Beta Adrenergic Receptors of Polymorphonuclear

Particulates in Bronchial Asthma

STANLEYP. GALANT, LAKSHMI DURISETI, SHARONUNDERWOOD,SANDRAALLRED,and PAUL A. INSEL, Department of Pediatrics, University of California, Irvine,Irvine, California 92717; Department of Pediatrics, University of Utah, Salt LakeCity, Utah 84132; Division of Pharmacology, Departmetnt of Medicine, Univiersityof California, San Diego, San Diego, California 92093

A B S T RA C T Wehave tested the beta adrenergic re-ceptor theory of bronchial asthma by determining thenumber and affinity of binding sites of the beta adren-ergic radioligand [3H]dihydroalprenolol (DHA) and theactivity of adenylate cyclase in broken cell preparationsof polymorphonuclear leukocytes (PMN). We studied31 control subjects (group 1), 30 asthmatics receivingno systemic adrenergic medication (group 2), and 17asthmatics receiving adrenergic agonists systemically(group 3). Control subjects and asthmatics taking noadrenergic drugs bound similar amounts of DHA at0.5 nM and 30 nM DHAand had about 900 bindingsites per PMN. In contrast, asthmatics receiving adren-ergic agonists had a >70% decrease in their numberof DHAbinding sites per PMN(254+±57). In a subsetof our three groups of subjects (eight from group 1,six from group 2, and five from group 3) we measuredDHAbinding at several DHAconcentrations and foundsimilar values (0.4-0.7 nM) for the dissociation con-stant of DHAamong these subjects.

In further studies we examined the interaction of theagonist (-)-isoproterenol with beta adrenergic recep-tors in 8 normal subjects and 10 asthmatics not receiv-ing adrenergic medication. Wetested the ability of iso-proterenol to compete for DHAbinding sites and tostimulate adenylate cyclase in sonicates prepared fromPMNand examined under identical conditions. Thedissociation constants for the competition of isoprotere-nol for DHAbinding sites in normal and asthmaticsubjects were virtually identical (-1.0 ,M). In addi-

This work was presented in part at the Annual NIeeting ofthe American Federation for Clinical Research, 5-7 MaN1979, Washington, D. C.

Dr. Insel is an Established Investigator of the AmericanHeart Association. Dr. Galant is a recipient of an AllergicDiseases Academic Award (AI00304 from the NationalInstitute of Allergy and Infectious Diseases).

Received for publication 2 July2 1979 atnd in revised fortm8 October 1979.

577

tion, the (activation constant) values for stimulation ofadenylate cyclase were similar (0.16-0.19 u.tM) in thetwo groups of subjects.

Thus, these data suggest that asthma per se is notassociated with alteration in either the number or af-finity of beta adrenergic receptors in PMN. Our findingsindicate that previous reports of abnormal beta adren-ergic receptor function in asthmatic patients may inpart be explained by prior treatment of such patientswith adrenergic agonists. Because the asthmatics whoreceived adrenergic agonists in our study tended to bemore ill and to receive additional medication com-pared to subjects in group 2, we cannot rule out un-equivocally that severe asthma may be associated withdecreased binding to beta adrenergic receptors. Never-theless, we conclude that beta adrenergic receptorson PMNfrom asthmatics are relatively normal unlesssuch patients are treated with adrenergic agonists.

INTRODUCTION

In 1968, Szentivanyi (1) postulated that the under-lying defect in atopic diseases, particularly bronchialasthma, is an imbalance of the autonomic nervous sys-tem. He suggested that beta adrenergic receptor func-tion is decreased in asthmatic patients and that thisresults in increased alpha adrenergic and cholinergicresponses to a variety of stimuli. Certain evidence hasbeen reported that appears to confirm the possibilityof beta adrenergic blockade in pulmonary (2, 3) andextrapulmonary tissues in asthmatics (4). A consequenceof this blockade would be decreased cellular cyclicAMP, which in the lung would promote bronchospasmand mediator release (5, 6). Because it is difficult tostudy samples of lung in human subjects, several in-vestigators have used either peripheral blood leuko-cytes or lymphocytes to show that asthmatics have de-creased stimulation of cyclic AMPby beta adrenergicagonists (7, 8). Two problems must be considered in

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. (0021-9738/80/03/0577109 $1.00Volumlle 65 March 1980 577-585

assessing these studies: (a) beta adrenergic responsesand receptors in T and B lymphocytes may be quitedifferent (9, 10), and thus changes in adrenergic re-sponse in asthmatics might reflect altered distributionof cells in lymphocyte subpopulations; and (b) prioradministration of beta adrenergic agonists reportedlydiminishes subsequent beta adrenergic stimulation ofleukocyte cyclic AMP(11-13). Thus, changes in re-sponsiveness of asthmatics might arise from therapy aswell as from the disease itself. Some authors (7) haveattempted to consider these problems in studies withlymphocytes.

