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Non-myelinated vagal lung receptors and their reflex effects on respiration in rabbits
63
Respiration Physiology (1984) 55, 63-79 Elsevier
N O N - M Y E L I N A T E D VAGAL LUNG R E C E P T O R S A N D THEIR REFLEX EFFECTS O N R E S P I R A T I O N IN RABBITS
D I A N A T R E N C H A R D I, N. J. W. R U S S E L L 2 and H E L E N E. RAYBOULD 2 IMidburst Medical Research Institute, Midhurst, Sussex GU29 OBL, and 2Bioscience 11 Department, ICI Pharmaceuticals Division, Alderley Park, Macclesfield, Cheahire SKIO 4TG, U.K.
Abstract. The response characteristics of non-myelinated vagal lung receptors have been studied in
the anaesthetized rabbit. The results indicate that the behaviour of these endings strongly resembles those found in cats and dogs and that they can be classified into 'pulmonary', 'bronchial' and 'pulmonarybronchial' groups depending on their accessibility from either circulation. Experiments involving perieardial block with local anaesthetic to exclude responses from cardiac receptors and the use of sodium dithionite as a novel stimulus to 'pulmonary' endings alone, have shown that the predominant effect of these endings in the anaesthetized rabbit is to increase respiratory frequency. Reflex responses from "bronchial' endings similar provided sufficient amount of the activating chemical was given. Bronchus Heart Lung Pulmonary receptors
Receptors Sodium dithionite Vagus nerve Ventilation
Pulmonary chemoreflexes were first investigated in detail over thirty years ago (see review by Dawes and Comroe, 1954). These reflex changes in both the respiratory and cardiovascular systems were elicited by injection of a variety of chemicals, including phenyl diguanide (PDG) into the pulmonary circulation. The afferent part of the reflexes was shown to be mediated by vagal fibres of small diameter, probably non-myclinated. When non-myelinated vagal lung afferent fibres (J receptors) were first described a few years later in cats by Paintal (1955) and subsequently in dogs (Coleridge et al., 1965) it was concluded that they provided the afferent pathway for the pulmonary chemoreflexes, since these nervc endings in the lung responded to the same chemicals that activated the chemoreflexes, with a comparable latency of onset of activity. Chemoreflexes were also described in the rabbit (Dawes et al., 1951) and although Accepted for publication 15 October 1983 0034-5687/84/S03.00 f© 1984 Elsevier Science Publishers B.V.
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D. T R E N C H A R D et al.
no detailed studies of the sensory receptors giving rise to these reflexes have been carried out in this species, Guz and Trenchard (1971) showed, using differential anodal block of the vagus, that the reflexes were at least mediated by non-myelinated thoracic vagal afferent axons. The sensory innervation of the rabbit lung is similar to other species with respect to pulmonary stretch and irritant receptors, but apart from a brief mention of the existence of vagal receptors with non-myelinated fibres responding to P D G (Sellick and Widdicombe, 1970), there has been no detailed investigation of the characteristics of these receptors in the rabbit. The present study was designed to look at the response characteristics and distribution of these nerve endings in the rabbit, and the reflexes arising from their stimulation. Preliminary reports of some of these results have been published Russell and Trenchard, 1980, 1981). Methods
Surgical and recording techniques Adult rabbits weighing 2.5-4.0 kg were used in these studies. Initial surgery was performed under halothane (2 3%) anaesthesia in 50% N20/ 50% 02, which was replaced by intravenous sodium pentobarbitone (30 mg/kg) at least 30 min betbre any recordings were made. Throughout each study the animals were kept hyperoxic (Pao, > 300 mm Hg) by the addition of oxygen to the inspired air, and rectal temperature was maintained at normal levels by external heating lamps. A tracheal cannula was inserted and connected to a pneumotachograph (Fleisch No. 0) and differential strain gauge (Statham, PM 15) for measurement of air flow; subsequent integration provided a recording of tidal volume. Airway pressure was measured from the can.nula between trachea and pneumotachograph, and airway [CO2] analysed with an infra-red CO2 analyser (Beckman LB2). Catheters were inserted to the region of the vena caval/right atrial junction via an external jugular vein; to the root of the aorta via the left carotid artery; and into the abdominal aorta via a femoral artery. The latter catheter was used for monitoring arterial pressure, the two tbrmer catheters for injection of test chemicals. In the studies recording single nerve fibre activity in open-chested animals (see later), catheters were inserted directly into the left atrium, instead of the aortic root. However, for simplicity in the text, all injections into the left side of the heart (left atrium or aortic root) are referred to as left atrial (LA). The two sites were sufl'iciently close to make no significant difference to the injection-response times measured in these experiments. The positions of the catheter tips were always confirmed post mortem. In 6 rabbits, a catheter was also inserted into the pericardium through a small incision and sealed into place with a purse-string suture. This catheter was exteriorized through the left-side thoracotomy before closing the rib space and reinflating the lung. This catheter was subsequently used for injection of local anaesthetic (2~o lignoeaine) into the pericardium, to block cardiac receptors (see later).
NON-MYELINATED L.UNG RECEPTORS
65
In all animals the oesophagus was sectioned below the diaphragm through a midline abdominal incision which was subsequently sewn up. Since the abdominal vagi form a plexus in the wall of the oesophagus at this level it ensured that there would be no interference from non-myelinated abdominal vagal receptors, which can also respond to the test chemicals.
Single-fibre recording technique The cervical vagus nerves were cxposed through a mid-line incision in the neck, either for sectioning later in the experiment or, when single-fibre recordings were to be made, one nerve usually the right - was sectioned rostrally. Functional single-unit recordings wcre made from fine filaments of the desheathed right cervical vagus nerve via bipolar platinum electrodes connected to a preamplifier (Neurolog NL103) and high gain AC amplifier (Neurolog NLI06 or Medelec AA6). The skin in the neck region formed a pool filled with liquid paraffin which was maintained between 35 and 37°C. Action potentials were displayed on an oscilloscope (Medelec MS6). All analogue signals were recorded on magnetic tape (Racal Store 7D) for later analysis. The conduction velocity of single units under investigation in this type of experiment was measured. Stimulating pulses (1 msec duration, l/sec) derived from a Devices Digitimer and isolated stimulator (Devices, type 2533), were delivered to the whole vagus nerve via bipolar Ag/AgCI electrodes situated 26 38 mm caudal to the recording electrodes. In the animals in which single-fibre recordings wcre made, the chest was opened through the sternum to permit location of the receptors in the lungs by gently prodding the lungs with a fine glass rod.
Test chemicals Chemicals injected into the cardiovascular system consisted of phenyl diguanide (PDG, 1 mg/ml) and sodium dithionite (NaD, 100 mg/ml). Phenyl diguanide has come to be used as the standard test chemical for eliciting both reflex responses from vagal non-myelinated endings in the lung, and also activity in single-fibre recordings from these endings. Injections of sodium dithionite into the carotid artery have previously been used to activate carotid chemoreceptors in anaesthetized dogs (Critchley and Ungar, 1974; Cross et al., 1979). It was originally intended to use NaD in the present experiments for this function, so that the timing of carotid chemoreceptor stimulation could be distinguished from the timing of vagal lung receptor stimulation by PDG. However, it was found that a right atrial injection of NaD (10-30 mg/kg) itself produced a vagally mediated reflex respiratory response, that originated from receptors accessible from the pulmonary circulation, but which differed from the response to P D G (see Results). This reflex response to NaD was therefore subjected to further study, and the response in single-fibre recordings from these endings was also recorded. Since NaD is a reducing agent and readily absorbs oxygen from the atmosphere, it was stored under liquid paraffin and ,samples taken for injection when needed.
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D. T R E N C H A R D et al.
The doses injected were in the range 30-150/~g/kg tbr PDG, and 10-30 mg/kg for NaD. Injection volumes of the test chemicals were kept small (0.1-0.5 ml) to ensure that the chemical could be injected as a bolus tbr accurate estimation of injection-response times. The bolus of test chemical was placed in the catheter and then rapidly flushed in with 1.5 ml of 0.9~,,, saline. The recorded event marker was activated simultaneously with the beginning of this rapid injection. Control injections of this volume of saline alone, never elicited any response.
Reflex studies Sodium dithionite.
Since NaD is known to stimulate carotid chemoreceptors in other species, it was necessary to determine how much of the response to NaD~A could be attributed to such stimulation. As well as comparing the reflex responses to NaD with that to PDG, the responses to a range of doses of NaDRA (usually 10-30 mg/kg) were also recorded, and repeated during, and after recovery from, a temporary occlusion of both carotid arteries.
Phenyl diguanide: dose-adjustment studies. It has previously been found (e.g., Jain et al., 1973) that when equal doses of P D G are injected into the left and right atria of anaesthetized rabbits, the respiratory responses usually differ from each other (e.g., see fig. 3). One possible explanation is that receptors with similar characteristics are activated by the two injections, but that greatly differing amounts of the test chemical may reach the two groups, due to dilution within the bloodstream. Accordingly, the PDGL^ dose was increased or conversely the PDGR~ dose was decreased from standard doses of 250 pg and the responses compared.
Pericardialblock.
