Expert / Consultant Submissions RegardingETS to Regulatory Agencies on Behalf of Philip Morris
Working DraftAugust 11, 1998
(1190809.01 1
http://legacy.library.ucsf.edu/tid/vbh77a00/pdf
tConfidential
Attorney Work ProductAttorney-Client Privilege
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)Moore, Richard E . (AAL)(3-1085)Reasor, Mark J . (WVU-HSC)(3-1071)
Will. James A .Robertson, Gray (HBI)(L3-1182)Roe. Francis J.C. 0(3-515)Switzer, Paul (SU)(3-1066)The Tobacco Institute (3-1086, L3-1188)Witorsch, Philip (GWU)(3-1045)Wu. Joseph M. (NYMC)(3-1080)
Tab B: OSHA Notice of Proposed Rulemaking - Public (NPR)(04/15/94)
Ashford, John R. (EHIS)(9-26467)(06/00/94)Barnes. P.J . (9-105472)Benda, George (9-47596)Bridges, J .W. (RIIEHS)(9-40331 )Caldwell, John (St . Mary's Hosp .)(9-101053, 9-8672, 9-59160, 9-47799)Chanter, Dennis 0. (Bookwood Stats., Ltd.)(9-26010, 9-26026)Chappell, Willard R. (UC-Denver)(9-27972 )Cline, Martin J. (LICR)(9-62029, 9-27687)Constangy, Brooks & Smith (9-2202, 9-7175, 9-24993, 9-91211, 9-22884,9-100505, 9-84161 . 9-85545, 9-69816)
Smith, David L.Tyson, Patrick R.Wasser. Neil H.
Devine, Thomas (RMT)(9-47508)Robinson, H . ColemanSkipper, Steven
deWolff Frederik (Univ. Amsterdam)(9-105467)Fletcher, George (Fletcher Group)(9-27972)Gratt, Lawrence B . (IWG Corp.)(9-27972)Greenfield, Stanley (SAI)(9-1612 ; 9-27989)
* 16 Comments that are particularly useful examples .
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PROFESSOR PETER J . BARNESSELECTED BIOGRAPHICAL INFORMATION
• Professor of Thoracic Medicine, University of London
• Chairman, Department of Thoracic Medicine and HonoraryConsultant Physician, Royal Brompton Hospital, London
• MA, DM, DSC, Fellow of the Royal College of Physicians
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PROFESSOR PETER J . BARNESSELECTED QUOTATIONS
Pulmonary Diseases and Conditions :
• "I have several concerns about the interpretation of theevidence quoted in this document . While there is naturallyconcern about the effects of ETS in the workplace, many of thestudies purporting to show adverse effects on airway functioncould be interpreted differently and there are severalrelevant studies that have not been referred to . The OSAAreport reviews an extensive literature on the effects [of] ETSon airway function and has reached a conclusion that appearsto extend beyond the available data ." (Written submission, p .1)
• "In the review by Tredaniel et al, 6 studies which show noeffect of ETS exposure on lung function are discussed, whereasthe OSHA document refers to only two of these studies ;although of the positive studies cited in this review, 7/8 arereferred to in the OSHA document . This appears to indicate abiased selection of references ." (Written submission, p . 1)
• "There is no convincing evidence for a dose-responserelationship between the amount of [ETS] exposure and the riskof airway disease and there is little quantification of theamount of exposure in the workplace ." (Written submission, p .2)
• "There is certainly a pressing need for large longitudinalstudies to address these issues and accurately quantify anyrisk of ETS in the workplace . Until such evidence isavailable, it may be premature to conclude that exposure toETS in the workplace is significantly increasing the risks ofairway disease ." (Written submission, p . 2)
October 19`h-22" d 1998Lainston House, Sparsholt, Winchester, UK
Anna-Lisa Fisher (Secretary)Mike DixonEian MasseyDavid O'ReillyGraham SmithChristopher ProctorAntonio Augusto Rodrigues
Welcome and Introduction - RB welcomed all members. The objectives of thismeeting were :•
To assess the progress on projects currently funded by the SRG and consider newareas for funding.
•
To review the scientific content of the new position papers•
To be updated on topics relevant to smoking and health issues•
To hear the views of external experts in smoking and health areas in the form of amini-symposium
Matters Arising•
In response to RB's communication, Erwin Kausch, had indicated that it would beappropriate for updates of relevant projects funded by the Verband derCigarettenindustrie to be presented at future SRG meetings .
•
The previous work performed by BAT on the effect of humectants on lungretention has been re-analysed . Further work is likely to be undertaken inSouthampton and/or B&W .
BAT Position Papers on Smoking and HealthThe currently available position papers are :•
Smoking and lung cancer,
t
(Action ES-W)
(Action MD/SA)
•
The TMA funded Covance study on nicotine retention is due to next month . Thestudy is expected to take approximately two months . If the study,is completed, theresults will be presented at the next SRG meeting .
(Action MD)
•
Following publication of a paper on ammonia/nicotine chemistry by Pankow et al,1997, John Lauterbach (B&W) has written a chemical critique of this paper . TheSRG recommends that this critique should be submitted for publication in anappropriate journal .
(Action SA)
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aQ1lcg
•
Smoking and respiratory disease,•
Smoking and coronary heart disease,•
British American Tobacco's research and the "safer" cigarette,•
Cigarette tobacco ingredients,•
Smoking and "addiction",•
Environmental Tobacco Smoke .These have been updated and distributed world-wide .Three proposed new position papers have been considered :•
Smoking and reproduction (in draft form)•
Mortality statistics•
Smoke constituentsAP reviewed the new Reproduction Position Paper . There was general agreement withthe views expressed by Professor Jean Golding (see Mini-Symposium) . AP will checkreferences and send the final version to the SRG members .
(Action AP)A request for a "Smoke Constituents" position paper from the Board has not beenminuted . This position paper will not be progressed until a firm mandate is given .
Current SRG Projects - AF presented updates of some of the projects currentlyfunded by the SRG. DO reviewed the project on the putative tumour suppressor gene,DUTTI, by Sundaresan . This was considered an important area of science . Success inthis project will be defined by determining whether DUTTI has a tumour suppressorrole, particularly in lung cancer . Sundaresan has also requested extra funds (£45k) topurchase some specialised equipment (a FISH workstation) . It was agreed that theSRG would fund this equipment but Sundaresan would need to advise the SRG onhow he wished the equipment purchase to proceed .
(Action AF/DO'R)
Due to unforeseen difficulties, the nicotine protection project by Gray was unable tocontinue. It was decided that funding for this project would no longer be provided orredeployed to Gray's second project on nicotine modulation of pre-pulse inhibition . Itwas agreed that the nicotine modulation project would continue to its naturalconclusion . In view of Gray's imminent retirement, it was agreed that his future roleas a consultant to the SRG would be explored .
(Action AF)Tovee made a request for extra consumables (£4k) . MD will assess the protocol andjustification for these consumables .
(Action MD)It was decided that AF should send out the project briefs and full detailed projectupdates in advance of future SRG meetings .
(Action AF)Ingredient Toxicity TestingSA described B&W's intention to set up facilities to perform biological and toxicitytesting on all cigarette ingredients not otherwise covered by some other body . TheSRG recommend that there should be a co-ordinated approach across BAT .[Following further discussion at the Smoke Science Team meeting on 22/23 October1998, it was subsequently agreed that SA and GS would develop appropriateprotocols for testing additives] .
2
(Action SAGS)
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It was recommended that SA would make a presentation of the proposed protocols tothe TSC/REC meeting in Macon in November 1998 .
SRG IssuesSM gave an overview of Federal Regulations in British Columbia regarding toxicconstituents and described his communication to ????? of information resulting fromthe monitoring of a reduced toxicity cigarette .
AP reviewed the current perspectives in COPD and Asthma . AP had visited twoexperts in these fields, Professors Peter Barnes and Clive Page . PB works at theNational Heart and Lung Institute and is the leading world expert on asthma whowould be willing to consult for the SRG and may submit a proposal to investigate "thegenetic influences on susceptibility to COPD" . CPg, at Kings College, London, is aleading world expert on asthma. As well as being willing to provide consultationservices to the SRG, CPg may also submit a research . These proposals (if submitted)will be assessed at the next SRG meeting .
Project Proposals1 .
PJ presented his project proposals :•
"Developmental and Respiratory function follow-up of infants fromantenatal study" by Paul Johnson . It was agreed that this project was ofrelevance to the SRG portfolio and of sufficiently high quality to fund. AFwill inform Johnson and make the necessary arrangements to commencefunding .
(Action AF)•
"An Interventionist Study of nutrition supplementation during pregnancyto see whether foetal and postnatal development can be altered in smokers"by Paul Johnson. Due to several problems associated with this projecthighlighted by an expert in this area who reviewed the project, it wasdecided that this project could not be funded in its present form. AF toinform Johnson of the SRG's decision .
(Action AF)2 .
"Ni cotininc-Muscarinic interaction in the modulation of dopamine release inrat striatal preparations" by Sue Wonnacott . This project has been favourably peerreviewed. Since this project was not discussed at the SRG meeting, a decision will bemade in the next two weeks by contacting SRG members by lotus notes/fax/telephoneas to whether this project will be funded by the SRG .
(Action AF)3 .
"Intra-Uterine effects on adult non-insulin dependent diabetes in South India ;a risk factor for coronary hear disease" by Caroline Fall and David Barker .Unfortunately this project was submitted too late for SRG review . It will beconsidered at the next meeting .
3
(Action AF)
32149497 1
To :
Graham Read/Southampton/GB/BATCo@BAT, Graham R .Smith/Southampton/GB/BATCo@BAT, DavidO'ReillyfSouthampton/GB/BATCo@BAT, EianMassey/SouthamptonlGB/BATCo@BAT, MikeDixon/SouthamptonlGB/BATCo@BAT, Scott Appleton/Macon/US/BAT@BAT,Stewart Massey/ITL@ITL @ BATCOEXTERNAL, EvaSchumacher-Wittkopf/Bayreuth/DE/BAT@BAT, Leopoldo Caruso/ComplexoAmorim/BR/SouzaCruz@BAT, Christopher Proctor/Staines/GB/BATCo@BAT,Derek Irwin/Southampton/GB/BATCo@BAT, RichardBaker/Southampton/GB/BATCo@BAT, Antonio Augusto Rodrigues/ComplexoAmorim/BR/SouzaCruz@BAT
cc :
Adrian Payne/Staines/GB/BATCo@BAT, Linda Rudge/Pagewood/AU/BAT@ BATFrom:
Anna-Lisa Fisher/SouthamptonlGB/BATCoDate :
18/11 /98 16 :48 :02Subject :
Molecular Genetics of COPD
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 bepublished 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, associatedfactors and what/why further research may be important .Professor Barnes is considering submitting a proposal to the SRG next year on genetic markersof susceptibility to developing COPD .
Regards
. . .Anna-Lisa
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24 September 199 9
Department of Thoracic MedicineNational Heart and Lung InstituteDovehouse StreetLONDON SW3 6LY
Dear Professor Barnes
I
As a recognised expert in this field, we would like to invite you to write areview on mechanisms of chronic obstructive lung disease (COPD) withspecial reference to smoking. The review will be your property to publishif you wish, and it is not a requirement on our part to acknowledge thesource of funding (although some journals may of course require this) . Forour purposes, this review would be a reference document for BritishAmerican Tobacco scientists in the context of current awareness ofscientific opinion in smoking and health research, as part of our duty ofcare responsibility .
Although we do not want to influence or set specific guidelines for thecontent of the review, issues that we are particularly interested in include :
• Concise Review of the Most Recent Literature and the impact on CurrentScientific Opinion• Current views on important Risk Factors and Possible DiseaseMechanisms
• Emerging Trends in the Understanding of Possible Genetic Influences• Gaps in what is known about Disease Mechanisms and SuggestedResearch that could address thes e
We will, of course, provide a mutally agreed appropriate sum tocompensate you for the time spent on this project and any reasonableexpenses that you incur . There is no particular deadline for this review tobe finished, but a guideline would be about six months .
I would be grateful if you could contact me to confirm that thisproposition in acceptable to you in principle and, if so, to discuss thelength of the review and remuneration .
32526181 5
&11c'. US DO', Philip Moms
My contact details are :direct line 01703 793309email [email protected]
Best regards
. . .Anna-Lisa Fisher
32526181 6
PATfl, I11 DO,] v Philir Mnmc
Professor Peter J Barnes MA, DM, DSc, FRCPProfessor and Head of Thoracic Medicin eHonorary Consultant Physician, Royal Brampton Hospital
Direct Lime: 01713518174 Fax: 01713515675Email : p j [email protected]
Department of Thoracic MedicineImperial College School of MedicineNational Heart & Lung InstituteDovehouse Street, London SW3 SL Y
6 October 1999
Imperial CollegeOF SCIENCE, TECHNOLOGY AND MEDICIN E
Anna-Lisa FisherR&D CentreRegents Park RoadMillbrook
Southampton SO15 8TL
Dear Ms Fisher
Thank you for asking me to write a report on mechanisms of COPD with special reference tosmoking. I will try to let you have this within the next six months . I briefly discussed thiswith Adrian Payne who suggested that an article of approximately 20 pages with referenceswould be ideal .
Best wishes .
Yours sincerely
e c"-,
Peter J Barnes MA DM DSc FRCPProfessor of Thoracic Medicine
t--bLo'L Prvj . Q c~.~r-c S
t'l6se .-e.
Imperial College School of Medicine
325261906
FLAT.-' U% 0(U v Philip -
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Professor Peter J Barnes MA, DM, DSc, FRCPProfessor and Head of Thoracic Medicin eHonorary Consultant Physician, Royal Brompton Hospital
Direct Line : 01713518174 Fax: 0171 351 5675Email : p .j [email protected]
Department of Thoracic MedicineImperial College School of MedicineNational Heart & Lung InstituteDovehouse Street, London SW3 6LY
22 December 1999Imperial CollegeOF SCIENCE, TECHNOLOGY AND MEDICIN E
Dr E 0 GreggScientific and Regulatory Studies ManagerBritish American Tobacc oR & D CentreRegents Park Road
• MillbrookSouthampton SO15 8TL
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 isimportant and Dr Young has expertise in molecular genetics . This is an area where severalother larger labs are already actively involved. I am surprised about the concern aboutconfidentiality since these precise studies are already underway in at least two locations andthese are very obvious studies to do !
• I think that the project is worthy of funding .
Best wishes and happy New Year .
Yours sincerely
Peter J Barnes MA DM DSc FRCPProfessor of Thoracic Medicine
Enc.Imperial College School of Medicine
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Grant Application of Dr R P Young
The genetic predisposition to COPD and emphysema is an important area of researchwhich is currently attracting a lot of interest . Only a small proportion (approx. 15%)of smokers develop COPD, suggesting that genetic factors are important . Sinceseveral studies have shown that certain matrix metalloproteinases and neutrophilelastase are released in increased amounts in patients with emphysema, it is possiblethat gene polymorphisms of MMPs and their tissue inhibitors (MMP) may play a rolein predisposing to this disease . The approach taken by Dr Young is therefore logical(although several other groups are already looking at this same area). Most genepolymorphisms in common diseases are in the promoter region and affecttranscriptional control of gene expression, so the emphasis on promoterpolymorphisms is sensible . However variations in sites on the promoter may alsoaffect transcription by changing the conformation of DNA .
The patient selection is sensible and it is clearly important to closely match smokin g• history in emphysema and normal subjects .
Dr Young proposes to use heteroduplex analysis, but most investigators believe thatSSCP may detect more polymorphisms.
It will be important to establish that any polymorphisms detected more frequently inemphysema are of functional importance, using a gene expression system .
Preliminary data to show the existence of polymorphisms in the MMP/MMPpromoters would be useful before embarking on this project .
Dr Young has a background of research in molecular genetics and is familiar with themethodology .
The funding requested is reasonable for the proposed work.
0
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O. B. Cohan
' » '*-»»%*1 %*» IV»«KU«a»i».l *\st K \ ( l k m ) \ ) U | » » P i » t » i » * • I * * * - . " * " f**U*» l*«r ti*a**« raS«nni- f f^jim-*.•}••„» i*M9>%ra>l»z*mt*S<al lAaV.nwi**
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Characterization of Beta Adrenoceptor Subtypes in Canine Airway Smooth Muscle by Radioligand Binding and Physiological Responses1
PETER J. BARNES/ JAY A. NADEL. BENGT-ERIC SKOOGH/ and JAMES M. ROBERTS' Cardiovascular Research taitiMn and Departments of Matiicmo and Physiology. University of CaMorma. Sail Francisco. CaUtomm Accepted for puWcaton FaDrusry 22.1983
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.
Using selective beta antagonists, wa detennined the ratio of bttO'i/bBta-2 receptors in tracheal smooth muscle membranes to be 1-4. The relaxation response of tracheal smooth rnuteie stnps to exogenous tats agomsts was mediated by Oata-2 receptors, with a very small coninbutton from befe-1 receptors. However, the relaxation response to electrical field stimulation of sympathetic nerves was mediated predomtnantty by Dett-1 receptors- Our results suggest that most Data receptors * i dog tracheal smooth muscle are of the oera-2 subtype and meoiate responses to circulating catecholamines, but there w a small concentration of oafa-l receptors which mediate the response to neuraiy released norepinephrine.
Airway* an? relaxed both in vitnt and in avn hy beta adrenergic aguni»t*. 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 in 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
Hn»«^l"r |>uMHoli .ml^-nJ«-f .'V |SJU *Tlii« «•*!• * * » MipiH-ntil in pun In NslH>n»l I f tMi l i iWnf Hrsl lh I 'mir im
1'itwni l.Mtii H l . - i t l m and rntann M i l I rum i b r Ceonnl l«r Toliirro f t r w n b
* 1 ' i o r n i « W » « - M I l r I V I M - I B«rB». l>»p«iimfni.4MfdK>«>».H»ini7wr«Hiih Hn-mlnl Ducal* H<wl. | j *H- i» U I . ' I K K K V H M I I «il • Mrdiml HorMrh Citonul »l t.ftv* I I H L H I I f ratrHmr l>*"*»hiH
' N J | I | « ' M M I hi « mum Irrnn ihr S»rtliO> Nmrir»I A » « K I * > H I H A K » I I I « H M H flnrtfhM t>murr*Jinrtlnain/in1tnwn('i)i<l1uirffl»mjlii.ili*ui»|j>in\ iSummtt. V I i
*Hrc>|wni »l Satmnul lr»iiiu<r. til Mrslik K r v i K h l » r i r l Ih-trltpntrni
of.. 1975: Omini ft a/.. 1979: lakovidiH vt <if. 1980: Jnhnnamm and WaldccJt. 1981) and cat (LulR-h rt at., una}. suggesting the presence of/n'.o-l receptors in addition to fx'tn-2 rereptiirs in airway smoot h muscle.
Direct binding studies using labeled beta adrenoceptor antagonists! hove confirmed the coexistence of brto-l *n£ thta-'J receptors in homogenotc* of king in se.'crat specirs iKujig rl o/.. 1978: Minnemnn el al., 197Jln; Kngrl. 19H1I, and using selective beta agonists it has been possible to determine the ratio of beta-Xfbeia-'l receptors. Hut lung contains o»er -ti> different cell types and we have recently shmvn that ihi> \m\ majnriiy of beta receptor* in lung are asuodated with alvenlar walls rather than with nirway smooth muscle (Haines »r nl.. 1932). As the lung has a heterogeneous cell population, it is not known whether some cell types of erfo-l rerepinrs. whereas other* have oc/a-2 receptiHs. it in not pnsslhlr to study' intra-pulmonary airway smooth muscle by direct binding assav. as insufficient tissue would be available to prepare membrAm* hamogenatr.H. We have therefore invextigated trm-henl smooth muscle of the dag /is thin can be disserted free or surrounding tiSHues and have used ('HJDHA to study ihr charnrtprimiiv »f beta receptnrs in hnmngenates of thin tisnue. For ciimpnrixiui. we a1*> stiidiitl m ri'fn* beta adrcnergir resjionses in the wiim-tiHsup lining Imth exogenoun beta agunism nnd etn-tricai stun-ulmion t f vvrnpnlhrtir nerves.
AMHEVIS-TION: DHO o.nyofoaOfenoiol
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19S3 Airway BUM Receptor Subtype* 457
Methods
Do» tracbeatis t w h w i i f p n f i n i l n . We obtaiwd tracheae tiom Ayp- which were being used in other etpenrarm* which did net imvhe (he useol drug* other than anesthetic*. Dogs were anesthf tired nuh pewuhartnwl sodium t.10rng/kg t.v.t.and the trachea was rapidly removed. The posipnor membrane portion, of ihe trachea containing the trachealw muscle was dissected free of loou connective tissue and the epiihelium »as stopped awry. The tracheal*! muscle was the* separated and finely mmred with *ct*»or* in 10 vulume* of ice-cold incubation buffer i50 mM Tri* HO. i«H «.•!!. thru homogenized in a Polytron tissue homogenizer (Bnhkmann Instruments. Inc, U'estbury. NVi at setting d tor A x IX tec period*. The resulting homogenate wa* filtered through two layer* of cheesecloth 10 remove unbroken cell* and connective tissue. The supernatant was centrifuged at aojOOD x « for 15 min aad the pellet worked and rccentrifuged in buffer at a concentration of OA to l.o ma of protein* per ml. Thi* particulate preparation wax either used directly in ihe binding away or stored at —TOT for up to .1 month* without change in binding characteristics. Protein wa* determined by the method of Bradford 119781. using buviiir serum alrmmtn as the standard. Particulate* were aim prepired from peripheral lunjj of the umr animals after dissecting sway majur atrwoy* and blond vessels, using the seme procedure.
I'HIDHA binding assay. Membrane* lapproximately IdO vie of protein per a»«ay> were incubated with | 'H]DHA in a final volume of 025 ml. Nonspecific binding wait determined by inrubntion* in the pretence or I *M /-propranolol. Each data point wis determined in duplicate, and duplicate* did not vary by >ltici. Equilibrium incuba-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/ C filter*. toNowed by two further Aral washes. Fiher* were counted by Squid scintillation spectrometry- Specific binding, which was determined from the difference between total and nonspecific count* bound to the fitter*, comprised 60tnHo', ol total bindingat ligand concentration* ol leu* than 2 n\l. For equilibrium binding, concent rat HWK of I'HIDHA varying irom O.l 108 nM were used, and Inr competition and kinetic studie*. a concentration hrtwern I and 2 nM wan u*ed.
J* vitro beta adrenergic responses. For physiological studies the excited canine trachea wa* immediately immersed in Krehs-Henseleit solution with the billowing composition: NaCI. I ID mM: KCI. $.9 mM: CaCI-. 3.* mM: MgSO.. 1.3 mM; NaH.PO.. 1.2 mM: NaHCO.. 2W» mM: and glucose. 3.6 mM. which was gassed with 9l*» O, and tf» CO,. Alter separation ol the epithelium the iracheatit muscle was cut trans-vcrsel.v into strips - t o 3 mm wide which were mounted vertically in glass chambers filled with |A ml of Krebs-Henwleit solution, main-tained at 3i*C and aerated with 94'« O, and 6!7 CO,. l*o»etric tension was measured with strain gauges <0rass model FT 0.03 lorre-dlsplace* mem transducer) and recorded continuously (Grass model <D polygraph*. The tissue baths were fitted with platinum elecirndes for electrical field stimulation using bipharic pulses (supramaximal voltage tt* msec duration. 12 Hr frequency! for 20 sec. Strips were allowed to eiaiilibrate lor 1 hr and retting tension was adjusted to 10 R. which was optimal fur determining changes iti tension. Only strip* that developed a tension ol titeaier than 10 g to electrics! field stimulation at 12 H* far 20 sec srere wed in the sludv. ,
Muscle >tnri» were ranirarted by 5 MM acetylrhutine. taeii. when cent radium, were stable, a cumulative dose-responne was performed tn iMfproterenti) tiMii-lini «Ml ut terbutaline il-.l »M). using a 2-min expmure tmte to each concentratibn. Theelfecl ol selective orio adre. nergic Wockade on these agonwt dW-Ksponse turn* was determined h\ preincubation with erther the fti-»o-l selective antagonist prartolnl in s«Ml «r the orfa-2 selrrthe amagoniM IPS X» «0.1 »M». These conrentratwn* ol antagani*! were chosen on the basis of cimpelitiDn studVk »nh I "HIDHA hindmg. so that seleciivitv wiHild be retained.
