~
~ - ~
soi
}0 -
.0
60
ep
D
-e 1
iW
nV ADRENERGIC .AGONISTS
1)ISnprdarenol
1IEOnaphnne
Terbuta4ne
i JNOrepInephr.ne
l-)Proprsnolor
/IOroprendot
. .
07 . t o"
223 06 x t0's
6 A= 06
1 I x D 6
Rnsez
10`
110'
05 n
IU`*
pI
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1,
109
.10
.0
IANTAGOMSTI
(M]
F,g 3 IMID,tmn ql speMs ('HIDHA Dmeingfi anlated,,ntaCl tlOg maslocyt9rnacens br adrenerg,c agena rho
mncenlrgponofr'NIDNAIntfresestudosrang9tlfrpnO8tolSnM L6npBng/ InhibltqnbYagOmgs ~-Jqoprjte,arpr
(cloBeDCrTJ96J . (-)ep inephnne lopan CllLhs). temutal,ne lOp9n (nanpl96) . (-)norepmephnne (C/oap, mLngys)
Conlrol refers ro,SOpm[arenpl CpmpaGaen AlgMpan9r
Inhibition
I6asl-squares C4mputer program . For (-)proP2nptol Me tlala were Llled by assum,ng an o hiDihon ot binding
612 3aes . ThedBla shown are rmm I expenm9nl . perfprmeU m Gupl,cate. which w5s typiCal pf 3 such eapenmenry
K, (M)T_
19 z
'.A
(AGONIST)
INHI131TIOn OF SPECKIC
I'HIDIIavDROALPRENOLOI 61NDIN0 TO
IgOtLATEO DOG M1bASTOCYTOMA CELLS
Agenl
\Y
:5
,6
ie
. 10'
tn'M~W ~w r Iam
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
carinie
cells .
cholinergic
receptors on these
Phprmpcologx Studies
The adrenergic agonist terbutaline was
evaluated for its modulat ion of histamine
release induced by both ragwecd antjgcn
and compound 48/80. In each of 6 experiment s, t erbutahne (10-' M) altered the
ragweed antigen dose-response curves,
one example of which is shown in felgure
4 . The maximal response was reduced
and the concentration of antigen needed
to induce a response that was 50% of
maximum was increased . 7ivo-way analysis of variance using replicate values
-1
!
wa .wn +.ny .n wy~m
PUBLICATIONS "rTmR1T
10333402
CM~WICrERIZ~noM
pr
VeRmFD
DOG
W,SrOLVIOMa
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
CEris
NY,ew,o F.y.9r
release obtained in the presence of antiIt. om .uu
gen alone, and when a11 4 experiments
,oor
were considered as a group, no significant differences were found . In a sepae
rate experiment, the use of (+)propranolol failed to alter the inhibitory effect
of isoproterenol on antigen-in'duced
histamine release .
The alpha-adrenergic agonist pheny6
ephrine ( IO-' M) was evaluated for modulation of histamine release induced by
ragweed antigen in 5 experiments and
modulation of histamine release induced
o . . nw)
by compound 48/80 in 2 experirments .
rig 7 HiSlam~ne .9leasetnryyceLlqrpgn,,a6eanl
Phenylephrine (10" M) inhibited hista(50 regrmr) m me presenee bt mnyrent tmnpentrn'
OI ~dOpml9renol (541W Dar3) }iipre3aee a5 B p9ree
mine release induced by antigen by 18 .3
a malChed eGMml wtnGYl Imeprotprenol ?Te BHe
m 23.7% and enhanced histamine release
,nCUbaeon w .th botE 10" M prApranolel aOE lD-i N
induced .by compound 48/80 by 6.3 m
proterenol bebre the atl6i(Icin ol fagweEd y,4gr
4 .0%u at the ED,o for each mast cell actishpWn q Ine hal[rlatl pa1 . The rd5ul16 are eepM :
as mean z SD TTe aponreniwus release w99 3vator, respectively. Neifher overal0, nor
a 1394 H1stam,ne releese was induced Gy 50,rgrr
in an individual experintent, could Jphenragw64tl anl .gen cakulffied to g.vo spprOamatnly I
ylephrine be demonstrated to cause a sigmal,mal hlstaamme releasB (adunl releaYe achle
nificant change in the dose-response
2ar s 004%)
curve.