Neutrophilic polymorphonuclear cells (PMN)1 maybe more suitable than lymphocytes for clinical studiesof beta adrenergic receptors, because PMNappear tobe a more homogeneous population than are lympho-cytes. In addition, PMNhave well-defined beta adren-ergic responses that have been reported to be decreasedin asthmatics (14-16). Recently we have used [3H]di-hydroalprenolol (DHA), a radiolabeled beta adrenergicantagonist to characterize beta adrenergic binding sites(i.e., receptors) on PMNparticulates prepared fromnormal subjects (17). In this study we have comparedDHAbinding of control subjects with binding of asth-matics who either were or were not receiving adren-ergic agents. In addition, we have compared the abilityof the agonist isoproterenol to compete for DHAbind-ing sites and to stimulate adenylate cyclase in brokenPMNfrom controls and asthmatics. Wefind that asth-matics have only minimal alterations in PMNbetaadrenergic receptors unless the patients are receivingtherapy with beta adrenergic agonists.

METHODS

SubjectsSUBJECTSWEREDIVIDED INTO THREECATEGORIES

Group 1: control population. These were 31 healthyadults without evidence of atopic disease or asthma. The ageranged from 19 to 65 yr (mean age, 34) with 13 males and18 females.

Group 2: asthmatics receiving no beta adrenergic medica-tion. There were 30 asthmatics in this category who hadreceived no beta adrenergic drugs systemically for at least2 wk before the study (Table I). Three patients (Nos. 20, 28,and 29) used a preparation of inhaled metaproterenol infre-quently and had not used it for 48 h before testing. Patientsin this group were allowed theophylline, cromolyn sodium,or corticosteroids as necessary. This group was comprised of15 males and 15 females with an age range from 15 to 64(mean age, 36). The duration of asthma ranged from 1 to 40yr (mean, 16 yr).

Group 3: asthmatics receiving beta adrenergic medication.This group consisted of 17 patients who were receiving betaadrenergic medication at therapeutic dosage in addition to

1Abbreviations used in this paper: DHA, [3H]dihydro-alprenolol; PMN, polymorphonuclear cells.

the medication mentioned in group 2 (Table II). There werenine males and eight females with an age range of 18 to 60(mean age, 35 yr). The duration of asthma ranged from 1 to 58yr (mean, 17 yr).

Selection of criteriaThe subjects in all three groups were carefully screened

to exclude individuals with active viral infections, those re-ceiving birth control medication, cardiac glycosides, anti-hypertensive agents, or other sympathomimetic containingmedication. All asthmatics in this study had previously dem-onstrated reversible airway disease by pulmonary functiontesting.

Grading of asthma severity and activitySeverity of disease was graded on the basis of the overall

course of asthma with the following criteria: (a) steroid usage,(b) requirement for bronchodilation therapy to control asthma,(c) frequency and persistence of asthma exacerbations, and(d) hospitalization. From these criteria asthmatics were identi-fied as mild, moderate, or severe. Mild asthmatics could becontrolled with intermittent bronchodilator therapy. Patientsin the moderate category required daily bronchodilator ther-apy and short courses of steroids for acute exacerbations. Thesevere asthmatics required frequent treatment (more than onecourse per month) with steroids, daily bronchodilators, andhad persistent wheezing with frequent exacerbations of asthmaoften requiring hospitalization. The activity of disease wasscored as 0 to 3, where 0 meant that no wheezing had oc-curred within 3 mo, 1 indicated wheezing between 14 d and3 mo, 2 signified wheezing within 2 wk, and 3 indicated acuteasthma at the time of the study.

Preparation of PMNparticulates60 ml of heparinized blood was obtained from each subject

in the study. Patients on adrenergic therapy were sampledgenerally 2-4 h after taking their medication. Blood was cen-trifuged on a Ficoll-Hypaque (Pharmacia Fine Chemicals,Piscataway, N. J.) density gradient according to the methodof Boyum (18). Particulates from PMN(90±3.5% SD of finalcells obtained) were prepared as described (17) or by sonicat-ing (Heat Systems-Ultrasonics, Inc., Plainview, N. Y.) cellsgently at a setting of two for 5 s. Freshly prepared particulatesor sonicates were used immediately in the binding studies andsonicates were used immediately in adenylate cyclase assays.

