Thorax 1984;39:561-567

Editorial

The third nervous system in the lung:physiology and clinical perspectivesThe autonomic nervous system controls manyaspects of pulmonary function' and, until recently, itwas thought that this was achieved entirely by meansof classical cholinergic and adrenergic mechanisms.This view has recently been challenged by thedemonstration of a third nervous system in the air-ways (fig). Although the identities of the neuro-transmitters in this newly recognised nervous systemare not certain, there is increasing evidence that theyare peptides. This third nervous system is of particu-lar interest as it is the predominant inhibitory nerv-ous pathway to human airway smooth muscle andthis raises the possibility that a functional defect inthis nervous system might underlie bronchial hyper-reactivity in asthma. The recognition that a thirdcomponent of the autonomic nervous system regu-lates airway smooth muscle tone and other aspectsof lung physiology has stimulated great interest,since it may provide new insights into diseases suchas asthma and chronic obstructive lung disease, andmay also lead to novel therapeutic approaches.

A third component of the autonomic nervous system

The existence of a nervous system in the gastrointes-tinal tract which is neither adrenergic norcholinergic has been established for many years.Non-adrenergic, non-cholinergic (NANC) nervescontrol gut motility and secretions and have beendemonstrated in vertebrates from fish to man.23Indeed, the presence of NANC nerves in primitivevertebrates indicates that this nervous systemdeveloped early in evolution. The third nervous sys-tem has also been demonstrated in the urogenitaltract, the eye, and the cardiovascular system.3Because the airways develop embroyologically fromthe foregut it is not surprising to find that NANCnerves are also present in the lung.

NANC nerves in the airways

Inhibitory nerves which relax toad lungs and which

Address for reprint requests: Dr Peter J Barnes, Department ofMedicine (Respiratory Division), Royal Postgraduate MedicalSchool, Hammersmith Hospital, London W12 OHS.

are neither adrenergic nor cholinergic weredescribed by Campbell in 1971.4 Since then NANCnerves which relax airway smooth muscle have beenfound in several species, including man.55 In thesestudies airway strips are mounted in an organ bathand stimulated by an electrical current that selec-tively activates nerves. In guinea pig tracheal stripselectrical field stimulation produces an initial con-traction that is blocked by the anticholinergic drugatropine and therefore due to acetylcholine releasefrom cholinergic nerves. The initial contraction isfollowed by a relaxation, which is only partiallyinhibited by ,3 adrenergic blockers.56 Thus, althougha component of the inhibitory response isadrenergic, there is a component that is non-adrenergic. The nerve toxin tetrodotoxin abolishesthese responses to field stimulation and proves thatthey are mediated by nerves (which must be post-ganglionic), and not due to a direct effect on smoothmuscle. Thus guinea pig trachea appears to haveexcitatory cholinergic nerves and inhibitory nerveswhich are both adrenergic and non-adrenergic. Insimilar studies on human airway smooth muscle acholinergic contractile component is also demon-strable, but the inhibitory response is unaffected byadrenergic blockade.78 This indicates, rather surpris-ingly, that there is no functional sympathetic inner-vation of human airway smooth muscle and thatinhibitory nerves are non-adrenergic. The absenceof sympathetic nerves in human airway smoothmuscle is supported by histological studies showingan absence of adrenergic nerve terminals withinsmooth muscle,79 by studies showing a lack of effectof cocaine (a neuronal uptake inhibitor) on norad-renaline dose-response curves,'0 and by the lack ofeffect of tyramine (which directly releases norad-renaline from nerve terminals) on airway function inman." There are, of course, adrenergic receptors inairway smooth muscle,'2 and these are presumablyregulated by circulating adrenaline.'3 NANC nervesappear to be the only neural inhibitory pathway inhuman airway smooth muscle from the trachea tothe smallest bronchi,7 and for this reason there isparticular interest in their physiological role andregulation.NANC nerves have also been demonstrated in

