Autoantibodies to β2-adrenergic receptors with antiadrenergic activity from patients with allergic asthma

Autoantibodies to &adrenergic receptors with antiadrenergic activity from patients with allergic asthma Gerd Wallukat, Berlin-Buch,

Dr. rer. nat., and Albert

Wollenberger,

PhD

Germany

The serum y-globulin fraction from patients with allergic bronchial asthma (818), in contrast to the fraction from nonatopic control subjects (101 IO), was found to inhibit the positive chronotropic action of the &selective adrenergic agonist, clenbuterol, on pyruvate- or lactate-treated cultured neonatal rat heart myocytes. No inhibition was exerted on the positive chronotropic response to prenalterol, which acted via P,-adrenergic receptors. The inhibitory effect of the asthmatic y-globulins was concentration dependent. It could nearly be abolished by immunoprecipitation with antihuman y-globulin and antihuman IgG, but not with antihuman IgM. This finding means that the inhibitory immunoglobulins of the patients with asthma were chiefly autoantibodies of the IgG isotype, capable of cross-reacting with chronotropic &-adrenergic receptors on the cultured rat cardiomyocytes. These findings provide support for the notion that autoimmunity plays a role in the impairment of /3-adrenergic responsiveness in patients with allergic bronchial asthma. (J ALLERGYCLIN IMMUNOL 1991:88:581-7.) Key words: Asthma, &adrenergic clenbuterol

receptors, serum y-globulins, cultured heart cells,

In 1968Szentivanyi’ proposedthe (3-adrenergictheory of BP,in which a reduced P-adrenergicsensitivity of bronchial smooth muscle and mucous glands leads to a shift in autonomic control favoring the bronchoconstrictor effects of acetylcholine and o-adrenergic receptor stimulation.2, 3 This theory appearedto be supportedby studies4-‘jdemonstratingreduced CAMP responsesto P-adrenergic agonists in peripheral leukocytes from subjectswith asthma. However, desensitization.causedby P-adrenergicbronchodilator therapy may have been a decisive factor in weakeningthe action of the P-agonists.? A new aspect of the problem of P-adrenergic hyposensitivity in subjects with asthma and also in patients with AR was presented by Venter et a1.8 and Fraser et a1.9who detected, in atopic patients, circulating auto-Abs that were directed against the p2-_ From the Division of Cellular and Molecular Cardiology, Institute for Cardiovascular Research, Berlin-Buch, Germany. Received for publication Nov. 27, 1990. Revised May 20, 1991. Accepted for publication May 22, 1991. Reprint requests: G. Wallukat, Dr. rer. mat., Bereich fiir zellullre und m,olekulare Kardiologie, Robert-Riissle-Str., 10, O-1115 Berlin-Buch, Germany. l/1/31218

Abbreviations used

BA: [‘Z51]IHYP: Ab: CAMP: AR:

Bronchial asthma [‘Z51]Iodohydroxybenzylpindolol Antibody Cyclic adenosinemonophosphate Allergic rhinitis

adrenergic receptors of lung membranesand that interfered with the specific binding of a high-affinity padrenergicligand, [“‘I]IHYP, to thesemembranes.A role of these auto-Abs as P-receptor antagonistswas suggestedby the finding of Harrison et al.‘* that diluted heat-inactivated serum of a patient with asthma inhibited the CAMP responseof a human lung cell line. It cannot be overlooked, however, that of the 17 allergic subjects with asthma investigated by Venter et a1.8and Fraser et al.,’ only four subjectsrevealed evidence of possessinganti-p,-receptor auto-Abs in the serum detectableby the [‘251]IHYPbinding assay. in a larger study encompassing 376 children with asthmaand again with [‘251]IHYPas P-ligand, about 5% only of this population were found to possessa P-blocking auto-Ab.” In another study with 76 subjects with asthma and 24 nonatopic control subjects, 581

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I. Effect of the serum y-globulin fraction (dilution, 1 : 20) of allergic subjects with asthma and of a patient with AR on the positive chronotropic response of cultured neonatal rat heart myocytes to 2 lJ,mol/L of clenbuterol

TABLE

Age Condition

Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic asthma Allergic rhinitis Allergic rhinitis Normal nonatopic control subjects(N = 10) Mean

Patient

(yrl

Sex

H. F. S. G. W. J. A. L. H. P. D. W. Mean u. 0. R. V. R. ‘I. G. W. G. W.

51 46 37 44 56 50 46 41 40 40 47 47

F F M F F M -

49

7F, 3M

F M M M M

Severity of disease

+++ +++ +++ +++ +++ +++ +++ ++ + +++ 0

0

Increase in the rate of beating in % of increase in the absence of the y-globulin fraction

