Afferent pathways involved in reflex regulation of airway smooth muscle

Pharmac. Ther.Vol. 42, pp. 1-63, 1989

0163-7258/89 $0.00 + 0.50 Copyright ~ 1989 Pergamon Press pie

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Associate Editor: I. W. RODGEg

AFFERENT PATHWAYS INVOLVED IN REFLEX REGULATION OF AIRWAY SMOOTH MUSCLE H. M.

COLERIDGE,

J. C. G. COLERIDGEand H. D. SCHULTZ

Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, California 94143-0130, U.S.A.

1. INTRODUCTION The prominent role played by the extrinsic nerve supply in the regulation of airway smooth muscle might perhaps be regarded as surprising. The electrophysiological characteristics of this muscle are not those usually accepted as typical of smooth muscle subject to a major degree of neural control. Motor nerve endings are relatively scarce, and close contacts between nerve and muscle rare, in central airways such as the bovine and dog trachea, which have been used frequently in both in vitro and in vivo studies of the neural regulation of bronchial smooth muscle (Cameron and Kirkpatrick, 1977; Suzuki et aL, 1976; Stephens and Kroeger, 1980; Gabella, 1987). They are also scarce in the human trachea (Daniel et al., 1986). Nevertheless neurally mediated smooth muscle tone is present throughout the airways during quiet breathing, and this tone appears to depend entirely on volleys of impulses passing down the vagus nerves. If the cervical vagus nerves are cut in animals, or if atropine is administered intravenously to animals or man, the airways dilate fully, airflow resistance decreases, and no residual airway tone can be demonstrated (Severinghaus and Stupfel, 1955; Cabezas et al., 1971; Hahn et al., 1976; Nadel, 1980). The influence of the parasympathetic bronchomotor nerves responsible for this baseline tone has been shown to extend from the trachea to the small bronchi in animals and humans (Olsen et al., 1965; Hahn et al., 1976; Douglas et al., 1979b), and indeed there is evidence in dogs and humans that it extends to the terminal lung units (De Troyer et al., 1979) to influence not only airflow resistance but also dynamic lung compliance (Loring et al., 1981). The existence of this neurally-mediated bronchial smooth muscle tone provides the possibility of extrinsic modification by a variety of reflexes, and it is with the afferent arm of these reflexes that this article is concerned. The sensory mechanisms discussed exert a bronchomotor reflex function by increasing or decreasing activity in the excitatory vagal pathway described above. Involvement of the reciprocal, inhibitory, sympathetic fl-adrenergic pathway appears to be minor in most mammals, including man, although there is evidence that this inhibitory pathway plays an important role in lower airway reflexes in guinea pigs. The two other efferent pathways, an excitatory sympathetic ~-adrenergic pathway, and an inhibitory vagal pathway whose transmitter is still uncertain, have been demonstrated mainly by nerve stimulation in in vitro preparations of airway smooth muscle in which muscarinic and ~-adrenergic effects have been prevented by pharmacological blockade. The physiological role of these alternative pathways in the reflex regulation of airway smooth muscle tone is unknown. Although recent studies of the bronchomotor effects of stimulating laryngeal mechanoreceptors in man (Michoud et al., 1987) and laryngeal mechanoreceptors and lower airway C fibres in cats (Szarek et al., 1986; Ichinose et al., 1987) indicate that an inhibitory vagal pathway is, indeed, engaged, the net reflex effect of stimulating these sensory inputs when motor pathways are intact is a vagal muscarinic bronchoconstriction. The motor pathways to airway smooth muscle are discussed in another article in this series, and are mentioned here only in so far as their physiological and pharmacological blockade is commonly used

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H.M. COLERIDGEet al.

to establish that an experimentally-induced change in bronchomotor tone is neurally mediated and to characterize the efferent arm of the reflex arc. However, because the influence of the various afferent inputs is superimposed on an existing degree of vagal bronchoconstrictor tone, a brief discussion of the factors that maintain this baseline tone seems warranted. Activity in the vagal preganglionic bronchomotor fibres and ganglionic motoneurones responsible for resting airway smooth muscle tone has been recorded in dogs and cats (Widdicombe, 1961a, 1966; Mitchell et al., 1987), and has been found to wax and wane rhythmically with respiration. In cats whose lungs were ventilated artificially at high rates and low tidal volumes, so that the rhythmic bursts of phrenic discharge reflected the intrinsic slow rate of the central rhythm generator, a rhythmic increase in vagal bronchomotor tone occurred with each phrenic burst, leading the investigators to postulate that medullary inspiratory neurones and central vagal bronchomotor neurones were driven by a common generator (Mitchell et aL, 1985). This does not seem to be the case, however, and the numerous afferent inputs that affect bronchial smooth muscle frequently drive inspiratory activity and bronchomotor activity in opposite directions (see below). The common stimulus linking inspiratory neural output and bronchomotor output is CO2, acting on the central medullary chemoreceptors. Although central mechanisms are not usually considered as the starting point for reflex arcs, an exception can be made here, for the central chemoreceptors may be held to initiate a bronchomotor reflex whose afferent arm is entirely within the central nervous system, and whose efferent arm is in the vagus nerve. In both anaesthetized and conscious dogs, neurally-mediated bronchomotor tone disappears when arterial CO2 is reduced by hyperventilation, and is restored by adding CO2 to the inspired air (Stein and Widdicombe, 1975; Sorkness and Vidruk, 1986). In addition, as judged from observations of tracheal muscle tone in sleeping dogs, vagal bronchomotor tone is influenced by 'central state', and fluctuates widely during periods of REM sleep (Sullivan et al., 1979). Nevertheless, although central factors play a major role in maintaining background activity in vagal muscarinic motor nerves, experiments in anaesthetized animals suggest that the basal tone of airway smooth muscle is also influenced by a balance of bronchoconstrictor and bronchodilator reflex influences from the periphery. These peripheral afferent inputs appear to arise mainly from sensory endings in the respiratory tract itself, although there is increasing evidence that input from sensory areas remote from the lungs and airways also contributes to bronchomotor reflexes. Studies of the reflex regulation of airway smooth muscle began at the end of the last century. Details of these early studies, with a critical account of the methods then in use, can be found in an early review by Dixon and Brodie (1903). Already, Einthoven and other investigators had recognized from experiments in anaesthetized animals that smooth muscle tone was present in the airways, that it was maintained by activity in vagal efferent nerves, that it was largely dependent on the drive produced by CO2 acting centrally at the level of the medulla, and that reflex changes in the calibre of the trachea or large bronchi could be triggered by a variety of mechanical or chemical stimuli applied to the upper airways. In studies carried out in the first half of the present century, the major emphasis was on bronchomotor reflexes initiated by stimulation of the upper airways in laboratory animals (Widdicombe, 1963). As Widdicombe pointed out, the volumetric methods used in some of these early experiments to assess bronchomotor effects did not allow changes resulting from alterations in airway smooth muscle tone to be distinguished clearly from those arising from alterations in the pulmonary vascular bed. Even so, in some experiments stimuli applied to the nares or larynx were found to induce constriction of the trachea or a bronchus, hence there seems little doubt that the excitatory smooth muscle effects described were genuine. Studies in human subjects of bronchomotor reflexes triggered by stimulation of the airways effectively began with the development of the technique of whole body plethysmography for measuring airway resistance (DuBois et al., 1956). Results of experiments in

Bronchomotor reflexes

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humans arc sometimes difficult to interpret because stimulant gases or aerosols administered through a mouthpiece may affect nerve endings in both upper and lower airways, and attempts to limit the stimulus to the upper airways may involve complex respiratory manoeuvres, which may themselves alter the final reflex response. Moreover, because flow resistance may be affected by reflex relaxation or contraction of laryngeal muscles, the assessment in human subjects of changes in bronchomotor tone from changes in airflow resistance measured from the mouth may be misleading unless atropine is used to confirm that the change in airway resistance has a muscarinic component. Studies in man have gone hand in hand with detailed analyses in laboratory animals of the bronchomotor reflexes elicitable from the respiratory tract and of the afferent properties of the relevant sensory nerves. Nevertheless, experiments in man present a more integrated picture of the bronchomotor responses, and supplement the more analytical reflex and afferent studies that are possible only in anaesthetized animals. The afferent inputs capable of influencing bronchomotor tone are of many different types and are more widespread than was once thought. Sensory inputs with reflex bronchomotor effects originate not only from endings in the upper and lower respiratory tract, but also from arterial chemoreceptors and baroreceptors, and from afferent endings in the heart, gut, skin and skeletal muscle. The effects of input from regions other than the respiratory tract have been studied mainly in experiments in anaesthetized animals. Indeed most of what we know of the afferent mechanisms that give rise to changes in airway smooth muscle tone stems from animal studies in which investigators have been able to confine the applied stimulus to a specific region, and sometimes to a particular group of sensory receptors. In discussing the reflex control of a system it is customary to pay at least lip-service to its functional implications, and if possible to assign a certain 'usefulness' to the effects described. Often, however, it is not easy to make a strong case for the utility of reflexes involving airway smooth muscle, and, indeed, the functional significance of airway smooth muscle itself is still a matter for debate. Taking the extreme view, some authorities claim that airway smooth muscle is only of unequivocal functional relevance in the bronchospasm of asthma, when clearly it functions in an adverse sense to increase the work of breathing (Otis, 1983). However, neurally-induced contraction of airway smooth muscle certainly brings about two opposing functional changes, and both can be demonstrated physiologically: it decreases ventilatory dead space and at the same time increases airflow resistance (Stcphens and Hoppin, 1986). Reflex control may function to adjust the balance between these two effects according to functional needs. Thus during quiet breathing, when resistance to airflow is not an important factor in the work of breathing, background activity in afferent fibres may contribute to the maintenance of a degree of vagal tone sufficient to keep anatomical dead space, and thus the elastic work of breathing, within limits. During exercise, on the other hand, when tidal airflow increases and dead space becomes a smaller fraction of minute volume, reflex influences may bring about withdrawal of vagal tone, so that a neurally-evoked dilation of the airways decreases airflow resistance, which has now become a more considerable fraction of the total work of breathing. The reflex bronchomotor role of sensory receptors in the respiratory tract itself is, perhaps, easiest to interpret with respect to function, and is of interest not only in physiological circumstances but also in lung disease. Bronchomotor reflexes originating in the respiratory tract fall naturally into two categories: regulatory and protective or defensive. Regulatory reflexes are exemplified by the bronchomotor response to the increased breathing of exercise; when ventilation increases, stimulation of pulmonary stretch receptors promotes bronchodilatation, an inhibitory influence that probably combines with input from exercising muscle to reduce the resistive work of breathing. By contrast, when irritants are inhaled or the respiratory tract is threatened by disease, stimulation of other vagal endings in the airways evokes a bronchoconstriction that may be sufficiently severe to be characterized as 'bronchospasm'. The bronchoconstriction is only one component of a pattern of reflex changes collectively termed the 'respiratory

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H . M . COLERmGE et al.

defense reflex', which include constriction of the larynx, coughing, increased airway secretion, and disturbances of breathing. In these circumstances, human subjects often complain of sensations of tickling or retrosternal rawness and burning; such sensations depend on the integrity of the vagus nerves (Guz, 1977). These sensations are quite unlike those aroused from other visceral structures but resemble the sensations evoked by cutaneous inflammation. Moreover, the tickling sensations aroused by airway irritation are relieved by coughing in much the same way as the sensations of cutaneous itch are relieved by scratching. The bronchoconstriction is thought to stabilize the airways in the face of the wide swings in transpulmonary pressure that occur during coughing, thus increasing the efficiency of coughing and contributing to the effective expulsion of accumulated secretions. In addition, narrowing of the bronchi, particularly when accompanied by rapid breathing, may help to promote turbulent flow in the airways, and deposition of particles and droplets at the more central airway bifurcations. Thus bronchoconstriction would help to limit the further entry of an inhaled irritant into the more peripheral airways and its access to the bloodstream. Our purpose in the present review is to give an account of the various sensory regions from which reflex effects on airway smooth muscle are known to arise, with some reference to the nature of the sensory structures involved, where this is known. We deal first with intrinsic afferent inputs--intrinsic in the sense that they arise from the airways themselves, although for convenience the term has been extended to include the upper airways that do not contain airway smooth muscle---and then with extrinsic afferent inputs that arise from regions remote from the airways and lungs. 2. BRONCHOMOTOR INFLUENCE OF AFFERENTS IN UPPER AIRWAYS Stimulation of afferent nerve endings in the upper airways has both excitatory and inhibitory effects on airway smooth muscle (reviewed by Widdicombe, 1963, 1986). The nares, the epipharynx, and the larynx have each been investigated for their reflexogenic potency, with results suggesting that the direction of the reflex bronchomotor response depends on the region of airway stimulated. A withdrawal of vagal bronchomotor tone is evoked by stimulating nerve endings in the epipharynx, and an increase in tone by stimulating those in the larynx. By contrast, both bronchoconstrictor and bronchodilator reflexes have been described in response to stimuli applied to the nasal passages. Investigation of upper airway reflexes is complicated by the fact that the primary bronchomotor changes may be accompanied by stereotyped ventilatory responses, such as sneezing, coughing or gasping, that in turn evoke secondary reflex changes in airway smooth muscle tone by stimulating stretch receptors in the lower airways. Such secondary reflex effects can be avoided in anaesthetized animals by paralysis of the respiratory muscles and artificial ventilation of the lungs. 2.1. NASALAFFERENTS

The general sensory nerve supply of the nares is through the maxillary branch of the trigeminal nerve, the fibres of which subserve several modalities of sensation, including light touch, temperature, tickling, and stinging or burning pain. Electron-microscopic studies have demonstrated the presence of terminals of sensory appearance between the epithelial cells of the human nasal mucosa (Cauna et al., 1969), and electroneurographic studies in animals have shown that large numbers of thermal receptors, particularly cold receptors, are present in the skin of the nostrils (Hensel, 1973). The possibility that the special sense of olfaction is also associated with reflex bronchomotor effects has not been explored. Bronchomotor reflexes initiated by stimulation of nasal and nasopharyngeal afferent nerve endings have been examined in anaesthetized dogs, cats, rabbits and guinea pigs. The methods used to stimulate these afferents have varied widely, and have included the introduction of irritant gases (ammonia, bromine) or smoke into the nares, direct

Bronchomotor reflexes

5

application of chemical irritantsto the nasal mucosa, mechanical or electricalstimulation of the mucosa, and application of cold. In most early studies in animals, stimulation of afferentnerve endings in the nose evoked bronchoconstriction.A fullbibliography of these early studies is given by Widdicombc (1963), and is not included here. In general, the evidence indicated that the afferentpathway for these bronchoconstrictor reflexeswas in the trigeminal nerve and the efferent pathway in the vagus nerve. In one study on dccerebratc dogs, however, application of ice water or chloroform vapour to the nares evoked a small bronchoconstriction after atropine had been administered (Rall et al., 1945). The atropine-resistantbronchoconstriction was abolished by ergotamine, and hence was thought to bc duc to withdrawal of sympathetic vasodilator tone. Reflcx bronchoconstriction of nasal origin has been demonstrated in man, in studies in which the subjects firsttook a deep breath, and then exhaled while a saline aerosol containing silicaparticlcswas directed into the nares (Kaufman and Wright, 1969). This ventilatory manocuvrc made it reasonably certain that the aerosol was confined to the nares and nasopharynx, and that sensory endings in the larynx and lower airways were not stimulated. A n increase in airway resistance,measured by whole body plethysmography, was observed in all subjects. Exposure to the saline aerosol alone during a similar vcntilatorymanoeuvre was without effect.The incrcascd airway resistancedid not involve laryngeal constrictionbut was ascribed solelyto an increase in vagal muscarinic bronchomotor tone, since it could no longer be evoked after atropine. A similar procedure was used to demonstrate a nasally-evoked bronchoconstriction in subjectswho had undergone unilateral section of the maxillary division of thc trigcminal nerve for intractable tic doulourcux (Kaufman et al., 1970). The aerosol containing silicaparticles evoked an increase in airway rcsistance when delivercd to the nostril on the side opposite the trigcminal section, but was ineffectivewhcn dclivcrcd to the dencrvated side. The effects of delivering aerosol to the innervated nostril were abolished by atropine. The presence of a nasal bronchoconstrictor reflexin man was also described by Konno et al. (1983),who instructed theirsubjectsto take a deep breath, and to cxhale through the nostrilsas pepper was applied directlyto the nasal muscosa in an amount sufficientto cause a sensation of burning. Performance of the respiratorymanoeuvre alone had no significanteffecton the airways. Taken together,the studiesof Kaufman and Wright (1969),Kaufman et al. (1970) and Konno et al. (1983) provide good evidence that irritant stimulation of trigeminal affercnt endings in thc nose causes reflex vagal bronchoconstriction. The existence of a naso-bronchial constrictor rcflcx has aroused clinical interest bccausc of its possiblc relevance to patients with asthma, in w h o m bronchoconstriction is known to have a Pcflcx component. A marked increase in airway resistance, mcasurcd by an oscillatorymethod, was demonstrated by Nolte and Bergcr (1983) in asthmatic patients when metered doses of a cold propellant were delivered into the narcs (Fig. I, top). The bronchoconstriction was greatly diminished after the subjects inhaled an aerosol of the antimuscarinic agent ipratropium bromide into the lower airways (Fig. I, bottom). The response was present in laryngectomizcd patients in w h o m the upper and lower airways were not connected. It was thought to be specificto patientswith asthma because it could not be evoked in healthy individuals.Reflex bronchoconstriction has also been evoked by applying histamine to the nasal mucosa of asthmatics with seasonal rhinitis(Yan and Salome, 1983). However, the intensity of the bronchomotor response varied widely in individualpatients,and bore littlerelationto the degree of lower airway reactivityto histamine; moreover the response could not bc demonstrated in all patients. Hence the relevance of a naso-bronchial reflex to asthmatic attacks remains uncertain. Although most studies in animals and man have found that stimulation of sensory endings in the nose evokes bronchoconstriction,there are reports in anaesthetized animals that stimulation has eitherno effect(Nadel and Widdicombe, 1962b), or evokes bronchodilatation (Tomori and Widdicombc, 1969). Thus Tomori and Widdicombc described a decrease in lower airway resistancein anaesthetizedcats when the anteriorpart of the nasal passage was tapped gently and repeatedly with a thin nylon catheter.This nasally-evoked

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FIG. 1. Above:reflex bronchoconstriction evoked in a laryngectomized asthmatic patient by delivery of a metered dose of a freon-propelled aerosol into the nares. Cold stimulation occurred when the propellant passed from the liquid to the gaseous state. Ros, airway resistance measured continuously by a forced-oscillation technique. Below:the bronchoconstrictor response to cold stimulation of the nares was considerably reduced after the anticholinergic drug ipratropium bromide was inhaled into the lower respiratory tract. The response was abolished by local anaesthesia of the nares. (Nolte and Bergcr, 1983.)

bronchodilatation seems, at least in some experiments, to have been an accompaniment of the complex respiratory act of sneezing, which consisted of several deep inspirations preceding an explosive expiration, bronchodilatation occurring during the 2-3 subsequent eupnoeic breaths. Large inspirations are known to be associated with reflex relaxation of airway smooth muscle (Widdicombe and Nadel, 1963), so that the bronchodilatation associated with sneezing (Tomori and Widdicombe, 1969) may have been secondary to an increase in pulmonary stretch receptor discharge (see below). Nevertheless, Tomori and Widdicombe found that a decrease in airflow resistance could also be evoked by nasal stimulation in paralysed, artificially ventilated cats, precluding the influence of changes in pulmonary stretch receptor input. Mechanical stimulation of the anterior nasal mucosa has also been found to evoke relaxation of tracheal smooth muscle in cats (Baker and Don, personal communication). A bronchodilator response to nasal stimulation has also been described in dogs (Konno, 1976). The reflex bronchodilatation evoked by nasal stimulation is thought to be due largely to withdrawal of vagal efferent activity (Tomori and Widdicombe, 1969), and there is no evidence that an adrenergic inhibitory influence on the airways is involved (Baker and Don, personal communication). The most likely explanation for the diametrically opposite bronchomotor responses to nasal stimulation reported by different investigators is that an innocuous stimulus, such as that provided by lightly touching the nasal mucosa, recruits one population of afferents causing bronchodilatation (Tomori and Widdicombe, 1969; Konno, 1976; Baker and Don, personal communication), whereas cold and potentially noxious stimuli, such as silica particles, recruit other afferents causing bronchoconstriction (Kaufman and Wright, 1969; Kaufman et aL, 1970; Konno et al., 1983; Nolte and Berger, 1983; Yah and Salome, 1983). 2.2. EPIPHARYNGEAL AFFERENTS

Mechanical stimulation of the epipharyngeal mucosa with a fine nylon fibre in anaesthetized cats evokes a decrease in airflow resistance in the lower airways, with a parallel decrease in impulse frequency in vagal bronchoconstrictor efferent fibres (Tomori and Widdicombe, 1969). An electroneurographic study of afferent fibres in the pharyngeal branch of the glossopharyngeal nerve of cats has identified mechanoreceptors in the dorsal wall of the epipharyngeal region, which are probably responsible for this inhibitory bronchomotor reflex (Nail et al., 1969).

