to in and - thorax · thorax 1994;49:26-32 peripheral mononuclearleucocyte adrenoceptors...

Thorax 1994;49:26-32

Peripheral mononuclear leucocyteadrenoceptors and non-specific bronchialresponsiveness to methacholine in young andelderly normal subjects and asthmatic patients

Martin J Connolly,* Joseph J Crowley, Christopher P Nielson, Nirmal B Charan,Robert E Vestal

ClinicalPharmacology andGerontology ResearchUnitM J ConnollyC P NielsonE Vestal

Pulmonary MedicineSectionJ J CrowleyN B Charan

Department ofVeterans AffairsMedical Center, 500W. Fort Street, Boise,Idaho 83702, USAand

Departments ofMedicine andPharmacology,University ofWashington School ofMedicine, Seattle,Washington 98195,USA

Reprint requests to:Dr R E Vestal.

* Present position:Senior Lecturer in Care ofthe Elderly and Medicine,University of Manchester,Barnes Hospital, Cheadle,Cheshire SK8 2NY, UKReceived 8 February 1993Returned to authors12 May 1993Revised version received9 August 1993Accepted for publication6 October 1993

AbstractBackground - As P adrenoceptor dys-function occurs in both the normalelderly subject and in young asthmaticpatients, the hypothesis was examinedthat age related P adrenoceptor changesare important in the pathogenesis of lateonset asthma in old age.Methods - Subjects were non-smokerswho comprised 17 young normal subjectsof mean (SE) age 29'4 (1-3) years, 17elderly normal subjects of 67-2 (1-3)years, seven young asthmatic patients of31P0 (2 8) years, and 17 elderly asthmaticpatients of 68-5 (14) years. All asthmaticpatients withheld inhalers for 12 hoursand oral treatment for 24 hours beforeeach study day. Subjects underwent aninhaled methacholine challenge (New-castle dosimeter method) on two non-consecutive days. The slope of the flow at50% of the vital capacity (FEF,O) dose-response curve was derived from the per-centage fall in FEF50 divided by metha-choline dose (sFEF50). Beta-adrenoceptordensity (Bmax) and affinity (%KH) weredetermined with (125I)iodocyanopindololas the radioligand in membranes pre-pared from mononuclear leucocytes.Results - Log sFEF50 was shown to bereproducible (repeatability coefficient0-41) on the two study days and was in-versely related to %KH but not to Bmax.Multiple regression analysis (all 58 sub-jects, overall R2=0 57) revealed an in-verse relation between log sFEFSO and%KH, and between log sFEF,4 and Bmax.The inverse relation between log sFEFSOand %KH was preserved whereas thatbetween log sFEF50 and Bmax was lostwhen young asthmatic subjects or whenall asthmatic subjects were excludedfrom multiple regression analysis.Conclusions - The P adrenoceptor dys-function observed in late onset asthmamay be similar to that seen during age-ing. Thus late onset asthma may repres-ent the extreme of a spectrum of ageassociated P adrenoceptor dysfunction.

(Thorax 1994;49:26-32)

In elderly patients asthma has a prevalence ofat least 6-5% and is often unrecognised.' 2

Although most elderly asthmatic patients firstdevelop asthma in adulthood,' the pathogen-esis of late onset asthma is poorly understood.Reduced ,B adrenoceptor responsiveness isseen in young asthmatic patients' and innormal elderly subjects.9'3 Increased non-spe-cific bronchial responsiveness, determined bymethacholine or histamine challenge, is char-acteristic of asthma, its degree correlating withthe severity of the asthma.' '6 Beta-receptordysfunction has long been postulated as acause of increased non-specific bronchialresponsiveness and of asthma itself.'7 Thisshould imply an increasing prevalence of highlevels of non-specific bronchial responsivenesswith increasing age, and a relation between thedegree of 1 adrenoceptor function and the levelof non-specific bronchial responsiveness.However, evidence of the former is confus-ing,1827 and the latter hypothesis has been littleinvestigated.2728We have examined the hypothesis that age

related 1 adrenoceptor dysfunction occurs inlate onset asthma by assessing: (1) mononuc-lear leucocyte membrane P adrenoceptor dens-ity and affinity in elderly, late onset asthmaticand elderly non-asthmatic subjects, and inyoung asthmatic and non-asthmatic subjects;and (2) the relation between non-specific bron-chial responsiveness to methacholine and theabove in vitro indices of 1 adrenoceptor func-tion in all four subject groups.

