Mechanisms of Ageing and Development, 54 (1990)29--42

29

ElsevierScientificPublishers Ireland Ltd.

BETA-ADRENERGIC RESPONSES AND AIRWAY REACTIVITY IN H E A L T H Y ADULTS

P A M E L A B. DAVIS a and P A M E L A J. BYARD b Departments of Pediatrics"~, Medicine*, and Epidemiology and Biostatistics~, Case Western Reserve University School of Medicine at Rainbow Babies and Childrens Hospital, Cleveland, OH (U.S.A.)

(ReceivedJune 17th, 1989) (RevisionreceivedNovember21st, 1989) SUMMARY Healthy adults aged 18--90 years were tested for lymphocyte and granulocyte cyclic A M P responses to isoproterenol and prostaglandin E 1 (PGE1), beta-adrenergic receptor density and antagonist binding properties, and for airway reactivity to methacholine. Our hypothesis was that reduced beta-adrenergic responses occur with aging and are associated with increased airway reactivity. This hypothesis was not supported by the data. Lymphocyte stimulation ratios (cyclic A M P level with stimulation/baseline cyclic AMP level) at higher concentrations of isoproterenol and PGE~ increased significantly with age. There were no significant age trends for any of the other variables. None of the beta-adrenergic responses or receptor properties correlated with airway reactivity to methacholine. Beta-adrenergic responses in lymphocytes and granulocytes from the same subject were weakly correlated at high concentrations. Prior studies which suggest that reduced betaadrenergic responses and increased airway reactivity are concomitants of normal aging may differ from the present study in subject selection. In healthy older subjects, there appears to be no reduction in leukocyte beta-adrenergic responses or receptor properties and no change in airway reactivity.

K e y words: Beta-adrenergic responses; Airway reactivity; Ageing

INTRODUCTION Because beta-adrenergic stimulation causes relaxation of airway smooth muscle, investigators have looked for associations between impaired beta-adrenergic responAddress correspondence to: Pamela J. Byard, Ph.D., Department of Pediatrics, 2101 Adelbert Road,

Cleveland, OH 44106, U.S.A. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990ElsevierScientificPublishers Ireland Ltd.

30 ses and increased airways obstruction or airways reactivity. Kaliner e t al. [1] showed that beta-adrenergic sensitivity is negatively associated with airway reactivity to methacholine. Reduced beta-adrenergic responses or receptor concentration have been associated with increased airway reactivity in animal models [2--5] and in human subjects [1,6--10]. Several laboratories have found reduced cardiovascular or metabolic responses to exogenous beta-adrenergic agents associated with increased airway reactivity [6,7,9,19]. The relationship between leukocyte betaadrenergic responses and airway reactivity is more controversial. Although some investigators have found reduced lymphocyte or granulocyte beta-adrenergic responses in patients with increased airway reactivity (asthmatics) [ 11--13], others have not [8,14--18]. Prior medication usage by asthmatics, different rates of receptor desensitization and recovery, and subclinical disease in controls may account for the variable results among laboratories. The leukocyte beta-adrenergic system has been used as a model for beta-adrenergic systems of the lung. Because the receptor and coupling proteins are highly conserved, alterations in one or the other should be reflected in any tissue in which they are expressed. Moreover, recent data indicate that the lymphocyte beta2-adrenergic receptor system is a good model for beta2-adrenergic receptor systems on less accessible solid tissues, including the lung [! 9,20]. Beta-adrenergic responses are difficult to assess directly in the lungs of living human subjects. Ordinarily, attempts to do so involve measurement of airflow before and after administration of an aerosol containing a beta-adrenergic agent. However, this is a poor test in a healthy population, for fully normal airflow cannot be improved even if there is excellent betaadrenergic response, and the variability of the test itself (at least 5%) may be large compared to the change induced by the beta-adrenergic agent. Beta-adrenergic responses are reported to change with age: older subjects are less responsive to exogenous beta-adrenergic agents but also respond less well to endogenous stimuli, suggesting a failure of the beta-adrenergic system p e r s e [21--28]. The cardiovascular system shows less chronotropic response to isoproterenol in the elderly, and the leukocyte cyclic AMP response to beta-adrenergic agents is reported to be reduced in the aged. These findings are in contrast to the situation in the alphaadrenergic system of the pupil in older subjects, in which endogenous responses (e.g. pupillary dilation in darkness) are blunted, but responses to exogenous agonists such as phenylephrine are heightened [29]. Because beta-adrenergic responses decrease [21--28] and airway reactivity increases [30,31] with age, and because of the evidence that the beta-adrenergic system contributes to airway reactivity [1--13], we examined age-related changes in beta-adrenergic responses and airway reactivity. We began this study with the hypothesis that the reduction in beta-adrenergic response with age is related to the increase in airway reactivity. Determining the relationship between beta-adrenergic responses, airway reactivity, and aging in a healthy population sample is important because the beta-adrenergic system can be modified pharmacologically. If airway