Bronchomotorreflexes

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2.3. LARYNGEALAFFERENTS

Because the larynx is located immediately below a section of upper airway common to the functions of both respiration and alimentation, its innervation constitutes a first line of defense to the respiratory exchange region, triggering reflexes that guard against the accidental inhalation of liquids or solids during swallowing or regurgitation. The larynx is richly supplied by afferent fibres of the superior laryngeal branch of the vagus nerve. The powerful airway defense reflexes originating in the larynx include apnoea, adduction of the vocal chords, coughing and bronchoconstriction. These can be evoked in human subjects by stimulating the central end of the superior laryngeal nerve (Ogura and Lam, 1953), and in animals by mechanically stimulating the larynx or by stimulating the laryngeal mucosa with water and chemicals such as ammonia and sulphur dioxide (Widdicombe, 1954a; Tomori and Widdicombe, 1969; Boushey et al., 1972; Storey and Johnson, 1975; Kovar et al., 1979). Most of the afferent fibres in the superior laryngeal nerve are small and myelinated; relatively few (about 25%) are C fibres (Dubois and Foley, 1936; Ogura and Lam, 1953). Studies with the light microscope have identified a number of different forms of sensory ending (Koizumi, 1953; Feindel, 1956; Van Michel, 1963). Occasional endings are encapsulated and may be cold receptors (Hensel, 1973), but the majority correspond to the complex unencapsulated endings of myelinated fibres that are typical of many visceral sensory structures. These unencapsulated endings, which have been likened to the arterial baroreceptors of the carotid sinus and aortic arch, are found in the mucosa of the vocal chords and in association with the intrinsic muscles of the larynx (Van Michel, 1963). In addition, taste buds are described in the aryepiglottic region (Koizumi, 1953; Feindel, 1956), and also small, nonmyelinated fibres with delicate beaded arborizations ending in the superficial layers of the epithelium (Feindel, 1956). Sensory endings are plentiful on the laryngeal surface of the epiglottis (Feindel, 1956), which is strategically placed to guard the entrance to the larynx. Action potential studies of the afferent innervation of the larynx indicate that the endings are of several different sensory modalities, and include: mechanoreceptors variously sensitive to touch, movement of the vocal chords and transmural pressure (Andrew, 1956; Boushey et al., 1974; Harding et al., 1978; Sant'Ambrogio et al., 1983); receptors stimulated by water but not by isotonic saline (Storey and Johnson, 1975; Harding et al., 1978); receptors (some of which are thought to be taste buds in the aryepiglottic region above the vocal folds) stimulated by salts, acids and sugars (Harding et al., 1978); and receptors sensitive to chemical irritants such as ammonia, sulphur dioxide and cigarette smoke (Boushey et al., 1974; Schultz et al., 1982). In addition, there are nerve endings that were once thought to be sensitive to airflow but have since been shown to be thermal receptors sensitive to cold (Sant'Ambrogio et al., 1985). Cold receptors of this type may be present throughout the central airways, but only those in the larynx have been investigated systematically. The cold sensitivity of the upper airways is dealt with at greater length below. In spite of the wide range of afferent modalities revealed by electrophysiological studies, sensation from the larynx seems to be confined to a sense of poorly defined local irritation and dull or burning pain. The limited range of laryngeal sensation has been confirmed in human patients, undergoing surgery for carcinoma of the upper respiratory tract, by electrically stimulating the central end of the superior laryngeal nerve with pulses of gradually increasing strength and duration (Ogura and Lam, 1953). Moreover, regardless of the type of stimulus applied to the larynx, an atropine-sensitive contraction of airway smooth muscle seems to be the primary bronchomotor response (see below). It is a matter of common observation that acute 'asthma-like' reactions can be triggered in human patients by aspiration of gastric contents during anaesthesia (Mendelson, 1946), and from what is known of the reflex bronchomotor response to laryngeal stimulation in animals (see below), it is reasonable to assume that a major component of this bronchoconstriction in humans is triggered from the larynx. A similar mechanism is thought to

8

H.M. COLERIDGE et al.

be involved in nocturnal asthmatic attacks in children (Martin et al., 1982). In human subjects, inhalation of aerosols of chemical irritants including capsaicin, a chemical which stimulates afferent C fibres (Coleridge and Coleridge, 1984), causes cough and a transient increase in airflow resistance, the latter being abolished by ipratropium bromide (Fuller et aL, 1985). It seems likely that at least a part of the afferent input for these bronchoconstrictor responses arises from chemosensitive nerve endings in the larynx, although a role for chemosensitive C fibres in the lower airways cannot be excluded. In tracheostomized animals, stimuli can be confined with certainty to the laryngeal interior, and mechanical stimulation of the vocal chords with a soft nylon probe, or the introduction of irritants such as ammonia, sulphur dioxide and cigarette smoke into the larynx, evoke an increase in lower airway resistance and tracheal smooth muscle tone, which are abolished by cutting the superior laryngeal or vagus nerves, or by administering atropine (Nadel and Widdicombe, 1962b; Tomori and Widdicombe, 1969; Boushey et al., 1972; Schultz et aL, 1982). Even the mechanical stimulation provided by the accumulation of secretions in the larynx is sufficient to trigger reflex excitatory effects on airway smooth muscle (Fig. 2D), which disappear when the accumulated secretions are aspirated (Fig. 2E) (Coleridge et aL, 1982a). Laryngeal stimulation not only evokes reflex contraction of airway smooth muscle, it also triggers a marked increase in secretion by airway submucosal glands (Phipps and Richardson, 1976; Davis et al., 1982). Nadel and Widdicombe (1962b) pointed out that the large increase in airflow resistance produced in their experiments by mechanical stimulation of the larynx was unlikely to have been due to a reflex increase in secretion in the lower airways, because airway resistance returned promptly to control when laryngeal stimulation ceased. Moreover, stroking the interior of the larynx evokes a powerful reflex contraction of an open tracheal segment (Fig. 3B) (Coleridge et al., 1982a).

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FIG. 2. Effect of stimulating various afferent inputs on the smooth muscle tension of an upper tracheal segment in an anaesthetized dog. The chest was open and the lungs were ventilated artificially. The segment was innervated only by the superior laryngeal nerve (the recurrent and pararecurrent laryngeal nerves were cut at the beginning of the experiment). A: tracheal relaxation evoked by hyperinflating the lungs (FRC + 3 VT). B: tracheal contraction evoked by gently stroking the larynx with a cotton-tipped applicator. C: tracheal contraction evoked by stimulating the arterial chemoreceptors (200 #g/kg NaCN injected i.v.). The cervical vagus nerves were intact in A and B: they were cut between B and C. D: irregular tracheal contractions caused by secretions collecting in the larynx. E: contractions ceased when the secretions were aspirated. ABP, arterial blood pressure; Tr tension, tracheal tension in grams above the baseline tension, which was set at 75 g; PT, tracheal pressure. The tracheal tension calibration in A is on the left, that in B and C on the right. (Coleridge et al., 1982a.)

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FIG. 3, Changes in lung resistance (RI) evoked by mechanical stimulation of the larynx (indicated by the filled circles) in an anaesthetized, artificially ventilated cat. The larynx was stimulated by gently stroking its dorsal mucosa just below the vocal chords with a fine probe. Initially, the larynx was stimulated under basal conditions (far left). Thereafter, baseline airway tone was increased by a continuous infusion of 5-hydroxytryptaminc (5HT). Laryngeal stimulation now evoked a brief bronchoconstriction followed by prolonged bronchodilatation; the latter was due to activation of the nonadrcnergic, noncholinergic vagal inhibitory pathway to the airways. (Szarek et al., 1986.)

The reflex bronchoconstrictor effects described above were demonstrated in artificially ventilated, and sometimes paralysed, animals, as well as in spontaneously breathing animals. Hence they were a direct consequence of stimulation of sensory nerve endings in the larynx, and were not secondary reflexes induced by changes in pulmonary afferent discharge resulting from alterations in breathing. Although an atropine-sensitive bronchoconstriction appears to be the primary response to stimulation of afferent nerve endings in the larynx, not all the reflexly recruited vagal bronchomotor activity is necessarily bronchoconstrictor. There is evidence in both cats (Szarek et al., 1986) and human subjects (Michoud et al., 1987) that activity in the nonadrenergic, noncholinergic inhibitory vagal bronchomotor system also increases after stimulation of the vocal chords. Thus stimulation of mechanoreceptors in the dorsal laryngeal mucosa evoked a biphasic bronchomotor response in anaesthetized cats that had received an infusion of serotonin to raise basal airway smooth muscle tone (Szarek et al., 1986). The response consisted of a transient increase in lower airway resistance, followed by a prolonged decrease (Fig. 3). The bronchoconstriction was abolished by atropine. The relaxation, though obviously reflex in origin because it was blocked reversibly by vagal cooling, persisted after both atropine and propranolol but was abolished by ganglionic blockade with hexamethonium. Stimulation of mechanoreceptors on the vocal chords in human subjects triggers a similar combination of vagal excitatory and vagal inhibitory bronchomotor effects (Michoud et al., 1987). The inhibitory effects were manifest when airway smooth muscle tone was first increased by the inhalation of histamine aerosol, muscarinic and ~-adrenergic effects being blocked by atropine and propranolol. The function of this vagal bronchodilator reflex pathway in the normal control of lower airway smooth muscle remains uncertain, however, because it does not seem to be capable of reversing muscarinic tone (Szarek et al., 1986). 2.4. REFLEXES FROM COLD RECEPTORS

Asthmatic subjects often wheeze when inhaling cold dry air. In the laboratory, exposure to cold dry air has been shown to evoke an increase in airflow resistance in normal subjects as well as in asthmatics (Wells et al., 1960; Melville and Morris, 1972). The sensory field for the reflex bronchoconstriction produced by inhalation of cold air is likely to be confined to the upper airways, which are very effective in warming and humidifying inhaled air. Even during mouth breathing, inhaled cold air is warmed to within a degree or so of core temperature by the time it reaches the tracheal bifurcation, provided the subject is at rest (Moritz and Weisiger, 1945; Cole, 1953). The conclusion that the major input for the bronchoconstriction evoked by breathing cold air comes from the upper respiratory tract is confirmed by the observation that in a group of asthmatic children the response was prevented by anaesthesia of the nose and pharynx (Rodriguez-Martinez et al., 1973). Cooling of the upper respiratory tract may be a stimulus for the bronchoconstriction evoked by exercise in asthmatics and other susceptible subjects, because breathing cold air

10

H.M. COLEmDGEet al.

potentiates the development of exercise-induced bronchoconstriction and breathing warm air can prevent it (for references see McNally et al., 1979). In asthmatic children exercising on a treadmill, local anaesthesia of the posterior oropharynx was found to attenuate exercise-induced bronchoconstriction but did not abolish it (McNally et al., 1979). There is evidence that the receptive field for the bronchoconstrictor response to cold also extends to the skin of the face, because an increase in airflow resistance, attributable in part to bronchoconstriction and in part to constriction of the larynx, has been evoked in both normal and asthmatic subjects (Josenhans et al., 1969; Melville and Morris, 1972), and also in laboratory animals (Melville, 1972), by applying cold packs to the face. A number of investigators have suggested that receptors sensitive to drying of the upper airway mucosa may contribute to the bronchoconstrictor effects of inhaling cold air. However, humidification of cold air does not prevent the reflex response (RodriguezMartinez et al., 1973). Studies of the behaviour of cold receptors in the laryngeal mucosa in dogs may go some way to explain why the inhalation of cold dry air is such an effective stimulus to reflex bronchoconstriction, especially in human subjects with hyperreactive airways. Sant'Ambrogio et al. (1985) found that the inspiratory flow of 100% humidified cold air through the larynx was a less effective stimulus to the cold receptors than the same inspiratory flow of 50% humidified cold air. The greater stimulation by the dryer air was directly related to the greater decrease in laryngeal mucosal temperature, i.e., to the greater evaporative heat loss produced by the dryer air (Fig. 4). It may not, therefore, be necessary to postulate the existence of a separate set of mucosal receptors that are sensitive to drying in order to explain the increased bronchoconstrictor effectiveness of dry cold air.

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t 2s FIG. 4. Effect of changing the temperature and humidity of the inhaled air on the impulse activity of laryngeal 'flow' (cold) receptors in anaesthetized dogs breathing spontaneously through the upper airways. A.P., action potentials recorded from a filament of the superior laryngeal nerve; P,, oesophageal pressure (kPa, kiloPascal); ¢¢ (l/s), airflow in liters/see; Lar. Temp., laryngeal temperature recorded by a thermistor. Above: response of a cold receptor to inhalation of warm air (between the arrows); note that the respiratory modulation of firing disappeared when laryngeal temperature during inspiration was raised to the expiratory level. Below: response of a cold receptor when laryngeal temperature was varied by changing the relative humidity of the inhaled air from 100% to 55% (at the arrow); note increased afferent discharge associated with the greater decrease in laryngeal temperature during inspiration. (Sant'Ambrogio et al., 1985.)

Bronchomotorreflexes

11

3. BRONCHOMOTOR INFLUENCE OF AFFERENTS IN LOWER AIRWAYS 3.1. LOWERRESPIRATORYAFFERENTS The respiratory tract below the larynx receives its major afferent supply from the vagus nerves. Stimuli applied to the lower airways can evoke either inhibitory or excitatory vagal bronchomotor reflex effects by engaging different types of vagal afferent, and the net input from the lower airways probably plays an important role in determining the resting level of airway smooth muscle tone. Only a relatively brief description of the afferent innervation of the lower airways can be given here, and we make no attempt to provide a comprehensive bibliography. The reader is referred to several review articles for more detailed accounts (Widdicombe, 1964, 1974; Fillenz and Widdicombe, 1972; Coleridge and Coleridge, 1984, 1986; Sant'Ambrogio, 1982, 1987). In most mammalian species the reflex changes evoked from the lower airways are abolished by blocking conduction in vagal afferent fibres, and in man even pain and irritant sensations from the lower airways seem to depend on the integrity of the vagus nerve (Guz, 1977). However, some afferent fibres from the lower airways and lungs pass directly to the spinal cord. A small reflex bronchoconstrictor effect that depended on the integrity of the upper thoracic sympathetic chain and rami was observed when sulphur dioxide was insufflated into the lower airways of cats whose vagus nerves had been cut in the neck (Widdicombe, 1954a). Even so, the spinal contribution to irritant bronchoconstrictor reflexes in cats is small and difficult to demonstrate, and in dogs bronchoconstrictor reflexes of lower airway origin probably depend entirely on vagal input (Russell and Lai-Fook, 1979; Roberts et al., 1981; Coleridge et al., 1982a). In any event, nothing is known about the spinal afferents that respond to the commonly used chemical irritants such as capsaicin, histamine and sulphur dioxide. By contrast, the sensory characteristics of the various types of specialized nerve ending supplied by vagal fibres have been investigated in detail, and the fibres responsible for both inhibitory and excitatory bronchomotor reflexes have been identified with reasonable certainty. When the vagus nerves are cut above the nodose ganglion, and the preganglionic motor fibres allowed to degenerate, the vagal bronchial and pulmonary branches can be seen to contain large numbers of both myelinated and nonmyelinated afferent fibres, the latter greatly outnumbering the former. Indeed, of the 5000 or so afferent fibres distributed to the lungs and lower airways by each vagus nerve in cats, about 4000 are nonmyelinated (Agostoni et al., 1957), and more recent studies put the proportion of nonmyelinated fibres even higher (Jammes et al., 1982). Myelinated fibres whose cell bodies are in the vagal nodose ganglion supply mechanoreceptors (stretch receptors) that innervate both the extrapulmonary and intrapulmonary airways. The receptors are classified as slowly or rapidly adapting on the basis of their response to a static lung inflation. However, it seems likely that both slowly and rapidly adapting receptors respond primarily to tension within the airway walls as the lungs inflate, rather than to stretch or inflation per se, because they can be sensitized to a given degree of inflation by factors, such as contraction of airway smooth muscle and decreased lung compliance, that increase airway tension (Davenport et al., 1981; Richardson et al., 1984; Jonzon et al., 1986; Yu et al., 1987). The myelinated fibres of slowly and rapidly adapting receptors have conduction velocities ranging from 11 to 66 m/sec; both have average velocities of about 32 m/sec (Jonzon et al., 1988). They are termed A fibres because they contribute to the A-wave of the compound action potential evoked by electrically stimulating the vagal trunk. Nonmyelinated afferents have conduction velocities of less than 2.5 m/sec; they are termed C fibres because their activity contributes to the C-wave of the evoked compound action potential. 3.1.1. Slowly Adapting Pulmonary Stretch Receptors

Low-threshold, slowly adapting stretch receptors were first examined systematically by Adrian in his classical electroneurographic study (Adrian, 1933). Their afferent

12

H. M. COLERIDGE et al.

input is associated with the Hering-Breuer inflation inhibitory reflex and is of major importance in determining resting breathing pattern in animals, but appears to be less so in man. Slowly adapting stretch receptors probably correspond to the histologically identified terminal arborizations of vagal myelinated afferent fibres closely associated with airway smooth muscle (Elftman, 1943; Fisher, 1964; Fillenz and Woods, 1970). The terminals, which are bound to connective tissue elements between the lamina propria and the smooth muscle layer, are oriented in the long axis of the bronchus (Von During et al., 1974). The receptors are particularly numerous at the tracheal bifurcation and in the extrapulmonary bronchi, those in the trachea being confined to the smooth muscle of the posterior membranous wall (Bartlett et al., 1976). The relative distribution of the receptors in the extrapulmonary and intrapulmonary airways is controversial, some investigators claiming that a majority of receptors are in the extrapulmonary airways, others that more than 80% are intrapulmonary (for references see Coleridge and Coleridge, 1986). On balance, action potential studies suggest that intrapulmonary stretch receptors are virtually confined to the walls of the conducting airways, whereas morphological studies provide convincing evidence that the endings extend as far peripherally as the respiratory bronchioles and alveolar ducts. In dectroneurographic studies, slowly adapting receptors appear to be more numerous than their rapidly adapting counterparts (Sant'Ambrogio, 1982, 1987), and their waxing and waning discharge during normal breathing is the most prominent component of vagal afferent input from the lower respiratory tract. The receptors are capable of signalling changes in lung volume or transpulmonary pressure with considerable precision, and this seems to be their most important sensory function. Individual receptors discharge at frequencies of 100 impulses/sec or more at the peak of inflation, and many remain active throughout normal deflation, at functional residual capacity. Feedback from slowly adapting stretch receptors inhibits the central inspiratory neurones, their mounting discharge in inspiration providing an 'off-switch' to the centres controlling inspiratory muscles, and their tonic input in expiration lengthening the expiratory pause. Their feedback also inhibits the vagal bronchomotor neurones, causing withdrawal of vagal muscarinic effects on airway smooth muscle, resulting in bronchodilatation (Fig. 2A). Whereas a wide variety of chemicals stimulate rapidly adapting receptors and lower airway C fibres (see below), few chemicals are known to excite slowly adapting receptors, and none to do so selectively. Low doses of veratrum alkaloids sensitize stretch receptors, and higher doses cause continuous high frequency firing, but these alkaloids evoke repetitive firing from many types of afferent nerve ending. Chemicals, such as capsaicin, phenyl diguanide, bradykinin and the prostaglandins, which have been used extensively to stimulate airway C fibres, have virtually no direct effect on slowly adapting stretch receptors. Though stimulated by few chemicals, slowly adapting receptors appear more susceptible than other pulmonary afferents to inhibition by chemicals. Thus high concentrations of certain volatile anaesthetics first increase and then abolish receptor discharge (Coleridge et al., 1968); and, in rabbits, inhalation of sulphur dioxide abolishes stretch receptor firing selectively--an effect that has been used to assess the contribution of these afferents to certain reflexes (Davies et al., 1978). Thus, in general, while chemicals have proved to be a valuable experimental tool with which to explore the reflex functions of rapidly adapting receptors and C fibres, they have not been widely used to investigate the functional role of slowly adapting receptors. 3.1.2. Rapidly Adapting Receptors

Rapidly adapting receptors, first described in detail by Knowlton and Larrabee (1946) in cats, differ from slowly adapting receptors in having a higher volume threshold, a more rapid rate of adaptation and a more irregular pattern of discharge. Unlike slowly adapting receptors, rapidly adapting receptors have excitatory effects on breathing (Larrabee and

Bronchomotor reflexes

13

Knowleton, 1946). Rapidly adapting receptors remained of somewhat academic interest until Widdicombe (1964) suggested that their activation evoked cough and protective reflexes from the tracheobronchial tree. Subsequently, Widdicombe and his colleagues (see below) found that rapidly adapting receptors were stimulated not only by the inhalation of chemical and particulate irritants but also by a variety of pathological conditions of the lung, and they introduced the alternative title of 'irritant receptor'. These observations led to the notion that irritant receptors were principally responsible for the airway defense reflexes, and that indeed this was their principal function. However, a number of observations made during the past 15 years have led to a reappraisal of the 'irritant' role of rapidly adapting receptors and have also served to re-emphasize the mechanoreceptive aspects of their behaviour (e.g., Sampson and Vidruk, 1975; Pack and Delaney, 1983; Jonzon et al., 1986). With the renewed interest in the mechanosensitive properties of the endings, investigators have tended increasingly to return to the original nomenclature of 'rapidly adapting receptors' introduced by Knowlton and Larrabee (1946). Rapidly adapting receptors probably correspond to the epithelial nerve endings that have been identified histologically in the airways of several mammalian species. These endings represent the terminal arborizations of myelinated fibres that penetrate the bronchial wall and ramify in the tracheobronchial mucosa (Elftman, 1943; Fisher, 1964; Fillenz and Woods, 1970). The fine terminals appear to pass between the columnar cells of the epithelium towards the mucosal surface. In general, rapidly adapting receptors appear to be more loosely arranged and extensive than the endings identified as slowly adapting receptors, indeed the parent axons of endings in the trachea and bronchi branch extensively to supply end organs over a wide area of mucosa. Electrophysiological studies suggest that the terminals of a single axon may be distributed to both superficial (epithelial) and deeper layers of the airway (Sant'Ambrogio et al., 1978). The receptors are particularly numerous at the carina and at bronchial bifurcations. Endings of myelinated fibres have been identified in the mucosa of the intrapulmonary airways, but these more peripheral structures are hard to categorize and many of the descriptions could apply equally to the endings of both types of myelinated pulmonary afferent. Rapidly adapting receptors in the intrathoracic airways have a generally scanty discharge during quiet breathing, and they often fire no more than two or three impulses during the normal ventilatory cycle--indeed the threshold to inflation of some receptors is well above resting tidal volume, so that they are completely inactive in the majority of cycles under control conditions (Mills et al., 1969; Sampson and Vidruk, 1975; Jonzon et al., 1986). Because of their rapidly adapting character their discharge is sporadic rather than continuous. Nevertheless, conventional action potential studies may provide a false impression of the normal level of afferent input arising from these receptors. The lungs are usually hyperinflated at the beginning of an experiment in order to confirm the rapidly adapting nature of the receptor. The mechanical state of the lungs is an important determinant of rapidly adapting receptor firing, and the increased lung compliance resulting from this initial hyperinflation reduces the subsequent level of activity, and is often enough to silence the receptor (Jonzon et al., 1986; Yu et al., 1987). Thereafter, the level of spontaneous firing slowly increases as lung compliance gradually returns to its original level. Such observations suggest that afferent input from rapidly adapting receptors may be of functional significance not only when the endings are stimulated by a variety of physiological or pharmacological interventions but also during normal ventilation. Like many dynamically sensitive mechanoreceptors, rapidly adapting receptors are sensitive to distortion regardless of its sign, and they are readily excited by collapse of the lung. Thus their activity increases in pneumotharax (Sellick and Widdicombe, 1969), when lung volume is reduced below functional residual capacity, and they usually fire with each lung deflation in open-chest animals when the expiratory resistance is removed from the outlet port of the ventilator and the lungs are allowed to collapse to atmospheric pressure in the deflation phase of the cycle (Armstrong and Luck, 1974; Jonzon et al., 1986). Their response to deflation distinguishes them clearly from all other types of pulmonary afferent