The etlolinergic agonist ACh (1d- M)
was evaluated for its effect on histamine
ragweed antigen (1 to, 100 pg/ml) w
release induced by ragweed antigen in 5
examined and no significant effect of
experiments and on histamine release int her agonist was obser.+ed, suggesting r
duced by compound 48/80 in 2 experihancement of nonimmunologic or i
ments . Overall- ACh caused enhancemunologic activation of Ihe mast cel
ment of histamine release at ED,e of 11 .4
Incubation times with ACH and betl
- 7-6% (or ragweed-an[igen-induced renecol were varied frorh I to 5 min .
lease exp'eriments and 8 .5 x 4 .74'u for
In these series of experiments, t
compourld-48/80-induced release . Howmean histamine content of the cells w
ever, in rtio experiment did Ach cause a
6.5 x 3 .6 pg/cell and the mean spom
significant change in the dose-response
neous release was 4.3% (range, 0.6
curve by two-way analysis of variance .
8 .5oJo) . The mean maxiunalhistamine .
In another experiment, the molar conlease induced by ragweed antigen w
centration ofACh was varied from 10-10
3fi-4t7n (ra nge, 17 .R to 68', 3%) and by coi
M to 10- M in tenfold increments and
pound 48/80was 65 .5% (range, 47-8
its effect on histamine release induced by
74 .40/0) . Cell suspensions yielded a hi,
compound 48/80 (0 .2 pg/ml) was exampercentage of mast cells (93 .8 m 3-4ined . No significant differences were oband cell viability, as assessed by trypi
served in histamine release in the presblue staining, was always in excess
ence of ACh at any concentration com92% . None of the auto I nomic agents
pared to the release with compound
releasing agents caused autofluorescen
48/80alone. The effect of both bethaneat the concentrations used, affected I
col (2 experiments) and ACh (2 experimeasurement of known histamine sta
ment s) over fu l I dose-response cu : ves for
dards with the autoanalyter, or alterl
compounds 48/80 (0 .01 to 1)<g/ml) and
significantly the rate of spontaneohistamine release
. There was no diffe
ence in histamine released by antigen
preparations from which amphoteric
B(fungazone) was removed by washil
or from preparations in which no ar
photericin B was added (n=5). indica
ing that amphotericin B not removed I
washing had no effect on histamine r
lease itt these cells .
~
~
PUBLICATION$
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Diacussion
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1024
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
is one report that beta-adrenergic stimulation of rat peritoneal cells in vivo appeared to inhibit mediator secretion (24),
most other studies using cells that had
been purified by centrifugation through
density gradients failed to demonstrate
inhibition of mediator telease (25), afthough increases in cyclic AMP may occur (26). It appears that in rat peritonea]
mast cells the adenylate cyclase stimulated by beu-adrenergic agonists may not
activate the cyclic-AMP-dependent protein kinases that affect mediator release
(27) . Furthermore, Sullivan and coworkers (26) found that the effects of a varfety of catecholamines on rat peritoneal
mast cell cyclic AMP were inconsistent
and did not fit into the generally accepted
pattern of alpha-adrenergic and betaadrenergic receptors . Thus, epinephrine
and norepinephrine stimulated cyclic
AMP production and inhibited mediator release, whereas isoproterenol had little effect on either cyclic AMP levels or
compound-48/80-induced histamine release. Nevertheless, there is evidence from
radioligand binding studies that betaadrenergic receptors are present on rat
mast cells (28-3 1) . Thus, Marquardt and
Wasserman (31) demonstrated the presence of these adrenergic receptors on rat
serosal mast cells, and showed that betaadrenergic agonists caused a rise in cyclic AM P levels, but were unable to demonstrate anyeffect on IgE-mediated mast
cell histamine release
-fhus, pharmacologic studies alone, or
radiol igand b inding studies alone arc inadequate for characterization of receptors on mast cells- In addition to radioligand binding studies, we therefore
undertook pharmacologic studies to examine the modulation of mediator release. In this study, we have shown that
beta-adrenergic agonists can inhibit immunologically and nonimmunologically
induced histamine release and that this
action is affected through a stereoselective site that can be blocked by (-)proptanolol but not by (+)propranoloL This
finding indicates the presence of functional beta-adrenergic receptors and substantiates the conclusions drawn from
studies of heterogeneous human lung tissue and confirms reports recently made
on purified human mast cells (32, *3) .