Beta adrenergic receptor assayDHABinding assays contained in a total volume of 0.15 ml:

DHA, PMNmembranes (-0.440 mg protein in which 1 mgprotein corresponds to 1.7 x 107 cells), 50 mMTris-HCl, pH7.5, 10 mMMgCl2, 1 mMascorbic acid, 0.3 mMcatechol, and0.1 mMphentolamine. Ascorbic acid was included to blockoxidation of catecholamines, and catechol and phentolaminewere added to inhibit nonspecific binding (17). These agentsdid not affect specific binding. Samples were incubated at37°C for 15 min. Incubations were terminated by rapidly dilut-ing samples with 2 ml of ice-cold incubation buffer and thenimmediately filtering the mixture through a glass fiber filter(Gelman A/E, Gelman Instruments Co., Ann Arbor, Mich.).The filters were presoaked in incubation buffer containing0.1 mM(±)-propranolol to block nonspecific binding of radio-activity to the filters. The filters were rapidly (10 s) washedwith 15 ml of ice-cold incubation buffer and the radioactivity

578 S. P. Galant, L. Duriseti, S. Underwood, S. Allred, and P. A. Insel

TABLE IAsthmatics not Receiving Beta Adrenergic Drugs (Group 2)

Subject Age Sex Duration Severity Activity Medications

1 50 M

2 34 M3 46 M4 30 F5 23 F6 28 M7 45 F8 40 F9 31 F

10 31 XI11 33 NI12 36 F13 15 F14 52 F15 28 F16 50 F17 25 M18 28 M19 29 M20 31 F

21 41 M22 28 F23 32 M24 39 M25 50 M

26 38 F

27 41 F28 29 F29 44 M

30 64 NI

yr

25 Severe

30 Mild40 Mild17 Moderate

8 Moderate2 Moderate

40 Mild20 Moderate30 Moderate30 Moderate10 Mild30 Moderate14 Moderate4 Severe

25 Mild1 Moderate6 Mild

20 Mild24 Mild

1 Moderate

18 Mild9 Mild

20 Moderate

3 Theophylline, beclomethasonedipropionate aerosol

1022221221222220133

331

NoneNoneTheophyllineTheophyllineTheophylline, cromolyn sodiumNoneTheophyllineTheophyllineTheophyllineNoneTheophyllineTheophyllineTheophyllineNoneTheophyllineNoneNoneNoneTheophylline, metaproterenol inhaler,

beclomethasone dipropionate inhalerNoneNoneTheophylline

1 Mild 0 None6 Moderate 1 Theophylline beclomethasone

dipropionate inhaler20 Moderate 1 Theophylline, beclomethasone

dipropionate10 Severe 2 Theophylline, prednisone20 Moderate 2 Theophylline, metaproterenol inhaler

4 Severe 2 Theophylline, prednisone,

2 Severe

metaproterenol, beclomethasonedipropionate

3 Theophylline, beclomethasonedipropionate

of the dried filters was determined in a liquid scintillation taining Tris-HCl, pH 7.5,50 mM; MgCl2, 5mniM; dithiothreitol,system (3H efficiency = 45%). Specific binding represents the 1 mM; [a-32P]ATP, 1 mMand 1-2 x 106 cpm; cyclic AMP,total amount of radioactive DHAbound minus the amount 0.5 mM; 3-isobutyl-1-methylxanthine, 0.2 mM; phosphoenol-of DHAbound in the presence of 1 ,uM (+)-propranolol; spe- pyruvate, 16 mM; pyruvate kinase, 50 ,ug/ml; ascorbic acid,cific binding was 50-80% of the total amount bound at all DHA 1 mM; catechol, 0.3 mM; phentolamine, 0.1 mM; DHA, 10 nM;concentrations. All data shown in this paper are specific bind- PMNsonicate protein, 0.30-0.45 mg, and various additionsing. Specific binding was linear with protein over the con- as indicated. Routinely, samples were incubated with all re-centration range used in these experiments. All samples were actants except [32P]ATP (but including 1 mMnonradioactiverun as duplicates and these differed from each other by < 15%. ATP) for 15 min at 370C, at which time equilibrium of DHAProtein was measured by the method of Lowry et al. (19) binding was achieved. [32P]ATP was then added to the sampleswith bovine serum albumin standards. in some experiments and the incubation was continued for

an additional 15 min. Samples for DHAbinding were thenfiltered and washed as described above, and in those experi-