The ellert «il selective Mit adrenergic antsgitaists on the beta adre-neriiir telataiiim re»n«im>e to electrical field Mimutoliim was also determined. The cholim-rgn re»|Hin»ew»«Hlicl(edb\ 1 »tM atrtipine. and ihea^ihaaJreaergn r»»iKmM«h> lt)«M pheniiilsmme The muwle wa»
then contracted with serotonin tOilaMl ami» heft the contravtinn WHM stable t.lD rain!, the retasation response to eectrical field stumiUuvn was determmed. This response was inhibited by I iiM propranolol. The effect of seleciivr brio adrenergic agonists on the response tr> tlehl stimulation was determined by prior mcubatiun 130 nun) with either prxctolul t:lp.M) or IPS :i&) 10.1 uMl.
Data analyale. Result* are espresied as mtin * S £ . Statistk-al comparisons were made by unpaired Student** l test. Binding data were analyzed by a nonlinear least.squire* cutve fitting computer program. For competition studies parameters were chosen to yield the best fit ol data, as determined by the mmunal variance of experimental data about a curve generated by these parameters iMurlasrtaf.. I9B2J. Parameters Tor one and two affinity site interactions were tested. The inhibitory dissociation constant l Ki I was determined from the tela! iun-ship-
K. I C
I + |L]/K„
where U\. is the concentration of drug causing -Wi inhdiition of spenfir i'HIDHA bmdinK. IM is the concentration of I'HIDHA used and Kit the dissociation constant of |'H|DHA determined in equilibrium binding assay*. Saturation isotherm* were analyted arcoriiing l o a one siteor two site interaction and the best tit determined by triimmal variance. The estimated dissociation constant I KM I for antagonist* tn the physiological studies wa* determined from the relationship:
KM< J!L 1LVL - I)
where | l | is the concentration of amagonsn. L' is the EDv.of agoniat in the presence of antagonist and L t>e EDv. of agonist alone."
Drugs and chemical*. |'H|DHA <>prcific activHy 101 ri/mmoll was obtained from New England Nuclear (Boston. MAI. Drug* ware obtained from the following sources: f-isoproterenril. /-eiwirphrihr hydrochloride, f-nurepmephnnrliydrochloride.atfopinesuluic. serinomn creatinine sulfate. acetylcholinrrhloride.oY.propranolol iSigmn Chemical Co.. iit. Umi*. MO): practolol. d- and f-proprahiilnl lAyerst Laboratories. New York. NYl: IPS.139 hydrochwrid* < AB Hassle, (ioteborx. Sweden): terbutaline sulfate (Astra Pharmaceutical Pmdurm. Inc.. Worcester. MAI: and phentolamine mesylate tCiba-l»eigy Corp..Summit. NJ). All drugs were made up freshly in distilled water immedf stely before use. Catecholammes ware made up in O.l mM ascorbic acid.
Results t ^ i D H A biNdlng anturatlan atudlet. Specific binditiR or
I'HIDHA to don trachealiB membranes won saturable and of high offinity. Saturation iaothemta were beat defined by inter* action of | *H|DHA with a single populnlion of hindinit otic* and Scatchitrd analysis save an equilibrium dinsticiatiiin con-Mant (Kul of 1.0 ± 0.00 nM <n •> 61. which was very simitar t o that determined in peripheral lung membranes from the same animals (0.98 ± 0.0ft nM, n «• fil (fix. 1)., The maximum concentration of binding; sites ( B ^ , I to tracheal smooth muscle membranen was 9A.6 ± 4.7 fmol/mg of protein, whiih wan considerably less than that determined in whole lunu membranen {R32 ± 48 fmol/mg of protein).
Kinet ic studiea. Specific bindinx was rapid « T , . »'.S min), reachinx equilibrium al tn min. and was revemiUIrun aildition orf•propranolol i T , u 3Jt mm). The kinetic Ki. calralatrd from the ratKi o f the reverse rale curjHtnnt tn the forward rate constant wait calculated as 0.7(1 ± 0.1.1 nM (n •» .*!). which was in ttood ngreement with the Ki. deirrminrrl tn equilibrium Murlir*.
Compel l t ion aludlea. Sprrinr liindinK to f rm-hrnlis mem-branes was stereoselective, with / pmpMnoNI opiwoHiinntely
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4 » Basnet at at.
8max - ' ' • 8 fmoi/ina crorsm
W.225
R 9 . I . BKXJmsoM'HPHAtoacatncfiaaismooinrxrtda membfarts. UHpanatspaoiCtxtxfngi*) art nonieaolie bmomg mine presence ot 1 *M£propranoioi{0)are shown. Right panti: Seaward antfyas snowing a smgia daw ol binding s>t* with equ*bhu«i 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, /-aoprottf-•not ( • ) . /-epintpnrine (O). Mwrnpncprmna (A) and taroutaime (A). Right p a w inhibition by /propranolol (•)) and tf-propranoioi p ) . Each pant 11 from a angle experiment performed m duplicate and * typical of three) auch expenmeme.
•r -9 -S (OB (AGONIST) CM)
•9 -8 -7 -6 log < ANTAGONIST)
TABLE 1. DiaaartaHon oonstaats WW ot adrenergic aganta lor mhibNon of specJflcfolQHAbhxk^ 1 0 0 ^ t r a v e l emcothrnuecte
t>anolsesfttceusffiQ9p%iflfttst*Qnof apaonc|*H)DHA twiOAQ fram a computer cts^aring program. fc)i»B^ concentration ol j'H|0MAui<>d«» the away (1-2 fiiMl and Ko it Mia oissoothon constant from oojueonum among {l 0 oM)
• for aw sweckve aniagonau K. * the Ossoeeten o»j*a hish-aftrtiy ana and K, «• tftt lewarftnmi ite TM otta shown are meanslt $ t ot tnraa to «vo asperats •xperanantE.
MO timet more potent than •'•propranolol (fig. 2: tabte 11. Among agonists the rank order of potency wae /-isoproterenol > /-epinephrine > /-norepinephrine, indicating that tht majority of beia receptor* were of the bcta-'2 subtype. The beta-2 wleriheaapniatterrwtalinewaKapproxtrnaieiyequipoteniMiith rMrepinephrine which 1* similar In the finding of other* using lane tarmhranes which have predominantly brfa-2 receptor* (Minntman el ol.. 19T91i». Inhibition ot specific ( HJDHA bind-
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 site of lower affinity which U presumably the btto-\ receptor. With practolol. the converse pattern waa seen with most specific J H IDHA binding <78.0 ± 2.4rc*. n . - 4) to a low-affinity aite (the 6t»/o-2 receptor). As | 'HJDHA binding has equal affinity to 6rta*l and frefa-2 rereptora. the ratio of tota-l/bcta-i aitea using either antagonist waa therefore approximately 1:4.
ltt vitro response*; 6e/a aajoniatt. Isoproterenol caused a relaxation of dog trachealis strips, which had been contracted with acetylcholine, with an ED*, of 4.2 ± 0.7 **M (n •• 8). Thtre was no evidence for aignificant beta receptor deaensitiiation during the cumulative dose-response to isoproterenol, aa in preliminary atudiea we found that the final cumulative doae <10u * M 1 caused a similar relaxation in strips not exposed to progressive increases in concentration. IPS 339 (O.t «M> gave a marked parallel shift to the right in the isoproterenol dose* response curve <ED-» 78 ± 1.5 pM) with a K» of 4.6 n M which waa in good agreement with its K i for the high-affinity site 11.0 nM) determined from competition with | "HJDH/*. binding (fig. 4). Practolol (3 pM) produced only a small »hift in the dose-response curve to isoproterenol (ED. , 9.7 ± 2.S «iM) with a KM of 2.2 *iM which la intermediate between the high- and low. affinity binding sites determined by competition with J 'HIDHA binding. Thia may suggest interaction with both ftr/o-1 and rxfa-2 recrptors physiologicallv. With trrbutalinetED^ IrV) ± 27 »iM) IPS 339 gave a matked inhibition tED. . :l.VH> ± lutu nM) with a K H of 4 ^ nM. which was similar to that found with isoproterenol. Practolol, however, had almoM no elfect <>n the
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Kg. 3. Wvci'jon ot soecr&e I HJDHA taMing to 009 tracheal muse*) membranes by setae* far* bsfa adrenoceptor antagonists. Lett panet-inhotMo by (PS 339 (MM-2 selective}. The data pomia ar» Bast wsed by computer-generated curve which, assumes two sites. The calculsted Association constants for the high ( IU- and low K X l l M y sues and the per-ceoiagect total sites toreec* are snown. Right panel: mrsbrtioo by pracioJot (oeta-1 selective} showng a two-site interaction Each point » thamaanotdupueate'datefnunaiionsinasingle expenmem and is type*) ot four to fcve sucn expenmants.
tB0<
so.
I t / "
\
• 10 -a -a -7 -a tea tms » •> oio
- • -a - r -a -» toe <an*CTOLOU u »
toer .«<£
Fig. 4. In vitro relaxation response ot dog tracheal smooth muscle strips to beta adrenoceptor agonists. Left panel: response to /-aoprcierenoi a>one (•> ana in the presence- of 3 « M practotoi (O) and O.t »M IPS 339 <•). Right panel: response to tertxitabr» alone ( • ) and in the presence ot practotoi K3» and IPS 339 (•>. The response is measured, as percentage ot maximum control response. Each pent is the mean * S.C. ol tnree to sat separate strips.
100
so
OL
LOB llSOPnOlERENOL) (Ml LOO (TEflBUtALINE) <M)
terbutaline dose-response (ED-.. 198 ± 24 «iM) wi th a !!•> of 12 / i M . suggesting interaction at the low-affinity site which corresponds to the beia-2 receptor. Taken together, these results suggest that the physiological response to exogenous 6cfo agonists is mediated predominantly by beta-2 receptors with only a minor contribution from beta-l receptors.
E lect r ica l f i e l d a t lma la t lon . Electrical field stimulation, after cholinergic and alpha adrenergic blockade, produced a relaxation o f serotonin-induced contraction which was inhibited by 1 «iM propranolol. This response waa significantly inhibited by both 3 pM practolo! ( P < .001} and by O.I ph i I P S 339 (P < .01) . but the inhibition was significantly greater ( P < .OODwithpractotoI if ig.5h This suggent* that the beta receptors mediating the response to endogenously released norepinephrine are predominantly o f the or/a-1 subtype.
DlaeusBjon
Specific binding of I 'HJDHA to canine tracheal smooth . muscle membranes had the characteristics expected o f interactions w i th bcla receptors, {finding wan o f high affinity and similar to that determined in peripheral lung membranes from the same animals and also in hing membranen of other specie* fRugg *t at.. 1978; Barnes tt at.. 1979: Barnes c/ of., 1980). Binding was stereoselective and the rank order of potency among agonists suggested that the beta receptors in tracheal smooth muscle were predominantly nf the beta-2 subtype. The oero-2 selective agonist terbutaline Uad a low potency which was simitar to that reported in king membranes (Minnerann rt
at. 1979h>. Competition Tor f TOJDHA binding by the beta-2 selective antagonist I P S 339 revealed two classes of binding site. Most, of the binding was to a high-affinity site which in presumably beta-2 receptor and the remainder t o a lower-affinity aite which in presumably the btta-i receptor. T h e binding affinities correspond well with those determined for the beta-1 a n d 6rto-2 receptors in cot and guinea-pig heart, with the same compound rHedberg el aL, 19801. Competition with the 6cfn-1 selective antagonist practotoi similarly gave a n inhibitory curve which was best characterized by two binding sites but With the converse pattern, as most of the binding waa to a tow-affinity site <tbe beta-2 receptorl. As the results with both antagonists are complementary, we estimate that the ratio of beta- \/beta-2 receptors in dog tracheitis smooth musrte is approximately 1:4.
Our physiological studies on dog trachealia muscle supported the evidence f rom the binding studies that bcta-2 receptors are predominant, but in addition showed ttuW 6e/a- l receptors contribute t o the relaxation response, particularly when induced by sympathetic nerve stimulation. IPS 339 had • potent inhibitory effect on isoproterenol- and terbutaline-induced relaxation, with a n apparent dissociation constant ( K M ) which is in good agreement with the K» determined in the binding studies for the beta-2 receptor aite. By contrast, practotoi had atmnst no effect on terbutaline-indured relaxation, with a K » which was similar to the Ki . of the low-affinity bcto-2 receptor site. Practolul was more potent against the isoproterenol re-sponnf with a K * which waa intermediate between its high- and
low-affinity binding. This suggests that, although the relaxa-' tion response to isoproterenol is mediated predominantly by
beta-i receptor*, a small component of the response may be due to activation of Arte-1 receptors.
Field stimulation using the electrical parameters described causes the release cf neurotransmitters from nerve terminals within the smooth muscle atrip (Russell. 1978). After blockade of cholinergic and alpha adrenergic receptors, electrical stimulation produced a relaxation response which was blocked by propranolol, suggesting that it was due to activation of beta receptors by neurally released norepinephrine. Practoioi, in the same concentration that had little effect on exogenous beta agonists, caused significantly more inhibition of the relaxation reaponseiofield stimulation than IPS 339. This is an indication that tVra-l receptors predominate in the bet a adrenergic response to sympathetic nerve stimulation, in contrast to the beta-'Z receptor predominance in responsa to exogenous beta agonists. Inhibition or presynaptic bela-2 adrenoceptors could theoretically reduce norepinephrine released by nerve stimu-lation. but under the conditions of the experiment, this effect is likely to be minimal (Langer. 1980).
Our findings support the suggestion that beta-l receptors, which Have a high sensitivity to norepinephrine, are related to sympathetic innervation, whereas 6cto-2 receptors are unrelated to innervation and respond to circulating epinephrine (Ariens, I9SI I. As the pattern of sympathetic innervation varies considerably among species, it might he predicted that the ratio of brta'l/beta'2 receptors show a similar variation. Some evidence in support ril this hypothesis in provided by the (lading that in both guinra pig and cat. which have a dense adrenergic nerve supply to tracheal smooth muscle (Richardson. 1979). tracheal smooth muscle shows pronounced tVta-l receptor-mediated effect* (Furchgott el erf. 1975; Omini <*f of.. 1979; lakovidisrr of.. !<*8ih .Johansson and Watdeck. 1981: l.ulich cl at.. 197B1. In contrast, the »V/e-I receptor-mediated ejects, in dog appear to be relatively minor, an observation which in
! Vol 2 »
consistent with the sparse distribution of >ympeeh«w nerve* in this sjiecies (Suzuki ft at.. 1976). In human irarhr.il .-mouth muscle, which is only sparsely innervated by cympuihwic nerveB. we would predict that tnta-I receptors rnuzht play only a minor role, but thin has nut yet been examined. The den*it\ of sympathetic innervation decreases in more prripnerdl mi-ways, suggesting that beta-l receptor-mediated effects may nl*<> be less in smaller airways. In cm peripheral lung strips, the relaxation response to beta agonists is predominantly mediated by bcta-Z receptors (presumably in smooth rauwle a*? small airways), whereas in tracheal smooth muscle. brto-\ receptor* are predominant (Lulichrr at.. 19761.
The relevance of tVfo-1 receptors in airway smooth muscle in human asthma is not certain. In asthmaticsubjerts, elective beta-] adrenoceptor antagonists, such as practoioi and atenolol, may cause bronchoconstnetion. although this is lex* likely than with nonselective antagonist* such as propranolol iThirinper andSvedmyr. 1976: firihhin «*/ at.. 19791. This brunt huc«n>t rii'-tor response to 6rta-l selective antagonists may indicate the presence of tVra-l receptors in human, airway*, but is more likely to reflect the lack oi "selectivity of these drugs in clinically used doses. The fccia-1 selective agonist prenalterol is reported to have no bronchodilating effect in asthmatic subject* at rest, which argues against an important role tor btia-l receptor* in human airways (Lofdahl and Svedmyr. 19821.
We have demonstrated by direct binding assay that beta receptors of dog airway smooth muscle are predominantly of the beta-2 subtype, and mediate relaxation in response to exogenous agonist sand presumably circulating cat echolnniine*. In addition, there is a small population lapproximaielv 2uN» of beta-l receptors which appear to mediate the relaxation response to sympathetic nerve stimulation. It ii> proposed that the proportion of orlo-1 receptors may lie directly relmed to the density of sympathetic innervation oi airway smooth muscle. It is possible that tVra-l and bcta-'i receptors may be differentially regulated and there is some evidence to suggest that ftefn-1 receptors in guinea-pig tracheal may be more resist-anttodesensitizatkmthanbpro-S receptors (Omini era/.. 1979). Acknowtoitsmenu
W> Ikaiik Beth C M and Pailv Snfll h't Ifcrir * U » I M C * in ptfpanne ihw mamnenpt.
Rafarrarrt AtLMi. B.. CASUSON. E. AMI Ks. L.: f*ttaNnar»l<wiral Mudio «l t«» nrw
rstduvrirrlivr adtenenir tw«-m*|rto» amajBiiu^n. Ltlr Sri 12: t»* 114. ISTS.
BaMtt*. P. J„ BASSAI-M. r . B-. KAIIEL. J . A. A M I RiWKHr*. J M_ l..».»!»»«tinn • I tola adrr ntxtptor* in mammalian tuna •>> iMthi M nwific atHoMcliuara-»hy. Natar* ItuniLl S9»: 444- 44:. I9H2.
BMNES.C . J.. KAMUMRM.J.X. AMilHMXiJtV.r. T.: Human lunxai!mii*-r|H«r» nuaVrd hv radiuliaand ramltaic tint. Sri SH: *X- 4*1. l:«"i
BothES.P.KULIDI.H..I. HAMILTON.C A. AM>Dalll.MV.«' llmiim^i.mm er alphacMrrmiiTptiira in auawa piie tunic tinny lUbtmicm. Lttr >ii 35> 120T.IJ14.1»m
Bnaiaca.J.R.AmrMKH.r..i;umrci.i.t, I F AM»VIAHI> . I 1 .M* I« -> .M ihr naurrtlbfuM-hulhtia-adrtawYBiiii'. Kur .1 rhnrm»n>l IS; IB) li«< l*>"l
fiauirtiMi. M M : A rapid sail wimivt nirl hm? l«r iMaamiini wi-J at* tnttxm quii)l>lt«.i»l pnH«n Aflat.ttnrhrn 7 t : J l * J ^ . IHTH
CASLSkOH. K.AHLAK, B , BHtMH>TMOH. A. AMlt*«Mt»»tlV B ' !M!rr, tlL.tlrH MurkaaV <>1 Ike cSmni>1fii*K HlrrK ••! %mti«u» dibrnrraK Mimoli in ilw <<i httn LH* Sri. 11:9VI.9M1 IS7.'
EM;u_ti Satirla><r«ti|hai»adrrniirr|>tor%—AiiiMntiiaiitrr^inuuiinioihri.i, and hrWi-adiriiiirratwr* m mnara | M * and hwixjo Inn* I'mijiuil \M I A?: aapul 1.7T-H.1.1!*)
Pt-arm.oir. K. r*. WAHAWK-T D.Siniarr. R A i M i M n l i k I "» l^I«f rrsi*«•!tio'hIrlai anrll*»aJ-m*r'«>r*"• tttiirxmI«KirjiiK.il-!«»-ilinraiIt aarf ihe lanaiHtn al lit* but, Ivia^raDo in iMlrtrM aiiinut- i.\'»ir.i>t> t«.' fnt 3 d 7U. I<J»
Cauauc H. R , BAI D M S . C. 4. AKnT*rrtM*m*. A. S* QuaMrtMivr am*** • K M of htaarhal hMa-odnrimtpiarblarkadtin man. Br J. CEn. Pfcarmanl. 7:A!M-VW.IKIL
H n W K . A , M I M C O U N . K: f». A M Moturor. P. B : IMtr,-rntn) rfntnbuMa of httn-l aael hna-2 arfiantrjor imftat* la cat and guinrtpi* fcrari. J. Phamaol. Eaa. Thtr. S IS: Mt-iHA. lantt
I A K C U M * . D , MALTA. E . StcPmauDV C . A. AND RANCH. d la viuo artinty of R O t t l a to Ui-admoctptor iwltrln* agon*!. Br. J . PhtmatoL M i Sit'
«h» point* W uarhn: EastrinwnU wrtb prrmkaraJ. a ariaratlrctivt adrany actpiaraooaia.4.PhanB.P»aria»roL3S:3U-a*Ubi. '
LANIM. A. M . Awrout. A . McAtUrr.4. P.. L C M C K A . P. P. AND BHOWX. T. 6 , J*.- DiMrnmnatiea of ttctptor (vttnaa anivattrf by *ymp«tkaMitnttk aainta. Narutt OaadlRldj M7-M*. INT.
LAKCKK. S. Zi Ptttymftte mndanon of ih* ttkaao « f cattehoIaBHM*. Ffcat-atacol. Rtv. a t : 337-ltt. JMO.
Lor&MtL. C..C AM> S t l u m i . Ji_- EfTnrt» of nrtaakriol in atihrnauc patlanu. Eur. 4. CKa. Phaimrol, M : 397-303.1M2.
LVUCH.K. M_ MncNStU H. W. AHD!>nuUB>M. M. P J Threat hint atrip a* m in viitppupaiation 1 prnphtrit airway*: A <wnpaii»on of otta-adftnoctp-teraiamMi^aMocaidiaadaaaVhyianircaaUtiiitreaitlwIuaiiimpanduacfata. Br. J. Pharmaml. M l 7t-T». 1S7&
MINNKMANV K. P.. HntmuNn. L, JL AM> MOUNorr. P. B.: Simuliantoiu dn tnntnaiioa of b»ia.| and btta-3 a*tn»nrir m*pion> in timet coniumnjt both ttcrptorKihtypt*. Moi. Pharmacol. ! • : M H C I9tfta.
fllraiaii Bala Bar a M ^ i J I IMIMO 411 M'MMHAht. K. P.. HKOSTIUND. L. R. Akp M O U M W . P. B-: Tht aktwrtrmto*-
tea* saanflcky of «M and o" * admwrf* trrrpuM* ia am (wan aai tana » viuo Mol PkaraarftL Ifc 31-tl. iron.
MI-MA*. C NAOXL. J. A. AN* Roicm, J M^ TJ» . fencarinnr mtotHi a. «Jrmiy amooUi tauatlt: Thtir chrranamitien ia »«n{ 4. Appl. PayHofc U I I8M-IW). UU.
OMm. C SoNuniux, A. R. Fotro, C. C ROMDM. n AMI Fvuttf. U-tflMracusa tetwrtn b»i»i- and otUradmMrtptari in iha iatttattd I trachea. Pharmacol. Rr*. Caataiaa. I I t ill-tU, l ITt .
RKMAMMOTt. J . B-: SUM of I W an. Nrnt a«a«(y is Iha hull*. Aim. Rtv. Raipir. C i * . l I » : 7 A 3 - » M i m
R u n . C. L.. B M W I T . D. B. ami NAKOMW. S. f»_- Csniuanc* of Wu, «ai •HataaVanoctotaraiaaNK«alianIut^fNidmc*fr»MdiraetlM*diataiiidi»t. MaLPnirnacal.14tPM-IO0S.ltM.
RttAnx, J. A.: Raaponn of iaolaitd ctniaa mm** w alteirinl attawiladoa •ndar«yJtho»»i»t.J.A»pLPh»-»»ol.48i«a»-»*»,iB7i.
Sctirnt. R . MomrA. K. ANA KvmMatl. H^ Inamauan aad ptoiNltlt* of Iha MKXh nuirla of <ba dot tMrhta. Jtfn. J. Phjmol. t « i 393-310. ISM.