31

reactivity contributes to the development and progression of obstructive airways disease [32], and beta-adrenergic responsiveness contributes to airway reactivity, then one might be able to retard progression of airways obstruction by modifying beta-adrenergic reactivity. We examined the lymphocyte and granulocyte cyclic AMP response to isoproterenol, beta-adrenergic receptor concentration and antagonist binding properties, and airway reactivity to methacholine in subjects 18--90 years of age to test the hypothesis that variation in beta-adrenergic responsiveness is related to variation in airway reactivity, and that both systems change with age in healthy subjects. To further define the cause of the expected reduction in cyclic AMP response to beta-adrenergic agents, we measured cyclic AMP responses to PGEt as well, reasoning that a general defect in receptor-cyclase coupling, adenylate cyclase itself, or G proteins would be reflected in reduced PGE~-stimulated cyclic AMP production as well as in reduced beta-adrenergic responses, but a receptor-specific deficit would be manifest in the beta-adrenergic, but not the P GEt, response. MATERIALS AND METHODS

Subjects A total of 85 subjects (36 males and 49 females) was used in this analysis. The frequency distribution is shown in Table I. The mean age was 48 years (S.E.M. 2). Subjects less than 60 years of age were recruited by advertisement in the University community. They professed to be healthy and to have taken no medications except acetaminophen, hormone replacement, and/or birth control pills for the preceding 2 weeks. They specifically denied a history of lung disease (such as asthma, cystic fibrosis, or chronic obstructive pulmonary disease) in themselves or their firstdegree relatives. All subjects had a baseline FEV~ >t 80°7o of that predicted by age, sex, and height according to the equations of Knudsen et al. [33]. Subjects over 60 years of age were recruited in conjunction with the Volunteer Registry of the Teaching Nursing Home Project at Case Western Reserve Univer-

TABLE I A G E DISTRIBUTION O F T H E S A M P L E

Age cohort

Males

Females

18--27 28--37 38--47 48--57 58--67 68 +

6 7 5 5 7 6

9 7 It 8 6 9

32 sity. Most of these subjects were recruited from active seniors clubs in community centers, and were usually retired professional workers. They were all ambulatory, mobile, non-institutionalized people who professed to be healthy and specifically denied any history o f lung disease in themselves or their first-degree relatives. Of the 27 elderly subjects recruited, 19 had taken no medications for at least 14 days prior. In this group, one reported a myocardial infarction 20 years prior and two complained of arthritis, but refrained from taking analgesics for 2 weeks prior to study. The remaining subjects were taking medications at the time of the study which were unlikely to have affected their beta-adrenergic or airway responses. Two subjects took hormone replacement (one conjugated estrogens and one levo-thyroxine), and six subjects took other medications as follows: one took isosorbide dinitrate (and reported myocardial infarction 10 years prior but denied angina in the last year), one took piroxicam, one tolbutamide, one dipyridamole, and two took thiazide diuretics for mild hypertension. All elderly subjects had normal chest examination and normal baseline pulmonary function (FEV 1 >1 80% o f predicted for age, sex, and height by the equations of Knudsen et aL [33]). Though subjects were recruited to participate in all phases of the study, some failed to complete one part or the other or did not permit the additional blood to be drawn for receptor assays. However, the age distribution of subjects who participated in each test did not differ from that of the overall group. Lymphocyte and granulocyte studies Blood was drawn from fasting subjects between 0800 h and 1000 h. Lymphocytes and granulocytes were separated as described previously [34] from whole blood (anticoagulated with acid-citrate-dextrose) by the histopaque gradient technique. Stimulation of cyclic A M P production was done as previously described [34]. Assay of cyclic A M P was performed by either the competitive protein binding method of Brown et al. [35] if samples contained 0.75-8 pmol cyclic AMP, or by radioimmunoassay of acetylated samples [36] if samples contained 20--400 fmol cyclic AMP. For both assays to be acceptable, B0/total counts ratio was 0.2--0.5, and only values with B/B 0 o f 0.1--0.9 were used. Non-specific binding was less than 507o. For the Brown assay, standard curves were linear (r > 0.98) over the concentration range used. For the radioimmunoassay, a spline curve fitting program was used and the fitting factor was close to zero for each acceptable assay. For both assays, duplicates were within 5°7o of each other, lnterassay reliability was assessed by three external standards at the high, low, and midranges of the curve, two of which were required to fall within 1.675 S.D. of their mean for an assay to be accepted. Cell counts were done in Coulter Counter Model ZF. The basal cyclic A M P content was taken as the cyclic A M P content of cells exposed to theophylline but no other agonists, expressed as pmol cyclic AMP/106 cells. Stimulated cyclic AMP content was expressed as pmol cyclic AMP/106 ceils in the presence of isoproterenol or PGE 1. Further analysis was conducted on the ratio of stimulated cyclic AMP con-