19.7 6.9 10.9 37.9 19.4 9.3 17.3 ? 4.6 41.6 77.0 9.1* 18.8 103.1

113.9 + 6.3

+ + + , Severe;+ + , moderate,+ , mild; 0, no atopy. *Dilution of y-globulin fraction, 1: 10.

the [‘251]IHYPbinding test revealedthat no more than -20% of the sera from subjectswith asthmayielded binding inhibition values outside the 99% confidence limit for serafrom the nonatopiccontrol group. I2More recently it was reportedI that diluted seraof children with severe asthma caused an inhibition of [‘251]iodocyanopindololbinding to guinea pig lung membranesaveraging 40%, as comparedto 24% for serafrom control subjects,whereasserafrom subjects with mild asthma revealed values for binding inhibition that did not deviate from values of the sera from control subjects. The low frequency with which auto-Abs against P-adrenergic receptors were detected in asthma has nourished doubts concerning a possible pathogenetic role of these Abs in this disease.I4 In a discussion of some of these findings, it was pointed out’5’I6that the sensitivity of the binding assay may have been insufficient to detect anti&-receptor Abs that may have been present in the serum of the negatively reacting subjects with asthma and that a bioassay would have a better chance to reveal the presenceof these Abs, since the biologic responseto a hormone occurs in many instancesat considerably lower concentrations than concentrationsrequired to inhibit binding of a ligand in a receptor assayand also concentrationsneededto stimulate CAMP formation. A casein point is the relationship betweenthe potency

of isoproterenol as a stimulant of chronotropism in cultured neonatalrat heart myocytes on the one hand, and as a stimulant of adenylate cyclase in membrane suspensionsfrom the cultured heart cells and a competitor of labeled propranolol for binding to these membranesuspensionson the other hand.” Since we had foundI that cultured neonatal rat heart myocytes contain, in addition to their P,-adrenergic receptors, a subpopulation of chronotropic &receptors that, after treatment with pyruvate or L( -t-)-lactate, become accessible for immunoglobulins from patients with asthma, it occurred to us that this phenomenoncould perhapsbe usedas a basis for a sensitive biologic test for these immunoglobulins. As demonstratedby the results discussedbelow, this finding proved indeed to be the case. Some of these results have briefly been discussed.I9 MATERIAL AND METHODS Culture and use of beating heart cells Hearts were removed aseptically from l- to 2-day-old Wistar rats. Single cells were dissociatedfrom the minced ventricles with a 0.2% solution of trypsin and were cultured at 37” C for 4 or 8 days as monolayerson the 67 cm2bottom of Mueller flasks (1.2 x lo6 seededcells) in 3 ml of glucose-containingHalle SM 20-1 medium” equilibrated with humidified air and supplementedwith 10% heat-inactivated calf serum, 0.1 mU of insulin, and 2 x 106mol/L of fluorodeoxyuridine. The flasks were attachedto a plate that was

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continuously rocked back and forth at two cycles of ? 30” each per minute to ensure an adequate gas and metabolite exchange.” The medium was changed every second or third day. On the day of the experiment, the cells were washed with medium and incubated at 37” C for 2 hours with continued rocking in 2 ml of this medium to which 1 mmol/L of pyruvate or 3 mmol/L of L( +)-lactate had been added. The flasks were then transferred to the heated stage of an inverted Zeiss (Carl Zeiss, Inc., Oberkochen, Germany) (Jena) microscope, in which 10 small circular fields of the cell layer were inspected at 37 ? 0.2” C through the perforations of a metal template. Unless it is otherwise stated, the number of beats of a selected isolated myocardial cell or a cluster of synchronously contracting cells in each of the 10 fields was counted for 15 seconds each time, first before and 5 minutes after addition of adrenergic agonist, usually the lipophilic &-selective agent, clenbuterol, and then, after cumulative addition of aliquots of the serum yglobulin fraction (see below), to reach, after 30 minutes, a final dilution of 1: 20, reckoned in terms of the serum volume from which the fraction had been prepared. As a rule, this procedure was repeated twice in different cultures to yield results representing a total of 30 cells or cell clusters. The basal rate of beating was in the range of 120 to 160 beats/ min. No difference was noted between the 4- and 8day cultures in the responses to the adrenergic agents and immunoglobulins.