An alternative explanation for the different respiratory responses to PDGRA and PDGLA injections is that two separate types of receptor are activated from each injection site, which give different reflex responses. In cats it has been shown that a large part of the reflex response to PDGLA arises from cardiac receptors - the coronary chemoreflex (Dawes and Comroe, 1954) - since it was virtually, if not completely, abolished by a pericardial block with local anaesthetic (Anand and Paintal, 1980). The same technique was applied in the present studies, using the catheter that had been inserted into the pericardium at prior surgery. After establishing the control responses to PDGRA, PDG~,A and NaDRA, small volumes of local anaesthetic ( 2 ~ lignocaine) were injected into the pericardium, and the responses to the same test chemicals recorded. After recovery from the pericardial block (approximately 30 rain) the responses were again recorded. There was a possibility of leakage of the local anaesthetic into the thoracic cavity with a subsequent direct block of the vagus nerves, which would have made interpretation of results difficult. For this reason, all studies were performed after unilateral (left) vagotomy the catheter was inserted into the left side of the pericardium. This had the effect of producing weaker, but still unequivocal, responses in the control situation, than had previously been found with both vagus nerves intact. Addition-
NON-MYELINATED LUNG RECEPTORS
67
ally, at the end of each study and prior to killing the rabbits, Evans Blue dye was injected into the pericardial catheter in exactly the same manner and volumes as had been used for the local anaesthetic. Inspection of the chest cavity at post mortem confirmed that the dye had been retained within the pericardium, with no evidence of leakage. At this stage, a small leak was deliberately introduced at the site of insertion of the catheter into the pericardium, and although further injection of the dye resulted in its appearance in the left thoracic cavity, no trace was found in the contralateral side. This indicated the extreme unlikelihood of" any of the results with pericardial block being attributable to a direct action of the local anaesthetic on the right vagus nerve within the thorax.
Results SINGLE-FIBRE RECORDINGS
Responses to phenyl diguanide A total of twenty functional single-unit recordings have been made from nonmyelinated afferent fibres with conduction velocities in the range 0.6-3.25 m • sec ~. Of these twenty fibres, nineteen had conduction velocities less than 1.5 m . sec -~. The receptors have been classified into three groups - 'pulmonary', 'bronchial' and 'pulmonary-bronchial' - on the basis of their responses to cardiovascular injections of P D G (30 150 pg/kg), and an example of each group is illustrated in fig. 1. The ' p u l m o n a r y ' group (n = 11) responded within a few seconds (0.3--4.0 sec, mean 2.2 sec) to a right atrial injection of P D G , but not to a left atrial injection, indicating accessibility from the pulmonary circulation. The 'bronchial' group (n = 6) responded later to right atrial injections (3.0--5.8 sec, mean 4.5 scc), and also responded to a left atrial injection (1.6-2.3 sec, mean 2.0 sec). If the right-to-left heart circulation time is taken into account, it indicates that this group of receptors is only accessible from the bronchial circulation. The 'pulmonary-bronchial' group (n = 3) gave a biphasic response to right atrial injections. The first phase began within 1.3 4.3 sec (mean 2.3 sec) and the second phase within 3.5-5.2 sec (mean 4.2 sec). They also responded to left atrial injections with a monophasic response within 1.3-3.0 sec (mean 2.2 see), with the timing corresponding to the second phase of the right atrial response. This group of receptors appears to be accessible from both the pulmonary and bronchial circulations. Receptors from each of the three groups have been directly located in the lung tissue by tactile stimulation. Receptor fields were estimated as approximately 4 m m 2, and appeared to be located randomly throughout the lung tissue from the hilum to the edge of the lung ipsilateral to the site of recording. The responses to the mechanical stimuli were very variable, probably reflecting both the non-standardization of the stimuli, as well as the variable depth of location of the receptor field within the lung tissue. For example, one receptor responded with 74 impulses over a
68
D. I R E N C H A R D
et al.
'PULMONARY'
~a
i
i
I
:ZIIL2_TZ21ILZ2_LW la
'BRONCHIAL'
ra
,
,
[a
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'PULMONARY--
r
a
h
BRONCHIAL'
~
. . . . . . . . . . . . . . . . . . . . . .
la
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..
-~
.
.
.
.
~l.l~llll?li3)',Lt'l~tu.k~hllj!!l
bLiJJ,[Itlt_
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Fig. 1. Typical responses in three single non-myelinated affcrent fibres of the cervical vagus nerve~, in rabbits, to similar injections of phenyl diguanide given either into the right atrium (r a) or th~ left atrium (1 a). The fibres have been assigned to one of three groups 'pulmonary', 'bronchial" oi 'pulmonary-bronchial" according to the pattern of response obtained, as described in the text. In thi., figure and in fig. 2, the test chemicals were given as a bolus ( <0.5 ml) followed by saline (I.5 ml) during the time indicated by the event marker.
p e r i o d o f 4.5 sec to a s t i m u l u s o f d u r a t i o n
0.4 sec: the f r e q u e n c y o f d i s c h a r g e
d e c r e a s e d f r o m 23 H z in the first s e c o n d to 11 H z in the last s e c o n d . A r e p e a t e d a p p l i c a t i o n o f the s a m e s t i m u l u s elicited 32 i m p l u s c s in 2.8 sec, d e c r e a s i n g f r o m 18 H z in t h e first s e c o n d to 7 H z in the last s e c o n d . O n the o t h e r h a n d a d i f f e r e n t r e c e p t o r in a n o t h e r r a b b i t r e s p o n d e d to a s i m i l a r s t i m u l u s o f 0.4 sec d u r a t i o n w i t h 19 i m p u l s e s r a n d o m l y d i s t r i b u t e d o v c r a p e r i o d o f 4.3 sec. N o q u a n t i f i c a t i o n of t h e s e r e s p o n s e s to m e c h a n i c a l stimuli are t h e r e t b r e p r e s e n t e d , but it was c o n s i s t e n t l y o b s e r v e d that all fibres r e s p o n d e d w i t h i n 0 . 2 - 0 . 4 sec o f the a p p l i c a t i o n o f the
NON-MYEHNATED I.UNG RECEPTORS
69
stimulus, and the response always outlasted the duration of the stimulus, usually for several seconds. No effects of cardiovascular injections of P D G were ever seen on fibres from pulmonary stretch receptors (n = 14), or irritant receptors (n = 7). It was important to distinguish the irritant receptors from the non-myelinated endings, since activity in some of the very small diameter myelinated fibres from the former may give a superficial resemblance to the latter. Accordingly, the characteristic shape of the impulses in the neural discharge under investigation, both spontaneous and activated by the test chemicals, was always carefully compared at a recording speed of at least 200 c m , sec ~, with the components of the compound action potentials elicited to measure the conduction velocities. No irritant receptor was detected with a conduction velocity less than 5 m • sec-~; as reported above, 19 out of the 20 non-myelinated fibres conducted at less than 1.5 m • s e c |, while the remaining one was 3.25 m - sec -~. Responses to sodium dithionite When tested on single fibres (n = 7), NaDRA consistently activated the same ' p u l m o n a r y ' non-myelinated vagal nerve endings as PDGRA (fig. 2). The response characteristics of the nerve endings differed in that a higher frcquency of impulses was attained in a much shorter burst duration with NaDRA than with PDGRA. The same doses of the two test chemicals had previously produced reflex changes in breathing similar to those illustrated in figs. 3 and 4 (see later), in the same animals prior to the single-fibre recordings, at a time when the animals were breathing spontaneously. No cffect of NaDRA on pulmonary stretch or irritant receptors was ever observed. (a)
__I
|
(b)
i'
I, I
j
] sec Fig. 2. Comparison of the responses in a single non-myclinated vagal afferent fibre of the cervical vagus nerve (conduction velocity 1.3 m/see), to right atrial injections of 80 pg/kg pheny] diguanidc (a) and ]0 mg/kg sodium dithionite (b). Reproduced from J. PhysioL (London) with permission of the editors.
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D. T R E N C H A R D et al.
R E F L E X STUDIES
There is a large problem in the presentation of quantitative data on the reflex respiratory responses to the test chemicals, since, although each rabbit gave reproducible responses, there was a great variability in the pattern of response between animals. This is illustrated in fig. 3, which represents the two extremes of variability of the control responses. The details of the patterns of response will be dealt with subsequently, but it can clearly be seen that the responses in the rabbit on the left are apparently much weaker than those on the right, although the same doses of each test chemical were given to both rabbits. Various factors could explain this difference, acting either alone or in combination. These include different receptor sensitivities in the two animals, different levels of anaesthesia, or variation in cardiac output affecting the delivery of the test chemicals to the receptor sites. Two particular problems were encountered in attempts at quantification. Firstly, whether the latency of" onset of the responses and the peak effect should be measured, or whether it should be the magnitude of the response at a pre-determined time after injection. Secondly, the changes in tidal volume presented a particular difficulty, since they are various combinations of a decrease in end-inspiratory level, an increase in functional residual capacity, together with an unknown contribution from integrator 'drift'. Different ways of presenting the data for reflex responses have been examined, but each was rejected since it was felt that none gave an accurate representation. It was also fbund that experimental manoeuvres such as pericardial block produced unequivocal effects in each rabbit. The subsequent reflex responses have therefore been presented as qualitative results illustrated by typical examples. A
V 1 {ml} (in I )
+
'
"°°'° o ]AD't/L/c/t/t/t/t/t/Tt/t #,
B
,itiTtitfTtf(','tlTt41t4t44!(ttl 4' seconds
Fig. 3. Reflex respiratory responses in 2 rabbits (A,B) to similar injections of tcst chemicals, illustrating the extremes of the range of responses encountered. Phenyl diguanide (PDG. 83 #g/kg) was injected into either the right (ra) or left (la) atria: sodium dithionite (NaD, 10 mg/kg) was injected into the right atrium only. All injections commenced at arrow.
