Pharmac. Ther. Vol, 48, pp. 295-322, 1990 Printed in Great Britain. All rights reserved

0163-7258/90 $0.00 + 0.50 © 1991 Pergamon Press pie

Associate Editor: I. W. RODGER

ADRENOCEPTORS IN AIRWAY SMOOTH MUSCLE R o Y G . GOLDIE, JAMES W. PATERSON a n d KARMELO M . LULICH Department of Pharmacology, University of Western Australia, Perth, Nedlands, 6009, Australia Abstract--This review examines the roles and functional significance of a and fl-adrenoceptor subtypes in airway smooth muscle, with emphasis on human airway function and the influence of asthma. Specifically, we have examined the distribution of fl-adrenoceptors in lung and the influence of age, the epithelium, respiratory viruses and inflammation associated with asthma on airway smooth muscle fl-adrenoceptor function. Sites of action, fl2-selectivity, efficacy and tolerance are also examined in relation to the use of fl2-agonists in man. In addition, a-adrenoceptor function in airway smooth muscle has been reviewed, with some emphasis on comparing observations made in airway smooth muscle with those in animal models. CONTENTS 1. Introduction 2. fl-Adrenoceptors 2.1. Subclassification of fl-adrenoceptors 2.2. The molecular nature of the fl-adrenoceptor complex 2.2.1. The fl-adrenoceptor 2.2.2. Regulatory proteins 2.2.3. Adenylate cyclase and smooth muscle relaxation 2.2.4. fl-Adrenoceptor desensitization 2.3. Density and distribution of fl-adrenoceptors in the lung 2.3.1. Radioligand binding studies 2.3.2. Autoradiography 2.4. Airway smooth muscle 2.4.1. Site of action of fl-agonists 2.4.2. fl-Adrenoceptor subtypes 2.4.3. Relaxant activity against different spasmogens 2.4.4. Efficacy 2.4.5. Ageing and fl-adrenoceptor function 2.4.6. The epithelium and airway smooth muscle fl-adrenoceptor function 2.4.7. Interaction between respiratory viruses and fl-adrenoceptors 2.4.8. fl-Adrenoceptor function in asthma 2.5. Clinical actions of/~-agonists in lung 2.5.1. fl-Agonist drugs 2.5.2. Use of fl-agonists in infants 2.5.3. Sites of action in vivo 2.5.4. Tolerance to fl-agonists 2.5.5. fl-Agonists and hyperreactivity 2.5.6. Role of inflammation 3. ~t-Adrenoceptors 3.1. Subclassification of ~-adrenoceptors 3.2. Mechanism of action of ~t- and ct2-adrenoceptors 3.3. In vivo studies of lung ~t-adrenoceptors in asthma 3.3.1. ~t-Agonists 3.3.2. ~-Adrenoceptor antagonists 3.3.3. Exercise-induced asthma 3.3.4. Usefulness of the information obtained 3.4. In vitro studies of airway ~t-adrenoceptors 3.4.1. Radioligand binding and autoradiographic studies 3.4.2. Functional studies 4. Conclusions References

1. I N T R O D U C T I O N Ahlquist (1948) compared the rank order of potencies of different agonists mediating various adrenergic JPr 483-B

295 296 296 297 297 297 297 297 298 298 298 299 299 299 299 300 300 301 301 302 304 304 305 305 306 3O6 306 306 306 307 308 308 308 309 309 3O9 309 310 313 314

responses and on this basis divided adrenoceptors into two subtypes, ct and /$. Subsequently Lands et al. (1967) showed that/$-adrenoceptors which mediated bronchodilatation (/$2) were pharmacologically 295

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different to those that mediated cardiac stimulation (fit). ~t-Adrenoceptors have also been subdivided as ~tt- and cq-adrenoceptors (Berthelsen and Pettinger, 1977; Hoffman and Lefkowitz, 1980). Asthma is a disease in which there is reversible airway obstruction (Paterson et al., 1979; Norn and Skov, 1980) and nonspecific bronchial hyperreactivity to bronchoconstrictor stimuli (Boushey et al., 1980; Paterson et al., 1984; Woolcock, 1986). Airway obstruction in asthma is produced by a combination of bronchospasm and inflammation (Woolcock, 1986; Barnes, 1987). The inflammatory component is characterized by mucosal and bronchial wall edema, lymphocyte and eosinophil infiltration, desquamation of the airway epithelium and the presence of mucus plugs within the airway lumen (Reed, 1986). fl-Adrenoceptor agonist bronchodilators have been the principal treatment for asthma for many years (Paterson et al., 1979). Agents selective for fl2-adrenoceptors can be delivered by inhalation and provide rapid and effective reversal of acute airway obstruction caused by bronchoconstriction without causing significant side-effects (Lulich et al., 1986; Barnes, 1987). The demonstration that fl-adrenoceptors are widely distributed through the lung, raises the possibility of therapeutically useful actions of fl-agonists at sites other than airway smooth muscle (Barnes, 1984, 1986). Despite this, it is believed that fl2-agonists reverse airway obstruction primarily by relaxing airway smooth muscle. The effects of fl2-agonists on mucus production and transport, tracheobronchial microvessels and cholinergic nerves may be useful, but the therapeutic relevance of these effects still remains to be established (Goldie et al., 1990b). Castro de la Mata et al. (1962) showed that in canine bronchial smooth muscle, noradrenaline caused fl-adrenoceptor-mediated relaxation, but :tadrenoceptor-mediated contraction after fl-adrenoceptor blockade. Tracheal preparations from several

other animal species demonstrate a similar response profile (Takagi et al., 1967; Kasses et al., 1968; Fleisch et al., 1970; Left and Munoz, 1981a,b). Furthermore, in bronchi obtained from human lung samples resected at surgery, adrenaline which normally produced relaxation, caused contraction in the presence of the fl-adrenoceptor antagonist propranolol (Mathe et al., 1971). These contractions were abolished by phentolamine suggesting mediation via a-adrenoceptors. It has been proposed that bronchial obstruction in asthma may be partly mediated by airway ct-adrenoceptors (Griffin et al., 1972; Patel and Kerr, 1973; Snashall et al., 1978). Szentivanyi subsequently suggested that an increase in pulmonary ct-adrenoceptor activity coupled to a decrease in fl-adrenoceptor function in the lung may be an important cause or contributing factor in asthma (Szentivanyi, 1980; Szentivanyi et al., 1984). This review examines the role of ~t- and fl-adrenoceptors in airway smooth muscle, including that from human nondiseased and asthmatic lung. Both pharmacological and therapeutic aspects of adrenoceptor function in airway smooth muscle are considered. 2. fl-ADRENOCEPTORS 2.1. SUBCLASSIFICATIONOF fl-ADRENOCEPTORS

Lands et al. (1967) implied that various organs contained either fit- or fl2-adrenoceptors. However, it is now well established that both fit- and fl2-adrenoceptors are present in airway smooth muscle (Levy and Apperley, 1978; Barnes et al., 1983c; O'Donnell and Wanstall, 1983; Zaagsma et al., 1983), cardiac muscle (Ablad et al., 1974; Heitz et al., 1983; Robberecht et al., 1983; Corea et al., 1984) and in several other tissues. A pharmacological profile of fit- and fl2-adrenoceptor function is summarized in Table 1.

TABLE1. Properties of fit and fl2-Adrenoceptors fit-Adrenoceptors ]~2-Adrenoceptors I Responses produced

Cardiac stimulation: chronotropic and inotropic Lipolysis

Bronchodilation Vasodilation Inhibition of histamine release Skeletal muscle tremor Hyperglycemia and hypokalemia

II Relative potencies of fl-adrenoceptor agonists EC:

ISO > NOR > ADREN > SALB* ISO > ADREN > SALB > NOR 1 5-20 10~0 >500 1 3-15 10-35 60-400 III Potencies of fl-adrenoceptor antagonists

Propranolol (nonselective) Practolol (#:selective)

PA2

pA2

8.3-8.8

8.3-9.4

6.5~5.9

4.6-5.1

ISO =(-)-isoprenaline; NORf(-)-noradrenaline; ADREN =(-)-adrenaline; SALB = ( - ) - s a l b u t a m o l . EC = equipment concentration. *Partial agonist in vitro with respect to (-)-isoprenaline.

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Adrenoceptors in airway smooth muscle 2.2. THE MOLECULARNATUREOF THE

fl-ADRENOCEPTORCOMPLEX The ]]-adrenoceptor is a cell membrane glycoprotein (Stiles et al., 1984). The agonist/]]-adrenoceptor complex interacts with membrane bound adenylate cyclase via guanyl nucleotide binding regulatory proteins, inducing the production of the second messenger substance 3"5" cyclic adenosine monophosphate (c-AMP) (Fig. 1).

2.2.3.

Adenylate Relaxation

2.2.1. The ]]-Adrenoceptor

]]j- and ]]2-adrenoceptor subtypes are the products of different genes and hence are distinct polypeptides (Caron et al., 1985; Strader et el., 1987). The ]]2adrenoceptor isolated from hamster lung has a molecular weight of approximately 64,000 (Caron et al., 1985; Dixon et al., 1986) and is a single polypeptide chain consisting of 418 amino acids (Caron et al., 1988). It has 7 regions of hydrophobic residues which constitute the transmembrane spanning section of the receptor. The N-terminus is on the extracellular face of the cell membrane and the C-terminus is on the cytoplasmic side. These workers have also shown that there is a high degree of homology between the ]]z-adrenoceptors found in the hamster and in man. 2.2.2. Regulatory Proteins Agonist-bound ]]-adrenoceptors (]]t and ]]2) stimulate adenylate cyclase indirectly through guanyl nucleotide binding stimulatory proteins (Benovic et al,, 1985; Caron et al., 1985). The Gs proteins are heterotrimers, with subunits designated ~, ]] and y (Caron et al., 1985). The ~-subunit of Gs contains the binding site for guanosine 5'-triphosphate (GTP). The ]],y-subunit complex inhibits the activation of Gs (Gilman, 1987). The binding of agonist alters the conformation of the ]]-adrenoceptor so that it can

f ATP

form a complex with Gs (Stiles et al., 1984). This facilitates the displacement of GDP by GTP from the ~t-subunit of the Gs trimer and results in the ~t-subunit shedding the inhibitory ]],~,-subunit complex (Gilman, 1987). The activated form of Gs (Gs(ct)-GTP) can then stimulate adenylate cyclase. The activation cycle is stopped by the enzymatic hydrolysis of ct-subunit-bound GTP to GDP (Caron et al., 1985; Gilman, 1987).

cAMP stimulation of protein Idnases

Cyclase

and

Smooth

Muscle

Adenylate cyclase appears to be a single polypeptide glycoprotein (Gilman, 1987). It converts ATP to c-AMP which then acts as an intracellular messenger by activating specific kinases that are composed of tetramers containing 2 regulatory and 2 catalytic subunits (R2C2); Lohmann and Walter, 1984). c-AMP-dependent protein kinases are activated as a result of c-AMP binding to the regulatory subunits causing them to dissociate from the catalytic subunits as illustrated. R2 (22(INACTIVE) + 4-c-AMP --* 2(R-(c-AMP)2) + 2C(ACTIVE). fl-Agonists relax airway smooth muscle by elevating intracellular c-AMP and activating the c-AMPdependent protein kinase that phosphorylates myosin light chain kinase (MLCK) (Stull et al., 1980; Silver and Stull, 1982). Phosphorylated MLCK has less affinity for the calmodulin calcium complex causing a reduction in myosin phosphorylation and a decrease in actin/myosin coupling (Cauvin et al., 1984; Lulich et al., 1988). However, ]]-agonists may also inhibit airway contraction mainly by enhancing the sequestration of free calcium ions into intracellular storage sites and/or extrusion of calcium ions into the extracellular space (Ito and Itoh, 1984). ]]-Adrenoceptor activation may also result in smooth muscle relaxation following inhibition of calcium entry into the cells (Cauvin et al., 1984). Thus, the elevation in intracellular c-AMP produced by ]]-adrenoceptor stimulation can relax airway smooth muscle by increasing the phosphorylation of MLCK and/or decreasing the free calcium ion concentration as illustrated in Fig. 1.