14

H.M. COLERIDGEet al.

and it can serve as a distinguishing feature in the case of myelinated pulmonary afferents whose adaptation rate is in the intermediate range (Yu, Jonzon, Pisarri, Coleridge and Coleridge, unpublished observations). As might be expected from their rapidly adapting character, the receptors are sensitive to the rate of inflation (Pack and Delaney, 1983). However, they are unlikely to function as true 'air flow' receptors, but instead appear to be sensitive to the rate of change of airway pressure (Yu et al., 1987). Their activity increases if lung compliance decreases, and they fire a brief, high frequency burst of impulses with each normal inflation when the lungs become stiffer (Jonzon et al., 1986; Yu et al., 1987), probably because the stiffened lung parenchyma exerts traction on the bronchial walls in which the receptors lie (Mills et al., 1969). Rapidly adapting receptors are stimulated by a variety of chemicals with a bronchoconstrictor action, such as histamine and serotonin, the bronchoconstrictor prostaglandin F2~, and the muscarinic agonists acetylcholine and methacholine (Mills et al., 1969; Coleridge et al., 1976, 1978; Dixon et al., 1979a). Receptors in the central airways are stimulated when particulate irritants are inhaled. The effects of various chemicals and inhaled irritants on rapidly adapting receptors are described in detail in later sections of this review. A major difficulty in investigating the afferent characteristics and reflex functions of rapidly adapting receptors is that all the naturally occurring chemicals known to stimulate the receptors are themselves powerful bronchoconstrictors, acting directly on airway smooth muscle. In some species, receptor stimulation by bronchoconstrictor chemicals is thought to be due mainly to contraction of bronchial smooth muscle and stimulation is often abolished when direct effects on the muscle are prevented by appropriate pharmacological blockade (Mills et al., 1969; Widdicombe, 1974). Autocoids such as bradykinin that do not contract smooth muscle directly have little effect on rapidly adapting receptors. Chemically-induced muscle contraction appears to sensitize the receptors to inflation rather than to stimulate them directly. Thus, administration of histamine by intravenous injection or aerosol evokes regular bursts of firing at the peak of inflation that can be abolished simply by switching off the ventilator (Coleridge et aL, 1978). The bursts can also be reduced or abolished by hyperinflating the lungs to restore compliance to its initial level (Sampson and Vidruk, 1975). These observations suggest that the chemically-evoked increase in rapidly adapting receptor firing is largely the result, rather than the cause, of bronchoconstriction (Mills et al., 1969; Fillenz and Widdicombe, 1972). Nevertheless, the receptor response to histamine may not be due to smooth muscle effects alone. Evidence for a direct stimulating effect has been obtained in dogs by applying histamine solutions directly to the receptive fields of rapidly adapting receptors in the trachea and major bronchi through a bronchoscope (Vidruk et al., 1977). For many years, major interest in the afferent and reflex properties of rapidly adapting receptors undoubtedly stemmed from the observation that the impulse activity of receptors in the intrapulmonary airways increased in experimental models of lung disease such as histamine-induced bronchoconstriction, congestion and embolism (Mills et al., 1969; Sellick and Widdicombe, 1969; Armstrong et aL, 1976; Roberts et al., 1986a). These findings, coupled with the widespread use of the persuasive term 'irritant receptor', led to the assumption that rapidly adapting receptors were largely responsible for the neural component of obstructive airway diseases such as asthma (Nadel, 1980) and played the major role in irritant reflexes evoked from the respiratory tract below the larynx (Boushey et al., 1980; Nadd, 1980). However, this identification of the sensory mechanisms responding to airway irritants exclusively with intrapulmonary rapidly adapting receptors is clearly too narrow, for autocoids such as bradykinin and the bronchodilator prostaglandins, which have little if any effect on these afferents, cause powerful vagallymediated contractions of airway smooth muscle and other manifestations of irritant airway reflexes, all of which can be demonstrated when the vagus nerve is cooled to temperatures that block conduction in myelinated fibres. Even in the case of histamine, which undoubtedly stimulates these endings, the reflex bronchoconstrictor response can no longer

Bronchomotorreflexes

15

be assumed to be due entirely to rapidly adapting receptors, for the reflex bronchoconstriction evoked by inhalation of histamine aerosol still occurs after myelinated vagal axons are blocked selectively by cooling (Jammes et aL, 1985). Even so, there is no doubt that the afferent input from rapidly adapting receptors plays an important part in many circumstances, especially in lung disease. Rapidly adapting receptors are known to have powerful excitatory effects on the inspiratory centers of the medulla causing augmented inspiratory efforts (Widdicombe, 1954c) that may be of major importance when the lungs become stiffer (Jonzon et al., 1986). In addition, there is strong, though circumstantial, evidence (Mills et al., 1969) that rapidly adapting receptor input is excitatory to the vagal bronchomotor centre. Moreover, recent evidence suggests that the tracheal contraction and submucosal gland secretion evoked in anaesthetized dogs by high frequency oscillatory ventilation may be due to stimulation of rapidly adapting receptors (Pisarri et al., 1987; Schultz et al., 1987a). 3.1.3. C Fibres Morphological and electroneurographic studies indicate that nonmyelinated vagal afferents, like their myelinated counterparts, innervate all levels of the lower respiratory tract. Electronmicroscopic studies have identified nonmyelinated fibres in the alveolar walls and alveolar ducts of mouse lung (Hung et al., 1973a) and in the alveolar walls of human lung (Fox et al., 1980); several fibres have been traced to axonal enlargements having a sensory appearance (Hung et al., 1973a). A characteristic feature of these intrapulmonary endings is that most of the axons or axon bundles within the interstitial tissue are surrounded by collagen fibres--an observation that may be of some functional significance (Paintal, 1970; see pulmonary oedema below). Nonmyelinated afferent fibres have also been identified in the conducting airways. Naked nerve terminals of sensory appearance have been described between the epithelial ceils of the human tracheal mucosa, and are thought to correspond to the afferent endings of nonmyelinated fibres innervating most epithelia (Rhodin, 1966). Nonmyelinated fibres in the lamina propria of the airway mucosa have been seen to penetrate the outer layers of the basement membrane and appear to give rise to the intra-epithelial endings. Nonmyelinated fibres with endings of a sensory type have also been identified between the epithelial ceils of intrapulmonary airways (Hung et al., 1973b). Results of action potential studies indicate that C fibres arising from the lower respiratory tract may be classed as 'pulmonary' or 'bronchial' according to whether their endings are accessible from the pulmonary or bronchial circulations (Coleridge and Coleridge, 1977b). Their properties have been reviewed in detail by Coleridge and Coleridge (1984). The two types of C fibre ending differ not only in their location and vascular accessibility to chemicals injected at various sites in the blood stream, they also differ in their responses to certain mechanical and chemical stimuli, and in some of the reflex effects evoked by their stimulation (see below). Both pulmonary and bronchial C fibres have a sparse and irregular spontaneous discharge, and their firing frequency is usually less than 1 impulse/see under control conditions. Consequently, the electroneurographic investigation of these C fibres has relied heavily on injection of chemicals to stimulate their endings, and to reveal the presence of C fibres in what often appear to be inactive nerve strands. Pulmonary C fibres are usually identified initially by their prompt response to chemicals (capsaicin in dogs and cats; phenyldiguanide in cats) injected into the right atrium or pulmonary artery, and by their lack of response to chemicals injected into the left atrium, downstream to the pulmonary circulation. Their location can be further defined by examining the response to chemicals injected into the different lobar arteries (Coleridge et al., 1965). The intrapulmonary location of the endings is confirmed by exploring the lung with a fine probe to find the sensitive point from which impulses can be evoked by mechanical stimulation (Coleridge et al., 1965). Since the endings accessible from the pulmonary circulation were promptly stimulated by volatile anaesthetics delivered via the JFT 42ti--B

16

H . M . COLEI~IDGEet al.

airways, Paintal (1969) concluded that they were located in the most peripheral lung divisions, a region that appears to lack innervation by myelinated fibres. To acknowledge what seemed to be a juxta-pulmonary capillary location, Paintal (1969) called the endings type J receptors. In Paintal's view, the primary function of J receptors is to act as interstitial stretch receptors in the alveolar walls (see pulmonary oedema, below). (The terms pulmonary C fibre and J receptor are interchangeable, although the former is now more widely used in order to distinguish pulmonary from bronchial C fibres.) Pulmonary C fibres are also stimulated by large lung inflations (2 or 3 VT) (Coleridge et al., 1965; Coleridge and Coleridge, 1977b; Kaufman et al., 1982a), and hence were at one time called high threshold inflation receptors to distinguish them from the low threshold slowly adapting pulmonary stretch receptors (Coleridge et al., 1968); however, the term is no longer used. Pulmonary C fibres are relatively insensitive to lung autocoids---they are stimulated by large doses of PGE2, but not by bradykinin, and rarely by histamine (see below). However, they are stimulated by certain inhaled substances (volatile anaesthetics, cigarette smoke, sulphur dioxide). Bronchial C fibres innervate the conducting airways supplied by the bronchial circulation. Their endings in the larger airways can be stimulated by gently stroking the mucosa with a fine probe or bristle after the airways have been opened (Coleridge and Coleridge, 1977b). Whereas pulmonary C fibres are stimulated after an interval of 1-2 sec by capsaicin injected into the right atrium or pulmonary artery, bronchial C fibres are stimulated after a long delay (6-9 sec) by injection into the right heart, and after a shorter delay (3-5 sec) by injection into the left. Bronchial C fibres arising from endings in the lungs are also stimulated by injection of small doses of chemicals into the bronchial artery (Kaufman et al., 1980b). Endings with similar afferent properties have been identified in the trachea and extrapulmonary bronchi, where they are accessible to chemicals injected into the systemic circulation (Coleridge et al., 1983; Roberts et al., 1985b). Bronchial C fibres are stimulated by hyperinflation but they are less sensitive than pulmonary C fibres to changes in lung volume (Coleridge and Coleridge, 1977b; Kaufman et aL, 1982a). However they are much more sensitive than pulmonary C fibres to autocoids, such as histamine, bradykinin, and prostaglandins, that are known to be released in the airway walls (see below). In this respect they resemble the chemosensitive C fibres of the skin, and like the C fibres of the skin, may play a part in so-called 'neurogenic inflammation'. They are also stimulated by the inhalation of some irritant substances, and by severe congestion and interstitial oedema of the lungs (see below). In general, pulmonary and bronchial C fibres have similar reflex effects on respiratory mechanisms, causing rapid shallow breathing (often preceded by apnoea), contraction of bronchial smooth muscle and increased secretion by tracheal submucosal glands: they also cause cardiovascular depressor effects (Coleridge and Coleridge, 1984). The combination of immediate reflex effects evoked when pulmonary C fibres are stimulated selectively by chemicals injected into the pulmonary artery is called the pulmonary chemoreflex. The combination of effects evoked when bronchial C fibres are stimulated selectively by chemicals injected into the bronchial artery can be called the bronchial chemoreflex. Pulmonary C fibres appear to have the greater effect on the circulation, producing bradycardia and systemic hypotension, whereas bronchial C fibres appear to have the greater effect on the airways (Coleridge et aL, 1982a; Coleridge and Coleridge, 1984). Indeed, the cardio-inhibitory effects of stimulating bronchial C fibres may be difficult to demonstrate in spontaneously breathing dogs, probably because of the opposing influence of other reflexes set in train by increased breathing (Coleridge et al., 1983). There is as yet no convincing evidence that bronchial C fibres affect peripheral vascular resistance; and selective stimulation of bronchial C fibres by injection of small doses of chemicals into the bronchial artery may evoke marked effects on the airways, breathing and heart rate without having any obvious effect on arterial blood pressure. The systemic hypotension sometimes observed when bronchial C fibres are stimulated by bradykinin can probably be accounted for by the powerful direct vasodilator action of the chemical (Roberts et al., 1981). The role of pulmonary and bronchial C fibres in the

Bronchomotor reflexes

17

bronchomotor effects evoked by chemical agents and pathological changes in the lung is discussed below. 3.2. METHODSFOR INVESTIGATINGBRONCHOMOTORREFLEXES The bronchomotor effects elicited by stimulating afferent vagal endings in the lower airways will be dealt with, as far as possible, according to the stimulus that evokes them. The ideal stimulus is one that acts on a single type of afferent ending, but such selective stimulation has rarely been achieved in experimental studies on the lower airways. Consequently, several of the bronchomotor reflexes found to originate in the lower respiratory tract result from changes in afferent input from more than one type of sensory ending. For example, the reflex bronchoconstrictor effects of histamine, which formerly were attributed entirely to input from rapidly adapting receptors, now appear to include a contribution from bronchial C fibres (see below). Even lung inflation, which would seem to be a relatively straightforward stimulus to the lower airways, excites all four types of pulmonary vagal afferent. In addition, determination of the mechanism by which an experimental stimulus acts on a particular type of lung afferent may present problems. For instance, a bronchoactive chemical such as histamine may stimulate sensory nerves in the airways directly (i.e., the afferent terminals are stimulated chemically), or indirectly as a consequence of its bronchoconstrictor action (i.e., the terminals are stimulated mechanically). Moreover, selective stimulation of a single type of respiratory afferent may set in train reflex effects that in their turn stimulate other respiratory afferents whose input contributes to the final bronchomotor changes. Thus, although changes in airway smooth muscle tension or total pulmonary resistance can be demonstrated when a variety of stimuli are applied to the lower airways, the identity of the responsible afferent input may be difficult to determine. Indeed, even the reflex nature of the bronchomotor effects may be difficult to establish conclusively. For example, large inflations and deflations of the lung not only stimulate receptors in the airways, they also have direct effects on the smooth muscle itself. Many of the foreign and endogenous chemicals used to stimulate sensory nerve endings in the bronchial walls also have direct smooth muscle effects. We next describe two techniques used in animal studies to facilitate the analysis of vagal bronchomotor reflexes arising from the lower airways. Investigators have examined bronchomotor responses in a region of the airways remote from the applied stimulus; and they have combined afferent stimulation with graded cold blockade of the vagus nerves to determine whether the reflexogenic input is carried in myelinated or nonmyelinated axons. 3.2.1. Examination of Bronchomotor Responses Some of the complications described above may be avoided by applying a stimulus to the more distal airways and recording changes in tension or volume in an isolated, innervated, tracheal segment that has not been exposed to the stimulus (Loofbourrow et al., 1957; Roberts et al., 1981). An alternative approach, which takes advantage of the fact that afferents from the lung travel mainly in the ipsilateral vagus nerve, has been to ventilate the lungs separately and to apply the stimulus to one lung only, recording the reflex bronchomotor changes in the opposite lung (Gold et al., 1972). An additional refinement of this method has been to deliver an irritant stimulus (ozone) into the lung periphery through a bronchoscope wedged in a peripheral bronchus, and to measure airflow resistance downstream and in nonstimulated segments (Gertner et al., 1983, 1984). Results of these latter experiments suggest that vagal reflex arcs can be relatively localized in the periphery and that, provided the stimulus is small and circumscribed, bronchomotor reflex effects are confined to the ipsilateral lung (Gertner et al., 1984).

18

H.M. COt~rODOEet al.

The measurement of changes in tracheal smooth muscle tension remains a popular method for studying bronchomotor reflexes originating in the lower airways. The method has been adapted for use in unanaesthetized dogs, both awake and sleeping, tracheal pressure being measured in a distensible water-filled cuff surrounding a tracheostomy tube (Sullivan et aL, 1979; Sorkness and Vidruk, 1986). In anaesthetized animals, the technique of recording reflexly-evoked tracheal responses has been further refined by using the tracheal segment immediately below the larynx for measurement of smooth muscle tone (Fig. 5), Since this upper tracheal segment receives most o f its bronchomotor innervation from the superior laryngeal nerves, the recurrent and pararecurrent laryngeal nerves can be cut before the experiment, allowing anatomical separation of the afferent and efferent arms of a vagal bronchomotor reflex arc originating in the lower respiratory tract. Hence cutting or cooling the lower cervical vagus nerves during the experiment will eliminate input in vagal afferent pathways arising from the lower airways and lungs without interrupting the vagal efferent supply to the tracheal segment (Roberts et aL, 1981; Coleridge et al., 1982a). 3.2.2. Cold Block o f Afferent Inputs Several methods for producing reversible neural blockade have been used over the past hundred years to examine respiratory reflexes (see Coleridge and Coleridge, 1984). Only nerve cooling is dealt with here, because it is the only nerve blocking technique used so far to investigate bronchomotor reflexes. Cooling the cervical vagus nerves to 0°C produces total reversible blockade ('reversible vagotomy'), which is much more satisfactory in reflex studies than simply cutting the vagus nerves. Because the saltatory

SLN

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;

FIo. 5. Innervated tracheal segment (TS) for examiningthe reflexcontrol of airway smooth muscle in dogs. The upper trachea is incised ventrally in midline, and each cut edge is retracted laterally and attached to a light plastic bar, one bar being anchored to a fixedmetal rod, the other attached to an isometric force transducer (FT) mounted on a rack and pinion. The segment is stretched initially to a baseline tension of 50-75 g. Recurrent (and pararecurrent) laryngeal nerves (RLN) are cut so that the tracheal segmentis innervatedonly by superiorlaryngealnerves(SLN). Cervical vagus nerves (VN) are placed on cooling platforms (CP). Right bronchial artery (Br A) lies dorsal (dotted line) to right lung root. The smallerdiagram on left depictsright bronchial artery stemming from an intercostalartery (Int A), which arises from thoracicaorta (Ao). Bradykinin(BK) or other chemicals injected retrogradely into intercostal artery pass into bronchial artery. (Roberts et al., 1981.)

Bronchomotorreflexes

19

conduction in myelinated axons renders them more susceptible to cooling (Franz and Iggo, 1968), nerve cooling is also used to determine whether the afferent fibres responsible for a given bronchomotor reflex are myelinated or nonmyelinated. This is done by comparing the blocking temperature of the various pulmonary afferents, established in electroneurographic studies, with the blocking temperature of the reflex itself. Nerve cooling must be used with an awareness of its limitations. Tension on the nerve trunk can cause transmission block, particularly in myelinated axons. Hence even slight tension accidentally exerted by the placement of the nerve on the cooling device adds to the effects of cooling, so that nerve block may occur at a temperature several degrees higher than would otherwise be the case (Paintal, 1965). One might expect the effects of such tension to be especially troublesome in reflex studies in conscious dogs in which the exteriorized vagus nerves, enclosed in skin loops, are placed on cooling devices. In evaluating the effects of nerve cooling on reflexes it is important to make a clear distinction between the temperature required to block all impulse activity in a particular group of fibres and the temperature required to block the increase in activity evoked by a given stimulus. For example, the average blocking temperature of slowly adapting stretch receptor axons is several degrees lower than the temperature required to block the Hering-Breuer reflex (Widdicombe, 1954c). Jonzon et al. (1988) compared the attenuating effects of vagal cooling on the impulse activity evoked in the various types of pulmonary fibre by hyperinflating the lungs, activity recorded rostral to the cooling platform representing the input to the medullary centres. Progressive cooling attenuated the impulse traffic in both myelinated (Fig. 6) and nonmyelinated (Fig. 7A) fibres, at each temperature attenuation being less marked in the latter (Fig. 8). Before cooling, hyperinflation evoked a lower input in C fibres than in the fibres of either slowly or rapidly adapting receptors. When the nerve was cooled to 7°C, however, firing in C fibres began to dominate the activity transmitted across the cooled region of nerve, input in myelinated fibres being only

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FIG.6. Vagal coolingprogressivelyattenuates conductionin an afferentfibrearising from a rapidly adapting receptor in the lung of an anaesthetized dog. The chest was open and the lungs were ventilated artificially.The vagus nerve was cooled caudal to the recordingelectrodes.The rapidly adapting receptor was stimulated at intervals of 1-2 rain by brieflyhyperinflatingthe lungs (3 VT), as indicatedby the increasesin trachealpressure(PT)"Note the virtual abolitionof recordedactivity at 7°C and the restoration of activityas the nerve was rewarmed. PCO2, tidal PCO2; ABP, arterial blood pressure; temp (needle),temperature of vagus nerve recorded by a needle thermistor; temp (platform), temperature of the cooling platform; IF, impulse frequencyrecorded by a ratemeter. Between 28°C and 6°C, the vagus nerve was cooled at the rate of 1.8°C/min. Beforevagal cooling, the rapidly adapting receptorwas stimulatedby injectinghistamine(20 ~g/kg) into the right atrium; when the vagus nerve had been cooled to 7-6°C, injectionof histamine (hist: note decrease in ABP and increase in PT) had no effect on rapidly adapting receptor activity recorded rostral to the cooling platform. (Jonzon et al., 1988.)