TtttilNGCA. G. ANB SvKotavn. N. : tajtractie* of orallv adaHnftitrad mrtoareM. ' mctslol and pwpianalol with ttepnnallttt M aMhmaiif*. Bur. J . Clla. Phar>
«*<*). l « i 1S3-IT0. IKS.
• " ' i^«—i- •———-— Sand rtprtnt raautata tat Dr. Prttr J. tatnii. Orpanattnl of Mtdkino. HanNnrnunith Hatptttt. Dwrant Road. LaadoR W.|3. U.K.
FAMILIAL FACTORS IN BLADDER CARCINOMA 451 11' Fraunwni. J F , Jr. and Thomas. L. B.: Malignant bladder turscrs
. in a man and his three sons. J A M A.. 201: SOT. 1967. 12. McCulIoufth. D. I», Lamm. D L, McLaughlin. A P.. III and Cities,
R. F-! Familial transit tonal cdl carcinoma of the bladder. J. Urol. 113: 629. 1975.
13. Petfcova-Bocharova. T.. Chemozemsky. !. N_ Nikolov. L G. and Stoyanov. L & Families with multiple caae* of urinary system tumors: brief communtcatioa. J. Natl. Cancer Inst, 59: 1419, 1877.
14. Chemozemsky. L N-. Stoyanov, I. S - Pctkova-Bocharoia, T. 1C Nirolov. I. C , Draganov. I. V.. Stoichev, 1. L. Tancbev. Y„ Naidenov. D. and Kalcheva, N. D.: Geographic correlation between the occurrence of endemic nephropathy and urinary tract tumours in Vratza district. Bulgaria. Intern. J. Cancer, 19: 1, 1977.
15. Morganti. C Gianferrari, L., Crrsseri. A., Arngoni, G. a.id Lovati, G~ Recherches clinico-statistiques et gehetiques sur lea neoplas i a dels vesste. Acta Genets fi: 306. I954J.
16. Harvald, B. and Haute. M.: Heredity o f cancer elucidated by a
study of ureelected twins. J.A..M A.. 1S8: 749.1063. 17. Chan. H. and Pratt. C. B J A new famtlial cancer syndrome? A
spectrum of malignant and benign tumors including retinoblastoma, carcinoma of the bladder, and other genitourinary turgors thyroid adenoma, and a probable case of multifocal est eosarcorra. J. Natl. Cancer Inst, 5«: 205,1977.
i a Ahcrne. G- Retinoblastoma associated nilh other primary malir,. nanl tumours. Trans. Ophthalmol. Soc. UK. 94: 938,1974.
19. Smith, 4 L . & Histotory and spontaneous regression of retinoblastoma. Trans. Ophthalmol. Soc UK. 94: 953,1974.
20. Lynch. H. T.. Mutcahy. G. M , Karris, R. B.. Guirns, H. A. and Lynch, J. F- Genetic and pathologic findings, in a kindred with hereditary sarcoma, breast cancer, brain tumors. leukemia, tunc, laryngeal, and adrenal cortical carcinoma. Cancer, 41:2055.1978.
21. Zincke, H.. Aguilo. J. J„ Farrow. G. M„ Utz. D. C. and Kahn. A. U-Signiftcance of urinary cytology in the early detection of transi-tional cell cancer of the upper urinary tract. J. Urol, 118: 781, 1977.
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t` -Ctr Acknowledged
D. B. Cohert +¢°4s- C7ti~~ Autoradiographic Localization o~ Autonomic Receptors. In Airway Smooth Muscle
Marked Differences Betweorl Large and Small AirwaysI-3
PETER J. BARNES; CAROL B. BASBAUM, and JAY A. NADEL
IntroductionAirway smooth muscle tone is regu-lated in pan by rhe autonomic nervoussystem acting on specific receptors :muscarinic and alpha-adrenergic recep-tors, which are excitatory, and beta-adrenergie and possibly pepaidergicreceptors, which are inhibitory . In air-way disease, particularly asthma, atuo-nomic control appears to be abnormal,and an imbalance between excitatoryand inhibitory effects may contributeto bronchial byperreactivity . /Suto-nomic receptors in airway smooth mus-cle have previously been studied invitro by measuring tension in isolaledstrips of muscle exposed to autonomicdrugs, and in vivo by making directand indirect measurements of airwaycaliber after administration of auto-nomic drugs systemically or by inhala-tion (1). However,it is not possible tostudy smooth muscle of small airwaysby these methods. The smallest airwaysthat have been isolated are small bron-chi (2) . Il has been suggested thal thebronchioles are the sites of earlychanges in airway disease (3) . and theyare a major site or obstruction in ex-perimensal asthma (4), yet these air-wcyshavebeendifficult tosludybyex-isting methods. One approach has beenthe measurement of tension in Isolatedlung parenchymal strips (S-8) . Becausethese strips contain multiple types ofcontractile elements (including bron-chioles, alveolar ducts, small bronchi,blood vessels, and interstitial cells), theinterpretation or tension measurementsIn these preparations is difficult .Another approach to the study of
autonomic receptors is the use of directreceptor binding assays. Autonomic re-ceptors have been Identified in lung ho-mogenates or several species by directreceptor binding, using radiolabeledadrenergie and cholinergic antagonists
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aaMMARY Auroracltqnpruemrprod, war.uaMtodaumnnrlrMdlatribulionolaulon0mlonle pptnA in airway fmooth muscle ol tanN rrnrn traehea 10 IamMMI brplcMain : PWldlhytlrnrF4
penaol . f•uguaraala and PN/quinudidlnyl iumltata w .n used to nbal Ortwmannak . alpna•adraMple, and murerNnlc ncaptnrs, ra.paenraty- using aeperimental cprdllluns that aarama.rmal specific eaerptor hindlnp wsraad dlner.ncaa wnrr rnund In Iha longitudinal disldGdionel .aCh qe.plor and en dirrelbutlun of the rarlous neaplan In .eeh pan0er alnray. aala•ROaPtots rran present in Mah density th .ouanaullM alrwaya . r.ah Iha hlpMat Wrn11y In MnneNnVar.Alplu•r.e.ptnrs wan eprrau In large sinraya, but numereur In small bronehlo4s, whareat ehoeinerale eAn.ptaf w.n nWrNroua b1 WOneMN .mooB, muscee, apana /n prorlnla) Wa1eMnN1,and almost Nu.nt rrom dlual Maremotea . TNr malhod mpy be useful In atudyma anaranons a/auloncmlc racainors dirldbutlon in aman and 4rar anwarr atter oayMmantal manlpulatlnn and
indi.eaaa. su Rnr naann on /aax ranrM.ra
(radioligands) (9-12). Because lungs nized glus microscope sections . Thesv slidecare made up of over 40 different cell were eilher used directly in the binding as-lypes, assays alone are inadequate to say or stored al -70• C .determine receptor distribution in spe- Renpro B/ndingcific intrapulmonary structures, such Incubation and washing conditiom hasras airway smooth muscle. We have re- pfeviousiy been shown to produce optimalcensly shown that autonomic receptors speciflc receptor binding ror each radio-can be labeled specifically in frozen )igmd (13-1!); I'Hldihydroalprenololsections 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, 20have now studied the distribution of Cf/mmol ; New England Nuclear) was usedalpha-adrenergic, beta-adrenergic, and to label alpha-recepton, and Irrt)qulnucli-muscarinic receptors in smooth muscle dinylbenrilale(IIH)QNR)(speciRcanivily,of airways from trachea to terminal 33 Ci/mmop wa~ used to labe)muscarinic
bronchides. receptors. With ) H)DHA d 11HIPZ, see-
tions wele incubated at 256 C for 20 min,
Methoda (Mnnvd 6r oNNffN/Wen Onoan rJ, rpl2 orrd
Tissur PnparettoA in rerurd/onn feb.wry a. 19a)
IFerrets (Musrelo parorius) were anesthetized • From the Cardlovascular Research InstituteAvilh pentobarbilal sodium (35 mg/kg by In- and the Depanmmis of Analomy and Medicine .craperiloneal inJeclionh and the trachea Univershy of Californla- San Francisco, Sanand thoracic contents werequickly removed . Francisco. Califomia.Endividual lobes of Ihe lung werr separated, e Supponed /n pan by Propram Project Granteannulated- and inOMed wlth tissueambed- No. HL.24136 from Ihe National Inunule, ofding fluid (OCT: Lab-Tek Produqs, Naper- Heabh and by Contran No . 1111 from rhevllle, IL) diluted 1 :4 with phosphale-buf- Council for Tobaao ResearcRUSA . Ine .fered saline . The infiated lobe and tracheal ' Requeus tor reprinn should be addrcaed to
rinas 5 mm in length w~ere rapidly froten in ~' R J. Barnn, Depanmenl of Medicine .
Hammerrmith 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 bomstat at 1S° C and thaw-moumed onto aelati- the Medical Research Counca of Great Bnlnn .
d and with l'H/QNB, for 60 min . Concenna-lionsof radloligand were used that were ap•
- prorimately equal to the dissociation con-stanl (Ka) for binding to ferret lung sections .as previously delermined (13-15). Nonspe-cific binding was,dnumined by incubationof adjacenl secrions from the same animalin ihe same eoncentration of radioligandbut with an exceas of unlabeled anlagonin,so that «ccptrtr binding was competitivelyinhibited. For /tiHIDHA we used I PM(-)propranolol, for pH)PZ. IOvM Dhen-tolamine, 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 wererapidly dried in a stream of cold air to pre-venl diffusion of tadioligand, and stored Ina dessicalor overrbight .
ArrrorodrogmphyAutoradiogmphy was performed using themethod of Young and Kuhar (16) to retainreversibly bound (i .e ., diffusible) mdioli-gands at reeeploo• sites. Glass coverslips .which had previously been eoaled in pisoto•graphic emulsion, were pieced over the sec-tions and 6xed to one end of the slide withcyanoacrylme adhesive. The emulsion washeld in contact whh the section by binderclips, then stored in light-proof boxes at4' C. Optimal exposure times were foundio be 3 months for pHJDHA, 4 months forlrHIPZ, and S months for pHJQNB. Afterexposure, the cov'erslip was partially sepa•raled from the slide so that the emulsioncould be developed and the section stainedwith 20a cresyl vuolet . The sections weremounted and viewed under brightfield anddartfield illuminsition. Autoradiographicgrain counts were performed using a cali•brated eyepiece and a x 100 objective lens .Grains were counted over areas of smoothmuscle in several airways from each section,and the airway dimensions were recorded .There is some eonfusion over the definitionor "small airways," particularly when dif-ferenl species are compared . In our ssudy,imrapulmonary airways were categorized ascither O) cartilaginous airways (bronchi)measuring I to 2 min in diameter and corra•+ponding to subsegmental bronchi ofsourth or fs(Ih gene,ration, or (?) noncanila•ginous airways (bkanehiotes). Bronchiolesaere arbitrarily defined as proximal (> 0 .3mm) or dislal (< 0.3 mm). Distal bronehi•ales Included terminal bronchioles and respiratory bronchioles.
Dald Ana)ys&Resuhs are expressed as means m SE . Spe-afrc grain counts were determined byc,+unming grains/unit area. then subtractingbackground counts and nonspecific countsflom the same area in an adjacent section.R:ceptor density was then determined bya+rreclion for specific activity of the radio-hgand and the time of exposure using thefprmula :
It = (gd/1) x (A/5.C) x 2
7>9
TABLE I
DISTRIBUTION OF 6ETA-ADRENEROIC. ALPHA#DRENERGIC . AND CHOLINERGIC RECEPTORSIN AIRWAY SMOOTH MUSCLE OF FERRET
Tbacnea 6 1D7 s4 -1 T9 :0.0 - 263 x BaBronohN 1-2 BO-0 ar 6.1 9.1 29-9 7852 96Pioxlmat aronchiolas O.y-09 122 a 20 99 s 7.t 139 a 77D/slar erpnemotes e 0.3 160 z 15 163 : 6.9 102 o tl
- RKrptp dM41Y eNlrieiryn hym .n .el,nn eefCxMn ,n MR"npf Rtlyln e,e nyyn r eF 110m e, Nen ] 4•re,efrMn Nch yl 3 an,marr
where R is the number of binding sites/unharea of tissue, g is the number of specificgrains in the same area, d is the number ofdecays required to give one developed grain(a 7lms section - 34.01), t is the exposuretime (days) . A is Avogadro's number (6.023X 1020 moleculeslmmo)), S is the specificactivity of the radioligand (Ci/mmoq, andC is a disintegration constant (3 .2 x l0'de,eays/day/Ci) (17) . Because the concen-tration of each radioligand uxed was ap-proximasely equal to its dissociation eon-stanl, approximately 30% of total bindingsites should have been occupied, Therefore,the total receptor concentration was ealcu•iated by multiplying the producl by 2.
RatullsThere was a striking difference in thepattern of labeling of airway smoothmuscle with the different radioligands
(table 1). With [rH)DHA, smoothmuscle was heavily labeled in all air-ways (figure 1), but the density of la-beling was inversely related to the sizeof airway. With 1sH)PZ, there was lit-tle specific labeling of smooth musclein large airways, but there was a highdensity of labeling in bronchioles, par-licularly distally (figure 2). The(sH)QNB showed the converse patternwith the greatest densny of labeling insmooth muscle of large airways, parlic-ular)y in the intrapulmonary bronchi .There was much less labeling of proxi-mal bronchioles and almost no labelingof distal bronchioles. The distribuyonof each autonomic receptor in airwaysmooth muscle, therefore, depends onthe generation of the airway, and eachcaliber airway shows a distinctive re-
~~1aYe'''a •F/g. 1 . 6oCa11raUpi o1lMtaidranoraesplON in snwar amootll muscle 01 (aMet (al Darallaia llluminertioqatqna the distribution or auloraalo9raphie e1rMt qralna 66 br/pht datla in an inlralnrtmpnary bran-ehu9: PHIDNA deneely labela sm qth muaete ISM). (b) . B/lahlliate view 61 the aba shown InteL (e) OarM-natQ waw ot 7ha sune araa In an adlarant section incubated wnh PM)DNA In the preunea ar an areesaor pFopranol4l, showinp rq speeilie IaIN1rnB . (ol Dsr4hald view Ol a ametl nranehlo4 ahowinp danNIabeling of sn1oolh musete . Note the rarlehn0 ot aunoundinB aN/plir wana . (ar Br/qhthete Yiew or Inaatea ahown ln (d1-(1) Deraaatd view ot the aame anta trCm an adjacent aaction Incu Wletl wnn l'MJOMAand Dropranolol. Sule bat . 100 rrn.
Fig. a Lorallranon ot aipna•atlunocePlon ..• arnsy rmootn muecN 01 ferral Is) Duldbrd vrww o1 an/myprununuy broncttus. arowina /abs4na by plriprarosrn of eonlrobum (EP)oul arewsf no labeuna ofampoth muscro taM). Baeeprouns rs conautlerably hlaner than,n Npura I ID) . ananerom wew of the er.aShown In (s) (e) . Darkllettl view of tM same araa nom an adlaceni seenon incubaleP with PMloraroNnantl 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 .
ceptor pattern. Thus, for trachea andtnt2apulmonary bronchi, the rankirder of receptor der.sity was muscar•ini¢ > beta-adrenergic >> alpha-adren-ergir, for proximal bronchioles beta-adrenergic > alpha-adrenergic > mus•carinic, whereas for distal bronchiolesbeta-adrenergic = alpha-adrenergic>y muscarinic.
olneuuknAutonomic regulation of airwaysmooth muscle has been studied mainlyin trachea and major bronchi, becauseh is possible to isolate and test the ef-fects of drugs on these airways . Theperipheral lung strip preparation wasintroduced to study the effects of phar-macologic agents on smooth muscle orairways that were too small to be iso•lated (5) . In that preparation, however,other contractile elements are presentto a variable extent, making interpreta-tion of contractile responses difficult.For example, eontractile responses ofperipheral lung strips in response tonotepinephrine and other alpha-agon•ists have been taken to indicate thepresence of alpha-receptors in "periph-eral airway" smooth muscle (18-20),but contraction of vascular smoothmuscle and contractile interstitial cells(21) could explain these responses (22)equally well .We have used a different approach
to studyitdg autonomic rei-eptors in air-way smooth muscle using autoradio-graphic localization of specific radioli-gands . Wc have previously establishedthat specific binding of each radioli-gand had the characteristics expectedof interaction with its receptor in lungsections, and were able to determinethe experimental conditions ner :essaryto produce maximal specific binding. Itmay be difficult to precisely determinereceptor densities from autoradio-graphic grain counts, but it is possibleto obtain estimates of relative reoeptordensity (17). Such estimates involveseveral as'sumptions, but berarrse theseapply equally to each radioligand, it Isvalid to compare both the relative dis-tribution of different retxptors in thesame structure as well as the relativedensity of each reecptor in differantstructures .
Beta-adrenoceptors were numerousin smooth muscle of all airways, butthe density increased al airways be-came smaller, with the smallest bron-chioles having a very high density ofbeta-receptors. This is in agreementwith the finding that beta-agonists pro-duce relaxation of lung parenchymalstrips as well as isolated tracheal andbronchial smooth muscle (3-B) . Alpha-adrenergic contractile responses havebeen described in smooth muscle stripsin many species (23-23), but they may
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only become evident after beta-adren-ergic blockade or after precontractionof the muscle strio . In large airways•alltha•receptors were present in smoothmuscle but were far outnumbeved bybeta-reccptors, whieh may explain w•hybeia-blockade is necessary to see thealpha-adrenergic contractik response .The density of alplha•receptors waship.her in bronchi than in trachea,wFiich is consistent with the findingth>tt alpha-adrenergic responses aregreater in the bronchi than in the tra-chca or dogs (26) . There was a surpris•ingly high density of qlpha-receptors insmall airways, particularly in the distalbronchioles where the, density of alpha-receptors was equal to that of beta-re-ceptors. This suggests that at least partof the contractile response to lung pa-remchymal strips may be due to con-traction of small airways .
The significance ol' the high densityof alpha-receptors in small airways isuna;ertain . Alpha-adrenergic responsesin airway smooth muscle may only be-come apparent after beta-adrenergicbltlckade, after prgcontracting themuscle with histamine, serotonin orpotassium chloride, or in the presenceof airway disease (25,; 27). We have re-eeatly shown th3t the activation ofalpha-adrenergic responses is not dueto a change in smooth muscle alpha-re•ceptors, but is due to some postrecep•tor mechanism (28) . In the presence ofmediators or airway disease, the alpha-receptors that are alr,eady present be-come capable of initiating a contractileresponse. The high density of alpha-re-eeptors in small airways may be of im-portance in diseases, such as asthma, inwhich mediators such as histemine maybe released in the vicinity of small air-ways and "turn on" alpha-adrenergicresponses. The physiologic role ofthese peripheral alpha-receptors ls ob-scure . It is possible that they are in-volved in matching of ventilation toperfusion In peripheral lung units .Adrenergic axons releasing norepi-nephrine may produce both constric-tion of pulmonary arterioles and (bydiffusion to alpha-receptors in nearbybronchioles) bronehoconstriction, sothat ventilation is reduced in thc sameareas that have a redqsced blood flow .Such a mechanism would be an advan-tage in the case of pulqtonary embolism•
Muscarinic cholinerJ{ic receptors weremost numerous in large airways ; ofthese. the smooth muscle of intrapul-monary bronchi had a higher density
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Fig. 3. LOCatiranbn of muiear/nie rKeptora in aineay smooth muscle ol tana,t . ia) DVkiield view pl anmrmpidmanary siOnenus showing dense labeling by rNioNa of arnootn muaclo {arA) Di- erialltaeidvl.o of ineatN rhown in (a).OCI. DarMiald vifw o11M aanNarra fromanad/acpnt .action incutNUdwitnrlqnNa and an auce9{ of atropille. (d). Damturld vlew o/ a pro}linai broncniole Showing aparsN labelingof 691001111 musnr Bldl . (e) ariamludd view ot 1M area shown in (dl dt Darknald vUnr ot iM same areaIrom an tidReenl paction inoubalUd wtth pH1DNa and an a.oe.as of atroplna . (a). Darkfleid view of d4,ai6ronciuete 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 recep-Smooth muscle of proximal bronchi- tors should be closely related to cholin-oles had a much lower density, and the ergic axoas . This is in marked contrastdistal bronchioles had almost no cho- to the distribution of adrenergic recep-linergic receptors. This distribution is tors . In most species, direct sympathe-consistent with physiolegic studies in tic Bsnervation of airway smooth mus-animals showing that the greatest bron- cle is rather sparse in large airways andchoconstriction after va$al stimulation absent from bronchioles (33) . Adrener-or cholinergic agonists Is in large rather gic axons in ferret lung show a similarthan in small airways, both in vivo (29, distribution (determined by fluores-3D) and (n vitro (31). Similarty, in asth- cence histochemistry) (13) . Adrenergicmatie subjects, antichtilinergie drugs receptors in small airway smooth mus-appear to cause dilation mainly in cle would, therefore, be regulated bybronchi, whereas beta-qgonisas dilate circulating calecholamines .all airways (32). The pattern of distri- Although there may be marked spe-bution of cholinergic reorpton Is prob- des differences in the response of air-ably related to parasympathetic inner- way smooth muscle to autacolds andvation, which Is dense in bronchial prostaglandins, differences in responsessmooth muscle but sparser in bronchi- to autonomic drugs are much less ap-olar smooth muscle and vinually ab- parrnt (34) . Our findings in ferret air-sent from terminal bronchioles (33) . ways can, therefore, probably be cx-This Is not surprising because acetyl- trapolated to other species . Includingcholine, when released from choliner- humans . Littte Is known about thegic a\ons, is rapidly Inactivated by cho- function of small airways in humanlineslerase and does not function as a disease. Several physiologic tests ofcirculating hormone. small airway function have been advo-
re+
eated, but ahey may be difficult ro in-terpret in the face of coc%ictenl ab-normalities in large airway function .Studies on peripheral lung strips snffer.*•om the problem of helerogencity incontractile efements, as di.cussedabove. The autoradiographic method .however, has the advantage thatsmooth mttscle of Ihe smallest airwac+may be studied. This method may beuseful in delermining the c%istencc ofabnormalities in airway autonomic re-ceptors in disease. /n a guinea pipmodel of aslhma, a decrease in pulmo-nary beta-receptors was accompaniedby an increase in alpha-receptors (35) .and in chronic airway obstruction therewas an apparent increase in alpha-re-ceptors (11).
Because these studies involved radio-ligand binding to homogenates ofwhole lung, it was uncertain whetherthe changes occurred in airway smoothmuscle, or In other lung components.The autoradiographic method we havedescribed should provide answers tothese important questions. Althoughwe have studied adrenergic and cholin-ergic receptors in the airways, the tech-nique is equally applicable to other rt-ceptors (e .g., for histamine, serotonin,prostaglandins. and regulatory pep-tides) . This technique offers a new stra-tegy for the study of regulation ofsmall airways and other structureswithin the lung. The demonstration ofspecific binding sites does not necessar-ily indicate that tbey are functionallysignifieant, and in the future it will beimportant to correlate the presence orchanges in binding sites in a tissue.with some measurement of receptorfunction in that tissue.
AeknowkdamrNrThe writers wish lo Ihank Leona Lauriceltafor expen photographic assistance, andBeth Cost and Palty Snell for their assit-tanee in preparina the manuuript .
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Phmmacol 1971 ; 23:9oS-10.2S. Kneussl MP. Richardson JR . Alpha.adren.eryie recepton in human and canine tracheal andbronchial mmmh muscle. J Appl Phyxiol 1978 :45 :307-/1 .