I 2.2.4. fl-Adrenoceptor Desensitization ~, formation of MLCK-P (Inactive)

free Intracellular Ca 2~"

in enzyme afflnl~ for calmodulin-Ce z" complex

# calmodulln-Ca 2÷ complex formation

I

I ~' formation of myosin-P

RELAXATION

FIG. 1. Intracellular events leading to relaxation of airway smooth muscle following activation of fl-adrenoeeptors.

Prolonged exposure to a fl-adrenoceptor agonist may result in fl-adrenoceptor desensitization (Stiles et al., 1984). The reduction in the number of functional fl-adrenoceptors appears to result from phosphorylation of agonist-bound fl-adrenoceptors, which is mediated by the cytosolic enzyme fl-adrenoceptor kinase (BARK) (Benovic et al., 1986, 1988). BARK is also able to phosphorylate other adenylate cyclase coupled receptors (Strasser et al., 1986), but specific fl-adrenoceptor desensitization occurs because only agonist occupied receptors are acted upon by this kinase (Lefkowitz et al., 1986). Agonist binding to the fl-adrenoceptor changes the conformation of the receptor so that normally encrypted phosphorylation sites on the cytoplasmic face of the cell membrane

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become exposed to BARK. Phosphorylation of the fl-adrenoceptor initially results in uncoupling of the receptor from adenylate cyclase. Continued exposure to fl-adrenoceptor agonist leads to the sequestration of fl-adrenoceptors within the cell and the result is a decrease in the number of fl-adrenoceptors in the cell membrane. Sequestered fl-adrenoceptors may be dephosphorylated, recycled to the plasma membrane surface and recoupled to adenylate cyclase to become fully functional once again (Lefkowitz et aL, 1986; Strasser et aL, 1986). 2.3. DENSITY AND DISTRIBUTION OF

fl-ADRENOCEPTORSIN TIlE LUNC 2.3.1. Radioligand Binding Studies The availability of the highly specific and selective fl-antagonist radioligands [3H]-dihydroalprenolol (DHA) (Caron and Lefkowitz, 1976) and [125I]-iodocyanopindolol (I-CYP) (Engel et aL, 1981) has facilitated the study of pulmonary fl-adrenoceptors. High levels of binding of DHA to pulmonary fl-adrenoceptors were first demonstrated using rat and rabbit lung membranes (Rugg et al., 1978) and human lung membranes (Szentivanyi, 1979). Subsequently, high numbers of fl-adrenoceptors were revealed in tissues from guinea-pig (Barnes et aL, 1980b; Engel et al., 1981), canine (Barnes et al., 1982b) and hamster lung (Benovic et al., 1983). Peripheral lung tissue was found to contain mixed populations of fit- and fl2-adrenoceptors in the approximate proportions 20% and 80% respectively in the guinea-pig (Engel et al., 1981; Carswell and Nahorski, 1983), hamster (Benovic et al., 1983), rat (Dickinson et al., 1981; Minneman et al., 1979) and human lung (Brown et al., 1985). In sharp contrast, fll-adrenoceptors predominated in rabbit lung with estimates varying between 60% (Rugg et aL, 1978; Barnett et al., 1978) and 80% (Brodde et aL, 1983). However, no information about the cellular locations of these receptors was obtained from studies using membrane homogenates. 2.3.2. Autoradiography Light-microscopic autoradiography (Young and Kuhar, 1979) has enabled assessments of fl-adrenoceptor numbers and subtypes on different cell types in both central and peripheral lung. fl-Adrenoceptors have been localized in mammalian trachea and lung from various species including ferret (Barnes et al., 1982a), guinea-pig (Goidie et aL, 1986a), rabbit (Barnes et al., 1984), mouse (Fig. 2; Henry et aL, 1990), rat (Finkel et aL, 1984), pig (Goldie et al., 1986b) and man (Carstairs et al., 1985; Spina et aL, 1989a). 2.3.2.1. Trachea. Large numbers of fl-adrenoceptors were detected in mouse tracheal smooth muscle and epithelium (Henry et aL, 1990). In ferret trachea, fl-adrenoceptors were associated with epithelial cells, submucosal glands and airway smooth muscle (Barnes and Basbaum, 1983). The highest density of fl-adrenoceptors was associated with the

FIG. 2. (a) Dark-field photomicrograph of the autoradiographic distribution of total binding sites for [~25I]-iodocyanopindolol (I-CYP) (70 pr,l) in 10 #m frozen transverse sections of mouse trachea. (b) Light-fieldphotomicrograph of the above section showing the epithelium (Epi), airway smooth muscle (SM) and cartilage (C). Bar= 100#m. (c) Dark-field photomicrograph showing the distribution of I-CYP binding sites in the presence of 1 # i (--)-propranolol, i.e. nonspecific binding. Reproduced from Henry et al. (1990) with permission of the copyright holder, the Macmillan Press, Ltd, Hampshire. epithelium > mucous gland cells > serous gland cells > airway smooth muscle. In guinea-pig trachea, the binding of I-CYP to fl-adrenoceptors was greatest over epithelium and submucosal cells with significantly lower numbers associated with airway smooth muscle (Goldie et al., 1986a). 2.3.2.2. Parenchyma. fl-Adrenoceptors were found in greatest density in alveolar septae in mouse (Henry et al., 1990), rat (Finkel et al., 1984; Conner and Reid, 1984), guinea-pig (Carswell and Nahorski, 1983), pig (Goidie et al,, 1986b) and human lung (Carstairs et al., 1984), with high numbers also detected in ferret

Adrenoceptors in airway smooth muscle lung (Barnes et al., 1983a). In contrast, rabbit bronchioles contained greater numbers of fl-adrenoceptors than aiveoli in rabbit lung (Barnes et al., 1984). Such studies have also reported significant numbers of i-adrenoceptors in bronchial and bronchiolar epithelium and smooth muscle and in vascular endothelium and smooth muscle. In human lung, approximately 78% of the total lung tissue volume consists of alveolar tissue, 8% vascular smooth muscle and 3% airway smooth muscle. The remaining 11% consists of connective tissue and cartilage (Bertram et aL, 1983). t-Adrenoceptor numbers are 3 times higher over alveolar cells than over bronchial smooth muscle (Carstairs et al., 1984). Thus, approximately 96% of the fl-adrenoceptor population is located in alveolar tissue. Similarly, approximately 95% of pig lung i-adrenoceptors exists in alveoli (Goldie et al., 1986b). This is consistent with data in rat lung, showing that 97% of the t-adrenoceptor population resides over alveolar tissue (Conner and Reid, 1984). 2.4. AIRWAYSMOOTHMUSCLE 2.4.1. Site o f Action o f i - A g o n i s t s i-Adrenoceptor agonists reverse airway obstruction in asthmatics, primarily by relaxing airway smooth muscle (Paterson et al., 1979; Barnes, 1986). i-Agonists are potent relaxants of human isolated bronchial preparations (Svedmyr et al., 1976; Goldie et al., 1984; Zaagsma et al., 1984; Spina et al., 1989a), bronchioles (Zaagsma et al., 1984) and peripheral lung strips (Goidie et aL, 1982a,b). Thus in man, functional i-adrenoceptors are found in airway smooth muscle from the upper airways to terminal bronchioles. This is consistent with autoradiographic studies in human and animal lung which have shown the presence of i-adrenoceptors in airway smooth muscle from trachea to terminal bronchioles (Barnes et al., 1983a; Carstairs et al., 1985). 2.4.2. fl-Adrenoceptor Subtypes The original subdivision of i-adrenoceptors suggested that airway smooth muscle contained only 12-adrenoceptors (Lands et al., 1967). However, it is now clear that i~-adrenoceptors also exist in airway smooth muscle and may even be the dominant subtype depending on the airway model examined. In guinea-pig tracheal smooth muscle, i-agonistinduced relaxation was mediated by both i : and i2adrenoceptors (Levy and Apperley, 1978). Carswell and Nahorski (1983) found that there was a mixture of these subtypes in the proportions 15:85, respectively in this tissue. In the guinea-pig, the proportion of fl~-adrenoceptors decreases progressively from the laryngeal end of the trachea to the bronchial end (Zaagsma et al., 1987) with only i2-adrenoceptors mediating relaxation of the lung parenchyma strip (Carsweli and Nahorski, 1983; Zaagsma et aL, 1984). As with guinea-pig trachea, in canine tracheal smooth muscle, the ratio of il- to i2-adrenoceptors was shown to be about 1:4 (Barnes et al., 1983c). However, feline tracheal smooth muscle contains

299

mostly t~-adrenoceptors (Lulich et al., 1976; O'Donnell and Wanstall, 1983). In addition, ill" adrenoceptors predominate in rabbit trachea (Bristow et al., 1970; Toda et al., 1978), pig bronchus (Goldie et al., 1983) and mouse trachea (Henry and Goldie, 1990; Henry et al., 1990). The i-adrenoeeptor subtypes in mouse trachea have recently been characterized in a very comprehensive manner. Functional organ bath studies demonstrated that i~-adrenoceptors mediated i agonist-induced relaxation, with a minor contribution from i2-adrenoceptors (Fig. 3; Henry and Goldie, 1990). In line with these findings, quantitative autoradiography revealed that mouse tracheal smooth muscle contained a mixed population of i~" (69%) and i2-adrenoceptors (31%). In contrast, the overlying tracheal epithelium contained mostly i2adrenoceptors (71%) (Henry et al., 1990). Functional studies indicate that /~2-adrenoceptors are responsible for i-agonist induced bronchodilatation in man. Thus, intravenous prenalterol (i~-selective) increased the pulge rate of asthmatics of 25 beats per minute but failed to cause significant bronchodilatation (Lofdahl and Svedmyr, 1982). Conversely, terbutaline (ia-selective) caused pronounced bronchodilatation in these subjects. Functional studies in vitro (Harms, 1976; Zaagsma et al., 1983; Goldie et al., 1984) and autoradiographic studies have confirmed that human bronchial smooth muscle i-adrenoceptors are entirely of the i2-subtype (Carstairs et al., 1985). 2.4.3. Relaxant Activity Against Different Spasmogens fl-Agonists reverse bronchoconstriction caused by several different spasmogens (Barnes, 1986). However, on canine isolated bronchi, isoprenaline reversed contractions induced by histamine more effectively than those produced by acetylcholine (Russell, 1984). In guinea-pig isolated trachea, i agonists were more effective in reversing contractions induced by leukotriene D4 or PGF2~ than those induced by cholinergic agonists (Torphy, 1984; Heaslip et al., 1986).