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F]O. 8. Comparison of the effects of vagal cooling on transmitted impulse frequency (IF) in the fibres of three types of lung afferent stimulated by hyperintiating the lungs (3 VT) in anaesthetized dogs. The vagus nerve was cooled caudal to the recording electrodes. Data are means + SE: hatched bars, 42 slowly adapting pulmonary stretch receptors; stippled bars, 43 rapidly adapting receptors; open bars, 38 lung C fibres. A: results obtained over the complete range of vagal temperatures; calibration for pulmonary stretch receptors on the left, that for rapidly adapting receptors and C fibres on the right. B: results obtained over the lower range of vagal temperatures, plotted on a common expanded calibration scale. Oonzon et aL, 1988.)

Bronchomotor reflexes

21

a small fraction of that at 37°C. Indeed at 7°C, C fibre input exceeded the combined transmitted input in slowly and rapidly adapting fibres (Fig. 8). Below 7°C, attenuation was virtually complete in both types of myelinated fibre, although occasional impulses were transmitted in a few fibres down to 1°C. By contrast, even at 3°C, C fibres still transmitted some of the activity evoked by hyperinflation, the average input passing the cold block amounting to about 20% of that transmitted at 37°C (Figs 7A, 8). Complete blockade of conduction in all myelinated fibres occurs over the same temperature range (Franz and Iggo, 1968); above the final blocking temperature, however, effects of cooling on slowly and rapidly adapting fibres differ (Jonzon et al., 1988). Thus, between 12°C and 7°C, input from slowly adapting receptors was attenuated significantly more than was input from rapidly adapting receptors (Fig. 8). Effects of cooling are frequency-dependent--the greater the impulse frequency the greater the relative attenuation--probably because cooling increases the refractory period of axons. It follows, therefore, that the effects of cooling will also depend to some extent on the temporal pattern of activity characteristic of the particular type of fibre, and on the scatter of interspike intervals. This explains why the hyperinflation-evoked activity in slowly adapting fibres, which are already discharging at high and regular frequencies under control conditions, is blocked more readily by cooling than the lower, more irregular frequencies evoked in rapidly adapting fibres, which have a typically low control discharge. That vagal cooling has corresponding effects on the reflexes evoked by stimulating these two types of myelinated afferent is shown by the observation that the inspiration-inhibitory Hering-Breuer reflex evoked by a large inflation is abolished at a vagal temperature several degrees higher than that required to abolish the gasp or augmented breath attributed to stimulation of rapidly adapting receptors (Widdicombe, 1954c). Not only does the mode of conduction in C fibres make them less susceptible to cooling, but their low-frequency, irregular pattern of discharge affords them an additional margin of protection against the attenuating effects of cooling. This explains why at least some of the augmented activity evoked by large lung inflations (Fig. 7A) or by injection of capsaicin (Fig. 7B) is transmitted at temperatures as low as 2-3°C (Jonzon et al., 1988). Although there is good reason to conclude that a vagally-mediated reflex bronchoconstriction remaining after the vagi are cooled below 7°C is mediated by afferent C fibres, it is important to remember that this surviving response does not represent the total contribution of C fibres to the response evoked at 37°C. At 7°C, only 40% of the activity evoked by stimulating the sensory terminals of pulmonary C fibres is transmitted across the cooled region of nerve to reach the medullary centres (Jonzon et al., 1988). If reflex effects are attenuated in proportion to the attenuation of afferent input, it seems reasonable to expect that cooling will produce a corresponding reduction in the bronchomotor response. In the event, the reflex increase in tracheal tension evoked by stimulating pulmonary (Coleridge et al., 1982a) or bronchial (Roberts et al., 1981) C fibres, after myelinated afferent input had been blocked by cooling to 6-7°C, was about 40% of that evoked before the vagus nerves were cooled.

3.3. REFLEX INFLUENCE OF LUNG VOLUME ON AIRWAYS

3.3.1. Tonic Effects of Lung Afferents During Normal Ventilation Even during normal ventilation, input from slowly adapting stretch receptors in the lower airways exercises a tonic inhibitory influence on bronchomotor mechanisms. In anaesthetized dogs with lungs ventilated artificially at normal volumes, tracheal smooth muscle tone increases if the cervical vagus nerves are cooled to 7-12°C to block input from slowly adapting stretch receptors 0Viddicombe and Nadel, 1963; Roberts et al., 1988); such vagal cooling has also been shown to decrease lung conductance in rabbits (Karczewski and Widdicombe, 1969a). Since cooling the lower cervical vagus nerves to 8°C had no effect

22

H . M . COt~Rn)~E et al;

on basal airway tone when the pulmonary branches of the vagus nerves were cut, the inhibitory input was entirely of pulmonary origin (Roberts et al., 1988). Although the inhibitory bronchomotor influence of slowly adapting stretch receptors is usually prepotent during normal ventilation--prepotent in the sense that the airways constrict when this inhibitory influence is attenuated by cooling--there is evidence to suggest that another pulmonary afferent input contributes to basal bronchomotor tone. Cutting the cervical vagus nerves or cooling them below 8°C causes airway smooth muscle to relax (Widdicombe and Nadel, 1963; Karczewski and Widdicombe, 1969a; Roberts et aL, 1988), an effect attributed in the first two of these studies to interruption of efferent nerves to the airways. However, in the experiments of Roberts et al., vagal cooling did not affect the efferent innervation of their tracheal smooth muscle preparation, which was supplied solely by the superior laryngeal nerves. In two-thirds of their experiments, tracheal tension decreased as the vagi were cooled below 8°C, reaching a steady level just above, at, or slightly below the initial baseline when the temperature reached 5-2°C. The most likely explanation for the final relaxation is that cooling attenuated an excitatory C fibre input to the bronchomotor centre. Since the effects of cooling were abolished by cutting the pulmonary vagal branches, the excitatory input originated in the lungs. In some experiments, however, tracheal tone did not return to the baseline when the vagi were cooled below 8°C, indicating that tonic input in C fibres was not solely responsible for maintaining vagal bronchomotor tone, as Jammes and Mei (1979) suggested on the basis of experiments in cats. It is possible that the excitatory bronchomotor influence unmasked in these latter experiments when stretch receptor input was blocked was due to a central drive from medullary chemoreceptors maintained by the level of COe (Loofbourrow et al., 1957; Mitchell et al., 1985). There are parallels between the effects of vagal cooling on baseline bronchial tone and those on breathing. In anaesthetized dogs, a tonic vagal C fibre input from the lungs continues to exert significant stimulation of breathing frequency at temperatures as low as 3°C (Pisarri et aL, 1986). Background input from rapidly adapting receptors may possibly contribute an additional tonic excitatory influence on the airways when vagal conduction is intact. Even after stretch receptor input is blocked by cooling to 8°C, a small surviving input from rapidly adapting receptors (Jonzon et al., 1988) may supplement the excitatory input from C fibres. But below 7°C, tonic input from rapidly adapting receptors is negligible (Jonzon et aL, 1988). 3.3.2. Bronchodilatation Evoked by Increasing Lung Volume The dominant inhibitory influence of slowly adapting stretch receptors on bronchomotor mechanisms is demonstrated most effectively by hyperinflating the lungs. In normal human subjects, a deep breath causes a reduction in airflow resistance that lasts 1-2 rain and is attributed, at least in part, to the reflex consequence of an increase in slowly adapting stretch receptor discharge (Nadel and Tierney, 1961; Nadel, 1980). This observation is supported by the results of experiments in anaesthetized dogs and cats, in which lung inflation reduces the impulse activity in vagal efferent nerves supplying the lungs and trachea (Widdicombe, 1961a, 1966), and relaxes smooth muscle in isolated in situ tracheal segments (Fig. 2A) (Loofbourrow et al., 1957; Widdicombe and Nadel, 1963; Coleridge et aL, 1982a; Roberts et al., 1988). This inflation-evoked reduction of airway smooth muscle tone seems to be due entirely to withdrawal of vagal bronchomotor activity. The motor side of the reflex arc has been examined in studies on conscious dogs with a permanent tracheostomy (Bowes et aL, 1984). The tracheal relaxation induced in these dogs by a 1 liter inflation of the lungs was unaffected by p-adrenergic blockade but abolished by atropine, and no further evidence of a bronchomotor effect could be obtained after atropine, even when smooth muscle tone was restored by administration of serotonin. An active relaxation of the airways often occurs in physiological circumstances in which breathing is increased. This is thought to represent a neural regulatory phenomenon

Bronchomotorreflexes

23

that helps to reduce the flow-resistive work of increasing minute volume (Stein and Widdicombe, 1975). An increase in input from slowly adapting stretch receptors is believed to be responsible, and to prevail in most circumstances when tidal volume increases. For example, experiments in both anaesthetized and conscious animals show that the excitatory bronchomotor reflex evoked by stimulating central and peripheral chemoreceptors can be partly or completely overcome by an increased inhibitory input from the lower airways (Stein and Widdicombe, 1975; Sorkness and Vidruk, 1986). In conscious dogs, hypoxia caused bronchoconstriction if ventilation was kept constant, but not when the dogs were allowed to breathe spontaneously and hyperventilated (Sorkness and Vidruk, 1986). Thus the bronchomotor influence of pulmonary stretch receptors appears to be of some functional utility, in the sense that the receptors act to increase airway conductance in circumstances that require an increase in ventilation. 3.3.3. Paradoxical Bronchoconstriction Evoked by Large Inflations Lung inflation does not invariably cause reflex bronchodilatation. In asthmatic patients, deep breaths often evoke an atropine-sensitive bronchoconstriction (Simonsson et al., 1967; Gayrard et al., 1975). Hyperventilation, also, has bronchoconstrictor effects in asthmatic subjects (Tam et al., 1985), and in Basenji-Greyhound dogs bred for the hyperreactivity of their airways (Davidson et al., 1987). In addition, large lung inflations evoke an increase, rather than a decrease, in tracheal smooth muscle tension in approximately 5% of apparently normal, anaesthetized dogs (Roberts et al., 1988). Some insight into a possible reflex mechanism contributing to these paradoxical bronchomotor effects is provided by the observation that the usual inhibitory response to lung inflation is reversed by vagal cooling (Figs 9, 10) (Roberts et al., 1988). If lung inflations are sufficiently large, the resulting reflex relaxation of bronchial smooth muscle appears to be the net result of the interaction of both inhibitory and excitatory inputs from the lower airways. Thus, although a large lung inflation (3 VT) evokes tracheal relaxation in a majority of anaesthetized dogs when the vagus nerves are at body temperature, relaxation is followed by contraction in some dogs, and contraction is the sole response in a few (Fig. 10). The afferents responsible for the bronchoconstrictor effects of these large lung inflations probably include both rapidly adapting receptors and pulmonary C fibres, since both are stimulated by hyperinflation and both are excitatory to the airways. The influence of these two afferent pathways can be separated by cooling the vagus nerves to block conduction in myelinated fibres. The tracheal relaxation evoked by a large lung inflation is progressively reduced as stretch receptor input is attenuated by cooling the lower cervical vagus nerves below 15°C, and is replaced by reflex contraction (Figs 9, 10) (Roberts et al., 1988). Contraction, which is the sole response at 8°C, is abolished by cooling to 0°C. Rapidly adapting receptors with myelinated fibres probably play some part in the reversal of the response at intermediate temperatures, since their fibre blocking temperature is somewhat lower than that of slowly adapting receptors (Jonzon et al., 1988). However, contraction can still be evoked at a vagal temperature of 5-6°C (Fig. 9), when conduction in myelinated fibres is virtually blocked. Hence although rapidly adapting receptors may contribute to the excitatory smooth muscle effects observed at higher vagal temperatures, C fibres are clearly responsible for the reflex contraction evoked at temperatures of 5-6°C or less. The 'paradoxical' excitatory effect of lung inflation on airway smooth muscle that is unmasked by vagal cooling appears to be the bronchomotor counterpart of the 'paradoxical' effect on breathing observed by Head (1889) in rabbits when the vagus nerves were cooled sufficiently to abolish the usual Hering-Breuer inhibition of breathing. Although Head's paradoxical reflex is often ascribed to stimulation of rapidly adapting receptors, it can be demonstrated at a temperature as low as 3°C (Whitteridge and Bulbring, 1944) when input in myelinated afferents is blocked. Hence Head's paradoxical reflex, like the paradoxical bronchomotor response described by Roberts et al. (1988), is triggered by nonmyelinated afferents.

24

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FIG. 9. Reversal of the reflex tracheal response to lung inflation as the lower cervical vagus nerves were gradually cooled in an anaesthetized dog. Smooth muscle tension recorded from an upper tracheal segment innervated solely by the superior laryngeal nerves. A, B: tracheal relaxation; C, D: biphasic response consisting of relaxation followed by contraction; E, F: contraction only; (3: contraction virtually abolished. In F and G, note small relaxation; this could still be evoked by hyperinflating the lungs after the vagus nerves were cut, and was possibly due to stimulation of afferents travelling along sympathetic pathways to the spinal cord. H: restoration of original tracheal response by rewarming the vagus nerves. PCOz, partial pressure of CO2 in tidal air; ABP, arterial blood presure; Tr tension, tracheal tension in grams above the baseline tension, which was set at 75 g; PT, tracheal pressure. (Roberts et al., 1988.)

Nevertheless, although rapidly adapting receptors and lower airway C fibres are known to be stimulated by large inflations, and their reflex effects correspond to those observed in asthmatics, the reason why inhibitory input from slowly adapting receptors does not prevail in all individuals remains obscure. Studies of hyperventilation-induced bronchoconstriction in Basenji-Greyhound dogs suggest that release of prostaglandins plays some part (Davidson et al., 1987), and it is at least conceivable that if autocoids are released by hyperinflation, hyperreactive airways are deficient in the enzymes that rapidly degrade them. The excitatory reflex input from rapidly adapting receptors and lower airway C fibres, the latter being particularly susceptible to lung autocoids, would then become prepotent. In any event, bronchoconstrictor reflexes of lower airway origin are associated with increased activity in rapidly adapting receptors and in bronchial and pulmonary C 2O J

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VAGAL TEMPERATURE ('(3) Fie. 10. Reversal of the tracheal response to lung hyperinflation (3-4 VT) as the lower cervical vagus nerves were cooled progressively in 18 anaesthetized dogs (vagal temperature measured to the nearest 0.5°C). Smooth muscle tension recorded in an upper tracheal segment innervated solely by the superior laryngeal nerves; hence cooling the lower cervical vagus nerves had no direct effect on the efferent nerve supply to the segment. Stippled bars, number of tests of lung inflation in each temperature range in which the tracheal segment relaxed; hatched bars, tests in which relaxation was followed by contraction (biphasic response); solid bars, tests in which contraction was the only response; open bars, tests in which there was no vagally-mediated tracheal response to hyperinflation. Note that contraction could still be evoked in one test when the vagus nerves were cooled to 1.5--0°C; it was abolished by vagotomy. (Roberts et al., 1988.)

Bronchomotor reflexes

25

fibres, and it is with the bronchoconstrictor responses evoked by chemical and irritant stimulation of these lower airway afferents that we are next concerned.

3.4. BRONCHOMOTOR EFFECTS OF PULMONARY AND BRONCHIAL CHEMOREFLEXES The bronchoconstrictor role of lower airway C fibres in irritant airway reflexes was first demonstrated convincingly in the laboratory by stimulating the afferent endings with two foreign chemicals that cause sensations of itch and burning pain when applied to blister-base preparations of human skin. Phenyldiguanide, injected into the right atrium of rabbits, stimulates pulmonary C fibres and evokes a reflex increase in airway resistance (Karczewski and Widdicombe, 1969b). Capsaicin, injected into the right atrium of dogs, stimulates pulmonary C fibres (Coleridge et al., 1965) and evokes reflex contraction of tracheal smooth muscle (Fig. 11A) (Coleridge et al., 1982a) and reflex narrowing of the intrathoracic airways, revealed by the tantalum bronchogram technique (Russell and Lai-Fook, 1979). Injected in small doses into the bronchial circulation of dogs, capsaicin stimulates bronchial C fibres and again evokes tracheal contraction (Fig. 11C) (Coleridge et al., 1982a). The bronchoconstrictor effects of stimulating the two groups of lower airway C fibres are accompanied by a vagally-mediated increase in tracheal submucosal gland secretion (Davis et al., 1982; Schultz et al., 1985). It should be emphasized that injecting capsaicin into the bloodstream does not invariably cause bronchoconstriction--for the bronchomotor effects vary with the site of injection. Thus capsaicin injected into the left atrium most frequently causes tracheal relaxation (Fig. liB), even though some of the injected chemical undoubtedly passes into the bronchial circulation and stimulates bronchial C fibres. The relaxation appears to be due to stimulation of muscle afferents (discussed in a later section) and can also be evoked by injecting capsaicin directly 150 r i & ~ ~

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26

H, M. COLERIDGEet al.

into the femoral artery (Fig. 11D). Injection into the femoral vein, i.e., downstream to the hindlimb, once again stimulates pulmonary C fibres and evokes tracheal contraction (Fig. liE). Just as the bronchoconstrictor response to hyperinflation can be demonstrated when conduction in myelinated fibres is blocked selectively by cooling the vagus nerves to 6-7°C (see above), so the excitatory bronchomotor effects produced by chemical stimulation of pulmonary and bronchial C fibres are still present when input from slowly and rapidly adapting receptors is blocked (Roberts et ai., 1981; Coleridge et al., 1982a). Small doses of capsaicin have been injected into the right atrium in human subjects ('~Vinning et al., 1986) but the chemical was poorly tolerated because of the burning substernal and rectal sensations it evoked. Although no respiratory effects were observed in three of the subjects, paroxysms of coughing were evoked in the fourth. Substernal sensations and coughing are also evoked in human subjects by intravenous or pulmonary arterial injection of lobeline (Bevan and Murray, 1963; Jain et al., 1972), which is known to stimulate pulmonary C fibres in animals. Since bronchoconstriction invariably accompanies the cough reflex, it seems reasonable to conclude that stimulation of pulmonary C fibres evokes bronchoconstriction in man also. The constellation of reflex effects evoked by stimulating pulmonary and bronchial C fibres are seen in their most striking form when a chemical solution is injected as a bolus into the pulmonary or bronchial artery. Since the pulmonary and bronchial chemoreflexes are triggered by the abrupt and simultaneous stimulation of a large population of C fibres, they might be considered experimental artifacts encountered only in the laboratory. On the contrary, there is now evidence to suggest that abrupt chemoreflex-like responses are not necessarily confined to the laboratory: for example, the pulmonary chemoreflex can be triggered by the inhalation of a single breath of cigarette smoke (see below). The role of pulmonary and bronchial C fibres in the bronchomotor effects evoked by chemical agents and pathological changes in the lung is discussed below.

3.5. BRONCHOCONSTRICTIONEVOKEDBY INHALEDIRRITANTS 3.5.1. Defense Reflex Stimulation of sensitive nerve endings in the nose, pharynx and larynx by the inhalation of atmospheric irritants or the aspiration of regurgitated gastric contents triggers powerful protective responses that serve to expel the irritants or to delay their entry into the lower respiratory tract (see above). The effectiveness of these responses is limited by the subject's need to take a breath. Once the first line of defense is breached and the irritants pass through the larynx, a second line of protective mechanisms is engaged by stimulation of afferent nerve endings in the lower airways and lungs. Effects are generally similar to those triggered by the upper airway afferents, and include cough, bronchoconstriction, increased airway secretion, breathing disturbances (gasps, apnoea and tachypnoea in various combinations), and bradycardia and hypotension (Widdicombe, 1977; Coleridge and Coleridge, 1981, 1985, 1986). In human subjects, these changes are accompanied by substernal sensations of tickling, rawness or burning, according to the intensity of the irritant stimulus (Guz, 1977). The afferent pathways for the reflex effects, and the irritant sensations that accompany them, are in the vagus nerves. (An afferent component has been shown to travel with sympathetic nerve branches to enter the spinal cord, but it probably plays no more than a minor part in the lower airway defense reflexes.) In experiments in human subjects, the irritants are usually inhaled through a mouthpiece, and upper airway receptors undoubtedly contribute to the reflex responses. In anaesthetized animals, the irritants are usually delivered directly through a tracbeostomy so that stimulation is confined to sensory mechanisms in the lower airways. Cough can rarely be elicited in anaesthetized dogs, although the cough reflex in cats does not seem to be so susceptible to inhibition by anaesthesia.