Adenylate cyclase assays ments in which [32P]ATP had been added, the total filter-bound radioactivity was corrected for counts of 32P retained

In this study, adenylate cyclase assays were performed con- on the filters (generally <100 cpm). For samples in whichcurrently with DHAbinding studies under identical condi- adenylate cyclase activity was measured, 0.15 ml of 0.33 Ntions. Samples were incubated in a total volume of 0.17 ml con- HCl was added and cyclic AMPwas then isolated by sequen-

Neutrophil Beta Adretnergic Receptors in Asthma79579

TABLE IIAsthmatic Receiviing Beta Adrentergic Drugs (Group 3)

Subject Age Sex Duration Severity Activity Medications

1 47 M 1 Severe2 37 M 35 Severe

3 32 M 2 Severe

4 60 F 58 Severe

5 31 F 30 Severe

6 37 M 20 Mild7 47 M 18 Severe8 47 M 10 Severe

9 20 F 3 Severe

10 24 M 20 Severe11 30 F 22 Severe

12 36 F 23 Severe

13 35 M 7 Severe

2 Terbutaline 5 mg/q6h, theophylline3 Terbutaline 2.5 mg q6h, theophylline,

prednisone2 Terbutaline 5 mg q6h, theophylline,

cromolyn sodium, prednisone qod3 Terbutaline 5 mg tid, metaproterenol inhaler

(2) tid, theophylline, methylprednisolone2 Terbutaline 5 milg q8h, theophylline,

prednisone (lod, beclomethasonedipropionate aerosol

2 Terbutaline 2.5 mg bid2 Terbutaline 2.5 mg qid2 Ephedrine 25 mg (16h, theophylline,

cortisone daily, beclomethasonedipropionate

2 Ephedrine 25 mg (lid, metaproterenol inhaler(1-2) qid

2 Terbutaline 2.5 mg q6h, theophylline2 Metaproterenol (20 mg) 1 tid isoetharine

inhaler bid, theophylline, beclomethasonedipropionate aerosol

2 Terbutaline 5 mig tid, theophylline,beclomethasone dipropionate aerosol

3 Terbutaline 5 mig tid, fenoterol inhaler tid,metaproterenol inhaler tid, theophylline,beclomethasone dipropionate inhaler,prednisone

14 50 M 8 Moderate 2 Terbutaline 5 mg tid, prednisone15 18 F 10 Moderate 3 Terbutaline 5 mg tid, beclomethasone

dipropionate inhaler, metaproterenol16 18 F 5 Moderate 2 Terbutaline 5 mg tid, beclomethasone

dipropionate inhaler, theophylline17 25 F 23 Severe 3 Terbutaline 5 mg tid, prednisone,

theophylline

tial chromatography on Dowex AG 50 WX4and neutral alu-mina, as described by Salomon et al. (20). Recovery of cyclic[32P]AMP (determined by addition of cyclic [3H]AMP) wasgenerally 70-90%. Radioactivity was measured in eluatesfrom the alumina column counted in Aquasol-2 solution (NewEngland Nuclear, Boston, Mass.) using a Beckman LS 8000scintillation counter (Beckman Instruments, Inc., Spinco Div.,Palo Alto, Calif.). Adenylate cyclase activity was linear for30 min at 37°C at the concentrations of protein used in theseexperiments. All experiments were performed in duplicate,and the variability between samples was usually between10 and 15% or less. Under the conditions used, the maximallevel of isoproterenol-stimulated adenylate cyclase activitywas generally 100-150 pmol cyclic AMPgenerated/min permgprotein and the specific activity of DHAbound was 10- 15fmol/mg protein.

Competition of isoproterenol for DHAbinding sites wasexpressed as a percentage of that competed by 1 AM(+)-pro-pranolol (i.e., 100% specific DHAbinding). The dissociationconstant for isoproterenol (Kd isoproterenol) was calculatedas described by Cheng and Prusoff (21) with the equation:

Kd isoproterenol = 150( d DHAS + Kd DHA

where h means the concentration of isoproterenol competingfor 50% of the specific DHAbinding sites, Kd DHAwas as-sumed to be 0.37 and 0.39 nM in asthmatics and controls,respectively (see below), and S was 10 nM, the DHAconcen-tration used in these experiments.

The potency of (-)-isoproterenol in stimulating adenylatecyclase activity was determined graphically and expressedas the Kac., where the observed concentration producing a50% maximal stimulation (EC50) was corrected for the DHAincluded in the incubation by the equation shown above.

