Lung (1992) 170:125-141

Review

Beta2 Adrenergic Receptors in Asthma: A Current Perspective Tony R. Bai Pulmonary Research Laboratory, St. Paul's Hospital, Universityof British Columbia, Vancouver, British Columbia V6Z IY6

Abstract. The role of the/32-adrenergic receptor in both the pathogenesis and treatment of asthma has been a subject of intense speculation and investigation for 25 years. The physiological effects of endogenous circulating catecholamines and exogenous adrenergic agonists in the lung are mediated by the/32-adrenergic receptor, which is present on a variety of cell types. Documented effects of/32-adrenergic receptor activation in the human lung include smooth muscle relaxation, inhibition of acetylcholine release from cholinergic nerve terminals, stimulation of serous and mucous cell secretion, increases in ciliary beat frequency, promotion of water movement into the airway lumen by stimulation of ion secretion across the apical membrane of epithelial cells, increase in bronchial blood flow, reduction in venular permeability, and inhibition of mediator release from some, but not all, inflammatory cells. /32-Adrenergic receptors are present in normal or increased numbers on asthmatic airway smooth muscle but are uncoupled in severe asthma, leading to functional hyporesponsiveness, probably due to the effects of inflammatory mediators. There is also evidence for dysfunction of/32-adrenergic receptors on circulating inflammatory cells following mediator release, However, dysfunction of the receptors on airway smooth muscle and inflammatory cells is unlikely to be of primary importance in the pathogenesis of asthma. There is increasing concern that regular/32-adrenergic receptor agonist use in the therapy of asthma is deleterious. Although a number of theories have been advanced to explain such an effect, none is well established and further research is urgently required. Key words: Asthma--Betaz-adrenergic sis--Asthma, treatment.

receptors--Asthma,

pathogene-

Historical Aspects Long before Langley [45] and Dale [21] developed the concept that the specific biological effects of hormones, neurotransmitters, and drugs result from high-

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affinity, stereospecific interactions with tissues, the English physician, Henry Salter, reported in 1859 what is probably the first account in modern times of the beneficial effects of activation of beta adrenergic receptors (/3AR) in asthma, when he wrote that "asthma is immediately cured in situations of either sudden alarm or violent fleeting excitements" [60]. Endogenous levels of circulating catecholamines, in particular epinephrine, are now known to influence airway caliber in asthmatic patients by stimulating/32 adrenergic receptors on airway smooth muscle, and possibly cholinergic nerves [7]. It is likely that Salter was describing sympathoadrenal release of epinephrine following emotional triggers. At the turn of the century, the vasodilator hypothesis of asthma had considerable support in both Germany and the United States. This hypothesis stated that airway obstruction was caused by swelling of the bronchial mucosa secondary to vasodilatation. The other major hypothesis at that time was that asthma was due to "the spasm of the circular muscles of the bronchi." Thus, in 1900, SolisCohen, encouraged by reports that adrenal extracts caused vasoconstriction, gave large oral doses of dessicated adrenal glands to asthmatic subjects with success, which he interpreted to be consistent with his view that asthma was a "vasomotor ataxia of the relaxing variety" [68]. However, it is unlikely the epinephrine content of the adrenal could have survived the oral route as an active drug and, indeed, the slow onset of action of the extract treatment reported in this paper is now thought to be more likely the demonstration of the beneficial effects of glucocorticosteroids (62). Soon after this, epinephrine became available as a pure substance and in 1903 Bullowa and Kaplan successfully gave injections of it to asthmatic patients [10]. They too thought this success was consistent with the vascular hypothesis of asthma, but in 1907 epinephrine was shown to relax airway smooth muscle [37]. In 1924, ephedrine was introduced to Western medicine, although the plant from which it is derived has been used for more than 5000 years in China for respiratory conditions. Ephedrine, although mainly an c~-adrenergic agonist with a weak /3 agonist activity, and adrenaline (epinephrine) were widely used over the ensuing decades in the treatment of asthma. In 1941, Konzett isolated isoprenaline [41], the first/3AR agonist devoid of c~-adrenergic effect. Ahlquist in 1948 used isoprenaline to partition sympathomimetic effects into c~ and/3 based on physiological responses in isolated tissues [1]./3-Adrenergic responses are stimulated by isoproterenol more potently than by epinephrine or norepinephrine. Evaluation of a large volume of data generated in the study of/3-adrenergic pharmacology enabled Lands in 1967 [44] to suggest a further division of the/3AR response into subtypes termed/31 and/32. This distinction was based on the relative potency of the naturally occurring catecholamines, epinephrine and norepinephrine. /3~ Responses are equally sensitive to these two agonists;/32 responses are more potently stimulated by epinephrine. Generally, but not invariably,/31 responses appear to be initiated by the neurotransmitter, norepinephrine, in innervated tissues, whereas/32 responses are triggered by the circulating hormone epinephrine [56]. Subsequently, a third subtype of /33AR was defined in nonpulmonary tissues [24]. Both salbutamol and terbutaline, the first of the current generation of/32AR