26. Left A. Palhophysioloey of anhmauirbrrtrnchocarstdclion . Chen 1982: 82:133-215 .27. Ohno Y, Waranabe M. Knuri Y. Ma~:.rra•tation of Inent alpha.e∎ciulory responst inlhecanine tracheal smooth muscle preparation. Re•lat,ion to basal tone. Arch Im Pharmacodfn Thn1981 : 231 :20AI6.28. Barnes PJ . Skoogh BB•E. Brown JK. Nrdel1R. . Anisalion of alpha-adrmergk responses ineriMeal smooth musde: a posr-reeepmr meeha•nism. J Appl Physiol, in press.29. Coleb.reh HJH. Olsen CR. Nadel JA. Ef•fect of histamine. serotonin and acnyieholine onpeHpheral airways. J Appl Physbl 1966: 21 :217
. "; Nadel JA. Cabeaas GA. Austin JHM . Invisp roentgenographic eaamination of parasfm•paahnk innerralion of slllkll airways : Yse ofpo;rdered unwlum and a fine foal spot .-raytube. In.esl RadWl 1971 : 6 :9-i7.31 ; Drarm JM, Schneider MW. -Comparadveresponses of tracheal spirals and parenchymalsrrips to histamine and carbachol in vitro . J OinIn~va 1978 ; 61 :1M1-8 .32. McFadden ER Jr. Ingram RH Jr.•'HayaesAL. N'ellman JJ . Predominant sile U Row gmi•rmton and mechanisms of poneaneional asthma .J Appl Physiol 1977; 42 :746-52 .33., Richardson 1B. Nerve supply tonhe lurqr .Am Rev Respir Dis 1979; 119:785-802. ,34. Fielsch JH . Pharmaeologinl aspects of air-svaq smooth musde. In : Nadel JA . ed . Physiol•ogl• and phmmacotogY of t:ae airways . NerYmk: Marcel Dekker, 1980:191-216
gTREPTOfACCUS PNEUMONIAP CELL LYSATE IIOfIBITS HUHANNEUTROPHIL ELt5TA5E. T.g. Collins and R .A. SantlhausDepartment of Medicine, aclona ev an Nospita and tbeUnleerslty of Colorado School of Medicine . genver . CO.
StreptocoCpW pOeumnNae l5trep Pn) inducea a markedsutrophil (PMN) influx into the lung . Coplte thie avail-
. •illty of large amounts of pttagocyte protelnenee, the` reumonltla aeldgm reaulta in paronchymal necrosia or
residual eephyaematoUa changes . To test the hypothesis thatthis sparing or the lung parenchyma 13 due to inhlbitlon or
a clinical Ssolate ofInssn P!!1 elutaase cell lyeate frwmStrsp Po type 11 was tested [or ira ability to Inhibite3aeCOlylla by porcine pancrtetic slmetaae (PE) and by hlmanP18t granular extract (CE) . Stspnylococcm aurcua ATCC 259z3(Staph) vaa used u the anntrol . In>aluble- crltleted .bov)ne ligamentm nuceoe elaatln eerved u LDe substrate .Purified PE, and Ge prepared frem p/4( of normal auman volun-teepa aeewed ae the elaateae sources . Hlaetaea activity v.acaleuleted tr® label x)uD(lfzed during 5 hours of Sneub .-
. tion. Toe bacteria vere grown at 37oC in trypticase soybroth vontalning /f dextrose . Staph was harvested at ahours and Strap Pn at the end of Sta erponentlal grovthphaae. The ce11a vera uashed, spun, and ground withalw(nus oxide in the cold . The resultant lysatea were apunat 20,000 a a for 20 m3nutes and dialysed vs . O.1M TrSs HCl,pH 7.6- We round that neither lysate had endogenouselaetase sotivlty. Nelther lysete Snhiblted P6 Dut therevsre algnlflesnt differencen In their SnhibittOn of Pa9Velastaee . Strep Pn reached greater than S0i inhibition(731) at 6 to 10 mg proteta/ml, the lowest concentrationtegted, vhSle Staph reached 50% inhlbitfon at 65 to 75 mgfrotainlal . In one experlment . the lysate frOm a nCCro-tiaing type 3 Strep Pn failed to inhib .t P10( elastase . Lnsuamary . lynete ot Strep Pn type 1t appears to contain oneor eore non-dialyzable oonst3tuents which inhlDit humsn PIQ(elastase but not P8. Staph cell ]yspte inhietta PfD+ alas-tase poorly at <omparable protein concentrations and alsofaila to inhibit PC. This lnhlbitory phenomenon may helpexplain vby Strep Pa pnemonla ael.aom result . In parmchymalnecrosis or reaidual itmg tlamage . (Supported by NIH HL-o1U01 and yR-D7og5)
'kcNOLINtRCIC RESPONSES IN HUNAN LUNG - ASTkUCTUHt/FOdCTION CURNELaT1UN .J .L . glack . N .3 .6. Ptnney snd N . Herend,Departreot of Ph.rwcology, and the Deparrmenc ofNedaclna, Coocord gospltal, un3veteicy of Sytsey,N.S .V .e anatTalla .
ln contrast to the reproducible reapooses ofbusao lung paranchymi strlps (HLPS) el/cited vithaaaeiata aucN as noradrenallne (Ilack et al . Eur JPLarucoy 72- 83, 1951) reepooeer to cerbacnol tn[hfa preparation were found to be highly varfable .The marked vr.latiee 10 masSaal coqtvactileresponse to arbachol between dtffereot 11123 couldnot be etplsined by dlfferences in the veight ofthe preparations (c - 0 .06 P> 0 .05 0- 53) .To datmrmine if contracttou to carbachol vas afunction of a varying proportion of a tlssuecosponeat, a ateueture/fuoctlon correlation studytqs performed. Pittaeo dI.PS uere aelected forsorphosetrie snalysi> so tamt a wide range ofccotractile responses to urDschnl Sa v[tro vaatapresented . Comp>oeots of the gf3S vereu[agori.ed p etthar 1) alrray 2) blood veaeela3) parenchyaa 4) noo-parenchysa . Proportione byvolume of these tisnue eoeponentm oere determinedby rsne of a pofnt-cuut systen (Duani)1, Thorax17, 720, 1962) . Pifteen histological sectloneuere taken tros esch atrip and 5,000 - 10,000poiets aere tounted to sach strip . There eaa aetgniftunt correlation (r - 0 .77 P < 0.01)between the percentage volgae of slrwy Sm a stripand the masbum Coatractllt respcnae of that atrlpto eaebachol . 'Ihera rraa vo slgoiElGUtcoyrelmeion betveen the percentage volume of anyother structure Category sea the mawlaumvontxactioo . It is concluded that sirvay mmootb
~ euscle Sg the component of the at-P5 chieflyeeponaiele for the contractile mffactm of
.arbachnl in vltto . Supported 2 . part by theNat3ooel Hulth and Madieel Reaaarrb Counetl ofAuetralll .
VTV, flzet tsolated from porcine Intestine, alao ealsts inpostgsng11og1c autonomic axone iu mnny eisaues . 8ee,ve (aberseontaining i®ugoreactive VIP have been demonstracad in therespiratory tract of guinen plgs . rabblts, ots . doga, aaghuamns . Recenrly. VIP nev been implicated In the regulat!eoof respiratory mucus secretioo . a pretess involving release ofmacromleculee frou esocrlne celle, and transport of fons andvater aeross mucosa . Hecauee aubaueosel gbnda ond mucosaboth cousiet of sised Cell populations, it vee raot clear uhicbspeclflo ulls poaeessed VIP receptors and eon[rlbti;ed to Vly-eooked responses. Since VIP 1s known to accivate ac)enyl Lyc-lase, ve used an 1menmcytocLemical Probe for rJ4tP (0 loaotifythoae celln acfauLted by VIP. Dog tracheal poaterlor mem-brane and ferret tracheal rlage vere equ111bra[ed x 60 mio inottyBeveted LLam'• F-11 medium conC a1n!ng indosethoelq. (10-6)1)and becirreclo (Img/nl), ehes traesfarred to bea$enq cantelq-1ag fresh medium~ VIP (3 x 10-7tn, or VIP (3 a 10-7)t) plusproPrenolel (10-0l1), pbaocalaadna (10-6M), atrop).ne (10`6M),and et[mdotoaln (3 a 10-yM) . After 5 .1.0, Clseuer verefraseo and cryCSt.t >ecriona luined fbr Saun>reetRive cA`P,u>ittg the unlabeled antibody eneyme mathod . In fer;ec trache ;VIP lacreased imuunoreectlve cAHP 1v both sarous arlQ mcoueeellc tn submucossl glands, and this Sne .use veb enx inMab-Ited Ly the blockers tested . VIP produced 1it¢le f4 anyImcrease in cAt(P In ciliated eplthellal eella of the ferret-In dog tracheal eplphellum, VIP markedly increased cAMP Inc111oted and basel cells, but not Sn goblec cella, and thiain<reasa vse not blocked by the antagonfate Ce>ied : Yhoe . uehave .ueed levunocytochemical localizecenn of cAMP i a markerfor VIP receptor activation In heterogeneous tia0uufn whichcellespeclfle receptors cennet be meaaurad d3rectlj, ; WecoaClude that seroua and mucVVa [ella of ferret tracheal sub-mucodal glande and e111ated end basel cello of the dogcracheal ep(thelium contain VIP receptors, and ate .responsiblefor i+edisting physiological responses evoked by VIP ;(Supqorted by USPNS PPC (0.-24136 and Council for 7obatcoResesrch-USn . Inc . Grant l327R1)
CFFC(.T OF TNIOG1YCOy.1TE ON HafL9lER 11/NG MACROPMACE .~VrfIGin
N, Nacler and d .D . araln. Department of Thysinlnqy, Harvacd
School of Public Health . Boston. nA
we have produc .d n nronoclonal antibody which ieAccs vit .`, arell xusface antiqen on bamatea lung eucrophaqea birt not vsthother lung cells- eirc~elating eronocytea, or peritoneal vucro-phag9s . Pluorescein tagqed antlbody can be used to~auanti .'ythe 8nount of antigen using flov cytometry . Nhen the asountof tluo[eecent anubody un the cell aurtac* vas quhntitatedand plotted aa a histogram ef eell number versus amount offluorescence (xr cell, we found that hamater lung tsacrophegesheve a broed sy®etrieal Gaussian dlstriluRion . In previouswork . we noted that the length of time tllat mncrephnqes bavmbeen In the lung In an important daterminant Of thb quantltyef antlgen. In tS,is study we ccmpare chengea in the numberand e(re of lung mecrophagea and their aonunt of antigen aftera single intiacrecheal inst111at1on of 0-15 ml of 6e thlo-qlyc-olate 1TG) . One day after SC, the mmOer of Pagrophagesrecovered by repe$ted lavage increased fros 2 .4 a 10 in can-trols to 8.1 a 10 r thesa levels pereiecad for 30 days . Flowc)tometry analysls of macrophaqes frust TG animals Indicated aninltial dacrease in mean cell eit4 ss a.easured by kl[1tl lightlosa one day after instilletion from a control vm14e af 51 .0:2 .2 to '/H .5a1 .7- By the third day and thereafter, the siseof the 9G macrophaqes Increased to 88 .624.0 (not aiqnificantlydifferent from control) . Nean fluorescence ef the mectaopnsqapopulatlon fe decteaead from the eontrol value of 51-7s1 .015 .0. of the mean of 6 animals) to 19-6e) .3 In the TG treatedaaisele an day 1 . Menn fluorrcance per cell mcaa0ilyincreased to 65.9lT.1 on dey 10 at which time it surpassedcontrol voluea, Ostng the ratio of fluorescence to assallight 1ose, this sane trend ras seen. On day 1, thm ratiodecreased from the control velue of ]0.7!1 .0 to d-9ta.2. 'rMratio lncreased to 42 .1:6.6 at day 10 . Rhese dlta suggevtthat TG sevrvits inantwa nbcrophagee which lack aucfaceaneiqen into the lung, . With t Ue. theme cells nature andincretse both in srre and Sn the number of eati}enic siteaon the cell 9urface. Tbua this sonoclonnl antibody may beuseful In studies of the mturation of alveolas macropneqea .(SUpported rry NIH Grante Nt.-]7]ee and r5-aUnn] .)
Characterization of Purified Dog Mastocytoma Cells4P
Autonomic Membrane Receptors and Pharmacologic Modulationof Histamine Release' -`
MARTIN J. PHILLIPS,' PETER d. BARNES,' and WARREN M. GOLD
' Intlnducttona Mast cells appear to play a cent ral role
in many innammatory and immunologicreactions in the lung because they releasediverse chcimicals capabic of initiatingand modulating such reacuons (1)- As-sem and Schild (2) showed that a varietyof beta-adrenergic agonists co W d intubilantigen-induced release of histaminefrom passively sensitized human lung tis-sue, presumably by their effects on lungmast cells. Since then, the autonoroicreceptors and biochemical mechanismsby which vanous neurol ransout ters, hormones, and pharmacologic agents modu-
~te release of mast cell mediators haveeen the subject of intense investigation
but controversial conclusions . This situ-alion appears to be due to expelvoenausing heterogeneous lung iissue and beo-erogeneous cells, and differences in be-havior hetween purified cells from differ-ent smlrce-a (ng ., rodent mast cells andmast cell tumors, human basophits andhuman lung mast cells). These discrepan-cies might be due to differenees in mastcells from different species ar from differ-ent locations in the same species, or theymight be due to effetts on the mast cellscaused by ttie puriGcation techniques, Al-ternal ively, m a heterogeaeous ceU popu-lation such as the lung, pharmacologlceffects on mediator release may he in-direct, acuag via other cells present fnthe essue.
We have studied dog mastocytomaeaUs for several reasons-the dog has
. been used Jddely for the study of bron-chial reactivity and is the only mammalother than Aumans that develops a nat u-
i rally occurring respiratory poUeuosts .However, charactcrizaaon of dog mast
~cells has depended on studies of hetero-ncous tissucs such as lurlg(3)and bron-
chl (J). or ceUs harvested by bronchiallavage (5), and is Ihus subjeet to uncer-tainties inherent in all studies of m1Redcell popula8ions. Dog mauocyeoma cells
9UMMM.riY TM .q la conlllQlny atvyanra u to whlcn oulon4o•~C .eeeprCr . rnaat CMM PnaMa .
anG wn.rMr nM1lNpi0lf anfJprpia orr1lGEWamrp netllaryr LMOa vI. nMeuMNY CCa maaro4y
loma celY nn'auM tMr a . . Ml4ale In 4Nf numWra In a MnMty yun tom1 . unlrar npmal yty
maal GYRk. Maalorytoma nYCUIp rNm a YYp .wn i¢IaM anE aLayysQ+rwd wM . cellaypvaa to
P•nvlos a an fuapenawnot maatorytwna Ma er ~ 93M wntr. TM pw.~ ef aurunomk waP
rw curmdanl. rMw Yey ma.foryton~ eell. MO a nqn e.elry of pey..ac.pb.a lZf .5nn :
3,aGG: mean. SE ML-nppMNpll . n-31 et+tneh IM p.aGOmIMm aubrrpa.pparaU m tw Eau,.
No Mtlenoew..kunCbrtMpns.~+w W .Ipnfee..r.a .qb e .r~ .dl .r.px ~epow..Nw IM dirscl
.aeayro . olnWnp ot oy tinv acvone on nlp.Nn. ..kur
.r nrv .as.a os .rs:
closely resemble normal dog mast cells(6) and may be obtafned in large num-bers in a relatively pure form without theneed for extensive pWPcation procc-duru. Study of these cells may aid in un-derstauding the physiology of bronchialbyperrcactiviry and the role of the maetcell in this phenomenon as well as thegeneral features of the phalmacologiccontrol of mast cell function . Moreover,these oeLLs appear to have advantages overrodent mast ceU tumors because they con-tain similar mediators to human mastceLls (including a large amount of hism-mine rather than of serotonn), bind 1gE,and releasc theu mediatorswith both im-munologic and nonimmunologc chal-len ge- features that are preunt only vari-ably in rodent mast cell tumors (7) .Funhermo[e, prrvious studies have
tended to test for the presencc of phar-macologic receptors by evaluating the ef-fect of specific agonistsand antagonistson ceU function (eg-, change in cyclicAMP concentration or modulation ofmediator release) . Converscly, otherworkers have assumed that the prescnce
of receptors (by figand binding studies)or the increase in cyclic AMP implied aneffecl on release of mast cell mediators.Ho.vever-the presence of receptors can-not be assumed to correlate itrvariably
-' From Ne OMiwaseWar Reavrm tusutuleaodtkpanmun of Med4cine . Uw+avty of Californsa-San Fraoos<o. San FrannsttA C•Jiforaia.
• Supporud in p•rt by C+rant Nn ffL-7A1]6 fromthe Natwoel Hean . Lungaod Blood Imorutef, byGraar No. 133]R from the fouod for lbbre<oIkseartb-tiSA . lo4 and by graau num ITe SrmotlMd]fcal Resea/ea Fund (Amerinn tuny Assada-uuu of Sea Fuocnce). the Um.erary of CaWor-nur Rzua1N Ewluaoon and Alloonoo Cammir-uz, aod tne California Rcealeb and MedlealEducauon Fund (Ameriean LueB Associadnn ofCal3fornfa) .
' Prrseotetl m preliminsry form m W e AunualMetnng of Wa Amerum Thoreoc Sooery. IDS An-gelea- CA. May 18. 7952 .
Aequen5 tor Rpnnu shoWd be addressed toWarreo M-Gold, M .R, Caedo.meutar Reeeuc .
losuruu: UIS•M. Umwrsry of Califomia. 6anF.anoscu sen Frano.co. CA 94143.
• Rmprcol of a Thvenlna FeBmstlip frum IheMedical geearch Counci7 of Grrat Bntau,
totY-
PUBL ICflTIONS
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with functional effects on secretion. Inthis study, we have used radioligand bind-ing techniques as well as evaluation oft he functional effects of pharmacologicagonists on histamine release to test forthe presence of beta-adrenergic, alpha-adrenergic, and cholinergic receptors ondog mastocytoma cells.
Method9
Mmerrats
The radioligands used .vere: ('Hldihydroal-
prenolol ll'H)DHA), (specific activity, 101Ci/mmol); I'HJprazosin (specificanivity. 20Ci/mmoU; f 1-IJquinuclidinyl benzilate(['HJQNB) (specific aclivity, 33 Ci/mmol) ;EconoBuoA scintillation-counting fluid : allwere obtained from Ihe New England NuclearCorp., Boston, MA .
The drugs and chemicals used were : ace-tylcholine chloride (ACh); arropine sulfate,bovine serum albumimfracuon V (BSA),compound 48/B0, ( x )epinephrine hydrochlo-ride, histamine diphosphate. ( m hsoprotere-nol, ( x )arcerenol (norepinephrine hydrochlo-ride), (x )propnnotol hydrbchloride, penicil-lin O, streptomycin (Sigma Chemical Co., St .Louis, MO), phentolaminr mesylale (Ciba-Giegy Co., Summit, NJ), terbutaline sulfate(Astra Pharmaceutical Products, Inc. . Wor-cester, MA). (-)pherlylephnne hydrochloride(Winthrop Laboratories, New York, NY),shon ragweed (ombrosia arremnifolra) pol-len extract (Hollister-Stier Laboratories, Spo-kane, WA), acepromazine maleate (AverstIzboratories, Ina, New York, NY), sodiumthiamylal (Bio-Ceutic Laborarories, Inc . St .Joseph, MO), Dulbecco's modified Eaglemedium / DME), mrnunum essent tal medium(Eaglel-JOKLIK-modified (MEM-IOKLIK),4-( 2-hydroxyelhyl)-1-piperazine<I han esul-fonic acid (HEPES) (Boehfmger MannheimBiochemicals, Indianapolis; IN) . felal calf se-rum (Hyclone Sterile Systems, Logan, UT),amphotericin B with deoxycholate (Funga-zone; E. R- Squibb Co., Mirad a, CA ), trypanblue (GIBCQ. Grand Island, NY), toluidineblue (Fisher ScientificCa . Pittsburgh, PA),protease-free cotlagenase (CPLSA, Worthrng-ton Chemical Co., Freehold . NJ), and deox-yribonuclease (DNAase) (Millipore Corpo-ration, Bedford, MA).
The following reagents were used forfluorometric histamine analysis; perch7oricacid, hydrochtoric acid . mctbanol IJ . 7: BakerChemical Ca. Phillipsburg; NJ), n-heptane,I-butanol alcohol (MCB ManufacturingChemists, Inc, Cincinnati, OH) . o-phthal-aldehyde (Dionex Co., Sunnyvale, CA), so-dium hydroxide (Maliinckrudr Inc, Paris, KY),and phosphoric acid (Fisher Scientific Co .,Pittsburgh, PA) .
The experimenlal procedure followed ad-hered to Ihe published "Gaiding Principlesin the Care and Use of Animals" approved
by the Council of The American Physiological Society and specific protocols approvedby the Committee on Animal Care of IheUniversity of Califorma, San Francisco
owrwn. a~wrca un pqn
Masmcyrorna Biopsy and Disaggregarion for 20 min ; for saturation binding studies,Ma-stocytonia nodules were excised from a cnncentrationsrapgedfrom0.1 to2OnMand4-yr-oldGerdnanShepherddogthaAhadnrul- forcompcthionsAudiesfrorrilto2nM .Notiplesutrcutaneouslesionslocatedneainlyover specificbinding .~asmeasuredbyincubatijethe abdomen, chorax, and hindquarters . irstheprosenceoflµM(-)propranoloLWhenThese tumors gretv slowly, doubling in size catecholamines ivere used in competitionover the course of I yr . The nodules ranged studies . 100 µM ascorbic acid was added tofrom 5 to 25 mm in diameter at the time of tbeincubation tubes topreve,nt oxldattorr . ABbiopry. A total of 22 nodules were excised over samples were run in duplicate or triplicate .a 12-month period . On each occasion, thedog Atpha-adrenergic binding was determinedwas frrst anesthetized by a subcutaneous in- byincubationwith0-1 to 5 nM I'Hlprazosin,jection of 0.25 mg/lb of acepromazine fol- usingthesameexperimenral conditions . Non-lowed b,v an intravenous infusion of sodium specifSc binding was defined by incubationIhiamylal (2a1a wt/vol) until no response to with 10µM phentolamineor 100µM (-)nor-pain could be elicited. A mastocytoma nod- epinephrine Cholinergic reeeptor binding wasule was excised, dissected free of sdbcutane- determined by incubation with 0 .1 to 2 nMous tissue, and washed in calcium-free PBS 1'f 1QNB for 60 ririn, and nonspecific bind-containing penicillin (100 U/ml), streptomy- ing was defined by incubation with 1 .rM atro-cin (100 pg/ml), and fungazone (2 .5 ag/mI) pinato counter any bacterial or fungal contami-nation.Thenodulewaslhenmincedintofine Dara Analysrsfragments (approximately I rnm diamrter) Datawasexpressedasmeans m SE . Bindingwith scissors (6. 8, 9). curveswere fitted bya nonlinear least-squares
These fragments were disaggregated enzy- curve fittingcompurer program (IO) . Satura-marically in an Ehrlenmeyer ffask try a solu- lion curves were ilnalyzed by estimating the
lion consisting of 50 U/ml high purity equilibrium dissociation constant, and comprotease-free cullagenase, 2% BSA and 25 petition curves by eslimating the inhibitorymM HEPES in calcium-free DME,adjusted dissociation constant . Parametcrs were oho-toapHof7-4andanosmolantyof290mos- sentogivethebesAdarafitaideterniinedbymoles.Disaggregationwascarrieddutunder minimalvarianceofexperimenraldataabout95% O, and 5% CO, at 37° C in a gyrotory thecurve generated by these phirameters. Datawater bath. At 2(}mm intervals supernatant fit was compared at increasing number ofwas removcd from the disaggregation solu- parameters (N) thdt resulted inastatisticalt,tion, which was then replenished with fresh sigmficanr improvement in lit over (N-q . •solut ion . A fter fi0 to 90 min, t he dlsaggrega-tion was discontinued and the remaining so- Pharma'rologic Studieslu]ion was filtered through cheesecloth . Cell These studies evaluated the modulation ofsuspensions obtained from disaggregation h-stamme release by beta-adtenergiq alpha-were centrifuged (200 x g for 10 rd .in), and adrenergic, and chblinergic agonisu and an-the resultant cell pellet was washed tvSice with tagonists. Histamiote release .vas induced ei-caldum-free 7yrode's buffer before resuspen- ther nommmunologically by challenge withsioninanappropriatemedium .Mastocytoma compound 48/80, or immunologieaUy bycells were identified by their characteristic challenging passively sensit'urtd cells with an-granules. whicl, stained merachromatically tigea For pharmacologic studies requiringwithtoluidineblue(O.2ol.solutionatpH3.0, compound4g/80.inducedhistaminerelease,dilutedll0withthecellsuspension)andwere cellswereresuspendedin7yrode'sbuffercan-quantitaled by countingtn a hemacytometer taining 25 mM HEPES and 0 .lwo BSA at a(American Optical Scientific Instmments, concentration of approximately 3 x 10'Buffalo, NY) (cell count ranging fronr 47 to cells/ml . These cells were then reacted with2)0)_ appropriate concenrrations of compound
Cells were resuspended in 7yrode s buffer con- For studies involving immtdnologic hism-tainrng 25 mM HEPES at a concentration of mine release, cells were first sensitized pas-approximately 0 .5 x]0° cells/ml (range, 0.3 sively to ragweed antigen byy resuspendingto 1 0 x 10° cells/m4, then DNAasc (0 .1 them in I ml of MEM-JOKLIK solution andpg/m1) was added to the cell suspcnsions to incubating them with I ml of dog IgE-richprevent clumping. The cell suspension con- hyperimmune antishort mgweed serum . Af-tained95.1 ±2.a3cfomastcellsln=7). Cells terpassivesensitization,thecegswerewashed(approximately I x lo° cells/assay tube in twice in calcium-free Tyrode's buffer anda fmal volume of 0 .25 rtd) were incubated with resuspended in Tyrode s buffer containing 25radioligand at 25°C, and incubations were ter- mM HEPES andO.10lo BSA in a manner idea-minated by dilution with 5 ml ice-cold PBS tical to that used for nonimmunologic chat-and filtration under vacuum through What- lrnge and at similar concenlmlions
. omanGF/Cglasshberblters.Fdterswerethen immunoglobulin E-nch, hyperimmunwashed twice with 5 ml PBS and, after addi- anti short ragweed serumwas obtained frorntion of scimdlahon Buid, were counted by acolonyofdogsthalweresenntizedtoshonliquid scnntillation spectrometry at an effi- ragweed antigens by repeated parenteral in-ciency of 5O°lo . For rdennficanon of beta)ectionsafterexposuretoliveauenuateddis-adrenergic receptors, 1'HJDHA was incubated trmprr virus (11) . Serum used for passive sen-
CHARACTERIHInDN OF PYRIFIHD DDD rrAarnC/TGYA Cr1E5
sitiiation was obtained from dogs that hadbeen shown to have marke-0 cutaneous andhronchral reacs ions to short ragweed antigcnssc+ciated with high levels of specific IgE
RAST serurn levels (12,97 t 1 .18, mean ~.SEM) (12).