Patients Six subjects with severe and one subject with moderate allergic BA (Table I) were admitted to the clinic of the Institute for Lung Research and Tuberculosis, Berlin-Buch. The subject with mild allergic BA and the subject with AR (Table I) were observed for ambulant treatment. The diagnosis of allergy was confirmed by the clinical history and a positive skin prick test. Diagnosis of asthma was based on history and evidence of a subnormal FEV, without medication as well as by bronchial responsiveness to challenge with inhaled histamine and &agonist. The subject with AR had a history of seasonal nasal symptoms and demonstrated no signs of asthma. The nonatopic control subjects were healthy nonsmokers who had been free of respiratory disease for at least 1 year.

Preparation and handling y-globulin fraction

of the serum

The serum y-globulin fraction of the subjects with allergic BA was isolated by the ammonium sulfate precipitation methods2 from thawed serum samples that had been kept frozen at - 20” C The purified immunoglobulins were taken up in pho:jphate-buffered saline (pH 7.2) before being added to the cultures at the appropriate dilution. In some experiments, aliquots of the dissolved immunoglobulins were mixed in a centrifuge tube with an excess of goat antihuman immunoglobulin, IgM or IgG preparations, from which sodium azide had been removed by dialysis. The precipitated immune complex was centrifuged off and the supematant was dilutl:d with the phosphate-buffered saline to the desired value, again referring to the serum of origin.

to p2 receptors

in asthma

583

f

-

I -

Prenalteroi,

10-‘M

FIG. 1. Lack of inhibitory influence of the serum fraction of a patient with allergic asthma (E. chronotropic response of cultured neonatal myocytes to prenalterol; leti column, control; right columns, y-globulin fraction at dilution 1 : 20, respectively.

y-globulin P.) on the rat cardiocenter and 1 : 100 and

Statistics The results are expressed as means ‘-c SEM. Student’s paired and unpaired t tests were used, when these were appropriate, to compare variables between groups.

Drugs The following substances were received as kind gifts: clenbuterol HCI from Dr. Karl Thomae GmbH, Biberach, Germany; prenalterol HCI from HIssle Pharmceuticals, Molndal, Sweden; ICI 118, 55 1 from Imperial Chemical Industries Ltd., Macclesfield, England; acebutolol from Bayer AG, Leverkusen, Germany; goat antihuman y-globulin was obtained from Staatliches Institut fiir Immunprgparate and Medien, Berlin; goat antihuman IgM and IgG from Behringwerke AG, Marburg, Germany; sodium pyruvate and sodium L( + )-lactate from Boehringer Mannheim GmbH, Mannheim, Germany; and trypsin (crude preparation) from Leidholdt Biochemica, Kleinmachnow, Germany. All other chemicals were commercial products of highest purity grade.

RESULTS

The serum y-globulin fraction of all eight patients with asthmathat were investigated inhibited the positive chronotropic effect of the &selective adrenergic agonist, clenbuterol (2 kmol/L), on the spontaneously beatingcultured rat heart myocytespretreated with pyruvate or L( +)-lactate (Table I). In contrast, the clenbuterol effect was not significantly altered by the serum y-globulins from any of the 10 nonatopic control subjects. The inhibition, measuredat a twentyfold dilution of the y-globulin fraction, was very

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CLIN. IMMUNOL. OCTOBER 1991

IL r? r

$5100 co) z 0 $6

“2 75 bO 0, .E %a L @ 50 .E i!L gj .E a,..- 25 LO

tis 0

Control 1:lOOO 1:lOO boy-GF) Dilution of y-globulin

1: 20

1:2c

fraction

FIG. 2. Inhibitory effect of the y-globulin fraction (yGF) of a patient (E. P.) with allergic asthma on the positive chronotropic response of pyruvate-pretreated neonatal rat heart myocytes to 2 Fmol/L of clenbuterol; right column, no pyruvate pretreatment; **p < 0.001 and *p < 0.01, respectively, versus control.