~o-9

~o~

lo `7

lo-S

lo-S

[ 8-ADRENOCEPTOR AGONIST ] ( M ) FIG, 3. Mean cumulative concentration-effect curves to isoprenaline (O ; nonselective), RO363 (O ; /~:selective),

noradrenaline (A; fit-selective),adrenaline (A; fl2-selective), procaterol (I-q; fl2-selective)and fenoterol ( I ; fl2-selective) in carbachol-contracted mouse isolated trachea. Shown are the mean of 8-15 observations from separate preparations; vertical bars show SE mean. Reproduced from Henry and Goldie (1990) with permission of the copyright holder, the Macmillan Press, Ltd, Hampshire.

300

R.G. GOLDIEet al.

Contractions induced by histamine, serotonin or PGF2, in airway smooth muscle were largely dependent upon extracellular calcium, while those induced by acetylcholine were mainly dependent upon intracellular calcium stores (Kirkpatrick et al., 1975; Farley and Miles, 1978; Creese and Denborough, 1981; Russell, 1984; Ahmed et al., 1985). In addition, in vitro studies in human bronchial smooth muscle have demonstrated that blockade of voltage dependent calcium influx with nifedipine inhibited histamine induced contractions to a greater extent than contractions induced by acetylcholine (Henderson et al., 1983). Thus it is posible that//-agonists selectively antagonize histamine or serotonin rather than acetylcholine because inhibition of extracellular Ca 2÷ influx is an important part of their bronchodilator action. This needs to be further explored. Alternatively, ~-agonists may be less effective in inhibiting contractions produced by muscarinic agonists such as methacholine because these agonists produce intracellular biochemical changes which negate the relaxant response to //-adrenoceptor agonists. In canine tracheal muscle, it has been demonstrated that increasing the concentration of methacholine reduced the relaxation produced by isoprenaline and that this was accompanied by inhibition of fl-agonist-mediated cyclic-AMP accumulation and cyclic AMP-dependent protein kinase activation (Torphy et al., 1983). These results indicate that the sensitivity of airway smooth muscle to ~-agonists is not only affected by the level of tone, but also by the spasmogen used. Accordingly, Russell (1984) suggested that ~2-agonists would inhibit bronchoconstriction in asthmatic airways produced by immunological mechanisms involving histamine or leukotrienes more effectively than the same level of bronchoconstriction resulting from acetylcholine released from the vagus. However, caution should be exercised when extrapolating from canine isolated airways to asthmatic patients. For example, fl-agonists produce much more effective relaxation when high levels of cholinergic tone are present in human isolated bronchi than is the case in canine isolated bronchi (Advenier et al., 1988). 2.4.4. Efficacy Efficacy is a measure of the capacity of a drug to initiate an effect once it combines with the receptor (Stephenson, 1956). Thus, a full agonist has a higher efficacy than a partial agonist and a pure competitive antagonist has an efficacy equal to zero. For some full agonists with very high efficacy, there may be a significant functional receptor reserve i.e. spare receptors (Waud, 1968). The higher the efficacy of such a full agonist, the greater is the proportion of spare receptors since a lower receptor occupancy is required to produce its effect. In general, in airway smooth muscle there is an inverse relationship between the initial level of tone induced by a spasmogen and the ability of a p-agonist to produce relaxation (Van der Brink, 1973; Buckner and Saini, 1975; O'Donnell and Wanstall, 1978; Torphy et aL, 1983; Advenier et al., 1988). As the amount of spasmogen induced airway tone increases

a greater proportion of the population of fl-adrenoceptors needs to be stimulated to produce relaxation. O'Donnell and Wanstall (1978) have demonstrated that the relative efficacies of ~2-agonists were determined more accurately when high levels of airway tone were induced in guinea-pig isolated trachea. They found that with low levels of carbachol induced tone the maximal relaxation produced by saibutamol was similar to that produced by fenoterol. In contrast, with higher levels of carbachol induced tone, maximal relaxation induced by fenoterol was more than twice that produced by salbutamol. When studying the influence of spasmogen-induced tone on the relaxant effects of ~-agonists, the choice of airway model is critical. Supramaximal concentrations of spasmogen reduce the maximal relaxation produced by a fl-agonist much less in human isolated bronchi and guinea-pig trachea than in bovine, canine or rat trachea (Henry et al., 1982; Advenier et al., 1988). Thus, studies of functional antagonism in guinea-pig trachea appear to more closely reflect the response of human bronchial muscle than those in bovine, canine or rat trachea. In theory, a fl2-seleetive agonist with a high efficacy should be advantageous in severe asthma where there may be both marked bronchoconstriction (Paterson et al., 1984) and some down-regulation of fl-adrenoceptor function (Goldie et al., 1986e; Spina et al., 1989a). Fenoterol has a greater bronchodilator efficacy than salbutamol (O'Donnell and Wanstall, 1978). A potential disadvantage is its higher efficacy at cardiac fl-adrenoceptors (Brittain et al., 1976). Hockley and Johnson (1983) observed that nebulized fenoterol was superior to nebulized salbutamol with regard to peak effect and duration of action. However, little difference was detected between these two drugs clinically (Madsen et al., 1979; Hockley and Johnson, 1983). Thus, the clinical importance of high //:-adrenoceptor efficacy for the treatment of asthma is yet to be established. 2.4.5. Ageing and [3-Adrenoceptor Function It has been recognized for many years that tissue responsiveness to some drugs may be increased, decreased or remain unaltered as a function of age (Lasagna, 1956). Age-related changes in drug metabolism account for some of these effects (Nadel, 1973), although altered receptor function has also been demonstrated (Fleisch et al., 1973). Age-related changes in ~-adrenoceptor function in a wide range of tissue types, including pulmonary tissue, occur in two phases. Firstly, tissue maturation between fetal and early postnatal life is accompanied by marked increases in fl-adrenoceptor number. This has been described for rat and rabbit lung (Whitsett et al., 1981a,b), with a similar postnatal increase occurring in rat cerebral cortex (Harden et al., 1977). Other studies have shown lower densities of 8" adrenoceptors in newborn than in adult guinea-pig lung (Gatto et al., 1984), rabbit lung (Barnes et al., 1984), rabbit heart (Schumacher et al., 1984) and human lymphocytes (Doyle et al., 1982). Pretreatment with a glucocorticoid has been shown to increase the density of fetal rabbit lung ~-adrenoceptors to levels approaching those seen in mature

Adrenoceptors in airway smooth muscle rabbit lung (Barnes et al., 1984). Whitsett et al. (1981b) also showed that while adult rabbit lung had a greater density of fl-adrenoceptors than did fetal rabbit lung, both tissues contained mixed populations of ill- and fl2-adrenoceptors in similar proportions. This maturation phase has also been associated with increased adenylate cyclase activity in rat brain (Perkins and Moore, 1973) and rat lung (Nijjar, 1979). The second phase of age-related change in fladrenoceptor function occurs in adulthood. Hyashi and Toda (1978) demonstrated age-related increases in isoprenaline-induced relaxation in rabbit aorta from 2-30 day old animals, with decreased responsiveness occurring between 30 and 360 days of age. A similar profile was observed in rat aorta (Schoeffter and Stoclet, 1982, 1983). Relaxant responsiveness to the fl-adrenoceptor agonist isoprenaline has also been shown to significantly decrease in rat trachea (Aberg et al., 1973; Frossard and Landry, 1985) and guinea-pig trachea (Aberg et al., 1973; Duncan et al., 1982) as a function of animal age. Such decreased responsiveness in aged tissue has been attributed to fl-adrenoceptor desensitization (Ebstein et al., 1985; Heinsimer and Lefkowitz, 1985) in response to agerelated increases in plasma catecholamines (Krall et al., 1981; Lake et al., 1977) and to decreased adenylate cyclase activity (Williams and Thompson, 1973; Ebstein et al., 1985). Andersson et al. (1978a) found that basal levels of c-AMP were 5-fold higher in bovine trachea from young animals than from old animals. Similarly, in rat lung both basal levels of adenylate cyclase and the maximum adenylate cyclase response to fl-adrenoceptor stimulation, have been shown to be decreased as a function of age (Scarpace and Abrass, 1983). Krall et al. (1981) and O'Connor et al. (1983) have shown that ageing results in reductions in both G protein levels and adenylate cyclase activity, although attenuation of function would be expected to be the result of reduced adenylate cyclase activity, since G protein is present in excess. In addition, decreased fl-adrenoceptor density has been associated with age-related decreases in responsiveness to fl-adrenoceptor agonists in cerebral microvessels (Kobayashi et al., 1982) and in brain tissue (Greenberg and Weiss, 1978; Cimino et al., 1984). However, the influence of postnatal animal ageing on fl-adrenoceptor density in airway smooth muscle is as yet unknown. 2.4.6. The Epithelium and Airway Smooth Muscle fl-Adrenoceptor Function It is now well established that the airway epithelium has a modulatory influence on the spasmogenic potency of a wide variety of agonists in airway smooth muscle preparations from several species including man (Raeburn et al., 1986; Goldie et al., 1990a). Holroyde (1986) has suggested that the epithelium can act as a barrier to drug diffusion. Depending upon the agonist in question, the epithelium can also act as a site of loss (uptake and/or metabolism) (Farmer et al., 1986; Advenier et al., 1988; Fine et al., 1989; Frossard et al., 1989) or may release spasmolytic inhibitory factors (Flavahan et al., 1985;