MaterialsMost reagents were obtained from sources listed previously

(17). Dithiothreitol, phosphoenolpyruvate, cyclic AMP, ATP,and neutral alumina were purchased from Sigma ChemicalCo. (St. Louis, Mo.) Dowex AG5OWx4, other reagents were

580 S. P. Galant, L. Duriseti, S. Underwood, S. Allred, and P. A. Insel

purchased as follows: Bio-Rad Laboratories (Richmond, Calif.);pyruvate kinase, Boehringer Mannheim Biochemicals (Indian-apolis, Ind.); [32P]ATP (10 Ci/mmol) and [3H]cyclic AMP(37.7Ci/mmol), New England Nuclear, and 3-isobutyl-1-methyl-xanthine, Aldrich Chemical Co., Inc. (Milwaukee, Wis.).

RESULTS

Clinical data. The clinical data of asthmatic pa-tients on nonadrenergic drugs (group 2) is shown inTable I, whereas data for those taking adrenergic andnonadrenergic drugs (group 3) is in Table II. The pa-tients appeared to be reasonably well-matched for age,sex, and duration of illness, as noted above. Subjectsin group 3 had more severe disease (13 of 17 subjectsvs. 5 of 22 in group 2) and greater activity of theirasthma than those in group 2 (disease activity scoresof 1.77+0.87, mean+SD, for group 2 and 2.29±0.47 forgroup 3, one-tailed P < 0.05). 12 patients in group3, and 7 in group 2 received corticosteroids, and 12 of17 patients in group 3 and 19 of 30 patients in group 2received theophylline derivatives. The most commonlyused adrenergic agent by the patients in group 3 wasterbutaline, which 14 of the 17 patients ingested atdoses ranging from 5 to 20 mg/d. Other adrenergic drugsused by patients in group 3 included ephedrine, meta-proterenol, isoproterenol, fenoterol, and isoetharine.

DHAbinding studies. To determine properties ofbeta adrenergic receptors in the three groups of sub-jects, we conducted studies to examine receptor bind-ing (affinity and number of receptors per PMN) as wellas receptor function (activation of adenylate cyclase).In studies of receptor affinity we performed DHAbind-ing experiments in eight control subjects, six asthmaticsreceiving no adrenergic medication (subjects 17-22,Table I), and 5 asthmatics receiving adrenergic agonists(subjects 13-17, Table II). Mean data for these threegroups are shown in Fig. 1. Specific binding was sat-urated and could be resolved into one class of bindingsites (r by least squares fit = 0.94, 0.99, and 0.96 forgroups 1, 2, and 3, respectively) on Scatchard analysis(Fig. 1, inset). The mean data shown in Fig. 1 yielda dissociation constant for DHAof 0.39, 0.30, and 0.49nM for groups 1, 2, and 3, respectively. When individ-ual studies on the 19 subjects were analyzed by Scatch-ard plots (all r values for regression lines were -0.80)and the individual dissociation constants pooled forthe three groups, the population values were 0.41±0.05,0.38±0.06, and 0.72+0.22 nM (mean±SEM). Thevalues for the dissociation constant for the subjectsin group 3 were not statistically different when com-pared to those for subjects in group 1 or 2 (P > 0.05).These values for the dissociation constant for DHAare somewhat lower than we reported previously (1-5nM [17]) and may reflect the fact that the data in Fig. 1was obtained using sonicates rather than with mem-

[DHAI nM

FIGURE 1 Saturability of DHAbinding to PMNparticulates.Specific DHAbound (femtomoles per milligram protein) toPMNparticulates from eight control subjects (A), six asth-matics not receiving systemic adrenergic agonists (0), and fiveasthmatics receiving adrenergic agonists (0) is plotted on theordinate, and the total DHAconcentration in each sample ison the abscissa. The data shown are the mean±SEM. Theinset shows a Scatchard plot of DHAbound (femtomoles permilliliter) using the mean data from the figure for each groupof subjects. The lines in the inset are the regression linesdetermined by least squares fit for each group (values statedin text).

brane particulates, which were used in our earlier studyand in the studies shown in Fig. 2.

Data for maximal number of DHAbinding sites inthese subjects were obtained in two ways: (a) Scatchardanalysis of data for the subjects depicted in Fig. 1 and(b) DHAbinding determined in a larger group of 26controls, 22 subjects in grouip 2 (subjects 1-22), and17 subjects in group 3 at a single concentration of DHA(30 nM). This concentration appears to be close to sat-urating for these three groups of subjects (Fig. 1 and [17]).