561

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NON-ADRENERG IC,ADRENERGIC CHOLINERGIC NON-CHOLINERGIC

sympathetic nerve

c2 Iv vagus

cholinergic efferent

adrenal medulla ik non -adrenergic,

afferent

ACh NVIP

smooth muscle

epithelium

mnt

airway lumen

Innervation ofhuman airway smooth muscle: the three components oftheautonomic nervous system. In addition to classical adrenergic andcholinergic pathways, there is a third component to the autonomic nervoussystem, shown by the dashed lines. The non-cholinergic, non-adrenergicpathway is inhibitory to airway smooth muscle, the neurotransmitter ofwhich is likely to be vasoactive intestinal peptide (VIP). There may also bean excitatory non-cholinergic pathway, which may consist ofcollateralbranches ofafferent nerves which release substance P. Specific receptorsfor these neurotransmitters present on airway smooth muscle cells:3,-8 adrenergic, M-muscarnic cholinergic, V-VIP, and P-substanceP receptors. ACh-acety1choline.

cats and guinea pigs in vivo by electrical stimulationof the vagus nerve after cholinergic and adrenergicblockade.'4-'6 Stimulation of this pathway producespronounced and long lasting bronchodilatation. Thisresponse can be inhibited by ganglion blockers, sug-gesting that these NANC nerves are preganglionicas well as postganglionic.'4 It has not yet been poss-ible to establish whether NANC nerves mediatebronchodilation in human airways in vivo, althoughsuch studies should be possible with pharmacologi-cal blockade and reflex stimulation.NANC nerves also regulate secretion of airway

mucus in animals. Stimulation of the vagus nerves

promotes mucus secretion in cat trachea, which isreduced but not abolished by cholinergic andadrenergic blockers"; and electrical field stimula-tion of ferret tracheal segments in vitro also indi-cates that NANC nerves stimulate mucus secre-tion.'8 NANC nerves may also mediate relaxation ofpulmonary blood vessels.'9

The search for the neurotransmitter

Although it has been possible to demonstrate theexistence of NANC nerves in the lung both in vitroand in vivo, it is difficult to investigate the physio-

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logical role of this nervous system until the neuro-transmitter has been identified and a specific blockerdeveloped. Burnstock originally proposed that theneurotransmitter might be a purine nucleotide suchas adenosine triphosphate (ATP) or adenosine,since in the gut these purines are released on nervestimulation and both exogenous ATP and adenosinemimic some of the effects of NANC nerve stimula-tion.2 These nerves were therefore termedpurinergic. The evidence is, however, against apurine as the neurotransmitter of NANC nerves inthe airways. Although ATP relaxes isolated guineapig airway smooth muscle,202' its antagonistquinidine does not block NANC relaxation either invitro or in vivo,202223 nor does the purine uptakeinhibitor dipyridamole enhance non-adrenergicbronchodilation.2'22

More recent studies suggest that the neurotrans-mitter may be a regulatory peptide, so that NANCnerves are probably "peptidergic." There is nowrather convincing evidence that several regulatorypeptides may be neurotransmitters orneuromodulators in the gut.24 Electron microscopyhas revealed different types of neurosecretorygranules in autonomic nerve terminals in the gut25:small clear vesicles which contain acetylcholine andsmall dense granules containing catecholamines re-present cholinergic and adrenergic nerves. In addi-tion, there are large dense vesicles resemblingneurosecretory granules in the central nervous sys-tem that are known to contain peptides, and whichwere therefore called "p" type granules.25 Ultra-structural immunocytochemical techniques havenow confirmed that peptides such as substance P andvasoactive intestinal peptide (VIP-see below) arelocalised to these granules.24 Similar "p" typegranules have also been described in nerves of the

26airways, suggesting that peptides may function asneurotransmitters ofNANC nerves in the lung. Sev-eral regulatory peptides have recently beenidentified by radioimmunoassay in lung tissue ofseveral species (including man); they include VIP,substance P, bombesin, cholecystokinin, andsomatostatin.2728 Immunocytochemical studies havedemonstrated that some of these peptides are local-ised to nerves within the airway.