301

Hay et al., 1987; Fernandes et al., 1989; Fernandes and Goldie, 1990). The airway epithelium contains an abundance of fl-adrenoceptors (Goldie et al., 1986a,b). Epitheliumderived inhibitory factors (EpDIF) may also play a significant role with respect to modulating the relaxant effects of /~-adrenoceptor agonists on airway smooth muscle. Deliberate removal of the epithelium from isolated airway preparations has been reported to reduce the relaxant potency and/or maximal relaxant effect of isoprenaline in canine bronchus (Flavahan et al., 1985; Stuart-Smith and Vanhoutte, 1987), bovine trachea (Barnes et al., 1985), guinea-pig trachea (Goldie et al., 1986a) and pig bronchus (Stuart-Smith and Vanhoutte, 1988). In the dog and pig, the influence of this putative inhibitory factor on fl-adrenoceptor agonist-induced smooth muscle relaxation increases as the diameter of the airways decreases (Vanhoutte, 1988). In guinea-pig trachea however, the epithelium has also been reported to act as an extraneuronal uptake site for catecholamines, thereby reducing fl-agonist potency (Farmer et al., 1986). Conversely, in human bronchus, epithelium removal had no significant effect on relaxation responses to isoprenaline (Aizawa et al., 1988). 2.4.7. Interactions between Respiratory Viruses and fl-Adrenoceptors Viral particles from different classes utilize different means to engage the surface of target cells prior to their being internalized. It has recently been shown that reovirus-3 particles contain a surface binding site with many of the properties of mammalian fl-adrenoceptors (Co et al., 1985). Reovirus-3 may also possess a site which recognizes fl-adrenoceptors on cell membranes (Co et al., 1985). Interaction between the virus with fl-adrenoceptors may be one way that these particles attach to the cell surface before they are internalized. Rhinovirus-16, parainfluenza and influenza-A viruses trigger wheeze in asthmatic children and adults (Hudgel et al., 1979; Tario et al., 1979). fl-Adrenoceptor antagonists also precipitate asthma symptoms in asthmatics (Zaid and Beall, 1966; Richardson and Sterling, 1969; Paterson et al., 1982) due to unopposed parasympathetic nerve activity causing bronchoconstriction. While upper respiratory tract viral infections induce airway inflammation and epithelial damage (Hers, 1966) which may contribute to asthma symptoms including bronchial hyperreactivity (Empey et al., 1976), virally precipitated bronchospasm in asthma may also result from reduced airway fl-adrenoceptor function. Several studies provide evidence for the attenuation of fl-adrenoceptor function by respiratory viruses. Viral particles including influenza and rhinovirus-16 have been shown to reduce the inhibitory effect of isoprenaline on lysozymal enzyme release from human granulocytes (Bush et al., 1978; Busse et al., 1980). Furthermore, fl-adrenoceptor-mediated release of fl-glucuronidase from polymorphonuclear leukocytes (PMN) was reduced in the presence of influenza virus as was isoprenaline-induced PMN rosette formation with erythrocytes (Lee, 1980). Parainfluenza-3 virus infection was also shown to

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reduce fl-agonist-mediated inhibition of ovalbumininduced contractions of guinea-pig trachea (Buckner et al., 1981). Such data raise the possibility that respiratory viral infections initially induce bronchial smooth muscle fl-adrenoceptor hypofunction, which then exacerbates bronchial obstruction. 2.4.8. fl-Adrenoceptor Function in Asthma It has been proposed that diminished responsiveness of the fl-adrenoceptor adenylate cyclase system was the key defect in asthma (Szentivanyi, 1968). Airway responsiveness to salbutamol in asthma is not reduced in mild asthmatics (Tattersfield et al., 1983), but diminished responsiveness can be positively correlated with disease severity, as assessed by the level of baseline function (Barnes and Pride, 1983). The interpretation of such studies is difficult, since in asthma there may be a reduction in access of the inhaled salbutamol in proportion to the degree of airway obstruction (Barnes and Pride, 1983) and/or an elevation of bronchial tone sufficient to reduce its bronchodilator effectiveness (Barnes, 1986). In vitro studies allow more direct investigations of fl-adrenoceptor function in asthma. 2.4.8.1. Functional studies in human isolated bronchi. Svedmyr et al. (1976) and Whicker et al. (1988) reported no significant difference in responsiveness to isoprenaline in bronchial preparations from nonasthmatic and asthmatic lungs. The bronchial preparations tested by Svedmyr et al. (1976) were from asthmatics undergoing surgery for lung cancer. As these asthmatic patients were sufficiently healthy to undergo surgery, they presumably had mild stable asthma. Similarly, the bronchi tested by Whicker et al. (1988) were obtained from mild asthmatic subjects. Thus, bronchial fl-adrenoceptor hypofunction does not appear to occur in mild asthma. In contrast, Cerrina et al. (1986) showed that the effective concentration of isoprenaline producing 50% of the maximal relaxant response, was 10-fold greater in bronchi from asthmatics than in preparations from nondiseased patients. In addition, a negative correlation was demonstrated between asthma severity (as measured by airway reactivity to inhaled histamine in vivo) and bronchial responsiveness to isoprenaline in vitro.

Goldie et al. (1986c) demonstrated that the relaxant potencies of isoprenaline, fenoterol and terbutaline were significantly attenuated in bronchial preparations from lung tissue from patients who had died with severe asthma. Conversely, relaxant responsiveness to the non-fl-adrenoceptor agonist, theophylline, was similar in bronchi from nondiseased and asthmatic lung, suggesting selective dysfunction of the fl-adrenergic system in adults with severe asthma (Fig. 4). Since sensitivity to isoprenaline in bronchi from two asthmatic subjects who did not receive regular fl-agonist medication was significantly attenuated, the hypofunction appeared to be related to disease rather than to inhalation of fl-adrenoceptor agonists. 2.4.8.2. Allergen studies in lymphocytes. Koeter et al. (1982) demonstrated that prior to inhalation of house

dust mite allergen, lymphocyte c-AMP responses to isoprenaline in asthmatics were similar to those in nondiseased individuals. In contrast, 24 hr after inhalation of allergen, the maximal fl-adrenoceptor mediated c-AMP increase in lymphocytes was reduced in asthmatics experiencing a late phase inflammatory reaction, but not in those with no late phase reaction and also not in nonasthamtic patients. This suggests that fl-adrenoceptor dysfunction in lymphocytes from asthmatics occurred as a consequence of inflammation. As asthma becomes more severe, the number of lymphocyte fl-adrenoceptors falls (Brooks et al., 1979). Meurs et al. (1982) found that the allergen induced decrease in lymphocyte fl-adrenoceptor function appeared to involve an uncoupling of the fl-adrenoceptor from the guanine nucleotide regulatory protein and/or adenylate cyclase, as well as a reduction in membrane receptor number. Further study by Meurs and his coworkers demonstrated that the fl-adrenoceptor response could be reduced by 51% after allergen challenge even though the reduction in fl-adrenoceptor number was only 19% (Meurs et aL, 1987). Thus allergen-induced reduction in fl-adrenergic function in asthmatic lymphocytes appears to be principally due to receptor uncoupling rather than receptor downregulation. A similar reduction in fl-adrenoceptor coupling in human airway smooth muscle may contribute towards the observed fl-adrenoceptor hypofunetion in asthmatic bronchi. 2.4.8.3. Radioligand binding studies and autoradiography in lung. Szentivanyi (1979) has reported that the amount of specific DHA (12 riM) bound, was significantly less in lung from asthmatic patients than in lung from healthy subjects. In a later study Barnes et al. (1980b) showed that the maximum specific number of sites binding (Bmax) for DHA in tissue from a healthy individual was well within the range of values obtained for lung tissue from six chronic bronchitic patients with mild obstructive disease. Van Koppen et al. (1989) also examined bronchial tissue obtained from normal subjects and from those with chronic airflow obstruction. They found that while the fl-adrenoceptor numbers were not reduced in airway smooth muscle of patients with chronic airflow obstruction, there appeared to be less effective coupling between the fl-adrenoceptor and adenylate cyclase in bronchial tissue from these subjects. Moreover, the number of DHA binding sites has been found to increase in patients with chronic obstructive lung disease compared with that in nondiseased lung (Raaijmakers et al., 1985). We have used lung samples obtained postmortem from healthy individuals and from subjects who suffered and/or died as a result of severe asthma to determine whether or not reduced responsiveness of bronchi from asthmatic lung to fl-adrenoceptor agonists was reflected in a decrease in fl-adrenoceptor number in the lung in general and in bronchial smooth muscle in particular (Goldie et al., 1987; Spina et al., 1989a). Data from these studies showed that in healthy human lung, specific I-CYP binding was saturable, involving a homogeneous class of

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sites with high affinity and the KD value for I-CYP binding was not significantly altered in asthmatic lung compared with healthy lung. Bmax estimates in the two asthmatic lung samples were 81 and 149 fmol/mg protein, while nmax in nondiseased human lung was 56 + 7 fmol/mg protein (n = 8). These data suggest that nmax was not reduced and may even be increased in severe asthma. The absence of reduced fl-adrenoceptor density in the two asthmatic lungs studied, is consistent with the notion that fl-adrenergic hypofunction in asthma is not necessarily associated with a reduction in fladrenoceptor number. An increase in the density of autoradiographic grains representing specific I-CYP binding sites was observed over smooth muscle in bronchi from a severe asthmatic. However, in bronchial preparation from the same lung, isoprenaline and fenoterol were 10-13 and l l-13-fold

less potent than in nondiseased lung. The relaxant potency of theophyUine was unaltered. These data are also consistent with uncoupling of the fl-adrenoceptor/-adenylate cyclase system in severe asthma (Meurs et al., 1987; Lulich et al., 1988). 2.4.8.4. The effects of PLA2 and inflammatory chemical mediators on [3-adrenoceptor function. The membrane-bound enzyme phospholipase A 2 is responsible for the mobilization of arachidonic acid and platelet activating factor (PAF) from membrane phospholipids (Lulich et al., 1988). Taki et al. (1986) examined the influence of antigen challenge on phospholipase A2 activity and /~-adrenoceptor function in guinea-pig lung membranes. Allergen inhaled for 7-8 min for 10 successive days increased phospholipase Az activity by 50% and decreased/~-adrenoceptor number and function by 37% and 54%

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respectively. They also showed that the addition of phospholipase A2 to isolated lung membranes produced a decrease in fl-adrenoceptor number and function. Thus phospholipase A 2 activation is not only linked to inflammation by the release of PAF, prostaglandins and leukotrienes but also by a reduction in fl-adrenoceptor function. Mallorga et al. (1980) have suggested that both lysosphatidylcholine and arachidonic acid metabolites released by the activation of phospholipase A2 can cause uncoupling between the fl-adrenoceptor and adenylate cyclase. The addition of PAF to the guinea-pig isolated trachea has been shown to decrease the relaxant potency of isoprenaline in this preparation (Agrawal et al., 1987). PAF also decreased the sensitivity to isoprenaline of lung parenchymal strips taken from endotoxin-treated guinea-pigs (Touvay et aL, 1988). Agrawal and Townley (1987) reported that PAF caused a decrease in the density of fl-adrenoceptors but did not alter their affinity in human lung. They also found that PAF shifted isoprenaline dose-response curves to the right in both human trachea and lung parenchyma. However, other workers found that PAF did not produce fl-adrenoceptor dysfunction (Barnes et al., 1987; Chand et al., 1988). Barnes et al. (1987) demonstrated that pretreatment with PAF did not reduce the effectiveness of isoprenaline in relaxing guinea-pig isolated trachea or reduce fl-adrenoceptor numbers of guineapig lung. Chand et al. (1988) reported that PAF did not produce any down regulation of the fladrenoceptor in rat or guinea-pig isolated trachea. Raaijmakers et al. (1986) found that while PAF caused a small reduction in fl-adrenoceptor number in guinea-pig lung, LTB4 produced a much greater reduction. Abbracchio et al. (1986) proposed that arachidonic acid metabolites might reduce coupling by modulating inositol phospholipid turnover. This leads to activation of protein kinase C which inactivates the fl-adrenoceptor by promoting its phosphorylation. In addition, Meurs et al. (1987) indicated that receptor uncoupling induced by allergen challenge may involve protein kinase C activation. Muscarinic agonists such as carbachol are able to produce fl-adrenoceptor down-regulation and uncoupling in bovine