MethodsSUBJECTSHealthy young (18-50 years) and elderly (60-85 years) non-asthmatic subjects, and young(22-42 years) and elderly (60-85 years) asth-matic subjects who were otherwise healthywere recruited from the community. Sixteenof the healthy young subjects had participatedin a previous study using an identical proto-col29 and their data are included for compar-ison with the other groups. In addition, dataon the sensitivity of the neutrophil respiratoryburst to inhibition by isoproterenol in many ofthe subjects have been reported previously.30Table 1 gives subject characteristics for eachgroup. Asthmatic subjects were those withvariable respiratory symptoms and docu-mented 20% variability in forced expiratoryvolume in one second (FEVI), either spontan-eously or following a single dose of inhaled 1agonist. Non-asthmatic (normal) subjects had

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Beta-adrenoceptors and asthma in old age

Table 1 Mean (SE) characteristics of study subjects

Subject n Age Duration of Baseline FEV,group (M:F) (years) asthma (years) (%predicted)*

Young normal 17 29-4 (1 3) - 105 9 (2-6)(8:9)

Young asthmatic 7 31-0 (2 8) 11 9 (5-1) 100-8 (3-7)(3:4)

Elderly normal 17 67-2 (1-3) - 109-1 (34)(11:6)

Elderly asthmatic 17 68-5 (1 4) 18 8 (5 4) 80-9 (3-0)(11:6)

* Determined according to standards published by Morris.58

no respiratory or atopic history and normalbaseline lung function. Exclusion criteria forall groups included: cognitive impairment;pregnancy; cardiac disease; thyroid disorder;current cigarette smoking or smoking historyof > 10 pack years; respiratory infection,severe wheeze, medication change, or antihis-tamine treatment within six weeks; past or

present treatment with adrenergic antagon-ists, calcium antagonists or angiotensin con-

verting enzyme (ACE) inhibitors; baselineFEV, (each study day) less than 60% of pre-dicted.

Volunteers were screened by history, phys-ical examination, and electrocardiography. Apregnancy test was performed where appropri-ate. Subjects refrained from drinks containingcaffeine and oral medication for 24 hours andfrom inhalers for at least 12 hours before eachstudy period (48 hours for long acting medica-tion).Of the 17 elderly asthmatic patients four

were receiving no drugs and 13 were takinganti-asthma medication (some on multiplemedication). Four subjects used occasionalinhaled agonists only (less than once daily),nine used regular inhaled agonists, seven

used methylxanthines, four inhaled steroids,and five received inhaled cromoglycate. Of theseven young asthmatic patients six usedinhaled agonists regularly and two regularlyinhaled steroids.

STUDY PROTOCOL

Subjects were studied on non-consecutivedays within a seven day period according to a

protocol detailed elsewhere.29 Briefly, the pro-tocol comprised the following:Day 1: Subjects rested semirecumbent for

30 minutes, following which they sat up andinhaled methacholine was administered (initialdose 1-5 .g methacholine chloride) by a modi-fied Newcastle dosimeter method." A singlemeasurement of maximal expiratory flow at50% of vital capacity (FEF50) was obtainedimmediately before each dose. Altemative endpoints were 35% fall in FEF50 or administra-tion of the maximum cumulative dose of6-4 mg methacholine. FEF50 was monitoreduntil it returned to normal. A bronchodilatorwas not given. The output of 12 Acorn "Sys-tem 22" Turbo Nebulisers (Medic-Aid Ltd,Bognor Regis, West Sussex, UK) was main-tained at 10 Ili per nebulisation (standard devi-ation 0 9-2 5%) by computer controlled vari-ation of nebulisation time.Day 2: The protocol was identical to that for

Day 1 except that a blood sample was obtainedimmediately before methacholine challenge.Plasma was separated and frozen (- 70'C)

for catecholamine and IgE assays and mono-nuclear leucocytes were harvested from wholeblood for assay of mononuclear leucocyte 1adrenoceptor characteristics.

All subjects gave written informed consentto participate in the study which was approvedby the Human Subjects Committee of theUniversity of Washington and the Researchand Development Committee of the BoiseDepartment of Veterans Affairs MedicalCenter.