It is of interest that dog mastocytoma
cells in this respect appear similar tohuman mast cells . in contradistinction to
rat peritoneal mast cells or murine
mastocyioma cells .
The demonstration of beta-adrenergic
PUBLICRTICNs
10333404
I
tors (21,500 33UD receptors/cell) was
high, although greater numbers of bind
g sites havp been reported in other inact cells including fat cells (15), cultured
mllsclecells (36),and tat peritoneal mast
cells (28 . 31) . The high density of betaadrenergic receptors on these cells may
indicate the presence of spare receptors,
which are a means by which a cell
achieves a high sensitivity to agomsts . as
stimulation of only a fraction of Ihe
receptors results in a maximal response .
This might also account for the dis
crepancy between thecffect of isoproterenolon binding, and its funnional effecL
The presence of alpha-adrenergic
receptors on mast cells was initially
postulated in studies of heterogeneous
human lung tissne in which alphaadrenergic stimulation by phenylephrine
or norepi nephrine (n the pmsence of propmnolol) resulted in enhancement of
anligcn-induced mediator release (37) .
Opposite results were obtained by Nm
and Bloom (38) in rat pentoneal mast
cells . They found that norepinephrine in
the presence of the beta-adrenergic antagonists propranolol or practolol inhibited histamine release, and this effect
was dimimshed by phentolaminc {ndiing that alpha-adrenergic stimulation
~'nhib{ted mediator releasa We were unable to find any evidence for alphaadrenergic receptors in these dog
mastocyloma cells based on radioliga.rd
binding asszys or pharmacologic effects.
lb our knowledge, radioligand bindinR
assays have not been used before to look
for alpha-adtenergic receptors in purified
mast cell preparations .
The presence of cholinergic receptors
on mast cells was also first inferred from
studies on human lungussue (37) . Most
studies on purified cells were based on
the rat peritoneal mast cell, and once
again confliaing results exist . Some
workers (39, 40) reported cho6nergicinduced histamine release in W istar rats,
but others (41-43), using dif feren l strains
of mts, could not demonstrate arry such
ef fe<u. Masini and ooworkers (44) later
reported variable results even within the
same species, and could find no correlation between ACh-induced histamine release and the high af6ruty binding of
/'H]QNB, a specific cholinergic muscarinic ligand . to rat mast cell membranes. These discrepancies might be due
o o diffcn:nces m the species, to differcnces
n purification lechniques, or, in hcterogeneous tissue such as lung, t he choliuer .
gic agonist may be acting indirectly
though other cdl types
. This laaer possibility is thought to he the case in the
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
. Sctmruvla W Comyar~bve nudies of maat ceUr from normel (nonrmmiuiitN) and acvvely uanuud dogs . Agcnts
Anioni 1982: 12192-8.
4 . Go,ld W\1, Meysn GL . Dam DS Md1er RL.
Boume HR . Changn m airway rnast ttnsand hissa
rmnt caused by aotigen aerosol,n anergtc dogs3 Appf Physnol 1977 : .f 3 :271-5 .
PUBLICBTIOVS
10333405
014416
rozs
-IMPbannamlogral speoRarS-olbaa-1 and bere .2
adreaergic rvoeptnn in rn hean and lung in vrma
Mot Phumacol 1979 . 16di-H.
1] . Rugg EL. Barnett DB. Nabonki SR . Corzsstcncr of Beu. and Betar adresoceptors in mam .
mUian lung . evtdence frOmdirtR bindmg 51 udies .
Mol Pharma<ol 197& 14 :996-l0p1 .
18. Bames Pl . Karfiner J& Doilery CF Human
lung adtnoorerepton studied by mtlroligand bind .
1985; 212399-107 .
http://legacy.library.ucsf.edu/tid/hez49c00/pdf
physical stimug- J tmmunol 1975 : 1I4 :1a93-9. stana of anapMlanu from human h.ag, ly. P,ar
28- Donlon M- Hunr WA-Cavavaa GN, Ka3inee Menaawm PE Modulation of the spuntaneouy
M. A eRaraeunration of bua-ad .energic reC<plors huumtne releasc by,aNenergic and chobnape
onccVularandpengranularmembrenaofrAtpen- dmgs Agenis Aaions l9]8 : 8'3a-f_Sg.
toneal maat cells . Lrfe Sc . 1982: 31 :411-6 . 40 Bmndina P. Fanrdm R . Mannaioni PF, Masmf
mme release Naunyn Srhmtedcbcrgs Arch PhmFadur< of aKlycholine lo release huumioe fmm
macol 1982 ; }21
:p1-0- tal mast calls- Agents Actions 198P 101-3 .