Bronchomotor

reflexes

27

According to the size of their particles, dusts and chemical aerosols will tend to precipitate out at more proximal airway bifurcations, a process that may be facilitated by rapid shallow breathing. Gases will penetrate to the more distal lung divisions. Consequently, the reflex responses to an inhaled irritant will depend not only on the afferent susceptibilities of the various endings but also on their anatomical distribution along the airways. Reflex responses to mechanical stimulation by foreign bodies and chemically inert dusts and particles delivered below the larynx appear to be triggered largely by stimulation of sensory nerve endings in the central (extrapulmonary) airways; whereas the cough and bronchoconstriction induced by chemical irritants seem to be dependent upon stimulation of sensory nerve endings located more distally in the respiratory tract (Widdicombe, 1954a). In this regard, electroneurographic studies indicate that rapidly adapting receptors in the larger airways are most susceptible to mechanical stimulation, and that C fibre endings in the intrapulmonary airways are most susceptible to chemical stimulation (see below). Certainly, there is now good evidence that both the atropine-sensitive bronchoconstriction and the pulmonary chemoreflex-like response evoked, respectively, by delivery of sulphur dioxide or cigarette smoke to the lower trachea are due largely to stimulation of C fibre endings in the lung itself (see below). Moreover, stimulation of bronchial C fibres almost certainly contributed to the cough, retrosternal discomfort and transient bronchoconstriction evoked in human subjects by inhalation of a single breath of capsaicin aerosol (Fuller et al., 1985), although it seems reasonable to assume that at least a part of the reflexogenic input arose from nerve endings in the upper respiratory tract. Nevertheless, the division of inhaled irritants into chemically inert dusts and particles that stimulate afferent endings mechanically, and aerosolized droplets and gases that stimulate the endings chemically may be misleading, because solid particles may damage the airway mucosa, causing release of chemical mediators that stimulate the endings. 3.5.2. Dusts and Particulate Irritants Widdicombe et al. (1962) demonstrated that cough and an atropine-sensitive bronchoconstriction were readily evoked in human subjects inhaling charcoal dust through the mouth, and in anaesthetized cats when charcoal dust was introduced into an isolated, innervated segment of the trachea. They also showed that charcoal dust stimulated rapidly adapting receptors in the isolated tracheal segment, and it seems likely that similar receptors in the central airways played a part in the reflex effects described in man. Rapidly adapting receptors in the extrapulmonary airways have been designated 'cough receptors' (Fillenz and Widdicombe, 1972), and are known to be stimulated strongly when powdered talc, starch, or carbon particles are blown into the lower airways (Fig. 12) (Widdicombe, 1954b; SeUick and Widdicombe, 1971). Cough and bronchoconstriction can also be evoked in anaesthetized cats when these proximal regions of the airways are stimulated mechanically with a soft nylon catheter (Widdicombe, 1954a). 3.5.3. Sulphur Dioxide Sulphur dioxide is a common air pollutant in industrial cities, and concentrations of 4-6 ppm cause cough, sensations of pharyngeal and substernal irritation, and an atropinesensitive bronchoconstriction in normal individuals (Nadel et al., 1965). Much of the sensory input for the irritant effects of sulphur dioxide probably arises from receptors in the larynx and pharynx (Boushey et al., 1974; Schultz et al., 1982). Nevertheless, the observation that inhalation of sulphur dioxide in human subjects causes rapid shallow breathing (Amdur et al., 1953) strongly suggests that lower airway C fibres are stimulated also. Sulphur dioxide causes an atropine-sensitive bronchoconstriction when administered to the lower airways of cats (Nadel et al., 1965). Sulphur dioxide (200-500 ppm) delivered to the lower trachea in dogs causes a marked increase in the smooth muscle tension of an upper tracheal segment, accompanied by increased airway submucosal gland secretion; both effects are abolished by cooling the vagus nerves to 0°C (Roberts et al., 1982). Roberts

28

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FIG. 12. Stimulation of a rapidly adapting receptor by inhalation of carbon dust in an anaesthetized, paralyzed and artificially ventilated rabbit; both vagus nerves were sectioned. Upper record, control discharge showing sparse and irregular pattern of firing; lower record, 20 sec after start of inhalation of carbon dust, showing maximal stimulation of the receptor. Traces from above down: arterial blood pressure (BP), transpulmonary pressure (Par), and action potentials recorded from the afferent vagal fibre. (Sellick and Widdicombe, 1971.)

et al. also found that afferent vagal C fibres in the lower airways were vigorously stimulated

by sulphur dioxide, the evoked discharge sometimes occurring in slow, irregular waves that were synchronous with irregular increases in smooth muscle tension. Slowly and rapidly adapting receptors were relatively insensitive to sulphur dioxide. It seems reasonable to conclude, therefore, that the bronchoconstrictor effects of sulphur dioxide delivered to the lower airways are mediated mainly by stimulation of C fibres, and that these afferents contribute to the atropine-sensitive bronchoconstrictor effects of sulphur dioxide in asthmatic subjects. 3.5.4. Ozone Ozone is also an acute airway irritant and causes an atropine-sensitive bronchoconstriction in man as well as in laboratory animals (Nadel, 1980). In anaesthetized dogs, delivery of 0.1 ppm ozone by bronchoscope into one lung caused a bronchoconstriction that was abolished by vagotomy (Gertner et al., 1984). If delivery was restricted to a single lung lobe, vagally-mediated bronchoconstriction remained unilateral, but if two or more lobes of the lung were exposed to ozone, a vagally-mediated bronchoconstriction also developed in the opposite lung. In the laboratory, a longer exposure to ozone is often employed to produce a late inflammatory response and bronchial hyperreactivity, i.e., an increased bronchoconstrictor response to agents such as histamine and prostaglandin F2~ (Holtzman et aL, 1983). Exposure of conscious dogs, breathing through a tracheostomy, to 0.65 ppm ozone for 2 hr caused rapid, shallow breathing that reached a maximum 1 to 3 hr after the exposure to ozone, and was abolished by cooling the vagus nerves to 0°C (Lee et al., 1979). Delivery of ozone to the trachea in anaesthetized dogs caused an increase in the bronchomotor response to inhaled histamine aerosol, the ozone-induced hyperreactivity being abolished by atropine and by vagal cooling (Lee et aL, 1977). It was concluded that vagal bronchopulmonary receptors were responsible for both the respiratory and the bronchomotor changes. In dogs, no evidence was found that intrapulmonary rapidly adapting receptors were stimulated by exposure to ozone (Sampson et aL, 1978). Since rapid shallow breathing and bronchoconstriction are associated with stimulation of bronchial (Coleridge et al., 1983) and pulmonary (Green et aL, 1984) C fibres, it seems reasonable to postulate that bronchopulmonary C fibres were involved in the responses to ozone.

Bronchomotor reflexes

29

3.5.5. Cigarette S m o k e Inhalation of cigarette smoke causes bronchoconstriction in human subjects (Nadel and Comroe, 1961; Sterling, 1967; Kagawa and Kerr, 1970). Since the bronchoconstrictor response was abolished by atropine (Sterling, 1967), and appeared to be unaltered by extraction of volatile substances from the smoke or by varying the nicotine content of the cigarettes (Nadel and Comroe, 1961), it was thought to be triggered reflexly by the irritant action of smoke particles on receptors in the tracheobronchial tree (Nadel and Comroe, 1961; Sterling, 1967). Support for this notion was provided by the observations that inhalation of chemically inert carbon particles stimulates rapidly adapting receptors and evokes a vagally-mediated bronchoconstriction (Widdieombe et al., 1962), and that inhalation of cigarette smoke also stimulates rapidly adapting receptors (Sellick and Widdicombe, 1971). Subsequent studies stressed the importance of the nicotine content of the cigarettes in evoking bronchoconstriction. Hartiala et al. (1984) observed an increase in both lung resistance and tracheal smooth muscle tension in anaesthetized dogs after delivery of 2 or 3 tidal volumes of cigarette smoke to the lower trachea (Fig. 13). They concluded that the increased lung resistance was due in part to a direct effect of smoke on airway smooth muscle, in part to stimulation of vagal afferents in the lung, and in part to excitatory effects of blood-borne materials on the bronchomotor centre. Hartiala et al. (1985) compared the effects of injecting nicotine into the vertebral circulation with those of injecting blood collected during cigarette smoking, and examined the responses after nicotine receptors in the central nervous system and airway parasympathetic ganglia had been blocked. They concluded that nicotine is the main cause of cigarette smoke-induced bronchoconstriction, producing central respiratory stimulation and having a direct excitatory effect on airway parasympathetic ganglia. Although Hartiala et al. played down the importance of a vagal reflex mechanism, the fact that bronchoconstriction was accompanied in their experiments by vagally-dependent bradycardia, systemic hypotension and an initial phrenic 'apnoea' (Fig. 13) suggests that a lower airway reflex probably played an important part.

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FIG. 13. Effects on tracheal tension, blood pressure (BP), expiratory CO2, phrenic motor nerve output and lower airway pressure of inhaling two tidal volumes of cigarette smoke into the lower trachea in an anaesthetized, artificially ventilated dog; vagus nerves intact. The bradycardia, systemic hypotension and initial phrenic 'apnoea' were abolished by vagotomy; see text for effect of vagotomy on bronchomotor responses. (Hartiala et aL, 1984.)

30

H.M. COLERIDGEet al.

Lee and his colleagues (for references see Lee et al., 1987) confirmed that nicotine is largely responsible for the effects of cigarette smoke, and they re-emphasized the importance of lung reflexes in these effects. They found in both conscious and anaethetized dogs that inhalation of a single breath of cigarette smoke immediately evoked apnoea (followed by hyperpnoea), bradycardia and systemic hypotension; they did not examine bronchomotor effects. They attributed the immediate triad of effects to the operation of the pulmonary chemoreflex; the secondary hyperpnoea was thought to be due in part to stimulation of carotid chemoreceptors by nicotine. Reflex responses could still be evoked by inhaling smoke when myelinated vagal fibres were blocked by cooling to 8~°C, but were abolished when nonmyelinated fibres were blocked by cooling to 0°C (Lee et al., 1987). It remains to be seen whether a single breath of cigarette smoke evokes a vagally-mediated reflex bronchoconstriction, although it might be expected to do so since bronchoconstriction is an integral part of the pulmonary chemoreflex. Electroneurographic studies suggest that nonmyelinated lung afferents are indeed responsible for the vagally-mediated effects evoked by a single breath of cigarette smoke (Lee et al., 1988). In experiments on anaesthetized, artificially ventilated dogs, pulmonary C fibres were stimulated within 1-2 sec of the delivery of 200cc smoke generated by high-nicotine cigarettes, activity increasing twentyfold on average, the evoked discharge usually lasting 3-5 sec; other lung afferents were virtually unaffected. Smoke generated by low-nicotine cigarettes caused only a slight increase in pulmonary C fibre firing. Prolonged delivery (10-120 see) of lower concentrations of cigarette smoke caused delayed but sustained stimulation of some pulmonary and bronchial C fibres and rapidly adapting receptors; slowly adapting stretch receptors were not significantly affected. Since pulmonary C fibres are stimulated by nicotine injected into the blood stream (Paintal, 1955), it seems likely that they are stimulated by nicotine delivered in the cigarette smoke. 3.6. STIMULATIONBY AUTOCOIDS

The influence of lower airway afferents on bronchomotor mechanisms can also be demonstrated by administering certain naturally occurring autocoids, to which bronchial C fibres are particularly susceptible. Thus, bronchial C fibres are stimulated by bradykinin, histamine and serotonin, chemicals that have little or no effect on pulmonary C fibres. Both types of C fibre are stimulated by the prostaglandins, but bronchial C fibres are sensitive to much smaller doses. We deal first with bradykinin and prostaglandin; the effects of histamine on lower airway afferents and bronchomotor mechanisms are discussed in a later section. 3.6.1. Bradykinin Bradykinin, which has little or no direct effect on bronchial smooth muscle in dogs (Waaler, 1961; Roberts et aL, 1981) or man (Newball et aL, 1975), has been a particularly useful chemical tool for investigating the bronchomotor reflex role of bronchial C fibres because in low doses it vigorously stimulates bronchial C fibres (Fig. 14) but has little or no effect on pulmonary C fibres or slowly and rapidly adapting receptors (Kaufman et al., 1980a; Coleridge et al., 1983). Bradykinin is produced in the lower airways, but is rapidly broken down in the pulmonary circulation (Levine et al., 1973) and has little or no respiratory effect when injected intravenously in small doses (Coleridge et al., 1983). When low concentrations of bradykinin are delivered as an aerosol to the lower airways of dogs, or when small doses are injected or infused into the left atrium or bronchial artery, bronchial C fibres are stimulated selectively and evoke contraction of tracheal smooth muscle (Fig. 15) and an increase in submucosal gland secretion (Roberts et al., 1981; Davis et al., 1982; Coleridge et aL, 1983). A few rapidly adapting receptors are activated when larger doses of bradykinin are given repeatedly, but the evoked discharge has an obvious respiratory modulation and is reduced or abolished by hyperinflation; thus it appears to be due to the reflex changes in lung compliance rather than to direct chemical stimulation

Bronchomotor reflexes

31

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FIG. 14. Effect of bradykinin on two airway endings in the left lung in an anaesthetized, artificially-ventilated dog with open chest. Impulses recorded from strand of left vagus nerve: one fibre (large spikes; conduction velocity, 21.9 m sec-[) arose from a rapidly adapting receptor, the other (small spikes; conduction velocity, 1.1msec -[) from a bronchial C fibre ending. A, bradykinin 1/~gkg-' was injected into the left atrium at the signal (note decrease in arterial blood pressure). Records A and B are continuous. AP, action potentials; ABP, arterial blood pressure; PT, tracheal pressure. (Kaufman et al., 1980b.) of the receptors themselves (Coleridge et al., 1983). Strong contractions of tracheal smooth muscle can be evoked when the dogs breathe spontaneously, even though tachypnoea is a prominent feature of the total reflex response. Hence any increase in the inhibitory input from pulmonary stretch receptors that results from the tachypnoea is not sufficient to counteract the excitatory effect of the bradykinin-evoked increase in bronchial C fibre input (Coleridge et al., 1983). Cutting or cooling the vagus nerves or administering atropine abolishes all bronchoconstrictor effects of bradykinin. The reflex nature of the bronchom o t o r response has been demonstrated in a striking fashion in anaesthetized dogs by recording smooth muscle tension in an upper tracheal segment and administering bradykinin aerosol to the lower airways while the lower cervical vagus nerves were cooled to 0°C (Coleridge et al., 1983). Delivery of the bradykinin aerosol (0.1% solution) for as long as 7 min had no effect on tracheal tension, provided vagal conduction was blocked. But as soon as afferent input from the lower airways to the medullary centres was restored by rewarming the vagus nerves, there was an immediate and dramatic increase in tracheal tension and a profound decrease in heart rate. Arterial blood pressure was virtually

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F]O. 15. Stimulation of airway C fibres by administration of bradykinin aerosol (0.01% solution) to the lower trachea in an anaesthetized dog evokes reflexcontraction of an upper tracheal segment (aerosol delivery signalled by the black bar). The chest was open and the lungs were ventilated by positive pressure. The trachial response was subsequently abolished by cutting the vagus nerves. PCO2, tidal CO2; ABP, arterial blood pressure; HR, heart rate; Tr tension, tracheal tension in grams above a baseline set at 75 g; PT, pressure in the lower trachea. (Coleridge and Coleridge, 1984.) 42/I--C

32

H.M. Commie et al.

unchanged throughout, indicating that bradykinin, which is a powerful vasodilator, had not passed into the systemic circulation. Bradykinin has also been used in studies in human subjects, and when administered as an aerosol evokes an atropine-sensitive bronchoconstriction accompanied by cough and irritant substernal sensations, both in asthmatic subjects (Simonsson et aL, 1973; Fuller et al., 1987a) and in some normal subjects (Simonsson et al., 1973). In these studies, in which aerosol was inhaled through a mouthpiece, sensory endings in the upper airways may well have contributed to the reflex response. However, bronchoconstriction, cough and increased airway secretion have been reported in normal subjects given captopril, an angiotensin-converting enzyme inhibitor that prevents the rapid degradation of bradykinin in the pulmonary circulation (Semple and Herd, 1986; Popa, 1987). Bronchoconstriction in these subjects was unlikely to have been initiated by stimulation of afferents in the upper airways, and was probably a consequence of stimulation of C fibres in the intrapulmonary airways. 3.6.2. Prostaglandins Prostaglandins have powerful effects on airway calibre, some prostaglandins causing bronchoconstriction, others bronchodilatation. The observation that prostaglandins of the E series relax airway smooth muscle (Spannhake et al., 1981) and that PGI2 reverses the bronchospasm induced by histamine and other autocoids (Wasserman et al., 1980) raises the possibility that these prostaglandins might have a place in the treatment of human asthma. However, cough, irritation of the upper respiratory tract, retrosternal soreness, and a paradoxical bronchoconstriction have been reported in both normal and asthmatic subjects after administration of aerosols of PGI2 and PGE~, asthmatic subjects being particularly prone to these effects (see Roberts et aL, 1985b). Since the elicitation of cough and irritant sensations indicates that sensory nerves were stimulated, it is conceivable that the bronchoconstriction also had a neural component. Both lower airway C fibres and rapidly adapting receptors are stimulated by injection of PGF2, into the blood stream in dogs (Coleridge et al., 1976). The bronchodilator prostaglandins PGI~ and PGE~ stimulate both pulmonary and bronchial C fibres, effects on pulmonary C fibres being difficult to demonstrate unless very large doses are injected; effects on rapidly adapting receptors are much less pronounced, even when large doses are injected (Coleridge et al., 1976; Roberts et aL, 1985b). Inhalation of aerosols of PGI2 and PGE~ is a powerful stimulant of C fibre endings, both those within the lung itself and those located in the trachea and large extrapulmonary bronchi. Selective stimulation of bronchial C fibres by injection of PGI2 or PGE2 into a bronchial artery, and a more widespread engagement of lower respiratory tract C fibres by aerosols of PGI2 and PGE2 delivered to the lower trachea, triggered reflex contraction of smooth muscle in an upper tracheal segment (Roberts et al., 1985b). Tracheal contraction could still be evoked when myelinated fibres in the lower cervical vagus nerves were blocked by cooling to 5-7°C but was abolished by cooling to 0°C. That the reflex responses were not due to passage of the administered prostaglandin into the general circulation was demonstrated by the fact that vagally-mediated bronchoconstriction could be evoked when PGI2 was injected into the pulmonary artery of dogs whose pulmonary and systemic circulations were independently pump perfused (Roberts et aL, 1985b). On the basis of these afferent and reflex studies in dogs, Roberts et aL (1985b) postulated that stimulation of afferent vagal C fibres in the respiratory tract is responsible for the paradoxical bronchoconstriction and wheezing that is seen occasionally in human subjects, and particularly in asthmatic patients, when bronchodilator prostaglandins are given as an aerosol or infused into a peripheral vein (Smith and Cuthbert, 1976). From the latter observations one might conclude that in susceptible subjects the threshold concentration of bronchodilator prostaglandin required to stimulate airway C fibres, and hence to evoke reflex contraction of airway smooth muscle, is lower than the concentration required for a direct inhibitory effect on the smooth muscle itself.

Bronchomotor reflexes

33

3.7. BRONCHOCONSTRICTIONAND THE AXON REFLEX

There is increasing interest in the possibility that axon reflexes analogous to the weal and flare reaction in the skin and to the local (intrinsic) reflexes in the wall of the alimentary tract also occur in the airways, and that peptides released from the terminals of bronchial C fibres cause bronchoconstriction, as well as 'neurogenic inflammation' (Lundberg and Saria, 1987). Lundberg and Saria (1982) demonstrated that stimulation of the cut, peripheral end of the vagus nerve in guinea pigs induced a pronounced increase in tracheobronchial resistance to airflow that was unaffected by atropine. This effect could not be obtained, however, if the afferent C fibre system had been destroyed by systemic injection of neurotoxic doses of capsaicin soon after birth. Lundberg and Saria (1983) also demonstrated that in rats exposed to airway irritants such as cigarette smoke or ether, the integrity of the capsaicin sensitive afferent C fibre innervation was essential, both for the avoidance behaviour of the rats, and for the inflammatory response in the tracheal mucosa. The possibility that bronchoconstriction is a component of the airway axon reflex is now being vigorously pursued (Barnes, 1986a,b). An axon reflex depends only on the integrity of the peripheral axon, and does not require participation of a central reflex arc. Although there is, strictly speaking, no clear experimental evidence for what constitutes an axon reflex, it was initially postulated that some of the peripheral branches of afferent C fibres were in fact efferent in function, their terminals containing vasodilator autocoids. Thus a wave of depolarization passing centrally in response to stimulation of the afferent terminals would cause antidromic depolarization of the efferent ones, with local release of a vasodilator peptide, identified as Substance P (Lundberg and Saria, 1987). Judging from the rich distribution of peptide-containing C fibre branches in the airway mucosa of mammals, including man (Lundberg et al., 1984), it now seems equally likely that Substance P and other neural peptides are present in the afferent terminals themselves, reaching the terminals by axonal transport from the cell bodies in the nodose ganglion (Brimijoin et al., 1980), and that peptides are released when the sensory nerve terminals are depolarized by a stimulus. Hence, although the term 'axon reflex' continues to be used, it may in fact be something of a misnomer. The afferent C fibres involved in axon reflex phenomena in both visceral and somatic sensory systems are associated with reflexes of a nociceptive nature, particularly those evoked by irritant chemicals, including capsaicin in low doses. Both visceral and somatic C fibre components are destroyed in new born animals by subcutaneous injection of large doses of capsaicin, 103-105 times those required to evoke profound reflex effects (Jancso et al., 1977), or, in the case of vagal C fibres, by applying high concentrations of capsaicin directly to the vagus nerve (Jancso and Such, 1983). The possible bronchoconstrictor effect of neural peptides has been examined in several mammalian species. The peptides identified so far appear to have little effect on airway smooth muscle in rats; however, they are potent bronchoconstrictors in guinea pigs, and much of the work on airway axon reflexes has been carried out in this species. For example, the bronchoconstriction evoked in guinea pigs by intravenous injection of small doses of capsaicin appears to be due to the release of bronchoactive peptides at C fibre endings in the lower airways and lungs, without the participation of a vagal muscarinic reflex arc (Biggs and Goel, 1985). Thus, the increase in airflow resistance evoked by small doses of capsaicin was unaffected by vagotomy, by atropine, by the adrenergic antagonist bethanidine, by the ganglionic blocking agent mecamylamine, by the histamine antagonist mepyramine, or by sodium cromogiycate. However, the bronchoconstriction was significantly reduced, although not abolished, by a Substance P antagonist and by morphine, the latter being thought to act by reducing the release of peptides from C fibre endings. A recent finding of particular interest is that destruction of the sensory C fibre system in guinea pigs by systemic administration of capsaicin abolishes the bronchial hyperreactivity induced by exposure to toluene diisocyanate, a polyurethane precursor and industrial air pollutant that causes bronchial hyperreactivity in man (Thompson et al., 1987).