Hrsramrne Release Jrom Cells
Histamine release induced by compound48/$0or ragveed anttgen was carried out byincubating Ihe cells with the releasing dgentat a final reaction volume of I ml for 3d minat 37' C in polypropylene t ubes W hen ph ar-macologicagonisss were used, the) were firstinclibated wilh the cells for the tollowmg
times: ]-min Incubauonwith ACh, 5-min in-cubation with isoproterenol. lerbutalina and
phenylephrine. When the pharmacologic an-tagonists piopranolol, phentolamine, andanopi ne were used, t hey were incubated wit ht he cells for 5 min before the addition of theagohlst.
At the end of the release experiment, thereaction was stopped in irx, the cells were cen-trifuged at 450 x g for 10 min, and the su-
pernatanl was separated from the pellet byaspirat ion . perchlonc acid was added to bot hcell and supkmatant fractions to a frnal con-centrauon of 0 .2 M in order to lysc Ihe cells .and Ihe samples were stored at -70° C untilassayed for histamine
Experfinenral Desrgn
Pharmacologlc modulaaon of histamine re-lease was evaluated by 2 methods: In one, asingle concentration of the autonomic agentwas examined for its effect on histamir6e re-lease induced by differenl concentrations ofthe releasing agent ti .e., a dose-responsecurvefor the histamine-releasrng agent). Concen-trationsof ragweed antigen used for a dose-responsecutvemcluded : 1, 5, 10. 25 .50, 1(Cf,250; and 5f10 trg/mI : concentrations of com-pound 48/80 included : 0 .01, 0 .02, 0.05, 0 .1,0.2 . 015 . I, and 10 pg/ml . A few of these con-centratlons were omitted in some dose-response cuFves, but all curves consisted ofat least 6 concentratiuns, each in duplicateor triplicate. Each dose-response curve alwaysincluded control tubes t hat received cells butno releasing agent so t hal spontaneous hista-mine release might be determined . For eachespAnment, parallel doseresponse curves foreach histamine releasing agent to which theautonomic agenl or agents were added werealso analyzed . These dose-response curvesalso included tubes to whlch no releasingagehl was added but which contained the au-tonomfc agent in order todelermine whetherthe agent had any direct effect on histaminerelease In the other method of evaluatfon,the concenttation of the autonomic agent wasvaried, white the concentration of the hutarnrrie-releasing agent was unchanged . Thc:oncentrauon of the hhs ammeaeleasmg agentwas chosen to cause apprortmately 500?a o(the maxsmal histamine release observed prcvlnttslv in doseresponu curves wnl
: Ihatagent. Each concentration of the auronomrcagent was examined in Uuadruphcate and
masched with 41p bes to which only the releas-ing agent was added . In addition, spontane-ous histamine release was also examined inquadruplicate iri tubes containing eells, butneither releasing agent nor autonomrcagent .
G(ara Analysrs
Histamine release was calculated as theamount of hfssainine present in the superna-tant fmctson expressed as a percent of the to-tal histamine present in bot h supernaa ns andpellet fractions, The effect of an autonomlcagent on a ragweed antigen or compound48/80 dose-response curve was analyzedstatisucally fur each experiment bytwo-wayanalysis of varlance using replicate .aluhs lodetermine whether significant differences ex-isted in hislamine release between the dose-response curve containing the releasing agentalone and the curve to which the autonomicagent had also been added . In order to e>:am-me statistically all the experiments with a par-licular autonomic ageni as a group, the hista-mine release values were normalized by disig-natmg the maximal release as 100% andexpressing all other values as a percent of themaximal release. The extreme ends of thedose-response curve probably provide art un-reliable basis for desermining the inhibitoryor enhancing effect on histamine release ofan autonomic agenl. For this reason, modu-lation produced by an autonomic agent wasestimated on the basis of its effeclon thatpart of the curve at which 50% of maximalhistamine releaae occurred (the ED .n for hista-mine release) . This effect was expressed intermsof percent inhibition (or enhancement)according to the formula :
comrol - eaperi__ental°~a inhibition = control x tm
wherecontrollsthepercent hrstaminereleasedby the releasing agenl and experimental is thepercent histamine released by the releasingagent in the presenceof the autonomic agent .
The values to be inserted in this formulawere obtained as follows: t he percent releasewit h t he releasing agent cor responding t o theE Ds° was calculated by subtracting rhe spon-taneous release from the maximal release ob-tained and dividing by 2. In order to dcter-mtne the corresponding percent release in thepresence of the autonomic agent. dose-re-sponse curves were drawn graphically. Thepoint on rhe dose-response curve fa t he re-leasing agent corresponding to the ED,o,'aluewas found by adding t he spontaneous releaseto the F Dso value. From this poi nl, a perpen-dicular line was dropped to the abscissa . Thepercent telease was noted at the pointat whichthis line cut the dose-response curve con-structed for the autonuntic agent. The re-quired value for the formula was then calcu-lated b} subtracting the spontaneous releasein the presence of the autonomic agent fromthe value obtained from the graph, ai de-scribed prevtously. Experiments with differ-ent concentrations of the autonomic agentswcre analyvrd individually by Student'ss testand s a group by 1woway analysis of varl-ance using rrplf :ate values .
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F 9 1 T,ma Coume Or Epe<inC 1'HIDHA b,nOmg 10 i60neteG . •nraor oog masrocytoma eells. Bmaenq v.a5 per .brmea a125° C at a hgnna eonpentranon Of 09 nMAss«~anon was ramtl vnth a L„ of 25 m,n, (-)propranOlo
1 (r PM)was added arthe anow 3and d iesooauon r.ol-lowea for 30 m,n Data Irom i Ya{ponmenr pe.fo•mrMin O°plsata, wM1rCn were typ¢atlor 3 such espenmenta .e .e snswn
Histamine AssayHistamfne was determined by the o-phthal-aldehyde spectrofluorometric procedure ofShore and coworkers (13) . but modified forautoanalysis (14) with an automated spec-trofluorometric analyzer (Alpkem Corp .,Clakamas, OR). The autonomic agents, com-pound 48/80 and ragweed antigen, wereeachtested in the concentrations used in these ex-periments for auto0uorescenceand for theirpossible effects on the determ-inauon ofknown histamine standards-
Reaulte
Radro(igand Binding1'HJDHA binding to dog mastocytomacells was displaced by I f,M (- )propran-olol. Binding was rapid at 25°C, reach-ing equilibrium at 15 min, with a meanhalf-ti me of 1-9 min (n = 3), and was par-tially displaced on addition of 1 ytM(-)propranolol (figure 1). Scatchardanalysis of propranolol-displaceablebinding revealed a curvilinear plot, andcomputer analysis showed that the datawere best fitted by assuming 2 differentbindingsites (figure 2) . The high affinitybi nd ing site had a mean dissociation con-stant (Ko) of 0-90 ± 0.17 nM (n=5),with a maximal concentration of bind-ing sites (Bmax) of 35 .7 t 5.5 fmol/10°cells, corresponding to 21,500 :t 3,300high-affinity binding sites/cell (n=S)-The second binding site did not appearto saturate, and it was therefore not pos-sible to estimate Ka or Bmax- Bindingto this nonsaturable site was reduced byapproximately 15%u when 100 µM phen-tolamine was included in the incubation,as described by others (1S) .Catecholamines competed for /aH1
DHA binding with the rank order ofpotency isoproterenol > epinephrine >norepinephrinc, indicating binding tobeta-adrenergic receptors (table 1 and fig-ure 3). The beta,-adrenergic agonist hada relatively low affinity, as noted byothers in receptor-binding studies (16) .
Iated, ,maCl dog masmcytoma L911s. TTIe d,tf6renCe be,
aveen Imel binding antl binding in the presence ol 1yM (-)pmprarqlol s ITIOnerl- Tne wrve wee ened bynonGnear Isasf-squarea CUmpuler enalysls. The dota
.were oesl o9scnUee Lry.asSum,ng a mv-sne rmeratoonThe high efen,lY sila 6 rapre59Mr/ by tha steepstra,gntnne . Tne slope Of wn,ch,9,yeJ the d050CIaLOn Con9tanl
(Kal. and the imerceprw4h The aU5os9a 9w991he manImal CpnpeOireaon pf binding yes ndmaxJ The non.
saturBCle sne ie repte5AMe0 py me nDraontal line Thedate shown afe fmm 1®tpenm9la perfnrmep ,n puplnrate giving a KO of I I nM anC Bmsa of 42 u frnoVtO'Ce118 (25:.,ag MnD Fng SuteNC91q TTB was typlta/ pf 5separeroe.penmentsg,vmgameanKOO1090 x 0.77nM I s sE)antl emae o12r, .A0 s 3.30Dbrnamg snOSCelI
The (-)isoproterenol displaced approx-imately 30% of total binding, which isequivalent to 60Oio of propranolol displaceable binding at a.similar concentra-tionof!'HjDHA~ Competition for /'H[DHA by (-)propianolol showed a com-plex curve which was best fitted by as-suming 2 binding sites . The first, com-peted by low concentrations of (-)pro-pranolol had a high d.ffiniry (inhibitorydissociation constant K, = 1 .6 ± 0.5 x10-° M; n- 3) and accounted for approx-imately 60% of the ['H[DHA binding .This is similar to the k, values reportedfor propranolol in membrane prepara-
TADLE I
INHI131TIOn OF SPECKICI'HIDIIavDROALPRENOLOI 61NDIN0 TOIgOtLATEO DOG M1bASTOCYTOMA CELLS
nV ADRENERGIC.AGONISTS•
Agenl K, (M)T_
. .
1•)ISnprdarenol 19 z 07 . t o"1•IEOnaphnne 223 06 x t0'sTerbuta4ne 6 A= 06 z 10`i• JNOrepInephr.ne 1 I x D 6 x 110'l-)Proprsnolor Rnsez 05 n IU`*
Kl4 2x23x 10-'4/•IOroprendot 132 06 . 10'
' vaues "s mean u SE v1 3 veon,nx e .o.nmenuI 4,ne .IOh OR54c .a1Wn (p,gl9ni (I(~ .vM oel.nn~nqe no~n
Ira e,a.alm `ir' ~a
.,ne .e ,C .. ..a. nu conOSnnncn u,
apen,[eWw(QSpib.nno,pn01I'MIDrUaMn9. (llvy IM1p
[urcwv, .woo w 1'*IDn . mv ny .e,p,0~ N 9 nsp- en0 ee"++~M nean u.ne0c~a,m (enslnrn
~ n . .uuee ~w ,- IO .nn~edvwr m, n .p.. rrwr an ... .q v,entn'•M~W ~w r Iam A qW 1o - ,°.In,Ia^,nJV6n9s
F,g 3 IMID,tmn ql speMs ('HIDHA Dmeingfi anlated,,ntaCl tlOg maslocyt9rnacens br adrenerg,c agena rhomncenlrgponofr'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 by antagonl5la (-Jprppr9nol01(NOSetl squyyp)and(+proprannlol(Cppnsqu9rsd) Cpmrolsr91prm1-)propranololCOmpehbon .CurveawerelleeohyaMnl,npa,I6asl-squares C4mputer program . For (-)proP2nptol Me tlala were Llled by assum,ng an o hiDihon ot binding612 3aes . ThedBla shown are rmm I expenm9nl . perfprmeU m Gupl,cate. which w5s typiCal pf 3 such eapenmenry
tions(17, 18) . The second site had a muchlower affinity, being approximatel)I 1,000times less than the first . Competitionwith (+)propranolol . however, showedonly the single low affinity site . Thls sug-gests that the high afOnity, site, which isstereoselective, probably represenlS dis-placement from beta-receptors . whereasthe low affinity site, which is not sdereo-selective, corresponds to the fraction ofbinding that is nonsaturable as i)eter-mi ned by arla[ysis of the sat urat ion eurve.
With ('Hjprazosin . approximately 10to 15% of total counts bound were dis-placed by 10 or 100 pM phentolarnine,but Scatchard analysis revealed no highaffinity binding in 3 experiments, therewas no inhibition of binding to l00 pM(-)norepinephrine, indicating Ihat al-pha-l-adrenergic receptors could not bedetected on these cells .
With I'HIQNB using concentrationsto 2 nM, there was very little binding af-ter 60 m i n a125° C(300 to 1,200 codnts/l0° cells) and no inhibition of bindingin the presence of I µM atropine, Thissuggests an absence of detectable nms-carinie cholinergic receptors on thesecells .
Phprmpcologx StudiesThe adrenergic agonist terbutaline wasevaluated for its modulat ion of histaminerelease induced by both ragwecd antjgcnand compound 48/80. In each of 6 ex-periment s, t erbutahne (10-' M) altered theragweed antigen dose-response curves,one example of which is shown in felgure4. The maximal response was reducedand the concentration of antigen neededto induce a response that was 50% ofmaximum was increased . 7ivo-way anal-ysis of variance using replicate values
showed that this effect was significant ineach individual experiment (p < 0:01 in5 experiments and < 0 .02 in I e,iperi-ment), and also when all experitttentswere taken as a group after normaliza-tion of values (p < 0.001). The percentinhibition caused by the drug at ED,° forhistamine release was 67 .7 :t 15 .4 (mean± SD). Incubation with (-)propraelolol(10-' M) blocked the inhibitory effect oterbutaline and resulted in a dosresponse curve l hat was not significantlydifferent from the dose-response curvewith ragweed antigen (figure 4). ,
The effect of terbutaline (10'' M) oncompound 48/80 dose-response curveswas examined in 6 experiments, I ezam-ple of which is shown in figure 5 . In 5experiments, terbutaline reduced the re-sponse to compound 48/80 at all doses(p < 0.01). In I experiment, no signifr-cant change could be demonstrated byanalysis of variance because of crossoverof the curves in the plateau region, but
wa.wn +.ny .n wy~m
F,y 4 Ibetrrasponaa cuTros showrng LM pa 'hgtami ne relea6e in respCn6e to different CanCBnla'
tMna of ragweed ant,gen . with ragwsed ant'qen aNne(c1psB0 farGas) . m the pres9nce of 1P' M lerqra/me(opOn m9ng/es), and in the presenCe Uf 10" M tsrelNIme ano lD > M propranolol /opm+ Grcks)
nistamme r916b6e in reaponse ]o Udterenl CpnGemra-
1•ons ol Compoend 48180. ,.ith 1Ompo, .n0 aa/aOalone
(C1p148d C~K'I65) . ~n the pre66nCe of 1e1 M Ierbulalino
(opsn mengrest
. ane ,nrne breaence ai to` M rarUUlaM1ne antl 10" M prop/enolUl (open p/e/9s)
in this experimenl terbutaline did causeinhibition at the lowerconcentrations ofcompound 48/80, with a percent inhibi-tion at the ED,> for histamine release of66.4%. The percent inhibitic,n for thegroup was 57.7 ± 9.2. iivo-way analysisof variance on normalized values for t hegroup also showed that terbutaline (10-'M) signit icam ly aherrd t he dose-responsecurve (p < 0 .025). Incubation with(-)proptanolol (10-' M) again blockedthe inhibitory effect of terbutaline andesulted in a dose-response curve that wasnot significantly different from t he dose-response curve with compound 48/80alone (figure 5) .
The beta-adrenergic agonist isoproter-enol was also evaluated for its modula-t ion of histamine release induced by rag-weed antigen. In each of 2 experiments,isoproterenol (10-' M) markedly inhibitedthe ragweed antigen dose-responsecurve,an example of which is shown in figure6. The percent inhibition caused by tso-proterenol at ED,/ for histamine releasewas 85 .2 ~ 12 .5 . In 4 additional expcri-ments, each in quadruplicate, the effectof different concentrations of isoproter-enol on histaminerelease induced by 50pg/ml ragweed antigen was examined(figure 7). In each experiment, all con-centrations of isoproterenol significantlyinhibited release of histamine (p < 0 .01)when compared with their preassignedcontrols, and similar significant differ-ences were also found when all 4 experi-ments were considered as a group . Incu-bation with (- )propranolol (10" M) partially blocked the inhibition of'coproterenol (10" M) on ragweed-
ntigen-induced histamine release (figure 7). ln 2 of the 4 experimems, hista-
mine release in the presence of( - )plopranolol and isoproterenol wasnot significant ly different from histamine
release obtained in the presence of anti-gen alone, and when a11 4 experimentswere considered as a group, no signifi-cant differences were found . In a sepa-rate experiment, the use of (+)propran-olol failed to alter the inhibitory effectof isoproterenol on antigen-in'ducedhistamine release.
The alpha-adrenergic agonist pheny6ephrine ( IO-' M) was evaluated for modu-lation of histamine release induced byragweed antigen in 5 experiments andmodulation of histamine release inducedby compound 48/80 in 2 experirments .Phenylephrine (10" M) inhibited hista-mine release induced by antigen by 18 .3m 23.7% and enhanced histamine releaseinduced .by compound 48/80 by 6.3 m4.0%u at the ED,o for each mast cell acti-vator, respectively. Neifher overal0, norin an individual experintent, could Jphen-ylephrine be demonstrated to cause a sig-nificant change in the dose-responsecurve.
The etlolinergic agonist ACh (1d-° M)was evaluated for its effect on histaminerelease induced by ragweed antigen in 5experiments and on histamine release in-duced by compound 48/80 in 2 experi-ments. Overall- ACh caused enhance-ment of histamine release at ED,e of 11 .4- 7-6% (or ragweed-an[igen-induced re-lease exp'eriments and 8 .5 x 4.74'u forcompourld-48/80-induced release. How-ever, in rtio experiment did Ach cause asignificant change in the dose-responsecurve by two-way analysis of variance .In another experiment, the molar con-centration ofACh was varied from 10-10M to 10-° M in tenfold increments andits effect on histamine release induced bycompound 48/80 (0.2 pg/ml) was exam-ined. No significant differences were ob-served in histamine release in the pres-ence of ACh at any concentration com-pared to the release with compound48/80alone. The effect of both bethane-col (2 experiments) and ACh (2 experi-ment s) over fu l I dose-response cu : ves forcompounds 48/80 (0.01 to 1)<g/ml) and
Fig 6 Oose-response-c.nves`show.ng the pememngtam .ne release n responga to dArerenl COneentrarions of mgweeo am
.gen . -rth ragwaee anogen alonptcrose0 nrcros I ana ~n me vresenCe ol to' ° M~soprpre~-ena lopen rnangres)
PUBLICATION$
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rig 7 HiSlam~ne .9leasetnryyceLlqrpgn,,a6eanl
(50 regrmr) m me presenee bt mnyrent tmnpentrn'
OI ~dOpml9renol (541W Dar3) }iipre3aee a5 B p9ree
a malChed eGMml wtnGYl Imeprotprenol ?Te BHe
,nCUbaeon w .th botE 10" M prApranolel aOE lD-i N
proterenol bebre the atl6i(Icin ol fagweEd y,4gr
shpWn q Ine hal[rlatl pa1. The rd5ul16 are eepM :as mean z SD TTe aponreniwus release w99 3-
a 1394 H1stam,ne releese was induced Gy 50,rgrr
ragw64tl anl .gen cakulffied to g.vo spprOamatnly I
mal,mal hlstaamme releasB (adunl releaYe achle
2ar s 004%)
ragweed antigen (1 to, 100 pg/ml) wexamined and no significant effect oft her agonist was obser.+ed, suggesting rhancement of nonimmunologic or imunologic activation of Ihe mast celIncubation times with ACH and betlnecol were varied frorh I to 5 min .
In these series of experiments, tmean histamine content of the cells w6.5 x 3.6 pg/cell and the mean spomneous release was 4.3% (range, 0.68.5oJo). The mean maxiunalhistamine .lease induced by ragweed antigen w3fi-4t7n (ra nge, 17 .R to 68', 3%) and by coipound 48/80was 65 .5% (range, 47-874.40/0). Cell suspensions yielded a hi,percentage of mast cells (93 .8 m 3-4-and cell viability, as assessed by trypiblue staining, was always in excess92%. None of the auto I nomic agentsreleasing agents caused autofluorescenat the concentrations used, affected Imeasurement of known histamine stadards w•ith the autoanalyter, or alterlsignificantly the rate of spontaneohistamine release
. There was no diffeence in histamine released by antigenpreparations from which amphotericB(fungazone) was removed by washilor from preparations in which no arphotericin B was added (n=5). indicaing that amphotericin B not removed Iwashing had no effect on histamine rlease itt these cells .
DiacussionThe presence of autonomic receptoin purified mast cell preparations hi
proved tb be a contentious issue In thisstudy, we demonstmtea the presence ofbeta-adrenergic receptors but no alpha-adrenergic or cho6nergic receptors. Thesefindings are based on both radioligandstudies and pharmacologic studies that+.vere in accord with each other. In com-rnotd with all purified mast cell prepara-tions, rhk results are subject to the criti-cisrri that the prooedute used for the iso-latidn of the cells may have causeddamage to cell membrane receptors. Wehelieve that this potential hazard has beenminimized in this study by the use ofa gentle disaggregation process, usinghighly purified collagenase, and more im-portantlN by the fact that purifrcation didhot require density centrifugation, a tech-niqtie known to result in a loss of 50 to80% of IgE-receptorbinding activity(19). We have also shown, in anotherstudy, a close similarity in the morpho-logic characteristics of mastocytoma cellsin situ inn the tumor and those in cultureaftet disaggregation (20) . These resultsconl irm those of other workers (3) whocompared the effect of collagenase andpronase disaggregation procedures ondog lung and mcsenteric tissue, andfound that collagenase had advantagesover pronase and produced a high yieldof mast cells with ultrnstructures alwaysintar_t .