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tracheal smooth muscle and this may be because muscarinic agonists activate protein kinase C which promotes fl-adrenoceptor phosphorylation (Grandordy and Barnes, 1987). 2.5. CLINICALACTIONSOF fl-AGONISTSIN LUNG Inhaled fl-agonists act quickly, are very potent in relaxing airway smooth muscle and can produce significantly more bronchodilatation acutely than therapeutic blood levels of theophylline administered intravenously (McFadden, 1985). In addition, fl-agonists are effective on airway smooth muscle throughout the lung and have a high therapeutic index (Lulich et al., 1986; Goldie et al., 1990b). Thus, inhaled fl-agonists are and should be the drugs of choice for quickly reversing acute asthmatic attacks. 2.5.1. fl-Agonist Drugs Isoprenaline (Fig. 5) is a fl-adrenoceptor agonist which appears to have no stimulating action on -adrenoceptors. Although it is potent, it has a short duration of action (Paterson et al., 1979; Chu, 1984). It is usually given by inhalation and considerable O-methylation can occur during absorption from the airways, due to the presence of catechol-O-methyltransferase (COMT) in the lung (BlackweU et al., 1974). Kinetic studies and investigations using radiolabeled tracers indicate that approximately 10% of the dose from a pressurized aerosol reaches the lung (Davies, 1975). The majority of the inhaled drug is swallowed and inactivated. fl2-Selective bronchodilators such as salbutamol, terbutaline and fenoterol were introduced in an attempt to reduce cardiac stimulation (Paterson et al., 1983; Chu, 1984; Reed, 1985). Studies using isolated tissues from the guinea-pig suggest that fl2-agonists are highly selective for airway smooth muscle in preference to cardiac muscle (Brittain et al., 1976; Paterson et al., 1983). However, because there is a significant population of flz-adrenoceptors in the human heart (Ablad et al., 1974; Heitz et al., 1983; Robberecht et al., 1983; Corea et aL, 1984) a reduction in airway selectivity of fl2-agonists is predicted when they are administered to humans in vivo.

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Adrenoceptors in airway smooth muscle Thus some cardiac stimulation is expected even with very selective/~2-agonists. In most cases, the severity of cardiac side-effects can be effectively minimized by administration of the /~2-agonists by inhalation (Paterson et al., 1983; Lulich et al., 1986). Terbutaline has a resoreinol nucleus while salbutamol has a methylene group inserted at the 3hydroxyl position to form a saligen derivative (Fig. 5). Both drugs are rapidly absorbed from airways and no metabolism occurs in the lung (Davies, 1975; Shenfield et al., 1976). The two drugs have virtually equal fl2-selectivity. The relatively greater fl2-activity appears to be due to substitution of the tertiary butyl group in the amine head. Both compounds are effective orally, by inhalation and by intravenous injection. Both salbutamol and terbutaline however remain substrates for conjugating enzymes and in man mainly sulfate conjugates are formed (Evans et al., 1973; Nilsson et al., 1972). Fenoterol (Fig. 5) has a resorcinol nucleus and like other compounds in this group is metabolized by 5-sulfo-conjugation, but sufficient free drug is absorbed so that it is effective when given by the oral route. It can be given also by injection or inhalation and its fiE-selectivity depends on the large substituent group on the amine head. These three orally active flE-agonists are used in various inhalation formulations. With these, the aim is to give a small but highly effective dose to the lung. The small doses delivered to the lung by pressurized multidose aerosol give the desired therapeutic effect within 5-10rain and have a duration of action of up to 6 hr (Chu, 1984; Tattersfield, 1986). Following inhalation of such small doses, plasma concentrations of the active drug will be negligible. The properties of other clinically useful selective fl2-agonists and prodrugs are discussed in the following reviews (Paterson et al., 1979; Chu, 1984; Goldie et al., 1990b). 2.5.2. Use o f fl-Agonists in Infants Lenney and Milner (1978) have shown that while nebulized salbutamol reduced airway obstruction in most wheezing infants over 20 months of age, it did not improve lung function in children between 7 and 18 months of age. Prendiville et al. (1987b) have provided evidence that in the airways of infants aged 3-12 months, airway /~2-adrenoceptor function can provide protection against histamine challenge. The protective effect of nebulized salbutamol in infants may be due to its action on fl-adrenoceptors in airway smooth muscle, although its action on fl-adrenoceptors elsewhere in the lung needs investigation (Silverman and Prendiville, 1987). Prendiville et al. (1987a) have also studied the effect of nebulized salbutamol on lung function in infants who were only 3-15 months of age and who had recurrent wheeze. Nebulized salbutamol did not affect peak expiratory flow (a measure of specific airways resistance), but did decrease maximal flow at functional residual capacity (VmaxFRC). This reduction is potentially detrimental, since in acute severe obstruction, infants use forced expiration during tidal breathing (Prendiville et al., 1987a). Silverman and Prendiville (1987) proposed that in

305

very young infants, airway obstruction was mainly caused by inflammation and edema and that airway smooth muscle tone probably helped to splint the airways. Any reduction in tone caused by inhaled fl-agonists would thus increase airway compliance and decrease flow as described. Alternatively, fladrenoceptor function may be poorly developed in such young infants (see Section 2.4.5). In either case, the use of fl-agonist$ in wheezing infants under the age of 18 months is not recommended. 2.5.3. Sites o f Action In Vivo Despas et al. (1972) investigated the site of airway obstruction in asthma by measuring maximal expiratory flow with air and a helium-oxygen (He/O2) mixture and obtained results indicating that in asthma, the major site of obstruction was either the large or small airways. Antic and Macklem (1976) obtained data with He/O2 in asthmatics that suggested that inhaled salbutamol acted predominantly at the airway smooth muscle in the smaller more peripheral airways. Ingram et al. (1977) reported that while muscarinic cholinoceptor antagonists produced selective dilatation at the larger upstream airways in normal subjects, isoprenaline preferentially dilated the smaller airways. Hensley et al. (1978) also found that/~-agonists acted predominantly on the smaller, more peripheral airways in human patients. However, others have found that fl-agonists acted mainly on the central airways (Sybrecht et al., 1980; Nassari et al., 1981). Fairshter and Wilson (1980) have indicated that the site of airflow limitation is important when fl-agonists are delivered by inhalation. The higher the airway resistance, the more centrally will the aerosol be deposited. Hence asthmatics with mainly small airway obstruction respond by dilatation of the peripheral airways, while those who have a predominant obstruction of the central airways respond mainly by dilatation of the larger airways. Tashkin et al. (1980) compared the airway sites acted upon by 0.5 mg terbutaline given by nebulizer and subcutaneous injection in twelve asthmatics. They found that while inhaled terbutaline selectively dilated the larger airways, subcutaneous terbutaline acted on both large and small airways in most subjects. In contrast, Pierce et al. (1981) used higher doses of nebulized terbutaline to show it acted on both the large and smaller airways. Lower doses of inhaled fl-agonists act more selectively on the central airways than higher doses because the higher doses are absorbed and recirculuted to the peripheral airways (De Troyer et al., 1978). /~-Agonists given orally produce bronchodilatation more selectively in the peripheral airways than when they are inhaled (Larsson and Svedmyr, 1977; Fairshter et al., 1981). Pierce et al. (1981) found there was no difference between intravenous and nebulized terbutaline with regard to the bronchodilatation produced. Thus, the principal site of action of fl-agonists in asthma may be either the central or peripheral airways, or both levels may be affected to a similar extent depending upon the principal site of airway obstruction, dose of inhaled drug or route of administration (De Troyer et al., 1978; Fairshter and Wilson, 1980; Chu, 1984).

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2.5.4. Tolerance to fl-Agonists

2.5.6. Role in Inflammation

The clinical response to fl-agonist therapy may diminish as asthma becomes more severe (Paterson et al., 1979, 1984). Conolly et al. (1971) proposed that inhalation of excessive amounts of isoprenaline may cause desensitization of airway smooth muscle fladrenoceptors, resulting in attenuation of bronchodilator response to fl-agonists. Furthermore, the development of desensitization of fl2-adrenoceptors in airway smooth muscle might be a greater problem with the longer acting, orally active, selective fl2agonists, such as salbutamol, terbutaline and fenoterol. However, inhaled fl2-agonists may also become less effective in severe asthma primarily due to an alteration in the nature of the bronchial obstruction rather than as a consequence of decreased airway fl-adrenoceptor function (Paterson et al., 1979; Rossing et al., 1983). The manner in which fl-agonists can produce fl-adrenoceptor desensitization has been described in Section 2.2.4. Long-term exposure to fl2-agonists can cause significant desensitization of fl-adrenoceptors in normal human airways, but it has been found that asthmatic airways are less susceptible to fl2-agonist induced desensitization of fl-adrenoceptors (Harvey and Tattersfield, 1982; Tashkin et al., 1982; Lipworth et al., 1989). fl2-Adrenoceptors in asthmatic airways are resistant to fl-agonist induced desensitization possibly because there is a relatively large population of 'spare fl2-adrenoceptors' in asthmatic airway smooth muscle. Alternatively, fl2-adrenoceptors in asthmatic airways may be more resistant to down-regulation. Thus, the bronchodilatation produced by a fl2-agonist should not be reduced by agonist-induced desensitization if recommended oral and/or inhaled doses of fl2-agonists are used in the prophylactic treatment of asthma.

fl2-Agonists appear to be ineffective in suppressing or controlling airway inflammation in asthmatics (Goldie et al., 1990b). They do not suppress late asthmatic reactions (Booij-Noord et al., 1972; Hegardt et al., 1981; Cockcroft and Murdoch, 1987), nor do they decrease bronchial hyperreactivity when taken on a long term basis (Kraan et al., 1985; Kerrebijn et al., 1987). This draws attention to the danger of relying exclusively on fl2-agonist bronchodilators for the prophylactic treatment of asthma. Their powerful bronchodilator action may mask the onset and/or exacerbation of airway inflammation in asthmatics, resulting in the possible under utilization of effective antiinflammatory agents. Thus, there should be a primary emphasis on the use of drugs with anti-inflammatory properties, such as corticosteroids, cromoglycate and perhaps theophylline for the prophylactic treatment of asthma (Paterson et aL, 1988; Goldie et al., 1990b). Nevertheless, it is important to stress that if adequate treatment with anti-inflammatory prophylactics is maintained, it is beneficial to use long acting inhaled fl2-agonists to produce supplementary bronchodilation. In fact, the results of Vathenen et al. (1988) indicate that it is inadvisable to stop the administration of high doses of fl-agonist suddenly because temporary rebound nonspecific hyperreactivity to bronchoconstrictor stimuli may result and thus exacerbate the asthma.