VIP and NANC neurotransmintter

There is now considerable evidence in favour of VIPas a neurotransmitter of NANC nerves in the air-way. VIP is a 28 amino acid peptide which was orig-inally discovered by Said as a vasoactive substancein lung extracts,29 and was later shown to relax air-way smooth muscle in vitro.30 More recently VIP hasbeen found in both animal and human lungs local-

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ised to neurones and nerve terminals in airwaysmooth muscle (particularly in the upper airways),around submucosal glands, beneath airway epi-thelial cells, and in bronchial and pulmonaryvessels. 273132 In addition, VIP is localised in airwayganglia in both neurones and preganglionic nerveterminals (CB Basbaum, PJ Barnes, unpublishedobservations). VIP produces prolonged relaxationof airway smooth muscle in vitro, which is unaf-fected by adrenergic or cholinergic blockers.2'2230Moreover, VIP mimics the electrophysiologicalchanges in airway smooth muscle produced byNANC nerve stimulation.2' 22 In vivo inhaled VIPprotects against histamine induced bronchoconstric-tion in dogs'9 and guinea pigs,33 and infused VIPreverses serotonin induced bronchoconstriction incats.34 In isolated human airways VIP has a smallrelaxant effect8 and inhaled VIP has only a weakprotective effect against histamine induced bron-choconstriction compared with a /8 agonist.35 VIPgiven by infusion has a small bronchodilator effectand protects against histamine induced wheeze inasthmatics,36 although the cardiovascular actions ofthe infused peptide may have accounted for theseeffects. The relative lack of effect of VIP in humanairways by no means excludes it as a possibleneurotransmitter as the exogenous peptide mayhave relatively poor access to airway smooth musclecompared with peptide released from nerves withinthe muscle.

Electrical field stimulation of tracheobronchialpreparations releases VIP into the bathing mediumand the release is blocked by tetrodotoxin, indicat-ing that VIP is derived from nerve stimulation.223'Furthermore, the amount of VIP released is relatedto the magnitude of NANC relaxation.3' Unfortu-nately no specific blocker of VIP is yet available, butprolonged incubation of airway smooth muscle withVIP reduces subsequent responses to VIP(tachyphylaxis) and also reduces the magnitude ofNANC nerve relaxation in cats.2' Preincubation ofguinea pig trachea with a specific antibody to VIPalso reduces the NANC relaxation response.37Taken together, all this evidence points strongly toVIP as a likely candidate for the role of neurotrans-mitter of non-adrenergic inhibitory nerves. Conclu-sive proof, particularly in human airways, awaitsdevelopment of a specific blocker.

Other VIPergic nerves in lung

VIPergic nerves may regulate other aspects of pul-monary function, since they are widely distributedwithin the lung. VIPergic nerves are closely associ-ated with airway submuscosal glands27 32 and in vitroVIP stimulates secretion of mucus from tracheal

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glands in ferrets." VIP is therefore a possibleneurotransmitter of non-adrenergic, non-cholinergic nervous secretion of mucus. VIP has anunexpected inhibitory effect on secretion of mac-romolecules from isolated human airways, how-ever.39 VIPergic nerves are also present in closeassociation with the airway epithelium and VIP is apotent stimulant of active ion transport and there-fore water secretion across airway epithelium indogs.40 Thus VIPergic nerves may regulate secretionof both mucus and water, and so influence mucocili-ary transport in the airways.VIP is a potent vasodilator in the pulmonary cir-

culation, and the finding of a rich supply of VIPergicnerves in pulmonary vessels indicates that it is quitelikely to be the mediator for NANC nervous vasodi-lation in the lung.'9 VIPergic nerves may also mod-ulate cholinergic neurotransmission in airway gang-lia. VIP inhibits antigen induced release of his-tamine from sensitised guinea pig lung fragments invitro4' and this raises the possibility that VIPergicnerves also regulate mast cell mediator secretion.