We believe these mastocytoma cel ls aresimilar to, and reasonably representativeof, thormal mast cells on the followinggrounds: (!) They are morphologicallyand histochemically very similar to nor-mal dog mast cells (20) . (2) They appearfunctionally viable and similar to otherspecies in that the cell histaminecontentis similar to that of mast cells in dogsand other species (20) ; they bind IgE andcan be triggered to release histamine byimmunologic and nonimmunologic stim-uli (8, 9) ; they are capable of generatingleukotrienes (8), and the autonomicreceptors and pharmacologic modulationof secretion is similar to that describedfor dog lung in vitro (21) and in vivo (22)and to isolated human lung mast cells(23).
The presence of beta-adrenergic n-cep-iors on mast cells was first inferred fromstudies of heterogeneous lung tissue inwhich beta-adrenergic agonists caused in-hibitionof mediator release. However, itcannot be assumed that beta-adrenergicreceptors on mast cells that have been pu-rified and are from other species neces-sanly modulate mediator release. Indeed,studies of purified rat peritoneal mastcells and murine mastocytoma eel ls havefailed to show such a link . Although t here
is one report that beta-adrenergic stimu-lation of rat peritoneal cells in vivo ap-peared to inhibit mediator secretion (24),most other studies using cells that hadbeen purified by centrifugation throughdensity gradients failed to demonstrateinhibition of mediator telease (25), af-though increases in cyclic AMP may oc-cur (26). It appears that in rat peritonea]mast cells the adenylate cyclase stimu-lated by beu-adrenergic agonists may notactivate the cyclic-AMP-dependent pro-tein kinases that affect mediator release(27). Furthermore, Sullivan and cowork-ers (26) found that the effects of a varf-ety of catecholamines on rat peritonealmast cell cyclic AMP were inconsistentand did not fit into the generally acceptedpattern of alpha-adrenergic and beta-adrenergic receptors . Thus, epinephrineand norepinephrine stimulated cyclicAMP production and inhibited media-tor release, whereas isoproterenol had lit-tle effect on either cyclic AMP levels orcompound-48/80-induced histamine re-lease. Nevertheless, there is evidence fromradioligand binding studies that beta-adrenergic receptors are present on ratmast cells (28-3 1) . Thus, Marquardt andWasserman (31) demonstrated the pres-ence of these adrenergic receptors on ratserosal mast cells, and showed that beta-adrenergic agonists caused a rise in cy-clic AM P levels, but were unable to dem-onstrate anyeffect on IgE-mediated mastcell histamine release
-fhus, pharmacologic studies alone, orradiol igand b inding studies alone arc in-adequate for characterization of recep-tors on mast cells- In addition to radio-ligand binding studies, we thereforeundertook pharmacologic studies to ex-amine the modulation of mediator re-lease. In this study, we have shown thatbeta-adrenergic agonists can inhibit im-munologically and nonimmunologicallyinduced histamine release and that thisaction is affected through a stereoselec-tive site that can be blocked by (-)pro-ptanolol but not by (+)propranoloL Thisfinding indicates the presence of func-tional beta-adrenergic receptors and sub-stantiates the conclusions drawn fromstudies of heterogeneous human lung tis-sue and confirms reports recently madeon purified human mast cells (32, *3) .It is of interest that dog mastocytomacells in this respect appear similar tohu-man mast cells. in contradistinction torat peritoneal mast cells or murinemastocyioma cells .
The demonstration of beta-adrenergicreceptors by radioligand binding assaysin our study correlated with the inhibi-
PUBLICRTICNs
wowos. rwwee. ua oota
tion of histamine release by beta-adsen-ergic receptor agonists. ['HJDHA boufgto intact dog mastocytoma cells anddisplaced by (-. )propranolol. Scatch:6analysis of displaceable bi nding revealeda component that had an affinity simi-lar to that e.xpected for 1'HJDHA bind-ing to beta-receptors in broken mem-brane preparations (l7, 18) and other iso-lated cells (34)- There was a secondbinding site, which was not satorable.Similar nonreceptor binding that is dis-placeable by propranolol has also beendescribed in isolated fat cells (15) and inrat peritoneal mast cells (28) and nSay bedue to the solubility of ['HJDHA in thecell membrane. Further e.idence lhat thehigh affinity binding represents bihdingto beta-receptors is provided by thedemonstration that high affinity,bind-ing accounts for approximately 60v/o ofdisplaceable binding and isoprorerenolcompetes for a similar proportion ofbinding (60°Io of propranolol displace-able binding;. Funhermore, the propor-tion of binding that is stereoselective isalso similar to the component of dis-placeable bindingthat is of high af6inity.All this evidence suggests that high af-frnity ['HJDHA binding to dog mastocy-toma cells labels beta-receptors . Agonis~competed for binding in the rank ordof potency isoproterenol > epinephrine> norepinephrine, suggesting that thepredominant beta-adrenergic receptorsubtype on these dog mastocytoma cellsis beta,. The affinities of adrenergicagonistswere lower than expected for in-teraction with beta-adrenergic receptors,as determined by competition studies inthe cell membrane preparations. How-ever, this discrepancy has been describedin other isolated cell preparations (34)and may be due to lhe presence of guano-sine triphosphate in whole cells, whichreduces agonist affinity, either becauseof desensit ization of beta-adrenergic re-ceptors during incubation with agonists(35) or because of metabolic breakdownor uptake of catecholamines . The beta,-selective agonist terbutaGne similarty hadlow potency, but a low affinity and ab-sence of beta,-selectivity of this agonisthave been described previously. Thesefindings suggest that the selectivity thatis seen physiologically may be due todifferential activation of adenylate cy-cl ase (16) . The af finity of binding of pro-pranolol to t he stereoselective binding swas in good agreement with that detmined from competition experi4nentswith whole cells (34) and membranepreparations (17, IS).
tors (21,500 ¢ 3•3UD receptors/cell) washigh, although greater numbers of bind•
g sites havp been reported in other in-act cells including fat cells (15), cultured
mllsclecells (36),and tat peritoneal mast
cells (28 . 31) . The high density of beta-adrenergic receptors on these cells mayindicate the presence of spare receptors,which are a means by which a cellachieves a high sensitivity to agomsts . asstimulation of only a fraction of Ihereceptors results in a maximal response .
This might also account for the dis•crepancy between thecffect of isoproter-enolon binding, and its funnional effecLThe presence of alpha-adrenergic
receptors on mast cells was initiallypostulated in studies of heterogeneoushuman lung tissne in which alpha-adrenergic stimulation by phenylephrineor norepi nephrine (n the pmsence of pro-pmnolol) resulted in enhancement ofanligcn-induced mediator release (37) .
Opposite results were obtained by Nmand Bloom (38) in rat pentoneal mastcells . They found that norepinephrine inthe presence of the beta-adrenergic an-tagonists propranolol or practolol in-hibited histamine release, and this effectwas dimimshed by phentolaminc {ndi-
ing that alpha-adrenergic stimulation~'nhib{ted mediator releasa We were un-
able to find any evidence for alpha-adrenergic receptors in these dogmastocyloma cells based on radioliga.rdbinding asszys or pharmacologic effects.lb our knowledge, radioligand bindinRassays have not been used before to lookfor alpha-adtenergic receptors in purifiedmast cell preparations .The presence of cholinergic receptors
on mast cells was also first inferred fromstudies on human lungussue (37) . Moststudies on purified cells were based onthe rat peritoneal mast cell, and onceagain confliaing results exist . Someworkers (39, 40) reported cho6nergic-induced histamine release in W istar rats,but others (41-43), using dif feren l strainsof mts, could not demonstrate arry suchef fe<u. Masini and ooworkers (44) laterreported variable results even within thesame species, and could find no correla-tion between ACh-induced histamine re-lease and the high af6ruty binding of/'H]QNB, a specific cholinergic mus-carinic ligand . to rat mast cell mem-branes. These discrepancies might be due
o o diffcn:nces m the species, to differcncesn purification lechniques, or, in hctero-geneous tissue such as lung, t he choliuer .gic agonist may be acting indirectlythough other cdl types
. This laaer possibility is thought to he the case in the
autonomic regulation of type 11 pneu-mocyles w here chotinergic agonisu causesecretion of surfactant but not in virro(45. 46) . An alternative source of varia-tion in rat mast cells was suggested bySchmutz)er and coworkers (47), whofound that cholinesgic-induced histaminerelease occurred only sn sensitized cells .A similar rffect was noted in dispersef] ;dog lung cells, wherechoGnergioinduced
histamine release could oNy be demonstmted in cells from actively sensitizeddogs (3)- These effects, however, appeatto be relatively small . In our study, withdog mastocytoma cells taken from an an-imal that had not been actively sensitized,we were unable to find any evidence forcholinergic receptors based on radio-ligand binding assays or pharmacologiceffects.
It is apparent that a confusing pictureexists witb regard to the autonomic coM-trol of mast cells. Some of Ihis confre-sion may be attributable to studies thatwere performed on heterogeneous tissueor cell suspensions . While the need forpure mast cell preparations has been ap-preciated, it has proved difficult toachieve thls goal without extensive puri fr-cation procednres that may, in them-selves, be deleterious to the cells- Themost readily available pure mast cellpreparationscome from rodent sources .Most rodent mastocytoma cell lines suf-fer from limitations in their histaminecontent or the releasability of their hista-mine, while the rat peritoneal mast cellappears to differ significantly from thereporled characteristies of human mastcells . Thus a further element of confu-sion may be due to species differences .More recently, Bienenstock and cowork-ers (48) suggested that mast cells mayhave marked differences in morphology,histochemistry, and function within thesame tissue, further compGcating at-tempts to characterize mast cells of eventhe sarne species . If this is the case, it isimportant to have spenfrc knowledgeabout the behavior of mast cells in thelaboratory animals being studied, andeven the behavior of mast cells withinspecific tissues. Knowledge about dosmast cells is relevant because dogs havebeen used widely to study bronchial ro~anivity. The dog mastocytoma cells ca-ami ned in this study are morphologic2llysimtlar to normal dog mast cells and havethe advantage of being readily availablcin a relatively pure form . Our frndingsthat these cells possessed beta-adrMergicreceptors but no alpha-adrenergic, oreholinerg7c receptors was Strengthened byconcordant results from radio6gllnd
binding assays and pharmaeologicstudiect, whereas many previous invtsti-gators have relied on evidence from onesource olily.
AcknowlcdgmenrThe vnters wish to thank Ryckie ttragner/Hall foi her assistance in preparing thi9 mau•uscript, Mary Helen Briseoe for the illussra-tions, and Denise Bcnkek Roben L Stuelds-Andnw D. KeOer• and Lassrence McCabe fortheir technical 25sutaslce.
References
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. Sctmruvla W Comyar~bve nudies of maat ceUr from normel (non-rmmiuiitN) and acvvely uanuud dogs . AgcntsAnioni 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 dogs-3 Appf Physnol 1977 : .f 3 :271-5 .
5 Pa4rersoo R, Tomrla Y, Oh SH, husel.o IM.Pruzarnky 1J . Aespwrory mml cHa and basopnil-md cef]s. I . E+.dence t hat t hq are secreted mto rhebronctual lumen, morphology, degnnubuon andMstan4ne release Chn kap Immunol 1974 :16 :2231-3 .6 Plrsaipa MJ . Calomco L, Gold WM . Morpho-lotPcal and phartrutcologrcal Bnranerizauon oldogmasra :poma cens (ab .arun. An- Rev Respv Dt61992. 125 .63 .l . Taurog JR Mepdov GR Hoot WA
. SinBsruan F13. MeVger H . Nonrytutavc IgE-metlutedrelease of tunamrnr nnd sermmun from munnemauacyloma cells. J Immunol 19"n; 119 :n5'l4.l .
8 . Ph.asps MJ . Gold 9VM, Goctz) FJ . IgE•deperkdens and .onophorr.mduced genaralion ofIru4occieaa by dog Ituxacymme oeLLV 3 Immunol1983; 131 :906r110.9. GocvJ EJ, PhsWps MJ .Oo1d wM- Stimvtvsslxof.fory of the gmerauoa of leukotnrnes try dogsnasRicytoma cxlls. J Eap MM 1933; 15g :'131-7.10 . Murlas C . Nadd rA, Hoberts JM . The mus-cannic nxepwn of airway smooN musdr thencharearnuuon m,.nm. I Apd Phystol 1982,52:10%-91 .I f . Fru 4 OL- Brooks pL . lnununoglabuan E an-bbodws to Pouens atspneured In dogs by vuns vac.inn. AID J vel lia 1983; ~ :ar0-5 .12. Mepp G I lanqla h Fnck OL- Slueldr RL .Gold wM. Auway respoos,venas ro rnhaled anu-gen, luatnmsne and methschohoe 1n tpared,ragwrrd-xos,4ud ao[ts Am Rev Rupsr ps 19E5 :132:292-8.13. Shore PA- Buckhalt[r AR, Cohn VH . Amethod for tne nuoremetnc a5say of hirtarmne in3uec J Phannacol F p Thn 1959; 127 :182-6.14 . 5uaganun RP. An autotnarad<oonnuous-Hwtynem fur the cxrracvoo aod fWtometric analysnof hwaawoe Anal Blochem 191a; 57383-94.15. Caben : RJ• Malbon CC . Clunctewuon of(-)-I'Fllathydroalprcnolol bMndng vtes on niIsted 2t fat cdls. J B,ol Chrm 1979 ; 25a_8903116 M,nneman KP. Hegsuand l.R, Mobnotf P6
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-IMPbannamlogral speoRarS-olbaa-1 and bere .2adreaergic rvoeptnn in rn hean and lung in vrmaMot Phumacol 1979. 16di-H.
1] . Rugg EL. Barnett DB. Nabonki SR . Corzss-tcncr of Beu. and Betar adresoceptors in mam .mUian lung . evtdence frOmdirtR bindmg 51 udies .Mol Pharma<ol 197& 14:996-l0p1 .
19. Coutb SM. Nehnog RE Jr. lanwafa NU.Purification of rat pemmncal mul cells : nccvpa .6Un of IgE-ncCptpn by 16 prevents loss of therectpmrs. J Jmmunol 1980: 12a23g9-15 .
2U. Ca]onico LD, Phillips MJ,!NcDorlald DM,Gold WM . An ullrastrvnutal analyers of dogmasswyroma <clh and nomul rnul celh . Anal Rec
.1985; 212399-10721 . Ftef MJ, Cbcsrown SE, Rmi BR-er of- Im .munological release of hummme Gom dog lungenmpanson of,n vrvo end
22 . MjbrMal 7D. Chesrown SE, Fm Ml . ReetlRR. Winrs SC . Gold WM. E/fecx of heta-atlnnergie stimulation on eapmmenrnl ~nranaphylaus .n mvo. J AlIergyCbn lmrnunol 1983 ;11:62-N1.23. Schlnmer RP. MacGlau .an OW Jr . Peten SP,NaG Inflammetarym[d:etonmdmaRensmsoimleasr from purified human basophils and ma .Wee8s. J Allergy Chn Immunol 1984. 74 <v3-81 .
24 . Koopman WI, Omoge RP. Auuen KF. Im-munoehenumi and broloetc popenies of ®r IgE .ill. Modolanon of the IgE-nsemaled nkax of slowreaettog subtanee of ansphylam hy agenu m-Rurnong ihe level of cycbc 3 .5 ''-adenoasne mono-phospAate / Immunol 19]0; 105 :1096-102 .
25 . Jonmon AR. Moran NC InWbruon of therNW a of bnmmme from ra mau cdls: me effeotuf cold and etlrenerg,e drugson t6e rNeaseof ]usta-minebycompoundGB/80anGanugeu J Pharma .col Eap Tha l9"JR 175 :632ao.26 . Sulhvan Tl . Parker KL . Srevsoo W. ParkerCW. MadWwon o( cy>:bc AMP m panfscd ral mast[eRS I . Ropousef to Pharcnambjae memuwe and
physical stimug- J tmmunol 1975 : 1I4 :1a93-9. stana of anapMlanu from human h.ag, ly. P,ar22 . HolgartST-W-m1ov.CM,LewisRA-'Auatm haattmmt by choghergec and alpha-adreptrgligKF. Ff7erta of prnsmgaodirn D, and theophyWne stimulatson . J F~P Med 19]2; 13&556-7 .on ra, icroul mast cella : d .yicordantz brrwern .n- 38- Alm PE. Bloom G11 What-if asy-ycreaud cellular Icvels of cyclic AMP and activa role of adrenergic medunigms ip hiaumin<uonofpcbcAMP4ryen.dcnAPrurrfnlinaw_J Im- fmm mau «sas' Agcucs Amions 1981, 11 :6p-6&munol 1981 : 121:1536-3 . . 39. Fmtory R . Mworu F, Masmi E, Blandin. g28- Donlon M- Hunr WA-Cavavaa GN, Ka3inee Menaawm PE Modulation of the spuntaneouyM. A eRaraeunration of bua-ad.energic reC<plors huumtne releasc by,aNenergic and chobnapeonccVularandpengranularmembrenaofrAtpen- 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, Masmf29 Mar9uardl DL. MorulskyH], Waurmian SI . E. CTaractersration o(histartineteleaseevoked byRat lung <holmergu rKeptor: chanaennbon and acrtylchuline in isolatxW rat man a1La J Phydolreguletion by roaicosremrdz J APpI Physros 1982 ; 198Q 3012 81-9 3.53-231-46. <I. Rieman JA. Effect of knwn and suspamd30. Maa~ni L-Fanm~ R, gl`and .na P. Hnl Pres- nesunennsmraleo subsunecs and of some nudeo-enm of funaqmng anrve bcta-adrenoccpsors m tsdes on isolated man ttlls. Eapenrmia 19/2;mr mast celh. Conelauon berween (-)PHldr 2gb53-5.Aydroalpnnolol binding and inhibition of htsta- 12 . f(ansai~k W . Adamas a MauliMks Ci,mme release Naunyn Srhmtedcbcrgs Arch PhmFadur< of aKlycholine lo release huumioe fmmmacol 1982 ; }21:p1-0- tal mast calls- Agents Actions 198P 101-3 .31 . MarquardtDL .WassermanSlChamcrenn- 43 EriavecR-Histamdnereleaeefmmmancdsuon of mt masr ceu beuadrencrgsc rxtptor rn rcM- bypllysrologlnRy oaudring subsunca Agmu pa-m8 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.a3d33 . Pcten SP ScFUlman ES. MarGlashan DW Jr- ,5~ Mannaioni PF. ConKlasionbetueea[hnhnerpeSchlermnRP,Ne*haBHH,L:.chtensusnLM . Dis- hsstaminereleaseandpmnudidsnyi-bennlaletFH}pened Ruman lung mast cells : pharmarologrc QNB3bir
.dinginmulceBm<mbrspaAgentfAC) aepecu and comparuon snlh hvman lung lusuc trom 1981 ; 11 :55-9.ftagmasts . Am Rev Resmr Du 198k 126.ID34-9. 45. Bmav 1 a G LonBmore WJ Adrtnag4 and33 . Sahu3manE5 .MacGlashanDWJr,PetcnSP. choboergsregulauondflongsWfaeunfsxrrtrnnScMmmerRP,NmvhallHH . LirhtcmtnnLM Hv- intheisolatedperfusedratlungandiotheelveolarman lung masl erJls : purif.caNuon and charactcr- 1ype 11 cegmculture J B&olChern 1981 :255:66-TLsrannn. 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 mmWa-rng to beta odrenergi<recePtors of mtast cullured uon of secreuon is mdireee Am I Phlstol 198s5a9 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 Knon of lugh-affSniry agon6l, binding tU bela- $choenftld W. ResPOGSr ro i111mnnOlO,Wl orad .energicreceptorson~mm~ceRa .FadProcf98Z chohn[rgsestiauulauomufieplatedmasice8sfrom91:1535 an, gutoea-Piga sad ra,te. CIA sympoaunk Ne .36 . Atlas D, Ha.uki E. Lec-nzta A. Eghty Ihou- [Jrleans . MoetoB ABtryy 1978 ; 14W9-56,sand betaa atlrenorecepton in a S+nglr «B. Nature 48. BitoconocY J, Befya Aq Penroe F, Dm6uq1977 . 268 :1au-6. 1-GoodaneR.Mastce4hetc.rogcndty.denvaaon37 . Kal,nerM .OrangeRP,AussenKF.lmmunp andNncnon .wieRcmpquuontneiotutinalACIo8ical ydcax of Ruramloc and s/ow reaenng suD- lergy Cho lmmunol 19g2 ; 7D:407-12.
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~ cAMP immunocytochemistry provides evidencefor functional VIP receptors in trachea
STEPHEN C. LAZARUS. CAROL B. BASBAUM, PETER J- BARNES.AND WARREN M:GOLDCardiouascalar Reseorch Institute and Deportmenta of Medictne andAnamrny,Untvers+ry of Cohforrirn. San Franc+sco- CaliJomia 94143
LAZARU$. STEPHEN C .. CAROL B . BASRAUM. PETER J-BARNES, AND WARREN M. GOLD . eqAfh imrnunotytoclyrmstrypromdes 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 mtes-tine (S . 1 . Said and V . Mutt . Scirna-e Was), DC 169: 1217-1218-1970), has been identified in postgenglionic autonomic axonsof many ti .c.nrs. VIP has potent regulamry effrots on thefunction of various cell typfs within these ti58ues, ranging fromrelaxation of smooth muscle to ion transport. Recently, VIPhas been implicated in the regulation of mucus secretion in therespiratory tract, a process involvmg releas.e of macromoleculesfrom exocrine cells and transport of ions and water across theairway mucoss . However, because airway glands and mucoseboth consist of mixed cell populattons- it was unclear whichspecific cells contained VIP receptors and contributed to VIP-evoked responses. We idenufied these specific ce0s by usingimmunoeywehemical techniques to monitor concentrationchanges in odenosine 3'-5'-cyclic monophosphate (cAMP). theintracellular compound known to mediate VIP reaponaea . Se-rous and mucous cells of ferret trecheal submucosal glands andcihated and basal cells of dog trsebeal mucose all increusedcAMP in resportse to VIP stimulation . We conclude that thesecell types nossess VIP receptors and thus participate in VIP-stimulated responses. In contrast ferret tracheal epitbe(iumand dog epPtbelial goblet cells sbowed little or no reactivityafter VIP, and tbuswe believe that these rells lack VIP recep-tors .
ion transport : mucus secretion ; cilisted epitbelial cell ; submu-eosal gland: dog: ferret
The biologic effocts of VIP in the respiratory tractappear to occur at sites close to nerve fibera containingimmunoreactive VIP. VIP produces dilatation of bloodvessels in the nnsal mucosa (14) and lung (15), causesrelaxation of guinea pig tracheal smooth muscle in vitro(19, 25, 35, 36), protects against histamine and prosta-glandin Fye-induced hronchocnnstriction in dogs in vivo(24), and increases short-circuit current and ion trans-port in dog tracheal epithelium (I6) . It has also beenreported to both stimulate (18) and inhibit (4) the releaseof radiolabeied macromolecules from ferrM and humantracheal esplants, respectively .
It is believed that VIP-evoked effects are mediated viaadenosine 3',5'-cyclic monopbosphate (cAMP), sinceV IP stimulates adenylate cyclase in a number of systems(2, 3, 7, 9, 11, 22, 27) . In rat intestinal epithelium, VIPnot only stimulates adenylate cyclase but also at.imulatesion transport (6) . binds to specific receptors (21). andstimulates cAMP-dependent protein kinases I1 . 9, 101 .Furthermore, cAMP phosphorylates specific proteins ofrat intestinal membranes associated with ion transport(28) .