71
N O N - M Y E I . I N A T E D LUNG RECEPTORS
Sodium dithionite Right atrial injections of sodium dithionite (NaDRA) and phenyl diguanide (PDGRA) in 29 rabbits, both produced reflex changes in breathing with the same latency (0.5-1.5 sec), and usually showing similar increases in both end-expiratory lung volume and the frequency of breathing (fig. 4). However, with NaD there was only a little, or no, reduction in tidal volume, while with PDG there was a significant decrease in tidal volume. One other major distinction between the reflex responses was the non-appearance, following NaDRA injections, of the profound and rapid hypotension and bradycardia invariably associated with PDGRA injections. All respiratory and cardiovascular responses to the test chemicals were abolished by vagotomy. Increasing the dose of NaD produced a more marked rapid, shallow breathing within the first few breaths of the response, and at the highest dose produced a second prominent component consisting of very large breaths (fig. 5). That this second component was due to carotid chemoreceptor stimulation is illustrated in fig. 6, where this part of the response disappeared when the carotid arteries were temporarily occluded in six rabbits. The doses of NaD used subsequently for lung receptor stimulation were always well below this level that stimulated carotid chemoreceptors. (al
(in ~' ) 1(30- ~
Bp J
(mm Hg
t t
0
(b) VI(ml)(in t ) ~O] JL/~/~/L~LI~]~/~L~V~/I~
,
t lO0(mm Hg)
t 0
(c)
t I)s~l Fig. 4. Typical reflex responses in one rabbit to right atrial injections of (a) phenyl diguanide (62.5 ug/ kg) and (b,c) sodium dithionite (10 mg/kg). Vagus nerves intact in a and b, sectioned in c. The test chemicals wcre given as a bolus at the times indicated by the arrows. In this and subsequent figures, V I = tidal volu,nc, BP = arterial pressure.
72
D. T R E N C H A R D et al.
VT (ml) (in f )
t ]Omgl kg
f
12.5mg/kg
t
15mglkg
/
t
r~l
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2 sec
l':i 8. 5. The effect of increasing the dose of sodium dithionitc on the respiratory response to right atrial injections (given at arrows).
(a) VT (ml) (in ¢ )
t BP Hg)
~mm
0 (b) V T (ml) (in t )
t 150
BP
t
(mm Hg) 0
i
i 5 s~:
Fig. 6. The reflex responses to right atrial injections of sodium dithionite (20 mg/'kg, given at arrows) in the control situation (a) and after temporary occlusion of both carotid arteries (b).
73
NON-MYELINATED LUNG RECEPTORS
VT(ml) Z} {in 1') t
BP 100't Hg) 0 6 PETC02°/° 1 0
t
(ram
1
i
f sec,
Fig. 7. Typical change in expired CO2 concentration (PEIco~) accompanying the reflex responses to right atrial injections of sodium dithionite (12.5 mg/kg).
Coincident with the respiratory response to NaDRA, it was consistently observed that there was a transient increase in end-tidal CO2 concentration for a few seconds duration (fig. 7). From control values of PET(o: in the range 3.8 4.9~o (mean 4.5~o), the peak effect was in the rang~: 5.1-7.6~ (mean 6.50/o), for an injection of 10 mg/kg NaD. When comparable doses of NaD were injected via the left atrium, there were marked movements of the animal, primarily due to leg and abdominal muscle contractions, similar to that seen with a nociceptive stimulus. These movements produced effects on breathing that masked any reflex respiratory responses that may have occurred, and the total effects were unaltered by vagotomy. Reducing the dose of NaD so that these movements were not present, also eliminated any respiratory response. PDG 'dose-adjustment' experiments Fig. 3 compared the vagal reflex responses arising from right and left atrial injections of a standard dose of 250 pg of PDG. Left atrial injections consistently produced respiratory responses which at first sight appeared quite distinct from equivalent right atrial injections. It was found however, that the respiratory responses to PDGRA could be matched by an increase of the PDGL,~ dose, and vice versa where a reduced PDGRA dose matched the reflex response to PDGLA (figs. 8A and 9A). In the nine rabbits in this series of experiments, similar responses to right and left atrial injections were usually produced when the left atrial dose was approximately five times greater than the right atrial dose. Pericardial block This technique was applied on 15 occasions in six rabbits in the present studies, in order to block any responses from cardiac receptors to injections of the test
74
D. TRENCIIARD
et al.
(a)
Vllint, lm')
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~~¢li,
iIf!~ll! IL~l ! !i.It'I![ ~!1 t
] 0 0 |~
.,,,.,.,.,'......,.,~¥ ~ ."~
~ ....
(mmHg} 0
(b)
,,or,
BP
(ramHg) 0
Fig. 8. Reflex responses to right atrial injections of phenyl diguanide (100 #g) given before (a) and during (b) a pericardial block of cardiac receptors.
chemicals. The results were unequivocal in spite of the weaker control responses after unilateral vagotomy. In every case the respiratory response to NaDRa was unchanged, the response to PDGRa was considerably reduced and virtually limited to a small increase in respiratory frequency (fig. 8B). The response to 250 /~g PDGI. A was abolished completely, but when higher doses of PDGLA were administered, then a small recognisable respiratory responsc remained during the pericardial block (fig. 9B). Additionally, the hypotensive response to both PDGRA and PDGLA were usually abolished or profoundly diminished there were no cardiovascular responses to NaDRa in the control situation. It should be noted that the residual response to PDGRA during pericardial block has a strong resemblance to the NaD~A response, in that it is primarily an effect on the frequency of breathing alone, with little or no effect on tidal volume or blood pressure. In four of the six rabbits, the same volume of local anaesthetic that had previously been used to establish the pericardial block - usually 0.4 ml was injected intravenously. No effects were produced on the reflex responses to P D G and NaD, indicating that systemic absorption of the local anaesthetic had not contributed to any of the effects seen with pericardial block.
75
N O N - M Y E I . I N A T E D LUNG RECEPTORS (a)
t
BP
lO01
" ~"~7
," .
(ramHg) 0
t
(b)
BP (mmHg)
' ' " ~' ' o
15s ~
I
Fig. 9. Reflex responses to left atrial injcctions of phenyl diguanide (1250 /~g) given before (a) and during (b) a pericardial block of cardiac rcceptors. Note the similarity of the control responses to left and right (fig. 8a) atrial injections, when the dose of the formcr is increased.
Discussion
The response characteristics of the non-myelinated vagal afferent nerve endings in the lungs of anaesthetized rabbits, with respect to their pharmacologi~l activation, resemble those o f cat and dog. In addition, their separation into three groups on the basis of their accessibility from the pulmonary or bronchial circulations is similar to the situation in cats and dogs, with the exception that the 'pulmonarybronchial' group have not yet been described in dogs (Coleridge and Coleridge, 1977; Dclpierre et al., 1980). The results can give no indication of the relative numbers of these nerve endings within the three groups. The accessibility of these endings from either the pulmonary or bronchial circulations does not imply clear separation of location within the airways, but the response latencies must reflect the passage of the drug to sites at varying distances from the point of injection. it is best concluded that accessibility from one or other or both circulations reflects a widespread distribution of these endings within the lungs, in contrast to the original idea that these non-myelinated vagal afferent endings (at that time called
76
D. T R E N C H A R D et al.
J receptors) might be predominantly peripheral, and accessible only from the pulmonary circulation (Paintal, 1969). To underline this conclusion that the vagal non-myelinated afferent innervation of the lungs is one overall group, it was shown that when appropriate measures were taken, "pulmonary' and 'bronchial' endings can subserve identical respiratory reflex functions. Thus it was originally assumed that the reflex responses to left atrial injections ofphenyl diguanide were mediated exclusively by the 'bronchial' receptors and that they appeared to evoke a different change in the pattern of respiration from 'pulmonary' receptors (fig. 3). However, the 'dose-adjustment' series of experiments suggested that the different responses to the same dose of phenyl diguanide injected via the right or left atrium may be purely a reflection of the dilution of the drug from the latter injection before reaching the receptors (figs. 8A and 9A). The subsequent studies with pericardial block, however, showed that in the rabbit a large proportion of the reflex respiratory response to phenyl diguanide injected into the right or left atrium arises from stimulation of cardiac receptors. The cardiovascular responses to both PDGRA and PDGLA were abolished by pericardial block, which implies that they must also arise from stimulation by PDG of cardiac receptors. This conclusion can be reached since block of vagal efferent transmission by atropine alone, results in abolition of the bradycardia response to PDG whilst sparing the transient vagal afferent-dependent hypotension. A significant proportion of the respiratory responses to PDGRA and PDG~.A must therefore also be produced by stimulation of cardiac receptors, since both of these were reduced during pericardial block. However, if sufficient PDGLA is administered (fig. 9B) to overcome the dilution problem mentioned above, there is a recognisable residual respiratory response which must presumably originate from 'bronchial" non-myelihated vagal receptors. The PDGLA response produced under these conditions is qualitatively similar to PDGRA alter pericardial block. Quantitative comparisons are difficult to make, but this result strongly suggests that 'pulmonary' and "bronchial' receptors in the rabbit may have the same reflex effect on respiration. 'Pulmonary" and 'bronchial" C-fibres in the dog have identical bronchoconstrictor reflex effects (Roberts" et al., 1981) and indeed it would be surprising if these two groups of receptors had been found to be functionally distinct, since all the evidence suggests that sensory endings with non-myelinated vagal afferents constitute a widespread nociceptive system within the lungs, with different accessibilities from pulmonary or bronchial capillaries depending on their particular location. The only argument against this generalisation is the apparent difference in the chemosensitivity, to bradykinin in particular, of 'pulmonary' and 'bronchial' C-fibre endings in the dog (Kaufman et al. 1980). The studies with NaD in the rabbit present novel evidence to suggest that in appropriate doses, NaDRA provides a specific stimulus to 'pulmonary' vagal nonmyelinated endings, and that the reflex responses elicited by these endings consist of an increase in both the frequency o1" breathing and functional residual capacity: no reflex effects are produced on either tidal volume, heart rate or blood pressure.