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agonists used in the treatment of asthma, were synthesized and characterized before the report of Lands. Since the 1960s, a number of/3eAR agonists have been developed as therapeutic agents. The key substitutions to the beta-phenylethylamine parent have been to the catechol ring, or related structure, to make the compounds resistant to metabolism by endogenous methyl transferases and monoamine oxidase, and addition of an ethanolamine side chain of varying length. Such alterations prolong half-life and increase the selectivity of these agents for/32 receptors.

Receptor Localization and Physiological Effects in the Human Lung Prior to 1974, the/3-adrenergic receptors were known only indirectly as entities that responded to drugs in a selective manner to mediate a variety of physiologically important responses. Then a variety of high-affinity ~25I-labeled radioligands selective for these receptors were developed that lead to experiments utilizing direct binding assays to establish the biochemical properties of the receptor protein. This technique, when coupled with efficient methods for detergent solubilization, formed the basis of receptor purification using affinity chromatography, and when coupled with autoradiographic methods led to the cellular localization and quantification of/3-adrenergic receptors on thin sections of tissues [71]. Organ bath and autoradiographic studies have demonstrated that the airway smooth muscle relaxant effect of/3AR agonists is largely via/32 receptors directly on the muscle surface [2, 8, 13, 54]. This is not unexpected, given that/3~ receptors are found at sites of sympathetic innervation responding to norepinephrine release and there is no direct sympathetic innervation of human airway smooth muscle [8, 22]. The receptors on mucous and serous glands and inflammatory cells are likewise largely of the /32 type [81 9]. /32 receptors also predominate on epithelium, type I and II pneumocytes, and vascular smooth muscle so that they make up 70% of the/3AR's in the human lung, the other 30% being/3~ on alveolar walls./3-adrenergic receptors are, in general, a low-abundance receptor (500-5000 sites/cell) but the density of/32 receptors increases from the large to small airways and is much greater on alveolar walls than other structures in the lung [13]. Although in vivo the most obvious and therapeutic effect of/3-adrenergic stimulation is bronchodilation mediated by airway smooth muscle relaxation, a number of other effects also occur (Table 1). Beta agonists promote secretion from serous cells and, to a lesser extent, mucous cells, in mucous glands. Serous cell stimulation produces antibacterial proteins such as lyzozymes and lactoferrin. This effect has been demonstrated convincingly in vitro only, using human tracheal explants at relatively high concentrations of/3 agonists [9]. However, theoretical calculations of luminal/32AR agonist concentrations following inhalation indicate that such levels may be achieved in vivo [39]. Furthermore,/32AR agonists increase chloride ion transport through apical membranes of epithelial cells via an increase in cyclic AMP. Sodium follows passively via paracellular channels and water by osmosis. The next effect is to increase periciliary fluid [79, 83]. The