Many investigatnts have now identified aod character-ized receptors for VIP on cell membranes (2). However,there is presently no method for detecting functionallycoupled VIP receptors on individual cells in a heteroge-neous tiasue such as the trachea. Even if the various celltypes could be successfully isolated. disaggregation tech-niques might alter the characteristics of receptors. Wehave taken advantage of the physiological coupling be-tween VIP and cAMP and have used intracellular cAMPas a marker for VIP receptor modulation of specific cells .Because of the recent studies at ou> institution by Na-thanson et al . (16) and Peatfield et al . (18) showingphysiological effectsa of VIP in dog and fernt trachea, weexamined tracheal tissue from these species .
niques to demonstrate immunoreactive VIP in specific MErHODScell types. Nerve f5bers containing immunoreactive VIPhave since been demonstrated in the respiratory tract of These esperiments were conducted according to theguinea pig3- rabbits- cats, dogs, and humans (5 . 33 . 34). published gttidelines of the American f'hysiological So-These VIP nerves are distributed around submucosal ctery for the care and use of laboratory animals . Thegland acini in the nasal and tracheobronchia] mucosa, in specific protocols were approved by the Animal Researchthe subepithelial connective tissue of the nose, and Committee of the University of California. San Fran-around blood vessels and airway smooth muscle of both cisco .intra- and eatrapulmonary mrways . Dogs and ferrets were anesthetized with pentobarbital
VASOACTIVE INTESTINAL pErrlnE (VIP), thought to beresponsible for nonadrenergic, noncholinergic neuro-transmission, has been found not only in the gastroin-tcstinal t.rart but in a number of additlonal sitvs in thecentral and peripheral nervous system of many species .Polak et al . (20) frrst used itnmunocytocbernical tech-
sodium (25-35 mg/kg)- Although nerves containing im-munoreactive VIP have been demonstrated in the respi-ratory tract of many ~pecies including dngs, no studieshave described the distribution in ferret trachea- Todemonstrate the presence of ViP-containing nerves, fer-ret tracheae were rewoved, intmersed in frsative, andprocessed for immunocytochemistry to localize VIP (seebelow). Dog and ferret tissues for itnmunocytochemistryto localize cAMP were imme-qed in oxygenated Ham'sF-12 medium containing penicillin/streptomycin (1 U/100 ml). Indomethacin (10-° M) was included to preventstimulation of intrackllular cAMP by cycloosygenaseproducts of arachidonic acid generated as a result of themechanical traume associated with dissection (13) . In-domethacin does not interfere with the subsequent tnaa-imal response of the tissue to direct pbarmacologicalstimulation (29). Bacitracin (1 mg/ml), a protease irthib-itor, was used to prevent breakdown of VIP during in-cubations (16. 18) . After equilibration for 60 min, tissueswere transferred to beakers containing fresh culture me-dium, isoproterenol (10~ M) . VIP (3 x 10-r M), orpmpranolol (10' M), phentolamine 110~ M), atropine(30's M), and tetrodotoain (3 x 10'' M) for 15 min,followed by VIP (3 x 10' M). After 5 min, tissues werefroten in liquid Freon and liquid N~ and processed forcAMP immunocytochemistry .VIP immunoeyrochemiatry. Antiserums to VIP were
obtained from Drs . S. Said and J. Walsh. These antise-mms have been characterized extensively and show neg-ligible or no cross-reactivity with other peptides (S . 23) .Ferret tracheal rings were fixed in 4% parafortnelde-
hyde in 0 .1 M phosphate buffer, pH 74 (2 h, 4°C) thencryoprotected by incubation for 18 b in 30% sucrose .Tissues were then frozen in embedding molds containingOptimal Cutting Temperature compound (OCT) (Lab-Tek Products), Sections (10 pm thick) were made usinga Bright cryostat and inelted onto gelatinized glass slides .Slides were briefly washed in pbosphatr-buffered saline(PBS) to remove OCC and were blocked in PBS contain-ing 3''ro normal goat srrum (NGS. Antibodies) and 0 .3%7titon. VIP antiserum was diluted 1 :1,000 with PBScontaining 1% NGS and 0 .3% Triton . The diluted senunwas applied to the seccfons for 48 h at 4'C . Sections werewashed in PBS containing 1% NGS and 0 .3% Triton.They were then blocked as above and incubated withgoat anti-rabbit immdmoglobulin (Ig) G fluorescein iso-thiocyanate (Cappel Laboratoriea ; 1 :40) for 30 tnin atroom temperature. After that they were washed in PBSand covered with glycerin-PBS (3:1) and glass cover afips .Slides were viewed with a Zeiss fluorescence nvcroacope .The absorption control was prepared as descnbed above,e:cept that anti-VIP serum waa incubated with 50 ug/ml pure synthetic VIP (Peninsula Laboratories) for 18 hand was centrifuged before use for stainingP cAMP imntunocytochemiacry
. For cAMP immunocy-toebernintry, ferret tracheae were divided into rings 1 .5cm ia length. To obtain samplee of dog tracheal epitbe-lium, the posterior membranaus portion of trachea wasdissected free of its 4artilaginous attachments and wasdivided into pieces 1 .5 cm in length. Froren tissues wereembedded in OCT, equilibrated in a cryostat at -20°C,
and sectioned at thicknesses of 6-8 um . Cryoatatwere mounted on gelatinized glass slidea . These sectiewere stained for cAMP using the unlabeled anenzyme (horseradish perozidase-antiborseradish pero>Ddase. PAP) method (30, 32), as we have describedviously (12). Anti-cAMP antiserum was producedimmunizing New Zealand White rabbits with 2'-O-mon-osuccinyl-cAMP (Sigma Chemical) conjugated to humaasetun albumin, Fraction V (US Biochemical)- Tlrefraction of this antiserum was incubated for 12 h with;human serum albutnin (I%), centrifuged at 10,000g,anpassed through a 22-Nm nitmcellulose filter (Millipore)Testing of this antiserum in a radioimmunoassay (31)and by Ouchterlony agar double diffusion (17) demoastrared high affinity for cAMP without significant cross-reactivity with other nucleotides or with the carrier pre-tein, human setum albumin . Cryostat sections werewashed for 10 m.in at room temperature in PBS (pH 7.4)then incubated for 30 min with 3% normal goat aetuaLThe slides were blotted to remove excess goat serum, andtissues were incubated at 4°C for iS h with anti-cAMPantiserum diluted (1 :800) in PBS containing 1% NGS .After thorough washing with PBS containing 1% NGB ;sections were incubated for 30 min with goat anti-tabIgG (Cappel l.eboratoriea ; 1:20), washed again, andcubated for 30 tnin with horseradish peroaidaesanhorseradish peroridase complex (Cappel Lsboratori1 :300). Sections were washed with PBStris(hydrnsymetbyl)aminomethane buffer (0 .05 M.7.6) and reacted with a mixture of diaminobenzi(Sigma Chemical; 0.05'k) and hydrogen peroxide (0for 5 min- Slides were examined using convenbright-field miernacopy. Staining controls includedsubstitution of preimmune for immune serum, oof first or second antiserum, and liquid phase absorpttof anti-cAMP serum with cAMP . No sr, iniug waswith any controls. Wben necessary to confirm thetity of immtmoreactive cell types, aome tissuescounterstained with Alcian blue-periodic acid-Schifrdescribed previously (12)-
REBVLTS
Studies were performed in duplicate on tiesueatained from two ferrets and two dogs. Immunical localization of VIP revealed nerve fibers conimmunoeeactive VIP surrounding submucoeal g]andeferret trachea (Fig . 1) . In ferret trachea, incubationVIP (3 x 10' M) increased immunoreactiveboth serous and mutous cells in submucosal glands2). The iucrease in staining aftrr VIP was comwith that produced by isoproterenol (10-a M) andnot inhibited wheu tissues were preincubated with,pranolol (10,M).phentolamine (10' M), atropine (1M), and tetrodotoxin (3 x 10-' M) . In contrastpronounced effect on tracheal submucoeal glanda,produced little if any incteease in immunoreactivein ciliated epithelial celle of the ferret trachea In thetrachea, incubation with VIP produced a marked inin immunoreactive cAMP in ciliated and baeal oelhrthe epithelium, and, as in the ferret, this incre,ee
P3G . I . A' Ouoreatence pbotoovcrog}-aph of ferret tracbesl eubmu .rnssl glsnde sratned mtb annVI}' eePUm, ehoaemg unmunorracaveVIP rn ner,res Lo~/ surroundtng submucosal glends (SG)- H : sen+oonaned mth ennVIP aervm preatlsarbed mth pure VIP (50 rg/ml)_Bar. 40 emm
not blocked by propranolol, pheninlam3ne, atropine, andtetrodototin (Fig . 3). Goblet cells in the dog tracheacontained little Immunoreactive cAMP under baaal con-ditions and did not respond to exogenous VIP-
OISCUSSIOT:
In this study. we utilized iminunocyt.ochemical technlquea to demonatrateVIP in ferret tracheal tissues and
to localize cAMP wlthin specific cells in these tissues .We have demonstrated immunoreactive nerves near fer-ret tracheal glande and have localized cAMP in specificcells in theseti»ues . The submucoaal glands of ferretsand the epithelium of dog tratheae have heen shownpreviously to secrete sulfated macromolecules and chlo-nde, respectively, in response to VIP (16, 18) . We nowreport tbat serous and mucous gland cells in the fenettrachea and ciliated epithehal 'cells in the dog tracheashow elevated cAMP levels in response to VIP . Thusimmunocytochemtatry enables us to attnbute specificfunctions to specific cells. Because the rombrnation ofpropranolol, phentnlamine, atropine, and tetvodotoxindid not mhlbit the mcrease in cAMP produced by VIP,
C117
ne. 2. Bnght-6eld phnrnm.crogrepho of ferret trubeal eubmucoealglands etawed for immuooreetu'r tM1P- A : cuotrol tusue- B
: uoprotereool (10'• M). C. VlP (3 x]0-' M). D: VIP (3 x 10-' M) .proprmo)ol (10~ M) + phentelamme 00-~7) . euopme 110~ M) +iewJuwun 13 x 10'' M). Note thnt VIP 'u .ervnue.l nutuunureeetiwcAMP m aubmueoeal glend mW oad lhat ttus mereme m" not in .h.bned by bloc4ere restad. Boe, 50,m.
rlo . 3 . Bnght5eld pbotomtcrogrephe of dog tracbeel epitheliumslaad for unmunoreective cAMP A' controL & rsoptoterenol (10-M) C: VIP (3 x 10-' M) . D- VIP 13 x 10" M 1+ propnnolot (10-' M)a Pbenwlam .oe ( l0- M) + stropme (10 ' '-N ) atetrodorna.n (3 x 10-M) Nole that ViP increesed unmunortact .ve cAMP in eprtSrlium andwnt tbu mereaee w not mhubited by blochera teeted Bos, 30 ..m
we believe that VIP was acting directly by way of apecifrcV IP receptors on individual secret .ory target cella, ratherthan by causing release of other mediators or activationof neural pathways .
The dietribution of innaunoreactive VIP in theee ferrettracheal tissues waa similar In that previously describedin studies of other respiratory tract tissues (5, 33, 34) .Nerves containing VIP bavebeen identified around bron-chial submucosal glands of the cat, dog, rabbit, and
human: around submucosal glands in the 6asa1 mucossand trachea of the human, rabbit, and catl and close tothe epithelium in the subepitbelial connective tissue ofthe human nose and the cat and rabbit broncbus . In ourstudies, the nerves containing immunoreaetive V IP ter-minated sufficiently close to the cells activated by esog-enous VIP to suggest that endogenously released VIPmight easily reach these sites by diffusion .
The Increase in cAMP staining ohserved in tissuesstimulated with VIP was qualitatively siinilar to thatproduced by isoproterenol (10-e M) . Although in thesestudies no attempt was made to quantitate the increasein cAMP observed comparison with a ptevious study(12) may allow a semiquantitative estimate . In thatstudy, tissues incubated with isoproterenol (10-6 M) in-creased total tissue levels of cAMP approximately four-fold and could be stained for cAMP by the PAP methodusing anti-cAMP antiserums at a dilution of 1 :1,000. Inthe current study . immunoreactive cAMP in stimulatedtissue was visualized using the same antiserum diluted1 :800, suggesting that cAMP levels may have been inapproximately the same range as in the previous study.
Recent studies have clearly established d role for VIPin the regulation of mucus secretion in the respiratorytract. However, even the newly developed techniques fordetection of VIP receptors do not permit tlssessment ofreceptor function on individual cells in a beterogeneoustissue such as the trachea . By taking advantage of thephysiological coupling between V1P and cAMP, immu-nocytochemical localization of cAMP permits identifi-cation of cells activated by VIP and serves as a markerfor the presence of VIP receptors on these cells. Ourresults are consistent with studies which demonstratethat V IP stimulates ion transport in dog tracheal epithe-liuen (16) and release of macromolecules from ferrettrachea (18), and suggest that ciliated epithelial ceUs areinvolved in the former and that both serous and mucoussubmucosal gland cells are involved in the latter. Ourresults also support the observation that VIP does notstimulate ion transport in ferret trachea (18) .
We thsn4 Dts Samr Smd endJobn Walsh for their generoue ginaof snti-VlP anueenmu
This study wee supporud .n pan by National Heart, Lung, andBlood Institute Pmgam prolu-t Grent HL24138 end by Grant 1327from the Council for Tobacco Reee.erchUSA S. C . lararue aae thetenpieat of Natioeal Hean, Ltmg, and Blood lnenmrae New Inveeu•getor Award FIIr29877 and grmte 6nm the Ftance S . North, Sr.,Found.uon and the Strobel Medical Research Flmd of the AmencanLung Awaorieuon of San Francisco. P . J . Bamee wes supported by aMedical Heseerch Counrd TYaveBing FeBome6rp .
Current addrese of P . J- Henoes The Candouroranc lneutvte.London SW36HP- UK.
The raaulu of tha amdy were preeented m prelimwery form et theAnnual Meetmg of the Amenean Thoraoc Society. May 10. 1983.Kanses Gry. MO.
Received 31 August 1984 : atzwpt4d in final form 11 Pebrnary 1986 .
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7. KrrAUUaA~ S . . Y . IsanultA, AND S. 1. SA1D . EBea of VIP, pb,norybenmmrne• and predaieolooe on cyclic nucleotide mot4ar ofieulaud guinea•p.g lung and trachea . 6ur. J. Phornwcut 67: 2W223.1988.
& KRws, G . J- . J- H . W AteN . S. G . MoaAwssr. Areu J . S . Foaaawta .IatractaDle diarrhea: int<eunal per[upion scudiee and pleema VIPtenerotreuons in peuenta witL paocratic cholera syndrmne .ad®rrrptiuoue ingesnon of Isaativee and diuretiee . Am . J. D'y. Di.22 : 2ga292, 1977 .
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10. Lt9neifm . M . . J . C . Paxero . B. AaqWVOn. C. DuPOnr, D. ytNBon HoA. AND G. Roes® .ln . lnterafuon of vaeuec-uve iat.atmdpepnde a.th ieoleted mteetmel epahelisl eells from ret- 2 Chsr-aeterlraaoo and evucnnal requveml•ot of the simlulewry eBectof vmoactive mteatanal prptide on producuon of odeaoeiae 3'S'-monophoephate. Eur J . B.cchem 93;239-24g, 1979-
11 . Iwsuarae, M . .. M- Roussrr, C . BO1ssAaD. G. C1mvAt®t A.ZwmuuM, AND G. RUaae]JN . Vpoe<tive mtacunul peplidx apotem eumWwmr of adenoeme 3' :5'-cyclic monophoephate amt•malsaon m gm canlnome cell linn m cvltwe . P~ NatL Aoa1Scc USA 75: 2T72-2775, 1978.
12 . Wzerrus . S . C., C. 8. SAsa.uu. ANq W . M. GOLD. Lonli:atiouof cAhff u1 dog and cat nschen eHecte of S-edtaner®c ageaistaAm J PhyewL 241 tCeR PhpioL 161 :,C327-C334 . 1984.
13 . L.aneus. S. G. C. B . BASaAum . AND W . M. GOLD . Pmstagleodinand ~ntmceWiler cyclic AMP in reepimtory secretory callw An .Rev . Rz.pv. Du . 130. 262-266 . 198i.
14 . MAta . L, . P, SONDLan, AND R Uoauwn . Effecu of vanacta.mtmmel polypeptide (VIPI on rnmuace and eapecitana .roewbin the nsesl mucoeo . Arm OcobryngoL tStockA .l 90: 3M-808, 1999.
15. MaASAD. M . . S . I . SAm. AraD A . L. HrnrAN . Vanoecuv<intenitWpepude /VIPI dilatee pulmonery vessels in anesthetized eets tAbbetrseU . C4n Rra 28: 89M, 19g0.
16 . NAreAecsoN. 1.,J. H. Wmmcora[. AND P . J . BARNES. 69ettdvasoattrve mteaetnal pepude on ,on trmsport ecroea dog uachedepitbeBum. J Appl. PhyamL 55: 18f4-IBI8. 1963
17 . Oucmwrt.onr, 0 . Diff.loo-io-gel nmthode for immunelopeJcnelyua . Im Prog.ea. m ARergy. Soel Harger. 1968 . voL S . p . 1-7b .
18. PaArPmtu, A . C . P . J. BAaxas. C. Baxrc~ J- A. N .Dal ., ANDB- DAVIS . Vaaeetteve intenunal pepade stimulates tracheal ab-mumeal gland aenetlon m ferret A ... Reo. Reapv Du l2& 8B-93, 1983
19. Pam P. J . . S . 1 . SAm, AND J . R VANt Bffecte on emooth mmdrpieparemne of amdeatifial vaaWecve pepridee from inta .4ne andlung. Nmure Innd 22S 1144-1118.197D.
20 . Pous, J. M. . A. G . E Pacase, J . C. GARAw. AND S. R Bl.oar.CaBNer locafvetion uf a vaeoacnve mtritiml peptide m the m .m-loelim and evien psuptateattual tmec Car 15: 72o-724, 1974.
21 . Patsro. J . C. . M . LASunrtts: AND G . Rosela.m. luteractien ofvaeoecnva mleeanel prpude wtth ieolaad inteetinal epitheBal otlbfrom ra6 1 . Cberartenaauoa Quaotiunve aepecte aud acmcrort .ln~uirtmenb of bmdlvg utea. Eur. J. RiacAern. 96 : 229-'Li7. 197A
22 RnaaaaacNt, P, T. P . CONLON. AND J . D. GMUNVt. Iau~etVOaof poteine vesoective mteetmel pepude wnb diapera¢d psnme .ltc
acdner eells from tbe (oiba pi6 etruodval reGuire/nenta tor effect,of vaeoectivt mleetiael ixptide snd seoetion on cellular ederueine3' :5' .monophoapNte . d BwL CAem 251 : 4635-46i39. 1976
23. SAiD . S. I . Vaeoective intat.uul polypepude (VIP) ae a mediatorof the wetery durrhen eyndrome. Wor)d J Surg. 3: 559-563 . 1979 .
24 . SAOD. 5. 1 ., A . Gauwel: uvo N. HAUA. 8roncboiiiletor effect ofVW in vivo : proteMton against broncbocvnetric#ion induced bybutamine or proetagleedio Ft. . In : Voroanur InuannoE Pepnde,edited by S. L Said New York: Raven, 1982.
25 . SAID, S . L, S. RTTMfURA, T. YOSHIDA. J. Pnrartrlr. .wu L D.HDt.DSrw . Humoral raotrol of euweye. Ann NY Acad Sci 22t :10 ~1- 1 Lf, 1974 .
26 . SuD, S. 1-, u+D V . Mttlm . Polypeptade with broad biolugirel activ .ny iaoletiao from rmall inrertinr- Sc4oce WoeA DC 169 : 1217-121& 1970.
Y) . Sctiw .urrz, C . J . D . V. KlMemtc, H. 8. Sn~. M. Ptel.p . cr+nS. SAID. VLa4acGve intGft1O91 peptide ltimulatjon of edenyllra
and eclive elemolyte aetretion in :nteaunel muenaa J.Ctirt I,umr 54: 53C.544, 1974 .
2B. SHyA7z. L . J . . D . V. glneeeaD, AND K . A . C .rrabu. Pboephntyle-nory of specific at mtatmal mtcrovillue and bmal•Iaterel mem•brepe prmrine by cyclic oucleotida . Gaer .Dentembp76: 293-298.1979 .
29 . Sntrrw, P- L-, M- J . WeESH. J . S . Sroer, Ar+D R. A. Pntazeu_Chloride secrttion by cenme trechea) epitbelYUm . I. Role of intre•telluler cAMP levele. J Memhr Biot 70 : 217-226. 1962.
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30. $~ert, A . L, S. H . ONG . an9 H . J. W®ww- Cyclic eueleutid ._ immunochemietry. In : Aduancn m Cyrlic Nuc4otde Reuarch .
edited by P . Greengerd end G . A Robieon. New York: Reren, V o17, 1976.
31. STSINBn. A . L, C. W. PutRElt. r,ND D. M . K.vrna Radioimmu-noeaey for cycEc nucleotidea 1 . Prepetauon of epubodiee andiodineted cyclic nucleoudea. J. Biot Ctrem 247 : 11f16-1113, 1972.
32. SrrnwaexGea, L A, P . H. HAnpv. Jm . J . J . CDcws, .wD H- C.MBYP.x. The unlabeled antibody enryme method of immunohino-chendetry: preparation and properua of soluble entisen-enmbadyeomplea (boreeradieh perotidax•anuhnreexdieh peroad.ee) er•dits use in idenciSauon of apirochetee. J . Hietochern Cyto-Aen. 18:315--333, 1970 .
33 . UDDrawrv . R., J . Ar_UMerg . O . Derrsem, R . HArtwrrsnx. AND P.Surrut .crt . Oecurrence and dietdbution of VIP nervee in the nualmucone and ereehtobronchial well. Acro Orolnry.gol (Stor4A .) 66~443i48. 1978 .
34. UDOHAN. R, AND F. SUNDLM VBeOtetnY intestinal pOtypepddeDerva in humen upper respiratory trecr_ ORL 41 : 221-226, 1979.
35. VstrucopAww, C . S, S. 1 . S .aD, AND J . M . Dx .zan 8ffeeta ofvaeoertive intestinal peptide (VIP) and isuprowrenol (ISO) on theguinea pig tracheal pouch relsaatroo induced by vegel and eympe•thetic taunk sfimulntioa (Atewacs) . P/rymoLpist 2N4) : 83. 1961 .
36. W t,sssrWArv, M . A . . R . L GtetlnN, AND P. E. Mwtu . Comperetivein vitro trechesl relssent egeru ofpoxme pod hen VIP. In :Va.oortwp Inteatind Pepude, edited by S . I . Sud. Ne. York :Revet4 1984 p. 177-154 .
Copyright @ERS Journals Ltd 1995Eur Respir J, 1995, a, 1458-1464 European Respiratory JournalPnnted in UK - all rights reserved ISSN 0903 - 1936
(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•
IF -
riboiing :phosemb
celi
Hicle c
NEF
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 inflam-mation in the respiratory tract . It is of interest to determine which respiratory resi-dent cells express NEP .
Trachea and bronchi from seven nonsmoking, nonasthmatic subjects were exam-ined. NEP messenger ribonucleic acid (mRNA) was characterized by Northern blothybridization of cultured human tracheobronchial epithelial and smooth musclecells, and reverse traascriptase-polymerase chain reaction (RT-PCR) in trachea andbronchi. ln situ hybridization with biotin- and 35S-labelled antisense complemen-tary ribonucleic acid (cRNA) probes was used to determine the distribution of NEPmRNA in human bronchial mucosa. NEP-immunoreactive material was detectedusing MEK10 murine monoclonal antibodies and the immunogold method with sil-ver enhancement.NEP mRNA was 4.5 kb in size in the cultured human smooth muscle and epithe-
lial cells by Northern blot analysis . No evidence was found by RT-PCR for trun-cated, alternatively spliced NEP mRNAs, such as del exon 16 or del exons 5-18 inhuman bronchus. NEP mRNA was detected by in situ hybridization in epithelialcells, submucosal glands, bronchial smooth muscle and endothelium . NEP-immuno-reactive material was identified in the epithelium, submucosal glands, bronchialsmooth muscle, and endothelium, demonstrating an excellent correlation betweenthe distribution of NEP mRNA and the cell surface protein . NEP mRNA andimmunoreactive material were excluded from epithelial goblet cell and submuco-sal gland mucous cell vacuoles.