NON-MYELINATED LUNG RECEPTORS
77
However, before presenting the evidence in favour of this conclusion, it is important to discuss the use of sodium dithionite, since it has been used as a novel stimulus in these studies. The mode of action of this chemical remains unclear. At first it was assumed that NaD injection might be providing a hypoxic stimulus, (since NaD is a reducing agent), as had previously been suggested for carotid chemoreceptor stimulation (Critchley and Ungar, 1974; Cross et al., 1979). This was rejected since the rabbits in the present studies were kept hyperoxic (Pao: > 300 mm Hg), and carotid artery occlusion did not alter the pattern of response. Additionally, comparable in vitro addition of NaD to rabbit blood samples tonometered at 37°C to give blood gas and pH values comparable to those found in vivo, never reduced Po~ below 150 mm Hg. The in vitro studies did, however, increase Pco2 by 10.-20 mm Hg, which corresponded closely to the effects seen in vivo where there was a transient increase in PETco: by 1 - 3 ~ after injection of NaDRA (fig. 7). (It should be noted that similar effects on Po:, Pco: and pH can also be elicited in vitro by the addition of NaD to a bicarbonate solution that had been tonometred to the same values as the blood samples.) It is therefore more probable that this increase in CO2 (or accompanying increase in H ÷) induced by NaDE~A, provided an indirect stimulus to the non-myelinated nerve endings, by releasing a substantial amount of CO2 from the blood in the pulmonary circulation. NaD does not appear penetrate cell membranes readily, and would tend to remain in the intravascular compartment (Burns and Shepard, 1979); this reinforces the suggestion that it is the CO:-releasing properties of NaD that provides the stimulus to non-myelinated endings. The reasons why we believe that the stimulus provided by smaller doses of NaD does not survive beyond the pulmonary circulation are as follows. The prominent increase in end-tidal Pco: as the test chemical passes through the pulmonary circulation, clearly indicates that a large amount of the released CO2 has escaped from the blood. The possibility remains that a small CO2 stimulus will survive through the pulmonary veins into the left heart, to stimulate receptors not of pulmonary origin. However, the evidence that the response to NaD~A is unaffected by pericardial block strongly suggests that this is not the case. In addition, the total dependence of the reflex response on the integrity of the vagus nerves, and the appearance at higher doses of an obvious peripheral chemoreceptor stimulation, supports the conclusion that provided the dose is small enough, the origin of the reflex is limited to vagal endings in the pulmonary circulation. The residual response to PDGRA during pericardial block undoubtedly originates from pure 'pulmonary' receptor stimulation, and qualitatively is indistinguishable from the responses to NaDRa. This lends further support to the conclusion that NaDRA can serve as a specific stimulus to 'pulmonary' vagal non-myelinated endings. Although the evidence from the present studies has suggested that these 'pulmonary' nerve endings are stimulated by increases in CO2 or H ÷, it is recognised that this has been demonstrated under abnormal conditions. It would therefore be incorrect to make conclusions about a natural CO2-sensor role for these endings
78
D. TRENCHARD et al.
on the basis of the present results alone. However, p r e l i m i n a r y results from current investigations in anaesthetized r a b b i t s are available ( R a y b o u l d a n d Russell, 1982), which show that n o n - m y e l i n a t e d vagal afferent nerve fibres o f thoracic origin can mediate the reflex increase in the frcquency o f breathing in response to hypercapnia. In conclus&n, the present results indicate that the characteristics of the n o n myelinated e n d i n g s in the lungs o f anaesthetized rabbits, strongly resemble those f o u n d in cats a n d dogs. The use o f s o d i u m dithionite as a novel stimulus to the e n d i n g s only accessible from the p u l m o n a r y circulation, c o m b i n e d with pericardial block, has indicated that in rabbits most of the respiratory response a n d all ot the cardiovascular responses classically a t t r i b u t e d to chemoreflexes initiated from these receptors, is in fact due to s t i m u l a t i o n of cardiac receptors. A c t i v a t i o n ot ' p u l m o n a r y ' endings produces o n l y a n increased frequency o f b r e a t h i n g in anaesthetized r a b b i t s : ' b r o n c h i a l ' e n d i n g s a p p a r e n t l y give a similar response provided a sufficient a m o u n t of the activating chemical is given.
Acknowledgement The a u t h o r s arc indebtcd to Professor A . S . Paintal for d c m o n s t r a t i n g the technique o f pericardial block.
References Anand, A. and A.S. Paintal (1980). Reflex effects following selective stimulation of J receptors in the cat. J. Physiol. (London) 299: 553-572. Burns, B. and R.H. Shcpard (1979). Dlo2 in excised lungs pcrfused with blood containing sodium dithionitc (Na2S204). J. Appl. Physiol. 46: 100-.I10. Coleridge, H.M.. J.C.G. Coleridge and J.C. Luck (1965). Pulmonary afferent fibres of small diameter stimulated by capsaicin and by hyperinflation of the lungs. J. Physiol. (l,ondon) 179: 248 262. Coleridge, 14. M. and J. C. G. Coleridge (1977). Impulse activity in afferent vagal C-fibres with endings in the intra-pulmonary airways of dogs. Respir. Physiol. 29: 125-142. Critchlcy, J.A.J.H. and A. Ungar (1974). A chemical method of lowering the Po2 of blood in experimental studies of arterial chemoreceptor reflexes. J. Physiol. (London) 244: 12- 13P. Cross, B.A., B.J.B. Grant, A. Guz, P.W. Jones, S.J.G. Semplc and R.P. Stidwell (1979). Dependence of phrenic motoneuronc output on the oscillatory component of arterial blood gas composition. J. Physiol. (London) 290:163 184. Dawes, G.S., J.C. Mott and J.G. Widdicombe (1951). Respiratory and cardiovascular reflexes from heart and lungs. J. Physiol. (London) 115: 258-291. Dav,es, G.S. and J. t4. Comroe (1954). Chemoreflexes from the heart and lungs. Physiol. Rev. 34: 167-201. Delpierre, S., Y. Jammes and N. Mei (1980). Effects of hypercapnia, hypoxia and increase of tidal volume on vagal bronchopulmonary C fibres in cat. J. Physiol. (London) 298: 48- 49P. Delpierre, S., C. Grimaud, Y. Jammes and N. Mei (1981). Changes ill activity of vagal bronchopulmonary C fibres by chemical and physical stimuli in the eat. J. Physiol. ILondon) 316: 61.-74. Dickinson, C.J. and A.S. Paintal (1970). Stimulation of type-J pulmonary receptors in the cat by carbeon dioxide, tiT&. Sci. 38: 33P.
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Guz, A. and D. Trenchard (1971). The role of non-myelinated vagal afferent fibres in the genesis of tachypnoea in the rabbit. J. Physiol. (London) 213:.2,45 371. Jain, S.K., D. -l'renchard, F. Reynolds, M.I.M. Noble and A. Guz (1973). The effect of local anaesthesia of the airway on respiratory reflexes in the rabbit. Clin. Sci. 44:519 538. Kaufman, M.P., D.G. Baker, H.M. Coleridge and J. C.G. Coleridge (1980). Stimulation by bradykinin of afferent vagal C-fibres with chemosensitive endings in the heart and aorta of the dog. Circ. Res. 46:476 484. Paintal, A.S. (1955). Impulses in vagal afferent fibres from specific deflation receptors. The response of these receptors to phenyl diguanide, potato starch, 5-hydroxytryptamine and nicotine, and their role in respiratory and cardiovascular reflexes. Q. J. Exp. Physiol. 40: 8% 111. Paintal, A.S. (1969). Mechanism of stimulation of type J pulmonary receptors. J. PhysioL (London) 203:511 532. Raybould, H.E. and N.J.W. Russell (1982). Afferent activity in pulmonary vagal C-fibres reflexly increases respiratory rate during hypercapnia in the anaesthetized rabbit. J. PhysioL (London) 326:60 61P. Roberts, A.M., M.P. Kaufman, D.G. Baker, J.K. Brown, H.M. Coleridge and J.C.G. Coleridge (1981). Reflex tracheal constriction induced by stimulation of bronchial C-fibres in dogs. J. Appl. Physiol. 51 : 485~1.93. Russell. N.J.W. and D. "Frcnchard (1980). Non-myelinated vagal lung receptors in the rabbit. J. Physiol. (London) 300: 31P. Russell, N.J.W. and D. Trenchard (1981). Chemoreflexes of pulmonary origin elicited by sodium dithionite in the anaesthetized rabbit. J. Physiol. (London) 310: 63-64P. Sellick, H. and J.G. Widdicombe (1970). Vagal deflation and inflation reflexes mediated by lung irritant receptors. Q. J. Exp. Physiol. 55:153 163.