31 . MarquardtDL .WassermanSlChamcrenn- 43 EriavecR-Histamdnereleaeefmmmancds
uon of mt masr ceu beuadrencrgsc rxtptor rn rcM- bypllysrologlnRy oaudring subsunca Agmu pam8 nnd sttmulated arllsby mdioliyand bmdmg . J sions 1981 : 11 :71-2 .
Im.nunol 1982; 129:2122-'1
.
m. Masmi E, Blanditu P, Famom R Brmelle .a3d
33 . Pcten SP ScFUlman ES. MarGlashan DW Jr- ,5~ Mannaioni PF . ConKlasionbetueea[hnhnerpe
SchlermnRP,Ne*haBHH,L :.chtensusnLM . Dis- hsstaminereleaseandpmnudidsnyi-bennlaletFH}
pened Ruman lung mast cells : pharmarologrc QNB3bir
.dingmulceB<brspaAgntfC)ecuadomprnslhvaugsctrom198 ; 11 :55-9.
ftagmasts . Am Rev Resmr Du 198k 126.ID34-9
. 45 . Bmav 1 a G LonBmore WJ Adrtnag4 and
33 . Sahu3manE5 .MacGlashanDWJr,PetcnSP . choboergsregulauondflongsWfaeunfsxrrtrnn
ScMmmerRP,NmvhallHH . LirhtcmtnnLM Hv- intheisolatedperfusedratlungandiotheelveolar
man lung masl erJls : purif.caNuon and charactcr- 1ype 11 cegmculture J B&olChern 1981 :255:66-TL
srannn . J Immunol 1982; 1292662-`7 .
.6, Massaro D. Clerch L . Maadno GD. Sur(a[34 . InSel PA . Stoolman LM . Ratlrobgand bind- tam senesson : evidena that chobnerpe mmWarng to beta odrenergi<recePtors of mtast cullured uon of secreuon is mdireee Am I Phlstol 198s
5a9 cclls . Mul Pharmacol 19"J8 : 1<Sa9-61 .
243 :39-45 .
35 . Toews ML. Hard[n TK . PRrkuu 1P. Iletec4'l . SchmmrJer W, Po4lete-Frrundl G, Raucb K
non of lugh-affSniry agon6l, binding tU bela- $choenftld W. ResPOGSr ro i111mnnOlO,Wl or
ad .energicreceptorson~mm~ceRa .FadProcf98Z chohn[rgsestiauulauomufieplatedmasice8sfrom
91 :1535
an,
gutoea-Piga
sad
ra,te . CIA sympoaunk Ne .
36 . Atlas D, Ha.uki E. Lec-nzta A. Eghty Ihou- [Jrleans . MoetoB ABtryy 1978 ; 14W9-56,
sand betaa atlrenorecepton in a S+nglr B . Nature 48 . BitoconocY J, Befya Aq Penroe F, Dm6uq
1977 . 268 :1au-6 .
1-GoodaneR .Mastce4hetc.rogcndty.denvaaon
37 . Kal,nerM .OrangeRP,AussenKF.lmmunp andNncnon .wieRcmpquuontneiotutinalAC
Io8ical ydcax of Ruramloc and s/ow reaenng suD- lergy Cho lmmunol 19g2 ; 7D:407-12 .
PUBLICRTIONS
10333406
014417
LAZARU$ . STEPHEN C . . CAROL B . BASRAUM . PETER JBARNES, AND WARREN M . GOLD . eqAfh imrnunotytoclyrmstry
promdes evidence )or funcrional VIP receptora in trachea Am .