2.5.5. fl-Agonists and Hyperreactivity After acute administration, fl2-agonists cause a dose-dependent reduction in nonspecific bronchial reactivity to spasmogens which is larger than that produced by other currently used antiasthmatic drugs (Tattersfield, 1987). Thus the powerful bronchodilatation produced by fl2-agonists can mask the exacerbation of airway inflammation associated with bronchial hyperreactivity. When fl2-agonists are given chronically and measurements are made 12-16hr after the last administration, there is no decrease in bronchial hyperreactivity (Kraan et aL, 1985; Kerribijn et al., 1987). Furthermore, Vathenen et al. (1988) showed that terbutaline administered continuously for two weeks appeared to produce a rebound increase in reactivity after its bronchodilator action declined. Vathenen et aL (1988) proposed that continuous exposure to terbutaline produced densensitization of airway fl-adrenoceptors. They suggested that while fl-agonist-induced desensitization was not sufficient to reduce the response to inhaled fl-agonist it appeared to be sufficient to reduce the protective effect of endogenous catecholamines in lung and thus cause rebound bronchial hyperreactivity.

3. a-ADRENOCEPTORS 3.1. SUBCLASSIFICATION OF 0~-ADRENOCEPTORS

ct-Adrenoceptors may be subclassified as a~ or ~t2 subtypes depending on the selectivity of specific agonists and antagonists (Berthelsen and Pettinger, 1977; Starke and Langer, 1979; Hoffman and Lefkowitz, 1980). Initially, it was proposed that the pre- and postsynaptic receptors be called ~t2- and ~t~-adrenoceptors respectively (Langer, 1974). Prejunctional ~t2-adrenoeeptors not only inhibit the release of noradrenaline from adrenergic nerve terminals (Langer, 1974; Starke, 1977; Westfall, 1977), but also neurotransmitters from other types of nerve terminals (Langer, 1981). ~t~-Adrenoceptors are the classic postsynaptic ~t-adrenoceptors, but it is now well established that a2-adrenoceptors are also located on smooth muscle cells and mediate smooth muscle contraction (Doxey and Easingwood, 1978; Docherty et al., 1979; Drew and Whiting, 1979; Timmermans et al., 1979). Thus, ~t-adrenoceptors should not be subclassified in relation to their location, but according to their affinities for agonists and antagonists. For example, ~-adrenoceptors are selectively stimulated by phenylephrine and selectively antagonized by prazosin while clonidine is a selective ~t2-agonist and yohimbine is a selective • 2-antagonist (Langer, 1981; Docherty, 1989; Minneman, 1988). Some agonists and antagonists selective for ~t~- or ~t2-adrenoceptors are indicated in Table 2.

Adrenoceptors in airway smooth muscle TABLE2. *t-Adrenoceptor Agonist and Antagonist AGONISTS ~q selective ,t2 selective nonselective phenylephrine clonidine noradrenaline methoxamine UK14304' adrenaline cirazoline guanabenz ANTAGONISTS prazosin yohimbine phentolamine BE22541" rauwolscine dihydroergotamine WB4101 * idazoxan *UK14304: 5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline. tBE2254: 2-[fl-(4-hydroxyphenyl)-ethyl aminomethyl]teralone. .+WB4101: 2-(2,6-dimethoxyphenoxyethyl)aminomethyl1,4-benzodioxane HCI.

307

determine a functional subclassification that will stand the test of time (Docherty, 1989). 3.2. MECHANISM OF ACTION OF gj- AND (x2-ADRENOCEPTORS

Activation of g2-adrenoceptors appears to produce contraction of smooth muscle by inhibition of adenylate cyclase through guanine nucleotide inhibitory protein (Gi) (Homcy and Graham, 1985; Bylund, 1988; Exton, 1988; Docherty, 1989; Fig. 6). Current evidence indicates that ~t-adrenoceptors mediate phosphatidylinositol breakdown through a G protein (G) that still remains to be specifically identified (for reviews see Homcy and Graham, 1985; Exton, 1988; Minneman, 1988). Phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis appears to be the initial step by which g~-adrenoceptor activation produces contraction of smooth muscle. Agonist-receptor interaction activates phospholipase C, which catalyses the hydrolysis of PIP2 in the plasma membrane to form inositol-1, 4, 5-trisphosphate (IP3) and diacylglycerol (DG). IP3 acts as a second messenger by triggering the release of calcium from intracellular stores such as sarcoplasmic reticulum. DG activates the Ca 2+ and phospholipid-dependent protein kinase C and promotes arachidonic acid metabolism. The protein kinase C activated by DG may contribute towards smooth muscle contraction by increasing the sensitivity of the contractile proteins to Ca 2÷ (Bulbring and Tomita, 1987). It appears that u~-adrenoceptor subtypes may work through distinct mechanisms. Thus, while ~j,adrenoceptors initiate signals through inositol phospholipid breakdown, g~b-adrenoceptors primarily act on nifedipine-sensitive calcium channels to promote the influx of extracellular Ca 2+ (Minneman, 1988; Docherty, 1989). The mechanisms by which gl~-

cq-Adrenoceptors have been further subclassified as ~qa and ~]b (Bylund, 1988) on the basis of different affinities for the ~tl-selective antagonist WB4101 (Docherty, 1989). It has been proposed that "the cq selective antagonist WB4101 has a high affinity for ~t]a-adrenoceptors while it has a low affinity for Cqb-adrenoceptors (Morrow and Creese, 1986; Han et al., 1987). Binding data suggest that while the hippocampus and vas deferens contained both ~1,- and cqb-adrenoceptor subtypes, liver and spleen had only the ~lb subtype (Minneman, 1988). The molecular weight of the ~l-adrenoceptor has been variously estimated at 78,000-85,000Da (Exton, 1988) and 77,000-91,000 Da (Docherty, 1989). However, there is no conclusive evidence from molecular weight studies for distinct ,q-adrenoceptor subtypes (Docherty, 1989). More binding, functional and molecular cloning data are required before a generally accepted subclassification of cq-adrenoceptors is determined (Docherty, 1989). ~t2-Adrenoceptors have also been further subclassified as ct2a and ~t2bon the basis of their affinity Phenylephdne Clonldine for prazosin. Neonatal rat lung contains ct2b-adrenoceptors (prazosin affinity of 5 riM) and the human platelet is the standard preparation for the detection ~1 ~2 of cq,-adrenoceptors (prazosin affinity of 250 nM) toG toG I (Bylund, 1988). While prazosin is selective for Ct2badrenoceptors, oxymetazoline is selective for ~%adrenoceptors (Bylund et al., 1988). The molecular weight of the human platelet ct2-adrenoceptor subtype has been estimated to be approximately 64,000 Da while the ~2-adrenoceptor subtype in neonatal rat lung has a molecular weight of about 44,000 Da Mobilization of Entry of extracellular Inhibition of (Lanier et al., 1988). ~2-Adrenoceptors from opposintracellular calcium calcium ( a t= ) adenylate cyclase sum kidney derived OK cells (Murphy and Bylund, (inositol phosphate ( (x 2=' (= 2b' (= ~ ? ) production ( ~ l b ) ) 1988) may represent a third subtype designated as ct2c (Bylund, 1988). Molecular cloning techniques also support the presence of at least three subtypes of ct2-adrenoceptors and they have been designated as ct2 CI0, C4 and C2 receptors because their Contraction of smooth muscle DNA coding sequences reside on chromosomes 10, 4 and 2, respectively (Kobilka et al., 1987; Bylund, 1988). Nevertheless, more research is required before the exact number of ct2-adrenoceptor subtypes is G I : guanine nucleotide binding inhibitorypmmin established. G : guanine nucleotlde binding protein still to be characterized In summary, presently there are differing viewpoints with regard to how ~q- and ~q-adrenoceptors FIG. 6. IntraceUular events leading to contraction of airway should be further subclassified. The problem is to smooth muscle following activation of ~-adrenoceptors.

I

I

R. G. C-OLDIEet al.

308

and ~]b-adrenoceptors may produce contraction of smooth muscle are illustrated in Figs 7 and 8.

Noradrenaline

3.3. IN VIVO STUDIESOF LUNG~x-ADRENOCEPTORS IN ASTHMA PLC..--)~ PIP2--->- D A G ~

3.3.1. ~-Agonists Several studies in man have shown that asthmatics may experience bronchial obstruction in response to inhaled ~-adrenoceptor agonists, while healthy volunteers do not (Patel and Kerr, 1973; Snashall et al., 1978; Black et aL, 1982). Furthermore, the ~-agonist phenylephrine has been shown to be a significantly more potent constrictor of cutaneous blood vessels and pupillary dilator in allergic asthmatics than in nonasthmatic subjects (Henderson et al., 1979). These data suggest that in asthma, enhanced u-adrenoceptor function is not restricted to airway smooth muscle. In contrast, Larsson (1985) reported that ~-adrenoceptor-mediated vasoconstriction was not enhanced in asthmatic subjects. Phenylephrine-induced airway obstruction could only be demonstrated in asthmatics after/~-adrenoceptor blockade with propranolol (Patel and Kerr, 1973). In the absence of this treatment, phenylephrine caused marked bronchodilatation in asthmatics but had no such effect in nonasthmatic subjects, suggesting the higher levels of resting airway tone in the asthmatics. In nontreated subjects, u-adrenoceptor function was clearly subordinate to/~-adrenoceptor function even in asthmatics. Methoxamine-induced bronchial obstruction was antagonized by ipratropium bromide (Black et aL, 1985) indicating the involvement of parasympathetic nerves. This raises the possibility of a nonspecific effect of inhaled nebulized methoxamine solution on exposed irritant receptors in asthmatic airways. While methoxamine-induced obstruction may have been mediated via airway smooth muscle ~-adrenoceptors in asthmatics, enhanced ~-adrenoceptor function in asthmatics compared with normals has not been unequivocally demonstrated. The interpretation of the observations is complicated by differences in the levels of baseline airway obstruction and in airway sensitivity to inhaled irritant stimuli between the normals and the asthmatics (Ind and Doilery,

Ca 2+

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: Guanine nuclechde binding protein associated with the OGlb-adrenoceptor that still remains to be characterized : Phospholipase C : Phosphattdylinositol - 4,5 - bisphosphate : Inositol - 1,4,5 - tdsphosphate : Diacylglycerol : C a 2+ and phospholipM-depenent protein kinase C : Endoplasmic reticulum