Respiration Physiology (1984) 55, 63-79 Elsevier
N O N - M Y E L I N A T E D VAGAL LUNG R E C E P T O R S A N D THEIR REFLEX EFFECTS O N R E S P I R A T I O N IN RABBITS
D I A N A T R E N C H A R D I, N. J. W. R U S S E L L 2 and H E L E N E. RAYBOULD 2 IMidburst Medical Research Institute, Midhurst, Sussex GU29 OBL, and 2Bioscience 11 Department, ICI Pharmaceuticals Division, Alderley Park, Macclesfield, Cheahire SKIO 4TG, U.K.
Abstract. The response characteristics of non-myelinated vagal lung receptors have been studied in
the anaesthetized rabbit. The results indicate that the behaviour of these endings strongly resembles those found in cats and dogs and that they can be classified into 'pulmonary', 'bronchial' and 'pulmonarybronchial' groups depending on their accessibility from either circulation. Experiments involving perieardial block with local anaesthetic to exclude responses from cardiac receptors and the use of sodium dithionite as a novel stimulus to 'pulmonary' endings alone, have shown that the predominant effect of these endings in the anaesthetized rabbit is to increase respiratory frequency. Reflex responses from "bronchial' endings similar provided sufficient amount of the activating chemical was given. Bronchus Heart Lung Pulmonary receptors
Receptors Sodium dithionite Vagus nerve Ventilation
Pulmonary chemoreflexes were first investigated in detail over thirty years ago (see review by Dawes and Comroe, 1954). These reflex changes in both the respiratory and cardiovascular systems were elicited by injection of a variety of chemicals, including phenyl diguanide (PDG) into the pulmonary circulation. The afferent part of the reflexes was shown to be mediated by vagal fibres of small diameter, probably non-myclinated. When non-myelinated vagal lung afferent fibres (J receptors) were first described a few years later in cats by Paintal (1955) and subsequently in dogs (Coleridge et al., 1965) it was concluded that they provided the afferent pathway for the pulmonary chemoreflexes, since these nervc endings in the lung responded to the same chemicals that activated the chemoreflexes, with a comparable latency of onset of activity. Chemoreflexes were also described in the rabbit (Dawes et al., 1951) and although Accepted for publication 15 October 1983 0034-5687/84/S03.00 f© 1984 Elsevier Science Publishers B.V.
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D. T R E N C H A R D et al.
no detailed studies of the sensory receptors giving rise to these reflexes have been carried out in this species, Guz and Trenchard (1971) showed, using differential anodal block of the vagus, that the reflexes were at least mediated by non-myelinated thoracic vagal afferent axons. The sensory innervation of the rabbit lung is similar to other species with respect to pulmonary stretch and irritant receptors, but apart from a brief mention of the existence of vagal receptors with non-myelinated fibres responding to P D G (Sellick and Widdicombe, 1970), there has been no detailed investigation of the characteristics of these receptors in the rabbit. The present study was designed to look at the response characteristics and distribution of these nerve endings in the rabbit, and the reflexes arising from their stimulation. Preliminary reports of some of these results have been published Russell and Trenchard, 1980, 1981). Methods
Surgical and recording techniques Adult rabbits weighing 2.5-4.0 kg were used in these studies. Initial surgery was performed under halothane (2 3%) anaesthesia in 50% N20/ 50% 02, which was replaced by intravenous sodium pentobarbitone (30 mg/kg) at least 30 min betbre any recordings were made. Throughout each study the animals were kept hyperoxic (Pao, > 300 mm Hg) by the addition of oxygen to the inspired air, and rectal temperature was maintained at normal levels by external heating lamps. A tracheal cannula was inserted and connected to a pneumotachograph (Fleisch No. 0) and differential strain gauge (Statham, PM 15) for measurement of air flow; subsequent integration provided a recording of tidal volume. Airway pressure was measured from the can.nula between trachea and pneumotachograph, and airway [CO2] analysed with an infra-red CO2 analyser (Beckman LB2). Catheters were inserted to the region of the vena caval/right atrial junction via an external jugular vein; to the root of the aorta via the left carotid artery; and into the abdominal aorta via a femoral artery. The latter catheter was used for monitoring arterial pressure, the two tbrmer catheters for injection of test chemicals. In the studies recording single nerve fibre activity in open-chested animals (see later), catheters were inserted directly into the left atrium, instead of the aortic root. However, for simplicity in the text, all injections into the left side of the heart (left atrium or aortic root) are referred to as left atrial (LA). The two sites were sufl'iciently close to make no significant difference to the injection-response times measured in these experiments. The positions of the catheter tips were always confirmed post mortem. In 6 rabbits, a catheter was also inserted into the pericardium through a small incision and sealed into place with a purse-string suture. This catheter was exteriorized through the left-side thoracotomy before closing the rib space and reinflating the lung. This catheter was subsequently used for injection of local anaesthetic (2~o lignoeaine) into the pericardium, to block cardiac receptors (see later).
NON-MYELINATED L.UNG RECEPTORS
65
In all animals the oesophagus was sectioned below the diaphragm through a midline abdominal incision which was subsequently sewn up. Since the abdominal vagi form a plexus in the wall of the oesophagus at this level it ensured that there would be no interference from non-myelinated abdominal vagal receptors, which can also respond to the test chemicals.
Single-fibre recording technique The cervical vagus nerves were cxposed through a mid-line incision in the neck, either for sectioning later in the experiment or, when single-fibre recordings were to be made, one nerve usually the right - was sectioned rostrally. Functional single-unit recordings wcre made from fine filaments of the desheathed right cervical vagus nerve via bipolar platinum electrodes connected to a preamplifier (Neurolog NL103) and high gain AC amplifier (Neurolog NLI06 or Medelec AA6). The skin in the neck region formed a pool filled with liquid paraffin which was maintained between 35 and 37°C. Action potentials were displayed on an oscilloscope (Medelec MS6). All analogue signals were recorded on magnetic tape (Racal Store 7D) for later analysis. The conduction velocity of single units under investigation in this type of experiment was measured. Stimulating pulses (1 msec duration, l/sec) derived from a Devices Digitimer and isolated stimulator (Devices, type 2533), were delivered to the whole vagus nerve via bipolar Ag/AgCI electrodes situated 26 38 mm caudal to the recording electrodes. In the animals in which single-fibre recordings wcre made, the chest was opened through the sternum to permit location of the receptors in the lungs by gently prodding the lungs with a fine glass rod.
Test chemicals Chemicals injected into the cardiovascular system consisted of phenyl diguanide (PDG, 1 mg/ml) and sodium dithionite (NaD, 100 mg/ml). Phenyl diguanide has come to be used as the standard test chemical for eliciting both reflex responses from vagal non-myelinated endings in the lung, and also activity in single-fibre recordings from these endings. Injections of sodium dithionite into the carotid artery have previously been used to activate carotid chemoreceptors in anaesthetized dogs (Critchley and Ungar, 1974; Cross et al., 1979). It was originally intended to use NaD in the present experiments for this function, so that the timing of carotid chemoreceptor stimulation could be distinguished from the timing of vagal lung receptor stimulation by PDG. However, it was found that a right atrial injection of NaD (10-30 mg/kg) itself produced a vagally mediated reflex respiratory response, that originated from receptors accessible from the pulmonary circulation, but which differed from the response to P D G (see Results). This reflex response to NaD was therefore subjected to further study, and the response in single-fibre recordings from these endings was also recorded. Since NaD is a reducing agent and readily absorbs oxygen from the atmosphere, it was stored under liquid paraffin and ,samples taken for injection when needed.
66
D. T R E N C H A R D et al.
The doses injected were in the range 30-150/~g/kg tbr PDG, and 10-30 mg/kg for NaD. Injection volumes of the test chemicals were kept small (0.1-0.5 ml) to ensure that the chemical could be injected as a bolus tbr accurate estimation of injection-response times. The bolus of test chemical was placed in the catheter and then rapidly flushed in with 1.5 ml of 0.9~,,, saline. The recorded event marker was activated simultaneously with the beginning of this rapid injection. Control injections of this volume of saline alone, never elicited any response.
Reflex studies Sodium dithionite.
Since NaD is known to stimulate carotid chemoreceptors in other species, it was necessary to determine how much of the response to NaD~A could be attributed to such stimulation. As well as comparing the reflex responses to NaD with that to PDG, the responses to a range of doses of NaDRA (usually 10-30 mg/kg) were also recorded, and repeated during, and after recovery from, a temporary occlusion of both carotid arteries.
Phenyl diguanide: dose-adjustment studies. It has previously been found (e.g., Jain et al., 1973) that when equal doses of P D G are injected into the left and right atria of anaesthetized rabbits, the respiratory responses usually differ from each other (e.g., see fig. 3). One possible explanation is that receptors with similar characteristics are activated by the two injections, but that greatly differing amounts of the test chemical may reach the two groups, due to dilution within the bloodstream. Accordingly, the PDGL^ dose was increased or conversely the PDGR~ dose was decreased from standard doses of 250 pg and the responses compared.