pronounced, on the average, 83% in the six patients who were suffering from severeasthma, 58% in the patient with moderate asthma, and only 23% in the patient with mild asthma. The basal rate of beating was unaffected by the immunoglobulins. A marked inhibition of the chronotropic responseto clenbuterol was also causedby the serum y-globulin fraction of a patient with AR when the serum was obtained at the height of the allergic symptoms. When the serum was obtained in the following winter, after the AR had subsided, the inhibitory action of the immunoglobulins had likewise disappeared(Table I). That the chronotropic effect of clenbuterol on the cultured rat heart cells was mediated by stimulation of P,-adrenergic receptors with which the human asthmatic immunoglobulins were obviously cross-reacting,is suggested becauseit could be suppressedby the highly p,-selective adrenergic antagonist, ICI 118, 551 (0.1 pmol/L), but was unopposed by the p,-selective blocker, acebutolol(O.1 p.mol/ L) . In contrast, asdocumented in Fig. 1, the serum y-globulin fraction of the subjects with asthmahad no significant influence on the increasein the rate of beating produced by the P,-selective23 adrenergic agonist, prenalterol (10 p,mol/L). Its action on the cultured beating heart cells could be opposedby acebutolol, but not by ICI 118, 55 1. This finding could mean that there was no crossreaction with P,-adrenergic receptors. In Fig. 2 is presentedan example of how the beat accelerating action of clenbuterol on pyruvate-pretreated cultured neonatal heart cells was inhibited by the serum-y-globulin fraction of allergic subjectswith

asthmain a concentration-dependentfashion. The action of clenbuterol on the beating cardiomyocytes in cultures not pretreated with pyruvate (Fig. 2) (also for L( + )-lactate) remainedvirtually unaffectedby the immunoglobulins of subjects with asthma, even at a dilution of only 1: 20. In Fig. 3 is depicted a dose-responsecurve for the positive chronotropic action of clenbuterol on the pyruvate-treatedcultured cardiomyocytesas affectedby the serumy-globulin fraction (1: 100)of a patient with asthma. The curve was shifted to the right by a wide margin. The maximum was depressed,and the shift was far from being parallel, indicating that the antagonism by the y-globulins was not competitive. The intrinsic activity of clenbuterol closely approachedor equaled that activity found previously by us under identical experimental conditions for isoprotereno1,18 which meansthat in our system, in contrast to adult rat atria,24clenbuterol may be a full agonist. The time courseof the chronotropic action of clenbuterol on pyruvate-treatedcardiomyocytes, as influenced by the y-globulin fraction of another patient with asthma,is depicted in Fig. 4. In this experiment, the cultures were incubated for 30 minutes in the absence or presence of the y-globulin fraction (1:20) before clenbuterol was added. The action of clenbuterol was observed to be greatly retarded in the presence of the immunoglobulins and remained partly inhibited. In the experiment on lactate-pretreatedcultures presentedin TableII, a 95% inhibition of the chronotropic action of clenbuterol by the serum y-globulin fraction

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to p2 receptors

in asthma

I

5

0

-

.I-

A__.-’

-10 -9 Cknbuterol,

~.I_

-L-I-

-6 -5 -0 -7 log molar concentration

*

-4

FIG. 3. Dose-response curve for the chronotropic action of clenbuterol on beating cultured neonatal rat heart myocytes in the absence (0) and presence (Cl) of the y-globulin fraction (dilution, 1 : 100) of a patient (H. P.) with allergic asthma.

TABLE

II. Influence

of immunoprecipitation

with

Patient

Pretreatment

of cultures

antihuman

H. P. H. P. W. J. W. J.

3 mmol/L L( +)-lactate 3 mm01/L of L( + )-lactate 1 mmol/L of pyruvate 1 mmol/L of pyruvate

Precipitating

y-globulin, fraction

neonatal

immunoglobulin

Antihumany-globulin AntihumanIgG AntihumanIgM

of the patient with asthma, E. P., was completely eliminated by immunoprecipitation of the fraction with goat antihuman y-globulin, strengtheningthe belief that the inhibitory action was due to auto-Abs presentin the serumof subjectswith asthma.To identify the classof the auto-Abs possessingthe inhibitory activity, the y-globulin fraction of anothersubjectwith asthma (I. I.) was treated with antihuman IgG and antihuman IgM. As presentedin Table II, the inhibitory action of the fraction on the chronotropic responseof the cultured heart cells (pretreatedwith pyruvate) was maintained after immunoprecipitation with antihuman IgM, but was lost by immunoprecip-

15

20 25 Minutes

30

35

I _

40

FIG. 4. Effect of the serum y-globulin fraction (dilution, 1 :20) of an allergic subject with asthma (W. J.) on the time course of action of 1 PmollL of clenbuterol on the rate of beating of cultured neonatal rat heart myocytes. Each symbol represents 5 to 10 cells or cell clusters. Clenbuterol was added after an additional 30-minute incubation in culture medium (see METHODS) without (V-V)and with (e-e) the y-globulins.