33 tent to basal cyclic AMP content (the stimulation ratio) because it has higher intraclass correlation coefficients than other measures; that is, it is more reproducible from day to day in the same subject [37]. Membranes were prepared from washed, separated lymphocytes and granulocytes as previously described [34]. Whole lymphocytes or granulocytes were suspended in normal saline containing 50 mmol/l Tris (pH 7.4) and 8 mmol/l theophylline and incubated for 5 rain at room temperature. Inhibition of phosphodiesterase with theophylline was routine in order to isolate the stimulatory effects of agonists from the combined effects of cAMP synthesis and destruction. The cell suspension (70/al) was then added to tubes containing either PGE 1 (10- 8 10-5 mol/1), isoproterenol (10-8--10 -4 mol/l), isoproterenol and propranolol, or buffer (total volume, 100 gl), and incubated 5 min at 37 °C. The reaction was terminated by boiling for 2 min. The samples were diluted with 400/al distilled water and frozen for later assay in duplicate for cyclic cAMP. Washed lymphocytes were resuspended in distilled water and allowed to sit on ice at least 10 min. They were then lysed by Polytron action (15 at setting of 8), and the resulting suspension centrifuged at 4°C for 10 min at 39 000 g. The pellet was washed in buffer containing 50 mol/1 Tris pH 7.4, and 10 mmol/1 MgCI2, and finally resuspended in this buffer with a motor-driven Teflon pestle for use in the receptor binding assays. Binding of [3H] dihydroalprenolol ([3H]DHA), a beta-adrenergic antagonist, to lymphocyte or granulocyte particulates was conducted at 37 °C in buffer containing 50 mmol/1 Tris (pH 7.4) and 10 mmol/l MgCI 2 in total volume 500/al as described by Williams et ai. [39]. Non-specific binding was taken as that not displaced by 10-5 mol/1 DL-propranolol, and ranged from 20--50°70 of total binding for lymphocyte particulates and 30--60°70 for granulocyte particulates. At the end of the incubation period, the mixture was diluted with 3 ml cold buffer, and bound ligand was separated from free by rapid (< 15 s) filtration onto Whatman GF/C filters (Whatman, Inc., Clifton, N J) under vacuum, followed by washing of the filters with 20 ml icecold buffer. Filters were then dried and counted in a Searle liquid scintillation counter with efficiency for 3H of 40--44% (Searle Radiographics, Inc., Des Plaines, IL). Specific binding increased linearly with added protein over the range used in these assays (50--300/ag for lymphocytes, 50--500/ag for granulocytes). Protein determinations were by the Bradford method [38]. For equilibrium binding studies, [3H]DHA (at least five concentrations, 0.5-5 nmol/l) was incubated for 30 min with the particulates. KD, the binding constant for [3H]DHA and Bmax,the theoretical maximum binding, were calculated by Scatchard analysis [40]. Materials for [3H]cyclic AMP assays, and ACS scintillant, [3H]-DHA (30--60 Ci/ mmol) were obtained from Amersham Corp., Arlington Hts., IL. Theophylline, (-)-isoproterenol, DL-propranolol, PGE~, and other laboratory chemicals were purchased from Sigma Chemical Co., St. Louis, MO.