Mode of action of VIP

VIP is released from nerve terminals and stimulatesspecific receptors, which have been demonstrated inlung membranes by radioligand binding techniqueswith labelled VIP.42 Preliminary results with auto-radiography of radiolabelled VIP indicate that thereceptors are widely distributed within the lung (ANimmo et al, unpublished observations). Stimula-tion of VIP receptors activates adenylate cyclase,thereby increasing the cellular concentration ofcyclic AMP. Thus VIP increases the cyclic AMPcontent of lung and trachea,43 and this increase incyclic AMP has been localised immunocytochemi-cally to epithelial cells, submucosal glands, and air-way smooth muscle cells.44 Beta adrenoceptor agon-ists also activate adenylate cyclase and thephysiological effects of VIP on the airways are simi-lar to those of m agonists. It is possible that theremay be a deficiency in VIP receptors in the airwaysof asthmatic patients, in the same way that (8adrenoceptor dysfunction has been postulated as anunderlying mechanism of asthma.45 The protectiveeffect of VIP against histamine induced bron-choconstriction is not, however, correlated with thedegree of bronchial hyperreactivity, which arguesagainst a defect in VIP receptors that contributes tothis phenomenon."

VIPergic nerves are usually distributed withcholinergic nerves. Ultrastructural evidence suggeststhat VIP may coexist in the same nerve terminals asacetylcholine and may therefore function as a co-transmitter.46 Possibly VIP, in addition to a direct

effect on VIP receptors of target cells, also has aprejunctional effect on acetylcholine release or, asdemonstrated in submaxillary glands,47 an effect onpostjunctional cholinergic receptors. The relation-ship between cholinergic and VIPergic neurotrans-mission in the lung is worthy of further exploration.

Substance P and non-cholinergic excitatory nerves

Substance P, an 11 amino acid peptide, is localisedto afferent nerves in the airways of several species,including man.2748 Substance P immunoreactivenerves in the airway are found beneath and withinthe airway epithelium, around blood vessels, and toa lesser extent within airway smooth muscle. Treat-ment of animals with capsaicin, a pungent extract ofred peppers, releases substance P from sensorynerves and depletes the lung of substance P.49 Sub-stance P contracts airway smooth muscle of severalspecies, including man.4850 Moreover, capsaicin iscapable of inducing a similar contraction, indicatingrelease of substance P from intrinsic nerves withinairway smooth muscle.50 In rats substance P andcapsaicin induce oedema of the airway mucosa byincreasing vascular permeability, and depletion ofsubstance P nerves by neonatal capsaicin pretreat-ment prevents irritants such as cigarette smoke fromcausing mucosal oedema, suggesting that substanceP nerves mediate this effect.5' Substance P alsostimulates airway mucus secretion.5' Electricalstimulation of guinea pig bronchi in vitro produces acomponent of bronchoconstriction which is notinhibited by atropine,5253 but which is blocked by asubstance P antagonist.52 In human airways, how-ever, this non-cholinergic non-adrenergic excitatorynervous pathway was found in only one out of eightairways tested.50 In man inhalation of capsaicininduces cough and transient bronchoconstrictionwhich is seen in both normal and asthmatic sub-jects,54 and substance P is a bronchoconstrictorwhen given by inhalation (R Fuller et al, unpub-lished observations).