Pericardialblock.
An alternative explanation for the different respiratory responses to PDGRA and PDGLA injections is that two separate types of receptor are activated from each injection site, which give different reflex responses. In cats it has been shown that a large part of the reflex response to PDGLA arises from cardiac receptors - the coronary chemoreflex (Dawes and Comroe, 1954) - since it was virtually, if not completely, abolished by a pericardial block with local anaesthetic (Anand and Paintal, 1980). The same technique was applied in the present studies, using the catheter that had been inserted into the pericardium at prior surgery. After establishing the control responses to PDGRA, PDG~,A and NaDRA, small volumes of local anaesthetic ( 2 ~ lignocaine) were injected into the pericardium, and the responses to the same test chemicals recorded. After recovery from the pericardial block (approximately 30 rain) the responses were again recorded. There was a possibility of leakage of the local anaesthetic into the thoracic cavity with a subsequent direct block of the vagus nerves, which would have made interpretation of results difficult. For this reason, all studies were performed after unilateral (left) vagotomy the catheter was inserted into the left side of the pericardium. This had the effect of producing weaker, but still unequivocal, responses in the control situation, than had previously been found with both vagus nerves intact. Addition-
NON-MYELINATED LUNG RECEPTORS
67
ally, at the end of each study and prior to killing the rabbits, Evans Blue dye was injected into the pericardial catheter in exactly the same manner and volumes as had been used for the local anaesthetic. Inspection of the chest cavity at post mortem confirmed that the dye had been retained within the pericardium, with no evidence of leakage. At this stage, a small leak was deliberately introduced at the site of insertion of the catheter into the pericardium, and although further injection of the dye resulted in its appearance in the left thoracic cavity, no trace was found in the contralateral side. This indicated the extreme unlikelihood of" any of the results with pericardial block being attributable to a direct action of the local anaesthetic on the right vagus nerve within the thorax.
Results SINGLE-FIBRE RECORDINGS
Responses to phenyl diguanide A total of twenty functional single-unit recordings have been made from nonmyelinated afferent fibres with conduction velocities in the range 0.6-3.25 m • sec ~. Of these twenty fibres, nineteen had conduction velocities less than 1.5 m . sec -~. The receptors have been classified into three groups - 'pulmonary', 'bronchial' and 'pulmonary-bronchial' - on the basis of their responses to cardiovascular injections of P D G (30 150 pg/kg), and an example of each group is illustrated in fig. 1. The ' p u l m o n a r y ' group (n = 11) responded within a few seconds (0.3--4.0 sec, mean 2.2 sec) to a right atrial injection of P D G , but not to a left atrial injection, indicating accessibility from the pulmonary circulation. The 'bronchial' group (n = 6) responded later to right atrial injections (3.0--5.8 sec, mean 4.5 scc), and also responded to a left atrial injection (1.6-2.3 sec, mean 2.0 sec). If the right-to-left heart circulation time is taken into account, it indicates that this group of receptors is only accessible from the bronchial circulation. The 'pulmonary-bronchial' group (n = 3) gave a biphasic response to right atrial injections. The first phase began within 1.3 4.3 sec (mean 2.3 sec) and the second phase within 3.5-5.2 sec (mean 4.2 sec). They also responded to left atrial injections with a monophasic response within 1.3-3.0 sec (mean 2.2 see), with the timing corresponding to the second phase of the right atrial response. This group of receptors appears to be accessible from both the pulmonary and bronchial circulations. Receptors from each of the three groups have been directly located in the lung tissue by tactile stimulation. Receptor fields were estimated as approximately 4 m m 2, and appeared to be located randomly throughout the lung tissue from the hilum to the edge of the lung ipsilateral to the site of recording. The responses to the mechanical stimuli were very variable, probably reflecting both the non-standardization of the stimuli, as well as the variable depth of location of the receptor field within the lung tissue. For example, one receptor responded with 74 impulses over a
68
D. I R E N C H A R D
et al.
'PULMONARY'
~a
i
i
I
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ra
,
,
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.
.
.
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Fig. 1. Typical responses in three single non-myelinated affcrent fibres of the cervical vagus nerve~, in rabbits, to similar injections of phenyl diguanide given either into the right atrium (r a) or th~ left atrium (1 a). The fibres have been assigned to one of three groups 'pulmonary', 'bronchial" oi 'pulmonary-bronchial" according to the pattern of response obtained, as described in the text. In thi., figure and in fig. 2, the test chemicals were given as a bolus ( <0.5 ml) followed by saline (I.5 ml) during the time indicated by the event marker.
p e r i o d o f 4.5 sec to a s t i m u l u s o f d u r a t i o n
0.4 sec: the f r e q u e n c y o f d i s c h a r g e
d e c r e a s e d f r o m 23 H z in the first s e c o n d to 11 H z in the last s e c o n d . A r e p e a t e d a p p l i c a t i o n o f the s a m e s t i m u l u s elicited 32 i m p l u s c s in 2.8 sec, d e c r e a s i n g f r o m 18 H z in t h e first s e c o n d to 7 H z in the last s e c o n d . O n the o t h e r h a n d a d i f f e r e n t r e c e p t o r in a n o t h e r r a b b i t r e s p o n d e d to a s i m i l a r s t i m u l u s o f 0.4 sec d u r a t i o n w i t h 19 i m p u l s e s r a n d o m l y d i s t r i b u t e d o v c r a p e r i o d o f 4.3 sec. N o q u a n t i f i c a t i o n of t h e s e r e s p o n s e s to m e c h a n i c a l stimuli are t h e r e t b r e p r e s e n t e d , but it was c o n s i s t e n t l y o b s e r v e d that all fibres r e s p o n d e d w i t h i n 0 . 2 - 0 . 4 sec o f the a p p l i c a t i o n o f the
NON-MYEHNATED I.UNG RECEPTORS
69
stimulus, and the response always outlasted the duration of the stimulus, usually for several seconds. No effects of cardiovascular injections of P D G were ever seen on fibres from pulmonary stretch receptors (n = 14), or irritant receptors (n = 7). It was important to distinguish the irritant receptors from the non-myelinated endings, since activity in some of the very small diameter myelinated fibres from the former may give a superficial resemblance to the latter. Accordingly, the characteristic shape of the impulses in the neural discharge under investigation, both spontaneous and activated by the test chemicals, was always carefully compared at a recording speed of at least 200 c m , sec ~, with the components of the compound action potentials elicited to measure the conduction velocities. No irritant receptor was detected with a conduction velocity less than 5 m • sec-~; as reported above, 19 out of the 20 non-myelinated fibres conducted at less than 1.5 m • s e c |, while the remaining one was 3.25 m - sec -~. Responses to sodium dithionite When tested on single fibres (n = 7), NaDRA consistently activated the same ' p u l m o n a r y ' non-myelinated vagal nerve endings as PDGRA (fig. 2). The response characteristics of the nerve endings differed in that a higher frcquency of impulses was attained in a much shorter burst duration with NaDRA than with PDGRA. The same doses of the two test chemicals had previously produced reflex changes in breathing similar to those illustrated in figs. 3 and 4 (see later), in the same animals prior to the single-fibre recordings, at a time when the animals were breathing spontaneously. No cffect of NaDRA on pulmonary stretch or irritant receptors was ever observed. (a)
__I
|
(b)
i'
I, I
j
] sec Fig. 2. Comparison of the responses in a single non-myclinated vagal afferent fibre of the cervical vagus nerve (conduction velocity 1.3 m/see), to right atrial injections of 80 pg/kg pheny] diguanidc (a) and ]0 mg/kg sodium dithionite (b). Reproduced from J. PhysioL (London) with permission of the editors.
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D. T R E N C H A R D et al.
R E F L E X STUDIES
There is a large problem in the presentation of quantitative data on the reflex respiratory responses to the test chemicals, since, although each rabbit gave reproducible responses, there was a great variability in the pattern of response between animals. This is illustrated in fig. 3, which represents the two extremes of variability of the control responses. The details of the patterns of response will be dealt with subsequently, but it can clearly be seen that the responses in the rabbit on the left are apparently much weaker than those on the right, although the same doses of each test chemical were given to both rabbits. Various factors could explain this difference, acting either alone or in combination. These include different receptor sensitivities in the two animals, different levels of anaesthesia, or variation in cardiac output affecting the delivery of the test chemicals to the receptor sites. Two particular problems were encountered in attempts at quantification. Firstly, whether the latency of" onset of the responses and the peak effect should be measured, or whether it should be the magnitude of the response at a pre-determined time after injection. Secondly, the changes in tidal volume presented a particular difficulty, since they are various combinations of a decrease in end-inspiratory level, an increase in functional residual capacity, together with an unknown contribution from integrator 'drift'. Different ways of presenting the data for reflex responses have been examined, but each was rejected since it was felt that none gave an accurate representation. It was also fbund that experimental manoeuvres such as pericardial block produced unequivocal effects in each rabbit. The subsequent reflex responses have therefore been presented as qualitative results illustrated by typical examples. A
V 1 {ml} (in I )
+
'
"°°'° o ]AD't/L/c/t/t/t/t/t/Tt/t #,
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,itiTtitfTtf(','tlTt41t4t44!(ttl 4' seconds
Fig. 3. Reflex respiratory responses in 2 rabbits (A,B) to similar injections of tcst chemicals, illustrating the extremes of the range of responses encountered. Phenyl diguanide (PDG. 83 #g/kg) was injected into either the right (ra) or left (la) atria: sodium dithionite (NaD, 10 mg/kg) was injected into the right atrium only. All injections commenced at arrow.