34

Methacholine challenge testing These tests were performed as previously described [8]. The best of 3--5 spirograms performed on a 13.5-1 water sealed spirometer (Warren E. Collins, Braintree, MA) was taken for baseline values. Aerosol was generated by a deVilbiss model 561 nebulizer (deVilbiss Co., Somerset, PA). Subjects inhaled 5 breaths from functional residual capacity to total lung capacity, with 5s breathhold, of aerosol of 0.1, 0.3, 1, 3, 10, and 30 mg/ml methacholine chloride prepared in normal saline and kept frozen in aliquots until use; 3 min after the last breath, spirometry was repeated. The test was terminated when the highest dose had been inhaled or when the forced expiratory volume in one second (FEVm) had decreased by 20o70 or more. Because we studied a healthy sample, few subjects had FEV~ fall by 20°70 even at the highest dose. Therefore, expressing results as the PC20FEV ~, the concentration of methacholine (mg/ml) required to produce a 20070 fall in baseline FEV 1 would not have given an adequate description of the airways responsiveness for the majority of subjects. Therefore, we used the method of O'Connor et al. [41], calculating the slope of a line between the first and last points on the methacholine dose-response curve. Because the distribution of the slopes was highly skewed, a logarithm transformation was performed before statistical analysis. Statistical analysis Data were entered on an IBM P C / A T computer using the SPSS/PC + data entry package, and verified. The relationship between beta-adrenergic responses, airway reactivity to methacholine, and age was assessed using Pearson's product-moment correlation coefficient (r). TABL E II D E S C R I P T I V E STATISTICS FOR L E U K O C Y T E C Y C L I C A M P LEVELS (pmol/106 CELLS)

L ymphocytes Mean

Granulocytes S.E.M.

N

Mean

S.E.M.

N

Baseline Isoproterenol 10-8 m o l / l 10-7 mol/1 10"~ mol/1 10-5 m o l / l 10-~ m o l / l

5.66

0.57

65

0.98

0.23

50

6.69 8.74 14.83 19.47 20.12

0.70 0.82 1.58 1.99 2.00

64 64 63 63 60

0.99 1.18 1.85 2.46 2.66

0.24 0.24 0.30 0.38 0.38

50 50 50 50 49

Prostaglandin E t 10-8 m o l / l 10-7 m o l / l 10-~ m o l / l 10-5 m o l / l

7.71 14.16 34.79 49.49

0.80 1.30 3.45 5.01

64 65 64 63

1.07 1.56 2.96 4.01

0.20 0.25 0.46 0.58

49 49 49 45

35

RESULTS

Beta-adrenergic responses The mean cyclic A M P contents of lymphocytes and granulocytes under basal conditions and upon stimulation with isoproterenol or P G E 1 are shown in Table II. Intracellular cyclic AMP content increases with increasing concentrations o f isoproterenol and P G E r Stimulation of cyclic AMP production by isoproterenol was rapid (maximal reached in 3--5 min), almost entirely (>/85°/0) propranolol-inhibitable, and dose-related. Correlations with age for baseline cyclic AMP and stimulation ratios are presented in Table III. There are no changes in the lymphocyte stimulation ratios with age at isoproterenol concentrations 10-8 mol/l, 10-7 tool/l, 10-6 mol/l or 10-5 mol/1. The lymphocyte cyclic AMP response to isoproterenol at 10-4 mol/l is weakly but significantly positively correlated with age. The ECs0 for isoproterenol was not significantly correlated with age (r = - 0.15; P = 0.23). There is a weak but significant positive correlation with age for the lymphocyte cyclic AMP response to PGEI at 10-7 mol/l to 10-5 mol/l. Mean lymphocyte stimulation ratios for PGE1 at 10-5 mol/l and for isoproterenol at 10-4 tool/1 are plotted at 10 year age cohorts in Fig. 1. For PGE~, the rise with age begins with the 48--57-year-old cohort. For isoproterenol, the relationship is less regular, but is seen primarily in the last two age cohorts. Plots of the mean stimulation ratios by age cohort for the other concentrations of

T A B L E III CORRELATION

BETWEEN LEUKOCYTE CYCLIC AMP RESPONSES AND AGE

Lymphocytes

Granulocytes

r

n

- 0.04

65

0.15

50

10-B tool/1 10-7 m o l / l 10~ m o l / 1 10 -s tool/1

0.05 0.02 0.18 0.21

64 64 63 63

0.04 - 0.12 - 0.01

50 50 50 50

10 -4 m o l / l

0.32*

60

- 0.03

49

0.03 0.32** 0.38** 0.40**

64 65 64 63

-

49 49 49 45

Baseline cyclic A M P Isoproterenol stimulation ratio

P r o s t a g l a n d i n E~ s t i m u l a t i o n r a t i o 10 -s t o o l / ! l 0 -7 m o l / l l 0 "~ m o l / l l 0 -s tool/1 *P< 0.05. **P< 0.01.

r

7/

0.13

0.22 0.13 0.02 0.02

36 20.