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Table 1. Documented physiological effects of fl2-adrenergic receptor stimulation in human lung Airway smooth muscle relaxation Prejunctional inhibition of aceytlcholine release from parasympathetic neurons in airway smooth muscle Stimulation of mucous and serous cell secretion Stimulation of chloride ion secretion across the apical membrane of airway epithelial cells Increase in ciliary beat frequency Stimulation of surfactant secretion from alveolar type II cells Inhibition of mediator release from lung mast cells and neutrophils ? Reduction in microvascular permeability (animal models) ? Increase in bronchial blood flow (animal models)

combined effects of stimulation of mucous glands and chloride channels, together with an increase in ciliary beat frequency [79], is to increase mucocilary clearance, which has been demonstrated in vivo using radio tracer methods, although the clinical relevance of this enhancement in patients with asthma is unknown [79]. fizAR agonists also stimulate the secretion of surfactant from alveolar type II cells in vitro, although the magnitude of the effect is modest [50]. In animal models of inflammation, mediators increase microvascular permeability by contracting postcapillary venular endothelial cells so that spaces form between the cells. In such models/32AR agonists relax endothelial cells and therefore reduce permeability [61]. However,/32AR agonists also increase bronchial blood flow by acting as vasodilators of bronchial arterioles [38]. The net effect of these 2 opposing effects on exudation or transudation of fluid into the lumen and wall of inflammed human airways is unclear. A report that nebulized epinephrine was no more effective than a nebulized fiAR agonist that lacked an alpha-adrenergic vasoconstrictor effect in producing bronchodilatation in acute asthma suggests that potential alterations in bronchial blood flow induced by/32AR agonists do not adversely affect fluid shifts across the lumen wall [19]. Moreover, the lack of additional benefit by epinephrine suggests that the potential decrease in lumen area produced by mucosal vasodilation induced by fiAR agonists is not an important component of airflow resistance in asthma. fl2-Adrenergic receptors are present on a variety of inflammatory cells that passage through, or are resident in, the lung. Circulating lymphocytes and neutrophils have low numbers of receptors, which appear to be relatively poorly coupled to second messenger pathways in that they are easily downregulated [36]. Studies using circulating lymphocytes or neutrophils as a marker of pulmonary fiAR function can therefore be misleading (vide infra). However, 1 study showed a strong relationship between fiAR densities on circulating mononuclear leukocytes and of lung tissue obtained at thoracotomy [46]. Human alveolar macrophages contain 5000 fiARs per cell [47] but fizAR agonists do not prevent mediator release from activated human alveolar macrophages [27]. There is some evidence that beta agonists reduce the release of histamine from mast cells and that this is part of their mechanism of action in abating the early

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(M) Fig. 1. Effect of increasing concentrations of fenoterol (FEN) and isoprenaline (ISO) -+ the/32 antagonist ICI 118, 551 (10nM) (ICI/ISO) on cholinergic contractile responses to electrical field stimulation (EFS) at 5Hz ( e e ) or comparable contractions induced by acetylcholine (ACh) ( e . . . . e ) in postmortem human tracheal strips. The concentrations of ISO (56nM) and FEN (165nM) required to inhibit EFS (SHz) by 50% (IC50) were significantly less than those required to inhibit ACh responses to a comparable degree and the maximum inhibition of EFS was greater. Following ISO + ICI, ICS0s were not different for EFS and ACh, demonstrating that the prejunctional inhibition was mediated by the/3~ adrenergic receptor, n = number of subjects in each group [3].