FIG. 8. Interactions between a~b-adrenoceptors, G regulatory protein and the inositolphosphate pathway, leading to calcium mobilization and contraction of airway smooth muscle. 1983). Following the suggestion that the pH of inhaled solutions might affect airway responsiveness (Cockcroft and Berscheid, 1982), Thomson et al. (1982) used buffered solutions when testing the effects of the ~-adrenoceptor agonist phenylephrine by inhalation. They showed that phenylephrine had no greater effect on lung function than buffered saline alone. 3.3.2. ~-Adrenoceptor Antagonists Studies of the effects of ~-adrenoceptor antagonists in asthma are also difficult to interpret. Phentolamine and thymoxamine have been shown to improve resting airway conductance in asthmatics (Griffin et al., 1972; Patel and Kerr, 1975; Patel, 1976). However, such antagonists can induce significant falls in systemic blood pressure (Taylor et al., 1965) and thus may activate the sympatho-adrenal system to release bronchodilator catecholamines. Furthermore, ~blockers such as phentolamine have antihistamine activity (Bianco et al., 1972; Gaddie et al., 1972), directly relax smooth muscle and can modify catecholamine release and uptake (Ind and Dollery, 1983; Svedmyr, 1984; Walden et al., 1984). Inhaled prazosin (0.5-2 mg), which does not have these properties (Barnes et al., 1981a,b), did not alter resting airway tone in asthmatics. This indicates that ~adrenoceptors do not play an important role in determining the level of airway obstruction (Jenkins et al., 1985; Barnes et al., 1981a,b), an idea consistent with the fact that ~-blockers have proved to be ineffective in the treatment of most forms of asthma

309

Adrenoceptors in airway smooth muscle (Ind and Dollery, 1983; Svedmyr, 1984; Utting, 1979). 3.3.3. Exercise-Induced Asthma Although ~-blockers have proved to be ineffective in the treatment of asthma in general, prazosin has been shown to reduce exercise-induced asthma (Barnes et al., 1981a,b). Inhalation of cold air resulting in airway hypothermia is thought to induce bronchospasm. Cooling of the airways during exercise might also activate ~-adrenoceptors in airway smooth muscle resulting in bronchoconstriction (Walden et al., 1984; Bleecker et al., 1983). In a small number of bronchial preparations from nonasthmatic lung, noradrenaline-induced contractions were enhanced by reduction of the organ bath temperature from 37°C to 20°C (Black, 1986). However, this was not a consistent finding. As yet unpublished data from our laboratory has shown that reducing the incubation temperature from 37°C to 24°C had no potentiating effect on the level of ~-adrenoceptor function in bronchi isolated from human asthmatic lung. This does not support the concept of hypothermia-induced increases in airway smooth muscle ~-adrenoceptor function. While some studies have demonstrated the clinical efficacy of phentolamine and prazosin in exerciseinduced asthma (Walden et al., 1984; Barnes et al., 1981a,b; Bleecker et al., 1983), inhalation of the • t-selective agonist methoxamine can reduce exercise induced asthma in some cases (Dinh Xuan et al., 1989). Thus, the role of airway c¢-adrenoceptors in exercise-induced asthma is difficult to ascertain from in vivo studies in asthmatics. 3.3.4. Usefulness o f the Information Obtained In addition to airway smooth muscle contraction, the responses of several other pulmonary tissues to ~-agonists and ~-antagonists, may alter airway caliber and airflow resistance (Goldie et al., 1988). These include enhancement of chemical mediator release from mast cells (Kaliner et al., 1972; Coffey and Middleton, 1973), stimulation of airway mucus secretion (Phipps et al., 1980, 1982), modification of neurotransmitter release (Grundstrom and Andersson, 1985a,b; Andersson et al., 1986) and constriction of vascular smooth muscle (Advenier and Floch-Saint-Aubin, 1984; Miller and Vanhoutte, 1985). The distribution and function of ~-adreno-

ceptors in lung is summarized in Table 3. ~-Adrenoceptor stimulation can produce effects which either exacerbate airway obstruction (e.g. contraction of airway smooth muscle, increase glandular secretions), or improve airflow (e.g. reduce bronchoconstrictor nerve traffic). In vivo, the nett change in lung function in response to c~-agonists, results from the sum of such opposing effects (Goidie et al., 1988). Therefore, in vivo studies in asthmatics are of very limited use for investigating the role of ~-adrenoceptors in asthmatic airway smooth muscle. 3.4. IN VITRO STUDIESOF AIRWAY~-ADRENOCEPTORS

3.4.1. Radioligand Studies

Binding

and Autoradiographic

The very few binding studies that have been published documenting ~-adrenoceptor density or distribution in human lung, largely support the notion of disease-induced increases in c¢-adrenoceptor function. Szentivanyi (1979) used [3H]-dihydroalprenolol (DHA) to identify /~-adrenoceptors and [3H]-dihydroergocryptine (DHE) to detect ~-adrenoceptors in membrane fragments from both nondiseased and asthmatic human lung. The ratio of D H A : D H E binding sites was approximately 6:1 in nondiseased lung but was 1:1 in asthmatic lung, indicating a relative increase in ~-adrenoceptor density. Similar results were obtained using lymphocyte membranes (Szentivanyi, 1979, 1980). However, DHE also binds to receptors for 5-hydroxytryptamine and dopamine (Barnes et al., 1980b). The more specific, g~-selective ligand [3H]-prazosin has been used in studies in guinea-pig and human lung. In guinea-pigs, maximum prazosin binding was increased by 2-fold by ovalbumin sensitization, while DHA binding to fl-adrenoceptors was significantly reduced (Barnes et al., 1980a). In lung tissue from patients with chronic bronchitis, g-adrenoceptor density was approximately 10 times greater than in tissue from a patient with no airway obstruction (Barnes et al., 1980b). Raaijmakers et al. (1984) examined [3H]-prazosin binding in lung tissue membranes from 24 normal patients and 12 with chronic obstructive lung disease and found that the number of g~-adrenoceptors appeared to be increased in preparations from diseased lung. A highly significant correlation was also shown between specific [3H]-prazosin binding sites and the severity of disease as measured by lung function tests in vivo. In membranes isolated from ferret lung, the

TABLE3. ct-Adrenoceptor Function in the Lung Predominant Effect on Site subtype Function airflow Airway smooth muscle a~* contraction decrease Autonomic nerves a2 decrease neurotransmitter releaser increase Secretory glands ~t increase secretions decrease Mast cells ? increase mediator release decrease Vascular smooth muscle a~ contraction increase or decrease:l: *Postsynaptic a2-adrenoceptors appear to predominate in canine tracheal smooth muscle. tlnhibitory effect on bronchoconstrictor nerves predominates. :[:Predominant stimulation of arteriolar ~¢-adrenoceptors would prevent edema, while predominant stimulation of venular ~-adrenoceptors would exacerbate edema.

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number of ~2-adrenoceptors (specific [3H]-yohimbine binding sites) was much less than the number of ctladrenoceptors ([3H]-prazosin binding sites) (Barnes et al., 1983b). cq-Adrenoceptors also predominated in dog lung (Barnes, 1984). Membrane binding studies cannot provide information concerning the cellular locations of pulmonary ct-adrenoceptors, but specific binding to lung sections can be localized using light microscopic autoradiography. In ferret lung, prazosin binding was detected over vascular, bronchiolar and bronchial smooth muscle as well as over airway epithelial structures and alveolar walls (Barnes et al., 1983b), with bronchiolar smooth muscle being the most heavily labeled, of the airway components. Very sparse binding was observed elsewhere. An extremely small number of cq-adrenoceptors was detected in bronchioles in rat lung (Xue et al., 1983). However, the sites at which the increases in ~-binding occurred in a guinea-pig model of asthma (Barnes et al., 1980a) and in bronchitic human lung (Barnes et al., 1980b), have not been established. We have examined the autoradiographic distribution of ~-adrenoceptors in nondiseased human lung using the ~i-selective radioligand [125I]-BE2254 (Engel et al., 1981). Only very low levels of specific binding were detected throughout the lung, with no greater binding detected in bronchiolar tissue than in other airway sites (Goldie et al., 1988). The low density of ~t-ligand binding differed greatly from the high densities of binding of [~25I]-iodocyanopindolol to fl-adrenoceptors in bronchiolar and alveolar tissue from the same lung. We have also demonstrated that low levels of specific [3H]-prazosin (H-PZ) binding was present in both human nondiseased and asthmatic bronchus, even though a high level of I-CYP binding was detected (Spina et al., 1989b). The distribution of H-PZ and I-CYP bindings sites in asthmatic human bronchus is illustrated in Fig. 9. In addition, very low H-PZ binding was found in all of the airway structures in nondiseased or asthmatic parenchyma (Spina et al., 1989b). These data again suggest the dominance of fl- over ~t-adrenoceptor function in human lung. Furthermore, they emphasize that extrapolations of data from animal lung studies to man must be made with great care. The number and distribution of ~land ~tz-adrenoceptors in human lung is still to be determined. It is difficult to ascertain the importance of a change in the number of specific binding sites for ct-ligands without relating this to the mechanical response of airway smooth muscle (Goldie et al., 1985). Even though maximum prazosin binding in guinea-pig lung was shown to be increased 2-fold by ovalbumin sensitization (Barnes et al., 1980a), contractions produced by noradrenaline in guinea-pig lung strips were not changed by antigen sensitization (Turner et al., 1983). The maximal tension generated, threshold concentrations, ECs0 and EC~00 for noradrenaline in lung strips from sensitized animals, were not significantly different from those observed in nonsensitized guinea-pig lung strips. This suggests that the additional ~-adrenoceptors produced in response to ovalbumin were located at sites other than smooth muscle and raises questions about the