A / O

/

16.

, m

I

o

rY

J

12.

PGE

~

.A

a i

O

° i

A ~

o

A----A ~

8.

A---__ A

E 00

4.

18-'27 28L37 38'-47 48'-57 58'-67 Age Cohort

6/;+

Fig. 1. Mean lymphocyte stimulation ratio for prostaglandin E~ at 10-~ mol/1 (PGE) and isoproterenol at 10"4 m o l / l (ISO), by 10 year age cohort.

P G E 1 and isoproterenol revealed no hidden non-linear relationships with age. There is no correlation between age and stimulation ratio for isoproterenol or PGE~ in granulocytes. There were no sex differences in the stimulation ratios for either cell type at any age. A m o n g the subjects over 65 years of age, 15 had taken no medications but hormone replacement for at least 2 weeks but six had taken some medications (see methods) not thought to interfere with beta-adrenergic responses. Elimination of these six subjects f r o m the sample did not affect the results. Moreover, comparison of the unmedicated and medicated elderly subjects revealed no difference in basal or stimulated cyclic A M P values or isoproterenol stimulation ratios. Within each cell type, the cyclic A M P responses to different concentrations of isoproterenol are highly intercorrelated at higher concentrations (10-6--10 -4 mol/1 isoproterenol, r = 0.78--0.84 for lymphocytes and r = 0.78--0.90 in granulocytes). Weaker but still highly significant correlations occur within cell type among the cyclic A M P responses to lower concentrations (10 -8 mol/l and 10-7 mol/1) and the higher (r = 0.33--0.59, p < 0.01). Similarly, cyclic A M P responses to PGE~ at the different concentrations are intercorrelated (r = 0.29--0.94 in lymphocytes; r = 0.32--0.90 in granulocytes, P < 0.01). Statistically significant correlations exist among lymphocyte cyclic A M P responses to PGE~ and isoproterenol at higher concentrations (r = 0.44--0.80, P < 0.01), that is, the subjects with greater cyclic A M P response to isoproterenol also had greater cyclic A M P response to PGE~. The responses to PGE1 and isoproterenol at higher concentrations are positively correlated in granulocytes as well (r = 0.54--0.74, P < 0.01). There are no significant

37 TABLE IV CORRELATION BETWEEN LEUKOCYTE BETA-ADRENERGIC RECEPTOR PROPERTIES A N D AGE

Lymphocytes

Granulocytes

r

Receptor density Antagonist binding

n

0.13 - 0.34

31 32

r

- 0.49 - 0.16

n

13 13

correlations for stimulation ratios across cell type when different agonists were used (i.e., lymphocyte isoproterenol with granulocyte PGE~) or for P G E r The lymphocyte and granulocyte responses to high concentrations of isoproterenol are weakly correlated (r = 0.35 for 10-5 mol/l; r = 0.37 for 10-4 mol/l, P < 0.05). In both lymphocytes and granulocytes, the stimulation ratios for isproterenol and for PGE~ are significantly negatively correlated with the basal cyclic A M P level; that is, those with higher basal cyclic A M P levels tend to have lower stimulation ratios. Receptors The mean receptor density is 44.7 (S.E.M. 5.5, n = 31) f m o l / m g protein in lymphocytes and 46.6 f m o l / m g protein in granulocytes (S.E.M. 7.4, n = 13). The K o for [3H]DHA is 2.1 nmol/l in lymphocytes (S.E.M. 0.3, n = 32) and 2.4 nmol/l in granulocytes (S.E.M. 0.5, n = 13) These values are comparable to those reported previously [34]. Neither the K o for [3HIDHA nor the receptor density in lymphocyte or granulocyte membranes is significantly correlated with age (Table IV).

5.

C o co u 32

4 3. 2. II b

I ~-~ 0 o_ o-I

•

•

••

,6

e•

~-~--2 C-3 I0

2'0 3'0 4'0 5'0 6'0 7'0 8'0 9'0 Age in Years

I()0

Fig. 2. Plot of In(slope)methacholine by age (n = 72).