ASSAY METHODSThe assay methods are described in detailelsewhere.29 Plasma catecholamine concentra-tions were measured by the radioenzymaticmethod of Peuler and Johnson,'2 and plasmaIgE was determined by an amplified immuno-radiometric assay. Mononuclear cells wereisolated by density gradient centrifugationwith Ficoll-Hypaque at 4'C,33 counted, andthen lysed.29 Prepared membranes were storeddry at - 70'C. The Bradford method was usedfor protein analysis.34 Beta-adrenoceptor den-sity and affinity in mononuclear leucocytemembranes were determined with (251I)iodo-cyanopindolol (ICYP, 2200 Ci/mmol, NENResearch Products, Boston, Massachusetts,USA), as previously described.29 Receptordensity (Bmax) and affinity for antagonist(KD) were determined from saturation iso-therms by computer analysis using Lundon I(Lundon Software Inc, Cleveland, Ohio,USA).35 Agonist competition curves were ana-lysed with a two site model and InPlot (Graph-Pad Software, San Diego, California, USA),which employs the Marquardt algorithm fornon-linear regression.'3

STATISTICAL ANALYSISThe following information was derived foreach subject: (1) simplified slope37 of FEF50(sFEF50) dose-response curve to methacholine(in duplicate). This is a measure of non-specific bronchial responsiveness with unitsexpressed as the percentage fall in FEF50/mgmethacholine. In this analysis slope is posi-tively related to non-specific bronchialresponsiveness; (2) total number of [ adreno-ceptors/mg cell membrane protein (Bmax);(3) dissociation constants for isotope and iso-proterenol; and (4) percentage of receptors inhigh affinity state (%KH).The following analyses were performed: (1)

reproducibility of sFEF50 by the method ofBland and Altman;38 (2) Student's unpaired ttests comparing IgE and catecholamine levelsbetween groups; (3) one way analysis of vari-ance (ANOVA) to compare in vitro resultsbetween groups, allowing for sex differences;and (4) linear and multiple regression analysisemploying sFEF50 as the dependent variable.Unless otherwise stated, results are expressedas mean (SE). Statistical significance wasdefined at the 5% level.

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Figure 1 Repeatability of log sFEF,0 to methacholine (Day 1 v Day 2).Repeatability is expressed as the ratio of the slope on Day I to that on Day 2 aplotted against the geometric mean of the pair. The ratios did not differ signific6from unity. The solid horizontal line indicates the overall geometric mean and tbroken horizontal lines represent the geometric mean plus or minus two standarcdeviations which constitute the 95% limits for repeat sFEF,0 determination.

ResultsRESPONSE TO METHACHOLINEElderly asthmatic subjects had a lower bNFEV1 (% predicted) than young normajects (p < 0-0001), elderly normal su(p < 00001), and young asthmatic su(p < 0-001). To evaluate reproducibilityresponse to methacholine, for each subjratio of the simplified slope of FEF50.(sFEF50.Day 1) to sFEF50.Day 2 on a lo,(equivalent to difference of the logs on ascale) was plotted against the geometricof the pair on a log scale (equivalent tolog sFEF50 on a linear scale). The ratios cdiffer significantly from unity (fig 1)geometric means of sFEF50 on Days 1were 237 8% fall/mg (95% confidencefor mean 113-3 to 499 5°% fall/mg) and 2fall/mg (95% confidence limits for meanto 442-8% fall/mg), respectively. The gcric mean of the ratios of sFEF50.Da3sFEF50.Day 2 was 1-110 (95% conflimits for mean 0 982 to 1-254). Coefficrepeatability38 was 0 408. Thus, the 950/fidence limits for repeat sFEF50 determiwere 0*39 x initial sFEF50 to 256 xsFEF50.The geometric mean of sFEF50.Day

6 47% fall/mg (95% limits for mean 4

Table 2 Mean (SE) plasma levels of IgE and catecholamines

Subject group IgE (units/ml) Adrenaline (pmol/l) Noradrenaline

Young normal 43-2 (16-5) 217 (37) 1-82 (0-12)Young asthmatic 212-6 (140-2) 191 (56) 1-97 (0-37)Elderly normal 53-1 (17-4) 220 (31) 2-22 (0-23)Elderly asthmatic 100-0 (38-7) 258 (79) 2-85 (0-32)

Table 3 Characteristics of 6 adrenoceptors in membranes of peripheral mononleucocytes

Subject group Bmax (fmol/mg) %KH KDH (nmol/l) KDL (JIM

Young normal 45-57 (6 25) 6068 (3 76) 218 4-79(138-344) (2-68-8-5

Young asthmatic 26-63 (477) 49-21 (9-54) 134 3-98(58-308) (1-23-12

Elderly normal 33-84 (4-31) 45-26 (5 83) 95 2-27(47-192) (1*31-3-9

Elderly asthmatic 43-26 (5-65) 31 13 (4-30) 38 1-84(17-85) (1-14-2-9

Data are mean (SE) except for KDH and KDL which are geometric means and for whichconfidence limits are shown in parentheses.