Data derived from the Scatchard analyses yieldvalues of 24.2±3.2, 23.1+2.0, and 10.1±1.2 finol/mg

cX cz 2

In-

2 3 2 3GROUP GROUP

J

n

z

m

FIGURE 2 DHAbindinig to PMIN particulates at 0.5 nM (leftpanel) and 30 nM DHA (right panel). DHA bindinig toPMNparticulates is plotted as either femntomiioles per milli-gram protein or as bindling sites per cell for 26 control sub-jects, 22 asthmatics receiving n1o systemic adlrenergic therapy,and 17 asthmatics receiving aclrenergic agonists. The datashown are meanl +SEM for each group.

Neutrophil Beta Adrenergic Receptors in Asthla 58581

PMNparticulate protein (mean±SEM) in groups 1, 2,and 3, respectively. The number of binding sites ingroup 3 was significantly lower (P < 0.005) than thatin either group 1 or 2.

The values for the DHAbinding studies in each ofthe three groups of subjects performed at 30 nM DHA(Fig. 2, right panel) were 25.1±1.8, 27.6+1.7, and7.6±1.7 (mean±SEM) for controls, asthmatics takingno adrenergic agonists, and asthmatics receiving adren-ergic agonists. These values correspond to 838±60,922+57, and 254±57 binding sites per PMNin the threegroups. Values for control subjects and asthmatics re-ceiving no adrenergic medication were similar (P> 0.1), whereas asthmatics receiving asthmatic agonistshad significantly less binding at 30 nMDHA(P < 0.001compared to both of the other groups). The amount ofDHA bound at 30 nM DHA was not significantlycorrelated with duration of asthma in patients in eithergroup 2 or 3 (P > 0.05). Subjects in group 2 whoreceived theophylline derivatives bound equivalentamounts of DHA(29.6±2.1 fmol/mg) compared to thosenot receiving theophylline (25.9±2.7 fmol/mg, P > 0.1).Further analysis of these data with respect to sex or ageof subjects in the groups yielded no significantdifference between males and females (P > 0.1) andinsignificant correlation of binding at 30 nMDHAwithage (P > 0.05) in both male and female subjects in allthree groups.

When the number of DHAbinding sites in group 3was determined separately for subjects who receivedor did not receive therapy with corticosteroids, thebinding in the 12 subjects receiving steroids was 8.3± 1.8 fmol/mg, a value not significantly different fromthe amount of DHAbound in the five subjects notreceiving steroids ([5.8±3.9 fmol/mg], P > 0.2).

As an additional experiment to determine if a popula-tion of asthmatic subjects might have differences inbinding of DHA, we measured binding at 0.5 nMDHAin the same 26 control subjects, 22 subjects in group 2,and 17 subjects in group 3 in whomwe measured bind-ing at 30 nM DHA(Fig. 2, left panel). Again, subjectsin group 1 and 2 showed nearly identical amounts ofDHAbinding, whereas subjects in group 3 bound sig-nificantly (P < 0.01) less DHAthan those in either ofthe two other groups.

Although the data presented thus far indicated in-significant differences for binding of the beta adren-ergic antagonist DHAin control subjects and asthmaticsnot receiving adrenergic therapy, it was important todetermine if asthmatics might show an altered affinityfor a beta adrenergic agonist. We compared 8 asth-matics from group 2 (subjects 23-30) and 10 controlsubjects (Fig. 3A). Competition for DHAbindings sitesby the beta adrenergic agonist (-)-isoproterenol wasvirtually identical in controls and asthmatics. The dis-sociation constant for isoproterenol calculated from theresults for individual studies of the subjects presented

10 100[(-)-ISOPROTERENOL], FM

FIGuRE 3 (A) Competition for DHAbinding sites by (-)-isoproterenol. (B) Stimulation of adenylate cyclase by (-)-isoproterenol in control and asthmatic subjects. DHAbindingand adenylate cyclase were examined concurrently as de-scribed in Methods. Data shown are mean±SEMfrom studieson 8 asthmatic (0) and 10 control subjects (0).

in Fig. 3 was 1.06+0.20 uM for control and 1.00+0.20,M for the asthmatic subjects.