response to allergic bronchial challenge, in addition to their role as functional antagonists of the airway smooth muscle contraction induced by release of mediators [8, 12]./3 agonists may also inhibit mediator release from basophils [8]. Human neutrophils possess 900-1800/32ARs per intact cell and mediator release is inhibited in a dose-dependent manner by isoproterenol [11]. In contrast, mediator release from the human eosinophil, although possessing a greater density and affinity of/32ARs (5000 sites per cell), is not inhibited by isoproterenol [87]. Both alveolar macrophages and eosinophils are thought to be important effector cells in the pathogenesis of asthma and the lack of influence of/3AR agonists on these cell types may explain in part the poor efficacy of these agents as monotherapy in asthma (vide infra). /3ARs are present in peribronchial parasympathetic ganglia, which receive direct sympathetic innervation [8]. /32ARs are also present on cholinergic nerve terminals in airway smooth muscle and act here to inhibit acetylcholine release (prejunctional inhibition, Fig. 1), thereby reducing the cholinergic component of bronchoconstriction [3, 64]. It is possible that propranolol induces asthma attacks not only by reducing the tonic effect of epinephrine on airway smooth muscle in maintaining airway patency but also by blocking the effect of epinephrine on cholinergic nerve terminals leading to the exuberant release of acetylcholine. In support of this hypothesis, cholinergic antagonists have been shown to reverse partially propranolol-induced bronchoconstriction [30].

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Molecular Biology

Improvements in receptor isolation techniques in the first half of the last decade led to the availability of substantial amounts of purified/32-adrenergic receptor that allowed determination of the molecular mass and amino acid sequence of part of the receptor. This new information led in turn to the production of polynucleotide probes and eventually to cloning of the receptor gene and determination of the complete primary sequence of the receptor protein [26, 71, 72]. The/32 adrenergic receptor gene maps to chromosome 5 and encodes a protein of 413 amino acids, only 54% of which are shared with/3~AR receptors [16, 25, 40, 72]. In addition, the gene for a/33AR has now been cloned and there is even some pharmacologic evidence for subtypes of/33ARs in nonpulmonary tissues [24]. Adrenergic receptors belong to the G-protein-linked rhodopsin-related receptor superfamily, 1 of at least 3 cell membrane receptor superfamilies. The current model of this cell-membrane-associated receptor indicates 7 transmembrane segments connected by alternating intracellular and extracellular loops. Homology among all the members of the 7 transmembrane regions (serpentine) receptor family is greatest in the transmembrane spanning domains. Genetic and biochemical manipulation of the/32AR has identified that the ligand-binding domain is a pocket buried within the membrane bilayer with agonists interacting specifically with transmembrane helices III and V [26, 72]. G proteins are membrane-associated heterotrimers composed of o~,/3, and 7 subunits. Interaction with a receptor causes the release of GDP from the subunit of the G protein, allowing GTP to bind and leading to the dissociation of the activated a subunit from the receptor and from the/37 complex. Various G proteins activate or inhibit different effector enzymes, modulating the levels of intracellular second messengers. In the case of the /32AR, binding of an agonist to the receptor, coupled to the stimulatory guanine-nucleotide binding protein, G~, catalyzes the release of GDP from the c~ subunit of the G protein (%), allowing the binding of GTP; this in turn leads to the direct activation of adenylyl cyclase by %-GTP. Adenylyl cyclase catalyzes the formation of the classic second messenger cyclic AMP so that levels of cAMP up to 400 times over basal can occur within minutes of exposure to agonist [26, 35, 49, 71, 72]. Upon removal of agonist, the activation of adenylyl cyclase persists until the intrinsic GTPase activity of % hydrolyzes the bound nucleotide [26, 35]. The mature mRNA transcript for the/32AR is 2.2 kilobases. Studies of the regulation of gene transcription are incomplete, but in cell culture glucocorticoids increase mRNA levels by increasing the rate of gene transcription and isoproterenol decreases mRNA levels by decreasing stability of the mature mRNA [17, 31]. Hamid et al. [32] have recently reported the distribution of /32AR mRNA in human lung by in situ hybridization and correlated this with receptor autoradiographic distribution. They report striking differences between the density of labeling with the 2 techniques in different cell types, although this was not quantitated. Pulmonary vascular and airway smooth muscle showed a high intensity of mRNA but only a low density of receptors and the converse was reported for the alveolar epithelium. These investigators speculate that the

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differences may be due to either a rapid rate of/32AR synthesis or high stability of mRNA in the airways and this observation may explain the difficulty in demonstrating desensitization in airway smooth muscle [34, 82].