IgM on the inhibitory effect of the serum y-globulin with allergic asthma on the chronotropic response of cultured 2 kmol/l. of clenbuterol antihuman

10

585

antihuman (dilution,

IgG, and 1 : 20) of patients

rat heart myocytes

to

Increase in rate of beating % of increase without immunoprecipitation

5.4 +- 4.4 106.7 ‘- 7.1 109.7 k 6.8 12.4 ‘_ 5.2

itation with antihuman IgG. This finding may indicate that the inhibitory immunoglobulins were auto-Abs of the IgG isotype, directed’ against &-adrenergic receptors. DISCUSSION Lau et al.*’ and Karliner et a1.26reported, basedon radioligand binding assays,that the P-adrenergicreceptors on cultured neonatal rat heart myocytes were of the &-subtype. Using a functional criterion, namely, the chronotropic response, Etaiche et a1.27 presentedevidencethat P,-receptorswere also present on these cells. This finding, suggestinga higher sen-

586

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and Wollenberger

sitivity of the functional as compared to the binding assay,was confirmed and extendedin our laboratory. In rocked cultures of neonatal rat heart cells, the chronotropic &-adrenergic receptorswere found to be maskedand to require exposureto pyruvate or L( + )lactate for about 45 minutes to becomeaccessibleto hydrophilic ligands and macromolecules, like immunoglobulins’8 (Fig. 2). This phenomenon, associated with a pronounced potentiation of the beataccelerating action of the p, and P,-adrenergic agonist , isoproterenol, I8 was likely the outcome of a reaction sequenceset in motion by lactate or pyruvate, including, as intermediary steps, the activation of phospholipaseAZand 15-lipoxygenase.28Theseevents allowed us to conduct the present experiments. The effect of the y-globulin fraction from a patient with asthmaon the dose-responserelationship for the chronotropic response of the beating cultured heart myocytes to clenbuterol (Fig. 3) indicates a noncompetitive antagonism, compatible with the existenceof different binding sitesof clenbuterol and the asthmatic anti-&receptor auto-Abs on the P,-receptor. The ligand binding site of the cell membranespanning /3adrenergic receptors has been localized to the transmembraneregion,29.3owhereas, according to a recent study dealing with the human P-receptors,amino acid sequencesof the second extracellular loop were the immunogenic regions.31In view of the very high degree of homology of the human P,-adrenergic receptorjZ with those of the &receptor of rodents,33.34it appearsnot unlikely that in the cultured neonatal rat heart myocytes an extracellular sequenceconstituted the immunogen with which the anti-P,-receptor autoAbs of the subjectswith asthmawere able to form an immune complex, resulting, presumably, in a conformational change of the receptor that hindered clenbuterol from exerting its chronotropic action. This action was slow to developand remainedpartly inhibited when clenbuterol was administered after a 30-minute exposure of the cultured cardiomyocytes to an asthmatic y-globulin fraction. If this is applicable to the clinical setting, this finding may indicate that, in the presenceof circulating asthmaticanti-g,-receptor Abs, &agonists would be unable to exert their full effect. Although the presentinvestigation was confined, in addition to a patient with AR, to only eight patients with allergic BA and 10 nonatopic control subjects, the results are sufficiently uniform (Table I) to permit the conclusion that the existence of circulating autoAbs to the P,-adrenergic receptor is a more common phenomenon in human allergic asthma than can be gathered from existing data of receptor binding assaysg. ‘l-l3 in which positive results were obtained

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only in a few patients, as indicated earlier. Furthermore, none of the healthy control subjectsof the present study produced, with the chronotropic test, anti&adrenoceptor auto-Abs, whereasinhibition by sera of radioligand binding to receptorsof lung membranes occurred in 16% and 24%, respectively, of normal subjectsin the studies of Fraser et a1.9and Potter and Dakers.13It would obviously be desirable to extend the presentexperimentsto a larger number of subjects with asthmaand normal control subjects;nevertheless, the results obtained can be regarded as providing acceptableevidence for a role of an autoimmunemechanism asa possiblepathogeneticfactor in BA. ‘3loSuch a mechanism could contribute, in conjunction with inflammatory changes,I4to an unfavorable imbalance in the autonomic control of bronchial function, in accordance with the hypothesis originally proposed by Szentivanyi.’ Note added in proof. As this article goes to press, we find, using the serum y-globulin fraction of four of our patients with asthma(H. F., W. J., A. L., and U. B.), that the immunogenic region, against which the inhibitory anti-&-receptor auto-Abs of these patients are specifically directed, can be localized to the third extracellular loop of the P,-adrenoceptor. We are indebtedto Dr. Ingrid Dehnertfor providingus with the sera of the patients with asthma and with information on their clinical status. REFERENCES