71
N O N - M Y E I . I N A T E D LUNG RECEPTORS
Sodium dithionite Right atrial injections of sodium dithionite (NaDRA) and phenyl diguanide (PDGRA) in 29 rabbits, both produced reflex changes in breathing with the same latency (0.5-1.5 sec), and usually showing similar increases in both end-expiratory lung volume and the frequency of breathing (fig. 4). However, with NaD there was only a little, or no, reduction in tidal volume, while with PDG there was a significant decrease in tidal volume. One other major distinction between the reflex responses was the non-appearance, following NaDRA injections, of the profound and rapid hypotension and bradycardia invariably associated with PDGRA injections. All respiratory and cardiovascular responses to the test chemicals were abolished by vagotomy. Increasing the dose of NaD produced a more marked rapid, shallow breathing within the first few breaths of the response, and at the highest dose produced a second prominent component consisting of very large breaths (fig. 5). That this second component was due to carotid chemoreceptor stimulation is illustrated in fig. 6, where this part of the response disappeared when the carotid arteries were temporarily occluded in six rabbits. The doses of NaD used subsequently for lung receptor stimulation were always well below this level that stimulated carotid chemoreceptors. (al
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t I)s~l Fig. 4. Typical reflex responses in one rabbit to right atrial injections of (a) phenyl diguanide (62.5 ug/ kg) and (b,c) sodium dithionite (10 mg/kg). Vagus nerves intact in a and b, sectioned in c. The test chemicals wcre given as a bolus at the times indicated by the arrows. In this and subsequent figures, V I = tidal volu,nc, BP = arterial pressure.
72
D. T R E N C H A R D et al.
VT (ml) (in f )
t ]Omgl kg
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l':i 8. 5. The effect of increasing the dose of sodium dithionitc on the respiratory response to right atrial injections (given at arrows).
(a) VT (ml) (in ¢ )
t BP Hg)
~mm
0 (b) V T (ml) (in t )
t 150
BP
t
(mm Hg) 0
i
i 5 s~:
Fig. 6. The reflex responses to right atrial injections of sodium dithionite (20 mg/'kg, given at arrows) in the control situation (a) and after temporary occlusion of both carotid arteries (b).
73
NON-MYELINATED LUNG RECEPTORS
VT(ml) Z} {in 1') t
BP 100't Hg) 0 6 PETC02°/° 1 0
t
(ram
1
i
f sec,
Fig. 7. Typical change in expired CO2 concentration (PEIco~) accompanying the reflex responses to right atrial injections of sodium dithionite (12.5 mg/kg).
Coincident with the respiratory response to NaDRA, it was consistently observed that there was a transient increase in end-tidal CO2 concentration for a few seconds duration (fig. 7). From control values of PET(o: in the range 3.8 4.9~o (mean 4.5~o), the peak effect was in the rang~: 5.1-7.6~ (mean 6.50/o), for an injection of 10 mg/kg NaD. When comparable doses of NaD were injected via the left atrium, there were marked movements of the animal, primarily due to leg and abdominal muscle contractions, similar to that seen with a nociceptive stimulus. These movements produced effects on breathing that masked any reflex respiratory responses that may have occurred, and the total effects were unaltered by vagotomy. Reducing the dose of NaD so that these movements were not present, also eliminated any respiratory response. PDG 'dose-adjustment' experiments Fig. 3 compared the vagal reflex responses arising from right and left atrial injections of a standard dose of 250 pg of PDG. Left atrial injections consistently produced respiratory responses which at first sight appeared quite distinct from equivalent right atrial injections. It was found however, that the respiratory responses to PDGRA could be matched by an increase of the PDGL,~ dose, and vice versa where a reduced PDGRA dose matched the reflex response to PDGLA (figs. 8A and 9A). In the nine rabbits in this series of experiments, similar responses to right and left atrial injections were usually produced when the left atrial dose was approximately five times greater than the right atrial dose. Pericardial block This technique was applied on 15 occasions in six rabbits in the present studies, in order to block any responses from cardiac receptors to injections of the test
74
D. TRENCIIARD
et al.
(a)
Vllint, lm')
20]
~~¢li,
iIf!~ll! IL~l ! !i.It'I![ ~!1 t
] 0 0 |~
.,,,.,.,.,'......,.,~¥ ~ ."~
~ ....
(mmHg} 0
(b)
,,or,
BP
(ramHg) 0
Fig. 8. Reflex responses to right atrial injections of phenyl diguanide (100 #g) given before (a) and during (b) a pericardial block of cardiac receptors.
chemicals. The results were unequivocal in spite of the weaker control responses after unilateral vagotomy. In every case the respiratory response to NaDRa was unchanged, the response to PDGRa was considerably reduced and virtually limited to a small increase in respiratory frequency (fig. 8B). The response to 250 /~g PDGI. A was abolished completely, but when higher doses of PDGLA were administered, then a small recognisable respiratory responsc remained during the pericardial block (fig. 9B). Additionally, the hypotensive response to both PDGRA and PDGLA were usually abolished or profoundly diminished there were no cardiovascular responses to NaDRa in the control situation. It should be noted that the residual response to PDGRA during pericardial block has a strong resemblance to the NaD~A response, in that it is primarily an effect on the frequency of breathing alone, with little or no effect on tidal volume or blood pressure. In four of the six rabbits, the same volume of local anaesthetic that had previously been used to establish the pericardial block - usually 0.4 ml was injected intravenously. No effects were produced on the reflex responses to P D G and NaD, indicating that systemic absorption of the local anaesthetic had not contributed to any of the effects seen with pericardial block.
75
N O N - M Y E I . I N A T E D LUNG RECEPTORS (a)
t
BP
lO01
" ~"~7
," .
(ramHg) 0
t
(b)
BP (mmHg)
' ' " ~' ' o
15s ~
I
Fig. 9. Reflex responses to left atrial injcctions of phenyl diguanide (1250 /~g) given before (a) and during (b) a pericardial block of cardiac rcceptors. Note the similarity of the control responses to left and right (fig. 8a) atrial injections, when the dose of the formcr is increased.
Discussion
The response characteristics of the non-myelinated vagal afferent nerve endings in the lungs of anaesthetized rabbits, with respect to their pharmacologi~l activation, resemble those o f cat and dog. In addition, their separation into three groups on the basis of their accessibility from the pulmonary or bronchial circulations is similar to the situation in cats and dogs, with the exception that the 'pulmonarybronchial' group have not yet been described in dogs (Coleridge and Coleridge, 1977; Dclpierre et al., 1980). The results can give no indication of the relative numbers of these nerve endings within the three groups. The accessibility of these endings from either the pulmonary or bronchial circulations does not imply clear separation of location within the airways, but the response latencies must reflect the passage of the drug to sites at varying distances from the point of injection. it is best concluded that accessibility from one or other or both circulations reflects a widespread distribution of these endings within the lungs, in contrast to the original idea that these non-myelinated vagal afferent endings (at that time called
76
D. T R E N C H A R D et al.
J receptors) might be predominantly peripheral, and accessible only from the pulmonary circulation (Paintal, 1969). To underline this conclusion that the vagal non-myelinated afferent innervation of the lungs is one overall group, it was shown that when appropriate measures were taken, "pulmonary' and 'bronchial' endings can subserve identical respiratory reflex functions. Thus it was originally assumed that the reflex responses to left atrial injections ofphenyl diguanide were mediated exclusively by the 'bronchial' receptors and that they appeared to evoke a different change in the pattern of respiration from 'pulmonary' receptors (fig. 3). However, the 'dose-adjustment' series of experiments suggested that the different responses to the same dose of phenyl diguanide injected via the right or left atrium may be purely a reflection of the dilution of the drug from the latter injection before reaching the receptors (figs. 8A and 9A). The subsequent studies with pericardial block, however, showed that in the rabbit a large proportion of the reflex respiratory response to phenyl diguanide injected into the right or left atrium arises from stimulation of cardiac receptors. The cardiovascular responses to both PDGRA and PDGLA were abolished by pericardial block, which implies that they must also arise from stimulation by PDG of cardiac receptors. This conclusion can be reached since block of vagal efferent transmission by atropine alone, results in abolition of the bradycardia response to PDG whilst sparing the transient vagal afferent-dependent hypotension. A significant proportion of the respiratory responses to PDGRA and PDG~.A must therefore also be produced by stimulation of cardiac receptors, since both of these were reduced during pericardial block. However, if sufficient PDGLA is administered (fig. 9B) to overcome the dilution problem mentioned above, there is a recognisable residual respiratory response which must presumably originate from 'bronchial" non-myelihated vagal receptors. The PDGLA response produced under these conditions is qualitatively similar to PDGRA alter pericardial block. Quantitative comparisons are difficult to make, but this result strongly suggests that 'pulmonary' and "bronchial' receptors in the rabbit may have the same reflex effect on respiration. 'Pulmonary" and 'bronchial" C-fibres in the dog have identical bronchoconstrictor reflex effects (Roberts" et al., 1981) and indeed it would be surprising if these two groups of receptors had been found to be functionally distinct, since all the evidence suggests that sensory endings with non-myelinated vagal afferents constitute a widespread nociceptive system within the lungs, with different accessibilities from pulmonary or bronchial capillaries depending on their particular location. The only argument against this generalisation is the apparent difference in the chemosensitivity, to bradykinin in particular, of 'pulmonary' and 'bronchial' C-fibre endings in the dog (Kaufman et al. 1980). The studies with NaD in the rabbit present novel evidence to suggest that in appropriate doses, NaDRA provides a specific stimulus to 'pulmonary' vagal nonmyelinated endings, and that the reflex responses elicited by these endings consist of an increase in both the frequency o1" breathing and functional residual capacity: no reflex effects are produced on either tidal volume, heart rate or blood pressure.