9 00% fall/mg) in young normal subjects,115-6% fall/mg (95% limits 43-3 to 308-6%fall/mg; p < 0-0001 v young normal subjects) in

*+2 SD elderly normal subjects, 844 9% fall/mg (95%limits 123-6 to 577 6% fall/mg; p<0-0001 vyoung normal subjects) in young asthmaticpatients, and 8030% fall/mg (95% limits 4638to 13903% fall/mg; p<0-0001 v elderly nor-

|-2 SD mal subjects) in elderly asthmatic patients.The range of sFEF50.Day 2 was 1-72 to 23-5%fall/mg in young normal subjects, 1 15 to2020% fall/mg in elderly normal subjects,23-0 to 19329% fall/mg in young asthmaticpatients, and 1380 to 46 272% fall/mg in

ind elderly asthmatic patients.antlyrhed IGE AND PLASMA CATECHOLAMINES

As shown in table 2, elderly asthmatic patientshad higher resting noradrenaline levels thanyoung normal subjects (p<0005). ANOVA(sex and subject group as categorising vari-

aseline ables) confirmed the difference in noradren-al sub aline levels among all four groups (F1,56 = 4-21,abjects p<0-01), and between elderly asthmatic andubjects young normal subjects (F,,32=776, p <0.01),of the with similar trends between other groupsect the except young normal v young asthmatic sub-Day 1 jects, and elderly normal v young asthmaticig scale subjects. Plasma levels of IgE and adrenalinelinear did not differ significantly between groups.

c meanmean

did not CHARACTERISTICS OF 13 ADRENOCEPTORS

The Since absolute values for dissociation con-

and 2 stants of high (KDH) and low affinity (KDL)limits receptors for isoproterenol were not normally!11*1%/ distributed, these values were logarithmically100.6 transformed before analysis to ensure a normal100et distribution. As shown in table 3, for all

eometo four subject groups there was a significantidence intergroup difference in %KH (F1,s6= 6i07,ient of p=0-001), and in the dissociation constant;/ con- (KDH) of the high affinity site (F=56 5 18,nation p < 0 005), but no difference in Bmax or in theinitial dissociation constant (KDL) of the low affinity

site. Young normal subjects had significantly2 was higher %KH values (F1,32 = 5 10, p < 0 05) and4.65 to KDH values (F,,32 = 5-02, p < 0 05) than elderly

normal subjects. Other variables were not sig-nificant. Although there was a trend towards ahigher %KH in elderly normal subjects than in

(nmolll) elderly asthmatic patients, the difference didnot reach statistical significance. ANOVAcomparing young normal with elderly asth-matic subjects showed differences for %KH(F1,32 = 25-93 p <0 0001), KDL (F1,32 = 8-62,p < 0-01), and KDH (F1,32= 15-46, p < 0-0005),but not for Bmax. Young asthmatic subjects

'uclear had lower Bmax values than young normalsubjects (F1,22 = 7-06, p < 0 02) or elderly asth-

mol/l) matic patients (F122 = 423, p< 0-05). Youngasthmatic patients had higher %KH values

5S) than elderly asthmatic patients (F1 22= 5-67,*-92) p <0 05). KDH and KDL values did not differ

between young asthmatic subjects and other95) groups. ANOVA revealed no sex differences in96) any analysis.the 95% Although there was no relation between log

sFEF50 and Bmax, log sFEF,0 was inversely

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Figure 2 Linear regression analysis of bronchial responsiveness (sFEF50.Day 2) vpercentage of fi adrenoceptors in the high affinity state (%KH).

related to %KH (fig 2). To evaluate furtherrelative importance of factors that mightexpected to influence the response to metcholine, multiple regression analysis was pformed with log sFEF50 as the dependent v;able (table 4). Data from all 58 subjects wused for these analyses. Log sFEF50 showesignificant inverse relation with both %(p<0005) and Bmax (p<0005), with a hcoefficient of multiple regression.When multiple regression analysis i

repeated in normal subjects only (table 5),inverse relationship between log sFEF50 .%KH was preserved (p<0 05), but tbetween log sFEF50 and Bmax was lost. Siilar results were obtained with multiple regrsion in young and elderly normal subjects .elderly asthmatic subjects (that is, excludyoung asthmatic subjects, table 6).