We conclude that the various sites of NEP protein and mRNA expression corre-late with the locations of peptide receptors and NEP enzyme function, and are con-sistent with the hypothesis that NEP may regulate peptide-induced inflammation inhuman bronchi.Eur Respir J., 1995, 8, 1458-1464.
Neutral endopeptidase (NEP) is a 749 amino acid, zinc-containing, membrane-bound enzyme, which may playa key role in regulating peptide-induced inflammatoryevents [1-3] . NEP, also known as E.C.3 .4.24.11, enkephali-nase, common acute lymphoblastic leukaemia antigen(CALLA), CD10, and gplOO, has been cloned fromhuman, rat and rabbit tissues [4-9]. Functional studiesindicate that NEP activity is present on epithelial, glan-dular, smooth muscle and vascular cells which possesspeptide receptors [1, 2] . Loss of NEP activity may sig-nificantly contribute to the hypersecretion, vascular per-meability, bronchoconstriction and other pathologicalchanges seen in respiratory inflammation by permittingunopposed, prolonged actions of inflammatory peptides,such as tachykinins and bradykinin .
In the present study, in situ hybridization was used todetermine the distribution of NEP gene expression in
I •Division of Rheumatology, Immunologyand Allergy, Georgetown Medical Center,Wacttington, DC, USA. •'Ikpt of Otolaryo-gology, Nippon Medical School, Tokyo,Japan. 'Dept of Thorac:ic Medicane, NationalHeart and Lung Institute, London, UK .-Department of Respiratory Medicine . St .Banholotnew's Hospital, Lordon, UK. 'Deqof Pulmonary Medicine, SL Thomas Hospi-tal, London, UK. "Asthma and AllergyInstitute, Washington Hospital Center,Washington. DC, USA. loivision ofImmunology . Hospital for Sick Children,Toronto, Ontario, Canada
Correspondence : J .N . Baraniuk, Divisionof Rheumatology, Immunology and AllergyGL-020 Gorman Building, GeorgetownUniversity Medical Center, Washington,
The research was supported by Schering-Plough Research, Kenilworth, NJ, USA,Boehringer-Ingelheim, Bracknell, UK, andthe National Asthma Campaign, London,UK. JNB has been awarded the EdwardLivingston Trudeau Scholar Award by theAmerican Lmg Association, and is a TobaccoCouncil for Research Scholar. -
human bronchial mucosa, whilst the identity of NEP mes-senger ribonucleic acid (mRNA) was confirmed byNorthern blotting . NEP immunoreactive material wasdetected by immunohistochemistry .
Human Tissue
Methods
Human trachea and large, cartilaginous bronchi wereobtained from four organ specimens collected at hearttransplantation and at autopsy of three victims of motorvehicle accidents. None of the subjects were known tobe smokers or asthmatic . Tissue was transported in 4°CKrebs solution, and : 1) frozen in liquid nitrogen for
ribonucleic acid (RNA) extraction and cryostat section-ing; and 2) fixed in 4% paraformaldehyde in pH 7 .4phosphate buffered saline (PBS) for 4 h at 4°C beforeembedding in paraffin.
Cell culture
Human bronchial epithelial cells [10] and smooth mus-cle cells [11] were cultured as described previously .
NEP complementary ribonucleic acid (cRNA)
An NEP insert [7] coding for bases 642 to 2223 (1,581bases) was obtained by Eco RI digestion of human NEPcomplementary deoxyribonucleic acid (cDNA) clones[6,7] and inserted into a M13+ Bluescript plasmid (Strata-gene, San Diego, CA, USA) . This insert codes for theregion stretching from amino acid 210 in exon 8 to aminoacid 735 in exon 24 [6, 81 .
Antisense NEP cRNA probes for Northern blot analy-sis and in situ hybridization were prepared by lineariz-ing the plasmid with Ava II and transcribing with RNApolymerase T3. The antisense cRNA coded for the re-gion from base 2029 to 2223 . Sense cRNA probes wereprepared by linearizing with Pvu l1 and transcribingwith RNA polymerase T7 . The sense cRNA coded forthe region between bases 642 and 1099 . cRNA probesfor Northern blots were labelled by incorporating 32P-uridine triphosphate (UTP) (Amersham, Inc ., Amersham,UK) during transcription. Probes for in situ hybridiza-tion were labelled by incorporation of 35S-UTP (Amer-sham, Inc ., Amersham, UK) for radioactive detection, orbiotin-5-UTP (Sigma, Poole, UK) for nonradioactivedetection. Probes were purified from unincorporatednucleotides using G-50 Sephadex columns eluted withpH 8, 10 mM Tris (hydmxymethyl) aminomethane (TRIS),l mM ethylenediamine tetra-acetic acid (EDTA), 0 .1%sodium dodecyl sulphate (SDS) . The yield of biotin-5-UTP-labelled probe was determined from the OD26o,(optical density at 260 nm) .
Northern blots
RNA samples for Northern blot analysis were preparedfrom cultured cells scraped from culture flasks using theacid, guanidinium thiocyanate, phenol-chloroform extrac-tion method [12] . RNA integrity was confirmed by theappearance of the 28S and 18S recombinant ribonucleicacid (rRNA) bands [13] after denaturing 1% agarose gelelectrophoresis in TAE (Tris-Acetic acid-EDTA) . TheRNA was transferred from the gel to Hybond nylonfilters (Amersham, UK) by capillary action with 20xstandard sodium citrate (SSC), and fixed by ultravioletlight (UV) for 4 min. The filters were prehybridized in50% formamide, SxSSC, 0.1% SDS, 5 mM EDTA, 100pgmL-1 denatured salmon sperm DNA, 0.5 pg•mL-t yeasttotal RNA, 5x Denhardt's solution for 16 h at 50°C . 32P-antisense NEP cRNA probe (106 cpm per 10 cmz in I
1459
mL hybridization buffer) was added, incubated 16 h at50°C, and washed in 0.5xSSC at 55°C before exposureto radiographic film . The filter was stripped in 50% for-mamide, pH 6.5, 10 mM Na phosphate for 60 min at65°C. After prehybridization, the filter was hybridizedwith sense NEP cRNA and washed using the same con-ditions .
In situ hybridization
Cryostat tissue sections (10 pm) from seven subjectswere thaw mounted onto gelatin-coated slides, allowedto dry for 12 h at 37°C, and then post-fixed in freshlyprepared 4% paraformaldehyde in pH 7 .4, 0.1 M PBSfor 30 min . Paraffin sections were dewaxed and hydrat-ed in PBS for 5 min. Cryostat and paraffin sections weresubsequently treated in identical fashion [14, 15] . Sectionswere permeabilized in 0.3% Triton X-100 in PBS for 10min and then proteinase K (l pg mL'i) in 0.1 M TRIS,50 mM EDTA for 30 min at 37°C . After treatment in4% paraformaldehyde in PBS for 5 min, nonspecificbinding sites were blocked in 0.25% acetic anhydride,0.1 M triethanolamine for 10 min . Slides were prehy-bridized in 0 .3 M NaCl, 30 mM Na citrate (2xSSC), 50%formamide for 30 min at 50°C . Slides were hybridizedfor 16 h at 50°C . 35S-cRNA probe in hybridization bufferwas added at 106 cpm per slide . Biotin-cRNA probeswere added at 200 ng•mL-J . After hybridization, slideswere washed in 4xSSC. Unincorporated, single-strand-ed cRNAs were degraded in 20 pg mL I ribonuclease(RNase) A, 0 .5 M NaC1. 10 mM TRIS, and I mM EDTAfor 30 min at 42°C. Washing was continued in decreas-ing concentrations of SSC to 0 .2xSSC at 50'C .
35S-labelled slides were dehydrated in 70% ethanol,air dried, and coated with Ilford K-5 photographic emul-sion melted at 42°C . Coated slides were exposed for14-21 days, developed in Kodak reagents, and stainedwith haematoxylin .
Biotin-labelled slides were immersed in Lugol's iodinefor 2 min, decolourized in 2 .5% Na thiosulphate, washedin PBS [15, 16], and nonspecific binding sites blockedwith 0.8% bovine serum albumin (BSA), 0 .1% gelatin,5% nonimmune goat serum, 2 mlvl Na azide in PBS for1 h at room temperature. Anti-biotin goat serum labelledwith I nm colloidal gold particles (Amersham, Amersham,UK) diluted 1/10 with blocking solution was added, andthe slides incubated overnight at 4°C . Slides were washedtwice for 5 min in PBS followed by distilled water . Silverenhancing solution (Amersham, Amersham, UK) wasadded to pairs of sense and antisense slides and staindevelopment observed under darkfield illumination . Slideswere washed in water for 5 min, 2.5% Na thiosulphatefor 3 min, and dehydrated .
Primers
NEP primers coding for mRNA splice sites wereidentified from published cDNA sequences [5, 6, 8] andwere synthesized by the Lombardi Cancer Center Core
http://legacy.library.ucsf.edu/tid/tyg16d00/pdf
1460 J.N. BARANIUK
Laboratory of Georgetown University, Washington, DC .NEP primers were chosen to identify mRNAs contain-ing deletions of exon 16 (del 16) [17] and deletion ofexons 5-18 (del 5-18) [18]. A single antisense primerwas used that coded for the exon 19-exon 20 splice site(5'GTTTCCGCrsPUCECATTGTCATCGAA) . When pairedwith a sense primer coding for the exon 3-exon 4 splicesite (5'ATGCAACCTACGATGSPWMATGGTAT), sev-eral possible NEP mRNA RT-PCR products could begenerated, including one from full length mRNA (1,641nucleotides), one for del 16 (1,563 nucleotides), and onefor del5-18 (177 nucleotides) . When paired with a senseprimer coding for the exon 14-exon 15 splice site(5'AGTAAACATGTC,/sPUCE~GTCGAGGAT), full lengthNEP mRNA would generate a RT-PCR product 477nucleotides long, and 399 nucleotides long for the del16 variant. Deletion of exons 5-18 would generate noproduct. The annealing temperatures (55°C) were cal-culated according to MenaKOnt and WAFn. [19] .
(3-actin [20, 21 J primers were purchased from Clontech(Palo Alto, CA, USA) . They generated an RT-PCR pro-duct 661 bases long from mRNA, but 867 bases longfrom genomic DNA because of the presence of a shortintron .
Reverse transcriptase-polymerase chain reaction
RNA (5 pg), reverse transcriptase/antisense primers,and Perkin Elmer RT-PCR reagents (Norwalk, CN, USA)were mixed according to manufacturer's recommenda-tions at 4°C in a Perkin Elmer thermocycler . MineralMicroliter oil (70 pL) was added, and then the temper-ature increased to 42°C to permit annealing of the RT/antisense primer to specific mRNA sequences . After 60min at 42°C, the solution was denatured at 99°C for 5min, and then cooled to 60°C . Perkin Elmer taq, otherPCR reagents, and sense primers were preheated to 60°Cand added to each reversely transcribed tube . In thisway, "Hot-Start" conditions that reduce nonspecific pri-ming were produced . To permit efficient annealing andextension for this first cycle of PCR, the temperature wasthen appropriately adjusted to the annealing temperature(55°C) [19], and maintained for 5 min. This was fol-lowed by 2 min at 70°C. Then the thermocycler wasset to cycle for a total of 45 cycles at 94°C for I min,55°C for I min, and 70°C for 1 min . In preliminaryexperiments, it was found that most samples generatedpositive bands after 35 cycles, but some samples withminimal RNA required 45 cycles . Since the additional10 cycles did not cause generation of superfluous bands,45 cycles was adopted as a standard method .PCR products were mixed with TRIS-borate-EDTA
(TBE) loading buffer and run in 0.5xIBE on 2% agarose(FMC, Rockland, ME, USA) 1 .5 h at 120 V. Clon-Tech(Palo Alto, CA, USA) DNA molecular weight stan-dards (1353, 1078, 872, 603, 310, 281, 271, 234, 194,118 and 72 nucleotides) were run on each gel. Gelswere stained with ethidium bromide, bands visualized byUV-fluorescence, and photographed with Polaroid 667film .
Immunohistochemistrv
Paraffin embedded sections from seven human largebronchi were dewaxed, rehydrated in PBS, and incubat-ed in blocking solution [15] . Murine immunoglobulinG(IgG) monoclonal antibody to NEP (MEKIO, courtesyof S. Shak, Genentech, South San Francisco, CA, USA)diluted 1 :200 with blocking solution was aliquoted ontothe slides and incubated for 20 h at 4°C . After wash-ing in PBS, the monoclonal antibody was detected bythe immunogold method with silver enhancement [15] .Nonspecific staining was determined by preadsorption ofthe antibody with 200 pg•mL-t recombinant human NEP(courtesy of S . Shak, Genentech, South San Francisco,CA, USA) .
Results
Northern blot analysis
Cultured human tracheal smooth muscle cells and cul-tured human bronchial epithelial cells contained a singleNEP mRNA band at 4 .5 kb (fig . 1) .
RT-PCR
A single NEP mRNA band was identified indicatingthe presence of full length mRNA coded by exons 4-20(fig . 2). No variant NEP mRNA could be amplified,suggesting that truncated mRNA (del exon 16 or delexons 5-18) were not present in human tracheobronchialtissues from nonasthmatic, nonsmoking subjects .
In situ hybridization
The biotin-labelled antisense NEP cRNA probe detect-ed NEP mRNA in epithelial cells and submucosal glands(fig . 3) . Staining was less intense, but still present, overbronchial smooth muscle and endothelial cells of sub-epithelial capillaries/postcapillary venules and deeper,larger venous vessels . This may suggest that the NEPmRNA concentration was lower in smooth muscle andendothelial cells than epithelium and glands .
20 10 5Smooth Muscle
20 10 5Epithelium
Fig. 1 . - Northem blot of 32P-antisense NEP cRNA binding to 20,10 and 5 pg of total RNA obtained from cultured human smooth mus-cle [111 and epithelial cells [ 10] . The bands are at 4 .5 kb . The largeaevowhead identifies the location of the 28S rRNA band (4 .8 kb), andthe small arrowhead the 18S rRNA band (1 .8 kb) [131 . NEP: neu-tral endopeplidssc ; RNA: ribonucleic acid; cRNA: complementaryRNA; rRNA: recombinant RNA.
ic
Ft
e
E
t
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NEP mRNA IN HUMAN BRONCHI
A B C
r r. .. ' ...
AY
Y
Fig . 2 . - Reverse transcriptase-polmeritation chain reaction (RT-PCR) for NEP mRNA in human bronchus total RNA . The eaons19-20 splice site antisense (RT) primer and e.waa 14-15 splice sitesense primer generated a band at 477 nucleolides corresponding to thefull length mRNA coded from the exons 14-20 (t .anes A) . No trun-cated variant NEP mRNAs were found. Lane B shows markers at1353 (top), 1078, 872, 603 and 310 (bottom) nucleotides . Lane Cshows the ~-actin mRNA RT-PCR product (661 nucleotides) . LanesA: NEP; B: markers ; C: (S-actin. mRNA: messenger ribonucle-ic acid . For further abbreviations see legend to figure 1 .
0
eA
A
100Nm r.
Fig . 3 . - Nonradioactive in situ hybtidiTation of NEP in human• bronchus . A) Biotin-labelled antisense NEP mRNA was detected bythe immunogold method with silver enhancement as the intense blackstain over the epithelium (e) and submucosal glands (g) . Slightly lessintense staining was detected over bronchial smooth muscle (m),endothelium of subepithelial capillarieslpost-capillary venules and larg-er vessels (arrowheads), and chondrocytes (c) . B) Biotin-labelled senseNEP eRNA did not bind to the sections . For abbreviations see leg-ends to figures I and 2 . (Internal scale bar=100 µm) .
This distribution of NEP mRNA-containing cells wasconfirmed using the 35S-labelled cRNA probes . Theepithelium, vessels, smooth muscle, and glands containedNEP mRNA. The epithelium had a high silver graindensity. The nuclei of basal and other cells, and thevacuoles of goblet cells were devoid of NEP mRNA(fig . 4) . The serous cells of submucosal glands werepositive (fig. 5) . The vacuoles of mucous cells containedno NEP mRNA, but the cytoplasmic rims of mucouscells appeared to contain some silver grains suggestingthe presence of NEP mRNA . Endothelial cells of sub-epithelial capillary/post-capillary venules contained NEPmRNA (figs 3 and 4), as did the smooth muscle of a
>
1461
Fig . 4 . - NEP in sim hybridization in epithelium . Brightlield (A and
C and darkfield (B and D) images of 35S-antisense NEP binding toepithelium shows dense silver grains over epithelial cell cytoplasm
excluding nuclei (small arrowheads) and goblet cell vacuoles (G) .Superficial vessel endothelial cells (large arrowheads) have a low den-sity of silver grains when viewed with darkfield illumination . The
bronchial lumen (L) is shown . Serial sections treated with sense probes
had no binding (not shown) . Bar Iine=50 pm .
deeper periglandular artery (not shown) . NEP mRNAwas detected in bronchial smooth muscle . The distrib-ution was the same in all specimens .
Neither the biotin- nor the 'SS-labelled sense probeshybridized with the tissue (figs 3 and 5) indicating thespecificity of the antisense probe .
Immunoh isrochemistn,
NEP immunoreactive material was detected in epithe-lial cells, submucosal glands, smooth muscle and endo-thelium of human bronchus (figs 6 and 7). In submucosal
sibly mucous cell cytoplasm, but not mucous cell vacuoles, demon-
strate silver grains . B) Brightfield image showing submucosal glandacini stained with haematoxylin . C) The sense probe did not bind to
the gland, confirming the specificity of the antisense binding seen in
(A). cRNA : complementary ribonucleic acid. Bar line=50 pm .
glands, the NEP appeared to be at the edges of glandcells rather than in secretory granules, suggesting thatthe NEP was membrane-associated . The same impres-sion is obtained for bronchial smooth muscle . Endothe-lium of vessels immediately below the epithelium werepositive (fig . 6A). The cellular distribution of NEPimmunoreactive material was the same as that of NEPmRNA. The distribution was identical in all specimens .Adsorbed NEP antibodies did not stain the tissue,indicating the specificity of the immunolocalizationmethod.
Fig. 6. - Neutral endopeptidase (NEP) immunohistochemistry . A)NEP immunoreaclive material was detected with MEK10 antibodiesand the immunogold method, and appears as the densely stained black
material . Goblet cells and glandular cells appear grey with the methylpyronin green counterstain . NEP was detected in the basal region ofthe epithelium (B), and the outer rims of submucosal gland cells (G) .Vacuoles of goblet cells and submucosal gland mucous cells did not
contain the black stain indicative of NEP, but still appear grey fromthe counterstain . Smooth muscle (Sm) also contained NEP. The NEPappeared to be localized to the surfaces of these cells in a rim pauem .
B) MEKIO antibodies adsorbed with recombinant human NEP did notbind to this serial tissue sectiooa Only methyl pyronin green counter-
stained cells can be seen . (Internal scale bar-100 µm) .
Fig . 7. - Neutral endopeptidase (NEP) immunohistochemisny . A)NEP immunoreactive material (black stain, immunogold method) isshown with methyl pyronin green counterstain. NEP immunore.artivematerial was detected in the outer rims of glandular acini (G) in semuscells, mucous cells, and possibly mycepithelial cells. Smooth muscle(Sm) and the endothelium of a venule (V) contained NEP . B) MEK10antibodies adsorbed with recambinant human NEP did not bind to thisserial tissue section . Only the methyl green pyronin counterstain (grey)is apparent. (Internal scale bar-40 µm) .
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NEP mRNA IN HUMAN BRONCHI 1463
Discussion
Cell surface NEP may limit the actions of many of thepeptides that are active in human tracheobronchial mucosa[23-25], including tachykinins and calcitonin gene-relat-ed peptide (CGRP) released by axon response mecha-nisms from nociceptive sensory netuones during neurogenicinflammation, vasoactive intestinal peptide (VIP) releasedby parasympathetic reflexes, bradykinin generated inmany types of allergic and nonallergic inflammation, andcirculating peptides, such as endothelin and atrial natriu-retic peptide (ANP) [26-28] . Decreases in NEP activity[22] may underlie the increased responses of respira-tory mucosa found during viral infections [29-31], afterexposure to cigarette smoke [32], ozone [33], hypochlor-ous acid, [34], and high doses of toluene diisocyanate[35]. The release of peptides into areas with reducedNEP activity could lead to enhanced peptide-inducedepithelial cell function, glandular secretion, vascular per-meability and smooth muscle contraction [1-3, 26, 27] .Each of these proinflammatory processes occurs in siteswhere NEP mRNA and protein are found : epithelial cells,submucosal gland cells, bronchial smooth muscle, endothe-lium and arterial smooth muscle (table 1) . Apparent dif-ferences in the numbers of gland and epithelial cellscontaining NEP mRNA and immunoreactive materialsbetween specimens were due to different proportions ofgoblet and mucous gland cells (figs. 3-7). These mucouscell vacuoles did not contain NEP . Other epithelial cellsand glandular serous cells did contain NEP mRNA andimmunoreactive material.These locations also correlate with the sites of NEP
enzyme activity detected in guinea-pig tracheal epithe-lium, glands, and vessels by fluorescent zymographicmicroscopy [36]. Enzyme activity was also detected inthe perichondrium and chondrocytes, but was not detect-ed in guinea-pig tracheal smooth muscle cells [361 .Expression of NEP in lung parenchyma has been inves-tigated by JotwsoN et al . [371 .
Whilst changes in NEP distribution or expression ordecreased 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 confirmthis contention . In fact, RotsMAN et al . [38] suggestedan increase in NEP activity in lungs of asthmatic sub-jects . Modulation of NEP activity in disease or after
Table 1 . - Distribution and relative intensity of expres-sion of NEP mANA and immunoreactive material in humantrachea and large bronchi
*: mRNA and irtununoreactive material were excluded fromepithelial goblet cell and submucosal gland mucous cell vac-uoles. NEP: neutral endopeptidase ; mRNA: messengerribonucleic acid. +: present; ++: intense stain .
oxidant exposure may be more complex in humans invivo than animal and in vitro models [l-3] would leadus to believe. The current investigation of NEP distribu-tion was not designed to determine whether destructionof NEP activity is a primary event in airway inflamma-tion that permits exaggerated neurogenic inflammation,but does indicate that changes in expression by residentcells is a feasible hypothesis .
An additional level of complexity in NEP expressionis the regulation of NEP gene transcription . The NEPgene is complex, with 24 miniexons and multiple poly-adenylation sites [6, 8] . NEP mRNAs of several sizesare generated by post-transcriptional processing . The useof alternate polyadenylation sites accounts for some ofthe variation [6] . However, alternate splicing with thedeletion of certain exons may also lead to mRNA sizeand protein product diversity . ItmrtA et al. [17] havedetected a 3 .2 kb mRNA which lacks exon 16. RT-PCR(fig. 2) identified mRNA coding for exons 14-20, butdid not identify any for the del exon 16 variant . Anothertruncated NEP gene product detected by PCR in rat thy-roid, intestine and whole brain [18] excludes exons 5-18,and is postulated to generate a 255 amino acid protein .No evidence was found to support the expression of thisvariant (data not shown) .These data indicate that NEP mRNA and immuno-
reactive materials are widely distributed on airway epi-thelium, glands, vessels and smooth muscle, sites knownto possess receptors for many peptide mediators. Thedistribution of NEP indicates its critical role in limitingthe effects of peptides in human airways in vivo .
Acknowledgements : The authors wish to thankL Roman, 1. Tabachnik and I . Adcock for their sup-port. Recombinant NEP and MEK-10 antibodies werekindly donated by S . Shak of Genentech, South SanFrancisco, CA. USA .
6. Shipp MA, Richardson NE, Sayre PH, et al. Molecularcloning of the common acute lymphoblastic leukemiaantigen (CALLA) identifies a type of type II integralmembrane protein . Proc Nat! Acad Sci USA 1988 ; 85 :4819-4823 .
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1464 J.N. BARANIUK
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