NON-MYELINATED LUNG RECEPTORS
77
However, before presenting the evidence in favour of this conclusion, it is important to discuss the use of sodium dithionite, since it has been used as a novel stimulus in these studies. The mode of action of this chemical remains unclear. At first it was assumed that NaD injection might be providing a hypoxic stimulus, (since NaD is a reducing agent), as had previously been suggested for carotid chemoreceptor stimulation (Critchley and Ungar, 1974; Cross et al., 1979). This was rejected since the rabbits in the present studies were kept hyperoxic (Pao: > 300 mm Hg), and carotid artery occlusion did not alter the pattern of response. Additionally, comparable in vitro addition of NaD to rabbit blood samples tonometered at 37°C to give blood gas and pH values comparable to those found in vivo, never reduced Po~ below 150 mm Hg. The in vitro studies did, however, increase Pco2 by 10.-20 mm Hg, which corresponded closely to the effects seen in vivo where there was a transient increase in PETco: by 1 - 3 ~ after injection of NaDRA (fig. 7). (It should be noted that similar effects on Po:, Pco: and pH can also be elicited in vitro by the addition of NaD to a bicarbonate solution that had been tonometred to the same values as the blood samples.) It is therefore more probable that this increase in CO2 (or accompanying increase in H ÷) induced by NaDE~A, provided an indirect stimulus to the non-myelinated nerve endings, by releasing a substantial amount of CO2 from the blood in the pulmonary circulation. NaD does not appear penetrate cell membranes readily, and would tend to remain in the intravascular compartment (Burns and Shepard, 1979); this reinforces the suggestion that it is the CO:-releasing properties of NaD that provides the stimulus to non-myelinated endings. The reasons why we believe that the stimulus provided by smaller doses of NaD does not survive beyond the pulmonary circulation are as follows. The prominent increase in end-tidal Pco: as the test chemical passes through the pulmonary circulation, clearly indicates that a large amount of the released CO2 has escaped from the blood. The possibility remains that a small CO2 stimulus will survive through the pulmonary veins into the left heart, to stimulate receptors not of pulmonary origin. However, the evidence that the response to NaD~A is unaffected by pericardial block strongly suggests that this is not the case. In addition, the total dependence of the reflex response on the integrity of the vagus nerves, and the appearance at higher doses of an obvious peripheral chemoreceptor stimulation, supports the conclusion that provided the dose is small enough, the origin of the reflex is limited to vagal endings in the pulmonary circulation. The residual response to PDGRA during pericardial block undoubtedly originates from pure 'pulmonary' receptor stimulation, and qualitatively is indistinguishable from the responses to NaDRa. This lends further support to the conclusion that NaDRA can serve as a specific stimulus to 'pulmonary' vagal non-myelinated endings. Although the evidence from the present studies has suggested that these 'pulmonary' nerve endings are stimulated by increases in CO2 or H ÷, it is recognised that this has been demonstrated under abnormal conditions. It would therefore be incorrect to make conclusions about a natural CO2-sensor role for these endings
78
D. TRENCHARD et al.
on the basis of the present results alone. However, p r e l i m i n a r y results from current investigations in anaesthetized r a b b i t s are available ( R a y b o u l d a n d Russell, 1982), which show that n o n - m y e l i n a t e d vagal afferent nerve fibres o f thoracic origin can mediate the reflex increase in the frcquency o f breathing in response to hypercapnia. In conclus&n, the present results indicate that the characteristics of the n o n myelinated e n d i n g s in the lungs o f anaesthetized rabbits, strongly resemble those f o u n d in cats a n d dogs. The use o f s o d i u m dithionite as a novel stimulus to the e n d i n g s only accessible from the p u l m o n a r y circulation, c o m b i n e d with pericardial block, has indicated that in rabbits most of the respiratory response a n d all ot the cardiovascular responses classically a t t r i b u t e d to chemoreflexes initiated from these receptors, is in fact due to s t i m u l a t i o n of cardiac receptors. A c t i v a t i o n ot ' p u l m o n a r y ' endings produces o n l y a n increased frequency o f b r e a t h i n g in anaesthetized r a b b i t s : ' b r o n c h i a l ' e n d i n g s a p p a r e n t l y give a similar response provided a sufficient a m o u n t of the activating chemical is given.
Acknowledgement The a u t h o r s arc indebtcd to Professor A . S . Paintal for d c m o n s t r a t i n g the technique o f pericardial block.
References Anand, A. and A.S. Paintal (1980). Reflex effects following selective stimulation of J receptors in the cat. J. Physiol. (London) 299: 553-572. Burns, B. and R.H. Shcpard (1979). Dlo2 in excised lungs pcrfused with blood containing sodium dithionitc (Na2S204). J. Appl. Physiol. 46: 100-.I10. Coleridge, H.M.. J.C.G. Coleridge and J.C. Luck (1965). Pulmonary afferent fibres of small diameter stimulated by capsaicin and by hyperinflation of the lungs. J. Physiol. (l,ondon) 179: 248 262. Coleridge, 14. M. and J. C. G. Coleridge (1977). Impulse activity in afferent vagal C-fibres with endings in the intra-pulmonary airways of dogs. Respir. Physiol. 29: 125-142. Critchlcy, J.A.J.H. and A. Ungar (1974). A chemical method of lowering the Po2 of blood in experimental studies of arterial chemoreceptor reflexes. J. Physiol. (London) 244: 12- 13P. Cross, B.A., B.J.B. Grant, A. Guz, P.W. Jones, S.J.G. Semplc and R.P. Stidwell (1979). Dependence of phrenic motoneuronc output on the oscillatory component of arterial blood gas composition. J. Physiol. (London) 290:163 184. Dawes, G.S., J.C. Mott and J.G. Widdicombe (1951). Respiratory and cardiovascular reflexes from heart and lungs. J. Physiol. (London) 115: 258-291. Dav,es, G.S. and J. t4. Comroe (1954). Chemoreflexes from the heart and lungs. Physiol. Rev. 34: 167-201. Delpierre, S., Y. Jammes and N. Mei (1980). Effects of hypercapnia, hypoxia and increase of tidal volume on vagal bronchopulmonary C fibres in cat. J. Physiol. (London) 298: 48- 49P. Delpierre, S., C. Grimaud, Y. Jammes and N. Mei (1981). Changes ill activity of vagal bronchopulmonary C fibres by chemical and physical stimuli in the eat. J. Physiol. ILondon) 316: 61.-74. Dickinson, C.J. and A.S. Paintal (1970). Stimulation of type-J pulmonary receptors in the cat by carbeon dioxide, tiT&. Sci. 38: 33P.
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79
Guz, A. and D. Trenchard (1971). The role of non-myelinated vagal afferent fibres in the genesis of tachypnoea in the rabbit. J. Physiol. (London) 213:.2,45 371. Jain, S.K., D. -l'renchard, F. Reynolds, M.I.M. Noble and A. Guz (1973). The effect of local anaesthesia of the airway on respiratory reflexes in the rabbit. Clin. Sci. 44:519 538. Kaufman, M.P., D.G. Baker, H.M. Coleridge and J. C.G. Coleridge (1980). Stimulation by bradykinin of afferent vagal C-fibres with chemosensitive endings in the heart and aorta of the dog. Circ. Res. 46:476 484. Paintal, A.S. (1955). Impulses in vagal afferent fibres from specific deflation receptors. The response of these receptors to phenyl diguanide, potato starch, 5-hydroxytryptamine and nicotine, and their role in respiratory and cardiovascular reflexes. Q. J. Exp. Physiol. 40: 8% 111. Paintal, A.S. (1969). Mechanism of stimulation of type J pulmonary receptors. J. PhysioL (London) 203:511 532. Raybould, H.E. and N.J.W. Russell (1982). Afferent activity in pulmonary vagal C-fibres reflexly increases respiratory rate during hypercapnia in the anaesthetized rabbit. J. PhysioL (London) 326:60 61P. Roberts, A.M., M.P. Kaufman, D.G. Baker, J.K. Brown, H.M. Coleridge and J.C.G. Coleridge (1981). Reflex tracheal constriction induced by stimulation of bronchial C-fibres in dogs. J. Appl. Physiol. 51 : 485~1.93. Russell. N.J.W. and D. "Frcnchard (1980). Non-myelinated vagal lung receptors in the rabbit. J. Physiol. (London) 300: 31P. Russell, N.J.W. and D. Trenchard (1981). Chemoreflexes of pulmonary origin elicited by sodium dithionite in the anaesthetized rabbit. J. Physiol. (London) 310: 63-64P. Sellick, H. and J.G. Widdicombe (1970). Vagal deflation and inflation reflexes mediated by lung irritant receptors. Q. J. Exp. Physiol. 55:153 163.