Desensitization /32-Adrenergic receptor desensitization--waning of the stimulated response in the face of continuous agonist exposure--can occur by several mechanisms [35]. Rapid desensitization is mediated by an alteration in the function of the beta receptor: it becomes uncoupled from the stimulatory G protein, Gs. This uncoupling phenomenon involves phosphorylation of the receptor in its terminal intracellular segment by at least 2 different kinases, protein kinase A, and a/3ARassociated kinase (BARK), which are activated under different desensitization conditions. The decreased efficiency of coupling of the BAR to Gs leads to decreased adenylyl cyclase activity and hence decreased cAMP levels. Desensitization can also occur by intracellular sequestration of the receptor complex or by "downregulation," which refers to agonist-induced decrease in receptor number, that occurs upon prolonged exposure to agonists and results in degradation of the receptor, presumably via a lysozymal pathway. Both uncoupling and sequestration (internalization) occur within minutes of exposure to micromolar concentration of/3AR agonists and the process is essentially complete within 30 min. Downregulation is evident after only several hours of exposure [35]. It has been proposed that the rapid desensitization mechanisms involving phosphorylation of the/3AR (uncoupling) may be operative mainly for nonneural /32ARs that respond to circulating concentrations of epinephrine in the nanomolar range [8, 56, 71]. Downregulation is also mediated by a decrease in mature /32AR mRNA caused by a decrease in mRNA stability, rather than a decreased rate of transcription. Phosphorylation and therefore uncoupling of the beta receptor can also be induced by stimulation of adjacent receptors ("receptor crosstalk") such as cholinergic muscarinic receptors. Activation of muscarinic receptors leads to stimulation of phosphatidylinositol pathways with secondary activation of protein kinase C by diacylglycerol, which, in turn, can phosphorylate and uncouple the BAR [49]. Glucorticosteroids have been demonstrated in vitro to reverse desensitization [23] and this is probably due to increased/32AR gene transcription, and possibly increased coupling, and may be an important mechanism of action of glucocorticosteroids in the treatment of asthma.

Receptor Expression and Function in Asthma The notion that a defective/3-adrenergic receptor system or imbalance between ~- and/3-adrenergic receptors might be a primary causal abnormality in asthma was first proposed by Szentivanyi in 1968 [75]. Currently, however, there is relatively little evidence to suggest that alterations in the properties of/3ARs are of criticalimportance in the pathogenesis of asthma. The usual effectiveness of/3AR