functional significance of reported increases in ~ sites in lung membranes (Zaagsma et al., 1987). 3.4.1.1. Influence o f animal age on airway ot-adrenoceptor density. Few studies have documented the effect of ageing on ct-adrenoceptor density in airway tissue. Latifpour et al. (1982) and Latifpour and Bylund (1983) have demonstrated that ~tl-adrenoceptor density in rat lung increased to peak at 3 weeks of age. In contrast, during the first week of life, ~:-adrenoceptor density decreased sharply in rat lung, to be all but absent in the adult. 3.4.2. Functional Studies 3.4.2.1. Animal airway smooth muscle preparations. Noradrenaline produced contractions in tracheal preparations isolated from guinea-pig (Takagi et al., 1967; Everitt and Cairncross, 1969; Fleisch et al., 1970), rabbit, cat and in old but not young rats (Fleisch et al., 1970), but only after fl-adrenoceptor blockade, confirming the dominance of relaxant fladrenoceptor function over contractile ~-adrenoceptor activity in airway smooth muscle. Many workers have used canine airway preparations in order to study airway ct-adrenoceptors (Pandya, 1977; Kneussl and Richardson, 1978; Beinfield and Seifter, 1980; Left and Munoz, ! 981 a,b; Ohno et aL, 1981; Barnes et al., 1983d,e; Brown et al., 1983). Pandya (1977) has reported that canine tracheal preparations isolated from new born animals contracted to noradrenaline and phenylephine and the response declined with the age of the animals. At 25-55 days of age, these ~t-agonists did not produce increases in basal tone, but caused contractions when tone was induced with acetylcholine. Kneussl and Richardson (1978) found that fl-blocked canine tracheal preparations did not contract to noradrenaline unless they were precontracted with histamine or KCI. In agreement, Ohno et al. (1981) reported that in propranolol pretreated isolated preparations of canine trachea the prior addition of K + or methacholine resulted in ct-agonists being able to produce contractions. In contrast to tracheal isolated muscle,/~-blocked canine tracheal muscle in situ can contract in response to ~-agonists without the prior addition of a spasmogen (Beinfield and Seifler, 1980; Left and Munoz, 1981a,b; Brown et al., 1983). Even so, Brown et al. (1983) reported that exposure to histamine, serotonin, acetylcholine and carbachol potentiated the responsiveness of canine trachealis muscle in situ to the ~-agonists noradrenaline and phenylephrine. Histamine augmented the contractile response to the -agonists to a greater extent than cholinergic agonists and the observed potentiation was maintained for at least 20 min after histamine-induced contraction. Barnes et al. (1983d) found that histamine and serotonin were very effective in potentiating contractions mediated by ct-adrenoceptors in canine tracheal smooth muscle both in vitro and in situ. In agreement with Brown et al. (1983), they observed that acetylcholine was much less potent than histamine in potentiating ~t-mediated contractions. Barnes et al. (1983d) also used [3H]-yohimbine and [3H]-prazosin to demonstrate that neither the number nor affinity of

Adrenoceptors in airway smooth muscle

311

FiG. 9. Photomicrographs of 10#m thaw-mounted frozen sections of human asthmatic bronchus. (a, d) Light-field photomicrographs of 2 serial sections of human asthmatic bronchus showing smooth muscle (SM), damaged epithelium (E) and a luminal mucous plug (mp) infiltrated with cells. Dark-field photomicrographs of the above sections showing the distribution and localization of autoradiographic grains derived from [3H]-prazosin (H-PZ; 1 nM) and (e) [125I]-iodocyanopindolol(I-CYP; 50 pM) binding. Dark-field photomicrograph showing the distribution of nonspecific autoradiographic grains in respective serial sections incubated with (c) H-PZ (I nM) and phentolamine (10 #M) or (f) I-CYP (50pM) and isoprenaline (200 #i). An artifact seen as bright areas associated with the epithelial basement membrane appears in each dark-field photomicrograph. Bar = 100/~m. Reproduced from Spina et al. (1989) with permission of the copyright holder, the Macmillan Press, Ltd, Hampshire. ~q- and ~t2-adrenoceptors were altered by preincubation with histamine. This indicates that the observed potentiation produced by histamine in canine tracheal smooth muscle involved a postreceptor mechanism. It was suggested that histamine potentiated ~z-adrenergic activity by activation of the Ca 2+ channels (Barnes et al., 1983d; Brown et al., 1983). However, other workers who used both phenylephrine and noradrenaline were unable to confirm that histamine pretreatment enhanced ct-adrenoceptor activity in canine trachea in vivo (Left et al., 1981b). Goldie et al. (1985) also demonstrated that an EC30 concentration of histamine (2-5 x 10 -6 M) failed to potentiate contractions produced by noradrenaline in 20 preparations from 5 guinea pigs in

which fl-adrenoceptors were blocked with 10-5M propranolol. In canine tracheal smooth muscle in situ, both phenylephrine and clonidine produced dosedependent contractions (Left and Munoz, 1981a). Phenylephrine (~l-agonist) was selectively antagonized by prazosin, while clonidine (~2-agonist) was selectively antagonized by yohimbine. Thus, both ~t- and ~2-adrenoceptors mediated contractions in canine trachea. In addition, the observation that the maximal contraction produced by noradrenaline (~land ~2-agonis0 was reduced to a greater extent by yohimbine than by prazosin indicated that ~2-adrenoceptors appeared to predominate over cq-adrenoceptors. Barnes et al. (1983e) further investigated the

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role of ~1- and ~2-subtypes in canine trachea using radioligand binding and examining the in vitro responsiveness of tracheal smooth muscle to exogenous -agonists. They reported that the number of specific [3H]-yohimbine sites in tracheal smooth muscle membranes was 51.4+4.9mol/mg of protein, while the number of specific prazosin binding sites was only 11.1 +2.9mol/mg of protein. The order of potency for producing contractions was clonidine > noradrenaline > phenylephrine and the contractions produced by noradrenaline were weakly inhibited by prazosin and markedly inhibited by yohimbine. Thus, both in situ (Left and Munoz, 1981a) and in vitro (Barnes et al., 1983e) functional studies show that postsynaptic ~2-adrenoceptors appear to predominate over postsynaptic ~l-adrenoceptors in canine tracheal smooth muscle. In addition, Barnes et al. (1983e) found that neuronally released noradrenaline acted on ~2 rather than ~l-adrenoceptors to contract canine tracheal smooth muscle. This is quite different to vascular smooth muscle, in which neuronally released noradrenaline selectively activates postjunctional ~adrenoceptors, while circulating catecholamines act on the postjunctional ~2-adrenoceptors (McGrath, 1983). In the presence of propranolol, contractions produced by noradrenaline were much stronger in canine bronchus than trachea (Left et al., 1983a,b). Left's team measured tracheal and bronchial responses /n situ in the same dogs. After B-blockade, the maximal in situ tracheal response to noradrenaline was 27.3 + 4.2% of the maximum contraction to acetylcholine, while the maximal in situ bronchial contraction to noradrenaline was greater than the maximal acetylcholine response (Left et aL, 1983a). Thus, Left et al. (1983a,b) concluded that the use of canine tracheal models of airway smooth muscle (Pandya, 1977; Kneussl and Richardson, 1978; Ohno et al., 1981) leads to a substantial underestimation of the importance of contractile ~-adrenoceptors in the resistance airways of lung. Nevertheless, a more important question is: how relevant are canine airways as a model of ~-adrenoceptor function in human airway smooth muscle? 3.4.2.2. H u m a n airway smooth muscle preparations. Guirgis and McNeill (1969) found no evidence for • -adrenoceptor-mediated contractile responses in human bronchial preparations challenged with adrenaline in the presence or absence of propranolol. In contrast, Mathe et al. (1971) showed that in the presence of propranolol, adrenaline caused phentolamine-sensitive contractions of human isolated bronchial preparations, although these responses were weak. In the absence of B-adrenoceptor blockade, adrenaline caused relaxation of these preparations. Similarly, phenylephrine produced phentolamine-sensitive contractions of human isolated bronchial strips, but only after B-blockade (Simonsson et al., 1972). Both noradrenaline and adrenaline were also shown to cause small increases in bronchial tone after B-blockade in bronchi isolated from nondiseased lung obtained postmortem (Kneussel and Richardson, 1978). These responses were enhanced in the presence of either histamine or KCI. In addition, bronchi from subjects with lung

disease including pneumonia, chronic obstructive lung disease and pulmonary fibrosis, contracted in response to noradrenaline without prior exposure to histamine or KC1, suggesting that airway ~-adrenoceptor function was enhanced in disease. These data supported the concept of asthma-induced increases in airway ~-adrenoceptor activity. Consistent with results from other workers, evidence from our laboratory indicated that only very weak contractions of nondiseased human bronchi obtained postmortem can be induced by noradrenaline or phenylephrine after pretreatment with a /~adrenoceptor antagonist (Goidie et al., 1984, 1985). Even then, the effect of the ~-agonist was highly variable with no response occurring in most preparations (Goldie et al., 1982a, 1984; Spina et aL, 1989b). In the absence of propranolol, phenylephrine caused partial relaxation of carbachol-contracted bronchus, mediated via /~-adrenoceptor stimulation (Fig. 10). A similar pattern of responsiveness to phenylephrine was observed in human bronchi obtained from asthmatic lung (Goldie et al., 1985; Spina et al., 1989b). No evidence of asthma-induced enhancement of ~-adrenoceptor function was observed. Furthermore, histamine failed to increase contractile responsiveness to noradrenaline in nondiseased human bronchi (Goldie et al., 1985). Consistent with the lack of [3H]-prazosin binding sites observed in either human nondiseased or asthmatic bronchus (Spina et al., 1989b) and the findings of other workers (Andersson et al., 1986; Black and Armour, 1986), our in vitro functional data indicate that ~-adrenoceptor function was not significant either in nondiseased or asthmatic human isolated bronchi. Postjunctional g:-adrenoceptors have been reported to be more active than postjunctional ~tadrenoceptors with respect to contraction of canine isolated tracheal smooth muscle (Barnes et al., 1983e). In contrast, human airways seem to be quite different. Postjunctional ~2-adrenoceptors do not appear to be present in nonasthmatic human isolated bronchial smooth muscle (Grundstrom and Andersson, 1985a). Although we did not directly investigate the ~-adrenoceptor subtypes mediating contractions in asthmatic bronchial smooth muscle, noradrenaline was used at concentrations up to 100/tM, which was more than enough to stimulate contractile ~2-adrenoceptors if they were present (Goldie et al., 1985). There was no evidence of ~2-adrenoceptor function in asthmatic bronchial smooth muscle. However, some recent studies have suggested that the ~2-agonist clonidine may have beneficial effects on airway caliber. Inhalation of clonidine (75/~g) improved basal airway function in man without affecting systemic blood pressure and also reduced the airflow obstruction induced by antigen challenge of between 42 and 65% (Lindgren et al., 1986). Clonidine may improve airway function in asthma by activating prejunctional ~2-adrenoceptors and thus reduce cholinergic and/or nonadrenergic/noncholinergic bronchoconstrictor function (Grundstrom and Andersson, 1985b; Andersson et al., 1986). Clonidine may also partly improve bronchial obstruction by an ~2-adrenoceptor-mediated anti-iriflammatory action (Kulkarni et al., 1986) or via a nonspecific action

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4. CONCLUSIONS fl~- and fl2-adrenoceptors have been demonstrated to coexist or to be present in largely homogeneous populations in airway smooth muscle from several animal species. In man, only fl2-adrenoceptors have been detected in airway smooth muscle from the

trachea to terminal bronchioles. These receptors mediate the relaxant effect of potent, selective ~ agonists such as fenoterol and salbutamoi which remain the bronchodilators of choice in asthma. Theoretically, a fl2-agonist with high efficacy at the receptor should be clinically more effective in severe asthma in which there is both marked bronchoconstriction and reduced fl-adrenoceptor function, although this remains to be established. Present evidence suggests that fl~-agonists do not exert a significant part of their bronchodilator action

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