34

H.M. COLEmDGEet al.

The hypothesis that bronchial hyperreactivity in animals and bronchial hyperreactivity and asthma in humans involves an 'axon reflex' mechanism is an attractive one (Barnes, 1986a). The hypothesis is supported by the above observation that, at least in the guinea pig, the integrity of the C fibre system is essential for the induction of airway hyperreactivity. It is also supported by the finding that Substance P and calcitonin gene-related peptide, which is also present in C fibre terminals (Lundberg and Saria, 1987), have excitatory effects on human bronchial smooth muscle in vitro, calcitonin gene-related peptide being particularly effective (Palmer et al., 1987). So far, however, effects of the axon reflex type have not been demonstrated in the lower airways of animals other than rodents, nor have neural peptides been shown to cause bronchoconstriction in human subjects (Lundberg and Saria, 1987). In normal and asthmatic subjects, inhalation or intravenous infusion of Substance P in sufficient concentration to produce marked systemic vasodilatation was found to have little or no effect on airway function (Fuller et al., 1987b). Indeed a small but significant bronchodilatation was observed at high infusion rates, and a similar bronchodilatation in response to calcitonin gene-related peptide was briefly reported by the same investigators. Nevertheless, this area of investigation continues to be of great interest, and studies of potential axon reflex phenomena in the lower airways may prove to be a turning point in our understanding of the abnormal behaviour of bronchial smooth muscle in airway disease. 3.8. VAGALAFFERENTSAND BRONCHOCONSTRICTIONOF LUNG DISEASE Reviews by Boushey et al. (1980) and more recently by Barnes (1986b) provide excellent and comprehensive accounts of the significance of neural factors in the increased airflow resistance in lung disease. In view of the demonstrated sensitivity of the various pulmonary receptors to a wide range of mechanical and chemical stimuli, it is inevitable that the pattern of vagal afferent input will be greatly altered by lower airway disease. Changes in afferent input have been examined in several animal models of lung disease including histamine- and antigen-induced bronchoconstriction to simulate human asthma, acute pulmonary inflammation and bronchitis, pulmonary microembolism, and pulmonary congestion and interstitial oedema (see below). In most earlier studies, emphasis was placed on stimulation of rapidly adapting (irritant) receptors as the origin of the reflex bronchoconstriction of lung disease (Nadel, 1980), but more recent evidence suggests that stimulation of afferent vagal C fibres is also an important factor. There is a growing appreciation of the potential importance of the afferent C fibre innervation of the airways, not only because of its sensitivity to autocoids that are known to be released in lung disease but also because some of these C fibres release peptides at their terminals, which may have both vasoactive and bronchoactive effects (see above). The possible role of changes in slowly adapting stretch receptor discharge has generally received less attention, in spite of the demonstrated importance of these receptors in setting the baseline tone of airway smooth muscle. The atropine-sensitive increase in airflow resistance reported in several diseases, taken together with other reflex manifestations such as cough and changes in breathing pattern, is a clear indication of increased sensory input from the diseased airways. In some cases, however, the contribution of reflexes to the bronchoconstriction of lower airway disease may be difficult to demonstrate because bronchoconstrictor autocoids released by the damaged lung may produce powerful direct effects on airway smooth muscle that overshadow any neurally-mediated effects; moreover, the disease process itself may cause a narrowing of the airways that obscures any reflex component. Nevertheless, even in the absence of clear evidence of reflex bronchoconstriction, the presence of cough and changes in breathing provide incontrovertible evidence that sensory endings are stimulated. 3.8.1. Asthma, Histamine and Bronchial Hyperreactivity In discussing the contribution of nervous pathways to the bronchoconstriction of lower airway diseases, we begin with asthma, a disease characterized by an increased

Bronchomotorreflexes

35

responsiveness of the trachea and bronchi to various stimuli and manifested by a widespread narrowing of the airways that changes in severity either spontaneously or as a result of therapy (American Thoracic Society, 1962). The existence of a neural component in the bronchial hyperreactivity of asthma has long been suspected. Barnes (1986b) quotes a passage from a treatise on asthma written by Salter more than a hundred years ago, in which the author refers to the 'perverse sensibility' of the nerve supply to the asthmatic airways as being the primary causative factor. That the vagal innervation of the airways has a causative role in the immediate pathogenesis of asthma is indicated by the therapeutic effectiveness of muscarinic blocking agents (for references see Gross and Skorodin, 1984a). Thus in asthmatic children atropine decreases air flow resistance to normal levels (Cropp, 1975), and aerosols of the atropine congener ipratropium bromide are effective in the treatment of acute asthmatic attacks (Ward et al., 1981). Taken alone, however, such evidence does not necessarily establish the 'perverse sensibility' of sensory receptors as the source of the increased muscarinic bronchoconstrictor tone in asthma. Central factors may play an important role; an abnormality of the motor pathway is equally possible (for bibliography see Boushey et al., 1980). Thus emotional stress may precipitate asthmatic attacks in some individuals, and lower airway stimuli that have either no effect or cause reflex bronchodilatation in normal individuals may cause reflex bronchoconstriction in asthmatics (Simonsson et al., 1967; Nadel, 1980). The dysfunction may involve the motor pathway by potentiating vagal ganglionic transmission. There is evidence in dogs with allergic asthma that the airway smooth muscle itself may be hyperresponsive to muscarinic transmitters (Antonissen et al., 1979), although this has not been confirmed in vitro studies of bronchial smooth muscle from patients with hyperresponsive airways (Vincenc et al., 1983). Finally, abnormalities in the production and degradation of chemical mediators that could act at sites along the reflex pathway, either at sensory or motor terminals, vagal ganglia, or smooth muscle membrane, might have a causative role (Boushey et al., 1980; Barnes, 1986a,b). Regardless of the locus of hyperreactivity along the reflex arc, however, the fact remains that afferent nerve endings are the natural starting point for all reflex activity. In most asthmatics baseline airway tone is within normal limits, but the airways are hyperreactive in the sense that various bronchomotor challenges known to increase the activity of afferent vagal fibres supplying the lower airways (e.g., histamine, acetylcholine, methacholine) evoke a vagal muscarinic bronchoconstriction that is exaggerated compared with that in normal subjects (Simonsson et al., 1967; Boushey et al., 1980; Nadel, 1980). Since asthmatic individuals are known to be highly sensitive to the bronchoconstrictor and irritant properties of histamine, administration of histamine to the lower airways is often regarded as providing a useful experimental model of human asthma. If one subscribes to this view, electroneurographic studies of the effect of histamine on pulmonary afferent input in animals might be expected to provide some insight into the causative mechanisms of human asthma. Administration of histamine by intravenous injection or aerosol greatly increases the impulse frequency of rapidly adapting receptors in rabbits, cats and dogs, and, after a short period of irregular activity, most receptors begin to fire in regular bursts at the peak of each inflation (Mills et al., 1969; Sellick and Widdicombe, 1971; Armstrong and Luck, 1974; Sampson and Vidruk, 1975; Coleridge et al., 1978). Whether the histamine-evoked increase in rapidly adapting receptor activity is due to direct chemical stimulation of the endings or is merely secondary to changes in lung mechanics induced in part by chemical stimulation of bronchial C fibres, it will undoubtedly contribute to the final reflex effect. Slowly adapting stretch receptors are sensitized by histamine, resulting in an increase in the impulse activity evoked by inflation (Widdicombe, 1961b). Such an increase in stretch receptor firing during inflation might be expected to shorten the inspiratory period and promote tachypnoea, but not to promote bronchoconstriction. That C fibres contribute to the reflex component of the bronchomotor response to histamine has been demonstrated in rabbits and dogs in which myelinated afferent fibres were blocked selectively by vagal cooling (Jammes et al., 1985). Bronchial C fibres are

36

H.M. COLEPJDGE et al.

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vigorously stimulated by histamine injected into the blood stream or inhaled as an aerosol (Fig. 16) (Coleridge and Coleridge, 1977b; Coleridge et al., 1978). The evoked discharge is irregular, it has no relation to the ventilatory changes in airway pressure, and it is unaffected by turning off the ventilator; hence it is not secondary to changes in lung mechanics. Afferent studies indicate that pulmonary C fibres are generally insensitive to histamine (for references see Coleridge and Coleridge, 1984). The relative contributions of bronchial C fibres and rapidly adapting receptors to the reflex component of histamineinduced bronchoconstriction remain to be determined. Even so, the fact that the bronchoconstriction evoked by small doses of histamine is greatly reduced by vagal cooling and often abolished by vagotomy, indicates that the reflex component is important (Karczewski and Widdicombe, 1969b; Jammes et al., 1985). A role for bronchial C fibres in the bronchoconstriction of asthma can also be postulated on the basis of the sensitivity of these nonmyelinated afferents to bradykinin, another autocoid known to be released in asthma (Garcia Leme, 1978). Bradykinin has been found in concentrations of 1-12 ng ml-1 in forearm mixed venous blood in patients with severe asthma (Abe et aL, 1967): hence concentrations at the site of release in the lung may be much higher. Injection of bradykinin in doses of only 0.002-1.5/ag into the bronchial artery in dogs evokes a vagally-mediated increase in airway smooth muscle tone (Roberts et al., 1981). It seems likely that these small amounts of injected bradykinin produce concentrations in the vicinity of the C fibre endings in the airways within the range of those reached during endogenous bradykinin production in asthma. In some respects, bradykinin would seem to be a more useful experimental tool than histamine for studying animal models of human asthma. Unlike histamine, bradykinin administered in small doses has no direct effect on airway smooth muscle in dogs (Waaler, 1961; Roberts et aL, 1981) or humans (Newball et aL, 1975); moreover, it stimulates bronchial C fibres selectively (Kaufman et al., 1980b; Coleridge et al., 1983).

Bronchomotor reflexes

37

Because asthma is often regarded as an immunologic disease, an alternative model of human asthma has been used in which bronchoeonstriction was produced by administering allergen to sensitized rabbits (Karczewski and Widdicombe, 1969c) and dogs (Gold et al., 1972). In rabbits, the reflex increase in total lung resistance was diminished by cooling the vagus nerves to 8°C, and further reduced by vagotomy (Karczewski and Widdicombe, 1969c), suggesting that both rapidly adapting receptors and C fibres contributed to the reflex bronchoconstriction. In dogs, aerosols of antigen administered to one lung caused a vagally mediated increase in total lung resistance in the opposite lung (Gold et al., 1972). From the effects produced by cooling the vagus nerve supplying the lung receiving the aerosol, the investigators concluded that the reflex bronchoconstriction was due to stimulation of rapidly adapting receptors. Electrophysiological studies have shown that rapidly adapting receptors are stimulated by anaphylaxis induced in rabbits previously sensitized to egg albumen (Mills et al., 1969). The afferent responses of lung C fibres to antigen have not, so far, been examined in single fibre studies, although the general direction of such responses might be inferred from the sensitivity of bronchial C fibres to mediators of anaphylaxis such as bradykinin and histamine (Kaufman et al., 1980b; Coleridge and Coleridge, 1977b). These observations are compatible with the hypothesis that in human asthma the increased firing of rapidly adapting receptors and bronchial C fibres provides a positive feedback to the vagal bronchomotor centre that culminates in the asthmatic paroxysms. To put a complex matter at its simplest, one possible sequence of events is that as the result of a chemical challenge or the release of lung autocoids, bronchial C fibres are stimulated, their augmented input evoking a vagally-mediated bronchoconstriction, which in turn stimulates rapidly adapting receptors, adding further to the bronchomotor response. Although pulmonary C fibres are capable of evoking vagally-mediated bronchoconstriction, their contribution to the reflex component of human asthma is probably less important, pulmonary C fibres being less susceptible than bronchial C fibres to stimulation by naturally released substances such as histamine, bradykinin, prostaglandins and serotonin. The asthmatic attack has all the hallmarks of a response to a sensory stimulus originating in the airways, because the bronchoconstriction is accompanied by other vagal reflex disturbances such as coughing, that do not involve muscarinic motor pathways. It is also accompanied by irritant substernal sensations that can only result from increased activity in sensory nerves. Indeed one report indicates that the acute asthmatic attack and all its reflex manifestations can be abolished by local anaesthesia of the airways (Petit and Delhez, 1970). Additional, though circumstantial, evidence for a neural component is that the basic defect in asthma leads not only to excessive contraction of airway smooth muscle but also to excessive secretion in the bronchial tree. It seems likely that the lower airway afferents responsible for the reflex component of the bronchoconstriction also contribute to the bronchosecretion. Thus, selective stimulation of bronchial C fibres by injection of bradykinin into the bronchial artery evokes both bronchoconstriction and an immediate vagally-mediated increase in secretion by tracheal submucosal glands (Davis et al., 1982), and recent evidence indicates that rapidly adapting receptors also are capable of triggering a reflex increase in secretion (Schultz et al., 1987a). 3.8.2. Sodium Cromoglycate The drug sodium cromoglycate (eromolyn) is used frequently in the prophylactic treatment of bronchial asthma. Of several suggested mechanisms of action, one is that cromolyn acts on reflex mechanisms influencing airway calibre, and so interferes with the reflex component of certain bronehoconstrictor responses. Dixon et al. (1980) suggested that cromolyn acts in part by reducing the excitability of afferent C fibres that cause reflex bronchoconstriction. Their suggestion arose from the observation that intravenous injection of cromolyn reduced the stimulation of pulmonary C fibres by capsaicin in dogs. Attempting to confirm these observations, Coleridge et al. (1982b) found that eromolyn

38

H.M. COLEPaDOEet al.

reduced neither the stimulation of pulmonary C fibres by capsaicin nor the reflex effects of such stimulation (bradycardia, hypotension, bronchoconstriction and changes in breathing). In addition, cromolyn had no effect on the stimulation of lower airway C fibres by inhaled sulphur dioxide, bradykinin, prostaglandins or histamine, nor had it any effect on the reflex changes evoked by these chemicals. Moreover, cromolyn itself evoked reflex bradycardia and hypotension which appeared to be due to stimulation of chemosensitive C fibre endings accessible from the coronary circulation. A recent study by Fuller et al. (1985) indicated that the bronchoconstriction evoked in human subjects by inhalation of capsaicin aerosol was not affected significantly by inhalation of cromolyn, although the capsaicin-induced bronchoconstfiction was undoubtedly dependent on a vagal cholinergic reflex because it was reduced by inhalation of ipratropium bromide aerosol. Taken together, the afferent studies in animals (Coleridge et al., 1982b) and the reflex studies in humans (Fuller et al., 1985) suggest that the primary action of cromolyn in preventing asthma is unlikely to be due to inhibition of capsaicin-sensitive C fibres, but they do not necessarily exclude all possibility of a neural action. Cromolyn has no effect on the dose-dependent coughing evoked in normal and asthmatic subjects by inhalation of capsaicin aerosol; the coughing appears to be due to stimulation of capsaicin-sensitive laryngeal afferents since it is abolished by local anaesthesia of the vocal chords (Collier and Fuller, 1984). Cromolyn abolishes the bronchoconstriction evoked in asthmatic subjects by inhalation of sulphur dioxide, but the effect is possibly due to inhibition of the release of mediators from mast cells (Sheppard et aL, 1981). 3.8.3. Inflammation Atropine and its congeners have been found to relieve the increase in airflow resistance in chronic inflammatory conditions of the lower airways in human diseases such as bronchitis, emphysema and cystic fibrosis (Douglas et al., 1979a; Gross and Skorodin, 1984b; Larsen et al., 1979). The relief had a rapid onset, and was therefore thought to be due to reversible effects on bronchial smooth muscle, rather than to a decrease in accumulated secretions. Atropine was also found to abolish the bronchial hyperreactivity to histamine observed in human subjects with acute upper respiratory tract infections (Empey et al., 1976). In general, the atropine-sensitive changes in airway resistance were attributed to stimulation of rapidly adapting receptors. Studies in animal models have been confined to the effects of acute airway inflammation and to the possible involvement of rapidly adapting receptors. In dogs, the acute bronchitis and bronchiolitis of kennel cough (Dixon et al., 1979b) and the inflammatory changes produced by exposure to ozone (Holtzman et al., 1983) are associated with bronchial hyperreactivity. In both conditions, hyperreactivity is mediated by the vagus nerves. In dogs with kennel cough, an increased bronchoconstrictor response to histamine was associated with an increase in the histamine sensitivity of rapidly adapting receptors; hence the bronchial hyperreactivity was ascribed to sensitization of these receptors (Dixon et al., 1979b). In dogs exposed to ozone, however, there was no evidence that rapidly adapting receptors were sensitized to bronchoconstrictor agents; indeed, the histamine-sensitivity of these receptors after ozone exposures was less than in control dogs (Sampson et aL, 1978). The possibility that afferent C fibres are implicated in the vagally°mediated hyperreactivity evoked by kennel cough or exposure to ozone has not been investigated. However, evidence of involvement of lower respiratory tract C fibres was obtained in one of the earliest electroneurographic studies employing an animal model of lung disease (Frankstein and Sergeeva, 1966). Acute pulmonary inflammation was induced in cats by instilling small volumes of hot water into the fight lung; effects on myelinated and nonmyelinated fibres in the right vagus were estimated from changes in the evoked compound action potential. The tonic input in afferent C fibres was increased by inflammation, whereas the phasic input in myelinated afferent fibres was reduced. Results of a number of reflex studies also indicate that afferent C fibres are stimulated by inflammation of the lung lobes (see Coleridge and Coleridge, 1984).

B r o n c h o m o t o r reflexes

39

3.8.4. E m b o l i s m The afferent and reflex effects of pulmonary embolism have interested respiratory physiologists for many years. Indeed Paintal's classical studies on the afferent C fibre innervation of the lungs began with a search for the pulmonary afferents responsible for the vagaUy-dependent tachypnoea produced by embolism and oedema (Paintal, 1955). Paintal pointed out that earlier investigators had found little to confirm the hypothesis that the tachypnea was caused by sensitization of slowly adapting stretch receptors. Instead he attributed the tachypnea to the small-spike afferent activity recorded from multifibre vagal bundles in cats when potato starch was injected into the right atrium. These small fibre afferents were subsequently identified as J receptors (Paintal, 1969) or pulmonary C fibres (Coleridge et al., 1965; Coleridge and Coleridge, 1977b). Rapidly adapting receptors in rabbits were also found to be stimulated by pulmonary embolism (Mills et al., 1969). The changes in pulmonary afferent vagal input induced by embolization were examined in detail by Armstrong et al. (1976) in experiments in which several small aliquots of potato starch or plastic spheres were injected successively into the inferior vena cava in open chest, artificially ventilated cats. They found that both pulmonary C fibres (Fig. 17) and rapidly adapting receptors were often stimulated vigorously by pulmonary embolization, thus confirming the observations of Paintal (1955) and Mills et al. (1969). They also found that slowly adapting stretch receptors were sensitized rather than stimulated by embolization, peak discharge frequencies increasing on average by about 10%, and firing becoming concentrated in the inflation phase. From what is known of the reflex properties of slowly adapting receptors, this change in firing pattern might be expected to promote more rapid, shallow breathing, but would be unlikely to contribute to reflex bronchoconstriction. Nevertheless, since pulmonary C fibres and rapidly adapting receptors are I

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FIG. 17. Stimulationof a type J receptor(pulmonaryC fibre)by emboli.Afferentimpulsesrecorded from the right vagusnervein an anaesthetizedcat; receptor locatedin right upper lobe; chest open, lungs ventilatedartificially.From above down in each panel; action potentials; ITP, intratrachcal pressure; RVP, right ventricularpressure; BP, systemicarterial blood pressure. A, the first of five 0.1 g aliquots of 25-50 #g potato starch was injectedinto the inferiorvena cava at the horizontal signalbar (aliquotsinjectedat 5 rain intervals);intervalof 83 sec betweenA and B. The fifthaliquot was injected at the signal mark in C; interval of 150 sec between C and D. D, phenyldiguanide (150 #g) was injected into the vena cava at the signal. (Armstronget ai., 1976.)

40

H . M . COLEPd~.E et al.

stimulated by embolization, and since both are known to evoke vagally-mediated contraction of airway smooth muscle, one might expect embolization to evoke reflex bronchoconstriction. In the event, experimental evidence that pulmonary embolism evokes a vagally-mediated bronchoconstriction has been hard to find. Pulmonary embolism in animals induces complex changes in lung mechanics, including an often transient increase in airflow resistance and a marked and prolonged decrease in pulmonary compliance (Halmagyi and Colebatch, 1961). A vagally-mediated constriction of the central airways, visible on serial bronchography has been observed in dogs with lungs embolized by barium sulphate (Jesser and de Takats, 1942). Some investigators have obtained evidence for the contribution of a vagal reflex component in the embolism-induced increase in airflow resistance (Boyer and Curry, 1944), but others have not (Halmagyi and Colebatch, 1961; CahiU et al., 1961; Nadel et al., 1964). Nevertheless, there is general agreement that the bradycardia, apnoea and subsequent rapid shallow breathing induced by embolism are of vagal reflex origin (for references see Coleridge and Coleridge, 1984). These reflex effects are characteristic of pulmonary C fibre stimulation. Thus although embolization certainly stimulates pulmonary C fibres and rapidly adapting receptors, both of which undoubtedly provide an excitatory drive to the vagal bronchomotor centre, most investigators have failed to identify a vagally-mediated reflex bronchomotor component. This is probably due to the fact that the increase in airflow resistance induced by pulmonary embolism is initiated mainly by the release of bronchoconstrictor autocoids (Halmagyi and Colebatch, 1961), the direct effects of these chemicals on airway smooth muscle completely overshadowing any reflex bronchoconstrictor responses, making the latter impossible to demonstrate. 3.8.5. Congestion and Oedema Afferent vagal endings in the lung are thought to be largely responsible for initiating the dyspnoea and bronchoconstriction of acute left ventricular failure in humans. Support for this hypothesis was provided by several early studies reporting that acute congestion of the pulmonary vascular bed in dogs and cats evoked vagally-mediated tachypnoea, bradycardia and systemic hypotension (for references see Roberts et al., 1986a). Not all subsequent investigators have been able to confirm this observation. Nevertheless, Jones et al. (1978) found that acute pulmonary congestion in dogs induced a vagally-mediated bronchoconstriction; and Chung et al. (1983) reported a vagally-mediated bronchoconstriction in dogs at a late stage of infusion-induced pulmonary oedema when intrapulmonary vascular pressures had reverted to control and when significant blood gas changes were absent. In human patients with pulmonary congestion resulting from left heart disease, inhalation of an atropine aerosol caused a marked reduction in closing volume, whereas atropine had no effect on closing volume in healthy subjects (Collins et aL, 1975). These observations further supported the notion that pulmonary congestion might be associated with a vagally-mediated bronchoconstriction. The effects of pulmonary congestion and oedema on vagal afferent input from the lungs have been studied extensively--particular attention being paid to the responses of nonmyelinated fibres. In most animal studies acute pulmonary congestion has been produced by increasing pressure in the left side of the heart (either by momentary obstruction of left ventricular outflow or by stepwise inflation of a balloon in the left atrium) or by rapid infusion of large volumes of Krebs-Henseleit solution as a stage in the production of interstitial pulmonary oedema; pulmonary congestion and oedema have also been produced by administration of irritant chemicals. Paintal (1969, 1970) postulated that pulmonary J receptors (pulmonary C fibres) were interstitial stretch receptors whose natural stimulus was an increase in interstitial pressure or volume resulting from an increase in pulmonary capillary pressure. He suggested that the afferent terminals were surrounded by collagen fibres that in the presence of the increased interstitial fluid of pulmonary oedema would swell and distort the nerve endings.