Other regulatory peptides in lung

Several other peptides have recently been found inlung. Peptide histidine isoleucine (PHI) is structur-ally similar to VIP and is localised to VIPergicnerves.55 PHI, like VIP, relaxes airway smooth mus-cle but stimulates specific PHI receptors. It is there-fore another candidate for the role of neurotrans-mitter of NANC nerves and probably functions inconjunction with VIP. A newly discovered peptide,neuropeptide Y (NPY), has also been localised tonerves in the lung and is associated with sympatheticnerves, so that it is found in blood vessels rather

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than airway smooth muscle in human airways.2856Cholecystokinin and somatostatin, which function asneurotransmitters in the gut, have also beenidentified in airways, although in concentrations toolow to permit localisation at present.27 Another pep-tide, bombesin, causes bronchoconstriction in highconcentrations,5' but is localised to neuroendocrinecells rather than nerves.27 As assay techniques andisolation procedures improve it is almost certain thatother peptides will be identified in the respiratorytract.

Clinical significance of NANC nerves in lung

The recent discovery of NANC innervation in thelung has important clinical implications and maylead to further understanding and new treatment ofseveral pulmonary diseases. Asthmatic subjectsshow exaggerated bronchoconstrictor responses to awide variety of stimuli, and, because changes in air-way tone are rapid, it is suggested that abnormalnervous control of the airway is the underlyingmechanism of bronchial hyperreactivity, with a pre-ponderance of excitatory (cholinergic and a

adrenergic) or a deficiency of inhibitory (,8adrenergic) control. Although minor abnormalitiesin classical autonomic mechanisms have been foundin asthma, they are insufficient to account for bron-chial hyperreactivity.' The recent discovery of non-adrenergic inhibitory nerves in the airways hastherefore raised the possibility that this nervous sys-tem is defective in asthma, particularly since it is theonly neural inhibitory system in human airways.Non-adrenergic inhibitory nerves may exert a brak-ing effect on bronchoconstriction, and a functionaldefect would presumably lead to exaggeratedresponses to constrictor stimuli. Unfortunately theabsence of specific blocking drugs for these nervesmakes it difficult to evaluate this possibility at pres-ent, although it seems likely that specific inhibitorsof putative neurotransmitters such as VIP will bedeveloped soon. There are analogies betweenasthma and other conditions in which there isabnormal smooth muscle control and in whichabnormalities of peptidergic nerves have beenfound. In Hirschprung' s disease there is contractionof colonic smooth muscle and an absence of gangliaand peptidergic nerves in the colon wall.58 A similarreduction in VIPergic nerves has also been found inthe wall of unstable bladders.58The demonstration of substance P containing

nerves in human airways is also clinically relevantsince this peptide can produce bronchoconstriction,bronchial mucosal oedema, and mucus hypersecre-tion, all of which are features of asthma. In humanskin substance P produces a weal and flare

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response when injected intradermally48 and causesthe direct release of histamine from human skinmast cells in vivo (D Heavey et al, unpublishedobservations). If this were to occur in the airwaythen stimulation of substance P nerves could lead toa local axon reflex, with release of substance P,resulting in bronchial smooth muscle contractionand mucosal oedema. It is tempting to speculate thatin asthma these substance P nerves may be sensi-tised by inflammation, or that airway mast cells maybe more sensitive to substance P, so that constrictorresponses become exaggerated.The clinical implications of NANC innervation in

the lungs are only now being appreciated, butabnormal function of non-adrenergic inhibitorynerves (which are probably VIPergic and PHIergic)or of non-cholinergic excitatory nerves (probablysubstance P containing) could underly bronchialhyperreactivity in asthma. NANC nerves alsoinfluence airway mucus secretion and pulmonaryvascular tone, so that there are important implica-tions for chronic bronchitis and for pulmonary vas-cular disease. Future development of novel drugswhich interact with NANC neurotransmissionshould lead to new forms of treatment for these dis-eases. It is certain that much more will be heard ofNANC nerves and their neurotransmitters in thelungs as their physiology and pathophysiology areunravelled.

PETER J BARNESDepartment of Medicine

(Respiratory Division)Royal Postgraduate Medical School

Hammersmith HospitalLondon

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