1. Szentivanyi A. The beta-adrenergictheory of the atopic abnormality in bronchial asthma. J ALLERGY1968;42:203-32. 2. Szentivanyi A. Die Flexibilitiit in der Konfiguration der Adrenozeptorenund die konstitutionelle Grundlage der Atopie. Triangel 1979;18:109-16. 3. Barnes PJ. Neural control of human airways in health and disease.Ann Rev Respir Dis 1986;134:1289-1314. 4. Logston PA, Middleton E, Coffey RG. Stimulation of leukocyte adenyl cyclase by hydrocortisone and isoproterenol in asthmatic and non-asthmatic subjects. J ALLERGYCLIN IMMUNOL1972;50:45-56. 5. Parker LW, Smith JW. Alterations in cyclic adenosinemonophosphatemetabolism in human bronchial asthma. I. Leukocyte responsivenessto P-adrenergic agents. J Clin Invest 1973;52:48-59. 6. Alston WC, Pate1KR, Kerr JW. Responseof leucocyte adenyl cyclase to isoprenaline and effect of alpha blocking drugs in extrinsic bronchial asthma. Br Med J 1974;1:90-3. 7. Conolly ME, GreenacreJK. The lymphocyte beta-adrenoceptor in normal subjectsandpatientswith bronchial asthma:effect of different forms of treatment. J Clin Invest 1976;58:130716. 8. Venter JC, Fraser LM, Harrison LC. Autoantibodies to p2adrenergic receptors: a possible cause of adrenergic hyporesponsiveness in asthma and allergic rhinitis. Science 1980;207:1361-3. 9. Fraser CK, Venter JC, Kaliner M. Autonomic abnormalities

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24. Cohen ML, Wiley KS, Bemis KG. Analysis of the beta, and beta, adrenoceptor interactions of the partial agonist, clenbuterol (NAB 365), in the rat jugular vein and atria. Naunyn Schmiedebergs Arch Pharmacol 1982;320: 145-51. 25. Lau YH, Robinson RB, Rosen MR, Bilezikian JP. Suhclassification of P-adrenergic receptors in cultured rat cardiac myoblasts and fibroblasts. Circ Res 1980:47:41-S. 26. Karliner JS, Simpson PC, Taylor JE, Honbo N, Woloszyn W. Adrenergic receptor characteristics of cardiac myocytes cultured in serum-free medium: comparison with serumsupplemented medium. Biochem Biophys Res Commun 1985; 128:376-82. 27. Etaiche M, Athias P, Vario N, Klepping J. Characterization of postjunctional adrenoceptor subtypes in isolated rat myocardial cells in culture. Can J Physiol Pharmacol 1985;63: 1221-7. 28. Wallukat G, Nemecz G, Farkas T, Kuehn H. Wollenberger A. Modulation of the beta-adrenergic response in cultured rat heart cells. I. Beta-adrenergic supersensitivity is induced by lactate via a phospholipase A1 and 15-lipoxygenase involving pathway. Mol Cell Biochem 1991;102:35-47. 29. Strader CD, Sigal IS, Register RB, Candelore MR, Rands E, Dixon RAF. Identification of residues required for ligand binding to the P-adrenergic receptor. Proc Nat1 Acad Sci USA 1987;84:4384-8. 30. Dohlman HG, Bouvier M, Benovic JL, Caron ML, Lcfkowitz RJ. The multiple membrane spanning topography of the p?adrenergic receptor. J Biol Chem 1987:242:1482-S. 31. Magnusson Y, H(iyer S, Lengagne R, et al. Antigenic analysis of the second extra-cellular loop of the human beta-adrenergic receptor. Clin Exp Immunol 1989;78:42-8. 32. Emorine LJ, Marullo S, Delavier-Klutchko C, Kaveri S, Durier-Trautmann 0, Strosberg AD. Structure of the gene for human P,-adrenergic receptor: expression and promoter characterization. Proc Nat1 Acad Sci USA 1987;84:6995-9. 33. Dixon RAF, Kobilka BK, Strader DJ. et al. Cloning of the gene and cDNA for mammalian P-adrenergic receptor and homology with rhodopsin. Nature 1986;321:75-9. 34. Allen JM, Baetge EE, Abrass BA, Palmiter RD. Isoproterenol response following transfection of the mouse &adrenergic receptor gene into Yl cells. EMBO J 1988:7:133-S.