38

M e t h a c h o l i n e challenge tests

The natural logarithm transformed slope of the methacholine dose-response relationship is plotted by age in Fig. 2. The airway responsiveness of elderly subjects is comparable to that of the young. The correlation between age and airway reactivity (r = 0.01) is not significantly different from zero (P = 0.96, n = 73). Neither betaadrenergic responses nor receptor properties of lymphocytes or granulocytes correlate significantly with airway reactivity. DISCUSSION

The cyclic AMP response to isoproterenol in lymphocytes and granulocytes does not decline with age in our sample. In fact, the lymphocyte responses to high concentrations of receptor agonists correlate positively with age (Table III). This result is unexpected, because several laboratories have reported reduced cyclic AMP response to isoproterenol in the lymphocytes and granulocytes of elderly subjects [1,15,24,25,26,32], and because the elevated circulating catecholamines reported in the elderly [42] would be expected to produce down regulation of leukocyte betaadrenergic systems. Small sample size or systematic laboratory bias is unlikely to have produced these results for 27 subjects over 65 years of age were studied (more than in most other studies), and young and elderly subjects were studied simultaneously. Our basal [22,23,43] and stimulated [23,42] cyclic AMP values (Table I) are in the range reported by others, suggesting that our assay system is comparable to those of laboratories who found reduced responses to isoproterenol in the elderly. Absolute cellular cyclic AMP content also does not change with age in our sample, so the difference between our study and others cannot be attributed to the manner of data expression. It is possible that our elderly subjects, all of whom were ambulatory and living independently, were healthier than those studied by others, some of whom were institutionalized or required "day care". Indeed, evidence has been presented for reduced beta-adrenergic responses in ill elderly people [28]. Our subject group may display a "survivor effect", that is, elderly people who meet our criteria for entry into the study (ambulatory, free of interfering medication, normal baseline pulmonary function) may be especially physically fit. We suggest, therefore, that aging p e r se does not affect lymphocyte beta-adrenergic responses, or that wellmaintained beta-adrenergic responses may be a marker of successful aging. Our findings are in agreement with other studies which found no difference in lymphocyte cyclic AMP responses to isoproterenol between healthy young and elderly subjects [23,43]. We also found no decline with age in the cyclic AMP response to PGE~ in lymphocytes or granulocytes (Table III), in contrast to previous reports [42,44]. Subject selection is the most likely explanation for this discrepancy as well. There is a positive but weak correlation (r = 0.35--0.37) between lymphocyte and granulocyte cyclic AMP responses to high concentrations of isoproterenol in

39 this sample. Although each of these systems has been used as a model for the biochemistry of less accessible systems, their relationship to one another has not been established previously. Our data suggest that results in one system may not reflect results in the other, at least for the cyclic AMP response. Since both receptor systems are of the beta2-adrenergic class, the absence of a stronger correlation was unexpected. Nevertheless, the two beta-adrenergic systems differ both in the maximal cyclic AMP response achieved (greater in lymphocytes, when expressed as a percent of baseline) and the lower ECs0 for lymphocytes. Differences in the susceptibility of the beta-adrenergic receptor to down regulation by circulating catecholamines, or differences in counter-regulatory systems for intracellular cyclic AMP may account for the lack of close correlation between lymphocyte and granulocyte beta-adrenergic responsiveness. The latter possibility seems most attractive; basal cyclic AMP levels are "set" lower in granulocytes than in lymphocytes. However, our results also suggest that for either lymphocytes or granulocytes, the cyclic AMP response at either 10-4 mol/l, 10-5 mol/l or 10-6 mol/1 isoproterenol is a good index of maximal response to beta-adrenergic agents, but that submaximal responses need to be measured independently. In this sample of healthy adults, there is no relation between lymphocyte cyclic AMP response to isoproterenol and airway reactivity. In parents of children with cystic fibrosis, reduced beta-adrenergic responses are correlated with increased bronchial reactivity to methacholine [7]. However, for asthmatics and their healthy controls (under 45 years of age), no relationship could be established between leukocyte beta-adrenergic responses and airway reactivity [8]. The present study shows that, for a sample from a healthy population, intrinsic variations in beta-adrenergic responses as can be assessed in leukocytes are not significantly related to airway reactivity. This may be because leukocytes are a poor model for the beta-adrenergic contribution to airway reactivity or because the autonomic contributions to airway reactivity in the absence of disease are minimal. Even if there is no correlation of airway reactivity with leukocyte cyclic AMP responses to beta-adrenergic agents, beta-adrenergic receptor concentration might still be related. In leukocytes, the number of beta-adrenergic receptors is greatly in excess of that required for maximal adenylate cyclase stimulation [15,34] and thus may vary considerably without substantially changing the cyclic AMP response. Betaadrenergic receptor concentration, more than cyclic AMP response of leukocytes, has been shown to correlate with the beta-adrenergic responses (and receptor properties) of internal organs [19,20,45,46]. Nevertheless, beta-adrenergic receptor concentration in lymphocytes or granulocytes is not significantly correlated with airway reactivity in this sample. This study was undertaken to see if the reported decline in beta-adrenergic responses of the elderly [21--28] is associated with the reported increase in airway reactivity [30,31]. However, we found no reduction in beta-adrenergic responses in granulocytes or lymphocytes, nor is airway reactivity increased in these healthy eld-