Table 4 Multiple regression analysis of non-specific bronchial responsiveness (logsFEF50.Day 2) against f, adrenoceptor parameters, catecholamines, and IgE in all.subjects

b SE t p

Intercept 6 35 1 00 6-35 < 0 0001%KH -0013 0006 -2 17 <005Baseline FEV, (%) -0 041 0-008 - 5-28 < 0 0001Age 0022 0007 316 <005Bmax -1-125 0056 -202 <005

b = partial regression coefficient; SE = standard error; t = Student's t test; coefficient of mulregression (R') adjusted for degrees of freedom = 0-57.KDH, KDL, catecholamines, and IgE were not significant.

Table S Multiple regression analysis of non-specific bronchial responsiveness (logsFEF50.Day 2) against ,B adrenoceptor parameters, catecholamines, and IgE in youand elderly normal subjects only

b SE t p

Intercept 0 617 0-486 1-270 >0-2%KH -0012 0006 -2 16 <005Age 0-030 0-006 4 89 <0 0001IgE 00018 00009 209 <005

b = partial regression coefficient; SE = standard error; t = Student's t test; coefficient of mulregression (R ) adjusted for degrees of freedom = 0-56.Bmax, KDH, KDL, catecholamines, and % baseline FEV, were not significant.

Table 6 Multiple regression analysis of non-specific bronchial responsiveness (logsFEF5O.Day 2) against f, adrenoceptor parameters, catecholamines, and IgE(excluding young asthmatic subjects)

b SE t p

Intercept 3-76 0-98 3-85 <0 001%KH -0-013 0-006 -2-34 0-02Baseline FEVy (%) -0 032 0-007 -4-72 <0 001Age -0 033 0-007 -5 06 <0 001

b = partial regression coefficient; SE = standard error; t = Student's t test; coefficient of mulregression (R2) adjusted for degrees of freedom = 0 74.Bmax, KDH, KDL, catecholamines, and IgE were not significant.

Since ANOVA comparing elderly normaland elderly asthmatic subjects had onlydemonstrated a trend towards an intergroupdifference in %KH, a further multiple regres-sion analysis was performed for these 34 sub-jects only. It showed significant relationsbetween log sFEF50 and baseline FEV1(p < 00005) and %KH (p < 005), but notbetween log sFEF50 and Bmax.No overall relation was shown between any

- in vitro variable and baseline lung function.100 For young or elderly asthmatic patients there

was no relation between any in vitro variablethe and either duration of asthma or age of onset or

between log sFEF50 and asthma duration orage of onset.

thebe

:ha- Discussionper- This study has confirmed the reduction inari- mononuclear leucocyte membrane 3 adreno-rere ceptor affinity with preservation of receptord a density in normal elderly men and women.KH More importantly it has shown that, in com-igh parison with young and elderly normal sub-

jects, mononuclear leucocytes from elderly,was late onset asthmatic patients have furtherthe reductions in affinity without a reduction inand receptor density. Indeed, there was an inversethat correlation between non-specific bronchial re-mi- sponsiveness (a marker of asthma severity) andres- receptor affinity, but not between non-specificand bronchial responsiveness and receptor density.ling These abnormalities in receptor affinity were

not seen in young asthmatic patients, althoughin this latter group the previously describedreductions in receptor density compared with

58 young normal subjects were confirmed. Youngasthmatic patients were also found to havelower receptor density than elderly asthmaticpatients.

Multiple regression analysis employing anindex of bronchial responsiveness as the de-pendent variable showed an inverse relationbetween responsiveness and both receptor

Itiple affinity and receptor density when all subjectswere included in the analysis. When youngasthmatic subjects were excluded, however,the relation between responsiveness and dens-

nity was lost but the inverse relation betweenresponsiveness and affinity was preserved.This was also true if only normal subjects wereincluded in the analysis, strongly suggestingthat the relation is a function of age (or ageing)rather than of asthma.Taken together these results suggest that the

tiple mechanism or mechanisms responsible for 1adrenoceptor dysfunction in late onset asthmaare similar to those underlying age-associated,B adrenoceptor dysfunction, and distinct fromthose associated with juvenile onset asthma. Iftrue, then late onset asthma may represent thefar end of a spectrum of age-associated Iadrenoceptor dysfunction.Although there is controversy as to the ade-

quacy of the mononuclear leucocyte model interms of the degree to which it reflects changesin airway j adrenoceptors, in vitro and in vivo

ltiple abnormalities of f adrenoceptor function arewell recognised in young asthmatics'8 and