Bronchomotor reflexes

41

Morphological studies have, indeed, confirmed that nonmyelinated fibres with sensory enlargements are present in the interstitium of the alveolar walls and are surrounded by collagen fibres (Hung et al., 1973a; Fox etal., 1980). Paintal's hypothesis was based largely on his observation that lung C fibres were stimulated by intravenous injection of alloxan and insufflation of chlorine, chemicals that cause pronounced pulmonary congestion and oedema. Reservations about this chemically-induced model of pulmonary congestion and oedema were expressed in a subsequent study which suggested that the increased C fibre activity resulted from chemical irritation of the terminals rather than from mechanical stimulation secondary to pulmonary vascular changes (Coleridge and Coleridge, 1977a). Nevertheless, most investigators now agree that lung C fibres are indeed stimulated by the mechanical effects of acute pulmonary congestion. This was first demonstrated by Paintal (1969) himself who reported that J receptor activity increased slightly when the aorta was briefly occluded in cats. The effects of pulmonary congestion on lung C fibres have been studied in a more quantitative fashion in dogs by inflation of a balloon in the left atrium to produce small stepwisc increments in left atrial pressure (Coleridge and Coleridge, 1977a; Kappagoda etal., 1987). Some C fibres were stimulated significantly when left atrial pressure was increased by as little as 5 mm Hg. In one study pulmonary C fibres were found to be more sensitive and to have a lower average threshold to congestion than bronchial C fibres (Coleridge and Coleridge, 1977a, 1984); in another, the reverse was reported to be the case (Kappagoda et al., 1987). Rapidly adapting receptors are stimulated more consistently and more vigorously than slowly adapting receptors by pulmonary congestion induced by inflating a balloon in the left atrium (Marshall and Widdicombe, 1958; Sellick and Widdicomb¢, 1969; Kappagoda etal., 1987) or by infusing large volumes of Krebs-Henseleit solution (Roberts etal., 1986a). Their discharge frequency increased threefold when left atrial pressure was raised to 15mmHg (Kappagoda et al., 1987), and eightfold when pressure was raised to 35 mm Hg (Fig. 18A, B) (Roberts etal., 1986a). The augmented discharge often had a pronounced respiratory rhythm, firing being maximal during inflation (Fig. 18B). Since the ventilatory discharge was abolished and the overall firing frequency markedly reduced by turning off the ventilator, receptor stimulation appeared to be due in large part to changes in lung mechanics (Roberts et al., 1986a). Slowly adapting receptors have the most varied response to acute congestion of any of the pulmonary afferents. Investigators have described an augmentation of firing during inflation that varied from the trivial to the modest but significant, and also an increase A 2801

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Bronchomotor reflexes

43

pulmonary afferent in severely oedematous lung was significantly less than in normal lung (Roberts et al., 1986a). These results suggest that the vagally-mediated bronchoconstriction demonstrated at a late stage of infusion-induced oedema in dogs when pulmonary vascular pressures have reverted to control (Chung et al., 1983) is almost certainly due to stimulation of lung C fibres. 4. BRONCHOMOTOR INFLUENCE OF AFFERENTS REMOTE FROM AIRWAYS Afferent endings remote from the respiratory tract are known to influence airway smooth muscle tone by increasing or decreasing vagal bronchomotor activity. These inputs are of various types and travel in somatic as well as visceral afferent pathways. Two examples of each serve to illustrate the diverse nature of these extra-respiratory inputs. Bronchomotor effects triggered by somatic afferents include an increase in airway resistance in normal human subjects when facial cold receptors are stimulated by application of ice packs (Josenhans et al., 1969; Melville and Morris, 1972) and a decrease in airway resistance in anaesthetized dogs when muscle afferents are activated by electrically-evoked muscular contraction (Kaufman et al., 1985). Bronchomotor effects triggered by extra-respiratory visceral afferents in anaesthetized dogs include a reflex bronchoconstriction evoked by stimulating arterial chemoreceptors (Nadel and Widdicombe, 1962a), and a reflex bronchodilatation evoked by stimulating arterial baroreceptors (Schultz et aL, 1986). As in the case of most intrinsic airway reflexes described above, the bronchomotor influence of extra-respiratory sensory mechanisms has been demonstrated in 'open-loop' preparations in anaesthetized animals with lungs ventilated artificially at constant rate and tidal volume. Hence the functional significance of these reflex connections is not always easy to assess. Nevertheless, such animal studies have clearly established the existence of a number of extrinsic afferent inputs capable of initiating bronchomotor reflexes. The airway responses evoked by engagement of these extra-respiratory inputs accompany primary reflex respiratory and cardiovascular changes, which are often themselves of obvious functional importance and which may in turn act to modulate the bronchomotor response. Studies of the extrinsic bronchomotor reflexes (e.g., Sorkness and Vidruk, 1986) provide further evidence to refute the notion that the vagal bronchomotor centres controlling airway calibre and the medullary centres controlling breathing are necessarily subject to a common central drive, so that increased vagal bronchomotor activity invariably goes hand in hand with increased inspiratory discharge, and withdrawal of vagal bronchomotor activity with decreased inspiratory discharge (Mitchell et al., 1985). Some of the extra-respiratory afferent inputs to be considered here certainly exert functionally parallel effects on bronchomotor tone and ventilation. For example, carotid body chemoreceptors are excitatory to breathing, and, at least in animals, are also excitatory to airway smooth muscle; carotid sinus barorec~ptors inhibit both breathing and airway smooth muscle tone. By contrast, groups III and IV muscle afferents inhibit airway smooth muscle tone but stimulate breathing. In the latter instance the reflex drives to the bronchomotor and ventilatory centres of the medulla are clearly independent: in other words, the cell bodies in the region of the nucleus ambiguous that provide the excitatory drive to airway smooth muscle are not driven by the same pattern generators that drive the phrenic and inspiratory intercostal motoneurones. By the same token, the reflex drives to the vagal motor centres that regulate heart rate and airway calibre are not invariably interdependent, in the sense that stimulation of carotid chemoreceptors evokes a primary increase in both vagal cardiomotor and bronchomotor activity, whereas stimulation of carotid baroreceptors evokes an increase in the former and a decrease in the latter. We deal first with the visceral afferent inputs arising from two reflexogenic sites classically associated with respiratory and cardiovascular regulation: namely, the peripheral arterial chemoreceptors and the arterial baroreceptors.

44

H.M. COLERUmEet al. 4.1. ARTERIALCHEMORECEPTORS

It has been known almost since the turn of the century that the level of CO2, acting through a central neural mechanism, influences the baseline tone of airway smooth muscle, an influence now thought to originate in the chemoreceptors of the ventral medulla (see Section 1). Interest in a comparable role for the peripheral chemoreceptors that signal changes in the arterial blood gases followed naturally. Certainly, an influence on bronchomotor mechanisms would be consistent with the known importance of the peripheral chemoreceptors in ventilatory control. The specialized chemoreceptors of the carotid bodies, and their counterparts in the aortic bodies, which are generally thought to be much less important in respiratory regulation, are stimulated by hypoxia, hypercapnia and acidemia (Fidone and Gonzalez, 1986; Fitzgerald and Lahiri, 1986). The weight of evidence suggests that the carotid body chemoreceptors have a primary bronchoconstrictor effect. However, as we shall see, the primary vagal reflex action of the carotid chemoreceptors on bronchial smooth muscle is, in one important respect, similar to their primary vagal reflex action on heart rate--rarely are either of these effects evoked by hypoxia in spontaneously breathing animals or human subjects. Early studies in artificially ventilated dogs and cats, in which carotid and aortic chemoreceptors were stimulated by lobeline or cyanide and changes in bronchomotor tone were assessed from changes in lung volume, appeared to indicate that both groups of chemoreceptors evoked bronchodilatation (Daly and Schweitzer, 1951). These results have not been confirmed. Indeed it is now quite clear that arterial chemoreceptor stimulation evoked in anaesthetized dogs by injecting nicotine into the isolated carotid sinus region (Nadel and Widdicombe, 1962a), or cyanide into the systemic circulation (Coleridge et al., 1982a), results in contraction of tracheal smooth muscle (Fig. 2C) and an increase in lower airway resistance. Loofbourrow et al. (1957) observed a striking increase in tracheal tension in anaesthetized dogs during the asphyxia brought about by ventilating the lungs with expired air, but concluded that this was due principally to the central action of CO2, systemic hypoxia having only a weak bronchoconstrictor effect. Nadel and Widdicombe (1962a) extended these studies in anaesthetized dogs, and examined changes in both tracheal smooth muscle tone and lower airway resistance produced by ventilating the lungs with hypoxic or hypercapnic gas mixtures. Reducing arterial oxygen tension to a mean of 37 mmHg, increasing arterial PCO: to a mean of 66 mmHg, and injecting nicotine into an isolated carotid sinus, had similar bronchomotor effects--decreasing the volume of an isolated tracheal segment by approximately 12% and increasing total pulmonary resistance by 50-60%. The bronchomotor effects of both systemic hypoxia and nicotine were prevented by ligating the glossopharyngeal nerves central to the origin of the sinus nerves, and hence were attributed to the reflex action of carotid body chemoreceptors. Because the aortic nerves remained intact in these experiments, Nadel and Widdicombe concluded that aortic chemoreceptors made no contribution to the reflex bronchomotor effects of systemic hypoxia, a conclusion in keeping with the minor influence of the aortic bodies on ventilatory mechanisms in general (Fitzgerald and Lahiri, 1986). The reflex reponses to chemoreceptor stimulation were also abolished by cooling the cervical vagus nerves, and therefore could be attributed to activation of a vagal bronchomotor pathway. The bronchomotor effects of systemic hypercapnia persisted after the glossopharyngeal nerves were ligated, confirming the generally accepted view that the neurally-mediated effects of hypercapnia on airway smooth muscle are due mainly to stimulation of the central medullary chemoreceptors. The bronchoconstrictor effects of systemic hypoxia were confirmed in a subsequent study in anaesthetized dogs (Green and Widdicombe, 1966), although weak bronchomotor responses were found to persist after administration of atropine or elimination of the efferent nerve supply to the airways, suggesting a minor local excitatory effect of systemic hypoxia on airway smooth muscle itself. Nevertheless, the functional significance of what seems at first sight to be an important bronchomotor reflex is far from clear. Studies in both anaesthetized (Stein and

Bronchomotorreflexes

45

Widdicombe, 1975) and unanaesthctized dogs (Sorkness and Vidruk, 1986) provide good evidence that the reflex hyperventilation induced by systemic hypoxia sets in train secondary changes that act to oppose the primary bronchoconstrictor response to carotid body stimulation. Thus hyperventilation not only reduces arterial PCO2 and hence the neural drive from central chemoreceptors, it also evokes a powerful volume-related input from pulmonary stretch receptors that exerts an inhibitory effect on airway smooth muscle tone. In conscious dogs trained to accept artificial ventilation at constant rate and tidal volume, hypoxia invariably evoked a tracheal constrictor response when ventilation was kept constant (Fig. 20C), but hypoxia no longer increased tracheal tone if the dogs breathed freely through their tracheostomies (Fig. 20A) (Sorkness and Vidruk, 1986). However, a tracheal constrictor response to hypoxia could sometimes be demonstrated in freely breathing, conscious dogs if the decrease in arterial PCO2 was prevented by adding CO2 to the inspired air (Fig. 20B). Studies of possible bronchoconstrictor effects of hypoxia in healthy human subjects have yielded conflicting results. Some investigators found that airway resistance was unchanged by hypoxia, even when isocapnia was maintained and arterial haemoglobin oxygen saturation was kept at the mixed venous level for several minutes (Milic-Emili and Petit, 1960; Nielsen and Pedersen, 1977; Goldstein et al., 1979). Others reported a small, and, in one study, significant, increase in airway resistance during relatively severe systemic hypoxia (Sterling, 1968; Saunders et al., 1977), but the results of pharmacological blockade were inconclusive, and a direct bronchoconstrictor effect of hypoxia at the level of the airways could not be ruled out (Sterling, 1968). Although the bronchoconstrictor effects of hypoxia can rarely be demonstrated in healthy human subjects (possibly owing to the opposing influence of reflexes favouring bronchodilatation), administration of oxygen-rich mixtures to patients with hypoxia secondary to chronic obstructive lung disease often reduces the accompanying

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FIO. 20. Effect of hypoxia on tracheal smooth muscle tone in three awake dogs. Pressure changes in a water-filled cuff in an isolated portion of the cervical trachea reflect changes in tracheal tone. Each data point is the mean 5: SD of 4-12 paired trials. P ~ , average tracheal cuff pressure; PETCO2, average end-tidal CO2; VT, average tidal volume. A, B, dogs breathing spontaneously; in B, CO2 added to the inspired hypoxic gas. C, dogs ventilated mechanically. *Significant change (P < 0.05) in cuff pressure from normoxia to hypoxia. (Sorkness and Vidruk, 1986.)

46

H.M. COt~RIDCEet aL

bronchoconstriction (Astin, 1970; Libby et al., 1981). The relief of bronchoconstriction may be in part a direct effect on airway smooth muscle, since hypoxia itself appears to have a mild bronchoconstrictor action (Green and Widdicombe, 1966; Sterling, 1968). Even so, atropine has been found to be as effective as oxygen administration in these hypoxic patients (Libby et al., 1981), suggesting that a reflex triggered by stimulation of the arterial chemoreceptors plays a major role. These observations on human subjects, taken together with the animal studies of Nadel and Widdicombe (1962a) described above, appear to provide some rationale for the surgical removal of the carotid bodies (glomectomy) in the treatment of asthmatic bronchospasm (Nakayama, 1961; Overholt, 1961; Winter, 1974). Whether the procedure, which has had considerable vogue, is of real benefit remains controversial, for several studies have been characterized by a lack of objective measurement (Fitzgerald and Lahiri, 1986). Although some remain convinced of the value of the procedure (Winter, 1974), supporters and detractors of glomectomy appear to be equally matched. On balance, the available evidence indicates that the efficacy of either unilateral or bilateral glomectomy in the treatment of obstructive airway disease remains unproven. In general, a causal role for the carotid bodies in the muscarinic bronchoconstriction of lung disease is not always easy to establish. For example, though the airways of asthmatics are thought to be particularly prone to muscarinic bronchoconstrictor effects, Fisher et al. (1970) could find no evidence of increased bronchoconstriction in a group of asthmatic human subjects breathing hypoxic gas mixtures at rest and during exercise. Moreover, a recent study of asthmatic human subjects by Tam et al. (1985) showed that the decrease in airway resistance caused by hyperventilation with a hypoxic gas mixture was no greater than that caused by an equivalent hyperventilation with air. Thus, although hypoxic stimulation of the arterial chemoreceptors undoubtedly exerts a bronchoconstrictor influence in anaesthetized animals, and in certain conditions in conscious animals as well, the reflex role of the chemoreceptors in the regulation of bronchomotor mechanisms in human subjects has yet to be established. As to the functional utility of a chemoreceptor-evoked bronchoconstriction, there seems no doubt that during hypoxia or hypercapnia a reduction of dead space would be beneficial because ventilation of dead space is wasteful from the point of view of gas exchange (Stein and Widdicombe, 1975). One might surmise that if hypoxia were present and ventilation itself were limited by disease, a chemoreceptor-induced bronchoconstriction would represent the only means of improving gas exchange. As to the reversal of a chemoreceptorevoked bronchoconstriction by the resulting hyperpnoea, Stein and Widdicombe (1975) have argued persuasively that the bronchoconstrictor influence of the central and peripheral chemoreceptors is balanced by a bronchodilator influence, related to the minute volume of ventilation, that is important in adjusting airway calibre to reduce the resistive work of breathing as minute volume increases (Nadel, 1980). 4.2. ARTERIALBARORECEPTORS Arterial baroreceptors, located in the carotid sinus and the aortic arch and at the junction of the brachiocephalic and right subclavian arteries, respond to changes in blood pressure and evoke reflex adjustments that are of obvious functional importance in the regulation of arterial blood pressure, peripheral blood flow and heart rate. The reflex effects of stimulating carotid sinus baroreceptors are not confined to the cardiovascular system but also involve breathing and bronchomotor tone. In general, excitation of carotid sinus baroreceptors by increasing carotid sinus pressure inhibits breathing, and decreasing sinus pressure stimulates it (for references see Heymans and Neil, 1958; Brunner et al., 1982). There has been less agreement about the effects of baroreceptors on bronchomotor tone. In electroneurographic studies in dogs and cats, efferent nerve activity recorded from small vagal branches to the trachea and bronchi increases in response to carotid arterial occlusion, and decreases in response to the hypertension induced by injection of

Bronchomotorreflexes

47

catecholamines (Widdicombe, 1961a, 1966), suggesting that baroreceptor unloading causes bronchoconstriction, and baroreceptor stimulation bronchodilatation (Widdicombe, 1963). Nevertheless, results of reflex studies of the bronchomotor influence of the arterial baroreceptors have often been conflicting and hard to interpret. Some early investigators found that bronchomotor responses were difficult to elicit from the carotid sinus and that when they did occur they were very weak; others reported that baroreceptor stimulation produced bronchoconstriction and concluded that baroreceptors had a role in the reflex maintenance of bronchomotor tone in normal conditions (for references see Widdicombe, 1963). However, several of the earlier studies did not allow the investigators to distinguish between changes in airflow resistance and changes in lung compliance or lung volume. Nadel and Widdicombe (1962a) examined the effects of carotid baroreceptors on bronchomotor tone, varying pressure in the perfused carotid sinuses of anaesthetized dogs and measuring changes in tracheal volume and total lung resistance. Increasing sinus pressure in a single step from 20 mmHg to 200 mmHg caused a small increase in tracheal volume, reducing sinus pressure again had the opposite effect; total lung resistance was not affected. The functional significance of these observations was difficult to assess because the effects observed at the two extremes (20 and 200 mmHg) of the baroreceptor stimulus-tracheal response curve gave little indication of the bronchomotor changes likely to occur when blood pressure varies around the normal arterial setpoint of about 100 mmHg. Schultz et al. (1986, 1987b) recently examined the influence of carotid sinus baroreceptors on tracheal smooth muscle tension and total airway resistance in anaesthetized dogs, varying perfusion pressure in the vascularly-isolated carotid sinuses above and below a setpoint of 100 mmHg. Input from the aortic baroreceptors was abolished by cutting the aortic (depressor) nerves. Results showed clearly that the carotid baroreflex has an atropine-sensitive influence on airway smooth muscle, and that this influence extends over the normal range of arterial pressures. Increasing carotid sinus pressure in steps above 100 mmHg decreased tracheal tension, heart rate, and arterial pressure (Fig. 21A-C), and decreasing sinus pressure below 100 mmHg had the opposite effects (Fig. 21D-F) (Schultz et al., 1987b). The relationship of sinus pressure to each of these three variables displayed the characteristic sigmoidal baroreflex stimulus-response curve, operating linearly across the setpoint, and flattening at pressures below 50 mmHg and above 175 mmHg (Fig. 22). Stepwise changes in mean carotid sinus pressure of as little as 10 mmHg above and below the setpoint were sometimes sufficient to evoke bronchomotor effects. The sensitivity of the response was also demonstrated by increasing and decreasing sinus pulse pressure around a constant mean of 100 mmHg, which respectively decreased and increased tracheal smooth muscle tension. The carotid baroreflex was found to exert a tonic restraining influence on bronchomotor tone. Thus, cooling the sinus nerves to 0°C, while sinus pressure was held constant at the setpoint, significantly increased tracheal tone, an effect reversed by rewarming the nerves (Fig. 23). Impulses arising from carotid sinus baroreceptors travel in myelinated (A) and nonmyelinated (C) fibres (Coleridge et al., 1987). A fibre baroreceptors are active at a sinus pressure of 100 mmHg, and their firing increases and decreases as pressure is varied around the setpoint. C fibre baroreceptors have a much higher threshold and are virtually inactive at a pressure of 100 mmHg: therefore they are probably of little functional importance at normal arterial pressure but are increasingly brought into play as pressure exceeds the setpoint. Hence the bronchomotor effects produced by decreasing carotid sinus pressure below 100 mmHg (Figs 21, 22) or by cooling the sinus nerves when sinus pressure is held constant at 100mmHg (Fig. 23) result solely from a reduction of A fibre baroreceptor input. However, C fibre baroreceptors undoubtedly contribute to the bronchodilatation produced by increasing sinus pressure above the setpoint, because relaxation of airway smooth muscle can still be evoked after the sinus nerves are cooled to 7°C, a temperature at which conduction in baroreceptor A fibres is blocked selectively (Coleridge et al., 1987). JPT (2/I--D

48

H.M. COLE~I~E et al.