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agonists in the treatment of stable asthma is one obvious argument against a major defect in BARs in asthma [54]. There is a large literature on this topic and many of the studies have utilized circulating leukocyte BAR function as a marker of intrapulmonary BAR function. Results of these studies are confounded by prior beta agonist use or elevated circulating catecholamines in many but not all reports. In the other studies, it may be that the abnormalities are indicative of the atopic state rather than asthma per se. In addition, leukocyte BARs are very easily desensitized compared to airway smooth muscle BARs [34, 36, 82]. For a detailed review of studies of atopy and asthma utilizing leukocytes, the reader is referred to the recent review by Insel [36]. Illustrative reports include those by Muers et al. [51,52] and Connolly et al. [ 18]. Muers et al. observed that challenge with allergen caused a variety of changes in the B2AR-adenylyl cyclase system of lymphocyte membranes of allergic asthmatic patients including uncoupling and downregulation as well as nonspecific refractoriness ofadenylyl cyclase to nonhormonal stimuli. Connolly et al. noted that the density of BARs in peripheral blood lymphocytes (Bmax) and affinity (Kd) was inversely correlated to the severity of asthma, as judged by lung function tests, in drug-naive subjects with normal circulating catecholamine levels. Furthermore, Szefler et al. [74] have demonstrated, in asthmatic subjects with nocturnal symptoms, a circadian rhythm in BAR leukocyte density characterized by a 33% decrease at 4 a.m., and impaired leukocyte responses to isoproterenol in cells sampled at that time, but no such changes in subjects free of nocturnal asthma (asthmatic or normal). The usual nocturnal fall in epinephrine occurred 4 hr earlier and these investigators concluded the change was unlikely to be related to this and was more likely due to nocturnal mediator release. However, subjects with nocturnal asthma had used more inhaled BAR agonists so that downregulation may have resulted from prior drug treatment. More studies of betaadrenergic function in other cell types involved in the inflammatory response of asthma, such as airway mucosal mast cells and dendritic cells, would be of interest. fiRAR agonists inhibit antigen-induced release of mast cell mediators from human lung fragments [12]. The infusion of therapeutic concentrations of epinephrine in asthmatic subjects lowers plasma histamine levels [7, 8], suggesting that/32 adrenergic receptors on mast cells and related cell types are functional and are capable of being influenced by circulating BAR agonists. Studies of BARs utilizing lung tissues from asthmatic patients have yielded conflicting results. Szentivanyi reported decreased ability to synthesize cAMP in response to BAR stimulation and a decreased number of BARs in homogenized membrane preparations of lung tissue from 12 subjects with "reversible obstructive lung disease" (including an unknown number of asymptomatic asthmatics) [76]. Three groups of investigators have systematically examined BAR-mediated relaxant responses in airway smooth muscle in patients with mild to moderate asthma and in 2 of these studies there was no evidence of abnormal BAR responsiveness [14, 73, 81]. In the third study Cerrina et al. [14] reported decreased relaxant responses in some subjects with stable asthma. The decreased response correlated with airway hyperresponsiveness to inhaled histamine in vivo, suggesting a possible relationship between BAR function and asthma severity. In an ideal study, both precontracted airway smooth muscle

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and muscle under intrinsic tone should be evaluated since subtle defects in the relaxant pathway may not be elicited when tissues are only studied under conditions of intrinsic tone. Van Koppen has provided evidence for this phenomenon in airway smooth muscle from subjects with chronic obstructive pulmonary disease (COPD). Without precontraction with methacholine, no differences were seen in/3AR agonist-medicated relaxation. However, following precontraction airways from patients with COPD but not normal airways showed a one-half log-dose decrease in responsiveness [77]. Whicker et al. [81] studied airway smooth muscle under conditions of intrinsic tone only, whereas Svedmyr et al. [73] and Cerrina et al. [14] precontracted tissues with cholinergic agonists and histamine, respectively. Although there is conflicting evidence for impairment of/3AR-mediated airway smooth muscle relaxation in mild or moderate asthma, the data are more consistent in severe asthma. During exacerbations of asthma, bronchodilation in response to inhaled or parenteral/3 agonists is impaired [63]. Although the explanation of this observation is multifactorial, and probably explained in large part by the fact that the obstruction is due to airway wall edema and intraluminal exudate, it is possible that impaired relaxation of smooth muscle plays a part. The results of 2 systematic studies [4, 5, 29] have shown impaired/3AR-mediated relaxant responses in the airway smooth muscle from patients dying relatively suddenly of asthma outside hospital. In total, 13 asthmatic patients have been compared, with 63 control subjects following sudden nonpulmonary deaths. The increase in IC50 (effective concentration of/3AR agonist required to relax the muscle by 50% of maximum) in the asthmatic airway tissue ranged from one-half to 1 log-dose in these studies (Fig. 2). The confounding effect of/0AR desensitization by prior drug therapy does not appear to be the only factor involved in the decreased/3AR responsiveness, since 2 subjects who died in status asthmatius and demonstrated decreased in vitro/3AR responsiveness, were not receiving /OAR agonist therapy [29]. Furthermore, the decreased/3AR function noted by Cerrina et al. [14] is unlikely to have been due to prior drug treatment, since all /3AR agonists had been withdrawn for 2 weeks prior to thoracotomy. The relaxant effect of theophylline was unaffected in the studies of fatal asthma cited above. The cause of this impaired response is unknown but inflammatory cytokines may induce selective bata-adrenergic hyporesponsiveness, possibly by activation of phospholipase A2, which can uncouple the/3AR [85]. As an alternative, other adjacent receptor-mediated events, acting via protein kinase C, may be involved, that is, via receptor crosstalk (see section on densensitization). Furthermore, the /3AR-mediated relaxant abnormality may not be unique to asthma, since similar changes were seen in airway smooth muscle from subjects with COPD in the study by Van Koppen discussed above [77]. We have examined the hypothesis that the decreased fiAR responsiveness in severe asthma could be explained by a decreased number of cell-surface receptors by studying/3AR affinity and density by radioligand binding techniques, including autoradiography, on tissue sections of asthmatic airway smooth muscle adjacent to those tissues shown to be hyporesponsive in the organ bath [6]. Comparison has been made to normal airways. The results