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have been suggested as a cause of asthma.'7Similar in vitro and in vivo abnormalities havebeen described in normal elderly subjects9-'3and may be a consequence of the effects ofageing on the I adrenergic receptor adenylylcyclase system. Nevertheless, the limitations ofthe mononuclear leucocyte model must beacknowledged.Lymphocytes and lung membranes ofyoung

asthmatic subjects have reduced numbers(density) of f3 adrenoceptors.568 In contrast,normal elderly subjects have normal lympho-cyte membrane receptor density'339 41 butreduced receptor affinity for agonist.'3 This isthought to represent functional uncoupling ofthe receptor from the adenylyl cyclase com-plex, and is associated with impaired receptor-mediated adenylyl cyclase activity.4243Impaired adenylyl cyclase activity is commonto asthma and normal ageing.4' '244 The possibility that late onset asthma represents theextreme end of a spectrum of ageing effects onthe 3 adrenoceptor pathway has not previouslybeen examined, and in vitro studies on 0receptors of elderly asthmatic subjects havenot been reported.

It has been suggested that P adrenoceptordysfunction in ageing is due to downregulationby endogenous catecholamines."1 13 The higherresting noradrenaline levels in the elderly nor-mal and elderly asthmatic subjects in thisstudy are in general agreement with previousliterature. '1345 Our failure to show a relationbetween noradrenaline levels and either recep-tor affinity or non-specific bronchial respons-iveness level argues against the downregula-tion hypothesis.The present finding of a greater degree of

bronchial responsiveness in elderly normalthan in young normal subjects is entirely con-sistent with the hypothesis of an associationbetween late onset asthma (and thereforehyperresponsiveness) and age-associated ,Badrenoceptor dysfunction. There is, however,disagreement in the literature as to the effect ofage on non-specific bronchial responsiveness.Some studies have indeed suggested a higherlevel of responsiveness in the elderly19-22 whileothers have found no effect of age.'8 2336 Insome of these latter cases, however, few or nosubjects over 65 years were studied.'82335Others2426 examined the percentage of subjectswith hyperresponsiveness (as opposed to abso-lute level of responsiveness) and at least one24showed a trend towards hyperresponsivenessat the extreme end of the age range. A recentpaper which again failed to show an age-associated increase in airway reactivity in-cluded only two of 85 subjects over 70 years.27The young normal subjects in this same studydisplayed a surprisingly large range of non-specific bronchial responsiveness, measured asFEVI slope (over 400 fold, as opposed to 14fold in the present study), and yet repeatabilityof their non-specific bronchial responsivenessmeasurement was not assessed. Two recentlarge studies4647 report conflicting data on theeffects of age on bronchial responsiveness innon-smokers.There are three potential pitfalls with the

novel hypothesis of the model of late onsetasthma as an age-associated receptor-mediateddisease. Firstly, we were unable to study largenumbers of drug naive elderly asthmatic sub-jects. Thus, chronic treatment with inhaled fagonists may be implicated in downregulationof 3 adrenoceptors. While this may partiallyexplain our results, it would not explain whythe receptor abnormality shown was exclu-sively one of reduced affinity, whereas similarstudies on treated young asthmatics haveshown reduced receptor density.8 Further-more, it has been shown in older patients withchronic obstructive pulmonary disease thattreatment with ,B agonists leads to a fall indensity but not in agonist affinity in mononuc-lear leucocytes.48