Adenylate cyclase studies. As a further measure ofchanges in beta adrenergic receptors in asthma, wealso determined basal and isoproterenol-stimulatedadenylate cyclase activity in sonicates prepared fromPMNand measured under conditions identical to thoseused to measure competition of (-)-isoproterenol forDHAbinding sites (Fig. 3B). When plotted as percentmaximal adenylate cyclase activity, the curves are simi-lar for the two groups, and the Kact for (-)-isoproterenolcalculated to be 0.16±0.03 for control and 0.19±0.02for asthmatic subjects (P > 0.1). Although the absoluteactivity of basal adenylate cyclase (Fig. 4) is lower incontrol than in asthmatic subjects (42.0±4.9 vs. 53.5±6.8 pmol cAMPgenerated/min per mg, this differenceis not statistically significant [P > 0.1]) and bothgroups of subjects show about a threefold maximal in-crease in adenylate cyclase activity in response to iso-proterenol and similar responses at each concentrationtested. By comparison, the only subject from group 3(subject 14) whomwe tested had a lower basal activityand a blunted response to isoproterenol.

DISCUSSION

These data indicate that patients with bronchial asthmawho are not receiving systemic therapy with adrenergic

582 S. P. Galant, L. Duriseti, S. Uncderwvood, S. Allred, and P. A. Insel

175H

150

0-1 125

< C7.