J . Physiol . 251 (Cell Physiol . 20)e C115-C119, 5986 .-Vasoat .
tive mtrstinal pepdde (V1P)- fvst isolated from porcine mtestine (S . 1 . Said and V . Mutt . Scirna-e Was), DC 169: 1217-12181970), has been identified in postgenglionic autonomic axons
of many ti .c.nrs. VIP has potent regulamry effrots on the
function of various cell typfs within these ti58ues, ranging from
relaxation of smooth muscle to ion transport . Recently, VIP
has been implicated in the regulation of mucus secretion in the
respiratory tract, a process involvmg releas .e of macromolecules
from exocrine cells and transport of ions and water across the
airway mucoss . However, because airway glands and mucose
both consist of mixed cell populattons- it was unclear which
specific cells contained VIP receptors and contributed to VIPevoked responses. We idenufied these specific ce0s by using
immunoeywehemical techniques to monitor concentration
changes in odenosine 3'-5'-cyclic monophosphate (cAMP) . the
intracellular compound known to mediate VIP reaponaea . Serous and mucous cells of ferret trecheal submucosal glands and
cihated and basal cells of dog trsebeal mucose all increused
cAMP in resportse to VIP stimulation . We conclude that these
cell types nossess VIP receptors and thus participate in VIPstimulated responses. In contrast ferret tracheal epitbe(ium
and dog epPtbelial goblet cells sbowed little or no reactivity
after VIP, and tbuswe believe that these rells lack VIP receptors .
ion transport : mucus secretion ; cilisted epitbelial cell ; submueosal gland: dog: ferret
(28) .
Many investigatnts have now identified aod character-
types could be successfully isolated. disaggregation techniques might alter the characteristics of receptors . We
have taken advantage of the physiological coupling between VIP and cAMP and have used intracellular cAMP
have since been demonstrated in the respiratory tract of These esperiments were conducted according to the
guinea pig3- rabbits- cats, dogs, and humans (5 . 33 . 34) . published gttidelines of the American f'hysiological SoThese VIP nerves are distributed around submucosal ctery for the care and use of laboratory animals . The
gland acini in the nasal and tracheobronchia] mucosa, in specific protocols were approved by the Animal Research
the subepithelial connective tissue of the nose, and Committee of the University of California . San Franaround blood vessels and airway smooth muscle of both cisco .
intra- and eatrapulmonary mrways . Dogs and ferrets were anesthetized with pentobarbital
U3Gt-6143/86 31 .50 Copyngbt &~ 1988 tbe Amencm PhyetolocsJ Sooery C71 S
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PUBL IC19TIONS
10333325
014336
Clio
VEP
RECEPTORS
QJ
DOG
wND
PsRItEr
sodium (25-35 mg/kg)- Although nerves containing immunoreactive VIP have been demonstrated in the respiratory tract of many ~pecies including dngs, no studies
1Rwctnrw
were transferred to beakers containing fresh culture medium, isoproterenol (10~ M) . VIP (3 x 10-r M), or
cAMP immunocytochemistry .
http://legacy.library.ucsf.edu/tid/tfz49c00/pdf
PUBL ICHTINS
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C117
CV-
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pUBLICRTIONS
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014338
'Clt8
VIP
RECEPTORS
IN
DOG
AND
FSRREP
REFERENCES
1 . AaumNon. B . M LAauNrNa . C DuvoNr AND G . RosaauNCbarectertvuoo of a vaaoenive mteatwd pepude-eeoetuve adenylate t-ydaae m rat mteatwd emtbelial cell membranes. B+ucMm
Bwphya. Arrn 544 : 474 481, 197&
http://legacy.library.ucsf.edu/tid/tfz49c00/pdf
1TtAC11EA
humeo e.r.oaye ,n v.uo. An. Ren. Redpp. . Dis. 124: 531S3B, igey'
5. D6Y, R D., W. A. SNANNON, JR . . AND 5. f . SAm . 1 . ..ItN .Vlp .mmlmoreanive nervea in wirweye and pulmonary vesy
doga. cetsm and humen eublecte . Ceff Twae Ree. 22R 231-Sig, lW
6. GA61Nel .u . T. S .. K . A Hueet, ANO't M . O'Doruslo . E8ee1r uf
,v. .voective intestiml pepude on mreeaml ehfonde secmnnn . fo :
Vvmmve lacettioo( PePnde . edited by S . I . Sai1 New York
Raven, 1982.p - 211-222.
7. KrrAUUaA~ S . . Y . IsanultA, AND S. 1. SA1D . EBea of VIP, pb,
norybenmmrne and predaieolooe on cyclic nucleotide mot4ar of
ieulaud guineap.g lung and trachea . 6ur. J. Phornwcut 67: 2W
223.1988.