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indicate, surprisingly, a 3-fold increase in the number of receptors compared to that of controls (Figs. 3, 4). Affinity was also increased in the asthmatic group [6]. These studies confirm and extend results by Spina et al. [69] and Sharma and Jeffery [67], reporting increased and normal numbers of BAR's, respectively, pointing clearly to a more distal defect in the BAR pathway in asthma. Such studies are clearly in contrast with at least 5 reports of reduced BAR density in animal models of allergic airway inflammation [reviewed in 36, 53] and the report of Szentivanyi [76]. Furthermore, we have recently reported increased levels of fi2AR mRNA in lung homogenates from subjects with severe asthma compared to those with COPD and normal lungs [15]. This increase may be attributable to corticosteroid therapy or upregulation of receptor expression by growth factors and is further evidence that receptor expression is unlikely to be decreased in asthma and may explain the in vitro and in vivo resistance of asthmatic airways to BAR agonist desensitization.

Potential Adverse Effects of Beta-Adrenergic Agonist Therapy B-Agonists have been widely used for many years in asthma treatment but recent reports have raised the possibility that regular beta agonist use is hazardous [28,

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Fig. 3. Distribution of/3-adrenergic receptors in human nonasthmatic (a,b) and asthmatic trachea (d,e). b, e. Bright field photomicrographs showing the epithelium (Ep), smooth muscle (SM), and submucosal glands (G) after being stained with 1% cresyl fast violet, a, d. Dark field photomicrographs of adjacent sections showing the distribution of autoradiographic grains after incubation with 25pM iodocyanopindolol (ICYP). There are fewer grains over the epithelium in asthmatic subjects because of epithelial disruption, but grain density over smooth muscle was increased 3fold [6].

42, 59, 66, 70, 78]. In my assessment, the published evidence does indicate that frequent use of/3AR agonists contributes to both morbidity and mortality in asthma. Although some studies have suggested that adverse effects are limited to specific agents such as fenoterol and isoprenaline [28], more recent evidence supports a class effect [70]. A number of speculations and hypotheses have been advanced to explain these recent studies (Table 2). One explanation is that excessive reliance on potent bronchodilators leads to delay in presentation for more effective therapy such as corticosteroids and oxygen and hence more severe or even fatal asthma attacks. This is a well-recognized risk of ~AR agonist use [65]. A second hypothesis is that prolonged bronchodilation resulting from regular/3AR agonist use leads to an increase in the amount of antigen or irritant inhaled into the lung. Normally, bronchoconstriction serves as a protective reaction to restrict entry of noxious materials into the airways. Furthermore, the bronchodilation can mask the usual warning signs of an attack (the immediate