Secondly, the methacholine challenge itself(Day 1) may have affected in vitro ,B adreno-ceptor parameters (Day 2). Exposure of guineapigs to high doses of inhaled acetylcholine forone week reduces 3 adrenoceptor density inthe lung membrane.49 However, affinity wasunchanged and systemic I adrenoceptors werenot examined.49 Thus, even if - as seems un-likely - systemic P adrenoceptors were affectedby one methacholine challenge two days beforesampling, changes in density rather than affin-ity would be expected, with the greatestchanges occurring in subjects receiving thelargest methacholine dose (young normals).Such an effect would blunt rather than pro-duce or exaggerate the differences seen in ourstudy.The third and most important criticism of

the suggestion that I adrenoceptor ageingplays a major part in the pathogenesis of lateonset asthma is quantitative rather than quali-tative. Although acute intravenous f blockadepotentiates non-specific bronchial responsive-ness in young non-asthmatic subjects,50 thepotentiation is small (up to fourfold), whereasour elderly asthmatic patients possessed a de-gree of non-specific bronchial responsiveness2000 times that of young normal subjects.However, this is likely to represent an over-simplification of the pathophysiology ofchronic f adrenoceptor dysfunction. The ,Badrenergic system also modulates cholinergicneurotransmission at a prejunctional level witheffects upon endogenous acetylcholine sensiti-vity up to 100 times more potent than itseffects on response to exogenous cholinergicagents.5' Further, acute intravenous 3 block-ade would not produce immediate effects viaother f adrenergic-dependent mechanismssuch as lung microvascular leakage, alveolarpermeability, surfactant secretion, inhibitionof mast cell mediator release and neutrophillysozyme secretion, and stimulation of ciliatedepithelial function.52 Modulation of any ofthese systems could be expected to result inchanges in bronchial responsiveness. Indeed,late asthmatic responses, which are paralleledby increases in non-specific bronchial respons-iveness,53 are associatd with bronchial oedemaand the release of prostaglandins, leukotrienes,and other mediators from mast cells and othersources. Furthermore, increases in epithelialpermeability do enhance non-specific bron-

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chial responsiveness,54 and chronic asthma isassociated with neutrophil accumulation in theairway and chronic oedema.55We also showed the value of the methacho-

line challenge approach for the study of airwayfunction in ageing. The methacholine chal-lenge technique used for this study is wellestablished.3' The use of FEF50 as the ventil-atory parameter was dictated by the need toobtain measurable bronchoconstriction innon-asthmatic subjects, and it is the smallairway parameter which changes least withage.56 Expression of results as the simplifiedslope of the dose-response curve has beenvalidated previously for FEV,.37 The presentstudy has shown its use with FEF50 to producereproducible results (comparing favorablywith more conventional indices using the samedosimeter in both young and elderly sub-jects31 57), which discriminated well betweensubject groups.

Our overall conclusion is that age-associatedreduction in adrenoceptor affinity for agonist(in the absence of any reduction in adreno-ceptor density usually associated with juvenileonset asthma) plays a part in the aetiology oflate onset asthma either by direct effects uponthe adrenoceptor system in the airway or,more possibly, by modulation of the responseof airway inflammatory cells, mediator release,or other indirect mechanisms. Although it islikely that late onset asthma is an age-relatedphenomenon, additional studies will be neces-sary to fully elucidate the mechanismsinvolved.

The authors thank Beth Musser and Bob Fritts for theirinvaluable technical help and advice.Dr Connolly was supported by the British Geriatrics Society/

Nuffield Foundation Travelling Fellowship. Dr Crowley is therecipient of a Faculty Development Award in Clinical Pharma-cology from the Pharmaceutical Manufacturers' AssociationFoundation. The project was supported in part by the Depart-ment of Veterans Affairs (Office of Research and Development,Medical Research Service) and by grants from the AmericanHeart Association of Idaho and Glaxo Inc, USA.

1 Burr ML, Charles TJ, Roy K, Seaton A. Asthma in theelderly: an epidemiological survey. BMJ 1979;1:1041-4.

2 Banerjee DK, Lee GS, Malik SK, Daly S. Underdiagnosisof asthma in the elderly. Br Dis Chest 1987;81:23-9.

3 Cookson DU, Reed CE. A comparison of the effects ofisoproterenol in the normal and asthmatic subject. AmRev Respir Dis 1963;88:636-43.

4 Parker CW, Smith JW. Alterations in cyclic adenosinemonophosphate metabolism in human bronchial asthma.1. Leukocyte responsiveness to beta-adrenergic agents.Clin Invest 1973;52:48-59.

5 Kariman K, Lefkowitz RJ. Decreased beta-adrenergicreceptor binding in lymphocytes from patients withasthma. Lung 1980;158:41-51.

6 Brooks SM, McGowan K, Bernstein LI. Relationshipbetween numbers of beta-adrenergic receptors in lym-phocytes and disease severity in asthma. Allergy ClinImmunol 1979;63:401-6.

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