& KRws, G . J- . J- H . W AteN . S. G . MoaAwssr. Areu J . S . Foaaawta .
IatractaDle diarrhea : int<eunal per[upion scudiee and pleema VIP
tenerotreuons in peuenta witL paocratic cholera syndrmne .ad
rrrptiuoue ingesnon of Isaativee and diuretiee . Am . J. D'y. Di.
22 : 2ga292, 1977 .
9. L..etmrNS, M ., P . btwccnr, G . M.u+atts-MouasN . wso G. ROSseuN . Activation of cyclic AMP-0epeadent protein kiaeses by
vaw.eaw inteatinel peptide ( VIP) in uolaud inteatmal epitb .~Wl
celle fmm rat Le Sc. 25 : 1931-1938, 1979.
10. Lt9neifm . M . . J . C . Paxero . B . AaqWVOn. C. DuPOnr, D . ytN
Bon HoA. AND G. Roes .ln . lnterafuon of vaeuec-uve iat.atmd
pepnde a.th ieoleted mteetmel epahelisl eells from ret- 2 Chsraeterlraaoo and evucnnal requvemlot of the simlulewry eBect
of vmoactive mteatanal prptide on producuon of odeaoeiae 3'S'monophoephate. Eur J . B.cchem 93 ;239-24g, 1979-
PUBLICATIONS
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C179
25 . SAID, S . L, S. RTTMfURA, T.
http://legacy.library.ucsf.edu/tid/tfz49c00/pdf
34 .
pig
tracheal
by
vegel
and
eympe
'
(M)
Localization of neutral endopeptidase (NEP)
mRNA in human bronchi
J .N . Baraniuk*, K . Ohkubo**, O .J . Kwon+, J . Mak+, M . Ali*, R . Davies,
C. Twort*, M . Kaliner##, M . Letartet, P.J . Barnes
Localization of neutral endopeptidase (NEP) mRNA in human 6ronchi . J.N. Baraniuk,
K. Ohkubo, 03. Kwon, J. Mak, M. Ali, R. Davies, C Twort, M. Kaliner, M. Lerarte,
P .J. Barnes. (PERS Journals Ltd 1995 .
ABSTRACT : Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract . It is of interest to determine which respiratory resident cells express NEP .
Trachea and bronchi from seven nonsmoking, nonasthmatic subjects were examined . NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot
hybridization of cultured human tracheobronchial epithelial and smooth muscle
cells, and reverse traascriptase-polymerase chain reaction (RT-PCR) in trachea and
bronchi. ln situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP
mRNA in human bronchial mucosa . NEP-immunoreactive material was detected
using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement.
NEP mRNA was 4 .5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis . No evidence was found by RT-PCR for truncated, alternatively spliced NEP mRNAs, such as del exon 16 or del exons 5-18 in
human bronchus . NEP mRNA was detected by in situ hybridization in epithelial
cells, submucosal glands, bronchial smooth muscle and endothelium . NEP-immunoreactive material was identified in the epithelium, submucosal glands, bronchial
smooth muscle, and endothelium, demonstrating an excellent correlation between
the distribution of NEP mRNA and the cell surface protein . NEP mRNA and
immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles .
We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function, and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in
human bronchi .
Eur Respir J., 1995, 8, 1458-1464 .
Neutral endopeptidase (NEP) is a 749 amino acid, zinccontaining, membrane-bound enzyme, which may play
a key role in regulating peptide-induced inflammatory
events [1-3] . NEP, also known as E.C.3 .4.24.11, enkephalinase, common acute lymphoblastic leukaemia antigen
(CALLA), CD10, and gplOO, has been cloned from
human, rat and rabbit tissues [4-9] . Functional studies
indicate that NEP activity is present on epithelial, glandular, smooth muscle and vascular cells which possess
peptide receptors [1, 2] . Loss of NEP activity may significantly contribute to the hypersecretion, vascular permeability, bronchoconstriction and other pathological
changes seen in respiratory inflammation by permitting
unopposed, prolonged actions of inflammatory peptides,
such as tachykinins and bradykinin .
In the present study, in situ hybridization was used to
determine the distribution of NEP gene expression in
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human bronchial mucosa, whilst the identity of NEP messenger ribonucleic acid (mRNA) was confirmed by
Northern blotting . NEP immunoreactive material was
detected by immunohistochemistry .