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FIG. 21. Modulation of airway smooth muscle tension by carotid sinus baroreceptors. Smooth muscle tension was recorded in an upper tracheal segment of an anaesthetized dog (baseline tension was set at 75 g), and baroreceptor input was varied by changing pressure in the vascularly isolated carotid sinuses. Changes in tracheal tension (TT), heart rate (HR) and systemic arterial blood pressure (ABP) were evoked by graded alterations in mean carotid sinus pressure (CSP) above (A-C) and below (D-F) the setpoint pressure of 100 mmHg. Carotid sinus pulse pressure was kept constant throughout. (Schultz et al., 1987b.) 4O

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FIG. 22. Inhibitory effects of carotid sinus baroreceptors on bronchomotor tone, heart rate and blood pressure. Average changes in tracheal tension (ATT, g), heart rate (AHR, beats/rain), and systemic arterial blood pressure (AABP, mmHg) evoked by increasing and decreasing mean carotid sinus pressure (CSP) in six dogs (carotid sinus pulse pressure was held constant). Baseline values for tracheal tension, heart rate, and systemic arterial blood pressure at a mean carotid sinus pressure of 100 mmHg were respectively: 120 + 12 g, 178 + 13 beats/rain, 132 + 5 mmHg. (Schultz et al., 1987b.)

Bronchomotor reflexes

ABP

49

zoo

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I rain FIG. 23. Carotid sinus baroreceptors exert a tonic inhibitory effect on airway smooth muscle. Smooth muscle tension in an anaesthetized dog was recorded from an upper tracheal segment innervated by the superior laryngeal nerves. The vascularly-isolated carotid sinuses were distended with a constant pulsatile pressure; each carotid sinus nerve was placed on a cooling platform. Tracheal tension increased when the sinus nerves were cooled. ABP, arterial blood presure; HR, heart rate; TT, tracheal tension (baseline tension set at 75 g); CSNT, carotid sinus nerve temperature, measured with a thermistor on the cooling platform; CSP, carotid sinus pressure; PCO2, tidal CO2 (Schultz et al., 1987b.)

Raising pressure in the vascularly isolated carotid sinuses also caused a reflex decrease in total pulmonary resistance, and reducing pressure an increase (Schultz et al., 1986). The effects were present over a normal range of arterial pressures when the dogs breathed spontaneously, as well as when the lungs were ventilated artificially. In spontaneously breathing dogs, an increase in carotid sinus pressure depresses ventilation and a decrease in pressure excites it, so that arterial CO2 is likely to change in the same direction as carotid sinus pressure. Such secondary changes in arterial CO2, acting on the central medullary chemoreceptors, would tend to oppose the primary effects of the baroreflex on the airways. Nevertheless, the baroflex was prepotent in spontaneously breathing dogs, carotid sinus distension invariably producing bronchodilatation (Schultz et al., 1986). Thus, although the carotid baroreflex was once thought to have only a minor influence on airway smooth muscle (Nadel and Widdicombe, 1962a), the results of Schultz et al. (1986, 1987b) provide strong support for the notion that arterial baroreceptors exert a tonic influence on airway smooth muscle, increasing and decreasing baseline smooth muscle tension as arterial blood pressure varies around the normal setpoint. Indeed, the changes in airway smooth muscle tone evoked by the carotid baroreflex are of a comparable magnitude to those produced in the same experiment by stimulating other afferent inputs (such as pulmonary stretch receptors, pulmonary C fibres, hind-limb muscle afferents and laryngeal receptors) that are generally agreed to have powerful effects on airway smooth muscle tone (Schultz et al., 1987b). It is not known whether aortic baroreceptors modulate bronchomotor tone but it seems reasonable to assume that their effects, if any, would be in the same direction as those resulting from the carotid baroreflex. 4.3. CAV.DIACAFFERENTS Impulses arising from afferent endings in the heart and atrio-venous junctions are carded in myelinated and nonmyelinated fibres that travel in the vagus nerves to reach the medullary centres and in sympathetic nerve branches to reach the spinal cord. The endings are of two types: mechanoreceptors responding to changes in intracardiac pressure and volume, and chemosensitive endings responding to changes in the chemical environment. The role of these afferents in cardiovascular regulation has attracted most attention over

50

H.M. C o t ~

et al.

the past 40-50 years, but recent evidence suggests that sensory endings in the heart may also have an influence on respiratory mechanisms. The afferent characteristics and reflex properties of these cardiac receptors are discussed in several recent reviews (Coleridge and Coleridge, 1979, 1980; Abboud and Thames, 1983; Bishop et al., 1983; Daly, 1986). 4.3.1. Cardiac Mechanoreceptors Although mechanoreceptors in the ventricles, atria and atrio-venous junctions are readily stimulated by increases in cardiac pressure and volume, it is often hard to determine which afferent endings are responsible for a given reflex effect--in large part because of the technical difficulty of confining the stimulus to a particular cardiac chamber. For example, reflex effects triggered by receptors in the left atrium may not be easy to distinguish from those triggered by receptors in the lungs, because the methods used to increase left atrial pressure often increase pressure upstream and result in pulmonary congestion. Such difficulties are often glossed over by using the terms 'cardiopulmonary receptors' to describe cardiovascular receptors other than the arterial baroreceptors and 'cardiopulmonary reflexes' to describe effects evoked by these receptors--as if all 'cardiopulmonary' receptors had similar functions. On the contrary, 'cardiopulmonary' embraces a variety of receptors of widely different function and afferent susceptibility: receptors in the left atrium and ventricle, pulmonary stretch receptors, rapidly adapting receptors, pulmonary C fibres and mechanoreceptors in the right heart--all of which are influenced by intravascular pressure. Some of these receptors act to increase heart rate, others to decrease it; some inhibit breathing and dilate the airways, others stimulate breathing and constrict the airways. It is not surprising that the reflex effects of increasing central intravascular pressures should often be hard to analyze. Distension of a balloon in the left atrial appendage in anaesthetized dogs has been found to evoke a vagally-mediated contraction of an upper tracheal segment, an effect attributed to stimulation of left atrial receptors (Roberts et aL, 1986b). A similar vagally-mediated tracheal response to left atrial distension in dogs was reported by Teo et al. (1985), although these authors attributed the tracheal contraction to stimulation of afferent endings in the lung by the resultant pulmonary venous congestion. The most convincing evidence to date that cardiac mechanoreceptors exert a reflex influence on bronchomotor tone was obtained by Lloyd (1980), who found that distension of the isolated left heart in dogs on cardiopulmonary bypass produced small increases in tracheal wall tension, the responses being eliminated by vagotomy. Another technique worth exploring would be that used by Ledsome and Hainsworth (1970) to assess the role of atrial reflexes on breathing: namely, to distend the left atrial-pulmonary vein junctions with small balloons, so that atrio-venous receptors could be stimulated selectively without producing pulmonary congestion. 4.3.2. Cardiac Chemosensitive Afferents A second group of vagal afferent endings in the heart have a sparse and irregular discharge and are generally insensitive to changes in cardiac pressure or volume within the physiological range (Coleridge et aL, 1964, 1973). They are thus easily distinguished from atrial and ventricular mechanoreceptors, which fire with an obvious cardiac modulation and are extremely sensitive to changes in pressure and volume. Although not chemoreceptors in the conventional sense, for they are not stimulated by hypoxia or cyanide, these irregularly firing vagal afferents are stimulated by capsaicin and other chemicals that have no direct effect on cardiac mechanoreceptors. They are called 'chemosensitive endings' to distinguish them from the chemoreceptors of the aortic and carotid bodies. Most chemosensitive endings are supplied by C fibres, a few by slowly conducting A fibres. In anaesthetized dogs with open chest and lungs ventilated artificially, stimulation of these chemosensitive endings by injection of capsaicin into the circumflex or anterior

Bronchomotor reflexes

51

descending coronary artery decreases blood pressure and heart rate (Bezold-Jarisch reflex; coronary chemoreflex) and evokes a reflex, atropine sensitive contraction of tracheal smooth muscle (Roberts et al., 1984). Depressor and tracheal responses are abolished by cutting or cooling (0°C) the lower cervical vagus nerves. Since the bronchoconstriction evoked by stimulating cardiac chemosensitive C fibres is frequently, although not invariably, accompanied by a marked decrease in arterial blood pressure, and since baroreceptor unloading is known to cause bronchoconstriction, the question naturally arises whether the cardiac afferents or the arterial baroreceptors are the prime initiators of the bronchomotor response. In the event, bronchoconstriction can still be evoked after changes in baroreceptor input have been eliminated by distending the carotid sinuses at constant pressure and cutting the aortic nerves (Roberts, Schultz, Pisarri, Coleridge and Coleridge, unpublished observations). When arterial baroreceptor mechanisms are intact, however, they obviously contribute to the bronchoconstrictor response. This cardiac-bronchomotor reflex mechanism is probably of little functional significance in normal physiological conditions. Nevertheless, cardiac chemosensitive endings are stimulated not only by foreign chemicals such as capsaicin but also by substances such as bradykinin (Kaufman et al., 1980a) and the prostaglandins (Roberts et al., 1980) that are known to be formed and released in the myocardium in response to ischaemia and gross cardiac distension. Recent evidence also indicates that cardiac chemosensitive C fibre endings are stimulated by myocardial ischaemia, resulting in an atropine-sensitive bronchoconstriction (Pisarri, Schultz, Clozel, Coleridge and Coleridge, unpublished observations). By contrast, application of solutions of capsaicin or bradykinin to the epicardial surface of the heart produces not contraction but relaxation of airway smooth muscle, relaxation being accompanied by an increase in blood pressure and heart rate (Roberts et al., 1985a). These effects can still be evoked after the lower cervical vagus nerves have been cut or cooled to O°C, but are abolished by cutting the stellate ganglia. Electroneurographic studies have shown that sympathetic afferents in the heart are stimulated by epicardial application of capsaicin or bradykinin (Baker et al., 1980). Thus chemicals acting on sensory endings in the heart engage two opposing reflex mechanisms, stimulating vagal afferents to evoke bronchoconstriction and sympathetic afferents to evoke bronchodilatation. The functional interplay of these reciprocal reflex mechanisms in various pathophysiological circumstances remains to be elucidated. 4.4. AFFERENTSFROM SKELETAL MUSCLE AND DIAPHRAGM

Several studies have shown that exercise dilates the airways of normal human subjects (McIlroy et al., 1954; Kagawa and Kerr, 1970; Warren et al., 1984). The increase in airway conductance is proportional to the work load (Kagawa and Kerr, 1970). The bronchodilatation, which results from a reflex inhibition of vagal hronchomotor tone, is sufficiently powerful to cause rapid reversal of the bronchoconstriction evoked by inhalation of cigarette smoke (Kagawa and Kerr, 1970). There appears to be little or no contribution from the sympathetic nervous system in man (Warren et al., 1984), even though sympathetic outflow is greatly increased during exercise. Although an increase in the inhibitory input from slowly adapting pulmonary stretch receptors is likely to contribute to the reflex hronchodilatation of exercise, recent evidence suggests that input from muscle afferents plays a major role in the bronchodilator response (see below). Results of experiments involving selective anodal blockade of afferent fibres in cats indicate that the excitatory cardiovascular-ventilatory responses to isometric contraction of hindlimb muscles is evoked by an increase in the discharge of fine myelinated (group III) and nonmyelinated (group IV) muscle afferents; input from spindle or tendon organ afferents (groups I and II) plays no part (McCloskey and Mitchell, 1972). This small fibre afferent input is thought to contribute to the prompt cardiovascular and ventilatory changes that occur at the onset of exercise, and is also likely to be involved in the bronchodilatation of exercise.

H.M. COLERIDG]~et al.

52

The properties of group III and group IV muscle afferents have been examined in electrophysiological studies in cats and dogs. Some fibres in both groups were equally sensitive to isometric muscle contraction and to algesic chemicals such as capsaicin, bradykinin and KCI, but in general group III afferents appeared to be more responsive to mechanical stimulation and group IV afferents to chemicals (Kaufman et al., 1983; Rybicki et al., 1985; Kaufman and Rybicki, 1987). Although the response of group IV afferents to muscular contraction was potentiated by ischaemia (Kaufman and Rybicki, 1987), the metabolites responsible for the chemical stimulation of these endings under physiological conditions have not yet been identified. Nevertheless the net input from these two groups of muscle afferents provides a graded signal related to the vigour of contraction (Kaufman and Rybicki, 1987). The inhibitory bronchomotor reflex has been demonstrated in anaesthetized dogs and cats as a decrease in tracheal smooth muscle tension when chemicals (bradykinin, capsaicin, KC1), known to stimulate group III and group IV muscle afferents, are injected in small amounts into the arterial supply of hindlimb muscles (Kaufman et al., 1982b; Baker and Don, 1988). A similar decrease in tracheal tension (Kaufman and Rybicki, 1984; Longhurst, 1984) and in total pulmonary resistance (Kaufman et al., 1985) has been demonstrated in dogs and cats during isometric contraction of hindlimb muscles evoked by stimulating the appropriate ventral roots (Fig. 24A). The relaxation of tracheal smooth muscle and the decrease in airflow resistance are abolished by administering atropine or by interrupting the afferent input (Fig. 24B) (Rybicki and Kaufman, 1983; Kaufman et al., 1985). It seems reasonable to suggest that the vagal bronchomotor A Tension

(kg) HR (bpm) AP (mmHg)

4[ 0 240 [ '150

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~

L_____

L 30sac

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Fn(~. 24. Static contraction of hind-limb muscles reflexly decreases total pulmonary airflow resistance in an anaesthetized, artificially ventilated dog with open chest. A: static muscular contraction induced by stimulating the peripheral ends of the cut L~-L7 ventral roots at 50 Hz; note bronchodilatation. B: static contraction induced again by stimulating L r L 7 ventral roots after L4-S 3 spinal roots had been sectioned to abolish sensory input from the contracting muscles; note absence of bronchodilatation. Tension, hind-limb muscle tension recorded by attaching the calcaneal tendon to a force transducer; HR, heart rate; AP, arterial pressure; TPR, total pulmonary resistance; Cdy,, dynamic compliance; VT, tidal volume; P~, tracheal pressure. (Kaufman et al., 1985.)

Bronchomotorreflexes

53

inhibitory reflex evoked by stimulation of muscle afferents subserves a useful regulatory function in exercise, by helping to reduce the flow-resistive work of breathing as ventilation increases. There is no evidence that sympathetic bronchodilator mechanisms are engaged by muscle afferents. Thus in a study of the tracheal relaxation evoked by chemical stimulation of muscle afferents in cats, tracheal tone was first abolished by atropine, then restored by administration of serotonin (Baker and Don, 1988). Even under these conditions, stimulation of muscle afferents failed to activate sympathetic bronchoinhibitory pathways. Bronchomotor tone also appears to be susceptible to the influence of afferent input arising from the diaphragm. Thus, electrical stimulation of groups III and IV phrenic nerve afferents evoked a reflex decrease in total lung resistance in anaesthetized, open chest dogs (McCallister et al., 1986). Bronchodilatation was due to withdrawal of cholinergic muscarinic tone because it was unaffected by propanolol or phentolamine but was abolished by atropine. Since the chest was open, bronchodilatation was not secondary to ventilatory-induced increases in pulmonary stretch receptor discharge. The functional significance of the bronchodilatation evoked by increased phrenic afferent input is not clear, in large part because the physiological stimulus to groups III and IV phrenic afferents is uncertain, although it is probably of metabolic origin. However, McCallister et al. (1986) suggested that the afferents might be stimulated in conditions, either physiological or pathological, in which the diaphragm contracted more forcefully or frequently. Thus phrenic afferent input might be stimulated during exercise and also in any pathological condition that resulted in bronchoconstriction and an increase in the work of breathing. In both circumstances increased feedback from phrenic afferents would reflexly dilate the airways, thereby decreasing the work of breathing. 4.5. AFFERENTSFROMALIMENTARYTRACT Electroneurographic studies have identified vagal endings sensitive to acid in the oesophageal mucosa of cats (Harding and Titchen, 1975). The association of gastrooesophageal reflux with noctural wheezing in asthmatic children (Martin et al., 1982) and adults (Spaulding et al., 1982) directed attention to the possibility that a reflex triggered by stimulation of acid-sensitive oesophageal nerve endings might be responsible (Boyle et al., 1985). In human studies infusion of 0.1 N HC1 into the oesophagus was found to cause bronchoconstriction in asthmatic but not in normal subjects (Spaulding et al., 1982). An oesophageal-bronchoconstrictor reflex has also been described in animals. In dogs with oesophagitis, which had been induced by daily oesophageal infusions of dilute acid, infusion of 100 ml of 0.1 N HC1 caused a significant increase in lower airway resistance that was no longer present after vagotomy (Mansfield et al., 1981). In cats not subjected to previous oesophageal infusions, the introduction of 10 ml of 0.2 N HCI was found to evoke a rather small and inconstant increase in pulmonary resistance (Tuchman et al., 1984). The small effects of oesophageal infusions of acid were in marked contrast to the prominent vagally-mediated bronchoconstriction that was invariably evoked when much smaller volumes of acid solution were introduced into the trachea. Tuchman et al. (1984) therefore conclude that although bronchoconstriction can indeed be evoked by stimulating vagal chemosensitive afferents in the oesophagus, nocturnal gastro--oesophageal reflux in humans is more likely to cause bronchoconstriction if the acid stomach contents are inhaled. Whereas stimulation of vagal endings in the oesophageal mucosa evokes bronchoconstriction, stimulation of sympathetic afferent endings on the serosal surface on the abdominal viscera evokes bronchodilatation; however, both effects are mediated by changes in vagal efferent bronchomotor activity. Thus, application of capsaicin or bradykinin to the serosal surface of the stomach and small intestine evokes an atropinesensitive relaxation of tracheal smooth muscle tone in dogs (Rybicki and Kaufman, 1983; Rybicki et al., 1983). The effects appear to be due to stimulation of the afferent endings of myelinated and nonmyelinated fibres that travel in the splanchnic nerves and enter the

54

H . M . COLERIDGE et al.

spinal cord (Rybicki et al., 1983; Longhurst et al., 1984). Although this viscerobronchomotor reflex was thought by Rybicki et al. (1983) to represent one of the components of a constellation of autonomic responses triggered by visceral pain and inflammation, the visceral sites from which the reflex could be evoked appeared to be limited, and little or no bronchomotor response was produced by applying capsaicin or bradykinin to the surface of the liver or gall bladder. 5. CONCLUSIONS Unless one is to concede that a physiological role for bronchial smooth muscle has never been convincingly demonstrated (Otis, 1983), one can continue to advance arguments for the potential usefulness of a neural control of airway smooth muscle tone. One can point, for example, to the bronchoconstrictor component of the airway defense reflexes that limit the entry of noxious substances into the lung. One can also point to the bronchoconstriction that accompanies coughing and helps to stabilize the airways during the explosive expiratory event. The functional utility of the bronchomotor reflexes triggered by stimulation of afferent nerves remote from the respiratory tract may appear less obvious. Even so, one can argue that during haemorrhagic hypotension a baroreflexinduced reduction in airway dead space is beneficial in that it tends to increase alveolar ventilation, and that during exercise a reflex bronchodilatation evoked by stimulation of muscle and diaphragmatic afferents helps to reduce the flow-resistive work of breathing. We agree, however, that not all bronchomotor reflexes demonstrated in the laboratory are of obvious physiological benefit. But, useful or not, bronchomotor reflexes are important because they are often engaged by disease, particularly of the lower respiratory tract. They may be involved in initiating major components of a disease or in aggravating an already existing disease and the discomfort associated with it. It is important to bear in mind that one usually attempts in the laboratory to examine the bronchomotor response evoked by selective stimulation of a single group of sensory endings, isolating the reflex arc as far as possible from the influence of other afferent inputs. In such controlled experiments, one is rarely able to do more than demonstrate the reflex potentialities of a given afferent input. In most physiological and pathophysiological circumstances outside the laboratory, bronchomotor reflexes are not evoked in isolation but are part of a cascade of interacting primary and secondary reflexes, some of which promote bronchoconstriction, others bronchodilatation. In some instances, the secondary reflexes act to amplify the bronchomotor response to the primary stimulus, as when reflex bronchoconstriction is accompanied by a decrease in blood pressure. In others, the secondary reflexes act to oppose the primary bronchomotor effect, as when bronchomotor responses are evoked by carotid chemoreceptor stimulation in freely breathing animals. Perhaps the most that can usefully be said at present about the possible utility of such a system of supporting and opposing reflexes is that it confers some measure of stability on the regulation of airway smooth muscle tone. Much remains to be discovered about the bronchomotor reflexes, especially in regard to interaction between the various reflexes. There is also much that we do not know about the full range of sensory nerves supplying the airways. Over the past fifty years action potential studies have identified several types of afferent ending in the upper and lower airways and have provided a great deal of information about their sensory characteristics. Even so, the survey is probably far from complete, especially in the case of nonmyelinated fibres, which account for 80-90% of the vagal afferents arising from the lower airways and lungs. The possibility that the peptide content of afferent C fibres in the airways gives them a special significance in bronchomotor regulation now engages the interest of many investigators. And, even though 'axon reflex' type phenomena have so far been demonstrated only in rodents, and a bronchomotor component of axon reflexes only in guinea pigs, the presence of peptide-containing afferent C fibres in the airways of several mammalian species including man undoubtedly opens up an exciting area for exploration.

Bronchomotorreflexes

55

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