d E 100

~3 r

E

!i 6 75Ji

E

> -az )50

25

- ~~~/e/

/_

0.1 10 100 1000

[H - ISOPROTERENOL], ; M

FIGURE 4 Adenylate cyclase activity in PMIN particulatesfrom control and asthmatic subjects. Adenylate cyclase activityassayed as described in Methods is plotted for basal ac-tivity (intercept on the ordinate) or in response to (-)-isoproterenol. Data shown are mean-SENI for 8 asthmaticson no systemic adrenergic therapy (0), 10 controls (0) and1 asthmatic (A) who received terbutaline.

agonist have beta adrenergic receptors on their PMNwith the following similar properties compared to con-trol subjects: affinity for the radioligand DHAand forthe agonist isoproterenol, number of beta adrenergicreceptors, and beta adrenergic stimulated adenylatecyclase activity. In contrast, asthmatics receivingadrenergic agonists systemically have decreased num-bers of beta adrenergic binding sites, and decreasedisoproterenol-stimulated adenylate cyclase activity inthe single subject in whom we measured enzymaticactivity. Even though beta adrenergic binding sites aredecreased about 70% by adrenergic therapy, the af-finity of the remaining binding sites for DHAis similarto the value found in controls and asthmatics not re-ceiving adrenergic agonists. The preferential decreasein number of DHAbinding sites in subjects receivingadrenergic agonists is consistent with studies showingthat the number, but not the affinity, of radioligandbinding sites is decreased after treatment of animalsor cells with beta adrenergic agonists (22-26).

Our data offer further evidence that systemic treat-ment of patients with beta adrenergic agonists appearsto "down regulate" receptors for beta adrenergicamines (27). Wehave recently shown that asthmaticswith moderate to severe asthma, comparable to group3 in this study, have normal binding 1 wk after adren-ergic therapy is discontinued (27). This would suggestthat it is the adrenergic therapy rather than the severityor duration of asthma, which accounts for the decreasedbeta adrenergic binding in the asthmatics in group 3compared to those in group 2 with milder asthma orcompared to the control population. Because we havefound that asthma in patients withdrawn from adren-ergic medication worsened, we did not feel justifiedin repeating this maneuver with the present subjects (27).

Therefore, because we did not withdraw therapyfrom subjects in group 3 and because our subjects ingroup 3 were more ill than those in group 2 and werereceiving additional medication (e.g., 0 of 17 subjectsin group 3 but 11 of 30 subjects in group 2 receivedno medication for their asthma), we cannot state un-equivocally that therapy with catecholamine agonists,rather than disease severity, is exclusively responsiblefor the decreased binding in the subjects in group 3.Furthermore, although our findings indicate that treat-ment with corticosteroids produced no significant in-crease in the number of beta adrenergic receptors inthe asthmatic patients receiving adrenergic agonists,the number of subjects examined was small. More com-plete studies involving treatment and withdrawal ofcorticosteroids from normals and asthmatics will berequired to ascertain whether such treatment altersproperties of beta adrenergic receptors in granulocytes.

It is difficult to be certain that our findings for betaadrenergic receptors in PMNderived from asthmaticsnecessarily reflect the status of receptors from othertissues involved in the pathophysiology of asthma.However, Conolly and Greenacre (28) have shown thathuman lymphocytes and human lung parenchyma havebeta2-type adrenergic responses to several adrenergicagonists and antagonists and have suggested that periph-eral blood lymphocytes may be a reasonable marker tis-sue for receptors in the lung. Beta adrenergic receptorson human PMNalso appear to be of the beta2 type(17). If peripheral blood cells can be considered validindicators of the state of adrenergic receptors in thebronchopulmonary tree, then our results would suggestthat asthmatics may also have normal numbers of pul-monary beta adrenergic receptors. Further proof of thiswould require direct study of receptors in pulmonarytissue in humans; such studies are not currentlyfeasible.

In an earlier study, Sokol and Beall (29) reportedthat asthmatics and control subjects had similar num-bers of beta adrenergic receptors. In that study theauthors identified beta adrenergic receptors by using[3H]epinephrine, a method that was later shown to pre-dominantly represent interaction of oxidized catechol-amine derivatives with tissue macromolecules (23).Thus, our results offer more definitive evidence of thesimilarity of beta adrenergic receptor number in asth-matics and controls.

In addition our results indicate that beta adrenergicreceptor affinity and function (i.e., activation of adenyl-ate cyclase) are similar in PMNfrom asthmatic andcontrol subjects. In this regard, we should note therelationship between the Kd isoproterenol and Kact iso-proterenol in our subjects. Wefind that the agonist ismore potent in activating adenylate cyclase than incompeting with DHA for the receptors. It has beenproposed that if binding to receptors and activationof adenylate cyclase are examined under identical con-

Netitrophil Beta Acirenergic Receptors in Asthmna

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ditions, the relationship between Kd and Kact offers anindex of the efficiency of coupling between beta adren-ergic receptors and adenylate cyclase (23). Wethere-fore attempted to perform these two assays concurrentlywith the same conditions. Our findings indicate thatbeta adrenergic receptors in the PMNmembrane arecoupled with high efficiency to adenylate cyclase witha Kd/Kact in control subjects of 6.6 and in asthmaticsnot receiving systemic adrenergic medication of 5.3.These values for PMNparticulates are much higherthan has been observed for most other tissues withbeta adrenergic receptor coupled adenylate cyclase,and are similar to values observed for S49 lymphomacells, a model system demonstrating highly efficientcoupling (23, 30). An alternative way of interpretingthe relationship between Kd and Kact in PMNis thepresence of spare receptors such that only a small frac-tion of receptors need to be occupied to produce fullresponse.

Our data on asthmatic subjects conflict with two re-cent reports (31, 32), which showed that lymphocytesfrom asthmatics have decreased DHAbinding even inthe absence of adrenergic therapy. Additionally, ourfinding of statistically insignificant changes in numberof PMNbeta adrenergic receptor with increasing agecontradict the findings of Schocken and Roth (33) whoobserved a decrease in number of DHAbinding siteson lymphocytes as a function of increasing age. Be-cause recent evidence indicating differences in betaadrenergic receptors in lymphocyte subpopulations(10), it is possible that the data in lymphocytes maybe explained by differences in distribution of lympho-cyte subclasses in asthmatics and older people. Al-ternatively, we have to consider the possibility thatdata obtained with PMNmay not necessarily applyto other beta adrenergic receptors in the body.

The preliminary findings recently reported by Sanoet al. (34) suggest that this theoretical possibility may,in fact, apply. Using a different radioligand ([1251]iodo-hydroxybenzylpindolol), Sano et al. found resultsidentical to ours in granulocytes but noted that lympho-cytes showed a decrease in number of beta adrenergicreceptors even in asthmatics who did not receive adren-ergic agonists. More complete characteriztion of betaadrenergic receptors in other tissues will be necessaryto determine the validity of results obtained usingvarious types of blood cells.

With respect to asthma and PMN, Busse and Sosman(35) have reported that intact PMNfrom asthmatic sub-jects who have been withdrawn from adrenergic therapyfor at least 2 wk have decreased histamine and iso-proterenol-stimulated cyclic AMPaccumulation (16,35). Busse and Sosman's results thus indicate thatPMNare altered in asthma independent of adrenergictherapy. In reconciling Busse and Sosman's findingsand other reports in the literature with those in this

study, we suggest that if beta adrenergic hyporespon-siveness occurs in intact leukocytes from patients withbronchial asthma, this decreased response is not at-tributable to defective beta adrenergic receptor bind-ing as detected in disrupted PMN. In light of recentevidence indicating differences in properties of betaadrenergic receptors in intact and broken cells (30, 36,37), it may prove fruitful to perform more detailed stud-ies comparing adrenergic receptors and adrenergicresponse in intact PMNprepared from asthmatic andcontrol subjects.

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