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ribonucleic acid (RNA) extraction and cryostat sectioning ; and 2) fixed in 4% paraformaldehyde in pH 7 .4
phosphate buffered saline (PBS) for 4 h at 4C before
embedding in paraffin.
1459
Cell culture
Human bronchial epithelial cells [10] and smooth muscle cells [11] were cultured as described previously .
In situ hybridization
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1460
J .N . BARANIUK
http://legacy.library.ucsf.edu/tid/tyg16d00/pdf
Immunohistochemistrv
ic
20 10 5
Smooth Muscle
20 10 5
Epithelium
1461
A B C
r r
. .
. ' . ..
Y
>
Fig . 2 . - Reverse transcriptase-polmeritation chain reaction (RTPCR) for NEP mRNA in human bronchus total RNA . The eaons
19-20 splice site antisense (RT) primer and e.waa 14-15 splice site
sense primer generated a band at 477 nucleolides corresponding to the
full length mRNA coded from the exons 14-20 (t .anes A) . No truncated variant NEP mRNAs were found. Lane B shows markers at
1353 (top), 1078, 872, 603 and 310 (bottom) nucleotides . Lane C
shows the ~-actin mRNA RT-PCR product (661 nucleotides) . Lanes
A : NEP ; B : markers ; C : (S-actin . mRNA: messenger ribonucleic acid . For further abbreviations see legend to figure 1 .
A
100Nm r.
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NEP immunoreactive material was detected in epithelial cells, submucosal glands, smooth muscle and endothelium of human bronchus (figs 6 and 7) . In submucosal
1462
1 .N . BARANIUK
: :.
t_
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NEP
mRNA
Discussion
Cell surface NEP may limit the actions of many of the
peptides that are active in human tracheobronchial mucosa
[23-25], including tachykinins and calcitonin gene-related peptide (CGRP) released by axon response mechanisms from nociceptive sensory netuones during neurogenic
inflammation, vasoactive intestinal peptide (VIP) released
by parasympathetic reflexes, bradykinin generated in
many types of allergic and nonallergic inflammation, and
circulating peptides, such as endothelin and atrial natriuretic peptide (ANP) [26-28] . Decreases in NEP activity
[22] may underlie the increased responses of respiratory mucosa found during viral infections [29-31], after
exposure to cigarette smoke [32], ozone [33], hypochlorous acid, [34], and high doses of toluene diisocyanate
[35] . The release of peptides into areas with reduced
NEP activity could lead to enhanced peptide-induced
epithelial cell function, glandular secretion, vascular permeability and smooth muscle contraction [1-3, 26, 27] .
Each of these proinflammatory processes occurs in sites
where NEP mRNA and protein are found : epithelial cells,
submucosal gland cells, bronchial smooth muscle, endothelium and arterial smooth muscle (table 1) . Apparent differences in the numbers of gland and epithelial cells
containing NEP mRNA and immunoreactive materials
between specimens were due to different proportions of
goblet and mucous gland cells (figs . 3-7). These mucous
cell vacuoles did not contain NEP . Other epithelial cells
and glandular serous cells did contain NEP mRNA and
immunoreactive material .
These locations also correlate with the sites of NEP
enzyme activity detected in guinea-pig tracheal epithelium, glands, and vessels by fluorescent zymographic
microscopy [36] . Enzyme activity was also detected in
the perichondrium and chondrocytes, but was not detected in guinea-pig tracheal smooth muscle cells [361 .
Expression of NEP in lung parenchyma has been investigated by JotwsoN et al . [371 .
Whilst changes in NEP distribution or expression or
decreased activity have been postulated to contribute to
-changes in airway reactivity to selected stimuli in vivo
[ 1, 2], there are as yet few data from humans to confirm
this contention . In fact, RotsMAN et al . [38] suggested
an increase in NEP activity in lungs of asthmatic subjects . Modulation of NEP activity in disease or after
Table 1 . - Distribution and relative intensity of expression of NEP mANA and immunoreactive material in human
trachea and large bronchi
Site
Epithelium*
Endothelium
Submucosal glands*
Smooth muscle
In situ
hybridization
Immunoreactive
material
++
+
++
+
++
+
++
+
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IN
HUMAN
BRONCHI
1463
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