40 erly subjects. Malo e t al. [47] also found no age-related change in airway reactivity in a study o f healthy adults. Burney e t al. [30] found a substantial age-related increase in airway reactivity, largely among smokers. However, this study accepted subjects with poorer pulmonary function (FEV~ /> 60°70 predicted) than in our sample. Since older smokers are more likely to have reduced FEV~, and many studies have shown airway reactivity to be significantly correlated with baseline FEV~ (the lower the FEV~, the more likely bronchial reactivity), the age effect may have been confounded by the effect of reduced pulmonary function. H o p p e t al. [31] reported that in a nonatopic non-smoking population (but one in which baseline pulmonary function was not an exclusion criterion), airway reactivity was increased in the young and the old (over age 65 particularly). However, only 11 subjects over age 65 were studied, and though 148 subjects in all were enrolled, only 57 were over age 20. Our study was restricted to subjects with baseline FEV~ >i 8007o predicted (i.e., normal) and thus would not be expected to be confounded by baseline FEV~, but did not control for current or remote smoking (which may affect airway reactivity [48]) or for atopy (which, in the absence o f frank asthma, probably does not affect airway reactivity [48]). Study o f a larger number of healthy elderly subjects may clarify this issue. Our results suggest, however, that parallel changes in beta-adrenergic and airway reactivity need not occur with aging alone. ACKNOWLEDGMENTS We thank Gaye Paget, Kathy Vargo, Kristin McNelis, Susan Durda, and Andrea Billups for excellent technical assistance. This study was supported in part by grants from the Veterans Administration and the National Institutes of Health (AG04391, HL25830, HL28386, DK27651). REFERENCES 1 M. Kaliner, J. Shelhamer, L. Smith, P.B. Davis and J.C. Venter, NIH Conference. Autonomicnervous systemabnormalities and allergy. Ann. Intern. Med., 96 (1982) 349--357. 2 V.S. Douglas, P. Ridgwayand C. Brink, Airwayresponses of the guinea pig in vivo and in vitro. J. PharmacoL Exp Ther., 202 (1977) 116--124. 3 C.A. Hirschman, Bronchial inhalation challenge testing in dog models of asthma. In S.L. Spector (ed.), Provocative Challenge Procedures, Vol II; Bronchial Oral and Exercise, CRC Press, Boca Raton, Florida, 1983, pp. 13--32. 4 G.A. Rinard, A.M. Pucket, A.D. Jensen, K.L. Minneman, T.J. Torphy and S.E. Mayer, Pulmonary hyperreactivity of greyhound dogs to methacholine is related to beta-adrenergic-cholinergic sensitivity imbalance of airway smooth muscle. Am. Rev. Resp. Dis., 131 (1985) A340(Abstract). 5 G.A. Rinard, T.J. Torphy, A.D. ]ensen, A.M. Puckett, K.L. Minneman and S.E. Mayer, Pulmonary reactivity to ascaris is inversely related to tracheal smooth muscle beta receptor density and isoproterenol sensitivity, responsiveness and cyclic AMP dependent protein kinase in greyhound dogs. Am. Rev. Resp. Dis., 131 (1985)A340(Abstract). 6 J. Apold and L. Aksnes, Correlation between increased bronchial responsiveness to histamine and diminished plasma cyclic adenosine monophosphate response after epinephrine in asthmatic children. J. Allerg. Clin. lmmunol., 59 (1977) 343.

41 7 8 9

10

11 12

13

14

15 16

17 18

19

20

21 22 23 24 25 26

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