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Table 2. Mechanisms of adverse effects of /3-adrenergic agonist treatment: hypotheses and speculations Excess reliance on bronchodilators leads to delay in seeking more appropriate care Bronchodilation increases allergen/irritant load Regular use leads to desensitization of/3-adrenergic receptors and rebound airway hyperresponsiveness Stimulation of cardiac/3-adrenergic receptors, in the setting of hypoxemia and hypokalemia, causes cardiac arrhythmias Inhibition of mast-cell mediator release prevents release of anti-inflammatory autocoids Regular use promotes airway secretions, exacerbating air flow obstruction Dilatation of the bronchial vasculature worsens obstruction by thickening airway walls

allergic response) so that the usual avoidance measures are not used. Although there is little direct evidence for this possibility, it may be an important mechanism by which asthma can worsen [43]. The third possibility, that excessive BAR agonist use leads to clinically significant desensitization of/3AR, has been extensively investigated. There is evidence in some studies [80] but not others [33] of a small decrease in peak bronchodilator effect and duration of action in patients with stable mild asthma but not in peak bronchodilator effect in more severely asthmatic patients [48]. The recent study by Lipworth et al. [48] and many others have only examined peak bronchodilator effects, and if duration of action is important, as suggested by Nelson et al. [55], such studies need to be repeated. The majority of positive reports have used oral/3 agonists. This is

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in contrast to studies in normal subjects in whom densensitization can be more easily demonstrated both in the lung and in nonpulmonary/3-adrenergic systems [33]. Small increases in airway responsiveness have been detected following cessation of regular/3 agonists, which have hypothesized as being due to desensitization of airway smooth muscle/3ARs [42, 78]. Overall, the importance of desensitization as a hazardous effect of/3AR agonist treatment remains unclear. The effect of desensitization of/3AR's on cell types other than smooth muscle requires further study. A fourth hypothesis is that stimulation of cardiac/32ARs or, in the case of more nonselective agents,/3~ARs, by increasing heart rate and inotropy, in the setting of the hypokalemia induced by/3AR agonists and hypoxemia, gives rise to fatal cardiac arrhythmias. There is little direct evidence to support this hypothesis [20, 86]. Additional speculations include that inhibition of mast cell mediator release by/3AR agonists gives rise to an abnormal prolongation of inflammation by preventing the release of natural anti-inflammatory substances such as heparin and other proteoglycans, which are normally released along with other mediators following mast cell activation [57]; promotion of airway secretions by /3AR agonists leads to worsening airflow obstruction [66, 84]; and dilatation of the bronchial vasculature induced by/3AR agonists worsens obstruction, both by a vascular engorgement and thickening of airway walls, and perhaps by increasing airway wall and luminal edema [58]. In conclusion, much of the data suggesting that /3AR-agonist therapy is hazardous can be explained by such treatment modifying patient behavior so as to place him or her at greater risk of illness by both inducing delay in obtaining more appropriate treatment and suppressing the early asthmatic response. However, additional "class effects" may be operative and should be a research priority.

Summary Stimulation of/32 adrenergic receptors mediates a multitude of effects in the lung. Most current evidence does not suggest an important defect in flARs on airway smooth muscle in mild or moderate asthma, but dysfunction of the receptor with increasing severity of asthma, due to uncoupling, does appear to occur and may, in part, explain the poor response to/3AR agonists in patients with acute severe asthma, fiAR dysfunction is also present on circulating leukocytes in asthma and cannot always be explained by prior drug use or elevated levels of circulating catecholamines, but rather is probably secondary to release of inflammatory mediators in the lung. However, the importance of leukocyte or other inflammatory cell/3AR dysfunction in the asthmatic diathesis remains unclear. There is increasing concern that regular/3AR agonist therapy is deleterious in asthma, which, together with recognition of the inflammatory nature of the disorder, has led to a revision of treatment protocols. Further research on potential mechanisms of adverse effects of/3AR agonist therapy is urgently required.

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T . R . Bai

Acknowledgments. The author's current research is supported by the B.C. Health Research Foundation, B.C. Lung Association, and Glaxo Canada. Kent Webb prepared the manuscript and Dr. Peter Par6 provided critical review.

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Accepted for publication: 16 January 1992